THE PROTECTIVE EFFECT OF BEETROOT AND CARROT JUICES ON DOXORUBICIN-INDUCED CARDIAC INJURY AND ASSOCIATED INFLAMMATORY RESPONSE

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1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Amir et l. World Journl of Phrmy nd Phrmeutil Sienes SJIF Impt Ftor Volume 6, Issue 12, Reserh Artile ISSN THE PROTECTIVE EFFECT OF BEETROOT AND CARROT JUICES ON DOXORUBICIN-INDUCED CARDIAC INJURY AND ASSOCIATED INFLAMMATORY RESPONSE Shim El-Msry nd Amir Ad El-Rhmn* Deprtment of Biohemistry nd Nutrition, Fulty of Women for Arts, Siene nd Edution A in Shms University. Artile Reeived on 08 Otoer 2017, Revised on 28 Otoer 2017, Aepted on 18 Nov DOI: /wjpps *Corresponding Author Dr. Amir Ad El-Rhmn Deprtment of Biohemistry nd Nutrition, Fulty of Women for Arts, Siene nd Edution A in Shms University. ABSTRACT Doxoruiin (Dox), hs n importne s ntitumor ntiioti, ut it s using is limited y the development of rdiomyopthy. Free rdils re involved in ute doxoruiin-indued toxiity nd inflmmtory ondition in rdi tissue. The study imed to determine the protetive effet of eetroot nd/or rrot juies in ute Dox indued rdi injury in dult mle rts. Animls were rndomly seprted into 5 groups, G1ontrol reeived norml sline I.P, G2 reeived single dose of Dox 20 mg/kg I.P, the other three groups lso reeived doxoruiin 20 mg/kg I.P s single dose fter four weeks umultive doses of eetroot juie (10 mg/kg), rrot juie (10 mg/kg) nd mixture of equl volumes of two juies respetively. Serum CK-MB, LDH, C- RP, Homoysteine nd rdi troponin-i were exmined y ELIZA. Hert tissue histopthology, DNA dmge of rdi ell nd oxidtive stress nd inflmmtory mrkers were determined in ll rts fter 48hrs from doxoruiin injetion. Doxoruiin indued rdi injury ws evidened y signifint elevtion in serum mrkers of hert funtion (CK-MB, LDH, Homoysteine nd rdi troponin-i), oxidtive stress nd inflmmtory mrkers (MDA, CAT, IL-6 nd CRP). Also rdi ell s DNA dmge ws indited y signifint hnges in omet ssy prmeters. All these results ws onfirmed y hert tissue histopthology. While, dministrtion of eetroot nd rrot juies protet hert tissue from dmge y oxidtive nd inflmmtory ondition. Moreover mixing of two juies ws more effetive. 109

2 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes KEYWORDS: Doxoruiin, eetroot, rrot, oxidtive stress, inflmmtory response, DNA dmge, CK-MB, LDH, C-RP, Homoysteine nd rdi troponin-i. 1. INTRODUCTION Doxoruiin (Dox) is potent nthryline ntiioti tht hs een used s nti-ner for mny yers. It used in tretment tumors suh s heptolstom., one sroms, lymphom, Wilm s tumor, soft tissue, neurolstom nd leukemi (El-Syed et l., 2011). There is evidene suggest tht oxidtive stress with inresed genertion of Retive Oxygen Speies (ROS) medites Dox-indued rdiotoxiity. Prodution of ROS fter Dox tretment my results due to lipid peroxidtion, with inresing mlondildehyde (MDA) levels. Dox injetion lso hve een implited in the inidene of rdiotoxi effets using redution of ntioxidnt enzyme levels (Xin et l., 2011). Bet vulgris rur, lso known s red eetroot, is potentil her used in rdiovsulr onditions, the juie of eetroot is lso used s nturl therpy to expel ldder nd kidney stones (Shrm et l., 2011). It ws lso reported tht Bet vulgris extrts possess ntihypertensive, hypoglyemi, ntioxidnt (Ninfli nd Angelino, 2013), ntiinflmmtory (Jn nd Shrm, 2011) nd heptoprotetive tivities (El Gml et l., 2014). Beetroot is mong the highest nitrte-umulting vegetles (Hos et l., 2012). Nitrte (NO3 ) nd nitrite (NO2 ), present in eetroot nd in other food soures, were relted to rdiovsulr enefits (El-Ri et l., 2016). The limited NO iovilility is the min mehnism involved in endothelil dysfuntion, whih is ruil for the development of CVD (Ci nd Hrrison, 2000). Beetroot is known to e powerful ntioxidnt. Betlin pigments (etynins nd etxnthins) hve speifilly een shown to possess vrious ntioxidnt funtions. Beets lso ontin smll mounts of lium, Vitmin A, Vitmin C, potssium, mgnesium, opper, folte, phosphorus nd iron (Wettsinghe et l., 2002). Crrot (Duus rot L.) is root vegetle from Apiee fmily. Crrots were used s food fter long term use for medil purposes (Simon, 2000). It is rih with flvonoids, rotenoids, polyetylenes, minerls nd vitmins whih re of gret nutritionl nd helth enefits nd t s, ntirinogens, ntioxidnts nd immune-enhners (Niolle et l., 2003). Other enefits of rrots hd een reported inluding, nti-inflmmtory, holesterol lowering, Anti-dieti, nti-hypertensive, heptoprotetive nd wound heling enefits (Silv Dis, 2014). Redution of oxidtive DNA dmge ws oserved following orl intke of rrot juie (Olshnsky et l., 2005). 110

3 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes Therefore, the present study ws onduted to evlute the protetive effets of eetroot nd/or rrot juies on rdi injury nd ssoited inflmmtory response indued y doxoruiin in rts. 2. MATERIALS AND METHODS 2.1. Plnt mterils Beetroot nd rrot were purhsed from the lol mrket, El-Slheyi, Ciro, Egypt. The juies were prepred from hopped eetroots nd rrot y household juie extrtor. The mixture of two juies ws prepred y mixing equl volumes of two juies Chemils Doxoruiin drug ws otined from El-Ezy Phrmy, Ciro, Egypt Animls Forty eight dult mle lino rts Sprgue Dwely weighed g, kept in wellventilted niml house of the Medil Reserh Center, Fulty of Mediine, Ain Shms University. Rts hd een kept in the room for 1 week prior to the eginning of the experiment for limtiztion. They hd ess to 12h yle of light/drk nd provided with stndrd diet prepred y AIN (1993) nd tp wter d liitum Experimentl design Rts were divided into five groups 8 rts / group exept for group (2) ontin 16 rts euse of elevted mortlity used y doxoruiin. Group 1: (ontrol group) rts were injeted intrperitonely (i.p) y norml sline. Group 2: (Dox. group) rts were injeted i.p y 20 mg/kg Dox s single dose ording to Hdi, et l., (2012) Group 3: (Dox.+ BJ) reeived eetroot juie (10 ml/kg ody weight/dy) ording to Reh (2015) orlly using orl gvge for four weeks followed y doxoruiin injetion (20 mg/kg). Group 4: (Dox.+CJ) reeived rrot juie (10 ml/kg ody weight/dy) ording to Potter et l., (2011) orlly using orl gvge for four weeks followed y doxoruiin injetion (20 mg/kg). Group 5: (Dox.+BJ+CJ) reeived mixture of equl volumes of eetroot nd rrot juies orlly using orl gvge for four weeks followed y intrperitonil injetion of Dox (20 mg/kg) s single dose. 111

4 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes 2.5. Smple olletion At the end of experimentl period (4weeks), ll nimls were srified, lood smples were olleted from hepti portl vein. After lood lotting, serum ws seprted y entrifugtion t 3000 r.p.m. for 10 min t 4 C nd kept for iohemil nlysis. Herts were removed, wshed with sline solution, dried nd weighed for its reltive weight. Then hert tissue smples were tken for histopthologil exmintion, determintion of DNA dmge in hert ell y omet ssy nd iohemil estimtions Determintion of Antioxidnt Content in Beetroot nd rrot juies Totl phenoli ontent ws determined ording to the Folin-Ciolteu proedure nd expressed s mg of glli id equivlent (GAE) per g of smple. Totl flvonoid ontent ws determined ording to Zili et l., (2012) nd expressed s mg of tehin equivlent (CE) per g of smple. Determintion of rdil DPPH svenging tivity (Antioxidnt tivity) ws determined using the stle 1, 1-Diphenyl-2-piryl-hydrzyl (DPPH) ording to Hwng nd Do Thi., (2014) Determintion of DNA dmge in hert tissue 0.5 g of rushed smples were trnsferred to 1 ml ie-old PBS., stirred for 5 min then filtered. Cell suspension ws mixed with low-melting grose (0.8% in PBS). 100 μl of this mixture ws spred on pre-oted slides. The oted slides were immersed in lyses uffer (0.045 M TBE, ph 8.4, ontining 2.5% SDS) for 15 min. The slides were pled in eletrophoresis hmer ontining the sme TBE uffer, ut devoid of SDS. The omets tils lengths were mesured from the middle of the nuleus to the end of the til with 40x inrese for the ount nd mesure the size of the omet. For visuliztion of DNA dmge, oservtions re mde of EtBr-stined DNA using 40x ojetive on fluoresent mirosope ording to Singh et l., (1988) Determintion of oxidtive stress nd inflmmtory iomrkers Crdi Ctlse Ativity ws estimted y using Colorimetri/Fluorometri Assy Kit No.: #K Lipid Peroxidtion yprodut, (MDA) ws estimted in hert tissue using Colorimetri/Fluorometri Assy Kit No.: # K Interleukin-1α (IL-1α) ws estimted s pro-inflmmtory mrker using ELISA Kit No.: ERIL1A. Serum C - retive protein [CRP] ws estimted y using ELISA Kit No.:

5 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes 2.9. Determintion of serum hert funtion iomrkers Estimtion of serum Cretine Kinse MB (CK-MB) ws performed y using ELISA Kit No.: ER0841, serum L-Ltte dehydrogense (L-LDH) y using ELISA Kit No. : CSB-E11324r, rdi troponin-i in serum using ELISA Kit No. KT-478 nd serum Homoysteine (Hy) using ELISA Kit No.: CSB-E13376r Histopthologil Exmintion of hert Crdi tissues were immersed in formlin solution (10%), dehydrted in lohol nd emedded in prffin. Setions of 5 μm were otined, deprffinized then stined with hemtoxylin nd eosin (H nd E) then exmined y stndrd light mirosopy (Bnroft et l., 1996) Sttistil nlysis Results were expressed s men ± Stndrd devition (S.D.) of the men. Differenes mong mens were tested for sttistil signifine y one-wy nlysis of vrine using SPSS pkge version 19. Sttistil signifine ws onsidered when P < RESULTS Tle (1) shows the ontent of totl phenols, totl flvonoids nd the ntioxidnt pity of red eetroot nd rrot. The hemil nlysis reveled tht red eetroot hd higher mount of totl phenols nd totl flvonoids ompred with rrot. The DPPH whih mesure the reltive ntioxidnt ilities of the two smples to svenge free rdils generted in the ssy system demonstrted tht eetroot hd higher ntioxidnt tivity ompred with rrot. The results in tle (2) indited tht hert weight nd reltive hert weight were signifintly (p<0.05) deresed in doxoruiin treted rts tht reorded (0.625±0.046) nd (0.282±0.001) ompred to ontrol tht reorded (0.712±0.099) nd (0.380± 0.001) respetively. Administrtion of eetroot or (eetroot + rrot) juies efore doxoruiin injetion used signifint inrese in hert weight nd reltive hert weight when nd their reorded vlues were (0.750±0.092), (0.367±0.008), (0.737±0.074), (0.355±0.001) respetively. On the other hnd, rrot juie dministrtion efore doxoruiin injetion showed non-signifint hnge t (p<0.05) on the hert weight nd reltive hert weight lthough its mens were notiely higher thn in the doxoruiin group. Doxoruiin injetion indued 30% mortlity in rts, 113

6 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes this ws mrkedly deresed to 10% in rts dministered rrot juie efore injetion nd olished in rts treted with eetroot or (eetroot + rrot) juies efore injetion. Comet ssy is sensitive method for the detetion of DNA dmge. Dox injetion use dmge in rdiomyoyte DNA the perent of dmge (Tiled DNA%) rehed (36.812±0.60) in Dox group ompred to ontrol (4.962±0.64). Moreover Dox injetion led to inrese the levels of omet prmeters (% til DNA, til length nd til moment) nd the perentges of inrese were (208.27%, % nd %) ompred to the ontrol group tle (3). Beetroot nd /or rrot juies pretretment signifintly redued the Doxindued rdiomyoyte DNA dmge s shown from % of tiled DNA (12.775±0.28), (14.850±0.23) nd ( ±0.75) respetively when ompred with (36.812±0.60) in Dox group. Also it ws notied tht there ws signifint improvement in omet prmeters in groups pretreted with BJ nd/or CJ when ompred with Dox group. The results tulted in tle (3) were onfirmed y figure (1) tht showed pperne nd perentges of dmged DNA (tiled DNA) in ll groups. The dt in Tle (4) represent the effet of eetroot nd/or rrot juies on Dox-indued hnges in rdi oxidtive sttus nd inflmmtion mrkers. The results demonstrted tht Dox injetion extremely inresed mlondildehyde level in hert tissue s mrker for lipid peroxidtion ompring to ontrol group, the elevtion of MDA ws from ( ±0.090) to ( ±0.084) (P<0.05) nd the perentge of inrement in MDA ws (203.13%). Pretretment with eetroot nd/or rrot juies uses signifint redution MDA level, with perentges (160.73%, % nd %) respetively ompred to (203.13%) with doxoruiin lone. There ws mrked redution in tlse CAT tivity Dox-injeted rts (9.088±0.089) ompred to ontrol (14.685±0.035). And the perentge of derement ws (61.88%) when ompred to ontrol. While pretretment with eetroot nd/or rrot juies resulted in signifint inrese in rdi CAT enzyme tivity the perentges of hnge in CAT tivity were (87.44%, 83.74% nd 88.11%) respetively ompred to (61.88%) with doxoruiin lone. These results indite tht eetroot nd rrot down regulte the level of rdi MDA nd up regulte the rdi CAT tivity so they hve ntioxidnt tivity. From the results in tle (4) it ws lso notied tht Dox injetion produed signifint inrese in the rdi inflmmtory iomrker IL-1α ompred to ontrol rts nd the perentge of inrement ws % ompred to ontrol. However, the hnge in IL-1α 114

7 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes levels were signifintly deresed in eetroot nd/or rrot treted groups nd men vlues rehed (6.885±0.057), (6.495±0.037) nd (6.278±0.033) respetively ompred to Dox group men vlue (11.231±0.039). Serum C-retive protein mrker of ute phse of inflmmtion showed mrked inrese in Dox injeted group (805.58±0.12) ompred to ontrol (398.38±0.07). While the pretretment with BJ, CJ nd BJ+CJ modulte this inrement to reh (428.09±0.04), (433.57±0.06) nd (405.28±0.04) respetively. These results indite tht eetroot nd rrot down regulte levels of the rdi IL-1α nd serum CRP so they hve nti-inflmmtory tivity. Moreover, we n notie tht the ntioxidnt nd nti-inflmmtory effets of eetroot preeded the effets of rrot while their omintion together ws the est. Prmeters of hert funtion re listed in tle (5), inluded serum (LDH, CK-MB, Troponin- 1 nd Homoysteine). From the results we n oserve tht Dox injetion used signifint inrese of serum LDH level (2.321±0.030) to (4.627±0.056) with % of rise ompred to seline vlue (P< 0.05). While the pretretment with BJ, CJ nd BJ+CJ modulted the inresed serum LDH levels indued y Dox with rise perentge %, % nd % respetively. We n oserve signifint inrese of serum CK-MB level from (11.493±0.048) in ontrol to ( ±0.040) in Dox injeted group with perentge of rise % ompred to seline vlue. While, pretretment with BJ, CJ nd BJ+CJ redued the inresed CK-MB level. In present study we notied signifint inrese in the group injeted with doxoruiin from (0.286±0.026) to (0.608±0.036), s shown in tle (5). While the orl dministrtion of eetroot nd/or rrot juies signifintly redued the level of serum troponin- I p<0.05 to pproh the norml level espeilly in group treted with (BJ+CJ) the men vlues were (0.311±0.023), (0.372±0.017) nd (0.292±0.024) respetively. Serum homoysteine level reorded signifint inrese in Dox injeted group when ompred to ontrol the perentge of inrese ws % ompred to seline vlue (P<0.05). While pretretment with eetroot nd/or rrot juies improved this inrement of serum homoysteine signifintly the perentges of rise were (187.27%, % nd %) ompred to the perentge of Dox lone %. From the results of hert funtion prmeters we n notie tht pretretment with two juies together hd the est effet on enhnement of hert funtion. 115

8 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes Tle (1): Totl phenols, totl flvonoids nd ntioxidnt tivity ontent of red eetroot nd rrot. Prmeters Totl Phenols (GAE mg/100g) Totl Flvonoids (CE mg/100g) DPPH (AAE mg/100g) Smples Red Beetroot Crrot GAE: Glli id equivlent, CE: Cthin equivlent, AAE: Asori id equivlent. Tle (2): Effet of eetroot nd/or rrot juies on doxoruiin-indued hnges in reltive ody weight, hert weight nd perent of mortlity ompred with ontrol group. Prmeters Reltive hert Perent of Hert weight Groups weight mortlity (g) (%) (%) Control ± ±0.001 Doxoruiin ± ±0.001 Dox.+ B.J ± ±0.008 Dox. +C.J ± ±0.001 Dox.+B.J.+C.J ± ±0.001 Vlues re represented s men ± SD, There ws no signifint differene etween mens hve the sme letter in the sme olumn (P<0.05). Tle (3): Effet of eetroot nd/or rrot juies on doxoruiin-indued hnges in DNA dmge mrkers (omet ssy) in hert tissue ompred with ontrol group. Prmeters Groups Tiled (%) Untiled (%) Til DNA (%) Til Length (µm) Til Moment (Units) Control d 4.962± ± ± ± ±0.006 Doxoruiin d d ± ± ± ± ±0.003 Dox.+ B.J.,, ± ± ± ± ±0.003 Dox. +C.J ± ± ± ± ±0.003 Dox.+B.J.+C.J ± ± ± ± ±0.004 Vlues re represented s men ± SD, There ws no signifint differene etween mens hve the sme letter in the sme olumn (P<0.05). 116

9 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes Tle (4): Effet of eetroot nd/or rrot juies on doxoruiin-indued hnges on oxidtive stress nd inflmmtion mrkers in hert tissue nd serum ompred with ontrol group. Prmeters MDA nmol/mg CAT mu/mg IL-1 α pg/mg Serum CRP ng/ml Groups Control ± ± ± ±0.07 Doxoruiin d ± ± ± ±0.12 Dox.+ B.J ± ± ± ±0.04 Dox. +C.J ± ± ± ±0.06 Dox.+B.J.+C.J., ± ± ± ±0.04 Vlues re represented s men ± SD, There ws no signifint differene etween mens hve the sme letter in the sme olumn (P<0.05). Tle (5): Effet of eetroot nd/or rrot juies on doxoruiin-indued hnges in serum mrkers for hert funtion ompred with ontrol group. Prmeters LDH mu/ml CKMB pg/ml TROP-I ng/ml HOMOCYST nmol/ml Groups Control 2.321± ± ± ±0.041 Doxoruiin d 4.627± ± ± ±0.410 Dox.+ B.J., 3.212± ± ± ±0.058 Dox. +C.J ± ± ± ±0.057 Dox.+B.J.+C.J ± ± ± ±0.084 Vlues re represented s men ± SD, There ws no signifint differene etween mens hve the sme letter in the sme olumn (P<0.05). Histopthologil exmintion of hert tissue The histopthologil exmintion reveled tht rdi tissues of ontrol group rts showed norml rdi myoytes figure (2), menwhile, hert of rts injeted with doxoruiin showed dilttion nd ongestion of myordil lood vessel, mononuler ells infiltrtion etween rdi myoytes nd fol nerosis of rdi myoytes figure (3). Menwhile, pretretment with eetroot juie signifintly mintined the norml histologil pperne 117

10 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes of myordium exept ongestion of myordil lood vessel in some setions figure(4). Also, some exmined setions from rrot juie pretreted rts showed ongestion of myordil lood vessels wheres, other setions from this group reveled no histopthologil hnges figure (5). The est histopthologil results were lerly showed in rts whih pretreted with oth eetroot nd rrot juies together, this ws indited in figure (6) whih reveled no histopthologil hnges. Fig. (1): The omets tils lengths were mesured from the middle of the nuleus to the end of the til with 40x inrese for the ount nd mesure the size of the omet. Fig. (2): Hert of rt from group 1 showing norml rdi myoytes (H & E X 400). Fig. (3): Hert of rt from group 2 showing dilttion nd ongestion of myordil lood vessel, mononuler ells infiltrtion etween rdi myoytes nd fol nerosis of rdi myoytes (H & E X 400). 118

11 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes Fig. (4): Hert of rt from group 3 showing redued Dox-indued degenertive hnges in myordil lood vessels nd mintined norml histologil pperne of myordium (H & E X 400). Fig. (5): Hert of rt from group 4 showing redution in ongestion of myordil lood vessels nd fewer mononuler ells infiltrtion etween rdi myoytes (H & E X 400). Fig. (6): Hert of rt from group 5 showing no histopthologil hnges nd mintined norml histologil pperne of myordium (H & E X 400). 3. DISCUSSION From the results in tle (1) it is ler tht smples of red eetroot nd rrot hve effiient mounts of totl phenols nd totl flvonoids, eetroots hve higher mount of totl phenols 119

12 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes nd totl flvonoids followed y rrot. The DPPH ssy, tht mesure the ntioxidnt ilities of eetroot nd rrot to svenge free rdils showed tht red eetroots hve good ntioxidnt properties nd these results were in greement with Váli et l. (2007) who mentioned tht eetroots ontin importnt iotive gents (etins nd polyphenols), whih hve wide rnge of physiologi effets. Pl et l., (2010) reported the presene of flvonoids, rohydrte, etins nd nthoynin pigments in, Bet vulgris (eetroot). In the phytohemil studies the ntioxidnt tivity determined y the DPPH method exhiited 40% nd 78% tivity in methnol extrts of rrot nd eetroot respetively (Shyml nd Jmun, 2010). So, the results from hemil nlysis suggest tht red eetroot nd rrot n e exploited for their ntioxidnt omponents nd used for vlue ddition in food formultions. Dox is known effetive hemotherpeuti gent used ginst different types of ners. However, it hd limited use due to its toxi effet on rdi musles. )Kontny et l., 2013). Our results hve shown Dox injeted rts possess inresed mortlity, 30% of the nimls died. Crdiomyopthy mke the most importnt ontriution to the mortlity. An oserved redution in hert/ody weight rtio indited severe rdi performne dysfuntion (El- Syed et l., 2011). While deresing the perentge of mortlity in group tht onsumed rrot juie to 10% onfirms the rdio-protetive effets of this juie (Potter t l., 2011). Moreover disppering of mortlity (0%) in groups onsumed eetroot juie nd eetroot with rrot juies onfirms the etter effet of eetroot if lone or ompound with rrot juie )Hos et l., 2012). The oxidtive dmge of rdiomyoyte DNA my led to of rdiotoxiity nd deth) Clifford et l., 2015). In the present study, the Comet prmeters (til DNA, til extent moment nd til length) in rdi ell were distured in Dox group rts ompred with ontrol rts. While in groups tht pretreted with eetroot juie nd /or rrot juie omet prmeters showed signifint enhnements when ompred with Dox group. Our results greed with the results of Lee et l., (2009) who exmined the effet of red eetroot on oxidtive dmge of DNA nd they found n effetive redution in lymphoytes dmged DNA mnifested s signifint derese in Comet prmeters (p<0.05). Moreover, Reh, (2015) onfirmed tht vitmin C nd E were strong ntioxidnts used to redue the rdiotoxiity (Ayz et l., 2004). Our results were in greement with studies showing reltion etween ntioxidnt effets of plnts nd rdi injury) Erog et l., 2016). In 120

13 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes previous reports it ws onfirmed tht eetroot hs eet pigments known s etlins these pigment re of higher ntioxidnt power )Lee et l., 2005). These results go hnd in hnd with our results in determintion of ntioxidnt (DPPH) tht showed potent ntioxidnt tivity, whih further supports its rdil svenging tivity. The oserved ntioxidnt nd rdio protetive effet is onsidered to e relted to the phyto-onstituents of eetroot) frnk et l., 2005). They lso exert nti-rinogeni tivities, redue inflmmtory insult, nd modulte immune response) Silv Dis, 2014). The oxidtive stress prmeters nd inflmmtory mrkers tested in our study (MDA, CAT. IL1α nd CRP) were ffeted y Dox-injetion tht ssoited with prodution of ROS. Dox produe free rdil y two different wys, the first is the formtion of semiquinone free rdil whih gives superoxide rdils (Singl et l., 2000) nd the seond wy produes Fe 2+ Dox omplex (DeBeer et l., 2001) tht n redue oxygen to H2O2 nd other tive speies (Shinell et l., 2002). In our study, elevted MDA levels ws oserved fter Dox tretment ut the pretretment with eetroot nd rrot juies either single or ompound together ttenuted lipid peroxidtion evoked y doxoruiin in hert of rts nd led to deresed MDA level. The inresed MDA levels n e explin the ft tht, hydroxyl rdils (OH ) gretly enhnes the NADHdependent mirosoml lipid peroxidtion nd thus led to oxidtive dmge to ell memrnes. MDA ws signifintly redued y the effet of BJ nd CJ this n e explined y tht ntioxidnt in these juies llows free rdils to ttrt hydrogen tom from the ntioxidnt rther thn ttking from polyunsturted ftty ids, this will rek the free rdil retions (Hdi et l., 2012). Erog et l., (2016) indited the redued tivity of CAT in Dox group. A study y Suurmn et l., (2014) exmined the nringen protetive effet of ginst Dox-indued rdiotoxiity, Dox injetion redued CAT tivity. Similr redution in CAT tivity ws oserved in the study y Alpsoy et l., (2013) in whih Dox ws dministered t dose of 30 mg/kg. In line with previous reports, Dox ws ssoited with redued levels of CAT, while dministrtion of BJ, CJ were ssoited inresed levels of CAT. Moreover their omintion together hd the est effet. Elevtion of rdi IL-1α in Dox injeted rts is n inditor of ellulr inflmmtion sine IL-1α is produed minly y tivted mrophges, s well s neutrophils, epithelil ells nd endothelil ells. Serum CRP level inresed signifintly in Dox group tht reflet the inflmmtory ondition our y Dox. While this inflmmtion improved in other groups due to pretretment with nturl juies this results greed with Potter et l., (2011) who noted tht the derese in the C- retive protein, s n index for inflmmtion my hve een relted to drinking rrot juie whih is good soure of -rotenes nd α-rotenes. 121

14 Amir et l. World Journl of Phrmy nd Phrmeutil Sienes Hert tissue were very sensitive to ROS indued dmge this my due to its fewer ntioxidnt defenses ompred to other orgns this explin the inrese of LDH nd CK onentrtion in the serum of nimls injeted with doxoruiin. The LDH nd CK mehnism for relese seems to e from oxidtive dmge of Dox to rdi tissue nd the susequent relese of its ontents into irultion (Rskovi et l., (2011). While normliztion of CK-MB nd LDH elevted levels in groups onsumed eetroot juie nd rrot juie onfirms the rdioprotetive effets of these juies. These results re on the sme line with the previous studies reported y Ngi nd Mnsour, (2000) tht demonstrte signifint inrese in serum enzymes, LDH nd CK fter Dox injetion due rdi dmge y doxoruiin whih s result of of nulei id inhiition nd protein synthesis (Hdi et l., 2012). Serum troponin I results of the urrent were oinided with the work of Hdi et l., (2012) who reported mrked mrophge umultion with dysfuntion of rdi endothelium. Serum homoysteine showed mrked inrese in Dox injeted rts. Hrdening of rteries due to the umultion of ftty plques my our due to high levels of homoysteine. Vegetles s eetroots nd rrots ontin vitmins mong them is pyridoxine (B6) whih is neessry for rohydrte, ft nd protein metolism. Pyridoxine nd folte inhiits the of homoysteine formtion nd redues the hert disese risk (Silv Dis, 2014). Histologil exmintion y light mirosopy n prove doxoruiin toxiity, ut it n only revel gross ellulr hnges. By using eletroni mirosopy more ommon ultr-struturl hnges suh s srotuulr edem nd disorders of the fine mitohondril struture n e found (Rskovi et l., 2011). In our study the struturl nd funtionl hnges, demonstrted mirosopilly in the hert tissue of doxoruiin intoxited rts, showed dilttion nd ongestion of myordil lood vessel, mononuler ells infiltrtion etween rdi myoytes nd fol nerosis of rdi myoytes ssoited with mononuler ells infiltrtion. These results go hnd in hnd with inresed serum levels of troponin I, homoysteine nd inresed ROS ondition tht were oserved in Dox group. While pretretment with eetroot nd /or rrot juies signifintly redued the Dox-indued histopthologil hnges in the rdi tissue nd mintined the norml histologil pperne of myordium this effet my e due to the highly ntioxidnt tivities of these nturl juies )Inj et l., 2009). 122

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