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doi: 10.1016/j.foodres.2014.01.057
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Please cite this article as: Cheok, C.Y., Salman, H.A.K. & Sulaiman, R., Extraction
and quantication of saponins: A Review, Food Research International (2014), doi:
10.1016/j.foodres.2014.01.057
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Extraction and quantification of saponins: A Review
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Abstract
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Saponins, a second metabolites mainly derived from plant materials, have been used
extensively in drug-related industry due to the pharmaceutical properties. These have driven the
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emergence of various new extraction technologies with the main purpose to optimize the yield in
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order to accommodate the recent need. The plants contain saponins is discussed, and its
pharmaceutical properties and applications in food are highlighted. This review focuses on the
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saponins extraction with emphasis on conventional and green technologies techniques employed
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in previous works by relating to their specific objective in each study. The quantification
methods of saponins yield, ie., spectrophotometric and chromatographic, are summarized and
discussed. In addition, this review aims to provide a point of reference to researchers who wish
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Contents
Introduction ........................................................................................................................................... 4
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Quantification of Saponins.................................................................................................................. 20
6.2.
7.
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6.1.
Conclusions ......................................................................................................................................... 24
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References ................................................................................................................................................... 25
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1. Introduction
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Saponins are second metabolites which widely distributed in plant kingdom. It acts as a
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chemical barrier or shield in the plant defense system to counter pathogens and herbivores
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(Augustin, Kuzina, Anderson, & Bak, 2011). Therefore, it is found in plant tissues that are most
vulnerable to fungal or bacterial attack or insect predation (Wina, Muetzel, & Becker, 2005).
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Saponins divided into two major classes which are triterpenoid and steroid glycosides which
their structure characterization are varied by the numbers of sugar units attached at dierent
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positions (Hostettmann & Marston, 1995). The classification and occurrence of saponins in the
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Saponins, which derived from soapwort (Saponaria officinalis L.), has been widely used for
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centuries as household detergent (Sparg, Light, & van Staden, 2004) due to its amphiphilic
nature with presence of a lipid-soluble aglycone and water-soluble chain(s) in their structure
(Gl-ntnda & Mazza, 2007). The seeds of Barringtonia asiatica Kurz (Lecythidaceae)
which have known to contain saponins, have been used traditionally by native Asian and Pacic
sherman as fish poison to enhance their catches (Sparg et al., 2004). Saponin-containing plant
materials, i.e., Yucca schidigera, alfafa, were used as feed additives to increase growth, milk or
wool in ruminant production (Wina et al., 2005). The molluscicidal saponins derived from
soapnut (Sapindus mukorossi Gaerth) has been found having inhibitory effects against golden
apple snail, which is the major pests of rice and other aquatic crops in Asian countries (Huang,
Liao, Kuo, Chang, & Wu, 2003).
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The discovery of biological activities of saponins is not only limited to the traditional uses,
but more recently, in pharmaceutical applications (Gl-ntnda & Mazza, 2007; Sparg et al.,
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2004). Saponins have been found having pharmaceutical properties of haemolytic, molluscicidal,
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drugs in pharmaceutical industry. Sheng and Sun (2011) reviewed the clinical significance of
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The pharmaceutical properties discoveries, especially anticancer, have intensified the seeking
of saponins from plant materials. These have driven the emergence of various new extraction
technologies with the main purpose of maximizing the yield in order to accommodate the recent
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need. Saponins are also known possessing mineral complexes of iron, zinc, and calcium (Milgate
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& Roberts, 1995). The beneficial effect of saponins intake in plasma cholesterol for human is
another important factor contributes to the continuous sorting of saponins (Milgate & Roberts,
1995). Besides anticancer (Cheng et al., 2011; Man, Gao, Zhang, Huang, & Liu, 2010; Waheed
et al., 2012), saponins have been discovered scientifically having pharmaceutical properties of
antioxidant (Chan, Khong, Iqbal, & Ismail, 2013; Dini, Tenore, & Dini, 2009; Li et al., 2010b),
immunological adjuvant activities (Estrada, Katselis, Laarveld, & Barl, 2000; Sun, Chen, Wang,
& Zhou, 2011; Verza et al., 2012), and haemolytic activities (Hassan et al., 2010; Sun et al.,
2011).
Since saponins are currently the most interested subject of their potential for industrial
processes and pharmacology, a correct selection of extraction technique through a review of
appropriate literature is essential. Researchers from a variety of scientific backgrounds are often
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challenged by the initial extraction process prior to isolation and identification of specific
saponins responsible for biological activities. The aim of this review is to summarize the
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selection of extraction methods from previous literature in respect to research focus in order to
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Saponins are mainly derived from various plant materials (Sparg et al., 2004; Vincken et al.,
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2007), but several of them are found in sea cucumber and starfish (Augustin et al., 2011;
Demeyer et al., 2014). The most widely studied plant material that found having saponins is
ginseng (Kwon, Blangar, Pare, & Yaylayan, 2003; Qian, Lu, Gao, & Li, 2009; Vongsangnak,
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Gua, Chauvatcharin, & Zhong, 2004; Wu, Lin, & Chau, 2001; Zhang & Cheng, 2006; Zhang,
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Liu, Qi, Li, & Wang, 2013b), even though saponins derived from alfafa has been carried out as
early by Van Atta et al. (1961). Other plant materials which have been discovered containing
saponins were soymilk (Lai, Hsieh, Huang, & Chou, 2013), sugar beet (Ridout, Price, Parkin,
Dijoux, & Lavaud, 1994), soy and chickpea (Serventi et al., 2013), asparagus (Vzquez-Castillo
et al., 2013), marion blackberry, strawberry, and plum fruit (Yoon & Wrolstad, 1984).
Saponins distribution has been found to vary in individual plant parts. For example, the roots
of Medicago truncatula (Huhman, Berhow, & Sumner, 2005) and Allium nigrum L. (Mostafa et
al., 2013) have been revealed containing the greatest total amount of saponins accumulation. The
yam tuber cortex has been discovered possess the highest amount of saponins of 582.53 g/g dw
which was about 2.55 times higher than tuber flesh of 227.86 g/g dw (Lin & Yang, 2008).
However, the total saponins concentration has been reported contain highest level in leaves from
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four varieties of Swithchgrass (Lee et al., 2009) and greenhouse grown Maesa lanceolata
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(Theunis et al., 2007). Table 1 tabulates the saponins derived from different plant parts.
Since saponins fall in two categories which on water-soluble sugar units attached to a lipophilic
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steroid (C27) or triterpenoid (C30) moiety (Challinor & De Voss, 2013; Gl-ntnda &
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Mazza, 2007; Harborne & Baxter, 1999), therefore, the isolation and structure elucidation of
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triterpenoid (Connolly & Hill, 2010) and steroidal (Challinor & De Voss, 2013) saponins have
been reviewed. Sparg et al. (2004) reviewed a list of plant species from which saponins have
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been isolated by categorizing them into triterpenoid and steroidal in period from 1998 to 2003.
However, recent review on the triterpenoid and steroidal saponins derived from various plants is
shown in Table 2. The elucidation and characterization of saponins structure are conducted on
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resonance) data, such as in Ipomoea batatas (Dini et al., 2009), Aralia taibaiensis (Bi et al.,
2012), and Allium ampeloprasum var. porrum L. (Ado et al., 2011).
Saponins rich in pharmaceutical properties and recently many studies focus on saponins
ability to increase immune responses (Estrada et al., 2000; Sun, 2006; Sun et al., 2011; Verza et
al., 2012), possess of antibacterial (Hassan et al., 2010; Mostafa et al., 2013; Iorrizzi, Lanzotti,
De Marino, Ranalli, & Zollo, 2002; Teshima et al., 2013), antioxidant (Bi et al., 2012; Chan et
al., 2013; Dini et al., 2009; Li et al., 2010b; Lin, Yang, & Lin, 2011), anticancer (Cheng et al.,
2011; Man et al., 2010), antidiabetic and anti-obesity (Joseph & Jini, 2013; Kimura, Ogawa,
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Katsube, Yokota, & Jisaka, 2008; Yun, 2010). Thus ginseng, which contains saponins, is
included in most of the Chinese Medicinal Prescriptions, for example, Bianxia Xiexin decoction
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in treating gastroenteritis diseases (Wang et al., 2014). Seven structurally consecutive saponins
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derived from Platycodon grandiflorum have been discovered having haemolytic activities and
adjuvant potentials on the immune responses to Newcastle disease virus-based combinant avian
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inuenza vaccine in mice (Sun et al., 2011). Both Verza et al. (2012) and Sun (2006) revealed
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that saponin fractions derived from Chenopodium quinoa seeds and Bupleurum chinense
enhanced haemolytic activities and adjuvant potentials on immune responses of mice against
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ovalbumin. Saponins obtained from Polygala senega L. were also suggested as potential vaccine
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Hassan et al. (2010) reported that 100% methanol fraction of saponin-rich extracts from guar
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A number of previous literature reported that saponins rich fraction have antioxidant
properties. There were derived from root bark of Aralia taibaiensis (Bi et al., 2012), deffated rice
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bran (Chan et al., 2013), Ipomoea batatas tubers (Dini et al., 2009), yellow horn (Li et al.,
2010b), stems and fruits of Momordica charantia (Lin et al., 2011). The sprouts of soybean,
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mung bean (Vigna radiata L.), and alfafa (Medicago sativa L.) which are rich in saponins, have
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been suggested as a good supplement of bioactive compound in daily diet with health-promoting
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Man et al. (2010a) highlighted saponins possess significant anticancer properties and the
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Gynostemma pentaphyllum leaves (Cheng et al., 2011) and a novel steroidal saponin glycoside
derived from Fagonia indica (Waheed et al., 2012) have been discovered having
antiproliferation and apoptosis against prostate, breast and colon cancer cells. In a recent study,
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two new steroidal saponins, fruticoside H and fruticoside I, derived from Cordyline fruticosa
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leaves have been found having moderate cytotoxic activity against human breast, colon, and
melanoma cell lines (Fouedjou et al. 2014).
The lack of physical activity in daily routines and increase in high-calorie fast food intake
have led to a number of health-related consequences such as obesity and diabetes, where 80% of
type 2 diabetes patients are linked to obesity (Yang et al., 2010). Hence, studies on identification
of anti-obesity property from plant materials have become a popular trend. In a recent review,
saponins separated from plant materials of Panax japonicas, Platycodi radix, Kochia scoparia
fruits, Thea sinensis leaf, Scabiosa tschiliensis Grun., and Acanthopanax sessiliflorous were
suggested having anti-obesity property in inhibited pancreatic lipase (Yun, 2010). The possibility
of obesity treatment with saponins derived from leaves of Acanthopanax sessiliflorus
(Yoshizumi et al., 2006) and saponins from Japanese horse chestnut (Aesculus turbinate
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BLUME) after the treatment with wood ashes (Kimura et al., 2008) have been discussed. Panax
notoginseng saponins have both anti-hyperglycemic and anti-obese effects which may beneficial
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to type 2 diabetic patients by improving insulin sensitivity and decreasing leptin resistance (Yang
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et al., 2010). A typical triterpenoid saponin of charantin derived from Momordica charantia is a
well-known anti-diabetic bioactive compound (Joseph & Jini, 2013; Raman & Lau, 1996). Four
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new triterpenoid saponins isolated from the root bark of Aralia taibaiensis exhibited moderate
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effects on antioxidant and antiglycation activities which could be correlated with treatment of
diabetes mellitus (Bi et al., 2012). Both Liu et al. (2012b) and Zheng et al. (2012) demonstrated
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that total saponins from Rhizoma Anemarrhenae and Entada phaseoloides L. were able to
ameliorate diabetes-associated cognitive decline in rats. Saponin rich fractions from Bryonia
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Laciniosa (Patel, Patel, Vyas, Singh, Shah, & Gandhi, 2012a), Momordica charantia (Keller et
al., 2011) and Polygonatum odoratum (Deng et al., 2012) have been proven having anti-diabetic
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Other therapeutic properties of saponins were reported in previous literature. There were
cardioprotective effects of saponins from Panax japonicas (He et al., 2012), anti-thrombotic
activity from Dioscorea zingiberensis (Li et al., 2010a), anti-inflammatory and antiulcerogenic properties from the bulbs of Allium ampeloprasum (Ado, da Silva, & Parente,
2011), anti-HIV activity from Momordica charantia (Chen et al., 2008; Chen et al., 2009), and
antiurolithiatic activity from fruit of Solanum xanthocarpum (Patel et al., 2012a).
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consumer preference for natural substance, Quillaja saponin has been used as a natural small
molecule surfactant in beverage emulsions in replacing synthetic surfactant of Tweens
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(Piorkowski & McClements, 2013). The effectiveness of the natural surfactant isolated from the
bark of the Quillaja saponaria Molina tree for forming and stabilizing emulsions with a synthetic
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surfactant (Tween 80) has been compared by Yang et al. (2013). After comparing the inuence
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of homogenization pressure, number of passes, and emulsier concentration on the particle size
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produced from these two surfactants, they suggested that the natural surfactant is an effective
surfactant that may be able to replace synthetic surfactants in food and beverage products. This
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natural surfactant has been further proven its stability and effectiveness at forming edible
Vitamin E delivery systems, thus it is recommended for functional food encapsulation and
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Due to its natural foam-like characteristic, the application of saponins as a natural bio-surfactant
to improve the surface properties of food is intensively studied recently. Wojciechowski et al
(2014) have conducted a study to evaluate the surface activity between Quillaja bark saponin
with -casein of bovine milk protein. From their results obtained, they suggested that the
Quillaja bark saponin can be used as a natural low molecular weight bio-surfactant. A recent
study indicated that banana cellulose micro and nano bres obtained by steam explosion process
which soaked with saponin, a surfactant extracted from soapnut fruit, showed differences in the
degree of modication and morphology of the cellulose bres (Cordeiro et al., 2013). The results
clearly show that the saponin can provide a continuous path of hydrogen bonds between the bre
surfaces which will thus enhance the hydrophobic and the acidbase nature of the bre surface.
This behaviour will lead to better polymer/bre interaction during the composite preparation.
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Andreuccetti et al. (2010) evaluated the incorporation of hydrophobic plasticizers in a matrix of
gelatin, using the saponin extracted from Yucca schidigera (yucca) as emulsifier, in the
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production of biodegradable emulsified films using the casting technique. Their results showed
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that the gelatin-based films produced have good mechanical resistance, low values of water
vapor permeability and reduced drying times, even though the films presented limited
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elongation, considerable solubility and opacity. Therefore, they suggested that the possibility of
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using this natural surfactant may allow for new applications of biodegradable emulsified films.
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The use of saponins as a natural biochemical substance in inactivation of food-borne viruses has
been reviewed by Li et al. (2013). The saponins-extract from Sapindus saponaria combined with
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heat-treatment was recommended to inactivate Alicyclobacillus acidoterrestris, a spoilagecausing bacterium, in orange juice (Alberice et al., 2013). Tea saponin, a tea seed-derived natural
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surfactant, combining with Bacillus amyloliquefaciens (Hao et al., 2011) and imazalil and
prochloraz (Hao et al., 2010), have been used for postharvest treatment of Mandarin fruit and
results showed that the incidence of green and blue mold and sour rot were reduced.
5. Extraction techniques
The recent advances in extraction of bioactive compound from plant material have been
intensively reviewed (Azmir et al., 2013; Wang & Weller, 2006) and this might due to the
increase in public awareness of preventative health care which could be promoted through the
consumption of plant material extract. In general, the extraction techniques employed in saponins
extraction can be classified into two categories, the conventional and the green technologies. The
conventional extraction techniques are maceration, Soxhlet, and reflux extraction, where the
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green technologies are ultrasound-assisted, microwave-assisted, and accelerated solvent
extraction (Heng, Tan, Yong, & Ong, 2013). The conventional extraction is relied on the
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solubility of solute from plant materials into solvent. Therefore, it often utililizes a large quantity
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of solvent to extract the desired solute, even though sometimes is aided with elevated
temperature by heating, and mechanical stirring or shaking. On the other hand, the green
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extraction techniques involved less hazardous chemical synthesis, safer chemicals used, energy
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efciency, use of renewable feedstock, and pollution prevention (Azmir et al., 2013). The design
of green extraction technologies are governed under these measurements. Consequently, water is
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used as extraction solvent by manipulating the extraction system pressure and temperature, as in
pressurized liquid extraction.
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output to cope with the increasing demand. Therefore, a synthesis of previous literature in
extraction technique selection may provide useful information in related processing industry.
Fig. 1 clearly demonstrates that researchers are more inclined to selection of the conventional
extraction techniques (70%) which include the subsequent methods, compared to the green
technologies (30%), even though the green techniques use minimal solvent. The selection of
these methods usually was governed by the research focus of the studies being conducted. To
further analyze the selection of extraction technique made by researchers, Table 3 presents an
overview of extraction techniques in accordance to their research objectives. For isolation of new
saponins and pharmaceutical properties studies, 78% and 91% of the previous works were using
the conventional extraction methods. However, in works focused on quantification and
optimization studies, 58% and 67% of the previous works have selected green extraction
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technologies. It is also noteworthy that the ultrasound-assisted extraction is the most selected
green extraction technologies in quantification studies which gave an implication of its capability
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(solute) inside the plant material is extracted by soaking the plant material in a specific solvent
for a period of time (Takeuchi et al., 2009). The efficacy of maceration process is determined by
two main factors, solubility and effective diffusion. The solubility is governed by basic rule of
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like dissolves like where indicated that a polar compounds dissolve in polar solvents, and
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nonpolar compounds dissolve in nonpolar solvents (Reichardt & Welton, 2011). The rate of
dissolution of a solute in the extraction solvent is determined by the rate of mass transfer of a
solute from the plant material to the solvent (Takeuchi et al., 2009). Due to the concentration
gradient in the solid-liquid interface, the transfer of the solute inside the plant material occurs
showing an effective diffusion takes place (Takeuchi et al., 2009).
No complicated utensil and equipment are needed for the set-up of a maceration extraction
system has made it a popular choice for researchers. The only paramount factor to be paid
attention in enhancing extractability is the knowledge of similarity of bioactive compound
interest and solvent polarity. Table 4 summarizes the maceration extraction procedure carried out
in previous literature in respect to their objective(s).
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Ethanol and methanol were the extraction solvents used to extract saponins from plant
material, and ethanol preferred better probably due to environment friendly concern. The
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duration of extraction time is long and sometimes takes up to weeks using this method, therefore,
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maceration extraction often aided with mechanical shaker (Cheng et al., 2011; Huhman et al.,
2005; Lee et al., 2009; Sylwia, Bogumil, & Wieslaw, 2006) or magnetic stirring (Verza et al.,
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Due to the similar working principle of Soxhlet and reflux extractions, the discussion is
carried out under the same sub-title. The only different between reflux and Soxhlet is that
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Soxhlet apparatus consists of a thimble to house the plant material. Reflux and Soxhlet extraction
involved distillation process which widely used in food and non-food industrial and laboratories.
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The process involves heating a solution to boiling and then returning the condensed vapours to
the original flask (Bart, 2011). The disadvantage of reflux and Soxhlet extractions is time
consuming where it required at least one hour for an extraction. Table 5 presents a summary of
saponins extraction from plant materials using reflux and Soxhlet extraction method. Ethanol is
still the most used solvent in reflux extraction, although there are few used methanol as
extraction solvent. The extraction duration of reflux extraction was varied from 1 to 4 hours,
while for Soxhlet was 24 to 72 hours.
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highly purify the extract before subjected to HPLC analysis for isolation and identification
saponins compound from plant material. Table 6 presents the application of two subsequent
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extraction methods in obtaining the specific saponins from various plant materials. For Soxhlet
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and reflux subsequent method, the Soxhlet extraction is carried out first to remove the lipid of
the plant material using solvent such as chloroform (Bialy, Jurzysta, Mella, & Tava, 2004; Bialy
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Jurzysta, Mella, & Tava, 2006; Oleszek et al., 2001; Tava et al., 2009) and hexane (Ncube,
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microjet to denature the plant cell wall when the bubbles collapse at rarefraction resulted in a
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greater extraction yield of biaoactive compounds. Few researchers have reviewed the ultrasound
effect on the technological properties and bioactivity of food (Soria & Villamiel, 2010), the
applications of ultrasound-assisted extraction on bioactive principles from herbs (Vinatoru,
2001), food industry and processing (Mason, 1998; Vilkhu, Mawson, Simons, & Bates, 2008).
Although ultrasound-assisted extraction is commonly employed in many bioactive compounds
extraction (Cheok, Chin, Yusof, Taib, & Law, 2013; de Koning, Janssen, & Brinkman, 2009;
Jadhav, Rekha, Gogate, & Rathod, 2009; Zhang et al., 2008), only few has been found in
saponins extraction (Table 7).
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Microwaves are non-ionizing electromagnetic waves with a frequency range from 0.3 to
300 GHz (Heng et al., 2013; Takeuchi et al., 2009). Recently, MAE has drawn attention in
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bioactive compound extraction from plant material due to short extraction time, minimal solvent
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usage, and its special heating mechanism (Heng et al., 2013). The recent applications of MAE of
plant secondary metabolites such as flavonoids, quinones, phenylpropanoids, terpenoids,
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alkaloids and saponins have been reviewed (Zhang, Yang, & Wang, 2011). Microwaves are able
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to penetrate into biomaterials and generate heat by interacting with polar molecules such as water
inside the materials. The penetration depth of microwaves into plant matrix depends on dielectric
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constant, moisture content, temperature, and the frequency of the electrical field (Takeuchi et al.,
2009). The water contained in a plant material is responsible for the absorption of microwave
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energy which led to internal superheating and cell structure disruption, and consequently,
facilitates the diffusion of bioactive compound from the plant matrix (Takeuchi et al., 2009). The
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efficacy of MAE is relied on the effect of microwave on extraction solvent and plant matrix cell
structure (Takeuchi et al., 2009).
Although the potential application of microwave extraction for flavonoids has been reviewed
thoroughly (Routray & Orsat, 2011), only few works of saponins extraction using MAE has been
mentioned in previous reviews (Zhang et al., 2011; Gl-ntnda & Mazza, 2007). Table 8
synthesizes an up-to-date literature of using MAE in saponins extraction in which the content
was not covered in those two reviews. The superiority of MAE in saponins extraction in
comparison with other extraction methods, in terms of higher yield (Chen, Xia, & Gong, 2007b;
Li et al., 2010b; Mandal & Mandal, 2010; Xu et al., 2012) and shorter extraction time (Chen et
al., 2007b; Kwon et al., 2003; Mandal & Mandal, 2010; Xu et al., 2012), has been found in
previous literature.
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Accelerated solvent extraction has been regarded as a green technique in plant material
sample preparation prior to chromatographic analysis (Heng et al., 2013; Azmir et al., 2013).
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This technique was introduced by Dionex Corporation in 1995. It is also known as pressurized
liquid extraction, pressurized solvent extraction, and enhanced solvent extraction. Sometime it is
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referred as pressurized hot water extraction, sub-critical water extraction or superheated water
extraction, when water is used as solvent (Mustafa & Turner, 2011). It is an automated rapid
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extraction technique that uses minimal solvent at elevated temperature and pressure. The merit of
using increased temperature is to enhance the solubility and mass transfer of solute to solvent,
and elevated pressure keeps the solvent below its boiling point, enabling fast, safe, and efcient
extraction of target analytes from plant materials into the extraction solvent (Mottaleb & Sarker,
2012). An extraction process is usually completed in 15-25 minutes using only 15-45 ml
consumption of solvent. Therefore, it has been widely applied in the fields of environmental,
food, polymer, and pharmaceutical researches.
ASE comprises of two main set-ups, there are the static and dynamic instruments
(Mustafa & Turner, 2011). Static setup is replacement of the solvent between cycles if the
extraction process consists of one or several extraction cycles. A high pressure pump is required
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to pump the extraction solvent through the sample vessel continuously in the dynamic setup. The
parameters affecting ASE efficiency are temperature, pressure, type and composition of solvents,
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modifiers and additives, matrix composition, and extraction mode (Sun, Ge, Lv, & Wang, 2012).
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The most commonly applied operating temperature and pressure for ASE are 100 C at 1500 psi
(Mottaleb & Sarker, 2012). Although ASE is a green technology in extracting bioactive
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compound from plant material, the application in saponins extraction is still scarce. Table 9
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summarizes extraction of saponins using ASE from plant materials. Worth noted that this method
is used mostly to extract ginsenoside from ginseng (Qian, Lu, Gao, & Li, 2009; Wan, Zhang, Ye,
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& Wang, 2008; Wan et al., 2006; Zhang, Liu, Qi, Li, & Wang, 2013) could be due to the
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The efficacy of ASE in saponins extraction has been studied and compared with other
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extraction methods. A higher saponin yield has been obtained from cow cockle seeds using
accelerated solvent extraction compared to ultrasonic-assisted extraction in pure and aqueous
solvents of ethanol and methanol (Gl-ntnda, Balsevich, & Mazza, 2007). Similarly, the
pressurized hot water system extracted a greater yield of ginsenosides (11.2 mg/g) compared to
ultrasound-assisted method (7.2 mg/g) from Panax quinquefolium (Engelberth et al., 2010).
Although slight increase in saponins yields of escin Ia, escin Ib, isoescin Ia and isoescin Ib were
obtained in extraction from Aesculus chinensis Bunge using ASE, it required shorter extraction
time of 7 minutes compared to reflux and sonication extraction of 1 hour and 30 minutes,
respectively (Chen et al., 2007a). Pressurised liquid extraction showed distinctive advantages of
yielding total amount of saponins of 7.36% over other green extraction methods of ultrasound of
5.77%, and conventional extractions of Soxhlet of 6.99% and maceration of 6.00%, in the
extraction of saponins from Panax notoginseng (Wan et al., 2006).
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6.
Quantification of Saponins
Prior to the quantification of total saponins of a plant source, it is appropriate to carry out a
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simple procedure to test the presence of saponins. This can be done by putting the plant material
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into a test tube filled with distilled water and vigorously shaken for 2 minutes (Ncube et al.,
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2011). The appearance of stable and persistent foam on the liquid surface for 15 minutes
indicated the presence of saponins. The quantification of plant saponins is usually carried out by
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Spectrophotometric method
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Two works (Patel et al., 2012a and Ncube et al., 2011) were found using the conditions of
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60 C and 10 minutes to allow the mixture to have full colour development following procedure
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in Hiai et al. (1976). However, other researchers used 70 C for 15 minutes (Chen et al., 2007b)
and 20 minutes (Li et al., 2010b) to allow full colour development. This inconsistency should be
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standardized because the reaction time may directly attribute to the final absorbance value which
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later translates into quantity. As presented in Table 10, the standards of oleanolic acid,
soyasaponin, Quillaja saponin, and ginsenoside are grouped in triterpenoid saponins, where
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diosgenin and sarsasapogenin are categorized in steroid saponins (Harborne & Baxter, 1999).
Since total saponins method is to quantify total saponins from the reaction of oxidized triterpene
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saponins with vanillin (Li et al., 2010b), these inevitably raise a question whether the selection of
standard to be used in spectophotometeter is essential to express the correct saponins group from
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the plant source. Unfortunately, the related information on selection of the standards is rarely
found. No explanation is stated in previous works on the selection of wavelength, but most
researchers selected wavelength of 544 nm is observed (Table 10). However, the selected
wavelengths are falled within the range of 480-610 nm, except 473 nm (Mostafa et al., 2013) and
283 nm (Liu et al., 2012b), most probably due to the maximum absorption of purple colour falls
within this range (Bruice, 2007).
Despite total saponins, total steroidal sapogenin is employed to quantify specifically the
steroidal saponins content of plant material (Baccou, Lambert, & Sauvaire, 1977). This method
is also based on colour reactions with anisaldehyde (or vanillin), sulphuric acid and ethyl acetate
measured at maximum wavelength (max) of 430 nm (Baccou et al., 1977). The difference
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between total saponin and total steroidal sapogenins is the solvent used to prepare the reagents.
For total saponins, vanillin reagent is prepared by diluting with ethanol and sulphuric acid with
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water, whereas for total steroidal sapogenins, both the anasaldehyde and sulphuric acid are
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diluted with ethyl acetate. Total steroidal sapogenins has been proven stable and reproducible
with a number of standards, ie., diosgenin, tigogenin, hecogenin, smilagenin, yonagenin,
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tokorogenin, etc. without interference from sugars, sterols, fatty acid and vegetable oil (Baccou
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et al., 1977).
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(consists of 0.5 ml anisaldehyde and 99.5 ml ethyl acetate) and 1 ml of reagent C (consists of 50
ml concentrated sulphuric acid and 50 ml ethyl acetate). The test tube with mixtures was placed
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in a water-bath at 60 C for 10 minutes to allow full colour development. Then, it was cooled for
10 minutes at room temperature before measuring at 430 nm using a spectrophotometer. It has
been employed in recent researches to quantify total steroidal saponins from micropropagated
Tulbaghia violacea which obtained 10.03 mg DE/ml (Ncube et al., 2011), and the extract from
defatted rice bran in n-butanol fraction which yielded 5.70 mg DE/g (Chan et al., 2013). Qin et
al. (2009) carried out the total steroidal sapogenin determination of Dioscorea zingiberensis with
some modifications. They used perchloric acid instead of sulphuric acid. The test tube was
placed in a water bath maintained at 70 C for 15 min to develop colour fully, then allowed to
cool for 2 min in 0 C water bath and metered volume to 25 ml with glacial acetic acid. After 30
minutes of stabilization, only then the test tube was subjected to measure its absorbance at 454
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nm using a spectrophotometer and yielded total steroidal saponins of 28.34% (w/w) of
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lyophilized powder.
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the reaction of saponins with blood reagent to release oxy-hemoglobin which results a
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dissolved in distilled water and 100 l of this solution were incubated with 1 ml fresh EDTAblood at 30 C for 30 min. After centrifugation for 10 min, haemoglobin was quantied in the
supernatant photometrically at 545 nm and the result was expressed in haemolytic saponins.
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They revealed that white bitter gourd varieties were found to contain signicantly lower saponin
6.2.
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Chromatograhic method
Saponins are separated and purified from plant materials using chromatographic methods in
many studies to identify a specific saponins compound (Ado et al., 2011; Liu et al., 2012a) and
investigate its pharmaceutical property (Gupta et al., 2010; He et al., 2012; Zheng et al., 2012).
The most common chromatographic methods employed are high performance liquid
chromatography (HPLC) (Bi et al., 2012; He et al., 2012; Liu et al., 2012b; Mostafa et al., 2013)
and thin layer chromatography (TLC) (Ado et al., 2011; Liu et al., 2012a; Patel et al., 2012a).
The chromatographic determination of plant saponins for period from 2002 to 2005 has been
reviewed by Oleszek and Bialy (2006). Therefore, the present review looks into the most recent
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works of chromatographic method specifically in quantitation study of plant saponins where
HPLC was found to be the most commonly used method. A summary of quantification of plant
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saponins using HPLC is presented in Table 11. As noted the quantification of specific saponin
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compound is the primary objective of all the studies which apply HPLC method. The specific
saponins content detected not only serve as a good data reference source to future researchers,
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but as a strong scientific reference to drug-related manufacturer who is interested to process the
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particular plant source further. Besides HPLC, ultra pressure liquid chromatography (UPLC)
(Foubert et al., 2010; Ha et al., 2014; Serventi et al., 2013; Verza et al., 2012) was also employed
Conclusions
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7.
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to quantify saponins.
Saponins are important secondary metabolites derived from various plant sources because
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of its invaluable pharmaceutical properties. This review focuses on two different extraction
techniques (conventional and green technology) employed in previous works to obtain crude
saponins extract prior to further analysis. Moreover, spectrophotometric and chromatographic
methods in saponins quantification are described. This synthesis of the range and diversity of
previous studies already active in the field serves as important information to researchers who
wish to embark a new project. This may provide an overview and quick reference for future labscale experimental design. The knowledge of extraction technique employed in respect to
objective is vital and can be extended to address the rising food processing challenges over time.
After highlighting the lack of green extraction technologies utilization in lab-scale saponins
extraction, more attention should be paid by researchers for these technologies exploration in
future.
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Verza, S.G., Silveira, F., Cibulski, S., Kaiser, S., Ferreira, F., Gosmann, G., Roehe, P.M., &
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Wang, W., Li, P., Wang, X., Jing, W., Chen, L., & Liu, A. (2012). Quantication of saponins
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Wang, Y., Xu, R., Xiao, J., Zhang, J., Wang, X., An, R., & Ma, Y. (2014). Quantitative analysis
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Zheng, T., Shu, G., Yang, Z., Mo, S., Zhao, Y., & Mei, Z. (2012). Antidiabetic effect of total
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List of Figure
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Fig. 1. Current extraction techniques employed in extraction of saponins from plant materials
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Maceration (36%)
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Ultrasound-assisted (14%)
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Reflux (22%)
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Conventional extractions
Fig. 1. Current extraction techniques employed in extraction of saponins from plant materials
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List of Tables
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Table 2. Triterpenoid and steroidal saponins derived from various plant materials
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Table 5. Summary of studies focusing on saponins extraction using reflux and Soxhlet extraction
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Table 6. Summary of studies focusing on saponins extraction using two subsequent extractions
Table 7. Summary of studies focusing on saponins extraction using ultrasound-assisted
extraction
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Reference(s)
Seed
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Root
Entada phaseoloides
Leguminous species
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Plant part
Glycyrrhiza inflate,
Glycyrhiza glabra,
Glycyrrhiza uralensis
Glycyrrhiza yunnanensis
Gypsophila trichotoma
Maesa lanceolata
Medicago hybrid
Momordica charantia
Paris polyphylla var.
chinensis
Paris polyphylla var.
yunnanensis
Pulsatilla chinensis
Panax notoginseng
Panax quinquefolius
Platycodon grandiorum
Polygonatum odoratum
Radix Astragali
Vigna radiata L.
Yucca gloriosa L.
Leaf
Acanthopanax sessiliflorus
Allium nigrum L.
Ji et al. (2014)
Voutquenne-Nazabadioko et al. (2013)
Theunis et al. (2007)
Bialy et al. (2006)
Chen et al. (2008)
Liu et al. (2013); Zhang et al. (2010)
Zhang et al. (2010)
Xu et al. (2011)
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Tuber
Flower
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Pericarp
Bark
Stem
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Fruit
Tribulus terrestris L.
Vigna radiata L.
Ziziphus jujube, Z. jujuba
var. spinosa
Allium nutans L.
Allium nigrum L.
Crocus sativus
Gleditsia sinensis Lam.
Momordica charantia
Solanumxanthocarpum
Tribulus terrestris L.
Caryocar villosum
Momordica charantia
Silphium asteriscus L.
Tribulus terrestris L.
Vigna radiata L.
Sapindus mukorossi
Yucca schidigera Roezl
Harpullia austro-caledonica
Ipomoea batatas
Agave offoyana
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Bulb
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Antonia ovate
Beaucarnea recurvata
Cordyline fruticosa
Gymnema sylvestre
Maesa lanceolata
Silphium asteriscus L.
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Table 2. Triterpenoid and steroidal saponins derived from various plant materials
Reference(s)
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Plant material
Triterpenoid saponins
Antonia ovata
Aralia taibaiensis
Bacopa monnieri
Caryocar villosum
Momordica charantia
medicago truncatula
medicago arabica L.
medicago hydriba
Caulophyllum thalictroides
Chenopodium quinoa
Chiococca alba
Flos Lonicerae
Genista ulicina Spach
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Skhirtladze et al. (2011)
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CE
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Yucca gloriosa L.
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Quantification studies
Maceration
Processing
parameters/optimization
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AC
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Conventional
extractions:
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Saponins isolation
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Subsequent
methods
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Soxhlet
Ultrasoundassisted
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Green
extraction
techniques:
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Micowaveassisted
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ED
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AC
Accelerated
solvent
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Xanthoceras sorbifolia
Bunge (Li et al. 2010b)
Dioscorea zingibernsis (Li et
al. 2010a)
Panax ginseng
(Vongsangnak et al. 2004)
Bupleurum chinense (Hu et
al. 2008)
Gymnema sylvestre (Mandal
& Mandal 2010)
Aesculus chinensis Bunge
(Chen et al. 2007a)
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Table 4. Summary of studies focusing on saponins extraction using maceration extraction
Pretreatment and extraction condition
1.16
kg/5 L
methanol
72 hours
Room
temperature
10 kg/ -
70%
ethanol
1 kg/10
L
80%
methanol
30 g/150
ml
-.
SC
Ado et al.
(2011)
Three novel
cholestane-type
steroidal glycosides
and three known
steroidal glycosides
saponins have been
isolated and
identified.
Bai et
(2014)
Six triterpene
saponins have been
isolated.
Boutaghane
et al. (2013)
3 hours
60 C
Cheng et al.
(2011)
Ethanol
extract
partitioned with nbuthanol showed
the highest antidiabetic potential.
Two new saponins
have been isolated
and identified.
Spectrophotometric
method detected
yield: 200.01
mg/100 g. HPLCDAD: saponin1:
161.20 mg/100 g;
saponin 2: 14.67
mg/100 g.
The antioxidant
activities tested of
total phytochemical
fraction and of
single saponins
were moderate in
relation to
commercial
standards.
Deng et al.
(2012)
NU
To investigate
the
antiproliferation
and apoptosis
mechanism of
saponin and
flavonoid
fractions of
Gynostemma
pentaphyllum.
Powder
Polygonatum
odoratum
(Mill.) Druce
To characterize
the anti-diabetic
active fractions
in this herb.
Dried
5 kg/40
L
80%
ethanol
2 hours
80 C
Ipomoea
batatas tubers
To isolate,
identify and
quantify
triterpenes
glycosides.
To evaluate
their antioxidant
properties.
Flour
6 g/ -
80%
methanol
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Identified a new
saponins.
Showed haemolytic
effects in vitro and
demonstrated antiinammatory
activity and
gastroprotective
property in vivo.
Boiling point
2 hours
Gynostemma
pentaphyllum
ethanol
Reference
Mechanical
aid
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Extraction
Temperature
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To isolate
steroidal
saponins.
Extraction
duration
Polygonatum
odoratum
Solvent
used
Air-dried
powder
To isolate a
new steroidal
saponin.
To investigate
its antiinflammatory
and
antiulcerogenic
properties in
vitro and in
vivo.
Solid/
solvent
Air-dried
roots
Allium
ampeloprasum
var. porrum
State of
input
material
Fresh
bulbs
Outcome(s)
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Objective(s)
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Plant
material(s)
Shaking
62
al.
Dini et al.
(2009)
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To isolate new
steroidal
saponins and
investigate their
cytotoxic and
antimicrobial
activity.
Dried
pulverize
d leaves
3 kg/ -
methanol
24 hours
Panax
quinquefolius
To investigate
the extraction
efficiency of
three solvent
systems on
triterpene
saponins from
root of Panax
quinquefolius.
Dried
roots
1:5
50%
ethanol; a
mixture
of 20%
ethanol,
40%
glycerin,
and 40%
water; or
65%
glycerin
6 weeks
Momordica
charantia
To investigate
antileishamanial
activity of the
aqueous extract
in vitro and in
vivo.
Air dried
5 kg/ -
To isolate and
identify
trinocucurbitane
and cucurbitane
triterpenoids
from roots.
To investigate
the anti-HIV
activity.
Air-dried
To quantify the
saponin extract,
the lipid extract,
and the
hydrophilic
extract in
bittergourd
varieties.
Powder
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48 hours
Room
temperature
1.9 kg/5
L
methanol
6 hours
60 C
10 g/
150 ml
Ethyl
acetate
2 hours
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water
63
Fouedjou et
al. (2014)
Gafner et al.
(2004)
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Cordyline
fruticosa
Gupta et al.
(2010)
Chen et al.
(2008)
Habicht et
al. (2011)
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extract contained
high amounts of
conjugated linoleic
and linolenic acids
(up to 65.89%).
To characterize
saponins of nine
different species
of Leguminous
using UPLC.
Dried
pulverise
d seeds
-/10 ml
80%
methanol
24 hours
25 C
A total of twenty
saponins
were
characterized.
Ha et
(2014)
Sapindus
mukorossi
To investigate
the
molluscicidal
effects of
saponins from
Sapindus
mukorossi
against the
golden apple
snail.
Powder
35.5 g/ -
methanol
72 hours
Bioassay
data
revealed that the
seven
isolated
saponins
from
soapnut
were
molluscicidal,
causing 70-100%
mortality at 10 ppm
against the golden
apple snail.
Huang et al.
(2003)
Medicago
truncatula
To quantify
saponins in M.
truncatula root,
leaves, stem,
seedpod and
seeds.
Lyophili
zed
powder
10
mg/0.5
ml
80%
methanol
2 hours
Room
temperature
Shaking
Roots (5924
ng/mg/dw)
contained the
greatest total
amount of saponins
followed by leaf
(1064 ng/mg/dw)
and seed (991
ng/mg/dw),
respectively.
Huhman et
al. (2005)
Glycyrrhiza
yunnanensis
To isolate
triterpene
saponins.
Powder
18 kg/30
L
70% and
95%
ethanol
2 hours
Ji et
(2014)
al.
Panicum
virgatum L.
To isolate,
characterize,
and quantify of
steroidal
saponins in
Switchgrass
(Panicum
virgatum L.)
Dried
100
mg/5 ml
methanol
30 minutes
Shaking
Three types of
steroidal saponins
have been isolated
from 4 types of
Switchgrass
variety. Differences
in the relative
concentrations of
different saponins
were observed
between
switchgrass
cultivars and plant
parts.
Lee et
(2009)
al.
Freezedried
40 g/1 L
methanol
24 hours
25 C
Results showed
that the contents of
saponins were
decreased along
with increasing
cooking
temperature and
time except for the
steaming treatment.
None of the
steamed yam slices
Lin et
(2006)
al.
AC
CE
P
TE
MA
NU
SC
RI
PT
Leguminous
species
Dioscorea
To investigate
pseudojaponica
the effects of
Yamamoto
domestic
processing on
steroidal
saponins and
furostanol and
spirostanol
glycosides.
64
al.
ACCEPTED MANUSCRIPT
Freezedried
600 g/6
L
methanol
24 hours
Room
temperature
Commin
uted
1 kg/8 L
75%
ethanol
1 hour
80 C
Silphium
asteriscus L.
To isolate
saponins and
investigate their
antiproliferative
activity.
Dried
leaves
and
stems
500 g/ -
CH2Cl2methanol,
methanol,
50%
methanol
48 hours
Solanum
xanthocarpum
To establish the
scientic
rationality of
Solanum
xanthocarpum
fruit saponin
rich fraction on
antiurolithiatic
activity using in
vitro calcium
oxalate (CaOx)
crystallization
and in vivo
ethylene glycol
induced
urolithiasis in
the male albino
Wistar rats.
Fruit
powder
100 g/
500 ml
SC
NU
TE
MA
Rhizoma
Anemarrhenae
RI
To isolate and
identify
steroidal
saponins.
To investigate
the cognitionenhancing
effects of total
saponins.
PT
significantly
change their initial
compositions or
quantities of
furostanol and
spirostanol
glycosides.
50%
ethanol
24 hours
AC
CE
P
Dioscorea
zingiberensis
To determine
the biochemical,
hematological
and
histopathologica
l toxicity of
steroidal
saponins from
Dioscorea
zingiberensis
rhizome after
Yang et al.
(2003)
Eighteen saponins
with highly
hydroxylated
oleanane-type
aglycones were
isolated. The
isolated
compounds showed
antiproliferative
activity against
human epitheloid
cervix carcinoma,
leukaemic T-cell
line, and colorectal
adenocarcinoma.
Masullo et
al. (2014)
The antiurolithiatic
activity in Solanum
xanthocarpum is
mediated possibly
through the
inhibition of CaOx
crystal formation
and its effect on the
urinary
concentration of
stone-forming
constituents and
nephrolithiasis
inducing factors.
Patel et al.
(2012a)
Liu et
(2012b)
al.
Dried
powder
0.5 kg/ -
70%
ethanol
48 hours
Room
temperature
Prez et al.
(2013)
Air-dried
600 g/2
L
ethanol
12 hours
Room
temperature
The steroidal
saponins did not
show any sign of
toxicity up to oral
dose of 562.5
mg/kg in mice. No
signicant changes
of biochemical and
hematological
parameters in rats
(except at 510
Qin et
(2009)
65
al.
ACCEPTED MANUSCRIPT
acute oral
dosing in mice
and sub-chronic
oral
administration
in rats.
70%
ethanol
Powder
200 g/ -
80%
methanol
Room
temperature
1 g/100
ml
70%
methanol
PT
3.5 kg/ -
To investigate
the effect of
high and low
saponin lines of
alfalfa on pea
aphid
performance.
To evaluate the
differences in
saponins within
the studied lines
and estimate an
inuence of
some isolated
compounds on
the pea aphid
probing
behavior.
Freezedried
Chenopodium
quinoa
Seeds
To investigate
the
immunoadjuvan
t activity,
toxicity assays.
To determine
triterpenic
saponins using
UPLC/Q-TOFMS.
Dried
To isolate and
characterize of
twelve
Dried
1 hour
60 C
Shaking
AC
CE
P
TE
Medicago
sativa L.
MA
NU
SC
Dried
RI
To isolate five
steroidal
saponins
mg/kg/day).
100 g/ 1
L
40%
hydroetha
nol
solution
1 hour
4 kg/ -
90%
ethanol
3 days
50 C
Stirring
66
Room
temperature
Five steroidal
saponins have been
isolated and
identified.
Five steroidal
glycosides
including three
spirostane, one
furostane and one
cholestane
glycosides, along
with seven known
compounds have
been isolated and
characterized, The
dried extract
obtained more than
25% w/w of
glycosides.
Zhang et al.
(2013a)
The high-saponin
line of alfalfa
differed from the
low-saponin one by
the presence of
zanhic acid
tridesmoside and a
higher level of 3GlcA,28AraRhaXyl
medicagenic acid
glycoside.
The saponins
incorporated into
sucroseagarose
gels signicantly
reduced number of
the aphid probes
into the gels and
extended their
duration in
comparison to the
control gels
(without tested
compounds).
Sylwia et al.
(2006)
Verza et al.
(2012)
Twelve triterpene
saponins have been
isolated, and their
Zhu et
(2002)
Skhirtlardze
et al. (2011)
al.
ACCEPTED MANUSCRIPT
structures were
characterized on
the basis of
hydrolysis and
spectral data,
especially NMR
evidence.
PT
triterpene
saponins.
Harpullia
austrocaledonica
To isolte
triterpenoid
saponins.
Dried
stem
bark
powder
1.110
kg/10 L
20%
methanol
17 hours
and boiled
3 hours
Fagonia indica
To isolate a
novel saponin
glycoside.
To perform the
apoptosis and
necrosis test on
three cancer cell
lines.
Dried
powder
200 g/1
L
ethanol
14 days
Room
temperature
Panax
notoginseng
To investigate
the mechanisms
of antihyperglycemic
and anti-obese
effects of Panax
notoginseng
saponins (PNS)
in KK-Ay mice.
Powder
75 g/1 L
75%
ethanol
4 hours
Salicornia
herbacea
To isolate
triterpene
saponins and
investigate their
antiproliferative
activity.
Fresh
parts
22.6 kg/
-
80%
acetone
24 hours
Ambient
temperature
Entada
phaseoloides
L.
To evaluate the
potential
therapeutic
effects of total
saponins from
Entada
phaseoloides
(TSEP) in
experimental
type2 Diabetes
mellitus
(T2DM) rats.
Dried
powder
70%
ethanol
2 hours
RI
NU
SC
AC
CE
P
TE
MA
67
Voutquenne
et al. (2005)
A novel steroidal
saponin has been
isolated.
It was able to
induce apoptosis or
necrosis in cancer
cells depending on
the cell type.
Waheed et
al. (2012)
Yang et al.
(2010)
Two new
noroleanane-type
triterpene saponins,
Salbige A and
Salbige B, have
been isolated. Both
compounds
exhibited potent
antiproliferative
activities against
cancer cells.
Total saponins
demonstrated both
hypoglycemic and
hypolipidemic in
type 2 diabetic rats
which indicating
possess antidiabetic property.
Zhao et al.
(2014)
Zheng et al.
(2012)
ACCEPTED MANUSCRIPT
Table 5. Summary of studies focusing on saponins extraction using reflux and Soxhlet extraction
Objective(s)
State of
input
material
Solid/
solvent
solvent used
duration (hr)
Reflux
extraction:
To isolate
triterpenoid
saponins
Dried
powder
400
g/3.5 L
methanol
Allium nutan L.
Isolation of four
steroidal
saponins from
underground
parts of A.
nutans.
Oven-dried
powder
300
g/500
ml
80%
methanol
Aralia
taibaiensis
To isolate four
new triterpenoid
saponins.
To investigate
the four newly
derived saponins
on antioxidant
and antiglycation
activities.
Air-dried
powder
1.2 kg/1
L
Defatted rice
bran
To fractionate
with different
solvent systems.
To investigate
the antioxidant
activity.
Oven-dried
Flos Lonicerae
To determine
triterpenoid
saponins in five
specicies of Flos
Lonicerae both
qualitatively and
quantitatively.
Xanthoceras
sorbifolia
To isolate and
quantify saponins
from parts of
husks, twig bark
and xylem, seed
coats and
kernels, flowers,
and leaves.
Alabdul
Magid et al.
(2006)
Four steroidal
glycosides including
deltoside and
nolinofuroside D and
two novel saponins
were isolated.
Akhov et al.
(1999)
70% ethanol
Bi
et
(2012)
al.
10
g/150
ml
50% ethanol
Chan et
(2013)
al.
Oven-dried
pulverized
2.0 g/
25 ml
60% ethanol
Chai et
(2005)
al.
Dried
1 kg/1
L
70% ethanol
Ling et
(2011)
al.
Oven-dried
powder
-/250
ml
70% ethanol
Liu et
(2013)
al.
NU
MA
D
TE
AC
CE
P
Reference
Seven triterpenoid
saponins have been
isolated and identified.
SC
Caryocar
villosum
Finding(s)
PT
Plant material(s)
Conditions
RI
Extraction
68
ACCEPTED MANUSCRIPT
Momordica
charantia
50% ethanol
Saponins of Paris VI
was found majorly in
Paris fargesii (19.520
mg/g), and Trillium
tschonoskii (11.770
mg/g).
Crude saponin-rich guar
meal extract was
puried by RP-HPLC
eluting 20%, 60% and
100% methanol
fractions with 2.04
0.32%, 0.91 0.16%
and 1.55 0.15% DM
of crude saponin-rich
GM extract,
respectively.
Results indicated that
only 100% methanol
fraction and its 16 min
peak sub-fraction
exhibited both
haemolytic and
antibacterial activities
against Staphylococcus
aureus, Salmonella
Typhimurium and
Escherichia coli, but
20% and 60% MeOH
fractions stimulated
Lactobacillus spp.
growth.
Man et
(2010b)
RI
25
g/250
ml
1.5
SC
80% ethanol
MA
Ground
0.5 g/50
ml
NU
Oven-dried
powder
AC
CE
P
Guar meal
To determine
major saponins in
Paris and
Trillium plant
qualitatively and
quantitatively.
To isolate
saponin rich
extract.
To evaluate its
fractions for
haemolytic and
antibacterial
activities against
two gram
positive bacteria
(Lactobacillus
spp. and
Staphylococcus
aureus) and two
gram negative
bacteria
(Escherichia coli
and Salmonella
Typhimurium) in
a dose dependent
manner.
TE
Paris and
Trillium plants
kinds of saponins
inuenced the
mechanism and
cytotoxicity. Also the
results of the cytotoxic
and hemolytic activities
of plants suggested that
the existed correlation
of the steroidal saponins
affected the activities.
PT
saponins.
al.
Hassan et al.
(2010)
Dried
pieces
1 kg/4
L
60% ethanol
He et
(2012)
al.
To investigate
the chemical
constituents from
fresh fruit.
Fresh fruits
3 kg/24
L
90% ethanol
1.5
Liu et
(2012a)
al.
To isolate and
identify new
triterpenoid
saponins.
To investigate
Air-dried
powder
30
kg/100
L
ethanol
Identified
fourteen
cucurbitane
triterpenoids.
Exhibited weak antiHIV activity.
Chen et
(2009)
al.
69
ACCEPTED MANUSCRIPT
their anti-HIV
activity in vitro.
methanol
Saponins containing
extract reduced
preadipocyte
proliferation and lipid
accumulation of the
adipocyte.
Popovich
al. (2010)
et
200
mg/10
ml
1.5
Oleszek
(1998).
methanol
Qi et al.
(2006)
30%
methanol
NU
Freezedried
powder
SC
RI
PT
TE
Lyophilized
pieces
MA
To investigate
two classes of
saponins, ie.,
cucurbitane and
oleanane type
triterpenoids, for
the potential to
reduce
preadipocyte
viability, lipid
accumulation and
adiponectin
expression in
3T3-L1 cells.
Radix Astragali
To isolate four
main saponins.
Bupleurum
chinense
To evaluate the
haemolytic
activities of
Bupleurum
chinense
saponins and its
adjuvant
potentials on the
immune
responses of ICR
mice against
ovalbumin.
Powder
1 kg/ -
70% ethanol
Glycyrrhiza
inflate,
Glycyrhiza
glabra,
Glycyrrhiza
uralensis
To isolate and
quantify
triterpenoid
saponins from
species of
Glycyrrhiza.
Dried
powder
0.5 g/30
ml
50%
methanol
Tao et
(2013)
Maesa
lanceolata
To determine
saponins using
LC-UV method.
Dried
powder
1.5 g/70
ml
50%
methanol
Compounds of oleanane
type triterpenes were
identified.
Theunis et al.
(2007)
Polygala
japonica
To isolate and
quantify
triterpenoid
saponins.
Dried
powder
1.0 g/50
ml
methanol
Six triterpenoid
saponins have been
isolated. The total
saponins found were
4.45-27.32 mg/g.
Wang et al.
(2007)
AC
CE
P
Oven-dried
powder
1.5 g/60
ml
70
Sun (2006)
al.
ACCEPTED MANUSCRIPT
Air-dried
powder
10
kg/80 L
Hot water
To identify and
determine
biological
activities of
triterpenoid
saponins.
Dried
powder
900 g/ -
petroleum
ether,
methanol
Yoshizumi et
al. (2006)
72
Woldemichael
&
Wink
(2001)
24
Saponins produced by
mungbean plants added
to the soil enhanced the
growth
of
new
mungbean plants as an
allelochemical
plant
growth regulator.
Waller et al.
(1999)
MA
NU
SC
Soxhlet
extraction:
Chenopodium
quinoa seed
PT
To isolate four
saponins.
To determine
their inhibitory
activities on
pancreatic lipase
in vitro and in
vivo.
RI
Acanthopanax
sessiliflorus
leaves
Chloroform,
80% ethanol
TE
Fresh plant
AC
CE
P
71
ACCEPTED MANUSCRIPT
Table 6. Summary of studies focusing on saponins extraction using two subsequent extractions
Objective(s)
Pretreatment/Procedures
Finding(s)
References
Soxhlet reflux
al.
al.
Bialy et
(2004)
Medicago
hybrid roots
Fourteen triterpene
saponins have been
isolated.
Bialy et
(2006)
Yucca
schidigera
Roezl
Oleszek et al.
(2001)
Tava et
(2009)
SC
NU
MA
TE
Medicago
To acquire further evidence of the
arabica (L.)
previously reported compounds in
leaves
M. arabica, and elucidate the
chemical structure of these newly
identied saponins.
RI
Medicago
Arabia L.
PT
Plant
materials
al.
Maceration reflux
Six pentacyclic
triterpenoid saponins,
named antoniosides EJ
along with two known
alkaloids, were isolated.
Inhibited the growth of
oral epithelium carcinoma
cell.
Alabdul Magid
et al. (2012)
Tribulus
terrestris L.
Dinchev et al.
(2008)
Medicago
truncatula
Kapusta et al.
(2005)
AC
CE
P
Antonia
ovata leaves
72
ACCEPTED MANUSCRIPT
A chemical ngerprint
method was rstly
established and validated
to quantify and
standardize rhizomes
including parvioside,
protodeltonin,
protodioscin,
protogracillin,
zingiberensis saponin,
deltonin, dioscin and
trillin.
The results indicate that
total steroidal saponins
could inhibit thrombosis
by both improving the
anticoagulation activity
and inhibiting platelet
aggregation action,
suggesting that total
steroidal saponins from
Dioscorea zingiberensis
rhizomes have the
potential to reduce the risk
of cardiovascular diseases
by antithrombotic action.
Li et al. (2010a)
Extracts of
micropropagated plants
and outdoor grown plants
showed good antibacterial
activity in vitro.
Ncube
(2011)
PT
TE
AC
CE
P
Tulbaghia
violacea
Soxhlet maceration
MA
NU
SC
RI
Dioscorea
To characterize the major active
zingiberensis
constituents.
To evaluate the anti-thrombotic
activity.
73
et
al.
ACCEPTED MANUSCRIPT
Findings
Reference
Avula
(2011)
et
al.
RI
Pretreatment/Extraction
procedure
Dry plant samples were weighed
and sonicated in 2 ml of methanol
for 30 minutes.
NU
SC
Plant
Objectives
material(s)
Caulophyllum To compare
thalictroides
chromatographic
performance of HPLC and
UPLC in determining
saponins from the roots.
PT
Borges
(2013)
et
al.
Maesa
lanceolata
Foubert
(2010)
et
al.
Bacopa
monnieri
TE
Ganzera et al.
(2004)
Ha et al. (2006)
AC
CE
P
Ziziphus
To perform simultaneous
jujuba,
qualitative and quantitative
Z.jujuba var.
analysis in the leaves.
spinosa
Platycodi
Radix
MA
Chiococca
alba
74
ACCEPTED MANUSCRIPT
minutes.
Li et al. (2005)
MA
NU
SC
RI
PT
Panax
notoginseng
Gleditsia
To determine the contents
sinensis Lam.
of saponins.
AC
CE
P
TE
Soy
and To investigate the stability
chickpea
during breadmaking and in
vitro bioaccessibility of
saponins from soy and
chickpea.
Serventi
(2013)
et
al.
et
al.
Allium
nigrum L.
Mostafa
(2013)
Dioscorea
panthaica
Wang
(2012)
75
et
al.
ACCEPTED MANUSCRIPT
TE
MA
NU
SC
RI
AC
CE
P
Paris
polyphylla
var.
yunnanensis,
Paris
polyphylla
var.
chinensis
37435.1 g/g.
PT
76
Zhang
(2010)
et
al.
ACCEPTED MANUSCRIPT
Finding
To develop a novel
MAE method, and
to evaluate MAE
and conventional
extraction
techniques for the
extraction of
triterpenoid
saponins from
Ganoderma atrum.
Panax
notoginseng
To compare the
efficiency of MAE
with the
conventional
extraction methods
Reference
Chen et al.
(2007b)
Kwon et al.
(2003)
Vongsangnak
et al. (2004)
PT
Objectives
AC
CE
P
TE
MA
NU
SC
RI
Plant
material(s)
Ganoderma
atrum
To find an efficient
extraction method
and optimize the
MAE for saponin
from cultured cells
of Panax
notoginseng.
To evaluate the
efficiency of high
pressure MAE as a
fast extraction of
ginsenosides from
Panax notoginseng.
77
ACCEPTED MANUSCRIPT
the extraction was carried out continuously at the
preset pressure.
Xanthoceras
sorbifolia
Bunge.
kernel
To optimize the
microwave-assisted
extraction
procedure for
triterpene saponins
from the defatted
residue of yellow
horn kernel.
Dioscorea
zingibernsis
Gymnema
sylvestre
To evaluate the
applicability of
MAE on oleanolic
acid extraction from
Gymnema sylvestre
leaves.
Pulsatilla
turczaninovii
To develop a
method for
quantifying and
Hu et
(2008)
al.
Li
et
(2010b)
al.
An improved MAATPE
Li
et
(2010a)
al.
Mandal
Mandal
(2010)
&
Xu et
(2012)
al.
PT
AC
CE
P
TE
MA
NU
SC
RI
To study the
relationships
between the
extraction yields of
saikosaponins and
the operating
parameters of
MAE.
Bupleurum
chinense
78
ACCEPTED MANUSCRIPT
PT
SC
RI
TE
MA
To compare six
different extraction
methods, i.e.,
maceration,
Soxhlet, heat reflux
extraction,
ultrasonic
extraction,
microwave
extraction, and
accelerated solvent
extraction, on
oleanolic acid and
ursolic acid from
Lamii albi flos.
AC
CE
P
Lamii albi
flos
NU
comparing the
triterpenoid
saponins in nine
different seasonal
batches of P.
turczaninovii from
the Liaoning
province.
79
WjciakKosior et al.
(2013)
ACCEPTED MANUSCRIPT
Plant
material(s)
Aesculus
chinensis
Bunge
Objectives
Pretreatment/Extraction conditions
To identify and
quantify four major
saponins using HPLC
method.
To introduce a new
extraction process,
accelerated solvent
extraction, to optimize
the extraction yield.
Folium
Ginseng and
Radix Ginseng
To develop a rapid
pressurized liquid
extraction and rocket
column HPLC
analysis method for
the determination of
one avonoid
(panasenoside), nine
saponins of
ginsenoside and two
polyacetylenes
(panaxydol and
panaxynol) in Folium
Ginseng and Radix
Ginseng.
Panax ginseng
To quantify and
separate
protopanaxatrial and
protopanaxadiol
saponins with
macroporous resins.
To determine nine
types of saponins
from Panax ginseng.
To compare the
extraction efficiency
of methods
pressurized liquid,
ultrasonication, and
Soxhlet extraction.
To demonstrate a
novel simpler and
faster three-stage
temperature gradient
ASE coupled with
HPCCC for the
systematic extraction
and online separation
of saponins with a
broad range of
polarity from the raw
plant materials.
PT
Reference
Chen et al.
(2007a)
Qian et al.
(2009)
Wan et al.
(2008)
Wan et al.
(2006)
Zhang et al.
(2013b)
AC
CE
P
TE
MA
NU
SC
RI
80
ACCEPTED MANUSCRIPT
AC
CE
P
TE
MA
NU
SC
RI
PT
plant P. notoginseng.
81
ACCEPTED MANUSCRIPT
Table 10. Spectrophotometric method in quantifying total saponins from various plant materials
0.2ml of 5%
vanillin-acetic
acid + 1.2ml
perchloric acid.
70 C, 15 min
0.5ml 8%
vanillin
in ethanol + 5
ml of 72%
suiphuric acid
in water.
60 C, 20 min
0 C, 5 min
Xanthoceras
sorbifolia Bunge
kernel
0.2ml 5%
vanillin-acetic
acid + 1.2ml
70% perchloric
acid
70 C, 20 min
Running water, 2
min
Bryonia Laciniosa
0.5ml 8%
vanillin + 5ml
72% sulphuric
acid
60 C, 10 min
Allium nigrum L.
Tulbaghia violacea
soymilk
Selected
wavelength
(nm)
Oleanolic acid
Oleanolic acid
Type of
spectrophotometer
PT
Ganoderma atrum
Standard used
550
References
Chen et al.
(2007b)
544
550
Unico 2100
Spectrophotometer
(Shanghai Unico
Equipment Co. Ltd.,
China
The optimum
microwave-assisted
extraction parameters of
51 C, 7 min, 900 W, 32
ml/g, 42% ethanol and 3
cycles yielded the
highest extraction of
triterpene saponins
reached 11.62 0.37% of
defatted kernel.
Li et al. (2010b)
538
MA
Oleanolic acid
15 g/mg
Patel et al.
(2012b)
PT
ED
CE
Yield obtained
Double beam
RI
Cooling
conditions before
UV/vis
measurement
Running water, 2
min
SC
Conditions to allow
full colour
development
NU
Reagents
mixture
AC
Plant material
Oleanolic acid
diosgenin
diosgenin
473
U-2001
Spectrophotometer
(Hitachi, Tokyo,
Japan)
Mostafa et al.
(2013)
250l of
vanillin reagent
(8g/100ml
ethanol) +
2.5ml of 72%
sulphuric acid.
60 C, 10 min
diosgenin
544
Ncube et al.
(2011)
0.5ml of 8%
60 C, 10 min
Ice water
Soyasaponin
544
multidetection
82
ACCEPTED MANUSCRIPT
microplate reader
(SynergyTM HT,
BIO-TEK, GA,
USA).
Soyasaponin
Seeds of Achyranthus
aspara, Tribulus
terrestris, and Albizia
lebbeck
Rhizoma
anemarrhenae
Roots of Panax
quinquefolium and
Panax ginseng
544
0.25 ml of
vanillin reagent
(8%, w/v in
99.9% ethanol)
+ 2.5 ml of 72%
(v/v) sulphuric
acid.
60 C, 10 min
Quillaja saponin
6.0ml of H2SO4
60 C, 30 min
Cool water
0.2ml 5%
vanillin-acetic
acid + 0.8ml
perchloric acid
60 C, 15 min
544
sarsasapogenin
283
Purity 65.3%
ginsenoside
560
Wu et al. (2001)
AC
CE
PT
ED
MA
NU
SC
RI
PT
vanillin solution
(in ethanol), and
5mL of 72%
sulfuric acid.
83
ACCEPTED MANUSCRIPT
Column
Caulophyllum
thalictroides
Waters
Alliance, 996
photodiode
array
Agilent 1100, -
Phenomenex LC18
guard
Solvent system
detection
Ammonium acetateacetonitrile
Zorbax SB C18
Acetonitrile-acetic acid
Agilent 1300,
DAD 1300
diode array
Agilent 1100,
UV-Vis diode
array
Zorbax SB C18
Agilent 1100
series, G1315B
photodiode
array
Waters
Associates, -
Gemini C18
HwelettPackard 1100
series,
Photodiode
SC
Water-acetic
acid/methanolacetonitrile-acetic acid
Acetonile
/0.1%phosphoric acid
0.1%formic acid
solution/acetonitrile
Tribulus
terrestris L.
Dinchev et al.
(2008)
Ipomoea batatas
tubers
Hewlett-Packard HP5
n-BuOH/HOAc/H2O
HPLC-DAD:
Saponin1: 161.20 mg/100g dw;
Saponin2: 14.67 mg/100g dw
HPLC-MS:
MA
PT
ED
Gynostemma
pentaphyllum
Sinochrom ODS-BP
C18
CE
Aesculus
chinensis
AC
Defatted rice
bran
Reference
RI
NU
Flos Lonicerae
Saponins yields
PT
Source
84
ACCEPTED MANUSCRIPT
array
Waters
Alliance, 996
photodiode
array
Luna C8(2)
Water-methanol
Seeds of
Vaccaria
segetalis Garcke,
Saponaria
Vaccaria L.,
Vaccaria
pyramidate
Phasedus
vulgaris L.
Zorbax SB-C18
Agilent 110,
diode array
Zorbax SB-Aq
Ganzera et al.
(2004)
PT
ED
MA
NU
SC
RI
PT
Bacopa monnieri
Trifluoroacetic acid
solution/acetonitrile
CE
Platycodi Radix
Waters
Alliance,
photodiode
array
Hitachi L-6200,
-
Gl-ntnda et
al. (2007)
Guajardo-Flores et
al (2012)
AC
Ziziphus jujuba,
Z.jujuba var.
spinosa
Sunfire C18
Acetonitrile/0.2%
acetic acid
Water/acetonitrile
Ha et al. (2006)
85
ACCEPTED MANUSCRIPT
Water-acetonitrile
Huhman et al.
(2005)
Panicum
virgatum L.
-, Thermo
Finnigan LCQ
ion trap mass
spectrometer
Betasil C-8
1%formic acidacetonitrile
Dioscorea
zingiberensis
Shimadzu,
Photodiode
array
Shim-pack VP-ODS
C18
Water/acetonitrile
Li et al. (2010a)
Gleditsia
sinensis Lam.
Agilent 1100,
UV
ODS-2 Hypersil
Acetonitrile-0.1%
acetic acid/water-0.1%
acetic acid
Xanthoceros
sorbifolia
Shimadzu 2010
series, -
Diamonsil C18
Methanol/0.05% formic
acid
Paris and
Agilent 1200,
Kromasil RP-C18
Water/acetonitrile
PT
-, photodiode
array
AC
CE
PT
ED
MA
NU
SC
RI
Medicago
truncatula
86
ACCEPTED MANUSCRIPT
Allium nigrum L.
-, Diode array
AQUASIL SS-1251120
0.005% trifluroacetic
acid buffer (TFA)
Folium ginseng,
Radix ginseng
Prevail C18
Water-acetonitrile
Yucca gloriosa
L.
Agilent Series
1200, Diode
array
Agilent 1100,
UV
Maesa
lanceolata
Agilent 1100,
Diode array
300 monomeric
C18
Asparagus
Waters Alliance,
Mediterranea Sea18
PT
ELSD
Mostafa et al.
(2013)
SC
RI
Trillium plants
Water-0.05%
triflouroacetic
acid/acetonitrile-0.05%
triflouroacetic acid
Skhirtladze et al.
(2011)
0.05%HCO2H/CH3CN0.05%HCO2H
Theunis et al.
(2007)
Water-1%formic
acid/acetonitrile1%formic acid
Vzquez-Castilla
et al. (2013)
AC
CE
diode array
PT
ED
MA
NU
Polygala
japonica
Shimadzu, ELSD
Discovery C18
Acetonitrilemethanol/0.05%
trifluoroacetic acid
Panax
notoginseng
Dynamic
Extractions
Spectrum,
photodiode
Acetonitrile/0.05%
phosphoric acid in
water
Zhang et al.
(2013b)
87
ACCEPTED MANUSCRIPT
Agilent 1100,
UV
Zorbax SB C18
Acetonitrile/0.001%
formic acid
Agilent 1100,
ELSD
Kromasil RP-C18
Li et al. (2005)
RI
Waters Symmetry
C18
CE
Water-0.1% formic
acid/acetonitrile
AC
Paris polyphylla
var. yunnanensis,
Paris polyphylla
var. chinensis
PT
ED
MA
NU
SC
Agilent 1100,
photodiode
array
PT
array
88
ACCEPTED MANUSCRIPT
Highlights
PT
RI
SC
NU
MA
D
TE
Plant materials contain saponins and its pharmaceutical properties are highlighted.
Presented an overview of extraction techniques employed in different field of research
focus.
Review on conventional and green extraction techniques used in saponins extraction.
Spectrophotometric and chromatographic quantification methods for saponins are
synthesized.
AC
CE
P
89