Tropical Journal of Natural Product Research

ABSTRACT


Introduction
Epilepsy is ranked the fourth most common and serious brain disorder in the world, with about 50 million of the world population affected. 1,2A significant number of persons are diagnosed yearly with epilepsy globally.About 6 in 1000 people are affected in the developed countries, while about 10 in 1000 people are victims in the developing world. 3Although the mechanisms underlying the development of epilepsy, its progress and the intriguing nexus with other central nervous system (CNS) disorders are poorly understood, 4 oxidative stress has been identified as one of the intrinsic factors involved in the pathogenesis of CNS disorders, including neurodegenerative diseases, 5,6 which has been linked with the manifestation of epilepsy.Poor repair capacity, increased oxygen requirement, presence of lipids and massive mitochondria have been adduced as reasons for the susceptibility of the brain to the deleterious effects of reactive oxygen species. 7Chang and Yu 8 reported that © 2020 the authors.This work is licensed under the Creative Commons Attribution 4.0 International License
Plant material and extraction C. afer leaves were obtained from Aiyepe Town, Odogbolu Local Government Area, Ogun State, Nigeria in August, 2018.Prof. J.D. Olowokudejo of the Department of Botany, Faculty of Science, University of Lagos, Nigeria, identified and authenticated the plant material which was assigned an institutional herbarium voucher specimen number LUH8018.Freshly collected leaves of C. afer were air-dried until constant weight was obtained, after which the leaves were grinded, weighed (510 g) and macerated in 2 L of hydroethanol (1:1) for 72 h.Thereafter, decanting and sieving of the extract using muslin cloth and subsequently with Whatman filter paper was done.These processes (extraction, decanting and filtration) were repeated 2 more times with the residues obtained.Evaporation of the cumulative filtrates to dryness was achieved in a laboratory oven set at 40 o C (Gallenhamp ® , Leicestershire, UK) to give a dark brown solid with yield of 10.8%.The extract was dissolved in distilled water prior to oral administration to experimental animals.The doses of the extract used in this study were selected based on results of preliminary investigations.

Experimental animals
Mice (25-30 g) of either sex were procured from the Laboratory Animal Centre of the Faculty of Pharmacy, Olabisi Onabanjo University, Ago Iwoye, Ogun State, Nigeria.The animals were kept under hygienic conditions in well ventilated compartments and housing.They were maintained under standard environmental conditions with access to standard rodent feed (Livestock Feeds PLC, Ikeja, Lagos, Nigeria) and water ad libitum.Fourteen days period of acclimatization was observed before the commencement of the experiments.Ethical approval was obtained from the Health Research Ethics Committee (HREC) of the College of Medicine, University of Lagos, Nigeria (CMUL/HREC/12/17/330).

Preliminary phytochemical screening
The extract was screened for the presence of various phytoconstituents using established procedures. 19,20urier-transform infrared spectroscopy (FT-IR) analysis FT-IR analysis of the extract for the presence of various functional groups was done as previously described. 21

Strychnine-induced convulsion test
In this experiment, animals were divided into 6 groups of 5 mice each.The first group served as control and received distilled water 10 mL/kg p.o.The second group served as standard group and received diazepam 3 mg/kg p.o.The third to sixth groups received C. afer at 4 different doses (25, 50, 100 and 200 mg/kg p.o.), respectively.One hour later, mice in all the groups received strychnine 4 mg/kg i.p.The latency and duration of convulsion were recorded for each mouse.Animals that did not convulse after 30 min were considered protected. 22,23crotoxin-induced convulsion test The same procedure described above in the strychnine model was followed, except that convulsion was induced with picrotoxin 5 mg/kg i.p. 24 Traction (muscle relaxant) test Each mouse was screened by placing their forepaws on a small twisted wire rigidly supported above a bench top.Those that were able to grasp the wire with the forepaws and place at least one hind foot on the wire within 5 sec, when allowed to hang free, were considered eligible for the test.A response longer than 5 sec was considered as failure. 25,26Screened mice were randomly divided into 6 groups of 5 mice each.These groups were separately treated orally with distilled water (10 mL/kg), C. afer (25, 50, 100 and 200 mg/kg) and diazepam (5 mg/kg).Each animal was subjected to the traction test 1 h posttreatment and the reaction time for each mouse was recorded.

Inclined screen (muscle relaxant) test
This test was carried out in accordance with the method of Adebesin et al. 26 with little modification.Mice were grouped and treated as mentioned above in the traction test.One hour post-treatment, each mouse was subjected to the inclined screen test.Mice were in turn placed on a glass plane inclined at 35 o and the time taken for each mouse to slide off the screen was recorded.

Total antioxidant capacity determination
The total antioxidant capacity (TAC) of the extract was evaluated using established procedure. 27Three millilitre of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) and 0.3 mL of the extract (100 µg/mL) were mixed together.For 90 min, the capped tubes were incubated in a water bath containing boiling water at 95 o C. The samples were allowed to cool off to room temperature and measurement at 695 nm of the absorbance of each of the aqueous solution was taken.The TAC was expressed as equivalent of ascorbic acid.

Total phenols content determination
With little modification using gallic acid as standard, the Folin-Ciocateu reagent model 28 was used to determine the total phenols content.For 15 min, the mixture of 0.1 mL of Folin-Ciocalteu reagent (0.5 N) and 0.5 mL of C. afer (100 µg/mL) were incubated at room temperature.Sodium carbonate solution (2.5 mL, 7.5% w/v) was thereafter added to the mixture and incubated at room temperature for another 30 min.The measurement of absorbance was done at 760 nm.Total phenols content was expressed as gallic acid equivalent.

Total flavonoids content determination
Using quercetin as standard 29 total flavonoids content of C. afer was evaluated with aluminium chloride.One millilitre of 100 µg/mL of the extract was added to the mixture of methanol (3 mL), aluminium chloride (0.2 mL of 10%), potassium acetate (0.2 mL of 1 M) and distilled water (5.6 mL) and this was incubated at room temperature for 30 min.The absorbance was measured at 415 nm.

In-vitro antioxidant activity 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay
Following established procedure, 30,31 the DPPH free radical scavenging activity of the extract was investigated.Different concentrations (20, 40, 60, 80, and 100 μg/mL) of the extract (0.5 mL) in ethanol (95%) were mixed with the reagent solution (2.0 mL; 0.004 g of DPPH in 100 mL methanol).The control had only DPPH solution in place of the sample, while methanol was used as the blank.After intense shaking, the mixture was left at room temperature.The absorbance was measured at 517 nm after 30 min.Ascorbic acid was used as standard.The scavenging activity was calculated using the expression: Ax Where Ax is the absorption of the control and Ay is the absorption of the extract or standard.

Nitric oxide scavenging assay
Different concentrations (20, 40, 60, 80, and 100 μg/mL) of the extract (4 mL) were placed in different test tubes and sodium nitroprusside (1 mL; 5 mM in phosphate buffered saline) solution was added into the test tubes.The mixtures were incubated at room temperature for 2 h.Two millilitres of the sample was taken from the mixture and mixed with 1.2 mL of Griess reagent (1% sulphanilamide, 0.1% naphthylethylenediaminedihydrochloride in 2% H3PO4).The absorbance was measured at 550 nm. 32Ascorbic acid was used as standard.© 2020 the authors.This work is licensed under the Creative Commons Attribution 4.0 International License Ax Where Ax is the absorbance of the control and Ay is the absorbance of the extract or standard.

Reducing power assay
Varying extract concentrations (20, 40, 60, 80, and 100 μg/mL) were mixed with sodium phosphate buffer (2.5 mL of 200 mmol/L; pH 6.6) and potassium ferricyanide (2.5 mL of 1%).Incubation was done at 50 o C for 20 min.Trichloroacetic acid (2.5 mL of 10% w/v) was later added to the mixture which was centrifuged at 650 rpm for 10 min.The upper layer was mixed with deionized water (2 mL) and ferric chloride (1 mL of 0.1%).The absorbance was measured at 517 nm. 30scorbic acid was used as standard.

Lipid peroxidation assay
The lipid peroxidation activity was investigated in accordance with the method of Buege and Aust. 33Fresh rat liver was cut and homogenized to obtain 10% homogenate in cold 150 mM KCl-Tris-HCl buffer.The mixture contained liver homogenate, Tris-HCl buffer (20 mM; pH 7.0), FeCl2 (2 mM), ascorbic acid (10 mM), and extract (0.5 mL) at various concentrations (20, 40, 60, 80, and 100 μg/mL) in a final volume of 1 mL.The mixture was incubated at room temperature for 1 h.Lipid peroxidation was measured as malondialdehyde (MDA) equivalent.The mixture was later mixed with thiobarbituric acid (TBA) -trichloroacetic acid (TCA) reagent (2 mL) and boiled on water bath for 15 min.Upon cooling, the precipitate was removed by centrifugation.Malondialdehyde absorbance was determined spectrophotometrically at 535 nm.Ascorbic acid was used as standard.

Hydrogen peroxide scavenging assay
The hydrogen peroxide scavenging activity of the extract was determined using the procedure of Ruch et al. 34 Varying concentrations of the extract (20, 40, 60, 80, and 100 μg/mL) were mixed with hydrogen peroxide solution (0.6 mL, 40 mM; pH 7.4 buffer).Hydrogen peroxide absorbance was measured at 230 nm for 10 min.Ascorbic acid was used as standard.

Inhibition (%) = [Ax-Ay] × 100
Ax Where Ax is the absorbance of the control and Ay is the absorbance of the extract or standard.

Statistical analysis
The data generated in this study were expressed as mean ± standard error of mean (S.E.M.).One-way ANOVA (followed by Dunnett's and Tukey's multiple comparison tests) using GraphPad Prism 6 Software (GraphPad Software Inc., CA, USA) was used for data analysis.Results were considered significant at p < 0.05.

Results and Discussion
The central nervous system is a complex, sophisticated entity that regulates and coordinates body activities.Disorders of this system can lead to some neurological abnormalities which are manifested as seizures, insomnia, muscle spasm, neurodegenerative diseases, and are managed largely by orthodox medicines.As a result of paradigm shift, the use of ethnobotanicals in treating some of these disorders is gaining more acceptance.Accordingly, this study was conducted to investigate the anticonvulsant, muscle relaxant and in-vitro antioxidant activities of the hydroethanol leaf extract of Costus afer sequel to the claims by traditional medicine practitioners that the plant can be used to manage convulsion, muscle spasm and oxidative stress related diseases.Strychnine-and picrotoxin-induced convulsion tests are two widely used animal models to identify the antiepileptic-like activity of drug substances.Strychnine is a neurotoxin/chemical convulsant which blocks both glycine and acetylcholine receptors. 35It binds to the glycine receptor, thereby preventing the inhibitory effects of glycine on the postsynaptic neuron in the spinal cord.The extract did not elicit any significant change (p > 0.05) in seizure latency and duration compared with the control in the strychnine model.However, diazepam produced significant increase (p < 0.001) in seizure latency with no significant change (p ˃ 0.05) in seizure duration (Figure 1).The lack of significant ameliorative effects of C. afer in respect of the onset and duration of convulsion in this model suggest a lack of interaction with glycine receptors.A similar effect was obtained with diazepam in the strychnine-induced convulsion test, except for the significant increase in the onset of convulsion.Diazepam is known to elicit its anxiolytic, sedative-hypnotic, anticonvulsant, and muscle relaxant effects via positive allosteric modulation of the GABAAreceptor, 26 gamma-aminobutyric acid (GABA) being a major inhibitory neurotransmitter in the CNS. 35n the picrotoxin model, C. afer at 50 mg/kg significantly increased (p < 0.05) the seizure latency and decreased (p < 0.001) the seizure duration.However, diazepam only significantly decreased (p < 0.05) the seizure duration.C. afer at doses of 25 and 200 mg/kg also significantly decreased (p < 0.01) the seizure duration (Figure 2).The mechanism of epileptogenic action of picrotoxin (a GABAA-receptor antagonist) has been generally reported to be by inhibiting gammaaminobutyric acid (GABA) neurotransmission and blocking chlorideion channels linked to GABAA receptors. 36Augmentation of GABAergic neurotransmission has been reported to prevent, block or attenuate seizures, while its antagonism enhances and facilitates seizures. 37Enhancement of GABAergic neuron and interaction with GABAA receptor by the extract may therefore be suggested as the possible mechanism of antiepileptic action of C. afer.Muscle relaxant-like actions of drug substances are widely investigated using traction and inclined screen tests. 26In the traction test, mice with proper muscular coordination are those that are able to grasp the horizontally hanged twisted wire with their forepaws and place at least one hind foot on the wire within 5 sec when allowed to hang freely.This capability could be altered in animals with relaxed muscles.An increase in the reaction time of mice in the traction test suggests muscle relaxant activity. 26C. afer (25-200 mg/kg) elicited non-significant (p > 0.05) increase in the reaction time of mice at all the doses used in this study compared to control, suggesting a trend towards inherent muscle relaxant effect.Diazepam, an established muscle relaxant, produced significant increase (p ˂ 0.01) in the reaction time of mice compared to the control (Figure 3).Possession of muscle relaxant property by the extract was however established in the inclined screen test.Adebesin et al. 26 reported that reduction in the post-treatment sliding latency relative to the corresponding pre-treatment sliding latency suggest muscle relaxant activity.C. afer (25-200   Diazepam produced a significant (p ˂ 0.001) reduction in the posttreatment sliding latency relative to the pre-treatment latency value, thus confirming its well-known muscle relaxant activity (Figure 4).Oxidative stress is an imbalance between the destructive reactive oxygen species and protective/defensive antioxidant mechanisms. 38ice-Evans 39 reported medicinal plants as natural source of antioxidants.These natural antioxidants protect and reduce the vulnerability of the body to certain diseases such as cardiovascular diseases and cancer. 40DPPH, NO, lipid peroxidation, H2O2 and reducing power assays are widely used in-vitro tests to investigate the antioxidant properties of natural products. 41C. afer extract in a concentration-dependent fashion scavenged generated radicals in the DPPH (IC50 = 40.00µg/mL vs. 36.23µg/mL for ascorbic acid; Figure 5), NO (IC50 = 13.50 µg/mL vs. 21.41µg/mL for ascorbic acid; Figure 6), and H2O2 (IC50 = 30.66µg/mL vs. 18.06 µg/mL for ascorbic acid; Figure 7) assays.The extract also elicited concentration-dependent reducing power (IC50 = 36.58µg/mL vs. 37.20 µg/mL for ascorbic acid; Figure 8) and anti-lipid peroxidation (IC50 = 42.11µg/mL vs. 35.23 µg/mL for ascorbic acid; Figure 9) effects.In respect of the NO assay, the IC50 value for the extract was lower than that of ascorbic acid, while values were comparable in the reducing power assay.For the DPPH, H2O2, and lipid peroxidation assays, the established © 2020 the authors.10).Qualitative phytochemical screening of the extract revealed the presence of flavonoids, steroids, glycosides, phenols, alkaloids, terpenoids and tannins.Some of these chemical constituents have been reported to be responsible for anticonvulsant, muscle relaxant and antioxidant activities of medicinal plants. 42

Conclusion
The findings in this study suggest that the hydroethanol leaf extract of Costus afer possess anticonvulsant, muscle relaxant and antioxidant activities.The anticonvulsant and muscle relaxant activities of the extract are possibly mediated via positive modulation of the GABAA receptor-chloride channel complex.The outcome of this study presents the extract as a candidate for standardized herbal remedy development for the treatment of convulsion, muscle spasm and diseases linked to oxidative stress.

This work is licensed under the Creative Commons Attribution 4.0 International License antioxidant
used as standard (ascorbic acid) had lower IC50 values relative to C. afer.The in-vitro antioxidant activities demonstrated by the extract in this study indicate potential therapeutic usefulness in diseases linked to oxidative stress.This finding is supported by the values obtained with the extract in respect of total antioxidant capacity, total phenols and total flavonoids contents (31.78 ± 0.24 mg/100 g, 17.60 ± 0.97 mg/100 g and 25.34 ± 0.64 mg/100 g equivalent of standards, respectively).In respect of the FT-IR analysis,