Antidepressant activity of standardized extract of Bacopa monniera in experimental models of depression in rats.

Summary

Bacopa monniera Wettst. (syn. Herpestis monniera L.; Scrophulariaceae) is a commonly used Ayurvedic drug for mental disorders. The standardized extract was reported earlier to have significant anti-oxidant effect, anxiolytic activity and improve memory retention in Alzheimer's disease. Presently, the standardized methanolic extract of Bacopa monniera (bacoside A - 38.0 - [+ or -] 0.9) was investigated for potential antidepressant activity in rodent models of depression. The effect was compared with the standard antidepressant drug imipramine (15 mg/kg, ip). The extract when given in the dose of 20 and 40 mg/kg, orally once daily for 5 days was found to have significant antidepressant activity in forced swim and learned helplessness models of depression and was comparable to that of imipramine.

Key words: Bacopa monniera, rasayanas, bacoside A, antidepressant activity

Introduction

Bacopa monniera Wettst. (syn. Herpestis monniera L.; Scrophulariaceae) is commonly mentioned as a rasayana in Ayurvedic materia medica and is advocated to be useful in clinical mental disorders akin to modern classification of anxiety disorders, including generalized anxiety syndrome, obsessive compulsive disorders, panic attacks, hysteria, epilepsy and insomnia (Udupa and Singh, 1995). The Ayurvedic concept of rasayana consists of specialized class of drugs which prevent ageing, increase longevity, impart immunity, improve mental functions and add vigor and vitality to the body (Sharma, 1994) and are akin to modern classification of adaptogens. In a related report, Bacopa monniera (BM) has been shown to have mast cell stabilizing activity (Samiulla et al., 2001). Further it is sub-classed as a Medhya rasayana, a class of plant drugs used to promote mental health and improve memory and intellect (Udupa and Singh, 1995). Bacopa monniera (BM) has been reported to have several neuropsycopharmacological activitie s. BM has been reported to posses memory enhancing effect (Dhawan and Singh, 1996) and to protect against phenytoin-induced cognitive deficit (Vohora et al., 2000). BM has been reported to posses anxiolytic activity with an advantage over the widely used benzodiazipine anxiolytics, that it promotes cognition unlike the amnesic action of the latter (Bhattacharya and Ghosal, 1998). Antioxidant activity in different brain regions (Bhattacharya et al, 2000) and in gastric mucosal incubates (Sairam et al., 2001) have been reported. Anxiety occurs along with depression and is treated with anxiolytics, anti-depressants or both (Casacalenda and Boulenger, 1998; Stahl, 1999). There have been several reports of drugs possessing both anxiolytic and antidepressant activities (Yocca, 1990; Labrid et al., 1992; Murphy et al., 1995) including natural drugs (Kumar et al., 1999, 2000). These reports lead us to further investigate BM for potential antidepressant activity. The evaluation was done by using forced swim and learne d helpless tests, the two extensively validated and widely used models of experimentally induced depression in rats (Weiss and Kitts, 1998; Bhattacharya et al., 1999a).

Phytochemical investigations have shown the presence of several saponins (Chatterjee et al., 1965; Rastogi et al., 1994) including bacoside A (Fig. 1) and its optical isomer, bacoside B (Garai et al., 1996a, b). Other new saponins have also been reported, but their activities are relatively unexplored (Mahato et al., 2000). Bacoside A has been reported to be responsible for facilitation of memory (Rastogi et al., 1994), and extracts standardized to bacoside A, have been reported to have anxiolytic activity (Bhattacharya and Ghosal, 1998a), antiulcer activity (Sairam et al., 2001) and reverse cognitive deficits in animal models of Alzheimer's disease (Bhattacharya et al., 1999b). Hence, the present study was conducted with BME standardization to bacoside-A content.

The study, thus, investigates the anti-depressant activity of standardized extract of Bacopa monniera in models of experimentally induced depression in rats.

Materials and Methods

Animals

The antidepressant activity of Bacopa monniera was studied on inbred Charles-Foster (CF) albino rats (130-180 g), of either sex, obtained from the central animal house of Institute of Medical Sciences, Banaras Hindu University, Varanasi. They were kept in the departmental animal house at 26 [+ or -] 2 [degrees]C and relative humidity 44-56%, light and dark cycles of 10 and 14 h respectively for 1 week before and during the experiments. Animals were provided with standard rodent pellet diet (Hind liver) and water was allowed ad libitum. 'Principles of laboratory animal care' (NIH publication no. 82-23, revised 1985) guidelines were followed.

Drug treatment

Whole plants of cultivated variety of Bacopa monniera (Ayurvedic Gardens, Banaras Hindu University) were collected in the month of March and were identified with the standard sample preserved in the department of Dravyaguna, Institute of Medical Sciences, Varanasi.

The fresh whole plants of Bacopa monniera were size reduced and macerated with methanol for 7 days. The extract was filtered, vacuum dried and stored in a refrigerator until further use. The yield was 1.2%. The methanolic extract was subjected to HPTLC analysis (CAMAG TLC system; evaluation software; CATS 3.16/Scanner II v 3.14) as described earlier (Chatterjee et al., 1965; Bhattacharya and Ghosal, 1998a; Sairam et al., 2001) for estimation of bacoside-A (Fig. 1). The solvent systems used were n-butanol acetic acid-water (4:1:1) and ethyl acetate-acetic acid-formic acid-water (100:11:11:27); mode of detection, quenching at [lambda] 260 nm; staining reagent-2,4-dinitrophenyl-hydrazine; reflectance spectra: [lambda] max 278 nm. The percentage of bacoside A was 38.0 [+ or -] 0.9. BME (suspended in 1% carboxymethyl cellulose in distilled water) in doses of 20 and 40 mg/kg was administered orally once daily for 5 days and imipramine (Sun Pharma, India, IMP) in the dose of 15 mg/kg, ip, was administered 30 minutes before the experiment. Control group of animals received suspension of 1% carboxymethyl cellulose in distilled water. Experiments were conducted on day 6, one hour after last drug administration.

Experimental methods

The following experimental models of depression were used.

* Behavioral despair test: The rat was placed in a cylinder (45 x 20 cm) containing 38 cm water (25 [+ or -] 2 [degrees]C), so that the rat could not touch the bottom of the cylinder with its hind limb or tail, or climb over the edge of the chamber. Two swim session were conducted, an initial 15 mm. pretest, followed by a 5 mm. test 24 h later. Drugs were administered after pretest. The period of immobility (remained floating in water without struggling and making only those movements necessary to keep its head above water) during 5 min. test period was noted (Willuer, 1984).

* Learned helplessness test:

(a) Inescapable shock pretreatment: Electric foot shocks was delivered in 20 x 10 x 10 cm chamber with plexiglass walls and cover. The floor was made of steel grids to deliver electric shock. A constant current shocker was used to deliver 60 scrambled, randomized inescapable shocks (15 s duration, 0.8 mA, every min. + 15 s to grid floor. Control rats were placed for 1 h identical chambers but no shocks were administered. Inescapable shock pretreatment was performed in the morning (Sherman and Petty, 1982).

(b) Conditioned avoidance training: In order to evaluate escape and avoidance performance, avoiding training was initiated 48 h after inescapable shock pretreatment in the Sidman jumping box (Techno, Lucknow, India). The jumping box was divided into two equal chambers (27 x 29 x 25 cm) by a plexy glass partition with a gate providing access to the adjacent compartment through a 14 x 17 cm space. Animals were placed singly in one of the chambers of jumping box and were allowed to habituate to the test environment for 5 mm. (for the first session only) and then were subjected to 30 avoidance trails (inter-trial intervals being 30 s). During the first 3 s of each trial, a light signal was presented, allowing the animals to avoid shocks. If a response does not occur within this period, a 0.8 mA shock (3 s duration) was applied via the grid floor. In case no escape response occurs within this period, shock and "light conditioned stimulus" were terminated. Avoidance sessions performed for 3 consecutive days (day 3, 4 and 5) in the morning, and the number of escape failures, referred as no crossing response during shock delivery, was recorded.

* Statistical analysis: Data were analyzed by one-way ANOVA followed by Newman-Keuls test.

* Results

Behavioral despair test

BME, 20 and 40 mg/kg given orally once daily for five days, significantly decreased the period of immobility induced by behavioral despair due to restrictive swimming by the rats. Imipramine (IMP) also showed a similar profile of activity.

Learned helplessness test

BME (20 and 40 mg/kg) significantly decreased escape failures after 5 days of pretreatment and increased avoidance responses in contrast to control rats with prior experiences of inescapable shocks, which exhibited marked increase in escape failures. IMP also showed a similar activity.

* Discussion

BME showed significant anti-depressant activity in the most commonly used paradigms of models depression namely, the forced swim and learned helplessness tests. In the forced swim test, BME significantly reduced immobility period suggesting anti-depressant activity and the activity was comparable to the reference drug 1PM. Immobility is a state of lowered mood or hopelessness, which the rats experience when they are allowed to swim in a restricted space from which they cannot escape. This is thought to reflect either a failure to persist in escape directed behavior after persistent stress or the development of passive behavior that disengages the animal from active forms of coping with stressful stimuli (Lucki, 1997). This behavioral immobility reflecting a state of despair is reduced by a broad spectrum of antidepressant drugs (Porsolt et al., 1978; Borsini and Meli, 1988).

In learned helplessness test, rodents exposed to inescapable and unpreditable electric shock, at one situation fail to escape even when escape is possible. This is termed as escape failures. Potential anti-depressant drugs decrease escape failures. The animals are also able to avoid the impending danger by escaping to the safe chamber, which is termed as avoidance responses, which are concomitantly increased. BME and IMP significantly decreased escape failures and increased avoidance responses indicating anti-depressant activity.

The wide variety of neuropharmacological actions of BM opens up interesting avenues for further research. The activity of BME both as an anxiolytic (Bhattacharya and Ghosal, 1998a) and anti-depressant needs further evaluation. This offers new perspectives in the treatment of these diseases, as there is compelling evidence that, symptoms of anxiety and depression overlap with one another (Shader and Greenblatt, 1995). Many anti-depressants have been reported to be of use in anxiety disorders and anxiolytics in depression (Haefely, 1992). This suggests that there is some etiological similarity in the development of anxiety and depression. Several hypotheses have been proposed to explain this aspect. The serontonergic theory hypothesis that, acute and chronic behavioral and biochemical consequences of antidepressant are in the direction of reduced serontonergic neurotransmission and that depression and anxiety may result from excessive functioning of the serontonergic punishment system (Deakin, 1983). There are several reports pertaining to both anxiolytic and antidepressant of serontonergic drugs (Yocca et al., 1990; Labrid et al., 1992; Murphy et al., 1995). Another theory proposes that GAB Anergic, which forms the basis of action of anxiolytic activity of many drugs, may also be involved in the antidepressant activity (Lloyd et al., 1989). Many GABA-mimetic drugs have been reported to be effective antidepressant as well (Lloyd et al., 1989). These reports show that anxiety and depressant may share some common etiological factors and drugs showing both anxiolytic and antidepressant activities are to be extensively studied for their therapeutic beneficial uses.

Thus, the present observations indicate the antidepressant activity of BME. Further, neurochemical studies would unravel the possible mechanisms involved in the antidepressant activity of BME.

Table 1

Effect of methanolic extract of Bacopa monniera (BME) and imipramine
(IPM) on behavioral despair model of depression in rats (data are mean
[+ or -] SEM, n = 8 in each group).

Groups   Dose                  immobility period
         (mg/kg, po x 5 days)  (Seconds)

Control  -                     112.2 [+ or -] 8.4
IPM      10                     61.0 [+ or -] 6.9 **
BME      20                     69.0 [+ or -] 7.7 **
BME      40                     51.7 [+ or -] 3.8 **

** P < 0.01 compared to control.
Table 2

Effect of methanolic extract of Bacopa monniera (BME) and Imipramine
(IPM) on learned helplessness model of depression in rats (data are mean
[+ or -] SEM, n=8 in each group).

Groups       Day 1
(mg/kg, po,
x5 days)     Escape                Avoidance
             failures              responses
             (N)                   (N)

Control      26.2 [+ or -] 0.9      1.5 [+ or -] 0.5
IPM 10        9.0 [+ or -] 0.5 **  17.0 [+ or -] 0.7 **
BME 20        9.8 [+ or -] 0.9 **  11.9 [+ or -] 0.9 **
BME4O         8.3 [+ or -] 0.6 **  13.3 [+ or -] 1.1 **

Groups       Day 2
(mg/kg, po,
x5 days)     Escape                Avoidance
             failures              responses
             (N)                   (N)

Control      14.3 [+ or -] 1.3      9.7 [+ or -] 1.4
IPM 10        2.3 [+ or -] 0.4 **  22.0 [+ or -] 1.0 **
BME 20        3.2 [+ or -] 0.4 **  21.3 [+ or -] 0.9 **
BME4O         2.2 [+ or -] 0.7     23.0 [+ or -] 1.3

Groups       Day 3
(mg/kg, po,
x5 days)     Escape            Avoidance
             failures          responses
             (N)               (N)

Control      1.7 [+ or -] 0.2  25.3 [+ or -] 0.6
IPM 10       1.3 [+ or -] 0.2  27.7 [+ or -] 0.3
BME 20       1.8 [+ or -] 0.3  26.0 [+ or -] 0.7
BME 4O       1.1 [+ or -] 0.3  27.8 [+ or -] 0.3

** P < 0.01 compared to control.

Acknowledgement

RKG is thankful to Indian Council of Medical Research for grant-in aid. The authors are thankful to, Indian Herbs Ltd (IH), Saharanpur, India, for technical facilities provided for chemical standardization of the extract.

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