[Probe (1995): (XXXIV), 2, 124-139] Pharmacological Profile of BR-16A (Mentat) Kulkarni, S.K. and Verma, A. Department of Pharmaceutical Sciences, Panjab University, Chandigarh, India. ABSTRACT The present review elaborates the effectiveness of BR-16A (Mentat) as a nootropic agent, in preventing the development of tolerance and dependence to morphine and its deaddiction liabilities against ethanol withdrawal reactions. It was found to have nootropic effect in higher doses, to reverse scopalamine-induced dementia and to facilitate learning and memory. It also reversed the amnesia produced by electroconvulsive shock and prevented the development of tolerance and addiction to morphine. It controlled ethanol-induced withdrawal reactions. INTRODUCTION BR-16A (Mentat), a herbal psychotropic preparation has been reported to have beneficial effect in cases of mental retardation and cerebral deficit. Clinical trials have shown this multicomponent herbal preparation to be effective in behavioural disorders following postnatal organic lesions of central nervous system, in cases of minimal brain dysfunction as well as in cases of nocturnal enuresis 1,2,3. BR-16A contains the following indigenous ingredients, reputed in the ancient system of Ayurvedic medicine, to be of value in the management of nervous disorders: Brahmi (Hydrocotyl asiatica), Ashvagandha (Withania somnifera), Vacha (Acorus calamus), Shatavari (Asparagus racemosus), Amla (Emblica officinalis), Shankhapushpi (Evolvulus alsinoides), and Triphala. Preliminary pharmacological investigations indicated it to be a safe preparation with central action, and anticonvulsant and adaptogenic activity. The present paper describes the extensive study done on BR-16A to establish the pharmacotherapeutics of this herbal preparation especially for its nootropic profile in animals with the ultimate objective of human use. The nootropic profile was investigated on mice and rats. The effectiveness of BR-16A in improving short-term memory in naïve mice or in reversing memory deficits induced by scopolamine and after acute or chronic treatment with electroconvulsive shock, was evaluated on a passive avoidance paradigm and elevated plusmaze. The effectiveness of BR-16A in preventing the development of tolerance and dependence to morphine was investigated in mice. The deaddiction liability against ethanol was determined by studying the effect of BR-16A against alcohol withdrawal reactions as reduction in pentylenetetrazole (PTZ) threshold (convulsions) and increase in anxiety response as measured on elevated plus-maze.
MATERIALS AND METHODS Animals: Albino mice (Balb c strain) and rats (Porton strain) of either sex, bred in the central animal house facility of the Panjab University, weighing 20-25 g and 200 250 g, respectively were used. The animals were housed under standard light/dark cycle with food and water provided ad libitum. The experiments were preformed between 9.00 and 17.00 hrs. Drugs: The drugs used in the present study were obtained from the following sources: BR-16A (The Himalaya Drug Co., Bangalore), Scopolamine HBr (Merck and Co. Inc., Rahway, NJ, USA), Physostigmine (C.H. Boehringer, Sohn Ingelheim Am Rhein, Germany), GABA (Sigma, St. Louis, MO, USA). Aniracetam (F. Hoffman-La Roohe, Basel, Switzerland), Morphine (Government Analytic Laboratory, Chandigarh, India), Pentylenetetrazole (Sigma, St. Louis, MO, USA) and Ethanol (Bengal Chemicals and Pharmaceuticals Ltd., Calcutta, India). BR-16A was dispersed in water and administered per orally while the aqueous solutions of the remaining drugs were given intraperitoneally in a constant volume of 1 ml/100 g of body weight. a) Evaluation of nootropic effect apparatus: Passive avoidance step-down paradigm: The apparatus consisted of an electric grid (24 cm x 30 cm) with a shock-free zone (SFZ; 2 cm x 3 cm x 1 cm) in the centre and the entire grid having a perflex enclosure. Elevated plus-maze: An elevated plus-maze consisting of two open arms (16 cm x 5 cm) and two enclosed arms (16 cm x 5 cm x 12 cm) was used. The maze was elevated to a height of 25 cm. Electroconvulsive shock: Electroconvulsive shock (ECS; 10 ma, 0.2 sec) was applied through ear clip electrodes. The animals received either a single shock (acute treatment) or a series of 6 shocks at 24 h interval (chronic treatment). Procedure Experiment 1: Effect of BR-16A on passive avoidance acquisition and retention/retrieval (short-term memory) was studied. The mice were put individually on the electric grid and allowed to explore for 1 minute. The stimulus (20 v) was then applied and latency to reach SFZ recorded three consecutive times as basal readings. Animals that reached the SFZ in 2 minutes in the first trial were selected for the study. After 1 hour of training, each animal was put on the electric grid again and the latency to reach SFZ (acquisition) and the number of mistakes (descents) the animal made in 15 minutes were recorded. Drugs were administered either immediately or 30 minutes after passive avoidance training.
Experiment 2: The procedure and the apparatus were identical to those described in experiment 1 except that instead of number of mistakes in 15 minutes, the latency to climb SFZ measured 24 hours after training served as a parameter for retention. The animals were divided into 4 groups. One group received ECS through the ears and another group was subjected to the same procedure but without ECS (non-ecs: control group). In the ECS experiment, ECS was applied immediately after the training. Latency to reach SFZ was then recorded 1 hour and 24 hour after training. BR-16A (50 and 100 mg/kg) or vehicle was administered 30 minutes following the application of ECS. Experiment 3: The procedure and the apparatus were identical to those described in experiment 1, except that treatment with ECS was repeated every 24 hours over 6 successive days. On the seventh day, the mice received BR-16A (20-500 mg/kg) or saline and returned to their home cages. 30 minutes later, latency to reach SFZ was recorded. In 2 other groups, BR-16A (50 and 100 mg/kg) was administered 30 minutes prior to training on the 1 st day. Electroconvulsive shock was applied immediately after the training. Latency to reach SFZ was measured 1 hour and 24 hour after training. On the 2 nd day, after the measurement of retention latency, administration of BR-16A was followed 1 hour later by application of ECS. On the third and successive days the mice received BR-16A followed by ECS for a total of 6 days. On the seventh day, 24 hour after the last shock, the latency to reach SFZ was measured. Experiment 4: Elevated plus-maze was employed for the measurement of transfer latency (TL). The mice were placed individually at the end of one open arm facing away from the central platform and the time it took to move from open arm to either of the enclosed arms (TL) was recorded. Transfer latency was the time lapse between the time the animal was placed in the open arm and the time when it fully entered (all the four paws in) the enclosed arm. On the 1 st day, the mouse was allowed to explore the plus-maze for 20 seconds after the measurement of TL. The mice were returned to their home cages after the 1 st trail. Twenty four hours later, the mice were placed on the plus-maze as before and TL was recorded again. Transfer latency measured on 1 st and 2 nd day served as parameters for acquisition and retrieval, respectively. All the drugs were administered 30 minutes prior to the first trial, either alone or in combination. b) Deaddiction profile Measurement of analgesic response: Analgesic response was measured by recording tailflick latency 4,5. Measurement of ethanol withdrawal-induced anxiety: Elevated plus-maze was used to measure ethanol withdrawal-induced anxiety. The plus-maze used for rats and mice was same as described earlier 6,7. Briefly, the apparatus consisted of two open arms (16 cm x 5
cm for mice and 50 cm x 10 cm for rats) and two enclosed arms (16 cm x 5 cm x 12 cm for mice and 50 cm x 10 cm x 40 cm for rats). The maze was elevated to a height of 25 cm in case of mice and 50 cm in case of rats, respectively. An anxiogenic response was defined as decreased number of entries and time spent in the open arm. There was reduction in percent preference for the open arm in animals, with anxiogenic response. Measurement of ethanol withdrawal-induced sensitisation of pentylenetetrazole (PTZ)- induced convulsions: The onset of body jerks, clonic convulsions followed by tonic convulsions and death were recorded following PTZ challenge in naïve and ethanol withdrawn animals. Each animal was observed individually for 2 hours for acute response. Reduction in the dose of PTZ to produce full blown convulsions in ethanol withdrawn animals was considered as with withdrawal-induced reduction in the convulsion threshold. The protective effect of BR-16A following acute as well as chronic treatment was noted in rats and mice. Drug administration: In chronic morphine administration study, mice received saline or BR-16A (20-500 mg/kg) followed 30 minutes later by saline or morphine (10 mg/kg). The analgesic response was assessed by the tail-flick test 60 minutes after the second injection. For the induction of tolerance to morphine, mice received the opiate injection twice daily (9.00 and 16.00 hours) for 9 days. On days 1, 3 and 9 analgesic response was assessed by the tail-flick test 60 minutes after morphine injection. In chronic ethanol administration study, mice received 2 g/kg of ethanol (10% w/v), intragastrically, twice a day on 1 st day and once daily on successive days for a total of 6 days. On the 7 th day i.e., 24 hours after the last dose of ethanol, the rats/mice were tested for withdrawal reaction. The other treatment groups included (pretreatment:treatment): i) Saline: Saline ii) Saline: Morphine/ethanol iii) BR-16A: Morphine/ethanol iv) BR-16A: Saline. On the 10 th day of morphine study or on 7 th day of ethanol study, the treatments were reversed so that the animals that had received BR-16A followed by morphine on day 1 through 9 or ethanol on days 1 through 6 were challenged with saline followed by morphine or ethanol, respectively. In addition, the animals that had been treated with BR-16A followed by saline were challenged with Br-16A followed by morphine and the animals that had been treated with saline followed by ethanol received only BR-16A. Immediately after tail-flick test on day 10, mice received injection of naloxone (2 mg/kg) to precipitate withdrawal. The withdrawal syndrome was assessed by placing mice in a 45 cm high plexiglass box and the incidence of escape jumps were recorded for 15 minutes.
The withdrawal reaction to ethanol was assessed by studying the anxiogenic reaction on elevated plus-maze or decreased threshold to PTZ (convulsive response to an otherwise non-convulsive dose in naïve animals). Statistical analysis: The data expressed as Mean ± SEM was analysed by one-way analysis of variance (ANOVA) followed by Student s t test. p<0.05 was considered significant. RESULTS a) Nootropic profile: Effect of Br-16A on passive avoidance acquisition and retrieval in mice. Retention latency, measured 1 hour after training was not significantly affected by BR-16A (50 and 100 mg/kg). The retention latency was reduced by the higher doses (250 and 500 mg/kg). Significant decrease in latency was observed only with 500 mg/kg dose of BR- 16A as compared to the control. The lower dose of BR-16A (50 mg/kg) failed to elicit any significant reduction in the number of mistakes, but the higher doses produced significant decrease as compared with the control (Table 1). Table 1: Effect of various doses of BR-16A on a) latency to reach SFZ and b) number of mistakes (descents) made in 15 minutes in passive avoidance step-down paradigm in mice Treatment (mg/kg) Latency to reach SFZ in sec (a) No. of mistakes (descents) in 15 minutes (b) Vehicle 9.00 ± 2.25 23.00 ± 4.00 BR-16A (50) 9.50 ± 1.50 19.50 ± 2.50 BR-16A (100) 10.25 ± 1.75 12.50 ± 0.75 BR-16A (250) 8.00± 3.00 10.00 ± 1.25 BR-16A (500) 4.25 ± 1.00* 6.75 ± 1.25* Values are Mean ± SE Control n=7 and n=5-7 in other treatment groups. *p<0.05 as compared with vehicletreated control group. Effect of the combination of BR-16A and GABA on passive avoidance responding: GABA (100 mg/kg) produced a significant decrease in retention latency (3.8 ± 0.51) as well as number of mistakes (5.8 ± 0.48) as compared with the control. The lower dose of GABA (50 mg/kg), however, failed to produce a significant effect on retention latency but reduced the number of mistakes as compared with the control. The combination of BR-16A (50 and 100 mg/kg) with the subeffective doses of GABA (50 mg/kg produced a further decrease in the number of mistakes. GABA also increased the effectiveness of BR-16A (100 mg/kg) in reducing retention latency 8.
Effect of the combination of BR-16A and aniracetam on passive avoidance responding: Aniracetam (50 and 100 mg/kg) significantly increased the latency to reach SFZ but the higher dose (300 mg/kg) produced a significant decrease in retention latency. Aniracetam (100 and 300 mg/kg) also produced a significant decrease in the number of mistakes. The combination of BR-16A (50 and 100 mg/kg) and aniracetam (50 mg/kg) produced a significant decrease in the retention latency as compared with the effect of BR-16A alone and a further decrease in the number of mistakes 8. Effect of BR-16A on passive avoidance performance in scopolamine-treated mice: Scopolamine (0.3 mg/kg) significantly increased the latency to reach SFZ and the number of mistakes as compared with the control. BR-16A (50-500 mg/kg) reversed scopolamine-induced delay in retention latency and reduced the number of mistakes. Significant effect was not obtained with the lower doses of BR-16A (Table 2). Table 2: Effect of various doses of BR-16A on a) latency to reach SFZ and b) number of mistakes (descents) made in 15 minutes in passive avoidance step-down paradigm in scopolamine-treated mice Treatment (mg/kg) Latency to reach SFZ in sec (a) No. of mistakes (descents) in 15 minutes (b) Vehicle 9.00 ± 2.25 23.00 ± 4.00 Scopolamine (0.3) 36.30 ± 3.00*** a 71.00 ± 9.00*** a Scopolamine (0.3) + BR-16A (50) 40.00 ± 8.12 65.00 ± 8.00 Scopolamine (0.3) + BR-16A (100) 29.00 ± 6.00 52.00 ± 9.00* Scopolamine (0.3) + BR-16A (250) 21.08 + 5.03* 38.00 ± 6.01** Scopolamine (0.3) + BR-16A (500) 17.21 ± 5.11** 25.00 ± 4.01*** Values are Mean ± SE Control n=10 and n=6-10 in other treatment groups. *p<0.05, **p<0.01, ***p<0.001 as compared with scopolamine treated group. *** a p<0.001 as compared with the vehicle-treated group. Effect of BR-16A on passive avoidance performance in mice receiving acute treatment with ECS: ECS, applied immediately after training produced a significant increase in latency to reach SFZ, measured 1 hour and 24 hours after training, as compared with the non-ecs control. Pretreatment with BR-16A (50 and 100 mg/kg) significantly reduced the latency to reach SFZ, measured 1 hour and 24 hours after training as compared with the ECS-treated group (Table 3).
Table 3: Effect of pretraining administration of BR-16A on amnesia produced by acute treatment with ECS (10 ma, 0.2 S) Treatment Latency (sec) measured after 1 Latency (sec) measured hour after 24 hours Non-ECS 5.30 ± 0.53 3.13 ± 0.62 ECS 9.13 ±0.75** 9.45 ±0.85** BR-16A (50) 10.15 ± 1.24 8.83 ± 1.25 BR-16A (100) 5.70 ±0.12* 3.90 ± 0.55* Values are Mean ± SE, *p<0.05 as compared with the ECS-treated group. **p<0.01 as compared with non-ecs control. Effect of BR-16A on passive avoidance performance in mice receiving chronic treatment with ECS: Chronic treatment with ECS for 6 successive days at 24 hours intervals produced a significant prolongation of latency to climb SFZ, measured on the 7th day after training as compared with the non-ecs control. Daily administration of BR-16A (50 and 100 mg/kg) 30 minutes prior to the application of ECS, for 6 successive days, prevented any delay in latency to reach SFZ on the 7th day after training as compared with ECS-treated control group. The latency of the non-ecs group in the 7th day was same as that in the group receiving concurrent treatment with ECS and BR-16A (100 mg/kg) (Table 4). Table 4: Effect of daily administration of BR-16A on amnesia produced by chronic treatment with ECS (10 ma, 0.2 Sec) over 6 days in mice Treatment (mg/kg) Latency (sec) to reach SFZ on 7th day Non-ECS 7.43 ± 0.42 ECS 16.85 ± 1.52** a BR-16A (50) 10.66 ± 1.02* BR-16A (100) 6.59 ±0.68** Values are Mean ± SE, n=6-7. ** a p<0.01 as compared with non-ecs group. *p<0.05, **p<0.01 as compared with non-ecs-treated group. The data are expressed as Mean ± SE. Effect of BR-16A on TL measured on plus-maze: The TL on 2 nd day was shorter than that on 1 st day in the control group. Scopolamine (0.3 mg/kg) produced a significant increase in TL on 1 st day but not on the 2 nd day as compared with the control. Scopolamine-induced increase in TL was, however, reduced by the prior treatment with BR-16A (50 and 100 mg/kg). Aniracetam (50 mg/kg), though produced a significant increase in TL on 1 st day as compared with the control, reversed scopolamine effect. The combination of BR-16A (50 mg/kg) and aniracetam further reduced the TL on the 1 st day but the same could not be observed on the 2 nd day (Table 5).
Table 5: Effect of BR-16A on transfer latency (TL) as studied on elevated plus-maze in scopolaminetreated mice Treatment (mg/kg) n Transfer latency (TL) in sec. (Mean ± SE) 1 st day 2 nd day Vehicle 8 36.25 ± 9.50 28.87 ± 3.56 Scopolamine (0.3) 7 101.43 ± 10.89*** a 39.97 ± 7.04 Scopolamine (0.3) + BR-16A (50) 8 58.25 ± 7.20* 37.25 ± 4.82 Scopolamine (0.3) + BR-16A (100) 8 29.50 ± 5.64*** 25.50 ± 3.06 Aniracetam (50) 9 72.22 ± 11.15* 40.44 ± 6.72 Aniracetam (50) + Scopolamine (0.3) 6 39.2 ± 7.01* 28.00 ± 7.70 Aniracetam (50) + Scopolamine (0.3) + BR-16A (50) 6 27.83 ± 3.20** 23.30 ± 3.30 *p<0.05, **p<0.01, ***p<0.001 as compared with the respective control values. b) Deaddiction profile Effect of chronic treatment with BR-16A on development of tolerance to the analgesic effect of morphine: Mice receiving chronic treatment with morphine (10 mg/kg) displayed maximal analgesia on days 1 and 3. In saline-treated mice, the analgesic response to morphine displayed rapid development of tolerance, reaching baseline latencies by days 9 and 10. Mice treated repeatedly with BR-16A (20-500 mg/kg) showed significant analgesic response on days 1 and 3 of morphine (10 mg/kg) treatment. Pretreatment with BR-16A prevented with development of tolerance to the analgesic response of morphine on day 9 of testing. The animals that had been treated with BR-16A and morphine on days 1 through 9, then given morphine alone on day 10 also displayed considerable analgesia on day 10 (Fig. 1 and 2). Chronic administration of BR-16A (20-500 mg/kg) following by saline failed to elicit any analgesic response on days 1 and 3 in mice. Animals treated with BR-16A followed by saline displayed tail-flick latencies no greater than saline-pretreated animals on days 1 through 9. Challenging with BR-16A and morphine (10 mg/kg) on day 10 produced significant analgesia; a response comparable to morphine effect on day 1.
Figure 1: Effect of various doses of BR-16A on morphine analgesia on day 1 and 3 of the treatment. Values are mean ± SE, n=10-21. ***p<0.001 as compared with Sal: Sal. treated control. Sal: Saline; Mor: Morphine. Figure 2: Effect of various doses of BR-16A on development of tolerance to analgesic effect of morphine on days 9 and 10 of testing. *p<0.001 as compared with Sal: Sal. treated control. *p<0.05, **p<0.01, ***p<0.001 as compared with respective Sal: Mor-treated groups. Sal: Saline; Mor: Morphine. (Other details are same as in Figure 1)
Effect of chronic treatment with BR-16A and morphine on naloxone-precipitated withdrawal jumps: Mice that had received repeated treatment with saline followed by morphine (10 mg/kg) displayed numerous escape jumps in response to an injection of naloxone (2 mg/kg) on day 10. In contrast, mice treated with BR-16A (20-500 mg/kg) and morphine (10 mg/kg) on days 1 through 8, then with saline and morphine on day 10 displayed significantly fewer jumps after naloxone (2 mg/kg) administration (Fig. 3). Figure 3: Effect of various doses of BR-16A on naloxoneprecipitated morphine-withdrawl. Effect of BR-16A on ethanol *p<0.05, ***p<0.001 as compared with the withdrawal-induced anxiety: Sal-Mor-treated control. Sal: Saline; Mor: Morphine. Acute administration of ethanol (Other details are same as Figure 1). (2 gm/kg); 10% w/v) in mice produced significant increase in the duration in open arms as compared to the vehicletreated control group. Percent preference for closed arm entry was also reduced following acute treatment with ethanol as compared with the vehicle-treated group in mice. On the other hand, mice withdrawn from chronic ethanol treatment showed greater preference for closed arm entry and significant reduction in the duration of time spent in open arms as compared with the vehicle-treated control group. Mice receiving chronic treatment with BR-16A (100 mg/kg) followed by ethanol failed to show any withdrawal-induced anxiety. There was a significant decrease in the duration in closed arms. The duration and the number of entries in open arms increased significantly as compared with the ethanol withdrawn group. Mice treated repeatedly with saline followed by ethanol for 6 days and then challenged with BR-16A on the 7 th day displayed significant increase in preference for closed arm entry and the time spent in closed arms. The duration in open arms reduced significantly as compared with the vehicle-treated control (Table 6).
Effect of BR-16A on PTZ threshold in ethanol withdrawn animals: PTZ (80 mg/kg) induced severe tonic-clonic seizures followed by 100% mortality in naïve mice. A lower dose (60 mg/kg) induced mild tonic-clonic seizure with no mortality, whereas a still lower dose failed to cause any observable behavioural change in naïve mice. However, in ethanol-withdrawn mice PTZ (60 mg/kg) produced severe tonic-clonic seizures and 60% mortality. Acute administration of BR-16A (100 mg/kg) to ethanol withdrawn mice showed protection against PTZ (60 mg/kg) convulsions. Animals showed only mild clonic convulsions followed by recovery. Chronic administration of BR-16A (100 mg/kg) followed by ethanol for 6 days exhibited only mild clonic seizures with delayed onset and 25% mortality following administration of PTZ (60 mg/kg) on day 7 in mice. Table 6: Effect of BR-16A on ethanol (EtOH) withdrawal-induced anxiety in mice Treatment n % preference for closed arm entry Vehicle 8 50 EtOH, 10% (2 g/kg; Acute) 6 20 EtOH, 10% (2 g/kg; Chronic) W/D 10 80 EtOH, 10% (2 g/kg; Chronic) W/D + BR-16A (100 mg/kg; Acute) EtOH, 10% (2 g/kg; Chronic) W/D + BR-16A (100 mg/kg; Chronic) 7 100 7 50 Duration (sec) 177.50 ± 21.33 150.00 ± 17.73 199.50 ± 10.96 Closed arm Entries (mean) 8.25 ± 1.93 10.00 ± 1.78 10.90 ± 1.62 Duration (sec) 34.25 ± 8.03 87.75 ±16.11* 15.20 ± 1.09* Open arm Entries (mean) 3.75 ± 0.63 4.00 ± 0.74 1.90 ± 0.55 243.28 7.00 7.00 1.14 ± 16.10* a ± 0.72* a ± 1.32* a ± 0.45 158.43 9.86 ± 11.35* a ±1.37 All values are Mean ± SE *p<0.05 as compared with the vehicle-treated group. * a p<0.05 as compared with the ethanol (EtOH)-withdrawal (W/D) group. 46.28 4.14 ± 6.30* a ± 0.74* a DISCUSSION The present study demonstrates that in a paradigm of short-term memory, BR-16A improves passive avoidance acquisition and memory retrieval. The memory improving effect of BR-16A manifested as decrease in latency to reach SFZ and number of mistakes (descents) the animal made in 15 minutes. A deficient cholinergic system has been implicated for the progressive decline of learning and memory in various neuropsychiatric disorders 9. Scopolamine-induced amnesia has been used as a pharmacological tool in various clinical and experimental paradigms 10,11. In the present study, scopolamine, an anticholimergic agent, produced deficits in learning as well as memory retention as the animals showed a delay in reaching SFZ and increase in the number of mistakes BR-16A
(50-500 mg/kg) reversed scopolamine-induced deficits in acquisition and retrieval. In another study in which latency measured 24 hours after training served as parameter for memory retention, acute treatment with ECS, immediately after training, produced a significant increase in latency measured 1 hour and 24 hours after training. This suggests that ECS impairs acquisition as well as retention of learned passive avoidance tasks. To substantiate the claim for the memory improving effect of BR-16A. The influence on two different parameters i.e., number of mistakes in 15 minutes and latency to reach SFZ, 24 hours after training was studied. Pretraining administration of BR-16A (50 and 100 mg/kg) prevented increase in the latency to reach SFZ, measured 1 hour and 24 hours after the application of shock since the treatment such as scopolamine or ECS cause retrograde amnesia by interfering with memory consolidation process 12,13, the above studies suggest an effectiveness of BR-16A in improving short-term memory in naïve as well as amnestic mice. The above claim is substantiated by the finding that physostigmine enhanced the reversal effect of BR-16A against scopolamine-induced disruption of short-term memory. Hence a role of cholinergic modulation in the memory enhancing effect of BR-16A can be speculated. However, the role of other neurotransmitter systems particularly GABA cannot be ruled out, as GABA and aniracetam displayed significant improvement in retention performance and also protected against disruption of learning and working memory produced by scopolamine. Aniracetam, a well-established nootropic agent, also measured the efficacy of BR-16A in improving acquisition and memory retention in GABA as well as scopolamine-treated mice. There is a general agreement that pyrrolidinone class of nootropic compounds (particularly piracetam and aniracetam) do not interact directly with any of the major neurotransmitter systems, although there have been some speculations about their indirect effects on both cholinergic and GABA ergic systems; our findings being consistent with these predictions. Additional evidence for the nootropic action of BR-16A was obtained from the studies on elevated plus-maze. Mice show a natural aversion to open and high spaces and therefore, spend more time in enclosed arms. Itoh et al., suggested that TL might be shortened if the animal had previously experienced entering the open arms 14. The shortened TL could be related to memory. In our study, BR-16A reversed scopolamine-induced delay in TL on 1 st day; the effectiveness of BR-16A being enhanced by concomitant administration with aniracetam. The observation that TL on the 2 nd day in all the treatment groups was not significantly different from that of control group suggests the failure to retain the learning task in the absence of any forceful motivation. Chronic application of ECS disrupts acquisition, retention and consolidation of a learned task as evidenced by increase in latency to reach SFZ measured on the 7 th day following application of ECS for 6 days. Concurrent administration of ECS and BR-16A, however, prevented any impairment in memory consolidation. Daily administration of BR-16A for 6 days produced
significant attenuation of amnesic effect of chronic application of ECS. The latency measured on the 7 th day was of same magnitude as of non-ecs group. These results supplement the effectiveness of BR-16A in improving cognitive functions in acute as well as chronic amnesic models in mice. This proposition is strengthened by the observation that single dose administration of BR-16A on the 7 th day, 24 hours after the last treatment with ECS, produced a significant reduction in latency to reach SFZ. Since dementia and cognitive dysfunctions are known to occur in narcotic addicts, the effectiveness of this herbal preparation in preventing the development of tolerance and dependence to morphine was investigated. Mice receiving chronic treatment with morphine (10 mg/kg) displayed maximal analgesia on days 1 and 3 but displayed rapid development of tolerance to morphine, reaching baseline latencies by days 9 and 10. On the other hand, animals treated with BR-16A displayed less tolerance, maintaining an analgesic response throughout the testing period. Pretreatment with BR-16A prevented the development of tolerance to the analgesic response of morphine on day 9 of testing. The substitution of BR-16A with saline on day 10 also displayed significant analgesia following morphine administration. The increased analgesia seen in BR-16A treated group was not attributable to an analgesic action of BR-16A alone or to an acute analgesic interaction between BR-16A and morphine. Acute treatment with BR-16A did not elicit any significant alteration in tail-flick latency demonstrating lack of effect of BR-16A on pain responsiveness per se. The substitution of saline with morphine on day 10 in the group that had been receiving BR-16A followed by saline on days 1 through 9 produced significant analgesia. This demonstrates the lack of effect of chronic treatment with BR-16A on the expression of morphine analgesia. BR-16A, therefore, inhibits the development of tolerance to the analgesic effect of morphine. In mice receiving morphine (10 mg/kg) on days 1 through 9, naloxone (2 mg/kg) precipitated numerous withdrawal jumps when tested on day 10 of testing. Pretreatment with BR-16A (20-500 mg/kg) dose-dependently reduced the number of jumps after naloxone administration in groups chronically treated with morphine. Mice that had received BR-16A and morphine on days 1 through 9 displayed few jumps despite the fact that they did not receive BR-16A on day 10 prior to the naloxone-precipitated withdrawal. The above data, therefore, suggest that BR-16A administered during repeated treatment with morphine interferes with the development of tolerance and dependence to opiates. Withdrawal from chronic treatment with ethanol (10%, 2 gm/kg) showed severe anxiogenic profile as demonstrated by greater preference for closed arm entry and significant reduction in the duration of time spent in open arms as compared with vehicle-treated group in mice.
However, animals treated chronically with BR-16A followed by ethanol for 6 days displayed significant reversal of withdrawal-induced anxiety on the 7 th day. Acute administration of BR-16A on the 7 th day to ethanol withdrawal animals also displayed considerable anxiety on the 7 th day. Reversal of anxiogenic response following substitution of BR-16A with saline in ethanol-withdrawn animals suggests that BR-16A need not be present during testing to observe anxiety in chronically BR-16A treated animals (Table 2 and 3). In ethanol-withdrawn mice lower doses of PTZ, a chemoconvulsant, were required to elicit severe tonic seizures and mortality. BR-16A significantly inhibited PTZ convulsions (60 mg/kg) when given repeatedly for 6 days. Acute BR-16A (100 mg/kg) administration following ethanolwithdrawal also produced significant attenuation of PTZ convulsions (60 mg/kg) (Table 4). The reversal of ethanol withdrawal sensitised convulsant response to PTZ suggests the effectiveness of this herbal preparation in suppressing ethanol withdrawal syndrome, whether given acutely or chronically. The development of physical dependence and tolerance with repeated use or intake is a characteristic feature of all opioid drugs and ethanol. Alcoholism and withdrawal to chronic ethanol intake are grave social and medical problems. There is no single therapy that helps the patients in overcoming alcoholism. BR-16A has been found to be a safe preparation having no apparent response per se on reward system. Chronic administration of this multicomponent herbal preparation may help prevent development of tolerance and dependence to ethanol and morphine. ACKNOWLEDGEMENT The financial assistance by The Himalaya Drug Co., Bangalore, India, is thankfully acknowledged. REFERENCES 1. Bai, K., Sastry, V.N. Probe (1991): 30, 247. 2. D Souza, B.D., Chavda, K.B. Probe (1991): 30, 227. 3. Mehta, U.R. Probe (1991): 30, 233. 4. D Armour, F.E., Smith, D.I. J. Pharmacol. Exp. Ther. (1941): 72, 74. 5. Kulkarni, S.K. Life Sci. (1980): 27, 185. 6. Pellow, S., Chopin, P., File, S.E., Briley, M. Neurosci. Meth. (1985): 14, 149. 7. Lister, R.G. Psychopharmacology (1987): 92, 180. 8. Kulkarni, S.K., Verma, A. Indian Drugs (1993): 30, 97.
9. Smith, G. Brain Res. Rev. (1988): 13, 103. 10. Flood, J., Cherkin, A. Behav. Neural. Biol. (1986): 45, 169. 11. Preston, G.C., Ward, C.E., Lines, C.R., Poppletion, P., Haigh, J.R.M., Trauf, M. Psychopharmacology (1989): 98, 487. 12. Cumin, R., Bandle, E.F., Gamzu, E., Haefely, W.E. Psychopharmacology (1982): 78, 104. 13. Spangler, E.L., Rigby, P., Ingram, D.K. Pharmacol. Biochem. Behav. (1986): 25, 673. 14. Itoh, J., Nabeshima, T., Kameyama, T. Eur. J. Pharmacol. (1991): 194, 71.