Soybean Saponin and Isoflavonoids - ACS Symposium Series (ACS

Chapter 26

Soybean Saponin and Isoflavonoids Structure and Antiviral Activity against Human Immunodeficiency Virus In Vitro 1

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Kazuyoshi Okubo , Shigemitsu Kudou , Teiji Uchida , Yumiko Yoshiki , Masaki Yoshikoshi , and Masahide Tonomura 3

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Downloaded by CORNELL UNIV on July 25, 2016 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch026

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Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori, Amamiyamachi, Aoba-ku, Sendai 981, Japan Kanesa Company, Ltd., 202 Hamada, Tamagawa, Aomori 030, Japan Nestle Japan, Ltd., 1-16, 7-Chome, Goko-Dori, Chuo-ku, Kobe 651, Japan 2

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Nine kinds of isoflavone glycosides were isolated from the hypocotyls of soybean seeds. Three were proved to be malonylated soy bean isoflavones named 6"-O-malonyldaizin, 6"-O-malonylglycitin and 6"-O-malonylgenistin. Soyasaponins are divided into three groups according to their respective type of aglycone, soyasapogenol A , Β and E. Bb, major constituent of group Β saponins, completely inhibited HIV-induced cytopathic effects and virus -specific antigen expression 6 days after infection at concentration greater than 0.25 mg/ml, but had no direct effect on HIV reverse transcriptase activity. Bb also inhibited HIV-induced cell fusion in the MOLT-4 cell system. Soybean seeds have been consumed by humans for thousands of years because they are rich in protein and their oil is of good nutritional quality. Most East Asians consume soybean seeds regularly from childhood via a variety of soybean products. The incidence of breast and colon cancer in Oriental people is considerably lower than in those living in Western countries (1), who seldom eat soybean products. Additionally, vegetarians, who are also at decreased risk of breast and colon cancer, frequently consume soybean-based meat substitutes (2). These associations suggest that soybeans may play a role in reducing breast and colon cancer risk. Relative to other food stuffs, soybean seeds contain high levels of four classes of compound with demonstrated anticancer activity: glycosides, phytosterols, protease inhibitors, and phytic acid. Soybean seeds contain about 2% glycosides, which are composed of several kinds of soyasaponins and isoflavonoids (3). These two groups of glycosides will be the focus of this presentation, especially their chemical structures and their antiviral activity — such as against human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV).

0097-6156/94/0546-0330$06.00/0 © 1994 American Chemical Society Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Soybean Saponin and Isoflavonoids

Chemical Structure and Distribution


Saponins. Among the various edible legume beans, soybeans contain the highest amount of saponins (3). Depending upon the chemical nature of their aglycones, saponins can be divided into triterpene saponins and steroidal saponins. Furthermore, depending upon the skeleton of the aglycone, triterpene saponins are subdivided into oleanane, urasane, dammarane and cycloartane types (4). Soyasaponins are oleanane-type triterpene saponins. Two nomenclature systems (from Kitagawa and Okubo, Table I) have been used to identify various soyasaponins. For the sake of consistency, only the Okubo system will be used here. Table I. Soyasaponin Nomenclature Assigned by the Research Groups of Kitagawa and Okubo Kitagawa

Okubo

acetylA4 acetyl A1 — — acetylA5 acetylA2 acetylA6 acetylA3

Aa Ab Ac Ad Ae Af Ag Ah

A4 Al A5 A2

deacetyl A a deacetyl Ab deacetyl Ae deacetyl A f

A6 A3

deacetyl A g deacetyl A h

V I II III IV

Ba Bb Be Bb' Be

3

A group — acetylated glc-gal-glcUA-A-ara- xyl (2,3,4-triAc) glc-gal-glcUA-A-ara- glc (2,3,4,6-tetraAc) rha-gal-glcUA-A-ara- glc (2,3,4,6-tetraAc) glc-ara-glcUA-A-ara- •glc (2,3,4,6-tetraAc) gal-glcUA-A-ara- •xyl (2,3,4-triAc) gal-glcUA-A-ara- •glc (2,3,4,6-tetraAc) ara-glcUA-A-ara-•xyl (2,3,4-triAc) ara-glcUA-A-ara--glc (2,3,4,6-tetraAc) a

A group — deacetylated glc-gal-glcU A-A-ara-xyl (2,3,4-triAc) glc-gal-glcUA-A-ara-glc (2,3,4,6-tetraAc) gal-glcUA-A-ara-xyl (2,3,4-triAc) gal-glcUA-A-ara-glc (2,3,4,6-tetraAc) rha-gal-glcUA-A ara-glcUA-A-ara-xyl (2,3,4-triAc) ara-glcUA-A-ara-glc (2,3,4,6-tetraAc) b

c

Β group glc-gal-glcUA-B rha-gal-glcUA-B rha-ara-glcUA-B gal-glcUA-B ara-glcUA-B

7

d

Ε group glc-gal-glcUA-E rha-gal-glcUA-E a b c d

Bd Be

Sugar chains on the left of A are linked to 3-0 and those on the right to 22-0. Isolated by Curl et al and given the name soyasaponin A3. Soyasapogenol Β contains a hydroxyl moiety at C-22. Soyasapogenol Ε contains a ketone moiety at C-22.

Recently, we have determined the structures of genuine saponins in soybean seeds, which are named soyasaponins oc , β , β , y and y and are characterized as g

β

3

g

a

Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES

the aglycones of soyasaponins Ba, Bb, Be, Bb' and Be', respectively, with 2,3dihydro-2,5-dihydroxy-6-methyl-4//-pyran-4-one (DDMP) attached by acetal link age to C-22 (see Kudou et al, Chapter 27). The structures of typical soyasaponins are shown in Figure 1 ; these are not only confined to soybeans but may also be found in other beans and peas, and in alfalfa. Soyasaponins are subdivided into three groups — A , Β and Ε — depending upon whether the aglycone is olean-12-en-3,21,22,24-tetraol (soyasapogenol A ) , olean-12-en-3,22, 24-triol (soyasapogenol B), or olean-12-en-3,24-diol-22-one (soyasapogenol E). Group A saponins which contain two ether-linked sugar chains attached to C-3 and 22 are termed bis-desmosides, and groups Β and Ε which contain sugars attached to C-3 alone are thus mono-desmosides. Five kinds of soyasaponins (Bb, Be, Bb', deacetylated Ab and deacetylated Af) were isolated and completely characterized by Kitagawa et al (5-7) in 1985. More recently, using a different method (without saponification), Okubo et al (89) isolated and identified 11 kinds of intact soyasaponins (six group A and five group B) from the hypocotyl of the seeds.

OR

R

Ri

2

R

2

A group Aa Ab Ac Ad Ae Af Ag Ah

Glc(1->2)gal(1->2)glcUAGlc(1->2)gal(1->2)glcllA-

-OH -OH

2,3,4-tri Ac.xyl(1 ->3)ara2,3,4,6-tetra Ac.glc(1-»3)ara-

Rha(1 ->2)gal(1 H>2)glcllA-

-OH

2,3,4,6-tetra Ac.glc(1->3)ara-

Glc(1-»2)ara(lH>2)glcUA-

-OH -OH -OH -OH -OH

2,3,4,6-tetra Ac.glc(1-»3)ara2,3,4-tri Ac.glc(1->3)ara2,3,4,6-tetra Ac.glc(1->3)ara2,3,4,6-tetra A c . g l c ( 1 - » 3 ) a r a 2,3,4-tri Ac.xyl(1 ->3)ara-

Gal(1->2)glcUAGal(lH>2)glcUAAra(1->2)glcllAAra(1->2)glcUA-

Β group Ba Bb Be Bb' Be'

Glc(1-*2)gal(1->2)glcUARha(1^2)gal(1->2)glcUARha(1 ->2)ara(1->2)glcUAGal(1->2)glcUAAra(1->2)glcUA-

-H -H -H -H -H

Figure 1. Structures of group A and Β saponins.


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The sugars found in the oligosaccharides of soyasaponins are D-galactose, L-arabinose, L-rhamnose, D-glucose, D-xylose and D-glucuronic acid. Chain lengths of 2 or 3 saccharide units are the most common. The terminal monosaccharide of the C-22 sugar chain of intact soyasaponin A is usually acetylated (10). The majority of the hypocotyl saponins were found to be group A soyasaponins. Aa and Ab are the major soyasaponins in group A , whereas Ba and Bb are two major soyasaponins in group B. In contrast to group A and Ba, which are present only in the hypocotyl, groups Β (except Ba) and Ε are present in both the hypocotyl and the cotyledon. On an equal weight basis, the hypocotyl contains a much greater amount of soyasaponins than the cotyledon (77). The hull does not contain soyasaponins (72). Isoflavonoids. Kudou et al. (13) isolated and characterized nine kinds of iso flavonoids from soybean hypocotyl. They are daizin, glycitin, genistin, acetylated daizin, acetylated glycitin, acetylated genistin, malonylated daizin, malonylated glycitin and malonylated genistin (Figure 2). The malonylated glucosides were found to be heat labile; at 80°C, the majority of the glucosides were converted to daizin, glycitin and genistin. The non-malonylated compounds and their aglycones were stable at that temperature. Eldridge and Kwolek (14) reported six kinds of isoflavonoids in soybean seed. They did not observe the malonylated glucosides, presumably because heat was applied in the extraction procedure. Isoflavonoids, like soyasaponins, are concentrated in the hypocotyl; the isoflavonoid content of the soybean hull is very low. On an equal weight basis, the hypocotyl contains 5 to 6 times more total isoflavonoids than the cotyledon.

CHaOFh J π

( /

r 2

HO

I Η OH

Compound Daizin Glycitin Genistin 6"-0-Malonyldaizin 6"-OMalonylglycitin 6"-0-Malonylgenistin 6"-OAcetyldaizin 6"-0-Acetylglycitin 6"-OAcetylgenistin

π

π

Τ T ÎÎ R

ο

3

1^

Ri

R

-H -H -H -COCH COOH -COCH COOH -COCH COOH -COCH -COCH3 -COCH3

-H -OCH3 -H -H -OCH3 -H -H -OCH3 -H

2

2

2

3

2

R

3

-H -H -OH -H -H -OH -H -H -OH

Figure 2. Structures of the isoflavones in soybean seeds.


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FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES

Factors Affecting Saponin and Isoflavonoid Content


The total and relative amounts of soyasaponins vary, depending upon the variety of soybean. Kitagawa et al. (15-16) found one Chinese variety to be richer in total soyasaponins (0.3% Bb plus deacetylated Ab') than those from Japan, Canada and the United States. The amounts of group A and Β saponins in the hypocotyl were 0.36-3.41% and 0.26-2.75% respectively. In a detailed study by Shiraiwa et al. (77), 457 varieties of soybeans were analyzed, and classified into seven types according to their hypocotyl group A saponin composition. Of the types, three were predominant: the A a type, with soyasaponin A a (16.6%); the Ab type, with soyasaponin Ab (76.2%); and the Aa-Ab type, with soyasaponins A a and Ab (5.5%). Two kinds of group A saponin, A a and Ab, are present as the main constituent in the hypocotyl of soybean seed. The saponin composition and content in F l and F2 seeds derived from the crossing of Aa and Ab types were analyzed. The group A saponin was of Aa-Ab type in all the F l seeds, and the ratio of Aa type:Aa-Ab type:Ab type was 1:2:1 in the F2 seeds. From these results, it appears that Aa and Ab were controlled by codominant allelic alternatives at a single locus (18). The saponin level and composition in the seed is different from the iso flavonoid level and composition varies tremendously not only by variety but also by year harvested and geographic location, even among the same variety. One study reported a three-fold variation in total isoflavonoid content among four varieties of soybeans. There are few studies concerning the effects of cooking or processing on soyasaponins and isoflavonoids. Fermentation was found to reduce the saponin content (79), due to enzymatic degradation by microorganisms (20). Germination of soybean seeds changed the composition of soyasaponins, especially in the sprout (27). Germination under light caused a slight increase in the amount of soyasaponin Bb in the cotyledon, while in the sprout, the amounts of soyasaponins Ba, Bb and Be (especially Bb) increased greatly. Germination under a 12 hr light/dark cycle caused more dramatic changes. For example, the content of soyasaponin Bb in the sprout was increased 12-fold (on dry weight basis) in 8 days. Kitagawa et al. analyzed soy products for soyasaponin content. On a dry weight basis, tofu, dried tofu and soymilk were found to contain more soyasaponins, 0.301-0.407%, than soybeans (0.3%), while the fermentation products miso and natto contained less, 0.148% and 0.264%, respectively. Liener (22) reported the same results.

Anticancer Activity Isoflavonoids have activity against tyrosine protein kinase (23), D N A topoisomerase (24) and ribosomal S6 kinase (25) and induce specific cytochrome P450s (26). In one animal study, carcinogen-induced mammary tumorigenesis was inhibited by feeding the animals isoflavonoid-rich soy products. In postmenopausal



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women with low levels of estrogen, however, isoflavonoids were found to have estrogenic effect (27). The saponins most extensively investigated for anticancer activity have been glycyrrhizin and its aglycone (28). More recently, oleanolic acid (29-31), ursolic acid and saponins isolated from other plants (32) have also been shown to have anticancer activity. Dietary saponins were found to enhance natural killer cell activity (33), presumably with an initial effect on the mucosal immune system due to its poor absorption (34). Saponins were also found to be cytotoxic to sarcoma 37 cells (35), to inhibit D N A synthesis in tumor cells (36), and to decrease the growth of human epidermal carcinoma cells (37) and human cervical carcinoma cells (38). Glycyrrhetinic acid has also been found to inhibit the specific binding of 12-0-tetradecanoylphorbol-13-acetate to mouse epidermal membrane receptors in a dose- and time-dependent manner associated with increased K

Soybean Saponin and Isoflavonoids - ACS Symposium Series (ACS

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