Nigerian Journal of Basic and Clinical Sciences

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 19  |  Issue : 1  |  Page : 20--28

Anticonvulsant effect of flavonoid-rich fraction of ficus platyphylla stem bark on pentylenetetrazole induced seizure in mice


Madinat Hassan1, Sunday Z Bala2, Aisha M Gadanya2,  
1 Biology Department, Faculty of Science, Airforce Institute of Technology, Kaduna State, Nigeria
2 Biochemistry Department, Faculty of Basic Medical Science, Bayero University Kano, Kano State, Nigeria

Correspondence Address:
Madinat Hassan
Airforce Institute of Technology, Mando, Kaduna, Kaduna State
Nigeria

Abstract

Context: Epilepsy is characterized by recurrent spontaneous seizures. Several antiepileptic drugs have been used over the years and these drugs have shown serious side effects, thereby prompting the use of medicinal plants to avert the resultant side effects of anti-epileptic drugs. Aim: To evaluate the anticonvulsant effect of the flavonoid-rich fraction (FRF) of Ficus platyphylla stem bark (FPSB) on pentylenetetrazole (PTZ) induced seizures in mice. Study Design: Experimental cohort study. Subjects and Methods: We evaluated the anticonvulsant effect of the flavonoid-rich fraction (FRF) of Ficus platyphylla stem bark (FPSB) on pentylenetetrazole (PTZ) induced seizures in mice by measuring its antioxidant activity in vivo and in vitro and identify possible flavonoids present via Liquid Chromatography Mass Spectroscopy (LC MS) and Fourier Transform Infrared Spectroscopy (FTIR). Statistical Analysis: One way analysis of variance (ANOVA) was used to determine the level of significance at a 95% confidence interval followed by Tukey's multiple comparison test using SPSS software. Result: The FRF of FPSB exhibited weak anticonvulsant activity against PTZ-induced seizure in mice. Maximum anticonvulsant activity (25% protection) was observed at a dose of 100 mg/kg and 200 mg/kg with a delay in the meantime of onset of myoclonic jerks and latency to tonic seizure. The effect of the fraction was found to be dose-independent. The FRF contains a flavanone Astilbin (flavonoid 3 O glycosides) which may have effectuated the high antioxidant activity against 2,2 diphenyl 1 picrylhydrazyl (DPPH) and nitric oxide (NO) while increasing brain glutathione content and decrease in malondialdehyde content. Conclusion: Although the anticonvulsant capacity of FRF on PTZ-induced mice was minimal, this further requires an exploration of other seizure models to ascertain its mechanism of action.



How to cite this article:
Hassan M, Bala SZ, Gadanya AM. Anticonvulsant effect of flavonoid-rich fraction of ficus platyphylla stem bark on pentylenetetrazole induced seizure in mice.Niger J Basic Clin Sci 2022;19:20-28


How to cite this URL:
Hassan M, Bala SZ, Gadanya AM. Anticonvulsant effect of flavonoid-rich fraction of ficus platyphylla stem bark on pentylenetetrazole induced seizure in mice. Niger J Basic Clin Sci [serial online] 2022 [cited 2022 Aug 14 ];19:20-28
Available from: https://www.njbcs.net/text.asp?2022/19/1/20/350715


Full Text

 Introduction



Plants have been explored and utilized since antiquity for several purposes in the life of mankind, specifically, as food for nutritional benefits and medicines for the treatment of diseases. Plants are utilized in all cultures of the world and have been relied upon for several millennia to support, promote and restore human health.[1] They form a vital component of traditional medicine (TM) and their use for the maintenance of health and well-being is a common practice in many societies across the globe. They are used as remedies for the prevention and treatment or management of myriad disease conditions. The use of plant parts in African folk medicine has gained cultural acceptability for several centuries and has since been relatively accessible and affordable.[2] The rationale for the medicinal use of plants is due to the presence of mixtures of different biologically active plant constituents called phytochemicals. These phytochemicals have been generally classified into six major categories based on their chemical structures and characteristics, which include; carbohydrate, lipids, phenolics, terpenoids, alkaloids, and other nitrogen-containing compounds.[3] These categories undergo further division based on biogenesis to gives rise to different subcategories consisting of alkaloids, flavonoids, saponins, glycosides, tannins, lignans, coumarins, triterpenes, carotenoids, etc., that may act solely or synergistically to manifest an effect which may be useful or harmful to health.[3]

Flavonoids, a sub-class of polyphenols, are a group of phytochemicals that are among the most potent and abundant antioxidants in our diet[4] and have been utilized in medicinal, nutraceutical, pharmaceutical and cosmetic industries because of their various reported biological activities such as anti-inflammatory, nephro-protective, anti-cancer, anti-bacterial, hepato-protective, hypolipidemic, immuno-enhancing effects etc.[4] Flavonoids are classified into several subgroups such as flavone, flavanone, flavonol, isoflavonoid, catechins, anthocyanidin, and chalcones,[5] in which the group flavonol and catechins have been reported to possess the highest antioxidant activity amongst the subgroups.[6] Current research trend to ascertain bioactive functions of flavonoids involves its isolation, identification and characterization.[6]

The preliminary phytochemical screening of FPSB was reported to contain phytochemical components such as flavonoid, tannins and saponins.[7] Ficus platyphylla Del-Holl (Family: Moraceae) is a deciduous plant found mainly in the savannah regions of the West African coast. It grows widely in the Northern part of Nigeria, up to 60 ft high. It is popularly called Gamji in the Hausa Language and belongs to the family of Fig trees. The plant parts have been used in Nigerian traditional medicine to treat psychosis, depression, epilepsy, pain, infertility and inflammation for many years and its efficacy is widely acclaimed among the Hausa communities of northern Nigeria.[8]

Epilepsy is the term used for a group of disorders characterized by recurrent spontaneous seizures and involving hyper-excitable neurons.[9] Molecular investigations reveal an imbalance between inhibitory Gamma-Amino Butyric Acid (GABA) mediated and excitatory glutamate-mediated neurotransmission.[9],[10] It is commonly associated with brain dysfunctions leading to several behavioral comorbidities.[11] Seizures can be limited to one hemisphere of the brain, known as a focal seizure, or they can affect both hemispheres of the brain, known as a generalized seizure.

The World Health Organization (2019) reported epilepsy as one of the most common neurological diseases affecting around 50 million people worldwide.[12] The estimated proportion of the general population with active epilepsy (i.e., continuing seizures or with the need for treatment) at a given time is between 4 and 10 per 1000 people.[12] Globally, an estimated 5 million people are diagnosed with epilepsy each year. In high income countries, there is an estimated number of 49 per 100,000 people diagnosed with epilepsy each year.[12] In middle-and low-income countries, this figure can be as high as 139 per 100,000. This is likely due to the increased risk of endemic conditions such as malaria or neurocysticercosis, the higher incidence of road traffic injuries, birth related injuries and variations in medical infrastructure, availability of preventive health programmes and accessible care.[12] Three quarters of people with epilepsy living in low-income countries do not get the treatments they need, thereby increasing the incidence of premature death.[12] Interestingly, several Anti-Epileptic Drugs (AED) have been explored to manage epilepsy. However, some of these drugs show serious side effects like ischemia, hepatotoxicity, depression, cognitive disorders, and motor disability.[13] Additionally, 20-30% of those patients are resistant to treatments with synthetic drugs.[13] Therefore, it is necessary to discover new treatments by exploring the use of medicinal plants to reduce the complications or side effects associated with antiepileptic drugs. Many extracts of medicinal plant parts have been evaluated for anti-convulsion properties. However, the individual or synergistic activity of the bioactive principles of the plant sources responsible for the anticonvulsant property were not fully explored. Hence, the extraction of the flavonoid-rich fraction of FPSB to determine its antioxidant activity in vivo, in vitro and identify possible flavonoids present via Liquid Chromatography-Mass Spectroscopy (LC-MS) and Fourier-Transform Infrared Spectroscopy (FTIR) as well as evaluate its anticonvulsant potential.

 Materials and Methods



Collection, identification and preparation of plant material

Ficus platyphylla (Moracea) stem bark was obtained from Karau-Karau village, Zaria, Kaduna state, Nigeria. Mallam U. S. Gallah of Ahmadu Bello University's Biology Department in Zaria, Kaduna State, collected, identified, and authenticated the plant material. A sample with voucher number 7719 was deposited in the Herbarium. The stem bark of the plant was air dried for fourteen days and milled in a mortar using a pestle. This was subsequently sieved with a local sieve of approximately 2 mm pore size to obtain fine powder and weighed. Before use, the weighed powder was stored in an airtight container and labeled.

Preparation of crude extract of Ficus platyphylla stem bark

Extraction was achieved via microwave-assisted extraction (MAE) as reported by Hossain et al.[14] In a nutshell, the powdered stem bark of the plant material (1500 g) was dissolved in 3500 mL of 70% aqueous ethanol and microwaved for two minutes at 80oC. The extract was then filtered using a Whatman No. 41 filter paper and the filtrate was concentrated under reduced pressure using a rotary evaporator.

Phytochemical screening

The phytochemical constituents of FPSB was screened qualitatively for presence or absence of saponin (foam test), flavonoids (lead acetate test), phenols (ferric chloride test), tannin (gelatin test), alkaloids (Dragendorff's reagent), glycosides and steroids using methods described by Shah et al.[15]

Preparation of flavonoid-rich fraction of Ficus platyphylla stem bark extract

The crude extract of 70% ethanol was partitioned using different solvents of increasing polarity index-petroleum ether, chloroform, ethyl acetate and methanol with the aid of a Soxhlet apparatus at a temperature of 40°C for 2-3 hours.[14] Each fraction obtained was concentrated under reduced pressure by a rotary evaporator and subsequently labelled.

Determination of total flavonoid content (TFC)

The total flavonoid content of each fraction was determined by Aluminum chloride colorimetric assay with slight modifications as reported by Abdulqayoom et al.[16] and quercetin was used as a standard to construct the calibration curve. Briefly, quercetin (25 mg) was dissolved in 25 mL of aqueous ethanol (1 mg/mL stock solution) and then diluted to 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL with ethanol. About 20 μL each of the fractions (0.1 g in 10 mL aqueous ethanol) and standard solution (0.2 to 1.0 mg/mL) were mixed with 15 μL of sodium nitrite (0.5% NaNO2, w/v) solution separately in a 96 well plate and incubated for 6 minutes at room temperature (25°C). Thereafter, 15 μL of (1% AlCl3, w/v) solution was added to each reaction well and allowed to stand for a further 6 min before the addition of 80 μL of sodium hydroxide (0.4% NaOH, w/v) to each well. The mixtures were incubated for another 15 minutes at room temperature (25°C) and absorbance was taken at 510 nm.

Flavonoid content of each fraction was estimated using the linear regression equation obtained from the standard curve of quercetin and total flavonoid content was calculated as mean ± SD (n = 3) and expressed as mg/g of quercetin equivalent of dry FPSB extract using the formula below.

y = 0.578x + 0.0954, r2 = 0.9938

Where y = Absorbance of each fraction, x = concentration of quercetin from calibration curve

[INLINE:1]

C = Concentration of quercetin from calibration curve in mg/mL, V = volume of extract in ml and M = weight of extract in grams.

Determination of total phenolic content (TPC)

Folin-Ciocalteu reagent was used to determine the total phenolic content of the various fraction as described by Al-Owaisi, et al.[17] Gallic acid was used as a reference standard to construct the calibration curve. The content of total phenolic compounds was calculated as mean ± SD (n = 3) and expressed as mg/g of gallic acid equivalent of dry FPSB extract.

The Polyphenol content of the fractions were estimated using the linear regression equation obtained from the standard curve of gallic acid and calculated using the formula below.

y = 1.3x + 2.1434, r2 = 0.9882

Where y = Absorbance of the fractions and x = concentration of gallic acid from calibration curve,

[INLINE:2]

C = concentration of gallic acid from calibration curve in mg/ml

V = volume of extract in ml

M = weight of extract in grams.

Determination of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity

The DPPH radical scavenging activity was assessed using the IC50 value- which is the concentration of antioxidant required to scavenge 50% DPPH radicals in the specified time. The fractions exhibited a dose-dependent activity implying that, the DPPH scavenging activity of the fraction increased proportionally to the increase in concentration of the fractions. The smaller the IC50 value, the higher the antioxidant activity of the fractions. The free radical scavenging activity of different concentrations of the fractions of FPSB and of standard ascorbic acid solution was evaluated using the DPPH radical scavenging method as reported by Al Owaisi, et al.[17] with slight modifications. A 0.01 mM DPPH radical solution was prepared by dissolving 3.94 mg of DPPH in 10 mL of methanol. Fractions and ascorbic acid were prepared at various concentrations of 20, 40, 60, 80, 100, 200 and 400 μg/mL in a 96 well plate. 180 μL of DPPH solution was added to 12 μL of ascorbic acid and sample wells, rapidly mixed and allowed to stand in the dark at 37°C for 30 min. The blank was prepared in a similar way without extract or ascorbic acid. The decrease in the absorbance of each solution as measured at the 517 nm wavelength reader. The percentage of radical scavenging activity of tested extracts and positive control ascorbic acid was calculated using the formula below.

[INLINE:3]

Where Ac = Absorbance of control at 517 nm and As = Absorbance of sample.

The concentration of sample required to scavenge 50% of DPPH free radical (IC50) was determined from the curve of percent inhibitions plotted against the respective concentration.

Determination of free nitric oxide scavenging activity

The nitric oxide radical scavenging assay was carried out following a slightly modified method of Sakat and Juvekar.[18] The stock of each fraction and ascorbic acid were prepared (100 mg/mL) in methanol. These were then serially diluted to make concentrations of 20, 40, 60, 80, 100, 200 and 400 microgram. Griess reagent was prepared by mixing equal amounts of 2% sulphanil amide with 5% phosphoric acid and 0.1% naphthyl ethylenediamine dihydrochloride immediately before use. A volume of 50 μL of 10 mM sodium nitroprusside (0.29 g/100 mL) in 0.1 M phosphate buffered saline was mixed with 50 μL of each concentration prepared in a 96 well plate and incubated at 25°C for 180 minutes. Griess reagent (100 μL) was added to the solution above. A control sample without the extracts but with an equal volume of methanol was prepared in a similar manner as was done for the test samples. The absorbance was measured at 542 nm using a UV-Vis microplate reader.

The percentage nitrite radical scavenging activity of the fractions and ascorbic acid were calculated using the following formula:

Calculations

[INLINE:4]

Characterization of FRF of FPSB using liquid chromatography mass spectroscopy (LC-MS) and fourier-transform infrared spectroscopy (FTIR)

Protocol for LCMS Analysis (Generic Method) using LC Waters e2695 separation module with W2998 PDA and coupled to ACQ-QDA MS. The methanol fraction of FPSB was analyzed using liquid chromatography (LC) tandem mass spectrophotometer (MS) as described by Piovesana et al.[19] with some modifications. The extracted samples were reconstituted in methanol and filtered through a polytetrafluoroethylene (PTFE) membrane filter with a 0.45 μm size. After filtration, the filtrate (10.0 μL) was injected into the LC system and allowed to separate on Sunfire C18 5.0 μm 4.6 mm x 150 mm column. The run was carried out at a flow rate of 1.0 mL/min. sample and column temperature at 25oC. The mobile phase consists of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) with a gradient. From a ratio of A/B 95:5 this ratio was maintained for a further 1 min, then A/B 5:95 for 13 min, to 15 min. Then A/B 95:5 to 17 min, 19 min and finally 20 min. The PDA detector was set at 210-400 nm with a resolution of 1.2 nm and a sampling rate of 10 points/sec. The mass spectra were acquired with a scan range from m/z 100 – 1250 after ensuring the following settings: ESI source in positive and negative ion modes; capillary voltage 0.8 kv (positive) and 0.8 kv (negative); probe temperature 600oC; flow rate 10 mL/min; nebulizer gas, 45 psi. MS set in automatic mode applying a fragmentation voltage of 125 V. The data was processed with Empower 3. The compounds were identified on the basis of the following information: elution order, and retention time (tR), fragmentation pattern, and Base m/z.

The Protocol for FTIR spectroscopy was done by encapsulating 10 mg of the dried methanol extract in 100 mg of potassium bromide (KBr) pellet, in order to prepare translucent sample discs. The powdered sample of the fraction was loaded into the FTIR Spectroscope (FT-IR Agilent carry 630), with a scan range of 400 to 4000 cm-1 with a resolution of 4 cm-1. Literature search for the functional group range and assignment was correlated with that reported by Nandiyanto et al.[20]

Experimental animals

Forty mice obtained from the Department of Pharmaceutical science of Kaduna State University were used for this study and kept in well ventilated cages under standard condition. The animals were acclimatized for two weeks and maintained under standard conditions of temperature (23oC ± 2oC), controlled humidity and a 12-hour light/dark cycle. The mice were fed standard feed and water was given to them ad libitum. The animal handling protocols were adapted based on the guidelines of the National Institute of Health (NIH,1985) for laboratory animal care. Ethical clearance was approved with reference number BUK/CHS/REC/112 by the Research Ethics Committee of Bayero University Kano.

Acute toxicity (LD50) study

The lethal dose (LD50) of the flavonoid-rich fraction of FPSB was determined as described by Lorke.[21] Briefly, nine mice were divided into 3 groups of 3 mice each in the first phase, which were then orally administered 10, 100 and 1000 mg/kg of the fraction, respectively. The mice were monitored for any signs of distress or death for 24 hours. In the absence of death or distress, the doses were increased to 1600, 2900 and 5000 mg/kg in the second phase involving one mouse per group.

Screening for anticonvulsant activity of the flavonoid-rich fraction

Anticonvulsant screening was conducted using the method reported by Calderon et al.[13] in which the animals were divided into five groups, each consisting of four mice and treated as shown below for seven days.

Group I-received normal saline orally (2 mg/kg)

Group II-received the standard drug diazepam (1 mg/kg, intraperitoneal).

Groups III, IV and V-received methanol fraction at oral doses of 100, 200 and 400 mg/kg body weight respectively. On the fourth day, thirty minutes after administration of the last dose of normal saline, flavonoid-rich fraction (methanol fraction) and diazepam. Seizure was induced in mice by intraperitoneal injection of PTZ (80 mg/kg body weight). The onset of myoclonic jerks, latency to tonic seizures, duration of tonic seizures and percentage of animals protected against seizures were recorded within an hour period using a stop watch timer.

Brain tissue assay

Tissue homogenate preparation-Approximately 700 mg of brain tissues was homogenized with a mortar and pestle in 4 mL of cold buffer solution (50 mM potassium phosphate, pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA), then centrifuged at 4000 rpm for 15 minutes. The supernatant was kept at a low temperature until used for analysis.[22]

Reduced Glutathione (GSH) assay

Reduced glutathione was determined according to Ellman's method.[23] Briefly, to 150 μL of tissue homogenate in cold buffer (50 mM potassium phosphate, pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA), 1.5 mL of 10% tricarboxylic acid (TCA) was added and centrifuged at 1500 rpm for 5 minutes. 1 mL of the supernatant was treated with 0.5 mL of Ellman's reagent and 3 mL of phosphate buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm. The quantity of GSH was obtained from the graph of the GSH standard curve. The Glutathione content of the brain cell in PTZ induced seizure mice model was estimated using the linear regression equation obtained from the standard curve of glutathione.

y = 0.0047x + 0.0062, r2 = 0.9998

Where y = Absorbance of glutathione and x = concentration of glutathione from the calibration curve.

Lipid peroxidation assay

Lipid peroxidation was determined by measuring the thiobarbituric acid reactive substances (TBARS) produced during lipid peroxidation. Briefly, tissue homogenate (150 μL) was treated with 2 mL of tris boric acid (TBA)-tricarboxylic acid (TCA) – hydrochloric acid (HCL) reagent (1:1:1 ratio) and placed in a water bath at 90oC for 60 minutes. The mixture was cooled and centrifuged at 3000 rpm for 5 minutes and the absorbance of the pink supernatant (TBA-Malondialdehyde complex was then measured at 535 nm. Malondialdehyde formed was calculated using the Molar extinction coefficient of 1.56 x 10 5 cm-1M-1, and was expressed as μM/g of tissues.[24]

Statistical analysis

Data analysis was carried out in triplicates and values were reported as mean ± standard deviation. One-way analysis of variance (ANOVA) was used to determine the level of significance at 95% confidence interval followed by Tukey's multiple comparism test using SPSS 2014 software.

 Results



Phytochemicals are plant components that have shown potent characteristics in the protection and cure of certain pathological conditions. The result below [Table 1] reveals the phytochemical constituent of FPSB ethanolic extract (70% aq.).{Table 1}

Total flavonoid and total phenolic content

The methanol fraction of FPSB was found to contain the highest flavonoid content at concentration of 537.30 mg compared to ethyl acetate, petroleum ether and chloroform fractions. However, it was determined that methanol fraction contained the highest phenolic content at 87.73 mg in comparison with ethyl acetate, chloroform and petroleum ether [Table 2].{Table 2}

Radical scavenging activity

Methanol fraction exhibited the highest scavenging activity (84.89 ± 0.04) at concentration of 140 μg/mL with IC50 of 59.34 μg/mL, although less than the standard- ascorbic acid (94.55 ± 0.03) at concentration of 140 μg/mL with IC50 of 27.43 μg/mL [Table 3]. The Nitric oxide (NO) scavenging activity of respective fractions of FPSB shows a dose dependent inhibition of NO radicals in proportion to increased concentration. The methanol fraction shows an inhibition (82.34 ± 0.05) at 20 μg/mL compared to other fractions as shown in the table below although lower than ascorbic acid (96.48 ± 0.05) at 20 μg/mL [Table 4].{Table 3}{Table 4}

Liquid chromatography-mass spectroscopy (LC-MS) analysis

The flavonoid-rich fraction (methanol fraction) of FPSB was chosen for LC-MS analysis on the basis of its high flavonoid and polyphenol content as well as its high DPPH and NO activity. The LC-MS analysis reveals the retention time of the possible compounds as shown in [Figure 1] and the fragmentation pattern revealing the relative abundance of the identified compound (astilbin) at 451 au as shown in [Figure 2].{Figure 1}{Figure 2}

Fourier transform infrared radiation (FTIR) analysis of methanol fraction from ethanolic extract of Ficus platyphylla stem bark

The numerous peaks shown in [Figure 3] reveal that the analyzed molecule is a complex molecule. The peak contains a single bond area (2500-4000 cm-1) and reveals a broad absorption band at 3302 cm-1 informing the presence of a hydrogen bond in the molecule. There is no narrow band at above 3000 cm-1 indicating the absence of unsaturated compounds. There is a sharp band at around 2922 and 2855 cm-1 revealing the presence of an aliphatic compound. No triple bond region (2000 – 2500 cm-1) was detected, implying the absence of CC bond in the molecule. Regarding the double bond region (1500-2000 cm-1), there is a narrow peak at about 1736 cm-1 revealing the presence of a carbonyl compound, which could be an aldehyde. A sharp band was observed at 1606 cm-1 and 1517 cm-1 informing the presence of a double bond or aromatic compound. In the fingerprint region (400 – 1500 cm-1), an aromatic compound was present at strong visible bands of 767 cm-1 for ortho and 820 cm-1 for para. A strong visible band was observed at 1144, 1095 and 1043 cm-1 revealing the presence of an alcohol component in the molecule. Details of each peak is shown in [Table 5]. The FTIR spectra reveals the presence of a hydroxyl group, an aromatic ring, a long aliphatic chain, a double bond, absence of triple bond, an aldehyde and alcohol related components in the molecule. The above correlations were made in correspondence to the frequency range and functional group assignment reported by Nandiyanto et al.[20]{Figure 3}{Table 5}

Acute toxicity test

Treatment of mice with the different concentrations of the flavonoid-rich fraction showed no signs of toxicity, distress or death. Hence, 100, 200 and 400 mg kg-1 doses were chosen in the present study. This indicates that the fraction was found to be safe up to the dose levels studied. Since, all the mice survived at a dose of 5000 mg/kg body weight, the LD50 of the fraction will be >5000 mg/kg body weight. No major behavioral changes were observed during the period of study.

Anticonvulsant activity

The methanol fraction of FPSB exhibited a weak anticonvulsant activity against pentylenetetrazole (PTZ) induced seizure in mice. Maximum anticonvulsant activity (25% protection) was observed at a dose of 100 and 200 mg/kg with a significant delay in mean time of onset of myoclonic jerks and latency to tonic seizure and the effect of the fraction was found to be independent of dose as shown in [Table 6].{Table 6}

Brain tissue assay

The effect of methanol fraction on brain glutathione (GSH) and malondialdehyde (MDA) concentration at doses of 100 and 200 mg/kg showed a decrease in the MDA and an increase in GSH level compared with the control group as shown in [Table 7]. Although a reduction of GSH is observed at dosage of 400 mg/kg. The activity of the fraction was dose independent.{Table 7}

 Discussion



The total phenolic and flavonoid content of methanol fraction analyzed, contained the highest flavonoid content with a percentage yield of 10.71% compared to ethyl acetate (10.36%), petroleum ether (2.97%) and chloroform (0.44%) fractions. However, it was determined that methanol fraction (0.07%) contained the least phenolic content in comparison with ethyl acetate (1.68%), chloroform (2.03%) and petroleum ether (6.96%). The outcome of the radical scavenging activity against DPPH and NO radicals was statistically significant at P < 0.05 revealing a strong antioxidant capacity of each fraction and it was observed that the DPPH and nitric oxide inhibition capacity of the fractions increased in a dose-dependent manner in proportion to concentration which correlates to the report of Sheidu et al.[25] The radical scavenging activity of methanol fraction was at 85% against DPPH radicals and 82% against nitric oxide respectively, compared to the other fractions, indicating a high radical scavenging activity of the fraction as shown in [Table 3] and [Table 4]. Evidently, the amount of polyphenol and flavonoid of the fraction contributes to the antioxidant capacity of the methanol fraction observed in this study. The LC-MS analysis of the methanol fraction reveals the presence of flavonoid-astilbin (Flavonoid-3-O-glycosides) in the positive mode of LC-MS at 451 au. The FTIR spectra reveals the presence of a hydroxyl group, an aromatic ring, a long aliphatic chain, a double bond, absence of triple bond, an aldehyde and alcohol related components in the molecule. Astilbin belongs to the flavanone group, established to possess several pharmacological properties. Among which are; anticancer, anti-inflammatory, antibacterial, anti-oxidative, immuno-enhancing and hepato-protective activity.[26] The methanol fraction exhibited a weak anticonvulsant activity against PTZ-induced seizure in mice against that reported by Chindo et al.[8] This may result from differences in phytochemical constituent analyzed and possibly difference in solvent-system used. Maximum anticonvulsant activity (25% protection) was observed at a dose of 100 and 200 mg/kg with a significant delay in mean time of onset of myoclonic jerks and latency to tonic seizure and the effect of the fraction was found to be dose-independent as shown in [Table 6]. The anticonvulsant effect of the methanol fraction was less compared with the standard anti-epileptic drug diazepam (1 mg/kg) which completely antagonized the seizures produced by PTZ. The anticonvulsant property of methanol fraction might be minimal but evidently, the fraction contains psychoactive compounds that are imperative to the management of convulsive disorders. And this is verifiable by the significant delay in the onset of myoclonic jerks as well as tonic seizure exhibited during the anticonvulsant screening. Additionally, the antioxidant activity of the methanol fraction exhibited against DPPH and nitric oxide radicals, as well as an increase in brain glutathione and decrease in MDA levels might be linked to the anticonvulsant effect of the fraction. It is assumed that the flavonoid identified in this study as astilbin may not possess the neuroactivity or maybe insufficient in quantity to traverse the blood-brain barrier in other to facilitate an appreciable protection against seizure produced by PTZ. The effect of methanol fraction on brain glutathione (GSH) and malondialdehyde (MDA) at doses of 100 and 200 mg/kg showed a decrease in the MDA and an increase in GSH level compared with the control group. A reduction of GSH is observed at dosage of 400 mg/kg, which indicates that there was an increased generation of free radicals and that GSH was depleted during the process of combating oxidative stress. The increase in brain GSH level indicates that methanol fraction exerted a good antioxidant effect.

Prolonged evaporation of the solvent to obtain dried extract in rotary evaporator and water bath, as well as series of heating stages via microwave and Soxhlet apparatus to obtain extracts, are some limitations encountered during the experimental procedure. These stages might have caused a reduction in the actual quantity or activity of the flavonoid-rich fraction. These limitations can be circumvented, using more appropriate techniques and procedures that will improve the yield and activity of the fraction. This will help in adequate determination of anticonvulsant potential of the active compounds as well as their mechanism of action will be uncovered in order to reduce complications or side effects associated with anti-epileptic drugs (AEDs).

 Conclusion



The anticonvulsant effect of flavonoid-rich fraction from FPSB was assessed on PTZ-induced seizure in mice as well as its in vitro and in vivo antioxidant capacity. The result shows a maximum dose dependent radical scavenging activity in vitro via DPPH and NO assay and reveals a minimal anticonvulsant activity in vivo which may be due to the less activity of the identified flavonoid (Astilbin) to cross the blood-brain barrier in order to exert its function on the brain tissue. Although exploration of other seizure models to ascertain its potential and mechanism of action is encouraged.

Ethical approval

All authors hereby declare that the principles of laboratory animal care (NIH publication No. 85-23, revised 1985) were followed as well as specific national laws where applicable. All experiments have been examined and approved by the appropriate ethics committee.

Acknowledgements

The authors express their acknowledgement to Dr Bashir Musa of the Centre for Dryland Agriculture, Bayero University Kano for his contribution towards the technical aid in the analysis and interpretation of the characterized compounds and Muhammad Abdullahi Umar for his support during the inception and completion of this project.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Mensah MLK, Komlaga G, Forkuo AD, Firempong C, Anning AK, Dickson RA. Toxicity and safety implications of herbal medicines used in Africa. Herbal Medicine. Philip F. Builders, IntechOpen; 2019.
2Bala SZ, Hassan M, Sani A. Effect of Flavonoid-rich fraction of Irvingia gabonensis seed extract on Tetrachloromethane (CCL4)-induced liver damage in mice. Int J Sci Glob Sustain 2021;7:102-9.
3Huang Y, Xiao D, Burton-Freeman BM, Edirisinghe I. Chemical changes of bioactive phytochemicals during thermal processing. reference module in food. Science 2016:1-9.
4Tungmunnithum D, Thongboonyou A, Pholboon A, Vangsabai A. Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: An overview. Medicines (Basel) 2018;5:93.
5Harborne JB. Phytochemical Methods, a Guide to Modern Techniques of Plant Analysis. 3rd ed. New Delhi, India: Springer Pvt. Ltd; 1998.
6Panche AN, Diwan AD, Chandra SR. Flavonoids: An overview. J Nutr Sci 2016;5:e47.
7Chindo BA, Schroder H, Becker A. Methanol extract of Ficus platyphylla ameliorates seizure severity, cognitive deficit and neuronal cell loss in pentylenetetrazole-kindled mice. Phytomedicine 2015;22:86-93.
8Chindo BA, Anuka JA, McNeil L, Yaro AH, Adamu SS, Amos S, et al. Anticonvulsant properties of saponins from Ficus platyphylla stem bark. Brain Res Bull 2009;78:276-82.
9De Almeida RN, de Sousa DP, N'obrega FFDF, de Sousa Claudino F, Araujo DAM, Leite JR, et al. Anticonvulsant effect of a natural compound α, β-epoxycarvone and its action on the nerve excitability. Neurosci Lett 2008;443:51–5.
10Grosso C, Valent˜ao P, Ferreres F, Andrade PB. The use of flavonoids in central nervous system disorders. Curr Med Chem 2013;20:4694–719.
11Singh B, Singh D, Goel RK. Dual protective effect of Passiflora incarnata in epilepsy and associated post-ictal depression. J Ethnopharmacol 2012;139:273–9.
12WHO. Epilepsy. Available from: https://www.who.int/news-room/fact-sheets/detail/epilepsy. [2019 Jun 20].
13Calderon OH, Santiva ~ nez-Acosta R, Pari-Olarte B, Enciso-Roca E, Montes VMC, Acevedo JLA. Anticonvulsant effect of ethanolic extract of Cyperus articulatus L. leaves on pentylenetetrazol induced seizure in mice. J Tradit Complement Med 2018;8:95–9.
14Hossain MA, Shah MD, Sakari M. Gas chromatography-mass spectrometry analysis of various organic extracts of Merremiaborneensis from sabah. Asian Pac J Trop Med 2011;4:637–41.
15Shah MD, Hossain MA. Total flavonoids content and biochemical screening of the leave of the tropical endemic medicinal plant Merremia borneensis. Arab J Chem 2014;7:1034–6.
16Abdulqayoom L, Shahabuddin M, Aisha N, Abdulhafeez L. Extraction, identification and antioxidative properties of the flavonoid-rich fractions from leaves and flowers of Cassia angustifolia. Am J Analyt Chem 2011;2:871–8.
17Al-Owaisi M, Al-Hadiwi N, Khan SA. GC-MS analysis, determination of total phenolics, flavonoid content and free radical scavenging activities of various crude extracts of Moringa peregrine (Forssk.) fiori leaves. Asian Pac J Trop Biomed 2014;4:964–70.
18Sakat SS, Juvekar AR. Comparative study of Erythrina indica Lam. (Febaceae) leaves extracts for antioxidant activity. J Young Pharm 2010;2:63–7.
19Piovesana A, Rodrigues E, Norena CPZ. Composition analysis of carotenoids and phenolic compounds and antioxidant activity from Hibiscus calyces (Hibiscus sabdariffa L) by HPLC-DAD-MS/MS. Phytochem Anal 2018;1:435.
20Nandiyanto ABD, Oktiani R, Raghadita R. How to read and interpret FTIR spectroscope of organic material. Indones J Sci Technol 2019;4:97-118.
21Lorke D. New approach to practical acute toxicity testing. Arch Toxicol 1983;54:275-87.
22Dayan FE, Owens DK, Corniani N, Lima Silva FM, Watson SB, J'Lynn H, et al. Biochemical markers and enzyme assays for herbicide mode of action and resistance studies. Weed Sci 2015;63:23-63.
23Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-7.
24Buege J, Aust S. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302–10.
25Sheidu AR, Umar ZA, Abubakar A, Ahmed CB, Garba MM, Ogere AI, et al. Antioxidant and hepatoprotective potentials of methanol extract of Ficus platyphylla stem bark delile (Moraceae) in wistar rats. Trop J Nat Prod Res 2020;4:91-7.
26Sharma A, Gupta S, Chauhan S, Nair A, Sharma P. Astilbin: A promising unexplored compound with multidimensional medicinal and health benefits. Pharmacol Res 2020;158:104894.