PREVENTIVE EFFECT OF BIO-AQ ON CARDIAC MARKERS, LIPIDS, AND MEMBRANE BOUND ENZYMES IN ISOPROTERENOL - INDUCED MYOCARDIAL INFARCTION IN RATS

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1 Aemi Sienes Asin Journl of Phrmeutil n Clinil Reserh Vol 5, Suppl 2, 212 ISSN Reserh Artile PREVENTIVE EFFECT OF BIO-AQ ON CARDIAC MARKERS, LIPIDS, AND MEMBRANE BOUND ENZYMES IN ISOPROTERENOL - INDUCED MYOCARDIAL INFARCTION IN RATS ABSTRACT S.T. SHARMILA, M. RAJADURAI* Deprtment of Biohemistry, Muthymml College of Arts & Siene, Rsipurm , Tmil Nu, Ini. Emil: rjuripi@gmil.om Reeive:23 Jnury 212, Revise n Aepte:22 April 212 Bio-Aq, is n Inin Ayurvei herominerl preprtion of selete ingreients, whih provies signifint protetion ginst ishemi n hypertension. However, there is lk of informtion regring the effet of Bio-Aq on the ri hnges ssoite with isoproterenol (ISO)- inue myoril infrtion (MI). Ojetives: This mnusript reports the preventive effet of Bio-Aq on lipi profile, ri mrker enzymes n memrne oun enzymes in ISOinue riotoxi mle Wistr rts. Mterils n Methos: Bio-Aq (25 n 5 mg/kg) ws issolve in imethyl sulfoxie (DMSO) ministere orlly s pretretment to Wistr rts ily for 28 ys. After pretretment, rts were inue with suutneous injetion of ISO (85 mg/kg) t n intervl of 24 h for two ys to proue MI. Vrious iohemil mrkers of loo n tissue origin were estimte. Results: ISO-inue rts showe signifint inrese in the tivities of mrker enzymes in serum n there y susequent erese in the hert. ISO-inue rts lso showe signifint ltertions in the tivities of memrne oun ATPse in the hert of ISO-inue rts. In ition, inrese in the levels of lipis in serum n hert. A rise in the levels of phospholipis (PL), LDL n VLDL n erese HDL were lso oserve in the serum n erese in the level of high ensity lipoprotein-holesterol (HDL) in serum n PL levels, in the hert of ISO-inue rts ws oserve. Pretretment with Bio-Aq (25 n 5 mg/kg) positively ltere the tivities of mrker enzymes n other iohemil prmeters in ISO-inue rts. Conlusion: The present stuy onlue tht Bio-Aq posses rioprotetive effet in ISO-inue MI in rts. Keywors: Bio-Aq, ri mrkers, isoproterenol, lipis, lipoproteins, memrne oun enzymes, myoril infrtion. INTRODUCTION In the 21st entury, there is n ongoing effort to integrte omplementry n lterntive meitions into the prtie of onventionl meiine, for the tretment of riovsulr iseses (CVD) suh s ishemi hert iseses (IHD). Epiemiologil stuies inite tht IHD, espeilly myoril infrtion (MI), will onstitute the mjor isese-uren worlwie in the yer Myoril ishemi ours, when myoril oxygen emn exees oxygen supply n s result it uses ell injury known s MI, whih is one of the most lethl mnifesttions of CVD 2. Isoproterenol (ISO), syntheti teholmine n β-renergi gonist, hs een reporte to use severe stress in the myorium resulting in infrt like nerosis of the hert musle 3. The inution of MI in experimentl nimls y ISO is proly ue to its tion on the srolemml memrne, stimultion of enylte ylse, tivtion of N + n C 2+ hnnels, exggerte C 2+ inflow n energy onsumption leing to ellulr eth 4. ISO-inue MI serves s well-stnrize moel to stuy the vrious effets of mny rugs n hert funtion 5, 6. Bio-Aq is herl meiine s well s herl foo enrihe with protein, terpenois, flvonois, tnnin, vitmins n minrls. It is only herl rug whih ts on the enorine system. Bio-Aq stimultes the pnres to proue more insulin n reue the high loo gluose level. Bio-Aq prevents ute mnifesttions of hyperglyemi n llevites the symptoms of exessive thirst, frequent urintion, weight loss, ietes ketoios n long term omplitions suh s retinopthy, neuropthy, nephropthy, mro vsulr iseses n reues yslipiemi. Sine oxitive stress hs een implite in the evelopment of CVD n Bio-Aq plys n importnt protetive role ginst oxitive mge, we evlute the preventive effet of Bio-Aq on ri mrkers, lipis n memrne oun enzymes in norml n ISO-trete MI in mle lino Wistr rts. MATERIALS AND METHODS Experimentl nimls All the experiments were rrie out with mle lino Wistr rts weighing g, were otine from Venkteshwr enterprises, Bnglore, Ini. They were house in polypropylene ges (47 m 34 m 2 m) line with husk, renewe every 24 h uner 12:12 h light rk yle t roun 22 C n h free ess to tp wter n foo. The rts were fe on stnr pellet iet (Prnv Agro Inustries Lt., Mhrshtr, Ini). The pellet iet onsiste of 22.2% rue protein, 4.25% rue oil, 3.25% rue fire, 7.5% sh, 1.38% sn sili,.8% lium,.6% phosphorus, 2.46% gluose, 1.8% vitmins n 56.17% nitrogen-free extrt (rohyrtes). The iet provie metolisle energy of 36 kl. The experiment ws rrie out oring to the guielines of the Committee for the Purpose of Control n Supervision of Experiments on Animls (CPCSEA), New Delhi, Ini. Drugs n hemils ( ) Isoproterenol hyrohlorie ws purhse from Sigm Chemil Compny, St. Louis, MO, USA. Bio-Aq ws purhse from Agsthir sih Dietes Reserh Centre, Ngeroil, Tmilnu, Ini. Cretine kinse-mb (CK-MB), retin kinse (CK), Ltte ehyrogense (LDH), sprtte trnsminse (AST), lnine trnsminse (ALT), ltte ehyrogense (LDH), triglyeries (TG) n HDL holesterol kits were purhse from Agppe Dignostis, Kerl, Ini. ATP ws purhse from Himei lortory, Mumi, Ini. All other hemils use in the stuy were of nlytil gre. Inution of experimentl myoril infrtion Isoproterenol (85 mg/kg) issolve in norml sline ws injete suutneously to rts t n intervl of 24 h for two ys to inue experimentl MI 7. Experimentl esign A totl of 3 rts ivie in to 5 groups of 6 rts in eh. Bio-Aq ws issolve in.2% imethyl sulfoxie (DMSO) n ministere to rts orlly using n intrgstri tue ily for perio of 28 ys. Group 1: Norml ontrol rts Group 2: Norml + Bio-Aq (5 mg/kg) Group 3: ISO-ontrol rts (85 mg/kg) Group 4: Bio-Aq (25 mg/kg) + ISO

2 mg IU/L Rjuri et l. Asin J Phrm Clin Res, Vol 5, Suppl 2, 212, Group 5: Bio-Aq (5 mg/kg) + ISO At the en of the experimentl perio, fter 12 h of seon ISOinjetion, ll the rts were nesthetize with soium pentoritl (35 mg/kg, i.p.) n srifie y ervil epittion. Bloo ws ollete; plsm ws seprte n use for vrious iohemil estimtions. The hert tissue ws exise immeitely from the nimls, wshe off loo with ie-hille physiologil sline n use for vrious iohemil estimtions. A known weight of the hert tissue ws homogenize in pproprite uffer solution. The homogente ws entrifuge n the superntnt ws use for the estimtion of vrious iohemil prmeters. BIOCHEMICAL ASSAYS Assy of ri mrker enzymes Cretine kinse n retine Kinse-MB levels were estimte y Witt n Trenelenurg (1982) 8 methos using ommeril kit (Prout No n Prout No ). Ltte ehyrogense tivities ws estimte y Wei Bhr (1975) 9 metho using ommeril kit (Prout No ). Asprtte mino trnsferse n Alnine mino trnsferse were ssye y the methos of Clim (1976) 1 n Thefel et l. (1994) 11 respetively (Prout No n 11491) were otine from Agppe Dignostis, Kerl, Ini. Estimtion of lipis n lipoprotein Lipis were extrte y the metho of Folh et l. 12 The levels of totl holesterol n triglyeries (TGs) were estimte y the methos of Zltkis 13 n Shettler n Nussel (1975) 14 metho using ommeril kit (Prout No ) otine from Agppe Dignostis, Kerl, Ini. The levels of free ftty is (FFAs) n phospholipis (PLs) in serum n hert tissue were estimte oring to the methos of Flholt et l. 15 n Zilversmit n Dvis 16 respetively. HDL levels 25 2 ) were estimte y Assmnn (1979) 17 metho using ommeril kit (Prout No. 1111). Serum lowensity lipoproteins (LDL) n very lowensity lipoproteins (VLDL) were lulte s VLDL=triglyeries/5 n LDL=totl holesterol (HDL holesterol+vldl holesterol) respetively. Assy of memrne oun enzymes The tivity of N + /K + -ATPse ws ssye oring to the proeure of Bonting (197) 18. The ontent of phosphorus lierte ws estimte s esrie y Fiske n Surow (1925) 19. The tivity of C 2+ -ATPse ws ssye oring to the metho of Hjerken n Pn (1983) 2.The tivity of Mg 2+ -ATPse ws ssye y the metho of Ohnishi et l. (1982) 21. Totl Protein levels were etermine y the metho of Lowry et l. (1951) 22. Sttistil nlysis Sttistil nlysis ws performe using one-wy nlysis of vrine (ANOVA) followe y Dunn s multiple rnge test (DMRT) using SPSS softwre pkge 9.5. Results were expresse s men ± S.D. from six rts in eh group. p vlues <.5 were onsiere s signifint. RESULTS Effet of Bio-Aq on ri mrker enzymes Fig. 1 (,), 2 (,) &3 (,) represent the effet of Bio-Aq on hert weight n ri mrker enzymes suh s (CK,CK-MB, LDH, AST n ALT) in serum n hert of norml n ISO-inue rts. Rts inue with ISO, showe signifint inrese in hert weight n the tivities of these ri mrker enzymes in serum with susequent erese in the hert, when ompre with norml rts. Pretretment with Bio-Aq (25 n 5 mg/kg) for perio of 28 ys signifintly erese the hert weight n the tivities of these mrker enzymes in serum with signifint inrese in the hert of ISO-inue rts CK-MB 5 ) Hert weight 25 Figure 1 (, ). Effet of Bio-Aq on the tivity of serum retine kinse-mb (CK-MB) n hert weight in norml n isoproterenol (ISO)- inue myoril infrtion (MI) in rts. Eh vlue is men ± S.D. for 6 rts in eh group Vlues not shring ommon supersript (-) iffer signifintly with eh other (P<.5, DMRT). ) 18

3 µmoles of pyruvte lierte/min/mg protein * Unit IU/L IU/L Rjuri et l. Asin J Phrm Clin Res, Vol 5, Suppl 2, 212, CK-serum LDH-serum ) AST-serum ALT-serum 1 Figure.2. Effet of Bio-Aq on the tivity of serum retine kinse (CK), ltte ehyrogense (LDH), sprtte trnsminse (AST) n lnine trnsminse (ALT) in norml n isoproterenol (ISO)-inue myoril infrtion (MI) in rts. Eh vlue is men ± S.D. for 6 rts in eh group Vlues not shring ommon supersript (-) iffer signifintly with eh other (P<.5, DMRT) ) CK-Hert ALT-Hert 5 ) LDH-Hert AST-Hert 2 Figure 3 (,). Effet of Bio-Aq on the tivity of hert retine kinse (CK), ltte ehyrogense (LDH), sprtte trnsminse (AST) n lnine trnsminse (ALT) in norml n isoproterenol (ISO)-inue myoril infrtion (MI) in rts. *CK tivity: µmol of phosphorus lierte/min/mg protein, *ALT: nmol of pyruvte lierte/min/mg protein. Eh vlue is men ± S.D. for 6 rts in eh group Vlues not shring ommon supersript (-) iffer signifintly with eh other (P<.5, DMRT). 19

4 mg/g wet tissue mg/l mg/g wet tissue mg/l Rjuri et l. Asin J Phrm Clin Res, Vol 5, Suppl 2, 212, ) Serum holesterol Serum PL 3 ) Hert holesterol Hert PL Figure 4 (,). Effet of Bio-Aq on the levels of serum n hert totl holesterol, n phospholipis (PL) in norml n isoproterenol (ISO)-inue myoril infrtion (MI) in rts. Eh vlue is men ± S.D. for 6 rts in eh group Vlues not shring ommon supersript (-) iffer signifintly with eh other (P<.5, DMRT). ) S. TGL S. FFA 2 ) Hert TGL 2 11

5 mg/l mg/l mg/g wet tissue Rjuri et l. Asin J Phrm Clin Res, Vol 5, Suppl 2, 212, ) Hert FFA.2 Figure 5 (,,). Effet of Bio-Aq on the levels of serum n hert triglyeries (TG), free ftty is (FFA) in norml n isoproterenol (ISO)-inue myoril infrtion (MI) in rts. Eh vlue is men ± S.D. for 6 rts in eh group Vlues not shring ommon supersript (-) iffer signifintly with eh other (P<.5, DMRT) HDL VLDL LDL 5 Figure 6 (,). Effet of Bio-Aq on the levels of serum high-ensity lipoprotein (HDL), low ensity lipoprotein (LDL) n very low ensity lipoprotein (VLDL) in norml n isoproterenol (ISO)-inue myoril infrtion (MI) in rts. Eh vlue is men ± S.D. for 6 rts in eh group Vlues not shring ommon supersript (-) iffer signifintly with eh other (P<.5, DMRT). Effet of Bio-Aq on lipoproteins Figures 6 (,) shows the levels of low ensity lipoprotein (LDL), very low ensity lipoprotein (VLDL) n high ensity lipoprotein (HDL) in serum of norml n ISO-inue rts. Rts trete with ISO, showe signifint inrese in LDL n VLDL levels with signifint erese in HDL levels when ompre to norml ontrol rts. Pretretment with Bio-Aq to ISO-inue rts showe signifintly minimize the ltertions in the lipoproteins levels. Effet of Bio-Aq on memrne oun enzymes in tissues Tle 1 illustrtes the effet of Bio-Aq on the tivities of soium potssium-epenent enosine triphosphtse (N + /K + -ATPse), lium-epenent enosine triphophtse (C 2+ -ATPse) n mgnesium-epenent enosine triphophtse (Mg 2+ -ATPse) in norml n ISO-inue rts. The tivity of N + /K + -ATPse ws erese n the tivities of C 2+ n Mg 2+ -ATPses were inrese signifintly in the hert of ISO-inue rts. Bio-Aq 111

6 Rjuri et l. Asin J Phrm Clin Res, Vol 5, Suppl 2, 212, pretretment to ISO-inue rts signifintly minimize the ltertions in the tivities of these memrne oun enzymes in myorium when ompre to ISO-lone inue rts. For ll the prmeters stuie Bio-Aq t ose of 25 n 5 mg/kg to ISO-inue rts showe signifint effets. However the higher ose (5 mg/kg) giving etter effet thn lower ose (25 mg/kg). Bio-Aq t 5 mg/kg to norml rts in t show ny signifint effet. Tle 1: Effet of Bio-Aq on the tivities of soium potssium epenent enosine triphosphtse (N + /K + -ATPse), lium epenent enosine triphosphtse (C 2+ -ATPse) n mgnesium epenent enosine triphophtse (Mg 2+ -ATPse) in the hert in norml n isoproterenol (ISO)-inue myoril infrtion (MI) in rts. N + /K + -ATPse C 2+ -ATPse Mg 2+ -ATPse Units*/mg protein Units*/mg protein Units*/mg protein Norml ontrol.61 ± ± ±.49 Norml + Bio-Aq (5 mg/kg).63 ± ± ±.48 ISO (85 mg/kg) ontrol.35 ± ± ±.7 Bio-Aq (25 mg/kg) + ISO.47 ± ± ±.62 Bio-Aq (5 mg/kg) + ISO.54 ± ± ±.51 * Ativity expresse s units: μmol of phosphorus lierte/min/mg protein Eh vlue is men S.D. for 6 rts in eh group. Vlues not shring ommon supersript (-) iffer signifintly with eh other (P<.5, DMRT). DISCUSSION Isoproterenol is well known riotoxi gent ue to its ility to estrut myoril ells. In this stuy, signifint eline ws shown in the tivities of ri mrkers suh s retine kinse (CK), ltte ehyrogense (LDH), sprtte trnsminse (AST) n lnine trnsminse (ALT) in the hert of ISO-trete rts, whih is in onsistent with erlier report 23. Derese tivities of theses enzymes in hert oul e ue to the relese of the enzyme from mge myorium into irultion y ISO. This oul e lso ue to mge of the myofiril egenertion n myoyte nerosis. Prior ministrtion of Bio-Aq (25 n 5 mg/kg) for perio of 28 ys, signifintly prevente the ISO-inue elevtion in the tivities of ignosti mrker enzymes in serum, n signifintly inrese the tivities of these mrkers in the hert. Bio-Aq on the myorium, reuing the ri mge therey restriting the lekge of these enzymes. Bio-Aq is omplex herl preprtion n some of the ingreients like Terminli rjun, Curum long, Phyllnlhus niruri, Withni sominifer n Emli offiinlis hs een reporte to prevent riovsulr isorers in Inin system of meiine 24. The oserve inrese in the oy weight In ISO-inue rts is possily ue to the umultion of wter ontent in the intrmusulr spe n y nerosis of ri musle fires. Pretretment with Bio-Aq signifintly erese the hert weight in ISO-inue rts; this oul e ue to reution in the proess of nerosis offere y Bio-Aq. Lipis ply n importnt role in the pthogenesis of MI. ISO-trete riotoxiity is ssoite with inrese levels of irultory lipis. Hyperholesterolemi n hypertriglyeriemi re the risk ftor for the evelopment of MI. In this stuy, we oserve inrese levels of totl holesterol, TG, FFA in the serum n myorium of ISO-ontrol rts. Inrese levels of loo holesterol n their umultions in the hert re well ssoite with myoril mge 25. The min trget ville for ROS for ttk is polyunsturte ftty is (PUFA), whih is the preursor for lipi peroxie formtion. Lipi peroxie elevtion oul e ttriute to the umultion of lipis in the hert 26. The oserve inrese in TG fter MI my e ue to elevte flux of ftty is n impire removl of very low ensity lipoprotein (VLDL) from the serum. Pretretment with Bio-Aq erese the levels of TG n holesterol in MI rts. ISO-inue rts lso showe n inrese in the levels of PL n FFA in serum. The FFA lierte from ipose tissue lso enters into the myorium, n the proess is proportionl to the FFA onentrtion in the oronry sinus. Though the hert n utilize FFA for its energy requirements, the exess FFA my e use for the synthesis of TG, resulting in hypertriglyeriemi. PL is essentil omponents for the integrity of ellulr memrne n suellulr orgnelles. Mny ftty is re sustrtes for the iosysthesis of PL 27. Pretretment with Bio-Aq ereses the levels of serum PL n FFA in ISO-trete rts. This oul e ue to lipi lowering property of the rug. Altertions in lipi metolism iretly reflet the omposition of lipoproteins in ISO-inue riotoxi rts. HDL n LDLholesterols re signifint vriles for CHD. Prior tretment with Bio-Aq erese the inrese levels of serum LDL-holesterol, VLDL-holesterol n inrese the lowere levels of serum HDLholesterol in ISO- trete riotoxi rts. Inrese levels of serum HDL holesterol oserve in ISO-inue rts pretrete with Bio- Aq, filitte the trnsport of holesterol from peripherl tissues to the liver for tolism n exretion from the oy. Experiments onute with the rk of Arjun hve een shown to possess hypolipiemi, hypoholesterolemi, hypotensive, tivities 28. Altertions in the tivities of memrne oun enzymes ffet the funtion of the hert. Vjreswri n Nrynrey, 29 (1992) hve shown tht the filure of the ell memrne to mintin norml trnsmemrne ioni istriution through ion pumps is onsiere to e mjor event in the pthogenesis of ishemi n rrhythmi. A signifint erese tivity of N + /K + -ATPse n signifint inrese tivities of Mg 2+ n C 2+ -ATPse in the hert were oserve in ISO-inue rts. Derese tivity of N + /K + -ATPse oul e ue to enhne lipi peroxition y ISO 5. Derese tivity of N + /K + -ATPse n le to erese in soium efflux, therey ltering memrne permeility 3. C 2+ -ATPse regultes the lium pump tivity 31. Enhne C 2+ -ATPse tivity oserve in ISO-trete rts is ue to the tivtion of enylte ylse y ISO. Mg 2+ -ATPse tivity is involve in other energy requiring proess in the ell n its tivity is sensitive to lipi peroxition. 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