Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1-1-2000
Isolation and quantitative analysis of soybean saponins by high performance liquid chromatography
Jiang Hu Iowa State University
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This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Isolation and quantitative analysis of soybean saponins by high
performance liquid chromatography
by
Jiang Hu
A thesis submitted to the graduate faculty
in partial fulfillment of the requirement for the degree of
MASTER OF SCIENCE
Major: Toxicology
Major Professors: Dr. Patricia A. Murphy and Dr. Suzanne Hendrich
Iowa State University
. Ames, Iowa
2000 11
Graduate College Iowa State University
This is to certify that the Master's thesis of Jiang Hu has met the requirement of Iowa State University
Signatures have been redacted for privacy iii
TABLE OF CONTENTS
ABSTRACT iv
GENERAL INTRODUCTION 1
CHAPTER 1. LITERATURE REVIEW 3
1.1 Saponins 3
1.2 Soybean Saponins 14
CHAPTER 2. MATERIALS AND METHODS 34
2.1 Materials 34
2.2 Preparation and Isolation of the Group B Soybean Saponins 35
2.3 Quantitative Analysis of the Group B Soybean Saponins by HPLC 39
CHAPTER 3. RESULTS AND DISCUSSION 43
3.1 Isolation and Purification of the Group B Soybean Saponins 43
3.2 Chemical Identification of Isolated Saponins 46
3.3 Thermostabilty of Purified DDMP-conjugated Saponins 52
3.4 Quantitative Analysis of the Group B Soybean Saponins,by HPLC 53
3.5 Saponin Contents in Soybeans and Soy Products 64
CHAPTER 4. CONCLUSIONS 76
APPENDIX A. MASS SPECTRA OF ISOLATED SOYASAPONINS 79
APPENDIXB. SOYASAPONIN CONTENTS IN 50 VARIETIES OF 83 SOYBEANS
LITERATURE CITED 86
ACKNOWLEDGEMENTS 96 iv
ABSTRACT
Dietary soybean saponins have been proposed the potential role in plasma
cholesterol-lowering, colon cancer inhibiting and immune-stimulating effects to human
health. To evaluate the biological properties of soybean saponins, saponin concentrations in
soy products must be determined. This has led to the need for sensitive and accurate assays
of saponins for food matrices. Methods for isolation and quantitative determination of the
group B soybean saponins by high performance liquid chromatography (HPLC) were
developed. 2, 3-Dihydro- 2, 5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated
soybean saponin ag, ~g, ~a, and their non-DDMP counterparts I, II, and V were isolated and
identified by electrospray mass spectrometry and ultraviolet absorbance spectrometry in
HPLC profile. The yield of purified soyasaponin was > 0.017% (w/w), and the purity was >
95%. The molar extinction coefficients for the group B saponins were ranged from 4500 to
6000 M- 1cm-1 at 292 and 205 nm. The purified DDMP-conjugated saponin standards were
stable at -20°C for at least 15 days or at 30°C for 3 hours without observable decomposition.
The coefficient of variation of saponin quantitation analysis by HPLC was 9.8% for the
within-day assay and <14.3% for the between-days assay. The external recovery of saponin
analysis showed >93% recovery for soyasaponin I analysis. Formononetin was demonstrated to be a good intemaf standard in soyasaponin analysis. The soyasaponin contents were evaluated in soybeans and soy products. The group B saponin concentration in soybeans ranged from 2.50 to 5.85 µmol/g and varied among different varieties of soybeans. The saponin content and composition in soy ingredients (soybean seeds, toasted soy hypocotyls, soy protein isolates, textured vegetable protein, soy protein concentrates, and Novasoy®), V and the typical soy foods (commercial soymilk, tofu, and tempeh) ranged from 0.47 to
114.02 µmol/g. In the commercial soy products, the ratio of total isoflavone to saponin on a molar basis was ranged from 0.2 to 20.4 depending on the production process. 1
GENERAL INTRODUCTION
Saponins are the triterpenoid or steroid glycosides naturally occurring in plants and
some marine organisms. Relatively high concentrations of saponins have been found in
soybeans and soy products. The primary saponins in soybeans· are the bisdesmosidic group
A saponins and the monodesmosidic group B saponins. The amount of saponins on a dry
weight basis has been reported ranging from 0.22 to 0.24 g/lO0g in soybeans (Tsukamoto, et
al. 1995), up to 0.76 g/lO0g in soy ingredients (Ireland, et al. 1986), and from 0.15 to 0.30
g/l00g in tofu, miso, and tempeh (Kitagawa, et al. 1984). Whole soybean seeds contain
about 74% group B saponins and 25% group A saponins by weight according to the profile
reported by Ireland, et al. (1986). The 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one
(DDMP) conjugated saponins have been identified as the genuine group B saponins in raw
soybeans, and their non-DDMP counterparts can be produced by heating during soybean
processing (Kudou, et al. 1993). Five types ofDDMP-conjugated group B soybean
saponins, pg, pa, ya, yg and a.g were isolated and characterized by Kudou, et al. in 1994.
Many studies have shown that soybean saponins possess potential health beneficial
properties. Soybean saponins have been considered as the major active components
contributing to the cholesterol-lowering effect of soy products (Potter, et al. 1995). Soybean
saponins inhibited various tumor developments in vivo and in vitro, particularly development
of colon cancer (Rao and Sung, 1995). The inhibitory effect of the group B soybean
saponins on human immunodeficiency virus (HIV) replication and infection in vitro has been reported (Hayashi, et al. 1996). The anti-hepatotoxic activity of soyasaponin I has been
observed in in vitro studies (Kim, et al. 1997; Miyao, et al. 1997). 2
Isolation and quantitation of soybean saponins have been the main challenge in
saponin research. The major DDMP-conjugated soyasaponins can be extracted by aqueous
alcoholic solvents at or below room temperature (Kudou, et al. 1992), but the further
separation of individual soyasaponins involved complicated procedures and usually gave low
yield. Different methods have been deve~oped for the qualitative and quantitative
determination of saponins, including hemolysis, spectrophotometry, thin layer
chromatography (TLC), gas chromatography (GC), and HPLC. However, most of the
methods only provided qualitative determination or quantitative estimation of limited types
of soybean saponins.
To evaluate the biological properties of soybean saponins, saponin concentrations in.
soy products needs to be measured. This has led to the need for sensitive and accurate assays
of saponins in food matrices. There is no method reported to quantify all the group B
saponins, including both DDMP-conjugated forms and their non-DDMP counterparts in one
chromatogram. Therefore, in the present study, the overall objective was to isolate and
purify the group B soybean saponins to prepare standards for quantification analysis; and to
develop the practical HPLC methods to quantify soybean saponins in various soy products
accurately and precisely. The information obtained in this research project would be useful to evaluate the potential of dietary saponins as human-health enhancing agents, and to provide the general public with quantitative information on soybean saponins in dietary soy products. 3
CHAPTER I
LITERATURE REVIEW
1.1 SAPONINS
The saponins are a family of steroid or triterpenoid glycosides occurring in nature.
The name saponin is derived from the soapwort plant (Saponaria officinalis) due to its distinctive foaming characteristics. Saponins have been used as natural detergents, as well as fish poisons by humans since ancient times. Some plants containing high concentration of saponins have been extensively used as herbal drugs in oriental medicine for centuries.
Although there has been great interest in their biological properties, no saponins were fully characterized before the 1930s because few isolation and structure characterization approaches were available. With advances in modern analytical techniques in chromatography, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, the structures of over a thousand saponins have been elucidated to date (Hostettmann and
Marston 1995).
1.1.1 Classification and structure
There are thousands of different types of saponins present in a wide variety of plants.
The basic structure of the compounds consists of a triterpenoid or steroid aglycone attached to one or more sugar chains, resulting in the amphiphilic nature of saponins (Hostettmann and Marston 1995). The aglycones of saponins are called the sapogenins. Depending on the of aglycone present, saponins can be divided into three main classes: triterpene glycosides, steroid glycosides, and steroid alkaloid glycosides (figure 1.1 ). Modified aglycone skeletons 4
28
Triterpene class HO
.. ... 27
Steroid class
HO
. '' ''
Steroid alkaloid class
HO
Figure 1.1. Aglycone structures found in three principal classes of saponins 5 have been identified for some individual saponins. Monodesmosidic saponins are the type of saponins having a di- or tri-saccharide chain attached at C-3 position of aglycone through an ether linkage. Bisdesmosidic saponins are those having two di- or tri-saccharide chains, one attached at C-3 of the ring structure, another attached through ester linkage to C-28
(triterpene saponins) or C-26 (steroid saponins). Tridesmosidic saponins are rarely found in nature. Glucose, arabinose, glucuronic acid, and xylose are the sugars most frequently found in the polysaccharide chains of saponin structure.
1.1.2 Occurrence and distribution
Saponins are widely distributed throughout the plant kingdom. Over 90 plant families have been reported to contain different kinds of saponins (Oakenfull 1980). Some of plants are used as human foods or livestock feeds, such as alfalfa, soybeans, chickpeas, lentils, spinach, oats, and yams (Oakenfull 1970; Price, et al. 1986; Ireland, et al. 1986), and some used as herbal medicines, such as licorice (Gibson 1978) and ginseng (Shibita 1986).
The saponin content in plants is affected by variety, cultivation, plant tissue location and the physiological age of the plant. It is known that immature plants contain more saponins than do mature ones (Alder, et al. 1985). The distribution of saponins is variable in different organs of the plant. In soybeans, for instance, the saponin level was the highest in the hypocotyl at 0.93% by weight, followed by the root hair at 0.63% and the leaf at 0.43%
(Shimoyamada, etal. 1990). In alfalfa (Medicago sativa L.), zanhic acid saponins are present exclusively in green plant tops while medicagenic acid saponins and soyasaponins are found in the roots (Oleszak 1996). The plant material often contains saponins in considerable amounts. Primula roots contain saponin at 5 to10% by weight. Licorice root 6
. contains 2-12% while Quillaja bark has up to 10% saponins. Ginsenoside levels are 6% by weight in Panax ginseng root hairs (Koizumi, et al. 1982).
Triterpene saponins are the most abundant structure of saponins. The pentacyclic structure of triterpene was first published in 1949 (Bischof, et al. 1949). Triterpene glycosides containing oleananic acid or hederagenin as aglycones are found most commonly.
These saponins are found mainly in the dicotyledon families such as the Leguminoseae,
Araliaceae, Caryophyllaceae, Asteraceae, Primulaceae, Chenopodiaceae, and Sapindaceae.
Steroid saponins are less widely distributed than are the triterpenoid type. Monocotyledons are the main source of steroid saponins, such as Liliaceae, Dioscoreaceae, Agavaceae, and
Palmae. Of the dicotyledons, Scrophlariaceae digitalis and Leguminosae trigonella have been found to contain certain steroid saponins. More recently, saponins have been isolated in marine organisms, mainly in the Echinoderms, such as sea cucumbers, sea urchins, starfish, and sea sponge (Habermehl, et al. 1990). These compounds may be synthesized by the organisms and/or taken from their diet.
1.1.3 Biological activities of saponins
The function of saponins in plants has often been questioned, and there is not always a good explanation for their wide distribution and high content (up to 30%) in some species.
These amphiphilic compounds have been considered as secondary metabolites of plants to protect against insects, bacteria, fungi, and herbivores.
The divergent effects of saponins against microorganisms have been observed in many plants. Saponin extracts from plants demonstrated highly inhibitory activity against some fungi, including plant pathogens such as Fusarium solani in corn, Candida insidiosum 7
in alfalfa (Timbekova, et al. 1996; Levy, et al. 1986), and human pathogenic fungi such as
Cryptococcus neoformans (Polacheck, et al. 1986). The mechanism of the activity might
involve the interaction of saponins with sterols of cell membrane and the reduction of
membrane integrity to cause the death of the pathogens. Bisdesmosidic saponins are
virtually inactive against fungus compared to monodesmosides. It has been proposed that the
water-soluble bisdesmosidic saponins may exist as a transportable form from the plant organs
not at risk to the parts under attack by microorganisms. The monodesmosidic derivatives are
then released by enzymatic reactions and provide a defense against microbial damage
(Hostettmann and Marston 1995).
Although relatively antifungicidal, the saponins have weak antibacterial activity.
Certain alfalfa saponins are inhibitory to some phytopathogenic bacteria (Agrobacterium
tumefaciens, Corynebacterium michiganese) (Timbekova, et al. 1996). Zablotowicz, et al.
(1996) found that soil fungal populations were more sensitive to saporiins exuded by soybean
seedlings than were the rhizobacteria, therefore, favoring the growth of rhizosphere
microflora, which is beneficial to the nitrogen-fixing plants.
The antiviral activity of saponins has been investigated. In vitro antiviral action of
aescine, saikosaponin A and glycyrrhizin against influenza, measles and herpes viruses have been documented (Rao, et al. 1974; Ushio and Abe 1992). Soyasaponins (Hayashi, et al.
1997) and glycyrrhizin (Ito, et al. 1987) were found to inhibit the replication of human immunodeficiency virus (HIV) and cytomegalovirus in vitro. The mechanism is not well understood. Amoros, et al. ( 1988) found that the inhibition of virus-host cell attachment by saponins might be involved against virus infection in vivo. Some in vitro experiments demonstrated that virus inactivation effect of saponins might be involved (Hayashi, et al. 8
1997).
The protective function of plants against insects has been attributed to saponin
constituents. Mono- and bisdesmosidic saponins repel termites. Alfalfa saponin mixtures
have been tested against summer fruit tortrix moth and European grape moth (Oleszek 1996).
Feeding a saponin-containing diet to grass grub, locusts, and flour beetles resulted in a rise of
larvae mortality and decrease of body weight (Tava and Odoardi 1996). However, many
insects have shown adaptation to this defense mechanism and may even sequester the
compounds for their own use to protect themselves from predators (Renwick 1996).
Numerous saponin-containing plants are toxic to molluscs and organisms with gills,
such as frogs, fish, and snails. Their molluscicidal activity has been applied in the local
control of schistosomiasis effectively by killing the intermediate host, the fresh water snail
(Hostettmann, et al. 1996). Some saponins may have allelopathic effect on the growth of
competitive plants. One example is that alfalfa saponins affected germination and seedling
growth of wheat, coffeeweed, cotton, and pigweed (Oleszek 1996).
The production of saponins by plants has apparently evolved as a defense mechanism
against herbivores. Swine, poultry, and cattle displayed an aversion to dietary saponins
(Cheek 1994). Cattle were observed to prefer low-saponin alfalfa meal to alfalfa with high
saponin content (Cheek 1983). Lowered feed-intake caused by the anti-palatability
properties of saponins has been observed in both livestock and laboratory animals. However,
growth retardation caused by saponins in feeds, observed in mammalian livestock, was not
consistent in rats, mice, and poultry. In the previous literature, 1 to 10% quillaja or
soyasaponins in diet were used to fed rats, rabbits, chicken and there were no observable growth depression in these animals (Oakenfull, et al. 1984; Ueda, et al. 1996; Pathirana, et 9
al. 1981).
The major biological effects of saponin may generally occur in the digestive tract
because dietary saponins are considered to be poorly absorbed. The saponins influence
rumen fermentation. Lu and Jorgensen ( 1987) reported that alfalfa saponins reduced
microbial fermentation and nutrient digestion in the rumen of cattle. Dietary saponins
reduced volatile fatty acids and microbial protein synthesis in the rumen. Saponins caused
bloating in ruminants by killing protozoa and inhibiting forestomach motility (Klita, et al.
1996). In non-ruminants, the primary digestive effects of saponins occur in the small
intestine. Johnson, et al. (1986) indicated that 0.3 to 8 mM Gypsophylla saponins or
Saponaria saponins increased the permeability of intestinal mucosa! cells, thereby inhibiting
active nutrient transport and possibly facilitating the uptake of substances that the gut would
normally be impermeable. There is evidence that 0.5 to 1.0 mM alfalfa saponins caused the
loss of activity of mucosa! membrane-bound enzymes and changed the transmural potential
difference in vitro (Oleszek, et al. 1994). Saponins have been implicated in increased fecal
excretion of minerals and associated with the chronic induction of negative mineral balance
(Southon, et al. 1988; Foley, et al. 1995). Shimoyamada, et al. (1998) observed that endogenous soybean saponins suppressed the chymotryptic hydrolysis of soybean proteins in vitro.
Recently, saponins have been related to the incidence of secondary photosensitization. Lajis, et al. (1993) reported that steroid saponins from plants could induce photosensitization in ruminant animals. For example, puncture vine (Tribilus terrestris) caused a photosensitization condition known as geeldikkop (yellow thick head) to sheep in South Africa. Diosgenin and yamogenin found in these plants were metabolized to lithogenic glucuronides of epismilagnin and episasapogenin. These glucuronides were shown to be deposited in the bile ducts and caused photosensitization in sheep (Miles, et al.
1994). However, this effect appeared to be strongly structure-dependent, accounting for the sporadic incidence of the disease. The mechanism for photosensitization by these compounds is not clear.
1.1.4 Saponins and human health
Saponins reached the human body in relatively large amounts through consumption foods and pharmaceuticals rich in saponins. The saponins are thought to be beneficial to human health in spite of the adverse effects on livestock (Cheeke 1996).
The hypocholesterolemic effect of saponins has been reported in the literature for the past two decades. Saponin extracts appeared to lower total blood lipids and cholesterol levels of laboratory animals such as chicks, rats, and rabbits (Oakenfull, et al. 1984).
Quillaja saponins at 20 mg/ml were reported to form high molecular weight complexes with casein in the diet in vitro, and addition of 10% quillaja or soybean saponins to casein diet resulted in 32% decrease of total plasma cholesterol level and 44% decrease of LDL- cholesterol level in comparison to the casein diet alone in gerbils (Potter, et al. 1993).
Subchronic dietary administration of steroid saponins at 0.042 mg/body weight/day from fenugreek decreased total plasma cholesterol in normal and streptozotocin diabetic rats
(Sauvaire, et al. 1996). Formation of insoluble saponin-cholesternl complexes in the intestines could decrease the absorption of dietary cholesterol and fatty acids. Increased excretion of bile acids led to increased synthesis of bile acids from endogenous cholesterol.
All these mechanisms may be involved in the cholesterol lowering activity of saponins (Rao 11
and Kendall, 1986; Sidhu and Oakenfull, 1986). There is no direct evidence of cholesterol
lowering effect reported in human subjects.
Rao and Sung (1995) reviewed the potential anticarcinogenic effects of saponins.
The cytotoxic and antitumor activities of saponins against various human malignant tumors have been confirmed in both in vitro and in vivo experiments. Some examples are listed in table 1.1. The proposed mechanisms for the anticarcinogenicity of the saponins included
direct cytotoxicity, immune-modulatory effects, bile acid binding and normalization of carcinogen-induced cell proliferation (Rao & Sung 1995).
The non-specific immunomodulatory activity of saponins has been observed for more than two decades. Saponins extracted from quillaja trees have demonstrated adjuvant activity for many antigens when administered orally or intraperitoneally (Richou, et al.
1964). So, et al. (1997) reported that 10 µg quillaja saponin extract addition to 2 µg hepatitis B surface antigen significantly increased immune response of mice to recombinant hepatitis B. Saponins could enhance both humoral and cell-mediated responses. Ginseng saponins administered orally stimulated phagocytosis in the reticuloendothelial system in the normal and tumor-bearing mice and promoted antibody production and natural killer cell activity (Kenarova, et al. 1990). Tea-leaf saponins, ginsenosides, and soyasaponins exhibited high neutrophil stimulating activity and low cytotoxicity to retinoic acid differentiated promyelocytic leukemia cells in vitro (Uemura, et al. 1995). However, in these studies, mixtures of saponin extracts were used and the concentrations used were not clearly stated. Table 1.1 C~totoxic and antitumor activit~ of sa~onins Sa~onins Dosage Tumor or Cell line Results of sa~onin treatments Reference soybean saponins 0-1200ppm colon carcinoma (HCT-15) ..J.. cell growth and cell viability, t cell Sung, etal. In and gypsophilla membrane permeability. 1995 vitro saponins ginsenoside 10-60 µmol/ml human myeloid leukemia (HL- IC50 of growth inhibitory activity were 24.3, Lee, et al. metabolite (20-0- 60), pulmonary 25.9, 56.6 and 24.9 µM against HL-60, PC- 1999 beta -D- adenocarcinoma (PC-14), 14, MKN-45 and HepG2 cell lines, glucopyranosyl- gastric adenocarcinoma respectively. 20(S)- (MKN-45) and hepatoma protopanaxadiol (HepG2) cell lines (IH-901)) asparagus crude 50-200 µg/ml human leukemia HL-60 cells ..J.. synthesis of DNA, RNA and protein at 50 Shao, et al. saponins µg/ml, in HL-60 cells by 84, 68 and 59%, 1996 respectively; ..J.. cell growth and macromolecular s~nthesis at 75-100 f:!g/ml . thalictrum saponins 30 mg/kg body breast cancer PMKl, pliaa- ..J.. tumor growth in rats. Semenov, et In weight lymphatic sarcoma, and al. 1996 ...... vivo Walker carcinoma N steroid and 0.02-2 mg/site mouse skin tumor ED50 against TPA-induced lesion was 0.1-3 Yasukawa, triterpene saponins µmol; ..J.. TPA-induced ornithine et al. 1991 decarboxylase accumulation . soybean saponins 3 g/100 g diet colon cancer ..J.. the incidence of aberrant crypts in mice Koratkar, et induced with azoxymethane . al. 1997 gleditsia japonica 82 nmol/site skin papillomas ..J.. the growth of mouse skin tumor induced Tokuda, et and promoted by DMBA and TPA al. 1991 respectively. ginsenoside Rh2 0.4-1.6 mg/kg human ovarian cancer cells Peritoneal (p.o) treatment resulted in Nakata, et body weight (HRA) implanted into nude apoptosis in the tumor cells and t the al. 1998 mice natural killer activity in spleen cells from tumor-hearin.[ nude mice. 13
1.1.5 Toxicity
The toxicity of saponins is an important issue because of their widespread
occurrence in foods. As mentioned above, saponins are considered deleterious to livestock because of the unfavorable effects on animal performance. Prolonged exposure of livestock to excessive amounts of saponins to livestock has been associated with growth retardation, vomiting, bloating, loss of appetite, and paralysis. Generally the glycoalkaloids are more toxic than other saponins. The ability of saponins to cause hemolysis in vitro has been reported since 1887. High concentrations of saponins, for instance, 0.5 to .3.0 mg/ml alfalfa saponins, are capable of lysing erythrocyte membranes and releasing hemoglobin (Oleszek
1990). It could be a threat to animals if the saponins enter the circulation system directly.
Hemolytic activity varies with the structure of saponins. The bisdesmosidic saponins and aglycones appeared inactive in the hemolysis assay.
The safety of saponins for human consumption is of concern as well. The average human daily intakes vary from 10 mg/day to 200 mg/day for the Western style diet to Asian vegetarians (Ridout, et al. 1988). The oral toxicity of saponins to warm-blood animals is relatively low. LD50 ranged from 50 to 3000 mg/kg body weight (George, et al. 1965). In addition, the saponin-containing materials humans consume are usually processed or heat- treated, which may alter the composition, content, and even the native structures of saponins in the food. These changes in the processed foods might affect the biological effects of foods on humans. Few negative effects have been observed in the populations with long- term consumption of saponins from edible plants. Saponins from quillaja and yucca are widely used as food additives, immune adjuvants, and antidermatophytic ingredients in 14
cosmetics, and they are classified as 'generally recognized as safe (GRAS)' by the U.S FDA
(Osamu, et al. 1996).
Parenteral (especially intravenous) injection of saponins can have severe effects.
The intravenous LDso of saponins was reported a thousand times lower than the oral LD50 in
rats (Oakenfull 1980). Once saponins enter the blood, liver damage, hemolysis, respiratory
failure, convulsions, and coma could occur.
Saponins may have significant effects on all phases of animal digestion from
ingestion to excretion. Despite the considerable amount of work done, there is no consensus
regarding the usefulness and importance of saponins in agriculture and in human health.
The uncertainty seems to be due to the enormous diversity of the naturally occurring
saponins and the difficulty in isolating pure compounds from the crude plant extracts.
Mixtures of saponins with unknown purity and content have often been used in the previous
studies. There is, thus, a need for further work to elucidate these biological effects with
single, well-defined compounds.
1.2. SOYBEAN SAPONINS
Soybean seeds or their products have been consumed by humans and animals for two thousand years because they are rich in high quality protein and oil. Soybean seeds contain about 2% glycosides mainly in the form of isoflavones and saponins (Tsukamoto, et al. 1995).
Soybean saponins are oleanene-type triterpenoid glycosides. Most of them have been isolated and characterized. Soybean saponins are divided into three nomenclature 15
groups A, B, and E, according to their respective aglycones, sapogenol A, B, or E
(figurel.2). They have a common structure that contains a glucuronic acid residue attached
to C-3 position of the aglycone through an ether bond. Different nomenclature systems
soyasapogenol A soyasapogenol B soyasapogenol E
Figure 1.2. Structures of soyasapogenols
have been used for the soybean saponins by two groups, Kitagawa and Okubo, who have
published most extensively in this area. For consistency, the Okubo's nomenclature system
will be used in the thesis.
The group A saponins are bisdesmoside saponins with two different polysaccharides
attached to the C-3 and C-22 position of sapogenol A (figure 1.3). Eight kinds of the group
A saponins, temporarily named Aa, Ab, Ac, Ad, Ae, Af, Ag, and Ah according to their
elution order from reverse phase high performance liquid chromatography (HPLC), were
detected in soybeans and characterized by Shiraiwa, et al. (1991). Saponins Aa, Ae, and Ag
contain a xylose residue at the C-22 position of sapogenol A whereas the others contain a
glucose residue at this position. The xylose or glucose hydroxyls of the sugar chains linked to C-22 of sapogenol A of saponin Ab, Ac, Ad, Af, and Ah were completely acetylated 16
(Tsukamota, et al. 1994). Although acetylated and un-acetylated group A saponins have
been characterized by Kitagawa s' and Okubo's research groups, Shiraiwa, et al. (1991)
suggested that all group A saponins existed in the acetyl form in soybean seeds whereas the
non-acetyl form may be the artifacts of extraction or the intermediates of biosynthesis.
The group Band E saponins are monodesmoside saponins with olean-12-en-3!3, 2213,
24-triol (sapogenol B) and olean-12-en-3!3, 24-diol-22-one (sapogenol E) as the aglycones
(figure 1.2). The group B saponins isolated from soybeans were named soyasaponin I, II, ill, IV, and V. Kudou, et al. (1993) reinvestigated the structure of the native group B saponins in soybean seeds extracted at ambient temperature instead of traditional heating extraction conditions. They isolated a soybean saponin (BeA) which contains a 2,3- dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) moiety attached to C-22 of soyasaponin I. Eventually, five different kinds of saponins with DDMP moieties were isolated, and were named saponin j3g, j3a, ya, yg and ag (figure 1.4), which correspond to the
DDMP conjugated forms of soyasaponin I, II, ill, IV and V, respectively (Kudou, et al.
1994). They showed that DDMP-conjugated saponins had maximal absorbance at 292 nm due to the DDMP moiety. DDMP-conjugated saponins were detected as the major saponin constituents in soybeans using cold or room temperature extraction procedures, whereas soyasaponin I - V were normally found when extracting at 80°C or under refluxing solvent conditions (Kudou, et al. 1993). Therefore, it appears that the DDMP conjugated saponins may be the genuine saponins in intact soybeans (Kudou, et al. 1994).
The DDMP saponins are not stable and are easily converted into soyasaponin I - V during heated extraction procedures, in cooking processes, in the presence of Fe3+, or upon storage in an alcoholic solution at room temperature for prolonged period of time (Yoshki, 17
OH
0
CH3
0 0
H I~~o"' OAc H
Rl R2 R3 Soy saponin Aa CH2OH 13-D-Glc H Soy saponin Ab CH2OH j3-D-Glc CH2OAc Soy saponin Ac CH2OH a-L-Rha CH2OAc Soy saponin Ad H j3-D-Glc CH2OAc Soy saponin Ae CH2OH H H Soy saponin Af CH2OH H CH 2OAc Soy saponin Ag H H H Soy saponin Ah H H CH2OAc
Figure 1.3. Structures of group A soybean saponins (Y oshiki, et al. 1998) Glc: glucosyl; Rha: rhamnosyl; · Ac: acetyl 18
~------, 0
OH
Qi Cfi3 ·: I I I I ~------J DDMP Cfi3 HO O H 0 ~,,,, GI20H
H~ ~C
0
R2I
RI R2 DDMP Soy saponin J3g CH20H a-L-Rha y Soy saponin I CH20H a-L-Rha N Soy saponin J3a H a-L-Rha y Soy saponin II H a-L-Rha N Soy saponin yg CH20H H y Soy saponin ill CH20H H N Soy saponin ya H H y Soy saponin IV H H N Soy saponin ag CH20H J3-D-Glc y Soy saponin V CH20H J3-D-Glc N
Figure 1.4. Structures of group B soybean saponins (Kudou, et al.. 1994) Rha: rhamnosyl; Gk: glucosyl; DDMP: 2,3-dihydro-2, 5-dihydroxy- 6-methyl-4H-pyran-4-one; Y: yes; N: no. 19 et al. 1998). Kudou, et al. (1994) reported that heating DDMP-conjugated saponins produced rnaltol which rnay contribute to the sweet aroma of soybean products detected during cooking. ·
The group E saponins, Be, and Bd, have structures similar to soyasaponin I and V, respectively, except there is a ketone group at C-22 instead of a hydroxyl group. The purified ~g soyasaponin fraction produced saponin Be and I after heating at 80°C in aqueous alcohol (Tsukamoto, et al. 1994). Yoshiki, et al. (1998) suggested that the group E saponins might be one of the decomposition products of the DDMP-conjugated saponins and generated by heating or the presence of metal ions in the extraction solution.
1.2.2. Distribution and content of soybean saponins
The saponin content and distribution in soybeans have been investigated by sorne research groups. Shirnoyarnada, et al. (1990) reported the composition of saponins on a wet weight basis in six varieties of soybean plants harvested in 1986. They reported that the group A soybean saponins and soyasaponin ag were only detected in the seed hypocotyl at concentrations ranging frorn 1.2 to 2.9 g/100 g. The other group B soybean saponins were detected in all organs of the soybean plants. Soyasaponin I levels were high in the hypocotyl at 0.5 to 1.9 g/100 g, in nodule at 0.11 to 0.46 g/100 g and in leaf at 0.15 to 0.76 g/100 g. The concentration of soyasaponin I was low in stern at 0.03 to 0.13 g/100 g, in pod shell at 0.01 to 0.25 g/100 g, and in rnain root at 0.06 to 0.11 g/100 g. These data indicated that the group B saponins, especially soyasaponin ~g, were high in plant tissues which were physiologically active, but low in the structural support parts (Shirnoyarnada, et al. 1990). 20
Whole soybean seeds contains about 74% the group B saponins and 25% the group
A saponins according to the profile reported by Ireland, et al. (1986). Shiraiwa, et al.
( 1991) reported saponin levels are higher in the hypocotyl than in the cotyledons of the
seeds. They reported the group A acetyl-saponins occurred only in hypocotyl. The group A
saponin content in the hypocotyl of soybean seeds may range from 0.5 to 3.3 g/100 g on a
dry weight basis. The hypocotyl contained soysaponin ag and f3g as the main constituents
of the group B saponins at a ratio of 1:2. The cotyledons contained mainly the group B ·
saponins f3g and f3a at a ratio of 5:2, whereas saponin yg and ya were present as minor
constituents.
Wild soybeans were reported to contain higher concentrations of saponins in the
hypocotyl at a concentration of 4.30% (w/w) for the group A saponins and 4.35% (w/w) for
the DDMP-conjugated saponins whereas cultivated soybeans had concentrations of 1.62%
(w/w) for the group A and 1.83% for the DDMP saponins (Tsukamoto, et al. 1994). The
content of each saponin on dry weight basis in soybean seeds increases during maturation
(Shimoyamada, et al. 1991). It is not clear yet whether environmental factors affect saponin
composition in soybeans. It has been reported that the saponin composition in soybean
seeds was more dependent on the variety than on the cultivation temperatures (Shiraiwa, et al. 1991; Tsukamoto, et al. 1995). Interestingly, saponin contents were found to be higher in soybeans grown with light-irradiated germination but lower when light was excluded
(Shimoyamada, et al. 1991).
The saponin content of defatted soy flour and some commercial soy products were estimated by measuring the sapogenins by HPLC-MS (Ireland, et al. (1986). They reported that there was slightly higher percentage of soyasapogenol A in heat-treated soy flour and 21 isolated soy protein than in unprocessed soybean flour, but showed the opposite distribution for soyasapogenol B content. No sapogenol A was detected in soymilk. Saponin contents in some soy ingredients and soy foods reported in the literature were given in table 1.2.
Table 1.2. Soyasaponin contents in some soy ingredients and soy foods Soyasaponin Reference content (g/l00g) Soybean seeds* 0.22-0.24 Tsukamoto, et al. 1995 Soybean fl.our (British Soya products, UK)# 0.47 Toasted, defatted soy fl.our (British Arkady Co. Ltd., UK/ 0.67 Isolated soy protein (British Arkady Co. Ltd., UK/ 0.76 Ireland, et al. 1986 Soymilk (Granose Food Ltd., UK/ 0.022-0.026 Soy protein concentrate (British Arkady Co. Ltd., UK)# Undetectable Tofu* 0.30 Miso * 0.15 Kitagawa, et al. 1984 Natto * 0.25 *: dry weight #: wet weight
Soyasaponins are not only found in soybeans but also in other legumes, such as mung beans, cowpeas, scarlet runner beans, lentils, chickpeas, kidney beans and alfalfa
(Price, et al. 1986; Tsukamoto, et al. 1994; Oleszek 1998). Soyasaponin contents in the leguminous seeds examined are listed in table 1.3. Okubo's group (1996) investigated the composition of DDMP-conjugated soyasaponins in leguminous seeds. Legume seeds were classified into six_saponin-containing types according to the composition pattern of DDMP- conjugated saponins (table 1.4). This classification has been considered to be a good agreement with genus or subgenus of Leguminoseae. Table 1.3. Soybean saEonin contents in the leguminous seeds Variety Methods used Soybean saponin contents Reference (g/l00g) Kidney beans soy sapogenols were measured by GC- total saponins, 0.35* Price et al. 1986 MS
Runner bean soy sapogenols were measured by GC- total saponins, 0.34* Price et al. 1986 MS
Soybean RP-HPLC at 205nm and 292nm DDMP-conjugated saponins, Tsukamoto et al. 0.22-0.24 1995 Cowpeas reflux extraction with methanol, RP- soyasaponin I and V, 0.12 Kinjo et al.1998 HPLC at 205nm
N Garden peas reflux extraction, RP-HPLC at 205nm soyasaponin I, 0.04-0.06 Kinjo et al. 1998 N
Peanuts reflux extraction, RP-HPLC at 205nm soyasaponin I, 0.10 Kinjo et al. 1998
Broad beans reflux extraction, RP-HPLC at 205nm soyasaponin I, 0.05 Kinjo et al. 1998
Chickpeas extraction below 30°C, RP-HPLC at soyasaponin I and pg, 0.071- Ruiz et al. 1996 205nm 0.075
Lentils extraction below 30°C, RP-HPLC at soyasaponin I and pg, 0.070- Ruiz et al. 1996 205nm 0.114
*: Saponin content was calculated based on the sapogenol analysis assuming aglycon: carbohydrate ratio was 1:1. 23
Table 1.4. Classification of leguminous seeds by DDMP saponin composition (Okubo and Y oshiki 1996) DDMP-conjugated soyasaponins Type Leguminous seeds ex,g aa 13g 13a yg X Phaseolus vulgaris • • • • Phaseolus lunatus • • • • I Pueraria lobata • • Pycnospora lutescens • • Glycine max • • • Glycine soja • • • Aeschynomene indica • Amorpha fruticosa • • • Apios americana • • Dunbaria villosa • Flemingia prostrata • Galactia tashiroi • • Glycine tabacina • • Indigofera trifoliata • Lupinus luteus • II M edicago sativa • Pisum sativum • • Ruduaaurea • Stypholobim fomentosa • Viciasepium • Vigna vexillata • • Wisteria floribunda • • Canavalia gladiata • III Canavalia maritirna • Christia obcordata • • • Desmodium heterophyllum • • • Desmodium triflorum • • • • N Desmodium uncinatum • • • • Phaseolus coccineus • • • • Vigna sinensis • • • • • • Dolichos lablab • • • Lupinus hirstus • Macroptilium atropurpureum • • • V Stylosanthes guyanensis • • Uraria lagopodioides • • Vigna angularis • • • • Atylosia scarabaeolides Cassia occidentalis VI Cajanus cajan No DDMP-conjugated saponins detected Caesalpinia crista Cassia tora 24
1.2.3 Isolation of soyasaponins
It is necessary to isolate saponins in sufficient quantity and purity for biological
testing of the pure compounds to be performed. Saponin isolation has been a challenge.
Difficulties are encountered with the labile substituents of saponins, the presence of
compounds with closely related structures, and the co-extraction of other substances in the
plant material such as sugars and phenolic compounds like isoflavonoids.
The correct choice of isolation procedure is very important because the original
structures of saponins might be altered by the solvents and/or the extraction conditions. The
major genuine soybean saponins, the DDMP-conjugated soyasaponins, can be extracted by
aqueous alcoholic solvents at or below room temperature (Kudou, et al. 1992). Extraction
at high temperature results in the loss of the DDMP group of the soyasaponins. The most recent procedures used to obtain soybean saponins were developed by Okubo's group
(Okubo, et al. 1991). Soybean flour or the hypocotyls were extracted with 70% aqueous ethanol at room temperature or at 80°C depending on the final products desired. The concentrated extracts were dispersed in butanol-water (1: 1) to extract saponins into the butanol layer. The crude saponin mixtures were separated to the individual saponins by sequentially fractionating with different chromatographic columns. Sephadex LH-20 gel filtration was usually the first step, which separated the group A saponin fraction, the group
B saponin fraction, and the non-saponin components with 50% aqueous ethanol elution.
Each saponin fraction was chromatographied on LiChroprep RP:-18 column by using a gradient of methanol-water mobile phase to obtain individual saponin fractions. Final purification was achieved by semi-preparative C18 reverse-phase HPLC with isocratic acetonitrile-water mobile phase. The pure soybean saponins can be obtained in milligram 25
quantities through these procedures. Modifications of this procedure have been noticed in
some studies. Different concentrations of methanol were used as extraction solvent by
Price, et al. (1986), Burrows, et al. (1987) and Kudou, et al. (1993). Shimoyamada, et al.
(1990) and Shiraiwa, et al. (1991) used methanol-1-propanol-water-acetic acid as the mobile
phase for preparative or semi-preparative chromatography. Butanol-water dispersion has
been skipped in saponin isolation procedures published by Kudou' s group since 1993
(Kudou, et al. 1993).
Two methods have been patented in the United States for obtaining soybean
saponins in large scale for nutritional applications. Arichi, et al. (1983) filed the first patent.
This method was similar to the isolation procedure described above but on a large scale.
The procedures involved were redundant with very low yield for industrial uses, and it has
been now abandoned. Liu (1999) documented another method for extracting saponins by
repeated ethanol and diethyl ether extractions on the residue from soybean oil extrusion in
another invention. The final product of the procedure may contain isoflavones in addition to
saponins. The method recovers extraction solvents and utilizes soybean residue, the by-
product of soybean oil extraction as the material. This method has great economic and
environmental value compared to the Okubo's methods (Okubo, et al. 1991).
1.2.4 Qualitative and quantitative analysis of soyasaponins
Different methods have been developed for the qualitative and quantitative determination of saponins, including hemolysis, T. viride assay, spectrophotometry, thin layer chromatography (TLC), gas chromatography (GC) and HPLC. The ability of saponins to rupture erythrocytes has been used historically as a detection and quantification method. 26
Various hemolysis methods have been reviewed by Birk, et al. (1969). The hemolytic index is defined as standard unit of activity. The activity strongly relies on the structure and purity of the saponin. Colorimetric methods were used to determine saponins as well. Different reagents used mostly reacted with aglycones of saponins to give various colored products depending on the structure of aglycones. Soybean saponins give purple color due to their triterpene aglycones when reacting with aromatic aldehydes or with acetic anhydride and sulfuric acid (Hostettmann and Marston 1995). The colorimetric reactions can be employed in TLC to detect saponins as well.
The qualitative analysis of saponins by TLC has been of great importance for saponin investigations. The most frequently used solvent system for soybean saponin analysis on
TLC is chloroform-methanol-water (65:35: 10) for normal phase TLC (Shimoyamada, et al.
1990; Shiraiwa, et al. 1991), and methanol-water (3:2) (Price, et al. 1986; Curl, et al. 1988) was used for developing reverse phase TLC plates (Karikura, et al. 1991; Hasegawa, et al.
1996). By using densitometery and with appropriate standards, TLC can quantify saponins by comparing the density of the sample and the standard spots.
Identification and quantification of the soyasapogenols have been carried out by using
GC-FID (Flame Ionization Detector) (Meyer, et al. 1990) or GC-mass spectrometry analysis
(Price, et al. 1984 and 1986; Burrows, et al. 1987). The saponins had to be converted into sapogenins by acid or basic hydrolysis. The sapogenins could then be analyzed by GC analysis. Therefore, the saponin content had to be estimated from the sapogenin content by using the sapogeniri: sugar ratio. The ratio of sapogenin to sugar by mass varies depending on the individual saponin composition in the sample. For soybeans and soy products, the ratio is approximately 1: 1 (Ireland, et al. 1986) 27
The analytical methods based on HPLC may be ideal for analysis of saponins because of its adaptability and sensitivity. Bisdesmosidic saponins elute earlier than monodesmosidic saponins, and glucuronides of saponins eluted earlier than other glycosides of saponins on reverse phase HPLC. However, the use of the analytical HPLC has been limited primarily due to the difficulties in the detection of triterpene saponins because of their low extinction coefficients and the lack of appropriate standards. There have been some attempts to overcome the detection problems including detecting saponins at 190-210 nm or by using a mass detector. These modes of detection, however, had some limitations as well.
Absorbance detection at low ultraviolet wavelengths can lead to unstable baselines.
Currently, acetonitrile is preferred to methanol because of its lesser ultraviolet absorbance.
HPLC-mass spectrometry has been successfully applied to the analysis of non-volatile thermally labile molecules of soybean saponins (Ireland and Dziedic 1985) but the high cost limits its use as the routine analysis. Oleszek, et al. (1990) developed a saponin analytical method that involved pre-column derivatizing saponins with 4-bromophenacyl bromide to increase the sensitivity of HPLC method. The carboxyl group of the glucuronic acid of soyasaponins reacts with 4-bromophenacyl bromide to produce uv-absorbing compounds which can be separated by reverse phase HPLC and detected at 260 nm. Phenanthrene was used as the internal standard in this method. However,.thisprocedure was time-consuming and the reagents used were highly toxic under long-term exposure. Due to the problems in quantitative determination of saponins, there are no well-established saponin profiles for food plants.
Currently, the most common procedures used for soybean saponins analysis by HPLC is conducted on an octadecylsilica C1s analytical column and elution with aqueous 28 acetonitrile. The group A soybean saponins have been separated by the reverse phase HPLC by using acetonitrile-2-propanol-water-acetic acid (34:6:59.9:0.1, v/v) solvent system at flow rate 1.0 ml/min and detection at 205 nm (Shiraiwa, et al. 1991). A mixture of DDMP- conjugated soybean saponins has been analyzed with acetonitrile-water-trifluoroacetic acid
(42:59.5:0.05, v/v) solvent system at flow rate 1.0 ml/min and detection at 292 nm
(Tsukamoto, et al. 1995). a-Hederin was used as the internal standard in an analysis procedure for soyasaponin I and g (Ruiz, et al. 199 5). There is no methods reported to· analyze all DDMP-conjugated soybean saponins and their non-DDMP counterparts in one chromatogram.
The characterization of pure saponins is difficult because of the lack of crystalline material. Melting points of soyasaponins are imprecise and often occur with decomposition.
TLC and HPLC may be a better technique to examine the purity of a saponin but still may not be specific enough. Fast atom bombardment (FAB) mass spectrometry is commonly employed to obtain molecular weights and sugar sequence information for soybean saponins.
Few investigators have used electrospray ionization (ESI) mass spectrometry for saponin characterization. ESI gives a series of characteristic fragment ions, which are useful for determination of aglycone structure and the sugar sequences (Lee, et al. 1996). 1H and 13C
NMR are fast and non-destructive for structure identification. Shiraiwa, et al. (1991) and
Kudou, et al. ( 1994) summarized the NMR spectra of the entire group A and the DDMP soybean saponins. However, all these techniques require pure compounds. 29
1.2.4 Biological properties of soybean saponins
In addition to the general biological activities of the saponins described in 1.1.3, the
specific functional properties of soybean saponins have been investigated as well. The use
of soybeans and soy products has been limited in western countries due to the undesirable
tastes and causing a gas feeling of the stomach of soy consumption. The acetylated group A
saponins have been linked to the bitter and astringent taste of soybeans (Kitagawa, et al.
1988). The undesirable taste depends on the aglycone structure of saponins. The astringent
and bitter characteristics in soybeans are in the order of soyasapogenol A> soyasapogenol E
> soyasapogenol B (Okubo, et al. 1992). Thus, one of the major problems with soybean
consumption may be attributed to the group A saponins in soybeans. Fortunately, the group
A saponins are present only in the hypocotyl of soybeans and may be controlled by
• genetical modification of saponin synthesis genes (Tsukamoto 1994).
The interaction between saponins and nutrients can be important in characterizing
physiological properties of saponins. Ikedo, et al. (1996) reported that soybean saponins
extracted from soybean hypocotyls interacted with bovine serum albumin (BSA) and
decreased sensitivity of BSA to chymotrypsin hydrolysis in vitro. Shioyamada, et al. (1998)
observed that the endogenous saponins in soybean plants apparently interacted with soybean
proteins and inhibited chymotryptic hydrolysis of the proteins. This effect of saponins was greater for glycinin than for f3-conglycinin. Acidic polypeptides of glycinin became more resistant to hydrolysis when incubated with saponins. The f3-subunit of f3-conglycinin was found to be more susceptible to saponin interaction and more easily changed on its highly ordered structure than was the a or a' subunits. These results might provide some enlightment on the hypocholesterolemic effect of soy since the complex of saponin and 30 peptide fragments could be potentially active. Another nutritional problem associated with saponins in the diet is mineral absorption. It has been observed that soyasaponin I in the diet did not affect Fe and Zn absorption in rats, whereas gypsophila saponins did although the author did not state the concentration used (Southon, et al. 1988).
Little information is available concerning the role of soybean saponins on cholesterol metabolism although it was believed saponins have cholesterol-lowering activity. The results from the few studies available are conflicting. Oakenfull, et al. (1984) reported that 1% soybean saponins addition to the 1% cholesterol-containing diet increased bile acid and neutral sterol excretion in rats. Sidhu and Oakenfull (1986) reported that isolated soybean saponins reduced the absorption rate of bile salts in rat intestines in vitro by forming micelles with bile acids. Therefore, it is possible that saponins may be a major contributor of cholesterol-lowering ability of the soy-based diet. However, two confounding phenomena have been observed by several investigators. When 1.5 to 10% soy or quillaja saponins were added to the casein-based diets, regardless of fat level in the diet, the significant decrease of LDL cholesterol and LDL/HDL ratios could be observed in gerbils, rats, and rabbits, along with the increased bile excretion. When saponins were washed away from the soy-based diets, or added to the intact soy-based diets, no significant change in cholesterol level was observed in these animals (Oakenfull, et al. 1984; Potter, et al.; 1993 Ueda, et al.1996). Secondly, the addition of 1 to 10% soybean saponins in diet was effective on lowering serum and liver cholesterol and triglyceride levels in rats and chicks fed the high cholesterol (1 % ) diets, but not in the animals fed the low fat diets
(Oakenfull, et al. 1984; Ueda, et al. 1996). Pathirana, et al. (1981) observed that no significant total blood cholesterol decrease in the rabbits fed low fat casein or isolated soy 31 protein diet at 5% energy from fat and supplemented with 1% (w/w) soybean saponins.
Calvert and Blight (1981) fed the hypercholesterolemic human patients intact soy flour or saponin-depleted soy flour for 4 weeks and observed no significant changes in blood lipid profile or bile acid excretion. Based on these observations, it would be difficult to conclude that saponins are the cause of the hypocholesterolemic effect of soy products at this point.
The epidemiological data from Asian countries has suggested that the consumption of soy products may be associated with reduced risk of both hormone and non-hormone dependent cancers (Messina, et al. 1994). Among the bioactive constituents of soy, saponins have been shown to significantly suppress carcinogenesis in in vitro -and in vivo studies. Soybean saponins at 10-50 µg/ml showed an inhibitory effect on the DNA- aflatoxin B 1 adduct formation in human colon and liver cells in culture (Joen and Sung
1999). This result indicates that saponins might be effective in reducing cellular DNA damages caused by carcinogens. The saponin fraction from soybeans repressed the genotoxicity of the dietary carcinogen 2-amino-3-methyl-imidio-(4,5-f)quinoline in human lymphocytes (Plewa, et al. 1999). This saponin fraction suppressed 2- acetoxyacetylaminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary
(CHO) cells (Plewa, et al. 1999). Berhow, et al. (2000) demonstrated that sapogenol B exhibited the most potent effect among the group B saponins in this protocol. These reports were the first demonstrations of the anti-mutagenic activity of soyasaponins in mammalian cells. Konoshima, et al. (1996) observed that soyasaponin ~g treatment delayed the formation of papillomas and decreased the numbers of tumors in TPA-promoted mouse skin tumor model. Soyasaponins inhibited the expression of oncogenic EB virus genome in vitro
(Tokuda 1988). From these observations, it is evident that soybean saponins might be the 32 effective antitumor agents in the promotion stage of carcinogenesis. The effect of soybean saponins on the colon cancer development has been studied since saponins taken orally might not be absorbable and could remain in the intestinal tract. Sung, et al. (1995) investigated the effect of soybean saponins on the growth and viability of HCT-15 colon carcinoma cells. Their data showed soybean saponins at 10 to 600 ppm decreased cell growth and viability. Koratkar and Rao (1997) showed that dietary intake of 3% soybean saponins extract reduced the incidence of preneoplastic lesions on the colon mucosal side in mice initiated by azoxymethane. The mechanism of anti-carcinogenesis of soybean saponins on colon cancer is not well understood but it is likely that interaction between saponins and bile acids might be involved. Bile acids are considered the colon cancer promoters (Sugezawa and Kaibara 1991).
The inhibitory effect of the group B soybean saponins on HIV replication and infection in vitro has been reported. Nakashima, et al. (1989) demonstrated that 250 to 500
µg/ml soyasaponin I completely reduced HIV-induced cytopathic effects in MT-4 cells and virus-specific antigen expression 6 days after infection. Soyasaponin II showed similar effects but was less potent. Soyasaponin I inhibited cell infusion in the MOLT-4 cell system in this study, although saponins had no direct effect on the reverse transcriptase activity of
HIV. Soyasaponin II was found to inhibit the replication of human cytomegalovirus, influenza virus, and HIV-1, probably via virucidal activity. Soysaponin II did not interfere with the fusion between virus envelope and host cell membrane (Hayashi, et al. 1997).
The anti-hepatotoxic activity of soyasaponin I has been observed in in vitro studies.
Soyasaponin I at doses of 50 to 500 µg/ml reduced the amount of elevation in glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) activities 33
induced by CC14 in the primary cultured rat hepatocytes (Miyao et al. 1998). The structure-
hepatoprotective relationship of soybean saponins was investigated using ALT as the
indicator of liver cell injury (Kinjo, et al. 1997). There was no significant protective effect
for soyasaponins I, II or IV below 200 µM, whereas soyasaponin III showed a strong effect
at 30 µM level.
The recent studies have shown various results on the anti-oxidation activity of
soybean saponins. A number of experiments have demonstrated that DDMP moiety of
saponins was the actual free radical scavenger (Yoshiki and Okubo 1995; Tsujino, et al.
1994). Soyasaponin j3g at 0.1 mM concentration inhibited the 2,2'-azobis(2- ·
amidinopropane) dihydro-chloride-induced oxidation of soybean phosphatidylcholine
liposomes by 70.8% compared with the controls. Yoshikoshi's group investigated whether
non-DDMP saponins had ability to protect the cells from oxidative damage (1996). They
tested soyasaponin I and Ab in vitro, and observed 20 µM saponins exhibited even greater
inhibition on the cytotoxicity induced by 5 µM hydrogen peroxide than soyasaponin j3g in
the cultured mouse fibroblasts. More information is needed to elucidate and verify this
property of saponins under in vivo conditions.
Anti-nutritional properties of soybean saponins have been reported. Saponins have
.long been known for their hemolytic activity due to their affinity to cell membrane sterols.
In a study done by Khalil and El-Adaway (1994), saponins extracted from soybeans caused
the least blood cell rupture effects compared with saponins from peas (Pisum sativum) and
common beans (Phaseolus vulgaris). Saponins are known as the contributor to the bloated
or full feeling of stomach following soy product consumption (Yoshiki, et al. 1998). 34
CHAPTER2
MATERIALS AND METHODS
2.1. MATERIALS
Vinton 81 soybean seeds from the 1994 crop year were used to extract the crude
soybean group B saponins. Soybean hypocotyls were used to extract soyasaponin ag.
Soybean seeds were generously provided by the Iowa State University Department of
Agronomy, and soy hypocotyls were provided by Schouten USA, Inc. Certified grade 200 proof ethanol (Chemistry Stores, Iowa State University) was used a~ soyasaponin extraction solvent. The solvents used for high performance liquid chromatography (HPLC) analysis including acetonitrile, methanol, trifuoroacetic acid, were HPLC grade and purchased from
Fisher Scientific (Fair Lawn, NJ). Milli-Q system (Millipore Co., Bedford, MA)-HPLC grade water was used to prepare all the mobile phase of HPLC analysis. Formononetin, used as an internal standard for the saponin assay, was purchased from Indofine Chemical
Company Inc. (Somerville, NJ). The fifty varieties of soybeans analyzed in this study were grown in Iowa in 1999, and were provided by Dr. C. R. Hurburgh, Department of Agriculture and Biosystems Engineering, Iowa State University. The analyzed soy ingredients were donated by Archer Daniels Midland Company (ADM) (Decatur, IL) for soy protein concentrates and Novasoy®, and by Protein Technologies International (PTI) (St Louis, MO) for soy protein isolates. Commercial soy products, tofu (firm, Mori-nu, Torrance, CA), tempeh (Quong Hop & Company, San Francisco, CA) and soymilk (plain, White Wave, Inc.,
Boulder, CO) were purchased from the local grocery store. Tofu sample analyzed during the 35
recovery experiment was produced by Dr. L. A. Wilson, Department of Food Science and
Human Nutrition, Iowa State University.
2.2. PREPARATION AND ISOLATION OF THE GROUP B SOYBEAN SAPONINS
2.2.1. Extraction and isolation of soyasaponins
Soybean seeds or hypocotyls were first ground to flour with a household coffee mill
(Krups North America, Inc., Denver, CO). One hundred grams of soy flour were extracted
with lliter of 70% aqueous ethanol. The mixture was stirred for 3 hours at room
temperature. The ethanol extract was removed by water aspirator vacuum filtration using a
Buchner funnel and Whatman No. 1 filter paper. The resulting solids were discarded. The
ethanol extract was evaporated to 100 ml with a rotary evaporator (BUCHNER-Brinkman, R-
114, Switzerland) at reduced pressure and< 30°C. The concentrated extract was loaded on a
C18 Lobar column (Merck Lichroprep RP-18, 40-63 µm, 310x25 mm ID, EM Science Co.,
Gibbstown, NJ) equilibrated with acetonitrile-water (10:90, v/v), and was fractionated with a
gradient mobile phase from 30% to 100% aqueous acetonitrile at flow rate 3 ml/min. The
fractions containing DDMP-conjugated saponins were collected and evaporated to dryness at
< 30°C. The residue was redissolved with 50 ml of 80% aqueous methanol to obtain crude
DDMP-conjugated saponin solution. Further purification was performed on a semi-
preparative HPLC system composed of a Beckman model 1 lOA pump, model 163 UV detector and a YMC-ODS-AM-303 column (RP-18, 5 µm, 250x10 mm ID, YMC, Inc.,
Wilmington, NC) using acetonitrile-water-trifluoroacetic,acid mobile phase (40:59.95:0.05) at a flow rate 2.6 ml/min with absorbance monitored at 292 nm. The eluted individual 36
saponins were collected separately, evaporated to remove organic solvents, and freeze-dried
by a freeze drying apparatus (Labconco Co., Kansas City, MI). The purity of saponins was
calculated based on the percentage of peak area in the HPLC chromatogram. The whole
procedure is shown in figure 2.1.
To isolate soyasaponin I, II, and V, soybean flour was extracted following the same
procedure as above but the extraction solution was heated at 100°C while stirring. The
purification process by the semi-preparative HPLC system was monitored at 205 nm, and an
acetonitrile-water-trifluoroacetic acid (36:63.95:0.05) mobile phase was used. The rest of
steps followed the same as described above.
2.2.2. Chemical identification of purified soyasaponins
The DDMP-conjugated saponins have maximal absorbance at 292 nm and specific
ultraviolet spectra at 190-350 nm. The purified DDMP-conjugated saponins were identified
by HPLC analysis according to their ultraviolet spectra and retention times.
Purified soyasaponins I, II, V, ag, pg, and pa, were evaluated by high-resolution electro-
spray ionized mass spectrometry (ESI). One milligram of the solid compound was dissolved
in 1 ml methanol. Electrospray ionization was performed on a Finnigan TSQ 700 triple
quadrupole mass spectrometer (Finnigan MAT, San Jose, CA) fitted with a Finnigan ESI
interface. The saponin solutions were introduced into the electrospray interface through an untreated 50 µm I.D. and 190 µm O.D fused-silica capillary tube. The mass spectrometer was used in the positive ion QlMS mode and the scan range was maintained from rn/z 100 to mfz 2000 with a scan rate of 3 s per scan. The voltage on the ESI needle tip was 5 KV in all experiments. The mass spectrum of each soyasaponin was determined. 37
Soybean flour 100 g
1000 ml 70% aaueous ethanol
Stir 3 hours at room temperature
Discard solid residue
Suoernatant i Concentrate to 100 ml at reduced pressure, 30°C
Load on C18 Lobar column and elute with 30-100% acetoniltrile/water mobile phase, collect eluted fractions
Isoflavones and other imourities i Crude soybean saponins i Semi-preparative HPLC with acetonitrile/water/trifluoroacetic acid (40:60:0.05)
Soyasaponin ag* (29mg),L f3g (63mg), f3a (28mg)
Figure 2.1 Isolation of the group B soybean saponins *: ag was isolated from soy hypocotyls. 38
The molar extinction coefficients (c) of pure saponins were determined. Purified compound was dissolved in methanol, and the standard concentrations were prepared accurately by series dilutions in volumetric glassware to give the absorbance from 0.1 to 1.0 at 205 nm. The absorbance of the solutions was measured on the spectrophotometer (Model
250, Gilford Instrument Laboratories Inc.) at 292 nm and 205 nm for DDMP-saponins. The soyasaponin I, Il, III, IV, and V were measured at 205 nm only. The molar extinction coefficient (crnethano1) was calculated according to the Beer-Lambert equation.
2.2.3. Determination of thermostability of purified DDMP-conjugated saponins
Purified soyasaponin f3g was examined as a representative to evaluate the stability of isolated compounds. Pure soyasaponin f3g was dissolved in methanol to give 1.2 mg/ml concentration in a 25 ml volumetric flask. The solution was stored at -20°C for 22 days. A l ml aliquot was taken from the solution daily and analyzed by the HPLC condition described in 2.3.1. The f3g peak areas were recorded and the percentages of change in concentrations of f3g were calculated. All the samples were taken and analyzed in duplicate.
The stability of soyasaponin f3g was evaluated at 30°C and 65°C. The solutions of f3g in methanol were prepared accurately. The solutions were heated in a water bath (Isotemp
Water-bath) at 30°C or 65°C for 3 hours. An aliquot was taken every 30 minutes, cooled down to room temperature, then analyzed on the HPLC condition described in 2.3.1. The percentages of concentration change of f3g were calculated and plotted. All the samples were taken and analyzed in duplicate. 39
2.3. QUANTITATIVE ANALYSIS OF THE GROUP B SOYBEAN SAPONINS BY
HPLC
2.3.1. Analysis of soybean saponins
Four grams of dried, ground soybean sample was extracted with 100 ml of 70% aqueous ethanol for 2.5 hours at room temperature. The extract was filtered through
Whatman No. 42 filter paper. The filtrate was evaporated to dryness with the rotary evaporator at reduced pressure and < 30°C. The residue was redissolved in 80% HPLC grade aqueous methanol and made up to 10.0 ml in the volumetric flask. An aliquot was filtered through 0.45 µm PTFE filter (Alltech Associates Inc., Deerfield, IL) and analyzed by
HPLC.
HPLC analysis of soybean saponins was performed on a system consisting of two
Beckman model 11 OB pumps, a Beckman model 420 micro-controller, a Waters 991 photodiode array detector (PDA) and a NEC Power Mate SXP Plus computer (NEC,
Boxborough, MA) equipped with a Waters 990 PDA data processing program (Millipore
Corp. 1990). A YMC-ODS-AM-303 column (RP-18, 5 µm, 4.6x250 mm, YMC, Inc.,
Wilmington, NC) was used. The mobile phase consisted: 0.05% trifluoroacetic acid in water
(solvent A), and acetonitrile (solvent B). The following gradient program was used:
Table 2.1 HPLC gradient program for soyasaponin analysis Run time (min) B% A% Duration of increase (min) 0 37 63 0 3 40 60 12 15 48 52 25 40 37 63 1 50 37 63 End of program 40
Injection volume was 50 µ1 per sample and the flow rate was 1 ml/min. The UV
absorbance was monitored at 190-350nm. The retention factors, peak asymmetry factor and
resolution of HPLC assay were calculated based on the peak on the chromatogram of soy
samples to assess the quality of separation (Snyder, et al. 1997).
Retention factor k = (tR - t0)/t0 for the first and last analyte peaks in the chromatogram
Peak asymmetry factor As = BlA IO%
Resolution between two adjacent peaks Rs= (t2 -t1)/[1.7x0.5x(W112,1 + W1,2,2)]
Six soyasaponin standards, V, I, II, ag, J3g, and J3a, were used to prepare calibration·
curves for HPLC assay. The soyasaponin stock solutions of each individual saponin were
prepared by dissolving pure standards in methanol followed by serial dilutions. The standard
curves were obtained by plotting the saponin concentration as a function of peak area
obtained from HPLC analysis. The concentration of standard solutions ranged from 40 to
1700 µmol/L. The calibration curves were used to calculate the concentrations of saponins in
soybeans and soy products.
2.3.2. Eval11ation of precision and accuracy
Vinton 81 soybean seeds and textured vegetable protein (U-118, minced 180, ADM) were analyzed to determine precision of HPLC assay. To determine the within-day variation of the assay, 5 replications of each sample were extracted and analyzed in duplicate within 24 hours. The between-days variation was determined by analyzing soy samples in duplicate for five different days. The means and standard deviations of the saponin contents and the 41
coefficients of variation of within-day and between-days assay were calculated for each
individual saponin in the soy samples.
To evaluate the accuracy of HPLC analysis, a recovery study using internal and
external standards was carried out on soybean seeds, textured vegetable protein, and freeze-
dried tofu. Formononetin (7-hydroxy-4' -methoxyisoflavone} was used as a reference peak to
aid in peak identification and saponin quantification. Five hundred microliters of the internal
standard, formononetin in methanol at 1.50 µmol/ml, and different amounts (depending on
the soy sample) of the external standard, soyasaponin I in methanol at 19.74 µmol/ml, were
added to 3.0000 g of dry soy sample. The samples were mixed well and then stored at room
temperature until methanol has evaporated. Extraction and analysis were performed as same
as described above. Each soy sample was duplicated for the unspiked sample and replicated
four times for the spiked sample. The recovery (%) was calculated as
1OOx(amount of saponin I in spiked group - amount of saponin I in unspiked group) (µmol/g soy sample) Recovery(%)= ------amount of saponin I added (µmol/g soy sample)
2.3.3. Quantification of saponin content in soybeans and soy products
The saponin content of 50 different varieties of soybean seeds grown in Iowa in 1999
and five different types of commercial soy products: toasted soy hypocotyl (Schouten USA,
Inc.), soymilk (plain, White Wave), soy protein isolates (500E and Supro670, PTI), soy
protein concentrates (ADM), Novasoy® (ADM), tempeh (Quong Hop & Company) and tofu
(Mori-nu), were analyzed with the method described. Soybeans, soy hypocotyls, soy protein · concentrate, and soy protein isolate were analyzed on 'as is' weight basis. Soymilk, tofu and 42 tempeh were freeze-dried and analyzed. The results were reported on 'as is' weight basis after adjusting moisture contents.
The saponin content and composition were determined by HPLC analysis and calculated with adjustment of recoveries. The isoflavone concentration of these samples was analyzed by using the method published by Murphy, et al. (1999). The total saponin and isoflavone contents of these samples were compared.
A standard solution consisting of formononetin and saponin I was run daily before soy samples were analyzed. Any significant deviations (>5%) from the original concentrations of these reference compounds suggested that there might be the error.on the
HPLC system. The system would be inspected and corrected prior to further sample analysis.
2.3.4. Statistical Analysis
Two or four replications were preformed each sample. The data are presented as means and standard deviations for the precision and accuracy experiments. Coefficients of variation were calculated to estimate precision of the analytical method. Linear regression was used to obtain the calibration curves of the saponin standards. The soyasaponin ~g thermostability and the comparison of soyasaponin concentrations between Round-up Ready soybeans and conventional soybeans were analyzed by Student's t-test. 43
CHAPTER3
RESULTS AND DISCUSSION
3.1. ISOLATION AND PURIFICATION OF THE GROUP B SOYBEAN SAPONINS
The DDMP-conjugated group B soyasaponins ag, Bg, Ba, and their non-DDMP counterparts, soyasaponin I, II and V, were isolated from soybeans and soybean hypocotyls by following the procedures described in 2.2. Soyasaponins with high purity(> 95%) could be obtained. The purity was determined based on the percentage of the soyasaponin peak area in HPLC chromatogram at 205 nm. The yields of individual saponins ranged 0.017% to 0.063% (table 3.1), and were higher compared to the yield reported by Kudou, et al.
(1993). The yield of DDMP-conjugated saponins from the starting materials was relatively low. It is probably because of the loss of DDMP moiety of the compounds during the multiple-step isolation and purification process. Overall, this method was reproducible and effective to yield the group B soybean saponins with high purity and in sufficient quantities to prepare standards for quantitative analysis.
The isolation methods developed by Okubo's group (Okubo, et al. 1991) and
Kudou's group (Kudou, et al. 1993) have been used to isolate the group B soybean saponins. A number of different organic solvents were used, and the whole process required considerable time due to multiple extractions and chromatographies using their methods.
Using their protocol, Kudou, et al. (1992) reported the yield of ~g was 26 mg/100 g ground soybean hypocotyls. In another paper published by Kudou, et al. (1993), the yield of each individual saponin ranged from 4 mg to 36 mg/100 g ground soybean flour. The 1: 1 butanol-water extraction and Sephadex LH-20 gel filtration were employed in Okubo's 44
Table 3.1. The yield of the group B saponin purification Saponin Amount (mg) obtained Amount (mg) obtained Yield of the from 100 g soy flour from 100 g soy flour by modified rel!orted by Kudou (1993) the modified method method(%l at 3 29 5.7 ya 17 5.5 f3g 36 63 22.8 I 49 31.2 f3a 12 28 40.2 II 22 52.7
8 : saponin a.g and V were isolated from soy hypocotyls. b: recovery(%) was calculated as: 100 x amount of saponin purified/amount of saponin in the starting material method in order to reduce impurity contents in the crude saponin extract before the preparative HPLC was initiated. These processes were time-consuming and might increase the loss of desired products. We modified Kudou's methods as shown in figure 2.1. The concentrated ethanol extract of saponins was chromatographied directly through the C18
Lobar column using the modified acetonitrile/water gradient instead of butanol extraction and LH-20 gel filtration. This approach appeared to be sufficient for separating saponins from undesired impurities, such as isoflavones, which were the major alcoholic co-extracts from soybeans. Acetonitrile was used in the mobile phase of the reverse phase chromatography instead of methanol to reduce the system pressure and baseline noise of the ultraviolet detection. The final purified saponins were obtained through a semi-preparative
C18 HPLC system. Figure 3.1 shows a representative chromatogram of the crude DDMP- conjugated soyasaponin fraction running through the semi-preparative HPLC system. By collecting each fraction, individual soyasaponin can be obtained with relatively high purity. bg
•::I· :·- 1·--· .. •j::• •1:: I -:. ···:
·•::1·,---.. •-••·· e •-.C = -::J N .---· 1:·-. ,. ... :a:·-. t -=-· ·=·-. ·== ·•=- ·=·- •-•· ,.Q= .__ ,: I\., 0 [ll ,--. ,.Q .j:::,. < eog~ Vl •---- ~:!:·=--- ::-=~ ;:::~ •--.1: ...J ~· .·=···---- ,---.-·==• =~~- :==~= !:~;:, ·==--· ==--~ i:il!;: }(;:,: ;::'.:,, ~:;:fb~. u::i::2.::: ::z::; •=1::: ;~:~; Retention time (min)
Figure 3.1. HPLC profile of the partially purified saponin fraction from soybeans on the semi-preparative HPLC system. The chromatogram was obtained with reverse phase HPLC using a YMC-ODS-AM-303 column (RP-18, 5 µm, 250x10 mm ID) and an acetonitrile-water-trifluoroacetic acid mobile phase (40:59.95:0.05) at flow rate 2.6 ml/min with absorbance monitored at 292 nm. 46
3.2. CHEMICAL IDENTIFICATION OF ISOLATED SAPONINS
The isolated soyasaponins were identified on reverse phase HPLC with the
absorbance monitored over 190 to 350 nm. Figure 3.2 presents the example chromatograms of isolated soyasaponin pg and I. There was a small saponin I peak observed in the chromatogram of isolated pg (figure 3.2a) due to the decomposition of pg to saponin I during storage. The DDMP-conjugated saponins showed the maximal absorbance at 292 nm due to DDMP moiety (figure 3.3a). Soyasaponin I, II, and V showed the non-specific ultraviolet spectra with the absorbance at 205 nm due to the C-C bonds (figure 3.3b).
The intensity of the detection signal at the chosen wavelength is proportional to the molar extinction coefficients of the analytes of interest. Kudou, et al (1992) reported that the extinction coefficient s292methanoI of pg was 3700 M- 1cm-1• No information is available on the molar extinction coefficients of the other group B soyasaponins. In the present study, the molar extinction coefficients ( EmethanoI) of the isolated soyasaponins were determined at
292 nm and 205 nm. As shown in table 3.2, Emethanol of these compounds ranged 4500 to
6000 M-1cm-1• The absorbance of these saponins was relatively low compared with that of isoflavones in soybeans, which have been reported 5-fold higher molar extinction coefficients at their maximum absorbance wavelength (Murphy, et al. 1999). Moreover, the absorbance of the analytes was monitored at 205 nm in order to detect the non-DDMP saponins, which led to baseline interference by the mobile phase. The small extinction coefficients of soyasaponins and low wavelength absorbance monitoring decreased the detection sensitivity in the HPLC analysis.
The identity of purified soyasaponins was confirmed by their high-resolution electro- 47
(a)
(b)
Figure 3.2 Chromatograms of purified soyasaponins. (a) purified soyasaponin ~g; (b) purifi~d soyasaponin I ,lf>MJ •. ·!)
(a) (b)
Figure 3.3. The ultraviolet spectra of the purified soyasaponins at 190-350 nm (formononetin in (a) is used as the reference peak to aid peak identification) 49
Table 3.2. Molar extinction coefficients and ESI-MS molecular ions of soyasaponins
Saponin Molecular Calculated ESI-MS Emethanol formula molecular molecular ions (M -1cm-1) weight (m/z) 292nm 205nm ag Cs4Hs4O22 1085.24 1086 4838 5946 V C4sH78O19 959.13 959 4971 j3g Cs4Hs4O21 1069.24 1069 4504 6267 I C4sll79O1s 944.13 944 5278 j3a Cs3Hs2O20 1039.21 1039 4795 5645 II C41H16OI7 913.10 913 5078
spray ionized (ESI) mass spectra. The primary ion peaks given by ESI-MS (positive-ion mode) for soyasaponin ag were the molecular ion at mlz 1086 [M+Ht, the fragment ion at m/z 959 [M+H-DDMPt and the fragment ion at m/z 520 [M+H-Glu-Glc-Glct (figure 3.4).
The spectrum of soyasaponin j3g showed the molecular ion at m/z 1069 M+ and the fragment ion at mlz 943 [M+H-DDMPt. The spectrum of soyasaponin j3a showed the molecular ion at mlz 1039 M+ and the fragment ion at m/z 925 [M+H+C-DDMPt. The ESI mass spectra of non-DDMP saponins were characterized by the molecular ion [M+Ht and the fragment ion of [aglycone+CHt as well. The spectrum of soyasaponin I showed the molecular ion at mlz 944 [~+Ht and the fragment ion at m/z 453 [aglycone+CHt (figure 3.5). The spectrum of soyasaponin II showed the molecular ion at m/z 913 M+. The spectrum of soyasaponin V showed the molecular ion at m/z 959 M+ and the fragment ion at m/z 505
[aglycone+OHt. The sodium adduct with the molecular ions [M+H+Na] + or the fragment ions also appeared in the mass spectra of the saponins. All the DDMP-saponins gave the non-DDMP fragment ions [M+H-DDMPt due to the breakdown of the DDMP structure , St>EC: -Hn9'9'5.5$18 Elaps<'.ll r 01:05.4 34 Stmip: al:Phll. Start 112 Corom; ll!SI pos Ql, loop ~ode! RSI ~QlMS LMR. UP PROF Study: routine Opel;"~ k.,imel Client: H1,1 Inlet. : .ease~ 319.l Inte11. 2409472 Ma.a:ses: 20.0 :,- 1200 Norm: 1085.6 RIC 742302 Ul;,)eaks: 2 Peak; 1000. 00 :mmu Smth: 7+7+7
10815 100 E+ 0.S [M+Ht r d.Sd
80 ESI :P08 of ~lph~ g [M+H-DDMPt 959 60 V, 0
40 520I
[M+H+Nat 1108 I 20 - 48-0 I 634
:soo 1000 12:DO
Figure 3.4. ESI positive mass spectrum of soyasaponin ag !iii'f!fX:: :ftnSf:~S:£.:9:t ··;,3~pJR!t>-')Hl :Rl a:~'"'r,1, : OS.. & ;L $<)p soy .,z.ap'l'.. ·st.a.:i~t $6, ,rHJ l Coro.nt t-4$.¢10 ~;i ~,giMzt '.iLlMR AVM:R 't)ii? i?'RbF st.,;,~d;i t xout.it':te, c~er F.mne.t Cl.:ient :. .H.u :J;:nJl,i'ii!t. :· Bas$: 943,7 sa:2:s,1s. t,f~f;Ji:t,H;\S 10() > 12(}0 lll'~i:ma· 943'.7 ..... axe :h;:i>Bo:a JIP®*ke.i 2 ·?·11'/•Kiki :t OCH). (HJ rtl't%1
llii4:3, 11 1-00 '!\)•!<0$. [Mt 6 .:$3
eo
60 -V, [M+Nat
.'l :to 4.$:$ .,. J .'& .1.
20ti 800 1000
Figure 3.5. ESI positive mass spectrum of soyasaponin I 52 during mass spectroscopy, but no fragment ion at mlz 127 [DDMP+Ht was observed. The mass spectrometry data obtained here are consistent with the data reported in previous studies using the high resolution FAB-MS (Shiraiwa, et al. 1991; Kudou, et al. 1994; Kinjo, et al. 1998).
3.3 THERMOSTABILITY OF PURIFIED DDMP-CONJUGATED SAPONINS
The stability of purified saponins is an important issue because it determines the storage conditions, shelf-life of the purified compounds, and sample preparation for the
HPLC analysis. Massiot, et al. (1996) and Okubo, et al. (1996) investigated decomposition of soyasaponin pg in various conditions. Okubo, et al. (1996) reported that soyasaponin pg converted into soyasaponin I in 0.1 M NaOH solution within 24 hours, and the decomposition rate increased with increase of NaOH concentration at ambient temperature; soyasaponin pg degraded to soyasaponin I in 2.5 mM FeCh solution. Massiot, et al. (1996) observed that soyasaponin pg converted into soyasaponin I in acidic or basic solutions, or just simply standing in alcoholic solution at ambient temperature. However, the information is limited on the stability of soyasaponin pg at different temperatures. Kudou, et al. (1992) observed that soyasaponin ag and pg converted into soyasaponin V and I, respectively, when the alcoholic extract of soy hypocotyls was heated at 80°C for 5 hours. Okubo, et al.
(1996) reported that soyasaponin pg was comparatively stable at temperatures below 90°C but did not indicate the sample standing condition. Therefore, we evaluated the stability of purified soyasaponin pg in methanol at different temperatures.
Figure 3.6 shows the decomposition of soyasaponin pg in methanol at-20°C, 30°C, 53 and 65°C. Methanol was used as the solvent because it was commonly used in saponin isolation and analysis. Soyasaponin Bg was relatively stable in the methanol solution for 15 days at -20°C, but began to decompose to soyasaponin I after 16 days. No significant decrease in the concentration of soyasaponin Bg was observed over three hours at 30°C.
The concentration of soyasaponin Bg significantly decreased when heated at 65°C, and the concentration of soyasaponin I increased correspondingly. However, the increase of soyasaponin I concentration was 0.15 µmol/ml less than the decrease of soyasaponin Bg concentration after heating at 65°C for 3 hours. It is probably because that some Bg molecules might break down to even smaller molecules, which could not be detected on
HPLC due to the very low concentration. These results suggested the purified DDMP- conjugated saponins could be stored at -20°C without significant change in composition for at least 15 days. The samples containing DDMP-conjugated saponins can be processed in the alcoholic solution at room temperature for 3 hours without significant loss of DDMP moiety. However, the rapid decomposition of DDMP-conjugated saponins would be expected at > 60°C. The caution should be taken during the sample preparation for soyasaponin isolation or analysis.
3.4. QUANTITATIVE ANALYSIS OF THE GROUP B SOYBEAN SAPONINS BY
HPLC
The HPLC conditions revealed well-resolved individual group B soybean saponins.
Separations of a mixture of soybean saponins in soybean seeds and textured vegetable protein are shown in figure 3.7 and figure 3.8, respectively. The retention times of 54
1.2 ~------.
0 5 10 15 20 25 days (a)
1.2 -.------, I 1.0 J----I--- _.,. _ ------:E-- _____ -1: 0 E 0.8 a 6 0.6
!- 0.4 8gC ,..______C 0.2 0.0 --1-----,-----,------,----- 0 50 100 150 200 time (minute) (b)
1.4 -- 1.2 i0 E::, ...... 0.8 C 0 ; 0.6 E 0.4 cGI u * C * 0 0.2 u * * 0 0 50 100 150 200 time (minute)
(c)
Soyasaponin I ------Soyasaponin J3g
Figure 3.6. Thermostability of soyasaponin f3g (n = 2) (a). f3g in methanol at-20°C; (b) f3g in methanol at 30°C; ( c). f3g in methanol at 65°C. *: The concentration of saponin f3g is statistically significantly different from the concentration at time 0 (p < 0.05). 55 soyasaponin V, I, II, ag, Bg, Ba, yg and ya on HPLC profile were 18.1, 19.8, 22.7, 28.5,
30.8, 33.4, 35.9, and 38.4 min, respectively. The retention times shifts depending on the variation of temperature and fl9w rate. When running the HPLC system at the described conditions the variation of the retention time was usually < 1.0 min. The quality of separation for the HPLC program was evaluated according to Snyder, et al. (1997). The retention factors k were 6.47 and 18.87 for the first (soyasaponin V) and last peak
(soyasaponin ya), respectively, in the chromatogram. With the k value at 2 < k < 20 range, the peaks of interest were well dispersed, and the analysis time was not excessive. The peak asymmetry factors As of the saponins ranged 0.64 to 1.2. Although the ideal peak shape is symmetrical with As values around 1, some of the saponin peaks were slightly tailing or fronting, which indicated that more than one retention mechanism might be involved in chromatographic separation (Dolan, 1999). The resolution value of the least separated two peaks of interest on the HPLC profile, soyasaponin V and I, was greater than 3.0, which satisfied the recommended quality of separation with the minimum resolution of 1.7 to 2.0
(Snyder, et al. 1997). The good resolution between peaks of interest ensured the accurate peak-area measurements and the precise saponin quantification.
Shiraiwa, et al. (1991) reported that soyasaponin yg and ya were minor constituents of saponins in soybeans, and it was also confirmed by the chromatogram of soybeans we obtained. Therefore, in the present study, only soyasaponin V, I, II, ag, Bg and Ba were isolated to establish the standard curves for quantitative analysis. The least square regression gave linear calibration curves of these compounds with R2 > 0.98 (table 3.3 and figure 3.9).
The working range of calibration curves was 40 to 1700 µmol/L. The detection range for WatOl'::!lill i3o1220 3A~D.• 3J'DJ • 2M1 • 2M1 . O~U- 3M ,0.,,..,.
"",;::; c:,...... • c,,• =r;"II
'l:,Q 0:::: .....4l- 3:" ,;,; • .1-- ,;.,.,,,; ~· .. 41'.,,,.,. ... ~,; .. J',;.,..,.. ',1,',..1- ,.41',;4/'J,; ,~ -4/'J,; .. 41' I I • • L --, . .I .• .P .L .I I • • • I • I I • II I I V, I I • lo L I : I "i :- L :: 0\ . .I ..I .• ..I .L .. :::lo •, 1 l < = : i: ;: ;f: ! ! : : : I : I I : :: : I : : I I :I ;;:I LI>, : •• - .... .i:. ... - • - - •• - -;- .... •• - • • .; ••• - • - • - • ...... - ..... - .- • - ... ·,· - •• - -;- • • • ·: •• "" C' : i : : i : t i : ;, ,.,., .,._ • I • I I • I • j formono~etij ~g j ==<;"I I If • !I • &f& • .., i• !• -i • I P I I , • • I I • I I .. I • a i I ; I :: A: : .: ...... : • . :• •I /!,/g= ..,,a• -L • 0 ! _..._ !: ! !" . yg i,... . __._ . . .1· • - •• - ...... !· . . . ._;..-.~.:...:::::.-,"'-''"r-""' a ' . 0 1.0 20 ::ro 40 (min) Figure 3.7. Chromatogram of soyasaponins in Vinton 81 soybeans Chromatogram was obtained with reverse phase HPLCusinga YMC-ODS-AM-303 column (RP-18, 5 µm, 250x4.6 mm ID) and a gradient mobile phase of acetonitrile-water-trifluoroacetic acid·at flow rate 1.0 ml/min with absorbance monitored at 205 nm. W'O~ON> .ia,:i::i,o, "'r;; ,0 LI:'>.,,.., c::, 0 = .. "' .c.., e & "" :.e .....t §:'. :£1 o_~..,.a ~- . r"IJj rJ=r-!irr=, ... -1= ·>=...... I "" \~ . . .,.,,,.. ... • ;,_.,. ;.--.... ~-.,) ,/·' .., . .-- .,• .,.,.,,. .-, . .,..,. .,.•""'"' ,,, .,. . \ .,.•"' .,.,•"' .,,,..,. .,.,"""' .. · ,,.. . ,.,"' ,.,,•,.,,..,.... ,~.,.. r .,.,.,,,,. ,.,..-· .,.... .,,,.. .,,.- ·~ .. ,-., -:· r.,"' VI .....:i '::!I< < ..... - ,. I .. .. "': . - . -- .;- --- -: - . - . - . - .... ' -- i - -;-.- . - - - -;:- - . - - •i- ... . la! I ., -...'--"" A I I r · -· -· 1· - ·· ·· · · i i: ; I r• : • •• I I : to- Ion r·-··-·-·r-:· I : c;> Cl fctrmbn~netin i\ i ' <:"'I --r ' : [~tr ! : . • • 'l' l : 0 ... . !I I 1:1#I hI ' . ' : : . .I . I • I • I I I J.la ! l!!0 0 a~ ; ygj .... ,._.,, a r-r ••I• . . ._.:._ .... --...... J 10 '2:0 :3(1 40 ( :rt: i C Figure 3.8. Chromatogram of soyasaponin in textured vegetable protein. Chromatogram was obtained with reverse phase HPLC using a YMC-ODS-AM-303 column (RP-18, 5 µm, 250x4.6 mm ID) and a gradient mobile phase of acetonitrile-water- trifluoroacetic acid at flow rate 1.0 ml/min with absorbance monitored at 205 nm. 58 0.6 • S3IX>n:b.I • S3IX>n:b. JI A S3IX>n:b. V • l:g ::t( l:a • ag 0.5 Lirer(g;p'.)n:b.V) Lirer (5:p'.)n:b.]) - - - _ Lirer(g;p'.)n:b.Jl l1)a • - - •••. Lirer t::g) Lirerl:a) - - - _Lirer(:g) 0 , N .w co 0.4 u ...:i P-i ::r::: 0.3 0 co (J) 0.2 H co co (J) 0.1 P.i 0 0.0000 0.0005 0.0010 0.0015 0.0020 concentration (rrol/L) Figure 3.9. Calibration curves of soyasaponin standards Table 3.3. Calibration equations for soyasaponin standards Compound Calibration equation R1 a,g Y = 268.58x + 0.0022 0.9996 V Y = 238.04x + 0.0033 0.9951 pg Y = 230.41x + 0.0068 0.9984 I Y = 209.19x + 0.0027 0.9984 ~a Y = 285.02x + 0.0013 0.9995 II Y = 202.20x + 0.0007 0.9855 59 saponin analysis in soy products was 0.11 to 4.86 µmol/g sample. The high minimum detection level of the HPLC assay was mainly due to the low extinction coefficients of the soyasaponins and high baseline noises at 205 nm. Three to five grams of regular soy food sample were required in order to detect the analytes on the HPLC profile precisely. During the HPLC analysis of soyasaponins, the retention times of analytes sometimes shifted due to the variation of temperature, flow rate or mobile phase makeup. A reference peak in the chromatography can be useful to aid in peak identification; Few saponin studies have used the reference compound in saponin quantification. Alpha-hederin was used as the reference peak and internal standard in an analysis procedure for soyasaponin j3g and I in lupine seeds (Ruiz, et al. 1995). Alpha-hederin eluted later than the latest analyte (soyasaponin j3g) in that HPLC program and consequently prolonged the analysis time for each sample. Formononetin was chosen as a reference compound in our saponin assay. The chemical structure and its calibration curve are shown in figures 3.10 and 3.11, respectively. The compound is not found in soybeans and soy products. It is stable and available commercially. The retention time of formononetin was J 7.2 min under the HPLC conditions described in 2.3.1. It did not overlap with any other analytes and eluted earlier than the first analyte, soyasaponin V. Using formononetin as the reference, soyasaponins can be easily identified according to their retention times relative to the reference peak. The internal standard can compensate for the changes in sample sizes or concentration due to instrumental variations, especially for the samples requiring significant pretreatments, such as extraction, filtration, and reaction. Formononetin was used as the internal standard in the saponin assay to evaluate the losses of the analytes during analysis 60 HO 0 0 OCH3 Formononetin (7-hydroxy-4' -methoxy-isoflavone) Figure 3.10. Chemical structure of formononetin 0.6 ~------~ y(205nm) = 1613.2x - 0.0206 0.5 R2 = 0. 9972 ro 0.4 Q) y(292nm) = 688.15x - 0.0085 H R2 0 .9988 ro 0.3 = ,!, 0 -1--~,r;;=---,-----,----,------,-----,---,----...,.----1 0 DODOO OD0005 0 DOOJD OD0015 0 D0020 0 D0025 0 D0030 0 D0035 0 D0040 concentration mol/L peakarea.205 i peakarea.292 ---L:hear peak area 205) - - L:hearpeak:area.292) Figure 3.11. Calibration curve of formononetin on HPLC 61 in our study. A known amount of formononetin was added to different types of soy products. A calibration curve was constructed by plotting the peak ratio of a known saponin to the internal standard as a function of the concentration of the saponin obtained from HPLC assay (figure 3.12). A linear relationship indicates that the internal standard interacts with the different sample matrices similarly to the saponins and the fraction of internal standard lost during extraction is proportional to the fraction of the saponins lost. The ratio of the unknown saponins and formononetin concentration remains the same. This calibration curve can be used to estimate the concentrations of saponins in the unknown soy samples when formononetin is added prior to extraction. The saponin concentrations from " different soy samples obtained by using the internal standard method were compared· with the results obtained from HPLC after adjusted by recovery. This indicated the extraction method works well for different types of soy products. Vinton 81 soybean seeds and textured vegetable protein with different saponin concentrations were analyzed to evaluate the precision of the HPLC assay. The results are presented in table 3.4. The saponin assay variability within-day was< 9.8% CV (n = 5) and between-days was <14.3% CV (n = 2/day) for all the saponins analyzed. The variation of soyasaponin ag was larger because of its low concentration in the samples. This range of coefficients of variation is generally acceptable for food analysis and indicates good precision of the analytical method. The external recovery study was carried out to evaluate the accuracy of the soyasaponin assay. Soyasaponin I was used as the external standard because it is stable compared to the DDMP-conjugated saponins. Soybean seeds, textured vegetable protein and freeze-dried tofu were analyzed with known amount of formononetin and soyasaponin I 62 1.6..------, 1.4 y = 0.0752x - 0.013 1.2 b"l R2 = 0.9966 1.0 0 S 0.8, 0.6 b"l .Q 0.4 0.2 0.0e,c'------.------,------,------i 0.0 5.0 10.0 15.0 20.0 bg/fonnononetin (a) 6.0 b"l 5.0 y = 0.0767x - 0.0706 • '- r-1 2 0 R = 0.9966 f=i 4.0 ;j 3.0 H i:: ·.-l 2.0 i:: 0 0. 1.0 (tj Ul 0.0 0.0 20.0 40.0 60.0 80.0 saponin I/fonnononetin (b) Figure 3.12. Internal standard calibration curves of soyasaponins for six soy product matrices: soybeans, dried tofu, dried soymilk, soy protein isolates, textured vegetable protein and tempeh. (a) soyasaponin f3g/formononetin; (b) soyasaponin I/formononetin 63 Table 3.4. Precision analysis of HPLC measurement of saponins in soy foods sample saponin Content(umol/g) Coefficient of variation ( % ) Mean±SD Within day Between days (n=5) (n=2/day for 5 days) ag 0.14 ± 0.02 7.1 14.3 Soybean flour V 0 Vinton 81,'94 pg 2.23 ± 0.16 3.6 7.3 crop I 0 pa 0.48 ±0.04 4.8 8.3 II 0 ag 0 Textured V 0 vegetable pg 1.45 ± 0.16 6.9 11.1 protein I 2.03,± 0.14 4.4 6.8 (ADM) pa 0.42±0.04 9.8 10.2 II 1.03 ± 0.13 6.7 12.2 Table 3.5. Recovery of soyasaponin I and formononetin from the spiked soy samples External standard Internal standard Sample Saponinl Formononetin Recovery(%) CV Recovery ( % ) CV Mean±SD (%) Mean±SD (%) Soybean flour (Vinton 81, '94 crop) 120.9 ± 3.63 3.0 98.4 ± 2.54 2.6 Textured vegetable protein (ADM) 103.2 ± 9.71 9.4 99.5 ± 2.73 2.7 Dried Tofu (Iowa State University) 93.9 ± 8.99 9.6 97.5 ± 1.86 1.9 *: Each soy sample was duplicated for the control and replicated four times for the spiked. 64 added prior to extraction. The amount of the external standards recovered from the procedure indicated the actual amounts of the analytes, which could be extracted and identified by the method. The recovery results in table 3.5 show that soyasaponin I had varied recoveries in different samples while formononetin recovery was consistent among different soy samples. This is probably because soyasaponins bind to the food matrices differently. The recovery of soyasaponin I in the raw soybeans was 120.9% and the variation was large. This high percentage of recovery and high variation of saponin I in soybeans have occurred because there was virtually no soyasaponin I in the raw soybeans (Kudou, et al. 1994). Usually the response of the analyte naturally present in the sample before spiking should be high enough to provide a reasonable signal/noise ratio (usually >10) to ensure a precise measurement (Snyder, et al. 1997). The recovered soyasaponin I might partially come from the soyasaponin j3g decomposition during the sample preparation. Therefore, non-DDMP conjugated saponins might not be a good external standard for the DDMP-conjugated forms in the raw soy material. Overall, more than 93% of soyasaponins could be recovered from the soy samples. It indicated the method for soyasaponin analysis is accurate. The recovery of formononetin was close to the recovery of soyasaponin I. It · indicated soyasaponins and the internal standard could be extracted by this procedure with similar efficacy, and formononetin was, therefore, a proper choice as an internal standard. 3.5. SAPONIN CONTENTS IN SOYBEANS AND SOY PRODUCTS Soybean saponin analysis was performed by the HPLC method described in 2.3. The standard solution consisting of formononetin and saponin I was run daily to confirm that HPLC system operated correctly (figure 2.13). Any significant deviation (>5%) from 65 the running average of the standard concentration indicated that a malfunction might be occurring on the HPLC system. For example, in figure 3.13, the positive deviation that occurred in March was the result of a pump failure. No sample quantitation would be considered valid until the problem was corrected. Fifty varieties of soybeans were evaluated for saponin concentrations. These soybeans were grown in Iowa, U.S. A. in 1999. The results are shown in Appendix B. The mean of total saponin content in soybeans was 4.04 ± 0.9lµmol/g, the range was from 2.50 to 5.85 µmol/g among different varieties. DDMP-conjugated soyasaponins consisted 85 to 94% of the total group B soyasaponins in soybeans. The minor amounts ofnon-DDMP saponins might be the decomposition products of DDMP saponins during sample preparation. Soyasaponin content varied among different soybeans varieties. Tsukamoto, et al. (1995) reported DDMP-conjugated soyasaponin contents in thirteen Japanese varieties of soybeans. They found the total content of soyasaponin ag, f3g, and f3a ranged from 1.39 to 3.25 µmol/g in these soybean seeds. The level and composition of soyasaponins we measured in Iowa soybeans were similar to Tsukamoto's results. Among these soybean varieties, twenty-one varieties were genetically modified to resist the herbicide 'Round-up'. The soyasaponin concentrations in these varieties were compared with other soybean varieties. There were significantly lower content of soyasaponin f3g, J3a, and total soyasaponins in these 'Round-up Ready' soybeans than the .. other conventional soybean varieties (figure 3.14). Perhaps these soybeans produced less soyasaponins probably because they experienced less environmental competition of other plants. Similarly, Tsukamoto, et al. (1994) reported that two folds higher saponin contents in wild-type soybeans were observed compared with the cultivated soybeans. saponin standard mix 25 .------, .!<: co Q) 20 formononetin 0. -+- pump failure 4--l saponin I -0- 0 15 Q) bl co H Q) 10 :> co Q) 5 ..., ...... c:: .µ co s QJ O H 0 H co 4--l s:: -5 J ...... 0 ·rl .µ 0\ co 0\ ·rl -10 :> Q) 'D -15 .j.) s:: Q) CJ H -20 Q) p.. -25 1/30..l...... ------2/29 3/30 4/29 5/29 6/28 7/28 8/27 9/26 Date Figure 3.13. Quality control analysis of standards in routine saponin assay. 67 Round-up Ready (n=21) Com..entional varieties (n=29) 5.0 4.5 * C) 4.0 -::::, 0 3.5 E ::s 3.0 C -0 :;:::: 2.5 ca .. 2.0 C -G) 0 1.5 C 0 0 1.0 0.5 0.0, ag bg ba total soyasaponins Figure 3.14. Comparison of soyasaponin contents between 'Round-up Ready' and conventional soybean varieties. *: The differen~e between Round-up Ready soybeans and conventional soybean varieties was statistically significant (p < 0.05) 68 The soyasaponin contents of some representative commercial soy products examined in the present study are shown in table 3.6. These products include soy ingredients (soybean seeds, soy protein isolates, soy protein concentrates, textured vegetable protein, Novasoy®, and toasted soy hypocotyls), and typical soy foods (soymilk, tofu, and tempeh). The results were adjusted based on the recoveries and reported on 'as is ' weight basis. The total saponin concentrations in these soy products ranged from 0.47 to 114.02 µmol/g. Direct comparisons of total saponin contents might not be proper between the soy products reported because the soybean varieties used to produce each product were unknown, and the saponin contents varied depending on the sources of soybeans. However, it was evident that the composition of saponins was different among the soy products depending on the processing conditions of the products. The DDMP-conjugated soyasaponins (a.g, j3g and j3a) were about 85% of total group B saponins detected in the raw soybean seeds. Soyasaponin I and II were the major forms in the processed soy products due to heat treatment during processing. A high content of soyasaponin V and a.g were detected in the toasted soy hypocotyls but no soyasaponin j3a was observed. The ratio of soyasaponin a.g to j3g was approximately 1:2 in soy hypocotyls, in good agreement with results reported by Shiraiwa, et al. (1991). The level of saponins in the two types of ADM soy protein isolates was significantly higher than in the soybean flour. Saponins were undetectable in ethanol-washed soy protein concentrate but relatively high concentration in acid-washed soy protein concentrate and similar to soy protein isolates. Soy protein isolate is commercially prepared by isoelectric precipitation of soy proteins from the aqueous extract of defatted soy flakes. The protein concentrate is usually made from defatted soy Table 3.6. Soiasal?onin contents and coml?osition in the commercial soi l?roducts Moisture The groul! B soiasal!onin content {~mol/g} Sample content V I II a.g pg pa total (n = 2 per product) (%). Soybean flour 6.43 0.00 0.28 0.21 0.17 2.19 0.47 3.31 Tofil 86.87 0.00 0.31 0.13 0.01 0.11 0.03 0.59 Tempeh3 59.65# 0.00 0.76 0.39 0.01 0.28 0.09 1.53 Isolated soy protein 500E4 4.87 0.87 5.73 2.39 0.10 1.20 0.31 10.60 Isolated soy protein Supro 6704 4.56 0.00 5.59 2.50 0.07 1.01 0.33 9.51 Soymilk5 90.75 0.00 0.22 0.12 0.00 0.09 0.04 0.47 Soy hypocotyl6 3.55 4.41 5.80 0.00 4.71 12.53 0.00 27.46 O'I Textured vegetable protein7 5.66 0.00 1.89 0.87 O.ll 1.26 0.38 4.51 \.0 Acid-washed soy concentrates7 5.80# 0.00 2.41 1.05 0.19 4.90 0.86 9.41 Ethanol-washed soy concentrates7 5.80# 0.00 0.08 0.12 0.00 0.00 0.00 0.20 Novasoy7 3.75 0.00 77.55 36.48 0.00 0.00 0.00 114.02 Vinton81,- 1994 crop beans 2p-rrm, M on-nu . 3 Quong Hop & Company 4 Protein Technology International 5 White Wave Inc. 6 Toasted, Schouten USA Inc 7 Archer Daniels Midland Company # Obtained from USDA food composition database * All samples were analyzed in duplicates. Saponin content reported on an 'as is' weight basis and the data were corrected according to internal standard. 70 flakes by washing with aqueous alcohol or water to remove the soluble carbohydrates. The alcohol-wash resulted in the loss of saponins in soy concentrates. Novasoy® is produced by drum-drying the alcoholic extracts from soy protein concentrate production, and is a very concentrated source of both isoflavones and non-DDMP saponins. Soymilk, tempeh and tofu appeared to be low in saponin content compared to the raw soybeans on an 'as is' weight basis. However, the saponin contents in these soy foods were 3.79 µmol/g in tempeh, 4.49 µmol/g in tofu, and 5.06 µmol/g in soymilk on dry weight basis, which were close or higher than in the raw soybean flour. Heat processing might not be extreme in the production of these soy foods compared with Novasoy® evidenced by the relatively small amount of DDMP-conjugated saponins present. Previous literature provided little information on the saponin contents in various soy products. Ireland, et al. (1986) determined saponin contents in soy protein isolate, soy protein concentrate and soymilk by measuring soyasapogenol concentrations. They found the total saponin contents were 0.76 g/lO0g in soy protein isolate, undetectable in soy protein concentrate, and 0.022 to 0.026 g/lOOg in soymilk on 'as is' basis. The results of the present study were expected to be lower because only the group B soyasaponins were measured while Ireland, et al.(1986) measured total soyasaponins including the group A and the group B saponins. However, the saponin levels found in the present study were 1.04 g/lOOg for soy protein isolate and 0.048 g/l00g for soymilk, both of which were higher than Ireland's data though the variation among the different sample sources must be considered. These results suggest that using the sugar/sapogenol ratio to estimate total saponin concentrations in soy products may not be accurate. Kitagawa, et al. (1984) reported the total saponin levels were 0.3 g/lOOg in tofu on a dry weight basis. We observed the similar 71 concentrations of saponins in the commercial tofu we examined. In general, the values reported here were comparable to the data reported in the literature. Isoflavones are another important secondary metabolites in soybean plants. Numerous studies have shown the health beneficial properties of soy isoflavones, including inhibition of bone resorption in postmenopausal women (Bahram, et al. 1996), prevention of hormone-related cancers such as breast cancer and prostate cancer (Peterson, et al. 1991), the lowering cholesterol activity and decrease in the risk of cardiovascular diseases (Anthny, et al. 1996). In the present study, the isoflavone contents in soybeans and the commercial soy products were evaluated and compared to the composition and concentrations of soyasaponins. For example, figure 3.15 shows the levels of isoflavones and soyasaponins iu 10 varieties of soybeans randomly chosen from all 50 soybean varieties. In the figure, the maximum, minimum and the average levels of total soyasaponins and isoflavones of these 50 soybean varieties were also listed. The ratio of total isoflavone level to soyasaponin level was 0.9 to 2.9 on a mole basis. The relationship between total isoflavones and total soyasaponins was plotted as shown in figure 3.16. The randomly dispersed plots indicated that there was no apparent correlation between isoflavone and soyasaponin content in the raw soybean seeds (r = 0.1285, P > 0.05). In the commercial soy products, the ratio of total isoflavone to saponin on a mole basis was ranged from 0.2 to 20.4 in soybean seeds, soymilk, tofu, soymilk, texturized vegetable protein, soy protein isolates and concentrates (figure 3.17a), soy hypocotyls, and Novasoy® (figure 3.17b), depending on the production process. The isoflavone concentrations were slightly higher than saponin concentrations in raw soybean seeds, while the ratio of isoflavones to saponins changed after production processing. A higher isoflavone 12 Max I ~!--It.•------I <'!Iii 10 -Cl -0 8 E::, -C 0 :.:; 6 l! ....C Cl) u 1 -....l \!i?~- ~:'. .'• :Jr:,,, 1IA'i1 %:: N C 4i II 1. IWLt:. ll;I. 11~1i~· "'"' l~lil~I I 0 l 2 0 A~ .,.,,I)>"' r+,,._d' iP 11#" °'#" 4~ ~o~tJ # _,,n,,"' ,t,o/ 91#" IY ~-4' n; !\, -.; ~")# O 1}"' #,f .,~o. r/' V .i,• °" ...f""' ,u- ,? ~o O" rf ~• -0:-0 rP" 0~ --t,& ~'l ,<;<>o variety Figure 3.15. Isoflavone and soyasaponin levels in 10 representative soybean varieties and the maximum, minimum and average for 50 soybean varieties. 73 7.0 6.0 - • •• • 0 5.0 - • • E • • .. • :::s •.I • • 0 • • • •• .5- 4.0 • • C ...... • 0 C. • C'CI 3.0 ,: •• 0 • C'CI • •• ••• 0 2.0 - ca 0 - 1.0 - r=0.1285, p>0.05 0.0 I I I I I 0.0 2.0 4.0 6.0 8.0 10.0 12.0 total isoflavones (umoVg) Figure 3.16. Correlati,on of total isoflavones to total group B soyasaponins in 50 varieties of soybeans 12 ------~ 1000 Iii total isoflavone 10--1 I I II total saponin 800 11 8 600 J2> 0 6 -!2> E 0 ::::, E :::, 4 400 111 I I i 200 2 0 0 .4>~\ (\0-10-so"i ~'f. eVc- e~ o/' \_O~ 11,1() ,.o"i""-f< \_0~9 9t~ '1;0..,{ ~(\<§>()~ a.e"' .,o'l • t:,'1>9{0 ~o'-0 e(\,s'I>' a.es :o\0 ,.ooJ,9 cP(\o c,,(\'- (a) (b) Figure 3.17. Isoflavone and soyasaponin levels in commercial soy products (n = 2) (a) soy ingredients-and foods; (b) nutraceutical soy products 75 to soyasaponin ratio was observed for soymilk, tofu, soymilk and textured vegetable protein but more saponins than isoflavones were detected in soy protein isolates and acid-washed soy protein concentrates. The concentrations of isoflavones and saponins in ethanol-washed soy protein concentrate were both low compared with other soy products. It is possibly because soybean saponins are more hydrophobic and might bind protein more tightly in these high protein products while isoflavones might be leached out during the acidic or alkaline washing process. The soy protein isolates from Protein Technologies International have been used to investigate the cholesterol lowering effects of soy protein in a number of studies. Since we found significant amount of saponins present in these protein products, the conclusions from these previous cholesterol studies might need to be evaluated carefully because of the potential hypocholesterolemic activity of saponins. Isoflavone contents were more than twenty times higher than saponin contents in soy hypocotyls and eight times higher in Novasoy® (figure 3.17b). In summary, the group B soybean saponins in soybeans, soy foods, and ingredients vary in concentration and composition depending upon the variety of soybeans and the processing conditions used to produce a particular product. Soybean saponins have shown potential beneficial effects humans including cholesterol-lowering, colon cancer-inhibiting, immune-modulating, and anti-oxidative activities. A database on saponin concentrations of soy products will be very useful for evaluating the biological activities of these compounds and clarifying the health-promoting effects of soy products. 76 CHAPTER4 CONCLUSIONS Soy saponins have attracted attention recently because these compounds have been associated with potentially beneficial human health effects, such as lowering cholesterol, anticarcinogenic, modulating immune function and inhibiting virus infection. However, the mixtures of saponins with unknown purity and content have often been used in the previous studies. The purified soyasaponins are needed for elucidation of the biological activities of saponins. Sensitive and accurate assays for soybean saponin quantification in soy products are needed as well to evaluate their biological activities and provide the general public dietary information on soybean saponins. The saponin isolation method developed in the current study can be used to produce the DDMP-conjugated soyasaponin ag, j3g, j3a, and the non-DDMP soyasaponin I, II, and V with 0.017% to 0.063% yield and> 95% purity. The method is practical and reproducible. Our study demonstrated that the purified DDMP-conjugated soyasaponins decompose to the non-DDMP saponins when standing in alcoholic solution, and the decomposition rate increased along with the temperature increased. The analysis of saponins is difficult because of their structural diversity and low ultraviolet absorbance. There have been considerable efforts to quantify the saponin content in various food products. The HPLC method of saponin assay described in the present study was the first one which could separate and quantify the group B soybean saponins including DDMP-conjugated forms and non-DDMP forms in soy products specifically. The accuracy of the quantification assay was > 93% for both soybean seeds and the soy foods. The 77 variation of the assay was< 10% for within-day analysis and< 13% for analysis performed over different days. Formononetin (7-hydroxy-4'-methoxy-isoflavone) has been demonstrated to be a good internal standard for accurate and precise soyasaponin analysis. The results of the internal standard experiment also demonstrated that this analytical method works well for a wide range of soy ingredients and soy foods. The DDMP-conjugated soyasaponins were found as the primary saponins in soybean seeds. The total saponin contents varied among different varieties of soybean seeds. There was no significant correlation between saponin levels and isoflavone levels in these soybean seeds. The soyasaponin concentration and composition vary in different soy products depending on the production processing. In the heat-treated soy products, significant concentrations of non-DDMP saponins were present. The ratio of total isoflavone to total soyasaponin changed in various soy products due to the processing. Because some soy products, especially soy protein isolates, have been used to investigate the cholesterol- lowering effect of soy, the factor of saponins and the ratio of isoflavone/saponin should be taken account to justify the results. Although saponins have partially been attributed to the health beneficial effects of soy consumption, there is no direct evidence in vivo for many of the biological activities of saponins. The metabolism of saponins in humans and animals is poorly understood because of the lack of reliable analytical methods. Therefore, future research efforts will focus on the fate of the soyasaponins in humans and animals and identify the primary active forms of soyasaponins participating in improved human health. The·information will be useful for further elucidation of their biological mechanisms and properties. The information provided 78 in the current study will be useful for the qualitative and quantitative analysis of soyasaponins in biological specimens. APPENDIX A. MASS SEPCTRA OF ISOLATED SOYASAPONINS-1 SPEC :f :in99 S 9'-6"1 DBRI.'1/BD S.PEC'!'RUM ,¥ 9 Samp bati!lig- st~rt : 220 Comm ESIMS pos Mode ESi +Q1NS ~MR U~ PROF Study: rou~ine Ope;i:- x~mel Client• L~ Inlet : Base !06:9. 6 :en ten 55-1l.11.:L2 M~$ses: 200 > 1200 ~Ol;')'a 106~-6 ~IC 3.S.3.152803 :ll-peaks: 1683 Peak i000.00 mmu Dat.a ~/190>211 - /52>67 1069.6 100 R+ 0'1 . [M+Ht S.Si,. l:l:S.:! poa. 80 -....) \0 -60 [M+Nat 40 :Lflfil- - 6 [M+H-DDMPt :20 9d.3.0 !!23---. _4 I 200 4;()0 600 800 1.000 1200 ESI positive mass spectrum of soyasaponin ~g APPENDIX A. MASS SEPCTRA OF ISOLATED SOYASAPONINS - 2 SPEC fin995990 23-SEP-99 Elapse; oo:o.s.s 1. $amp Ba St.art. J.0:39:16 1. c;!o:mm ES:t pos !:-lode ES'I +Ql.MS LMR AVER UP PROF Stuo.y routine Oper Kamel. Client:: HU :rnlet Base 520.2 Intan 1371:83-6 Masses 100 > :1.200 Norm 520 .2 RIC 781339 '#pe.a>.ks 2 Paak 1000.00 mmu ~2!)_3 100 E+ 06 1.3-"7 trisaccharide chain so 00 0 oO 40 33\2 rMt 485...,..S. 20 si2s. e; :i.o::rn. 7 '947 .6 ! 445.....JL • S9S-.l i I I , :2 00 400 600 800 1000 l200 ESI positive mass spectrum of soyasaponin ~a APPENDIX A. MASS SEPCTRA OF ISOLATED SOYASAPONINS - 3 SPEC: firt006~52 J.0-FEB-00 S:lapae: 00:07.5 J. Sampr Sap V Start : 11.: 52 :1.:9 1 COM:!i'I~ ESI pos. Ioode: ESI +Q1MS LMR AVBR U~ ~RO~ Study E$I/M$ Ope.rt KH Client: HU In.let Base: 13S.6 Int:.en 384'178 Masaea J.00 > J..200 Norm: 981.6 RIC 84.!l-115,(i ff.i;,eaks 2 Peak: 1000.ao mmu. 982 100 E+ 04 [M+Nat 7.95 rM+Hl;00 I 80 :ESl :bo:s ...... 00 60 40 906 I 1055 I :911 ~ss l 20 1 832, 8531 192:91 990 }({>61r :~~. ,I~ ~- aoo sso 9 oo 1000 ioso 1100 ESI positive mass spectrum of soyasaponin V APPENDIX A. MASS SEPCTRA OF ISOLATED SOYASAPONINS - 4 .SPE)C: t'in99 5240 08-DEC~9:9 Blaps.e~ 00r06.3 l Samp: Sap II s::art , l4,40:48 1 Comm: ES! pos Mede: ESI +Q~US LMR AVER UP PROP St:1.1-d.y ; routine Oper: K;;Lm,a;I. Client.: Hu Inlet; :a~se; 351.0 Inten 1.H'.'sS81 MaSSC!e:S: 200 > 1100 Noi:-m; 913.5 R!C 2 858-tl 00 I N ·· ,~~\41111~1~1~l#~~~f1~w~A~~N\~~i~~~~1tV~,~ 40 20 800 esa 900 950 ESI positive mass spectrum of soyasaponin II 83 APPENDIX B. SOYASAPONIN CONTENTS IN FIFTY VARIETIES OF SOYBEANS The Grou2 B Soybean Sa2onin Content (~mol/g) Variety V I II ag Qg Qa total Praire Brand 2630 RR 992221006 0.00 0.34 0.10 0.09 1.69 0.28 2.50· AgriPro 3083 RR 992201046 0.00 . 0.62 0.26 0.06 · 1.24 0.35 2.53 FS2786RTRR 9·92257018 0.00 0.57 0.25 0.06 1.41 0.32 2.61 Producers 237 RR 992234018 0.00 0.35 0.19 0.13 1.75 0.31 2.73 Garst 261 RR 992209044 0.00 0.34 0.17 0.09 1.80 0.34 2.74 Trelay 227STS 992153027 0.00 0.58 0.30 0.09 1.41 0.41 2.78 Fontanelle F8933 RR 992234013 0.00 0.44 0.13 0.14 1.85 0.29 2.85 Stine 2698-4A RR 992153018 0.00 0.33 0.12 0.12 2.04 0.36 2.97 Dynagro 3281W 992257093 0.00 0.05 0.07 0.17 2.17 0.57 3.03 Latham 656 RR 992234001-201 0.00 0.42 0.21 0.10 1.96 0.37 3.06 Midwest 1985 992209081 0.00 0.58 0.14 0.12 1.94 0.33 3.11 Thompson seeds Ex8704 - 992266042 0.00 0.27 0.10 0.18 2.23 0.37 3.14 Mycogen 5242 RR 992209019 0.00 0.26 0.15 0.17 2.25 0.38 3.21 Pioneer 92B05 RR 992234001 0.00 0.08 0.04 0.13 2.47 0.54 3.26 (No name) 992201047 0.00 0.56 0.17 0.13 2.11 0.41 3.37 Dekalb CX 285 RR 992201040 0.00 0.34 0.04 0.18 2.58 0.47 3.60 Exce18278 RR 992221046 0.00 0.17 0.09 0.19 2.54 0.64 3.63 Trelay268 992153031 0.00 0.08 0.03 0.12 2.39 0.55 3.64 AgriPro 2329 992237017 0.00 0.30 0.28 0.17 2.36 0.57 3.69 Garst 198 RR 992234001-211 0.00 0.30 0.13 0.20 2.68 0.44 3.76 Latham696 RR 99257074 0.00 0.22 0.04 0.21 2.91 0.43 3.82 84 The Grou2 B Soybean Sa2onin Content (~mol/g) Variety V I II ag ~g ~a total Oekalb cx285 RR 992153010 0.00 0.09 0.06 0.18 2.80 0.70 3.83 Gold county Kardi 992209064 0.00 0.11 0.09 0.20 2.92 0.53 3.86 Jacobsen Hybrid Check 774 - 992221117 0.00 0.08 0.06 0.19 2.81 0.77 3.91 Trelay 268 992207046 0.00 0.16 0.00 0.20 3.05 0.53 3.94 AgriPro 2329 992237014 0.00 0.09 0.03 0.17 2.53 0.62 4.17 Latham L437 RR 992221058 0.00 0.08 0.00 0.22 3.35 0.54 4.20 Excel 8261 RR 992153024 0.00 0.29 0.29 0.21 2.75 0.71 4.26 MWSeed Genetic 2210 RR - 992234029 0.00 0.29 0.10. 0.23 ·3,03 0.73 4.38 LG6288 992207049 0.00 0.11 0.03 0.25 3.37 0.73 4.48 Croplan RT 2856 RR 992153019 0.00 0.20 0.00 0.21 3.54 0.57 4.52 AgriPro 2329 992237018 0.00 0.32 0.13 0.22 3.20 0.74 4.61 Mycogen 5261 992153040 0.00 0.09 0.12 0.29 3.33 0.84 4.67 NC+2A39RR 992257027 0.00 0.09 0.07 0.29 3.41 0.82 4.68 LG Seeds 6245 992266036 0.00 0.12 0.03 0.27 3.60 0.75 4.76 Wilson 2630 992237005 0.00 0.12 0.10 0.31 3.41 0.87 4.80 HY-Vigor 2375 992221118 0.00 0.37 0.07 0.29 3.45 0.65 4.82 Garst D330 992201025 0.00 0.10 0.05 0.31 3.59 0.78 4.83 NK-X99223 992209048 0.00 0.10 0.04 0.31 3.66 0.76 4.87 Stine 2788 992207063 0.00 0.14 0.04 0.24 3.74 0.77 4.92 K2727A 992237020 0.00 0.29 0.08 0.32 3.52 0.73 4.94 Mycogen 5261 992257092 0.00 0.32 0.05 0.35 3.58 0.69 4.99 Jacobsen Hybrid J-7SO -992221120 0.00 0.31 0.07 0.26 3.68 0.72 5.04 Novartis S21Al 992299036 0.00 0.08 0.04 0.35 4.01 0.76 5.24 85 The Grour B Soybean Saronin Content (t!:mol/g) Variety V I II ag ~g ~a total Wilson 2832 RR 992201042 0.00 0.19 0.06 0.32 4.19 0.87 5.63 Wilson 2630 992221094 0.00 0.14 0.05 0.32 4.31 0.87 5.69 AgriPro 2761 SCN 992299023 0.00 0.14 0.04 0.38 4.39 0.91 5.85 Mean±SD 0.00±0.0 0.24±0.1 0.10±0.1 0.21±0.1 2.87±0.8 0.60±0.2 4.04±0.9 * All the soybeans were analyzed in duplicates, the data reported on 'as is' weight basis, and the data were corrected by recovery. 86 LITERATURE CITED Anderson, R.L. andWolf, W.J. 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Cancer 1992; 20:635-638. 96 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my major professor. Dr. Patricia A. Murphy for her knowledge, guidance, patience and encouragement throughout my graduate study at Iowa State University. She not only provided support on knowledge and expertise but also mentored me to become a person optimistic and prepared in the face of challenges. The experiences I gained here will greatly benefit my future career. I would like also to · extend my gratitude to Dr. Suzanne Hendrich who served as my co-major professor, gave very valuable comments, and challenged me in my research as well. I also enjoyed and was inspired by her creative thoughts in scientific research. I would like to thank Dr. Donald C. . Beitz for being my committee member and for his helpful advice towards my study and thesis writing. I appreciate the great help and the friendship of Dr. Murphy's group: Dr. Song, Dr. Wu, Yun, Dave, Kobita, Vanessa and Cathy. I wish to thank the staff, faculty and colleagues at the Interdepartment of Toxicology and Department of Food Science & Human Nutrition for their support and services. At the last but not the least, my sincerely gratitude goes to my family, my husband, parents and sister. Without their encouragement and support, it would not be possible for me to complete my three-year study abroad.