Phytochemistry 154 (2018) 94–105

Contents lists available at ScienceDirect

Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Antimicrobial peptides from different plant sources: Isolation, T

characterisation, and purification
Swee-Seong Tanga,∗, Zakaria H. Prodhana,b, Sudhangshu K. Biswasa, Cheng-Foh Lec,
Shamala D. Sekarand
a
Division of Microbiology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
b
Department of Agronomy, Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China
c
School of Biosciences, Faculty of Science, The University of Nottingham Malaysia Campus, Semenyih, Selangor, Malaysia
d
Faculty of Medicine, MAHSA University, Saujana Putra Campus, 42610, Jenjarum, Selangor, Malaysia

A R T I C LE I N FO A B S T R A C T

Keywords: Antimicrobial peptides (AMPs), the self-defence products of organisms, are extensively distributed in plants.
Antimicrobial peptides They can be classified into several groups, including thionins, defensins, snakins, lipid transfer proteins, glycine-
Therapeutic agent rich proteins, cyclotides and hevein-type proteins. AMPs can be extracted and isolated from different plants and
Isolation plant organs such as stems, roots, seeds, flowers and leaves. They perform various physiological defensive
Purification
mechanisms to eliminate viruses, bacteria, fungi and parasites, and so could be used as therapeutic and pre-
Plant source
servative agents. Research on AMPs has sought to obtain more detailed and reliable information regarding the
selection of suitable plant sources and the use of appropriate isolation and purification techniques, as well as
examining the mode of action of these peptides. Well-established AMP purification techniques currently used
include salt precipitation methods, absorption-desorption, a combination of ion-exchange and reversed-phase
C18 solid phase extraction, reversed-phase high-performance liquid chromatography (RP-HPLC), and the sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. Beyond these traditional methods,
this review aims to highlight new and different approaches to the selection, characterisation, isolation, pur-
ification, mode of action and bioactivity assessment of a range of AMPs collected from plant sources. The in-
formation gathered will be helpful in the search for novel AMPs distributed in the plant kingdom, as well as
providing future directions for the further investigation of AMPs for possible use on humans.

1. Introduction
Antimicrobial peptides (AMPs), also known as host defense peptides
(HDPs), constitute part of the innate immune system found in almost all
classes of life including microorganisms, arthropods, plants and animals
(Bulet et al., 1999; Carvalho and Gomes, 2009; Zasloff, 2002). These
AMPs are potent, broad-spectrum antibiotics against pathogenic bac-
teria (Gram-negative and Gram-positive), fungi, enveloped viruses and
other parasites (Reddy et al., 2004).
Plant AMPs are an abundant group of proteinaceous compounds
produced in plants (Jenssen et al., 2006). The first reported plant AMP
was purothionin from wheat flour (Triticum aestivum) (De Caleya et al.,
1972). Several groups of plant AMPs with antimicrobial activity have
since been identified, characterised and purified, including defensins,
snakins, puroindolines, glycine-rich proteins, cyclotides, hevein-type
proteins, and lipid transfer proteins (Pelegrini and Franco, 2005;
Witkowska et al., 2007). Such AMPs have been isolated from different
plants and plant organs such as the stem, root, seed, flower and leaf.
They exhibit potent microbicidal activities against viruses, bacteria,
fungi, parasites and protozoa (Nawrot et al., 2014). Plant AMPs have
become important candidates for developing potential new techniques
for controlling crop losses as well as novel antibiotics for treating var-
ious infections in humans (Pelegrini et al., 2008).
The adverse effects of chemical pesticides, the frequent emergence
of drug-resistant bacteria and the failure of some traditional antibiotics
have all led to an urgent search for new antimicrobial agents
(Nordström and Malmsten, 2017). Although more than 5000 AMPs
have been identified from different sources so far (Hu et al., 2016; Zhao
et al., 2013), only just over 2400 AMPs have been deposited in the AMP
Database (http://aps.unmc.edu/AP/main.php), of which 343 are from

∗
Corresponding author.
E-mail addresses: sstang@um.edu.my, sstang2107@hotmail.com (S.-S. Tang), rajugenetics2003@gmail.com (Z.H. Prodhan),
shu_genetics@yahoo.com (S.K. Biswas), chengfoh.le@nottingham.edu.my (C.-F. Le), shamaladevi@mahsa.edu.my (S.D. Sekaran).

https://doi.org/10.1016/j.phytochem.2018.07.002
Received 5 May 2018; Received in revised form 3 July 2018; Accepted 7 July 2018
Available online 17 July 2018
0031-9422/ © 2018 Elsevier Ltd. All rights reserved.
S.-S. Tang et al. Phytochemistry 154 (2018) 94–105

Table 1
Main families of plant antimicrobial peptides with representative peptides and their sources.
Family: Plant organ Plant Reference
Representative peptide

Cyclotides: Leave & flowers Oldenlandia affinis (Craik, 2001; Nawrot et al., 2014; Selitrennikoff, 2001; Stec, 2006)
Kalata B1 and B2
Impatiens: Seeds Impatiens balsamina (Stepek et al., 2005; Tailor et al., 1997)
Ib-AMPs
Knottin-peptides: PAFP-S Seeds Phytolacca americana, Mirabilis jalapa (Marcus et al., 1999; Nawrot et al., 2014; Terras et al., 1995)
Lipid transfer proteins (LTPs): Seeds Zea mays (Nawrot et al., 2014; Pelegrini et al., 2007; Stec, 2006)
LTP1s and LTP2s
Defensins: Seeds Phaseolus vulgaris (García-Olmedo et al., 1998; Mendez et al., 1990; Ramos et al., 2014)
Peptide PvD1 Hordeum vulgare
γ-hordothionin
Puroindolines: Endosperm Triticum aestivum (Liu et al., 2000; Nawrot et al., 2014; Tailor et al., 1997)
PINA and PINB
Snakins: Tubers Solanum tuberosum (Fujimura et al., 2003; Nawrot et al., 2014; Terras et al., 1995)
StSN1 and StSN2
Thionein: Endosperm Triticum aestivum (De Caleya et al., 1972; Nawrot et al., 2014)
α-1-purothionin
Vicilin-like AMPs: Seeds Macadamia integrifolia (Marcus et al., 1999)
PMAPI
Hevein-like AMPs: leaves Broussonetia papyrifera syn. Morus papyrifera L. (Zhao et al., 2011)
PMAPI
Others: Seeds Cicer arietinum (Ramos et al., 2014; Tailor et al., 1997; Ye et al., 2002)
Arietin
Ay-AMP Seeds Amaranthus hypochondriacus (Rivillas-Acevedo and Soriano-García, 2007)
Cicerin Seeds Cicer arietinum (Souza et al., 2011; Ye et al., 2002)
Shepherins Roots Capsella bursa-pastoris (Marcus et al., 1999; Remuzgo et al., 2014)
Hispidulin Seeds Benincasa hispida (Sharma et al., 2014)
Lunatusin Seeds Phaseolus lunatus (Wong and Ng, 2005a)
Peptidesa Seeds Brassica napus (del Mar Yust et al., 2004)
Vulgarinin Seeds Phaseolus vulgaris (Wong and Ng, 2005b)

plants (Liu et al., 2017). Many of these identified AMPs have not been
tested in clinical trials, and a number of those that have reached the
clinical phase have failed due to unacceptable toxicity or lack of effi-
cacy.
There is thus a need to search for new AMPs to widen the range of
AMPs potentially available for therapeutic and similar uses. However,
variations in screening, identification and purification methods, as well
as the wide variety of different plant organs and species involved, have
made the discovery process complicated and time-consuming. A com-
prehensive study involving the isolation, characterisation and pur-
ification of AMPs together with an analysis of their antimicrobial ac-
tivity and cytotoxicity should help to identify novel AMPs with
improved antimicrobial activity and reduced cytotoxicity, thereby ac-
celerating the application of new AMPs for clinical and agricultural uses
(Zhao et al., 2013).
This study summarises and updates current information on the
isolation, identification and purification strategies for AMPs from plant
sources. In addition to reviewing existing methods, this study also looks
at a wide range of possible options for obtaining novel AMPs from the
plant kingdom. Above all, this review aims to provide insights to help
guide researchers interested in the isolation, identification, evaluation
and how to test antimicrobial activity of novel AMPs from plant
sources.
2. Isolation of antimicrobial peptides from plants
2.1. Selection of plant materials
The immense biodiversity in plant species means that there is en-
ormous scope to explore new prospective AMPs for drug development
in human welfare and other potential applications in agriculture.
However, that very diversity also means that enormous efforts are re-
quired to select, identify and characterise plant materials in the search
for AMPs. The availability of morphological, physiological and mole-
cular data on plant species has made them suitable targets for research
into the collection of AMPs. Data derived from molecular techniques
designed to characterise and identify genes responsible for AMP
synthesis and regulation, including the in silico analysis of AMPs along
with the gene products responsible for inhibiting pathogens, is required
to validate the functional aspects of AMPs (Clara Pestana-Calsa et al.,
2010). The increasing number of plant species being deposited in public
databases, together with the rapid growth in information available on
proteins, genomes, transcripts and other molecular data, has made
plants attractive as potential sources of AMPs.
Plant species that exhibit a strict ecological relationship with a pa-
thogen and produce AMPs in a symbiotic context might be particularly
suitable for use as sustainable biological controlling agents for pests and
pathogens (Udwary et al., 2011). The first identified plant-derived AMP
was purothionin, which was found to be active against Xanthomonas
phaseoli, Pseudomonas solanacearum, Erwinia amylovora, Cor-
ynebacterium flaccumfaciens, C. michiganense and C. fascians (De Caleya
et al., 1972). Several other plant AMPs have subsequently been dis-
covered and characterised through a range of morphological, physio-
logical and molecular analyses. The major AMPs collected from dif-
ferent plant species have been categorised into several groups,
including defensins, cyclotides, lipid transfer proteins and thionins
(Nawrot et al., 2014; Pelegrini et al., 2007; Stec, 2006; Udwary et al.,
2011), as well as less common groups such as the impatiens, pur-
oindolines, vicilin-like, glycine-rich, shepherins, snakins and heveins
(Berrocal-Lobo et al., 2002; Fujimura et al., 2003; Liu et al., 2000;
Marcus et al., 1999; Zottich et al., 2011).
AMPs have been isolated from whole plants, such as lunatusin col-
lected from Phaseolus lunatus L. (the Chinese lima bean), as well as from
seeds such as vulgarinin from Phaseolus vulgaris (haricot beans), hispi-
dulin from a medicinal plant (Benincasa hispida), and cicerin and arietin
from Cicer arietinum (chickpea) (Rivillas-Acevedo and Soriano-García,
2007; Wong and Ng, 2005a; b). Other AMPs have been obtained from
the thick cell walls of Spinacia oleracea cv. Matador (spinach leaves)
(Segura et al., 1998). The names of known AMPs, plant species, and the
related references are summarised in Table 1 below.

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S.-S. Tang et al. Phytochemistry 154 (2018) 94–105

Plant material
Crushed and blended
Frozen and thawed

Supernatant Centrifugation Residue Discard

Ammonium
sulfate
precipitation

Centrifugation Pellet Supernatant Dialysis

Dissolve in deionized water

Dialysis and SDS-PAGE

Gel filtration chromatography

Fractions collected
Dialysis of peak

Purified AMPs

Amino acid sequence

Similarity search in databanks

Peptide identification

Fig. 1. Diagram of different strategies used for isolation, purification and identification of the antimicrobial peptide from the plant source.

Plant AMPs have been isolated from different plants and plant or-
gans, including the stems, roots, seeds, flowers and leaves from a wide
variety of plant species (Montesinos, 2007). The number of AMPs iso-
lated from plants is extremely high, with some plant species possessing
hundreds of different AMPs or more. In sum, this wide range of plant
organs and plant species represent a potentially huge source of AMPs
which might be used as drugs or food preservatives.
2.2. Isolation and extraction of plant AMPs
The first step in obtaining AMPs is to perform a series of processes to
extract and purify them from potential source plant materials. Although
AMPs have been successfully isolated directly from crude plant extracts
in several plant species (Kovaleva et al., 2009), fractionating com-
pounds and isolating AMPs from plant materials generally require ef-
ficient and sophisticated techniques (Moreira et al., 2011). The ex-
traction and purification of potential new AMPs, followed by
sequencing and searching for similar AMPs in databases, is an effective
way to identify novel AMPs, as described by Odintsova et al. (2009)
(Fig. 1).
In fact, a number of AMPs have already been identified using the
above techniques. For example, AMPs were isolated directly from crude
plant extract followed by functional assays and characterisation from
the plant in Lippia sidoides Cham. (rosemary pepper) flowers through
Octyl-Sepharose hydrophobic column separation (Moreira et al., 2011).
Similarly, an antiviral peptide (2 kDa) purified from sorghum (Sorghum
bicolor) seeds by gel filtration, ion exchange and high-performance li-
quid chromatography showed potent inhibitory effects on the herpes
simplex virus type 1 and bovine herpes virus (Camargo Filho et al.,
2008). Two other AMPs with anti-yeast properties were isolated from
the seeds of Capsicum annuum (pepper) and identified by amino acid
sequencing (Ribeiro et al., 2011). Other AMPs isolated from Sesamum
indicum (sesame) kernel flour have been shown to exhibit anti-bacterial
activity against Klebsiella sp. (Costa et al., 2007). The direct isolation of
AMPs from plants has also been observed in Crotalaria pallida (the
African legume), a widely dispersed weed in South America, producing
a novel peptide structurally similar to defensin (Pelegrini et al., 2008).
Some antibacterial and antifungal peptides (passiflin, Pg-AMP1, and 2S
albumin-like) have been isolated from the seeds of Psidium guajava
(guava) and Passiflora edulis (passion fruit) (Lam and Ng, 2009). Some
studies have also reported the successful isolation of AMPs from
common vegetables and spices, including Pisum sativum L. (garden pea)

96
S.-S. Tang et al.
(Diz et al., 2006; Ngai and Ng, 2004).
Water is the most commonly used solvent to extract AMPs from
dried plants such as teas, either by steeping the plants in hot water or
boiling suspensions of the parts using steam. Dried plant parts can also
be added to oils or petroleum jelly and poultices (concentrated teas or
tinctures) (Brantner and Grein, 1994).
All of the above components extracted and identified from plants
are active against microorganisms; most of them are aromatic or satu-
rated organic compounds, often obtained through ethanol or methanol
extraction (Zhang and Lewis, 1997). Different plant parts may also
contain active antimicrobial components, such as saponins in the root
of Ginseng (Panax ginseng) plants, essential oils and tannins in Eu-
calyptus (Eucalyptus spp) leaves, and phenolic compounds in the bark,
leaves and shoots of Balsam poplar (Populus balsamifera) (Cichewicz
and Thorpe, 1996; Taylor et al., 1996).
In sum, research has shown that AMPs can be isolated and extracted
from whole plants as well as from the seeds and flour of plant parts.
3. Purification and characterisation of antimicrobial peptides
from plants
3.1. Concentrating the supernatant of AMPs
Following the extraction of AMP from plant materials as described
in Section 2.2 above, it is necessary to purify and characterise the re-
levant antimicrobial peptide. Plant extract contains much lower levels
of AMPs than other compounds (He et al., 2010), so it is essential to
concentrate the AMP solution in order to recover the AMPs present.
Many procedures have been developed to concentrate AMP containing
supernatant, but two of the most common are ammonium sulphate
precipitation and organic solvent extraction.
3.1.1. Ammonium sulphate precipitation
AMPs are short peptides, which can be concentrated through a
salting-out method with ammonium sulphate as a common reagent.
Solid ammonium sulphate or 75% ammonium sulphate is added to the
extracted plant sample until the desired saturation level of ammonium
sulphate is reached. For this, the hulled seeds or other relevant parts of
a plant sample are homogenised with sodium acetate buffer and stirred
overnight, then centrifuged. The supernatant is concentrated under
reduced pressure and saturated with ammonium sulphate (Yokoyama
et al., 2008). Sometimes AMPs are precipitated at lower ammonium
sulphate concentrations (< 70%). In such cases, it is essential to
monitor and determine the right concentration of salt to isolate and
concentrate the AMPs. The concentrated AMP suspension can then be
desalted by dialysis against a phosphate buffer using benzoylated
membranes or with dialysis cassettes (Pingitore et al., 2007; Yokoyama
et al., 2008). Plant extract may also contain tannin, which needs to be
eliminated before salt precipitation. To achieve this, plant extract is
dissolved in water and stirred for 30 min, then centrifuged for 15 min at
3144 × g to remove insoluble polysaccharide excipients. The solution
then needs to be dried down using a Genevac sample concentrator
under reduced pressure at 30 °C, and re-suspended at a concentration of
10 mg/mL in water and methanol (1:1 v/v). About 300 mg of polyvinyl
pyrrolidone is added, and the solution is stirred for 30 min and cen-
trifuged. Finally, the supernatant is removed and dried down to yield
de-tanninized plant extracts (Miyasaki et al., 2013; Pingitore et al.,
2007).
3.1.2. Organic solvent extraction
Organic solvent extraction from crude extract is a successful pro-
tocol for concentrating AMPs from free-cell supernatant that has been
treated with polar solvents (acetone, methanol, etc.) stored at −30 °C
overnight. The extracted AMPs are centrifuged to recover the peptide
fraction in the pellet, and the suspension is subjected to ion exchange
chromatography (Pingitore et al., 2007).
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Phytochemistry 154 (2018) 94–105
3.2. Purification and characterisation of AMPs
Ammonium sulphate precipitation and solvent extraction are gen-
erally used for the initial partial purification of plant AMPs. Such AMPs
then need to be subjected to further purification using other methods,
such as Ion exchange chromatography (IEXC), Sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE), MALDI-TOF MS/MS,
2D-gel electrophoresis, Reversed-phase (RP) HPLC and Mass
Spectrometry (Fig. 1).
3.2.1. Ion exchange chromatography (IEXC)
Ion exchange chromatography (IEXC) can be used for both cation-
and anion-charged AMPs that require separation by their electric
charge at a defined pH. For this, the crude extract needs to be loaded
onto a cellulose column, equilibrated with ammonium acetate, pH 5.2,
and washed with buffer. The fractions are collected, re-suspended in
acetic acid, lyophilized, and stored at −20 °C. Alternatively, crude ex-
tract can be dialysed into ammonium acetate, pH 5.2, using spectra-
mesh dialysis tubing with exclusion and aliquots loaded onto a cation
exchange column. In both processes, the plant extract is passed through
the column and washed with phosphate buffer containing NaCl, and the
eluted AMPs are collected (Duvick et al., 1992; Pingitore et al., 2007).
Ion exchange protein purification can be applied to purify plant
AMPs because most plant AMPs contain non-zero net electrostatic
charges at all pHs except the pH equal to pI (isoelectric point). When
the pH is higher than its pI, an AMP becomes negatively charged (an
anion), while at a pH lower than its pI the AMP becomes positively
charged (a cation). Ion exchange chromatography works for two rea-
sons: (i) the electrostatic attraction between buffer-dissolved charged
AMPs; and (ii) the oppositely charged binding sites on a solid ion ex-
change adsorbent. In sum, the charge (both net and local), stability and
conformation of the charged form of the buffer can all play an im-
portant role in the purification of plant AMPs by the ion exchange
technique (Khan, 2012).
3.2.2. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE)
Fractions containing AMPs can be analysed by sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using 15%
gel. After completing the electrophoresis, the gel is sensitised using a
sodium thiosulfate (Na2S2O3) solution, followed by staining with silver
nitrate at room temperature for 2 h. Sodium carbonate containing for-
malin in water can be used as the developer. Finally, the gel needs to be
placed in a solution of acetic acid in water to stop the staining process
(Schägger and von Jagow, 1987). An SDS-PAGE analysis helps to se-
parate effective antibacterial substances which are peptides with a low
molecular weight.
3.2.3. Reverse-phase (RP) HPLC
Reverse-phase high-performance liquid chromatography (RP-HPLC)
is the most useful analytical technique for the purification of AMPs from
plant and other sample sources. Plant AMPs are often resistant to var-
ious organic solvents used as mobile phases, making RP-HPLC a suitable
option for the purification of plant AMPs (Dolezilkova et al., 2015;
Duvick et al., 1992). In this method, samples are lyophilized and dis-
solved in a MilliQ water solution of trifluoro-acetic acid (TFA), then
separated using HPLC. HPLC is a frequently used semi-preparative
technique. Elution of pure AMP fraction from a column can be carried
out with a gradient of solvent or a solution with constant water-solvent
composition. Previous studies have reported that the elution times of
hydrophilic and hydrophobic fractions are 45 and 75 min respectively.
The absorbance is monitored at 214 nm. The fractions can then be
collected manually, reconstituted and lyophilized to obtain a final
concentration in MilliQ water prior to analysis for antimicrobial activity
(Dolezilkova et al., 2015; Pingitore et al., 2007).
RP-HPLC is also useful for the separation and removal of plant
S.-S. Tang et al.
pigments before proceeding to purification. For example, an HPLC
based on a reversed-phase C8 column and mobile phases containing
pyridine was developed by Yacobi et al. (1996) for the simultaneous
separation of plant pigments such as chlorophylls and carotenoids. The
benefits of RP-HPLC in terms of speed and sensitivity as well as high
resolution in separating plant pigments are particularly dependent on
the type of column material used. Different reverse-phase columns,
such as C8, C18, C18 monolithic, π-NAP and cholester, can be used for
the separation of pigments at several specific states of the mobile phase
as well as at different temperatures. Of the range of columns available,
the cholester column is most suitable for separating not only hydro-
phobic pigments in a simple mobile phase but also pigments at a broad
range of polarities (Shioi et al., 2015).
3.2.4. Circular dichroism spectral (CDS) analysis
Circular dichroism spectral (CDS) analysis is a useful way to predict
the secondary structure of a purified protein. The purified AMP and
buffer are subjected to both 250–350 nm (Near UV) and 190–250 nm
(Far UV) light in order to obtain spectral scans. For the spectral mea-
surement, a volume of 300 μl of Tris-HCl buffer and purified peptide is
used, with Tris-HCl buffer as blank for the scan. Depending on the
concentration of AMP, the antimicrobial protein can be diluted two- or
three-fold with Mili-Q water before measurement. CDS analysis can
then be carried out using a CD Spectropolarimeter to obtain the
structural information of the purified AMP (Sakthivel and Palani,
2016).
3.2.5. 2-Dimensional gel electrophoretic analysis
2-D gel electrophoresis is another useful technique for analysing the
secondary structure of purified protein. A native-PAGE electrophoresis
is first carried out to obtain an initial separation of the protein. After
that, the gel slice containing separated protein is incubated with SDS
sample buffer, but without beta-mercaptoethanol, for 10–15mins. SDS-
separating gel electrophoresis is then carried out for the second phase of
protein separation. This analysis is particularly useful to verify whether
the purified AMP is in a monomeric form or otherwise (Sakthivel and
Palani, 2016).
3.2.6. Amino acid analysis
High performance liquid chromatography (HPLC) can also be used
to carry out an amino acid analysis of purified AMPs. First, the purified
peptide is subjected to chromatography separation with an HPLC
column. Next, the amino acids eluted from the column are detected
with a photodiode array detector at 280 nm. The amino acids are
identified based on the retention times of their corresponding standards
and the calibration curves obtained from with respective amino acid
standards which have undergone the same process of derivatization as
the AMP samples. The internal standard method is applied based on the
areas of the peaks of the derivatives. Finally, the chromatograms for the
amino acids from AMP samples, along with blank, are obtained. Amino
acids sequence in AMP can also be further studied using a MALDI-TOF
MS/MS analysis (Sakthivel and Palani, 2016) – described below.
3.2.7. MALDI-TOF MS/MS analysis
A matrix-assisted laser desorption-ionization time-of-flight tandem
mass spectrometer (MALDI–TOF MS/MS) that uses a pulsed nitrogen
laser is a useful tool to measure the molecular mass and peptide se-
quence of AMPs. A matrix solution containing saturated acid in acet-
onitrile is put on a sample plate, then air dried completely. Mass spectra
are collected using a linear delayed extraction mode (DE mode) and
positive ion mode (PI mode), with accelerated voltage, grid voltage,
guide wire voltage, low mass gate, and laser intensity. The signals from
the excitation pulses are accumulated and averaged to yield each re-
corded mass spectrum. The spectra are externally calibrated using
matrix ion peaks as per international standards. The data obtained from
the mass spectrometer are put through a database search (NCBI
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Phytochemistry 154 (2018) 94–105
nonredundant/SwissProt) and analysed using software. A programme
such as MASCOT (http://matrixscience.com) is useful for such analysis,
as it allows up to one missed trypsin cleavage and a monoisotopic mass
tolerance of 1.2 Da. The calibrated peptide masses are searched with
200 ppm mass accuracy. Blast2Go and UniProtKB software (http://
uniprot.org) can then be used for functional classification. The simi-
larity of mass spectra, tandem MS data and information on peptide
sequences in the database are used in combination in order to detect
AMPs with an appropriate molecular weight (Dos Santos et al., 2017;
Sakthivel and Palani, 2016; Yokoyama et al., 2008).
3.3. Common properties of plant AMPs
Most plant AMPs are basic in nature, have a molecular weight be-
tween 2 and 10 kDa, contain 4 to 12 cysteine residues, and form dis-
ulphide bonds that give them structural and thermodynamic stability
(García Olmedo et al., 2001). Plant AMPs are therefore generally clas-
sified based on their amino acid sequences and the number and position
of cysteines forming disulphide bonds (de Souza Cândido et al., 2011;
Marcus et al., 1997). These distinctive disulfide bond patterns de-
termine their tertiary structure folding and thermal stability. Plant
AMPs contain 2-6 intra-molecular disulfide bonds which bolster struc-
tural stability without affecting the general scaffold. The cross-bracing
of multiple disulfide bonds in plant AMPs makes them structurally
compact, with high thermal, chemical, and enzymatic stability. These
properties of plant AMPs make them suitable for purification by
warming, homogenizing, adjusting the pH, and separating the target
protein/peptide from other components by chromatography, including
affinity-based methods and salt precipitation (Naderi et al., 2015; Tam
et al., 2015).
AMPs also display peptide promiscuity – meaning that a single
peptide is able to exert multiple biological functions, often containing
three domains (N- and C-terminal pro-domains and a mature AMP
domain) (Tam et al., 2015). Plant AMPs also demonstrate antifungal
and antibacterial activity; are active against oomycetes and herbivorous
insects; exhibit enzyme inhibitory activities; have a heavy metal and
abiotic stress tolerance; and display anticancer activity against different
cancer cells (Allen et al., 2008; Koike et al., 2002; Kong et al., 2004).
These unique characteristics and broad antimicrobial coverage make
AMPs superior to synthetic or chemical antibiotics.
4. Antimicrobial activity of plant AMPs on targeted organisms
AMPs of plant origin can be tested against a variety of different
microorganisms, including bacteria, fungi, viruses, protozoa and hel-
minths. An initial assay for potential antimicrobial compounds derived
from plants can be performed with either pure substances (Afolayan
and Meyer, 1997) or crude extracts (Freiburghaus et al., 1996). Crude
plant extracts demonstrating antimicrobial activities have been shown
to be rich in plant AMPs. For example, crude Indian herbal extract
(ethanolic extract) from Piper longum fruits has counteracted Entamoeba
infections, while chloroform extract from the legume Milletia thonningii
has been shown to prevent the establishment of Schistosoma mansoni
infection on the skin of mice (Ghoshal et al., 1996; Sohni et al., 1995).
Among other studies, various extracts of the bark of Pteleopsis suberosa
against stomach ulcers caused by Helicobacter pylori, papaya latex
against infections with the helminth Heligmosomoides polygyrus, and
Santolina chamaecyparissus essential oil against candidal infections, have
all been observed as promising (He et al., 2010; Suresh et al., 1997).
The procedures generally used by researchers for antiviral, anti-
bacterial and antifungal tests on AMPs are described below.
4.1. Antiviral activity
Previous studies have reported that both RNA and DNA viruses can
be controlled by antiviral AMPs (Horne et al., 2005). Antiviral AMPs
S.-S. Tang et al.
neutralise such viruses by incorporating themselves into the viral en-
velope or host cell membrane. This causes membrane instability and
disruption. As a result, the viruses are unable to infect host cells
(Robinson et al., 1998). AMPs such as defensins also can reduce the
binding sites of viruses to their host cells; for example, by binding to
viral glycoproteins and inhibiting the binding of herpes simplex viruses
(HSV) to the surface of their host cells (Yasin et al., 2004). Some an-
tiviral AMPs prevent the entry of the viral particles into their host cells
by occupying specific receptors (Song et al., 2001). For example, de-
fensins bind to viral glycoproteins, preventing HSV from binding to the
surface of host cells and blocking virus-receptor interactions (Andersen
et al., 2004; WuDunn and Spear, 1989). Some AMPs cannot compete
with viral glycoproteins in binding to the heparan sulphate receptors on
the cell surface, but can cross the cell membrane and localise in the
cytoplasm. These types of AMPs can cause changes in the gene ex-
pression profile of the host cells and stimulate the host defence system
to fight against viruses or modulate cellular pathways to block viral
gene expression, thereby inhibiting viral replication. Both lunatusin and
vulgarinin were shown to inhibit HIV-1 reverse transcriptase and HIV-1
gene expression in a cell-free rabbit reticulocyte lysate system, which
suggests that antimicrobial activity might be linked to gene expression
and protein synthesis (Wong and Ng, 2005a; b). In some cases, AMPs
can also serve multiple roles to exert anti-viral effects. For instance,
Malformin A1, a cyclic penta-peptide containing one disulphide bond
isolated from an endophytic fungus, has been found to strongly inhibit
both infection and the replication of the Tobacco mosaic virus (TMV) in
host plants (Tan et al., 2015).
Several methods can be used to evaluate the antiviral activity of
plant AMPs. For viruses which form plaques in suitable cell systems,
there are two basic approaches: plaque reduction assays and titer re-
duction assays. In plaque reduction assays, a constant number of viral
particles are used together with a range of substance concentrations.
The level of antiviral activity is determined by measuring the reduction
in the size or number of plaques formed in a cell monolayer. Titer re-
duction assays, on the other hand, use a fixed amount of test substance
but a varying number of viral particles. By serial dilution of the virus
suspension to a concentration that renders a number of viral plaques
small enough to be counted, an obvious lower virus titer (compared to
the virus titer determined in the assays without the test substance) can
be detected, showing antiviral activity (Abou-Karam and Shier, 1990).
For viruses that induce cytopathic effects but do not readily form
plaques in cell cultures, inhibition assays of virus-induced cytopathic
effect and virus yield reduction assays can be carried out. Antiviral
activity is detected by determining a virus-induced cytopathic effect in
cell monolayers cultured in liquid medium, infected with a limited dose
of virus, and treated with a nontoxic dose of the test substance (Cowan,
1999; Vlietinck and Vanden Berghe, 1991).
Other common in vitro antiviral assays are virus yield reduction
assays and end-point titer determination tests. Antiviral activity is
measured by determining the virus yield in tissue cultures infected with
a given amount of virus(es) and treated with a nontoxic dose of the test
substance. Virus titer determination is carried out, after virus multi-
plication, by a plaque test. For end-point titer determination assays,
antiviral activity can be measured by determining the level of virus titer
reduction in the presence of two-fold dilutions of the test substance(s).
This technique was designed especially for the antiviral screening of
crude extracts (Chattopadhyay and Naik, 2007; Vlietinck and Vanden
Berghe, 1991).
For viruses that do not induce cytopathic effects and do not form
plaques in cell culture either, assays based on the measurement of
specialised functions and viral products can be used. Antiviral activity
is detected by determining virus specific parameters, such as the in-
hibition of cell transformation (Epstein-Barr virus), heamagglutination
and hemadsorption tests (myxoviruses), and immunological tests de-
tecting antiviral antigens in cell cultures (Epstein-Barr virus, HIV, HSV,
cytomegalovirus). Antiviral activity can also be expressed as a
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Phytochemistry 154 (2018) 94–105
reduction or inhibition of the synthesis of virus-specific product/poly-
peptides in infected cell cultures, such as viral nucleic acids, determi-
nation of the uptake of radioactive isotope-labelled precursors, or viral
genome copy numbers (Cowan, 1999; Lee et al., 1996; Paris et al.,
1993; Vlietinck and Vanden Berghe, 1991).
In sum, the common endpoints for antiviral activities are cytopathic
effects, plaque formation, and transformation or proliferative effects on
cell lines. Viral replication can be assayed by detecting viral products
such as DNA, RNA or polypeptides.
However, Vlietinck and Vanden Berghe (1991) noted that the
methods used for assaying antiviral substances in various laboratories
were not standardised, and so the results from different such tests could
not reliably be compared. They also pointed out that further research is
needed to explain the differences between the merely toxic effects of
agents on host cells and actual antiviral properties in plant AMPs
(Esimone et al., 2005; Vlietinck and Vanden Berghe, 1991). There is
therefore a need to develop alternative techniques, such as in vitro
pharmaco-dynamic screening. This type of method can contribute not
only to the rapid screening of bioactive plant compounds, but also to
the standardization and pharmacokinetic-pharmaco-dynamic profiling
of bioactive components. Various reported gene-based antiviral
screening assays have been shown to be flexible, rapid, unbiased and
amendable to high throughput (Johansen et al., 2004; Kimpton and
Emerman, 1992; Marschall et al., 2000; Proffitt and Schindler, 1995).
Esimone et al. (2005) reported using a vector-based assay technique for
an in vitro pharmacodynamic evaluation of antiviral medicinal plants,
while the cytotoxicity of the extracts was evaluated in parallel on target
cell lines.
4.2. Antibacterial activity
Antibacterial AMPs are the most studied cationic AMPs. Many of
them target the bacterial cell membranes and cause disintegration of
the lipid bilayer structure (Shai, 2002). They are also amphipathic in
nature, containing both hydrophilic and hydrophobic domains. Both
domains provide the capability of binding to lipid (hydrophobic region)
and phospholipid groups (hydrophilic region) (Brogden, 2005). Some
AMPs can kill bacteria at low concentrations without affecting mem-
brane integrity (Shai, 2002). They do so by inhibiting some important
pathways inside the cell such as DNA replication and protein synthesis
(Brogden, 2005; Jenssen et al., 2006; Le et al., 2017).
The most frequently used methods for screening AMPs to determine
their antimicrobial susceptibility are the disc diffusion assay or agar
well diffusion assay (Jenkins and Schuetz, 2012), and the agar and
broth dilution assays (Citron and Goldstein, 2011; Jaskiewicz et al.,
2016). For the disc diffusion as well as agar and broth dilution assays,
standardised guidelines detailing microbial strains, culture media, in-
cubation conditions, quality control procedures, and criteria for inter-
preting results have been published by organisations such as the Clin-
ical and Laboratory Standards Institute (CLSI) and European Committee
on Antimicrobial Susceptibility Testing (EUCAST).
The disc diffusion assay is the most common method used for an-
timicrobial susceptibility testing, largely due to its suitability for testing
a wide range of aerobic and anaerobic microorganisms and anti-
microbial compounds. It is also a simple experiment without the need
for specialised equipment, and capable of testing a relatively large
number of antimicrobial compounds at the same time. This method
involves the innoculation of nutrient agar with a standardised suspen-
sion of the test organism, followed by the application of a filter paper
disc containing the test compound on the surface of the agar plate. The
agar plate is then incubated at the appropriate temperature and dura-
tion for the test organism. The antimicrobial agents diffuse through the
agar to inhibit the growth of the bacteria, giving rise to a clear zone of
inhibition for which there is no bacterial growth. The diameter of the
zone of inhibition is then measured and reported in millimetres. It is
generally recommended to perform quality control testing for each test
S.-S. Tang et al.
organism using a disc containing a known antibiotic so as to ensure that
the zone of inhibition falls within acceptable limits, before interpreting
the results.
The agar well diffusion assay is a variant based on the disc diffusion
assay. Wells of about 6–10 mm in diameter are formed on the in-
oculated nutrient agar plates using a sterile cork borer. The wells are
then filled with a solution of the antimicrobial compound (100 μl). The
plates are kept at room temperature for a short period of time to allow
diffusion of the antimicrobial agents, before being incubated at suitable
conditions for microbial growth.
The above two methods have been reported to produce generally
consistent results with each other for certain bacterial species
(Luangtongkum et al., 2007). Although agar diffusion methods are
popular due to their low cost and simplicity, they do have some dis-
advantages, including difficulties in determining the minimum in-
hibitory concentrations (MICs) – defined as the minimum concentra-
tions of antimicrobial agents required to inhibit the growth of the
microorganism.
One good example of how to conduct antimicrobial tests using agar
well diffusion tests was shown by Tadeg et al. (2005). They carried out
experiments to screen the antimicrobial activities of some traditional
Ethiopian medicinal plants used in the treatment of various skin dis-
orders through agar well diffusion assays at three concentration levels
(100, 50 and 25 mg/mL) for crude extracts and at two concentration
levels (25 and 5 mg/mL) for fractions. During this experiment, 80%
methanol, 100% methanol, chloroform and distilled water were used as
negative controls for bacteria and fungi. The results were the averages
of triplicate tests, and the zones of inhibition included the diameters of
the wells.
The agar and broth dilution tests (macrodilution or microdilution)
are routinely used to determine the MICs of new antimicrobial agents
(Wiegand et al., 2008). In the agar dilution assay, the solution con-
taining the compound of interest is serially diluted at 10 × the intended
test concentration and added to 9 parts of molten agar, before being
poured into petri dishes to achieve an agar height of 3–4 mm (EUCAST,
2000). The standardised inoculum is then aliquoted onto the surface of
the agar to give 104 CFU per 5–8 mm diameter spot using inoculation
loops, micropipettes or inoculum replicators. Following incubation, the
MIC is taken as the lowest concentration of antimicrobial agent that
inhibits discernible microbial growth, often without taking into account
the presence of a single remaining colony or a slight haze in the in-
oculated spot. A major advantage of this method is that it permits the
simultaneous study of different bacterial strains on a single agar plate.
In the broth dilution method, a series of dilutions (typically two-fold)
are prepared. 1 mL is added into a test tube in the macrodilution
method, while 0.1 mL is added into each well of a 96-well round bottom
plate in the microdilution method. The standardised inoculum is added
into the macrodilution test tubes or microdilution well plates, which are
then incubated at approximately 35 °C for 16–20 h. The MIC is again
regarded as the lowest concentration of antimicrobial agent that com-
pletely inhibits the growth of the test organism – determined either by
viewing with the naked eye or by comparing spectrophotometric
readings (O.D. 600 nm) before and after incubation.
Both the agar and broth dilution methods do have some limitations.
These include the relatively laborious and expensive processes in-
volved; also the fact that the end results can be greatly affected by
experimental variables. As such, these experimental methods and the
associated conditions must be tightly controlled to ensure that the re-
sults obtained are reproducible and can be interpreted accurately.
4.3. Antifungal activity
Antifungal AMPs can kill fungi by targeting the cell wall or in-
tracellular components, while some antifungal AMPs can bind to chitin
(major components of fungal cell walls) (Pushpanathan et al., 2012;
Yokoyama et al., 2009). Such binding helps antifungal AMPs to target
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Phytochemistry 154 (2018) 94–105
fungal cell walls and kill target cells by disrupting the integrity of fungal
membranes (Terras et al., 1992; van der Weerden et al., 2010). Al-
though antifungal AMPs have polar and neutral amino acids in their
structures, there is no correlation between the structure of an antifungal
AMP and the type of cells (Jiang et al., 2008; Lee et al., 2003).
As with antibacterial testing, agar diffusion and broth dilution are
both commonly used methods for the evaluation of the antifungal ac-
tivity of new compounds. The main difference lies in the type of culture
medium or agar used as well as the incubation time. As a typical ex-
ample, a fungal strain is removed from the stock and suspended in 2 mL
of non-selective medium such as dextrose broth medium. This suspen-
sion needs to be uniformly spread on Petri plates containing dextrose
agar media, using sterile swabs. Wells are formed by the same tech-
nique as for bacteria. The plates are incubated at 25 °C for 3 days in the
case of Candida albicans and Aspergillus niger, and for 7 days in the case
of Trichophyton mentagrophytes. The inhibition zones are measured and
compared to a positive control (De Lucca et al., 1998; De Lucca and
Walsh, 1999).
The broth macrodilution and microdilution methods have been used
for the determination of MIC values in yeasts and moulds. Due to the
consistency of results between the two methods and the fact that the
microdilution method is easier to carry out than the macrodilution
method, the former has been the preferred choice for many laboratories
for use to determine antifungal susceptibilities.
For yeasts, the antifungal agent is prepared in water or a suitable
solvent and serially diluted in a RPMI-1640 growth medium. One
hundred microliters of each of the dilutions is then added in replicates
to each well of a 96-well plate. The yeast strain is inoculated onto a
nutritive agar medium such as Sabouraud dextrose agar or potato
dextrose agar, and allowed to incubate at 35 °C. The inoculum is pre-
pared by picking five representative colonies (≥1 mm) and suspending
them in sterile saline or distilled water. The density of the suspension is
then adjusted spectrophotometrically at 530 nm to match that of a 0.5
McFarland, giving an approximate concentration of 1–5 × 106 CFU/
mL. An equal volume (0.1 mL) of the standardised inoculum (typically
between 5 × 102 to 5 × 105 CFU/mL depending on the guideline being
followed) is then added to each well. The microdilution plates are in-
cubated at 35 °C without shaking, usually up to 72 h depending on the
yeast strain. MIC determination is less clear cut than in the case of
bacterial testing, being defined as the minimum concentration at which
a certain degree of inhibition of growth occurs compared to in the drug-
free control. This is typically defined as ≥ 90% inhibition for ampho-
tericin B and ≥50% inhibition for flucytosine, azoles, and echino-
candins. A relevant control antifungal agent is therefore normally in-
cluded to compare antifungal potency in the evaluation of new
antifungal agents. The MIC can be determined either by viewing with
the naked eye or spectrophotometrically, although the interpretation of
results can be highly variable if only visual observations of turbidity are
made.
To determine the MIC for moulds, the fungus is inoculated onto an
appropriate agar medium and allowed to sporulate at 35 °C. Water
supplemented with 0.1% Tween 20 is added to cover the colonies, and
then a sterile swab is used to transfer the conidia to a sterile tube
containing 5 mL of water with Tween 20. After vortexing to disperse
clumps and removing a significant amount of any hyphae present by
filtering through a 11 μm filter, the conidia is counted under a hae-
mocytometer and diluted using sterile water to a working concentration
of 2–5 × 105 conidia/mL. An equal volume of the conidia (0.1 mL) is
then added to the serially diluted antifungal agent in the 96-well plate.
The microdilution plates are incubated without agitation, typically for
24–72 h. The MIC is regarded as the lowest concentration at which no
growth is observed by eye.
In addition to these assays, antifungal phytochemicals can be eval-
uated by a spore germination assay. This involves adding plant extracts
to fungal spores collected from solid cultures, placing these on glass
slides, and incubating them at an appropriate temperature (25 °C) for
S.-S. Tang et al.
24 h. The slides are fixed in lactophenol-cotton blue, and observed
under the microscope for spore germination assessment (Rana et al.,
1997).
4.4. Antiparasitic activity
Screening plant extracts against protozoa and helminths is more
complicated than screening against bacteria, fungi or viruses, and the
assays involved need to be specific to the microorganisms concerned
(Freiburghaus et al., 1996). For example, two specific methods against
Trypanosoma brucei were developed by Freiburghaus's group to evaluate
the trypanocidal effectiveness of compounds extracted from African
medicinal plants (Freiburghaus et al., 1996). First, a 66-h cultured
tissue of trypanosomes was exposed to various concentrations of ex-
tracts. Then the MICs to which the lowest concentration of extract
capable of completely inhibiting the growth of the trypanosomes was
determined with an inverted microscope. Second, a fluorescence assay
was carried out to assess trypanosome viability in micro-titer wells. In
this fluorescence assay, the lipophilic and methanolic plant extract as
well as water extract was suspended in 10% dimethyl sulfoxide
(DMSO), 10% Methanol, and filtered sterilised water respectively.
Three-fold serial dilutions ranging from 500 to 0.07 μg/mL were per-
formed on each extract, with complete culture medium in a 96-well
microtiter plates (at least 2 times in duplicate). After 66 h of incubation
at 37 °C in a humidified incubator, the MIC of each extract was de-
termined. For extracts that showed MIC values ≤ 56 μg/mL, a 100 μl of
4 μM BCECF-AM (2′,7′-bis(carboxyethyl)-5 (6)-carboxyfluorescein-pen-
taacetoxy-methylester) was added to each well and the plate was fur-
ther incubated for another 45 min before fluorescence units were read
with a fluorescence plate reader at 485 nm and 530 nm wavelengths.
The concentration of plant extracts that inhibited the growth of try-
panosomes by 50% (IC50) was then calculated (Freiburghaus et al.,
1996).
Plant AMPs which have anti-infective properties against the
Plasmodium species are of interest to scientists. Parasitized erythrocytes
are incubated both with and without test substances, and after the in-
cubation period the numbers of Plasmodium organisms are quantified
(François et al., 1996). In one study, ethanol extracts from nine dif-
ferent Bidens species were collected. Tests showed that seven of the nine
extracts inhibited parasite growth in vitro by 65–91%. An HPLC analysis
using a photo diode-array detector showed the presence of phenyl
acetylene and flavonoids in the ethanol extract, while chloroform
fractions caused 86% inhibition of parasite growth in vitro containing 1-
phenyl-1,3-diyn-5-en-7-ol-acetate (Brandão et al., 1997).
There are not many studies investigating the antiparasitic property
of plant-derived AMPs. The first evidence for such antiparasitic (leish-
manicidal) activity in plant-derived AMPs was presented by Berrocal-
Lobo and co-researchers (2009). In their experiment, thionin, a barley
lipid transfer protein derived from wheat and potato derived defensins,
tested positive against Leishmania donovani (a human pathogen) at a
low range of concentrations. Both AMPs managed to collapse ionic and
pH gradients across this parasite plasma membrane. The mode of action
of antiparasitic peptides is broadly the same as other AMPs: most kill
cells directly by interacting with the cell membrane to form pores (Park
et al., 2004).
Further proof of leishmanicidal activity in plant-derived AMPs has
been published by a number of Brazilian researchers (Carvalho et al.,
2006; Carvalho et al., 2001; dos Santos et al., 2010; Souza et al., 2013).
They found evidence that a plant defensin (Vu-Def), isolated from the
seeds of Cowpea (Vigna unguiculata L. Walp), was able to negatively
affect the growth of Leishmania amazonesis promastigotes. In addition to
the cloning and overexpression of recombinant defensin from Cowpea
seeds (Vu-Defr) in Escherichia coli, they also carried out various mod-
ifications and improvements to the expression and purification pro-
cesses of Vu-Defr. They conducted experiments to compare the leish-
manicidal activity of Vu-Defr to the natural defensin Vu-Def and the
101
Phytochemistry 154 (2018) 94–105
results demonstrated that recombinant Vu-Defr was as biologically ac-
tive as its counterpart, and retained its full level of biological activity
after improved recombinant production and purification (Souza et al.,
2013). These results are further evidence of the potential of plant-de-
rived AMPs to be used as new antiparasitic agents.
Separately, in 2015, a group led by Gomes successfully isolated a
plant AMP from the seeds of Perola (Phaseolus vulgaris), which they
called PvD1, and which displayed inhibitory activity against protozoan
L. amazonesis promastigotes (do Nascimento et al., 2015). In addition to
evaluating the effect of PvD1 on the proliferation of this protozoan, they
also characterised the antiparasitic mechanisms of this plant-derived
AMP. Their results proved that the defensin PvD1 was able to cause
membrane permeabilization, the formation of cytoplasmic vacuoles and
cytoplasmic fragmentation, thus inhibiting the proliferation of L. ama-
zonesis promastigotes cells. Interestingly, they also found the presence
of this plant-derived AMP within L. amazonesis cells – which suggests
that the toxicity of these peptides may not be restricted to the plasma
membrane, but may also be acting on intracellular targets. This finding
opens new perspectives on the antiparasitic mechanisms of plant-de-
rived AMPs, offering new avenues for future research (do Nascimento
et al., 2015).
5. Conclusions and prospects
Antimicrobial peptides (AMPs) of plant origin have a variety of
amino acid compositions and structures, many of which exhibit potent
broad spectrum antimicrobial activities and prove capable of killing
microbes rapidly. Plant AMPs are thus a potentially valuable natural
alternative to chemical antibiotics for use in both human healthcare
and in agriculture to protect plants and animals from disease. Many
transgenic plants have been developed to express AMPs that confer
different degrees of protection against disease (Nawrot et al., 2014). It
has been shown that a number of transgenic plants containing AMPs
can protect themselves from pathogen attack, such as the Mj-AMP1
(jalapa defensin) in tomato against Alternaria solani (Schaefer et al.,
2005), Rs-AFP2 (radish defensin) in tobacco and tomato against Alter-
naria longipes (Terras et al., 1995), hevein Pn-AMP in tobacco against P.
parasitica (Koo et al., 2002), and alfalfa defensin in potato for V. dahliae
(Gao et al., 2001). Similarly, thionins, cyclotides, protein transduction
domains and cell-penetrating peptides are other important peptides for
the development of transgenic plants. Genetic improvements in plants
to increase their pathogenic resistance and reduce crop losses in agri-
culture could eventually lead to a reduction in pesticide use.
In sum, AMPs could play an important role in agriculture as plant
protection products. However, there are problems to overcome. First of
all, transgenic plants are not generally released as commercial cultivars
due to regulatory limitations and social concerns. In addition, the in-
trinsic toxicity, low stability and high production costs of some AMPs
are further obstacles to the commercial use of plant AMPs. There is thus
a need to identify and develop commercial plant AMPs that are less
toxic, more stable and can be produced at lower cost.
Several options for improving the quality, selectivity, durability and
safety of AMPs have already been explored, including improving their
functional and immunological properties by partial hydrolysis (Salas
et al., 2015). AMPs in hydrolysate form can be used as a food additive
for beverages and infant formulae, as a food texture enhancer, and as a
pharmaceutical ingredient. They can also be computationally modelled,
genetically manipulated, and expressed in different systems to serve
various practical pharmacological and pesticidal purposes.
In conclusion, the progress made in developing peptide libraries, as
well as the development of sophisticated proteomics, bioinformatics
and modification strategies, have made plant AMPs promising anti-
microbial agents. The further discovery and development of novel plant
AMPs with potent biological properties should provide excellent op-
portunities to expand further their use in the treatment of human, an-
imal and plant diseases.
S.-S. Tang et al.
Conflicts of interest
None.
Acknowledgements
This article and the associated research were produced with the fi-
nancial support of the University of Malaya, Malaysia (research grant
number RG347-15AFR).
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(LTP) from Coffea canephora seeds with α-amylase inhibitor properties. Biochim. of the plant, and he is familiar with metabolomic analysis of natural compounds (ex-
Biophys. Acta Gen. Subj. 1810, 375–383. traction, identification, and quantification of volatile compounds from plants). Zakaria
has obtained 1st class in both the M.Sc. (Thesis) and B.Sc. (Honors) examinations and his
research theme was on the effect of plant extracts on salivary gland- and oocyte chro-
mosomes of Musca domestica L. (Diptera: Muscidae). Dr. Zakaria is an author of twelve
scientific publications, and his main research interests are in genetics, breeding and
molecular biology of plants. He is the Editor in chief of the Scholarstic Editing Service and
is a reviewer for a prominent international journal (Rice Research journal).

Dr Swee-Seong Tang is a Senior Lecturer at the Faculty of Science, University of

Malaya, Malaysia. He received his B.Sc (Hons) in Biochemistry in 2000 and M.Sc in
Microbiology in 2004 from the University of Malaya. He was awarded a tutorship by the
University of Malaya and SLAI fellowship (University academic training scheme) by the
Ministry of Higher Education Malaysia to pursue his PhD studies at the Australian
National University, Australia. He obtained his PhD in Molecular Microbiology in 2013.
He has since returned to Malaysia to take up a tenure track lectureship position at the
University of Malaya. He has been playing a key role in several research projects colla-
borations, and leads the project ‘Studies on novel genes involved in synthesizing unique
antimicrobial peptides’. His current research includes chemical & structural elucidation of
antimicrobial peptide from medicinal plants and Lactic Acid Bacteria, pharmacognosy,
Sudhangshu Kumar Biswas is an Assistant professor in the department of
Biotechnology and Genetic Engineering, Islamic University, Bangladesh and current PhD
student at the Institute of Biological Sciences, University of Malaya, Malaysia. He ob-
tained his degree and Masters in Genetics and Breeding both in first class from University
of Rajshahi, Bangladesh. His research interests are related to antimicrobial activity of
different plants extract, microbial biochemistry, multidrug-resistant bacteria, Molecular
biology of bacteria and virus, bacteriophage biology and phage therapy with an aim to
purify a unique broad host-range bacteriophage as well as to develop phage-cocktail
against multidrug-resistant bacterial pathogen.

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Dr Cheng-Foh Le is an Assistant Professor at the University of Nottingham Malaysia
Campus. He obtained his PhD from the University of Malaya in 2013 for his research
study on designing and development of antimicrobial peptides as alternative anti-
microbials against Streptococcus pneumoniae. The significance of his works had been re-
cognized in a number of conferences and symposia. In 2013, He received the Young
Investigator Award in the 9th International Symposium on Antimicrobial Agents and
Resistance (ISAAR-9) for his research on the design of a series of novel AMPs which
successfully cured the mice from lethal pneumococcal infection. His major interest is
grounded on antimicrobial drug discovery, particularly against the clinical pathogens.
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Phytochemistry 154 (2018) 94–105
Prof Dr. Shamala Devi Sekaran began her career centring in immunology which
then expanded to the field of virology, bacteriology, diagnostic microbiology, anticancer
as well as anti-microbial drug/ peptide discovery and lately endothelium dysfunctions in
infectious diseases. Dengue was her primary focus during her Ph.D. programme and has
remained her stronghold particularly lie in the field of diagnostics. She and her team have
also developed various molecular diagnostic kits and have been actively involved in
evaluation projects of dengue diagnostic kits conducted by the WHO and collaborating
partners around the world as well as with companies that are commercializing these kits.
The lack of anti-dengue drugs and vaccines has prompted Prof Shamala to further venture
into antiviral drug development. Her projects include (i) the application of siRNA to in-
hibit entry of dengue virus into target cells; (ii) use of natural products such as Phyllanthus
plant species to inhibit dengue virus infections; and more recently (iii) using combina-
torial bioinformatics to design antimicrobial peptides (AMPs) against dengue virus.
Besides dengue, Prof Shamala and her team have begun to design and develop novel
synthetic AMPs as standalone or combination antimicrobial agents against the bacteria
Streptococcus pneumoniae, and the fungus Candida albicans. The research conducted in this
area involve in vitro and in vivo antimicrobial and toxicity evaluation of the designed
peptides which eventually will be investigated in appropriate animal models for ther-
apeutic efficacy and clinical usefulness. In few such endeavours, her team had designed a
series of hybrid peptides showing potent antipneumococcal activity with broad spectrum
antibacterial activity against various gram-positive and gram-negative clinically im-
portant bacteria. Among the series, one of the peptides displayed significant therapeutic
efficacy in murine lethal pneumococcal infection model and could potentially a promising
candidature for further development.