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CHEMISTRY RESEARCH AND APPLICATIONS

ADVANCES IN NATURAL
PRODUCTS DISCOVERY

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CHEMISTRY RESEARCH AND APPLICATIONS

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CHEMISTRY RESEARCH AND APPLICATIONS

ADVANCES IN NATURAL
PRODUCTS DISCOVERY

ANA RITA GOMES, TERESA ROCHA-SANTOS


AND ARMANDO DUARTE
EDITORS

New York
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Library of Congress Cataloging-in-Publication Data

Names: Gomes, Ana Rita, editor. | Rocha-Santos, Teresa, editor. | Duarte,


Armando C., editor.
Title: Advances in natural products discovery / editors, Ana Rita Gomes,
Teresa Rocha-Santos, and Armando Duarte (Department of Chemistry,
University of Aveiro, Campus de Santiago, Aveiro, Portugal).
Description: Hauppauge, New York : Nova Science Publishers, Inc., [2016] |
Series: Chemistry research and applications | Includes bibliographical
references and index.
Identifiers: LCCN 2016038135 (print) | LCCN 2016043908 (ebook) | ISBN
9781536100884 (hardcover) | ISBN  H%RRN
Subjects: LCSH: Natural products.
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615.3--dc23
LC record available at https://lccn.loc.gov/2016038135

Published by Nova Science Publishers, Inc. † New York


CONTENTS

Preface vii
Chapter 1 Bioactive Natural Compounds: Biological Significance and Clinical
Implementation in Organ Pathophysiology 1
Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil
Chapter 2 Natural Therapeutics Against Alzheimer’s Disease: Conventional
Treatment Versus Phytotherapy 55
Abhijit Dey and Anuradha Mukherjee
Chapter 3 Bioactive Constituents from Artocapus 79
Rohaya Ahmad and Mohd Nazrul Hisham Daud
Chapter 4 Maize (Zea mays L.) – An Ethnopharmacological Review 133
Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira,
Simone Kobe de Oliveira, Rosendo Augusto Yunes, Shirley Kuhnen
and Marcelo Maraschin
Chapter 5 Antitumor Sesterterpenoids 167
Lishu Wang, Junfeng Wang and Yonghong Liu
Chapter 6 Recent Studies of Polar Steroids from Starfish: Structures,
Biological Activities and Biosynthesis 191
N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko and V. A. Stonik
Chapter 7 Strategies Based on Microbial Metabolites for Microbial Control in
Industrial Water Systems 225
Vera Lúcia dos Santos and Andrea Sousa Monteiro
Chapter 8 Strategies Based on Microbial Metabolites for Microbial Control in
Agriculture 255
Vera Lúcia dos Santos and Andrea Souza Monteiro
Index 285
PREFACE

The largely unexplored natural world harbors a great biodiversity and provides a unique
and rich source of natural products with interesting pharmaceutical activities and potential
application for environmental protection. In the last years, much attention has been paid to
unraveling the structural, compositional and sequential properties of bioactive compounds,
but the exploring of natural resources needs to be developed wisely and keeping in mind
sustainability principles.
Natural products have often been used in medicine, agriculture, food, fragrances, and pest
control. Probably, due to their easy accessibility, terrestrial plants have been the major source
of medicinal products, especially for traditional or folk medicine. However, only 10% of over
250,000 plants have been investigated for biological activity. On the other hand, the marine
environment contains over 80% of world’s plant and animal species. In recent years, several
bioactive compounds have been extracted from various marine organisms, such as tunicates,
sponges, starfish, soft corals, bryozoans, sea cucumbers among others. The search for new
metabolites from marine organisms has resulted in the isolation of more than 10,000
metabolites, many of which are endowed with pharmacodynamic properties. These natural
products are of high-value commercial due of their natural source, complete biodegradable
properties, lower or no toxicity, and in most cases lower cost compared to those synthetic
chemicals. Despite the biodiversity in the marine environment overcoming that of the
terrestrial environment, the research into the use of marine natural products as pharmaceutical
agents and for environmental applications is still in its early stages.
In this context, this book highlights some of the most recent advances in natural product
discovery from the past years.
In: Advances in Natural Products Discovery ISBN: 978-1-53610-088-4
Editors: Ana Rita Gomes, Teresa Rocha-Santos et al. © 2017 Nova Science Publishers, Inc.

Chapter 1

BIOACTIVE NATURAL COMPOUNDS: BIOLOGICAL


SIGNIFICANCE AND CLINICAL IMPLEMENTATION IN
ORGAN PATHOPHYSIOLOGY

Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil*


Division of Molecular Medicine, Bose Institute. Calcutta-700054, West Bengal, India

ABSTRACT
Nature serves as an infinite source of novel bioactive agents for the exploration in
the field of drug discovery and therapeutic application. Nature-derived compounds are
the most convenient sources in the pharmaceutical industry. The immense diversity in the
chemical structures of several bioactive natural components directs the way to focus on
these natural products as the subject of higher prospect and drug design oriented research.
In the area of new drug discovery, the advancement of technologies such as chemical
compound libraries, modern spectroscopic techniques and highly sophisticated screening
process combined with the computer-graphic model building are the golden opportunities
to ensure the rapid identification of new pharmacologically active biological compounds
from natural product pool. With the aid of modern technological tools, the natural
component based new drug formulations are demanding world-wide attention because of
their lower or no toxicity, complete biodegradable properties, natural source, and, in most
cases lower cost compared to those xenobiotic compounds. Furthermore, extensive
researches on nature derived bioactive compounds are an urgent issue for increasing the
consciousness regarding the protection of biodiversity from the severe destruction of
rain-forest and wild habitats. According to the molecular structure, the biologically active
natural compounds are broadly classified into two categories such as macromolecular and
micromolecular. A variety of herbal proteins are macromolecular compounds. Among the
micromolecular compounds amino acids and herbal secondary metabolites are
remarkable for eliciting pharmacological or toxicological impact on human. Many
proteins, identified from herbal sources, (e.g., Cajanus indicas, Phyllanthus niruri,
Opuntia ficus-indica, Ulmus davidiana Nakai, Morus indica Linne, Cudrania tricuspidata
Bureau, etc.) play beneficial roles in combating oxidative insults. Apart from the plant

*
Corresponding author. Division of molecular medicine Bose Institute P-1/12, CIT Scheme VII M, Calcutta-
700054 West Bengal, INDIA. E-mail: parames@jcbose.ac.in/ parames_95@yahoo.co.in.
2 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

proteins, conditionally essential amino acid (taurine), flavonoids (morin, quercetin) and
xanthones (mangiferin) have sound reputation for multitude of therapeutic applications in
the prevention of various diseases such as hypertension, diabetes, asthma, nephropathy,
atherosclerosis, Rheumatoid, Alzheimer, Parkinson and cardiovascular diseases. The
possible signaling mechanism(s) involved in the ameliorative pathways of their biological
activities has been discussed in the light of experimental studies. Clinical trials are
usually carried out to determine the consequence of investigation. In typical physiological
prominence, the endogenous antioxidants maintain the balance of the magnitude of ROS
formation. A variety of drugs and environmental toxin exposure to the cells results in
oxidative stress. The cells undergo through a considerable impact that is leading to
cellular malfunction, or disease. Therefore, a better unconventional medication is
indispensable to serve as clinical tools against oxidative stress-related complications.
These natural bioactive compounds can aid as complementary and alternative medicine in
the clinical prospect. So, this chapter has aimed to be a practical guide to evaluate the
perspective and opportunities of natural bioactive molecules in the field of organ
pathophysiology.

Keywords: oxidative stress, reactive oxygen species, antioxidant, signaling mechanism,


organ pathophysiology, therapeutic applications

INTRODUCTION
From the historical aspect, the medical science was practically set up since the
manifestation of civilization. The utility of nature derived medicines around the world can be
traced to the ancient eras. Likewise, the therapeutically multidirectional utilization of plants is
found to be described in the “Rig Veda”, “Susruta Samhita”, “Charaka Samhita”, “Nei
Ching”, “De Materia Medica”, etc. having been written around 4500-1600 BC. However, in
India various plants are therapeutically employed since the Ayurvedic and Unani formulations
of medicine. At the initiation stage, those plant based formulations were applied mostly in the
crude form of extracts which served as the basis for novel drug discovery. With the aid of
modern scientific tools, recent research trends in this field throughout the globe are highly
optimistic for discovering efficacious nature derived bioactive molecules due to their less
toxicological impact along with the source of complementary and alternative medicine [1-7].
These are the renewable source of medicine, and therefore their applications for the treatment
of several diseases have been enhanced day by day. However, it is to be mentioned that the
rapid destruction of rain forests are the serious threats for the sustainable uses and future
discovery of bioactive compounds. In this context, a worldwide awareness of protecting the
biodiversity is needful not only to save the natural bioactive ingredients but also for the
progress of nontoxic drug discovery in future. The global demand has been raised up to
develop nontoxic alternative medications. As a consequence, this may enrich the focus of
pharmaceutical research on nature derived bioactive molecules for the new possibilities of
drug development as well as favourable for the large scale industrial production. In this
regard, more sophisticated technological tactics are also necessary for the development of
nature originated ingredients into innovative drugs. To date, in research oriented therapeutic
approaches of the novel bioactive ingredients (plant derived proteins, amino acids, flavonoids
Bioactive Natural Compounds 3

and xanthones) against multiple organ pathophysiology (diabetes, drug toxicity, metal
toxicity, etc.) clearly marks a significant advancement in the clinical implementation of those
natural compounds [8-50].
This book chapter comprises some brief discussion on the clinical efficacy of nature born
bioactive molecules to combat various ailments. Furthermore, this chapter could be a valuable
resource for the investigators to explore an overview of modern trends leading to emerging
pharmaceuticals in this remarkable field. The possible signaling pathways involved in their
biological activities have been emphasized to deliberate the significant exploration.

MACROMOLECULAR BIOACTIVE INGREDIENTS:


BENEFICIAL ROLE AGAINST OXIDATIVE STRESS
A variety of proteins and glycoproteins has been isolated from herbal plants and has been
proven to be effective therapeutic strategies for the amelioration of certain ailments.
Researchers reported that the medicinal properties are generally attributed to the structural
variability, which relies on the molecular mass, the degree of branching, and the conformation
of sugar residues of different glycoproteins [51, 52]. However, these proteins
and glycoproteins could be expected to be a therapeutic promise in alternative medicine
(Figure 1A).

Figure 1A. Different targets and signaling pathways modulated by glycoprotein.


4 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

Background of Opuntia ficus-indica (OFI) Glycoproteins

The Opuntia ficus-indica is a species of cactus that has long been utilized as a traditional
health food and folklore medicines. In search of natural alternative medicines, the
investigators have isolated a 90kDa (OFI) glycoproteins from the plant. The protein
comprises of 37.54% carbohydrate contents and 62.46% protein contents [53]. The
investigators also checked the biological activity of the OFI glycoprotein and found that the
glycoprotein had scavenging activities against oxygen radicals in cell-free systems [53].
Moreover, to investigate the clinical significance of OFI glycoprotein, it was applied against
oxygen radical-induced NIH/3T3 cells and found that the 90kDa OFI glycoprotein possesses
cytoprotective along with anti-apoptotic activities [53]. That incident caught the attention of
the investigators, and further researches were conducted to find out the biological effect of
OFI glycoprotein in animal models.

Protective Effect of OFI Glycoprotein against Triton WR-1339 Intoxication

Triton WR-1339 administration elevated the levels of plasma cholesterol and triglyceride.
In the year 2006, Oh and Lim conducted their study with this non-ionic detergent and found
that intoxication with Triton WR-1339 induces the influx of plasma [54]. Furthermore, Triton
WR-1339 administration in mice reduced the antioxidant enzyme activities (SOD, CAT,
GPx) in association with the increased TBARS and NO levels [54]. Treatment with OFI
glycoprotein before the toxin exposure could maintain normalcy in the abovementioned
parameters. The authors also confirmed that OFI glycoprotein administration itself did not
alter the level of plasma NO and other parameters. It is noteworthy to mention that the
authors found OFI glycoprotein administration could upregulate and appeared to elevate the
activities of CAT and GPx more than that of the SOD. These findings indicated the efficacy
of the inhibitory effect of OFI glycoprotein on lipid peroxidation during Triton WR-1339-
induced hyperlipidemia. Later, the same group cultured BNL CL.2 cells (murine embryonic
liver cells) and induced oxidative stress using the glucose/glucose oxidase (G/GO) system.
Treatment with the OFI glycoprotein improves the cell viability as suggested from their
experimental evidence. Moreover, the authors were convinced that treatment with OFI
glycoprotein resulted in the inhibition of G/GO system-induced ROS production in BNL
CL.2 cells in a dose-dependent manner [54]. All together data from both in vitro and in vivo
studies supported that OFI glycoprotein has the beneficial role against various toxicant
exposures.

Anti-Allergic Efficacy of OFI Glycoprotein

Most herbal products have the characteristic of modulating inflammation, immunity, and
allergy-related factors. The antioxidant properties of OFI glycoprotein motivated the
investigators to take experimental approaches to find out other favourable effects of this
glycoprotein. In 2010, Kye-Taek Lim designed a study to check the anti-allergic properties of
Bioactive Natural Compounds 5

OFI [55]. There are many synthetic compounds which can act as allergens by eliciting
allergic reactions in cells. Hypersensitivity reactions occurred via mast cells in response to
IgE-dependent or independent stimuli. Likewise, in a study Lim utilized compound 48/80
which could interact with mast cells via phospholipase D and heterotrimeric GTP-binding
proteins [56, 57]. In response to the allergens, many pro-inflammatory cytokines and
chemokines are released via the activation of basophils and mast cells. Lim observed that
therapeutically utilization of OFI glycoprotein against allergic hypersensitivity conferred
protection in BALB/c mice in a significant dose-dependent manner [55]. Rat mucosal mast
cells and RBL-2H3 cells are not sensitive to the compound 48/80. Therefore, the author
cultured the RBL-2H3 cells and carried out the subsequent experimental studies. The result
suggested that treatment with OFI glycoprotein effectively lower the release of LDH in those
cells. Moreover, the OFI glycoprotein therapy was found to be effective against allergen-
mediated histamine release and β-hexosaminidase (degranulation) in BRL-2H3 cells. All
these data showed that the authors appear to be successful with the probable anti-allergic
efficacy of OFI glycoprotein in the animal system. The outcome from in vivo studies clearly
depicted the anti-allergic immunomodulatory response of this OFI glycoprotein. Attenuation
of immediate allergic reactions along with inhibition of IL-4 expression in mice supported the
expectation of the authors [55].

Molecular Signaling of the Anti-Allergic Efficacy of OFI Glycoprotein

The outcome of the earlier studies helped the authors to investigate the molecular
signaling behind this anti-allergic response. First of all, the author shaded light on the effect
of OFI glycoprotein on the transcription factor NFκB, the key regulator of pro-inflammatory
responses. Allergen mediated activation of NFκB interacts with specific target genes, such
as COX-2 leading to increased inflammatory processes. Moreover, the activated NFκB
influences i-NOS to synthesize NO in order to enhance the oxidative stress. In this
connection, the MAPKs also played a crucial role and regulated the expression of
inflammatory cytokines. The author elucidated the effectivity of OFI protein against these
inflammatory mediators and found that OFI glycoprotein could attenuate the activation of
NFκB, COX-2, i-NOS and ERKs. The western blot data supported the amelioration efficacy
of OFI glycoprotein against these pro-inflammatory inducers [55]. The overall study showed
that OFI could modulate the pro-inflammatory response via,

(i) Inhibition of histamine, β-hexosaminidase release, lactate dehydrogenase (LDH), and


interleukin 4 (IL-4) in the compound 48/80-treated ICR mice serum.
(ii) Inhibition of the transcription factor NFκB along with attenuation of downstream
signaling cascades (COX-2, i-NOS and ERKs).

The author concluded that OFI glycoprotein could be used for the prevention or treatment
of mast cell-dependent allergic diseases.
6 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

Phytomedicinal Role of Ulmus davidiana Nakai and Isolation


of UDN Glycoprotein

Ulmus davidiana Nakai has been widely distributed in the region of China, Japan and
Korea. The leaves and bark extracts have been used as traditional medicines for many years.
Lee et al. (2004) isolated and identified a noble 116kDa glycoprotein from Ulmus davidiana
Nakai (UDN glycoprotein) to homogeneity [58]. They determined the molecular weight of
UDN using SDS-PAGE. Primarily, the authors found that the glycoprotein has strong free
radical scavenging power. Furthermore, they designed a study where they speculated that
UDN glycoprotein has anti-apoptotic property against 12-O-tetradecanoylphorbol 13-acetate
(TPA)-stimulated NIH/3T3 cells. The data from western blot analysis, electrophoretic
mobility shift assays (EMSA) and NO assays confirmed that UDN treatment could modulate
PKCα translocation, NF-κB DNA binding activity, NO production, and apoptosis in TPA-
stimulated NIH/3T3 cells but failed to regulate the DNA binding activity of AP-1 [58].

Modulation of Cellular Injury: Scavenging Role against ROS and RNS

A couple of years later, a group of investigators found that UDN glycoprotein therapy
could ameliorate 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated intracellular
injuries in BNL CL.2 cells [59]. They used TPA because TPA could act as a promoter of the
iNOS expression in activated macrophages and hepatocytes [60]. UDN treatment could
ameliorate the TPA-induced cellular viability as evidenced from LDH assay [59]. With
increasing the dose of UDN concentration the LDH activity was reduced. Besides, this
glycoprotein could modulate TPA-induced ROS and RNS generation in a dose-dependent
manner. Both the outcomes from DCFDA assay and measurement of NO production showed
satisfactory results for UDN therapy in the cells. The investigators did not explore the
complete molecular mechanism behind the incident. They reported that UDN treatment could
down regulate the expression level of the p50 subunit of NFκB in a dose-dependent manner
for the cytoprotection [59].

Amelioration against Cadmium Chloride Mediated Cell Cycle


(G0/G1) Arrest

A recent study from Lee and Lim (2011) speculated that therapeutic application of UDN
against cadmium chloride intoxication confers cell cycle progression besides inhibition of
ROS in mouse primary cultured myelocytes [61]. UDN could promote cellular protection by
maintaining the metabolic activity of the cells, as supported from the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, the authors depicted the
inhibitory effect of UDN glycoprotein on the mobilization of intracellular Ca2+ in primary
cultured mouse myelocytes exposed to cadmium chloride. The intracellular Ca2+ level was
upregulated up to 1.40 fold in cadmium chloride intoxicated cells, whereas UDN therapy
could maintain normalcy in those cells. The DCF fluorescence intensity data represented that
UDN could scavenge ROS extensively in a dose-dependent manner. Intracellular ROS and
Bioactive Natural Compounds 7

Ca2+ level played a critical role in cell cycle arrest at the entrance from G0/G1 phase by
inhibiting the activity of CDKs. The S phase depends on p21 and p53 as CKIs. Therefore, the
authors checked the effect of this glycoprotein therapy regarding the cell cycle issue. The
percentage of cell number obtained from FACS data reported that UDN glycoprotein could
ameliorate cell cycle arrest. Besides, the data from immunoblot analysis clearly depicts the
beneficial activity of UDN glycoprotein on cyclin D1 and CDKs expression level.
Interestingly, the authors also checked the protective effects of UDN on the expression level
of p21, p27 and p53 proteins. Overall data suggest that UDN provides cytoprotection via
inhibition of cadmium chloride mediated cell cycle (G0/G1) arrest [61].

Background of Cajanus indicus and Basics of Cajanus indicus Protein (CI)


Isolation, Purification

Since ancient times, many plants are therapeutically employed for the formulation of
herbal medicines. Among them, Cajanus indicus L is a popular one, and it is cultivated
mostly in the region of Asia and Africa for the seeds. Seeds are used as pulses and are rich in
proteins. In rural culture, the aqueous extract of the leaves of Cajanus indicus is
therapeutically utilized as an antidote of jaundice and hepatomegaly. A group of researchers
(Dutta et al. 2001) isolated a protein molecule from the leaves of this plant and further
confirmed its phytomedicinal role against carbon tetrachloride and beta galactosamine-
induced hepatic injury [62-64]. A few years later, another group of researchers (Sarkar et al.,
[65]) isolated another protein from the same source and with the help of some biological
assays they purified the protein to homogeneity. Furthermore, with the evidence from SDS-
PAGE they confirm the molecular mass of this hepatoprotective protein as approximately 43
kDa [65]. During the analysis of the amino acid sequence of this protein, it was found that
some part of its sequence partially matched with plastocyanin while another part showed a
few amino acid sequence similarity with a protein present in tomato. As the structural detail
of the former was not known, it is still uncertain whether these two proteins the same or not.

Amelioration of Chemical Toxicity: CI Protein as Antioxidant Remedy

Most of the chemical agents, used for industrial purpose, are the potent inducer of
cytotoxicity. It is noteworthy that CI protein played positive impact against various chemical
induced toxicity [66-71]. These chemicals may produce free radicals during their
biotransformation. Once formed, these free radicals could accelerate a series of oxidation
reactions and produce detrimental effects on living systems. The investigators found that CI
protein could scavenge free radicals in cell-free systems [72]. Following the result, the
researchers utilize this property of CI protein in the biological system under various
pathophysiological conditions. The investigators found that CI protein confers its
hepatoprotection against chloroform and thioacetamide mainly by up-regulating the activities
of antioxidant enzymes (such as SOD, CAT, GST, GPx, etc.) and scavenging ROS both in
vivo and in vitro. Furthermore, from the histopathological evidence it was clearly established
that these types of chemical induced widespread tissue damages were less severe in the
murine livers when treated with CI protein prior and post to the toxin exposure [66, 70].
8 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

Besides hepatoprotection, the investigators also showed that this protein possesses the ability
to ameliorate renal protection [73]. Comparable shielding action of CI protein was observed
against galactosamine induced nephrotoxicity [73]. Both prior and post treatment with CI
protein against the toxin administration showed beneficial effects in the experimental mice.
The protein has also been found to play beneficial role against metal (sodium fluoride,
mercuric chloride, cadmium chloride) induced cytotoxicity [35, 67, 73, 74]. Moreover, the
antioxidant property of this protein was successfully compared with various renowned
antioxidants e.g., Vitamin E, N-acetyl cysteine, etc. To explore the mechanism of its
protective action, the investigators found that CI protein could prevent the release of marker
enzymes (ALP, ALT, Creatinine, Urea, etc.) in the cytosol and could scavenge ROS [35, 67,
73, 74]. Furthermore, CI protein could enhance cellular antioxidants power and normalizing
the effect of cytokines (different cytokines like TNF-α, IL-6 and IL-1B) [35, 67, 73, 74]. With
these findings, the authors conclude that CI protein acts as an antidote against environmental
toxic substances.

CI Protein as an Antidote against Drug Toxicity:


Beneficial Role in Organ Pathophysiology

Drug metabolism is closely related to clinical trials. The outcome of the clinical trials
determines the fate of the drugs regarding its mode of action, efficacy and toxic effects (if
any) [48]. Acetaminophen is a well-known drug, previously prescribed very often for the
analgesic purpose. With the advancement of clinical prosecutions, it has been established that
during the metabolism of this drug via cytochrome p450 enzymes, a highly reactive
secondary metabolite, N-acetyl-p-benzoquinone imine (NAPQI) is produced [48, 75-77].
Once formed, NAPQI could bind with the proteins and form acetaminophen-protein adducts
[78]. As a result, cellular expiry occurs [12, 15, 20, 79-84]. With the grace of modern science,
the investigators detected that both in humans and in experimental animals the administration
of acetaminophen produces reactive oxygen species (ROS) and reactive nitrogen species
(RNS) [85-88]. These ROS and RNS progressively result in hepatic as well as renal
pathophysiology [85-88].
Attempts were, therefore, made to clarify the segments of this type of drug bioactivation.
Continuous effort revealed that treatment with CI protein could scavenge NAPQI mediated
ROS and RNS formation. However, the preliminary experimental outcomes showed that CI
protein has positive impact on different marker enzymes (namely, glutamate pyruvate
transaminase and alkaline phosphatase, creatinine and blood urea nitrogen) and activities of
different antioxidant enzymes (namely, superoxide dismutase, catalase, glutathione-S-
transferase) [89]. Furthermore, CI protein could modulate the lipid peroxidation end-products
and glutathione in both liver and kidney [89]. Furthermore, histological evidence revealed
that acetaminophen-induced necrotic tissue damages were protected with the simultaneous
treatment of CI protein. This observation was justified through flow cytometry, DNA
fragmentation analysis and TUNEL assay. Moreover, the drug may induce apoptosis in
certain doses in hepatocytes, and that could also be effectively modulated through CI protein
[89]. After achieving this ameliorative efficacy of CI protein against acetaminophen-induced
intoxication, a question comes, “how does CI protein confer its cytoprotective role in this
pathophysiological situation?” Data suggested that CI protein could modulate acetaminophen-
Bioactive Natural Compounds 9

induced up-regulation of CYPs, TNF-α, serum nitrite; counteracted the acetaminophen-


induced loss in mitochondrial membrane potential, reduction in cellular adenosine tri
phosphate (ATP) level and the increase in calcium level. Moreover, the intracellular signaling
based studies showed that the protein exerts its ameliorative action via activation of NFκB,
Akt and down-regulation of STAT-1 pathways [89]. In summary, supplementation with CI
protein warranted complementary and alternative medication against acetaminophen mediated
organ pathophysiology.
Doxorubicin (DOX) is one of the most common anthracycline antitumor drugs. Its
medical utilization has now been constrained due to numerous serious acute and chronic side
effects. Recently, a couple of studies were carried out to check the medicinal efficacy and
therapeutic implementation of CI protein against DOX-mediated detrimental effects [90, 91].
The outcome of both of the studies established neuro as well as nephro protective role of CI
protein. DOX exposure could enhance ROS formation and thereby inducing neurological
impairments in the brain tissue. Furthermore, DOX administration altered the brain specific
coenzymes (like acetyl coenzyme, monoamine oxidase, etc.), ATPases (like Na(+)/K(+),
Ca(2+), etc.) and brain biogenic amines levels. The molecular mechanistic approach
suggested that CI protein could convey its ameliorative action via attenuation of DOX-
mediated ROS formation, nuclear translocation of NFκB, maintenance of physiological
balance of Bcl-2 family proteins and inhibition of all the apoptotic indices (decreased
mitochondrial membrane potential, cytochrome c release in the cytosol, increased levels of
Apaf1, caspase-9/3, cleaved PARP protein) [90]. Histopathological studies also supported the
neuro-protective role of CI protein [90]. The same group also established the renal protective
role of this CI protein against DOX exposure. Their study showed that CI protein could
significantly ameliorate DOX-induced renal dysfunction via maintaining physiological
normalcy in blood urea nitrogen (BUN), creatinine, uric acid, TNF-α, urinary γ-glutamyl
transpeptidase (γ-GT) activity, total urinary protein and urinary glucose level. Besides, the
data suggested that CI protein applied a protective influence against DOX-induced all the
programmed cell death (PCD) stimulus like, MAPKs activation, NFκB translocation,
alteration of ATPase, mitochondrial membrane potential along with caspase activities [91].
Combining, results suggest that CI protein might act as a remedial supplementation in Dox-
induced organ pathophysiology (Figure 1B).

Background of Phyllanthus niruri

Phyllanthus niruri (P. niruri) is a traditional herbaceous plant of Euphorbiaceae family,


found in different regions of India and some other Asian countries, has a sound reputation as
traditional herbal medicine systems like Indian Ayurveda, Traditional Chinese Medicine, etc.
for over 2000 years. Phyllanthus niruri is a well-known herb having phytomedicinal activities
against various pathological states [92-99]. Since early days the herb has been used widely for
the treatment of jaundice [92, 97-99] and these evidence suggest that Phyllanthus niruri is
beneficial for hepatic problems. Besides, no side effects were found after its therapeutic
utilization in clinical trials [93, 94, 98]. All these evidence made Phyllanthus niruri a subject
of the research topic in modern medical sciences. The researchers were trying to explore the
responsible bioactive molecule for its phytomedicinal property. In connection with those two
10 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

active ingredients, phyllanthin [95] and corilagin [96] were isolated and well characterized
from the organic extracts of this herb.

Isolation and Purification of a Protein Molecule from Phyllanthus niruri

From previous decades, enormous investigations were carried out to identify the basic
bioactive molecule(s) from the herb Phyllanthus niruri. The investigators explored that not
only the aqueous extract but also the protein isolate of this herb retain antioxidant activity [97,
98, 100-103]. All these information played a motivational influence on the laboratory
researchers, and further innovative researches were carried out on the herb. Ultimately, after a
long period of time, our group was successful to ascertain a 35 kD novel antioxidant protein
molecule (PNP), isolated and purified from the aqueous extract of this herb to homogeneity.
After that, its amino acid sequence was partially determined by tryptic digestion along with
subsequent MALDI-TOF as well as LC-MS analyses of the four peptide fragments [104]. It
was found from NCBI non-redundant protein database analysis, that the protein molecule is
unique and retains no match with any other proteins [104].

Figure 1B. Schematic diagram of the doxorubicin (DOX) induced nephrotoxicity and its modulation by
treatment with CI protein. DOX increased ROS production phosphorylates MAPKs. The activated
MAPKs can alter the balance of proapoptotic and anti-apoptotic members of the Bcl-2 family in favour
of the proapoptotic proteins, promoting the mitochondrial membrane permeability transition (MPT)
pore opening. The MPT eventually results in loss of mitochondrial membrane potential, the release of
cytochrome c and cell death. Both intrinsic and extrinsic apoptotic pathways are involved. CI treatment
ameliorates these events.
Bioactive Natural Compounds 11

Phyllanthus niruri Protein (PNP) against Environmental Chemical Toxin

Many chemical toxins (like carbon tetrachloride, chloroform, mercuric chloride, sodium
fluoride, thioacetamide, tertiary butyl hydro peroxide, etc.) are utilized in the modern society
for industrial purpose. The biotransformation of these chemicals to highly reactive
metabolites could initiate cellular toxicity. Investigators observed that one such chemical,
tertiary butyl hydro peroxide (TBHP), could persuade oxidative insult in different organs, like
liver [105], testes [106], etc. mainly via the mobilization of arachidonic acid (AA) from
membrane phospholipids under cytotoxic conditions. Thus TBHP exposure leading to an
increase in intracellular AA and malondialdehyde formation and ultimately cell death.
However, Sarkar and Sil [107] found that PNP treatment of hepatocytes exposed to TBHP
improved cell viability and inhibited LDH leakage to confer its shielding effect against
TBHP induced cytotoxicity. Their study further revealed that TBHP exposure reduced the
GSH/GSSG ratio, antioxidant enzyme activities, caused injury in cellular mitochondria,
disrupted mitochondrial membrane potential by altering the levels of Bcl-2 family proteins
and facilitated cytochrome c release in the cytosol. In addition, DAPI staining, flow
cytometric analyses and caspases activities determination depicted that TBHP introduced
apoptosis in hepatocytes. PNP treatment, however, efficaciously encountered these alterations
in cellular level to maintain normalcy in hepatocytes.

Prophylactic Activity of PNP against Iron Overload


Induced Oxidative Stress

In a living system, iron is present as heme and nonheme complexes of biomolecules like
hemoglobin [108], myoglobin, cytochromes and numerous iron-containing enzymes. Both
deficiency and excessive iron produce various pathophysiological circumstances (iron-deficit
anemias, hereditary hemochromatosis, thalassemia, etc.) in the human biological system.
These pathophysiological situations are associated with the overproduction of free radicals.
Whenever, Hydroxyl radicals formed via Fenton reaction, extravagantly induces oxidative
stress by causing oxidation of lipids and proteins that ultimately leads to cell death [109]. A
group of investigators found that exposure of hepatocytes to iron caused enhanced reactive
oxygen species (ROS) production, lipid peroxidation, protein carbonylation, depleted
intracellular glutathione, reduced the intracellular antioxidant power (FRAP) and leading to
loss of cell viability [110]. They further explored the signaling mechanism and found that iron
overload disrupted mitochondrial membrane potential to cause apoptosis mainly following the
intrinsic pathway (via the down-regulation of IкBα with a connected up-regulation of NF-
kB). Furthermore, Mitogen Activated Protein Kinases (MAPKs) are closely involved in this
circumstances. The investigators found that iron-induced ROS formation caused the
phosphorylation of ERKs and p38 MAP kinases. On the other hand, PNP neutralizes iron-
induced oxidative stress, suppresses caspase-3 activation, regulates the balance of Bcl-2
family proteins and protects hepatocytes from apoptotic cell death.
Another essential serine/threonine protein kinase that plays a vital role in cell survival
pathways by inhibiting apoptosis is Akt. Bad could be phosphorylated by Akt to form
homodimer Bad-(14-3-3) and lost the pro-apoptotic property. Activation of Akt requires the
activation of PI3K and might promote cellular survival [111]. It was found that phospho-Bad
12 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

level has been enhanced in the hepatocytes upon PNP treatment and thereby eliminating the
risk of iron-induced apoptotic pathways [110]. Activation of Bim is also associated with iron-
overload-induced oxidative insults. Accumulation of Bim to mitochondria depends on ROS
production. ROS may directly affect Bim interaction with microtubules, leading to its
translocation to neighbouring mitochondria [112]. These result in the activation of Bax and
Bak, which further leads to the permeabilization of the mitochondrial outer membrane
(MOMP). MOMP releases cytochrome c and subsequently activates the caspase cascade. On
the other hand, PNP activates PI3k/Akt pathway, inhibits Bim as well as Bax activation and
thereby protects hepatocytes from iron-intoxicated apoptotic death. Therefore, PNP could
confer its amelioration effect against iron overload by the combination of (i) scavenging free
radicals thereby inhibiting ROS formation; (ii) enhancing the cellular defense mechanism
against iron-mediated cytotoxicity; (iii) inhibiting the activation of MAPKs; (iv) maintaining
the physiological balance of Bcl-2 family proteins and inhibiting the subsequent
mitochondrial permeabilization and (v) blocking the apoptotic signaling by activating the
PI3K/Akt pathway.

Phytomedication with PNP: Modulation of Drug Induced


Organ Pathophysiology

Conventional (Non-Steroidal Anti-Inflammatory Drugs) NSAIDs mediated ROS


formation may be clinically utilized to suppress the risk of gastrointestinal-related cancers
[113, 114]. Nowadays, modulation of redox biochemistry denotes successful approach in
cancer anticipation. According to literature, regular use of aspirin (ASA) reduces the risk
of cancers, modulating mast cell degranulation, COX expression, the release of pro-
inflammatory cytokines and induces apoptotic death in hepatocellular carcinoma cells [115,
116]. Moreover, a majority of drugs are metabolized in the liver. Clinically suitable drugs
cause severe cellular damage through metabolic activation of the compound to highly reactive
substances such as free radicals. Liver and spleen are the vital organs which are very much
susceptible to ASA-mediated varied damages. In recent years attention has been focused in
search of a safe antidote which could combat ASA-mediated apoptotic complications without
hindering its target mediated efficacy. A very recent study is highly significant in this regard
[117]. Aspirin administration in mice enhanced serum marker (ALP) levels, reactive oxygen
species (ROS) generation, reduced antioxidant power and altered oxidative stress related
antioxidant enzyme activities (SOD, CAT, GST, GPX, GSH, etc.) in both the liver and spleen
tissues. At the molecular level, the apoptotic/necrotic death of hepatocytes was investigated
using Fluorescence-Activated Cell-Sorting (FACS) technique. Data suggest that majority of
hepatocytes followed apoptotic pathway in ASA exposure. Besides, TUNEL-positive
hepatocyte’s nuclei were also detected in the terminal deoxynucleotidyl transferase mediated
dUTP nick end-labeling (TUNEL) assay in ASA-administrated mice. Furthermore, the
histopathological evidence supported that ASA-induced hepatic, as well as splenic tissue
damages, are apoptotic in nature. On the other hand, PNP possesses amelioration effect in all
over the study without exhibiting any side effects.
After determining the ameliorative action of PNP against NSAIDs, the investigators
aimed to explore what signaling pathways PNP could utilize to convey its therapeutic
applications. The results show that PNP could protect organs by i) scavenging free radicals
Bioactive Natural Compounds 13

thereby inhibiting drug-mediated ROS formation; ii) enhancing the antioxidant enzyme
activities and maintaining the proper glutathione balance ratio; iii) ameliorating ASA-
mediated inhibition of NF-B, activation of anti-apoptotic Bcl-2 proteins as well as inhibition
of MAPKs activation; and iv) activating Akt/PI3k mediated cell survival signaling pathways.
Combining, the outcome of all these studies clearly suggests that the protein could
efficiently prevent the alterations of the ASA-induced liver as well as splenic damages and
maintains the normalcy of both the organs (Figure 1C) [117].

Figure 1C. Schematic diagram of the aspirin (ASA) mediated hepatotoxicity and its prevention by PNP
treatment. ASAinduced ROS production phosphorylates MAPKs. The activated MAPKs can alter the
balance of proapoptotic and anti-apoptotic members of the Bcl-2 family in favour of the proapoptotic
proteins, promoting the mitochondrial membrane permeability transition (MPT) pore opening. The
MPT eventually results in loss of mitochondrial membrane potential, the release of cytochrome c and
cell death. Both intrinsic and extrinsic apoptotic pathways are involved. PNP treatment prevents these
events by activating PI3k/Akt cell survival signaling pathways.

Phytoglycoprotein (75 kDa) Isolated from Cudrania tricuspidata Bureau

The plant Cudrania tricuspidata Bureau has been utilized as an alternative herbal
medicine for the treatment of inflammation and cancer since ancient times. Various parts of
the plants were clinically employed since earlier times. Likewise, the extract form of the fruits
and barks were employed therapeutically to cure diseases like inflammation, lumbago,
hemorrhages, and contusions [118, 119]. With the advancement of science, the researchers
14 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

wanted to explore the bioactive molecule present within the crude material of the herbal
drugs. Ultimately, the investigators have isolated a 75 kDa glycoprotein (CTB) from the
plant. This CTB glycoprotein was found to be the major biofunctional molecule, responsible
for the medicinal property of that plant [120, 121].

Prophylactic Effect of CTB against Inflammation

In 2009, Shim and Lim found that CTB glycoprotein regulated the inflammatory reaction
stimulated by bisphenol A (BPA) in human mast cells (HMC-1). Further studies of the group
showed that CTB could modulate the BPA-mediated pro-inflammatory signaling cascades via
the attenuation of NFκB and AP-1; inhibition of MAPKs (mainly ERKs and JNKs) family
proteins expression along with degranulation of histamines [120]. In addition, the gene
expression of cytokines (IL-4, IFN-γ, IL-1β and COX-2) were also regulated by CTB
treatment. Another group of researcher found that CTB (75 kDa) has strong potential against
allergic inflammation caused by bisphenol A (BPA) in both BALB/c mice and RBL-2H3
cells [121]. BPA-stimulated the IgE-dependent allergic diseases [122]. CTB treatment could
effectively neutralize this IgE production as evidenced from Enzyme-linked immunosorbent
assay (ELISA). The authors conducted β-hexosaminidase secretion and histamine release
assay to check the anti-inflammatory responses of CTB treatment in animal models. The
result showed that CTB could modulate the production of both of these parameters.
Moreover, CTB therapy could protect the cell viability along with inhibition of NO
production in RBL-2H3 cells. It is notable that CTB could modulate the BPA-induced
activation of COX-2, ERKs and P38 MAPKs. The Reverse transcription-polymerase chain
reaction (RT-PCR) and Real-time polymerase chain reaction data confirm that CTB
efficiently down-regulated the BPA-stimulated release of cytokines (IL-6, IL-1β) along with
Tumor Necrosis Factor alpha (TNF-α) expression level [121]. Overall results suggest that
bioactive molecule CTB glycoprotein possesses both anti-inflammatory and cytoprotective
activities.

CTB Glycoprotein as Antioxidant Supplement against Oxidative Stress

Recently, the investigators evaluated the antioxidant property of CTB [123] by


investigating its role against di(2-ethylhexyl) phthalate (DEHP)-induced differentiation of T
helper (Th) type 2 cells in primary cultured thymocytes. The results showed that CTB could
promote the percentage of cell viability along with inhibition of DEHP mediated intracellular
ROS production. The DCF signal supported the result. Later, the authors extended their study
to determine the molecular mechanism behind the protection. They found that CTB could
attenuate the P38 MAPK along with GATA binding protein-3 activation dose-dependently.
Furthermore, the RT-PCR analysis suggested that CTB treatment could ameliorate the
DEHP-mediated cytokines responses (IL-4, IL-10 and IFN-γ) in primary thymocytes. On the
basis of these confirmations, the authors concluded that CTB glycoprotein supplementation
could be a useful antidote for prevention of Th2 cell response-related immune diseases [123].
Bioactive Natural Compounds 15

Background of Morus indica L Glycoproteins

The demands of natural herbal based medications are continuously increasing day by day.
These are one type of complementary as well as safe medicines having positive effects on
health with little or no side effects. In this connection, Morus indica L (MIL), belonging to
the family of Moraceae, was paid increased attention of the investigators since a couple of
decades. In the year of 2000, Doi et al., for the first time, reported the free radical scavenging
activity of the crude plant extract [124]. After that, another group of researchers explored the
anti-diabetic effect of this plant extract [125, 126]. On the basis of these findings, research
was extended to find out the bioactive molecule responsible for its protective action.
Recently, a group of scientists purified a 32 kDa glycoprotein from MIL consisting of 59.97%
proteins and 40.03% carbohydrates [127]. Further investigations were performed by the same
group to check the anti-inflammatory and antioxidant properties of the MIL glycoprotein.

Immunomodulatory Effect of Morus indica L Glycoproteins

Oh et al. conducted their study to evaluate the immunomodulatory role of MIL


glycoprotein against carbon tetrachloride (CCl4)-induced hepatic pathophysiology [127]. The
data from their study suggested that MIL glycoprotein treatment could ameliorate CCl4-
mediated up-regulation of alanine aminotransferase (ALT), lactate dehydrogenase (LDH),
and thiobarbituric acid-reactive substances (TBARS). Moreover, it was further observed that
MIL glycoprotein could down-regulate the expression of pro-inflammatory proteins including
cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in CCl4-
exposed mice. From these findings, it was confirmed that MIL glycoprotein possesses anti-
inflammatory and hepatoprotectiveactivities against CCl4-induced oxidative stress.

Amelioration Mechanism behind the Protection

After achieving the preliminary ameliorative response with MIL glycoprotein against
CCl4-induced oxidative stress, the same group further extended their study to evaluate the
possible anti-oxidant mechanism responsible for the MIL glycoprotein-induced
hepatoprotective effect [127]. Carbon tetrachloride-intoxication generates trichloromethyl
free radical (.CCl3) via microsomal cytochrome P450-dependent monooxygenase system and
thereby induces oxidation of proteins and lipids. Post-treatment with the 32 kDa MIL
glycoprotein could prevent hepatic damage through scavenging free radicals with its
antioxidant function. In the next step, they revealed that MIL glycoprotein could modulate the
up-regulation of pro-inflammatory cytokines (TNF-α and IL-1β), which are prompted from
stimulated Kupffer cells and act on numerous surrounding cells (endothelial cells, hepatic
stellate cells and hepatocytes). As a consequence, MIL treatment could block the subsequent
inflammatory responses along with recruitment of the immune cells. In addition, their study
showed that MIL-mediated inhibition of COX-2 activity and cytokine expression may be
therapeutically successful for the anticipation and cure of the oxidative stress-induced
16 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

inflammatory ailments [127]. Furthermore, the data revealed that CCl4-induced accumulation
of substantial ROS triggered SAPK/JNK activation to carry out the hepatic death.
MIL treatment blocked the JNK/AP-1 pathway and conquered the subsequent toxic scenario.
The western blot studies clearly depicted that MIL administration could modulate the
expression of JNKs, AP-1 (c-jun) and AP-1 (c-fos) dose-dependently with no alterations in
the expression of the house keeping protein (α-tubulin). All the results suggest that the MIL
glycoprotein has hepatoprotective role by preventing CCl4-induced pro-inflammatory
expression including COX-2, TNF-α and IL-1β via inhibition of both SAPK/JNK and AP-1 in
murine liver [127].

MICROMOLECULAR BIOACTIVE INGREDIENTS: AMELIORATIVE


ROLE AGAINST ORGAN PATHOPHYSIOLOGY
Taurine

Taurine (2-aminoethanesulfonic acid), referred to as a conditionally amino acid, (Figure


2A) is present in mammalian tissues in high concentrations and is also obtained from the
seafood in abundance. It is highly soluble in water and has an amino group that is paired with
two methylene groups to a sulphonic acid group. Taurine has a structural resemblance to that
of peptide neurotransmitters such as dopamine, etc. (Figure 2). Taurine exhibits
cytoprotective and osmoregulatory effects, stabilizes membrane, possesses antioxidant and
anti-inflammatory properties [128-131]. Taurine is also known to regulate the intracellular
levels of Ca2+ concentrations, neurotransmission and ion movement [132, 133]. The sulphur
containing molecule is synthesized endogenously from cysteine and methionine in the liver,
freely exists in the cytosol and is broadly distributed in blood, heart, brain, retina, etc.
Nowadays, taurine is widely used as a supplement in health drinks viz. Red Bull, Rockstar,
etc.; but there lacks any evidence on the effect of taurine on physical activities. Recent in
vitro and in vivo studies reflect the ameliorative effect of taurine against several toxins and
drug induced multiple organ pathophysiology as well as diabetes [16, 30, 130, 134, 135]. The
review summarizes the prophylactic action of taurine against such complications.

Figure 2A. The structure of Taurine.


Bioactive Natural Compounds 17

Metabolism and Transportation of Taurine in Humans and Level


of Taurine in Foods

In the liver, a magnesium-catalyzed methylation of methionine initiates the synthesis of


taurine to form homocysteine. However, this step can be reversed by the vitamin B12 and the
folate-dependent enzyme, methionine synthetase [136]. Homocysteine then donates its sulfur
group to form cystathionine, and that can be broken down to cysteine under the influence of
pyridoxal-5′phosphate (P5P) cystathionine. In the next step, cysteine combines with dioxygen
to become cysteine sulfinic acid by cysteine deoxygenase. Decarboxylation of cysteine
sulfinic acid then occurs by cysteine sulfinic acid decarboxylase (CSAD) and P5P to
hypotaurine that is further oxidized to taurine by hypotaurine dehydrogenase. An alternative
pathway of the formation of taurine is achieved via the oxidation of cysteine sulfinic acid to
cysteic acid followed by the decarboxylation of cysteic acid by P5P [137]. Humans mostly
obtain taurine from dietary intake as they have a low level of CSAD. The level of taurine is
low in dairy products, whereas, in shellfish, turkey, chicken, etc. the level of taurine is high.
The absorption of taurine, thus obtained from food, takes place in the small intestine. After
absorption, taurine moves to enterocytes by the carrier-mediated active transport in the brush
border membrane and is then delivered to the portal vein [138]. After that, it is transported to
the liver, released into circulation and with the help of taurine transporter (TauT), can enter
cells. Once inside the cell, it can respond to the concentration of taurine in cells [139]. If the
concentration of taurine is high, the down-regulation of TauT occurs, and taurine is excreted
from the body through urine. Conversely, TauT is up-regulated at its low concentration and
via the renal tubules in the kidney, taurine is reabsorbed into circulation.

Biological Significance: Heart and Taurine

In the myocardium, taurine exhibits antioxidant activities probably because of its huge
concentration (25–50% of the amino acid pool). Metal induced toxicity is associated with
cardiac dysfunction. Arsenic (As), an environmental toxin, affects an organism’s health by its
contact with skin and consumption of As-contaminated drinking water. Literature suggests
that As-toxicity results in ROS production/cardiac oxidative stress, overload in Ca2+
concentration, oxidation of proteins, DNA and lipid; and promotes apoptosis and/or necrosis
[11, 140, 141]. As-intoxication is associated with NF-κB-p65 phosphorylation, activation of
JNK, p38, MAPKs but not ERK pathway to that level. Inhibitor studies have shown that PS-
1145, an IKK inhibitor could prevent As-mediated cleavage of caspase-3 and PARP protein
in primary murine cardiomyocytes. Pre-treatment with SP600125, a JNK inhibitor and
SB203580, a p38 MAPK inhibitor could attenuate IKK and NF-κB phosphorylation, thus
indicating that p38 and JNK play an important role in As-mediated NF-κB activation [11].
Taurine has the potential to combat against such anomalies by attenuating NF-κB activation
through p38, JNK and MAPK pathways [11] and ameliorates such cardiac oxidative insults.
Cadmium (Cd), a noxious agent, brings in hazards to life via drinking water, diet, cigarette
smoking, etc. and symptoms associated with the metal exposure are dyspnea, polyuria,
dysuria, fatigue, chest pain, dizziness, etc. [142]. The incidence of cardiovascular diseases
and atherosclerotic alterations are associated with Cd exposure [143]. The hallmarks of Cd
toxicity in the myocardium are oxidative stress and altered levels of ROS [144]. Cd disturbs
18 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

the intracellular antioxidant defense levels [145-148], alters the metabolic processes, and
brings about an alteration in the structure and function of membranes [149]. Cd also modifies
proteins containing thiol groups [150] and DNA structures [151]. However, oral
administration of taurine has been observed to decrease the accumulation of Cd in hearts,
ameliorates the altered levels of cardiac impairment specific markers in serum, reduces
DNA damage and oxidative insults in animal models [152]. Doxorubicin, a potent
chemotherapeutic drug is used to cure leukemia, lymphoma and several other carcinomas
[153]. However, the profound use of the drug is being restricted to its cardiotoxicity that is
imparted via cellular stress viz. oxidative stress and results in cell death [154]. Literature
suggests the ameliorative effect of taurine against doxorubicin-induced oxidative insults and
apoptotic cell death. Studies with numerous inhibitors such as PS-1145, SP600125, SB-
203580, LY294002 (PI3K/Akt inhibitor), etc. reflect on the cardioprotective action of the
molecule through the activation of PI3K/Akt pathway and inhibition of JNK, p53, p38 and
NF-κB [155]. In rabbits, taurine supplementation has been shown to restore doxorubicin-
mediated altered left ventricular diastolic pressure, Ca2+ concentration in the myocardium and
SERCA2a mRNA level [131, 156]. Cardiovascular complications are common in diabetic
patients [157]. However, the literature suggests taurine be a potential candidate in improving
insulin-mediated transportation of glucose, ameliorating cardiac oxidative insults and
apoptotic cell death in experimentally induced diabetic animals [19, 158]. Taurine ameliorates
the altered levels of plasma triglycerides, cholesterol, LDL/HDL and total cholesterol/HDL
ratios, cardiac marker, pro-inflammatory cytokines and significantly reduces cellular stress in
alloxan induced diabetic rats. It restores the translocation of GLUT4 to the plasma membrane
and alters the insulin signaling pathways to improve cardiac functions [19]. Taurine also
protects the heart from apoptosis and DNA damage under experimental conditions in vivo
[19, 158]. It exerts antioxidant effects by binding to free ions and metalloproteins that act as
pro-oxidants in cases on high blood glucose thus modulating membrane fluidity and
morphology [159, 160]. In the cardiac tissue, this molecule imparts protection against
ischaemia-reperfusion injury, activates angiogenesis via PI3K/Akt, FAC/Src, MEK/ERK
signaling pathways and accelerates the proliferation of endothelial cells via CyclinD1/B
[161]. A survey has shown that taurine supplementation (1500 mg taurine for two consecutive
weeks) improves the blood flow and restores the altered parameters in type 1 diabetic
smokers suffering from endothelial dysfunction [162]. Evidence, both in vivo (animals and
human studies) and in vitro shed some light on the beneficial role of taurine against coronary
heart disease due to anti-inflammatory and anti-oxidative properties together with its ability to
regulate blood pressure and conjugate bile acids [163].

Diabetic Complications and Taurine

Severe organ pathophysiology is associated with persistent hyperglycemia.


Hyperglycemia generates free radicals that in turn results in the development of diabetes and
its associated complications such as nephropathy, cardiac complications, retinopathy, etc.
Some studies have shown that taurine increases that secretion of insulin in streptozotocin-
induced diabetic animals [164] while another study has shown that the hypoglycemic effect of
the molecule is not mediated via an enhanced release of insulin [165], thus showing a varied
mode of its anti-diabetic effect. The involvement of taurine in glucose homeostasis has been
Bioactive Natural Compounds 19

observed in some studies [166, 167], while some have reported that taurine administration in
diabetic rats did not alter the increased plasma glucose level [168, 169]. Given the
hypoglycemic effect of the molecule, the possible reasons include improved sensitivity
towards insulin [170], β-cells protection [167], and reduced absorption of glucose from the
gastrointestinal tract [171]. Diabetic nephropathy is a common complication associated with
patients suffering from diabetes [172]. Oxidative stress is a key factor in such complications,
and recent reports suggest the nephroprotective action of taurine is mediated through
suppression of ROS, advanced glycation end product (AGEs), altered level of Na+-K+-
ATPase, xanthine oxidase, p47phox and CYP2E1 expression in diabetic animals. Thus,
taurine is ameliorating such oxidative insults [27, 166, 173]. Taurine exhibits an anti-
inflammatory effect in attenuating the levels of pro-inflammatory cytokines, combats against
nitrosative stress, ROS-mediated up-regulation of MAPKs and PKC pathways to protect
apoptosis in the renal tissue of diabetic rats [27]. Diabetic neuropathy is associated with
increased accumulation of sorbitol and leads to various osmolyte depletion (viz. taurine)
[174]. However, taurine supplementation prevents such anomalies under experimental
diabetic conditions [174]. Taurine improves the impaired handling of calcium in sensory
neurons [175], the decreased nerve growth factor [168] in streptozotocin-induced diabetic
murine models. In experimentally induced diabetic retinopathy, it effectively attenuates
activation of glial fibrillary acid protein and retinal glial cells apoptosis [176, 177], reduces
the elevated levels of retinal glutamate concentration and restores the anomalies in retinal
vascular functioning [177]. Taurine also plays a beneficial role in endothelial dysfunction in
experimentally induced diabetic models by regulating the expressions of ICAM-1, VCAM-1,
Lectin-like oxidized LDL receptor-1, etc. [178, 179].

Oxidative Stress, Cell Death and Taurine

Taurine interferes with the iNOS expression and prevents nitric oxide mediated cellular
damage [180]. It indirectly takes part in preventing ROS-mediated cellular stress by restoring
the altered levels of antioxidant enzymes viz. Superoxide dismutase, thioredoxin reductase,
etc. [181, 182]. Taurine imparts a protection on mitochondria, especially preventing the
calcium overload, thus interfering with ROS production by damaged mitochondria [183]. The
molecule has the potential in efficiently maintaining the mitochondrial protein translation and
combating mitochondrial ROS production [184]. It has been observed that a depletion in the
level of cellular taurine results in a decrease in specific mitochondrial proteins related to
structure and function. Literature suggests that, in renal cells, overexpression of taurine
transporter (TauT) protects the cells against cisplatin-mediated cell death [185]. Yasunaga et
al. have reported that in colorectal carcinoma cells, TauT knockdown increases the sensitivity
of the drug and attenuates the survival of the cells [186]. The activity of TauT is affected
following increased activity of p53 that is either due to the interference of p53 with TauT
promoter or because of p53 triggered AMP-dependent kinase recruitment, inhibition of
mTOR followed by suppression of TauT. In Ehrlich ascites tumor cells and Ehrlich Lettrẻ
ascites mouse fibroblast carcinoma, cisplatin resistance complies with modulation in the
expression of TauT [187]. TauT knockout mice show a retarded exercise capacity, neural
activity, photoreceptor functioning of the retina [188], alterations in the development and
function of renal tissues, cardiomyopathy, liver fibrosis [189, 190], apoptosis in erythrocytes
20 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

and generation of T-cell memory [191]. In the pancreatic islets of streptozotocin-induced


diabetic animals, taurine administration reduces apoptosis and downregulates the expressions
of Bax and death receptor ligand-Fas (FasL) [192]. Under hypoxic conditions, taurine blocks
apoptosis, mitochondrial dysfunction and the release of cytochrome c in retinal ganglion cells
of rat [193]. This bioactive molecule also reduces apoptosis in retinal glial cells exposed to
high glucose [194]. Taurine ameliorates CD3/IL-2 mediated apoptosis of Jurkat T cells by
down-regulating FasL protein [195]. Under the experimental ischemic condition, taurine
reduces/inhibits apoptosis, elevated Bcl-2 expression and formation of Apaf-1/caspase 9
apoptosome [196, 197]. Das et al. have shown that taurine attenuates doxorubicin-mediated
oxidative stress by regulating the Ca2+ concentration, preventing apoptosis and the activation
of p53 [198]. The molecule attenuates mitochondria-mediated hepatic apoptosis by
suppressing PKCδ-JNK signalling pathway [134], effectively counteracts As-mediated
apoptosis of testicular cells by regulating Akt, p38, EK1/2 and NF-κB [9], Taurine also
prevents As-mediated cerebral oxidative stress in vivo [130]. Manna et al. [13] have shown
that taurine treatment prevents Cd-induced oxidative insults in the renal tissues of mice and
thus holds a promise against nephrotoxicity [13]. Researchers have shown that taurine pre-
treatment prevents Cd-mediated hepatic damages [21], neurological disorders [199] in a
murine model, thus ameliorating cellular stress. Furthermore, the molecule imparts a
cytoprotective role against sodium fluoride induced cell death in primary hepatocytes [128]
and combats against tertiary butyl hydroperoxide-mediated hepatic injury by regulating
PI3K/Akt and intrinsic pathways [200]. By literature, Taurine also protects against
acetaminophen mediated renal [84] and hepatic oxidative damage [12].
Recent findings suggest that taurine holds a promise as a potential candidate in
preventing various metal/drug induced organ pathophysiology and diseases viz. Diabetes and
the complications associated with it (Figure 2B).

Figure 2B. Various biological activities of taurine.


Bioactive Natural Compounds 21

Flavonoids: Morin

Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one) was


discovered in the twentieth century as a bioflavonoid constituent of many herbs and fruits.
This natural flavonoid is ubiquitously distributed in almond, mill, old fustic, osage orange,
figure and other family members of moraceaes [201, 202]. Apart from being a dietary
flavonoid, morin also serves as a beneficial molecule in herbal medicines and possesses metal
ion chelation and antioxidant capacity in addition to its free radical scavenging activity [203,
204]. This unique bioactive molecule also acts as a chemopreventive agent against
carcinogenesis [202]. Experimental findings indicate that morin exhibits anti-inflammatory
[205], anti-hypertensive [206], cardioprotective activities [207] and holds a promise as an
anti-virulent therapeutic agent. For these reasons, the therapeutic potential of morin
supplementation has been tested in several pathophysiological scenarios like diabetes and its
associated organ dysfunctions, drug-induced disorders that concern oxidative stress and/or
other related complications. Following summarization provides insight on the long term
scientific explorations of the modulatory effects of this intriguing molecule in regulating
metabolism to understand its functions and applications.

Source

Morin (Figure 3A), a natural yellow coloured crystalline bioflavonoid, was originally
isolated from the members of Moraceae family viz. white mulberry (Morus alba) [208]. The
polyphenol is ubiquitously distributed in guava (Psidium guajava) [209], almond (Prunus
dulcis) [201], mill (Prunus dulcis), figure (Cholorophora tinctoria), osage orange (Maclura
pomifera), sweet chestnut (Castanea sativa), etc. It is also found in several herbs and red wine
[210].

Figure 3A. The structure of morin.


22 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

Chemical Structure and Analysis

The phenyl ring represents the hydrophobic part of the amphipathic molecule whereas,
the hydroxyl groups constitutes the hydrophilic part. The oxygen atom of benzo-c-pyrene
behaves as hydrogen bond acceptor while the hydroxyl group behaves as hydrogen bond
donor and/or acceptor. In aqueous medium, morin is sparingly soluble because of its low
polarity. The structure of morin is an isomeric form of quercetin, differing only in the
hydroxylation pattern on B-ring [211]. The hydroxyl group at position 3 and 7 are more likely
to participate in reacting with metal ions in all solvents [212]. Sequential proton loss electron
transfer (SPLET) begins with the deprotonation of the hydroxyl group in position preceded by
a hydrogen atom transfer (HAT) mechanism that forms the phenoxyl radical ref. Studies on
the effect of ROS on flavone photo-stability clearly shows that morin is degraded by singlet
molecular oxygen and superoxide anion under working conditions [213]. Thermal hysteresis
(DTM) associated with the morin transition and field dependence of the Morin temperature
(TM) are observed in warming-cooling cycles (DTM = 25 and 13 K for H = 0.1 and 3 T,
respectively) because of the first-order phase transition [214]. The specific fluorescence
properties of morin shows that it has an almost planar molecular structure in the S1 state
because of very low rotational energy barrier around the interring bond between B and the A,
C rings (Figure 3A). It has been found in various CH3OH–H2O and CH3CN–H2O mixed
solvents although the dihedral angle is large in the S0 state [215]. During the formation of
morin-protein complex, hydrogen bonds and van der Waals forces are the predominant
intermolecular interactions responsible for the binding [216, 217]. Spectroscopic studies on
the interaction of enzyme-ligand complex, e.g., morin with trypsin, shows that the fluorescent
intensity of trypsin decreases because of the quenching action of morin [218].

Beneficial Effects of Morin: Inflammation and Morin

The immunomodulatory activity of morin has been reported in murine macrophages in


vitro. Morin prompts the proliferation of splenocytes, enhances the phagocytic capacity of
macrophages, blocks both nitric oxide and cytokine production following lipopolysaccharide
(LPS) mediated autophagy and also inhibits the complement system, thus protecting the
macrophages from LPS-mediated autophagic cell death [219]. Besides, it has the potential to
ameliorate LPS-induced acute lung injury in mice [220]. Tianzhu et al. have shown that morin
effectively suppresses inflammatory cell numbers in the bronchoalveolar lavage fluid,
downregulates the level of inflammasome protein NLRP3 and inhibits myeloperoxidase
activity, thus inhibiting neutrophil infiltration in the lung tissue [220]. Jung et al. have shown
that treatment with morin increased the exudate volume in carrageenan-induced air pouches
following dexamethasone exposure [221]. Nitrite level and polymorphonuclear leukocyte cell
numbers were decreased in the pouches following morin treatment [221]. Generation of free
radicals, nitric oxide and leukotrienes are involved in intestinal inflammation [222]. Morin
efficiently inhibits nitric oxide, myeloperoxidase, leuckotriene-b4 synthesis [223] and
interleukine-1β (IL-1β) expression [224]. In chronic inflammatory disorder, namely Crohn’s
disease and ulcerative colitis, there is a surge of reactive oxygen species, platelet activating
factors and cytokines [225]. Colitis was induced in rats by a single injection of hapten
trinitrobenzene sulphonic acid and after that when treated orally with morin (25 mg kg-1 body
Bioactive Natural Compounds 23

weight for four weeks), colitis insults were found to get minimized. Inhibition of IL-1 β
synthesis decreased nitric oxide synthase and generation of free radicals associated with the
inflammatory cascade could be attributed to the anti-inflammatory property of morin [226]. In
hepatic inflammation, SphK1/S1P signaling cascade plays a crucial role [227]. Morin
alleviates high fructose-mediated liver inflammation and accumulation of lipid in rats by
modulating SphK1/S1P pathway via the downregulation of the activity of SphK1, S1p
production, levels of S1PR1, S1PR3, and SphK1 protein, followed by NF-κB activation and
production of inflammatory cytokines [205].

Gastropathy and Morin

Prostaglandin biosynthesis deregulation, severe inflammation, oxidative stress and


apoptosis are the causative factors that mark the pathogenesis of nonsteroidal anti-
inflammatory drug (NSAID)-induced gastropathy. Morin, prescribed widely to treat cases of
inflammation and pain, has been reported that patients taking NSAIDs suffer gastrointestinal
complications ascribed to erosions or ulcers. Oxidative stress resulting in mitochondrial
dysfunction is associated with inflammation [228, 229]. Sinha et al. have shown that
pretreatment with the antioxidant, morin, at a dose of 50 mg kg−1 body weight, prior to
indomethacin (IND) exposure (48 mg kg−1 body weight) can impart the desired anti-ulcer
efficacy against IND, a well-known NSAID drug [230]. Morin was found to provide
protection to gastric mucosa against IND-mediated insult by acting as a free radical scavenger
and Fe2+ chelators. Aggravated ROS production and reduction in the activity of antioxidant
enzymes viz. SOD2, catalase and GST in the gastric tissue was found to be compensated by
the pre-treatment of morin, thus restoring the balance to the antioxidant system in IND-
mediated gastropathy [230]. The study also showed that morin could suppress the iNOS
production, an inflammatory parameter in the gastric inflammation, pro-inflammatory
cytokines, cell adhesion molecules and chemokines that contribute to the diminished
infiltration of neutrophils at the site of gastric lesion due to inhibition of the myeloperoxidase
activity. Besides, morin also inhibits the degradation of IκBα and NF-κB activation in an
IND-mediated gastric injury that in turn brings about the transcriptional inhibition of an array
of genes concerned with inflammation [230]. HSP70 is activated during severe stress
following IND-induced gastropathy. Sinha et al. showed the efficacy of morin to combat such
up-regulation during cellular stress [230]. NSAIDs are known to inhibit prostaglandin
synthesis catalyzed by COX-1 and COX-2, cyclooxygenase enzymes [231]. The former
inhibits the enzyme COX-1 in stomach whereas, induces COX-2 at the inflammation sites.
Morin is potent enough to regulate the COX-2 level in an independent manner without
altering COX-1 and PGE2 levels to protect the gastric tissue against IND-mediated stress
[230].

Oxidative Stress and Morin

Cellular defense system viz. heme oxygenase-1 (HO-1) protects cells and tissues against
oxidative insults by catalyzing the oxygen-dependent breakdown of heme to iron, biliverdin
and carbon monoxide. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor
24 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

of HO-1, plays a crucial role in phase II detoxifying antioxidant enzymes expression. In


human lens epithelial cells (HLE-B3), the anti-oxidant response of morin regarding HO-1 has
been elucidated [232]. Studies using HO-1 inhibitor ZnPP in HLE-B3 cells conferred that the
cytoprotective action of morin was mediated through the induction of HO-1. Morin treated
cells showed activation of extracellular regulated kinase (ERK), nuclear translocation and
increased antioxidant response element (ARE) binding capacity of Nrf2 while ERK inhibitor
U0126 attenuated the morin-induced expression of Nrf2, DNA binding capacity and the level
of HO-1 [232]. Subash et al. have shown that oral administration of morin (30 mg kg-1
body weight) can significantly ameliorate oxidative insult in ammonium chloride induced
hyperammonemia rats (100 mg kg-1 body weight; i.p.) by regulating the levels of antioxidants
viz. SOD, catalase, GPX, GSH, etc. [233]. The scavenging potential of morin following ROS
generation by γ-irradiation in Chinese hamster lung fibroblast V79-4 was confirmed through
DCF-DA and also provides protection against DNA damage and membrane lipid peroxidation
[234]. Morin could combat against oxidative stress-induced apoptotic cell death via the
inhibition of SEK1-JNK-AP-1 cascade [234]. Kim et al. suggested the possible suppression
of reactive species induced NF-κB activation through modulation of p38 MAPKs and ERK
signaling pathways by the antioxidant activity of morin in t-BHP treated rat endothelial cells
[235]. The antioxidant effect of morin was also evaluated against deoxycorticosterone
acetate-salt hypertensive rats. The study suggested that the effective attenuation of the levels
of lipid hydroperoxides, thiobarbituric acid reactive substances, conjugated dienes, nitrate and
nitrite in tissues and plasma and restoration of antioxidant levels occurred following morin
supplementation (50 mg kg-1 body weight, orally, every day for six weeks) [236].
Cyclophosphamide, an effective anticancer and immunosuppressant drug, induces oxidative
insults which in turn results in adversities viz. Hemorrhagic cystitis [237, 238]. The natural
flavonoid effectively imparts a protection against such cyclophosphamide triggered adverse
effects by attenuating the antioxidant levels (superoxide dismutase, malondialdehyde,
glutathione concentrations) [239]. The anti-peroxidative efficacy of morin on
cyclophosphamide/flutamide mediated lipid peroxidation in vivo has been exploited by Ray et
al. [240].
In liver fibrosis, chronic hepatic disorder, oxidative stress is associated with uncontrolled
inflammation. Focusing on the therapeutic potential of morin against carbon tetrachloride-
mediated liver fibrosis in rats, the molecule, effectively reduces the elevated parameters
related to oxidative stress (glutathione, malondialdehyde), inflammation (NF-κB, iNOS,
TNF-α) and fibrosis (hydroxyproline) [204]. The anti-fibrotic property of morin has been
elucidated in experimental liver fibrosis, where the molecule inhibited the proliferation of
hepatic stellate cells (HSCs) by Wnt/β-catenin pathway suppression [241]. In context to the
anti-fibrotic property of morin, suppression or inhibition of the HSCs proliferation or
activation by inhibiting NF-κB activation was studied by Madan Kumar et al. in vitro in LX-2
cells (HSCs) and diethylnitrosamine-induced experimental fibrosis in vivo [242]. Morin
induces apoptosis in experimental liver fibrosis by downregulating Bcl-2, up-regulating
cytochrome c and Bax, activation of caspases and phosphatidylserine translocation to the
outer membrane [242].
Bioactive Natural Compounds 25

Cancer and Morin

In search of an affordable and safe yet effective molecule against cancer, the potentiality
of morin has been tested against human breast cancer cells, neck and head carcinoma cells
and myeloma cells. The flavonoid imparts its anti-cancer potential by suppressing the
constitutively activated and inducible signal transducer and activator of transcription 3
(STAT3) and blocking its subsequent translocation to the nucleus that in turn regulates the
cancer metastasis. Such suppression of STAT activation is due to the inhibition of JAK1,
JAK-2 and Src by morin but silencing of SHP1 induced STAT3 phosphorylation, thus
indicating that morin imparts such beneficial effect on STAT3 through SHP1 [243]. In
diethylnitrosamine mediated hepatocellular carcinoma in a murine model, morin, as an anti-
cancer and anti-inflammatory molecule favoured the suppression of hepatocarcinogenesis by
regulating the expression of COX-2, NF-κB-p65 and matrix metalloproteinases (MMP-2,
MMP-9) and thus confirming its role in preventing angiogenesis [244]. Evidence shows that
morin (50 µM) effectively suppresses the growth and invasion of MDA-MB-231, a metastatic
breast cancer cell line. Literature suggests that morin provides an inhibition to the epithelial to
mesenchymal transition process by reversing the mesenchymal cellular morphology to
epithelial one, decreasing MMP-9, downregulating the mesenchymal marker and partly
suppressing Akt activation [245]. [245]. Morin induces caspase-dependent apoptosis through
the mitochondria-dependent pathway in U397 human leukemic cells in a dose-dependent
manner by upregulating the release of cytochrome c, proapoptotic proteins viz. BAX, BAD
and downregulating antiapoptotic protein like Bcl-2 [246]. Oral supplementation of morin (50
mg kg-1 body weight) augments anti-cancer efficacy against 7,12-dimethylbenz(a)-
anthracene mediated mammary carcinogenesis in rats by restoring the levels of enzymatic and
non-enzymatic antioxidants, the markers for lipid peroxidation and tumor (AFP, CA 15-3 and
CEA). The molecule downregulates the expression levels of proliferating cell nuclear antigen-
positive (PCNA) cells and the number of Ag-nucleolar organizer regions/nuclei [247]. Morin
suppresses the activity of base excision repair enzyme, N-methylpurine-DNA glycosylase,
known to play a critical role in carcinogenesis [248].

Neurodegenerative Diseases and Morin

The debilitating Alzheimer’s disease (AD) is linked with the deposition and aggregation
of amyloid β-peptide (Aβ) in the neural tissue. Atomistic, explicit molecular dynamics
simulations (MD) have shown the interaction of morin with Aβ monomers and dimers.
Though morin cannot completely block β-strand formation or Aβ aggregation, MD simulation
confirms its effect on Aβ40 and Aβ42 in the monomeric and dimeric states, thus altering the
quaternary and tertiary structures to produce “off-pathway” aggregates [249]. Lemkul et al.
performed MD simulations to identify Aβ fibril destabilization by the flavonoid and reported
that morin binds to the fibrillar ends and inhibits the incoming peptide attachment by
penetrating hydrophobic core and disrupting the Asp23-Lys28 Salt bridges that ultimately
interfere with backbone hydrogen bonding [250]. Hyperphosphorylation and accumulation of
26 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

Tau protein in the neural tissue is an important event in the process of dementia since
mutation of the protein results in frontotemporal lobe dementia [251]. Of the kinases that are
associated with tau phosphorylation, glycogen synthase 3β (GSK 3β) is linked with the AD
[252]. Morin inhibits the activity of this kinase and blocks/reduces GSK 3β mediated tau
phosphorylation both in vitro and in vivo [253]. As evident from light scattering and
transmission electron microscopy, morin inhibits the formation of amyloid by islet amyloid
polypeptide (IAPP) and disaggregates preformed IAPP amyloid fibers, thus acting as an
inhibitor of the IAPP amyloid [254]. Morin provides neuroprotection against 1-methyl-4-
phenylpyridinium ion induced apoptosis and generation of ROS in PC-12 neuronal cells (5-50
µmol/L) and attenuates dopaminergic neuronal death, depletion of striatal dopamine and
behavioural defects in 1-methyl-4-phenyl-1,2,3,6-tetrahydrooyridine induced experimental
Parkinson’s disease in vivo [253].

Allergy and Morin

In hepatic inflammation, SphK1/S1P signaling cascade plays a crucial role [227]. Morin
alleviates high fructose-mediated liver inflammation and accumulation of lipid in rats by
modulating SphK1/S1P pathway. The molecule downregulates the activity of SphK1, S1P
production, levels of S1PR1, S1PR3 and SphK1 protein, followed by NF-κB activation and
production of inflammatory cytokines [205].

Diabetes and Morin

Type-2 diabetes, a major health concern, is characterized by insulin resistance that occurs
as a result of the alterations in several receptors and/or post-receptor events concerned with
insulin signaling. Molecular docking studies and in vitro experiments in HepG2 cells have
shown the potentiality of morin as a non-competitive inhibitor of protein-tyrosine
phosphatase 1B (PTP1B) which plays a key role in negatively regulating insulin signaling
[255]. The molecule upregulates Akt and the phosphorylation of insulin receptor IR-β, blocks
gluconeogenesis and increases the synthesis of glycogen [255]. In streptozotocin-induced
experimental diabetes mellitus, oral administration of morin (25 and 50 mg kg-1 body
weight for 30 days) restores the blood glucose and serum insulin levels and attenuates
hepatic enzymes concerned with carbohydrate metabolism (hexokinase, glucose-6-phosphate
dehydrogenase). The molecule can thus be regarded as an effective candidate for the
treatment of diabetes [256]. It alleviates experimentally induced diabetic osteopenia by
regulating oxidative stress and inflammation. Trabecular bone mineral density and bone-
specific alkaline phosphatase, osteocalcin, deoxypyridinoline cross-links levels conferred the
beneficial role of morin in ameliorating such disorder [257]. This dietary flavonoid modulates
apoptosis induced by high levels of glucose in primary rat hepatocytes via an intrinsic
pathway through oxidative stress intervention [258].
Bioactive Natural Compounds 27

Heart and Morin

Myocardial infarction (MI), cardiovascular disorder, is associated with severe


abnormalities viz. Calcium overload and accumulation, up-regulation of cAMP, altered
myocardial permeability and metabolism, etc. [259]. Isoproterenol [1-(3,4-dihydroxyphenyl)-
2-isopropylaminoethanol hydrochloride], a β-adrenergic and synthetic catecholamine, when
administered in excessive doses results in MI [259]. Morin acts as a potential candidate for
providing cardioprotection against isoproterenol induced myocardial infarction (MI) in
experimental rats by regulating the anomalies in electrocardiography and cardiac biomarkers
such as creatine kinase, troponin I, lactate dehydrogenase, lipid peroxidation, etc. [260]. Pre-
treatment with morin imparts protection against isoproterenol-mediated MI by ameliorating
the altered levels of expression of glycoproteins (sialic acid, fucose, hexose, hexosamine) and
adenosine triphosphatases due to its antioxidant property, free radical scavenging activity
(attributed by the phenolic group) and membrane stabilizing capacity [261]. Morin restores
the levels of both enzymatic and non-enzymatic antioxidants, thus reversing the altered
biochemical parameters in experimentally induced cardiotoxicity [262]. Also, morin
possesses a dose-dependent antihypertensive activity against deoxycorticosterone acetate-salt-
induced hypertension in experimental rats by regulating the systolic and diastolic pressure and
restoring the enzymatic activities of hepatic and renal functions markers viz. Alanine
aminotransferase, alkaline phosphatase, urea, creatinine, etc. [207].
Cumulative evidence suggests that morin can be regarded as a potential candidate in
combating several human disorders (Figure 3B). Over the years, several in vitro and in vivo
experiments have been conducted to elucidate the exact molecular mechanism of its
protection yet detailed pharmacodynamics and pharmacokinetic studies together with in-
depth signaling mechanism should be explored thoroughly followed by clinical trials to
establish its usefulness in mitigating several human complications.

Figure 3B. Protective role of morin.


28 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

Flavonoids: Quercetin

Flavonoids are the large heterogeneous group of benzo-γ-pyrone derivatives and are
found in a wide variety of fruits, vegetables and medicinal herbs with some structural
diversity. One of them, quercetin, (3,3’,4’,5,7-pentahydroxyl-flavone) (Figure 4A) is an
important dietary flavonoid found in the most plants, fruits and vegetables. Quercetin is
consumed daily by the millions of people in their diet [263]. A multitude of biological
activities of quercetin (including anti-inflammatory effects, atherosclerosis, thrombosis,
hypertension, and arrhythmia as well as modulation of cancer-related multidrug resistance)
have been reported by the investigators [264]. It is noteworthy that quercetin also has
hormetic properties because it may act both as an antioxidant or pro-oxidant depending on its
concentration used for the purposes [265].

Anticancer Efficacy of Quercetin

The hormetic nature of quercetin marks it complementary to use in cancer prevention


efforts. As a consequence, quercetin has been utilized therapeutically against different types
of cancer cell lines and tumors [264, 266-273]. Besides, medical researchers suggest that
application of quercetin is beneficial against colon cancer [274], melanoma growth, invasion
and meta-static potential [275]. Evidence from all the reports supported the anti-oncogenic
property of quercetin.
Quercetin can donate an electron to ROS and thereby lower the risk of DNA damage
[263]. This type of mechanism is the preliminary mechanism of quercetin to exhibit both
antioxidants as well as chemo-preventive applications. Quercetin concentrations in the range
of 1-40 µM showed these types of effects. Beyond this range, quercetin could induce
oxidative stress and cytotoxicity in carcinogenic cells [276-278]. The mitochondrial mediated
cell-death pathway has been proposed as one of the mechanisms quercetin follows to induce
cellular apoptosis [277].

Figure 4A. The structure of quercetin.


Bioactive Natural Compounds 29

The second mechanism probably explains the anticancer efficacy of quercetin. This
mechanism relies on the interaction of quercetin with different cell cycle regulatory proteins.
Quercetin can influence the expression of p53 [276-278]. It has been reported that in human
cervical cancer (HeLa) cells, the activation of the p53 tumor suppressor protein could elicit a
G2/M phase cell cycle arrest. This pathway has been suggested to be a potential target for
cancer therapy [279]. However, Tan et al. explored the status of a human hepatocellular
carcinoma cell line after treatment with 40-120 µM dosages of quercetin and found that
quercetin upregulated p53 while declining the same for the anti-apoptotic Bcl-2 proteins
[276]. A similar effect of quercetin was found in human breast cancer cells (MDA-MB-231)
[280]. Besides, quercetin (10 µM) has been found to constrain the expression of the p21-ras
oncogene in cultured colon cancer cell lines [281].
Other pathways through which quercetin can also provoke its anticancer efficacy is its
interaction with Topoisomerase II (TopoII) [282, 283] and quercetin directed protein
chaperone inhibition [284, 285]. In Jurkat cells (immortalized T-lymphocytic cells) quercetin
is found to prevent the kinase activity of casein kinase 2 (CK2) and calcium/calmodulin
kinase II (CamKII). These may trigger subsequent diminution of HSP70 expression along
with cumulative tumor sensitivity to radiation [285].

Quercetin as Protective Mediator against Organ Pathophysiology

Literature suggests that quercetin which induces apoptosis in various cancer cells also act
as an anti-apoptotic i.e., cytoprotective agent against various oxidants [286, 287]. It has been
found that quercetin supplementation could shield central nervous system against oxidative
stress induced by different cytotoxic agents [288-290]. For this particular function, it may
enter into the nucleus via the cell membrane penetration and initiates the interactions between
cytosolic and nuclear molecules in the cultured neurons [264, 266-275, 279, 282, 283, 288-
291]. Quercetin could ameliorate oxidative injuries of some cells via modulation of
mitochondrial dysfunction and inhibition of caspase activity [292]. It is found to be effective
to cure reperfusion ischemic tissue damage [293] and LDL oxidation in vitro [294]. Thus
quercetin is also able to protect atherosclerosis. Moreover, quercetin deliberates intracellular
protection by restraining xanthine oxidase activity [295], pro-inflammatory cytokines
expression [296] along with membrane stabilization [297]. Besides the inflection on pro-
inflammatory cytokines, quercetin is found to be effective in reduction of histamine secretion
from basophils [298] and catalysis the transformation of arachidonic acid to its metabolites
[299]. Quercetin-facilitated chelation therapy is another outstanding approach to extravagance
the metal induced oxidative ailments [300, 301].
From all the biological activities of quercetin, it may be concluded that quercetin is the
focus of intense clinical research for its antioxidant, anti-inflammatory and anti-cancer
accomplishments (Figure 4B).
30 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

Figure 4B. A possible mechanism of biological activities of quercetin.

Xanthones: Mangiferin

Another important group of phytonutrients are xanthones. They are polyphenolic


compounds having the molecular formula C13H8O2. Recently, xanthones are a notable subject
of research because of their extensive prominence in biological and pharmacological fields.
They exist in trace amount throughout nature. One of the common resources of xanthone
glucoside is mangiferin (2-C-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone; molecular
formula C19H18O11) (Figure 5A) [302]. Mangiferin has been found from the barks, leaves,
roots and fruits of Mangifera indica L., (mango, family Anacardiaceae) [303]. After isolation,
the purification and homogeneity were confirmed by HPLC, mass, NMR (1H, 13C)
spectroscopy and reverse-phase HPLC. Literature suggests that mangiferin has been widely
utilized therapeutically for the treatment of several disorders [304-314].

Figure 5A. The structure of mangiferin.


Bioactive Natural Compounds 31

Mangiferin as Antioxidant Agent

DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging properties of mangiferin


suggests its strong antioxidant activity [315]. Besides, mangiferin was found to
ameliorate 12-O-tetradecanoylphorbol-13-acetate (TPA, a stimulator of ROS production)
induced oxidative damage [305]. Mangiferin treatment is found to be beneficial against
D-galactosamine mediated oxidative impairments. It is effective to modulate the
D-galactosamine-induced cellular alterations (ALT, triglycerides, total cholesterol, etc.
increased ROS and NO production and decreased levels of total proteins, albumin and cellular
GSH). The molecular signaling studies revealed that it can modulate NF-κB, iNOS, Bax and
caspase 3/9 proteins to show renal and hepato-protection [19, 316]. In addition, GSH
enhancing the ability of mangiferin confirms its antioxidant action [317].

Amelioration of Metal Induced Oxidative Impairments

Extensive research is being carried out to explore the prophylactic role of mangiferin
against metal-induced oxidative complications and organ pathophysiology. The results from
the studies showed the cellular protective efficacy of mangiferin against various metal
induced ROS formation along with alterations of several oxidative stress-related parameters
[36, 318-320]. It has been found that mangiferin can ameliorate iron (Fe) mediated
mitochondrial swelling and loss of mitochondrial transmembrane potential [318]. The study
also suggested that mangiferin could ameliorate Fe induced cytotoxicity by the formation of a
stable Fe3+–mangiferin complex (related to its iron-chelating properties) and scaveng the free
radicals. Another group of investigators highlighted the cytoprotective role of mangiferin
against mercuric chloride (HgCl2) induced toxicity [320]. They have confirmed the protective
efficiency of the xanthone through the results from antioxidant enzyme activities, ROS
scavenging assay, colony formation assay, fluorescence microscopy, flow cytometric DNA
analysis and DNA fragmentation pattern assay. A recent study by Pal et al. (2013) explored
the amelioration mechanism of mangiferin against metal toxicity by performing both in vitro
and in vivo studies [36]. In their study, the authors found that mangiferin supplementation is a
significant therapeutic tool to restore the lead nitrateinduced alterations of ALT, ALP,
antioxidant enzyme activities, activation of MAPKs, NFĸB translocation, and apoptotic cell
death [36]. These results are of pharmacological relevance and mangiferin could be
considered as a potential candidate for quenching of the metal induced ROS in the cells
because of oxidative stress, restoration of mitochondrial membrane potential and
normalization of various cellular antioxidant activities.

Anti-Inflammatory and Immunomodulatory Effects

Inducible isoforms of NO synthase (iNOS) and cyclooxygenase (COX-2) are potent


inducers of pro-inflammatory responses. The investigators have found that mangiferin
administration is efficient to reduce/inhibit NO production and iNOS mRNA levels in
activated macrophages [321, 322]. It is noteworthy that NF-κB which serves as a transcription
factor for both COX-2 and iNOS genes, the inhibition of NF-κB activation by mangiferin
32 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

appears to be the anti-inflammatory mechanisms of action [323]. Besides, in the year of 2012,
Das et al. showed that mangiferin implementation has an immunomodulatory response
against galactosamine (GAL) mediated TNF-α, IFN-γ, IL-1β, IL-6, IL-12, IL-18 mRNA
expression levels [19]. Another study from Leiro et al. [313] characterized the
immunomodulatory activity of mangiferin on thioglycollate-elicited mouse macrophages
which were stimulated with lipopolysaccharide (LPS) and gamma interferon (IFN-γ).
Evidence from microarray analysis suggests that mangiferin treatment could modulate
interleukin 1 (IL-1) at the level of TNF receptor-associated factor 6 and inhibit tolllike
receptor proteins and a series of pro-inflammatory cytokines (IL-1α, IL-1, IL-6, IL-12, TNF-
α, granulocyte and macrophage colony-stimulating factors, A2) along with vascular adhesion
molecules (VCAM- 1) [313].

Modulation of Hyperglycemia: Mangiferin as Anti-Diabetic Agent

Researchers have investigated the role of mangiferin in streptozotocin (STZ) induced


diabetic rats [324]. Mangiferin therapy to the diabetic animals significantly decreased the
level of blood glucose, glycosylated hemoglobin as well as increased level of insulin and
hemoglobin. Apart from that, in mangiferin treated diabetic rats the activities of other proteins
and enzyme levels (hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase,
glycogen synthase, and glycogen content level) have been restored. In addition, activities of
certain diabetic related enzymes (lactate dehydrogenase, glucose-6-phosphatase, fructose-1,6-
diphosphatase and glycogen phosphorylase) were also significantly declined in liver tissue of
the diabetic rats. These findings validated the antidiabetic activity mangiferin. A very recent
study by Pal et al. (2014) speculated the mechanistic approaches of mangiferin through which
mangiferin could attenuate STZ induced diabetic nephropathy [325].

Figure 5B. Different biological modulation by mangiferin.


Bioactive Natural Compounds 33

Overall, the results demonstrated that mangiferin possesses various biological activities
(Figure 5B) and therefore could be considered as a potent bioactive nature derived
complementary and alternative medicine.

CONCLUSION
We hope that this book chapter can offer an overview of the substantial aids of various
bioactive molecules derived from natural sources and concurrently enlighten the application-
based clinical advancement of the same throughout the world. The multifunctional beneficial
efficacy and signaling properties of these bioactive compounds reveal that they are eco-
friendly outstanding promising templates for future drug discovery and development.

ACKNOWLEDGMENTS
The authors are grateful to Chirajyoti Guha and Krishnendu Sinha for extending their
helping hand whenever required.

ABBREVIATIONS
ALT alanine transaminase
ALP alkaline phosphatase
ALX Alloxan monohydrate
APAP Acetaminophen
AR Aldose reductase
ATP adenosine tri phosphate
CAT catalase
CI protein Cajanus indicus protein
DPPH, 2 2-diphenyl-1-picrylhydrazyl
DSL D-saccharic acid-1,4-lactone
ERK extracellular signal-regulated kinases
GAL D-galactosamine
GSH glutathione
GST glutathione S-transferase
GPx Glutathione Peroxidase
HO-1 heme oxygenase-1,
HDL high density lipoprotein
JNK c-Jun-NH2-terminal protein kinase
LDH Lactate dehydrogenase
LDL low density lipoprotein
MAPKs mitogen-activated protein kinases
MPT mitochondrial membrane permeability transition
NAPQI N-acetyl-p-benzoquinone imine
34 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

NF-B nuclear factor kappa B


NO nitric oxide
NOS nitric oxide synthase
iNOS inducible nitric oxide synthase
Nrf2 nuclear erythroid 2-related factor 2
PARP Poly (ADP-ribose) polymerase
PI3K Phosphatidylinositol 3-kinases
PKC protein kinase
c PNP Phyllanthus niruri protein
ROS reactive oxygen species
SOD Super Oxide Dismutase
STZ streptozotocin
TBHP tertiary butyl hydroperoxide
TNF  tumor necrosis factor alpha.

REFERENCES
[1] Balunas, MJ; Kinghorn, AD. Drug discovery from medicinal plants. Life Sci, 2005,
78(5), 431-41.
[2] Newman, DJ; Cragg, GM; Snader, KM. Natural products as sources of new drugs over
the period 1981-2002. J Nat Prod, 2003, 66(7), 1022-37.
[3] Chin, YW; Balunas, MJ; Chai, HB; Kinghorn, AD. Drug discovery from natural
sources. The AAPS journal, 2006, 8(2), E239-E53.
[4] Paterson, I; Anderson, EA. The renaissance of natural products as drug candidates.
Science, 2005, 310(5747), 451-3.
[5] Newman, DJ; Cragg, GM; Snader, KM. The influence of natural products upon drug
discovery. Nat Prod Rep, 2000, 17(3), 215-34.
[6] Jones, WP; Chin, YW; Kinghorn, AD. The role of pharmacognosy in modern medicine
and pharmacy. Curr Drug Targets, 2006, 7(3), 247-64.
[7] Koehn, FE; Carter, GT. The evolving role of natural products in drug discovery. Nature
reviews Drug discovery, 2005, 4(3), 206-20.
[8] Manna, P; Sinha, M; Sil, PC. Aqueous extract of Terminalia arjuna prevents carbon
tetrachloride induced hepatic and renal disorders. BMC Complement Altern Med, 2006,
6(1), 33.
[9] Das, J; Ghosh, J; Manna, P; Sinha, M; Sil, PC. Taurine protects rat testes against
NaAsO2-induced oxidative stress and apoptosis via mitochondrial dependent and
independent pathways. Toxicol Lett, 2009, 187(3), 201-10.
[10] Manna, P; Sinha, M; Sil, PC. Arsenic-induced oxidative myocardial injury: protective
role of arjunolic acid. Arch Toxicol, 2008, 82(3), 137-49.
[11] Ghosh, J; Das, J; Manna, P; Sil, PC. Taurine prevents arsenic-induced cardiac oxidative
stress and apoptotic damage: Role of NF-κB, p38 and JNK MAPK pathway. Toxicol
Appl Pharmacol, 2009, 240(1), 73-87.
Bioactive Natural Compounds 35

[12] Das, J; Ghosh, J; Manna, P; Sil, PC. Acetaminophen induced acute liver failure via
oxidative stress and JNK activation: protective role of taurine by the suppression of
cytochrome P450 2E1. Free Radic Res, 2010, 44(3), 340-55.
[13] Manna, P; Sinha, M; Sil, PC. Taurine plays a beneficial role against cadmium-induced
oxidative renal dysfunction. Amino Acids, 2009, 36(3), 417-28.
[14] Manna, P; Das, J; Ghosh, J; Sil, PC. Contribution of type 1 diabetes to rat liver
dysfunction and cellular damage via activation of NOS, PARP, IκBα/NF-κB, MAPKs,
and mitochondria-dependent pathways: Prophylactic role of arjunolic acid. Free Radic
Biol Med, 2010, 48(11), 1465-84.
[15] Ghosh, J; Das, J; Manna, P; Sil, PC. Acetaminophen induced renal injury via oxidative
stress and TNF-α production: Therapeutic potential of arjunolic acid. Toxicology, 2010,
268(1), 8-18.
[16] Das, J; Ghosh, J; Manna, P; Sil, PC. Taurine suppresses doxorubicin-triggered
oxidative stress and cardiac apoptosis in rat via up-regulation of PI3-K/Akt and
inhibition of p53, p38-JNK. Biochem Pharmacol, 2011, 81(7), 891-909.
[17] Ghosh, J; Das, J; Manna, P; Sil, PC. Cytoprotective effect of arjunolic acid in response
to sodium fluoride mediated oxidative stress and cell death via necrotic pathway.
Toxicol In Vitro, 2008, 22(8), 1918-26.
[18] Manna, P; Sinha, M; Sil, PC. Protection of arsenic-induced testicular oxidative stress by
arjunolic acid. Redox Report, 2008, 13(2), 67-77.
[19] Das, J; Ghosh, J; Roy, A; Sil, PC. Mangiferin exerts hepatoprotective activity against
D-galactosamine induced acute toxicity and oxidative/nitrosative stress via Nrf2–NFκB
pathways. Toxicol Appl Pharmacol, 2012, 260(1), 35-47.
[20] Ghosh, J; Das, J; Manna, P; Sil, PC. Arjunolic acid, a triterpenoid saponin, prevents
acetaminophen (APAP)-induced liver and hepatocyte injury via the inhibition of APAP
bioactivation and JNK-mediated mitochondrial protection. Free Radic Biol Med, 2010,
48(4), 535-53.
[21] Sinha, M; Manna, P; Sil, PC. Induction of necrosis in cadmium-induced hepatic
oxidative stress and its prevention by the prophylactic properties of taurine. J Trace
Elem Med Biol, 2009, 23(4), 300-13.
[22] Manna, P; Sinha, M; Sil, PC. Protective role of arjunolic acid in response to
streptozotocin-induced type-I diabetes via the mitochondrial dependent and
independent pathways. Toxicology, 2009, 257(1), 53-63.
[23] Das, J; Vasan, V; Sil, PC. Taurine exerts hypoglycemic effect in alloxan-induced
diabetic rats, improves insulin-mediated glucose transport signaling pathway in heart
and ameliorates cardiac oxidative stress and apoptosis. Toxicol Appl Pharmacol, 2012,
258(2), 296-308.
[24] Sinha, M; Manna, P; Sil, PC. Aqueous extract of the bark of Terminalia arjuna plays a
protective role against sodium-fluoride-induced hepatic and renal oxidative stress. J Nat
Med, 2007, 61(3), 251-60.
[25] Ghosh, J; Das, J; Manna, P; Sil, PC. The protective role of arjunolic acid against
doxorubicin induced intracellular ROS dependent JNK-p38 and p53-mediated cardiac
apoptosis. Biomaterials, 2011, 32(21), 4857-66.
[26] Manna, P; Sinha, M; Sil, PC. Cadmium induced testicular pathophysiology:
prophylactic role of taurine. Reprod Toxicol, 2008, 26(3), 282-91.
36 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

[27] Das, J; Sil, PC. Taurine ameliorates alloxan-induced diabetic renal injury, oxidative
stress-related signaling pathways and apoptosis in rats. Amino Acids, 2012, 43(4), 1509-
23.
[28] Manna, P; Ghosh, J; Das, J; Sil, PC. Streptozotocin induced activation of oxidative
stress responsive splenic cell signaling pathways: protective role of arjunolic acid.
Toxicol Appl Pharmacol, 2010, 244(2), 114-29.
[29] Sinha, M; Manna, P; Sil, PC. Cadmium-induced neurological disorders: prophylactic
role of taurine. J Appl Toxicol, 2008, 28(8), 974-86.
[30] Das, J; Ghosh, J; Manna, P; Sil, PC. Taurine protects rat testes against doxorubicin-
induced oxidative stress as well as p53, Fas and caspase 12-mediated apoptosis. Amino
Acids, 2012, 42(5), 1839-55.
[31] Bhattacharya, S; Manna, P; Gachhui, R; Sil, PC. D-saccharic acid 1, 4-lactone protects
diabetic rat kidney by ameliorating hyperglycemia-mediated oxidative stress and renal
inflammatory cytokines via NF-κB and PKC signaling. Toxicol Appl Pharmacol, 2013,
267(1), 16-29.
[32] Pal, S; Pal, PB; Das, J; Sil, PC. Involvement of both intrinsic and extrinsic pathways in
hepatoprotection of arjunolic acid against cadmium induced acute damage in vitro.
Toxicology, 2011, 283(2), 129-39.
[33] Sarkar, K; Ghosh, A; Sil, PC. Preventive and curative role of a 43kD protein from the
leaves of the herb Cajanus indicus L on thioacetamide-induced hepatotoxicity in vivo.
Hepatology research, 2005, 33(1), 39-49.
[34] Pal, PB; Pal, S; Das, J; Sil, PC. Modulation of mercury-induced mitochondria-
dependent apoptosis by glycine in hepatocytes. Amino Acids, 2012, 42(5), 1669-83.
[35] Sinha, M; Manna, P; Sil, PC. Attenuation of cadmium chloride induced cytotoxicity in
murine hepatocytes by a protein isolated from the leaves of the herb Cajanus indicus L.
Arch Toxicol, 2007, 81(6), 397-406.
[36] Pal, PB; Sinha, K; Sil, PC. Mangiferin, a natural xanthone, protects murine liver in Pb
(II) induced hepatic damage and cell death via MAP kinase, NF-kappaB and
mitochondria dependent pathways. PLoS One, 2013, 8(2), e56894.
[37] Bhattacharjee, R; Sil, PC. Protein isolate from the herb Phyllanthus niruri modulates
carbon tetrachloride-induced cytotoxicity in hepatocytes. Toxicol Mech Methods, 2007,
17(1), 41-7.
[38] Bhattacharyya, S; Ghosh, J; Sil, PC. Iron induces hepatocytes death via MAPK
activation and mitochondria-dependent apoptotic pathway: beneficial role of glycine.
Free Radic Res, 2012, 46(10), 1296-307.
[39] Ghosh, S; Banerjee, S; Sil, PC. The beneficial role of curcumin on inflammation,
diabetes and neurodegenerative disease: A recent update. Food Chem Toxicol, 2015, 83,
111-24.
[40] Sinha, K; Pal, PB; Sil, PC. Mangiferin, a naturally occurring xanthone C-glycoside,
ameliorates lead (Pb)-induced murine cardiac injury via mitochondria-dependent
apoptotic pathways. Signpost Open Access J Org Biomol Chem, 2013, 1, 47-63.
[41] Rashid, K; Sil, PC. Curcumin enhances recovery of pancreatic islets from cellular stress
induced inflammation and apoptosis in diabetic rats. Toxicol Appl Pharmacol, 2015,
282(3), 297-310.
Bioactive Natural Compounds 37

[42] Ghosh, S; Bhattacharyya, S; Rashid, K; Sil, PC. Curcumin protects rat liver from
streptozotocin-induced diabetic pathophysiology by counteracting reactive oxygen
species and inhibiting the activation of p53 and MAPKs mediated stress response
pathways. Toxicology Reports, 2015, 2, 365-76.
[43] Rashid, K; Sil, PC. Curcumin ameliorates testicular damage in diabetic rats by
suppressing cellular stress-mediated mitochondria and endoplasmic reticulum-
dependent apoptotic death. Biochimica et Biophysica Acta (BBA)-Molecular Basis of
Disease, 2015, 1852(1), 70-82.
[44] Sarkar, K; Sil, PC. Studies on a hepatoprotective protein from a herbal source: Bose
Institute, Kolkata, 2005.
[45] Ghosh, M; Pal, S; Sil, PC. Taurine attenuates nano-copper-induced oxidative hepatic
damage via mitochondria-dependent and NF-κB/TNF-α-mediated pathway. Toxicology
Research, 2014, 3(6), 474-86.
[46] Pal, PB; Ghosh, S; Sil, PC. Beneficial Effect of Naturally Occurring Antioxidants
against Oxidative Stress–Mediated Organ Dysfunctions. Bioactive Natural Products:
Chemistry and Biology, 2015.
[47] Saha, S; Sadhukhan, P; C Sil, P. Genistein: A Phytoestrogen with Multifaceted
Therapeutic Properties. Mini Rev Med Chem, 2014, 14(11), 920-40.
[48] Bhattacharyya, S; Sinha, K; C Sil, P. Cytochrome P450s: Mechanisms and Biological
Implications in Drug Metabolism and its Interaction with Oxidative Stress. Current
drug metabolism, 2014, 15(7), 719-42.
[49] Ali, SS; Kasoju, N; Luthra, A; et al. Indian medicinal herbs as sources of antioxidants.
Food Research International, 2008, 41(1), 1-15.
[50] Narendra, K; Swathi, J; Sowjanya, K; Satya, A. Phyllanthus niruri: a review on its
ethno botanical, phytochemical and pharmacological profile. J Pharm Res, 2012, 5(9),
4681-91.
[51] Neville, Jr. D; Glossmann, H. Molecular weight determination of membrane protein
and glycoprotein subunits by discontinuous gel electrophoresis in dodecyl sulfate.
Methods Enzymol, 1973, 32, 92-102.
[52] c Ooi, VE; Liu, F. Immunomodulation and anti-cancer activity of polysaccharide-
protein complexes. Curr Med Chem, 2000, 7(7), 715-29.
[53] Oh, PS; Lee, SJ; Lim, KT. Antioxidative activity of 90 kDa glycoprotein isolated from
Opuntia ficus-indica var. saboten Makino. Food Science and Biotechnology, 2004.
[54] Oh, PS; Lim, KT. Glycoprotein (90 kDa) isolated from Opuntia ficus-indica var.
saboten MAKINO lowers plasma lipid level through scavenging of intracellular
radicals in Triton WR-1339-induced mice. Biol Pharm Bull, 2006, 29(7), 1391-6.
[55] Lim, KT. Inhibitory effect of glycoprotein isolated from Opuntia ficus-indica var.
saboten MAKINO on activities of allergy-mediators in compound 48/80-stimulated
mast cells. Cell Immunol, 2010, 264(1), 78-85.
[56] Senyshyn, J; Baumgartner, RA; Beaven, MA. Quercetin sensitizes RBL-2H3 cells to
polybasic mast cell secretagogues through increased expression of Gi GTP-binding
proteins linked to a phospholipase C signaling pathway. The Journal of Immunology,
1998, 160(10), 5136-44.
38 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

[57] Chahdi, A; Fraundorfer, PF; Beaven, MA. Compound 48/80 activates mast cell
phospholipase D via heterotrimeric GTP-binding proteins. J Pharmacol Exp Ther,
2000, 292(1), 122-30.
[58] Lee, SJ; Heo, KS; Oh, PS; Lim, K; Lim, KT. Glycoprotein isolated from Ulmus
davidiana Nakai inhibits TPA-induced apoptosis through nuclear factor-kappa B in
NIH/3T3 cells. Toxicol Lett, 2004, 146(2), 159-74.
[59] Oh, PS; Lee, SJ; Lim, KT. Glycoprotein (116 kD) isolated fromUlmus davidiana Nakai
protects from injury of 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated BNL CL. 2
cells. Pharmacol Rep, 2006, 58(67), 67-74.
[60] Hortelano, S; Genaro, A; Bosca, L. Phorbol esters induce nitric oxide synthase activity
in rat hepatocytes. Antagonism with the induction elicited by lipopolysaccharide. J Biol
Chem, 1992, 267(35), 24937-40.
[61] Lee, J; Lim, KT. Normalizing effect of plant-originated glycoprotein (116 kDa) on
G0/G1 arrest in cadmium chloride-induced primary cultured mouse myelocytes.
Naunyn-Schmiedeberg’s archives of pharmacology, 2011, 383(2), 109-18.
[62] Datta, S; Sinha, S; Bhattacharyya, P. Effect of a herbal protein, CI-1, isolated from
Cajanus indicus on immune response of control and stressed mice. J Ethnopharmacol,
1999, 67(3), 259-67.
[63] Datta, S; Sinha, S; Bhattacharyya, P. Hepatoprotective activity of a herbal protein CI-1,
purified from Cajanus indicus against β-galactosamine HCl toxicity in isolated rat
hepatocytes. Phytother Res, 1999, 13(6), 508-12.
[64] Datta, S; Bhattacharyya, P. Effect of a herbal protein CI-1, purified from Cajanus
indicus on the ultrastructural study of hepatocytes, in models of liver failure in mice. J
Ethnopharmacol, 2001, 77(1), 11-8.
[65] Sarkar, K; Ghosh, A; Kinter, M; Mazumder, B; Sil, PC. Purification and
Characterization of a 43 kD Hepatoprotective Protein from the Herb Cajanus indicus L.
The protein journal, 2006, 25(6), 411-21.
[66] Ghosh, A; Sarkar, K; Sil, PC. Protective effect of a 43 kD protein from the leaves of the
herb, Cajanus indicus L on chloroform induced hepatic-disorder. J Biochem Mol Biol,
2006, 39(2), 197.
[67] Ghosh, A; Sil, PC. A protein from Cajanus indicus Spreng protects liver and kidney
against mercuric chloride-induced oxidative stress. Biol Pharm Bull, 2008, 31(9), 1651-
8.
[68] Manna, P; Sinha, M; Sil, PC. Galactosamine-induced hepatotoxic effect and
hepatoprotective role of a protein isolated from the herb Cajanus indicus L in vivo. J
Biochem Mol Toxicol, 2007, 21(1), 13-23.
[69] Sinha, M; Manna, P; Sil, PC. Taurine protects the antioxidant defense system in the
erythrocytes of cadmium treated mice. BMB reports, 2008, 41(9), 657-63.
[70] Sarkar, K; Ghosh, A; Sil, PC. Preventive and curative role of a 43kD protein from the
leaves of the herb Cajanus indicus L on thioacetamide-induced hepatotoxicity in vivo.
Hepatology research: the official journal of the Japan Society of Hepatology, 2005,
33(1), 39-49.
[71] Sarkar, K; Sil, PC. A 43kDa protein from the herb Cajanus indicus L. protects
thioacetamide induced cytotoxicity in hepatocytes. Toxicol In Vitro, 2006, 20(5), 634-
40.
Bioactive Natural Compounds 39

[72] Sarkar, K; Sil, PC. Cajanus indicus leaf protein: Beneficial role in experimental organ
pathophysiology. A review. Pathophysiology, 2011, 18(4), 295-303.
[73] Sinha, M; Manna, P; Sil, PC. Amelioration of galactosamine-induced nephrotoxicity by
a protein isolated from the leaves of the herb, Cajanus indicus L. BMC Complement
Altern Med, 2007, 7(1), 11.
[74] Sinha, M; Manna, P; Sil, PC. A 43kD protein from the herb, Cajanus indicus
L., protects against fluoride induced oxidative stress in mice erythrocytes.
Pathophysiology, 2007, 14(1), 47-54.
[75] Patten, CJ; Thomas, PE; Guy, RL; et al. Cytochrome P450 enzymes involved in
acetaminophen activation by rat and human liver microsomes and their kinetics. Chem
Res Toxicol, 1993, 6(4), 511-8.
[76] Thummel, KE; Lee, CA; Kunze, KL; Nelson, SD; Slattery, JT. Oxidation of
acetaminophen to N-acetyl-p-aminobenzoquinone imine by human CYP3A4. Biochem
Pharmacol, 1993, 45(8), 1563-9.
[77] Chen, W; Koenigs, LL; Thompson, SJ; et al. Oxidation of acetaminophen to its toxic
quinone imine and nontoxic catechol metabolites by baculovirus-expressed and purified
human cytochromes P450 2E1 and 2A6. Chem Res Toxicol, 1998, 11(4), 295-301.
[78] Mitchell, JR; Jollow, DJ; Potter, WZ; Gillette, JR; Brodie, BB. Acetaminophen-induced
hepatic necrosis. IV. Protective role of glutathione. J Pharmacol Exp Ther, 1973,
187(1), 211-7.
[79] Nelson, SD. editor Molecular mechanisms of the hepatotoxicity caused by
acetaminophen. Semin Liver Dis, 1990.
[80] Weis, M; Kass, G; Orrenius, S; Moldeus, P. N-acetyl-p-benzoquinone imine induces
Ca2+ release from mitochondria by stimulating pyridine nucleotide hydrolysis. J Biol
Chem, 1992, 267(2), 804-9.
[81] Ray, SD; Kamendulis, LM; Gurule, MW; Yorkin, RD; Corcoran, GB. Ca2+ antagonists
inhibit DNA fragmentation and toxic cell death induced by acetaminophen. The FASEB
journal, 1993, 7(5), 453-63.
[82] Donnelly, PJ; Walker, RM; Racz, WJ. Inhibition of mitochondrial respiration in vivo is
an early event in acetaminophen-induced hepatotoxicity. Arch Toxicol, 1994, 68(2),
110-8.
[83] Salas, VM; Corcoran, GB. Calcium-dependent DNA damage and adenosine 3′, 5′-
cyclic monophosphate-independent glycogen phosphorylase activation in an in vitro
model of acetaminophen-induced liver injury. Hepatology, 1997, 25(6), 1432-8.
[84] Das, J; Ghosh, J; Manna, P; Sil, PC. Taurine protects acetaminophen-induced oxidative
damage in mice kidney through APAP urinary excretion and CYP2E1 inactivation.
Toxicology, 2010, 269(1), 24-34.
[85] Wrights, N; Prescott, LF. Potentiation by previous drug therapy of hepatotoxicity
following paracetamol overdosage. Scott Med J, 1973, 18(2), 56-8.
[86] Ameer, B; Greenblatt, DJ. Acetaminophen. Ann Intern Med, 1977, 87(2), 202-9.
[87] Mudge, GH; Gemborys, MW; Duggin, GG. Covalent binding of metabolites of
acetaminophen to kidney protein and depletion of renal glutathione. J Pharmacol Exp
Ther, 1978, 206(1), 218-26.
[88] Srivastava, A; Maggs, JL; Antoine, DJ; et al. Role of reactive metabolites in drug-
induced hepatotoxicity. Handb Exp Pharmacol, 2010, 196, 165-94.
40 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

[89] Ghosh, A; Sil, PC. Anti-oxidative effect of a protein from Cajanus indicus L against
acetaminophen-induced hepato-nephro toxicity. J Biochem Mol Biol, 2007, 40(6),
1039-49.
[90] Pal, S; Ahir, M; Sil, PC. Doxorubicin-induced neurotoxicity is attenuated by a 43-kD
protein from the leaves of Cajanus indicus L. via NF-kappaB and mitochondria
dependent pathways. Free Radic Res, 2012, 46(6), 785-98.
[91] Pal, S; Sil, PC. A 43 kD protein from the leaves of the herb Cajanus indicus L.
modulates doxorubicin induced nephrotoxicity via MAPKs and both mitochondria
dependent and independent pathways. Biochimie, 2012, 94(6), 1356-67.
[92] Chopra, R; Varma, B; Chopra, I. Supplement to glossary of Indian medicinal plants:
Publications and Information Directorate-CSIR; 1986.
[93] Thyagarajan, S; Jayaram, S; Valliammai, T; et al. Phyllanthus amarus and hepatitis B.
The Lancet, 1990, 336(8720), 949-50.
[94] Thyagarajan, S; Thirunalasundari, T; Subramanian, S; Venkateswaran, P; Blumberg, B.
Effect of Phyllanthus amarus on chronic carriers of hepatitis B virus. The Lancet, 1988,
332(8614), 764-6.
[95] Harish, R; Shivanandappa, T. Antioxidant activity and hepatoprotective potential of
Phyllanthus niruri. Food chemistry, 2006, 95(2), 180-5.
[96] Cheng, JT; Lin, TC; Hsu, FL. Antihypertensive effect of corilagin in the rat. Can J
Physiol Pharmacol, 1995, 73(10), 1425-9.
[97] Chatterjee, M; Sil, PC. Hepatoprotective effect of aqueous extract of Phyllanthus niruri
on nimesulide-induced oxidative stress in vivo. Indian J Biochem Biophys, 2006, 43(5),
299.
[98] Chatterjee, M; Sarkar, K; Sil, PC. Herbal (Phyllanthus niruri) protein isolate protects
liver from nimesulide induced oxidative stress. Pathophysiology, 2006, 13(2), 95-102.
[99] Chatterjee, M; Sil, PC. Protective role of Phyllanthus niruri against nimesulide induced
hepatic damage. Indian J Clin Biochem, 2007, 22(1), 109-16.
[100] Bhattacharjee, R; Sil, PC. The protein fraction of Phyllanthus niruri plays a protective
role against acetaminophen induced hepatic disorder via its antioxidant properties.
Phytother Res, 2006, 20(7), 595-601.
[101] Sarkar, MK; Sil, PC. Hepatocytes are protected by herb Phyllanthus niruri protein
isolate against thioacetamide toxicity. Pathophysiology, 2007, 14(2), 113-20.
[102] Bhattacharjee, R; Sil, PC. Protein Isolate from the Herb Phyllanthus niruri Modulates
Carbon Tetrachloride-Induced Cytotoxicity in Hepatocytes. Toxicol Mech Methods,
2007, 17(1), 41-7.
[103] Bhattacharjee, R; Sil, PC. Protein isolate from the herb, Phyllanthus niruri L.
(Euphorbiaceae), plays hepatoprotective role against carbon tetrachloride induced liver
damage via its antioxidant properties. Food Chem Toxicol, 2007, 45(5), 817-26.
[104] Sarkar, MK; Kinter, M; Mazumder, B; Sil, PC. Purification and characterisation of a
novel antioxidant protein molecule from Phyllanthus niruri. Food Chemistry, 2009,
114(4), 1405-12.
[105] Cawthon, D; McNew, R; Beers, K; Bottje, W. Evidence of mitochondrial dysfunction
in broilers with pulmonary hypertension syndrome (ascites): Effect of t-butyl
hydroperoxide on hepatic mitochondrial function, glutathione, and related thiols. Poult
Sci, 1999, 78(1), 114-24.
Bioactive Natural Compounds 41

[106] Kaur, P; Bansal, MP. Upregulation of AP1 by tertiary butyl hydroperoxide induced
oxidative stress and subsequent effect on spermatogenesis in mice testis. Mol Cell
Biochem, 2008, 308(1-2), 177-81.
[107] Sarkar, MK; Sil, PC. Prevention of tertiary butyl hydroperoxide induced oxidative
impairment and cell death by a novel antioxidant protein molecule isolated from the
herb, Phyllanthus niruri. Toxicol In Vitro, 2010, 24(6), 1711-9.
[108] Wilson, MT; Reeder, BJ. Oxygen-binding haem proteins. Exp Physiol, 2008, 93(1),
128-32.
[109] Um, HD; Orenstein, JM; Wahl, SM. Fas mediates apoptosis in human monocytes by a
reactive oxygen intermediate dependent pathway. The Journal of Immunology, 1996,
156(9), 3469-77.
[110] Bhattacharyya, S; Pal, PB; Sil, PC. A 35kD Phyllanthus niruri protein modulates iron
mediated oxidative impairment to hepatocytes via the inhibition of ERKs, p38 MAPKs
and activation of PI3k/Akt pathway. Food Chem Toxicol, 2013, 56, 119-30.
[111] Wang, J; Ito, T; Udaka, N; et al. PI3K–AKT pathway mediates growth and survival
signals during development of fetal mouse lung. Tissue Cell, 2005, 37(1), 25-35.
[112] Khawaja, NR; Carre, M; Kovacic, H; Esteve, MA; Braguer D. Patupilone-induced
apoptosis is mediated by mitochondrial reactive oxygen species through Bim
relocalization to mitochondria. Mol Pharmacol, 2008, 74(4), 1072-83.
[113] Giardina, C; Inan, MS. Nonsteroidal anti-inflammatory drugs, short-chain fatty acids,
and reactive oxygen metabolism in human colorectal cancer cells. Biochimica et
Biophysica Acta (BBA)-Molecular Cell Research, 1998, 1401(3), 277-88.
[114] Mingatto, FE; Santos, AC; Uyemura, SA; Jordani, MC; Curti, C. In Vitro Interaction of
Nonsteroidal Anti-inflammatory Drugs on Oxidative Phosphorylation of Rat Kidney
Mitochondria: Respiration and ATP Synthesis. Arch Biochem Biophys, 1996, 334(2),
303-8.
[115] Suzuki, Y; Inoue, T; Ra, C. NSAIDs, mitochondria and calcium signaling: Special
focus on aspirin/salicylates. Pharmaceuticals, 2010, 3(5), 1594-613.
[116] Hossain, MA; Kim, DH; Jang, JY; et al. Aspirin enhances doxorubicin-induced
apoptosis and reduces tumor growth in human hepatocellular carcinoma cells in vitro
and in vivo. Int J Oncol, 2012, 40(5), 1636-42.
[117] Bhattacharyya, S; Ghosh, S; Sil, PC. Amelioration of aspirin induced oxidative
impairment and apoptotic cell death by a novel antioxidant protein molecule isolated
from the herb Phyllanthus niruri. PLoS One, 2014, 9(2).
[118] Joo, HY; Lim, K,T. Glycoprotein isolated from Cudrania tricuspidata Bureau inhibits
iNO and COX-2 expression through modulation of NF-κB in LPS-stimulated RAW
264.7 cells. Environ Toxicol Pharmacol, 2009, 27(2), 247-52.
[119] Medica, CM. Jiangsu New Medical College, Ed. Shanghai People’s Pub House:
Shanghai. 1977.
[120] Shim, JU; Lim, KT. Inhibitory effect of glycoprotein isolated from Cudrania
tricuspidata bureau on expression of inflammation-related cytokine in bisphenol A-
treated HMC-1 cells. Inflammation, 2009, 32(4), 211-7.
[121] Lee, J; Lee, SJ; Lim, KT. CTB glycoprotein (75kDa) inhibits IgE releasing; TNF-α and
IL-6 expressed by bisphenol A in vivo and in vitro. Food Chem Toxicol, 2012, 50(6),
2109-17.
42 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

[122] Yurino, H; Ishikawa, S; Sato, T; et al. Endocrine disruptors (environmental estrogens)


enhance autoantibody production by B1 cells. Toxicol Sci, 2004, 81(1), 139-47.
[123] Oh, PS; Lim, KT. Phytoglycoprotein (75 kDa) isolated from Cudrania tricuspidata
Bureau inhibits expression of interleukin-4 in the presence of di (2-ethylhexyl)
phthalate via modulation of p38 mitogen-activated protein kinase in primary-cultured
mouse thymocytes. J Appl Toxicol, 2009, 29(6), 496-504.
[124] Doi, K; Kojima, T; FUJIMOTO, Y. Mulberry leaf extract inhibits the oxidative
modification of rabbit and human low density lipoprotein. Biol Pharm Bull, 2000,
23(9), 1066-71.
[125] Andallu, B; Suryakantham, V; Srikanthi, BL; Reddy, GK. Effect of mulberry (Morus
indica L.) therapy on plasma and erythrocyte membrane lipids in patients with type 2
diabetes. Clin Chim Acta, 2001, 314(1), 47-53.
[126] Andallu, B; Varadacharyulu, NC. Antioxidant role of mulberry (Morus indica L. cv.
Anantha) leaves in streptozotocin-diabetic rats. Clin Chim Acta, 2003, 338(1), 3-10.
[127] Oh, PS; Lim, K; Lim, KT. Phytoglycoprotein (75 kDa) inhibits expression of
interleukin-1β stimulated by DEHP in human mast cells. Cell Biochem Funct, 2010,
28(5), 352-9.
[128] Das, J; Ghosh, J; Manna, P; Sil, PC. Taurine provides antioxidant defense against NaF-
induced cytotoxicity in murine hepatocytes. Pathophysiology, 2008, 15(3), 181-90.
[129] Manna, P; Sinha, M; Sil, PC. Cadmium induced testicular pathophysiology:
prophylactic role of taurine. Reprod Toxicol, 2008, 26(3-4), 282-91.
[130] Das, J; Ghosh, J; Manna, P; Sinha, M; Sil, PC. Arsenic-induced oxidative cerebral
disorders: protection by taurine. Drug Chem Toxicol, 2009, 32(2), 93-102.
[131] Das, J; Ghosh, J; Manna, P; Sil, PC. Taurine suppresses doxorubicin-triggered
oxidative stress and cardiac apoptosis in rat via up-regulation of PI3-K/Akt and
inhibition of p53, p38-JNK. Biochem Pharmacol, 2011, 81(7), 891-909.
[132] Uchida, S; Kwon, HM; Yamauchi, A; et al. Molecular cloning of the cDNA for an
MDCK cell Na(+)- and Cl(-)-dependent taurine transporter that is regulated by
hypertonicity. Proc Natl Acad Sci, 1992, 89(17), 8230-4.
[133] Bouckenooghe, T; Remacle, C; Reusens, B. Is taurine a functional nutrient? Curr Opin
Clin Nutr Metab Care, 2006, 9(6), 728-33.
[134] Das, J; Ghosh, J; Manna, P; Sil, PC. Protective role of taurine against arsenic-induced
mitochondria-dependent hepatic apoptosis via the inhibition of PKCdelta-JNK pathway.
PLoS One, 2010, 5(9), e12602.
[135] Das, J; Ghosh, J; Manna, P; Sinha, M; Sil, PC. Taurine protects rat testes against
NaAsO(2)-induced oxidative stress and apoptosis via mitochondrial dependent and
independent pathways. Toxicol Lett, 2009, 187(3), 201-10.
[136] Birdsall, TC. Therapeutic applications of taurine. Altern Med Rev, 1998, 3(2), 128-36.
[137] Huxtable, RJ. Expanding the circle 1975-1999: sulfur biochemistry and insights on the
biological functions of taurine. Adv Exp Med Biol, 2000, 483, 1-25.
[138] O'Flaherty, L; Stapleton, PP; Redmond, HP; Bouchier-Hayes, DJ. Intestinal taurine
transport: a review. Eur J Clin Invest, 1997, 27(11), 873-80.
[139] Tappaz, ML. Taurine biosynthetic enzymes and taurine transporter: molecular
identification and regulations. Neurochem Res, 2004, 29(1), 83-96.
Bioactive Natural Compounds 43

[140] Yamauchi, H; Aminaka, Y; Yoshida, K; et al. Evaluation of DNA damage in patients


with arsenic poisoning: urinary 8-hydroxydeoxyguanine. Toxicol Appl Pharmacol,
2004, 198(3), 291-6.
[141] Bustamante, J; Nutt, L; Orrenius, S; Gogvadze, V. Arsenic stimulates release of
cytochrome c from isolated mitochondria via induction of mitochondrial permeability
transition. Toxicol Appl Pharmacol, 2005, 207(2 Suppl), 110-6.
[142] Wittman, R; Hu, H. Cadmium exposure and nephropathy in a 28-year-old female
metals worker. Environ Health Perspect, 2002, 110(12), 1261-6.
[143] Bhatnagar, A. Environmental cardiology: studying mechanistic links between pollution
and heart disease. Circ Res, 2006, 99(7), 692-705.
[144] Szuster-Ciesielska, A; Stachura, A; Slotwinska, M; et al. The inhibitory effect of zinc
on cadmium-induced cell apoptosis and reactive oxygen species (ROS) production in
cell cultures. Toxicology, 2000, 145(2-3), 159-71.
[145] Yazihan, N; Kocak, MK; Akcil, E; Erdem, O; Sayal, A. Role of midkine in cadmium-
induced liver; heart and kidney damage. Hum Exp Toxicol, 2011, 30(5), 391-7.
[146] Jin, T; Nordberg, M; Frech, W; et al. Cadmium biomonitoring and renal dysfunction
among a population environmentally exposed to cadmium from smelting in China
(ChinaCad). Biometals, 2002, 15(4), 397-410.
[147] Ahn, DW; Kim, YM; Kim, KR; Park, YS. Cadmium binding and sodium-dependent
solute transport in renal brush-border membrane vesicles. Toxicol Appl Pharmacol,
1999, 154(3), 212-8.
[148] Casalino, E; Calzaretti, G; Sblano, C; Landriscina, C. Molecular inhibitory mechanisms
of antioxidant enzymes in rat liver and kidney by cadmium. Toxicology, 2002, 179(1-
2), 37-50.
[149] Muller, L. Consequences of cadmium toxicity in rat hepatocytes: mitochondrial
dysfunction and lipid peroxidation. Toxicology, 1986, 40(3), 285-95.
[150] Li, W; Zhao, Y; Chou, IN. Alterations in cytoskeletal protein sulfhydryls and cellular
glutathione in cultured cells exposed to cadmium and nickel ions. Toxicology, 1993,
77(1-2), 65-79.
[151] Coogan, TP; Bare, RM; Waalkes, MP. Cadmium-induced DNA strand damage in
cultured liver cells: reduction in cadmium genotoxicity following zinc pretreatment.
Toxicol Appl Pharmacol, 1992, 113(2), 227-33.
[152] Manna, P; Sinha, M; Sil, PC. Amelioration of cadmium-induced cardiac impairment by
taurine. Chem Biol Interact, 2008, 174(2), 88-97.
[153] Hortobagyi, GN. Anthracyclines in the treatment of cancer. An overview. Drugs, 1997,
54 (Suppl 4), 1-7.
[154] Orhan, B. Doxorubicin cardiotoxicity: growing importance. J Clin Oncol, 1999, 17(7),
2294-6.
[155] Liu, TJ; Yeh, YC; Ting, CT; et al. Ginkgo biloba extract 761 reduces doxorubicin-
induced apoptotic damage in rat hearts and neonatal cardiomyocytes. Cardiovasc Res,
2008, 80(2), 227-35.
[156] Huang, XM; Zhu, WH; Kang, ML. Study on the effect of doxorubicin on expressions of
genes encoding myocardial sarcoplasmic reticulum Ca2+ transport proteins and the
effect of taurine on myocardial protection in rabbits. Journal of Zhejiang University
Science, 2003, 4(1), 114-20.
44 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

[157] El-Seweidy, MM; Sadik, NA; Shaker, OG. Role of sulfurous mineral water and sodium
hydrosulfide as potent inhibitors of fibrosis in the heart of diabetic rats. Arch Biochem
Biophys, 2011, 506(1), 48-57.
[158] Li, C; Cao, L; Zeng, Q; et al. Taurine may prevent diabetic rats from developing
cardiomyopathy also by downregulating angiotensin II type2 receptor expression.
Cardiovasc Drugs Ther, 2005, 19(2), 105-12.
[159] Trachtman, H; Del Pizzo, R; Futterweit, S; et al. Taurine attenuates renal disease in
chronic puromycin aminonucleoside nephropathy. Am J Physiol, 1992, 262(1 Pt 2),
F117-23.
[160] Wu, QD; Wang, JH; Fennessy, F; Redmond, HP; Bouchier-Hayes, D. Taurine prevents
high-glucose-induced human vascular endothelial cell apoptosis. Am J Physiol, 1999,
277(6 Pt 1), C1229-38.
[161] Baek, YY; Cho, DH; Choe, J; et al. Extracellular taurine induces angiogenesis by
activating ERK-, Akt-, and FAK-dependent signal pathways. Eur J Pharmacol, 2012,
674(2-3), 188-99.
[162] Moloney, MA; Casey, RG; O’Donnell, DH; et al. Two weeks taurine supplementation
reverses endothelial dysfunction in young male type 1 diabetics. Diab Vasc Dis Res,
2010, 7(4), 300-10.
[163] Wojcik, OP; Koenig, KL; Zeleniuch-Jacquotte, A; Costa, M; Chen, Y. The potential
protective effects of taurine on coronary heart disease. Atherosclerosis, 2010, 208(1),
19-25.
[164] Pandya, KG; Patel, MR; Lau-Cam, CA. Comparative study of the binding
characteristics to and inhibitory potencies towards PARP and in vivo antidiabetogenic
potencies of taurine, 3-aminobenzamide and nicotinamide. J Biomed Sci, 2010, 17
(Suppl 1), S16.
[165] Kulakowski, EC; Maturo, J. Hypoglycemic properties of taurine: not mediated by
enhanced insulin release. Biochem Pharmacol, 1984, 33(18), 2835-8.
[166] Winiarska, K; Szymanski, K; Gorniak, P; Dudziak, M; Bryla, J. Hypoglycaemic,
antioxidative and nephroprotective effects of taurine in alloxan diabetic rabbits.
Biochimie, 2009, 91(2), 261-70.
[167] Gavrovskaya, LK; Ryzhova, OV; Safonova, AF; Matveev, AK; Sapronov, NS.
Protective effect of taurine on rats with experimental insulin-dependent diabetes
mellitus. Bull Exp Biol Med, 2008, 146(2), 226-8.
[168] Obrosova, IG; Minchenko, AG; Marinescu, V; et al. Antioxidants attenuate early up
regulation of retinal vascular endothelial growth factor in streptozotocin-diabetic rats.
Diabetologia, 2001, 44(9), 1102-10.
[169] Li, F; Obrosova, IG; Abatan, O; et al. Taurine replacement attenuates hyperalgesia and
abnormal calcium signaling in sensory neurons of STZ-D rats. Am J Physiol Endocrinol
Metab, 2005, 288(1), E29-36.
[170] Wu, N; Lu, Y; He, B; et al. Taurine prevents free fatty acid-induced hepatic insulin
resistance in association with inhibiting JNK1 activation and improving insulin
signaling in vivo. Diabetes Res Clin Pract, 2010, 90(3), 288-96.
[171] Kim, HW; Lee, AJ; You, S; Park, T; Lee, DH. Characterization of taurine as inhibitor
of sodium glucose transporter. Adv Exp Med Biol, 2006, 583, 137-45.
Bioactive Natural Compounds 45

[172] Yan, HD; Li, XZ; Xie, JM; Li, M. Effects of advanced glycation end products on renal
fibrosis and oxidative stress in cultured NRK-49F cells. Chin Med J (Engl), 2007,
120(9), 787-93.
[173] Higo, S; Miyata, S; Jiang, QY; et al. Taurine administration after appearance of
proteinuria retards progression of diabetic nephropathy in rats. Kobe J Med Sci, 2008,
54(1), E35-45.
[174] Stevens, MJ; Lattimer, SA; Kamijo, M; et al. Osmotically-induced nerve taurine
depletion and the compatible osmolyte hypothesis in experimental diabetic neuropathy
in the rat. Diabetologia, 1993, 36(7), 608-14.
[175] Eguia, L; Materson, BJ. Acetaminophen-related acute renal failure without fulminant
liver failure. Pharmacotherapy, 1997, 17(2), 363-70.
[176] Yu, X; Xu, Z; Mi, M; et al. Dietary taurine supplementation ameliorates diabetic
retinopathy via anti-excitotoxicity of glutamate in streptozotocin-induced Sprague-
Dawley rats. Neurochem Res, 2008, 33(3), 500-7.
[177] Zeng, K; Xu, H; Mi, M; et al. Dietary taurine supplementation prevents glial alterations
in retina of diabetic rats. Neurochem Res, 2009, 34(2), 244-54.
[178] Wang, LJ; Yu, YH; Zhang, LG; et al. Taurine rescues vascular endothelial dysfunction
in streptozocin-induced diabetic rats: correlated with downregulation of LOX-1 and
ICAM-1 expression on aortas. Eur J Pharmacol, 2008, 597(1-3), 75-80.
[179] Ulrich-Merzenich, G; Zeitler, H; Vetter, H; Bhonde, RR. Protective effects of taurine
on endothelial cells impaired by high glucose and oxidized low density lipoproteins.
Eur J Nutr, 2007, 46(8), 431-8.
[180] Gurujeyalakshmi, G; Wang, Y; Giri, SN. Suppression of bleomycin-induced nitric
oxide production in mice by taurine and niacin. Nitric Oxide, 2000, 4(4), 399-411.
[181] Yildirim, Z; Kilic, N; Ozer, C; Babul, A; et al. Effects of taurine in cellular responses to
oxidative stress in young and middle-aged rat liver. Ann N Y Acad Sci, 2007, 1100, 553-
61.
[182] Vohra, BP; Hui, X. Taurine protects against carbon tetrachloride toxicity in the cultured
neurons and in vivo. Arch Physiol Biochem, 2001, 109(1), 90-4.
[183] El Idrissi, A; Trenkner, E. Growth factors and taurine protect against excitotoxicity by
stabilizing calcium homeostasis and energy metabolism. J Neurosci, 1999, 19(21),
9459-68.
[184] Schaffer, SW; Azuma, J; Mozaffari, M. Role of antioxidant activity of taurine in
diabetes. Can J Physiol Pharmacol, 2009, 87(2), 91-9.
[185] Han, X; Yue, J; Chesney, RW. Functional TauT protects against acute kidney injury. J
Am Soc Nephrol, 2009, 20(6), 1323-32.
[186] Yasunaga, M; Matsumura, Y. Role of SLC6A6 in promoting the survival and multidrug
resistance of colorectal cancer. Sci Rep, 2014, 4, 4852.
[187] Tastesen, HS; Holm, JB; Moller, J; et al. Pinpointing differences in cisplatin-induced
apoptosis in adherent and non-adherent cancer cells. Cell Physiol Biochem, 2010, 26(6),
809-20.
[188] Lotsch, J; Hummel, T; Warskulat, U; et al. Congenital taurine deficiency in mice is
associated with reduced sensitivity to nociceptive chemical stimulation. Neuroscience,
2014, 259, 63-70.
46 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

[189] Ito, T; Oishi, S; Takai, M; et al. Cardiac and skeletal muscle abnormality in taurine
transporter-knockout mice. J Biomed Sci, 2010, 17(Suppl 1), S20.
[190] Han, X; Chesney, RW. Knockdown of TauT expression impairs human embryonic
kidney 293 cell development. Adv Exp Med Biol, 2013, 776, 307-20.
[191] Kaesler, S; Sobiesiak, M; Kneilling, M; et al. Effective T-cell recall responses require
the taurine transporter Taut. Eur J Immunol, 2012, 42(4), 831-41.
[192] Lin, S; Yang, J; Wu, G; et al. Inhibitory effects of taurine on STZ-induced apoptosis of
pancreatic islet cells. Adv Exp Med Biol, 2013, 775, 287-97.
[193] Chen, K; Zhang, Q; Wang, J; et al. Taurine protects transformed rat retinal ganglion
cells from hypoxia-induced apoptosis by preventing mitochondrial dysfunction. Brain
Res, 2009, 1279, 131-8.
[194] Zeng, K; Xu, H; Mi, M; et al. Effects of taurine on glial cells apoptosis and taurine
transporter expression in retina under diabetic conditions. Neurochem Res, 2010,
35(10), 1566-74.
[195] Maher, SG; Condron, CE; Bouchier-Hayes, DJ; Toomey, DM. Taurine attenuates
CD3/interleukin-2-induced T cell apoptosis in an in vitro model of activation-induced
cell death (AICD). Clin Exp Immunol, 2005, 139(2), 279-86.
[196] Takahashi, K; Ohyabu, Y; Takahashi, K; et al. Taurine renders the cell resistant to
ischemia-induced injury in cultured neonatal rat cardiomyocytes. J Cardiovasc
Pharmacol, 2003, 41(5), 726-33.
[197] Takatani, T; Takahashi, K; Uozumi, Y; et al. Taurine inhibits apoptosis by preventing
formation of the Apaf-1/caspase-9 apoptosome. Am J Physiol Cell Physiol, 2004,
287(4), C949-53.
[198] Das, J; Ghosh, J; Manna, P; Sil, PC. Taurine protects rat testes against doxorubicin-
induced oxidative stress as well as p53, Fas and caspase 12-mediated apoptosis. Amino
Acids, 2012, 42(5), 1839-55.
[199] Sinha, M; Manna, P; Sil, PC. Cadmium-induced neurological disorders: prophylactic
role of taurine. J Appl Toxicol, 2008, 28(8), 974-86.
[200] Roy, A; Sil, PC. Taurine protects murine hepatocytes against oxidative stress-induced
apoptosis by tert-butyl hydroperoxide via PI3K/Akt and mitochondrial-dependent
pathways. Food Chemistry, 2012, 131(4), 1086-96.
[201] Wijeratne, SS; Abou-Zaid, MM; Shahidi, F. Antioxidant polyphenols in almond and its
coproducts. J Agric Food Chem, 2006, 54(2), 312-8.
[202] Gopal, JV. Morin Hydrate: Botanical origin, pharmacological activity and its
applications: A mini-review. Pharmacognosy Journal, 2013, 5(3), 123-6.
[203] Caillet, S; Yu, H; Lessard, S; et al. Fenton reaction applied for screening natural
antioxidants. Food Chemistry, 2007, 100(2), 542-52.
[204] Heeba, GH; Mahmoud, ME. Therapeutic potential of morin against liver fibrosis in rats:
modulation of oxidative stress, cytokine production and nuclear factor kappa B.
Environ Toxicol Pharmacol, 2014, 37(2), 662-71.
[205] Wang, X; Zhang, DM; Gu, TT; et al. Morin reduces hepatic inflammation-associated
lipid accumulation in high fructose-fed rats via inhibiting sphingosine kinase
1/sphingosine 1-phosphate signaling pathway. Biochem Pharmacol, 2013, 86(12),
1791-804.
Bioactive Natural Compounds 47

[206] Prahalathan, P; Kumar, S; Raja, B. Effect of morin, a flavonoid against DOCA-salt


hypertensive rats: a dose dependent study. Asian Pacific journal of tropical
biomedicine., 2012, 2(6), 443-8.
[207] Al-Numair, KS; Chandramohan, G; Alsaif, MA. Pretreatment with morin, a flavonoid,
ameliorates adenosine triphosphatases and glycoproteins in isoproterenol-induced
myocardial infarction in rats. J Nat Med, 2012, 66(1), 95-101.
[208] Aggarwal, BB; Shishodia, S. Molecular targets of dietary agents for prevention and
therapy of cancer. Biochem Pharmacol, 2006, 71(10), 1397-421.
[209] Rattanachaikunsopon, P; Phumkhachorn, P. Contents and antibacterial activity of
flavonoids extracted from leaves of Psidium guajava. Journal of Medicinal Plants
Research, 2010, 4(5), 393-6.
[210] Sreedharan, V; Venkatachalam, KK; Namasivayam, N. Effect of morin on tissue lipid
peroxidation and antioxidant status in 1, 2-dimethylhydrazine induced experimental
colon carcinogenesis. Invest New Drugs, 2009, 27(1), 21-30.
[211] Janeiro, P; Brett, AMO. Solid state electrochemical oxidation mechanisms of morin in
aqueous media. Electroanalysis, 2005, 17(9), 733-8.
[212] Markovic, Z; Milenkovic, D; Dorovic, J; et al. PM6 and DFT study of free radical
scavenging activity of morin. Food chemistry, 2012, 134(4), 1754-60.
[213] Montaña, MP; Massad, WA; Criado, S; Biasutti, A; García, NA. Stability of Flavonoids
in the Presence of Riboflavin-photogenerated Reactive Oxygen Species: A Kinetic and
Mechanistic Study on Quercetin, Morin and Rutin. Photochem Photobiol, 2010, 86(4),
827-34.
[214] Suber, L; Imperatori, P; Mari, A; et al. Thermal hysteresis of Morin transition in
hematite particles. Phys Chem Chem Phys, 2010, 12(26), 6984-9.
[215] Park, HR; Im, SE; Seo, JJ; et al. Spectroscopic properties of morin in various CH3OH-
H2O and CH3CN-H2O mixed solvents. Photochem Photobiol, 2015, 91(2), 280-90.
[216] Xie, MX; Long, M; Liu, Y; Qin, C; Wang, YD. Characterization of the interaction
between human serum albumin and morin. Biochim Biophys Acta, 2006, 1760(8), 1184-
91.
[217] Qi, ZD; Zhang, Y; Liao, FL; et al. Probing the binding of morin to human serum
albumin by optical spectroscopy. J Pharm Biomed Anal, 2008, 46(4), 699-706.
[218] Zhang, HM; Wang, YQ; Zhou, QH. Investigation of the interactions of quercetin and
morin with trypsin. Luminescence, 2009, 24(5), 355-62.
[219] Jakhar, R; Paul, S; Chauhan, AK; Kang, SC. Morin hydrate augments phagocytosis
mechanism and inhibits LPS induced autophagic signaling in murine macrophage. Int
Immunopharmacol., 2014, 22(2), 356-65.
[220] Tianzhu, Z; Shihai, Y; Juan, D. The effects of morin on lipopolysaccharide-induced
acute lung injury by suppressing the lung NLRP3 inflammasome. Inflammation, 2014,
37(6), 1976-83.
[221] Jung, HJ; Kim, SJ; Song, YS; Park, EH; Lim, CJ. Evaluation of the antiangiogenic,
anti-inflammatory, and antinociceptive activities of morin. Planta Med, 2010, 76(3),
273-5.
[222] Galvez, J; Coelho, G; Crespo, ME; et al. Intestinal anti-inflammatory activity of morin
on chronic experimental colitis in the rat. Aliment Pharmacol Ther, 2001, 15(12), 2027-
39.
48 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

[223] Hogaboam, CM; Jacobson, K; Collins, SM; Blennerhassett, MG. The selective
beneficial effects of nitric oxide inhibition in experimental colitis. Am J Physiol, 1995,
268(4 Pt 1), G673-84.
[224] Salzman, A; Denenberg, AG; Ueta, I; O’Connor, M; Linn, SC; Szabo, C. Induction and
activity of nitric oxide synthase in cultured human intestinal epithelial monolayers. Am
J Physiol, 1996, 270(4 Pt 1), G565-73.
[225] Stenson, WF. Inflammatory mediators in inflammatory bowel disease. Current Opinion
in Gastroenterology, 1994, 10(4), 384-9.
[226] Yamada, Y; Marshall, S; Specian, RD; Grisham, MB. A comparative analysis of two
models of colitis in rats. Gastroenterology, 1992, 102(5), 1524-34.
[227] Jolly, PS; Bektas, M; Watterson, KR; et al. Expression of SphK1 impairs degranulation
and motility of RBL-2H3 mast cells by desensitizing S1P receptors. Blood, 2005,
105(12), 4736-42.
[228] Pal, C; Bindu, S; Dey, S; et al. Gallic acid prevents nonsteroidal anti-inflammatory
drug-induced gastropathy in rat by blocking oxidative stress and apoptosis. Free Radic
Biol Med, 2010, 49(2), 258-67.
[229] Bindu, S; Mazumder, S; Dey, S; et al. Nonsteroidal anti-inflammatory drug induces
proinflammatory damage in gastric mucosa through NF-κB activation and neutrophil
infiltration: Anti-inflammatory role of heme oxygenase-1 against nonsteroidal anti-
inflammatory drug. Free Radic Biol Med, 2013, 65, 456-67.
[230] Sinha, K; Sadhukhan, P; Saha, S; Pal, PB; Sil, PC. Morin protects gastric mucosa from
nonsteroidal anti-inflammatory drug, indomethacin induced inflammatory damage and
apoptosis by modulating NF-kappaB pathway. Biochim Biophys Acta, 2015, 1850(4),
769-83.
[231] Hawkey, CJ. Non-steroidal anti-inflammatory drug gastropathy: causes and treatment.
Scand J Gastroenterol Suppl, 1996, 220, 124-7.
[232] Park, JY; Kang, KA; Kim, KC; Cha, JW; Kim, EH; Hyun, JW. Morin Induces Heme
Oxygenase-1 via ERK-Nrf2 Signaling Pathway. Journal of cancer prevention, 2013,
18(3), 249-56.
[233] Subash, S; Subramanian, P. Morin a flavonoid exerts antioxidant potential in chronic
hyperammonemic rats: a biochemical and histopathological study. Mol Cell Biochem,
2009, 327(1-2), 153-61.
[234] Zhang, R; Kang, KA; Kang, SS; Park, JW; Hyun, JW. Morin (2',3,4',5,7-
pentahydroxyflavone) protected cells against gamma-radiation-induced oxidative stress.
Basic Clin Pharmacol Toxicol, 2011, 108(1), 63-72.
[235] Kim, JM; Lee, EK; Park, G; et al. Morin modulates the oxidative stress-induced NF-
kappaB pathway through its anti-oxidant activity. Free Radic Res, 2010, 44(4), 454-61.
[236] Prahalathan, P; Kumar, S; Raja, B. Morin attenuates blood pressure and oxidative stress
in deoxycorticosterone acetate-salt hypertensive rats: a biochemical and
histopathological evaluation. Metabolism, 2012, 61(8), 1087-99.
[237] Abraham, P; Rabi, S. Protective effect of aminoguanidine against cyclophosphamide-
induced oxidative stress and renal damage in rats. Redox report: communications in
free radical research, 2011, 16(1), 8-14.
Bioactive Natural Compounds 49

[238] Korkmaz, A; Topal, T; Oter, S. Pathophysiological aspects of cyclophosphamide and


ifosfamide induced hemorrhagic cystitis; implication of reactive oxygen and nitrogen
species as well as PARP activation. Cell Biol Toxicol, 2007, 23(5), 303-12.
[239] Merwid-Lad, A; Trocha, M; Chlebda-Sieragowska, E; et al. The impact of morin, a
natural flavonoid, on cyclophosphamide-induced changes in the oxidative stress
parameters in rat livers. Advances in clinical and experimental medicine: official organ
Wroclaw Medical University, 2014, 23(4), 505-9.
[240] Ray, S; Chowdhury, P; Pandit, B; Ray, SD; Das, S. Exploring the antiperoxidative
potential of morin on cyclophosphamide and flutamide-induced lipid peroxidation and
changes in cholesterol profile in rabbit model. Acta Pol Pharm, 2010, 67(1), 35-44.
[241] MadanKumar, P; NaveenKumar, P; Manikandan, S; Devaraj, H; NiranjaliDevaraj, S.
Morin ameliorates chemically induced liver fibrosis in vivo and inhibits stellate cell
proliferation in vitro by suppressing Wnt/beta-catenin signaling. Toxicol Appl
Pharmacol, 2014, 277(2), 210-20.
[242] MadanKumar, P; NaveenKumar, P; Devaraj, H; NiranjaliDevaraj, S. Morin, a dietary
flavonoid, exhibits anti-fibrotic effect and induces apoptosis of activated hepatic stellate
cells by suppressing canonical NF-kappaB signaling. Biochimie, 2015, 110, 107-18.
[243] Gupta, SC; Phromnoi, K; Aggarwal, BB. Morin inhibits STAT3 tyrosine 705
phosphorylation in tumor cells through activation of protein tyrosine phosphatase
SHP1. Biochem Pharmacol, 2013, 85(7), 898-912.
[244] Sivaramakrishnan, V; Niranjali Devaraj, S. Morin regulates the expression of NF-
kappaB-p65, COX-2 and matrix metalloproteinases in diethylnitrosamine induced rat
hepatocellular carcinoma. Chem Biol Interact, 2009, 180(3), 353-9.
[245] Jin, H; Lee, WS; Eun, SY; et al. Morin, a flavonoid from Moraceae, suppresses growth
and invasion of the highly metastatic breast cancer cell line MDA-MB‑231 partly
through suppression of the Akt pathway. Int J Oncol, 2014, 45(4), 1629-37.
[246] Park, C; Lee, WS; Go, SI; et al. Morin, a Flavonoid from Moraceae, Induces Apoptosis
by Induction of BAD Protein in Human Leukemic Cells. International journal of
molecular sciences, 2014, 16(1), 645-59.
[247] Nandhakumar, R; Salini, K; Niranjali Devaraj, S. Morin augments anticarcinogenic and
antiproliferative efficacy against 7,12-dimethylbenz(a)-anthracene induced
experimental mammary carcinogenesis. Mol Cell Biochem, 2012, 364(1-2), 79-92.
[248] Dixon, M; Woodrick, J; Gupta, S; et al. Naturally occurring polyphenol, morin hydrate,
inhibits enzymatic activity of N-methylpurine DNA glycosylase, a DNA repair enzyme
with various roles in human disease. Bioorg Med Chem, 2015, 23(5), 1102-11.
[249] Lemkul, JA; Bevan, DR. Morin inhibits the early stages of amyloid β-peptide
aggregation by altering tertiary and quaternary interactions to produce “off-pathway”
structures. Biochemistry, 2012, 51(30), 5990-6009.
[250] Lemkul, JA; Bevan, DR. Destabilizing Alzheimer’s Abeta(42) protofibrils with morin:
mechanistic insights from molecular dynamics simulations. Biochemistry, 2010, 49(18),
3935-46.
[251] Gong, EJ; Park, HR; Kim, ME; et al. Morin attenuates tau hyperphosphorylation by
inhibiting GSK3beta. Neurobiol Dis, 2011, 44(2), 223-30.
50 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

[252] Stoothoff, WH; Johnson, GV. Tau phosphorylation: physiological and pathological
consequences. Biochim Biophys Acta, 2005, 1739(2-3), 280-97.
[253] Zhang, ZT; Cao, XB; Xiong, N; et al. Morin exerts neuroprotective actions in Parkinson
disease models in vitro and in vivo. Acta Pharmacol Sin, 2010, 31(8), 900-6.
[254] Noor, H; Cao, P; Raleigh, DP. Morin hydrate inhibits amyloid formation by islet
amyloid polypeptide and disaggregates amyloid fibers. Protein Sci, 2012, 21(3), 373-
82.
[255] Paoli, P; Cirri, P; Caselli, A; et al. The insulin-mimetic effect of Morin: a promising
molecule in diabetes treatment. Biochim Biophys Acta, 2013, 1830(4), 3102-11.
[256] Vanitha, P; Uma, C; Suganya, N; et al. Modulatory effects of morin on hyperglycemia
by attenuating the hepatic key enzymes of carbohydrate metabolism and beta-cell
function in streptozotocin-induced diabetic rats. Environ Toxicol Pharmacol, 2014,
37(1), 326-35.
[257] Abuohashish, HM; Al-Rejaie, SS; Al-Hosaini, KA; Parmar, MY; Ahmed, MM.
Alleviating effects of morin against experimentally-induced diabetic osteopenia.
Diabetol Metab Syndr, 2013, 5(1), 5.
[258] Kapoor, R; Kakkar, P. Protective role of morin, a flavonoid, against high glucose
induced oxidative stress mediated apoptosis in primary rat hepatocytes. PLoS One,
2012, 7(8), e41663.
[259] Bloom, S; Cancilla, PA. Myocytolysis and mitochondrial calcification in rat
myocardium after low doses of isoproterenol. Am J Pathol, 1969, 54(3), 373-91.
[260] Pogula, BK; Maharajan, MK; Oddepalli, DR; et al. Morin protects heart from beta-
adrenergic-stimulated myocardial infarction: an electrocardiographic, biochemical, and
histological study in rats. J Physiol Biochem, 2012, 68(3), 433-46.
[261] Al-Numair, KS; Chandramohan, G; Alsaif, MA. Pretreatment with morin, a flavonoid,
ameliorates adenosine triphosphatases and glycoproteins in isoproterenol-induced
myocardial infarction in rats. J Nat Med, 2012, 66(1), 95-101.
[262] Al-Numair, KS; Chandramohan, G; Alsaif, MA; Veeramani, C; El Newehy, AS. Morin,
a flavonoid, on lipid peroxidation and antioxidant status in experimental myocardial
ischemic rats. Afr J Tradit Complement Altern Med, 2014, 11(3), 14-20.
[263] Metodiewa, D; Jaiswal, AK; Cenas, N; Dickancaite, E; Segura-Aguilar, J. Quercetin
may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and
quinoidal product. Free Radic Biol Med, 1999, 26(1-2), 107-16.
[264] Dajas, F. Life or death: neuroprotective and anticancer effects of quercetin. J
Ethnopharmacol, 2012, 143(2), 383-96.
[265] Wätjen, W; Michels, G; Steffan, B; et al. Low concentrations of flavonoids are
protective in rat H4IIE cells whereas high concentrations cause DNA damage and
apoptosis. The Journal of nutrition, 2005, 135(3), 525-31.
[266] Braganhol, E; Zamin, LL; Canedo, AD; et al. Antiproliferative effect of quercetin in the
human U138MG glioma cell line. Anticancer Drugs, 2006, 17(6), 663-71.
[267] Xie, X; Yin, J; Jia, Q; et al. Quercetin induces apoptosis in the methotrexate-resistant
osteosarcoma cell line U2-OS/MTX300 via mitochondrial dysfunction and
dephosphorylation of Akt. Oncol Rep, 2011, 26(3), 687-93.
[268] Vidya Priyadarsini, R; Senthil Murugan, R; Maitreyi, S; et al. The flavonoid quercetin
induces cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer
Bioactive Natural Compounds 51

(HeLa) cells through p53 induction and NF-kappaB inhibition. Eur J Pharmacol, 2010,
649(1-3), 84-91.
[269] Hsieh, TC; Wu, JM. Targeting CWR22Rv1 prostate cancer cell proliferation and gene
expression by combinations of the phytochemicals EGCG, genistein and quercetin.
Anticancer Res, 2009, 29(10), 4025-32.
[270] Choi, EJ; Bae, SM; Ahn, WS. Antiproliferative effects of quercetin through cell cycle
arrest and apoptosis in human breast cancer MDA-MB-453 cells. Arch Pharm Res,
2008, 31(10), 1281-5.
[271] Priego, S; Feddi, F; Ferrer, P; et al. Natural polyphenols facilitate elimination of HT-29
colorectal cancer xenografts by chemoradiotherapy: a Bcl-2-and superoxide dismutase
2-dependent mechanism. Mol Cancer Ther, 2008, 7(10), 3330-42.
[272] Duraj, J; Zazrivcova, K; Bodo, J; Sulikova, M; Sedlak, J. Flavonoid quercetin, but not
apigenin or luteolin, induced apoptosis in human myeloid leukemia cells and their
resistant variants. Neoplasma, 2004, 52(4), 273-9.
[273] Kang, JW; Kim, JH; Song, K; et al. Kaempferol and quercetin, components of Ginkgo
biloba extract (EGb 761), induce caspase-3-dependent apoptosis in oral cavity cancer
cells. Phytother Res, 2010, 24 Suppl 1, S77-82.
[274] Murakami, A; Ashida, H; Terao, J. Multitargeted cancer prevention by quercetin.
Cancer Lett, 2008, 269(2), 315-25.
[275] Caltagirone, S; Rossi, C; Poggi, A; et al. Flavonoids apigenin and quercetin inhibit
melanoma growth and metastatic potential. Int J Cancer, 2000, 87(4), 595-600.
[276] Tan, J; Wang, B; Zhu, L. Regulation of Survivin and Bcl-2 in HepG2 Cell Apoptosis
Induced by Quercetin. Chem Biodivers, 2009, 6(7), 1101-10.
[277] Zhang, Q; Zhao, XH; Wang, ZJ. Flavones and flavonols exert cytotoxic effects on a
human oesophageal adenocarcinoma cell line (OE33) by causing G2/M arrest and
inducing apoptosis. Food Chem Toxicol, 2008, 46(6), 2042-53.
[278] Zhang, Q; Zhao, XH; Wang, ZJ. Cytotoxicity of flavones and flavonols to a human
esophageal squamous cell carcinoma cell line (KYSE-510) by induction of G 2/M
arrest and apoptosis. Toxicol In Vitro, 2009, 23(5), 797-807.
[279] Haupt, S; Berger, M; Goldberg, Z; Haupt, Y. Apoptosis-the p53 network. J Cell Sci,
2003, 116(20), 4077-85.
[280] Chien, SY; Wu, YC; Chung, JG; et al. Quercetin-induced apoptosis acts through
mitochondrial-and caspase-3-dependent pathways in human breast cancer MDA-MB-
231 cells. Hum Exp Toxicol, 2009, 28(8), 493-503.
[281] Ranelletti, FO; Maggiano, N; Serra, FG; et al. Quercetin inhibits p21-RAS expression
in human colon cancer cell lines and in primary colorectal tumors. Int J Cancer, 2000,
85(3), 438-45.
[282] Bandele, OJ; Clawson, SJ; Osheroff, N. Dietary polyphenols as topoisomerase II
poisons: B ring and C ring substituents determine the mechanism of enzyme-mediated
DNA cleavage enhancement. Chem Res Toxicol, 2008, 21(6), 1253-60.
[283] López-Lázaro, M; Willmore, E; Austin, CA. The dietary flavonoids myricetin and
fisetin act as dual inhibitors of DNA topoisomerases I and II in cells. Mutation
Research/Genetic Toxicology and Environmental Mutagenesis, 2010, 696(1), 41-7.
[284] Aalinkeel, R; Bindukumar, B; Reynolds, JL; et al. The dietary bioflavonoid; quercetin;
selectively induces apoptosis of prostate cancer cells by down-regulating the expression
of heat shock protein 90. The Prostate, 2008, 68(16), 1773-89.
52 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

[285] Wang, RE; Kao, JL; Hilliard, CA; et al. Inhibition of heat shock induction of heat shock
protein 70 and enhancement of heat shock protein 27 phosphorylation by quercetin
derivatives. J Med Chem, 2009, 52(7), 1912-21.
[286] Oršolić, N; Benković, V; Horvat-Knežević, A; et al. Assessment by survival analysis of
the radioprotective properties of propolis and its polyphenolic compounds. Biol Pharm
Bull, 2007, 30(5), 946-51.
[287] Benković, V; Knežević, A; Đikić, D; et al. Radioprotective effects of quercetin and
ethanolic extract of propolis in gamma-irradiated mice. Archives of Industrial Hygiene
and Toxicology, 2009, 60(2), 129-38.
[288] Dajas, F; Arredondo, F; Echeverry, C; et al. Flavonoids and the brain: Evidences and
putative mechanisms for a protective capacity. Curr Neuropharmacol, 2005, 3(3), 193-
205.
[289] Jakubowicz-Gil, J; Rzeski, W; Zdzisinska, B; Dobrowolski, P; Gawron A. Cell death
and neuronal arborization upon quercetin treatment in rat neurons. Acta Neurobiol Exp
(Wars), 2008, 68(2), 139.
[290] Ossola, B; Kääriäinen, TM; Männistö, PT. The multiple faces of quercetin in
neuroprotection. 2009.
[291] Arredondo, F; Echeverry, C; Abin-Carriquiry, JA; et al. After cellular internalization,
quercetin causes Nrf2 nuclear translocation, increases glutathione levels, and prevents
neuronal death against an oxidative insult. Free Radic Biol Med, 2010, 49(5), 738-47.
[292] Park, C; So, HS; Shin, CH; et al. Quercetin protects the hydrogen peroxide-induced
apoptosis via inhibition of mitochondrial dysfuntion in H9c2 cardiomyoblast cells.
Biochem Pharmacol, 2003, 66(7), 1287-95.
[293] Santos, AC; Uyemura, SA; Lopes, JL; et al. Effect of naturally occurring flavonoids on
lipid peroxidation and membrane permeability transition in mitochondria. Free Radic
Biol Med, 1998, 24(9), 1455-61.
[294] Kerry, NL; Abbey, M. Red wine and fractionated phenolic compounds prepared from
red wine inhibit low density lipoprotein oxidation in vitro. Atherosclerosis, 1997,
135(1), 93-102.
[295] Chang, WS; Lee, YJ; Lu, FJ; Chiang, HC. Inhibitory effects of flavonoids on xanthine
oxidase. Anticancer Res, 1992, 13(6A), 2165-70.
[296] Calamia, KT. Current and future use of anti-TNF agents in the treatment of
autoimmune, inflammatory disorders. Adamantiades-Behçet’s Disease: Springer, 2003.
p. 545-9.
[297] Busse, WW; Kopp, DE; Elliott, M. Flavonoid modulation of human neutrophil
function. J Allergy Clin Immunol, 1984, 73(6), 801-9.
[298] Middleton, E; Drzewiecki, G; Krishnarao, D. Quercetin: an inhibitor of antigen-induced
human basophil histamine release. The Journal of Immunology, 1981, 127(2), 546-50.
[299] Yoshimoto, T; Furukawa, M; Yamamoto, S; Horie, T; Watanabe-Kohno, S. Flavonoids:
potent inhibitors of arachidonate 5-lipoxygenase. Biochem Biophys Res Commun, 1983,
116(2), 612-8.
[300] Ferrali, M; Signorini, C; Caciotti, B; et al. Protection against oxidative damage of
erythrocyte membrane by the flavonoid quercetin and its relation to iron chelating
activity. FEBS Lett, 1997, 416(2), 123-9.
Bioactive Natural Compounds 53

[301] Sorata, Y; Takahama, U; Kimura, M. Protective effect of quercetin and rutin on


photosensitized lysis of human erythrocytes in the presence of hematoporphyrin.
Biochimica et Biophysica Acta (BBA)-General Subjects, 1984, 799(3), 313-7.
[302] Aritomi, M; Kawasaki, T. A new xanthone C-glucoside, position isomer of mangiferin,
from Anemarrhena asphodeloides Bunge. Tetrahedron Lett, 1969, 10(12), 941-4.
[303] Singh, S; Kumar, Y; Kumar, SS; et al. Antimicrobial evaluation of mangiferin
analogues. Indian J Pharm Sci, 2009, 71(3), 328.
[304] Martınez, G; Delgado, R; Pérez, G; et al. Evaluation of the in vitro antioxidant activity
of Mangifera indica L. extract (Vimang). Phytother Res, 2000, 14, 424-7.
[305] Sánchez, GM; Re, L; Giuliani, A; et al. Protective effects of Mangifera indica L.
extract, mangiferin and selected antioxidants against TPA-induced biomolecules
oxidation and peritoneal macrophage activation in mice. Pharmacol Res, 2000, 42(6),
565-73.
[306] Sairam, K; Hemalatha, S; Kumar, A; et al. Evaluation of anti-diarrhoeal activity in seed
extracts of Mangifera indica. J Ethnopharmacol, 2003, 84(1), 11-5.
[307] Anila, L; Vijayalakshmi, N. Flavonoids from Emblica officinalis and Mangifera
indica—effectiveness for dyslipidemia. J Ethnopharmacol, 2002, 79(1), 81-7.
[308] Aderibigbe, A; Emudianughe, T; Lawal, B. Antihyperglycaemic effect of Mangifera
indica in rat. Phytother Res, 1999, 13(6), 504-7.
[309] Garcia, D; Escalante, M; Delgado, R; Ubeira, F; Leiro, J. Anthelminthic and
antiallergic activities of Mangifera indica L. stem bark components Vimang and
mangiferin. Phytother Res, 2003, 17(10), 1203-8.
[310] Bairy, I; Reeja, S; Rao, PS; Bhat, M; Shivananda, P. Evaluation of antibacterial activity
of Mangifera indica on anaerobic dental microglora based on in vivo studies. Indian J
Pathol Microbiol, 2002, 45(3), 307-10.
[311] Guha, S; Ghosal, S; Chattopadhyay, U. Antitumor, immunomodulatory and anti-HIV
effect of mangiferin, a naturally occurring glucosylxanthone. Chemotherapy, 1996,
42(6), 443-51.
[312] Yoshimi, N; Matsunaga, K; Katayama, M; et al. The inhibitory effects of mangiferin, a
naturally occurring glucosylxanthone, in bowel carcinogenesis of male F344 rats.
Cancer Lett, 2001, 163(2), 163-70.
[313] Leiro, J; Arranz, JA; Yánez, M; et al. Expression profiles of genes involved in the
mouse nuclear factor-kappa B signal transduction pathway are modulated by
mangiferin. Int Immunopharmacol, 2004, 4(6), 763-78.
[314] Makare, N; Bodhankar, S; Rangari, V. Immunomodulatory activity of alcoholic extract
of Mangifera indica L. in mice. J Ethnopharmacol, 2001, 78(2), 133-7.
[315] Dar, A; Faizi, S; Naqvi, S; et al. Analgesic and antioxidant activity of mangiferin and
its derivatives: the structure activity relationship. Biol Pharm Bull, 2005, 28(4), 596-
600.
[316] Ghosh, M; Das, J; Sil, PC. D(+) galactosamine induced oxidative and nitrosative stress-
mediated renal damage in rats via NF-kappaB and inducible nitric oxide synthase
(iNOS) pathways is ameliorated by a polyphenol xanthone; mangiferin. Free Radic Res,
2012, 46(2), 116-32.
[317] Manna, SK; Kuo, MT; Aggarwal, BB. Overexpression of gamma-glutamylcysteine
synthetase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear
transcription factor-kappa B and activator protein-1. Oncogene, 1999, 18(30), 4371-82.
54 Sudip Bhattacharyya, Sayantani Chowdhury and Parames C. Sil

[318] Andreu, GP; Delgado, R; Velho, JA; Curti, C; Vercesi, AE. Iron complexing activity of
mangiferin, a naturally occurring glucosylxanthone, inhibits mitochondrial lipid
peroxidation induced by Fe 2+-citrate. Eur J Pharmacol, 2005, 513(1), 47-55.
[319] Hansen, JM; Zhang, H; Jones, DP. Differential oxidation of thioredoxin-1, thioredoxin-
2, and glutathione by metal ions*. Free Radic Biol Med, 2006, 40(1), 138-45.
[320] Agarwala, S; Mudholkar, K; Bhuwania, R; Rao, S. Mangiferin, a dietary xanthone
protects against mercury-induced toxicity in HepG2 cells. Environ Toxicol, 2012, 27(2),
117-27.
[321] Garcıa, D; Delgado, R; Ubeira, F; Leiro, J. Modulation of rat macrophage function by
the Mangifera indica L. extracts Vimang and mangiferin. Int Immunopharmacol, 2002,
2(6), 797-806.
[322] Leiro, JM; Alvarez, E; Arranz, JA; Siso, IG; Orallo, F. In vitro effects of mangiferin on
superoxide concentrations and expression of the inducible nitric oxide synthase, tumour
necrosis factor-α and transforming growth factor-β genes. Biochem Pharmacol, 2003,
65(8), 1361-71.
[323] Aggarwal, BB; Shishodia, S; Sandur, SK; Pandey, MK; Sethi, G. Inflammation and
cancer: how hot is the link? Biochem Pharmacol, 2006, 72(11), 1605-21.
[324] Sellamuthu, PS; Muniappan, BP; Perumal, SM; Kandasamy, M. Antihyperglycemic
effect of mangiferin in streptozotocin induced diabetic rats. Journal of Health science,
2009, 55(2), 206-14.
[325] Pal, PB; Sinha, K; Sil, PC. Mangiferin attenuates diabetic nephropathy by inhibiting
oxidative stress mediated signaling cascade, TNFα related and mitochondrial dependent
apoptotic pathways in streptozotocin-induced diabetic rats. PLoS One 2014; DOI:
10.1371/ journal.pone.0107220.
In: Advances in Natural Products Discovery ISBN: 978-1-53610-088-4
Editors: Ana Rita Gomes, Teresa Rocha-Santos et al. © 2017 Nova Science Publishers, Inc.

Chapter 2

NATURAL THERAPEUTICS AGAINST ALZHEIMER’S


DISEASE: CONVENTIONAL TREATMENT VERSUS
PHYTOTHERAPY

Abhijit Dey1, and Anuradha Mukherjee2


1Department
of Biological Sciences, Presidency University,
Kolkata, India
2Biological Sciences, MMHS, West Bengal, India

ABSTRACT
Since antiquity, botanicals have been prescribed in the treatment of a number
of human ailments throughout the world. Even in modern society, phytotherapy is
used as complementary and alternative therapeutics against mortality and morbidity.
Alzheimer’s disease (AD) is a severe progressive neurodegenerative disorder affecting
the brain functionality in elderly people. Conventional treatment was found to provide
symptomatic relief in order to provide limited effectiveness. Hence a number of plants
and plant derived bioactive constituents have been investigated for anti-AD properties in
a number of preclinical and clinical studies. Scientific databases such as Pubmed, Google
Scholar, Scopus, Sciencedirect, Springer etc. were searched with relevant search strings
to find references of scientific studies involving use of plants and phytochemicals against
AD. This chapter elucidates the molecular mechanisms of action of natural products
against various aspects of disease pathogenesis and their possible role as neuroprotectants
against dementia and as cognitive enhancers. It also discusses the role of traditional
Chinese and Indian medicine as never ending resources in the discovery of lead
compounds against neurodegenerative disorder such as AD.

Keywords: Alzheimer’s disease, natural products, medicinal plants, phytotherapy,


neuroprotectants


Corresponding author. Department of Biological Sciences, Presidency University, 86/1 College Street, Kolkata-
700073. E-mail: abhijitbio25@yahoo.com.
56 Abhijit Dey and Anuradha Mukherjee

INTRODUCTION
Alzheimer's disease (AD) has been considered as one of the deadliest diseases of human
beings especially affecting elderly populations [34]. Alzheimer's disease is an age-associated,
irreversible, progressive neurodegenerative disease characterized by dementia, unusual
behavior, personality changes, and a loss in cognitive function [34, 39]. The progressive
decline in cholinergic synapses in hippocampus and neocortex and generation and
accumulation of amyloid-beta peptide (Aβ) are implicated in the pathogenesis of Alzheimer's
disease (AD) [1]. Alzheimer's disease (AD) is caused by malfunctioning of an array of
complex and varied intracellular and extracellular biochemical processes leading to neuron
death [11, 46]. Pathology of AD is dependent on the failure or defect in one or more such
biochemical pathways including beta-amyloid (Aβ) protein metabolism, abnormalities of
glutamatergic, adrenergic, serotonergic and dopaminergic neurotransmission, with possible
involvement of inflammatory, oxidative and hormonal mechanisms [11]. In addition,
mutations in the presenilin (PS) subunit of γ-secretase producing increased amounts of highly
amyloidogenic Aβ42 isoform has been known to cause early onset familial Alzheimer disease
(FAD) [42].
Unfortunately AD is non-curable, and the conventional medications suffer from limited
effectiveness [39]. A number of intense investigations have been carried out to elucidate the
molecular events occurring during AD pathogenesis and to explore novel therapeutic options
against AD. However, due to heterogeneity of the disease, very limited treatment strategies
have been reported. Hence, therapeutics acting on multiple levels of the pathology has been
proposed to combat heterogeneous diseases like AD [9].
A number of approved and non-approved therapies including pharmaceutical (AChE
and BChE inhibitors, secretase inhibitors, prolyl endopeptidase inhibitors, selective
phosphodiesterase inhibitors, antihypertensive agents,nonsteroidal anti-inflammatory drugs,
insulin resistance drugs, aggregation inhibitors, etanercept, brain-derived neurotrophic factor,
and immunization), nutritional and botanical (various plant extracts and phytochemicals,
vitamins and supplements) and stimulatory (physical exercise, cognitive training, music, and
socialization) therapies have been reported and many of these are considered as effective
treatment strategies with their own benefits and drawbacks [11, 34, 46]. Other non-
cholinergic strategies include the use of transition metal chelators, growth factors, hormones,
lipid-lowering agents, neurotransmitter enhancing agents and various receptor [e.g., nicotinic,
muscarinic, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA), gamma-
aminobutyric acid (GABA), N-methyl-D-aspartate (NMDA)] modulators [11] (. Among
these, NMDA receptor antagonist memantine deserves a special mention since it is already
approved in Europe to treat moderately severe to severe AD [11]. Besides inhibiting
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) responsible for break down
acetylcholine and butyrylcholine respectively, inhibition of secretase enzyme family of
protease type in the amyloidogenic pathway responsible for generation of beta-amyloid
peptide formed by the beta- and gamma-secretase is considered as another effective treatment
strategy against AD [34]. Plants and phyto-constituents used in traditional systems of
medicine such as including Ayurvedic, Chinese, European and Japanese medicine have been
reported as sources of effective leads either directly or as templates against various
neurodegenerative and cognitive disorders including AD [16]. Phytotherapy and plant derived
Natural Therapeutics Against Alzheimer’s Disease 57

chemicals have been known to provide pleiotropic protective properties effective on multiple
levels of the pathology associated with complex and heterogeneous diseases such as AD [9].
Medicinal plants have been depicted as a prolific source of anti-AD phytochemicals such as
lignans, flavonoids, tannins, polyphenols, triterpenes, sterols, alkaloids etc. active against a
number of pathological processes associated with AD via their anti-inflammatory, anti-
amyloidogenic, anti-cholinesterase, hypolipidemic, anti-apoptotic, oestrogenic and
antioxidant abilities [16, 39]. Plant secondary metabolites with their ability to inhibit
cholinesterase, prolyl endopeptidase, and secretase enzymes are considered as potential
treatment strategies against AD [34]. Efficacy and safety of herbal formulations as either
monotherapy or as adjunct to conventional medications effective against AD have also been
depicted and multi-location trials, large sample size, high qualities of methodology and
standardized herbal formulations needed to be studied prior to any clinical recommendations
of administering plant products against AD [31].
Acetylcholinesterase (AChE) (the key enzyme needed to breakdown acetylcholine)
inhibition, is recognized as a well-accepted strategy to treat neurological disorders such as
Alzheimer's disease, senile dementia, ataxia and myasthenia gravis [33]. Dementia associated
with AD is primarily attributed to the reduced level of the neurotransmitter, acetylcholine
(ACh). Hence, restoration of ACh via inhibiting two major types of cholinesterase
[Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)] reported to be an effective
treatment strategy against AD associated dementia [1]. In addition, effective, natural, or safer
alternative medications against dementia have got some momentum by some successful large,
multicenter clinical trials of AChE inhibitors providing symptomatic relief against AD [35].
However, such alternative disease modifying agents are yet to be characterized fully
considering their side effects and possible interactions with the conventional drugs [35].
However, cholinesterase inhibitors are known to provide only symptomatic treatment instead
of being curative medications [34]. Botanicals are considered as a promising source of this
AChE inhibitors [33]. In addition natural products with diverse structures and pleiotropic
profile have been reported to modulate γ-secretase activity which might serve as an exciting
aid in anti-AD drug development since mutations in the presenilin (PS) subunit of γ-secretase
has been reported to cause early onset familial Alzheimer disease (FAD) [42]. Table 1
represents anti-AD properties of plant derived natural products. Figure 1 depicts the chemical
structures of anti-AD compounds derived from http://www.chemspider.com.

ANTI-AD ACTIVITY OF PLANT


DERIVED NATURAL PRODUCTS
4-O-Methylhonokiol

4-O-methylhonokiol isolated from Magnolia officinalis at adose of 1.0 mg/kg protected


Swedish AβPP AD model (AβPPsw) mice via preventing memory impairment, neuronal cell
death, Aβ1-42 accumulation and oxidative damage, downregulating brain beta-site AβPP
cleaving enzyme (BACE1), upregulating Aβ degradation enzymes, insulin degrading enzyme
and neprilysin, low density lipoprotein receptor-related protein-1 and glutathione levels in the
brain and also inhibiting carbonyl protein, lipid peroxidation, caspase-3 and BAX expressions
58 Abhijit Dey and Anuradha Mukherjee

[7]. 4-O-methylhonokiol reduced oxidative stress and neuronal cell death via inhibiting p38
MAP kinase pathway in Aβ(1-42) induced mice pretreated with 0.2, 0.5 and 1.0 mg/kg
followed by Aβ(1-42) infusion in the animals. Since, p38 MAP kinase inhibitor attenuated the
inhibitory effects of 4-O-methylhonokiol in culture cortical neurons; the role of the compound
was attributed to the inhibition of p38 MAP kinase pathway [24].

Catalpol

Catalpol extracted from the roots of Rehmanniaglutinosa prevented beta-amyloid (1-42)


induced toxicity in cortical neuron-glia cultures at a concentration of 500 μM via exhibiting
anti-inflammation and reducing neurotoxicity and glial activation [20]. Catalpol at a dose of
0.5 mM prevented Abeta(1-42)-induced toxicity in primary cortical neuron cultures via
attenuating neuronal apoptosis, reversal of intracellular ROS accumulation, Bax level,
mitochondrial membrane potential and cytochrome c release and also by controlling of
caspase-3 and caspase-9 activity and cleavage [27].

Curcuminoids (A Mixture of Bisdemethoxycurcumin, Demethoxycurcumin


and Curcumin)

Various components of curcuminoids (a mixture of bisdemetho-xycurcumin,


demethoxycurcumin and curcumin) at doses of 30 mg/kg (curcuminoids mixture) and 3-30
mg/kg (individual components) enhanced spatial memory via upregulating PSD-95,
synaptophysin and camkIV expressions [2]. Curcuminoids decreased hippocampal IL-1β,
GFAP, caspase-3 and FasL levels at different doses (30 and 10mg/kg; 3mg/kg) in an Aβ plus
ibotenic acid-infused rat model [3].

(-)-epigallocatechin-3-gallate (EGCG)

The green tea catechin (-)-epigallocatechin-3-gallate (EGCG) at a dose of 50 mg/kg


daily, alone or in combination with voluntary exercise, enhanced nest building behavior and
spatial learning in TgCRND8 (Tg) mice via reducing the levels of soluble Aβ1-42 in the
cortex and hippocampus [43]. EGCG at a dose of 1.5 or 3 mg/kg prevented memory
dysfunction, reduced the levels of brain beta- and gamma-secretases and suppressed the
activation of extracellular signal-regulated kinase and nuclear transcription factor-kappaB and
inhibited apoptotic neuronal cell death in Abeta(1-42) induced mice. Moreover, at a dose of 3
mg/kg body weight EGCG enhanced memory function and brain alpha-secretase activity and
reduced Abeta levels besides declining brain beta- and gamma-secretase activities in
preseniline 2 (PS2) mutant AD mice. In addition, it prevented Abetafibrillization in vitro
exhibiting a half maximal inhibitory concentration of 7.5 mg/L [24].
Natural Therapeutics Against Alzheimer’s Disease 59

Melatonin

Melatonin at doses of 0.1, 1, and 10 mg/kg enhanced learning and memory functions and
prevented the massive glial reaction and the loss of neurons in cortex and hippocampus of
Aβ25-35 induced elder rats [41]. Melatonin at doses of 0.1 and 1 mg/kg enhanced learning
and memory and reduced the levels of IL-1alpha and C1q in hippocampus of Abeta25-35
induced rats [40].

Resveratrol

Resveratrol prevented memory loss, reduced amyloid burden, enhanced the level of
mitochondrial complex IV protein via inducing sirtuin 1 and AMPK pathways and promoting
IL1β and TNF gene expression in amyloid-β protein precursor/presenilin 1 (AβPP/PS1)
mouse model of Alzheimer's disease (AD) [38]. Resveratrol enhanced spatial memory,
reduced cellular levels of iNOS and lipid peroxidation and enhanced the level of heme
oxygenase-1 (HO-1) in Aβ induced adult Sprague-Dawley rats [18].

Silibinin

Silibinin extracted from milk thistle ameliorated memory deficits via reducing the
nitrotyrosine level and prevented overexpression of iNOS and TNF-alpha mRNA at
concentrations of 2, 20, and 200 mg/kg in the hippocampus and amygdala in Aβ(25-35)
induced mice [29]. Silibinin at a dose of 2, 20 and 200 mg.kg(-1) attenuated memory
impairment via prevented the increase in malondialdehyde and decline in glutathione levels
and prevented oxidative damage in Abeta(25-35) induced mice [30].

Xanthoceraside

Xanthoceraside isolated from the husk of Xanthocerassorbifoliaprotected SH-SY5Y cells


from Aβ25 35 induced toxicity by enhancing cell viability, decreasing apoptosis, ROS
accumulation, MMP dissipation, intracellular calcium burden and caspase-3 activity at doses
of 0.01 and 0.1 μM [5]. Xanthoceraside prevented learning and memory impairment in
Aβ(25-35) induced mice via enhancing the levels of superoxide dismutase, glutathione
peroxidase and acetylcholinesterase and declining the level ofmalondialdehydein Aβ impaired
mice [6].

Xylocoside G

Xylocoside G extracted from Itoaorientalis protected Aβ-induced SH-SY5Y cells and


primary neurons from Aβ-induced toxicity by increasing cell viability, modulating Bax and
60 Abhijit Dey and Anuradha Mukherjee

Bcl-2 expression and apoptosis and downregulating the expressions of caspase-3, tumor
necrosis factor-α, prostaglandin E2, cyclooxygenase-2, NF-κB p65 translocation and probably
by inhibiting JNK phosphorylation [48].

Piperine

Piperine isolated from the fruits of Piper nigrum at doses of 5, 10 and 20mg/kg protected
ethylcholineaziridinium ion (AF64A) induced adult male Wistar rats via preventing memory
impairment and neurodegeneration, reducing the levels of lipid peroxidation and
acetylcholinesterase activity besides exhibiting neurotrophic effect in hippocampus [8].

Salidroside

Salidroside from Rhodiolarosea protected Aβ(25-35)-induced SH-SY5Y human


neuroblastoma cells via elevating cell viability, inducing antioxidant enzymes, thioredoxin
(Trx), peroxiredoxin-I (PrxI) and heme oxygenase-1 (HO-1), downregulating Bax,
upregulating Bcl-X(L), restoring mitochondrial membrane potential (MMP) and reducing the
level of intracellular reactive oxygen species (ROS). In addition, the neuroprotective
mechanisms of the compound was attributed to its ability to inhibit Aβ(25-35)-
inducedphosphorylation of mitogen-activated protein (MAP) kinases without interfering with
extracellular signal-regulated kinase1/2 (ERK1/2) [50].

Nicotine

Nicotine hydrogen tartrate salt administered towards (Aβ) 25-35 induced rats at a dose of
1mg/kg prevented cognitive impairment and tau phosphorylation at Ser-202 and Thr-231 in
the hippocampus of the animals [10].

l-Theanine

l-theanine isolated from green tea (Camellia sinensis) at doses of 2 and 4 mg/kg
prevented memory impairment, decreased Aβ(1-42) levels and neuronal cell death via
downregulating ERK/p38 and NF-kappaB, reducing the enhanced glutathione level and
protecting the oxidative protein and lipid damage to the brain of Aβ(1-42)-induced mice [21].

Cryptotanshinone

Cryptotanshinone from Salvia miltiorrhiza induced alpha-secretase enzyme activity in


cortical neurons overexpressing Swedish mutant human APP695 via inducing
Natural Therapeutics Against Alzheimer’s Disease 61

phosphatidylinositol 3-kinase (PI3K) pathway [32]. In addition,cryptotanshinoneenhanced the


levels of sAβPPα and C-terminal fragment-α (CTF-α) produced from AβPP besides
enhancing the level of a disintegrin and metalloproteinase-10 (ADAM10) via PKC-α and
ADAM10 pathways [13].

Huperzine A

Huperzine A (at a dose range of 0-10 microM) enhanced alphaAPPs release and
membrane-coupled APP CTF-C83 possibly involving protein kinase C (PKC) in human
embryonic kidney 293 cells transfected with human APP bearing the Swedish mutation
(HEK293 APPsw) [36]. Huperzine A (at a dose range of 0-10 microM) enhanced the levels of
alphaAPPs and ADAM10, induced phosphorylation of p44/p42 mitogen-activated protein
(MAP) kinase via possibly inducing muscarinic acetylcholine receptors, PKC and MAP
kinase in human neuroblastoma SK-N-SH cells overexpressing wild-type human APP695
[37].

Gastrodin

Gastrodin extracted from the rhizome of Gastrodiaelata reduced memory impairment via
alleviating Aβ deposition and glial activation in Tg2576 mice AD model at a dose of
60 mg/kg via anti-inflammatory and anti-amyloidogenic properties [17].

Berberine

Berberine from Coptischinensis at doses of 25 mg/kg and 100 mg/kg prevented learning
deficits, reduced plaque load and β-amyloid deposition and improved spatial memory via
downregulating glycogen synthase kinase (GSK)3 and tau phosphorylation in TgCRND8
mice AD models. In addition, the compound decreased the C-terminal fragments of APP,
prevented hyperphosphorylation of APP and tau via involvement of Akt/glycogen synthase
kinase 3 signaling pathway in N2a mouse neuroblastoma cells stably expressing human
Swedish mutant APP695 (N2a-SwedAPP) [12].

Morin

Morin suppressed GSK3β and attenuated GSK3β-induced tau phosphorylation in vitro.


Moreover, it inhibited Aβ-induced tau phosphorylation and protected against Aβ cytotoxicity
in human neuroblastoma cell. Further, in 3xTg-AD mice it reduced tau pathology via
preventing tau hyperphosphorylation [14].
62 Abhijit Dey and Anuradha Mukherjee

Table 1. Anti-AD activity of plant derived natural products

Phytochemicals Plant source AD models Dose Mechanism of action Refe-


rences
4-O- Magnolia Aβ(1-42) 0.2, 0.5 and ↓oxidative stress, ↓neuronal cell [24]
methylhonokiol officinalis induced mice, 1.0 mg/kg death, ↓p38 MAP kinase pathway
culture cortical
neurons
4-O- Magnolia Swedish AβPP 1.0 mg/kg ↓memory impairment,↓neuronal [7]
methylhonokiol officinalis AD model cell death, ↓Aβ1-42 accumulation,
(AβPPsw) mice ↓oxidative damage, ↓brain beta-
site AβPP cleaving enzyme
(BACE1),↑Aβ degradation
enzymes,↑insulin degrading
enzyme and neprilysin, ↑low
density lipoprotein receptor-
related protein-1, ↓carbonyl
protein,↓lipid peroxidation,
↑glutathione, ↓caspase-3, ↓BAX
Catalpol Root of Aβ (1-42) 500 μM ↑anti-inflammation, [20]
Rehmanniaglu induced cortical ↓neurotoxicity, ↓glial activation
tinosa neuron-glia
cultures
Catalpol Root of Abeta(1-42)- 0.5 mM ↓neuronal apoptosis, ↑reversal of [27]
Rehmanniaglu induced primary intracellular ROS accumulation,
tinosa cortical neuron Bax level, mitochondrial
cultures membrane potential and
cytochrome c release, ↑control of
caspase-3 and caspase-9 activity
and cleavage
Curcuminoids (a Turmeric Aβ peptide- 30 mg/kg ↑spatial memory, ↑PSD-95, [2]
mixture of (Curcuma infused rat (Curcuminoid ↑synaptophysin, ↑camkIV
bisdemethoxy- longa) hippocampus s mixture), 3- expression
curcumin, 30 mg/kg
demethoxycurcu (individual
min and components)
curcumin)
Curcuminoids (a Turmeric Aβ plus ibotenic 30 and 10 hippocampal IL-1β, hippocampal [3]
mixture of (Curcuma acid-infused rat mg/kg; 3 GFAP, hippocampal caspase-3,
bisdemethoxy- longa) model mg/kg hippocampalFasL
curcumin,
demethoxycurcu
-min and
curcumin)
(-)- Green tea TgCRND8 (Tg) 50 mg/kg ↑nest building, ↑spatial learning, [43]
epigallocatechin (Camellia mice ↓Aβ1-42 levels
-3-gallate sinensis)
(EGCG)
(-)- Green Abeta(1-42) 1.5 or 3 ↓memory dysfunction, ↓brain [23]
epigallocatechin tea(Camellia induced mice, mg/kg, half beta- and gamma-secretase,
-3-gallate sinensis) preseniline 2 maximal ↓extracellular signal-regulated
(EGCG) (PS2) mutant inhibitory kinase,↓nuclear transcription
AD mice, Abeta concentration factor-kappaB, ↓apoptotic
= 7.5 mg/L neuronal cell death, ↑brain alpha-
secretase, ↓Abeta levels,
↓Abetafibrillization
Natural Therapeutics Against Alzheimer’s Disease 63

Phytochemicals Plant source AD models Dose Mechanism of action Refe-


rences
Melatonin Many plants Aβ(25-35) 0.1, 1, and 10 ↑learning, ↑memory, ↓massive [41]
induced elder mg/kg glial reaction ↑number of neurons
rats in cortex and hippocampus,
Melatonin Many plants Abeta(25-35) 0.01, 0.1, and ↑learning and memory, ↓IL- [40]
induced rats 1 mg/kg 1alpha, ↓C1q
Resveratrol Many plants Aβ protein - ↓memory loss, ↓amyloid burden, [38]
precursor/presen ↑mitochondrial complex IV
ilin 1 protein, ↑sirtuin 1 and AMPK
(AβPP/PS1) pathways, ↑IL1β and TNF gene
mouse model of expression
AD
Resveratrol Many plants Aβ induced adult - ↑spatial memory, ↓cellular levels [18]
Sprague-Dawley of iNOS,↓lipid peroxidation,
rats ↑heme oxygenase-1 (HO-1)
Silibinin Milk thistle Aβ(25-35) 2, 20, and 200 ↓memory deficits, ↓nitrotyrosine, [29]
(Silybummaria induced mice mg/kg ↓overexpression of iNOS and
num) TNF-alpha mRNA
Silibinin Milk thistle Abeta(25-35) 2, 20 and 200 ↓memory impairment, [30]
(Silybummaria induced mice mg.kg(-1) ↓malondialdehyde, ↑glutathione,
num) ↓oxidative damage
Xanthoceraside Husk of Aβ(25-35) - ↓learning and memory [6]
Xanthocerasso induced mice impairment, ↑superoxide
rbifolia dismutase, ↑glutathione
peroxidase, ↑acetylcholinesterase,
↓malondialdehyde
Xanthoceraside Husk of Aβ25-35 0.01 and 0.1 viability, ↓apoptosis, ↓ROS, [5]
Xanthocerasso induced SH- μM ↓MMP dissipation, ↓intracellular
r-bifolia SY5Y cells calcium burden, caspase-3
XylocosideG Itoaorientalis Aβ-induced SH- - ↑cell viability, modulation of Bax [48]
SY5Y cells and and Bcl-2 expression, ↓caspase-3,
primary neurons apoptosis control, ↓tumor necrosis
factor-α, ↓interleukin-1β,
↓prostaglandin E2,
↓cyclooxygenase-2, ↓NF-κB p65
translocation, ↓JNK
phosphorylation
Piperine Fruit of Piper ethylcholineaziri 5, 10 and ↓memory impairment, [8]
nigrum dinium ion 20mg/kg ↓neurodegeneration, ↓lipid
(AF64A) peroxidation,
induced adult ↓acetylcholinesterase,
male Wistar rats ↑neurotrophic effect
Salidroside Rhodiolarosea Aβ(25-35)- - ↑cell viability, ↑antioxidant [50]
inducedSH- enzymes, ↑thioredoxin (Trx),
SY5Y human ↑heme oxygenase-1 (HO-1),
neuroblastoma ↑peroxiredoxin-I (PrxI), ↓Bax,
cells ↑Bcl-X(L), ↑mitochondrial
membrane potential (MMP),
↓intracellular reactive oxygen
species (ROS), ↓phosphorylation
of mitogen-activated protein
(MAP) kinases
64 Abhijit Dey and Anuradha Mukherjee

Table 1. (Continued)

Phytochem- Plant source AD models Dose Mechanism of action Refe-


icals rences
Nicotine Members of Aβ (25-35) induced 1mg/kg ↓cognitive impairment, ↓tau [10]
Solanaceae rats phosphorylation at Ser-202 and
Thr-231
l-theanine Green tea Aβ(1-42)-induced 2 and 4 ↓memory impairment, ↓Aβ(1- [21]
(Camellia mice mg/kg 42) levels, ↓neuronal cell death,
sinensis) ↓ERK/p38 and NF-kappaB,
↓oxidative protein and lipid
damage, ↓glutathione
Cryptotan- Salvia Cortical neurons - ↑alpha-secretase, [32]
shinone miltiorrhiza overexpressing ↑phosphatidylinositol 3-kinase
Swedish mutant (PI3K) pathway
human APP695
Cryptotan- Salvia N2 a mouse - ↑sAβPPα, ↑C-terminal [13]
shinone miltiorrhiza neuroblastoma cells fragment-α (CTF-α), a
stably expressing disintegrin and
human metalloproteinase-10
SwedishAβPP (N2a- (ADAM10)
SwedAβPP)
Huperzine A Huperzia sp. Human embryonic 0-10 µM ↑alphaAPPs release, [36]
kidney 293 cells ↑membrane-coupled APP CTF-
transfected with C83, ↑protein kinase C (PKC)
human APP bearing
the Swedish
mutation (HEK293
APPsw)
Huperzine A Huperzia sp. Human 0-10 µM ↑alphaAPPs, ↑ADAM10, [37]
neuroblastoma SK- ↑phosphorylation of p44/p42
N-SH cells mitogen-activated protein
overexpressing wild- (MAP) kinase,
type human APP695 ↓acetylcholinesterase activity,
↑PKC and MAP kinase
Gastrodin Rhizome of Tg2576 mice 60 mg/kg ↓memory impairment, ↓Aβ [17]
Gastrodiaelata deposition, ↓glial activation,
↑anti-inflammatory, ↑anti-
amyloidogenic
Berberine Coptischinensis TgCRND8 mice; 25 mg/kg, ↓learning deficits, ↑spatial [12]
N2a mouse 100 mg/kg memory, ↓plaque load, ↓β-
neuroblastoma cells amyloid, ↓glycogen synthase
stably expressing kinase (GSK)3, ↓tau
human Swedish phosphorylation, ↓C-terminal
mutant APP695 fragments of APP,
(N2a-SwedAPP) ↓hyperphosphorylation of APP
and tau, ↑Akt/glycogen
synthase kinase 3 signaling
pathway
Morin many plants 3xTg-AD mice, - ↓GSK3β, ↓GSK3β-induced tau [14]
human phosphorylation, ↓Aβ-induced
neuroblastoma cells tau phosphorylation, ↓Aβ
cytotoxicity, ↓tau pathology
Reserpine Rauvolfia sp. AD model in - ↓paralysis, ↑longevity [4]
Caenorhabditise-
legans
Natural Therapeutics Against Alzheimer’s Disease 65

Phytochem- Plant source AD models Dose Mechanism of action Refe-


icals rences
Withana- Withaniasomnife beta-amyloid (25- - ↓fibril formation [19]
mides A and ra fruit 35) induced PC-12
C cells and rat
neuronal cells
Allicin Allium sp. Aβ(1-42) induced - ↓learning and memory [26]
mice impairment, ↑superoxide
dismutase (SOD),
↓malondialdehyde (MDA),
↓Aβ,↓p38MAPK
Puerarin Puerarialobata Aβ(1-42) induced - ↓cognitive impairment, [25]
rats ↓apoptosis,
↑Akt,↑phosphorylation of Bad
Puerarin Puerarialobata Aβ(25-35) induced - ↓Aβ-toxicity, ↓apoptosis, ↑P- [47]
PC12 cells Akt, ↑Bcl-2, ↑p-Bad, ↓Bax,
↓cytochrome c
Linarin Menthaarvensis Aβ(25-35) induced 0.1, 1.0 and ↑cell viability, [28]
and PC12 cells 10 μM ↓apoptosis,↓acetylcholinesteras
Buddlejadavidii e, ↑phosphorylation of Akt,
↑phosphorylation of glycogen
synthase kinase-3β (GSK-3β),
↑Bcl-2
Hesperidin Citrus fruits Aβ(25-35) induced - ↓apoptosis, ↓mitochondrial [44]
PC12 cells dysfunction, ↓ROS, ↓VDAC1
phosphorylation, ↓glycogen
synthase kinase-3b,
↑hexokinaseI, ↓cytochrome c
release, ↓caspase-9 and caspase-
3
z-ligustilide Radix Angelica Abeta(25-35) 40 mg/kg ↓Abeta, ↓amyloid precursor [22]
sinensis induced rats protein, ↓phosphorylated Tau
immunoreactivity, ↓TNF-alpha,
↓activated NF-kappaB
4',5- Artemisia Abeta-induced PC12 - ↓ROS, ↓Abeta toxicity, [15]
dihydroxy- asiatica cells ↓oxidative stress
3',6,7-
trimethoxy-
flavone
Acteoside Lamiales order Abeta(25-35) - ↑viability, ↓apoptotis, ↓ROS, [45]
induced SH-SY5Y ↓mitochondrial dysfunctions,
cells ↓Bax/Bcl-2 ratio, ↓cytochrome
c release, ↓cleavage of caspase-
3
Genistein Many plants Abeta(25-35) 100 nM, 40 ↑estrogen receptor-mediated [49]
induced cultured µM pathway, ↑antioxidation,
hippocampal ↓neuronal apoptosis
neurons
66 Abhijit Dey and Anuradha Mukherjee

Reserpine

Reserpine decreased paralysis and increased longevity in an AD model in


Caenorhabditiselegans without any change in the Abeta transcript and expression levels [4].

Withanamides A and C

Withanamides A and C isolated from Withaniasomnifera fruit protected beta-amyloid


(25-35) induced PC-12 cells and rat neuronal cells via inhibiting fibril formation via binding
to the active motif of beta-amyloid (25-35) [19].

Allicin

Allicin protected Aβ[1-42] induced mice via preventing learning and memory
impairment, elevating the level of superoxide dismutase (SOD) and reducing
malondialdehyde (MDA) level and Aβ and p38MAPK expressions [26].

Puerarin

Puerarin from Puerarialobata protected Aβ(1-42) induced rats via preventing cognitive
impairment and apoptosis by inducing Akt and phosphorylation of Bad [25]. The compound
also reduced Aβ-toxicity and apoptosis via upregulating P-Akt, Bcl-2 and p-Bad and
downregulating Bax and cytochrome c expressions in Aβ(25-35) induced PC12 cells [47].

Linarin

Linarin from Menthaarvensis and Buddlejadavidii at doses of 0.1, 1.0 and 10 μM


enhanced cell viability, decreased apoptosis and acetylcholinesterase activity via inducing
phosphorylation of Akt and phosphorylation of glycogen synthase kinase-3β (GSK-3β) and
Bcl-2 in Aβ(25-35) induced PC12 cells [28].

Hesperidin

Hesperidin protected Aβ25-35-induced PC12 cells via preventing apoptosis,


mitochondrial dysfunction, ROS accumulation via downregulation of VDAC1
phosphorylation, glycogen synthase kinase-3b, cytochrome c release, caspase-9 and caspase-3
and upregulation of hexokinase I [44].
Natural Therapeutics Against Alzheimer’s Disease 67

Z-ligustilide

Z-ligustilide at a dose of 40 mg/kg prevented accumulation of Abeta and amyloid


precursor protein, downregulated phosphorylated Tau immunoreactivity and reduced the
levels of TNF-alpha, and activated NF-kappaB in Abeta(25-35) induced rats [22].

4',5-dihydroxy-3',6,7-trimethoxyflavone

4',5-dihydroxy-3',6,7-trimethoxyflavone isolated from Artemisia asiatica and vitamin E


protected Abeta-induced PC12 cells via preventing ROS accumulation and oxidative stress
and reducing Abeta toxicity [15].

Acteoside

Acteoside protected Abeta(25-35)-induced SH-SY5Y cells via increasing viability,


reducing apoptotis, ROS level, mitochondrial dysfunctions, Bax/Bcl-2 ratio, cytochrome c
release and cleavage of caspase-3 [45].

Genistein

Genistein protected Abeta25-35-induced cultured hippocampal neurons via attenuating


neuronal apoptosis mediated via estrogen receptor-mediated pathway (at a dose of 100 nM)
and via inducing antioxidation (at a dose of 40 microM) [49].

4',5-dihydroxy-3',6,7-trimethoxyflavone

4-O-methylhonokiol
68 Abhijit Dey and Anuradha Mukherjee

Acteoside

Allicin

Berberine

Bisdemethoxycurcumin

Catalpol
Natural Therapeutics Against Alzheimer’s Disease 69

Cryptotanshinone

Curcumin

Dimethoxycurcumin

Epigallocatechin-3-gallate

Gastrodin
70 Abhijit Dey and Anuradha Mukherjee

Genistein

Hesperidin

Huperzine

Linarin
Natural Therapeutics Against Alzheimer’s Disease 71

l-theanine

Melatonin

Morin

Nicotine

Piperine
72 Abhijit Dey and Anuradha Mukherjee

Puerarin

Reserpine

Resveratrol

Salidroside
Natural Therapeutics Against Alzheimer’s Disease 73

Silibinin

Withanamide A

Xylocoside G

z-ligustilide
74 Abhijit Dey and Anuradha Mukherjee

CONCLUSION
AD is one of the deadliest and commonest diseases mostly affecting the elderly people.
Besides providing systematic relief, none of the present medications either prevent the onset
of the disease or cure the disease. However, present pharmaceutical, botanical, nutritional and
stimulatory therapies have been popularized as conventional, complementary, alternative or
adjunctive therapies because of their ability to reduce the symptoms of AD. Since time
immemorial, plant derived extracts, semi-purified fractions, isolated compounds and plant
based traditional formulations have been used as effective anti-AD therapeutics which has
been continued in a number of pre-clinical and clinical trials investigating the scientific
rationale of using them. Less side effects, wide acceptability and inexpensiveness have
popularized the use of herbal therapy in developing as well as developed countries. In
addition, synergistic effects among the herbal constituents in plant extracts and traditional
formulations were found to be effective against complex and heterogeneous diseases such as
AD with complex underlying mechanisms of pathogenesis modulating an array of
biochemical and molecular signaling pathways. Therefore, the herbal products with multiple
constituents with effective synergistic interactions may provide valuable therapeutic option
against multi-factorial diseases like AD affecting multiple and complex pathogenesis.

Conflicts of Interest

The authors declare no conflict of interest.

REFERENCES
[1] Ahmed F, Ghalib RM, Sasikala P, Ahmed KK. Cholinesterase inhibitors from
botanicals. Pharmacogn Rev 2013; 7(14): 121-30.
[2] Ahmed T, Enam SA, Gilani AH. Curcuminoids enhance memory in an amyloid-infused
rat model of Alzheimer's disease. Neuroscience 2010; 169(3): 1296-306.
[3] Ahmed T, Gilani AH. A comparative study of curcuminoids to measure their effect on
inflammatory and apoptotic gene expression in an Aβ plus ibotenic acid-infused rat
model of Alzheimer's disease. Brain Res 2011; 1400: 1-18.
[4] Arya U, Dwivedi H, Subramaniam JR. Reserpine ameliorates Abeta toxicity in the
Alzheimer's disease model in Caenorhabditis elegans. Exp Gerontol 2009; 44(6-7): 462-
6.
[5] Chi TY, Wang LH, Ji XF, Shen L, Zou LB. Protective effect of xanthoceraside against
β-amyloid-induced neurotoxicity in neuroblastoma SH-SY5Y cells. J Asian Nat Prod
Res 2013; 15(9): 1013-22.
[6] Chi TY, Wang LH, Qu C, Yang BZ, et al. Protective effects of xanthoceraside on
learning and memory impairment induced by Abeta(25-35) in mice. J Asian Nat Prod
Res 2009; 11(12): 1019-27.
[7] Choi IS, Lee YJ, Choi DY, et al. 4-O-methylhonokiol attenuated memory impairment
through modulation of oxidative damage of enzymes involving amyloid-β generation
Natural Therapeutics Against Alzheimer’s Disease 75

and accumulation in a mouse model of Alzheimer's disease. J Alzheimers Dis 2011;


27(1): 127-41.
[8] Chonpathompikunlert P, Wattanathorn J, Muchimapura S. Piperine, the main alkaloid of
Thai black pepper, protects against neurodegeneration and cognitive impairment in
animal model of cognitive deficit like condition of Alzheimer's disease. Food
ChemToxicol 2010; 48(3): 798-802.
[9] Davinelli S, Sapere N, Zella D, Bracale R, Intrieri M, Scapagnini G. Pleiotropic
protective effects of phytochemicals in Alzheimer's disease. Oxid Med Cell Longev
2012; 2012: 386527.
[10] Deng J, Shen C, Wang YJ, et al. Nicotine exacerbates tau phosphorylation and cognitive
impairment induced by amyloid-beta 25-35 in rats. Eur J Pharmacol 2010 10; 637(1-3):
83-8.
[11] Doraiswamy PM. Non-cholinergic strategies for treating and preventing Alzheimer's
disease. CNS Drugs. 2002; 16(12): 811-24.
[12] Durairajan SS, Liu LF, Lu JH, et al. Berberine ameliorates β-amyloid pathology, gliosis,
and cognitive impairment in an Alzheimer's disease transgenic mouse model. Neurobiol
Aging 2012; 33(12): 2903-19.
[13] Durairajan SS, Liu LF, Lu JH, et al. Stimulation of non-amyloidogenic processing of
amyloid-β protein precursor by cryptotanshinone involves activation and translocation
of ADAM10 and PKC-α. J Alzheimers Dis 2011; 25(2): 245-62.
[14] Gong EJ, Park HR, Kim ME, et al. Morin attenuates tau hyperphosphorylation by
inhibiting GSK3β. Neurobiol Dis 2011; 44(2): 223-30.
[15] Heo HJ, Cho HY, Hong B, et al. Protective effect of 4',5-dihydroxy-3',6,7-
trimethoxyflavone from Artemisia asiatica against Abeta-induced oxidative stress in
PC12 cells. Amyloid 2001; 8(3): 194-201.
[16] Howes MJ, Perry NS, Houghton PJ. Plants with traditional uses and activities, relevant
to the management of Alzheimer's disease and other cognitive disorders. Phytother Res.
2003; 17(1): 1-18.
[17] Hu Y, Li C, Shen W. Gastrodin alleviates memory deficits and reduces neuropathology
in a mouse model of Alzheimer's disease. Neuropathology 2014; 34(4): 370-7.
[18] Huang TC, Lu KT, Wo YY, Wu YJ, Yang YL. Resveratrol protects rats from Aβ-induced
neurotoxicity by the reduction of iNOS expression and lipid peroxidation. PLoS One
2011; 6(12): e29102.
[19] Jayaprakasam B, Padmanabhan K, Nair MG. Withanamides in Withania somnifera fruit
protect PC-12 cells from beta-amyloid responsible for Alzheimer's disease. Phytother
Res 2010; 24(6): 859-63.
[20] Jiang B, Du J, Liu JH, Bao YM, An LJ. Catalpol attenuates the neurotoxicity induced
by beta-amyloid(1-42) in cortical neuron-glia cultures. Brain Res 2008; 10; 1188: 139-
47.
[21] Kim TI, Lee YK, Park SG, et al. l-Theanine, an amino acid in green tea, attenuates beta-
amyloid-induced cognitive dysfunction and neurotoxicity: reduction in oxidative
damage and inactivation of ERK/p38 kinase and NF-kappaB pathways. Free RadicBiol
Med 2009; 47(11): 1601-10.
[22] Kuang X, Du JR, Chen YS, Wang J, Wang YN. Protective effect of Z-ligustilide against
amyloid beta-induced neurotoxicity is associated with decreased pro-inflammatory
markers in rat brains. Pharmacol Biochem Behav 2009; 92(4): 635-41.
76 Abhijit Dey and Anuradha Mukherjee

[23] Lee JW, Lee YK, Ban JO, et al. Green tea (-)-epigallocatechin-3-gallate inhibits beta-
amyloid-induced cognitive dysfunction through modification of secretase activity via
inhibition of ERK and NF-kappaB pathways in mice. J Nutr. 2009; 139(10): 1987-93.
[24] Lee YK, Choi IS, Ban JO, et al. 4-O-methylhonokiol attenuated β-amyloid-induced
memory impairment through reduction of oxidative damages via inactivation of p38
MAP kinase. J Nutr Biochem 2011; 22(5): 476-86.
[25] Li J, Wang G, Liu J, et al. Puerarin attenuates amyloid-beta-induced cognitive
impairment through suppression of apoptosis in rat hippocampus in vivo. Eur J
Pharmacol 2010; 649(1-3): 195-201.
[26] Li XH, Li CY, Xiang ZG, Zhong F, Chen ZY, Lu JM. Allicin can reduce neuronal death
and ameliorate the spatial memory impairment in Alzheimer's disease models.
Neurosciences (Riyadh) 2010; 15(4): 237-43.
[27] Liang JH, Du J, Xu LD, et al. Catalpol protects primary cultured cortical neurons
induced by Abeta(1-42) through a mitochondrial-dependent caspase pathway.
Neurochem Int 2009; 55(8): 741-6.
[28] Lou H, Fan P, Perez RG, Lou H. Neuroprotective effects of linarin through activation of
the PI3K/Akt pathway in amyloid-β-induced neuronal cell death. Bioorg Med Chem
2011; 19(13): 4021-7.
[29] Lu P, Mamiya T, Lu LL, et al. Silibinin attenuates amyloid beta(25-35) peptide-induced
memory impairments: implication of inducible nitric-oxide synthase and tumor necrosis
factor-alpha in mice. J Pharmacol Exp Ther 2009; 331(1): 319-26.
[30] Lu P, Mamiya T, Lu LL, et al. Silibinin prevents amyloid beta peptide-induced memory
impairment and oxidative stress in mice. Br J Pharmacol 2009; 157(7): 1270-7.
[31] Man SC, Durairajan SS, Kum WF, et al. Systematic review on the efficacy and safety of
herbal medicines for Alzheimer's disease. J Alzheimers Dis 2008; 14(2): 209-23.
[32] Mei Z, Situ B, Tan X, et al. Cryptotanshinione upregulates alpha-secretase by activation
PI3K pathway in cortical neurons. Brain Res 2010; 1348: 165-73.
[33] Mukherjee PK, Kumar V, Mal M, Houghton PJ. Acetylcholinesterase inhibitors from
plants. Phytomedicine 2007; 14(4): 289-300.
[34] Orhan IE. Current concepts on selected plant secondary metabolites with promising
inhibitory effects against enzymes linked to Alzheimer's disease. Curr Med Chem 2012;
19(14): 2252-61.
[35] Ott BR1, Owens NJ. Complementary and alternative medicines for Alzheimer's disease.
J Geriatr Psychiatry Neurol 1998 Winter; 11(4): 163-73.
[36] Peng Y, Jiang L, Lee DY, Schachter SC, Ma Z, Lemere CA. Effects of huperzine A on
amyloid precursor protein processing and beta-amyloid generation in human embryonic
kidney 293 APP Swedish mutant cells. J Neurosci Res 2006; 84(4): 903-11.
[37] Peng Y, Lee DY, Jiang L, Ma Z, Schachter SC, Lemere CA. Huperzine A regulates
amyloid precursor protein processing via protein kinase C and mitogen-activated
protein kinase pathways in neuroblastoma SK-N-SH cells over-expressing wild type
human amyloid precursor protein 695. Neuroscience 2007; 150(2): 386-95.
[38] Porquet D, Griñán-Ferré C, Ferrer I, et al. Neuroprotective role of trans-resveratrol in a
murine model of familial Alzheimer's disease. J Alzheimers Dis 2014; 42(4): 1209-20.
[39] Rao RV, Descamps O, John V, Bredesen DE. Ayurvedic medicinal plants for
Alzheimer's disease: a review. Alzheimers Res Ther 2012; 4(3): 22.
Natural Therapeutics Against Alzheimer’s Disease 77

[40] Shen Y, Zhang G, Liu L, Xu S. Suppressive effects of melatonin on amyloid-beta-


induced glial activation in rat hippocampus. Arch Med Res 2007; 38(3): 284-90.
[41] Shen YX, Wei W, Yang J, Liu C, Dong C, Xu SY. Improvement of melatonin to the
learning and memory impairment induced by amyloid beta-peptide 25 - 35 in elder rats.
Acta Pharmacol Sin. 2001; 22(9): 797-803.
[42] Tan Y, Zhang Q, Wong SG, Hua Q1. Anti-Alzheimer Therapeutic Drugs Targeting γ-
Secretase. Curr Top Med Chem 2016; 16(5): 549-57.
[43] Walker JM, Klakotskaia D, Ajit D, et al. Beneficial Effects of Dietary EGCG and
Voluntary Exercise on Behavior in an Alzheimer's Disease Mouse Model. J Alzheimers
Dis 2015; 44(2): 561-72.
[44] Wang DM, Li SQ, Zhu XY, Wang Y, Wu WL, Zhang XJ. Protective effects of
hesperidin against amyloid-β (Aβ) induced neurotoxicity through the voltage dependent
anion channel 1 (VDAC1)-mediated mitochondrial apoptotic pathway in PC12 cells.
Neurochem Res 2013; 38(5): 1034-44.
[45] Wang H, Xu Y, Yan J, et al. Acteoside protects human neuroblastoma SH-SY5Y cells
against beta-amyloid-induced cell injury. Brain Res 2009; 1283: 139-47.
[46] Wollen KA. Alzheimer's disease: the pros and cons of pharmaceutical, nutritional,
botanical, and stimulatory therapies, with a discussion of treatment strategies from the
perspective of patients and practitioners. Altern Med Rev. 2010 Sep;15(3):223-44.
[47] Xing G, Dong M, Li X, et al. Neuroprotective effects of puerarin against beta-amyloid-
induced neurotoxicity in PC12 cells via a PI3K-dependent signaling pathway. Brain
Res Bull 2011; 85(3-4):2 12-8.
[48] Yu Y, Zhou L, Sun M, et al. Xylocoside G reduces amyloid-β induced neurotoxicity by
inhibiting NF-κB signaling pathway in neuronal cells. J Alzheimers Dis 2012; 30(2):
263-75.
[49] Zeng H, Chen Q, Zhao B. Genistein ameliorates beta-amyloid peptide (25-35)-induced
hippocampal neuronal apoptosis. Free RadicBiol Med. 2004; 36(2): 180-8.
[50] Zhang L, Yu H, Zhao X, et al. Neuroprotective effects of salidroside against beta-
amyloid-induced oxidative stress in SH-SY5Y human neuroblastoma cells. Neurochem
Int. 2010; 57(5): 547-55.
In: Advances in Natural Products Discovery ISBN: 978-1-53610-088-4
Editors: Ana Rita Gomes, Teresa Rocha-Santos et al. © 2017 Nova Science Publishers, Inc.

Chapter 3

BIOACTIVE CONSTITUENTS FROM ARTOCAPUS

Rohaya Ahmad1,2, and Mohd Nazrul Hisham Daud1,3


1
Faculty of Applied Sciences, Universiti Teknologi MARA, Selangor, Malaysia
2
Atta-ur-Rahman Institute for Natural Product Discovery,
Universiti Teknologi MARA, Selangor, Malaysia
3
Food Safety and Forensic, Malaysian Agricultural Research
and Development Institute, Selangor, Malaysia

ABSTRACT
The genus Artocarpus belongs to the tribe Artocarpeae (family Moraceae) and comprises
approximately 50 species of trees and shrubs of Southeast Asian and Pacific origin. A.
heterophyllus and A. champeden are two species well known for their edible fruits and food
products derived from them. Rinds and other waste parts of the fruits have high value as a
nourishing feed for livestock. Traditionally, A. heterophyllus tree has been used in the
preparations of various Ayurvedic and Unani medicines and consumed to prevent excessive
formation of bile and to strengthen the body and increase virility, among others. Aqueous
extracts of mature leaves some plants of the genus such as A. heterophyllus are used by
traditional medical practitioners in Sri Lanka and India for the treatment of diabetes while its
roots are useful in treating various skin diseases and for asthma and diarrhea. Ashes produced
by burning bark of A. heterophyllus are believed to heal abscesses and ear problems. In
Traditional Chinese Medicine, some Artocarpus species are found to be of use in overcoming
the influence of alcohol. Some notable bioactivities of Artocarpus species include
antimalarial activity from aerial parts of A. champeden, antioxidant activities from heartwood
and cortex of A. altilis, antiplatelet activity from roots of A. commnunis and anti-cancer from
tree bark of A. lanceifolius. Phytochemical investigation on constituents of the plants have
yielded several new classes of flavonoids, stilbenes and heterocyclic compounds including
benzofurans, benzoxanthones and aurones, some of which possess interesting biological


Corresponding author. Faculty of Applied Sciences, Universiti Teknologi MARA 40450 Shah Alam, Selangor,
Malaysia and Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA,43600
Bandar Puncak Alam, Selangor, Malaysia G.B. E-mail: rohayaahmad@salam.uitm.edu.my.
80 Rohaya Ahmad and Mohd Nazrul Hisham Daud

properties. This chapter reviews the chemistry as well as biological and pharmacological
properties of the plants and its constituents and discusses emerging and potential innovative
uses of the plant.

Keywords: Artocarpus, constituents, chemistry, biological activity, pharmacology

INTRODUCTION
Artocarpus belongs to the tribe Artocarpeae (family Moraceae) and comprises
approximately 50 species of trees and shrubs of Southeast Asian and Pacific origin,.
Artocarpus is the third largest genus in the Moraceae family [1]. Seventeen common
representatives including A. heterophyllus, A. altilis, A. integer, A. anisophyllus, A. dadah, A.
elasticus, A. lakoocha, A. sarawakensis, A. kemando, A. lanceifolius, A. odoratissimus, A.
reticulates, A. rigidus, A. sericicarpu, A. nitidus, A. hirsutus and A. hypargyreus, with edible
fruits have been reported. Table 1 shows the distribution of Artocarpus species (along with
their common names) worldwide.

Table 1. Common representatives of Artocarpus species and their distribution

No Species Common/Local Name Geographical Distribution


A. altilis/
1 A.communis/ Breadfruit, sukun South East Asia and South Pacific
A.camansi
Tawak, Entawak, Bintawak, Kelidang, Borneo, Peninsula Malaysia and
2 A. anisophyllus
Bakil, Puan Sumatra Indonesia
3 A. dadah Dadak, Tampang and Selanking dadak Sunda shelf and Thailand
Mendi, Tekalong, Benda, Terap Togop,
4 A. elasticus Sunda shelf
Ahbat, Jerami, Ho, Aw, Ka aw
India, South East Asia (Malaysia,
5 A. heterophyllus Jackfruit, nangka
Thailand, Vietnam, Indonesia etc.)
6 A. hirsutus Anjili, hebhalsina and ran-phunnas South-west India
7 A. hypargyreus Kwaimuk Guandong and Hainan, China
A. integer/ Sunda Shelf, Sulawesia and Irian
8 Cempedak, temedak and nakan pudau,
A.champeden Jaya
South Borneo, South Sumatra and
9 A. kemando Pudu, selibut and kudu
South Peninsula Malaysia
Monkey Jack, Lakuch, Barhal, Oahu, East and North India, Burma,
10 A. lakoocha
Myauklok, Hat lom, Hat non, Thailand and Laos
Peninsula Malaysia, North Sumatra
11 A. lanceifolius Keledang, klidang and keliput
and East Borneo
Selanking, butong, empatah, sinojoh, ma Sunda, North Philippines, Bali and
12 A. nitidus
hat sampor, cay chay Timor-Leste
13 A. odoratissimus Marang, terap, madang Nort Borneo and South Philippines
14 A. reticulatus Utu, maumbi Sulawesi and Moluccas
pujan, perian, tampuni, pussar, pala
15 A. rigidus Sunda shelf and South Indochina
musoh
16 A. sarawakensis Pingan Sarawak, Malaysia
17 A. sericicarpus Pedalai, belalai, gumihan and terap Borneo, Philippines and Sulawesi,
Among the common representatives, A. heterophyllus Lam (Jackfruit) and A. altilis or
breadfruit have become the two most economically important plants cultivated throughout the
Bioactive Constituents from Artocapus 81

tropics and Pacific Islands mainly for their fruits [2, 3]. Besides its ripe fruit, the young fruits
and seeds of A.heterophyllus are often used as vegetables [4]. The plant is commonly referred
to as “poor man’s food’” for its availability, low-cost and abundance during the season and
has served the nutritional needs of rural communities to the people of Southeast Asia,
Indonesia, western part of Java and India [5].
India, the biggest producer for A. heterophyllus in the world, is considered to be the
motherland of jackfruit, followed by Bangladesh [2]. Although both countries produce a huge
amount of jackfruit, there is a lack of effort in commercializing its production due to lack of
government support. However, through the Export Promotion Bureau, the Bangladesh
government has agreed to supply jackfruits which will be processed in Malaysia for re-export
[6]. Malaysia being one of the fast growing countries driven by its agricultural sector, has an
export value from agricultural production was worth RM 106.21 billion in 2013 [7] A.
heterophyllus has been identified as one of the major fruits to be exported under agriculture.
Up to 2013, via the Economic Transformation programme, almost 4000 hectares of land has
been planted with the plant in the peninsular. The production of A.heterophyllus fruits in the
country in 2013 was reported to be 20,989 tones with a productivity of 9.7 (tonne/hectare)
generating a total income of about RM 8.4 million. These values are expected to increase
significantly by 2020 [8]. Table 2 shows the production status of the economically important
A. heterophyllus in countries which are major producers of the fruit.

Table 2. Major Asia-Pasific Country with Production of A. heterophyllus [6, 7, 9]

Country Year Area (‘000 hectare) Production (‘000 tonne) Productivity (tonne/hectare)
Bangladesh 2006 10.0 926.0 8.20
India 1992 102.0 1436.0 11.40
Indonesia 1987 50.0 340.0 9.0
Malaysia 2013 4.0 20.9 9.7
Nepal 2010 1.60 18.97 11.89
Sri Lanka 2011 50.0 - -
Thailand 1987 37.0 392.0 10

ISOLATION PROCEDURES
To date, an increasing awareness among consumers between diet and health has
exponentially increased scientific studies into biological effect of secondary metabolite
products in living systems [10]. Studies on secondary metabolite have been become
increasingly important with increasing evidence that plants provide a source of therapeutic
agents for defense or protection against various diseases [11]. Besides this, the innovative use
of plants as nutraceuticals, health foods or supplements to promote good health and growth
are continuously growing.
In the search for bioactive compounds from plants, the choice and availability of plants
for evaluation are important. Its traditional use, ethnopharmacology, chemotaxonomy or
ecological observations are amongst the important criteria for selection. Since traditional or
ethnomedicinal use is a major indicator of activity, the isolation of active compounds may
very well result in interestingresults, although the activity could be due to synergistic effects
82 Rohaya Ahmad and Mohd Nazrul Hisham Daud

of its constituents compounds [12, 13]. Based on their ethnomedical use or random
screening, plant extracts are usually biologically screened in pharmacologically relevant
assays or panel of assays [14]. Primary biological screening for promising extracts usually
involves simple and inexpensive but rapid bioassays in order to cope with the large number of
samples and sensitive enough although in small concentration [15]. Plant extracts are usually
dissolved in polar solvents such as methanol, ethanol or aqueous ethanol for the isolation of
polar constituents [5, 16-20] ‘Active’ extracts will then be selected for further work in which
the selected plants may either undergo bioassay-guided isolation of active constituents or
subjected to phytochemical investigation followed by structural elucidation and biological
testing of the isolated compounds.

BIOLOGICAL ACTIVITIES AND CONSTITUENTS OF ARTOCARPUS


The most recent review on the traditional uses, phytochemistry and pharmacology
of the genus Artocarpus was by Jagtap and Bapat, 2010 [5]. The authors gave an overview of
the phytochemistry of the fruits and the seeds of the genus with a special focus on the
on jacalin, the lectin isolated from the seeds. Up to 2010, a total of 18 biological activities
have been reported for the crude extracts and constituents of the genus. The structures
of 64 constituents including flavones, flavanone, flavan-3-ol, dihydrobenzoxanthone,
furanodihydro-bennzoxanthone, pyranodihydrobenzoxanthone as well as stilbenes and
arylbenzofurans, among others, along with its source and selected biological activity were
given. In a later study, Baliga et al., 2011 [21] reviewed the phytochemistry, nutritional and
pharmacological properties of the most economically important species A. heterophyllus Lam.
and its 22 constituents highlighting on recent research trends towards the plant’s economic
potential and innovative uses [20, 22].
In this review, the morphology of the genus will not be discussed on which readers can
refer to previous literature [5, 17]. However, the distribution of 17 common representatives
along with their common and local names is given (Table 1) followed by the economic
significance of A.heterophyllus, as indicated by the production of its fruits by major producers
(Table 2). The biological importance of the Artocarpus plants will then be re-evaluated by i)
looking at recent biological screening of the crude extracts of Artocarpus species (Table 3),
ii) systematically enlisting 113 phytochemical constituents of the genus based on a more
systematic classification of natural product such as flavonoids (including prenylated
flavonoids and related structures), stilbenoids and other main classes of compounds (Table 4)
and iii) presenting documented biological activities in a chronological manner and identifying
the constituents responsible for the their biological activities. The section will conclude with a
summary of the bioactivities of the common representatives of Artocarpus excluding the
much reviewed A.champeden and discuss their potential economic significance.

CRUDE EXTRACTS
Anti-cancer, antioxidant, anti-inflammatory, antidiabetic and tyrosinase-inhibitory
bioassays have been important tools to evaluate the biological potential of crude extracts of
Bioactive Constituents from Artocapus 83

Artocarpus [5]. Most of the biological studies reported have mostly focused on in-vitro assays
except for anti-inflammatory work for inhibition of carrageenan induced paw edema in rats.
Amongst the in-vitro assays conducted, anti-cancer using cancer cell-lines and antioxidant
assays based on radical scavenging ability have been dominantly used as screening tools.
Both assays were of the colorimetric type, involving the determination of colored compounds
in tetrazolium dye (MTT) for anti-cancer and 1,1-diphenyl-2-picrylhydrazyl (DPPH) or
ABTS radical scavenging assays for antioxidants, respectively. It is interesting to note that
anti-cancer and antioxidant assays continued to dominate biological work on Artocarpus
crude extracts which has led to the corresponding biological evaluation of their isolated
constituents. However, in recent years, there has been an increase in the number of anti-
inflammatory, antidiabetic and tyrosinase-inhibitory screening of the crude extracts.
In a recent study, the ethanolic leaf extract of A. camansi. showed selective cytotoxic
activities on MCF-7 breast cancer cell line with an IC50 value of 9.58 μg/ml [19]. In a
separate study, the heartwood extract of A.communis (a synonym for A. camansi) showed
anti-hepatoma activity against HepG2 and PLC/PRF/5 hepatocellular carcinoma cell lines
with IC50 value between 16-17 μg/ml [20]. Interestingly, the polar (diethy ether) extract of the
heartwood of A. altilis was reported to possess cytotoxic properties against breast cancer cells
T47D in a concentration- dependent manner with an IC50 value of 6.19μg/ml [23]. The
aqueous ethanolic stembark extract of the plant was found to possess good DPPH radical
scavenging activity and inhibited paw edemea with 61.29% inhibition [30] indicating polar
antioxidant and anti-inflammatory constituents. The methanolic leaf extract showed
tyrosinase-inhibitory and α-glucosidase inhibitory activities [25].
Evaluation of the in vitro antibacterial activities of jackfruit have been reported earlier [5,
21]. As shown in Table 3, only moderate activity was recorded for the EtOAC fraction of the
leaves. Antibacterial activity of A.heterophyllus seed extracts against wound isolates of Multi
Drug Resistant-Methicillin Resistant Staphylococcus aureus (MDR-MRSA) showed that the
seed extracts did not show good inhibitory activity although the plant is traditionally used for
antibacterial activity. This indicated that the active compounds could be mainly distributed in
aerial parts, roots and rhizomes, but not in the seeds [26]. Recent biological screening of the
pulp of jackfruit found only moderate cytotoxic activity against murine B-cell lymphoma
cancer cell line M12.C3.F6 with IC50 of 49.2 μg/ml. More significantly, DPPH radical
scavenging screening of the aqueous acetone shell powder extract of A.heterophyllus found
strong activities of 0.61 μg/ml indicating the presence of polar antioxidant constituents [18].
Furthermore, the antibacterial effects of seeds can be further explored for the development of
anti-bacterial products.
Table 3 summarizes the major biological activities including anti-cancer/cytotoxic,
antioxidant, anti-inflammatory, tyrosinase-inhibitory and anti-diabetic activities of the crude
extracts of common Artocarpus representatives including jackfruit (A.hetrophyllus),
breadfruit (A.communis/A.altilis/A.camansi) and cempedak (A.champeden/A.integer).
Table 3. Major biological activities of Artocarpus extracts

Biological Species Extract (part of plant) Activities Reference


activity
Highly selective cytotoxic activity against breast cancer cell line
(MCF-7): IC50= 9.58 μg/ml
Moderate cytotoxic activity against: human colon carcinoma [19]
A. camansi Ethanol (leaves)
HCT116, : human lung non-small cell carcinoma A549, and Chinese
hamster ovary AA8 cell lines with IC50 values ranging from 39.6-
48.9 μg/ml
Methanol (heartwood) and
fractions: AM: Crude Anti-hepatoma activity against HepG2 and PLC/PRF/5
Anticancer/
methanol; AH: hexane hepatocellular carcinoma cell lines; IC50=16.1 μg/ml, 17.2 μg/ml,
Cytotoxic A. communis [20]
AD: dichlromethane; respectively
AE: ethyl acetate; AB: n- AD>AM>AB>AE>AH
butanol
Cytotoxic effect on breast cancer cells T47D in a concentration-
A. altilis Diethylether (wood) [23]
dependent manner: IC50 = 6.19μg/ml.
A. heterophyllus Methanolic extract of seed Showed maximum cytotoxicity on HEp2 cells up to 1:4 dilution [24]
Cytotoxic activity against cancer cell line M12.C3.F6 (murine B-cell
A. heterophyllus Methanol (pulp) [27]
lymphoma) with IC50 = 49.2 μg/ml
80% acetone aqueous (shell
A. heterophyllus Strong antioxidant activity (DPPH); IC50 = 0.61 μg/ml [18]
powder)
Antioxidant
A. heterophyllus Aqueous (pulp) Strong hydroxyl radical scavenging activity [16]
Free radical scavenging activity increased from 28 to 69% after 24
A. heterophyllus Aqueous (fully ripened pulp) [5]
hour incubation
A. heterophyllus Aqueous (leaves) Radical scavenging activity: IC50 = 73.5 μg/ml [17]
A. heterophyllus Ethanolic (dried mature fruits) DPPH radical scavenging IC50= 410 μg/ml [28]
A.gomezianus MeOH (stem bark) 90% DPPH reduction at 100 μg/ml [29]
A. altilis 70% ethanol (stem bark) Radical scavenging activity; IC50= 19.03 [30]
Dose-dependent decrease in foot edema (61.29% inhibition) at a dose
A. altilis 70% ethanol (stem bark) of 300 mg/kg comparable to diclofenac and dexamethasone (69.56 [30]
and 68.74%, respectively)
Anti-
Dose-dependent anti-inflammatory activity (49% inhibition) against
inflammatory A. heterophyllus Methanol (bark) [31]
carrageenan induced paw edema in rats at dose 400mg/kg
Inhibition of the development of collagen-induced arthritis (CIA):
A. tonkinensis Ethyl acetate (leaves) [32]
minimum significant dose=50 mg/ml.
Biological Species Extract (part of plant) Activities Reference
activity
Tyrosinase-
A.altilis Methanol (leaves) Tyrosinase-inhibitory activity with IC50 values < 100 μM [25]
inhibitory
Inhibit haemoglobin glycation via hydroxyl radical scavenging
A.heterophyllus Aqueous (pulp) [16]
Anti-diabetic activity
A.altilis Methanol (leaves) Potent α-Glucosidase inhibitory activity, IC50 = 4.9-5.4 μM [25]
Butanol fractions of root bark and fruit inhibit Bacillus cereus,
Methanol (all parts) and their
A. heterophyllus Bacillus megaterium, Lactobacillus casei, Micrococcus luteus and [33]
organic fractions
Streptococcus faecalis
A. heterophyllus Methanolic (seeds) Extracts and fractions not active against MDR-MRSA [26]
Anti-bacterial
Moderate antibacterial activities against some foodborne pathogens
including E. coli, Listeria monocytogenes, Salmonella typhimurium,
A. heterophyllus Aqueous (leaves) [17]
Salmonella enterica, Bacillus cereus, Enterococcus faecalis, and
Staphylococcus aureus
86 Rohaya Ahmad and Mohd Nazrul Hisham Daud

PHYTOCHEMICAL CONSTITUENTS OF ARTOCARPUS


The plants of the genus contain secondary metabolites which may be grouped into main
classes of compounds. Figure 1 shows the chemical structures of 113 Artocarpus constituents
arranged according to its class and related structures: β-carotenes, flavonoids, chalcones and
benzoxanthones, stilbenes, other phenolics and triterpenes.

β-carotene (1)

β-carotene (9Z) (2).

β-carotene (13Z) (3).

β-carotene (15Z) (4).


Bioactive Constituents from Artocapus 87

Artocarpin/Artocarpine (5).

Norartocarpetin (6).

6-prenylapigenin (7).

Artocarpanone (8).
88 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Albanin A (9).

Catechin (10).

Artoheterophyllin (11).

Heteroflavanone A (12).
Bioactive Constituents from Artocapus 89

Cudraflavone B/Mulberrochromene (13).

Cudraflavone C/Mulberrin (14).

Brosimone I (15).
90 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Kuwanon C (16).

Cycloartocarpin (17).

Steppogenin (18).
Bioactive Constituents from Artocapus 91

Arcommunol A (19).

Cyclocommunol (20).

Cyclocommunin (21).

Dihydroisocycloartomunin (22).
92 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Isolespeol (23).

5′-geranyl-2′,4′,4-trihydroxychalcone (24).

3,4,2′,4′-tetrahydroxy-3′-geranyldihydrochalcone (25).

Xanthoangelol (26).

2-[6-hydroxy-3,7-dimethylocta-2(E),7-dienyl]-2’,3,4,4’-tetra Hydroxydihydrochalcone (27).


Bioactive Constituents from Artocapus 93

1-(2,4-dihydroxyphenyl)-3-9-(hydroxy-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-6H-dibenzo[b,d]
pyran-5-yl)-1-propanone,1-(2,4-dihydroxyphenyl)-3-[3,4-dihydro-3,8-dihydroxy-2-methyl-2-(4-
methyl-3-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone (28).

Artoindonesianin A (29).

Artoindonesianin A-2 (30).


94 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Artoindonesianin A-3 (31).

Artoindonesianin B (32).

Artocommunol CC (33).

Chaplasin (34).
Bioactive Constituents from Artocapus 95

Artoindonesianin E (35).

Artoindonesianin F (36).

Artoindonesianin G (37).

Artoindonesianin H (38).
96 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Artoindonesianin I (39).

Artoindonesianin L (40).

Cycloartobiloxanthone (41).

Artoindonesianin P (42).
Bioactive Constituents from Artocapus 97

Artoindonesianin Q (43).

Artoindonesianin R (44).

Artoindonesianin S (45).

Artoindonesianin T (46).
98 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Artoindonesianin U (47).

Artoindonesianin V (48).

Artelasticin (49).

Artobiloxanthone (50).
Bioactive Constituents from Artocapus 99

Artonol A (51).

Artonol B (52).

Artonin A (53).

Artonin B (54).
100 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Artonin E (55).

Artonin F (56).

Artonin M (57).

Artonin O (58).
Bioactive Constituents from Artocapus 101

2-hydroxynaringenin 4’-O-β-D-glucopyranoside (59).

Styracifolin A (60).

Styracifolin B (61).

Artoheterophyllin B (62).
102 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Heterophyllin (63).

Cycloheterophyllin (64).

Dihydroartomunoxanthone (65).

Cyclomulberrin (66).
Bioactive Constituents from Artocapus 103

Artocarpetin (67).

Artocarpetin A (68).

Hydroxyartoflavone A (69).
104 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Artoflavone A (70).

Artogomezianone (71).

Artelastoheterol (72).
Bioactive Constituents from Artocapus 105

Artochamin B (73).

Dihydroisocycloartomunin (74).

Cycloartelastoxanthone (75).
106 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Bonannione A (76).

5,7,4’-trihydroxy-6-geranylflavanone (77).

Artocarpesin (78).

Morachalcone A (79).

Isocycloartobiloxanthone (80).
Bioactive Constituents from Artocapus 107

Artogomezianol (81).

Andalasin A (82).

Resveratrol (83).

3-(γ, γ -dimethylallyl)resveratrol (84).


108 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Trans-4-(3-methyl-E-but-1-enyl)-3,5,2’,4’-tetrahydroxystilbene (85).

Artocarbene (86).

Isochlorophorin (87).

Artocarpene (88).
Bioactive Constituents from Artocapus 109

Chlorophorin (89).

Artolacuchin (90).

Artoxanthol (91).

Alboctalol (92).
110 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Atilisin H (93).

Atilisin I (94).

Artilisin J (95).
Bioactive Constituents from Artocapus 111

Artostyracin A (96).

Artostyracin B (97).

Artostyracin C (98).
112 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Artoheterophyllin A (99).

Artoindonesianin X (100).

Artoindonesianin Y (101).
Bioactive Constituents from Artocapus 113

Artoindonesianin O (102).

Artotonkin (103).

β-sitosterol (104).

Ursolic acid (105).


114 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Cycloartenone (106).

Cycloartenol (107).

24-methylenecycloartanone (108).
Bioactive Constituents from Artocapus 115

24-methylenecycloartanol (109).

Cycloeucalenol (110).

Glutinol (111).
116 Rohaya Ahmad and Mohd Nazrul Hisham Daud

Betulinic acid (112).

Heterophylol (113).

Figure 1. Constituents of Artocarpus a) β-carotenes (1-4) b) flavonoids, chalcones and benzoxanthones


(5-80) c) stilbenes (81-90) d) other phenolic compounds (91-95), benzofurans (96-103) and e)
triterpenes and sterols (104-113).

BIOLOGICAL ACTIVITIES OF CONSTITUENTS


Biological screening of the crude extracts of Artocarpus and its constituents has mostly
employed in vitro assays for the evaluation of anticancer, antioxidant and anti-inflammatory
properties. The phytochemicals are usually dissolved in a polar solvent such as ethanol or
methanol. The biological activities observed have been generally attributed to the flavonoids
as major constituents, especially of the prenylated type [34, 35] but more recently prenylated
chalcones such as geranyl chalcones from A. altilis have been found to be responsible for the
bioactivities. However, other polyphenolic constituents such as resveratrol (83), trans-4-(3-
methyl-E-but-1-enyl)-3,5,2’,4’-tetrahydroxystilbene (85), cycloartelastoxanthone (75), and
artoindonesianin H (38) are believed to be responsible for anticancer, antioxidant and
antimalarial properties [36, 37]. From 1991-2010, the most explored biological
propertyreported for Artocarpus was anti-cancer followed by antioxidant and anti-
inflammatory properties and the trend has not changed much since then. However, the
tyrosinase-inhibitory property and inhibition of melanin production of constituents of
A.heterophyllus, A. altilis, A. xanthocarpus and A. gomezianus have yielded promising
Bioactive Constituents from Artocapus 117

results. Interestingly, the antimalarial properties of A. integer was reported to be due to its
stilbene derivative.

ANTICANCER ACTIVITIES
Cancer is one of the major targets in many research involving groups of constituents from
Artocarpus. Most of the assays employed for anti-cancer screening purpose involved in-vitro
methods using various human cancer cell lines including colon cancer cell, breast cancer,
melanoma cancer cells, tumor cell, lung cancer, hepatitis (Hep2 and HepG2) and human
fibroblast cell.
Flavonoids, an ubiquitous class of phenolic compounds in natural products, populates in
many parts of Artocarpus species and possess a wide range of anti-cancer applications. Early
cytotoxic studies of the bark and heartwood of Artocarpus campeden and A. lanceifolius from
1999-2004 was by Hakim’s group on a series of prenylated flavonoids named artoindonesians
aginst P-388 murine leukemia cell line on a series of prenylated flavonoids named
Artoindonesians [1]. Syah et al (2001 & 2004) [36, 38] evaluated a group of isoprenylated
flavonoids artoindonesianins G (37), H (38), I (39), U (47), V (48) and artelasticin (49) for
their cytotoxic effects against P-388 tumor cell. From the results, artoindonesianins V (48)
exhibited the lowest IC50 value followed by artoindonesianin G (37), H (38), I (39) and U (47)
and artelasticin (49) 3.0 μg/ml indicating artoindonesianin V (48) as a potential anti-cancer
agent. In a related study, Suhartati et al (2001) [39] reported that five compounds,
artoindonesianin L (40), artonins M (57) and E (55), cycloartobiloxanthone (41) and artonin
O (58) isolated from root bark of A. rotunda to possessed significant cytotoxic effect against
the same cell line with low IC50 values between 0.06-7.9μg/ml. The same group (Hakim et al.,
2002) later reported that the prenylated flavones, artoindonesianin P (42), artobiloxanthone
(50), cycloartobiloxanthone (41) and artonol B (52) significantly exhibited cyctotoxic effect
against murine P388 leukemia cells with IC50 values between 1.7-100 μg/ml. Interestingly,
artonol A (51) isolated from root bark of A. elasticus showed ED50 value of 1.1 μg/ml [40]
against A549 human cancer cell line.
Later studies (2006-2015) on the heartwood of A. heterophyllus, focusing on the
heartwood of the plant found strong activity of its flavonoid constituents against selected
human cancer cell lines with artocarpin (5) exhibiting the strongest activity. Arung et al.
(2010) [34] conducted the search for anti-cancer constituents by evaluating the cytotoxic
effects of nine flavonoids including artocarpin (5), cudraflavone C (14), 6-prenylapigenin (7),
kuwanon C (16), norartocarpetin (6), albanin A (9), cudraflavone B (13), brosimone I (15)
and artocarpanone (8), against B16 melanoma cancer cells. The results indicated that the
isoprenoid moiety in flavonoids could enhance the cytotoxic effect on the melanoma cancer
cells. Bonannione A (-) (76) showed significant cytotoxicity effect with IC50 value 29.9 μM
against PANC1 cancer cell line together with ten other constituents that as reported by Arai
et al., (2015) [41]. All of the compounds were isolated from the ethyl acetate fraction of A.
communis methanolic leaf extract via bioassay-guided isolation. A strong cytotoxic
compound Arcommunol A (19) isolated from chloroform fraction of the fruits of the same
species showed an IC50 value 2.05 μM against SK-Hep-1 cell , as reported by Hsu et al (2011)
[42]. The cytotoxicity effect of 2-hydroxynaringenin 4’-O-β-D-glucopyranoside (59) against
118 Rohaya Ahmad and Mohd Nazrul Hisham Daud

human lung cancer cell line has been evaluated by Ti et al (2011) [43]. This compound
isolated from stems of A. nitidus showed moderate cytotoxic effect with IC50 at 47.3 μg/ml.
From the root bark of A. fretessi, the cytotoxic effect in brine shrimp (A. salina) of
artoindonesianin X (100) was found to be stronger than for artoindonesianin Y (101) with
LC50 values of 78.7μg/ml and 294 μg/ml, respectively [44]. However, the compounds were
not subjected to any of the previously tested cell lines.
Compounds of the geranyl dihydrochalcones type (24)-(28) isolated from ethyl acetate
soluble fraction of the methanol extract from A. altilis leaves was found to possess cytotoxic
effect against selected human cancer cells as reported by Wang et al (2007) [45]. From three
compounds tested, [20] showed the most potent activity against SPC-A-1, SW-480 and
SMMC-7721 human cancer cells. From all the documented findings, it can be concluded that
further evaluation using in-vivo model need to be carried out to justify its effectiveness for
prevention of human cancer.
The leaves of the complex plant A. communis involving three species of breadfruit: A.
altilis, A. mariannensis, and A. camansi have yielded chalcones but there was no report of this
type of compound in the leaves of other species. Similarly, studies on the fruits of Artocarpus
are rather limited but the isolation of β-carotenes (1) from A. heterophyllus fruits has been
reported. β-carotenes belong to the carotenoids, a class of constituents that react as preventer
agent from several chronic diseases such as cancer [46] and hence have been reported to be
important anti-cancer constituent of the plant [47].

ANTIOXIDANT ACTIVITIES
Antioxidants play an important role in preventing a variety of diseases and arresting
disease progression because they help the body protect itself against damage caused by
reactive oxygen species and degenerative diseases. The preventive action of antioxidants can
be divided into 3 levels. The first line of defense is the suppression of the formation of free
radicals or Reactive Oxygen Species (ROS) such as superoxide anions, hydrogen peroxide,
hydroxy radicals, hypochlorous acid and peroxynitrites. The second line of defense involves
radical scavenging and the final line of defense involves the antioxidative enzymes such as
phospholipases, protease, DNA repair enzymes and transferases. Thus, antioxidants are
known to act at different levels in the oxidative sequence involving lipid molecules. They
may act by decreasing oxygen concentration, intercepting singlet oxygen, preventing first-
chain initiation by scavenging radicals, binding metal ion catalysts, decomposing primary
products to non-radical compounds and chain-breaking to prevent continued hydrogen
abstraction from substrates.
Most of the antioxidant studies on Artocarpus evaluated how plant antioxidants are
involved in the second line of defense which involves radical scavenging. Early work on
antioxidant screening was reported by Ko et al., (1998) [48] who evaluated the scavenging
ability of prenylflavones isolated from A. heterophyllus. The authors found that,
cycloheterophyllin (64), artonins A (53) and B (54) serve as powerful antioxidants agents
with IC50 values of 2.1, 2.4 and 1.8 μM respectivelyHowever, none of these prenylflavones
were subsequently tested for anti-cancer activities preventing any correlations to be made
between these properties. As mentioned in the earlier section 2.31, there seems to be a shift in
the interest towards their cytotoxic properties of the prenylflavones..
Bioactive Constituents from Artocapus 119

Investigation on heterocyclic compounds from other Artocarpus species such as


artobiloxanthone (50) and cycloartobiloxanthone (41) as antioxidants agent was reported by
Lin et al (2009) [37]. These compounds were reported to possess good radical scavenging
ability with IC50 30.0 and 13.4μM. Both compounds could be found in the bark of A. nobilis
and A. gomezianus. Cycloartobiloxanthone (41) isolated from roots of A. communis and A.
elasticus along with artoflavone A (70), cycloartelastoxanthone (75), and artelastoheterol (72)
also showed promising antioxidant potential via scavenging activity [1, 49, 50]. None of the
compounds were subjected to anti-cancer activity but artonol A (51) isolated from A. elasticus
had earlier showed strong cytotoxic effect against A549 human cancer cell line. In a related
study, Lan et al (2013) [51] evaluated the antioxidant activity of 14 flavonoids isolated from
heartwood and cortex of A. altilis using DPPH, ABTS+ and O2– assays and concluded that the
flavonoids hydroxyartoflavone A (69), isocycloartobiloxanthone (80) and artoflavone A (70)
isolated from heartwood and cortex of A. altilis showed significant effects. This suggests that
they are good candidates as antioxidant agents but further investigation is required in order to
determine their mechanisms of action. It is also worth mentioning that none of these
compounds were subjected to any cytotoxic evaluation. Hence, further investigations are
required in order to determine their mechanisms of action.
The role of stilbene constituents of Artocarpus in their antioxidant properties have been
evaluated by some researchers. From their study, Lorenz et al (2003) [52] concluded that the
stilbene group of compound known as resveratrol (83) and 3-(γ, γ -dimethylallyl) resveratrol
(84) found in A. dadah possess antioxidants agents with IC50 values of 28.9μM and 38.5 μM,
respectively, as measured by the DPPH assay. Other evaluation on antioxidant ability of
stilbene group was conducted much later by Ti et al (2011) [43] with the isolation of
artocarpene (88) from the stems of A. nitidus. However, the compound only showed moderate
antioxidant activity with IC50 of 116.15 μg/ml.

ANTI-INFLAMMATORY ACTIVITIES
Compared to anti-cancer and antioxidant activities, the anti-inflammatory activity of
Artocarpus constituents have not been reported much. Only flavonoids isolated from A.
heterophyllus and A. communis have been subjected to anti-inflammatory assays yielding
promising results. From A. dadah resveratrol (83) and its derivative showed COX inhibition.
These findings warrant further anti-inflammatory work on the compounds.
Artocarpanone (8) from A. heterophyllus significantly inhibited the release of lysozyme
from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP). Along with
cycloheterophyllin (64), artonin B (54), and artocarpanone (8) significantly inhibited
superoxide anion formation in fMLP-stimulated rat neutrophils. Artocarpanone (8) exhibited
significant inhibitory effect on NO production and iNOS protein expression in RAW 264.7
cells [53]. The potent inhibitory effect of artocarpanone on NO production in
lipopolysaccharide (LPS)-activated macrophages, was suggested to be via the suppression of
iNOS protein expression. Wei et al., (2005) [53] evaluated an anti-inflammatory effect of
fifteen flavonoids isolated from A. heterophyllus and A. communis by determining their
inhibitory effect on chemical mediators released from mast cells, neutrophils and
macrophages. The authors found that dihydroisocycloartomunin (74) significantly inhibited
120 Rohaya Ahmad and Mohd Nazrul Hisham Daud

the release of beta-glucuronidase and histamine from rat peritoneal mast cells stimulated with
p-methoxy-N-methylphenethylamine.

TYROSINASE-INHIBITORY ACTIVITY AND INHIBITION


OF MELANIN PRODUCTION

Besides the targeted anticancer and antioxidant activity which are dominant for the
genus, other biological evaluations include tyrosine inhibitory activity and suppression of
melanin production. Earlier work by Arung and co-researchers (2005) [54] on Artocarpus
woods which led to the identification of a stilbene, chlorophorin from A. heterophyllus as a
strong tyrosinase inhibitor. Recently, chlorophorin along with the more common stilbenes
resveratrol and resveratrol (83) were found to be potent tyrosinase inhibitors along with
arthoxanthol (91), alboctalol (92), steppogenin (18), and norartocarpetin (6) isolated from
A.xanthorcarpus [49]. Interestingly, a stilbene-type compound known as artocarbene (86)
isolated from heartwood of A. incisus showed tyrosinase inhibitory activities [55].
The role of flavones in inhibiting melanin production was also studied. Cudraflavone A
and artonin M was found to inhibit melanin production by strongly suppressing tyrosinase
activity while artocarpin (5) reduced the melanin content without inhibiting tyrosinase
activity [51]. In a separate study, it was found that morachalcone A (79) isolated from A.
heterophyllus methanolic wood extract was 3000 times more active than the positive control
kojic acid along with compounds (6), (8), (78), and (89). These activities indicate the strong
potential of the plant as a skin-whitening agent and the economic significance of the plant in
the cosmetic industry [56].

OTHER BIOLOGICAL ACTIVITIES


Following the screening of the methanolic leaf extract of A. altilis, the isolated geranyl
aurones atilisin H (93), I (94) and J (95) displayed potent α-glucosidase inhibitory effects
with IC50 values ranging from 4.9 to 5.4 μM against acarbose [25] supporting the traditional
use of the plant for diabetes. Besides this species, no other species have been tested for their
α-glucosidase inhibitory activities. From the less explored A. styracifolius, unusual flavonoids
isolated from stem bark A. styracifolius [57] named styracifolins A (60) and B (61),
artoheterophyllin A (99) and B (62), artonins A (53), B (54) and F (56), and heterophyllin
(63) exhibited strong antiplasmodial activities with IC50 values from 1.1μM to 13.7 μM.
Furthermore, styracifolin A (60) and B (61) also showed significant anti-trypanosomal
activities. Besides non-prenylated and prenylated flavonoids, an antimalarial activity-guided
isolation from aerial parts of A. integer conducted by [58] yielded a prenylated stilbene as an
active major constituent. This compound showed potential antimalarial activity with an EC50
of 1.7 μg/ml against Plasmodium falciparum. The biological activity of a stilbene-type
compound artocarbene (88) has been reported by Jennifer et al (2012) [59] in a separate
study. In a recent study, Qiao et al (2015) [60] suggested that artoindonesianin O (102)
isolated from the tree bark A. gomezianus [1] is suitable and a possible candidate for the
development of general food type neuroprotection on AD by protecting against brain damage
Bioactive Constituents from Artocapus 121

and memory impairment. A study on the inhibitory effects on respiratory burst of rat
neutrophils for artostyracin A (96), B (97), and C (98) isolated from the roots of A.
styracifolius demonstrate significant results as reported by Ren et al (2014) [61] with IC50
values 1.42, 11.56 and 1.91μM, respectively. Table 4 highlights the bioactivity of constituents
isolated from Artocarpus categorized according to i) type of bioactivity tested, ii) the type of
species with the part of the plant they were isolated from, given in parentheses.

8
CONCLUSION
The anti-cancer and cytotoxic properties of A. champeden as well as the pharmacological
properties of A.heterophyllus has been reviewed before [5, 21]. To understand the
economic significance of the genus, in this section, we shall only highlight the recent and
significant findings on the most explored Artocarpus species including A. heterophyllus,
A.communis/A.altilis/A.camansi, A. xanthocarpus and A. styracifolius.
Jackfruit (A. heterophyllus): Artocarpin (5) and cudraflavone C (14) from the heartwood
of A.heterophyllus (jackfruit) possessed cytotoxic activities against various human cancer cell
lines with potent activity against various cancer cell lines. Morachalcone A (79) isolated
from the wood of the plant was 3000 times more active as a tyrosinase inhibitor than kojic
acid with IC50 of 44.6 μM. Norartocarpetin (6) and artocarpesin (78) from the twigs displayed
strong tyrosinase inhibitory properties. β-carotenes from the fruits showed cytotoxic activities
against SW 620 human colon cancer cell and antioxidant radical scavenging activities as
measured by α-TEAC, FRAP and chemiluminescence assays, with respect to α-tocopherol.
Breadfruit (A. communis/A. altilis/A. camansi): Recent studies on A.camansi found the
leaves extract to be highly selective cytotoxic activity against breast cancer cell line MCF-7
but moderately cytotoxic against: human colon carcinoma HCT116, human lung non-small
cell carcinoma A549 and Chinese hamster ovary AA8 cell lines. Geranyl chalcones (24-28)
from the leaves of from the leaves of A.communis and the related A.altilis, respectively,
played a significant role in cytotoxic activity against various human cancer cells. Bonannione
A (76), also from the leaves of the former was moderately cytotoxic against pancreatic cancer
cell line supporting early reports on the bioactivities of the leaves of the plant. Atilisin H, I
and J (93-95) from the leaves showed potent α-glucosidase inhibitory effects. From the
heartwood of A.communis extracts and fractions, good anti-hepatoma activity against HepG2
and PLC/PRF/5 hepatocellular carcinoma cell lines was recorded with the DCM extract
showing highest activity compared to the other organic fractions and the diethyl ether wood
extract was cytotoxic against breast cancer cells T47D cells. From the heartwood and cortex
of the related A. altilis, isocycloartobiloxanthone (80) showed the strong ABTS+ activity
while 5,7,4’-trihydroxy-6-geranylflavanone (77) showed a great inhibitory effect on gene
expression of RAGE and down-regulated both TNF-R and IL-1β secretion and gene
expression. Artoflavone A (70) from the roots of the plant showed good scavenging activity.
Other activities include antiplatelet effects of prenylated flavonoids in A.altilis rootbarks and
strong cytotoxic activity against SK-Hep-1 cell of Arcommunol A (19) from the fruits of A.
styracifolius.
Table 4. Biological activities of constituents of Artocarpus

Biological
Species (part) Constituents Results Notes Reference
activity
IC50 value of (5), (11), (17), and (14) against
Artocarpin/Artocarpine (5), human cancer cell lines MCF-7 11.3, 26.4, 20.7
A. heterophyllus Artoheterophyllin (11) and 10.8 μM, SMMC-7721 liver cancer cell 25.3,
Active [34, 48, 70]
(wood) Cycloartocarpin (17) 15.9, 23.7 and 12.1 μM and against lung cancer
Cudraflavone C/Mulberrin (14) cell NCI-H460 11.0, 15.8, 20.7 and 5.2 μM,
respectively
A. heterophyllus Cytotoxic against T47D breast cancer cells:IC50
Artocarpin (5) Active [35]
(wood) value (2) 12.6 μM
Artocarpin (5)
Cudraflavone C (14) Cytotoxic against B16 melanoma cancer cells
Kuwanon C (16), Norartocarpin IC50 values:
Active
(6), Cudraflavone B/ (5) 0.3μM, (14) 9.2 μM, μM, (16) 14.2 μM, (6)
A. heterophyllus
Mulberrochromene (13), 7.8 μM, (13) 12.5 μM, (15) 10.7 μM [35, 61]
(wood)
Brosimone I (15)
(7) 32.5, (9) 84.7 and (8) 122.2 μM
6-Prenylapigenin (7) Moderately
Isoprenoid moiety substitutions in flavonoids
Albanin A (9) Artocarpanone (8) Active
Anticancer enhance cytotoxic activities
A. heterophyllus (1) showed most effective dosage at 5.8 μM
β-Carotene (1) Active [46]
(fruits) against SW 620 human colon cancer cell
A. communis IC50 value for (76) against PANC1 cancer cell
Bonannione A (76) Active [41]
(leaves) line 29.9 μM
A. communis (19) showed IC50 value 2.05 μM against SK-Hep-
Arcommunol A (19) Active [42]
(fruits) 1 cell
Isolespeol (23), Cytotoxic test conducted against human cancer
5′-geranyl-2′,4′,4- cells (SW 872, HT-29, COLO 205, Hep3B,
A. communis trihydroxychalcone (24), PLC5, Huh7, and HepG2 cells). All constituents
Active [22]
(leaves) 3,4,2′,4′-tetrahydroxy-3′- showed most strong inhibition for human cancer
geranyldihydrochalcone (25) cell SW 872 with IC50 value (23) 3.8, (24) 4.1,
and xanthoangelol (26) (25) 4.3 and (26) 4.4 μM
(27) showed strongest cytotoxic effect with IC50
A. altilis values against SPC-A-1, SW-480 and SMMC-
Geranyl chalcones (27) and (28) Active [44]
(leaves) 7721 human cancer cells at 28.1, 34.6 and 49.9
μM, respectively.
Biological
Species (part) Constituents Results Notes Reference
activity
Artoindonesianin A-2 (30 )
Artoindonesianin A-3 (31)
A. champeden (30 ), (31), (63) and (14) showed strong cytotoxic
Artonin B (54) Active [62]
(heartwood) properties against murine leukemia P-3888 cells
Heterophyllin (63)
Cudraflavone C (14)
IC50 value with (47)] 2.0 μg/ml and (48) 0.5
A. champeden Artoindonesianin U (47)
Active μg/ml cytotoxic affect against P-388 tumor cell. [38]
(heartwood) Artoindonesianin V (48)
A potential anti-cancer agent.
Both (29) and (32) exhibit cytotoxic activity (IC50
A. champeden Artoindonesianin A (29)
Active 21.0 and 3.9 μg/mL, respectively) against murine [63]
(root bark) Artoindonesianin B (32)
leukemia (P-388) cells
Artoindonesianin G (37)
IC50 values: (37) 0.7 μg/ml, (38) and (39)
A. lanceifolius Artoindonesianin H (38)
Active 1.8ug/ml; (49) 3.0 μg/ml cytotoxic affect against [36]
(heartwood) Artoindonesianin I (39)
P-388 cells
Artelasticin (49)
Artoindonesianin P (42)]
Exhibited IC50 values at 5.9 (42), 1.7 (50), 4.6
A. lanceifolius Artobiloxanthone (50)
Moderate (80) and 100 (52) μg/ml against murine P388 [1]
(bark) Cycloartobiloxanthone (41)
leukemia cells
Artonol B (52)
Artoindonesianin L (40) Artonin
IC50 0.6 (40), 7.9 (55), 0.06 (41) and 0.9 (58)
A. rotunda M (57) and E (55)
Active μg/ml cytotoxic affect against P-388 leukimia [39]
(root bark) Cycloartobiloxanthone (41)
cells
Artonin O (58)
A. nitidus 2-Hydroxynaringenin 4’-O-β-D- (59) showed cytotoxic effect with IC50 value 47.3
Moderate [43]
(stem) glucopyranoside (59) μg/ml against human lung cancer cell line
A. elasticus (51) showed IC50 value of 1.1 μg/ml against
Artonol A (51) Active [40]
(root bark) A549 human cancer cell line
A. fretessi Artoindonesianin X (100) Brine shrimp (A. salina) test: LC50 value: (100)
Moderate [64]
(root bark) Artoindonesianin Y (101) 78.7μg/ml (101) 294 μg/ml
Styracifolin A (60)
Styracifolin B (61) Cytotoxic test conducted against human
A. styracifolius Artoheterophyllin (11) fibroblast cell line (MRC-5) with IC50 values:
Active [56]
(bark) Artoheterophyllin B (62) (60) 31.7, (61) 4.7, (11) 13.5, (62) 94.3, (63)
Artonin A (53) 97.0, (54) 8.2, (56) 72.2 and (63) 3.8 μM
Artonin B (54)
Table 4. (Continued)

Biological
Species (part) Constituents Results Notes Reference
activity
Artonin F (56) Heterophyllin
(63)
(all-E)-β-carotene, (9Z)-β- Radical scavenging activities of β-carotene
A. heterophyllus
carotene, (13Z)-β-carotene, Active isomers with respect to α-tocopherol 0.8 (1), 0.8 [46, 72]
(fruits)
(15Z)-β-carotene (1-4) (2), 0.8 (3) and 0.5 (4) mol α-TE/mol β-carotene
Cycloheterophyllin (64) Artonin
A (53)
(64), (53) and (54) scavenged DPPH and peroxyl
A. heterophyllus Artonin B (54)
Active and hydroxyl radicals against lipid peroxidation. [47]
(root bark) Artocarpin (5)
No activity for (5), (67) and (68)
Artocarpetin (67) Artocarpetin A
(68)
Artolacuchin (90)
A. lakoocha Isochlorophorin (87) Scavenged DPPH with IC50 values : (90) 37.5,
Active [49]
(seed) Chlorophorin (89) Artotonkin (87) 34.3, (89) 38.6, (103) 12.5 μM respectively
(103)
Antioxidant
A. gomezianus Artonin E (55) artobiloxanthone Scavenged DPPH with IC50 values: (55) 13.5 (50)
Active [29]
(stem bark) (50) catechin (10) 6.3 and (10) 13.6 μM respectively
Hydroxyartoflavone A (69) DPPH radical-scavenging with IC50 values : (69)
A. altilis
Isocycloartobiloxanthone (80) 20.9 (80) 33.9 and (70) 53.5 μM; ABTS+ radical
(heartwood and Active [51]
Artoflavone A (70) scavenging: IC50 (80) 7.2 μM; O2– assay: IC50
cortex)
Artogomezianone (71) (71) 39.7 μM.
A. communis (70) showed significant scavenging activity with
Artoflavone A (70) Active [37]
(root) IC50 values 24.2 μM.
Cycloartelastoxanthone (75)
A. elasticus (75), (72) and (41) showed scavenging activity
Artelastoheterol (72) Active [37]
(root) with IC50 values 18.7, 42.2 and 26.8 μM
Cycloartobiloxanthone (41)
A. nitidus (5) showed scavenging activity with IC50 of
Artocarpin (5) Moderate [43]
(stem) 116.15 μg/ml
(22) inhibited the release of β-glucuronidase and
Anti- A. communis histamine from rat peritoneal mast cells
Dihydroisocycloartomunin (22) Active [53]
inflammatory (root bark) stimulated with P-methoxy-N-methylphenethyl
amine at 13.2 μM
Biological
Species (part) Constituents Results Notes Reference
activity
Low levels of (77) (≤2.5 μM) showed a great
5,7,4’-trihydroxy-
A. communis inhibitory effect on gene expression of RAGE [65]
6-geranylflavanone (77) Active
(fruit) and down-regulated both TNF-R and IL-1β
secretion and gene expression
(78) show inhibitory effects on the production of
A. heterophyllus proinflammatory mediators in lipopolysaccharide
Artocarpesin (78) Active [73]
(pulp) (LPS)-activated RAW 264.7 murine macrophage
cells with 25 μM
Resveratrol (83)
A. dadah (83) and (84) showed IC50 for COX-1, 1.1 and
3-(γ, γ -dimethylallyl) Active [66]
(bark) 0.61 μM, COX-2, 1.3 and 9.5 μM, respectively
resveratrol (84)
A.heterophyllus (89) showed 97% inhibition at 130μM on the
Chlorophorin (89) Moderate [53]
(wood) oxidation of l-DOPA by tyrosinase
(79) has IC50, 3000 times more active as a
A. heterophyllus
Morachalcone A (79) Most active tyrosinase inhibitor than (0.013 μM) than kojic [55]
(wood)
acid (44.6 μM)
A. heterophyllus (8) inhibit mushroom tyrosinase activity with
Artocarpanone (8) Active [61]
Tyrosinase (sapwood) IC50 values of 80.8 μM
inhibitory A. heterophyllus Norartocarpetin (6) Artocarpesin (6) and (78) showed tyrosinase inhibitory activity
Active [67]
(twigs) (78) with IC50 values 0.9 and 1.7 μM
Artoxanthol (91) (91), (92), (18), (6), (83) and (89) potently
Alboctalol (92) Steppogenin inhibited mushroom tyrosinase activity with IC50
A. xanthocarpus
(18) Norartocarpetin (6) Active values 5.7, 6.4, 1.9, 0.9, 4.9, 1.0 and 2.5 μM [48]
(root)
Resveratrol (83) respectively that were all far stronger than the
Chlorophorin (89) positive control kojic acid (IC50, 63.7 μM)
A. gomezianus Artogomezianol (81) Andalasin Moderately (81) and (82) showed tyrosinase inhibitory
[68]
(root) A (82) Active activity with IC50 values of 68 and 39 μM
Inhibitory
effect on A. styracifolius The IC50 for values of (96), (97) and (98) were
Artostyracin A, B and C (96-98) Active [60]
respiratory (root) 1.42, 11.56 and 1.91 μM, respectively
burst
(93-95) showed potent α-glucosidase inhibitory
α-Glucosidase A. altilis
Atilisin H, I and J (93-95) Active effect with IC50 values ranging from 4.9 to 5.4 [25]
inhibitory (leaves)
μM
Table 4. (Continued)

Biological
Species (part) Constituents Results Notes Reference
activity
(65), (73) and (33) demonstrated antiplatelet
Dihydroartomunoxanthone (65) effect mainly owing to an inhibitory effect on
A. communis
Antiplatelet Artochamin B (73) Active thromboxane formation. Percent aggregations in [69]
(root bark)
Artocommunol CC (33) human PRP range from 46.8 to 65.6% compared
to aspirin as control (29.5%)
Trans-4-(3-methyl-E-but-1-
A. integer (85) showed antimalarial activity with an EC50
Antimalarial enyl)-3,5,2’,4’- Active [58]
(aerial parts) 1.7 μg/ml against Plasmodium falciparum.
tetrahydroxystilbene (85)
Bioactive Constituents from Artocapus 127

A. styracifolius: The flavonoids styracifolin B (61), artoheterophyllin A (99), artonin B


(54), and heterophyllin (63) from the bark of the plant showed strong cytotoxic effects against
human fibroblast cell line (MRC-5). From the roots of the plant, artostyracin A , B, and C
(96-98) was found to have an inhibitory effect on respiratory burst.
A. xanthocarpus: The flavonoids artoxanthol (91), alboctalol (92), steppogenin (18),
norartocarpetin (6) and the stilbenoids resveratrol (83), and chlorophorin (89) from the roots
of A. xanthocarpus potently inhibited mushroom tyrosinase activity and were all far stronger
than the positive control kojic acid.
There is enough evidence for the potential innovative uses of A.heterophyllus (jackfruit),
A.altilis/A.communis/A.camansi (breadfruit), A. styracifolius, A. xanthocarpus. Other
Artocarpus species including A. lakoocha, A. gomezianus, A. elasticus and A. nitidus and its
constituents displayed moderate to strong radical scavenging activities. Based on the reported
activities and abundance, jackfruit and breadfruit and cempedak may be amongst the potential
plants to be explored further for the development of economically useful functional
foods/herbal products as antioxidants or to treat inflammatory diseases or bacterial infections.
While A. champeden constituents are rather well-explored for its anti-cancer and cytotoxic
properties, other cytototoxic constituents of from jackfruit, breadfruit or from A. styracifolius
should be subjected to in-vivo studies and clinical trials for drug development. Similarly,
strong tyrosinase-inhibitory properties of the flavonoids and stilbenoids of roots of A.
xanthocarpus as well as flavonoids and morachalcone A (79) of A.heterophyllus should be
further explored for their potential development of the extracts as skin whitening agents for
the cosmetic industry.

Conflicts of Interest

The authors declare no conflict of interest.

REFERENCES
[1] Hakim EH, Aimi N, Kitajima M, Takayama H. Artoindonesianin P. A new prenylated
flavone with cytotoxic activity from Artocarpus lanceifolius. Fitoterapia 2002; 73(7):
668-73.
[2] Taylor M. Strengthening of plant genetic resources for food and agriculture:
Conservation and utilization in the Pacific. Asia-Pacific Association of Agricultural
Research Institutions, Bangkok, Thailand: APAARI, June 2011.
[3] Zerega NJ, Supardi MN, Motley TJ. Phylogeny and recircumscription of Artocarpeae
(Moraceae) with a focus on Artocarpus. Syst Bot 2010; 35(4): 766-82.
[4] Jagtap UB, Waghmare SR, Lokhande VH, Suprasanna P, Bapat VA. Preparation and
evaluation of antioxidant capacity of Jackfruit (Artocarpus heterophyllus Lam.) wine
and its protective role against radiation induced DNA damage. Ind Crops Prod 2011;
34(3): 1595-601.
[5] Jagtap U, Bapat V. Artocarpus: A review of its traditional uses, phytochemistry and
pharmacology. J Ethnopharmacol 2010; 129(2): 142-66.
128 Rohaya Ahmad and Mohd Nazrul Hisham Daud

[6] Sidhu AS. Jackfruit improvement in the Asia-Pacific Region–A status report. Asia-
Pacific Association of Agricultural Research Institutions, Bangkok, Thailand: APAARI,
June 2012.
[7] Arshad FM, Noh KM, Saari MY. Policies and strategies for the development of small
and medium scale food processing enterprises in Malaysia, in Policy Measures for.
micro, small and medium food processing enterprises (MSMFEs) in the Asian Region,
FAO RAP Publication 2014/27; pp. 151-75.
[8] Ali R, Dardak RA. Competitiveness of Malaysian fresh agricultural and agro-based
products in global markets: A case study in the United Arab Emirates. Econ Technol
Manage Rev 2014; 9b: 93-101.
[9] Hossain A, Haq N. Jackfruit: Artocarpus heterophyllus. In: Williams JT, Smith RW,
Dunsiger Z, Eds. Tropical fruit trees. Southampton UK: Southampton Centre for
Underutilised Crops, University of Southampton 2006; pp. 127-139.
[10] Tiwari U, Cummins E. Factors influencing levels of phytochemicals in selected fruit
and vegetables during pre-and post-harvest food processing operations. Food Res Int
2013; 50(2): 497-506.
[11] Valli M, Pivatto M, Danuello A, et al. Tropical biodiversity: has it been a potential
source of secondary metabolites useful for medicinal chemistry? Quím Nova 2012;
35(11): 2278-87.
[12] Lahlou M. The success of natural products in drug discovery. Pharmacol Pharm 2013;
4(3A): 17-31.
[13] Patwardhan B, Vaidya AD, Chorghade M. Ayurveda and natural products drug
discovery. Curr Sci India 2004; 86(6): 789-99.
[14] Balunas MJ, Kinghorn AD. Drug discovery from medicinal plants. Life Sci 2005; 78(5):
431-41.
[15] Hostettmann K. Strategy for the biological and chemical evaluation of plant extracts.
Pure Appl Chem 1998; 70(11): 1-9.
[16] Biworo A, Tanjung E, Iskandar K, Suhartono E. Antidiabetic and antioxidant activity of
Jackfruit (Artocarpus heterophyllus) Extract. J Med Bioeng 2015; 4(4): 318-23.
[17] Loizzo M, Tundis R, Chandrika U, Abeysekera A, Menichini F, Frega N. Antioxidant
and antibacterial activities on foodborne pathogens of Artocarpus heterophyllus
Lam.(Moraceae) leaves extracts. J Food Sci 2010; 75(5): M291-M5.
[18] Sharma A, Gupta P, Verma A. Preliminary nutritional and biological potential of
Artocarpus heterophyllus L. shell powder. J Food Sci Technol 2015; 52(3): 1339-49.
[19] Tantengco OAG, Jacinto SD. Cytotoxic activity of crude extracts and fractions from
Premna odorata (Blanco), Artocarpus camansi (Blanco) and Gliricidia sepium (Jacq.)
against selected human cancer cell lines. Asian Pac J Trop Biomed 2015; 5(12): 1037–
1041.
[20] Tzeng C-W, Tzeng W-S, Lin L-T, et al. Artocarpus communis induces autophagic
instead of apoptotic cell death in human hepatocellular carcinoma cells. Am J Chin Med
2015; 43(03): 559-79.
[21] Baliga MS, Shivashankara AR, Haniadka R, Dsouza J, Bhat HP. Phytochemistry,
nutritional and pharmacological properties of Artocarpus heterophyllus Lam (jackfruit):
A review. Food Res Int 2011; 44(7): 1800-11.
Bioactive Constituents from Artocapus 129

[22] Fang S-C, Hsu C-L, Yu Y-S, Yen G-C. Cytotoxic effects of new geranyl chalcone
derivatives isolated from the leaves of Artocarpus communis in SW 872 human
liposarcoma cells. J Agric Food Chem 2008; 56(19): 8859-68.
[23] Arung ET, Wicaksono BD, Handoko YA, Kusuma IW, Yulia D, Sandra F. Anti-cancer
properties of diethyl ether extract of wood from sukun (Artocarpus altilis) in human
breast cancer (T47D) cells. Tropical J Pharm Res 2009; 8(4): 317-24.
[24] Amponsah IK, Annan K, Kofluor G, Sarkodie JA, Umerie IJ, Osei-wusu S. Anti-
inflammatory and antioxidant properties of the ethanolic stem bark extract of
Artocarpus altilis (Parkinson) Fosberg (Moraceae). Pharm Lett 2014; 6(3): 211-7.
[25] Mai NTT, Hai NX, Phu DH, Trong PNH, Nhan NT. Three new geranyl aurones from
the leaves of Artocarpus altilis. Phytochem Lett 2012; 5(3): 647-50.
[26] Karthy E, Ranjitha P, Mohankumar A. Antimicrobial potential of plant seed extracts
against multidrug resistant methicillin resistant Staphylococcus aureus (MDR-MRSA).
Int J Biol 2009; 1(1): 34.
[27] Ruiz-Montañez G, Burgos-Hernández A, Calderón-Santoyo M, et al. Screening
antimutagenic and antiproliferative properties of extracts isolated from Jackfruit pulp
(Artocarpus heterophyllus Lam). Food Chem 2015; 175: 409-16.
[28] Titov S. Antioxidant activities of some local Bangladeshi fruits (Artocarpus
heterophyllus, Annona squamosa, Terminalia bellirica, Syzygium samarangense,
Averrhoa carambola and Olea europa). Chin J Biotechnol 2007; 23(2): 257-61.
[29] Sritularak B, Tantituvanont A, Chanvorachote P, et al. Flavonoids with free radical
scavenging activity and nitric oxide inhibitory effect from the stem bark of Artocarpus
gomezianus. J Med Plants Res 2010; 4(5): 387-92.
[30] Amponsah IK, Annan K, Koffuor GA, et al. Anti-inflammatory and antioxidant
properties of the ethanolic stem bark extract of Artocarpus altilis (Parkinson) Fosberg
(Moraceae). Pharm Lett 2014; 6(3): 211-7.
[31] Lakheda S, Devalia R, Jain UK, Gupta N, Raghuwansi AS, Patidar N. Anti-
inflammatory activity of Artocarpus heterophyllus bark. Der Pharmacia Sinica 2011;
2(2): 127-30.
[32] Ngoc D, Catrina A, Lundberg K, et al. Inhibition by Artocarpus tonkinensis of the
development of collagen‐induced arthritis in rats. Scand J Immunol 2005; 61(3): 234-
41.
[33] Khan M, Omoloso A, Kihara M. Antibacterial activity of Artocarpus heterophyllus.
Fitoterapia 2003; 74(5): 501-5.
[34] Arung ET, Yoshikawa K, Shimizu K, Kondo R. Isoprenoid-substituted flavonoids from
wood of Artocarpus heterophyllus on B16 melanoma cells: cytotoxicity and structural
criteria. Fitoterapia 2010; 81(2): 120-3.
[35] Arung ET, Wicaksono BD, Handoko YA, et al. Cytotoxic effect of artocarpin on T47D
cells. J Nat Med 2010; 64(4): 423-9.
[36] Syah YM, Achmad SA, Ghisalberti EL, Hakim EH, Makmur L, Mujahidin D.
Artoindonesianins GI, three new isoprenylated flavones from Artocarpus lanceifolius.
Fitoterapia 2001; 72(7): 765-73.
[37] Lin K-W, Liu C-H, Tu H-Y, Ko H-H, Wei B-L. Antioxidant prenylflavonoids from
Artocarpus communis and Artocarpus elasticus. Food Chem 2009; 115(2): 558-62.
130 Rohaya Ahmad and Mohd Nazrul Hisham Daud

[38] Syah YM, Achmad SA, Ghisalberti EL, Hakim EH, Mujahidin D. Two new cytotoxic
isoprenylated flavones, artoindonesianins U and V, from the heartwood of Artocarpus
champeden. Fitoterapia 2004; 75(2): 134-40.
[39] Suhartati T, Achmad SA, Aimi N, et al. Artoindonesianin L, a new prenylated flavone
with cytotoxic activity from Artocarpus rotunda. Fitoterapia 2001; 72(8): 912-8.
[40] Ko H-H, Lu Y-H, Yang S-Z, Won S-J, Lin C-N. Cytotoxic prenylflavonoids from
Artocarpus elasticus. J Nat Prod 2005; 68(11): 1692-5.
[41] Arai MA, Uchida K, Sadhu SK, et al. Hedgehog inhibitors from Artocarpus communis
and Hyptis suaveolens. Bioorg Med Chem 2015; 23(15): 4150-4.
[42] Hsu C-L, Shyu M-H, Lin J-A, Yen G-C, Fang S-C. Cytotoxic effects of geranyl
flavonoid derivatives from the fruit of Artocarpus communis in SK-Hep-1 human
hepatocellular carcinoma cells. Food Chem 2011; 127(1): 127-34.
[43] Ti H, Wu P, Lin L, Wei X. Stilbenes and flavonoids from Artocarpus nitidus subsp.
lingnanensis. Fitoterapia 2011; 82(4): 662-5.
[44] Wang Y, Xu K, Lin L, Pan Y, Zheng X. Geranyl flavonoids from the leaves of
Artocarpus altilis. Phytochemistry 2007; 68(9): 1300-6.
[45] Krinsky NI, Landrum JT, Bone RA. Biologic mechanisms of the protective role of
lutein and zeaxanthin in the eye. Annu Rev Nutr 2003; 23(1):171-201.
[46] De Faria A, De Rosso V, Mercadante A. Carotenoid composition of jackfruit
(Artocarpus heterophyllus) determined by HPLC-PDA-MS/MS. Plant Foods Hum Nutr
2009; 64(2): 108-15.
[47] Ko FN, Cheng ZJ, Lin CN, Teng CM. Scavenger and antioxidant properties of
prenylflavones isolated from Artocarpus heterophyllus. Free Radic Biol Med 1998;
25(2):160-8.
[48] Jin Y-J, Lin C-C, Lu T-M, et al. Chemical constituents derived from Artocarpus
xanthocarpus as inhibitors of melanin biosynthesis. Phytochemistry 2015; 117: 424-35.
[49] Maneechai S, De-Eknamkul W, Umehara K, Noguchi H, Likhitwitayawuid K.
Flavonoid and stilbenoid production in callus cultures of Artocarpus lakoocha.
Phytochemistry 2012; 81: 42-9.
[50] Lan W-C, Tzeng C-W, Lin C-C, Yen F-L, Ko H-H. Prenylated flavonoids from
Artocarpus altilis: antioxidant activities and inhibitory effects on melanin production.
Phytochemistry 2013; 89: 78-88.
[51] Lorenz P, Roychowdhury S, Engelmann M, Wolf G, Horn TF. Oxyresveratrol and
resveratrol are potent antioxidants and free radical scavengers: effect on nitrosative and
oxidative stress derived from microglial cells. Nitric Oxide 2003; 9(2): 64-76.
[52] Wei B-L, Weng J-R, Chiu P-H, Hung C-F, Wang J-P, Lin C-N. Antiinflammatory
flavonoids from Artocarpus heterophyllus and Artocarpus communis. J Agric Food
Chem 2005; 53(10): 3867-71.
[53] Arung ET, Kusuma IW, Iskandar YM, Yasutake S, Shimizu K, Kondo R. Screening of
Indonesian plants for tyrosinase inhibitory activity. J Wood Sci 2005; 51(5): 520-5.
[54] Shimizu K, Kondo R, Sakai K. A stilbene derivative from Artocarpus incisus.
Phytochemistry 1997; 45(6): 1297-8.
[55] Nguyen NT, Nguyen MHK, Nguyen HX, Bui NKN, Nguyen MTT. Tyrosinase
Inhibitors from the wood of Artocarpus heterophyllus. J Nat Prod 2012; 75(11): 1951-
5.
Bioactive Constituents from Artocapus 131

[56] Bourjot M, Apel C, Martin M-T, et al. Antiplasmodial, antitrypanosomal, and cytotoxic
activities of prenylated flavonoids isolated from the stem bark of Artocarpus
styracifolius. Planta Med 2010; 76: 1600-04.
[57] Boonlaksiri C, Oonanant W, Kongsaeree P, Kittakoop P, Tanticharoen M,
Thebtaranonth Y. An antimalarial stilbene from Artocarpus integer. Phytochemistry
2000; 54(4): 415-7.
[58] Jennifer C, Stephie C, Abhishri S, Shalini B. A review on skin whitening property of
plant extracts. Int J Bio Sci 2012; 3(4): (B) 332-47.
[59] Qiao A, Wang Y, Zhang W, He X. Neuroprotection of brain-targeted bioactive dietary
artoindonesianin O (AIO) from mulberry on rat neurons as a novel intervention for
Alzheimer’s Disease. J Agric Food Chem 2015; 63(14): 3687-93.
[60] Ren G, Xiang HY, Hu ZC, et al. Inhibitory effects of phenolic compounds from
Artocarpus styracifolius on respiratory burst of rat neutrophils. Pharm Biol 2014; 52(8):
944-50.
[61] Arung ET, Shimizu K, Kondo R. Inhibitory effect of artocarpanone from Artocarpus
heterophyllus on melanin biosynthesis. Biol Pharm Bull 2006; 29(9): 1966-9.
[62] Syah YM, Juliawaty LD, Achmad SA, Hakim EH, Ghisalberti EL. Cytotoxic
prenylated flavones from Artocarpus champeden. J Nat Med 2006; 60(4): 308-12.
[63] Hakim EH, Fahriyati A, Kau MS, et al. Artoindonesianins A and B, two new prenylated
flavones from the root of Artocarpus champeden. J Nat Prod 1999; 62(4): 613-5.
[64] Soekamto NH, Achmad SA, Ghisalberti EL, Hakim EH, Syah YM. Artoindonesianins
X and Y, two isoprenylated 2-arylbenzofurans, from Artocarpus fretessi (Moraceae).
Phytochemistry 2003; 64(4): 831-4.
[65] Lin J-A, Fang S-C, Wu C-H, Huang S-M, Yen G-C. Anti-inflammatory effect of the 5,
7, 4′-trihydroxy-6-geranylflavanone isolated from the fruit of Artocarpus communis in
S100B-induced human monocytes. J Agric Food Chem 2010; 59(1): 105-11.
[66] Su B-N, Cuendet M, Hawthorne ME, et al. Constituents of the bark and twigs of
Artocarpus dadah with cyclooxygenase inhibitory activity. J Nat Prod 2002; 65(2):
163-9.
[67] Zheng Z-P, Chen S, Wang S, et al. Chemical components and tyrosinase inhibitors from
the twigs of Artocarpus heterophyllus. J Agric Food Chem 2009; 57(15): 6649-55.
[68] Likhitwitayawuid K, Sritularak B. A new dimeric stilbene with tyrosinase inhibitiory
activity from Artocarpus gomezianus. J Nat Prod 2001; 64(11): 1457-9.
[69] Weng J-R, Chan S-C, Lu Y-H, Lin H-C, Ko H-H, Lin C-N. Antiplatelet
prenylflavonoids from Artocarpus communis. Phytochemistry 2006; 67(8): 824-9.
[70] Soekamto NH, Nafie, LN, Mandey, WM, Garson, M. Norartocarpetin flavone
derivatives from leaves of Artocarpus fretessi. Indo J Chem 2009; 9(2): 328-31.
[71] Lin C-N, Shieh W-L. Pyranoflavonoids from Artocarpus communis. Phytochemistry
1992, 31 (8), 2922-4.
[72] Mueller L, Boehm V. Antioxidant Activity of β-Carotene Compounds in Different in
Vitro Assays. Molecules 2011; 16: 1055-69
[73] Fang SC, Hsu CL, Yen GC. Anti-inflammatory Effects of Phenolic Compounds
Isolated from the Fruits of Artocarpus heterophyllus. J Agric Food Chem 2008; 56,
4463–8.
In: Advances in Natural Products Discovery ISBN: 978-1-53610-088-4
Editors: Ana Rita Gomes, Teresa Rocha-Santos et al. © 2017 Nova Science Publishers, Inc.

Chapter 4

MAIZE (ZEA MAYS L.) –


AN ETHNOPHARMACOLOGICAL REVIEW

Priscilla Maria Menel Lemos, Beatriz Veleirinho,


Aline Pereira, Simone Kobe de Oliveira,
Rosendo Augusto Yunes, Shirley Kuhnen
and Marcelo Maraschin
Plant Morphogenesis and Biochemistry Laboratory –
Core of Natural Products, Federal University of Santa Catarina – CCA,
Florianopolis, SC, Brazil

ABSTRACT
Archaeological records show that maize (Zea mays L.) was cultivated in Central
America as far back as 6,250 years before the present. Nowadays, maize is the second
most important staple food worldwide, but since a long time ago its grains, flowers
(tassels), and leaves have also been used to prepare traditional medicines. This review
covers ethnopharmacological information about maize, considering initially its centre of
origin in Mexico, then its dispersion over America thousands of years ago and throughout
the world in more recent times. The majority of the information obtained refers to the
utilisation of maize stigmas mainly as a diuretic, but also as an anti-inflammatory and for
treatment of gastrointestinal disorders, gynaecological problems, and urogenital cancers.
Beneficial effects on human health could be associated with the presence of a significant
content of antioxidants, like (poly)phenolic and carotenoid compounds in maize stigmas
and eventually grains. It is assumed that these biomasses could be an interesting source of
such compounds and thus, could be profitably used to make dedicated by-products with
nutraceutical properties, or to obtain raw extracts or isolated compounds for the food
industry, as antioxidants and colours, for example, as well as for health care and the
cosmetics industry. In fact, traditional knowledge might be used with improved results in
association with modern scientific studies to generate, for instance, a new kind of
knowledge about maize and its uses, addressing interesting questions to some of the


Corresponding author. Plant Morphogenesis and Biochemistry Laboratory – Core of Natural Products, Federal
University of Santa Catarina, Florianopolis, SC, Brazil. E-mail: m.maraschin@ufsc.br.
134 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

contemporary human challenges regarding the treatment of some pathophysiologies.


Besides, it is also important to recognise the stringent association between traditional
medicines prepared from Z. mays and the usage of Creole or landrace varieties of that
species in several countries, mostly in the southern hemisphere. Ethical use of the
traditional knowledge associated with the ethnopharmacological data should recognise
the local people’s rights, promoting the use and development of autochthonous resources
and populations. Such an issue is thought to be relevant for both ethnopharmacological
purposes and for food security because of the urgent need of conservation of those
genetic resources.

Keywords: Zea mays, maize landraces, traditional knowledge, urinary disorders

1. INTRODUCTION
Plants have served as food and provided relief to physical suffering for human beings
even before crossing the limits of the Garden of Eden. In reality, humans always found in
nature all the resources to supply their basic needs such as shelter, clothing, food, and
treatment for illnesses [1, 2]. The folk medicine understanding regarding useful plants and
their properties was acquired by means of trial and error during hundreds or thousands of
years and built the foundation of allopathic modern medicine [3-5]. People probably
empirically learned how to distinguish useful plants with beneficial effects from those that
were non-active or toxic, and which plant processing or combination shall be used to obtain
optimal results, such as Brazilian Indians who distinguish between poisonous plants
important for hunting (‘curare’) and those for treating health problems.
The first record on the utilisation of plants for the purpose of illness treatment occurred in
Mesopotamia at about 4,600 A.D. and refers to the use of Cedrus spp. (cedar), Cupressus
sempervirens (cypress), Glycyrrhiza glabra (liquorice), Commiphora spp. (myrrh), and
Papaver somniferum (poppy) oils, indicated for the treatment of various ailments ranging
from coughs and colds to parasitic infections and inflammatory processes. Egyptians reported
for vitiligo treatment the use of Ammi majus (bishop’s weed) and ancient Greek people
organised a precise herbal text containing plant-based drug descriptions [5]. During the
Middle Ages, the ancient knowledge was preserved through monastic scriptoria and, in some
cases, it was dynamically altered, reflecting the accumulation of experience in plant remedies
during this period. Fortunately, those herbal medicine documents remain preserved, at least in
part, until today [4]. In fact, throughout human history the preservation of this kind of
wisdom has made possible the genesis, growth, and maintenance of an important part of
natural medicine knowledge as it is widely acknowledged nowadays [4-7]. Nevertheless, as a
function of several factors, the transmission of the traditional empirical knowledge over
generations sometimes does not occur in an adequate manner. Thus, preservation of this kind
of information could not always be assured [4, 8, 9].
Throughout centuries, traditional medicine evolved depending on the local flora, culture,
and religion and has served as a lead for many important pharmaceutical drugs, such as
vincristine, vinblastine, atropine, digoxin, morphine, quinine, salicylic acid, and artemisinin.
In the 80s, an inventory identified 120 chemical compounds from about 95 plant species that
were used in Western medicine and 72% of them were clinically indicated for similar
purposes in the related ethnobotanical record [5, 10]. Nowadays, derivatives of natural by-
Maize (Zea mays L.) – An Ethnopharmacological Review 135

products represent more than 50% of all clinically used drugs [5, 11] and novel molecules
have continuously been developed based on natural products. In the past 25 years, about half
of the new chemical entities that came to the market were natural products, derivatives of
natural products, or synthetic analogues of natural products [12, 13]. Despite the considerable
research activity in natural product identification, this field has not been explored enough. An
estimative indicates that approximately 5,000 plant species have been studied for possible
medical utilisation all over the world, only a minor fraction (~ 1.7 - 2%) of the 250,000–
300,000 plant species estimated to exist so far [14]. In Brazil, a country of recognized huge
biodiversity, estimations point to a number even lower; only 0.4% of the 55,000 plant species
have been accessed in a bioprospective way [5].
Discovery of a single new pharmaceutical agent originating from plant material is a well-
known time consuming and expensive process. In a random collection for pharmacological
screening, most of the plant extracts tested (95% or more) are inactive and a great number of
those active extracts contain already known compounds. If 5,000 compounds are tested at the
start of the process, perhaps one will become a clinically useful drug. Estimations point out
that from its discovery until its commercialisation, drug development might cost about
US$800 million and take a long time, e.g, 20 years, like taxol, for example [12, 15]. If
ethnopharmacological or ethnobotanical research is used to provide initial information,
success in screening is significantly improved, with 20–60% of plant extracts tested showing
some pharmacological activity [10, 16].
Following an ethnopharmacological and ethnobotanical approach, there is much
interesting documented information on the traditional knowledge associated with the usage
of maize (Zea mays L.) for the purpose of disease treatment. Indeed, besides the current use
of maize as the most nutritional representative cereal for several countries, other profitable
uses of the biomasses of this species might be found worldwide and will be further discussed
in this review. Focus is placed on the usage of maize flowers, grains, and leaves for
traditional medicine preparations to treat human health disorders. The occurrence of bioactive
secondary metabolites in maize tissues is described in connection with their nutritional and
therapeutic/prophylactic effects. Furthermore, some light is shed on the uses of maize
landraces by rural and indigenous populations, genotypes that are out of the global market
and seem to be an under-explored source of bioactive compounds. Information of such a
nature could aid in formulation of scientific hypotheses about the biological activity and
pharmacological mechanisms attributed to tissue extracts of Z. mays biomasses, as well as a
value-added strategy to under-utilised biomasses such as female flowers and leaves.

2. ETHNOPHARMACOLOGY OF MAIZE (ZEA MAYS)


The history of maize begins at the dawn of human agriculture when ancient people from
Mexico took the first steps in domesticating maize as they simply chose which kernels (seeds)
to plant according to desirable characteristics. Humans are considered the main diffuser agent
of Z. mays. Several studies indicate the Pacific coastal region of Mexico as the centre of
origin of that species, suggesting that its domestication process was associated with the
usage by local pre-historic people. Ancestral forms of maize were produced probably after
136 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

thousands of years of human selection from one or more species of teosinte that grow
naturally in Mexico and Guatemala [17].
Archaeological records show that maize was present in Central America as far back as
6,250 before present (BP) [18-21]. More recent genetic evidences showed ancient
domesticated populations of maize in highlands of the Balsas River, western Mexico.
Besides, archaeological evidences of early maize in Mexico are cobs found in Guilá Naquitz
(State of Oaxaca) and in a series of caves in the Tehuacán highlands (State of Puebla)
dated to about 4,700 BP [22]. A description of interesting information regarding
ethnopharmacological data of maize in several countries is summarised in Table 1.
Indigenous communities of Mexico preserve until today their practices of maize culture,
growing local varieties and maintaining, at least in part, traditional knowledge of their
millenary medicinal systems. As example, the Zapotec people, who belong to the largest
group of the indigenous population in Oaxaca, has a peculiar medical system that includes
specialists in curing different types of health problems. The most important among them are
the healers (‘curanderos’) who treat ailments by means of ritual ceremonies, but there are also
midwives (‘parteras’) who help women during pregnancy and some individuals known as
‘hierberos’, who are specialists in manipulating and prescribing medicinal plants without
performing healing sessions [48]. Thus, a clear hierarchy in the social system of ancient
indigenous communities is found with consequences in the medicinal usage of Z. mays.
Ethnopharmacological information about maize in those medical systems refers to the use
of unripe ears of corn for treatment of female and male genito-urinary complaints and the
husks of grains to treat gastrointestinal disorders, as well as hepatic problems [49]. In
communities from Zapotitlán de las Salinas, situated in the Valley of Tehuacán-Cuicatlán
(Puebla, Mexico), stigmas of maize (‘elote’) are boiled prepared and used by oral
administration to treat gastrointestinal diseases, mainly diarrhoea [50]. In turn, the Macro-
Mayan people mentions Z. mays for different treatment purposes and the antiquity of this
medicine knowledge was indicated by Leonti et al. (2003) in a study of medicinal plant uses
in the Isthmus of Tehuantepec, Mexico [39]. This region corresponds to the homeland of the
Olmecs people (about 3,500 BP), responsible for some of the earliest permanent monumental
constructions in Mesoamerica, whose culture influenced subsequent local traditions. Leonti et
al. (2003) compared the pharmacopoeias of linguistically related Lowland Mixe and Zoque-
Popoluca people known to have divergent cultures and separated by, perhaps, 2,000 years
[39]. The Zoque-Popoluca people reported the usage of maize (‘mok’) to treat gynaecological
and urological problems and Lowland Mixe communities used maize (‘meek’) for treatment
of gastrointestinal ailments. Although maize was named in a similar manner by both people,
different health care purposes were observed (Table 1).
Taking the bulk of the ethnopharmacological information worldwide about the uses
of Z. mays, from a clinical viewpoint the usage of its stigmas as a diuretic has been reported
in 93 phytomedicinal books, published in 13 countries [51]. The majority of the
ethnopharmacological information usually refers to the utilisation of maize as a diuretic to
pass kidney stones, to cure bladder ailments, as a renal antispasmodic, and also as anti-
inflammatory in the urinary system in general. Other current uses include treatment for
hypertension, constipation, gastrointestinal disorders, and gynaecological problems.
In the Mesoamerican culture area, maize dispersion tended to occur occasionally and
along the lines of pre-existing social contacts, inside and outside local communities [52, 53].
The eventual occurrence of catastrophic events caused sudden massive migrations of human
Maize (Zea mays L.) – An Ethnopharmacological Review 137

populations, accelerating the dynamics of the maize’s dispersion process in Central America
[54]. From Mexico, Z. mays started to spread northward to Canada and southward along the
Pacific coast until the Patagonia region of Argentine, a movement that occurred before the
arrival of Columbus in America [19, 21, 55]. Despite suggestions of widespread maize
consumption on the eastern Canadian Prairies between approximately 3,000–3,600 BP [56],
no reports were found in the literature about its use for purposes of treatment of health
disorders by native populations such as the Navajo, Hopi, or Zuni people in the past or
nowadays.

Central and South America

Moving towards the south, not much has been found about ethnopharmacological data of
Z. mays utilisation, a surprise, taking into account the close association established between
maize and many great cultures in America, for instance, Inca, Maya, and Aztec civilizations.
Archaeological and paleoecological evidences support the introduction of maize into southern
Central America and northern South America in late pre-ceramic times, between 7,000 and
5,000 BP [57]. Considering the medicinal usage preserved until today in Central America,
folk medicine of eastern Cuba mentions maize as a constituent of a herbal mixture prescribed
for renal afflictions [58]. A herbal treatment reported in ethnobotanical literature of the
Dominican Republic and prescribed by Dominican healers living nowadays in New York,
USA shows the use of ‘barba de maiz’ (tassels) to treat hot flashes, menorrhagia, and uterine
fibroids, and to regulate menses. Healers also mention the usage of maize in herbal mixtures
to reduce cramps caused by fibroids [34]. Longuefosse and Nossi (1996) performed an
ethnobotanical survey within the population of Martinique, considering that its popular
pharmacopoeia is the expression of a Creole pan-Caribbean culture [40]. This study found
folk medicine reports of a maize grain decoction orally administered to relief measles
symptoms. Ethnobotanical information on the diuretic activity of medicinal plants popularly
used in Mesoamerica (Guatemala) for urinary ailments refers to the aqueous extract of Z.
mays stigmas. Besides, the aqueous extract is also mentioned as a kidney anti-infective as
Guatemalan folk medicine prescribes it for cystitis and nephrosis [35, 59].
Staller (2003) considered that the early role of maize for ancient inhabitants of coastal
Ecuador and Andes was, in general, more important as a ceremonial plant associated with
exchange rituals where it was consumed in the form of fermented beer or ‘chicha’, than as an
economic staple food [22]. Moreover, that ritual and ceremonial significance associated with
Andean cultural traditions led to a rapid spread of maize through the low- and highlands of
the region. The early distinction established by indigenous people considered maize varieties
with red and purple grains better for ceremonial utilisation performed by shamans, while
yellow and white ones were preferably chosen for food preparation [60]. Indeed, more recent
studies focusing on Mesoamerican varieties show that even today, the colour of seeds is the
most important criteria for identifying and classifying maize as observed in the highlands of
Mexico (Chiapas), where red maize seeds are associated with indigenous agricultural rituals
as mentioned above, while white and yellow ones are clearly preferred for human and animal
nutrition [61, 62].
Table 1. Ethnopharmacological data of maize (Zea mays L.) according to geographical regions, local name of the species, part of
the plant used, and therapeutic utilisation

Region/Community Local name Plant parts used Therapeutic uses References


Anti-inflammatory, for Intestinal
Milho, barbas de milho
and bladder ailments, renal
Arrábida – Natural Park of Serra de (retrós de milho, cabelos Styles, stigmas, and
antispasmodic, urinary system [23]
São Mamede (Portugal) de milho, linho de young leaves
troubles (anti-inflammatory,
maçaroca de milho)
diuretic, and cystitis)
Arbërësh community
Grandinjë, Garëdin Stigma Purgative and blister [24]
(southern Italy)
Female flower or corn Anti-hypertensive, antilithiasic,
Bafia (Cameroon) Bazeùh [25]
beard and diuretic
Bolivia Maíz Stigma Retention of urine [26]
Anti-sickness, bladder diseases,
Female flower and chicken pox, cholagogue, cystitis, [28]
Brazil Milho, Abati, Avati
purple straw diarrhoea, diuretic,
fever, and hypoglycaemia
Bulamogi (Uganda) Not specified Stem Toothache (‘false teeth’) [29]
Bulgaria Zarevisa Stigmas Cholagogue and astringent [30]
Castelmezzano (Basilicata region, Anti-septic, diuretic, and
Not specified Kernel and stigma [31]
southern Italy) reconstituent
Chieti town (Abruzzo, central Italy) Rantign Stigmas filaments, flour Diuretic [30]
China Not specified Styles and stigma Diabetes (hyperglycaemia) [32, 33]
Dolomiti Lucane Stigma, kernel, and
Grandinie/Pupē Diuretic [24]
(inland, southern Italy) flour
Uterine fibroids and regulate
Dominic Republic Barba de Maíz Not specified [34]
menses
Ginestra (Basilicata region, southern Anti-septic, diuretic, and
Not specified Kernel and stigma [31]
Italy) reconstituent
Urinary anti-infective and/or to
Guatemala Maíz Stigmas [35]
ally burning, cystitis, and nephrosis
Region/Community Local name Plant parts used Therapeutic uses References
Urinary system, kidney stones,
Golan Heights and West Bank
Not specified Kernel and fibre blood pressure, prostate cancer, [9]
(Israel)
joint inflammation, and weight loss
Diuretic, depurative, sudorific, and
Italy Granoturco, Mais Stigmas [30]
hypotensive
Common cold, obesity, oedema,
Jordan Not specified Corn silk constipation, kidney sand and [6]
stones
Kadiogo province (Burkina Faso) Not specified Flowers Toothache [36]
Kingdom of Jordan Not specified Pestle Diuretic [9]
Korean Buddhist (Korea) Oksusu Kernel Constipation [37]
La Paz and El Alto (Bolívia) Pelo de choclo Styles and stigmas Diuretic and kidney stone [38]
Lowland Mixe community
Mëëk Not specified Gastrointestinal [39]
(México)
Martinique Mayi Kernel Measles [40]
Northwestern Patagonia (Mapuche
Maíz Not specified Urinary [41]
community)
Hypertension and diabetes
Orient Not specified Style [42]
(hyperglycaemia)
Palestine – West Bank (Jenin,
Diuretic, antispasmodic, urinary
Tulkarm, Qalqilya, Nablus, Salfit,
Not specified Kernel tract antiseptic, and for the [43]
Ramalha, East Jerusalem,
treatment of hair loss and dandruff
Bethlehem, and Hebron)
Sakarya province Stigma
Misir püskülü Diuretic [44]
(Turkey)
Sile (Turkey) Misir Stigma Prostatitis and diuretic [45]
Taurus Mountains (Turkey) Mekke püskülü Style Kidney stone [46]
Turkmen Sahrs (North of Iran) Jewen Stigma Kidney stone [47]
Zoque-Popoluca community
Moc Not specified Gynaecological and urological [39]
(México)
140 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

In parallel with the great importance of maize as a staple food for South America, some
traditional people still use it to prepare medicines. In Peru, people consume a typical drink
made after decoction of purple corn grains called ‘chicha morada’, which is believed by
folklore to be effective as a stimulant, as well as to improve health [63]. In Altiplano, the
highlands of Bolivian Andes, two indigenous cultures were found, Aymara and Quechua.
Their descendents living in La Paz and El Alto cities report the use of ‘pelo de choclo’
(tassel) after decoction as a diuretic to treat and relief kidney disorders [38]. Similarly, the
Qollahuaya (Callawaya) Andean herbalists in Bolivia recognise the therapeutic properties of
Z. mays aqueous extract (maté) as a diuretic and use it to treat the retention of urine [26].
Considering the spread of maize along the Andean region towards the south, its use was cited
in a study performed with the Curruhuinca community settled in Chilean northwestern
Patagonia. The interviewed families, descendents of the Mapuche people that are ancient
inhabitants of the southern Andean region, mentioned usage of maize to treat urinary
disorders [41].
In turn, the arrival of maize in Brazil occurred through two major Tupi-Guarani
migration events, i.e., in the northern region towards Amazonia and the second one in the
southern region. Related to this, an archaeological survey at the Orinoco Valley-Amazon
basin/Venezuela revealed the importance of maize as food, with this plant species and bitter
manioc (Manihot esculenta) mentioned as the two most common staple food resources at
approximately 3,000 years ago [64]. The Brazilian coastal region was the geographical
domain of Tupi-Guarani linguistic groups, distributed from Amazon to the Prata River, in the
vicinity of Paraguay. Before Portuguese colonisation, the Tupi people, mostly spanning the
northern region, subsisted from bitter manioc-based agriculture. Nonetheless, they also
cultivated maize to a lesser extent, mainly a variety with dark orange flint grains that was
early adopted by Portuguese colonisers and denominated Cateto [65].
Guarani people, in turn, cultivated maize mostly for subsistence and occupied
preferentially southern regions of Brazil [66]. Interestingly, maize plays a central role in
social and religious structure up to now for that people. With regard to this, it was observed
within Guarani communities in the Chaco region of Bolivian lowlands the occurrence of
many myths to explain the excellence of maize as food and its divine origin [67]. The Guarani
people cultivated several varieties of maize that probably existed before the pre-colonisation
period; one was the generally named Avati-Moroti that had floury light yellow grains and
another, named Cristal, with flint white grains and used for ‘canjica’ preparation [65].
Guarani communities usually eat maize cobs, both before and after complete maturation of
grains. The latter could be threshed or triturated with a pestle and used to prepare porridge or
cakes. Chewing the grains, the Guaranis also used maize to make a fermented beer named
‘chicha’ [66]. Since Guarani people were known as a ‘maize’ civilisation, it is intriguing that
Z. mays was not mentioned for health treatment purposes, even in a study about its
pharmacopoeia [67], nor in the literature consulted in the present work. Interestingly, some
reports on the usage of maize for treatment of bladder diseases, chicken pox, cystitis,
diarrhoea, fever, and hypoglycaemia were found in rural and urban Brazilian communities
[27, 28], but not directly related to the Guarani people.
Maize (Zea mays L.) – An Ethnopharmacological Review 141

Europe, Mid East, and Asia

When Columbus returned to Europe in 1493 after his first contact with America, he
brought with him some seeds of Z. mays, a yet unknown cereal, and introduced it in
Mediterranean countries. After a hundred years, European businessmen and navigators
distributed it over the five continents, mainly during the colonisation period [19, 55]. Maize
arrived in Europe through the Iberia Peninsula and since then, its stigmas have been used for
treatment of health problems. An example is the usage of maize stigmas by the traditional
people living in the Natural Park of Serra de São Mamede (Portugal). This group of people
named the stigmas ‘barbas-de-milho’, ‘retrós-de-milho’, ‘cabelos-de-milho’, and ‘linho-de-
maçaroca-de-milho’ and utilise them as a diuretic to treat urinary dysfunction, cystitis, and
prostate disorders [23]. Considering other countries, in southern Italy, maize preparations are
used as a purgative and blister, as indicated in an ethnobotanical survey performed with
traditional Arbe ‘reshe’ people [24]. In Castelmezzano (southern Italy), folk pharmacopoeia
reports the use of maize grains and stigmas as an antiseptic, diuretic, and reconstituent [31].
Another study about traditional medicine in Italy reports that stigmas of Z. mays (i.e.,
‘zareviza’, ‘granoturco’, or ‘mais’) are used after decoction, infusion, or tincture preparation
as a diuretic, cholagogue, astringent, depurative, sudorific, and hypotensive agent [30].
Besides, these authors explain that grain flour was commonly used by Italian people in
‘polenta’ preparation, which reinforces the anticholesterol properties of the oils present in
maize grains (Table 1).
In Ireland, the aqueous extract of tassel-like tufts (stigmas or green pistils) of Indian
maize (i.e., Zea mays) had been introduced into medicine as a demulcent and diuretic to treat
inflammation of the bladder and kidneys even by 1885 [68]. The effectiveness of that
phytomedicine was associated with the utilisation of fresh biomass for infusion preparation as
a diuretic of the mildest and least irritating kind, with positive effects in treatment of the
nocturnal urinary incontinence. Interestingly, the physiological actions of the drug upon
administration are reported, e.g., regular pulse and increase of arterial tension as the veins
diminish. The drug is reported to be tolerated by the urinary system, and in chronic cases its
administration may be continued for a month or 6 weeks without inconvenience. No
disturbing effects were found on the nervous system or digestive organs. Besides, the author
describes that distinct results have been found concerning the effects on the patients' urinary
system, because the fluid extract of the drug appears to vary in strength according to the
nature of the soil, climate, time and mode of picking, and the manner in which the stigmas are
dried. Of course, the effects of ecological factors and post-harvest management on the quality
of raw material of medicinal plants, i.e., their pharmacological effects, have been largely
recognised. Thus, technical precautions are recommended when scaling up the production of
Z. mays medicinal biomasses.
Market activities led maize to the Mid-East during the early 16th century and since then, it
has been used in folk medicine in various countries. In Turkey, traditional people of Istanbul
currently use maize styles (‘misir’) after decoction, taken alone as a diuretic and to treat
prostatitis, or in association with young branches of Juglans regia, taken after breakfast to
relief eczema cases [45]. Nowadays, Turkish folk medicine reports usage of Z. mays to
combat parasitic infections and in this case, the grains are cooked together with dried white
beans and swallowed. The potent antihelmintic activity of the aqueous extract of maize grains
taken alone has also been described [69]. Furthermore, an essay on folk medicine in the inner
142 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

region of the Taurus Mountains in south Anatolia, Turkey revealed the utilisation of Z. mays
(‘mekke püskülü’) stigmas to treat kidney stone [46].
A study carried out in 1998–1999 at Jordanian markets to observe plant-derived material
sold there identified that corn pestle was used and commercialised as a diuretic [9]. In the
Syrian Arab Republic, the rural population and Bedouins usually collect the local flora to
prepare traditional medicines, following the Unani system of medicine. This system is based
on Hippocratic theory of the four humours: blood, phlegm, yellow bile, and black bile. Galen
and Avicenna built theoretical foundations of this medical system in the past and presently,
regions of the near east and northern Africa remain under their influence. In a study
performed for identification of species composition of six multicomponent Unani medicines,
maize appeared in four of the medicines. Stigmas of maize (‘dhurah-safra’) alone are
prescribed mainly for treatment of problems in the urinary system and also as a general
panacea for relief of digestive problems, ailments in the osteomuscular system, as well as
when the patient shows badly defined symptoms and conditions [7].
In Palestine, several medicinal plants have been described for treatment of diseases and
herbal medicine is considered an integral part of Palestinian culture, playing a pivotal and
indispensable role in current public health care. An ethnopharmacological survey of natural
products used in healing diseases covering all major regions in the West Bank of historic
Palestine (Jenin, Tulkarm, Qalqilya, Nablus, Salfit, Ramalha, East Jerusalem, Bethlehem, and
Hebron) revealed that maize oil was used in folk medicine as a diuretic, antispasmodic, and
urinary tract antiseptic, as well as for the treatment of hair loss and dandruff [43].
The Vietnamese pharmacopoeia contains more than 250 monographs and shows the
importance of traditional medicine in that Asian country. Z. mays has been used individually
or combined with other plant species as a traditional herbal remedy to produce an increase of
diuresis [70].
Considering the movement of maize into the African continent and its use as medicine,
an ethnobotanical register was found for Cameroon, where the stigmas are prescribed in
association with watermelon peel and banana cut into pieces as an antilithiasic and diuretic.
Furthermore, some traditional healers use maize stigmas for hypertension treatment, with a
controlling action rather than a curative one in this case [25]. Another study on the
phytomedicines in Africa reports the usage of corn silk extract in folk medicine to treat
urinary infections, cystitis, and prostrate disorders, e.g., frequent urination caused by irritation
of the bladder and urethral walls [71].
It is especially noteworthy that the most cited medicinally useful part of the maize plant
is the stigma of female flowers, commonly referred to as corn silk or ‘hair of maize.’ Extracts
of corn silk have been shown to be a chemically complex matrix rich in phenolic compounds,
e.g., p-coumaric acid, vanillic acid, protocatechuic acid, anthocyanins, and derivatives of
quercetin and hesperidin [72], with significant effects on human health, for instance, as an
antibiotic [73], antidiabetic [74], or antioxidant [75], or for immune enhancement [76] or
antitumour effects [77]. Uncomplicated urinary tract infections (UTI) are one of the most
common infectious diseases, caused in 90% of all cases by uropathogenic Escherichia coli
strains (UPEC). A bioassay guided fractionation of Z. mays L. stigmata by EtOH-water
extraction followed by chromatography on Sephadex LH20 afforded two active fractions (I
and XI) against UPEC antiadhesive and antibacterial activity. Interestingly, the fraction I
revealed the presence of significant amounts of the biocide benzethonium chloride, an illegal
contaminant. Benzethonium chloride is not part of contaminant list for which the content
Maize (Zea mays L.) – An Ethnopharmacological Review 143

has to be monitored in herbal materials [78]. The fraction I of Z. mays stigmata samples
did not show an interesting antiadhesive and antibacterial activity. In turn, the fraction
XI, was chromatographed by MPLC and preparative HPLC, leading to a still complex
subfraction XIG, which was analyzed by UHPLC/+ESI-QTOF-MS. Advanced data
processing and species-metabolite relationship database revealed tentatively the existence of
the unusual C-glycosidic flavones derhamnosylmaysin, 3′-deoxyrhamnosylmaysin, 3′-O-
methylderhamnosylmaysin, as well as apiferol, alternanthin, gibberelin A12, piperine, α-
linolenic acid, linoleic acid, palmitic acid, and oleic acid. In conclusion, the antiadhesive
activity of the hydroalcoholic extract from Z. mays L. stigmata could be attributed exclusively
to the fraction XI (49% at 1 mg/mL and 37% at 0.5 mg/mL). Besides, the antiadhesive
potential of Z. mays L. stigmata extracts validates the traditional use of this herbal material
for UTI. However, in vivo studies and clinical investigations have to be performed to
registered phytopharmaceuticals based on this herbal material [79].
In this regard, however, the use of aqueous extract of stigmas/styles of maize flowers as a
phytomedicine to treat urinary disorders seems be common worldwide [71, 80], regardless of
the ethnical group that is the source of the information and its geographical region of
occurrence. Such information is meaningful as one envisages the development of a medicine
that meets the rules of health regulatory agencies, because the positive effect of
ethnopharmacological knowledge on optimisation of the drug development process is very
well known. In fact, an increasing body of evidence point to corn silks as source of secondary
metabolites with a meaningful effect on the urinary system [6, 30, 31, 38, 44]. More recently,
a study performed in Brazil demonstrated that the administration of corn silk aqueous extract
to anaesthetised Wistar rats caused a diuretic effect, without leading to potassium or marked
sodium loss. Such findings suggest that the corn silk aqueous extract is not a loop diuretic,
helping to elucidate its mechanism of pharmacological action [81].
Finally, it is worth mentioning that despite the well-known oriental pharmacopoeia, with
thousands of plant species and phytomedicines currently used all over the world, records
found about the usage of maize to treat ailments of any nature are scarce [32], and a possible
reason for that are the routes of dispersion of that cereal over the continents as previously
discussed. A survey of natural medicines used in the traditional Chinese medical system for
diabetes therapy reported that a simple recipe of Z. mays styles and stigmas was very
effective, as the anti-hyperglycaemic effect of stigma extract has been previously confirmed
by a pharmacological test [33].

3. ZEA MAYS AS A SOURCE OF BIOACTIVE COMPOUNDS


Maize is recognised worldwide as a staple food with unequivocal importance for food
security, but concomitantly, it is a plant species that is poorly recognised as a source of
bioactive compounds. An interesting hypothesis to this under-representation as a medicinal
plant is tentatively explained by the apparency theory [82]. Maize, similar to other cereals
(wheat, rice, barley, sorghum, rye, and oat, for instance), is highly apparent and rather than
relying on qualitative defences, i.e., producing important secondary metabolites as chemical
responses to pathogens, it relies on quantitative ones such as silica content [83]. Besides,
taking into account the continuous genetic breeding of such species over the past decades
144 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

using conventional and biotechnological tools and the resulting decrease of its genetic
variability, one could expect a more restricted chemical profile regarding bioactive secondary
metabolites in modern varieties of maize spread out over the world. Consequently, in modern
societies maize is rarely used as a source of medicinal compounds [84], instead, along with
rice and wheat, it is one of the most important human food plants, accounting for over 50% of
the daily global requirement of proteins and 60% of the total calorific intake [85].
On the other hand, some landraces and varieties of Z. mays show differences in grain
colour, ranging from red, purple, blue, and variegated to white (Figure 1), in spite of the most
common type observed being the yellow one. In a similar manner, other parts of the maize
plant might show distinct colour patterns, reflecting significant quantitative and qualitative
differences in content of pigments, i.e., secondary metabolites. The most important pigments
observed in maize associated with colour variation are the secondary metabolites known as
(poly)phenolic and carotenoid compounds (Figure 2) [51, 86, 87].

Figure 1. Discrepancies in grain colours of local maize varieties developed and cultivated by small
farmers in southern Brazil. The variegated trait in some grains might be noted. Typically, the grain’s
colour patterns are associated with their contents of the secondary metabolites carotenoids and
anthocyanins. MG = Mato Grosso Palha Roxa, RX = Roxo, LP = Língua de Papagaio, AM = Amarelão,
MP = MPA1, and FO = Fortuna.

Phenolic compounds form a chemical group with broad distribution and play an
important role in plant physiology, since they are involved in development and reproduction
processes, pathogen resistance, and herbivory defence [88]. More than 8,000 chemical
Maize (Zea mays L.) – An Ethnopharmacological Review 145

structures of phenolic compounds are known so far, which have in common at least one
benzenic ring linked to a hydroxyl group. This functional group (-OH) confers the antioxidant
activity against reactive oxygen species (ROS) of those secondary metabolites [89-92]. The
basic chemical structure of (poly)phenolics might be modified in a great variety of ways,
producing compounds like gallic acid and its derivatives [92], phenylpropanoids, such as
caffeic acid, normally related to pathogen defences, and flavonoids, the major class of the
(poly)phenols [88]. Phenolic compounds were also observed in oligomeric forms, like galates
and epicatechin galate, also known as tannins [92, 93].

Figure 2. Chemical structures of polyphenolic (A–F) and carotenoid (G–J) compounds commonly
found in Zea mays tissues. Gallic acid (A), quercetin (B), apigenin (C), taxifolin (D), cyanidin (E),
pelargonidin (F), β-carotene (G), cryptoxanthin (H), lutein (I), and zeaxanthin (J).

The antioxidant effect of (poly)phenols is so remarkable that Seo et al. (2003) report the
utilisation of these compounds in industry, replacing synthetic antioxidants like tert-butyl-4-
hydroxytoluene (BHT) and tert-butyl-4-hydroxyanisole (BHA), both already known to be
carcinogenic agents [91]. Indeed, consumers’ concern has come to focus on the toxicity and
potential health hazards of synthetic antioxidants, as plant phenolics, flavonoids, tannins, and
anthocyanidins are safe and have also shown important pharmacological effects [75]. In
Japan, purple maize grains are used as a source of food colourants by industry and pigments
146 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

extracted from them were associated with a decrease in carcinogenesis in the rectal colon of
rats [94]. A study performed in Serbia analysed extracts of mature silks of 15 maize hybrids
and demonstrated the occurrence of variable contents of phenolic compounds, ranging from
880 to 2937 mg of catechin equivalent/100 g dry weight. The observed differences were
directly related to the maize variety considered and higher contents seemed to be a
determinant of pharmacological efficiency, since antioxidant activity increased proportionally
to the polyphenol content [87]. El-Ghorab et al. (2007) demonstrated the high antioxidant
activity exhibited by several extracts obtained from Egyptian corn silks [80]. These extracts
included volatile, aqueous, petroleum ether, and ethanol extracts and, among them, the
ethanol extract showed the highest antioxidant activity, inhibiting DPPH (2, 2-diphenyl-1-
picrilhidrazil) activity at level of 400 ug/mL by 84%. Recently, Bai et al. (2010) demonstrated
the antioxidant effect of ethanol extract of maize silk against oxidative damage in vivo [95].
Intragastric administration of maize silk ethanolic extract to mice at 75, 150, and 300 mg/kg,
30 min before radiation, and then every day for 10 days ameliorated the radiation-induced
oxidative tissue damage effectively. The extract seemed not to be as effective in the kidney as
in liver tissue and its protective role was partially via up-regulation of the Nrf2 gene, as it up-
regulated the hepatic expression of Nrf2 protein dose-dependently and also increased the
activities of Nrf2-related antioxidant enzymes. Nrf2 is a critical transcription factor that binds
to the antioxidant response element in the promoter region of a number of target genes, which
encode many detoxifying antioxidant enzymes such as SOD (superoxide dismutase),
CAT (catalase), and GR (glutathione reductase), providing multiple layers of protection
during oxidative stress. As the authors claim, the results may significantly improve the
understanding of maize silks with respect to therapeutic approaches to diseases associated
with oxidative stress. However, no data on the chemical composition of the studied extract
were provided, which are needed to draw further conclusions on the biologically active
compound(s).
Other pharmacological properties attributed to phenolic compounds are chelation of
metallic ions [75, 92] and anti-inflammatory [96], antiviral [97], antitumoural [98-100],
antiproliferative [90, 101], and antimutagenic effects [102-106], justifying the increasing
interest in these compounds and their regular inclusion in the diet as a strategy for prevention
of health disorders. Moreover, Farsi et al. (2008) showed that silk extracts (40 ug/mL) from
the Mexican landrace Zapalote chico and modern inbred lines of maize inhibited non-
enzymatic glycation in vitro [107]. The antiglycation activity of the silk extracts was highly
associated with their total phenolic content and the most active maize genotype CO441 (44.8
mg total phenolics/g extract) displayed an IC50 of 9.5 ug/mL, being even more effective than
aminoguanidine, a known inhibitor of glycation. On the other hand, the Zapalote chico
landrace, with its high maysin content, showed only moderate inhibitory activity compared
with the modern maize genotypes, suggesting that the C-glycosyl flavone does not reduce
AGE (advanced glycation endproducts) formation as efficiently as the other phenolics present
in corn silk. According to the authors, modern resistant and high phenolic maize inbreds are
attractive candidates for development as a therapeutic for diabetic complications or the
degenerative effects of ageing, but further studies on the isolation and structural elucidation
are required to identify the phenolic compounds effective in preventing AGE formation. Non-
enzymatic glycation and the accumulation of AGEs have been associated with diabetes and
ageing. These results show the potential of phenolic compounds from maize inbreds as
natural AGE inhibitors for prevention and treatment of diabetic and ageing complications.
Maize (Zea mays L.) – An Ethnopharmacological Review 147

In China, folk remedies containing corn silk extract have been used as an oral antidiabetic
agent for decades [108]. According to the authors, corn silk extract orally administered to
alloxan-induced hyperglycaemic mice demonstrated to reduce hyperglycaemia by increasing
insulin levels and recovery of injured β cells, although the compounds involved in this action
remain to be elucidated. Interestingly, the doses studied, i.e., 0.5, 1.0, 2.0, and 4.0 g/kg body
weight of corn silk extract, seemed to be quite high for further pharmacological studies, since
toxic effects might be predicted. In fact, the rationale for biological assay designs should
always take into account the effect of doses so that eventual further pharmacological and
toxicological studies are meaningful in drug development processes for human health.
Other studies report the hypoglycaemic effect and improved carbohydrate tolerance
associated with the saponin [109, 110] and polysaccharide fractions of maize extracts [111].
The latter authors, based on the fact that corn silk is well known and frequently used in
traditional Chinese herbal medicines, evaluated the hypoglycaemic effect of polysaccharides
extracted from that biomass using streptozotocin-induced diabetic rats as the study model.
Daily intragastric administration of 100–500 mg/kg body weight of the polysaccharides in the
diabetic rats led to a significant decrease in the animal’s glycaemic index in the fourth week
and reduced serum lipid levels, including total cholesterol and total triglycerides. Despite the
interesting results, it would be worth mentioning the eventual effect of the doses investigated
in further clinical assays, because extrapolation of the tested values seems not to be feasible,
hampering future developments based on the experimental conditions described.
Another interesting effect of the polyphenolic compounds from corn silk is the anti-
fatigue activity, recently reported [112]. The study demonstrated that oral administration of
purified flavonoid extract from corn silk (at 100 and 400 mg/kg) exhibited anti-fatigue
activity by inhibiting the production of blood lactic acid, retarding the formation of blood
urea nitrogen, and increasing hepatic glycogen concentration. Besides the need to profile the
chemical constituents of the flavonoid extract, the mechanism of the action on fatigue and/or
exercise durability seems to be a relevant issue for further investigation, as the studied extract
concentrations are thought to be unfeasible for clinical assays due to eventual toxic effects.
Besides corn silk, the nutritional composition related to the phenolic profiles and antioxidant
effect of pollen from maize genotypes are also important as a functional food ingredients, as
well as dietary supplement with therapeutic effects. Sweet maize pollen samples have shown
to contain relevant amounts of phenolic compounds (9933.01 ± 85.65 mg GAE/kg dry matter)
and flavonoids (15,001.09 ± 912.69 mg CE/kg dry matter), also exhibiting high antioxidant
activity (104.38 mmol trolox eq/kg). Quercetin diglycoside was the most abundant flavonoid
in all the investigated pollen samples[113].
Flavones are another class of flavonoids found in Z. mays tissues. Important biological
effects have been associated with these compounds, such as the activity against the
corn earworm (Helicouerpa zea Boddie) attributed to the C-4’’-hydroxy derivatives
of maysin and 3’-methoxymaysin, i.e., flavone glycosides present in corn silks [75, 114,
115]. Incorporation of these compounds into silks by breeders could enhance the resistance of
corn to this important pest [116]. Further examples demonstrating the enormous potential of
maize as a source of bioactive compounds are the two novel flavone glycosides found in corn
silk 2’’-O-α-L-rhamnosyl-6-C-3’’-deoxyglucosyl-3’-methoxyluteolin and 6, 4’-dihydroxy-3’-
methoxyflavone-7-O-glucoside, not previously identified in any other natural source [117].
Xu et al. (2008) have detected a novel flavone (6-acetyl-luteolin) and two urea glycosides
(rhamnosyl urea and 1, 3-dirhamnosyl urea). Wang et al. (2010) have isolated a new
148 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

flavonoid (4’, 5, 7-trihydroxy-3’, 5’-dimethoxyflavone-7-O-[b-D-apiofuranosyl (1->2)]-b-D-


glucopyranoside) from the bracts of Z. mays L. and Liu et al. (2011) have identified the
flavone glycoside isoorientin-2’’-O-L-rhamnoside from the style of Z. mays [75, 118, 119].
Anthocyanins are flavonoids, usually responsible for the red, pink, purple, and blue
colours of plant tissues. They protect plant cells from ultraviolet (UV) radiation damage and
play an important role in attraction of pollinators and disperser agents. The most common
anthocyanins in nature are cyanidin, pelargonidin, peonidin, delphinidin, petunidin, and
malvidin [120]. Considering different varieties of Z. mays, distinct contents of anthocyanins
and their derivatives could be observed in grains, mainly in the aleurone layer, but also in
pericarp tissue, female and male flowers, roots, straw, and leaves [60]. In Mexican maize
varieties with blue grains, cyanidin and malvidin were the most common anthocyanins
observed, and for the red grain varieties, pelargonidin, cyanidin, and malvidin were
predominantly found [121]. Considering purple grains of maize varieties currently cultivated
in Peru, pelargonidin, peonidin, and cyanidin have been identified as the main compounds,
with an interesting total content of anthocyanins, ca. 1600 mg of cyanidin-3-glucoside
equivalent/100 g dry weight [122, 123]. Anthocyanins also act as antioxidant compounds [63,
124, 125], besides showing antimutagenic [63, 126] and antitumoural activitiesl [63, 127-
129]. Analysis of Z. mays stigmas from Limoeiro do Norte (Ceará state, northeastern Brazil)
showed different concentrations of flavonoids, pro-anthocyanins, and cinnamic derivatives
[51]. In addition, more recent work has demonstrated the application of liquid
chromatography, nuclear magnetic ressonance (NMR) spectroscopy, and chemometric
analysis for the determination of metabolic fingerprint and pattern recognition of silk extracts
of maize landraces cultivated in southern Brazil [130]. In that metabolomic study, emphasis
was given to the not yet completely exploited potential of maize landraces developed and
cultivated by small farmers in southern Brazil as a source of bioactive compounds for the
purpose of nutritional improvement of human populations, as well as in breeding programmes
of that cereal. The major differences in chemical composition were found in carbohydrate
constituents, as well as anthocyanin and polyphenol contents. A combination of NMR and
principal component analysis (PCA) revealed to be a useful tool for the discrimination of
maize silks with respect to their chemical composition, including rapid authentication of the
raw material of current pharmacological interest, pointing out three distinct metabolic
profiles. Another approach used attenuated total reflection Fourier transform infrared
spectroscopy (ATR-FTIR) and chemometrics for discrimination of landrace maize flours.
Amylose/amylopectin ratio and protein composition were shown to be the major contributors
for the discrimination of the twenty-six studied maize landraces [131, 132]. Further studies
performed on the anthocyanin contents of grains, leaves, and silks [133] for an F1 population
of eight genotypes of maize landraces cultivated in southern Brazil corroborated previous
results on the heterogeneous amounts of those phenolic compounds among the studied
genotypes and parts of the plant as shown in Figure 3 [131]. Indeed, maize landraces are
recognised to have a high genetic variability so that one can expect finding concomitant
heterogeneous metabolic profiles [132].
Maize (Zea mays L.) – An Ethnopharmacological Review 149

Figure 3. Anthocyanin content (ug/mL) of grains, leaves, and silks of maize landraces (G1–G8)
cultivated in southern Brazil using a low input agro-ecological production system.

Purple corn (Zea mays L.) is traditionally used in Peru in the preparation of desserts and
juices. Purple corn cob and kernel extracts are rich in anthocyanins and phenolic compounds.
Purple corn cob, whole grain, ground grain, and pericarp extracts were obtained by
supercritical fluid technology in a fixed bed at 50°C and 400 bar, in a sequential extraction
process using supercritical carbon dioxide (scCO2) as a solvent in the first step, ethanol in a
second step, and water in a third step. The HPLC analysis showed high yields of
anthocyanins, e.g., cyanidin-3-glucoside (cob: 71.52%; pericarp: 73.62% from 2nd step
extraction, respectively and cob: 75.28%; pericarp: 77.27% from 3rd step extraction,
respectively), peonidin-3-glucoside (cob: 10.41%; pericarp: 10.48% from 2nd step extraction,
respectively and cob: 8.55%; pericarp: 9.27% from 3rd step extraction, respectively), and
pelargonidin-3-glucoside (cob: 18.07%; pericarp: 15.50% from 2nd step extraction,
respectively and cob: 16.16%; pericarp: 13.46% from 3rd step extraction, respectively). Purple
corn cob and pericarp also have higher amounts of phenolics and antioxidant activity in their
aqueous extracts. This sequential extraction stands out as an effective technique to increase
the extraction yield of compounds of interest, also allowing to obtain extracts with different
chemical composition [134].
In plant kingdom, carotenoids are widely distributed, with some playing an important role
in photosynthesis (i.e., β-carotene), helping light absorption and protecting cell apparatus
against UV radiation damage [135, 136]. These pigments are responsible for yellow, orange,
and red colours of plant tissues. Chemically, carotenoids are a tetraterpenic arrangement (40
carbon atoms) that could be altered, generating more than 600 known compounds, and are
characterised by a high hydrophobicity [92, 136-138]. Approximately 50 kinds of carotenoids
are thought to be important in human nutrition, with at least 40 of them acting as precursors in
retinoid pathway biosynthesis [92]. Retinoid compounds are bioactive forms of vitamin A and
150 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

have been successfully used in cancer therapy as cell modulators, re-coordinating cellular
differentiation events typically found in uncontrolled cancer cells [37, 139-141].
The most common carotenoids in the grains of commercial and landrace varieties of Z.
mays are the xanthophylls lutein and zeaxanthin [86, 130, 142], with minor amounts of α- and
β-carotene. In humans, these compounds are concentrated in sun-exposed areas like the eyes
and skin, probably for photoprotection, and they also participate in an effective manner in the
maintenance of the retina’s health, preventing injury from excess light and oxidative stress. In
fact, they may be found at very high concentrations in the retina’s central region (macula
lutea). Their presence was directly related to the low incidence of macular degeneration and
cataract development, in addition to their already observed anticancer activity [92, 136, 142-
144]. A series of studies have shown appreciable contents of carotenoids (zeaxanthin and
lutein) in grains, leaves, and silks of commercial and landrace varieties of Z. mays cultured in
southern regions of Brazil, determined by high performance liquid chromatography and UV-
visible spectrophotometry, as shown in Figure 4 [133, 142, 145]. It is worth mentioning the
high heterogeneity of carotenoid contents in grains of the maize landraces studied by those
authors, as clearly depicted in Figure 4. Additionally, silk tissues seem to be an interesting
source of those secondary metabolites for most of the genotypes studied. The genotypes also
varied widely in their silk’s carotenoid contents, in agreement with the results found in grains,
as leaf tissues seemed to vary in that pigment content to a smaller extent. Taken together,
such findings reinforce the importance of maize genotypes with little and exclusively local
commercial importance as a source of bioactive compounds in tissues other that not grains,
emphasising the relevance of the silks and leaves as attractive biomasses due to their
prominent amounts of carotenoids. More importantly, it is technically feasible to manage the
plant population in the field in order to collect silks, leaves, and grains over the growth cycle,
without any penalty in the yield of the latter.
In another series of studies, the quantification of lutein and zeaxanthin in grains and silks
of local maize varieties from southern Brazil also revealed meaningful discrepancies
according to the genotype; lutein and zeaxanthin contents are an important trait in the
nutritional and medicinal value of those biomasses [133, 142, 145]. In fact, daily consumption
of the non-provitamin A carotenoids zeaxanthin and lutein seems to have desirable health-
related effects, e.g., enhancement of immune function [146], blockade of tumour growth
[147], and protection against age-related macular degeneration, a human disorder similar to
cataracts that causes early blindness [136]. Additionally, it has been demonstrated that the
xanthophyll-rich grain extract of maize displays antivasculo/angiogenic properties, suggesting
a potential role of compounds such as zeaxanthin and lutein in the prevention of diseases
related to uncontrolled vessel formation [142].
The antioxidant activity conferred by maize compounds such as polyphenols or
carotenoids may have an effect on a variety of biological pathways. For instance, Ren et al.
(2005) have shown that the flavone glycosides AX-5″-methane-3’-methoxymaysin and AX-
4″-OH-3′-methoxymaysin isolated from corn silk have a potent antioxidant activity against
lipid peroxidation [117]. Likewise, Bai et al. (2008) have demonstrated the dose-dependent
free radical scavenging and antilipoperoxidant activities of maydis stigma extract, as well as
the protective effect of maize silk ethanolic extract on the radiation-induced oxidative stress
[148]. Another interesting activity of maize compounds with antioxidant activity is the
protective effect against oxidation damage in silkworms (Bombyx mori) conferred by the
maize plumule extract [149]. Sepehri et al. (2011) reported the protective effect of corn silk
Maize (Zea mays L.) – An Ethnopharmacological Review 151

extract on nephrotoxicity induced by prolonged use of aminoglycoside antibiotics such as


gentamicin [150]. Plasma creatinine and urea levels were significantly increased in animals
intraperitoneally treated with gentamicin, and corn silk administration (200 and 300 mg/kg) in
association with the antibiotic significantly decreased serum creatinine, but not urea,
compared with gentamicin-treated Wistar rats. Additionally, the authors reported the
occurrence of acute tubular necrosis, hyaline casts in tubular lumen, interstitial nephritis, and
glomeruli in the gentamicin-treated group. Co-treatment of corn silk with gentamicin
decreased the interstitial nephritis, but not the acute tubular necrosis or hyaline cast
formation. Interestingly, a high dose of corn silk caused hyaline cast formation, apoptosis,
congestion, and swelling of renal tubules, findings that typically give rise concerns about the
meaning of the doses studied, because they seem not to be of interest for further clinical
studies with human subjects. The authors claimed that corn silk might ameliorate nephropathy
during prolonged therapeutic use of gentamicin and related aminoglycosides.

Figure 4. Total content of carotenoids (ug/mL) of grains, leaves, and silks of eight (G1–G8) maize
landraces cultivated in southern Brazil using an agro-ecological production system.

Besides all the above-mentioned biological properties of maize components, there are
other interesting bioactivities that have been suggested, including the immunostimulant and
antimicrobial effects of corn silk [151-153]. Authors have suggested the potential of maize
primary and secondary metabolites in cancer therapy. Indeed, in vitro studies revealed the
potential of stigma maydis extract in reducing the viability of both leukaemia cells and gastric
carcinoma cells [154]. Moreover, Lu et al. (2006) have shown that a polysaccharide from
stigma maydis not only inhibits the proliferation of human hepatocellular carcinoma, but also
induces its apoptosis [155]. Despite the preliminary nature of the studies aforementioned, they
demonstrate the enormous potential of maize as a source of bioactive compounds and the
need for further studies on this subject. Indeed, for the purpose of usage in human health upon
152 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

the recognition of official regulatory agencies, despite the claimed pharmacological effects of
maize extracts, further studies are necessary, e.g., to isolate the bioactive compound(s) and/or
to standardise extracts according to a given biochemical marker (if any) for a certain
pathophysiology, to determine the toxicological effects and the eventual presence of
pesticides in raw materials, as well as to perform pre-clinical and clinical assays and
bioavailability measurements. More recently, by using a metabolomic approach, corn silk
extracts have been proved to be complex matrices with distinct chemical profiles according to
genotypes [130, 131], suggesting that their safe use as a medicine requires minimum
procedures of quality control of that raw material in producing areas, post-harvest, and
industrial processing so that the pharmacological properties are guaranteed. In fact, despite
the powerful analytical tools available for phytochemical studies, it seems that scant attention
has been paid to elucidate the chemical features of maize genotypes for pharmacological
purposes, with the exception of the content of β-carotene in grain tissues.
A second important and critical ethnopharmacological issue to be addressed concerns the
maintenance of the Z. mays germoplasm that is associated with the biological effects
described for maize tissue extracts worldwide. In most cases, and more commonly in
developing countries, the genetic diversity of that cereal and the corresponding phytochemical
potential have been preserved for small farmers with no or minimum acknowledgment of the
social and economic importance of doing so. The conservation of strategic genotypes such as
Creole and landrace varieties of maize, not only for the purpose of food security, but in a
wider view for the discovery of new and relevant compounds for the treatment of human
diseases seems to be urgent. For example, over the past years studies performed in southern
Brazil on the genetic diversity and chemical characterisation of maize landraces [133, 145,
156-158] have reported an ongoing genetic erosion process regarding those genotypes, mostly
because of their replacement by hybrid and transgenic varieties. Unfortunately, it is possible
to envisage such a scenario in other parts of the world due to the commercial predominance of
genetically improved/modified varieties of that cereal, which continuously increases the risk
of losing the Creole and landrace genotypes and their medicinal properties accordingly. On
the other hand, it is argued that the commercial exploitation of maize biomasses as a source of
phytochemicals of interest, in parallel with grain production, would be an interesting value-
added strategy to help preserving local genotypes in the developing world, with obvious
ecological and social gains.
More recently, some interesting issues have been reviewed, showing the connections
between ethnopharmacology, food production, and biodiversity conservation [159], as well as
arguing for the consequences of the lack of communication and compartmentalisation among
them with respect to the usage and conservation of plant species with medicinal and
nutritional functions. Obviously, maize landraces are typical examples to be considered in this
context, since it is notorious that the distinction between food and medicines for some
indigenous peoples and traditional communities is meaningless or, at least, poorly
understood. Unfortunately, that seems not to be the perception of most people living in urban
areas, i.e., the main consumers of maize. Thus, it has been thought that whether the general
public becomes aware of the food-medicine linkage claimed by ethnopharmacological studies
of maize landraces, a more favourable scenario would emerge to increase their production,
lowering the risk of loss of that germoplasm of ultimate importance.
Maize (Zea mays L.) – An Ethnopharmacological Review 153

4. PRESENT AND FUTURE PERSPECTIVES


The impact of Ethnopharmacology on public health and its role as a link between
allopathic and traditional medicine appear to be poorly considered. Nowadays, in most
societies these two distinct health care systems occur side by side in a complementary way.
The former is usually adopted for the treatment of serious and acute physiopathological
disorders, while the latter is adopted to treat chronic illness, to reduce symptoms, and to
improve the quality of human life [5, 10]. There is a common understanding that the benefits
of modern medicine are mostly available in developed countries, whereas traditional
medicines are predominantly used by countries in development and in most parts of rural
societies. Herbal medicine is a valuable resource of pharmacologically active compounds and
a necessity where an efficient basic health system is absent [3, 10, 160]. Modern medical
attention is based on the paradigm of single target-single compound drugs and services that
are expensive, while traditional medicines and medical consults of this type have a much
lower or no cost [13, 38]. In accordance with the World Health Organization (WHO), about
80% of human beings still choose plant remedies as their first therapeutic alternative for
illness relief [5, 161], and there seems to be a renewed interest in traditional forms of
medicine resulting from the perception of pharmacologists that the concept one disease-one
target-one drug does not always lead to cures, particularly of chronic and degenerative
diseases, due to their multifactorial nature [13]. Nevertheless, in most parts of the world there
is no regulation or safe control of phytomedicines, which highlights the requirement for
further studies related to the elucidation of biological mechanisms, as well as the toxicity,
safety, and efficacy of these plant-based end-products [10].
The relationship between some nutritional habits and appearance or prevention of
diseases directly impacts the improvement in quality of life of human populations.
Worldwide, health problems like cardiac diseases, cancer, and diabetes have increased in
frequency in modern societies and are undoubtedly influenced by unhealthy dietetic patterns
[162]. On the other hand, a number of foods are currently recognised as important agents for
prevention of human degenerative diseases, due to the existence in their composition of
compounds that display chemical protection [91, 100, 141, 163]. As an example, natural by-
products like polysaccharides extracted from some seaweed, mushroom, and plant species
such as the Aloe genus, besides soybean isoflavones and polyphenolic compounds (e.g.,
catechin and resveratrol) of several plant materials have been investigated for their biological,
nutritional, and pharmacological properties [164-168].
Concerning the control of diseases with a bad prognostic such as cancer, conventional
therapies are related to undesirable side effects due to their toxicity and, in general, only
extend the lifespan of patients by a few years. Thus, by combining studies of determining
chemical profiles of plant extracts with biological activity assays in an oriented-manner for a
certain pharmacological application (prophylactic and/or therapeutic) might be instrumental
in providing new phytomedicines or alternative therapeutic tools that combine selectivity,
efficacy, and low toxicity, eventually diminishing the unwanted side effects of current drugs
[169].
In this way, it is assumed that Z. mays could be an interesting and strategic resource due
to its bioactive compounds, mainly carotenoids, anthocyanins, and (poly)phenols. This is
because it has been cultivated and consumed worldwide, being a strategic staple food mostly
154 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

in developing countries that still maintain its genetic diversity. For example, high pigmented
maize grains could be utilised to make flours and bakery by-products that might have
nutraceutical properties, at least in relation to their significant antioxidant content [20]. In
addition, raw extracts or isolated compounds obtained from maize biomasses could be of
interest to the food industry as antioxidants, pigments, and flavourings, as well as to cosmetic
and health care products sectors [94, 121, 122]. Taking into account the information
previously reported regarding Z. mays utilisation as traditional medicine, the consumption of
aqueous extract of stigmas, i.e., tea by patients suffering from renal afflictions seems to be the
most prominent form of usage worldwide. Thus, further studies might be performed to isolate
and identify the bioactive compound(s) present in maize stigma extracts, allowing, for
instance, the standardisation of stigma-derived products that are already marketed. Nowadays,
the investigation focusing on the isolation and identification of bioactive compounds of maize
tissues associated with evidence of the pharmacological activity claimed by healers in
ethnopharmacological surveys seem to be in its infancy. Additionally, regardless of the
biological activity of interest, the assays in pre-clinical phases should be designed with
careful attention to the doses to be administered to animals, so that further clinical tests might
be performed using treatments at doses that make sense in physiological conditions for human
beings, for instance. Such approaches could give rise to insights on the pharmacological
mechanisms associated with the already known biological effects, improving the ongoing
therapeutic process. Additionally, it is worth mentioning that one should keep in mind the
usage of maize stigmas for the treatment of some neoplasic processes, which is certainly an
exciting issue.
Finally, the long-known content of carotenoids in maize grains might be thought as a
strategy to address issues regarding the incidence of blindness associated with poverty in
developing countries. Maize is the basic staple food of more than 1.2 billion consumers in
sub-Saharan Africa and Latin America, and vitamin A deficiency, often sub-clinical, is still
very common in these areas [170]. For example, prevention or mitigation of this disease
might be pursued by stimulating the selection and cultivation by local farmers of maize
landrace genotypes with a superior content of pro-vitamin A carotenoids in their grains, a
low-cost effort with ecological, social, and political merits. This would be, without doubt, a
counter revolution to the expansion of genetically modified food towards underdeveloped
countries, such as golden rice. In fact, traditional knowledge such as ethnopharmacological
information might be used with improved results in association with modern interdisciplinary
scientific studies to generate, for instance, a new kind of knowledge about maize, addressing
very interesting questions to some of the contemporary human challenges.

5. CONCLUSION
In conclusion, even with new technologies available to find new pharmaceuticals, it
appears that one of the best sources to improve drug discovery programmes is still the
healer’s pouch, because it contains several plant species that have been tested by generations
of autochthonous people. If so, it is necessary to recognise the sovereign right of States over
their natural resources so that our endeavour to discover new medicines from the diverse life
around us must take into account the international protocols established for protection and
sharing of national biological resources, e.g., local maize varieties, and traditional knowledge,
Maize (Zea mays L.) – An Ethnopharmacological Review 155

as stated by The 1992 Convention on Biological Diversity. In fact, in some cases the long-
term efforts to improve primary health care in poor regions could be strongly improved in
connection with concerns on the preservation of local knowledge (ethnopharmacology) and
the conservation of its biodiversity (ethnobotany). Unfortunately, due to several reasons and
in diverse ways, traditional knowledge and local biodiversity have been severely threatened
as local people’s rights are not a matter of concern in several cases of ethnopharmacological-
based bioprospecting all over the world. This scenario has been a critical constraint for the
claimed rational (sustainable) exploitation of maize as a source of bioactive compounds,
especially for indigenous and small farmer communities that have developed, cultivated, and
preserved many of the Z. mays races that most of humanity probably will ever know.

CONFLICTS OF INTEREST
The authors declare no conflict of interest.

6. REFERENCES
[1] Pirages D. Diversity and social progress in the next millennium: an evolutionary
perspective. Futures 2000; 32(6): 513-23.
[2] Verpoorte R, Maraschin M. Engenharia do metabolismo de plantas medicinais. In:
Yunes RACJB, Eds. Plantas medicinais sob a ótica da química medicinal moderna.
Florianópolis: Argos Editora Universitária 2001; pp. 381-432. [Metabolic engineering
of medicinal plants. Florianopolis: Argos Universitary Press 2001; pp. 381-432].
[3] Raskin I, Ribnicky DM, Komarnytsky S, et al. Plants and human health in the twenty-
first century. Trends Biotechnol 2002; 20(12): 522-31.
[4] Buenz EJ, Schnepple DJ, Bauer BA, Elkin PL, Riddle JM, Motley TJ. Techniques:
Bioprospecting historical herbal texts by hunting for new leads in old tomes. Trends
Pharmacol Sci 2004; 25(9): 494-8.
[5] Gurib-Fakim A. Medicinal plants: traditions of yesterday and drugs of tomorrow. Mol
Aspects Med 2006; 27(1): 1-93.
[6] Abu-Irmaileh BE, Afifi FU. Herbal medicine in Jordan with special emphasis on
commonly used herbs. J Ethnopharmacol 2003; 89(2-3): 193-7.
[7] Carmona MD, Llorach R, Obon C, Rivera D. “Zahraa,” a Unani multicomponent herbal
tea widely consumed in Syria: Components of drug mixtures and alleged medicinal
properties. J Ethnopharmacol 2005; 102(3): 344-50.
[8] Anyinam C. Ecology and ethnomedicine: exploring links between current
environmental crisis and indigenous medical practices. Soc Sci Med 1995; 40(3): 321-9.
[9] Lev E, Amar Z. Ethnopharmacological survey of traditional drugs sold in the Kingdom
of Jordan. J Ethnopharmacol 2002; 82(2-3): 131-45.
[10] Cordell GA, Colvard MD. Some thoughts on the future of ethnopharmacology. J
Ethnopharmacol 2005; 100(1-2): 5-14.
[11] Butler MS. The role of natural product chemistry in drug discovery. J Nat Prod 2004;
67(12): 2141-53.
156 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

[12] Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25
years. J Nat Prod 2007; 70(3): 461-77.
[13] Verpoorte R, Crommelin D, Danhof M, et al. Commentary: “A systems view on the
future of medicine: inspiration from Chinese medicine?.” J Ethnopharmacol 2009;
121(3): 479-81.
[14] Verpoorte R. Secondary metabolism. In: Verpoorte R, Alfermann A, Eds. Metabolic
engineering of plant secondary metabolism. Dordrecht: Kluwer Academic Publishers
2000; pp. 1-29.
[15] Newman DJ, Cragg GM, Snader KM. Natural products as sources of new drugs over
the period 1981-2002. J Nat Prod 2003; 66(7): 1022-37.
[16] Soejarto DD, Fong HHS, Tan GT, et al. Ethnobotany/ ethnopharmacology and mass
bioprospecting: Issues on intellectual property and benefit-sharing. J Ethnopharmacol
2005; 100(1-2): 15-22.
[17] Staller JE, Tykot RH, Benz BF. Histories of maize: Multidisciplinary approaches to the
prehistory, linguistics, biogeography, domestication, and evolution of maize. California:
Left Coast Press 2006.
[18] Brush SB. Genes in the field: on-farm conservation of crop diversity. New York: Lewis
Publishers 2000.
[19] Freitas FO. Estudo genético-evolutivo de amostras modernas e arqueológicas de milho
(Zea mays L.) e feijão (Phaseolus vulgaris L.). Tese de doutorado. São Paulo:
Universidade de São Paulo 2001. [Genetic and evolutionary study of modern and
archaeological samples of maize (Zea mays L.) and bean (Phaseolus vulgaris L.). PhD
thesis. São Paulo: University of São Paulo 2001].
[20] Hallauer AR. Specialty Corns. 2nd Edition. Florida: CRC Press 2000.
[21] Freitas FO, Bendel G, Allaby RG, Brown TA. DNA from primitive maize landraces and
archaeological remains: implications for the domestication of maize and its expansion
into South America. J Archaeol Sci 2003; 30(7): 901-8.
[22] Staller JE. An examination of the palaeobotanical and chronological evidence for an
early introduction of maize (Zea mays L.) into South America: A response to Pearsall. J
Archaeol Sci 2003; 30(3): 373-80.
[23] Camejo-Rodrigues J, Ascensao L, Bonet MA, Valles J. An ethnobotanical study of
medicinal and aromatic plants in the Natural Park of “Serra de Sao Mamede”
(Portugal). J Ethnopharmacol 2003; 89(2-3): 199-209.
[24] Pieroni A, Nebel S, Quave C, Munz H, Heinrich M. Ethnopharmacology of liakra:
traditional weedy vegetables of the Arbereshe of the Vulture area in southern Italy. J
Ethnopharmacol 2002; 81(2): 165-85.
[25] Noumi E, Houngue F, Lontsi D. Traditional medicines in primary health care: plants
used for the treatment of hypertension in Bafia, Cameroon. Fitoterapia 1999; 70(2):
134-9.
[26] Bastien JW. Pharmacopeia of Qollahuaya Andeans. J Ethnopharmacol 1983; 8(1): 97-
111.
[27] Panizza S. Plantas que curam: cheiro de mato. São Paulo: Ibrasa 1998. [Healing plants:
smell of wood. São Paulo: Ibrasa 1998].
[28] Souza NN, Silva AFC, Martins FS, Ferreira GS, Ramos FM, Pereira RO. Plantas
medicinais: etnobotânica na várzea do Mamirauá. In: Rocha SFR; Scarda FM, Eds.
Mamirauá: Instituto de Desenvolvimento Sustentável Mamirauá 2003. [Medicinal
Maize (Zea mays L.) – An Ethnopharmacological Review 157

plants: Ethnobotany at Mamirauá Valley. Mamirauá Institute for Sustainable


Development Mamirauá 2003].
[29] Tabuti JRS, Lye KA, Dhillion SS. Traditional herbal drugs of Bulamogi, Uganda:
plants, use and administration. J Ethnopharmacol 2003; 88(1): 19-44.
[30] Leporatti ML, Ivancheva S. Preliminary comparative analysis of medicinal plants used
in the traditional medicine of Bulgaria and Italy. J Ethnopharmacol 2003, 87, 123-142.
[31] Pieroni A, Quave CL. Traditional pharmacopoeias and medicines among Albanians and
Italians in southern Italy: A comparison. J Ethnopharmacol 2005; 101(1-3): 258-70.
[32] Li WL, Zheng HC, Bukuru J, De Kimpe N. Natural medicines used in the traditional
Chinese medical system for therapy of diabetes mellitus. J Ethnopharmacol 2004;
92(1): 1-21.
[33] Li W, Chen YL, Yang M. Experimental study on antihyperglycemic effect of Stigma
Maydis. Chinese Traditional and Herbal Drugs 1995; 26: 305-11.
[34] Ososki AL, Lohr P, Reiff M, et al. Ethnobotanical literature survey of medicinal plants
in the Dominican Republic used for women's health conditions. J Ethnopharmacol
2002; 79(3): 285-98.
[35] Caceres A, Giron LM, Martinez AM. Diuretic activity of plants used for the treatment
of urinary ailments in Guatemala. J Ethnopharmacol 1987; 19(3): 233-45.
[36] Tapsoba H, Deschamps JP. Use of medicinal plants for the treatment of oral diseases in
Burkina Faso. J Ethnopharmacol 2006; 104(1-2): 68-78.
[37] Liu R, Takayama S, Zheng Y, et al. Interaction of BAG-1 with retinoic acid receptor
and its inhibition of retinoic acid-induced apoptosis in cancer cells. J Biol Chem 1998;
273(27): 16985-92.
[38] Macia MJ, Garcia E, Vidaurre PJ. An ethnobotanical survey of medicinal plants
commercialized in the markets of La Paz and El Alto, Bolivia. J Ethnopharmacol 2005;
97(2): 337-50.
[39] Leonti M, Sticher O, Heinrich M. Antiquity of medicinal plant usage in two Macro-
Mayan ethnic groups (Mexico). J Ethnopharmacol 2003; 88(2-3): 119-24.
[40] Longuefosse JL, Nossin E. Medical ethnobotany survey in Martinique. J
Ethnopharmacol 1996; 53(3): 117-42.
[41] Estomba D, Ladio A, Lozada M. Medicinal wild plant knowledge and gathering
patterns in a Mapuche community from North-western Patagonia. J Ethnopharmacol
2006; 103(1): 109-19.
[42] Yin MH, Kang DG, Choi DH, Kwon TO, Lee HS. Screening of vasorelaxant activity of
some medicinal plants used in Oriental medicines. J Ethnopharmacol 2005; 99(1): 113-
7.
[43] Jaradat NA. Ethnopharmacological survey of natural products in Palestine. An-Najah
Univ J Res 2005; 19(1): 13-67.
[44] Uzun E, Sariyar G, Adsersen A, et al. Traditional medicine in Sakarya province
(Turkey) and antimicrobial activities of selected species. J Ethnopharmacol 2004; 95(2-
3): 287-96.
[45] Tuzlaci E, Tolon E. Turkish folk medicinal plants, part III: Sile (Istanbul). Fitoterapia
2000; 71(6): 673-85.
[46] Yesilada E, Honda G, Sezik E, et al. Traditional Medicine in Turkey. V. Folk Medicine
in the Inner Taurus Mountains. J Ethnopharmacol 1995; 46(3): 133-52.
158 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

[47] Ghorbani A. Studies on pharmaceutical ethnobotany in the region of Turkmen Sahra,


north of Iran - (Part 1): General results. J Ethnopharmacol 2005; 102(1): 58-68.
[48] Morehart CT, Eisenberg DTA. Prosperity, power, and change: Modeling maize at
Postclassic Xaltocan, Mexico. J Anthropol Archaeol 2010; 29(1): 94-112.
[49] Frei B, Baltisberger M, Sticher O, Heinrich M. Medical ethnobotany of the Zapotecs of
the Isthmus-Sierra (Oaxaca, Mexico): Documentation and assessment of indigenous
uses. J Ethnopharmacol 1998; 62(2): 149-65.
[50] Hernandez T, Canales M, Avila JG, et al. Ethnobotany and antibacterial activity of some
plants used in traditional medicine of Zapotitlan de Las Salinas, Puebla (Mexico). J
Ethnopharmacol 2003; 88(2-3): 181-8.
[51] Velazquez DV, Xavier HS, Batista JE, de Castro-Chaves C. Zea mays L. extracts
modify glomerular function and potassium urinary excretion in conscious rats.
Phytomedicine 2005; 12(5): 363-9.
[52] Bonzani RM, Oyuela-Caycedo A. The gift of the variation and dispersion of maize –
social and technological context in Amerindian societies. In: Staller JE, Tykot RH, Benz
BF, Eds. Histories of maize: Multidisciplinary approaches to the prehistory, linguistics,
biogeography, domestication, and evolution of maize. California: Left Coast Press
2006; pp. 343-56.
[53] Staller JE, Tykot RH, Benz BF. Histories of maize in Mesoamerica: multidisciplinary
approaches. California: Left Coast Press 2010.
[54] van Etten J. Molding maize: the shaping of a crop diversity landscape in the western
highlands of Guatemala. J Hist Geogr 2006; 32(4): 689-711.
[55] Machado CTT, Paterniani MLS. Origem, domesticação e difusão. In: Soares AC, Ed.
Milho crioulo: conservação e uso da biodiversidade. Rio de Janeiro: AS-PTA 1998;
pp.17-27. [Origin, domestication, and diffusion. In: Soares AC, Ed. Maize landrace:
conservation and use of the biodiversity. Rio de Janeiro: AS-PTA 1998; pp.17-27].
[56] Boyd M, Surette C, Nicholson BA. Archaeobotanical evidence of prehistoric maize
(Zea mays) consumption at the northern edge of the Great Plains. J Archaeol Sci 2006;
33(8): 1129-40.
[57] Piperno DR. A few kernels short of a cob: on the Staller and Thompson late entry
scenario for the introduction of maize into northern South America. J Archaeol Sci
2003; 30(7): 831-6.
[58] Cano JH, Volpato G. Herbal mixtures in the traditional medicine of Eastern Cuba. J
Ethnopharmacol 2004; 90(2): 293-316.
[59] Arnason T, Uck F, Lambert J, Hebda R. Maya medicinal plants of San Jose Succotz,
Belize. J Ethnopharmacol 1980; 2(4): 345-64.
[60] Stafford HA. Teosinte to maize — some aspects of missing biochemical and
physiological data concerning regulation of flavonoid pathways. Phytochemistry 1998
Set; 49(2): 285-93.
[61] Benz B, Perales H, Brush S. Tzeltal and Tzotzil farmer knowledge and maize diversity
in Chiapas, Mexico. Curr Anthropol 2007; 48(2): 289-300.
[62] Brush SB, Perales HR. A maize landscape: Ethnicity and agro-biodiversity in Chiapas
Mexico. Agric Ecosyst Environ 2007; 121(3): 211-21.
[63] Pedreschi R, Cisneros-Zevallos L. Phenolic profiles of Andean purple corn (Zea mays
L.). Food Chem 2007; 100(3): 956-63.
Maize (Zea mays L.) – An Ethnopharmacological Review 159

[64] Perry L. Starch analyses reveal the relationship between tool type and function: an
example from the Orinoco valley of Venezuela. J Archaeol Sci 2004; 31(8): 1069-81.
[65] Patterniani E, Nass LL, Xavier dos Santos M. O valor dos recursos genéticos de milho
para o Brasil: uma abordagem histórica da utilização do germoplasma. In: Udry CV,
Duarte W, Filho AB, Eds. Uma história brasileira do milho: o valor dos recursos
genéticos. Brasília: Paralelo 15 2000; pp. 11-42. [The value of maize genetic resources
for Brazil: a historical approach of the germplasm usage. In: Udry CV, Duarte W, Filho
AB, Eds. A Brazilian history of maize: the value of genetic resources. Brasília: Paralelo
15 2000; pp. 11-42].
[66] Felipim AP. O sistema agrícola guarani mbyá e seus cultivares de milho: um estudo de
caso na aldeia Guarani da Ilha do Cardoso, município de Cananéia. MSc dissertation.
Piracicaba: Universidade de São Paulo 2001. [The guarani mbyá crop system and its
maize cultivars: a study case in the Guarani village at Cardoso island, Cananéia county.
MSc dissertation. Piracicaba: University of São Paulo 2001].
[67] Bourdy G, Chavez de Michel LR, Roca-Coulthard A. Pharmacopoeia in a shamanistic
society: the Izoceno-Guarani (Bolivian Chaco). J Ethnopharmacol 2004; 91(2-3): 189-
208.
[68] George G. Stigmata maidis, or corn silk, in the treatment of vesical catarrh.: with notes
of two cases. Lancet 1885; 126(3244): 798-9.
[69] Kozan E, Kupeli E, Yesilada E. Evaluation of some plants used in Turkish folk
medicine against parasitic infections for their in vivo anthelmintic activity. J
Ethnopharmacol 2006; 108(2): 211-6.
[70] Du Dat D, Ham NN, Khac DH, et al. Studies on the individual and combined diuretic
effects of four vietnamese traditional herbal remedies (Zea mays, Imperata cylindrica,
Plantago major and Orthosiphon stamineus). J Ethnopharmacol 1992; 36(3): 225-31.
[71] Steenkamp V. Phytomedicines for the prostate. Fitoterapia 2003; 74(6): 545-52.
[72] Ebrahimzadeh MA, Pourmorad F, Hafezi S. Antioxidant Activities of Iranian Corn Silk.
Turk J Biol 2008; 32: 43-9.
[73] Maksimovic ZA, Kovacevic N. Preliminary assay on the antioxidative activity of
Maydis stigma extracts. Fitoterapia 2003; 74(1-2): 144-7.
[74] Rau O, Wurglics M, Dingermann T, Abdel-Tawab M, Schubert-Zsilavecz M. Screening
of herbal extracts for activation of the human peroxisome proliferator-activated
receptor. Pharmazie 2006; 61(11): 952-6.
[75] Liu J, Wang CN, Wang ZZ, Zhang C, Lu SA, Liu JB. The antioxidant and free-radical
scavenging activities of extract and fractions from corn silk (Zea mays L.) and related
flavone glycosides. Food Chem 2011; 126(1): 261-9.
[76] Kim KA, Choi SK, Choi HS. Corn silk induces nitric oxide synthase in murine
macrophages. Exp Mol Med 2004; 36(6): 545-50.
[77] Habtemariam S. Extract of corn silk (Stigma of Zea mays) inhibits the tumour necrosis
factor-alpha- and bacterial lipopolysaccharide-induced cell adhesion and ICAM-1
expression. Planta Med 1998; 64(4): 314-8.
[78] Pharmacopoea Europaea 7.0, Allgemeine Methoden, Pestizid-Rückstände. Deutscher
Apotheker Verlag, Stuttgart 2011; 1: 345–6.
[79] Rafsanjany N, Sendker J, Lechtenberg M, Petereit F, Scharf B, Hensel A. Traditionally
used medicinal plants against uncomplicated urinary tract infections: Are unusual,
flavan-4-ol- and derhamnosylmaysin derivatives responsible for the antiadhesive
160 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

activity of extracts obtained from stigmata of Zea mays L. against uropathogenic E. coli
and Benzethonium chloride as frequent contaminant faking potential antibacterial
activities? Fitoterapia 2015; 105:246-53.
[80] El-Ghorab A, El-Massry KF, Shibamoto T. Chemical composition of the volatile extract
and antioxidant activities of the volatile and nonvolatile extracts of Egyptian corn silk
(Zea mays L.). J Agric Food Chem 2007; 55(22): 9124-7.
[81] Pinheiro ACS, Pais AA, Tardivo ACB, Alves MJQF. Efeito do extrato aquoso de cabelo
de milho (Zea mays L.) sobre a excreção renal de água e eletrólitos e pressão arterial
em ratos Wistar anestesiados. Rev Bras plantas Med 2011; 13(4): 375-81. [Effect of the
aqueous extract of maize silks (Zea mays L,) on the renal excretion of water and
electrolytes and arterial pressure in anesthetized Wistar rats. Braz. J. Med. Plants 2011;
13(4): 375-81].
[82] Feeny PP. Plant apparency and chemical defense. In: Wallace JW; Mansell RL, editors.
Recent advances in phytochemistry. New York: Plenum Press 1976. pp. 1-40.
[83] Stepp JR, Moerman DE. The importance of weeds in ethnopharmacology. J
Ethnopharmacol 2001; 75(1): 19-23.
[84] Jaenicke H, Höschle-Zeledon I, Crops ICFU. Strategic framework for underutilized
plant species research and development: with special reference to Asia and the Pacific,
and to Sub-Saharan Africa: International Centre for Underutilised Crops, Colombo, Sri
Lanka and Global Facilitation Unit for Underutilized Species. Rome 2006.
[85] Food and Agriculture Organization of the United Nations. The state of food insecurity
in the World 2005: Eradicating World hunger - key to achieving the millennium
development goals. Rome 2005.
[86] Aman R, Carle R, Conrad J, Beifuss U, Schieber A. Isolation of carotenoids from plant
materials and dietary supplements by high-speed counter-current chromatography. J
Chromatogr A 2005; 1074(1-2): 99-105.
[87] Maksimovic Z, Malencic D, Kovacevic N. Polyphenol contents and antioxidant activity
of Maydis stigma extracts. Bioresource Technol 2005; 96(8):873-7.
[88] Ross JA, Kasum CM. Dietary flavonoids: Bioavailability, metabolic effects, and safety.
Annu Rev Nutr 2002; 22: 19-34.
[89] Curvelier ME, Richard H, Berst C. Comparison of antioxidant activity of some acid-
phenolic: structure-activity relationship. Biosc Biotech Biochem 1992; 56(2): 324-5.
[90] Rodriguez-Caso C, Rodriguez-Agudo D, Sanchez-Jimenez E, Medina MA. Green tea
epigallocatechin-3-gallate is an inhibitor of mammalian histidine decarboxylase. Cell
Mol Life Sci 2003; 60(8): 1760-3.
[91] Seo H-C, Suzuki M, Ohnishi-Kameyama M, et al. Extraction and identification of
antioxidant components from Artemisia capillaris herba. Plant Foods for Hum Nutr
2003; 58(3): 1-12.
[92] Grune T, Schröder P, Biesalski H. Low molecular weight antioxidants. In: Grune T, Ed.
Oxidants and antioxidant defense systems. Berlin: Springer Berlin Heidelberg 2005;
pp.77-90.
[93] Tsao R, Deng ZY. Separation procedures for naturally occurring antioxidant
phytochemicals. J Chromatogr B Analyt Technol Biomed Life Sci 2004; 812(1-2): 85-
99.
[94] Hagiwara A, Miyashita K, Nakanishi T, et al. Pronounced inhibition by a natural
anthocyanin, purple corn color, of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
Maize (Zea mays L.) – An Ethnopharmacological Review 161

(PhIP)-associated colorectal carcinogenesis in male F344 rats pretreated with 1,2-


dimethylhydrazine. Cancer Lett 2001; 171(1): 17-25.
[95] Bai H, Hai C, Xi M, Liang X, Liu R. Protective effect of maize silks (Maydis stigma)
ethanol extract on radiation-induced oxidative stress in mice. Plant Foods Hum Nutr
2010; 65(3): 271-6.
[96] Maggi-Capeyron MF, Ceballos P, Cristol JP, et al. Wine phenolic antioxidants inhibit
AP-1 transcriptional activity. J Agr Food Chem 2001; 49(11): 5646-52.
[97] King PJ, Ma G, Miao W, et al. Structure-activity relationships: analogues of the
dicaffeoylquinic and dicaffeoyltartaric acids as potent inhibitors of human
immunodeficiency virus type 1 integrase and replication. J Med Chem 1999; 42(3):
497-509.
[98] Rao CV, Desai D, Simi B, Kulkarni N, Amin S, Reddy BS. Inhibitory effect of caffeic
acid esters on azoxymethane-induced biochemical changes and aberrant crypt foci
formation in rat colon. Cancer Res 1993; 53(18): 4182-8.
[99] Jeong WS, Kim IW, Hu R, Kong ANT. Modulation of AP-1 by natural chemopreventive
compounds in human colon HT-29 cancer cell line. Pharm Res 2004; 21(4): 649-60.
[100] Zieliński H. Contribution of low molecular weight antioxidants to the antioxidant
screen of germinated soybean seeds. Plant Foods Hum Nutr 2003; 58(3): 1-20.
[101] Seeram NP, Adams LS, Henning SM, et al. In vitro antiproliferative, apoptotic and
antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract
are enhanced in combination with other polyphenols as found in pomegranate juice. J
Nutr Biochem 2005; 16(6): 360-7.
[102] Middleton EJ, Kandaswami C. The impact of plant flavonoids on mammalian biology:
implications of immunity, inflammation and cancer. In: Harborne JB, Ed. The
flavonoids. London: Chapman and Hall 1994; pp. 619-52.
[103] Rice-Evans C, Miller N, Paganga G. Antioxidant properties of phenolic compounds.
Trends Plant Sci. 1997; 2(4): 152-9.
[104] Sugihara N, Arakawa T, Ohnishi M, Furuno K. Anti- and pro-oxidative effects of
flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in
cultured hepatocytes loaded with alpha-linolenic acid. Free Radic Bio Med 1999;
27(11-12): 1313-23.
[105] Yang CS, Landau JM, Huang MT, Newmark HL. Inhibition of carcinogenesis by
dietary polyphenolic compounds. Annu Rev Nutr 2001; 21: 381-406.
[106] Orsolic N, Sver L, Terzic S, Tadic Z, Basic I. Inhibitory effect of water-soluble
derivative of propolis and its polyphenolic compounds on tumor growth and
metastasizing ability: a possible mode of antitumor action. Nutr Cancer 2003; 47(2):
156-63.
[107] Farsi DA, Harris CS, Reid L, et al. Inhibition of non-enzymatic glycation by silk
extracts from a Mexican land race and modern inbred lines of maize (Zea mays).
Phytother Res 2008; 22(1): 108-12.
[108] Guo JY, Liu TJ, Han LN, Liu YM. The effects of corn silk on glycaemic metabolism.
Nutr Metab (Lond) 2009; 6:47.
[109] Miao M, Sun Y, X. J, Liu H. Effects of saponin extracted from Zea mays L. (ZMLS) on
pathogenic diabetic mouse model caused by administration of alloxan and glucose.
China Journal of Traditional Chinese Medicine and Pharmacy. 2007; 22: 181-3.
162 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

[110] Miao MS, Miao YY, Ji XN, Liu HL. Effect of total saponin of stigmata maydis on
carbohydrate tolerance reducing. China Journal of Traditional Chinese Medicine and
Pharmacy 2009; 24: 1071-3.
[111] Zhao WZ, Yin YG, Yu ZP, Liu JB, Chen F. Comparison of anti-diabetic effects of
polysaccharides from corn silk on normal and hyperglycemia rats. Int J Biol Macromol
2012; 50(4): 1133-7.
[112] Hu QL, Zhang LJ, Li YN, Ding YJ, Li FL. Purification and anti-fatigue activity of
flavonoids from corn silk. Int J Phys Sci 2010; 5(4): 321-6.
[113] Žilić S, Vančetović J, Janković M, Maksimović V. Chemical composition, bioactive
compounds, antioxidant capacity and stability of floral maize (Zea mays L.) pollen. J
Func Foods 2014; 10: 65-74.
[114] Widstrom NW, Snook ME, Wilson DM, Cleveland TE, McMillan WW. Silk maysin
content and resistance of commercial corn [maize] hybrids to kernel contamination by
aflatoxin. J Sci Food Agric 1995; 67(3): 317-21.
[115] Hasanudin K, Hashim P, Mustafa S. Corn silk (Stigma maydis) in Healthcare: a
phytochemical and pharmacological review. Molecules 2012; 17(8): 9697-715.
[116] Snook ME, Widstrom NW, Wiseman BR, Byrne PF, Harwood JS, Costello CE. New C-
4''-Hydroxy derivatives of maysin and 3'-Methoxymaysin isolated from corn silks (Zea-
Mays). J Agr Food Chem 1995; 43(10): 2740-5.
[117] Ren SC, Liu ZL, Ding XL. Isolation and identification of two novel flavone glycosides
from corn silk (Stigma maydis). J Med Plants Res 2009; 3(12): 1009-15.
[118] Xu Y, Liang JY, Zhou ZM, Yang JS. A novel flavone and two urea glycosides from the
style of Zea mays L. Acta Chim Sinica 2008; 66(19): 1235-8.
[119] Wang Y, Liu YY, Yang XH, Chen D, Peng C, Wang GS. A new flavonoid from the bract
of Zea mays L. Chinese Chem Lett 2010; 21(11): 1350-1.
[120] Escribano-Bailon MT, Santos-Buelga C, Rivas-Gonzalo JC. Anthocyanins in cereals. J
Chromatogr A 2004; 1054(1-2): 129-41.
[121] Cortes GA, Salinas MY, San Martin-Martinez E, Martinez-Bustos F. Stability of
anthocyanins of blue maize (Zea mays L.) after nixtamalization of seperated pericarp-
germ tip cap and endosperm fractions. J Cereal Sci 2006; 43(1): 57-62.
[122] Aoki H, Kuze N, Kato Y. Anthocyanins isolated from purple corn (Zea mays L.). Food
Foods Ingredients J Jpn 2002; 199: 63-5.
[123] Cevallos-Casals BA, Cisneros-Zevallos L. Stoichiometric and kinetic studies of
phenolic antioxidants from Andean purple corn and red-fleshed sweetpotato. J Agr
Food Chem 2003; 51(11): 3313-9.
[124] Tsuda T, Shiga K, Ohshima K, Kawakishi S, Osawa T. Inhibition of lipid peroxidation
and the active oxygen radical scavenging effect of anthocyanin pigments isolated from
Phaseolus vulgaris L. Biochem Pharmacol 1996; 52(7): 1033-9.
[125] Gabrielska J, Oszmianski J, Komorowska M, Langner M. Anthocyanin extracts with
antioxidant and radical scavenging effect. Z Naturforsch C 1999; 54(5-6): 319-24.
[126] Yoshimoto M, Okuno S, Yoshinaga M, Yamakawa O, Yamagushi M, Yamada J.
Potential mechanism of cancer chemoprevention by anthocyanins. Biosc Biotech
Biochem 1999; 63: 358-62.
[127] Koide T, Hashimoto Y, Kamei H, Kojima T, Hasegawa M, Terabe K. Antitumor effect
of anthocyanin fractions extracted from red soybeans and red beans in vitro and in vivo.
Cancer Biother Radiopharm 1997; 12(4): 277-80.
Maize (Zea mays L.) – An Ethnopharmacological Review 163

[128] Fimognari C, Berti F, Nusse B, Cantelli-Forti G, Hrelia P. Induction of apoptosis in two


human leukemia cell lines as well as differentiation in human promyelocytic cells by
cyanidin-3-O-beta-glucopyranoside. Biochem Pharmacol 2004; 67(11): 2047-56.
[129] Hou DX, Tong X, Terahara N, Luo D, Fujii M. Delphinidin 3-sambubioside, a Hibiscus
anthocyanin, induces apoptosis in human leukemia cells through reactive oxygen
species-mediated mitochondrial pathway. Arch Biochem Biophys 2005; 440(1): 101-9.
[130] Kuhnen S, Ogliari JB, Dias PF, et al. Metabolic fingerprint of brazilian maize landraces
silk (stigma/styles) using NMR spectroscopy and chemometric methods. J Agr Food
Chem 2010; 58(4): 2194-200.
[131] Kuhnen S, Ogliari JB, Dias PF, et al. ATR-FTIR spectroscopy and chemometric
analysis applied to discrimination of landrace maize flours produced in southern Brazil.
Int J Food Sci Tech 2010; 45(8): 1673-81.
[132] Maraschin M, Kuhnen S, Lemos PMM, et al. Metabolomics and chemometrics as tools
for chemo(bio)diversity analysis - maize landraces and propolis. In: Varmuza K, editor.
Chemometrics in practical applications. Rijeka: InTech 2012; pp. 253-70.
[133] Uarrota VG. Perfil metabólico primário (proteínas, amido e lipídios) e secundário
(carotenóides, antocianinas e ácidos [poli]fenólicos) de grãos de oito variedades
crioulas de milho. MSc dissertation. Florianópolis: Universidade Federal de Santa
Catarina 2011. [Primary (protein, starch, and lipids) and secondary (carotenoids,
anthocyanins, and (poly)phenolics) metabolic profiles of eight maize landraces. MSc
dissertation. Florianópolis: Federal University of Santa Catarina 2011].
[134] Monroy YM, Rodrigues RAF, Sartoratto A, Cabral FA. Extraction of bioactive
compounds from cob and pericarp of purple corn (Zea mays L.) by sequential extraction
in fixed bed extractor using supercritical CO2, ethanol, and water as solvents. J
Supercritic Fluids 2016; 107: 250-9.
[135] Walton NJ, Brown DE. Chemicals from plants: perspectives on plant secondary
products. London: Imperial College Press 1999.
[136] Ahmed SS, Lott MN, Marcus DM. The macular xanthophylls. Surv Ophthalmol 2005;
50(2): 183-193.
[137] Bartley GE, Scolnik PA. Plant carotenoids: pigments for photoprotection, visual
attraction, and human health. Plant Cell 1995; 7(7): 1027-38.
[138] Howitt CA, Pogson BJ. Carotenoid accumulation and function in seeds and non-green
tissues. Plant Cell Environ 2006; 29(3): 435-45.
[139] Agadir A, Shealy YF, Hill DL, Zhang X. Retinyl methyl ether down-regulates activator
protein 1 transcriptional activation in breast cancer cells. Cancer Res 1997; 57(16):
3444-50.
[140] Lin BZ; Chen GQ; Xiao DM, et al. Orphan receptor COUP-TF is required for induction
of retinoic acid receptor beta, growth inhibition, and apoptosis by retinoic acid in cancer
cells. Mol Cell Biol 2000; 20(3): 957-70.
[141] Hashimoto Y. Structural development of synthetic retinoids and thalidomide-related
molecules. Cancer Chemother Pharmacol 2003; 52 Suppl 1:S16-23.
[142] Kuhnen S, Lemos PMM, Campestrini LH, Ogliari JB, Dias PF, Maraschin M.
Antiangiogenic properties of carotenoids: A potential role of maize as functional food. J
Funct Foods 2009; 1(3): 284-90.
164 Priscilla Maria Menel Lemos, Beatriz Veleirinho, Aline Pereira et al.

[143] Snodderly DM. Evidence for Protection against Age-Related Macular Degeneration by
Carotenoids and Antioxidant Vitamins. Am J Clin Nutr 1995; 62(6 Suppl): 1448S-
1461S.
[144] Wrona M, Rozanowska M, Czuba-Pelech B, Sarna T. Antioxidant action of zeaxanthin
in protection of human RPE cells against oxidative damage. Invest Ophth Vis Sci 2001;
42(4): S756-S756.
[145] Kuhnen S. Metabolomics and bioprospection of maize creole and landraces (Zea mays):
PhD dissertation. Florianópolis: Universidade Federal de Santa Catarina 2007.
[146] Chew BP, Park JS. Carotenoid action on the immune response. J Nutr 2004; 134(1):
257S-261S.
[147] Chew BP, Brown CM, Park JS, Mixter PF. Dietary lutein inhibits mouse mammary
tumor growth by regulating angiogenesis and apoptosis. Anticancer Res 2003 Jul-Aug;
23(4): 3333-9.
[148] Bai HC-X, Liang X, Zhao H-L, Miao-miao XI. Free radical scavenging and
antilipoperoxidant properties of maydis stigma extract. Teratogen Carcin Mut 2008; 20:
36-40.
[149] Zhang L, Ouyang W, Hu Y, Yang B, Bai K, Li H. Pathology observation of protection
effect of oxidation damage resistance on Bombyx mori by feeding extracts of maize
plumule. Acta Sericologica Sinica 2005; 31: 458-62.
[150] Sepehri G, Derakhshanfar A, Yazdi Zadeh F. Protective effects of corn silk extract
administration on gentamicin-induced nephrotoxicity in rat. Comp Clin Pathol 2011;
20: 89-94.
[151] Tang L, Ding X, You L, Gu W, Yu F. Bio-active substances from corn silk
polysaccharide (CSPS) and its immunological enhancing function. Journal of Wuxi
University of Light Industry 1995; 4: 319-24.
[152] Ji L, Fan Y. Study on bacteriostasis of maize silk extracts for food preservation. Food
Sci 2000; 21(12): 131-4.
[153] Ji L, Tan R. Antimirobial activities of maize silk extracts against food spoilage and
food-borne pathogens. Sheng Ming Ke Xue Yan Jiu 2001; 5(1): 68-72.
[154] Hong M, Ling G. Effects of extract of stigma maydis on K562 and SGC cells. Journal
of Nanjing University of Traditional Chinese Medicine 1998; 14.
[155] Lu DX, Wang XL, Wei FX, Luo JB, Zhang, Zhang SH. Stigma maydis polysaccharide
induces apoptosis of human hepatocellular carcinoma SMMC-7721 cells. Heilongjiang
Medicine and Pharmacy 2006; 29.
[156] Canci A, Vogt GA, Canci I. A diversidade das espécies crioulas em Anchieta-SC:
diagnóstico, resultado de pesquisa e outros apontamentos para a conservação da
agrodiversidade. São Miguel do Oeste: McLee 2004. [The diversity of creoule species
in Anchieta-SC: diagnosis, research outputs, and other notes for the agrobiodiversity
conservation. São Miguel do Oeste: McLee 2004].
[157] Ogliari JB, Alves AC. Manejo e uso de variedades de milho em comunidades de
agricultores de Anchieta como estratégia de conservação. In: De Boef W, Thijssen MH,
Sthapit B, Eds. Biodiversidade e agricultura: fortalecendo o manejo comunitário. Porto
Alegre: L&PM 2007; pp. 226-234. [Management and use of maize varieties in small
stakeholders communities at Anchieta county as strategy for conservation. In: De Boef
W, Thijssen MH, Sthapit B, Eds. Biodiversity and agriculture: strenghtening the
communitary management. Porto Alegre: L&PM 2007; pp. 226-234].
Maize (Zea mays L.) – An Ethnopharmacological Review 165

[158] Lemos PMM. Análise do metaboloma foliar parcial de variedades locais de milho (Zea
mays) e dos efeitos anti-tumoral in vitro e na morfogênese embrionária de Gallus
domesticus. PhD dissertation. Florianópolis: Universidade Federal de Santa Catarina
2010. {Analysis of the leaf partial metabolome of maize landraces and of the in vitro
antitumoral effect and on the morphogenesis of Gallus domesticus. PhD dissertation.
Florianópolis: Federal University of Santa Catarina 2010].
[159] Heywood VH. Ethnopharmacology, food production, nutrition and biodiversity
conservation: Towards a sustainable future for indigenous peoples. J Ethnopharmacol
2011; 137(1): 1-15.
[160] Jang MS, Cai EN, Udeani GO, et al. Cancer chemopreventive activity of resveratrol, a
natural product derived from grapes. Science 1997; 275(5297): 218-20.
[161] Morales R. Farmacologia y farmacognósia como fuentes de validación y contraste en
etnobotánica. Monografías del jardín botánico. Córdova: Universidad de Córdoba -
Servicio de Publicaciones 1996; pp. 93-98.
[162] German JB, Watkins SM, Fay LB. Metabolomics in practice: Emerging knowledge to
guide future dietetic advice toward individualized health. J Am Diet Assoc 2005;
105(9); 1425-32.
[163] Wu LW, Chiang YM, Chuang HC, et al. Polyacetylenes function as anti-angiogenic
agents. Pharm Res 2004; 21(11): 2112-9.
[164] Igura K, Ohta T, Kuroda Y, Kaji K. Resveratrol and quercetin inhibit angiogenesis in
vitro. Cancer Lett 2001; 171(1): 11-6.
[165] Lim SN, Cheung PCK, Ooi VE, Ang PO. Evaluation of antioxidative activity of
extracts from a brown seaweed, Sargassum siliquastrum. J Agr Food Chem 2002;
50(13): 3862-6.
[166] Dias PF, Siqueira JM, Jr., Vendruscolo LF, et al. Antiangiogenic and antitumoral
properties of a polysaccharide isolated from the seaweed Sargassum stenophyllum.
Cancer Chemother Pharmacol 2005; 56(4): 436-46.
[167] Camelini CM, Maraschin M, de Mendonca MM; Zucco C, Ferreira AG, Tavares LA.
Structural characterization of beta-glucans of Agaricus brasiliensis in different stages of
fruiting body maturity and their use in nutraceutical products. Biotechnol Lett 2005;
27(17): 1295-9.
[168] Schuldt EZ, Bet AC, Hort MA, et al. An ethyl acetate fraction obtained from a Southern
Brazilian red wine relaxes rat mesenteric arterial bed through hyperpolarization and
NO-cGMP pathway. Vasc Pharmacol 2005; 43(1): 62-8.
[169] Reddy L, Odhav B, Bhoola KD. Natural products for cancer prevention: a global
perspective. Pharmacol Ther 2003; 99(1): 1-13.
[170] Hulshof PJM, Kosmeijer-Schuil T, West CE, Hollman PCH. Quick screening of maize
kernels for provitamin A content. J Food Compos Anal 2007; 20(8): 655-61.
In: Advances in Natural Products Discovery ISBN: 978-1-53610-088-4
Editors: Ana Rita Gomes, Teresa Rocha-Santos et al. © 2017 Nova Science Publishers, Inc.

Chapter 5

ANTITUMOR SESTERTERPENOIDS

Lishu Wang1,2, Junfeng Wang1 and Yonghong Liu1, 3,


1
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology/Guangdong Key
Laboratory of Marine Materia Medica/RNAM Center for Marine Microbiology, South
China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China
2
Jilin Provincial Academy of Chinese Medicine Sciences, Changchun, China
3
South China Sea Bio-Resource Exploitation and
Utilization Collaborative Innovation Center

ABSTRACT
The sesterterpenoids are a group of pentaprenyl terpenoids whose structures are
derivable from geranylfarnesyl diphosphate. Although sesterterpenoids are a relatively
small group of terpenoids, their sources are widespread, and they have been isolated from
terrestrial fungi, lichens, higher plants, insects, and various marine organisms, especially
sponges. The structural conciseness and diverse bioactivity of sesterterpenoids have made
them attractive targets for both biomedical and synthetic purposes. This review covers the
literature published from 1986 to June 2015 with 116 citations. The emphasis is on
sesterterpenoids together with their antitumor activity, source organisms, and country of
origin, including linear, monocarbocyclic, bicarbocyclic, tricarbocyclic, tetracarbocyclic,
and miscellaneous sesterterpenoids.

Keywords: sesterterpenoids, antitumor, cytotoxicity

1. INTRODUCTION
The sesterterpenoids are a group of pentaprenyl terpenoids whose structures are derivable
from geranylfarnesyl diphosphate. Although sesterterpenoids are a relatively small group of


Corresponding author. Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea
Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510-301, China. E-mail:
yonghongliu@scsio.ac.cn.
168 Lishu Wang, Junfeng Wang and Yonghong Liu

terpenoids, their sources are widespread, and they have been isolated from terrestrial fungi,
lichens, higher plants, insects, and various marine organisms, especially sponges [1-5]. For
example, the terrestrial fungal metabolite ophiobolin was the first naturally occurring
sesterterpenoid identified in 1958. The structural conciseness and diverse bioactivity of
sesterterpenoids have made them attractive targets for both biomedical and synthetic
purposes. Sesterterpenoids exhibit diverse biological properties such as anti-inflammatory [6],
cytotoxic [7], anticancer [8-10], antimicrobial [11, 12], antitubercular [13, 14], and anti-
biofilm activities [15]. For example, the famous anti-inflammatory sesterterpenoid manoalide
was first isolated from the Pacific sponge Luffariella variablis. So far, only a few reviews
have dealt with the class of sesterterpenoids: “Sesterterpenoids” [1-5], “Heterocyclic
terpenes: linear furano- and pyrroloterpenoids” [16, 17], “Occurrence, biological activity and
synthesis of cheilanthane sesterterpenoids” [18], “Research advances in the biologically
active scalarane sesterterpenoid” [19], “Targeting cancer with sesterterpenoids: the new
potential antitumor drugs” [20], “Bioactive sesterterpenes and triterpenes from marine
sponges: occurrence and pharmacological significance” [21], and “Synthetic approaches
toward sesterterpenoids” [22]. This chapter covers the literature from 1986 to 2015 and is
devoted to the anticancer sesterterpenoids [1-5] and describes 265 sesterterpenoids from 116
articles.

2. LINEAR SESTERTERPENOIDS
The C25 highly branched isoprenoid alkenes 1-6 were isolated from cultures of the marine
diatom Haslea ostrearia (Simonsen) and Rhizosolenia setigera. 1-6 appear to possess
cytostatic effects against human lung cancer cells in vitro [23]. A norsesterterpene rhopaloic
acid A 7 was isolated from a marine sponge Rhopaloeides sp. 7 exhibited potent
cytotoxicities in vitro against human myeloid K-562 cells, human MOLT-4 leukemia cells,
and marine L1210 leukemia cells [24]. Aikupikoxide A 8 was isolated from the lipophilic
extract of the Red Sea sponge D. erythraenus, and has cytotoxic activity [25]. Cytotoxic
furanosesterterpenoids 9 and 10 were isolated from a Sarcotragus sp. [26, 27] The
norsesterterpenoid irciformonin I 11 was isolated from I. formosana (E. Taiwan), 11 was
found to inhibit peripheral blood mononuclear cell proliferation [28]. Two acyclic manoalide-
related sesterterpenoids hippolides A 12 and B 13 were isolated from the South China Sea
sponge Hippospongia lachne (Yongxing Is., China). 12 exhibited cytotoxicity against A549,
HeLa, and HCT-116 cell lines with IC50 values of 5.22×10-2, 4.80×10-2, and 9.78 μM, 13
showed moderate cytotoxicity against the HCT-116 cell line with IC50 value of 35.13 μM
[29]. Two C22 furanosesterterpenoids, 15-acetylirciformonin B 14 and 10-acetylirciformonin
B 15, were isolated from the sponge Ircinia sp (Orchid Is., Taiwan). 14 and 15 exhibited
significant cytotoxic activity against K562, DLD-1, HepG2, and Hep3B cancer cell lines [30].
Bioactivity-guided fractionation of the ethyl acetate extract of a marine sponge, Xestospongia
sp (Sikao Sea, Trang Province, Thailand), led to the isolation of a thiophene-S-oxide acyclic
sesterterpenoid 16, which showed weak cytotoxicity against Vero cells [31]. Furospinosulin-1
17, a marine sponge-derived furanosesterterpenoid, exhibited selective antiproliferative
activity against DU145 human prostate cancer cells under hypoxic conditions in
concentrations ranging from 1 to 100 μM. 17 also demonstrated antitumour activity at 10-50
mg/kg in oral administration to a mouse model inoculated with sarcoma S180 cells.
Antitumor Sesterterpenoids 169

Mechanistic analysis revealed that 17 suppresses transcription of the insulin-like growth


factor-2 gene (IGF-2), which is selectively induced under hypoxic conditions through
prevention of the binding of nuclear proteins to the Sp1 consensus sequence in the IGF-2
promoter region [32]. A concise synthesis of 17 has been developed, and some structurally
modified analogues were prepared. Biological evaluation of them revealed that the whole
chemical structure was important for the hypoxia-selective growth inhibitory activity of 17.
Among the compound that were prepared, the desmethyl analogue showed excellent hypoxia-
selective inhibitory activity similar to that of 17 and also exhibited in vivo anti-tumour
activity in oral administration [33]. A specimen of S. officinalis from La Caleta, Cádiz, Spain
contained two minor C21 furanoterpenes, the weakly cytotoxic furospongin-5 18 and
cyclofurospongin-2 19 [34]. A moderately cytotoxic norsesterterpenoid sarcotin N 20 was
isolated from a Korean Sarcotragus species [35]. Two C22 sesterterpenes irciformonins C 21
and D 22 have been isolated from the marine sponge Ircinia formosana, collected off the
coast of eastern Taiwan. 21 and 22 exhibited significant cytotoxicity against human colon
tumor cells [36].
Three norsesterterpenoids 23-25 isolated from an Okinawa Ircinia species, were found to
be moderately cytotoxic against KB cells [37]. Cytotoxic norsesterterpenoid sarcotin O 26 and
ent-kurospongin 27 were isolated from a Korean Sarcotragus species [35]. A
norsesterterpenoids sarcotin P 28 was isolated from a Korean Sarcotragus species [38]. Both
the 8S,21S,22S,23R and 8R,21S,22S,23R isomers of okinonellin B 29, which is a cytotoxic
and antispasmodic agent from Spongionella sp., have been synthesized but neither has the
same optical rotation as the natural product [39]. Cytotoxic furanosesterterpenes isopalinurin
30 was reisolated from a marine sponge Psammocinia sp. collected from Ulleung Island,
Korea [40]. Three cytotoxic furanosesterterpenoids sacotins A–C 31-33 were reported from a
specimen of Sarcotragus sp. collected at Cheju Island, Korea [41]. Cytotoxic
sesterterpenoids, epi-sacotin A 34, sarcotins F 35 and M 36, were isolated from same Korean
Sarcotragus species [42] epi-Sacotin F 37 [35] was found in two Korean Sarcotragus species.
Two cytotoxic trinorsesterterpenes sarcotins I 38 and J 39 [42] were isolated from Korean
sponge Sarcotragus sp. Two sesterterpenoids 40 and 41, isolated from an Okinawa Ircinia
species, were found to be moderately cytotoxic [37]. Ten cytotoxic furanosesterterpenes
psammocinins A1 42, A2 43, B 44, palinurin 45, isopalinurin 46, 7E,12E,18R,20Z-variabilin
47, 8E,13Z,18R,20Z)-strobilinin 48, 7E,13Z,18R,20Z-felixinin (also known as
7E,13Z,18R,20Z-variabilin) 49, 8Z,13Z,18R,20Z)-strobilinin 50, 7Z,13Z,18R,20Z-felixinin 51
were isolated from a Korean marine sponge Psammocinia sp. 42-51 were cytotoxic against a
small panel of five human tumor cell lines [40]. Cytotoxic bisfuranosesterterpenoids ircinins
1 52 and 2 53, sacotins D 54, E 55 [41], G 56, and H 57 [42] were reported from a specimen
of Sarcotragus sp. collected off Cheju Island, Korea. Ircinin-1 52 induces cell cycle arrest and
apoptosis in SK-MEL-2 human melanoma cells [43]. A cytotoxic sesterterpenoid
cacospongionolide D 58 was isolated from sponge Fasciospongia cavernosa from the Bay of
Naples [44]. An Acanthodendrilla species (Indonesia) provided the cytotoxic acantholide C
59 [45]. Extracts of the marine sponge Thorectandra sp. have been found to contain cytotoxic
luffarins R 60 and V 61 [46]. Cytotoxic pyrrolosesterterpenes sarcotrines A 62, B 64, C 66, D
68, epi sarcotrines A 63, B 65, and C 67 [35, 42] were isolated from the marine sponge
Sarcotragus sp..
170 Lishu Wang, Junfeng Wang and Yonghong Liu
Antitumor Sesterterpenoids 171
172 Lishu Wang, Junfeng Wang and Yonghong Liu

3. MONOCARBOCYCLIC SESTERTERPENOIDS
The diacarnoxides A 69 and B 70 were isolated from Diacarnus levii (Papua New
Guinea). 69 and 70 displayed cytotoxic properties and increased activity under hypoxic
conditions [47]. The cytotoxic aplysinoplides A–C 71-73 were isolated from Aplysinopsis
digitata (Oshima-shinsone, Kagoshima Pref., Japan) [48]. The extract of marine sponge
Hyrtios communis was found to inhibit activation of the transcription factor hypoxia-
Antitumor Sesterterpenoids 173

inducible factor-1 (HIF-1) in T47D human breast tumor cells. Bioassay-guided isolation led
to the identification of luffariellolide 74, 74 exhibited a significant level of cytotoxicity that
mirrored its HIF-1 inhibitory activity [49]. An Okinawan Luffariella species yielded two new
luffariolides H 75 and J 76 that were found to be cytotoxic [50]. An Acanthodendrilla species
(Indonesia) provided the cytotoxic acantholides A 77 and B 78 [45]. A new derivative of
manoalide, 24-n-propyl-O-manoalide 79 was isolated from Luffariella sp., showed significant
cytotoxicity against HCT-116 cell line by MTT assay [51]. The marine sponge Diacarnus cf.
spinopoculum has provided a series of compounds of the muqubilin 80 class (norsesterterpene
peroxides) or the nuapapuin class (norditerpene peroxides). These compounds were evaluated
for their cytotoxic properties using a soft agar assay system and the NCI’s 60 cell-line screen.
Compounds without peroxide functionality were active. Three cytotoxic norsesterterpenoids,
tasnemoxides A–C 81–83, have been isolated from a Red Sea D. erythraenus [52]. Specimens
of D. cf. spinopoculum from the Solomon Islands and Papua New Guinea yielded ent-
muqubilin A 84, ent-epimuqubilin A 85, nuapapuin B 86, epi-nuapapuin B 87, muqubilin B
88, and epi-muqubilin B 89, all of which were evaluated for cytotoxicity [53]. An
Acanthodendrilla sp. (Indonesia) provided the cytotoxic acantholides D 90 and E 91 [45]. The
unusual spirosesterterpenoids alotaketal A 92 and B 93 were isolated from a Hamigera sp
(Milne Bay, Papua New Guinea) and found to activate the cAMP cell signalling pathway
[54]. The closely related, moderately cytotoxic phorbaketals A-C 94-96 were isolated from
Phorbas sp (Gageo Is, Korea) [55]. The sesterterpenoid phorbaketal N 97 was isolated from a
marine sponge of the genus Phorbas. 97 showed potent cytotoxicity against human pancreas
cancer cells (IC50 = 11.4µM) [56].

4. BICARBOCYCLIC SESTERTERPENOIDS
Cacospongionolide B 98 has been isolated from the Adriatic sponge Fasciospongia
cavernosa, 98 shows cytotoxic activity [57]. Two norsesterterpene cyclic peroxides
mycaperoxide A 99 and B 100 have been isolated from a Thai sponge of the genus Mycale,
99 and B 100 exhibit significant cytotoxic activity [58]. A sponge from the Great Barrier
Reef, a Coscinoderma sp. has been to produce a cytotoxic and antibacterial sesterterpenoid
quinol, coscinoquinol 101 [59]. Two sesterterpenoids 102 and 103 were isolated from the
sponge Coscinoderma sp (Chuuk Is., Micronesia). 102 and 103 exhibited moderate
cytotoxicity against the K562 cell line [60]. Stoeba extensa (Japan) gave the cytotoxic
halisulfate 7 104 [61]. Two new sesterterpene sulfates, hipposulfate A 105 has been isolated
from an Okinawan sponge, Hippospongia cf. metachromia. Both compounds contain an
enolsulfate functionality. 105 showed moderate cytotoxicity [62]. A Palauan species of
Thorectandra yielded the cytotoxic thorectandrols A 106 and B 107 [63]. All compounds
were found to inhibit the protease activity of human RAS converting enzyme (hRCE1) and
are the first natural products reported with this activity. Three cytotoxic sesterterpenes
thorectandrols C–E 108-110 were isolated from a Thorectandra sp. collected in Palau [46].
The sponge Stoeba extensa (Japan) gave the cytotoxic furanosesterterpenoid shinsonefuran
111 [64]. Dysidea etheria from the Caribbean contained the sesterterpene, dysidiolide 112
that inhibited the cdc25A protein phosphatase [65]. The stereochemistries of sesterterpenes
cladocorans A 113 and B 114 have been revised to 115 and 116 respectively by total
174 Lishu Wang, Junfeng Wang and Yonghong Liu

synthesis [66], while preparation and testing of related stereoisomers indicated the series
exhibits cytotoxicity towards a panel of human tumor cell lines [67].
Antitumor Sesterterpenoids 175

The cytotoxic kohamaic acids A 117 and B 118 were isolated from an Ircinia sp. from
Okinawa [68]. Bilosespens A 119 and B 120, which were isolated as an inseparable mixture
from an Eritrean sample of Dysidea cinerea, are cytotoxic sesterterpenes having an
unprecedented carbon skeleton [69]. Kohamaic acid A 121 is a powerful DNA polymerase
inhibitor isolated from the Okinawan marine sponge Ircinia sp. A series of structurally
simplified analogs of 121 were synthesized with the aim of evaluating structure-activity
relationships [70]. Kohamaic acid A 121 derivatives can prevent the growth of human cancer
cells (promyelocytic leukemia cell line, HL-60) with the same activity as the inhibition of
mammalian pols [71].

5. TRICARBOCYCLIC SESTERTERPENOIDS
Cytotoxic a-hydroxybutenolides spongianolides A 122 and C 123 have been isolated
from a Spongia species collected off the coast of Florida [72]. Six sesterterpenoids 124-129
were isolated from the sponge Coscinoderma sp (Chuuk Is., Micronesia). 124-129 exhibited
moderate cytotoxicity against the K562 cell line [60]. Five isomalabaricane-derived natural
products globostelletins C–G 130-134 have been isolated from the marine sponge
Rhabdastrella globostellata (Hainan Is., China). The inhibitory activities of 130-134 against
human tumor cell lines were evaluated [73]. Cytotoxic isomalabaricane-type norterpenoids
jaspiferals C-F 135-138 were isolated from an Okinawan marine sponge Jaspis stellifera [74].
Chemical examination of the fungus Aspergillus ustus isolated from the Mediterranean
sponge Suberites domuncula yielded the five ophiobolin-type sesterterpenoids 139-143. All
compounds were evaluated for their cytotoxicity against the murine lymphoma cell line
L5178Y [75]. The novel agent ophiobolin O induces apoptosis and cell cycle arrest of MCF-7
cells through activation of MAPK signaling pathways [76]. Ophiobolin A, also exerts
anticancer activity through ion channel targeting [77]. Fusicoccin-A selectively induces
apoptosis in tumor cells after interferon- priming [78]. The purpose of this study was to
identify and characterize fungal natural products (NPs) with in vitro bioactivity towards
leukemia cells. We based our screening on a combined analytical and bio-guided approach of
LC-DAD-HRMS dereplication, explorative solid-phase extraction (E-SPE), and a co-culture
platform of CLL and stromal cells. A total of 289 fungal extracts were screened and we
tracked the activity to single compounds in seven of the most active extracts. The novel
ophiobolin U was isolated together with the known ophiobolins C, H, K as well as 6-
epiophiobolins G, K and N from three fungal strains in the Aspergillus section Usti.
Ophiobolins A 144, B 145, C 146, and K 147 displayed bioactivity towards leukemia cells
with induction of apoptosis at nanomolar concentrations. The remaining ophiobolins were
mainly inactive or only slightly active at micromolarconcentrations. Dereplication of those
ophiobolin derivatives possessing different activity in combination with structural analysis
allowed a correlation of the chemical structure and conformation with the extent of
bioactivity, identifying the hydroxy group at C3 and an aldehyde at C21, as well as the A/B-
cis ring structure, as indispensible for the strong activity of the ophiobolins [79].
176 Lishu Wang, Junfeng Wang and Yonghong Liu

Five ophiobolane sesterterpenes, ophiobolins P-T 148-152, 6-epi-21-O-


dihydroophiobolin G 153, 6-epi-ophiobolin G 154, and 6-epi-ophiobolin K 155, were isolated
from the acetone extract of the endolichenic fungus Ulocladium sp. by using OSMAC
method. Ophiobolin T 152 and 6-epi-ophiobolin G 154 exhibited the most potent cytotoxic
activity against HepG2 with IC50 of 0.24 and 0.37 µM, respectively. Compounds 148-155
Antitumor Sesterterpenoids 177

showed strong cytotoxicity against two cell lines including KB (human nasopharyngeal
carcinoma cell line) and HepG2 (human hepatocarcinoma cell line) in comparison with the
positive control etoposide. In particular, compounds 152 and 154 exhibited stronger cytotoxic
activities against HepG2 than etoposide with IC50 values of 0.24 and 0.37 µM, respectively
[80]. Cytotoxic sesterterpene 6-epi-ophiobolin N 156 was isolated from marine derived
fungus Emericella variecolor GF10 [81]. 6-epi-ophiobolin A 157, 3-anhydro-6-epi-
ophiobolin A 158, and ophiobolin I 159 were isolated from culture broth of Bipolaris sp. as
cytotoxic agents against human tumor cells. Both 6-epi-ophiobolin A 157 and 3-anhydro
derivatives 158 demonstrated significant cytotoxicity, the activity of ophiobolin I 159 was,
however, very weak compared with those of 6-epi-ophiobolin A 157 and etoposide [82].
Lintenolides F 160 and G 161 are two additional antiproliferative sesterterpenes from the
Caribbean sponge Cacospongia cf. linteiformis [83]. The Japanese nudibranch Chromodoris
inornata contained a cytotoxic sesterterpenes, inorolide C 162 [84]. Petrosaspongiolide L 163
was isolated as cytotoxic metabolite of Petrosaspongia nigra from New Caledonia [85]. The
sponge Hyatella intestinalis from the Gulf of California contains the new scalarane-related
sesterterpenes hyatolide A 164. Hyatolide A 164 has shown activity as growth inhibitors of
several tumor cell lines [86].

6. TETRACARBOCYCLIC SESTERTERPENOIDS
The scalarane sesterterpenes 12-deacetoxy-23-hydroxyscalaradial 165, 12-dehydroxy-23-
hydroxyhyrtiolide 166, 12-O-acetyl-16-deacetoxy-23-acetoxyscalarafuran 167, 12-deacetoxy-
23-hydroxyheteronemin 168, 12-deacetoxy-23-acetoxy-19-O-acetylscalarin 169, 12-
deacetoxy-23-O-acetoxyheteronemin 170, and 12-deacetoxyscalaradial 171 were isolated
from a Korean marine sponge, Psammocinia sp. 165-171 exhibited cytotoxicity against
intractable human cancer cell lines A498, ACHN, MIA-paca and PANC-1, with an IC50 range
of 0.4-48µM [87].
The scalarane sesterterpenoids eight 20,24-bishomoscalaranes, carteriofenones D–K 172-
179, were obtained from the marine sponge Carteriospongia foliascens collected from the
South China Sea. 172-179 showed cytotoxicity against the mouse lymphocytic leukemia cell
line (P388, HT29, and A549) [88]. Seven scalarane sesterterpenoids 180-186 were isolated
from the marine sponge Scalarispongia sp. 180-186 showed significant in vitro cytotoxicity
(GI50 values down to 5.2 µ M) against six human cancer cell lines [89]. Two cytotoxic
bishomoscalaranes 187 and 188 have been isolated from a Dictyoceratid sponge,
Strepsichordaia lendenfeldi [90]. Hyatella intestinalis (S. E. Queensland, Australia) yielded
the cytotoxic norsesterterpenoid mooloolabenes A-E 189-193 and sesterterpenoid
mooloolaldehyde 194 [91]. Sponge Phyllospongia papyracea collected in Hainan, China,
yielded cytotoxic 195 [92]. The cytotoxic scalarane-type sesterterpenoids 196 and 197 were
obtained from Hyrtios erectus (Kavieng, Papua New Guinea) [93]. A series of cytotoxic
scalarane sesterterpenoids 198-207 were obtained from a Smenospongia sp (Soheuksan Is.,
Korea) [94].
178 Lishu Wang, Junfeng Wang and Yonghong Liu
Antitumor Sesterterpenoids 179

The scalaranoid 208 was obtained from Hyrtios gumminae (Similan Is., Thailand). 208
was weakly cytotoxic [95]. The cytotoxic phyllofenones D 209 and E 210 were isolated from
Phyllospongia foliascens (Yongxing Is., China) [96]. Five sesterterpenoids 211-215 were
isolated from the sponge Hyatella sp (Soheuksan-do, Korea). 211-215 exhibited moderate
cytotoxicity [97]. The scalarane sesterterpenoid hippospongide B 216 was isolated from a
180 Lishu Wang, Junfeng Wang and Yonghong Liu

sponge Hippospongia sp (Tai-tung, Taiwan). 216 exhibited significant cytotoxicity against


DLD-1, HCT-116, T-47D, and K562 cancer cell lines [98]. Heteronemin 217, a spongean
sesterterpenoid, inhibits TNF -induced NF-κB activation through proteasome inhibition and
induces apoptotic cell death [99, 100]. Heteronemin is a bioactive marine sesterterpene
isolated fromthe sponge Hyrtios sp. Previous reports have shown that heteronemin possesses
anticancer activity. Here, heteronemin displayed cytotoxic effects against three human cancer
cell lines (A549, ACHN, and A498) and exhibited potent activity in A498 human renal
carcinoma cells, with an IC50 value of 1.57 µM by MTT assay and a GI50 value of 0.77 µM by
SRB assay. Heteronemin initiates apoptotic cell death by downregulating Bcl-2 and Bcl-xL
and upregulating Bax, leading to the disruption of the mitochondrial membrane potential and
the release of cytochrome c from the mitochondria. These effects were associated with the
activation of caspase-3/caspase-8/caspase-9, followed by PARP cleavage. Furthermore,
heteronemin inhibited the phosphorylation of AKT signaling pathway and ERK and activated
p38 and JNK. The specific inhibition of the p38 pathway by SB203580 or p38 siRNA
treatment reversed the heteronemin-induced cytotoxicity and apoptotic signaling.
Heteronemin also induced autophagy in A498 cells, and treatment with chloroquine
(autophagy inhibitor) or SP600125 (JNK inhibitor) inhibited autophagy and increased
heteronemin-induced cytotoxicity and apoptotic signaling. Taken together, this study
proposes a novel treatment paradigm in which the combination of heteronemin and autophagy
inhibitors leads to enhanced RCC cell apoptosis [101]. The scalaranes are amongst the most
common sesterterpenoids and were found in a number of marine sponges, particularly from
the order Dictyoceratida. They form a closely related series of compounds. In a number of
instances, these sesterterpenoids have also been isolated from a nudibranch that is associated
with a sponge, and hence the sesterterpenoid in the nudibranch may have a dietary origin.
Four cytotoxic scalarane sesterterpenes 218-221 were obtained from a Japanese specimen of
H. erecta [102]. A specimen of H. erecta from the Maldives contained the cytotoxic
sesterterpenes sesterstatins 1–5 222-226 [103, 104]. H. erecta collected from the Egyptian
Red Sea was found to contain salmahyrtisol B 227, 3-acetyl- and 19-acetyl-sesterstatin 228
and 229, all of which showed significant cytotoxicity in human cancer cell lines [105]. A
Spongia species collected in Japan yielded three cytotoxic pentacyclic sesterterpenoids 230-
232 [106].
A new scalarane-type pentacyclic sesterterpene, sesterstatin 6 233, was isolated from the
Republic of Maldives marine sponge H. erecta. The structure was elucidated by analyses of
HRMS and high-field 2-D NMR spectra. Sesterstatin 6 showed significant cancer cell growth
inhibition against murine P388 lymphocytic leukemia and a series of human tumor cell lines
and proved to be the most inhibitory of the series [107]. The sponge Hyatella intestinalis from
the Gulf of California contains the new scalarane-related sesterterpenes hyatelones A 234 and
B 235, hyatolide C 236. The compounds have shown activity as growth inhibitors of several
tumor cell lines [86]. The sponge Hyatella intestinalis from the Gulf of California contains the
new scalarane-related sesterterpene hyatelactam 237. Hyatelactam has shown activity as
growth inhibitors of several tumor cell lines [86]. Petrosaspongiolide K 238 was isolated as
cytotoxic metabolites of Petrosaspongia nigra from New Caledonia [85]. The sponge
Hyatella intestinalis from the Gulf of California contains deacetylnorscalaral B 239. 12-O-
deacetylnorscalaral B has shown activity as growth inhibitors of several tumor cell lines [86].
Scalarane-type sesterterpenes, PHCs 2–7 240-245, which have been isolated from a marine
spongePhyllospongia chondrodes, which was collected at Yaeyama Islands, Okinawa, Japan,
Antitumor Sesterterpenoids 181

increased hemoglobin production in human chronic myelogenous leukemia cell line K562.
These sesterterpenes were found to induce erythroid differentiation in K562 cells [108].
Strepsichordaia aliena from Indonesia contained honulactones A–D 246-249, were shown to
be cytotoxic [109, 110]. Biology and HRESIMS-guided screening of the dichloromethane
fraction of the marine sponge Phyllospongia lamellosa collected from the Red Sea resulted in
the isolation and characterization of scalarane sesterterpenes; phyllospongins B-E 250-253,
12α-acetoxy-24,25-epoxy-24-hydroxy-20,24-dimethylscalarane 254, 250-254 showed
cytotoxic activity against HCT-116 with compound 253 as potent as doxorubicin. 254 showed
cytotoxic activity against MCF-7 comparableto doxorubicin. All the isolated compounds were
less active against the HePG-2 cell line when compared to doxorubicin [111].
182 Lishu Wang, Junfeng Wang and Yonghong Liu

Hyrtios erecta collected from the Egyptian Red Sea was found to contain salmahyrtisol A
255, which showed significant cytotoxicity in human cancer cell lines [112]. A marine fungal
isolate, tentatively identified as Fusarium heterosporum, has been found to produce a series
of structurally novel sesterterpene polyols, the mangicols A–G 256-262. The mangicols,
which possess unprecedented spirotricyclic skeletal components, show only weak to modest
cytotoxicities toward a variety of cancer cell lines in in vitro testing [113]. Three novel
sesterterpenes, neomangicols A 263 and B 264 were isolated from the mycelial extract of a
marine fungus belonging within the genus Fusarium. The carbon skeleton of the
neomangicols is undescribed and constitutes a new class of C25 rearranged sesterterpenes. 263
Antitumor Sesterterpenoids 183

and 264 are cytotoxic against HCT-116 human colon carcinoma in in vitro evaluation, while
neomangicol B inhibits the growth of the Gram-positive bacterium Bacillus subtilus with
potency similar to that of the antibiotic gentamycin [114].

7. MISCELLANEOUS SESTERTERPENOIDS
Terpestacin 265 inhibited tumor angiogenesis by targeting UQCRB of mitochondrial
complex III and suppressing hypoxia-induced reactive oxygen species production and cellular
oxygen sensing [115]. 265 has been reported to have only modest antimicrobial activity,
suggesting that it is not an indiscriminate cytotoxin and may therefore be a useful lead
compound for the development of anticancer as well as anti-AIDS chemotherapeutics [116].

CONCLUSION
Sesterterpenoid are a small group of terpenoids showing a number of interesting
pharmacological properties. Interestingly, many sesterterpenoids from natural sources have
reported to exhibit strong cytotoxicities by inhibiting cancer cell proliferation and/or inducing
cell death. These sesterterpenoids are attracting more interest and may represent new
promising compounds in cancer therapy. Although many sesterterpenoids have reported to
exhibit significant cytotoxicities in vitro, few studies have provided insights into their
molecular targets and mechanisms. Thus, it is necessary to explore studies on signal
transduction involved in cancer pathways, the in vivo physiological roles and the systematic
structure–activity relationships of these compounds. Sesterterpenoids may use in combination
with other chemotherapeutic drugs to increase effectiveness and decrease doses of individual
compounds, therefore reducing side effects.
184 Lishu Wang, Junfeng Wang and Yonghong Liu

CONFLICTS OF INTEREST
The authors declare no conflict of interest.

REFERENCES
[1] Hanson JR. Sesterterpenoids. Nat Prod Rep 1986;3(2):123-32.
[2] Hanson JR. The sesterterpenoids. Nat Prod Rep 1992;9(5):481-9.
[3] Hanson JR. The sesterterpenoids. Nat Prod Rep 1996;13(6):529-35.
[4] Liu Y, Wang L, Jung JH, Zhang S. Sesterterpenoids. Nat Prod Rep 2007;24(6):1401-
29.
[5] Wang L, Yang B, Lin X-P, Zhou X-F, Liu Y. Sesterterpenoids. Nat Prod Rep
2013;30(3):455-73.
[6] Schumacher M, Juncker T, Schnekenburger M, Gaascht F, Diederich M. Natural
compounds as inflammation inhibitors. Genes Nutr 2011;6(2):89-92.
[7] Villa FA, Gerwick L. Marine natural product drug discovery: Leads for treatment of
inflammation, cancer, infections, and neurological disorders. Immunopharm Immunot
2010;32(2):228-37.
[8] Sithranga Boopathy N, Kathiresan K. Anticancer drugs from marine flora: an
overview. J Oncol 2010;2010:214186-.
[9] Jain R. Marine life: New hope for cancer drugs. Indian J Cancer 2009;46(3):243-4.
[10] Chakraborty C, Hsu CH, Wen ZH, Lin CS. Anticancer drugs discovery and
development from marine organisms. Curr Top Med Chem 2009;9(16):1536-45.
[11] Baquero F, Coque TM, De La Cruz F. Ecology and evolution as targets: The need for
novel eco-evo drugs and strategies to fight antibiotic resistance. Antimicrob Agents
Chemother 2011;55(8):3649-60.
[12] Laport MS, Santos OCS, Muricy G. Marine sponges: Potential sources of new
antimicrobial drugs. Curr Pharm Biotechnol 2009;10(1):86-105.
[13] Garcia A, Bocanegra-Garcia V, Palma-Nicolas JP, Rivera G. Recent advances in
antitubercular natural products. Eur J Med Chem 2012;49:1-23.
[14] Thengyai S, Maitarat P, Hannongbua S, Suwanborirux K, Plubrukarn A. Probing the
structural requirements for antitubercular activity of scalarane derivatives using 2D-
QSAR and CoMFA approaches. Monatsh Chem 2010;141(6):621-9.
[15] Stowe SD, Richards JJ, Tucker AT, et al. Anti-biofilm compounds derived from marine
sponges. Mar Drugs 2011;9(10):2010-35.
[16] Liu Y, Zhang S, Abreu PJM. Heterocyclic terpenes: linear furano- and
pyrroloterpenoids. Nat Prod Rep 2006;23(4):630-51.
[17] Wang B, Wang L, Li Y, Liu Y. Heterocyclic terpenes: linear furano- and
pyrroloterpenoids. Rsc Adv 2014;4(24):12216-34.
[18] Ungur N, Kulcitki V. Occurrence, biological activity and synthesis of cheilanthane
sesterterpenoids. Tetrahedron 2009;65(19):3815-28.
[19] Liu Y, Wang Z, Meng X, et al. Research advances in the biologically active scalarane
sesterterpenoid. J Chin Pharm Univ 2010;41(4):289-98.
Antitumor Sesterterpenoids 185

[20] Zhang C, Liu Y. Targeting cancer with sesterterpenoids: the new potential antitumor
drugs. J Nat Med 2015;69(3):255-66.
[21] Ebada SS, Lin W, Proksch P. Bioactive sesterterpenes and triterpenes from marine
sponges: Occurrence and pharmacological significance. Mar Drugs 2010;8(2):313-46.
[22] Hog DT, Webster R, Trauner D. Synthetic approaches toward sesterterpenoids. Nat
Prod Rep 2012;29(7):752-79.
[23] Rowland SJ, Belt ST, Wraige EJ, et al. Effects of temperature on polyunsaturation in
cytostatic lipids of Haslea ostrearia. Phytochemistry 2001;56(6):597-602.
[24] Ohta S, Uno M, Yoshimura M, Hiraga Y, Ikegami S. Rhopaloic acid A: A novel
norsesterterpene from a marine sponge, Rhopaloeides sp, which inhibits gastrulation of
starfish embryos. Tetrahedron Lett 1996;37(13):2265-6.
[25] Youssef DTA, Yoshida WY, Kelly M, Scheuer PJ. Cytotoxic cyclic norterpene
peroxides from a Red Sea sponge Diacarnus erythraenus. J Nat Prod
2001;64(10):1332-5.
[26] Barrow CJ, Blunt JW, Munro MHG. Autooxidation studies on the marine sesterterpene
tetronic acid, variabilin. J Nat Prod 1989;52(2):346-59.
[27] Barrow CJ, Blunt JW, Munro MHG, Perry NB. Variabilin and related-compounds from
a sponge of the genus Sarcotragus. J Nat Prod 1988;51(2):275-81.
[28] Shen Y-C, Shih P-S, Lin Y-S, et al. Irciformonins E-K, C(22)-trinorsesterterpenoids
from the sponge Ircinia formosana. Helv Chim Acta 2009;92(10):2101-10.
[29] Piao S-J, Zhang H-J, Lu H-Y, et al. Hippolides A-H, acyclic manoalide derivatives
from the marine sponge Hippospongia lachne. J Nat Prod 2011;74(5):1248-54.
[30] Su J-H, Tseng S-W, Lu M-C, et al. Cytotoxic C(21) and C(22) terpenoid-derived
metabolites from the sponge Ircinia sp. J Nat Prod 2011;74(9):2005-9.
[31] Pedpradab P, Suwanborirux K. A new acyclic thiophene sesterterpene from the Sikao
Bay sponge, Xestospongia sp. J Asian Nat Prod Res 2011;13(9):879-83.
[32] Arai M, Kawachi T, Setiawan A, Kobayashi M. Hypoxia-selective growth inhibition of
cancer cells by furospinosulin-1, a furanosesterterpene isolated from an Indonesian
marine sponge. ChemMedChem 2010;5(11):1919-26.
[33] Kotoku N, Fujioka S, Nakata C, et al. Concise synthesis and structure-activity
relationship of furospinosulin-1, a hypoxia-selective growth inhibitor from marine
sponge. Tetrahedron 2011;67(35):6673-8.
[34] Garrido L, Zubia E, Ortega MJ, Salva J. New furanoterpenoids from the sponge
Spongia officinalis. J Nat Prod 1997;60(8):794-7.
[35] Liu YH, Mansoor TA, Hong JK, et al. New cytotoxic sesterterpenoids and
norsesterterpenoids from two sponges of the genus Sarcotragus. J Nat Prod
2003;66(11):1451-6.
[36] Shen Y-C, Lo K-L, Lin Y-C, et al. Novel linear C-22-sesterterpenoids from sponge
Ircinia formosana. Tetrahedron Lett 2006;47(24):4007-10.
[37] Issa HH, Tanaka J, Higa T. New cytotoxic furanosesterterpenes from an okinawan
marine sponge, Ircinia sp. J Nat Prod 2003;66(2):251-4.
[38] He W, Lin X, Xu T, et al. A new norsesterterpenoid from the sponge species
Sarcotragus. Chem Nat Comd 2012;48(2):208-10.
[39] Schmitz WD, Messerschmidt NB, Romo D. A beta-lactone-based strategy applied to
the total synthesis of (8S,21S,22S,23R)- and (8R,21S,22S,23R)-okinonellin B. J Org
Chem 1998;63(7):2058-9.
186 Lishu Wang, Junfeng Wang and Yonghong Liu

[40] Choi K, Hong JK, Lee CO, et al. Cytotoxic furanosesterterpenes from a marine sponge
Psammocinia sp. J Nat Prod 2004;67(7):1186-9.
[41] Liu YH, Bae BH, Alam N, et al. New cytotoxic sesterterpenes from the sponge
Sarcotragus species. J Nat Prod 2001;64(10):1301-4.
[42] Liu YH, Hong JK, Lee CO, et al. Cytotoxic pyrrolo- and furanoterpenoids from the
sponge Sarcotragus species. J Nat Prod 2002;65(9):1307-14.
[43] Choi HJ, Choi YH, Yee SB, et al. Ircinin-1 induces cell cycle arrest and apoptosis in
SK-MEL-2 human melanoma cells. Molecular Carcinogenesis 2005;44(3):162-73.
[44] DeRosa S, DeGiulio A, Crispino A, Iodice C, Tommonaro G. Further bioactive
sesterterpenes from the tyrrhenian sponge Fasciospongia cavernosa. Nat Prod Lett
1997;10(4):267-74.
[45] Elkhayat E, Edrada RA, Ebel R, et al. New luffariellolide derivatives from the
Indonesian sponge Acanthodendrilla sp. J Nat Prod 2004;67(11):1809-17.
[46] Charan RD, McKee TC, Boyd MR. Thorectandrols C, D, and E, new sesterterpenes
from the marine sponge Thorectandra sp. J Nat Prod 2002;65(4):492-5.
[47] Dai J, Liu Y, Zhou Y-D, Nagle DG. Hypoxia-selective antitumor agents:
Norsesterterpene peroxides from the marine sponge Diacarnus levii preferentially
suppress the growth of tumor cells under hypoxic conditions. J Nat Prod
2007;70(1):130-3.
[48] Ueoka R, Nakao Y, Fujii S, van Soest RWM, Matsunaga S. Aplysinoplides A-C,
cytotoxic sesterterpenes from the marine sponge Aplysinopsis digitata. J Nat Prod
2008;71(6):1089-91.
[49] Li J, Du L, Kelly M, Zhou Y-D, Nagle DG. Structures and potential antitumor activity
of sesterterpenes from the marine sponge Hyrtios communis. J Nat Prod
2013;76(8):1492-7.
[50] Tsuda M, Endo T, Mikami Y, Fromont J, Kobayashi J. Luffariolides H and J, new
sesterterpenes from a marine sponge Luffariella species. J Nat Prod 2002;65(10):1507-
8.
[51] Zhou G-X, Molinski TF. Manoalide derivatives from a sponge, Luffariella sp. J Asian
Nat Prod Res 2006;8(1-2):15-20.
[52] Youssef DTA. Tasnemoxides A-C, new cytotoxic cyclic norsesterterpene peroxides
from the red sea sponge Diacarnus erythraenus. J Nat Prod 2004;67(1):112-4.
[53] Sperry S, Valeriote FA, Corbett TH, Crews P. Isolation and cytotoxic evaluation of
marine sponge-derived norterpene peroxides. J Nat Prod 1998;61(2):241-7.
[54] Forestier R, Merchant CE, De Voogd NJ, et al. Alotaketals A and B, sesterterpenoids
from the marine sponge Hamigera species that activate the cAMP cell signaling
pathway. Org Lett 2009;11(22):5166-9.
[55] Rho JR, Hwang BS, Sim CJ, et al. Phorbaketals A, B, and C, sesterterpenoids with a
spiroketal of hydrobenzopyran moiety isolated from the marine sponge Phorbas sp.
Org Lett 2009;11(24):5590-3.
[56] Lee Y, Wang W, Kim H, et al. Phorbaketals L-N, cytotoxic sesterterpenoids isolated
from the marine sponge of the genus Phorbas. Bioorg Med Chem Lett
2014;24(17):4095-8.
[57] deRosa S, Crispino A, deGiulio A, et al. Cacospongionolide B, a new sesterterpene
from the sponge Fasciospongia cavernosa. J Nat Prod 1995;58(11):1776-80.
Antitumor Sesterterpenoids 187

[58] Tanaka J, Higa T, Suwanborirux K, et al. Bioactive norsesterterpene 1,2-dioxanes from


a Thai sponge, Mycale sp. J Org Chem 1993;58(11):2999-3002.
[59] Alea GV, Carroll AR, Bowden BF. Coscinoquinol, a new cytotoxic sesterterpene from
a Dictyoceratid sponge, Coscinoderma sp. Aust J Chem 1994;47(1):191-4.
[60] Bae J, Jeon JE, Lee YJ, et al. Sesterterpenes from the tropical sponge Coscinoderma sp.
J Nat Prod 2011;74(8):1805-11.
[61] Fu X, Ferreira MLG, Schmitz FJ, Kelly M. Halisulfate 7, a new sesterterpene sulfate
from a sponge, Coscinoderma sp. J Nat Prod 1999;62(8):1190-1.
[62] Musman M, Ohtani, II, Nagaoka D, Tanaka J, Higa T. Hipposulfates A and B, new
sesterterpene sulfates from an Okinawan sponge, Hippospongia cf. metachromia. J Nat
Prod 2001;64(3):350-2.
[63] Charan RD, McKee TC, Boyd MR. Thorectandrols A and B, new cytotoxic
sesterterpenes from the marine sponge Thorectandra species. J Nat Prod
2001;64(5):661-3.
[64] Phuwapraisirisan P, Matsunaga S, van Soest RWM, Fusetani N. Shionefuran, a
cytotoxic furanosesterterpene with a novel carbon skeleton, from the deep-sea sponge
Stoeba extensa. Tetrahedron Lett 2004;45(10):2125-8.
[65] Gunasekera SP, McCarthy PJ, KellyBorges M, Lobkovsky E, Clardy J. Dysidiolide: A
novel protein phosphatase inhibitor from the Caribbean sponge Dysidea etheria de
Laubenfels. J Am Cheml Soc 1996;118(36):8759-60.
[66] Miyaoka H, Yamanishi M, Kajiwara Y, Yamada Y. Total synthesis of cladocorans A
and B: A structural revision. J Org Chem 2003;68(9):3476-9.
[67] Marcos IS, Pedrero AB, Sexmero MJ, et al. Synthesis of bioactive sesterterpenolides
from ent-halimic acid. 15-epi-ent-cladocoran A and B. J Org Chem 2003;68(19):7496-
504.
[68] Kokubo S, Yogi K, Uddin MJ, et al. Kohamaic acids A and B, novel cytotoxic
sesterterpenic acids, from the marine sponge Ircinia sp. Chem Lett 2001(2):176-7.
[69] Rudi A, Yosief T, Schleyer M, Kashman Y. Bilosespens A and B: Two novel cytotoxic
sesterpenes from the marine sponge Dysidea cinerea. Org Lett 1999;1(3):471-2.
[70] Takikawa H, Kamatani N, Nakanishi K, et al. Synthetic studies on kohamaic acids:
synthesis of structurally simplified analogs of kohamaic acid A. Biosci Biotechnol
Biochem 2008;72(11):3071-4.
[71] Mizushina Y, Manita D, Takeuchi T, et al. The inhibitory action of kohamaic acid a
derivatives on mammalian DNA polymerase beta. Molecules 2009;14(1):102-21.
[72] He HY, Kulanthaivel P, Baker BJ. New cytotoxic sesterterpenes from the marine
sponge Spongia sp. Tetrahedron Lett 1994;35(39):7189-92.
[73] Li J, Xu B, Cui J, et al. Globostelletins A-I, cytotoxic isomalabaricane derivatives from
the marine sponge Rhabdastrella globostellata. Bioorg Med Chem 2010;18(13):4639-
47.
[74] Kobayashi J, Yuasa K, Kobayashi T, Sasaki T, Tsuda M. Jaspiferals A-G, new
cytotoxic isomalabaricane-type nortriterpenoids from Okinawan marine sponge Jaspis
stellifera. Tetrahedron 1996;52(16):5745-50.
[75] Liu HB, Edrada-Ebel R, Ebel R, et al. Ophiobolin sesterterpenoids and pyrrolidine
alkaloids from the sponge-derived fungus Aspergillus ustus. Helv Chim Acta
2011;94(4):623-31.
188 Lishu Wang, Junfeng Wang and Yonghong Liu

[76] Yang T, Lu Z, Meng L, et al. The novel agent ophiobolin O induces apoptosis and cell
cycle arrest of MCF-7 cells through activation of MAPK signaling pathways. Bioorg
Med Chem Lett 2012;22(1):579-85.
[77] Bury M, Girault A, Wauthoz N, et al. Ophiobolin A, a fungal phytotoxin, exerts
anticancer activity through ion channel targeting. Ann Oncol 2011;22:34-.
[78] De Vries-van Leeuwen IJ, Kortekaas-Thijssen C, Nzigou Mandouckou JA, et al.
Fusicoccin-A selectively induces apoptosis in tumor cells after interferon-alpha
priming. Cancer Lett 2010;293(2):198-206.
[79] Bladt TT, Duerr C, Knudsen PB, et al. Bio-activity and dereplication-based discovery
of ophiobolins and other fungal secondary metabolites targeting leukemia cells.
Molecules 2013;18(12):14629-50.
[80] Wang Q-X, Bao L, Yang X-L, et al. Ophiobolins P-T, five new cytotoxic and
antibacterial sesterterpenes from the endolichenic fungus Ulocladium sp. Fitoterapia
2013;90:220-7.
[81] Wei HX, Jasoni RL, Shao HW, Hua JL, Pare PW. Anti-selective and regioselective
aldol addition of ketones with aldehydes using MgI2 as promoter. Tetrahedron
2004;60(51):11829-35.
[82] Ahn JW, Lee MK, Choi SU, Lee CO, Kim BS. Cytotoxic ophiobolins produced by
Bipolaris sp. J Microbiol Biotechnol 1998;8(4):406-8.
[83] Carotenuto A, Fattorusso E, Lanzotti V, et al. Antiproliferative sesterterpenes from the
Caribbean sponge Cacospongia cf. linteiformis. Compar Biochem Physiol C-
Pharmacol Toxicol Endocrinol 1998;119(2):119-23.
[84] Miyamoto T, Sakamoto K, Amano H, et al. New cytotoxic sesterterpenoids from the
nudibranch Chromodoris inornata. Tetrahedron 1999;55(30):9133-42.
[85] Paloma LG, Randazzo A, Minale L, Debitus C, Roussakis C. New cytotoxic
sesterterpenes from the New Caledonian marine sponge Petrosaspongia nigra
(Bergquist). Tetrahedron 1997;53(30):10451-8.
[86] Hernandez-Guerrero CJ, Zubia E, Ortega MJ, Carballo JL. Sesterterpene metabolites
from the sponge Hyatella intestinalis. Tetrahedron 2006;62(23):5392-400.
[87] Hahn D, Won DH, Mun B, et al. Cytotoxic scalarane sesterterpenes from a Korean
marine sponge Psammocinia sp. Bioorg Med Chem Lett 2013;23(8):2336-9.
[88] Cao F, Wu Z-H, Shao C-L, et al. Cytotoxic scalarane sesterterpenoids from the South
China Sea sponge Carteriospongia foliascens. Org Biomol Chem 2015;13(13):4016-24.
[89] Lee Y-J, Lee J-W, Lee D-G, et al. Cytotoxic sesterterpenoids isolated from the marine
sponge Scalarispongia sp. Int J Mol Sci 2014;15(11):20045-53.
[90] Bowden BF, Coll JC, Li HT, et al. New cytotoxic scalarane sesterterpenes from the
Dictyoceratid sponge Strepsichordaia lendenfeldi. J Nat Prod 1992;55(9):1234-40.
[91] Somerville MJ, Hooper JNA, Garson MJ. Mooloolabenes A-E, norsesterterpenes from
the Australian sponge Hyattella intestinalis. J Nat Prod 2006;69(11):1587-90.
[92] Lan W-J, Li H-J. New sesterterpenoids from the marine sponge Phyllospongia
papyracea. Helv Chim Acta 2007;90(6):1218-22.
[93] Diaz-Marrero AR, Matainaho T, van Soest R, Roberge M, Andersen RJ. Scalarane-
based metabolites isolated from the antimitotic extract of the marine sponge Hyrtios
erectus. Nat Prod Res 2008;22(15):1304-9.
[94] Song J, Jeong W, Wang N, et al. Scalarane sesterterpenes from the sponge
Smenospongia sp. J Nat Prod 2008;71(11):1866-71.
Antitumor Sesterterpenoids 189

[95] Mahidol C, Prawat H, Sangpetsiripan S, Ruchirawat S. Bioactive scalaranes from the


Thai sponge Hyrtios gumminae. J Nat Prod 2009;72(10):1870-4.
[96] Zhang H-J, Fang H-F, Yi Y-H, Lin H-W. Scalarane sesterterpenes from the Chinese
sponge Phyllospongia foliascens. Helv Chim Acta 2009;92(4):762-7.
[97] Jeon J, Bae J, Lee KJ, Oh K-B, Shin J. Scalarane sesterterpenes from the sponge
Hyatella sp. J Nat Prod 2011;74(4):847-51.
[98] Chang Y-C, Tseng S-W, Liu L-L, et al. Cytotoxic sesterterpenoids from a sponge
Hippospongia sp. Mar Drugs 2012;10(5):987-97.
[99] Schumacher M, Cerella C, Eifes S, et al. Heteronemin, a spongean sesterterpene,
inhibits TNF alpha-induced NF-kappa B activation through proteasome inhibition and
induces apoptotic cell death. Biochem Pharmacol 2010;79(4):610-22.
[100] Schumacher M, Cerella C, Eifes S, et al. Heteronemin, a spongean sesterterpene,
inhibits TNF alpha-induced NF-kappa B activation through proteasome inhibition and
induces apoptotic cell death (vol 79, pg 610, 2010). Biochem Pharmacol
2010;79(12):1837-.
[101] Wu S-Y, Sung P-J, Chang Y-L, Pan S-L, Teng C-M. Heteronemin, a spongean
sesterterpene, induces cell apoptosis and autophagy in human renal carcinoma cells.
Biomed Res Int 2015.
[102] Tsuchiya N, Sato A, Hata T, et al. Cytotoxic scalarane sesterterpenes from a sponge,
Hyrtios erecta. J Nat Prod 1998;61(4):468-73.
[103] Pettit GR, Tan R, Melody N, et al. Antineoplastic agents 397: Isolation and structure of
sesterstatins 4 and 5 from Hyrtios erecta (the Republic of Maldives). Bioorganic and
Medicinal Chemistry Letters 1998;8(16):2093-8.
[104] Pettit GR, Cichacz ZA, Tan R, et al. Antineoplastic agents. 386. Isolation of
sesterstatins 1-3 from the marine sponge Hyrtios erecta. J Nat Prod 1998;61(1):13-6.
[105] Roy MC, Tanaka J, de Voogd NJ, Higa T. New scalarane class sesterterpenes from an
Indonesian sponge, Phyllospongia sp. J Nat Prod 2002;65(12):1838-42.
[106] Tsukamoto S, Miura S, van Soest RWM, Ohta T. Three new cytotoxic sesterterpenes
from a marine sponge Spongia sp. J Nat Prod 2003;66(3):438-40.
[107] Pettit GR, Tan R, Cichacz ZA. Antineoplastic agents. 542. Isolation and structure of
sesterstatin 6 from the Indian ocean sponge Hyrtios erecta. J Nat Prod
2005;68(8):1253-5.
[108] Aoki S, Higuchi K, Isozumi N, et al. Differentiation in chronic myelogenous leukemia
cell K562 by spongean sesterterpene. Biochem Biophys Res Commun 2001;282(2):426-
31.
[109] Jimenez JI, Yoshida WY, Scheuer PJ, et al. Honulactones: New bishomoscalarane
sesterterpenes from the Indonesian sponge Strepsichordaia aliena. J Org Chem
2000;65(21):6837-40.
[110] Jimenez JI, Yoshida WY, Scheuer PJ, Kelly M. Scalarane-based sesterterpenes from an
Indonesian sponge Strepsichordaia aliena. J Nat Prod 2000;63(10):1388-92.
[111] Hassan MHA, Rateb ME, Hetta M, et al. Scalarane sesterterpenes from the Egyptian
Red Sea sponge Phyllospongia lamellosa. Tetrahedron 2015;71(4):577-83.
[112] Youssef DTA, Yamaki RK, Kelly M, Scheuer PJ. Salmahyrtisol A, a novel cytotoxic
sesterterpene from the Red Sea sponge Hyrtios erecta. J Nat Prod 2002;65(1):2-6.
190 Lishu Wang, Junfeng Wang and Yonghong Liu

[113] Renner MK, Jensen PR, Fenical W. Mangicols: Structures and biosynthesis of a new
class of sesterterpene polyols from a marine fungus of the genus Fusarium. J Org
Chem 2000;65(16):4843-52.
[114] Renner MK, Jensen PR, Fenical W. Neomangicols: Structures and absolute
stereochemistries of unprecedented halogenated sesterterpenes from a marine fungus of
the genus Fusarium. J Org Chem 1998;63(23):8346-54.
[115] Jung HJ, Shim JS, Lee J, et al. Terpestacin inhibits tumor angiogenesis by targeting
UQCRB of mitochondrial complex III and suppressing hypoxia-induced reactive
oxygen species production and cellular oxygen sensing. J Biol Chem
2010;285(15):11584-95.
[116] Rezanka T, Siristova L, Sigler K. Antiviral sesqui-, di- and sesterterpenes. Anti-
infective Agents Med Chem 2009;8(3):169-92.
In: Advances in Natural Products Discovery ISBN: 978-1-53610-088-4
Editors: Ana Rita Gomes, Teresa Rocha-Santos et al. © 2017 Nova Science Publishers, Inc.

Chapter 6

RECENT STUDIES OF POLAR STEROIDS FROM


STARFISH: STRUCTURES, BIOLOGICAL ACTIVITIES
AND BIOSYNTHESIS

N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko


and V. A. Stonik
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of Russian
Academy of Sciences, Vladivostok, Russia

ABSTRACT
Starfish contain a great number of polar natural products, especially steroids, many
of which have no counterpart in the entire animal kingdom. This review covers the
starfish polar steroid compounds described since 2008. The emphasis is made on new
structures, taxonomic distribution, biological activities and biosynthesis of these
compounds.
The analysis of information, reported during last years, suggests the studies on
starfish polar steroids are still in progress. More then 70 new polar steroids were isolated
from over 30 starfish species, some of them have rare or unique structural features. A
new data about anticancer, immunomodulatory, anti-inflammatory, neuritogenic and
neuroprotective activities of these compounds, including mechanisms of actions, were
obtained. Some synthesis of biological active polyhydroxysteroids and asterosaponins has
been developed for the first time. A metabolite profiling of starfish polar steroids was
investigated for the first time by LC-ESI MS/MS approach with new information
concerning structures, content and biosynthesis of polar steroids obtained. For the first
time it was experimentally established that the dietary cholesterol and cholesterol sulfate
are biosynthetic precursors of polyhydroxysteroids and related low molecular weight
glycosides in starfish.


Corresponding author. G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of Russian
Academy of Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, Russia. E-mail:
ivanchina@piboc.dvo.ru.
192 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

Keywords: starfish, polar steroids, polyhydroxysteroids, steroid glycosides, asterosaponins,


cyclic glycosides, biological activities, biological functions, biosynthesis, metabolite
profiling

INTRODUCTION
Starfish, belonging to the class Asteroidea (phylum Echinodermata), contain a wide
diversity of polar steroids with unique structural features that are products of unusual
biosyntheses. These substances usually present in very complicated mixtures which are
difficult to separate into individual compounds. Starfish steroid metabolites, especially
glycosides, have been reported to show a wide spectrum of biological activities, including
cytotoxic, antiviral, antibacterial, antibiofouling, neuritogenic, antifungal, cancer preventive,
anti-inflammatory and other effects [1–6].
On the basis of their chemical structures, the starfish polar steroids may be subdivided
into polyhydroxysteroids, related mono- and biosides (rare trioside), oligoglycosides named
as asterosaponins and rare glycosides with cyclic carbohydrate chains. Polyhydroxysteroids
are compounds containing from four to nine hydroxyl groups in steroid nucleus and side
chains. Related glycosides are substances having a polyhydroxylated steroid nucleus and, as a
rule, one or two monosaccharides units which are attached to steroid moiety, to side chains or
to steroid nucleus and side chain simultaneously. These compounds are found in the both
sulfated and non-sulfated forms. Asterosaponins are steroid oligoglycosides, containing
3,6-dihydroxysteroid aglycone with a 9(11)-double bond, a sulfate group at C-3 and a
carbohydrate chain, consisting, as a rule, of four to six sugar units attached to C-6 of
aglycone. Cyclic glycosides, so far found only in two species of the genus Echinaster, have a
trisaccharide chain cyclized between C-3 and C-6 of the 7-3,6-dihydroxysteroid aglycone.
Polar steroid compounds like starfish polar steroids were not found in other classes of
echinoderms (Crinoidea, Ophiuroidea, Echinoidea, and Holothurioidea), so these compounds
are taxonomic markers of class Asteroidea.
Studies on polar steroids from starfish carried out before 1993 were reviewed by Minale
et al. [1]. The corresponding data published over next years were discussed in the reviews by
us [2, 4, 5], Iorizzi et al. [3], and Dong et al. [6], some last data about bioactive compounds
from echinoderms were described by Gomes et al. [7]. This chapter presents the literature
data concerning the studies of structures, biological activities and biosynthesis of polar
steroids from starfish covering the period 2008-2015.

ISOLATION PROCEDURES
The isolation of the polar steroids is a rather difficult experimental task because these
compounds usually occur only in minute amounts and present in animals as complex mixtures
of close related substances. To obtain individual compounds usually used a low pressure
chromatography on Amberlite XAD-2, Polychrom-1, Sephadex LH-20, florisil, silica gel and
a high pressure liquid chromatography (HPLC). For example Malyarenko et al. applied
following isolation procedures in the separation of twelve compounds from the starfish
Asteropsis carinifera [8]. The fresh animals were cut into small pieces and extracted twice
Recent Studies of Polar Steroids from Starfish 193

with EtOH at 20C. The H2O/EtOH layer was evaporated, and the residue was dissolved
in H2O. The H2O-soluble materials were passed through an Amberlite XAD-2 column,
eluted with distilled H2O until a negative chloride ion reaction was obtained, and eluted
with EtOH. The combined EtOH eluate was evaporated to yield a brownish material.
This total fraction was chromatographed on a Si gel column using CHCl3/EtOH (stepwise
gradient, 4:1 to 1:6) to yield ten fractions, 110, which were then analyzed by TLC on
Si gel plates in the eluent system BuOH/EtOH/H2O (4:1:2). Fractions 3 and 4 mainly
contained the free polyhydroxysteroids, and fractions 58 mainly contained the glycosides of
polyhydroxysteroids. Fractions 58 were further purified on a florisil column using
CHCl3/EtOH (stepwise gradient, 5:1 to 1:2) to yield ten subfractions. HPLC separation of
these subfractions on a Diasfer-110-C18 column with EtOH/H2O (65:35) as an eluent system
and further HPLC separation of obtained subsubfractions on a Discovery C18 column with
MeOH/H2O/1 M NH4OAc (75:24:1) as an eluent system yielded pure six new cariniferosides
A–F and six previously known glycosides [8].

OH

H H
+Na-O SO
3
H

H3C O
O
H HO
HO O H OH
O H
H3C H O H
HO O
HO H H
HO O O
H
O H
H3C
H OH
H3C H
H OH
HO HO HO
H
O
H
O

H NOE
H OH
13C)
HO HMBC (1H
HO H

Figure 1. Key NOESY and НМВС correlations of carbohydrate chains of hippasterosides A-D.

STRUCTURE ELUCIDATION
Structures of new starfish polar steroids usually were established by extensive NMR and
MS techniques, and chemical transformations. The application of 1D and 2D NMR
experiments, including DEPT, 1H-1H COSY, HSQC, HMBC, NOESY, ROESY, H2BC, and
1D TOCSY, allowed the assignment of all the carbon and proton chemical shifts. As an
194 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

example, the deducing of the attachment of the carbohydrate chain to steroid aglycone and
positions of interglycosidic linkages of hippasterosides A–D was done from long-range
correlations in the NOESY and HMBC spectra (Figure 1) [9]. Acid hydrolysis of glycosides
with 2 M TFA and alcoholysis of the mixture of sugars by L()-2-octanol, followed by
acetylation, GC analysis and comparison with the corresponding derivatives of standard
monosaccharides were carried out to ascertain the stereochemical series of its
monosaccharide residues.
Determination of absolute stereochemistry is a key stage of natural products structure
elucidation. One of the widespread approaches to solve this problem is the chemical
transformation of the compound with asymmetric reagents followed by analysis of the NMR
spectra of the obtained derivatives. The most frequently Mosher’s method basing on
comparison of the NMR spectra of (R)- and (S)-2-methoxy-2-phenyl-2-(trifluoromethyl)-
acetates (MTPA-esters), products of the reaction of hydroxy group in chiral center with
MTPA-chlorides is used [10–12]. However, in the cases when compound is obtained in minor
amount, for example less than 1 mg, the application of the traditional Mosher’s method is
either not possible or rather difficult.
Recently Kicha et al. propose a method for assignment of C-23 absolute configuration in
(20R)-23-hydroxycholestane side chains of steroid compounds by extensive 1H and 13C NMR
spectroscopy based on a comparison of the spectra with those of model compounds [13]. The
model (20R,23R)- and (20R,23S)-3,6,23-trihydroxy-5-cholest-9(11)-enes (2a and 2b)
were synthesized from known aglycone of asterosaponins isomarthasterone (1) (Figure 2), the
absolute configuration of C-23 in their side chain were established by Mosher’s method. 1H-
1H COSY-45, HSQC, HMBC and NOESY experiments led to the assignment of all proton

and carbon signals of these steroids in the two solvents, C5D5N and СD3OD, which are
usually used for the study of NMR spectra of starfish polar steroids, including asterosaponins.
Careful examination of the obtained resonances exhibited that there are some significant
differences in the chemical shifts of protons and carbons in the saturated side chains of
epimers 2a and 2b in the presence of a C-23 hydroxyl function. These data were used for
determination of C-23 absolute configurations of two steroids isolated earlier from starfish
Lethasterias nanimensis chelifera and Lethasterias fusca without application of Mosher’s
method [13].

21 26
20 23
18 1
11 17 27
19 O
H 14
1 9
3 6 H 2a
RO OH
H
OR
2b
OH

Figure 2. Structures of compounds 1, 2a and 2b.


Figure 3. ESI MS/MS spectrum of [M – Na]– precursor ion at m/z 1213 of ophidianoside F.
196 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

Another approach, developed by Makarieva et al. [14], was based on the idea that sugars,
as components of conjugated natural products such as glycosides, may also be considered as
chiral substituents. A study was done by the 1H NMR spectra of peracetylated -
glucopyranosides and -arabinopyranosides obtained by reaction of D- and L-glucoses, and
L- and D-arabinoses with either (R)- or (S)-2-octanols. The obtained and literature data
showed that 1H NMR spectra may be used to determine the absolute configuration of the
aglycone moieties of some alicyclic glycosides without the need to synthesize derivatives
with chiral reagents, as long as the absolute configuration of their monosaccharide moiety is
known or vice versa. Spectra of marine steroid glycosides and their acetates containing
glycosylated side chains were also examined. It was shown that analysis of 1H NMR spectra
is more applicable for the determination of the absolute configuration in the cases when
glycosides have the same substitution in the D-ring of the aglycone moiety [14].
Mass spectrometry, especially electrospray ionization (ESI) tandem mass spectrometry,
has been playing an important role in the structural analysis of starfish polar steroids. As an
example, the characteristic fragmentations in ESI MS/MS spectrum of parent [M – Na]– ion
of asterosaponin ophidianoside F give information about type of aglycone, presence of sulfate
group, sugar sequences as well as branching (Figure 3) [15]. This method in combination with
liquid chromatography (LC-ESI MS/MS) was also used for metabolomic analysis of
complicated mixture of steroids of the Far Eastern starfish Aphelasterias japonica (see below)
[15].

STRUCTURES OF NEW COMPOUNDS


Polyhydroxysteroids

Starfish polyhydroxysteroids are compounds usually containing from four to nine


hydroxyl groups in steroid nucleus and side chains. It is of special interest that all the groups
were found only in positions restricted within some limits. The hydroxyl groups usually
occupy 3, 6 (or ), 8, 15 (or ), 16 positions in steroid nucleus, although they may be
occasionally found at 4, 5, 7 (or ), and rarely at 14 positions. Side chains of these
compounds are very diverse, but in the majority of the polyhydroxysteroids, hydroxyl groups
in side chains are attached to C-24 either C-26 or to C-28 either C-29 for steroids with
ergostane (24-methyl-cholestane) or stigmastane (24-ethyl-cholestane) series. Starfish
polyhydroxysteroids occur in the both sulfated and non-sulfated forms.
Two new steroid hexaols 3 and 4 and one steroid heptaol 5 were isolated from the
tropical starfish Asteropsis carinifera. The (24R,25S)-configurations of their side chains were
established by Mosher’s method [16]. The investigation of steroids from the starfish
Archaster typicus collected in shallow waters of Quang Ninh province (Vietnam) led to
isolation of five new polyhydroxysteroids 6–10, two of them 6 and 7 are 27-nor-cholestane
derivatives and other two 8 and 9 are 24,26-dihydroxycholestane derivatives [17, 18]. Three
new sulfated polyhydroxysteroids 11–13 were found by Yang et al. from the commercially
available same starfish species, wherein compound 11 has the same rare ∆25(27)-24,26-
dihydroxy cholestane side chain as 9 [19]. All steroids from Archaster typicus contain a rare
14-hydroxyl group. A new steroid hexaol (14) also having a 24,26-dihydroxylated side
Recent Studies of Polar Steroids from Starfish 197

chain was isolated from the starfish Solaster endeca, inhabiting in the Sea of Okhotsk [20].
The study of the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis gave a
new 24-O-sulfated tetrahydroxylated steroid (15) [21] (Figure 4).

OH OH

OH
OH
OH
OH OH OH
OH OH OH
OH OH
HO HO HO
3R=H 6R=H OH 8
R OH 7 R = OH OH OH
R OH 4 R = H, 22

5 R = OH, 22

OH OH

OH OH
O
OH OH OH
OSO3Na OSO3Na OH
OH OH OH
HO 9 HO 10 HO OH 11
OH
OH OH OH OSO3Na
OH OSO3Na

OH OH

OH OH
OSO3Na OH OH
OH
HO 12 R = OH HO 14 HO 15
13 R = H OH OH
R OH

Figure 4. Structures of polyhydroxysteroids 3–15.

O
OH
OH
OH OH
HO OH 16 17
OH HO CHO

OH HO
O O
HO O 18 HO 19 HO 20
H O

Figure 5. Structures of compounds 16–20.


198 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

The investigation of the starfish Asterina pectinifera, which in recent years attracted
much attention due to its large scale outbreak in Chinese coastal areas, allowed to obtain a
new unique polyhydroxysteroid ester, (25S)-5-cholestane-3,6,7,8,15,16-hexaol-26-
O-14’Z-eicosenoate (16). The structure of this ester was proved also by alkali hydrolysis [22].
Four new less polar unusual steroids, astropectenols A–D (17–20), were isolated from the
methanol extract of the starfish Astropecten polyacanthus collected in Vietnamese waters
(Figure 5). Possible hypothetic pathways for biosynthesis of these compounds were proposed
[23].
The first convergent synthesis of certonardosterol D2, (22E,24R)-24-methyl-5-cholest-
22-ene-3,6,15,24-tetraol, isolated from the starfish Certonardoa semiregularis in 2004,
was carried out from the natural diosgenin via the C22-steroid 23,24-bisnorcholanic lactone,
which was derived from sapogenins with peroxyacetic acid in the presence of catalytic
concentration of H2SO4 and iodine. The side chain was facilely stereoselective installed via
Julia olefination with a chiral alkyl sulfone [24].
The efficient and highly stereoselective convergent synthesis of certonardosterol D3,
(24R)-24-ethyl-5-cholestane-3,6,15,24-tetraol, also isolated earlier from Certonardoa
semiregularis, has been achieved from commercially available (+)-dehydroepiandrosterone.
The combination of an ene reaction and improved allylic oxidation was proved to be efficient
to construct the C15 functional group in sterol skeleton. The highly selective Julia olefination
was used to couple the chiral side chain for steroid [25].
Thus, the eighteen new polyhydroxylated compounds were isolated from different
starfish species in the last years. Compounds with rare side chains, unique ester of
polyhydroxysteroid and low polar steroids with two or three oxygen atoms were found. The
convergent syntheses of two bioactive polyhydroxysteroids were described.

Glycosides of Polyhydroxysteroids

Glycosides of polyhydroxysteroids are characteristic metabolites of the majority of


starfish. They have a polyhydroxylated steroid nucleus and one or two (rarely three)
monosaccharide units attached to a polycyclic system, either to side chains or to the steroid
nucleus and side chain simultaneously. They also may occur in the sulfated and non-sulfated
forms.
Six new steroid biglycosides, cariniferosides A–F (2126), were isolated from the
fraction of glycosides of polyhydroxysteroids of the starfish Asteropsis carinifera [8]. The
study of the alcoholic extract of the starfish Asterina pectinifera collected from the Yellow
Sea (China) near the Dalian coast gave three new steroid glycosides, pectiniosides H–J (27–
29), at that compounds 28 and 29 differed from each other in position of a sulfate group only
[26] (Figure 6).
A new polyhydroxysteroid xyloside 30 was found from the starfish Pentaceraster
chinensis collected from the Sanya Bay of the South China Sea [27]. The investigation of the
Far Eastern starfish Leptasterias fisheri led to isolation a new fisherioside A (31) with rare
(24S,25S)-22E,26-amide ergostane side chain [28]. Four new steroid biglycosides, plancisides
A–D (32–35), were obtained from the ethanolic extract of the crown-of-thorns starfish
Acanthaster planci collected from Van Phong Bay near Nha Trang (Vietnam). Compounds 33
Recent Studies of Polar Steroids from Starfish 199

and 35 are the first steroid glycosides containing an -L-fucopyranose unit found from
starfish, glycoside 34 belongs to a rare group of triglycosides [29, 30]. A new steroid bioside,
novaeguinoside Y (36), was isolated from Culcita novaeguineae (juvenile) collected in the
Seychelles area [31] (Figure 7).
The study on minor components of the total steroid fraction from the Far Eastern starfish
Aphelasterias japonica gave new steroid glycoside aphelasteroside E (37). A detailed analysis
of the fragmentation of the glycoside using tandem ESI mass spectrometry confirmed
completely the proposed structure of 37 [32]. Recently new steroid glycoside capelloside A
(38) with rare 3,6,8,15-tetrahydroxy 15-O-sulfate steroid nucleus was isolated from the
starfish Ogmaster capella collected at a depth of 90-100 m by dredging at the Mansfield bank
in the South China Sea [33]. Three new sulfated steroid monoglycosides, leptaochotensosides
A–C (39–41), together with described above steroid 15 were obtained from the Far Eastern
starfish Leptasterias ochotensis. Glycosides 40 and 41 have a rare 4-O-sulfate-β-xylose unit
[21] (Figure 7).
Therefore, during the last few years 21 new polyhydroxysteroid glycosides were found in
starfish, all of them are mono- and biosides except planciside D (35) with three sugar units.
At the first time a -L-fucopyranose moiety was found in starfish glycosides.

HO
HO
R2O HO O OCH3
O O
HO O O H3CO O
O HO O
OCH3 OR3

OH
OH 24,25
OH
OH OH
OH OH OH
O O
HO HO H3CO O
HO O
R1 OH R1 OR2 OCH3
21 R1 = H, R2 = CH3 24 R1 = R2 = H, R3 = CH3
22 R1 = R2 = H, 22 25 R1 = R2 = R3 = H
26 R1 = OH, R2 = SO3Na 26
23 R1 = OH, R2 = CH3

NaO3SO

OCH3
O
HO
O
O
OR1 OH

OH 28 R = H, R1 = NaO3SO OH
OH OH
OH OH O
OH
HO OH 29 R = SO3Na, R1 =
HO 27
OR HO OH
OH

Figure 6. Structures of steroid glycosides 21–29.


200 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

OH

O
SO3-Na+
N
OH H
OH
OH OH
OSO3Na
OH
HO O O HO O
30 O 31
HO HO
OCH3 OH OH OH
OH
OH
OH O
O HO
HO H3CO HO
O O HO
A OH H3C B H3C O
O
O OH O O
HO HO R1O HO O
C HO
OH HO OH
OH
H3CO O O
HO O
OCH3 OH OH
OH OH
OH OH
HO O
HO HO O
35
OCH3 OH
OH OR 32 R = H, R1 = A
33 R = H, R1 = B
34 R = SO3Na, R1 = C

H3CO O O
O
HO HO O
OCH3 OCH3

OSO3Na
OH
OH
OH OH

HO 36 HO O O 37
HO OH
OH OH OH OH
R3 O
HO O
HO O OH
O
H3CO
OH

R2
OH
OSO3Na HO
R1
OH
HO 38
39 R1 = H, R2 = OSO3Na, R3 = OH
OH 40 R1 = H, R2 = OH, R3 = OSO3Na
41 R1 = OH, R2 = OH, R3 = OSO3Na

Figure 7. Structures of steroid glycosides 30–41.


Recent Studies of Polar Steroids from Starfish 201

Asterosaponins and Their Aglycones

Asterosaponins, characteristic secondary metabolites from starfish, are steroid


oligoglycosides having the 3-O-sulfated 9(11)-3,6-dihydroxysteroid aglycones with
various side chains and carbohydrate chains containing, as a rule, from four to six sugar units
attached to C-6 of aglycone. These compounds have general architectures with the same β-
1,3, β-1,4 and β-1,2 sequences of glycosidic bonds in the linear part of carbohydrate chains
and β-1,2-bond in the branching at the second monosaccharide. It is known that majority of
asterosaponins have the quinovoses as the first monosaccharide unit and branching
monosaccharide unit, attached to the second monosaccharide, and quinovose or xylose as the
second monosaccharide unit in main chain. Asterosaponins were discovered before other
groups of starfish polar steroids, but to date isolation of new representatives of this structural
group is continued.
Two new aglycones of asterosaponins, lysaketotriol (42) and lysaketodiol (43), were
isolated from the ethanolic extract of ambulakrums separated from the arms of the starfish
Lysastrosoma anthosticta collected in the Posyet Bay (Sea of Japan). The steroid 43 contains
23-oxo-cholane side chain, previously known only in an artificial product isolated as a result
of acid hydrolysis of some asterosaponins [34]. A new asterogenin mithrotriol (44) with the
hydroxyl function at C-20 was isolated from the Pacific starfish Mithrodia clavigera collected
near the islands of Maldive archipelago [35]. A new 5-cholesta-9(11),24-dien-3,6,20-
triol-23-one 3-O-sulfate (45) was isolated from the Chinese Archaster typicus [19] (Figure 8).
Two new aglycones, (23S)-3,6,23-trihydroxy-5-cholesta-9(11), 20(21)-diene 3-O-
sulfate (46) and (20E)-3,6-dihydroxy-5-cholesta-9(11), 20(22)-dien-23-one 3-O-sulfate
(47), were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias
ochotensis. The asterogenin 47 was found in the native sulfated form for the first time, as
desulfated asterosapogenin it was earlier obtained from hydrolysates of some asterosaponins
[36] (Figure 8).
Also six new asterosaponins, leptasteriosides A–F (4853), were obtained from
Leptasterias ochotensis. The type of carbohydrate chain of glycosides 49–52 was not found in
the other asterosaponins. The leptasterioside D (51) has rare aglycone 47 as a steroid moiety.
Two new asterosaponins, hylodoside A (54) and asteropsiside A (55), were isolated earlier
from the starfish Leptasterias hylodes reticulata collected in the Sea of Okhotsk [31] and
from the tropical starfish Asteropsis carinifera, respectively [37] (Figure 9).

OH
42
OH 45
O O
43 46
O OH
NaO3SO OH O
OH 44 47

Figure 8. Structures of aglycones of asterosaponins 42–47.


202 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

OH
49 R1 = H, R2 = CH3
OH

NaO3SO OH O
O 48 R1 = H, R2 = CH3
Fuc or Gal Glc or Xyl H3C O
50 R1 = CH2OH, R2 = CH3
HO R1 O HOO OH 52 R1 = CH2OH, R2 = CH3
R2 O O
O Qui
HO O
HO O
O OH
OH
Fuc O
OH H3C 51 R1 = CH2OH, R2 = CH3
H3C Qui O
HO 54 R1 = CH2OH, R2 = CH2OH
HOHO 55 R1 = H, R2 = CH2OH 53 R1 = CH2OH, R2 = CH3
HO

OH
Fuc or Gal
HO R Qui or Xyl NaO3SO
2 O O
O R 1 H3C
O O 56 R1 = CH3, R2 = CH3
HO O HO 59 R1 = H, R2 = CH2OH
O HO O O 57 R1 = CH3, R2 = CH2OH
Fuc O O OH
O Qui
H3C OH
HO OH H3C OH
HO Qui O OH
HOHO
58 R1 = CH3, R2 = CH2OH 60 R1 = H, R2 = CH2OH

Figure 9. Structures of asterosaponins 48–60.

A new minor asterosaponin archasteroside С (56) was isolated from the Vietnamese
starfish Archaster typicus, its oligosaccharide moiety includes 6-deoxy sugars only [38]. Two
new asterosaponins, diplasteriosides A and B (57 and 58), bearing the identical
oligosaccharide chains linked to the C-6 atom of the known 3-O-sulfates of thornasterols A
and B, respectively, were isolated from the Antarctic starfish Diplasterias brucei [39]. New
lethasteriosides A and B (59 and 60) were obtained from the ethanolic extract of the Far
Eastern starfish Lethasterias fusca. Glycoside 60 has aglycone with a rare 23-hydroxy-
cholestane side chain [40], the (23R)-configuration was suggested by analogy with the known
nipoglycoside D and later determined by comparing NMR data with those of model
compounds [13] (Figure 9).
Three new asterosaponins 61–63 having the same oligosaccharide chains along with four
known glycosides have been found from the starfish Asterias amurensis collected off the
coast of Pohang, Korea, at that compounds 62 and 63 were obtained as a mixture (1 : 1) [41]
(Figure 10). For known thornasteroside A, versicoside A, anasteroside B, and
asteronylpentaglycoside sulfate, the complete nuclear magnetic resonance (NMR) assignment
was accomplished using 600 MHz high magnetic field NMR [42].
Recent Studies of Polar Steroids from Starfish 203

61
O
NaO3SO
Gal
HO OH Qui 62
OH OH
O H3C
O O O
HO O
O HO O O
Fuc O O OH 63
O Gal
H3C
HO OH H3C OH O
HO Qui
HOHO

Figure 10. Structures of asterosaponins 61–63.

Four new asterosaponins, astrosteriosides A−D (64−67), were isolated from the methanol
extract of the starfish Astropecten monacanthus collected in Cat Ba, Haiphong, Vietnam
(Figure 11). Astrosterioside D (67) has an unusual 20-hydroxy-23,25-diketocholestane side
chain, the (20R)-configuration was suggested on the basis of the marked difference between
the 13C NMR chemical shifts at C-20 and C-21 of 67 with those of 65 and related compounds
having a (20S)-configuration [43].
We would also like to mention particularly an article of Chinese scientists about
investigation of the starfish Asterias rollestoni Bell (in title of article the name of starfish was
erroneous Asterias rollentoni) collected in the Yellow Sea near the shore of Jiangsu Province
[44]. This article described the isolation of two new triterpene glycoside, rollentoside A and
B. The structure of rollentoside A was established as 3-O-{3-O-methyl--D-xylopyranosyl-
(1→3)-O--D-glucopyranosyl-(1→4)-O--D-quinopyranosyl-(1→2)-O--D-xylopyranosyl}-
16,23S-diacetoxy-holost-7-ene, the rollentoside B is its 23-non-acetoxy analog. This fact has
caused the doubts, because the structures of isolated triterpene glycosides were very similar to
those of glycosides of sea cucumbers but not starfish that contain steroid glycosides. There is
significant similarity between triterpene glycosides isolated from Asterias rollestoni [44] and
glycosides isolated from sea cucumbers Eupentacta spp. Moreover rollentoside B from the
starfish is absolutely identical by the structure to cucumarioside A15 from sea cucumber
Eupentacta fraudatrix [45]. It is known that some starfish are predators of sea cucumbers.
The chemical defense of sea cucumbers, namely the presence of toxic triterpene glycosides, is
not effective against starfish because starfish contain their own toxic steroid glycosides that
have a similar mechanism of action. Hence, the presence of triterpene glycosides in Asterias
rollestoni found by Zhan et al. [44] may be explained by feeding of the starfish on sea
cucumbers of the genus Eupentacta. The model experiment for finding of holothurian
triterpene glycosides in starfish fed by a sea cucumber was done in our laboratory. LC-ESI
MS identification of a series of triterpene glycosides from Eupentacta fraudatrix in starfish
Patiria pectinifera fed these sea cucumbers confirmed our suggestion [46].
204 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

64

OH
NaO3SO 65
Gal
HO OH Xyl OH OH
O H3C O
O HO O 66
HO O
O HO O O O O
Fuc O O OH
O Qui
H3C
HO OH H3C OH
Qui NaO3SO 67
O HOHO Fuc
Araf O HO Qui
H3C OH
OH O H3C
O HO O
HO O
HO O HO O O
OH Fuc O O OH
O Glc
H3C
HO OH H3C OH
HO Qui
HOHO

Figure 11. Structures of asterosaponins 64–67.

The first total syntheses of asterosaponins were developed in the last years. The total
synthesis of goniopectenoside B, asterosaponin from the starfish Goniopecten demonstrans,
has been achieved in a total of 70 steps starting from cheap materials, with a convergent
linear sequence of only 21 steps and in 4.3% overall yield from adrenosterone [47]. Recently
the total synthesis of astrosterioside A (64) from Astropecten monacanthus with a convergent
linear sequence of 24 steps and in a high 6.8% overall yield was described. The synthesis
features stereoselective HWE olefination to construct the steroid (20(22)E)-ene-23-one side
chain, regioselective glycosylation of the xylopyranoside 4-OH, and highly efficient gold(I)-
catalyzed coupling of the aglycone with a hexasaccharide (cyclopropylethynyl)benzoate
donor [48]. Moreover, the first synthesis of the pentasaccharide fragment of thornasteroside
A, the first asterosaponin isolated from Acanthaster planci L. in 1978, has been achieved.
Initially, a [3+2] convergent strategy was attempted, but the β(1→4) glycosidic linkage
between galactopyranose (sugar IV) and xylopyranose (sugar II) was formed with a low
stereoselectivity and in low yield. Subsequently, a [3+1+1] strategy was adopted. A
galactopyranosyl donor equipped with a neighboring participating Lev (levulinoyl) group at
the 2-position was first coupled with a trisaccharide acceptor to construct the β(1→4)
glycosidic bond. Then the Lev group was selectively removed, and subsequent glycosylation
with a perbenzoylated D-fucopyranosyl Schmidt donor efficiently gave the desired
pentasaccharide [49]. Given the conserved nature of the structures of asterosaponins, these
works offer the possibilities to access many more members of this class of marine natural
products.
Thus, 20 new asterosaponins and six new native aglycones of asterosaponins were
isolated from starfish in the last years. All of them have the same steroid nuclear and differ
from each other in side and oligosaccharide chains. Glycosides with oligosaccharide moiety
including 6-deoxy sugars only, and rare and unique side chains were found. The total
Recent Studies of Polar Steroids from Starfish 205

syntheses of two asterosaponins and pentasaccharide moiety of thornasteroside A were


described in the first time.

Cyclic Steroid Glycosides

A rare group of starfish steroid glycosides are cyclic glycosides. This group of glycosides
discovered by Italian scientists more than 30 years ago from two species of the genus
Echinaster was previously represented by five glycosides [1]. These unique compounds have
several unusual structural features, including a trisaccharide chain cyclized between C-3 and
C-6 of the ∆7-3,6-dihydroxysteroid aglycone and the presence of a glucuronic acid unit in
the carbohydrate moiety. Recently five new cyclic glycosides, luzonicosides B–F (69–73),
along with known luzonicoside A (68) were obtained from the starfish Echinaster luzonicus,
collected at Van Phong Bay in the South China Sea near the coast of Vietnam [50] (Figure
12).
Luzonicosides D–F (71–73) have the -D-glucose residue instead -D-galactose residue
in luzonicosides A–C (68–70), the type of cyclic carbohydrate chain found in 71–73 has not
previously been identified in other starfish steroid glycosides. The luzonicoside D (72)
contains a unique 7-hydroxy-8,9-epoxy pattern in the steroid nucleus, which is found for
the first time in polar steroids of marine origin. The luzonicoside F (73) is open carbohydrate
chain glycoside, its linear carbohydrate chain structurally related to that of 71 and 72, but is
not cyclic was also described for the first time [50].

O
68 R1 = OH R2 = H (Gal)
71 R1 = H R2 = OH (Glc)
O
GlcA
O O
HOOC O
O 69 R1 = OH R2 = H (Gal)
HO R2
HO O O O
R1
O OH
Ara
HO Gal or Glc
HO 70 R1 = OH R2 = H (Gal)
HO

O O

O
72 73
GlcA O OH GlcA O
O
HOOC O O
HOOC O
O OH OH
HO O OH
HO HO
O O HO O
OH O
Ara O Ara OH
HO Glc O Glc
HO HO
HO HO
HO

Figure 12. Structures of cyclic steroid glycosides 68–73.


206 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

BIOLOGICAL ACTIVITIES OF STARFISH POLAR STEROIDS


Polar steroid compounds from starfish attract the attention of researchers is not only by
their diverse structures, but also by the wide spectrum of their biological activities, including
cytotoxic, antiviral, antibacterial, antibiofouling, neuritogenic, antifungal, cancer preventive,
anti-inflammatory and other effects. In the last time new knowledge about different biological
activities of new and known starfish polar steroid compounds were obtained. These data are
very important for study on structure-activity relationships and mechanism of action of some
active compounds.

Anticancer Activity

New polyhydroxysteroids 11–13 and 44 along with 13 known compounds from


Archaster typicus collected in the Chinese waters were tested for anticancer activities against
MDA-MB-435 breast cancer and Colo205 human colon adenocarcinoma cell lines. The
thornasterol A 3-O-sulfate showed weak activities with IC50 values of 58.3 and 47.1 g/mL,
respectively, while 27-nor-5-cholestane-3,4,5,6,7,8,14,15,24-nonaol exhibited
positive effect only on MDA-MB-435 with an IC50 value of 42.3 g/mL [19].
An inhibition of nonspecific esterase, a cytoplasmic enzyme from mouse Ehrlich
carcinoma cells and cytotoxicity against HeLa human epithelial carcinoma cells were
investigated for fourteen known steroids from two starfish species of the Evasterias genus
and new hexaol (14) from Solaster endeca. The tested steroids were not highly toxic towards
tumor cells and did not stimulate the activity of a well known p53 tumor suppressor, except
two steroids, which demonstrated low cytotoxicities, but stimulated p53 activity in a yeast
two hybrid test system [20].
Cytotoxic effects of five known steroids, including three sulfated polyhydroxysteroids,
non-sulfated hexaol and Δ7-sitosterol, isolated from the methanol extract of the starfish
Ctenodiscus crispatus, were evaluated toward two human carcinoma cell lines, human
hepatocellular carcinoma (HepG2) and human glioblastoma (U87MG). Non-sulfated (25S)-
5-cholestane-3,5,6,15,16,26-hexaol showed cytotoxicity against these cells via
inhibition of cell growth and induction of apoptosis. Induction of apoptosis by this compound
was demonstrated by cell death, DNA fragmentation, increased Bax/Bcl-2 protein ratio and
the activation of caspase-3, caspase-9 and poly (ADP-ribose) polymerase (PARP) [51].
New polyhydroxysteroid esters (16) together with seven known steroids from the starfish
Asterina pectinifera were evaluated for their cytotoxicity against HepG2 cell line in vitro. The
(25S)-5-cholestane-3,4,6,7,8,15, 16,26-octaol and cholest-7-ene 3-O-sulfate
exhibited cytotoxicity against HepG2 cells with IC50 values of 0.2 and 1.6 M, respectively
[22].
The cytotoxic effects of methanol extract, CH2Cl2 fraction, four new astropectenols A–D
(17–20) and three known low-polar steroids from the starfish Astropecten polyacanthus were
examined against three human cancer cells as HL-60 (leukemia cells), PC-3 (prostate cancer
cells), and SNU-C5 (colorectal cancer cells) using a MTT assay [23]. The CH2Cl2 fraction
and known 5-cholest-7,9(11)-diene-3-ol exhibit potent cytotoxic effects against HL-60
cells with the IC50 of 8.29 g/mL and 2.70 M, respectively. When HL-60 cells were treated
Recent Studies of Polar Steroids from Starfish 207

with these fraction or steroid, several apoptosis events like chromatin condensation and the
increase of the population of sub-G1 hypodiploid cells were observed. Investigations for the
possible mechanism underlying the induction of apoptosis showed that tested CH2Cl2 fraction
and 5-cholest-7,9(11)-diene-3-ol induced apoptosis through down-regulation of Bcl-2, up-
regulation of Bax, cleavage of caspase-9, cleavage of caspase-3 and cleavage of poly(ADP-
ribose) polymerase in HL-60 cells. The apoptosis induction of HL-60 cell by CH2Cl2 fraction
or this compound was attended by the decreasing of phospho-extracellular signal-regulated
kinase (ERK) 1/2 and C-myc. These results indicated that the CH2Cl2 fraction and 5-
cholest-7,9(11)-diene-3-ol induced apoptosis via the down-regulation of ERK 1/2 pathway
and C-myc in HL-60 cells [23].
New pectiniosides H–J (27–29) along with known asterosaponin P1, (25S)-5-
cholestane-3,4,6,7,8,15,16,26-octaol and (25S)-5-cholestane-3,4,6,7,8,15,
16,26-octaol from Asterina pectinifera were tested for the cytostatic activity in leukemia
HL-60 cell line, pectiniosides H–J and asterosaponin P1 were not cytostatic below 100 mM,
while octaols showed moderate cytostatic activity with the IG50 values of 80.3 and 40.5 mM,
respectively [26].
The new polyhydroxysteroid glycoside 30 and new asterosaponin obtained from
Pentaceraster chinensis exhibited strong cytotoxicity against tumor cell lines K-562 (human
erythromyeloblastoid leukemia), BEL-7402 (human hepatoma) and U87MG (human
glioblastoma). The mechanism of asterosaponin inducing U87MG cells apoptosis was
modulated by up-regulation of Bax protein a down-regulation of Bcl-2 protein [27, 52].
A cytotoxic activities against human skin melanoma cells SK-MEL-28, SK-MEL-5, and
RPMI-7951 for new mithrotriol (43) and known thornasterol A 3-O-sulfate, echinasteroside
B, granulatoside A, linckoside K, forbeside L and cholesterol 3-O-sulfate isolated from
Mithrodia clavigera were determined using MTS reagents. Monoglycosylated forbeside L
with the IC50 from 75 to 84 M was the most active among the all investigated compounds
[35].
A cytotoxic activity against human promelocytic leukemia cells HL-60 of known
asterosaponins thornasteroside A and versicoside A isolated from Asterias amurensis were
evaluated, these compounds exhibited moderate cytotoxic activity with IC50 values ranging
from 21.3 to 81.5 M [41].
The cytotoxic activities of the MeOH extract and four new astrosteriosides A−D (64−67)
and two known asterosaponins, psilasteroside and marthasteroside B, from Astropecten
monacanthus were evaluated on three human leukemia HL-60, prostate PC-3 and colorectal
SNU-C5 cancer cell lines. The MeOH extract (with IC50 values ranging from 0.84 to 3.96
g/mL) and astrosterioside D (67) (with IC50 values ranging from 4.3 to 5.2 M) exhibited
potent cytotoxic effects against all three tested cell lines. In addition, the MeOH extract and
astrosterioside D (67) have an effect on leading to apoptosis. They induced apoptosis via
activation of caspase-3, which resulted from increase of Bax and/or decrease of Bcl-2. The
treatment with the MeOH extract or compound 67 resulted in a decrease in C-myc levels
along with down-regulation of phospho-ERK 1/2 and phospho-AKT. These results suggested
that the induction of apoptosis by the MeOH extract and astrosterioside D was also
accompanied by the inactivation of phosphatidyl inositol 3-kinase PI3K/AKT and
extracellular signal-regulated kinase ERK 1/2 mitogen-activated protein kinase (MAPK)
pathways and the down-regulation of C-myc in HL-60, PC-3, and SNU-C5 cells [53].
208 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

It was shown previously that asterosaponin 1 from the starfish Culcita novaeguinea is
capable of inhibiting growth and inducing apoptosis in human glioblastoma U87MG cells
[54]. However, the role of asterosaponin 1 in the process of apoptosis in lung cancer cells has
not been determined. The potential antiproliferative and pro-apoptotic activity of
asterosaponin 1 in A549 human lung cancer cells, as well as the potential mechanisms was
investigated by Zhao et al. [55]. The results showed that asterosaponin 1 inhibited the
proliferation of A549 cells in a dose-dependent manner, and the cytotoxicity was attributable
to apoptotic cell death. Asterosaponin 1 increased endoplasmic reticulum (ER) dilation and
cytosolic Ca2+ concentration, and enhanced the expression of the ER molecular chaperones
GRP78 and GRP94 in a dose- and time-dependent manner. In addition, asterosaponin 1
treated A549 cells exerted increased expression and activity of CHOP, caspase-4 and JNK,
three essential ER-associated apoptotic molecules. In summary, these results demonstrated
that asterosaponin 1 inhibits the proliferation of A549 cells through induction of ER stress-
associated apoptosis [55].
The bioactivity of asterosaponin novaeguinoside II, isolated early from the Chinese
starfish Culcita novaeguinea was investigated on human U87MG glioblastoma cells. The
results showed that novaeguinoside II significantly suppresses U87MG cell proliferation in a
time and concentration-dependent manner. Flow cytometric analysis of DNA in U87MG cells
showed that novaeguinoside II induces the prominent appearance of an S phase peak in the
cell cycle suggestive of apoptosis that is identical to the result of an annexin V/PI assay.
Fluorescence and electron microscopy revealed apoptotic change of U87MG cells. The
electrophoresis of DNA showed a typical “ladder” that is consistent with apoptotic DNA
fragmentation. Cytofluorometry showed a decreased mitochondrial transmembrane potential
in novaeguinoside II-treated U87MG cells. Western blot showed that novaeguinoside II
increased the expression of cytochrome-c and caspase-3 protein. These data suggest that
novaeguinoside II can induce apoptosis of U87MG cells by a mitochondrial apoptotic
pathway [56].
A series articles about anticancer properties of isolated steroids against human colon
cancer cell line HCT-116, human breast cancer cell line T-47D, and human melanoma cancer
cell line RPMI-7951 was published. The cytotoxicities of studied compounds were evaluated
by the MTS method, all of them were non-toxic or exhibited slight cytotoxicity. The effects
of these compounds at a non-cytotoxic concentration on the proliferation of HCT-116, T-47D,
and RPMI-7951 cell lines and reduced colony formation of these cells were also examined.
Cariniferosides A–F (2126) and six known compounds isolated from Asteropsis
carinifera did not show any apparent cytotoxicity against HCT-116, T-47D, and RPMI-7951
cells, but the majority of the tested steroid glycosides displayed a capability to inhibit in vitro
colony formation of T-47D and RPMI-7951 cancer cells. The sulfated compounds
cariniferoside F (26), previously known halityloside A 6-O-sulfate and 4''-O-
methylhalityloside A 6-O-sulfate exhibited best inhibitory effects from the studied glycosides,
these compounds reduced the numbers of the T-47D cell colonies by 38, 64, and 67%,
respectively, and the numbers of the RPMI-7951 cell colonies by 57, 60 and 50%,
respectively [8].
New planciside A (32) from Acanthaster planci at a non-cytotoxic concentration of 15
M inhibited the proliferation of T-47D cells after 72 h by 35%, and the proliferation of
Recent Studies of Polar Steroids from Starfish 209

RPMI-7951 cells after 48 h by 27%, but did not show effect on colony formation of these
cells in a soft agar clonogenic assay [29].
The investigation of cytotoxic activities of leptasteriosides A–F (4853) and sulfated
genine 47 from Leptasterias ochotensis against cancer cell lines RPMI-7951 and T-47D
showed slight or moderate cytotoxic activities. The asterosaponins 48–50 demonstrated a
significant inhibition of RPMI-7951 and T-47D cell colony formation in soft agar clonogenic
assay in nontoxic doses. The treatment of T-47D cells by 48, 49 and 50 at non-toxic
concentrations 7, 1, and 10 M reduced the number of colonies on 51, 76, and 34%,
respectively. The results obtained for the RPMI-7951 cells indicated that compounds 48, 49
and 50 at a concentration 10 M effectively reduced the number of colonies on 81, 93, and
15%, respectively [36].
Known asterosaponins regularoside A and thornasteroside A isolated from Asteropsis
carinifera showed slight cytotoxicities against T-47D, RPMI-7951, and HCT-116 cancer
cells. Regularoside A inhibited formation of the T-47D cell colonies by 57% and the RPMI-
7951 cells by 26%, whereas thornasteroside A suppressed formation of the T-47D and the
RPMI-7951 cell colonies by 53 and 71%, respectively [37].
The study of cytotoxic properties of new diplasteriosides А and B (57 and 58) and known
asteriidoside А isolated from Diplasterias brucei on HCT-116, T-47D, and RPMI-7951 cell
lines exhibited that unlike pentaosides 57 and 58 hexaoside asteriidoside А was not toxic in
all the tested cell cultures, only diplasterioside A (57) was toxic on HCT-116 cells. Both new
asterosaponins 57 and 58 showed moderate toxicity toward T-47D cells. They were most
toxic against RPMI-7951 cells and had similar toxicities (IC50 67 and 60 M, respectively)
[39].
New lethasteriosides A and B (59 and 60), and known asterosaponins thornasteroside A,
anasteroside A, and luidiaquinoside obtained from Lethasterias fusca did not show any
apparent cytotoxicity against cancer cell lines T-47D, RPMI-7951, and HCT-116, but
glycoside 59 at concentration of 20 M demonstrated considerable inhibition of the T-47D
(97%), RPMI-7951 (90%) and НСТ-116 (90%) cell colony formations in a soft agar
clonogenic assay [40].
The biological action of new leptaochotensosides A–C (38–40) and sulfated steroid (15)
from Leptasterias ochotensis were examined using the EGF-induced colony formation of
normal mouse epidermal JB6 Cl41 cells and the growth and colony formation of RPMI-7951
and T-47D cancer cells. The glycoside 38 significantly reduced colony formation of T-47D
cells (the percentage of inhibition was 48% at concentration of 50 M) and the EGF-induced
colony formation of JB6 Cl41 cells (decreased EGF-induced colony number on 44% of
control at non-toxic concentration of 200 M). It was shown that compound 38 demonstrated
its antiproliferative effects in part through the inhibition of phosphorylation of MAP kinases,
namely through the inhibition of EGF-induced phosphorylation of ERK1/2 and MSK-1
kinases [21].
The anticancer properties of ethanol, petroleum ether, ethylacetate, and butanol fractions
of the crown-of-thorns starfish Acanthaster planci were investigated. The butanol fraction
was especially shown to inhibit human malignant melanoma A375.S2 cells’ proliferation,
which is involved in the apoptotic progression. This fraction could induce apoptosis and even
necrosis in A375.S2 cells as evidenced by double staining with an Annexin V-FITCand PI
assay and DNA fragmentation analysis [57].
210 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

Recently the results of the in silico screening of 182 previously reported starfish polar
steroids along with their 282 derivatives prepared by in silico transformations against Bcl-2
and CDK-4/Cyclin D1 - two important targets of progression and proliferation of cancer cells
- were published [58]. Their physico-chemical properties, druglikeliness, binding potential
with the selected targets, ADMET (absorption, distribution, metabolism, toxicity) were
predicted. Furthermore, the results were compared with those of existing steroid and non
steroid drugs and inhibitors of Bcl-2 and CDK-4/Cyclin D1. It was established that marine
polyhydroxysteroids certonardosterol D5 and certonardosterol Q3 known from Certonardoa
semiregularis have excellent potential to become leads for the development of orally safe
potent drugs for the treatment of various cancer types [58].

Immunomodulatory and Anti-Inflammatory Activities

The inhibitory effect of crude extracts, four new astropectenols A–D (17–20) and three
earlier known low-polar steroids from the starfish Astropecten polyacanthus on pro-
inflammatory cytokine (interleukin-12 (IL-12) p40, interleukin-6 (IL-6), and tumor necrosis
factor  (TNF-)) production in lipopolysaccharide (LPS)-stimulated bone marrow-derived
dendritic cells (BMDCs) was studied using enzyme-linked immunosorbent assays (ELISA).
Among those tested, known 5-cholest-7-ene-3,6-diol and 5-cholest-7,9(11)-diene-3-ol
showed potent inhibitory effects on the production of all three pro-inflammatory cytokines
with IC50 values ranging from 1.82 to 7.00 M. Potent inhibitory activities were also
observed for astropectenol A (17) on the production of IL-12 p40 and IL-6 with values of
3.96 and 4.07 M, respectively, and for astropectenols C and D (19 and 20) on the production
of IL-12 p40 with values of 6.55 and 5.06 M, respectively. In addition, astropectenol B (18)
(IC50 = 34.86 M) and 5-cholest-8(14)-ene-3,6-diol (IC50 = 79.05 M) exhibited
moderate inhibitory effects on the production of IL-12 p40, whereas astropectenol C (19)
(IC50 = 22.80 M) and astropectenol D (20) (IC50 = 16.73 M) moderately inhibited the
production of TNF- and IL-6, respectively [59].
The same activity of the MeOH extract of Astropecten monacanthus, n-BuOH fraction,
and four isolated new asterosaponins astrosteriosides A−D (64−67) as well as two known
compounds, psilasteroside and marthasteroside B, were evaluated by measuring the
production of IL-12 p40, IL-6, and TNF- in LPS-stimulated BMDCs. The n-BuOH fraction
exhibited potent inhibitory effects on the production of all three pro-inflammatory cytokines.
Astrosterioside D (67), having two ketone groups in the side chain, exhibited potent
inhibitory effects on the production of IL-12 p40, IL-6, and TNF- with IC50 values of 0.60,
3.51, and 1.21 M, respectively, comparable to that of the positive control. In addition,
astrosterioside A (64) and marthasteroside B showed significant inhibitory effects on IL-6
production with IC50 values of 3.17 and 4.37 M [43].
In addition, the inhibitory effects of a methanolic extract, dichloromethane fraction, water
layer, three polyhydroxylated steroids and one steroid bioside nodososide isolated from the
Vietnamese starfish Protoreaster nodosus on IL-12 p40, IL-6, and TNF- production in LPS-
stimulated BMDCs were examined. As result, the methanolic extract and dichloromethane
fraction exerted potent inhibitory effects on the production of all three pro-inflammatory
cytokines with IC50 values ranging from 0.60 to 26.19 g/mL. Potent inhibitory activities
Recent Studies of Polar Steroids from Starfish 211

were also observed for (25S)-5-cholestane-3,4,6,7,8,15,16,26-octaol on the


production of IL-12 p40 and IL-6 (IC50 = 3.11 and 1.35 M), and for (25S)-5-cholestane-
3,6,8,15,16,26-hexaol and (25S)-5-cholestane-3,6,7,8,15,16,26-heptaol on the
production of IL-12 p40 (IC50 = 0.01 and 1.02 M). Moreover, nodososide exhibited
moderate inhibitory effects on IL-12 p40 and IL-6 production [60].
The immunomodulatory activities of cyclic glycosides luzonicosides А and D (68 and 71)
from Echinaster luzonicus, including lysosomal stimulation, intracellular ROS (reactive
oxygen species) level elevation, and nitric oxide (NO) synthesis up-regulation, on the RAW
264.7 murine macrophage cells were investigated [50]. At non-cytotoxic doses, luzonicoside
A (68) regulated the lysosomal activity of the RAW 264.7 cells in a dose-dependent and bell-
shaped manner. This compound significantly stimulated the lysosomal activity in the
concentration range of 0.001 – 1.0 M with maximal stimulation at 0.1 M up to about 50%
in comparison with control cells. Compound 71 was less effective in this experiment.
Luzonicoside A (68) induced ROS formation in the same cells in the concentration range of
0.01  10.0 M and maximally increased ROS level about two times at the dose of 0.01 M.
This compound induced an increase in NO production in macrophages in a dose-dependent
and bell-shaped manner similar to that of the lysosomal activity. The most effective
concentrations that up-regulated NO generation in RAW 264.7 cells up to 15-30% when
compared with control level were of 0.01 and 0.1 M, respectively. In comparing with LPS
from Escherichia сoli, glycoside 68 at a concentration of 0.1 M was more effective in
lysosomal activity stimulation than LPS from E. сoli at the dose 1 g/mL and had a similar
effect to LPS in stimulating NO synthesis in RAW 264.7 murine macrophage cells.
Stimulation of NO production in the cells is a relatively rare phenomenon for marine low
molecular weight natural products. Based on these observations, luzonicoside А (68) seems to
be promising for further investigation as a potent immunomodulatory agent [50].
Lysaketotriol (41) from Lysastrosoma anthosticta produced moderate stimulation of
lysosomal activity in mouse splenocytes (increase lysosomal activity by up to 25-30% at a
maximal concentration of 6.25 g/mL). The compounds 41 and 42 showed
immunomodulatory activity, the increase of ROS formation in mouse macrophages by up to
50 and 20% was observed in response to application of compounds at concentrations of 6.25
and 12.5-50 g/mL, respectively [34].
A new capelloside A (36) along with the previously known coscinasteroside B from
Ogmaster capella decreased intracellular ROS levels in murine macrophages of the RAW
264.7 cell line at induction by LPS from E. coli by 60 and 41%, correspondingly, at a non-
cytotoxic doses of 10 M [33].
Three new asterosaponins 61–63 and four known asterosaponins, thornasteroside A,
versicoside A, anasteroside B, and asteronylpentaglycoside sulfate, from Asterias amurensis
were tested for their anti-inflammatory activity in RAW 264.7 cells. In case of compounds
61–63, anasteroside B and asteronylpentaglycoside sulfate, a concentration-dependent activity
was not observed due to their cytotoxicity toward the RAW 264.7 cells. Compared to control
RAW 264.7 cells, treated with lipopolysaccharide, the treatment with thornasteroside A
reduced the production of NO by 6.08, 8.13, 10.35, 13.86, and 19.72% at 0.1, 0.5, 1, 2, and 4
M, respectively. The versicoside A reduced the production of NO by 5.52, 8.29, 12.55,
14.59, and 41.11% at 0.5, 1, 2, 4, and 8 M, respectively [41].
212 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

Total fractions of starfish saponins from Asterias amurensis and Asterina pectinifera
were investigated as adjuvants to find new sources of compounds capable to stimulate both
Th1 immune response and production of cytotoxic T cells for using as vaccine components
against intracellular pathogens. Crude starfish saponins had hemolytic activities (EC50 = 10 to
100 g/mL) and thin layer chromatography indicated heterogeneity of their constituents.
When starfish saponins were subcutaneously injected into mice with ovalbumin (OVA),
OVA-specific IgG, especially IgG2a instead of IgG1 was produced in mouse blood,
suggesting starfish saponins stimulated Th1 type immunity and they were potential sources of
new adjuvants [61]. The anti-inflammatory activities of methanolic extracts, aqueous extract
and two functional bioactive compound fractions from the starfish Asterina pectinifera were
investigated, the methanolic extract had the strongest activity in reduction of LPS-induced
inflammatory mediators in murine macrophage [62].

Neuritogenic and Neuroprotective Activities

Small molecule weight compounds with neurotrophic and neuroprotective properties may
be useful in the treatment of brain disorders characterized by neurodegeneration, neuronal cell
loss, and deficiencies in synaptic connectivity. Recently, a review concerning data about a
large number of natural and synthetic neuritogenic compounds was published [63]. These
compounds have various structures, including terpenoids, lipids, alkaloids, steroid glycosides,
small peptides, and so on. Some of them possess not only neurotrophic properties but also
neuroprotective activities. The structures and activities of steroid glycosides linckosides A-K
and granulatoside A from the starfish Linckia laevigata were also included in this review
[63].
In continuation of the investigation of the neurotrophic activities of starfish steroid
compounds, the study of the neuritogenic and neuroprotective effects of six starfish polar
steroids: asterosaponin Р1, (25S)-5-cholestane-3,4,6,7,8,15,16,26-octaol, and (25S)-
5-cholestane-3,6,7,8,15,16,26-heptaol, designated as PP1, PP2, and PP3, from
Patiria (=Asterina) pectinifera, and distolasterosides D1–D3, designated as D1, D2, and D3,
from Distolasterias nipon was made [64]. The activities of these compounds were analyzed
using the mouse neuroblastoma (NB) C-1300 cell line and an organotypic rat hippocampal
slice culture (OHSC). All of these steroids enhanced neurite outgrowth in NB cells. Dose-
dependent responses to compounds PP1, PP2, and PP3 were observed within the
concentration range of 10–100 nM, and dose-dependent responses to glycosides D1, D2, and
D3 were observed at concentrations of 1–50 nM. The neuritogenic effects of starfish steroids
on NB cells were synergistic with the effects of the neurotrophic nerve growth factor (NGF, 1
ng/mL) or brain-derived neurotrophic factor (BDNF, 0.1 ng/mL), the simultaneous treatment
of NB cultures with low concentrations of studied compounds and the ineffective
concentrations of the neurotrophic factors significantly increased neuronal differentiation.
Using NB cells and OHSCs, it was shown for the first time that starfish steroids act as
neuroprotectors against oxygen-glucose deprivation (OGD) by increasing the number of
surviving cells. Altogether, these results suggest that neurotrophin-like neuritogenic and
neuroprotective activities are most likely common properties of starfish polyhydroxysteroids
Recent Studies of Polar Steroids from Starfish 213

and the related glycosides, although the magnitude of the effect depended on the particular
compound structure [64].

Other Activities

The toxic effects of isolated from the Vietnamese starfish Archaster typicus compounds
7–10, monoside typicusoside A and three known polyhydroxysteroids on fertilization of eggs
of the sea urchin Strongylocentrotus intermedius and early embryonic development were
measured. The known (24E)-5-cholest-24-ene-3,4,5,6,7,8,14,15,26-nonaol 6-O-
sulfate was found to be most active of these substances with an effective inhibitory
concentration of EC50 = 12 g/mL in the sperm-test and EC50 = 23 g/mL in the 8-blastomere
test [18].
New polyhydroxysteroid esters (16) together with seven known steroids from
Asterina pectinifera were evaluated for their antiviral activity against herpes simplex
virus type 1 (HSV-1). Known (25S)-5-cholestane-3,4,6,7,8,15,16,26-octaol,
(25S)-5-cholestane-3,4,6,7,8,15, 16,26-octaol, cholest-7-ene 3-O-sulfate, (24S)-5-
cholestane-3,6,8, 15,24-pentaol, and asterosaponin P2 exhibited antiviral activity against
HSV-1 virus with the minimal inhibitory concentration (MIC) values of 0.2, 0.05, 0.2, 0.22,
and 0.07 M, respectively [22].
New bioside novaeguinoside Y (36) and asterosaponin hylodoside A (53) along with
known steroid hexaol and pentaol and three biosides from the starfish Leptasterias hylodes
reticulata and Culcita novaeguineae (juvenile) were tested for antimicrobial activity against
E. coli, Staphylococcus aureus and Candida albicans, at that only known 5-cholestane-
3,6,8,15,16,24-hexaol inhibited the growth of S. aureus up to 10% from the control at a
concentration of 1 mg/mL. New compounds and known steroid pentaol showed moderate
hemolytic activities in the mouse erythrocytes assay. Known hexaol and two biosides
displayed pH-depended hemolytic properties [31].
The action of steroid biosides halitylosides D and A and asterosaponin regularoside A,
isolated from the starfish Asteropsis carinifera, with the control heteroauxin on growth of
sprouts’ root and stem of soya, buckwheats, corns, and rice was studied. It was shown that the
investigated compounds stimulated the growth of sprouts’ root of testing agricultural plants.
Between the stimulating doses the “dead zones” have been discovered, effect of which was
equal or considerably lower to control. The halityloside D exhibited the most stimulating
activity on the growth of stem of corn in the concentrations of 10–12 and 10–8 g/mL,
accordingly the doze–effect curve had bimodal character [65].
A series of studies on the biological activities has been made on the extracts of various
starfish. The contractile and relaxant activity of the smooth muscles and the antimicrobial and
antioxidant activity of six different tissues (muscle, gut, liver, tube feet, gonads, and body) of
the starfish Asterias amurensis were investigated by Korean scientists [66]. The crude
fractions of Stellaster equestris from the Southeast coast of India showed remarkable
antimicrobial activities against some human bacterial pathogens [67]. Together with
anticancer properties, the antioxidant activities of the ethanol, petroleum ether, ethylacetate
and butanol fractions of Acanthaster planci were examined. Among them the ethanol fraction
contained the highest antioxidant effects [57].
214 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

Thus, in the last time the new data about biological activities of starfish polar steroid
compounds were obtained. The more studied activities are anticancer and immunomodulatory
effects. Moreover, the mechanisms of actions of some steroids were investigated. It was
shown for the first time that starfish steroids act as neuroprotectors against oxygen-glucose
deprivation by increasing the number of surviving cells. These results suggest that starfish are
a rich resource for obtaining the biologically active substances.

BIOLOGICAL FUNCTIONS OF STARFISH POLAR STEROIDS


The biological functions of starfish polar steroids are still in need of clarification. It is
assumed that the biological roles of the steroid metabolites may be related to tissue
distribution. Thus, early investigation of distribution of free sterols, polyhydroxysteroids and
steroid glycosides in different body components of the Far Eastern starfish Patiria
(=Asterina) pectinifera revealed that all the body components of this starfish contain free
sterols and asterosaponins but polyhydroxysteroids and related mono- and biosides were
found predominantly in the digestive organs (stomach and pyloric caeca) only [68].
It is generally believed that oligoglycoside asterosaponins in starfish have different
biological functions such as defense of starfish against predatory fish, reproduction, and
chemical signaling [1]. For example, although asterosaponins were found in all the studied
body components of Patiria pectinifera, hemolytic activities of polar steroids from body
walls (where only oligoglycoside asterosaponins were identified) were higher in comparison
with those from inner organs. Moreover, the differences in levels of toxic asterosaponins from
different body components, especially body walls and gonads were observed. It may be
connected with implementation of different biological functions by asterosaponins [68].
Recently the asterosaponin contents of five different organs, namely the aboral body wall,
the oral body wall (including tube feet), the stomach, the pyloric caeca and the gonads of the
starfish Asterias rubens were studied by MALDI–MS, MALDI–MS/MS, LC–MS and LC–
MS/MS, and the seventeen compounds have been detected [69]. It was observed that the five
different organs are each characterized by specific saponin contents and that between animals
there are also qualitative and quantitative variability of the saponin contents. The body wall
and the gonads are the organs containing specific saponin congeners. In these two organs the
highest hemolytic activity was measured, although once again large differences between
individuals were noted [69].
Further, Demeyer et al. used MALDI-MSI (MALDI mass spectrometry molecular
imaging) for determination the spatial distribution of saponins directly on the tissue of the
starfish Asterias rubens [70]. This modern method can be performed directly on the tissue
without the need of time-consuming extraction and purification procedures. MALDI imaging
methods also provide data on the distribution of saponin molecules within the organs.
MALDI-MSI performed at different spatial resolutions revealed that the inter- and intra-organ
distributions of saponin congeners are not homogeneous. These data are important for
structure/activity relationship investigations. It was determined that saponin molecules are
located not only inside the body wall of the animals but also within the mucus layer, where
they probably protect the animal against external aggressions [70].
Recent Studies of Polar Steroids from Starfish 215

The participation of some oligoglycoside asterosaponins in regulation of oocyte


maturation and action as cofactors for acrosomal reaction in starfish were previously
established [1]. Recently the mechanism of action of asterosaponin named cofactor for
acrosome reaction-inducing substance (Co-ARIS) known from the starfish Asterias amurensis
was investigated. It is known that in Asterias amurensis the acrosome reaction (AR) is
induced by the cooperative action of egg coat components (ARIS, Co-ARIS, and other
asterosaponins). The membrane dynamics involved in the action of Co-ARIS were elucidated
by Naruse et al. [71]. It was found that cholesterol specifically inhibited the Co-ARIS activity
for AR induction and detected the binding of labeled compounds with sperm using
radioisotope-labeled Co-ARIS. Co-ARIS treatment did not reduce the content of sperm
sterols, however, the condition was changed and localization of GM1 ganglioside on the
periacrosomal region disappeared. Moreover, a caveola-breaking assay, a novel method to
detect the effect of chemicals on microdomains of cell culture, was developed to confirm the
disturbance of somatic cell caveolae in the presence of Co-ARIS. The revelation that Co-
ARIS colocalized with GM1 clusters on the microdomains was done by atomic force
microscopy observations and surface plasmon resonance measurements using an artificial
membrane. A capacitation-like event for AR in starfish sperm was established [71].
Polyhydroxysteroids and related mono- and biosides in contrast with asterosaponins, play
another biological role. Proceeding from allocation of these compounds in digestive organs of
the starfish Patiria pectinifera and their definite structural resemblance to bile alcohols of
hagfishes and amphibians, their participation in digestion of food have been assumed [68].
The investigation of seasonal variations in the levels of polyhydroxysteroids and related
glycosides in the digestive tissues of Patiria pectinifera confirmed this hypothesis. The
maximal contents of these steroids in digestive organs more or less coincide with periods of
active feeding and their contents decrease when active feeding decreases after spawning. The
high content of these compounds in pyloric caeca, where semi-digested food is utilized by
digestive cells, may show that low molecular weight polar steroids are important for this stage
of digestion in the starfish [72, 73].

BIOSYNTHESIS AND METABOLOMIC STUDIES


OF STARFISH POLAR STEROIDS

The study of biosynthesis of biological active metabolites is very important for


fundamental research and also for the development of novel biotechnological approaches to
the production of these compounds. Until recently the biogenesis of starfish polar steroids
was unknown, although a hypothesis that dietary sterols are biogenetic precursors of these
steroids was proposed [72]. There are two main approaches to study biosynthesis of
complicated natural products. The first of them is based on using of proposed labeled
biosynthetic precursors and estimation of incorporation of labeled atoms in the target
compounds. The second approach is based on isolation and structural identification of series
compounds from investigated species, including minor metabolically active substances, and
construction of hypothetical schemes of biosynthesis.
Experimentally the biosynthesis of starfish polar steroids remained also to be
insufficiently studied. Only a few data were obtained in in vivo experiments by Mackie et al.,
216 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

in which aglycones of asterosaponins were shown to include some radioactivity from [2-
14
C]mevalonic acid and [4-14C]cholesterol [74], and low inclusion into asterosaponins was
indicated in experiments with homogenates of different starfish body components, when
radioactive [7-3H] and [4-14C]cholesterol and [4-14C]6-hydroxycholesterols were used as
precursors [75]. Until recently, there was no information regarding the biosynthesis of
polyhydroxysteroids and related low molecular weight steroid glycosides in starfishes.
The study on possible biosynthetic pathways of polyhydroxysteroids and related
glycosides in the Far Eastern starfish Patiria (=Asterina) pectinifera was done by our group
[76]. We have carried out the feeding experiments with labeled by stable isotopes precursors,
including [25,26,26,26,27,27,27-2H7]cholesterol and [2,2,3,4,4,6-2H6]cholesterol 3-O-sulfate.
The fractions of free sterols, asterosaponins and three pure compounds, steroid monoside
asterosaponin Р1, and polyhydroxysteroids (25S)-5-cholestane-3,4,6,7,8,15,16,26-
octaol and (25S)-5-cholestane-3,6,7,8, 15,16,26-heptaol, from digestive organs and
gonads of Patiria pectinifera were isolated and analyzed. The data concerning isotope
incorporation and distribution in these steroids were obtained using GC-MS, ESIMS,
ESIMS/MS, and NMR spectroscopy.
In these experimental conditions, we have experimentally established for the
first time, that polyhydroxysteroids and related low molecular weight steroid glycosides
are biosynthesized from dietary cholesterol and cholesterol sulfate. These deuterium labeled
precursors were converted into partly deuterated 5-cholestane-3,6,7,8,15,16,26-
heptaol, 5-cholestane-3,4,6,7,8,15,16,26-octaol, and steroid monoside astero-
saponin P1 (74–76). Scheme of the first stages of biosynthesis of polar steroids in these
animals was suggested on the basis of inclusion of three from six deuterium atoms and
determination of their positions in biosynthetic products, when [2,2,3,4,4,6-2H6]cholesterol 3-
O-sulfate was used as precursor (Figure 13). It was determined that transformations in rings A
and B include oxidation into corresponding 3-keto-derivatives and participation of 4(5)-
unsaturated ketones as biosynthetic intermediates. In addition, results of investigation of free
sterol fractions confirmed that starfishes are capable of converting dietary cholesterol into
lathosterol (5-cholest-7-ene-3-ol), but do not transform it into alkylated in side chain
sterols [76].

D D D
D
D
D desulfation D D [O] D
-
O3SO -SO3- HO -D O
D
D D D D D D
D
-D
D D D
H H [H] H H
D D [O] D
HO O O
H DH OH D OH -D D D
(74, 76) [O]
D
-H OH H
D
HO
H DH OH
(75)

Figure 13. The hypothetical pathway of A,B-rings fragments biosynthesis of [2,2,4-2H3]labeled polar
steroids 74–76 from [2,2,3,4,4,6-2H6]cholesterol 3-O-sulfate.
Recent Studies of Polar Steroids from Starfish 217

Another approach to biosynthetic studies, analysis of structures of isolated starfish polar


steroids, including minor compounds, allows to offer hypothetical biogenesis pathway of
these compounds. For example, the investigation of minor polyhydroxysteroids from the
Vietnamese starfish Archaster typicus and analysis of structural information concerning all
fifteen polyhydroxylated steroids of this starfish allowed to propose a biogenesis pathway for
the unusual side chain of 27-nor-cholestane derivatives [18].
In general, the analysis on more than 500 structures of polar steroid compounds from 64
different starfishes shows that, whereas mosaic character of their biosynthesis, hydroxylations
of these polar steroids during their biosynthesis from dietary sterols or sterol sulfates [76]
generally follows the next order: at the position 6, then at position 24 or 26 (for stigmastane
derivatives at the position 29), then at position 15 or 8, then at position 16 and other positions
[77].
In the last time metabolite profiling studies of secondary metabolites is widely
developing. This approach is very useful for searching minor compounds and for comparing
metabolomic profiles of different species and populations for ecological, dietary and
biosynthesis studies. Moreover, these data may allow further isolation of new individual polar
steroids for bioassays to be performed. The metabolomic studies on total fractions of starfish
polar steroids with indication of previously unknown constituents and the determination of
distribution of individual compounds within this class of animals are of great interest. As
mentioned above, steroid metabolites in starfish extracts from very complicated mixtures
difficult for separation into pure compounds by chromatographic and other modern methods.
Many minor metabolites remain to be largely unstudied, although knowledge about their
chemical structures is important for understanding of the biosynthesis of polar steroids in
these invertebrates.
Recently the first study of metabolite profiling of starfish polar steroid constituents was
carried out by our group [15]. We investigated the Far Eastern starfish Aphelasterias
japonica, which is a rich source of different steroid glycosides and polyhydroxysteroids. For
detailed analysis of complicated mixture of steroids from this species, isolated by solid-phase
extraction, a liquid chromatography – electrospray tandem mass spectrometry (LC-ESI
MS/MS) approach was applied. The characteristic fragmentations in ESI MS/MS spectra of
steroid glycosides allowed determining types of aglycones, presence of sulfate groups, sugar
sequences as well as branching. In addition, main structural features of polyhydroxylated
polar steroids including position of hydroxylation and level of sulfation were also established.
As results, the MS and MS/MS spectra of 33 asterosaponins including 3 previously isolated
compounds and of 35 polyhydroxysteroid compounds including 13 previously isolated
compounds were described. Although in many cases stereochemistry and some details of
exact structures cannot be deduced from mass spectra, reasonable proposals for new
glycoside structures can be given as it was demonstrated for 33 asterosaponins. A big series
of polyhydroxylated steroid compounds were detected and their exact or preliminary
structures were also proposed on the basis of their MS spectra and chromatographic behavior
[15].
Analysis of all structures of Aphelasterias japonica allowed to make conclusion about
characteristic feature of biosynthesis in this species, such as retardation of hydroxylation at
the position 8 and early dihydroxylation of a part of metabolites at the positions 5 and 6 in
combination with hydroxylation at the side chain. It was shown that absolute majority of
218 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

metabolites belonging to polyhydroxylated derivatives from Aphelasterias japonica contain


sulfate groups. It means that sulfation terminates further biosynthetic transformations. The
theoretical biosynthetic pathways of polyhydroxylated steroids of this starfish were proposed
[15].

CONCLUSION
Despite more than forty-year history of the study on starfish polar steroids, the analysis of
information, reported during last years, suggests the studies on these secondary metabolites
are still in progress. More then 70 new polar steroids were isolated from more then 30 starfish
species. Some of isolated steroids have rare or unique structural fragments. A new data about
anticancer and immunomodulatory activities of these compounds, including mechanisms of
actions were obtained. For the first time, the neuroprotective action of polyhydroxysteroids
and related glycosides against oxygen-glucose deprivation was described. Due to the high
biological activities, some syntheses of polyhydroxysteroids and asterosaponins have been
developed.
Studies of biological functions of starfish polar steroids are continued in the last years, at
that application of modern methods, for example, MALDI and MALDI-MSI open new
possibilities for investigation of functions of these compounds in different organs. For the
first time, the metabolite profiling of starfish polar steroids, including polyhydroxysteroids,
related glycosides and asterosaponins, was obtained by LC-ESI MS/MS. This approach may
be used for searching new minor compounds, evaluating the content of steroids, their further
isolation, bioassays as well as for comparing metabolomic profiles of different species and
populations along with or ecological, dietary and biosynthesis studies.
For the first time it was experimentally established that the dietary cholesterol and
cholesterol sulfate are biosynthetic precursors of polyhydroxysteroids and related low
molecular weight glycosides in starfishes using the deuterium labeled precursors in the
feeding experiments.

ACKNOWLEDGMENTS
This work was supported by the Grant No. 14-04-00341 from the Russian Foundation for
Basic Research.

CONFLICTS OF INTEREST
The authors declare no conflict of interest.
Recent Studies of Polar Steroids from Starfish 219

REFERENCES
[1] Minale L, Riccio R, Zollo F. Steroidal oligoglycosides and polyhydroxysteroids from
Echinoderms. Fortschr Chem Org Naturst 1993; 62: 75–308.
[2] Stonik VA. Marine polar steroids. Russ Chem Rev 2001; 70(8): 673–715.
[3] Iorizzi M, De Marino S, Zollo F. Steroidal oligoglycosides from the Asteroidea. Curr
Org Chem 2001; 5(9): 951–73.
[4] Stonik VA, Ivanchina NV, Kicha AA. New polar steroids from starfish. Nat Prod
Commun 2008; 3(10): 1587–610.
[5] Ivanchina NV, Kicha AA, Stonik VA. Steroid glycosides from marine organisms.
Steroids 2011; 76(5): 425–54.
[6] Dong G, Xu TH, Yang B, et al. Chemical constituents and bioactivities of starfish.
Chem Biodivers 2011; 8(5): 740–91.
[7] Gomes AR, Freitas AC, Rocha-Santos TAP, Duarte AC. Bioactive compounds derived
from echinoderms. RSC Adv 2014; 4(56): 29365–82.
[8] Malyarenko TV, Kicha AA, Ivanchina NV, et al. Cariniferosides A-F and other
steroidal biglycosides from the starfish Asteropsis carinifera. Steroids 2011; 76(12):
1280–7.
[9] Kicha AA, Kalinovsky AI, Ivanchina NV, et al. Four new asterosaponins,
hippasteriosides A, B, C, and D, from the Far Eastern starfish Hippasteria kurilensis.
Chem Biodivers 2011; 8(1): 166–75.
[10] Dale JA, Dull DL, Mosher HS. -Methoxy--trifluoromethyl-phenylacetic acid, a
versatile reagent for the determination of enantiomeric composition of alcohols and
amines. J Org Chem 1969; 34(9): 2543–9.
[11] Dale JA, Mosher HS. Nuclear magnetic resonance enantiomeric reagents.
Configurational correlations via nuclear magnetic resonance chemical shifts of
diastereomeric mandelate, O-methylmandelate and -methoxy--
trifluoromethylphenylacetate (MTPA) esters. J Am Chem Soc 1973; 95(2): 512–9.
[12] Ohtani I, Kusumi T, Kashman Y, Kakisawa H. High-field FT NMR application of
Mosher’s method. The absolute configurations of marine terpenoids. J Am Chem Soc
1991; 113(11): 4092–6.
[13] Kicha AA, Kalinovsky AI, Antonov AS, et al. Determination of C-23 configuration in
(20R)-23-hydroxycholestane side chain of steroid compounds by 1H and 13C NMR
spectroscopy. Nat Prod Commun 2013; 8(9): 1219–22.
[14] Makarieva TN, Shubina LK, Guzii AG, et al. Determination of absolute
stereochemistry of natural alicyclic glycosides by 1H NMR spectroscopy without
application of chiral reagents - an indication. Nat Prod Commun 2011; 6(5): 673–6.
[15] Popov RS, Ivanchina NV, Kicha AA, Malyarenko TV, Dmitrenok PS, Stonik VA.
Metabolite profiling of polar steroid constituents in the Far Eastern starfish
Aphelasterias japonica using LC−ESI MS/MS. Metabolomics 2014; 10(6): 1152−68.
[16] Malyarenko TV, Kicha AA, Ivanchina NV, Kalinovsky AI, Dmitrenok PS, Smirnov
AV. Three new polyhydroxysteroids from the tropical starfish Asteropsis carinifera.
Russ J Bioorg Chem 2010; 36(6): 755–61.
[17] Huong TTT, Truyen CQ, Long PQ, et al. A new polyhydroxysteroid from the
Vietnamese starfish Archaster typicus. J Chem 2009; 47: 374–8.
220 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

[18] Ivanchina NV, Kicha AA, Huong TTT, et al. Highly hydroxylated steroids of the
starfish Archaster typicus from the Vietnamese waters. Steroids 2010; 75(12): 897–
904.
[19] Yang XW, Chen XQ, Dong G, et al. Isolation and structural characterisation of five
new and 14 known metabolites from the commercial starfish Archaster typicus. Food
Chem 2011; 124(4): 1634–8.
[20] Levina EV, Aminin DL, Kovalchuk SN, et al. Polar steroids from Solaster endeca
starfish and the physiological activity of polar steroids from three starfish species. Russ
J Bioorg Chem 2010; 36(2): 233–9.
[21] Malyarenko TV, Malyarenko (Vishchuk) OS, Ivanchina NV, Kalinovsky AI, Popov
RS, Kicha AA. Four new sulfated polar steroids from the Far Eastern starfish
Leptasterias ochotensis: structures and activities. Mar Drugs 2015; 13(7): 4418–35.
[22] Peng Y, Zheng J, Huang R, et al. Polyhydroxy steroids and saponins from China Sea
starfish Asterina pectinifera and their biological activities. Chem Pharm Bull 2010;
58(6): 8568.
[23] Thao NP, Cuong NX, Luyen BTT, et al. Steroidal constituents from the starfish
Astropecten polyacanthus and their anticancer effects. Chem Pharm Bull 2013; 61(10):
1044–51.
[24] Jiang B, Shi HP, Tian WS, Zhou WS. The convergent synthesis of novel cytotoxic
certonardosterol D2 from diosgenin. Tetrahedron 2008; 64(3): 469–76.
[25] Jiang B, Shi HP, Xu M, Wang WJ, Zhou WS. Stereoselective synthesis of
certonardolsterol D3. Tetrahedron 2008; 64(41): 9738–44.
[26] Li ZQ, Chen G, Lu X, Wang HF, Feng BM, Pei YH. Three new steroid glycosides
from the starfish Asterina pectinifera. Nat Prod Res 2013; 27(20): 1816–22.
[27] Pan MX, Wu JH, Yi YH, et al. Studies on saponins constituents on Pentaceraster
Semper. Chin J Nat Med 2006; 4(5): 344–6.
[28] Kicha AA, Ivanchina NV, Malyarenko TV, Kalinovsky AI, Dmitrenok PS.
Fisherioside A  a new steroidal glycoside from the starfish Leptasterias fisheri. Chem
Nat Comp 2012; 48(5): 806–9.
[29] Kicha AA, Dinh TH, Ivanchina NV, et al. Three new steroid biglycosides, plancisides
A, B, and C, from the starfish Acanthaster planci. Nat Prod Commun 2014; 9(9):
1269–74.
[30] Kicha AA, Ivanchina NV, Malyarenko TV, et al. Minor steroid triglycoside, planciside
D, from the tropical starfish Acanthaster planci. Chem Nat Comp 2014; 50(6): 1032–6.
[31] Levina EV, Kalinovsky AI, Dmitrenok PS, Martyyas EA, Stonik VA. Two new
steroidal saponins, hylodoside A and novaeguinoside Y, from the starfish Leptasterias
hylodes reticulata and Culcita novaeguineae (juvenile). Nat Prod Commun 2010;
5(11): 1737–42.
[32] Popov RS, Ivanchina NV, Kicha AA, Malyarenko TV, Kalinovskii AI, Dmitrenok PS.
Minor steroidal glycosides from the Far-East starfish Aphelasterias japonica. Chem
Nat Comp 2013; 49(2): 286–90.
[33] Ivanchina NV, Kicha AA, Malyarenko TV, et al. The influence on LPS-induced ROS
formation in macrophages of capelloside A, a new steroid glycoside from the starfish
Ogmaster capella. Nat Prod Commun 2015; 10(11): 1937–40.
Recent Studies of Polar Steroids from Starfish 221

[34] Levina EV, Kalinovsky AI, Dmitrenok PS, Aminin DL. Bioactive steroidal sulfates
from the ambulakrums of the Pacific starfish Lysastrosoma anthosticta. Nat Prod
Commun 2009; 4(8): 1041–6.
[35] Levina EV, Kalinovskii AI, Ermakova SP, Dmitrenok PS. Steroid compounds from
Pacific starfish Mithrodia clavigera and their toxicity to human melanoma cells. Russ J
Bioorg Chem 2012; 38(5): 520–5.
[36] Malyarenko TV, Kicha AA, Ivanchina NV, et al. Asterosaponins from the Far Eastern
starfish Leptasterias ochotensis and their anticancer activity. Steroids 2014; 87:
119−27.
[37] Malyarenko TV, Kicha AA, Ivanchina NV, Kalinovskii AI, Dmitrenok PS, Ermakova
SP. Asteropsiside A and other asterosaponins from the starfish Asteropsis carinifera.
Russ Chem Bull 2012; 61(10): 1986–91.
[38] Kicha AA, Ivanchina NV, Huong TTT, Kalinovsky AI, Dmitrenok PS, Long PQ.
Minor asterosaponin archasteroside C from the starfish Archaster typicus. Russ Chem
Bull 2010; 59(11): 21336.
[39] Ivanchina NV, Malyarenko TV, Kicha AA, Kalinovsky AI, Dmitrenok PS, Ermakova
SP. Structures and cytotoxic activities of two new asterosaponins from the Antarctic
starfish Diplasterias brucei. Russ J Bioorg Chem 2011; 37(4): 499–506.
[40] Ivanchina NV, Kalinovsky AI, Kicha AA, et al. Two new asterosaponins from the Far
Eastern starfish Lethasterias fusca. Nat Prod Commun 2012; 7(7): 853–8.
[41] Hwang IH, Kim DW, Kim SJ, et al. Asterosaponins isolated from the starfish Asterias
amurensis. Chem Pharm Bull 2011; 59(1): 78–83.
[42] Hwang IH, Kulkarni R, Yang MH, et al. Complete NMR assignments of undegraded
asterosaponins from Asterias amurensis. Arch Pharm Res 2014; 37(10): 1252–63.
[43] Thao NP, Cuong NX, Luyen BTT, et al. Anti-inflammatory asterosaponins from the
starfish Astropecten monacanthus. J Nat Prod 2013; 76(9): 1764−70.
[44] Zhan YC, Sun Y, Li W, Lin Y, Sha Y, Pei YH. A new triterpene glycoside from
Asterias rollentoni. J Asian Nat Prod Res 2006; 8(7): 631−6.
[45] Silchenko AS, Kalinovsky AI, Avilov SA, et al. Triterpene glycosides from the sea
cucumber Eupentacta fraudatrix. Structure and biological action of cucumariosides
A1, A3, A4, A5, A6, A12 and A15, seven new minor non-sulfated tetraosides and
unprecedented 25-keto,25-norholostane aglycone. Nat Prod Commun 2012; 7(4):
517−25.
[46] Popov RS, Avilov SA, Silchenko AS, et al. Cucumariosides F1 and F2, two new
triterpene glycosides from the sea cucumber Eupentacta fraudatrix and their LC-ESI
MS/MS identification in the starfish Patiria pectinifera, a predator of the sea
cucumber. Biochem System Ecol 2014; 57: 191–7.
[47] Xiao G, Yu B. Total synthesis of starfish saponin goniopectenoside B. Chemistry 2013;
19(24): 7708–12.
[48] Dai Y, Yu B. Total synthesis of astrosterioside A, an anti-inflammatory asterosaponin.
Chem Commun 2015; 51(72): 13826–9.
[49] Xiong JL, Lu ZC, Ding N, Ren SM, Li YX. Synthesis of the pentasaccharide moiety of
thornasteroside A. Eur J Org Chem 2013; (27): 6158–66.
[50] Kicha AA, Kalinovsky AI, Malyarenko TV, et al. Cyclic steroid glycosides from the
starfish Echinaster luzonicus: structures and immunomodulatory activities. J Nat Prod
2015; 78(6): 1397–405.
222 N. V. Ivanchina, A. A. Kicha, T. V. Malyarenko et al.

[51] Quang TH, Lee DS, Han SJ, et al. Steroids from the cold water starfish Ctenodiscus
crispatus with cytotoxic and apoptotic effects on human hepatocellular carcinoma and
glioblastoma cells. Bull Korean Chem Soc 2014; 35(8): 233541.
[52] Tian XR, Tang HF, Lin HW, Cheng G, Wang SW, Zhang X. Saponins: the potential
chemotherapeutic agents in pursuing new anti-glioblastoma drugs. Mini Rev Med Chem
2013; 13(12): 170924.
[53] Thao NP, Luyen BTT, Kim EJ, et al. Asterosaponins from the starfish Astropecten
monacanthus suppress growth and induce apoptosis in HL-60, PC-3, and SNU-C5
human cancer cell lines. Biol Pharm Bull 2014; 37(2): 315–21.
[54] Cheng G, Zhang X, Tang HF, et al. Asterosaponin 1, a cytostatic compound from the
starfish Culcita novaeguineae, functions by inducing apoptosis in human glioblastoma
U87MG cells. J Neurooncol 2006; 79(3): 235–41.
[55] Zhao YC, Zhu CL, Li XF, et al. Asterosaponin 1 induces endoplasmic reticulum stress-
associated apoptosis in A549 human lung cancer cells. Oncol Rep 2011; 26(4): 919–
24.
[56] Zhou J, Cheng G, Cheng GA, Tang HF, Zhang XA. Novaeguinoside II inhibits cell
proliferation and induces apoptosis of human brain glioblastoma U87MG cells through
the mitochondrial pathway. Brain Res 2011; 1372: 22–8.
[57] Lee CC, Hsieh HJ, Hsieh CH, Hwang DF. Antioxidative and anticancer activities of
various ethanolic extract fractions from crown-of-thorns starfish (Acanthaster planci).
Environ Toxicol Pharmacol 2014; 38(3): 761–73.
[58] Saikia S, Kolita B, Dutta PP, et al. Marine steroids as potential anticancer drug
candidates: in silico investigation in search of inhibitors of Bcl-2 and CDK-4/Cyclin
D1. Steroids 2015; 102: 7–16.
[59] Thao NP, Cuong NX, Luyen BTT, et al. Anti-inflammatory components of the starfish
Astropecten polyacanthus. Mar Drugs 2013; 11(8): 2917−26.
[60] Thao NP, Luyen BTT, Koo JE, et al. Anti-inflammatory components of the
Vietnamese starfish Protoreaster nodosus. Biol Res 2015; 48: 12.
[61] Kawase O, Goo YK, Jujo H, Nishikawa Y, Xuan XN. Starfish, Asterias amurensis and
Asterina pectinifera, as potential sources of Th1 immunity-stimulating adjuvants. J Vet
Med Sci 2011; 73(2): 227–9.
[62] Jo WS, Choi YJ, Kim HJ, et al. Methanolic extract of Asterina pectinifera inhibits
LPS-induced inflammatory mediators in murine macrophage. Toxicol Res 2010; 26(1):
3746.
[63] Qi J, Luo Y, Gao L. Structural diversity of neuritogenic substances and their
application perspective. Mini Rev Med Chem 2011; 11(8): 658–77..
[64] Palyanova NV, Pankova TM, Starostina MV, Kicha AA, Ivanchina NV, Stonik VA.
Neuritogenic and neuroprotective effects of polar steroids from the Far East starfishes
Patiria pectinifera and Distolasterias nipon. Mar Drugs 2013; 11(5): 1440–55.
[65] Anisimov MM, Chaikina EL, Malyarenko TV, Kicha AA, Ivanchina NV. Efficiency of
the steroid glycosides from the starfish Asteropsis carinifera and heteroauxin to an
increase in the sprouts of the agricultural plants. Agrokhimia 2012; (3): 41–7.
[66] Go HJ, Jo MJ, Kim TY, et al. Biological activity of extracts of the starfish Asterias
amurensis. J Life Sci 2014; 24(5): 4917.
Recent Studies of Polar Steroids from Starfish 223

[67] Prabhu K, Bragadeeswaran S. Antibacterial activity of starfish Stellaster equestris


from Southeast coast of India. J Coast Life Med 2013; 1(3): 2106.
[68] Kicha AA, Ivanchina NV, Gorshkova IA, Ponomarenko LP, Likhatskaya GN, Stonik
VA. The distribution of free sterols, polyhydroxysteroids and steroid glycosides in
various body components of the starfish Patiria (=Asterina) pectinifera. Comp
Biochem Physiol B 2001; 128(1): 43–52.
[69] Demeyer M, De Winter J, Caulier G, Eeckhaut I, Flammang P, Gerbaux P. Molecular
diversity and body distribution of saponins in the sea star Asterias rubens by mass
spectrometry. Comp Biochem Physiol B 2014; 168: 1–11.
[70] Demeyer M, Wisztorski M, Decroo C, et al. Inter- and intra-organ spatial distributions
of sea star saponins by MALDI imaging. Anal Bioanal Chem 2015; 407(29): 8813-24.
[71] Naruse, M; Suetomo, H; Matsubara, et al. Acrosome reaction-related steroidal saponin,
Co-ARIS, from the starfish induces structural changes in microdomains. Dev Biol
2010; 347(1): 147–53.
[72] Kicha AA, Ivanchina NV, Stonik VA. Seasonal variations in the levels of
polyhydroxysteroids and related glycosides in the digestive tissues of the starfish
Patiria (=Asterina) pectinifera. Comp Biochem Physiol B 2003; 136(4): 897–903.
[73] Kicha AA, Ivanchina NV, Stonik VA. Seasonal variations in polyhydroxysteroids and
related glycosides from digestive tissues of the starfish Patiria (=Asterina) pectinifera.
Comp Biochem Physiol B 2004; 139: 581–5.
[74] Mackie AM, Singh HT, Owen JM. Studies on the distribution, biosynthesis and
function of steroidal saponins in echinoderms. Comp Biochem Physiol B 1977; 56(4):
9–14.
[75] Kapustina II, Stonik VA, Levina EV. In vitro biosynthesis of asterosaponins from
cholesterol and other sterols by homogenates of the gonads and pyloric caeca of
starfish. Khim Prirod Soedin 1985: 414–5.
[76] Ivanchina NV, Kicha AA, Malyarenko TV, Kalinovsky AI, Dmitrenok PS, Stonik VA.
Biosynthesis of polar steroids from the Far Eastern starfish Patiria (=Asterina)
pectinifera. Cholesterol and cholesterol sulfate are converted into polyhydroxylated
sterols and monoglycoside asterosaponin P1 in feeding experiments. Steroids 2013;
78(12-13): 1183–91.
[77] Kicha AA, Ivanchina NV, Kalinovsky AI, Dmitrenok PS, Stonik VA. Steroidal
monoglycosides from the Far Eastern starfish Hippasteria kurilensis and hypothetic
pathways of polyhydroxysteroid biosynthesis in starfish. Steroids 2009; 74(2): 238–44.
In: Advances in Natural Products Discovery ISBN: 978-1-53610-088-4
Editors: Ana Rita Gomes, Teresa Rocha-Santos et al. © 2017 Nova Science Publishers, Inc.

Chapter 7

STRATEGIES BASED ON MICROBIAL METABOLITES


FOR MICROBIAL CONTROL IN INDUSTRIAL
WATER SYSTEMS

Vera Lúcia dos Santos1, and Andrea Sousa Monteiro2


1
Department of Microbiology, Institute of Biological Science,
Universidade Federal de Minas Gerais, Belo Horizonte-MG, Brazil
2
Laboratory of Parasitic Biology – Universidade Ceuma, MA, Brazil

ABSTRACT
The control of microorganisms and their negative effects on performance of water
systems, such as in distribution pipelines, membrane-filtration processes and cooling
towers, is a serious operational challenge in all water sectors. Microorganisms and their
products form a matrix of protective and adhesive extracellular polymeric substances
(EPSs), mainly polysaccharides, lipids and proteins. Biofilm formation can lead to
decreased efficiency of heat exchangers, membrane reactors, and potable water
distribution systems, in addition to increasing the risk of occurrence of microbiologically
influenced corrosion. Cells show greater resistance to environmental challenges,
including biocidal agents, than their free-living counterparts, mainly due to polymeric
matrix barrier formation. Conventional disinfection and cleaning strategies do not
proficiently address biofilm-related problems, such as the persistence of microorganisms
and the generation of harmful disinfection products. Due to these limitations, ecologically
safe and more efficient alternatives are being sought to control biofilms in water systems,
such as the use of microbial hydrolytic enzymes, surface actives compounds, and phages,
as well as the use of quorum sensing (QS) inhibitors and energy uncoupling. These
biologically based microbial control strategies seem to constitute a promising component
of an efficiently integrated control program because they help to overcome the current
problems of biofilm control. Microbial metabolites can be effective in preventing
adhesion to surfaces, bacterial differentiation and matrix elimination and control of
planktonic microbiota. Furthermore, they are biodegradable, and their commercial

 Corresponding author. Laboratoy of Applied Microbiology, Department of Microbiology, ICB-UFMG, C.P. 486,
31270-901, Belo Horizonte, Brazil. E-mail: vlsantos@icb.ufmg.br.
226 Vera Lúcia dos Santos and Andrea Sousa Monteiro

production is cost-effective because the culture media components and substrates utilized
can be obtained from less expensive sources, such as agro-industrial waste. In this
review, we explore aspects of biofilm characteristics in water systems and examine the
potential of microbial metabolites for microbial control.

Keywords: biofilm, resistance, water system, microbial metabolites, surface actives


compounds

INTRODUCTION
Aquatic microbial communities occur as planktonic assemblages that develop in the water
columns of either natural or artificial environments and also as attached biofilm communities.
While planktonic and biofilm communities share similar biogeochemical and organic carbon
processing functions, they have markedly different physical structures and biotic interactions
that can lead to different responses to changes in the supply of resources and physical
environmental factors [1]. The ability of this diverse microbiota to attach to surfaces and to
develop into multispecies biofilm appears to be an ancient and integral feature of
microorganisms, which over evolutionary time has enabled them to optimize growth and
survival in adverse environments, such as flowing environments [1-3]. Thus, microorganisms
are able to colonize natural compartments, attached to plant and animal debris and submerged
or fluctuant macrophytes in water columns or attached to rocks immersed in the water of
rivers and marine environments, and artificial compartments, such as water-contact equipment
surfaces in diverse industrial sectors, including cooling towers, pipeline distribution systems
and water storage facilities or membrane reactors.
Biofilms comprise highly complex communities including bacteria, archaea, fungi, algae
and protozoa living among a protective matrix of extracellular polymeric substances (EPS),
comprised on polysaccharides, DNA, protein, particulate material and detritus [4]. A mature
biofilm has interwoven fluid channels that enable the transportation and transformation of
nutrients, gases and their associated waste products throughout the structure of the biofilm [5-
6]. Quorum sensing (QS) is bacterial density-dependent cell-to-cell communication using
small molecules produced and recognized by microbes. QS has been shown to regulate gene
expression, mediating some bacterial behaviors, such as the production of soluble microbial
products, secretion of EPS and extracellular enzymes, virulence factor production and biofilm
formation [7- 9].
Biofilm communities play important roles in the functioning of aquatic ecosystems. They
act as sites of essential ecological processes, such as current primary production, carbon and
nitrogen fixation and cycling of key nutrients, including phosphorus and nitrogen in
freshwaters. It is believed that less than 1% of microbial carbon cycling occurs within the
water columns of aquatic ecosystems [10].
The lifestyle of biofilm benefits microorganisms in various ways, including the efficient
use of resources due to the diverse metabolic capabilities of the different members of the
biofilm [11], the recycling of secreted end products, e.g., the usage of organic carbon
produced by autotrophs by heterotrophic bacteria [12-13], buffering against nutrient
limitations [14], protection from shear stress, oxygen radicals, and pH changes [3, 15-16] and
protection from predation [17]. Conversely, biofilms act as a basal food resource in aquatic
Strategies Based in Microbial Metabolites … 227

ecosystems, playing a major role in the bottom-up supply of nutrients to organisms at a higher
trophic level, potentially affecting food webs throughout the stream ecosystem. Thus, the
specific microbial composition of the biofilm can modify the nutritional quality of the
material for grazing species [18].
This pattern is also beneficial in artificial environments, in which it forms the
fundamental basis of treatment technologies, such as fluidized and fixed bath reactors or
membrane bioreactors (MBRs). However, in several industrial sectors, microorganisms can
accumulate in equipment, resulting in biofouling and biocorrosion both actively (due to the
consumption of oxygen by aerobic bacteria and/or the formation of a mass of occlusion that
creates an oxygen gradient) and passively (by silt deposition on the metal surface) [19]. Many
bacteria exhibit corrosive effects through the action of their metabolic end products, and
among these bacteria are the sulfate-reducing bacteria (SBR), acid-producing bacteria, such as
Thiobacilus, and hydrogen-utilizing sulfate reducers and iron bacteria, such as Gallionella,
which oxidize ferrous ions to ferric ions that can bind free chloride ions in solution to form
the ferric chlorides that are deposited as corrosion products. Microbiologically influenced
corrosion and associated biofouling have been implicated in high profile failures across a
wide range of environments, ranging from oil and water pipelines and nonmetallic materials,
such as concrete and machinery, to biomedical devices. The economic impact of corrosion is
significant given the need to replace corroded equipment, as well as repairs and attempts to
prevent corrosion [20-22].
Another important aspect is that biofilms formed on industrial equipment can include the
growth of microorganisms of sanitary interest, such as Legionella pneumophila in cooling
towers, which can be released into the environment through aerosol generated in the tower,
causing pneumonia-like symptoms in immunocompromised patients [23-24]. In systems of
potable water distribution, biofilm results in water flow contamination by biomass detachment
of opportunistic pathogens, such as Mycobacterium avium, Pseudomonas aeruginosa,
Klebsiella spp., Legionella spp., Flavobacterium spp., enteric viruses, Bacillus spores or
Cryptosporidium oocysts [25-33]. Biofilms in water systems also result in unpleasant taste
and odor [34]. In the beverage and food industry, biofilms on equipment surfaces act as
potential sources of product contamination with foodborne pathogens and spoilage
microorganisms [35]. Biofilms can also be important in the clinical setting when they develop
on medical devices, such as implants, catheters, and contact lenses, leading to life-threatening
infections [36].
Another problem is based on biofilm cells showing greater resistance to biocides and
stress environments than their planktonic counterparts, enabling the proliferation of biofilm in
industrial environments, presenting problems in removing them once established. Many
different mechanisms of biofilm resistance have been discussed in the literature, reflecting the
different manners in which biofilm organisms withstand biocides. These mechanisms include
physical and chemical diffusion-reaction barriers in the biofilm restricting biocide penetration
of the biofilm [37-38], the slow growth rate of biofilm cells due to nutrient limitations [39],
activation of general stress response genes [40], the emergence of a biofilm-specific
phenotype and the presence of persister cells [41-42].
It has been suggested that the EPS matrix can restrict the mass transfer of antimicrobial
agents for biofilm microorganisms by acting as a diffusion barrier or by the interaction of the
biocide with matrix components. Most traditional chlorinated oxidant biocides, such chlorine,
are consumed by reactions with corrosion products and deposits [43-44], pipe materials [45-
228 Vera Lúcia dos Santos and Andrea Sousa Monteiro

47] and biofilm constituents, including cells and EPS [6, 48]. In fact, field observations and
laboratory studies have indicated that biofilms seem to be recalcitrant to the killing and
disrupting activity of chlorine in concentrations that are relevant in practice, whereas
planktonic cells are more easily inactivated by the biocide. Planktonic control cells of
Streptococcus thermophiles were effective after exposure to 20 mg/L of sodium hypochlorite
for 30 min (pH 6.8–7.0), whereas viability of this organism grown as a biofilm on stainless
steel was still detected after treatment with up to 1,000 mg/L of sodium hypochlorite [49].
Mathieu et al. (2009) showed that biofilms grow and harbor active bacteria, even in
continuously chlorinated water systems with residual levels of chlorine ranging from 0.1 to
0.4 mg Cl2/L [50].
Several studies have also shown that chlorine had a slight weakening effect on P.
aeruginosa biofilms [51], but it was more effective in weakening the mechanical cohesiveness
of S. epidermidis biofilm and eroding the attached biomass [37], and it induced detachment
and killing of Pseudoalteromonas ruthenica at 1 g/L [52]. Tachikawa et al. showed an
apparent decrease in EPS in the biofilm matrix exposed to halogenated oxidants or ozone, as
well as a clear relationship between the removal of EPS and the bacterial inactivation rate
[53].
Chlorine-based biocides, monochloramine and chlorosulfamates were reported to
penetrate better into biofilms than free chlorine because they seem to have a lower capacity to
react with biofilm constituents [38]. As a result, these compounds can exhibit similar or
superior efficacy when tested against biofilms, despite being weaker biocides than chlorine
[54]. For example, it was demonstrated that, for equivalent chlorine concentrations,
monochloramine initially penetrated biofilm 170 times more rapidly than free chlorine [38].
Concentrations as low as 1 ppm were able to penetrate biofilm matrices, such as those in
cooling towers [54], and residuals of 2-3 mg/L of Cl2 were needed to control biofilms
effectively in cooling systems using tertiary treated wastewater as makeup water [55].
Subsequently, it was shown that EPS quantity and composition significantly affected
monochloramine penetration, biofilm inactivation, and detached cell viability due to the
selective reactivity of monochloramine [56]. Biocide has selective reactivity with proteins
over polysaccharides. Thus, the presence of monochloramine-reactive or monochloramine-
nonreactive components in biofilm matrix can have great influence on monochloramine
transport, as well as disinfection efficacy. Recently, Stewart [57] examined the tolerance of
microorganisms in biofilms to antimicrobials through a meta-analysis of data from the
literature. It was discussed that the biofilm tolerance variation was not explained by consider
the size and chemistry of the antimicrobial, the substratum material, or the microbial species
composition. The antimicrobial was partially correlated with the cell density of the biofilm at
the time of treatment and the age of the biofilm as grown in a particular experimental system,
suggesting that there is something occurring during biofilm maturation, either physical or
physiological, that is essential for full biofilm tolerance.
Another problem is that the greater reactivity of chlorine with natural organic matter can
lead to the formation of by-products, such as trihalomethanes or haloaceto-nitriles. As a
result, chlorinated effluents can have adverse impacts on human health and aquatic
ecosystems because the effluent can contaminate downstream drinking water sources.
The aforementioned points show clearly that biofilms can potentiate various problems
related to the presence of microorganisms in different environments. Thus, microbial control
programs in industry should be directed to prevent the adhesion of microorganisms to surfaces
Strategies Based in Microbial Metabolites … 229

and/or to prevent colonizing organisms from building up to problematic levels or to break up


the biofilm EPS matrix to disperse the biofilm or alter the elastic properties of biofilms and
allow for biocides to diffuse rapidly and kill microbial cells.
A variety of strategies for biofilm control has been used, including periodic cleaning
using physical methods (rinsing, brushing, ultrasonic), application of chemical agents
(oxidants, alkali, surfactants, enzymes, complexing substances, dispersants) to kill and detach
biofilm cells and limitations of nutrients in water systems to minimize microbial growth.
Considering the inefficacy and products generated, the use of innovative strategies that are
less harmful to the environment to optimize the biofilm control has been evaluated. In this
regard, biologically based anti-biofilm strategies to enhance the effectiveness of currently
applied cleaning protocols in industry have been proposed. These strategies include the use of
metabolites that target a specific molecule or mechanism that is responsible for the
attachment, communication, motility or growth of microbial cells and the biofilm matrix
structure. Therefore, this chapter sheds light on the state of the art of potential strategies to
control biofilms based on hydrolytic enzymes, biosurfactants, QS inhibitors and phages.
However, because most of the biologically based anti-biofilm strategies are still under
development, the challenges and limitations will also be discussed.

BIOLOGICAL ANTI-BIOFILM APPROACHES TO PREVENT OR


REMOVE BIOFILMS
A large number of new biological approaches have been developed in the last few years
for biofilm control. They include: bioactive microbial metabolites, such as biosurfactants,
exopolysaccharides and bacteriocins [58-59]; enzymes that dissolve biofilms by
depolymerizing polysaccharides, proteins or extracellular DNA [60-61]; uncoupling energy
[62]; the use of QS inhibitors [63]; and lytic phages [64]. These approaches can in some
situations have the advantages of greater efficiency, lower toxicity, greater sustainability and
less bacterial resistance relative to other control methods.

Strategies Based on Biosurfactants

Microorganisms are able to produce a wide diversity of surface-active compounds


(SACs) that present both hydrophilic and hydrophobic moieties, and these molecules can
interact with surfaces, lowering surface and interfacial tensions, and form micelles, which are
emulsifying immiscible substances. The microbial SACs can be distinguished in terms of size,
and they include: (1) biosurfactants that are low-molecular-weight surfactants; (2)
amphiphilic polymer that are high-molecular-weight surface-active polymers with one
hydrophobic region at one end of the molecule, such as lipopolysaccharides, lipoteichoic
acids and lipoglycans on bacterial cellular walls; and (3) polyphilic polymers that are high-
molecular-weight surface-active polymers with hydrophobic groups distributed across the
entire molecule, identical to hydrophobically modified, comb-type polymers, such as emulsan
and hydrophobic polysaccharides [65].
230 Vera Lúcia dos Santos and Andrea Sousa Monteiro

Another criterion for categorizing microbial SACs is the chemical nature of the
molecules. The major classes include varied structures, such as glycolipids, lipopeptides,
polysaccharides or protein complexes, phospholipids, fatty acids and neutral lipids. These
biomolecules can be excreted into the extracellular medium or remain attached to the cell
surface, denominated as particulate biosurfactants [66].
Microbial SACs have been recognized for some time in a diverse array of potential
applications in industries including agriculture, food, cosmetics, and the pharmaceutical and
petroleum industries. The surface and interfacial tension-reducing properties of biomolecules
provide excellent detergency, emulsification, foaming and dispersing traits, making them
some of the most versatile products in chemical processes. In addition, they have shown their
specificity, low toxicity, high biodegradability, widespread applicability and effectiveness at
extremes of pH and temperature [66-70]. In the biomedical field, biosurfactants are receiving
more attention for their anti-biofilm and antimicrobial activities, due to their lower toxicity to
plants and animals, high biodegradability, and low irritancy to and compatibility with human
skin [71].
The physiological roles of these biomolecules are often unclear, but many of them are
essential for the survival of microorganisms in the environment, including increased surface
area and bioavailability of hydrophobic substrates, antagonistic activity, binding to heavy
metals, bacterial pathogenesis, QS, biofilm formation and desorption surfaces [72]. These
tensoactive molecules can spontaneously adsorb to surfaces, altering properties such as
wettability and charge or even changes in bacterial cell surface structures, thus modeling
bacterial adhesion to surfaces [65, 73]. A loss of cell outer membrane lipopolysaccharide
protein complexes was observed in P. aeruginosa treated with mono-rhamnolipids (RLs),
resulting in increased cell surface hydrophobicity [74]. RL biosurfactants are important in
maintaining transport channels, and they directly influence biofilm structure, namely the
creation of mushroom-like filaments, and exclude other invasive species from the biofilm
structure [75-76]. Other example is a 546-KDa exopolysaccharide (A101) isolated from a
marine Vibrio that inhibited initial adhesion of both Gram-negative and Gram-positive
bacteria, selectively affected cell-to-cell interactions and induced biofilm dispersion of P.
aeruginosa [77].
Because of their tensoactive properties, many SACs show antibacterial, antifungal and
antiviral activities. These properties are the result of the action of these molecules, increasing
membrane permeability and causing metabolite leakage by altering physical membrane
structures or by disrupting proteins, thus, interfering with important membrane functions,
such as energy generation and transport [78]. This finding has also been reported to lead to
toxicity, lysis, pyrogenicity, mitogenicity and immunogenicity, among other effects. Several
studies have demonstrated that, under certain conditions, SACs can be more effective than
many traditional biofilm inhibition and/or disruption strategies.
This antimicrobial property of SACs has been extensively explored with a view toward
clinical application, but there have been few studies focusing on applications of these
biomolecules in the water industry. RLs were successively used to clean ultrafiltration
membranes fouled by proteins. The strategy could largely remove fouling from polysulfone,
polyacrylonitrile and polysulfone-g-poly-ethylene glycol membranes and restore the water
flux to approximately 94% of the initial level, performing much better than the flux recovery
of 50–70% for Tween 20 and SDS [79]. BS also exhibited superior properties over sodium
hydrate (NaOH) and commercial membrane cleaner, considering the cleaning efficiency and
Strategies Based in Microbial Metabolites … 231

operation mildness. RLs were also able to disrupt preformed biofilms of Ps. aeruginosa [80].
A commercially available RL was assessed for its ability to inhibit adhesion and disrupt
Bacillus pumilus pre-formed biofilms isolated from the surfaces of titanium coupons
immersed in seawater. Titanium surfaces are generally used for the manufacture of
condensers and other heat exchangers in power plants, which are subjected to microbial
corrosion. The biomolecule inhibited the planktonic growth of B. pumilus cells at
concentrations >1.6 mM, and concentrations from 0.05 to 100 mM inhibited the adhesion of
cells to polystyrene microtiterplates, wherein the effectiveness ranged from 46 to 99%. The
rhamnolipid was effective against pre-formed biofilms, acting on the removal of biofilm-
matrix components and disruption of biofilms [81]. RLs at 10 and 200 mg/L reduced the
initial attachment of the bacteria P. aeruginosa, P. putida, and Escherichia coli and of
Bacillus subtilis on hydrophilic glass and hydrophobic octadecyltrichlorosilane (OTS)-
modified glass under continuous-flow conditions. For Staphylococcus epidermidis, the effect
occurred only on hydrophobic surfaces [82].
Apparently, treatment of biofilms with biosurfactants that modify the structure of biofilm
matrix would make them an attractive choice as chemical agents that can enhance the efficacy
of antibacterial agents, for example, in cooling water systems for enhancing the efficacy of
chlorinated biocides in microbial control. RLs from Pseudomonas spp. enhanced the
disinfection effects of NaOCl and peracetic acid/hydrogen peroxide on stainless-steel surfaces
contaminated with Listeria monocytogenes [83]. Synergistic enhancement was reported of the
killing effects of silver ions and lipopeptide BSs against an E. coli biofilm population to
achieve complete biofilm eradication [84]. The authors also hypothesized that V9T14
lipopeptide interacts with the bacterial membrane, leading to pore formation and alteration of
membrane integrity; this effect increases the entry of several antimicrobials into the cells and
their effectiveness against uropathogenic E. coli CFT073 biofilm [85]. Our group showed that
the combination of sub-inhibitory concentrations of the Lactobacillus jensenii P6A
biosurfactant and benzazole compounds presented synergic effects that were concentration-
dependent against E. coli and C. albicans.
Qin et al. (2012) evaluated a novel submerged membrane bioreactor (SMBR) combined
with rhamnolipids (RSMBR) to treat frying oil wastewater and to control the problem of
membrane fouling [86]. RSMBR not only exerted high removal efficacy of oil of up to 90%
in a short hydraulic time, but it also exhibited 10 times greater membrane permeability than
the reactor without RLs. The presence of rhamnolipids greatly enhanced the contact and
reaction between the microorganism and oil molecules. Great improvement in membrane
filterability was associated with an increase in the hydrophobicity of flocs, as well as the
increase in particle size from 53.06 to 145.54 µm. The oil strongly adhered to the surfaces of
flocs by rhamnolipids, and it consequently prevented larger oil droplets from directly
depositing on the membrane surface.

Strategies Based on Enzymes

Enzymes can affect the colonization and adhesion of microorganisms to surfaces and can
result in biofilm control in several manners. They can affect the adhesion to surfaces of
settling organisms, thus preventing settlement events [87-88]. Second, specific enzymes can
hinder intercellular communication during colonization of a surface [89]. Finally, hydrolytic
232 Vera Lúcia dos Santos and Andrea Sousa Monteiro

enzymes can effectively promote the matrix degradation of EPS in multi-structured biofilms,
altering the physical properties of biofilm. The most commonly used are amylases,
hydrolases, glycosidases, lipases, acidic and alkaline proteases and deoxyribonucleases [90-
93]. This approach has the advantages of being non-toxic and less susceptible to the
development of bacterial resistance, which are mechanisms commonly observed for many
anti-biofilm agents.
Microbial EPS have different substituent groups, such as ketal-linked pyruvate or ester-
linked acetyl groups, and the removal of these groups can affect the physical properties of
exopolysaccharides. Intracellular carboxylesterase (EC 3.1.1.1), isolated from Arthrobacter
viscosus, removed acetyl residues from xanthan, alginate, glucose penta acetate, cellobiose
octaacetate, exopolysaccharide produced by A. viscosus, deacetylated p-nitrophenyl
propionate, naphthyl acetate, isopropenyl acetate and triacetin [94]. Pectin esterase,
originating from Trichoderma viride, could deacetylate a polysaccharide in Pseudomonas
fluorescens biofilm matrix, rendering it softer and more porous [95-96].
A crude cellulase preparation from Trichoderma viride was effective in the degradation
of dephosphorylated and partially derhamnosylated EPS of Lactococcus lactis subsp.
cremoris B40 [97]. Cellulase from Penicillium funiculusum was effective in degrading mature
biofilms of P. aeruginosa and the exopolysaccharides of P. fluorescens [98-99]. The enzyme
reduced the molecular weight of the polymers in assays using purified biofilm EPS.
Actinobacillus actinomycetemcomitans produces an hexoaminidase known as dispersin B
that hydrolyses the glycosidic linkages of polymers that contain β-1,6-N-acetyl-D-
glucosamine (poly-β-1,6-GlcNAc) (PIA/PNAG), which serves as an Escherichia coli and
Staphylococcus epidermidis biofilm adhesin, the formation of which requires the pgaABCD
and icaABCD loci, respectively. The hydrolysis of this polymer by this enzyme disrupts
biofilm formation by these species and also by Yersinia pestis and P. fluorescens, which
possess pgaABCD homologues [60]. In general, S. epidermidis biofilms are dispersin B
susceptible and DNase I resistant, whereas S. aureus biofilms are DNase I susceptible and
dispersin B resistant, suggesting that PNAG is a major matrix adhesin in S. epidermidis
biofilms and a minor component of S. aureus biofilms [100]. Precoating polyurethane and
Teflon catheters with this enzyme prevented S. epidermidis biofilm formation; the modified
polyurethane catheters retained enzyme activity for at least 30 days when stored at room
temperature [92]. The combined use of dispersin B and rifampicin was effective in eradicating
the S. epidermidis biofilm that developed in poly(dimethylsiloxane)(PDMS) microfluidic
devices [101].
Proteolytic enzymes have been employed to remove established biofilms. Proteinase K
caused 98% detachment of 53 biofilm-positive clinical S. haemolyticus isolates [102].
Addition of this enzyme or DNase I to culture medium also inhibited biofilm formation by
nontypeable Haemophilus influenzae. Both enzymes also caused significant detachment of
pre-formed biofilms of this strain, indicating that both proteinaceous adhesins and
extracellular DNA contribute to biofilm cohesion [103]. Leroy et al. (2008) investigated the
antifouling potential of savinase (subtilisin) on adhesion of Pseudoalteromonas sp. D41
[104]. The enzyme should be more effective in preventing initial microbial adhesion than
disrupting established biofilm because the IC50 of subtilisin was found to be 38 times lower
for the prevention of microbial adhesion than for the detachment of adhered bacteria.
Extracellular DNA (eDNA) could play important roles in biofilm development, including
supply substrates for sibling cells, maintaining the three-dimensional structure of biofilms and
Strategies Based in Microbial Metabolites … 233

enhancing the exchange of genetic material [61, 105]. Thus, disruption of eDNA would lead
to the detachment or dispersal of biofilms. Whitchurch et al. (2002) showed that
deoxyribonuclease I could inhibit the development of biofilm and dissolve established
biofilms of P. aeruginosa PAO1 [106]. Alterations in the biomass, architecture, morphology,
and numbers of colony-forming units (CFU) of biofilms formed by diverse bacteria (Gram-
positive and Gram-negative bacteria) were observed in the presence of DNase [107]. The
addition of this enzyme also enhanced the effects of antibiotics, resulting in decreased biofilm
biomass and numbers of CFU, probably because the cleavage of extracellular DNA led to the
formation of an altered biofilm that permitted the increased penetration of antibiotics.
Some hydrolytic enzymes of bacterial cell walls have been described by their
antimicrobial activities. An example is lysozyme, which targets the 1,4 glycosidic bonds that
link the N-acetylglucosamine (NAG) and N-acetylmuramic (NAM) acid moieties that
compose the bacterial cell wall peptidoglycan. The enzyme immobilized on the surface of
polyethylene and polyvinylalcohol films exhibited activity against Micrococcus lysodeikticus
[108-109]. This enzyme also reduced by 28.2–69.6% the metabolic activity of Candida
biofilms on acrylic dentures [110]. Lysozyme combined with the antifungals nystatin,
amphotericin B, and ketoconazole resulted in effective synergistic killing of biofilm of
Candida. Combination with imidazole lanoconazoles resulted in synergistic antifungal
activity against C. albicans blastopores [111]. Another example consists of endolysin SAL-1
and LysK, which are produced by phages [112].
There are two main alternatives for developing efficient enzymatic products for biofilm
control. The first concerns the identification of the polysaccharides present in the biofilm and
the choice of specific enzymes capable of degrading them. The other aims to identify the
active compounds in commercial products, followed by evaluation of their effects on biofilm.
In this latter approach, the specificity in the enzyme modes of action makes it a complex
technique, increasing the difficulty of identifying enzymes that are effective against all of the
different types of biofilms. Therefore, formulations containing several different enzymes
seem to be fundamental for a successful biofilm control strategy.
An example of the first case is the study by Marcato-Roamin et al. (2012), based on the
characterization of the EPS extracted from six industrial biofilms from the paper industry and
the subsequent evaluation of the effectiveness of eight hydrolytic enzymes in reducing them
[113]. The EPS were mainly proteins, and the protein-to-polysaccharide ratio ranged from 1.3
to 8.6 depending on where the sampling point was situated in the paper-making process.
Glycosidases and lipases were inefficient or only slightly efficient for biofilm reduction, while
proteases were more efficient. After treatment for 24 h with pepsin, Alcalase® or Savinase®,
the removal exceeded 80%. When tested on an industrial biofilm sample, Savinase® led to a
significant release of proteins from the EPS matrix, indicating its potential efficacy on an
industrial scale. This enzyme was successively used in combination with shear stress
treatment. The combined treatment led to an increase of 80% in biofilm mass removal (COD)
compared enzymatic treatment alone, and it removed a large proportion of the basal layer of
the biofilm, with 80% reduction observed in the support coverage [114]. It was shown that
colanic acid, commonly produced by some enterobacterias and observed in biofilms from
different paper mills, could be hydrolyzed to its corresponding hexasaccharide repeating unit
by β(1,4)-fucanosyl hydrolase [115]. Other example is levan, which is a β-2,6-linked polymer
of fructose that is normally present in biofilms of several species of Bacillus and
234 Vera Lúcia dos Santos and Andrea Sousa Monteiro

Pseudomonas. Levan could be hydrolyzed by the enzyme levan hydrolase to low-molecular-


weight polymers that are water soluble, thereby cleaning the slime out of the system [116].
Chaignon et al. (2007) investigated the susceptibility of biofilms of clinical
staphylococcal strains to a range of enzyme preparations containing dispersin B, pancreatin,
proteinase K, Pectinex Ultra SP, periodate and trypsin [117]. Whereas dispersin B was the
most effective against strains that contained N-acetylglucosamine as the major component in
the extracellular biofilm matrix, these biofilms were not affected by protease treatment. The
proteases were effective against strains that lacked N-acetylglucosamine. Therefore, a
recommendation for the complete removal of biofilms formed by a range of staphylococcal
strains from inert surfaces could be the combined use of dispersin B and protease.
Torres et al. (2011) evaluated the efficacy of 17 non-specific, commercial enzymatic
mixtures for the prevention and control of biofilm formed by bacteria isolated from paper
mills on laboratory and pilot plant scales [118]. Pectinex Smash® and its fraction
Novoshape® were the best formulations in the prevention of biofilm formation, and they are
predominantly composed by the pectin methylesterase. This enzyme was able to reduce
biofilm formation by 71%, compared to control tests.
In another study, Leroy et al. (2008) evaluated the antifouling potential of four proteases,
seven glycosidases and one lipase in the adhesion of marine Pseudoalteromonas sp. D41
[119]. Savinase (subtilisin) was the most effective hydrolase in preventing bacterial adhesion
and removal of the adhered bacteria. Proteases were also evaluated to remove biofouling from
ultrafiltration membranes for wastewater treatment [120]. Enzymatic treatment showed
greater efficiency in removing biofouling, leading to complete recovery of clean water flux at
low temperatures (25-30°C), compared to traditional cleaning methods based on alkaline
chemicals.
Molobela et al. (2010) tested selected commercial proteases (savinase, everlase and
polarzyme) and amylases (myloglucosidase and bacterial amylase novo) for their
effectiveness in the degradation and removal of EPS in P. fluorescens biofilm [121]. Savinase
(subtilisin) and everlase were the most effective for degradation of P. fluorescens EPS, while
the protease polarzyme was ineffective. The reason for the inefficiency of polarzyme could be
attributed to its lack of action on the protein structural components of the biofilm EPS that
was evaluated. In addition, Lequette et al. (2010) also evaluated the activity of
polysaccharidases and proteolytic enzymes against the biofilms of 16 bacterial species found
in food industry processing lines, using a microtiter plate model [93]. The two serine
proteases removed the biofilms of a broader range of bacterial species, while the amylase S1
totally removed the biofilms of three Pseudomonas strains. The authors also evaluated the
effects of two serine proteases, amylase S1, and polysaccharides mix A in the removal of
biofilms formed on surfaces of stainless steel slides in a CIP procedure. The efficacy of
enzymes depended on the bacterial species; proteases were more efficient than
polysaccharidases in removing Bacillus biofilms, while polysaccharide-degrading enzymes
were more efficient in removing P. fluorescens biofilms.
Despite the potential use of enzymes to control microbial biofilm, some drawbacks
inherent to the method could limit its large-scale application. Enzyme activity would be
reduced or even totally lost in operations that lack the optimal pH and temperature values
required by the enzyme. To circumvent this problem, various approaches are being used to
increase the stability of enzymes, including enzyme modification, protein engineering and
medium engineering. New techniques, such as self-immobilization of enzymes, the
Strategies Based in Microbial Metabolites … 235

immobilization of enzymes using nanoscale structures, and the production of single-enzyme


nanoparticles, are also currently gaining a great deal of attention for this purpose [122].
Because EPS secreted by microorganisms is a mixture of macromolecules, its efficient
removal by enzymatic disruption will depend on the availability of various enzymes. Another
alternative is the combined use of enzymes and diverse chemical and physical agents for the
complete removal and/or elimination of microbial cells associated to the biofilm. Many
studies and patents have focused on these approaches.
The patents involve: mixtures of enzymes and a surface active agent, preferably anionic
[123]; at least one enzyme belonging to the polysaccharidases, proteases, lipases and glycol
proteases or lipases and a short-chained glycol component [124]; enzyme mixing, consisting
of 2 parts cellulose to 1 α-amylase to 1 protease utilized in 2-100 parts per million [125]; at
least one mannanase, optionally in combination with at least one enzyme from the group
consisting of carbohydrases, proteases, lipases, glycoproteases [126]; composition for
removing biofilm from a surface, comprising an enzyme mixture with at least two different
enzymes selected from protease, cellulase, esterase, mannanase, glucanase, phospholipase and
amylase [127]; methylene-bis-thiocyanate, dimethyl dithiocarbamate or disodium ethylene-
bis-dithiocarbamate as a biocide and amylase, as well as a dextran-degrading enzyme or a
levan hydrolase as the polysaccharide-degrading enzyme [128]; application of biocides, such
as chlorine, hypochlorite, bromine, hydrogen peroxide, in concentrations of 0.5-500 ppm and
trypsin and/or endo-protease and/or chymotrypsin in approximately 0.01-1,000 units to inhibit
the growth of filamentous organisms [129]; an acellular dispersant produced by P.
aeruginosa, which induces dispersion of sessile bacterial cells, and an additive (biocide,
surfactant, antimicrobial, antiseptic, detergent, chelating agent or virulence factor inhibitor)
[130]; a patented compound and an enzyme selected from oxidoreductase (oxidative/reductive
enzyme), transferase (transferring enzyme), hydrase (hydrolytic enzyme), lyase (isomerizing
enzyme), and isomerase (isomerizing enzyme) and a surfactant [131]; a mixture of enzymes
(at least one protease, one esterase and one amylase) and an alkaline detergent [132]; and an
EDTA chelating agent, a N-acetyl cysteine antioxidant or derivative salts, and an optional
carrier [133].
Oulahal-Lagsir et al. (2003) investigated the removal of biofilms formed by E coli from a
stainless steel surface using combined ultrasonic and enzymatic treatment (proteolytic or
glycolytic enzymes) [134]. The combined treatment was more effective in removing all
biofilms than the application of ultrasound alone. Similarly, Oulahal et al. (2007) reported that
application of ultrasound alone was not effective in removing all biofilms formed by two meat
spoilage microorganisms (E. coli and S. aureus) from a stainless steel surface [87]. When
ultrasound was used in combination with EDTA and/or enzymes, approximately 75% of E.
coli and 100% of S. aureus biofilms were removed.

Strategies Based on Phages

Phages are viruses that infect bacteria, presenting several morphologies, including
filamentous, icosahedral with tails, tail-phage, phage with lipoprotein envelope or phage with
lipid reservoirs [135]. Considering the course of infection, viruses can exhibit several life
cycles, including lytic, chronic, lysogenic and pseudolysogenic [136]. Infection initiates by
attachment of the virion to a susceptible bacterial cell by binding to the receptors of the
236 Vera Lúcia dos Santos and Andrea Sousa Monteiro

phages, such as lipopolysaccharides (LPSs) and proteins of the outer membrane, fimbriae,
pilli, flagella, capsular and slime polysaccharides, followed by injection of the virion or its
nucleic acid into the cell. Then, the cell biosynthetic apparatus is altered so that virally
encoded enzymes and genetic material are produced for the assembly of the capsid shells and
packaging of the nucleic acids within them. The mature virions are released from the cell by
either nondestructive budding or lysis, characterizing a chronic or lytic cycle, respectively.
The specificity of the receptors determines the host range of the phages. These monovalents
are able to adsorb to the specific bacterial species or strains, while polyvalent phages can
infect different bacterial species or genera. In the lysogenic cycle, the viral genome integrates
into the host chromosome, remaining in a state of prophage until induction of the lytic cycle
through the activation of a suitable set of cellular triggers in response, for example, to the
nutrient levels, oxidative stress caused by UV radiation and the presence of hydrogen
peroxide [137-138]. However, environmental signals and molecular mechanisms that control
prophage induction/excision under biofilm conditions remain elusive for most species [139].
The pseudolysogenic cycle is an intermediary state between lytic and lysogenic stages, in
which an extrachromosomal virus replicates in synchrony with the host chromosome, such as
an episome.
Due to the their nature, the phages are good candidates for biofilm control, with particular
reference to the following characteristics: broad spectrum of lytic activity; generation of
minimal numbers of resistant mutants; relatively simple preparation at high titers; long-term
storage; rapid reproduction: minimal latent period with high yield; absence of lysis inhibition;
lack of transduction; phage capacity for mutations or recombinant formation to overcome
simultaneously several types of resistance; and capacity of phages from the same mixture for
the cross-lysis of mutants [140-141]. In addition, phages can replicate at the site of an
infection, thereby increasing in numbers where they are most required, and some phages
produce enzymes that degrade the EPS matrix of biofilms. The phages are also non-toxic to
animals and plants.
The effectiveness of the use of phages in microbial control depends on several factors,
including the bacteriophage-to-target bacteria ratio, the mode of treatment, the age of the
biofilm during treatment, the neutralization of phages and the accessibility to target bacteria
[140, 142-143]. The biocidal activity of phages must be investigated in environmental
conditions relevant to their potential applications, considering that the lytic activity and
stability of phages are also variable depending upon the conditions of the environment, such
as pH and temperature.
The use of bacteriophages in microbial control has been studied using two different
approaches: passive or active treatment. In the passive approach, the bacteriophages are
applied at a dose sufficient to ensure that all target bacteria are infected and lysed in a short
period of time. In contrast, active treatment relies on the addition of a relatively small dose of
phages because most bacteria are killed by secondary infections due to replication and
transmission from neighboring cells. This approach is dependent on the phages being able to
spread between the target hosts, which could be weakened by the biochemical and physico-
chemical characteristics of the surrounding system, such as viscosity, or by the presence of a
greater number of inert bacteria.
The bacteriophage-to-bacteria ratio is explained by the term MOI (multiplicity of
infection), which expresses the number of viruses that are added per cell during infection.
This term is used only in fluid systems with large numbers of host cells, and most in vitro and
Strategies Based in Microbial Metabolites … 237

in vivo assays of phages against bacteria apply MOIs between 0.01 and 100. Considering that
not all phages replicate and survive in the same Fashion, a correct definition of the
bacteriophage-to-bacteria ratio requires the determination of the lytic cycle replication and
phage resistance under different environmental conditions [140]. The change in viral ratio can
improve the treatment efficacy, as observed in a study conducted with P. aeruginosa [143]. At
concentrations of 400 and 4x107 FPU/mL, the phages inhibited P. aeruginosa biofilm
formation by 45± 15% and 73±8%, respectively. For pre-existing biofilm treatment, similar
results were obtained; biofilm removal efficiency was increased in rate from 45± 9% to
75±5% when the phage dose increased from 6x103 to 6 x107 PFU/mL.
Weld et al. (2004) showed that targeted bacteria concentrations and seeded phage density
affected the success of infection [144]. Growth of T4 only occurs when the bacterial
concentration is greater than 20,000 (CFU/mL) because the phages cannot contact bacteria
efficiently for propagation. The growth phase of host bacteria can also affect infection
efficacy. Because the isolated Pseudomonas phages were RNA ones [143], it was expected
that they would infect bacteria by first attaching to pili and then by entering the bacterial cells
though F-pilus retraction [145-146]. Because F piliation reaches a maximum in the mid-
exponential phase and disappears in the late-exponential phase [147], the growth status of
target bacteria can affect the success of phage treatment as well. In addition to host cell
concentrations, nutrient limitations, competition from other phages and the presence of phage-
resistant cells can also affect replication [148].
Other studies have discussed the effect of biofilm age on the efficacy of phage lysis.
Ganegama Arahchi et al. (2013) explored the potential of individual phages, along with a
three-phage cocktail, to clear L. monocytogenes mixed-strain biofilms adhering to stainless
steel coupons, including ones contaminated with fish proteins at low temperatures (15°C)
[142]. Multi-log10 removal, including to the point of apparent sterilization, was observed with
younger biofilms, but partial physical removal of biofilm bacteria from surfaces was required
for similar results in the treatment of older, more mature biofilms. This fact was attributed to
the shielding effect of the biofilm matrix and/or the phage resistance of biofilm cells deep in
the biofilm matrix. Soni and Nannapaneni [149] also observed that the efficacy of phage lysis
was affected by the age of the biofilm. The 24 h treatment of phage P100 (9 log10 PFU/ ml) in
2- and 7-day biofilms formed by five strains of L. monocytogenes on stainless steel coupon
(initial cell counts of 7 and 6.6 log10 CFU/cm2, respectively) resulted in decreases in the
biofilm cells by 5.4 and 3.5 log units, respectively, at 22°C.
The temperature would be a sensitive parameter in both the growth of bacteria and
infection of the bacteria with the phage. A previous study showed that, whereas the burst size
of E. coli phage PR4 was approximately 40 from 30 to 42°C, the burst size at 20°C was less
than 3 [150]. When the temperature dropped to 0°C, approximately 80% of phage infectivity
was lost [151]. In contrast, a high temperature (37°C) inhibited phage infection of both
planktonic and biofilm cells of P. fluorescens [152].
Biofilm control using phages can involve phage application prior to biofilm formation for
control planktonic cells, application to already formed biofilms, which impacts the biofilm
structurally by lysis of producing EPS cells, and matrix rupture by the depolymerase enzyme
produced by phages [153-154]. This control results in the release of new phage virions that
can potentially reach and then infect adjacent bacteria. It was proposed that the progeny phage
would propagate radially through a biofilm by cycles of replication and cell lysis. At least in
238 Vera Lúcia dos Santos and Andrea Sousa Monteiro

theory, a single phage dose should be capable of treating a biofilm infection because the
progeny phage infects adjacent cells and degrades the biofilm matrix [155-156].
A model ultrafiltration (UF) continuous recycled system fed with two previously
sterilized source effluents was experimentally inoculated with three bacterial species
(Pseudomonas aeruginosa, Acinetobacter johnsonii and Bacillus subtilis) (separately and
combined) and specific lytic bacteriophages [157]. The seeded phages’ lytic activity reduced
membrane biofouling by an average of 40% to >60%, compared to controls. The concentrated
phage numbers increased accordingly, and some were found in the permeate, but inoculated
bacteria were not found in the permeate.
To penetrate EPS layers, some phages carry EPS depolymerases as tail spikes or tail
fibers as part of the viral particle to enable them to reach the bacterial cell wall [158].
Consequently, phages cause biofilm and capsule disruption by cell infection and lysis, as well
as by EPS degradation. The group includes alginate lyases, amylases, cellulases, dextranases,
endohexosaminidases, exopolygalacturonic acid lyases, galactosidases, glucosidases,
guluronan lyases, hyaluronate lyases, and pullulanases. The breakdown of EPS has the
potential to increase phage penetration into biofilms, thereby improving phage acquisition of
target bacteria.
A polysaccharide depolymerase of bacteriophages promoted substantial degradation of
mono species biofilms of Enterobacter agglomerans GFP that were phage-susceptible [159],
and 60 min of treatment with a polysaccharase caused a 20% reduction in dual-species
biofilm adhesion [160]. Cornelissen et al. (2011) investigated the in vitro biofilm degradation
capacity of a lytic P. putida phage φ15 with associated EPS depolymerase on different aged
mono-species biofilms of P. putida strains, PpG1 and RD5PR2 [161]. The phage was able to
infect seven strains of P. putida in a group of 53, in addition acting differentially in breaking
down PpG1 RD5PR2 biofilms. It was hypothesized that EPS material serves as a primary
bacterial receptor for phage adsorption and that specific adsorption to and disruption of this
receptor by depolymerase is necessary to accomplish the phage replication cycle [161].
Belgini et al. (2014) evaluated the effects of four bacteriophages isolated from activated
sludge on biofilm formation by bacteria from the feed water of a reverse osmosis system
[162]. The vB_AspP-UFV1 (Podoviridae) interfered in the biofilm formation of most tested
bacteria, causing no decrease in bacterial growth, suggesting that its interference in biofilm
formation might be due to the action of depolymerase or infection of the cell without
necessarily causing cell lysis.
Some studies have suggested that the EPS breakdown might also allow for increased
penetration of non-phage materials, including certain antibiotics. Klebsiella pneumoniae
specific phage KPO1K2 depolymerase possesses anti-biofilm potential and improves
gentamicin’s efficacy against K. pneumoniae by dispersing the capsular polysaccharide of this
bacteria, facilitating antibiotic penetration across the biofilm [163]. The combined use of
cobalt ions (as an iron antagonist) and depolymerase-producing bacteriophages resulted in a
significantly greater reduction in bacterial numbers in the younger, as well as older, K.
pneumoniae biofilms, compared to when either of the agents was used alone [164]. Zhang and
Hu (2013) observed similar results in an equivalent system using phages alone [143]. Greater
biofilm removal, however, was seen with chlorine treatment following phage treatment, while
chlorine treatment alone was somewhat less effective in eradicating these P. aeruginosa
biofilms.
Strategies Based in Microbial Metabolites … 239

In addition, treatment with bacteriophages is suitable for systems in which the selective
removal of one group of bacteria without affecting the other groups is required. An example is
phage application in wastewater filtration systems for selectively remove P. aeruginosa while
not affecting the ammonia-oxidizing bacterial community inside the biofilters [141].

OTHER APPROACHES
Because QS systems control bacterial biofilm differentiation and maturation, inhibiting
quorum sensing will make more difficult or prevent biofilm formation. In Gram-negative
bacteria, there are different methods to control QS: (1) inhibition of N-acyl homoserine
lactone (AHL) production; (2) inactivation of AHL signal molecules; and (3) blocking the
signal receptor (quorum quenching) [165]. Such approaches directly disrupt communication
between the microorganisms contained in the biofilm, thus impeding their ability to
coordinate their actions to replenish, expand and maintain the matrix and ultimately leading to
decomposition of the matrix.
The degradation of quorum-sensing signaling molecules can be achieved by quorum-
quenching enzymes (QQ), including acylases, which cleave the acyl side chain from the HSL
ring, lactonases that open the homoserine (HSL) ring, and oxidoreductases that catalyze the
oxidation or reduction of acyl side chain [166-169].
Production of AHL lactonases has been described in bacteria and fungi. The most
promising AHL lactonase-producing bacteria belong to the genus Bacillus, such as B. cereus,
B. subtilits, B. thuringiensis and B. mycoides [170-171]. The other producing bacteria are P.
aeruginosa PAI-A [172], Arthrobacter sp., K. pneumoniae [173], Agrobacterium tumefaciens
[174], Acidobacteria sp. [175], Ochrobactrum sp. T63 [176], and Rhodococcus sp. [177]. The
fungal quorum-quenching enzyme gluconolactonase has been reported from Aspergillus niger
IAM 2094 [178].
Several bacteria, including species of Comamonas [179], Streptomyces [180], Ralstonia
and Variovorax genera, have been reported to produce QQ acylase [181-182]. Bacteria such
as V. paradoxus and P. aeruginosa PAO1 are able to proliferate with AHLs as the sole source
of energy and carbon and nitrogen mediated by the action of amino acylase, which cleaves the
peptide bond of the signal molecule [172, 181]. CYP102A1, a widely studied cytochrome
P450 from B. megaterium, is also capable of very efficient oxidation of AHLs, and their
lactonolysis products acyl homoserines [166].
Patents for QS control using inhibitor or QQ have been published. They suggest the use
of solutions containing enzymes, cell culture or cell extract solutions. Although this method is
still effective in some application areas, such as preventing plant pathogens, it also has
difficulties in being applied in systems with continuous fluid flow, such as water cooling,
MBR or pipelines, which require the periodic supply of biocides. Most of the application
examples were conducted under laboratory conditions with relatively short time periods. An
important issue in the industrial use of enzymes is maintaining enzyme activity for as long as
possible. In this context, immobilization would be expected to provide the stability required to
bring the technique closer to being a practical solution to the biofouling problem in the
engineering field.
240 Vera Lúcia dos Santos and Andrea Sousa Monteiro

Yeon et al. (2009) reported a magnetic enzyme carrier (MEC) prepared by immobilizing
the quorum-quenching enzyme (acylase) on magnetic particles to overcome the technical
limitations of free enzyme [183]. The MEC showed no activity decrease under either
continuous shaking for 14 days or 29 iterative cycles of reuse. Kim et al. (2011) coupled
acylase directly on nanofiltration membrane (NF) surfaces for water treatment [184]. They
showed that the newly developed membrane with quorum-quenching activity could inhibit
quorum sensing between microorganisms in the membrane biocake, thereby reducing
biofouling.
Jiang et al. (2013) immobilized acylase into sodium alginate and observed a significant
improvement in membrane permeability without any negative impact on effluent quality
[185]. The authors reported that a QQ enzyme might change the mixed liquor properties, such
as sludge settleability, protein and polysaccharide concentrations, and viscosity. In particular,
it was claimed that the size of the microbial flocs in the QQ MBR became smaller than that of
the microbial flocs in the normal MBR, possibly due to QQ activities.
To achieve economic feasibility for the use of QQ in anti-biofouling of MBRs, enzymatic
QQ has been replaced by bacterial QQ using a microbial vessel (CMV) or cell-entrapping
beads (CEB). A recombinant E. coli that produces N-acyl homoserine lactonase or a quorum-
quenching Rhodococcus sp. isolated from a real MBR plant was encapsulated inside the
lumen of a microporous hollow fiber membrane, which was placed in submerged MBR to
alleviate biofouling over 80 days of MBR operation [186]. In another study, Oh et al. (2013)
encapsulated the QQ bacteria Rhodococcus sp. BH4, which produced acylase and was
isolated from a real MBR plant, in a microbial vessel [187]. Kim et al. (2013) prepared free-
moving beads by entrapping the same QQ bacteria, BH4, in alginate beads [188]. Cheong et
al. (2013) also isolated the natural AHL-degrading Pseudomonas sp. 1A1 strain from a real
municipal MBR plant to prepare a microbial vessel [189]. In all of these studies, the QQ
effect of CEBs or CMV on microbial cells in the biofilm generated fewer extracellular
polymeric substances and thus formed a loosely bound biofilm, which enabled it to slough off
from the membrane surface more easily. In the study of Kim et al. (2013), it was suggested
that biofouling was controlled not only by biological action but also by the physical action
provided by bombardment of beads onto the membrane surface [188]. However, they also
found that biofouling was unavoidable because the calcium alginate matrix was gradually
decomposed during long-term MBR operations, although the feed to MBR was relatively mild
synthetic wastewater. To increase the chemical and physical stability of the alginate matrix in
a biological environment, Kim et al. (2015) enclosed the QQ bacteria-containing alginate core
with a porous polymeric membrane layer using various commercial polymers, such as
poly(vinylidene) fluoride (PVDF), polyethersulfone (PES) and polysulfone (PSf) and phase
inversion methods [190]. The macrocapsules were capable of maintaining QQ activity more
safely than previously reported with alginate beads under harsher environmental conditions,
such as in real wastewater or in the presence of a chelating agent (EDTA), which can
disintegrate alginate matrix. In particularly, the PSf-coated membrane layer was more
effective in preventing QQ bacteria from leaking outside the macrocapsules.
Further improvement of this approach was attained by Cheong et al. (2014), who
designed a quorum-quenching MBR with a ceramic microbial vessel (CMV), prepared using a
monolithic ceramic microporous membrane and the AHL-degrading QQ bacterium
Pseudomonas sp. 1A1 [191]. The authors applied an inner flow-feeding mode under which
fresh feed was supplied to the MBR only through the center lumen, enabling the CMV to
Strategies Based in Microbial Metabolites … 241

maintain greater bacterial QQ activity through the facilitated nutrient transfer. In the QQ
MBR with the CMV, the concentrations of EPS were substantially decreased in the biocake
on the membrane surface, compared with those in the conventional MBR. The system also
showed little loss of its initial AHL degradation activity over 30 days of MBR operation.
Weerasekara et al. (2014) investigated the synergistic control of membrane fouling in an
MBR when QQ was coupled with two different physical cleaning methods: air backpulsing
and relaxation [192]. The effects of QQ bacteria on mixed liquor properties and on membrane
fouling control and energy consumption were evaluated at different aeration intensities. QQ
achieved a substantial reduction in membrane fouling, particularly when combined with
relaxation. This approach enabled the stable operation of an MBR at a lower extreme in
aeration, and it minimized the energy consumption for filtration and aeration. QQ bacteria
could hamper the formation of a biofilm on the membrane surface, but the mixed liquor
properties and treatment performances were not affected by the QQ activity.
Approaches based on metabolic interventions that alter the development and
differentiation of biofilms have been evaluated. In vitro experiments showed that both iron
depletion (<1 µM) and iron repletion (>100 µM) retarded biofilm formation [193]. A range
of synthetic iron-chelating molecules (2,2-dipyridyl/2DP, diethylenetriaminepentacetic
acid/DTPA, ethylenediamine-N,N9-diacetic acid/EDTA) and the biologically occurring
chelator lactoferrin were reported to reduce the biofilm formation of P. aeruginosa under
anaerobic conditions [194]. The iron chelator lactoferrin stimulates twitching motility and
prevents biofilm formation by this bacterium [195]. Another approach consists of replacing
iron, which has redox potential, with metabolically inactive ions, such as Sc3+, In3+ or Ga3+,
which are chemically similar to iron. The ions efficiently affect iron uptake and inhibit P.
aeruginosa growth and biofilm formation, and they also kill planktonic and biofilm bacteria
in vitro [196]. In addition, the inhibition of the enoyl-acyl carrier protein reductase from the
type II fatty acid synthesis pathway by the green tea Epigallocatechin gallate was shown to
reduce both QS and the biofilm development of P. aeruginosa. Type II fatty acid synthesis
intermediates are substrates for the LuxI family of autoinducer synthases [197]. Xu and Liu
(2011) demonstrated that disruption of energy metabolism and subsequent production of QS
signaling molecules effectively controlled membrane biofouling [62].

CONCLUSION
In conclusion, as discussed, the demand for research in green technologies for biofilm
control is urgent. Although many technological and financial barriers to biological strategies
remain, they might represent a breakthrough in biofilm control, with innovative designs
considering molecular and biochemical aspects of biofilm formation and resistance to
conventional treatments based on drugs and chemical biocides. However, most of the
strategies are still in the development phase, and many of them face particular challenges and
limitations.
For most of the SACs, there are still no standardized, large-scale methods of production
and purification, and others can present high production costs, which could be reduced with
production strategies based on recombinant microorganisms and the use of industrial
coproducts, such as growth substrate. With SACs, the enzymatic method is nontoxic and
242 Vera Lúcia dos Santos and Andrea Sousa Monteiro

environmentally friendly, but enzymes are unstable and are highly pH-, temperature-, and salt
concentration-sensitive. Phages with viral-attached EPS depolymerases should preferably be
selected for microbial control because they are able to break down biofilms, attacking their
main components, bacterial cells and EPS matrix. However, the high specificity of phage-
associated EPS depolymerases can restrict the host range of the virus. The limitations
associated with specificity can be overcome by the application of phage cocktails directed at
various strains of the target species.
In the case of QQ, care should be taken so that desirable metabolic processes undertaken
by the microbiota are not affected by a particular quorum-quenching strategy. In some
situations, such as MBRs, it is necessary to inhibit biofilm without interfering with the growth
of bacteria that conduct organic charge removal, which is difficult to obtain with oxidant
biocides, for example, which are nonspecific and consequently toxic to non-target organisms.
In this context, strategies that block the expression of biofilm-forming phenotypes, such as
QQ, compared those that kill or inhibit the growth of bacteria are promising. It has been
reported that a variety of natural compounds from plants, quenching enzymes or bacteria
showed considerable QQ activity against biofouling bacteria without interfering with its
growth.
Despite the success of recent studies presented in this chapter, more research is needed to
examine the scaling up of these results to real scale systems and to validate their effectiveness
using real wastewater and its physical and chemical conditions. Another challenge to be
overcome is to validate their applicability to specific operational conditions in different
engineering fields, alone or as part of an integrated microbial control approach, because
diverse studies have shown that combined approaches might offer greater potential for
effectively limiting biofilm problems than single control methods. Moreover, it is important to
learn more about the physical and chemical structures of biofilm, as well the functional and
taxonomical diversity of biofilm microbial communities.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

ACKNOWLEDGMENTS
The authors gratefully acknowledge support from the UFMG, CEUMA,
CENPES/PETROBRAS, Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq),
Fundação do Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) and Comissão de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

REFERENCES
[1] Stoodley P, Sauer K, Davies DG, Costerton JW. Biofilms as complex differentiated
communities. Ann Rev Microbiol 2002; 56: 187-209.
Strategies Based in Microbial Metabolites … 243

[2] Purevdorj B, Costerton JW, Stoodley P. Influence of hydrodynamics and cell signaling
on the structure and behavior of Pseudomonas aeruginosa biofilms. Appl Environ
Microbiol 2000; 68(9): 4457-4464.
[3] Hödl I, Mari L, Bertuzzo E, et al. Biophysical controls on cluster dynamics and
architectural differentiation of microbial biofilms in contrasting flow environments.
Environ Microbiol 2014; 16(3): 802–812.
[4] Sutherland IW. Biofilm exopolysaccharides: a strong and sticky framework.
Microbiology 2001; 147(Pt 1): 3–9.
[5] De Beer D, Stoodley P, Roe F, Lewandowski Z Effect of biofilm structures on oxygen
distribution and mass transport. Biotechnol Bioeng 1994a; 43(11): 1131–1138.
[6] De Beer D, Stoodley P, Lewandowski Z. Liquid flow in heterogeneous biofilms.
Biotechnol Bioeng 1994b; 44(5): 636–641.
[7] Von Bodman SB, Farrand SK. Capsular polysaccharide biosynthesis and pathogenicity
in Erwinia stewartii require induction by an N-acylhomoserine lactone autoinducer. J
Bacteriol 1995; 177(17): 5000–5008.
[8] Hammer BK, Bassler BL. Quorum sensing controls biofilm formation in Vibrio
cholerae. Mol Microbiol 2003; 50(1): 101–114.
[9] Parsek MR, Greenberg EP. Sociomicrobiology: the connections between quorum
sensing and biofilms. Trends Microbiol 2005; 13(1): 27–33.
[10] Buesing N, Gessner MO. Benthic and fungal productivity and carbon turnover in a
freshwater marsh. Appl Environ Microbiol 2006; 72: 596-605.
[11] Jefferson KK. What drives bacteria to produce a biofilm? FEMS Microbiol Lett 2004;
236: 163-173.
[12] Romani AM, Sebater S. Effect of primary producers on the heterotrophic metabolism of
a stream biofilm. Freshwater Biol 1999; 41(4): 729-736.
[13] Roseselers G, Van Loosdrecht MCM, Muyzer G. Heterotrophic pioneers facilitate
phototrophic biofilm development. Microb Ecol 2007; 54(3): 578-585.
[14] Freeman C, Lock MA. The biofilm polysaccharide matrix – a buffer against changing
organic substrate supply. Limmol Oceanogr 1995; 40(2): 273-278.
[15] Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the Natural
environment to infectious diseases. Nature Rev Microbiol 2004; 2: 95-108.
[16] Rickard AH, McBain AJ, Stead AT, Gilbert P. Shear rate moderates community
diversity in freshwater biofilms. Appl Environ Microbiol 2004; 70(12): 7426-7435.
[17] Matz C, Kjelleberg S. Off the hook – how bacteria survive protozoan grazing. Trends
Microbiol 2005; 13(7): 302-307.
[18] Sheldon F, Walker KF. Changes in biofilms induced by flow regulation could explain
extinctions of aquatic sanils in the lower River Murray, Australia. Hydrobiologia 1997;
347: 97-108.
[19] Hero HM, Port RD. The Nalco guide to cooling water system failure analysis, McGraw
Company, New York, 1993.
[20] Coniglio MA, Melada S, Yassin MH. Monochloramine for controlling Legionella in
biofilms: how much we know? J Nature Sci 2015; 1(2):e44.
[21] Akid R, Wang H, Smith T, Greenfield D, Earthman J. Biological functionalization of a
sol-gel coating for the mitigation of microbial-induced corrosion. Adv Funct Mat 2008;
18(2): 203–211.
244 Vera Lúcia dos Santos and Andrea Sousa Monteiro

[22] Li KH, Whitfield M, Van Vliet KJ. Beating the bugs: Roles of microbial biofilms in
corrosion, Corrosion Rev 2013; 31: 73-84.
[23] Murga R, Forster TS, Brown E, et al. Role of biofilms in the survival of Legionella
pneumophila in a model potable-water system. Microbiology 2001; 147: 3121–3126.
[24] EPA. 2002. Health Risks from Microbial Growth and Biofilms in Drinking Water
Distribution Systems. Office of Ground Water and Drinking Water, Washington, D.C
(http://www.epa.gov/ogwdw/ disinfection/tcr/pdfs/whitepaper_tcr_biofilms.pdf).
[25] Langmark J, Storey MV, Ashbolt NJ, Stenstrom TA. Accumulation and fate of
microorganisms and microspheres in biofilm formed in a pilot-scale water distribution
system. Appl Environ Microbiol 2005; 71(2): 706-712.
[26] Berry D, Xi C, Raskin L. Microbial ecology of drinking water distribution systems.
Curr Opin Biotechnol 2006; 17: 297-302.
[27] Morrow JB, Almeida JL, Fitzgerald LA, Cole KD. Association and decontamination of
Bacillus spores in a simulated drinking water system. Water Res 2008; 42(20): 5011-
5021.
[28] Helmi K, Skraber S, Gantzer C, et al. Interactions of Cryptosporidium parvum, Giardia
lamblia, vaccinal poliovirus type 1, and bacteriophages phiX 174 and MS2 with a
drinking water biofilm and a wastewater biofilm. Appl Environ Microbiol 2008; 74(7):
2079-2088.
[29] Paris T, Skali-Lami S, Block JC. Probing young water biofilms with hard and soft
particles. Water Res 2009; 43(1): 117-126.
[30] Altman SJ, McGrath LK, Souza CA, Murton JK, Camper AK. Integration and
decontamination of Bacillus cereus in Pseudomonas fluorescens biofilms. J Appl
Microbiol 2009; 107(1): 287-299.
[31] Feazel LM, Baumgartner LK, Peterson KL, et al. Opportunistic pathogens enriched in
showerhead biofilms. PNAS 2009; 106(38): 16393-16399.
[32] Wingender J, Flemming H-C. Biofilms in drinking water and their role as reservoir for
pathogens. Int J Hyg Environ Health 2011; 214(6): 417-423.
[33] Pelleieux S, Bertrand I, Skali-Lami S, et al. Accumulation of MS2, GA, and Qb phages
on high density polyethylene (HDPE) and drinking water biofilms under flow/non-flow
conditions. Water Res 2002; 46919): 6574-6584.
[34] Kerr CJ, Osborn KS, Rickard AH, Robson GD, Handley PS. Biofilms in water
distribution systems. In: Mara D, Horan NJ, Eds. Water and wastewater engineering.
London: Academic Press 2003; pp. 757-776.
[35] Shi X, Zhu X. Biofilm formation and food safety in food industries. Trends Food Sci
Technol 2009; 20(9): 407-413.
[36] Talsma SS. Biofilms on medical devices. Home Healthc Nurse 2007; 25(9): 589-594.
[37] Davison WM, Pitts B, Stewart PS. Spatial and temporal patterns of biocide action
against Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother 2010;
54(7): 2920-2927.
[38] Lee WH, Wahman DG, Bishop PL, Pressman JG. Free chlorine and monochloramine
application to nitrifying biofilm: comparison of biofilm penetration, activity, and
viability. Environ Sci Technol 2011; 45(4): 1412-1419.
[39] Walters MC, Roe F, Bugnicourt A, Franklin MJ, Stewart PS. Contributions of antibiotic
penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas
Strategies Based in Microbial Metabolites … 245

biofilms to ciprofloxacin and tobramycin. Antimicrob Agents Chemother 2003; 47(1):


317-323.
[40] Cochran WL, McFeters GA, Stewart, PS. Reduced susceptibility of thin Pseudomonas
aeruginosa biofilms to hydrogen peroxide and monochloramine. J Appl Microbiol
2000; 88: 22–30.
[41] Mah TFC, O’Toole GA. Mechanisms of biofilm resistance to antimicrobial agents.
Trends Microbiol. 2001; 9(1): 34-39.
[42] Denkard E, Ausubel FM. Pseudomonas biofilm formation and antibiotic resistance are
linked to phenotypic variation. Nature 2002; 416(6882): 740–743.
[43] Zhang H, Andrews SA. Catalysis of copper corrosion products on chlorine decay and
HAA formation in simulated distribution systems. Water Res 2012; 46(8): 2665-2673.
[44] Wang H, Hu C, Hu X, Yang M, Qu JH. Effects of disinfectants and biofilm on the
corrosion of cast iron pipes in a reclaimed water distribution system. Water Res 2012;
46(4): 1070-1078.
[45] Hallam NB, Westy JR, Forster CF, Powell JC, Spencer I. The decay of chlorine
associated with the pipe wall in water distribution systems. Water Res 2002; 36(14):
3479-3488.
[46] Lethola MJ, Miettinen IT, Lampola T, et al. Pipeline materials modify the effectiveness
of disinfectants in drinking water distributions systems. Water Res 2005; 39(10): 1962-
1971.
[47] Hubbard H, Poppendieck D, Corsi RL. Chlorine dioxide reactions with indoor materials
during building disinfection: surface uptake. Environ Sci Technol 2009; 43(5): 1329-
1335.
[48] Xue Z, Sendamangalam VR, Gruden CL, Seo Y. Multiple role of extracellular
polymeric substances on resistance of biofilm and detached clusters. Environ Sci
Technol 2012; 46(24), 13212-13219.
[49] Flint SH, van den Elzen H, Brooks JD, Bremer PJ. Removal and inactivation of thermo-
resistant streptococci colonizing stainless steel. Int Dairy J 1999; 9(7): 429–436.
[50] Mathieu L, Bouteleux C, Fass S, Angel E, Block J-C. Reversible shift in the a-, b- and
g-proteobacteria populations of drinking water biofilms during discontinuous
chlorination. Water Res 2009; 43: 3375-3386.
[51] Jones WL, Sutton MP, McKittrick L, Stewart PS. Chemical and antimicrobial
treatments change the viscoelastic properties of bacterial biofilms. Biofouling 2011;
27(2): 207-215.
[52] Saravanan P, Nancharaiah YV, Venugopalan VP, Rao TS, Jayachandran S. Biofilm
formation by Pseudoalteromonas ruthenica and its removal by chlorine. Biofouling
2006; 22(5-6): 371-381.
[53] Tachikawa M, Yamanaka K, Nakamuro K. Studies on the disinfection and removal of
biofilms by ozone water using an artificial microbial biofilm system, Ozone: Sci Eng
2009; 31(1): 3-9.
[54] Turetgen I. Comparison of the efficacy of free residual chlorine and monochloramine
against biofilms in model and full scale cooling towers. Biofouling 2004; 20(2): 81–85.
[55] Chien S-H, Dzombak DA, Vidic RD. Comprehensive Evaluation of Biological Growth
Control by Chlorine-Based Biocides in Power Plant Cooling Systems Using Tertiary
Effluent. Environ Eng Sci 2013; 30(6): 324–332.
246 Vera Lúcia dos Santos and Andrea Sousa Monteiro

[56] Xue Z, Lee WH, Coburn KM, Seo Y. Selective reactivity of monochloramine with
extracellular matrix components affects the disinfection of biofilm and detached
clusters. Environ Sci Technol 2014; 48(7): 3832-3839.
[57] Stewart PS. Antimicrobial Tolerance in Biofilms. Microbiol Spectr 2015; 3(3):
10.1128/microbiolspec.MB–0010–2014.
[58] Campos FF, Johann S, Santos VL. Natural Products as a Source of Potential Drugs for
the Treatment of Fungal Infections. In: Leon V. Berhardt. (Org.). Advances in Medicine
and Biology. 1ed. New York: Nova Science Publishes v39 2012, pp. 49-83.
[59] Sumi CD, Byung WY, Yeo IC, Hahm YT. Antimicrobial peptides of the genus
Bacillus: a new era for antibiotics. Can J Microbiol 2014; 61(2): 93-103.
[60] Itoh Y, Wang X, Hinnebusch BJ, Preston JF, Romeo T. Depolymerization of β-1,6-N-
acetyl-d-glucosamine disrupts the integrity of diverse bacterial biofilms. J Bacteriol
2005; 187(1): 382–387.
[61] Spoering AL, Gilmore MS. Quorum sensing and DNA release in bacterial biofilms.
Curr Opin Biotechnol 2006; 9(2): 133-137.
[62] Xu H, Liu Y. Control and cleaning of membrane biofouling by energy uncoupling and
cellular communication. Environ Sci Technol 2011; 45(2): 595-601.
[63] Lönn-Stensrud J, Landin MA, Benneche T, Petersen FC, Scheie AA. Furanones,
potential agents for preventing Staphylococcus epidermidis biofilm infections? J
Antimicrob Chemother 2009; 63(2): 309-316.
[64] Carson L, Gorman SP, Gilmore BF. The use of lytic bacteriophages in the prevention
and eradication of biofilms of Proteus mirabilis and Escherichia coli. FEMS Immunol
Med Microbiol 2010; 59(3): 447–455.
[65] Neu TR. Significance of bacterial surface-active compounds in interaction of bacteria
with interfaces. Microbiol Rev 1996; 60(1): 151-166.
[66] Desai JD, Banat IM. Microbial production of surfactants and their commercial potential.
Microbiol Mol Biol Rev 1997; 61(1): 47–64.
[67] Monteiro AS, Bonfim MRQ, Domingues VS, et al. Identification and characterization
of bioemulsifier-producing yeasts isolated from effluents of a dairy industry. Bioresour
Technol 2010; 101(14): 5186-5193.
[68] Monteiro AS, Domingues VS, Souza MVD, et al. Bioconversion of biodiesel refinery
waste in the bioemulsifier by Trichosporon mycotoxinivorans CLA2. Biotechnol
Biofuels 2012; 5: 29.
[69] Santos VL, Monteiro AS, Domingues VS, Júlio ADL. Microbial Bioemulsifiers and
Environmental Applications Potential. In: Tannen KM, Ed. Emulsification: Processes,
New Technology and Current Applications. 3nd ed. New York: Nova Science Publishers
2013.
[70] Coutinho JOPA, Silva MPS, Moraes PM, et al. Demulsifying properties of extracellular
products and cells of Pseudomonas aeruginosa MSJ isolated from petroleum-
contaminated soil. Bioresour Technol 2013; 128: 646-654.
[71] Cameotra S, Makkar R. Recent applications of biosurfactants as biological and
immunological molecules. Curr Opin Microbiol 2004; 7: 262–266.
[72] Singh P, Cameotra S. Potential applications of microbial surfactants in biomedical
sciences. Trends Biotechnol 2004, 22(3): 142–146.
Strategies Based in Microbial Metabolites … 247

[73] Monteiro AS, Miranda TT, Lula I, et al. Inhibition of Candida albicans CC biofilms
formation in polystyrene plate surfaces by biosurfactant produced by Trichosporon
montevideense CLOA72. Colloids Surf B Biointerfaces 2011; 84(2): 467-476.
[74] Al-Tahhan RA, Sandrin TR, Bodour AA, Maier RM. Rhamnolipid-induced removal of
lipopolysaccharide from Pseudomonas aeruginosa: Effect on cell surface properties and
interaction with hydrophobic substrates. Appl Environ Microbiol 2000; 66(8): 3262–
3268.
[75] Davey ME, Caiazza NC, O’Toole GA Rhamnolipid surfactant production affects
biofilm architecture in Pseudomonas PAO1. J Bacteriol 2003; 185(3): 1027–1036.
[76] Pamp SJ, Tolker-Nielsen T. Multiple roles of biosurfactants in structural biofilm
development by Pseudomonas aeruginosa. J Bacteriol 2007;189(6): 2531–2539.
[77] Jiang P, Li J, Han F, et al. Anti-biofilm activity of an exopolysaccharide from marine
bacterium Vibrio sp. QY101. PLoS One 2011; 6(4): e18514.
[78] Thimon L, Peypoux F, Wallach J, Michel G. Effect of the lipopeptide antibiotic iturin
A, on morphology and membrane ultrastructure of yeast cells. FEMS Microbiol Lett
1995; 128(2): 101-106.
[79] Long X, Meng Q, Zhang G. Application of biosurfactant rhamnolipid for cleaning of
UF membranes. J Memb Sci 2014; 457: 113-119.
[80] Boles BR, Thoendel M, Singh PK. Rhamnolipids mediate detachment of Pseudomonas
aeruginosa from biofilms. Mol Microbiol 2005; 57: 1210-1223.
[81] Dusane DH, Nancharaiah YV, Zinjarde SS, Venugopalan VP. Rhamnolipid mediated
disruption of marine Bacillus pumilus biofilms. Colloids Surf B Biointerfaces 2010;
81(1): 242-248.
[82] Sodagari M, Wang H, Newby BM, Ju LK. Effect of rhamnolipids on initial attachment
of bacteria on glass and octadecyltrichlorosilane-modified glass. Colloids Surf B
Biointerfaces 2013; 103: 121–128.
[83] Meylheuc T, Renault M, Bellon-Fontaine MN. Adsorption of a biosurfactant on
surfaces to enhance the disinfection of surfaces contaminated with Listeria
monocytogenes. Int J Food Microbiol 2006; 109(1-2): 71-78.
[84] Rivardo F, Martinotti MG, Turner RJ, Ceri H. The activity of silver against Escherichia
coli biofilm is increased by a lipopeptide biosurfactant. Can J Microbiol 2010; 56(3):
272–278.
[85] Rivardo F, Martinotti MG, Turner RJ, Ceri H. Synergistic effect of lipopeptide
biosurfactant with antibiotics against Escherichia coli CFT073 biofilm. Int J Antimicrob
Agents 2011; 37(4): 324–331.
[86] Qin L, Zhang G, Meng Q, et al. Enhanced submerged membrane bioreactor combined
with biosurfactant rhamnolipids: Performance for frying oil degradation and membrane
fouling reduction. Bioresour Technol 2012; 126: 314–320.
[87] Oulahal N, Martial-Gros A, Bonneau M, Blum LJ. Removal of meat biofilms from
surfaces by ultrasounds combined with enzymes and/or a chelating agent. Innov Food
Sci Emerg Technol 2007; 8(2): 192–196.
[88] Longhi C, Scoarughi GL, Poggiali F, et al. Protease treatment affects both invasion
ability and biofilm formation in Listeria monocytogenes. Microb Pathog 2008; 45(1):
45-52.
[89] Kristensen JB, Meyer RL, Laursen BS, et al. Antifouling enzymes and the biochemistry
of marine settlement. Biotechnol Adv 2008; 26(5): 471- 481.
248 Vera Lúcia dos Santos and Andrea Sousa Monteiro

[90] Johansen C, Falholt P, Gram L. Enzymatic removal and disinfection of bacterial


biofilms. Appl Environ Microbiol 1997; 63(9), 3724-3728.
[91] Augustin M, Ali-Vehmas T. Assessment of enzymatic cleaning agents and disinfectants
against bacterial biofilms. J Pharm Pharmaceut Sci 2004; 7(1): 55-64.
[92] Kaplan JB, Ragunath C, Velliyagounder K, Fine DH, Ramasubbu N. Enzymatic
detachment of Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother
2004; 48(7): 2633–2636.
[93] Lequette Y, Boels G, Clarisse M, Faille C. Using enzymes to remove biofilms of
bacterial isolates samples in the food industry. Biofouling 2010; 26(4): 421-431.
[94] Cui W, Winter WT, Tanenbaum SW, Nakas JP. Purification and characterization of an
intracellular carboxylesterase from Arthrobacter viscosus NRRL B-1973. Enzyme
Microb Technol 1999; 24(3): 200-208.
[95] Orgaz B, Kives J, Pedregosa AM, et al. Bacterial biofilm removal using fungal
enzymes. Enzyme Microb Technol 2006; 40(1): 51-56.
[96] Orgaz, B., Neufeld, R.J., Sanjose, C. Single-step biofilm removal with delayed release
encapsulated pronase mixed with soluble enzymes. Enzyme and Microbial Technology
2007; 40: 1045–1051.
[97] van Casteren WHM, Dijkema C, Schols HA, Beldman G, Voragen AGJ.
Characterisation and modification of the exopolysaccharide produced by Lactococcus
lactis subsp. cremoris B40. Carbohyd Polym 1998; 37(2): 123–130.
[98] Loiselle M, Anderson KW. The use of cellulose in inhibiting biofilms formation from
organisms commonly found on medical implants. Biofouling 2003; 19(2): 77-85.
[99] Vickery K, Pajkos A, Cossart Y. Removal of biofilm from endoscopes: evaluation of
detergent efficiency. Am J Infect Control 2004; 32(3): 170–176.
[100] Izano EA, Amarante MA, Kher WB, Kaplan JB. Differential roles of poly-N-
acetylglucosamine surface polysaccharide and extracellular DNA in Staphylococcus
aureus and Staphylococcus epidermidis biofilms. Appl Environ Microbiol 2008; 74(2):
470–476.
[101] Lee JH, Kaplan JB, Lee WY. Microfluidic devices for studying growth and detachment
of Staphylococcus epidermidis biofilms. Biomed Microdevices 2008; 10(4): 489-498.
[102] Fredheim EGA, Klingenberg C, Rohde H, et al. 2009. Biofilm Formation by
Staphylococcus haemolyticus. J Clin Microbiol 2009; 47(4): 1172-1180.
[103] Izano EA, Shah SM, Kaplan JB. Intercellular adhesion and biocide resistance in
nontypeable Haemophilus influenzae biofilms. Microb Pathog 2009; 46(4): 207–213.
[104] Leroy C, Delbarre C, Ghillebaert F, Compere C, Combes D. Influence of subtilisin on
the adhesion of a marine bacterium which produces mainly proteins as extracellular
polymers. J Appl Microbiol 2008;105(3): 791-799,
[105] Molin S, Tolker-Nielsen T. Gene transfer occurs with enhanced efficiency in biofilms
and induces enhanced stabilisation of the biofilm structure. Curr Opin Biotechnol 2003;
14(3): 255-261.
[106] Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS. Extracellular DNA required
for bacterial biofilm formation. Science 2002; 295(5559): 1487.
[107] Tetz GV, Artemenko NK, Tetz VV. Effect of DNase and Antibiotics on Biofilm
Characteristics. Antimicrob Agents Chemother 2009; 53(3): 1204-1209.
Strategies Based in Microbial Metabolites … 249

[108] Conte A, Buonocore GG, Bevilacqua A, Sinigaglia M, Del Nobile MA. Immobilization
of lysozyme on polyvinylalcohol films for active packaging applications. J Food Prot
2006; 69(4): 866-870.
[109] Conte A, Buonocore GG, Sinigaglia M, et al. Antimicrobial activity of immobilized
lysozyme on plasma-treated polyethylene films. J Food Prot 2008; 71(1): 119-125.
[110] Samaranayake YH, Cheung BP, Parahitiyawa N et al. Synergistic activity of lysozyme
and antifungal agents against Candida albicans biofilms on denture acrylic surfaces.
Arch Oral Biol 2009; 54:115-126.
[111] Nishiyama Y, Nakoaka C, Hiratani T, Abe S, Uchida K,Yamaguchi H (2001). Synergy
of lysozyme and lanoconaz-ole on the morphology of Candida albicans. J Electron
Microsc; 50:41–49.
[112] Jun SY, Jung GM, Son J-S, et al. Comparison of the Antibacterial Properties of Phage
Endolysins SAL-1 and LysK. Antimicrob Agents Chemother 2011; 55(4): 1764–1767.
[113] Marcato-Romain CE, Pechaud Y, Paul E, Girbal-Neuhauser E, Dossat-Létisse V.
Removal of microbial multi-species biofilms from the paper industry by enzymatic
treatments. Biofouling 2012; 28(3): 305-314.
[114] Pechaud Y, Marcato-Romain C-E, Girbal-Neuhauser E, et al. Combining hydrodynamic
and enzymatic treatments to improve multi-species thick biofilm removal. Chem Eng
Sci 2012; 80: 109-118.
[115] Verhoef R, Schols HA, Blanco A, et al. Sugar composition and FT-IR analys is of
exopolysaccharides products by microbial isolates from paper mill slime deposits.
Biotechnol Bioeng, 2005; 91:91-105.
[116] Hatcher H, Lechner T, Mcduff C, Truda R. Slime control in industrial waters. US
Patent 3824184, 1974.
[117] Chaignon P, Sadovskaya I, Ragunah C, et al. Susceptibility of staphylococcal biofilms
to enzymatic treatments depends on their chemical composition. Appl Microbiol
Biotechnol 2007; 75: 125-132.
[118] Torres CE, Lenon G, Craperi D, Wilting R, Blanco A. Enzymatic treatment for
preventing biofilm formation in the paper industry. Appl Microbiol Biotechnol 2011;
92(1): 95-103.
[119] Leroy C, Delbarre C, Ghillebaert F, Compere C, Combes D. Effect of commercial
enzymes on the adhesion of a marine biofilm forming bacterium. Biofouling 2008;
24(1): 11-22.
[120] Poele S, van der Graaf J. Enzymatic cleaning in ultrafiltration of wastewater treatment
plant effluent. Desalination 2005; 179: 73-81.
[121] Molobela P, Cloete TE, Beukes M. Protease and amylase enzymes for biofilm removal
and degradation of extracellular polymeric substances (EPS) produced by Pseudomonas
fluorescens bacteria. Afr J Microbiol Res 2010; 4(14): 1515-1524.
[122] Richards M, Cloete TE. Biofilm Removal using Nanozymes In: Nanotechnology in
Water Treatment Applications. Eds. Cloete TE, Kwaadsteniet M, Botes M, López-
Romero JM. Publisher: Caister Academic Press 2010; pp 196.
[123] Hollis CG, Terry JP, Jaquess PA. Methods for removing biofilm from or preventing
buildup thereof on surfaces in industrial water systems. USA Patent 5411666, 1995.
[124] Eyers ME, Van Pee KLI, Van Poele J, Schuetz JF, Schenker AP. Composition and
process for the avoidance of slime formation and/or for the removal of biofilm in water-
bearing systems. US Patent 5789239-A, 1998.
250 Vera Lúcia dos Santos and Andrea Sousa Monteiro

[125] Wiatr CL. Application of cellulase to control industrial slime. US Patent 4936994,
1990.
[126] Ignatius Van PKL, Jozef Van P, Speybroeck MMP Van. Use of mannanases as slime
control agentes. US Patent 1996036569 A1, 1996.
[127] Kumar, M. Enzymatic prevention and control of biofilm. US Patent 20080019956 A1,
2008.
[128] Pedersen DE, Hatcher HJ. Two component biocidal process. US Patent 4684469, 1987.
[129] Robertson LR, LaZonby JG, Krolczyk JJ, Melo HR. Treatment of Process Waters to
Destroy Filamentous Bacteria. US Patent 5324432, 1994.
[130] Davies D. Induction of a physiological dispersion response in bacterial cells in a
biofilm. US Patent 2007/0207095 A1, 2007.
[131] Isobe K, Okano T, Iwasaki S, Okauchi Y. Composition of biofilm control agent. US
Patent 20090123449, 2009.
[132] Marion K, Sanchez T. Method for eliminating biofilm. US Patent 20050079594 A1,
2005.
[133] Aggarwal D, Shah CB. Antimicrobial and anticoagulant compositions and methods. US
Patent 20100022652-A1, 2010.
[134] Oulahal-Lagsir N, Martial-Gros A, Bonneau M, Blum LJ. “Escherichia coli-milk"
biofilm removal from stainless steel surfaces: synergism between ultrasonic waves and
enzymes. Biofouling 2003; 19(3): 159-168.
[135] Ackermann HW. Bacteriophage observations and evolution. Res Microbiol 2003;
154(4): 245–251.
[136] Weinbauer M. Ecology of procaryotic viruses. FEMS Microbiol Rev 2004; 28(2):127–
181.
[137] Paul JH. Prophages in marine bacteria: dangerous molecular time bombs or the key to
survival in the seas? ISME J 2008; 2(6): 579–589.
[138] Maranger R, del Giorgio PA, Bird DF. Accumulation of damaged bacteria and viruses
in lake water exposed to solar radiation. Aquat Microb Ecol 2002; 28(3): 213–227.
[139] Binnenkade L, Teichmann L, Thormann KM. Iron Triggers λSo Prophage Induction
and Release of Extracellular DNA in Shewanella oneidensis MR-1 Biofilms. Appl
Environ Microbiol 2014; 80: 5304–5316.
[140] Ly-Chatain MH. The factors affecting effectiveness of treatment in phages therapy.
Front Microbiol 2014; 5: 51.
[141] Zhang Y, Hunt HK, Hu Z. Application of bacteriophages to selectively remove
Pseudomonas aeruginosa in water and wastewater filtration systems. Water Res 2013;
47(13): 4507-4518.
[142] Ganegama Arachchi GJ, Cridge AG, et al. Effectiveness of phages in the
decontamination of Listeria monocytogenes adhered to clean stainless steel, stainless
steel coated with fish protein, and as a biofilm. J Ind Microbiol Biotechnol 2013;
40(10): 1105-1116.
[143] Zhang Y, Hu Z. Combined treatment of Pseudomonas aeruginosa biofilms with
bacteriophages and chlorine. Biotechnol Bioeng 2013; 110(1): 286–295.
[144] Weld RJ, Butts C, Heinemann JA. Models of phage growth and their applicability to
phage therapy. J Theor Biol 2004; 227(1): 1–11.
[145] Bradley DE. A study of pili on Pseudomonas aeruginosa. Genet Res 1972; 19(1): 39–
51.
Strategies Based in Microbial Metabolites … 251

[146] Bradley DE. Shortening of Pseudomonas aeruginosa pili after RNA-phage adsorption.
J Gen Microbiol 1972; 72: 303–319.
[147] Biebricher CK, Duker E-M. Light-microscopic visualization of F and type 1 pili. J Gen
Microbiol 1984; 130(4): 941-949.
[148] Woody MA, Cliver DO. Replication of coliphage Qβ as affected by host cell number,
nutrition, competition from insusceptible cells and non- FRNA coliphage. Journal of
Appl Microbiol 1997; 82(4): 431–440.
[149] Soni KA, Nannapaneni R. Removal of Listeria monocytogenes biofilms with
bacteriophage P100. J Food Prot 2010; 73(8): 1519-1524.
[150] Sands JA, Auperin D. Effects of temperature and host cell genetic characteristics on the
Replication of the Lipid-Containing Bacteriophage PR4 in Escherichia coli. J Virol
1977; 22(2): 315-320.
[151] Adams MH. Abortive infection With phage T2 at low temperatures. Virology 1955;
1:335-346.
[152] Sillankorva S, Oliveira R, Vieira MJ, Sutherland I, Azeredo J. Pseudomonas
fluorescens infection by bacteriophage ΦS1: the influence of temperature, host growth
phase and media. FEMS Microbiol Lett 2004; 241(1): 13-20.
[153] Abedon ST. Spatial vulnerability: Bacterial arrangements, microcolonies, and biofilms
as responses to low rather than high phage densities. Viruses 2012; 4(5): 663–687.
[154] Shen Y, Mitchell MS, Donovan DM, Nelson DC. Phage-based enzybiotics. In: Hyman
P, Abedon ST, Eds. Bacteriophages in Health and Disease. Wallingford: CABI Press,
2012; pp. 217–239.
[155] Doolittle MM, Cooney JJ, Caldwell DE. Tracing the interaction of bacteriophage with
bacterial biofilms using fluorescent and chromogenic probes. J Ind Microbiol 1996;
16(6): 331-341.
[156] Hughes KA, Sutherland IW, Clark J, Jones MV. Bacteriophage and associated
polysaccharide depolymerases – novel tools for study of bacterial biofilms. J Appl
Microbiol 1998; 85(3): 583–590.
[157] Goldman G, Starosvetsky J, Armon R. Inhibition of biofilm on UF membrane by use of
specific bacteriophages. J Membr Sci 2009; 342(1-2): 145–152.
[158] Bayer ME, Thurow H, Bayer MH. Penetration of the polysaccharide capsule of
Escherichia coli (Bil 62/42) by bacteriophage K29. Virology 1979; 94: 95–118.
[159] Hughes KA, Sutherland IW, Jones MV. Biofilm susceptibility to bacteriophage attack:
the role of phage-borne polysaccharide depolymerase. Microbiology 1998; 144(Pt 11):
3039–3047.
[160] Skillman LC, Sutherland IW, Jones MV. The role of exopolysaccharides in dual species
biofilm development. J Appl Microbiol 1999; 85 (Suppl. 1): 13S–18S.
[161] Cornelissen A, Ceyssens P-J, T’Syen J, et al. The T7-Related Pseudomonas putida
Phage ϕ15 Displays Virion-Associated Biofilm Degradation Properties. PLoS ONE
2011; 6(4) e18597.
[162] Belgini DRB, Dias RS, Siqueira VM, et al. Culturable bacterial diversity from a feed
water of a reverse osmosis system, evaluation of biofilm formation and biocontrol using
phages. World J Microbiol Biotechnol 2014; 30: 2689 -2700.
[163] Bansal S, Harjai K, Chhibber S. Aeromonas punctata derived depolymerase improves
susceptibility of Klebsiella pneumoniae biofilm to gentamicin. BMC Microbiology.
2015;15:119.
252 Vera Lúcia dos Santos and Andrea Sousa Monteiro

[164] Chhibber S, Nag D, Bansal S. Inhibiting biofilm formation by Klebsiella pneumoniae


B5055 using an iron antagonizing molecule and a bacteriophage. BMC Microbiol 2013;
13:174.
[165] Kalia VC. Quorum sensing inhibitors: An overview. Biotechnol adv 2013; 31(2): 224 -
245.
[166] Chowdhary PK, Keshavan N, Nguyen HQ, et al. Bacillus megaterium CYP102A1
oxidation of acyl homoserine lactones and acyl homoserines. Biochemistry 2007; 46:
14429–14437.
[167] Augustine N, Kumar P, Thomas S. Inhibition of Vibrio cholerae biofilm by AiiA
enzyme produced from Bacillus spp. Arch Microbiol 2010; 192(12): 1019–1022.
[168] Bijtenhoorn P, Schipper C, Hornung C, et al. BpiB05, a novel metagenome-derived
hydrolase acting on N-acylhomoserine lactones. J Biotechnol 2011; 155: 86–94.
[169] Lade H, Paul D, Kweon JH. Quorum Quenching Mediated Approaches for Control of
Membrane Biofouling. Int J Biol Sci 2014; 10(5): 550–565.
[170] Dong YH, Gusti AR, Zhang Q, Xu JL, Zhang LH. Identification of quorum-quenching
N-acyl homoserine lactonases from Bacillus species. Appl Environ Microbiol 2002;
68(4):1754-1759.
[171] Lee SJ, Park SY, Lee JJ, et al. Genes encoding the N-acyl homoserine lactone-
degrading enzyme are widespread in many subspecies of Bacillus thuringiensis. Appl
Environ Microbiol 2002; 68(8): 3919-3924.
[172] Huang JJ, Han JI, Zhang LH, Leadbetter JR. Utilization of acyl-homoserine lactone
quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1.
Appl Environ Microbiol 2003; 69(10)5941-5949.
[173] Park SY, Lee SJ, Oh TK, et al. AhlD, an N-acylhomoserine lactonase in Arthrobacter
sp., and predicted homologues in other bacteria. Microbiology 2003; 149(Pt 6): 1541-
1550.
[174] Carlier A, Uroz S, Smadja B, et al. The Ti plasmid of Agrobactetium tumefaciens
harbors an attM paralogous gene, aiiB, also encoding N-acyl homoserine lactonase
activity. Appl Environ Microbiol 2003; 69: 4989-4993.
[175] Riaz K, Elmerich C, Moreira D, et al. A metagenomic analysis of soil bacteria extends
the diversity of quorum-quenching lactonases. Environ Microbiol 2008; 10(3): 560–
570.
[176] Mei GY, Yan XX, Turak A, et al. AidH, an alpha/beta-hydrolase fold family member
from an Ochrobactrum sp. strain, is a novel N-acylhomoserine lactonase. Appl Environ
Microbiol 2010; 76: 4933–4942.
[177] Park SY, Hwang BJ, Shin MH, et al. N-acylhomoserine lactonase producing
Rhodococcus spp. with different AHL-degrading activities. FEMS Microbiol Lett 2006;
261(1): 102-108.
[178] Ogawa K, Nakajima-Kambe T, Nakahara T, et al. Coimmobilization of
gluconolactonase with glucose oxidase for improvement in kinetic property of
enzymatically induced volume collapse in ionic gels. Biomacromolecules 2002; 3(3):
625–631.
[179] Uroz S, Oger P, Chhabra SR et al. N-Acyl homoserine lactones are degraded via an
amidolytic activity in Comamonas sp. strain D1. Arch. Microbiol 2007; 187(3):249–
256.
Strategies Based in Microbial Metabolites … 253

[180] Park SY, Kang HO, Jang HS, et al. Identification of extracellular N-acylhomoserine
lactone acylase from a Streptomyces sp and its application to quorum quenching. Appl
Environ Microbiol 2005; 71(5): 2632-2641.
[181] Leadbetter JR, Greenberg EP. Metabolism of acyl-homoserine lactone quorum-sensing
signals by Variovorax paradoxus. J Bacteriol 2000; 182(24): 6921-6926.
[182] Lin YH, Xu JL, Hu JY, et al. Acyl-homoserine lactone acylase from Ralstonia strain
XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol
Microbiol 2003; 47(3): 849-860.
[183] Yeon KM, Lee CH, Kim J. Magnetic enzyme carrier for effective biofouling control in
the membrane bioreactor based on enzymatic quorum quenching. Environ Sci Technol
2009; 43(19): 7403–7409.
[184] Kim J, Choi D, Yeon K, Kim S, Lee C. Enzyme-immobilized nanofiltration membrane
to mitigate biofouling based on quorum quenching. Environ Sci Technol 2011; 45(4):
1601-1607.
[185] Jiang W, Xia S, Liang J, Zhang Z, Hermanowicz S. Effect of quorum quenching on the
reactor performance, biofouling and biomass characteristics in membrane bioreactors.
Water Res 2013; 47(1): 187-196.
[186] Oh H-S, Yeon KM, Yang CS, et al. Control of membrane biofouling in MBR for
wastewater treatment by quorum quenching bacteria encapsulated in microporous
membrane. Environ Sci Technol 2012; 46(9): 4877-4884.
[187] Oh H-S, Kim SR, Cheong WS, Lee CH, Lee J-K. Biofouling inhibition in MBR by
Rhodococcus sp. BH4 isolated from real MBR plant. Appl Microbiol Biotechnol 2013;
97(23):10223-10231.
[188] Kim SR, Oh H-S, Jo SJ, et al. Biofouling control with bead-entrapped quorum
quenching bacteria in membrane bioreactors: physical and biological effects. Environ
Sci Technol 2013; 47(2): 836-842.
[189] Cheong W-S, Lee C-H, Moon Y-H, et al. Isolation and identification of indigenous
quorum quenching bacteria, Pseudomonas sp. 1A1, for biofouling control in MBR. Ind
Eng Chem Res 2013; 52(31): 10554-10560.
[190] Kim S-R, Lee K-B, Kim J-E, et al. Macroencapsulation of quorum quenching bacteria
by polymeric membrane layer and its application to MBR for biofouling control. J
Memb Sci 2015; 473: 109–117.
[191] Cheong WS, Kim SR, Oh HS, et al. Design of quorum quenching microbial vessel to
enhance cell viability for biofouling control in membrane bioreactor. J Microbiol
Biotechnol 2014; 24: 97-105.
[192] Weerasekara NA, Choo K-H, Lee, C-H. Hybridization of physical cleaning and quorum
quenching to minimize membrane biofouling and energy consumption in a membrane
bioreactor. Water Res 2014; 67: 1-10.
[193] Musk DJ, Banko DA, Hergenrother PJ, Iron salts perturb biofilm formation and disrupt
existing biofilms of Pseudomonas aeruginosa. Chem Biol 2005; 12(7): 789-796.
[194] O'May CY, Sanderson K, Roddam LF, et al. Iron-binding compounds impair
Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions. J
Medic Microbiol 2009; 58: 765-773.
[195] Singh PK, Parsek MR, Greenberg EP, Welsh MJ. A component of innate immunity
prevents bacterial biofilm development. Nature 2002; 417(6888): 552–555.
254 Vera Lúcia dos Santos and Andrea Sousa Monteiro

[196] Kaneko Y, Thoendel M, Olakanmi O, Britigan BE, Singh PK. The transition metal
gallium disrupts Pseudomonas Aeruginosa iron metabolism and has antimicrobial and
antibiofilm activity. J Clin Invest 2007;117(4): 877-888.
[197] Yang L, Liu Y, Sternberg C, Molinptide S. Evaluation of Enoyl-Acyl Carrier Protein
Reductase Inhibitors as Pseudomonas aeruginosa Quorum-Quenching Reagents.
Molecules 2010;15(2): 780-792.
In: Advances in Natural Products Discovery ISBN: 978-1-53610-088-4
Editors: Ana Rita Gomes, Teresa Rocha-Santos et al. © 2017 Nova Science Publishers, Inc.

Chapter 8

STRATEGIES BASED ON MICROBIAL


METABOLITES FOR MICROBIAL CONTROL IN
AGRICULTURE

Vera Lúcia dos Santos1, and Andrea Souza Monteiro2


1
Departament of Microbiology, Institute of Biological Science,
Universidade Federal de Minas Gerais, Belo Horizonte-MG, Brazil
2
Laboratory of Parasitic Biology – Universidade Ceuma, MA, Brazil

ABSTRACT
The control of microorganisms and their negative effects on the performance of
water systems, such as in distribution pipelines, membrane-filtration processes and
cooling towers, is a serious operational challenge in all water sectors. Microorganisms
and their products form a matrix of protective and adhesive extracellular polymeric
substances (EPSs), mainly polysaccharides, lipids and proteins. Biofilm formation can
lead to decreased efficiency of heat exchangers, membrane reactors, and potable water
distribution systems, in addition to increasing the risk of occurrence of microbiologically
influenced corrosion. Cells show greater resistance to environmental challenges,
including biocidal agents, than their free-living counterparts, mainly due to polymeric
matrix barrier formation. Conventional disinfection and cleaning strategies do not
proficiently address biofilm-related problems, such as the persistence of microorganisms
and the generation of harmful disinfection products. Due to these limitations, ecologically
safe and more efficient alternatives are being sought to control biofilms in water systems,
such as the use of microbial hydrolytic enzymes, surface actives compounds, and phages,
as well as the use of quorum sensing (QS) inhibitors and energy uncoupling. These
biologically based microbial control strategies seem to constitute a promising component
of an efficiently integrated control program because they help to overcome the current
problems of biofilm control. Microbial metabolites can be effective in preventing
adhesion to surfaces, bacterial differentiation and matrix elimination and control of
planktonic microbiota. Furthermore, they are biodegradable, and their commercial

 Corresponding author. Laboratory of Applied Microbiology, Department of Microbiology, ICB-UFMG, C.P. 486,
31270-901, Belo Horizonte, Brazil. E-mail: vlsantos@icb.ufmg.br.
256 Vera Lúcia dos Santos and Andrea Souza Monteiro

production is cost-effective because the culture media components and substrates utilized
can be obtained from less expensive sources, such as agro-industrial waste. In this
review, we explore aspects of biofilm characteristics in water systems and examine the
potential of microbial metabolites for microbial control.

Keywords: biofilm, resistance, water system, microbial metabolites, surface actives


compounds

INTRODUCTION
Aquatic microbial communities occur as planktonic assemblages that develop in the
water columns of either natural or artificial environments and also as attached biofilm
communities. While planktonic and biofilm communities share similar biogeochemical and
organic carbon processing functions, they have markedly different physical structures and
biotic interactions that can lead to different responses to changes in the supply of resources
and physical environmental factors [1]. The ability of this diverse microbiota to attach to
surfaces and to develop into multispecies biofilm appears to be an ancient and integral feature
of microorganisms, which over evolutionary time has enabled them to optimize growth and
survival in adverse environments, such as flowing environments [1-3]. Thus, microorganisms
are able to colonize natural compartments, attached to plant and animal debris and submerged
or fluctuant macrophytes in water columns or attached to rocks immersed in the water of
rivers and marine environments, and artificial compartments, such as water-contact
equipment surfaces in diverse industrial sectors, including cooling towers, pipeline
distribution systems and water storage facilities or membrane reactors.
Biofilms comprise highly complex communities including bacteria, archaea, fungi, algae
and protozoa living among a protective matrix of extracellular polymeric substances (EPS),
comprised on polysaccharides, DNA, protein, particulate material and detritus [4]. A mature
biofilm has interwoven fluid channels that enable the transportation and transformation of
nutrients, gases and their associated waste products throughout the structure of the biofilm [5-
6]. Quorum sensing (QS) is bacterial density-dependent cell-to-cell communication using
small molecules produced and recognized by microbes. QS has been shown to regulate gene
expression, mediating some bacterial behaviors, such as the production of soluble microbial
products, secretion of EPS and extracellular enzymes, virulence factor production and biofilm
formation [7- 9].
Biofilm communities play important roles in the functioning of aquatic ecosystems. They
act as sites of essential ecological processes, such as current primary production, carbon and
nitrogen fixation and cycling of key nutrients, including phosphorus and nitrogen in
freshwaters. It is believed that less than 1% of microbial carbon cycling occurs within the
water columns of aquatic ecosystems [10].
The lifestyle of biofilm benefits microorganisms in various ways, including the efficient
use of resources due to the diverse metabolic capabilities of the different members of the
biofilm [11], the recycling of secreted end products, e.g., the usage of organic carbon
produced by autotrophs by heterotrophic bacteria [12-13], buffering against nutrient
limitations [14], protection from shear stress, oxygen radicals, and pH changes [3, 15-16] and
protection from predation [17]. Conversely, biofilms act as a basal food resource in aquatic
Strategies Based in Microbial Metabolites … 257

ecosystems, playing a major role in the bottom-up supply of nutrients to organisms at a higher
trophic level, potentially affecting food webs throughout the stream ecosystem. Thus, the
specific microbial composition of the biofilm can modify the nutritional quality of the
material for grazing species [18].
This pattern is also beneficial in artificial environments, in which it forms the
fundamental basis of treatment technologies, such as fluidized and fixed bath reactors or
membrane bioreactors (MBRs). However, in several industrial sectors, microorganisms can
accumulate in equipment, resulting in biofouling and biocorrosion both actively (due to the
consumption of oxygen by aerobic bacteria and/or the formation of a mass of occlusion that
creates an oxygen gradient) and passively (by silt deposition on the metal surface) [19]. Many
bacteria exhibit corrosive effects through the action of their metabolic end products, and
among these bacteria are the sulfate-reducing bacteria (SBR), acid-producing bacteria, such
as Thiobacilus, and hydrogen-utilizing sulfate reducers and iron bacteria, such as Gallionella,
which oxidize ferrous ions to ferric ions that can bind free chloride ions in solution to form
the ferric chlorides that are deposited as corrosion products. Microbiologically influenced
corrosion and associated biofouling have been implicated in high profile failures across a
wide range of environments, ranging from oil and water pipelines and nonmetallic materials,
such as concrete and machinery, to biomedical devices. The economic impact of corrosion is
significant given the need to replace corroded equipment, as well as repairs and attempts to
prevent corrosion [20-22].
Another important aspect is that biofilms formed on industrial equipment can include the
growth of microorganisms of sanitary interest, such as Legionella pneumophila in cooling
towers, which can be released into the environment through aerosol generated in the tower,
causing pneumonia-like symptoms in immunocompromised patients [23-24]. In systems of
potable water distribution, biofilm results in water flow contamination by biomass
detachment of opportunistic pathogens, such as Mycobacterium avium, Pseudomonas
aeruginosa, Klebsiella spp., Legionella spp., Flavobacterium spp., enteric viruses, Bacillus
spores or Cryptosporidium oocysts [25-33]. Biofilms in water systems also result in
unpleasant taste and odor [34]. In the beverage and food industry, biofilms on equipment
surfaces act as potential sources of product contamination with foodborne pathogens and
spoilage microorganisms [35]. Biofilms can also be important in the clinical setting when
they develop on medical devices, such as implants, catheters, and contact lenses, leading to
life-threatening infections [36].
Another problem is based on biofilm cells showing greater resistance to biocides and
stress environments than their planktonic counterparts, enabling the proliferation of biofilm in
industrial environments, presenting problems in removing them once established. Many
different mechanisms of biofilm resistance have been discussed in the literature, reflecting the
different manners in which biofilm organisms withstand biocides. These mechanisms include
physical and chemical diffusion-reaction barriers in the biofilm restricting biocide penetration
of the biofilm [37-38], the slow growth rate of biofilm cells due to nutrient limitations [39],
activation of general stress response genes [40], the emergence of a biofilm-specific
phenotype and the presence of persister cells [41-42].
It has been suggested that the EPS matrix can restrict the mass transfer of antimicrobial
agents for biofilm microorganisms by acting as a diffusion barrier or by the interaction of the
biocide with matrix components. Most traditional chlorinated oxidant biocides, such chlorine,
are consumed by reactions with corrosion products and deposits [43-44], pipe materials [45-
258 Vera Lúcia dos Santos and Andrea Souza Monteiro

47] and biofilm constituents, including cells and EPS [6, 48]. In fact, field observations and
laboratory studies have indicated that biofilms seem to be recalcitrant to the killing and
disrupting activity of chlorine in concentrations that are relevant in practice, whereas
planktonic cells are more easily inactivated by the biocide. Planktonic control cells of
Streptococcus thermophiles were effective after exposure to 20 mg/L of sodium hypochlorite
for 30 min (pH 6.8–7.0), whereas viability of this organism grown as a biofilm on stainless
steel was still detected after treatment with up to 1,000 mg/L of sodium hypochlorite [49].
Mathieu et al. (2009) showed that biofilms grow and harbor active bacteria, even in
continuously chlorinated water systems with residual levels of chlorine ranging from 0.1 to
0.4 mg Cl2/L [50].
Several studies have also shown that chlorine had a slight weakening effect on P.
aeruginosa biofilms [51], but it was more effective in weakening the mechanical
cohesiveness of S. epidermidis biofilm and eroding the attached biomass [37], and it induced
detachment and killing of Pseudoalteromonas ruthenica at 1 g/L [52]. Tachikawa et al.
showed an apparent decrease in EPS in the biofilm matrix exposed to halogenated oxidants or
ozone, as well as a clear relationship between the removal of EPS and the bacterial
inactivation rate [53].
Chlorine-based biocides, monochloramine and chlorosulfamates were reported to
penetrate better into biofilms than free chlorine because they seem to have a lower capacity to
react with biofilm constituents [38]. As a result, these compounds can exhibit similar or
superior efficacy when tested against biofilms, despite being weaker biocides than chlorine
[54]. For example, it was demonstrated that, for equivalent chlorine concentrations,
monochloramine initially penetrated biofilm 170 times more rapidly than free chlorine [38].
Concentrations as low as 1 ppm were able to penetrate biofilm matrices, such as those in
cooling towers [54], and residuals of 2-3 mg/L of Cl2 were needed to control biofilms
effectively in cooling systems using tertiary treated wastewater as makeup water [55].
Subsequently, it was shown that EPS quantity and composition significantly affected
monochloramine penetration, biofilm inactivation, and detached cell viability due to the
selective reactivity of monochloramine [56]. Biocide has selective reactivity with proteins
over polysaccharides. Thus, the presence of monochloramine-reactive or monochloramine-
nonreactive components in biofilm matrix can have great influence on monochloramine
transport, as well as disinfection efficacy. Recently, Stewart [57] examined the tolerance of
microorganisms in biofilms to antimicrobials through a meta-analysis of data from the
literature. It was discussed that the biofilm tolerance variation was not explained by consider
the size and chemistry of the antimicrobial, the substratum material, or the microbial species
composition. The antimicrobial was partially correlated with the cell density of the biofilm at
the time of treatment and the age of the biofilm as grown in a particular experimental system,
suggesting that there is something occurring during biofilm maturation, either physical or
physiological, that is essential for full biofilm tolerance.
Another problem is that the greater reactivity of chlorine with natural organic matter can
lead to the formation of by-products, such as trihalomethanes or haloaceto-nitriles. As a
result, chlorinated effluents can have adverse impacts on human health and aquatic
ecosystems because the effluent can contaminate downstream drinking water sources.
The aforementioned points show clearly that biofilms can potentiate various problems
related to the presence of microorganisms in different environments. Thus, microbial control
programs in industry should be directed to prevent the adhesion of microorganisms to
Strategies Based in Microbial Metabolites … 259

surfaces and/or to prevent colonizing organisms from building up to problematic levels or to


break up the biofilm EPS matrix to disperse the biofilm or alter the elastic properties of
biofilms and allow for biocides to diffuse rapidly and kill microbial cells.
A variety of strategies for biofilm control has been used, including periodic cleaning
using physical methods (rinsing, brushing, ultrasonic), application of chemical agents
(oxidants, alkali, surfactants, enzymes, complexing substances, dispersants) to kill and detach
biofilm cells and limitations of nutrients in water systems to minimize microbial growth.
Considering the inefficacy and products generated, the use of innovative strategies that are
less harmful to the environment to optimize the biofilm control has been evaluated. In this
regard, biologically based anti-biofilm strategies to enhance the effectiveness of currently
applied cleaning protocols in industry have been proposed. These strategies include the use of
metabolites that target a specific molecule or mechanism that is responsible for the
attachment, communication, motility or growth of microbial cells and the biofilm matrix
structure. Therefore, this chapter sheds light on the state of the art of potential strategies to
control biofilms based on hydrolytic enzymes, biosurfactants, QS inhibitors and phages.
However, because most of the biologically based anti-biofilm strategies are still under
development, the challenges and limitations will also be discussed.

BIOLOGICAL ANTI-BIOFILM APPROACHES TO


PREVENT OR REMOVE BIOFILMS
A large number of new biological approaches have been developed in the last few years
for biofilm control. They include: bioactive microbial metabolites, such as biosurfactants,
exopolysaccharides and bacteriocins [58-59]; enzymes that dissolve biofilms by
depolymerizing polysaccharides, proteins or extracellular DNA [60-61]; uncoupling energy
[62]; the use of QS inhibitors [63]; and lytic phages [64]. These approaches can in some
situations have the advantages of greater efficiency, lower toxicity, greater sustainability and
less bacterial resistance relative to other control methods.

Strategies Based on Biosurfactants

Microorganisms are able to produce a wide diversity of surface-active compounds


(SACs) that present both hydrophilic and hydrophobic moieties, and these molecules can
interact with surfaces, lowering surface and interfacial tensions, and form micelles, which are
emulsifying immiscible substances. The microbial SACs can be distinguished in terms of
size, and they include: (1) biosurfactants that are low-molecular-weight surfactants; (2)
amphiphilic polymer that are high-molecular-weight surface-active polymers with one
hydrophobic region at one end of the molecule, such as lipopolysaccharides, lipoteichoic
acids and lipoglycans on bacterial cellular walls; and (3) polyphilic polymers that are high-
molecular-weight surface-active polymers with hydrophobic groups distributed across the
entire molecule, identical to hydrophobically modified, comb-type polymers, such as emulsan
and hydrophobic polysaccharides [65].
260 Vera Lúcia dos Santos and Andrea Souza Monteiro

Another criterion for categorizing microbial SACs is the chemical nature of the
molecules. The major classes include varied structures, such as glycolipids, lipopeptides,
polysaccharides or protein complexes, phospholipids, fatty acids and neutral lipids. These
biomolecules can be excreted into the extracellular medium or remain attached to the cell
surface, denominated as particulate biosurfactants [66].
Microbial SACs have been recognized for some time in a diverse array of potential
applications in industries including agriculture, food, cosmetics, and the pharmaceutical and
petroleum industries. The surface and interfacial tension-reducing properties of biomolecules
provide excellent detergency, emulsification, foaming and dispersing traits, making them
some of the most versatile products in chemical processes. In addition, they have shown their
specificity, low toxicity, high biodegradability, widespread applicability and effectiveness at
extremes of pH and temperature [66-70]. In the biomedical field, biosurfactants are receiving
more attention for their anti-biofilm and antimicrobial activities, due to their lower toxicity to
plants and animals, high biodegradability, and low irritancy to and compatibility with human
skin [71].
The physiological roles of these biomolecules are often unclear, but many of them are
essential for the survival of microorganisms in the environment, including increased surface
area and bioavailability of hydrophobic substrates, antagonistic activity, binding to heavy
metals, bacterial pathogenesis, QS, biofilm formation and desorption surfaces [72]. These
tensoactive molecules can spontaneously adsorb to surfaces, altering properties such as
wettability and charge or even changes in bacterial cell surface structures, thus modeling
bacterial adhesion to surfaces [65, 73]. A loss of cell outer membrane lipopolysaccharide
protein complexes was observed in P. aeruginosa treated with mono-rhamnolipids (RLs),
resulting in increased cell surface hydrophobicity [74]. RL biosurfactants are important in
maintaining transport channels, and they directly influence biofilm structure, namely the
creation of mushroom-like filaments, and exclude other invasive species from the biofilm
structure [75-76]. Other example is a 546-KDa exopolysaccharide (A101) isolated from a
marine Vibrio that inhibited initial adhesion of both Gram-negative and Gram-positive
bacteria, selectively affected cell-to-cell interactions and induced biofilm dispersion of P.
aeruginosa [77].
Because of their tensoactive properties, many SACs show antibacterial, antifungal and
antiviral activities. These properties are the result of the action of these molecules, increasing
membrane permeability and causing metabolite leakage by altering physical membrane
structures or by disrupting proteins, thus, interfering with important membrane functions,
such as energy generation and transport [78]. This finding has also been reported to lead to
toxicity, lysis, pyrogenicity, mitogenicity and immunogenicity, among other effects. Several
studies have demonstrated that, under certain conditions, SACs can be more effective than
many traditional biofilm inhibition and/or disruption strategies.
This antimicrobial property of SACs has been extensively explored with a view toward
clinical application, but there have been few studies focusing on applications of these
biomolecules in the water industry. RLs were successively used to clean ultrafiltration
membranes fouled by proteins. The strategy could largely remove fouling from polysulfone,
polyacrylonitrile and polysulfone-g-poly-ethylene glycol membranes and restore the water
flux to approximately 94% of the initial level, performing much better than the flux recovery
of 50–70% for Tween 20 and SDS [79]. BS also exhibited superior properties over sodium
hydrate (NaOH) and commercial membrane cleaner, considering the cleaning efficiency and
Strategies Based in Microbial Metabolites … 261

operation mildness. RLs were also able to disrupt preformed biofilms of Ps. aeruginosa [80].
A commercially available RL was assessed for its ability to inhibit adhesion and disrupt
Bacillus pumilus pre-formed biofilms isolated from the surfaces of titanium coupons
immersed in seawater. Titanium surfaces are generally used for the manufacture of
condensers and other heat exchangers in power plants, which are subjected to microbial
corrosion. The biomolecule inhibited the planktonic growth of B. pumilus cells at
concentrations >1.6 mM, and concentrations from 0.05 to 100 mM inhibited the adhesion of
cells to polystyrene microtiterplates, wherein the effectiveness ranged from 46 to 99%. The
rhamnolipid was effective against pre-formed biofilms, acting on the removal of biofilm-
matrix components and disruption of biofilms [81]. RLs at 10 and 200 mg/L reduced the
initial attachment of the bacteria P. aeruginosa, P. putida, and Escherichia coli and of
Bacillus subtilis on hydrophilic glass and hydrophobic octadecyltrichlorosilane (OTS)-
modified glass under continuous-flow conditions. For Staphylococcus epidermidis, the effect
occurred only on hydrophobic surfaces [82].
Apparently, treatment of biofilms with biosurfactants that modify the structure of biofilm
matrix would make them an attractive choice as chemical agents that can enhance the efficacy
of antibacterial agents, for example, in cooling water systems for enhancing the efficacy of
chlorinated biocides in microbial control. RLs from Pseudomonas spp. enhanced the
disinfection effects of NaOCl and peracetic acid/hydrogen peroxide on stainless-steel surfaces
contaminated with Listeria monocytogenes [83]. Synergistic enhancement was reported of the
killing effects of silver ions and lipopeptide BSs against an E. coli biofilm population to
achieve complete biofilm eradication [84]. The authors also hypothesized that V9T14
lipopeptide interacts with the bacterial membrane, leading to pore formation and alteration of
membrane integrity; this effect increases the entry of several antimicrobials into the cells and
their effectiveness against uropathogenic E. coli CFT073 biofilm [85]. Our group showed that
the combination of sub-inhibitory concentrations of the Lactobacillus jensenii P6A
biosurfactant and benzazole compounds presented synergic effects that were concentration-
dependent against E. coli and C. albicans.
Qin et al. (2012) evaluated a novel submerged membrane bioreactor (SMBR) combined
with rhamnolipids (RSMBR) to treat frying oil wastewater and to control the problem of
membrane fouling [86]. RSMBR not only exerted high removal efficacy of oil of up to 90%
in a short hydraulic time, but it also exhibited 10 times greater membrane permeability than
the reactor without RLs. The presence of rhamnolipids greatly enhanced the contact and
reaction between the microorganism and oil molecules. Great improvement in membrane
filterability was associated with an increase in the hydrophobicity of flocs, as well as the
increase in particle size from 53.06 to 145.54 µm. The oil strongly adhered to the surfaces of
flocs by rhamnolipids, and it consequently prevented larger oil droplets from directly
depositing on the membrane surface.

Strategies Based on Enzymes

Enzymes can affect the colonization and adhesion of microorganisms to surfaces and can
result in biofilm control in several manners. They can affect the adhesion to surfaces of
settling organisms, thus preventing settlement events [87-88]. Second, specific enzymes can
hinder intercellular communication during colonization of a surface [89]. Finally, hydrolytic
262 Vera Lúcia dos Santos and Andrea Souza Monteiro

enzymes can effectively promote the matrix degradation of EPS in multi-structured biofilms,
altering the physical properties of biofilm. The most commonly used are amylases,
hydrolases, glycosidases, lipases, acidic and alkaline proteases and deoxyribonucleases [90-
93]. This approach has the advantages of being non-toxic and less susceptible to the
development of bacterial resistance, which are mechanisms commonly observed for many
anti-biofilm agents.
Microbial EPS have different substituent groups, such as ketal-linked pyruvate or ester-
linked acetyl groups, and the removal of these groups can affect the physical properties of
exopolysaccharides. Intracellular carboxylesterase (EC 3.1.1.1), isolated from Arthrobacter
viscosus, removed acetyl residues from xanthan, alginate, glucose penta acetate, cellobiose
octaacetate, exopolysaccharide produced by A. viscosus, deacetylated p-nitrophenyl
propionate, naphthyl acetate, isopropenyl acetate and triacetin [94]. Pectin esterase,
originating from Trichoderma viride, could deacetylate a polysaccharide in Pseudomonas
fluorescens biofilm matrix, rendering it softer and more porous [95-96].
A crude cellulase preparation from Trichoderma viride was effective in the degradation
of dephosphorylated and partially derhamnosylated EPS of Lactococcus lactis subsp.
cremoris B40 [97]. Cellulase from Penicillium funiculusum was effective in degrading mature
biofilms of P. aeruginosa and the exopolysaccharides of P. fluorescens [98-99]. The enzyme
reduced the molecular weight of the polymers in assays using purified biofilm EPS.
Actinobacillus actinomycetemcomitans produces an hexoaminidase known as dispersin B
that hydrolyses the glycosidic linkages of polymers that contain β-1,6-N-acetyl-D-
glucosamine (poly-β-1,6-GlcNAc) (PIA/PNAG), which serves as an Escherichia coli and
Staphylococcus epidermidis biofilm adhesin, the formation of which requires the pgaABCD
and icaABCD loci, respectively. The hydrolysis of this polymer by this enzyme disrupts
biofilm formation by these species and also by Yersinia pestis and P. fluorescens, which
possess pgaABCD homologues [60]. In general, S. epidermidis biofilms are dispersin B
susceptible and DNase I resistant, whereas S. aureus biofilms are DNase I susceptible and
dispersin B resistant, suggesting that PNAG is a major matrix adhesin in S. epidermidis
biofilms and a minor component of S. aureus biofilms [100]. Precoating polyurethane and
Teflon catheters with this enzyme prevented S. epidermidis biofilm formation; the modified
polyurethane catheters retained enzyme activity for at least 30 days when stored at room
temperature [92]. The combined use of dispersin B and rifampicin was effective in
eradicating the S. epidermidis biofilm that developed in poly(dimethylsiloxane)(PDMS)
microfluidic devices [101].
Proteolytic enzymes have been employed to remove established biofilms. Proteinase K
caused 98% detachment of 53 biofilm-positive clinical S. haemolyticus isolates [102].
Addition of this enzyme or DNase I to culture medium also inhibited biofilm formation by
nontypeable Haemophilus influenzae. Both enzymes also caused significant detachment of
pre-formed biofilms of this strain, indicating that both proteinaceous adhesins and
extracellular DNA contribute to biofilm cohesion [103]. Leroy et al. (2008) investigated the
antifouling potential of savinase (subtilisin) on adhesion of Pseudoalteromonas sp. D41
[104]. The enzyme should be more effective in preventing initial microbial adhesion than
disrupting established biofilm because the IC50 of subtilisin was found to be 38 times lower
for the prevention of microbial adhesion than for the detachment of adhered bacteria.
Extracellular DNA (eDNA) could play important roles in biofilm development, including
supply substrates for sibling cells, maintaining the three-dimensional structure of biofilms and
Strategies Based in Microbial Metabolites … 263

enhancing the exchange of genetic material [61, 105]. Thus, disruption of eDNA would lead
to the detachment or dispersal of biofilms. Whitchurch et al. (2002) showed that
deoxyribonuclease I could inhibit the development of biofilm and dissolve established
biofilms of P. aeruginosa PAO1 [106]. Alterations in the biomass, architecture, morphology,
and numbers of colony-forming units (CFU) of biofilms formed by diverse bacteria (Gram-
positive and Gram-negative bacteria) were observed in the presence of DNase [107]. The
addition of this enzyme also enhanced the effects of antibiotics, resulting in decreased biofilm
biomass and numbers of CFU, probably because the cleavage of extracellular DNA led to the
formation of an altered biofilm that permitted the increased penetration of antibiotics.
Some hydrolytic enzymes of bacterial cell walls have been described by their
antimicrobial activities. An example is lysozyme, which targets the 1,4 glycosidic bonds that
link the N-acetylglucosamine (NAG) and N-acetylmuramic (NAM) acid moieties that
compose the bacterial cell wall peptidoglycan. The enzyme immobilized on the surface of
polyethylene and polyvinylalcohol films exhibited activity against Micrococcus lysodeikticus
[108-109]. This enzyme also reduced by 28.2–69.6% the metabolic activity of Candida
biofilms on acrylic dentures [110]. Lysozyme combined with the antifungals nystatin,
amphotericin B, and ketoconazole resulted in effective synergistic killing of biofilm of
Candida. Combination with imidazole lanoconazoles resulted in synergistic antifungal
activity against C. albicans blastopores [111]. Another example consists of endolysin SAL-1
and LysK, which are produced by phages [112].
There are two main alternatives for developing efficient enzymatic products for biofilm
control. The first concerns the identification of the polysaccharides present in the biofilm and
the choice of specific enzymes capable of degrading them. The other aims to identify the
active compounds in commercial products, followed by evaluation of their effects on biofilm.
In this latter approach, the specificity in the enzyme modes of action makes it a complex
technique, increasing the difficulty of identifying enzymes that are effective against all of the
different types of biofilms. Therefore, formulations containing several different enzymes
seem to be fundamental for a successful biofilm control strategy.
An example of the first case is the study by Marcato-Roamin et al. (2012), based on the
characterization of the EPS extracted from six industrial biofilms from the paper industry and
the subsequent evaluation of the effectiveness of eight hydrolytic enzymes in reducing them
[113]. The EPS were mainly proteins, and the protein-to-polysaccharide ratio ranged from 1.3
to 8.6 depending on where the sampling point was situated in the paper-making process.
Glycosidases and lipases were inefficient or only slightly efficient for biofilm reduction,
while proteases were more efficient. After treatment for 24 h with pepsin, Alcalase® or
Savinase®, the removal exceeded 80%. When tested on an industrial biofilm sample,
Savinase® led to a significant release of proteins from the EPS matrix, indicating its potential
efficacy on an industrial scale. This enzyme was successively used in combination with shear
stress treatment. The combined treatment led to an increase of 80% in biofilm mass removal
(COD) compared enzymatic treatment alone, and it removed a large proportion of the basal
layer of the biofilm, with 80% reduction observed in the support coverage [114]. It was
shown that colanic acid, commonly produced by some enterobacterias and observed in
biofilms from different paper mills, could be hydrolyzed to its corresponding hexasaccharide
repeating unit by β(1,4)-fucanosyl hydrolase [115]. Other example is levan, which is a β-2,6-
linked polymer of fructose that is normally present in biofilms of several species of Bacillus
and Pseudomonas. Levan could be hydrolyzed by the enzyme levan hydrolase to low-
264 Vera Lúcia dos Santos and Andrea Souza Monteiro

molecular-weight polymers that are water soluble, thereby cleaning the slime out of the
system [116].
Chaignon et al. (2007) investigated the susceptibility of biofilms of clinical
staphylococcal strains to a range of enzyme preparations containing dispersin B, pancreatin,
proteinase K, Pectinex Ultra SP, periodate and trypsin [117]. Whereas dispersin B was the
most effective against strains that contained N-acetylglucosamine as the major component in
the extracellular biofilm matrix, these biofilms were not affected by protease treatment. The
proteases were effective against strains that lacked N-acetylglucosamine. Therefore, a
recommendation for the complete removal of biofilms formed by a range of staphylococcal
strains from inert surfaces could be the combined use of dispersin B and protease.
Torres et al. (2011) evaluated the efficacy of 17 non-specific, commercial enzymatic
mixtures for the prevention and control of biofilm formed by bacteria isolated from paper
mills on laboratory and pilot plant scales [118]. Pectinex Smash® and its fraction
Novoshape® were the best formulations in the prevention of biofilm formation, and they are
predominantly composed by the pectin methylesterase. This enzyme was able to reduce
biofilm formation by 71%, compared to control tests.
In another study, Leroy et al. (2008) evaluated the antifouling potential of four proteases,
seven glycosidases and one lipase in the adhesion of marine Pseudoalteromonas sp. D41
[119]. Savinase (subtilisin) was the most effective hydrolase in preventing bacterial adhesion
and removal of the adhered bacteria. Proteases were also evaluated to remove biofouling from
ultrafiltration membranes for wastewater treatment [120]. Enzymatic treatment showed
greater efficiency in removing biofouling, leading to complete recovery of clean water flux at
low temperatures (25-30°C), compared to traditional cleaning methods based on alkaline
chemicals.
Molobela et al. (2010) tested selected commercial proteases (savinase, everlase and
polarzyme) and amylases (myloglucosidase and bacterial amylase novo) for their
effectiveness in the degradation and removal of EPS in P. fluorescens biofilm [121]. Savinase
(subtilisin) and everlase were the most effective for degradation of P. fluorescens EPS, while
the protease polarzyme was ineffective. The reason for the inefficiency of polarzyme could be
attributed to its lack of action on the protein structural components of the biofilm EPS that
was evaluated. In addition, Lequette et al. (2010) also evaluated the activity of
polysaccharidases and proteolytic enzymes against the biofilms of 16 bacterial species found
in food industry processing lines, using a microtiter plate model [93]. The two serine
proteases removed the biofilms of a broader range of bacterial species, while the amylase S1
totally removed the biofilms of three Pseudomonas strains. The authors also evaluated the
effects of two serine proteases, amylase S1, and polysaccharides mix A in the removal of
biofilms formed on surfaces of stainless steel slides in a CIP procedure. The efficacy of
enzymes depended on the bacterial species; proteases were more efficient than
polysaccharidases in removing Bacillus biofilms, while polysaccharide-degrading enzymes
were more efficient in removing P. fluorescens biofilms.
Despite the potential use of enzymes to control microbial biofilm, some drawbacks
inherent to the method could limit its large-scale application. Enzyme activity would be
reduced or even totally lost in operations that lack the optimal pH and temperature values
required by the enzyme. To circumvent this problem, various approaches are being used to
increase the stability of enzymes, including enzyme modification, protein engineering and
medium engineering. New techniques, such as self-immobilization of enzymes, the
Strategies Based in Microbial Metabolites … 265

immobilization of enzymes using nanoscale structures, and the production of single-enzyme


nanoparticles, are also currently gaining a great deal of attention for this purpose [122].
Because EPS secreted by microorganisms is a mixture of macromolecules, its efficient
removal by enzymatic disruption will depend on the availability of various enzymes. Another
alternative is the combined use of enzymes and diverse chemical and physical agents for the
complete removal and/or elimination of microbial cells associated to the biofilm. Many
studies and patents have focused on these approaches.
The patents involve: mixtures of enzymes and a surface active agent, preferably anionic
[123]; at least one enzyme belonging to the polysaccharidases, proteases, lipases and glycol
proteases or lipases and a short-chained glycol component [124]; enzyme mixing, consisting
of 2 parts cellulose to 1 α-amylase to 1 protease utilized in 2-100 parts per million [125]; at
least one mannanase, optionally in combination with at least one enzyme from the group
consisting of carbohydrases, proteases, lipases, glycoproteases [126]; composition for
removing biofilm from a surface, comprising an enzyme mixture with at least two different
enzymes selected from protease, cellulase, esterase, mannanase, glucanase, phospholipase and
amylase [127]; methylene-bis-thiocyanate, dimethyl dithiocarbamate or disodium ethylene-
bis-dithiocarbamate as a biocide and amylase, as well as a dextran-degrading enzyme or a
levan hydrolase as the polysaccharide-degrading enzyme [128]; application of biocides, such
as chlorine, hypochlorite, bromine, hydrogen peroxide, in concentrations of 0.5-500 ppm and
trypsin and/or endo-protease and/or chymotrypsin in approximately 0.01-1,000 units to
inhibit the growth of filamentous organisms [129]; an acellular dispersant produced by P.
aeruginosa, which induces dispersion of sessile bacterial cells, and an additive (biocide,
surfactant, antimicrobial, antiseptic, detergent, chelating agent or virulence factor
inhibitor) [130]; a patented compound and an enzyme selected from oxidoreductase
(oxidative/reductive enzyme), transferase (transferring enzyme), hydrase (hydrolytic
enzyme), lyase (isomerizing enzyme), and isomerase (isomerizing enzyme) and a surfactant
[131]; a mixture of enzymes (at least one protease, one esterase and one amylase) and an
alkaline detergent [132]; and an EDTA chelating agent, a N-acetyl cysteine antioxidant or
derivative salts, and an optional carrier [133].
Oulahal-Lagsir et al. (2003) investigated the removal of biofilms formed by E coli from a
stainless steel surface using combined ultrasonic and enzymatic treatment (proteolytic or
glycolytic enzymes) [134]. The combined treatment was more effective in removing all
biofilms than the application of ultrasound alone. Similarly, Oulahal et al. (2007) reported
that application of ultrasound alone was not effective in removing all biofilms formed by two
meat spoilage microorganisms (E. coli and S. aureus) from a stainless steel surface [87].
When ultrasound was used in combination with EDTA and/or enzymes, approximately 75%
of E. coli and 100% of S. aureus biofilms were removed.

Strategies Based on Phages

Phages are viruses that infect bacteria, presenting several morphologies, including
filamentous, icosahedral with tails, tail-phage, phage with lipoprotein envelope or phage with
lipid reservoirs [135]. Considering the course of infection, viruses can exhibit several life
cycles, including lytic, chronic, lysogenic and pseudolysogenic [136]. Infection initiates by
attachment of the virion to a susceptible bacterial cell by binding to the receptors of the
266 Vera Lúcia dos Santos and Andrea Souza Monteiro

phages, such as lipopolysaccharides (LPSs) and proteins of the outer membrane, fimbriae,
pilli, flagella, capsular and slime polysaccharides, followed by injection of the virion or its
nucleic acid into the cell. Then, the cell biosynthetic apparatus is altered so that virally
encoded enzymes and genetic material are produced for the assembly of the capsid shells and
packaging of the nucleic acids within them. The mature virions are released from the cell by
either nondestructive budding or lysis, characterizing a chronic or lytic cycle, respectively.
The specificity of the receptors determines the host range of the phages. These monovalents
are able to adsorb to the specific bacterial species or strains, while polyvalent phages can
infect different bacterial species or genera. In the lysogenic cycle, the viral genome integrates
into the host chromosome, remaining in a state of prophage until induction of the lytic cycle
through the activation of a suitable set of cellular triggers in response, for example, to the
nutrient levels, oxidative stress caused by UV radiation and the presence of hydrogen
peroxide [137-138]. However, environmental signals and molecular mechanisms that control
prophage induction/excision under biofilm conditions remain elusive for most species [139].
The pseudolysogenic cycle is an intermediary state between lytic and lysogenic stages, in
which an extrachromosomal virus replicates in synchrony with the host chromosome, such as
an episome.
Due to the their nature, the phages are good candidates for biofilm control, with particular
reference to the following characteristics: broad spectrum of lytic activity; generation of
minimal numbers of resistant mutants; relatively simple preparation at high titers; long-term
storage; rapid reproduction: minimal latent period with high yield; absence of lysis inhibition;
lack of transduction; phage capacity for mutations or recombinant formation to overcome
simultaneously several types of resistance; and capacity of phages from the same mixture for
the cross-lysis of mutants [140-141]. In addition, phages can replicate at the site of an
infection, thereby increasing in numbers where they are most required, and some phages
produce enzymes that degrade the EPS matrix of biofilms. The phages are also non-toxic to
animals and plants.
The effectiveness of the use of phages in microbial control depends on several factors,
including the bacteriophage-to-target bacteria ratio, the mode of treatment, the age of the
biofilm during treatment, the neutralization of phages and the accessibility to target bacteria
[140, 142-143]. The biocidal activity of phages must be investigated in environmental
conditions relevant to their potential applications, considering that the lytic activity and
stability of phages are also variable depending upon the conditions of the environment, such
as pH and temperature.
The use of bacteriophages in microbial control has been studied using two different
approaches: passive or active treatment. In the passive approach, the bacteriophages are
applied at a dose sufficient to ensure that all target bacteria are infected and lysed in a short
period of time. In contrast, active treatment relies on the addition of a relatively small dose of
phages because most bacteria are killed by secondary infections due to replication and
transmission from neighboring cells. This approach is dependent on the phages being able to
spread between the target hosts, which could be weakened by the biochemical and physico-
chemical characteristics of the surrounding system, such as viscosity, or by the presence of a
greater number of inert bacteria.
The bacteriophage-to-bacteria ratio is explained by the term MOI (multiplicity of
infection), which expresses the number of viruses that are added per cell during infection.
This term is used only in fluid systems with large numbers of host cells, and most in vitro and
Strategies Based in Microbial Metabolites … 267

in vivo assays of phages against bacteria apply MOIs between 0.01 and 100. Considering that
not all phages replicate and survive in the same Fashion, a correct definition of the
bacteriophage-to-bacteria ratio requires the determination of the lytic cycle replication and
phage resistance under different environmental conditions [140]. The change in viral ratio can
improve the treatment efficacy, as observed in a study conducted with P. aeruginosa [143].
At concentrations of 400 and 4x107 FPU/mL, the phages inhibited P. aeruginosa biofilm
formation by 45± 15% and 73±8%, respectively. For pre-existing biofilm treatment, similar
results were obtained; biofilm removal efficiency was increased in rate from 45± 9% to
75±5% when the phage dose increased from 6x103 to 6 x107 PFU/mL.
Weld et al. (2004) showed that targeted bacteria concentrations and seeded phage density
affected the success of infection [144]. Growth of T4 only occurs when the bacterial
concentration is greater than 20,000 (CFU/mL) because the phages cannot contact bacteria
efficiently for propagation. The growth phase of host bacteria can also affect infection
efficacy. Because the isolated Pseudomonas phages were RNA ones [143], it was expected
that they would infect bacteria by first attaching to pili and then by entering the bacterial cells
though F-pilus retraction [145-146]. Because F piliation reaches a maximum in the mid-
exponential phase and disappears in the late-exponential phase [147], the growth status of
target bacteria can affect the success of phage treatment as well. In addition to host cell
concentrations, nutrient limitations, competition from other phages and the presence of
phage-resistant cells can also affect replication [148].
Other studies have discussed the effect of biofilm age on the efficacy of phage lysis.
Ganegama Arahchi et al. (2013) explored the potential of individual phages, along with a
three-phage cocktail, to clear L. monocytogenes mixed-strain biofilms adhering to stainless
steel coupons, including ones contaminated with fish proteins at low temperatures (15°C)
[142]. Multi-log10 removal, including to the point of apparent sterilization, was observed
with younger biofilms, but partial physical removal of biofilm bacteria from surfaces was
required for similar results in the treatment of older, more mature biofilms. This fact was
attributed to the shielding effect of the biofilm matrix and/or the phage resistance of biofilm
cells deep in the biofilm matrix. Soni and Nannapaneni [149] also observed that the efficacy
of phage lysis was affected by the age of the biofilm. The 24 h treatment of phage P100 (9
log10 PFU/ ml) in 2- and 7-day biofilms formed by five strains of L. monocytogenes on
stainless steel coupon (initial cell counts of 7 and 6.6 log10 CFU/cm2, respectively) resulted in
decreases in the biofilm cells by 5.4 and 3.5 log units, respectively, at 22°C.
The temperature would be a sensitive parameter in both the growth of bacteria and
infection of the bacteria with the phage. A previous study showed that, whereas the burst size
of E. coli phage PR4 was approximately 40 from 30 to 42°C, the burst size at 20°C was less
than 3 [150]. When the temperature dropped to 0°C, approximately 80% of phage infectivity
was lost [151]. In contrast, a high temperature (37°C) inhibited phage infection of both
planktonic and biofilm cells of P. fluorescens [152].
Biofilm control using phages can involve phage application prior to biofilm formation for
control planktonic cells, application to already formed biofilms, which impacts the biofilm
structurally by lysis of producing EPS cells, and matrix rupture by the depolymerase enzyme
produced by phages [153-154]. This control results in the release of new phage virions that
can potentially reach and then infect adjacent bacteria. It was proposed that the progeny
phage would propagate radially through a biofilm by cycles of replication and cell lysis. At
268 Vera Lúcia dos Santos and Andrea Souza Monteiro

least in theory, a single phage dose should be capable of treating a biofilm infection because
the progeny phage infects adjacent cells and degrades the biofilm matrix [155-156].
A model ultrafiltration (UF) continuous recycled system fed with two previously
sterilized source effluents was experimentally inoculated with three bacterial species
(Pseudomonas aeruginosa, Acinetobacter johnsonii and Bacillus subtilis) (separately and
combined) and specific lytic bacteriophages [157]. The seeded phages’ lytic activity reduced
membrane biofouling by an average of 40% to >60%, compared to controls. The concentrated
phage numbers increased accordingly, and some were found in the permeate, but inoculated
bacteria were not found in the permeate.
To penetrate EPS layers, some phages carry EPS depolymerases as tail spikes or tail
fibers as part of the viral particle to enable them to reach the bacterial cell wall [158].
Consequently, phages cause biofilm and capsule disruption by cell infection and lysis, as well
as by EPS degradation. The group includes alginate lyases, amylases, cellulases, dextranases,
endohexosaminidases, exopolygalacturonic acid lyases, galactosidases, glucosidases,
guluronan lyases, hyaluronate lyases, and pullulanases. The breakdown of EPS has the
potential to increase phage penetration into biofilms, thereby improving phage acquisition of
target bacteria.
A polysaccharide depolymerase of bacteriophages promoted substantial degradation of
mono species biofilms of Enterobacter agglomerans GFP that were phage-susceptible [159],
and 60 min of treatment with a polysaccharase caused a 20% reduction in dual-species
biofilm adhesion [160]. Cornelissen et al. (2011) investigated the in vitro biofilm degradation
capacity of a lytic P. putida phage φ15 with associated EPS depolymerase on different aged
mono-species biofilms of P. putida strains, PpG1 and RD5PR2 [161]. The phage was able to
infect seven strains of P. putida in a group of 53, in addition acting differentially in breaking
down PpG1 RD5PR2 biofilms. It was hypothesized that EPS material serves as a primary
bacterial receptor for phage adsorption and that specific adsorption to and disruption of this
receptor by depolymerase is necessary to accomplish the phage replication cycle [161].
Belgini et al. (2014) evaluated the effects of four bacteriophages isolated from activated
sludge on biofilm formation by bacteria from the feed water of a reverse osmosis system
[162]. The vB_AspP-UFV1 (Podoviridae) interfered in the biofilm formation of most tested
bacteria, causing no decrease in bacterial growth, suggesting that its interference in biofilm
formation might be due to the action of depolymerase or infection of the cell without
necessarily causing cell lysis.
Some studies have suggested that the EPS breakdown might also allow for increased
penetration of non-phage materials, including certain antibiotics. Klebsiella pneumoniae
specific phage KPO1K2 depolymerase possesses anti-biofilm potential and improves
gentamicin’s efficacy against K. pneumoniae by dispersing the capsular polysaccharide of this
bacteria, facilitating antibiotic penetration across the biofilm [163]. The combined use of
cobalt ions (as an iron antagonist) and depolymerase-producing bacteriophages resulted in a
significantly greater reduction in bacterial numbers in the younger, as well as older, K.
pneumoniae biofilms, compared to when either of the agents was used alone [164]. Zhang and
Hu (2013) observed similar results in an equivalent system using phages alone [143]. Greater
biofilm removal, however, was seen with chlorine treatment following phage treatment, while
chlorine treatment alone was somewhat less effective in eradicating these P. aeruginosa
biofilms.
Strategies Based in Microbial Metabolites … 269

In addition, treatment with bacteriophages is suitable for systems in which the selective
removal of one group of bacteria without affecting the other groups is required. An example
is phage application in wastewater filtration systems for selectively remove P. aeruginosa
while not affecting the ammonia-oxidizing bacterial community inside the biofilters [141].

OTHER APPROACHES
Because QS systems control bacterial biofilm differentiation and maturation, inhibiting
quorum sensing will make more difficult or prevent biofilm formation. In Gram-negative
bacteria, there are different methods to control QS: (1) inhibition of N-acyl homoserine
lactone (AHL) production; (2) inactivation of AHL signal molecules; and (3) blocking the
signal receptor (quorum quenching) [165]. Such approaches directly disrupt communication
between the microorganisms contained in the biofilm, thus impeding their ability to
coordinate their actions to replenish, expand and maintain the matrix and ultimately leading to
decomposition of the matrix.
The degradation of quorum-sensing signaling molecules can be achieved by quorum-
quenching enzymes (QQ), including acylases, which cleave the acyl side chain from the HSL
ring, lactonases that open the homoserine (HSL) ring, and oxidoreductases that catalyze the
oxidation or reduction of acyl side chain [166-169].
Production of AHL lactonases has been described in bacteria and fungi. The most
promising AHL lactonase-producing bacteria belong to the genus Bacillus, such as B. cereus,
B. subtilits, B. thuringiensis and B. mycoides [170-171]. The other producing bacteria are P.
aeruginosa PAI-A [172], Arthrobacter sp., K. pneumoniae [173], Agrobacterium tumefaciens
[174], Acidobacteria sp. [175], Ochrobactrum sp. T63 [176], and Rhodococcus sp. [177]. The
fungal quorum-quenching enzyme gluconolactonase has been reported from Aspergillus niger
IAM 2094 [178].
Several bacteria, including species of Comamonas [179], Streptomyces [180], Ralstonia
and Variovorax genera, have been reported to produce QQ acylase [181-182]. Bacteria such
as V. paradoxus and P. aeruginosa PAO1 are able to proliferate with AHLs as the sole source
of energy and carbon and nitrogen mediated by the action of amino acylase, which cleaves the
peptide bond of the signal molecule [172, 181]. CYP102A1, a widely studied cytochrome
P450 from B. megaterium, is also capable of very efficient oxidation of AHLs, and their
lactonolysis products acyl homoserines [166].
Patents for QS control using inhibitor or QQ have been published. They suggest the use
of solutions containing enzymes, cell culture or cell extract solutions. Although this method is
still effective in some application areas, such as preventing plant pathogens, it also has
difficulties in being applied in systems with continuous fluid flow, such as water cooling,
MBR or pipelines, which require the periodic supply of biocides. Most of the application
examples were conducted under laboratory conditions with relatively short time periods. An
important issue in the industrial use of enzymes is maintaining enzyme activity for as long as
possible. In this context, immobilization would be expected to provide the stability required
to bring the technique closer to being a practical solution to the biofouling problem in the
engineering field.
270 Vera Lúcia dos Santos and Andrea Souza Monteiro

Yeon et al. (2009) reported a magnetic enzyme carrier (MEC) prepared by immobilizing
the quorum-quenching enzyme (acylase) on magnetic particles to overcome the technical
limitations of free enzyme [183]. The MEC showed no activity decrease under either
continuous shaking for 14 days or 29 iterative cycles of reuse. Kim et al. (2011) coupled
acylase directly on nanofiltration membrane (NF) surfaces for water treatment [184]. They
showed that the newly developed membrane with quorum-quenching activity could inhibit
quorum sensing between microorganisms in the membrane biocake, thereby reducing
biofouling.
Jiang et al. (2013) immobilized acylase into sodium alginate and observed a significant
improvement in membrane permeability without any negative impact on effluent quality
[185]. The authors reported that a QQ enzyme might change the mixed liquor properties, such
as sludge settleability, protein and polysaccharide concentrations, and viscosity. In particular,
it was claimed that the size of the microbial flocs in the QQ MBR became smaller than that of
the microbial flocs in the normal MBR, possibly due to QQ activities.
To achieve economic feasibility for the use of QQ in anti-biofouling of MBRs, enzymatic
QQ has been replaced by bacterial QQ using a microbial vessel (CMV) or cell-entrapping
beads (CEB). A recombinant E. coli that produces N-acyl homoserine lactonase or a quorum-
quenching Rhodococcus sp. isolated from a real MBR plant was encapsulated inside the
lumen of a microporous hollow fiber membrane, which was placed in submerged MBR to
alleviate biofouling over 80 days of MBR operation [186]. In another study, Oh et al. (2013)
encapsulated the QQ bacteria Rhodococcus sp. BH4, which produced acylase and was
isolated from a real MBR plant, in a microbial vessel [187]. Kim et al. (2013) prepared free-
moving beads by entrapping the same QQ bacteria, BH4, in alginate beads [188]. Cheong et
al. (2013) also isolated the natural AHL-degrading Pseudomonas sp. 1A1 strain from a real
municipal MBR plant to prepare a microbial vessel [189]. In all of these studies, the QQ
effect of CEBs or CMV on microbial cells in the biofilm generated fewer extracellular
polymeric substances and thus formed a loosely bound biofilm, which enabled it to slough off
from the membrane surface more easily. In the study of Kim et al. (2013), it was suggested
that biofouling was controlled not only by biological action but also by the physical action
provided by bombardment of beads onto the membrane surface [188]. However, they also
found that biofouling was unavoidable because the calcium alginate matrix was gradually
decomposed during long-term MBR operations, although the feed to MBR was relatively
mild synthetic wastewater. To increase the chemical and physical stability of the alginate
matrix in a biological environment, Kim et al. (2015) enclosed the QQ bacteria-containing
alginate core with a porous polymeric membrane layer using various commercial polymers,
such as poly(vinylidene) fluoride (PVDF), polyethersulfone (PES) and polysulfone (PSf) and
phase inversion methods [190]. The macrocapsules were capable of maintaining QQ activity
more safely than previously reported with alginate beads under harsher environmental
conditions, such as in real wastewater or in the presence of a chelating agent (EDTA), which
can disintegrate alginate matrix. In particularly, the PSf-coated membrane layer was more
effective in preventing QQ bacteria from leaking outside the macrocapsules.
Further improvement of this approach was attained by Cheong et al. (2014), who
designed a quorum-quenching MBR with a ceramic microbial vessel (CMV), prepared using
a monolithic ceramic microporous membrane and the AHL-degrading QQ bacterium
Pseudomonas sp. 1A1 [191]. The authors applied an inner flow-feeding mode under which
fresh feed was supplied to the MBR only through the center lumen, enabling the CMV to
Strategies Based in Microbial Metabolites … 271

maintain greater bacterial QQ activity through the facilitated nutrient transfer. In the QQ
MBR with the CMV, the concentrations of EPS were substantially decreased in the biocake
on the membrane surface, compared with those in the conventional MBR. The system also
showed little loss of its initial AHL degradation activity over 30 days of MBR operation.
Weerasekara et al. (2014) investigated the synergistic control of membrane fouling in an
MBR when QQ was coupled with two different physical cleaning methods: air backpulsing
and relaxation [192]. The effects of QQ bacteria on mixed liquor properties and on membrane
fouling control and energy consumption were evaluated at different aeration intensities. QQ
achieved a substantial reduction in membrane fouling, particularly when combined with
relaxation. This approach enabled the stable operation of an MBR at a lower extreme in
aeration, and it minimized the energy consumption for filtration and aeration. QQ bacteria
could hamper the formation of a biofilm on the membrane surface, but the mixed liquor
properties and treatment performances were not affected by the QQ activity.
Approaches based on metabolic interventions that alter the development and
differentiation of biofilms have been evaluated. In vitro experiments showed that both iron
depletion (<1 µM) and iron repletion (>100 µM) retarded biofilm formation [193]. A range of
synthetic iron-chelating molecules (2,2-dipyridyl/2DP, diethylenetriaminepentacetic
acid/DTPA, ethylenediamine-N,N9-diacetic acid/EDTA) and the biologically occurring
chelator lactoferrin were reported to reduce the biofilm formation of P. aeruginosa under
anaerobic conditions [194]. The iron chelator lactoferrin stimulates twitching motility and
prevents biofilm formation by this bacterium [195]. Another approach consists of replacing
iron, which has redox potential, with metabolically inactive ions, such as Sc3+, In3+ or Ga3+,
which are chemically similar to iron. The ions efficiently affect iron uptake and inhibit P.
aeruginosa growth and biofilm formation, and they also kill planktonic and biofilm bacteria
in vitro [196]. In addition, the inhibition of the enoyl-acyl carrier protein reductase from the
type II fatty acid synthesis pathway by the green tea Epigallocatechin gallate was shown to
reduce both QS and the biofilm development of P. aeruginosa. Type II fatty acid synthesis
intermediates are substrates for the LuxI family of autoinducer synthases [197]. Xu and Liu
(2011) demonstrated that disruption of energy metabolism and subsequent production of QS
signaling molecules effectively controlled membrane biofouling [62].

CONCLUSION
In conclusion, as discussed, the demand for research in green technologies for biofilm
control is urgent. Although many technological and financial barriers to biological strategies
remain, they might represent a breakthrough in biofilm control, with innovative designs
considering molecular and biochemical aspects of biofilm formation and resistance to
conventional treatments based on drugs and chemical biocides. However, most of the
strategies are still in the development phase, and many of them face particular challenges and
limitations.
For most of the SACs, there are still no standardized, large-scale methods of production
and purification, and others can present high production costs, which could be reduced with
production strategies based on recombinant microorganisms and the use of industrial
coproducts, such as growth substrate. With SACs, the enzymatic method is nontoxic and
272 Vera Lúcia dos Santos and Andrea Souza Monteiro

environmentally friendly, but enzymes are unstable and are highly pH-, temperature-, and salt
concentration-sensitive. Phages with viral-attached EPS depolymerases should preferably be
selected for microbial control because they are able to break down biofilms, attacking their
main components, bacterial cells and EPS matrix. However, the high specificity of phage-
associated EPS depolymerases can restrict the host range of the virus. The limitations
associated with specificity can be overcome by the application of phage cocktails directed at
various strains of the target species.
In the case of QQ, care should be taken so that desirable metabolic processes undertaken
by the microbiota are not affected by a particular quorum-quenching strategy. In some
situations, such as MBRs, it is necessary to inhibit biofilm without interfering with the growth
of bacteria that conduct organic charge removal, which is difficult to obtain with oxidant
biocides, for example, which are nonspecific and consequently toxic to non-target organisms.
In this context, strategies that block the expression of biofilm-forming phenotypes, such as
QQ, compared those that kill or inhibit the growth of bacteria are promising. It has been
reported that a variety of natural compounds from plants, quenching enzymes or bacteria
showed considerable QQ activity against biofouling bacteria without interfering with its
growth.
Despite the success of recent studies presented in this chapter, more research is needed to
examine the scaling up of these results to real scale systems and to validate their effectiveness
using real wastewater and its physical and chemical conditions. Another challenge to be
overcome is to validate their applicability to specific operational conditions in different
engineering fields, alone or as part of an integrated microbial control approach, because
diverse studies have shown that combined approaches might offer greater potential for
effectively limiting biofilm problems than single control methods. Moreover, it is important
to learn more about the physical and chemical structures of biofilm, as well the functional and
taxonomical diversity of biofilm microbial communities.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

ACKNOWLEDGMENTS
The authors gratefully acknowledge support from the UFMG, CEUMA,
CENPES/PETROBRAS, Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq),
Fundacao do Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) and Comissao de
Aperfeicoamento de Pessoal de Nivel Superior (CAPES).

REFERENCES
[1] Stoodley P, Sauer K, Davies DG, Costerton JW. Biofilms as complex differentiated
communities. Ann Rev Microbiol 2002; 56: 187-209.
Strategies Based in Microbial Metabolites … 273

[2] Purevdorj B, Costerton JW, Stoodley P. Influence of hydrodynamics and cell signaling
on the structure and behavior of Pseudomonas aeruginosa biofilms. Appl Environ
Microbiol 2000; 68(9): 4457-4464.
[3] Hödl I, Mari L, Bertuzzo E, et al. Biophysical controls on cluster dynamics and
architectural differentiation of microbial biofilms in contrasting flow environments.
Environ Microbiol 2014; 16(3): 802–812.
[4] Sutherland IW. Biofilm exopolysaccharides: a strong and sticky framework.
Microbiology 2001; 147(Pt 1): 3–9.
[5] De Beer D, Stoodley P, Roe F, Lewandowski Z Effect of biofilm structures on oxygen
distribution and mass transport. Biotechnol Bioeng 1994a; 43(11): 1131–1138.
[6] De Beer D, Stoodley P, Lewandowski Z. Liquid flow in heterogeneous biofilms.
Biotechnol Bioeng 1994b; 44(5): 636–641.
[7] Von Bodman SB, Farrand SK. Capsular polysaccharide biosynthesis and pathogenicity
in Erwinia stewartii require induction by an N-acylhomoserine lactone autoinducer. J
Bacteriol 1995; 177(17): 5000–5008.
[8] Hammer BK, Bassler BL. Quorum sensing controls biofilm formation in Vibrio
cholerae. Mol Microbiol 2003; 50(1): 101–114.
[9] Parsek MR, Greenberg EP. Sociomicrobiology: the connections between quorum
sensing and biofilms. Trends Microbiol 2005; 13(1): 27–33.
[10] Buesing N, Gessner MO. Benthic and fungal productivity and carbon turnover in a
freshwater marsh. Appl Environ Microbiol 2006; 72: 596-605.
[11] Jefferson KK. What drives bacteria to produce a biofilm? FEMS Microbiol Lett 2004;
236: 163-173.
[12] Romani AM, Sebater S. Effect of primary producers on the heterotrophic metabolism of
a stream biofilm. Freshwater Biol 1999; 41(4): 729-736.
[13] Roseselers G, Van Loosdrecht MCM, Muyzer G. Heterotrophic pioneers facilitate
phototrophic biofilm development. Microb Ecol 2007; 54(3): 578-585.
[14] Freeman C, Lock MA. The biofilm polysaccharide matrix – a buffer against changing
organic substrate supply. Limmol Oceanogr 1995; 40(2): 273-278.
[15] Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the Natural
environment to infectious diseases. Nature Rev Microbiol 2004; 2: 95-108.
[16] Rickard AH, McBain AJ, Stead AT, Gilbert P. Shear rate moderates community
diversity in freshwater biofilms. Appl Environ Microbiol 2004; 70(12): 7426-7435.
[17] Matz C, Kjelleberg S. Off the hook – how bacteria survive protozoan grazing. Trends
Microbiol 2005; 13(7): 302-307.
[18] Sheldon F, Walker KF. Changes in biofilms induced by flow regulation could explain
extinctions of aquatic sanils in the lower River Murray, Australia. Hydrobiologia 1997;
347: 97-108.
[19] Hero HM, Port RD. The Nalco guide to cooling water system failure analysis, McGraw
Company, New York, 1993.
[20] Coniglio MA, Melada S, Yassin MH. Monochloramine for controlling Legionella in
biofilms: how much we know? J Nature Sci 2015; 1(2):e44.
[21] Akid R, Wang H, Smith T, Greenfield D, Earthman J. Biological functionalization of a
sol-gel coating for the mitigation of microbial-induced corrosion. Adv Funct Mat 2008;
18(2): 203–211.
274 Vera Lúcia dos Santos and Andrea Souza Monteiro

[22] Li KH, Whitfield M, Van Vliet KJ. Beating the bugs: Roles of microbial biofilms in
corrosion, Corrosion Rev 2013; 31: 73-84.
[23] Murga R, Forster TS, Brown E, et al. Role of biofilms in the survival of Legionella
pneumophila in a model potable-water system. Microbiology 2001; 147: 3121–3126.
[24] EPA. 2002. Health Risks from Microbial Growth and Biofilms in Drinking Water
Distribution Systems. Office of Ground Water and Drinking Water, Washington, D.C
(http://www.epa.gov/ogwdw/ disinfection/tcr/pdfs/whitepaper_tcr_biofilms.pdf).
[25] Langmark J, Storey MV, Ashbolt NJ, Stenstrom TA. Accumulation and fate of
microorganisms and microspheres in biofilm formed in a pilot-scale water distribution
system. Appl Environ Microbiol 2005; 71(2): 706-712.
[26] Berry D, Xi C, Raskin L. Microbial ecology of drinking water distribution systems.
Curr Opin Biotechnol 2006; 17: 297-302.
[27] Morrow JB, Almeida JL, Fitzgerald LA, Cole KD. Association and decontamination of
Bacillus spores in a simulated drinking water system. Water Res 2008; 42(20): 5011-
5021.
[28] Helmi K, Skraber S, Gantzer C, et al. Interactions of Cryptosporidium parvum, Giardia
lamblia, vaccinal poliovirus type 1, and bacteriophages phiX 174 and MS2 with a
drinking water biofilm and a wastewater biofilm. Appl Environ Microbiol 2008; 74(7):
2079-2088.
[29] Paris T, Skali-Lami S, Block JC. Probing young water biofilms with hard and soft
particles. Water Res 2009; 43(1): 117-126.
[30] Altman SJ, McGrath LK, Souza CA, Murton JK, Camper AK. Integration and
decontamination of Bacillus cereus in Pseudomonas fluorescens biofilms. J Appl
Microbiol 2009; 107(1): 287-299.
[31] Feazel LM, Baumgartner LK, Peterson KL, et al. Opportunistic pathogens enriched in
showerhead biofilms. PNAS 2009; 106(38): 16393-16399.
[32] Wingender J, Flemming H-C. Biofilms in drinking water and their role as reservoir for
pathogens. Int J Hyg Environ Health 2011; 214(6): 417-423.
[33] Pelleieux S, Bertrand I, Skali-Lami S, et al. Accumulation of MS2, GA, and Qb phages
on high density polyethylene (HDPE) and drinking water biofilms under flow/non-flow
conditions. Water Res 2002; 46919): 6574-6584.
[34] Kerr CJ, Osborn KS, Rickard AH, Robson GD, Handley PS. Biofilms in water
distribution systems. In: Mara D, Horan NJ, Eds. Water and wastewater engineering.
London: Academic Press 2003; pp. 757-776.
[35] Shi X, Zhu X. Biofilm formation and food safety in food industries. Trends Food Sci
Technol 2009; 20(9): 407-413.
[36] Talsma SS. Biofilms on medical devices. Home Healthc Nurse 2007; 25(9): 589-594.
[37] Davison WM, Pitts B, Stewart PS. Spatial and temporal patterns of biocide action
against Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother 2010;
54(7): 2920-2927.
[38] Lee WH, Wahman DG, Bishop PL, Pressman JG. Free chlorine and monochloramine
application to nitrifying biofilm: comparison of biofilm penetration, activity, and
viability. Environ Sci Technol 2011; 45(4): 1412-1419.
[39] Walters MC, Roe F, Bugnicourt A, Franklin MJ, Stewart PS. Contributions of antibiotic
penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas
Strategies Based in Microbial Metabolites … 275

biofilms to ciprofloxacin and tobramycin. Antimicrob Agents Chemother 2003; 47(1):


317-323.
[40] Cochran WL, McFeters GA, Stewart, PS. Reduced susceptibility of thin Pseudomonas
aeruginosa biofilms to hydrogen peroxide and monochloramine. J Appl Microbiol
2000; 88: 22–30.
[41] Mah TFC, O’Toole GA. Mechanisms of biofilm resistance to antimicrobial agents.
Trends Microbiol. 2001; 9(1): 34-39.
[42] Denkard E, Ausubel FM. Pseudomonas biofilm formation and antibiotic resistance are
linked to phenotypic variation. Nature 2002; 416(6882): 740–743.
[43] Zhang H, Andrews SA. Catalysis of copper corrosion products on chlorine decay and
HAA formation in simulated distribution systems. Water Res 2012; 46(8): 2665-2673.
[44] Wang H, Hu C, Hu X, Yang M, Qu JH. Effects of disinfectants and biofilm on the
corrosion of cast iron pipes in a reclaimed water distribution system. Water Res 2012;
46(4): 1070-1078.
[45] Hallam NB, Westy JR, Forster CF, Powell JC, Spencer I. The decay of chlorine
associated with the pipe wall in water distribution systems. Water Res 2002; 36(14):
3479-3488.
[46] Lethola MJ, Miettinen IT, Lampola T, et al. Pipeline materials modify the effectiveness
of disinfectants in drinking water distributions systems. Water Res 2005; 39(10): 1962-
1971.
[47] Hubbard H, Poppendieck D, Corsi RL. Chlorine dioxide reactions with indoor materials
during building disinfection: surface uptake. Environ Sci Technol 2009; 43(5): 1329-
1335.
[48] Xue Z, Sendamangalam VR, Gruden CL, Seo Y. Multiple role of extracellular
polymeric substances on resistance of biofilm and detached clusters. Environ Sci
Technol 2012; 46(24), 13212-13219.
[49] Flint SH, van den Elzen H, Brooks JD, Bremer PJ. Removal and inactivation of thermo-
resistant streptococci colonizing stainless steel. Int Dairy J 1999; 9(7): 429–436.
[50] Mathieu L, Bouteleux C, Fass S, Angel E, Block J-C. Reversible shift in the a-, b- and
g-proteobacteria populations of drinking water biofilms during discontinuous
chlorination. Water Res 2009; 43: 3375-3386.
[51] Jones WL, Sutton MP, McKittrick L, Stewart PS. Chemical and antimicrobial
treatments change the viscoelastic properties of bacterial biofilms. Biofouling 2011;
27(2): 207-215.
[52] Saravanan P, Nancharaiah YV, Venugopalan VP, Rao TS, Jayachandran S. Biofilm
formation by Pseudoalteromonas ruthenica and its removal by chlorine. Biofouling
2006; 22(5-6): 371-381.
[53] Tachikawa M, Yamanaka K, Nakamuro K. Studies on the disinfection and removal of
biofilms by ozone water using an artificial microbial biofilm system, Ozone: Sci Eng
2009; 31(1): 3-9.
[54] Turetgen I. Comparison of the efficacy of free residual chlorine and monochloramine
against biofilms in model and full scale cooling towers. Biofouling 2004; 20(2): 81–85.
[55] Chien S-H, Dzombak DA, Vidic RD. Comprehensive Evaluation of Biological Growth
Control by Chlorine-Based Biocides in Power Plant Cooling Systems Using Tertiary
Effluent. Environ Eng Sci 2013; 30(6): 324–332.
276 Vera Lúcia dos Santos and Andrea Souza Monteiro

[56] Xue Z, Lee WH, Coburn KM, Seo Y. Selective reactivity of monochloramine with
extracellular matrix components affects the disinfection of biofilm and detached
clusters. Environ Sci Technol 2014; 48(7): 3832-3839.
[57] Stewart PS. Antimicrobial Tolerance in Biofilms. Microbiol Spectr 2015; 3(3):
10.1128/microbiolspec.MB–0010–2014.
[58] Campos FF, Johann S, Santos VL. Natural Products as a Source of Potential Drugs for
the Treatment of Fungal Infections. In: Leon V. Berhardt. (Org.). Advances in Medicine
and Biology. 1ed. New York: Nova Science Publishes v39 2012, pp. 49-83.
[59] Sumi CD, Byung WY, Yeo IC, Hahm YT. Antimicrobial peptides of the genus
Bacillus: a new era for antibiotics. Can J Microbiol 2014; 61(2): 93-103.
[60] Itoh Y, Wang X, Hinnebusch BJ, Preston JF, Romeo T. Depolymerization of β-1,6-N-
acetyl-d-glucosamine disrupts the integrity of diverse bacterial biofilms. J Bacteriol
2005; 187(1): 382–387.
[61] Spoering AL, Gilmore MS. Quorum sensing and DNA release in bacterial biofilms.
Curr Opin Biotechnol 2006; 9(2): 133-137.
[62] Xu H, Liu Y. Control and cleaning of membrane biofouling by energy uncoupling and
cellular communication. Environ Sci Technol 2011; 45(2): 595-601.
[63] Lönn-Stensrud J, Landin MA, Benneche T, Petersen FC, Scheie AA. Furanones,
potential agents for preventing Staphylococcus epidermidis biofilm infections? J
Antimicrob Chemother 2009; 63(2): 309-316.
[64] Carson L, Gorman SP, Gilmore BF. The use of lytic bacteriophages in the prevention
and eradication of biofilms of Proteus mirabilis and Escherichia coli. FEMS Immunol
Med Microbiol 2010; 59(3): 447–455.
[65] Neu TR. Significance of bacterial surface-active compounds in interaction of bacteria
with interfaces. Microbiol Rev 1996; 60(1): 151-166.
[66] Desai JD, Banat IM. Microbial production of surfactants and their commercial
potential. Microbiol Mol Biol Rev 1997; 61(1): 47–64.
[67] Monteiro AS, Bonfim MRQ, Domingues VS, et al. Identification and characterization
of bioemulsifier-producing yeasts isolated from effluents of a dairy industry. Bioresour
Technol 2010; 101(14): 5186-5193.
[68] Monteiro AS, Domingues VS, Souza MVD, et al. Bioconversion of biodiesel refinery
waste in the bioemulsifier by Trichosporon mycotoxinivorans CLA2. Biotechnol
Biofuels 2012; 5: 29.
[69] Santos VL, Monteiro AS, Domingues VS, Júlio ADL. Microbial Bioemulsifiers and
Environmental Applications Potential. In: Tannen KM, Ed. Emulsification: Processes,
New Technology and Current Applications. 3nd ed. New York: Nova Science Publishers
2013.
[70] Coutinho JOPA, Silva MPS, Moraes PM, et al. Demulsifying properties of extracellular
products and cells of Pseudomonas aeruginosa MSJ isolated from petroleum-
contaminated soil. Bioresour Technol 2013; 128: 646-654.
[71] Cameotra S, Makkar R. Recent applications of biosurfactants as biological and
immunological molecules. Curr Opin Microbiol 2004; 7: 262–266.
[72] Singh P, Cameotra S. Potential applications of microbial surfactants in biomedical
sciences. Trends Biotechnol 2004, 22(3): 142–146.
Strategies Based in Microbial Metabolites … 277

[73] Monteiro AS, Miranda TT, Lula I, et al. Inhibition of Candida albicans CC biofilms
formation in polystyrene plate surfaces by biosurfactant produced by Trichosporon
montevideense CLOA72. Colloids Surf B Biointerfaces 2011; 84(2): 467-476.
[74] Al-Tahhan RA, Sandrin TR, Bodour AA, Maier RM. Rhamnolipid-induced removal of
lipopolysaccharide from Pseudomonas aeruginosa: Effect on cell surface properties and
interaction with hyd