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Extraction and quantification of saponins: A Review

Choon Yoong Cheok, Hanaa Abdel Karim Salman, Rabiha Sulaiman

PII: S0963-9969(14)00074-X

DOI: doi:10.1016/j.foodres.2014.01.057

Reference: FRIN 5055

To appear in: Food Research International

Received date: 1 October 2013

Accepted date: 19 January 2014

Please cite this article as: Cheok, C.Y., Salman, H.A.K. & Sulaiman, R., Extractionand quantification of saponins: A Review, Food Research International (2014), doi:10.1016/j.foodres.2014.01.057

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http://dx.doi.org/10.1016/j.foodres.2014.01.057http://dx.doi.org/10.1016/j.foodres.2014.01.057http://dx.doi.org/10.1016/j.foodres.2014.01.057http://dx.doi.org/10.1016/j.foodres.2014.01.057

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Extraction and quantification of saponins: A Review

Choon Yoong Cheok, Hanaa Abdel Karim Salman, Rabiha Sulaiman*

Department of Food Technology

Faculty of Food Science and Technology

Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

* Corresponding author: Tel.: +60-3-89468520; Fax: +60-3-89423552;

Email address:[email protected]

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Abstract

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 theemergence of various new extraction technologies with the main purpose to optimize the yield in

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

saponins extraction with emphasis on conventional and green technologies techniques employed

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

to design experiment to suit their particular objective in swift.

Keywords: Saponins; conventional extraction; green extraction technologies; quantification;

spectrophotometric; chromatographic.

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Contents

1. Introduction ........................................................................................................................................... 4

2. Plant materials contain saponins ........................................................................................................... 6

3. Pharmaceutical properties of saponins .................................................................................................. 7

4. Applications of saponins in foods ....................................................................................................... 105. Extraction techniques .......................................................................................................................... 12

5.1. Conventional extraction techniques............................................................................................ 14

5.2. Green extraction technologies .................................................................................................... 16

6. Quantification of Saponins.................................................................................................................. 20

6.1. Spectrophotometric method ........................................................................................................ 20

6.2. Chromatograhic method ............................................................................................................. 23

7. Conclusions ......................................................................................................................................... 24

References ................................................................................................................................................... 25

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1. Introduction

Saponins are second metabolites which widely distributed in plant kingdom. It acts as a

chemical barrier or shield in the plant defense system to counter pathogens and herbivores

(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).

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 different

positions (Hostettmann & Marston, 1995). The classification and occurrence of saponins in the

plant kingdom is reviewed in detail by Vincken et al. (2007).

Saponins, which derived from soapwort (Saponaria officinalisL.), has been widely used for

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 Pacific

fisherman 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.,

2004). Saponins have been found having pharmaceutical properties of haemolytic, molluscicidal,

anti-inflammatory, antifungal or antiyeast, antibacterial or antimicrobial, antiparasitic, antitumor,

and antiviral (Sparg et al., 2004). It employs as a starting point for the semi-synthesis of steroidal

drugs in pharmaceutical industry. Sheng and Sun (2011) reviewed the clinical significance of

triterpene saponins in prevention and treatment of metabolic and vascular disease.

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

need. Saponins are also known possessing mineral complexes of iron, zinc, and calcium (Milgate

& 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

selection of extraction methods from previous literature in respect to research focus in order to

provide a quick reference in future experimental design.

2. Plant materials contain saponins

Saponins are mainly derived from various plant materials (Sparg et al., 2004; Vincken et al.,

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,

Gua, Chauvatcharin, & Zhong, 2004; Wu, Lin, & Chau, 2001; Zhang & Cheng, 2006; Zhang,

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

ofMedicago truncatula(Huhman, Berhow, & Sumner, 2005) andAllium nigrumL. (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

(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

steroid (C27) or triterpenoid (C30) moiety (Challinor & De Voss, 2013; Gl-ntnda &

Mazza, 2007; Harborne & Baxter, 1999), therefore, the isolation and structure elucidation of

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

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

the basis of EI-MS (electrospray ionization-mass spectra), 1H and 13C NMR (nuclear magnetic

resonance) data, such as in Ipomoea batatas (Dini et al., 2009), Aralia taibaiensis (Bi et al.,

2012), andAllium ampeloprasumvar.porrumL. (Ado et al., 2011).

3. Pharmaceutical properties of saponins

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

in treating gastroenteritis diseases (Wang et al., 2014). Seven structurally consecutive saponins

derived from Platycodon grandiflorum have been discovered having haemolytic activities and

adjuvant potentials on the immune responses to Newcastle disease virus-based combinant avian

influenza vaccine in mice (Sun et al., 2011). Both Verza et al. (2012) and Sun (2006) revealed

that saponin fractions derived from Chenopodium quinoa seeds and Bupleurum chinense

enhanced haemolytic activities and adjuvant potentials on immune responses of mice against

ovalbumin. Saponins obtained fromPolygala senegaL. were also suggested as potential vaccineadjuvants to increase specific immune responses (Estrada et al., 2000).

Hassan et al. (2010) reported that 100% methanol fraction of saponin-rich extracts from guar

meal exhibited antibacterial activities against Staphylococcus aureus, Salmonella Typhimurium

and Escherichia coli, however the results showed 20% and 60% methanol fractions stimulated

Lactobacillus spp. growth. Aginoside saponins extracted from Allium nigrum L. roots had

significant antifungal activity (Mostafa et al., 2013). Saponins isolated from seeds of Capsicum

annum L. showed higher antimicrobial activity against yeasts compared to common fungi

(Iorrizzi et al., 2002). The n-butanol extract of shallot basal plates and roots exhibited antifungal

activity against plant pathogenic fungi (Teshima et al., 2013). Fruticoside I, a new steroidal

saponins derived from Cordyline fruticosa leaves, has been found showing moderate

antibacterial activity against the Gram-positiveEnterococcus faecalis(Fouedjou et al., 2014).

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,

mung bean (Vigna radiataL.), and alfafa (Medicago sativaL.) which are rich in saponins, have

been suggested as a good supplement of bioactive compound in daily diet with health-promoting

antioxidant (Silva et al., 2013).

Man et al. (2010a) highlighted saponins possess significant anticancer properties and the

structure-function of saponins influenced the antitumor mechanism. Saponins derived from

Gynostemmapentaphyllumleaves (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,

two new steroidal saponins, fruticoside H and fruticoside I, derived from Cordyline fruticosa

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

notoginsengsaponins have both anti-hyperglycemic and anti-obese effects which may beneficial

to type 2 diabetic patients by improving insulin sensitivity and decreasing leptin resistance (Yang

et al., 2010). A typical triterpenoid saponin of charantin derived from Momordica charantiais a

well-known anti-diabetic bioactive compound (Joseph & Jini, 2013; Raman & Lau, 1996). Four

new triterpenoid saponins isolated from the root bark of Aralia taibaiensisexhibited moderate

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

that total saponins from Rhizoma Anemarrhenae and Entada phaseoloides L. were able toameliorate diabetes-associated cognitive decline in rats. Saponin rich fractions from Bryonia

Laciniosa(Patel, Patel, Vyas, Singh, Shah, & Gandhi, 2012a), Momordica charantia(Keller et

al., 2011) andPolygonatum odoratum(Deng et al., 2012) have been proven having anti-diabetic

property and suggested to use in the treatment of diabetes.

Other therapeutic properties of saponins were reported in previous literature. There werecardioprotective effects of saponins from Panax japonicas (He et al., 2012), anti-thrombotic

activity from Dioscorea zingiberensis (Li et al., 2010a), anti-inflammatory and anti-

ulcerogenic 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).

4. Applications of saponins in foods

Apart from pharmaceutical applications, saponins have been used in foods as natural surfactant

and serve as preservative in controlling microbial spoilage of food. More recently, due to

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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

(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

surfactant (Tween 80) has been compared by Yang et al. (2013). After comparing the influenc e

of homogenization pressure, number of passes, and emulsifier concentration on the particle size

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

natural surfactant has been further proven its stability and effectiveness at forming edibleVitamin E delivery systems, thus it is recommended for functional food encapsulation and

beverage applications (Yang & McClements, 2013).

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 thatbanana cellulose micro and nano fibres obtained by steam explosion process

which soaked with saponin, a surfactant extracted from soapnut fruit, showed differences in the

degree of modification and morphology of the cellulose fibres (Cordeiro et al., 2013). The results

clearly show that the saponin can provide a continuous path of hydrogen bonds between the fibre

surfaces which will thus enhance the hydrophobic and the acidbase nature of the fibre surface.

This behaviour will lead to better polymer/fibre 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

production of biodegradable emulsified films using the casting technique. Their results showed

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

elongation, considerable solubility and opacity. Therefore, they suggested that the possibility of

using this natural surfactant may allow for new applications of biodegradable emulsified films.

The use of saponins as a natural biochemical substance in inactivation of food-borne viruses hasbeen reviewed by Li et al. (2013). The saponins-extract from Sapindus saponariacombined with

heat-treatment was recommended to inactivate Alicyclobacillus acidoterrestris, a spoilage-

causing bacterium, in orange juice (Alberice et al., 2013). Tea saponin, a tea seed-derived natural

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

solubility of solute from plant materials into solvent. Therefore, it often utililizes a large quantity

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

extraction techniques involved less hazardous chemical synthesis, safer chemicals used, energy

efficiency, 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

used as extraction solvent by manipulating the extraction system pressure and temperature, as inpressurized liquid extraction.

Based on the importance of saponins as pharmaceutical properties especially in countering

cancer has provoked the invention of new extraction methods in order to obtain the maximum

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

and efficacy in obtaining significant saponins yields.

5.1. Conventional extraction techniques

5.1.1. Maceration extraction

The maceration extraction is a solid-liquid extraction where the bioactive compound

(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

like dissolves like where indicated that a polar compounds dissolve in polar solvents, and

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

duration of extraction time is long and sometimes takes up to weeks using this method, therefore,

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.,

2012) to shorten the extraction time.

5.1.2. Reflux and Soxhlet extractions

Due to the similar working principle of Soxhlet and reflux extractions, the discussion iscarried out under the same sub-title. The only different between reflux and Soxhlet is that

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.

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.

5.1.3. Subsequent extraction

There were numerous studies carried out on extraction of saponins using two extraction

methods subsequently. The purpose of using two extraction methods subsequently might due

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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

extraction methods in obtaining the specific saponins from various plant materials. For Soxhlet

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

Jurzysta, Mella, & Tava, 2006; Oleszek et al., 2001; Tava et al., 2009) and hexane (Ncube,

Ngunge, Finnie, & Staden, 2011).

5.2. Green extraction technologies

5.2.1. Ultrasound-assisted extraction (UAE)

The phenomenon of ultrasound in creating cavitation bubbles in the solvent by acting as a

microjet to denature the plant cell wall when the bubbles collapse at rarefraction resulted in a

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).

5.2.2. Microwave-assisted extraction (MAE)

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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

bioactive compound extraction from plant material due to short extraction time, minimal solvent

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,

alkaloids and saponins have been reviewed (Zhang, Yang, & Wang, 2011). Microwaves are able

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

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

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

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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5.2.3. Accelerated solvent extraction (ASE)

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).

This technique was introduced by Dionex Corporation in 1995. It is also known as pressurizedliquid extraction, pressurized solvent extraction, and enhanced solvent extraction. Sometime it is

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

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 efficient

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,

modifiers and additives, matrix composition, and extraction mode (Sun, Ge, Lv, & Wang, 2012).

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

compound from plant material, the application in saponins extraction is still scarce. Table 9

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,

& Wang, 2008; Wan et al., 2006; Zhang, Liu, Qi, Li, & Wang, 2013) could be due to theprecious value of the product.

The efficacy of ASE in saponins extraction has been studied and compared with other

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 chinensisBunge 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 fromPanax 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

simple procedure to test the presence of saponins. This can be done by putting the plant material

into a test tube filled with distilled water and vigorously shaken for 2 minutes (Ncube et al.,

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

spectrophotometric and chromatographic methods. The difference quantitative expression

between the two methods is the spectrophotometrics gives a total saponins value while the

chromatographics quantifiesspecific saponins compound.

6.1. Spectrophotometric method

The spectrophotometric technique has become a popular method in the quantification of

saponins from plant materials could be because it is simple, fast and inexpensive to operate.

Total saponins also known as vanillin-sulphuric acid assay, is the most commonly selected

spectrophotometric method in plant saponins quantification (Table 10). However few factors,

such as selection of standards, wavelength, and others should be considered before using this

method. The basic principle of this method is the reaction of oxidized triterpene saponins with

vanillin (Li et al., 2010b). Sulphuric acid is used as oxidant and the distinctive colour of this

reaction is purple (Hiai, Oura, & Hakajima, 1976) and sometimes perchloric acid is used (Chen

et al., 2007b; Li et al., 2010b; Wu et al., 2001). Due to differences in selection of reagent,

condition to allow full colour development, standard, and wavelength from previous researches

as presented in Table 10, it is hard to compare the results in terms of yields. However, it provides

a good reference for future experimental design.

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Two works (Patel et al., 2012a and Ncube et al., 2011) were found using the conditions of

60 C and 10 minutes to allow the mixture to have full colour development following procedure

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

standardized because the reaction time may directly attribute to the final absorbance value which

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

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

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

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

water, whereas for total steroidal sapogenins, both the anasaldehyde and sulphuric acid are

diluted with ethyl acetate. Total steroidal sapogenins has been proven stable and reproducible

with a number of standards, ie., diosgenin, tigogenin, hecogenin, smilagenin, yonagenin,

tokorogenin, etc. without interference from sugars, sterols, fatty acid and vegetable oil (Baccou

et al., 1977).

The procedure to quantify total steroidal sapogenin is by weighing 0-40 g of crudeextract and dissolved in 2 ml of ethyl acetate in the test tube. Then mixed with 1 ml of reagent A

(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

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 ofDioscorea zingiberensiswith

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

lyophilized powder.

Haemolytic method is another spectrophotometric method uses to quantify saponins

content of a plant material (Barve, Laddha, & Jayakumar, 2010). The principle of this method is

the reaction of saponins with blood reagent to release oxy-hemoglobin which results a

measurable colour for spectrophotometer. The saponins concentrations in bittergourd varieties

were quantified using haemolytic method (Habicht et al., 2011). The saponin extract was

dissolved in distilled water and 100 l of this solution were incubated with 1 ml fresh EDTA -blood at 30 C for 30 min. After centrifugation for 10 min, haemoglobin was quantified in the

supernatant photometrically at 545 nm and the result was expressed in haemolytic saponins.

They revealed that white bitter gourd varieties were found to contain significantly lower saponin

concentrations (0.25%) compared to green varieties (0.67%).

6.2. 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

saponins using HPLC is presented in Table 11. As noted the quantification of specific saponin

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,

but as a strong scientific reference to drug-related manufacturer who is interested to process the

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

to quantify saponins.

7. Conclusions

Saponins are important secondary metabolites derived from various plant sources because

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 lab-

scale 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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