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Antioxidant potential of processed Vigna umbellate (L.) seeds: An Indian

underutilized legume

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International Journal of Chemical Studies 2017; 5(4): 1407-1412

P-ISSN: 2349–8528
E-ISSN: 2321–4902
IJCS 2017; 5(4): 1407-1412 Antioxidant potential of processed Vigna
© 2017 IJCS
Received: 08-05-2017 umbellate (L.) seeds: An Indian underutilized
Accepted: 10-06-2017
legume
Savita Rani
Department of Chemistry and
Biochemistry, CCS Haryana Savita Rani and M Khabiruddin
Agricultural University, Hisar,
Haryana, India Abstract
The raw and processed seeds of rice bean variety (RBL-6) grown in India were examined for bioactive
M Khabiruddin compounds (total phenolic content, hydrophilic and hydrophobic phenols, o-dihydric phenols,
Department of Chemistry and flavonoids) and antioxidant potential (free radical scavenging capacity by DPPH, total antioxidant
Biochemistry, CCS Haryana
activity using ferric thiocyanate assay). One-way ANOVA revealed significant differences in
Agricultural University, Hisar,
Haryana, India
phytochemical constituents in different methanolic extracts. Results concluded that phenolic contents in
seed parts as well as in cooked cotyledons correlated (p<0.05) significantly with antioxidant properties in
most of the extracts. Hence, the methanolic extracts of seed coat showed highest phenolics (103.62 mg
GAEg-1) and highest scavenging efficiency against DPPH (0.17 mg/ml) as a consequence. The present
study demonstrated that ricebean, an unexploited legume endowed with high bioactive constituents as
well as antioxidant potential and thus serve as future nutraceutical source.

Keywords: Vigna umbellate (L.), Indian underutilized legume, processed seeds, ferric thiocyanate assay

Introduction
Several studies have shown a highly significant correlation between uptake of foods with high
antioxidant potential such as fruits, vegetables, legumes and decreasing occurrence of severe
diseases namely cancer, cardiovascular diseases, hypocholesterolemic effects, diabetes etc.
(Nithiyanantham et al., 2012) [11]. Based on the mechanism of antioxidant activity, poly
phenolics are categorized as chain breaker, oxygen scavenger, metal in activator and peroxide
decomposer. Legumes which play an important role as human food or animal feed are widely
utilized for health perspective. Rice bean (Vigna umbellate L.) is one of the unexploited and
underutilized legume crop widely grown in rice fields after the harvesting of paddy on residual
soil moisture. Kaur and Kapoor (1991) [6] have studied the nutrient profiles in rice bean and
reported that rice bean is comparable to commonly-consumed pulses such as black gram and
green gram. The red rice beans are used to relieve oedema in some illnesses and are an integral
part of traditional Chinese therapy. Rice bean as a grain legume is attracting attention
throughout the world as a potential source of high-quality protein for the future for bridging
the ‘protein gap’. Hence, it is one example of the ‘underutilized crops’ or ‘orphan crops’ that
have received attention over the last few years due to the still narrower species and genetic
base in the global food supply. Although the nutritional value of rice bean seeds has been
discussed in previous literature still information regarding phenolic content and antioxidant
profile of seed material is scanty.
Most of the legumes need to be processed before consumption to improve their palatability. In
spite of that, the processing may affect their antioxidant potential of food. Hence, the objective
of this study was to evaluate the profile and content of phenolic compounds and their relation
to the antioxidant capacity as affected by processing like de hulling, cooking etc. in rice bean
grown in Haryana (India). Therefore, the present study has been aimed at:
1. To determine the chemical composition of whole seeds, seed coats, de-coated raw and
cooked dal of rice bean.
Correspondence 2. To evaluate the anti-radical and antioxidant activity of the methanolic extracts of the
Savita Rani above samples.
Department of Chemistry and
Biochemistry, CCS Haryana This study will provide much beneficial information for the food and nutraceutical industry
Agricultural University, Hisar,
from underutilized legume crop and serve as a good base for other researchers to investigate
Haryana, India
legume antioxidants in future research.
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International Journal of Chemical Studies

Materials and Methods used (mg). A blank was also prepared by following same
Seed material aforementioned procedure without extract.
For experimental analysis, healthy seeds of rice bean var.
RBL-6 were provided by the Pulses section, Department of Determination of flavonoids
Genetics and Plant Breeding, CCS Haryana Agricultural The aluminum chloride colorimetric assay, as described by
University (India). These seeds were cleaned to remove Zhishen et al. (1999) [20] with modification was used. Briefly,
foreign materials, damaged seeds and immature seeds. After 1 ml of diluted (1:4) extracts were added. A blank solution
sorting, amajor portion of seeds from each variety wasde- using doubled distilled water was prepared. Then 0.3 ml 5%
husked to obtain seed coat and dal. Dehusked dal was divided NaNO2 was added to the testing samples, followed by 0.3 ml
into two sets. The first set was kept for extraction which 10% AlCl3, 2 ml of 1M NaOH and the total volume was
constitutes intact dehusked seeds and thesecond part was made10ml with dilution and mixed thoroughly. Then the
cooked using distilled water in 1:10 (w/v) ratio at 100◦C, dried absorbance was measured at 510 nm against blank. Total
at 55 ◦C. Thus, we obtained four samples of rice bean, 100 gm flavonoid contents were expressed as mg catechin equivalent
each for further analysis. per gram of the extract (mg CAE/g).

Preparation of extracts 2, 2’-Diphenyl-1-picrylhydrazyl (DPPH) free radical

For the preparation of extracts, the samples were ground using scavenging assay
an electric grinder and converted into fine powder. Powdered 2, 2’-Diphenyl-1-picrylhydrazyl (DPPH) free radical
samples were then extracted separately by refluxing for six scavenging assay was used to evaluate the antioxidant activity
hours using methanol. Extracts produced after refluxing were of the both the genotypes. The antioxidant response of
filtered and the filtrates were concentrated under reduced extracts against DPPH free radical was estimated by the
pressure on rotating evaporator at 40◦C. These concentrated method of Hatano et al, 1988. Methanol treated extracts were
filtrates were stored at 5 ◦C for analyses of phytoconstituents. dried completely and weighed. The dried mass of methanol
extracts was re dissolved in required volume of methanol to
Determination of total phenolic content make the stock solution (1 mg/ml).
Determination of total phenolic content was done by Folin- Different concentrations (0.1-1 mg/ml) were made by
Ciocalteu reagent using gallic acid as standard (Shahidi and appropriate dilutions with 100% methanol from the stock
Naczk, 2001).To a 50 ml volumetric flask 1.0 ml extract, 1.0 solution. In extracts of different concentrations, 2.0 ml of
ml Folin–ciocalteau reagent (50% v/v) and 2.0 ml of Na2CO3 DPPH solution (0.025 g L-1 in 50% methanol) was added and
(20%w/v) were added and mixed and final volume was made the mixture was shaken and absorbance was measured at 515
to 50 ml. The mixture was allowed to settle for 30 minutes nm at every 5 minutes interval until the reaction subsided.
and then centrifuged at 6000 rpm for 5-7 minutes. After After 2 hours, the percent absorbance was declined
centrifugation, thesolution was measured colorimetrically at corresponding to the percentage of DPPH scavenged which
730 nm using Shimadzu UV-Vis spectrophotometer (UV- was an expression of antioxidant activity. During this process,
2600). A blank was also prepared by following same butylated hydroxyanisole (BHA) and butylated
aforementioned procedure without a sample. After hydroxytoluene (BHT) with conc. 0.1-1.0 mg/ml were used as
multiplication with the dilution factor, the concentration of standard solutions having asame preparatory method.
phenolic content was expressed as equivalent to milligrams of Similarly, a control sample was also prepared. By using
gallic acid per gram of extract (mg GAEg-1) by using the Microsoft Excel Software, a quadratic regression equation (y
standard plot. = ax2+bx+c) was obtained. By putting y = 50% in the
equation y = ax2+bx+c; it was converted to the form ax2+bx+c
Determination of hydrophilic and hydrophobic phenolic = 0. IC50 was calculated from the equation ax2+bx+c = 0 by
contents using the formula:
For the determination of hydrophilic and hydrophobic
phenolic contents, 50 ml of crude extract was fractionated to
- b  b 2 - 4ac
obtain constituting hydrophilic and hydrophobic components. x
It was done by mixing the extract with deionized water and n- 2a
butanol (100ml each) in separating funnel as per
Wettasinghe’s method (2000).The mixture was then allowed where, x = IC50 (mg/ml)
to stand until separate layers visible. Separated layers were
then concentrated using rotavapor at 40◦Ctemperature. After Calculation
measuring the weight of each fraction, the phenolic content of The calculation for DPPH scavenged (% DPPH*sc) was done
each fraction was determined as per Shahidi and Naczk by following formulae:
method.
Acontrol - Asample
Preparation of standard curve of o-dihydric phenols % DPPH * sc   100
Preparation of standard curve was done by estimating o-
Acontrol
dihydric phenols in methanol extracts by Arnow’s method
Where Acontrol and Asample represents the absorbance of control
(Kim et al., 2003) using catechol as standard. 0.4 ml of
and sample. Based on the results obtained after calculation a
extracted solutions were added to 1 ml 0.5N HCl, 1 ml
graph was made by plotting percent DPPH free radical
Arnow’s reagent and 2 ml 1N NaOH. The final volume was
scavenging activity or inhibition percentage (y-axis) against
made to 10 ml using double distilled water. The intensity of
extract concentration (x-axis).
resulting orange red color was measured colorimetrically at
515 nm using aspectrophotometer. A standard curve was
made by plotting absorbance against the amount of catechol
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International Journal of Chemical Studies

Ferric thiocyanate (FTC) method Statistical analysis

The FTC method was given by Kikuzaki and Nakatani (1993) Four replications of each sample were used for statistical
[7]
. This method was used for the evaluation of antioxidant analysis for minimizing random experimental error. Values
activity of the extracts under study. After making required obtained were expressed as mean± S.E. Both one way and
dilutions with methanol, 0.1 to 1 mg/ml of samples were two-way analysis of variance (ANOVA) and F-test were
made. After that, samples were mixed with 5 ml of linoleic carried out to assess significant differences in between means
acid emulsion and final volume was made to 10 ml using 0.2 (p<0.05). Correlation analyses of polyphenolic composition
M phosphate buffer (pH 7.0) and incubation was done at 37◦C and their antioxidant activities were carried out using Pearson
for 96 hours (4 days). After incubation aliquots of 0.1 ml were correlation program in Online Statistical Analysis (OPSTAT
drawn from the incubated mixture after 24 hours interval and www.hau.ernet.in).
mixed with 30% ammonium thiocyanate, 20 mM ferrous
chloride in 3.5% HCl and final volume was made to 10 ml Estimation of Phyto-constituents and Antioxidant
with 75% ethanol and allowed to stand for 10 minutes. The Activities
color developed was measured colorimetrically at 500 nm Estimation of total phenolic content
using aspectrophotometer. For this experiment, butylated As per results analyzed (Table 1) for estimation of total
hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) phenols in ricebean extracts, seed coat contained the highest
were used as standard solutions and by using quadratic amount of total phenolics i.e. 103.62 mg GAEg-1. Rest of the
regression equation (y = ax2+bx+c) calculations were done as extracts contained asignificantly reduced amount. Raw dal
described earlier. A control mixture was prepared extract contained 45.10 mg GAEg-1, cooked dal extract
simultaneously following same procedure without the test contained 21.95 mg GAEg-1 and whole seed contained 52.15
sample. mg GAEg-1 phenolic content. For 100g sample, total
phenolics in samples of the seed coat, raw dal, cooked dal and
Calculation whole seed were 0.550 g, 0.271 g, 0.067 g, 0.480 g
Observed antioxidant activity was expressed as: respectively.
Antioxidant activity (%) = {1- (increase in abs. of sample/
increase in abs. of control)} x 100
Table 1: Phytochemical contents and antioxidant activity of ricebean extracts.
Parameters Seed Coat Dehusked Dal Cooked Dal Whole Seed BHT as Standard
Yield (%) of methanolic extracts 5.31 6.03 3.09 9.21 _
Total phenolics
103.62 45.10 21.95 52.15 _
(mgGAE/g)
Total o-dihydric phenols
42.35 30.57 9.92 32.32
mg COEg-1
Hydrophilic phenols
53.09 32.23 9.82 31.26 _
(mgGAE/g)
Hydrophobic phenols (mgGAE/g) 50.53 12.87 12.12 20.88 _
Flavonoids (mgCAE/g) 25.32 33.66 15.80 30.19 _
DPPH radical scavenging assay EC50 (mg/g of extract) 0.17 0.35 0.43 0.25 0.41
Ferric thiocyanate (FTC) assay EC50 (mg/g of extract) 0.29 0.65 0.69 0.57 0.63
Values are mean of four replicates ± standard deviation and means are different from each other by F-test (p<0.05)
mg GAE/g- milligrams gallic acid equivalent/g of the extract
mg CAE/g- milligrams catechin equivalent/g of the extract

Estimation of ortho - dihydric phenolic content hydrophilic phenols (ratio of approx. 1:1, w/w) respectively
Analysis of variance of means (p<0.05) showed that there was (Fig. 1). In thecase of raw dal extract, it contained 12.87 mg
thesignificant statistical difference between theo-dihydric GAEg-1 hydrophobic and 32.23 mg GAEg-1 hydrophilic
content of all the extracts. It was observed that ortho-dihydric phenols which constituted 29% (w/w) and 71% (w/w) of total
phenolic content was highest in seed coat extract (42.35 mg phenolics (45.10 mg GAEg-1). Similarly, cooked dal extract
COEg-1) analogous with theresult obtained for total phenolics. possessed 12.12 mg GAEg-1 hydrophobic (55%, w/w) and
Corresponding values for o-dihydric phenolic content in raw 9.82 mg GAEg-1 hydrophilic phenols (45%, w/w). Whole
dal, cooked dal, whole seed extracts were 30.57, 9.92, 32.32 seed methanolic extract had total phenol with the value of
mg COEg-1 which were statistically different from each other. 52.15 mg GAEg-1 and it exhibited 60% and 40% (w/w) for
For 100 g sample, the o-dihydric phenols were observed as hydrophilic (31.26 mg GAEg-1) and hydrophobic (20.88 mg
0.22 % in seed coat extract, 0.18 % in raw dal extract, 0.03 % GAEg-1) phenols respectively. Graphical representation for
in cooked dal extract and 0.29 % in whole seed extract. This total hydrophobic and hydrophilic phenolic content (mg
data was in close agreement with total phenolics (305 GAEg-1) in ricebean extracts is given in Fig 1.
mg/100g) determined by Gujral et al., 2011 who reported
25% decrease in total phenolic on cooking.

Estimation of hydrophobic and hydrophilic phenolic

content
In ricebean extracts, it was observed that total phenolics
reported in seed coat extract was 103.62 mg GAEg-1and it
consisted of 49% (w/w) and 51% (w/w) of hydrophobic and
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International Journal of Chemical Studies

Fig 3: Inhibition % of rice bean extracts by ferric thiocyanate (FTC)

method

Fig 1: Total hydrophobic and hydrophilic phenolic content (mg

Relationship between various phytochemicals and
GAEg-1) in rice bean extracts antioxidant activities of ricebean extracts
To know the implication of polyphenolic compounds in the
antioxidant activities measured, the correlations were
evaluated. The correlative values were obtained between
DPPH free radical scavenging activity, antioxidant activity by
ferric thiocyanate (FTC) method, in terms of IC50 value and
total phenols, o-dihydric phenols, hydrophilic and
hydrophobic phenols and flavonoids in all the four extracts of
the seed coat.
In seed coat extracts, total phenols were highly significantly
correlated with hydrophobic phenols and IC50 value by DPPH
method with correlation coefficient value 0.990*,
0.966*respectively (where ‘*’ shows significant at 5% level).
A significant correlation (r =0.961*) was analyzed between
IC50 values obtained by both the antioxidant activity methods.
The corresponding correlation values obtained for raw dal
Fig 2: Inhibition % of rice bean extracts by DPPH free radical extract showed thattotal phenols were highly significant with
scavenging method hydrophobic phenols, flavonoid, IC50 value by DPPH method
with correlation coefficient value 0.986*, 0.982*, 0.983*
Estimation of flavonoid content in ricebean extracts respectively. There was a high and positive correlation
Here also, flavonoid content was highest in seed coat (25.32 observed between IC50 value by DPPH method and
mg CAEg-1) analogous with theresult obtained for total hydrophobic phenol (r =0.988*). The correlation between
phenolics. Corresponding values for flavonoid content in raw flavonoid content and IC50 value by FTC method was also
dal, cooked dal, whole seed extracts were 33.66, 15.80, 30.19 positive and highly significant as r =0.973*.
mg CAEg-1. The correlation values determined for cooked dal extracts
found thattotal phenols were highly significant with
Evaluation of antioxidant activity in ricebean extracts hydrophilic phenols and hydrophobic phenols with correlation
Evaluation of DPPH free radical scavenging activity coefficient value 0.985*, 0.989*respectively. Hydrophilic
IC50 values obtained by DPPH method in ricebean extracts phenols and IC50 value by FTC method were correlated with
were 0.17, 0.35, 0.43, 0.25 mg/ml for seed coat, raw dal, high positive and significant value (r =0.952*). There was a
cooked dal, whole seed respectively. Statistically, IC50 values high and positive correlation observed between IC50 value by
for all the four extracts varied each other (Table 1). The DPPH method and IC50 value by FTC method (r =0.980*).
corresponding maximum antioxidant activity (%) was 82.62, The respective correlation values obtained for whole seed
73.34, 67.66, 77.60 as shown in Fig 2. extract concluded that total phenols were highly significant
with o-dihydric phenols and IC50 value by FTC method with
Evaluation of antioxidant activity by ferric thiocyanate correlation coefficient value 0.974* and 0.958* respectively.
(FTC) method Hydrophilic phenols were observed as significantly correlated
Similar to DPPH method’s observations, here also IC50 value with IC50 value by FTC method (r =0.978*). Rest of the
of all the treatments of ricebean varied widely. The % of correlation values obtained were found to be non-significant.
inhibition or antioxidant activity of methanolic extracts in As per previous work is concerned, Yao et al., (2012) [19]
theseed coat, raw dal, cooked dal, thewhole seed was 76.60, investigated the individual phenolic acids, the total phenolic
65.80, 62.30 and 71.00 respectively. Corresponding IC50 content (TPC), the total flavonoid content (TFC), and the
values for all extracts were 0.29, 0.65, 0.70, 0.57 antioxidant and antidiabetic potential of 13 varieties of rice
mg/ml.Graphical representation of percentage inhibition value beans from China. Eight phenolic compounds (catechin,
obtained by FTC method for all extracts of ricebean is shown epicatechin, p-coumaric acid, ferulic acid, vitexin, isovitexin,
in Fig 3. sinapic acid, quercetin) were analyzed. In present studies,
total phenolics including o-dihydric phenols, hydrophobic and
hydrophilic phenols were observed to be decreased on
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International Journal of Chemical Studies

cooking. Segev et al., 2010 [14], reported 85% decrease in total effects of processing on the phenolic compounds and
phenolics by cooking in Kabuli chickpea. Similarly, Hwang et antioxidant activities of an underutilized pulse crop
al., 2012, found that boiling and steaming significantly ‘Ricebean’. In this context, it was concluded that
decreased the ascorbic acid content, total phenolics, and phytochemical constituents and antioxidant activity were
antioxidant levels compared with the other cooking methods. dependent on the processing method. Ricebean are found to
Reduced total phenolics in boiled or steamed foods have been be a better source of antioxidative phytochemicals. Therefore,
attributed to the dissolution of phenolic compounds into the suitably processed underutilized legume grains could be
cooking water (Zhuang et al., 2012) [21]. Previous literature envisaged as a dietary ingredient in the formulation of
showed the phenolic composition of the seed coats and supplementary foods with therapeutic values.
cotyledons of red lentils (Duen˜as et al., 2002) [2] and the
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