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Anticonvulsant activity of Carissa edulis (Vahl) (Apocynaceae) root bark extract
Journal of Ethnopharmacology 120 (2008) 255–258
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Anticonvulsant activity of Carissa edulis (Vahl) (Apocynaceae) root bark extract J. Ya’u a,∗ , A.H. Yaro a , M.S. Abubakar b , J.A. Anuka a , I.M. Hussaini a,c a
Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria, Nigeria Department of Pharmacognosy and Drug development, Ahmadu Bello University, Zaria, Nigeria c Department of Pathology, University of Virginia, School of Medicine, Charlottesville, VA 22908, USA b
a r t i c l e
i n f o
Article history: Received 8 March 2008 Received in revised form 4 July 2008 Accepted 25 August 2008 Available online 4 September 2008 Keywords: Anticonvulsant activity Carissa edulis Benzodiazepine
a b s t r a c t Aim of the study: To investigate the anticonvulsant activity of root bark extract of Carissa edulis. Materials and methods: The median lethal dose (LD50 ) of Carissa edulis extract was determined using Lork’s method (1983). The anticonvulsant activity of the extract was assessed in pentylenetetrazole (PTZ)induced convulsion in mice and maximal electroshock test (MEST) in chicks, with benzodiazepine and phenytoin as standard drugs, respectively. While mechanistic studies were conducted using both flumazenil, a GABAA -benzodiazepine receptor complex site antagonist and naloxone a non-specific opioid receptor antagonist. Results: The median lethal dose (LD50 ) of Carissa edulis was 282.8 mg/kg and over 5000 mg/kg following intraperitoneal and oral administration, respectively. Carissa edulis produced 40% and 20% protection against convulsion at 5 and 20 mg/kg, respectively, compared with 100% protection with benzodiazepine. The mean onset and percentage protection against convulsion in Carissa edulis extract-treated mice were reduced by flumazenil and naloxone. Carissa edulis exhibited dose-dependent inhibition of the convulsion induced by MEST with 20 mg/kg providing 90% protection while phenytoin (20 mg/kg) produced 100% protection. Conclusion: These results suggest that Carissa edulis possesses biologically active constituent(s) that have anticonvulsant activity which supports the ethnomedicinal claims of the use of the plant in the management of epilepsy. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Carissa edulis (CE) belongs to the family Apocynaceae. It was formerly known as C. pubescence (Irvine, 1961; Hutchison and Dalziel, 1963). The plant is commonly known among Hausa people in Northern Nigeria as ‘cizaki’, and in Somalia as ‘adishawel’. The English name of the plant is Arabic num-num. Other common names include; ‘endelkoring-noeminoem’ (Africana), ‘agam’ (Tigrigna and Amharic), ‘emir’ (Arabic), ‘muyonza’ (Luganda), ‘mlanoa-mboo’ (Swahili) and ‘mpambala myoloko’ (Sofowora, 1986). The plant parts are used in ethnomedicine for wide variety of illnesses, such as epilepsy (Malam Suleiman, personal communica-
tion), headache, chest complaints, gonorrhea, syphilis, rheumatism, rabies and as well as a diuretic (Nedi et al., 2004). Other folkloric uses of Carissa edulis include fever, sickle cell anaemia and hernia (Ibrahim, 1997). Carissa edulis is used as a source of dye (Oliver, 1960; Irvine, 1961; Burkill, 1985; Banker and Verma, 1987; Omino and Kokwaro, 1993). The chemical constituents of Carissa edulis include steroids, terpenes, tannins, flavonoids and cardiac glycosides (Ibrahim, 1997), benzenoids, phenylpropanoid, lignans, sesquiterpenes and cumarins (Achenbach et al., 1983; Bentley et al., 1984). This study was undertaken to investigate the pharmacological basis for the folkloric use of Carissa edulis as an anticonvulsant agent. 2. Materials and methods
Abbreviations: AEDs, antiepileptic drugs; BDZ, benzodiazepine; CE, Carissa edulis; Flu, flumazenil; GABA, gamma amino butyric acid; IP, intraperitoneal; LD50 , mean lethal dose; MEST, maximal electroshock test; Nal, naloxone; PH, phenytoin; PTZ, pentylenetetrazole; Sc, subcutaneous; SEM, standard error of the mean; THLE, tonic hind limb extension. ∗ Corresponding author. Tel.: +234 8032415294. E-mail address: [email protected] (J. Ya’u). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.08.029
2.1. Plant materials and preparation of the extract Samples of root bark of Carissa edulis were collected in July 2006 from Basawa Barracks, Samaru, Zaria, Nigeria. The plant was identified and authenticated by Malam Musa and Umar Gallah of the
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J. Ya’u et al. / Journal of Ethnopharmacology 120 (2008) 255–258
Herbarium section in the Department of Biological Sciences, A. B. U. Zaria, Nigeria. A voucher specimen number 601 was deposited at the herbarium. The root bark was scraped and washed with distilled water. The root bark was air dried under the shade until a constant weight was obtained. It was then powdered using wooden pestle and mortar and sieved with 20-mesh sieve size. The powdered root bark (100 g) was extracted with 700 ml of 60% (v/v) aqueous ethanol using maceration extraction method. The extract was concentrated to obtain an average weight. The extract was then stored in desiccators and solutions of the extract were prepared freshly for each study. The aqueous ethanol extract of the root bark of Carissa edulis was brown in colour and had a pleasant smell. The yield was 25.77% (w/w). 2.2. Animals Swiss Albino mice of either sex (weighing 15–24 g) were obtained from animal house of the Department of Pharmacology and Clinical Pharmacy, A. B. U. Zaria. White ranger cockerels, a day old of either sex (weighing 30–41 g) purchased from National Animal Production Research Institute (NAPRI), Shika, Zaria, Nigeria. The animals were maintained in a well-ventilated room, fed on Excel Feeds (Feeds Masters, Ilorin) and water ad libitum. All studies using mice and chicks were approved by the Department’s Animal Use Committee. 2.3. Drugs/chemicals and equipments Pentylenetetrazole was purchased from Sigma–Aldrich Chemical Co (St Louis MO, USA), phenytoin sodium is a product of Parke-Davis and Co Ltd. (Detroit, MI) and sodium valproate was bought from Sanofi synthelabo (Onslow st. Surrey, UK). Flumazenil and naloxone are products of BIOMOL Research Lab (Plymouth Meeting PA, USA). Ugo Basile current electroshock machine (Model 7800 with corneal electrodes) was purchased from Ugo Basile (Comerio VA, Italy). 2.4. Acute toxicity studies The median lethal dose (LD50 ) determination was performed using the method of Lorke (1983) for intraperitoneal (IP) and oral routes in mice. The method consisted of two phases. In the first stage, three groups of three mice each were injected with the Carissa edulis extract at doses of 10, 100 and 1000 mg/kg body weight IP and observed for signs of toxicity and death within 24 h. In the second stage, four groups of one mouse each were treated with four more specific doses of the extract based on the result of the stage 1. The LD50 value was determined by calculating the geometric means of the lowest dose that caused death and the highest dose for which the animals survived. 2.5. Screening for anticonvulsant activity 2.5.1. Pentylenetetrazole (PTZ)-induced convulsion in mice The method of Swinyard et al. (1989) was employed to induce convulsion in mice using PTZ. Thirty mice were divided into six groups each containing five mice. The first group received normal saline 10 ml/kg body weight IP, the second, third and fourth groups received 5, 10 and 20 mg/kg body weight IP of ethanol extract of Carissa edulis, while the fifth and sixth group were injected with diazepam 1 and 0.5 mg/kg body weight IP. Thirty minutes after treatment, mice in all the groups received PTZ 90 mg/kg subcutaneous (Sc). Mice were observed over a period of 30 min, absence of an episode of clonic spasm of at least 5 s duration indicated
the extract or a compound’s ability to abolish the effect of PTZ on seizure threshold. 2.5.2. Maximal electroshock-induced convulsion in chicks The method described by Swinyard and Kupferberg (1985) as modified by Sayyah et al. (2002) was used in the study. Fifty (day-old) white cockerels were randomly divided into five groups each containing 10 chicks. The first group received normal saline 10 ml/kg body weight IP, second group, third and fourth groups were treated with 5, 10 and 20 mg/kg of the extract IP, and the fifth group was injected with 20 mg/kg of phenytoin IP. Thirty minutes later, maximal electroshock was administered to induce seizure in the chicks using Ugo Basile electroconvulsive machine (model 7801) connected to Clande Lyons stabilizer with corneal electrodes placed on the upper eyelid of the chicks. The shock duration, frequency, current and pulse width were set and maintained at 1.6 s, 150 pulse/s, 80 mA and 0.8 ms, respectively. A current of about 80 mA which produced tonic seizures in 90% of the negative control chicks was used throughout the study. Seizures were manifested as tonic hind limb extension (THLE) (Swinyard, 1969). The ability to prevent this feature or prolong the latency and/or onset of the THLE was considered as an indication of anticonvulsant activity (Swinyard, 1969; Sayyah et al., 2002). 2.6. Statistical analysis Results were expressed as percentage (%) protection and mean ± SEM where applicable. Statistical significance was tested using Student’s t-test. The difference was taken to be statistically significant at P < 0.05. 3. Results 3.1. Acute toxicity studies The median lethal dose (LD50 ) of the Carissa edulis extract in mice was found to be 282.8 mg/kg body weight administered intraperitoneally. At 1000 mg/kg dose there was 100% mortality and the minimum dose that killed a rodent was 100 mg/kg. The oral LD50 value for Carissa edulis was above 5000 mg/kg. 3.2. Screening for anticonvulsant activity The anticonvulsant activities of ethanol extract of Carissa edulis were determined using chemically induced (PTZ) and electrically induced (MEST) convulsion in mice and chicks, respectively. In PTZ-induced convulsion, Carissa edulis protected the rodents but the response was not dose-dependent (Table 1). At 5.0 mg/kg Table 1 Effect of hydro-alcoholic root bark extract of C. edulis, diazepam and flumazenil on pentylenetetrazole (PTZ)-induced seizure mouse model. Drugs (mg/kg)
Mean onset (min)
%Protection against seizures
Control CE (5) CE (20) DZ (0.5) DZ (1) DZ (3) CE (5) + Flu (10) DZ (0.5) + Flu (10)
8.61 ± 1.21 6.67 ± 1.76 12.0 ± 2.08 13.0 ± 2.00 0.0 0.0 4.00 ± 0.84* 9.67 ± 2.19
0.0 40.0 20.0 60.0 100 100 0.0 40.0
Control = normal saline, DZ = diazepam and CE = Carissa edulis were administered 30 min intraperitoneally before injection of PTZ (90 mg/kg); Flu = flumazenil was administered 15 min before CE or DZ; values are the mean ± SEM for five mice; (*) P < 0.05 (Student t-test).
J. Ya’u et al. / Journal of Ethnopharmacology 120 (2008) 255–258 Table 2 Effect of naloxone on the anticonvulsant activity of -alcoholic root bark extract of C. edulis in Pentylenetetrazole (PTZ)-induced seizure mouse model. Drugs (mg/kg)
Mean onset (min)
Control CE (5) CE (5) + Nal (0.3) Nal (0.3)
5.40 5.75 7.40 8.00
± ± ± ±
0.87 0.63 1.21 1.52
%Protection against seizure 0.0 20.0 0.0 0.0
Nal (Naloxone) was administered 15 min before CE (Carissa edulis). PTZ (90 mg/kg) was injected 30 min after the administration of CE; control: normal saline; values are the mean ± SEM of five mice.
the extract provided 40% protection (P < 0.05). The mean onset (6.67 ± 1.76) of convulsion in the mice treated with 5.0 mg/kg that had seizure was not statistically significant from control (8.61 ± 1.21 min). In contrast, the mean onset of convulsions in PTZinduced convulsion was increased by 39 % with 20 mg/kg of Carissa edulis extract (Table 1). Diazepam (0.5 mg/kg) increased the mean onset of convulsion by approximately 51% and offered 60% protection of seizure (Table 1). When we increased the dose of diazepam to 1 mg/kg none of the mice convulsed. In order to elucidate the mechanism involved in Carissa edulisinduced partial protection of mice from PTZ-induced seizure, we administered flumazenil (10 mg/kg), antagonist of benzodiazepine (BZD) site in the GABAA -BZD receptor complex or naloxone (0.3 mg/kg), an opioid receptor antagonist prior to injection of Carissa edulis extract (5 mg/kg) or diazepam (0.5 mg/kg). Flumazenil (10 mg/kg) reduced the onset of convulsion in diazepam-treated mice by 25.6% (from 13 ± 2.0 to 9.67 ± 2 .19 min) and reduced the protection against seizure from 60% to 40% (33.3% reduction). Similarly, the same dose of flumazenil reduced the onset of seizure in Carissa edulis extract-treated mice from 6.67 ± 1.76 to 4.0 ± 0.8 min (40% reduction). In addition, flumazenil completely blocked the protection provided Carissa edulis against seizure (Table 1). In contrast, Naloxone (0.3 mg/kg) increased the onset of seizure induced by Carissa edulis extract from 5.57 ± 0.63 to 7.40 ± 1.12 min. However, the protection against seizure in Carissa edulis extract-treated mice was abolished in the presence of naloxone (Table 2). To determine whether Carissa edulis would be more effective against seizures in chicks that have little or no blood–brain-barrier, we tested the extract in electrically induced convulsion. Carissa edulis extract was more effective in protecting the chicks from electrically-induced convulsion as shown in Table 3. The lowest dose (5 mg/kg) provided 60% protection against seizures, while 20 mg/kg protected the chicks by 90%. The onset of convulsion was delayed by 60.6% (from 4.26 ± 0.31 in control to 6.84 ± 0.0 min in 20 mg/kg extract). Phenytoin (20 mg/kg) administration 30 min prior to electrical induction of seizure completely protected the chicks from convulsion. 4. Discussion We have used two different animal model experiments that characteristically described three types of seizures activity. These Table 3 Effect of Carissa edulis root bark ethanol extract on MEST-induced convulsions in chicks. Dose (mg/kg)
Mean onset of seizures (min)
%Protection against seizure
Control 5.0 (CE) 20.0 (CE) 20 (PH)
4.26 ± 0.31 3.54 ± 0.54 6.84 ± 0.0* 0.0
0.0 60.0 90.0 100.0
Values were presented as mean ± SEM, n = 10, CE = Carissa edulis and PH = phenytoin. * P < 0.05 (Student’s t-test).
257
were demonstrated by the activity against PTZ-induced seizures which correlate with anti-absence activity and activity against electrically induced seizures signifies activity against generalized tonic–clonic and partial seizures (Delgado and Remers, 1998). The Carissa edulis extract had little protection on the animals used in the screening for anticonvulsant activity using PTZ animal model, but it increased seizures latency (Table 1). However, these effects were not dose-dependent. In MEST-induced convulsion, the Carissa edulis extract significantly protected the animals against seizures, increased the onset and reduced the duration of seizures (Table 3). The current available antiepileptic drugs (AEDs) that are clinically effective in the management of generalized tonic–clonic and partial seizures such as carbamazepine, phenytoin, primidone, phenobarbital, valproate and lamotrigine all suppress hind limb tonic extension (HLTE) in MEST (Browning, 1992; Rho and Sankar, 1999). Protection against HLTE also indicates the ability of a testing material to inhibit or prevent seizures discharge within the brainstem seizure substrate (Browning, 1992). The ability of the extract to inhibit the HLTE in MEST as compared to phenytoin (100% protection) in the model suggests anticonvulsant activity for the management of generalized tonic–clonic and partial seizures. AEDs effective in the therapy of generalized seizures of (absence or myoclonic) petit mal type such as phenobarbitone, valproate, ethosuximide and benzodiazepines exhibit dose-dependent suppression of various seizure pattern induced by PTZ (Loscher et al., 1991). PTZ-induced seizures are similar to the symptoms observed in the absence seizures and drugs such as valproate and ethosuximide which are useful in the management of absence seizures inhibit PTZ-induced seizures (McNamara, 2001). At cellular level, one of the basic mechanisms of actions of AEDs such as ethosuximide and valproate is the suppression of T-type calcium currents in thalamic neurons (Macdonald and Kelly, 1994; Meldrum, 1996; Rho and Sankar, 1999). In PTZ-induced convulsions, the extract of Carissa edulis had only increased the latency but not the incidence of seizures as compared to diazepam (Table 1). This extract might not be useful in the management of absence seizures. The observed effects might be in agreement with the findings of Swinyard et al. (1952) and Swinyard (1969) that not all antiepileptic drugs have protective value against Sc-PTZ induced convulsions. The biphasic activity observed in PTZ-induced studies may probably be due to possible interaction between constituents of the crude extract. The majority of currently available antiepileptic drugs fall into one of two pharmacological classes, those that modulate neuronal voltage-gated sodium channels (e.g. carbamazepine, phenytoin, lamotrigine, and topiramate) and those that modulate inhibitory GABAergic neurotransmission (e.g. benzodiazepine, vigabatrin and tiagabine). While, small number of AEDs such as ethosuximide, gabapentin and possibly levetiracetam, may exert their effects via an interaction with voltage-operated calcium channels (Wickenden, 2002). The ability of the extract to exhibit activity against these two types of seizures suggests that it may act through different mechanisms to elicit its anticonvulsant effects, such as voltage-gated sodium, calcium, and potassium or GABAergic pathway. The fact that flumazenil reduced seizure onset and attenuated the protection against seizure provided by Carissa edulis suggest that the extract may contain substance(s) that interacts with the benzodiazepine site in the GABAA -benzodiazepine receptor complex. The results of the study have demonstrated that Carissa edulis possessed anticonvulsant activity on the animal models investigated and this provides a rationale for its use in traditional medicine for the management of epilepsy. Further work to establish the active chemical constituent(s) of the extract and the exact mechanism of action is currently going on in our laboratory.
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References Achenbach, H., Weibal, R., Addae-Mensah, I., 1983. Lignan from Carissa edulis. Phytochemistry 76, 30837–38545 (in Biological Abstract). Banker, G.J., Verma, S.K., 1987. Preliminary studies of Flowering and Fruiting in Carissa edulis. Progressive Horticulture 19, 163–166. Bentley, M.D., Brackett, S.R., Chapya, A., 1984. 2-Hydroxyacetophenone, Principal Root Volatile of the East African Medicinal Plant, Carissa edulis. Journal of Natural Products 47, 1056–1057. Browning, R., 1992. The electroshock model, neuronal network and antiepileptic drugs. In: Faingold, C.L., Fromm, G.H. (Eds.), Drugs for Control of Epilepsy: Actions on Neuronal Networks in Seizure Disorders. CRC Press, Boca Raton, FL, pp. 195–211. Burkill, H.M., 1985. The Useful Plants of West Africa, 1., Second ed. Families A-D Royal Botanical Gardens, Kew, p145. Delgado, J.N., Remers, W.A. (Eds.), 1998. Wilson and Gisvold’s Textbook of Organic and Medicinal Pharmaceutical Chemistry. Lippincott-Raven, U.S.A.. Hutchison, J., Dalziel, J.M., 1963. Flora of West Tropical Africa. Vol. II. Crown Agents for Oversea Government and Administrations. Millbank, London, S.W.I. pp. 51–54. Ibrahim, H., 1997. Pharmacognostic and Biological (Analgesic Activity) Studies of Carissa edulis Vahl. Ph.D. Thesis. Ahmadu Bello University, Zaria, Nigeria, pp. 232. Irvine, F.R., 1961. Woody Plants of Ghana. Oxford University Press, London, p. 618. Lorke, D., 1983. A new approach to acute toxicity testing. Archives of Toxicology 54, 275–287. Loscher, W., Honack, D., Fassbender, C.P., 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. Macdonald, R.L., Kelly, K.M., 1994. Mechanisms of action of currently prescribed and newly developed antiepileptic drugs. Epilepsia 35, S41–S50.
McNamara, J.O., 2001. Drugs effective in the therapy of the epilepsies. In: Gilman, A.G., Rall, T.W., Nies, A.S., Taylor, P. (Eds.), The Pharmacological Basis of Therapeutics. Permagon Press, New York, U.S.A., p. 521. Meldrum, B.S., 1996. Update on the mechanism of action of antiepileptic drugs. Epilepsia 37, S4–S11. Nedi, T., Mekonnen, N., Urqa, K., 2004. Diuretic effect of the crude extract of Carissa edulis in rats. Journal of Ethnopharmacology 95, 57–61. Oliver, B., 1960. Medicinal Plants in Nigeria published as a Private Edition by the Nigerian College of Art, Science & Technology. pp. 52. Omino, E.A., Kokwaro, J.O., 1993. Ethnobotany of Apocynaceae specie in Kenya. Journal of Ethnopharmacology 4, 167–180. Rho, J.M., Sankar, R., 1999. The pharmacological basis of antiepileptic drug action. Epilepsia 40, 1471–1483. Sayyah, M., Saroukhari, G., Peirovi, A., Kamalinejad, M., 2002. Analgesic and anti-inflammatory activity of the leaf essential oil of Lauraus nobilis Linn. Phytotherapy Research 17, 733–736. Sofowora, A., 1986. The State of Medicinal Plant Research in Nigeria, First ed. University Press Ltd. Ife, Nigeria, p. 338. Swinyard, E.A., 1969. Laboratory evaluation of antiepileptic drugs: Review of laboratory methods. Epilepsia 10, 107–119. Swinyard, E.A., Kupferberg, H.J., 1985. Antiepileptic drugs: detection, quantification and evaluation. Federation Procedings 44, 3. Swinyard, E.A., Brown, W.C., Goodman, L.S., 1952. Comparative assays of antiepileptic drugs in mice and rats. Journal of Pharmacology and Experimental Therapy 106, 319–330. Swinyard, E.A., Woodhead, J.H., White, H.S., franklin, M.R., 1989. General principles: experimental selection, quantification and evaluation of anticonvulsants. In: Levy, R., Mattson, R., Meldium, B., Penry, J.K., Dreifuss, F.E. (Eds.), Antiepileptic Drugs. Raven Press, New York, pp. 233–239. Wickenden, A.D., 2002. Potassium channels as antiepileptic drug targets. Neuropharmacology 43, 1055–1060.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Anticonvulsant activity of Carissa edulis (Vahl) (Apocynaceae) root bark extract J. Ya’u a,∗ , A.H. Yaro a , M.S. Abubakar b , J.A. Anuka a , I.M. Hussaini a,c a
Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria, Nigeria Department of Pharmacognosy and Drug development, Ahmadu Bello University, Zaria, Nigeria c Department of Pathology, University of Virginia, School of Medicine, Charlottesville, VA 22908, USA b
a r t i c l e
i n f o
Article history: Received 8 March 2008 Received in revised form 4 July 2008 Accepted 25 August 2008 Available online 4 September 2008 Keywords: Anticonvulsant activity Carissa edulis Benzodiazepine
a b s t r a c t Aim of the study: To investigate the anticonvulsant activity of root bark extract of Carissa edulis. Materials and methods: The median lethal dose (LD50 ) of Carissa edulis extract was determined using Lork’s method (1983). The anticonvulsant activity of the extract was assessed in pentylenetetrazole (PTZ)induced convulsion in mice and maximal electroshock test (MEST) in chicks, with benzodiazepine and phenytoin as standard drugs, respectively. While mechanistic studies were conducted using both flumazenil, a GABAA -benzodiazepine receptor complex site antagonist and naloxone a non-specific opioid receptor antagonist. Results: The median lethal dose (LD50 ) of Carissa edulis was 282.8 mg/kg and over 5000 mg/kg following intraperitoneal and oral administration, respectively. Carissa edulis produced 40% and 20% protection against convulsion at 5 and 20 mg/kg, respectively, compared with 100% protection with benzodiazepine. The mean onset and percentage protection against convulsion in Carissa edulis extract-treated mice were reduced by flumazenil and naloxone. Carissa edulis exhibited dose-dependent inhibition of the convulsion induced by MEST with 20 mg/kg providing 90% protection while phenytoin (20 mg/kg) produced 100% protection. Conclusion: These results suggest that Carissa edulis possesses biologically active constituent(s) that have anticonvulsant activity which supports the ethnomedicinal claims of the use of the plant in the management of epilepsy. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Carissa edulis (CE) belongs to the family Apocynaceae. It was formerly known as C. pubescence (Irvine, 1961; Hutchison and Dalziel, 1963). The plant is commonly known among Hausa people in Northern Nigeria as ‘cizaki’, and in Somalia as ‘adishawel’. The English name of the plant is Arabic num-num. Other common names include; ‘endelkoring-noeminoem’ (Africana), ‘agam’ (Tigrigna and Amharic), ‘emir’ (Arabic), ‘muyonza’ (Luganda), ‘mlanoa-mboo’ (Swahili) and ‘mpambala myoloko’ (Sofowora, 1986). The plant parts are used in ethnomedicine for wide variety of illnesses, such as epilepsy (Malam Suleiman, personal communica-
tion), headache, chest complaints, gonorrhea, syphilis, rheumatism, rabies and as well as a diuretic (Nedi et al., 2004). Other folkloric uses of Carissa edulis include fever, sickle cell anaemia and hernia (Ibrahim, 1997). Carissa edulis is used as a source of dye (Oliver, 1960; Irvine, 1961; Burkill, 1985; Banker and Verma, 1987; Omino and Kokwaro, 1993). The chemical constituents of Carissa edulis include steroids, terpenes, tannins, flavonoids and cardiac glycosides (Ibrahim, 1997), benzenoids, phenylpropanoid, lignans, sesquiterpenes and cumarins (Achenbach et al., 1983; Bentley et al., 1984). This study was undertaken to investigate the pharmacological basis for the folkloric use of Carissa edulis as an anticonvulsant agent. 2. Materials and methods
Abbreviations: AEDs, antiepileptic drugs; BDZ, benzodiazepine; CE, Carissa edulis; Flu, flumazenil; GABA, gamma amino butyric acid; IP, intraperitoneal; LD50 , mean lethal dose; MEST, maximal electroshock test; Nal, naloxone; PH, phenytoin; PTZ, pentylenetetrazole; Sc, subcutaneous; SEM, standard error of the mean; THLE, tonic hind limb extension. ∗ Corresponding author. Tel.: +234 8032415294. E-mail address: [email protected] (J. Ya’u). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.08.029
2.1. Plant materials and preparation of the extract Samples of root bark of Carissa edulis were collected in July 2006 from Basawa Barracks, Samaru, Zaria, Nigeria. The plant was identified and authenticated by Malam Musa and Umar Gallah of the
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Herbarium section in the Department of Biological Sciences, A. B. U. Zaria, Nigeria. A voucher specimen number 601 was deposited at the herbarium. The root bark was scraped and washed with distilled water. The root bark was air dried under the shade until a constant weight was obtained. It was then powdered using wooden pestle and mortar and sieved with 20-mesh sieve size. The powdered root bark (100 g) was extracted with 700 ml of 60% (v/v) aqueous ethanol using maceration extraction method. The extract was concentrated to obtain an average weight. The extract was then stored in desiccators and solutions of the extract were prepared freshly for each study. The aqueous ethanol extract of the root bark of Carissa edulis was brown in colour and had a pleasant smell. The yield was 25.77% (w/w). 2.2. Animals Swiss Albino mice of either sex (weighing 15–24 g) were obtained from animal house of the Department of Pharmacology and Clinical Pharmacy, A. B. U. Zaria. White ranger cockerels, a day old of either sex (weighing 30–41 g) purchased from National Animal Production Research Institute (NAPRI), Shika, Zaria, Nigeria. The animals were maintained in a well-ventilated room, fed on Excel Feeds (Feeds Masters, Ilorin) and water ad libitum. All studies using mice and chicks were approved by the Department’s Animal Use Committee. 2.3. Drugs/chemicals and equipments Pentylenetetrazole was purchased from Sigma–Aldrich Chemical Co (St Louis MO, USA), phenytoin sodium is a product of Parke-Davis and Co Ltd. (Detroit, MI) and sodium valproate was bought from Sanofi synthelabo (Onslow st. Surrey, UK). Flumazenil and naloxone are products of BIOMOL Research Lab (Plymouth Meeting PA, USA). Ugo Basile current electroshock machine (Model 7800 with corneal electrodes) was purchased from Ugo Basile (Comerio VA, Italy). 2.4. Acute toxicity studies The median lethal dose (LD50 ) determination was performed using the method of Lorke (1983) for intraperitoneal (IP) and oral routes in mice. The method consisted of two phases. In the first stage, three groups of three mice each were injected with the Carissa edulis extract at doses of 10, 100 and 1000 mg/kg body weight IP and observed for signs of toxicity and death within 24 h. In the second stage, four groups of one mouse each were treated with four more specific doses of the extract based on the result of the stage 1. The LD50 value was determined by calculating the geometric means of the lowest dose that caused death and the highest dose for which the animals survived. 2.5. Screening for anticonvulsant activity 2.5.1. Pentylenetetrazole (PTZ)-induced convulsion in mice The method of Swinyard et al. (1989) was employed to induce convulsion in mice using PTZ. Thirty mice were divided into six groups each containing five mice. The first group received normal saline 10 ml/kg body weight IP, the second, third and fourth groups received 5, 10 and 20 mg/kg body weight IP of ethanol extract of Carissa edulis, while the fifth and sixth group were injected with diazepam 1 and 0.5 mg/kg body weight IP. Thirty minutes after treatment, mice in all the groups received PTZ 90 mg/kg subcutaneous (Sc). Mice were observed over a period of 30 min, absence of an episode of clonic spasm of at least 5 s duration indicated
the extract or a compound’s ability to abolish the effect of PTZ on seizure threshold. 2.5.2. Maximal electroshock-induced convulsion in chicks The method described by Swinyard and Kupferberg (1985) as modified by Sayyah et al. (2002) was used in the study. Fifty (day-old) white cockerels were randomly divided into five groups each containing 10 chicks. The first group received normal saline 10 ml/kg body weight IP, second group, third and fourth groups were treated with 5, 10 and 20 mg/kg of the extract IP, and the fifth group was injected with 20 mg/kg of phenytoin IP. Thirty minutes later, maximal electroshock was administered to induce seizure in the chicks using Ugo Basile electroconvulsive machine (model 7801) connected to Clande Lyons stabilizer with corneal electrodes placed on the upper eyelid of the chicks. The shock duration, frequency, current and pulse width were set and maintained at 1.6 s, 150 pulse/s, 80 mA and 0.8 ms, respectively. A current of about 80 mA which produced tonic seizures in 90% of the negative control chicks was used throughout the study. Seizures were manifested as tonic hind limb extension (THLE) (Swinyard, 1969). The ability to prevent this feature or prolong the latency and/or onset of the THLE was considered as an indication of anticonvulsant activity (Swinyard, 1969; Sayyah et al., 2002). 2.6. Statistical analysis Results were expressed as percentage (%) protection and mean ± SEM where applicable. Statistical significance was tested using Student’s t-test. The difference was taken to be statistically significant at P < 0.05. 3. Results 3.1. Acute toxicity studies The median lethal dose (LD50 ) of the Carissa edulis extract in mice was found to be 282.8 mg/kg body weight administered intraperitoneally. At 1000 mg/kg dose there was 100% mortality and the minimum dose that killed a rodent was 100 mg/kg. The oral LD50 value for Carissa edulis was above 5000 mg/kg. 3.2. Screening for anticonvulsant activity The anticonvulsant activities of ethanol extract of Carissa edulis were determined using chemically induced (PTZ) and electrically induced (MEST) convulsion in mice and chicks, respectively. In PTZ-induced convulsion, Carissa edulis protected the rodents but the response was not dose-dependent (Table 1). At 5.0 mg/kg Table 1 Effect of hydro-alcoholic root bark extract of C. edulis, diazepam and flumazenil on pentylenetetrazole (PTZ)-induced seizure mouse model. Drugs (mg/kg)
Mean onset (min)
%Protection against seizures
Control CE (5) CE (20) DZ (0.5) DZ (1) DZ (3) CE (5) + Flu (10) DZ (0.5) + Flu (10)
8.61 ± 1.21 6.67 ± 1.76 12.0 ± 2.08 13.0 ± 2.00 0.0 0.0 4.00 ± 0.84* 9.67 ± 2.19
0.0 40.0 20.0 60.0 100 100 0.0 40.0
Control = normal saline, DZ = diazepam and CE = Carissa edulis were administered 30 min intraperitoneally before injection of PTZ (90 mg/kg); Flu = flumazenil was administered 15 min before CE or DZ; values are the mean ± SEM for five mice; (*) P < 0.05 (Student t-test).
J. Ya’u et al. / Journal of Ethnopharmacology 120 (2008) 255–258 Table 2 Effect of naloxone on the anticonvulsant activity of -alcoholic root bark extract of C. edulis in Pentylenetetrazole (PTZ)-induced seizure mouse model. Drugs (mg/kg)
Mean onset (min)
Control CE (5) CE (5) + Nal (0.3) Nal (0.3)
5.40 5.75 7.40 8.00
± ± ± ±
0.87 0.63 1.21 1.52
%Protection against seizure 0.0 20.0 0.0 0.0
Nal (Naloxone) was administered 15 min before CE (Carissa edulis). PTZ (90 mg/kg) was injected 30 min after the administration of CE; control: normal saline; values are the mean ± SEM of five mice.
the extract provided 40% protection (P < 0.05). The mean onset (6.67 ± 1.76) of convulsion in the mice treated with 5.0 mg/kg that had seizure was not statistically significant from control (8.61 ± 1.21 min). In contrast, the mean onset of convulsions in PTZinduced convulsion was increased by 39 % with 20 mg/kg of Carissa edulis extract (Table 1). Diazepam (0.5 mg/kg) increased the mean onset of convulsion by approximately 51% and offered 60% protection of seizure (Table 1). When we increased the dose of diazepam to 1 mg/kg none of the mice convulsed. In order to elucidate the mechanism involved in Carissa edulisinduced partial protection of mice from PTZ-induced seizure, we administered flumazenil (10 mg/kg), antagonist of benzodiazepine (BZD) site in the GABAA -BZD receptor complex or naloxone (0.3 mg/kg), an opioid receptor antagonist prior to injection of Carissa edulis extract (5 mg/kg) or diazepam (0.5 mg/kg). Flumazenil (10 mg/kg) reduced the onset of convulsion in diazepam-treated mice by 25.6% (from 13 ± 2.0 to 9.67 ± 2 .19 min) and reduced the protection against seizure from 60% to 40% (33.3% reduction). Similarly, the same dose of flumazenil reduced the onset of seizure in Carissa edulis extract-treated mice from 6.67 ± 1.76 to 4.0 ± 0.8 min (40% reduction). In addition, flumazenil completely blocked the protection provided Carissa edulis against seizure (Table 1). In contrast, Naloxone (0.3 mg/kg) increased the onset of seizure induced by Carissa edulis extract from 5.57 ± 0.63 to 7.40 ± 1.12 min. However, the protection against seizure in Carissa edulis extract-treated mice was abolished in the presence of naloxone (Table 2). To determine whether Carissa edulis would be more effective against seizures in chicks that have little or no blood–brain-barrier, we tested the extract in electrically induced convulsion. Carissa edulis extract was more effective in protecting the chicks from electrically-induced convulsion as shown in Table 3. The lowest dose (5 mg/kg) provided 60% protection against seizures, while 20 mg/kg protected the chicks by 90%. The onset of convulsion was delayed by 60.6% (from 4.26 ± 0.31 in control to 6.84 ± 0.0 min in 20 mg/kg extract). Phenytoin (20 mg/kg) administration 30 min prior to electrical induction of seizure completely protected the chicks from convulsion. 4. Discussion We have used two different animal model experiments that characteristically described three types of seizures activity. These Table 3 Effect of Carissa edulis root bark ethanol extract on MEST-induced convulsions in chicks. Dose (mg/kg)
Mean onset of seizures (min)
%Protection against seizure
Control 5.0 (CE) 20.0 (CE) 20 (PH)
4.26 ± 0.31 3.54 ± 0.54 6.84 ± 0.0* 0.0
0.0 60.0 90.0 100.0
Values were presented as mean ± SEM, n = 10, CE = Carissa edulis and PH = phenytoin. * P < 0.05 (Student’s t-test).
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were demonstrated by the activity against PTZ-induced seizures which correlate with anti-absence activity and activity against electrically induced seizures signifies activity against generalized tonic–clonic and partial seizures (Delgado and Remers, 1998). The Carissa edulis extract had little protection on the animals used in the screening for anticonvulsant activity using PTZ animal model, but it increased seizures latency (Table 1). However, these effects were not dose-dependent. In MEST-induced convulsion, the Carissa edulis extract significantly protected the animals against seizures, increased the onset and reduced the duration of seizures (Table 3). The current available antiepileptic drugs (AEDs) that are clinically effective in the management of generalized tonic–clonic and partial seizures such as carbamazepine, phenytoin, primidone, phenobarbital, valproate and lamotrigine all suppress hind limb tonic extension (HLTE) in MEST (Browning, 1992; Rho and Sankar, 1999). Protection against HLTE also indicates the ability of a testing material to inhibit or prevent seizures discharge within the brainstem seizure substrate (Browning, 1992). The ability of the extract to inhibit the HLTE in MEST as compared to phenytoin (100% protection) in the model suggests anticonvulsant activity for the management of generalized tonic–clonic and partial seizures. AEDs effective in the therapy of generalized seizures of (absence or myoclonic) petit mal type such as phenobarbitone, valproate, ethosuximide and benzodiazepines exhibit dose-dependent suppression of various seizure pattern induced by PTZ (Loscher et al., 1991). PTZ-induced seizures are similar to the symptoms observed in the absence seizures and drugs such as valproate and ethosuximide which are useful in the management of absence seizures inhibit PTZ-induced seizures (McNamara, 2001). At cellular level, one of the basic mechanisms of actions of AEDs such as ethosuximide and valproate is the suppression of T-type calcium currents in thalamic neurons (Macdonald and Kelly, 1994; Meldrum, 1996; Rho and Sankar, 1999). In PTZ-induced convulsions, the extract of Carissa edulis had only increased the latency but not the incidence of seizures as compared to diazepam (Table 1). This extract might not be useful in the management of absence seizures. The observed effects might be in agreement with the findings of Swinyard et al. (1952) and Swinyard (1969) that not all antiepileptic drugs have protective value against Sc-PTZ induced convulsions. The biphasic activity observed in PTZ-induced studies may probably be due to possible interaction between constituents of the crude extract. The majority of currently available antiepileptic drugs fall into one of two pharmacological classes, those that modulate neuronal voltage-gated sodium channels (e.g. carbamazepine, phenytoin, lamotrigine, and topiramate) and those that modulate inhibitory GABAergic neurotransmission (e.g. benzodiazepine, vigabatrin and tiagabine). While, small number of AEDs such as ethosuximide, gabapentin and possibly levetiracetam, may exert their effects via an interaction with voltage-operated calcium channels (Wickenden, 2002). The ability of the extract to exhibit activity against these two types of seizures suggests that it may act through different mechanisms to elicit its anticonvulsant effects, such as voltage-gated sodium, calcium, and potassium or GABAergic pathway. The fact that flumazenil reduced seizure onset and attenuated the protection against seizure provided by Carissa edulis suggest that the extract may contain substance(s) that interacts with the benzodiazepine site in the GABAA -benzodiazepine receptor complex. The results of the study have demonstrated that Carissa edulis possessed anticonvulsant activity on the animal models investigated and this provides a rationale for its use in traditional medicine for the management of epilepsy. Further work to establish the active chemical constituent(s) of the extract and the exact mechanism of action is currently going on in our laboratory.
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References Achenbach, H., Weibal, R., Addae-Mensah, I., 1983. Lignan from Carissa edulis. Phytochemistry 76, 30837–38545 (in Biological Abstract). Banker, G.J., Verma, S.K., 1987. Preliminary studies of Flowering and Fruiting in Carissa edulis. Progressive Horticulture 19, 163–166. Bentley, M.D., Brackett, S.R., Chapya, A., 1984. 2-Hydroxyacetophenone, Principal Root Volatile of the East African Medicinal Plant, Carissa edulis. Journal of Natural Products 47, 1056–1057. Browning, R., 1992. The electroshock model, neuronal network and antiepileptic drugs. In: Faingold, C.L., Fromm, G.H. (Eds.), Drugs for Control of Epilepsy: Actions on Neuronal Networks in Seizure Disorders. CRC Press, Boca Raton, FL, pp. 195–211. Burkill, H.M., 1985. The Useful Plants of West Africa, 1., Second ed. Families A-D Royal Botanical Gardens, Kew, p145. Delgado, J.N., Remers, W.A. (Eds.), 1998. Wilson and Gisvold’s Textbook of Organic and Medicinal Pharmaceutical Chemistry. Lippincott-Raven, U.S.A.. Hutchison, J., Dalziel, J.M., 1963. Flora of West Tropical Africa. Vol. II. Crown Agents for Oversea Government and Administrations. Millbank, London, S.W.I. pp. 51–54. Ibrahim, H., 1997. Pharmacognostic and Biological (Analgesic Activity) Studies of Carissa edulis Vahl. Ph.D. Thesis. Ahmadu Bello University, Zaria, Nigeria, pp. 232. Irvine, F.R., 1961. Woody Plants of Ghana. Oxford University Press, London, p. 618. Lorke, D., 1983. A new approach to acute toxicity testing. Archives of Toxicology 54, 275–287. Loscher, W., Honack, D., Fassbender, C.P., 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. Macdonald, R.L., Kelly, K.M., 1994. Mechanisms of action of currently prescribed and newly developed antiepileptic drugs. Epilepsia 35, S41–S50.
McNamara, J.O., 2001. Drugs effective in the therapy of the epilepsies. In: Gilman, A.G., Rall, T.W., Nies, A.S., Taylor, P. (Eds.), The Pharmacological Basis of Therapeutics. Permagon Press, New York, U.S.A., p. 521. Meldrum, B.S., 1996. Update on the mechanism of action of antiepileptic drugs. Epilepsia 37, S4–S11. Nedi, T., Mekonnen, N., Urqa, K., 2004. Diuretic effect of the crude extract of Carissa edulis in rats. Journal of Ethnopharmacology 95, 57–61. Oliver, B., 1960. Medicinal Plants in Nigeria published as a Private Edition by the Nigerian College of Art, Science & Technology. pp. 52. Omino, E.A., Kokwaro, J.O., 1993. Ethnobotany of Apocynaceae specie in Kenya. Journal of Ethnopharmacology 4, 167–180. Rho, J.M., Sankar, R., 1999. The pharmacological basis of antiepileptic drug action. Epilepsia 40, 1471–1483. Sayyah, M., Saroukhari, G., Peirovi, A., Kamalinejad, M., 2002. Analgesic and anti-inflammatory activity of the leaf essential oil of Lauraus nobilis Linn. Phytotherapy Research 17, 733–736. Sofowora, A., 1986. The State of Medicinal Plant Research in Nigeria, First ed. University Press Ltd. Ife, Nigeria, p. 338. Swinyard, E.A., 1969. Laboratory evaluation of antiepileptic drugs: Review of laboratory methods. Epilepsia 10, 107–119. Swinyard, E.A., Kupferberg, H.J., 1985. Antiepileptic drugs: detection, quantification and evaluation. Federation Procedings 44, 3. Swinyard, E.A., Brown, W.C., Goodman, L.S., 1952. Comparative assays of antiepileptic drugs in mice and rats. Journal of Pharmacology and Experimental Therapy 106, 319–330. Swinyard, E.A., Woodhead, J.H., White, H.S., franklin, M.R., 1989. General principles: experimental selection, quantification and evaluation of anticonvulsants. In: Levy, R., Mattson, R., Meldium, B., Penry, J.K., Dreifuss, F.E. (Eds.), Antiepileptic Drugs. Raven Press, New York, pp. 233–239. Wickenden, A.D., 2002. Potassium channels as antiepileptic drug targets. Neuropharmacology 43, 1055–1060.