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Free Radicals and Antioxidants xxx (2013) 1e6

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Free Radicals and Antioxidants

journal homepage: www.elsevier .com/locate/ f ra

Original article

Antioxidant and DNA damage protecting activities of Eulophia nudaLindl.

Vinay Kumar a,*, Melissa Lemos a, Mansi Sharma a, Varsha Shriram b

aDepartment of Biotechnology, Modern College of Arts, Science and Commerce, Pune 411 053, IndiabDepartment of Botany, Prof. Ramkrishna More College of Arts, Commerce and Science, Pune 411 044, India

a r t i c l e i n f o

Article history:Received 7 May 2013Accepted 11 July 2013Available online xxx

Keywords:AntioxidantsDNA damage protectionFenton’s reagentLipid-peroxidationReactive oxygen species

* Corresponding author. Tel.: þ91 20 25634021; faxE-mail addresses: vinaymalik123@gmail.com, vi

(V. Kumar).

2231-2536/$ e see front matter Copyright � 2013, Schttp://dx.doi.org/10.1016/j.fra.2013.07.001

Please cite this article in press as: Kumar V,Antioxidants (2013), http://dx.doi.org/10.101

a b s t r a c t

Introduction: In recent years, natural antioxidants have seen an unprecedented importance and demandin bio-pharmaceuticals, nutraceuticals besides their use as food additives. Antioxidants act as potentialprophylactic and therapeutic agents against various diseases caused by free-radicals. Plants offertremendous source of antioxidants and are therefore being evaluated for their potentials. Eulophia nudais an important medicinal plant used by local healers in India; however its antioxidant properties havenot yet been investigated.Methods: Aqueous (AqE), methanol (ME), aqueousemethanol (AqME) and acetone (AE) extracts of shadedried tubers were obtained and were concentrated in vacuo. Total phenols, flavonoids, ascorbic acid andcarotenoids were estimated from all extracts using standard methods. Antioxidant activities of extractswere determined by total antioxidant activity, FRAP, ABTS, DPPH, and OH radical scavenging assaysbesides lipid peroxidation inhibition. Extracts were evaluated for protection of Fenton’s reagent inducedDNA damage.Results: The results confirmed the plant as a rich source of phenols, flavonoids, vitamin C and carotenoids.Among four extracts, AqME showed highest antioxidant activities as evidenced by maximum scavengingof ABTS (98%), DPPH (87%), and OH radicals (99%) at 1 mg ml�1 concentration and showed maximalinhibition of lipid peroxidation. All extracts protected the DNA from hydroxyl-radical-induced damage.Again, AqME was proved to be best in providing protection to DNA against damage caused by free-radicals.Conclusion: The results provides scientific basis for its traditional usage as natural antioxidant and phyto-therapeutic agent. The plant possesses high amount of phenolic compounds and showed a broad-spectrum antioxidant properties including DNA protection.Copyright � 2013, SciBiolMed.Org and Phcog.Net, Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction

Oxidative stress, induced by the generation of reactive oxygenspecies (ROS) consisting of both free radical compounds such assuperoxide anions (O2

�), hydroxyl radicals (�OH), as well as non-free-radical compounds hydrogen peroxide (H2O2), organicperoxide (ROOH), ozone (O3) and singlet oxygen (1O2) are consid-ered as major causative factors of many of today’s diseasesincluding diabetes and cardiovascular diseases.1e3 These ROS areknown as exacerbating factors in DNA damage and mutations,cellular injury, oncogenesis (as many mutagens and carcinogensacts through the ROS) and ultimately the aging process.4,5 Further,

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et al., Antioxidant and DNA d6/j.fra.2013.07.001

ROS are considered to cause cancer and several neurodegenerativediseases such as Alzheimer’s disease, Parkinson’s disease, Down’ssyndrome, inflammation, viral infection and various other digestivedisorders including ulcer and gastrointestinal disorders.6,7

Antioxidants are considered to play an imperative role inproviding protection against ROS-driven oxidative damage andassociated lipid peroxidation, and DNA strand breaking.3 Eventhoughmany antioxidants of synthetic origin are available and usedquite frequently, especially in food industry for preservation andprolonging the shelf-lives of food products, however, they are oftenbeing associated with quality deterioration, nutritional losses andoff-flavor development.8 Furthermore, available synthetic antioxi-dants have been reported to exhibit toxic and mutagenic effects.9

On the other hand, their natural counterparts have an edge overthem for being less- or non- toxic and hence can serve as potentialdrug and dietary molecules. Therefore, in recent years the

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amage protecting activities of Eulophia nuda Lindl., Free Radicals and

V. Kumar et al. / Free Radicals and Antioxidants xxx (2013) 1e62

antioxidants of phyto-origin have seen an unprecedented demandin bio-pharmaceuticals, nutraceuticals besides their use as foodadditives.

Medicinal plants offer an excellent source of various phyto-chemicals such as phenols, flavonoids, vitamins, tannins, many ofwhich have potent antioxidant activities and therefore can beexploited in drug discovery programs as well as in the preparationof foods and pharmaceutical products.10

Eulophia nuda Lindl. (Orchidaceae), a medicinally importantperennial orchid with underground tubers, is found in central andSoutheast Asian regions. In India, this plant is found in tropicalHimalayas, from Nepal to Assam, and in Deccan from Konkansouthwards. The tubers are reported to be used against tumors,scrofulous glands of the neck, bronchitis, blood diseases and asvermifuge.11e15 Raw tubers are eaten for curing rheumatoidarthritis.16 Earlier our group has reported anti-proliferative activ-ities of a phenanthrene derivative compound 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol isolated from this plant againsthuman cancer cells.17 The tubers are eaten raw and therefore makeit a perfect candidate for its exploration as potential antioxidativesource.

However, in spite of the fact that various medicinal values havebeen described by folklore and is extensively used by local healersin different parts of India, till date no scientific validation has beenevidenced for antioxidant potential of this plant. This is the firstreport dealing with the phytochemical analyses, antioxidant po-tentials and oxidative DNA damage preventive activities of variousorganic and aqueous extracts of E. nuda tubers.

2. Materials and methods

2.1. Plant material

Fresh tubers of E. nuda Lindl. (synonym: Eulophia spectabilis)were collected from Belgaum region (N 15� 41.0990, E 74� 25.0260,elevation 723 m) of Western Ghats, India. The botanical identifi-cation of the plant was carried out at the Botanical Survey of India,Pune 411001, India (Ref. No. BSI/WC/Tech/2012/244).

2.2. Preparation of plant extracts

Shade dried tubers of E. nuda were finely powdered with auto-mix blender. One Kilogram dry powder of bulbs were soaked in 3 Lacetone, methanol, aqueous methanol (1:1) solvents (Merck, India)or distilled water separately. The crude extract was prepared bycold percolation for 24 h at room temperature (26 � 2 �C). Thefiltrate was concentrated in vacuo at 40, 40, 56 and 60 �C to getacetone, methanol, aqueous methanol and aqueous extractsrespectively. This process was repeated thrice to get total extracts.The extracts were labeled as AE (Acetone extract), ME (Methanolextract), AqME (Aqueous Methanol extract) and AqE (Aqueousextract), and were obtained as reddish/brown solid residues with2.44%, 1.84%, 4.20% and 3.14% yield, respectively.

2.3. Chemicals

All the chemicals were of analytical grade and obtained fromHiMedia, Merck or Fisher while standard antioxidants were pro-cured from SigmaeAldrich.

2.4. Determination of total phenols

The total soluble phenolic content was determined by FolineCiocalteu (FC) method.18 In brief, 10 ml of extract was taken and thefinal volumewasmade 2ml with distilled water. To this 0.5ml of FC

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reagent was added and sample was incubated for 3 min, followedby addition of 2 ml of Na2CO3 and samples were placed in boilingwater for 1 min. The reaction mixture was allowed to cool and thenabsorbance was recorded at 765 nm on Chemito Spectrascan UV-2600 spectrophotometer. The total phenolic content wasexpressed as mg gallic acid equivalents (GAE) g�1 extract calculatedusing standard gallic acid calibration graph.

2.5. Determination of total flavonoids

Total flavonoids were estimated using modified Marinova et al19

method. Briefly, 10 ml plant extract was added to distilled water tomake a final volume of 2 ml and kept at room temperature for3min. To this, 3ml of 5% NaNO2 and 0.3ml of AlCl3 was added. After6 min incubation, 2 ml 1 M NaOHwas added and volumewas made10ml with distilled water. Absorbancewas taken at 510 nm and theconcentration of flavonoid compounds was expressed as mgquercetin equivalents per g extract.

2.6. Determination of total ascorbic acid

Total ascorbic acidwas estimated by 2,4-dinitrophenylhydrazine(DNPH)method as described earlier.20 Tenmicroliter of each extractwas separately taken and total volume was made to 2 ml withdistilled water. To this 2 ml of DNPH and 1 drop of 10% thioureawasadded, themixturewasheated in aboilingwater bath for 15min andcooled to room temperature. Five microliter of 80% (v/v) H2SO4 wasadded to themixture at 0 �C in an ice bath. Absorbancewas taken at521 nm and ascorbic acid was used as standard.

2.7. Determination of total carotenoid content

For estimation of total carotenoids, 10 microliter plant extractwas added to distilled water to make a final volume of 2 ml. To this,3.75 ml of 10% methanolic KOH and 3.75 ml diethyl ether wasadded. The reaction mixture was washed with 5% ice cold salineand dried over anhydrous Na2SO4 for 2 h. The absorbance of thefiltrate was taken at 450 nm and concentration of carotenoids wasexpressed as mg b-carotene equivalents per g extract.21

2.8. Antioxidant activities

2.8.1. Total antioxidant activity (TAA)Total antioxidant activity was determined using modified

phosphomolybdenum method.22 The assay is based on the reduc-tion of Mo(VI) to Mo(V) by sample compound and formation ofgreen colored phosphate/Mo(V) complex at acidic pH (4.0). 0.1 mlof extract from varying concentrations (200e1000 mg ml�1) wasadded to 1 ml reagent solution (0.6 M H2SO4, 28 mM sodiumphosphate and 4 mM ammonium molybdate). The mixture wasincubated at 95 �C for 90 min and the absorbance was measured at695 nm after cooling the samples. Total antioxidant capacity wasexpressed as GAE.

2.8.2. FRAP (ferric reducing antioxidant power)The antioxidant capacity of E. nuda extracts was estimated

spectrophotometrically.23 The method is based on reduction ofFe3þ- tetra (2-pyridyl) pyrazine (TPTZ) complex to Fe2þ-tripyr-idyltriazine formed by action of electron donating antioxidants atlow pH. FRAP reagent was prepared by the addition of 300 mMacetate buffer 10 ml TPTZ dissolved in 40 mM HCl and 20 mMFeCl3.6H2O in the ratio 10:1:1. Five hundred microliter of standardwas added to 1 ml of FRAP and the mixture was incubated at 37 �Cfor 30 min. Absorbance was recorded at 593 nm against blank. The

amage protecting activities of Eulophia nuda Lindl., Free Radicals and

Table 1Total phenols, flavonoids, ascorbic acid and carotenoid contents of E. nuda extracts.

Extract Total phenols*

(mean � SE)Flavonoids^

(mean � SE)Ascorbic acid#

(mean � SE)Carotenoids$

(mean � SE)

AE 23.3 � 0.8b 16.4 � 0.5b 29.1 � 2.1a 0.8 � 0.1a

ME 24.5 � 0.6c 22.2 � 1.8d 75.6 � 3.9d 3.0 � 0.3c

AqME 24.2 � 1.1c 19.9 � 0.2c 50.0 � 1.8c 2.7 � 0.2b

AqE 16.2 � 0.8a 6.6 � 0.2a 40.4 � 1.5b 2.5 � 0.2b

*Gallic acid; ^quercetin; #ascorbic acid and $b-carotene equivalents mg g�1 extract.Each value represents the mean of three replications � SE. Means within a columnfollowed by different superscript letters were significantly different from each otheraccording to Duncan’s Multiple Range Test (DMRT) at P � 0.05.

V. Kumar et al. / Free Radicals and Antioxidants xxx (2013) 1e6 3

values of FRAP were expressed as GAE for varying concentrations ofthe extracts (200e1000 mg ml�1).

2.8.3. ABTS radical scavenging assayFree radical scavenging activity of plant extracts was deter-

mined by ABTS radical decolorization assay.24 In brief, ABTS�þ (2,20-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid cation radical)was produced by the reaction between 7 mM ABTS in water and2.45 mM potassium persulfate in water (1:1). This reaction mixturewas stored in dark at room temperature for 16e20 h. This ABTS�þ

solutionwas then diluted with methanol to get absorbance of 0.7 at734 nm. Five microliter of plant extract was added to 3.995 ml ofABTS�þ solution and incubated for 30 min and absorbance wasmeasured at 734 nm. The results were expressed as percent scav-enging effect of plant extracts.

2.8.4. DPPH radical scavenging activityThe antioxidant activity of the plant extract was examined on

the basis of the scavenging effect on the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical activity as described by Bracaet al.25 Ethanolic solution of DPPH 0.05 mM (300 ml) was added to40 ml of extract of different concentrations (200e1000 mg ml�1).After 5 min, absorbance was measured spectrophotometrically at517 nm against extract blank. The radical scavenging activity of theplant extract, expressed as percent inhibition was calculated ac-cording to the following equation:

Percent inhibition of DPPH radical ¼½ðabsorbance control� absorbance testÞ=absorbance control� � 100

2.8.5. Hydroxyl radical scavenging activityHydroxyl radical scavenging activity was measured by studying

the competition between deoxyribose and test extract for hydroxylradical generated by Fenton’s reaction.26 The reaction mixturecontained deoxyribose (2.8 mM in KH2PO4eKOH buffer, pH 7.4),FeCl3 (0.1 mM), EDTA (0.1 mM), H2O2 (1 mM), ascorbate (0.1 mM),and various concentrations of the sample extracts (200e1000 mg ml�1) in a final volume of 1.0 ml. The reaction mixture wasincubated for 1 h at 37 �C. Deoxyribose degradation was measuredusing thiobarbituric acid (TBA) assay. One ml of TBA (1%) and 1 mlof TCA (2.8%) was added to above mixture and incubated at 100 �Cfor 20min. The development of pink color was measured at 532 nmand percent inhibition was calculated.

2.8.6. Determination of inhibition of lipid peroxidationLipid peroxidation inhibition potential of E. nuda extracts were

evaluated in vitro by following the modified Halliwell and Gutter-idge27 method. Briefly, freshly excised goat liver was minced usingglass Teflon homogenizer in cold phosphate buffered saline, pH 7.4.Ten percent homogenate was prepared and then was filtered toobtain a clear homogenate. The process of homogenization andfiltrationwas carried on ice. Different concentrations of the extracts(200e1000 mg ml�1) in water were added to the liver homogenate.Lipid peroxidation was initiated by adding 100 ml of 15 mM ferroussulfate solution to 3 ml of the tissue homogenate. After 30 min,100 ml of this reactionmixturewas taken in a tube containing 1.5 mlof 10% TCA. After 10 min, tubes were centrifuged and supernatantwas separated and mixed with 1.5 ml of 0.67% TBA in 50% aceticacid. Themixturewas heated for 30min in a boiling water bath. Theintensity of the pink colored complex wasmeasured at 535 nm. Thedegree of lipid peroxidation was assayed by estimating the TBARS(TBA-reactive substances) content. The results were expressed aspercentage inhibition using the formula:

Please cite this article in press as: Kumar V, et al., Antioxidant and DNA dAntioxidants (2013), http://dx.doi.org/10.1016/j.fra.2013.07.001

Percent inhibition of lipid peroxidation ¼½ðabsorbance control� absorbance testÞ=absorbance control� � 100

2.9. DNA protection activity

The ability of different extracts to protect DNA (pBR322, Merck,India) from damaging effects of hydroxyl radicals generated byFenton’s reagent was assessed by DNA nicking assay28 with minormodifications. The reaction mixture contained 2.5 ml of DNA(0.25 mg) and 10 ml Fenton’s reagent (30 mMH2O2, 500 mM ascorbicacid and 800 mM FeCl3) followed by the addition of 5 ml extract andthe final volume of the mixture was brought up to 20 ml withdistilled water. The reaction mixture was then incubated for 45minat 37 �C and followed by addition of 2.5 ml loading buffer (0.25%bromophenol blue, 50% glycerol). The results were analyzed on0.8% agarose gel electrophoresis by staining with ethidium bro-mide. Quercetin was used as positive control.

2.10. Statistical analyses

All experiments were conducted in triplicate to check thereproducibility of the results obtained. The graphs were plottedusing Microcal Origin 6.0. The results are presented asmeans � standard error (SE) and means were compared usingDuncan’s Multiple Range Test (DMRT) at P � 0.05, using MSTAT-Cstatistical software.

3. Results and discussion

Quantitative evaluation of phytochemicals, known for theirroles in providing antioxidant properties to plants, from tuber ex-tracts of E. nuda indicated the plant as a rich source of phenolics,flavonoids, ascorbic acid and carotenoids, though with solventdependent variations in their contents (Table 1). Several reportshave shown a correlation between higher amounts of total poly-phenols in plants and correspondingly higher antioxidant poten-tial.18,28e32 Our results also largely supported these conclusions. Inthe present study, overall, methanol extract (ME) showedmaximum amounts of total phenols, flavonoids, ascorbic acid andcarotenoids closely followed by aqueous methanol (AqME) extract.Total phenol content in tuber extracts expressed as gallic acidequivalent (GAE) were found in the range of 16.2e24.5 mg GAE g�1

extract (Table 1). Total flavonoids were comparably in lesserquantities than total phenols in all the extracts, where ME showedhighest concentrations (22.2 mg AAE g�1) followed by AqME with19.9 mg AAE g�1. Similar patterns were seen in case of ascorbic acidand carotenoids as evidenced from the results presented in Table 1.

Even though free radical generation and oxidation process areintrinsic in energymanagement of all living organisms and are keptunder strict control by several cellular mechanisms,33 however, if

amage protecting activities of Eulophia nuda Lindl., Free Radicals and

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Fig. 1. ABTS radical scavenging activities of various extracts of E. nuda. The values inparenthesis represent the concentration of gallic acid (20e100 mg ml�1). Each valuerepresents the mean of three replications � SE. The bars with different letters aresignificantly different from each other at P � 0.05 according to Duncan’s MultipleRange test.

V. Kumar et al. / Free Radicals and Antioxidants xxx (2013) 1e64

the supply of oxygen is in excess or its reduction is insufficient, ROSare generated that causes cellular injuries and initiate peroxidationof polyunsaturated fatty acids in biological membranes. In recentyears, the scientific community has strongly advocated explorationand use of plant-origin natural effective antioxidants, especiallyfrom edible plants and their parts as they have less or no side ef-fects than their synthetic counterparts.10,34 Therefore, in the pre-sent study, E. nuda known for its enormous medicinal properties infolklore was selected for the first time for evaluating its antioxidantproperties.

Antioxidant activity is generally attributed to phytochemicalspresent, a number of mechanisms in plants and the synergies be-tween them. Thus, antioxidant activity of plant extracts cannot beevaluated by a single method.10,35 Hence, in order to explore andunderstand these possible mechanisms, several antioxidant assaysincluding TAA, FRAP, ABTS, DPPH and OH radical scavenging assayswere performed for evaluating antioxidant activities of E. nuda. Theresults confirmed that this plant has a broad range of antioxidantproperties, including substantial inhibition of lipid peroxidation.The results of TAA and FRAP scavenging activity are summarized inTable 2. Amongst all the four extracts, again ME showed highestTAA (expressed as mg GAE g�1 extract). TAA gradually increasedwith the concentration of extract from 200 to 1000 mg ml�1 irre-spective of extract nature (Table 2). FRAP is an important indicatorof reducing potential of an antioxidant which is associated with thepresence of compounds responsible for breaking the free radicalchain through donation of hydrogen atom.36 The results showednoticeable antioxidant potential (in terms of FRAP measured asGAE) of all the extracts of E. nuda tubers, which was graduallyincreased with increasing concentrations of samples. The FRAPassay provides a reliable method for evaluation of antioxidant ac-tivities of various plant extracts and compounds as antioxidantcapacity is directly correlated with its reducing capacity23 and ourresults are in conformity of these findings.

Fig. 1 depicts the results of ABTS radical scavenging activities ofall the extracts as well as gallic acid (standard antioxidant com-pound). AqME showed highest capacity to scavenge ABTS cationradical amongst all the extracts, however the activities were infe-rior to gallic acid, and therefore the standard was used at compa-rably low range (20e100 mg ml�1) than the extracts (200e1000 mgml�1), respectively. Proton radical scavenging is consideredas an important attribute of antioxidants. ABTS, a protonatedradical, has characteristic absorbance maxima at 734 nm whichdecreases with the scavenging of the proton radicals.8 The magni-tude of free-radical quenching was dose-dependent and steadilyincreased with increase in plant sample concentrations (Fig. 1).

DPPH is a stable free radical and the DPPH assay based on itsreduction by antioxidant is most commonly used model system forstudying antioxidant capacity of plant extracts or pure compoundsto act as free radical scavengers or hydrogen donors.8,37 All theextracts showed significantly high tendency to quench the DPPHradicals, as indicated by the dose-dependent increase in inhibition

Table 2FRAP and total antioxidant activities (TAA) of E. nuda extracts.

Conc. of extracts(mg ml�1)

TAA of various extracts (GAE) (mean � SE)

AE ME AqME AqE

200 64 � 2.1a 70 � 3.3a 27 � 1.6a 21 �400 66 � 2.4a 95 � 3.1b 41 � 2.3b 23 �600 68 � 2.7a,b 132 � 4.7c 54 � 3.1c 26 �800 70 � 3.1b,c 138 � 4.4d 63 � 2.8d 30 �1000 75 � 3.4d 160 � 5.1e 88 � 3.6e 35 �

GAE: gallic acid equivalents. Each value represents the mean of three replications � SE. Msignificantly different from each other according to Duncan’s Multiple Range Test (DMR

Please cite this article in press as: Kumar V, et al., Antioxidant and DNA dAntioxidants (2013), http://dx.doi.org/10.1016/j.fra.2013.07.001

percentage (Fig. 2). AqME showed strongest radical scavengingactivity in comparison with other extracts, and its percentage in-hibition reached 87.29% at 1 mg ml�1 concentration (IC50 value:330 mg ml�1), which was significantly higher even than the stan-dard (ascorbic acid, 68.81%) at the same concentration and an IC50value of 804 mg ml�1. Similar to our findings, several authors haveattributed the antioxidant potential of plants to higher DPPHradical scavenging activity.18,30,32,38

Among the four extracts used, acetone extract did not showinhibition of OH radical generation at concentrations ranging from200 to 1000 mg ml�1 (Fig. 3). AqME again showed notably higherhydroxyl radical scavenging activity (IC50 value: 230 mg ml�1) thanother extracts as well as the standard (ascorbic acid, IC50 value:617 mg ml�1). The radical scavenging activity was found to beconcentration dependent and was increased with concentrations ofall the extracts and standard antioxidant as well (Fig. 3). Hydroxylradical is an extremely reactive ROS which initiates auto-oxidationand attacks almost every biological molecule causes damage toDNA, protein and lipids leading to mutagenesis, carcinogenesis andaging.28,30,33 The plants with higher OH radical scavenging holdsgreat importance as their consumption can result in controlling andmitigating the devastating effects of oxidative stresses.18,38 Thecurrent results are of great importance as the extracts especiallyAqME exhibited great potential in scavenging the OH radicals.

The inhibition of lipid peroxidation is considered as the mostimportant index of antioxidant activity as reported by various

FRAP of various extracts (GAE) (mean � SE)

AE ME AqME AqE

1.1a 200 � 2.5a 272 � 2.9a 251 � 4.5a 189 � 3.5a

1.4a 233 � 3.2b 301 � 5.4b 258 � 5.2b 201 � 5.4b

1.1b 251 � 2.9c 330 � 3.8c 259 � 5.0b 210 � 5.5c

1.5c 272 � 3.8d 353 � 3.7d 271 � 4.7c 222 � 5.8d

1.8d 290 � 4.3e 381 � 4.9e 292 � 3.9d 231 � 4.6e

eans within a column for each extract followed by different superscript letters wereT) at P � 0.05.

amage protecting activities of Eulophia nuda Lindl., Free Radicals and

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Fig. 2. DPPH radical scavenging activities of various extracts of E. nuda. Each valuerepresents the mean of three replications � SE. The bars with different letters aresignificantly different from each other at P � 0.05 according to Duncan’s MultipleRange test.

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Fig. 4. Inhibition of lipid peroxidation in goat liver by various extracts of E. nuda.Each value represents the mean of three replications � SE. The lines with the samecolor and symbol with different letters are significantly different from each other atP � 0.05 according to Duncan’s Multiple Range test.

V. Kumar et al. / Free Radicals and Antioxidants xxx (2013) 1e6 5

investigators.18,31,38 In the present investigation, in vitro lipid per-oxidation was induced in goat liver by FeSO4 which takes placethrough OH radical generation by Fenton’s reaction. E. nuda tuberextracts showed significant inhibition of lipid peroxidation ratemeasured in terms of TBARS, an indicator of malondialdehyde(MDA) content which is a degradation product of lipid peroxidation(Fig. 4). The results hold great significance as AqME showedtremendous potential in terms of inhibition percentage of lipidperoxidation, and showed 100% inhibition at 1000 mg ml�1 con-centration followed by ME (96% inhibition) against standardascorbic acid with 90% inhibition at similar concentration (Fig. 4).AqME thus offered a good degree of protection against the bio-logical end-point of oxidative damage. Lipid peroxidation inducescellular damage that eventually leads to many human diseases.31,34

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Fig. 3. Hydroxyl radical scavenging activities of various extracts of E. nuda. Eachvalue represents the mean of three replications � SE. The lines with the same color andsymbol with different letters are significantly different from each other at P � 0.05according to Duncan’s Multiple Range test.

Please cite this article in press as: Kumar V, et al., Antioxidant and DNA dAntioxidants (2013), http://dx.doi.org/10.1016/j.fra.2013.07.001

Antioxidant may offer resistance against the oxidative stress byscavenging the free radicals, inhibiting the lipid peroxidation andthus prevent diseases.

All the extracts were evaluated for their oxidative damageprotective activity against a model DNA pBR322. Hydroxyl radicalsgenerated by Fenton’s reaction are known to cause DNA damage, asDNA band is absent in Fig. 5, lane 3 and only a smear of degradedDNA can be observed. Even though all the extracts effectivelymitigated the oxidative stress and protected the DNA fromhydroxylradicals generated by Fenton’s reaction, as confirmed by the pres-ence of DNA bands, aqueous methanol extract (Fig. 5, lane 6) seemsto be comparably most effective in maintaining the DNA intactfollowed by aqueous (Fig. 5, lane 4), acetone (Fig. 5, lane 7) andmethanol extract (Fig. 5, lane 5), respectively. Standard antioxidantcompound quercetin was also used for comparison with plant ex-tracts (50 mg ml�1 each) for DNA protection efficacy. Free radicalsare known for DNA strand breaking and damage which eventuallycontributes to carcinogenesis, mutagenesis and cytotoxicity.3,5

Various researchers have reported the similar results and usedplant extracts and fractions for DNA protection against oxidativedamage.3,5,39

Fig. 5. DNA damage protecting effect of aqueous, aqueous methanol, methanoland acetone extracts of tubers of Eulophia nuda against hydroxyl radicals inducedDNA damage of pBR322. Lane 1: native pBR322 plasmid DNA; Lane 2: DNA þ Fenton’sreagent þ quercetin (50 mg ml�1, positive control); Lane 3: DNA þ Fenton’s reagent(DNA damage control); Lane 4: DNA þ Fenton’s reagent þ AqE; Lane 5: DNA þ Fenton’sreagent þ ME; Lane 6: DNA þ Fenton’s reagent þ AqME; Lane 7: DNA þ Fenton’sreagent þ AE.

amage protecting activities of Eulophia nuda Lindl., Free Radicals and

V. Kumar et al. / Free Radicals and Antioxidants xxx (2013) 1e66

4. Conclusion

It can be concluded that E. nuda contains considerable amount oftotal phenols, flavonoids, vitamin C and carotenoids and showedsolvent-dependent variations in their quantities. Various in vitroantioxidant assays clearly indicated that the plant extracts exhibitedbroad spectrum of antioxidant properties mediated by effectivescavenging of various free radicals and subsequently inhibited thelipid peroxidation. Amongst all the extracts AqME showed consider-ably higher antioxidant activities than other extracts. The plant ex-tracts successfully protected the DNA from damage caused by freeradicals. Overall, methanol and aqueous methanol extracts showedgreat promise and should therefore be used further for fractionationand isolation of pure compound responsible for antioxidant activities.

Conflicts of interest

All authors have none to declare.

Acknowledgments

Authors thank the Principals of Modern College, Ganeshkhind,Pune and Prof. Ramkrishna More College, Akurdi, Pune forencouragement and support to carry out this work.

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