NEUROPROTECTIVE ACTIVITY OF Valeriana officinalisAGAINST GLOBAL CEREBRAL ISCHEMIA / REPERFUSION AND ALUMINIUM FLUORIDE INDUCED OXIDATIVE STRESS IN RAT MODEL

Protocol of Dissertation Submitted

By

Mr. SANTOSH PRASAD MAURYA

To

RajivGandhiUniversity of Health Sciences

Bangalore, Karnataka.

Under the guidance of

Dr. I. S. MUCHCHANDI

Principal and Professor

Department of Pharmacology

HANAGALSHRIKUMARESHWARCOLLEGE OF PHARMACY

BAGALKOT-587101, KARNATAKA

(2012-2013)

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

KARNATAKA-BANGALORE

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR

DISSERTATION

1. / Name of the candidate and address / SANTOSH PRASAD MAURYA
Department of Pharmacology,
H.S.K.College of Pharmacy,
B.V.V.S campus,
Bagalkot-587101, Karnataka.
2. / Name of institution / H.S.K.College of Pharmacy,
B.V.V.S campus,
Bagalkot-587101, Karnataka.
3. / Course of study and subject / Master of Pharmacy inPharmacology.
4. / Date of admission to course / 25/ 06/ 2012.
5. / Title of topic:Neuroprotective activity of Valeriana officinalisagainst Global cerebralischemia/ reperfusion and Aluminium fluoride induced oxidative stressin ratmodel.
6.
7.
8. /

BRIEF RESUME OF THE INTENDED WORK

6.1Need for the study:
In western countriesCerebrovascular diseases (CVD) is third most common cause of death and second most common cause of neurologic disability after Alzheimer’s diseases. The major types of cerebrovascular diseases are cerebral insufficiency caused due to transient disturbance or infraction due to embolism or thrombosis due to intra cranial or extra cranial arteries1. Cerebral ischemia is caused by a reduction in blood flow that last longer than several seconds. When the blood flow is quickly restored, brain tissue can be recovered fully and patient’s symptoms are only transient, so it is called as transient ischemic attack. The stroke or ischemic stroke has occurred, if the neurological signs and symptoms last for several hours. In more severe instances global hypoxic ischemia causes wide spread brain injury and infractions in various regions of brain2.
Neuronal death is a complex event involving failure of metabolic process, exocitotoxicity, loss of calcium homeostasis among the other factors and fluoride contamination causes severe exocitotoxicity and organ oxidative stress. Due to oxidative stress, the activated intracellular metabolic events lead to generation of oxygen free radicals, which overcomes through antioxidant defenses and here you require proper antioxidant drugs to reduce the oxidative stress.3
At the present, state of knowledge of treatment of ischemic stroke is not adequate and the allopathic drugs for treatment of strokes are not sufficient and not much benefited. So, natural products (medicinal plants based products) probably represent an ideal source to develop safe and effective agents for management of stroke and desire scientific proofs. The scientifically validated flavonoids4 and Gingko biloba5, Ginseng6and have reported successfully recovery from ischemia / reperfusion injuries in the brain and oxidative stress. Author has fascinated with this background and thepresent study will be propose to know the safe and potential neuroprotective effect ofValeriana officinalisagainst global cerebral ischemia and Aluminium fluoride induced oxidative stress in pre-clinical model studies will be carried out.
6.2 REVIEW OF LITERATURE
Plant Profile-
Title of the plant :Valeriana officinalis
Family : Valerianaceae
Synonyms : Garden Valerian, Garden Heliotrope, All-heal, Bala Hrivera, Tagar.
Habitat : Native to Europe and parts of Asia, valerian has been introduced into NorthAmerica. It is consumed as food by the larvae of some Lepidoptera (butterflyandmoth) species including Grey Pug.
Parts used : Root and rhizome (fresh or dried)7
Chemical constituents :
This plant contain alkaloids are valeranine, chatinine, alpha-methyl pyrrylketone, actinidine, skyanthine, naphthyridylmethylketone8-11& volatile essential oil (0.2 – 02.8%) include bornyl isovalerenate, bornyl acetate, valerenic, valeric, isovaleric, acetoxyvalerenic acids, valerenal, valeranone, cryptofaurinol.12Iridoid valepotriates (0.5% -2.0%) are valtrates, isovaltrate, didrovaltrate, valerosidate13
6.3 Medicinal uses :
Valeriana officinalisis used in disorder ofspinal marrow and the nerves; nervous debility and failing reflexes; also as hypotonic, and in spastic disorders like chorea, gastrospasms etc. Valeriana officinalis belongs to the principle remedies of insomnia, especially where due to nervous exhaustion and mental overworkand recent researchers have found it useful in neurosis in epilepsy and also in scorpion-sting.14Valerian root has been used as a popular anticonvulsant remedy in Europe and Iran in the past centuries.15
Pharmacological actions :
It act asstimulant and antispasmodic. Valerian is not only a nervine in the sedative and hypnotic sense but also useful analeptic stomachic and calmative.16
6.4 OBJECTIVES OF THE STUDY :
The traditionally well-known medicinal plants Valeriana officinaliswill be subjected to extraction with polar and non-polar solvents and dried extract was used for the present study.
The objectives of present study are;
  1. Neuroprotective activity ofValeriana officinalisextracts against global cerebral ischemia / reperfusion in rat models.
  2. Neuroprotective activity ofValeriana officinalis extracts against Aluminium fluoride Induced oxidative stress in rat models.

MATERIALS AND METHODS:

7.1 Source of data:
All the data will be collected from the animal experiments and standard parameters for the study. The selection of doses will be based on earlier literature acute toxicity studies17.
7.2 Materials :
Chemicals : 2-Thiobarbituric acid,Trichloroacitic acid, Aluminium fluoride.
Animals : Sprague dawley rats of either sex.
Instruments : Surgical microscope, Refrigerator centrifuge, Tissue homogenizer, UV-Spectrophotometer.
7.3 Method:
7.3.1 Global cerebral ischemia/reperfusion model :
The animal will be divided into different groups:
Group-I : Normal saline (10 ml/kg), orally. (n=6).
Group-II : Sham control (10 ml/kg), normal saline orally. (n=6).
Group-III : Normal saline (10 ml/kg,) oral, BCA occlusion for 30 min,followedby reperfusion for 24 hr. (n=6).
Group-IV: V.O.extract, low dose (100 mg/kg), BCA occlusion for 30 min, followed by reperfusion for 24 hr. (n=6).
Group-V : V.O.extract, moderate dose (200 mg/kg), BCA occlusion for 30 min,followed by reperfusion for 24 hr. (n=6).
Group-VI :V.O.extract, high dose (300 mg/kg), BCA occlusion for 30 min, followed by reperfusion for 24 hr. (n=6).
7.3.2 Experimental protocol for Aluminium fluoride induced oxidative stress
The animal will be divided into different groups:
Group-I : Normal saline (10 ml/kg, orally) (n=6)
Group-II : Aluminium fluoride (600 ppm through their drinking water) (n=6)
Group-III : V.O. extract (100 mg/kg orally) + Aluminium fluoride (n=6)
Group-IV : V.O. extract (300 mg/kg orally) + Aluminium fluoride (n=6)
Group-V : V.O. extract (500 mg/kg orally) + Aluminium fluoride (n=6)
7.4 Methods :
7.4.1 Preparation of plant extract:
The roots are authenticated and collected in ideal conditions, will be air dried, sieved and powdered to a fine powder and passed through a sieve # 44. The collected powder will be extracted using polar and non polar solvents.
7.4.2 Global Cerebral ischemia:
Animals will be subjected to bilateral carotid artery occlusion under Ketamine anesthesia (45 mg/kg, i.p.). Animals has to be placed on back, both carotid artery will be exposed and clamped by using atraumatic micro-vascular clamp. Temperature will be maintained around 37 ± 0.5ºC throughout the surgical procedure19.
7.4.3 Effect of extract on Aluminium fluoride induced oxidative stress:
To determine at which doses the extract shows maximum activity for neuroprotective. The different groups of rats were separately treated with different doses of extract (low, moderate and high) according to body weight for 10 days prior to Aluminium fluoride treatment through their drinking water (7days at a dose of 600 ppm). 24 hours after the final dose of Aluminium fluoride administration, all the rats were sacrificed, the brain tissue is homogenated and collected and stored in (-4ºC) cold condition18.
7.4.4 Effect of extract on biochemical profile:
In ischemia reperfusion model, the brain from each animal will be removed and subjected for brainhomogenate (10% w/v) in cold phosphate buffer (pH 7.4). The homogenate will be centrifuged at 10,000 rpm for 20 min at 4ºC and the supernatant has to be used for lipid peroxidation assay, total protein estimation20, glutathione estimation21, superoxide dismutase22 and catalase23 will be analyzed using spectrophotometric method.
7.4.5 Histopathological examination:
Two animals from each group will be sacrificed on the day of blood withdrawal and brain will be isolated and tissue will be immersed in 10 % formalin solution for histopathological studies. Sample will be embedded in paraffin sectioned and stained with haemotoxollin and eosin24.
7.4.6 Statistical analysis:
All the data is has to express in mean ± SEM. The significance of differences in mean between control and treated animals for different parameters determined by one way ANOVA followed by Dunnett’s multiple comparison test. Significance for difference between groups will be evaluated forstudent’s t-test to come to final conclusion.
7.5Does the study require any investigations or interventions to be conducted on patients or other humans/animals? If so please describe briefly.
Yes, the above study requires to be carried out in rats. So for this study Sprague dawley rats will be use.
7.6Has ethical clearance been obtained from your institution in case of 7.2 and 7.3?
Yes, the study is cleared from Institutional Animal Ethics Committee. The copy is enclosed with this protocol.
REFERENCES:
  1. Pereiar MH, Beers MD, Berkow R. The Merck manual 17th Ed. Merck ResLabs 1999;1417-1466.
  2. Wade SS, JohnsonSC, Easton JD. Cerebrovascular disease principle’s of internal medicine. McGraw Hill2:2372.
  3. Spittle B. Comments on the findings of toxin-induced blood vessels inclusions andalteration in neuronal and cerebrovascular integrity following the chronic administration ofaluminium fluoride and sodium fluoride. Fluoride31(2):89-90.
  4. Dajas F, Rivera-Megret F, Blasina F. Neuroprotection by flavonoids, BrazJ Medl Biol Res2003;36:1613-1620.
  5. Lee EJ, Chen HY, Wu TW, Chen TY, Ayoub IA, Maynard KI, (2002). Acuteadministration of Ginkgo bilobaextract (Egb 761) affords Neuroprotection againstpermanent and transient focal cerebral ischemia in Sprague-Dawley rats. JNeurosci Res68:636-645.
  6. Shah ZA, Gilani RA, Sharma P, Vohora SB. Cerebroprotective effect of Korean ginsengtea against global and focal models of ischemia in rats, JEthnopharmacol 1012005; 299-307.
  7. Joseph IB and Angela MN. Safety Issues with Herbal Medicine: Common Herb MedPharmacother2000;20(3).
  8. Franck B, Petersen U, Huper F. Valerianie, a tertiary monoterpene alkaloid from valerian (1). Angew Chem Int Ed Engl 1970;9:891.
  9. Janot MM, Guilhem J, Contz O, Venera G, Cionga E. Contribution to the study of valerian alkaloids: actinidine and naphthyridylmethylketone, a new alkaloid. Ann Pharm Fr1979;37:413-20.
  10. Duke JA. CRC handbook of medicinal herbs. Boca Raton: CRC Press 1985.
  11. Torssell K, Wahlberg K. Isolation, structure and synthesis of alkaloids from Valerianaofficinalis L. Acta Chem Scand 1967;21:53-62
  12. Morazzoni P, Bombardelli E. Valeriana officinalis: traditional use and recent evaluations of activity. Fitoterapia 1995;66:99-112.
  13. Becker H, Chavadej S. Valepotriate production of normal and colchicine-treated cell suspension cultures ofValeriana wallichii. J Nat Prod 1985;48:17-21.
  14. Nandakarni KM. Indian Materia Medica. Popular Prakashan. Mumbai. India2007;2(1):1260-1261.
  15. Gorji A,Khaleghi M. Ghadiri History of epilepsy in Medieval Iranian medicineNeurosci Biobehavl Revs 2001;25:455-461
  16. Nandakarni KM. Indian Materia Medica. Popular Prakashan. Mumbai. India.2007;2(1):1260
  17. Joshi PV, Shirkhedkar A, Prakash K. Antidiarrheal activity, chemical and toxicity profile ofBerberis aristata. Pharm Biol 2010;123.
  18. Sinha M, Manna P, Parames C. Aqueous extract of the bark of Terminalia arjunaplays a protective role against sodium- fluoride -induced hepatic and renal stress, J Nat Med 2007;6:251-260.
  19. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke American Heart Association 1989;20:84-91.
  20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-Phenol reagent. J BiolChem1951;193:265-272.
  21. Jollow DJ, Mitchell JR, Zampaglione N, Gillete JR. Bromobenzene induced liver necrosis: protective role of glutathione and evidence for 3, 4-bromobenzene as the hepatotoxicmetabolite. J Pharmacol 1974;11:151-169.
  22. Beauchamp C, Fridovich I. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971;44:276-287.
  23. Claiborne A. Catalase activity. In: Greenwald. USA CRC Hand Book of Methods for Oxygen Radical Research. CRC Press Florida1985;283-284.
  24. Carbonell LF, Salazai FJ, Garcia EJ, Ubeda M, Quesada T. Normal homodyna mice parameters in conscious rats. Rev Exp Physiol 1985;4:437-442.

9 / SIGNATURE OF THECANDIDATE / SANTOSH PRASAD MAURYA
10 / REMARKS OF THE GUIDE / Valeriana officinalishas been mentioned as very good medicinal plant in various literatures. The present study will be evaluated for its neuroprotective activity scientifically was recommended.
11 / NAME AND DESIGNATION OF THE GUIDE / Dr. V.M. Chandrashekhar
(Associate Professor)
12 / SIGNATURE
13 / CO-GUIDE / ------
14 / SIGNATURE / ------
15 / HEAD OF THE DEPARTMENT / Dr. V.M. Chandrashekhar
(Associate Professor)
16 / SIGNATURE
17 / REMARKS OF THE PRINCIPAL / The above mentioned information is correct and I recommended the same for approval.
18 / NAME OF THE PRINCIPAL / Dr. I.S. MUCHCHANDI
19 / SIGNATURE

OFFICE OF THE INSTITUTIONAL ANIMAL ETHICS COMMITTEE (IAEC)

HANGAL SHRIKUMARESHWARCOLLEGE OF PHARMACY,

BAGALKOT-587101, KARNATAKA.

REG NO.821/01/a/CPCSEA, Dated:6th AUG 2004 UNDER THE RULES 5(a) OF THE

“BREEDING OF AND EXPERIMENTS ON ANIMAL (control and supervision)

RULES 1998”

Ref: HSK CP/IAEC, Clear/2011-12/1-8

CERTIFICATE

This is to certify that Mr. Santosh Prasad Maurya, A student of first M.Pharm ispermitted to carry out experiments on animal for the dissertation / thesis work entitled as “Neuroprotective activity of Valeriana officinalisagainst Global cerebral ischemia and Aluminium fluoride induced oxidative stress in rat model.” as per details mentioned and after observing the usual formalities lay down by IAEC as per provision made by CPCSEA.

Animal house in charge Chairman

Form B

See rule [6 (a) and 8 (a)]

PART A

1] / Name and address of the establishment: / H.S.K.COLLEGE OF PHARMACY,
BAGALKOT, KARNATAKA.
2] / Date and registration Number of the
Establishment: / 821 /01/a CPCSEA.
3] / Name, address and registration NO. of the
Breeder from whom acquired and the date of
Acquisition: / OFFICE OF CPSEA,
MINISTRY OF ENVIRONMENT AND
FOREST, 3RD SEAWARD ROAD,
VALMIKINAGAR,THRIRUVANMIYUR,
CHENNAI-600041.
4] / Place where the animals are presently kept: / ANIMAL HOUSE,
H.S.K.COLLEGE OF PHARMACY,
BAGALKOT, KARNATAKA.
5] / Place where experiment is to be performed: / DEPARTMENT OF PHARMACOLOGY,
H.S.K.COLLEGE OF PHARMACY,
BAGALKOT, KARNATAKA.
6] / The date on which the experiment is to be commence and the duration of the experiment: / 15th June 2013 6 Month

The protocol form for the research proposal - PART B in the case of experiments using other than non-human primate animals for a few projects, PART C for use of non-human primate for new projects and PART D for use of non-human primate for extension of ongoing projects-should be duly filled, signed and annexed with this form.

Dated: Signature

Place: (NAME AND DESIGNATION)

PART –B
Protocol form for research proposal to be submitted to the committee on use of small animals / animals other than non-human Primate in biomedical research for ONGOING / NEW PROJECTS.
1. / Project title: / “Neuroprotective activity ofValeriana officinalisagainst Global cerebral ischemia and Aluminium fluoride induced oxidative stress in rat model.”
2. / Investigation (s):
Designation / Dr. I. S. MUCHCHANDI
(Principal)
3. / Department(s): / Department of Pharmacology,
H.S.K.College of Pharmacy,
Bagalkot, Karnataka.
S
4. / (a) Funding source (s) : if any / ----
(b) Are sufficient funds available for purchase and maintenance of the animals / Yes.
(c) / Duration of present project:
(1) Number of months: / 6 months.
(2) Date of start of the project:
( Experiment) / 15th June 2013.
(3) Date of termination of the project: / 15th December 2013.
5. / Date by which approval is needed in case the project is to be funded by outside agency (If less than six weeks from the date of admission, please justify below). / ----
6. / Summary of project briefly summarize in laymen’s term the background, the objective and the experimental approach.
(a)Background: / Enclosed.
(b)Objectives: / Enclosed.
(c)Experimental procedure: / Enclosed.
7. / (a) / Name of species / Sprague- Dawley rats.
Age / Sex / Weight
4-8 weeks / Either sex / 200-250 g
(b) / Rationale for selection
Approximate number of animals required during the first 12 weeks. / 66
Justification of number (define treatment group and number per group) / Eleven groups, each group containing six animals.
Number of animals housed per week / 16
8. / List all invasive Non-Surgical Animals Procedures and Potentially stressful Non-invasive procedures to be used (example: IM injection, foot pad injection, venapunctures). / Invasive surgical animal procedures.
Procedure and approximate frequency: / Enclosed.
Anaesthetic and /or Analgesic and dosage: / Ketamine hydrochloride 45 mg/kg, i.p.
Test substance injected and /or applied: / Test substance will administer orally.
9. / Does the protocol prohibit the use of anaesthetic and analgesic for the conduct of painful procedures?
No.
10. / With surgical procedure/Experimental procedure be performed?
Yes.
(a)Will the animal be sacrificed after surgery?
Yes.
(b)Give anticipated post-operative survival time:
Yes.
11. / Will hazardous agent such as radio isotopes, carcinogens, radiation exposure, microbial and parasitic agent be administered to animals?
No.

DATE: INVESTIGATOR SIGNATURE

1