ArticlePDF AvailableLiterature Review

Abstract and Figures

Saponins are a diverse group of compounds widely distributed in the plant kingdom, which are characterized by their structure containing a triterpene or steroid aglycone and one or more sugar chains. Consumer demand for natural products coupled with their physicochemical (surfactant) properties and mounting evidence on their biological activity (such as anticancer and anticholesterol activity) has led to the emergence of saponins as commercially significant compounds with expanding applications in food, cosmetics, and pharmaceutical sectors. The realization of their full commercial potential requires development of new processes/processing strategies to address the processing challenges posed by their complex nature. This review provides an update on the sources, properties, and applications of saponins with special focus on their extraction and purification. Also reviewed is the recent literature on the effect of processing on saponin structure/properties and the extraction and purification of sapogenins.
Content may be subject to copyright.
Critical Reviews in Food Science and Nutrition, 47:231–258 (2007)
Copyright
C
Taylor and Francis Group, LLC
ISSN: 1040-8398
DOI: 10.1080/10408390600698197
Saponins: Properties, Applications
and Processing
¨
OZLEM G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G and GIUSEPPE MAZZA
Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, British Columbia, Canada V0H 1Z0
Saponins are a diverse group of compounds widely distributed in the plant kingdom, which are characterized by their structure
containing a triterpene or steroid aglycone and one or more sugar chains. Consumer demand for natural products coupled
with their physicochemical (surfactant) properties and mounting evidence on their biological activity (such as anticancer
and anticholesterol activity) has led to the emergence of saponins as commercially significant compounds with expanding
applications in food, cosmetics, and pharmaceutical sectors. The realization of their full commercial potential requires
development of new processes/processing strategies to address the processing challenges posed by their complex nature. This
review provides an update on the sources, properties, and applications of saponins with special focus on their extraction
and purification. Also reviewed is the recent literature on the effect of processing on saponin structure/properties and the
extraction and purification of sapogenins.
Keywords Triterpenes, sapogenins, ginsenosides, health products, surfactants, extraction
INTRODUCTION
Saponins, glycosides widely distributed in the plant king-
dom, include a diverse group of compounds characterized by
their structure containing a steroidal or triterpenoid aglycone and
one or more sugar chains. Their structural diversity is reflected
in their physicochemical and biological properties, which are
exploited in a number of traditional (as soaps, fish poison, and
molluscicides) and industrial applications (Price et al., 1987;
Oakenfull, 1981; Fenwick et al., 1991; Hostettmann and
Marston, 1995; Oakenfull and Sidhu, 1989). While plant ex-
tracts containing saponins have been widely used in food and
other industrial applications mainly as surface active and foam-
ing agents (San Martin and Briones, 1999); saponins in foods
have traditionally been considered as “antinutritional factors”
(Thompson, 1993) and in some cases have limited their use due
to their bitter taste (Ridout et al., 1991). Therefore, most of
the earlier research on processing of saponins targeted their re-
moval to facilitate human consumption (Khokhar and Chauhan,
1986; Ridout et al., 1991). However, food and non-food sources
of saponins have come into renewed focus in recent years
Address correspondence to Dr. Giuseppe (Joe) Mazza, Pacific Agri-Food Re-
search Centre, Agriculture and Agri-Food Canada, Box 5000, 4200 Highway 97,
Summerland, British Columbia, Canada V0H 1Z0. E-mail: MazzaG@agr.gc.ca,
mazzag@shaw.ca
due to increasing evidence of their health benefits such as
cholesterol lowering and anticancer properties (Gurfinkel and
Rao, 2003; Kim et al., 2003b). Recent research has established
saponins as the active components in many herbal medicines
(Liu and Henkel, 2002; Alice et al., 1991) and highlighted
their contributions to the health benefits of foods such as soy-
beans (Kerwin, 2004; Oakenfull, 2001) and garlic (Matsuura,
2001).
The commercial potential of saponins has resulted in the de-
velopment of new processes/processing strategies and reevalu-
ation of existing technologies (Muir et al., 2002) for their ex-
traction/concentration (Rickert et al., 2004b). The objective of
this review is to provide a timely update on the sources, prop-
erties and applications of saponins with special focus on their
extraction and purification.
SOURCES
The presence of saponins has been reported in more than
100 families of plants, and in a few marine sources such as star
fish and sea cucumber (Hostettmann and Marston, 1995). The
steroidal saponins are mainly found in monocotyledons (such
as Agavaceae, Dioscoreaceae and Liliaceae), and triterpene
saponins are predominantly present in dicotyledons (Legumi-
nosae, Araliaceae, Caryophyllaceae) (Sparg et al., 2004). While
the main dietary sources of saponins are legumes (soybeans,
231
232
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
(A) AGLYCONES
Triterpene aglycones
21
22
28
16
23
30
3
11
24
Steroid aglycone
Diosgenin
Aglycone -OH =O -COOH
Glycyrrhetinic acid 3
11 30
Gypsogenin 3 23 28
Oleanolic acid 3
28
Quillaic acid 3
, 16 23 28
Soyasapogenol A 3
, 21 , 22 , 24
Soyasapogenol B 3
, 22 , 24
Soyasapogenol E 3
, 24 22
HO
O
O
(B) SOYASAPONINS
Soyasaponin Soyasapogenol Structure
a
Group A
Aa A glc(1 2)gal(1 2)glcUA(1 3)A(22 1)ara(3 1)xyl(2,3,4-tri-O-Acetyl)
Ab A glc(1
2)gal(1 2)glcUA(1 3) A(22 1)ara(3 1) glc(2,3,4,6-tetra-O-Acetyl)
Ac A rha(1
2)gal(1 2)glcUA(1 3) A(22 1)ara(3 1) glc(2,3,4,6-tetra-O-Acetyl)
Ad A glc(1
2)ara(1 2)glcUA(1 3) A(22 1)ara(3 1) glc(2,3,4,6-tetra-O-Acetyl)
Ae A gal(1
2)glcUA(1 3) A(22 1)ara(3 1)xyl(2,3,4-tri-O-Acetyl)
Af A gal(1
2)glcUA(1 3) A(22 1)ara(3 1) glc(2,3,4,6-tetra-O-Acetyl)
Ag A ara(1
2)glcUA(1 3) A(22 1)ara(3 1)xyl(2,3,4-tri-O-Acetyl)
Ah A ara(1
2)glcUA(1 3) A(22 1)ara(3 1) glc(2,3,4,6-tetra-O-Acetyl)
Group B
Ba B glc(1
2)gal(1 2)glcUA(1 3)B
Bb B rha(1
2)gal(1 2)glcUA(1 3)B
Bc B rha(1
2)ara(1 2)glcUA(1 3)B
Bb’ B gal(1
2)glcUA(1 3)B
Bc’ B ara(1
2)glcUA(1 3)B
Group E
Bd E glc(1
2)gal(1 2)glcUA(1 3)E
Be E rha(1
2)gal(1 2)glcUA(1 3)E
DDMP
g B
DDMP
b
glc(1 2)gal(1 2)glcUA(1 3)B
DDMP
g B
DDMP
b
rha(1 2)gal(1 2)glcUA(1 3)B
DDMP
a B
DDMP
b
rha(1 2)ara(1 2)glcUA(1 3)B
DDMP
g B
DDMP
b
gal(1 2)glcUA(1 3)B
DDMP
a B
DDMP
b
ara(1 2)glcUA(1 3)B
DDMP
a
glc:D-glucose, ara:L-arabinose, gal:D-galactose, glcUA:D-glucuronic acid, xyl:D-xylose, rha: L-rhamnose
b
B
DDMP
: DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one) attached through an acetal linkage
to the C-22 hydroxyl of soyasapogenol B
Figure 1 Structure of (A) aglycones (Hostettman and Marston, 1995), (B) soyasaponins (Berhow et al., 2002; Gu et al., 2002), (C) ginsenosides (Li et al., 1996),
(D) glycyrrhizic acid (Ong and Len, 2003), and (E) quillaja saponins (Reprinted from Nord and Kenne, 2000, Copyright (2002) with permission from Elsevier).
(Continued)
SAPONINS 233
(C) GINSENOSIDES
O
OH
O
R2
R1
R3
glc: D-glucose, ara(p): L-arabinopyranose, ara(f): L-arabinofuranose, rha: L-rhamnose
(D) GLYCYRRHIZIC ACID (GLYCYRRHIZIN)
GlcUAGlcUAO
COOH
O
Ginsenosides R1 R2 R3
Rb
1
-glc[2 1]glc -glc[6 1]glc -H
Rb
2
-glc[2 1]glc -glc[6 1]ara(p) -H
Rc -glc[2
1]glc -glc[6 1]ara(f) -H
Rd -glc[2
1]glc -glc -H
Re -H -glc -O-glc[2
1]rha
Rf -H -H -O-glc[2
1]glc
Rg
1
-H -glc -O-glc
Figure 1 (Continued)
chickpeas, mungbeans, peanuts, broad beans, kidney beans,
lentils), they are also present in oats, allium species (leek, garlic),
asparagus, tea, spinach, sugarbeet, and yam (Price et al., 1987).
Soap bark tree (Quillaja saponaria), fenugreek (Trigonella
foenum-graceum), alfalfa (Medicago sativa), horse chestnut
(Aesculus hippocastanum), licorice (Glycyrrhiza species such as
Glycyrrhiza glabra), soapwort (Saponaria officinalis), Mojave
yucca (Yucca schidigera), gypsophila genus (such as Gypsophila
paniculata), sarsaparilla (Smilax regelii and other closely re-
lated species of Smilax genus) and ginseng (Panax genus) are
the main non-food sources of saponins used in health and indus-
trial applications (Hostettmann and Marston, 1995; Balandrin,
1996).
A single plant species may contain a complex mixture of
saponins. For example, the characterized soybean saponins in-
clude three groups of compounds: soyasaponins A, B and E
categorized according to the soyasapogenol in their structure
(Figure 1B). Similarly ginseng contains a mixture of saponins
(ginsenosides), the main components of which are Rb
1
,Rb
2,
Rc,
Rd, Re, Rf, and Rg
1
(Figure 1C). Commonly used plant sources
and their main saponins are presented in Table 1.
The saponin content of plant materials is affected by the plant
species, genetic origin, the part of the plant being examined, the
environmental and agronomic factors associated with growth
of the plant, and post-harvest treatments such as storage and
processing (Fenwick et al., 1991) (Table 2).
234
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
Figure 1 (Continued)
STRUCTURE AND PROPERTIES
Structure
Saponins are glycosides containing one or more sugar chains
on a triterpene or steroid aglycone backbone also called a
sapogenin (Figure 1). They are categorized according to the
number of sugar chains in their structure as mono, di-, or
tridesmosidic. Monodesmosidic saponins have a single sugar
chain, normally attached at C-3. Bidesmosidic saponins have
two sugar chains, often with one attached through an ether
linkage at C-3 and one attached through an ester linkage at
C-28 (triterpene saponins) or an ether linkage at C-26 (furas-
tanol saponins). The most common monosaccharides include:
D-glucose (Glc), D-galactose (Gal), D-glucuronic acid (GlcA),
D-galacturonic acid (GalA), L-rhamnose (Rha), L-arabinose
(Ara), D-xylose (Xyl), and D-fucose (Fuc). The nature of the
aglycone and the functional groups on the aglycone backbone
and number and nature of the sugars can vary greatly resulting
in a very diverse group of compounds (Figure 1; Price et al.,
1987; Hostettmann and Marston, 1995).
Properties
Physicochemical Properties
The structural complexity of saponins results in a number
of physical, chemical, and biological properties, only a few of
which are common to all members of this diverse group. Prop-
erties of a few selected aglycones and saponins are summarized
in Table 3.
Due to the presence of a lipid-soluble aglycone and water-
soluble sugar chain(s) in their structure (amphiphilic nature),
saponins are surface active compounds with detergent, wet-
ting, emulsifying, and foaming properties (Wang et al., 2005;
Sarnthein-Graf and La Mesa, 2004; Mitra and Dungan, 1997;
Ibanoglu and Ibanoglu, 2000). In aqueous solutions surfac-
tants form micelles above a critical concentration called criti-
cal micelle concentration (cmc). Saponins, including soybean
saponins, saponins from Saponaria officinalis, and Quillaja
saponaria, form micelles in aqueous solutions, the size and
structure of which are dependent on type of saponin (Oaken-
full, 1986). The micelle forming properties (cmc and the aggre-
gation number (number of monomers in a micelle)) of quillaja
SAPONINS 235
Table 1 Selected plant sources and their constituent saponins
Source Aglycone Saponin Reference
Soybean Soyasapogenol A Acetyl soyasaponins A
1
(Ab), A
2
(Af),
A
3
,A
4
(Aa), A
5
(Ae), A
6
,A
c
,A
d
Yoshiki et al., 1998
Soyasapogenol B Soyasaponin DDMP
a
conjugated Yoshiki et al., 1998
I (Bb) βg
II (Bc) βa
III (Bb
) γ g
IV (Bc
) γ a
V (Ba) αg
Soyasapogenol E Soyasaponin Be, Bd Yoshiki et al., 1998
Chickpea Soyasapogenol B DDMP
a
conjugated saponins Kerem et al., 2005; Price et al., 1988
Quillaja Quillaic acid QS 1-22, S1-12 Kensil and Marciani, 1991; Nord and Kenne,
2000
Horse chestnut Protoescigenin, barringtogenol C Aescin (escin): β-aescin, cryptoaescine,
α-aescine
World Health Organization, 2001
Alfalfa Medicagenic acid I-XV Oleszek, 1995
Hederagenin XVI-XIX Oleszek, 1995
Soyasapogenol B, E XX- XXVI Oleszek, 1995
Zanhic acid XXV-XXVI Oleszek, 1995
Licorice Glycyrrhetic acid Glycyrrhizic acid
b
World Health Organization, 1999a
Ginseng 20(s)-protopanaxadiol Ra
13
,Rb
13
, Rc, Rc
2
, Rd, Rd
2
,Rh
2
World Health Organization, 1999b
20(s)-protopanaxatriol Re
2
,Re
3
, Rf, Rg
1
,Rg
2
,Rh
1
World Health Organization, 1999b
Quinoa Phytolaccagenic acid Quinoa saponins Mizui et al., 1990
Oleanolic acid
Hederagenin
Oat Nuategenin Avenacoside A, B
¨
Onning et al., 1994
Yam (Dioscoera species) Diosgenin Dioscin Hostettmann and Marston, 1995
Fenugreek Diosgenin, yamogenin, tigogenin,
neotigogenin, yuccagenin,
lilagenin, gitogenin, neogitogenin,
smilagenin, sarsasapoenin
Trigofoenoside A-G, Trigonelloside B
(C)
Sauvaire et al., 1995
a
2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one.
b
Synonyms: glycyrrhizin, glycyrrhizinic acid.
saponins were affected by temperature, salt concentration, and
pH of the aqueous phase (Mitra and Dungan, 1997). At 25
C, the
values of cmc of quillaja saponins were in the range of 0.5 and
0.8 g/L. It increased with temperature and pH but decreased with
increasing salt concentration. The incorporation of cholesterol
Table 2 Saponin content of some selected plant materials
Source Saponin content (%) Reference
Soybean 0.22–0.47 Fenwick et al., 1991
Chickpea 0.23 Fenwick et al., 1991
Green pea 0.18–4.2 Price et al., 1987
Quillaja bark 9–10 San Martin and Briones, 1999
Yucca 10 Oleszek et al., 2001
Fenugreek 4–6 Sauvaire et al., 2000
Alfalfa 0.14–1.71 Fenwick et al., 1991
Licorice root 22.2–32.3 Fenwick et al., 1991
American ginseng
(P. quinquefolium L).
Young leaves 1.42–2.64 Li et al., 1996
Mature leaves 4.14–5.58 Li et al., 1996
Roots (4 year old) 2.44–3.88 Li et al., 1996
Oat 0.1–0.13 Price et al., 1987
Horse chest nut 3–6 Price et al., 1987
Sugar beet leaves 5.8 Price et al., 1987
Quinoa 0.14–2.3 Fenwick et al., 1991
into the saponin micelles increased their cmc, size, viscosity, and
the aggregation number (Mitra and Dungan, 2000) resulting in
the solubility enhancement of cholesterol as much as a factor of
10
3
at room temperature (Mitra and Dungan, 2001).
Quillaja saponins also had a solubilizing effect on phenan-
therene, and fluoranthene, which increases linearly with saponin
concentration at values higher than cmc (Soeder et al., 1996). A
similar linear relationship has been observed between the con-
centration of the saponin extract from Sapindus mukurossi and
aqueous solubility of hexachlorobenzene and naphthalene up to
a surfactant concentration of 10% (Kommalapati et al., 1997;
Roy et al., 1997).
Solubility enhancement has also been observed for Yellow
OB (Nakayama et al., 1986), and progesterone (Nakayama et al.,
1986) in the presence of bidesmoside saponins from Sapindus
mukurossi, and for α-tocopherol, and oleanolic acid in the pres-
ence of glucoside and glucuronide esters of glycyrrhizic acid
(Sasaki et al., 1988). Purified saponins and saponin mixtures
resulted in both enhancements and reductions in water solubility
of test compounds quercetin (Sch¨opke and Bartlakowski, 1997),
digitoxin (Walthelm et al., 2001), rutin (Walthelm et al., 2001),
and aesculin (Walthelm et al., 2001), the extent of which was
determined by concentration of saponin and the model com-
pound. Solubility enhancement of quercetin obtained by pure
236
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
Table 3 Physical properties of some selected aglycones and saponins (Adapted from Budavari et al., 1996; Biran and Baykut, 1975)
Compound Formula Solubility Source MW MP
Aglycone
Oleanolic acid C
30
H
48
O
3
Insoluble in water, sol in 65 parts ether,
106 parts 95% alcohol, 35 parts boiling
95% alcohol, 118 parts chloroform,
180 parts acetone, 235 parts methanol.
Quinoa 457 310
Quillaic acid C
30
H
46
O
5
Soluble in alcohol, ether, acetone, ethyl
acetate, glacial acetic acid
Quillaja 487 292–293
Diosgenin C
27
H
42
O
3
Soluble in the usual organic solvents, in
acetic acid
Dioscorea, fenugreek,
yam
415 204–207
Glycyrrhetic acid C
30
H
46
O
4
Licorice 471 298–300
Saponin
Glycyrrhizic acid
(Glycyrrhizin)
C
42
H
62
O
16
Freely soluble in hot water, alcohol,
practically insoluble in ether
Licorice 823
Escin Horse chestnut
α-escin Very soluble in water and methanol, only
slightly soluble in acetone, insoluble in
ether and hydrocarbons
225–227
β-escin Readily soluble in methanol, slightly
soluble in acetone, practically
insoluble (very little solubility) in
water, insoluble in ether and
hydrocarbons
222–223
Gypsophia saponin C
35
H
61
O
24
Soluble in water (0.5147 g/100 mL
at 25
C)
Gypsophia 863 221–227
saponins at concentrations > cmc values can be attributed to
micellar solubilization, whereas solubilization effect of some
saponin mixtures at concentrations < cmc points to an alterna-
tive mechanism (Sch¨opke and Bartlakowski, 1997).
Purified saponins or saponin mixtures may also have a
solubilizing effect on other saponins. Solubility enhancement
of monodesmosides (such as monodesmosides of Sapindus
mukurossi (Nakayama et al., 1986; Kimata et al., 1983), Bu-
pleuri radix (saikosaponins) (Kimata et al., 1985; Morita et al.,
1986; Watanabe et al., 1988) and soyasaponins Bb, Bb
and B-G
(Shimoyamada et al., 1993)), which have very low water solubil-
ity, in the presence of bidesmoside saponins is well documented.
The extent of the enhancement is dependent on the structure of
the monodesmoside saponin, and the composition/concentration
of the saponin bidesmosides. Solubility of Sapindus mukurossi
monodesmosides was enhanced in the presence of mukurossi
bidesmoside saponins containing hederagenin (Y1, Y2, X)
(Nakayama et al., 1986; Kimata et al., 1983). However,
mukurossi bidesmosides did not affect the solubility of saikos-
aponins (Kimata et al., 1985), which was enhanced by oleanolic
acid bidesmosides with a glucuronide moiety such as ginseno-
sides (chikusetsusaponin-V (ginsenoside Ro) and IV) (Kimata
et al., 1985; Watanabe et al., 1988), Hemsleya macrosperma (cu-
curbitaceae) bidesmosides (Ma2 and Ma3) (Morita et al., 1986),
and cyclic bidesmoside tubeimoside I isolated from tubers of
Bolbostemma paniculatum Franquet (Kasai et al., 1986b).
The solubility of saikosaponin-a in water at 37
C (0.14
mg /mL) increased with concentration of ginsenosi de Ro
reaching a value of 4.08 mg/mL at a bidesmoside concentration
of 1.4 mg/mL (Kimata et al., 1985). A significant decrease in
the solubilizing effect on saikosaponin-a was observed upon
methylation or reduction of the glucuronide carboxyl group of
ginsenoside-Ro indicating the role of the glucuronide moiety in
the observed effect (Tanaka, 1987). A greater extent of enhance-
ment was obtained for Hemsleya macrosperma (cucurbitaceae)
bidesmosides Ma2 and Ma3, which are structurally similar to
ginsenoside Ro with similar cmc values, at a concentration of
0.1% resulting in saikosaponin-a solubilities of 5–8.7 mg/mL
compared to 3.4 mg/mL for ginsenoside Ro (Morita et al.,
1986). The solubility enhancement of saikosaponin-a became
apparent near the cmc of these bidesmosides (Kimata et al.,
1985; Morita et al., 1986; Nakayama et al., 1986).
The solubility of diene saponin saikosaponin-b1 produced by
heating or mild-acid treatment of saikosaponin-a was increased
by malonyl-ginsenosides and to a lesser extent by ginsenoside
Ro (Zhou et al., 1991). The effect of malonyl-ginsenosides on
saikosaponin-a has also been demonstrated (Zhou et al., 1991).
While neutral dammarane ginsenosides did not have a solubi-
lizing effect on saikosaponins by themselves, they enhanced the
solubilizing effect of ginsenoside Ro (Watanabe et al., 1988) and
dammarane ginsenosides (Zhou et al., 1991).
Solubility enhancement of saikosaponin-a has also been ob-
served in the presence of glycyrrhizic acid, which is the glu-
curonide monodesmoside saponin of licorice (Sasaki et al.,
1988). The decrease in the degree of enhancement observed
at high glycyrrhizic acid concentrations was attributed to the
increase in solution viscosity (Sasaki et al., 1988). A solubiliz-
ing effect was also observed for the 30-β-glucoside (isolated
SAPONINS 237
from licorice roots) and glucuronide esters of glycyrrhizic
acid at higher concentrations (Sasaki et al., 1988). In addi-
tion to bidesmosides, co-occuring compounds such as acyclic
sesquiterpene oligoglycosides have also been shown to have a
solubilizing effect on monodesmosides of Sapindus mukurossi
(Kasai et al., 1986a) and Sapindus delavayi (Wong et al., 1991).
Solubility enhancement may have important implications for
the bioactivity and processing of saponins. Monodesmosides,
while poorly soluble in water in purified form, can be extracted
readily due to the solubilizing effect of co-occuring compounds
(Kimata et al., 1983). Micellar solubilization by saponins can be
exploited for the development of micellar extraction processes
or to affect the solubilization of ingredients in cosmetic, phar-
maceutical or food formulations (Shirakawa et al., 1986).
Solubility of saponins is also affected by the properties of
the solvent (as affected by temperature, composition, and pH).
While water, alcohols (methanol, ethanol) and aqueous alcohols
are the most common extraction solvents for saponins, solubil-
ity of some saponins in ether, chloroform, benzene, ethyl ac-
etate, or glacial acetic acid has also been reported (Hostettmann
and Marston, 1995). In the ethanol concentration range of
30–100%, solubility of soyasaponin Bb (soyasaponin I) was
maximum in 60% ethanol (Shimoyamada et al., 1993). Solu-
bility of gypsophia saponin in water increased with temperature
from 7.4 g/100 mL at 30
Cto18.0 g/100 mL at 70
C (Biran and
Baykut, 1975). A sharp increase was observed in the solubility
of soyasaponin Bb, which was very low in the acidic region,
in the pH range 6.5–7.3 (Shimoyamada et al., 1993). The de-
gree of partitioning of components of crude 70% ethanol extract
of soybeans between water and butanol was dependent on the
concentration of the extract and pH of the aqueous phase (Shi-
moyamada et al., 1995). The highest recovery of soyasaponin I in
the butanol layer was obtained using 0.04 g/mL of crude extract
in the acidic region (about pH 4) (Shimoyamada et al., 1995).
While bitterness is the most common sensory attribute asso-
ciated with saponins (Price et al., 1985), the occurrence of sweet
saponins is also well known (Kennelly et al., 1996). For example,
the sweetness of licorice is attributed to its main saponin, gly-
cyrrhizic acid (Figure 1), which is 50 times sweeter than sugar
(Muller and Morris, 1966).
The complex structure of saponins may undergo chemi-
cal transformations during storage or processing which in turn
may modify their properties/activity. The glycosidic bond (be-
tween the sugar chain and the aglycone), and the interglycosidic
bonds between the sugar residues can undergo hydrolysis in
the presence of acids/alkali, due to hydrothermolysis (heating
in presence of water) or enzymatic/microbial activity resulting
in the formation of aglycones, prosapogenins, sugar residues
or monosaccharides depending on the hydrolysis method and
conditions (Hostettmann and Marston, 1995). Complete acid
hydrolysis yields the constituent aglycone and monosaccha-
rides, whereas under basic hydrolysis conditions, cleavage of
O-acylglycosidic sugar chains results in the formation of pros-
apogenins (Hostettmann and Marston, 1995). The solubility
behavior of the parent aglycone can be markedly different
than the saponin due to its lipophilic nature (Table 3). DDMP
(2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one) conju-
gated saponins, which were determined to be the genuine
saponins in intact soybeans, are hydrolyzed into Group B and
E saponins upon heating, in alkaline solutions, and in the pres-
ence of iron (Kudou et al., 1993; Okubo and Yoshiki, 1995).
Soyasaponin βg, which was stable in acidic solution and at tem-
peratures < 90
C, was converted into soyasaponin Bb at basic
pH and upon heating at 90–100
C (Okubo and Yoshiki, 1995).
In the presence of FeCl
3
,itwas degraded into soyasaponin Be
and Bb in a ratio of 3:2 (Okubo and Yoshiki, 1995). Deacyla-
tion of quillaja saponins was observed upon storage in aqueous
solution at pH > 6 (Okubo and Yoshiki, 1995).
The interaction of sterols (Gestetner et al., 1971, 1972; Walter
et al., 1954; Shany et al., 1970), minerals (West et al., 1978), and
proteins (Potter et al., 1993; Tanaka et al., 1995) with saponins
may result in the modification of the physicochemical properties
and biological activity of these compounds. Steroid saponins
(such as digitonin (Gestetner et al., 1972), alfalfa saponins
(Walter et al., 1954)), and triterpenoid saponins (such as lucerne
(Gestetner et al., 1971, 1972; Shany et al., 1970)) form water-
insoluble addition products with cholesterol and phytosterols
such as sitosterol and stigmasterol. Interaction of sterols and
lucerne saponins was dependent on the structure of the saponin
and sterols (Gestetner et al., 1971, 1972). While cholesterol and
β-sitosterol formed complexes with lucerne saponins contain-
ing medicagenic acid, which possess carboxyl groups at C23 and
C28 positions, saponins with soyasapogenol aglycones did not
precipitate (Gestetner et al., 1971). Insoluble complexes were
also formed between ammoniated glycyrrhizic acid and alfalfa
root saponins and the minerals zinc and iron (West et al., 1978).
The nature and effect of the saponin-protein interaction were
dependent on the type of protein (Potter et al., 1993) and the
type of the saponin mixture (Tanaka et al., 1995). Upon heating
at 78
C (upto 26 min) quillaja saponin interacted with casein to
form high molecular weight complexes, whereas soybean pro-
teins formed insoluble aggregates independent of saponin ad-
dition (Potter et al., 1993). Similarly, while heating salt soluble
proteins from walleye pollack meat at 40–100
C for upto 10 min
in the presence of quillaja saponins increased protein aggrega-
tion, tea seed saponins inhibited the aggregation of the protein
(Tanaka et al., 1995). Complex formation between beet saponin
and protein (as evidenced by turbidity and interfacial tension
measurements) and destabilization of a model dispersion of su-
crose, oil, saponin, and protein in acidic conditions point to the
role of beet saponin and protein in the formation of acid beverage
floc in sucrose-sweetened carbonated soft drinks and acidified
syrups (Morton and Murray, 2001).
The interaction of saponins and proteins also resulted in mod-
ifications of protein properties such as heat and enzyme stability
(Ikedo et al., 1996; Shimoyamada et al., 1998), and surface prop-
erties (Chauhan et al., 1999). Heat stability of bovine serum al-
bumin (BSA) (Ikedo et al., 1996), and resistance of BSA (Ikedo
et al., 1996) and soybean protein (Shimoyamada et al., 1998)
to chymotryptic hydrolyses improved upon addition of soybean
238
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
saponins. The stability of whey proteins to chymotryptic hy-
drolyses however decreased upon addition of soybean saponins
(Shimoyamada et al., 2000). Similarly, unlike soybean protein
whose sensitivity to tryptic hydrolysis improved, whey pro-
teins showed higher sensitivity in the presence of soya saponins
(Shimoyamada et al., 2000). The influence of soybean saponin
on the trypsin hydrolysis of bovine milk α-lactalbumin was at-
tributed to the modification of the protein’s tertiary structure
(Shimoyamada et al., 2005). Desaponization of quinoa protein
increased water hydration capacity and lowered the fat binding
and buffer capacity, and total nitrogen solubility (Chauhan et al.,
1999). Removal of saponins reduced the emulsion and foaming
capacity of the proteins but increased the stability of the foams
and emulsions (Chauhan et al., 1999).
Biological Activity
Saponins have been reported to possess a wide range of bi-
ological activities, which are summarized and listed alphabet-
ically in Table 4 (Hostettmann and Marston, 1995; Lacaille-
Dubois and Wagner, 1996; Milgate and Roberts, 1995; Francis
et al., 2002). While crude isolates, extracts, and saponin-
containing plants have been utilized in the investigation of bi-
ological activity, especially in the earlier studies, developments
in the isolation/purification and characterization techniques have
enabled the investigation of the bioactivity of well characterized
saponins and led to the emergence of structure and bioactivity
relationships (Oda et al., 2000; Gurfinkel and Rao, 2003).
The ability of saponins to swell and rupture erythrocytes caus-
ing a release of haemoglobin (the in vitro haemolytic activity)
has been one of the most investigated properties of saponins
(Oda et al., 2000). However, even for this activity, which has
been related to the saponin structure (type of aglycone and the
presence of sugar side chains), there is no apparent consistency
between members of this diverse group (Oda et al., 2000).
The toxicity of saponins to insects (insecticidal activity), par-
asite worms (anthelmintic activity), molluscs (molluscicidal),
and fish (piscidal activity) and their antifungal, antiviral, and
antibacterial activity are well documented (Lacaille-Dubois and
Wagner, 1996; Milgate and Roberts, 1995; Francis et al., 2002).
Toxicity of saponins to warm blooded animals is dependent on
the method of administration, source, composition, and con-
centration of the saponin mixture (George, 1965; Oakenfull
and Sidhu, 1990). While they show toxicity when given in-
travenously, their toxicity is much lower when administered
orally which has been attributed to their low absorption and
the much reduced haemolytic activity in the presence of plasma
constituents (Fenwick et al., 1991; George, 1965; Oakenfull and
Sidhu, 1990). The results of in vivo studies with rats (Yoshikoshi
et al., 1995; Gestetner et al., 1968), mice (Gestetner et al., 1968),
and rabbits (Gestetner et al., 1968) suggested that saponins are
not absorbed in the alimentary channel but hydrolyzed to sa-
pogenins by enzymatic action. A study on the bioavailability of
soyasaponins in humans showed that ingested soyasaponins had
low absorbability in human intestinal cells and seem to be metab-
Table 4 Reported biological activities of saponins
(Hostettmann and Marston, 1995; Lacaille-Dubois and
Wagner, 1996; Milgate and Roberts, 1995; Francis et al.,
2002)
Biological Activity
Adaptogenic
Adjuvant
Analgesic activity
Antiallergic
Antiedematous
Antiexudative
Antifeedant
Antifungal
Antigenotoxic
Antihepatotoxic inhibitory effect on ethanol absorption
Anti-inflammatory
Antimicrobial
Antimutagenic
Antiobesity
Antioxidant
Antiparasitic
Antiphlogistic
Antiprotozoal
Antipsoriatic
Antipyretic
Antispasmodic
Antithrombotic (effect on blood coagulability)
Antitussive (relieving or preventing cough)
Antiulcer
Antiviral
Chemopreventive
Cytotoxic
Diuretic
Effect on absorption of minerals and vitamins
Effect on animal growth (growth impairment), reproduction
Effect on cognitive behavior
Effect on ethanol induced amnesia
Effect on morphine/nicotine induced hyperactivity
Effects on ruminal fermentation
Expectorant
Haemolytic
Hepaprotective
Hypocholesterolemic
Hypoglemic
Immunostimulatory effects
Increase permeability of intestinal mucosa cells
Inhibit active nutrient transport
Molluscicidal
Neuroprotective
Reduction in fat absorption
Reduction in ruminal ammonia concentrations
Reductions in stillbirths in swine
Ruminant bloat
Sedative
olized to soyasapogenol B by human intestinal microorganisms
in vivo and excreted in the feces (Hu et al., 2004).
The safety of saponins of commonly used food and feedstuffs
such as soybeans (Ishaaya et al., 1969), and alfalfa (Malinow
et al., 1981) has been established by animal toxicology studies.
The safety of saponins (such as glycyrrhizic acid) or saponin-
containing extracts (such as quillaja extracts) that are used as
SAPONINS 239
Table 5 Lethality of quillaja saponins to CD-1
mice (Kensil and Marciani, 1991)
Dose (µg) Quil-A QS-7 QS-18 QS-21
125 1/5 0/5 4/5 0/5
250 2/5 0/5 5/5 0/5
500 4/5 0/5 5/5 1/5
Results are expressed as number of deaths per group
of five mice within 72 h after intradermal injection
of saponins.
food additives has been the subject of thorough reviews (Joint
FAO/WHO Expert Committee on Food Additives, 2004, 2005a;
Eastwood et al., 2005). Toxicological recommendations for gly-
cyrrhizic acid are based on its effect of increasing mineralocorti-
coid activity, which in turn results in electrolyte imbalance due to
sodium retention and potassium excretion, and water retention.
This effect though reversible can lead to elevated blood pres-
sure if sustained (Joint FAO/WHO Expert Committee on Food
Additives, 2005a). Safety evaluation of quillaja extracts takes
into consideration the chemical composition of the extracts (such
as saponin content, qualitative, and quantitative information on
non-saponin constituents) (Joint FAO/WHO Expert Commit-
tee on Food Additives, 2004). Quillaja extracts are classified
as type 1 and type 2 based on their saponin content, 20–26%
and 75–90% respectively (Joint FAO/WHO Expert Committee
on Food Additives, 2004), and Acceptable Daily Intake (ADI)
values are based on the saponin content of the extracts (Joint
FAO/WHO Expert Committee on Food Additives, 2005b). Pu-
rification of a saponin extract may result in production of highly
potent saponin fractions with varying degrees of toxicities as
observed for quillaja saponins (QS-7, QS-18, and QS-21) pro-
duced by the purification of an aqueous quillaja extract (Quil-A)
(Table 5) (Kensil and Marciani, 1991).
Saponins can impact the immune system through their ad-
juvant activity, their ability to improve effectiveness of orally
administered vaccines by facilitating the absorption of large
molecules, and their immunostimulatory effects (Cheeke, 1999).
The ability of saponins to act as immunological adjuvants by
enhancing the immune response to antigens has been recog-
nized since 1940s (Bomford et al., 1992; Francis et al., 2002).
In addition to quillaja saponins, which have been almost exclu-
sively used in the production of saponin adjuvants (Bomford
et al., 1992), adjuvant activity of soyasaponins, lablabosides, ju-
jubosides, quinoa, gypsophila, and saponaria saponins has also
been reported (Bomford et al., 1992; Oda et al., 2000; Estrada
et al., 1998).
Cholesterol-lowering activity of saponins, which has been
demonstrated in animal (Oakenfull and Sidhu, 1990; Matsuura,
2001) and human trials (Oakenfull and Sidhu, 1990; Kim et al.,
2003b; Bingham et al., 1978), has been attributed to inhibition of
the absorption of cholesterol from the small intestine, or the reab-
sorption of bile acids (Oakenfull and Sidhu, 1990). Feeding ani-
mals (poultry, rats, monkeys) diets containing purified saponins
or concentrated extracts containing saponins such as digitonin (a
steroid saponin obtained from Digitalis purpurea), saikosaponin
(triterpenoid saponins obtained from roots of Bupleurum falca-
tum L. and related plants), saponaria, soya, chick pea, yucca, al-
falfa, fenugreek, quillaja, gypsohila, and garlic saponins resulted
in reductions in the plasma and in some cases liver cholesterol
concentrations (Oakenfull and Sidhu, 1990; Matsuura, 2001).
Recent research highlighted the role of saponins in addition to
isoflavones on the hypocholesterolemic effect of soy protein
(Lucas et al., 2001; Oakenfull, 2001). The cholesterol lowering
effect of dietary saponins in humans is also supported by ecolog-
ical studies (Chapman et al., 1997). The low incidence of heart
disease in the Batemi and Maasai populations of East Africa de-
spite a saturated fat/cholesterol diet, has in part been attributed
to the use of plant dietary additives containing saponins in ad-
dition to polyphenols, phytosteroids and water-soluble dietary
fibre (Chapman et al., 1997).
Anticancer activity has been reported for a number of triter-
pene and steroid saponins including but not limited to soyas-
aponins (Rao and Sung, 1995; Kerwin, 2004; Berhow et al.,
2000; Plewa et al., 1998), ginsenosides (Huang and Jia, 2005;
Liu et al., 2000), saikosaponin-d (Hsu et al., 2004), diosgenin
(Raju et al., 2004), and glycyrrhizic acid (Hsiang et al., 2002).
Although the potential of soybean saponins as anticarcinogens
has been studied in recent years, animal studies are rather lim-
ited and most of the evidence comes from cell culture stud-
ies (Kerwin, 2004). Methyl protoneogracillin (Hu and Yao,
2003), methyl protogracillin (Hu and Yao, 2001) (steroidal
saponins isolated from the rhizomes of Dioscorea collettii), pro-
toneodioscin (Hu and Yao, 2002a), and protodioscin (Hu and
Yao, 2002b) (furastanol saponins isolated from the rhizomes of
Dioscorea collettii)have been identified as potential anticancer
agents by the National Cancer Institute’s (NCI) anticancer drug
screen program. Anticancer activities of saponin containing
plants such as ginseng and licorice are also being investigated
(Wang and Nixon, 2001; Yun and Choi, 1998; Shin et al., 2000).
While the cancer preventive effects of ginseng have been demon-
strated in experimental models and in epidemiological studies,
the evidence on its effect on humans is not conclusive (Shin
et al., 2000).
The aglycones, which might be naturally present in the plants
or formed by hydrolysis of saponins in vivo or during storage
and/or processing of the plant material, may have biological
activity which is absent or present in a lower degree in their
corresponding saponins. The study of the relationship between
chemical structure and colon anticancer activity of soybean
saponins (as indicated by their ability to suppress the growth
of a colon cancer cell line) revealed that the soyasapogenols
were more bioactive than the glycosidic saponins (Gurfinkel
and Rao, 2003). Other aglycones with anticancer activity in-
clude dammarane sapogenins from ginseng (Huang and Qi,
2005), betulinic acid (Yogeeswari and Sriram, 2005; Wick et
al., 1999), and oleanolic acid (Liu, 1995; Hsu et al., 1997).
Oleanolic acid, one of the most common triterpene saponin agly-
cone, has also been reported to possess anti-viral (anti-HIV),
anti-inflammatory, hepatoprotective, anti-ulcer, antibacterial,
240
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
hypoglycaemic, anti-fertility, and anticariogenic activity (Liu,
1995). Anti-viral (anti-HIV), anticancer, antibacterial, anti-
malarial, anti-inflammatory, anthelmintic, and antioxidant prop-
erties have been demonstrated for betulinic acid and its deriva-
tives (Yogeeswari and Sriram, 2005). The conversion of saponins
to their aglycones may also result in the loss of activity. For ex-
ample the hydrolysis of saponins by ruminal bacteria results
in the loss of antiprotozoal activity, which requires the intact
saponin structure (Cheeke, 1999). Similarly, the deacylation of
quillaja saponins decreased their adjuvant activity (Marciani
et al., 2002).
COMMERCIAL APPLICATIONS
The diverse physicochemical and biological properties of
saponins have been successfully exploited in a number of com-
mercial applications in food, cosmetics, agricultural and phar-
maceutical sectors. Market trends towards the use of natural
ingredients, and increasing evidence of their biological activ-
ity have increased the demand for saponins in recent years
(Brown, 1998; Malcolm, 1995). As natural non-ionic surfac-
tants, they find widespread use as emulsification and foam-
ing agents, and detergents (San Martin and Briones, 1999;
Balandrin, 1996). Other investigated/proposed applications of
saponins and saponin containing plants include as feed addi-
tives (Cheeke, 1999; Zhan, 1999; Aoun et al., 2003; Jensen and
Elgaard, 2001), as bacterial (Henderson, 2001) and vegetable
growth regulators (Yamauchi et al., 2000), and for soil reme-
diation (Roy et al., 1997). While the two major commercial
sources of saponins are Quillaja saponaria and Yucca schidig-
era extracts (San Martin and Briones, 1999; Balandrin, 1996), a
number of other plant materials such as horse chestnut (Indena,
2005), tea seed (Zhan, 1999), and soybeans (Organic Technolo-
gies, 2005) are being utilized/evaluated for use as commercial
sources of saponins. Pharmaceutical applications of saponins
include as raw materials for production of hormones (Blunden
et al., 1975), immunological adjuvants (Kensil et al., 2004),
and as drugs (Panagin Pharmaceuticals Inc., 2005; Panacos,
2005). Saponins have also been reported to be the active ingre-
dients in various natural health products, such as herbal extracts
(Balandrin, 1996).
Food Applications
Yucca (Mohave yucca, Yucca schidigera Roezl Fla) and quil-
laja (quillaia, soap bark, Quillaja saponaria Mol Fla) are clas-
sified as food additives in the US under section 172.50 (Natural
Flavoring Substances and Natural Substances Used in Conjuc-
tion with Flavors) (US Food and Drug Administration, 2003).
The food additives from natural origins containing saponins used
in Japan include enzymatically modified soybean saponin, Pfaf-
fia paniculata extract, quillaja extract, tea seed saponins, and
yucca foam extract (Japanese Ministry of Health and Welfare,
2005). Quillaja extract is classified by the European Union as a
foaming agent for use in water-based, flavored non-alcoholic
drinks (E 999; 200 mg/liter calculated as anhydrous extract)
(Office for Official Publications of the European Communities,
1996).
Although quillaja and yucca are not considered Generally
Recognized As Safe (GRAS) by the US Food and Drug Ad-
ministration (FDA), they have been given GRAS designation by
Flavor and Extract Manufacturers’ Association (FEMA) (FEMA
#2973, and 3120 respectively) (Ash and Ash, 2002). There is a
pending GRAS notice (GRN #165) received by FDA in 2005
from the American Beverage Association for quillaja extract
(type 2) to be used as a foaming agent in semi-frozen carbon-
ated and non-carbonated beverages at levels not exceeding 500
milligrams dry weight per kilogram beverage (US Food and
Drug Administration, 2005a).
Quillaja extract (type 1) is used in foods and beverages mainly
for its foaming properties at concentrations of 100 ppm (dry ba-
sis, undiluted extract) in soft drinks, and at concentrations up
to 250 ppm in frozen carbonated beverages (Joint FAO/WHO
Expert Committee on Food Additives, 2004). Quillaja extract,
type 2 is used in Japan as an emulsifier for preparations con-
taining lipophilic colors or flavors that are added to soft drinks,
fermented vegetables, and dressing (at claimed concentrations
<10 ppm) (Joint FAO/WHO Expert Committee on Food Addi-
tives, 2004). Licorice and licorice derivatives, which are consid-
ered as GRAS by FDA, are used in foods such as baked foods,
beverages, chewing gum, candy, herbs and seasonings, plant
protein products, and vitamin and mineral dietary supplements
as a flavoring agent only with specific limitations (U.S. Food
and Drug Administration, 2005b). Saponins have also been pro-
posed for use in foods as antimicrobial (Sogabe et al., 2003)
and anti-yeast agents (Ashida and Matsuda, 1999). Other com-
mercial saponin products for food applications include soybean
concentrates marketed as functional food ingredients and nu-
traceuticals (Organic Technologies, 2005), and a Korean ginseng
extract called saponia (Godwithus Co Ltd., 2005).
The presumed health benefits of oleanolic acid led to the
development of methods to fortify food products (such as olive
oil) with oleanolic acid (van Putte, 2002). Proposed applications
for oleanolic acid include as a flavoring agent to modify the
aftertaste/taste of the artificial sweetener (Kang et al., 1999) and
in fat blends as crystal modifier (Bhaggan et al., 2001).
The physicohemical properties of saponins can also be uti-
lized in food processing applications. Thus, while complex for-
mation of saponins with cholesterol has been used for the re-
moval of cholesterol from dairy products such as butter oil
(Micich et al., 1992; Richardson and Jimenez-Flores, 1994), the
interaction of saponins with cell membranes has been consid-
ered for the selective precipitation of fat globule membranes
from cheese whey (Hwang and Damodaran, 1994). In this
last application, saponins are used to increase the hydropho-
bicity of the fat membrane to facilitate flocculation and pre-
cipitation of the formed complexes (Hwang and Damodaran,
1994).
SAPONINS 241
Cosmetics
Due to their surface active properties, saponins are being uti-
lized as natural surfactants in cleansing products in the personal
care sector such as shower gels, shampoos, foam baths, hair con-
ditioners and lotions, bath/shower detergents, liquid soaps, baby
care products, mouth washes, and toothpastes (Indena, 2005;
Olmstead, 2002; Brand and Brand, 2004). Natural surfactants
containing saponins available commercially include Juazarine
from the bark of Zizyphus joazeiro tree (Anonymous, 2004),
horse chestnut saponins (Indena, 2005) and mixture of plant
saponins (Bio-Saponins, Bio-Botanica, Inc., 2005). Saponins
and sapogenins are also marketed as bioactive ingredients in
cosmetic formulations with claims to delay the aging process of
the skin (Yoo et al., 2003; Bonte et al., 1998), and prevent acne
(Bombardelli et al., 2001).
Pharmaceutical/Health Applications
Steroid saponin-containing plant materials gained commer-
cial significance in 1950s as raw materials for the production
of steroid hormones and drugs. The synthesis of progesterone
from the sapogenin diosgenin (Figure 1A) obtained from Mexi-
can yam by Marker et al. in 1940s (Marker et al., 1947) was the
beginning of a remarkable era in steroid research culminating
in the synthesis of the first oral contraceptive in 1951. Dios-
genin isolated from Dioscorea species and to a lesser extent
structurally similar sapogenins such as hecogenin from Agave
species have been widely used as raw materials by the steroid
industry (Blunden et al., 1975).
Saponins have been used as immunological adjuvants in vet-
erinary vaccine formulations due to their immune enhancing
properties since 1950s (Dalsgaard, 1974). Their use in human
vaccines, however, has been limited by their complexity and tox-
icity. Purification of the quillaja extract to yield fractions with
differing chemical and biological properties enabled the charac-
terization and thus reproducible production of the fractions for
optimal adjuvant activity and minimal haemolytic activity and
toxicity (Cox et al., 2002; Kensil and Marciani, 1991). Conse-
quently, there have been significant advances in the development
of saponins as human vaccine adjuvants in the last decade lead-
ing to the development of a new generation of vaccines against
cancer and infectious diseases which are at various phases of
clinical trials (Kensil et al., 2004). The use of quillaja extracts
(even at concentrations commonly used in foods) as oral ad-
juvants in human clinical tests requires supporting toxicology
and general safety data due to their non-GRAS status (Dirk and
Webb, 2005).
The wealth of information on the biological activity of
saponins and aglycones from a variety of sources is providing
leads for the development of drugs. The chemopreventive and
chemotherapeutic activities of ginseng dammarane sapogenins
have prompted the development of anticancer drugs which are
at various stages of development (Panagin Pharmaceuticals Inc.,
2005). A new class of HIV drugs called Maturation Inhibitors
(PA-457, in Phase 2 clinical trials) are being developed using
betulinic acid derivatives (Panacos, 2005).
Pharmaceutical compositions or plant extracts containing
saponins have been patented for the prevention and/or treat-
ment of a variety of conditions such as inflammation (Forse and
Chavali, 1997; Bombardelli et al., 2001), infection (Forse and
Chavali, 1997), alcoholism (Bombardelli and Gabetta, 2001),
pre- and post-menopausal symptoms (Bombardelli and Gabetta,
2001), cardiovascular and cerebrovascular diseases such as coro-
nary heart disease and hypertension (Yao et al., 2005; Hidvegi,
1994), prophylaxis and dementia (Ma et al., 2003), ultraviolet
damage including cataract, and carcinoma cutaneum (Satoshi
et al., 2004), gastritis, gastric ulcer, and duodenal ulcer (Kim
et al., 2003a). The use of saponins in pharmaceutical prepara-
tions as adjuvants to enhance absorption of pharmacologically
active substances or drugs has also been patented (Kensil et al.,
1996; Tanaka and Yata, 1985).
Saponin-containing plants such as ginseng, yucca, horse
chestnut, sarsaparilla, and licorice have been used in tradi-
tional medicine by various cultures for centuries for the pre-
vention/ treatment of various ailments (Liu and Henkel, 2002;
Hostettmann and Marston, 1995). Characterization of the medic-
inal plants and their extracts points to the role of saponins in con-
juction with other bioactive components such as polyphenols in
the observed health effects (Liu and Henkel, 2002; Alice et al.,
1991). Over 85% of the herbs most commonly used in Tra-
ditional Chinese Medicine were observed to contain saponins
(in addition to polyphenols) in significant detectable amounts,
while the herbal products in the eight best known and most com-
monly used formulae were explicitly rich in these components
(Liu and Henkel, 2002). It should be noted that while some of
the health benefits associated with these plants have been sup-
ported by clinical data or described in pharmacopeias and in
traditional systems of medicine, a variety of uses attributed to
these medicinal plants have not been substantiated (Table 6).
EXTRACTION AND PURIFICATION OF SAPONINS
AND SAPOGENINS
The recognition of the commercial significance of saponins
with expanding applications and increasing evidence of their
health benefits have prompted research on process development
for the production of saponins on a commercial-scale from natu-
ral sources. Existing food processing methods, such as soy pro-
tein production, are also being re-evaluated to obtain information
on the partitioning of saponins between different process streams
(Rickert et al., 2004a, 2004b), which is used to recover saponins
as separate fractions (Haokui, 2001), to maximize their retention
in the final product (Singh, 2004), and to identify potential raw
materials for the production of saponins (Rickert et al., 2004a).
Due to the abundance of saponins in nature, a wide range of
plant materials can be used as raw materials for commercial pro-
duction of saponins. A significant commercial opportunity lies
242
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
Table 6 Medicinal uses of licorice (Radix glycyrrhizae), ginseng (Radix ginseng), and horse chest nut (Semen hippocastani)(World Health Organization,
1999a, 1999b, 2001)
MEDICINAL USES Radix glycyrrhizae Radix ginseng Semen hippocastani
Supported by clinical data As a prophylactic and restorative
agent for enhancement of mental
and physical capacities, in case of
weakness, exhaustion, tiredness,
and loss of concentration, and
during convalescence.
For treatment of symptoms of chronic
venous insufficiency, including
pain, feeling of heaviness in legs,
nocturnal calf-muscle spasms,
itching and oedema.
For the symptomatic treatment of
chronic venous insufficiency,
sprains and bruises.
Described in pharmacopeias or in
traditional medicines
As a demulcent in the treatment of sore
throats, and as an expectorant in the
treatment of coughs and bronchial
catarrh. In the prophylaxis and
treatment of gastric and duodenal
ulcers and dyspepsia. As an anti-
inflammatory agent in the treatment of
allergic reactions, rheumatism and
arthiritis, to prevent liver toxicity, and
to treat tuberculosis and
adrenocorticoid insufficiency.
Treatment of diabetes, impotence,
prevention of hepatoxicity, and
gastrointestinal disorders such as
gastritis and ulcer.
Treatment of coronary heart disease.
Described in folk medicine, not
supported by experimental or
clinical data
As a laxative, emmenagogue,
contraceptive, galactagogue,
antiasthmatic drug, and antiviral
agent. In the treatment of dental caries,
kidney stones, heart disease,
consumption, epilepsy, loss of
appetite, appendicitis, dizziness,
tetanus, diphtheria, snake bite and
haemorrhoids.
Treatment of liver disease, coughs,
fever, tuberculosis, rheumatism,
vomiting of pregnancy,
hypothermia, dyspnoea, and
nervous disorders.
Treatment of bacillary dysentery and
fevers. Also as a haemostat for
excessive menstrual or other
gynaecological bleeding, and as a
tonic.
in the value-added processing of by-products for the concentra-
tion of saponins and/or aglycones such as soybean oil extraction
residue (Yoshiki et al., 2005), soy molasses (Dobbins, 2002),
asparagus waste (Schwarzbach et al., 2004), and sugarbeet pulp
(Sasazuka et al., 1995).
The development of an effective processing methodology
starts with the identification of process objectives/product spec-
ifications, which is in turn determined by end-product use. The
spectrum of saponins with commercial applications ranges from
crude plant extracts, which are commonly used for their foaming
properties, to high purity saponins with health applications such
as vaccine adjuvants, production of which requires a sequence
of purification steps. In addition to well-established analytical
methodologies, new technologies and approaches are also be-
ing investigated to overcome processing challenges posed by the
complex nature and diversity of this unique class of compounds.
While common trends can be identified, process development
is carried out for each raw material as the composition of the
plant material and the saponin mixture will affect the process
considerably.
Extraction of Saponins
The first step in the processing of saponins involves their
extraction from the plant matrix. As in any extraction process, the
extraction solvent, extraction conditions (such as temperature,
time, pH, solvent to feed ratio), and the properties of the feed
material (such as composition and particle size) are the main
factors that determine process efficiency and the properties of
the end product.
If a purified product is desired, the efficiency of the purifi-
cation steps needs to be considered while optimizing extraction
parameters. For example, conditions maximizing the extraction
yield can decrease the selectivity and thus, the purity of the
saponins, complicating further purification steps (Wanezaki et
al., 2005). The finding that malonyl isoflavones could be sep-
arated from soybean saponins easier than other soybean iso-
flavones due to their higher polarity led to the optimization of the
extraction of soybean saponins to be based on malonyl isoflavone
content of the extract (Wanezaki et al., 2005).
Sample Pretreatment
Pretreatment steps, which are carried out to increase the effi-
ciency of the extraction, include drying, particle size reduction,
and defatting (using a lipophilic solvent such as ethyl acetate
or hexane). Defatting can also be carried out after the extrac-
tion of saponins. Particle size reduction (grinding) is usually
carried out to increase the mass transfer efficiency of the ex-
traction. The variable qualitative and quantitative distribution
of saponins in plants enables the selection of the plant part to
be used as raw material considering efficiency of the process
SAPONINS 243
and/or extract properties. The efficiency of the separation is
improved by using part of the plant with the highest saponin
concentration. Selection of the raw material can also be used to
overcome processing challenges posed by the other components
present. For example, the use of quinoa hulls as raw material
for saponin extraction eliminated the problems associated with
swelling of starch during extraction of whole seeds (Muir et al.,
2002).
Extraction Methods
While traditional solvent extraction methods are commonly
used for the production of saponin extracts, recent research fo-
cuses on technologies that improve the extraction efficiency by
reducing extraction time and solvent consumption/waste without
compromising sample quality. Microwave (Vongsangnak, 2004;
Kwon et al., 2003a,b,c) and ultrasound (Wu et al., 2001) assisted
extractions involve disruption of the internal cell structure and
release of intracellular product to facilitate mass transfer, which
is achieved by rapid and selective heating of the raw material in a
solvent which is (partially) transparent to microwave energy (in
microwave extractions) (Kwon et al., 2003a,b,c; Vongsangnak,
2004) and the mechanical effects of acoustic cavitation (in ul-
trasonic extractions) (Wu et al., 2001).
Commercial applications of Microwave-Assisted Processes
(MAP
TM
, microwave technologies patented by Environment
Canada) are being currently developed for extraction of nat-
ural products such as oilseeds (in collaboration with Bunge
Canada, formerly CanAmera Foods, and BC Research) (Envi-
ronment Canada, 2005) and high value, low volume, natural ac-
tive ingredients for the pharmaceutical and nutraceutical markets
(Radient Technologies Inc., 2005). Ultrasonic liquid process-
ing devices are being used at production level in the pharma-
ceutical, chemical, petrochemical, and paint industry as well
as in the bioprocessing and food industries (Hielscher GmbH,
2005).
Lab-scale microwave and ultrasonic extractions were inves-
tigated for the extraction of ginsenosides from ginseng (Kwon
et al., 2003b; Vongsangnak, 2004), saponins from chickpeas
(Kerem et al., 2005) and glycyrrhizic acid from licorice root
(Pan et al., 2000). The ginsenoside yield and composition of a
80% methanol (50 mL) extract obtained from ginseng powder
(5 g) using MAP
TM
for 30 s (4×) (at 72.2
C) were comparable to
those of a 12 hr conventional reflux extraction carried out under
similar conditions (Kwon et al., 2003b). Similarly, a maximum
saponin yield of 7.4 mg/100 mg DW could be obtained in 6 min
by microwave-assisted extraction of ginseng (100 mg sample:
15 mL water-saturated n-butanol, 50
C) compared to 8 hr for
soxhlet extraction (7.7 mg/100 mg DW; 100 mg sample: 80 mL
methanol, 70
C), 6 hr for heat reflux extraction (6.7 mg/100 mg
DW; 100 mg sample: 15 mL methanol, 70
C), and 2 hr for ultra-
sonic extraction (7.6 mg/100 mg DW; 100 mg sample: 15 mL
water-saturated n-butanol) (Vong sangnak, 2004). Savings in
time and solvent consumption compared to traditional meth-
ods such as heat reflux, ultrasonic, Soxhlet extractions, and ex-
traction at room temperature were also achieved by microwave
assisted extraction of glycyrrhizic acid from licorice root (Pan
et al., 2000). Multi-stage counter-current extraction has also
been investigated to improve the efficiency of extraction of gly-
cyrrhizic acid from licorice (Wang et al., 2004).
Pressurized liquid extraction (PLE) involves the use of pres-
surized solvents at high temperatures. The high temperatures
made possible by the application of pressure results in improve-
ments in mass transfer properties of the solvent, hence improv-
ing extracting efficiency. The change in solvent polarity hence
solubility with temperature of the pressurized solvent coupled
with enhanced mass transfer properties makes PLE an attrac-
tive method for saponin processing; however, the applications
up to date have largely been limited to analytical procedures. In
their study on the PLE of medicinal plants, Benthin et al. (1999)
compared PLE of escin from CH
2
Cl
2
-defatted horse chestnut
using aqueous methanol (65%) at 140 bar and 100
C with tra-
ditional extraction procedure and achieved a higher escin con-
centration in the pressurized liquid extract (3.73%) than in the
traditional extract (2.63%). Extraction efficiency of ginsenosides
from Panax ginseng, American ginseng and health supplement
products using PLE (25–30 bar, 20 minute extraction, 20–25 mL
solvent used, 140
C) was comparable to Soxhlet extraction (Lee
et al., 2002). The ginsenoside yield of aqueous non-ionic sur-
factants was higher than that of water (at concentrations higher
than critical micelle concentration (0.01%)) and methanol at
lower temperatures (Choi et al., 2003). Efficiency of extraction
of glycyrrhizic acid from licorice using pressurized methanol
(Ong, 2002) (at 100
C, 20 min, 20–25 mL solvent) and pres-
surized water (Ong and Len, 2003) (at 95
C) was comparable
to or higher than that obtained with a multiple step ultrasonic
extraction using 70% methanol.
Extraction Solvent
Water, lower alcohols (methanol and ethanol), or water: al-
cohol mixtures have been widely used for extraction of saponins
from plant matrices (Kitagawa, 1986; Bombardelli and Gabetta,
2001). Other solvents investigated for extraction of saponins
include aqueous (Choi et al., 2003; Fang et al., 2000) and al-
coholic surfactant solutions (Choi et al., 2003), and glycerine
(Gafner et al., 2004). The addition of ammonia to solvents for
glycyrrhizic acid extraction is based on chemical complexation
of glycyrrhizic acid with ammonia, which results in an increase
in its extraction yield (Pan et al., 2000).
Supercritical CO
2
has been demonstrated to be a viable alter-
native to organic solvents for the processing of natural materials
with advantages such as ease of solvent removal, solvent free
products, and an oxygen free environment. However, the appli-
cation of SCCO
2
technology to the processing of polar solutes
such as polyphenolic and glycosidic compounds has been lim-
ited by the low solvent power of SCCO
2
for these solutes, which
can be improved by the addition of cosolvents (Hamburger et al.,
2004). The use of cosolvents, however, overrides one of the ma-
jor advantages of SCCO
2
processing: solvent-free processing.
244
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
Supercritical CO
2
extraction of ginsenosides from ginseng
(Wang et al., 2001), saikosaponins from Bupleurum chinense DC
(Ge et al., 2000) and glycyrrhizic acid from licorice (Chuanjing
et al., 2000; Kim et al., 2004) using cosolvents (ethanol (Wang
et al., 2001; Ge et al., 2000), methanol (Chuanjing et al., 2000),
and aqueous methanol (Chuanjing et al., 2000)) has been re-
ported. Wang et al (Wang et al., 2001) obtained an oil product
containing ginsenosides using SCCO
2
extraction of ginseng root
hair at 308–333 K and 10.4–31.2 MPa with ethanol. The addition
of ethanol to CO
2
(6 mol%) increased the SFE yield of ginseno-
sides in ginseng oil by a factor of 10 while increasing the yield of
the oil by a factor of 4 at 333 K and 31.2 MPa. The enrichment of
saponins in plant oils offer interesting product formulations, and
may warrant further research. Optimum conditions for recovery
of glycyrrhizic acid from licorice were 30 MPa and 60
C for
60 min using SCCO
2
+ 70% methanol (15% by volume) (Kim
et al., 2004).
Effect on Extraction Yield. The choice of solvent for a particu-
lar application will be based on the effect of solvent on saponin
yield and purity, and the composition of the saponin mixture.
Differences between yield and composition of extracts arise
from the varying selectivities of the solvents towards individ-
ual saponins and other feed components.
The saponin recovery obtained by aqueous alcohol extraction
(40–80%) of quinoa hulls was higher than that obtained by pure
water or alcohol extractions (Muir et al., 2002). Ultrasound-
assisted and Soxhlet extraction of ginseng using water-saturated
n-butanol gave higher ginsenoside yields than pure and 10%
methanol (Figure 2) (Wu et al., 2001). DDMP-saponin yield of
80% ethanol extraction of dehulled peas was higher than that of
pure methanol extraction, which was very low (Daveby et al.,
1998).
The yield of crude extract of Glinus lotoides seeds decreased
with the methanol content of aqueous methanol, and the high-
est crude extract yield (16.5%) was observed with pure water.
The highest yield of the n-butanol fraction (obtained by the
Figure 2 Total ginsenoside yield obtained by extraction of Chinese ginseng
root with water, water-saturated butanol, and 10% methanol in UB-ultrasound
cleaning bath, UP-ultrasound probe horn, and Sox-Soxhlet extractor (from Wu
et al., 2001, Copyright (2001), with permission from Elsevier).
0
2
4
6
8
10
12
14
16
18
0102030405060708090100
% Methanol in water
% Yield (w/w)
crude extract
n-butanol fraction
purified extract
Figure 3 Yield of crude extract, n-butanol fraction, and purified extract ob-
tained by extraction of Glinus lotoides seeds as a function of solvent composition
(Data from Endale et al., 2004).
partitioning of the crude extract between water and n-butanol)
and the purified saponins, however, was achieved by 20 and
60% methanol, respectively (Figure 3) (Endale et al., 2004).
The highest total extract yield of MAP
TM
extraction of ginseng
was obtained using 45–60% ethanol, whereas the saponin con-
tent increased with ethanol concentration reaching a maximum
at 60–75% ethanol (Kwon et al., 2003c). In red ginseng extrac-
tion (at 80
C, 5×8 hr), solids yield decreased whereas recovery
of ginsenosides increased with ethanol concentration (optimum
composition with 70% ethanol) (Sung and Yang, 1985).
The recovery of glycyrrhizic acid from licorice using mi-
crowave assisted extraction reached a maximum at 50–60%
ethanol (Pan et al., 2000). Addition of ammonia to the extraction
solvent, which reacts with glycyrrhizic acid to form glycyrrhizic
ammoniate, resulted in higher recoveries which were indepen-
dent of ethanol concentration in 0–60% ethanol range (Pan et al.,
2000). No significant difference in glycyrrhizic acid yield was
observed between the solvents pure water, 10% ethanol, and
0.5 wt% ammonia in water (Wang et al., 2004).
Effect on Composition of Saponins and Properties of
Extracts. The extraction solvent will also affect the composition
of the saponin extract. The ratio of neutral to malonyl ginseno-
sides in aqueous ethanol extract of American ginseng increased
with the proportion of ethanol in the solvent (Du et al., 2004).
While maximum extraction of neutral ginsenosides was obtained
with 70% ethanol, the highest yield of malonyl ginsenosides was
achieved using 40% ethanol resulting in the highest total gin-
senoside yield with 60% ethanol (Du et al., 2004). Differential
extraction of saponins from quinoa bran using pure water and
alcohol solvents was reflected in the differences in the saponin
composition of the extracts (Muir et al., 2002).
Extraction solvent has also been found to affect the physic-
ochemical properties of the saponin extracts, including parti-
cle size, size distribution, morphology, water uptake profiles,
sorption isotherms, densities, flow properties, and compaction
SAPONINS 245
Figure 4 Recovery (g/100 g seed dry weight) of DDMP-saponin obtained
by extraction of ground chickpea using microwave assisted extraction (three
serial 5-min extractions) and Soxhlet extraction with methanol (black), and 70%
ethanol (gray). Bars represent means ± standard deviation (n = 5); different
letters represent statistical significance level of p 0.01 (from Kerem et al.,
2005, Copyright Society of Chemical Industry. Reproduced with permission.
Permission is granted by John Wiley & Sons Ltd. on behalf of the SCI).
profiles, which are of great significance in pharmaceutical ap-
plications (Endale et al., 2004).
Effect of Temperature and Solvent:Feed Ratio on Extraction
Efficiency. While temperature was found to have no effect on the
microwave-assisted methanol extraction of chickpea saponins,
the saponin yield of ethanol:water extracts increased with tem-
perature (Figure 4) (Kerem et al., 2005). Solids (total extract)
yield of red ginseng extraction increased while saponin recovery
decreased with temperature (particularly at 100
C) (Sung et al.,
1985). The multi-stage counter-current extraction yield and gly-
cyrrhizic acid concentration both increased with temperature in
the range 30–70
C(Wang et al., 2004). The temperature effect
on the composition of aqueous licorice extract was reflected in its
flavor characteristics (Vora and Testa, 1997). The low tempera-
ture (65.6–82.2
C) extracts had significantly higher glycyrrhizic
acid, sugar content, and inorganic salt content, with a mild, sweet
flavor, whereas higher temperatures resulted in stronger licorice
character with balanced sweetness (Vora and Testa, 1997).
Glycyrrhizic acid concentration in the ethanol extract of
licorice decreased with increasing solvent/feed ratio from
339 mg/mL at 6 mL/g to 245 mg/mL at 10 mg/mL while extrac-
tion yield stayed in the range of 75–83% (Wang et al., 2004).
An increase in recovery of glycyrrhizic acid (%) with microwave
assisted extraction was observed with solvent/feed ratio (from
1.88% at 5:1 to 2.58% at 20:1) (Pan et al., 2000). The optimum
ratio for quinoa saponin extraction was determined to be 10–
15:1 considering extraction yield and practical considerations
such as ease of stirring (Muir et al., 2002).
Purification of Saponins
Purification of the crude saponin extract usually requires a
sequential approach. A common method for the preliminary
purification of saponins after the extraction step involves the
partitioning of saponins between aqueous extracts and a water
immiscible solvent such as n-butanol (Kitagawa, 1986). Fur-
ther purification can be carried out using solvent precipitation
(Kitagawa, 1986; Nozomi et al., 1986), adsorption (Giichi,
1987), ultrafiltration (Muir et al., 2002), and/or chromatogra-
phy (Kensil and Marciani, 1991). While chromatographic pro-
cedures such as open column chromatography, thin layer chro-
matography, flash chromatography, liquid chromatography (low,
medium and high pressure), and countercurrent chromatography
have been well established and widely used for analytical scale
purification of saponins (Hostettmann and Marston, 1995), their
feasibility for commercial scale processing of saponins needs to
be evaluated. The purification techniques used in the production
of saponins for a variety of applications are discussed below
with specific examples.
An aqueous extract of Quillaja saponaria bark was sepa-
rated into 22 fractions (QA1-22) with different adjuvant ac-
tivity and toxicity using a purification procedure involving
methanol extraction followed by silica gel and reverse phase
high pressure liquid chromatography (RP-HPLC) (Figure 5)
(Kensil and Marciani, 1991).
Due to their high volume of production and increasing evi-
dence on the biological activity of soyasaponins, soybeans (Dob-
bins, 2002; Giichi, 1987; Bombardelli and Gabetta, 2001), and
by-products of soybean processing (Yoshiki et al., 2005) have
great potential as raw materials for commercial saponin pro-
duction. The full realization of this potential in the marketplace
however requires development of processing schemes to effec-
tively tackle the associated processing challenges.
The patent “Process for isolating saponins from soybean-
derived materials” (Dobbins, 2002) exploits the temperature de-
pendence of solubility behavior of saponins in water:acetone
mixtures for the production of a soyasaponin concentrate. An
acetone:water (4:1) extraction step (56
Catatmospheric pres-
sure at pH >6.5) followed by cooling the extract led to the
precipitation of saponins resulting in a 70% saponin concentrate.
Further purification up to 90% was achieved by crystallization.
Asoyaextract containing 22.5% group B soyasaponin and
15% isoflavones was obtained by reflux extraction with pure
or aqueous aliphatic alcohols followed by hexane extraction
(for defatting purposes) (Figure 6) (Bombardelli and Gabetta,
2001). In an alternative approach, the defatted soya extract was
treated with polyethoxylated castor oil to dissolve the resinous
residues and adsorbed onto a polystyrene-based resin. Soya ex-
tract containing the isoflavones and saponins were then eluted
using 95% ethanol (Figure 6). The soya extract was fraction-
ated into group B saponins and isoflavones using solvent pre-
cipitation with aqueous alcohol and a water immiscible protic
solvent (such as ethyl acetate) (Figure 6). The fractionation of
soya extracts into isoflavone and saponin fractions can also be
achieved using an adsorption step (Giichi, 1987; Bombardelli
and Gabetta, 2001). The saponin fraction can be further puri-
fied using gel filtration and partition chromatography (Giichi,
1987).
Due to the unstable structure of soyasaponin βg, which adds
to the complexity and cost of the purification process, group
B and E saponins were identified as target compounds in the
246
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
Commercial aqueous Quillaja saponaria Molina extract
Quil-A (1 g)
Extract with methanol (75 mL
1
+ 50 mL
2
) at 60 °C
Filtrates 1+2
Evaporate to dryness on a rotoevaporator
Silica gel chromatography
Silica Si-60 column preequilibriated in 40 mM acetic acid
in chloroform:methanol:water (62/32/6, v/v/v)
Silica fractions (monitored by carbohydrate analysis, TLC, and HPLC)
Pooled and flash evaporated for further purification
Pooled fractions 19-30 31-60 85-104
Enriched in QA-21 QA-8 and 18 QA-7 and 17
Further purification by Reverse Phase HPLC (Vydac C4 column)
QA-21 QA-18 QA-7 and QA-17
isocratic separation in 40 mM methanol gradient in 40 mM acetic acid
acetic acid in 58% methanol 50-56% methanol/0-10 min 56-69% methanol, 10-79 min
Figure 5 Purification of quillaja saponins for use as adjuvants (adapted from Kensil and Marciani, 1991).
processing of a soybean by-product, the residue of oil extraction,
for the isolation of functional soybean saponins (Yoshiki et al.,
2005). A fractionation procedure for the production of Group B
and E saponin fractions was developed based on information on
the chemical characteristics of soyasaponin βg (Figure 7). The
soybean glycosides obtained by acidic precipitation were further
fractionated into an isoflavone-rich (supernatant) and a DDMP
saponin-Fe
2
complex rich fraction (precipitate) by dissolving
them in ethanol, mixing with FeCl
2
and allowing them to stand
overnight. Saponins were further purified by alkaline hydrolysis
to remove Fe-DDMP complex, followed by acidic precipitation
and partitioning of the precipitate between water and n-butanol.
SAPONINS 247
Oil-free soy flour (10 kg)
containing 0.2% of glucoside isoflavones, and 0.3% group B soyasaponins
Reflux extraction with 95% ethanol (30 L, X 5)
Concentration of the combined extracts (to 5 L)
Dilute with H
2
O (1.5 L)
Hexane extraction (5L, X 4)
Alcohol phase Hexane phase
Extraction with Treatment with polyethoxylated castor oil to
n-butanol (X4, 2.5 L) dissolve the resinous residues
Suspend in water (5 L)
Organic phase Adsorption to a polystyrene-based resin (XAD1180)
Concentrate/Dry Flush with water
Elution with 95% ethanol (10 L)
133 g
Concentrate/Dry
130 g
Soya extract: 22.5 % (w/w) group B soyasaponin
15 % (w/w) isoflavones
Figure 6 Production and purification of soya extract containing saponins and isoflavones (adapted from Bombardelli and Gabetta, 2001).
248
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
Suspend the extract (200 g) in 20% ethanol
Dilute with ethyl acetate (0.5 L)
Heat to complete dissolution
Precipitation at room temperature
Filter
Precipitate Liquid phase
Group B soyasaponins Organic phase
93 % purity, 38 g
Concentrate and dry
Isoflavones
81% purity, 37 g
Figure 6 (Continued)
Figure 7 Fractionation of soybean glycosides based on chemical characteristics of soybean saponin βg (from Yoshiki et al., 2005, Copyright (2005), with
permission from Elsevier).
SAPONINS 249
The evaporated and freeze-dried n-butanol fraction contained
Group B (>90%) and E saponins (>10%).
Soyasaponin-I has also been isolated from other legumes in-
cluding red and white clover, alfalfa, and lucerne using sol-
vent precipitation, adsorption, and heat treatment in an aqueous
lower aliphatic solution of an alkali hydroxide (Kitagawa, 1986).
One approach involved adsorption of the concentrated extract in
water or water:alcohol mixture (30%) using a porous, cross-
linked polystyrene resin, followed by subsequent elution (with
alcohol or alcohol:water mixture), concentration and purifica-
tion of the saponins by column chromatography on silica gel.
Alternatively, the crude saponins were recovered in n-butanol
by distributional extraction of the concentrated extract. The iso-
lation of soyasaponin-I from the n-butanol fraction was then
achieved by solvent precipitation (using a soyasaponin soluble
and insoluble solvent pair such as methanol and ethyl acetate)
followed by treatment with activated charcoal and crystallization
from a solvent mixture of chloroform:methanol:water. Alterna-
tively, the n-butanol extract was heated in an aqueous lower
aliphatic alcohol solution of an alkali hydroxide under reflux,
neutralized (by passing it through a column of an ion exchange
resin of strong acid type), concentrated, and further purified by
column chromatography on silica gel to obtain soyasaponin-I
(Kitagawa, 1986).
Quinoa saponin concentrates containing up to 85–90%
saponins were produced by ultrafiltration of aqueous alcohol
extracts (Muir et al., 2002). Individual saponins were then
recovered by Reversed Phase Solid Phase Extraction and prepar-
ative RP-HPLC with 98% purity. Solvent (water-n-butanol) par-
titioning, dialysis, and membrane filtration have also been in-
vestigated for the recovery of saponins from quinoa (Muir et al.,
2002).
A patented process for the isolation of escin from horse chest-
nut uses the ether extraction of the cholesterol-saponin adduct
obtained by treating an aqueous-alcoholic horse chestnut ex-
tract with cholesterol and separation of the resulting precipitate
(Wagner and Bosse, 1964). Further fractionation of escin into
its two isomers of high purity is achieved by converting it into
free acid form (by treating it with a cation exchange agent) and
heating (50–90
C) until one of the isomers is precipitated due
to low solubility in water (Wagner and Bosse, 1963).
Foaming properties of saponins have also been used for the
concentration of saponins from unfermented aqueous mixtures
(Barbour and Dibb, 1976). A 10–50 fold saponin concentration
in the aqueous extract was achieved by foam fractionation with
a suitable gas (air, nitrogen and carbon dioxide) (Barbour and
Dibb, 1976).
Effect of Processing on Saponin Structure/Properties
As the processing focus shifts from elimination of saponins
to their extraction/concentration or retention, information on
the effect of processing conditions (such as heat treatment)
on the content, structure and properties of saponins becomes
akey element in process development. Chemical modification
of saponins, as outlined in the section on their physicochemical
properties, can take place during processing and/or storage re-
sulting in a change in their total content, composition, and prop-
erties/biological activity which may or may not be desirable.
Information on the effect of processing conditions on saponins
is not only essential to product quality but can also be exploited
to customize the saponin properties for a specific application.
Earlier research on the effect of processing conditions on
saponins concentrated on the effects of food processing meth-
ods such as cooking, soaking, canning, and fermentation on
the saponin content of food plants or foods. The decrease in
saponin content of foods caused by these processes has been
well-documented for a variety of foods such as legumes and
quinoa (Anderson and Wolf, 1995; Zhou and Erdman, 1997;
Ridout et al., 1991).
The most widely investigated saponin group has been the gin-
senosides with a wealth of information available on the effect of
various processes such as drying (Du et al., 2004; Popovich et
al., 2005), microwave and conventional heating (Ren and Chen,
1999), steaming (Kim et al., 2000), chemical treatment (Kim
et al., 1998a), extraction parameters (Du et al., 2004), irradiation
(Kwon et al., 1990), and storage (Du et al., 2004) on the concen-
tration of individual ginsenosides and/or their biological activity.
The effects of heating, extraction, and storage on oat saponins
(
¨
Onning et al., 1994), alfalfa saponins (Tava et al., 2003), and
soyasaponins (Daveby et al., 1998) have also been documented.
The thermal stability of selected saponins has been inves-
tigated by process conditions (time, temperature, pH) and the
properties of the saponin. Oat saponins (avenacosides A and
B) were heated at 100 and 140
Catdifferent pH to study
the degradation during heat processing (
¨
Onning et al., 1994).
While they were stable up to 100
C for 3 hr at pH 4–7, heating
at 140
C especially at pH 4 lead to partial degradation. The
degradation rate was significantly increased at pH 4–6 in the
presence of catalytic amounts of iron and stainless steel. Drying
of American ginseng at temperatures above 40
C resulted in
a decrease in the total ginsenoside content (Reynolds, 1998;
Du et al., 2004) with a corresponding increase in the ratio of
neutral/malonly ginsenosides, which was attributed to the hy-
drolysis of malonyl to neutral ginsenosides (Figure 8) (Du et al.,
2004). The lower thermal stability of malonyl ginsenosides was
also documented during heating of American ginseng in 50%
ethanol and aqueous extracts (Ren and Chen, 1999). The effect
of microwave heating on ginsenoside degradation was the same
as conventional heating (Ren and Chen, 1999). A relatively
lower thermal stability was also observed for protopanaxadiol
than protopanaxatriol saponin (Sung et al., 1985).
Degradation can also occur during extraction and storage as
affected by time and temperature (Daveby et al., 1998; Tava
et al., 2003). Extraction temperature will be limited by the ther-
mal stability of the target compounds. For example, extraction
of glycyrrhizic acid using pressurized methanol was carried out
at 100
Casthe stability was impaired at temperatures higher
than 120
C (Ong, 2002). The malonyl saikosaponins a and d
250
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
Figure 8 Concentration of ginsenosides in ethanolic extracts obtained from
dried ginseng root powder (data from Du et al., 2004).
were hydrolyzed by heat and/or acid and the saikosaponins
were converted into hydroxylsaikosaponins during the decoc-
tion of Bupleurim falcatum roots (Ebata et al., 1996). DDMP-
conjugated soyasaponin I was converted into soyasaponin I dur-
ing extraction and storage of dehulled peas (Daveby et al., 1998).
Prolonged storage of Medicago sativa L. saponins in ethanol
resulted in artefact formation due to esterification of acidic
saponins with alcohol (Tava et al., 2003). Extraction solvent
also affects the properties of the product through its effect on
the content and composition of saponins as outlined in the sec-
tion on extraction solvent.
In the majority of the studies, the effect of processing on
saponins has been monitored using the total content or com-
position of the saponin mixture. Changes in the saponin con-
tent/composition, resulting from degradation of saponins present
in the raw material and production of new saponins, in turn affect
their properties such as bioactivity with significant implications
for product quality and product development.
The realization of the enhanced biological activity (antiox-
idant, anticancer activity) of heat-treated ginseng (such as red
ginseng produced by steaming and drying) has put the research
focus on the identification of trace compounds formed during
heating (Rh
2
,Rg
3
,Rg
5
,Rh
1
) and the investigation of their bi-
ological activity (Yun et al., 2001; Kim et al., 2000). Steaming
of raw ginseng at temperatures >100
C enhanced its biological
properties such as its vasodilating (Kim et al., 2000), radical
scavenging activity (Figure 9, Kim et al., 2000) and cytotoxicity
(Figure 10, Park et al., 2002). The enhanced activity was in turn
attributed to the changes in the composition of the ginsenoside
mixture induced by the heat treatment (Figure 11). These find-
ings have led to the use of processing as a means to enhance
the biological activity of ginsenosides (Park, 2005; Kim et al.,
1998b; An et al., 2005). A prodecure containing a series of dry-
ing and steaming steps has been used to improve the content of
ginsenosides with anticancer activity such as Rg
1
,Rg
2
,Rg
3
, and
Rf (An et al., 2005). A ginseng product (sun ginseng) (with a ra-
Figure 9 Radical scavenging activity of raw and steamed ginsengs (mean
± sem, n = 5) (from Kim et al., 2000. Copyright (2000) American Chemical
Society, and American Society of Pharmacognosy).
Figure 10 Cytotoxicity of (a) Methanol extracts of white ginseng (WG), red
ginseng (RG), processed ginseng (SG, 120
C, 3 h), and (b) Purified ginsenosides
(from Park et al., 2002, with permission).
SAPONINS 251
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
F4 Rg2 Rg1 Rg5Rg3 Rf Re Rd Rc Rb2 Rb1
Ginsenoside
Ginsenoside content (w/w, %)
raw ginseng
100 °C
110 °C
120 °C
Figure 11 Content (w/w%) of ginsenosides in raw ginseng and ginsengs steamed at 100, 110, and 120
C (data from Kim et al., 2000).
tio of ginsenoside (Rg
3
+Rg
5
)to(Rc+Rd+Rb
1
+Rb
2
) above 1)
produced by heat treatment at 120–180
C for 0.5–20 hours with
enhanced pharmacological effects such as antioxidant and va-
sodilation activity has also been patented (Kim et al., 1998b).
The hydrolysis of saponins to sapogenins can also mod-
ify their bioactivity (as discussed in the section on biological
activity). Ginseng sapogenins have been shown to have potent
anticancer activity making them the focus of drug development
efforts as discussed in the next section.
The biological activity of saponins can also be modified
by structural changes induced by activity of enzymes natu-
rally present in the plant material. Enzymatic hydrolysis of
bidesmodic saponins retained in the fruit pulp of Phytolacca
dodecandra berries upon crushing of the berries during aque-
ous extraction resulted in the formation of monodesmosides
with high molluscicidal activity (Ndamba et al., 1994). Simi-
larly, the fungitoxicities of the oat avenacosides were activated
by the cleavage of their C-26 bound glucose moiety by α-
glucosidase (avenacosidase) contained in oat leaves (Grunweller
and Kesselmeier, 1985).
Chemical modification of DDMP-conjugated soyasaponins
in soybeans can lead to changes in the quality of soybean foods.
Forexample, while hydrolysis of DDMP saponins can lead to
changes in flavor characteristics, the color of the product can be
modified by the formation of an insoluble brown complex in the
presence of iron (Okubo and Yoshiki, 1994).
Extraction and Purification of Sapogenins
The isolation of sapogenins from plant materials has been
widely investigated due to their commercial significance as
steroid precursors (Marker et al., 1947; Rothrock et al., 1957).
There is renewed interest on production of sapogenins arising
from evidence on their biological activities, which are being
exploited in a number of applications including pharmaceuti-
cals and cosmetics as described in the section on commercial
applications.
Sapogenins can be produced using chemical (Muir et al.,
2002; Rothrock et al., 1957), enzymatic (Isaac, 1977), or hy-
drothermal (Wilkins and Holt, 1958; Wilkins and Holt, 1961)
hydrolysis of saponins present in the plant material followed by
extraction with organic solvents (such as methanol, ether, ethy-
lene chloride, benzene, carbon tetrachloride, and ethyl acetate)
(Rothrock et al., 1957; Hershberg and Gould, 1956; Spensley,
1955) or supercritical fluids (Inada et al., 1990; De Crosta et al.,
1993). Alternatively, the hydrolysis can be carried out after sol-
vent extraction of saponins (Wall et al., 1952; Muir et al., 2002)
or after expressing the juice containing saponins (L¨oken, 1975;
Miramontes, 1959). Hydrolysis and extraction can take place
simultaneously utilizing supercritical fluids (De Crosta et al.,
1993; Inada et al., 1990). In their patent on the extraction of plant
materials using supercritical fluids, De Crosta et al. (De Crosta
et al., 1993) describe a procedure for the extraction of steroid
aglycones such as diosgenin and sarsapogenin from plants (bar-
basco root and Yucca seed, respectively) using CO
2
modified
with 10% chloroform and a pressure gradient of 100–300 atm at
250
C, which employs a hydrolysis step during or prior to the
supercritical fluid extraction.
Sample pretreatment steps such as incubation with
(Miramontes, 1959) or without (Gould and Hershberg, 1956) en-
zymes (carbohydrases such as cellulase and pectinase) and/or the
addition of steroid precursors, saturated hydrocarbons, and plant
growth regulators (Hardman, 1971) have been shown to increase
252
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
Crude ginseng extract (10 g) in 95% ethanol (40 mL)
Add NaOH (40 mL 5 N)
Reaction at 240
ο
C, 3.5 MPa, 1.5 hours
Add HCl (ph=7)
Add H
2
O (Total volume = 800 mL)
Extract with acetic ester (3 x 100 mL)
3.8 g dried extract
Dissolve ground extract in methanol (20 ml)
Silica gel chromatography
Ether:petroleum benzin (1:3, 60 mL) Chloroform:methanol (95:5, 90 mL)
PAM-120 (250 mg)
PAN 20 (50 mg)
PBM-100 (45 mg)
Glc-O
OH
OH
OH
OH
OH
OH
Figure 12 Production of dammarane sapogenins from ginseng (adapted from Huang and Qi, 2005).
SAPONINS 253
the yield of steroid sapogenins. Heating the fermented slurry ob-
tained as waste juice arising from decortication of leaves of the
plant Agave sisalan to temperatures above 140
C under pressure
facilitated the separation of the solids by filtration or centrifu-
gation (Wilkins and Holt, 1958; Wilkins and Holt, 1961).
A recent patent (Huang and Qi, 2005) describes the produc-
tion of sapogenins from ginseng by reacting a crude ginseng ex-
tract with water and a short chain alkali metal alcoholate solution
or hydroxide ethanol solution at high temperature (150–300
C)
and pressure (2.5–8.4 MPa) (Figure 12). Further purification of
the reaction mixture was achieved using silica gel column chro-
matography to yield novel sapogenins with anti-cancer activity
including PAM-120, PBM-110, PBM-100, PAN-20, and PAN-
30 (Huang and Qi, 2005).
The aglycones of saponin molecules, such as betulinic
acid and oleanolic acid, are also present in nature as isolated
molecules. In those cases, their isolation from the plant material
only necessitates extraction and purification steps. For example,
betulinic acid was extracted from the bark of trees such as Pla-
tanus acerifolia species using medium polarity solvents such as
dichloromethane, chloroform or diethylether followed by crys-
tallization from methanol (Draeger et al., 2001). An herbal ex-
tract containing betulinic acid with anticancer activity was pro-
duced from ground bark of Zizyphus jujuba by macerating the
bark in solvent (10–50% aqueous ethanol) (Mukherjee et al.,
2004). The recognition of the health benefits of oleanolic acid
resulted in the development of processes for the production of
extracts containing oleanolic acid from skins of fruits such as
apples, pears, cranberries, cherries, and prunes using organic
solvents for use in food formulations (Beindorff et al., 2001)
and for the fortification of food products such as olive oil with
oleanolic acid (van Putte, 2002).
CONCLUSIONS
Saponins include a diverse group of compounds characterized
by their structure containing a steroid or triterpenoid aglycone
and one or more sugar chains. Their physicochemical and bio-
logical properties, few of which are common to all members of
this diverse group, are increasingly being exploited in food, cos-
metics and pharmaceutical sectors. The full realization of their
commercial potential, which is driven by consumer demand for
natural products and increasing evidence of their health bene-
fits, requires development of commercially feasible processes
that can address processing challenges posed by their complex
nature, including their stability. Information on the composi-
tion (qualitative and quantitative) and properties of the saponins
present in the raw material, and the effects of processing on
their composition and properties are key elements of success-
ful process design. The abundance of saponins in nature and
their presence in significant quantities in processing by-products
(such as by-products of soybean processing) result in a wide
range of natural materials that can be exploited for commercial
production.
REFERENCES
Alice, C.B., Vargas, V.M.F., Silva, G.A.A.B., de Siqueira, N.C.S., Schapoval,
E.E.S., Gleye, J., Henriques, J.A.P., and Henriques, A.T. 1991. Screening of
plants used in south Brazilian folk medicine. J. Ethnopharmacol., 35:165–
171.
An, H.-S., Lee, T.-S., Shin, E.-M., and Kim, S.-H. 2005. Method of improving
the content of anticancer substances in ginseng. WO Patent 2005/020715.
Anderson, R.L., and Wolf, W.J. 1995. Compositional changes in trypsin in-
hibitors, phytic acid, saponins and isoflavones related to soybean processing.
J. Nutr., 125: 581S–588S.
Anonymous. 2004. The fine line. Soap, Perfumery & Cosmetics, July:57.
Aoun, M., Amiand, G., Garres, P., and Boide, P. 2003. Food supplement used
in feed formulations in ruminants. WO Patent 03/056935 A1.
Ash, M., and Ash, I. 2002. Handbook of Food Additives. Synapse Information
Resources, Inc., NY.
Ashida, T., and Matsuda, S. 1999. Food additive and its production. JP Patent
11,225,724.
Balandrin, M.F. 1996. Commercial utilization of plant-derived saponins: An
overview of medicinal, pharmaceutical, and industrial applications. In: G.
R. Waller, and K. Yamasaki, Eds., Saponins Used in Traditional Medicine.
Plenum Press, New York, pp. 1–14.
Barbour, J.B., and Dibb, D.N. 1976. Recovery of surface active saponins. GB
Patent 1,452,106.
Beindorff, C., Cain, F.W., Pierce, J.H., Schmid, U., Schweitzer, E., and van
Straalen, J.N.M. 2001. Blends of ursolic acid /oleanolic acid. EP Patent
1,161,879 A2.
Benthin, B., Danz, H., and Hamburger, M. 1999. Pressurized liquid extraction
of medicinal plants. J. Chromatogr. A, 837:211–219.
Berhow, M.A., Cantrell, C.L., Duval, S.M., Dobbins, T.A., Maynes, J.,
and Vaughn, S.F. 2002. Analysis and quantitative determination of group
B saponins in processed soybean products. Phytochem. Anal., 13:343–
348.
Berhow, M.A., Wagner, E.D., Vaughn, S.F., and Plewa, M.J. 2000. Characteriza-
tion and antimutagenic activity of soybean saponins. Mutat. Res., 448:11–22.
Bhaggan, K., Cain, F.W., Pierce, J.H., Rogers, J.S., and Schmid, U. 2001. Fat
blends with crystal modifiers. EP Patent 1,123,659 A1.
Bingham, R., Harris, D.H., and Laga, T. 1978. Yucca plant saponin in the treat-
ment of hypertension and hypercholesterolemia. J. Appl. Nutr., 30:127–136.
Bio-Botanica Inc. 2005. Bio-saponins
TM
The natural surface active agent.
http://www.bio-botanica.com/articles/articles.asp#, accessed 11/10/2005.
Biran, M., and Baykut, S. 1975. Physico-chemical properties of gypsophia
saponin. Chim. Acta Turc., 3:63–88.
Blunden, G., Culling, M.C., and Jewers, K. 1975. Steroidal sapogenins: a review
of actual and potential plant sources. Trop. Sci., 17:139–154.
Bombardelli, E., and Gabetta, B. 2001. Soya extract, process for its preparation
and pharmaceutical composition. US Patent 6,280,777.
Bombardelli, E., Morazzoni, P., Cristoni, A., and Seghizzi, R. 2001. Pharma-
ceutical and cosmetic formulations with antimicrobial activity. US Patent
Application 2001/0046525 A1.
Bomford, R., Stapleton, M., Winsor, S., Beesly, J.E., Jessup, E.A., Price, K.R.,
and Fenwick, G.R. 1992. Adjuvancity and ISCOM formation by structurally
diverse saponins. Vaccine, 10:572–577.
Bonte, F., Meybeck, A., and Massiot, G. 1998. Method of treatment for combat-
ting the effects of aging on the condition of skin and hair. US Patent 5,770,223.
Brand, H., and Brand, E. 2004. A weighty issue. Soap, Perfumery & Cosmetics
Asia, March:27–31.
Brown, R. 1998. The natural way in cosmetics and skin care. Chemical Market
Reporter, 254(2):FR8–9.
Budavari, S., O’Neil, M.J., Smith, A., Heckelman, P.E., and Kinneary, J.F. 1996.
The Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals.
Merck and Co., Inc. Rahway, New Jersey.
Chapman, L., Johns, T., and Mahunnah, R.L.A. 1997. Saponin-like in vitro
characteristics of extracts from selected non-nutrient wild plant food addi-
tives used by Masaai in meat and milk based soups. Ecol. Food Nutr., 36:1–
22.
254
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
Chauhan, G.S., Cui, W., and Eskin, N.A.M. 1999. Effect of saponin on the
surface properties of quinoa proteins. Int. J. Food Prop., 2:13–22.
Cheeke, P.R. 1999. Actual and potential applications of Yucca schidigera
and Quillaja saponaria saponins in human and animal nutrition. Proceed-
ings of the American Society of Animal Science, 1999. http://www.asas.org
/JAS/symposia/proceedings/0909.pdf, accessed 11/10/2005.
Choi, M.P.K., Chan, K.K.C., Leung, H.W., and Huie, C.W. 2003. Pressurized
liquid extraction of active ingredients (ginsenosides) from medicinal plants
using non-ionic surfactant solutions. J. Chromatogr. A, 983:153–162.
Chuanjing, S., Yunpeng, S., and Minghua, S. 2000. Supercritical CO
2
extraction
method for extracting glycyrrhizinic acid from licorice. CN Patent 1,272,501.
Cox, J.C., Coulter, A.R., Morein, B., Lovgren-Bengtsson, K., and Sundquist, B.
2002. Saponin preparations and use thereof in ISCOMS. US Patent 6,352,697.
Dalsgaard, K. 1974. Saponin adjuvants III. Isolation of a substance from Quillaja
saponaria Molina with adjuvant activity in foot-and-mouth disease vaccines.
Archiv fur die gesamte Virusforschung, 44:243–254.
Daveby, Y.D., Aman, P., Betz, J.M., and Musser, S.M. 1998. Effect of storage and
extraction on ratio of soyasaponin I to 2,3-dihydro-2,5-dihydroxy-6-methyl-
4-pyrone-conjugated soyasaponin I in dehulled peas (Pisum sativum L). J.
Sci. Food Agric., 78:141–146.
De Crosta, M. A., Kabasakalian, P., and Honold, Sr. J. F. 1993. Extraction of com-
pounds from plant materials using supercritical fluids. US Patent 5,252,729.
Dirk, D.D., and Webb, S.R. 2005. The next 15 years: Taking plant vaccines
beyond proof of concept. Immunol. Cell Biol., 83:248–256.
Dobbins, T. 2002. Process for isolating saponins from soybean derived materials.
US Patent 6,355,816.
Draeger, B., Galgon, T., Neubert, R., and Wohlrab, W. 2001. Method of produc-
ing betulinic acid. US Patent 6,175,035.
Du, X. W., Wills, R. B. H., and Stuart, D. L. 2004. Changes in neutral and
malonyl ginsenosides in American ginseng (Panax quinquefolium) during
drying, storage and ethanolic extraction. Food Chem., 86:155–159.
Eastwood, J., Vavasour, E., Baines, J. (First draft). 2005. WHO Food Additives
Series: 48 Safety evaluation of certain food additives and contaminants Quil-
laia extracts. http://www.inchem.org/documents/jecfa/jecmono/v48je03.htm,
accessed 11/10/2005.
Ebata, N., Nakajima, K., Hayashi, K., Okada, M., and Maruno, M. 1996.
Saponins from the root of Bupleurum falcatum. Phytochem., 41:895–901.
Endale, A., Schmidt, P. C., and Gebre-Mariam, T. 2004. Standardisation and
physicochemical characterisation of the extracts of seeds of Glinus lotoides.
Pharmazie, 59:34–38.
Environment Canada. 2005. Microwave-assisted process (MAP
TM
)
as an industrial energy-efficient extraction process for edible oils.
http://www.ec.gc.ca/press/globe00/000323
2 b e.htm., accessed 11/10/2005.
Estrada, A., Li, B., and Laarveld, B. 1998. Adjuvant action of Chenopodium
quinoa saponins on the induction of antibody responses to intragastric and
intranasal administered antigens in mice. Comp. Immunol. Microb., 21:225–
236.
Fang, Q., Yeung, H. W., Leung, H. W., and Huie, C. W. 2000. Micelle-
mediated extraction and preconcentration of ginsenosides from Chinese
herbal medicine. J. Chromatogr. A, 904:47–55.
Fenwick, G. R., Price, K. R., Tsukamoto, C., and Okubo, K. 1991. Saponins.
In: J.P.F. D’Mello, C.M. Duffus, and J.H. Duffus, Eds. Toxic Substances in
Crop Plants. The Royal Society of Chemistry, Cambridge, pp. 285–327.
Forse, R. A., and Chavali, S. 1997. Enternal formulations for treatment of in-
flammation and infection. US Patent 5,674,853.
Francis, G., Kerem, Z., Makkar, H.P.S., and Becker, K. 2002. The biological
action of saponins in animal systems: A review. Brit. J. Nutr., 88:587–605.
Gafner, S., Bergeron, C., McCollom, M. M., Cooper, L. M., McPhail, K. L.,
Gerwick, W. H., and Angerhofer, C. K. 2004. Evaluation of the efficiency
of three different solvent systems to extract triterpene saponins from roots
of Panax quinquefolius using high-performance liquid chromatography. J.
Agric. Food Chem., 52:1546–1550.
Ge, F.-H., Li, Y., Xie, J.-M., Li, Q., Ma, G.-J., Chen, Y.-H., Lin, Y.-C., and Li,
X.-F. 2000. Studies on technology of supercritical-CO
2
fluid extraction for
volatile oils and saikosaponins in Bupleurum chinense DC. China Journal of
Chinese Materia Medica, 25:149–153.
George, A. J. 1965. Legal status and toxicity of saponins. Food Cosmet. Toxicol.,
3:85–91.
Gestetner, B., Assa, Y., Henis, Y., Birk, Y., and Bondi, A. 1971. Lucerne saponins
IV. Relationship between their chemical constitution, and haemolytic and
antifungal activities. J. Sci. Food Agric., 22:168–172.
Gestetner, B., Assa, Y., Henis, Y., Tencer, Y., Rotman, M., Birk, Y., and Bondi,
A. 1972. Interaction of lucerne saponins with sterols. Biochim. Biophys. Acta,
270:181–187.
Gestetner, B., Birk, Y., and Tencer, Y. 1968. Soybean saponins:Fate of ingested
soybean saponins and the physiological aspect of their hemolytic activity. J.
Agric. Food Chem., 16:1031–1035.
Giichi, H. 1987. Production of saponin containing no isoflavone from soybean
embryo bud. JP Patent 62,005,917.
Godwithus Co., Ltd. 2005. Saponia [Korean Ginseng Oil].
http://godwithus.en.ec21.com/GC00513155/Saponia
[Korean Ginseng
oil].html, accessed on 11/10/2005.
Gould, D. H., and Hershberg, E. B. C. T. 1956. Process for the recovery of
saponins and sapogenins from vegetable matter. US Patent 2,774,713.
Grunweller, S., and Kesselmeier, J. 1985. Characterization of a membrane bound
β-glucosidase responsible for the activation of oat leaf saponins. Phytochem.,
24:1941–1943.
Gu, L., Tao, G., Gu, W., and Prior, R. L. 2002. Determination of soyasaponins
in soy with LC-MS following structural unification by partial alkaline degra-
dation. J. Agric. Food Chem., 50:6951–6959.
Gurfinkel, D.M., and Rao, A.V. 2003. Soyasaponins: The relationship between
chemical structure and colon anticarcinogenic activity. Nutr. Cancer, 47:24–
33.
Hamburger, M., Baumann, D., and Adler, S. 2004. Supercritical carbon dioxide
extraction of selected medicinal plants-Effects of high pressure and added
ethanol on yield of extracted substances. Phytochem. Anal., 15:46–54.
Haokui, J., Haisong, Z., and Yuerong, J. 2001. Continuous technological process
of extracting soybean and separating protein, isoflavone, oligosaccharide and
saponin. CN Patent 1,323,534.
Hardman, R. 1971. Extraction of steroidal materials from vegetable materials.
US Patent 3,620,919.
Henderson, G. M. 2001. Bio-enhancer. US Patent 6,228,265 B1.
Hershberg, E.B., and Gould, D. H. 1956. Process for the extraction of sapogenins
from plant materials. US Patent 2,774,714.
Hidvegi, M. 1994. Process for the preparation of a pharmaceutical composition
selectively lowering the blood-lipid level. US 5,277,910.
Hielscher GmbH. 2005. Hielscher—Ultrasound Technology. http://www.
hielscher.com/,accessed 11/10/2005.
Hostettmann, K., and Marston, A. 1995. Saponins. Cambridge University Press,
Cambridge, New York.
Hsiang, C.-Y., Lai, I.-L., Chao, D.-C., and Ho, T.-Y. 2002. Differential regulation
of activator protein 1 activity by glycyrrhizin. Life Sci., 70:1643–1656.
Hsu, Y.-L., Kuo, P.-L., and Lin, C.-C. 2004. The proliferative inhibition and
apoptotic mechanism of Saikosaponin D in human non-small cell lung cancer
A549 cells. Life Sci., 75:1231–1242.
Hsu, H.-Y., Yang, J.-J., and Lin, C.-C. 1997. Effects of oleanolic acid and ursolic
acid on inhibiting tumor growth and enhancing the recovery of hematopoietic
system postirradiation in mice. Cancer Lett., 111:7–13.
Hu, J., Reddy, M. B., Hendrich, S., and Murphy, P. A. 2004. Soyasaponin I and
sapongenol B have limited absorption by caco-2 intestinal cells and limited
bioavailability in women. J. Nutr., 134:1867–1873.
Hu, K., and Yao, X. 2001. Methyl protogracillin (NSC-698792): the spectrum
of cytotoxicity against 60 human cancer cell lines in the National Cancer
Institute’s anticancer drug screen panel. Anti-Cancer Drug., 12:541–547.
Hu, K., and Yao, X. 2002a. The cytotoxicity of protoneodioscin (NSC-698789),
a furostanol saponin from the rhizomes of Dioscorea collettii var. hypoglauca,
against human cancer cells in vitro. Phytomedicine, 9:560–565.
Hu, K., and Yao, X. 2002b. Protodioscin (NSC-698 796): its spectrum of cy-
totoxicity against sixty human cancer cell lines in an anticancer drug screen
panel. Planta Med., 68:297–301.
Hu, K., and Yao, X. 2003. The cytotoxicity of methyl protoneogracillin
(NSC-698793) and gracillin (NSC-698787), two steroidal saponins from the
SAPONINS 255
rhizomes of Dioscorea collettii var. hypoglauca, against human cancer cells
in vitro. Phytother. Res., 17:620–626.
Huang, W., and Jia, W. 2005. Use of ginsenosides Rh2 & Rg3, and aglycon gin-
senosides for the prevention of cancer. WO Patent Application 2005/034963
A1.
Huang, D., and Qi, D. F. 2005. Dammarane sapogenins, their use as anti-cancer
agents, and a process producing same. US Patent 6,888,014 B2.
Hwang, D.-C., and Damodaran, S. 1994. Selective precipitation of fat globule
membranes of cheese whey by saponin and bile salt. J. Agric. Food Chem.,
42:1872–1878.
Ibanoglu, E., and Ibanoglu, S. 2000. Foaming behavior of liquorice (Glycyrrhiza
glabra)extract. Food Chem., 70:333–336.
Ikedo, S., Shimoyamada, M., and Watanabe, K. 1996. Interaction between
bovine serum albumin and saponin as studied by heat stability and protease
digestion. J. Agric. Food Chem., 44:792–795.
Inada, S., Ogasawara, J., and Takahashi, M. 1990. Method for treating glycoside.
US Patent 4,968,787.
Indena. 2005. Horse chestnut saponins. http://www.indena.com/pdf/cosmleaf.
pdf, accessed 24/8/2005.
Isaac, O. 1977. Process for recovering the main sapogenins from the roots of
rhizomes of helleborus. US Patent 4,004,976.
Ishaaya, I., Birk, Y., Bondi, A., and Tencer, Y. 1969. Soyabean saponins IX.—
Studies of their effect on birds, mammals and cold-blooded organisms. J. Sci.
Food Agric., 20:433–436.
Japanese Ministry of Health and Welfare. 2005. List of Existing Food Ad-
ditives. http://www.ffcr.or.jp/zaidan/FFCRHOME.nsf/pages/list-exst.add, ac-
cessed 11/10/2005.
Jensen, J. M., and Elgaard, T. 2001. Natural feed additive, a method of preparing
said feed additive, a feed mixture containing the feed additive as well as a
method of breeding farm animals. EP Patent 1,129,627 A1.
Joint FAO/WHO Expert Committee on Food Additives. 2004. WHO Tech-
nical Report Series 922: Evaluation of certain food additives and con-
taminants. Sixty-first report of the Joint FAO/WHO Expert Committee on
Food Additives. http://whqlibdoc.who.int/trs/WHO
TRS 922.pdf, accessed
11/10/2005.
Joint FAO/WHO Expert Committee on Food Additives. 2005a. WHO Tech-
nical Report Series 928: Evaluation of certain food additives. Sixty-third
report of the Joint FAO/WHO Expert Committee on Food Additives.
http://whqlibdoc.who.int/trs/WHO
TRS 928.pdf, accessed 11/10/2005.
Joint FAO/WHO Expert Committee on Food Additives. 2005b. Sixty-
fifth meeting Geneva, 7–16 June 2005. Summary and Conclu-
sions. http://www.who.int/ipcs/food/jecfa/summaries/summary65.pdf, ac-
cessed 11/10/2005.
Kang, R. K. L., Zyzak, L. L., and Nakatsu, T. 1999. Flavored product additive
and method for using same. US Patent 5,948,460.
Kasai, R., Fujino, H., Kuzuki, T., Wong, W.-H., Goto, C., Yata, N., Tanaka, O.,
Yasuhara, F., and Yamaguchi, S. 1986a. Acyclic sesquiterpene oligoglyco-
sides from pericarps of Sapindus mukurossi. Phytochem., 25:871–876.
Kasai, R., Miyakoshi, M., Matsumoto, K., Nie, R.-L., Zhou, J., Morita, T., and
Tanaka, O. 1986b. Tubeimoside I, a new cyclic bidesmoside from chinese
cucurbitaceous folk medicine “Tu Bei Mu”, a tuber of Bolbostemma panicu-
latum. Chem. Pharm. Bull., 34:3974–3977.
Kennelly, E. J., Suttisri, R., and Kinghorn, A. D. 1996. Novel sweet-tasting
saponins of the cycloartane, oleanane, secodammarane, and steroidal types.
In: G.R. Waller, and K. Yamasaki, Eds., Saponins Used in Food and Agricul-
ture. Plenum Press, New York, pp. 13–24.
Kensil, C. A., and Marciani, D. J. 1991. Saponin adjuvant. US Patent 5,057,540.
Kensil, C. A., Soltysik, S., Marciani, D. J., and Recchia, J. 1996. Drug delivery
enhancement via modified saponins. WO Patent 96/38161.
Kensil, C. R., Mo, A. X., and Truneh, A. 2004. Current vaccine adjuvants: an
overview of a diverse class. Frontiers in Bioscience, 9:2972–2988.
Kerem, Z., German-Shashoua, H., and Yarden, O. 2005. Microwave-assisted
extraction of bioactive saponins from chickpea (Cicer arietinum L). J. Sci.
Food Agric., 85:406–412.
Kerwin, S. M. 2004. Soy saponins and the anticancer effects of soybeans and
soy-based foods. Curr. Med. Chem. - Anti-Cancer Agents, 4:263–272.
Khokhar, S., and Chauhan, B. M. 1986. Antinutritional factors in moth bean
(Vigna aconitifolia): Varietal differences and effects of methods of domestic
processing and cooking. J. Food Sci., 51:591–594.
Kim, D.-H., Bae, E.-A., Han, M.-J., Choo, M.-K., Park, E.-K., and Park, J.-H.
2003a. Novel use of the extract of processed panax genus plant and saponin
compound isolated therefrom. US Patent Application 2003/0190377 A1.
Kim, Y. W., Kim, J. M., Han, S. B., Lee, S. K., Kim, N. D., Park, M. K., Kim, C.
K., and Park, J. H. 2000. Steaming of ginseng at high temperature enhances
biological activity. J. Nat. Prod., 63:1702–1704.
Kim, H.-S., Lee, S.-Y., Kim, B.-Y., Lee, E.-K., Ryu, J.-H., and Lim, G.-B. 2004.
Effects of modifiers on the supercritical CO
2
extraction of glycyrrhizin from
licorice and the morphology of licorice tissue after extraction. Biotechnol.
Bioproc. E., 9:447–453.
Kim, D. S., Oh, S. R., Lee, I. S., Jung, K. Y., Park, J. D., Kim, S. I., and Lee, H.-K.
1998a. Anticomplementary activity of ginseng saponins and their degradation
products. Phytochem., 47:397–399.
Kim, S.-W., Park, S.-K., Kang, S.-I., Kang, H.-C., Oh, H.-J., Bae, C.-Y., and Bae,
D.-H. 2003b. Hypocholesterolemic property of Yucca schidigera and Quillaja
saponaria extracts in human body. Arch. Pharm. Res., 26:1042–1046.
Kim, N. D., Park, M. K., Lee, S. K., Park, J. H., and Kim, J. M. 1998b. Processed
ginseng product with enhanced pharmacological effects. US Patent 5,776,460.
Kimata, H., Nakashima, T., Kokubun, S., Nakayama, K., Mitoma, Y., Kitahara,
T.,Yata, N., and Tanaka, O. 1983. Saponins of pericarps of sapindus mukurossi
GAERTN. and solubilization of monodesmosides by bidesmosides. Chem.
Pharm. Bull., 31:1998–2005.
Kimata, H., Sumida, N., Matsufuji, N., Morita, T., Ito, K., Yata, N., and Tanaka,
O. 1985. Interaction of saponin of Bupleuri Radix with ginseng saponin:
solubilization of saikosaponin-a with chikusetsu-saponin V ( = ginsenoside-
Ro). Chem. Pharm. Bull., 33:2849–2853.
Kitagawa, I. 1986. Method of isolating soyasaponins. US Patent 4,594,412.
Kommalapati, R. R., Valsaraj, K. T., Constant, W. D., and Roy, D. 1997. Aqueous
solubility enhancement and desorption of hexachlorobenzene from soil using
a plant-based surfactant. Water Res., 31:2161–2170.
Kudou, S., Tonomura, M., Tsukamoto, C., Uchida, T., Sakabe, T., Tamura,
N., and Okubo, K. 1993. Isolation and structural elucidation of DDMP-
conjugated soyasaponins as genuine saponins from soybean seeds. Biosci.
Biotech. Biochem., 57:546–560.
Kwon, J.-H., B´elanger, M. R., and P´are, J. R. J. 2003a. Optimization of
microwave-assisted extraction (MAP) for ginseng components by response
surface methodology. J. Agric. Food Chem., 51:1807–1810.
Kwon, J.-H., B´elanger, J. M. R., P´are, J. R. J., and Yaylayan, V. A. 2003b.
Application of the microwave-assisted process (MAP
TM
)tothe fast extraction
of ginseng saponins. Food Res. Intl., 36:491–498.
Kwon, J.-H., B´elanger, J. M. R., Sigouin, M., Lanthier, J., Willmot, C., and Par´e,
J.R.J. 1990. Chemical constitutents of Panax ginseng exposed to γ irradiation
. J. Agric. Food Chem., 38:830–834.
Kwon, J.-H., Lee, G.-D., B´elanger, J. M. R., and Par´e, J. R. J. 2003c. Effect
of ethanol concentration on the efficiency of extraction of ginseng saponins
when using a microwave-assisted process (MAP
TM
). Int. J. Food Sci. Tech.,
38:615–622.
Lacaille-Dubois, M.A., and Wagner, H. 1996. A review of the biological and
pharmacological activities of saponins. Phytomedicine, 2:363–386.
Lee, H. K., Koh, H. L., Ong, E. S., and Woo, S. O. 2002. Determination of gin-
senosides in medicinal plants and health supplements by pressurized liquid
extraction (PLE) with reversed phase high performance liquid chromatogra-
phy. J. Sep. Sci., 25:160–166.
Li, T. S. C., Mazza, G., Cottrell, A. C., and Gao, L. 1996. Ginsenosides in roots
and leaves of American ginseng. J. Agric. Food Chem., 44:717–720.
Liu, J. 1995. Pharmacology of oleanolic acid and ursolic acid. J. Ethnopharma-
col., 49:57–68.
Liu, J., and Henkel, T. 2002. Traditional Chinese Medicine (TCM): Are polyphe-
nols and saponins the key ingredients triggering biological activities? Curr.
Med. Chem., 9:1483–1485.
Liu, W. K., Xu, S. X., and Che, C. T. 2000. Anti-proliferative effect of
ginseng saponins on human prostate cancer cell line. Life Sci., 67:1297–
1306.
256
¨
O. G
¨
UC¸L
¨
U-
¨
UST
¨
UNDA
˘
G AND G. MAZZA
Lucas, E. A., Khalil, D. A., Daggy, B. P., and Arjmandi, B. H. 2001. Ethanol-
extracted soy protein isolate does not modulate serum cholesterol in golden
syrian hamsters: a model of postmenopausal hypercholesterolemia. J. Nutr.,
131:211–214.
oken, B. 1975. Process for obtaining a crude sapogenin from agave leaves. US
Patent 3,895,999.
Ma, B., Dong, J., and Wang, B. 2003. Use of steroidal saponins for the propylaxis
or treatment of dementia, and novel steroidal saponin compounds. US Patent
6,593,301.
Malcolm, C. 1995. Teetertottering between synthetic and ‘natural’. DCI, 156
(5):50.
Malinow, M. R., McNulty, W. P., McLaughlin, P., Stafford, C., Burns, A. K.,
Livingston, A. L., and Kohler, G. O. 1981. The toxicity of alfalfa saponins in
rats. Food Cosmet. Toxicol., 19:443–445.
Marciani, D. J., Ptak, R. G., Voss, T. G., Reynolds, R. C., Pathak, A. K., Cham-
blin, T. L., Scholl, D. R., and May, R. D. 2002. Degradation of Quillaja
saponaria Molina saponins: Loss of the protective effects of a herpes simplex
virus 1 subunit vaccine. Int. Immunopharmacol., 2:1703–1711.
Marker, R. E., Wagner, R. B., Ulshafer, P. R., Wittbecker, G. P. J., and Ruof, C.
H. 1947. Steroidal sapogenins. J. Am. Chem. Soc., 69:2167–2230.
Matsuura, H. 2001. Saponins in garlic as modifiers of the risk of cardiovascular
disease. J. Nutr., 131:1000S–1005S.
Micich, T. J., Foglia, T. A., and Holsinger, V. H. 1992. Polymer-supported
saponins: An approach to cholesterol removal from butteroil. J. Agric. Food
Chem., 40:1321–1325.
Milgate, J., and Roberts, D. C. K. 1995. The nutritional and biological signifi-
cance of saponins. Nutr. Res., 15:1223–1249.
Miramontes, L. 1959. Procedure for obtaining sapogenins from natural un-dried
products. US Patent 2,912, 362.
Mitra, S., and Dungan, S.R. 1997. Micellar properties of quillaja saponin. 1.
Effects of temperature, salt and pH on solution properties. J. Agric. Food
Chem., 45:1587–1595.
Mitra, S., and Dungan, S.R. 2000. Micellar properties of quillaja saponin. 2.
Effect of solubilized cholesterol on solution properties. Colloid. Surface. B:
Biointerfaces, 17:117–133.
Mitra, S., and Dungan, S.R. 2001. Cholesterol solubilization in aqueous micellar
solutions of quillaja saponin, bile salts, or nonionic surfactants. J. Agric. Food
Chem., 49:384–394.
Mizui, F., Kasai, R., Ohtani, K., and Tanaka, O. 1990. Saponins from bran of
quinoa, Chenopodium quinoa WILLD. II. Chem. Pharm. Bull., 38:375–377.
Morita, T., Nie, R.-L., Fujino, H., Ito, K., Matsufuji, N., Kasai, R., Zhou, J.,
Wu, C.-Y., Noboru, Y., and Tanaka, O. 1986. Saponins from Chinese cucur-
bitaceous plants: Solubilization of saikosaponin-a with hemslosides Ma2 and
Ma3 and structure of hemsloside H
1
from Hemsleya chinensis. Chem. Pharm.
Bull., 34:401–405.
Morton, P.A.J., and Murray, B.S. 2001. Acid beverage floc: Protein-saponin in-
teraction and an unstable emulsion model. Colloid. Surface. B: Biointerfaces,
21:101–106.
Muir, A. D., Paton, D., Ballantyne, K., and Aubin, A. A. 2002. Process for recov-
ery and purification of saponins and sapogenins from quinoa (Chenopodium
quinoa). US Patent 6,355,249.
Mukherjee, R., Khattar, D., Jaggi, M., Singh, A. T., Kumar, M., and Bala, H.
2004. Method for treating cancer using betulinic acid rich herbal extract. US
Patent Application 2004/0116394.
Muller, R.E., and Morris, R.J. Jr. 1966. Sucrose-ammoniated glycyrrhizin sweet-
ening agent. US Patent 3,282,706.
Nakayama, K., Fujino, H., Kasai, R., Mitoma, Y., Yata, N., and Tanaka, O.
1986. Solubilizing properties of saponins from sapindus mukurossi GAERTN.
Chem. Pharm. Bull., 34:3279–3283.
Ndamba, J., Lemmich, E., and Mlgaard, P. 1994. Release of molluscicidal
saponins from Phytolacca dodecandra aqueous berry extracts as influenced
by the male plant and extraction conditions. Biochem. Syst. Ecol., 22:249–
257.
Nord, L.I., and Kenne, L. 2000. Novel acetylated triterpenoid saponins in a
chromatographic fraction from Quillaja saponaria Molina. Carbohyd. Res.,
329:817–829.
Nozomi, O., Haruo, S., Shisai, R., Fuku, S., Hikari, J., Toshi, H., and Bunshi,
K. 1986. Saikosaponin. JP Patent 61,282,395.
Oakenfull, D. 1981. Saponins in food-a review. Food Chem., 6:19–40.
Oakenfull, D. 1986. Aggregation of saponins and bile acids in aqueous solution.
Aust. J. Chem., 39:1671–1683.
Oakenfull, D. 2001. Soy protein, saponins and plasma cholesterol. J. Nutr.,
131:2971.
Oakenfull, D., and Sidhu, G.S. 1989. Saponins. In: Cheeke, P. R., Ed., Toxicants
of Plant Origin, Vol II Glycosides. CRC Pres, Inc. Boca Raton, Florida, pp.
97–141.
Oakenfull, D., and Sidhu, G.S. 1990. Could saponins be a useful treatment for
hypercholesterolaemia? Eur. J. Clin. Nutr., 44:79–88.
Oda, K., Matsuda, H., Murakami, T., Katayama, S., Ohgitani, T., and Yoshikawa,
M. 2000. Adjuvant and haemolytic activities of 47 saponins derived from
medicinal and food plants. Biol. Chem., 381:67–74.
Office for Official Publications of the European Communities. 1996. Com-
mission directive 96/77/EC of 2 December 1996 laying down specific
purity criteria on food additives other than colours and sweeteners.
http://europa.eu.int/eur-lex/en/consleg/pdf/1996/en
1996L0077 do 001.pdf,
accessed 11/10/2005.
Okubo, K., and Yoshiki, Y. 1994. Effect of DDMP saponin on the flavor and color
of soybean foods. Report of the Soy Protein Research Comittee, 15:36–40.
Okubo, K., and Yoshiki, Y. 1995. Oxygen-radical-scavenging activity of DDMP-
conjugated saponins and physiological role in leguminous plant. In: G.R.
Waller , and K. Yamasaki, Eds., Saponins Used in Food and Agriculture.
Plenum Press, New York, pp. 141–154.
Oleszek, W. 1995. Alfalfa saponins: structure, biological activity, and chemo-
taxonomy. In: Waller, G. R. and Yamasaki, K., Eds., Saponins Used in Food
and Agriculture. Plenum Press, New York, pp. 155–170.
Oleszek, W., Sitek, M., Stochmal, A., Piacente, S., Pizza, C., and Cheeke, P.
2001. Steroidal saponins of Yucca schidigera Roezl. J. Agric. Food Chem.,
49:4392–4396.
Olmstead, M. J. 2002. Organic toothpaste containing saponin. US Patent
6,485,711 B1.
Ong, E. S. 2002. Chemical assay of glycyrrhizin in medicinal plants by pres-
surized liquid extraction (PLE) with capillary zone electrophoresis (CZE). J.
Sep. Sci., 25:825–831.
Ong E.S., and Len, S. M. 2003. Pressurized hot water extraction of berberine,
baicalein and glycyrrhizin in medicinal plants. Anal. Chim. Acta, 482:81–
89.
Organic Technologies. 2005. Organic Technologies Products. http://www.
organictech.com/products/index.html, accessed 11/10/2005.
¨
Onning, G., Juillerat, M. A., Fay, L., and Asp, N.-G. 1994. Degradation of
oat saponins during heat processing-Effect of pH, stainless steel, and iron at
different temperatures. J. Agric. Food Chem., 42:2578–2582.
Pan, X., Liu, H., Jia, G., and Shu, Y. Y. 2000. Microwave-assisted extraction of
glycyrrhizic acid from licorice root. Biochem. Eng. J., 5:173–177.
Panacos. 2005. A new generation of anti-infective drugs. http://www.
panacos.com/product
2.htm, accessed 11/10/2005.
Panagin Pharmaceuticals Inc. 2005. http://www.panagin.com/index.htm, ac-
cessed 23/8/2005.
Park, I. H., Piao, L. Z., Kwon, S. W., Lee, J. Y., Cho, S. Y., Park, M. K., and
Park, J. H. 2002. Cytotoxic dammarane glycosides from processed ginseng.
Chem. Pharm. Bull., 50:538–540.
Park, M. H. 2005. Method for processing ginseng and the uses of extract of
processed ginseng. US Patent Application 2005/0031711 A1.
Plewa, M. J., Wagner, E. D., Kirchoff, L., Repetny, K., Adams, L. C., and
Rayburn, A. L. 1998. The use of single cell gel electrophoresis and flow
cytometry to identify antimutagens from commercial soybean by-products.
Mutat. Res.-Fund. Mol. M., 402:211–218.
Popovich, D. G., Hu, C., Durance, T. D., and Kitts, D. D. 2005. Retention of
ginsenosides in dried ginseng root: comparison of drying methods. J. Food
Sci., 70:S355–358.
Potter, S. M., Jimenez-Flores, R., Pollack, J., Lone, T. A., and Berber-Jimenez,
M. D. 1993. Protein-saponin interaction and its influence on blood lipids. J.
Agric. Food Chem., 41:1287–1291.
SAPONINS 257
Price, K. R., Eagles, J., and Fenwick, G. R. 1988. Saponin composition of 13
varieties of legume seed using fast atom bombardment mass spectrometry. J.
Sci. Food Agric., 42:183–193.
Price, K. R., Griffiths, N. M., Curl, C. R., and Fenwick, G. R. 1985. Undesirable
sensory properties of the dried pea (pisum sativum). The role of saponins.
Food Chem., 17:105–115.
Price, K. R., Johnson, I. T., and Fenwick, G. R. 1987. The chemistry and bio-
logical significance of saponins in foods and feeding stuffs. CRC Crit. Rev.
Food Sci., 26:27–135.
Radient Technologies. 2005. http://www.radientinc.com/, accessed 24/8/2005.
Raju, J., Patlolla, J.M.R., Swamy, M. V., and Rao, C. V. 2004. Diosgenin, a steroid
saponin of Trigonella foenum graecum (Fenugreek), inhibits azoxymethane-
induced aberrant crypt foci formation in F344 rats and induces apoptosis in
HT-29 human colon cancer cells. Cancer Epidem. Biomar., 13:1392–1398.
Rao, A.V., and Sung, M.-K. 1995. Saponins as anticarcinogens. J. Nutr.,
125:717S–724S.
Ren, G., and Chen, F. 1999. Degradation of ginsenosides in American ginseng
(Panax quinquefolium)extracts during microwave and conventional heating.
J. Agric. Food Chem., 47:1501–1505.
Reynolds, L. B. 1998. Effects of drying on chemical and physical characteristics
of American ginseng (Panax quinquefolius L.). Journal of Herbs, Spices &
Medicinal Plants, 6:9–21.
Richardson, T., and Jimenez-Flores, R. 1994. Process to remove cholesterol
from dairy products. US Patent 5,326,579.
Rickert, D. A., Johnson, L. A., and Murphy, P. A. 2004a. Improved fractionation
of glycinin and ß-conglycinin and partitioning of phytochemicals. J. Agric.
Food Chem., 52:1726–1734.
Rickert, D. A., Meyer, M. A., Hu, J., and Murphy, P. A. 2004b. Effect of extraction
pH and temperature on isoflavone and saponin partitioning and profile during
soy protein isolate production. J. Food Sci., 69:C623–C630.
Ridout, C. L., Price, K. R., DuPont, M. S., Parker, M. L., and Fenwick, G.
R. 1991. Quinoa saponins-Analysis and preliminary investigations into the
effects of reduction by processing. J. Sci. Food Agric., 54:165–176.
Rothrock, J. W., Hammes, P. A., and McAleer, W. J. 1957. Isolation of diosgenin
by acid hydrolysis of saponin. Ind. Eng. Chem. Res., 49:186–188.
Roy, D., Kommalapati, R. R., Mandava, S. S., Valsaraj, K. T., and Constant, W.
D. 1997. Soil washing potential of a natural surfactant. Environ. Sci. Technol.,
31:670–695.
San Martin, R., and Briones, R. 1999. Industrial uses and sustainable supply of
Quillaja saponaria (Rosaceae) saponins. Econ. Bot., 53:302–311.
Sarnthein-Graf, C., and La Mesa, C. 2004. Association of saponins in water and
water-gelatine mixtures. Thermochim. Acta, 418:79–84.
Sasaki, Y., Mizutani, K., Kasai, R., and Tanaka, O. 1988. Solubilizing proper-
ties of glycyrrhizin and its derivatives: solubilization of saikosaponin-a, the
saponin of Bupleuri Radix. Chem. Pharm. Bull., 36:3491–3495.
Sasazuka, T., Endo, M., Hiwatashi, K., and Suzuki, H. 1995. Extraction and
partial purification of beet saponin. Proceedings of the Research Society of
Japan Sugar Refineries Technologists, 43:57–62.
Satoshi, M., Erihi, O., and Satariyo, G. 2004. Composition for preventing or
ameliorating ultraviolet damage. JP Patent 2,004,131,431.
Sauvaire, Y., Baissac, Y., Leconte, O., Petit, P., and Ribes, G. 1995. Steroid
saponins from fenugreek and some of their biological properties. In: G.R.
Waller, and K. Yamasaki, Eds., Saponins Used in Food and Agriculture.
Plenum Press, New York, pp. 37–46.
Sauvaire, Y., Petit, P., Baissac, Y., and Ribes, G. 2000. Chemistry and pharma-
cology of fenugreek. In: Mazza, G., and Oomah, B. D., Eds., Herbs, Botani-
cals, & Teas.Technomic Publishing Company, Inc., Lancaster, PA, pp. 107–
129.
Schwarzbach, A., Schreiner, M., and Knorr, D. 2004. Secondary plant compo-
nent, food component and pesticide component. Saponins from asparagus as
health and plant protection agents. Gemuse Munchen, 40:30–31.
Sch¨opke, Th., and Bartlakowski, J. 1997. Effects of saponins on the water sol-
ubility of quercetin. Pharmazie, 52:232–234.
Shany, S., Gestetner, B., Birk, Y., and Bondi, A. 1970. Lucerne saponins III.
Effect of lucerne saponins on larval growth and their detoxification by various
sterols. J. Sci. Food Agric., 21:508–510.
Shimoyamada, M., Ikedo, S., Ootsubo, R., and Watanabe, K. 1998. Effects
of soybean saponins on chymotryptic hydrolyses. J. Agric. Food Chem.,
46:4793–4797.
Shimoyamada, M., Okada, Y.,