M. Pharm. Dissertation Protocol Submitted To

Neuroprotective Effect of Fenugreek Seeds in Transient Cerebral Ischemia (tMCAO) and long term cerebral hypoperfusion in rats

M. Pharm. Dissertation Protocol Submitted to

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

Ms. Chandana Baishya. B.Pharm.

Under the Guidance of

Dr. MANJUNATHA. P.M. M. Pharm, Ph.D. Assistant Professor

2012-13 Department of Pharmacology Acharya & B.M. Reddy College of Pharmacy, Acharya Dr. Sarvepalli Radhakrishnan Road, Soldevanahalli, Hesaraghatta Main Road, Chikkabanavara (Post), Bangalore – 560 090, Karnataka, India RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE.

ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1. Name of the candidate & Address. Ms. Chandana Baishya. Vill. Balikaria, Kharzara, New colony, Bye lane-13, Chilarai path. P. O.- Gopal Bazar-781335, (Dist.) -Nalbari. (State)-Assam. Acharya & B.M. Reddy College of Pharmacy 2. Name of the Institution. Acharya Dr. Sarvepalli Radhakrishnan Road, Soldevanahalli, Hesaraghatta Main Road, Chikkabanavara (Post), Bangalore-560 090. Phone No: 080-28396011 Ext. 2302/2303 Fax No: 080-28393541

3. Course of study M. Pharm (Pharmacology) & subject

4. Date of admission 13-07-2012

5. Title of the Topic Neuroprotective effect of Fenugreek seeds in transient cerebral ischemia (tMCAO) and long term cerebral hypoperfusion in rats.

2 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 Enclosed case of 7.3

8. List of references Enclosure VII

9. Signature of the candidate

3 10. Remarks of the guide Forwarded

11. 11.1 Name & Designation of Guide Dr. Manjunatha P. M M. Pharm, Ph. D. Assistant Professor Department of Pharmacology Acharya & B. M. Reddy College of Pharmacy, Acharya Dr. Sarvepalli Radhakrishnan Road, Soldevanahalli, Chikkabanavara (Post) Hesaraghatta main road, Bangalore – 560 090

11.2 Signature of Guide

11.3 Head of the Department Md. Asif Ansari M. Pharm., Ph. D. Professor and Head, Department of Pharmacology, Acharya & B.M. Reddy College of Pharmacy, Dr. Sarvapalli Radhakrishnan road, Soldevanahalli, Hesaraghatta Main Road, Chikkabanavara (Post) Bangalore – 560 090

11.4 Signature of HOD

4 12. Remarks of the Principal

12.1 Signature of the Principal PRINCIPAL

Dr. DIVAKAR GOLI M. Pharm., Ph. D. Acharya & B. M. Reddy College of Pharmacy, Acharya Dr. Sarvapalli Radhakrishnan Road, Soldevanahalli, Hesaraghatta main road, Chikkabanavara (Post), Bangalore-560090

5 ENCLOSURE - I

6. BRIEF RESUME OF INTENDED WORK:

6.1 INTRODUCTION AND NEED OF THE WORK: Cerebral ischemia is a condition in which there is insufficient blood flow to the brain to meet metabolic demand. This leads to poor oxygen supply or cerebral hypoxia and thus to the death of brain tissue or cerebral infarction / ischemic stroke.1 It has been third most common cause of death in developed countries. Among stroke patients, 80% suffer from cerebral infarction and 20% cerebral haemorrhage.2 According to World Health Organisation (WHO) stroke may be defined as, “rapidly developing signs of focal or global disturbance of cerebral function, lasting longer than 24 h (unless interrupted by death) with no apparent non-vascular cause”. Selective neuronal necrosis (SNN) denotes neuronal death with sparing of glial and vascular elements of the central nervous system. Transient ischemic attacks (TIA) and global ischemia following cardiac arrest are the most common causes of brief cerebral ischemia in humans.3 Transient MCAO model is one of the most widely and successfully used models of ischemic stroke. Under physiological conditions, reactive oxygen species such as superoxide

(•O2), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), play important roles in signalling and metabolic pathways. Superoxide dismutase prevents vasogenic brain edema

− after several kinds of injuries, suggesting that O2 is an important factor for disruption. Superoxide dismutase is a metalloenzyme that catalyze the dismutation of superoxide anion

− (O 2) into O2 and hydrogen peroxide (H2O2) in the cytosol, reduced to H2O by glutathione peroxidase in the cytosol, or by catalase in mitochondria and nucleus. Subsequently, H2O2 is the peroxisomes or in the cytosol. Another manifestation of central nervous system damage is the direct injury of neural cell including excitatory events that are induced by glutamate release after cerebral ischemia that is improved by normobaric hyperoxia preconditioning via upregulation of glutamate transporters. Glutamate elevates free calcium (Ca2+), which activates Ca2+ dependent enzymes and leads to free radical production.4 Evidence has accumulated over the past two decades showing that reactive oxygen species are involved in brain injuries caused by cerebral ischemia. Several components of reactive oxygen species (ROS) (superoxide, hydroxyl radical, hydrogen peroxide and peroxynitrite radical) that are generated after ischemia-reperfusion injury play an important

6 role in neuronal loss after cerebral ischemia.5 Several mechanisms are involved in the production of free radicals in cerebral ischemia reperfusion injury.

Reperfusion is the restoration of blood flow to the ischemic tissue. Despite the unequivocal benefit of reperfusion of blood to an ischemic tissue, reperfusion itself can elicit a cascade of adverse reactions that paradoxically injure tissue. Indeed, reperfusion injury has been well described in the literature to cause organ damage in the brain, heart, lungs, liver, kidneys and skeletal muscle. The susceptibility of tissue to ischemia reperfusion injury (IRI) is a major obstacle to both reperfusions after an infarct and successful organ transplantation. Reperfusion injury is a distinct entity from the primary ischemic injury; the oxygen arriving with blood circulation, although necessary for alleviating the ischemic status, may be harmful and worsen the damage. Excessive generation of reactive oxygen species (ROS) is believed to be the main culprit in the causation of reperfusion injury. Present study is focused on the effects of Fenugreek which prevents the oxidative stress during reperfusion injury as well as attenuates the behavioural deficits and histopathological alterations secondary to hypoperfusion. The present study will be also done to find out the neuroprotective effect of fenugreek seeds on transient cerebral ischemia (tMCAO) and long term hypoperfusion in rat. In-vivo scientific data is not available on neuroprotective activity of fenugreek seed extract in tMCAO model, hence the present research is planned to determine the neuroprotective activity of Hydroalcoholic extract of Trigonella foenum-graecum Linn. seeds in rats.

7 ENCLOSURE – II 6.2 REVIEW OF LITERATURE: Fenugreek (Trigonella foenum-graecum Linn.) is an annual leguminous herb that belongs to the family Fabaceae. The common names of the plant are Fenugreek, Methi, Greek hayseed and bird’s foot. It is grown extensively in Asian Middle Eastern and European countries. It is used as both culinary and medicinal herb. Fenugreek seeds are used as a spice in food preparations due to strong flavour and aroma.

Some of the therapeutic uses of Fenugreek include its use as hypoglycemic, antiulcerogenic, hypocholesterolemic and antihypertensive agent. In ayurvedic and unani system of medicine, fenugreek is used for treatment of epilepsy, paralysis, gout, piles and chronic cough.6

A fenugreek seed contains 45-60% carbohydrates, mainly mucilaginous fiber (Galactomannans); 20-30% proteins high in lysine and tryptophan; 5-10% fixed oils (lipids); Pyridine type Alkaloids, mainly Trigonelline (0.2-0.36%), Choline (0.5%), Gentianine and Carpaine; Flavonoids such as Apigenin, Luteolin, Orientin, Quercetin, Vitexin and Isovitexin; free Amino acids such as 4-hydroxyisoleucine (0.09%); Arginine, Histidine and Lysine. Glycosides yielding steroidal sapogenins on hydrolysis (Diosgenin, Yamogenin, Tigogenin, Gitongenin); Vitamins A,B,C and Nicotinic acid and 0.015% volatile oils (n- alkanes and sesquiterpenes). Further, fenugreek seeds contain the Saponins, known as Graecunins. Trigonelline degraded to nicotinic acid and related pyridines during roasting is responsible for flavour of the seed.7

The Fenugreek seed contains a central hard, yellow embryo surrounded by a corneous and comparatively large layer of white, semi-transparent endosperm, which contains the Galactomannans gum. A tenacious and dark brown husk surrounds the endosperm. The colour of the gum fraction depends upon the amount of outer husk (brown colour) and cotyledon (yellow colour). Dietary fiber consisting of non-digestible carbohydrates and lignin that are intrinsic and intact in traditional plants, has received much attention due to its health benefits. Soluble fiber lowers the absorption of released glucose. Consumption of dietary fiber is shown to reduce risk of diseases such as cardiovascular diseases, obesity etc. Soluble and insoluble dietary fibers are the storage and cell wall polysaccharides of plants

8 that cannot be hydrolyzed by human digestive system enzymes. Indeed the National advisory committee in Great Britain has recommended a fiber intake of 25-30 gm/day per person.7

Reported pharmacological activities of Fenugreek (Trigonella foenum-graecum Linn):

 Al-Hakeim MHH and co-researchers reported that aqueous extract of Fenugreek (Trigonella foenum-graecum Linn.) leaves possess hypoglycemic effect in normo-glycemic and alloxan induced hyperglycemic rats.8  In 2008 Bukhari SB revealed that all extracts of fenugreek exhibit antioxidant activity. This finding suggests that fenugreek extracts could act as potent source of antioxidants.9  Raisuddin S and co-authors reported that aqueous extract of Trigonella foenum-graecum Linn. modulates toxicity caused by concomitant exposure to CP and L-buthionine-SR-sulfoximine (BSO) by measuring lipid peroxidation and antioxidants in urinary bladder in mice.10  In 2011 Sharififar F reported antinociceptive and anti-inflammatory effects of methanolic extracts of fenugreek seeds.11  Immunomodulatory activity shown by ethanolic extract of Trigonella foenum- graecum leaves on mice was reported by Tripathi S and co-workers.12  Acetylcholinesterase enzyme inhibitory activity of standard extracts of Trigonella foenum-graecum Linn. was reported in 2010 by Mukharjee PK and co-researchers.13  Antifertility activity in female mice was shown using steroidal extract of Trigonella foenum-graecum (seeds) by Sharma JD and Anjula B.14  Petit PR and co-authors worked on steroid saponins from Fenugreek seeds: extraction, purification and pharmacological investigation on feeding behavior and plasma cholesterol. This study concludes that these saponins enhance food consumption and motivation to eat and reduce plasma cholesterol levels in rats.15  Nair MG and co-authors worked on compounds in functional food Fenugreek spice exhibit anti-inflammatory and anti-oxidant activities. Bioassay of Fenugreek seed extracts showed strong LPO, COX-1 and enzyme inhibitory activities.16

9  Hasnain SN and co researchers worked on Evaluation of the hypoglycaemic effect of Trigonella foenum-graecum Linn. in normal mice. This research proved that methanolic extract administered through oral route produced hypoglycaemic effect.17  Ahmadiani A et al., worked on Spinal seratonergic system is partially involved in anti-nociception induced by Trigonella foenum-graecum Linn. Leaf extract. This study confirms the central action of Trigonella foenum-graecum Linn. extract and that spinal 5-HT partially involved in the analgesia induced by it in the second phase of formalin test and also indicates for co-existence of other analgesic mechanisms.18

10 ENCLOSURE – III

6.3 AIM AND OBJECTIVE OF THE STUDY: AIM: To study the neuroprotective effect of Fenugreek seeds on transient cerebral ischemia in rats by using transient middle carotid artery occlusion and long-term hypoperfusion.

The main objective of the present study is: 1) To carry out Hydroalcoholic extract of fenugreek seeds. 2) Neuroprotective actions of Trigonella foenum-graecum Linn. (Fenugreek) seeds on tMCAO and long term cerebral hypoperfusion. 3) To explore the possible mechanisms involved in neuroprotective action of Trigonella foenum-graecum Linn. (Fenugreek) seed.

11 7. MATERIALS AND METHODS: Seed materials Fresh fenugreek seeds will be procured from herbal markets, Bangalore and will be authenticated by taxonomist. Extraction procedure: General maceration extraction procedure will be used for extraction of fenugreek seeds: Fenugreek seeds (moderately coarse powder)

Will be placed in a closed vessel

Whole of the selected solvent-Hydro-alcohol(ethanol:water,70:30) have to be added

Allowed to stand for seven days shaking occasionally

Liquid strained off

Solid residue (mark) will be pressed (recover as much as occluded solution)

(Strained and expressed liquids mixed)

Clarified by subsidence or filtration

Evaporation and concentration Drug solution: Hydroalcoholic extract [ethanol:water,70:30] of Fenugreek (Trigonella foenum-graecum Linn.) will be dissolved in distilled water. Two concentrations 100 and 200 mg/kg of Hydro- alcoholic extract of Fenugreek are chosen for oral administration route based on our earlier literature survay. Extract will be transferred to a dark bottle and stored in refrigerator until further analysis.19

12 Chemicals: 1,1,3,3-tetraethoxyproprane, NADH, nitroblue tetrazolium (NBT), phenazine methosulphate will be procured from Sigma- Aldrich chemicals Ltd, St. Louis, USA. 5, 5’-Dithiobis (2- nitrobenzoic acid) (DTNB), reduced glutathione will be obtained from Himedia Laboratories, Mumbai. All the other chemicals to be procured from Merck laboratories, nice chemicals, Loba chemie, Sd. fine chemicals and other chemicals proposed in this project will be of analytical grade.

Animals: 80 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, New Delhi and approved by the Institutional Animal Ethics Committee (IAEC) of Acharya & B. M. Reddy College of Pharmacy, Bangalore.

13 ENCLOSURE IV

7.1 SOURCE OF DATA: Data will be obtained from experiments which involves

A) Neuroprotective effect of Fenugreek using experimental animals by:  Evaluating various behavioural, biochemical and histopathological studies.

B) National and International Journals.

i) Indian journal of pharmacology

ii) European journal of pharmacology

iii) Journal of ethnopharmacology

iv) Nuerotoxicology research

C) Literature Survey, CD ROM, Chemical abstracts.

D) Text books.

E) Internet.

i) Www.pubmed.com

ii) Www.sciencedirect.com

iii) Www.intech.com

iv) Www.google.com

ENCLOSURE – V

7.2 METHOD OF COLLECTION OF DATA

14 7.2.1 Experimental protocol Animals will be divided into four groups of 10 rats each. To obtain maximum data from small number of animals, each group will be subjected to Locomotor activity, Beam Walking test and Hanging wire test before focal cerebral ischemia and after 24 h reperfusion; finally subjected to examine cerebral infarct volume, biochemical parameters and histopathological studies. The first group will be sham operated [rats will be subjected to surgical procedure, but do not suffer middle cerebral artery occlusion (MCAO), except for exposure of right internal carotid artery (ICA) and right external carotid artery (ECA)]. The second group served as control tMCAO group i.e., rats will be orally administered [simple syrup I.P 10 ml/kg] for 7 days (pre-treatment) before subjecting to 30 min MCAO followed by reperfusion for 24 h. Third and fourth group of rats, orally received (100 and 200 mg/kg) dose of fenugreek (Trigonella foenum-graecum Linn.) respectively. Fenugreek seed extract will be administered orally twice a day for 7 consecutive days prior to the experiment.

7.2.1 A. Induction of transient cerebral ischemia (tMCAO):

Transient cerebral ischemia will be produced by following the method. Rats will be anesthetized by giving thiopentone sodium (40 mg/kg) i.p. surgical technique and placed in a longitudinal incision of 1.5cm in length will be made in the midline of the ventral cervical skin. The right common carotid artery, ICA and ECA will be exposed and carefully isolated. A nylon monofilament (40mm in length and 0.24mm in diameter), its tip rounded by flame- heating, was inserted from the lumen of the ECA to that of the right ICA to occlude the origin of the right middle cerebral artery (MCA). The right MCA will be occluded for 30 min, and there after brain will be allowed to be reperfused with blood by withdrawing the nylon thread. 24 h after reperfusion, rats will be decapitated. Temperature will be maintained at 37±0.5°C throughout the surgical operation.20

7.2.1 B. Induction of chronic hypoperfusion: For chronic hypoperfusion experiments, animals will be divided into four groups (ten animals each). The first group served as sham-operated control. Second group served as control group, will be treated with Fenugreek (twice a day) for entire experimental period. Animals in the third group (100mg/kg) will be subjected to permanent BCCA occlusion for 15 days and received saline (hypoperfusion group). In the fourth group (200mg/kg), Fenugreek (Trigonella foenum-graecum Linn.) (orally) will be administered 60 min before

15 permanent BCCA occlusion. Fenugreek (Trigonella foenum-graecum Linn.) (twice a day) will be then continued up to the 15th postsurgical day. On day 15 (60 min after last dose of Fenugreek), all animals will be subjected to behavioural assessment to locomotor activity, Beam Walking test and Hanging wire test. Then, under overdose of Thiopentone sodium, animals will be sacrificed by decapitation and brain samples to be collected for infarct size, biochemical estimation and histopathological analysis.21

 Behavioural Procedures: 1. Locomotor activity The locomotor activity will be record by using actophotometer (Inco pvt.ltd. Ambala, India) before locomotor task; animals will be placed individually in the activity meter for 2 min for habituation. Thereafter, locomotor activity will be recorded using actophotometer for a period of 5 min (Kulkarni 1999).22

2. Beam walking test Beam walking test will be used to evaluate gross vestibule motor function. The apparatus consisted of a rod 120 cm in length and with a diameter of 2.3 cm. A wooden box (20 cm X 20 cm X 10 cm) will be set at one end of the rod as a nest for motivating the animal to cross the beam. The apparatus will be suspended 50 cm above a cushion, which protected the animals against fall injury. Rats will be trained twice daily for 2 days before BCCA occlusion and assessed for motor coordination after 24 h of reperfusion. The time taken to traverse the beam will be recorded. The cut-off time will be taken as 120s.23

3. Hanging test This Hanging wire test will be used to measure forelimb grip strength of the rats. In this test, animals will be suspended by the forelimbs on a wire (45 cm long and 0.3 cm diameter) stretched between two posts 40 cm above a foam pillow. The time(s) until the animal fell will be recorded. The cut-off time will be taken as 90 s.24

 Biochemical Estimations: 1. Measurement of Total Protein: The protein content of the brain homogenate will be determined by Lowry’s method using bovine serum albumin as standard the level of protein will be expressed as mg/protein/g of tissue.25

16 2. Measurement of Lipid Peroxidation: The extent of lipid peroxidation will be measured by estimating the amount of malanodialdehyde (MDA) formed, as described by Akhtar et al (2008). Briefly, to 0.1 ml homogenate, 1 ml of 10% (w/v) trichloroacetic acid (TCA) and 1 ml of 0.67% (w/v) thiobarbituric acid will be added and placed in a boiling water bath for 30 min. Then the mixture was placed in crushed ice for 10 min followed by centrifugation at 4000g for 10 min. The absorbance of the clear pink-coloured supernatant will be measured at 532 nm and the results to be expressed as n.mol MDA/mg protein.26

3. Measurement of Total Thiols: The total thiols content will be determined by the method described by Sedlak and Lindsay (1968), with slight modifications. To a mixture of 0.2 ml of homogenate and 0.36 ml of buffer, 0.04 ml of 10 mM DTNB and 1.5 ml of methanol will be added and mixed well. The mixture was centrifuged at 1500g for 5 min at -4°C. Then the intensity of the yellow colour developed was measured at 412 nm and the results to be expressed as n.mol/mg protein.27

4. Measurement of Glutathione: Glutathione-S-transferase (GST) activity will be measured as described by Habig et al. (1974). Briefly, 0.85 ml of Phosphate buffer (pH 7.4), 0.05 ml homogenate and 0.05 ml of 10 mM GSH will be added, to which 0.05 ml of 1 mM CDNB will be added to initiate the reaction. The rate of formation of GSH-CDNB complex will be monitored for 5min at 340nm and the results will be expressed as n.mol of CDNB conjugate formed/min/mg protein.28

5. Measurement of Brain Infarct Area: For the measurement of infarct area, two animals from each group will be decapitated under deep anaesthesia and the brains will be removed and sliced coronally into 2-mm-thick sections, incubated in phosphate-buffered saline (pH 7.4) containing 2% of 2,3,5-triphenyltetrazolium chloride (TTC) for 30 min at 370C and ﬁxed in 10% neutral-buffered formalin overnight29 expressed as a percentage of the total measured brain area.

 Histopathological Studies: A section of the brain will be ﬁxed with 10% formalin, embedded in paraffin wax and cut into sections of 5mm thickness. The sections will be stained with haematoxylin and eosin dye for histopathological observations.30

17  Statistical Analysis: The results will be expressed as mean ± SD for n= 6. The data obtained from the above study will be subjected to statistical analysis using analysis of variance (ANOVA) followed by Dunnets test. Differences with P<0.05 will be considered as statistically significant.

Total duration for the completion of whole project will be 9 months. I. Duration of experiment Eight months II. Literature survey Till the completion of the work 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 80 Wistar albino rats of either sex for antioxidant and neuroprotective activity of Trigonella foenum-graecum Linn.(Fenugreek) on cerebral ischemia.

18 7.4 Has ethical clearance been obtained from your institution in case of 7.3?

Enclosed

ENCLOSURE – VII

LIST OF REFERENCES:

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2. Amanullah, Nazir S, Shams-ur-Rehman, Salma A. Frequency of cerebral infarction and haemorrhage in the patients of stroke. J Ayub Med Coll Abbottabad 2009; 21(4):102-5.

19 3. Feigin VL et al. Epidemiology of ischaemic stroke and traumatic brain injury. Best Pract Res Clin Anaesthesiol 2010; 24(4):485-94.

4. Mohammad R B, Bahram R, Ali A M. In vivo normobaric hyperoxia preconditioning induces different degrees of antioxidant enzymes activities in rat brain tissue. Eur J Pharmacol 2009; 22–29.

5. Oliver CN, Starke-Reed PE, Stadtman ER, Liu GJ, Carney JM, Floyd RA, et al., Oxidative damage to brain proteins, loss of glutamine synthetase activity, and production of free radicals during ischemia/reperfusion-induced injury to gerbil brain. Proc Natl Acad Sci. USA 1990; 87:5144-7.

6. Verma SK, Mathur A, Singh SK. In vitro cytotoxicity of calotropis procera and Trigonella foenum-graecum against Human cancer cells. J Chem Pharm Res 2010; 2(4):861-5

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20 14. Sharma JD, Bhinda A. Antifertility activity of steroidal extract of Trigonella foenum- graecum (seeds) in female rats. Asian J Exp Sci 2005; 19(1):115-20. 15. Petit PR, Hillaire-buys DM, Sauvaire YD, Leconate OM, Baissac YG, Ponsin et al. Steroid saponins from fenugreek seeds: extraction, purification and pharmacological investigation on feeding behavior and plasma cholesterol. Butterworth Heinemann 1995; 60(10):674-80. 16. Nair MG, Liu Y, Kakani R. Compounds in functional food fenugreek spice exhibit anti-inflamatory and anti-oxidant activities. Food Chem 2012; 131(4):1187-92. 17. Hasnain SN, Zia T, Hasan SK. Evaluation of the hypoglycaemic effect of Trigonella foenum-graecum L (Methi) in normal mice. J Ethnopharmacol 2001; 75(2-3):191-5. 18. Ahmadiani A, Parvizpur A, Kamalinejad M. Spinal seratonergic system is partially involved in anti-nociception induced by Trigonella foenum-graecum leaf extract. J Ethnopharmacol 2004; 95:13-7 19. Modaresi M, Mahdian B, Jalalizand A. The effect of Hydro-Alcoholic Extract of Fenugreek Seeds on Female Reproductive Hormones in Mice. Int.Conf.App.Lif.Sci, 2012: 437:443 20. Iwasaky Y, et al. Forebrain ischemia induced by temporary bilateral common carotid artery occlusion in normotensive rats. J Neurol sci 1989; 90:155-65. 21. Pappas BA, et al. Choronic reduction of cerebral blood flow in the adult rat:late emerging CA1 cell loss and memory dysfunction. Brain Res 1996; 708:50-8. 22. Kulkarni SK. Hand book of experimental pharmacology. Vallabh Prakashan, New Delhi 1999; 117–9. 23. Song YN, et al. Histamine improves rat rota-rod and balance beam performances through H2 receptors in the cerebellar interpositus nucleus. Neurosci 2006; 140:33–43. 24. Hunter AJ, Hatcher J, et al. Functional assessments in mice and rats after focal stroke. Neuropharmacol 2000; 39:806–16. 25. Lowry OH, et al. Protein measurement with folin phenol reagent. J Biol chem 1991; 193:265-75 26. Akhtar M, Pillai KK, Vohora D. Effect of thioperamide on oxidative stress markers in middle cerebral artery occlusion model of focal cerebral ischemia in rats. Hum Exp Toxicol 2008; 27:761–7. 27. Sedlak J, Lindsay R. Estimation of total protein-bound and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 1968; 25:192–205.

21 28. Habig WH, Pabst MJ, Jarkoby WB. Glutathione. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974; 249:7130–9. 29. Bederson JB, et al. Evaluation of 2, 3, 5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infraction in rats. Ind J Pharmacol 2008; 40:215-20. 30. Thipeswamy BS, Nagakannan P. Protective Effect of Emblin against Transient Global Ischemia induced brain damaged, Neurotox Res 2011; 20:379-86.