Antidepressant effects of methanol extract and fractions of Bacopa monnieri

Abstract

Bacopa monniera (L.) Wettst (Scrophulariaceae), a reputed Ayurvedic medicinal plant of India, has been widely used as a brain tonic for centuries. In the present study, the methanol extract and different fractions of B. monniera were evaluated for antidepressant activity in the forced swimming test (FST) and tail suspension test (TST) in mice. The results showed that the methanol extract, EtOAc fraction, and n-BuOH fraction significantly reduced the immobility times both in FST and TST in mice after being administrated orally for 5 consecutive days. All tested samples, in the effective doses for FST and TST, showed no inhibitory effect against locomotor activity (LA) in mice.

Keywords::

• Bacopa monniera
• forced swimming test (FST)
• tail suspension test (TST)
• antidepressant activity

Introduction

Depression is a serious psychiatric disorder that affects 17–20% of the world population, and results in a great deal of social and economic problems (Li et al., 2007). Although many antidepressants have been used clinically for several decades, most of them inevitably have some serious adverse effects, such as cardiovascular disease, the narrow scope of remedial spectrum, and a short half-life (Zhu et al., 2006). Therefore, there is an urgent need to research and develop more effective antidepressants with lower adverse effects. In some developing countries, herbal preparations have been widely used to treat diseases. Some of them have been considered as effective alternatives in the treatment of depression, as in the case of Saint John’s wort, a western herb.

Bacopa monniera (L.) Wettst (Scrophulariaceae) is an Ayurvedic medicinal plant of India, clinically used for memory enhancing, epilepsy, insomnia, and as a mild sedative (Singh & Dhawan, 1982). Studies by Bhattacharya et al. (2001) showed that this plant is able to reduce the memory dysfunction in the rat model of Alzheimer’s disease and confirmed that saponins are the major active principles. However, there are no reports about the complete antidepressant evaluation of the different fractions of the titled plant. In our investigation, the methanol extract and different fractions of B. monniera were evaluated for antidepressant activities in the forced swimming test (FST) and tail suspension test (TST) in mice. Here we report the antidepressant screening of the methanol extract and different fractions of B. monniera.

Materials and methods

Animals

Male ICR mice weighing 18~25 g (Experimental Animal Center of Shanghai Pharmaceutical Graduate School, China Academy of Sciences) were housed with unlimited food and water in a room with a 12:12 h light-dark cycle for a week before the experiment. Mice (10 mice per group) were treated with methanol extract, CHCl₃ fraction, EtOAc fraction, n-butanol fraction once daily for five consecutive days, respectively. For the positive control group, mice were tested orally with fluoxetine hydrochloride (10 mg/kg) once on the fifth day. The control group received only 0.3% CMCNa once daily for 5 consecutive days. All animal treatments were strictly in accordance with the National Institutes of Health Guide of the Care and Use of Laboratory Animals. The experiments were carried out under the approval of the Committee of Experimental Animal Administration of the University.

Plant material

Bacopa monniera was collected in Zhangzhou, Fujian province, China, in November 2002, and identified by Han-cheng Zheng (Department of Pharmacognosy, College of Pharmacy, Second Military Medical University, Shanghai, China). A voucher specimen (No. 0211-11) is deposited at Herbarium of School of Pharmacy, Second Military Medical University, Shanghai, China.

Preparation of extract and fraction

The air-dried whole plants of B. monnieri (8 kg) were percolated with 100% methanol at room temperature three times to provide methanol extract (650 g), which was partitioned with CHCl₃, EtOAc and n-BuOH, respectively, to yield CHCl₃ fraction (95 g), EtOAc fraction (70 g), and n-BuOH fraction (308 g). The methanol extract (100 mg) is equivalent to 1.2 g dried plant.

Forced Swimming Test

Behavior despair was proposed as a model to assay antidepressant by Porsolt et al. (1977, 1978). Mice were placed individually and forced to swim for 6 min in a plexiglass cylinder (20 cm in height and 14 cm in diameter), which was filled with water (24 ± 3°C) to a height of  10 cm. The mouse was dropped into water 1 h after administration on the fifth day and was observed for 6 min. After the first 2 min of vigorous activity, each mouse assumed a typical immobile posture. The immobility time was recorded during the next 4 min by a video system.

Tail suspension test (TST)

The total duration of immobility induced by tail suspension was measured according to the method described by Steru et al. (1985). Mice were suspended by the tail from a lever (30 cm high) for 6 min with the movement of mouse being recorded by a video system. The observer was also unaware of drug treatment. The total duration of the test (6 min) can be divided into two periods of agitation and immobility. Animals were considered to be immobile when they did not show any movement of body and were hanging passively. The duration of immobility in the later 4 min was recorded as the “behavior despair” status.

Locomotor activity (LA)

LA of mice was monitored via a little animal locomotor activity meter (Y-2, made in Second Military Medical University, China). The distance traveled in the open field (25 cm in height, 35 cm in diameter) was automatically recorded by the arithmometer of Y-2 (Sanberg et al., 1985). Each animal was tested individually and used only once.

Drug administration

The methanol extract was tested at doses of 50, 100, 200 mg/kg, while the CHCl₃ fraction, EtOAc fraction, and n-BuOH fraction were tested at a dose of 100 mg/kg, respectively. All tested samples were freshly prepared in 0.3% carboxymethylcellulose sodium (CMCNa) at a concentration of 5 mg/mL each day before testing. All the animals were tested orally once daily for five consecutive days, and the tests of FST, TST, and LA were carried out 1 h after administration on the fifth day. Fluoxetine hydrochloride (Changzhou Huasheng Pharmaceuticals, Changzhou city, China) was also suspended in 0.3% CMCNa at a concentration of 0.5 mg/mL. Fluoxetine hydrochloride and 0.3% CMCNa were applied as positive and normal controls, respectively. The animals exposed to fluoxetine were only treated once on the fifth day, while other groups were treated for five consecutive days.

Data analysis

The results of FST, TST and LA were analyzed by one-way ANOVA (SPSS), followed by Dunnett’s t-test. The data were presented as means ± SEM. P < 0.05 was considered statistically significant.

Results and discussion

As shown in Table 1, the immobility times of forced swimming mice exposed to methanol extract for five consecutive days at the doses of 50, 100, and 200 mg/kg, were significantly reduced by 49.7%, 67.2%, and 71.3%, respectively, which showed the obvious dose-response relation. The immobility times of forced swimming mice administrated orally EtOAc fraction and n-BuOH fraction (100 mg/kg each) for five consecutive days were significantly reduced by 63.1% (P < 0.01) and 67.7% (P < 0.01, respectively. In the tail suspension test (Table 2), the immobility times of mice exposed to methanol extract, EtOAc fraction and n-BuOH fraction (100 mg/kg each) for five consecutive days were significantly reduced by 46.6% (P < 0.01), 47% (P < 0.01), and 45.1% (P < 0.01), respectively. The CHCl₃ fraction showed no obvious reduction in the immobility times both in forced swimming and tail suspension models. The spontaneous activity test (Table 3) showed that methanol extract, EtOAc fraction and n-BuOH fraction did not affect the spontaneous activity.

Table 1.  The effects of methanol extract, fractions from Bacopa monnieri in FST. Values are in mean ± SEM of 10 mice per group.

Treatment	Dose (mg/kg)	Immobility period (s)	Reduction (%)
Blank control	—	124.4 ± 8.5
Fluoxetine	10	24.9 ± 9.7^*	80.0
Methanol extract	50	62.5 ± 33.6^*	49.7
	100	40.8 ± 5.6^*	67.2
	200	35.7 ± 15.6^*	71.3
CHCl3 Fr.	100	99.5 ± 14.1	20.0
EtOAc Fr.	100	45.9 ± 5.2^*	63.1
n-BuOH Fr.	100	40.2 ± 3.8^*	67.7

^*P < 0.01 when compared with blank control group.

Table 2.  The effects of methanol extract, fractions from Bacopa monnieri in TST. Values are in mean ± SEM of 10 mice per group.

Treatment	Dose (mg/kg)	Immobility period (s)	Reduction (%)
Blank control	—	102.3 ± 9.3
Fluoxetine	10	28.3 ± 7.5^*	72.3
Methanol extract	100	54.6 ± 11.8^*	46.6
CHCl3 Fr.	100	90.2 ± 12.9	11.8
EtOAc Fr.	100	54.2 ± 14.8^*	47.0
n-BuOH Fr.	100	56.2 ± 10.8^*	45.1

^*P < 0.01 when compared with vehicle-treated (control) group.

Table 3.  The effects of methanol extract, fractions from Bacopa monnieri in LA. Data represent the means ± SEM of 10 mice per group.

Treatment	Dose (mg/kg)	Counting point	Comparison with the control group (P)
Blank control	—	650.1 ± 31.1
Fluoxetine	10	671.3 ± 70.2	> 0.05
Methanol extract	100	682.6 ± 53.5	> 0.05
CHCl3 Fr.	100	648.4 ± 61.2	> 0.05
EtOAc Fr.	100	657.7 ± 59.2	> 0.05
n-BuOH Fr.	100	669.7 ± 41.9	> 0.05

Forced swimming test and tail suspension test are two of the most commonly used animal models of depression for antidepressant screening. In the forced swimming test, the development of immobility when mice are placed into an inescapable cylinder of water reflects the cessation of persistent escape-directed behavior (Lucki, 1997). The tail suspension test is based on the fact that animals subjected to the short-term, inescapable stress of being suspended by their tail, will develop an immobile posture. Various antidepressants are able to reverse the immobility and promote the occurrence of escape-related behavior. Both models of depression are widely used to screen new antidepressants (Porsolt et al., 1977, 1978, 1979; Steru et al., 1985). These tests are quite sensitive to major antidepressant drugs including tricyclics, serotonin-specific reuptake inhibitors, MAO inhibitors, and atypical antidepressant (Porsolt et al., 1977; Steru et al., 1985; Detke et al., 1995).

In the Ayurvedic system of medicine of India, B. monnieri has been widely used to treat the diseases in brain for several centuries (Mukherjee et al., 1996), and modern pharmacological studies also confirmed several neuropsychopharmacological activities of this plant (Vohora et al., 2000). In the present study, the methanol extract, EtOAc fraction and n-BuOH fraction of B. monniera (100 mg/kg) produced a significant decrease in immobility times both in FST and TST, without modifying significantly the spontaneous motor activity. Sometimes, a reduction of immobility time in FST and TST may be caused by drug having a stimulation effect on the central nervous system. Our study showed that all fractions from B. monniera had no influence on LA of mice, which suggests the reduction in the duration of immobility was not due to stimulation effects on the central nervous system. From the above evidence, it was suggested that B. monniera may be a novel therapeutic approach to reduce or inhibit depression. Further work appears warranted to determine the mechanism of the antidepressant activity of the tested fractions at a cellular level.

Acknowledgements

The research work was supported by the Scientific Foundation of Shanghai (06DZ19717), and in part by the program for Changjiang Scholars and Inno vative Research Team in University (PCSIRT), NCET Foundation, NSFC (30725045), National 863 Program (2006AA02Z338), China Postdoctoral Science Foundation (20070410711), “973” program of China (2007CB507400), Shanghai Leading Academic Disci pline Project (B906), and the Scientific Foundation of Shanghai China (07DZ19728, 06DZ19005).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.