ANTIOXIDANT POTENTIAL OF DESI CHICKPEA VARIETIES COMMONLY CONSUMED IN PAKISTAN

ANTIOXIDANT POTENTIAL OF DESI CHICKPEA VARIETIESCOMMONLY CONSUMED IN PAKISTAN

MUHAMMAD ZIA-UL-HAQ1,6, SHAHID IQBAL2, SHAKEEL AHMAD3,MUHAMMAD IQBAL BHANGER4, WIESLAW WICZKOWSKI5 and

RYSZARD AMAROWICZ5

1Research Institute of Pharmaceutical SciencesDepartment of Pharmacognosy

University of KarachiKarachi-75270, Pakistan

2Department of ChemistryUniversity of Sargodha

Sargodha, Pakistan

3Department of AgronomyBahauddin Zakariya University

Multan, Pakistan

4National Center of Excellence in Analytical ChemistryUniversity of SindhJamshoro, Pakistan

5Division of Food ScienceInstitute of Animal Reproduction and Food Research

Polish Academy of SciencesOlsztyn, Poland

Submitted for Publication November 12, 2007Revised Received and Accepted March 12, 2008

ABSTRACT

Antioxidant potential of four Desi chickpeas (Cicer arietinum L.) variet-ies indigenous to Pakistan, namely Balksar 2000, CM98, Dasht and Winhar2000, was evaluated. All studied varieties exhibited appreciable total phenoliccontent (0.92–1.12 mg gallic acid equivalents/g), total flavonoid content(0.79–0.99 mg catechin equivalent [CAE]/g) and condensed tannin con-tent (0.58–0.69 mg CAE/g). In addition, antioxidant activities were testedusing 2,2-diphenyl-1-picrylhydrazyl radical (1.05–1.24 mmol trolox/g), 2,2′-

6 Corresponding author. TEL: +92-322-2506612; FAX: +92-48-3222121; EMAIL: [email protected]

Journal of Food Lipids 15 (2008) 326–342. All Rights Reserved.© 2008, The Author(s)Journal compilation © 2008, Wiley Periodicals, Inc.

326

azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (37.24–45.32 mmol trolox/g), ferric-reducing antioxidant power (0.73–0.90 mmol Fe2+ equivalents/100 g) and oxygen radical-absorbing capacity (8.58–11.4 mmol trolox/g). Allvarieties exhibited appreciable antioxidant potential and significant differ-ences (P < 0.05) were observed among the varieties in different systems ofantioxidant activity evaluation. The results of the present analytical studyshowed Desi chickpea (C. arietinum L.) indigenous to Pakistan to be a poten-tially valuable legume crop with high antioxidant potential.

PRACTICAL APPLICATIONS

The analytical findings of this study provide a regional database for thisvaluable legume crop, which has not been explored so far. The data obtainedwill be useful to both producers and consumers. Chickpeas may be used asfunctional ingredients for processing into health foods in the food industry.

INTRODUCTION

Free radical-induced oxidative damage is involved with various humandiseases such as cardiovascular diseases, neural disorders such as Alzheimer’sand Parkinson’s disease, diabetes and cancer (Shahidi 1996). Antioxidants canhelp in disease prevention by effectively quenching free radicals or inhibitingdamage caused by them (Halliwell et al. 1992). Synthetic antioxidants havebeen in use as food additives for a long time, but safety concerns and reportson their involvement in chronic diseases have restricted their use in foods.Attention has therefore been directed toward the development/isolation ofnatural antioxidants from botanical sources, especially edible plants. Evalua-tion of the antioxidant activity of leguminous seeds has been of interest inrecent years. Legumes including chickpea contain a wide range of polyphe-nolic compounds, including flavonols, flavone glycosides, flavanols and oli-gomeric and polymeric proanthocyanidins (White and Xing 1996; Sarma et al.2002; Singh et al. 2003). These are located essentially in their seed coat thatcan contribute to their antioxidant capacity. Phenolic compounds preset inleguminous seeds as typical antioxidants are capable of decreasing oxygenconcentration, intercepting singlet oxygen, preventing first-chain initiation byscavenging initial radicals such as hydroxyl radical, binding metal ion cata-lysts and decomposing primary products of oxidation to nonradical species(White and Xing 1996).

Chickpeas are the largest grown legume crop in Pakistan (Khokar et al.2001) and various varieties of chickpeas are popularly consumed as a source

327ANTIOXIDANT POTENTIAL OF DESI CHICKPEA VARIETIES

of dietary protein. Besides nutritional importance, chickpea seed has markedmedicinal properties. Seeds enrich the blood and cure skin diseases andinflammation of the ear (Caius 1989; Sastry and Kavathekar 1990; Agharkar1991; Warrier et al. 1995). They are used as tonic, appetizer, stimulant andaphrodisiac, and they also have anthelmintic properties (Sastry and Kavathekar1990). Among the food legumes, chickpeas are the most hypocholesterolemicagent and germinated chickpeas are reported to be effective in controllingcholesterol levels in rats (Geervani 1989). Dietary supplementation withchickpeas for at least 5 weeks resulted in significant reductions in serum totaland low-density lipoprotein cholesterols in adult woman and men (Pittawayet al. 2006).

The present study makes an attempt to screen and examine the anti-oxidant potential of Desi chickpea (Cicer arietinum L.) varieties of Pakistan.As antioxidant activity determination is reaction mechanism-dependent, thespecificity and sensitivity of a single method does not lead to complete exami-nation of all phenolic compounds in the extract. Therefore, a combination ofseveral tests that could provide a more reliable assessment of the antioxidantactivity profiles of foods has been used.

MATERIALS AND METHODS

Materials

All solvents used were of analytical grade. Methanol, acetone, ethanol,ammonium thiocyanate, ferrous chloride, linoleic acid, vanillin, gallic acid,Folin and Ciocalteau’s reagent, butylated hydroxytoluene (BHT) andhydroxyanisole (BHA), rutin, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH),2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-tri(2-pyridyl)-S-triazine (TPTZ) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were obtained from Sigma (St Louis, MO).

The seeds of four Desi chickpea (C. arietinum L.) varieties, namelyBalksar 2000, CM 98, Dasht and Winhar 2000, grown and harvested undersimilar environmental and agroclimatic conditions, were procured from theNuclear Institute for Agriculture and Biology, Faisalabad, Pakistan. Seeds ofall varieties were divided into groups for storage in stainless steel containers at4C prior to analysis.

Extraction

Chickpea samples were ground to flour with an IKA all-basic mill (IKAWorks Inc., Wilmington, NC) and were passed through a 60-mesh sieve. Theflour (0.5 g each) was accurately weighed into a set of centrifuge tubes, and

328 M. ZIA-UL-HAQ ET AL.

5 mL 50% acetone (v/v) was added. Then the capped tubes were placedhorizontally and shaken at 300 rpm at room temperature on an orbital shakerfor 3 h. The mixture was extracted for another 12 h by keeping the tubes in thedark overnight. The extracts were centrifuged at 2,000 ¥ g for 10 min. Residuewas reextracted with 5 mL of the respective extraction solvents. Two extractswere combined and stored at 4C in the dark. The extractions were conductedin triplicate for each individual variety.

Total Phenolics

The content of total phenolic compounds in extracts and each fractionwas estimated using the Folin and Ciocalteau’s phenol reagent (Singleton andRossi 1965). According to Heimler et al. (2005) and Xu and Chang (2007a),gallic acid was used as a standard in this work and results were reported asgallic acid equivalents (GAE)/g.

Total Flavonoid Content (TFC)

TFC was determined as reported previously (Jia et al. 1999). Accordingto Heimler et al. (2005) and Xu and Chang (2007a), the results were expressedas mg catechin equivalents (CAE) per g sample using the calibration curve of(+)-catechin. Linearity range of the calibration curve was 10 to 1,000 mg/mL(r = 0.99). The extraction was conducted in triplicate and extracts were dilutedto the linear range for determination.

Condensed Tannins

Condensed tannins (proanthocyanidins) were analyzed using acidifiedvanillin reagent (Broadhurst and Jones 1978). According to de Mejía et al.(2003) and Heimler et al. (2005), the results were as mg of CAE/g sampleusing the calibration curve of (+)-catechin. Linearity range of the calibrationcurve was 50 to 1,000 mg/mL (r = 0.99). For each specific sample, triplicateextractions were performed and used for analyses.

DPPH Radical Scavenging Activity

DPPH• scavenging capacity of chickpea extracts was evaluated accordingto the method described by Chen and Ho (1995). The absorbance of the sample(Asample) was measured using a UV 160 spectrophotometer (Shimadzu, Kyoto,Japan) at 517 nm against ethanol blank. A negative control (Acontrol) was takenafter adding DPPH· solution to 0.2 mL of the respective extraction solvent.

Antiradical activity was calculated from the equation


Antiradical activityAbsorbance

Absorbance% sample

control

= ⎛⎝⎜

⎞⎠⎠⎟

×100

Results were expressed as mmol of trolox equivalents (TE) per g oflegume as such (mmol TE/g) from the tests of the triplicate extracts using thecalibration curve of trolox. The linearity range of the calibration curve was 20to 1,000 mM (r = 0.99).

ABTS•+ Scavenging Assay

ABTS•+ scavenging assay was carried out following a reported modifiedmethod (Re et al. 1999). ABTS radical cation was prepared by passing a 5 mMABTS aqueous solution through the oxidizing reagent, manganese dioxide, onFisher Brand P8 filter paper (Anarkali, Lahore, Pakistan). Excess manganesedioxide was removed from the filtrate by passing the solution through a0.2-mm Fisher Brand membrane. The extracts were diluted in 5 mM phos-phate buffered saline (PBS; pH 7.4), to an absorbance of about 0.700 (�0.020)at 734 nm in a 1-cm cell. One mL of each of the extracts was added to 5 mLABTS•+ solution and the absorbance readings were taken 10 min after theinitial mixing at room temperature. PBS was used as the blank. The calibrationcurve of trolox standard was plotted. The antioxidant activities of chickpeaextracts were expressed as TE per 1.0 g of extract.

Ferric-reducing Antioxidant Power (FRAP) Assay

The FRAP assay was performed as described by Benzie and Strain(1999). The sample solution analyzed was first properly diluted with deionizedwater to fit within the linearity range of Fe2+. FRAP value was expressed asmmol of Fe2+ equivalents per 100 g of legume using the calibration curve ofFe2+. Linearity range of the calibration curve was 0.1 to 1.0 mM (r = 0.99).

Oxygen Radical-Absorbing Capacity (ORAC) Assay

Hydrophilic ORAC assay was carried out on a Gemini EM microplatespectrofluorometer (Molecular Devices, Sunnyvale, CA), which was equippedwith an incubator and wavelength adjustable fluorescence filters. The proce-dures were based on the previous reports (Prior et al. 2003; Wu et al. 2004).The kinetics of the fluorescence were recorded immediately by the softwareSoftMax Pro (Molecular Devices). The final ORAC values were calculatedusing a linear equation between the trolox standards or sample concentrationand net area under the fluorescein decay curve. The data were analyzed usingMicrosoft Excel (Microsoft, Roselle, IL). The area under the curve (AUC)


was calculated as: AUC = 0.5 + (R2/R1 + R3/R1 + R3/R1 + . . . + 0.5 Rn/R1),where R1 was the fluorescence reading at the initiation of the reaction and Rnwas the last measurement. The net AUC was obtained by subtracting the AUCof the blank from that of a sample or standard. The ORAC value was calcu-lated and expressed as mmol of TE/g legume using the calibration curve oftrolox. The linearity range of the calibration curve was 5.0 to 50 mM (r = 0.99).For each specific sample, triplicate extractions were analyzed.

Antioxidant Activity in Linoleic Acid System

The antioxidant activity of sample extracts was determined following themethod of Osawa and Namiki (1981). Sample extracts were added to a solu-tion mixture of linoleic acid (0.13 mL), 99.8% ethanol (10 mL) and 0.2 Msodium phosphate buffer (pH 7.0, 10 mL). The total volume was adjusted to25 mL with distilled water. The solution was incubated at 40C and the degreeof oxidation was measured according to the thiocyanate method (Yen and Duh1993) with 10 mL of ethanol (75%; v/v), 0.2 mL of an aqueous solution ofammonium thiocyanate (30%; m/v), 0.2 mL sample solution and 0.2 mL offerrous chloride (FeCl2) solution (20 mM in 3.5% [m/v] HCl) being addedsequentially. After 3 min of stirring, the absorption values of mixtures mea-sured at 500 nm were taken as peroxide contents. A control was performedwith linoleic acid but without the extracts. Synthetic antioxidants BHA andBHT were used as the positive controls. The percent inhibition of linoleic acidperoxidation to express antioxidative activity was calculated according toOsawa and Namiki (1981) from the equation

Antioxidative activityAbsorbance

Abso%

after hsample= −100360Δ

Δ rrbancecontrolafter h360100×⎛

⎝⎜⎞⎠⎟

High-Performance Liquid Chromatography (HPLC) Analysis

Phenolic constituents of extracts were analyzed using a Shimadzu HPLCsystem consisting of two LC-10AD pumps, SCTL 10A system controller andSPD-M 10A photodiode array detector. The chromatography was carried outusing a prepacked LiChrospher 100 RP-18 column (4 ¥ 250 mm, 5 mm;Merck, Darmstad, Germany). Elution for 50 min in a gradient system of5–40% acetonitrile in water adjusted to pH 2.5 with TFA was employed(Crozier et al. 1997). Detector was set at 280 nm; injection volume was 20 mLand the flow rate was 1 mL/min.

Acidic Hydrolysis of Phenolics from Extract

Acidic hydrolysis of phenolic compounds from Balksar 2000 extract wascarried out according to Crozier et al. (1997). Briefly, 50 mg of the fraction III


was dissolved in 5 mL of solution of 1.2 M HCl in 50% aqueous methanolcontaining 0.2% (m/v) of tert-butylhydroquinone. Solution was heated at 90Cfor 2 h. After hydrolysis the sample was adjusted to 25 mL with distilled water.

Liquid Chromatograph Mass Spectrometer (LC MS)

The Balksar 2000 extract dissolved in 80% (v/v) methanol and the sameextract after acidic were injected in a column of Shimadzu LC MS – QP 8000system. Condition of analysis: Cadenza CD-C18 (3 m, 2 ¥ 150 mm) column(Imtakt, Kyoto, Japan), flow rate 0.18 mL/min; injection loop 10 mL; elution ina gradient system: A, 3% (v/v) formic acid in water; B, 3% (v/v) formic acidin acetonitrile; gradient: 0 min = 10% B, 20 min = 30% B, 27 min = 80% B,45 min = 10%B; ionization mode: electrospray ionization (ESI); polarity:positive; curved desolvation line (CDL) temperature: 240C; CDL voltage:+15 V; probe voltage: +4.5 kV, defragmentation voltage: +35 V; detector gain1.8 kV; nebulizer gas flow: 4.0 mL/min.

Statistical Analysis

Analyses of variance and Tukey’s studentized test were performed at thelevel of P < 0.05 to evaluate the significance of differences among meanvalues. Data were analyzed by using the MSTATC statistical computerpackage. All analysis were triplicated and results were reported asmean � standard deviation.

RESULTS AND DISCUSSIONS

Chickpeas (C. arietinum L.) are the principal food legume used in Paki-stan. In perspective of global industrialization, ever increasing demand andscientific awareness regarding the nutritional and functional properties of food,the antioxidant potential of crops like chickpeas is of great importance in thedevelopment and commercialization of such crops, particularly for developingcountries like Pakistan.

According to literature data, the total phenolic content (TPC) is directlyassociated with antioxidant activity (Awika et al. 2003; Amarowicz et al.2004). TPC (expressed in mg GAE/g) of the antioxidant extracts from selectedchickpea varieties are presented in Table 1. A wide variation was observed forthe TPC and the varieties differed significantly with respect to this parameter.The highest TPC was obtained in the case of Balksar 2000 (1.12 mg GAE/g),which was followed by Winhar 2000 (1.08 mg GAE/g), whereas the lowestTPC was obtained in the case of CM-98 (0.92 mg GAE/g). Our values are inpartial agreement with those reported by other authors (Xu and Chang


2007a,b; Xu et al. 2007). The differences between current results and previousreport may be attributed to the differences in the sources of the samples.

In order to examine the potential role of flavonoids on the antioxidantactivity of selected chickpea varieties, the TFCs were analyzed and resultsexpressed in mg GAE/g samples are presented in Table 1. Significant differ-ences (P < 0.05) in TFC were found among studied varieties. The highest TFCwas obtained in the case of Balksar 2000 (0.99 mg GAE/g), whereas the lowestwas observed for CM 98 (0.79 mg GAE/g). Our findings are in agreement withresults reported by Xu et al. (2007). Growing location and year, and posthar-vest storage might contribute to the variations in flavonoid contents.

Condensed tannins are located mainly in the testa and play an importantrole in the defense system of seeds that are exposed to oxidative damage bymany environmental factors (Troszynska et al. 2002). Results in Table 1 showthat significant differences (P < 0.05) existed among studied varieties withrespect to this parameter. Balksar 2000 had the highest condensed tannincontent (0.69 mg CAE/g), whereas CM 98 had the lowest (0.58 mg CAE/g).Our results are supported by earlier studies (Xu et al. 2007). Differencesbetween our results and previous reports may be attributed partly to thedifferences in chickpea sources or determination methods.

The scavenging activity of chickpea extracts was determined by DPPH•

and ABTS•+ assays as shown in Table 2. Both of these radicals are commonlyused for assessment of antioxidant activity in vitro and are foreign to biologicalsystems. H-atom-donating capacity of polyphenols is an important biologi-cally significant property, in line with the ability of the plant antioxidants toconvert potentially damaging reactive oxygen species into nontoxic species(Goupy et al. 2003). In particular, DPPH• is increasingly used for quicklyassessing the ability of antioxidants to transfer the labile H atom to radicals.

The antiradical capacity values of chickpea varieties against DPPH•

ranged from 1.05 (CM 98) to 1.24 mmol trolox/g (Balksar 2000) (Table 2). Allvarieties differed significantly (P < 0.05) with respect to this parameter and the

TABLE 1.CONTENT OF TOTAL PHENOLICS, FLAVONOIDS, AND CONDENSED TANNINS IN

CHICKPEA VARIETIES

Content (mg/g) Balksar 2000 CM 98 Dasht Winhar 2000

Total phenolics 1.12 � 0.03a 0.92 � 0.01c 0.97 � 0.19bc 1.08 � 0.02ab

Total flavonoids 0.99 � 0.02a 0.79 � 0.01c 0.86 � 0.02b 0.95 � 0.11a

Condensed tannins 0.69 � 0.05a 0.58 � 0.13d 0.63 � 0.17c 0.67 � 0.04b

Data are expressed as means � standard deviations (n = 3) on dry weight basis.Values marked by the same letter in same column of same class are not significantly different(P > 0.05).


results are in line with values reported earlier (Xu and Chang 2007a,b; Xuet al. 2007). The antiradical capacity against ABTS•+ ranged from 37.2 (CM98) to 45.5 mmol trolox/g (Balksar 2000). Differences between our results andprevious reports may be attributed partly to the differences in the sources ofmaterials and in expressions based on dry weight or fresh weight basis calcu-lation. Although the DPPH• free radical is ubiquitously used to estimate thepotential free radical scavenging activity of natural products, the ABTS•+

cation radical is commonly used when issues of solubility or interference ariseand the use of DPPH•-based assays becomes inappropriate. The ABTS•+ scav-enging data (Table 2) suggest that the components within the extracts arecapable of scavenging free radicals via a mechanism of electron/hydrogendonation and should be able to protect susceptible matrices from free radical-mediated oxidative degradation. All varieties exhibited appreciable scavengingactivity against both the radicals and the same order of scavenging wasobserved by both assays. The antiradical activity of phenolic compoundspresent in the extracts of leguminous seeds against DPPH• and ABTS•+ werereported by several authors (Amarowicz et al. 2000, 2004, 2008a,b; Xu andChang 2007a,b).

The FRAP assay measures the antioxidant effect of any substance in thereaction medium as reducing ability. Antioxidant potential of the chickpeaseed extracts was estimated from their ability to reduce TPTZ-Fe (III) complexto TPTZ-Fe (II) complex. In contrast to other tests of total antioxidant power,the FRAP assay is simple, speedy, inexpensive and highly reproducible(Benzie and Strain 1999). The FRAP values of the antioxidant extracts fromselected chickpea varieties are presented in Table 2. Similar to that in DPPH•

analyses, high variations of FRAP values were observed and FRAP values of

TABLE 2.ANTIOXIDANT CAPACITY OF CHICKPEA VARIETIES

Assay Balksar 2000 CM 98 Dasht Winhar 2000

DPPH• scavenging capacity(mmol trolox/g)

1.24 � 0.3a 1.05 � 0.06c 1.15 � 0.19b 1.23 � 0.03ab

ABTS•+ scavenging capacity(mmol trolox/g)

45.3 � 1.1a 37.2 � 2.4b 42.2 � 0.8a 43.2 � 0.9a

FRAP (mmol Fe2+/g) 0.90 � 0.04a 0.73 � 0.02c 0.81 � 0.07bc 0.85 � 0.07ab

ORAC (mmol trolox/g) 11.4 � 0.15a 8.58 � 0.09d 9.63 � 0.32c 10.53 � 0.18b

Inhibition of linoleic acidperoxidation (%)

91.6 � 3.3a 79.5 � 2.3c 83.5 � 3.7bc 87.2 � 2.9b

Data are expressed as means � standard deviations (n = 3) on dry weight basis; values marked by thesame letter in same column of same class are not significantly different (P > 0.05).ABTS•+, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 2,2-diphenyl-1-picrylhydrazylradical; FRAP, ferric-reducing antioxidant power; ORAC, oxygen radical-absorbing capacity.


varieties ranged from 0.73 mmol Fe2+/g in (CM 98) to 0.90 mmol Fe2+/g(Balksar 2000). The varieties differed significantly with respect to this param-eter. The ability of leguminous seeds extracts (pea, beach pea, vetch, readbean, adzuki bean) to reduce Fe (III) was reported by Amarowicz et al. (2000,2008a,b) and Amarowicz and Troszynska (2003).

The ORAC method is usually employed to estimate antioxidant activityof foods and to evaluate in vivo responses to dietary antioxidant manipulation.The ORAC is the only method so far that combines both inhibition time anddegree of inhibition into a single quantity (Cao and Prior 1999). The food andnutraceutical industries have accepted the method to the point that somemanufacturers now include ORAC values on product labels (Bank andSchauss 2004). All chickpea extracts exhibited significant ORAC values andresults were expressed as TE (Table 2). Significant differences (P < 0.05) inORAC existed among chickpea varieties with values ranging from 8.58 mmolof trolox/g (CM 98) to 11.40 mmol trolox/g (Balksar 2000).

All the extracts of chickpea exhibited good antioxidant activity in thelinoleic acid peroxidation system (Table 2); the highest antioxidant activity

FIG. 1. CHROMATOGRAMS OF PHENOLIC COMPOUNDS OF CHICKPEA EXTRACTSRECORDED AT 280 nm

(A) Balkasar 2000. (B) CM 98. (C) Dasht. (D) Winhar 2000.


was observed for Balksar 2000, whereas the lowest value was for CM 98. At aconcentration of 0.2 mg/mL of linoleic acid solution, the chickpea extractsinhibited 79.56–91.57% peroxidation of linoleic acid after incubation for 360 h(15 days). This percentage was higher than that of the values for BHA (83.2%).The percent inhibition (91.57%) as exhibited by chickpea extracts was almostcomparable with that acquired for BHT (92.9%). The results obtained in thisstudy are in agreement with literature data. Antioxidant activity of the extractsof leguminous seeds (pea, bean, lentil, faba bean, broad bean, everlasting bean)in a b-carotene-linoleate model system has been reported in several studies(Amarowicz et al. 1996a,b, 2001, 2003; Madhujith et al. 2004).

The RP-HPLC chromatograms of the chickpea extracts recorded at280 nm were characterized by one dominant peak (1) with a retention time of12.23 min (Fig. 1). Ultraviolet (UV) spectrum of compound giving peak 1exhibited maximum at 272, 278 and 288 nm. Five peaks (2–6) with retentiontimes of 23.82, 25.73, 26.22, 27.03 and 28.70 were recorded at 350 nm(Fig. 2). UV spectra of compounds 2–6 showed maxima at 356, 356, 354, 336and 350 nm (Fig. 3). The chromatograms on Figs. 1 and 2 are recorded for

FIG. 2. CHROMATOGRAMS OF PHENOLIC COMPOUNDS OF CHICKPEA EXTRACTSRECORDED AT 350 nm

(A) Balkasar 2000. (B) CM 98. (C) Dasht. (D) Winhar 2000.


the same concentration (10 mg/mL) of the extract in the injected sample.Therefore, it is possible to compare the content of compounds 1–6 in theextracts. The highest content of compound 1 was noted for extracts obtainedfrom Balksar 2000 and Winhar 2000. The content of flavonoids in the extractswas in the order of Balksar 2000 > Winhar 2000 ~ Dasht > CM 98. These

FIG. 3. ULTRAVIOLET DIODE ARRAY DETECTION SPECTRA OF COMPOUNDSSEPARATED FROM CHICKPEA EXTRACTS USING HIGH-PERFORMANCE LIQUID

CHROMATOGRAPHY METHOD(1–6) Numbers of peaks corresponding to Figs. 1 and 2.


results are in accordance with the content of total flavonoids reported inTable 1.

The ESI mass spectrum in positive mode of compound 1 showed a[M+H]+ at an m/z of 205 (Fig. 4). Mass spectra indicated that compound hasa molecular weight of 204. Retention time of compound 1 (Fig. 1) wassimilar to those of vanillic, caffeic and gentisic acids. Most probably com-pound 1 was a derivative of phenolic acid with two hydroxyl groups in thebenzoic ring and with an attached high molecular weight aliphatic chain.The ESI mass spectra of compounds 2–6, because of the lack of dominantmolecular ions (results not shown), were difficult to interpret. After acidichydrolysis, peaks from compounds 2–6 were not observed on the chromato-gram (results not shown), which confirmed that compounds 2–6 were gly-cosided flavonoids. The main compound liberated after acidic hydrolysisshowed a [M+H]+ at an m/z of 384 (Fig. 5) that indicated that its molecularweight is 383.

150 200 250 300 350 400 450 500 550 600 m/z

0e3

100e3

200e3

300e3

400e3

Int. 205

206188 433409 455246 695503722 279 391 545173 365694433

FIG. 4. ELECTROSPRAY IONIZATION MASS SPECTRUM OF COMPOUND 1 FROMCHICKPEA EXTRACT

1, number of peak corresponding to Fig. 1.

150 200 250 300 350 400 450 500 550 600 m/z

0e3

250e3

500e3

Int.384

317 406299435324281 417 457363 595177 533551193362 884322205151

FIG. 5. ELECTROSPRAY IONIZATION MASS SPECTRUM OF MAIN COMPOUNDLIBERATED FROM CHICKPEA FLAVONOIDS AFTER ACIDIC HYDROLYSIS


CONCLUSIONS

Chickpeas could contribute significantly in the management and/orprevention of degenerative diseases associated with free radical damage, inaddition to their traditional role of preventing protein malnutrition. Thus,value-added chickpeas and chickpea-based products could expand into old andnew markets alike.

REFERENCES

AGHARKAR, S.P. 1991. Medicinal Plants of Bombay Presidency, pp. 62–63,India Scientific Publishers, Jodhpur, India.

AMAROWICZ, R. and TROSZYNSKA, A. 2003. Antioxidant activity of peaand its fractions of low molecular phenolics and tannins. Pol. J. FoodNutr. Sci. 53, 10–14.

AMAROWICZ, R., TROSZYNSKA, A., KARAMAC, M. andKOZLOWSKA, H. 1996a. Antioxidative properties of legume seedextracts. In Agri-Food Quality. An Interdisciplinary Approach(G.R. Fenwick, C. Hedley, R.L. Richards and S. Khokahr, eds.)pp. 376–379, The Royal Society of Chemistry, Cambridge, UK.

AMAROWICZ, R., KARAMAC, M., KMITA-GLAZEWSKA, H.,TROSZYNSKA, A. and KOZLOWSKA, H. 1996b. Antioxidant activityof phenolic fractions of everlasting pea, faba bean and broad bean.J. Food Lipids 3, 199–211.

AMAROWICZ, R., NACZK, M., ZADERNOWSKI, R. and SHAHIDI, F.2000. Antioxidative activity of condensed tannins of beach pea, canolahulls, evening primrose, and faba bean. J. Food Lipids 7, 195–205.

AMAROWICZ, R., KARAMAC, M. and WEIDNER, S. 2001. Antioxidativeactivity of phenolic fractions of pea (Pisum sativum). Czech J. Food Sci.19, 139–142.

AMAROWICZ, R., KARAMAC, M. and SHAHIDI, F. 2003. Antioxidativeactivity of phenolic fractions of lentil (Lens culinaris). J. Food Lipids 10,1–10.

AMAROWICZ, R., TROSZYNSKA, A., BARYLKO-PIKIELNA, N. andSHAHIDI, F. 2004. Extracts of polyphenolics from legume seeds – cor-relation between their total antioxidant activity, total phenolics content,tannins content and astringency. J. Food Lipids 11, 278–286.

AMAROWICZ, R., TROSZYNSKA, A., and PEGG, R.B. 2008a. Antioxida-tive and radical scavenging effects of phenolics from (Vicia sativum).Fitoterapia 79, 121–122.


AMAROWICZ, R., ESTRELLA, I., HERNÁNDEZ, T. and TROSZYNSKA,A. 2008b. Antioxidant activity of extract of adzuki bean and its fractions.J. Food Lipids 15, 119–136.

AWIKA, J.M., ROONEY, L.W., WU, X., PRIOR, R.L. and ZEVALLOS, L.C.2003. Screening methods to measure antioxidant activity of sorghum(Sorghum bicolor) and sorghum products. J. Agric. Food Chem. 51,6657–6662.

BANK, G. and SCHAUSS, A. 2004. Antioxidant testing: An ORAC update.Nutraceuticals World. http://www.nutraceuticalsworld.com/march042.htm

BENZIE, I.F.F. and STRAIN, J.J. 1999. Ferric reducing/antioxidant powerassay: Direct measure of total antioxidant activity of biological fluids andmodified version for simultaneous measurement of total antioxidantpower and ascorbic acid concentration. Methods Enzymol. 299, 15–27.

BROADHURST, R.B. and JONES, W.T. 1978. Analysis of condensed tanninsusing acified vanillin. J. Sci. Food Agric. 29, 788–794.

CAIUS, J.F. 1989. The Medicinal and Poisonous Legumes of India, pp. 27–28,India Scientific Publishers, Jodhpur, India.

CAO, G.H. and PRIOR, R.L. 1999. Measurement of oxygen radical absor-bance capacity in biological samples. Methods Enzymol. 299, 50–62.

CHEN, C.W. and HO, C.T. 1995. Antioxidant properties of polyphenolsextracted from green and black teas. J. Food Lipids 2, 35–46.

CROZIER, A., JENSEN, E., LEAN, M.E.I. and MCDONALD, M.S. 1997.Quantitative analysis of flavonoids by reverse-phase high performanceliquid chromatography. J. Chromatogr. A 761, 315–321.

DE MEJÍA, E.G., GUZMÍN-MALDOLNADO, S.H., ACOSTA-GALLEGOS, J.A., REYNOSO-CAMACHO, R., RAMÍREZ-RODRÍGUEZ, E., PONS-HERNÁNDEZ, J.L., GONZÍLEZ-CHAVIRA,M.M., CASTELLANOS, J.Z., and KELLY J.D. 2003. Effect of cultivarand crowing location on the trypsyn inhibitors, tannins, and lectins ofcommon beans (Phaseolus vulgaris L.) grown in the semiarid highlandsof Mexico. J. Agric. Food Chem. 51, 5962–5966.

GEERVANI, P. 1989. Utilization of chickpea in India and scope for novel andalternative uses. In Uses of Tropical Grain Legumes, Proceedings ofConsultant’s Meeting, 27–30 March 1989, pp. 47–54, ICRISAT Center,Patancheru, Andhra Pradesh, India.

GOUPY, P., DUFOUR, C., LOONIS, M. and DANGLES, O. 2003. Quanti-tative kinetic analysis of hydrogen transfer reactions from dietarypolyphenols to the DPPH radical. J. Agric. Food Chem. 51, 615–622.

HALLIWELL, B., GUTTERIDGE, J.M.C. and CROSS, C.E. 1992. Freeradicals, antioxidants, and human disease; where are we now? J. Lab.Clin. Med. 119, 598–619.


http://www.nutraceuticalsworld.com/march042

HEIMLER, D., VIGNOLINI, P., DINI, M.G. and ROMANI, A. 2005. Rapidtests to assess the antioxidant activity of Phaseolus vulgaris L. dry bean.J. Agric. Food Chem. 53, 3053–3056.

JIA, Z., TANG, M. and WU, J. 1999. The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals. FoodChem. 64, 555–559.

KHOKAR, S.N., MUZAFFAR, A.K. and CHAUDHRI, M.F. 2001. Somecharacters of chickpea-modulating Rhizobia native to thal soil. Pak. J.Biol. Sci. 4, 1016–1019.

MADHUJITH, T., NACZK, M. and SHAHIDI, F. 2004. Antioxidant activityof common beans (Phaseolus vulgaris L.). J. Food Lipids 11, 220–233.

OSAWA, T. and NAMIKI, M. 1981. A novel type of antioxidant isolated fromleaf wax of eucalyptus leaves. Agric. Biol. Chem. 45, 735–739.

PITTAWAY, J.K., AHUJA, K.D.K., CEHUN, M., CHRONOPOULOS, A.,ROBERTSON, I.K., NESTEL, P.J. and BALL, M.J. 2006. Dietarysupplementation with chickpeas for at least 5 weeks results in small butsignificant significant reductions in serum total and low-density lipopro-tein cholesterols in adult woman and men. Ann. Nutr Metab. 50, 512–518.

PRIOR, R.L., HOANG, H., GU, L.W., WU, X.L., BACCHIOCCA, M.,HOWARD, L., HAMPSCH-WOODILL, M., HUANG, D.J., OU, B.X.and JACOB, R. 2003. Assay for hydrophilic and lipophilic antioxidantcapacity (oxygen radical absorbance capacity (ORACFL) of plasma andother biological and food samples. J. Agric. Food Chem. 51, 3273–3279.

RE, R., PELLEGRINI, N., PROTEGGENTE, A., PANNALA, A., YANG, M.and RICE-EVANS, C. 1999. Antioxidant activity applying an improvedABTS•+ radical cation decolourisation assay. Free Radic. Biol. Med. 26,1231–1237.

SARMA, B.K., SINGH, D.P., MEFTA, S., SINGH, H.B. and SINGH, U.P.2002. Plant growth-promoting rhizobacteria-elicited alterations in phe-nolic profile of chickpea (Cicer arietinum) infected by Selerotium rolfsii.J. Phyopathol. 150, 277–282.

SASTRY, C.S.T. and KAVATHEKAR, K.Y. 1990. Plants for Reclamation ofWastelands, p. 684, Council of Scientific and Industrial Research, NewDelhi, India.

SHAHIDI, F. 1996. Natural antioxidants: An overview. In Natural Antioxi-dants. Chemistry, Health Effects, and Applications (F. Shahidi, ed.)pp. 1–11, AOCS Press, Champaign, IL.

SINGH, U.P., SARMA, B.K. and SINGH, D.P. 2003. Effect of plant growth-promoting rhizobacteria and culture filtrate of Selerotium rolfsii onphenolic and salicylic acid contents in chickpea (Cicer arietinum). Curr.Microbiol. 46, 131–140.


SINGLETON, V.L. and ROSSI, J.A. 1965. Colorimetry of total phenolics withphosphomolybdicphosphotungstic acid reagents. Am. J. Enol. Vitic. 16,144–58.

TROSZYNSKA, A., ESTRELLA, I., LÓPEZ-AMÓRES, M.L. andHERNÓNDEZ, T. 2002. Antioxidant activity of pea (Pisum sativum L.)seed coat acetone extract. Lebensm. Wiss. Technol. 35, 158–164.

WARRIER, P.K.W., NAMBIAR, V.P.K. and REMANKUTTY, C. 1995.Indian Medicinal Plants, pp. 70–73, Orient Longman, Chennai, India.

WHITE, P.J. and XING, Y. 1996. Antioxidants from cereals and legumes.In Natural Antioxidants. Chemistry, Hwalth Effects, and Application,(F. Shahidi, ed.) pp. 25–63, AOCS Press, Champaign, IL.

WU, X.L., BEECHER, G.R., HOLDEN, J.M., HAYTOWITZ, D.B., GEB-HARDT, S.E. and PRIOR, R. 2004. Lipophilic and hydrophilic antioxi-dant capacities of commonfoods in the United States. J. Agric. FoodChem. 52, 4026–4037.

XU, B.J. and CHANG, S.K.C. 2007a. A comparative study on phenolic pro-files and antioxidant activities of legumes as affected by extraction sol-vents. J. Food Sci. 72, S159–S166.

XU, B.J. and CHANG, S.K.C. 2007b. Comparative analyses of phenoliccomposition, antioxidant capacity, and color of cool season legumes asaffected by extraction solvents. J. Food Sci. 72, S167–S177.

XU, B.J., YUAN, S.H. and CHANG, S.K.C. 2007. Comparative studies on theantioxidant activities of nine common food legumes against copper-induced human low-density lipoprotein oxidation in vitro. J. Food Sci.72, S522–S527.

YEN, G.C. and DUH, P.D. 1993. Antioxidative properties of methanolicextracts from peanut hulls. J. Am. Oil Chem. Soc. 70, 383–386.


Documents

ANTIOXIDANT POTENTIAL OF DESI CHICKPEA VARIETIES COMMONLY CONSUMED IN PAKISTAN