Neuroprotective effect of alcoholic extract of Terminalia ... - Wseas.us

More documents

Recommendations

Info

Recent Researches in Modern Medicineimbalance of ionic gradients acrossmembrane, accumulates calcium, sodium,and reduces pH, which in turn altersmembrane transport, mitochondrial function,activates calcium-dependent enzymaticreactions, including DNA-breaking enzymesand generates excessive free radicals andtriggers lipid peroxidation and ultimatelycell apoptosis. Primary therapeutic strategyin treating cerebral ischemia is to restoreblood flow in the shortest time possible inorder to preserve neural tissues. Althoughreperfusion of ischemic brain tissue iscritical for restoring normal function, it canparadoxically result in secondary damage,called reperfusion injury. Secondaryapproach is to ameliorate pathophysiologicalconsequences of stroke caused by freeradicals generated during ischemia andreperfusion. Thus, antioxidants or freeradical scavenging agents are demonstratedto have protective effect in experimentalstroke [5-10]. It is important to note that ananti-ischemic agent aiming at both vascularand parenchymal effects would be morebeneficial.Terminalia arjuna (TA) is widelyused by Ayurvedic physicians for itscurative properties in organic/functionalheart problems including angina,hypertension and deposits in arteries. Itsstem bark possesses glycosides, largequantities of flavonoids, tannins andminerals. Flavonoids have antioxidant, antiinflammatoryand lipid lowering effectswhile glycosides are cardiotonic. Flavonoidsand Oligomeric proanthocyanidins provideantioxidant activity and vascularstrengthening [11]. It also has mild diuretic,antithrombotic, prostaglandin E 2 enhancingand hypolipidaemic activity [12].2 Materials and Methods2.1 Plant materialThe wet bark of T. arjuna was collectedfrom Tirumala hills during the month ofNovember, 2009 and was shade dried. Stembark was coarsely powdered and refluxedwith 90% v/v methanol for six hours. Theextract was filtered and the filtrate wasevaporated to dryness by rotary flashevaporator at low temperature under reducedpressure. A dark brownish-red shiny powder-like residue was obtained, which was storedin a desiccator..2.2 AnimalsMale albino rats weighing 200-250 g werechosen to avoid fluctuations due to estrouscycle. The rats were housed inpolypropylene cages under 12 hrs light /darkcycle, fed with standard laboratory chow(Hindustan Lever Limited, Mumbai) andwater ad libitum. Animals were acclimatizedto the laboratory conditions prior toexperimentation and all the experimentswere carried out according to the studyprotocol approved by the InstitutionalAnimal Ethical Committee.2.3 Induction of ischemiaTransient focal cerebral ischemia wasproduced by intra luminal middle cerebralartery occlusion procedure as reported byLonga et al.,[13] with slight modifications.Briefly the rats were anaesthetized withThiopentone Sodium (40mg/kg, i.p.). Amidline incision was made on ventral side ofthe neck and the right carotid bifurcationwas identified. The external carotid arterywas carefully isolated from accompanyingnerves and ligated. A small niche was givento the right common carotid artery (CCA)proximal to the carotid bifurcation and a 4-0monofilament nylon thread (Ethicon,Johnson & Johnson) with its tip rounded byheating quickly by bringing it near a flamewas introduced through it into the lumen ofinternal carotid artery and advanced until aslight resistance was felt which ensured theocclusion of the origin of middle cerebralartery. Two hours after the induction ofischemia, the filament was slowlywithdrawn and the animals were returned totheir cages for a reperfusion period of 22hrs, the body temperature was maintained atISBN: 978-960-474-278-3 282
Recent Researches in Modern Medicine37 0 C with a thermostatically controlledinfrared lamp. In sham rats the right CCAwas surgically prepared for the insertion ofthe filament, but the filament was notinserted.2.4 Study protocolRats (200-250g) were randomly divided intofive groups of 9 rats in each group, fed withdrug or vehicle for 14 days prior toexperiment. The first group served asnormal and received 1% tween 80 in waterorally. The second group served as shamcontrol, received 1% tween 80 in waterorally. The third group served as ischemiccontrol, received 1% tween 80 in waterorally. The fourth and fifth groups weretreated with TABE 500 and 1000mg/kg/p.o., respectively for 14 days prior toMCAO. After 24 hrs, neurologicalexaminations were performed in all groups.Then six rats in each group were decapitatedto obtain brain tissue samples forbiochemical analysis and remaining threebrains in each group were used forhistopathological studies.2.5 Neurological deficitNeurological deficit in the vehicle- anddrug-pretreated groups were determinedafter 24 hrs of induction. Neurologicalfindings were scored on a 5-point scale [13]as follows: no neurological deficit = 0,failure to extend left paw fully = 1, circlingto left = 2, falling to left = 3, did not walkspontaneously and had decreased levels ofconsciousness = 4. The above procedureswere performed in a blinded manner.2.6 Biochemical estimations24 h after induction of ischemia the animalswere killed by cervical dislocation and theirbrains were dissected out quickly. Theinfarcted area was homogenized in 50 mMphosphate buffer (pH 7.0) containing 0.1mM EDTA to give 5% w/v homogenate.The homogenate was centrifuged at 10,000rpm for 10 min at 0 o C in cold centrifuge andthe resulting supernatant was used forbiochemical estimations.2.6.1 Superoxide dismutaseSOD activity was measured according to themethod of Misra and Fridovich [14], at roomtemperature. 100 µl of supernatant wasadded to 880 µl of 0.05 M carbonate buffer(pH 10.2, containing 0.1 mM EDTA), and20 µl of 30 mM epinephrine (in 0.05%acetic acid) was added to the mixture, andthe optical density values were measured at480 nm for 4 min on a Systronics UV–Visible Spectrophoto-meter; activity wasexpressed as the amount of enzyme thatinhibits the oxidation of epinehrine by 50%which is equal to 1 unit.2.6.2 CatalaseCatalase activity was measured by a slightlymodified version of Aebi, [15] at roomtemperature. 100 µl of supernatant wasadded to 10 ul of 100% ethanol and placedin ice bath for 30 min. The tubes werebrought to room temperature followed bythe addition of 10 µl of Triton X-100. In acuvette containing 200 µl of phosphatebuffer and 50µl of above mixture, 250 µl of0.066M H 2 O 2 in phosphate buffer wasadded. The decrease in optical density wasmeasured at 240 nm for 60 sec in SystronicsUV Spectrophotometer. The molarextinction coefficient of 43.6 Mcm -1 wasused to determine catalase activity which isequal to the moles of H 2 O 2 degraded/mgprotein/min.2.6.3 Reduced GlutathioneReduced glutathione levels were measuredaccording to the method of Ellman [16] atroom temperature. 0.75 ml of supernatantwas mixed with 0.75 ml of 4%sulphosalicylic acid and then centrifuged at1200 rpm for 5 min at 4_C. From this 0.5 mlof supernatant was taken and added to 4.5ml of 0.01 M DTNB, and absorbance wasISBN: 978-960-474-278-3 283
Page 1: Recent Researches in Modern Medicin
Page 5 and 6: Recent Researches in Modern Medicin
Page 7: Recent Researches in Modern Medicin

Neuroprotective effect of alcoholic extract of Terminalia ... - Wseas.us

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?