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Yaro et al., Nig. Journ. Pharm. Sci., October, 2007, Vol. 6 No. 2, P. 22 ­ 27

Nigerian Journal of Pharmaceutical Sciences Vol. 6, No. 2, October, 2007, ISSN: 0189-823X All Rights Reserved

ANTICONVULSANT ACTIVITIES OF METHANOL EXTRACT OF CHRYSANTHELLUM INDICUM LINN. VATKE IN MICE AND CHICKS

1*

Yaro, A.H., 2Anuka, J.A., 3Salawu, O.A., 2Magaji, M.G

2

Department of Pharmacology, Faculty of Medicine, Bayero University, Kano, Nigeria Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria, Nigeria 3 National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria ([email protected], 234-806-155-6597)

1

*Correspondence Author:

ABSTRACT The anticonvulsant properties of Chrysanthellum indicum Linn. Vatke (Compositae) were studied on maximal electroshock test (MEST), pentylenetetrazole and strychnine-induced seizures model in chicks and mice. The intraperitoneal and oral acute toxicity values (LD50) in mice were also evaluated. The extract of Chrysanthellum indicum protected animals against maximal electroshock-induced convulsion by 80% and significantly (P<0.01) reduced the mean recovery time from convulsion. The extract had a weak anticonvulsant activity against pentylenetetrazole-induced convulsion and did not protect mice against strychnine-induced convulsions. The intraperitoneal and oral LD50 values were estimated to be 288.5 and 2154.0 mg/kg in mice respectively. These results suggest that the methanolic extract of Chrysanthellum indicum contains pharmacologically active substance(s) that may be valuable in the treatment of convulsive disorders, especially Grand mal epilepsy. Key words: Chrysanthellum indicum, epilepsy, strychnine, pentylenetetrazole, maximal electroshock test (MEST)

INTRODUCTION Epilepsy is one of the most common neurological disorders with an incidence of 3% in the general population (Annegers, 2001). Currently available Anti-epileptic drugs (AEDs) do not provide cure nor prevent relapse and they are often associated with serious side effects, including teratogenicity, chronic toxicity and adverse effects on cognition and behaviour (Samren et al., 1997). Consequently, many people living in developing countries still rely on herbal medicine for management of epilepsy. The number of plant-based crude drugs finding regular use in the management of epilepsy is put around 400 and the number is increasing. The clinical effectiveness, minimal side effect profile and relatively low costs of herbal drugs are the reason for their various applications in traditional medicine (Valiathan, 1998).

However, only limited efforts have been made to evaluate the potentials of such plants for their use in modern medicine or to scientifically justify their traditional use in the treatment of CNS disorders including epilepsy. Chrysanthellum indicum Linn. Vatke (Compositae) is a faintly aromatic herb that is widely distributed in the tropics. The plant is commonly known in Hausa as rariyar kasa (Kontagora), dunkufe (Zaria) and Goshin ba'ana and in southern Nigeria as Oyigi or Abilere in Yoruba (Dalziel, 1955). In Chinese traditional medicine (Sanskrit), the plant is used for antifebrile, detoxification and hypotensive purposes (Yu et al., 1992. Combination of its flowers and other herbs is used to treat childhood convulsions (Ludivina, 1996). In West Africa, the plant is used in

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treating maturing boils, fevers, jaundice and Gonorrhoea (Brasseur et al., 1987). In Northern Nigeria and Ghana, it is used in the treatment of hepatitis (in combination with Tamarindus indicas) and heart problems (Burkill, 1985). Locally, in Likoro village of Kaduna state of northern Nigeria, the plant is also claimed to be useful in scorpion sting and convulsion (Adamu Mohammed, personal communication). To our knowledge, there is no scientific report on the anticonvulsant activity of C. indicum. CI This study was therefore designed to evaluate the anticonvulsant activities of CI in order to scientifically justify its use in traditional medicine to treat epilepsy. MATERIALS AND METHODS Collection of plant materials The whole plant Chrysanthellum indicum was collected from Likoro Village, in Kudan Local Government Area, Kaduna State, Nigeria, in September 2003. The plant was identified and authenticated by Malam Musa Shehu and Umar Gallah of the Herbarium Section Department of Biological Sciences, A.B.U., Zaria. A voucher specimen (NO. 3110) was deposited at the Herbarium for future reference. Preparation of extract The plant material was cleaned, air dried for 7 days and then crushed into coarse powder with a pestle and mortar. 100 g of the powered plant was macerated with methanol for 48 hours with occasional shaking. The solvent was evaporated to give an average yield of 17.7%.w/w. Animals Swiss albino mice (18-25 g) of either sex obtained from the Animal House, Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria were used. Ranger cockerels were obtained from National Animal Production Research Institute (NAPRI), Shika. The animals were

maintained in a well ventilated room, fed on Excel feeds (Feed Masters, Ilorin) and water ad libitum. Phytochemical Test The methanolic extract of Chrysanthellum indicum was screened for the presence of alkaloids, glycosides, tannins, saponins and flavonoids according to standard procedure (Trease and Evans, 1983). Acute Toxicity Studies in Mice LD50 determination was conducted using the method of Lorke (1983). In the initial phase, 3 groups of three animals each were treated with the methanolic extract of the plant at doses of 10, 100 and 1000 mg/kg body weight i.p. and observed for 24 hours. In the second phase, 4 groups of one animal each were injected with the methanolic extract at doses of 140, 225, 370, and 600 mg/kg i.p. The LD50 value was determined by calculating the geometric mean of the lowest dose that caused death and the highest dose for which the animal survived (0/1and 1/1). The same procedure was repeated using the oral route of drug administration. Maximum electroshock-induced convulsion in chicks The modified method of Swinyard and Kufferberg (1985); and Browning, (1992) was employed. 50 two-day old ranger cockerels were randomly divided into five groups of ten chicks per group. The first group received normal saline (10ml/kg) i.p.; second, third and fourth groups were treated with 12.5, 25.0 and 50.0 mg extract per kg, i.p. and the last group was administered 20 mg phenytoin per kg, i.p. (positive control). Thirty minutes later, maximal electroshock was administered to induce seizure in the chicks using Ugobasile electroconvulsive machine (Model 7801) with corneal electrodes placed on the upper eyelids of the chicks. The current, shock duration, frequency and pulse width were set and maintained at 80 mA, 0.80 s, 100 pulse per

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second and 0.6 ms, respectively. Abolition of Hind limb tonic extension (HLTE) was considered as protection from electroshock (Porter et al., 1984; Swinyard, 1972).

Pentylenetetrazole-induced convulsion in mice

The method of Swinyard et al., (1989) was employed. Thirty mice were divided into five groups of six mice each. The first group received 10 mL normal saline per kg body weight i.p., the second group was given 200 mg valproic acid per kg body weight i.p., while the third, fourth and fifth groups received 12.5, 25.0 and 50.0 mg extract per kg body weight i.p. Thirty minutes later, mice in all the groups received 85 mg pentylenetetrazole per kg s.c. Mice were observed over a period of 30 minutes. Absence of an episode of clonic spasm of at least 5 seconds duration indicated a compound's ability to abolish the effect of pentylenetetrazole on seizure threshold. Subcutaneous Strychnine-induced convulsion in mice The method of Porter et al., (1984) was employed. Thirty mice were divided into five groups of six mice each. The first group received 10 mL normal saline per kg body weight i.p., the second group was given 200 mg valproic acid per kg body weight i.p., while the third, fourth and fifth groups received 12.5, 25.0 and 50.0 mg extract per kg body weight i.p. Thirty minutes later, mice in all the groups received 1.0 mg strychnine per kg, s.c. Abolition of tonic extensor jerks of the hind limbs was considered an indicator that the testing material could prevent strychnineinduced convulsions.

Statistical Analysis Results were expressed as Mean+Standard Error of Mean; Student's t-test was used to determine level of significance of all results obtained. Results were regarded as significant at P< 0.05. RESULTS The phytochemical screening of the methanolic extract of C. indicum revealed the presence of flavonoids, tannins, glycosides, alkaloids and steroids. The intraperitoneal and oral acute toxicity values (LD50) of the methanolic extract in mice were found to be 288.5 and 2154.0 mg/kg body weight, respectively. The extract (12.5, 25.0 and 50.0 mg/kg) protected the chicks against maximal electroshock-induced convulsions with highest percentage of protection observed at doses of 25 and 50mg/kg (Table 1). The extract also significantly reduced the mean recovery time of convulsed animals. Phenytoin (20 mg/kg), the standard anticonvulsant used produced 100% inhibition of hind limb tonic extension (HLTE) of maximal electroshock test (MEST). The extract significantly (P<0.01) delayed the onset of seizure induced by PTZ without exhibiting protection against threshold seizure (Table 2). Valproic acid (200 mg/kg), the standard anticonvulsant used protected the animals against threshold seizures. The extract had no activity in Sc-Strychnineinduced seizure test (Table 3). The extract exhibited no effect on onset of seizure or recovery time after convulsion. Valproic acid (200 mg/kg), the standard anticonvulsant drug used protected all the animals against ScStrychnine-induced convulsion.

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Table 1:

Effect of Methanolic Extract of C .indicum and Phenytoin on Maximal Electroshock-induced Seizures in Chicks.

Mean Onset of Seizures (sec.) 2.9 ± 1.1 3.4 ± 0.7 3.6 ± 0.8 3.7 ± 0.8 0.0 Mean Recovery Time (min.) 21.0 ± 2.7 7.8 ± 1.6c 5.6 ± 1.2 c 5.2 ± 1.1 c 0.0 Quantal Protection 1/10 6/10 8/10 8/10 10/10 % Protection 10.0 60.0 80.0 80.0 100.0 Mortality 0.0 0.0 0.0 0.0 0.0

Treatment (mg/kg) N/ Saline C.I.(12.5) C.I.(25.0) C.I.(50.0) Phenytoin (20.0)

c

P<0.01 (compared with control using Student's t- test); n = 10

Table 2:

Effect of Methanolic Extract and Valproic acid on Sc-pentylenetetrazoleinduced Convulsions in Mice

Mean Onset of Seizures (min) 3.8 ± 0.3 6.8 ± 0.7 b 7.6 ± 0.9b 7.4 ± 0.7c 0.0 Mean Duration of Seizures (min) 5.1 ± 1.6 4.2 ± 1.5 4.6 ± 1.7 3.8 ± 1.1 0.0 Quantal Protection 0/6 0/6 0/6 0/6 6/6 % Protection % Mortality

Treatment (mg/kg) N/ Saline C.I.(12.5) C.I.(25.0) C.I.(50.0) VA (200.0)

b,

0.0 0.0 0.0 0.0 100

0.0 0.0 0.0 0.0 0.0

and c are significantly different from control at P<0.01 and P< 0.001 respectively, Student's t- test. n=6 Table 3: Effects of the Extract and Valproic acid on Sc-Strychnine-induced Convulsions in Mice.

Mean Onset of Seizures (min) 12.2 ± 0.5 13.2 ± 0.7 11.4 ± 0.5 12.6 ± 0.3 0.0 Quantal Protection 0/6 0/6 0/6 0/6 6/6 % Protection 0.0 0.0 0.0 0.0 100 Mortality 3 4 4 5 -

Treatment (mg/kg) N/ Saline C.I.(12.5) C.I.(25.0) C.I.(50.0) VA (200.0)

Data Presented as Mean ±SEM,

n = 6 for all groups

DISCUSSION Generally, the data presented in this study suggest that C.I may contain psychoactive substance(s) with potential anticonvulsant properties. Protection against HLTE in the MEST by the extract predicts anticonvulsant activity of extract. The spread of the epileptic

seizure from an epileptic focus during seizure activity has been reported previously (DeLorenzo et al., 2001). The seizures generated in this model are consistent with the human grand mal epilepsy (Swinyard et al., 1989). AEDs that are effective in the treatment of generalized tonic clonic and

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partial seizures such as, phenytoin, carbamazepine, oxcarbazepine and lamotrigine suppress HLTE in MEST (Browning, 1992; Rho and Sankar, 1999). Protection against HLTE also indicates the ability of the extract to inhibit or prevent seizure discharge within the brainstem seizure substrate (Browning, 1992), suggesting that the extract of C. indicum may be useful for the treatment of generalized tonic-clonic and partial seizures. The extract possesses a weak anticonvulsant activity against pentyelenetetrazole-induced seizures in mice. PTZ is a known convulsant and anticonvulsant activity in scPTZ test identifies compounds that can raise the seizure threshold in the brain (White et al., 1998). AEDs effective in the therapy of generalized seizures of (absence or myoclonic) petit mal type such as pentobarbitone, valproic acid, ethosuximide and suppress various seizure patterns induced by PTZ (Löscher et al., 1991). AEDs such as ethosuximide and valproic acid suppresse T-type calcium currents in thalamic neurons (Rho and Sankar, 1999; Meldrum, 1996). Strychnine is a competitive antagonist of the inhibitory amino acid Glycine (Larson, 1969). The absence of anticonvulsant activity in the scSTN test suggests that the extract of C. indicum may not interact with glycine receptors. It may therefore be concluded, based on the data presented, that the use of Chrysanthellum indicum in traditional medicine for the treatment of epilepsy in Nigeria and other West African countries is justifiable scientifically. The plant extract may be valuable in convulsive disorders especially Grand mal epilepsy. Further research is going on in our laboratory to isolate the bioactive components responsible for the observed pharmacological activities.

REFERENCES

Annegers, J. F. (2001). The Epidimiology of Epilepsy. In Wyllre, Ed. The Treatment of Epilepsy: Principle and Practice. 3rd ed. Philadelphia: Lippincott Williams and Watking, Pp 131 ­ 138. Brasseur, T., Angenot, L., Pincemail, J. and Derby, C. (1987). Action antiradical aire de flavonoides et d' extract de Chrysanthellum indicum. Planta Medica. Phytother. 21:131-137. Browning, R. (1992); The electroshock model, neuronal network and antiepileptic drugs. In: Drugs for Control of Epilepsy: Actions on Neuronal Networks in Seizure Disorders, (Faingold, C.L. and Fromm, G.H. Eds.) CRC Press. Boca Raton, FL. pp. 195-211. Burkill, H.M. (1985). The Useful Plants of West Africa. Vol.1 2nd ed. Royal Botanical Gardens, Kew England. pp. 457. Dalziel, J. M. (1955). The useful plants of West Tropical Africa. Crown Agent for Overseas Government and Administration, London. pp417 DeLorenzo, R.J., Raza, M., Saheen, F., Choudhary, M.I., Suria, A., Attaur-Rahman and Sombati, S. (2001). Anticonvulsant Activities of the FS-1 Subfraction isolated from Roots of Delphinium denadatum. Phytotherapy Research. 15:426-430 Larson, M.D. (1969). An analysis of the action of strychnine on the recurrent IPSP and amino acid induced inhibitions in the cat spinal cord. Brain Research. 15:185-200. Lorke, D. (1983). A new approach to practical acute toxicity testing. Archives of Toxicology. 54: 275-287. Löscher, W., Hönack, D., Fassbender, C.P. and Nolting, B. (1991).The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. III. Pentylenetetrazole seizure models. Epilepsy Research. 8:171-189 Ludivina, S.P. (1996). Medicinal Plants, Herbal Medicine. (Technoguide Vol.1). Central Visayas Technology Packaging Project, Philippines. pp 115-120 Meldrum, B.S. (1996). Update on the mechanism of action of antiepileptic drugs. Epilepsia, 37:(S6), S4 ­ S11.

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Porter, R.J., Cereghino, J.J. and Gladding, G.D. (1984). Antiepileptic drug development program. Cleve. Clin. 51:293-305. Rho, J.M. and Sankar, R. (1999). The Pharmacologic basis of antiepileptic drug action. Epilepsia. 40: 14711483. Samren, E.B., Duijn, C.M., Koch, S. et al. (1997). Maternal use of antiepileptic drugs and the risk of major congenital malformations: a joint European prospective study of human teratogenesis associated with maternal epilepsy. Epilepsia 38: 981-990. Swinyard, E.A. (1972). Electrically induced convulsions. In: Experimental Models of Epilepsy. A manual for the Laboratory Worker, (Purpura, D.P., J.K., Tower, D.B., Woodbury, D.M. and Walter, R.D. Eds.). Raven Press: New York. pp. 433 ­ 458. Swinyard, E.A. and Kupferberg, H.J. (1985). Antiepileptic drugs: detection, quantification and evaluation. Federation Proceedings. 44:39-43.

Swinyard, E.A., Woodhead, J.H., White, H.S. and Franklin, M.R. (1989). General Principles: Experimental selection, quantification, and evaluation of anticonvulsants. In: Antiepileptic Drugs, 3rd eds. (Levy, R.H., Mattson, B., Melrum, J.K. and Dreifuss, F.E. Eds.) Raven Press. New York. pp. 85-103. Valiathan , M. S. (1998). Healing Plants. Current Science. 75 (10, 11): 1122- 1126. White, H.S., Wolf, H.H., Woodhead, J.H. and Kupferberg, H.J. (1998). The National Institute of Health anticonvulsant drug development program: Screening for efficacy. In: Antiepileptic Drug Development: Advances in Neurology, Vol. 76, (French, J., Leppik, I.E. and Dichter, M.A. Eds.). Lippincott-Raven Publishers: Philadelphia; 29-39. Yu, D. Q., Xie, F. Z., He., W. Y. and Liang, X. (1992). Application of 2D NMR techniques in the structure determination of Chrysantheriol. Acta pharmaceutica sinica, 27(3):191-196.

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