Claims:1. A composition for treatment, prevention or amelioration of epilepsy comprising chondroitin sulfate.

2. The composition as claimed in claim 1, wherein epilepsy is induced by pentylenetetrazole (PTZ).

3. The composition as claimed in claim 1, wherein epilepsy is induced by pilocarpine.

4. The composition as claimed in claim 1, wherein the composition is administered in a dose range of 1 mg/kg to 500 mg/kg of body weight each day.

5. The composition as claimed in claim 1, wherein the composition is administered orally, intracerebrally, transdermally, subcutaneously, intravenously, intraperitoneally, or intramuscularly.

6. The composition as claimed in claim 1, wherein the composition is administered as pills, tablets, capsules, lozenges, powders, granules, patches, suspensions, solutions, microparticles, nanoparticles, aerosols, or sustained release particles.

7. The composition as claimed in claim 1, wherein the composition further comprises a pharmaceutically acceptable excipient.

8. The composition as claimed in claim 1, wherein the pharmaceutically acceptable excipient is selected from dispersants, binders, buffering agents, preservatives, stabilizers, colouring agents, flavouring agents, sugars, diluents, or combinations thereof.
9. The composition as claimed in claim 1, wherein the composition is in the form of a medicament.

10. A formulation for treatment, prevention or amelioration of epilepsy comprising chondroitin sulphate.

Description:FIELD OF INVENTION
[0001] The present disclosure relates generally to the field of pharmaceuticals. Specifically, the present disclosure is directed to a composition for management of epilepsy comprising chondroitin sulfate.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Epilepsy is known as the second most common neurological condition characterized by sudden recurrent episodes of epileptic seizures unprovoked by any immediately identifiable cause and is manifested clinically by abnormal behavioural changes due to aberrant electrical discharges of set neurons in the brain (1). As per WHO report of 2018, approximately 50 million people are suffering from epilepsy worldwide, out of which nearly 80% belong to low and middle-income countries (2). An epileptic seizure is the clinical manifestation of an abnormal and excessive electrical discharge of a set of neurons in the brain (3). These abnormal electrical discharges are the biochemical processes promoting neuronal hyper synchronous neuronal excitability in brain measured by electroencephalography (EEG) which can begin at any age (4, 5).
[0004] The currently available pharmacotherapy for epilepsy is not permanently targeted in curing the disease but is effective as symptomatic treatment for different types of seizures by suppressing the occurrence time of these seizures which is recognized as symptomatology of epilepsy. Thus, the available antiepileptic drugs in many patients are quite successful in preventing the recurrence of these seizures shown to be as a remarkable improvement in the clinical condition of some patients (6, 7). As per the clinical reports, one third of the patients get refractory seizures to these medications. None of the available drugs conclusively address the problem of containing the progressive increase in the severity of seizure, a process by which a near normal brain turns epileptic.
[0005] The mechanistic approaches of these available antiepileptic drugs targeting various pathways have been elaborated by pharmacological evidence, to a considerable detail, and some of the important molecular signaling mechanisms underlying epileptogenesis (8). These various molecular signaling mechanisms involved in pathogenesis of epilepsy revolve around the neurotransmitters and its ionotropic glutamate receptors, metabotropic glutamate receptors, neurotrophin receptors, calcium regulated enzymes, reactive oxygen species, nitric oxide synthase and non-receptor tyrosine kinases and have been documented to be involved in orchestrating various biochemical events in brain, finally leading to precipitation of a multitude of severe epileptic conditions (9, 10, 11, 12). The available antiepileptic drugs according to these targets have not been shown to be effective in patients with refractory epilepsy.
[0006] Furthermore, the recent scenario in the area of epilepsy research is more likely to be engaged in exploring the natural therapy for epilepsy. Looking into the evidences from various research studies suggesting the alterations in the volume fraction (a) of the brain’s extracellular space (ECS) having the ability to prolong or even initiate seizures (13, 14). The ECS volume comprises the interstitial fluid and the extracellular matrix, the interstitial fluid is important for determining the concentration of ions, neurotransmitter and electric field interaction between neurons. The other extracellular matrix provides support to the neighbouring cells comprising a large number of molecules of proteoglycans- Hyaluronan, lecticans, tenascins, heparin sulfate and chondroitin sulfate (15, 13). In epilepsy, the shrinkage of brain causes the decrease of the extracelluar space leading to the shift of ionic concentration between intracellular region and extracellular region by increasing the intracellular space of ion concentration as compared to the extracellular region of the neuronal cell (16, 17, 18). The decreased extracellular space (ECS) increases the ephaptic interactions between excitatory neurotransmitter signalling leading to the excitability of the neuron (19).
[0007] The inventors have arrived at a composition for management of epilepsy based on natural component- Chondroitin sulphate- that provides a negative domain to the extracellular matrix and maintains the extracellular positive ion concentration and the extracellular space volume.

OBJECTS OF THE INVENTION
[0008] An object of the present disclosure is to provide a composition for treatment, prevention or amelioration of epilepsy.
[0009] Another object of the present disclosure is to provide a composition for attenuating kindling seizures induced by pentylenetetrazole (PTZ).
[0010] Yet another object of the present disclosure is to provide a composition for attenuating spontaneous seizures induced by pilocarpine.

SUMMARY OF THE INVENTION
[0011] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0012] The present disclosure increases the understanding of the role of a composition comprising chondroitin sulphate – a proteoglycan in epilepsy.
[0013] In an aspect, the present disclosure provides a composition for treatment, prevention or amelioration of epilepsy comprising chondroitin sulfate.
[0014] In an embodiment, the present disclosure provides a composition for treatment, prevention or amelioration of epilepsy comprising chondroitin sulfate and a pharmaceutically acceptable excipient.
[0015] In yet another aspect, the present disclosure provides use of a composition comprising chondroitin sulfate, for the treatment, prevention or amelioration of epilepsy.
[0016] In still another aspect, the present disclosure provides a method of treatment, prevention or amelioration of epilepsy by administering to a subject in need thereof, a therapeutically effective amount of a composition comprising chondroitin sulfate.
[0017] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0018] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
Figure 1 provides the statistical analysis of the effect of chondrotin sulfate on pentylenetetrazole-induced kindled seizures in mice, as per an embodiment of the present disclosure. Values are expressed as Mean ± SEM. a=P<0.05 vs Vehicle Control; b=P<0.05 vs PTZ Control.
Figure 2 provides the statistical analysis of the effect of chondrotin sulfate on pentylenetetrazole-mediated catalase activity in mice, as per an embodiment of the present disclosure.
Figure 3 provides the statistical analysis of the effect of chondrotin sulfate on pentylenetetrazole-mediated TBARS Levels in mice, as per an embodiment of the present disclosure.
Figure 4 provides the statistical analysis of the effect of chondrotin sulfate on pentylenetetrazole-mediated Reduced Glutathione Activity in mice, as per an embodiment of the present disclosure.
Figure 5 provides the effect of chondrotin sulfate pre-treatment in PTZ induced kindling model: (A) IL-6 concentration, (B) TNF-a, (C) NF?-ß concentrations and (D) IL-1ß concentration in different groups as measured by ELISA kits, as per an embodiment of the present disclosure.
Figure 6 provides the effect of chondrotin sulfate on pilocarpine-induced spontaneous recurrent seizures in mice, as per an embodiment of the present disclosure.
Figure 7 provides the effect of chondrotin sulfate on pilocarpine-mediated catalase activity in mice, as per an embodiment of the present disclosure.
Figure 8 provides the effect of chondrotin sulfate on pilocarpine-mediated TBARS levels in mice, as per an embodiment of the present disclosure.
Figure 9 provides the effect of chondrotin sulfate on pilocarpine-mediated reduced glutathione activity in mice, as per an embodiment of the present disclosure.
Figure 10 depicts the statistical analysis of the effect of chondrotin sulfate pretreatment in pilocarpine-induced spontaneous recurrent seizures model. (A) IL-1ß concentration, (B) IL-6 concentration, (C) TNF-a and (D) NF?-ß concentrations in different groups measured by ELISA kits. Each sample was analyzed in duplicate. One way ANOVA followed by Bonferroni post hoc analysis was done (n=6/group).

DETAILED DESCRIPTION OF THE INVENTION
[0019] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0020] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0021] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0022] In some embodiments, numbers have been used for quantifying weights, percentages, dosages, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0023] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0024] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0025] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0026] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[0027] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0028] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.
[0029] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0030] It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0031] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0032] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0033] The term ‘therapeutically effective amount’ generally refers to the amount that when administered to a subject is sufficient to affect the treatment intended by the drug.
[0034] The term, "pharmaceutically acceptable" as used herein refers to an excipient comprised of a material that is not biologically or otherwise undesirable.
[0035] The term, "subject" as used herein refers to an animal, preferably a mammal, and most preferably a human. The term "mammal" used herein refers to warm-blooded vertebrate animals of the class 'mammalia' , including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young, the term mammal includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig and human.
[0036] Chondroitin sulfate (proteoglycan) is widely distributed in the cartilages and tendons of animals and humans used by large scale industries in manufacturing veterinary supplements, pet food products or fertilizers (20).
[0037] In an embodiment, the present disclosure provides a composition for management of epilepsy comprising chondroitin sulphate.
[0038] In an embodiment, the present disclosure provides a composition for treatment, prevention or amelioration of epilepsy comprising chondroitin sulfate.
[0039] The disclosure also covers pharmaceutically acceptable derivatives of chondroitin sulfate.
[0040] In an embodiment of the present disclosure, epilepsy is induced by pentylenetetrazole (PTZ). In an embodiment, the composition attenuates kindling seizures induced by pentylenetetrazole (PTZ). In an embodiment, the composition attenuates kindling seizures induced by pentylenetetrazole (PTZ) for generalized tonic or clonic seizures.
[0041] In an embodiment of the present disclosure, epilepsy is induced by pilocarpine. In an embodiment, the composition attenuates spontaneous seizures induced by pilocarpine. In an embodiment, the composition attenuates spontaneous seizures induced by pilocarpine for status epileticus.
[0042] Without being bound to theory it is believed that, chondrotin sulfate provides the negative domain extracellular having a strong affinity for positive ions calcium, sodium and potassium which maintains the positive ions extracellular concentration and decreases the influx of calcium and sodium influx causing depolarization of membrane. The increase of the potassium extracellular induces an increase in the resting potential of a neuron. In this way, chondroitn sulfate maintains the extracellular space volume which seems to decrease due to the shrinkage of the epileptic brain. The chondroitin sulfate maintains the extracellular space by maintaining the ion concentration in the extracellular space and reduces the synchronized and frequent neuronal activity by decreasing the ephaptic interactions between excitatory neurotransmitter signalling.
[0043] In an embodiment, the composition may be administered in a therapeutically effective amount to a subject. This is determined based on the patient history, body weight and severity of the condition.
[0044] In an embodiment, the composition may preferably be administered in a dose range of about 1 mg/kg to about 500 mg/kg of body weight each day, more preferably in the range of about 100 mg/kg to about 400 mg/kg of body weight each day.
[0045] In an embodiment, the composition may be administered orally, intracerebrally, transdermally, subcutaneously, intravenously, intraperitoneally, or intramuscularly. Preferably, the composition may be administered orally.
[0046] The composition may be administered in any form known in the art and suitable for patient compliance including pills, tablets, capsules, lozenges, powders, granules, patches, suspensions, solutions, microparticles, nanoparticles, aerosols, or sustained release particles. In an embodiment, the composition may be delivered via injection or infusion.
[0047] In an embodiment, the composition further comprises a pharmaceutically acceptable excipient.
[0048] In an embodiment, the pharmaceutically acceptable excipient includes those generally known in the art and that do not affect the therapeutic activity of chondroitin sulfate. These excipients may be selected from dispersants, binders, buffering agents, preservatives, stabilizers, emulsifiers, surfactants, colouring agents, flavouring agents, sugars, diluents, combinations thereof.
[0049] In an embodiment, the pharmaceutically acceptable excipient may include but is not limited to mannitol, sorbitol, sucrose, microcrystalline cellulose, phospholipids, fatty acids, metals of fatty acids, cellulosic derivatives, polyacrylates, povidones, polyvinylpyrrolidone, starch, microcrystalline cellulose, ,colloidal silicon dioxide, precipitated silicon dioxide, sodium bicarbonate, sodium citrate, croscarmellose sodium, crospovidone, sodium starch glyconate, stearates, talc, kaolin, calcium carbonate, dextrates, carbopol, carbomer, sodium acrylate, EDTA, sodium gluconate, glycerine, potassium sorbate, water and the like.
[0050] In an embodiment, the composition may be in the form of a medicament. The medicament or composition may be administered to a subject along with alternative treatment methods for epilepsy.
[0051] In an embodiment, the present disclosure provides a formulation for treatment, prevention or amelioration of epilepsy comprising chondroitin sulphate.
[0052] In an embodiment, the present disclosure provides use of chondroitin sulfate for the treatment, prevention or amelioration of epilepsy.
[0053] In an embodiment, the composition may be used as a dietary supplement comprising the chondroitin sulfate as bioactive for epilepsy.
[0054] In an embodiment, the present disclosure provides use of a composition comprising chondroitin sulfate, for the treatment, prevention or amelioration of epilepsy.
[0055] In an embodiment, the present disclosure provides a method of treatment, prevention or amelioration of epilepsy by administering to a subject in need thereof, a therapeutically effective amount of chondroitin sulfate.
[0056] In an embodiment, the present disclosure provides a method of treatment, prevention or amelioration of epilepsy by administering to a subject in need thereof, a therapeutically effective amount of a composition comprising chondroitin sulfate.
[0057] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
[0058] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

MATERIAL AND METHODS:
[0059] Drugs and chemicals: Pentylenetetrazole (Sigma, St. Louis, USA) was dissolved in normal saline (which served as a vehicle for preparing all the drug solutions/dilutions). Diazepam (Calmpose inj., Ranbaxy) was used as standard drug. Synthetic chondrotin sulfate (CS) was obtained from Amulya Herbs Pvt. Limited, Panchkula. All drug solutions were freshly prepared before use.
[0060] Equipment: The equipment used for the current study included: cuvet, magnetic stirrer, centrifugator, IR-spectrophotometer, marker, incubator, and UV analyzer.
[0061] Animals: Male inbred Swiss albino mice weighing 25±2g maintained on standard laboratory diet (Ashirwad Pvt. Ltd., Chandigarh, India) having free access to tap water were employed in the present study. They were housed in the departmental animal house and were exposed to 12 hr cycle of light and dark. The experiments were conducted in a semi-sound proof laboratory. The experimental protocol was approved by the institutional animal ethics committee. Care of the experimental animals was carried out as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India.

EXPERIMENTAL PROTOCOL:
EXAMPLE 1:
1.1 Induction of pentylenetetrazole kindled model of epilepsy in mice
[0062] Pentylenetetrazole (PTZ) was administered in a subconvulsant dose (40 mg/kg, i.p.) to the groups of animals to induce tonic-clonic convulsions in mice (11). The treatment groups received Pentylenetetrazole (PTZ) (40 mg/kg, i.p.) after 45 minutes of administration or pre treatment with a lower dose of CS (150 mg/kg (p.o.)) and higher dose of CS (300 mg/kg (p.o.)). The standard group received diazepam (20 mg/kg, i.p.) which was administered 45 minutes prior to the administration of Pentylenetetrazole (PTZ). After each injection of Pentylenetetrazole (PTZ), occurrence of central nervous system excitation was recorded for 30 min by observing the animals in a Plexiglas chamber (30 X 24 X 22 cm). Occurrence of HLTE (Hind Limb Tonic Expens), the number of animals convulsing or not convulsing within the observation period and duration of seizures was then noted. The ability of the treatment with chondrotin sulfate to prevent or delay the onset of the hind limb extension exhibited by the animals was taken as an indication of anticonvulsant activity.
1.2 Induction of status epilepticus provoked spontaneous recurrent seizure activity in mice
[0063] Intraperitoneal application of pilocarpine (100 mg/ kg, i.p.) was repeated every 20 min until the onset of status epilepticus (11). In order to avoid peripheral cholinergic side effects, hyoscine butylbromide (1 mg/kg, i.p.) was administered 20 mins prior to the application of pilocarpine inducing status epilepticus. Status epilepticus was defined as a continuous seizure activity of the limbs, rearing and falling, Straub tail, repeated head twitches, jumping seizures and generalized clonic-tonic seizures. Status epilepticus was terminated, 40 min after the onset, using a single dose of diazepam (3mg/kg, i.p.). Normal saline was administered until complete recovery. A spontaneous recurrent seizure severity score (SRSSS) was then recorded as a measure of the quantitative assessment of progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus (SE). Treatment with a lower and higher dose of CS (150 mg/kg (p.o.) & 300 mg/kg (p.o.)) was started 6 days after the SE episode and was continued until day 37 (dosing was started 1 day prior to the first behavioural assessment day). Behavioural assessment was then performed prior to the drug administration (every third day until day 37).
[0064] In the present study a total of 8 groups were employed and each group comprised of 06 animals.
A. Pentylenetetrazole induced kindled seizure severity score assessment protocol
Group I (Vehicle control group-a): vehicle (10 ml kg-1, i.p.) every alternate day for 15 days + vehicle (10 ml kg-1 d-1, i.p.) for 16 days (day 0 to day 15).
Group II (Pentylenetetrazole (PTZ) control group): PTZ (40 mg kg-1, i.p.) every alternate day for 15 days + vehicle (10 ml kg-1 d-1, i.p.) for 16 days (day 0 to day 15).
Group III (Low dose (CS) + PTZ group): PTZ (40 mg kg-1, i.p.) every alternate day for 15 days + (CS) 150mg/kg (p. o.) for 16 days (day 0 to day 15).
Group IV (High dose (CS)+ PTZ group): PTZ (40 mg kg-1, i.p.) every alternate day for 15 days + (CS) 300mg/kg (p. o.) for 16 days (day 0 to day 15).
B. Pilocarpine induced spontaneous recurrent seizure severity score assessment protocol
Group I (vehicle control group-b): vehicle (10 ml kg-1d-1, i.p.) for 31 days period starting 6th day after SE + vehicle (10 ml kg-1, i.p. every 20 mins until the completion of 80 mins period followed by diazepam (3 mg kg-1, i.p.) 40 mins after last injection) on day 1.
Group II (pilocarpine (PC) control group): vehicle (10 ml kg-1 d-1, i.p.) for 31 days period starting 6th day after SE + PC (100 mg kg-1, i.p. every 20 mins until the onset of SE followed by diazepam (3 mg kg-1, i.p.) 40 min after SE) on day 1.
Group III (low dose (CS) 150mg/kg (p.o.) + PC group): CS 150 mg/kg (p.o.) for 31 days period starting 6th day after SE + PC (100 mg kg-1, i.p. every 20 mins until the onset of SE followed by diazepam (3 mg kg-1, i.p.) 40 min after SE) on day 1.
Group IV (high dose (CS) + PC group): CS 300 mg/kg (p.o.) for 31 days period starting 6th day after SE + PC (100 mg kg-1, i.p. every 20 min until the onset of SE followed by diazepam (3 mg kg-1, i.p.) 40 min after SE) on day 1.
EXAMPLE 2: STATISTICAL ANALYSIS
[0065] Data obtained from the study was statistically analyzed using one-way ANOVA followed by Tukey’s multiple range test as post-hoc analysis. Moreover, analysis of the time-course data of the study was also done using two-way ANOVA. A value of p<0.05 was considered to be statistically significant. The results are presented in Figures 1 to 10.
[0066] The chondroitin sulfate significantly ameliorated the PTZ and pilocarpine induced increased lipid peroxidation in animals. Endogenous antioxidants (TBARS, Glutathione, and Catalase) facilitated the combating of oxidative damage by neutralizing the effects of free radicals in the body. The excess production of reactive oxygen free radicals in seizures leads to overloading of these endogenous antioxidant mechanisms and results in oxidative damage to phospholipids, proteins and mitochondrial DNA (21). Results revealed a significant decline in endogenous antioxidants brain levels after the induction of PTZ and pilocarpine seizures in mice, and chondroitin sulfate treatment had significantly reinstated the endogenous antioxidant levels (Figures 2, 3, 4, 7, 8 and 9) in seizure animals.
[0067] The present study demonstrated the natural cure for epilepsy by using chondroitin sulfate at a dose of 300mg/kg, (p.o.) against kindling seizures induced by PTZ and spontaneous seizures induced by pilocarpine. The subconvulsive dose of PTZ (40 mg/kg, (i.p.)) and pilocarpine (100 mg/kg, (i.p.)) elicited a marked development of generalized seizures as assessed in terms of tonic–clonic seizures. From the above findings, the onset of seizures and severity of tonic–clonic seizures are reduced in the rodents receiving the treatment with (CS 300mg/kg+ PTZ 40mg/kg) as compared to disease (PTZ/ Vehicle) group (Figure 1). In pilocarpine model, the onset and severity of spontaneous seizures are reduced in the rodents receiving the treatment with (CS 300mg/kg+ pilocarpine 100mg/kg) as compared to disease (pilocarpine/ Vehicle) group (Figure 6). The standard drug diazepam is used as a positive control drug in the study. Thereby, chondroitin sulfate resulted in attenuating the kindling seizures time occurrence & mortality in rodents at a subconvulsive dose of PTZ (40 mg/kg, (i.p.)) was measured by evaluating the behaviour parameters and by reduction in the kindled seizure as compared to the (PTZ/Vehicle) group in terms of severity of generalized tonic–clonic seizures. The chondroitin sulfate also tends to be effective in attenuating the spontaneous recurrent seizure time occurrence & mortality in rodents at a subconvulsive dose of pilocarpine (100 mg/kg, (i.p.)) as compared to the (pilocarpine/ Vehicle) group in terms of status epilepticus.
[0068] Additionally, the histopathology evaluation by H&E staining showed the neuroprotection in the rodents of both treatment control groups PTZ (40 mg/kg, (i.p.)) and pilocarpine (100 mg/kg, (i.p.)) as compared to the disease groups (PTZ/Vehicle) and (Pilocarpine/ Vehicle).
[0069] The NF-kB ELISA estimation done by commercially available kits (Krishgen Biosystems Mumbai) showed a decrease in the expression of NF-kB of brain tissue sample of rodents of treatment groups (CS 300mg/kg + PTZ and CS 300 mg/kg + pilocarpine). The decreased expression of NF-kB indicates the reduction of the IL-6, and TNF-alpha and activating the Nrf2 controlling the production of reactive oxygen species induced due to the prolonged rapid electrical discharges in brain induced by PTZ and pilocarpine (Figure 5 and 10). Whereas, the biochemical estimation resulted in reduction of TBARS and increase of the Glutathione, Catalase levels in the brain confirmed the anti-oxidant, anti-inflammatory role of chondroitin sulfate.
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ADVANTAGES OF THE INVENTION:
[0070] The present disclosure provides a composition for treatment, prevention or amelioration of epilepsy.
[0071] The present disclosure provides a composition for attenuating kindling seizures induced by pentylenetetrazole (PTZ) and spontaneous seizures induced by pilocarpine.
[0072] The present disclosure provides a composition that increases catalase activity and decreases TBARS activity and glutathione activity.
[0073] The present disclosure provides a composition that decreases IL-1ß concentration, IL-6 concentration, TNF-a concentration and NF?-ß concentration.