(Zhang Et Al. 2011) Phenolic Composition and Antioxidant Activity in Seed Coats of 60 Chinese Black Soybean (Glycine Max L. Merr.) Varieties
(Zhang Et Al. 2011) Phenolic Composition and Antioxidant Activity in Seed Coats of 60 Chinese Black Soybean (Glycine Max L. Merr.) Varieties
(Zhang Et Al. 2011) Phenolic Composition and Antioxidant Activity in Seed Coats of 60 Chinese Black Soybean (Glycine Max L. Merr.) Varieties
pubs.acs.org/JAFC
ABSTRACT: Phenolics in black soybean seed coat (BSSC) are considered to be responsible for the health benefits of black
soybean. BSSCs of 60 Chinese varieties were examined for phenolic contents, anthocyanin profiles, and antioxidant activity. Total
phenolic and condensed tannin contents ranged from 512.2 to 6057.9 mg gallic acid equivalents/100 g and from 137.2 to 1741.1 mg
(þ)-catechin equivalents/100 g, respectively. Six anthocyanins (delphinidin-3-glucoside, cyanidin-3-galactoside, cyanidin-3-
glucoside, petunidin-3-glucoside, peonidin-3-glucoside, and malvidin-3-glucoside) were detected by HPLC. Total anthocyanin
contents (TAC) were from 98.8 to 2132.5 mg/100 g, and cyanidin-3-glucoside was the most abundant anthocyanin in all varieties,
with a distribution of 48.894.1% of TAC. Antioxidant properties detected by DPPH, FRAP, and ORAC methods all showed wide
variations ranging from 4.8 to 65.3 μg/100 mL (expressed as EC50), from 17.5 to 105.8 units/g, and from 42.5 to 1834.6 μmol
Trolox equivalent/g, respectively. Sixty varieties were classified into four groups by hierarchical clustering analysis, and group 4
consisting of nine varieties had the highest phytochemicals content and antioxidant activity.
KEYWORDS: black soybean, seed coat, anthocyanins, phenolics, antioxidant activity
r 2011 American Chemical Society 5935 dx.doi.org/10.1021/jf201593n | J. Agric. Food Chem. 2011, 59, 5935–5944
Journal of Agricultural and Food Chemistry ARTICLE
soybean are distributed all over China. The huge difference of HPLC Analysis of Anthocyanin Profiles. The analysis was
geographic and climatic conditions in different ecological regions performed according to a modified method of Baj et al.19 All of the
may have a great influence on phenolic profiles of black soybeans samples were analyzed on an Agilent 1200 HPLC system (Waldbronn,
cultivated there. However, very little is known about the phenolic Germany) equipped with an Agilent diode array detector and auto-
profiles and antioxidant effects of BSSC from different Chinese sampler, using a 4.6 mm 250 mm i.d., 5 μm, XBridge RP-18 column
varieties until now. Therefore, seed coats of 60 Chinese black fitted with a 4.6 mm 20 mm i.d. guard column of the same material
soybean varieties were analyzed in this study to determine the (Waters, Milford, MA).
contents of individual anthocyanins, total polyphenols, and Column temperature was maintained at 25 °C by an Agilent 1200
condensed tannins and antioxidant activity, to investigate corre- column oven. The mobile phase consisted of 10% formic acid in HPLC
lation among phenolic contents and antioxidant activity of grade water (solvent A) and methanol/acetonitrile/formic acid/water
samples tested, and to determine the black soybean varieties (25:25:10:40, v/v/v/v) (solvent B). The gradient was programmed as
with high phenolic contents and antioxidant activity. follows: 08 min, solvent B 1620%; 811 min, solvent B 2038%;
1120 min, solvent B 3846%. Other chromatographic conditions
included a constant flow rate of 1.0 mL/min, an injection volume of
’ MATERIALS AND METHODS 20 μL, and a run time of 20 min. Detection was set at 520 nm. Prior to
Chemicals. Reference standards of delphinidin-3-glucoside, cyanidin- analysis, all of the samples were filtered through a 0.25 μm membrane
3-galactoside, cyanidin-3-glucoside, petunidin-3-glucoside, peonidin-3- filter (Waters).
glucoside, and malvidin-3-glucoside were obtained from Polyphenols Identification of anthocyanins was primarily based on comparison of
Laboratories (Sandnes, Norway). Gallic acid, (þ)-catechin, 1,1-diphenyl- their retention times with known standards and consultation with the
2-picrylhydrazyl (DPPH), 2,20 -azobis(2-amidinopropane) dihydrochloride literature.6,12 Six anthocyanin compounds selected as standards were
(AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), delphinidin-3-glucoside, cyanidin-3-galactoside, cyanidin-3-glucoside,
and 30 ,60 -dihydroxyspiro[isobenzofuran-1(3H),90 -(9H)xanthene]-3-one, petunidin-3-glucoside, peonidin-3-glucoside, and malvidin-3-glucoside
disodium salt (FL), were obtained from Sigma-Aldrich Inc. (St. Louis, with >98% purity. They were used to identify and quantify anthocya-
MO). HPLC grade methanol, acetonitrile, and formic acid were nins in BSSC. All standards were dissolved in methanol to produce a
obtained from Fisher (Suwanee, GA). stock solution of 1.0 mg/mL. A portion of each stock solution was then
Soybean Materials. Sixty varieties of black soybeans (G. max L. diluted using acidified methanol into 1, 10, 50, 100, 200, and 500 μg/mL
Merr.) originating in different parts of China were donated by the of working solutions. Standard curves of anthocyanins were gener-
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences ated by injecting 0.021 μg of six anthocyanins in 20 μL of acidified
(Beijing, China). All varieties were grown in the experimental station of methanol. All anthocyanin standard solutions exhibited a linear
the Institute of Crop Germplasm Resources, Shanxi Academy of relationship within that range by plotting area response and injected
Agricultural Sciences, during MayOctober in 2008. Soybeans were amounts. The regression coefficients ranged from 0.9987 to 0.9999 for
air-dried and then stored at 20 °C in the dark until used. Seed coats a mixture of pure anthocyanins separated on the C18 column.
were freeze-dried using a Labconco FreeZone system (Kansas City, Anthocyanin contents were expressed as milligrams of anthocyanin
MO). All data were reported as the mean ( standard deviation (SD) of per 100 g of sample (mg/100 g). Spike recovery ranged from 92.6 to
experiments run in triplicate. Phenolic contents and antioxidant activity 96.3%. The lower detection limit for all anthocyanin standards was
were expressed on the basis of dry weight (DW) of BSSC. <20 ng/mL.
Extraction of Phenolic Compounds. Dried seed coats were DPPH Radical Scavenging Activity. DPPH scavenging capa-
ground into fine powder through a 60 mesh sieve. Phenolics were cities of BSSCE were evaluated by modification of previously reported
extracted using methods previously described16 and optimized in our methods.20 Briefly, 2.0 mL of serially diluted BSSCE solutions with 60%
laboratory. Briefly, 500 mg of powder was soaked in 10 mL of petroleum acidified methanol were added to 2.0 mL of 200 μmol/L of DPPH
ether for 24 h at room temperature to remove lipids. Degreased powder methanol solution. The absorbance at 517 nm was recorded after 30 min
was extracted twice by suspending in 10 mL of 60% acidified methanol of incubation in the dark at room temperature. The radical scavenging
extraction solvent (pH 2.5) for 2 h in a water bath shaker at 40 °C. capacity of each solution was calculated as the percent DPPH radical
Extracts were centrifuged at 2000g for 10 min, two supernatant aliquots scavenging effect: DPPH scavenging (%) = [1 (Abssample Absblank)/
were pooled to produce black soybean seed coat extract (BSSCE), and Abscontrol]M 100%, where methanol (2.0 mL) plus BSSCE (2.0 mL)
the extract was stored in the dark at 20 °C until analyzed. was used as a blank and DPPH solution (2.0 mL) plus methanol
Determination of Total Phenolic Content (TPC). TPC was (2.0 mL) was used as a negative control. The median effective
determined using a modified FolinCiocalteu assay.17 Briefly, 1.0 mL of concentration (EC50) value of each sample represents the concentration
BSSCE and FolinCiocalteu reagent were well mixed. Then, 2.0 mL of of samples at which 50% DPPH scavenging effect was obtained. EC50
10% Na2CO3 aqueous solution was added. The mixture was kept for 1 h was calculated by constructing the percentage of DPPH scavenging
at room temperature before measurement at 765 nm using a Shimadzu versus log(extract concentration expressed as BSSC weight) curves
UV-1800 UVvis spectrophotometer (Tokyo, Japan). The measure- (μg/100 mL).
ment was compared to a standard curve of prepared gallic acid solutions Ferric Reducing Antioxidant Power (FRAP) assay. A FRAP
and expressed as milligrams of gallic acid equivalents per 100 g of sample assay was performed according to the manufacturer's instruction by
(mg GAE/100 g). using a commercially available FRAP kit from Nanjing Jiancheng
Determination of Condensed Tannins Content (CTC). Bioengineering Institute (Nanjing, China). The FRAP value was expressed
Analysis of CTC was carried out according to a modified previous as the increased absorbance value of the reaction solution in 1 min by 1 g
method.18 To 0.5 mL of suitably diluted sample using methanol were (DW) of black soybean seed coat in the reaction system (units/g).
added 2.5 mL of 30 g/L aqueous vanillin solution and 2.5 mL of 30% Oxygen Radical Absorbance Capacity (ORAC) Assay. The
H2SO4 methanol solution. The mixture was kept for 20 min in the dark ORAC assay was conducted according to a previous method.21 Twenty
at 30 °C, and absorbance was measured at 500 nm against methanol as a microliters of blank, Trolox standard, or appropriately diluted BSSCE in
blank. Condensed tannin was calculated and expressed as milligrams of 75 mmol/L potassium phosphate buffer, pH 7.4 (working buffer), was
(þ)-catechin equivalents per 100 g of sample (mg CAE/100 g) using the added to a black clear-bottom 96-well microplate in triplicate. A volume
calibration curve of (þ)-catechin. of 200 μL of 0.96 μmol/L FL in working buffer was added to each well
Table 1. Total Phenolic Contents and Condensed Tannins Contents in Seed Coats of 60 Black Soybean Varietiesa
code variety total phenolic content (mg GAE/100 g)b condensed tannins content (mg CAE/100 g)c
Table 1. Continued
code variety total phenolic content (mg GAE/100 g)b condensed tannins content (mg CAE/100 g)c
55 Z09178 Daheidou 1884.6 ( 8.9 465.1 ( 24.2
56 Z09211 Chidingdou 1986.8 ( 18.6 461.1 ( 9.4
57 Z09201 Heidou 3111.5 ( 74.4 390.0 ( 12.6
58 Z09181 Zadou 1333.1 ( 5.4 342.6 ( 21.5
59 Z09204 Xiaoyoudou 4418.9 ( 23.4 1267.0 ( 41.6
60 Z17539 Xiaoheidou 3334.6 ( 112.7 804.8 ( 31.4
anthocyanin (mg/100 g)
1 Z09185 Gangbiandou 21.4 ( 0.7 5.6 ( 0.6 424.4 ( 43.2 38.4 ( 12.9 36.4 ( 12.8 21.6 ( 7.8 547.8 ( 64.3
2 Z17513 Heidou 92.0 ( 5.0 6.1 ( 0.6 591.1 ( 48.8 71.6 ( 15.1 19.1 ( 10.8 24.2 ( 6.5 804.1 ( 36.9
3 Z09173 Xiaoheidou 37.5 ( 1.1 8.6 ( 0.4 747.7 ( 23.6 70.2 ( 16.9 64.8 ( 15.0 30.5 ( 7.1 959.3 ( 6.7
4 Z09202 Heidou 6.7 ( 0.5 NDb 157.8 ( 19.4 9.3 ( 1.8 15.3 ( 5.2 7.5 ( 4.3 196.5 ( 19.1
5 Z09176 Daheidou ND 5.8 ( 0.4 614.9 ( 47.6 9.7 ( 0.8 22.8 ( 7.6 ND 653.2 ( 40.7
6 Z17501 Heidou ND 7.5 ( 0.2 1049.3 ( 49.3 3.9 ( 0.9 118.5 ( 14.1 ND 1179.2 ( 57.7
7 Z07161 Heijinyuan ND 6.0 ( 0.5 576.5 ( 65.4 17.1 ( 4.0 33.1 ( 10.2 ND 632.7 ( 79.7
8 Z17532 Heidou 212.9 ( 3.0 7.6 ( 0.3 782.6 ( 50.8 97.1 ( 16.6 19.6 ( 6.8 14.8 ( 3.9 1134.8 ( 58.5
9 Z09190 Biangandou 32.2 ( 0.5 6.2 ( 0.3 600.4 ( 46.1 54.3 ( 7.0 55.7 ( 15.5 26.3 ( 7.8 775.1 ( 62.3
10 Z17504 Heidou 228.1 ( 7.6 5.8 ( 0.2 438.4 ( 37.3 77.3 ( 12.3 16.4 ( 4.6 10.3 ( 2.8 776.3 ( 34.7
11 Z17494 Heidou 166.3 ( 3.3 13.3 ( 0.5 894.2 ( 68.6 85.6 ( 13.0 33.5 ( 8.7 27.7 ( 4.0 1220.6 ( 65.6
12 Z17508 Xiaoheidou 321.9 ( 2.8 11.6 ( 0.4 1058.7 ( 68.6 134.3 ( 19.5 29.6 ( 9.8 19.5 ( 7.0 1575.7 ( 82.9
13 Z17507 Xiheidou 213.9 ( 5.3 16.9 ( 0.4 1336.3 ( 76.1 149.0 ( 16.4 51.7 ( 12.2 47.8 ( 11.9 1815.6 ( 112.5
14 Z17496 Heidou 130.1 ( 2.9 7.1 ( 0.2 726.0 ( 40.3 113.0 ( 13.1 17.3 ( 3.5 31.3 ( 5.6 1024.8 ( 59.2
15 Z17492 Xiaoheidou 168.7 ( 7.9 4.4 ( 0.4 339.3 ( 38.9 66.1 ( 13.0 13.3 ( 3.7 9.5 ( 2.8 601.3 ( 39.5
16 Z17530 Heizaodou 98.9 ( 8.5 3.7 ( 0.2 303.9 ( 36.0 41.1 ( 5.9 11.2 ( 2.7 8.7 ( 2.4 467.6 ( 53.9
17 Z09214 Daheirangheidou 40.1 ( 3.4 7.4 ( 0.4 770.4 ( 58.2 62.1 ( 7.4 55.1 ( 10.7 24.9 ( 6.0 959.9 ( 38.7
18 Z09210 49.2 ( 1.7 4.9 ( 0.3 394.0 ( 43.8 58.9 ( 9.3 24.1 ( 4.9 31.6 ( 6.9 562.7 ( 36.1
Yaosanxiaodaheidou
19 Z17533 Heidou 233.0 ( 4.5 8.8 ( 0.3 663.8 ( 34.8 99.5 ( 13.4 18.1 ( 5.1 15.2 ( 2.9 1038.4 ( 9.4
20 Z07177 Fangzhengheidou 90.5 ( 1.0 4.0 ( 0.3 380.4 ( 34.1 49.3 ( 8.6 14.6 ( 4.2 6.7 ( 2.2 545.6 ( 28.5
21 Z09195 Daheidou 30.9 ( 1.3 ND 203.1 ( 27.4 50.5 ( 6.5 20.7 ( 6.5 31.5 ( 5.4 336.8 ( 21.0
22 Z09172 Xiaoheidou ND ND 106.6 ( 16.4 13.6 ( 3.7 18.4 ( 5.3 10.0 ( 2.7 148.7 ( 26.2
23 Z09215 Xiaoheidou 35.6 ( 3.2 5.5 ( 0.3 524.8 ( 17.7 57.5 ( 7.2 44.7 ( 11.2 22.5 ( 5.7 690.7 ( 6.6
24 Z09175 Xiaoheidou 13.6 ( 0.9 4.9 ( 0.3 495.5 ( 39.6 20.2 ( 4.5 23.0 ( 5.5 8.7 ( 3.0 565.9 ( 34.9
25 Z09183 Guangbiandou 17.6 ( 1.1 6.5 ( 0.3 564.4 ( 37.8 41.4 ( 4.7 41.4 ( 5.7 19.6 ( 4.3 690.9 ( 33.9
26 Z17538 Daheidou 170.5 ( 10.2 8.0 ( 0.5 742.3 ( 40.0 64.0 ( 8.5 20.2 ( 5.4 10.7 ( 3.1 1015.8 ( 47.1
27 Z17537 Heidou 93.4 ( 3.1 ND 312.0 ( 22.2 68.4 ( 5.6 6.0 ( 2.2 15.8 ( 4.8 495.6 ( 30.8
28 Z17502 Heihuangdou 233.1 ( 13.7 7.1 ( 0.4 949.7 ( 55.8 161.7 ( 18.3 32.3 ( 8.6 42.0 ( 7.0 1425.9 ( 56.4
29 Z09174 Xiaoheidou 21.6 ( 0.8 3.5 ( 0.3 292.4 ( 34.0 35.0 ( 4.9 24.7 ( 5.6 19.0 ( 5.9 396.2 ( 30.3
30 Z17510 Xiaoheidou 1.8 ( 0.1 7.7 ( 0.3 974.8 ( 83.7 3.8 ( 0.9 44.4 ( 8.0 4.9 ( 1.4 1037.5 ( 75.6
31 Z09182 Yangyanjing 15.0 ( 1.2 3.7 ( 0.2 298.4 ( 34.2 23.2 ( 4.4 24.4 ( 6.9 14.5 ( 3.5 379.2 ( 45.6
32 Z09199 Xiaoheidou 32.3 ( 1.3 3.3 ( 0.2 221.3 ( 25.4 40.1 ( 5.1 16.4 ( 4.4 15.2 ( 3.3 328.6 ( 17.9
33 Z17515 Heidou 139.9 ( 1.0 9.2 ( 0.6 784.1 ( 17.5 119.3 ( 18.3 27.5 ( 6.4 36.5 ( 6.0 1116.6 ( 15.6
34 Z09196 Xiaoheidou 22.5 ( 3.7 ND 279.8 ( 21.1 36.3 ( 5.9 28.7 ( 7.1 17.9 ( 5.6 385.2 ( 22.4
35 Z17525 Huangheidou 269.7 ( 18.3 6.1 ( 0.5 752.5 ( 44.3 125.7 ( 17.4 31.2 ( 7.1 15.3 ( 3.6 1200.3 ( 57.7
Table 2. Continued
anthocyanin (mg/100 g)
mean 94.5 ( 96.8 6.0 ( 6.2 556.5 ( 317.7 61.9 ( 41.4 31.8 ( 30.0 19.1 ( 17.1 769.7 ( 425.8
LSD0.05 7.7 0.8 78.8 18.7 13.5 8. 7 84.4
a
Data are expressed as the mean ( SD (n = 3) on a dry weight basis. b Not detectable.
antioxidant activityb
code variety FRAP (units/g) DPPH scavenging EC50 (μg/100 mL) ORAC (μmol TE/g)
Table 3. Continued
antioxidant activityb
code variety FRAP (units/g) DPPH scavenging EC50 (μg/100 mL) ORAC (μmol TE/g)
53 Z09194 Daheiyang 91.7 ( 1.6 4.8 ( 0.5 1834.6 ( 97.3
54 Z17540 Xiaoheidou 53.9 ( 0.7 27.6 ( 2.3 570.9 ( 26.7
55 Z09178 Daheidou 68.6 ( 2.5 18.6 ( 1.7 620.0 ( 32.1
56 Z09211 Chidingdou 60.5 ( 3.3 20.3 ( 1.2 674.1 ( 38.2
57 Z09201 Heidou 42.0 ( 2.8 18.7 ( 1.4 969.4 ( 52.4
58 Z09181 Zadou 30.6 ( 0.6 29.5 ( 2.7 413.1 ( 29.9
59 Z09204 Xiaoyoudou 60.5 ( 5.3 7.3 ( 0.8 1306.9 ( 66.7
60 Z17539 Xiaoheidou 74.5 ( 1.1 11.3 ( 1.2 1059.9 ( 44.3
Table 4. Phenolic Content and Antioxidant Activity of Black Soybean Seed Coats in Four Groups Classified by Hierarchical
Clustering Method and the Soybean Varieties Included in Each Groupa
TPC CTC TAC FRAP DPPH scavenging ORAC
group variety codes (mg GAE/100 g) (mg CAE/100 g) (mg/100 g) (units/g) EC50 (μg/100 mL) (μmol TE/g)
1 2, 4, 10, 15, 16, 21, 22, 27, 37, 1140.6 ( 347.9 a 246.7 ( 84.3 a 393.6 ( 228 a 30.8 ( 7.4 a 38.5 ( 10.8 a 424.0 ( 112.4 a
41, 43, 47, 48, 52, 58 (n = 15)
2 5, 8, 19, 26, 29, 30, 31, 32, 34, 38, 1765.4 ( 232.3 b 427.2 ( 98.5 b 704.7 ( 325.0 b 47.6 ( 12.8 b 21.7 ( 3.2 b 592.9 ( 66.0 b
42, 44, 45, 46, 54, 55, 56 (n = 17)
3 1, 7, 11, 12, 13, 18, 23, 24, 25, 28, 33, 2946.8 ( 527.0 c 671.0 ( 190.6 c 988.7 ( 398.8 c 64.3 ( 12.0 c 13.8 ( 3.4 c 901.4 ( 125.4 c
35, 36, 39, 40, 49, 51, 57, 60 (n = 19)
4 3, 6, 9, 14, 17, 20, 50, 53, 59 (n = 9) 4256.8 ( 797.8 d 1254.1 ( 221.2 d 1056.8 ( 444.1 c 78.2 ( 14.0 d 9.7 ( 3.0 d 1274.0 ( 246.5 d
a
Data are expressed as the mean ( SD on a dry weight basis of seed coats. Means in a row without a common letter are statistically different (p < 0.05).
TPC, total phenolic content; CTC, condensed tannins content; TAC, total anthocyanin content; FRAP, ferric reducing antioxidant power; DPPH
scavenging EC50, concentration of extract expressed as the dry weight of black soybean seed coat for 50% of DPPH scavenging; ORAC, oxygen radical
absorbing capacity.
Hierarchical Clustering Analysis of Black Soybean Vari- and that anthocyanins in BSSC displayed many health benefits.
eties. Sixty black soybean varieties were classified into four Therefore, our study chiefly focused on identifying the profiles of
groups by hierarchical clustering analysis with TAC, TPC, anthocyanins in various varieties. Six anthocyanins, including
CTC, and antioxidant properties of seed coat as variables. delphinidin-3-glucoside, cyanidin-3-galactoside, cyanidin-3-glu-
Detailed information of each group is presented in Table 4. coside, petunidin-3-glucoside, peonidin-3-glucoside, and malvi-
There were 15, 17, 19, and 9 black soybean varieties included in din-3-glucoside, were identified in this study. Anthocyanin
groups 14, respectively. Phenolic contents and antioxidant profiles of black soybeans reported in previous studies were
activity by all methods significantly increased from group 1 highly distinct. Among 10 Korean black soybean varieties, only
through 4 (p < 0.05), except that average TACs in groups 3 cyanidin-3-glucoside was detected in 2 varieties, whereas delphi-
and 4 were similar (p > 0.05). Group 4, consisting of Z09173 nidin-3-glucoside or petunidin-3-glucoside or both of them were
Xiaoheidou, Z17501 Heidou, Z09190 Biangandou, Z17496 also found in the other 8 varieties.13 Cyanidin-3-glucoside and
Heidou, Z09214 Daheirangheidou, Z07177 Fangzhengheidou, peonidin-3-glucoside were found by Xu et al. in 2 black soybean
Z09212 Bawangbian, Z09194 Daheiyang, and Z09204 Xiaoyou- varieties grown in the North DakotaMinnesota region and
dou, had higher levels of polyphenols and antioxidant capacities petunidin-3-glucoside, as well, was found in a commercially
compared to the other groups. available American variety.3,14 However, as many as 10 antho-
cyanins, consisting of 6 aglycones and 4 glycosides, were
identified in 1 of 5 varieties in another study.6 Lee et al.12 claimed
’ DISCUSSION the presence of 9 anthocyanins in black soybean, among
Anthocyanins are the second most important phytochemicals which catechin-cyanidin-3-glucoside, pelargonidin-3-glucoside,
in black soybean besides isoflavones. It has been shown that delphinidin-3-galactoside, and cyanidin were not detected in this
anthocyanins predominantly account for the difference in poly- study. Five of 6 anthocyanins detected in our experiment, except
phenols between black soybeans and other soybean cultivars4,22 malvidin-3-glucoside, were also found by them. Cyanidin-3-
5942 dx.doi.org/10.1021/jf201593n |J. Agric. Food Chem. 2011, 59, 5935–5944
Journal of Agricultural and Food Chemistry ARTICLE
galactoside was detected in most samples (48 varieties) despite together with FRAP and DPPH methods using the SET reaction
trace concentrations in this research. Only Lee et al.12 previously mechanism were utilized simultaneously in this study to estimate
reported its presence in black soybean. Varietal discrepancies of the antioxidant properties of BSSC. Correlation analysis indi-
anthocyanin profiles in these studies possibly contribute to the cated that all antioxidant assay methods were well-correlated,
difference in black soybean varieties subject to analysis and to which means these methods have good consistency in the
different sensitivities of methods by which anthocyanins were estimation of antioxidant activity of BSSC. It is difficult to
separated and characterized. compare our values of FRAP and DPPH with those reported
As revealed in previous studies, TAC in BSSC exhibited broad previously because different substrate concentrations and end
ranges of variation from <1.0 to 20.4 mg/g.3,14,15 Wide variation points were used.6,14 Using Trolox as a standard in the ORAC
was also found in our current study from 98.8 to 2135.2 mg/100 g. method makes it possible to compare the values obtained in
The highest TAC here is close to the highest content reported in different laboratories with each other. To the best of our knowl-
Japanese (20.4 mg/g)15 and Korean (20.18 mg/g)13 varieties. edge, only Xu et al.3 determined the antioxidant activity of BSSC
Three Japanese black soybeans analyzed in Yoshida’s research15 by the ORAC method and reported an ORAC value of approxi-
all exhibited high TAC (20.4, 16.9, and 15.3 mg/g). In contrast, mately 450 μmol TE/g in a commercially available American
Choung et al.13 found a much higher range of 1.5820.18 mg/ black soybean variety. Fifty-one of 60 analyzed varieties in this
100 g in 10 varieties, which is close to our data. Soybeans tested in study showed >450 μmol TE/g of antioxidant activity by ORAC,
other studies usually contained <5.0 mg/100 g of total which indicated most varieties in our study possessed higher
anthocyanin.3,6,14 In our current and the majority of previous antioxidant potential than this variety.
studies, cyanidin-3-glucoside was the predominant anthocyanin The correlation results between phenolic contents and anti-
in black soybean.3,6,15 Therefore, the high TAC in some black oxidant activities revealed that TPC, TAC, and CTC were well-
soybean varieties, to a large extent, was attributed to their high correlated with overall antioxidant activity of BSSC. This in-
cyanidin-3-glucoside content. Z09194 Daheiyang (1617.4 mg/ dicated that anthocyanins and condensed tannins were major
100 g) analyzed in this study contained more cyanidin-3-gluco- antioxidants in BSSC. Anthocyanins showed distinct antioxidant
side than all previously reported varieties with the exception of a capacity in many studies in vivo and in vitro;26,27 meanwhile, they
variety cultivated in Japan in Yoshida’s research.15 No other were the main components contributing to the many effects of
anthocyanins but 20.4 mg/g cyanidin-3-glucoside were detected black soybeans based on antioxidant activity. Condensed tannins
in this Japanese variety. have been proven to be antioxidative in many studies.28 As
In addition to anthocyanins, highly polymerized proantho- mentioned above, there were other minor phenolic compounds,
cyanidins, which are also known as condensed tannins, were such as phenolic acids, existing but not identified in BSSC in this
reported to be an important class of phenolics in soybean seed study.3
coat.23 Therefore, CTC and TPC were measured in this study. Grape seed and many other berry seeds have been used as
To our best knowledge, CTC and TPC were determined in valuable sources of antioxidants in nutraceutical production
whole black soybean but not seed coat in most previous studies.24 because of their plentiful phytochemicals and high antioxidant
However, in a study conducted by Xu et al.,3 they compared the activity. A study showed that Chardonnay grape seed flour
phenolic distribution in various black soybean seed parts and displayed the strongest antioxidant capacity (1076.4 μmol TE/g)
measured about 70 mg GAE/g of TPC and 50 mg CAE/g of by ORAC in tested fruit seed flours of raspberry, blueberry,
CTC. These values are even higher than the highest TPC cranberry, and grapes.29 According to clustering analysis results,
(6057.9 mg GAE/100 g) and CTC (1741.1 mg CAE/100 g) nine varieties with the highest phenolic contents and antioxidant
in the varieties analyzed in this study. In addition to varietal potential comprised one group (group 4) in this study. It is
differences, another possible reason why lower CTC and TPC noticeable that seven varieties in this group showed higher
but much higher TAC was determined in our varieties is that our antioxidant activity than the above-mentioned grape seed. These
extraction conditions were different from theirs. The structure of valuable properties make some black soybean varieties potential
condensed tannins was not characterized in this study, whereas sources for nutraceutical development. This study also provides
Todd et al.25 pointed out that procyanidin was the major subclass useful information for black soybean breeders. Varieties in group
of proanthocyanidin in BSSC. Takahata et al.23 demonstrated 4 can be used as parents for breeding new black soybean varieties
that the degree of polymerization (DP) of proanthocyanidins in with high bioactivity as well as high nutrition. Additionally, with
the brown or black soybean seed coat was as high as 30. It is increasingly more attention paid to food safety and health,
presently not clear whether there are differences in DP and consumers have many more concerns about the potential adverse
subclass of proanthocyanidins in diverse black soybean varieties. effects of synthetic food colorants than they ever had. Antho-
Significant positive correlation existed among TPC, TAC, and cyanins are popular natural colorants due to their vivid colors and
CTC, which indicates that both anthocyanins and condensed health benefits. TAC values in half of the tested black soybean
tannins are the main polyphenols in BSSC. Additional phenolic varieties are even higher than those in blueberries (7.2 ( 0.5 mg/g
compounds detected in BSSC in previous research such as DW),30 which have been considered a high-anthocyanin-contain-
phenolic acids and isoflavones3,6,14 also contributed to TPC. ing fruit. These black soybean varieties can be used to extract
Antioxidant activity is the basic bioactivity of phenolic com- anthocyanins for nutraceutical development or food colorants.
pounds and the main mechanism by which they exert health In conclusion, 6 anthocyanins including cyanidin-3-glucoside,
promotion effects. It is commonly known that antioxidants can etc. were detected in 60 black soybean varieties cultivated in
scavenge radicals by two major mechanisms: hydrogen atom China. Anthocyanin profiles, phenolic contents, and antioxidant
transfer (HAT) and single electron transfer (SET). Because no properties largely varied among tested varieties. Significantly
single present antioxidant activity assay is able to accurately correlative relationships existed between phenolic contents and
reflect all radical sources or all antioxidants in a mixed or complex antioxidant activity determined by FRAP, DPPH, and ORAC.
system, the ORAC method using a HAT reaction mechanism Clustering analysis classified nine varieties into one group with
5943 dx.doi.org/10.1021/jf201593n |J. Agric. Food Chem. 2011, 59, 5935–5944
Journal of Agricultural and Food Chemistry ARTICLE
more phytochemicals and stronger antioxidant capacities than damages incurred by ischemia and reperfusion in vivo. FEBS Lett. 2006,
the other three groups. These varieties have the potential to be 580, 1391–1397.
used in functional foods and black soybean breeding as well as (12) Lee, J.; Kang, N.; Shin, S.; Shin, S.; Lim, S.; Suh, D.; Baek, I.; Park,
food colorant development. K.; Ha, T. Characterisation of anthocyanins in the black soybean (Glycine
max L.) by HPLC-DAD-ESI/MS analysis. Food Chem. 2009, 112, 226–231.
(13) Choung, M. G.; Baek, I. Y.; Kang, S. T.; Han, W. Y.; Shin, D. C.;
’ AUTHOR INFORMATION Moon, H. P.; Kang, K. H. Isolation and determination of anthocyanins in
Corresponding Author seed coats of black soybean (Glycine max (L.) merr.). J. Agric. Food Chem.
*Phone: þ86-20-87237865. Fax: þ86-20-87236354. E-mail: 2001, 49, 5848–5851.
(14) Xu, B.; Chang, S. K. Characterization of phenolic substances
mwzhh@vip.tom.com. and antioxidant properties of food soybeans grown in the North
Funding Sources DakotaMinnesota region. J. Agric. Food Chem. 2008, 56, 9102–9113.
This work was supported by grants from the National Natural (15) Yoshida, K.; Sato, Y.; Okuno, R.; Kameda, K.; Isobe, M.;
Kondo, T. Structural analysis and measurement of anthocyanins from
Science Foundation of China (30700496) and the Natural Science
colored seed coats of Vigna, Phaseolus, and Glycine legumes. Biosci.,
Foundation of Guangdong Province of China (06025364). Biotechnol., Biochem. 1996, 60, 589–593.
(16) Chirinos, R.; Rogez, H.; Campos, D.; Pedreschi, R.; Larondelle,
’ ABBREVIATIONS USED Y. Optimization of extraction conditions of antioxidant phenolic com-
pounds from mashua (Tropaeolum tuberosum Ruíz & Pavon) tubers. Sep.
BSSC, black soybean seed coat; BSSCE, black soybean seed coat Sci. Technol. 2007, 55, 217–225.
extract; TPC, total phenolic content; CTC, condensed tannins (17) Ainsworth, E.; Gillespie, K. Estimation of total phenolic content
content; TAC, total anthocyanin content; DPPH, 2-diphenyl- and other oxidation substrates in plant tissues using FolinCiocalteu
1-picrylhydrazyl radical; FRAP, ferric reducing antioxidant reagent. Nat. Protoc. 2007, 2, 875–877.
power; ORAC, oxygen radical absorbing capacity; AAPH, 2,20 - (18) Zhang, L.; Mou, D.; Du, Y. Procyanidins: extraction and micro-
azobis(2-amidinopropane) dihydrochloride; AUC, area under encapsulation. J. Sci. Food Agric. 2007, 87, 2192–2197.
the curve; DP, degree of polymerization. (19) Baj, A.; Bombardelli, E.; Gabetta, B.; Martinelli, E. M. Qualita-
tive and quantitative-evaluation of Vaccinium myrtillus anthocyanins by
high-resolution gas-chromatography and high-performance liquid-chro-
’ REFERENCES matography. J. Chromatogr., A 1983, 279, 365–372.
(1) Omoni, A. O.; Aluko, R. E. Soybean foods and their benefits: (20) Sharma, O. P.; Bhat, T. K. DPPH antioxidant assay revisited.
potential mechanisms of action. Nutr. Rev. 2005, 63, 272–283. Food Chem. 2009, 113, 1202–1205.
(2) Xiao, C. W. Health effects of soy protein and isoflavones in (21) Wolfe, K. L.; Liu, R. H. Structureactivity relationships of
humans. J. Nutr. 2008, 138, 1244S–1249S. flavonoids in the cellular antioxidant activity assay. J. Agric. Food Chem.
(3) Xu, B.; Chang, S. K. Antioxidant capacity of seed coat, dehulled 2008, 56, 8404–8411.
bean, and whole black soybeans in relation to their distributions of total (22) Xu, B. J.; Chang, S. K. C. Total phenolics, phenolic acids,
phenolics, phenolic acids, anthocyanins, and isoflavones. J. Agric. Food isoflavones, and anthocyanins and antioxidant properties of yellow and
Chem. 2008, 56, 8365–8373. black soybeans as affected by thermal processing. J. Agric. Food Chem.
(4) Takahashi, R.; Ohmori, R.; Kiyose, C.; Momiyama, Y.; Ohsuzu, F.; 2008, 56, 7165–7175.
Kondo, K. Antioxidant activities of black and yellow soybeans against low (23) Takahata, Y.; Ohnishi-Kameyama, M.; Furuta, S.; Takahashi,
density lipoprotein oxidation. J. Agric. Food Chem. 2005, 53, 4578–4582. M.; Suda, I. Highly polymerized procyanidins in brown soybean seed
(5) Slavin, M.; Kenworthy, W.; Yu, L. L. Antioxidant properties, coat with a high radical-scavenging activity. J. Agric. Food Chem. 2001,
phytochemical composition, and antiproliferative activity of Maryland- 49, 5843–5847.
grown soybeans with colored seed coats. J. Agric. Food Chem. 2009, (24) Xu, B. J.; Chang, S. K. A comparative study on phenolic profiles
57, 11174–11185. and antioxidant activities of legumes as affected by extraction solvents.
(6) Kim, J. A.; Jung, W. S.; Chun, S. C.; Yu, C. Y.; Ma, K. H.; Gwag, J. Food Sci. 2007, 72, S159–S166.
J. G.; Chung, I. M. A correlation between the level of phenolic (25) Todd, J. J.; Vodkin, L. O. Pigmented soybean (Glycine max)
compounds and the antioxidant capacity in cooked-with-rice and seed coats accumulate proanthocyanidins during development. Plant
vegetable soybean (Glycine max L.) varieties. Eur. Food Res. Technol. Physiol. 1993, 102, 663–670.
2006, 224, 259–270. (26) Mauray, A.; Felgines, C.; Morand, C.; Mazur, A.; Scalbert, A.;
(7) Hertog, M. G.; Feskens, E. J.; Hollman, P. C.; Katan, M. B.; Milenkovic, D. Bilberry anthocyanin-rich extract alters expression of
Kromhout, D. Dietary antioxidant flavonoids and risk of coronary heart genes related to atherosclerosis development in aorta of apo E-deficient
disease: the Zutphen Elderly Study. Lancet 1993, 342, 1007–1011. mice. Nutr. Metab. Cardiovasc. Dis. 2010. (doi:10.1016/j.numecd.2010.04.011).
(8) Wu, A. H.; Yu, M. C.; Tseng, C. C.; Hankin, J.; Pike, M. C. Green (27) Kahkonen, M. P.; Heinonen, M. Antioxidant activity of antho-
tea and risk of breast cancer in Asian Americans. Int. J. Cancer 2003, cyanins and their aglycons. J. Agric. Food Chem. 2003, 51, 628–633.
106, 574–579. (28) Amarowicz, R.; Naczk, M.; Zadernowski, R.; Shahidi, F. Anti-
(9) Kwon, S. H.; Ahn, I. S.; Kim, S. O.; Kong, C. S.; Chung, H. Y.; Do, oxidant activity of condensed tannins of beach pea, canola hulls, evening
M. S.; Park, K. Y. Anti-obesity and hypolipidemic effects of black primrose, and faba bean. J. Food Lipids 2000, 7, 195–205.
soybean anthocyanins. J. Med. Food 2007, 10, 552–556. (29) Parry, J.; Su, L.; Moore, J.; Cheng, Z.; Luther, M.; Rao, J.; Wang, J.;
(10) Tsoyi, K.; Park, H. B.; Kim, Y. M.; Chung, J. I.; Shin, S. C.; Lee, Yu, L. Chemical compositions, antioxidant capacities, and antiproliferative
W. S.; Seo, H. G.; Lee, J. H.; Chang, K. C.; Kim, H. J. Anthocyanins from activities of selected fruit seed flours. J. Agric. Food Chem. 2006, 54, 3773–3778.
black soybean seed coats inhibit UVB-induced inflammatory cyloox- (30) Lohachoompol, V.; Srzednicki, G.; Craske, J. The change of
ygenase-2 gene expression and PGE2 production through regulation of total anthocyanins in blueberries and their antioxidant effect after drying
the nuclear factor-kB and phosphatidylinositol 3-kinase/Akt pathway. and freezing. J. Biomed. Biotechnol. 2004, 2004, 248–252.
J. Agric. Food Chem. 2008, 56, 8969–8974.
(11) Kim, H. J.; Tsoy, I.; Park, J. M.; Chung, J. I.; Shin, S. C.; Chang,
K. C. Anthocyanins from soybean seed coat inhibit the expression of
TNF-alpha-induced genes associated with ischemia/reperfusion in en-
dothelial cell by NF-kB-dependent pathway and reduce rat myocardial