Anticonvulsant and Antioxidant Effects of Methanol Extract of Stems of G. arborea Roxb

Ipomoea reniformis Chaos is claimed in Indian traditional medical practice to be useful in the treatment of epilepsy and neurological disorders. In the present study, pretreatment effect of methanolic extract of Ipomoea reniformis on epilepsy and psychosis was evaluated in rodents using standard procedures. Besides evaluating epileptic and behavioral parameters, neurotransmitters such as Gamma-Amino Butyric Acid (GABA) in epilepsy and in psychosis dopamine, noradrenaline and serotonin contents in the rodent brain were estimated. The extract pretreatment reduced maximal electro shock; Isoniazid (INH) and Pentylenetetrazole (PTZ) induced seizures and also significantly inhibited the attenuation of brain GABA levels by INH and PTZ in mice. These results suggested that the observed beneficial effect in epilepsy may be by enhancing the GABAergic system. The test drug also inhibited the apomorphine induced climbing and stereotyped behavior and showed significantly reduced levels of brain dopamine, noradrenaline and serotonin which may be due to blocking of central dopaminergic, noradrenergic and serotonergic pathways or by enhancing the GABAergic system. The results obtained in present study suggest that the title plant possesses antiepileptic and antipsychotic activities in rodents.


Introduction
Ipomoea reniformis (IR) also called as merremia emarginata (Burm. f.) is a procumbent herb belonging to the family convolvulaceae. In India, it is commonly known as Undirkana and Mushakparni. The plant is widely distributed in India, Sri Lanka, Philippines, Malaysia, Tropical Africa and mainly grows in rainy and winter season. In India, it is found in Southern part mainly counting Chennai, and some places of Andhra Pradesh [1]. Traditionally, IR has been used to treat diverse clinical conditions ranging from pain; fever to neurological disorders [2]. IR has been claimed to be useful for inflammation, headache, fever, cough, neuralgia, rheumatism and also in liver and kidney diseases [3]. The powder of leaves is used as a snuff during epileptic seizures. Juice acts as purgative and the root is having diuretic, laxative actions and applied in the disease of the eyes and gums [4].
The plant contains various neuroprotective chemical constituents such as caffeic, p-coumaric, ferulic and sinapic acid esters. Petroleum ether extract contains fats and fixed oil while aqueous extract contains amino acids, tannins (condensed and pseudo tannins) and starch [5]. IR has been reported to possess various pharmacological actions, mainly antidiabetic [6], antiinflammatory [7], nephroprotective [8], antibacterial [9], antioxidant and antimicrobial activity [10]. Further, the principle constituents of IR such as sinapic and ferulic acids have exhibited behavioural and pharmacological

Introduction
Epileptic seizures are traditionally characterized as the ultimate expression of monolithic, hypersynchronous neuronal activity arising from unbalanced runaway excitation [1]. It's the involuntary contraction of striated muscles repeatedly, characterized by many clinical disturbances of consciousness, movement and sensation. Convulsion arises due to sudden excessive and rapid discharge of cerebral neurons in the grey matter of the brain.
Epilepsy is the second most common neurological disorder after stroke, overall accounting for 1% of the world's burden of disease and affecting at least 50 million persons worldwide and 7 million people in India. Large numbers of antiepileptic drugs available for the treatment of different types of seizures are frequently reported with side effects like sedation, cognitive dysfunction, chronic toxicity and safety related issues. Further, many drugs are not able to cure the condition or prevent the relapse and repeatedly observed to cause drug interactions [2].
These findings have stimulated a considerable number of research efforts for development of drugs from natural sources for treatment of the epileptic seizures in order to find safe and effective lead compounds for the treatment of this debilitating neurological disorder. In this regard, medicinal plants with potential antiepileptic activity would be important source for development of newer drugs with possibly lesser side effects [2]. Natural products from folk remedies have contributed significantly in the discovery of modern drugs with better safety and efficacy profiles compared to the conventional medicines [2,3] [2,3].
Gmelina arborea Roxb. (Family: Verbenaceae) commonly known as 'Gambhari' , 'Candhar tree' or 'White teak' is a moderate sized, deciduous tree found distributed throughout the greater part of India up to an altitude of 1500 meters [4]. The roots and heartwood are used as bitter tonic, stomachic, laxative and prescribed in fever, indigestion, heart disease, piles and liver disorders. [5][6][7] Major chemical constituents of heartwood and stem include phenolics like paulownin acetate, epieudesmin and lignans such as arboreol, isoarboreol, gmelanone, ketolignans like arborone, hydroxylignan along with iridoid glycoside. [6][7][8][9] Heartwood of Gmelina is used as an ingredient in many well-known Ayurvedic and herbal preparations like Dashmula and Chyawanprasha and is therefore much used in a variety of diseases. [6,10] Pharmacological studies indicating antiinflammatory [11,12], antioxidant [13], antimicrobial [14], anti-ulcer [15] and wound healing [16] potential of different plant parts are well reported. The plant extracts are also reported for their hypolipidemic activity [17], antidiabetic activity [18], anti-diarrhoeal activity [19] and immunomodulatory activity [20]. The plant is described to be used to improve memory and as a remedy for hallucinations [8,9] but scientific study supporting the role of stems and heartwood of G. arborea in treatment of nervous system disorders like epilepsy or convulsions have not been reported. Therefore, present study has been undertaken to assess antioxidant and antiepileptic effect of methanol extract of stems of G. arborea in adult albino mice.

Drugs and Chemicals
All the chemicals and reagents used were of analytical grade. Pentylenetetrazole, strychnine and diazepam were acquired from MP Biomedicals Pvt. Ltd. (Mumbai, India), Sigma Aldrich Chemicals Pvt. Ltd. (Steinheim, Germany), and Ranbaxy Laboratories Limited (Mumbai, India) respectively.

Plant Material
Fresh stems were collected from fully-grown trees from fields near the outskirts of Ankleshwar city (Dist. Bharuch), during October 2010. The authenticity was established by comparing its morphological and microscopical characters with the available literature and by an ethanobotanist, Dr. Bhasker L. Punjani, of Smt. S.M. Panchal Science College, Talod (Dist. Sabarkantha). Voucher specimen [PL11NSAHKga001] was submitted to Institute of Pharmacy, Nirma University, Ahmedabad. Stems were shade dried and pulverized for further use.

Preparation of Extract
100 g powder of stems was subjected separately to continuous and sequential hot solvent extraction using 500 mL 50% methanol in a soxhlet apparatus for 48 h at 50 °C. Filtrate was dried under vacuum using rotary vacuum evaporator at temperature below 50 °C (percentage yield = 20 %w/w) which was stored in air tight container for further use upon drying.

Phytochemical Analysis
Preliminary phytochemical tests were performed to detect the presence of alkaloids, flavanoids, saponin glycosides, tannins, sterols, carbohydrates, phenolics, coumarins, fixed oils and fats. Total phenolic and flavonoid content of stems of G. arborea were determined in methanol extract by following methods [21,22].

Estimation of Phenolic Substances
One gram of air-dried powder of stems of G. arborea was extracted with 100 mL of methanol by maceration for 24 h to prepare methanol extract and then filtered. The final volume of the filtrate was adjusted to 100 mL. Five mL of the filtrate was diluted with an equal volume of methanol and was used for the estimation of phenolics. To 10 mL of stem extract, 10 mL of distilled water and 1.5 mL of diluted (1:2) Folin Ciocaulteau reagent was added and the mixture was kept for 5 min. After adding 4 mL of 20 %w/v Na 2 CO 3 solution, the final volume was adjusted to 25 mL using distilled water. The absorbance was measured at 765 nm at an interval of 30 min up to 2 h and distilled water was used as a blank. The data was compared with similarly prepared set of standard substance -gallic acid, in concentration range of 50 µg to 300 µg per 25 mL. Total phenolic content was calculated by the method described by Singleton and Rosi, 1965 [21].

Estimation of Flavonoids
One gram of air-dried powder of stems of G. arborea was extracted with 100 mL methanol by maceration for 24 h and filtered. The final volume of the filtrate was adjusted to 100 mL and one ml of the filtrate was diluted up to 10 mL with methanol and was used for the estimation of flavonoids. To 3 mL of this extract, 3 mL of methanolic AlCl 3 was added. After 10 min, the absorbance was read at 430 nm and distilled water was used as a blank. The data was compared with similarly prepared set of standard substance -quercetin, in concentration range of 25 µg to 300 µg per 25 mL. Total flavonoid content was calculated by the method described by Bahorun et al., 1996 [22].

Antioxidant activity MES of Gmelina arborea 2.5.1 DPPH Radical Scavenging Activity
The antioxidant activity of MES was assessed on the basis of the radical scavenging effect of the stable DPPH free radical [23]. MES (10-100 μL) was added to 3 ml of DPPH in methanol (0.33%) in a test tube. After incubation at 37 °C for 30 min, the absorbance of each solution was determined at 517 nm using UV visible spectrophotometer [24,25]. The corresponding blank reading was also taken and the remaining DPPH was calculated using following formula:

Hydrogen Peroxide Scavenging Potential
The ability of the MES to scavenge hydrogen peroxide was determined according to the method of Ruch et al. [26]. A solution of hydrogen peroxide (2 mM/L) was prepared in phosphate buffer (pH 7.4). MES (10-100 μg/mL) was added to hydrogen peroxide solution (0.6 mL) and absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide.

Reducing Power Assay
The reducing power of MES was determined according to the method of Gupta et al., 2007 [27]. Different concentrations of the MES (10-100 μg/mL) in 1.0 mL of deionised water were mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferrocyanide (2.5 mL, 1%). The mixture was incubated at 50 °C for 20 min. A portion of trichloroacetic acid (2.5 mL, 10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 mL) was mixed with distilled water (2.5 mL) and ferric chloride (FeCl 3 ) (0.5 mL, 0.1%) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing capacity of the sample. Institute of Pharmacy, Nirma University, Ahmedabad, India. Animals were maintained at 25 ± 2 °C , 50-60 % RH and kept under natural photoperiodic condition with free access to food and water. During the period of experiment the animals were fed with the standard diet (M/S Pranav Agro Tech Ltd. Vaghodiya, Vadodara) and water ad libitum. They were kept for one week in laboratories before the experiments for acclimatization to the laboratory conditions. The study was conducted after approval from the Institutional Animal Ethics Committee (IAEC) of Institute of Pharmacy and the protocol number given was IPS/PCOG/MPH1011/2016). Animal ethics guidelines and good laboratory practice guidelines were followed and precautions were taken to minimize pain and discomfort to the animals.

Preparation of Test Drug Material
Suspension of methanol extract of stems (MES) of G. arborea was prepared freshly using 0.5% carboxymethyl cellulose (CMC) in normal saline and was used for evaluation of anticonvulsant activity.

Experimental Protocol for Anticonvulsant Activity
The mice were randomly divided into four different groups with 6 mice (n = 6) in each group for PTZ and STR induced models (Table 1). Two different doses of MES were administered orally using oral gastric gavage tube one hour prior to the administration of the inducers viz. PTZ (60 mg/kg, s.c.) and STR (2 mg/kg, i.p.) [28]. The animals were observed for 1 h by placing in a separate cage. The onset of seizure and duration of tonic-clonic convulsions were recorded and the number of animals convulsing or not convulsing within the observation period were noted [29]. The latency for development of convulsions in mice and mortality rate were also noted. The ability of the plant extract to prevent or delay the onset of convulsions was taken as an indication of anticonvulsant activity [30]. The doses were decided on basis of literature review wherein these dose levels have been screened for other pharmacological activities of the extracts of same plant including the evaluation of anti diabetic activity of the roots and stems of G. arborea at the dose levels i.e. 250 and 500 mg/kg body weight in Wistar rats [31,32]. The toxicity studies have also been reported for aqueous and methanol extract of bark of G. arborea (ME). Acute toxicity study on Swiss albino mice at a dose range of 300-5000 mg/kg, p.o. and repeated dose toxicity study on Wistar rats at the doses of 300, 1000, and 2000 mg/kg/day, p.o. for 28 days have been reported with no symptoms of toxicity or mortality at the doses tested in both acute and repeated dose toxicity study [31,33,34].

Statistical Analysis
Data were expressed as mean ± S.E.M using Graph pad prism 5. Statistical significance between normal control and diseased control groups was tested using one way ANOVA followed by Post-hoc Dunnett's t-test.

Phytochemical Analysis
Results revealed that methanol extract of stems showed considerable amount of flavonoids and phenolics present along with presence of alkaloids, tannins, carbohydrates, sterols, lignans and saponin glycosides. Further estimation of total phenolics and flavonoids clearly indicated phenolic rich composition of stems with 0.634% of phenolics and 0.9% of total flavonoid content in the methanol extract of stems (Fig. 1).

DPPH Radical Scavenging Activity
Decrease in the absorbance was read to calculate % inhibition in the presence of extracts of stems of Gmelina   arborea. Results showed concentration dependent response relationship in DPPH radical scavenging activity and the activity was found increased with increased concentration of the extract. IC 50 value of MES was found 47.4733 µg/ml (Fig. 2).

Hydrogen Peroxide Scavenging Potential
The Scavenging ability of methanol extract of powdered stem of G. arborea is shown in Fig. 3. The extract was capable of scavenging peroxide radical in concentration dependent manner and the IC 50 value for scavenging of H 2 O 2 for methanol extract was found to be 97.3310 µg/ ml (Fig. 3).

Reducing Power Assay
The results of reducing power assay depicted increase in the absorbance observed with corresponding increase in the concentration of methanol extract as illustrated in the Fig. 4. The conversion of Fe 3+ to Fe 2+ in the presence of methanol extract could be considered as the reductive capability of constituents present in the extract. The results are suggestive of Fe 3+ reducing ability of the constituents of methanol extract of G. arborea stems which might be observed due to neutralizing effect on the free radical followed by forming a stable complex.

Evaluation of Anticonvulsant Activity of MES of G. arborea
The anticonvulsant activity of MES (250 and 500 mg/ kg) were studied in PTZ and STR induced seizure models. Our data suggests that methanol extract of stems showed significant dose dependant delay in the onset of seizures.

Pentylenetetrazole (PTZ) Induced Convulsion Model
Results of our study indicated that administration of MES at dose of 500 mg/kg body weight, one hour prior to injection of PTZ, significantly delayed the onset of convulsions (8.188 min, P<0.0001) which was found even higher than the standard group (3.494 min). However, it was observed that MES at the same dose level were not able to decrease the duration of clonic and tonic convulsions. MES at the dose of 250 mg/kg body weight was also found to be active due to their ability to delay the onset of convulsion by 2.408 min respectively. Further, pre-treatment with MES was found to prolong the latency of convulsions induced by PTZ dose dependently and protected the animals from mortality. MES at the dose of 250 mg/kg and 500 mg/ kg protected 60% and 80% of the animals while the standard antiepileptic drug, diazepam, offered complete protection to the animals from mortality (Table 2).

Strychnine (STR) Induced Seizure Model
In STR induced seizure model, mortality rate was found to be more than that in PTZ induced seizure model. MES at dose of 500 mg/kg was found to produce significant (P<0.0001) delay in onset of convulsion (11.81 min) while treatment with 250 mg/kg showed less increase in onset of convulsion. MES at dose of 250 mg/kg were found to have minimum protection against convulsion, 40%. MES at dose of 500 mg/kg showed 80% protection (Table 3).

Discussion
Results of phytochemical analysis revealed that methanol extract of stems showed considerable amount of flavonoids and phenolics which may be responsible for observed antioxidant and anticonvulsant activities of MES. Neuronal hyper excitability and unwarranted production of free radicals have been implicated in the pathogenesis of a substantial array of neurological disorders, including epilepsy [35]. The high rate of oxidative metabolism, coupled with the low antioxidant defences and the richness in polyunsaturated fatty acids, makes the brain highly vulnerable to free radical damage. Further many free radical scavenging assays are utilized to judge antioxidant potential of natural compounds and DPPH radical scavenging activity is considered as a basic and rapid screening method which demonstrated the antioxidant effect by decrease in the absorbance at 517 nm [36]. Free radical quenching has been recognized as one of the pivotal mechanism to prevent lipid peroxidation by various oxidants. Methanol extracts of powdered stem of G. arborea was able to reduce free DPPH with IC 50 value 47.4733 µg/ml. The effect of antioxidants on DPPH is thought to be due to their hydrogen donating ability. Through the DPPH radical scavenging activity, the study showed that the extracts have the proton-donating ability due to presence of phenolics and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants [36]. Hydrogen peroxide radical is not very much reactive, but can sometimes be noxious to cells due to generation of extremely reactive hydroxyl radicals in the cells which have been implicated as highly damaging reactive species. Scavenging of H 2 O 2 by methanol extract of stems might be attributed to their phenolic content, which can donate electrons to H 2 O 2 and thus neutralizing it to water [25]. The extracts were capable of scavenging hydrogen peroxide (IC 50 = 97.3310 µg/ml) in a concentrationdependent manner which is considered as an important antioxidant defence parameter in the cells.
Methanol extract of powdered stem of G. arborea showed the reductive capabilities in a concentrationdependent manner, as results indicated increase in absorbance in reducing power assay. MES could reduce the most Fe 3+ ions owing to its phenolic composition. Increased absorbance of the reaction indicated increased reducing capabilities [27]. Phytochemical screening and estimation of total phenolic content showed considerable amount of phenolics present in stems of this plant. This report suggests likely development of a mechanism behind the observed anticonvulsant activity of stems and the possible role of the polyphenols in anticonvulsant potential with underlying mechanisms like antioxidant activity.
The anticonvulsant activity of MES of G. arborea at two dose levels (250 and 500 mg/kg) was studied by PTZ and STR induced seizure model. PTZ is a noncompetitive Gamma Amino Butyric Acid (GABA) receptor antagonist and works by inhibiting the activity of GABA at GABA A receptor. On the other hand STR induces convulsions by antagonising glycine receptor and increases postsynaptic excitability and ongoing activity in dorsal neurons [37,38]. PTZ is the most popular and widely used chemically induced seizure model and it represents an effective model for human generalized myoclonic and also absence seizure while strychnine model is considered valid for primary generalized seizures [39].
Most of the anti-epileptic drugs act by enhancing GABA neurotransmission. In PTZ induced seizure model, PTZ produced clonic convulsions and lethality in mice, while pre-treatment with MES at two different dose levels has resulted in to delayed onset of convulsions, prolonged latencies of tonic seizures and reduction in lethality.
It has been well reported in literature that flavonoids and phenolics enhance GABA neurotransmission and GABA is the main inhibitory neurotransmitter which is suppressed in epilepsy [40]. Thus, the suppression of seizures by MES dose-dependently might be attributed to phenolics reach composition of the extract through enhancing GABA neurotransmission in brain or by blockade of glutamatergic neurotransmission mediated by the NMDA receptor. However effects of MES of G. arborea on glutamatergic neurotransmission was not studied in this study.
Strychnine has been demonstrated to have a welldefined mechanism of convulsant action by directly antagonizing the inhibitory spinal cord and brainstem reflexes of glycine and thus increasing spinal reflexes [41][42][43]. Glycine is an important inhibitory transmitter to motor neurons and interneurons in the spinal cord, and STR acts as a selective and competitive antagonist to block the inhibitory effects of glycine at all glycine receptors. STR sensitive synaptic inhibition in higher centres of the CNS is also mediated by glycine [28].
In the STR-induced seizure model, it is known that STR directly antagonises the inhibitory spinal reflexes of glycine [43] and suppression of seizures by MES at dose of 500 mg/kg may be due to, the glycine inhibitory mechanisms of constituents of MES. MES in dose of 500 mg/kg was significantly active against convulsion produced by PTZ and STR.

Conclusion
In present study, anti-oxidant activity and protective effects of methanol extract of stems against seizures induced by PTZ and STR were evaluated. The observed antioxidant and anticonvulsant activities are due to the presence of considerable amount of flavonoids and phenolics in the extract of stems. Increased oxidative load is directly implicated as seizures can cause imbalance in oxidant, antioxidant system of brain which leads to oxidation of lipids, DNA and protein ultimately resulting into neurodegeneration. Methanol extract of stems of G. arborea showed good anticonvulsant activity in PTZ as well as STR induced convulsions may be through the involvement of GABAergic and glutaminergic transmission and through glycine inhibitory property. However, further studies are needed to develop the exact underlying mechanism of anticonvulsant action of possible constituents of the plant after isolation of bioactives. Thus results of our study showed promising anti-oxidant and anticonvulsant effects of methanol extract of stems against both the toxicants and provided a scientific claim to the usefulness of this traditional plant in neurological disorders like epilepsy.