Anticonvulsant and free radical scavenging activity of Hybanthus enneaspermus: A preliminary screening

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1 Indian Journal of Traditional Knowledge Vol. 2(4), October 2003, pp Anticonvulsant and free radical scavenging activity of Hybanthus enneaspermus: A preliminary screening S Hemalatha 1 *, A K Wahi 1, P N Singh 1 and J P N Chansourii 1 Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi 2 Centre for Experimental Medicine and Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi. Received 10 May 2002; revised 23 May 2003 Anticonvulsant activity of aqueous (AQHE) and ethanolic (ETHE) extract of Hybanthus enneaspermus was studied using maximal electric shock (MES) and strychnine induced convulsions models. AQHE at the doses of 200 mg/kg and 400 mg/kg orally showed significant protection in both models. The activity was equipotent to phenobarbitone sodium (30 mg/kg; i.p.). The AQHE showed no neurotoxicity. Ethanolic extract did not show protection in these models. The AQHE exhibited free radical scavenging activity in an in vitro system using DPPH. Keywords: Hybanthus enneaspermus, MES, Strychnine, Anticonvulsant activity, Free radical scavenging activity, DPPH. Epilepsy is one of the most common neurological disorders and affects approximately 0.5-1% of population the world over 1 Number of plants have been reported to possess anticonvulsant activity, viz. Panax ginseni, Zizyphus xylopyra 3, Delphinium denudatum 4, Valeriana officinalis 5 and Withania somnifera 6. However, a search for the ideal anticonvulsant natural drug still continues. Hybanthus enneaspermus F. Muell. (Violaceae), a small perennial herb, found in the warmer parts of India and throughout the Deccan Peninsula 7 has been claimed to be effective in urinary calculi, pain and epileptic fits 8. * Correspondent author The plant has been reported to contain amino acids, sugars and flavanoids 9, alkaloid aurantioamide-oac, ~-sitosterol and iso arbornol 10 However the anticonvulsant activity of this plant has not yet been investigated systematically. In the present study the anticonvulsant activity of aqueous and ethanolic extract of H. enneaspermus has been evaluated using maximal electrical shock (MES) and strychnine induced convulsion models. Material and Methods Preparation of extracts The dried whole plant of H. enneaspermus purchased from market (Herbal vendors) in Chennai was

2 384 INDIAN J TRADITfONAL KNOWLEDGE, VOL 2, No.4, OCTOBER 2003 identified by the chief botanist T AMPCOL Anna Hospital Chennai. A voucher specimen (SH/HE/01) was kept in our department. Aqueous extract (AQHE) was prepared by boiling the powdered drug with distilled water repeated I y. The extract was concentrated and dried in vacuum desicator. Ethanolic extract (ETHE) prepared by continuous hot percolation process was concentrated under reduced pressure and dried. Distilled water and polyethylene glycol (PEG) were used as vehicle for AQHE and ETHE, respectively for experimental work. Animals Male albino rats ( g) were purchased from Zoological Animal Emporium, Varanasi. The rats were housed in polyacrylic cages under standard laboratory conditions with 12h± I h dark and light cycle. They were allowed free access to standard laboratory diet and tap water ad libitum. Drugs Strychnine hydrochloride (2 mg/kg; i.p.) was dissolved in distilled water. Phenobarbitone sodium (30 mg/kg, i.p.) was used as reference drug. Extracts were administered orally. The doses of extracts were selected by the response of MES induced convulsion. Maximal electric shock (MES) induced convulsion 1 1 The rats were placed in Perspex monitoring box for observation. MES was induced by using electro convulsion meter (TECHNO, India). The current of 150 rna for 0.2 seconds duration was delivered through ear clip electrodes by attaching them to the pinnae. Electrodes were dipped in normal saline before application. The rats were then observed for the presence or absence of hind limb tonic extension. Abolishment of hind limb extension was taken as protection. All the animals were screened for normal convulsions by MES. The experimental animals were divided into eight groups of 5 each. The rats of group I served as control and received vehicle. The animals belonging to group IT, III and rv were administered 200 mg/kg, 400 mg/kg and 600 mg/kg of AQHE p.o., respectively. The rats of group V, VI and VII were treated with different doses i.e. 200 mg/ kg, 400 mg/kg and 600 mg/kg; p.o. of ETHE, respectively. Group Vlll rats (reference group) received phenobarbitone sodium (30mg/kg; i.p.). The different extracts and reference drug were administered I hour before the electric shock (MES). The animals were examined I hr, 2 hr, and 4 hrs after drug administration. S I.. d d. 12 tryc uune tn uce se1zures The animals were divided into 2 groups of 5 animals each. The rats of group I were administered strychnine in the dose of 2 mg/kg; i.p. The group 11 animals were given AQHE at a dose of 400 mg/kg; p.o. one hour before strychnine (2 mg/kg; i.p.) administration. The animals were observed for the onset of convulsion and time of prolongation of life. The observed data were analysed by Student's t test.

3 HEMALATHA eta/: ANTICONVULSANT ACTIVITY OF HYBANTHUS ENNEASPERMUS 385 Neurotoxicity Minimal motor impairment was measured in rats by roto rod test 13. The rats were trained to stay on an accelerating roto rod that rotated at 10 revolutions per minute. The rod diameter was 3.2 cm 1 Trained animals were given AQHE (400 mg/kg; p.o.). Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least 1 min in each of three trials. Behavioural toxicity studies The AQHE in the doses of 200 mg/kg, 400 mg/kg and 600 mg/kg was given orally to animals and observed for any behavioral toxicity at one hour interval for 24 hours. Free radical scavenging activity- DPPH method To determine the free radical scavenging activity, a method 14 based on the reduction of a methanolic solution of the colored free radical DPPH was used. The decrease in absorption of DPPH at its absorption maximum of 517nm is proportional to the concentration of free radical scavenger added to the DPPH reagent solution. This activity was expressed as the effective concentration at 50% (EC 50 ) i.e. concentration of the test solution required to give a 50% reduction in absorbance of the test solution as compared to that of a blank solution. To a set of test tubes, containing 3 ml of methanol, 50 J..ll of DPPH reagent (2mg/ml) was added. The initial absorbance was measured at 517nm. To these tubes, methanolic solution of AQHE (2mg/ml, dried aqueous extract di ssolved in methanol) was added in the range of J..ll. Similarly in a different set of experiment in the place of drug ascorbic acid (0.5 mg/ml) was added in the range of J..ll(reference). The absorbance was measured after 30 minutes. The percentage reduction in absorbance was calculated from initial and final absorbance of each solution. The EC 50 value for the test material was calculated from the calibration curve of concentration of extract (J..ll) vs percentage reduction in absorbance. The results were subjected to linear regression analysis. Results and Discussion AQHE at the doses of 200 mg/kg and 400 mg/kg; p.o. showed 50% and 80% protection, respectively in MES screening. The AQHE in the doses of 600 mg/kg and ETHE (200 mg/kg; 400mg/kg and 600 mg/kg; po) did not show protection in MES induced convulsion. The anticonvulsant activity was compared with reference drug phenobarbitone sodium (30 mg/kg; i.p.) (Table 1). AQHE (400 mg/kg; p.o.) was also evaluated for anticonvulsant activity in strychnine-induced convulsions. The treated group of animals showed a significant increase in the survival time as compared to untreated rats (p<o.ool). There was a significant (p<o.ol) delay in the onset of convulsions in the treated group of rats. AQHE treatment also decreased the percentage of mortality (Table 2). No neurotoxicity was observed in animals treated with the effective

4 386 INDIAN J TRADITIONAL KNOWLEDGE, VOL 2, No. 4, OCTOBER 2003 Table 1 - Effect of AQHE and ETHE on MES induced convulsion No. of animals protected/no. of animals tested Group 1 hr 2 hr 4 hr %protection Control AQHE (200mg) AQHE (400mg) AQHE (600mg) ETHE (200mg) ETHE ( 400mg) ETHE (600mg) Phenobarbitone (30mg/kg;i.p.) 2/ /5 4/ % 2/5 50% 4/5 80% 0% 0% 0% 0% % Table 2---Effect of AQHE on strychnine induced convulsion in rats Group Onset of Convulsion :.:=.:.::..:..:._-,--- Time of death time in minutes % of mortality Control 4±0.4 AQHE400mg 10.6 ± 1.1 " 7.6 ± ± 1.2b 70 p<0.01, b<0.001 significantly differs from control. Each value of mean ± S.E. n = 5 Table 3- Free radical scavenging activity of AQHE by DPPH method Sample Scavenging activity EC 5 o r* I. AQHE 2 mg/ml j..ll 2. Ascorbic acid 0.5 mg/ml 25.5 j..ll 1.38 j..lg j..lg *Correlation coefficient. anticonvulsant dose (400 mg/kg; p.o.). The aqueous extract in different doses did not show any behavioural toxicity. In the present study the AQHE also exhibited free radical scavenging activity in an in vitro system (Table 3). Free radicals have been implicated in the development of seizures under pathological conditions The protective efficacy of antioxidant treatments against some types of seizures has also been reported Gupta et az2 have demonstrated the potential of antioxidants in epilepsy. Similarly anticonvulsant effect of H. enneaspermus against MES and strychnine-induced convulsions may possibly be attributed to its free radical scavenging property. However, other mechanisms cannot be ruled out. The AQHE antagonised the

5 HEMALATHA et al: ANTICONVULSANT ACTIVITY OF HYBANTHUS ENNEASPERMUS 387 strychnine-induced convulsions probably by enhancing GABAnergic activity and blocking multi neural pathways in the spinal cord 21 Similar results have also been observed in the case of Vitex negundo 22 and Clerodendron colebrookianum 23 This is the first report regarding the anticonvulsant activity and free radical scavenging activity of H. enneaspermus, which justifies the folk claim of its use in epileptic fits. Further work is in progress to isolate phytoconstituent of this drug, which may be responsible for the observed activity. References I Sander J W A S & Shrovan S D, Epidemiology of the epilepsies, J Neural Neurosurgery Psychiatry, 61 ( 1996) Gupta Y K, Sharma Monisha & Chaudhary Geeta, Antiepileptic activity of Panax ginseng against pentylenetetrazole induced kindling in rats, Indian J Physiol Pharmacal, 45(4) (2001) Rao Y B, Devi S, Singh J P & Pandey V B, Antinociceptive, anticonvulsant and antiinflammatory activities of Zizyphus xylopyra, Indian J Pharmacal, 19(1) (1987) Khan A B, Anti-convulsant activity of Jadwar Delphinium denudatum Wall., J Res Ayurveda Siddha, 3(3&4) ( 1982) Santos M S, Ferreira F, Faro C, Pires E, Carvalho A P, Cunha A P & Macedo T, The amount of GABA present in aqueous extracts of Valerian is sufficient to account for 3 [H] GABA release in synaptosomes, Planta Medica, 60(5) (1994) Kulkani S K, Sharma Alok, Verrna Anita & Ticku M K. GABA receptor mediated anticonvulsant action of Withania somnifera root extract, Indian Drugs, 30(7) (1993) Anonymous, The Wealth of India (Raw Materials), Vol. V, (Council of Scientific & Industrial Research, New Delhi), 1959, Kirtikar K R & Basu B D, Indian Medicinal Plants, Vol. I, reprint, (Bisen Singh Mahendra Pal Singh, Dehradun), 1984, Ravindra Retnam K & John DeBritto A, Preliminary phytochemical screening of three medicinal plants of Tirunelveli hills, J Econ Taxon Botany, 22(3) ( 1998) Majumader P L, Basu A & Mal D, Chemical constituents of Hybanthus enneaspermus F. Muell. (Violaceae), Indian J Chemistry, 178(3) (1979) 297. II Krall R L, Penry J K, White B G, Kupterberg H S & Swinyard E A, Antiepileptic drug development: II Anti convulsant drug screening, Epilepsia, 19 (1978) Turner R A, Screening Methods in Pharmacology, (Academic Press, New York & London), 1965, Meza-Toledo S E, Zenteno-Garcia M T, Juarez-Carvajal E, Martinez-Munoz D & Carvajal-Sandoval G, A new homologous series of anticonvulsant phenyl alcohol amides, synthesis and pharmacological evaluation, Arzeneim-Forsch./Drug Res, 40 (1990) Kato K, Terao S, Shimamoto N & Hirata M, Studies on scavengers of active oxygen species: I. Synthesis and biological activity of 2-0-alkyl ascorbic acids, J Med Chern, 31 ( 1988) Bruce A J & Baudry M, Oxygen free radical in rat limbic structures after kainate-induced seizures, Free Radical Bioi Med, 18 ( 1995) Singh R & Pathak D N, Lipid peroxidation and glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase and glucose-6-phosphate dehydrogenase activities in FeC1 3 -induced epileptogenic fits in the rat brain, Epilepsia, 31 ( 1990) Kabuto H, Yokoi I & Ogawa N, Melatonin inhibits iron-induced epileptic discharges in rats by suppressing peroxidation, Epilepsia, 39 (1998) Ogunmekan A 0 & Hwang P A, A randomized, double blind, placebo controlled, clinical trial to D-alpha tocopherol acetate (Vitamin E), as add on therapy for epilepsy in children, Epilepsia, 30 ( 1989) 84.

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