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Anticonvulsant effects of ethanol stem bark extract of Lannea barteri (Anacardiaceae) in mice and chicks
Journal of Ethnopharmacology 172 (2015) 227–231
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Anticonvulsant effects of ethanol stem bark extract of Lannea barteri (Anacardiaceae) in mice and chicks K. Garba a,n, A.H. Yaro a, J. Ya’u b a b
Department of Pharmacology, Bayero University, Kano, Nigeria Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria, Nigeria
art ic l e i nf o
a b s t r a c t
Article history: Received 12 May 2015 Received in revised form 17 June 2015 Accepted 19 June 2015 Available online 27 June 2015
Ethnopharmacological relevance: Preparation of Lannea barteri is used in the treatment of epilepsy, gastritis, childhood convulsions among other uses in northern Nigeria for many years. The popularity of its efficacy is well established among the Traditional Medical Practitioners. Aim of the study: The present study aimed at screening the ethanol stem bark extract of Lannea barteri for possible anticonvulsant action. Materials and method: Anticonvulsant screening was carried out using pentylenetetrazole (PTZ), strychnine (STN) and picrotoxin (PTC) induced seizures in mice while Maximal electroshock (MES) test was carried out in day old chicks. Preliminary phytochemical screening of the extract was performed on the extract. The intraperitoneal median lethal dose (LD50) was carried out in mice. Results: The intraperitoneal (i.p.) LD50 of the extract was estimated to be 567.70 mg/kg in mice. Lannea barteri (160 mg/kg) significantly (pr0.05) delayed the mean onset of seizures induced by PTZ when compared with normal saline treated group. Similarly, the extract at 160 mg/kg significantly (pr 0.05) prolonged the latency of convulsion induced by STN. Lannea barteri (40 mg/kg) significantly (pr 0.05) delayed the mean onset of seizures induced by picrotoxin in mice. The extracts at all the doses tested showed no observable effect in decreasing the mean recovery time of convulsed chicks in MEST. Flavonoids, alkaloids, tannins, saponins and glycosides were found present in the stem bark extract. Conclusion: Our findings revealed that the ethanol stem bark extract of Lannea barteri contained bioactive constituents that may be useful in the management of petit mal epilepsy and supports the ethnomedical claim for the use of its stem bark in the management of epilepsy. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Lannea barteri Epilepsy Maximal electroshock Pentylenetetrazole Strychnine Picrotoxin
1. Introduction Epilepsy ranked second after stroke, as the most common neurological disorder with severe morbidity (Ziyaurrahman and Patel, 2012). It is characterized by intermittent occurrence and recurrence of synchronous and often unpredictable discharges in the brain. The International League Against Epilepsy (ILAE) defines it as having two or more seizures occurring in more than one day, probability of having a similar attack within the span of ten years accompanied by epilepsy syndrome (Fisher et al., 2014). It is estimated that 50 million people are suffering from this neurological disorder globally (WHO, 1998). The prevalence in Nigeria is about 6.2 per 1000 (Banerjee et al., 2009). Several classes of drugs are used to treat epilepsy, and even with the introduction of highly efficacious Antiepileptic drugs (AED), control is still minimal (Picot n
Corresponding author. GSM: þ 234 803 297 0383. E-mail address: [email protected] (K. Garba).
http://dx.doi.org/10.1016/j.jep.2015.06.039 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
et al., 2008). It is against this background, that World Health Organization (WHO), welcomed alternative medical practice in the treatment of most ailments, since majority of people in developing countries depend on medicinal plants for their primary health care needs (WHO/PRO, 1998). Natural products have remain the cornerstone in drug discovery and will still maintain an important role in this area in the future (Newman and Cragg, 2012). Similarly, traditional medical practice is widely accepted in most communities in northern Nigeria. This stems from the fact that medicinal plants are cheap, easily accessible and presumed to cure all sorts of ailments (Muhammad and Awaisu, 2008). It is estimated that 11.9% of epileptic patients are on one form of alternative treatment before visiting the hospital in North Western Nigeria (Owolabi and Sale, 2011). This then makes research in medicinal plants with traditional evidence against this disorder worthwhile, to ascertain its efficacy or otherwise, among this community that hold unorthodox medical practice in high esteem. Animal models of seizures have been used for almost six
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K. Garba et al. / Journal of Ethnopharmacology 172 (2015) 227–231
decades in the screening of potential antiseizure drugs (Loscher, 2011). In addition, to their use in identifying new AED's, the animal models establish efficacy of the tested compound against different seizure models once anticonvulsant activity is detected. The pattern of epileptic seizures induced in animals correlates well with that of humans. Furthermore, animal models of epilepsy are easy to perform, saves time and are predictive of clinical usefulness in humans (Loscher, 2011). The primary gateways of anticonvulsant screening are the MES in chicks and the PTZ in mice. The MES and PTZ induced seizures produce epileptic seizures similar to Generalized tonic clonic and absence seizures in human respectively, while picrotoxin and strychnine induced seizures both in mice gives insight into the probable mechanism in which these agents act (Magaji and Abdurahman, 2012; Jamilu et al., 2007). These four models are adapted and used in this study Lannea barteri (Family: Anacardiaceae) is a common medicinal plant in Northern Nigeria that is used to treat variety of ailments. The bark is found useful in: Gastritis, epilepsy, childhood convulsions and inflammation (Burkill, 1985). Studies on the leaves of this plant revealed the presence of tannins, flavonoids and steroids with considerable antiviral activity (Njinga et al., 2014). Similarly, the leaves had shown broad spectrum activity against Gram þve and Gram –ve isolates (Adegoke et al., 2013). Despite its widespread usage and acceptability, no scientific evaluation of this plant was done for epilepsy, and this form the basis of this study.
2. Materials and methods 2.1. Plant material The plant material was collected in June, 2014 in Zaria, Nigeria. The plant was identified and authenticated by Taxonomist in the Herbarium Section of the Department of Biological Sciences, Ahmadu Bello University, Zaria, by comparing with a voucher number 1888. 2.2. Preparation of extract The stem bark of Lannea barteri was cleaned and air-dried at room temperature. The plant material was ground into a fine powder using pestle and mortar. Extraction was carried using cold maceration, where 612 g of the powder was soaked in 4 L of 70% v/ v ethanol for 72 h; it was occasionally stirred with a glass stirrer throughout this 72 h period. The resultant mixture was filtered using Whatman filter paper (No. 1) and the filtrate was concentrated to dryness in vacuo at 40 °C using rotary evaporator. 2.3. Animals Swiss albino mice (18–32 g) of either sex were obtained from the Animal Facility Centre (AFC) of Department of Pharmacology, Bayero, University, Kano, Nigeria, were used for this experiments. Day old cockerels were obtained from National Animal Production Research Institute (NAPRI), Zaria, Nigeria. Animals were maintained in well ventilated rooms. They were fed with standard feeds and water provided ad libitum. We certify that all experiments were carried out in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals {NIH Publications No. 80-23} revised in 1996. All efforts were made to minimize the number of mice and cockerels used and their suffering. 2.4. Phytochemical screening The phytochemical screening was performed on the dried
extract of Lannea barteri using standard procedure (Trease and Evans, 1997). 2.5. Acute toxicity testing The intraperitoneal LD50 was determined in mice. The study was carried out in two phases. In the first phase, nine mice of either sex were randomly divided into three groups of three mice each and were administered 10, 100 and 1000 mg/kg body weight of the extract intraperitonially. Mice were observed for signs and symptoms of toxicity, including death over a period of 24 h. In the second phase of the study, 200, 400, 800 and 1600 mg/kg of the extracts were given to four different group of one mouse each intraperitonially (i.p.) based on the result of the first phase. The LD50 was estimated by calculating the geometric mean of the lowest dose that caused death (1/1) and the highest dose that animal survived (0/1) (Lorke, 1983). 2.6. Anticonvulsant studies 2.6.1. Pentylenetetrazole induced seizures in mice Animals were divided into five groups of six mice each. Group 1 received normal saline (10 ml/kg), groups 2–4 received the extract at the doses of 40, 80 and 160 mg/kg of the extracts respectively, while group 5 received standard drug sodium valproate at a dose of 200 mg/kg intraperitonially. Thirty minutes later, all groups received 100 mg/kg pentylenetetrazole (PTZ) subcutaneously (s.c) (CD100). Animals were observed for the presence or absence of threshold seizures (an episode of clonic spasm of at least 5 s duration), mean onset of convulsion, quantal protection and percent protection were recorded (Swinyard et al., 1989). 2.6.2. Maximal electrical shock test in chicks The apparatus used was Ugo Basile Electroconvulsive Machine (Model 7801) with corneal electrodes placed on the upper eyelid of the chicks after dipping them in normal saline. The current, shock duration, pulse width and frequency were set and maintained at 80 mA, 0.6 s, 0.6 ms and 100 pulses per second respectively. Fifty day old cockerels were grouped into five groups of ten chicks each. Group 1 was pretreated with normal saline (10 ml/kg i.p.), groups 2–4 were administered with extract at doses of 40, 80, 160 mg/kg body weight intraperitonially, while group 5 was treated with phenytoin sodium 20 mg/kg body weight intraperitonielly. Thirty minutes post treatment, electroshock was administered to each animal to induce convulsion. Results were recorded as either positive or negative depending on whether tonic hind limb extension (THLE) was produced. The time of recovery of convulsed chicks were recorded and the percentage of convulsed animals calculated (Swinyard and Kupferberg, 1985). 2.6.3. Strychnine-induced seizures in mice Mice were grouped into 5 groups of 6 mice each. Group 1 received 10 ml/kg of normal saline, groups 2–4 were administered with the extract at doses of 40, 80 and 160 mg/kg of the extract respectively while group 5 received phenobarbitone at a dose of 30 mg/kg body weight intraperitonially. Thirty minutes later, mice were administered 1.2 mg/kg body weight of strychnine subcutaneously and observed for incidence of convulsions. Prevention of tonic hind limb extensor jerk was considered as protection against seizures induced by strychnine (Lehmann et al., 1988). 2.6.4. Picrotoxin induced seizures in mice Thirty mice were grouped into five each consisting of six mice. The mice in first grouped received normal saline (10 ml/kg), the second, third and fourth groups were injected with the doses of the extract 40, 80 and 160 mg/kg body weight respectively. The
K. Garba et al. / Journal of Ethnopharmacology 172 (2015) 227–231
fifth group received phenobarbitone 30 mg/kg, all via intraperitoneal route. Thirty minutes later, mice in all the groups were given picrotoxin subcutaneously 5 mg/kg. Animals were then observed for hind limb tonic extension over 30 min period. Absence of tonic hind limb extension or prolongation of the latency of the hind limb tonic extension was considered as an indication for anticonvulsant activity (Salih and Mustafa, 2008). 2.7. Mouse beam walking assay Mice were trained to walk from a start platform along a ruler (80 cm long, 3 cm wide) elevated 30 cm above the bench by metal sports to a goal box (enclosed hamster house). Three trials were performed for each mouse, and were designed such that the mice tested would be aware that there was a goal box that could be reached. Mice that failed the test were excluded from the experiment. A ruler was used because the mouse found this easy to cross, and at the same time, it induced minimum anxiety. Once the mice had been tested on the ruler, they moved immediately to the beam test. The beam was made of wood, 8 mm in diameter, 60 cm long and elevated 30 cm above the bench by metal supports. The mice were individually placed on the beam at the end and allowed to walk to the goal box. Mice that fell were returned to the position they fell from, with a maximum time of 60 s allowed on the beam. The measurements taken were time spent on the beam, the number of foot slips (one or both hind limbs slipped from the beam) and the number of falls (Stanley et al., 2005). Three groups of 6 mice each were used. The test was carried out 30 min after intraperitoneal treatment with stem extract at the dose that provided anticonvulsant effect (LB 160 mg/kg), normal saline (10 ml/kg) and diazepam (0.25 mg/kg) were used as negative and positive control respectively.
229
Table 1 The phytochemical constituents of ethanol stem bark extract of Lannea barteri. Group Flavonoids a. Sulfuric acid test
Lannea barteri
a. Lead acetate test
a. Shinoda test
þ
Tannins a. General test
þ
a. Ferric chloride test
þ
a. Phlobatannins
Saponins a. Frothing test
þ
Alkaloids a. Dragendorff's test
þ
a. Mayer's test
þ
a. Wagner's test
þ
Glycosides a. Salkowski's test
þ
a. Keller–Kelliani's test
Steroids/terpenoids a. Lieberman
a. Burchard test
2.8. Statistical analysis Anthraquinones
Results were expressed as Mean 7Standard Error of Mean (SEM). Statistical analysis was done by analysis of variance (ANOVA), a Dunnet's post hoc test was done, when statistically significant result was obtained with ANOVA. Values of p r0.05 were considered significant.
3. Results 3.1. Acute toxicity test The intraperitoneal LD50 were found to be 565.7 mg/kg. There is decreased physical and locomotive activity and respiratory depression before death. 3.2. Phytochemical studies Preliminary phytochemical studies revealed the presence of flavonoids, tannins, saponins, alkaloids and glycosides in the ethanolic stem bark extracts of Lannea barteri (Table 1). 3.3. Anticonvulsant studies The ethanol stem bark extracts of Lannea barteri significantly (p r0.05) delayed the mean onset of convulsions at the highest dose (160 mg/kg) against pentylenetetrazole induced seizures in mice when compared with the control group. Similarly, Sodium valproate (200 mg/kg) significantly (p r0.05) delayed the mean onset of seizures induced by PTZ. In the same vein, the extract protected 33% of the mice at a dose of 40 mg/kg (Table 2).
Key: þ present, absent
The extracts at all the doses tested did not delay the mean recovery of convulsed animals when compared with normal saline treated groups in Maximal Electroshock (MES) test. However, phenytoin (20 mg/kg) a standard drug completely abolished the seizures induced by MES (Table 3). The extracts of Lannea barteri significantly (p r0.05) delayed the mean onset of seizures at the highest dose (160 mg/kg). In the same manner, phenobarbitone (30 mg/kg) a standard drug protected all the mice against strychnine induced seizures during the 30 min observation period (Table 4). The stem bark extract significantly (pr 0.01) delayed the mean onset of seizures against picrotoxin at the lowest dose (40 mg/kg), this protection is the same as does the standard drug phenobarbitone (30 mg/kg), and similarly, it protected 50% of the animal at that dose (40 mg/kg) (Table 5). 3.4. Mouse beam walking assay The extract at the dose tested (160 mg/kg) did not produce foot slip, similar to the normal saline treated group. However, diazepam (0.25 mg/kg) a positive drug had effect on the foot slips. The extract (LB 160 mg/kg) had no effect on the time spent to reach the goal box (Table 6). 4. Discussions The findings of this study suggest that the ethanol stem bark
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K. Garba et al. / Journal of Ethnopharmacology 172 (2015) 227–231
Table 2 Effect of ethanol stem bark extracts of Lannea barteri against PTZ induced seizures in mice. Treatment
N/Saline LB 40 LB 80 LB 160 Sodium valproate
Dose (mg/ kg)
Onset of seizure (min)
Quantal protection
Mean no. of clonic spasm
Time to death (min)
10 ml/ kg 40 80 160 200
2.667 0.33
0/6
2.83 7 0.16
9.677 2.40
3.50 7 0.76 4.3370.42 5.667 0.66n 5.3370.42n
2/6 0/6 1/6 3/6
1.337 0.49 1.667 0.33 1.007 0.25 0.50 7 0.22
14.007 2.08 10.50 7 3.69 18.75 71.44 15.50 7 3.50
pr 0.05 one way ANOVA followed by Dunnette post hoc test.
Table 3 Effect of ethanol stem bark extract of Lannea barteri against MEST induced seizures in chick. Treatment Dose (mg/ kg)
Mean recovery time quantal protection (Min)
% Protection
N/Saline LB 40 LB 80 LB 160 Phenytoin
5.447 0.89 0/10 7.45 7 0.55 0/10 7.667 1.43 0/10 7.40 7 1.06 0/10 10/10
0.00 0.00 0.00 0.00 100.00
10 ml/kg 40 80 160 40
Data presented as Mean 7 SEM,% protection, n ¼10, LB ¼Lannea barteri, one way ANOVA, Table 4 Effect of ethanol stem bark extract of Lannea barteri in strychnine induced seizures in mice. Treatment
Dose
Mean Onset of Seizures (Min)
Quantal protection
N/saline LB 40 LB 80 LB 160 Phenobarbitone
10 ml/kg 40 80 160 30
3.667 0.21 3.83 70.54 5.167 0.40 5.50 70.56n –
0/6 0/6 0/6 0/6 6/6
Data expressed as Mean 7 SEM,% protection, n¼ 10, LB ¼Lannea barteri, n
Treatment
Dose (mg/kg)
No of foot slips
N/saline LB 160 Diazepam
10 (ml/kg) 160 0.25
– – 2.40 7 0.74
Time spent (sec) 6.337 1.66 10.83 72.30 10.667 1.80
Data expressed as Mean 7 SEM, n¼ 6, LB ¼ Lannea barteri, one way ANOVA
Data presented as Mean 7 SEM, % protection, n¼ 6, LB ¼ Lannea barteri, n
Table 6 Effect of ethanol stem bark extract of Lannea barteri on beam walk assay in mice.
pr 0.05, one way ANOVA followed by Dunnette post hoc test
Table 5 Effect of ethanol stem bark extract of Lannea barteri on picrotoxin induced seizures in mice. Treatment
Dose
Onset of seizures (Mean7 SEM)
Quantal protection
N/saline LB 40 LB 80 LB 160 Phenobarbitone
10 ml/kg 40 mg/kg 80 mg/kg 160 mg/kg 30 mg/kg
10.337 1.23 24.007 0.00n 14.337 0.99 14.007 1.65 29.00 70.00n
0/6 3/6 0/6 0/6 4/6
threshold in the brain (White, 1995). Consequently, they are effective in the management of petit mal and myoclonic seizures. AEDs such as valproic acid, phenobarbitone, ethosoxumide and benzodiazepines suppressed chemically induced seizures by scPTZ and are effective in the treatment of generalized seizures of petit mal or myoclonic (Jamilu et al., 2007). In a broader sense, PTZ induced seizures are abolished by agents that act by reducing T-type Ca þ þ currents or enhance GABAA receptor inhibitory neurotransmission such as ethosuximide and benzodiazepines respectively (Vogel, 2008). MES is a model that screen for molecules with potential effect against generalized seizures (Tripathi, 2006). Some of the drugs effective against this seizure model act by inhibiting sodium channel or block Aspartate receptors and they prevent the spread of seizures (Malawska, 2005; Magaji et al., 2013). Phenytoin, carbamazepine, lamotrigine, and felbamate are AEDs that act by blocking sodium channels, thereby stabilizing inactive state of sodium channels, thus, reducing high frequency firing of action potentials (Nestler et al., 2009). However, the extract afforded no effect against THLE induced by MES, this therefore suggested that it mediates its action via other mechanism but not through blocking sodium currents or aspartate receptors. Strychnine is a competitive antagonist of glycine an inhibitory neurotransmitter in the central nervous system (Biggio et al., 1992). Drugs that abolished chemically induced seizures by strychnine mediate their activity by enhancing inhibitory neurotransmission in the brain and the spinal cord. Probable mechanism of antiepileptic effect of this extract is by interacting with glycinergic receptors and enhancing its effect. The activity of the extract against picrotoxin induced seizures; further lend credence to its action via GABAergic pathway. Picrotoxin is a non-competitive antagonist of GABAA receptors. Drugs such as benzodiazepines, barbiturates, and sodium valproate produced their anticonvulsant effect via enhancing the GABAA interaction with picrotoxin sites in GABAA–chloride channel complex (McNamara, 2006). In conclusion, this study revealed that that the ethanol stem bark extract of Lannea barteri contained bioactive constituents that may be useful in the treatment of epilepsy or any condition of the same symptomatology and justify the use of this plant part in the traditional medicine for the treatment of epilepsy. Further studies on this plant part will involve isolation and identification of the bioactive constituent responsible for this observed anticonvulsant activity.
Data expressed as Mean 7 SEM, one way ANOVA, n¼ 6, LB ¼Lannea barteri one way ANOVA followed by Dunnette post hoc test n
pr 0.001
extract of Lannea barteri possess anticonvulsant effect. The LD50 of 567.1 mg/kg suggest that it is slightly toxic (Matsumura, 1985). Even though the doses employed in this study were lower than 30% of the LD50 values which have shown to be safe for ethnopharmacological assays. The ethanol stem bark extract protected the animals against scPTZ induced seizures. Agents that blocked scPTZ induced seizures in rodents are used to screen compounds that raises seizure
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2015.06.039.
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Anticonvulsant effects of ethanol stem bark extract of Lannea barteri (Anacardiaceae) in mice and chicks K. Garba a,n, A.H. Yaro a, J. Ya’u b a b
Department of Pharmacology, Bayero University, Kano, Nigeria Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria, Nigeria
art ic l e i nf o
a b s t r a c t
Article history: Received 12 May 2015 Received in revised form 17 June 2015 Accepted 19 June 2015 Available online 27 June 2015
Ethnopharmacological relevance: Preparation of Lannea barteri is used in the treatment of epilepsy, gastritis, childhood convulsions among other uses in northern Nigeria for many years. The popularity of its efficacy is well established among the Traditional Medical Practitioners. Aim of the study: The present study aimed at screening the ethanol stem bark extract of Lannea barteri for possible anticonvulsant action. Materials and method: Anticonvulsant screening was carried out using pentylenetetrazole (PTZ), strychnine (STN) and picrotoxin (PTC) induced seizures in mice while Maximal electroshock (MES) test was carried out in day old chicks. Preliminary phytochemical screening of the extract was performed on the extract. The intraperitoneal median lethal dose (LD50) was carried out in mice. Results: The intraperitoneal (i.p.) LD50 of the extract was estimated to be 567.70 mg/kg in mice. Lannea barteri (160 mg/kg) significantly (pr0.05) delayed the mean onset of seizures induced by PTZ when compared with normal saline treated group. Similarly, the extract at 160 mg/kg significantly (pr 0.05) prolonged the latency of convulsion induced by STN. Lannea barteri (40 mg/kg) significantly (pr 0.05) delayed the mean onset of seizures induced by picrotoxin in mice. The extracts at all the doses tested showed no observable effect in decreasing the mean recovery time of convulsed chicks in MEST. Flavonoids, alkaloids, tannins, saponins and glycosides were found present in the stem bark extract. Conclusion: Our findings revealed that the ethanol stem bark extract of Lannea barteri contained bioactive constituents that may be useful in the management of petit mal epilepsy and supports the ethnomedical claim for the use of its stem bark in the management of epilepsy. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Lannea barteri Epilepsy Maximal electroshock Pentylenetetrazole Strychnine Picrotoxin
1. Introduction Epilepsy ranked second after stroke, as the most common neurological disorder with severe morbidity (Ziyaurrahman and Patel, 2012). It is characterized by intermittent occurrence and recurrence of synchronous and often unpredictable discharges in the brain. The International League Against Epilepsy (ILAE) defines it as having two or more seizures occurring in more than one day, probability of having a similar attack within the span of ten years accompanied by epilepsy syndrome (Fisher et al., 2014). It is estimated that 50 million people are suffering from this neurological disorder globally (WHO, 1998). The prevalence in Nigeria is about 6.2 per 1000 (Banerjee et al., 2009). Several classes of drugs are used to treat epilepsy, and even with the introduction of highly efficacious Antiepileptic drugs (AED), control is still minimal (Picot n
Corresponding author. GSM: þ 234 803 297 0383. E-mail address: [email protected] (K. Garba).
http://dx.doi.org/10.1016/j.jep.2015.06.039 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
et al., 2008). It is against this background, that World Health Organization (WHO), welcomed alternative medical practice in the treatment of most ailments, since majority of people in developing countries depend on medicinal plants for their primary health care needs (WHO/PRO, 1998). Natural products have remain the cornerstone in drug discovery and will still maintain an important role in this area in the future (Newman and Cragg, 2012). Similarly, traditional medical practice is widely accepted in most communities in northern Nigeria. This stems from the fact that medicinal plants are cheap, easily accessible and presumed to cure all sorts of ailments (Muhammad and Awaisu, 2008). It is estimated that 11.9% of epileptic patients are on one form of alternative treatment before visiting the hospital in North Western Nigeria (Owolabi and Sale, 2011). This then makes research in medicinal plants with traditional evidence against this disorder worthwhile, to ascertain its efficacy or otherwise, among this community that hold unorthodox medical practice in high esteem. Animal models of seizures have been used for almost six
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decades in the screening of potential antiseizure drugs (Loscher, 2011). In addition, to their use in identifying new AED's, the animal models establish efficacy of the tested compound against different seizure models once anticonvulsant activity is detected. The pattern of epileptic seizures induced in animals correlates well with that of humans. Furthermore, animal models of epilepsy are easy to perform, saves time and are predictive of clinical usefulness in humans (Loscher, 2011). The primary gateways of anticonvulsant screening are the MES in chicks and the PTZ in mice. The MES and PTZ induced seizures produce epileptic seizures similar to Generalized tonic clonic and absence seizures in human respectively, while picrotoxin and strychnine induced seizures both in mice gives insight into the probable mechanism in which these agents act (Magaji and Abdurahman, 2012; Jamilu et al., 2007). These four models are adapted and used in this study Lannea barteri (Family: Anacardiaceae) is a common medicinal plant in Northern Nigeria that is used to treat variety of ailments. The bark is found useful in: Gastritis, epilepsy, childhood convulsions and inflammation (Burkill, 1985). Studies on the leaves of this plant revealed the presence of tannins, flavonoids and steroids with considerable antiviral activity (Njinga et al., 2014). Similarly, the leaves had shown broad spectrum activity against Gram þve and Gram –ve isolates (Adegoke et al., 2013). Despite its widespread usage and acceptability, no scientific evaluation of this plant was done for epilepsy, and this form the basis of this study.
2. Materials and methods 2.1. Plant material The plant material was collected in June, 2014 in Zaria, Nigeria. The plant was identified and authenticated by Taxonomist in the Herbarium Section of the Department of Biological Sciences, Ahmadu Bello University, Zaria, by comparing with a voucher number 1888. 2.2. Preparation of extract The stem bark of Lannea barteri was cleaned and air-dried at room temperature. The plant material was ground into a fine powder using pestle and mortar. Extraction was carried using cold maceration, where 612 g of the powder was soaked in 4 L of 70% v/ v ethanol for 72 h; it was occasionally stirred with a glass stirrer throughout this 72 h period. The resultant mixture was filtered using Whatman filter paper (No. 1) and the filtrate was concentrated to dryness in vacuo at 40 °C using rotary evaporator. 2.3. Animals Swiss albino mice (18–32 g) of either sex were obtained from the Animal Facility Centre (AFC) of Department of Pharmacology, Bayero, University, Kano, Nigeria, were used for this experiments. Day old cockerels were obtained from National Animal Production Research Institute (NAPRI), Zaria, Nigeria. Animals were maintained in well ventilated rooms. They were fed with standard feeds and water provided ad libitum. We certify that all experiments were carried out in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals {NIH Publications No. 80-23} revised in 1996. All efforts were made to minimize the number of mice and cockerels used and their suffering. 2.4. Phytochemical screening The phytochemical screening was performed on the dried
extract of Lannea barteri using standard procedure (Trease and Evans, 1997). 2.5. Acute toxicity testing The intraperitoneal LD50 was determined in mice. The study was carried out in two phases. In the first phase, nine mice of either sex were randomly divided into three groups of three mice each and were administered 10, 100 and 1000 mg/kg body weight of the extract intraperitonially. Mice were observed for signs and symptoms of toxicity, including death over a period of 24 h. In the second phase of the study, 200, 400, 800 and 1600 mg/kg of the extracts were given to four different group of one mouse each intraperitonially (i.p.) based on the result of the first phase. The LD50 was estimated by calculating the geometric mean of the lowest dose that caused death (1/1) and the highest dose that animal survived (0/1) (Lorke, 1983). 2.6. Anticonvulsant studies 2.6.1. Pentylenetetrazole induced seizures in mice Animals were divided into five groups of six mice each. Group 1 received normal saline (10 ml/kg), groups 2–4 received the extract at the doses of 40, 80 and 160 mg/kg of the extracts respectively, while group 5 received standard drug sodium valproate at a dose of 200 mg/kg intraperitonially. Thirty minutes later, all groups received 100 mg/kg pentylenetetrazole (PTZ) subcutaneously (s.c) (CD100). Animals were observed for the presence or absence of threshold seizures (an episode of clonic spasm of at least 5 s duration), mean onset of convulsion, quantal protection and percent protection were recorded (Swinyard et al., 1989). 2.6.2. Maximal electrical shock test in chicks The apparatus used was Ugo Basile Electroconvulsive Machine (Model 7801) with corneal electrodes placed on the upper eyelid of the chicks after dipping them in normal saline. The current, shock duration, pulse width and frequency were set and maintained at 80 mA, 0.6 s, 0.6 ms and 100 pulses per second respectively. Fifty day old cockerels were grouped into five groups of ten chicks each. Group 1 was pretreated with normal saline (10 ml/kg i.p.), groups 2–4 were administered with extract at doses of 40, 80, 160 mg/kg body weight intraperitonially, while group 5 was treated with phenytoin sodium 20 mg/kg body weight intraperitonielly. Thirty minutes post treatment, electroshock was administered to each animal to induce convulsion. Results were recorded as either positive or negative depending on whether tonic hind limb extension (THLE) was produced. The time of recovery of convulsed chicks were recorded and the percentage of convulsed animals calculated (Swinyard and Kupferberg, 1985). 2.6.3. Strychnine-induced seizures in mice Mice were grouped into 5 groups of 6 mice each. Group 1 received 10 ml/kg of normal saline, groups 2–4 were administered with the extract at doses of 40, 80 and 160 mg/kg of the extract respectively while group 5 received phenobarbitone at a dose of 30 mg/kg body weight intraperitonially. Thirty minutes later, mice were administered 1.2 mg/kg body weight of strychnine subcutaneously and observed for incidence of convulsions. Prevention of tonic hind limb extensor jerk was considered as protection against seizures induced by strychnine (Lehmann et al., 1988). 2.6.4. Picrotoxin induced seizures in mice Thirty mice were grouped into five each consisting of six mice. The mice in first grouped received normal saline (10 ml/kg), the second, third and fourth groups were injected with the doses of the extract 40, 80 and 160 mg/kg body weight respectively. The
K. Garba et al. / Journal of Ethnopharmacology 172 (2015) 227–231
fifth group received phenobarbitone 30 mg/kg, all via intraperitoneal route. Thirty minutes later, mice in all the groups were given picrotoxin subcutaneously 5 mg/kg. Animals were then observed for hind limb tonic extension over 30 min period. Absence of tonic hind limb extension or prolongation of the latency of the hind limb tonic extension was considered as an indication for anticonvulsant activity (Salih and Mustafa, 2008). 2.7. Mouse beam walking assay Mice were trained to walk from a start platform along a ruler (80 cm long, 3 cm wide) elevated 30 cm above the bench by metal sports to a goal box (enclosed hamster house). Three trials were performed for each mouse, and were designed such that the mice tested would be aware that there was a goal box that could be reached. Mice that failed the test were excluded from the experiment. A ruler was used because the mouse found this easy to cross, and at the same time, it induced minimum anxiety. Once the mice had been tested on the ruler, they moved immediately to the beam test. The beam was made of wood, 8 mm in diameter, 60 cm long and elevated 30 cm above the bench by metal supports. The mice were individually placed on the beam at the end and allowed to walk to the goal box. Mice that fell were returned to the position they fell from, with a maximum time of 60 s allowed on the beam. The measurements taken were time spent on the beam, the number of foot slips (one or both hind limbs slipped from the beam) and the number of falls (Stanley et al., 2005). Three groups of 6 mice each were used. The test was carried out 30 min after intraperitoneal treatment with stem extract at the dose that provided anticonvulsant effect (LB 160 mg/kg), normal saline (10 ml/kg) and diazepam (0.25 mg/kg) were used as negative and positive control respectively.
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Table 1 The phytochemical constituents of ethanol stem bark extract of Lannea barteri. Group Flavonoids a. Sulfuric acid test
Lannea barteri
a. Lead acetate test
a. Shinoda test
þ
Tannins a. General test
þ
a. Ferric chloride test
þ
a. Phlobatannins
Saponins a. Frothing test
þ
Alkaloids a. Dragendorff's test
þ
a. Mayer's test
þ
a. Wagner's test
þ
Glycosides a. Salkowski's test
þ
a. Keller–Kelliani's test
Steroids/terpenoids a. Lieberman
a. Burchard test
2.8. Statistical analysis Anthraquinones
Results were expressed as Mean 7Standard Error of Mean (SEM). Statistical analysis was done by analysis of variance (ANOVA), a Dunnet's post hoc test was done, when statistically significant result was obtained with ANOVA. Values of p r0.05 were considered significant.
3. Results 3.1. Acute toxicity test The intraperitoneal LD50 were found to be 565.7 mg/kg. There is decreased physical and locomotive activity and respiratory depression before death. 3.2. Phytochemical studies Preliminary phytochemical studies revealed the presence of flavonoids, tannins, saponins, alkaloids and glycosides in the ethanolic stem bark extracts of Lannea barteri (Table 1). 3.3. Anticonvulsant studies The ethanol stem bark extracts of Lannea barteri significantly (p r0.05) delayed the mean onset of convulsions at the highest dose (160 mg/kg) against pentylenetetrazole induced seizures in mice when compared with the control group. Similarly, Sodium valproate (200 mg/kg) significantly (p r0.05) delayed the mean onset of seizures induced by PTZ. In the same vein, the extract protected 33% of the mice at a dose of 40 mg/kg (Table 2).
Key: þ present, absent
The extracts at all the doses tested did not delay the mean recovery of convulsed animals when compared with normal saline treated groups in Maximal Electroshock (MES) test. However, phenytoin (20 mg/kg) a standard drug completely abolished the seizures induced by MES (Table 3). The extracts of Lannea barteri significantly (p r0.05) delayed the mean onset of seizures at the highest dose (160 mg/kg). In the same manner, phenobarbitone (30 mg/kg) a standard drug protected all the mice against strychnine induced seizures during the 30 min observation period (Table 4). The stem bark extract significantly (pr 0.01) delayed the mean onset of seizures against picrotoxin at the lowest dose (40 mg/kg), this protection is the same as does the standard drug phenobarbitone (30 mg/kg), and similarly, it protected 50% of the animal at that dose (40 mg/kg) (Table 5). 3.4. Mouse beam walking assay The extract at the dose tested (160 mg/kg) did not produce foot slip, similar to the normal saline treated group. However, diazepam (0.25 mg/kg) a positive drug had effect on the foot slips. The extract (LB 160 mg/kg) had no effect on the time spent to reach the goal box (Table 6). 4. Discussions The findings of this study suggest that the ethanol stem bark
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Table 2 Effect of ethanol stem bark extracts of Lannea barteri against PTZ induced seizures in mice. Treatment
N/Saline LB 40 LB 80 LB 160 Sodium valproate
Dose (mg/ kg)
Onset of seizure (min)
Quantal protection
Mean no. of clonic spasm
Time to death (min)
10 ml/ kg 40 80 160 200
2.667 0.33
0/6
2.83 7 0.16
9.677 2.40
3.50 7 0.76 4.3370.42 5.667 0.66n 5.3370.42n
2/6 0/6 1/6 3/6
1.337 0.49 1.667 0.33 1.007 0.25 0.50 7 0.22
14.007 2.08 10.50 7 3.69 18.75 71.44 15.50 7 3.50
pr 0.05 one way ANOVA followed by Dunnette post hoc test.
Table 3 Effect of ethanol stem bark extract of Lannea barteri against MEST induced seizures in chick. Treatment Dose (mg/ kg)
Mean recovery time quantal protection (Min)
% Protection
N/Saline LB 40 LB 80 LB 160 Phenytoin
5.447 0.89 0/10 7.45 7 0.55 0/10 7.667 1.43 0/10 7.40 7 1.06 0/10 10/10
0.00 0.00 0.00 0.00 100.00
10 ml/kg 40 80 160 40
Data presented as Mean 7 SEM,% protection, n ¼10, LB ¼Lannea barteri, one way ANOVA, Table 4 Effect of ethanol stem bark extract of Lannea barteri in strychnine induced seizures in mice. Treatment
Dose
Mean Onset of Seizures (Min)
Quantal protection
N/saline LB 40 LB 80 LB 160 Phenobarbitone
10 ml/kg 40 80 160 30
3.667 0.21 3.83 70.54 5.167 0.40 5.50 70.56n –
0/6 0/6 0/6 0/6 6/6
Data expressed as Mean 7 SEM,% protection, n¼ 10, LB ¼Lannea barteri, n
Treatment
Dose (mg/kg)
No of foot slips
N/saline LB 160 Diazepam
10 (ml/kg) 160 0.25
– – 2.40 7 0.74
Time spent (sec) 6.337 1.66 10.83 72.30 10.667 1.80
Data expressed as Mean 7 SEM, n¼ 6, LB ¼ Lannea barteri, one way ANOVA
Data presented as Mean 7 SEM, % protection, n¼ 6, LB ¼ Lannea barteri, n
Table 6 Effect of ethanol stem bark extract of Lannea barteri on beam walk assay in mice.
pr 0.05, one way ANOVA followed by Dunnette post hoc test
Table 5 Effect of ethanol stem bark extract of Lannea barteri on picrotoxin induced seizures in mice. Treatment
Dose
Onset of seizures (Mean7 SEM)
Quantal protection
N/saline LB 40 LB 80 LB 160 Phenobarbitone
10 ml/kg 40 mg/kg 80 mg/kg 160 mg/kg 30 mg/kg
10.337 1.23 24.007 0.00n 14.337 0.99 14.007 1.65 29.00 70.00n
0/6 3/6 0/6 0/6 4/6
threshold in the brain (White, 1995). Consequently, they are effective in the management of petit mal and myoclonic seizures. AEDs such as valproic acid, phenobarbitone, ethosoxumide and benzodiazepines suppressed chemically induced seizures by scPTZ and are effective in the treatment of generalized seizures of petit mal or myoclonic (Jamilu et al., 2007). In a broader sense, PTZ induced seizures are abolished by agents that act by reducing T-type Ca þ þ currents or enhance GABAA receptor inhibitory neurotransmission such as ethosuximide and benzodiazepines respectively (Vogel, 2008). MES is a model that screen for molecules with potential effect against generalized seizures (Tripathi, 2006). Some of the drugs effective against this seizure model act by inhibiting sodium channel or block Aspartate receptors and they prevent the spread of seizures (Malawska, 2005; Magaji et al., 2013). Phenytoin, carbamazepine, lamotrigine, and felbamate are AEDs that act by blocking sodium channels, thereby stabilizing inactive state of sodium channels, thus, reducing high frequency firing of action potentials (Nestler et al., 2009). However, the extract afforded no effect against THLE induced by MES, this therefore suggested that it mediates its action via other mechanism but not through blocking sodium currents or aspartate receptors. Strychnine is a competitive antagonist of glycine an inhibitory neurotransmitter in the central nervous system (Biggio et al., 1992). Drugs that abolished chemically induced seizures by strychnine mediate their activity by enhancing inhibitory neurotransmission in the brain and the spinal cord. Probable mechanism of antiepileptic effect of this extract is by interacting with glycinergic receptors and enhancing its effect. The activity of the extract against picrotoxin induced seizures; further lend credence to its action via GABAergic pathway. Picrotoxin is a non-competitive antagonist of GABAA receptors. Drugs such as benzodiazepines, barbiturates, and sodium valproate produced their anticonvulsant effect via enhancing the GABAA interaction with picrotoxin sites in GABAA–chloride channel complex (McNamara, 2006). In conclusion, this study revealed that that the ethanol stem bark extract of Lannea barteri contained bioactive constituents that may be useful in the treatment of epilepsy or any condition of the same symptomatology and justify the use of this plant part in the traditional medicine for the treatment of epilepsy. Further studies on this plant part will involve isolation and identification of the bioactive constituent responsible for this observed anticonvulsant activity.
Data expressed as Mean 7 SEM, one way ANOVA, n¼ 6, LB ¼Lannea barteri one way ANOVA followed by Dunnette post hoc test n
pr 0.001
extract of Lannea barteri possess anticonvulsant effect. The LD50 of 567.1 mg/kg suggest that it is slightly toxic (Matsumura, 1985). Even though the doses employed in this study were lower than 30% of the LD50 values which have shown to be safe for ethnopharmacological assays. The ethanol stem bark extract protected the animals against scPTZ induced seizures. Agents that blocked scPTZ induced seizures in rodents are used to screen compounds that raises seizure
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2015.06.039.
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