Abstract: Background and Objective: Tinospora cordifolia (Willd.) Hook.f. is employed in Ayurvedic system of medicine for the treatment of diabetes, jaundice, rheumatoid arthritis, metabolic disorders and likewise utilized as memory enhancer. In this study it tend to analyze in vivo sedative and anxiety potential in mice for T. cordifolia stems extracted in methanol. Materials and Methods: The crude extract of T. cordifolia was evaluated for its central nervous system depressant effect using rodent behavioral models; such as ‘Hole cross test’ and ‘Open field test’ for its sedative properties and an ‘Elevated plus maze test’ for its anxiolytic potential. Results: In sedative study, the extract at a dose of 200 and 400 mg kg–1 b.wt. showed a dose-dependent and statistically significant (p<0.05-0.01) suppression of motor activity and exploratory activity of the mice in both open field and hole cross test. The extract also showed increased percentage of entry into open arms at both doses in anxiolytic potential study. At a dose of 400 mg kg–1 b.wt. maximum anxiolytic activity (p<0.001) was found which was comparable to the standard Diazepam. Conclusion: This study unconcealed that the methanolic extract of Tinospora cordifolia has significant central nervous system depressant impact. More studies on active constituent of the extract will give approaches for therapeutic intervention.
INTRODUCTION
According to World Health report by WHO approximately 450 million people are suffering from mental or behavioral disorders. This amount is about 12.3% of the global burden of disease and will rise1,2 to 15% by 2020. Anxiety and depression are the most prevailing mental disorders and recognized to be symptomatically, psychologically and biologically heterogeneous3. Over 20% of the adult population suffers from these illnesses at some time during their lives4. Formerly, psychoactive synthetic drugs were recognized as most effective in the management of central nervous system (CNS) related disorders. Continuous and arbitrary use of these drugs has cause for various side effects affecting the allergic, endocrine, hematopoietic, autonomic and neurological systems of human body5. Thus, psychopharmacology has become an important area of research during last few decades6. Medicinal plants are the prominent source of secondary metabolites as well as bioactive compounds. It plays a pivotal role for the discovery of new drug molecules. Medicinal plants contain different essential bioactive compounds like alkaloids, phenols, flavonoids, terpenoids, tannins, saponins, steroids, polysaccharides and so on which are the important part of modern and traditional medicines7-9. Tinospora cordifolia (Willd.) Hook. f. (Fig. 1) commonly known as heart-leaved in English and Gulancha, Gurach, Gadancha in Bangla is a large, glabrous, deciduous climbing shrub belonging to the family Menispermaceae10. It is distributed throughout Bangladesh particularly in Chittagong Hill tract region different parts of tropical Indian subcontinent, Sir Lanka and China. The stems of T. cordifolia are succulent with long filiform fleshy aerial roots from the branches. The bark is creamy white to gray, leaves are membranous and cordate in shape, flowers are small and yellow or greenish yellow, seeds are curved and fruits are fleshy and single seeded. Flowers grow during the summer and fruits during the winter10. It is widely used in folk medicine and Ayurveda system of medicine as a tonic, vitalize and as a remedy for diabetes and metabolic disorders11. The T. cordifolia is reported for antidiabetic, immunomodulatory, cardiprotective, hypolipidemic, antineoplastic and antioxidant properties12-17. Butanol extract T. cordifolia (B-TCE) exhibited neuroprotective potential18 and 50% ethanol extract showed in ameliorating anxiety-like behavior induced in acute SD rats and resulting cognitive deficit and motor coordination19. Terpenoids, alkaloids, lignans, steroid compounds have been reported by photochemical investigation of this plant20-23. So far there has been no scientific report in literature about the sedative and anxiety activity (in vivo) of methanolic extract of T. cordifolia.
| Fig. 1: | Tinospora cordifolia |
Therefore, in this study, methanolic plant extract of T. cordifolia was evaluated to find its in vivo sedative and anxiety potential on some neuro pharmacological experimental model.
MATERIALS AND METHODS
Plant material: The plant Tinospora cordifolia was collected from Forests of Khagrachari, Chittagong Hill Tracts, Bangladesh in June, 2016 and authenticated by Dr. Shaikh Bokhtear Uddin, Associate Professor, Department of Botany, University of Chittagong, Chittagong 4331, Bangladesh. A voucher specimen (ACCN. 15327) has been deposited at the herbarium in the Department of Botany, University of Chittagong, Bangladesh.
Extraction method: Clean and small pieces of stems of Tinospora cardifolia were dried under subdue sun light with flow of air. After grinding powdered material (500 g) was macerated in methanol at room temperature for a period of 3 days with occasional shaking and stirring and filtered with clean cloth followed by filter paper. The solvent was removed by rotary vacuum evaporator (Lab Tech EV311) and the process was repeated for two times. The dried solid was preserved in a refrigerator at 2-8°C until further use.
Experimental animals: Animals Swiss albino mice of either sex, weighing between 18-28 g were maintained under standard environmental conditions [(24.0±1.0)°C, relative humidity 55-70% and 12 h light/dark cycle].
| Fig. 2: | Open field test |
| Fig. 3: | Hole cross test |
The animals were provided with standard laboratory food and water ad libitum. Prior to experimentation, the animals were acclimatized to laboratory condition for one week. All experiments were conducted under isolated and sound attenuated room. The research was carried out from May-November, 2018 at the Animal House and Pharmacology Lab, Department of Pharmacy, University of Science and Technology Chittagong (USTC), Chittagong 4202, Bangladesh and the research was approved by the Institutional Ethical Committee (Approval Number: USTMEBBC 18/05/70).
Sedative activity
Open field test: The method by Gupta et al.24 was adopted with minor modification. Briefly, half square meter of the floor was divided into a series of squares alternatively colored black and white with a wall of 40 cm high for an open field test (Fig. 2). The test groups received methanolic extract of T. cordifolia at a dose of 200 and 400 mg kg–1 b.wt. per oral and positive control received standard drug Diazepam at a dose of 1 mg kg–1 b.wt. per object. The number of squares traveled by animals was counted for 3 min at different time intervals (0, 30, 60, 90 and 120 min).
Hole cross test: The test carried out by the method described by Takagi et al.25. The apparatus was a cage of wood with dimension 30×20×14 cm. A steel partition fixed in the middle of the length, which divided the cage into two chambers and a hole was in the steel plate of 3.5 cm diameter at a height of 7.5 cm from the ground of the cage (Fig. 3). The test groups received T. cordifolia methanolic extract at a dose of 200 and 400 mg kg–1 b.wt. p.o. whereas, positive control received standard drug Diazepam at a dose of 1 mg kg–1 b.wt. p.o. The number of passages by the animals through the hole from one chamber to the other was counted for 3 min at 0, 30, 60, 90 and 120 min.
Anxiolytic activity
Elevated plus maze test: The test was employed according to the method by Lister RG with minor modifications26. The apparatus consists of two open arms (5×10 cm) and two close arms (5×10×15 cm) radiating from a platform (5×5 cm) to form a plus sign figure situated 40 cm above the floor. The height of open arms edges were 0.5 cm to keep the mice from falling and the closed-arms edges were 15 cm in height (Fig. 4). The test groups received T. cordifolia methanolic extract at a dose of 200 and 400 mg kg–1 b.wt. p.o. the control group received vehicle (1% Tween 80 in water) at a dose of 10 mL kg–1 p.o. and positive control received standard drug Diazepam at a dose of 1 mg kg–1 b.wt. p.o. 60 min after administration of test samples, each animal was individually placed in the center of the Elevated plus maze (EPM) and were allowed 5 min for free exploration. Next, the number of open and close arms entries and time spent on open and close arms were manually registered. The whole test was carried out in a sound attenuated room27. Entry into an arm was defined as the point when the mice placed all four paws onto the arm:
| Fig. 4: | Elevated plus maze test |
Statistical analysis: The data were expressed as mean±standard deviation (SD). Statistical comparisons were performed using One way ANOVA followed by Dunnett’s multiple comparison tests. The obtained values were compared with the vehicle control group and were considered statistically significant when p<0.05.
RESULTS
Open field test: The open field test for T. cordifolia treated groups (200 and 400 mg kg–1 b.wt.,) showed significant dose-dependent reduction of movement. The number of squares traveled by the mice and it was decreased significantly from its initial value at 0-120 min at a dose level of 400 mg kg–1 b.wt. (p<0.01) of the methanolic extract of T. cordifolia (Fig. 5).
Hole cross test: The number of hole cross from one chamber to another by mice of different groups and the control group was shown similar from 30-120 min. Hole cross test of T. cordifolia treated groups produced a significant (p<0.05) decrease of locomotion from its initial value during the period of the experiment at a dose of 400 mg kg–1 b.wt. which was comparable to the reference Diazepam (p<0.001) (Fig. 6).
| Fig. 5: | Effect of methanolic extract of T. cordifolia on open field test in mice |
Values are Mean±SD, (n = 5), *p<0.05, **p<0.01, ***p<0.001, Dunnett’s test as compared to control (vehicle = 10 mL kg–1) |
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| Fig. 6: | Effect of methanolic extract of T. cordifolia on hole cross test in mice |
Values are Mean±SD, (n = 5), *p<0.05, **p<0.01, ***p<0.001, Dunnett’s test as compared to control (vehicle = 10 mL kg–1) |
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Elevated plus maze test: The EPM test of T. cordifolia treated groups (200 and 400 mg kg–1 b.wt.) showed significant dose-dependent increment of percentage of entries and percentage of time spent in open arms. At a dose of 400 mg kg–1 b.wt. maximum anxiety activity was found as the maximum percentage of entries on open arms was displayed (p<0.001) which was comparable to the standard Diazepam (Fig. 7).
DISCUSSION
The present study exhibited that the infliction of methanolic extract of T. cordifolia stems at a dose of 200 and 400 mg kg–1 b.wt., showed potent sedative and anxiolytic properties.
| Fig. 7: | Effect of methanolic extract of T. cordifolia on EPM test in mice |
Values are Mean±SD, (n = 5), *p<0.05, **p<0.01, ***p<0.001, Dunnett’s test as compared to control (vehicle = 10 mL kg–1) |
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In comparison with control, both doses of methanolic extract decreased the movements of mice in locomotor studies that were measured by open field and hole cross tests. Spontaneous decrease of locomotor activity is a sign of sedative effect of this plant extract28, because the level of excitability of CNS is measured by locomotor activity29. Locomotion in mice has significantly decreased for both doses of T. cordiofolia methanolic extract. The locomotor lowering activity effect was observed at third observation (60 min) and it was continued up to the 5th observation period (120 min). The results were dose dependent and also statistically significant. However, anxiolytic effects of drugs in rodents were evaluated by the EPM test that has been recognized as a valuable model of anxiolytic effect30. The anxiolytic effect was observed when the experimental drug increases on open arm entries31. At both doses of T. cordifolia methanolic extract exhibited significant increase in the percentage of entries into open arms. But at the dose of 400 mg kg–1 b.wt. showed maximum increase in percentage of open arm entries which was comparable with Diazepam (Fig. 7). These results indicated an anxiolytic activity of the methanolic extract of Tinospora cordifolia stems.
GABAA-benzodiazepine receptors are the most abundant inhibitory receptor system32 in the CNS and binding of a benzodiazepine agonist to its recognizing site results in increased chloride ion flux33 which in turn hyperpolarizes the postsynaptic membrane at a level below that at which spike generation is possible and for this reason some GABAA agonists are frequently used for their sedative effects. The compounds identified from the T. cordifolia contain tinosporide, furanolactone diterpene, tinosporine, berberine, choline, giloinsterol and giloin23-26, act as GABAA agonists and this agonistic property could be attributed to the CNS depressant effect of T. cordifolia although there is no consensus about which substances are exactly responsible for these effects. However, further studies are necessary to evaluate the contribution of other substances that are isolated for the activity observed, because it still remains to be determined which components exactly were responsible for these effects.
CONCLUSION
The results from the experiments confirmed that the methanolic extract from T. cordifolia possesses a powerful sedative and anxiolytic potential that can be beneficial for the treatment of anxiety and related medicine disorders. However, more studies would be necessary to evaluate the contribution of compounds for the activity showed because it still remains to be determined that parts were precisely answerable for these effects. This study will help the researcher to uncover the critical areas of medicinal property of this plant that are related to anxiety and depression.
ACKNOWLEDGMENT
Authors are thankful to Department of Pharmacy, University of Science and Technology Chittagong (USTC) for provide facilities to carry out the research and Department of Chemistry, Chittagong University of Engineering and Technology (CUET) for financial support (grand number: CUET/DRE/2016-17/Chem007).