M. Pharm. Dissertation Protocol Submitted To s1

STUDY ON CEREBROPROTECTIVE ACTION OF HESPERETIN AGAINST CEREBRAL ISCHEMIA -REPERFUSION INDUCED CEREBRAL INFARCTION IN BOTH NORMAL AND STREPTOZOTOCIN-INDUCED DIABETIC RATS

M. Pharm. Dissertation Protocol Submitted to

Rajiv Gandhi University of Health Sciences, Karnataka Bangalore – 560 041

Mr. Sidramesh Hiremath B.Pharm.

Under the Guidance of

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

2010-11

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. Sidramesh Hiremath “Hiremath Center” Opp.Shankarmath, Kanchagar Galli,Hubli-580028, KARNATAKA.

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 M.Pharm (Pharmacology) & subject.

4. Date of admission. 06-07-2010

5. Title of the Topic STUDY ON CEREBROPROTECTIVE ACTION OF HESPERETIN AGAINST CEREBRAL ISCHEMIA -REPERFUSION INDUCED CEREBRAL INFARCTION IN BOTH NORMAL AND STREPTOZOTOCIN- INDUCED DIABETIC RATS 6. Brief resume of intended work

6.1 Need of the work Enclosure I

6.2 Review of Literature Enclosure II

6.3 Aim and Objective of the study Enclosure III

7. Materials & Methods 7.1 Source of data Enclosure IV

7.2 Methods of collection of data Enclosure V

7.3 Does the study require Enclosure VI investigation on animals? a. If yes give details

7.4 Has ethical clearance been obtained from your institution in Applied IAEC for animal ethical clearance case of 7.3

8. List of references Enclosure VII

9. Signature of the candidate

10. Remarks of the guide 11. 11.1 Name & Designation of Guide Mr. Mohammad Asif Ansari M.Pharm, (Ph.D). Assistant Professor Department of Pharmacology Acharya & B.M.Reddy College of Pharmacy, Soldevanahalli, Chikkabanavara (Post) Hesaraghatta main road, Bangalore – 560 090

11.2 Signature of Guide

Dr. Kalyani Divakar 11.3 Head of the Department M.Pharm., PhD. Professor and Head, Department of Pharmacology, Acharya & B.M.Reddy College of Pharmacy, Soldevanahalli, Chikkabanavara (Post) Bangalore – 560 090

11.4 Signature of HOD

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: The World Health Organization (WHO) defines stroke as a clinical syndrome typified by rapidly developing signs of focal or global disturbance of cerebral functions, without another apparent causes than of vascular origin. It is the third most common cause of death in developed countries, exceeded only by coronary heart disease and cancer, with 5 million people dying every year.1 Hyperglycaemia aggravates ischemic brain injury, possibly due to the activation of signalling pathways involving reactive oxygen species. Formation of ROS and RNS can occur in the cells by two ways: enzymatic and non-enzymatic reactions. Enzymatic reactions generating free radicals include those involved in the respiratory

2 chain, the phagocytosis, the prostaglandin synthesis and the cytochrome P450 system.

•ˉ) For example, the superoxide anion radical (O2 is generated via several cellular oxidase systems such as NADPH oxidase, xanthine oxidase, peroxidases. Once formed, it participates in several reactions yielding various ROS and RNS such as hydrogen peroxide, hydroxyl radical (OH•), peroxynitrite (ONOOˉ), hypochlorous acid (HOCl), etc. H2O2 (a non-radical) is produced by the action of several oxidase enzymes, including aminoacid oxidase and xanthine oxidase. The last one catalyses the oxidation of hypoxanthine to xanthine and of xanthine to uric acid. Hydroxyl radical (OH•), the most

•ˉ reactive free radical in vivo, is formed by the reaction of O2 with H2O2 in the presence of Fe2+or Cu+ (catalyst). This reaction is known as the Fenton reaction. Hypochlorous acid (HOCl) is produced by the neutrophil-derived enzyme, myeloperoxidase, which

• oxidizes chloride ions in the presence of H2O2. Nitric oxide radical (NO ) is formed in biological tissues from the oxidation of L-arginine to citrulline by nitric oxide synthase. Free radicals can be produced from non-enzymatic reactions of oxygen with organic compounds as well as those initiated by ionizing radiations. The non-enzymatic process can also occur during oxidative phosphorylation (i.e. aerobic respiration) in the mitochondria. 3 Many polyphenolic compounds from plants, including a large class of bioflavonoids, are known to offer health benefits to humans. The beneficial effects have been attributed to their anti-oxidant and anti-inflammatory properties.4 There is increasing evidence that besides the peripheral organs, bioflavonoids may help prevent tissue damage from oxidative stress in the brain. The brain is highly susceptible to oxidative stress and oxidative damages have been implicated in the pathology of a number of neurodegenerative diseases.5 Recent studies from our laboratory have provided evidence for the cerebroprotective activity of polyphenols such as hesperetin and naringenin. However the exact mechanism involved in the cerebroprotective actions of these drugs has not been elucidated. In this context we have planned to evaluate the mechanisms involved in cerebroprotective action of Heseperetin.

ENCLOSURE – II 6.2 REVIEW OF LITERATURE: XANTHINE OXIDASE (XO) INHIBITORS: The xanthine oxidoreductase catalysis the oxidation of hypoxanthine and xanthine in the process of purine catabolism forming uric acid. Xanthine oxido-reductase can exist in two inter convertible forms, either as xanthine dehydrogenase (XD) or xanthine oxidase (XO) and intermediate form. Xanthine oxidase produces uric acid and reactive oxygen species during the catabolism of purine, especially after Ischemia- reperfusion. Xanthine oxidase is a source of oxygen free radicals. In the reperfusion phase (ie, reoxygenation), xanthine oxidase reacts with molecular oxygen, there by releasing superoxide free radicals. Bioflavonoids inhibit xanthine oxidase activity, there by resulting in decreased oxidative injury.6

NITRIC OXIDE (NO) INHIBITORS: Nitric oxide is produced by several different types of cells, including endothelial cells and Macrophages. The activity of constitutive nitric-oxide synthase is important in maintaining the dilation of blood vessels, the much higher concentrations of nitric oxide produced by inducible nitric-oxide synthase in macrophages can result in oxidative damage. Nitric oxide reacts with free radicals, there by producing the highly damaging peroxynitrite. Nitric oxide injury takes place for the most part through the peroxynitrite route because peroxynitrite can directly oxidize LDL’s, resulting in irreversible damage to the cell membrane. When flavonoids are used as antioxidants, free radicals are scavenged and therefore can no longer react with nitric oxide, resulting in less damage. NOS inhibitor, L-nitro-arginine methyl ester (L-NAME), decreases the infract region to the level observed in normo glycemic rats. This indicates that nitric oxide production contributes significantly to the exacerbation of tissue damage observed in hyperglycemic ischemic reperfusion (I/R).7

COX INHIBITORS: Inflammation is a significant source of increased oxidative stress. COX-2 is believed to contribute to the ischemic damage via generation of superoxide radicals. Macrophages are also attracted to the post ischemic inflammatory site and produce both superoxide and nitric oxide which would favour peroxynitrite production. COX-1 is constitutively expressed in most tissues and is involved in maintenance of cellular homeostasis. COX-2, an inducible form, is expressed at low or undetectable levels but is readily up regulated by inflammatory stimuli such as cytokines and lypopolysaccharides. COX-3 was cloned recently from canine tissues. In rat brain, COX-3 is expressed in all cell types except neurons and the highest expressions in endothelial cells. Superoxide has pro-adhesive actions resulting in increased PMN leukocytes adhesion to the endothelium. PMN leukocytes may contribute to free radical production through two enzymes myeloperoxidase and NAD(P)H oxidase.8

ENCLOSURE – III 6.3 AIM AND OBJECTIVE OF THE STUDY: The main objective of the present study is to determine the, a) Cerebroprotective actions of Hesperetinin cerebral ischemia reperfusion induced cerebral infraction in rats. b) To explore the possible mechanisms of cerebroprotective action of Hesperetin.

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 Hesperetin using 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 Hesperetin, L-NAME, Allopurinol and Nimesulide will be collected from pharmaceutical company. Animals: 200 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. Induction of diabetes. Diabetes will be induced with a single dose of 45mg/kg i.p injection of Streptozotocin (STZ) in 10mM citrate buffer (pH 4.5) after an overnight fast. Three days after the STZ injection, rats with blood glucose levels greater than 250 mg/dl will be considered to be diabetic. 9 2. Ischemia and reperfusion. EXPERIMENTAL PROTOCOL: a) 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 1/2h 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.10 Evaluation 1. Non-Diabetic ischemic reperfused group:

 Group 1: Ischemic reperfusion (I/R)  Group 2: Sham  Group 3: Vehicle  Group 4: Hesperetin  Group 5: L-NAME  Group 6: L-NAME + Hesperetin  Group 7: COX Inhibitor  Group 8: COX Inhibitor + Hesperetin  Group 9: XO Inhibitor  Group 10: XO Inhibitor + Hesperetin Respective group will be treated with the respective inhibitors administered by i.p route 10 min before ischemia and respected group will receive Hesperetin dose (30mg/kg) by i.p route 10 min before reperfusion.

2. Diabetic ischemic reperfused group:

 Group 1: Ischemic reperfusion (I/R)  Group 2: Sham  Group 3: Vehicle  Group 4: Hesperetin  Group 5: L-NAME  Group 6: L-NAME + Hesperetin  Group 7: COX Inhibitor  Group 8: COX Inhibitor + Hesperetin  Group 9: XO Inhibitor  Group 10: XO Inhibitor + Hesperetin Respective group will be treated with the respective inhibitors administered by i.p route 10 min before ischemia and respected group will receive Hesperetin dose (30mg/kg) by i.p route 10 min before reperfusion. b) 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 10,000 rpm for 20 min at 4°C and post-mitochondrial supernatant will be kept on ice until assayed.11

3. Cerebroprotective activity of Hesperetin on ischemic reperfusedstreptozotocin induced diabetic 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. 12 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 nitroblue tetrazolium to blue coloured tetrazolium in presence of phenazine methosulphate and NADH by superoxide dismutase enzyme. The colour intensity of sample will be measured at 560 nm.13 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 at 460 nm.14 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 nmole/mg protein.15 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.16 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. 17 c) 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. I. Duration of experiment Seven and half month II. Literature survey One month III. 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 200 Wistar albino rats of either sex for cerebroprotective activity of Hesperetin on hyperglycemic cerebral ischemia.

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

Applied IAEC for animal ethical clearance. ENCLOSURE – VII LIST OF REFERENCES: 1. Mackay J, Mensah G. The atlas of heart disease and stroke, Avaliable at