research article antioxidant capacity and antimutagenic potential of murraya...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 263509 10 pageshttpdxdoiorg1011552013263509

Research ArticleAntioxidant Capacity and Antimutagenic Potential ofMurraya koenigii

Maryam Zahin12 Farrukh Aqil12 Fohad Mabood Husain1 and Iqbal Ahmad1

1 Department of Agricultural Microbiology Aligarh Muslim University Aligarh 202 002 India2 James Graham Brown Cancer Center University of Louisville Louisville KY 40202 USA

Correspondence should be addressed to Iqbal Ahmad ahmadiqbal8yahoocoin

Received 11 April 2013 Accepted 2 June 2013

Academic Editor Vincent Ribrag

Copyright copy 2013 Maryam Zahin et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

It is well known that the intake of antioxidants with increased consumption of fruits and vegetables andmedicinal herbs contributestowards reduced risk of certain diseases including cancers This study aims to evaluate the broad-spectrum antioxidant andantimutagenic activities as well as to elucidate phytochemical profile of an Indian medicinal plantMurraya koenigii (curry) leavesLeaves of the plant were successively fractionated in various organic solvents Benzene fraction demonstrated the highest phenoliccontent followed by petroleum ether The benzene fraction showed maximum antioxidant activity in all tested assays namelyphosphomolybdenum 22-diphenyl-1-picrylhydrazyl (DPPH) free radical ferric reducing antioxidant power (FRAP) and cupricreducing antioxidant capacity (CUPRAC) assays Based on the promising broad-spectrum antioxidant activity benzene fractionwas further evaluated for antimutagenic activity and showed a dose-dependent antimutagenic response in Ames Salmonellamutagenicity assay It inhibited 72ndash86 mutagenicity induced by sodium azide methyl methanesulfonate benzo(a)pyrene and 2-aminoflourene at the maximum tested concentration (100120583gmL) in Salmonella typhimurium tester strains At least 21 compoundswere detected by GCMS The findings clearly demonstrated that phenolic-rich benzene fraction has promising broad-spectrumantioxidant and antimutagenic property and needs further evaluation to exploit its therapeutic potential

1 Introduction

During the past decades it became obvious that mostdegenerative diseases are associated with reactive oxygenspecies (ROS) such as superoxide anion radicals hydroxylradicals and hydrogen peroxide [1] Under stress conditionsour bodies produce more ROS which creates homeostaticimbalance and generates oxidative stress and causes celldeath and tissue injury [2] There are endogenous as well asexogenous systems to protect cells from damage in animalsHowever in many conditions the endogenous antioxidantsare depleted and therefore exogenous supply of antioxidantsbecomes essential

Antioxidants play an important role in biological systems[3] by suppressing the formation of active oxygen species byreducing hydroperoxides (ROO∙) and H

2

O2

and scavengingfree radicals among others Antioxidants are classified asnaturalsynthetic exogenous or endogenous compounds thatcan play a role in the removal of free radicals scavenging ROS

or their precursors inhibiting formation of ROS and bindingmetal ions needed for catalysis of ROS generation and soforth [1] Plant-derived natural products could function asantioxidant especially singlet and triplet oxygen quenchersperoxide decomposers enzyme inhibitors or synergists [45]

Human exposure to genotoxic substances present inenvironment and the food is inevitable Study of mutagenesishas assumed importance due to the fact that mutations playan important role in carcinogenesis [6] Consequently fromcancer-preventing point of view an interest has also beenaroused in the presence of antimutagens in foodstuffs aswell as in traditionally used medicinal plants herbs andspices [7] On the other hand mechanism of mutagenesis iscomplex however many mutagens and carcinogens may actthrough the generation of ROS Therefore the discovery andexploration of plant extractsphytocompounds possessingboth antioxidant and antimutagenic properties are of greatpractical and therapeutic significance It has also been shown

2 BioMed Research International

that the increased consumption of fruits and vegetables hasgenerally been found protective against a variety of cancersThus it is presumed that plant with high antioxidant activitycould also show antimutagenic activity and counteract theeffect of the mutagens and carcinogens [8 9]

The screening of plant extractsphytocompounds forantimutagenic activity has been performed using severalmutagen assay systems in bacteria yeast and some plant andanimal cell cultures [10] Several authors have documentedthe antimutagenic activity of plant extracts associated withsecondary metabolites that act as antimutagen [11ndash13] Butconcerted efforts are needed to explore and exploit the Indianmedicinal plants in mutation-related carcinogenesis

Murraya koenigii (family Rutaceae) commonly knownas curry leaf is a native of India Sri Lanka and othersouth Asian countries and has been used in various cuisineas natural flavouring agent [14] In addition to its dietaryuse this plant has also been used in Indian system oftraditionalmedicine as tonic stomachic carminative and forhypoglycemic properties [15] The plant has been subjectedto intensive investigation and possesses various biologicalactivities [16]

We have previously reported that Indianmedicinal plantsare rich in novel compounds that possess antioxidant andantimutagenic potential [12 17 18] In this paper we discussthe fraction-based antioxidant and antimutagenic propertiesof curry leaves as well as their active constituents especiallyessential oils The broad-spectrum antioxidant activity usingvarious systems and its antimutagenic potential against bothdirect and indirect mutagens in vitro using Ames Salmonellatester strains has also been reported

2 Material and Method

21 Bacterial Strains and Chemicals The Salmonellatyphimurium strains TA97a TA98 TA100 and TA102were kindly provided by Professor B N Ames Universityof California Berkeley USA Sodium azide (NaN

3

)nicotinamide adenine dinucleotide phosphate sodium saltD-glucose-6-phosphate disodium salt sodium phosphateammonium molybdate neocuproine ferric chloride L-histidine monohydrate D-biotin and antioxidant standards(butylated hydroxy toluene (BHT) ascorbic acid andgallic acid) were purchased from Hi-Media Lab LtdMumbai India Potassium ferricyanide cupric chloride andammonium acetate were purchased from Qualigens FineChemicalsMumbai IndiaMethylmethanesulfonate (MMS)and trichloroacetic acid were purchased from Sisco-ResearchLaboratories Pvt Ltd Mumbai India while 11-diphenyl-2-picrylhydrazyl (DPPH) radicals 2-aminofluorene (2AF) and benzo(a)pyrene (BP) were purchased from SigmaChemical Co St Louis MO USA All the other reagentsused to prepare buffers and media were of analytical grade

22 Plant Material and Preparation of Extracts Leaves ofMurraya koenigii (L) were collected locally from the cam-pus of Aligarh Muslim University (AMU) Aligarh IndiaThe plant was further identified by a taxonomist in the

Department of Botany AMU Aligarh and voucher spec-imen (MBD-1406) was deposited in the Department ofAgricultural Microbiology AMU Aligarh India The leaveswere shade-dried ground finely and extract was preparedas described earlier [18] Briefly five hundred grams of dryleaf powder was soaked in 25 L of petroleum ether for 5days with intermittent shaking and finally the extract wasfiltered through Whatman filter paper no 1 (Whatman LtdEngland) to make a petroleum ether fraction The collecteddried powder was successively extracted with benzene ethylacetate acetone methanol and ethanolThe filtered fractionswere concentrated to dryness under reduced pressure onrotary evaporator at 40∘C and stored at 4∘C for future useThe yield of the dried fractions was calculated and extractswere reconstituted in minimum amount of DMSO (le01)to perform experiments

23 Antioxidant Assays Antioxidant potential of differentfractions from curry leaves was determined by DPPH freeradical scavenging assay the ferric reducing power assay(FRAP) and cupric ions (Cu2+) reducing ability (CUPRAC)assays as well as total antioxidant capacity was evaluated byphosphomolybdenum method

231 Determination of Total Antioxidant Capacity by Phos-phomolybdenum Method The total antioxidant capacity ofdifferent fractionswas evaluated by themethod of Prieto et al[19] An aliquot of 01mL of sample solution (containing 12525 50 and 100 120583gmL of respective fraction) was combinedwith 1mL of reagent (06M sulphuric acid 28mM sodiumphosphate and 4mM ammonium molybdate) Methanol(01mL) was used as blank in place of the sample Thetubes were capped and incubated in a boiling water bath at95∘C for 90min and then cooled at room temperature Theabsorbance of each solution was then measured at 695 nmagainst a blank in a double beam UV-Vis spectrophotometerUV570455 (EC Electronic Corporation of India Limited)For samples of unknown composition water-soluble antiox-idant capacity of the extract was expressed as equivalents ofascorbic acid (120583molg)

232 DPPH Radical Scavenging Assay Free radical scaveng-ing activity of different fractions against stable DPPH wasdetermined spectrophotometrically by the modified methodof Gyamfi et al [20] as described earlier [17] When DPPHreacts with an antioxidant which can donate hydrogenit is reduced The changes in color (from deep violet tolight yellow) were measured at 517 nm on a UV-Vis spec-trophotometer (Spectronic 20 D+ USA) Fifty microlitersof the solvent-dried leaf fractions yielding 125 25 50 and100 120583gmL respectively in each reaction was mixed with1mL of 01mM DPPH in methanol solution and 450120583Lof 50mM Tris-HCl buffer (pH 74) Methanol (50 120583L) wasused as a vehicle control in the experiment After 30min ofincubation at room temperature the reduction of the DPPHfree radicals was measured spectrophotometrically Ascorbic

BioMed Research International 3

acid and BHT were used as controls Percent inhibition wascalculated from the following equation

inhibition = (119886 minus 119887119886) times 100 (1)

where 119886 is the absorbance of control and 119887 is the absorbanceof test sample

233 FRAP Assay (Fe3+ Reducing Power Assay) The ferricreducing antioxidant power (FRAP) method of Oyaizu [21]with little modification [22] was adopted to measure thereducing capacity FRAP assay measured direct reductionof Fe3+(CNminus)

6

to Fe2+(CNminus)6

resulting in formation ofthe Perlrsquos Prussian Blue complex following the addition ofexcess ferric ions (Fe3+) Concentrations of extracts (125ndash100 120583gmL) in 075mL of distilled water were mixed with125mL of 02M (pH 66) sodium phosphate buffer and125mL of 1 potassium ferricyanide [K

3

Fe (CN6

)] After20min of incubation at 50∘C for 20min the reactionmixturewas acidified with 125mL of 10 trichloroacetic acid Finally05mL of 01 FeCl

3

was added and absorbance was mea-sured at 700 nm The increase in absorbance of the reactionmixture indicates greater reduction capability

234 CUPRAC Assay In order to determine the cupric ions(Cu2+) reducing ability of curry leaf extracts the methodof Apak et al [23] was used with a little modification asdescribed by Gulcin [24] Briefly 025mL CuCl

2

solution(001M) 025mL ethanolic neocuproine solution (75 times10minus3M) and 025mL CH

3

COONH4

buffer (1M) were addedto a test tube followed by mixing with different concentra-tions of extracts (125ndash100 120583gmL) The total reaction volumewas adjusted to 2mL with distilled water and the solutionwas mixed well The tubes were stoppered and kept atroom temperature for 30min and absorbance was measuredat 450 nm Increased absorbance of the reaction mixtureindicates increased reduction capability

24 Antimutagenicity Assay The Salmonella histidine pointmutation assay described by Maron and Ames [25] was usedto test the antimutagenic activity of the extracts as describedearlier [12 17] In the preincubation experiment a mixtureof solvent-dried curry leaves extracts and mutagen eachhaving a volume of 01mL of varying extract concentrationswas preincubated at 37∘C for 30min and then 01mL of 1times 107 CFUmL density of the bacterial culture was addedfollowed by the addition of 25mL of top agar (05NaCl and06 agar) supplemented with 05mM histidine and biotineach at 45∘C

The influence of metabolic activation of indirect actingmutagens BP and 2-AF was tested using 500 120583L of S9mixture(004mg proteinsmL of mix) The S9 microsome fractionwas prepared from the livers of rats treated with Aroclor 1254using standard protocols [25]The solution was vortex mixedand poured onto minimal glucose plates (40 glucose andVogel Bonner medium) The plates were incubated at 37∘Cfor 48 h after which the numbers of histidine-independentrevertant colonies were scored

Survival of the tester bacterial strains was routinelymonitored for each experiment To check the toxicity of thetest samples parallel controls were run with extracts alone atall concentrations tested with mutagens The concentrationsof the test sample for investigating the antimutagenicity were125 25 50 and 100 120583g solvent-dried extract01mLplateThese were tested against direct acting mutagens sodiumazide (15 120583g01mLplate) and MMS (1 120583g01mLplate) inTA97a TA98 TA100 and TA102 tester strains as well asagainst indirect acting mutagens BP and 2-AF All the testsamples and mutagens were dissolved in minimum volumeof DMSO In each case no toxicity was observed andthe numbers of spontaneous revertants were identical tothe DMSO vehicle control Nontoxic concentrations werecategorized as those where there was a well-developed lawnalmost similar size of colonies and no statistical differencein the number of spontaneous revertants in test and controlplates Triplicate plates were set up with each concentrationand the entire experiment was repeated twice that providedsix replicates per treatment Inhibition of mutagenicity wasexpressed as percentage decrease of reverse mutation andcalculated as

inhibition = (119886 minus 119887119886minus 119888) times 100 (2)

where 119886 is the number of histidine revertants induced bymutagen 119887 is the number of histidine revertants induced bymutagen in the presence of plant extract and 119888 is the numberof revertants induced in negative control

25 Total Phenolic Content of Plant Fractions The totalphenolic content of the plant fractions was determined usingFolin-Ciocalteau reagent by the method in [26] as describedearlier [18] Briefly to 05mL of each sample (containing125 25 50 and 100 120583gmL of the extract) 25mL of 110dilution of Folin-Ciocalteaursquos reagent and 2mL of Na

2

CO3

(75 wv) were added and incubated at 45∘C for 15minEach experiment was performed in triplicateThe absorbanceof all samples was measured at 765 nm using a UV-Visspectrophotometer Results are expressed as milligrams ofgallic acid equivalent per gram of extract dry weight (mgGAEg dw)

26 Gas Chromatography Mass Spectrometry (GC-MS) Theactive fraction of the extracts was subjected to GC-MSin order to identify the active constituents The GC wasdone on GCD 1800A Hewlett Packard coupled with HP-1 column (30m times 025mm times 025 120583m Thermo ScientificUSA) Injector and detector temperatures were 250∘C and280∘C respectively The carrier gas used was helium at1mLmin initial temperature of oven was 100ndash250∘C at therate of 10∘Cmin hold time was at 250∘C for 3min and finaltemperature was 250ndash280∘C at the rate of 30∘Cmin and holdtime at 280∘C for 2min The solvent used for making alldilutions was methanol

27 Statistical Analysis The results are presented as the meanof three experiments with triplicate platesdoseexperiment


The data were further analyzed for statistical significanceusing analysis of variance (one-way ANOVA) by Tukey testwhere treatment groups were compared with their respectivecontrolsThe regression analysis was carried out inMicrosoftExcel 2003 between percent inhibition of mutagenicity andlog values of concentrations of the plant extract

3 Results

Murraya koenigii has been reported to contain a variety ofphytochemicals of medicinal importance In this study weanalyzed different fractions isolated based on the polarity ofthe aqueous and organic solvents and compared their rela-tive antioxidant properties The curry leaves were extractedseparately in petrol ether benzene ethyl acetate acetonemethanol and ethanol Curry leaves yielded 10 12 12 1822 and 08 percent of the extract when fractionated in petrolether benzene ethyl acetate acetone methanol and ethanolrespectively

31 Antioxidant Activity of Curry The total antioxidantactivity of curry leaves fractions was tested by phospho-molybdenum method and compared with positive controlsascorbic acid and BHT All the tested fractions exhibitedconcentration-dependent antioxidant capacity with respectto ascorbic acid equivalents (Table 1) The benzene fractionshowed maximum antioxidant capacity (35104120583mol) at100 120583gmL followed by ethyl acetate (19823120583mol) petroleumether (19672120583mol) and acetone (17831 120583mol) fractionsHowever methanol and ethanol fractions displayed relativelyless antioxidant capacity (261ndash639 and 304ndash369 resp) at thetested concentrations of 125ndash100120583gmL

The free radical scavenging activity was measured asdecolorizing activity following the trapping of the unpairedelectron of DPPH as shown in Figure 1The free radical scav-enging activity of curry fractions by DPPHmethod exhibiteda concentration-dependent response (Figure 1) The benzenefractionwas found to be themost active free radical scavengerexhibited (883 decrease at a concentration of 100 120583gmL)followed by ethyl acetate (795) and petrol ether (787)fractions while positive controls (ascorbic acid and BHT) at aconcentration of 100120583gmL inhibited 931 and 865DPPHabsorption respectively Acetone methanol and ethanolfractions showed decolorization of 661 507 and 530respectively

The FRAP assay has been widely used in the evaluation ofantioxidant component in dietary polyphenolsThe reducingpotential of the curry leaf fractions was concentration-dependent (Figure 2(a)) The benzene fraction demonstratedmaximum reducing activity among the fractionsThe activitywas greater than the ascorbic acid and at par with BHT TheFRAP activity at 100 120583gmL concentration was in the orderof BHT = benzene gt ascorbic acid gt petroleum ether gt ethylacetate gt acetone gt ethanol and least in methanol fraction

Similarly the antioxidant activity by CUPRAC assaysindicated the highest reducing power potential in benzenefraction followed by petrol ether and ethyl acetate as shownin Figure 2(b) However ascorbic acid had superior activity

0

20

40

60

80

100

0 20 40 60 80 100 120

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT

Concentration (120583gmL)

Perc

ent d

ecol

oriz

atio

n (517

nm)

Figure 1 DPPH free radical scavenging activity of Murrayakoenigii leaf fractions (petroleum ether benzene ethyl acetateacetone methanol and ethanol) at different concentrations (125ndash100120583gmL)The activity is compared with commercial antioxidantsascorbic acid and BHT Each data point represents themean of threeexperiments Standard deviation (le4) is not shown for clarity ofthe data

compared to benzene fraction On the other hand benzenefraction had more CUPRAC activity than BHT

32 Antimutagenic Activity There are several reports sug-gesting the correlation of antioxidants with high antimuta-genic activity In this study benzene fraction demonstratinghighest antioxidant activity was tested to explore antimuta-genic potential of curry The benzene fraction at concentra-tions of 125 25 50 and 100 120583gplate was found nonmutagenicas well as non-toxic to Salmonella typhimurium strains eitheralone or in the presence of S9 mix Doses of 15 120583gplate ofsodium azide and 1 120583gplate ofmethylmethanesulfonate werechosen for the antimutagenicity study since these doses werenot toxic to the S typhimurium tester strains Similarly BPand 2-AF were tested at the concentration of 1 and 5120583gplatein the presence of S9 mix

The antimutagenic activity of curry leaves was found tobe dose dependent (Tables 2 to 5) At a dose of 100 120583gplateantimutagenic response was significant (119875 le 005) againstTA97a with a decrease in the NaN

3

-induced mutagenicity by849 followed by TA100 (844) TA98 (732) and TA102(722) (Table 2) The linear regression analysis betweenextract dose and antimutagenic response against respectivetest mutagen showed strong correlation with respect todose dependent response ranging from (095ndash10) respec-tively Similarly the decrease in number of His+ revertantswas significant (119875 lt 0005) for Salmonella typhimuriumTA102 (860) followed by TA100 (836) TA97a (800)and TA98 (741) against MMS-induced mutagenicity asdepicted in Table 3 Linear correlation between fraction dose


0

05

1

15

2

25

0 20 40 60 80 100 120

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Abso

rban

ce (7

00

nm)

(a)

0

05

1

15

2

25

3

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT

0 20 40 60 80 100Concentration (120583gmL)

Abso

rban

ce (4

50

nm)

(b)

Figure 2 Reducing power ofMurraya koenigii leaf fractions (petroleum ether benzene ethyl acetate acetone methanol and ethanol) Theextracts were tested in the range of 10ndash100120583gmL concentrations in FRAP (a) and CUPRAC (b) assays and compared with ascorbic acid andBHT Each data point represents the mean of three experiments Standard deviation (le4) is not shown for clarity of the data

Table 1 Antioxidant capacity of Murraya koenigii leaf fractions expressed as ascorbic acid equivalents (120583molg of extract) byphosphomolybdenum method

Concentration (120583gmL) Antioxidant capacity (120583molg of extract)Petrol ether Benzene Ethyl acetate Acetone Methanol Ethanol

125 9800d plusmn 183 16698d plusmn 211 9394d plusmn 434 7024d plusmn 262 2610d plusmn 158 3037d plusmn 13425 15316c plusmn 507 24595c plusmn 754 13992c plusmn 195 12338c plusmn 471 3985c plusmn 212 4193c plusmn 10150 16630b plusmn 569 31323b plusmn 972 15892b plusmn 669 14385b plusmn 394 5225b plusmn 126 5423b plusmn 166100 19672a plusmn 674 35104a plusmn 958 19823a plusmn 843 17830a plusmn 864 6387a plusmn 251 6688a plusmn 153LSD at 5 10901 11604 10206 8102 4389 793The above data are the mean of three experiments plusmn SD different letters in columns show significant difference in means LSD least significance difference

Table 2 Effect of benzene fraction ofMurraya koenigii on the mutagenicity induced by sodium azide (NaN3) using Salmonella typhimuriumstrains

Treatment Dose (120583gplate) Number of His+ revertants coloniesplateTA97a TA98 TA100 TA102

Spontaneous 1400 plusmn 40 310 plusmn 36 1287 plusmn 98 2457 plusmn 233Positive control (NaN3) 15 2463 plusmn 160 527 plusmn 21 3520 plusmn 330 3433 plusmn 333

aMurraya koenigii

125 1643 plusmn 123 460 plusmn 40 1917 plusmn 133 2867 plusmn 26325 1450 plusmn 155 373 plusmn 70 1723 plusmn 189 2600 plusmn 24150 1247 plusmn 91 307 plusmn 85 1530 plusmn 197 2453 plusmn 244100 1180 plusmn 105 240 plusmn 46 1270 plusmn 155 2283 plusmn 196

bMurraya koenigii + NaN3

125 2253 plusmn 336 (256) 513 plusmn 60 (200) 3163 plusmn 320 (223) 3323 plusmn 349 (194)25 2007 plusmn 227lowast (451) 453 plusmn 42 (478) 2580 plusmn 296lowast (523) 3130 plusmn 321 (364)50 1680 plusmn 223lowastlowast (644) 390 plusmn 82lowast (621) 2127 plusmn 266lowastlowast (700) 2927 plusmn 186 (517)100 1373 plusmn 127lowastlowastlowast (849) 317 plusmn 65lowastlowast (732) 1620 plusmn 210lowastlowast (844) 2603 plusmn 140lowast (722)

119865 value 18069 8288 33690 69171198772 value 0999 0953 0969 0996

The data represented in the table is the mean plusmn SD values of three replicates Mean values followed by asterisk are significantly different when compared topositive control at lowastP lt 005 lowastlowastP lt 0005 and lowastlowastlowastP lt 0001 The values in parentheses are the inhibition rates of mutagenicity () Positive control NaN3sodium azide aNegative control bpreincubation test 1198772 linear regression analysis


Table 3 Effect of benzene fraction ofMurraya koenigii on the mutagenicity induced by methyl methanesulfonate (MMS) using Salmonellatyphimurium strains


Spontaneous 1400 plusmn 40 310 plusmn 36 1287 plusmn 98 2457 plusmn 233Positive control (MMS) 1 4437 plusmn 199 510 plusmn 27 9527 plusmn 460 11953 plusmn 790

aMurraya koenigii


b119872119906119903119903119886119910119886 koenigii+ MMS

125 3883 plusmn 312 (198) 503 plusmn 40 (134) 8023 plusmn 571lowast (198) 8623 plusmn 588lowastlowast (366)25 3090 plusmn 308lowast (451) 450 plusmn 46 (439) 5650 plusmn 451lowastlowastlowast (497) 6753 plusmn 569lowastlowastlowast (556)50 2397 plusmn 202lowastlowast (639) 383 plusmn 40lowast (623) 4407 plusmn 486lowastlowastlowast (640) 5180 plusmn 321lowastlowastlowast (713)100 1830 plusmn 175lowastlowastlowast (800) 310 plusmn 56lowastlowast (741) 2623 plusmn 317lowastlowastlowast (836) 3637 plusmn 371lowastlowastlowast (860)

119865 value treatment 81635 9877 225881 1782551198772 value 0989 0958 0978 0996

The data represented in the table is the mean plusmn SD values of three replicates Mean values followed by asterisk are significantly different when compared topositive control at lowastP lt 005 lowastlowastP lt 0005 and lowastlowastlowastP lt 0001 The values in parentheses are the inhibition rates of mutagenicity () Positive control MMSmethyl methanesulfonate aNegative control bPre-incubation 1198772 Linear regression analysis

Table 4 Effect of benzene fraction of Murraya koenigii on the mutagenicity induced by benzo(a)pyrene with metabolic activation usingSalmonella typhimurium strains


Spontaneous 1437 plusmn 132 370 plusmn 30 1353 plusmn 95 3160 plusmn 145Positive control (BP) 1 7203 plusmn 285 1550 plusmn 76 6957 plusmn 170 6480 plusmn 171

a119872119906119903119903119886119910119886 koenigii


b119872119906119903119903119886119910119886 koenigii+ BP

125 5923 plusmn 445lowast (221) 1327 plusmn 192 (213) 5400 plusmn 501lowastlowast (277) 5603 plusmn 312lowast (229)25 5017 plusmn 366lowastlowast (387) 1010 plusmn 193lowast (481) 4353 plusmn 461lowastlowastlowast (482) 5017 plusmn 169lowastlowastlowast (419)50 3827 plusmn 356lowastlowastlowast (612) 807 plusmn 127lowastlowastlowast (619) 3523 plusmn 307lowastlowastlowast (651) 4360 plusmn 382lowastlowastlowast (652)100 2760 plusmn 265lowastlowastlowast (824) 533 plusmn 139lowastlowastlowast (801) 2537 plusmn 214lowastlowastlowast (849) 3820 plusmn 292lowastlowastlowast (860)


The data represented in the table is the mean plusmn SD values of three replicates Mean values followed by asterisk are significantly different when comparedto positive control at lowastP lt 005 lowastlowastP lt 0005 and lowastlowastlowastP lt 0001 The values in parentheses are the inhibition rates of mutagenicity () Positive control BPbenzo(a)pyreneaNegative control bPre-incubation test 1198772 Linear regression analysis

and antimutagenic response was highly significant in all thestrains (1198772 = 096 or more)

The antimutagenicity ofM koenigii benzene fraction hasalso been demonstrated against indirect acting mutagensbenzo(a)pyrene (BP) and 2-aminoflourene (2-AF) that inferstheir mutagenicity by microsomal activation At 100 120583gplatethe benzene fraction was found antimutagenic for BP-induced mutation at (119875 lt 0001) as evident from the datapresented in Table 4 The antimutagenic response was dose-dependent (1198772 ge 098) and it ranged from 801 to 860Similarly trend in antimutagenicity has been shown against2-AF as presented in Table 5 Further the linear regressionanalysis between extract dose and antimutagenic response

showed strong correlation ranging from 099 to 10 againstrespective tester strains

33 Phytochemical Analysis of Murraya koenigii (Leaf) Phy-tochemical analysis of fractions revealed the presence ofalkaloids phenolics and glycosides as major group of com-pounds (data not shown)The total phenolic content as gallicacid equivalent (mgg) ofMurraya koenigii fractions showedhighest polyphenolic content (1871 plusmn 63) in benzenefraction followed by petrol ether (1464 plusmn 63) ethyl acetate(1139 plusmn 30) acetone (1139 plusmn 27) ethanol (1103 plusmn 20)and methanol (1032 plusmn 31) fractions


Table 5 Effect of benzene fraction of Murraya koenigii on the mutagenicity induced by 2-aminofluorene (2-AF) with metabolic activationusing Salmonella typhimurium strains


Spontaneous 1437 plusmn 132 370 plusmn 30 1353 plusmn 95 3160 plusmn 145Positive control (2AF) 5 3280 plusmn 120 2623 plusmn 172 5020 plusmn 191 14303 plusmn 325

aMurraya koenigii


bMurraya koenigii+ 2-AF

125 2763 plusmn 311 (278) 2007 plusmn 170lowast (291) 4223 plusmn 327lowast (216) 11853 plusmn 770lowastlowast (210)25 2487 plusmn 280lowast (461) 1610 plusmn 125lowastlowast (461) 3463 plusmn 216lowastlowastlowast (449) 9607 plusmn 708lowastlowastlowast (414)50 2200 plusmn 250lowastlowast (678) 1147 plusmn 131lowastlowastlowast (650) 2807 plusmn 270lowastlowastlowast (663) 6550 plusmn 553lowastlowastlowast (700)100 2023 plusmn 142lowastlowastlowast (857) 700 plusmn 76lowastlowastlowast (821) 2050 plusmn 180lowastlowastlowast (909) 4620 plusmn 329lowastlowastlowast (888)


The data represented in the table is the mean plusmn SD values of three replicates Mean values followed by asterisk are significantly different when compared topositive control at lowastP lt 005 lowastlowastP lt 0005 and lowastlowastlowastP lt 0001 The values in parentheses are the inhibition rates of mutagenicity () Positive control 2-AF 2-aminofloureneaNegative control bPre-incubation test 1198772 Linear regression analysis

Further a total of 21 chemical components were iden-tified in the benzene fraction of the leaf extract byGC-MS analysis (Supplementary Figures 1ndash4 Table S1 inSupplementary Material found online at httpdxdoiorg1011552013263509) These numbers may be extended withthe help of chemo metric techniques The major com-pounds identified were caryophyllene (148) followedby 3-undecen-5-yne (Z)-(952) phytol (917) 2-methyl-3H-phenanthro[34-D] imida (890) caryophyllene oxide(661) propylparaben (611) D-limonene (601) Theremaining compounds were present in percentages of (106ndash572) as depicted in Table 6

4 Discussion

Antioxidants play an important role in biological systemsby suppressing the formation of active oxygen species byreducing hydroperoxides (ROO∙) and H

2

O2

and scavengingfree radicals among others Over the past few decadesscientific research has indicated a credible basis for someof the traditional ethnomedicinal uses of spices Thereforewe have focused our study to elucidate the broad-spectrumantioxidant potential of M koenigii leaf fractions by meansof four different in vitro tests including total antioxidantcapacity bymolybdenummethod free radicals scavenging byDPPH and reducing power by FRAP and CUPRAC assaysThe method to detect total antioxidant activity is based onthe reduction of Mo (VI) to Mo (V) by the antioxidantcompounds with the formation of a green Mo (V) complexwhich showsmaximumabsorption atsim700 nm [27]Whereasother two methods are based on scavenging of free radicals(DPPH) and reduction of Fe[(CN)6]3 to Fe[(CN)6]2 in FRAPassay and Cu2+ to Cu1+ in CUPRAC assay

This study demonstrated that the sequential fractionationof curry leaves in various solvents resulted in the extraction ofantioxidant bioactive compounds mainly in benzene petrol

ether acetone methanol and ethanol fractions The benzenefraction ofM koenigii was found to contain the highest totalphenolics and to be the most active fraction in all in vitroantioxidant assays tested Other fractions showed activity inorder of petrol ether gt ethyl acetate gt acetone gt methanolgt ethanol Polyphenols have been reported to have manybeneficial health effects including antioxidant activity In thisstudy the total phenolic content was the highest in benzenefraction followed by other suggesting the correlation betweenantioxidant and polyphenolics content A series of papershave shown similar correlation including few from this lab[12 17 18]

M koenigii leaves were found to have the highestantioxidant activities and total phenolic content in methanolextracts as studied in [28] These differences might be dueto the extraction of curry leaves in methanol only insteadof fractionating them into different solvents Our findingshighlight that M koenigii benzene fraction is the potentialsource of antioxidants It is probably the first report on thesequential fraction ofM koenigii leaves in respective solventsfor the evaluation of antioxidant activities However otherfractions might be having different levels and composition ofantioxidant compounds which needs further phytochemicalcharacterization

Moreover our results also indicated terpenes as themajor components in benzene fraction as identified by GC-MS analysis It is evident that the antioxidant activities ofM koenigii may be related to various carbazole alkaloidsandor phenolic compounds including monoterpenes [2930] Comparative studies of the major components have alsoindicated terpenes as the most versatile components in Mkoenigii leaves [29] whilemany carbazole alkaloids have beenisolated and identified from different parts of the plant [3132] Besides these phytochemicals the curry leaves are also arich source of nutrients including vitamin A and vitamin C[33]


Table 6 Components ofMurraya koenigii benzene fraction as identified by GC-MS analysis

Peak no Components Retention time Area ()(1) Caryophyllene 1119 148(2) 120572-Caryophyllene 1177 279(3) 1H-Cyclopropa[a] naphthalene 1a2 1237 293(4) Caryophyllene oxide 1324 661(5) Spiro[44]nonan-2-one 1360 249(6) 1010-Dimethyl-26-dimethylenebicy 1393 332(7) D-Limonene 1410 601(8) Propylparaben 1580 611(9) Salicylamide 1600 142(10) 9-Borabicyclo[331]nonane 9-hydr 1855 572(11) 12-Diphenylethanethione 1869 054(12) Phytol 1891 917(13) 1114-Eicosadienoic acid methyl e 1944 259(14) 3-Undecen-5-yne (Z)- 1958 952(15) Phenol 2-methoxy-4-[2-(4-hydroxyphenyl)] 2071 194(16) 26-Octadien-1-ol 37-dimethyl- 2241 106(17) Docosane 11-butyl- 2395 530(18) 2-Methyl-3H-phenanthro[34-D] imida 2402 890(19) Propanenitrile 2452 221(20) Pentatriacontane 2582 422(21) Vitamin A aldehyde 2657 233

On the basis of antioxidant potential the most activefraction (benzene) was tested for its antimutagenic potentialagainst S typhimurium tester strains Interestingly a signifi-cant antimutagenicity (119875 le 005) against direct and indirectacting mutagens in the presence of S9 was recorded SinceBP a known carcinogenic agent cannot be metabolized bySalmonella to an appreciable extent so a metabolizing systemof supernatant of liver homogenate from Aroclor inducedrats (S9) is included in the assay A number of reactiveintermediates like BP-45-oxide and 9-hydroxy-BP-45-oxideare formed by BP with this activating system that helpsin the binding of BP to bacterial DNA [34] Similarly theantimutagenic activity against 2-AF-induced mutagenicitywas tested in the presence of S9 fraction

In our study none of the tested extracts exhibited anymutagenic effect in the Ames test in the absence of enzymaticmetabolism This suggests that DNA does not seem tobe a relevant target for the extract tested and it did notproduce DNA lesions that block DNA synthesis leading tothe induction of the SOS system [35] It is evident from theresults of the present study that the antimutagenic behaviorof M koenigii is probably due to its antioxidant activitieswhichmay be related to various phenolicsmonoterpenes andcarbazole alkaloids and so forth [29 30]

The GC-MS analysis and total phenolic content revealedterpenes as the major active constituents in the tested frac-tion The studies on antimutagenic activity of plants haveindicated the involvement of chemical constituents whichcould act as nonspecific redox agents free radical scavengersor ligands for binding metals or toxic principles [36] Besides

these the curry leaves contained carotenoids and retinoidspreformed types of vitamin A which have been clearlyassociated with prevention of the induction of cancer andinhibition spontaneous and induced cancers [37] Recentlyterpenes are also reported to have antimutagenic effects inbacterial reverse mutation assay [38] as well as against UV-4NQO- and t-BOOH induced mutagenesis in E coli K12 andE coli WP2 by reversion assays [39] Therefore our studyhighlights that the benzene fraction of the curry leaf hasbroad-spectrum antimutagenic properties Further studiesare needed in order to isolate and characterize the activeconstituents present in it for their therapeutic efficacy

In summary we have shown for the first time that curryleaf yielding highest phenolics in benzene fractions uponsequential fractionation is a promising source of antioxidantcompounds that may be responsible for its antimutagenicproperties Further studies are needed to isolate the activeprinciples as well as to elucidate the role of various inter-acting phytocompounds in influencing therapeutic potentialand efficacy in vivo

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

Theauthors are thankful to theUGCNewDelhi for financialassistance in the form of Research Fellowship to MaryamZahin through AMU Aligarh They are also grateful to the


Director SAIF IIT Bombay India for the cooperation inchemical spectral analysis

References

[1] Y Gilgun-Sherki E Melamed and D Offen ldquoOxidative stressinduced-neurodegenerative diseases the need for antioxidantsthat penetrate the blood brain barrierrdquoNeuropharmacology vol40 no 8 pp 959ndash975 2001

[2] D Krishnaiah R Sarbatly and R Nithyanandam ldquoA review ofthe antioxidant potential of medicinal plant speciesrdquo Food andBioproducts Processing vol 89 no 3 pp 217ndash233 2011

[3] K H Cheeseman and T F Slater ldquoAn introduction to freeradical biochemistryrdquo British Medical Bulletin vol 49 no 3 pp481ndash493 1993

[4] R A Larson ldquoThe antioxidants of higher plantsrdquo Phytochem-istry vol 27 no 4 pp 969ndash978 1988

[5] A Mitrovic and J Bogdanovic ldquoEffect of gibberellic acidon total antioxidant activity during chenopodium rubrum lOntogenesis in vitrordquo Archives of Biological Sciences vol 61 no1 pp 49ndash55 2009

[6] K Yoshikawa K Inagaki T Terashita J Shishiyama S Kuoand D M Shankel ldquoAntimutagenic activity of extracts fromJapanese eggplantrdquoMutation ResearchmdashGenetic Toxicology vol371 no 1-2 pp 65ndash71 1996

[7] N S Sangwan S Shanker R S Sangwan and S Kumar ldquoPlant-derived products as antimutagensrdquo Phytotherapy Research vol12 no 6 pp 389ndash399 1998

[8] W M El-Sayed andW A Hussin ldquoAntimutagenic and antioxi-dant activity of novel 4-substituted phenyl-2 21015840-bichalcophenesand aza-analogsrdquo Journal of Drug Design Development andTherapy vol 7 pp 73ndash81 2013

[9] A Cardador-Martınez A Albores M Bah et al ldquoRelationshipamong antimutagenic antioxidant and enzymatic activities ofmethanolic extract from common beans (Phaseolus vulgarisL)rdquo Plant Foods for Human Nutrition vol 61 no 4 pp 161ndash1682006

[10] J Musarrat F Aqil and I Ahmad ldquoMutagenicity and antimuta-genicity ofmedicinal plantsrdquo inModern Phytomedicine TurningMedicinal Plants into Drug I Ahmad F Aqil and M OwaisEds pp 271ndash286 Wiley-VCH Weinheim Germany 2006

[11] S Bala and I S Grover ldquoAntimutagenicity of some citrus fruitsin Salmonella typhimuriumrdquoMutation Research vol 222 no 3pp 141ndash148 1989

[12] F Aqil M Zahin and I Ahmad ldquoAntimutagenic activity ofmethanolic extracts of four ayurvedic medicinal plantsrdquo IndianJournal of Experimental Biology vol 46 no 9 pp 668ndash6722008

[13] K Kaur S Arora S Kumar and A Nagpal ldquoAntimutagenicactivities of acetone and methanol fractions of Terminaliaarjunardquo Food and Chemical Toxicology vol 40 no 10 pp 1475ndash1482 2002

[14] S M Hiremath B B Madalageri and P W Basarkar ldquoCom-position of curry leaf (Murraya koenigii Spreng) oil during leafgrowthrdquo Indian Perfumer vol 42 no 2 pp 58ndash59 1998

[15] J Mishra A Yousuf R D Singh and A Aradhana ldquoPhy-tochemical investigation and in-vitro antioxidant potential ofleaves of Murraya koenigiirdquo International Journal of IntegrativeBiology vol 7 no 3 pp 171ndash174 2009

[16] S D Bonde L S Nemade M R Patel and A A PatelldquoMurraya koenigii (Curry leaf) ethnobotany phytochemistry

and pharmacologymdasha reviewrdquo International Journal of Pharma-ceutical and Phytopharmacological Research vol 1 pp 23ndash272011

[17] M Zahin I Ahmad and F Aqil ldquoAntioxidant and antimuta-genic activity of Carum copticum fruit extractsrdquo Toxicology inVitro vol 24 no 4 pp 1243ndash1249 2010

[18] M Zahin F Aqil and I Ahmad ldquoBroad spectrum antimuta-genic activity of antioxidant active fraction of Punica granatumL peel extractsrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 703 no 2 pp 99ndash107 2010

[19] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[20] M A Gyamfi M Yonamine and Y Aniya ldquoFree-radicalscavenging action of medicinal herbs from Ghana Thonningiasanguinea on experimentally-induced liver injuriesrdquo GeneralPharmacology vol 32 no 6 pp 661ndash667 1999

[21] M Oyaizu ldquoStudies on products of browning reactions antiox-idant activities of products of browning reaction prepared fromglucosaminerdquo Japaneese Journal of Nutrition vol 44 pp 307ndash315 1986

[22] F Aqil R Munagala M V Vadhanam et al ldquoAnti-proliferativeactivity and protection against oxidative DNA damage bypunicalagin isolated from pomegranate huskrdquo Food ResearchInternational vol 49 no 1 pp 345ndash353 2012

[23] R Apak K Guclu B Demirata et al ldquoComparative evaluationof various total antioxidant capacity assays applied to phenoliccompounds with the CUPRAC assayrdquo Molecules vol 12 no 7pp 1496ndash1547 2007

[24] I Gulcin ldquoAntioxidant activity of l-adrenaline a structure-activity insightrdquo Chemico-Biological Interactions vol 179 no 2-3 pp 71ndash80 2009

[25] D M Maron and B N Ames ldquoRevised methods for theSalmonella mutagenicity testrdquo Mutation Research vol 113 no3-4 pp 173ndash215 1983

[26] G A Spanos and R E Wrolstad ldquoInfluence of processingand storage on the phenolic composition of thompson seedlessgrape juicerdquo Journal of Agricultural and Food Chemistry vol 38no 7 pp 1565ndash1571 1990

[27] P S Negi G K Jayaprakasha and B S Jena ldquoAntioxidant andantimutagenic activities of pomegranate peel extractsrdquo FoodChemistry vol 80 no 3 pp 393ndash397 2003

[28] S Gupta and J Prakash ldquoStudies on Indian green leafy veg-etables for their antioxidant activityrdquo Plant Foods for HumanNutrition vol 64 no 1 pp 39ndash45 2009

[29] V S Rana J P Juyal R Rashmi andM A Blazquez ldquoChemicalconstituents of the volatile oil of Murraya koenigii leavesrdquoInternational Journal of Aromatherapy vol 14 no 1 pp 23ndash252004

[30] L J M Rao K Ramalakshmi B B Borse and B RaghavanldquoAntioxidant and radical-scavenging carbazole alkaloids fromthe oleoresin of curry leaf (Murraya koenigii Spreng)rdquo FoodChemistry vol 100 no 2 pp 742ndash747 2007

[31] Y Tachibana H Kikuzaki N H Lajis and N NakatanildquoAntioxidative activity of carbazoles from Murraya koenigiileavesrdquo Journal of Agricultural and Food Chemistry vol 49 no11 pp 5589ndash5594 2001

[32] R S Ramsewak M G Nair G M Strasburg D L DeWittand J L Nitiss ldquoBiologically active carbazole alkaloids from


Murraya koenigiirdquo Journal of Agricultural and Food Chemistryvol 47 no 2 pp 444ndash447 1999

[33] P R Bhandari ldquoCurry leaf (Murraya koenigii) or cure leafreview of its curative propertiesrdquo Journal of Medical Nutritionand Nutraceuticals vol 1 pp 92ndash97 2012

[34] M Osborne and N Crosby Metabolism Benzopyrenes Cam-bridge University Press New York NY USA 1987

[35] J M Arif M Kunhi Y M Siddiqui et al ldquoDifferentialmodulation of benzo[a]pyrene-derived DNA adducts in MCF-7 cells by marine compoundsrdquo International Journal of CancerPrevention vol 1 pp 259ndash268 2004

[36] D Sarkar A Sharma and G Talukder ldquoPlant extracts asmodulators of genotoxic effectsrdquo Botanical Review vol 62 no4 pp 275ndash300 1996

[37] J S Bertram and A L Vine ldquoCancer prevention by retinoidsand carotenoids independent action on a common targetrdquoBiochimica et Biophysica Acta vol 1740 no 2 pp 170ndash178 2005

[38] A di Sotto M G Evandri and G Mazzanti ldquoAntimutagenicand mutagenic activities of some terpenes in the bacterialreversemutation assayrdquoMutation ResearchmdashGenetic Toxicologyand Environmental Mutagenesis vol 653 no 1-2 pp 130ndash1332008

[39] BNikolic J Stanojevic DMitic B Vukovic-Gacic J Knezevic-Vukcevic and D Simic ldquoComparative study of the antimu-tagenic potential of Vitamin E in different E coli strainsrdquoMutation Research vol 564 no 1 pp 31ndash38 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


that the increased consumption of fruits and vegetables hasgenerally been found protective against a variety of cancersThus it is presumed that plant with high antioxidant activitycould also show antimutagenic activity and counteract theeffect of the mutagens and carcinogens [8 9]

The screening of plant extractsphytocompounds forantimutagenic activity has been performed using severalmutagen assay systems in bacteria yeast and some plant andanimal cell cultures [10] Several authors have documentedthe antimutagenic activity of plant extracts associated withsecondary metabolites that act as antimutagen [11ndash13] Butconcerted efforts are needed to explore and exploit the Indianmedicinal plants in mutation-related carcinogenesis

Murraya koenigii (family Rutaceae) commonly knownas curry leaf is a native of India Sri Lanka and othersouth Asian countries and has been used in various cuisineas natural flavouring agent [14] In addition to its dietaryuse this plant has also been used in Indian system oftraditionalmedicine as tonic stomachic carminative and forhypoglycemic properties [15] The plant has been subjectedto intensive investigation and possesses various biologicalactivities [16]

We have previously reported that Indianmedicinal plantsare rich in novel compounds that possess antioxidant andantimutagenic potential [12 17 18] In this paper we discussthe fraction-based antioxidant and antimutagenic propertiesof curry leaves as well as their active constituents especiallyessential oils The broad-spectrum antioxidant activity usingvarious systems and its antimutagenic potential against bothdirect and indirect mutagens in vitro using Ames Salmonellatester strains has also been reported

2 Material and Method

21 Bacterial Strains and Chemicals The Salmonellatyphimurium strains TA97a TA98 TA100 and TA102were kindly provided by Professor B N Ames Universityof California Berkeley USA Sodium azide (NaN

3

)nicotinamide adenine dinucleotide phosphate sodium saltD-glucose-6-phosphate disodium salt sodium phosphateammonium molybdate neocuproine ferric chloride L-histidine monohydrate D-biotin and antioxidant standards(butylated hydroxy toluene (BHT) ascorbic acid andgallic acid) were purchased from Hi-Media Lab LtdMumbai India Potassium ferricyanide cupric chloride andammonium acetate were purchased from Qualigens FineChemicalsMumbai IndiaMethylmethanesulfonate (MMS)and trichloroacetic acid were purchased from Sisco-ResearchLaboratories Pvt Ltd Mumbai India while 11-diphenyl-2-picrylhydrazyl (DPPH) radicals 2-aminofluorene (2AF) and benzo(a)pyrene (BP) were purchased from SigmaChemical Co St Louis MO USA All the other reagentsused to prepare buffers and media were of analytical grade

22 Plant Material and Preparation of Extracts Leaves ofMurraya koenigii (L) were collected locally from the cam-pus of Aligarh Muslim University (AMU) Aligarh IndiaThe plant was further identified by a taxonomist in the

Department of Botany AMU Aligarh and voucher spec-imen (MBD-1406) was deposited in the Department ofAgricultural Microbiology AMU Aligarh India The leaveswere shade-dried ground finely and extract was preparedas described earlier [18] Briefly five hundred grams of dryleaf powder was soaked in 25 L of petroleum ether for 5days with intermittent shaking and finally the extract wasfiltered through Whatman filter paper no 1 (Whatman LtdEngland) to make a petroleum ether fraction The collecteddried powder was successively extracted with benzene ethylacetate acetone methanol and ethanolThe filtered fractionswere concentrated to dryness under reduced pressure onrotary evaporator at 40∘C and stored at 4∘C for future useThe yield of the dried fractions was calculated and extractswere reconstituted in minimum amount of DMSO (le01)to perform experiments

23 Antioxidant Assays Antioxidant potential of differentfractions from curry leaves was determined by DPPH freeradical scavenging assay the ferric reducing power assay(FRAP) and cupric ions (Cu2+) reducing ability (CUPRAC)assays as well as total antioxidant capacity was evaluated byphosphomolybdenum method

231 Determination of Total Antioxidant Capacity by Phos-phomolybdenum Method The total antioxidant capacity ofdifferent fractionswas evaluated by themethod of Prieto et al[19] An aliquot of 01mL of sample solution (containing 12525 50 and 100 120583gmL of respective fraction) was combinedwith 1mL of reagent (06M sulphuric acid 28mM sodiumphosphate and 4mM ammonium molybdate) Methanol(01mL) was used as blank in place of the sample Thetubes were capped and incubated in a boiling water bath at95∘C for 90min and then cooled at room temperature Theabsorbance of each solution was then measured at 695 nmagainst a blank in a double beam UV-Vis spectrophotometerUV570455 (EC Electronic Corporation of India Limited)For samples of unknown composition water-soluble antiox-idant capacity of the extract was expressed as equivalents ofascorbic acid (120583molg)

232 DPPH Radical Scavenging Assay Free radical scaveng-ing activity of different fractions against stable DPPH wasdetermined spectrophotometrically by the modified methodof Gyamfi et al [20] as described earlier [17] When DPPHreacts with an antioxidant which can donate hydrogenit is reduced The changes in color (from deep violet tolight yellow) were measured at 517 nm on a UV-Vis spec-trophotometer (Spectronic 20 D+ USA) Fifty microlitersof the solvent-dried leaf fractions yielding 125 25 50 and100 120583gmL respectively in each reaction was mixed with1mL of 01mM DPPH in methanol solution and 450120583Lof 50mM Tris-HCl buffer (pH 74) Methanol (50 120583L) wasused as a vehicle control in the experiment After 30min ofincubation at room temperature the reduction of the DPPHfree radicals was measured spectrophotometrically Ascorbic






6

to Fe2+(CNminus)6


3

Fe (CN6


3



2


3

COONH4








2

CO3






3 Results






0

20

40

60

80

100

0 20 40 60 80 100 120

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Perc

ent d

ecol

oriz

atio

n (517

nm)





3



0

05

1

15

2

25

0 20 40 60 80 100 120

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Abso

rban

ce (7

00

nm)

(a)

0

05

1

15

2

25

3

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Abso

rban

ce (4

50

nm)

(b)








aMurraya koenigii




119865 value 18069 8288 33690 69171198772 value 0999 0953 0969 0996






aMurraya koenigii


b119872119906119903119903119886119910119886 koenigii+ MMS







a119872119906119903119903119886119910119886 koenigii


b119872119906119903119903119886119910119886 koenigii+ BP












aMurraya koenigii







4 Discussion


2

O2
















Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






6

to Fe2+(CNminus)6


3

Fe (CN6


3



2


3

COONH4








2

CO3






3 Results






0

20

40

60

80

100

0 20 40 60 80 100 120

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Perc

ent d

ecol

oriz

atio

n (517

nm)





3



0

05

1

15

2

25

0 20 40 60 80 100 120

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Abso

rban

ce (7

00

nm)

(a)

0

05

1

15

2

25

3

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Abso

rban

ce (4

50

nm)

(b)








aMurraya koenigii




119865 value 18069 8288 33690 69171198772 value 0999 0953 0969 0996






aMurraya koenigii


b119872119906119903119903119886119910119886 koenigii+ MMS







a119872119906119903119903119886119910119886 koenigii


b119872119906119903119903119886119910119886 koenigii+ BP












aMurraya koenigii







4 Discussion


2

O2
















Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3 Results






0

20

40

60

80

100

0 20 40 60 80 100 120

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Perc

ent d

ecol

oriz

atio

n (517

nm)





3



0

05

1

15

2

25

0 20 40 60 80 100 120

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Abso

rban

ce (7

00

nm)

(a)

0

05

1

15

2

25

3

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Abso

rban

ce (4

50

nm)

(b)








aMurraya koenigii




119865 value 18069 8288 33690 69171198772 value 0999 0953 0969 0996






aMurraya koenigii


b119872119906119903119903119886119910119886 koenigii+ MMS







a119872119906119903119903119886119910119886 koenigii


b119872119906119903119903119886119910119886 koenigii+ BP












aMurraya koenigii







4 Discussion


2

O2
















Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

05

1

15

2

25

0 20 40 60 80 100 120

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Abso

rban

ce (7

00

nm)

(a)

0

05

1

15

2

25

3

Petrol ether

Benzene

Ethyl acetate

AcetoneMethanol

Ethanol

Ascorbic acid

BHT


Abso

rban

ce (4

50

nm)

(b)








aMurraya koenigii




119865 value 18069 8288 33690 69171198772 value 0999 0953 0969 0996






aMurraya koenigii


b119872119906119903119903119886119910119886 koenigii+ MMS







a119872119906119903119903119886119910119886 koenigii


b119872119906119903119903119886119910119886 koenigii+ BP












aMurraya koenigii







4 Discussion


2

O2
















Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





aMurraya koenigii


b119872119906119903119903119886119910119886 koenigii+ MMS







a119872119906119903119903119886119910119886 koenigii


b119872119906119903119903119886119910119886 koenigii+ BP












aMurraya koenigii







4 Discussion


2

O2
















Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





aMurraya koenigii







4 Discussion


2

O2
















Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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