Isoflavone Composition within Each Structural Part of Soybean Seeds and Sprouts

RESEARCH ARTICLE
J. Crop Sci. Biotech. 11 (1) : 57 ~ 62
Isoflavone Composition within Each Structural Part
of Soybean Seeds and Sprouts
Siviengkhek Phommalth1, Yeon-Shin Jeong1, Yong-Hoon Kim2 and Young-Hyun Hwang1
1 School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Korea
2 N&B Co.,Ltd., Techno-Building 406, Kyungpook National University, Daegu 702-701, Korea
Abstract
Isoflavone content in various parts of six soybean cultivars and soybean sprout during germination was analyzed by high perform-
ance liquid chromatography. The parts analyzed were seed coat, cotyledon, and axis for seeds and whole sprout, root, hypocotyl, and
cotyledon for sprout. Two cultivars, Aga3 which is known to have the smallest seed size and the highest isoflavone content among
the Korean soybean cultivars and Pungsannamulkong which is the most widely being used as soy-sprout, were selected for sampling
from 1 to 10 days after germination. At the same weight, the order of isoflavone content increased from seed coat to cotyledon to
axis. The highest total isoflavone (isoflavone
dry weight) content was observed in the cotyledon and the lowest in the seed coat.
The cotyledon of the Aga3 variety had the highest total isoflavone content and the lowest was measured in the Pungsannamulkong
variety. The highest total isoflavone content, 10,788µg/g, was observed in whole sprouts (cotyledon+hypocotyl+root) on day 7 for
Aga3. After day 7, there was a decreasing trend in isoflavone content as the germination period increased. Total isoflavone content in
the cotyledon of Aga3 significantly increased after seed germination, whereas the isoflavone content in the cotyledon of
Pungsannamulkong decreased. However, total isoflavone content in the root of both varieties increased while isoflavone content in
the hypocotyls decreased after seed germination.
Keywords: Soybean, Soybean sprout, Isoflavone
Introduction
Soybean is a popular health food in many Asian countries.
including estrogenic activity as well as inhibition of topoiso-
Soybean is used in various forms such as soybean sprouts, soy
merase and protein kinases (Omoni and Aluko, 2005; Ososki
pastes, soymilk, soybean oil, and tofu as key ingredients in cul-
and Kennelly, 2003).
tural cuisines (Kim et al., 2006). Currently, there is an increasing
Isoflavones are categorized chemically according to their
consumption of soybean worldwide due to its nutritional proper-
functional groups. There are four subgroups including aglycones
ties and the beneficial characteristics of its constituent com-
(genistein, daidzein, and glycitein), glycosides (genistin,
pounds, like isoflavone. Consumption of isoflavone is associated
daidzin, and glycitin), malonyl glycoside (malonyl genistin, mal-
with human health benefits such as decreased risk of heart dis-
onyl daidzin, and malonyl glycitin), and acetyl glycosides
ease, menopausal symptoms, cardiovascular disease, and bone
(acetyl genistin, acetyl daidzin, and acetyl glycitin) (Eldridge,
resorption as well as breast, prostate, and colon cancers
1982; Kudou et al., 1991).
(Adlercreutz et al., 1992; Allred et al., 2005; Anderson and
Soybean sprouts is an important year-round vegetable con-
Gardner, 1997; Anthony et al., 1996; Cassidy et al., 1994; Kim
sumed for thousands of years in Korea, China, and Japan. They
et al., 2005; Kennedy, 1995; Messina, 2000). The physiological
are excellent source of nutrients as well as vitamins (Lee et.
function of isoflavone is mediated by a variety of mechanisms
2002). Annually, more than 500,000 tons of soybean sprouts are
consumed in soups, salads, and side dishes. Mass production of
soybean sprouts is an important agriculture business in Korea
* To whom correspondence should be addressed
Young-Hyun Hwang
(Hwang et al. 2004). Many studies have been conducted to
E-mail: hwangyh@knu.ac.kr.
improve the quality and quantity of soybean seeds and sprouts.
Tel:+ 82-53-950-5712
Journal of Crop Science and Biotechnology
57

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Siviengkhek Phommalth et al.
soaked in water at 20°C
for 4 hours to initiate
germination. The seeds
were then removed
from the water and
placed in an environ-
ment-controlled cham-
ber at 20°C with 80%
humidity and exposed to
light for 12 hours per
day. A submersible
pump connected to a
nozzle was placed
above the container and
was set to spray about 3
liters of water per
Fig. 1. Chemical structure of is isoflavone isomers in soybean.
minute for 4 minutes
every 3 hours.
The objective of this study was to evaluate the isoflavone con-
tent in each structure of the soybean seed and sprout to deter-
Isoflavone extract
mine the germination stage that contained the highest isoflavone
The extraction of isoflavone was modified from Rostageno et
content for soybean sprout consumption.
al (2003). Dry soybean powder 0.2g (75
75 µm) was added to
10 ml of 80% HPLC grade EtOH and incubated in an Ultrasonic
bath (Kodo Co., Korea) at 50°C for 1 hour. The samples were
Materials and Methods
then placed in a shaking (150 rpm) incubator at 50°C for 15
hours. Samples were then passed through a 0.45µm syringe filter
Seed materials and germination method
and collected for isoflavone analysis using high performance liq-
uid chromatography (HPLC).
The soybean varieties used in this study are shown in Table 1.
Seed germination method was according to Lee et al. (2007a).
Isoflavone analysis
Twenty grams of seeds of each variety were placed in a plastic
container (6.0 cm W
6.0 cm L
15.0 cm H) with several small
HPLC analysis of isoflavone was based on the work of Wang
perforations at the bottom for drainage. The samples were first
and Murphy (1994). The HPLC system consisted of a
TOTALCHROM V6.2.0.0.1 with LC Instrument control
(PerkinElmer series 200, USA) and A COL-CHOICE C18 column
Table 1. Hundred seed weight and seed coat color of six soybean varieties.
4.6 x 150 mm (5 µm) packed. A linear HPLC gradient utilized ace-
tonitrile (solvent A) and 0.1% of acetic acid in water (solvent B).
Variety
100seeds (g) Seed coat color
Remark
After injection of a 10-µl sample volume, solvent A was increased
Newly developed variety
Aga3
5.75
Green
known to have the highest
from 0 to 45% over 10.2 min. It further increased from 45% to
isoflavone content in the world
90% over 6 min, remained constant for 3.6 min, and then was
Aga4
6.69
Black
Newly developed variety
reduced from 90 to 0% over 15 min. The solvent flow rate was 1.0
A famous variety used for
Pungsannamulkong
13.94
Yellow
soy-sprout in Korea
ml/min. The elution was monitored by UV-absorption (Series 200
A famous variety used for
uv/vis Detector) at 260 nm. Identification of the isoflavone was
Taekwangkong
26.83
Yellow
soy-paste in Korea
based on comparisons with retention times of genuine standards
A famous variety used for
Hwangkeumkong
28.22
Yellow
including daidzein, genistein, and genistin (Sigma Chemical Co,
producing soy pastes in Korea
USA), as well as glycitin, daidzin, 6 -0-acetlygenistin, 6 -0-mal-
A cultivar known to have
Cheongjakong
32.14
Black
high content of isoflavone
onylgenistin, and 6 -0-acetyldaidzin (LC Laboratory, USA).
w w w . c r o p b i o . o r g
58

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Isoflavone Composition in Soybean Seeds
Statistical analysis
and Cheongjakong, respectively. The cotyledon of Aga3 had the
To identify significant treatment effects and interactions,
highest total isoflavone content and the lowest was in
analysis of variance (ANOVA) and multiple mean comparisons
Pungsannamulkong, which were 487 and 207µ g per seed,
were carried out on the data comparing isoflavone content with
respectively. The axis of Taekwangkong showed the highest
the general linear model (GLM) using Statistic Analysis System
total isoflavone content and the lowest was observed in
(SAS 9.1). Differences among mean values were determined
Hwangkeumkong, which were 64 and 20µg, respectively. The
using Duncan’s Multiple Range Test at P?0.05 when ANOVA
seed coat of Pungsannamulkong had the highest total isoflavone
indicated model and treatment significances.
content while the lowest was in Aga4, which were 7 and 1µg per
seed, respectively.
Results and Discussion
Table 2. The isoflavone content in different soybean seed components.
Seed
Dry weight
Isoflavone
Total
Variety
Total isoflavone content in seed components
components
(g)(A)
( /g)(B)
(AxB)
Radicle
0.002
20,304a
43b
The seed of soybean cultivar has a composition of 85.3-
Cotyledon
0.048
10,203b
487a
Aga3
91.6% cotyledon, 1.7-3.8% axis, and 6.4-12% seed coat.
Seed coat
0.006
617c
4c
Similarly, Ribeiro et al. (2006) reported that the BRS 213 soy-
Total
0.056
534A
Radicle
0.003
14,855a
45b
bean cultivar was composed of 86.8% cotyledons, 3.2% axis,
Cotyledon
0.042
5,524b
232a
and 10% seed coat. Content of the seed components depended
Aga4
Seed coat
0.009
94c
1c
on the size of seed. Big seeds had 91.3, 91.6, and 90.2% cotyle-
Total
0.054
278DE
don; 2.22, 1.7, and 2.0% axis; and 6.4, 8.7, and 7.9% seed coat
Radicle
0.005
10,154a
50b
Pungsannamul
Cotyledon
0.097
2,127b
207a
(Taekwangkong, Hwangkeumkong, and Cheongjakong, respec-
kong
Seed coat
0.014
494c
7c
tively). In the case of small and medium sized seeds, axis and
Total
0.116
264E
seed coat percentages were higher than big seeds, which were
Radicle
0.004
14,489a
64b
Cotyledon
0.202
1,502b
303a
3.8, 3.0, and 2.8% radicle and 11.0, 12.0, and 8.5% seed coat
Taekwangkong
Seed coat
0.018
213c
4c
(Aga3, Aga4, and Pungsannamulkong, respectively).
Total
0.224
371C
Nevertheless, the cotyledon of small and medium seeds was
Radicle
0.002
13,131a
20b
Cotyledon
0.238
1,608b
383a
smaller than that of big seeds, which were only 85.3, 84.9, and
Hwangkeumkong
Seed coat
0.006
266c
2c
88.8% for Aga3, Aga4, and Pungsannamulkong, respectively.
Total
0.248
405B
When compared within the same weight, the order of
Radicle
0.005
11,525a
52b
Cotyledon
0.207
1,035b
214a
isoflavone content increased from seed coat to cotyledon to axis
Cheongjakong
Seed coat
0.018
156c
3c
(Table 2). This observation was consistent with Kudou et al.
Total
0.230
269D
(1991), which reported the total isoflavone content of the
Variety
**
**
hypocotyls (containing the plumule and radicle) was 5.5 to 6.0
Seed component
times higher than that of the cotyledon. Isoflavone was not
** = Significant (P<0.01). + Value with different letters (capital letter A-F) is significantly
different at 5% level by DMRT between varieties.
found in the seed coat of soybean. However, our study found
+ Value with different letters (small letter a-c) is significantly different at 5% level by DMRT
isoflavone content in the seed coat, but only very small amount.
of seed component within a variety.
Total isoflavone content (isoflavone x dry weight per single
seed) in different seed components (axis, cotyledon, and seed
coat) and between varieties (Aga3, Aga4, Pungsannamulkong,
Total isoflavone content in different parts of germinat-
Taekwangkong, Hwangkeumkong, and Cheongjakong) were
ed soybean seeds
significantly difference (P<0.01) as shown in Table 6. In each
variety, the highest total isoflavone content was in the cotyle-
As shown in Table 3, the isoflavone content of Aga3 seeds, at
don, which was 487, 232, 207, 303, 383, and 214 µg per seed for
0 days after germination, was about 6 times higher than that of
Aga3, Aga4, Pungsannamulkong, Taekwangkong, Hwangkeumkong,
Pungsannamulkong when it was measured on the basis of
and Cheongjakong, respectively. The isoflavone content of seed
weight, µg/g. Aga3 and Pungsannamulkong were sampled from
coat was the lowest, which was 4, 1, 7, 4, 2, and 3µg per seed for
1 to 10 days after germination. Samples were taken from whole
Aga3, Aga4, Pungsannamulkong, Taekwangkong, Hwangkeumkong,
sprouts and their parts such as cotyledon, root, and hypocotyl.
Germinated soybean seed components were separated after 4 to
Journal of Crop Science and Biotechnology
59

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Siviengkhek Phommalth et al.
10 days because components were not distinguishable at 3 days.
greater than the roots, respectively.
Dry weights of whole sprout and cotyledons decreased after
Total isoflavone (isoflavone _ dry weight per single sprout)
seed germination. In contrast, hypocotyls and root dry weights
content in cotyledons of Aga3 significantly increased after seed
increased in Aga3 and Pungsannamulkong. Total isoflavone
germination, whereas in the cotyledons of Pungsannamulkong it
content in germinated soybean seed components and days after
decreased. However, total isoflavone content in roots of both
germination were significantly different (P<0.01) (Table 3-6).
varieties increased while the content in hypocotyls decreased
The isoflavone content of germinated soybean seeds at differ-
after seed germination (Table 4-6).
ent time intervals showed a significant change (P<0.05). The
Whole sprouts, cotyledons, and roots of Aga3 and whole
highest isoflavone content was observed in the whole sprout on
sprouts of Pungsannamulkong were observed to reach maximum
the day 7 for Pungsannamulkong and Aga3. After 7 days, there
isoflavone content at 7 days after seed germination, which were
was a decreasing trend in isoflavone content as the germination
578, 500, 68, and 335µ g per single sprout, respectively.
period increased (Table3). Terrence (1991) observed that soy-
Whereas, the highest total isoflavone content in the cotyledons
bean primary leaf tissues underwent a programmed shift from
of Pungsannamulkong was 294µg per single sprout at 2 days
isoflavonoid to flavonoid metabolism after 3 days of germina-
after seed germination. The maximum total isoflavone content in
tion and became largely dominated by glycosides of flavonols,
roots of Pungsannamulkong was 100µ g per sprout at 9 days
kampferol, quercetin, and isorhamnetin after 5 days. Regression
after seed germination. For hypocotyls of both varieties, total
analysis showed a quadratic polynomial trend for both Aga3 and
isoflavone content increased 2-3 days after seed germination and
Pungsannamulkong. The results followed a similar trend as
then progressively declined as shown in Table 6.
reported by Kim et al. (2002), which reported that isoflavone
increased 20-50% after 24 hours of germination. Danhua et al.
Table 3. Total isoflavone content in whole sprout.
(2005) reported that isoflavone content from seeds of 3-day-old
Aga3
Pungsannamulkong
Days after
Caviness and Hutcheson varieties demonstrated 21.61% and
Dry wt Isoflavone
Total
Dry wt Isoflavone Total
germination (g)(A) (µg/g) (B) (A + B) (g)(C) (µg/g) (D) (C + D)
11.51% increases in isoflavone content, respectively. In addi-
0
0.065
7,242
471e
0.100
1,236
124j
tion, Xu et al. (2005) showed that ascorbic acid in soybeans also
1
0.064
8,451
544bcd
0.100
1,353
135i
increased during germination primarily because of its increased
2
0.062
9,085
567a
0.098
1,875
185h
3
0.060
8,918
535d
0.097
2,819
275f
biosynthesis.
4
0.058
9,806
569a
0.097
2,962
287e
At the same weight, isoflavone content in root was higher
5
0.056
10,146
568a
0.096
2,970
285e
6
0.054
10,379
564ab
0.095
3,493
331b
than cotyledon, a gradual increase 1 to 6 days after germination
7
0.054
10,788
578a
0.094
3,556
335a
in Aga3, thereafter isoflavone content in the cotyledon was high-
8
0.053
10,545
559abc
0.092
3,440
317c
er than in the root. However, isoflavone content in the root was
9
0.052
10,364
542cd
0.091
2,892
262g
10
0.051
9,400
481e
0.089
3,253
289d
higher than in the cotyledon due to a gradual increase 10 days
+ Value with different letters in the same column within sprout component is significantly
after germination for Pungsannamulkong. Hypocotyl demon-
different at 5% level by DMRT.
strated the lowest isoflavone content. Our result agree with Kim
A and C = dry weight of single sprout’s components.
et al. (2003), which found that the isoflavone content of bean
sprouts increased gradually during the cultivation period and
Table 4. Total isoflavone content in cotyledon.
were the highest in the roots, then cotyledon, and then
Aga3
Pungsannamulkong
hypocotyl.
Days after
Dry wt Isoflavone Total
Dry wt Isoflavone Total
germination
Our data indicate that isoflavone content in different parts of
(g)(A)
(µg/g) (B) (A + B)
(g)(C)
(µg/g) (D) (C + D)
soybean sprouts were varied according to genotype and date of
0
0.046
10,196
473b
0.094
2,137
201de
1
0.045
10,255
461c
0.093
3,040
284a
germination. Similarly, Lee et al. (2007b) reported that in soy-
2
0.041
11,696
475b
0.086
3,426
294a
bean sprout at 5 days after germination the roots and hypocotyls
3
0.039
10,500
409e
0.082
3,265
267b
4
0.036
12,026
435d
0.078
2,863
223c
contained the highest and lowest isoflavone levels, respectivly.
5
0.035
12,551
434d
0.066
3,419
226c
Nevertheless, some varieties had isoflavone levels higher in
6
0.033
13,329
437d
0.069
3,342
229c
cotyledon than in roots. In agreement with this observation, Kim
7
0.031
15,930
500a
0.060
3,718
225c
8
0.027
14,601
397f
0.054
3,100
167f
et al. (2004) found that isoflavone content in roots was 2.9-fold
9
0.024
13,260
318g
0.055
3,852
211d
higher than cotyledons of Junjori, at 7 days post germination.
10
0.026
9,224
244h
0.053
3,708
197e
Whereas, for Myoungjoonamulkong and Chinese black soy-
+ Value with different letters in the same column within sprout component is significantly
different at 5% level by DMRT.
beans, isoflavone content in cotyledons were 1.38 and 4.18 fold
A and C = dry weight of single sprout’s components
w w w . c r o p b i o . o r g
60

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Isoflavone Composition in Soybean Seeds
gens on bone. Nutr. Res. 17: 1617-1632
Table 5. Total isoflavone content in root.
Anthony MS, Clarkson TB, Hughes C L. 1996. Soybean
Aga3
Pungsannamulkong
Days after
Dry wt Isoflavone Total
Dry wt Isoflavone Total
isoflavones improve cardiov-ascular risk factors without
germination (g)(A) (µg/g) (B) (A + B) (g)(C) (µg/g) (D) (C + D)
affecting the reproductive system of peripubertal rhesus mon
0
0.002
20,268
44d
0.003
9,492
30h
keys. J Nutr. 126: 43-50
1
0.001
28,417
40e
0.003
10,737
28h
Cassidy A, Bingham S, Setchell KDR. 1994. Biological effects
2
0.002
21,921
44d
0.004
12,133
44ef
3
0.003
9,788
33f
0.007
7,094
52cd
of isoflavones present in soy in premenopausal women:
4
0.002
19,129
46d
0.004
9,095
35gh
Implications for the prevention of breast cancer. Am J Clin
5
0.003
13,251
37e
0.005
7,817
39fg
6
0.004
14 ,100
51c
0.006
8,209
49de
Nutr. 60: 333-340
7
0.005
12,550
68a
0.007
9,489
70b
Danhua Z, Navam SH, Ronny H, Perngyin C. 2005.
8
0.007
8,849
60b
0.011
8,691
99a
9
0.006
9,293
59b
0.011
8,739
100a
Isoflavoine contents in germinated soybean seeds. Plant Food
10
0.007
6,317
47d
0.010
5,485
57c
for Human Nutrition. 60: 147-151
+ Value with different letters in the same column within sprout component is significantly
Eldridge AC. 1982. High-performance liquid chromatography
different at 5% level by DMRT.
A and C = dry weight of single sprout’s components.
separation of soybean isoflavones and their glucosides.
J Chromatography. A. 234: 494-496
Hwang YH, Jeong YS, Lee JD. 2004 Present status and future
Table 6. Total isoflavone content in hylpocotyl.
developmental direction of soy-related industries in Korea.
Aga3
Pungsannamulkong
Days after
Korea Soybean Dig. 21: 28-44
Dry wt Isoflavone
Total
Dry wt Isoflavone Total
germination
Kennedy AR. 1995. The evidence for soybean products as can
(g)(A)
(µg/g) (B) (A + B) (g)(C)
(µg/g) (D) (C + D)
cer preventive agents. J Nutr. 125: 733-743
0
0.002
20,268
44a
0.003
9,492
30c
1
0.001
28,417
40b
0.003
10,737
28c
Kim EH, Kim SH, Chung JI, Choi HY. 2006. Analysis of phe
2
0.002
21,921
44a
0.004
12,133
44b
nolic compounds and isoflavones in soybean seeds (Glycine
3
0.003
9,788
33c
0.007
7,094
52a
4
0.003
9,038
23d
0.006
3,179
18d
max (L.) Merrill) and sprout grown under different condi
5
0.004
4,221
17e
0.008
1,484
12e
tions. Eur Food Res Technol. 222: 201-208
6
0.008
1,921
16e
0.014
606
9f
7
0.008
1,100
9g
0.016
592
9f
Kim EM, Lee KJ, Chee KM. 2004. Comparison in isoflavone
8
0.009
1,697
15e
0.018
502
9f
contents between soybean and soybean sprouts of various
9
0.009
1,068
9g
0.019
606
12e
10
0.012
856
11f
0.022
337
7g
soybean cultivars. Korean J. Nutri. 37: 45-51
+ Value with different letters in the same column within sprout component is significantly
Kim JH, Lim HA, Lee JS, Sung MK, Kim YK, Yu RN, Kim JS.
different at 5% level by DMRT.
2005. Effect of low dose of genistein and equal on protein
A and C = dry weight of single sprout’s components.
expression profile in MCF-7 cells. Food Sci. Biotechnol.
14: 854-859
Kim JS, Yeo KE, Kim JK, Lee GB, Kim WJ. 2002. Effect of
Acknowledgement
germination on isoflavone contents in soybean. 2002 Annual
meeting and Food Expo-Anaheim, Clifornia. 15E-10
This work was supported by the Ministry of Commerce,
Kim YH, Hwang YH, Lee HS. 2003. Analysis off isoflavone
Industry and Energy (code 70000385).
for 66 varieties of sprout beans and bean sprouts. Korean J.
Food Sci. Technol. 35: 568-575
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