Abstract
Context: Hedranthera barteri (Hook.f.) Pichon (Apocynaceae) is used traditionally in the treatment of convulsion in Southern Nigeria.
Objective: The anticonvulsant activity of the methanol extract of H. barteri leaves was investigated.
Materials and methods: The anticonvulsant activity of the extract (50,100 and 200 mg/kg, p.o.) was investigated in seizures induced by picrotoxin and strychnine in mice. The total phenol and flavonoid contents were determined using a modified colorimetric method and the aluminium chloride method, respectively. The mineral composition of the plant was analyzed using atomic absorption spectrophotometry.
Results: The extract significantly prolonged the onset and reduced the duration of the seizures induced by picrotoxin (5 mg/kg, i.p.) and strychnine (1 mg/kg, i.p.). The response was, however, not dose-dependent. Phenobarbitone (2 mg/kg) completely protected the animals from seizures while diazepam (1 mg/kg) only prolonged the onset and reduced the duration of the seizures. The total phenol and flavonoid contents were 236.81 ± 0.05 mg gallic acid equivalent/g dry weight and 51.45 ± 0.01 mg quercetin equivalent/g dry weight, respectively. The elemental analysis revealed the presence of calcium 102.87 mg/g, magnesium 63.29 mg/mg, iron 54.16 mg/g, potassium 54 mg/g, sodium 48.95 mg/g, zinc 46.70 mg/g, manganese 41.72 mg/g, and copper 18.29 mg/g.
Discussion and conclusion: The results suggest that the methanol extract of H. barteri has anticonvulsant activity and may be exerting its effect by affecting the gabaergic and glycinergic mechanisms. The observed activity may be due to its flavonoid content and the appreciable amount of calcium present in the plant.
Introduction
A number of pharmaceutical anticonvulsant drugs are available in the management of convulsion; however, most of these drugs are not only expensive, but also inaccessible, and toxic. There is therefore a need to develop affordable, effective, and safe anticonvulsant drugs from natural products. One of such with a wide medicinal usage is Hedranthera barteri (Hook.f.) Pichon, Apocynaceae. It is a shrub of up to 2 m high found in the understory in damp situations of the closed-forest in Ghana, Northern and Southern Nigeria, Western Cameroons, and also in Zaire (CitationBurkill, 1985).
The flowers are large, white and tubular with a fragrant scent. The leaf decoction is drunk by the Igbo speaking people of Southern Nigeria for dizziness and the leaf is applied to tumors. It is also used in the treatment of convulsion. The fruit is taken in Nigeria for the treatment of gonorrhea and as a vermifuge. The plant is used to prevent miscarriage (CitationBurkill, 1985). The antidepressant, antimicrobial, anxiolytic, anti-inflammatory and antinociceptic activities of the leaves have been reported as well as the antiulcer and antioxidant activities of the roots (CitationOnasanwo & Elegbe, 2006; CitationDuru & Mbata, 2010; CitationOnasanwo et al., 2010a,Citationb).
Amataine, beninine, goziline, owerreine, subsessiline and isoquinoline have been reported in the plant (CitationOnasanwo, 2006). Vobtusine and callichiline have also been reported to be present in leaves, root bark and stem bark as well as traces of alkaloids in the seeds (CitationPatel & Rowson, 1965). The presence of cardenolides, saponins, flavonoids, and polyphenols has also been reported in the leaves and root (CitationDuru & Mbata, 2010).
This work investigated the anticonvulsant activity of H. barteri leaf extract in mice with a view to authenticating its traditional usage.
Materials and methods
Plant material and preparation of extract
Leaves of H. barteri (Hook.f.) Pichon (Apocynaceae) were collected in June 2010 from Ibadan, Oyo State, Nigeria. They were identified by Mr. Odewo and authenticated at the Herbarium of the Department of Botany and Microbiology, University of Lagos, Akoka, Lagos, Nigeria where a voucher specimen (LUH 3718) was deposited.
The plant was dried in a hot air oven at 40°C. The dried plant material (250 g) was extracted with 1 L methanol. The extract was filtered and concentrated in vacuo at 30°C using the rotary evaporator (Buchi, Switzerland). The extract obtained was brown in color and the crude yield was 29.9% (w/w). The extract was reconstituted in 0.05% Tween 80 to appropriate concentrations before administration to experimental animals.
Test animals
Swiss albino mice (20–30 g) of either sex were used in this study and they were obtained from the animal house facility of National Agency for Food and Drug Administration and Control (NAFDAC), Lagos. All the animals were maintained in well-ventilated and hygienic compartments under standard environmental conditions and fed with standard rodent pellets (Livestock Feed PLC, Lagos, Nigeria) and water ad libitum. Experimental protocols reported in this study were approved by the Experimentation Ethics Committee of the College of Medicine, University of Lagos (CM/COM/08).
Drugs
Strychnine (STR), picrotoxin (PCT), and diazepam (DZP) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), and phenobarbitone was purchased from Swipha Pharma Nig. Ltd. (Lagos, Nigeria). The drug solutions used in this work were freshly prepared and the solvents used were of analytical grade.
Acute toxicity study
The acute toxicity of the plant extract was evaluated using the method of CitationLorke (1983).
Five groups of six mice each were administered the plant extract at doses of 1, 2, 3 and 4 g/kg body weight orally. The animals were observed for signs of toxicity and mortality within 24 h. The animals that survived were further observed for 7 days for signs of delayed toxicity.
Assessment of anticonvulsant activity
The methods of CitationPorter et al. (1984) and CitationPerazzo et al. (2003) were used to assess the anticonvulsant effect of H. barteri. Seizures were induced with strychnine (1 mg/kg, i.p.) and picrotoxin (10 mg/kg, i.p.). In the experiment, the animals were divided into five groups of six mice each.
The first group served as the control and received 0.05% Tween 80 (10 mL/kg body weight i.p.), the second group served as standard and received the control drug [(Diazepam (DZP, 1 mg/kg i.p.); phenobarbitone (PBT, 2 mg/kg i.p.)], the third, fourth and fifth groups received the plant extract in three different doses (50, 100, 200 mg/kg body weight), respectively. Thirty minutes later, mice in all the groups received the convulsant agents. Animals which did not convulse after 30 min were considered as protected.
The latency to first convulsion and the duration of convulsions were recorded in the unprotected animals. The ability of the plant extract to prevent or delay the onset of the hind limb extension exhibited by the animals was used as an indication of anticonvulsant activity.
Total phenol content (TP)
The TP content of the extract was determined spectrophotometrically using a modified Folin-Ciocalteu method (CitationWolfe & Liu, 2003). Briefly, 0.5 mL (1 mg/mL) of the extract was mixed with 2.5 mL of the Folin-Ciocalteu phenol reagent and 2 mL (75 g/L) sodium carbonate. The mixture was allowed to stand for 30 min at room temperature after which absorbance was read at 765 nm. The results were expressed as mg of gallic acid equivalent (GAE)/g dry weight using the equation obtained from the calibration curve: y = 5.068x + 0.3755, R2 = 0.9247.
Total flavonoid content (TFC)
The TFC of the extract was determined using the method of CitationOrdonez et al. (2006). The extract [1 mL (0.1 mg/mL)] was mixed with 1 mL of 2% AlCl3 solution. The mixture was allowed to stand for 1 h at room temperature after which the absorbance was measured at 420 nm. The results were expressed as mg of quercetin equivalent (QE)/g dry weight using the equation obtained from the calibration curve: y = 55.232x − 0.4499, R2 = 0.955.
Mineral content
The powdered leaves (200 mg) were digested using 20 mL of concentrated HNO3. The solution was then filtered into a 100 mL volumetric flask and made up to the mark with distilled water. Calcium, sodium, potassium, manganese, zinc, iron, copper and magnesium were determined by atomic absorption spectrophotometry (Analyst 200, Perkin Elmer, Waltham, MA).
Statistical analysis
Results were expressed as mean ± standard error of mean (mean ± SEM) and percentage protection. Data were analyzed using one-way analysis of variance (ANOVA) followed by the Student’s t-test. Values of p < 0.05 were regarded as significant.
Results
Acute toxicity study
No death was recorded amongst the graded dose groups of mice up to a dose of 4 g/kg for the extract of H. barteri. No mortality was found up to 7 days. The results suggest that the plant extract is relatively safe in mice.
Anticonvulsant activity
In the PCT-induced convulsion, the methanol extract of H. barteri increased the seizure latency and reduced the duration of the seizures significantly (p < 0.01), but did not protect the animals from seizures. The response was, however, not dose-dependent. The highest activity was observed at 50 mg/kg after which there was a reduction in seizure latency and an increase in seizure duration. Phenobarbitone, however, completely protected the animals from seizures ().
Table 1. Effect of methanolic extract of Hedranthera barteri (HE) in strychnine (STR) and picrotoxin (PCT)-induced seizures in mice.
In addition, in the STR-induced convulsion, the plant extract increased the seizure latency and reduced its duration and did not protect the animals from seizures. The response was also not dose-dependent. However, unlike phenobarbitone, diazepam did not protect the animals from seizures, but increased the seizure latency and reduced its duration significantly (p < 0.01) ().
Total phenol and flavonoid content
The total phenol and the flavonoid contents of H. barteri were 236.81 ± 0.05 mg GAE/g dry weight and 51.45 ± 0.01 mg QE/g dry weight, respectively.
Mineral content
The mineral composition of the leaves of H. barteri is presented in . For the macroelements (K, Mg, Ca and Na), calcium occurred in the largest amount and sodium in the least and for the microelements (Fe, Mg, Zn and Cu), iron occurred in the largest amount and copper in the least.
Table 2. Mineral composition of Hedranthera barteri (mg/g of dry matter).
Discussion
The present study regarding the anticonvulsant activity revealed that the methanol extract of the leaves of H. barteri at 50 mg/kg significantly increased the seizure latency and reduced the seizure duration of PCT- and STR- induced convulsions, respectively.
Picrotoxin has been reported to cause convulsion by the blocking of GABAA receptor-linked chloride ion conductance into the brain cells (CitationLeonard, 2000; CitationNicoll, 2001) and strychnine by competitive antagonism of glycine, an inhibitory amino acid and neurotransmitter (CitationLarson, 1969; CitationParmer & Shiv Prakash, 2006). The results from this work suggest that the plant extract may have attenuated seizures induced by PCT and STR by affecting the GABAergic mechanism and the glycinergic pathway, respectively.
In this investigation, H. barteri possessed a considerable high level of phenols and flavonoid. The phytochemical screening results of CitationDuru & Mbata (2010) which reported the presence of saponins, alkaloids, flavonoids, polyphenols in the leaves of the plant support the result obtained in this work. We, however, report for the first time, the quantification of the phenolic and flavonoid contents of the plant.
Phenolic compounds, especially flavonoids, have been reported to exhibit a wide range of neuropharmacological properties such as modulation of neuronal oxidative metabolism, sedative and anticonvulsant effects (CitationAyoka et al., 2006). They have been reported to exert their anticonvulsant effect by modulating the GABAA-Cl complex as they are similar to benzodiazepines in structure (CitationAvallone et al., 2000; CitationPark et al., 2007). The flavonoids present in this plant may be responsible for the anticonvulsant activity observed in the plant however the involvement of other secondary metabolites present in the leaves of the plant cannot be completely ruled out.
All the elements investigated were found in the leaves with calcium occurring in the largest amount and copper in the least. Calcium is required for adequate bone formation and hypocalcaemia has been reported to be responsible for 20 to 34% of convulsions especially in children (CitationCockburn et al., 1973). This suggests that the leaves of H. barteri not only attenuate the seizures but also possess nutritional value.
The results obtained in this work justified the use of the leaves of H. barteri in the treatment of convulsion in Southern Nigeria. However, there is need for further investigation of the active constituents of the plant and their specific mechanism of action.
Declaration of interest
The authors report no conflicts of interest.
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