Amylase inhibitory paper 4

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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 abstract article info Article history: Received 13 August 2011 Accepted 24 December 2011 Keywords: Black gram (Vigna mungo L.) Nutrient composition Bioactive compounds Dietary ber 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 our 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 ber 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 our 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 (P b 0.05) to IC 50 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 benets. © 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, avonoids, phytosterols that possess health benets (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 our and jaggery were regarded as nutritious food in India. Whole black gram our paste either alone or in combination with sandalwood paste or fenugreek paste is used for skin or hair care, respectively. Incorporation of black gram our 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, ber, 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 ber are gaining impor- tance because of their health benets. Polyphenols, carotenoids and Food Research International 46 (2012) 370377 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: [email protected] (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
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Transcript of Amylase inhibitory paper 4

Page 1: Amylase inhibitory paper 4

Food Research International 46 (2012) 370–377

Contents lists available at SciVerse ScienceDirect

Food Research International

j ourna l homepage: www.e lsev ie r .com/ locate / foodres

Nutrient distribution, phenolic acid composition, antioxidant and alpha-glucosidaseinhibitory 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 Indiab Department of Grain Science and Technology, Central Food Technological Research Institute, Mysore, 570 020, India

⁎ Corresponding author at: Department of BiochemisTechnological Research Institute, Mysore, 570 020, Infax: +91 821 2517233.

E-mail address: [email protected] (U.

0963-9969/$ – see front matter © 2012 Elsevier Ltd. Alldoi:10.1016/j.foodres.2011.12.026

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 August 2011Accepted 24 December 2011

Keywords:Black gram (Vigna mungo L.)Nutrient compositionBioactive compoundsDietary fiberPhenolic acidsAntioxidantsα-glucosidase inhibition

Black gram belongs to the Leguminosae family. It is one of the less studied legumes, although it is widely usedin different parts of the world. Black gram in the form of cotyledon (dhal) is mainly used for the preparationof various food products. During milling of black gram into cotyledon, about 25% of the grain is removed aswaste by-products. In the present study, nutrient content, phenolic acid composition, antioxidant activityand α-glucosidase inhibitory properties of total black gram flour and its milled fractions were determinedwith 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 fatand protein, while seed coat and plumule fractions had the lowest (0.9%). Seed coat had the highest dietaryfiber content (78.5%) while cotyledon had the lowest (24.4%). Seed coat, plumule and aleurone layer enrichedin seed coat extracts showed a better antioxidant potential compared to other fractions and this may be dueto the quantitative and qualitative differences in phenolic acids. Extracts of seed coat, plumule and aleuronelayer 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 thesefractions 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 itsfractions 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 worldpopulation. It is also reported that legumes have certain phytochem-icals like polyphenols, flavonoids, phytosterols that possess healthbenefits (Kritchevsky & Chen, 2005; Sessa, 2004; Sreerama,Sashikala, & Pratape, 2010). Black gram or black gram lentil (Vignamungo L.) belongs to the Leguminosae family (Reddy, Salunkhe, &Sathe, 1982; Salunkhe, Kadam, & Chavan, 1985). It is one of the lessstudied 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 preparationof different food products. Dehusked cotyledon is used for the prepa-ration of fermented foods such as idli, dosa, and non-fermented foods

try and Nutrition, Central Fooddia. Tel.: +91 821 2514876;

J.S. Prasada Rao).

rights reserved.

like cooked dhal, hopper, papad and waries (spicy hollow balls)(Batra & Millner, 1974). Traditionally, sweets prepared with wholeblack gram flour and jaggery were regarded as nutritious food inIndia. Whole black gram flour paste either alone or in combinationwith sandalwood paste or fenugreek paste is used for skin or haircare, respectively. Incorporation of black gram flour was reported toimprove 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 dehuskedcotyledon essentially involves the removal of seed coat, germ, aleu-rone layer and plumule, and these fractions may consist of a varietyof nutrients. Currently, except cotyledon fraction, the other fractionsare 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

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371T.K. Girish et al. / Food Research International 46 (2012) 370–377

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 industrieswhich were traditionally treated as environmental pollutants arebeing recognized as good sources for obtaining valuable components.By-products from cereal, legume and fruit processing industries havebeen found to be rich and economically inexpensive sources of bioactivecompounds 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). Theseed 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 lipidswere shown to have cholesterol-reducing effect in both humans and ex-perimental animals (Saraswathi Devi & Kurup, 1972). Distribution ofbioactive compounds in plants varies in different tissues. In the presentstudy, 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 acidcomposition, 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), butylatedhydroxyanisole (BHA), α-amylase (Termamyl), pepsin, pancreatin,celite were purchased from Sigma Aldrich Chemical Co. (St. Louis,USA). Folin–Ciocalteu reagent was obtained from SR LaboratoriesLimited (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

Black gram

Milling

Cotyledon(dhal) (75%) seedcoat(husk) (9

Germ (6%)

Plumule (2%)

Fig. 1. Flow diagram for separation

60°C for 8 h. The black gram thus obtained after treatment was milledusing Versatile Dhal Mill according to the procedure described byNarasimha, Ramakrishnaiah, Pratape, Sasikala, and Narasimhan(2002). Black gram was milled into cotyledon, seed coat, and mixtureof germ, aleurone, seed coat powder, and plumule. Themixturewas fur-ther separated into different fractions by air classification as describedin Fig. 1 (Ajila & Prasada Rao, 2009).

2.3. Determination of proximate composition

Moisture, protein, fat, ash and crude fiber contents in whole blackgram flour and its milled fractions (BGMF) were determined by AOACmethods (2005). The total carbohydrate content was calculated bythe 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 with30 mL of either 80% acetone or 80% ethanol separately and were cen-trifuged for 15 min at 8000×g. The clear supernatants obtained weresubjected to total phenolic content estimation using the Folin–Ciocalteureagent following the procedure described by Swain and Hillis (1959).Gallic acid was used as a standard. The total polyphenol content in theextract was expressed as gallic acid equivalents (GAE).

2.5. Determination of anthocyanin content

Monomeric anthocyanin content of the black gram flour andBGMF acetone extracts were measured using a spectrophotometricpH differential method (Wolfe, Xianzhong, & Liu, 2003). Anthocyanincontent was expressed as mg cyanidin 3-glucosides equivalent/100 gsample for the triplicate extracts.

2.6. Determination of carotenoids

Black gram flour and BGMF (1 g) were homogenized with 40 mLof methanol containing 1 g KOH. The mixture was saponified

%) Mixture of germ, seed coat, plumuleand aleurone powder (16%)

Air classification

Air velocity 3.1 m/sec;

Feed rate 96 g/min

Plumule, aleurone, seed coat powder

Air classification

Air velocity 2.9m/sec;

Feed rate 184 g/min

aleurone layer enriched in seed coat(8%)

of black gram milled fractions.

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overnight and the saponified mixture was transferred to separatingfunnel containing 25 mL of hexane and gently shaken for 60 s, thephases were allowed to separate. The aqueous phase was separatedand was re-extracted in the separating funnel with 25 mL hexane.This was repeated until the hexane extract was colorless. The hexaneextracts were pooled, washed with water until free of alkali, driedover sodium sulfate and concentrated in a vacuum evaporator atroom temperature. The resulting solution was made up to a suitablevolume 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 smallamount comes from chlorophyll b and negligible absorbance fromchlorophyll 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 extractswas calculated by using the following formulae (Lichtenthaler, 1987).

Chlorophyll a Cað Þ ¼ 12;25A663:2−2:79A646:8Chlorophyll 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, BGMFand 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 (pH6.6) and mixed with 1 mL of potassium ferricyanide (0.1%) and themixture was incubated at 50 °C for 20 min. Trichloroacetic acid(500 μL, 10%) was added to the reaction mixture and centrifuged at3000×g for 10 min. The supernatant obtained was mixed with equalvolume of distilled water and 300 μL of 1% ferric chloride was addedand the absorbance was measured at 700 nm. Increased absorbanceof the reaction mixture indicated the increased reducing power. Theantioxidant activity of the extract was compared with BHA.

2.8. Measurement of free radical scavenging activity

Scavenging the stable DPPH radical is another widely usedmethodto evaluate antioxidant activity. DPPH is a stable free radical withcharacteristic absorption at 517 nm and antioxidants react withDPPH and convert it to 2,2-diphenyl-1-picrylhydrazine. The degreeof discoloration indicates the scavenging potential of the antioxidantextract, which is due to the hydrogen donating ability (Van Gadow,Joubert, & Hannsman, 1997).The effect of acetone extracts of blackgram flour and BGMF on DPPH radical was determined according tothe method described by Blois (1958) with modification describedby Brand-Williams, Cuvelier, and Berset (1995). A 100 μM solutionof DPPH inmethanolwasprepared andBGMF extracts (200 μL) contain-ing 1 to 5 μg GAE were mixed with 1 mL of DPPH solution. The mixturewas shaken vigorously and left in the dark at room temperature for20 min. The absorbance of the resulting solution was measured at517 nm. The control contained all the reagents except sample ex-tracts/BHA. The capacity to scavengeDPPH radicalwas 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 theabsorbance in the presence of sample extract/BHA. The results were

plotted as the % of scavenging activity against concentration of thesample. The half-inhibition concentration (IC50) was defined as theamount 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 outaccording 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 thereaction mixture was made up to 2.8 mL with sodium phosphatebuffer (pH 6.8; 50 mM). The reaction was initiated by adding 20 μLof α–glucosidase enzyme (2mg 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 theextract. The % of inhibition was calculated by the following equation.

Inhibition %ð Þ ¼ A405control−A405extract½ �A405control½ � 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 accordingto the method of Adom and Liu (2002) with some modifications.Briefly, fractions (1 g) were extracted with 20 mL of 80% acetone for1 h using magnetic stirrer. After centrifugation at 3000×g for20 min, the supernatant was removed and solution was extractedfive times with ethyl acetate phase separation followed by dryingwith 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) andinsoluble dietary fiber (IDF) contents. The samples were treatedwith thermo-stable α-amylase (Termamyl) and then digested withpepsin 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 overnightat 105 °C and were incinerated at 550 °C for 8 h and the weightswere determined. A control was performed following the same proce-dure. Total dietary fiber was then calculated as combined value of SDFand IDF.

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2.12. Statistical analysis

Three independent experiments were conducted in triplicate andthe data were reported as means±SD. Duncan's new multiplerange tests was used to determine the difference of means, andPb0.05 was considered to be statistically significant (Steel & Torrie,1980).

3. Results and discussion

3.1. Composition of nutrients in black gram and black gram milledfractions

The nutrient composition of black gram flour and BGMF is shownin Table 1. The total protein content in different fractions ranged from12 to 42%. The germ fraction had the highest amount of protein con-tent (42%) followed by whole black gram flour and the cotyledon. Thecrude fat content was the highest in the germ fraction, while it wasthe 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 leastin the cotyledon, while carbohydrate content was the highest in thecotyledon and the least in the germ. Ash content was the highest inthe germ followed by the aleurone layer enriched seed coat fraction.The fat, fiber, ash and total carbohydrate contents determined inwhole black gram flour are comparable to literature values. Kanthaand 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 reportedin black gram seeds by Salunkhe et al. (1985) and Kantha and Erdman(1987) varied between 21 and 31%, while in the present study theprotein 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 chickpea 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 thenutrient 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 ofdifferent milled fractions of black gram except for the cotyledon frac-tion. The protein, fat, ash, fiber and total carbohydrate contents in thecotyledon fraction were reported to be 24, 1.4, 3.2, 0.9 and 59.6%, re-spectively (Gopalan, Sastri, & Balasubramanian, 1996) and thesevalues are slightly different from the values obtained in the presentstudy (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 hadmore fat (2.6–7.8%) compared to other fractions of chickpea and

Table 1Proximate composition of black gram and its milled fractions (%).

Sample Protein Moisture Ash

Whole gram 26.75d±0.25 11.41b±0.08 3.29a±Cotyledon 24.33c±0.28 11.41b±0.18 3.24a±Germ 42.16e±0.14 11.40b±0.12 5.30c±Seed coat 12.41a±0.14 10.29a±0.31 3.03a±ALESC* 23.00b±0.25 11.96b±0.25 4.12b±Plumule fraction 12.25a±0.25 12.61c±0.31 3.00a±

Values are expressed on as is basis. All data are the mean±SD of three replicates. Mean foCarbohydrate content was calculated by subtracting % of protein, moisture, ash, fat and cru*ALESC, aleurone layer enriched in seed coat.

horse gram. However, these values are different from the compositionof black gram fractions reported in the present study. Among the ce-reals, composition of wheat milled fractions is known and wheatgerm 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 inseed coat fraction is rich in fiber as well as protein, and therefore,this fraction also can be used in various food formulations. Nowadaysimportance is given to consuming foods containing whole grain forhealth benefits. To get maximum benefits of nutraceuticals, thesefractions 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 andothers (Devries, Prosky, Li, & Cho, 1999; Lario et al., 2004). As canbe 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 andBGMF were determined using enzymatic gravimetric method. Asshown in Table 2, the total dietary fiber (TDF) content in differentfractions varied from 24.42% to 78.53%. Seed coat and plumule frac-tions had the highest value of fiber and cotyledon fraction had thelowest one compared to those of other fractions. The low value fortotal 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 insolubledietary fiber content in different fractions varied from 21 to 69%,while the soluble fiber content varied from 2.6 to 9.3%. Earlier reportsindicate that the soluble dietary fiber content in green gram, chickpeaand 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 ofhealth benefits, both IDF and SDF complement each other, and eachfraction has different physiological effect. Insoluble dietary fiber re-lates to both water absorption and intestinal regulation, whereasSDF associates with cholesterol in blood and diminishes its intestinalabsorption (Schneeman, 1987; Shinnick, Mathews, & Ink, 1991).

3.3. Total phenolic content and anthocyanin content in black gram andits fractions

Polyphenols are the major group of compounds that contribute tothe antioxidant properties. In the present study, black gram flour andits milled fractions were extracted with 80% (v/v) acetone or 80% (v/v) ethanol separately, and the total phenolic contents in the extractswere determined. Of the two solvents used, acetone showed a betterextraction 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

Fat Crude fiber Carbohydrates

0.17 1.44c±0.02 5.56b±0.16 51.53e±0.500.06 1.79d±0.03 1.20a±0.04 58.01f±0.270.03 3.42e±0.02 19.48c±0.24 18.21a±0.300.06 0.93a±0.01 33.72f±0.20 39.78c±0.160.04 1.04b±0.02 22.40d±0.14 37.46b±0.420.03 0.91a±0.01 23.30e±0.20 47.91d±0.49

llowed by different letters in the same column differs significantly (Pb0.05).de fiber from 100.

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Table 2Dietary 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±0.60Cotyledon 21.80a±0.70 2.62a±0.06 24.42a±0.67Germ 39.60c±0.40 5.20c±0.41 44.80c±0.11Seed coat 69.23f±0.66 9.30e±0.26 78.53f±0.55ALESC* 59.76d±0.20 7.93d±0.11 67.70d±0.10Plumule 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. Meanfollowed by different letters in the same column differs significantly (Pb0.05).*ALESC, aleurone layer enriched in seed coat.

Table 4Total 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±0.38Cotyledon 0.042b±0.0004 5.85a±0.41Germ 0.034a±0.0005 13.06c±0.41Seed coat 0.415f±0.0030 86.84f±0.59ALESC* 0.326e±0.0040 70.42e±0.58Plumule 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. Meanfollowed 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

aleurone layer enriched in seed coat fraction (72.83 mg GAE/g). Bothblack 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 (wholeflour) 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 oftotal polyphenol contents in different black gram cultivars using80% methanol and reported that polyphenol contents ranged from1.1 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 extractsof 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 maybe due to the variation in the cultivar as well as the extraction proce-dure followed. With respect to polyphenol content in different milledfractions, 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 andhorse gram and found the highest amount of polyphenols in seedcoat and lowest amount in cotyledon fraction. However, their resultsindicated that between chickpea and horse gram, the content of totalpolyphenols and their distribution in different fractions varieddepending on the type of pulse. Horse gram fractions had higheramount of polyphenols compared to that of chickpea, and amongthe fractions, seed coat had the highest total polyphenol content(Sreerama, Sashikala et al. (2010)).

As acetone extracts showed higher amount of total polyphenolcontent, further studies were carried out with acetone extracts.

Anthocyanins are a group of phenolic compounds present in theplant kingdom and they exhibit good antioxidant property. The an-thocyanin content in black gram flour and its milled fractions rangedfrom 6–87 mg/100 g (Table 4). Its content was proportional to thetotal polyphenol content in different fractions. Similar to total poly-phenol content, anthocyanin content was found to be the highest inseed coat fraction (87 mg/100 g). The anthocyanin contents in cookedblack 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

Table 3Total 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±0.01Cotyledon 0.87a±0.06 0.79a±0.02Germ 12.81c±0.45 5.00c±0.05Seed coat 134.66f±1.52 126.53f±1.36ALESC* 72.83d±1.23 37.68e±1.20Plumule 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.

of anthocyanins consumption are well known and anthocyanins aremainly present in blue and red colored fruits, vegetables includingred wine (Clifford, 2000). In developed countries consumption ofthese 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 inTable 4, the carotenoid content in the extracts of different fractionsranged from 0.042 to 0.415 mg/100 g. The seed coat was the richestin carotenoids followed by the aleurone layer rich husk fraction andthe germ fraction had the least value for carotenoids. The carotenoidcontents in different fractions of pulses and legumes have beenreported earlier by different workers. In general, legume seeds arepoor sources of carotenoids compared to their leaves, and also fruitsand vegetables. According to the studies of Fordham, Wells, andChen (1992) carotenoid content in different varieties of peas andbeans ranged from 0.003 to 0.037 mg/100 g, and 0.0002 to0.003 mg/100 g, respectively. Kantha and Erdman (1987) reportedcarotenoid content in peas, lima bean, soybean, and mung beanseeds and were found to be 0.0005 mg, 0.5 mg, 0.002 mg and0.004 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 foundto be different in different fractions. In all the fractions, ferulic acidwas the predominant phenolic acid followed by gentisic acid. Thefractions were also rich in gallic acid and protocatechuic acid. Seedcoat, aleurone layer enriched in seed coat and plumule fractionsshowed similar phenolic acid composition, except that seed coatand aleurone layer rich husk fractions did not show the presence ofvanillic acid, while phenolic acids like syringic and caffeic acidswere absent in plumule fraction. However, gentisic acid content wasthe highest in seed coat followed by aleurone layer enriched in seedcoat fraction, germ and plumule fractions, and it was very low inboth cotyledon and whole flour. Lopez-Amoros, Hernandez, andEstrella (2006) reported the presence of phenolic acids like ferulicacid, protocatechuic acid, p-coumaric acid, p-hydroxybenozoic acidand 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 thehighest in peas, and p-coumaric acid content was the highest in len-tils. Ferulic acid was the prominent phenolic acid reported in differentmilled fractions of chickpea (Sreerama, Sashikala et al., 2010). Ferulicacid is a major phenolic acid present in plant cell walls of variousseeds 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

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Table 5Phenolic 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±0.210Protocatechuic acid 0.510b±0.088 0.291a±0.007 1.412c±0.160 3.684d±0.316 8.591f±0.369 4.136e±0.090Gentisic acid 1.000a±0.091 2.357b±0.157 36.00d±1.043 69.71e±1.422 20.86c±0.645 88.20f±1.280Vanillic acid 0.052a±0.002 0.082b±0.001 ND ND 19.47c±0.523 NDSyringic acid 0.067a±0.003 ND ND 1.568b±0.176 ND 1.287b±0.172Caffeic acid 0.021a±0.002 ND ND 0.656b±0.110 ND 1.340c±0.097Ferulic 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).

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free radical scavenger and reported to possess antitumor properties(Kampa et al., 2004).

3.6. In vitro bioactive assays

3.6.1. Antioxidant activityPolyphenols 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 electrontransfer ability and may, therefore, serve as a significant indicator ofits antioxidant activity (Meir, Kanner, Akiri, & Hadas, 1995). Fig. 2shows the reducing power of the extracts from six different blackgram fractions. The reducing power increased with the concentrationof extracts of different fractions. At 15 μg GAE, plumule fractionshowed the highest antioxidant property compared to that of otherextracts. 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. Theantioxidant property of the extracts is mainly due to the presence ofpolyphenols and carotenoids in the extracts. It has been reportedthat 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 ofplumule, aleurone layer enriched in seed coat fraction and seed coatfraction 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 betterradical scavenging activity followed by aleurone layer enriched inseed coat fraction and husk fraction. Cotyledon extract showed the

Fig. 2. Reducing power of acetone extracts from black gram and its fractions.

highest IC50 value of 12 μg, indicating its lowest antioxidant activitycompared to the extracts from other fractions.

3.6.2. α-glucosidase inhibitory activity of different extractsType 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 hyperglycemiais to retard absorption of glucose through inhibition of carbohydratehydrolyzing enzymes. α-glucosidase is one of the key enzymes in-volved in the release of glucose from starch for the intestinal glucoseabsorption. The inhibition of this enzyme decreases the blood glucoselevels and thus it is an important strategy for the management oftype-2 diabetes (Plus, Keup, Krause, Thomos, & Hoffmeister, 1977).α-glucosidase inhibitors from natural food sources is an attractivestrategy 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 μglevel, seed coat, aleurone layer enriched in seed coat and plumulefractions inhibited 58–70% enzyme activity, while the other fractionsshowed 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 and8.75 μg GAE. Seed coat, plumule and aleurone layer enriched inseed coat fractions had low IC50 values of 1.85 μg, 1.90 μg and2.25 μg, respectively. Whole flour and germ had IC50 values of 3.8and 7 μg, respectively. Cotyledon fraction had the least inhibitoryproperties i.e., it had high value of IC50 (8.75 μg). Thus, whole flourextracts as well as seed coat fractions and plumule exhibited poten-tial antidiabetic properties.

3.7. Correlation of in vitro biological activities with bioactive compoundsin different extracts

As can be seen in Tables 3–6 and Figs. 1 and 2, total polyphenoliccontent, carotenoid content and phenolic acid composition, antiox-idant and α-glucosidase inhibitory potentials varied in differentextracts of black gram, and their milled fractions. The differencesin antioxidant and α-glucosidase inhibitory potentials in black

Table 6IC50 Values of antioxidant properties of acetone extract of black gram and its milledfractions (μg of GAE) as determined by DPPH method.

Samples IC50

Whole gram 5.36e±0.05Cotyledon 12.00f±0.05Germ 4.93d±0.02Seed coat 3.01c±0.07ALESC* 2.90b±0.02Plumule 2.27a±0.02

IC50 values were calculated from the dose responses curves. Values are expressed on asis basis.All data are the mean±SD of three replicates. Mean followed by different letters in thesame column differs significantly (Pb0.05). *ALESC, aleurone layer enriched in seedcoat.

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Fig. 3. α-glucosidase inhibition of acetone extracts from black gram and its fractions.

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gram flour and its milled fractions may depend on their bioactiveconstituents.

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 seedcoat and plumule, exhibited more enzyme inhibitory activities andantioxidant activities. This may be due to the differences in their phe-nolic acid composition and carotenoid contents. Extracts from seedcoat, plumule and aleurone layer enriched in seed coat fractions hadlower IC50 values and also they inhibited the enzyme more effectivelyat low concentrations compared to other fractions. These fractionshad very high amount of ferulic acid, gentisic acid and also goodamount of gallic and protocatechuic acid. Ferulic acid and gentisic acidare reported to have potent free radical scavenging activities (Astidateet al., 2005; Brand-Williams et al., 1995; Shahidi & Wanasundara,1992). Higher α-glucosidase inhibitory activity for seed coat, plumuleand aleurone layer enriched in seed coat fractions are also due to thechanges in phenolic acid composition. Although germ fraction hadhigher amount of phenolic acids compared to whole flour, it showedlow α-glucosidase inhibitory activity. The germ fraction is rich in ferulicacid and gentisic acid compared to whole flour, but it did not containcaffeic acid, vanillic acid and syringic acid (Table 5) and had low amountof carotenoids compared to whole flour (Table 3). Thus, the higher ac-tivities found inwhole flour and some of its fractionsmay be due to syn-ergistic effect of combination of the phenolic acids and carotenoidspresent in them. Earlier, Kwon et al. (2008) reported that caffeic acidand protocatechuic acid exhibited highα-glucosidase inhibitory activitycompared to other phenolic acids. Among the other fractions, cotyledonand germ showed lower α-glucosidase inhibitory activities comparedto whole flour as well as other fractions.

In the present study, correlation coefficients were determined forphenolic acids and IC50 values for enzyme inhibition and DPPH radicalscavenging activity. Protocatechuic acid, gallic acid, ferulic acid, genti-sic acid syringic acid and caffeic acid negatively correlated to IC50value 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 gallicacid showed negative correlation to IC50 values for radical scavengingactivity 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 anyof these activities. Contents of caffeic acid and syringic acid also didnot correlate to radical scavenging activity. It should be noted thathigh negative correlation values for IC50 indicate better inhibition ofeither free radical or enzyme activity. Thus, the present studies indi-cate that extracts containing ferulic acid, protocatechuic acid, gallicacid, gentisic acid, caffeic acid, syringic acid are potential inhibitor of

α-glucosidase enzyme while ferulic acid, protocatechuic acid, gallicacid, 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 andthese by-products are currently being wasted. The present study indi-cates that black gram and its milled fractions are rich in antioxidantcompounds and nutrients like protein and dietary fiber. Of the variousfractions, seed coat fraction, aleurone layer enriched in seed coat frac-tion and plumule fraction exhibited good antioxidant activities. Thepolyphenols, carotenoids and dietary fibers are mostly concentratedin the seed coat and aleurone layer enriched in seed fractions,which constitute about 17% of the seed. On the other hand, germand aleurone layer enriched in seed coat fractions, which are alsomajor 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 foodsto reduce complications associated with cellular oxidative stress andhyperglycemia-induced pathogenesis. Thus, this study provides eco-nomic importance to the black gram milled by-products.

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