Structural Elucidation and Physiological Properties of Genuine

Chapter 27

Structural Elucidation and Physiological Properties of Genuine Soybean Saponins 1

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Shigemitsu Kudou , Masahide Tonomura , Chigen Tsukamoto , Teiji Uchida , Masaki Yoshikoshi , and Kazuyoshi Okubo 2

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Downloaded by CORNELL UNIV on September 1, 2016 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch027

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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 Company, Ltd., 2-4-5 Azabudai, Minato-ku, Tokyo 106, Japan 2

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The composition and the structures of group Β saponins in native soybean seeds were investigated. Five kinds of saponins named soyasaponins α , β , β , γ and γ according to elution order from HPLC were isolated, and the structures were characterized as having a 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) group attached by acetal linkage to C-22 of the aglycones soyasaponin V , I, II, III and IV respectively. These D D M P con jugated saponin are considered to be the genuine saponins in native soybean seeds, and soyasaponins I, II, III, IV and V are artifacts produced by heating during the extraction or concentration process. Maltol produced by amino-carbonyl reaction was identified after heating an aqueous solution of D D M P conjugated saponins. In addition, soyasaponin β is same as the phytochrome killer chromosaponin I. D D M P conjugated saponins possess several biological activities. g

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g

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Many kinds of saponins have been isolated from soybean seeds. They are divided into three groups, group A , Β and Ε saponins, on the basis of their aglycone structures (soyasapogenol A , Β and E) (1-2). It has been reported that soybean saponins possess many physiological activities—hypolipidemic (3), antioxidative (3), anti-tumor promoting (4) and anti-HIV infection (5) properties. Soyasaponin I, II, III (6-7) and IV (8) from whole soybean seeds and soyasaponins (6-8) from hypocotyl are group Β saponins that have been isolated and their structures fully characterized (Figure 1). In a recent paper (9), we isolated a genuine saponin, BeA, and showed that the structure had an unusual sugar 2,3-dihydro-2,5-dihydroxy-6methyl-477-pyran-4-one (DDMP) attached by acetal linkage to C-22 of the aglycone of soyasaponin I. We further suggested that soyasaponin I, II, ΙΠ, IV and V also exist as DDMP conjugated forms in soybean seeds. Here we discuss our investigations of the composition of group Β saponins, and the structures of the soyasaponins α , β , β , γ and γ which contain DDMP, are described. Furthermore, the physiological properties of the unstable and reactive DDMP conjugated saponins are discussed. g

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0097-6156/94/0546-0340$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.

27. KUDOU ET AL.

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Properties of Genuine Soybean Saponins

Soyasapogenol A

Soyasapogenol B

Soyasapogenol E


ο

R

R

2

Maltol

R2

Rj Soyasaponin Soyasaponin Soyasaponin Soyasaponin Soyasaponin

DDMP

4

I CH OH II H III C H O H IV H V ΠΗ.ΩΗ 2

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^3

α-L-Rha α-L-Rha H H β-D-Glc

Soyasaponin Soyasaponin Soyasaponin Soyasaponin Soyasaponin

β CH OH B H 'g C H 0 H H α CH QH 9

2

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2

γ

α

α

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R4

a-L-Rha a-L-Rha H H β-D-Glc

Figure 1. Structures of the group B saponins in soybean seeds.

The Composition of Group Β Saponins in Whole Soybean Seeds The composition of group Β saponins in whole soybean seeds was analyzed by H P L C (Figure 2). Five peaks were detected that were presumed to correspond to D D M P conjugated saponins, because all of these compounds showed the same U V spectra with a maximum absorption at 292 nm due to the D D M P moiety. These compounds were named soyasaponins a and β (tentatively named BdA and BeA, respectively, in reference 9), β^ y and y according to their H P L C elution order. In our previous paper, we showed that the structure of soyasaponin β has the unusual sugar D D M P attached by acetal linkage to C-22 of the aglycone of soyasaponin I. g

g

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Structural Elucidation of Genuine Soybean Saponin Heating the soyasaponins a , β , β , y and y at 100°C for 1 hr to examine the effect of heating on them revealed that they are completely converted into g

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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

soyasaponins V, I, II, III and IV, respectively. This result shows that soyasaponins I, II, III, IV and V are artifacts produced by heating during the extraction or concentration process. It also strongly suggested that soyasaponins oc , β , β , Y and y are the DDMP conjugated soyasaponins V, I, II, III and IV, respectively. The five genuine saponins — a , β , β , y and y — were isolated to determine their chemical structures. Acid hydrolysis gave a glucose, a galactose and a glucuronic acid from oc ; a rhamnose, an arabinose and a glucuronic acid from β ; a galactose and a glucuronic acid from y ; an arabinose and a glucuronic acid from y ; and soyasapogenol Β was detected from all of these saponins. Mild alkaline hydrolysis of soyasaponins a , β , y and y gave corresponding soya saponins V , II, III and IV and another product. The product was identified by HPLC and a U V spectrum, which showed a maximum absorption at 274 nm, and was completely consistent with that of an authentic sample (Figure 3). The molecular formulas of soyasaponins oc , β , y and y were determined to be C 5 4 H 8 4 O 2 2 ( M 1084), C 3 H O 2 0 ( M 1038), C 4 8 H 7 4 O 1 7 ( M 922) and C 4 7 H 7 2 O I 6 ( M 892), respectively, by high resolution F A B - M S , indicating the conjugation of a C 6 H 6 O 3 fragment and the corresponding soyasaponins V , II, III and IV. F A B - M S (negative-ion mode) gave a molecular ion at m/z 1083 [M-H]and fragment ions m/z at 921 [M-H-Glc]- and 759 [M-H-Glc-Gal]- for soyasaponin oc ; a molecular ion at m/z 1037 [M-H]- and fragment ions at m/z 891 [M-H-Rha]and 759 [M-H-Rha-Ara]- for soyasaponin β ; a molecular ion at m/z 921 [M-H]and a fragment ion at m/z 759 [M-H-Gal]- for soyasaponin y ; a molecular ion at m/z 891 [M-H]- and a fragment ion at m/z 759 [M-H-Ara]- for soyasaponin y (Table I). None of the D D M P saponins gave a fragment ion due to the loss of DDMP, but a intense fragment ion at m/z 127 [DDMP-H20+H]- was observed. The assignments of soyasaponins oc , β , y and γ were established by ^ Η C O S Y spectra, COSY spectra, N O E and H M B C (10), and are compared with the spectral data of soyasaponins I, II, III and soyasaponin β (which has been already assigned in previous paper) in Tables II and III. The C - N M R spectra of soyasaponins oc , β , y and y showed six signals at 185.2, 152.5, 132.9, 96.6 or 96.7, 41.5 and 15.2. The *H-NMR also indicated the presence of one hydroxy group (7.45 or 7.44, 1H, br), one methane (5.35 or 5.38, 1H, dd), one unequivalent methylene group (2.93, 1H, dd; 2.35 or 2.36, 1H, dd) and one methyl group (1.90, 3 H , s). These C - and Ή - N M R data indicate the presence of D D M P moiety on soyasaponins oc , β , y and y as well as soyasaponin β . The structure of the D D M P moiety was further confirmed by the H M B C experiment and the NOE difference spectra of soyasaponin β . The H M B C experi ment of soyasaponin β* exhibited cross peaks C-2' and H-3', C-4' and H-2', C-4' and H-3', C-5' and H-7 , C-6 and H-2', C-6' and H-7', C-22 and H-2', respectively. When the signal at δ 2.36 (H-3'b) was irradiated, N O E was observed at δ 5.38 (H-2')and δ 2.93 (H-3'a). Irradiation at δ 2.93 (H-3'a) yielded NOE at δ 5.38 (Η-2') and δ 2.36 (H-3'b), and irradiation at δ 5.38 (Η-2') resulted in N O E at δ 3.35 (H-22), δ 2.93 (H-3'a), δ 2.36 (H-3'b) and δ 1,90 (Η-70· These results support that the compound linked to C-22 hydroxy group of soyasapogenol Β is a 2,3-dihydro2,5-dihydroxy-6-methyl-4//-pyran-4-one and is attached by acetal linkage to the C-22 position of soyasapogenol B. The chemical shifts (δ) of the aglycone moieties of soyasaponins oc , β , y and y were the almost same as that of soyasaponin $ . The chemical shifts (δ) of the sugar chain moiety linked to the oxygen at C-3 of the aglycone of soyasaponins oc and β were almost identical with those of soya saponin V and II, respectively. g

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g

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8 2

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

Properties of Genuine Soybean Saponins

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27. KUDOU ET AL.

{

A i

< Λ

J 10

Retention

T i m e (min)

Figure 3. HPLC analysis of a compound produced by alkaline hydrolysis of D U M P conjugated saponins. 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

Table I. Main Peaks in FAB-MS of DDMP Saponins

Positive-ion mode [M+H] [M+Na] [M+K] +

+


+

Negative-ion mode [M-H]" [(M-H)-146][(M-H)-162][(M-H)-132][(M-H)-146-162][(M-H)-162-162]" [(M-H)-146-132]-

Structural Elucidation and Physiological Properties of Genuine

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