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Extraction and quantification of saponins: A review

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DOI: 10.1016/j.foodres.2014.01.057

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Food Research International 59 (2014) 16–40

Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Review

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

a r t i c l e i n f o a b s t r a c t

Article history: Saponins, a second metabolites mainly derived from plant materials, have been used extensively in drug-related
Received 1 October 2013 industry due to the pharmaceutical properties. These have driven the emergence of various new extraction tech-
Accepted 19 January 2014 nologies with the main purpose to optimize the yield in order to accommodate the recent need. The plants con-
Available online 31 January 2014
taining saponins are discussed, and their pharmaceutical properties and applications in food are highlighted. This
review focuses on the saponin extraction with emphasis on conventional and green technology techniques
Keywords:
Saponins
employed in previous works by relating to their specific objective in each study. The quantification methods of
Conventional extraction saponins yield, i.e., spectrophotometric and chromatographic, are summarized and discussed. In addition, this re-
Green extraction technologies view aims to provide a point of reference to researchers who wish to design experiment to suit their particular
Quantification objective in swift.
Spectrophotometric © 2014 Elsevier Ltd. All rights reserved.
Chromatographic

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2. Plant materials contain saponins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3. Pharmaceutical properties of saponins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4. Applications of saponins in foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5. Extraction techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5.1. Conventional extraction techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.1.1. Maceration extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.1.2. Reflux and Soxhlet extractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.1.3. Subsequent extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.2. Green extraction technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.2.1. Ultrasound-assisted extraction (UAE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.2.2. Microwave-assisted extraction (MAE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.2.3. Accelerated solvent extraction (ASE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
6. Quantification of saponins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
6.1. Spectrophotometric method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
6.2. Chromatograhic method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

1. Introduction
Saponins are second metabolites which are widely distributed in
the plant kingdom. It acts as a chemical barrier or shield in the plant
defense system to counter pathogens and herbivores (Augustin,
⁎ Corresponding author. Tel.: +60 3 89468520; fax: +60 3 89423552.
E-mail address: rabiha@upm.edu.my (R. Sulaiman).
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 king-
dom are reviewed in detail by Vincken, Heng, de Groot, and Gruppen
(2007).

0963-9969/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2014.01.057
 Author's personal copy
C.Y. Cheok et al. / Food Research International 59 (2014) 16–40
Saponins, which are derived from soapwort (Saponaria officinalis L.),
have been widely used for centuries as household detergent (Sparg,
Light, & van Staden, 2004) due to its amphiphilic nature with the
presence of a lipid-soluble aglycone and water-soluble chain(s)
in their structure (Güçlü-Üntündağ & Mazza, 2007). The seeds of
Barringtonia asiatica Kurz (Lecythidaceae) which have known to contain
saponins, have been used traditionally by native Asian and Pacific fish-
erman 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 pro-
duction (Wina et al., 2005). The molluscicidal saponins derived from
soapnut (Sapindus mukorossi Gaerth) have 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).
The discovery of biological activities of saponins is not only limited
to the traditional uses, but more recently, also in pharmaceutical appli-
cations (Güçlü-Üntündağ & Mazza, 2007; Sparg et al., 2004). Saponins
have been found having pharmaceutical properties of hemolytic, mol-
luscicidal, 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 property 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 re-
cent 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 that 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 discov-
ered scientifically having pharmaceutical properties of antioxidant
(Chan, Khong, Iqbal, & Ismail, 2013; Dini, Tenore, & Dini, 2009; Li, Zu,
et al., 2010), immunological adjuvant activities (Estrada, Katselis,
Laarveld, & Barl, 2000; Sun, Chen, Wang, Wang, & Zhou, 2011; Verza
et al., 2012), and hemolytic 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 lit-
erature is essential. Researchers from a variety of scientific back-
grounds are often 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 selec-
tion of extraction methods from previous literature in respect to
research focus in order to provide a quick reference in future exper-
imental 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 was found having saponins
is ginseng (Kwon, Bélanger, 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, 2013),
even though saponins derived from alfafa have been carried out as
early by Van Atta, Guggolz, and Thompson (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
17
(Vázquez-Castilla et al., 2013), marion blackberry, strawberry, and
plum fruit (Yoon & Wrolstad, 1984).
Saponin 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 sa-
ponins accumulation. The yam tuber cortex has been discovered to
possess the highest amount of saponins of 582.53 μg/g dw which
was about 2.55 times higher than tuber flesh of 227.86 μg/g dw
(Lin & Yang, 2008). However, the total saponin concentration has
been reported to contain the highest level in leaves from the four va-
rieties of Swithchgrass (Lee et al., 2009) and greenhouse grown
Maesa lanceolata (Theunis et al., 2007). Table 1 tabulates the sapo-
nins 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; Güçlü-Üntündağ & Mazza, 2007;
Harborne & Baxter, 1999), therefore, the isolation and structure elucida-
tion 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 catego-
rizing them into triterpenoid and steroidal in period from 1998 to 2003.
However, recent review on the triterpenoid and steroidal saponins de-
rived from various plants is shown in Table 2. The elucidation and char-
acterization of saponins structure are conducted on the basis of EI-MS
(electrospray ionization-mass spectra), 1H and 13C NMR (nuclear mag-
netic resonance) data, such as in Ipomoea batatas (Dini et al., 2009),
Aralia taibaiensis (Bi et al., 2012), and Allium ampeloprasum var. porrum
L. (Adão, da Silva, & Parente, 2011).
3. Pharmaceutical properties of saponins
Saponins are 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),
and possession of antibacterial (Hassan et al., 2010; Iorizzi, Lanzotti,
De Marino, Ranalli, & Zollo, 2002; Mostafa et al., 2013; Teshima et al.,
2013), antioxidant (Bi et al., 2012; Chan et al., 2013; Dini et al., 2009;
Li, Zu, et al., 2010; Lin, Yang, & Lin, 2011), anticancer (Cheng et al.,
2011; Man, Gao, Zhang, Huang and Liu, 2010; Man, Gao, Zhang, Wang,
et al., 2010), antidiabetic and anti-obesity properties (Joseph & Jini,
2013; Kimura, Ogawa, Katsube, Yokota, & Jisaka, 2008; Yun, 2010).
Thus ginseng, which contains saponins, is included in most of the Chi-
nese 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 hemolytic 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 hemolytic
activities and adjuvant potentials on immune responses of mice against
ovalbumin. Saponins obtained from Polygala senega L. were also sug-
gested as potential vaccine adjuvants to increase specific immune re-
sponses (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 stim-
ulated Lactobacillus spp. growth. Aginoside saponins extracted from
A. 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 (Iorizzi 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
 Author's personal copy

18 C.Y. Cheok et al. / Food Research International 59 (2014) 16–40

Table 1
Saponins derived from different parts of plant materials.

Plant Plant material Reference(s)

part

Seed Aesculus chinensis Bunge Chen, Li, et al. (2007)

Aesculus turbinate BLUME Kimura et al. (2008)
Allium tuberosum Sang, Mao, Lao, Chen, and Ho (2001), Sang, Zou, et al. (2001)
Argania spinosa Alaoui et al. (2002)
Bryonia Laciniosa Patel, Santani, Shah, and Patel (2012)
Capsicum annum L. Iorizzi et al. (2002)
Chenopodium pallidicaule Rastrelli, De Simone, Schettino, and Dini (1996)
Chenopodium quinoa Dini, Schettino, Simioli, and Dini (2001), Verza et al. (2012), Zhu et al. (2002), Woldemichael and Wink
(2001)
Entada phaseoloides Zheng et al. (2012)
Leguminous species Ha et al. (2014)
Trigonella foenum graecum L. Petit et al. (1995)
Vaccaria segetalis Garcke, Saponaria Vaccaria L., Vaccaria Güçlü-Üntündağ et al. (2007)
pyramidate
Root Allium nigrum L. Mostafa et al. (2013)
Aralia taibaiensis Bi et al. (2012)
Bupleurum chinense Hu, Cai, and Liang (2008)
Caulophyllum thalictroides Avula et al. (2011)
Chiococca alba Borges, Valença, Lopes, Barbi, and Silva (2013)
Dioscorea panthaica Wang et al. (2012)
Glycyrrhiza inflate, Glycyrhiza glabra, Glycyrrhiza uralensis Tao et al. (2013)
Glycyrrhiza yunnanensis Ji et al. (2014)
Gypsophila trichotoma Voutquenne-Nazabadioko et al. (2013)
Maesa lanceolata Theunis et al. (2007)
Medicago hybrid Bialy et al. (2006)
Momordica charantia Chen et al. (2008)
Paris polyphylla var. chinensis Liu et al. (2013), Zhang et al. (2010)
Paris polyphylla var. yunnanensis Zhang et al. (2010)
Pulsatilla chinensis Xu et al. (2011)
Panax notoginseng Lau, Woo, and Koh (2003), Li et al. (2005), Wu et al. (2001)
Panax quinquefolius Engelberth et al. (2010), Gafner et al. (2004), Wu et al. (2001)
Platycodon grandiflorum Sun et al. (2011)
Polygonatum odoratum Bai et al. (2014), Deng et al. (2012)
Radix Astragali Qi et al. (2006)
Vigna radiata L. Waller et al. (1999)
Yucca gloriosa L. Skhirtladze et al. (2011)
Leaf Acanthopanax sessiliflorus Yoshizumi et al. (2006)
Allium nigrum L. Mostafa et al. (2013)
Antonia ovate Alabdul Magid et al. (2012)
Beaucarnea recurvata Eskander, Lavaud, and Harakat (2011)
Cordyline fruticosa Fouedjou et al. (2014)
Gymnema sylvestre Mandal and Mandal (2010)
Maesa lanceolata Foubert et al. (2010), Theunis et al. (2007)
Silphium asteriscus L. Masullo, Calabria, Gallotta, Pizza, and Piacente (2014)
Tribulus terrestris L. Dinchev et al. (2008)
Vigna radiata L. Waller et al. (1999)
Ziziphus jujube, Z. jujuba var. spinosa Guo et al. (2011)
Bulb Allium nutans L. Akhov, Musienko, Piacente, Pizza, and Oleszek (1999)
Allium nigrum L. Mostafa et al. (2013)
Crocus sativus Rubio-Moraga et al. (2011)
Fruit Gleditsia sinensis Lam. Lian and Zhang (2013)
Momordica charantia Li, Liang, Chen, Wang, and Zhao (2007), Lin et al. (2011), Liu, Lu, et al. (2012)
Solanumxanthocarpum Patel, Patel, et al. (2012a)
Tribulus terrestris L. Dinchev et al. (2008)
Stem Caryocar villosum Alabdul Magid et al. (2006)
Momordica charantia Lin et al. (2011)
Silphium asteriscus L. Masullo et al. (2014)
Tribulus terrestris L. Dinchev et al. (2008)
Vigna radiata L. Waller et al. (1999)
Pericarp Sapindus mukorossi Huang et al. (2003)
Bark Yucca schidigera Roezl Kowalczyk, Pecio, Stochmal, and Oleszek (2011)
Harpullia austro-caledonica Voutquenne et al. (2005)
Tuber Ipomoea batatas Dini et al. (2009)
Flower Agave offoyana Pérez et al. (2013)

from Cordyline fruticosa leaves, has been found showing moderate anti-
bacterial activity against the Gram-positive Enterococcus faecalis
(Fouedjou et al., 2014).
A number of previous literature reported that saponins rich fraction
have antioxidant properties. They were derived from the root bark of
Aralia taibaiensis (Bi et al., 2012), deffated rice bran (Chan et al., 2013),
Ipomoea batatas tubers (Dini et al., 2009), yellow horn (Li, Zu, et al.,
2010), stems and fruits of Momordica charantia (Lin et al., 2011). The
sprouts of soybean, mung bean (Vigna radiata L.), and alfafa (Medicago
sativa L.) which are rich in saponins, have been suggested as a good sup-
plement of bioactive compound in daily diet with health-promoting
antioxidant (Silva et al., 2013).
 Author's personal copy

C.Y. Cheok et al. / Food Research International 59 (2014) 16–40 19

Table 2 consequences such as obesity and diabetes, where 80% of type 2 diabetes
Triterpenoid and steroidal saponins derived from various plant materials. patients are linked to obesity (Yang et al., 2010). Hence, studies on
Plant material Reference(s) identification of anti-obesity property from plant materials have become
a popular trend. In a recent review, saponins separated from plant
Triterpenoid saponins
Antonia ovata Alabdul Magid et al. (2012) materials of Panax japonicas, Platycodi radix, Kochia scoparia fruits, Thea
Aralia taibaiensis Bi et al. (2012) sinensis leaf, Scabiosa tschiliensis Grun., and Acanthopanax sessiliflorous
Bacopa monnieri Ganzera, Gampenrieder, Pawar, Khan, and were suggested having anti-obesity property in inhibited pancreatic
Stuppner (2004)
lipase (Yun, 2010). The possibility of obesity treatment with saponins
Caryocar villosum Alabdul Magid et al. (2006)
Momordica charantia Chen et al. (2008), Li et al. (2007) derived from leaves of Acanthopanax sessiliflorus (Yoshizumi et al.,
Medicago truncatula Kapusta, Janda, Stochmal, and Oleszek (2005), 2006) and saponins from Japanese horse chestnut (Aesculus turbinate
Huhman et al. (2005) BLUME) after the treatment with wood ashes (Kimura et al., 2008)
Medicago arabica L. Bialy et al. (2004), Tava et al. (2009) have been discussed. Panax notoginseng saponins have both anti-
Medicago hydriba Bialy et al. (2006)
hyperglycemic and anti-obese effects which may be beneficial to
Caulophyllum thalictroides Avula et al. (2011)
Chenopodium quinoa Verza et al. (2012) type 2 diabetic patients by improving insulin sensitivity and decreasing
Chiococca alba Borges et al. (2013) leptin resistance (Yang et al., 2010). A typical triterpenoid saponin
Flos Lonicerae Chai, Li, and Li (2005) of charantin derived from M. charantia is a well-known anti-diabetic
Genista ulicina Spach Boutaghane, Voutquenne-Nazabadioko,
bioactive compound (Joseph & Jini, 2013; Raman & Lau, 1996). Four
Harakat, Simon, and Kabouche (2013)
Glycyrrhiza inflate, Glycyrhiza Tao et al. (2013) new triterpenoid saponins isolated from the root bark of A. taibaiensis
glabra, Glycyrrhiza uralensis exhibited moderate effects on antioxidant and antiglycation activities
Glycyrrhiza yunnanensis Ji et al. (2014) which could be correlated with treatment of diabetes mellitus (Bi
Gymnema sylvestre Mandal and Mandal (2010) et al., 2012). Both Liu, Zhu, et al. (2012) and Zheng et al. (2012) demon-
Gypsophila trichotoma Voutquenne-Nazabadioko et al. (2013)
strated that total saponins from Rhizoma Anemarrhenae and Entada
Harpullia austro-caledonica Voutquenne et al. (2005)
Lamii albi flos Wójciak-Kosior, Sowa, Kocjan, and Nowak phaseoloides L. were able to ameliorate diabetes-associated cognitive de-
(2013) cline in rats. Saponin rich fractions from Bryonia Laciniosa (Patel, Patel,
Pulsatilla chinensis Xu et al. (2011) Vyas, Shah & Gandhi, 2012), M. charantia (Keller et al., 2011) and
Salicornia herbacea Zhao, Wang, Wang, Liu, and Xin (2014)
Polygonatum odoratum (Deng et al., 2012) have been proven having
Panax quinquefolius Gafner et al. (2004)
Ganoderma atrum Chen, Xie, et al. (2007)
anti-diabetic property and suggested for use in the treatment of
Ipomoea batatas Dini et al. (2009) diabetes.
Xanthoceras sorbifolia Bunge Li, Zu, et al. (2010), Ling et al. (2011) Other therapeutic properties of saponins were reported in previous
Crocus sativus Rubio-Moraga et al. (2011) literature. There were cardioprotective effects of saponins from
Maesa lanceolata Foubert et al. (2010), Theunis et al. (2007)
P. japonicas (He et al., 2012), anti-thrombotic activity from Dioscorea
Polygala japonica Wang, Guo, Zhu, and Yu (2007)
Pulsatilla turczaninovii Xu et al. (2012) zingiberensis (Li, Huang, et al., 2010), anti-inflammatory and anti-
Acanthopanax sessiliflorus Yoshizumi et al. (2006) ulcerogenic properties from the bulbs of A. ampeloprasum (Adão et al.,
Steroidal saponins 2011), anti-HIV activity from M. charantia (Chen et al., 2008, 2009),
Agave offoyana Pérez et al. (2013)
and antiurolithiatic activity from fruit of Solanum xanthocarpum (Patel,
Allium ampeloprasum Adão et al. (2011)
Beaucarnea recurvata Eskander et al. (2011)
Patel, et al., 2012).
Cordyline fruticosa Fouedjou et al. (2014)
Dioscorea zingiberensis Li, Huang, et al. (2010), Qin et al. (2009), Zhang, 4. Applications of saponins in foods
Ito, et al. (2013)
Yucca schidigera Roezl Kowalczyk et al. (2011)
Apart from pharmaceutical applications, saponins have been
Dioscorea panthaica Wang et al. (2012)
Dioscorea pseudojaponica Lin, Liu, Chen, Chen, and Yang (2006), Lin and used in foods as natural surfactant and serve as preservative in con-
Yamamoto Yang (2008) trolling microbial spoilage of food. More recently, due to consumer
Asparagus officinalis L. Dawid and Hofmann (2012) preference for natural substance, Quillaja saponin has been used as
Panicum virgatum L. Lee et al. (2009)
a natural small molecule surfactant in beverage emulsions in replac-
Paris polyphylla var. chinensis Liu et al. (2013), Zhang et al. (2010)
Paris polyphylla var. yunnanensis Zhang et al. (2010)
ing synthetic surfactant of Tweens (Piorkowski & McClements,
Polygonatum odoratum Bai et al. (2014) 2013). The effectiveness of the natural surfactant isolated from the
Tribulus terrestris L. Dinchev et al. (2008) bark of the Quillaja saponaria Molina tree for forming and stabilizing
Fagonia indica Waheed et al. (2012) emulsions with a synthetic surfactant (Tween 80) has been com-
Yucca gloriosa L. Skhirtladze et al. (2011)
pared by Yang, Leser, Sher, and McClements (2013). After compar-
ing the influence 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 sur-
Man, Gao, Zhang, Huang, et al. (2010) highlighted that saponins factants in food and beverage products. This natural surfactant
possess significant anticancer properties and the structure–function has been further proven its stability and effectiveness at forming
of saponins influenced the antitumor mechanism. Saponins derived edible Vitamin E delivery systems, thus it is recommended for func-
from Gynostemma pentaphyllum leaves (Cheng et al., 2011) and a tional food encapsulation and beverage applications (Yang &
novel steroidal saponin glycoside derived from Fagonia indica McClements, 2013).
(Waheed et al., 2012) have been discovered having antiproliferation Due to its natural foam-like characteristic, the application of
and apoptosis against prostate, breast and colon cancer cells. In a re- saponins as a natural bio-surfactant to improve the surface proper-
cent study, two new steroidal saponins, fruticoside H and fruticoside ties of food is intensively studied recently. Wojciechowski, Kezwon,
I, derived from C. fruticosa leaves have been found having moderate Lewandowska, and Marcinkowski (2014) have conducted a study
cytotoxic activity against human breast, colon, and melanoma cell to evaluate the surface activity between Quillaja bark saponin with
lines (Fouedjou et al., 2014). β-casein of bovine milk protein. From their results obtained, they
The lack of physical activity in daily routines and increase in high- suggested that the Quillaja bark saponin can be used as a natural
calorie fast food intake have led to a number of health-related low molecular weight bio-surfactant. A recent study indicated that
 Author's personal copy
20 C.Y. Cheok et al. / Food Research International 59 (2014) 16–40
banana cellulose micro and nano fibers 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 fibers (Cordeiro, Faria, Abraham, &
Pothan, 2013). The results clearly show that the saponin can
provide a continuous path of hydrogen bonds between the fiber
surfaces which will thus enhance the hydrophobic and the acid–
base nature of the fiber surface. This behavior will lead to better
polymer/fiber interaction during the composite preparation.
Andreuccetti, Carvalho, and Grosso (2010) evaluated the incorpo-
ration 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 pro-
duced have good mechanical resistance, low values of water vapor
permeability and reduced drying times, even though the films pre-
sented limited elongation, considerable solubility and opacity.
Therefore, they suggested that the possibility of using this natural
surfactant may allow for new applications of biodegradable emulsi-
fied films.
The use of saponins as a natural biochemical substance in inactivation
of food-borne viruses has been reviewed by Li, Baert, and Uyttendaele
(2013). The saponins-extract from Sapindus saponaria combined
with heat-treatment was recommended to inactivate Alicyclobacillus
acidoterrestris, a spoilage-causing bacterium, in orange juice (Alberice,
Funes-Huacca, Guterres, & Carrilho, 2012). Tea saponin, a tea seed-
derived natural surfactant, combining with Bacillus amyloliquefaciens
(Hao, Li, Hu, Yang, & Rizwan-ul-Haq, 2011) and imazalil and prochloraz
(Hao et al., 2010), have been used for postharvest treatment of Manda-
rin 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 be 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
Ultrasound-assisted (14%)
Reflux (22%)
Green extraction technologies
Fig. 1. Current extraction techniques employed in extraction of saponins from plant materials.
extraction techniques employed in saponin extraction can be classi-
fied into two categories, the conventional and the green technolo-
gies. The conventional extraction techniques are maceration,
Soxhlet, and reflux extraction, where the green technologies are
ultrasound-assisted, microwave-assisted, and accelerated solvent
extraction (Heng, Tan, Yong, & Ong, 2013). The conventional extrac-
tion is relied on the solubility of solute from plant materials into sol-
vent. Therefore, it often utilizes 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 is governed
under these measurements. Consequently, water is used as extrac-
tion solvent by manipulating the extraction system pressure and
temperature, as in pressurized liquid extraction.
The importance of saponins as pharmaceutical properties espe-
cially in countering cancer has provoked the invention of new ex-
traction methods in order to obtain the maximum output to cope
with the increasing demand. Therefore, a synthesis of previous liter-
ature in extraction technique selection may provide useful informa-
tion in related processing industry. Fig. 1 clearly demonstrates that
researchers are more inclined to selection of the conventional ex-
traction 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 usu-
ally was governed by the research focus of the studies being conduct-
ed. 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 property studies, 78% and 91% of the
previous works were using the conventional extraction methods.
However, in works focused on quantification and optimization stud-
ies, 58% and 67% of the previous works have selected green extrac-
tion 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 saponin yields.
Maceration (36%)
Conventional extractions
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C.Y. Cheok et al. / Food Research International 59 (2014) 16–40
5.1. Conventional extraction techniques
5.1.1. Maceration extraction
The maceration extraction is a solid–liquid extraction where the bio-
active 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” which indicated
that polar compounds dissolve in polar solvents, and nonpolar com-
pounds 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 that 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 that has made it a popular choice for
researchers. The only paramount factor to be paid attention in enhanc-
ing extractability is the knowledge of similarity of bioactive compound
interest and solvent polarity. Table 4 summarizes the maceration ex-
traction procedure carried out in previous literature in respect to their
objective(s).
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 extrac-
tions, the discussion is carried out under the same sub-title. The only
difference 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 is widely used in food and non-
food industrial and laboratories. The process involves heating a solution
to boiling and then returning the condensed vapors 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 saponin extraction from plant materials using
reflux and Soxhlet extraction method. Ethanol is still the most used sol-
vent in reflux extraction, although there are few used methanol as ex-
traction solvent. The extraction duration of reflux extraction was
varied from 1 to 4 h, while for Soxhlet was 24 to 72 h.
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 be due to highly purify the ex-
tract before subjecting to HPLC analysis for isolation and identification
of saponin compound from the plant material. Table 6 presents the ap-
plication of two subsequent extraction methods in obtaining the specific
saponins from various plant materials. For Soxhlet and reflux subse-
quent 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, 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
21
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), and the applications of ultrasound-assisted extraction
on bioactive principles from herbs (Vinatoru, 2001), food industry and
processing (Mason, 1998; Vilkhu, Mawson, Simons, & Bates, 2008). Al-
though ultrasound-assisted extraction is commonly employed in
many bioactive compound 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 saponin
extraction (Table 7).
5.2.2. Microwave-assisted extraction (MAE)
Microwaves are non-ionizing electromagnetic waves with a fre-
quency range from 0.3 to 300 GHz (Heng et al., 2013; Takeuchi et al.,
2009). Recently, MAE has drawn attention in bioactive compound ex-
traction from plant material due to short extraction time, minimal sol-
vent usage, and its special heating mechanism (Heng et al., 2013). The
recent applications of MAE of plant secondary metabolites such as flavo-
noids, 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 penetra-
tion depth of microwaves into plant matrix depends on dielectric
constant, moisture content, temperature, and the frequency of the elec-
trical field (Takeuchi et al., 2009). The water contained in a plant mate-
rial 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 mi-
crowave 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 saponin extraction using MAE has been mentioned
in previous reviews (Güçlü-Üntündağ & Mazza, 2007; Zhang et al.,
2011). Table 8 synthesizes an up-to-date literature of using MAE in
saponin extraction in which the content was not covered in those
two reviews. The superiority of MAE in saponin extraction in com-
parison with other extraction methods, in terms of higher yield
(Chen, Xie & Gong, 2007; Li, Zu, et al., 2010; Mandal & Mandal, 2010;
Xu et al., 2012) and shorter extraction time (Chen, Xie, et al., 2007;
Kwon et al., 2003; Mandal & Mandal, 2010; Xu et al., 2012), has been
found in previous literature.
5.2.3. Accelerated solvent extraction (ASE)
Accelerated solvent extraction has been regarded as a green tech-
nique in plant material sample preparation prior to chromatographic
analysis (Azmir et al., 2013; Heng et al., 2013). This technique was
introduced by Dionex Corporation in 1995. It is also known as pres-
surized liquid extraction, pressurized solvent extraction, and en-
hanced solvent extraction. Sometimes 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 mini-
mal 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 boil-
ing 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 min using
only 15–45 ml consumption of solvent. Therefore, it has been widely
applied in the fields of environmental, food, polymer, and pharmaceuti-
cal researches.
ASE comprises of two main set-ups, there are the static and dynamic
instruments (Mustafa & Turner, 2011). Static setup is replacement of
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Table 3
Selection of saponins extraction technique based on research focus.
Extraction method Saponins isolation
Conventional extractions: Plant source (reference)
Maceration Allium ampeloprasum (Adão et al., 2011)
Momordica charantia (Chen et al., 2008)
Polygonatum odoratum (Bai et al., 2014; Deng
et al., 2012)
Ipomoea batatas (Dini et al., 2009)
Panicum virgatum L. (Lee et al., 2009)
Dioscorea pseudojaponica Yamamoto (Yang, Lu, &
Hwang, 2003)
Chenopodium quinoa (Verza et al., 2012)
Chenopodium quinoa (Zhu et al., 2002)
Fagonia indica (Waheed et al., 2012)
Yucca gloriosa (Skhirtladze et al., 2011)
Dioscorea zingiberensis (Zhang, Ito, et al., 2013)
Leguminous species (Ha et al., 2014)
Genista ulicina Spach (Boutaghane et al., 2013)
Glycyrrhiza yunnanensis (Ji et al., 2014)
Silphium asteriscus L. (Masullo et al., 2014)
Harpullia austro-caledonica (Voutquenne et al.,
2005)
Agave offoyana (Pérez et al. 2013)
Salicornia herbacea (Zhao et al., 2014)
Cordyline fruticosa (Fouedjou et al., 2014)
Reflux Allium nutans L. (Akhov et al., 1999)
Aralia taibaiensis (Bi et al., 2012)
Flos Lonicerae (Chai et al., 2005)
Momordica charantia (Chen et al., 2009)
Guar meal (Hassan et al., 2010)
Medicago sativa L. (Oleszek, 1998)
Momordica charantia (Liu, Lu, et al., 2012)
Maesa lanceolata (Theunis et al., 2007)
Acanthopanax sessiliflorus (Yoshizumi et al. 2006)
Radix Astragali (Qi et al., 2006)
22
Field of research focus Quantification studies Processing parameters/optimization
Pharmaceutical properties investigation
Allium ampeloprasum (Adão et al., 2011) Ipomoea batatas Panax quinquefolius (Gafner et al., 2004)
(Dini et al., 2009)
Momordica charantia (Chen et al., 2008) Momordica charantia Dioscorea pseudojaponica Yamamoto
(Habicht et al., 2011) (Lin et al., 2006)
C.Y. Cheok et al. / Food Research International 59 (2014) 16–40
Gynostemma pentaphyllum (Cheng et al., 2011) Medicago truncatula
(Huhman et al., 2005)
Polygonatum odoratum (Deng et al., 2012) Panicum virgatum L.
(Lee et al., 2009)
Ipomoea batatas (Dini et al., 2009) Yucca gloriosa
(Skhirtladze et al., 2011)
Momordica charantia (Gupta et al., 2010)
Rhizoma Anemarrhenae (Liu, Zhu, et al., 2012)
Solanum xanthocarpum (Patel, Patel, et al.,
2012; Patel, Santani, et al., 2012)
Dioscorea zingiberensis (Qin et al., 2009)
Chenopodium quinoa (Verza et al., 2012)
Fagonia indica (Waheed et al., 2012)
Panax notoginseng (Yang et al., 2010)
Entada phaseoloides (Zheng et al., 2012)
Cordyline fruticosa (Fouedjou et al., 2014)
Silphium asteriscus L. (Masullo et al., 2014)
Salicornia herbacea (Zhao et al., 2014)
Aralia taibaiensis (Bi et al., 2012) Flos Lonicerae (Chai et al., 2005) Defatted rice bran (Chan et al., 2013)
Defatted rice bran (Chan et al., 2013) Xanthoceras sorbifolia
(Ling et al., 2011)
Paris polyphylla var. chinensis (Liu et al., 2013) Paris and Trillium plants (Man, Gao, Zhang,
Wang, et al., 2010)
Momordica charantia (Chen et al., 2009) Glycyrrhiza inflate, Glycyrhiza glabra, Glycyrrhiza
uralensis
(Tao et al., 2013)
Guar meal (Hassan et al., 2010) Polygala japonica
(Wang et al., 2007)
Panax japonicus (He et al., 2012)
Medicago sativa L. (Oleszek, 1998)
Momordica charantia (Popovich, Li, & Zhang,
2010)
Bupleurum chinense (Sun, 2006)
Acanthopanax sessiliflorus (Yoshizumi et al.,
2006)

Extraction method
Saponins isolation
Soxhlet
Subsequent methods
Green extraction techniques:
Ultrasound-assisted
Micowave-assisted
Accelerated solvent
Author's personal copy
Field of research focus
Pharmaceutical properties investigation
Glycyrrhiza inflate, Glycyrhiza glabra, Glycyrrhiza
uralensis (Tao et al., 2013)
Polygala japonica (Wang et al., 2007)
Xanthoceras sorbifolia (Ling et al., 2011)
Caryocar villosum (Alabdul Magid et al., 2006)
Chenopodium quinoa (Woldemichael & Wink,
2001)
Medicago arabica L. (Bialy et al., 2004)
Medicago hybrid (Bialy et al., 2006)
Yucca schidigera Roezl.(Oleszek et al., 2001)
Medicago Arabica (Tava et al., 2009)
Antonia ovate (Alabdul Magid et al., 2012)
Medicago truncatula (Kapusta et al., 2005)
Dioscorea zingiberensis (Li, Huang, et al., 2010)
Allium nigrum L.(Mostafa et al., 2013)
Panax quinquefolium, Panax ginseng (Wu et al.,
2001)
Bacopa monnieri (Ganzera et al., 2004)
Chiococca alba (Borges et al., 2013)
Caulophyllum thalictroides (Avula et al., 2011)
Dioscorea panthaica (Wang et al., 2012)
Paris polyphylla var. yunnanensis, Paris polyphylla
var. chinensis (Zhang et al., 2010)
Platycodi Radix (Ha et al., 2006)
Ziziphus jujube, Z.jujuba var. spinosa (Guo et al.,
2011)
Aesculus chinensis Bunge (Chen, Li, et al., 2007)
Folium ginseng, Radix ginseng (Qian et al., 2009)
Panax notoginseng (Zhang, Liu, et al., 2013b)
Quantification studies
Chenopodium quinoa (Woldemichael & Wink,
2001)
Vigna radiata L. (Waller et al., 1999)
Antonia ovate (Alabdul Magid et al., 2012)
Dioscorea zingiberensis (Li, Huang, et al., 2010)
Tulbaghia violacea (Ncube et al., 2011)
Allium nigrum L.(Mostafa et al., 2013)
Soy and chickpea (Serventi et al., 2013)
Chiococca alba (Borges et al., 2013)
Processing parameters/optimization
Tribulus terrestris L. (Dinchev et al., 2008)
Caulophyllum thalictroides (Avula et al., 2011)
Gleditsia sinensis Lam.
(Lian & Zhang, 2013)
C.Y. Cheok et al. / Food Research International 59 (2014) 16–40
Maesa lanceolata
(Foubert et al., 2010)
Panax notoginseng (Lau et al., 2003; Li et al.,
2005)
Dioscorea panthaica (Wang et al., 2012)
Paris polyphylla var. yunnanensis, Paris
polyphylla var. chinensis (Zhang et al., 2010)
Platycodi Radix (Ha et al., 2006)
Ziziphus jujube, Z.jujuba var. spinosa (Guo et al.,
2011)
Pulsatilla turczaninovii (Xu et al., 2012) Xanthoceras sorbifolia Bunge (Li, Zu, et al.,
2010)
Dioscorea zingibernsis (Li, Huang, et al.,
2010
Panax ginseng (Kwon et al., 2003; Vongsangnak Panax ginseng (Vongsangnak et al., 2004)
et al., 2004; Wang et al., 2008)
Bupleurum chinense (Hu et al., 2008)
Gymnema sylvestre (Mandal & Mandal,
2010)
Aesculus chinensis Bunge (Chen, Li, et al., 2007) Aesculus chinensis Bunge (Chen, Li, et al.,
2007)
Panax quinquefolium (Engelberth et al., 2010)
23
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24
Table 4
Summary of studies focusing on saponins extraction using maceration extraction.

Plant material(s) Objective(s) Pretreatment and extraction condition Outcome(s) Reference

State of input Solid/solvent Solvent used Extraction Extraction Mechanical

material duration temperature aid

Allium ampeloprasum var. • To isolate a new steroidal saponin. Fresh bulbs 1.16 kg/5 l Methanol 72 h Room -. • Identified a new saponins. Adão et al.
porrum • To investigate its anti-inflammatory and temperature • Showed haemolytic effects in vitro and (2011)
antiulcerogenic properties in vitro and in vivo. demonstrated anti-inflammatory activity and
gastroprotective property in vivo.
Polygonatum odoratum • To isolate steroidal saponins. Air-dried roots 10 kg/– 70% ethanol 2h Boiling – • Three novel cholestane-type steroidal glyco- Bai et al.
point sides and three known steroidal glycosides sa- (2014)
ponins have been isolated and identified.
Genista ulicina Spach • To isolate triterpene saponins. Air-dried powder 1 kg/10 l 80% methanol – – – • Six triterpene saponins have been isolated. Boutaghane
et al. (2013)
Gynostemma pentaphyllum • To investigate the antiproliferation and Powder 30 g/150 ml ethanol 3h 60 °C Shaking • Both saponin and flavonoid fractions showed Cheng et al.
apoptosis mechanism of saponin and antiproliferation of prostate cancer cells (2011)
flavonoid fractions of Gynostemma effectively.

C.Y. Cheok et al. / Food Research International 59 (2014) 16–40

pentaphyllum.
Polygonatum odoratum • To characterize the anti-diabetic active frac- Dried 5 kg/40 l 80% ethanol 2h 80 °C – • Ethanol extract partitioned with n-buthanol Deng et al.
(Mill.) Druce tions in this herb. showed the highest anti-diabetic potential. (2012)
Ipomoea batatas tubers • To isolate, identify and quantify triterpenes Flour 6 g/– 80% methanol – – – • Two new saponins have been isolated and Dini et al.
glycosides. identified. Spectrophotometric method detected (2009)
• To evaluate their antioxidant properties. yield: 200.01 mg/100 g. HPLC–DAD: 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.
Cordyline fruticosa • To isolate new steroidal saponins and Dried pulverized 3 kg/– Methanol 24 h – – • Three new steroidal saponins, fruticoside H, Fouedjou
investigate their cytotoxic and antimicrobial leaves fruticoside I, and fruticoside J, have been isolated. et al. (2014)
activity. Fruticoside H and fruticoside I showed moderate
cytotoxic activity against human breast, colon, and
melanoma cell line. Fruticoside I showed moder-
ate antibacterial activity against the Gram-positive
Enterococcus faecalis.
Panax quinquefolius • To investigate the extraction efficiency of three Dried roots 1:5 50% ethanol; a mixture 6 weeks – – • The amount of total saponins was highest in the Gafner et al.
solvent systems on triterpene saponins from of 20% ethanol, 40% 50% ethanol extract (61.7 ± 0.1 mg/g dry root), (2004)
root of Panax quinquefolius. glycerin, and 40% although similar to the ethanol–glycerin–water
water; or 65% glycerin extract (59.4 ± 0.5 mg/g dry root).
Momordica charantia • To investigate antileishamanial activity of the Air dried 5 kg/– Water 48 h Room – Crude extract and momordicatin concentrations Gupta et al.
aqueous extract in vitro and in vivo. temperature of 16 mg/L and 0.4 mg/L were found inhibiting (2010)
100% parasites growth in vitro. In vivo, no
parasites were detected in hamster at doses of
300 mg/kg and 10 mg/kg of crude extract and
momordicatin.
• To isolate and identify trinocucurbitane and Air-dried 1.9 kg/5 l methanol 6h 60 °C – • Five new saponins have been isolated and Chen et al.
cucurbitane triterpenoids from roots. elucidated. (2008)
• To investigate the anti-HIV activity. • Possessed anti-HIV property.
• To quantify the saponin extract, the lipid Powder 10 g/150 ml Ethyl acetate 2h – – • White bitter gourd varieties were found to Habicht
extract, and the hydrophilic extract in contain significantly lower saponin concentrations et al. (2011)
bittergourd varieties. (0.25%) compared to green varieties (0.67%). The
lipid extract contained high amounts of conjugated
linoleic and linolenic acids (up to 65.89%).
Leguminous species • To characterize saponins of nine different Dried pulverized –/10 ml 80% methanol 24 h 25 °C – • A total of twenty saponins were characterized. Ha et al.
species of Leguminous using UPLC. seeds (2014)
Sapindus mukorossi • To investigate the molluscicidal effects of Powder 35.5 g/– Methanol 72 h – – • Bioassay data revealed that the seven isolated Huang et al.
saponins from Sapindus mukorossi against the saponins from soapnut were molluscicidal, (2003)
golden apple snail. causing 70–100% mortality at 10 ppm against
the golden apple snail.
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Plant material(s)

Objective(s) Pretreatment and extraction condition Outcome(s) Reference

State of input material Solid/solvent Solvent used Extraction duration Extraction temperature Mechanical
aid

Medicago truncatula • To quantify saponins in M. truncatula root, Lyophilized 10 mg/0.5 ml 80% methanol 2h Room Shaking • Roots (5924 ng/mg/dw) contained the greatest Huhman
leaves, stem, seedpod and seeds. powder temperature total amount of saponins followed by leaf et al. (2005)
(1064 ng/mg/dw) and seed (991 ng/mg/dw),
respectively.
Glycyrrhiza yunnanensis • To isolate triterpene saponins. Powder 18 kg/30 l 70% and 95% ethanol 2h – – • One new oleanane-type triterpenoid, seven Ji et al.
new triterpene saponins and four known sapo- (2014)
nins have been isolated.
Panicum virgatum L. • To isolate, characterize, and quantify of Dried 100 mg/5 ml methanol 30 min – Shaking • Three types of steroidal saponins have been Lee et al.
steroidal saponins in Switchgrass (Panicum isolated from 4 types of Switchgrass variety. (2009)
virgatum L.) Differences in the relative concentrations of
different saponins were observed between
switchgrass cultivars and plant parts.
Dioscorea pseudojaponica • To investigate the effects of domestic Freeze-dried 40 g/1 l Methanol 24 h 25 °C – • Results showed that the contents of saponins Lin et al.
Yamamoto processing on steroidal saponins and furostanol were decreased along with increasing cooking (2006)
and spirostanol glycosides. temperature and time except for the steaming
treatment. None of the steamed yam slices
significantly change their initial compositions or
quantities of furostanol and spirostanol glycosides.
• To isolate and identify steroidal saponins. Freeze-dried 600 g/6 l Methanol 24 h Room – • Six types of new steroidal saponins have been Yang et al.
temperature isolated and identified. (2003)

C.Y. Cheok et al. / Food Research International 59 (2014) 16–40

Rhizoma Anemarrhenae • To investigate the cognition-enhancing ef- Comminuted 1 kg/8 l 75% ethanol 1h 80 °C – • Fndings demonstrated that diabetes-associated Liu, Zhu,
fects of total saponins. cognitive declined. et al. (2012)
Silphium asteriscus L. • To isolate saponins and investigate their Dried leaves and 500 g/– CH2Cl2–methanol, 48 h – – • Eighteen saponins with highly hydroxylated Masullo
antiproliferative activity. stems methanol, 50% oleanane-type aglycones were isolated. The iso- et al. (2014)
methanol lated compounds showed antiproliferative ac-
tivity against human epitheloid cervix
carcinoma, leukaemic T-cell line, and colorectal
adenocarcinoma.
Solanum xanthocarpum • To establish the scientific rationality of Fruit powder 100 g/500 ml 50% ethanol 24 h – – • The antiurolithiatic activity in Solanum Patel, Patel,
Solanum xanthocarpum fruit saponin rich xanthocarpum is mediated possibly through the et al. (2012)
fraction on antiurolithiatic activity using in vitro inhibition of CaOx crystal formation and its effect
calcium oxalate (CaOx) crystallization and on the urinary concentration of stone-forming
in vivo ethylene glycol induced urolithiasis in constituents and nephrolithiasis inducing factors.
the male albino Wistar rats.
Agave offoyana • To isolate steroidal saponins. Dried powder 0.5 kg/– 70% ethanol 48 h Room – • Five unknown and six known steroidal Pérez et al.
temperature saponins have been isolated. (2013)
Dioscorea zingiberensis • To determine the biochemical, hematological Air-dried 600 g/2 L Ethanol 12 h Room – • The steroidal saponins did not show any sign of Qin et al.
and histopathological toxicity of steroidal temperature toxicity up to oral dose of 562.5 mg/kg in mice. (2009)
saponins from Dioscorea zingiberensis rhizome No significant changes of biochemical and
after acute oral dosing in mice and sub-chronic hematological parameters in rats (except at
oral administration in rats. 510 mg/kg/day).
• To isolate five steroidal saponins Dried 3.5 kg/– 70% ethanol – – – • Five steroidal saponins have been isolated and Zhang, Ito,
identified. et al. (2013)
Yucca gloriosa L. • To isolate and quantify steroidal saponins. Powder 200 g/– 80% methanol – Room – • Five steroidal glycosides including three Skhirtladze
temperature spirostane, one furostane and one cholestane et al. (2011)
glycosides, along with seven known compounds
have been isolated and characterized, The dried
extract obtained more than 25% w/w of glycosides.
Medicago sativa L. • To investigate the effect of high and low Freeze-dried 1 g/100 ml 70% methanol 1h 60 °C Shaking • The high-saponin line of alfalfa differed from Sylwia et al.
saponin lines of alfalfa on pea aphid the low-saponin one by the presence of zanhic (2006)
performance. acid tridesmoside and a higher level of 3-
• To evaluate the differences in saponins within GlcA,28-AraRhaXyl medicagenic acid glycoside.
the studied lines and estimate an influence of • The saponins incorporated into sucrose–agarose
some isolated compounds on the pea aphid gels significantly reduced number of the aphid
probing behavior. probes into the gels and extended their duration
in comparison to the control gels (without tested
compounds).
Dried 100 g/1 L 1h 50 °C Stirring

(continued on next page)

25
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26
Table 4 (continued)
Plant material(s) Objective(s) Pretreatment and extraction condition Outcome(s) Reference

State of input Solid/solvent Solvent used Extraction Extraction Mechanical

material duration temperature aid

Chenopodium quinoa • To investigate the immunoadjuvant activity, 40% hydroethanol • The two quinoa saponin fractions enhanced Verza et al.
Seeds toxicity assays. solution significantly the production of humoral and (2012)
• To determine triterpenic saponins using UPLC/ cellular immune responses to ovalbumin in mice.
Q-TOF-MS. • Two quinoa saponin fractions were obtained.
• To isolate and characterize of twelve triterpene Dried 4 kg/– 90% ethanol 3 days Room – • Twelve triterpene saponins have been isolated, Zhu et al.
saponins. temperature and their structures were characterized on the (2002)
basis of hydrolysis and spectral data, especially
NMR evidence.
Harpullia austro- • To isolate triterpenoid saponins. Dried stem bark 1.110 kg/10 l 20% methanol 17 h and - – • Eight new and one known acylated triterpenoid Voutquenne
caledonica powder boiled 3 h saponins were isolated. et al. (2005)
Fagonia indica • To isolate a novel saponin glycoside. Dried powder 200 g/1 l Ethanol 14 days Room – • A novel steroidal saponin has been isolated. Waheed
• To perform the apoptosis and necrosis test on temperature • It was able to induce apoptosis or necrosis in et al. (2012)
three cancer cell lines. cancer cells depending on the cell type.
Panax notoginseng • To investigate the mechanisms of anti- Powder 75 g/1 L 75% ethanol 4h - – • PNS possess anti-hyperglycemic and anti-obese Yang et al.
hyperglycemic and anti-obese effects of Panax activities by improving insulin- and leptin sen- (2010)

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notoginseng saponins (PNS) in KK-Ay mice. sitivity, and ginsenoside Rb1 is responsible for
the anti-hyperglycemic effect among the five
saponins in KK-Ay mice.
Salicornia herbacea • To isolate triterpene saponins and investigate Fresh parts 22.6 kg/– 80% acetone 24 h Ambient – • Two new noroleanane-type triterpene sapo- Zhao et al.
their antiproliferative activity. temperature nins, Salbige A and Salbige B, have been isolated. (2014)
Both compounds exhibited potent antiprolifera-
tive activities against cancer cells.
Entada phaseoloides L. • To evaluate the potential therapeutic effects Dried powder – 70% ethanol 2h – – • Total saponins demonstrated both Zheng et al.
of total saponins from Entada phaseoloides hypoglycemic and hypolipidemic in type 2 (2012)
(TSEP) in experimental type 2 Diabetes diabetic rats which indicating possess anti-
mellitus (T2DM) rats. diabetic property.
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C.Y. Cheok et al. / Food Research International 59 (2014) 16–40
the solvent between cycles if the extraction process consists of one
or several extraction cycles. A high pressure pump is required 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 tempera-
ture 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 saponin 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 et al., 2009;
Wan, Zhang, Ye, & Wang, 2008; Wan et al., 2006; Zhang, Liu,
Qi, Li, & Wang, 2013) could be due to the precious value of the
product.
The efficacy of ASE in saponin extraction has been studied and com-
pared 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 (Güçlü-Üntündağ et al., 2007). Simi-
larly, 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, Clausen, &
Carrier, 2010). Although a slight increase in saponin yields of escin Ia,
escin Ib, isoescin Ia and isoescin Ib was obtained in extraction from
Aesculus chinensis Bunge using ASE, it required shorter extraction
time of 7 min compared to reflux and sonication extraction of 1 h
and 30 min, respectively (Chen, Li, et al., 2007). Pressurized 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 P. notoginseng
(Wan et al., 2006).
6. Quantification of saponins
Prior to the quantification of total saponins of a plant source, it is ap-
propriate to carry out a simple procedure to test the presence of sapo-
nins. This can be done by putting the plant material into a test tube
filled with distilled water and vigorously shaken for 2 min (Ncube
et al., 2011). The appearance of stable and persistent foam on the liquid
surface for 15 min indicated the presence of saponins. The quantifica-
tion of plant saponins is usually carried out by spectrophotometric
and chromatographic methods. The difference between the quantitative
expression of the two methods is that the spectrophotometric's gives a
total saponin value while the chromatographic's quantifies specific sa-
ponin compound.
6.1. Spectrophotometric method
The reason why the spectrophotometric technique has become a
popular method in the quantification of saponins from plant mate-
rials could be because it is simple, fast and inexpensive to operate.
Total saponins also known as vanillin-sulfuric acid assay, is the
most commonly selected spectrophotometric method in plant sapo-
nin 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, Zu, et al., 2010). Sul-
furic acid is used as oxidant and the distinctive color of this reaction
is purple (Hiai, Oura, & Hakajima, 1976) and sometimes perchloric
acid is used (Chen, Xie, et al., 2007; Li, Zu, et al., 2010; Wu et al.,
2001). Due to differences in selection of reagent, condition to allow
full color development, standard, and wavelength from previous
27
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.
Two works (Ncube et al., 2011; Patel, Patel, et al., 2012) were found
using the conditions of 60 °C and 10 min to allow the mixture to have
full color development following the procedure in Hiai et al. (1976).
However, other researchers used 70 °C for 15 min (Chen, Xie, et al.,
2007) and 20 min (Li, Zu, et al., 2010) to allow full color development.
This inconsistency should be standardized because the reaction time
may directly attribute to the final absorbance value which later trans-
lates 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 catego-
rized in steroid saponins (Harborne & Baxter, 1999). Since total saponin
method is to quantify total saponins from the reaction of oxidized
triterpene saponins with vanillin (Li, Zu, et al., 2010), these inevitably
raise a question whether the selection of standard to be used in spectro-
photometer 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
fall within the range of 480–610 nm, except 473 nm (Mostafa et al.,
2013) and 283 nm (Liu, Zhu, et al., 2012), most probably due to the
maximum absorption of purple color which falls within this range
(Bruice, 2007).
Despite total saponins, total steroidal sapogenin is employed to quan-
tify specifically the steroidal saponins content of plant material (Baccou,
Lambert, & Sauvaire, 1977). This method is also based on color reactions
with anisaldehyde (or vanillin), sulfuric acid and ethyl acetate measured
at maximum wavelength (λmax) of 430 nm (Baccou et al., 1977). The dif-
ference between total saponin and total steroidal sapogenins is the sol-
vent used to prepare the reagents. For total saponins, vanillin reagent
is prepared by diluting with ethanol and sulfuric acid with water, where-
as for total steroidal sapogenins, both the anasaldehyde and sulfuric acid
are diluted with ethyl acetate. Total steroidal sapogenins has been prov-
en stable and reproducible with a number of standards, i.e., 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 μg of crude extract and dissolved in 2 ml of ethyl acetate in the
test tube. Then mixed with 1 ml of reagent A (consisting of 0.5 ml
anisaldehyde and 99.5 ml ethyl acetate) and 1 ml of reagent C
(consisting of 50 ml concentrated sulfuric acid and 50 ml ethyl ace-
tate). The test tube with mixtures was placed in a water-bath at 60
°C for 10 min to allow full color development. Then, it was cooled
for 10 min 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 yielded
5.70 mg DE/g (Chan et al., 2013). Qin et al. (2009) carried out the
total steroidal sapogenin determination of D. zingiberensis with
some modifications. They used perchloric acid instead of sulfuric
acid. The test tube was placed in a water bath maintained at 70 °C
for 15 min to develop color 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 min of stabilization, only then the test
tube was subjected to measure its absorbance at 454 nm using a
spectrophotometer and yielded total steroidal saponins of 28.34%
(w/w) of lyophilized powder.
Hemolytic method is another spectrophotometric method used
to quantify saponin content of a plant material (Barve, Laddha, &
Jayakumar, 2010). The principle of this method is the reaction of sa-
ponins with blood reagent to release oxy-hemoglobin which results a
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Table 5
Summary of studies focusing on saponins extraction using reflux and Soxhlet extraction.

Plant material(s) Objective(s) State of input Solid/solvent Extraction Conditions Finding(s) Reference
material
Solvent used Duration
(h)

Reflux extraction:

C.Y. Cheok et al. / Food Research International 59 (2014) 16–40

Caryocar villosum • To isolate triterpenoid saponins Dried powder 400 g/3.5 l Methanol 3 • Seven triterpenoid saponins have been isolated and identified. Alabdul Magid
et al. (2006)
Allium nutan L. • Isolation of four steroidal saponins from underground parts Oven-dried 300 g/500 ml 80% methanol 2 • Four steroidal glycosides including deltoside and nolinofuroside D Akhov et al.
of A. nutans. powder and two novel saponins were isolated. (1999)
Aralia taibaiensis • To isolate four new triterpenoid saponins. Air-dried 1.2 kg/1 l 70% ethanol – • Four new oleanane type triterpenoid saponins have been isolated. Bi et al. (2012)
• To investigate the four newly derived saponins on powder • Compounds exhibited moderate effects on antioxidant and
antioxidant and antiglycation activities. antiglycation activities which correlated with treatment of diabetes
mellitus.
Defatted rice bran • To fractionate with different solvent systems. Oven-dried 10 g/150 ml 50% ethanol 3 • The highest yield (236.14 ± 17.72 mg DE/g extract of fraction) was Chan et al.
• To investigate the antioxidant activity. obtained in n-buthanol fraction. (2013)
• Phenolic-saponins rich fraction showed higher antioxidant activity.
Flos Lonicerae • To determine triterpenoid saponins in five specicies of Flos Oven-dried 2.0 g/25 ml 60% ethanol 4 • Seven major saponins were found. Chai et al.
Lonicerae both qualitatively and quantitatively. pulverized • 53.7 mg/g of dipsacoside B was found in L.hypoglauca, more than (2005)
41.0 mg/g of macranthoidin B was found in L. macranthoides,
L. confuse, and L. similis.
Xanthoceras sorbifolia • To isolate and quantify saponins from parts of husks, twig Dried 1 kg/1 l 70% ethanol 2 • Two new triterpenoid saponins, sorbifoside A and B, were isolated. Ling et al.
bark and xylem, seed coats and kernels, flowers, and leaves. Among the parts, husk was found having the highest sorbifoside A (2011)
(157.0 μg/g) and B (58.33 μg/g).
Paris polyphylla var. • To evaluate cytotoxic and hemolytic activities of steroidal Oven-dried –/250 ml 70% ethanol 2 • The number of sugar chains of Paris steroidal saponins played a Liu et al. (2013)
chinensis saponins. powder crucial role in hemolysis, while the kinds of saponins influenced 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.
Paris and Trillium plants • To determine major saponins in Paris and Trillium plant Oven-dried 0.5 g/50 ml 80% ethanol 1.5 • Saponins of Paris VI was found majorly in Paris fargesii (19.520 mg/g), Man, Gao,
qualitatively and quantitatively. powder and Trillium tschonoskii (11.770 mg/g). Zhang, Wang,
et al. (2010)
Guar meal • To isolate saponin rich extract. Ground 25 g/250 ml 50% ethanol 3 • Crude saponin-rich guar meal extract was purified by RP-HPLC Hassan et al.
• To evaluate its fractions for haemolytic and antibacterial eluting 20%, 60% and 100% methanol fractions with 2.04 ± 0.32%, (2010)
activities against two gram positive bacteria (Lactobacillus spp. 0.91 ± 0.16% and 1.55 ± 0.15% DM of crude saponin-rich GM ex-
and Staphylococcus aureus) and two gram negative bacteria tract, respectively.
(Escherichia coli and Salmonella Typhimurium) in a dose • Results indicated that only 100% methanol fraction and its 16 min
dependent manner. peak sub-fraction exhibited both haemolytic and antibacterial ac-
tivities against Staphylococcus aureus, Salmonella Typhimurium and
Escherichia coli, but 20% and 60% MeOH fractions stimulated Lacto-
bacillus spp. growth.
Panax japonicus • To study the cardioprotective effects of saponins. Dried pieces 1 kg/4 l 60% ethanol 2 • Saponins from Panax japonicus exerted beneficially He et al. (2012)
cardioprotective effects on myocardial ischemia injury rats, mainly
scavenging oxidative stress-triggered overgeneration and accumu-
lation of reactive oxygen species, alleviating myocardial ischemia
injury and cardiac cell death.
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Plant material(s)

Objective(s) State of input Solid/solvent Extraction Conditions Finding(s) Reference

material
Solvent used Duration (h)

Momordica charantia • To investigate the chemical constituents from fresh fruit. Fresh fruits 3 kg/24 l 90% ethanol 1.5 • A new C30 sterol glycoside was isolated. Liu, Lu, et al.
(2012)
• To isolate and identify new triterpenoid saponins. Air-dried 30 kg/100 l Ethanol – • Identified fourteen cucurbitane triterpenoids. Chen et al.
• To investigate their anti-HIV activity in vitro. powder • Exhibited weak anti-HIV activity. (2009)
• To investigate two classes of saponins, i.e., cucurbitane and Lyophilized – Methanol 4 • Saponins containing extract reduced preadipocyte proliferation Popovich et al.
oleanane type triterpenoids, for the potential to reduce pieces and lipid accumulation of the adipocyte. (2010)
preadipocyte viability, lipid accumulation and adiponectin
expression in 3T3-L1 cells.
Medicago sativa var. • To determine the occurrence of individual saponins. Freeze-dried 200 mg/10 ml 30% methanol 1.5 • It was shown that monodesmosidic medicagenic acid glycoside was Oleszek (1998).
Boja • To verify early biological data on their quantification with powder synthesized after 4 days of germination and subsequently followed
new analytical techniques. by bidesmosidic saponin production.
• The total saponin concentration increased from 2.12 μmol/g of dry
matter at the beginning of germination to around 6 μmol/g after
8–16 days of seedling growth.
Radix Astragali • To isolate four main saponins. Oven-dried 1.5 g/60 ml Methanol 3 • Four main saponins have been isolated successfully. Qi et al. (2006)
powder
Bupleurum chinense • To evaluate the haemolytic activities of Bupleurum chinense Powder 1 kg/– 70% ethanol 2 • Bupleurum chinense saponins showed a slight haemolytic effect and Sun (2006)
saponins and its adjuvant potentials on the immune responses enhanced significantly a specific antibody and cellular response
of ICR mice against ovalbumin. against ovalbumin in mice.
Glycyrrhiza inflate, • To isolate and quantify triterpenoid saponins from species of Dried powder 0.5 g/30 ml 50% methanol 1 • Ten saponins have been isolated and the total saponins yields were Tao et al. (2013)
Glycyrhiza glabra, Glycyrrhiza. found in the range of 28.958–119.750 mg/g.

C.Y. Cheok et al. / Food Research International 59 (2014) 16–40

Glycyrrhiza uralensis
Maesa lanceolata • To determine saponins using LC–UV method. Dried powder 1.5 g/70 ml 50% methanol 1 • 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 6 • Six triterpenoid saponins have been isolated. The total saponins Wang et al.
found were 4.45–27.32 mg/g. (2007)
Acanthopanax • To isolate four saponins. Air-dried 10 kg/80 l Hot water 1 • Three known and one novel saponins have been isolated. Yoshizumi et al.
sessiliflorus • To determine their inhibitory activities on pancreatic lipase powder • Sessiloside and chiisanoside inhibited pancreatic lipase activity (2006)
leaves in vitro and in vivo. in vitro, and addition of the saponin-rich fraction to a high-fat diet
suppressed the body weight gain of mice.

Soxhlet extraction:
Chenopodium quinoa • To identify and determine biological activities of triterpenoid Dried powder 900 g/– Petroleum ether, 72 • Sixteen saponins were detected and isolated. Both bidesmosides Woldemichael
seed saponins. methanol and derived monodesmosides showed little or no antifungal activity, and Wink
whereas a comparatively higher degree of hemolytic activity could (2001)
be determined for monodesmosides.
Vigna radiata L. • To focus on plant adaptability, stages of growth, Fresh plant – Chloroform, 80% 24 • Saponins produced by mungbean plants added to the soil enhanced Waller et al.
concentration and type of allelochemical measurement of ethanol the growth of new mungbean plants as an allelochemical plant (1999)
biological activity, etc. to fully explore the allelochemical growth regulator.
response.

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30 C.Y. Cheok et al. / Food Research International 59 (2014) 16–40

Table 6
Summary of studies focusing on saponins extraction using two subsequent extractions.

Plant materials Objective(s) Pretreatment/procedures Finding(s) References

Subsequent methods employed: Soxhlet » reflux

Medicago Arabia L. • To isolate eight major triterpene saponins.
The powdered plant material was defatted • Eight major triterpene saponins have Bialy et al. (2004)
with chloroform in a Soxhlet apparatus and been isolated.
then extracted with 80% methanol under
reflux.
Medicago hybrid roots • To isolate fourteen triterpene saponins. The ground roots were defatted with • Fourteen triterpene saponins have been Bialy et al. (2006)
chloroform in a Soxhlet apparatus for 48 h isolated.
and then extracted with 95% methanol
under reflux for 24 h.
Yucca schidigera • To isolate and characterize the dominant Yucca powder (300 g) was defatted with • Eight steroidal saponins have been Oleszek et al. (2001)
Roezl saponins of yucca trunk. chloroform in a Soxhlet. After drying, it was isolated.
extracted three times by boiling with water
for half an hour.
Medicago arabica (L.) • To acquire further evidence of the Powdered leaves (500 g) were defatted • Fourteen known saponins have been Tava et al. (2009)
leaves previously reported compounds in with chloroform in a Soxhlet apparatus isolated, and five new saponins with a
M. arabica, and elucidate the chemical (fats 4.3% DM). Defatted material (200 g) hydroxy group at the 30-methyl position
structure of these newly identified saponins. was then extracted with 80% methanol of the triterpenic skeleton were identified.
under reflux for 24 h.

Subsequent methods employed: Maceration » reflux

Antonia ovata leaves • To isolate six new polyhydroxyoleanene
saponins.
• To test the effects on the growth of human
oral epithelium carcinoma (KB) cell line.
Tribulus terrestris L. • To quantify the distribution of steroidal
saponins of the plant from different
geographical regions.
Medicago truncatula • To identify and determine saponins in
aerial parts of barrel medic using LC–ESI/
MS/MS.
Dioscorea zingiberensis • To characterize the major active
constituents.
• To evaluate the anti-thrombotic activity.
Dried powdered leaves were macerated in
5 l of petroleum ether for 6 h and percolated
during 12 h.
The solvent was evaporated and the dried
extract was then macerated for another 2 h
with 5 l of 90% aqueous methanol and
further refluxed for 3 h.
One gram of the plant material (aerial parts,
fruits, leaves and stems) was extracted with
50 ml chloroform for 1 h, by shaking at room
temperature.
The extract was filtered and the plant
material was extracted again, three times by
refluxing with 50 ml of 70% ethanol for 1 h.
One gram of dried and finely powdered
barrel medic tops (leaves and stems) was
extracted overnight with 50 ml of 80%
methanol at room temperature.
The extract was filtered, and the residues
were additionally extracted twice by
refluxing with 50 ml of 80% methanol for 1 h.
An accurately weighed sample of 600 g
powder was put into a round bottom flask,
soaked with 2000 ml ethanol at room
temperature for 24 h, and then refluxed
with a mechanical stirrer at 78 °C for 2 h.
The heat-reflux extraction process was re-
peated twice.
• Six pentacyclic triterpenoid saponins, Alabdul Magid et al.
named antoniosides E–J along with two (2012)
known alkaloids, were isolated.
• Inhibited the growth of oral epithelium
carcinoma cell.
• Samples from Bulgaria were found to Dinchev et al. (2008)
contain a rather high percentage (0.245–
1.337%) of protodioscin, whereas samples
from China contained only small amounts
(0.063% and 0.089%) of this compound.
• Aerial parts of all three subspecies Kapusta et al. (2005)
contained 17 saponins previously identified
and also a substantial amount of
astragaloside VIII (3-GlcA-Xyl-Rha
soyasapogenol B), not previously reported
in M. truncatula.
• A chemical fingerprint method was firstly Li, Huang, et al. (2010)
established and validated to quantify and
standardize rhizomes including
parvifloside, protodeltonin, protodioscin,
protogracillin, zingiberensis saponin,
deltonin, dioscin and trillin.
• The results indicate that total steroidal sa-
ponins could inhibit thrombosis by both
improving the anticoagulation activity and
inhibiting platelet aggregation action, sug-
gesting that total steroidal saponins from
Dioscorea zingiberensis rhizomes have the
potential to reduce the risk of cardiovascular
diseases by antithrombotic action.

Subsequent methods employed: Soxhlet » maceration

Tulbaghia violacea • To compare the antimicrobial and
phytochemical properties of in vitro cultured
and outdoor grown Tulbaghia violacea plants
in the quest to validate the use of
micropropagated plants as alternatives to
outdoor grown plants in traditional
medicine.
The dried and ground plant samples were • Extracts of micropropagated plants and Ncube et al. (2011)
defatted with hexane in a Soxhlet apparatus outdoor grown plants showed good
for 3 h. antibacterial activity in vitro.
After air drying, saponins were extracted
twice from the defatted samples (10 g) in
100 ml of 50% aqueous methanol by
incubating at room temperature overnight
with continuous stirring.

measurable color for spectrophotometer. The saponin concentrations EDTA-blood at 30 °C for 30 min. After centrifugation for 10 min, he-
in bittergourd varieties were quantified using hemolytic method moglobin was quantified in the supernatant photometrically at 545
(Habicht et al., 2011). The saponin extract was dissolved in distilled nm and the result was expressed in hemolytic saponins. They re-
water and 100 μl of this solution was incubated with 1 ml fresh vealed that white bitter gourd varieties were found to contain

Table 7
Summary of studies focusing on saponins extraction using ultrasound-assisted extraction (UAE).
Plant material(s)
Caulophyllum thalictroides
Chiococca alba
Maesa lanceolata
Bacopa monnieri
Ziziphus jujuba, Z.jujuba var.
spinosa
Platycodi Radix
Panax notoginseng
Gleditsia sinensis Lam.
Soy and chickpea
Allium nigrum L.
Dioscorea panthaica
Paris polyphylla var.
yunnanensis, Paris
polyphylla var. chinensis
Objectives
• To compare chromatographic performance
of HPLC and UPLC in determining saponins
from the roots.
• To isolate five triterpenoid saponins from
the roots.
• To investigate their inflammatory activity
in vitro.
• To evaluate the efficiency of UPLC in quan-
tifying 14 saponins from the leaves.
• To separate and isolate triterpenoid
saponins.
• To perform simultaneous qualitative and
quantitative analysis in the leaves.
• To determine and quantify ten major
saponins.
• To analyze saponins in raw and steamed
P. notoginseng.
• To quantify six major active saponins using
HPLC.
• To determine the contents of saponins.
• To investigate the stability during
breadmaking and in vitro bioaccessibility of
saponins from soy and chickpea.
• To determine the content of cysteine
sulfoxides, total polyphenols, and total
saponins in different organs of A. nigrum by
using high-performance liquid chromato-
graph (HPLC) and spectral techniques.
• To isolate, quantify, and assess the antifungal
activity of the aginoside compound in a wide
range of phytopathogens.
• To isolate and quantify steroidal saponins.
• To isolate and quantify steroidal saponins.
Author's personal copy
Pretreatment/extraction procedure Findings Reference
Dry plant samples were weighed and sonicated in 2 ml of methanol • Eight triterpene saponins (cauloside H, leonticin D, cauloside G, cauloside D, cauloside Avula et al. (2011)
for 30 min. B, cauloside C, cauloside A and saponin PE) were separated within 35 min using HPLC
method and within 8.0 min using UPLC method with detection limits of 10 g/ml for
saponins.
The ethanolic extract of the pulverized dried roots of C. alba • Five types of triterpenoid saponins have been isolated and identified. Borges et al. (2013)
(1 kg) was obtained with the assistance of an ultrasonic bath, after • The saponin fractions have been observed against in vitro lipopolysaccharide-induced
extraction with hexane and methylene chloride. inflammation.
Hundred grams of dried plant material was sonicated in 5 ml of 50% • A rapid and sensitive UPLC–MS/MS method was developed to relatively quantify the Foubert et al. (2010)
methanol (v/v) for 1 h. amount of 14 individual maesasaponins present in the plant material of M. lanceolata.
One gram of finely powdered plant material was extracted three • Several B. monnieri samples (extract, plant material, commercial products) were Ganzera et al.
times with 3 ml methanol by sonication for 10 min. successfully analyzed, each of them containing at least four of the seven reference (2004)
compounds.
• Main components were either bacoside A3 or bacopaside II, least dominant showed to
be bacopasides IV and V.
• The total saponin content in the samples varied from 1.1 to 13.0%.
C.Y. Cheok et al. / Food Research International 59 (2014) 16–40
The dried powders of Z. jujube and Z. jujube var. spinosa leaves • Two saponins were separated and quantified having 1.41 mg/g of zizyphus saponins I Guo et al. (2011)
(1.0 g, 40 mesh) were extracted with 20 ml of 80% methanol by and 4.52 mg/g of zizyphus saponins II, respectively.
ultrasonication (40 kHz) at room temperature for 30 min.
Approximately 1 g of the finely powdered Platycodi Radix was extracted • Ten major saponins have been identified with the most majorly discovered were platycodin Ha et al. (2006)
three times with 10 ml of each solvent system (water, 30% MeOH, 50% D of 2431 μg/g, polygalacin D of 2116 μg/g, and platycoside E of 1088 μg/g.
MeOH, 70% MeOH and MeOH) by sonication for 10 min.
10 ml of 70% methanol was added to 1 g of the powdered sample. The • The contents of ginsenosides Rg1, Re, Rb1, Rd, and notoginsenoside R1 in most of the Lau et al. (2003)
suspension was ultrasonically (230 V) extracted for 20 min and raw samples were found to be higher than those in the corresponding steamed samples.
filtered.
To the dried powders of samples (40 mesh, 30 mg or so), 5.0 ml of • This HPLC assay performed using a reversed-phase C18 column with gradient elution of Li et al. (2005)
80% methanol solution with 1 ml internal standard solution acetonitrile and 0.01% formic acid in 30 min, provided good reproducibility and sensitivity
(2.5 mg/ml) was added and extracted in an ultrasonic bath for for the quantification of six saponins with overall intra- and inter-day precision and accu-
60 min. racy of less than 4.0% and higher than 90%, respectively.
• This assay is successfully applied to the determination of the six saponins in 23
notoginseng samples.
Each of the ground gleditsia materials (1.50–1.60 g) was placed in a • The established HPLC analytic method was successfully used to determine the concen- Lian and Zhang
250 ml flask with 50 ml of methanol and then by ultrasonic trations of 29 compounds including 19 gleditsia saponins and ten unidentified eight com- (2013)
treatment for three times (each 5 min). mercial gleditsia fruits from different sources. The results from this study suggested that
this newly developed HPLC method could be used for qualitative and quantitative analysis
of the saponins in the ingredients.
Entire loaves of breads were processed to a fine paste with a grinder • Saponin structure and food matrix affect the stability of saponins during processing and Serventi et al.
and aliquots (150 mg) were mixed with 3 ml of 70% ethanol in 4 ml digestion and that uptake of saponins by enterocyte-like cells is poor despite moderate (2013)
vials. apparent bioaccessibility.
Mixtures were sonicated for 2 min, passed through a 0.2 μm nylon
filter.
Fresh root–bulb basal stem of A. nigrum (80 g) was hand-cut and • The HPLC and spectral analyses of cysteine sulfoxides (CSOs), total polyphenols (TP), Mostafa et al.
air-dried at room temperature, and the final dry weight (30 g) was and total saponins revealed quantitative variations within the different organs of Allium (2013)
obtained and used for this study. nigrum L. A large accumulation of CSOs was detected in the bulb (0.367 mg/g fw), of TP
The dry weight was exhaustively extracted at room temperature in the leaf (116.05 mg CE/100 g fw), and of saponins in the root (19.38 mg/gdw).
with the following solvents: n-hexane and 70% methanol. • Aginoside saponins showed significant antifungal activity depending on the concen-
Each solvent extraction step was conducted for 1 day and repeated tration.
three times with 30 min of sonication.
The fine powder of plant (0.4 g) was accurately weighed and placed • Six steroid saponins were determined. The saponins found were in the range of Wang et al. (2012)
in a conical flask. After adding 20 ml of 75% ethanol to the flask, the 2571.3–37435.1 μg/g.
mixture was sonicated for 60 min.
Powder of plant material (0.5 g) was extracted in 50 ml of methanol • Ten and seven saponins were determined in P. polyphylla var. yunnanensis and Zhang et al. (2010)
with sonicated for 30 min. P. polyphylla var. chinensis, respectively, including four unknown compounds. Total
saponins for P. polyphylla var. yunnanensis was 19.62 mg/g and P. polyphylla var.
chinensis was 1.63 mg/g.
31
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32
Table 8
Summary of studies focusing on saponins extraction using microwave-assisted extraction (MAE).

Plant material(s) Objectives Extraction procedure Finding Reference

Ganoderma atrum • 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
• To find an efficient extraction method and
optimize the MAE for saponin from cultured 2450 MHz (P182, Whirlpool, USA). Fasks with 100 mg of dried cells and 15 ml of water- 7.4 mg/100 mg DW, compared to other methods that took 2–14 h.
cells of Panax notoginseng.
MAE was performed in the closed vessel unit MDS2002 (Xin'yi Microwave Extraction
Instrument Company, Shanghai) equipped with temperature sensor. Maximum oven power
for this system is 800 W. For the first series of experiments 3 g of the whole dried and ground
material were placed in 100 ml PFTE (CEM) extraction vessels and appropriate amount of
solvent was added (30 or 75 ml). The extraction temperature was set at different degrees due
to different solvents and programmed as follows: ramp to the temperature for 5 min step by
step, hold at temperature for a few minutes. Microwave power was 800 W (100%). After
extraction, the vessels were left for 30 min to cool down below 35 °C.
The extractions were performed using MAP extractor (Prolabo, France) at full power (300
W) and an emission frequency of 2450 MHz for 30 s on a mixture consisting of different
amounts of ginseng powders (2.5, 5.0, 10.0 g) and 50 ml of 80% methanol.
Microwave-assisted extraction was performed using a household microwave oven of
saturated n-butanol were exposed to the microwave (at a power of 90 or 125 W). A
• Compared with shaking extraction method, heat reflux extraction, Chen, Xie, et al.
supercritical fluid carbon dioxide extraction (SFE) and normal ultrasound- (2007)
assisted extraction (UAE), MAE only need 5 min to give the highest yield of
triterpenoid saponins at 0.968%, while the other extraction methods need
several hours or even more than 10 h and give lower yield.
• Results indicated that the MAP was more superior than the conventional Kwon et al.
method in its capability to extract target components without causing any (2003)
degradation. Additionally, it dramatically reduced the extraction time from
12 h to a few seconds, suggesting that it can be an alternative technique to
the time-consuming
conventional reflux method.
• The microwave-assisted extraction for 6 min led to a saponin yield of Vongsangnak
et al. (2004)
• The optimal extraction method was microwave-assisted extraction for

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temperature of 20, 50 or 80 °C was controlled by interrupting the irradiation every 15 or 4 min of extraction time at 50 °C of extraction temperature (at a radiation
30 s and cooling the irradiated samples in an ice-bath. power of 125 W) and 1:150 of the solid/liquid (w/v) ratio.
• To evaluate the efficiency of high pressure The high pressure MAE was performed in a WRT-C microwave preparation system with a • High pressure MAE took a shorter time of 10 min and obtained higher Wang et al.
MAE as a fast extraction of ginsenosides from pressure, temperature and time control system (Meicheng Technology Co., Ltd., Beijing, yields of ginsenoside Rb1 (8.74 mg/g DM), Rc (7.63 mg/g DM), Rb2 (2008)
Panax notoginseng. China). A sample powder of 1.00 g was accurately weighed, transferred into the liner (6.70 mg/g DM), and Rd (4.91 mg/g DM).
vessel and immersed by 40 ml extraction solvent. After the closed control and extraction
vessels were put into the microwave preparation system, the pressure was turned on and
began to gradually increase. When the pressure reached preset pressure, irradiation time
was counted and the extraction was carried out continuously at the preset pressure.
Bupleurum chinense • To study the relationships between the Experiments were carried out using a custom made microwave reactor with cylindrical • With ethanol concentration of 47–50%, the optimum MAE conditions of Hu et al. (2008)
extraction yields of saikosaponins and the cavity (model 961, Microwave Power Consultants, VIC, Australia). This microwave reactor 360–400 W microwave power, temperature of 73–74 °C, and extraction
operating parameters of MAE. is a continuously variable microwave power system with power output up to 1000 W and time of 5.8–6.0 min, yielded 96.18–96.91% saikosaponin a, 95.05–95.71%
a fiber optical temperature controller. saikosaponin c, and 97.05–97.25% saikosaponin d.
Xanthoceras sorbifolia • To optimize the microwave-assisted extrac- MAE is carried out using MASII Microwave Extraction Testing Equipment. One gram defatted • The optimum extraction parameters were 51 °C, 7 min, 900 W, 32 ml/g, Li, Zu, et al. (2010)
Bunge. kernel tion procedure for triterpene saponins from the residue was placed into the extraction vessel. 42% ethanol and 3 cycles, obtaining the highest extraction yield of
defatted residue of yellow horn kernel. The initial conditions were as follows: temperature 30 °C, duration 3 min, irradiation triterpene saponins of 11.62 ± 0.37% of defatted kernel.
power 300 W, ratio of solvent to material 20 ml/g, 10% ethanol and 1 extraction cycle.
For the subsequent condition optimization of MAE, the above parameters were chosen
differently according to the experimental design.
Dioscorea zingibernsis • To extract the total steroids in the culture Extraction of total steroid was performed using MAATPE. • An improved MAATPE could thoroughly extract the steroids from the Li, Huang, et al.
broth from the fermentation of Dioscorea Thirty milliliters of culture broth obtained from the fermentation of 1 g DZW powder was fermentation and subsequent application of three-liquid-phase system (2010)
zingibernsis using microwave-assisted mixed with water, (NH4)2SO and absolute ethanol in a flask and placed into a household with optimum conditions of 30% ethanol, 17% (NH4)2SO (w/w) and 40%
aqueous-two-phase extraction (MAATPE) and microwave oven refitted with a condensator outside, and irradiated at medium level power petroleum ether yielded top phase diosgenin recovery of 97.24%.
a novel three-liquid-phase system was devel- for 2 min.
oped to separate diosgenin from untrans- The mixture was cooled to room temperature and the top phase was taken for analysis.
formed steroidal saponins.
Gymnema sylvestre • To evaluate the applicability of MAE on One gram of homogenous leaf powder was mixed with 20 ml of ethanol. After allowing a • Under optimum conditions, 8 min of MAE produced a maximum yield of Mandal and
oleanolic acid extraction from Gymnema preleaching time of 5 min, the suspension was irradiated with a microwave extractor 7.6% (w/w) of oleanolic acid which was found to be 4 times, 3 times and 1.2 Mandal (2010)
sylvestre leaves. (CATAR, Catalyst Systems, Pune, India) at different experimental conditions. times more efficient than maceration, stirring extraction and heat reflux
extraction, respectively. Extracts obtained from 8 min of MAE showed better
antioxidant activity when compared to other conventional methods. No
degradation of the target analyte was observed at the optimum conditions as
evidenced from the stability studies performed with standard oleanolic acid.
Pulsatilla turczaninovii • To develop a method for quantifying and MAE was carried out in a QW-6HME (6000 W, 2450 MHz) microwave accelerated reaction • The total triterpenoid saponins content was the highest in April Xu et al. (2012)
comparing the triterpenoid saponins in nine system (KeWei Microwave energy Co., Ltd., Guangzhou, China). One gram of powder was (18328.6 μg/g), the seedling stage, and decreased from May (18295.9 μg/g)
different seasonal batches of P. turczaninovii weighed accurately into a round bottom flask (100 ml) and extracted under optimized to August (17157.6 μg/g). The contents of total triterpenoid saponins
from the Liaoning province. condition, including a solid/liquid ratio of 1:20 (g/ml), a microwave power of 500 W, an ex- decreased significantly after the blooming period.
traction temperature of 80 °C and an irradiation time of 3 min with 70% ethanol for one cycle.
Lamii albi flos • To compare six different extraction methods, MAE was carried out using Plazmotronika UniClever (350 W) BMZ I (Wroclaw, Poland). • MAE in closed system was the most effective technique. The best results Wójciak-Kosior
i.e., maceration, Soxhlet, heat reflux extraction, Plant material was placed into the extraction vessel and extracted with 40 ml of acetone for ursolic obtained (111.2 μg/g DM) with use of MAE in closed system for et al. (2013)
ultrasonic extraction, microwave extraction, using various generator powers (30%, 65%, 100%) during 10, 20 and 30 min. The obtained 10 min and 100% of generator power. Oleanolic acid (22.2 μg/g DM) was
and accelerated solvent extraction, on oleanolic extracts were filtered, evaporated, transferred into 5 ml flasks and made up to the mark better extracted with use of milder conditions (30% generator power and
acid and ursolic acid from Lamii albi flos. with acetone. 30 min).
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C.Y. Cheok et al. / Food Research International 59 (2014) 16–40 33

Table 9
Summary of studies focusing on saponins extraction using accelerated solvent extraction (ASE).

Plant material(s) Objectives Pretreatment/extraction conditions Findings Reference

Aesculus chinensis • To identify and quantify four major An ASE 100 System (Dionex, Sunnyvale, CA, • Four major saponins of escin Ia (17.3 ± 1.4 mg/g), Chen, Li,
Bunge saponins using HPLC method. USA) with 34-ml stainless steel ASE vessels escin Ib (10.4 ± 1.6 mg/g), isoescin Ia et al. (2007)
• To introduce a new extraction process, was used for the pressurized liquid extraction. (9.3 ± 2.5 mg/g) and isoescin Ib (5.9 ± 0.8 mg/g)
accelerated solvent extraction, to were extracted from seeds of A. chinesis Bunge using
optimize the extraction yield. ASE.
• The optimized ASE procedure employed 70% meth-
anol, 120 °C, 7 min of static extraction time, 60% flush
volume resulting extraction recoveries of the four
compounds nearly to 100% for two cycles.
Folium Ginseng and • To develop a rapid pressurized liquid The sample in the extraction cell was • A rapid pressurized liquid extraction (PLE) and Qian et al.
Radix Ginseng extraction and rocket column HPLC extracted using pressurized liquid extraction high-performance liquid chromatography coupled (2009)
analysis method for the determination on a Dionex ASE 200 system under the opti- with diode array detection and mass spectrometry
of one flavonoid (panasenoside), nine mum conditions: methanol; particle size, (HPLC–DAD–MS) method for the simultaneous de-
saponins of ginsenoside and two 0.30–0.45 mm; temperature, 150 °C; static termination of one flavonoid (panasenoside), nine
polyacetylenes (panaxydol and extraction time, 15 min; pressure 1500 psi; saponins (ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2,
panaxynol) in Folium Ginseng and Radix flush volume, 40%; static cycle, 1 and number Rb3 and Rd) and two polyacetylenes (panaxydol and
Ginseng. of extraction, 1. panaxynol) in Folium Ginseng and Radix Ginseng was
developed.
Panax ginseng • To quantify and separate The dried powder of P. notoginseng (90 g) was • The adsorption characteristics of PTS and PDS on four Wan et al.
protopanaxatrial and protopanaxadiol placed into six 33 ml-stainless steel extraction types of macroporous resins, including D-101, DA-201, (2008)
saponins with macroporous resins. cells. In brief, the optimized conditions were: DM-301 and DS-401, have been compared. Among
particle size, 0.3–0.45 mm; solvent, methanol; them, DS-401 resin showed the best separation
temperature, 150 °C; static time, 15 min; behaviors.
pressure, 6.895 × 10 3 MPa; static cycle, 1.
• To determine nine types of saponins Dried powder was placed into an 11 ml • Nine types of saponins have been identified. Wan et al.
from Panax ginseng. stainless steel extraction cell. The optimized • PLE has the highest extraction efficiency and re- (2006)
• To compare the extraction efficiency conditions were: particle size, 0.3–0.45 mm; peatability, which would be valuable on standardi-
of methods pressurized liquid, solvent, methanol; temperature, 150 °C; zation of sample preparation for quality control of
ultrasonication, and Soxhlet extraction. pressure, 6.895 × 103 MPa; static time, Chinese medicines.
15 min; and one static cycle and one
extraction times.
• To demonstrate a novel simpler and The extraction cells with notoginseng powder • More than nine saponins including notoginsenoside Zhang, Liu,
faster three-stage temperature gradient were placed into the ASE 150 system and the R6, notoginsenoside R1, ginsenoside Rb1, et al. (2013)
ASE coupled with HPCCC for the sys- extraction conditions and process were: first- notoginsenoside Spt1, ginsenoside F4, ginsenoside
tematic extraction and online separa- ly, static in 10 min, followed by a flush elution Rh4, ginsenoside 20S-Rg3, ginsenoside 20S-Rs3 and
tion of saponins with a broad range of with 60% volume, and followed by the nitro- ginsenoside Rk1 with the corresponding extraction
polarity from the raw plant materials. gen purge of 250 s, and extracted once. rates of 0.78 mg/g, 2.23 mg/g, 1.86 mg/g, 0.83 mg/g,
The extraction temperature and the extraction 1.23 mg/g, 1.46 mg/g, 2.47 mg/g, 2.22 mg/g and
solvents were optimized in the subsequent 1.84 mg/g were extracted and online isolated by ASE
experiments. coupled with HPCCC from the nature plant
P. notoginseng.

significantly lower saponin concentrations (0.25%) compared to interested to process the particular plant source further. Besides HPLC,
green varieties (0.67%). ultra pressure liquid chromatography (UPLC) (Foubert et al., 2010; Ha
et al., 2014; Serventi et al., 2013; Verza et al., 2012) was also employed
6.2. Chromatograhic method to quantify saponins.

Saponins are separated and purified from plant materials using chro-
matographic methods in many studies to identify a specific saponins
compound (Adão et al., 2011; Liu, Lu, et al., 2012) 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, Zhu, et al., 2012; Mostafa et al., 2013) and thin
layer chromatography (TLC) (Adão et al., 2011; Liu, Lu, et al., 2012;
Patel, Patel, et al., 2012). 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 re-
cent works of chromatographic method specifically in quantitation
study of plant saponins where HPLC was found to be the most common-
ly 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 saponin content detected not only
serves as a good data reference source to future researchers, but as a
strong scientific reference to drug-related manufacturer who is
7. Conclusions
Saponins are important secondary metabolites derived from
various plant sources because of its invaluable pharmaceutical proper-
ties. This review focuses on two different extraction techniques (con-
ventional and green technology) employed in previous works to
obtain crude saponin extract prior to further analysis. Moreover, spec-
trophotometric and chromatographic methods in saponin quantifica-
tion are described. This synthesis of the range and diversity of
previous studies already active in the field serves as important informa-
tion to researchers who wish to embark a new project. This may provide
an overview and quick reference for future lab-scale experimental de-
sign. The knowledge of extraction technique employed in respect to ob-
jective is vital and can be extended to address the rising food processing
challenges over time. After highlighting the lack of green extraction
technology utilization in lab-scale saponin extraction, more attention
should be paid by researchers for these technology explorations in the
future.
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34
Table 10
Spectrophotometric method in quantifying total saponins from various plant materials.

Plant material Reagents mixture Conditions to Cooling conditions Standard used Selected Type of spectrophotometer Yield obtained References
allow full color before UV/vis wavelength
development measurement (nm)

Ganoderma atrum 0.2 ml of 5% vanillin-acetic 70 °C, 15 min Running water, 2 min Oleanolic acid 550 Double beam 5.11% (the highest using MAE) Chen, Xie, et al.
acid + 1.2 ml perchloric acid. (2007)
Ipomoea batatas tuber 0.5 ml 8% vanillin 60 °C, 20 min 0 °C, 5 min Oleanolic acid 544 – 200.01 mg/100 g dry weight Dini et al. (2009)
in ethanol + 5 ml of 72%
sulfuric acid in water.
Xanthoceras sorbifolia Bunge 0.2 ml 5% vanillin-acetic 70 °C, 20 min Running water, 2 min Oleanolic acid 550 Unico 2100 The optimum microwave-assisted extraction Li, Zu, et al.

C.Y. Cheok et al. / Food Research International 59 (2014) 16–40

kernel acid + 1.2 ml 70% perchloric
acid
Bryonia Laciniosa 0.5 ml 8% vanillin + 5 ml 72%
sulphuric acid
Defatted rice bran – –
Allium nigrum L. 0.7% vanillin-60% sulphuric
acid reagent
Tulbaghia violacea 250 μl of vanillin reagent
(8 g/100 ml ethanol) + 2.5 ml
of 72% sulphuric acid.
Soymilk 0.5 ml of 8% vanillin solution 60 °C, 10 min
(in ethanol), and 5 ml of 72%
sulfuric acid.
Bian-Que bean soup – –
(consists of black soybean,
mung bean and adzuki bean)
Seeds of Achyranthus aspara, 0.25 ml of vanillin reagent
Tribulus terrestris, and Albizia (8%, w/v in 99.9%
lebbeck ethanol) + 2.5 ml of 72%
(v/v) sulphuric acid.
Rhizoma anemarrhenae 6.0 ml of H2SO4
Roots of Panax quinquefolium 0.2 ml 5% vanillin-acetic
and Panax ginseng acid + 0.8 ml perchloric acid
Spectrophotometer parameters of 51 °C, 7 min, 900 W, 32 ml/g, (2010)
(Shanghai Unico 42% ethanol and 3 cycles yielded the highest
Equipment Co. Ltd., China extraction of triterpene saponins reached
11.62 ± 0.37% of defatted kernel.
60 °C, 10 min Ice water bath, 15 min Oleanolic acid 538 - 15 μg/mg Patel, Santani,
et al. (2012)
– diosgenin – – 236.14 mg DE/g extract Chan et al.
(2013)
– – Diosgenin 473 U-2001 Spectrophotometer Yields (mg/g dw): Mostafa et al.
(Hitachi, Tokyo, Japan) Roots: 19.38 (2013)
Bulbs: 15.65
Leaves: 10.48
60 °C, 10 min Ice water bath, 3–4 min Diosgenin 544 – Yields (mg DE/ml): Ncube et al.
Micropropagated: 25.14 (2011)
Outdoor grown: 8.94
Ice water Soyasaponin 544 Multidetection microplate For both unfermented and fermented soymilk Lai et al. (2013)
reader (SynergyTM HT, extracted with 80% methanol gave the highest
BIO-TEK, GA, USA). yields of 247.44 and 167.21 mg saponin/g
extract, compared to 50% acetone and water.
– Soyasaponin 544 – Black soybean soup: 76.60–108.50 mg/g; Song, Xu, and Cai
adzuki bean soup: 103.18–129.49 mg/g; (2013)
nungbean soup: 32.84–39.23 mg/g.
60 °C, 10 min Ice water bath Quillaja saponin 544 – The total saponins content of seeds in chaff Goel, Sirohi, and
tree, gokhru and Siris were 45.75, 25.65 and Dwivedi (2012)
48.26% (w/w), respectively.
60 °C, 30 min Cool water Sarsasapogenin 283 – Purity 65.3% Liu, Zhu, et al.
(2012)
60 °C, 15 min – Ginsenoside 560 – It was found that UAE of ginseng saponins was Wu et al. (2001)
about three times faster than the traditional
extraction methods.
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Table 11
Quantification of plant saponins using HPLC.

Source Detector Column Solvent system detection Saponins yields Reference

Caulophyllum thalictroides Waters Alliance, 996 photodiode array Phenomenex LC18 guard Ammonium acetate–acetonitrile The quantities of saponins were found in the range of 5–25 μg/ml. Avula et al. (2011)
Flos Lonicerae Agilent 1100, – Zorbax SB C18 Acetonitrile-acetic acid 53.78 mg/g of dipsacoside B was found in L. hypoglauca. Chai et al. (2005)
45.65 mg/g, 46.22 mg/g, and 41.22 mg/g of macranthoidin B were found in
L. confusa, L. macranthoides, and L. similes, respectively.
Defatted rice bran Agilent 1300, DAD 1300 diode array Zorbax SB C18 Water-acetic acid/methanol–aceto- n-buthanol fraction yield: Chan et al. (2013)
nitrile–acetic acid 11.761 mg/g extract.
Aesculus chinensis Agilent 1100, UV–vis diode array Sinochrom ODS-BP C18 Acetonile/0.1%phosphoric acid Escin Ia of 17.3 mg/g was the most abundant saponin in the seeds of Chen, Li, et al. (2007)
Aesculus chinensis Bunge.
Gynostemma pentaphyllum Agilent 1100 series, G1315B Gemini C18 0.1% formic acid solution/ Total of 17 types of saponins detected: 1278 μg/ml. Cheng et al. (2011)
photodiode array acetonitrile
Tribulus terrestris L. Waters Associates, – Eurospher 100 C18 0.025% acetic acid-acetonitrile The results revealed distinct differences in the content of these compounds Dinchev et al. (2008)
depending on region of sample collection, plant part studied and stage of
plant development.
The samples from Bulgaria, Turkey, Greece, Serbia, Macedonia, Georgia and
Iran exhibited similar chemical profile and only some quantitative differ-
ence in the content with protodioscin and prototribestin as main

C.Y. Cheok et al. / Food Research International 59 (2014) 16–40

components.
Ipomoea batatas tubers Hwelett-Packard 1100 series, Photo- Hewlett-Packard HP-5 n-BuOH/HOAc/H2O HPLC–DAD: Dini et al. (2009)
diode array Saponin1: 161.20 mg/100 g dw;
Saponin2: 14.67 mg/100 g dw
HPLC–MS:
Saponin1:163.78 mg/100 g dw;
Saponin2: 15.60 mg/100 g dw
Bacopa monnieri Waters Alliance, 996 photodiode array Luna C8(2) Water–methanol Main components were either bacoside A3 or bacopaside II, least dominant Ganzera et al. (2004)
showed to be bacopasides IV and V.
The total saponin content in the samples varied from 1.1 to 13.0%.
Seeds of Vaccaria segetalis Garcke, Agilent, UV–vis diode array Zorbax SB-C18 Acetonitrile–water–formic acid/ The highest saponin yield of 42.2 g/100 g extract was obtained by 80% Güçlü-Üntündağ et al.
Saponaria Vaccaria L., Vaccaria acetonitrile–formic acid methanol using accelerated solvent extraction. (2007)
pyramidate
Phasedus vulgaris L. Agilent 110, diode array Zorbax SB-Aq Trifluoroacetic acid solution/ Hilum contained the highest concentration of saponins compared to Guajardo-Flores,
acetonitrile cotyledons or seed coats. García-Patiño, Serna-
The saponins concentration in hilum increased Guerrero, Gutiérrez-
2.3-fold after soaking from 455.95 mg/100 g to 1063.62 mg/100 g. Uribe, and Serna-
After the first day of germination, the saponin concentration in sprouts and Saldívar (2012)
cotyledons increased 1.9 and 2.1-fold, respectively.
Additional germination days decreased the amount of the most abundant
soyasaponins in black bean sprouts.
Ziziphus jujuba, Z.jujuba var. spinosa Waters Alliance, photodiode array Sunfire C18 Acetonitrile/0.2% acetic acid Zizyphus saponins I: 1.41 mg/g Guo et al. (2011)
Zizyphus saponins I: 4.52 mg/g
Platycodi Radix Hitachi L-6200, - Zorbax SB-Aq C18 Water/acetonitrile Platycodin D: 2431 μg/g Ha et al. (2006)
Polygalacin D: 2116 μg/g
Platycoside E: 1088 μg/g
Deapi-platycoside E: 744 μg/g
Deapi-platycodin D: 738 μg/g
Medicago truncatula -, photodiode array J.T Baker C18 Water–acetonitrile Total saponins (ng/mg/dw): Huhman et al. (2005)
Root: 5924
Stem: 417
Leaf: 1064
Seedpod: 30.8
Seed: 991
Panicum virgatum L. –, Thermo Finnigan LCQ ion trap mass Betasil C-8 1% formic acid-acetonitrile Total saponin concentrations were highest in leaves, lowest in the stems, Lee et al. (2009)
spectrometer and intermediate for the combined leaf and stem in all four varieties of
Switchgrass.
Dioscorea zingiberensis Shimadzu, Photodiode array Shim-pack VP-ODS C18 Water/acetonitrile Li, Huang, et al. (2010)

(continued on next page)

35

Table 11 (continued)
Source
Gleditsia sinensis Lam.
Xanthoceros sorbifolia
Paris and Trillium plants
Allium nigrum L.
Folium ginseng, Radix ginseng
Yucca gloriosa L.
Maesa lanceolata
Asparagus
Polygala japonica
Panax notoginseng
Paris polyphylla var. yunnanensis,
Paris polyphylla var. chinensis
Author's personal copy
36
Detector Column Solvent system detection Saponins yields Reference
The content ranges (g/100 g) were:
22.9858–44.5988 (parvifloside),
33.8612–57.7133 (protodeltonin),
0.1692–1.1125 (protodioscin),
1.9979–4.2469 (protogracillin),
4.2912–11.6437 (zingiberensis saponin), 7.0289–20.4885 (deltonin),
0.3783–1.3685 (dioscin),
0.1507–2.0500 (diosgenin diglucoside)
0.1117–1.1037 (trillin)
Agilent 1100, UV ODS-2 Hypersil Acetonitrile-0.1% acetic acid/water- Total of 19 saponins (μg/μg %): 4.137–11.479. Lian and Zhang (2013)
0.1% acetic acid
Shimadzu 2010 series, – Diamonsil C18 Methanol/0.05% formic acid Yields of sorbifoside A and B (μg/g): Ling et al. (2011)
Husks: 157.0; 58.33
Twig bark: 9.84; 30.14
Twig xylem: tr; 17.84
Seed coats: –; –
Seed kernels: –; 26.44
C.Y. Cheok et al. / Food Research International 59 (2014) 16–40
Flowers: 21.53; –
Leaves: –; –
Agilent 1200, ELSD Kromasil RP-C18 Water/acetonitrile Saponins of Paris VI was found majorly in Paris fargesii (19.520 mg/g), and Man, Gao, Zhang,
Trillium tschonoskii (11.770 mg/g). Wang, et al. (2010)
–, Diode array AQUASIL SS-1251-120 0.005% trifluoroacetic acid buffer Among different plant organs, the highest methiin content was detected in Mostafa et al. (2013)
(TFA) the bulb (0.17 mg/g fw), whereas a trace amount of methiin was detected
in the leaf (0.10 mg/g fw) and root (0.08 mg/g fw).
Agilent Series 1200, Diode array Prevail C18 Water-acetonitrile Saponins content was found in the range of 59.14–111.55 mg/g for both Qian et al. (2009)
ginseng varieties.
Agilent 1100, UV Waters Atlantis C18 Water-0.05% trifluoroacetic acid/ The dried extract obtained more than 25% w/w (236.81 mg/g) of steroidal Skhirtladze et al.
acetonitrile-0.05% trifluoroacetic acid glycosides saponins. (2011)
Agilent 1100, Diode array 300 Å monomeric C18 0.05%HCO2H/CH3CN-0.05%HCO2H The greenhouse grown M. lanceolata has been reported the highest Theunis et al. (2007)
percentage of 4.9% in total saponins.
Waters Alliance, Mediterranean Sea18 Water-1%formic acid/acetonitrile- Green spears contain from 0.024 mg/100 g fw at the top of the spears to Vázquez-Castilla et al.
diode array 1%formic acid 2.5 mg/100 g fw at the bottom cuts. (2013)
White spears content ranged from 1.4 to 5 mg/100 g fw depending on the
cultivar and portion of the spear.
The total content of saponin detected in Huétor asparagus ranged from 1.09
to 2.73 mg/100 g fw.
Shimadzu, ELSD Discovery C18 Acetonitrile-methanol/0.05% Total saponins found: 4.45–27.32 mg/g Wang et al. (2007)
trifluoroacetic acid
Dynamic Extractions Spectrum, Water SunfireTM C18 Acetonitrile/0.05% phosphoric acid Notoginsenoside R6: 0.78 mg/g Zhang, Liu, et al.
Photodiode array in water Ginsenoside R1: 2.23 mg/g (2013)
Ginsenoside Rb1: 1.86 mg/g
Notoginsenoside Spt1: 0.83 mg/g
Ginsenoside F4: 1.23 mg/g
Ginsenoside Rh4: 1.46 mg/g
Ginsenoside 20S-Rg3: 2.47 mg/g
Ginsenoside 20S-Rs3: 2.22 mg/g
Ginsenoside Rk1: 1.84 mg/g
Agilent 1100, photodiode array Waters Symmetry C18 Water/acetonitrile The contents of ginsenosides Rg1, Re, Rb1, Rd, and notoginsenoside R1 in Lau et al. (2003)
most of the raw samples were found to be higher than those in the
corresponding steamed samples.
Agilent 1100, UV Zorbax SB C18 Acetonitrile/0.001% formic acid Content of saponins (%): Li et al. (2005)
Notoginsenoside R1: 0.56–2.47
Ginsenoside Rg1: 2.15–5.00
Ginsenoside Rb1: 2.10–4.51
Ginsenoside Rg2: 0.05–0.31
Ginsenoside Rh1: nd-0.10
Ginsenoside Rd: 0.27–1.11
Agilent 1100, ELSD Kromasil RP-C18 Water-0.1% formic acid/acetonitrile Total saponins (mg/g) found for: Zhang et al. (2010)
Paris polyphylla var. yunnanensis = 19.62
Paris polyphylla var. chinensis = 1.63
 Author's personal copy
C.Y. Cheok et al. / Food Research International 59 (2014) 16–40
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