Neuroprotective activity of valeriana officinalis against parkinson’s disease and aluminium chloride induced oxidative stress in rat model




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NEUROPROTECTIVE ACTIVITY OF VALERIANA OFFICINALIS

AGAINST PARKINSON’S DISEASE AND ALUMINIUM CHLORIDE INDUCED OXIDATIVE STRESS IN RAT MODEL

Protocol of Dissertation Submitted

By

Mr. VINAY KUMAR DUBEY

To

Rajiv Gandhi University of Health Sciences



Bangalore, Karnataka.

Under the guidance of



Dr. V.M. CHANDRASHEKHAR

Associate Professor





Department of Pharmacology

HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY

BAGALKOT-587101, KARNATAKA

(2012-2013)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA-BANGALORE
ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR

DISSERTATION


1.

Name of the candidate and address




VINAY KUMAR DUBEY

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 in Pharmacology




4.

Date of admission to course


25/06/2012






5.

Title of topic : Neuroprotective activity of Valeriana officinalis against Parkinson’s disease



and Aluminium chloride induced oxidative stress in rat model.






6.

7.

8.



BRIEF RESUME OF INTENDED WORK
6.1. NEED FOR THE STUDY
Parkinson’s Disease (PD) is a progressive multifocal CNS degenerative disease. PD usually manifest with clinical feature that are the consequence of the degeneration of dopaminergic nigrostriatal neurons. These include the typical motor symptoms like bradykinesia, rigidity & tremor. The above are the degeneration of non dopaminergic as well as dopaminergic pathways. PD shortens life expectancy causes significant decline in quality of life for both patients & care givers and it is a significant economic burden on families & society.1 The etiology of Parkinson’s Disease are of two types- Primary PD caused due to pigmented neurons of substantia nigra, locus cerruleus and other brain stem dopaminergic cell group are lost. Secondary PD, results from loss of or interference with the action of dopamine in basal ganglia due to other idiopathic degenerative diseases. The mean age of onset is about 57 yr 2.

At the present state, knowledge of treatment of PD is not adequate and allopathic drugs for treatment of PD are not sufficient and not much benefited. In this concern, the natural products (medicinal plants based products) probably represent an ideal source to develop safe and effective agents for management of PD3.



Aluminium chloride caused oxidative stress, enhancement of free radicals and alterations in antioxidant enzymes in both in vivo and in vitro.4,5 Salts of aluminium may bind to DNA, RNA, inhibit such enzymes as hexokinase, acid and alkaline phosphates, phosphodiesterase and phosphooxydase6. Aluminium exposure caused impairments in glucose utilization, agonist-stimulated inositol phosphate accumulation, free radical-mediated cytotoxicity, lipid peroxidation, reduced cholinergic function, impact on gene expression and altered protein phosphorylation7.

The present study aims to know the safe and potential neuroprotective effect of Valeriana officinalis, in Parkinson’s Disease and oxidative stress pre-clinical rat models.



6.2. REVIEW OF LITERATURE

Plant Profile-

Title of the plant : Valeriana officinalis

Family : Valerianaceae

Synonyms : Garden Valerian, Garden Heliotrope, All-heal, Bala Hrivera, Tagar8

Habitat : Native to Europe and parts of Asia, it has been introduced into North

America. It is consumed as food by the larvae of some Lepidoptera

(Butterfly and moth) species including Grey Pug.



Parts used : Root and rhizome (fresh or dried)9

Chemical constituents : This plant contain alkaloids are valeranine, chatinine, alpha-methyl pyrrylketone, actinidine, skyanthine, naphthyridylmethylketone10-13 & volatile essential oil (0.2 – 02.8%) include bornyl isovalerenate, bornyl acetate, valerenic, valeric, isovaleric, acetoxyvalerenic acids, valerenal, valeranone, cryptofaurinol.14 Iridoid valepotriates (0.5% -2.0%) are valtrates, isovaltrate, didrovaltrate, valerosidate.15

Medicinal uses :

Valeriana officinalis is used in disorder of spinal marrow and the nerves, nervous debility and failing reflexes, also as hypotonic, and in spastic disorders like chorea, gastrospasms etc. It belongs to the principle remedies of insomnia, especially where due to nervous exhaustion and mental overwork and recent researchers have found it useful in neurosis in epilepsy and also in scorpion-sting.16

Valerian root has been used as a rather popular anticonvulsant remedy in Europe and Iran in the past centuries.17
Pharmacological actions :

It acts as stimulant and antispasmodic. Valeriana officinalis is not only a nervine in the sedative and hypnotic sense but that it is useful analeptic, stomachic and calmative18.



6.3. OBJECTIVE OF STUDY

The traditionally well known Valeriana officinalis (V.O.) will be extracted by using non-polar and polar solvents and extract will be used for the present study with following objectives-



  1. Neuroprotective activity of Valeriana officinalis extracts against 6-OHDA induced Parkinson’s

Disease in rat model.

2) Neuroprotective activity of Valeriana officinalis extracts against Aluminium chloride induced

oxidative stress in rat model.

MATERIALS AND METHODS:


    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 studies. 19

    2. Materials:

Chemicals : 6-OHDA, 2-Thiobarbituric acid, Trichloroacitic acid, Aluminium chloride

Animals : Sprague dawley rats of either sex.

Instruments : Surgical nylon thread, Surgical microscope, Refrigerator centrifuge,

UV-Spectrophotometer, Stereotaxic instrument



    1. Method:

      1. 6-OHDA induced Parkinson’s Disease:

The animals will be divided into different groups:

Group- I : Normal animals receive normal vehical orally (n=6).

Group-II : Sham receives 3µl of 0.2% L-ascorbic acid (L-AA) intra striatally & vehical orally (n=6).

Group-III : Control animals receive 6-OHDA in 0.2% L-AA intra striatally & vehical orally (n=6).

Group-IV : Treated receives 6-OHDA in 0.2% L-AA intra striatally & low dose of VO (n=6).

Group-V : Treated receives 6-OHDA in 0.2% L-AA intra striatally & moderate dose of VO (n=6).

Group-VI : Treated receives 6-OHDA in 0.2% L-AA intra striatally & high dose of VO (n=6).

7.3.2 Aluminium chloride induced oxidative stress:

The animal will be divided into different groups:

Group-I : Normal saline (10 ml/kg, orally) (n=6).

Group-II : Aluminium chloride (600 ppm through their drinking water) (n=6).

Group-III : V.O. extract (100 mg/kg orally) + Aluminium chloride (n=6).

Group-IV : V.O. extract (300 mg/kg orally) + Aluminium chloride (n=6).

Group-V : V.O. extract (500 mg/kg orally) + Aluminium chloride (n=6).

7.4. Methods:

7.4.1. Preparation of plant extract:

The plant will be authenticated and collected from NBRI, Lucknow in ideal conditions, will be air dried under the shade & powdered to a fine texture of uniform size by passing through the sieve # 44. The powder collected will be extracted with polar and non polar solvents and the extract obtained will be used for the present study.



7.4.2.1 6-OHDA induced Parkinson’s Disease: The dose will be selected on the basis of acute toxicity (OECD) guidelines. The different groups of rats will be separately treated with different doses of extract (low, moderate and high) according to body weight for a week before to 6-OHDA lesion. The striatal 6-OHDA lesions will be performed by using 28-gauge cannula was inserted vertically into the striatum to a depth of 5.5 mm at a rate of 1µl/min, 3.5 µl of 6-OHDA in 0.9% normal saline containing 0.2% ascorbic acid will be injected. The same treatment will be continued after the lesion for a week20.

7.4.2.2. Behavioural assessment: Deficits in forepaw adjusting steps in this PD rat model provided a simple and consistent behavior phenomenological similar to akinesia in PD. After the 6-OHDA lesion, rats will be held by the rear part of the torso and placed on the surface of treadmill so that its weight was on one forepaw. The treadmill will be set to move at a rate of 90 cm/12s in the direction opposite to the weight-bearing forepaw resulting in the outward lateral shifting of the torso relative to the weight-bearing forepaw. The number of adjusting steps, defined as the movement of weight-bearing forepaw towards the torso to compensate for the outward lateral movement of the body was counted. Each stepping test consisted of five trials for each forepaw, alternating between forepaws, and each trial lasted 12s. The average of the five trials for each forepaw will be used for analysis. Adjusting steps will be tested in each group of animals at the beginning of the experiment and 2, 4, 6 and 8 weeks after the lesion.21

7.4.2.3. Aluminium chloride induced oxidative stress: 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 chloride treatment through their drinking water (7days at a dose of 600 ppm). 24 hours after the final dose of Aluminium chloride administration, all the rats were sacrificed, the brain tissue is homogenated and collected and stored in (-4ºC) cold condition.22

7.4.2.4 Effect of extract on biochemical profile: The 10% homogenated brain tissue, in cold phosphate buffer (pH 7.4). The homogenate will be centrifuged at 10,000 rpm for 20 min at 4ºC and the supernatant will be used for lipid peroxidation assay, total protein estimation23, glutathione estimation24, superoxide dismutase25 and catalase26 will be analyzed using spectrophotometric method.

7.4.3 Histopathological examination: Two animals from each 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 eosin.27

7.4.4 Statistical analysis: All the data will be expressed in mean ± SEM. The significance of differences in mean between control and treated animals for different parameters determined by one way ANOVA followed by Turkey’s multiple comparison test. Significance for difference between groups were evaluated for student’s t-test to come to final conclusion.

7.5 Does the study require any investigations or interventions to be conducted on patients or other humans/animals? I so please describe briefly.

Yes, the above study requires to be carried out in rats. So, Sprague dawley rats will be used.

7.6 Has 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. Anthony HV, Schapira Jose Obeso. Timing of treatment initiation in Parkinson's disease: A need for reappraisal? Annals of Neurology. 2006;59:559–562.

  2. Pereira Mark H, Beers MD, Berkow R. The merck manual 17th edition Merck Research Labs. 1999;1417-1466.

  3. Marry AKK, Lloyd YY, Brian KA, Robin LC, Wayne AK. Applied therapeutics the clinical use of drug. WoltersKlawer / Lippincott Williams and Wilkins 9th edition. 2009;53-61.

  4. Yousef MI, El-Morsy AMA, Hassan ME. Aluminium-induced deterioration in reproductive performance and seminal plasma biochemistry of male rabbits: Protective role of ascorbic acid. Toxicology. 2005;215:97–107.

  5. Yousef MI, Kamel IK, El-Guendi IE, El-Demerdash FM. An in vitro study on reproductive toxicity of aluminium chloride on rabbit sperm: the protective role of some antioxidants. Toxicology. 2007;239:213–223.

  6. Ochmanski W, Barabasz W. Aluminium-occurrence and toxicity for organisms. Przegl. Lek. 2000;57:665–668.

  7. Strong MJ, Garruto RM, Joshi JG, Mundy WR, Shafer TJ. Can the mechanisms of aluminum neurotoxicity be integrated into a unified scheme? J. Toxicol. Environ. Health. 1996;48:599–613.

  8. Nandakarni KM. Indian Materia Medica. Popular Prakashan. Mumbai. India.2007;2(1):1259

  9. Joseph IB, Angela MN. Safety Issues with Herbal Medicine: Common Herb Med Pharmacotherapy. 2000;20 (3).

  10. Franck B, Petersen U, Huper F. Valerianie, a tertiary monoterpene alkaloid from valerian. Angew Chem Int Ed Engl 1970;9:891.

  11. Janot MM, Guilhem J, Contz O, Venera G, Cionga E. Contribution to the study of valerian alkaloids (Valeriana officinalis,L.):actinidine and naphthyridylmethylketone, a new alkaloid. Ann Pharm Fr 1979;37:413-20.

  12. Duke JA. CRC handbook of medicinal herbs. Boca Raton: CRC Press; 1985.

  13. Torssell K, Wahlberg K. Isolation, structure and synthesis of alkaloids from Valeriana officinalis L. Acta Chem Scand 1967;21:53-62.

  14. Morazzoni P, Bombardelli E. Valeriana officinalis: traditional use and recent evaluations of activity. Fitoterapia. 1995;66:99-112.

  15. Becker H, Chavadej S. Valepotriate production of normal and colchicines-treated cell suspension cultures of Valeriana wallichii. J Nat Prod. 1985;48:17-21.

  16. Nandakarni KM. Indian Materia Medica. Popular Prakashan. Mumbai. India. 2007;2(1):1260.

  17. Gorji A, Khaleghi M. Ghadiri. History of epilepsy in Medieval Iranian medicine Neuroscience and Biobehavioral Reviews. 2001;25:455–461.

  18. Nandakarni KM. Indian Materia Medica.Popular Prakashan. Mumbai. India. 2007;2(1):1260.

  19. Joshi PV, Shirkhedkar A, Prakash K. Antidiarrheal activity, chemical and toxicity profile of Berberis aristata. Pharm Biol. 2010;123.

  20. Kim MS, Lee JI, Lee WY, Kim SE. Neuroprotective effect of Ginkgo biloba L. extract in rat model of parkinson’s disease.Phytother Res. 2009;18:663-666.

  21. Chang JW, Wachtel SR, Young D, Kang UJ. Biomedical and anatomical characterization of forepaw adjusting steps in rat models of parkinson’s disease studies on medial forebrrain bundle and striatal lesions.Neurosci. 1999;88:617-628.

  22. Sinha M, Manna P, Parames C. Aqueous extract of the bark of Terminalia arjuna plays a protective role against sodium- fluoride -induced hepatic and renal stress.J Nat Med. 2007;61:251-260.

  23. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin- Phenol reagent.J BiolChem. 1951;193:265-272.

  24. Jollow DJ, Mitchell JR, Zampaglione N, Gillete JR. Bromobenzene induced liver necrosis: protective role of glutathione and evidence for 3,4-bromobenzene as the hepatotoxic metabolite. J Pharmacol. 1974;11:151-169.

  25. Beauchamp C, Fridovich I. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal Biochem. 1971;44:276-287.

  26. Claiborne A. Catalase activity. In: Greenwald. USA CRC Hand Book of Methods for Oxygen Radical Research.CRC Press Florida. 1985;283-284.

  27. Carbonell LF, Salazai FJ, Garcia EJ, Ubeda M, Quesada T. Normal homodyne mice parameters in conscious rats.Rev Exp Physiol. 1985;41:437-442.







9


SIGNATURE OF THE CANDIDATE




VINAY KUMAR DUBEY


10


REMARKS OF THE GUIDE



Valeriana officinalis has 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



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 SHRI KUMARESHWAR COLLEGE 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. Vinay Kumar Dubey, A student of first M.Pharm. is permitted to carry out experiments on animal for the dissertation / thesis work entitled as “Neuroprotective activity of Valeriana officinalis against Parkinson’s Disease and Aluminium chloride 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 CPCSEA,

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 :


15 June 2013, 6 months



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 of Valeriana officinalis against Parkinson’s Disease and Aluminium chloride induced oxidative stress in rat model.


2.

Investigation (S):

Designation





Dr. V.M. Chandrashekhar

Associate professor

3.

Department(S):


Department of Pharmacology

H.S.K.College of Pharmacy,

Bagalkot, Karnataka.

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)

Six groups, each group containing eight 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:
NO

11.

Will hazardous agent such as radio isotopes, carcinogens, radiation exposure, microbial and parasitic agent be administered to animals?
No.

DATE:

INVESTIGATOR SIGNATURE





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