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Effect of Bramhi Ghrita, an polyherbal formulation on learning and memory paradigms in experimental animals
G. Achliya, U. Barabde, S. Wadodkar, A. Dorle
ABSTRACT Department of Pharmaceutical Sciences, Nagpur University Campus, Amravati Road, Nagpur - 440033. India. Received: 12.7.2003 Revised: 12.9.2003 Accepted: 12.10.2003 Correspondence to: Girish S. Achliya
Objective: To investigate the neuropsychopharmacological effect of a polyherbal formulation Bramhi Ghrita (BG) on learning and memory processes in rats by elevated plus maze, and in mice by Morris water maze model. Material and Methods: BG contains Bacopa monneri (Bramhi), Evolvulus alsinoids, Acorus calamus, Saussurea lappa and cow’s ghee. Its effect (30, 50 and 100 mg/kg, p.o.) was tested on learning and memory processes. The activity of BG on memory acquisition and retention was studied using elevated plus maze model (EPM) in rats, and on spatial memory using Morris water maze model (MWM) in mice. The alcoholic extract of Bacopa monneri (40 mg/kg, p.o. ) was also administered to one group of animals. The results were compared with the vehicle-treated group. Results: Administration of Bramhi Ghrita (50 and 100 mg/kg, p.o.) showed significant reduction in transfer latency in EPM and escape latency in MWM as compared with the control group. Conclusion: BG may act as a memory enhancer formulation and may also be useful as a supportive adjuvant in the treatment of impaired memory functions. KEY WORDS: Bramhi ghrita, learning and memory, elevated plus maze, Morris water maze, Panchagavya
Introduction Bramhi Ghrita is one of the polyherbal formulations mentioned in Ayurveda (an Indian system of medicine) containing Bacopa monneri (8 g), Acorus calamus (4 g), Evolvulus alsinoids (4 g), Saussurea lappa (4 g) and cow’s ghee (80 g). This formulation has been used traditionally as a memory enhancer and for its anticonvulsant activity.1 Bacopa monneri is a wellknown nootropic plant reported for its tranquilizing,2 sedative,3 cognitive enhancer, 4-6 hepatoprotective7 and antioxidant8 actions. Acorous calamus is known for its carminative, 9 sedative and tranquilizing actions.10 Saussurea lappa has been reported to possess antiinflammatory, 11 and immunostimulant12 actions. Evolvulus alsinoids has been used traditionally as brain tonic, sedative, anthelmintic, antiepileptic and against leucoderma.13 Cow’s ghee (clarified butter fat) is believed to be useful as a memory enhancer, and antiinflammatory agent. 14 Learning is defined as the acquisition of information and skills, while subsequent retention of that information is called memory. One of the challenging tasks for neuroscientists is to elucidate the biochemical and molecular mechanisms under-
lying learning and memory. To assess the learning and memory paradigms in laborator y animals, mazes are used conventionally. There is a lack of scientific data regarding the effect of BG on learning and memory. The present study was, therfore, carried out for the authentication of traditional claims of BG as a memor y enhancer using two animal models, namely elevated plus maze (EPM) and Morris water maze task (MWM). Material and Methods Animals Male Swiss albino mice (25-30 g, body weight) and albino rats (150-200 g body weight) were used. The animals were housed in groups of five in standard laborator y conditions of temperature (23 ± 10 C), relative humidity (55 ± 5%), lighting (08:00-20:00 h) with food (Lipton India Ltd. pellets) and water freely available. The animals were transferred to the laborator y at least 1h before the start of the experiment. The experiments were perfor med between 08:00-16:00 h. The Institutional Animal Ethical Committee constituted for the purpose approved the protocol.
Indian J Pharmacol
| June 2004 | Vol 36 | Issue 3 | 159-162
which was prepared by an expert Ayurvedic practioner. The dried extract (yield 6 g) was used to compare the activity of BG on different learning and memory paradigms. The transfer latency was again recorded 24 h after the first exposure. Elevated plus maze Rats were divided into 5 groups of 6 animals each as follows: Group I animals served as control and received distilled water (10 ml/kg. Nagpur. The maze was elevated at a height of 50 cm from the ground. Statistical analysis The data obtained were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. The results were comparable with the alcoholic extract of BM. India. The level of significance was set at P<0. * * * * Figure 1: Effect of Bramhi Ghrita on spatial memory in elevated plus maze in rats *P<0. IV and V were fed orally with BG (30. No mortality was obser ved up to this dose. was noted as transfer latency. 2. Groups III.).Achliya G. Time to find the hidden platform is considered as escape latency. The test formula160 Indian J Pharmacol The rats showed a significant decrease in transfer latency in all groups (including control) on the second day. The apparatus used is a circular water tank (100 cm in diameter) filled to a depth of 30 cm with water (250C). The plant material was authenticated by the Department of Botany. The alcoholic extract of BM also showed a decrease which was not statistically significant when compared with control. Morris water maze The time required to reach the hidden platform in the water maze is illustrated in Figure 2. Group II animals received alcoholic extract of BM 40 mg/kg orally6 for comparison. The BG (100 mg/kg) treated animals reached the hidden platform in significantly lesser time than vehicle-treated animals. 2. 1. Animals received 4 trials for the first day and 8 trials per day with 5 min. p. The animals were placed individually 30 min af ter oral administration of either vehicle or test drug at the end of either of the open arms and the time taken by the animal to move from open to closed arm (Transfer latency) was noted on the first day. The ethanol was removed from the extract by distillation under reduced pressure to obtain a dried residue. The mice were released into the water and allowed 90 s to find the platform. 50 and 100 mg/kg). Groups III. Four points equally distributed along the perimeter of the tank ser ved as starting locations. The analysis revealed significant differences in transfer latency in EPM performance between BG (100 mg/kg) and vehicle-treated animals on both the days (P<0. Plant extract The aerial parts of Bacopa monneri (BM) were collected from the local forest of Deolapar and dried under shade. The for mulation was used as received in the present study. Group II animals received alcoholic extract of BM 40 mg/kg orally. IV and V received BG in doses of 30. The mice treated with BG (50 and 100 mg/ kg) showed a significant decrease in escape latency as compared with the control group (Figure 2). (Figure 1). Elevated plus maze15 tions were administered 30 min prior to the first trial daily.05 when compared to control (One-way ANOVA followed by Dunnett’s test) | June 2004 | Vol 36 | Issue 3 | 159-162 . The time elapsed between the time that the animal was placed on the open arm and the time at which all 4 legs were inside the enclosed ar ms. There was a significant difference between BG and vehicle-treated animals on Days 2-5 and 8.05. Deolapar Dist. Experimental The formulation was fed up to 2 g/kg to the animals (rats and mice) and they were observed for 24 h for mortality if any. inter-trial interval for 8 days until the performance was stable and the latency to find the platform was low (<10 sec). The platform remained in the same position during the training days. Results The formulation BG was safe on oral administration up to the dose of 2000 mg/kg. Nagpur. 6 and 7 did not show a significant difference. Data for Days 1. The plant material (750 g) was then subjected to defatting with petroleum ether (60-80) and extracted with ethanol (95% v/v) using Soxhlet extractor. 1. Morris water maze task16 Mice were grouped as 6 animals each in 5 groups as follows: Group I animals ser ved as control and received vehicle only. Nagpur University. The EPM apparatus used in the study consisted of two open and two closed arms facing each other. Bramhi Ghrita (BG) The for mulation BG was obtained as a gift sample for research from the Go-Vigyan Anusandhan Kendra. 50 and 100 mg/kg orally. The platform in the water maze was kept at the same position throughout the test to assess the effect of BG on spatial reference memory.05).o. et al. The tank was divided arbitrarily into four equal quadrants and a small platform (5 cm width) was located in the center of one of the quadrants.
15 BG significantly decreased the escape latency as compared with control. The alcoholic extract of BM (40 mg/kg. p. The water maze task was introduced by Morris (1981) and colleagues as a spatial localization or navigation task. age-associated changes in spatial navigation and the ability of nootropic agents to influence specific cognitive processes.o.) showed decreased escape latency though the efIndian J Pharmacol | June 2004 | Vol 36 | Issue 3 | 159-162 161 . and this shortened transfer latency has been shown to be related with memory processes.e.o.14 In EPM.05 compared to control (One-way ANOVA followed by Dunnett’s test) Discussion The elevated plus maze is used to measure the anxiety state in animals.) and BG (50 mg/kg. however transfer latency i. acquisition (learning) can be considered as transfer latency on first day trials and the retention/consolidation (memor y) is examined 24 h later. Recent studies of several nootropics and amnestic agents on EPM made this model a widely accepted paradigm to study learning and memory processes in rodents. The task has been used extensively to study the neurobiological mechanisms that underlie spatial learning and memory.Effect of Bramhi Ghrit a on learning and memory 90 Es ca p e la te ncy (se c) 70 60 50 40 30 20 10 0 0 1 2 3 4 Da ys 5 6 7 8 Es ca p e la te ncy (se c) 80 Cont rol BM 40mg/ kg 90 80 70 60 50 40 30 20 10 0 0 1 2 3 4 Days 5 6 7 Cont rol BG 30 mg/k g * * * * * 8 90 E sca pe l a te ncy ( se c) 80 70 60 50 40 30 20 10 0 0 1 2 3 4 Da ys 90 Co ntro l E sca pe l a te ncy ( se c) BG 5 0 m g/kg 80 70 60 50 40 30 20 10 0 0 1 2 3 4 Da ys 5 6 BG 1 00 m g/kg Co ntro l * * * * * * 7 8 * * * 5 6 7 * 8 Figure 2: Effect of Bramhi Ghrita on spatial memory in Morris water maze task in mice n = 6 in each group. which is an indication of the cognitive enhancer effect of BG in rodents. The animals treated with 100 mg/kg of BG showed a significant decrease in transfer latency as compared with the control group. the time elapsed between the movement of the animal from an open to an enclosed arm was markedly shortened if the animal had previously experienced entering open and closed arms. *P<0. p.
Sirsi M.in/icmrnews/wrk1. are necessary to evaluate the exact mechanism of the action of BG. Shukla R.34:523-6. 1994.399-407. Dhawan BN. Hand book of experimental pharmacology. a sesquiterpene lactone. Vol. Vajpayee S. Aithal HN. Govindaswamy S. Apart from memory enhancer activity these bacosides have the potential to modulate the activities of heat stock protein (HSP70) expression. Immunostimulant activity of inulin. The Authors are thankful to Dr.561. Chaurasia S. Veluchamy G. 29th November . Prakashan.292-4 Kirtikar KR. 3rd ed. Studies on formulation rotational and quality assessment of some indigenous medicinal preparations. M. place learning) before definitive conclusions can be reached. 17. (Mrs). NILIMA KSHIRSAGAR Department of Clinical Pharmacology. In vitro antiinflammatory effects of cynaropicrin. cytochrome P450 and superoxide dismutase in the rat brain. 1998. 12. fect was not significant statistically. 14. Subramanian S. Eur J Pharmacol 2000:398. 13. (Bramhi). More detailed and well-planned experimentation. Pande B.g. Pharmacological studies of Herpestis monneri. Pharmacological investigation of Herpestis monneri. Dehradun: International Book Distributors. Singh HK. 7. Parmar D.5:205-14. Sumathy T. age of animals. Phytother Res 2002:16:639-45. Ph.15:643-5. Panchal GM. J Ethnopharmacol 1982. 10.icmr. 9. et al. Indian Medicinal Plants. Building. Acknowledgements 2. 1996. New M. India. Sirsi M. Seth PK. Desai S. Upadhyay A.27. S. Phytother Res 2001. II.A.17 The bacosides of Bacopa monneri thus act as an antistress buffer in the brain of rodents. and memory processes. Wenk GL. Indian J Medical Res 1959. cued vs.D. Choudhari DK.nic. Jung JH. Basu BD.11th December 2004 Applications are invited from staff members of MCI recognized medical colleges who fulfill the eligibility criteria and who intend to develop Clinical Pharmacology at their own institutes. Certain factors need to be considered. Neuropsychopharmacological effects of the Ayurvedic Nootropic Bacopa monneri Linn.47:244-305 Prakash JC. Effect of Bacopa monniera Linn. India. 3rd ed. Bhatt HV. superoxide dismutase and cytochrome P450 activity in rat brain. 1998. Antistress effects of bacosides of Bacopa monneri: modulation of Hsp70 expression. Singh HK. III. Doctor RB. Mumbai . Balakrishna K. Bacopa monniera Linn as an antioxidant: mechanism of action. 1999. Tripathi E. J Sci Indust Res 1962. Current protocols in neuroscience. Kulkarni S. 16. 6. Tripathi YB.. Vanarasi: Chaukhamba Surbharati ICMR WORKSHOP IN CLINICAL PHARMACOLOGY Seth GS Medical College and KEM Hospital. 1st Floor. duration of treatment and experimental protocol employed (e.htm Applications should be sent on or before 31st July. Indian J Exp Biol 1989. isolated from Saussurea lappa roots. Park MH. As Bacopa monneri is one of the constituents of Bramhi Ghrita (BG). 8. Tripathi B. BG may also ser ve as a protective agent. Indian J Pharmcol 1961. Deepali M. Fulzele SV. Tel: 91 22 2413 3767/91 22 24174420 Fax: 91 22 2414 3435 162 Indian J Pharmacol | June 2004 | Vol 36 | Issue 3 | 159-162 . From the present study it can be concluded that BG may act as a memory enhancer formulation and may also be useful as a supportive adjuvant in the treatment of impaired memory functions. from Sassurea lappa. 4. Dubey GP. Comparative study of effects of Bramhi (Bacopa monneri) and chlorpromazine on motor learning in rats. References 1.Achliya G. (Bramhi) on avoidance responses in rat.D.S. 5. (Ayurveda) for her generous help. Dhawan BN. Indian J Pharm Sci 2001:63. 2004 For further information please contact: Dr. London: British Herbal Medicine Association. Kulkarni SK. Caraka Samhita. Bacosides are the main active nootropic principle present in the alcoholic extract of the plant. Parel. British Herbal Pharmacopoeia. Mumbai. which covers more parameters of learning. Thesis Nagpur University 2002. 11.23:2-5.400012. Protective role of Bacopa monniera on morphine-induced hepatotoxicity in rats. The plant Bacopa monneri is a well-known nootropic plant and a proven memory enhancer agent. Das PK. Srimal RC. Seth GS Medical College and KEM Hospital.M. Kakkar P. like species differences. Baik KV. New York: John Willey & Sons. Indian J Exp Biol 1996. providing a buffer against rapid age-related decline in mental function. Malhotra CL. Pharmacology of Acorus calamus Linn. Delhi: Vallabh Prakashan. Indian J Pharmacol 1997. 15.29:359-65.21:93-6. Chao JY. Vol. 3. Application Form can be downloaded from the ICMR website: http://www.