international journal of radiation research, january...

International Journal of Radiation Research, January 2014 Volume 12, No 1

Radioprotective efficacy of prunus avium fruit in mice brain with reference to in-vitro and in-vivo

studies

INTRODUCTION

During radiotherapy, ionizing radiationparticles interact with biological systems toinduceexcessiveoxygenfreeradicalsorreactiveoxygen species (ROS), which attack variouscellular components including DNA, proteinsand membrane lipids, thereby leading tosigni icant cellular damage. ROS also havenegative impact on the antioxidant defensemechanisms, which shift the balance betweenprooxidant and antioxidants in cell towards

former resulting in severe oxidative stress andultimatelycelldeath(1).Malondialdehyde(MDA)formation as an end product of lipidperoxidation is one of the important indices ofoxidative damage(2). One of the mechanism ofprotection, involving free‐radical scavenging, isbased on the assumption that free radicalsresulting from the radiolysis of water are themaincauseofradiationdamagetocells(3).Thus,scavenging free radicals and inhibiting lipidperoxidation are likely key target activities fordevelopingsuccessfulradioprotectionstrategies(4). Several molecular drugs of synthetic and

K.V. Sharma and R. Sisodia*

RadiationBiologyLaboratory,DepartmentofZoology,UniversityofRajasthan,Jaipur,India‐302055

ABSTRACT

Background: Radioprotec ve efficacy of Prunus avium fruit extract (PAE) rich in vitamin A, C, E and anthocyanin was studied against radia on induced biochemical altera ons in mice brain. Materials and Methods: In‐vitro assays were performed with PAE for its an oxidant studies; therea er for in‐vivo study, Swiss albino mice were divided into five groups. Group 1 (control) normal mice. Group 2 (PAE treated) PAE supplemented (450 mg/kg. b.wt/day) for 15 days. Group 3 (Irradiated) 5 Gy gamma‐radia on treated. Group 4 (PAE + Irradiated) was administered PAE once daily for 15 consecu ve days, therea er exposed to 5 Gy gamma‐radia on. Group 5 (Irradiated + PAE) was exposed to 5 Gy gamma‐radia on than PAE was administered for 15 consecu ve days. Brains were removed at various post treatment intervals for biochemical es ma ons. Results: The IC50 values of PAE for 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH·] scavenging assays, superoxide radical scavenging assays, inhibi on of in vitro lipid peroxida on assays and protein carbonyl forma on assay were 413 µg/ml, 7.63 µg/ml, 136.18 µg/ml and 16.94 µg/ml respec vely. Furthermore the total phenolic content in PAE was equivalent to 8.38 mg/ml of gallic acid. The rate of ·OH radical scavenging ac vity of PAE was 0.57 mes slower than SCN‐ derived from nanosecond pulse radiolysis studies. In‐vivo study also showed that radia on (5 Gy) induced augmenta on in the levels of lipid peroxida on and deple on in glutathione and protein level in mice brain were significantly ameliorated by PAE pre/post treatment. Conclusion: Results suggest that the radioprotec ve efficacy of PAE may be due its an oxidant property. Keywords: Prunus avium, in-vitro antioxidant scavenging assays, in-vivo studies, mice brain.

*Correspondingauthor:Dr.RashemiSisodia,Fax:+911412701137 E‐mail:

[email protected]

Submitted: Oct. 2012 Accepted: June 2013

Int. J. Radiat. Res., January 2014; 12(1): 21-31

► Original article

Sharma and Sisodia /Radioprotective efficacy of Prunus avium

natural origin are being tried in severalexperimental models to mitigate the radiationinjury (5). Among different molecularradioprotectors WR‐2721 and relatedcompounds have been found to be mostpromising but the side effects associated withthem have restrained their use (6). In view ofthis,searchfornewerandmoreeffectiveagentsisinevitable.

Indiahasa richheritageofmedicinalplants,many of which have been explored for thevarious bioactivities since ages, but theirradioprotective potential has hardly beenexplored. In thiscontext,Cherry(Prunusavium)commonly known as sweet cherry, wild birdcherry, found in JammuandKashmir,HimachalPradesh, Uttaranchal and hills of Tamil Nadu(7)rich inantioxidantswasselected toevaluate itsradioprotective ef icacy. According to Wang etal.(8), anthocyanin content in sweet cherry is350‐450mg/100gmof fruit.AccordingtoUSDANutrientDatabaseforStandardReference(9)100gramsofedibleportionoffruitsofPrunusaviumhasselenium0.6µg,ascorbicacid7mg,thiamin0.05 mg, ribo lavin 0.06 mg, niacin 0.4 mg,pantothenicacid0.127mg,vitaminB60.036mg,folate4.2µg,vitaminA214IU,vitaminA21µgREandvitaminE0.130mgATE.According to Pandey etal.(10) tender stem of

Prunusaviumhas ethnomedicinal value in heartdiseases. The fruit stalks are astringent,diureticandactas tonic.Adecoction isused inthe treatment of cystitis, oedema, bronchialcomplaints,loosenessofthebowelsandanemia(11). Fruits of Prunusavium have digestive andantispasmodicuses.Recentstudyreportedsomeevidence that cherry consumptionmight lowerlevels of urate in the blood (12). But theradioprotectivepotentialofthisfruithashardlybeenexplored.Brain is considered sensitive to oxidative

damage because brain is enriched in the moreeasily oxidizable polyunsaturated fatty acids asit has highest rate of aerobic glycolysis of alltissues. Human brain consumes 20% of bodyoxygen and glucose and for this reason it issensitivetohypoxia(13).Ontheotherhand,brainisnotenrichedinantioxidantdefensesystemasit contains relatively low levels of superoxide

Int. J. Radiat. Res., Vol. 12 No. 1, January 2014 22

dismutase, catalase, glutathione peroxidase (14).External supplementation through antioxidantsis recommended to protect cells from thedeleterious effects of such oxidative stressconditions. Ample researches indicate thatage‐relatedneuronal‐behavioraldecrementsarethe result of oxidative stress that may beamelioratedbyantioxidants(15).

So, we studied mitigation of deleteriouseffectsofionizingradiationinSwissalbinomiceby Prunusavium extract (PAE) with specialreference to brain along with free radicalscavenging activity of PAE by employingnanosecond pulse radiolysis, stopped‐ lowspectrometer and other assays to prove theradioprotective activity of anthocyanin andvitaminsrichPrunusaviumextract(PAE).

MATERIALSANDMETHODS

AnimalcareandhandlingSwiss albino mice, 6–8 weeks old weighing

23±2 gm, from an inbred colonywere used forthe present study. These animals weremaintained under controlled conditions oftemperature and light (light: dark, 10 hrs: 14hrs.). Four animals were housed in apolypropylene cage containing sterile paddyhusk (procured locally) as bedding throughoutthe experiment. They were provided standardmicefeed(procuredfromHindustanLeversLtd.,India) and water adlibitum. The departmentalanimalethicalcommitteeapprovedthisstudy.Chemicals

2,2‐diphenyl‐1‐picrylhydrazyl (DPPH*) waspurchased from Aldrich Chemicals, USA.Xanthine, Xanthine oxidase, Bovine SerumAlbumin (BSA), 2,2′‐dinitrophenyl hydrazine(DNPH), thiobarbituric acid (TBA),trichloroacetic acid (TCA), TetramethoxyPropane (TMP), glutathione (GSH), 5, 5dithio‐bis 2‐Nitrobenzoic acid (DTNB),Potassium superoxide were obtained fromSigma Chemicals, USA. All the other reagentsused were of the highest available purity.Nitrous oxide (N2O) and oxygen (O2) gases


obtainedfromIndianOxygenLtd.,Mumbaiwereof IOLAR grade purity. Nanopure water wasobtained from a Millipore Elix/A‐10 waterpuri ication system. Freshly prepared solutionswereusedforeachexperiment.

Extractpreparation(Drug) Fresh fruits of Prunusavium were washed,

shade dried and powdered after removal ofseeds.Methanolicextractwasthenpreparedbyre luxing for 48 hours (4×12) at 40oC. Theextractthusobtainedwasvacuumevaporatedsoas to get in powdered form. The extract wasre‐dissolved in doubled‐distilled water (DDW)just before the oral administration. For thevariousconcentrations,aknownamountofPAEwas suspended in DDW and 50 µl of PAEsuspension was given to each mouse by oralgavage.

A.Totalphenoliccontent

The total phenolic content present in PAEwasmeasuredbyusingamodi iedcolorimetricFolin‐Ciocalteu method (16). Brie ly, 0.5 mL ofwaterand0.125mLofthemethanolicextractofPAE (concentration range 15–30 μg/mL) wereaddedtoatesttube.TheFolin‐Ciocalteureagent(0.125 mL) was added to the solution andallowedtoreactfor6min.Tothat1.25mLofthesodium carbonate solution (7%) and 3 mL ofwater was added and allowed to stand for 90min. The absorbance of the solution wasmeasured at 760 nm. The measurements werecompared with the standard calibration curveplottedforthegallicacidsolution(2‐10μg/mL)and the total phenolic content in PAE wasexpressedas%gallicacidequivalents.

B. In‐vitro free radical scavenging studiesradicalscavengingassaysa.DPPHradicalscavengingDPPH· (2,2‐diphenyl‐1‐picrylhydrazyl) is a

stable freeradical (17).TheoddelectronpresentintheDPPHgivesastrongabsorptionmaximumat517nm.For steadystateDPPHexperiments,0.6mlof200mMDPPHinmethanolwasmixedwithPAE(200‐700mg/ml)inmethanolin3mlreaction mixture, and kept in dark for 20 min.Theabsorbanceat517nmwasmonitoredboth

in the presence and absence of PAE. Blankexperiment was also carried out to determinetheabsorbanceofDPPHbeforeinteractingwiththeextract.b.Superoxideradicalscavengingassay

The superoxide assay was studiedspectrophotometrically using xanthine/xanthine oxidase system (18). Brie ly, 3 mlsolution consists of 38 mM tris‐ HCl buffer pH7.4, 16 mM xanthine, 10 mM cytochrome C(Fe3+) and about 0.02 units/ml of xanthineoxidaseenzyme.ThedecreaseintheabsorbanceofFe2+wasmonitoredat550nmin theabsenceandpresenceof2‐25mg/mlofthePAE.c.Hydroxylradicalscavengingassay

Using nanosecond pulse radiolysistechnique, employing 500ns pulses of 7 MeVelectron, with an absorbed dose of 8 Gy, thereactionsof ·OHradicalswithPAEwerecarriedoutinmicrosecondtimescaleandthetransientsdetected by absorption spectrometry asdescribed by Sharma etal.(17). Rate constant forthe reaction of any antioxidant with a freeradical indicates its reactivity towards the freeradical. However, in the case of plant extractslikePAE,whichisamixtureofcomponentswithunknown concentrations, estimation of rateconstant is dif icult. However, it is possible toestimate their relative reactivity in comparisonwith a standard whose rate constant with ·OHradical is accurately known.Herewe employedpotassium thiocyanate (KSCN) as the referencesolute. In this method, ·OH generated byradiolysing water is made to react with 2 mMKSCN at pH 7, in the absence and presence ofvariousconcentrationsofPAE.IntheabsenceofPAE,·OHreactscompletelywithSCN–toproduce(SCN)2·‐. The absorbance was calculated bymonitoringthe formationof(SCN)2·‐at500nm.HoweverinthepresenceofPAE,decreaseintheabsorbance of (SCN)2·‐ was observed. From theextent of decrease, the rate of scavenging ofhydroxyl radical by PAE was calculated withrespect to SCN‐. The reactions involved in thiscompetitionkineticmethodare:

·OH+2SCN‐(SCN)2·–k1=1.1×1010M–1s–1,(1)



·OH+PAE Productsk2=? (2)The absorbancedue to (SCN)2·‐at500nm in

the absence (A0) and presence of varyingconcentrationsofPAEorPA(A),isrelatedtotherateconstantsbythefollowingequation

(3)

Slope of the linear plot for [(A0/A)–1] vs

[PAE]/[SCN‐],boththeconcentrationsexpressedin mg/ml, is used to estimate the comparativeability of the PAE to reactwith the ·OH radicalwithrespecttoKSCN.C.In‐vitroradioprotectiveactivitya.LipidperoxidationLipidperoxidation(LPO)studieswerecarried

out in phosphatidyl liposomal models inducedby the radiation from a 60Co g‐source.N2O/O2‐purged liposomal solutionwasexposedto g‐radiation in the absence and presence ofdifferent concentrations (25‐200mg/ml) of thePAEatphysiologicalpH7.4 (phosphatebuffer).The detailed methodology used for the lipidperoxidation is given in our earlier references(17). The extent of lipid peroxidation wasestimated in terms of thiobarbituric acidreactive substances (TBARS) using 15% w/vtrichloroacetic acid, 0.375% w/v TBA, 0.25 Nhydrochloric acid, 0.05% w/v BHT as TBAreagent measuring the absorbance at 532 nm(e532=1.56×105M‐1cm‐1).

b.ProteincarbonylestimationProtein oxidation was carried out in Bovine

Serum Albumin (BSA). The formation ofcarbonylgroupsduetoexposureing‐irradiationin the absence and thepresenceof (10‐50mg/ml) of PAE were assayed by a standardDNPH‐coupled Spectrophotometric method (17‐18). Samples were saturated with N2O andexposedtog‐radiationtoanabsorbeddoseof60Gyfrom60Cog‐sourcewithadoserateof30Gymin‐1 as measured by standard FrickeDosimetry. Percentage protection calculationsforlipidperoxidationandproteincarbonylation

calculatedbyusingequation4.Here ∆A (Test compound) and ∆A (Blank)

indicatetheabsorbanceeitherat532or370nminthepresenceandabsenceofPAErespectively.Fortheestimationtheconcentration(inmg/ml)required to inhibit lipid peroxidation andproteincarbonylation,by50%wasplottedasafunctionoftheconcentrationofPAEorandfromthe plot, the concentration required to reducetheactivityby50%wasidenti ied.

D.In‐vivoradioprotectivestudiesa.SourceofirradiationThe cobalt teletherapy unit (ATC‐C9) at

Cancer Treatment Center, RadiotherapyDepartment, SMSMedical College andHospital,Jaipur,Rajasthan,Indiawasusedforirradiation.Unanaesthestized animals were restrained inwell‐ventilated Perspex boxes and whole bodyexposedto5Gygammaradiation.b.DrugdoseselectionSingle dose of PAE at the rate of 450‐mg/kg

b.wt was given orally to mice one hour beforethe radiation exposure as used by Sisodia etal.(19)earlier.

c.ExperimentaldesignMice selected from an inbred colony were

dividedinto5groupsGroup 1. Control (vehicle treated): Mice ofthis group received only DDW water for 15days.

Group 2. PAE treated: Mice of this groupreceivedPAE(450mg/kgofb.wt./day)for15days.

Group 3. Irradiated: Mice received DDW(volume equal to PAE solution) one hourbefore the whole body exposure to 5 Gy ofgamma‐radiation.

Group4.PAEtreated+Irradiated:AnimalsinthisgroupwereorallyadministeredPAE(450mg/kg of b.wt./day) once daily for 15consecutive days before they were wholebody exposed to single dose of 5 Gygamma‐radiation.(4)



Group5.Irradiated+PAE treated:Themicewerewholebodyexposedtosingledoseof5Gy gamma‐radiation thereafter oraladministration of PAE (450mg/kg of b.wt./day)wasmadeoncedailyfor15consecutivedays.

RemovalofbraintissueMice were sacri iced by cervical dislocation.

Anincisionwasmadeatthesidesofthejawstoseparate the upper and the lower palates. Theupper palate was cut in the middle and afterhaving cleared of the surrounding tissue; brainwasexcisedandseparatedfromthespinalcordat the decussating of the pyramids. The intactwhole brain was then removed carefully andhomogenate was prepared and used forquantitativeestimations.Mice from each group were necropsied at

variousintervals, i.e.1,3,7,15,30and45dayspost irradiation. Whole brain was used toestimate various alterations in biochemicalparameters viz. glutathione, lipid peroxidationandproteincontent.d.Lipidperoxidation(LPO)assayLPOwasmeasured by themethod of Buege

andAust(20).Astandardcurvewaspreparedbyusing TMP. After comparison with standardcurve the LPO level were expressed in n moleTBARS/gtissue.e.Reducedglutathione(GSH)assayThe reduced glutathione (GSH) content of

tissue sampleswas determined in testis by themethod of Moron etal. (21). The results wereexpressedasnmolofGSH/100mgoftissue.f.ProteinassayEstimation of protein was based on the

methodproposedbyBradford(22).StatisticalanalysisThe results obtained in the present study

were expressed asmean ± SEM. The statisticaldifference between two groups were analyzedby the Student’s t‐testand difference betweenvarious group were analyzed by one wayANOVA with Graph Pad Prism‐5 software at

different autopsy intervals and the signi icancewas observed at the p <0.01, p<0.001 andp<0.000level.RESULTSANDDISCUSSION

Radiationprotectionisanimportantaspectin

radiotherapyof cancerwherenormalcellshaveto be protected while cancers are exposed toradiation (23). The interaction of ionizingradiation with biological system results in thegeneration of many highly reactive oxygenspecies (ROS) mainly due to the hydrolysis ofwater. These ROS attack cellularmacromolecules like DNA, RNA, proteins,membranes, etc., and cause its dysfunction anddamage. ROS increased the membrane lipidperoxidation, which in turn can alter theintegrity of membrane structure leading toinactivation of membrane‐bound enzymes, lossofpermeabilityof themembranesanddecreasein membrane luidity. Various diseaseconditions are also associated with free radicalinduced oxidative stress (24). As our previousstudyshowedthat5Gygammaradiationshowssigni icant change in brain protein and LPOlevelwhichalsocorrelateswithchange inbrainhistology alteration and behavioral imbalance[25], Similarly we found signi icant deleteriouschangeinprotein,LPOandGSHlevelingroup3i.e. irradiated only and at this exposure (5 Gy)mice nomortality has been noticed aswe haveshownalreadythatat10Gymicehardlysurvivebeyond10‐12days.

Antioxidantscanprotectagainstthedamageinducedby free radicals actingat various levels(26). Herbal drugs containing free radicalscavengers like phenolics, tannins andlavonoids are known for their therapeuticactivity (27). In the present study, preliminaryphytochemical testing showed the presence ofphenolics in PAE by Folin‐Ciocalteu method.Subsequent quanti icationwith respect to gallicacid revealed that the total phenolic content inPAEisequivalentto8.38mg/mlofgallicacid.

The reaction of DPPH radicalwith PAEwasstudied by steady state. Figure 1 shows



the change in absorbance of DPPH radical inmethanol at 517 nm in the absence andpresenceofdifferentconcentrationofPAE.Fromthis igure the IC50 value of PAE estimatedwas413mg/ml(table1).Xanthine oxidase is one of the main

enzymatic sources of reactive oxygen speciesin‐vivo. In this study PAE has been found topossess superoxide radicals scavengingproperty. The superoxide radicals generatedfrom xanthine/xanthine oxidase assay wereexamined for the scavenging by PAE incomparison with cytochrome C and thepercentage inhibition of superoxide radicalformation was estimated. Figure 2 shows thepercentage inhibition of superoxide radicals atdifferentconcentrationsofPAE.Fromthisstudy,the IC50 value of PAE for the inhibition ofsuperoxide radicals was calculated to be 7.63

Figure 1. Linear plot shows the scavenging ability of DPPH radical by different concentra ons (200μg/ml to 700 μg/ml)

of PAE (methanolic extract of Prunus avium fruits). The DPPH radical scavenging ac vity was measured by following

the decrease in absorbance at 517nm at DPPH concentra on of 100 μM.

Free radical scavenging assays

IC50 values (mg/ml)

DPPH scavenging assays 413 mg/ml

Superoxide radical scavenging assays

7.63 mg/ml

Lipid Peroxida on 136.18 mg/ml

Protein Carbonyl assays 16.94 mg/ml

Table 1. IC50 (i.e. 50% inhibi on concentra on) values of methanolic extract of Prunus avium fruits (PAE) obtained by various in‐vitro free radical scavenging and radioprotec ve

studies.

Figure 2. Inhibi on of the superoxide radical scavenging ac vity with increasing concentra ons (2 μg/ml to 25 μg/ml)

of PAE (methanolic extract of Prunus avium fruits).

mg/ml(table1).Here the activity for the inhibition of

superoxide radical can be either due to directscavenging of the superoxide radical or due tothe inhibition of the activity of xanthineoxidase.

Among the several free radicals, hydroxylradical (·OH) is the most potent oxidantproduced during radiation exposure as well asFenton reactions (17). It is a strong oxidizingradicalwith standardreductionpotentialof1.9V vs NHE at pH 7. Using nanosecond pulseradiolysis technique, the reactionof ·OHradicalwith PAEwas carried out inmicrosecond timescale and the transient spectrum obtained inthesereactionsisshownin igure3.Thespectraobtained from the reaction of ·OH radical withPAE showed a broad spectrum in the region of350 – 600 nm. The spectrum has nocharacteristic absorption bands, indicatingpresence of many unidenti ied species in PAEextract. The reactivity of the PAE towards ·OHradical was estimated by competition kineticsmethodsusingKSCNasareferencesolute.Insetof igure 3 shows the linear plot, for thevariation of the absorbance due to SCN2·‐ as afunction of the ratio of concentrations of PAEwithSCN‐accordingtotheequation3, fromtheslopeof this linearplot, calculated relative rateof scavenging ·OH radical by PAEwas found tobe0.57timesslowerthatofSCN‐.


Oxidative stress leads to lipid peroxidation,protein and carbohydrate oxidation andmetabolic disorders (28). The peroxyl radicalformed through lipid peroxidation attacksproteinmembraneandenzymesandreinitiateslipid peroxidation. The preservation of cellularmembrane integrity depends on protection orrepair mechanisms capable of neutralizingoxidative reactions such as lipid peroxidation.Inhibition of iron‐induced lipid peroxidation inegg phosphatidylcholine liposomes as assessedby the formation of TBARS by PAEwas shownconcentration dependent ( igure 4) and IC50value of PAE was found to be 136.18 μg/ml(table1).Thisresult indicates thatPAEhas theabilitytoinhibitthelipidperoxidationasshownbydecreasingamountofTBARScontent.

Lipid peroxidation product ( igure 5) asre lectedbyTBARSequivalent in in‐vivo studieswas signi icantly (p<0.001) higher after 5 Gygamma radiation exposure in Group 3 micebrain when compared to the control mice(Group 1) brain at all autopsy intervals. Suchincrease in lipidperoxidationproductwasnotstable and continuously decreased till the lastautopsy interval except at day 7 where slightincrease was noticed. TBARS content wassigni icantly lower (p<0.001) in Group 4 and 5as compared to Group 3 mice at all autopsyintervalsandvaluesalmostreached thecontrol

Figure 3. Difference in absorp on spectrum of the transient obtained on pulse radiolysis of N2O saturated

aqueous solu ons containing 100μg/ml of PAE (methanolic extract of Prunus avium fruits) at pH 7. Inset shows linear plot

for compe on kine cs between SCN2‐ and PAE towards hydroxyl radicals. Lines show fi ng according to equa on 3.

levels by the last autopsy interval i.e. day 45( igure 5). Statistically signi icant reduction(p<0.001) in the levelofTBARSequivalentwasseeninonlyPAEtreatedgroup(Group2).Whenallthefourgroupsi.e.groupI,III,IVandVwerecomparedtoeachotheroneachautopsyintervalby one‐way ANOVA, highly signi icantdifferences were observed on day 1(F3,44=792.11, p=0), 3 (F3,44=265.55, p=0), 7(F3,44=2002.69, p=0), 15 (F3,44=62.35, p=0), 30(F3,44=46.45,p=0)and45(F3,44=162.17,p=0)p.i.betweenallthegroups.The preservation of cellular membrane

integrity depends on protection or repairmechanisms capable of neutralizing oxidativereactions (17). Present study reveals that prior/post supplementation of PAE against radiationexposure shows bene icial effect against theformation of free lipid radicals and thuspreventstheformationofendoperoxidation.Exposureofmicewith5Gygammaradiation

declinedtheglutathione(GSH)contentuptoday7 thereafter recoverywasnoticed ( igure6). Ingroups(4and5)supplementedwithPAEprior/post irradiation magnitude of recovery fromoxidative damage was higher in comparison togroup 3 at all the autopsy intervals butsigni icant statistical difference was present atlater intervals only, whereas in Group 5(irradiated+PAE treated), afternon‐signi icant

Figure 4. Inhibi on of γ radia on (240 Gy) induced lipid peroxida on in phospha dyl choline liposome (1mg/ml) in

presence of different concentra ons of PAE (methanolic extract of Prunus avium fruits) (25μg/ml to 200μg/ml).



difference noticed at day 1, the values of GSHwere found signi icantly higher (p<0.001) thanGroup3micetillthelastautopsyintervalwherethevaluesreachednearcontrol level ( igure6).Statistically non‐signi icant change in GSHcontentwasnoted in group2 in comparison togroup1.Whenallthefourgroupsi.e.groupI,III,

Figure 5. Graph showing protec on of lipid peroxida on in mice brain a er 5 Gy gamma irradia on by prior and post administra on of PAE (methanolic extract of Prunus avium fruits). The lipid peroxida on was measured in terms of nM TBARS/g

ssue by LPO assay. Following groups were compared by student’s t‐test‐ a: Control v/s PAE treated b: Control v/s Irradiated, c: Irradiated vs PAE treated + Irradiated, d: Irradiated vs Irradiated + PAE treated. Symbols for p values of t‐test $<0.05,*<0.01,

**<0.005, ***<0.001, n= non significant.


IVandVwere compared toeachotheroneachautopsy interval by one‐way ANOVA, highlysigni icant differences were observed on day 1(F3,44=7.388, p=0.0004), 3 (F3,44=8.347,p=0.0002), 7 (F3,44=10.199, p=0.0000), 15(F3,44=6.45,p=0.001),30(F3,44=4.567,p=0.0072)

Figure 6. Graph showing protec on of glutathione content in mice brain a er 5 Gy gamma irradia on by prior/ post administra on of PAE (methanolic extract of Prunus avium fruits). The glutathione level was measured in terms of nM /100mg

ssue. Following groups were compared by student’s t‐test‐ a: Control v/s PAE treated b: Control v/s Irradiated, c: Irradiated vs PAE treated + Irradiated, d: Irradiated vs Irradiated + PAE treated. Symbols for p values of t‐test $<0.05,*<0.01, **<0.005, ***<0.001,

n= non significant.


which indicates the radioprotecting ability ofPAE. The IC50 value for PAE to protect 50%ofBSA in comparison to radiation control wasestimatedas16.94mg/mlforPAE(table1).

Protein estimated in in vivo studies alsoshowedstatisticallysigni icantdecreaseinmicebrain after radiation exposure. Such decline inprotein contentwasnotedcontinuously tillday30postirradiationatsigni icance(P<0.001)butat day 45 p.i. this reduction was signi icant atlevel p<0.05. In Group 4 recovery was evidentafterday7p.i.Proteincontentestimatedinmicebrainwassigni icantlyhigher(p<0.001)inboththe Groups (4 and 5) supplemented with PAE(prior/ post) irradiation in comparison toGroup 3 at all the autopsy intervals ( igure 8).PAEadministrationalonefor15dayscouldraisethebaselinevaluesofproteinconcentration.Theincrease was statistically signi icant (P<0.001).Whenallthefourgroupsi.egroupI,III,IVandVwere compared to each other on each autopsyinterval by one‐way ANOVA, highly signi icantdifferenceswereobservedonday1(F3,44=33.53,p=0),3(F3,44=45.85,p=0),7(F3,44=485.67,p=0),15 (F3,44=167.16, p=0), 30 (F3,44=132.31, p=0)and 45 (F3,44=32.42, p=0) p.i. between all thegroups. Increased protein concentrationrecorded in our study, shows that PAEsupplementation in irradiated mice is abene icialeffect.Prunusavium contains anthocyanin, vitaminC

etc as its constituents which are potentantioxidant. DelgadoVargas et al. (32)demonstrated that anthocyanins havescavenging properties against •OH and O‐2 andarebetteragentsagainstlipidperoxidationthanα‐tocopherol(uptoseventimes).Mechanismsofantioxidative action of vitamin C are directscavenging and blocking of ROS, as well asregenerationofotherantioxidative systems (33).Protective effects of vitamin C against ionizingradiation DNA damage have also beenextensively documented (34). Earlier studies inour laboratory showed that anthocyanin,vitamin C rich Grewia asiatica fruit possesradioprotective ef icacy in brain (35),cerebrum(31)], liver(36), blood(6) and testis(18)whensupplementedprior/postirradiation.

The results of the present investigation

and 45 (F3,44=3.32, p=0.028) post treatmentbetweenallthegroups.

Glutathione plays important role inantioxidant defense, nutrient metabolism, andregulation of cellular events (including geneexpression, DNA and protein synthesis, cellproliferationandapoptosis,signal transduction,cytokineproductionand immuneresponse,andproteinglutathionylation.Glutathionede iciencycontributestooxidativestress,whichplaysakeyroleinagingandpathogenesisofmanydiseases,including Alzheimer’s disease (AD) (29).Most ofthe researchers have observed that glutathionelevels decrease with ageing (30). The presentstudy demonstrates a signi icant reduction inGSH content in brain following radiationexposure. This could be due to the enhancedutilization of the antioxidant system as anattempt to detoxify the free radicals generatedbyradiation.OurstudiesareinlinewiththoseofSisodia et al. (31) who also showed theaugmentation of lipid peroxidation andreductioninGSHcontentinmicecerebrumafterradiation exposure. The increased GSH levelsuggests that protection by PAE may bemediated through the modulation of cellularantioxidantlevels.In‐vitroradioprotectingabilityofPAEwasalso

evaluated in terms of inhibition of radiationinducedproteincarbonylationbyDNPHmethod.igure7showsthattheformationofγradiation(60Gy) induced protein carbonylation in BSAdecreased as concentration of PAE increases


Figure 7. Inhibi on of γ radia on (60Gy) induced protein carbonyla on in BSA in presence of different concentra ons

(10‐ 50μg/ml) of PAE (methanolic extract of Prunus avium fruits).



Mr.Umang Singh Radiation& PhotochemistryDivision, Bhabha Atomic Research Centre,Trombay,Mumbai for their valuable suggestionsduring the courseof this study.Authorsarealsothankful to Dr. A. Chougle, Radiotherapy Unit,SMSMedicalCollegeandHospital, Jaipur (India)for providing irradiation facility and for help inradiationdosimetry.

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demonstrate that PAE pre/ post treatmentprotects the mice brain against radiationinduced damage by inhibiting the lipidperoxidation levels and ameliorating theglutathione and protein depletion. PAE alsoshows the free radical scavenging activity invariousin‐vitrostudies,whichmayberesponsiblefortheradioprotectingabilityofPAEnoticed.

CONCLUSIONSResults obtained from the present study

indicate that the constituents found in PAEsubstantially protect the brain from radiationinduced oxidative stress. Free radicalscavenging property of PAE, noticed in thepresent study, may be one of the mechanisticaspect responsible for its radioprotectiveef icacy.

ACKNOWLEDGEMENT

Authors humbly acknowledge Dr. K. I.Priyadarsini, Mr. Amit Kunwar and Late

Figure 8. Graph showing protec on of protein content in mice brain a er 5 Gy gamma irradia on by prior/ post administra on of PAE (methanolic extract of Prunus avium fruits). The protein level was measured in terms of mg/g ssue by Bradford method.

Following groups were compared by student’s t‐test‐ a: Control v/s PAE treated b: Control v/s Irradiated, c: Irradiated vs PAE treated + Irradiated, d: Irradiated vs Irradiated + PAE treated. Symbols for p values of t‐test $<0.05,*<0.01, **<0.005, ***<0.001,

n= non significant.


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