FIELD OF INVENTION
The field of invention relates to dietary supplements and discloses an oral dietary supplement useful in the management and treatment of epilepsy. More specifically it relates to an oral anti-epileptic composition and method thereof.
BACKGROUND OF THE INVENTION
The present invention discloses an oral anti-epileptic composition which is in the nature of a dietary supplement than a drug. Accordingly, the same are being defined below, to promote correct understanding in the context of present invention.
Dietary supplements and Drugs
Dietary supplements- The law defines dietary supplements in part as “products taken by mouth that contain a "dietary ingredient." Dietary ingredients include vitamins, minerals, amino acids and herbs or botanicals, as well as other substances that can be used to supplement the diet.
Ref: https://www.fda.gov/consumers/consumer-updates/fda-101-dietary-supplements

Drugs- Natural or synthetic substances which (when taken into a living body) affect its functioning or structure, and are used in the diagnosis, mitigation, treatment, or prevention of a disease or relief of discomfort. Also called legal drug or medicine. A legal or medicinal drug (such as amphetamines), however, can be harmful and addictive if misused.
Ref: http://www.businessdictionary.com/definition/drug.html

Epilepsy- Symptoms, Method of treatment and limitations

Symptoms- Epilepsy is a central nervous system (neurological) disorder in which brain activity becomes abnormal, causing seizures or periods of unusual behavior, sensations, and sometimes loss of awareness. Seizure signs and symptoms may include:
1. Temporary confusion
2. A staring spell
3. Uncontrollable jerking movements of the arms and legs
4. Loss of consciousness or awareness
5. Psychic symptoms such as fear, anxiety or Deja vu
Focal seizures- When seizures appear to result from abnormal activity in just one area of brain, they're called focal (partial) seizures and fall into two categories as below:
• Focal seizures without loss of consciousness- Once called simple partial seizures, these seizures don't cause a loss of consciousness. They may alter emotions or change the way things look, smell, feel, taste or sound. They may also result in involuntary jerking of a body part, such as an arm or leg, and spontaneous sensory symptoms such as tingling, dizziness and flashing lights.
• Focal seizures with impaired awareness- Once called complex partial seizures, these seizures involve a change or loss of consciousness or awareness.
Generalized seizures- Seizures that appear to involve all areas of the brain are called generalized seizures. Six types of generalized seizures exist.
i. Absence seizures- previously known as petit mal seizures, often occur in children and are characterized by staring into space or subtle body movements such as eye blinking or lip smacking. These seizures may occur in clusters and cause a brief loss of awareness.
ii. Tonic seizures- cause stiffening of muscles. These seizures usually affect muscles in back, arms and legs and may cause a person to fall to the ground.
iii. Atonic seizures- also known as drop seizures cause a loss of muscle control, which may cause a person to suddenly collapse or fall down.
iv. Clonic seizures- are associated with repeated or rhythmic, jerking muscle movements. These seizures usually affect the neck, face and arms.
v. Myo-clonic seizures- usually appear as sudden brief jerks or twitches of arms and legs.
vi. Tonic-clonic seizures- previously known as grand mal seizures, are the most dramatic type of epileptic seizure and can cause an abrupt loss of consciousness, body stiffening and shaking, and sometimes loss of bladder control or biting of the tongue.
https://www.mayoclinic.org/diseases-conditions/epilepsy/symptoms-causes/syc-20350093
Methods of treatment and their limitations
Many approaches have been developed to treat epilepsy. These include drugs as well as dietary supplements, as elaborated below:
Drugs used in treatment of epilepsy
Modern treatment of seizures started in 1850 with the introduction of bromides, which was based on the theory that epilepsy was caused by an excessive sex drive. In 1910, phenobarbital (PHB), which then was used to induce sleep, was found to have antiseizure activity and became the drug of choice for many years. A number of medications similar to PHB were developed, including primidone. In 1938, Houston Merrit and Tracy Putnam described animal models for screening multiple compounds for antiepileptic activity in the Journal of the American Medical Association. In 1940, phenytoin (PHT) was found to be an effective drug for the treatment of epilepsy, and since then it has become a major first-line antiepileptic drug (AED) in the treatment of partial and secondarily generalized seizures. In 1968, carbamazepine (CBZ) was approved, initially for the treatment of trigeminal neuralgia; later, in 1974, it was approved for partial seizures. Ethosuximide has been used since 1958 as a first-choice drug for the treatment of absence seizures without generalized tonic-clonic seizures. Valproate (VPA) was licensed in Europe in 1960 and in the United States in 1978, and now is widely available throughout the world. It became the drug of choice in primary generalized epilepsies and in the mid1990s was approved for treatment of partial seizures.These anticonvulsants were the mainstays of seizure treatment until the 1990s, when newer AEDs with good efficacy, fewer toxic effects, better tolerability, and no need for blood level monitoring were developed. A study of live-born infants in Denmark found that exposure to the newer-generation AEDs lamotrigine, oxcarbazepine, topiramate, gabapentin, and levetiracetam in the first trimester was not associated with an increased risk in major birth defects (Molgaard-Nielsen D, Hviid A. Newer-generation antiepileptic drugs and the risk of major birth defects. JAMA. 2011 May 18. 305(19):1996-2002). The new AEDs have been approved in the United States as add-on therapy only, with the exception of topiramate and oxcarbazepine (OXC); lamotrigine (LTG) is approved for conversion to monotherapy. A meta-analysis of 70 randomized clinical trials confirms the clinical impression that efficacy does not significantly differ among AEDs used for refractory partial epilepsy (Costa J, Fareleira F, Ascencao R, et al. Clinical comparability of the new antiepileptic drugs in refractory partial epilepsy: A systematic review and meta-analysis. Epilepsia. 2011 Jul. 52(7):1280-91). Ref: Antiepileptic Drugshttps://emedicine.medscape.com/article/1187334-overview

Mechanisms of Action of the drugs
Various molecular pathways are involved in epileptic seizures. Depending upon which parameter is upset in the pathway, drugs act on that parameter and help in alleviation of the symptoms. Molecular signaling mechanisms involved in pathogenesis of epilepsy revolve around the following:
i. Neurotransmitters and its ionotropic glutamate receptors
ii. Metabotropic glutamate receptors
iii. Neurotrophin receptors
iv. Calcium regulated enzymes
v. Reactive oxygen species
vi. Nitric oxide synthase
vii. Non-receptor tyrosine kinases
have been documented to be involved in orchestrating various biochemical events in brain, finally leading to precipitation of a multitude of severe epileptic conditions.
Limitations of existing AEDs (Anti-Epileptic Drugs)
Available antiepileptic drugs according to these targets have not been shown to be effective in patients with refractory epilepsy. There is an imperative need of identification and proposing a new molecular target with better knowledge in furtherance of developing new novel drugs accordingly with an aim to suppress/attenuate refractory seizures permanently. Furthermore, the recent scenario in the area of epilepsy research is more likely to be engaged in exploring the natural therapy for epilepsy. The current 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. As per the clinical reports, the one third of the patient gets refractory seizures to these medications. However, none of the available drugs conclusively addresses the problem of containing the progressive increase in the severity of seizure, a process by which a near normal brain turns epileptic.
Use of Dietary Supplements in the management of epilepsy
Food as part of disease treatment is not new and is now slowly being accepted by more and more doctors. During the first half of the 20th century, the impact of food on our bodies was one of the hottest scientific fields. Insulin- a natural hormone was discovered in 1921, and its commercial production meant survival for diabetics. In the 1930s, three scientists won a Nobel Prize for discovering that a substance in raw liver cured pernicious anemia, a disease that was almost always fatal. Eight Nobel prizes were awarded just for work related to vitamins. Dietary supplements in whole forms e.g. vitamins, minerals etc. have also shown to have beneficial effects in epileptic patients but studies in this area are very limited. A major reason could be lack of funding and financial support, as not many pharma companies would not like to invest in and promote a low-cost product in which profit margins are less.
Diets for Epilepsy control- Ketogenic diets and others
Ketogenic diet– it has shown promising results in control of epilepsy. It was first used as a medical treatment for epilepsy in the 1920s. The principles underlying the diet have been around since Hippocrates touched on them nearly 2,500 years ago. Starvation had long been one approach to treating epilepsy. Deny the patient food for, say, a week and often their seizures went away. But there were obvious limits on how long starvation could be used as a treatment. In the 1920s, researchers at the Mayo Clinic, looking for a way to treat diabetics, figured out that it was not fasting per se that helped control seizures. Rather, they found that it was what the body did during an extended fast that helped control them. Deprived of food, the human body starts burning body fat as fuel, and it was that process of ketosis that somehow had the antiepileptic effect. Trick the body into thinking it was starving by taking away its primary fuel of carbohydrates and forcing it to subsist on an all-fat diet, and you could create that antiepileptic effect as long as necessary. The diet was quickly adopted and widely used through the 1930s. And then, almost as fast as it had appeared, the keto diet disappeared. When Dilantin was first used as an antiepileptic drug in 1938, its success steered medical minds toward pharmaceutical solutions. A generation later, the diet had been all but forgotten. There was no scientific evidence that it worked, after all. More important, it was incredibly difficult to administer. Even in the 1990s, Millicent Kelly, Charlie Abrahams’s dietitian at Johns Hopkins, was planning menus with a calculator and a legal pad (Ref: https://www.nytimes.com/2010/11/21/magazine/21Epilepsy-t.html).
Other diets
i. Modified Atkins Diet (MAD), published by Johns Hopkins in 2003
ii. Thiele and Pfeifer’s Low Glycemic Index Treatment (L.G.I.T.), published in 2005.
Modified Atkins Diet (MAD) is more restrictive than the Atkins diet that people use for weight loss, but nonetheless a bit easier to follow than keto because it allows more protein.
Low Glycemic Index Treatment (L.G.I.T.) is easier than keto because it allows more carbs and protein as long as the carbs are like strawberries — which affect blood sugar slowly — and not like bread, potatoes or candy, which make it spike. There are volumes of clinical data supporting the effectiveness of these diets, but not yet the kind of randomized, controlled study that show these diets work as well as keto, and keto is still most often prescribed.
Scientific Evidence and Clinical Acceptance of Keto Diet for epilepsy control
By 2000, more people were asking about keto, but most pediatric neurologists still would not prescribe it. That bias seemed ridiculous to J. Helen Cross, the principal investigator of the 2008 randomized keto trial at University College London. “I’d been dealing with complex epilepsy cases for 10 years, and it was quite clear to me that certain children did respond to the ketogenic diet,” Cross says. “But we in our institution — and I know we weren’t alone — were coming up against barriers to get the resources to do it. They’d say there’s no evidence it works. It’s a quack diet. There is no controlled data. So I wanted to prove that it did work once and for all, and do it in a way so that people couldn’t argue with it.” It took five years to enroll and track enough patients to make the study credible and another two years to analyze the data and undergo the rigorous academic peer-review process. But since the study was published in 2008, it has answered doubts about keto’s clinical effectiveness. Keto has now attracted attention from all corners of the neurological community and studies are being carried out and published relating to this diet.

Various researches have shown the neuroprotective role of Low carbohydrate and high protein i.e. ketogenic diet in various neurodegenerative diseases by increasing the energy levels(Maalouf et al., 2009 Brain Res Rev. Mar. 59(2):293-315; Sada and Inoe 2018, Martin-McGill et al., 2018; Yang et al., 2019).
The ketogenic diet is a low cholesterol, carbohydrate and high protein diet, replacing the Glucose (carbohydrate) i.e. the primary source of energy with secondary source of fuel (ketone bodies) having role in reducing the calcium overload, opening of kATP channels, blocking the sodium voltage channel and increasing the mitochondrial uncoupling protein reducing the ROS production involved in neuronal excitability in epilepsy (Paoli et al., 2014; Williams et al., 2017; Barzegar et al., 2019). Therefore, the ketogenic diet tends to possess an anti- inflammatory activity in epilepsy by increasing the Nrf-2 transcriptional factor and inhibiting the NF-kB (Pinto et al., 2018; Yang et al., 2019; Barzegar et al., 2019). The ketone bodies decreases the low density lipoprotein (LDL) by increasing the High density lipoprotein (HDL) and decreasing the oxidative stress contributing to the increased cardio metabolic risks seem to be involved in epileptic patients (Lima et al., 2015).
Progress on use of keto diet as a therapeutic agent for epilepsy
Papers published in the past two years suggest that keto may slow the growth of a brain tumor in mice. A biotechnology company named Accera is marketing a high-fat powder to Alzheimer’s patients that is supposed to reproduce the effects of ketosis, without the dietary restrictions of keto.
A particularly impressive video of an epileptic patient who found the diet useful is that of Charlie Abrahams on YouTube. The video shares his speech to some 300 doctors, dietitians and researchers at the International Symposium on Dietary Therapy for Epilepsy and Other Neurological Disorders. When Charlie was a baby, his doctors’ diagnosed Lennox-Gastaut Syndrome, a particularly severe form of epilepsy that if not properly treated often leaves sufferers permanently brain damaged. By taking specific diet, he was able to manage the symptoms because drugs did nothing. Use of the diet is well-documented in an epilepsy book authored by Dr. John Freeman, at Johns Hopkins Medical Institution. In 1993 Freeman was the only doctor in the country using the diet consistently. He had been using it since 1969 and claimed that 30 percent of his patients became seizure-free after using it.
(Ref: https://www.nytimes.com/2010/11/21/magazine/21Epilepsy-t.html).
Vicente-Hernández M1 et al. 2007 have reviewed dietary supplements and alternatives available with primary focus on benefits of ketogenic diets (Therapeutic approach to epilepsy from the nutritional view: current status of dietary treatment, Neurologia. 2007 Oct. 22(8): 517-25).
Gaby AR (2007) has reviewed the use of diet, nutritional supplements, and hormones in the treatment of epilepsy. Nutrients that may reduce seizure frequency include vitamin B6, magnesium, vitamin E, manganese, taurine, dimethylglycine, and omega-3 fatty acids. Administration of thiamine may improve cognitive function in patients with epilepsy. Supplementation with folic acid, vitamin B6, biotin, vitamin D, and L-carnitine may be needed to prevent or treat deficiencies resulting from the use of anticonvulsant drugs. Vitamin K1 has been recommended near the end of pregnancy for women taking anticonvulsants. Melatonin may reduce seizure frequency in some cases, and progesterone may be useful for women with cyclic exacerbations of seizures. In most cases, nutritional therapy is not a substitute for anticonvulsant medications. However, in selected cases, depending on the effectiveness of the interventions, dosage reductions or discontinuation of medications may be possible. (Natural approaches to epilepsy. Altern Med Rev. 2007 Mar; 12(1):9-24).

Innovative Solution offered by the present invention
Present invention discloses an oral anti-epileptic composition consisting of concentrated, bone broth extract (COBBE) prepared by boiling mammalian bone powder in water and condensing the vapors followed by concentration of the same under low temperature and vacuum to give a clear solution. Analysis of the same by FTIR revealed that the composition is a synergistic mixture of specific nutrients viz. chondroitin sulfate, glucosamine, collagen like amino acids and other small molecules in synergistic ratios. Though horse bone was used in the experiments as example, any mammalian bone e.g. of cow, buffalo, sheep or goat or any other mammal, can be used for preparing the composition owing to molecular level similarity of bones. Hence, compositions prepared from bone of other animals, apart from horse may be considered within the scope of present invention.

Prior Art
Pandeya Surendra Nath, Kumar. Rajeev and Pathak. Ashish Kumar (2009) Natural Anticonvulsants: A Review. Research J. Pharm. and Tech.2 (4): Oct.-Dec. The article discloses several plants having antiepileptic properties and found to contain active ingredients when tested with modern bioassays for detecting anticonvulsive activities.
Manchishi. Stephen M (2018) Recent Advances in Antiepileptic Herbal Medicine
Current Neuropharmacology. 16 (1) Discloses herbal medicines having antiepileptic activities. Owing to the side effects Ayurvedic treatment for epilepsy is not so popular in use.
S. Miyata, H. Kitagawa (2016) Chondroitin sulfate and neuronal disorders. Front Biosci. (Landmark Ed). 2016 Jun 1;21 (1330-40)In this review, the authors have elaborated on the possible role of chondroitin sulphate proteoglycans (CSPGs) in several neuronal disorders including epilepsy, schizophrenia, Costello syndrome and Alzheimer’s disease. The article reviews the potential roles of CSPGs as therapeutic targets for neuronal disorders, with particular focus on structural changes of CS chains under pathological conditions. There is no disclosure about a dietary supplement or drug based on chondroitin sulphate which when given orally can alleviate the symptoms of such diseases or prove to be therapeutically useful in the treatment of neuronal disorders.

Wen Chen, Yan-Shuang Li1, Jing Gao, Xiao-Ying Lin, Xiao-Hong Li (2016) AMPA Receptor Antagonist NBQX Decreased Seizures by Normalization of Perineuronal Nets
PLOS ONE | DOI:10.1371/journal.pone.0166672 November 23, 2016AMPA receptor antagonist NBQX decreased the onset of epileptic seizures induced by PTZ through regulation of PNNs in the medial prefrontal cortex. Degradation of PNNs caused by ChABC in mPFC not only exacerbated seizures but also reversed the anti-epileptic effect of NBQX in PTZ-treated rats. These findings therefore revealed that PNNs in the medial prefrontal cortex is related to the anti-epileptic effect of NBQX and enhancement of PNNs may be effective for the treatment of epilepsy.

CA2143070 (A1) discloses Oral controlled release liquid suspension pharmaceutical formulation. The invention discloses, a controlled release oral formulation for use with a variety of drugs e.g. anti-Parkinsonian, cardiovascular and anti-epilepticdrugs are formed in liquid suspension form. The ingredients in the suspension are water, an edible oil and a stabilizer for the liquid suspension, at least one pharmaceutically active ingredient, at least two water soluble biodegradable polymers, and optionally with at least one antioxidant to prevent degradation and oxidation of the pharmaceutically active ingredients. A typical teaspoon dose of anti-Parkinson liquid suspension contains 15-150 mg carbidopa, 50-1500 mg levodopa, 100-300 mg of a combination of polyvinyl alcohol and polysucrose, 10-50 mg oil, 5-15 mg antioxidant, e.g. vitamin E, 5-20 mg stabilizer, 10-15 mg colorants, 10-15 mg natural flavoring agents and 5 ml water.
Indian Patents
A search of Indian Patent databases did not reveal disclosure of any horse bone derived aqueous extract for use as an oral anti-epileptic composition. From the above it is clear that none of the prior art discloses or anticipates the composition of present invention.

OBJECT OF THE INVENTION
In general it is an object of the invention to provide an oral anti-epileptic composition derived from mammalian bone.
One more object is to disclose that the composition contains nutrients like chondroitin sulfate, glucosamine, collagen like amino acids and other small molecules in synergistic ratios.
Another object of the invention to disclose its method of preparation.
Yet another object of the invention is to provide a safe and effective dosage form for oral anti-epileptic use.
SUMMARY OF INVENTION
Present invention discloses an oral anti-epileptic composition consisting of concentrated, bone broth extract (COBBE) prepared by boiling mammalian bone powder e.g. horse bone powder, in water and condensing the vapors followed by concentration of the same under low temperature and vacuum to give a clear solution. Analysis of the same by FTIR revealed that the composition is a synergistic mixture of specific nutrients viz. chondroitin sulfate, glucosamine, collagen like amino acids and other small molecules. COBBE when given orally, significantly prevented pharmacologically induced seizures in test animals (mice). It was 100% effective in preventing mortality in PTZ seizure induced mice, as evident from the fact that while all animals in control group died (no drug or COBBE given), not a single animal died in test group (COBBE given orally). Though horse bone was used in the experiments as example, any mammalian bone e.g. of cow, buffalo, sheep or goat or any other mammal, can be used for preparing the composition owing to molecular level similarity of bones. Data obtained from the study was statistically analyzed using one-way ANOVA followed by Turkey’s multiple range test as a post-hoc analysis. 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.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: FTIR spectrum of Test sample i.e. COBBE (Concentrated Bone Broth Extract)
Figure 2: Histopathological observations of the mice brain hippocampus (H & E stain X100) (A) Control group (with vehicle injection) without PTZ
(B) Control group (with vehicle injection)+ PTZ
(C) Diazepam group + PTZ
(D) COBBE Low Dose + PTZ
(E) COBBE High Dose + PTZ

DETAILED DESCRIPTION OF THE INVENTION
Present invention discloses an oral anti-epileptic composition consisting of concentrated, bone broth extract (COBBE) prepared by boiling mammalian bone powder e.g. horse bone, in water and condensing the vapors followed by concentration of the same under low temperature and vacuum to give a clear solution. Analysis of the same by FTIR revealed that the composition is a synergistic mixture of specific nutrients viz. chondroitin sulfate, glucosamine, collagen like amino acids and other small molecules in natural synergistic ratios. COBBE when given orally, significantly prevented pharmacologically induced seizures in test animals (mice) and also prevented mortality of the animals due to seizures.

Method of Preparation
The method consists of the following steps:
i. Mammalian bone e.g. horse bone is pulverized and boiled in water in ratio of 1:2 (1 part bone powder and 2 parts water, weight by weight) for about 20-30 minutes, optimally about 25 minutes.
ii. The vapors are condensed and the condensate is further concentrated at a temperature of 70 – 100 degree centigrade and vacuum to 1/10th the volume of condensate to give clear solution.
Time of boiling may vary depending upon quantity of horse bone used, size of vessel or the intensity of the flame being used for heating.
The concentrated bone broth extract (COBBE) (concentrated condensate) in the form of clear solution obtained after condensation of the vapors and their concentration, represents product of the invention. When given orally to subjects, it is amazingly very effective in preventing epileptic seizures. The method of preparation is exemplified in Example-1 given below.
Example-1
To 50 gram of dry horse bone powder, 100 ml of distilled water was added (1:2) and the mixture was boiled in a flask attached to a condenser. By circulating cold water in the condenser, the vapors arising from the horse bone broth, were condensed and collected in a separate container. The condensate was cooled and further concentrated to 1/10th volume, at low temperature and low pressure by using a rotary evaporator. About 50 ml condensate was obtained and concentrated to give 5 ml concentrated clear condensate.
Analysis of the extract
FTIR spectrum of COBBE revealed a strong peak at a wavelength of 3458.87 cm-1 indicative of hydroxide group and at 1650 cm-1indicative of –CONH i.e., the functional group of amide group coupling of carbonyl stretching. Strong IR spectra observed at a wavelength of 687.14cm-1, 1465.52 cm-1 confirmed the presence of Carboxylate associated with sulphate group. Therefore, the FTIR obtained spectra confirmed presence of functional groups of chondroitin sulfate present in horse bone broth extract as the major ingredient along with other minor ingredients (Fig. 1).
The FTIR obtained spectra confirmed the IR spectra of the functional groups of chondrotin sulfate.

The product of the invention is thus an oral anti-epileptic composition in form of clear solution consisting of aqueous distillate of mammalian bone powder containing specific nutrients viz. chondroitin sulfate, glucosamine, collagen like amino acids and other small molecules in natural synergistic ratios and wherein the composition has analytical FTIR fingerprint as given in Fig.1.

Experimental model for evaluating the oral anti-epileptic activity of Concentrated Bone Broth Extract (COBBE)
Pentylenetetrazole (PTZ) induced kindled seizure severity score assessment protocol was followed. Briefly, five groups of experimental animals (mice, 6 in each group) were used for experiments. The test groups (mice) first received treatments of standard anti-epileptic drug diazepam and also the test composition (COBBE). This was followed by treatment with seizure inducing compound i.e. pentylenetetrazole (PTZ).
The aim of the experiments was to confirm the effectiveness of new composition of present invention in preventing seizures in test animals- whether it was as good, or less or more than the standard drug diazepam given for preventing seizures.
Standard drug diazepam was given at a dose of 20 mg/kg, intraperitoneally (i.p.), while the test composition i.e. horse bone broth extract was given orally at low and high doses of 0.25 ml and 0.50 ml respectively. After 45 minutes, the seizure inducing compound i.e. PTZ or pentylenetetrazole (80 mg/kg, i.p.) was administered to the animals.
After each injection of Pentylenetetrazole (PTZ), occurrence of central nervous system excitation was recorded for 30 minutes 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 were noted and duration of seizures was also noted. The ability of COBBE to prevent or delay the onset of the hind limb extension exhibited by the animals was taken as an indication of anticonvulsant activity.
Details of the experimental groups are given in Table 1 below.

Table 1: Experimental Groups for testing anti-epileptic activity of the composition
Group Treatment Mice
Group-1 Control-1 (No PTZ i.e. no seizures induced)
Vehicle i.e. normal saline (10 mg/kg; i.p.) only.No PTZ. 6
Group-2 Control-2 (PTZ given intraperitoneally i.e. seizures induced)
Vehicle i.e. normal saline (10 mg/kg; i.p.) only + 80 mg/kg, i.p. PTZ 6
Group-3 Standard Drug (Diazepam) + PTZ
Standard drug diazepam given intraperitoneally (20 mg/kg, i.p.) + 80 mg/kg, i.p. PTZ 6
Group-4 Low oral dose of Concentrated Bone Broth Extract (COBBE) + PTZ
HBBE (0.25 ml, oral dose) + 80 mg/kg, i.p. PTZ 6
Group-5 High oral dose of COBBE + PTZ
HBBE (0.50 ml, oral dose) + 80 mg/kg, i.p. PTZ 6

Estimation of biochemical parameters in brain tissue
Thiobarbituric Acid (TBA), Glutathione and Catalase were estimated in brain tissue. For biochemical estimations, animals were sacrificed by cervical dislocation. The brains were removed and homogenized in phosphate buffer (pH 7.4, 10% w/v) using a homogenizer. The clear supernatant obtained after centrifugation at 3000 rpm for 15 min, was used for estimations as per details below:
i. Estimation of thiobarbituric acid reactive substances- The quantitative measurement of thiobarbituric acid reactive substances as an index of lipid peroxidation in brain was performed according to the method (Ohkawa et al., 1979).
ii. Estimation of reduced glutathione - The reduced glutathione in brain tissue was estimated using the method of (Beutler et al., 1963).
iii. Estimation of catalase - Catalase level in brain homogenate was estimated using method of (Aebi, 1984; Mishra et al., 2010).

Pathological examination of brain tissue
After completion of behavior tests, 3 mice in every group were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and the brains were transcardially perfused with phosphate buffer solution (PBS, pH 7.4), followed by 4% paraformaldehyde in PBS (pH 7.4). The brains were removed and kept overnight in PBS containing 4% paraformaldehyde at 4 °C (Brain tissue should not be frozen) and embedded in paraffin. Sections of 5-µm thickness were stained with hematoxylin and eosin. Hematoxylin is used to stain nuclei blue, while eosin stains cytoplasm and the extracellular connective tissue matrix pink. The lesions of brain tissues were observed with light microscope (NIKON E600, Japan) and the images were collected by image analysis system (Image Pro-Plus 7200, America, SONY 3CCD, Japan).

Statistical Analysis
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.

Results and Discussions
The effect of the composition on myo-clonic and tonic-clonic seizures was observed. As defined earlier, myo-clonic seizures usually appear as sudden brief jerks or twitches of arms and legs whereas tonic-clonic seizures (previously known as grand mal seizures) are the most dramatic type of epileptic seizures and can cause an abrupt loss of consciousness, body stiffening and shaking, and sometimes loss of bladder control or biting of the tongue.
Effect of Concentrated bone broth extract (COBBE) on Latency of Myo-clonic and Tonic- clonic seizures
COBBE exerted strong protective effect, greatly enhancing the time of seizures in animals whereas in animals not administered COBBE, seizures were observed at shorter time intervals. Though all animals in negative and treatment groups showed seizures after PTZ administration but the initiation time of myo–clonic and tonic-clonic seizures was much higher in test composition groups in comparison with PTZ control group i.e. the composition of present invention delayed the onset of seizures, indicating strong protective effect. This increase was significant at doses of 0.25 ml and 0.5 ml of concentrated horse bone broth extract (P<0.05) when compared with control group. Protective effect of higher dose was more.
Effect of Concentrated Bone Broth Extract (COBBE) in protecting test animals against mortality after PTZ -induced Seizures
The decrease in mortality after oral administration of COBBE was 100% i.e. none of the animals died, whereas in controls in which no treatment was given, all the animals died. The protective effect against mortality (convulsion survivors/ animals tested) of COBBE was 100% at oral doses
of 0.5 ml and 33% at a dose of 0.25 ml. as compared with PTZ control Group. This reduction was dose dependently significant (P<0.05) as compared with PTZ control Group. Diazepam completely inhibited PTZ-induced sei¬zures; therefore, the percent of survival of animals after treatment was 100%. Results are tabulated in Table 2 below:

Table 2 Effect of CHOBBE in reducing Pentylenetetrazole (PTZ) induced mortality in mice

Group Treatment Mice Mortality
Group-1 Control-1 (No PTZ i.e. no seizures induced)Vehicle i.e. normal saline (10 mg/kg; i.p.) only. No PTZ. 6 0
Group-2 Control-2 (PTZ given intraperitoneally i.e. seizures induced)Vehicle i.e. normal saline (10 mg/kg; i.p.) only + 80 mg/kg, i.p. PTZ 6 6
Group-3 Standard Drug (Diazepam) + PTZ
Standard drug diazepam given intraperitoneally (20 mg/kg, i.p.) + 80 mg/kg, i.p. PTZ 6 0
Group-4 Low oral dose of Concentrated Bone Broth Extract (COBBE) + PTZ
COBBE (0.25 ml, oral dose) + 80 mg/kg, i.p. PTZ 6 2
Group-5 High oral dose of COBBE + PTZ
COBBE (0.50 ml, oral dose) + 80 mg/kg, i.p. PTZ 6 0

Effect of Concentrated Bone Broth Extract (COBBE) against PTZ -induced Convulsions- Concentrated bone broth extract significantly decreased convulsions in mice. Data is given in Table 3 below:

Table3: Effect of Concentrated Horse Bone Broth Extract (CHOBBE) on Pentylenetetrazole (PTZ) induced convulsion in mice
Group Treatment Mice No. of animals convulsed % protection
Group-1 Control-1 (No PTZ i.e. no seizures induced) Vehicle i.e. normal saline (10 mg/kg; i.p.) only. No PTZ. 6 0 -
Group-2 Control-2 (PTZ given intraperitoneally i.e. seizures induced) Vehicle i.e. normal saline (10 mg/kg; i.p.) only + 80 mg/kg, i.p. PTZ 6 6 0
Group-3 Standard Drug (Diazepam) + PTZ
Standard drug diazepam given intraperitoneally (20 mg/kg, i.p.) + 80 mg/kg, i.p. PTZ 6 0 100
Group-4 Low oral dose of Concentrated Bone Broth Extract (COBBE) + PTZ
CHOBBE (0.25 ml, oral dose) + 80 mg/kg,
i.p. PTZ 6 3 50
Group-5 High oral dose of COBBE + PTZ
CHOBBE (0.50 ml, oral dose) + 80 mg/kg, i.p. PTZ 6 2 66

Protective Effect of Concentrated Bone Broth Extract (COBBE) on brain tissue of biochemical parameters of PTZ-induced epileptic mice
PTZ control group produced significant (P<0.05) increase in brain TBARS; and decrease in brain GSH levels when compared to the vehicle control group. Treatment with CHOBBE given orally to mice (0.25 ml and 0.5 ml, p.o.) and diazepam significantly (P<0.05) attenuated PTZ induced changes in brain biochemical parameters.

Protective Effect of Concentrated Bone Broth Extract (COBBE) on brain tissue of PTZ-induced epileptic mice
PTZ administration produced significant alterations in hippocampus, striatum and frontal cortex. Neurons appeared shrunken with a fusiform shape showing a typical picnotic conformation. Treatment with concentrated Horse bone broth extract and Diazepam attenuated the effects of PTZ on neuronal texture and configuration (Fig. 2). Results are tabulated in Table 4 below:

Table 4 Histopathological Observations relating to protective effect of COBBE on brain tissue of induced epileptic mice
Group Description Histopathological Observation
A Control group (with vehicle injection) without PTZ Hippocampus, striatum and frontal cortex normal. Neurons normal.
B Control group (with vehicle injection) + PTZ Hippocampus, striatum and frontal cortex abnormal. Neurons shrunken.
C Diazepam group + PTZ Hippocampus, striatum and frontal cortex normal. Neurons normal.
D COBBE Low Dose + PTZ -do-
E COBBE High Dose + PTZ -do-

Novelty, Inventive Step and Industrial Application
Novelty- The oral anti-epileptic composition of the present invention, consisting of concentrated, hydro-distillate of mammalian bone, is novel and neither anticipated nor disclosed in the prior art. It is a simple, easy to make and easy to consume oral composition with significant benefits in management and treatment of epilepsy.
Inventive Step- The technical advancement of knowledge lies in disclosing an oral anti-epileptic composition which is concentrated bone broth extract prepared by boiling powdered mammalian bone e.g. of horse, in water, condensing the vapors and concentration of the condensate to yield clear solution. The same contains nutrients like chondroitin sulfate, glucosamine, collagen like amino acids and other small molecules in natural synergistic ratios, exerting a powerful anti-epileptic effect, when given orally. The method of preparation is simple, fast and economical, not requiring sophisticated equipment or expensive chemicals at all, making commercial scale production very easy.
Note: Though horse bone was used in the experiments as example, any mammalian bone e.g. of cow, buffalo, sheep or goat or any other mammal, can be used for preparing the composition owing to molecular level similarity of bones. Hence, compositions prepared from bone of other animals, apart from horse may be considered within the scope of present invention.

Industrial Application- The composition can be easily manufactured at commercial scale by food and pharma industries and the raw material is easily available.

CLAIMS:WE CLAIM:
1. An oral anti-epileptic composition WHEREIN the same consists of aqueous distillate of mammalian bone powder in the form of clear solution and WHEREIN the composition has analytical FTIR fingerprint as given in Fig.1.

2. The oral anti-epileptic composition as claimed in claim 1 WHEREIN the same consists of chondroitin sulfate, glucosamine, collagen like amino acids and other small molecules in natural synergistic ratios.

3. The method of preparation of the oral anti-epileptic composition as claimed in claim 1 WHEREIN the same consists of the following steps:
i. Mammalian bone (e.g. horse bone) is pulverized and boiled in water in ratio of 1:2 (1 part bone powder and 2 parts water, weight by weight) for 20-30 minutes;
ii. The vapors are condensed and the condensate is concentrated at a temperature of 70 – 100 degree centigrade and vacuum to 1/10th the volume of condensate to give clear solution.