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Sedative and Anti-convulsant Effects of the Stem Bark Extract of Combretum hypopilinum in Laboratory Animals



Abubakar Kabiru, Usman Aminu, Yerima Musa, Muhammad Abubakaar Amali, Samaila Hassan, Abdullahi Suleiman, Rabiu Tijani Giaze and Abdulhamid Imam Tanko
 
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ABSTRACT

Background and Objectives: Limited efficacy and negative property of currently available antiepileptic drugs limit their use and cause difficulties in patient management. The aim of this study was to evaluate the sedative and anti-convulsant effects of Combretum hypopilinum. Combretum hypopilinum stem bark was extracted using 95% methanol. Preliminary phytochemical screening of the crude methanol stem bark extract was carried out using standard procedures. Materials and Methods: Diazepam induced sleep was employed for the sedative effect. The anti-convulsant effect was evaluated by using MEST, PTZ and strychnine induced seizure models. Motor coordination deficit was also evaluated by using the beam walk assay method. Results: The result of the phytochemical screening revealed the presence of secondary metabolites such as; steroids, flavonoids and alkaloids. Acute toxicity studies were carried out and oral LD50 were found to be 3807.89 and >5000 mg kg1 b.wt., respectively. The extract showed a dose dependent and statistically significant decrease in the onset of sleep. The extract was active in maximal electroshock seizure test at doses of 200 and 400 mg kg1. The extract also exerted significant activity in Pentylenetetrazole (PTZ) seizure test at a dose of 400 mg kg1. The extract had no effect on Strychnine (STC) induced seizure test. In the beam walk assay test, the extract revealed no significant motor deficit in all tested doses. Conclusion: From the results of the present work, it may be concluded that the Combretum hypopilinum extract contains bioactive principles with central nervous system depressant effect, this may also support the traditional use of the plant in treatment of epilepsy and other mental illnesses.

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Abubakar Kabiru, Usman Aminu, Yerima Musa, Muhammad Abubakaar Amali, Samaila Hassan, Abdullahi Suleiman, Rabiu Tijani Giaze and Abdulhamid Imam Tanko, 2020. Sedative and Anti-convulsant Effects of the Stem Bark Extract of Combretum hypopilinum in Laboratory Animals. Journal of Pharmacology and Toxicology, 15: 58-64.

DOI: 10.3923/jpt.2020.58.64

URL: https://scialert.net/abstract/?doi=jpt.2020.58.64
 
Received: January 15, 2020; Accepted: February 08, 2020; Published: March 15, 2020



INTRODUCTION

There are about 3.3 billion people in developing world who depend on plant-based traditional medicine for their healthcare needs. Almost 87% of the world’s inhabitants rely mainly on traditional medicine for their primary health care1. Epilepsy is a neurological disorder of the brain that affects people worldwide. It is identified as recurrent seizures, which are brief involuntary movements which may involve a body part or entire body. Some instances this is followed by loss of consciousness and control of bowel or bladder function2. It is the fourth most common neurological disorder in the United States after migraine, stroke and Alzheimer’s disease3.

Limited efficacy and negative property of currently available antiepileptic drugs limit their use and cause difficulties in patient management. Unwanted events, withdrawal symptoms, harmful interactions with other drugs and economic consequences, especially in developing countries limit the long term use of antiepileptic drugs4. Studies on economic consequences of epilepsy management have shown that in Northern Nigeria, an annual cost per patient was about $285.2 and in Southern Nigeria the cost was around $395 per individual patient5.

Several drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. For example, salicin from Salix alba Linn. (white willow) and emetine6. More than 355 species of medicinal plants have been evaluated and found to have anti-seizure activity using in vivo/in vitro models7. A lot of plants are now being used traditionally for the management of epilepsy and seizures, but till date only a handful have been documented8. Combretum hypopilium has been used by traditional medicine practitioners in Zuru, Kebbi state of Nigeria, for the treatment of epilepsy. Previous studies have shown that the plant possesses antimicrobial and antineoplastic properties9. Furthermore, the currently available antiepileptic drugs can provide only symptomatic relief as these drugs suppress seizures and have no effect on epileptogenesis10. These claims and the need for continued search and development of new drugs that are cheap, safe and effective for management of epilepsy from plants and other sources were part of the justification for this study

MATERIALS AND METHODS

Study area: The study was carried out at the Pharmacology and Toxicology Laboratory of the Faculty of Pharmaceutical Sciences, Usmanu Danfodiyo University, Sokoto from January-June, 2019.

Preparation of plant materials: A sample of the plant was collected in Zuru local government area, Kebbi state, Northern Nigeria. It was authenticated at the herbarium of the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Usmanu Danfodiyo, Sokoto, by Dr H. E. Mshelia. A voucher specimen number (PCG/D/US/Comb/0001) was deposited at the herbarium for future reference. Subsequently the stem bark of the plant was collected, washed with distilled water, shade-dried for one week and pulverized by using a pestle and mortar.

The powdered plant material (500 g) was continuously extracted with 2000 mL of 95% methanol by maceration for 3 days, extract obtained was filtered and concentrated in a rotary evaporator (Büchi Labortechnik AG, Switzerland) at 45°C. A brown product called methanol stem bark extract of Combretum hypopilinum was obtained. The extract was dried in an oven at a temperature of 40°C to remove any remaining moisture and stored at room temperature in an air tight container.

Phytochemical screening: Phytochemical screening was carried out on the plant by using standard procedures7.

Experimental animals: Five to six weeks old, male and female Swiss albino mice weighing (20-33 g), two day old ranger cockerels weighing 20-35 g were obtained from animal house of the Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria and Ojuanu Agricultural Enterprises, Sokoto, respectively. The animals were housed at the animal facility of the Department of Pharmacology and Toxicology, Usmanu Danfodiyo University, Sokoto. They were allowed free access to water and standard animal feed ad libitum. All procedures were performed according to the guidelines of care and use of laboratory animals as approved by the Animals Ethics Committee of the Department of Pharmacology and Toxicology, Usmanu Danfodiyo University, Sokoto, Nigeria.

Experimental design
Acute toxicity study: The method described by Lorke11 was used, three groups of three mice or chicks were treated with the methanol stem bark extract of Combretum hypopilinum at doses of 10, 100 and 1000 mg kg1 b.wt., orally and observed for signs of toxicity and death for 24 h. During second phase, the mice or chicks were divided into three groups of one mouse each. Group one, two and three received 1600, 2900 and 5000 mg kg1 b.wt., respectively. Signs of toxicity and death were observed. The LD50 values were calculated using the formula below:

LD50 = √ (D0×D100)

where, D0 is the highest dose that animal survived and D100 is the lowest dose that killed the animal.

Diazepam-induced sleep test in mice: Methanol stem bark extract of Combretum hypopilinum (CHME) was investigated for its Central Nervous System (CNS) effect using the diazepam-induced sleep test. Mice were divided into 4 groups of 6 each. Group (I-III) was treated with 100, 200 and 400 mg kg1 doses of Combretum hypopilinum methanol stem bark extract. Group IV (control group) was treated with normal saline (10 mL kg1, p.o). After 1 h, receiving of CHME and normal saline, each animal was injected with Diazepam 5 mg kg1 i.p. The criterion for sleep was the loss of righting reflex in which the mice cannot roll back when turned over12. The sleeping time was noted by recording the interval between the loss and regain of righting reflex13.

Maximal Electroshock Seizure (MES) test in chicks: The method was employed, two day old ranger cockerels were divided into 8 chicks per group. The test groups (I, II and III) were treated with 100, 200 and 400 mg kg1 doses of the extract. Group IV was administered Phenytoin (20 mg kg1, i.p), while group V received normal saline (10 mL kg1). Thirty minutes later, MES were induced in the chicks using the Ugo Basile Electro Convulsive Therapy (ECT) machine (Model 57800-001) with corneal electrodes placed on the upper eyelids of the chicks. The current, shock duration, frequency and the pulse width were set and maintained at 90 mA, 0.80 sec, 200 pulse sec1 and 0.8 msec, respectively14,15. Seizure was manifested as Tonic Hind Limb Extension (THLE)16. Ability to prevent this feature or decrease the recovery time post seizure was an indication of anti-convulsant activity15.

Pentylenetetrazole-induced seizure (PTZ) test in mice: Animals were divided into five groups of five mice each. Groups I, II and III received CHME 100, 200 and 400 mg kg1, p.o, respectively. Groups IV and V received diazepam (5 mg kg1) and 0.9% w/v of normal saline (10 mL kg1) i.p, respectively. Seizure was induced by administering 90 mg kg1 of sc PTZ14 as modified by Vellucci and Webster17. CHME was administered orally, 1 h before the PTZ administration. Absence of an episode of clonic spasm of at least 5 sec duration, hind limb extension or death indicated the extract‘s ability to abolish the effect of pentylenetetrazole on seizure threshold.

Strychnine (STN) induced seizure test in mice: Twenty five mice were divided into 5 groups (n = 5). Groups I, II and III received CHME 100, 200 and 400 mg kg1 p.o, respectively. Group IV received diazepam (5 mg kg1) i.p, as positive control, while Group V served as negative control received 0.9% w/v of normal saline (10 mL kg1). The extract was administered orally, 60 min prior to the administration of strychnine nitrate (2.5 mg kg1) i.p. The animals were observed for 30 min by placing them in separate cages. The onset of seizures (tonic-clonic convulsions) and time of death were recorded18.

Beam walking assay: Mice were trained to walk from a start platform along a ruler (80 cm long and 3 cm wide) elevated 30 cm above the bench by a metal support to a goal box. Three trials were performed for each mouse. Only the mouse that showed no neurological deficit and walked successfully along the ruler was admitted into the study and grouped into 5 groups of 6 mice each19. Mice in the first group were given normal saline (p.o), second, third and fourth groups received graded doses of the extract and the fifth group was given diazepam (0.5 mg kg1) (p.o). One hour later, each mouse was placed at one end of a beam (60 cm long, 8 mm in diameter and elevated 30 cm above the bench by a metal support) and allowed to walk to the goal box at the other end. The number of foot slips, which is a sensitive measure of motor coordination deficit19 was recorded for each mouse by using a tally counter.

RESULTS

Phytochemical constituents of methanol stem bark extract of Combretum hypopilinum: The methanol stem bark revealed the presence of steroids, glycosides, flavonoids, tannins, alkaloids, saponins, carbohydrates, reducing sugars and proteins (Table 1).

Table 1:Phytochemical screening of methanol stem bark extract of Combretum hypopilinum
+: Present, -: Absent

Acute toxicity studies (LD50): The median lethal dose (LD50) in mice was calculated to be 3807.89 mg kg1 b.wt., and in chicks it was found to be >5000 mg kg1 b.wt., orally as shown in Table 2.

Effects of Combretum hypopilinum methanol stem bark extract in the diazepam induced sleep test in mice: The result of diazepam induced sleep revealed a dose dependent decrease in onset of sleep in all treated groups. The decrease in sleep onset was statistically significant (p<0.05) compared with control (Table 3).

Anti-convulsant effect of Combretum hypopilinum on Maximal Electroshock-induced Seizure (MES) in chicks: The result of MEST showed dose dependent reduction in recovery time after Hind Limb Tonic Extension (HLTE) in chicks. The reduction was statistically significant (p<0.05) at doses of 200 and 400 mg kg1 b.wt., (Table 4). At highest dose, the extract offered 25% protection against the MEST. Phenytoin at 20 mg kg1, a known anti-convulsant agent showed significant (p<0.05) reduction in the recovery time and provided 75% protection against MEST.

Anti-convulsant effect of Combretum hypopilinum on pentylenetetrazole-induced seizures in mice: In the PTZ test Combretum hypopilinum showed dose dependent increase in onset of seizure. The increase was significant (p<0.05) at the highest dose (400 mg kg1 b.wt.,) of the extract. At the same dose, extract offered 20% protection against PTZ induced test. Diazepam (5 mg kg1) group offered 100% protection against PTZ induced seizure (Table 5).

Anti-convulsant effect of Combretum hypopilinum on strychnine-induced seizures in mice: The result of the STC test showed that the extract did not significantly increase the mean onset of seizure nor provide protection in STC induced seizure. Diazepam (5 mg kg1) significantly (p<0.05) increased seizure mean onset of the seizure and protected all the mice from death (Table 6).

Table 2:
Acute toxicity studies of methanol stem bark extract of Combretum hypopilinum

Table 3:
Effects of Combretum hypopilinum methanol stem bark extract and fractions in the diazepam induced sleep test in mice
CHMSE: Combrettium hypopilinium crude methanol stem bark extract, N/S: Normal saline, data was presented as mean±SEM, (n = 6), *p<0.05 compared to control, one way ANOVA followed by Dunnett‘s post hoc test

Table 4:Anti-convulsant activity of methanol stem bark extract of Combretum hypopilinum in Maximal Electroshock induced Seizure Test (MEST) in chicks
CHMSE: Combrettium hypopilinium methanol stem bark extract, values are expressed as Mean±SEM, n = 8, *p<0.05 statistically significant as compared with the negative control, one way ANOVA followed by Dunnett’s post hoc test

Table 5:
Anti-convulsant effect of Combretum hypopilinum on pentylenetetrazole-induced seizure in mice
Data presented as mean±SEM, N/S: Normal saline, CHMSE: Combretum hypopilinium crude methanol stem bark extract, (n = 5), *p<0.05, compared to control, One way ANOVA followed by Dunnetts‘ post hoc test

Table 6:
Anti-convulsant effect of Combretum hypopilinum on strychnine-induced seizure in mice (n = 5)
Data presented as mean±SEM, N/S: Normal saline, CHMSE: Combrettium hypopilinium crude methanol stem bark extract, (n = 5), *p<0.05, compared to control, One way ANOVA followed by Dunnett’s post hoc test

Table 7:
Effect of the methanol extract of Combretum hypopilinum on beam walking assay for motor coordination in mice
CHMSE: Combretum hypopilinium crude methanol stem bark extract, N/S: Normal saline, data presented as mean±SEM, (n = 5), *p<0.05, compared to control, One way ANOVA followed by Dunnett’s post hoc test

Effect of the methanol extract of Combretum hypopilinum on beam walking assay for motor coordination in mice: In the beam walk assay test, the extract revealed no significant motor deficit in all doses tested (Table 7).

DISCUSSION

The preliminary phytochemical screening of the crude methanol stem bark extract of Combretum hypopilinum revealed the presence of secondary metabolites which have been previously reported to have various neuropharmacological effects8,20. Flavonoids and steroids have been indicated in various pharmacological actions on central nervous system including anti-convulsant and anxiolytic activity21. Flavonoids have been implicated in central inhibitory and neuromodulatory effects22.

The LD50 values of crude methanol stem bark extract of Combretum hypopilinum in mice and chicks revealed that the plant is relatively safe23. Doses used in this work were selected to be less than one-quarter of the LD50 result for pharmacological safety24.

The extract of C. hypopilinum significantly reduced the onset of sleep and increased the duration of sleep in this study. This activity might be achieved through the inhibition of diazepam metabolism or an action through the central mechanisms involved in the regulation of sleep25,26. Studies have shown that the activation of GABAA receptors in the central nervous system enhances sleep27. Hypnotics have more depression effect on the CNS than sedatives and this effect can be achieved through increasing the dose of sedative-hypnotic drugs and this leads to reduction in sleep onset and increase sleep duration25.

In preliminary screening test for potential anti-convulsant agents, PTZ is among the most frequently used chemicals. PTZ is considered to act as an antagonist at GABAA receptor complex. Drugs that inhibit PTZ induced seizures are believed to be effective in controlling myoclonic and absence seizures in man28.

Result from this study indicated that the tonic convulsion produced by PTZ was attenuated by Combretum hypopilinum. Studies have shown that diazepam exerts its antiepileptic effects by enhancing GABA-mediated inhibition in the brain21. It is possible that diazepam and Combretum hypopililnum inhibit PTZ induced convulsion enhancing GABA neurotransmission. Since, the Combretum hypopililnum delay the onset of PTZ induced convulsion, it might be possible that the extract may be interfering with GABA aminergic mechanism to exert its anti-convulsant effect. This work is corroborated the findings of Rasilingam et al.21 and Tanko et al.29.

Strychnine has been known to exert its convulsing effect through antagonizing the inhibitory spinal cord and brain stem reflexes via glycine and thereby increasing the spinal reflexes30. Inability of the extract to inhibit strychnine induced seizure showed its lack of effect on the glycine receptors in the spinal cord.

The maximal electroshock test is use to characterize agents with activity against generalized tonic clonic seizures with reference to clinically established antiepileptic drugs. Seizure produced by MES-induced tonic extension can be prevented either by drugs that inhibit voltage-dependent Na+ channels, such as; phenytoin, valproate and lamotrigine or by drugs that block glutamatergic excitation mediated by the N-methyl-D-aspartate (NMDA) receptor, such as; felbamate28. The anti-convulsant activity indicated by the extract of Combretum hypopilinum in the MES model reveals that it might have blocked the seizure spread by inhibiting Na+ channels and/or glutamatergic excitation through NMDA receptor.

Studies have shown that the number of foot slips made by mice in the motor coordination test is a sensitive indicator for evaluating benzodiazepine induced motor coordination deficit and it is a good indicator of doses providing clinical sedation19. The methanol stem bark extract of Combretum hypopilinum showed no observable effects on motor coordination when compared with control implying that the inhibition effect observed in diazepam induced test might be initiated centrally not through peripheral neuromuscular blockade31. Thus, the sedative action of the extract observed was produced centrally. This work is similar to that of Nazifi et al.32.

CONCLUSION

The stem bark of Combretum hypopilinum demonstrated sedative and anti-convulsant activities in the models used, this study has provided a scientific backing for the traditional use of the plant in the treatment of epilepsy and other mental disorders.

SIGNIFICANCE STATEMENT

This study was able to demonstrate that the extract contains active constituents with anti-convulsant effects possibly mediated via GABAergic pathways (as observed in both the PTZ and maximum electroshock induced tests). The extract could not protect the animals against strychnine induced seizure and did not protect the animals against lethality induced by strychnine. This study will also provide a lead for researchers who may want to investigate the mechanism of its anti-convulsant action.

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