Black gram (Vigna mungo L.) is a legume widely consumed in parts of Asia and Africa. During milling of black gram into cotyledon for use in foods, about 25% of the grain is removed as by-products. This study analyzed the nutrient composition, phenolic acid profiles, antioxidant activity, and alpha-glucosidase inhibitory properties of black gram flour and its milled fractions. Protein and fat contents varied between fractions, with germ having the highest and seed coat the lowest. Seed coat had the highest fiber but lowest in cotyledon. Seed coat, plumule and aleurone layer showed better antioxidant activity than other fractions due to differences in phenolic acid quantities and types.

1. Nutrient distribution, phenolic acid composition, antioxidant and alpha-glucosidase
inhibitory potentials of black gram (Vigna mungo L.) and its milled by-products
T.K. Girish a
, V.M. Pratape b
, U.J.S. Prasada Rao a,
⁎
a
Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore, 570 020 India
b
Department of Grain Science and Technology, Central Food Technological Research Institute, Mysore, 570 020, India
a b s t r a c ta r t i c l e i n f o
Article history:
Received 13 August 2011
Accepted 24 December 2011
Keywords:
Black gram (Vigna mungo L.)
Nutrient composition
Bioactive compounds
Dietary fiber
Phenolic acids
Antioxidants
α-glucosidase inhibition
Black gram belongs to the Leguminosae family. It is one of the less studied legumes, although it is widely used
in different parts of the world. Black gram in the form of cotyledon (dhal) is mainly used for the preparation
of various food products. During milling of black gram into cotyledon, about 25% of the grain is removed as
waste by-products. In the present study, nutrient content, phenolic acid composition, antioxidant activity
and α-glucosidase inhibitory properties of total black gram flour and its milled fractions were determined
with a view to provide economic importance to these by-products. Protein content in black gram and its frac-
tions ranged from 12 to 42%, while fat content ranged from 0.9 to 3.4%. Germ had the highest content of fat
and protein, while seed coat and plumule fractions had the lowest (0.9%). Seed coat had the highest dietary
fiber content (78.5%) while cotyledon had the lowest (24.4%). Seed coat, plumule and aleurone layer enriched
in seed coat extracts showed a better antioxidant potential compared to other fractions and this may be due
to the quantitative and qualitative differences in phenolic acids. Extracts of seed coat, plumule and aleurone
layer enriched in seed coat extracts showed good α-glucosidase inhibitory activity. Black gram flour con-
tained phenolic acids like gallic, protocatechuic, gentisic, vanillic, syringic, caffeic and ferulic acids. However,
composition and content of these phenolic acids varied in different fractions. Ferulic acid was the major phe-
nolic acid in all the fractions. Protocatechuic acid, ferulic acid, gentisic acid and gallic acid contents in these
fractions negatively correlated (Pb0.05) to IC50 values of both free radical scavenging and α-glucosidase in-
hibitory activities indicating their potential antioxidant and antidiabetic properties. As black gram and its
fractions are rich in antioxidant compounds and nutrients, they may have potential applications as nutraceu-
ticals and functional food ingredients in various processed foods for the improvement of health benefits.
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Legume seeds are valuable sources of proteins and other nutrients,
and they are good source of nutrients for the majority of the world
population. It is also reported that legumes have certain phytochem-
icals like polyphenols, flavonoids, phytosterols that possess health
benefits (Kritchevsky & Chen, 2005; Sessa, 2004; Sreerama,
Sashikala, & Pratape, 2010). Black gram or black gram lentil (Vigna
mungo L.) belongs to the Leguminosae family (Reddy, Salunkhe, &
Sathe, 1982; Salunkhe, Kadam, & Chavan, 1985). It is one of the less
studied legumes although it is widely used in India, Pakistan, Iran,
Greece and East Africa (Bhattacharya, Latha, & Bhat, 2004;
Chaudhary & Ledward, 1988). Black gram is used for the preparation
of different food products. Dehusked cotyledon is used for the prepa-
ration of fermented foods such as idli, dosa, and non-fermented foods
like cooked dhal, hopper, papad and waries (spicy hollow balls)
(Batra & Millner, 1974). Traditionally, sweets prepared with whole
black gram flour and jaggery were regarded as nutritious food in
India. Whole black gram flour paste either alone or in combination
with sandalwood paste or fenugreek paste is used for skin or hair
care, respectively. Incorporation of black gram flour was reported to
improve the quality of buffalo meat burgers (Modi, Mahendrakar,
Narasimha Rao, & Sachindra, 2004) and beef sausages (Chaudhary &
Ledward, 1988) and the nutritional quality of biscuit (Patel &
Venkateswara Rao, 1995).
Whole black gram is a rich source of protein, fiber, several vita-
mins and essential minerals such as calcium and iron (Reddy et al.,
1982; Salunkhe et al., 1985). Processing of black gram into dehusked
cotyledon essentially involves the removal of seed coat, germ, aleu-
rone layer and plumule, and these fractions may consist of a variety
of nutrients. Currently, except cotyledon fraction, the other fractions
are discarded or used as animal feed. However, the distribution of nu-
trients and bioactive compounds in these fractions is not known.
Foods rich in nutraceuticals and dietary fiber are gaining impor-
tance because of their health benefits. Polyphenols, carotenoids and
Food Research International 46 (2012) 370–377
⁎ Corresponding author at: Department of Biochemistry and Nutrition, Central Food
Technological Research Institute, Mysore, 570 020, India. Tel.: +91 821 2514876;
fax: +91 821 2517233.
E-mail address: prasadarao_ummiti@yahoo.com (U.J.S. Prasada Rao).
Contents lists available at SciVerse ScienceDirect
Food Research International
journal homepage: www.elsevier.com/locate/foodres
0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.12.026

2. dietary fiber have a role in prevention of cardiovascular disease, can-
cer and diabetes (Lario et al., 2004; Scalbert, Manach, Morand, &
Remesy, 2005). By-products from different food processing industries
which were traditionally treated as environmental pollutants are
being recognized as good sources for obtaining valuable components.
By-products from cereal, legume and fruit processing industries have
been found to be rich and economically inexpensive sources of bioactive
compounds such as antioxidants, dietary fibers and enzymes (Ajila,
Bhat, & Prasada Rao, 2007; Ajila, Naidu, Bhat, & Prasada Rao, 2007;
Liyana-Pathirana & Sahidi, 2006; Moure et al., 2001; Sessa, 2004). The
seed coat (husk) of cereals and legumes possesses large quantities of en-
dogenous antioxidants such as phenolic compounds (Moure et al.,
2001; Tsuda, Ohshima, Kawakishi, & Osawa, 1994). Black gram lipids
were shown to have cholesterol-reducing effect in both humans and ex-
perimental animals (Saraswathi Devi & Kurup, 1972). Distribution of
bioactive compounds in plants varies in different tissues. In the present
study, the extract of black gram and its milled fractions viz., cotyledon,
seed coat, germ, aleurone layer enriched in seed coat fraction and plu-
mule were investigated for the nutritional composition, phenolic acid
composition, carotenoid content, and also their antioxidant and α-
glucosidase inhibition properties.
2. Materials and methods
2.1. Materials
Gallic acid, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), butylated
hydroxyanisole (BHA), α-amylase (Termamyl), pepsin, pancreatin,
celite were purchased from Sigma Aldrich Chemical Co. (St. Louis,
USA). Folin–Ciocalteu reagent was obtained from SR Laboratories
Limited (Mumbai, India). All other chemicals and solvents were of an-
alytical grade.
2.2. Milling of black gram and separation of milled products
Black gram (10 kg) was pitted in Versatile Dhal Mill (CFTRI design)
mixed with 30 mL of oil, kept overnight for tempering and dried at
60°C for 8 h. The black gram thus obtained after treatment was milled
using Versatile Dhal Mill according to the procedure described by
Narasimha, Ramakrishnaiah, Pratape, Sasikala, and Narasimhan
(2002). Black gram was milled into cotyledon, seed coat, and mixture
of germ, aleurone, seed coat powder, and plumule. The mixture was fur-
ther separated into different fractions by air classification as described
in Fig. 1 (Ajila & Prasada Rao, 2009).
2.3. Determination of proximate composition
Moisture, protein, fat, ash and crude fiber contents in whole black
gram flour and its milled fractions (BGMF) were determined by AOAC
methods (2005). The total carbohydrate content was calculated by
the difference method.
2.4. Extraction of total polyphenols and determination of total phenolics
Whole black gram flour (1 g) and BGMF (1 g) were extracted with
30 mL of either 80% acetone or 80% ethanol separately and were cen-
trifuged for 15 min at 8000×g. The clear supernatants obtained were
subjected to total phenolic content estimation using the Folin–Ciocalteu
reagent following the procedure described by Swain and Hillis (1959).
Gallic acid was used as a standard. The total polyphenol content in the
extract was expressed as gallic acid equivalents (GAE).
2.5. Determination of anthocyanin content
Monomeric anthocyanin content of the black gram flour and
BGMF acetone extracts were measured using a spectrophotometric
pH differential method (Wolfe, Xianzhong, & Liu, 2003). Anthocyanin
content was expressed as mg cyanidin 3-glucosides equivalent/100 g
sample for the triplicate extracts.
2.6. Determination of carotenoids
Black gram flour and BGMF (1 g) were homogenized with 40 mL
of methanol containing 1 g KOH. The mixture was saponified
Black gram
Milling
Cotyledon(dhal) (75%) seedcoat(husk) (9%) Mixture of germ, seed coat, plumule
and aleurone powder (16%)
Air classification
Air velocity 3.1 m/sec;
Feed rate 96 g/min
Germ (6%) Plumule, aleurone, seed coat powder
Air classification
Air velocity 2.9m/sec;
Feed rate 184 g/min
Plumule aleurone layer enriched in seed coat
(2%) (8%)
Fig. 1. Flow diagram for separation of black gram milled fractions.
371T.K. Girish et al. / Food Research International 46 (2012) 370–377

3. overnight and the saponified mixture was transferred to separating
funnel containing 25 mL of hexane and gently shaken for 60 s, the
phases were allowed to separate. The aqueous phase was separated
and was re-extracted in the separating funnel with 25 mL hexane.
This was repeated until the hexane extract was colorless. The hexane
extracts were pooled, washed with water until free of alkali, dried
over sodium sulfate and concentrated in a vacuum evaporator at
room temperature. The resulting solution was made up to a suitable
volume with acetone.
The total carotenoid content in these acetone extracts was esti-
mated using colorimetric method reported by Lichtenthaler (1987).
Carotenoids show good absorbance at 470 nm, however, a small
amount comes from chlorophyll b and negligible absorbance from
chlorophyll a. The concentration of total carotenoid content can,
therefore, be determined by subtracting the absorbance of chloro-
phyll a and b from the absorbance read at 470 nm followed by divi-
sion by the absorption coefficient of total carotenoids at 470 nm
(Lichtenthaler, 1987). The carotenoid contents in the acetone extracts
was calculated by using the following formulae (Lichtenthaler, 1987).
Chlorophyll a Cað Þ ¼ 12; 25A663:2−2:79A646:8
Chlorophyll b Cbð Þ ¼ 21:50A646:8−5:10A663:2
Total Carotenoid ¼
1000A470–1:82Ca–85:02 Cb
198
Ca and Cb represent absorbance of chlorophyll a and b, respectively.
‘A’ represents the absorbance at a particular wavelength.
2.7. Measurement of reducing power
The reducing power of acetone extracts of black gram flour, BGMF
and BHA was determined according to the method of Yen and Chen
(1995). Extracts containing 5 to 20 μg of gallic acid equivalents
(GAE) were made up to 500 μL with 0.2 M phosphate buffer (pH
6.6) and mixed with 1 mL of potassium ferricyanide (0.1%) and the
mixture was incubated at 50 °C for 20 min. Trichloroacetic acid
(500 μL, 10%) was added to the reaction mixture and centrifuged at
3000×g for 10 min. The supernatant obtained was mixed with equal
volume of distilled water and 300 μL of 1% ferric chloride was added
and the absorbance was measured at 700 nm. Increased absorbance
of the reaction mixture indicated the increased reducing power. The
antioxidant activity of the extract was compared with BHA.
2.8. Measurement of free radical scavenging activity
Scavenging the stable DPPH radical is another widely used method
to evaluate antioxidant activity. DPPH is a stable free radical with
characteristic absorption at 517 nm and antioxidants react with
DPPH and convert it to 2,2-diphenyl-1-picrylhydrazine. The degree
of discoloration indicates the scavenging potential of the antioxidant
extract, which is due to the hydrogen donating ability (Van Gadow,
Joubert, & Hannsman, 1997).The effect of acetone extracts of black
gram flour and BGMF on DPPH radical was determined according to
the method described by Blois (1958) with modification described
by Brand-Williams, Cuvelier, and Berset (1995). A 100 μM solution
of DPPH in methanol was prepared and BGMF extracts (200 μL) contain-
ing 1 to 5 μg GAE were mixed with 1 mL of DPPH solution. The mixture
was shaken vigorously and left in the dark at room temperature for
20 min. The absorbance of the resulting solution was measured at
517 nm. The control contained all the reagents except sample ex-
tracts/BHA. The capacity to scavenge DPPH radical was calculated by fol-
lowing equation.
Scavenging activity %ð Þ ¼ 1− As=A0ð ÞX100
Where A0 is the absorbance at 517 nm of the control and As is the
absorbance in the presence of sample extract/BHA. The results were
plotted as the % of scavenging activity against concentration of the
sample. The half-inhibition concentration (IC50) was defined as the
amount of GAE required for 50% of free radical scavenging activity.
The IC50 value was calculated from the plots as the antioxidant con-
centration required for providing 50% free radical scavenging activity.
2.9. α-glucosidase enzyme inhibition assay
The α-glucosidase enzyme inhibition assay was carried out
according to the method described by Kwon, Apostolidis, and Shetty
(2008). The enzyme inhibition assay mixture contained 50 μL p-
nitrophenyl-α-D-glucopyranoside (10 mg in 2 mL phosphate buffer),
different concentrations of acetone extract (inhibitor; 10 μL) and the
reaction mixture was made up to 2.8 mL with sodium phosphate
buffer (pH 6.8; 50 mM). The reaction was initiated by adding 20 μL
of α–glucosidase enzyme (2 mg in 1 mL of phosphate buffer; 5.7 U/mg;
Sigma Aldrich, USA). The reaction was monitored by increase in absor-
bance at 405 nm and compared with the enzyme reaction without the
extract. The % of inhibition was calculated by the following equation.
Inhibition %ð Þ ¼
A405control−A405extract½ Š
A405control½ Š
X100
A405 extract is absorbance at 405 nm in presence of acetone ex-
tract. IC50 values were calculated from the plots of % inhibition vs con-
centration of phenolic extract.
2.10. Identification of free phenolic acids
Free phenolic acids in different samples were extracted according
to the method of Adom and Liu (2002) with some modifications.
Briefly, fractions (1 g) were extracted with 20 mL of 80% acetone for
1 h using magnetic stirrer. After centrifugation at 3000×g for
20 min, the supernatant was removed and solution was extracted
five times with ethyl acetate phase separation followed by drying
with anhydrous sodium sulfite. Sodium sulfate was removed by filtra-
tion followed by evaporation to dryness, dissolved in 1 mL of metha-
nol and filtered through 0.45 μm membrane filter (Millipore, USA).
Phenolic acids were separated on a reverse phase Luna C18 column
(4.6x250 mm; 5 µm) using HPLC system (Agilent Model 1200 series)
coupled to a diode array detector (operating at 280 nm and 320 nm)
at room temperature (25 °C). A solvent system consisting of water:
methanol: acetic acid (83:15:2) was used as mobile phase (isocratic)
at a flow rate of 1 mL/min (Glowniak, Zgorka, & Kozyra, 1996).
Known quantities of phenolic acid standards such as caffeic acid,
chlorogenic acid, cinnamic acid, ferulic acid, gallic acid, gentisic acid,
protocatechuic acid, syringic acid, vanillic acid were used for identifi-
cation and quantification of phenolic acids present in the extracts.
2.11. Determination of total dietary fiber content
The dietary fiber estimation was done by an enzymatic gravimet-
ric method (Asp, Johnson, Hallmer, & Siljestroem, 1983). Sample
(0.25 g) was homogenized in 20 mL of sodium phosphate buffer
(0.1 M, pH 6.0) and was analyzed for soluble dietary fiber (SDF) and
insoluble dietary fiber (IDF) contents. The samples were treated
with thermo-stable α-amylase (Termamyl) and then digested with
pepsin and pancreatin. SDF and IDF were separated by filtration.
The filtrate obtained was subjected to alcohol precipitation and fil-
tered to obtain SDF and both the precipitates were dried overnight
at 105 °C and were incinerated at 550 °C for 8 h and the weights
were determined. A control was performed following the same proce-
dure. Total dietary fiber was then calculated as combined value of SDF
and IDF.
372 T.K. Girish et al. / Food Research International 46 (2012) 370–377

4. 2.12. Statistical analysis
Three independent experiments were conducted in triplicate and
the data were reported as means±SD. Duncan's new multiple
range tests was used to determine the difference of means, and
Pb0.05 was considered to be statistically significant (Steel & Torrie,
1980).
3. Results and discussion
3.1. Composition of nutrients in black gram and black gram milled
fractions
The nutrient composition of black gram flour and BGMF is shown
in Table 1. The total protein content in different fractions ranged from
12 to 42%. The germ fraction had the highest amount of protein con-
tent (42%) followed by whole black gram flour and the cotyledon. The
crude fat content was the highest in the germ fraction, while it was
the least in the seed coat and the plumule fractions. Crude fiber con-
tent was found to be the highest in the seed coat fraction and the least
in the cotyledon, while carbohydrate content was the highest in the
cotyledon and the least in the germ. Ash content was the highest in
the germ followed by the aleurone layer enriched seed coat fraction.
The fat, fiber, ash and total carbohydrate contents determined in
whole black gram flour are comparable to literature values. Kantha
and Erdman (1987) reported the protein content, fat, crude fiber,
ash and total carbohydrate contents in black gram seeds as 21%,
1.6%, 4.4%, 3.4%, and 63.4%, respectively, while Salunkhe et al.
(1985) reported lipid content as 1.64%. The protein content reported
in black gram seeds by Salunkhe et al. (1985) and Kantha and Erdman
(1987) varied between 21 and 31%, while in the present study the
protein content was 26%. Recently, Suneja, Kaur, Gupta, and Kaur
(2011) reported significant variations in alkali soluble protein con-
tents (17–28%) in different cultivars and advanced breeding lines,
and reported that protein content varies depending on the genotype.
The protein, fat, crude fiber, ash and carbohydrate contents in differ-
ent pulses (legumes) like horse gram, cowpea, mung bean and chick
pea were reported to range between 19 and 29%, 1.2–5.6, 2.5–-4.4,
3.1–4.2 and 54–62%, respectively (Kantha & Erdman, 1987) and the
nutrient contents of black gram are also comparable to these pulses.
Although, nutrient composition of whole black gram seed is available,
no reports are available with regard to the nutrient composition of
different milled fractions of black gram except for the cotyledon frac-
tion. The protein, fat, ash, fiber and total carbohydrate contents in the
cotyledon fraction were reported to be 24, 1.4, 3.2, 0.9 and 59.6%, re-
spectively (Gopalan, Sastri, & Balasubramanian, 1996) and these
values are slightly different from the values obtained in the present
study (Table 1). Recently, Sreerama, Neelam, Sashikala, and Pratape
(2010) reported the proximate compositions for cotyledon, embryon-
ic axis (germ) and seed coat fractions of two different pulses viz.,
chickpea and horse gram and they found that seed coat had low pro-
tein (7.3–9.1%) and more crude fiber (17.6–21.8%), and germ had
more fat (2.6–7.8%) compared to other fractions of chickpea and
horse gram. However, these values are different from the composition
of black gram fractions reported in the present study. Among the ce-
reals, composition of wheat milled fractions is known and wheat
germ had the highest content of protein (32%) and fat (12%) com-
pared to wheat grain and its other milled fractions (Bushuk, 1986).
As black germ has good amount of fiber, ash and protein, but low con-
tent in fat (Table 1) compared to cereal germs, it could be used in var-
ious food formulations as an ingredient. Aleurone layer enriched in
seed coat fraction is rich in fiber as well as protein, and therefore,
this fraction also can be used in various food formulations. Nowadays
importance is given to consuming foods containing whole grain for
health benefits. To get maximum benefits of nutraceuticals, these
fractions can be used as ingredients in different foods.
3.2. Dietary fiber content in black gram and its milled fractions
Dietary fiber plays an important role in prevention of various dis-
eases like cardiovascular diseases, cancer, diabetes, constipation and
others (Devries, Prosky, Li, & Cho, 1999; Lario et al., 2004). As can
be seen from the results of the proximate composition (Table 1),
some of the black gram milled fractions are rich in crude fiber. There-
fore, soluble and insoluble dietary fiber contents in black gram and
BGMF were determined using enzymatic gravimetric method. As
shown in Table 2, the total dietary fiber (TDF) content in different
fractions varied from 24.42% to 78.53%. Seed coat and plumule frac-
tions had the highest value of fiber and cotyledon fraction had the
lowest one compared to those of other fractions. The low value for
total dietary fiber in cotyledon may be due to the reason that cotyle-
don is devoid of seed coat portion. Between soluble and insoluble fi-
bers, insoluble fiber was higher in all the fractions. The insoluble
dietary fiber content in different fractions varied from 21 to 69%,
while the soluble fiber content varied from 2.6 to 9.3%. Earlier reports
indicate that the soluble dietary fiber content in green gram, chickpea
and pigeon pea was reported to range between 2.0 and 3.2% (Ramulu
& Udayasekhara Rao, 1997). However, in black gram milled fractions,
the soluble dietary fiber content ranged from 2.6 to 9.3%. In terms of
health benefits, both IDF and SDF complement each other, and each
fraction has different physiological effect. Insoluble dietary fiber re-
lates to both water absorption and intestinal regulation, whereas
SDF associates with cholesterol in blood and diminishes its intestinal
absorption (Schneeman, 1987; Shinnick, Mathews, & Ink, 1991).
3.3. Total phenolic content and anthocyanin content in black gram and
its fractions
Polyphenols are the major group of compounds that contribute to
the antioxidant properties. In the present study, black gram flour and
its milled fractions were extracted with 80% (v/v) acetone or 80% (v/
v) ethanol separately, and the total phenolic contents in the extracts
were determined. Of the two solvents used, acetone showed a better
extraction of total polyphenols (Table 3). The total polyphenol con-
tent in acetone extract was the highest in seed coat fraction
(134.66 mg GAE/g) followed by plumule (78.83 mg GAE/g) and
Table 1
Proximate composition of black gram and its milled fractions (%).
Sample Protein Moisture Ash Fat Crude fiber Carbohydrates
Whole gram 26.75d
±0.25 11.41b
±0.08 3.29a
±0.17 1.44c
±0.02 5.56b
±0.16 51.53e
±0.50
Cotyledon 24.33c
±0.28 11.41b
±0.18 3.24a
±0.06 1.79d
±0.03 1.20a
±0.04 58.01f
±0.27
Germ 42.16e
±0.14 11.40b
±0.12 5.30c
±0.03 3.42e
±0.02 19.48c
±0.24 18.21a
±0.30
Seed coat 12.41a
±0.14 10.29a
±0.31 3.03a
±0.06 0.93a
±0.01 33.72f
±0.20 39.78c
±0.16
ALESC* 23.00b
±0.25 11.96b
±0.25 4.12b
±0.04 1.04b
±0.02 22.40d
±0.14 37.46b
±0.42
Plumule fraction 12.25a
±0.25 12.61c
±0.31 3.00a
±0.03 0.91a
±0.01 23.30e
±0.20 47.91d
±0.49
Values are expressed on as is basis. All data are the mean±SD of three replicates. Mean followed by different letters in the same column differs significantly (Pb0.05).
Carbohydrate content was calculated by subtracting % of protein, moisture, ash, fat and crude fiber from 100.
*ALESC, aleurone layer enriched in seed coat.
373T.K. Girish et al. / Food Research International 46 (2012) 370–377

5. aleurone layer enriched in seed coat fraction (72.83 mg GAE/g). Both
black gram flour and cotyledon had very low polyphenol contents,
but between these two, whole flour had higher polyphenol content
(Table 3). Earlier, Saxena, Venkaiah, Anitha, Venu, and Raghunath
(2007) reported the total polyphenol content in black gram (whole
flour) as 0.59 mg GAE/g and cotyledon (dhal) as 0.26 mg GAE/g, re-
spectively. Recently, Suneja et al. (2011) reported the extraction of
total polyphenol contents in different black gram cultivars using
80% methanol and reported that polyphenol contents ranged from
1.1 to 3.2 mg/g seed, and these values are lower than the total poly-
phenol content (3.82 mg/g; Table 3) reported in the acetone extracts
of the present study. However, the phenolic content in the alcohol ex-
tract is comparable to the values reported by Suneja et al. (2011).
Thus, the differences in polyphenol content in different studies may
be due to the variation in the cultivar as well as the extraction proce-
dure followed. With respect to polyphenol content in different milled
fractions, no studies are available on black gram, however, few re-
ports are available on the milled fractions of other legumes (pulses).
Recently, Sreerama, Sashikala et al. (2010) have reported the pres-
ence of polyphenols in different milled fractions of chickpea and
horse gram and found the highest amount of polyphenols in seed
coat and lowest amount in cotyledon fraction. However, their results
indicated that between chickpea and horse gram, the content of total
polyphenols and their distribution in different fractions varied
depending on the type of pulse. Horse gram fractions had higher
amount of polyphenols compared to that of chickpea, and among
the fractions, seed coat had the highest total polyphenol content
(Sreerama, Sashikala et al. (2010)).
As acetone extracts showed higher amount of total polyphenol
content, further studies were carried out with acetone extracts.
Anthocyanins are a group of phenolic compounds present in the
plant kingdom and they exhibit good antioxidant property. The an-
thocyanin content in black gram flour and its milled fractions ranged
from 6–87 mg/100 g (Table 4). Its content was proportional to the
total polyphenol content in different fractions. Similar to total poly-
phenol content, anthocyanin content was found to be the highest in
seed coat fraction (87 mg/100 g). The anthocyanin contents in cooked
black bean and chickpea seeds varied from 1.5 to 4.8 mg/100 g (Silva-
Cristobal, Osorio-Diaz, Tovar, & Bello-Perez, 2010), while in the pre-
sent study the black gram contained 9.8 mg/100 g. Health benefits
of anthocyanins consumption are well known and anthocyanins are
mainly present in blue and red colored fruits, vegetables including
red wine (Clifford, 2000). In developed countries consumption of
these foods is very low and therefore, to improve nutraceutical con-
tent, seed coat fractions can be incorporated into commonly con-
sumed foods.
3.4. Carotenoid content in black gram and its milled fractions
Carotenoids exhibit potential antioxidant properties. As shown in
Table 4, the carotenoid content in the extracts of different fractions
ranged from 0.042 to 0.415 mg/100 g. The seed coat was the richest
in carotenoids followed by the aleurone layer rich husk fraction and
the germ fraction had the least value for carotenoids. The carotenoid
contents in different fractions of pulses and legumes have been
reported earlier by different workers. In general, legume seeds are
poor sources of carotenoids compared to their leaves, and also fruits
and vegetables. According to the studies of Fordham, Wells, and
Chen (1992) carotenoid content in different varieties of peas and
beans ranged from 0.003 to 0.037 mg/100 g, and 0.0002 to
0.003 mg/100 g, respectively. Kantha and Erdman (1987) reported
carotenoid content in peas, lima bean, soybean, and mung bean
seeds and were found to be 0.0005 mg, 0.5 mg, 0.002 mg and
0.004 mg carotenoids per 100 g seeds, respectively.
3.5. Phenolic acid composition of different fractions
As can be seen in Table 5, the phenolic acid composition was found
to be different in different fractions. In all the fractions, ferulic acid
was the predominant phenolic acid followed by gentisic acid. The
fractions were also rich in gallic acid and protocatechuic acid. Seed
coat, aleurone layer enriched in seed coat and plumule fractions
showed similar phenolic acid composition, except that seed coat
and aleurone layer rich husk fractions did not show the presence of
vanillic acid, while phenolic acids like syringic and caffeic acids
were absent in plumule fraction. However, gentisic acid content was
the highest in seed coat followed by aleurone layer enriched in seed
coat fraction, germ and plumule fractions, and it was very low in
both cotyledon and whole flour. Lopez-Amoros, Hernandez, and
Estrella (2006) reported the presence of phenolic acids like ferulic
acid, protocatechuic acid, p-coumaric acid, p-hydroxybenozoic acid
and vanillic acid in different legumes like pea, bean and lentils, how-
ever, their content varied depending on the legume. Ferulic acid con-
tent was the highest in beans, protocatechuic acid content was the
highest in peas, and p-coumaric acid content was the highest in len-
tils. Ferulic acid was the prominent phenolic acid reported in different
milled fractions of chickpea (Sreerama, Sashikala et al., 2010). Ferulic
acid is a major phenolic acid present in plant cell walls of various
seeds such as wheat, rice etc. In wheat bran, ferulic acid content var-
ied from 9.8 to 764.0 mg/100 g depending on the variety and species
(Onyeneho & Hettiarachchy, 1992; Yu, 2004). Ferulic acid is a potent
Table 2
Dietary fiber composition (%) of black gram and its milled fractions.
Samples IDF SDF TDF
Whole gram 36.76b
±0.55 3.13b
±0.05 39.90b
±0.60
Cotyledon 21.80a
±0.70 2.62a
±0.06 24.42a
±0.67
Germ 39.60c
±0.40 5.20c
±0.41 44.80c
±0.11
Seed coat 69.23f
±0.66 9.30e
±0.26 78.53f
±0.55
ALESC* 59.76d
±0.20 7.93d
±0.11 67.70d
±0.10
Plumule 65.93e
±0.11 5.93c
±0.11 71.86e
±0.11
Values are expressed on as is basis. All data are the mean±SD of three replicates. Mean
followed by different letters in the same column differs significantly (Pb0.05).
*ALESC, aleurone layer enriched in seed coat.
Table 3
Total polyphenol content of extracts from black gram and its milled fractions (mg GAE/g).
Samples Acetone (80%) Ethanol (80%)
Whole gram 3.82b
±0.24 2.11b
±0.01
Cotyledon 0.87a
±0.06 0.79a
±0.02
Germ 12.81c
±0.45 5.00c
±0.05
Seed coat 134.66f
±1.52 126.53f
±1.36
ALESC* 72.83d
±1.23 37.68e
±1.20
Plumule fraction 78.83e
±1.02 28.90d
±0.69
Values are expressed on as is basis. All data are the mean±SD of three replicates.
Mean followed by different letters in the same column differs significantly (Pb0.05).
*ALESC, aleurone layer enriched in seed coat.
Table 4
Total carotenoid and anthocyanin content in black gram and its milled fractions (mg/
100 g).
Samples Carotenoid Anthocyanin
Whole gram 0.052c
±0.0007 9.79b
±0.38
Cotyledon 0.042b
±0.0004 5.85a
±0.41
Germ 0.034a
±0.0005 13.06c
±0.41
Seed coat 0.415f
±0.0030 86.84f
±0.59
ALESC* 0.326e
±0.0040 70.42e
±0.58
Plumule fraction 0.128d
±0.0020 33.24d
±0.53
Values are expressed on as is basis. All data are the mean±SD of three replicates. Mean
followed by different letters in the same column differs significantly (Pb0.05).
*ALESC, aleurone layer enriched in seed coat.
374 T.K. Girish et al. / Food Research International 46 (2012) 370–377

6. free radical scavenger and reported to possess antitumor properties
(Kampa et al., 2004).
3.6. In vitro bioactive assays
3.6.1. Antioxidant activity
Polyphenols and carotenoids have the ability to scavenge free radicals
via hydrogen donation or electron donation (Shahidi & Wanasundara,
1992). The reducing power of a compound is related to its electron
transfer ability and may, therefore, serve as a significant indicator of
its antioxidant activity (Meir, Kanner, Akiri, & Hadas, 1995). Fig. 2
shows the reducing power of the extracts from six different black
gram fractions. The reducing power increased with the concentration
of extracts of different fractions. At 15 μg GAE, plumule fraction
showed the highest antioxidant property compared to that of other
extracts. Antioxidant property of BHA was comparable to that of ace-
tone extract of seed coat but lower than that of plumule extract. In ac-
etone extract, cotyledon showed the least antioxidant activity. The
antioxidant property of the extracts is mainly due to the presence of
polyphenols and carotenoids in the extracts. It has been reported
that polyphenols and carotenoids are electron donors and could re-
duce Fe3+
/ferricyanide complex to ferrous form (Chung, Chang,
Chao, Lin, & Chou, 2002; Yen & Chen, 1995).
The acetone extracts showed a concentration dependent scaveng-
ing of DPPH radical, which may be attributed to their hydrogen do-
nating ability. The total polyphenol contents in the extracts of
plumule, aleurone layer enriched in seed coat fraction and seed coat
fraction had IC50 values 2.27, 2.90 and 3.01 μg of GAE, respectively,
and these values were comparable to the IC50 value for BHA
(3.42 μg of GAE; Table 6). In all the extracts, plumule showed better
radical scavenging activity followed by aleurone layer enriched in
seed coat fraction and husk fraction. Cotyledon extract showed the
highest IC50 value of 12 μg, indicating its lowest antioxidant activity
compared to the extracts from other fractions.
3.6.2. α-glucosidase inhibitory activity of different extracts
Type 2 diabetes is caused by the impaired secretion of insulin
resulting in increased postprandial glucose level. One of the impor-
tant therapeutic approaches to decrease postprandial hyperglycemia
is to retard absorption of glucose through inhibition of carbohydrate
hydrolyzing enzymes. α-glucosidase is one of the key enzymes in-
volved in the release of glucose from starch for the intestinal glucose
absorption. The inhibition of this enzyme decreases the blood glucose
levels and thus it is an important strategy for the management of
type-2 diabetes (Plus, Keup, Krause, Thomos, & Hoffmeister, 1977).
α-glucosidase inhibitors from natural food sources is an attractive
strategy to manage postprandial hyperglycemia. The seed coat, aleu-
rone layer enriched in seed coat and plumule fractions showed a bet-
ter enzyme inhibition compared to the other three fractions. At 2.5 μg
level, seed coat, aleurone layer enriched in seed coat and plumule
fractions inhibited 58–70% enzyme activity, while the other fractions
showed 19–36% inhibition. Whole flour extracts showed better en-
zyme inhibitory activity compared to cotyledon and germ (Fig. 3).
The IC50 values for all the fractions ranged between 1.85 and
8.75 μg GAE. Seed coat, plumule and aleurone layer enriched in
seed coat fractions had low IC50 values of 1.85 μg, 1.90 μg and
2.25 μg, respectively. Whole flour and germ had IC50 values of 3.8
and 7 μg, respectively. Cotyledon fraction had the least inhibitory
properties i.e., it had high value of IC50 (8.75 μg). Thus, whole flour
extracts as well as seed coat fractions and plumule exhibited poten-
tial antidiabetic properties.
3.7. Correlation of in vitro biological activities with bioactive compounds
in different extracts
As can be seen in Tables 3–6 and Figs. 1 and 2, total polyphenolic
content, carotenoid content and phenolic acid composition, antiox-
idant and α-glucosidase inhibitory potentials varied in different
extracts of black gram, and their milled fractions. The differences
in antioxidant and α-glucosidase inhibitory potentials in black
Table 5
Phenolic acid content in black gram and its fractions (mg/100 g).
Sample Whole flour Cotyledon Germ Aleurone Plumule Seed coat
Gallic acid 0.221a
±0.005 0.782b
±0.030 1.082c
±0.100 3.425d
±0.191 9.701f
±0.357 3.848e
±0.210
Protocatechuic acid 0.510b
±0.088 0.291a
±0.007 1.412c
±0.160 3.684d
±0.316 8.591f
±0.369 4.136e
±0.090
Gentisic acid 1.000a
±0.091 2.357b
±0.157 36.00d
±1.043 69.71e
±1.422 20.86c
±0.645 88.20f
±1.280
Vanillic acid 0.052a
±0.002 0.082b
±0.001 ND ND 19.47c
±0.523 ND
Syringic acid 0.067a
±0.003 ND ND 1.568b
±0.176 ND 1.287b
±0.172
Caffeic acid 0.021a
±0.002 ND ND 0.656b
±0.110 ND 1.340c
±0.097
Ferulic acid 15.23a
±0.842 23.52b
±1.064 164.2d
±5.675 111.4c
±2.723 684.0f
±13.38 466.2e
±11.79
All data are the mean±SD of three replicates. Mean followed by different letters in the same row differs significantly (Pb0.05).
Table 6
IC50 Values of antioxidant properties of acetone extract of black gram and its milled
fractions (μg of GAE) as determined by DPPH method.
Samples IC50
Whole gram 5.36e
±0.05
Cotyledon 12.00f
±0.05
Germ 4.93d
±0.02
Seed coat 3.01c
±0.07
ALESC* 2.90b
±0.02
Plumule 2.27a
±0.02
IC50 values were calculated from the dose responses curves. Values are expressed on as
is basis.
All data are the mean±SD of three replicates. Mean followed by different letters in the
same column differs significantly (Pb0.05). *ALESC, aleurone layer enriched in seed
coat.
Fig. 2. Reducing power of acetone extracts from black gram and its fractions
.
375T.K. Girish et al. / Food Research International 46 (2012) 370–377

7. gram flour and its milled fractions may depend on their bioactive
constituents.
Black gram flour extracts showed good antioxidant properties
(IC50 5.36 μg GAE) and inhibited 80% of α-glucosidase activity. How-
ever, its fractions like seed coat, aleurone layer enriched in seed
coat and plumule, exhibited more enzyme inhibitory activities and
antioxidant activities. This may be due to the differences in their phe-
nolic acid composition and carotenoid contents. Extracts from seed
coat, plumule and aleurone layer enriched in seed coat fractions had
lower IC50 values and also they inhibited the enzyme more effectively
at low concentrations compared to other fractions. These fractions
had very high amount of ferulic acid, gentisic acid and also good
amount of gallic and protocatechuic acid. Ferulic acid and gentisic acid
are reported to have potent free radical scavenging activities (Astidate
et al., 2005; Brand-Williams et al., 1995; Shahidi & Wanasundara,
1992). Higher α-glucosidase inhibitory activity for seed coat, plumule
and aleurone layer enriched in seed coat fractions are also due to the
changes in phenolic acid composition. Although germ fraction had
higher amount of phenolic acids compared to whole flour, it showed
low α-glucosidase inhibitory activity. The germ fraction is rich in ferulic
acid and gentisic acid compared to whole flour, but it did not contain
caffeic acid, vanillic acid and syringic acid (Table 5) and had low amount
of carotenoids compared to whole flour (Table 3). Thus, the higher ac-
tivities found in whole flour and some of its fractions may be due to syn-
ergistic effect of combination of the phenolic acids and carotenoids
present in them. Earlier, Kwon et al. (2008) reported that caffeic acid
and protocatechuic acid exhibited high α-glucosidase inhibitory activity
compared to other phenolic acids. Among the other fractions, cotyledon
and germ showed lower α-glucosidase inhibitory activities compared
to whole flour as well as other fractions.
In the present study, correlation coefficients were determined for
phenolic acids and IC50 values for enzyme inhibition and DPPH radical
scavenging activity. Protocatechuic acid, gallic acid, ferulic acid, genti-
sic acid syringic acid and caffeic acid negatively correlated to IC50
value for α-glucosidase inhibitory activity and the coefficients were
−0.67, −0.56, −0.56, −0.55, −0.54, −0.51, respectively (Pb0.05).
Similarly, protocatechuic acid, ferulic acid, gentisic acid and gallic
acid showed negative correlation to IC50 values for radical scavenging
activity and the values were −0.69, −0.61, −0.59 and −0.59, re-
spectively (Pb0.05).Vanillic acid showed very low values of correla-
tion coefficient indicating that its content does not correlate to any
of these activities. Contents of caffeic acid and syringic acid also did
not correlate to radical scavenging activity. It should be noted that
high negative correlation values for IC50 indicate better inhibition of
either free radical or enzyme activity. Thus, the present studies indi-
cate that extracts containing ferulic acid, protocatechuic acid, gallic
acid, gentisic acid, caffeic acid, syringic acid are potential inhibitor of
α-glucosidase enzyme while ferulic acid, protocatechuic acid, gallic
acid, gentisic acid are potential inhibitor of free radicals.
4. Conclusions
Black gram in the form of cotyledon is widely used for the prepa-
ration various food products. During milling of black gram into coty-
ledon (dhal), about 25% of the grain is removed as by-products and
these by-products are currently being wasted. The present study indi-
cates that black gram and its milled fractions are rich in antioxidant
compounds and nutrients like protein and dietary fiber. Of the various
fractions, seed coat fraction, aleurone layer enriched in seed coat frac-
tion and plumule fraction exhibited good antioxidant activities. The
polyphenols, carotenoids and dietary fibers are mostly concentrated
in the seed coat and aleurone layer enriched in seed fractions,
which constitute about 17% of the seed. On the other hand, germ
and aleurone layer enriched in seed coat fractions, which are also
major by-products, are rich in protein and dietary fiber. Therefore, ei-
ther whole fractions or their extracts can be used as source of nutra-
ceuticals and functional food ingredients in various processed foods
to reduce complications associated with cellular oxidative stress and
hyperglycemia-induced pathogenesis. Thus, this study provides eco-
nomic importance to the black gram milled by-products.
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