|Year : 2011 | Volume
| Issue : 2 | Page : 58-61
Hydroethanolic leaf extract of Ficus religiosa lacks anticonvulsant activity in acute electro and chemo convulsion mice models
Damanpreet Singh1, Bikram Singh2, Rajesh Kumar Goel1
1 Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab, India
2 Natural Plant Products Division, Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India
|Date of Web Publication||25-Nov-2011|
Rajesh Kumar Goel
Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala - 147 002, Punjab
Source of Support: University Grant Commission, New Delhi, India,
for providing financial assistance [Vide F.No.: 34- 130/2008 (SR)] for
the project and project fellowship to Mr. Damanpreet Singh, Conflict of Interest: None
| Abstract|| |
Introduction: Ficus religiosa L. (Moraceae) has been of great medicinal value in traditional medicine and implicated in a wide variety of human and animal disorders. Its leaves have been used for the ethnomedical treatment of epilepsy. But its traditional antiepileptic use is not fully understood experimentally. Hence the present study was undertaken to explore the anticonvulsant effect of the leaves in experimental animal models of convulsion. Materials and Methods: The anticonvulsant effect of hydroethanolic leaf extract of F. religiosa was studied at 100, 250 and 500 mg/kg; intraperitoneally (i.p.) in maximal electroshock (MES), and at 100, 250, 500 and 600 mg/kg; i.p. doses in pentylenetetrazol (PTZ) test in mice. The duration of tonic hind limb extension(s) and latency to clonic convulsions (min) was noted in MES and PTZ tests, respectively. Phenytoin (25 mg/kg; i.p.) and diazepam (5 mg/kg; i.p.) served as reference standards in MES and PTZ tests, respectively. Percentage mortality was also noted. Results: There was no significant change observed after the extract treatment on the duration of tonic hind limb extension in MES test, and latency to clonic convulsions in PTZ test, as compared to their respective controls. Moreover, percentage mortality remained unaltered after the extract treatment. Conclusions: From the results of present study it is concluded that the hydroethanolic leaf extract of F. religiosa lacks anticonvulsant activity in MES- and PTZ-induced convulsion tests. Further studies are required from other regions and using different animal models to support these findings.
Keywords: Convulsions, epilepsy, Ficus religiosa L., maximal electroshock, pentylenetetrazol, traditional medicine
|How to cite this article:|
Singh D, Singh B, Goel RK. Hydroethanolic leaf extract of Ficus religiosa lacks anticonvulsant activity in acute electro and chemo convulsion mice models. J Pharm Negative Results 2011;2:58-61
|How to cite this URL:|
Singh D, Singh B, Goel RK. Hydroethanolic leaf extract of Ficus religiosa lacks anticonvulsant activity in acute electro and chemo convulsion mice models. J Pharm Negative Results [serial online] 2011 [cited 2019 Nov 12];2:58-61. Available from: http://www.pnrjournal.com/text.asp?2011/2/2/58/90212
| Introduction|| |
Plants have been a principal source of traditional medicine for more than 5000 years. Generally, therapeutic use of medicinal plant is justified by its long history of usage since antiquity. But, without experimental evidences, merely long traditional use does not assure that particular plant has therapeutic value.  Moreover, knowledge about the use of plants is obtained from ancient texts, which are thousands year old. Hence it is expected that with time and changes in climatic conditions, phytochemical composition of medicinal plants might have changed, leading to altered therapeutic effects.  Hence there is an unmet need of therapeutic validation of traditional herbal medicines using modern experimental techniques to determine their effectiveness, and acceptance globally.
In this regard, while searching some herbal remedies, we found literature reports pertaining to ethnomedical use of the leaves of Ficus religiosa L. (Moraceae) in treatment of epilepsy.  The leaves of F. religiosa have been of great therapeutic value in traditional medicine. Along with epilepsy, the leaves have been used for treatment of diarrhea, vomiting, helminths, inflammatory ulcers, asthma, earache, toothache, wounds, fever, gastric problems, scabies, gout, hematuria, constipation, migraine and skin diseases.  Phytochemical research carried out on the leaves had led to isolation of phytosterols (campesterol, stigmasterol, sitosterol and 28-isofucosterol), triterpene alcohols (α-amyrin, β-amyrin and lupeol), long-chain hydrocarbons (n -nonacosane and n-hentriacontane), aliphatic alcohols (n-hexacosanol and n-octacosanol), amino acids, fibers, tannins and minerals. ,,,,,
The anticonvulsant effect of other parts of F. religiosa has been thoroughly studied. In our previous study the fruit extract of F. religiosa showed anticonvulsant activity, which was found to be due to modulation of the brain serotonergic functions.  Similarly, the adventitious root extract of F. religiosa has shown anticonvulsant activity in experimental animal models.  But in spite of traditional use of the leaves, a limited experimental attempts have been made to explore and validate its use in epilepsy. Hence the present study was envisaged to investigate the anticonvulsant effect of the hydroethanolic leaf extract of F. religiosa using electro [(maximal electroshock (MES)] and chemo [(pentylenetetrazol (PTZ)] convulsion mice models.
| Materials and Methods|| |
Plant material, preparation of extract and dosing
The leaves of F. religiosa tree were collected from U.E. Phase-I, Patiala. The plant material was identified and authenticated by Prof. R.C. Gupta and specimens were deposited at the Herbarium, Department of Botany, Punjabi University, Patiala, Punjab, India. The leaves were cleaned, shade-dried, grounded to a moderately coarse powder. The leaf powder was subjected to repetitive extraction by 50% ethanol, using a percolator at room temperature, until exhausted. The filtered and combined percolate was dried under reduced pressure, using a rotavapor (Perfit, Ambala, India). The extract was then stored in a vacuum desiccator in refrigerator, until used.
Based on literature and results of some pilot studies carried out on limited number of animals, three doses were selected for MES test (100, 250 and 500 mg/kg) and four doses for PTZ test (100, 250, 500 and 600 mg/kg). Dosing was carried out by dissolving the extract in 10% DMSO (dimethyl sulfoxide) v/v in distilled water, freshly before use and was injected intraperitoneally (i.p.). The vehicle control groups were injected with equal volume of vehicle (10% DMSO) i.p. In all cases the injection volume was kept 10 mL/kg.
Preliminary phytochemical screening
The leaf extract was subjected to preliminary phytochemical tests to determine the presence of carbohydrates, glycosides, alkaloids, saponins, steroids, triterpenoids, tannins, flavonoids, proteins and amino acids. 
Male Swiss Albino mice, weighing 20-30 g obtained from CCS Haryana Agricultural University, Hisar were used in the present study. The animals were divided into different groups (n = 5) and were maintained at room temperature with natural day and night cycles. The animals were allowed free access to food (standard laboratory rodent's chow) and water during the study period. The experiments were conducted between 9:00 and 16:00 h. The experimental protocol was approved by the Institutional Animal Ethical Committee. All procedures were conducted according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, India.
Drugs and chemicals
PTZ (dissolved in normal saline) was obtained from Sigma Chemical Company (USA), DMSO from Spectrochem (Mumbai, India). Reference drugs diazepam and phenytoin were obtained locally from Jackson Laboratories Ltd (Amritsar, India) and Cadila Laboratories (Ahmedabad, India), respectively.
Maximal electroshock test
Mice were divided into different groups and treated with vehicle (control), varying doses of the extract (100, 200, and 500 mg/kg) and phenytoin (25 mg/kg). After 30 min of these treatments, convulsions in mice were induced by a calibrated [through a current calibrator (Rolex, Ambala, India)] transauricular current stimulus (56 mA, 50 Hz for 0.2 s), via a pair of crocodile ear clips, using a convulsiometer (Rolex, Ambala, India). The duration (s) of tonic hind limb extension (hind limbs of animals outstretched at 180° to the plane of body axis) was noted. Percentage of animals showing tonic hind limb extension was also observed. 
PTZ (75 mg/kg) was injected into 6 groups of mice pretreated 30 min prior with the varying doses of extract (100, 200, 500, and 600 mg/kg), diazepam (5 mg/kg), and vehicle (control). The latency to clonic convulsions was noted in all the groups. Percentage mortality was also observed. 
All the results were expressed as mean ± standard error (SEM) and were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. Statistical analysis for the percentage of mortality and animals showing tonic hind limb extension was performed using Chi-square test. The results were regarded as significant at P < 0.05.
| Results|| |
The extraction of leaf powder yielded 20.8% w/w of crude hydroethanolic extract. Preliminary phytochemical screening indicated the presence of carbohydrates, glycosides, sterols, flavonoids, tannins and amino acids in the extract.
In MES test, there was no protective effect observed after the extract treatment at any dose. The extract treated groups showed insignificant effect on the duration of tonic hind limb extension as compared to vehicle control [Table 1]. There was no significant effect observed on the percentage of animals showing tonic hind limb extension. Phenytoin treated group showed significant (P < 0.05) decrease in the duration of tonic hind limb extension as compared to vehicle control.
|Table 1: Effect of the leaf extract on maximal electroshock-induced convulsions |
Click here to view
Treatment with the extract showed slight dose-dependent increase in the latency to PTZ-induced clonic convulsions up to 500 mg/kg, however, the change observed in comparison to vehicle control was not significant. Further increment in the dose of extract up to 600 mg/kg again failed to prevent the PTZ-induced convulsion. There was no significant effect observed after the extract treatment on percentage mortality in comparison to vehicle control, when determined using Chi-square test. However no convulsions were induced in diazepam treated group [Table 2].
|Table 2: Effect of the leaf extract on pentylenetetrazol-induced convulsions |
Click here to view
| Discussion|| |
In spite of all the marvelous advancements in modern medicine, traditional medicines have always been practiced, because of their natural origin, easy availability, cost effectiveness, lesser side effects, better tolerability, etc. But, because of limited documented experimental evidences regarding their pharmacological effects, the use of traditional medicines remain restricted to a locality/region where they are being practiced traditionally, and not accepted globally. Current phytopharmacological research is focused on validating the therapeutic effects of traditional medicine in experimental studies. In the present study we selected F. religiosa, as it is a common and popular tree, and its leaves have been reported to be used in the ethnomedical treatment of epilepsy.  In the study, the leaf extract was found to be ineffective, as it failed to suppress experimentally induced electro and chemo convulsions in mice.
Several animal models are available that could potentially be used to screen for anticonvulsant activity, but in the present study MES- and PTZ-induced convulsion models were used, because both of these tests served as "gold standards" in early stages of drug testing.  Moreover, in previous anticonvulsant studies carried out on the other parts of F. religiosa, it was found to be effective in one of these two tests. , In MES test, the leaf extract showed no protection against convulsions at any dose. In PTZ test, the extract treatment showed slightly dose-dependent increase in the duration of seizure latency (but insignificant) up to 500 mg/kg. Hence it was thought that further increase in dose might show protection against PTZ-induced convulsions. But when tested no protective effect was observed at 600 mg/kg dose.
From the results of present study it can be concluded that the hydroethanolic leaf extract of F. religiosa lacks anticonvulsant activity in acute MES and PTZ mice models, indicating its ineffectiveness against generalized tonic-clonic convulsions. However, there are several clinically used drugs, such as levertiracetam, seleteracetam, etc., which are ineffective in acute MES and PTZ tests, but active in other models, such as, 6-Hz model, models of absence seizure, and various kindling models. , Hence further studies are required from other regions, using other models of convulsion to support or negate the traditional antiepileptic use of the leaves of F. religiosa.
| Acknowledgment|| |
We are thankful to the University Grant Commission, New Delhi, India, for providing financial assistance [Vide F.No.: 34-130/2008 (SR)] for the project and project fellowship to Mr. Damanpreet Singh. We are also grateful to Prof. R.C. Gupta of Department of Botany, Punjabi University, Patiala, Punjab for the authentication of plant material.
| References|| |
|1.||Mosihuzzaman M, Choudhary M. Protocols on safety, efficacy, standardization, and documentation of herbal medicine. Pure Appl Chem 2008;80:2195-230. |
|2.||Lewis WH, Elvin-Lewis MP. Medicinal plants as sources of new therapeutics. Ann Mo Bot Gard. 1995;82:16-24. |
|3.||Vyawahare NS, Khandelwal AR, Batra VR, Nikam AP. Herbal anticonvulsants. J Herb Med Toxicol 2007;1:9-14. |
|4.||Singh D, Singh B, Goel RK. Traditional uses, phytochemistry and pharmacology of Ficus religiosa: A review. J Ethnopharmacol 2011;134:565-83. |
|5.||Singh M. Effect of pipal leaf tannin and tannic acid on volatile fatty acids production in goat's rumen. Indian Vet J 1977;54:553-7. |
|6.||Desai HB, Desai MC, Patel BM., Patel BH, Shukla PC. Proximate and trace elements content of the forest tree leaves of Dangs district collected during summer season. Gujarat Agric Univ Res J 1980;6:34. |
|7.||Behari M, Rani KU, Matsumoto T, Shimizu N. Isolation of active-principles from the leaves of Ficus religiosa. Curr Agric 1984;8:73-6. |
|8.||Verma RS, Bhatia KS. Chromatographic study of amino acids of the leaf protein concentrates of Ficus religiosa Linn. and Mimusops elengi Linn. Indian J Hosp Pharm 1986;23:231-2. |
|9.||Williamson EM, Hooper PM. Major herbs of Ayurveda. London: Churchill Livingstone; 2002. p. 145-9. |
|10.||Niranjan PS, Srivastava V, Udeybir, Verma DN. Nutritional evaluation of peepal leaves in barbari kids. Indian J Anim Nutr 2007;24:128-9. |
|11.||Singh D, Goel RK. Anticonvulsant effect of Ficus religiosa: Role of serotonergic pathways. J Ethnopharmacol 2009;123:330-4. |
|12.||Patil MS, Patil CR, Patil SW, Jadhav RB. Anticonvulsant activity of aqueous root extract of Ficus religiosa. J Ethnopharmacol 2011;133:92-6. |
|13.||Trease G, Evans SM. Pharmacognosy. 15 th ed. London: Bailer Tindal; 2002. |
|14.||White HS, Johnson M, Wolf HH, Kupferberg HJ. The early identification of anticonvulsant activity: Role of the maximal electroshock and subcutaneous pentylenetetrazole seizure models. Ital J Neurol Sci 1995;16:73-7. |
|15.||Kaur M, Goel RK. Anti-convulsant activity of Boerhaavia diffusa: Plausible role of calcium channel antagonism. Evid Based Complement Alternat Med 2011 [In Press]. |
|16.||Shaikh BT, Hatcher J. Complementary and alternative medicine in Pakistan: Prospects and limitations. Evid Based Complement Alternat Med 2005;2:139-42. |
|17.||Rogawski MA. Diverse mechanisms of antiepileptic drugs in the development pipeline. Epilepsy Res 2006;69:273-94. |
|18.||Klitgaard H, Matagne A, Gobert J, Wülfert E. Evidence for a unique profile of levetiracetam in rodent models of seizures and epilepsy. Eur J Pharmacol 1998;353:191-206. |
[Table 1], [Table 2]