Preliminary Screening of a Classical Ayurvedic Formulation for Anticonvulsant Activity.

BACKGROUND: Epilepsy is a serious and complex central nervous system disorder associated with recurrent episodes of convulsive seizures due to the imbalance between excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmitters level in the brain. The available treatments are neither competent to control the seizures nor prevent progress of disease. Since ages, Herbal medicines have remained important sources of medicines in many parts of world which is evidenced through their uses in traditional systems of medicine i.e. Ayurveda, Siddha, Unani, Homeopathy and Chinese etc. AIM: A polyherbal formulation (containing Terminalia chebula Retz., Asparagus racemosus Willd., Embelia ribes Burm. F, Acorus calamus L., Tinospora cordifolia (Willd.) Miers, Convolvulus pluricaulis Choisy, Saussurea lappa C.B.Clarke, Achyranthes aspera L.) is mentioned in Ayurvedic classics Bhaiṣajya Ratnāvali. The aim of the study was to evaluate the anticonvulsant activity of the formulation in Maximum electroshock and Pentylenetetrazole induced convulsions in rats.
MATERIALS AND METHODS: In the present study, a polyherbal formulation was developed as directed by classical text and evaluated for the anticonvulsant activity using Maximal Electroshock Shock (MES) and Pentylenetetrazole (PTZ) induced convulsions in rats. Statistical comparison was done by one way ANOVA followed by the Tukey's multiple comparison test.
RESULTS: The obtained results showed that the PHF had a protective role on epilepsy. Treatment with PHF significantly improves antioxidant enzymes activities of superoxide dismutase (SOD) and glutathione (GSH) levels significantly as compared to controls. PHF also significantly decreased malonaldialdehyde (MDA) levels in the brain. Moreover, it also attenuated the PTZ-induced increase in the activity of GABA-T in the rat brain.
CONCLUSION: These findings suggest that PHF might have possible efficacy in the treatment of epilepsy.

Introduction

Epilepsy is one of the most widespread serious neurological conditions which affects approximately 1–2% of the world population with an annual prevalence of 50–100,000 per year.[1] More than 10 million people suffer from this disease in India.[2] The disease is characterized by brief episodes of seizures and excessive EEG discharge generally associated with the loss of consciousness with body movements (convulsions).[3] Epileptic seizures often cause temporary mutilation of perception, leaving the person at threat of physical damage.[45] Imbalance between excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmitters levels in the central nervous system (CNS) is widely accepted as the mechanism for pathogenesis of epilepsy.[6] Reduction in the concentration of GABA may result in many pathological alterations in the CNS and may manifest as convulsions.[7] Approximately 35% of the patients continue to have seizures and do not respond satisfactorily to regular pharmacological remedies using barbiturates, hydantoin and benzodiazapines that constitute the first line of treatment.[8] Typical antiepileptic treatments are unable to control seizures effectively and do not prevent progressive epileptogenic changes which are not well understood.[9] The available drugs are neither completely effective nor constantly safe and are usually associated with unwanted effects, toxicity and teratogenicity.[9] There is also a possibility of drug interaction with other drugs as epilepsy requires long term therapy. Epilepsy still continues as a neurological disorder in anticipation of safer drugs with enhanced therapeutic efficiency. World Health Organization (WHO) predicts that about 80% of world's epileptic patients live in developing countries and most of them do not get proper medical attention.[10] Different alternative therapies viz. herbal as well as yoga are also incorporated in the treatment of epilepsy along with mainstream therapy of drugs and in some cases, surgery. These are a few of the factors that create the need and hence the pursuit for innovative compounds of plant origin with fewer redundant side effects.[11] Several reports are available regarding anticonvulsant activity of different plants and efforts for further exploration of their activity is still going on.[12] Indian traditional medicine system which offers many potential medicinal plants for epilepsy either in raw form or in the form of formulations. In Ayurveda, plants such as Embelia ribes,[13] Bacopa monnieri, Ziziyphus nummularia, Cajanus cajan,[14] Mimosa pudica,[15] etc., have been suggested to be used to control epilepsy.

The present polyherbal formulation containing Terminalia chebula Retz., Asparagus racemosus Willd., Embelia ribes Burm. F, Acorus calamus L., Tinospora cordifolia (Willd.) Miers, Convolvulus pluricaulis Choisy, Saussurea lappa C.B. Clarke, Achyranthes aspera L. is indicated for memory enhancement and epilepsy in a popular Ayurvedic text Bhaiṣajya Ratnāvali, written by Govind Das Sen in 19th century.[16] The present study was undertaken to evaluate the anticonvulsant activity of the polyherbal formulation using Maximal Electroshock Shock (MES) and Pentylenetetrazole (PTZ) induced convulsions in rats.

Materials and Methods

Materials

The plants materials A. racemosus (root), T. cordifolia (stem), C. pluricaulis (whole plant) and A. aspera (whole plant) were collected from the Rajiv Gandhi South Campus, Banaras Hindu University, Barkachha, Mirzapur, Uttar Pradesh in the month of March-April 2014. T. chebula (fruit), E. ribes (fruit), S. lappa (root) and A. calamus (root) were procured from a local market in Mirzapur. The botanical authentication of the specimens was done by Prof. Anil Kumar Singh, Institute of Medical Science, Banaras Hindu University; Varanasi, India. For further reference, the voucher specimens (APRL/HERB/13-14/102-109) of plant materials were deposited in Rajiv Gandhi South Campus, Banaras Hindu University, Barkachha, Mirzapur (U.P.) India.

Preparation of formulation

The shade dried plant materials were made into coarse powder separately with the help of a mechanical grinder and then mixed in a definite proportion. Extraction was performed with 98% methanol by cold maceration technique. After that, the extract was concentrated in a vacuum evaporator and the dried extract was obtained (17% w/w). The dry polyherbal extract was suspended in 5% carboxy methyl cellulose (CMC) solution before oral administration to animals.

Chemical

Pentylenetetrazole, phenytoin and diazepam were purchased from Sigma Chemical Co. (Delhi, India). NADH (Sisco Research Laboratories Pvt. Ltd, Mumbai), NBT (Sisco Research Laboratories Pvt. Ltd, Mumbai) and were used for the experiments. ELISA kit (MBS939900; MyBioSource, Inc., San Diego, CA, USA) was used for the estimation of GABA-T activity. All the reagents used were of analytical grade.

Pharmacological evaluation

Animals

Adult Charles Foster albino rats (150 ± 20 g) of either sex were procured from the Central Animal House, Institute of Medical Sciences, Banaras Hindu University (Registration No-542/AB/CPCSEA); Varanasi. The animals were kept in a temperature–controlled room (22 ± 2°C) with humidity (55 ± 10%) and 12 h light dark cycle. The animals were provided with standard pelleted feed (Amrit Pvt. Ltd. Pune, India) and fresh water ad libitum. Rats were kept at standard laboratory environment for at least one week before the experiment. The study has been approved by the Institutional Animal Ethical Committee (IAEC) (Dean/2014/CAEC/858).

In vivo anticonvulsant screening

Maximal electric shock induced seizures

The electroshock was applied via ear-clip electrodes of rats by using an electroconvulsometer (Techno., India). Saline and other individual drugs were given at appropriate times. The 150 mA current stimulus with 50 Hz frequency was given to the animals for 0.2 s. The animals were observed for the occurrence of hind limb tonic extension (HLTE) within 60 sec following the stimulus. The electrodes were dipped in normal saline before electroshock delivery. The rats were divided into 5 groups, consisting of 6 animals in each group (n = 6). Group 1 received only carboxymethyl cellulose (0.5% CMC, p.o.); Group 2 Received Phenytoin (25 mg/kg, i.p.) whereas, Group 3-5 Received PHF 100, 200, 400 mg/kg. b.w. p.o. respectively. After 30 min, electrical stimulation was given to all the animals in all 5 groups. The absence of hind limb tonic extensor was considered as full protection in the model. The beginning of stupor, percentage of protection against mortality as well as time taken for death/recovery was also recorded.[17]

Pentylenetetrazole induced seizures

The PTZ-induced convulsion in animal model can be compared with myoclonic convulsions in human beings. The animals are divided into 6 subgroups each with 6 rats. Group I (normal control) received vehicle (0.5% CMC, p.o.) only. Group II (negative control) received PTZ (70 mg/kg, i.p.), whereas, Group III (standard treated group) received Diazepam (2 mg/kg, i.p.). Group IV, V, and VI (PHF treated groups) received PHF 100, 200, and 400 mg/kg, p.o. respectively. Animals treated with extracts and standard drug received single dose administration of PTZ after 30 min. The commencement of action showing forelimb and hind limb clonus, and percentage of protection against mortality as well as time taken for death/recovery was also recorded. Animals which showed no convulsions within 30 min were considered as protected and the percentage protection in each group was calculated. In unprotected animals, the latency of first convulsion was recorded. All the animals were carefully monitored for 30 min for any behavioral change related to convulsion. Finally after the observation, control and experimental rats were sacrificed by decapitation. The whole brain was dissected out and stored at −80°C for the estimation of GABA, GABA-T activity and in-vitro antioxidant activity.[18]

Estimation of GABA-T activity

GABA-T activity in the brain homogenate was measured spectrophotometrically.[19] at 450 nm using commercially available ELISA kit (MBS939900; MyBioSource, Inc., San Diego, CA, USA). GABA-T was expressed as pg/mg protein. 500 mg of tissue was rinsed with phosphate buffer solution (PBS, pH 7.2-7.4). Brain tissue was homogenized in 5.0 ml of PBS and stored overnight at −20°C. The homogenates were centrifuged for 5 min at 5000 ×g maintaining the temperature between 2 – 8°C. The supernatant obtained was assayed for the estimation of both GABA-T activity and oxidant stress markers.

Determination of GABA from the brain homogenate

For GABA quantification, 1 ml from each of the supernatant of brain homogenate and methanol were mixed together and centrifuged at 12000 rpm for 10 min. To a volumetric flask, 0.7 ml supernatant and 0.6 ml of borax buffer (pH = 8) were added, heated on a water bath at 80°C for 10 min and the final volume was adjusted to 5 ml with methanol. The 5 l solution was injected on phenomenex C18 and eluted with methanol: water (62:38 v/v) with a flow-rate of 1 ml/min. The concentrations were observed with the UV detector at 330 nm.[20]

Estimation of LPO activity

Lipid peroxidation [malondialdehyde (MDA)] was measured as per the method of Jainkang et al., (1990).[21] 1.0 ml of supernatant was added with 1.5 ml (20%, pH 3.5) acetic acid reagent, 1.5 ml thiobarbituric acid (0.8%) and 0.2 ml sodium dodecyl sulphate (8.1%). The reaction mixture was heated at 100°C for 60 min and then cooled under tap water. The mixture was then mixed with 1.0 ml of distilled water and 5.0 ml of n-butanol and pyridine mixture (15:1) and vortexed vigorously. The organic layer was separated after centrifugation at 4000 rpm for 10 min. Absorbance of organic layer was measured by using spectrophotometer at 532 nm and the concentration was expressed as nmol MDA/g tissue.

Estimation of GSH activity

Reduced glutathione activity was measured as per the method of Sedlak and Linsday, (1968).[22] Brain homogenate with an equal amount of trichloroacetic acid (50%) was centrifuged at 3000 g for 15 min. 1.5 ml of the supernatant was mixed with 4.0 ml of 0.4 M tris buffer and 0.1 ml of 5,5‘-dithio-bis-(2-nitrobenzoic acid) (DTNB). The mixture was vigorously vortexed and the absorbance was taken within 5 min after the addition of DTNB at 412 nm against reagent blank. The results were expressed as μg GSH/g of tissue.

Estimation of SOD activity

SOD activity was determined as per the method of Kakkar et al. (1984).[23] Sodium pyrophosphate buffer (1.2 mL, 0.052 M, pH 8.3), nitroblue tetrazolium (NBT) (0.3 mL of 300 μM), phenazine methosulphate (0.1 mL of 186 μM) and distilled water (0.8 mL) were added to the brain homogenate (0.4 mL). This mixture was added with 2.0 ml of 780 μM NADH solution and incubated at 30°C for 60 sec. After 60 sec, 1.0 mL of glacial acetic acid was added to the solution for termination of the reaction. The reaction mixture was then shaken strongly with 4.0 mL of n-butanol and later centrifuged at 3000 g for 5 min. Through this process the butanol layer was separated from rest of mixture. The absorbance was measured at at 560 nm against n-butanol as blank. A system devoid of enzyme served as control. SOD activity in the homogenate was expressed as unit of SOD activity/g of tissue.

Statistical analysis

All results are expressed as mean ± S.E.M (n = 6 in each group). Statistical comparison was done by one way ANOVA followed by the Tukey's multiple comparison tests using Graph Pad Prism Software Version 5.01, Inc. Fay Avenue, La Jolla, CA, USA.

Results

Effect of PHF on MES-induced convulsion

Oral administration of PHF (100, 200 and 400 mg/kg, p.o.) and phenytoin (25 mg/kg, i.p.) exhibited significant reduction in the duration of tonic convulsion when compared to negative control group. However, when compared to phenytoin (25 mg/kg, i.p.) treated group, PHF (200-400 mg/kg, p.o.) exhibited no significant difference in reduction of the duration of HLTE. This shows that the potency of the extract is comparable to that of phenytoin [Figure 1].

Figure 1

Effect of PHF (100–400 mg/kg, p.o.) on MES–induced convulsion in rats. Values are expressed as mean ± S.E.M (n = 6). Statistical comparison was analyzed by one way ANOVA followed by Tukey's multiple comparison test. aP < 0.05: Statistically significant as compared to negative control, bP < 0.05: Statistically significant as compared to phenytoin (PHT 25 mg/kg, i.p.)

Effect of PHF on PTZ-induced convulsion

Animal groups treated with PHF (200-400 mg/kg, p.o.) and Diazepam (2 mg/kg, i.p.) showed significant increase in the latency of tonic convulsions induced by PTZ (70 mg/kg, p.o.) as compared to negative control group. The percentage protection was observed to be as high as up to 66% and 100% when the rats were respectively treated with PHF and standard drug Diazepam [Figure 2].

Figure 2

Effect of PHF (100–400 mg/kg, p.o.) on PTZ–induced convulsion in rats. Values are expressed as mean ± S.E.M (n = 6). Statistical comparison was analyzed by one way ANOVA followed by Tukey's multiple comparison test. aP < 0.05: Statistically significant as compared to negative control, bP < 0.05: Statistically significant as compared to Diazepam (2 mg/kg, i.p.)

Effect of PHF on the level of oxidative stress markers

The results in Figure 3 indicate that rats administered with PTZ (70 mg/kg, i.p.) showed significant increase in the level of LPO, while significant reduction in the level of SOD and GSH was observed as compared to normal control group. Diazepam (2 mg/kg, i.p.) and PHF (400 mg/kg, p.o.) significantly produced an excellent reduction in the level of LPO and increased the level of SOD and GSH significantly. PHF (200 mg/kg, p.o.) also showed similar effect but was found to be ineffective in restoring the levels of SOD. However, PHF (100 mg/kg, p.o.) showed a significant recovery in the levels of LPO and GSH, but no effect was observed on SOD levels [Figure 3].

Figure 3

Effect of PHF (100–400 mg/kg, p.o.) on oxidative markers in PTZ–induced convulsion in rats, (a) effect on LPO level, (b) effect on GSH activity, (c) effect on SOD activity

Effect of PHF on GABA Level

PTZ (70 mg/kg, i.p.) administration in rats showed significant reduction in the level of GABA in the brain homogenate when compared to normal rats. Diazepam (2 mg/kg, i.p.) and PHF (200-400 mg/kg, p.o.) produced significant improvement in the level of GABA [Figure 4a].

Figure 4

Effect of PHF (100–400 mg/kg, p.o.) on GABA-T activity and GABA Level in brain in PTZ–induced convulsion in rats, (a) effect on GABA level, (b) effect on GABA-T activity

Effect of PHF on GABA-T activity

Rats administered with PTZ (70 mg/kg, i.p.) showed significant increase in the level of GABA-T enzymatic activity by 46% as compared to normal control group. Treatment of the animals with standard drug Diazepam (2 mg/kg, i.p.) and PHF (200-400 mg/kg, p.o.) produced a significant reduction in the level of GABA-T enzyme when compared to negative control group. However, the lower dose of PHF (100 mg/kg, p.o.) showed no significant effect in the reduction of enzyme activity [Figure 4b].

Discussion

MES induced seizures and subcutaneous PTZ induced seizures are the two most extensively studied and well established animal seizure models. Both tests provide models of single, acute seizures.[24] Inhibitions of inducible MES seizures or PTZ induced seizure by a PHF reported in the present study validates the persistent use of the formulation in the convulsive disorders.[15] Thus, chemical constituents with anticonvulsant activity and probable utility in the control of generalized convulsive disorders in epileptic patients can be isolated from different plants of this formulation. PHF (200 and 400 mg/kg, p.o.) dose-dependently reduced the convulsions in MES treated rats. Moreover, it also attenuated the PTZ-induced increase in the activity of GABA-T in the rat brain and reversed the PTZ-induced oxidative stress in brain.

The MES test identifies compounds which prevent seizure spread.[25] Therefore, it serves as a tool for detecting potential antiepileptic drugs effective against generalized tonic–clonic (grand mal) seizures.[26] Different underlying mechanisms are explained for the drugs effective in MES induced convulsion. For example phenytoin and rufinamide inhibit voltage-dependent Na+ channels whereas felbamate blocks N-methyl-d-aspartate (NMDA) receptors.[27] The present results indicate that neither phenytoin nor PHF (200 and 400 mg/kg, p.o.) protect the animal completely, but a significant reduction in the duration of tonic convulsion was observed. Lower dose of PHF (100 mg/kg, p.o.) showed no significant reduction in the duration of convulsion in MES exposed animals. Phenytoin is an effective and widely used standard drug in MES model.[27] which primarily acts by blocking voltage-dependent Na+ channels. Thus, the present study indicates that PHF (200 and 400 mg/kg, p.o.) has significant ability to slowdown the spread of seizure.

PTZ, an antagonist of GABA receptor complex, is a well known and established animal model for evaluating phytomedicine as well as other drugs against generalized absence seizures in humans.[24] The possible mechanism of action of effective drugs may be enhancement of GABAergic neurotransmission such as Phenobarbital, Diazepam and tiagabine.[28] or through T-type calcium (Ca2+) channels like ethosuximide.[29] In the present study, standard drug diazepam (2 mg/kg, i.p.) showed complete protection against PTZ-induced convulsion while PHF (200 and 400 mg/kg, p.o.) showed dose dependent effect against PTZ-induced convulsion. A catabolic enzyme GABA-T in the mammalian brain is responsible for the transfer of amino group from GABA to α-ketoglutarate which leads to the reduction in the level of GABA.[30] PTZ significantly increases the level of GABA-T activity in the rat brain. Administration of PHF (200-400 mg/kg, p.o.) and Diazepam (2 mg/kg, i.p.) significantly attenuated the enzyme activity indicating potent GABA-T inhibitory effect. It is well studied that free radicals are generated during convulsion and disturb the balance between GABA and glutamate activity in the brain.[31] Thus, in addition to GABA-T inhibitory activity, we have attempted to estimate the markers of oxidative damage in the brain of PTZ exposed rats. PTZ (70 mg/kg, i.p.) significantly increased the activity of LPO enzyme and reduced the activity of antioxidant enzymes such as SOD and GSH in the rat brain.[32] Both standard drug as well as PHF (200 and 400 mg/kg, p.o.) decreased the PTZ-induced increase in the activity of LPO. On the contrary, both increased the PTZ-induced decrease in the activities of SOD and GSH in the brain. This indicates that PHF (200 and 400 mg/kg, p.o.) can prevent oxidative stress in epileptic rats. Analogous effectiveness of the PHF to that of diazepam in PTZ induced seizure; indicate interaction of PHF with the GABAergic pathway and usefulness against human petit mal epilepsy.[32]

Bisdesmosidic saponins (I-III), 20 hydroxyecdysone, quercetin-3-O-ß-D-galactoside.[33] were isolated from aerial parts of A. aspera which have antioxidant activity and anticonvulsant activity.[34] T. chebula[35] and C. pluricaulis[36] have been reported for anticonvulsant activity in rats. A. calamus and S. lappa were also evaluated for their anticonvulsant activity.[2537]

Embelin.[13] isolated from E. ribes and berberine from T. cordifoli.[383940] were evaluated for their anticonvulsant activity. Moreover anthraquinones present in T. chebula and quercetin derivatives in A. aspera were also reported for antiepileptic effect.[414243]

Epilepsy is a chronic condition of spontaneous and recurrent seizures that are closely linked to the GABAA receptors in the brain. A seizure happens when there is an unexpected disproportion among the excitatory (Glutamate) and inhibitory (GABA) neurotransmitters in a neuron network. This in turn leads to hyper excitability. The condition also increases ROS in brain. Hence, all aspects of these two neurotransmitters are very important in search of an effective antiepileptic drug. Moreover, risk of oxidative damage of the brain may increase with an imbalance between the oxidant and antioxidant defenses. Antioxidant potential of drugs is also helpful in prevention of oxidative damage. PHF was able to avoid the enzymatic and non-enzymatic decreases in antioxidant defenses induced by the PTZ in all the brain tissues. It plays important roles in the brain's cellular defense against oxidative damage and is able to lower the risk of some neurological disorders.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.