Introduction
Bacopa monnieri is a small herb, belonging to Scrophulariaceae. In the traditional system of medicine the plant was noted as multifarious activities like laxative, carminative, digestive, anti-inflammatory, anti-convulsant, bronchodilator, febrifuge, and nervine tonic.1, 2, 3, 4, 5, 6 The plant shows the presence of various phytoconstituents namely, alkaloids, saponins, phenolic compound, flavonoids and mainly contains triterpenoid saponins called bacosides. Hersaponin, one of its active principle, is reported to have a sedative effect.7, 8, 9 The plant also shows cardioprotective, hepatoprotective, an aphrodisiac, effective in treating scabies and syphilis, purifies the blood, and having proven useful for diarrheas and paresis. 10, 11
The plant tissue culture techniques are more commonly used for the investigations of the secondary metabolites. Already 2000 plants have been reported to be regenerated through the plant tissue culture. It has also been shown that many of such plants can produce secondary metabolites in culture. Tissue culture (often called micropropagation) is a special type of asexual propagation where a very small part of tissue (shoot apex, leaf section, or even an individual cell) is excised (cut-out) and placed in aseptic culture in a test tube, petri-dish or tissue culture container containing a special culture medium. Micropropagation protocols are worked out for many plant species cultured in vitro to provide macro- and micro- mineral nutrients, vitamins, source of carbohydrates, appropriate environmental conditions (light intensity, photoperiod and temperature) and plant growth regulators required to obtain high regeneration rates. As such they are expected to facilitate commercially feasible micropropagation. Well-defined cell culture methods have also been developed to produce pharmacologically important secondary metabolites. Therefore, presently plant tissue culture has been incorporated as a significant tool under plant biotechnology.12, 13, 14, 15, 16
This research work was carried out to conduct antidepressant activity of natural and micropropagated plants of Bacopa monnieri.
Materials and Methods
Collection and authentication of plant
Bacopa monnieri was collected from the field of Jawaharlal Nehru Ayurvedic Medicinal Plant Garden and Herbarium Kothrud, Pune in the month of February 1998 and authenticate the plant from same institute. After collection plant material was washed thoroughly with water and kept for drying in the sunlight for 4-5 days. After drying, the plant material was broken into fine pieces and then passed through crusher mill, to obtain coarse powder. The powder was passed though sieve no.12.
Micropropagated tissue culture plant
Shoot tips and nodal segments of B. monnieri L. were cultured on Murashige and Skoogs (M. S.) basal medium supplemented with different concentration of BAP and IAA. 12
Table 1
Composition and preparation of Murashige and Skoog medium
Animals
This whole research work was done at Poona college of Pharmacy, Pune in the year 1998 which was already having approval of the animal experimentation by the official committee of Government of India, CPCSEA and all the projects used to approval of Institutional animal ethics committee. However, the certificate of those days is not traceable at present. All the experimental animals were procured from the laboratory of Institute. Animals were accommodated in clean polypropylene cages in groups with pellets for diet and distilled water in the laboratory maintained at standard conditions in 12:12 h light/dark cycle at 22–25°C temperature and 45–55% relative humidity. They were grouped into 6 mice in each group. Acclimatization of the animals to laboratory conditions before every procedure is done. Procedures were done from 9 a.m to 4 p.m daily. Institutional Committee for Ethical use of animals reviewed the study protocol and the study procedures was approved. Animals’ care and handling were followed as per guiding principles of the Committee for the Purpose of Control and Supervision of Experiments on Animals, India.
Chemicals and pharmaceuticals
Chlorpromazine, Tween 80. Chlorpromazine was procured with courtesy of Prof. Dr. S. L. Bodhankar, Department of Pharmacology, Poona college of Pharmacy, Pune in 1998.
Preparation of drug solution
Ethanolic extract of B. monnieri natural (BMN) plant, Ethanolic extract of B. monnieri micropropagated (BMM) plant and Ethanolic extract of B. Monnieri Standard (BMS) were prepared by dissolving required amount of BMN ethanolic extract in distilled water. A drop of tween 80 was used to prepare uniform suspension.
Chlorpromazine (2 mg/kg, ip): It was prepared by dissolving 0.2 mg of chlorpromazine in 1 ml of distilled water.
Pharmacological screening
Spontaneous motor activity
The spontaneous motor activity was measured by means of actophotometer. This device is used for the assessment of mild central depressant activity. It is based on measuring animals’ voluntary activity in an activity cage in terms of count.17 In the experiment thirty mice were divided in 5 groups of 6 mice in each group. The control group received a vehicle (tween 80, 0.5% v/v, ip) and other groups received BMN ethanolic extract, BMM ethanolic extract and BMS ethanolic extract, 100 mg/kg, ip of each. The readings were recorded at an interval of half an hour to a period of 2 hours. Chlorpromazine (2 mg/kg, ip) was used as a standard drug for comparison. The results were analyzed by applying student ‘t’ test. [Transfer latency TL] and the time for which the mice remained in the enclosed arm was noted down in seconds. To the one group BMN ethanolic extract, while other BMM ethanolic extract and third BMS ethanolic extract were administered in the dose of 100 mg/kg, ip of each. All the extracts were administered 30 min prior to the test. Fourth group received vehicle (tween 80, 0.5% v/v, ip). The results obtained were analyzed by student 't' test.
Statistical analysis
All the parameters recorded from the above models were tabulated and expressed as mean ± standard error of mean. For all inferential statistical tests, a P < 0.05 was considered to be statistically significant and P < 0.001 was considered to be extremely statistically significant. GraphPad InStat software was used for the analysis of data.
Results
Spontaneous motor activity
The results obtained in the present evaluation of all the extracts of B. Monnieri indicated inhibition of spontaneous motor activity in mice when tested in actophotometer (Table 2). The inhibition of spontaneous motor activity in case of BMN, BMS extract was found 291, 93.5 and 277, 93.66 count at interval of 30 and 60 min. respectively. But in case of BMM plant extract it was found 290, 87.33 and 146 count at 30, 60 and 120 min. interval respectively.
Table 2
Effect of ethanolic extract of B. Monnieri natural (BMN), micropropagated (BMM) and standard (BMS) on spontaneous motor activity
Discussion
Acute toxicity was determined in mice by employing various logarithmic doses and administered via intraperitoneal and oral routes. Each group contained six mice. Mice were injected with the ethanolic extract of BMN, BMM and BMS solution (1 g/kg, 2 g/kg, 3 g/kg and 5 g/kg by oral route and 25 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg, 400 mg/kg, 800 mg/kg, 1600 mg/kg, and 3200 mg/kg by intraperitoneal route) and kept in the plastic cages. When the drug was given by oral route at various doses effects were seen after 1 hour. Only slight depression was observed by oral route. When the drug was given by intraperitoneal route onset was faster that was within 30 minutes and effects were prominent at higher doses. Only slight depression at lower doses. The changes in the behavior were recorded at the interval of 30 minutes for 4 hours and also after 24 hours.
Strength and Limitations of the Study
The locomotor activity of the mice was recorded using an actophotometer. This effect mediated through stipulations or inhibition of neurotransmitters or their receptors of particular region or the whole brain needs to be determined. Such study with natural plant of B. monnieri have been confirmed by many researchers but the similar study on the micropropagated plants needs to be evaluated.
Conclusion
This study has demonstrated the antidepressant-like activity of natural and micropropagated plant extracts in animal behavior depression models. This extract may not be harmful as this does not cause any stimulation or increase in locomotor activity. However, future experimental studies are required to conclude whether these both plant extracts will produce a comparable beneficial effect in individuals with depression and also to interpret the cellular and molecular mechanism of action in depth.
