RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1 Name of the Candidate and GAJERA KALPESH DHARMASHI. KLE'S College of Pharmacy, Address P.B. No 1062, 2nd Block, Rajajinagar, Bangalore – 560010.
2 Name of the Institution KLE Society’s College of Pharmacy Bangalore
3 Course of Study and Subject M.Pharm (PHARMACOLOGY)
4 Date of Admission 11-06-07
5 Title of the Topic:
EVALUATION OF TRANQUIL -A POLYHERBAL FORMULATION FOR ANXIOLYTIC EFFECTS IN ANIMAL MODELS. 6. BRIEF RESUME OF INTENDED WORK:
6.1 NEED FOR STUDY
Several herbs have been used in folklore and by traditional healers in the treatment of variety of diseases/disorders for either treatment or management. Ayurveda-one of the traditional system of medicine in India is known to formulate several herbs to treat/ manage diseases/disorders as well to enhance the effect of another (synergistic effect). Combination of herbs is believed to be beneficial than single ingredient, therefore combination of several herbs are to derive therapeutic benefit. Even through several herbs have been reported to the effective in several animal models. Ingredients of this polyherbal formulation have been investigated and have proven neuropharmacological effects. The combination of them are not evaluated systematically, hence the study.
TRANQUIL- the polyherbal formulation to be investigated is indicated for anxiolytic conditions and the current study envisages the evaluation of the same in animal models of anxiety.
When the results are encouraging/ significant anxiolytic effects have been demonstrated will justify it's use in the management of anxiety state and normalize life functions.
6.2 REVIEW OF LITERATURE
TRANQUIL – a poly herbal formulation is a combination of Indian medicinal herbs of repute, the various ingredients of which have proven therapeutic properties. This proprietary formulation has been indicated for tranquillizing effect and contains following herbs and is being formulated for use by oral route (tablets) 1. Baccopa monnieri (bramhi) 2. Valeriana wallichi (tagara) 3. Nardostachys jatamansi (jatamansi) 4. Withinia somnifera (ashwaganda) 5. Terminalia arjuna (arjuna) 6. Roots of piper longum (pippali moola) 7. Myristca fragrans (jatiphala) 8. Aconus calamus (vacha) 9. Rauwolfia serpentina (sarpagandha) 10. Celastrus paniculatis (jyothismati) The above said herbs have spectrum of biological activity and only related to CNS have been reviewed.
Baccopa monnieri (bramhi)
Baccopa monnieri, a common medicinal plant having multiple uses in traditional system of medicine and is particularly used as memory enhancer and one of the ingredient in memory boosting proprietary formulation.
This herb has been subjected to several pharmacological investigations and of neuropharmacological interest is mentioned here.
Triterpene glycosides, isolated from whole plant possess antidepressant effect1 and Neuroprotective effect in epilepsy2.
Valeriana wallichi (tagara)
Valeriana wallichi (tagara) another ancient herb with tranquillizing effect have also been extensively investigated. Attempts have been made to isolate, characterize and to study pharmacological activities esp. on CNS. Following are some of them. Hesperidin and 6 methyl apigenin – a flavone glycoside, possess sedative and sleep enhancing properties3, 4. Antistress activity in human trials has shown encouraging result, therefore suggested for stress management5. Attenuation of ischaemia –reperfusion induced cerebral injury and an increase in short term memory6.
Nardostachys jatamansi (jatamansi)
Nardostachys jatamansi (jatamansi) a medicinally important herb of Indian origin is used for centuries in Ayurvedic and Unani system of medicine for several ailments. Several neuropharmacological and behavioral effects have been reported following investigations in lab animal models. Following are some of them In-vitro Acetylcholinestrase inhibitory activity effect of hydroalcholoic extract, hence a potential benefit in Alzheimer’s disease7. Facilitation of cholinergic neurotransmission in brain, thus memory enhancing and reversing aging induced amnesia in mice8. Ethanolic extract with antioxidant activity, biogenic amine enhancing property with ability to slow neuronal injury in animal model of Parkinson’s disease9. Anticonvulsant activity with minimal neurotoxicity in animal models10. Significant increase in levels of central monoamines and inhibitory amino acids after acute and sub chronic oral administration of extract11. Withinia somnifera (ashwagandha) Withinia somnifera (Ashwagandha) considered as Indian Ginseng, is classified as Rasayana –a rejuvenator and the use of which is expected to promote physical and mental health, rejuvenate the body in debilitated conditions and increase longevity, augments resistance of the body against disease and diverse environmental conditions Following are some CNS activity reported.
Root extract producing protective effect behavioral and biochemical alterations in sleep disturbed mice 12. Withinia somnifera and it’s combination with imipramine and Fluoxetine produced a dose dependent decrease in mean immobility time of mice in FST by altering central biogenic amine levels 13. Vinutha et.al have reported AchE inhibitory effect of methonolic and successive water extracts, indicating it’s potential to influence cognition 14. Oral administration of Withenolide IV, one of the constituents, ameliorates neuronal dysfunction in animal model of Alzheimer’s disease 15. Adoptogenic activity in rat models of chronic stress, dose dependent antistress activity of Withenolide –free aqueous extract from rats 16. Anxiolytic and antidepressant activity of Glycowithanolides, whose effect is comparable to imipramine 17. Terminalia arjuna (arjuna) Bark of Terminalia arjuna an indigenous plant used in ayurvedic medicine primarily as cardiotonic, is also used to treat diabetes, anemia, tumors and hypertension. Following are some of the pharmacological activities reported in studies involving animal models. Ulcer protective and gastro protective activity 18, 19. Antioxidant activity 20. Wound healing activity 21. Anti-inflammatory activity 22. Piper longum (pippali moola) Seeds of piper longum (pepper) are a common household spice in Indian home possessing diverse physiological activities. Many properties of pepper, its extracts and its major active principle have been reported. Enhancing digestion, antioxidant activity (in-vitro), anti-mutagenic and antitumor influence in animal models, enhancing bioavailability 23 and a significant influence on biomarkers of oxidative stress in fructose fed insulin resistant rats 24.
Myristca fragrans (jatiphala)
Myristca fragrans is well known spice in the Indian household and is reported to possess several neuropharmacological properties. Dhingra and Sharma have reported antidepressant like activity of n-hexane extract, such an effect involves adrenergic, dopaminergic and seratonergic system 25and an acetone insoluble portion of n- hexane extract possess anxiogenic properties 26Cholinesterase inhibiting properties 27hypnotic and weak analgesic activity activity 28 indicating wide spectrum of CNS effects. Acorus calamus s(vacha) Acorus calamus (vacha) is one of the ancient herbs of Asian continent. Roots and rhizomes of this have been subjected to extensive pharmacological investigations, especially for it’s influence on CNS. Following are some of the reported activities. Ethanolic extract of rhizomes possessing antidepressant activity29. Water soluble dried powder of alcoholic extract possessing wide variety of CVS and CNS effects such as antagonizing spontaneous motor activity, amphetamine induced hyperactivity, sedative and tranquillsing effect 30. Methanolic extract of rhizome improving memory and cognition 31. Neuroprotective effect of Ethanol: water extract of rhizome in rat model of Ischaemia 32. AchE inhibitory activity of hydroalcoholic extract 33 and protective effect of Ethonolic and Methanolic extract in noise induced stress 34. Rauwolfia serpentina (sarpagandha) Rauwolfia serpentina is a well-known herb with a established antihypertensive effect, also posses mild tranquillizing effect.
Celastrus paniculatis (jyothismati) Seed of Celastrus paniculatis has been extensively investigated for neuropharmacological activities. Celastrus oil from seeds is reported has significant anxiolytic effect mediated thro, seratonergic mechanism 35. Aqueous extract improved learning and memory in lab animals, possibly by virtue of its antioxidant properties 36. On chronic administration, seed oil reversed scopolamine induced task performance deficit 37and an overall reduction in monoamine levels in the brain indicating the involvement of aminergic system in the improvement in learning and memory 38. Invitro neuroprotective effect of seed oil and several organic extracts, modulating glutamate receptor function have been reported 39. 6.3 AIMS & OBJECTIVES:
AIM To evaluate the anxiolytic activity of TRANQUIL - a polyherbal formulation in animal models of anxiety.
OBJECTIVES To study and compare dose dependent anxiolytic effect in saline treated, test formulation treated and standard anxiolytic drug treated (single dose) animals using exteroceptive models of anxiety.(acute study) To study and compare dose dependent anxiolytic effect in saline treated, test formulation treated and standard anxiolytic drug treated animals for 15/21 days using exteroceptive models of anxiety.(chronic study)
7. MATERIALS AND METHODS 7.1 Source of data Source of data will be various parameters recorded in vehicle / test formulation / standard anxiolytic drug treated animals when subjected to various tests (models) to test anxiety levels. 7.2 Method of collection of data Data for study will be collected following single dose / 15 or 21 days of dosing in test animals when they will be subjected to various tests. i.e. Their behavior when placed in elevated plus maze, mirrored chamber etc, which measures their anxiety levels. e.g. When test animals are placed in elevated plus maze the time spent in open arms, latency to enter in open arm total number of entries in open arm etc will be recorded. Naïve Albino mice, of either sex, healthy, of appropriate weight range. will be employed for the study since this species have been extensively used by several pharmacologists world wide for similar studies and are believed to be sensitive to drugs acting on CNS. Following animal models will be employed for the study.
1. ELEVATED PLUS MAZE. Following single dose treatment, and15 or 21-day treatment with test formulation, vehicle, and standard anxiolytic drug, all animals in the group will be analyzed for anxiety levels using elevated plus maze. Following parameter in a 5 minutes session will be recorded 40. 1. Time spent in open arm. 2. Latency to enter in open arm. 3. Total number of entries in open arm. 4. First preference of animal (open/ closed). 2. MIRROR CHAMBER TEST. It is hypothesized that distortation of the appearance of the readily traversed environment by a compartment constructed of mirrored glass might produce an aversion to entry that is different from anxiety state / elicited by maze. All animals of groups either vehicle / test formulation/ anxiolytic drug treated will be placed individually in the chamber and following parameters are recorded in a 5 minutes session 41. 1. Time spent in chamber. 2. Latency to enter in chamber. 3. Total number of entries in chamber.
Elevated plus maze and mirrored chamber will have dimensions suggested by Kulkarni SK 42.
3. MARBLE BURYING BEHAVIOUR. In both natural and laboratory conditions rats and mice spontaneously use available bedding material to bury unpleasant sources of discomfort present in their home environment. Burying behavior consists of forward shoving the diggable material over the source of aversion using the snout and forepaws in order to avoid and protect from the localized threat43. Selective inhibition of objects burying behavior in rodents has been proposed as a test for anxiolytic and will be carried out as follows.
Female mice (23-35gm) with free access to food and water will be experimental room for 3 days prior to experiment. Mice will be taken out from stock cages and placed individually in polypropylene cage containing 20 clean glass marbles of 1.5 cm diameters evenly spaced on 5 cm deep sawdust without food and water. Number of marbles (at least 2/3rd) buried, will be calculated after 30 minute later 44.
4. HOLE BOARD TEST.
Laboratory animals like mice are naturally curious and benzodiazepine and benzodiazepine like drug reduces curiosity in them. Hole board test is one of the commonly used animals model of anxiety to evaluate anxiolytic drugs. Anxiety levels will be evaluated in mice using hole board apparatus (35cm X 35cm X 15 cm) one hour after oral treatment of vehicle / standard anxiolytic drug / test formulation. The walls of which are made up clean plexiglass and arena floor is divided in to 16 equal squares with 16 small holes (of 3.5 cm diameter ) the equipment is placed 50 cm above the floor. Each animal will be placed on the central square of the arena and number of crossed squares and holes poked will be recorded for 5 minutes 45.
5. LOCOMOTOR ACTIVITY. To determine whether inhibition of marble burying was due to alteration of locomotor activity, individual mice will be injected with 50% of dose that produced inhibition of burying behavior and their locomotor activity measured individually 30 min later 46. Locomotor activity will be measures using Actophotometer.
All experiments will be carried out between 10.00 AM to 4.00 PM.
All the animals used for these experiments will be maintained under standard animal house condition, temperature, relative humidity and light and dark cycle.
The animals will be maintained on standard laboratory pelleted diet and water ad libitum.
Data obtained during the above said experiments will be subjected to statistical analysis for significance
(ANOVA) following by multiple comparison tests.
7.3 Does the study require any investigation or any interventions to be conducted on patients, other humans or animals?
Yes, the study investigates the behavioral changes in the laboratory animals, when suggested to above said tests.
7.4 Has the ethical clearance been obtained from your institution in case of 7.3?
Proposal will be submitted to IAEC and clearance will be taken prior to experimentation 8. REFERENCES:
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27. SonavaneGS, Sarveiya VP, Kasture VS, KastureSB. Anxiogenic activity of Mystrica fragrens seeds. Pharmacol Biochem Behav 2002; 71(1-2) 239-44. 28. Dhingra D, Parle M, Kulkarni SK. Comparative brain cholinesterase activity of G.glabra, Mystrica fragrens, ascorbic acid and metrifonate in mice. J Med Food 2006; 9(2): 281-3. 29. Grover JK, Khandkar S, vats V, Dhunno Y, Das D. Pharmacological studies on Mystrica fragrens-antidiarrhoeal, hypnotic and hypodynamic (Blood pressure) parameters. Methods Find Exp Clin Pharmacol 2002; 24(10): 675-80. 30. Vohora SB, Shah SA, Dandiya PC. Central nervous system studies on an ethanol extract of Acorus calamus rhizomes. J Ethnopharmacol 1990; 28(1): 53-62. 31. Panchal GM, Venkatakrishna Bhatt H, Doctor RB, Vajpayee S. Pharmacology of Acorus calamus L. Indian J Exp Biol 1989; 27(6): 561-7. 32. Oh MH, Houghton PJ, Whang WK, Cho JH .Screening of Korean herbal medicines used to improve cognition function for anticholinesterase activity. Phytomedicine 2004; 11(6): 544-8. 33. Shukla PK, Khanna VK, Ali MM, Maurya R, Khan MY, Srimal RC. Neuroprotective effect of Acorus calamus against middle cerebral artery occlusion –induced ischaemia in rat. Hum Exp Toxicol 2006; 25(4): 187-94. 34. Mukherjee PK, Kumar V, Mal M, Houghton PJ. In-vitro acetyl cholinesterase inhibitory activity of essential oil from acorus calamus and its main constituents. Plant Med 2007; 73(3): 283-5. 35. Manikandan S, Srikumar R, Jayaparthasarthy N, Sheela Devi R. Protective effect of acorus calamus Linn., on free radical scavengers and lipid peroxidation in discrete regions of brain against noise stress exposed rats . Biol Pharm Bull 2005; 28(12): 2327-30. 36. Rajkumar R, kumar EP, Sudha S, Suresh B. Evaluation of anxiolytic potential of Celastrus oil in rat models of behavior. Fitoterpia 2007; 78(2): 120-4. 37. Kumar MH, Gupta YK. Antioxidant property of Celastrus paniculatus willd: a possible mechanism for enhancing cognition. Phytomedicine 2002; 9(4):302-11. 38. Gattu M, Boss KL, Terry AV Jr, Baccafusco JJ. Reversal of scopolamine induced deficits in navigational memory performance in seed oil of Celastrus paniculatus. Pharmacol Biochem Behav 1997; 57(4):793-9. 39. Nalini K, Karanth KS, Rao A, Aroor AR. Effects of Celastrus paniculatus on passive avoidance performance and amine turn over in albino rats. J Ethnopharmacol 1995; 47(2):101-8. 40. Kulkarni SK, Singh K, Bishnoi M. Elevated zero maze: A paradigm to evaluate anti anxiety effects of drugs. Methods Find Exp Clin Pharmacol 2007; 29(5): 343-8. 41. Toubas PL, abla KA, Cao W, Logam LG, Seale TW. Latency to enter a mirrored chamber: A novel behavior assay for anxiolytic agents. Pharmacol Biochem Behav.1986; 35(1): 121-6.
42. Kulkarni SK. Hand book of experimental pharmacology, 3rd Ed, Vallabh prakashan; New Delhi.1999. 43. Laurent BN, Yeter kolb, Eric PM Prinsses. A combined marble – burying locomotor activity test in mice: A practical screening test with sensitivity to different classes of anxiolytics and anti depressants. Eur J Pharmacol 2006; 547: 106-15. 44. Kung'U N, Shiela LH: Evaluation of marble burying behavior as a model of anxiety. Pharmacol Biochem Behav, 1991; 38: 63-7. 45. Mora S, Diazveliz G, Lungenstrass H, Garcia-Gonzalet M, Coto-Marales T, Poletti C et al. Central nervous system activity of the hydroalcoholic extract of casimiora edulis in rats and mice, J Ethnopharmacol 2005; 97: 191-7. 46. Kathryn SS, Martin Elliot J, Isabel calado M, Richard green A. The acute and long term neurotoxic effect of MDMA on marble burying behavior in mice. J Psychopharmacol 2006; 20(2): 264-71. 9 Signature of candidate 10 Remarks of the guide: Recommended for registration
11 Name and Designation of: 11.1 Guide: Prasanna GS Asst. Professor, Department of Pharmacology, KLE'S College of Pharmacy, Bangalore
11.2 Signature:
11.3 Co-Guide: NA 11.4 Signature: NA 11.5 Head of the Department: Dr. Purnima Ashok Department of Pharmacology,
11.6 Signature:
12. 12.1 Remarks of the Principal Recommended for registration 121.2 Signature:
Dr. Purnima Ashok Principal KLE Society’s college of Pharmacy Rajaji nagar 2nd Block, Bangalore 560010