Download Rajiv Gandhi University of Health Sciences, Karnataka

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.
PROFORMA FOR REGISTRATION OF SUBJECT (DRAVYAGUNA VIGNANA) FOR
DISSERTATION MD (AYURVEDA)
“EVALUATION OF NEUROPROTECTIVE EFFICACY OF
VARUNITAILA (Citrullus colocynthis Schard) IN KAMPAVATA W.S.R TO
PARKINSON’S DISEASE USING ROTENONE INDUCED ANIMAL
MODEL”
By
Dr. TAUSIF AHMED
I P.G. Scholar
Department of Post Graduate Studies in Dravyaguna Vignana
J.S.S. Ayurveda Medical College and Hospital,
Mysore, Karnataka.
GUIDE
Dr. SHIVAPRASAD HUDED
MD(AYU)
Reader
Department of Post Graduate Studies in Dravyaguna Vignana
J.S.S. Ayurveda Medical College and Hospital,
Mysore, Karnataka.
CO-GUIDE
Dr. SURESHA. R.N
Professor & HOD
Department of Pharmacology
JSS Medical College, Mysore,
Karnataka.
2012-2013
J.S.S. Ayurveda Medical College and Hospital
Mysore, Karnataka
1
From
Dr. TAUSIF AHMED
I P.G. Scholar
Department of Post Graduate Studies in Dravyaguna Vignana
J.S.S. Ayurveda Medical College,
Mysore
To
The Registrar
RajivGandhiUniversity of Health Sciences,
Bangalore, Karnataka.
Through
The Principal and Head of the Department of P.G. Studies in Dravyaguna,
J.S.S. Ayurveda Medical College,
Mysore
Respected Sir,
Sub: Submission of Completed Proforma for Registration of subject for Dissertation
I request you to kindly register the below mentioned subject against my name for the
submission of dissertation to the Rajiv Gandhi University of Health Sciences, Bangalore, for the partial
fulfillment of M.D. (Ayurveda) in Dravyaguna Vignana.
Title of the Dissertation:
“EVALUATION OF NEUROPROTECTIVE EFFICACY OF
VARUNITAILA (Citrullus colocynthis Schard) IN KAMPAVATA W.S.R TO
PARKINSON’S DISEASE USING ROTENONE INDUCED ANIMAL
MODEL”
Herewith I am enclosing completed Proforma for Registration of the Subject for dissertation
Thanking you,
Yours faithfully,
Date: 25-04-2013
Place: Mysore
(Dr. TAUSIF AHMED)
2
RAJIVGANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.
Name of the Candidate
Address
Dr. TAUSIF AHMED
2.
Name of the Institution
J.S.S. Ayurveda Medical College, Mysore
3.
Course of the Study and Subject
AYURVEDA VACHASPATHI
M.D. (Ayu) in Dravyaguna
4.
Date of Admission to Course
22-12-2012
5.
Title of the Topic
I P.G. Scholar
Department of Post Graduate Studies in Dravyaguna
Vignana,
J.S.S. Ayurveda Medical College &Hospital, Mysore.
“EVALUATION OF NEUROPROTECTIVE EFFICACY OF
VARUNITAILA (Citrullus colocynthis Schard) IN KAMPAVATA W.S.R TO
PARKINSON’S DISEASE USING ROTENONE INDUCED ANIMAL
MODEL”
3
6. Brief resume of the intended work:
6.1 Need for the study:
Parkinson’s disease (PD) is the most common form of a group of progressive neurodegenerative
disorders characterized by bradykinesia, rest tremor, muscular rigidity, shuffling gait, and flexed posture1.
Most of these features match with the Ayurvedic description of the disease KAMPAVATA2, 3.Worldwide
incidence of PD is estimated to be around 7 to 10 million4. Its peak age of onset is in the early 60s1,
progressively debilitating the affected individual.
Current drug therapies for human PD with Levodopa or various dopamine receptor agonists offer
symptomatic relief and appear to have little effect on the neurodegenerative process5. More than 50% of
patients with PD treated over 5 years with Levodopa will develop complications such as motor fluctuations
and dyskinesia’s. In this scenario, slowing the progression of PD through neuroprotective or restorative
therapy is a major focus of research. From a pharmacologic standpoint, current strategies involve
interrupting the cascade of biochemical events that leads to death of dopaminergic cells 1.Hence, the current
study aims at finding such an activity in an herb INDRAVARUNI – Citrullus colocynthis Schard (which is
prescribed in a classical Ayurvedic text for the management of KAMPAVATA6) using ROTENONE
induced PD animal model7, 8.
A Similar work has already been taken up to screen the antiparkinson’s activity of INDRAVARUNI
(Citrullus colocynthis), wherein OXOTREMORINE is used to induce Parkinsonism in mice.Oxotremorine
being a non-selective muscarinic acetylcholine receptor agonist produces temporary extrapyramidal signs
mimicking Parkinsonism9 -like tremor, ataxia and spasticity; which are antagonized by anticholinergic
drugs10.However, the tremor induced by such cholinergic agonists resembled more a condition of cholinergic
intoxication than parkinsonian tremor11. Hence, the antagonism of oxotremorine induced tremor can be more
suitably used to investigate the central antimuscarinic effect of a drug12.
6.2 Review of Literature:
In Charaka Samhita Vepathu has been described as one of the eighty types of Vataja nanatmaja vyadhi.
The term KAMPAVATA was explained for the first time in the text Basavarajeeyam3, with most of its
clinical features similar to that of PD. The main clinical feature of Kampavata is KAMPA2, 3 (Tremor),
wherein Tremor is particularly important in diagnosing PD, as it is present in 85% of patients with true
PD1.Also as explained in the text, Basavarajeeyam3 few more features of Kampavata can be compared with
that of PD;

Karapadatale kampa – Tremors in the hands and feet.

Dehabhramana – Postural instability.
Although PD defined clinically as a movement disorder, it is now widely appreciated that PD can be
accompanied by a variety of non-motor symptoms, including autonomic, sensory, sleep, cognitive, and
psychiatric disturbances1. These non-motor features can also be related with Ayurvedic description on
Kampavata as given in the text Basavarajeeyam3;

Nidrabhanga – sleep disturbances

Matiksheena – Dementia
Parkinson’s disease as such is characterized by loss of ∼50–70% of the dopaminergic neurons in the
substantia nigra pars compacta (SNc), a profound loss of dopamine(DA) in the striatum, and the presence of
intracytoplasmic inclusions called Lewy bodies (LB), which are composed mainly of α-synuclein and
ubiquitin13.
4
Oxidative stress appears to play an important role in the sporadic forms of PD. Endogenous sources of
oxidative stress include the free radicals produced by the metabolism of dopamine and melanin. Additional
stress may come from defects in mitochondrial complex I of the oxidative phosphorylation chain. Other
contributors to the selective dopamine neuron degeneration in PD are abnormal phosphorylation of proteins,
microglial activation, low-grade inflammation, and apoptosis; each represents a potential target for
therapeutic intervention1.
Varunitailawhich is prepared using the root of Indravarunihas been indicated in Kampavata6.
Indravaruni(Citrullus colocynthis) is a perennial, scabrid, prostrate or climbing herb with angular stems and
bified tendrils14. It has Tikta rasa; laghu, ruksha, tikshna guna; Ushna virya; katu vipaka; is
kaphapittahara, rechaka & garbhapataka; is indicated in udararoga, gulma, kosthabaddhata, plihodara,
krimi, kamala, sandhivata, amadosa, unmada & apasmara14.
Previous works done:
 Dhurv. Sanjay - A clinical study on Kampa-Vata (Parkinson’s disease) and its management with
Kapikacchu (Mucunapruriens), Gujarat University, Jamnagar. 2001
 Vijay Mahantesh Hiremath. A clinical study on Kampa-Vata (Parkinson’s disease) and its management
with Triguna rasa. RGUHS, Bengaluru. 2008
 Shivakumar B N, - Evaluation of kampavatahara effect of Triguna rasa, an experimental study,
RGUHS Bengaluru. 2010
 Rajamanickam E., GurudeebanS., Ramanathan T and Satyavani; Evaluation of anti-inflammatory activity
of Citrullus colocynthis;International J current research. March, 2010. Vol. 2, pp. 067-069,
 N. B. MARZOUKA, Z. MARZOUKB, FENINAB, A. BOURAOUIC, M. AOUNIA; Anti-inflammatory
and analgesic activities of Tunisian Citrullus colocynthisSchrad. Immature fruit and seed organic
extracts. European Review for Medical and Pharmacological Sciences; 2011; 15: 665-672
 Arshed Iqbal Dar, Ramesh Chandra Saxena, Suresh Kumar Bansal; Hepatoprotection: A Hallmark of
Citrullus colocynthis L. against Paracetamol Induced Hepatotoxicity in Swiss Albino Rats;American
Journal of Plant Sciences, 2012, 3, 1022-1027
 R. Jayaraman, Arihara shivakumar, t. Anitha, Vishal d. Joshi,Narahari n. Palei1; Antidiabetic effect of
petroleum ether extractof Citrullus colocynthis fruits againstStreptozotocin-induced hyperglycemic
rats. ROM. J. Biol. – plant biol., Volume 54, no 2, Bucharest, 2009, P. 127–134.
 Jeyanthi K.A, Mary Violet Christy.A; Antioxidant Effect ofCitrullus Colocynthis On Alloxan Induced
Diabetic Rats; International Journal of Pharmaceutical & Biological Archives 2011; 2(2):696-701
 B.B.Talole, D.G.Baheti, P.A.More; Antihistaminic effect of Citrullus colocynthis linn. Schard leaves;
Pharmacologyonline1: 468-472 (2011)
 BelsemMarzouk, ZohraMarzouk, MahaMastouri, Nadia Fenina and MahjoubAouni; Comparative
evaluation of the antimicrobial activity of Citrullus colocynthis immature fruit and seed organic
extracts; African Journal of Biotechnology Vol. 10(10), 14 March, 2011, P. 2130-2134.
 Nabila Benariba, RabehDjaziri, WafaaBellakhdar, NaceraBelkacem, Marcel Kadiata, Willy J. Malaisse et
al.; Phytochemical screening and free radical scavenging activity of Citrulluscolocynthis seeds
extracts; Asian Pac J Trop Biomed 2013; 3(1): 35-40
 ZohraMarzouk, BelsemMarzouk, Mohamed Ali Mahjoub, EhsenHaloui, ZineMighri, MahjoubAouni and
Nadia Fenina; Screening of the antioxidant and the free radical scavenging potential of Tunisian
Citrullus colocynthis Schrad. From Mednine. Journal of Food, Agriculture & Environment Vol.8 (2): 2 6
1 - 2 6 5. 2 0 1 0
5
6.3Aims & Objectives of the study To evaluate the neuroprotective efficacy of VARUNITAILA (Citrullus colocynthis Schard) in
KAMPAVATA (Parkinson’s disease).
 To compare the efficacy of VARUNITAILA in KAMPAVATA (PD) with different frequencies of
the dosage.
 To compare the efficacy of VARUNITAILA in KAMPAVATA (PD) with standard drug and vehicle
control groups.
7. Materials and Methods
7.1 Source of Data
1. Literary source: The literary aspect will be reviewed and data will be collected from authentic classical
Ayurvedic literatures, modern texts, recent medical journals, internet and other available sources.
2. Source of drug: Genuine, botanically identified Indravaruni moola will be collected from its natural
habitat.
3. Preparation: Varunitaila will be prepared as per the classical reference using Indravaruni moola and Tila
taila6.
4. Experimental source:
 Standard antiparkinsonian drug (L-Dopa) will be procured from recognized pharmaceutical
laboratory.
 Healthy male Albino rats of middle age group will be selected for the experiment and
maintained in the animal house under standard conditions8, 7.
 The Parkinson inducing drug rotenone and other chemicals& reagents required for the study
will be procured from authentic sources.
5. Place of work: - Dept. of Dravya Guna, JSS Ayurveda medical college, Mysore and JSS Medical College,
Mysore / DFRL, Mysore.
7.2 Method:A.
Study design:
Sample - 42male Albino rats of middle aged group will be selected for the study and will be separated
randomly into 7groups of 6 animals in each. Considering the 10% mortality associated with neurotoxic
model7 each group will be added with an extra animal.
6
Sl. No Groups
No. of Rats Inducing PD
Study
1
Normal Control
06+01
Not treated
2
Negative Control 06+01
Rotenone with vehicle Not treated
3
Standard Treated 06+01
Rotenone with vehicle Standard dose of L-Dopa
4
Test group A
06+01
Rotenone with vehicle Varunitaila OD dose
5
Test group B
06+01
Rotenone with vehicle Varunitaila Bid dose
6
Test group C
06+01
Rotenone with vehicle Varunitaila Tds dose
7
Vehicle Control
06+01
Rotenone with vehicle Tila Taila
Only vehicle
Each group will be housed in a separate rectangular polypropylene cage and the cages will be provided
with dust free paddy husk as a bedding material. The animals will be housed in a controlled conditions of
temperature (25 ± 30 C), humidity (50 ± 5%) with a 12h light/dark cycle. They will be maintained on a pellet
diet and tap water.
Dose –
Test drug (Varuni taila): 0.9ml/kg body wt6, 15.
Standard drug (L-dopa): 10mg/kg body wt/day16.
Mode of administration - orally.
Duration of the treatment – Treatment shall be continued till the animals develop debilitating phenotype /
till 11th day 8(whichever occurs first).
Preparation of Rotenone solution – Rotenone solution will be prepared as a stock for 3 days in 100%
Dimethylsulfoxide (DMSO) and diluted in medium chain triglyceride, Miglyol 812N to obtain a final
concentration of 3mg/ml rotenone in 98% Miglyol 812N & 2% DMSO. Vortexing the solution creates a
stable emulsion of the DMSO containing rotenone &Miglyol 812N. Fresh stock solution will be prepared
twice a week and stored in a vial protected from light. Vortexing of the vial several times before each
injection will be ensured to eliminate the possibility of settling7.
Schedule of the procedure –

Before inducing PD, the experimental animals will be acclimatized for three days and will be assessed
for their motor and behavioral scores.

The Rotenone solution thus prepared will be administered at 1ml/kg body weight/day intraperitoneally
to all the groups till 9th day8, except for the normal control group which receives only the vehicle
(DMSO+Miglyol 812N)7.
7

The respective treatment groups will receive their treatment from the day of starting of rotenone
injection8.

During daily handling, animals will be observed for the emergence of PD phenotype7, 8.

The animals will be assessed for their motor and behavioral scores on day 6, day 97 and day 118.

The trial and standard drug treatment will be continued till the animals develop debilitating phenotype
or till the 11th day8.

Animals developing debilitating phenotype – limiting their mobility, feeding or grooming; will be
sacrificed and brain samples will be procured from them for further analysis7.
Sample collection 
Terminally, rats will be sacrificed and brain samples shall be obtained from all the groups7, 8.
B.
Assessment criteria:
 Motor& Behavioral assessment
a) Open field test 17, 18
b) Pole test19
c) Rearing behavior20, 21
d) Postural instability test22
e) Rotarod test23, 24
f) Tail suspension test25

Time taken for the development of debilitating PD phenotype7.
 Oxidative stress assessment
a) Measuring the extent of lipid peroxidation (LPO) in brain homogenates26.
b) Measurement of reactive oxygen species (ROS) generation in the brain regions27, 28.
 Estimation of striatal Dopamine levels29
 Neuro Histopathological examination7.
a) Inclusion criteria: Healthy male Albino rats of middle age group will be selected7, 8.
b) Exclusion criteria: Others, which do not fulfill the above criteria.
7.3. Does the study require any investigation to be conducted on animals?
Yes, terminally histopathological examination of brain tissue will be performed.
Statistical analysis: Descriptive statistics, Wilcoxon test, ANOVA – Kruskal wallis contingency co-efficient
analysis using SPSS for windows.
7.4 Has ethical clearance obtained from your institution in case of 7.3?
Yes, obtained certificate enclosed.
8
8. List of reference:
1. DeLong MR, Juncos JL. Harrison’s principles of internal medicine – 17th edition; Chapter: 366; P. 2549-2557
2. Madhavakara. Ed Dr. Brahmanand Tripathi; Madhava Nidanam with Madhukoshateeka; Chaukambha
publications Vol. 1, Chapter. 22, P. 551
3. Basavaraja acharya. Basavarajeeyam; Chaukambha publications, 2005; Chapter 6th, P. 100
4. Parkinson’s disease foundation. Available from www.pdf.org
5. Giurgea CE. The Nootropic concept and its prospective implications. Drug Dev Res. 1982; 2:441-6
6. Sharangadhara acharya. Trans. Shrikantha Murthy KR. Sharangadharasamhita, Madhyamakhanda,
Snehakalpaadhyaya, Reprint. 2009. shloka. No. 111-112; P. 126
7. Jason. R. Cannon, et al., “A Highly Reproducible Rotenone Model of Parkinson’s disease” Neurobiol Dis.
2009 May; 34(2): 279-290
8. Sawsan. A. Zaitone, et al., “Piracetam and Vinpocetin ameliorate rotenone – induced Parkinsonism in rats”
Indian J Pharmacol. 2012 Nov-Dec; 44(6): 774-9
9. Tang C, Castoldi AF, Costa LG (April 1993). Effects of muscarinic agonist Oxotremorine on membrane
fluidity in rat lymphocytes. Biochem. Mol. Bio. Int 29(6): 1047-54
10. Vogel HG. Drug discovery & Evaluation. 2002. P. 577
11. Duvoisin, R.C: Parkinsonism: animal analogues of the human disorder. In: The Basal Ganglia. Ed: M.D. Yahr.
Raven press, New york,1976, P. 293-303
12. Brimblecombe, R.W & R.M. Pinder: Tremors &Tremorogenic agents. Scientechnica, Bristol, 1972
13. M.G. Spillantini, M.L. Schmidt et al “ α- synuclein in Lewy bodies” Nature Vol. 388, No. 6645, P. 839-840,
1997
14. Shastry JLN.“Dravya Guna Vijnana” Reprint. 2010.Vol. 2, P. 239-241
15. Table of Paget. GG & Barnes. JM (1964); Toxicity test in evaluation of drug activities: Pharmacometrics
16. Alam M, Schmidt WJ. 2004. L-DOPA reverses the hypokinetic behaviour and rigidity in rotenone-treated rats.
Behav. Brain Res. 153 (2), 439–446.
17. Correa M, Wisniecki A, Betz A, Dobson DR, O’Neill MF, O’Neill MJ, et al. The adenosine A2A antagonist
KF17837 reverses the locomotor suppression and tremulous jaw movements induced by haloperidol in rats:
Possible relevance to parkinsonism. Behav Brain Res. 2004;148:47–54.
18. Conceição IM, Frussa-Filho R. Effects of a single administration of buspirone on catalepsy, yawning and
stereotypy in rats. Braz J Med Biol Res. 1993;26:71–4.
19. Fernagut PO, Chesselet MF. Alpha-synuclein and transgenic mouse models. Neurobiol Dis. 2004;17: 123–30.
20. Fleming SM, Delville Y, Schallert T. An intermittent, controlled-rate, slow progressive degeneration model of
Parkinson's disease: antiparkinson effects of Sinemet and protective effects of methylphenidate. Behav Brain
Res. 2005;156:201–13.
21. Fleming SM, Zhu C, Fernagut PO, Mehta A, DiCarlo CD, Seaman RL, Chesselet MF. Behavioral and
immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone.
Exp Neurol. 2004; 187:418–29.
22. Woodlee MT, Kane JR, Chang J, Cormack LK, Schallert T. Enhanced function in the good forelimb of hemiparkinson rats: compensatory adaptation for contralateral postural instability? Exp Neurol. 2008;211:511–7.
23. Fleming SM, Mulligan CK, Richter F, Mortazavi F, Lemesre V, et al. (2011) A pilot trial of the microtubuleinteracting peptide (NAP) in mice overexpressing alpha-synuclein shows improvement in motor function and
reduction of alpha-synuclein inclusions. Mol Cell Neurosci 46: 597–606.
24. Duan W, Mattson MP (1999) Dietary restriction and 2-deoxyglucose administration improve behavioral
outcome and reduce degeneration of dopaminergic neurons in models of Parkinson's disease. J Neurosci Res
57: 195–206.
25. Cryan JF, O'Leary OF, Jin SH, Friedland JC et al (2004) Norepinephrine-deficient mice lack responses to
antidepressant drugs, including selective serotonin reuptake inhibitors. Proc Natl Acad Sci U S A 101:8186–
8191.
26. Ohkawa H, Ohnishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.
Anal Biochem 1979;95:351-58.
27. Driver AS, Kodavanti PS, Mundy WR. Age related changes in reactive oxygen species production in rat brain
homogenates. Neurotoxicol Teratol. 2000;22:175-81.
28. Shinomol GK, Muralidhara. Differential induction of oxidative impairments in brain regions of male mice
following subchronic consumption of khesari dhal (Lathyrus sativus) and detoxified khesari dhal.
Neurotoxicol 2007;28:798-806.
29. Dalpiaz A, Filosa K, de Capraris P, Conte G, Bortolotti F, Biondi C, Scatturin A, Prasad PD, Pavan B.
Molecular mechanism involved in the transport of a prodrug dopamine glycosyl conjugate. Intl J
Pharmaceutics 2007;336:133-9.
9
9.
Signature of the Candidate
10.
Remarks of the Guide
11.
Name and Designation of
11.1 Guide
Dr. SHIVAPRASADHUDED, M.D. (AYU)
Assistant Professor
Department of Post Graduate Studies in
DravyagunaVignana,
J.S.S. Ayurveda Medical College, Mysore.
11.2 Signature
11.3 Co-Guide
Dr. SURESHA. R.N
Professor & HOD
Department of Pharmacology
JSS Medical College, Mysore,
Karnataka.
11.4 Signature
11.5 Head of the Department
Dr.NAGAMANI. M.D. (AYU)
Professor and HOD
Department of Post Graduate Studies in
DravyagunaVignana,
J.S.S Ayurveda Medical College, Mysore.
11.6 Signature
12.
12.1 Remarks of the Chairman and Principal
12.2 Signature
10