Introduction

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.

Dillenia indica L.(DI) is a common evergreen tree that grows widely in tropical forests in the western peninsula, Bihar, SubHimalayan tracts, Assam, Bengal, and central and southern India from Sylhet to Sri Lanka. It has been grown in gardens for its handsome foliage and attractive flower as an ornamental plant. The plant is locally known as Karambel or Karmal in Marathi, Chalta in Hindi, and Ramphal in Nepal (8,9,10) .

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 Dillenia indica leaves is reported to possess central nervous system (CNS) depressant activity. Phytochemical studies showed the presence of the lupeol group of triterpene such as betulinic acid and betulin and flavonol such as myricetin. Flavonoids such as Kaempferol, Quercetin, Isorhamnetin, Naringenin,

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 Dillenia indica .

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 .

Animals

Ethical Review

Chemicals

Experimental models of anxiety

Hole-board test (15)

Open field test(15)

Elevated plus maze (EPM)(16)

Light-Dark exploration test (17)

Grouping

Statistical Analysis

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.

Figure 1

TABLE 1: TABLE SHOWING RESULTS FOR ELEVATED-PLUS MAZE TEST ENTRY O = number of entries in open arms , ENTRY C= number of entries in closed arms , TIME O = time spent in open arms , TIME C = time spent in closed arms , ENTRY T= total number of entries , 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

Figure 2

Figure 3

TABLE 2 : TABLE SHOWING RESULTS FOR LIGHT-DARK EXPLORATION TEST

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

Figure 5

ANOVA (one way ) :- ANOVA followed by Dunnett ‘t’ test is done

Figure 6

TABLE 3 : TABLE SHOWING RESULTS FOR HOLE BOARD TEST

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

Figure 7

ANOVA (one way ) :- ANOVA followed by Dunnett ‘t’ test is done.

Figure 8

TABLE 4 : TABLE SHOWING RESULTS FOR OPEN FIELD TEST

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

Figure 9

ANOVA ( one way ) :- ANOVA followed by Dunnett ‘t’ test is done.

{image:9}

Discussion

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 Dillenia indica extract increased rearing , assisted rearings and number of squares traversed at doses 200 and 400 mg/kg which was comparable with the standard drug diazepam

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 Dillenia indica leaves possessed potent anxiolytic activity which was evidenced by all the models as described above . At doses of 200 and 400 mg/kg it showed activity comparable to that of standard drug diazepam ( at doses of 2 mg/kg ip).

Conclusion

The present study elucidated the beneficial effects of Dillenia indica in various models of experimentally–induced anxiety in mice at doses of 200 mg/kg and 400 mg/kg . It does not give a statistically significant result at doses of 100 mg/kg . This appears to be a promising approach that may be considered to be a complementary remedy for anxiety.

Acknowledgements