Comparative anxiolytic activity of Mentat® and Shankhapushpi®

Vikas Kumar

The purpose of present study was to characterize the comparative anxiolytic effects of two marketed preparations, Mentat and Shankhapushpi using four validated models of anxiety viz. open field exploratory behaviour, elevated plus maze behaviour, elevated zero maze behaviour and novelty induced suppressed feeding latency test in rats.

Rats were treated orally with vehicle (0.3 % CMC suspension), Mentat, Shankhapushpi and Lorazepam (1 mg/kg; positive control), respectively, 1 h before evaluation of behavioural parameters. The results of the present study indicate that Mentat and Shankhapushpi demonstrated a significant anxiolytic effect in rats tested on all the four validated behaviour paradigms. Moreover, Shankhapushpi has shown more significant anxiolytic effect when compared to Mentat. However, the activity of these two formulations was found to be less marked than that of the common benzodiazepine anxiolytic agent, Lorazepam.

Introduction

Anxiety affects one-eighth of the total population world-wide and has become an important area of research interest in psychopharmacology during this decade. Benzodiazepines are the major class of compounds used in anxiety and they have remained the most commonly prescribed treatment for anxiety. However, the realization that benzodiazepines present a narrow safety margin between the anxiolytic effect and those causing unwanted side effects has prompted many researchers to evaluate new compounds in the hope that other anxiolytic drugs will have less undesirable effects ¹. Herbs such as St. John’s wort, kava, passion fruit, passion flower, valerian root etc., have been used as anxiolytics. There are herbal treatments that have been used in India for ages, including Brahmi and Shankhapushpi. After thousands of years of use in Indian Ayurvedic medicine, Brahmi has been proven to be an affective anxiolytic in over 30 clinical studies. There are no known side effects regarding the use of Brahmi and it does not contain any addictive ingredients. Shankhapushpi has also been used in India for hundreds of years for nervous disorders such as stress, anxiety and insomnia ².

Mentat, a herbal psychotropic preparation has been reported to have beneficial effect in cases of mental retardation and cerebral deficit. It 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 alsinoide), and Triphala. Preliminary pharmacological investigations indicated it to be a safe preparation with central action, and anticonvulsant and adaptogenic activity ³. Mentat improves the mental quotient, memory span, and concentration ability and stress threshold. It helps reduce the levels an endogenous Monoamine Oxidase inhibitor called tribulin that is elevated in various levels of anxiety. It also ameliorates attention fluctuations and behavioural disorders and exhibits significant anti-parkinsonian activity by enhancing the dopamine post-synaptic receptor activity. The sedative and tranquilizing effects of mentat offer protection against convulsions and are beneficial in insomnia. 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 ^4-6.

Shankhapushpi is used to treat various disorders related to nervous weakness, problems like insomnia, mental as well as physical fatigue, loss of memory etc. Primarily Shankhapushpi is used as a brain tonic. Shankhapushpi is one of the best and prominent natural medicine which helps in improving memory. From ancient times in India, people of all age groups (especially students, teachers, philosophers, etc.) always have tried Shankhapushpi. Shankhapushpi works as a stimulant for brain in a way to improve its capacity, ability etc. Natural chemical composition in the Shankhapushpi helps brain to calm down and relieves the tension, which is why Shankhapushpi is also used as tranquilizer for those who suffer from insomnia. Shankhapushpi is also used as one of the most important ingredient in treatment of disorders/syndromes such as hypertension, hypotension, anxiety neurosis, stresses etc ². Thus in the present study Anxiolytic effects of Mentat and Shankhapushpi was evaluated.

Materials And Methods

Animals

Adult Charles Foster strain albino rats (200 ± 20 g), of either sex, were obtained from the Central Animal House, Institute of Medical Sciences, Banaras Hindu University and were randomly distributed into different experimental groups. The rats were housed in groups of six in polypropylene cages at an ambient temperature of 25˚ C ± 1˚ C and 45-55 % RH, with a 12:12 h light/dark cycle. Animals were provided with commercial food pellets and water ad libitum unless stated otherwise. Experiments were conducted between 10:00-13:00hrs. Animals were acclimatized for at least one week before using them for experiments and exposed only once to every experiment. “Principles of laboratory animal care” (NIH publication number 85-23, revised 1985) guidelines were followed.

Drugs and chemicals

Following drugs were used in the present study:

Mentat syrup [BR-16A] (The Himalaya Drug Co.), Shankhapushpi syrup (Dabur India Ltd.) and Lorazepam.

Drug treatments

(a) Control group: Control animals were treated with 1ml of vehicle (0.3% CMC suspension) for 7 consecutive days.

(b) Standard group: Standard group was treated with Lorazepam (1 mg/kg, p.o.) in all the experiments. Behaviour of animals was observed after one hour of oral treatment.

(c) Mentat group: Mentat was diluted with water and administered per orally at a dose of 2 ml/kg of body weight of the rats twice a day for 7 consecutive days.

(d) Shankhapushpi group: Shankhapushpi was diluted with water and administered per orally at a dose of 3.5 ml/kg of body weight of the rats twice a day for 7 consecutive days.

Methods:

1. Elevated plus-maze (EPM):

The maze has two opposite arms, 50 x10 cm, crossed with two enclosed arms of the same dimension but having 40cm high walls. The arms were connected with a central square, 10x10cm, giving the apparatus shape of a plus sign. The maze was kept in a dimly-lit room and elevated 50cm above the floor. Naïve rats were placed individually in the centre of the maze, facing an enclosed arm. Thereafter, number of entries and time spent on the open and enclosed arms were recorded during the next 5min. An arm entry was defined when all four paws of the rat were in the arm. Observations were made by a neutral ‘blind’ observer ⁷.

2. Elevated zero-maze test (EZM):

The maze comprised of black Perspex annular platform (105cm in diameter, 10cm width) elevated to 65cm above the ground level, divided equally into four quadrants. the two opposite quadrants were enclosed by a black Perspex wall (27cm high) on both the inner and outer edges of the platform, while the remaining two opposite quadrants were surrounded by Perspex “lip” (1cm high) which served as a tactile guide to animals on these open areas. The apparatus was illuminated by dim white light arranged in such a manner as to provide similar lux levels in open and enclosed quadrants for 5min test period. Rats were placed on one of the enclosed quadrants for a 5min test period. The maze was cleaned with 5% ethanol/water solution and dried thoroughly between test sessions. Animals were scored as being in the open area when all four paws were in the open quadrants and in the enclosed area only when all four paws had passed the open-closed divide ⁸.

3. Open-field test (OFT):

The open-field apparatus was made of plywood and consisted of squares (61x61cm). The entire apparatus was painted black except for 6mm thick white lines which divided the floor onto 16 squares. Open-field was lighted by a 40W bulb focusing onto the field from a height of about 100cm. the entire room, except the open-field, was kept dark during the experiment. Each animal was centrally placed in the test apparatus for 5min and the following behavioural aspects were noted:

(a)    Ambulation: This was measured in terms of the number of squares crossed by the animal.

(b)   Rearing: Number of times the animal stood on its hind limbs.

(c)    Self-grooming: Number of times the animal groomed facial region, and licked/ washed/ scratched various parts of its body.

(d)   Activity in the centre: Number of central squares crossed by the animal; and

(e)    Fecal droppings: Number of fecal droppings excreted during this period ⁹.

4. Novelty induced suppressed feeding latency (FL) test:

The test apparatus was a wooden box (60 x 60 x 35 cm) with a solid floor placed in a dimly lit room. The floor of the wooden box was covered with 2cm layer of wooden chips, and laboratory chow pellet was placed on the floor. A similar arrangement was made in the home cages of the rats. Food was removed from the home cage 48hr prior to testing, but water was provided ad libitum. Naïve rates were placed individually in the test chamber and the latency to begin eating (defined as chewing of the pellet and not merely sniffing or playing with it), was recorded. If the rat had not eaten within 300sec, the test was terminated and latency score 300sec was assigned. Observations were made by a neutral ‘blind’ observer ¹⁰.

Statistical Analysis

The data are expressed as means + SEM for each treatment group. The data obtained from each response were subjected to one way analysis of variance (ANOVA) followed by Student-Newman-Keuls Test.

Results

1. Elevated plus-maze (EPM):

The rats treated with Mentat and Shankhapushpi showed anxiolytic activity in terms of significant increase in time spent in open arms, entries in open arms and closed arm and a significant decrease in the time spent in enclosed arms in comparison to the control rats. Time spent in the open arms was more than those treated with Shankhapushpi in rats treated with Mentat but the number of entries into the open arm of Shankhapushpi group was more. Lorazepam caused more anxiolytic effect in comparison to both Mentat and Shankhapushpi (Table 1). The percent open arm entries [100 * (open arm entries) / (open arm entries + closed arm entries)] have also indicated relative anxiolytic effect of these two formulations as shown in Figure 1.

Table 1: Effect of Mentat, Shankhapushpi and Lorazepam on elevated plus maze behaviour in rats

n=6. * P<0.05, ** P<0.01, *** P<0.001 vs. control.

Figure 1: Effect of Mentat, Shankhapushpi and Lorazepam on elevated plus maze behaviour in rats. (Shankhp = Shankhapushpi)

2. Elevated zero-maze test (EZM):

The rats treated with Mentat and Shankhapushpi showed anxiolytic activity in terms of significant increase in time spent in open arms, entries in open arms and a significant decrease in the time spent in enclosed arms and entries in enclosed arms in comparison to the control rats. Rats treated with Shankhapushpi, time spent in the open arms was more than rats treated with Mentat. In case of rats treated with Shankhapushpi, the number of entries into the open arm was more than rats treated with Mentat. As shown below, the ratio of open arm entries/total no. of entries also indicates the relative anxiolytic effect of these two formulations. (Table: 2, Figure: 2)

Table 2: Effect of Mentat, Shankhapushpi & Lorazepam on elevated zero maze behaviour in rats

n=6. * P<0.05, ** P<0.01, *** P<0.001 vs. control

Figure 2: Effect of Mentat, Shankhapushpi and Lorazepam on elevated zero maze behaviour in rats

Shankhp = Shankhapushpi

3. Open-field test (OFT):

Rats treated with Shankhapushpi and Mentat, showed significant increase in open field ambulation, rearing, self grooming and activity in the centre in comparison to the control. Shankhapushpi was found to be more effective than Mentat. A significant decrease in the fecal droppings was also observed in rats treated with Mentat and Shankhapushpi in comparison to the control. Rats treated with Lorazepam showed significant anxiolytic activity than Shankhapushpi and Mentat groups. Results are summarized in Table 3.

Table 3: Effect of Mentat, Shankhapushpi and Lorazepam on open field exploratory behaviour in rats

n=6. * P<0.05, ** P<0.01, *** P<0.001 vs. control.

4. Novelty induced suppressed feeding latency:

Both Mentat and Shankhapushpi treated rats showed significant decrease in latency time to feed in a novel environment. Rats treated with Lorazepam showed significant anxiolytic activity than Shankhapushpi and Mentat groups. However, Shankhapushpi was found to be more effective than Mentat. Results are summarized in Table 4.

Table 4: Effect of Mentat, Shankhapushpi and Lorazepam on novelty induced suppressed feeding latency behaviour in rats

n=6. * P<0.05, ** P<0.01, *** P<0.001 vs. control.

Discussion

Most of the animal models of anxiety now in use were developed for benzodiazepines (BZD) and, since these compounds also exhibit significant muscle relaxant and anticonvulsant effects, evaluation of anxiolytic activity, even with non- BZD compounds, invariably now includes test for these neuropharmacological actions ¹¹. The sedative, amnesic and ataxic effects of BZD are definite drawbacks when these drugs are used for the treatment of anxiety. However, since the question of reliability and validity is foremost in establishing animal test, recourse has to be taken to compare the pharmacological profile of activity of a putative anxiolytic agent with that elicited by a BZD ^{12 - 15}. As such, despite the additional effects that Lorazepam is known to have, it was used to validate the anxiolytic activity of Mentat^® and Shankhapushpi ^®.

In the open field test, when animals are taken from their home cage and placed in a novel environment, they express their anxiety and fear by decrease in ambulation and exploration, rearing and grooming behaviours, and increase in defecation due to heightened autonomic activity. These behavioural changes are attenuated by classical anxiolytics and augmented by anxiogenic agents ¹⁶. Likewise, the elevated plus maze and elevated zero maze tests are based on the principle that exposure to an elevated and open maze arm leads an approach- conflict that is considerably stronger than that of evoked by exposure to an enclosed arm of the maze. Thus, open/enclosed arm entries provide a measure of the fear-induced inhibition of exploratory activity. These responses are increased by anxiolytics and reduced by anxiogenic agents ¹⁷. Furthermore, anxiolytics decrease the feeding latency in the novelty induced suppressed feeding latency test in a novel environment.

Overall, the results of the present study indicate that Mentat and Shankhapushpi demonstrated a significant anxiolysis in rats tested on the validated behaviour paradigms viz. open field exploratory behaviour, elevated plus maze behaviour, elevated zero maze behaviour and novelty induced suppressed feeding latency tests. Moreover, Shankhapushpi has shown more significant anxiolysis when compared to Mentat. However, the activity of these two formulations was found to be less marked than that of the common BZD anxiolytic agent, Lorazepam.

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About Authors:

Dr. Vikas Kumar

Dr. Vikas Kumar is native to Faridabad, Haryana and currently serving as Reader in Pharmacology at Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi, U.P. India. Prior to joining BHU, Dr. Kumar was employed as Post Doctoral Research Associate at Texas Tech School of Pharmacy's Pharmaceutical Sciences Department. Dr. Kumar received Ph.D. (Pharmacology) from the IT, BHU, India in year 2001 after obtaining basic degrees in Pharmacy viz. M.Pharm., B.Pharm. and D.Pharm. Dr. Kumar have also worked at R&D Centre of Indian Herbs Ltd., Saharanpur as Scientist-C where he was instrumental in setting up of laboratory devoted to behaviour and other neuropharmacological studies in rodents. After working with India Herbs, Dr. Kumar joined Lupin Research Park, as Research Associate-II based at Pune. There he was one of active team members responsible for performing efficacy studies and documenting the reports for filing Investigational New Drug Application on LL-4218 (an isolated herbal fraction for the treatment of Psoriasis). Dr. Kumar has published more that 40 papers in peer reviewed national and international journals. Dr. Vikas has also published one chapter in an international book ‘Hypericum’ under a series ‘Medicinal and Aromatic Plants-Industrial Profile’ published by Taylor & Francis, London; simultaneously published by Taylor & Francis Inc., New York, USA and Canada (ISBN 0415369541). Dr. Vikas is also member of various reputed professional organisations in India and abroad as well. Dr. Kumar is recipient of Semi Khatib Gold Medal and “Servier Young Investigators’ Award” (1999) instituted by Institutet de Researches Internationales Servier, France. Beside USA, Dr. Vikas have also visited Canada, Germany and France to enhance his professional experience at various capacities. Dr. Kumar can be contacted through e-mail at vikask@bhu.ac.in or neuropharmacologist@rediffmail.com

Mr. G.M. Husain

Gulam Mohammed Husain is native to Najibabad, U.P. and currently pursuing M. Pharm. (Final Year) in Pharmacology from Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi, U.P. India. Email: gmhusain@gmail.com

Ms. S. Mridula

Swayampakula Mridula is native to Hyderabad, Andhra Pradesh and currently pursuing B.Pharm. (Final Year) from Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi, U.P. India. Email: smridula@gmail.com