Fitoterapia 76 (2005) 412 – 418 www.elsevier.com/locate/fitote CNS depressant activity of Lecaniodiscus cupanioides O.K. Yemitan, O.O. Adeyemi* Department of Pharmacology, College of Medicine, University of Lagos, Idi-Araba, P.M.B 12003, Lagos, Nigeria Received 4 December 2002; accepted in revised form 22 February 2005 Available online 13 June 2005 Abstract The aqueous root extract of Lecaniodiscus cupanioides was used to study the central nervous system depressant activity pattern of the plant. The extract protected mice from strychnine-induced convulsion at 400 mg/kg p.o. and 100 mg/kg i.p. A dose-dependent prolongation of seizure latency was produced at 400 mg/kg, p.o. and 100 mg/kg i.p. for strychnine-induced seizure; and at 400 mg/ kg p.o. and 100 mg/kg i.p. for picrotoxin-induced seizure. Moreover, the CNS depressant activity of the extract (200 mg/kg p.o. and 50 mg/kg i.p.) was demonstrated by a significant prolongation of 40 mg/kg, pentobarbitone sleeping time, and significant reduction in exploratory behavior of mice at a dose of 400 mg/kg p.o., with both effects comparable to effects produced by 4 mg/kg chlorpromazine. Acute oral toxicity test, up to 14 days, did not produce any visible signs of toxicity; however, acute (24 h) i.p toxicity test produced a dose-dependent mortality with LD50 of 455.2 mg/kg. D 2005 Published by Elsevier B.V. Keywords: Lecaniodiscus cupanioides; Anticonvulsant; CNS depressant activity 1. Introduction Lecaniodiscus cupanioides Planch. Ex Bth. (Sapindaceae) is a shrub widely distributed throughout deciduous and non-deciduous rain forests [1]. Parts of the plant have various * Corresponding author. Tel.: +234 1 5453760 74; fax: +234 1 5851432. E-mail address: cmul@rcl.nig.com (O.O. Adeyemi). 0367-326X/$ - see front matter D 2005 Published by Elsevier B.V. doi:10.1016/j.fitote.2005.02.010 O.K. Yemitan, O.O. Adeyemi / Fitoterapia 76 (2005) 412–418 413 applications in folk medicine for the treatment of boils, burns, wounds, oral hygiene, fever and abdominal swelling caused by liver abscess [1]. Moreover, the root decoction of the plant is claimed among the Yoruba people of Nigeria to control epilepsy and to enhance penile erection. In this study, we aimed at establishing any anticonvulsant effect of the aqueous root extract against pentylenetetrazole (PTZ), picrotoxin, and strychnine-induced convulsions. The direct central depressant activity and the acute toxicity were also investigated. 2. Experimental 2.1. Plant material L. cupanioides fresh root, collected in the wild forest of Ibadan, Nigeria, was identified by Mr. T.K. Odewo, Senior Superintendent of the Forestry Research Institute of Nigeria (FRIN) Ibadan, where a voucher specimen (FHI 106116) is preserved and by Prof. J.D. Olowokudejo of the Botany Department, University of Lagos. 2.2. Extract preparation Root, washed and macerated in water, was dried at r.t. and ground into fine powder. One hundred grams of powder was boiled in distilled water and left for 24 h at r.t. The sediment was filtered and the aqueous extract oven-dried at 40 8C giving a dried extract (yield: 5.2%). Dried extract was dissolved in distilled water before administration, in each test. Phytochemical screening revealed the presence of saponins, tannins as reported by Gill [1], flavonoids, phenols and reducing sugars. 2.3. Animals Swiss mice of either sex weighing 18–20 g, obtained from the Laboratory Animal Centre of the College of Medicine, University of Lagos, Lagos, Nigeria, were fed on standard diet and given water ad libitum. The mice were housed in polypropylene cages in a standard environmental conditions. 2.4. Strychnine-induced seizures Mice of either sex were randomly allocated to the different control and test groups (10 per group). They were treated with 50 and 100 mg/kg, i.p. or 200 and 400 mg/kg, p.o. of L. cupanioides root aqueous extract (LCRAE), respectively, 15 and 30 min before intramuscular injection of 4 mg of strychnine. Control group received only saline, while the reference group received 2 mg/kg i.m. of diazepam 15 min before strychnine. Mice that did not show clonic or tonic convulsion within 30 min of strychnine administration were considered protected. 414 O.K. Yemitan, O.O. Adeyemi / Fitoterapia 76 (2005) 412–418 2.5. Pentylenetetrazole(PTZ)-induced seizures Mice (10 per group) were treated with LCRAE at dose of 50 and 100 mg/kg i.p. or 200–800 mg/kg p.o., respectively, 15 and 30 min before PTZ. Phenobarbitone 10 mg/kg i.p. were used as reference. Seizure were induced by intraperitoneal injection of 80 mg/kg of pentylenetetrazole given 15 or 30 min after intraperitoneal or oral administration of the extract. Control mice were administered with normal saline 30 min before PTZ. Seizure stage and seizure latency were the two parameters used to evaluate antiepileptic activity of the drugs. Seizure stages were rated according to the following criteria [2,3]: Stage 0 No effect. Stage 1 Facial movements; hyperactivity; sniffing; forelimb cloni which is not successive and does not last (may or may not). Stage 2 Twitches of the face or body muscles; successive forelimb cloni. Stage 3 Forelimb and/or hindlimb cloni with rearing. Stage 4 Generalized tonic–clonic convulsion with rearing and falling down. Stage 5 Generalized convulsion with rearing, falling down, jumping and periods of tonus. The mice that had no seizures within 20 min after PTZ administration were considered protected. Seizure latency was defined as the time taken from the injection of PTZ to the first two myoclonic jerks of the forelimbs. This has been confirmed to be the first sign of the beginning of a seizure activity [2]. 2.6. Picrotoxin-induced seizures In this test, picrotoxin (5 mg/kg, i.p.) was used to induce seizure. Animals were treated with LCRAE at dose of 50 and 100 mg/kg i.p. or 200–800 mg/kg p.o., respectively, 15 and 30 min before picrotoxin. Phenobarbitone (10 mg/kg) was used as a standard drug. Seizure stage and seizure latency were the two parameters used to evaluate anticonvulsant activity of the drugs. All seizure stages were rated according to the following criteria: Stage 0 No effect. Stage 1 Initial reduced activity followed by hyperactivity and sniffing. Stage 2 Successive forelimb cloni with sharp noise. Stage 3 Generalized body tremor without rearing and falling down. Stage 4 Rearing, falling and periods of tonus. 2.7. Pentobarbitone sleeping time The method described by Dandiya and Collumbine [4] was used. Mice in different groups received 200 and 400 mg/kg, p.o. or 50 and 100 mg/kg, i.p. of LCRAE, while an equal volume (5 ml/kg) of saline served as the control. Chlorpromazine (1 mg/kg, i.p.) was used as standard drug. After 30 min mice received an intraperitoneal injection of pentobarbitone (40 mg/kg). The time between the loss and recovery of the righting reflex was taken as the sleeping time. 415 O.K. Yemitan, O.O. Adeyemi / Fitoterapia 76 (2005) 412–418 Table 1 Effect of L. cupanioides root aqueous extract (LCRAE) on strychnine-induced seizure in mice Seizurea latency (min), mean F S.E.M. Treatment Saline Diazepam 2 mg/kg i.m. LCRAE 50 mg/kg i.p. LCRAE 100 mg/kg i.p. LCRAE 200 mg/kg p.o. LCRAE 400 mg/kg p.o. 3.0 F 0.5 – 14.2 F 1.1* – 16.8 F 1.3* – Mice with convulsion Mice dead 10/10 0/10 10/10 10/10 0/10 0/10 10/10 0/10 6/10 0/10 8/10 0/10 a Seizure were induced by injection of 4 mg/kg i.m. of strychnine. * P b 0.05 significant as compared with control, Student’s t-test. 2.8. Exploratory activity This study was carried out by the hole board method using a white painted wooden board (40 cm  40 cm) with four equidistant holes (1 cm diameter  2 cm depth). The mouse was placed at one corner of the board and the animal moved about and dipped its head into the holes indicating exploratory behavior. The number of dips in 7.5 min was recorded [5]. The test was carried out 30 min after oral treatment with LCRAE at doses ranging from 100 to 800 mg/kg. Chlorpromazine (4 mg/kg, i.m.) was used as standard drug. 2.9. Acute toxicity study Fasted overnight mice (10 per group) were treated with LCRAE at doses up 20 g/kg orally, or at doses up to 800 mg/kg, intraperitoneally. The control mice were given normal saline. Mice were closely observed for toxic symptoms and behavioral changes for the first 2 h of administration and mortality recorded up to 14 days. LD50 was calculated using the method by Miller and Tainter [6]. Table 2 Effect of LCRAE on pentylenetetrazole-induced seizure in mice Treatment Seizurea latency (min), mean F S.E.M. Seizure stage (%) 0 1 2 3 Saline Phenobarbitone: 10 mg/kg i.p. LCRAE: 100 mg/kg i.p. 200 mg/kg i.p. 200 mg/kg (p.o.) 400 mg/kg (p.o.) 800 mg/kg (p.o.) 2.48 F 0.21 – – – – – 80 20 – – – – – 5.27 F 0.29* 5.36 F 0.17* 3.52 F 0.28 4.29 F 0.19* 5.52 F 0.28* – – – – – – – – – – – – – – – 20 40 20 40 60 80 60 80 60 40 – – – – – 40 60 0 20 60 a Seizure were induced by injection of 80 mg/kg i.p. of pentylenetetrazole. * P b 0.05 significant as compared with control, Student’s t-test. 4 5 100 Survival after convulsion (%) 0 416 O.K. Yemitan, O.O. Adeyemi / Fitoterapia 76 (2005) 412–418 Table 3 Effect of LCRAE on picrotoxin-induced seizure in mice Treatment Saline Phenobarbitone: 10 mg/kg i.p. LCRAE: 200 mg/kg p.o. 400 mg/kg p.o. 800 mg/kg p.o. 100 mg/kg i.p. 200 mg/kg i.p. Seizurea latency (min), mean F S.E.M. Mean seizure stage reached (%) Survival after convulsion (%) 0 1 2 3 4 – – – – 100 – – – – – – 100 8.02 F 0.13 14.06 F 0.21* 18.22 F 0.56* 14.58 F 0.36* 17.29 F 0.25* – – – – – – – – – – 20 20 80 20 60 60 80 20 80 40 20 – – – – 40 100 100 80 100 7.10 F 0.82 0 a Seizure were induced by injection of 5 mg/kg i.p. of picrotoxin. * P b 0.05 significant as compared with control, Student’s t-test. 2.10. Statistical analysis Results are reported as mean F S.E.M. Statistical analysis was carried out using Student’s t-test. 3. Results The oral treatment with the aqueous root extract of L. cupanioides (LCRAE) at doses from 200 to 800 mg/kg, significantly prolonged seizure latency, rating and increased survival after treatment with strychnine, pentylenetetrazole and picrotoxin (Tables 1–3). The same results were observed after imtraperitoneal treatment with 100–200 mg/kg of LCRAE. Moreover, LCRAE (200 and 400 mg/kg, p.o. and 50 and 100 mg/kg, i.p.) produced a dose-dependent potentiation of pentobarbitone sleeping time in mice (Table 4), and a dose-dependent suppression of exploratory behavior (Table 5). Table 4 Effect of LCRAE on pentobarbitone-induced sleeping time in mice Treatmenta Saline 5 ml/kg Chlorpromazine 1 mg/kg i.p. LCRAE: 200 mg/kg (p.o.) 400 mg/kg (p.o.) 50 mg/kg (i.p.) 100 mg/kg (i.p.) a Mean sleeping time (min) F S.E.M. 19.1 F 2.8 52.3 F 4.8* 128.3 F 5.7** 206.9 F 11.2*** 156.2 F 9.4** 190.6 F 8.8*** Sleeping was induced by injection of 40 mg/kg i.p. of pentobarbitone. * P b 0.05 significant as compared with control, Student’s t-test. ** P b 0.01 significant as compared with control, Student’s t-test. *** P b 0.001 significant as compared with control, Student’s t-test. 417 O.K. Yemitan, O.O. Adeyemi / Fitoterapia 76 (2005) 412–418 Table 5 Effect of LCRAE on exploratory activity in mice Treatment Dips in 7.5 mina Saline Chlorpromazine 4 mg/kg i.m. LCRAE: 100 mg/kg i.p. 200 mg/kg p.o. 400 mg/kg p.o. 800 mg/kg p.o. 17.8 F 1.08 6.4 F 0.66* 13.2 F 1.22 12.8 F 0.96 8.4 F 0.85* 6.9 F 0.56* a The study was carried out as reported in Section 2.8. * P b 0.05 significant as compared with control, Student’s t-test. No mortality was recorded after oral treatment with doses of extract up to 20 g/kg. A dose-dependent drowsiness was observed. However, when the extract was administered intraperitoneally, a dose-dependent mortality was observed with an LD50 of 455.2 mg/kg (390.7–510.4). 4. Discussion The aqueous root extract of L. cupanioides completely prevented seizure induced by strychnine, prolonged the latency and reduced severity of seizure induced by PTZ and picrotoxin. It is known that strychnine directly antagonize the inhibitory spinal reflexes of glycine [7]. LCRAE, therefore, might cause seizure suppression by acting on glycine inhibitory mechanisms. Although the mechanism of convulsant action of PTZ and picrotoxin is poorly understood [8–11], it is reported that they are able to inhibit chloride conductance by binding to picrotoxin sites of GABAA receptor complex [9]. LCRAE, therefore, might owe its seizure suppression activity, prolonged latency and reduced severity, to both GABAergic and glycine inhibitory mechanisms. Moreover, the prolongation of pentobarbitone sleeping time and suppression of exploratory activity confirm the central nervous system depressant activity of the LCRAE. Our results provide a justification for the use of the aqueous root extract of L. cupanioides by some herbalists to suppress some forms of epilepsy. The plant as used traditionally has been shown to be devoid of relevant sign of toxicity when given orally to mice at dose up to 20 g/kg. Acknowledgements The authors are grateful to Mrs. E.O. Ayorinde of the Pharmacology Department, Lagos State University, College of Medicine for technical assistance. References [1] Gill LS. Ethnomedical uses of plants in Nigeria. Benin City: UNIBEN Press; 1992. p. 95, 147. [2] Diehl RG, Smialowsky A, Gotwo T. Epilepsia 1984;25:506. 418 [3] [4] [5] [6] [7] [8] [9] [10] [11] O.K. Yemitan, O.O. Adeyemi / Fitoterapia 76 (2005) 412–418 Loscher W, Honack D, Fassbender CP, Noltig B. Epilepsy Res 1991;8:171. Dandiya PC, Collumbine H. 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