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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE.
M. PHARM SYNOPSIS
YEAR OF ADMISSION-JULY 2011
TITLE OF THE SYNOPSIS
“AN INVESTIGATION OF ANTI-EPILEPTIC AND ANTI-PSYCHOTIC ACTIVITIES OF SHILAJIT IN EXPERIMENTAL ANIMALS"
BYMr. SHARANBASAPPA
M. PHARM., PART-IDEPARTMENT OF PHARMACOLOGY
UNDER THE GUIDANCE OF
Mr. SYED MANSOOR AHMED M.Pharm., (Ph.D) Asst. Professor
DEPARTMENT OF PHARMACOLOGY
INSTITUTIONSREE SIDDAGANGA COLLEGE OF PHARMACY
B. H. ROAD, TUMKUR-572 102. KARNATAKA
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. NAME OF THE CANDIDATE AND ADDRESS
Mr. SHARANBASAPPA
IST M.Pharma.Department of Pharmacology,
Sree Siddaaganga College of Pharmacy,B.H.Road, Tumkur – 572 102
2. NAME OF THE GUIDE Mr. SYED MANSOOR AHAMEDM. Pharm.,(Ph.D)
Assistant Professor ,Department of Pharmacology.
3. NAME OF THE INSTITUTION
SREE SIDDAGANGA COLLEGE OF PHARMACYB. H. ROAD, TUMKUR- 572 102
4. COURSE OF STUDY AND SUBJECT
MASTER OF PHARMACY (PHARMACOLOGY)
5. DATE OF ADMISSION OF COURSE
23rd JULY 2011
6. TITLE OF THE TOPIC
“An investigation of anti-epileptic and anti-psychotic activities of
Shilajit in experimental animals”
2
7.0 BRIEF RESUME OF THE INTENDED WORK
7.1 - NEED FOR THE STUDY
The term Epilepsy, based on the Greek word “epilambaein” meaning “to seize” has
been first mentioned by Hippocrates.1 Recognised from the drawn of history as
‘disease of lightening’, it was correctly described by JH Jackson over a century ago.2
Epilepsy is one of the most common chronic neurological disorders with no age, racial,
social, sexual or geographical boundaries and affects about 50 million people
worldwide.3 The prevalence is high in tropical countries, particularly in Africa, where
it varies between 10 and 55 per 1000, with an estimated mean prevalence of 15.4 People
with epilepsy are at special risk for two life-threatening conditions: status epilepticus
and sudden unexplained death. 5
The term Psychosis, based on the Greek word “psyche” for mind/soul, and “-osis”, for
abnormal condition; means abnormal condition of the mind, and is a generic
psychiatric term for a mental state often described as involving a “loss of contact with
reality”. People suffering from psychosis are described as psychotic. Psychosis is given
to the more severe forms of psychiatric disorder, during which hallucinations,
delusions and impaired insight may occur. 6
Epilepsy and psychosis are among the disorders that are strongly associated with
significant psychological and social consequences for everyday living. CNS (Central
Nervous System) disorders often play a major role and will be a growing cause of
primary and secondary disability in the next 10 years. 7 According to WHO report one
in four people in the world will be affected by mental or neurological disorders at some
point in their lives. Around 450 million people currently suffer from such conditions,
placing mental disorders among the leading causes of ill-health and disability
worldwide. 8
Most CNS ailments follow a complex biology, and can have differing outcomes
depending on predisposing factors. 10 Though the available drugs fulfill the
requirements in the segment, there is a need of more effective drugs that are better
tolerated and cost effective to enhance long-term compliance. These diseases involve a
change in the basic physiology of the nervous system. It is difficult or sometimes
3
impossible to reverse these changes. Hence, the drugs are more palliative as compared
to curative. Besides, several neurological ailments are not entirely understood, hence
the treatments are not adequate, and even if they are well understood, therapy is
associated with limitations. 9
Many of the drugs merely treat the symptoms and do not provide cures. Current anti-
epileptic drugs are effective in controlling seizures in about 70% of patients, but their
use is often limited by side effects. 10 Hence there is a need for development of cheap,
effective and safe agents from plant, animals and mineral sources. The development of
new therapies has the potential to provide patients with significant improvements. In
folk medicine, Shilajit has been used to treat diverse clinical conditions ranging from
anaemia to nervous disorders. 11 Hence, the present project has been undertaken for
screening of the herbo-mineral drug Shilajit, 12 for anti-epileptic and anti-psychotic
activity in experimental animals, as Shilajit has not been studied for its anti-epileptic
and anti-psychotic activity despite of its use in treatment of nervous disorders in
traditional medicines. 11
7.2 REVIEW OF LITERATURE
Shilajit is a herbo-mineral drug ejected out of fissures in iron rich rocks, during hot
weather. 12
It is found to be produced naturally in mountainous areas, especially Himalayas,
Vindhya and other mountains in India and also in Nepal. It may also be found as a tar
in the earth crust, formed due to decomposition of vegetable substances. It is observed
that ‘Momia’ which occurs in Arabia and Persian mountains, resembles Shilajit. 12
The following forms of this drug have been reported in ancient Hindu literature, viz.
1. Iron Shilajit (blackish-brown variety)
2. Copper Shilajit (blue variety)
3. Silver Shilajit (white variety)
4. Gold Shilajit (red variety)
Synonyms
4
Sanskrit : Shilajit, Silajit, Silaras
Hindi : Silajita, Ral-yahudi
English : Asphalt, Mineral Pitch, Jew’s Pitch, Vegetable Asphalt
Latin : Asphaltum
Tamil : Perangyum, Uerangyum
Bengali : Silajatu
Gujarati : Silajita
In Sanskrit, Shilajit means ‘winner of rock’ (Mukherjee 1992). Another meaning is
“sweat of the rock” (Tirtha 1998).
Shilajit is a blackish-brown exudation, of variable consistency, obtained from steep
rocks of different formations found in the Himalayas at altitudes “between” 1000 to
5000 meter, from Arunachal Pradesh in the east to Kashmir in the west. It is also found
in Afghanistan, Nepal, Bhutan, Pakistan, China, Tibet and U.S.S.R. (Tien-shan, Ural
and Caucasus) (Jaiswal 1992). The black form of Shilajit is the most commonly used
medicinal form (Halpern 2003).
Medicinal uses
It is used as a general tonic stimulant, aphrodisiac 12, diabetes mellitus 13, nervous
disorders 11, obesity. 14 Shilajit of authentic quality is an important ingredient of
ayurvedic preparations meant for strengthening immunising system of human body. It
is a unique blend of herbal and mineral ingredients formed by a unique natural process.
Reported activities
Shilajit has been reported to be anti-anxiety 15, antioxidant 16, immuno-modulator 17,
asthma 18, adaptogenic agent 19, anti-ulcerogenic and anti-inflammatory. 20
7.3 OBJECTIVES OF THE STUDY
1. Collection of Shilajit from market as Shilajit powder.
2. To investigate the anti-epileptic activity of Shilajit by using maximal electro shock
induced seizures, isoniazid induced seizures and pentylenetetrazol induced seizures in
experimental animals.
3. To investigate the anti-psychotic activity of Shilajit by using apomorphine induced
stereotype behavior, pilocarpine induced purposeless chewing and apomorphine
induced climbing behavior in experimental animals.
MATERIALS AND METHODS
5
8.0 8.1 Materials – List of Chemicals and Instruments used
Shilajit, Caffeine, Tween 80
Ethyl acetate, Chloroform
Phenytoin, Diazepam, Haloperidol
Apomorphine, Pentylenetetrazol
Pilocarpine, Mecamylamine
Electro convulsometer
Melting point apparatus
8.2 Experimental animals
Adult albino wistar rats of either sex weighing approximately 180-200 g will be used.
They will be housed in standard laboratory conditions and fed with standard animal
feed and will be given water ad libitum.
8.3 Dose of Shilajit
Shilajit will be administered at dose of 25 and 50 mg/kg. 15
8.4 Grouping of animals
The albino wistar rat will be randomly distributed into four groups containing six
animals in each group.
Group I – control
Group II – suitable dose of standard
Group III – 25 mg/kg of Shilajit
Group IV – 50 mg/kg of Shilajit
8.5 Experimental procedure
8.51 Anti-epileptic study using maximal electro shock induced seizures
The albino wistar rat will be randomly distributed into four groups containing six
animals in each group. The test will be started 60 minutes after oral treatment with
suitable doses of Shilajit or the standard. An electro convulsometer with corneal or ear
electrodes will be used to deliver the stimuli. The incidence and duration of extensor
tonus will be noted. A complete abolition of hind limb tonic extension will be
considered as 100% protection. 21
8.52 Anti-epileptic study using isoniazid induced seizures
6
The albino wistar rat will be randomly distributed into four groups containing six
animals in each group. The groups will be treated with suitable doses of Shilajit or the
standard. 60 minutes after the treatment with Shilajit, animals will be injected with
subcutaneous doses of isoniazid. During the next 120 minutes the occurrence of clonic
seizures and tonic seizures will be recorded [17]. The percentage of seizures or deaths
occurring in the control group is taken as 100%. The suppression of these effects in
the treated group is calculated as percentage of controls.22
8.53 Anti-epileptic study using pentylenetetrazol (PTZ) induced seizures
The albino wistar rat will be randomly distributed into four groups containing six
animals in each group. The groups will be treated with suitable doses of Shilajit or the
standard. 60 minutes after the treatment with Shilajit, animals will be injected with
subcutaneous doses of PTZ. During the next 120 minutes the occurrence of clonic
seizures, tonic seizures and death is required. The percentage of seizures or deaths
occurring in the control group is taken as 100%. The suppression of these effects in
the treated group is calculated as percentage of controls. 23
8.54 Anti-psychotic study using apomorphine induced stereotypy
The albino wistar rat will be randomly distributed into four groups containing six
animals in each group. The suitable dose of Shilajit or the standard will be
administered 60 minutes prior apomorphine dosage. Apomorphine HCl will be injected
subcutaneous. The animals will be placed in individual plastic cages. A 10 second
observation period is used to measure the presence of stereotypic activity such sniffing,
licking and chewing 10 minutes after apomorphine administration. An animal is
considered protected if this behavior is reduced or abolished. 24
8.55 Anti-psychotic study using pilocarpine induced purposeless chewing
The albino wistar rat will be randomly distributed into four groups containing six
animals in each group. The groups will be treated with suitable doses of Shilajit or the
standard. 60 minutes after the treatment with Shilajit, animals will be injected with i.p.
doses of pilocarpine. Then the animals will be placed individually in a large glass
cylinder (height 30 cm, diameter 20 cm) at 21 ± 1oC and allowed to habituate for 15
minutes before injection of drugs. Numbers of chews are counted by direct observation
immediately after drug administration. The results will be presented as numbers of
chews in a 30 minute period.25
8.56 Anti-psychotic study using apomorphine induced climbing behavior
7
The albino wistar rat will be randomly distributed into four groups containing six
animals in each group. The groups will be treated with suitable doses of Shilajit or the
standard. 60 minutes after the treatment with Shilajit, animals will be injected with
haloperidol i.p. Then the animals are placed individually in wire- mesh stick cages; 30
minutes after the treatment is given. 10, 20 and 30 minutes after apomorphine
administration, they are observed for climbing behavior and scored as follows:
0 = four paws on the floor
1 = forefeet holding the vertical bars
2 = forefeet holding the bars 22
8.6 Experimental design
8.61 Anti-epileptic activity (single and multiple dose study)
a. Maximal electro shock (MES) induced seizures
GROUP
N=6
TREATMENT AND
DOSE
PARAMETERS
I Vehicle (1 ml/kg, p.o.) +
MES
Hind limb tonic extension
II Phenytoin (90 mg/kg, i.p.)
+ MES
III Shilajit (25 mg/kg, p.o.) +
MES
IV Shilajit (50 mg/kg, p.o.) +
MES
Multiple dose study is conducted for duration of 15 days.
b. Isoniazid (INH) induced seizures
8
GROUP
N=6
TREATMENT AND
DOSE
PARAMETERS
I Vehicle (1 ml/kg, p.o.) +
INH (300 mg/kg, s.c.)
Occurrence of tonic –
clonic seizure
II Diazepam (4 mg/kg, i.p.) +
INH (300 mg/kg, s.c.)
III Shilajit (25 mg/kg, p.o.) +
INH (300 mg/kg, s.c.)
IV Shilajit (50 mg/kg, p.o.) +
INH (300 mg/kg, s.c.)
Multiple dose study is conducted for duration of 15 days. INH is administered on 15 th
day.
c. Pentylenetetrazol (PTZ) induced convulsions
GROUP
N=6
TREATMENT AND
DOSE
PARAMETERS
I Vehicle (1 ml/kg, p.o.) +
PTZ (60 mg/kg, s.c.)
Occurrence of tonic –
clonic seizure
II Diazepam (4 mg/kg, i.p.) +
PTZ (60 mg/kg, s.c.)
III Shilajit (25 mg/kg, p.o.) +
PTZ (60 mg/kg, s.c.)
IV Shilajit (50 mg/kg, p.o.) +
PTZ (60 mg/kg, s.c.)
Multiple dose study is conducted for duration of 15 days. PTZ is administered on 15 th
day.
8.62 Antipsychotic activity (single and multiple dose study)
a. Apomorphine induced stereotype behavior
9
GROUP
N=6
TREATMENT AND
DOSE
PARAMETERS
I Vehicle (1 ml/kg, p.o.) +
Apomorphine (1.5 mg/kg,
s.c.)
10
Stereotyped behavior
II Standard drug (1 mg/kg,
i.p.) + Apomorphine (1.5
mg/kg, s.c.)
III Shilajit (25 mg/kg, p.o.) +
Apomorphine (1.5 mg/kg,
s.c.)
IV Shilajit (50 mg/kg, p.o.) +
Apomorphine (1.5 mg/kg,
s.c.)
Multiple dose study is conducted for duration of 15 days. Apomorphine is administered
on 15th day.
b. Pilocarpine induced purposeless chewing
GROUP
N=6
TREATMENT AND
DOSE
PARAMETERS
I Vehicle (1 ml/kg, p.o.) +
Pilocarpine (1mg/kg, i.p.)
Numbers of chewing in 30
minutes period
II Standard drug (1 mg/kg,
i.p.) + Pilocarpine
(1mg/kg, i.p.)
III Shilajit (25 mg/kg, p.o.) +
Pilocarpine (1mg/kg, i.p.)
IV Shilajit (50 mg/kg, p.o.) +
Pilocarpine (1mg/kg, i.p.)
Multiple dose study is conducted for duration of 15 days. Pilocarpine is administered on
15th day.
c. Apomorphine induced climbing behavior
GROUP
N=6
TREATMENT AND
DOSE
PARAMETERS
I Vehicle (1 ml/kg, p.o.) +
Apomorphine (3 mg/kg, 11
9.0s.c.)
Climbing behavior
II Standard drug (1 mg/kg,
i.p.) + Apomorphine (3
mg/kg, s.c.)
III Shilajit (25 mg/kg, p.o.) +
Apomorphine (3 mg/kg,
s.c.)
IV Shilajit (50 mg/kg, p.o.) +
Apomorphine (3 mg/kg,
s.c.)
Multiple dose study is conducted for duration of 15 days. Apomorphine is administered
on 15th day.
Statistical analysis:
The analysis of data will be done by using one - way ANOVA.
8.7 DOES THE STUDY REQUIRE ANY INVESTIGATION OR
INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER
HUMANS/ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY.
Yes, the above study requires investigation to be done on the adult albino wistar
rats of either sex for the anti-epileptic and anti-psychotic study.
8.8 HAS ANIMAL ETHICAL COMMITTEE CLEARNACE BEEN OBTAINED
FROM YOUR INSTITUTION IN CASE?
The study has been referred to the ethical committee of the institution and
clearance has been obtained (Ref: SSCPT/IAEC. Clear 104/11-12, Dated: 30-11-
11) Enclosed copy. Annexure – I.
REFERENCES
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2006. p. 158 (vol I).
2. Tripathi KD. Essentials of medical pharmacology. 6th ed. New Delhi: Jaypee
Brothers Medical Publishers; (P) Ltd. 2008. p. 401.
3. Munjal AM, Bharathi H, Ashok SK. Progress in neuroprotective strategies for
preventing epilepsy. Prog Neurobiol 2008;84:363-404.
12
4. Moshi MJ, Kagshe GA, Mbuuambo ZH. Plants used to treat epilepsy by
Tanzanian traditional healers. J Ethnopharmacol 2005; 97:327 – 36.
5. Seizures and Epilepsy: Hope through Research. [Online]. 2011 Oct 31 [cited
2011 Nov 25]; Available from:
URL:http://www.ninds.nih.gov/disorders/epilepsy/epilepsy.html
6. Gelder, Michael. Psychiatry. New York: Oxford University Press Inc; 2005.
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25]; Available from:
URL:http://www.who.int/nmh/publications/discussion_paper_en.pdf
8. Mental disorders affect one in four people. [Online]. 2011 Oct 4 [cited 2011 Nov
25]; Available from:
URL:http://www.who.int/whr/2001/media_centre/press_release/en/index.html
9. Centrally connected. [Online]. 2006 Jul 15 [cited 2011 Nov 25]; Available from:
URL:http://www.expresspharmaonline.com/20060715/research01.shtml
10. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th ed. Edinburgh:
Churchill Livingstone; 2003. p. 550.
11. Eugene W, Victor RG, Prasad DG, Petra K, Frauke M, Joyonto SF et al. Review
of Shilajit used in traditional Indian medicine. J Ethnopharmacol 2011;136:1-9.
12. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 41st ed. Vile-Parle (W)
Mumbai: Nirali Prakashan; 2008. p. 18.9.
13. Bhattacharya SK, Satyan KS, Chakrabarti A. Effect of trasina, an Ayurvedic
herbal formulation on pancreatic islet superoxide dismutase activity in
hyperglycemic rats. Indian J Exp Biol 1997;35: 297-9.
14. Prakash P, Pralhad P, Bhushan P. Ayurvedic treatment of obesity: A randomized
double - blind, placebo controlled clinical trial. J Ethnopharmacol 1990;29:1-11.
15. Jaiswal AK, Bhattacharya SK. Effects of Shilajit on memory, anxiety and brain
monoamines in rats. Indian J Pharmacol 1992;24: 12-17.
16. Kumar MR, Reddy AG, Anjaneyulu Y, Reddy GD. Oxidative stress induced
by lead and antioxidant potential of certain adaptogens in poultry. Toxicol Int
2010; 17:45-8.
13
17. Ghosal S. Chemistry of Shilajit, an immunomodulatory ayurvedic rasayan. Pure
and applied chemistry. 1990; 62:1285-1288.
18. Shilajit A material medica monograph [Online]. 2004 Aug 30 [2011 Nov 25];
Available from:
URL:http:// www.jivaka.com/org/docs/paper/Talbert%20Paper.pdf
19. Bhattacharya SK, Bhattacharya A, Chakrabarti A. Adaptogenic activity of
siotone, a polyherbal formulation of ayurvedic rasayanas. Indian J Exp Biol
2000; 38:119-28.
20. Goel RK, Banerjee, Acharya SB. Anti-ulcerogenic and Anti-inflammatory
studies with Shilajit. J Ethnopharmacol 1990;29(1):95-103.
21. Achliya GS, Wadodkar SG, Dorle AK. Evaluation of CNS activity of Brahmi
ghrita. Indian J Pharmacol 2005;37:33-6.
22. Pinelli A, Trivulzio S, Zefinetti GC, Tofanetti O. Anti-convulsant effects by
reduced glutathione and related amino acids in rats treated with isoniazid. J
Toxicol 1988;48:103-07.
23. Maheshwar HG, Despande SV, Pramaod HJ. Anti-convulsant activity of fruits
of Terminalia chebulia retz. Against MES and PTZ induced seizures in rats. J
Herb Med Toxicol 2010;4:123-6.
24. Vogel HG. Drug Discovery and Evaluation. 2nd ed. New York: Springer Berlin
Heidelberg. 2002. p. 488, 531-7.
25. Finn M, Jassen A, Baskin P, Salamone JD. Tremulous characteristics of the
vacuous jaw movements induced by pilocarpine and ventrolateral striatal
dopamine depletions. Pharmacol Biochem Behav 1995;57: 243-9.
10 Signature of the candidate
11 Remarks of the Guide
The above information and literature has been extensively investigated and verified. The
present study will be carried out under my supervision and guidance.
14
12 12.1 Name and Designation of Guide
Mr. SYED MANSOOR AHMEDM. Pharm., (Ph.D)
Asst. Professor DEPARTMENT OF PHARMACOLOGY
12.2 Signature
12.3 Co-Guide ( If any) N.A.
12.4 Signature N.A.
13 13.1 Head of the Department Dr. THIPPESWAMY B.S.,M. Pharm., Ph. D.Professor & Head
DEPARTMENT OF PHARMACOLOGY
13.2 Signature
14 14.1 Remarks of the Principal The above mentioned information is correct and I recommend the same for approval.
14.2 Signature
Dr. S. Badami M.Pharm., Ph.D.Principal
Sree Siddaganga College of Pharmacy B. H. Road, Tumkur-572 102.
15