STUDY ON CEREBROPROTECTIVE ACTION OF POLYHERBAL FORMULATION OF OXITARD AGAINST CEREBRAL ISCHEMIA REPERFUSION INJURY IN RATS

M. Pharm. Dissertation Protocol Submitted to

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore – 560 041

By

Mr. J.Viswanath B.Pharm.

Under the Guidance of

Mr. Mohammad Asif Ansari M.Pharm,(Ph.D).

Assistant Professor

2011-12

Department of Pharmacology

Acharya& B.M. Reddy College of Pharmacy,

Soldevanahalli, Chikkabanavara (Post)

Hesaraghatta Main Road, Bangalore – 560 090

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1. / Name of the candidate & Address. / Mr. J.Viswanath
“H.no.20-3-117/A”,lakshmitowers plot no:19,
yerramitta,near leelamahal,tirupati-517501.
ANDHRAPRADESH.
2. / Name of the Institution. / Acharya& B.M. Reddy College of Pharmacy.
Soldevanahalli, Hesaraghatta Road,
Chikkabanavara Post,
Bangalore-560 090.
Phone No: 080 65650815
Fax No: 080 28393541
3. / Course of study
& subject. / M.Pharm (Pharmacology)
4. / Date of admission. / 4-7-2011
5. / Title of the Topic / STUDY ON CEREBROPROTECTIVE ACTION OF POLYHERBAL FORMULATION OF OXITARD AGAINST CEREBRAL ISCHEMIA –REPERFUSION INJURY IN RATS
6. / Brief resume of intended work
6.1 Need of the work
6.2 Review of Literature
6.3 Aim and Objective of the study / Enclosure I
Enclosure II
Enclosure III
7. / Materials & Methods
7.1 Source of data
7.2 Methods of collection of data
7.3 Does the study require investigation on animals?
a. If yes give details
7.4 Has ethical clearance been obtained from your institution in case of 7.3 / Enclosure IV
Enclosure V
Enclosure VI
Applied
8. / List of references / Enclosure VII
9. / Signature of the candidate
10. / Remarks of the guide
11. / 11.1 Name & Designation of Guide
11.2 Signature of Guide
11.3 Head of the Department
11.4 Signature of HOD / Mr. Mohammad Asif AnsariM.Pharm, (Ph.D).
Assistant Professor
Department of Pharmacology
Acharya&B.M.Reddy College of Pharmacy, Soldevanahalli, Chikkabanavara (Post) Hesaraghatta main road,
Bangalore – 560 090
Dr. Kalyani DivakarM.Pharm., PhD.
Professor and Head,
Department of Pharmacology,
Acharya&B.M.Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara (Post)
Bangalore – 560 090
12. / Remarks of the Principal
12.1 Signature of the Principal / PRINCIPAL
Dr. DIVAKAR GOLI M.Pharm, PhD.
Acharya& B. M. Reddy College of Pharmacy,
Soldevanahalli, Hesaraghatta main road,
Chikkabanavara (Post),
Bangalore-560090

Enclosure - I

6. BRIEF RESUME OF INTENDED WORK:

6.1 INTRODUCTION AND NEED OF THE WORK:

Stroke is a major cause of death and disability worldwide. Among stroke patients, 80% suffer from cerebral infarction and 20% cerebral haemorrhage [1]. Cerebral infarction is an ischemic condition of the brain including thrombosis, embolism or systemic hemodynamic hypotension. Cerebral infarction is often caused by atherosclerosis of large and small arteries. The etiopathogenesis of atherosclerosis and stroke is related to inflammation, free radical productions, exocitotoxicity, disruption of sodium and calcium influx, enzymatic changes, endothelin release, activation of platelets and leukocytes, delayed coagulation and endothelial dysfunction and genetic factors. Given the few options currently available for patients following ischemic stroke and the recent disappointing failures of several large-scale Phase III clinical trials with neuroprotective drugs, the search for novel therapeutic approaches has become even more critical[2][3]. One such approach is the idea of combined therapy. Combinations of thrombolysis and a neuroprotecting agent or a combination of two neuroprotecting agents have been effective in experimental stroke. Herbal extracts or mixtures represent combinatorial chemistry of nature with vast repertoire of chemical entities that have a complex effect on numerous cellular components and functions. They have great potential in the multi-target approach to diseases. In recent years, there is considerable interest to investigate Antioxidative and anti-inflammatory effects of herbal drugs from different sources. A lot of work is being carried out to find the effectiveness of Indian and Chinese herbals in stroke. Both thrombolytic and neuroprotective properties of herbal drugs may be a novel strategy for effective stroke therapeutics. The Indian system of medicine, especially Ayurveda, has several medicinal plants with proven beneficial claims towards these pathological conditions. However, the potential of herbal drugs as defined therapeutic agents is undermined by the difficulty in standardization, pharmacodynamics and pharmacokinetics of these multi-component mixtures and also the lack of enough experimental data. Therefore,the potential of Indian herbals in the treatment of stroke needs to be further explored.

OXITARD

Oxitard is a poly herbal ayurvedic formulation which is evaluated for its antioxidant effects on various diseases like myocardial infarction [4], diabetes,and hepatotoxicity in rats; howeverOxitard is not yet evaluated for its cerebroprotective activity in cerebral ischemia- reperfusion injury.In the present study we will evaluate the Cerebroprotective actions of polyherbal formulation of Oxitard against cerebral ischemia–reperfusion injury in rats.

ENCLOSURE – II

6.2 REVIEW OF LITERATURE:

OXITARD:

Oxitard is a poly herbal formulation having the antioxidant activity.

Each gram of Oxitard contains:

  1. Mangifera indica Linn. (Anacardiaceae; bark, 340 mg),
  2. Glycyrrhiza glabra Linn. (Papilionaceae; rhizome, 100 mg),
  3. Syzygium aromaticum Linn. (Myrtaceae; flower bud, 100 mg),
  4. Vitis vinifera Linn. (Vitaceae; fruit, 21 mg),
  5. Emblica officinalis Linn. (Euphorbiaceae; fruit, 200 mg) and
  6. Daucus carota Linn. D. Vulgaris (Umbelliferae; root, 200 mg).

Previous work done on Oxitard:

In vitro and invivo studies proved that Oxitard is having a potent scavenging activity in rat ischemic heart [5].

Dr. R. Balaraman et al reported the antioxidant activity of Oxitard in rats by estimating the levels of SOD, CAT, reduced glutathione, lipid peroxidation [6].

Mitra, S.K,Venkataranganna et..al., reported the antioxidant effect of Oxitard in vitro and in vivo experiments in rats. [7].

Bafna,P.A. et al reported the antiulcer and antioxidant activity of Oxitard on ethanol-induced gastric mucosal injury in rats [8].

Epidemiological studies have been strongly suggesting that antioxidants can decrease the rate of many diseases

Present study was undertaken to evaluate the Cerebroprotective potential of Oxitard in cerebral ischemia induced reperfusion injury.

Enclosure – III

6.3AIM AND OBJECTIVE OF THE STUDY:

The main objective of the present study is to determine the,

a)Cerebroprotective actions of Oxitard cerebral ischemia reperfusion induced cerebral infraction in rats.

b)To explore the possible mechanisms of Cerebroprotective action of Oxitard.

Enclosure – IV

7. materials and methods:

7.1 SOURCE OF DATA:

Data will be obtained from experiments which involve-

A)The evaluation of Cerebroprotective effect of Oxitardusing experimental animals by:

  1. Various biochemical and enzymatic studies.

B)National and International Journals.

C)Literature Survey, CD ROM, Chemical abstracts.

D)Text books.

E)Internet.

Enclosure – V

7.2 METHOD OF COLLECTION OF DATA

Drugs:

Drugs such as Oxitard, and L-NAME, are procured from reliable sources.

Animals:

Healthy Wistar albino Rats (200-250g) of either sex will be procured from registered suppliers. The animals will be housed in standard environmental condition & provided with food & water ad libitum.All experimental animals will be conducted in accordance with the guidelines of CPCSEA.

Methodology:

1. Ischemia and reperfusion.

EXPERIMENTAL PROTOCOL:

Induction of global ischemia by Bilateral Common Carotid Artery Occlusion (BCCAO) followed by reperfusion:

Rats will be anesthetized by giving thiopentone sodium (40 mg/kg) i.p. surgical technique for the induction of cerebral ischemia will be adopted from the earlier published method of Natalie N. et al. 2005. Under anaesthesia midline incision will be given. Bilateral common carotid arteries will be identified and isolated carefully from vago-sympathetic nerve. Rats will be subjected to cerebral ischemia by occluding of bilateral Common carotid arteries with cotton thread for ½h and reperfusion will be allowed for 4h by removing the thread with the help of knot releasers. Body temperature will be maintained around 37 ± 0.5º C throughout the surgical procedure {9}

Experimental Design:

Ischemic reperfused group:

Group 1: Ischemic reperfusion (I/R)

Group 2: Sham

Group 3: Vehicle

Group 4: (I/R) –Dose 1

Group 5: (I/R) –Dose 2

Group 6: L-NAME

Group 7: L-NAME-Dose 1

Group 8: L-NAME-Dose 2

Respective group will be treated with the respective inhibitors administered by i.p route 10 min before ischemia and respected group will receive Oxitarddose (30mg/kg) by i.p route 10 min before reperfusion.

  1. Preparation of post-mitochondrial supernatant (PMS).

Following decapitation, the brain will be removed and washed in cooled 0.9% saline, will be kept on ice and subsequently blotted on filter paper, then weighed and homogenized in cold phosphate buffer (0.1 M, pH 7.4) using a homogenizer. Homogenization procedure will be performed as quickly as possible under completely standardized conditions. The homogenates will be centrifuged at 10,000rpmfor 20 min at 4°C and post-mitochondrial supernatant will be kept on ice until assayed. {10}

  1. Cerebroprotective activity of Oxitard on ischemic reperfused rats by measuring the following

I) Infract Size

After reperfusion period. The brain is frozen at -4°C. Frozen brain will be sliced into uniform sections of 1mm thickness. The slices will be incubated in 2% triphenyltetrazoliumchloride (TTC) at 37°C in phosphate buffered saline (pH 7.4). TTC is converted to red formazone pigment by NAD and dehydrogenase present in living cells. Hence viable cell will be stained deep red. Unstained areas shall be considered as ischemia lesions. {11}

ii) Superoxide Dismutase activity (SOD)

Superoxide dismutase is an enzyme that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. The principle of this method is the inhibition of reduction of nitrobluetetrazolium to blue coloredtetrazolium in presence of phenazinemethosulphateand NADH by superoxide dismutase enzyme. The color intensity of sample will be measured at 560 nm. [12]

iii) Myeloperoxidase activity (MPO)

Myeloperoxidase the most abundant protein in neutrophils (also found in monocytes), is the focus of inflammatory pathologies. Its ability to catalyze reaction between chloride and hydrogen peroxide (H2O2) to form hypochlorous acid.The samples will be measured at460nm. [13]

iv)Malondialdehyde Estimation(MDA)

The lipid peroxidation end product malondialdehyde (MDA) will be measured by the method of Okhawa et al. 0.1 ml of brain homogenate will be treated with 20% of 1.5 ml of acetic acid (pH 3.5), 1.5 ml thiobarbituric acid and 0.2 ml sodium dodecyl sulphate (8.1%). The mixture will be then heated at 100ºC for 60 min. The mixture will be cooled and 5 ml of n-butanol–pyridine mixture will be added followed by 1 ml of distilled water. The mixture will be shaken vigorously. After centrifugation of the mixture at 4000 rpm for 10 min, the organic layer was taken and its absorbance will be measured at 532 nm. The concentration of MDA formed will be expressed as nmol/mg protein. [14]

v) Catalase activity (CAT)

Catalase measurement was carried out by the ability of CAT to oxidize hydrogen

Peroxide (H2O2). Decomposition of H2O2 gives water and oxygen.The UV light absorption of hydrogen peroxide solution can be easily measured between 230 to 250 nm. On decomposition of hydrogen peroxide by Catalase,the absorption

Decreases its time. The enzyme activity could be arrived at this decrease. But this method is applicable only with enzyme solution which do not absorb strongly at 230-250 nm. [15]

VI) Protein estimation

The peptide bonds of protein react with the copper- II ions in alkaline solution to form a violet-blue complex (the so-called biuret reaction). The intensity of the colored complex of sample will be measured at 600-630 nm by using spectrophotometer and directly proportional to the absorbance reading and the concentration of the total protein in the sample. [16]

Statistical Analysis:

The data obtained from the above study will be subjected to statistical analysis using analysis of variance (ANOVA) followed by Dunnets test.

Total duration for the completion of whole project will be 9 months.

  1. Duration of experimentSeven and half month
  2. Literature surveyOne month
  3. Thesis writing One month

ENCLOSURE – VI

7.3 Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly.

The above study requires investigation on Wistar albino rats of either sex for Cerebroprotective activity of Oxitard cerebral ischemia.

7.4 Has ethical clearance been obtained from your institution in case of 7.3?

Applied

ENCLOSURE – VII

LIST OF REFERENCES:

  1. Gupta Y.K, Seema Briyal. Animal models of Cerebral Ischemia for evaluation of drugs. Indian J Pharmacol 2004; 48(4): 379-394.
  2. Caro JJ, Huybrechts KF, Duchesne I. Management patterns and costs of acute ischemic stroke, an international study. Stroke 2000; 31: 582–590.
  3. Siesjo .BK. Pathophysiology and treatment of Focal cerebral ischemia mechanisms of damage and treatment. J Neurosurg 1992; 77: 337–354.
  4. Mitra.SK,Venkataranganna .M.V, Sundarm.R, et al. Antioxidant activity of AO-8, an herbal formulation in vitroand in vivoExperimentalmodels. Phytotherapy Research 1999 ;( 13) 300-303.
  5. MitraS.K, VenkatarangannaM.V,SundarmR, Gopumadavan,et al.Phytotherapy research,Antioxidant activity of AO-8, Aherbal formulation in vitroand in vivoexperimental models 1999 ;( 13) 300-303.
  6. Balaraman.R, Pallavi A, Bafna1, Kolhapur S.A,et al. Journal of ethonopharmacologyAntioxidant activity of DHC-1– an herbalformulation 2004; 194:135-141.
  7. Venkataranganna V.K,Sundarm M.V.R,et al. Phytotherapy research,Phytotherapy research,Antioxidant activity of AO-8, A herbal formulation in vitroand in vivoexperimental models 1999;(13) 300-303.
  8. Bafna,P.A, Balaraman R,et al. Journal of ethonopharmacologyAntioxidant activity of DHC-1– A herbalformulation (2004); 90,123-127.
  9. Szabo M, Haines D, Garay E, Chiavaroli C, Jean-Claude F, Hannaert P et al. Antioxidant properties of calcium dobesilate in ischemic reperfused diabetic rat retina. Eur J Pharmacol2001;428:277-86.
  10. PoonamK, SitaA,Viswanathan P. Oxidative changes in brain of aniline-exposed rats. Arch Environ Con Tox1992;23:307-9.
  11. Watanabe Y, Takagi H, Shin-ichiro A, Sassa H. Prediction of cerebral infarct sizes by cerebral blood flow performed in the early acute stage. Ann Nucl Med 1999; 13(4):205-10.
  12. Mullane KM, Kraemer R, Smith B. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J Pharmacol Method 1985; 14:157-67.
  13. Sun C, Shan C, Gao X, Tan R. Peroxide-induced injury by EPS2, an exopolysaccharide from a marine filamentous fungus Keissleriella sp. YS4108. JBiotech2005;115(2):137-44.
  14. Roy S, Sehgal S, Padhy B, Kumar V. Antioxidant and protective effect of latex of calotropis procera against alloxan-induced diabetes in rats. Ethnopharmacol 2005; 102(31):470-3.
  15. Mohan R, Venkateswararao C, Rawat A, Pushpangadan P, Shirwaikar A. Antioxidant and Antihepatotoxic activities of hemidesmus indicus r. Br. Acta Pharmaceutical Turcica 2005;47:73-8.
  16. Peterson GL. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem1977; 83(2); 346-56.