Comparative study on antioxidant activity of different varieties of commonly consumed legumes in India

Food and Chemical Toxicology 49 (2011) 2005–2012

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Comparative study on antioxidant activity of different varieties of commonly consumed legumes in India Sushama A. Marathe ⇑, V. Rajalakshmi, Sahayog N. Jamdar, Arun Sharma Food Technology Division, Bhabha Atomic Research Center, Trombay, Mumbai 400 085, India

a r t i c l e

i n f o

Article history: Received 3 January 2011 Accepted 29 April 2011 Available online 12 May 2011 Keywords: Legumes Antioxidant activity Phenolics DPPH radical scavenging activity Fe2+ chelating activity

a b s t r a c t Legumes are rich source of proteins, dietary fiber, micronutrients and bioactive phytochemicals. Thirty different varieties of commonly consumed legumes in India, were screened for phenolic content and antioxidant activity using, radical scavenging [(1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,20 -azino-bis (3ethylbenz-thiazoline-6-sulfonic acid, (ABTS+)], Ferric Reducing Antioxidant Power (FRAP) and metal ion (Fe2+) chelation assays. Legumes varied largely in their antioxidant activity. Horse gram, common beans, cowpea (brown and red) and fenugreek showed high DPPH radical scavenging activity (>400 units/g), while lablab bean (cream and white), chickpea (cream and green), butter bean and pea (white and green) showed low antioxidant activity (<125 units/g). Green gram, black gram, pigeon pea, lentils, cowpea (white) and common bean (maroon) showed intermediate activity. Similar trend was observed when the activity was assessed with ABTS+ and FRAP assays. Thus most of the varieties having light color seed coat, except soybean exhibited low antioxidant activity. While legumes having dark color seed coat did not always possessed high antioxidant activity (e.g. moth bean, black pea, black gram, lentils). Antioxidant activity showed positive correlation (r2 > 0.95) with phenolic contents, in DPPH, ABTS+ and FRAP assays, whereas poor correlation (r2 = 0.297) was observed between Fe2+ chelating activity of the legumes and phenolic contents. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The nutritional value of legumes as a source of good quality proteins and micronutrients in the daily diet of Indians is very significant. This is mainly due to lower costs of legume proteins compared to the animal proteins. Supplementing cereal based diets with legumes improves overall nutritional status and is one of the best solutions to prevent protein calorie malnutrition observed in developing countries. Consumption of legumes is also correlated to number of positive health benefits (Messina, 1999) such as hypocholesterolemic, antiatherogenic, anticarcinogenic and hypoglycemic properties (Cardador-Martinez et al., 2002). These health benefits are known to be associated with dietary fiber and phytochemicals like polyphenols present in legumes. It is generally believed that antioxidants scavenge free radicals and reactive oxygen species and thus inhibit oxidative mechanisms which lead to degenerative diseases. Naturally existing biological antioxidant mechanisms though combat oxidative stress in the mammalian system; natural antioxidants from diets strengthen ⇑ Corresponding author. Tel.: +91 022 25593962; fax: +91 022 25505151. E-mail addresses: [email protected], [email protected] (S.A. Marathe). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.04.039

the endogenous antioxidant defenses (Benzie, 2003). An increasing number of epidemiological studies have shown an inverse correlation between the consumption of dietary antioxidants and incidence of various diseases including cancer and heart disease (Kris-Etherton et al., 2002). Polyphenols, particularly tannins have been considered as antinutrients, due to their complex formation with proteins, resulting in reduction of protein digestibility. However, more recently these compounds are considered as important dietary antioxidants (Bravo, 1998; Siddhuraju, 2006). They can act as reducing agents (free radical terminators), metal ion chelators and singlet oxygen quenchers (Rice-Evans et al., 1996) and thus prevent oxidative damage to biomolecules, such as DNA, lipids and proteins. Many scientists have documented antioxidant potential of polyphenols in fruits and vegetables and their correlation in reducing the incidence of degenerative diseases (Kaur and Kapoor, 2001). However, such studies in legumes are sparse, though, few short studies on screening of in vitro antioxidant activity of legumes have been documented (Latha and Daniel, 2001; Sreeramulu et al., 2009; Tsuda et al. ,1993). Also, the need of preparing phytochemical index of plant products has been felt (McCarty, 2004; Pennington, 2002; Rao, 2003). Therefore, it was planned to assess the antioxidant potential and its relationship with the phenolic content of commonly

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consumed legumes in India. Antioxidants may respond in a different manner to different radicals or oxidants due to multiple mechanisms involved, therefore, four different assay systems were used to evaluate antioxidant activity. 2. Materials and methods 2.1. Reagents and chemicals Gallic acid, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,20 -azino-bis (3-ethylbenz-thiazoline-6sulfonic acid) (ABTS) and ethylene diamine tetra acetic acid (EDTA) were purchased from Sigma Chemical Co. St Louis, MO, USA. 2,4,6-Tripridyl-s-triazine (TPTZ), Folin– Ciocalteu’s reagent and ferrozine were purchased from Himedia Laboratories, Mumbai, India. All other chemicals used were of analar grade and solvents were distilled prior to use. 2.2. Samples Different varieties of legumes were purchased from a three outlets of local grocery stores in Mumbai, India, which served as triplicate samples and 25 g of each legume was ground using kitchen grinder to get fine flour and sieved through 710 lm sieve. The flour was immediately used for extraction. 2.3. Extract preparation 1 g of flour was mixed with 15 ml of 80% aqueous methanol (V/V) and incubated at room temperature with continuous stirring for 2 h (using an orbital shaker, 150 rpm). After the incubation the mixture was centrifuged at 5000 rpm for 10 min. Supernatants were filtered through filter paper (Whatman No. 1) and the residue was re-extracted with 15 ml of 80% methanol for 1 h. The volume of pooled extracts was made up to 30 ml using 80% methanol. All the extracts were made in duplicate. Extracts were stored at 20 °C till analysis. 2.4. Estimation of phenolics The concentration of phenolics in legume extracts was measured using Folin– Ciocalteu assay (Hung and Yen, 2002). The assay mixture consisted of, appropriately diluted 0.2 ml extract, 0.1 ml Folin–Ciocalteu reagent (1 N), and 2.0 ml of 2.0% Na2CO3 solution. The absorbance of the mixture was measured after 30 min, at 750 nm, using a spectrophotometer (Varian DMS 100, USA). Standard curve was constructed using gallic acid. Phenolic contents were expressed as mg of gallic acid equivalent (GAE)/g legume. 2.5. Estimation of antioxidant activity of legume extracts 2.5.1. Estimation of DPPH scavenging activity Antioxidant activity was assessed using the stable free radical, 2, 2-Diphenyl-1picrylhydrazyl (DPPH), by the method of Brand-Williams et al. (1995), with some modifications. The methanolic stock solution of DPPH was prepared at concentration of 270 mg/L. A suitably diluted extract was added to working methanolic solution of DPPH and the volume was made to 1 ml. Since percent scavenging is dependent on initial DPPH concentration, in all the assays, the final concentration of DPPH was maintained at 75 lM. This concentration of DPPH gave an absorbance of 0.93 (as determined by calibration curve equation given by Brand-Williams et al. (1995)), where A515nm ¼ 12509  molar concentration  0:00258, which gives sufficient reaction capacity for higher content of antioxidants in extracts. The mixture was shaken vigorously and incubated in dark at room temperature, for 30 min. The absorbance of resulting solution was then measured at 515 nm. Using various concentrations of extracts, percent scavenging was determined and plot of percent scavenging versus concentration was constructed. EC50, the concentration of extract required to achieve 50% scavenging, was determined.

%Scavenging A ¼ ðAbsorbancecontrol  Absorbancesample =Absorbancecontrol  100 The antioxidant activity was expressed as units/g legume. A unit of DPPH scavenging activity was defined as the amount of antioxidant necessary to scavenge 50% of 75 nmol of DPPH. 2.5.2. Estimation of ABTS+ scavenging activity ABTS+ radical-scavenging activity of extracts was determined according to Re et al. (1999). The ABTS+ (cation radical) was produced by the reaction between 5 ml of 14 mM ABTS solution and 5 ml of 4.9 mM potassium persulfate (K2S2O8) solution, stored in the dark at room temperature for 16 h. Before use, this solution was diluted with distilled water to get an absorbance of 0.900 ± 0.020 at 734 nm. In a final volume of 1 ml, the reaction mixture comprised 0.8 ml of ABTS+ solution and 0.2 ml of the sample extract at various concentrations. The decrease in absorbance value was measured at 734 nm after 6 min. Standard solution of Trolox was used for

the preparation of calibration curve. The percent scavenging of ABTS+ (cation radical) was calculated as ðAbsorbancecontrol  Absorbancesample =Absorbancecontrol  100 and antioxidant activity was expressed as lmol TEAC (Trolox Equivalent Antioxidant Capacity)/g legume. 2.5.3. Estimation of FRAP (Ferric Reducing Antioxidant Power) The assay was carried out as described by Benzie and Strain (1996) with some modification. Freshly prepared FRAP reagent (0.8 ml) was mixed with 0.2 ml of appropriately diluted sample. The FRAP reagent was prepared by mixing 0.3 M acetate buffer (pH 3.6), aqueous 10 mM TPTZ in 40 mM HCl and 20 mM FeCl36H2O in the ratio of 10:1:1. After 10 min of incubation at room temperature, the absorbance was measured at 593 nm. Freshly prepared standard solution of FeSO47H2O was used for calibration and FRAP activity was expressed as lmol/g legume. 2.5.4. Estimation of metal ion (Fe2+) chelating activity The Fe2+ chelating activity of the extract was measured as reported by LiyanaPathirana and Shahidi (2007) with modification. The reaction mixture consisted 0.4 ml of extract, 0.285 ml of double distilled water, 0.275 ml of FeCl24H2O (0.2 mM) and 0.04 ml of ferrozine (5 mM). Absorbance was measured at 562 nm after 10 min, EDTA solution was used as the standard. All reagents were prepared in double distilled water. The lower absorbance of the reaction mixture indicated higher Fe2+ chelating ability. Fe2+ chelating activity (%) was calculated as follows and was expressed as lmol of EDTA equivalent/g legume using EDTA calibration curve.

Chelating activity ð%Þ ¼ ðAbsorbancecontrol  Absorbancesample =Absorbancecontrol  100

2.6. Statistical methods All data were expressed as mean ± standard deviation. Analysis of variance was performed by ANOVA procedures. P < 0.01 was considered to be statistically significant.

3. Results and discussion Legumes evaluated for antioxidant activity are listed in Table 1. 3.1. Phenolic contents in legumes Table 2 shows phenolic content of whole legumes. Phenolic content of legumes varied in the range of 0.325–6.378 mg GAE/g. The variation in phenolic content is known to be due to genetic factors, degree of maturity and environmental conditions. Secondly, extractability of phenolic compounds is governed by the type of solvent (polarity) used, degree of polymerization of phenolics, interaction of phenolics with other food constituents, as well as the extraction time and temperature. Thus, there is no uniform or completely satisfactory procedure that is suitable for extraction of all phenolics from plant materials (Naczka and Shahidi, 2004). However, 80% aqueous methanol has been reported to be most suitable solvent system (Zielinski and Kozlowska, 2000). Hence, the same solvent system has been used for the extraction of phenolics, in our studies. For convenience the legumes were categorized, depending on their phenolic content into three groups (Table 2), as low-L (<1.0 mg GAE/g), moderate-M (1.0–2.0 mg GAE/g) and high-H (>2.0 mg GAE/g). Lablab bean (white and cream), chickpea (cream, green and big-brown), moth bean, butter bean, lentil and pea (white and green), showed low phenolic content. While common bean (white and maroon), lablab bean (brown), pea (black), pigeon pea, green gram (green and yellow), chickpea (small-brown), cowpea (white) and black gram showed moderate phenolic content. Xu and Chang (2008) have reported phenolic content of green pea, yellow pea, and chickpea as 1.22, 1.38, and 1.44 mg GAE/g, respectively. These values are comparable with our data. Cowpea (red and brown), soybean, common bean (black, red, brown, and beige), peanut, fenugreek and horse gram showed high phenolic content (2.0–6.4 mg GAE/g). The outer layers of seed i.e. seed coat usually contain a higher amount of polyphenolic compounds as expected

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from their protective function in the plants. We tried to find out whether there is any correlation between seed coat color and phenolic content. Most of the varieties (except soybean) having light color seed coat contained low phenolics, while not all legumes with dark color seed coat contained high amount of phenolics. The legumes for example lentils, moth bean, black pea, lablab bean (brown) and pigeon pea, though having dark color seed coat, contained low to medium quantity of phenolics (<1.2 mg GAE/g). Among lablab beans, the highest content of phenolics was observed in brown colored beans followed by cream and white (P < 0.01). Similarly in pea with black colored seed coat exhibited highest phenolics content followed by pea with green and white seed coat (P < 0.01). Cowpea (brown) had high phenolics content (6.378 mg GAE/g); while white variety had moderate phenolics

Table 2 Phenolic contents and antioxidant activity of legumes. Legumesa

Synonyms

Botanical name

Indian name

Lablab bean (cream)

Dolichos lablab

Pawta, Sem

Dolichos lablab

Vaal, Sem

Dolichos lablab

Rangoon vaal

Pisum sativum

Pisum sativum

Mater, Vatana Mater, Vatana Mater,Vatana

Cicer arietinam

Kabuli Chana

Cicer arietinam

Chana

Cicer arietinam

Chana

Cicer arietinam

Chana

Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Vigna radiata

Rajma

Vigna radiata

Mung

Vigna mungo

Urad

Cowpea (brown)

Field bean, Hyacinth bean Field bean, Hyacinth bean Field bean, Hyacinth bean Garden pea, Field pea Garden pea, Field pea Garden pea, Field pea Bengal gram, Indian pea Bengal gram, Indian pea Bengal gram, Indian pea Bengal gram, Indian pea Kidney bean, French bean Pinto bean, French bean Pinto bean, French bean Navy bean, French bean Kidney bean, French bean Turtle bean, French bean Green bean, Mung bean Golden gram, Mung bean Urad bean, Black bean Black eyed pea

Chawli, Lobia

Cowpea(white)

Black eyed pea

Cowpea (maroon)

Black eyed pea

Moth bean (brown) Broad bean (white) Lentil (grey-brown) Fenugreek (yellow)

Mat bean Butter bean – –

Horse gram (brown)

Dolichos biflorus

Soybean (yellow) Pigeon pea (maroon) Peanut (pink)

Soya Red gram Groundnut

Vigna unguiculata Vigna unguiculata Vigna unguiculata Vigna aconitifolia – Lens culinaris Trigonella foenumgraecum Macrotylama uniflorum Glycine max Cajanus cajan Arachis hypogaea

Lablab bean (brown) Lablab bean (white) Pea (white) Pea (green) Pea (black) Chickpea (cream) Chickpea (bigbrown) Chickpea (smallbrown) Chickpea (green) Common bean (maroon) Common bean (beige-mottled) Common bean (brown-mottled) Common bean (white) Common bean (red) Common bean (black) Green gram (green) Green gram (yellow) Black gram (black)

Seed coat color is given in parenthesis.

Pisum sativum

ABTSd

FRAPe

Chelationf

L

L

L

L M L L

L L L L

L M M L

L

L

H

L

L

H

M L L

M L L

M L M

M

H

M

H

M M M

L M M

L

L

M M

H M

M

H

M

M

H H H

H H H

M H H

H

H

L

M H

M H

H M

H H

H H

H M

H H

H H

L H

B – Moderate phenolic contents (>1.0 and <2.0 mg GAE/g) Common bean 1.014 ± 0.020 L M (white) Lablab bean 1.017 ± 0.038 M M (brown) Pea (black) 1.040 ± 0.040 M M Pigeon pea 1.054 ± 0.011 M M Green gram 1.217 ± 0.030 M M (yellow) Chickpea (small 1.359 ± 0.046 M M brown) Cowpea (white) 1.480 ± 0.028 M M Common bean 1.600 ± 0.037 M M (maroon) Green gram 1.834 ± 0.016 M M (green) Black gram 1.878 ± 0.029 M M C – High phenolic contents (>2.0 mg GAE/g) Cowpea (red) 2.086 ± 0.058 H Soybean 2.170 ± 0.062 M Common bean 2.184 ± 0.036 H (black) Common bean 2.406 ± 0.022 H (brown) Peanut 2.448 ± 0.055 H Common bean 3.235 ± 0.061 H (beige) Horse gram 3.579 ± 0.072 H Common bean 3.583 ± 0.059 H (red) Fenugreek 4.298 ± 0.072 H Cowpea (brown) 6.378 ± 0.054 H

Rajma Rajma Rajma Kashmiri rajma Rajma

DPPHc

A – Low phenolic contents (<1.0 mg GAE/g) Lablab bean 0.325 ± 0.002 L (white) Chick pea (cream) 0.460 ± 0.053 L Moth bean 0.480 ± 0.010 L Chick pea (green) 0.491 ± 0.002 L Chick pea (big 0.514 ± 0.009 L brown) Butter bean 0.648 ± 0.036 L (white) Lablab bean 0.693 ± 0.017 L (cream) Lentil 0.914 ± 0.115 M Pea (white) 0.938 ± 0.039 L Pea (green) 0.966 ± 0.040 L

Table 1 List of legumes studied. Common name

Phenolics (mg GAE/g)b

a

Seed coat color is given in parenthesis. The values presented are mean ± SD computed from six replicates. DPPH activity. L, <125 units/g legume; M, 125–400 units/g legume; H, >400 units/g legume. d ABTS+ activity: L, <6.0 lmol TEAC/g legume; M, 6.0–12 lmol TEAC/g legume; H, >12.0 lmol TEAC/g legume. e FRAP activity: L, <8.5 lmol FRAP/g legume; M, 8.5–16.5 lmol FRAP/g legume; H, >16.5 lmol FRAP/g legume. f Metal ion chelating activity: L, <0.6 lmol EDTA eq/g legume; M, 0.6–1.0 lmol EDTA eq/g legume; H, >1.0 lmol EDTA eq/g legume. b

Mung

c

Chawli, Lobia Goa bean Moth, Matki Double bean Masur Methi Kulith Soya Arhar, Tur Mungphali

content (1.48 mg GAE/g). In case of common beans also white variety had the least amount of phenolics as compared to other color varieties. In chickpea varieties, one with cream color, known as Kabuli chana had least amount of phenolics followed by green and brown (P < 0.01). It was also observed that seed size could also significantly influence the total phenolics content and hence the antioxidant activity. For instance brown colored chickpea, though same in seed coat color varied in their phenolic content depending upon size of the seeds exhibiting lower content of phenolics (0.514 mg GAE/g), for bigger seeds and higher (1.359 mg GAE/g) for smaller sized seeds. To understand this phenomenon, the seed coats of both the varieties were isolated and weighed. The weights

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Fig. 1. DPPH free radical scavenging activity of legumes. Antioxidant activity is measured by DPPH free radical scavenging assay. Seed coat color is given in parenthesis. The values shows mean of three independent experiments ± SD. (A) <125 units/g legume; (B) >125 and <400 units/g legume; (C) >400 units/g legume.

of seed coat/g seeds were 96.01 and 132.48 mg, for big and small seeds, respectively. This could be due to the fact that the ratio of surface area (seed coat area)/g, of seeds vary inversely with seed size. Thus, bigger seeds have smaller seed coat area when explained per gram basis and so lesser amount of seed coat and phenolics. 3.1.1. DPPH free radical scavenging activity 2,2-Diphenyl-1-picrylhydrazyl (DPPH), radical is one of the few stable organic nitrogen radicals, which has a deep purple color. It is commercially available and unlike ABTS, it does not have to be generated before assay. The DPPH assay is considered to be mainly

based on an electron transfer reaction, and hydrogen-atom abstraction (Prior et al., 2005). This assay is based on the measurement of the reducing ability of antioxidants toward DPPH. The ability can be evaluated by measuring the decrease of its absorbance or electron spin resonance (EPR). DPPH method is interpreted on the basis of antioxidant amount required to decrease the initial DPPH concentration to 50% (EC50). Since percent scavenging is dependent on initial DPPH concentration, comparison between data of two laboratories becomes difficult. To make comparison easier, we have tried to interpret data in terms of units/g legume, using fixed initial concentration of DPPH (75 lM) and (EC50) value for this concentration. This gives more objective

S.A. Marathe et al. / Food and Chemical Toxicology 49 (2011) 2005–2012 Table 3 FRAP and ABTS+ radical scavenging activity of legumes. Common namea Lablab bean Lablab bean Lablab bean Pea Pea Pea Chickpea Chickpea Chickpea Chickpea Common bean Common bean Common bean Common bean Common bean Common bean Green gram Green gram Black gram Cowpea Cowpea Cowpea Moth bean Butter bean Lentil Fenugreek Horse gram Soya bean Pigeon pea Peanut a b

(Cream) (Brown) (White) (White) (Green) (Black) (White) (Big-brown) (Small-brown) (Green) (Maroon) (Beige) (Brown) (White) (Red) (Black) (Green) (Yellow) (Black) (Brown) (White) (Red) (Brown) (White) (Grey brown) (Yellow) (Brown) (Yellow) (Red) (Pink)

FRAP (lmol/g)b

ABTS+ (lmol TEAC/g)b

06.257 ± 0.128 09.242 ± 0.317 04.333 ± 0.098 08.273 ± 0.246 05.809 ± 0.249 09.000 ± 0.274 04.109 ± 0.228 05.424 ± 0.296 07.391 ± 0.562 05.366 ± 0.255 15.094 ± 0.347 33.117 ± 1.374 22.931 ± 0.523 10.221 ± 0.293 35.407 ± 1.203 30.983 ± 0.546 08.901 ± 0.207 10.111 ± 0.151 10.237 ± 0.159 68.030 ± 2.358 14.256 ± 0.607 22.797 ± 1.145 06.743 ± 0.088 05.908 ± 0.100 09.604 ± 0.256 37.852 ± 0.399 44.859 ± 1.789 17.743 ± 0.399 16.196 ± 0.276 10.047 ± 0.480

04.583 ± 0.247 06.901 ± 0.132 03.434 ± 0.050 05.482 ± 0.186 04.973 ± 0.027 06.178 ± 0.051 04.079 ± 0.290 05.599 ± 0.156 06.555 ± 0.123 04.365 ± 0.123 10.101 ± 0.242 22.678 ± 0.673 16.664 ± 0.737 06.039 ± 0.019 23.856 ± 1.323 20.464 ± 0.565 07.220 ± 0.063 09.000 ± 0.109 08.324 ± 0.181 46.261 ± 1.448 09.344 ± 0.217 17.070 ± 0.029 07.341 ± 0.066 04.277 ± 0.105 06.462 ± 0.048 20.209 ± 0.856 30.418 ± 0.861 12.748 ± 0.137 11.319 ± 0.497 11.508 ± 0.537

Seed coat color is given in parenthesis. The values present mean of three independent experiments ± SD.

comparison of the results by applying the same interpretation procedure with the unified standardized method. Since ABTS radical scavenging capacity is expressed as Trolox Equivalent Antioxidant Capacity, to avoid confusion, unit of DPPH free radical scavenging activity was based on original DPPH concentration. Our interpretation of data is comparable to Liyana-Pathirana and Shahidi (2007) who had interpreted the data in terms of lM of DPPH and used initial concentration of DPPH as 75 lM. The DPPH free radical scavenging activity of different varieties of legumes is shown in Fig. 1 and Table 2. The data was categorized depending on their DPPH free radical scavenging activity: low-L (DPPH units/g < 125), moderate-M (125–400, DPPH units/g) and high-H (>400 DPPH units/g). Lablab bean (white and cream), chickpea (cream, green and big brown), moth bean, butter bean, pea (white and green) and common bean (white), showed low antioxidant activity (Fig. 1A and Table 2). These legumes also had low phenolic content except common bean (white), which had phenolic contents in moderate range. Lablab bean (brown), pea (black), pigeon pea, green gram (green and yellow), chick pea (small-brown), cowpea (white), common bean (maroon), black gram, lentil and soybean exhibited moderate antioxidant activity (Fig. 1B and Table 2). All the legumes in this group showed phenolic content in the range of 1.0–2.0 mg GAE/g, except for lentils and soybean, which had marginally low and high phenolic content (0.914 mg GAE/g and 2.17 mg GAE/g), respectively. The third group (Fig. 1C and Table 2) with high antioxidant activity, comprised of, cowpea (red and brown), common beans (black, brown, beige and red), peanut, horse gram and fenugreek, also contained high amount of phenolics as well. 3.1.2. ABTS+ free radical scavenging activity The data on ABTS+ assay is given in Tables 2 and 3. ABTS+ is soluble in both aqueous and organic solvents and is not affected by

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ionic strength, so can be used in multiple media to determine both hydrophilic and lipophilic antioxidant capacities of extracts and body fluids (Prior et al., 2005). Legumes were grouped depending on their TEAC values (Tables 2 and 3), as low-L (<6.0 lmol TEAC/g), moderate-M (6.0–12.0 lmol TEAC/g) and high-H (>12.0 lmol TEAC/g). The legumes with low phenolic content like, lablab bean (white and cream), chickpea (cream, green and bigbrown), butter bean and pea (white and green) showed low antioxidant activity (<6.0 lmol TEAC/g). While moth bean and lentil, though low in phenolics, showed relatively higher antioxidant activity (7.34 and 6.46 lmol TEAC/g, respectively). Legumes with moderate phenolic content, such as common bean (white), lablab bean (brown), pea (black), pigeon pea, green gram (yellow and green), chickpea (small-brown), cowpea (white), common bean (maroon) and black gram showed moderate antioxidant activity (in the range 6.0–12 lmol TEAC/g). Legumes with high phenolic content like, cowpea (red and brown), soybean, common beans (black, brown, beige and red), horse gram and fenugreek showed high antioxidant activity (>12.0 lmol TEAC/g), while, peanut though with high phenolic group showed slightly low TEAC value (11.508 lmol TEAC/g). 3.1.3. FRAP (Ferric Reducing Antioxidant Power) The FRAP assay was originally developed by Benzie and Strain (1996), to measure reducing power in plasma, but the assay subsequently has also been adapted and used for the assay of antioxidants in botanicals. The reaction measures reduction of ferric 2,4,6-tripyridyl-s-triazine (TPTZ) to a colored product. Legumes were grouped depending on their FRAP values (Tables 2 and 3) as low-L (<8.5 lmol/g), moderate-M (8.5–16.5 lmol/g) and high-H (>16.5 lmol/g). The legumes with low phenolic content namely, lablab bean (white and cream), chickpea (cream, green and big-brown), moth bean, butter bean and pea (white and green) showed low FRAP values (<8.5 lmol/g). Lentil, though had low phenolic content, showed moderate FRAP values (9.60 lmol/g). Legumes with moderate phenolic content, such as common bean (white), lablab bean (brown), pea (black), pigeon pea, green gram (green and yellow), cowpea (white), common bean (maroon) and black gram showed moderate FRAP values. Chickpea (small-brown) though moderate in phenolic content showed low FRAP value (7.39 lmol/g). Legumes with high phenolic content namely, cowpea (red and brown), soybean, common beans (black, brown, beige and red), horse gram and fenugreek also had high of FRAP values, except peanut, which showed moderate FRAP value (10.05 lmol/g). 3.1.4. Metal ion chelating activity Fe2+ chelation is an important antioxidative mechanism which retards metal-catalyzed oxidation (Kehrer, 2000). The effective Fe2+ chelators afford protection against oxidative damage by removing Fe2+ that may otherwise participate in HO generating Fenton type reactions. This in turn can give protection against oxidative damage by inhibiting production of ROS and lipid peroxidation. The Fe2+ chelating capacity of various legumes was determined by measuring the iron–ferrozine complex and results are summarized in Table 2 and Fig. 2. Legumes were grouped depending on their antioxidant activity as low-L (<0.6 lmol EDTA eq/g), moderate-M (0.6– 1.0 lmol EDTA eq/g) and high-H (>1.0 lmol EDTA eq/g). Legumes with low phenolic content, lablab bean (white), chickpea (cream, and big-brown), and pea (white) had low metal ion chelating activity (<0.6 lmol EDTA eq/g). This data is comparable with antioxidant activity data assessed by other three assays. However, butter bean and lablab bean (cream) though had low antioxidant activity in other assays and low phenolic content, showed high metal ion chelating activity (1.329 and 1.087 lmol EDTA eq/ g, respectively). Similarly moth bean, chickpea (green) and pea

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Fig. 2. Metal ion chelating activity of legumes. Metal ion chelating activity was assessed as described in methods section. The values present mean of three independent experiments ± SD. Seed coat color is given in parenthesis. (A) <0.6 lmol EDTA eq/g legume; (B) 0.6–1.0 lmol EDTA eq/g legume; (C) >1.0 lmol EDTA eq/g legume.

(green), although low in phenolic content and antioxidant activity in other assays, exhibited moderate metal chelating activity. However, Pigeon pea, green gram (yellow), common bean (maroon) and black gram showed moderate antioxidant activity, assessed using other assay systems so also metal ion chelating activity. On the other hand, common bean (white), lablab bean (brown), cowpea (white) and green gram (green) although had moderate phenolic content and moderate antioxidant activity in other assay systems, showed comparatively high metal ion chelating activity. Pea black and chick pea (small-brown), though moderate in phenolic content and having moderate antioxidant activity as observed in other assays, showed weaker metal ion chelating activity. Soybean, common bean (black), horse gram, and cowpea (brown) showed high phenolic content, high antioxidant activity and had high metal ion chelating activity as well. However, common bean (brown) and fenugreek though had good amount of phenolics and antioxidant activity as observed in other three assays, showed low metal ion chelating activity. Peanuts on other hand with high phenolic content and high DPPH free radical scavenging activity, moderate ABTS and FRAP values, showed high metal ion chelating activity. Thus, horse gram, common beans (black) and cowpea (brown) showed excellent antioxidant activity in all four assays. While, lablab bean (white), chickpea (cream and big-brown) and pea (white) showed very weak antioxidant potential in all four assays.

3.2. Correlation of AOA with phenolic content and seed coat color Correlation between phenolic content and antioxidant activity is shown in Fig. 3A–D. Very good correlation was observed between phenolics with DPPH free radical scavenging activity, ABTS+ free radical scavenging activity and FRAP values with r2 values 0.952, 0.953 and 0.966, respectively. On the contrary, metal ion chelating activity of the legumes exhibited poor correlation with phenolic contents (r2 = 0.297). The results thus suggest that bioactive compounds other than polyphenols, which may be present in legumes, are responsible for metal ion chelating activity.

4. Conclusion Legumes contain varied amounts of polyphenols and possess wide range of antioxidant activity. Cowpea (brown and red), horse gram, common beans (black, red, brown and beige), soybean and fenugreek showed excellent antioxidant activity. While, chickpea (cream, green and big- brown), pea (white and green) and lablab bean (cream and white), showed very weak antioxidant potential. Thus, most of the varieties having light color seed coat, except soybean exhibited low antioxidant activity. While legumes having

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Fig. 3. Correlation of antioxidant activity with phenolic content. The plot of phenolic content, mg of gallic acid equivalent (GAE)/g legume, versus (A) DPPH units, (B) ABTS+ units, (C) FRAP value and (D) metal ion chelating activity of legume extracts.

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