A Study Of The Anxiolytic-Like Activity Of Dillenia Indica LINN. Leaves In Experimental Models Of Anxiety In Mice
M Lahkar, B Thakuria, P Pathak
anxiolytic activity, dillenia indica, elevated –plus, hole-board model, light-dark exploration test, maze, open-field test
M Lahkar, B Thakuria, P Pathak. A Study Of The Anxiolytic-Like Activity Of Dillenia Indica LINN. Leaves In Experimental Models Of Anxiety In Mice. The Internet Journal of Pharmacology. 2010 Volume 9 Number 2.
Anxiety affects one-eighth of the total population worldwide and has become a very important area of research interest in psychopharmacology during this decade. According to the World Health report (1), approximately 450 million people suffer from a mental or behavioral disorder, yet only a small minority of them receives even the most basic treatment. This amounts to 12.3% of the global burden of disease, and will rise to 15% by 2020 (2).
Anxiety, a state of excessive fear, is characterized by motor tension, apprehension sympathetic hyperactivity and vigilance syndromes. Benzodiazepines are considered the drug of choice in the treatment of anxiety that has been used for the last 45 years to treat several forms of anxiety(3). Benzodiazepines , barbiturates, alcohol and tricyclic antidepressants (TCA,s) have been used for long time to treat anxiety disorders (4,5). Although benzodiazepines had well-known benefits, their side-effects are prominent including sedation, muscle relaxation , anterograde amnesia and physical dependence(6). BZDs produce their therapeutic and undesirable effects by allosterically modulating the action of GABA via the central benzodiazepine receptor (CBR), located on the extracellular domain of the GABAa receptor(7).
In recent years, the development of new anxiolytics has been an area of interest. Various types of herbal medicines have been used as anxiolytic agents in different parts of the world.
The leaves, bark, and fruit of the plant are used in the indigenous system of medicine. The fruit juice is mixed with sugar and water and used as a cooling beverage in the treatment of fever. The fruit juice is used as a cardiotonic (11). The leaves and bark are used as a laxative and astringent.
Bruised bark is applied as a cataplasm for patients with arthritis(12). The alcoholic extract of the
and phenolic materials are also present (13,14). On the basis of the above considerations it was the purpose of this study to characterize the anxiolytic-like activity of a methanolic extract prepared from the leaves of
Materials And Methods Preparation Of Plant Material
The leaves of DI were collected from the rural districts surrounding Guwahati during the month of June 2010 and authenticated by Dr. T.R. Sarma , Lecturer in Botany , Swadeshi Academy Junior College , Guwahati-781005 . The fresh leaves were cleaned and washed thoroughly with water and rewashed with distilled water. Washed fresh leaves were dried under shade in clean dust-free environment, then grinded and powdered with a electric mixer and then stored in an airtight container . The powder ( around 250 grams) was then extracted with 500 ml of hydroethanol (50:50) in the Soxhlet extractor at a temperature of 40-50 degree centigrade . After complete extraction , the hydroethanolic extract was concentrated under vacuum to obtain a thick extract which was then used subsequently.
Acute toxicity studies were done with the extract and the animals were observed for gross behaviour and the median lethal dose (MLD). The No Observed Adverse Effect Limit (NOAEL) was subsequently found to be 2000 mg/kg/day . Three different doses of the extract ( 100 , 200 and 400 mg/kg ) were administered to the animals(11). The respective doses were administered to the animals for a period of 7 days .
Healthy adult Swiss albino mice of either sex weighing about 20-25 grams each were used for the study. The animals were divided into 5 groups of 6 animals each. The animals were housed in colony cages and maintained under standard environmental conditions, 25±2 centigrade temperature , RH 45-55 % and 12:12 h light: dark cycle . They were provided free access to food and water
All experiments were carried out during the light period (0800-1600 hrs.).
The study was conducted after obtaining approval from Institutional Ethics Committee, Gauhati Medical College, Guwahati (Regd. No.: 12/GMCH/CPCSEA/351).
Diazepam in a dose of 2 mg/kg i.p. was used as the standard drug . The respective doses were administered to the animals for a period of 7 days .
Experimental models of anxiety
For the evaluation of the anxiolytic activity of
the Elevated Plus Maze , the Light-Dark Exploration model , the Hole Board model and the Open Field test.
Hole-board test (15)
The hole board apparatus consists of a wooden box (40×40×25 cm) with 16 holes (each of diameter 3 cms ) evenly distributed on the base of the box . The apparatus was elevated to a height of 25 cms. Mice were fed orally with extract ( 100,200 and 400 mg/kg PO) or vehicle 30 minutes before they were placed in the apparatus . The number of head pokes and time of head dipping during a 5 minute period were recorded.
Open field test(15)
The apparatus consists of a wooden box (60×60×30 cms). The base of the box was divided into 16 squares (15×15 cms ). The apparatus was illuminated with a 40 W lamp suspended 100 cms above it. The mice were fed orally with the extract (100,200 and 400 mg/kg PO), vehicle or diazepam. After 30 mins. They were placed in one of the corner squares , the number of rearings, assisted rearings (forepaws touching the walls of the apparatus) and the number of squares crossed were counted for 5 mins.
Elevated plus maze (EPM)(16)
The EPM apparatus consists of two open arms (35×5 cms) crossed with two closed arms (35×5×20 cms.) . The arms were connected together with a central square of 5×5 cms . The apparatus was elevated to a height of 25 cms in a dimly illuminated room . Mice were treated with the extract doses (100,200 and 400 mg/kg), diazepam or vehicle 30 mins before being placed individually in the center of the EPM , facing a closed arm . The time spent in both open and closed arms , and the number of entries into both open and closed arms were counted for a period of 5 mins. An entry was defined as having all four paws within the arm .
Light-Dark exploration test (17)
The apparatus consists of two (25×25×25 cms.) joined together . One box was made dark by covering its top with plywood , whereas a 40 W lamp illuminated the other box . The light source was placed 25 cms above the open box . The mice were placed individually in the center of the lit box and observed for the next 5 mins for the time spent in the lit and dark boxes . The mice were orally administered with extract (100,200 and 400 mg/kg PO) , diazepam or vehicle 30 mins before being placed in the lit box .
A total of 30 animals were divided into 5 groups (I, II, III, IV and V). Group I: normal control group- will receive distilled water
Group II: positive control group- will receive diazepam 2mg/kg i.p.
Group III: will receive hydroethanolic extract of D.Indica leaves in dose of 100mg/kg p.o.
Group IV: will receive hydroethanolic extract of D.Indica leaves in dose of 200 mg/kg p.o.
Group V: will receive hydroethanolic extract of D.Indica leaves in a dose of 400 mg/kg p.o.
The respective doses will be administered to the animals for a period of 7 days . On the 7th day , 1 hour after administration of the last dose experiments were carried out on the mice in all the 4 different models.
The data was analyzed by one-way ANOVA followed by Dunnett-t test by using
Results And Observations
Data were expressed as mean ± SEM. Statistical calculations were done with SPSS 16.0 software. Results were analyzed by one way ANOVA P- values <0.05 were considered as significant.
ANOVA (one-way) :- ANOVA followed by Dunnett ‘t’ test is done
Df = degrees of freedom , F = F-value , P = P-value , ٭ = Pa < 0.05 when compared with control group , (±)=Pb < 0.05 when compared with std. drug ( Diazepam ) group , ® = Pb > 0.05 when compared with std. drug ( Diazepam ) group
ANOVA (one way ) :- ANOVA followed by Dunnett ‘t’ test is done
Df = degrees of freedom , F = F-value , P = P-value , ٭ = Pa < 0.05 when compared with control group , (±)=Pb < 0.05 when compared with std. drug ( Diazepam ) group , ®= Pb > 0.05 when compared with std. drug ( Diazepam ) group
ANOVA (one way ) :- ANOVA followed by Dunnett ‘t’ test is done.
Df = degrees of freedom , F = F—value , P = P-value , ٭ = Pa < 0.05 when compared with control group , (±)=Pb < 0.05 when compared with std. drug ( Diazepam ) group , ®= Pb > 0.05 when compared with std. drug ( Diazepam ) group
ANOVA ( one way ) :- ANOVA followed by Dunnett ‘t’ test is done.
Anxiety may be regarded as a particular form of behavioural inhibition that occurs in response to environmental events that are novel . It has been established that there are lots of plant secondary metabolites being employed in the treatment of psychotic disorders especially for anxiety in traditional medicine practice , most of which directly or indirectly affect the central nervous system , noradrenaline , serotonin, GABA and BZD neurotransmitter activities (18,19).
The hole-board model indicates that the head-dipping behaviour is sensitive to the emotional state of animals and suggests that the expression of the anxiolytic state in animals may be reflected by an increase in head-dipping behaviour(20).The Dillenia indica extract at doses 200 and 400 mg/kg (po) showed significant increase in the number of head poking and the time of head dipping comparable to that of the standard drug diazepam at a dose of 2 mg/kg (ip) .
In the Open Field Model, the confrontation with the situation induces anxiety behavior in mice. The anxiety behavior is triggered by two factors, i.e., individual testing as the animal was separated from its social group and agoraphobia, as the arena is very large, relative to the animals breeding or the natural environment. In such situations rodents show thigmotaxic behavior identified by spontaneous preference to the periphery of the apparatus and reduced ambulation(21). This model showed that the administration of the
The elevated plus maze (EPM) is a well established animal model for testing anxiolytic drugs(22,23). This test is based on a premise where the exposure to an EPM evokes an approach-avoidance conflict that is stronger than that evoked by an exposure to an enclosed arm(24).The decrease in aversion to the open arm is the result of an anxiolytic effect , expressed by the increase in time spent and entries into the open arm . The primary index is spatiotemporal in nature : it is reduced by anxiolytic drugs and can be increased by anxiogenic compounds(25) . Adminstration of extract in mice significantly increased the number of entries in open arm along with increase in duration in time spent at doses 200 and 400 mg/kg comparable to that of standard drug diazepam .
The light-dark test may be useful to predict the anxiolytic-like activity of drugs. Transitions have been reported to be an index of activity exploration because of habituation over time and the time spent in each compartment to be a reflection of aversion (19,26). The administration of Dillenia indica extract at doses 200 and
400 mg/kg showed significant increase in the time spent in the lit box , number of crossings and the time of latency with decrease in the time spent in the dark box .
Mechanism of anxiolytic action of plants may be by interaction with some of the natural endogenous mediators in the body as reported by various workers (27,28). Effect of most of the anxiolytic agents is to enhance the response to GABA , by facilitating the opening of GABA-activated chloride channels . Thus , the present study showed that the hydroethanolic extract of
The present study elucidated the beneficial effects of
I express my deepest acknowledgements and thanks to the all the faculty members and staff of the Department of Pharmacology , Gauhati Medical College for their invaluable suggestions and help during the course of the study. Besides I would like to thank Dr. Raktim Borgohain for assisting me with all the statistical work during the course of the study .