Anticonvulsant activity of methanolic extract of Withania cogulans in mice

Mental and neurological diseases including depression, Parkinson’s disease, dementia, epilepsy, anxiety disorders and bipolar disorders account for a considerable amount of the world’s disease burden. Unfortunately, drugs used in the treatment of neurological diseases are expensive, symptomatic and they produce undesirable side effects. People from different cultures prefer to use medicinal plants for the treatment of various ailments ranging from plain to perplex disorders because they are most affordable, cost effective and easily accessible source of treatment in the primary healthcare system throughout the world. Withania coagulans, an erect grayish under-shrub belongs to family Solanaceae. It is common in Pakistan, East India, Iran and Afghanistan. The objective of this study was to analyze the anti-seizure activity of crude methanolic extract of Withania coagulans fruits (MeWc). For screening of this activity, maximal electroshock seizures model (MES) and chemically-induced seizures models were used. In maximal electroshock seizures test MeWc showed significant dose dependent percent protection against hind-limb tonic extension; significant and dose-dependent increase in latency to myoclonic jerks and tonic clonic convulsions and decrease in seizures duration were observed in PTZ-induced seizures. In strychnine-induced convulsions MeWc significantly increased latency to hind-limb tonic extension and percent protection from death in a dose-dependent manner. Thus, it was inferred from the experiments that extract of Withania coagulans showed anticonvulsant activity.


Introduction
The term epilepsy was discovered from the Greek word "Epilepsia" which means "to take hold of", "seized" or "attacked". Epileptic seizure generates due to abnormal hyperactivity in the brain (Fisher et al. 2005). It is the widespread, chronic, serious neurological disease affecting 65 million people worldwide (Thurman et al. 2011) and its symptoms include disturbance of movements and sensation (hearing, vision and taste), unconsciousness, anxiety, depression, disturbed mood or other cognitive dysfunctions. Idiopathic epilepsy is the most common type affecting about 60% epileptic people. Other type is symptomatic or secondary epilepsy with known causes. The causes of symptomatic epilepsy may be severe brain injury, stroke, brain tumor, certain genetic disorders, brain infections and birth defects (Megiddo et al. 2016).
It has been reported that epilepsy affects approximately thirty thousand people every year and in every 20 people, one man develops epilepsy in his life. The ratio of new epilepsy cases is 20-70 cases/lakh population every year globally (Shorvon 1990). As compared to elder population, incidence rate of epilepsy is more in children and adults in developed countries, while in developing nations, range of active epilepsy in Asia is 1.5-14 per 1000, in South America 17-57 per 1000 and in African countries this ratio is 5.2-43 per 1000 population (Mac et al. 2007).
There are various pathologies involved in the brain that leads to the development of epileptic disorder such as increased hyperexcitability due to abnormal synaptic connectivity, suppressed GABAergic receptor function, dysfunction of potassium channels and sodium channels, increased neurotransmission of glutamate or decrease level of GABA level etc. (Wojda et al. 2009). Moreover, variety of models have been used for the screening of antiepileptic activity and for the investigating mechanistic pathway of the unknown compounds. These models include MES model (altering sodium influx and enhances glutamate level), PTZ induced seizures model (inhibiting GABA receptor), Kainic acid-induced seizure model, pilocarpine-induced seizure model (enhancing glutamate receptor activity), strychnineinduced seizure model (antagonism of glycine receptor) (Kupferberg 2001;Löscher 2017).
Conventionally, there are various classes of drugs available for the management of epilepsy such as Benzodiazpines, barbiturates, GABA analogs, succinimides, hydantoins, and carbamazepine (Goldenberg 2010). These AEDs are linked with various adverse effects, teratogenicity and dose-related chronic toxicity (Stefan et al. 2004). Drugs used in neurological diseases are expensive and they produce severe side effects which make the treatment difficult; so there is a demand for new drugs which are beneficial in neurological diseases and which are cost effective. One of the approaches is to search for naturally-occurring compounds (Vigil et al. 2005).
Withania coagulans belonging to family Solanaceae is mainly distributed in Pakistan, East India, Iran and Afghanistan (Maurya 2010). It is having globose-shaped smooth berries having diameter up to 6-8 mm and the seeds are somewhat ear-shaped having 2.5-3 mm diameter (Kirtikar and Basu 1918). Several phytochemicals like alkaloids, flavonoids, tannins have been extracted, characterized and isolated from aerial parts, roots and berries (Kapoor 2000). The antiepileptic activities of fruits of Withania coagulans has not been investigated adequately which influenced us to design this study which would help in exploring a novel agent from the plant source.
The objective of the study was to evaluate anticonvulsant activity and safety profile of MeWc in mice. Anticonvulsant activity was determined by using maximal electroshock test (MES) and chemical-induced seizure models like pentylenetetrazole (PTZ) and strychnine (STN). The safety profile of MeWc was investigated by acute toxicity and neurotoxicity tests.

Animals grouping
Male Balb/c mice weighing 20-30 g were used in each model. Animals were obtained from animal house of Department of Pharmaceutical Sciences, Abbottabad University of Science & Technology, Abbottabad. Animals were kept under standard conditions; 25 ± 2 °C and 12/12 h light/dark cycle and were fed with easily accessible food pellets and water.

Chemicals and plant material
Pentylenetetrazole and strychnine were purchased from Sigma Aldrich. Methanol was obtained from Musaji & sons, Rawalpindi while sodium valproate and Phenytoin were received as gift samples from PolyFine Chem Pharma (Pvt) Ltd. Peshawar and Adamjee Pharmaceuticals (Pvt) Ltd. Karachi. All the drugs and extracts were dissolved in the normal saline. Withania coagulans fruits were purchased from local herb market in Karak, Khyber Pakhtoonkhwa, Pakistan. Taxonomical identification of plant was performed by Dr. Syed Mujtaba Shah, Botany Department, Hazara University, Mansehra. A voucher specimen number HUP/S02 for Withania coagulans was submitted in the herbarium of said Department. Preliminary phytochemical analysis of the MeWc was carried out for the presence of phytochemicals such as steroidal compounds, phenolic compounds, alkaloids, saponin, flavonoids and tannins using standard procedures (Trease and Evans 2009).

Acute neurotoxicity (TD 50 )
Acute neurotoxicity of MeWc was determined by method developed by Coughenour andcolleagues in 1977 (Coughenour et al. 1977). It consisted of metal bars to which square platform was attached. The square platform was made of 0.6 cm wire mesh. One day prior to the experiment mice were pretrained on the apparatus. Those failing to climb the inverted screen were excluded. Testing was carried out 30 min after intraperitoneal administration of different doses of MeWc. Mice which were unable to climb inverted screen within sixty seconds were rated as failures.

Anticonvulsant activity of extract of Withania coagulans
Anticonvulsant screening of the extract was determined by maximal electroshock seizure test. Antiepileptic activity of the material was further validated by chemically induced antiepileptic models in mice. In each set of experiments, animals were randomly divided into five groups (n = 8) i.e. control group, reference standard group and 3 test groups. Experiments were blinded during the course of treatment and data collection.

Maximal electroshock seizure (MES) test
In MES test the electrical stimulus 72 mA for 0.2 s was applied to produce hind-limb tonic extension (HLTE) in control animals which served as group-I. The electrical stimulus was applied through transauricular electrodes (Kupferberg 2001). In group II phenytoin (20 mg/Kg) was intraperitoneally injected to mice and it served as reference standard group. (100-300 mg/Kg) MeWc were administered to group III-V and it served as test groups. All drugs and extract doses were injected intraperitoneally 30 min prior to the stimulus. Mice were considered protected if they were not displaying hind limb tonic extension.
Percent protection was calculated by using the following formula: Chemical induced seizure models

Pentylenetetrazole (PTZ) seizure test
In PTZ seizure test mice were divided randomly into five groups consisted of 8 mice each. Group-I received normal saline (10 ml/kg). Group-II was injected with 450 mg/Kg sodium valproate while group III-V were administered with (100 mg/Kg, 200 mg/Kg, 300 mg/Kg) MeWc. After 30 min of the drugs and plant extract treatment, PTZ (85 mg/Kg) was administered subcutaneously to all groups. Each animal was observed for a duration of 30 min for onset of myoclonic jerks and onset of tonic-clonic seizures. Total duration of seizures were also noted in 30 min observation. All doses were administered intraperitoneally except PTZ which was administered subcutaneously.

Strychnine (STN) seizure test
In STN seizure test 1.2 mg/Kg of STN was administered subcutaneously to group-I (Krall et al. 1978). Sodium valproate (550 mg/Kg) was injected to group II (positive control group). Three doses of MeWc (100 mg/Kg, 200 mg/Kg, and 300 mg/Kg) were injected to groups III-V. After administration of doses, STN was administered 30 min later to all groups and they were examined for protection against hind limb tonic extension (HLTE) and death.

Statistical analysis
All experimental data was analyzed statistically for significance by One-Way ANOVA using Origin 6 software. Values were expressed as mean ± SEM. P value of <0.05 was considered significant. Note that significant differences from control group were indicated as *** P < 0.001, ** P < 0.01 and * P < 0.05 by one-way ANOVA test.

Extraction and phytochemical analysis of the plant material
Dried fruits of Withania coagulans were ground to fine powder with the help of electric herb grinder and then extracted by maceration with methanol for 15 days. The process was carried out at room temperature with occasional shaking and left the mixture so that the plant material was maximally dissolved in organic solvent. After 15 days, it was filtered with white thin cloth. By repeating the same process thrice methanol soluble filtrate was obtained which was then concentrated with rotary evaporator at 40 °C and in this way crude methanolic extract of fruits was obtained. Results of the phytochemical analysis were depicted in Table 1. It has been reported in literature that 1 g of methanolic extract of Withania coagulans contains 55.9 mg of total phenolic contents (equivalent to gallic acid), 76.6 mg of total tannin contents (equivalent to tanic acid), 0.88 mg and 0.25 mg of total flavonoid and flavonol contents respectively (equivalent to rutin) (Prasad et al. 2010).

Acute neurotoxicity (TD 50 )
For assessment of acute neurotoxicity, inverted screen test was carried out after 30 min of administration of different doses to animals (600, 800, 1000, 1200 mg/Kg; n = 8; i.p.) of Withania coagulans extract. At 1000 mg/Kg dose all tested mice climbed the screen illustrating that MeWc didn't show any neurotoxic effects on the said dose (Fig. 1).

MeWc enhanced the percent protection in MES and increased the duration of seizures in PTZ induced model
Results presented in Fig. 2A and B showed that MeWc prevented percent protection against hind limb tonic extensor jerks in comparison to PTZ-treated animals. Figure 2A depicted that MeWc at 100, 200 and 300 mg/Kg protected animals from HLTE to 50%, 62.5% and 75% respectively compared to the control group (n = 8), while 20 mg/Kg phenytoin used as standard drug showed 100% protection in MES model of seizures. Similarly, MeWc extract has dose dependent effects on seizures duration. It has been observed that MeWc 100 mg/ Kg reduced the duration of seizures to 43 ± 3.35 s from 67.29 ± 3.84 s (PTZ treated group), while in 200 and 300 mg/ Kg tested groups the duration of tonic-clonic seizures was further reduced to 29.57 ± 1.75 s and 21.43 ± 1.68 s respectively as compared to the PTZ treated group (P < 0.001, Fig. 2B). Similarly, sodium valproate (450 mg/Kg) which was used as standard completely prevented the seizures in PTZ induced seizures (Fig. 2B).
MeWc increased the onset of myoclonic and tonic-clonic jerks in PTZ-induced seizures test.
MeWc delayed the onset of myoclonic and tonic-clonic jerks PTZ-induced seizure model in a dose dependent manner. It has been observed that PTZ (85 mg/Kg) caused the onset of myoclonic jerks in PTZ-treated group in 195.57 ± 7.32 s. MeWc in lowest tested dose (100 mg/Kg) significantly delayed the initiation of myoclonic jerks from 195.57 ± 7.32 s (control group) to 215.14 ± 5.16 s (Fig. 3A P < 0.05, F = 6.4). At 200 mg/Kg dose latency to myoclonic jerks was increased to 232.71 ± 9.37 s (p < 0.01, F = 12.8). Similarly, at 300 mg/Kg dose, latency to myoclonic jerks was further increased to 255.7 ± 9.60s (p < 0.001, F = 23.2). With increasing doses, an increase in latency was observed (Fig. 3A, n= 8 in each group). Furthermore, sodium valproate at a dose of 450 mg/Kg prevented the onset of myoclonic jerks in all the animals.
Moreover, our results shown that MeWc delayed significantly the initiation of tonic-clonic seizures in mice in   Fig. 2A) and reduced the duration of seizures in PTZ-induced seizure model (Fig. 2B). P*** < 0.001 vs. control group by applying one-way ANOVA. Note that NS = normal saline, MES = Maximal electroshock, Na Val = sodium valproate, PTN phenytoin, PTZ = pentylenetetrazole PTZ-induced seizure model. An increase in latency was observed with the incremental doses of plant extract in comparison to PTZ treated group (298 ± 7.93 s). MeWc at different doses (100, 200, 300 mg/Kg) significantly and dose-dependently raised the onset of tonic-clonic seizures in mice in PTZ-induced seizure model. It was noted that MeWc at 100 mg/Kg, latency to tonic-clonic seizures was increased to 354 ± 8.64 s (P < 0.001, F = 30.1). At 200 mg/ Kg, the latency to tonic-clonic seizures was increased to 386 ± 5.773 s (P < 0.001, F = 102.9) and at 300 mg/Kg the latency was further increased to 430 ± 13.42 s as compared to the PTZ-treated group (Fig. 3B, n = 8, P < 0.001, F = 98.7).

MeWc increased the percent protection and latency to HLTE and death in strychnine induced seizures
Hind limb tonic extension is considered as the last stage of the seizure scores that leads to the generalized seizures and ultimately cause death of the animals. It has been observed that MeWc significantly enhanced the % protection from HLTE and also delayed the onset of HLTE and death. It has been noted that HLTE was produced in all the animals by administration of strychnine (1.2 mg/Kg). MeWc at different doses (100, 200 and 300 mg/Kg) produced dose dependent and significant (Fig. 4A, n = 8, P < 0.05) percent protection from HLTE. Protection against strychnine induced seizures was observed 100% in sodium valproate 550 mg/Kg (standard group).
Similarly, MeWc delayed the time of onset of both HLTE and death in a dose dependent manner (Fig. 4B, n = 8, P < 0.05). In case of strychnine-treated group, latency to HLTE and death was 286.57 ± 11.70s and 302.28 ± 14.95 s respectively (Fig. 4B, n = 8). MeWc at 100 mg/Kg delayed latencies to HLTE and death to 346.85 ± 12.88 s and 352.85 ± 12.46 s respectively as compared to the control group. Similarly, MeWc 200 mg/Kg delayed the latencies to HLTE and death to 376.42 ± 10.12 and 382.85 ± 9.62 shown that MeWc delayed the occurrence of myoclonic seizures significantly. Figure 3B shown the delayed effects of MeWc on the onset of tonic clonic seizures caused by the administration of PTZ. Data obtained were represented as Mean ± SEM. P value was taken as significant P < 0.05, P** <0.01, P*** < 0.001 vs. control group by applying one-way ANOVA Fig. 4 MeWc enhanced the percent protection from HLTE (4A) in Strychnine-induced seizure model and delayed the onset of HLTE and death (4B) reduced the duration. Data were represented as Mean ± SEM and analyzed through one-way ANOVA in Origin statistical software. P value <0.05 was taken as significant. STN = strychnine respectively. Further, MeWc 300 mg/Kg delayed the latency to hind limb tonic extensions and latency to death further to 395 ± 10.05 and 402.42 ± 10.06 respectively. Moreover, sodium valproate prevented all the animals from HLTE and death as compared to the strychnine-treated group.

Discussion
Epilepsy being the second most common neurological disorder which affects approximately 80% people in developing countries (Yemedje et al. 2011). Synthetic drugs that are currently available for treatment of these disorders exhibited undesirable side effects. Therefore, to overcome this situation, search for new drugs from medicinal plants has progressed in the past decade. In the present study, anticonvulsant activity of MeWc was investigated through pentylenetetrazole (PTZ) seizure model, strychnine-induced seizure model and maximal electroshock seizure (MES).
It has been known that MES model potentiate influx of sodium and also raise the level of glutamate at the synaptic terminals. Glutamate then binds to the NMDA receptor that ultimately induced symptoms that mimics epileptic seizures in animals. Phenytoin and sodium valproate works by inhibiting NMDA-mediated opening of sodium influx and prevented the spread of the seizures and showed their anticonvulsant effects. The extract, MeWc, was not able to completely prevent HLTE in all mice at any dose but it increased the percent protection against MES induced HLTE with the increasing dose. Withania coagulans contains several phytochemicals including withanolides like withanolide A, withanolide H, withaferin A, withacoagulin H, withanolide K, and withanolide J etc. (Ihsan-ul-Haq et al. 2013;Ali et al. 2015). Withanolide A isolated from Withania somnifera has shown ameliorating effects in temporal lobe epilepsy and impaired learning and memory mediated by altered function of AMPA and NMDA receptors (Soman et al. 2012;Soman et al. 2013;Zhu et al. 2020). These findings indicate that withanolide A may modify conductance through NMDA and AMPA receptors coupled cationic channels and offer neuroprotection against epilepsy. Withanolides being relatively non polar compounds can enter in brain (Muhasaparur Ganesan et al. 2021). Similarly, in this study, protection may be due to presence of chemical constituents like wathanolide A in the extract that may prevent the spread of seizures by altering cationic conductance. Thus, it may be suggested that MeWc exerts its anticonvulsant effects through inhibiting sodium influx and prevented neuronal hyperexcitation further experimentation is needed to elaborate these effects of MeWc.
PTZ may exert its convulsant effects by potentiating calcium currents or by inhibiting GABA (inhibitory neurotransmitter) particularly at GABA A receptors (Huang et al. 2001). Anticonvulsant activity of an agent may be indicated when it completely stops or delays the onset of tonic-clonic convulsions induced by PTZ. As we evaluated in the current experiments that MeWc delayed the occurrence of high frequency seizures in mice and also decreased the duration of convulsions produced by the administration of GABA-A receptor antagonist (PTZ), it is inferred that MeWc may act through GABA receptor in the prevention of seizures.
Furthermore, strychnine antagonizes glycine receptors in the brain leads to the inhibition of hyperexcitation of the neurons that ultimately produces seizures (McGaraughty and Henry 1998). Our results demonstrated that administration of MeWc significantly increased the latency of seizures in strychnine-induced convulsions as compared to control group suggesting the possible involvement on glycinergic neurotransmission.

Conclusion
Extract of Withania coagulans have shown promising results in increasing percent protection in MES and STN induced models of seizures, delayed the occurrence of myoclonic and tonic-clonic seizures in PTZ-induced seizures and also delayed onset to HLTE and death in strychnine-induced seizure model in mice. Thus it was concluded from these experiments that Withania coagulans had a promising antiseizure activity. Further studies could provide the deep insight into the mechanistic pathway(s) involved in the prevention of epileptic seizures by Withania coagulans.