Original papers
Comparison of Anthocyanins, Proanthocyanidin Oligomers and Antioxidant Capacity between Cowpea and Grain Legumes with Colored Seed Coat
2019 Volume 25 Issue 2 Pages 287-294
Details
2019 Volume 25 Issue 2 Pages 287-294
The aim of this study was to investigate the composition and contents of anthocyanins and proanthocyanidins, and the antioxidant capacity of black and red cowpeas grown in Japan, with comparison to grain legumes (black kidney beans, black soybeans, and azuki beans) commonly consumed in Japan. This study revealed that black cowpeas contained seven anthocyanins, the 3–O–galactosides and 3–O–glucosides of cyanidin and delphinidin, and the 3–O–glucosides of malvidin, peonidin, and petunidin, at higher levels than other grain legumes. In addition, black and red cowpeas were rich in proanthocyanidin oligomers (monomers to hexamers) when compared with other grain legumes. Among these, black and red cowpeas showed potent hydrophilic oxygen radical absorbance capacity (H–ORAC), reaching 540.6 and 367.5 µmol–Trolox equivalents/g–dry matter, respectively. The contributions of anthocyanins and proanthocyanidin oligomers to H–ORAC values were 45.8% and 17.5% in black and red cowpeas, respectively.
Cowpeas (Vigna unguiculata) are one of the oldest and most widely consumed grain legumes in the world, and production amounted to about 7 million tons in 2016, which is 3.3 times the amount in 1990 (i). In addition, cowpeas are known to be a drought- and heat-tolerant grain legume with low input requirements. The main cultivation region of cowpeas is Africa, particularly Nigeria and Niger, which account for over 90% of the global cowpea production (i). In Japan, cowpeas are a food that has long been consumed, even though they are cultivated in a limited area (Sugita et al., 2017) and are recognized as minor grain legumes, despite their growing production worldwide.
Like other grain legumes, cowpeas are known to be a good source of protein and trace minerals (Prinyawiwaktkul et al., 1996). Cowpea seeds are usually boiled and eaten with stew or prepared in the form of porridge in Africa, and they may also be cooked, in addition to Cassava being eaten as a staple food (FAO, 1989). Cowpea seeds have also been widely used in food products such as bread and jam for traditional cakes in Asia (Chung et al., 1998). In Japan, cowpea seeds are utilized principally for celebratory meals, such as rice cakes, zenzai (sweet red bean soup with pieces of rice cake), and sekihan (steamed rice mixed with red beans) (Ohashi et al., 2013), in a similar manner to azuki beans (Vigna angularis), which belong to the same genus. Thus, red cowpeas have been used as an ingredient, like azuki beans of the same seed coat color, and are added to various dishes as an alternative to azuki beans. The seed coats of cowpeas are thought to be harder than those of azuki beans in Japan (ii), and thus cowpeas are more suitable for dishes that need to be heated or boiled for long periods.
Seed coat colors of cowpeas vary, and include black, brown, green, red, and white, as well as mottled colors; however, the cowpeas distributed in Japan are generally black or red. In addition, it has been reported that cowpeas with a red or black seed coat contain anthocyanins (Ha et al., 2010), flavonols (Cai et al., 2003), and proanthocyanidins (Hachibamba et al., 2013), which are a class of polyphenol compounds referred to as phytochemicals. In recent years, grain legumes with coats pigmented by anthocyanins and proanthocyanidins have been expected to possess various biological activities, including antioxidant (Siddhuraju et al., 2007; Starzyńska-Janiszewska et al., 2015; Wang et al., 2016), anti-inflammatory (Boudjou et al., 2013; Šibul et al., 2016), inhibit low-density lipoprotein oxidation (Cui et al., 2012; Yang et al., 2015), and α-glucosidase inhibition (Sreerama et al., 2012; Tan et al., 2017). However, little information is available on anthocyanins and proanthocyanidins besides their biological activity in cowpeas with black and red seed coats grown in Japan.
Among the biological activities, the antioxidant activity has attracted attention, because overproduction of reactive oxygen/nitrogen species in human body is involved in the pathogenesis of aging and many common diseases (Ames et al., 1993). Many methods for measuring antioxidant activity have been developed to date. Among these methods, the oxygen radical absorbance capacity (ORAC) method is likely to be of biological relevance, as the peroxyl radical induced by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) contributes to the oxidative degradation of biological molecules in the body, and is produced from AAPH at neutral pH. Furthermore, both hydrophilic–ORAC (H–ORAC) and lipophilic–ORAC (L–ORAC) methods were validated for measuring the antioxidant activities of lipophilic and hydrophilic compounds, respectively (Watanabe et al., 2012, Watanabe et al., 2016). In cowpeas, it has been demonstrated that values of H–ORAC were much higher than those of L–ORAC, similar to other edible plants (Wu et al., 2004). In addition, there have been several reports on antioxidant activities of cowpeas using the H–ORAC method. Thus, it is considered that application of the H–ORAC method makes it possible to facilitate the comparison with cowpeas grown in other countries than Japan. On the other hand, the contribution of phytochemicals such as anthocyanins and proanthocyanidins to the H–ORAC values of cowpeas with seed color of black and red remains unclear.
Therefore, this study aimed to clarify the composition and contents of anthocyanins and proanthocyanidins, as well as the antioxidant capacity, of black and red cowpeas grown in Japan, and to compare these data with those of other red or black grain legumes commonly consumed in Japan. In this study, we selected azuki beans for comparison with red cowpeas because of the usage as an alternative to azuki beans. Also, black soybeans and black kidney beans were selected for comparison with black cowpeas because of the same seed coat color, and black soybeans are more familiar to Japanese people. Furthermore, the contributions of anthocyanins and proanthocyanidins to overall antioxidant activities were evaluated in grain legumes.
Chemicals Cyanidin–3–O–galactoside, cyanidin–3–O–glucoside, delphinidin–3–O–galactoside, delphinidin–3–O–glucoside, malvidin–3–O–glucoside, peonidin–3–O–glucoside, and petunidin–3–O–glucoside were purchased from Tokiwa Phytochemical Co., Ltd. (Tokyo, Japan). 6–Hydroxy–2, 5, 7, 8–tetramethylchroman–2–carboxylic acid (Trolox), fluorescein sodium salt, and (+)–catechin hydrate were purchased from Sigma-Aldrich Co. (Tokyo, Japan). High-performance liquid chromatography (HPLC) grade acetic acid, acetonitrile, formic acid, and methanol were used as the mobile phase. All other reagents were of analytical grade (Wako Pure Chemical Industries, Osaka, Japan).
Plant materials Five samples of dried grain legumes were purchased from a local supermarket and used in this study; two types of cowpea (Vigna unguiculata) with seed coats of black or red, azuki beans (Vigna angularis) with a red seed coat, kidney beans (Phaselous vulgaria) with a black seed coat, and soybeans (Glycine max) with a black seed coat. Black cowpeas, azuki beans, and black soybeans were grown in north Japan. Red cowpeas and black kidney beans were grown in west and south-east Japan, respectively. Grain legumes were freeze-dried, and then ground using a Labo milser LM–PLUS (Osaka Chemical Co., Osaka, Japan) and stored at −80 °C until use. Moisture contents in black cowpeas, red cowpeas, black kidney beans, black soybeans, and azuki beans were 5.4%, 7.6%, 4.4%, 7.1% and 6.1%, respectively.
Extraction of anthocyanins One gram of grain legume sample was placed into a capped glass centrifuge tube, and 8 mL of 1% (v/v) hydrochloric acid-methanol was added. The tube was capped and sonicated for 30 min and allowed to stand for 48 hours at 5 °C. After centrifugation at 2,270 × g for 10 min, the supernatant was transferred into a 25−mL volumetric flask. This extraction procedure was repeated twice with 8 mL of extraction solvent, and the extract was made up to a final volume of 25 mL.
Extraction of proanthocyanidins One gram of grain legume sample was placed into a capped glass centrifuge tube, and 9 mL of extraction solvent (acetone : water : acetic acid = 70:29.5:0.5, v/v) was added. After vigorously shaking the sample using a vortex mixer, the tube was capped, immersed in a water bath at 37 °C, and sonicated for 5 min at an oscillating frequency of 39 kHz. Tubes were then incubated for 10 min in a water bath at 37 °C and centrifuged at 2,270 × g for 10 min. The supernatant was transferred into a 25–mL volumetric flask. This extraction procedure was repeated twice with 8 mL of extraction solvent, and the extract was made up to final volume of 25 mL.
Anthocyanin analysis of grain legume extract Anthocyanins were analyzed by HPLC (Prominence System, Shimadzu Co., Kyoto, Japan) equipped with a photodiode array (PDA) detector at a detection wavelength of 520 nm according to the method of Sawai et al. (2012) with slight modification. A Cadenza CD–C18 column (250 mm × 4.6 mm i.d., 3 µm, Imtakt, Kyoto, Japan) was used after warming to 35 °C in a column oven. Mobile phase A consisted of water and formic acid at a ratio of 97:3, v/v. Mobile phase B consisted of water, acetonitrile and formic acid at a ratio of 30:67:3, v/v. Elution was conducted with a linear gradient as follows: increase from 25% to 100% B from 0 to 90 min, at a flow rate of 0.6 mL/min. The extract from grain legumes was filtered through a 0.20−µm hydrophilic PTFE membrane prior to HPLC analysis with an injection volume of 10 µL. All stock solutions of anthocyanins were prepared at a concentration of 0.1 mg/mL in 1% (v/v) hydrochloric acid–methanol. Identification of anthocyanins targeted in this study was performed by comparing retention times and uv–vis absorption spectra (wavelength range from 190 to 800 nm) with anthocyanin standards. Contents in grain legumes were calculated based on the area of corresponding peaks, by interpolation based on calibration curves obtained using standards.
Proanthocyanidin analysis of grain legume extract Proanthocyanidins were analyzed by HPLC (Prominence System, Shimadzu Co) equipped with a fluorescence (FL) detector (excitation wavelength, 230 nm; emission wavelength, 312 nm; photomultiplier tube gain, × 4) according to the method reported by Obara et al. (2016). An Intersil WP300 Diol (250 mm × 4.0 mm i.d., 5.0 µm, GL Sciences Inc., Tokyo, Japan) was used and warmed to 30 °C in a column oven. Mobile phase A consisted of acetonitrile and acetic acid at a ratio of 98:2, v/v. Mobile phase B consisted of water, methanol, and formic acid at a ratio of 3:95:2, v/v. The applied gradient was 7% B from 0 to 3 min, 7% to 30% B from 3 to 60 min, and 30% to 100% B from 60 to 63 min, at a flow rate of 1.0 mL/min. The photomultiplier tube gain was set at × 16 from 36.5 to 60.0 min to detect proanthocyanidin pentamers to heptamers. Extract from legumes was filtered through a 0.45− µm PTFE membrane prior to HPLC analysis with an injection volume of 5 µL. Proanthocyanidin standards (monomer to heptamer) were isolated from apple and purified as described previously (Shoji et al., 2006). All stock solutions of proanthocyanidins were prepared in 70% (v/v) acetone solution containing 0.5% (v/v) acetic acid. Eluted peaks were identified as proanthocyanidins based on the retention time of proanthocyanidin standards according to a previous paper (Obara et al., 2016). The contents in grain legumes were calculated from the area of corresponding peaks, by interpolation based on calibration curves obtained using standards.
Determination of antioxidant capacity The antioxidant capacity of grain legume extracts was measured by hydrophilic oxygen radical absorbance capacity (H–ORAC) assay, as reported by Watanabe et al. (2012). H–ORAC values were expressed as Trolox equivalents (TE) per gram of dry matter (µmol–TE/g–DM).
Determination of total phenolic content Total phenolic contents of grain legume extracts were determined using the Folin-Ciocalteu spectrophotometric method (Yoshida et al., 2010). Total phenolic contents were expressed as gallic acid equivalents (GAE) per gram of dry matter (mg–GAE/g–DM).
Determination of total proanthocyanidin content Total proanthocyanidin contents of legumes were determined using the vanillin-sulfuric acid method (Sun et al., 1998). Total proanthocyanidin contents were expressed as catechin equivalents (CAE) per gram of dry matter (mg–CAE/g–DM).
Calculation of contribution to overall antioxidant activity The contribution percentage of each anthocyanin or proanthocyanidin oligomer to H–ORAC value in grain legume sample (Contr.) was calculated according to the following equation (Eq. 1):
 |
where, ORACstd is the H–ORAC value of each standard (µmol–TE/mg); Csample is the content of anthocyanin or proanthocyanidin oligomer in grain legume sample (mg/g–DM); ORACsample is the H–ORAC value of grain legume sample (µmol–TE/g–DM).
Statistical analysis Data are reported as means ± standard deviation. All tests were conducted in triplicate. Data were analyzed by a one-way ANOVA followed by a Tukey post-hoc test using the SPSS software (version 22.0 for Windows, SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant at P < 0.05.
Anthocyanin composition and contents in grain legumes Anthocyanins were identified by the corresponding retention time and uv−vis absorption spectra obtained from HPLC–PDA with anthocyanin standards commercially available. In cowpea with black seed coat, seven anthocyanins were identified as listed in Table 1. The chemical structures of anthocyanins were the 3–O–glucoside form of cyanidin, delphinidin, malvidin, peonidin, and petunidin, and the 3–O–galactoside form of cyanidin and delphinidin. The most abundant anthocyanin was cyanidin–3–O–glucoside (37.2%), followed by delphinidin–3–O–glucoside (20.0%), malvidin–3–O–glucoside (16.5%), and petunidin–3–O–glucoside (14.8%). On the other hand, four and three species of anthocyanins were detected in black kidney beans and soybeans, respectively. In black kidney beans, delphinidin–3–O–glucoside was the most predominant 60.0%), while cyanidin–3–O–glucoside was most abundant in black soybeans (98.6%). Total anthocyanin contents in black cowpeas (15.36 mg/g–DM) were about five times higher than in black kidney beans and black soybeans. On the other hand, red cowpeas and azuki beans contained low levels of anthocyanins.
| Sample | Black cowpea | Red cowpea | Black kidney bean | Black soybean | Azuki bean |
|---|---|---|---|---|---|
| Seed coat color | Black | Red | Black | Black | Red |
| Cyanidin–3–O–galactoside | 0.16 ± 0.01a | nd | nd | 0.02 ± 0.00b | nd |
| Cyanidin–3–O–glucoside | 5.71 ± 0.30a | 0.03 ± 0.00c | nd | 2.84 ± 0.15b | nd |
| Delphinidin–3–O–galactoside | 0.43 ± 0.03a | nd | 0.06 ± 0.01b | nd | nd |
| Delphinidin–3–O–glucoside | 3.08 ± 0.22a | nd | 1.71 ± 0.12b | nd | nd |
| Malvidin–3–O–glucoside | 2.53 ± 0.07a | 0.03 ± 0.01c | 0.4 ± 0.04b | nd | 0.01 ± 0.00c |
| Peonidin–3–O–glucoside | 1.18 ± 0.03a | nd | nd | 0.02 ± 0.01b | nd |
| Petunidin–3–O–glucoside | 2.28 ± 0.12a | 0.01 ± 0.00c | 0.68 ± 0.04b | nd | nd |
| Total anthocyanins | 15.36 ± 0.75a | 0.06 ± 0.01c | 2.85 ± 0.20b | 2.88 ± 0.15b | 0.01 ± 0.00c |
| Proanthocyanidins | |||||
| Monomers | 0.35 ± 0.06a | 0.23 ± 0.02b | 0.02 ± 0.00d | 0.13 ± 0.02c | 0.06 ± 0.00c,d |
| Dimers | 0.38 ± 0.06a | 0.38 ± 0.03a | 0.04 ± 0.01c | 0.09 ± 0.01c | 0.18 ± 0.01b |
| Trimers | 0.30 ± 0.05a | 0.22 ± 0.02b | 0.02 ± 0.00c | 0.06 ± 0.01c | 0.08 ± 0.01c |
| Tetramers | 0.38 ± 0.07a | 0.26 ± 0.01b | 0.02 ± 0.00c | 0.08 ± 0.01c | 0.09 ± 0.01c |
| Pentamers | 0.49 ± 0.09a | 0.26 ± 0.01b | 0.03 ± 0.00c | 0.11 ± 0.01c | 0.08 ± 0.01c |
| Hexamers | 0.39 ± 0.07a | 0.18 ± 0.01b | 0.02 ± 0.00c | 0.10 ± 0.01b,c | 0.07 ± 0.01c |
| Heptamers | 0.37 ± 0.07a | 0.14 ± 0.01b | 0.02 ± 0.00c | 0.10 ± 0.01b,c | 0.05 ± 0.01c |
| Total proanthocyanidin oligomers | 2.66 ± 0.46a | 1.67 ± 0.08b | 0.17 ± 0.02c | 0.68 ± 0.08c | 0.61 ± 0.02c |
Results are represented as the mean ± standard deviation (n = 3). Contents are expressed as per gram of dry matter after freeze–drying. Within each row, means followed by different lowercase letters are statistically different (P < 0.05). Abbreviation: nd, not detected.
Proanthocyanidin composition and contents in grain legumes Monomeric through heptameric proanthocyanidins were detected by HPLC–FL, and were identified in all five grain legumes. The contents are summarized in Table 1. Black cowpeas contained the highest total proanthocyanidin oligomers (calculated as the sum of monomers through heptamers), at 2.66 mg/g–DM. Red cowpeas also contained high levels of total proanthocyanidin oligomers (1.67 mg/g–DM), when compared to the other three grain legumes. In black cowpeas, pentamers were the most abundant (18.4%) proanthocyanidin, while trimers were the least (11.3%). In red cowpeas, the highest and lowest were dimers (22.8%) and heptamers (8.4%), respectively. Similarly, black kidney beans and azuki beans contained dimers with the highest levels of 23.5% and 29.5%, respectively. In black soybean, monomers were the most abundant (19.4%).
H–ORAC values and total phenolic and proanthocyanidin contents in grain legumes H–ORAC values and total phenolic and proanthocyanidin contents in grain legumes are summarized in Table 2. All legumes showed distinct H–ORAC data. Black cowpeas showed the highest H–ORAC values (540.6 µmol–TE/g–DM), followed by red cowpeas, black soybeans, azuki beans, and black kidney beans. In addition, black cowpeas showed the highest total phenolic contents; however, the rank order of total phenolic contents was not consistent with H–ORAC values (black cowpeas > red cowpeas > azuki beans > black soybeans > black kidney beans).
| Sample | H–ORAC (µmol TE/g–DM) |
Total polyphenol content (mg–GAE/g–DM) |
Total proanthocyanidin content (mg–CAE /g–DM) |
|---|---|---|---|
| Black cowpea | 540.6 ± 10.4a | 30.0 ± 1.2a | 14.5 ± 0.2a |
| Red cowpea | 367.5 ± 6.6b | 21.5 ± 0.7b | 8.0 ± 0.6b |
| Black kidney bean | 97.6 ± 0.7e | 5.3 ± 0.1d | 3.7 ± 0.1c |
| Black soybean | 186.3 ± 0.7c | 6.2 ± 0.3d | 1.1 ± 0.2d |
| Azuki bean | 148.6 ± 12.4d | 9.2 ± 0.6c | 2.1 ± 0.2d |
H–ORAC, Hydrophilic oxygen radical absorbance capacity; TE, Trolox equivalents; GAE, gallic acid equivalents; CAE, catechin equivalents. Results are represented as the mean ± standard deviation (n = 3). H–ORAC, total polyphenol content, and total proanthocyanidins content are expressed as per gram of dry matter after freeze–drying. Within each column, means followed by different lowercase letters are statistically different (P < 0.05). Total proanthocyanidin content of grain legumes was determined using the vanillin–sulfuric acid method.
Total proanthocyanidin contents of red and black cowpeas were higher than for other grain legumes, while total proanthocyanidin contents in black cowpeas were 1.8 times higher than in red cowpeas.
Contribution of anthocyanins and proanthocyanidins to H–ORAC values In order to estimate the contribution of anthocyanins and proanthocyanidins to H–ORAC values in grain legumes, H–ORAC values of individual anthocyanin and proanthocyanidin standards were determined. For anthocyanins, H–ORAC values of cyanidin–3–O–galactoside, cyanidin–3–O–glucoside, delphinidin–3–O–galactoside, delphinidin–3–O–glucoside, malvidin–3–O–glucoside, peonidin–3–O–glucoside, and petunidin–3–O–glucoside were 14.6, 11.5, 9.7, 10.0, 7.8, 9.7, and 10.3 µmol–TE/mg, respectively. For proanthocyanidins, H–ORAC values of monomers, dimers, trimers, tetramers, pentamers, hexamers, and heptamers were 43.3, 56.2, 44.8, 38.5, 30.7, 13.6, and 13.4 µmol–TE/mg, respectively.
Theoretical H–ORAC values generated by each compound were calculated, and the contribution was estimated, as shown in Table 3. Anthocyanins in black cowpeas and black kidney beans strongly contributed to H–ORAC values (black cowpeas, 29.1%; black kidney beans, 28.5%), while anthocyanins in red cowpeas and azuki beans had poor contribution to H–ORAC values. Regarding proanthocyanidin oligomers, higher contributions were observed in red cowpeas, black cowpeas, and azuki beans of 17.3%, 16.6%, and 16.0%, respectively, while the lowest was observed in black kidney beans (6.2%). In all grain legumes, the contribution of monomers, dimers, trimers, tetramers, or pentamers was more than twice that of hexamer or heptamers.
| Sample | Black cowpea | Red cowpea | Black kidney bean | Black soybean | Azuki bean |
|---|---|---|---|---|---|
| Seed coat color | Black | Red | Black | Black | Red |
| Cyanidin–3–O–galactoside | 0.42 ± 0.01f | nd | nd | 0.13 ± 0.03e | nd |
| Cyanidin–3–O–glucoside | 12.15 ± 0.44a | 0.10 ± 0.00e,f | nd | 17.57 ± 1.25a | nd |
| Delphinidin–3–O–galactoside | 0.76 ± 0.04f | nd | 0.57 ± 0.05d | nd | nd |
| Delphinidin–3–O–glucoside | 5.69 ± 0.34b | nd | 17.56 ± 1.14a | nd | nd |
| Malvidin–3–O–glucoside | 3.65 ± 0.03c,d | 0.05 ± 0.02e,f | 3.20 ± 0.28c | nd | 0.04 ± 0.03c |
| Peonidin–3–O–glucoside | 2.12 ± 0.01e | nd | nd | 0.11 ± 0.03e | nd |
| Petunidin–3–O–glucoside | 4.35 ± 0.19c | 0.02 ± 0.01f | 7.15 ± 0.41b | nd | nd |
| Total anthocyanins | 29.14 ± 1.04 | 0.17 ± 0.02 | 28.47 ± 1.85 | 17.81 ± 1.27 | 0.04 ± 0.03 |
| Proanthocyanidins | |||||
| Monomers | 2.81 ± 0.55d,e | 2.67 ± 0.21b | 1.08 ± 0.09d | 2.99 ± 0.36b | 1.68 ± 0.24b |
| Dimers | 3.96 ± 0.68c | 5.86 ± 0.38a | 2.25 ± 0.34c | 2.70 ± 0.31b,c | 6.98 ± 0.81a |
| Trimers | 2.51 ± 0.48e | 2.73 ± 0.27b | 0.84 ± 0.12d | 1.47 ± 0.18c,d | 2.41 ± 0.07b |
| Tetramers | 2.68 ± 0.55d,e | 2.71 ± 0.09b | 0.61 ± 0.05d | 1.61 ± 0.14c,d | 2.21 ± 0.20b |
| Pentamers | 2.80 ± 0.55d,e | 2.17 ± 0.06c | 0.86 ± 0.08d | 1.89 ± 0.20b,c,d | 1.57 ± 0.17b |
| Hexamers | 0.98 ± 0.19f | 0.66 ± 0.03d | 0.30 ± 0.03d | 0.74 ± 0.07d,e | 0.62 ± 0.06c |
| Heptamers | 0.91 ± 0.18f | 0.51 ± 0.02d,e | 0.28 ± 0.05d | 0.74 ± 0.05d,e | 0.46 ± 0.02c |
| Total proanthocyanidin oligomers | 16.64 ± 3.16 | 17.30 ± 0.48 | 6.23 ± 0.73 | 12.14 ± 1.30 | 15.92 ± 0.59 |
Results are represented as the mean ± standard deviation (n = 3). Within each column, means followed by different lowercase letters are statistically different (P < 0.05). Abbreviation: nd, not detected.
In Japan, cowpeas are classified as a minor grain legume, although they are consumed as a high-quality plant protein source in many parts of the world. In addition, cowpeas are known to be rich in phytochemicals, which appear to contribute to their health benefits. In order to obtain useful information on bioactive phytochemicals and promote their use as a functional food ingredient, we focused on anthocyanins and proanthocyanidins in cowpeas grown in Japan. This study confirmed that cyanidin–3–O–glucoside, delphinidin–3–O–glucoside, malvidin–3–O–glucoside, and petunidin–3–O–glucoside are the major anthocyanins in black cowpeas. These mono-glucoside anthocyanins accounted for approximately 90% of total anthocyanins. In addition, three minor anthocyanins were identified as cyanidin–3–O–galactoside, delphinidin–3–O–galactoside, and peonidin–3–O–glucoside in black cowpeas. Ha et al. (2010) and Ojwang et al. (2012) have already reported the same seven anthocyanins as being present in black cowpeas. Thus, the species of anthocyanins in black cowpeas grown in Japan were similar to those grown in other countries, although the composition ratio and content may vary with cultivation area. The anthocyanin contents in black cowpeas were about five times higher than in black soybeans. It has been reported that the anthocyanin contents in black soybeans vary according to cultivar and cultivation area in Japan (Oki et al., 2013); therefore, when compared to black soybeans, black cowpeas appear to have higher and/or comparable anthocyanin contents. On the other hand, the anthocyanin contents in red cowpeas were low, similar to red azuki beans.
With regard to proanthocyanidins, monomers through heptamers were detected and quantified in both black and red cowpeas grown in Japan. The sum of proanthocyanidin oligomers contents (degree of polymerization one to seven) in both black and red cowpeas was more than twice that in azuki beans, black soybeans, or black kidney beans. The composition ratio, on a weight basis, of each proanthocyanidin (monomers to heptamers) was slightly different between red and black cowpeas. The difference in the proanthocyanidin composition among the five grain legumes was relatively small, in contrast to the difference in anthocyanin composition. It was also demonstrated that proanthocyanidins were present in cowpeas by the vanillin-sulfuric acid method, which detects monomeric and polymeric proanthocyanidins. The total proanthocyanidin contents, determined by the vanillin-sulfuric acid method, in both black and red cowpeas were higher than in other grain legumes. On the other hand, the ratio of the sum of monomeric to heptameric proanthocyanidins to the total proanthocyanidin contents in black cowpeas (18.3%) was almost same as in red cowpea (20.9%). The highest was observed in black soybean (61.8%). This indicates that the proportion of oligomeric proanthocyanidins among total proanthocyanidins in cowpeas is lower than that in black soybeans.
Grain legumes are known to be a source of natural antioxidants (Amarowicz and Pegg, 2008). This study used the H–ORAC method to evaluate antioxidant capacities, and a comparison of H–ORAC vales was performed between cowpeas grown in Japan and grain legumes with black and red seed coat, which are commonly consumed in Japan. Black cowpeas were found to possess 2.9 and 5.5 times higher H–ORAC values than black soybeans and black kidney beans, respectively. In addition, H–ORAC values in red cowpeas were 2.5 times higher when compared with red azuki beans. Moreover, the H–ORAC values of cowpeas grown in Japan were higher than in those grown in Korea (Cho et al., 2007) and Mexico (Gutiérrez-Uribe et al., 2011). This might be caused by differences in variety, cultivation area, etc. In addition, total phenolic compounds in black and red cowpeas were higher than in black kidney beans, black soybeans, and red azuki beans. Thus, cowpeas appeared to have the highest antioxidant capacity, accompanied by higher phenolic contents, among legumes commonly consumed in Japan. It was also found that black soybeans showed higher H–ORAC values than azuki beans, although total polyphenol content was lower. The reasons are unclear, but isoflavones may be responsible, as soybeans are rich in isoflavones with higher H–ORAC values (Wang and Murphy, 1994).
We then examined the contributions of anthocyanins and proanthocyanidins, which are known to be potent antioxidants, to the overall antioxidant capacity. This study clarified that the contributions of anthocyanins and proanthocyanidins in black cowpeas were approximately 29.1% and 16.6%, respectively. In comparison with other black legumes, its contribution of anthocyanins was almost the same as in black kidney beans, and was higher than that in black soybeans. On the other hand, its contribution of proanthocyanidins was higher than both black kidney beans and black soybeans. In red cowpeas, the contribution of proanthocyanidins was almost the same as in black cowpeas (17.3%), while anthocyanins had nearly no contribution because of their low levels. In azuki beans with red seed coat, the estimated contribution was almost the same as in red cowpeas, while the antioxidant capacity of azuki beans was about 40% that of red cowpeas. In black cowpea, the contribution of proanthocyanidin oligomers to H-ORAC was more than half of that of anthocyanins, although the total content of proanthocyanidin oligomers was less than 20% of that of anthocyanins, and large difference was observed between the contribution to H–ORAC and the content in compar ison with proanthocyanidin oligomers and anthocyanins. This was thought to be because the H–ORAC values of each proanthocyanidin oligomer standard, particularly monomers to pentamers, were higher than those of each anthocyanin standard (monomers to pentamers > hexamers and heptamers ≥ anthocyanins). In addition, in grain legumes, leucoanthocyanidin has been known to be a precursor common to both anthocyanins and proanthocyanidins (Xie and Dixon, 2005), so it is presumed that legumes with red seed coat possess higher antioxidant activity than those with black seed coat due to the existence of proanthocyanidin oligomers. However, black legumes accumulated proanthocyanidins other than anthocyanins in the seed coat. Therefore, it is speculated that legumes with black seed coat, which contains both anthocyanins and proanthocyanidin oligomers, exhibit higher H–ORAC values, when compared to those with red seed coat.
In general, anthocyanins and proanthocyanidins are located in the seed coats of grain legumes and appear to be one of the most predominant polyphenols in red and black grain legumes. On the other hand, other polyphenols, such as flavonoids, with antioxidant capacity have been found in the embryo and cotyledon of grain legumes. This study suggests that the major candidates for antioxidant capacities would be anthocyanins and proanthocyanidins in black cowpeas and proanthocyanidins in red cowpeas, although other contributors would exist in the embryo and cotyledon. Cai et al. (2003) and Ojwang et al. (2012) have identified phenolic acid and flavonoids, such as protocatechuic acid, myricetin glycoside, and kaempferol glycoside, in cowpeas. It is possible that these flavonoids exhibited antioxidant capacity; however, the contributions of these compounds to antioxidant capacities are unclear, as this study focused on anthocyanins and proanthocyanidins in cowpeas and other grain legumes.
In conclusion, black cowpeas grown in Japan contained seven species of anthocyanins at higher levels, when compared with grain legumes with the same seed color (black soybeans and black kidney beans). These chemical structures were the 3–O–glucoside forms of cyanidin, delphinidin, malvidin, peonidin, and petunidin, and the 3–O–galactoside forms of cyanidin and delphinidin. Black and red cowpeas also contained proanthocyanidin monomers to heptamers with higher contents than red azuki beans, black soybeans, and black kidney beans. In addition, black and red cowpeas showed potent antioxidant capacity, accompanied by higher total phenolic content. In black and red cowpeas, the contributions of anthocyanins and proanthocyanidin oligomers to H–ORAC values were 45.8% and 17.5%, respectively. The results generated from this study may help to utilize cowpeas as a nutraceutical ingredient for promoting health. However, further study will be needed to clarify compounds other than anthocyanins and proanthocyanidin oligomers responsible for antioxidant activity in black and red cowpeas.
Acknowledgements This study was funded by a specific (Japanese) government grant for Okinawa No. 74.
catechin equivalents
DMdry matter
GAEgallic acid equivalents
HPLChigh-performance liquid chromatography
H–ORAChydrophilic oxygen radical absorbance capacity
TETrolox equivalents
