Eur Food Res Technol ( 2006 ) 224:259–270 DOI 10.1007/s00217-006-0377-y
ORIGINAL PAPER
A correlation between the level of phenolic compounds and the antioxidant capacity in cooked-with-rice and vegetable soybean (Glycine max L.) varieties J. A. Kim · W. S. Jung · S. C. Chun · C. Y. Yu · K. H. Ma · J. G. Gwag · I. M. Chung
Received: 2 January 2006 / Revised: 25 April 2006 / Accepted: 11 May 2006 / Published online: 1 September 2006 C Springer-Verlag 2006
Abstract This study was undertaken to determine the content of phenolic compounds contained in embryo, cotyledon and seed coat of nine soybean (Glycine max L.) varieties. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity was also measured to determine the correlation between the level of phenolic compounds and antioxidant capacity. A total of 10 anthocyanin constituents and 21 phenolic compounds was detected and quantified. In all nine varieties, the seed coat and cotyledon had the highest and lowest levels of phenolic compounds and anthocyanins, respectively despite the fact that all of them showed a wide variation in total amounts of phenolic compounds and anthocyanins in seed coat, embryo, and cotyledon. The seed J. A. Kim, W. S. Jung, and S. C. Chun are equally contributed to this work. J. A. Kim · W. S. Jung · I. M. Chung () Department of Applied Life Science, KonKuk University, Seoul 143-701, Korea e-mail:
[email protected] Tel.: +82-2-450-3730 Fax: +82-2-446-7856 S. C. Chun Department of Molecular Biotechnology, KonKuk University, Seoul 143-701, Korea C. Y. Yu Department of Bioresources Technology, Kangwon National University, Chunchon 200-701, Korea K. H. Ma · J. G. Gwag Bio-Resource Research, National Institute of Agricultural Biotechnology, Suwon 441-707, Korea
coat tissue, but not other seed parts, showed a strong correlation between the seed coat color and the content of both phenolic compounds and anthocyanins. The brown and black soybean seed coat contained much higher levels of phenolic compounds and anthocyanins in seed coat tissue than the yellow or green coat soybean. Among the individual phenolic compounds, syringic acid (214 µg g−1 ) and chlorogenic acid (31 µg g−1 ) were highest in seed coat and embryo, respectively. Myricetin was highest both in whole seed (16.7 µg g−1 ) and cotyledon (16.0 µg g−1 ), being equivalent to 20 and 30% of total phenolic compounds, respectively. Among the 10 anthocyanins, cyanidin-3-glucoside was found to accumulate at the highest level in the seed coat (1783 µg g−1 ), whole seed (106 µg g−1 ) and embryo (0.35 µg g−1 ), which correspond to 95, 96, and 40% of the total anthocyanin contents, respectively. The cotyledon accumulated pelargonidin-3-glucoside (0.39 µg g−1 ) at the highest level that is equivalent to 62% of total anthocyanin contents. DPPH activity was found to have a strong correlation (∗∗∗ probability < 0.001) with phenolic compounds (0.67∗∗∗ ) and anthocyanins (0.70∗∗∗ ). Keywords Soybean . Phenolic compounds . Anthocyanins . Cooked-with-rice . Vegetable soybean . HPLC
Introduction Soybean, which was originated in North and East Asia, is widely grown worldwide due to its nutritional benefits of a full range of amino acids and a high protein content. About 223 million tons of soybean were produced globally in 2004, and United States alone produced 85.5 million tons [1]. In Korea, soybean is an important highland crop that can be Springer
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cultivated on the levees around rice fields from summer to autumn. The gross production in Korea was 139,000 tons with 1,373,000 tons being imported, and soybean consumption per capita was 8.5 kg with total 393,000 tons during 2004 [2]. In Korea, soybean has been classified into five categories according to its food usage as bean paste, soybean curd or milk, bean sprout, cooked-with-rice and vegetable soybeans. Cooked-with-rice soybean varieties, which are usually consumed by being cooked along with boiled rice, have a medium-sized grain with a total weight of 25– 35 g for 100 grains. Colored soybeans have black or brown seed coat and an agreeable chewing texture. On the other hand, vegetable soybean varieties have a large grain seed with 100 grain weight of 30–40 g and a high concentration of sugar. Soybean is an important source of protein as well as useful secondary metabolites. It is especially rich in essential amino acids including lysine and tryptophan, which lack in rice and wheat. Secondary metabolites include phenolic compounds, isoflavones, saponins, phytic acids, and peptides. In plants, these compounds have various functions such as repellant to herbivorous insects and animals, protection against UV light and phytopathogens, nodulation signal, and attractant of pollinating animals [3]. Phenolic compounds have an aromatic ring bearing one or more hydroxyl groups. Biosynthesis begins with the conversion of phenylalanine to cinnamic acid, the first step of phenylpropanoid pathway that is catalyzed by phenylalanine ammonia lyase (PAL). Phenolic compounds are classified into three major groups based on the chemical structures; simple phenol and phenolic acid, hydroxycinnamic acid derivates, and flavonoids. The simple phenol and phenolic acid group include vanillic acid, gallic acid with other derivative forms. The hydroxycinnamic acid derivatives group contains ρ-coumaric acid, trans-cinnamic acid, caffeic acid, ferulic acid and their conjugated forms, ester or glycoside. Among the flavonoid group is catechin, anthocyanins, flavonol, flavone, and their glycosides [4]. The phenolic profiles of the plants may be changed depending on the conditions imposed by soil, season, climate, plant component, and other parameters [5]. Phenolic compounds are beneficial to human by exerting as an antioxidant that prevents the generation of harmful products by reactive oxygen species (ROS) during oxidative stresses [6]. Anthocyanins are water-soluble plant pigments belonging to a class of flavonoids that are mostly found in flowers and fruits. They play a role in attracting creatures for pollination, seed propagation, antibacterial activity, and UV protection. They are also beneficial to human due to the antioxidant activity, lowering blood cholesterol, prevention of heart diseases, anticancer activity, and protection from UV radiation. Anthocyanins are synthesized via phenylpropanoid pathway through the concerted activities of chalcone synthase, Springer
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chalcone isomerase, and dihydroflavonol reductase (DFR) [6]. They accumulate in the epidermis palisade layer of the seed coat, and the major anthocyanins in black seed coat of soybeans are cyanidin-3-glucoside, delphinidin-3glucoside, and pelargonidin-3-glucoside [8]. Concentrations of delphinidin-3-glucoside and cyanidin-3-glucoside, and oligosaccharide were reported to be changed during germination and years of storage [9, 10]. Petunidin-3-glucoside was also isolated from seed coat of black soybean [11]. Joo et al. [12] reported that the concentration of delphinidin-3glucoside and petunidin-3-glucoside was higher in the large grain soybean and yellow cotyledon soybean varieties than in the small grain soybean and green cotyledon varieties. In the present study, we determined the concentrations of phenolic compounds contained in each separated part of soybean seeds of nine varieties. We also investigated the relationship between the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and concentration of phenolic compounds.
Materials and methods Plant materials Nine varieties of soybean used in this experiment were Galmikong (GM), Geomjeongkong 2 (GJ), Geomjeongolkong (GO), Heugcheongkong (HC), Hwaeomputkong (HP), Ilpumgeomjeongkong (IG), Keunolkong (KO), Seoklyangputkong (SP), and Seonheukkong (SH). All the varieties have medium to large grain size, ranging from 25 to 35 g in weight for 100 grains. GM, GJ, GO, HC, and IG are all soybean varieties used for being cooked with rice, whereas the others are vegetable varieties. GO has a brown seed coat, whereas GJ, GO, HC, IG, and SH have a black seed coat. The seed coat of KO and SP is yellow and that of HP is green. The cotyledon of HC is green and that of all the other varieties is yellow (Table 1). The seed of HC was taken from the Gangwon Agricultural Research and Extension Services, and those of all the other varieties were obtained from the Youngnam Agricultural Research Institute. All of the soybean seeds were stored in a seed depository until use. HC was cultivated in the experimental field at the Chuncheon Agricultural Technology Center, South Korea on May 23, 2004, and the seed was harvested on October 24 of the same year. All other soybean varieties were grown in the experimental field of YARI at Milyang, South Korea on May 23, 2004, and the seeds were harvested on October 20. The planting arrangement was 60 × 15 cm per plot. Appropriate pesticides were used to control weeds, diseases and insects, and fertilizer was applied prior to plowing at the recommended rates of 8.8 and 12 kg per 1000 m2 for N, P2 O5 and K2 O, respectively. After drying below − 40 ◦ C, the whole grain
Eur Food Res Technol ( 2006 ) 224:259–270 Table 1 Characteristics of nine soybean varieties
a
Cooked-with-rice soybean
b
Vegetable soybean
261
Variety
100 seed weight (g)
Seed coat color
Cotyledon color
Seed use
Galmikong (GM) Geomjeongkong 2 (GJ) Geomjeongolkong (GO) Heugcheongkong (HC) Hwaeomputkong (HP) Ilpumgeomjeongkong (IG) Keunolkong (KO) Seoklyangputkong (SP) Seonheukkong (SH)
27.2 28.3 25.7 30.1 30.7 28.0 31.0 37.6 34.2
Brown Black Black Black Yellow Black Yellow Green Black
Yellow Yellow Yellow Green Yellow Yellow Yellow Yellow Yellow
CRa CR CR CR VSb CR VS VS VS
was crushed down lightly in mortar and then hand picked. The separated parts of the soybean seed were the embryo, cotyledon, and seed coat (Fig. 1). HPLC analysis of phenolic compounds Each soybean seed part was repeatedly freeze-dried using the lyophilizer (Labconco freeze dry system 4.5, Michigan, USA) and then ground with a small Angel mixer (Angel Co. Ltd., Seoul, Korea). Two grams of each powder were mixed
with 10 ml of acetonitrile (ACN) and 2 ml of 0.1 N HCl for 2 h at room temperature, filtered through No. 42 Whatman filter paper, and freeze-dried below − 40 ◦ C. Each sample was redissolved in 10 ml of 80% methanol (MeOH) and filtered through 0.45 µm syringe filter (TITAN, nylon) prior to HPLC injection. The HPLC system (Shimadzu, Tokyo, Japan) used was equipped with SPD-M10A Photo Diode Array Detector along with YMC ODS AM-303 (5 µm, 250 mm × 4.6 mm i.d.) and Midas autosampler (Spark Holland, Emmen, Netherlands). Each 20 µl sample was injected
Fig. 1 The separated seed components of nine soybean varieties. A Whole grain; B Cotyledon; C Embryo; D Seed coat
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Eur Food Res Technol ( 2006 ) 224:259–270
Fig. 2 HPLC chromatograms of phenolic compounds. A 21 phenolic compound standards; B GJ embryo; C HC cotyledon; D GM seed coat; E GM whole seed 2; 1, gallic acid; 2, pyrogallol; 3, homogentisic acid; 4, chlorogenic acid; 5, catechin; 6, vanillic acid; 7, caffeic acid; 8, syringic acid; 9, p-coumaric acid; 10, rutin; 11, ferulic acid; 12, naringin; 13, hesperidin; 14, o-coumaric acid; 15, myricetin; 16, quercetin; 17, trans-cinnamic acid; 18, naringenin; 19, kaempferol; 20, hesperetin; 21, biochanin A
at a flow rate of 1 ml min−1 and monitored at 280 nm. Solvent A was 0.1% glacial acetic acid in distilled water and solvent B was 0.1% glacial acetic acid in acetonitrile. The linear gradient solvent followed the method of Price et al. [13] as follows. Solvent B was increased from 8 to 10% in 2 min, then from 10 to 30% in 25 min, from 30 to 90% in 23 min, from 90 to 100% for 2 min, kept at 100% of B for 5 min, and returned to the initial condition. The column was equilibrated for 15 min before injection of the next sample. HPLC analysis of anthocyanins Two grams of each ground soybean seed part were mixed with 20 ml of 1% HCl in 80% MeOH for 24 h at 4 ◦ C without light. The samples were centrifuged at 13,000 rpm for 10 min, filtered through 0.45 µm syringe filter (TITAN, nylon), and used for HPLC analysis. The same HPLC system used for phenolic compound analysis was also used for anthocyanin analyses. Each 20 µl sample was injected at the flow rate of 1 ml min−1 and was monitored at 520 nm. Solvent A was 10% formic acid in distilled water and solvent B was pure MeOH. After injection of the sample, the linear gradient followed the method of Sun et al. [14] by increasing Springer
from an initial 20 to 40% in 5 min, from 40 to 60% in 15 min, from 60 to 100% in 5 min, keeping at 100% of solvent B (pure MeOH) for 5 min, and then returning to the initiation condition. Standard material and standard calibration curves Standard samples of 21 phenolic compounds and 10 anthocyanins were purchased from Sigma Aldrich (USA) and Extra Synthese (France), respectively. The phenolic standards were dissolved in dimethylsulfoxide (DMSO), and the anthocyanin standards were dissolved in 0.1% HCl solution in methanol (MeOH). The plotting standards were made at three concentrations of 1, 50, and 100 µg ml−1 , and a high linearity of r2 >0.998 was obtained for each curve. Sample peaks were identified by comparing their retention times with those of authentic standards. Concentrations of the identified compounds were calculated based on the peak areas of samples. DPPH radical scavenging activity DPPH radical scavenging activity was assayed according to the method of Chung et al. [15]. One gram of ground soy-
GM E C S W GJ E C S W GO E C S W HC E C S W HP E C S W IG E C S W KO E C S W SP E C S W
Part
6.27 0.00 27.91 18.87
0.00 0.00 41.53 6.77
5.79 3.90 24.04 1.35
0.00 12.75 33.40 10.67
0.00 0.85 0.00 5.06
0.00 0.00 21.95 7.53
0.00 0.00 16.36 11.14
0.00 8.77 0.00 5.27
0.12 0.46 5.26 0.80
1.32 0.08 6.96 1.00
0.53 0.54 4.93 0.41
0.00 0.89 5.53 0.61
0.00 0.22 2.15 0.25
0.00 0.10 5.95 0.74
0.95 0.01 1.63 0.82
0.01 0.55 0.50 0.41
PY2
15.6 6.47 2.9 2.39
2.19 2.90 19.20 5.00
21.60 9.04 25.88 6.55
5.31 0.00 18.53 0.01
0.00 0.18 0.00 4.36
VA6
4.98 4.72 0.66 1.13 78.21 41.47 4.97 0.00
14.21 7.87 0.49 0.16 76.93 12.78 4.34 6.54
4.66 0.90 3.21 0.65
7.29 2.01 7.62 0.89
1.83 1.92 38.75 2.86 7.91 1.07 0.05 1.16
0.83 1.00 3.12 1.50
8.17 7.94 0.84 0.29 49.93 13.42 3.07 2.35
12.95 2.20 7.19 2.06
95.79 4.12 3.12 20.96 1.95 1.13 155.26 257.53 53.36 9.77 2.32 1.82
21.02 6.65 39.68 23.25
48.64 4.53 24.42 20.02
13.13 1.73 28.48 6.83
0.00 0.00 14.41 4.41
CT5
35.90 0.45 1.06 0.00 5.51 2.20 516.23 181.75 34.41 0.01 9.00 14.30
CH4
0.00 0.00 4.51 0.38
0.00 0.00 19.15 2.14
1.77 5.70 18.16 3.57
0.00 0.00 17.45 5.78
0.00 0.17 12.75 2.74
HO3
28.38 0.16 0.50 0.30
2.22 0.88 3.15 0.39
32.36 0.96 12.97 0.00
12.75 1.25 0.45 0.97
25.34 1.10 53.59 0.88
29.04 0.23 59.16 2.96
42.61 0.85 16.83 12.84
7.48 1.77 28.84 5.20
CF7
PC9
7.58 0.72 0.17 0.71
1.44 0.56 0.32 1.73
0.72 12.29 0.95 1.00 0.08 0.14 1.60 1.58
0.01 1.13 0.60 1.70
0.95 11.77 0.01 0.78 159.05 14.38 2.33 2.03
0.51 0.00 0.94 1.07
0.01 11.89 0.01 2.57 791.07 29.14 1.33 1.69
0.81 9.65 0.99 1.76 366.77 24.68 2.04 2.31
0.00 17.70 0.01 0.66 99.45 6.67 0.00 4.88
11.29 2.33 2.88 0.93 239.24 28.51 3.31 2.57
SY8
FE11
NRl2
23.32 3.33 0.08 2.63
2.85 1.62 1.63 3.18
43.37 2.35 98.34 5.09
92.46 3.63 1.84 2.98
20.88 5.56 188.10 5.15
21.67 3.23 167.35 7.04
34.61 2.08 41.94 14.22
2.20 4.50 1.81 3.98
14.24 1.39 0.21 1.82
3.21 1.32 0.73 2.25
3.65 3.19 0.94 6.22
0.24 3.16 1.51 6.24
21.30 6.01 1.50 2.89 26.18 25.70 2.68 4.63
17.74 1.45 0.65 2.69
3.71 3.06 0.23 1.74
0.55 2.91 2.89 6.63
10.39 3.13 25.12 4.35
5.48 1.78 1.61 3.08
7.94 5.15 22.68 4.14
7.58 5.65 10.72 8.89
13.32 5.96 1.63 7.63 19.52 30.93 3.92 7.45 18.07 5.85 2.94 1.32 47.21 27.90 2.11 3.54
14.54 2.61 4.22 7.58
1.32 5.52 70.44 8.18
HS13
16.78 9.25 1.20 2.50 13.03 10.99 6.96 8.67
4.09 0.54 1.45 1.09 7.08 0.86 271.33 222.09 91.01 15.90 23.48 8.69
RU10
Concentration (µg g−1 ) of 21 phenolic compounds in soybean seed components of nine varieties
GA1
Table 2
7.43 1.09 0.08 1.32
0.84 0.79 0.24 1.70
6.59 0.52 1.31 0.91
5.53 0.51 0.13 0.61
10.36 2.85 2.98 1.95
8.05 0.98 4.59 1.83
16.71 0.59 0.97 3.82
2.21 4.41 31.26 1.66
OC14
22.65 16.4 0.92 16.5
5.08 15.71 6.32 13.05
29.56 10.74 8.55 11.42
30.92 11.80 1.47 17.77
25.80 33.29 17.74 20.04
15.88 22.17 18.86 35.47
29.58 11.15 15.43 0.01
1.55 0.37 45.19 26.37
2.17 1.10 0.01 0.74
0.35 2.84 0.01 0.84
1.69 0.30 9.37 2.24
3.58 2.09 0.14 0.46
2.87 0.82 10.77 0.78
2.94 2.06 15.93 2.29
5.00 0.50 12.81 2.72
0.44 17.55 86.51 6.51
MY15 QU16
0.44 0.19 0.01 0.29
0.04 0.37 0.01 0.27
0.56 0.07 0.71 0.36
0.42 0.36 0.01 0.31
0.57 0.32 0.63 0.45
0.49 0.85 0.53 0.66
0.73 0.10 0.89 1.11
0.03 1.40 5.94 0.65
CI17
13.03 5.12 0.17 2.94
3.05 3.33 0.70 3.41
26.86 2.25 0.27 7.07
23.17 3.01 0.52 2.95
7.43 6.45 0.86 5.66
8.32 4.91 0.47 7.64
22.86 2.01 0.71 7.83
0.00 0.64 7.57 6.37
NN18
0.32 0.42 0.06 0.68
0.00 0.38 0.13 0.58
1.20 0.12 0.12 0.63
0.34 0.46 0.07 0.43
0.90 0.00 1.04 0.39
1.25 0.69 0.06 0.65
1.21 0.04 0.37 1.57
0.00 4.39 6.22 1.56
KA19
0.57 0.27 0.03 0.19
0.00 0.18 0.07 0.17
0.61 0.07 0.08 0.32
0.32 0.23 0.02 0.16
1.04 0.07 0.12 0.31
1.53 0.39 0.10 0.34
1.50 0.05 0.48 0.01
0.00 0.64 2.40 0.49
HT20
1.51 0.88 0.27 2.44
0.27 0.80 0.67 2.59
1.69 0.90 0.09 2.63
4.09 0.74 0.21 1.50
3.74 0.64 0.59 3.25
1.70 2.00 0.25 2.27
1.96 0.74 0.16 1.90
0.00 0.35 1.73 1.73
BI21
165.24 57.6 14.8 55.47
25.95 41.81 112.45 70.46
237.93 36.86 517.90 66.94
237.83 38.43 55.58 54.90
245.72 100.77 1718.65 79.00
167.00 73.75 926.41 119.31
287.08 30.35 405.02 118.89
76.53 58.22 1916.59 158.39
TOT22
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(1) gallic acid, (2) pyrogallol, (3) homogentisic acid, (4) chlorogenic acid, (5) catechin, (6) vanillic acid, (7) caffeic acid, (8) syringic acid, (9) ρ-coumaric acid, (10) rutin, (11) ferulic acid, (12) naringin, (13) hesperidin, (14) o-coumaric acid, (15) myricetin, (16) quercetin, (17) trans-cinnamic acid, (18) naringenin, (19) kaempferol, (20) hesperetin, (21) biochanin A. (22) Total concentration. E, C, S, and W denote embryo, cotyledon, seed coat, and whole seed, respectively
65.56 11.36 229.97 32.28 0.58 0.17 0.14 0.42 0.27 0.13 0.14 0.1 0.25 0.27 0.29 0.49 0.76 0.32 0.91 0.25 1.65 0.87 2.25 2.11 8.75 0.23 9.31 0.22 3.74 0.07 3.97 0.32 4.33 0.32 3.14 0.46 0.94 0.17 2.09 2.12 1.71 1.40 0.68 0.36 39.85 124.84 6.56 1.50 0.38 0.13 0.68 0.70 0.61 1.42 5.48 4.82 0.19 0.14 0.56 0.26
1.34 2.15 2.92 0.67 20.67 13.47 8.40 2.61 0.41 0.33 3.87 0.58
5.65 0.83 4.37 2.08 10.43 1.43 16.11 4.85 1.34 0.29 2.10 0.31
1.55 19.11 0.54 2.19 1.33 7.17 0.91 3.86
1.75 0.57 5.12 0.41
0.17 0.23 0.12 0.13
0.71 0.20 0.44 0.23 0.65 0.33 0.97 0.73 12.83 3.62 1.36 5.03 13.75 4.66 6.58 1.43 3.49 3.26 38.32 22.57 15.77 5.43 5.56 5.41 29.28 3.25 92.0 7.87 6.48 4.54 22.32 1.59 9.24 1.41 0.83 0.94 0.45 1.03 97.12 20.37 22.84 214.36 12.39 3.33 3.21 2.72 1.54 2.10
0.01 0.01 272.1 0.51 27.91 4.31 2.74 20.68 1.95 0.39 0.05 0.13 27.8 176.17 21.5 30.1 12.14 0.46 0.58 0.99 0.00 12.28 0.00 0.00 20.83 16.24 8.90 0.07
SH E C S W Average E C S W LSD0.05 E C S W
0.75 0.07 1.91 0.16
31.31 6.64 93.32 9.55
7.74 0.64 3.99 4.14 18.58 7.30 0.56 0.74 15.27 12.34 2.97 0.63 20.31 0.37 57.41 14.65 8.48 0.13 7.46 1.41
7.18 0.87 4.70 1.60
6.89 41.37 0.10 5.11 0.78 9.91 0.63 9.76
22.49 2.34 15.99 1.28 13.82 16.13 16.71 1.95
0.44 0.34 1.03 0.48
0.8 0.14 0.67 0.12 10.79 1.12 0.97 1.44 2.01 0.46 9.60 0.95
0.67 0.14 0.51 0.19
0.64 0.20 0.67 0.07
1.79 0.93 0.46 2.12
1.15 0.87 0.16 0.78
TOT22 BI21 HT20 KA19 NN18 CI17 MY15 QU16 OC14 HS13 NRl2 FE11 RU10 PC9 SY8 CF7 VA6 CT5 CH4 HO3 PY2 GA1 Part
Continued Table 2
182.08 50.21 705.97 87.16
Eur Food Res Technol ( 2006 ) 224:259–270 195.41 13.18 686.39 61.55
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bean was mixed with 20 ml of 80% MeOH. The mixture was shaken for 24 h at room temperature, filtered through Whatman No. 42 filter paper, and was freeze-dried at − 40 ◦ C until use. Test samples were diluted to 1% in DMSO. DPPH reagent contained 30 mg of DPPH dissolved in 200 ml of pure ethanol and 200 ml of distilled water. For DPPH test, 2.5 ml of DPPH reagent was incubated with 250 µl sample (blank was DMSO) for 1 min, and the mixture was monitored at 517 nm. DPPH activity was calculated as follows: Abssample × 100 DPPH activity (%) = 1 − Absblank
Statistical analysis A statistical analysis was undertaken using the general linear model procedure (GLM) of statistical analysis program [16]. All of the experiments were replicated three times using a completely randomized design. The LSD (least significant difference) test was based on the 0.05 probability level. SAS program was used to test for the correlations between the DPPH radical scavenge activity and concentration of phenolic compounds and anthocyanins.
Results and discussion Content of phenolic compounds in the soybean seed of nine varieties As shown in Fig. 2, 21 out of 26 phenolic compounds were well separated by HPLC; five of them, mcoumaric acid, 3,4-dimethoxybenzoic acid, formononetin, o-hydroxyphenylacetic acid, and β-resorcylic acid were not detectable in any of the test samples. The quantified amounts of the individual compounds from nine varieties are listed in Table 2. Total amounts of phenolic compounds in seed coat, embryo, and cotyledon vary widely in nine varieties (Fig. 3). The average total concentration of phenolic compounds was 706 µg g−1 in the seed coat, 87 µg g−1 in the whole seed, 182 µg g−1 in the embryo, and 50 µg g−1 in the cotyledon. In only seed coat tissue, the total concentrations of the brown and black seed coat soybean were much higher than that of yellow or green coat soybean (Fig. 3). These results are consistent with the previous reports that there is a correlation between the level of phenolic compounds and seed coat color [9, 18]. In the seed coat tissue, GM (1917 µg g−1 ) and SP (15 µg g−1 ) contained the highest and the lowest amounts of phenolic compounds, respectively. The content of syringic acid (214 µg g−1 ) was highest, amounting to 30% of total phenolic compounds in the seed coat. In contrast, SH differs
Eur Food Res Technol ( 2006 ) 224:259–270 2000
Phenoliccompound concentration ( g g-1)
Fig. 3 The concentration of total phenolic compounds in soybean seed components of nine varieties. GM, Galmikong; GJ, Geomjeongkong 2; GO, Geomjeonggolkong; HC, Heugcheongkong; HP, Hwaeomputkong; IG, Ilpumgeomjeongkong; KO, Keunolkong; SP, Seoklyangnolkong; SH, Seonheukkong
265
Embryo Cotyledon Seed coat Whole seed
1500
1000
500
0 GM
GJ
GO
HC
HP
IG
KO
SP
SH
Soybean varieties
Fig. 4 HPLC chromatograms of anthocyanins. A 10 anthocyanin standards; B SH embryo; C SH cotyledon; D GJ seed coat; E GJ whole seed; 1, cyanidin-3-glucoside; 2, pelargonidin-3-glucoside; 3, peonidin-3-o-glucoside; 4, malvidin-3-glucoside; 5, delphinidin; 6, cyanidin; 7, petunidin; 8, pelargonidin; 9, peonidin; 10, malvidin
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266 Table 3
Eur Food Res Technol ( 2006 ) 224:259–270 Concentration (µg g−1 ) of anthocyanins in soybean seed components of nine varieties
Part GM E11 C12 S13 W14 GJ E C S W GO E C S W HC E C S W HP E C S W IG E C S W KO E C S W SP E C S W SH E C S W Average E C S W
C3G1
PL3G2
PO3G3
M3G4
DEL5
CYN6
PET7
PEL8
PEO9
MAL10
Total
nd nd 23.77 1.06
0.25 0.64 nd nd
0.54 0.02 11.40 0.48
nd 0.04 8.28 0.06
0.01 0.06 2.89 nd
0.20 0.05 0.61 0.01
0.17 nd 0.97 nd
nd nd 0.14 nd
nd 0.01 0.09 nd
nd nd nd 0.01
1.17 0.82 48.15 1.62
1.04 nd 4319.58 282.65
0.32 0.61 54.67 3.01
0.21 0.01 83.10 5.76
0.10 nd 98.66 6.36
nd 0.08 23.10 1.27
0.02 0.04 7.51 0.63
0.05 nd 1.46 0.26
0.01 nd 0.09 0.01
nd nd nd nd
nd nd 0.23 nd
1.75 0.74 4588.4 299.95
0.30 nd 2033.74 139.55
0.07 0.77 34.70 nd
0.31 nd 34.70 1.78
0.09 nd 42.43 2.62
0.10 0.02 9.51 0.27
0.07 0.16 3.27 0.29
nd nd 0.15 0.08
nd nd 0.04 nd
nd nd nd nd
nd nd 0.18 nd
0.94 0.95 2158.72 144.59
0.02 nd 2872.36 261.34
0.03 0.02 19.25 1.30
0.06 nd 23.95 1.77
nd nd 15.29 0.78
nd nd 13.03 0.81
nd nd 3.92 0.03
nd nd 0.35 0.01
nd nd 0.03 nd
nd nd 0.03 nd
nd nd 0.26 nd
0.11 0.02 2948.47 266.04
nd nd 0.14 nd
nd 0.27 nd 0.04
0.10 0.03 nd nd
0.03 nd nd nd
nd 0.08 nd nd
nd 0.08 nd nd
0.02 nd nd 0.01
0.03 nd nd nd
nd 0.01 nd 0.01
0.01 nd nd nd
0.19 0.47 0.14 0.06
0.10 0.20 2637.67 78.05
0.20 0.38 nd 0.60
0.18 nd 41.20 0.83
0.02 nd 65.20 0.78
nd 0.04 5.83 0.11
0.04 0.07 5.74 0.19
0.02 nd 1.50 nd
nd 0.02 0.07 nd
nd nd 0.04 nd
nd 0.03 0.38 nd
0.56 0.74 2757.63 80.56
nd nd 0.13 0.03
nd 0.28 nd nd
0.09 nd nd nd
nd nd nd nd
nd 0.05 0.02 0.03
0.02 0.13 nd 0.04
0.03 nd nd 0.01
nd nd nd 0.01
0.01 0.01 0.02 nd
0.04 nd nd nd
0.19 0.47 0.17 0.12
nd nd 0.08 nd
0.05 0.02 0.02 0.08
0.29 nd nd 0.01
0.03 nd nd nd
0.03 0.04 0.02 0.01
0.02 0.02 nd nd
0.05 nd nd nd
nd 0.01 nd nd
nd nd 0.01 nd
nd nd 0.02 nd
0.47 0.09 0.15 0.10
1.42 0.26 4163.69 194.74
nd 0.37 60.16 1.82
0.29 nd 98.21 3.91
nd0 0.40 14.35 0.11
nd 0.04 nd nd
0.07 0.04 6.91 0.51
0.08 nd nd nd
0.01 nd 0.5 nd
0.01 0.01 0.06 0.01
nd nd 0.24 nd
1.88 1.12 4344.12 201.09
0.35 0.05 1783.46 105.69
0.10 0.39 18.76 0.76
0.23 0.01 32.51 1.61
0.03 0.05 27.14 1.18
0.02 0.05 6.04 0.31
0.05 0.07 3.11 0.19
0.05 0.00 0.49 0.04
0.01 0.00 0.05 0.00
0.00 0.00 0.03 0.00
0.01 0.00 0.14 0.00
0.85 0.62 1871.73 109.78
from other vegetable soybean varieties (HP, KO, SP) in that it contained more than 272 times syringic acid. Total concentrations of phenolic compounds in the soybean embryo varied from 26 µg g−1 in KO to 287 µg g−1 in GJ. The Springer
average content of chlorogenic acid (31 µg g−1 ) was highest in the embryo, being followed by rutin, myricetin, and caffeic acid. They all together account for about 60% of total phenolic compounds in the embryo (Table 2).
Eur Food Res Technol ( 2006 ) 224:259–270 Table 3
267
Continued
Part
C3G1
PL3G2
LSD0.05 E C S W
0.04 0.05 217.10 47.36
0.13 0.69 2.57 0.31
PO3G3
M3G4
DEL5
0.06 0.02 2.91 0.87
0.03 0.02 2.69 0.23
0.02 0.08 1.52 0.06
CYN6
PET7
PEL8
PEO9
MAL10
0.03 0.14 0.62 0.11
0.04 0.00 0.59 0.03
0.02 0.01 0.09 0.01
0.01 0.01 0.02 0.01
0.02 0.01 0.17 0.00
Total
0.40 1.03 228.28 48.99
nd: not detected (1) cyanidin-3-glucoside, (2) pelargonidin-3-glucoside, (3) peonidin-3-o-glucoside, (4) malvidin-3-glucoside, (5) delphinidin, (6) cyanidin, (7) petunidin, (8) pelargonidin, (9) peonidin, (10) malvidin, (11) embryo, (12) cotyledon, (13) seed coat, (14) whole seed
Total phenolic concentrations of the whole seed range from 54 µg g−1 in HP to 158 µg g−1 in GM. In the cotyledon tissue, HC and SH contained the highest (101 µg g−1 ) and the lowest (13 µg g−1 ) levels of phenolic compounds, respectively. Among the individual phenolic compounds, myricetin was highest in the whole seed (17 µg g−1 ) as well as in the cotyledon (16 µg g−1 ), being equivalent to 20 and 30% of total phenolic compounds, respectively (Table 2). Our result confirms the previous report that soybean contains chlorogenic, caffeic, ferulic, p-coumaric, syringic, and vanillic acid [5]. Cheongjakong, a black seed coat of soybean variety, was reported to contain phenolic compounds in the order of benzoic acid (126.7 mg), p-coumaric (67.7 mg), salicylic (59.4 mg), gentisic (43.2 mg), ferulic (16.6 mg), and syringic (15.0 mg) acid [19]. Despite the similar types of phenolic acids obtained from this study, two major differences were noted from the previous reports [9]. Kim et al. [9] reported that among the 11 recognized compounds belonging to phenolic and flavonoid groups in soybean, main components were gentisic and salicylic acid, and that caffeic, chlorogenic, ferulic, hesperidin, naringin, myricetin, and syringic acids were detected either at a low concentration or undetectable. However, the latter seven flavonoids detected in this study made up of about 54% of total content in the whole soybean seed. In addition, it was reported that hydroxybenzoic acids and hydroxycinnamic acid are present in the cotyledon, whereas proanthocyanidins and glucosides of flavone are mainly found in the seed coat [20]. In this study, flavone glucosides are preferentially contained in the cotyledon rather than in the seed coat. Thus, the tissue-specific differences in the types and contents of phenolic compounds are dependent on the soybean varieties. Concentration of anthocyanins in the soybean seed of nine soybean varieties HPLC profiles of anthocyanins are shown in Fig. 4, and the contents of the individual compounds were quantified (Table 3). All nine varieties showed a wide variation in total amounts of anthocyanins in the seed coat, embryo, and
Table 4 DPPH free radical scavenging activity in embryo, cotyledon, seed coat, and whole seed Variety
Cotyledon Embryo (%) (%)
Seed coat (%)
GM GJ GO HC HP IG KO SP SH Average LSD0.05
13.7 17.3 12.0 15.0 17.4 11.2 15.5 12.4 9.2 13.8 2.3
79.4 62.9 48.7 67.6 2.7 56.1 4.9 2.4 53.4 42.0 1.1
4.7 8.3 8.1 6.2 6.1 9.2 7.0 8.5 6.2 7.1 3.0
Whole seed (%) 29.8 27.1 25.5 28.6 10.7 21.9 8.9 10.5 17.8 20.1 3.9
cotyledon. The average total concentration of anthocyanins was 1872 µg g−1 in the seed coat, 110 µg g−1 in the whole seed, 0.85 µg g−1 in the embryo and 0.62 µg g−1 in the cotyledon. Thus, anthocyanins are predominantly found in the seed coat. Consistent with the previous result [16], there appears to be a relationship between the seed coat color and the anthocyanin contents as the black soybean had far more anthocyanins in seed coat and whole seed than the brown, yellow, or green soybean. However, this relationship is not applied to the embryo and cotyledon. The levels of anthocyanins in the embryo and cotyledon were very low at between 1.90 and 0.02 µg g−1 (Fig. 4). Overall cyanidin-3glucoside was found to accumulate at the highest level in the seed coat (1783 µg g−1 on average), whole seed (106 µg g−1 ) and embryo (0.35 µg g−1 ). This corresponds to 95, 96, and 40% of the total anthocyanin contents in the respective tissue. The cotyledon accumulated pelargonidin-3-glucoside (0.39 µg g−1 ) at the highest level, which amounts to 62% of total anthocyanin contents (Table 3). In the five varieties of a black seed coat, i.e., GJ, GO, HC, IG, and SH, cyanidin-3-glucoside constituted more than 90% of the total anthocyanin contents. This result confirms the previous reports that a major anthocyanin of black seed coat soybean is cyanidin-3-glucoside [8, 11].
Springer
268 Table 5
Eur Food Res Technol ( 2006 ) 224:259–270 Correlation between DPPH activity and individual phenolic compound in the entire seed tissue DPPH
DPPH GA PY HO CH CT VA CF SY PC RU FE DPPH
GA1
PY2
HO3
CH4
CT5
VA6
CF7
SY8
PC9
RU10
FE11
0.72∗∗∗
0.62∗∗∗ 0.87∗∗∗
0.54∗∗∗ 0.84∗∗∗ 0.79∗∗∗
0.56∗∗∗ 0.48∗∗ 0.42∗ 0.34∗
0.54∗∗∗ 0.72∗∗∗ 0.74∗∗∗ 0.74∗∗∗ 0.63∗∗∗
0.65∗∗∗ 0.75∗∗∗ 0.73∗∗∗ 0.73∗∗∗ 0.57∗∗∗ 0.89∗∗∗
0.42∗ 0.50∗∗ 0.40∗ 0.55∗∗∗ 0.39∗ 0.62∗∗∗ 0.79∗∗∗
0.54∗∗∗ 0.70∗∗∗ 0.68∗∗∗ 0.70∗∗∗ 0.44∗∗ 0.91∗∗∗ 0.93∗∗∗ 0.68∗∗∗
0.62∗∗∗ 0.65∗∗∗ 0.51∗∗ 0.59∗∗∗ 0.67∗∗∗ 0.72∗∗∗ 0.86∗∗∗ 0.90∗∗∗ 0.74∗∗∗
0.72∗∗∗ 0.72∗∗∗ 0.62∗∗∗ 0.63∗∗∗ 0.80∗∗∗ 0.81∗∗∗ 0.88∗∗∗ 0.69∗∗∗ 0.76∗∗∗ 0.89∗∗∗
0.59∗∗∗ 0.49∗∗ 0.43∗∗ 0.36∗ 0.97∗∗∗ 0.60∗∗∗ 0.57∗∗∗ 0.39∗ 0.40∗ 0.67∗∗∗ 0.82∗∗∗
NR12
HS13
OC14
MY15
QU16
CI17
NN18
KA19
HT20
BI21
Total
– 0.63∗∗∗ 0.69∗∗∗ 0.89∗∗∗ 0.62∗∗∗ 0.69∗∗∗ 0.73∗∗∗ 0.43∗∗ 0.80∗∗∗
– – – – 0.45∗∗ – – 0.56∗∗∗ – –
0.67∗∗∗ 0.88∗∗∗ 0.83∗∗∗ 0.61∗∗∗ 0.79∗∗∗ 0.79∗∗∗ 0.71∗∗∗ – 0.66∗∗∗ 0.51∗∗ –
∗∗∗
DPPH NR HS OC MY QU CI NN KA HT BI Total
0.70
∗∗∗
0.64 0.95∗∗∗
∗
0.39 0.75∗∗∗ 0.82∗∗∗
– 0.82∗∗∗ 0.50∗∗ 0.63∗∗∗
∗∗∗
0.63 0.96∗∗∗ 0.93∗∗∗ 0.80∗∗∗ 0.44∗∗
∗∗∗
0.55 0.92∗∗∗ 0.93∗∗∗ 0.84∗∗∗ 0.52∗∗ 0.96∗∗∗
– – – 0.47∗∗ 0.59∗∗∗ – –
∗∗
0.49 0.85∗∗∗ 0.88∗∗∗ 0.85∗∗∗ 0.51∗∗ 0.89∗∗∗ 0.95∗∗∗ –
(1) gallic acid, (2) pyrogallol, (3) homogentisic acid, (4) chlorogenic acid, (5) catechin, (6) vanillic acid, (7) caffeic acid, (8) syringic acid, (9) ρcoumaric acid, (10) rutin, (11) ferulic acid, (12) naringin, (13) hesperidin, (14) o-coumaric acid, (15) myricetin, (16) quercetin, (17) trans-cinnamic acid, (18) naringenin, (19) kaempferol, (20) hesperetin, (21) biochanin A ∗
P
