1. INTRODUCTION:

In recent decades, it has become one of the principal pillars of a branch of science called cognitive neuroscience, an interdisciplinary link between cognitive and neuroscience.^1,2Dementia is a general term for the loss of memory and other intellectual abilities serious enough to interfere with daily life. Alzheimer’s disease (AD) is the most common cause of dementia, responsible for 60 to 80 percent of all dementia. AD causes severe suffering for patients, including progressive functional impairment, loss of independence, emotional distress, and behavioral symptoms.³ Families and caregivers often experience emotional and financial stress. AD is a progressive neurodegenerative disorder associated with loss of neurons in distinct brain areas and cord. Alzheimer’s is a disease of the brain that causes problems with memory, thinking and behavior. It is not a normal part of aging.

Age, stress and emotions are conditions that may lead to memory loss, amnesia, anxiety, high blood pressure, dementia, to more ominous threat like schizophrenia and AD.^4,5

Memory enhancers and cognitive enhancers are drugs, supplements, nutraceuticals and functional foods that are purported to improve mental functions such as cognition, memory, intelligence, motivation, attention and concentration.⁶ Nootropics are thought to work by altering the availability of the brain’s supply of neurochemicals (neurotransmitters, enzymes and hormones), by improving the brain’s oxygen supply, or by stimulating nerve growth. The natural memory enhancing drugs control the activity of AChE. AChE modulates ACh to proper levels by degradation. Accordingly excessive AChE activity results in ACh deficiency which leads to memory and cognitive impairments. These natural agents inhibit the excessive AChE activity and protect the people suffering with dementia. Dementia may be reversible when caused by drugs, alcohol, hormones, vitamin imbalance and depression or irreversible when caused by disease or injury. Since allopathic system of medicine is yet to provide a radical cure for dementia and it is worthwhile to look for new direction that is in ayurvedic system of medicine, which was reported with many drugs to minimize the memory loss seen in elderly patients.⁷

Herbal remedies which were used traditionally now significantly documented for the safety profile and as a therapy for some of the pathological conditions. From the last decade synthetic drugs have been combined with herbs which show some promising results. BRAVOBOL, a licensed polyherbal formulation contains the extracts of medicinal plants, but no scientific report available for its nootropic activity. Its efficacy in AD and dementia has not been validated using modern scientific parameters. Hence, the present study was aimed to investigate nootropic activity of licensed polyherbal formulation “Bravobol”. The main aim of this work was to investigate the nootropic activity of polyherbal formulation and the evaluation will conducted using various animal model

MATERIAL AND METHOD:

Drugs and chemicals:

Bravobol capsule is a poly herbal formulation manufactured by Ban Labs Ltd., C-4 UPSIDC, Selaqui, Dehradun, Piracetam (Micro B and B lab, India), Scopolamine (Cadila Health care Ltd, India) and Clonidine (Unichem Ltd, India) were used in the present study.

Animals:

Swiss albino mice having young (18gm i.e 8 weeks) and aged (25gm i.e 28 weeks) were used for the study. They were kept in clean dry cages week before the beginning of the experiment to acclimatize with the experimental conditions. The animals were fed with standard pelleted _{and distilled water ad libitum
was maintained at 21°C-23°C under a constant 12hrs light}and dark cycle. The animal care and experimental protocols were in accordance with CPCSEA /IAEC.

Acute oral toxicity study:

The aim of performing acute toxicity studies is to establishing the therapeutic index of a particular drug. Acute toxicity study is generally carried out by determination of LD₅₀ value in experimental animal.

The dose selection of Bravobol were based on acute toxicity studies. The acute toxicity of formulation was determined by using albino mice (20-30gm) those maintained under standard husbandry conditions. The animals were fasted three hours prior to the experiment. Animals were administered with single dose of the solution of Bravobol capsule and observed for its mortality up to 48 hr study period (short term toxicity). Based on the short term toxicity profile, the next dose was determined as per OECD guidelines NO 425. From the LD₅₀ dose 1/20, 1/10 and 1/5^th doses were selected and considered as low, medium and high dose respectively and used in the entire study.⁸

Animal models for testing learning and memory:

I. Elevated plus maze (Exteroceptive Behavioural Model):

This rodent model of anxiety has been extensively used for evaluation of novel anxiolytic agents and to investigate psychological and neurochemical basis of anxiety. This test has been proposed for selective identification of anxiolytic and anxiogenic drugs. Anxiolytic compounds, by decreasing anxiety, increase the open arm exploration time; anxiogenic compounds have the opposite effect. The primary measures are the proportion of entries into the open arms and the time spent on the open arm expressed as a percentage of the total time spent on both open and closed arms60.The maze is made of plywood and consists of two open arms 30 X 5cm and two enclosed arms 30X5X20cm. the arms extend from a central 5X5cm platform. The maze is dark brown in colour and mounted to the wooded base, raising at 30cm, above the flooring in a dark room. The light of 40W is illuminated above the maze.

Evaluation:

The above mentioned parameters of standard and test compounds were carefully evaluated and compared to find the anxiolytic activity of the test compound.

A. Exteroceptive Behavioral Model:

Method: Albino mice (young mice) of either sex were divided in to five groups of six mice in each group and were fasted in overnight, prior to the test but water was supplied ad libitum. The details of the experimental protocol was given below.

Group I: Control group:

Distilled water (10ml/kg) was administered p.o for 8 days. After 90 min of administration on 8^th day, TL was recorded. Retention of learned task was examined after 24hr i.e. on 9^th day.

Group II: Standard group:

Piracetam (200mg/kg) was administered p.o for 8 days. After 90 min of administration on 8^th day, TL was recorded. Retention of learned task was examined after 24 hr i.e. on 9^th day.

Group III:

PHFB (250mg/kg) was administered p.o for 8 days. Scopolamine (0.4mg/kg) was also administered i.p 45 mts before the administration of PHFB. After 90 min administration on 8^th day, TL was recorded. Retention of learned task was examined after 24 hr i.e. on 9^th day.

Group IV:

PHFB (500mg/kg)was administered p.o for 8 days. Scopolamine (0.4mg/kg) was also administered i.p 45 mts before the administration of PHFB. After 90 min administration on 8^th day, TL was recorded. Retention of learned task was examined after 24 hr i.e. on 9^th day.

Group V:

PHFB (750mg/kg)was administered p.o for 8 days. Scopolamine (0.4mg/kg) was also administered i.p 45 mts before the administration of PHFB. After 90 min administration on 8^th day, TL was recorded. Retention of learned task was examined after 24 hr i.e. on 9^th day.

B. Spatial learning in Morris water maze (MWM)

Spatial learning of rats can be tested in a water mazeas described by Morris and McNaughton and Morris.

Apparatus

The water maze consists of a circular tank with 100 cm diameter and a wall 20 cm above the water level. A circular platform (9 cm diameter, covered with white linen material for grip) is hidden 2 cm below the water level. The water is made opaque using titanium dioxide suspension and is kept at about 23 °C during the experiment. Training takes place on three consecutive days, with the rats receiving 4 consecutive trials per day with an inter trial interval of 6–10 min. Each trial is started from one of four assigned polar positions with a different sequence each day. The latency to find the platform is measured as the time of placement of the rat in the water to the time it finds the platform. If the animal fails to find the platform in any trial within 3 min it is placed on it for 10 s

Evaluation

On day before a probe test is performed. The platform is removed and the time spent in the target quadrant (the quadrant in the center of which the platform has been located) and the number of annulus crossings (across the actual location where the platform has been located) in the first 60 s of exposure are measured.

C. Escape latency test in young mace:

Method: Adult Swiss albino mice (young mice) devided in to six groups and each group containing six animals are fasted overnight, prior to the test but water was supplied ad libitum. The details of experimental protocol given below.

Group I: Control group:

Distilled water (10ml/kg) was administered p.o for 8 days. After 90 min of administration on 8^th day ELT (sec) and TSTQ (sec) are recorded and again after 24 hr i.e. on 9^th day.

Group II: Standard group:

Piracetam (200mg/kg) was administered p.o for 8 days. After 90 min of administration on 8^th day ELT (sec) and TSTQ (sec) was recorded and again after 24 hr i.e. on 9^th day.

Group III:

Scopolamine (0.4 mg/kg) was administered i.p on 8^th day. After 90 min administration on 8^th day, ELT(sec) and TSTQ (sec) was recorded and again after 24 hr i.e. on 9^th day.

Group IV:

PHFB (250mg/kg) was administered p.o for 8 days. Scopolamine (0.4mg/kg) was also administered i.p 45 mts before the administration of PHFB. After 90 min administration on 8^th day ELT(sec) and TSTQ (sec) was recorded and again after 24 hr i.e. 9^th on day.

Group V:

PHFB (500mg/kg) was administered p.o for 8 days. Scopolamine (0.4mg/kg) was also administered i.p 45 mts before the administration of PHFB. After 90 min administration on 8^th day ELT(sec) and TSTQ (sec) was recorded and again after 24 hr i.e. 9^th on day.

Group VI:

PHFB (750mg/kg) was administered p.o for 8 days. Scopolamine (0.4mg/kg) was also administered i.p 45 mts before the administration of PHFB. After 90 min administration on 8^th day ELT(sec) and TSTQ (sec) was recorded and again after 24 hr i.e. 9^th on day.^9,10

Escape latency test in aged mice:

Method: Adult Swiss albino mice (aged mice) devided in to five groups and each group containing six animals are fasted overnight, prior to the test but water was supplied ad libitum. The details of experimental protocol given below.

Group I: Control group:

Distilled water (10ml/kg) was administered p.o for 8 days. After 90 min of administration on 8^th day ELT (sec) and TSTQ (sec) are recorded and again after 24 hr i.e. on 9^th day.

Group II: Standard group:

Piracetam (200mg/kg) was administered p.o for 8 days. After 90 min of administration on 8^th day ELT (sec) and TSTQ (sec) was recorded and again after 24 hr i.e. on 9^th day.

Group III:

PHFB (250mg/kg) was administered p.o for 8 days. Scopolamine(1.0mg/kg) was also administered i.p 45 mts before the administration of PHFB. After 90 min administration on 8^th day, ELT(sec) and TSTQ (sec) was recorded and again after 24 hr i.e. on 9^th day.

Group IV:

PHFB (500mg/kg) was administered p.o for 8 days. Scopolamine(1.0mg/kg) was also administered i.p 45 mts before the administration of PHFB. After 90 min administration on 8^th day, ELT(sec) and TSTQ (sec) was recorded and again after 24 hr i.e. on 9^th day.

Group V:

PHFB (750mg/kg) was administered p.o for 8 days. Scopolamine(1.0mg/kg) was also administered i.p 45 mts before the administration of PHFB. After 90 min administration on 8^th day, ELT(sec) and TSTQ (sec) was recorded and again after 24 hr i.e. on 9^th day.¹¹

Statistical analysis

Data were presented as mean ± Standard Error of Mean (SEM). One-way Analysis Of Variance (ANOVA), followed by Dunnet’s multiple comparison test. For all test probability 0.05 or less was accepted as significance.

RESULTS:

A. Effect on transfer latency (using elevated plus maze)

In exteroceptive behavioural model in young mice (Table 1), low dose of PHFB (250mg/kg p.o) show slightly decreased TL on 8^th and 9^th day in young mice, when compared to control groups. Medium and higher dose of PHFB (500 mg/kg and750 mg/kg) show improved learning and memory of young mice reflected by decrease in TL on 8^th and 9^thday, when subjected to EPM (graph 1and2). PHFB pre-treatment for 8 days protected the young as well as the old mice against amnesia.

B. Effect on escape latency (using morris water maze)

In morris water maze test, the escape latency is studied in young and aged mice. The test drug alone is given in aged group whereas test drug along with scopolamine is given in young mice. A significant decrease in ELT and significant increase in TSTQ is observed for both the moderate (500mg) and higher doses (750) of PHFB compared to control on 8^th as well as 9^th day of treatment (Table No 3). The animals treated with piracetam alone showed a significant decrease in ELT and scopolamine alone showed significance increase in ELT in both young and aged mice.

Escape latency testy in young mice (Table 2) Morris water maze task performance (ELT) for learning of group I animals treated with control group was found to take averagely for 6 successful trials. Group II animals treated with piracetam was found to take 14.71±0.10 sec (on 8^th day) and 13.40±0.01 sec (on 9^th day) averagely for 6 successful trials. In group IV ,V and VI the ELT values was found to be significantly decreased both on 8^th and 9^th day. Among the test solution, tested aqueous solution at 750mg/kg p.o showed the value nearer to the standard drug piracetam. This clearly indicates that aqueous solution of PHFB significantly increased the learning and memory performance.

In young mice Chronic administration of Piracetam for eight days at the dose of 200mg/kg, has resulted in significant increased TSTQ value (78.00±1.06) on 8^th day and (89.88±0.62) on 9^th day as compared to normal group. PHFB resulted in a significant (P < 0.001) increase in TSTQ 750 mg/kg (81.00± 0.57)on 8^th day and (87.31±0.47) on 9^th day as compared to the normal group (Table 2). This shows that, the PHFB has potent nootropic activity similar to that of Piracetam.

The nootropic effects of PHFB aged mice presented in (Table3). When the Piracetam was administered for eight days at the dose of 200mg/kg, it has significantly (P < 0.001) decreased ELT value (18.00.±1.03) on 8^th day and (22.00±0.57) on 0^th day as compared to the Scopolamine treated group. It was observed that administration of PHFB extract at the dose of 750 mg/kg resulted in a significant decreased ELT value (20.00.±1.41) on 8^th day and (23.66±0.76) 0n 9^th day as compared to the Scopolamine treated group. It has shown effect similar to that of Piracetam.

In aged mice Chronic administration of Piracetam for eight days at the dose of 200mg/kg, has resulted in significant increased TSTQ value (80.00±0.57) on 8^th day and (84.00±0.73) on 9^th day as compared to normal group. PHFB resulted in a significant (P < 0.001) increase in TSTQ 750 mg/kg (78.00± 0.57)on 8^th day and (81.33±0.49) on 9^th day as compared to the normal group (Table 3). This shows that, the PHFB has potent nootropic activity similar to that of Piracetam.

Table 1: Effect of PHFB on EPM (Exteroceptive behavioural model) young mice

Sl. No	Treatment	TL (sec) on 8^th day	TL (sec) on 9^th day
1	Control	23.33 ±0 .88	23.50 ± 0.84
2	Piracetam	18.17 ± 0.47***	18 ± 0.51***
3	PHFB 250 mg	22.83 ± 0.74	23.17 ± 0.70
4	PHFB 500 mg	20.67 ± 0.61*	20.50 ± 0.56*
5	PHFB 750 mg	19.50 ± 0.67**	19.17 ± 0.60***

Data is expressed as a mean ±S.E.M for n=6, Statistical analysis by one-way ANOVA followed by Dunnett’s t test significance at *P<0.05, **P<0.01, ***P<0.001.

Figure 6: Effect of PHFB on MW on 9^th day young mice

Figure 7: Effect of PHFB on MW on 8^th day Aged mice

Figure 8: Effect of PHFB on MW on 8^th day Aged mice

Figure 9: Effect of PHFB on MW on 9^th day Aged mice

Figure 10: Effect of PHFB on MW on 9^th day Aged mice

DISCUSSION:

The purpose of current research was to investigate the therapeutic potential of the polyherbal formulation Bravobol on alzheimer’s model. Alzheimer’s disease is a progressive loss of memory and cognitive function in middle age individuals. Presently nootropic agents like piracetam, pramiracetam, aniracetam and choline esterase inhibitors like donazepil are used in this condition to improve memory, mood and behaviour. However the chemophobia and the adverse effects present with these agents have limited their use. Hence it is rational to explore natural sources that is plant kingdom, for better and newer drugs for the treatment of different cognitive disorders.¹²

Nootropics are the drugs better known as smart drugs, memory cognitive enhancers, suppliments, functional foods or neutraceuticals that improve mental functions, such as cognitive attention, concentration, motivation, intelligence and memory. It is thought that these drugs enhance memory by increasing brains supply of neurochemicals like neurotransmitters, enzymes and hormones that improve oxygen supply to brain or stimulating nerve growth. Bravobol capsule contains Shankapushpi (Convolvulus pruricaulis), Bhrahmi (Bacopa monnieri), Aswagndha (Withania somnifera), Malkangini (Celastrus paniculatus), Abhrak bhasma

Exact mechanism for memory dysfunction is not clear but some of the evidences like energy dependent inhibition of neuronal membrane function, formation of oedema, opening of voltage dependent Ca²⁺ channels (LandN type) and activation of NMDA receptors, Ca²⁺ influx with subsequent activation of NMDA receptor, Ca²⁺ influx with subsequent activation of Ca²⁺/ calmodulin dependent nitric oxide NO synthase and NO are demonstrated. This NO may react with superoxide anion to produce highly toxic peroxy nitrite, which is responsible for energy depletion. All the above mentioned are responsible for the impairment of memory. Still a controversial but a predominant role of cholinergic mechanisms long been stressed in learning and memory process. The rate of the central cholinergic system is well established in memory and deficiency of this is implicated in deficit in memory. Though a very good number of other receptor systems are also now reported to involve in the behavioural expression in dementia in animals and human beings as well and the role of these neurotransmitter system cannot be ignored.¹³

The literature review on screening on screening of herbs in AD revealed that constituents like flavonoids, tannins, alkaloids and saponins reported to be responsible for anti alzheimer’s activity. The active ingredients of Bravobol contains flavonoids, tannins, alkaloid, saponins and trace elements.

The exact mechanism underlying the anti alzheimers activity of Bravobol is not fully established, it is assumed that it may be due to flavonoids, tannins, alkaloids, saponins and various other ingredients present in them may contribute its neuroprotective, antioxidant, anticholinergic, dopaminergic agonism and NA agonism. Still further research is required in neurochemical level, include biochemical estimation of cholinergic, dopaminergic, nor adrenergic level in the brain, which plays a prominent role in learning and memory of a individual.

CONCLUSION:

In the present investigation, the polyherbal formulation Bravobol has shown a potent memory enhancing effect in interoceptive as well as exteroceptive animal model.

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Received on 22.05.2015 Accepted on 23.06.2015

Asian J. Res. Pharm. Sci. 5(2): April-June 2015; Page 86-90

DOI: 10.5958/2231-5659.2015.00015.6

Sl.No	Treatment	On 8^th day		On 9^thday
Sl.No	Treatment	ELT (sec)	TSTQ	ELT (sec)	TSTQ (sec)
1	Control	22.33±0.01	75.00±0.93	25.6±0.26	83.40±0.25
2	Piracetam	14.71±0.10***	78.00±1.06***	13.40±0.01***	89.88±0.62***
3	Scopolamine	26.33±0.00***	26.00±0.81***	56.03±0.14***	29.25±0.42***
4	PHFB 250 +S	21.50±0.12	73.83±0.70	24.80±0.11	82.67±0.54
5	PHFB 500 +S	20.00±0.57***	56.00±0.73***	20.00±0.57***	61.20±0.28***
6	PHFB 750 +S	16.50±0.64***	81.00±0.57***	15.30±0.05***	87.31±0.47***

Sl.No	Treatment	On 8^th day		On 9^th day
Sl.No	Treatment	ELT (sec)	TSTQ (sec)	ELT (sec)	TSTQ (sec)
1	Control	24.00±1.15	50.00±0.93	29.00±0.57	68.00±0.93
2	Piracetam	18.00±1.03***	80.00±0.57***	22.00±0.57***	84.00±0.73***
3	PHFB 250 gm	29.16±0.70	55.00±0.73***	32.00±0.81*	57.00±0.73***
4	PHFB 500 gm	22.00±0.57	65.16±0.70***	27.00±0.96	69.00±0.73
5	PHFB 750 gm	20.00±1.41**	78.00±0.57***	23.66±.76***	81.33±0.49***