Agric.

Biol.

Chem.,

55 (2),

577-579,

1991

577

Note

but S-II did not increase significantly (about O.ll mg/3 pairs of cotyledons). The S-III content was very low and
could not be accurately estimated. Furthermore, acetylsoyasaponins were absent. In sprouts that included

Variation in Saponin Contents in Germinating Soybean Seeds and

Effect of Light Irradiation
Makoto Shimoyamada and Kazuyoshi Okubo Department of Food Chemistry, Faculty of Agriculture,
Tohoku University, 1-1 Amamiyamachi- Tsutsumidori, Aoba-ku, Sendai 981, Japan Received July 9, 1990

epicotyl, hypocotyl, and root of germinating seed, mainly

sprouts, Fig. 2B). However, S-I, S-II, and S-V increased

S-I, S-II, S-V, and AS-A4, were detected. The AS-A4 content per 3 sprouts was constant (about 0.23mg/3

significantly during germination sprouts, Fig. 2B), especially S-I that of non-germinated seed. In S-V were probably synthesized

AS-A4 remained constant. Furthermore, it is interesting that AS-A4 was retained in an acetylated form. Little S-III was detected in the seed hypocotyl and this constituent

(0.74, 0.10, and 0.10 mg/3 increased by about 15 times the sprout, S-I, S-II, and during germination while

Several saponins have already been isolated from soybean seeds,1 ~ 8) and many physiological activities were also reported.9) In our previous paper,10) we analyzed the

was detected only at trace levels in the sprout. The distributions ofsaponins in epicotyl, hypocotyl, and root were separately examined. Eight days from sowing,

saponin composition of some varieties of soybean plants. We also noted significant variations of saponins in germinating seeds and have now analyzed the saponin

compositions during soybean germination. Soybean seeds (Glycine max (L.) Merrill cv. Keburi) were washed, imbibed, and placed on vermiculite-water (1 :2, w/v) and covered with the same wet vermiculite.
Seeds were germinated at 25C in continuous darkness.

Germinating soybean seeds were harvested 0, 2, 4, 6, or 8

days after sowing. Havested seeds were divided into cotyledon and sprout (the latter including epicotyl, hypocotyl, and root), then lyophilized and milled. Milled samples were extracted with 20 volumes of 70% ethanol. Each extract was analyzed by HPLCin a manner reported
in our previous paper.10) The equipment

used were a

Waters 510 pump, a Waters 484 UVdetector, and a Waters 741 data module. Authentic samples were isolated from
soybean seeds with HPLC.10) Saponin levels were

estimated with calibration curves that were constructed by plotting the concentrations of each saponin vs. the peak
areas. The fresh weight of the germinated soybean seeds

increased by more than 8 times that of the seed (nongerminated seed, 0.19g; after 8 days of sowing, 1.64g). On the other hand, the dry weight of the germinating

Fig. 1. Typical Liquid Chromatograms of 70%Ethanol Extract from Germinating Soybean Root after 8 Days of
Sowing. A: mobile phase, MeOH-2-PrOH-H2O-AcOH (70: 6 : 23.9:0.1); flow rate, 0.5ml/min. B: mobile phase, MeCN-2-PrOH-H2O-AcOH (34: 5 : 61 :0.1); flow rate,

soybean seed remained constant (about 0. 13 g) for 8 days.

Figure 1 shows typical liquid chromatograms
III (S-III), seeds. Soyasaponins V (S-V), I (S-I),

of 70%
and II

ethanol extract in the root part of the germinating soybean

(S-II),
separately.

l.O ml/min.

and acetyl-soyasaponin

A4 (AS-A4) were eluted

A4, acetyl-soyasaponin A4; I, soyasaponin I; II, soyasaponin II; III, soyasaponin III; V, soyasaponin V.
Column, YMC R-ODS-5 (5/mi, 250x4.6mm i.d.);

In germinating seed cotyledon (Fig. 2A), the S-I content increased slightly (8 days; 0.33 mg/3 pairs of cotyledons),
Abbreviations: S-I, soyasaponin I; S-II, soyasaponin

detection,
II; S-III,

UV205 nm; sensitivity,

soyasaponin III;

full scale 64mV.

V; AS-A4,

S-V, soyasaponin

acetyl-soyasaponin

A4.

578

M. Shimoyamada and K. Okubo

Fig. 2. Variation of Saponin Contents in Germinating Soybean Seed in the Dark. A, cotyledon; B, sprout, c, control, non-germinated seed. #. soyasaponin I; , soyasaponin II; soyasaponin V; O, acetyl-soyasaponin A4.

Fig. 3. Variation of Saponin Contents in Germinating Soybean Seed in the Light. A, cotyledon; B, sprout, c, control, non-germinated seed. #. soyasaponin I; , soyasaponin II; soyasaponin V; O acetyl-soyasaponin A4.
the epicotyl contained mainly S-I and S-III. The hypocotyl contained S-I and AS-A4, and the root tissue contained S-I, S-II, S-III, S-V, and AS-A4. Saponin compositions in
during germination were observed. Soybean seed was

the 3 parts of sprout were thus very different. In particular, it is interesting that only the root contained S-II, while the seed hypocotyl contained little S-II.
Then effects of light irradiation on saponin contents

germinated under a photoperiod of 12hr light and 12hr dark, the other conditions remaining constant. Variations of main saponins were estimated and compared with those
found in the dark (Fig. 3). In cotyledons, S-I and S-II

contents after 8 days of sowing were 0.48 and 0.18mg/3 pairs of cotyledons, respectively, and the S-I content was

Saponin Distribution

in Germinating Soybean Seed

References

579

significantly higher from 2 to 8 days after sowing than found in the dark. In the sprout, the AS-A4 content was almost constant and there were no significant differences between dark and light during germination. The S-I contents were almost equal to those in the dark after 2

I. Kitagawa, H. K. Wang, T. Taniyama and M. Yoshikawa, Chem. Pharm. Bull, 36, 153 (1988). J. C. Burrows, K. R. Price and G. R. Fenwick,
Phytochemistry,
FoodAgric, I. Kitagawa,

days of sowing, but significantly

after sowing (8 days, 1.26mg/3

sprouts).

higher from 4 to 8 days

Both S-II and

26, 1214

(1987).

C. L. Curl,

K. R. Price and G. R. Fenwick,

43, 101 (1988). M. Saito, T. Taniyama

/. Sci.
and M.

S-V contents were significantly higher under light irradiation (8 days, 0.15 and 0.21 mg/3 sprouts, respectively).
Hence, the AS-A4 content was little affected by light irradiation and was constant during germination; it is

Yoshikawa,
I.

Kitagawa,

Chem. Pharm. Bull,

M. Saito,

T.

33, 1069 (1985).

Taniyama and

M.

concluded that AS-A4 was likely to be very stable. On the other hand, S-I, S-II, and S-V contents were increased according to the increase of the dry weight of the sprouts;
these saponins are thus likely to be synthesized during plant growth, and light irradiation increases the saponin

Yoshikawa, Chem. Pharm. Bull.,33, 598 (1985). C. L. Curl, K. R. Priceand G. R. Fenwick, /. Natural
Prod., 51, 122 (1988).

I. Kitagawa, T. Taniyama, Y. Nagahama, K. Okubo, F. Yamauchi and M. Yoshikawa, Chem. Pharm.

Bull, 36, 2819 (1988).
Chem. Pharm. Bull, 36, 2829 (1988).

content increased during germination.

Acknowledgments. Wethank Mr. K. Harada of the National Institute of Agrobiological Resources for the supply of soybean seeds and the helpful discussion. We also thank Dr. G. R. Fenwick of the AFRCInstitute of Food Research for his useful suggestions in the preparation of the manuscript.

T. Taniyama, Y. Nagahama, M. Yoshikawa and I.

Kitagawa,

I. Kitagawa and M. Yoshikawa, Kagaku to Seibutu,

21, 224 (1983).

M. Shimoyamada, S. Kudo, K. Okubo, F. Yamauchi

and K. Harada, Agric. Biol. Chem., 54, 77 (1990).