DESC:FIELD OF THE INVENTION
The present invention relates to a composition for use in the prevention and management of neurodegenerative disorders. More specifically, the present invention relates to a synergistic composition of biomarkers extracted from different plant extracts of Dioscorea Bulbifera, Bacopa Monnieri, and Hippophae Rhamnoides.
BACKGROUND OF THE INVENTION
Neurodegenerative diseases refer to degenerative diseases occurring with aging, particularly in the brain. Neurodegenerative diseases may be classified depending on the main symptoms and the affected part of the brain. Neurodegenerative diseases and disorders are currently in the focus of various scientific and medical investigations. Due to unknown etiology, it is very difficult to cure such complex and multifactorial CNS diseases. They are emerging diseases occurring more frequently in an elderly population. As a consequence of the demographic development of our society to an older population, there is a high medical need to cure such diseases. Accordingly, there are various efforts made today to prevent or treat such diseases more efficiently. However to date the therapeutic options are limited to symptomatic treatment. Only drugs that reduce symptoms but no disease-modifying drugs are authorized for neurodegenerative diseases.

The currently available therapies for Neurodegenerative diseases include drug therapy, surgery, physical therapy, etc. For drug therapy, drugs that boost the depleted level of dopamine in the brain, prevent or delay the destruction of nerve cells by neutralizing the imbalance in neurotransmitters caused by the deficit of dopamine and control other symptoms such as depression, etc. are generally used. Along with drug therapy, phytopharmaceuticals have already been reported as effective medications in many diseases. The phytopharmaceutical drug is defined as a purified and standardized fraction with a defined minimum of four bio-active or phytochemical compounds (qualitatively and quantitatively assessed) of an extract of a medicinal plant or its part, for internal or external use of human beings or animals for diagnosis, treatment, mitigation, or prevention of any disease or disorder. Phytopharmaceuticals from Ginko Biloba have been mentioned as potential drugs for the treatment of dementia. However, the pharmaceutical efficacy is still put in jeopardy. Similarly, phytodrugs such as Resveratrol, Ginseng, and Green Tea Extract have been reported as drugs for the treatment of neurodegenerative disorders.

Thus, the need to present invention arises for further compositions for efficiently treating and/or preventing neurodegenerative diseases or disorders. The technical problem underlying the present invention can be seen as the provision of means and methods for complying with the aforementioned needs.
OBJECTIVE OF THE INVENTION
The main objective of the present invention is to provide a composition for the management of the neurodegenerative disorder.
Another objective of the present invention is to provide an added plant-based composition effective in improving the orientation, working memory, language, and communication skill among people having neurodegeneration.
Yet another objective of the present invention is to propose an herbal composition beneficial in improving the overall mental performance and maintenance of the general health status of people.
Yet another objective of the present invention is to provide a composition using the plant extracts of Dioscorea Bulbifera, Bacopa Monnieri, and Hippophae Rhamnoides.
Yet another objective of the present invention is to provide a composition from the active Phyto-constituents Bacoside-A3, Beta-Sitosterol, Luteolin, and Quercetin that are extracted from the dry leaf extract of Bacopa Monnieri; Diosgenin, Quercetin, Gallic Acid, and Vitamin-C that extracted from the dry leaf extract of Dioscorea Bulbifera; and Omega-3 Fatty acid, Vitamin-C, Quercetin, and Folic Acid that are extracted from the dry leaf extract of Hippophae Rhamnoides.
Yet another objective of the present invention is to elucidate the synergistic action of the active constituents extracted from the plant herb.

Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed invention are illustrated by way of example.

SUMMARY OF THE INVENTION
The present invention relates to a composition for use in the management of the neurodegenerative disorder. The said composition comprises of Bacoside A3 in the range of 0.5083 mg to 1.5248 mg, Diosgenin in the range of 0.0549mg to 0.1646 mg, Quercetin in the range of 0.0103mg to 0.0308mg, Vitamin C in the range of 0.0439mg to 0.1316mg, Gallic Acid in the range of 0.0914mg to 0.2742mg, Omega 3 fatty acids in the range of 0.0134mg - 0.0402mg, Luteolin in the range of 0.0019 mg to 0.0057mg, Beta-sitosterol in the range of 0.0081mg to 0.0242mg, Folic Acid in the range of 0.0119 mg to 0.0357mg, and a pharmaceutically acceptable carrier and/or diluent. Herein, the above-listed constituents are the active compounds separated chromatographically from the hydro-alcoholic extracts. The hydro-alcoholic extracts are selected from Dioscorea bulbifera, Hippophae rhamnoides, and Bacopa monnieri. The composition is a synergistic combination of the said active components having a definite action in restoring a neuromodulatory activity. The said neurodegenerative disease or disorder is amnesia, learning and memory deficits, amyotrophic lateral sclerosis, circumscribed brain atrophy, corticobasal degeneration, degeneration of nervous system due to alcohol, unspecified degenerative disease of the nervous system, degenerative syndrome, diabetic polyneuropathy, epilepsy, Friedreich's ataxia, frontotemporal dementia and Parkinsonism of Chromosome, Huntington's disease, mild cognitive impairment, Morbus Pick, non-classified degenerative diseases or disorders of the nervous system, progressive isolated aphasia, progressive supranuclear palsy, non-classified senile degeneration of the brain, spinocerebellar ataxia/degenerations, mental retardation, spinal cord injury, forgetfulness, and Parkinson's disease. The active Phyto-constituents Bacoside-A3, Beta-Sitosterol, Luteolin, and Quercetin are extracted from the dry leaf extract of Bacopa Monnieri; Diosgenin, Quercetin, Gallic Acid, and Vitamin-C are the target phytochemicals extracted from the dry leaf extract of Dioscorea Bulbifera; and Omega-3 Fatty acid, Vitamin-C, Quercetin, and Folic Acid are extracted from the dry leaf extract of Hippophae Rhamnoides. The present invention relates to a method for the preparation of the composition used in the management of the neurodegenerative disorder. The method comprises of the following steps, Step 1: a dry extract of Dioscorea Bulbifera, Bacopa Monnieri, and Hippophae Rhamnoides are sieve through no.30#, and Sift Microcrystalline Cellulose Plain and Maize Starch through Sieve No. 30#. Step 2: Mixing and Granulation: the sifted raw material is collected in an RMG and is mixed for 15 minutes at the slow speed of 40 RPM, resulting in a blended mixture, isopropyl Alcohol and PVPK-30 is added in a container along with continuous stirring to get a clear solution, and the clear solution is added slowly in the above-blended mixture in RMG to make granules. Step 3: Drying: the above-wet granules are loaded in FBD and are air-dried for 10 to 12 minutes, and the granules are dried in FBD at inlet temperature 50°C ± 5°C for approximately 30 minutes to achieve the LOD of between 2% to 3% w/w (by IR/halogen moisture analyzer at 105 °C in auto mode). Step 4: Dry Screening: Check the Integrity of Screen, Sift the dried granules through 16# mesh and collect in doubled polythene lined container, the oversized granules are passed through 2.0 mm screen with the help of multi mill, and after milling the granules are passed through 16 # mesh, and Collect the said granules in IPC/HDPE container, weigh and label it. Step 5: Blending & Lubrication: the sifted granules are loaded in Octagonal Blender, Sift Low-Substituted Hydroxypropyl Cellulose LH-11 through sieve No. 20# and then load in Octagonal Blender, Sift Crospovidone through Mesh No. 40# and load in Octagonal Blender and run the blender for 20 minutes at 7 RPM, Sift Colloidal Anhydrous Silica and Magnesium Stearate through 40# mesh. Add the above materials into the Octagonal blender and mix for 3 minutes at 7 RPM, Unload the lubricated granules in double polythene lined container, and Collect the said granules in IPC/HDPE container. Step 6: Compression: Compress the granules by using 20.9 mm X 9.9 mm punches on Compression Machine, and Maintain the temperature between 25ºC ± 2ºC and relative humidity 45% ± 5% RH. Step 7: Coating: The coating can be seal coating and Film coating. For Seal Coating: Take Isopropyl Alcohol and add Hydroxy Propyl Methyl Cellulose 15 cps with continuous stirring, Add Methylene Dichloride to above solution of step under continuous mixing, Stir continuously for 45 minutes until homogenous dispersion is obtained, Filter the coating solution through 100# filter cloth, and Load uncoated de-dusted solid dosage forms into Auto Coater and warm them for 10 min at 70°C ± 5°C then start their coating . For Film Coating: Take Isopropyl alcohol and add Novomix 80021 Red with continuous stirring, Add Methylene Dichloride to above solution of step under continuous mixing, Filter the coating solution through 100# filter cloth then start Color coating, and Sampling: Take a sample from the final Coated solid dosage forms & send for analysis to the laboratory. The said composition comprises at least one further pharmaceutically active ingredient selected from the group consisting of minerals, vitamins, salt, fillers, binders, and combinations thereof.

The main advantage of the present invention is that the present invention provides a composition for the management of the neurodegenerative disorder.
Another advantage of the present invention is that the present invention provides a plant-based composition effective in improving the orientation, working memory, language, and communication skill among people having neurodegeneration.
Yet another advantage of the present invention is that the present invention proposes a herbal composition beneficial in improving the overall mental performance and also maintenance of general health status of people.
Yet another advantage of the present invention is that the present invention provides a composition using the plant extracts of Dioscorea Bulbifera, Bacopa Monnieri, and Hippophae Rhamnoides.
Yet another advantage of the present invention is to provide a composition from the active Phyto-constituents Bacoside-A3, Beta-Sitosterol, Luteolin, and Quercetin that are extracted from the dry leaf extract of Bacopa Monnieri; Diosgenin, Quercetin, Gallic Acid, and Vitamin-C that extracted from the dry leaf extract of Dioscorea Bulbifera; and Omega-3 Fatty acid, Vitamin-C, Quercetin, and Folic Acid that are extracted from the dry leaf extract of Hippophae Rhamnoides.
Yet another advantage of the present invention is to elucidate the synergistic action of the active constituents extracted from the plant herb.

Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed invention are illustrated by way of example.

DETAILED DESCRIPTION OF THE INVENTION
While this invention is susceptible to embodiment in many different forms, there is shown in the drawings and will herein be described in detail specific embodiments, with the understanding that the present disclosure of such embodiments is to be considered as an example of the principles and not intended to limit the invention to the specific embodiments shown and described. In the description below, reference numerals are used to describe the same, similar, or corresponding parts in the several views of the drawings. This detailed description defines the meaning of the terms used herein and specifically describes embodiments for those skilled in the art to practice the invention.

Definition
The terms “a” or “an”, as used herein, are defined as one or as more than one. The term “plurality”, as used herein, is defined as two or more than two. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language). The term “coupled”, as used herein, is defined as connected, although not necessarily directly, and not necessarily mechanically. The term “comprising” is not intended to limit inventions to only claiming the present invention with such comprising language. Any invention using the term comprising could be separated into one or more claims using “consisting” or “consisting of” claim language and is so intended. The term “comprising” is used interchangeably used by the terms “having” or “containing”. Reference throughout this document to “one embodiment”, “certain embodiments”, “an embodiment”, “another embodiment”, and “yet another embodiment” or similar terms means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases or in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics are combined in any suitable manner in one or more embodiments without limitation. The term “or” as used herein is to be interpreted as an inclusive or meaning any one or any combination. Therefore, “A, B or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B, and C”. An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.
As used herein, the term "one or more" generally refers to, but is not limited to, singular as well as the plural form of the term.

The drawings featured in the figures are to illustrate certain convenient embodiments of the present invention and are not to be considered as limitations thereto. The term “means” preceding a present participle of an operation indicates the desired function for which there is one or more embodiments, i.e., one or more methods, devices, or apparatuses for achieving the desired function and that one skilled in the art could select from these or their equivalent is given the disclosure herein and use of the term “means” is not intended to be limiting.

The term "composition" as used herein refers to a mixture of extracts from, inter alia, biological sources such as plants which are further defined elsewhere herein. Preferably, the said composition can further comprise other ingredients or diluents. Preferably, such further ingredients can be stabilizing agents, wetting agents, pharmaceutical carriers, additional pharmaceutically active agents, release controlling agents, and the like. Preferred diluents encompass water, alcohols, physiological saline solutions, buffers, such as phosphate-buffered saline solutions, syrup, oil, water, emulsions, various types of wetting agents, and like.

The term "treating" as used herein refers to any improvement of the neurodegenerative disease or disorder that occurs in a treated subject compared to an untreated subject. Such an improvement can be a prevention of a worsening or progression of the said disease or disorder. Moreover, such an improvement may also be an amelioration or cure of the neurodegenerative disease or disorder or its accompanying symptoms. It will be understood that treatment may not be successful for 100% of the subjects to be treated.

Accordingly, preferred neurodegenerative diseases or disorders referred to in accordance with the present invention, are Alzheimer's disease, amnesia, learning and memory deficits, amyotrophic lateral sclerosis, circumscribed brain atrophy, corticobasal degeneration, degeneration of nervous system due to alcohol, unspecified degenerative disease of the nervous system, degenerative syndrome, diabetic polyneuropathy, epilepsy, Friedreich's ataxia, frontotemporal dementia and Parkinsonism of Chromosom, Huntington's disease, mild cognitive impairment, Morbus Pick, non-classified degenerative diseases or disorders of the nervous system, progressive isolated aphasia, progressive supranuclear palsy, non-classified senile degeneration of the brain, spinocerebellar ataxia/degenerations, mental retardation, spinal cord injury, forgetfulness, and Parkinson's disease. The symptoms accompanying the aforementioned diseases and disorders are well known to those skilled in the art and are described in more detail in standard textbooks of medicine.

The present invention relates to a composition for use in the management of the neurodegenerative disorder. The said composition comprises of Bacoside A3 in the range of 0.5083 mg to 1.5248 mg, Diosgenin in the range of 0.0549mg to 0.1646 mg, Quercetin in the range of 0.0103mg to 0.0308mg, Vitamin C in the range of 0.0439mg to 0.1316mg, Gallic Acid in the range of 0.0914mg to 0.2742mg, Omega 3 fatty acids in the range of 0.0134mg - 0.0402mg, Luteolin in the range of 0.0019 mg to 0.0057mg, Beta-sitosterol in the range of 0.0081mg to 0.0242mg, Folic Acid in the range of 0.0119 mg to 0.0357mg, and a pharmaceutically acceptable carrier and/or diluent. Herein, the above-listed constituents are the active compounds separated chromatographically from the hydro-alcoholic extracts.

In the preferred embodiment of the present invention, the hydro-alcoholic extracts are selected from Dioscorea bulbifera, Hippophae rhamnoides, and Bacopa monnieri.

In the preferred embodiment of the present invention, the active Phyto-constituents Bacoside-A3, Beta-Sitosterol, Luteolin, and Quercetin are extracted from the dry leaf extract of Bacopa Monnieri; Diosgenin, Quercetin, Gallic Acid, and Vitamin-C are the target phytochemicals extracted from the dry leaf extract of Dioscorea Bulbifera; and Omega-3 Fatty acid, Vitamin-C, Quercetin, and Folic Acid are extracted from the dry leaf extract of Hippophae Rhamnoides.

In an embodiment of the present invention, the composition is a synergistic combination of the said active components having a definite action in restoring a neuromodulatory activity.
In an aspect of the present invention, the said neurodegenerative disease or disorder is amnesia, learning and memory deficits, amyotrophic lateral sclerosis, circumscribed brain atrophy, corticobasal degeneration, degeneration of nervous system due to alcohol, unspecified degenerative disease of the nervous system, degenerative syndrome, diabetic polyneuropathy, epilepsy, Friedreich's ataxia, frontotemporal dementia and Parkinsonism of Chromosome, Huntington's disease, mild cognitive impairment, Morbus Pick, non-classified degenerative diseases or disorders of the nervous system, progressive isolated aphasia, progressive supranuclear palsy, non-classified senile degeneration of the brain, spinocerebellar ataxia/degenerations, mental retardation, spinal cord injury, forgetfulness, and Parkinson's disease.

The present invention relates to a method for the preparation of composition used in the management of neurodegenerative disorders. The method comprises of the following steps
Step 1:
i. a dry extract of Dioscorea Bulbifera, Bacopa Monnieri, and Hippophae Rhamnoides are sieve through no.30#, and
ii. Sift Microcrystalline Cellulose Plain and Maize Starch through Sieve No. 30#,
Step 2: Mixing and Granulation
i. the sifted raw material is collected in an RMG and is mixed for 15 minutes at the slow speed of 40 RPM, resulting in a blended mixture,
ii. isopropyl Alcohol and PVPK-30 is added in a container along with continuous stirring to get a clear solution, and
iii. the clear solution is added slowly in the above-blended mixture in RMG to make granules,
Step 3: Drying
i. the above-wet granules are loaded in FBD and are air-dried for 10 to 12 minutes, and
ii. the granules are dried in FBD at inlet temperature 50°C ± 5°C for approximately 30 minutes to achieve the LOD of between 2% to 3% w/w (by IR/halogen moisture analyzer at 105 °C in auto mode),
Step 4: Dry Screening
i. Check the Integrity of the Screen,
ii. Sift the dried granules through 16# mesh and collect in doubled polythene lined container,
iii. the oversized granules are passed through a 2.0 mm screen with the help of multi mill, and after milling the granules are passed through 16 # mesh, and
iv. Collect the said granules in IPC/HDPE container, weigh and label them,
Step 5: Blending & Lubrication
i. the sifted granules are loaded in an Octagonal Blender,
ii. Sift Low-Substituted Hydroxypropyl Cellulose LH-11 through sieve No. 20# and then load in Octagonal Blender,
iii. Sift Crospovidone through Mesh No. 40# and load in Octagonal Blender and run the blender for 20 minutes at 7 RPM,
iv. Sift Colloidal Anhydrous Silica and Magnesium Stearate through 40# mesh. Add above materials into Octagonal blender and mix for 3 minutes at 7 RPM,
v. Unload the lubricated granules in double polythene lined container, and
vi. Collect the said granules in IPC/HDPE container,
Step 6: Compression
i. Compress the granules by using 20.9 mm X 9.9 mm punches on Compression Machine, and
ii. Maintain the temperature between 25ºC ± 2ºC and relative humidity 45% ± 5% RH, and
Step 7: Coating
Seal Coating:
i. Take Isopropyl Alcohol and add Hydroxy Propyl Methyl Cellulose 15 cps with continuous stirring,
ii. Add Methylene Dichloride to the above solution of step under continuous mixing,
iii. Stir continuously for 45 minutes until homogenous dispersion is obtained,
iv. Filter the coating solution through 100# filter cloth, and
v. Load uncoated de-dusted solid dosage forms into Auto Coater and warm them for 10 min at 70°C ± 5°C then start their coating ,
Film Coating:
i. Take Isopropyl alcohol and add Novomix 80021 Red with continuous stirring,
ii. Add Methylene Dichloride to the above solution of step under continuous mixing,
iii. Filter the coating solution through 100# filter cloth then start Color coating, and
iv. Sampling: Take a sample from the final Coated solid dosage forms & send it for analysis to the laboratory.
In another embodiment of the present invention, the said composition comprises at least one further pharmaceutically active ingredient selected from the group consisting of minerals, vitamins, salt, fillers, binders, and combinations thereof.
EXPERIMENTAL DATA BASED ON STUDIES
In this composition extracts of Dioscorea Bulbifera, Bacopa Monnieri, and Hippophae Rhamnoides are used. Dioscorea bulbifera is a member of the Dioscoraceae family. The extract of the bulbil of the plant was used in this formulation. D. bulbifera extracts have shown hypolipidemic, anti-inflammatory, anti-hyperglycaemic and anti-obesity properties along with alphaglucosidase, amylase and lipase inhibitory effects, which may help prevent neurodegeneration caused by hyperglycaemia, hyperlipoproteinaemia and obesity. The targets of action are suggested to be pro-inflammatory cytokines- CRP, TNF-a, IL-6, adipokines, adiponectin, leptin, resistin, gherlin, and hyperinsulinaemia. Hippophase rhamnoides, belongs to the family Elaeagnaceae, and is commonly known as seabuckthorn. An extract of the leaves and fruit pulp has been used in this polyherbal formulation. The fruit of H. rhamnoides is rich in different flavonoids like vitamin P and quercetin, contains waterand fat-soluble vitamins along with essential amino acids folic acid, various fatty acids, phytosterols, a-tocopherol and phenolic compounds. The high concentration of folic acid in this plant can help in reducing elevated levels of homocysteine, which is a marker for neurodegeneration. Bacopa monnieri (BM) is traditionally well known for its activity as cognition enhancer. Clinically BM was demonstrated to improve cognitive function in various groups of people. BM increased cognition in elderly people. Developed formulation was performed for preclinical studies on below lines.
1. Behavioral Parameter
• Morris water maze (MWM) Test
2. Histopathological Studies
3. Biochemical Parameters:
• Estimation of MDA levels
• Estimation of SOD levels
• Estimation of GSH levels
• Estimation of Catalase levels
• Estimation of TNF-a
• Estimation of interleukine-6
• Estimation of Acetylcholinesterase
• Estimation of Amyloid beta
• Estimation of phosphorylated Tau
Morris water maze (MWM) Test
Tracking plot showing the efficacy of Polyherbal formulation on Morris water maze task while performing the behavioral experiment conducted for Probe trial on the 5th day of MWM test with reference to FIGURE 1.
Histopathological Studies:
Histochemical analysis of the morphological changes in the brain hippocampal neurons was analyzed with H&E staining. The brain sections of the high-dose group show numerous healthy neurons, which appeared spherical/ oval in shape with clear cytoplasm of the polyherbal formulation as shown in FIGURE 2.
Biochemical Parameters :
Estimation of MDA levels: The quantitative measurement of MDA, lipid peroxidation end product was done using the method described by Wills (Wills, 1966). In brief, 0.1 ml of brain homogenate was heated at 95°C for 1 h with 0.1 ml of 8.1% sodium dodecyl sulphate (SDS), 0.75 ml 20% glacial acetic acid, 0.75 ml of 0.8% thiobarbituric acid (TBA) and 0.3 ml of distilled water. The supernatant was collected by centrifugation at 10000 rpm for 10 min. The amount of MDA in the supernatant was estimated spectrometrically at 532 nm using Tetramethoxypropane as standard. (Epoch microplate spectrophotometer, Biotek, Winooski, US). The values are represented as a nanomole of MDA per milligram of protein. FIGURE 3 shows effect of test formulations on Lipid peroxidation activity in the brain of Amyloid-ß treated rats. Data presented as mean ± SEM; n = 6. **** P <0.0001, , ns: non-significant versus corresponding value in the disease control group.
Estimation of SOD: The estimation of SOD is performed based on autoxidation epinephrine at pH 10.4. Briefly, the supernatant of brain homogenate was mixed with 0.8ml of 50 mM glycine buffer (pH 10.4) and 0.02 ml of (-)-epinephrine was added. After 5 min the absorbance was measured at 480 nm. (UV-1800 Spectrophotometer, Shimadzu, Japan). The activity of SOD was represented as U/milligram of protein. FIGURE 4 shows Effect of test formulations on SOD activity in the brain of Amyloid-ß treated rats. Data presented as mean ± SEM; n = 6. **** P <0.0001, ** P < 0.05, ns: non- significant versus corresponding value in the disease control group.
Estimation of GSH levels: The reduced glutathione in brain homogenate was estimated by Ellman method. In brief, an equal volume of sulpho-salicylic acid (5%) and homogenate were mixed and cold digested for 1 h at 4 °C. Further, the supernatant was collected after centrifuging at 10000rpm for 10min at 4 °C. The 50µl of supernatant was mixed with 450µl of phosphate buffer and 1.5 ml of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) in 0.1 M phosphate buffer, (pH 8.0). After incubating the above reaction mixture for 10 min at 37 °C, the absorbance was measured immediately at 412 nm using a spectrophotometer. (Epoch microplate spectrophotometer, Biotek, Winooski, US). Results were represented as micromole per milligram protein. FIGURE 5 shows effect of test formulations on GSH activity in the brain of Amyloid-ß treated rats. Data presented as mean ± SEM; n = 6. **** P <0.0001, **** P < 0.001, ns: non- significant versus corresponding value in the disease control group
Estimation of Catalase: CAT is a hemeprotein, localized in the peroxisomes or the micro peroxisomes. This enzyme catalyzes the decomposition of H2O2 to water and oxygen and thus protecting the cell from oxidative damage by H2O2 and OH. CAT is a key component of the antioxidant defense system. Inhibition of these protective mechanisms results in enhanced sensitivity to free radical- induced cellular damage.0.1 ml of supernatant was added to cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7.0) Reaction was started by the addition of 1.0 ml of freshly prepared 30 mMH2O2.The rate of decomposition of H202 was measured spectrophotometrically from changes in absorbance at 240 nm using a spectrophotometer. (Epoch microplate spectrophotometer, Biotek,Winooski, US). FIGURE 6 shows Effect of test formulations on catalase activity in the brain of Amyloid-ß treated rats. Data presented as mean ± SEM; n = 6. **** P <0.0001, *** P < 0.001, ns: non- significant versus corresponding value in the disease control group.
Estimation of TNF-a: The level of pro-inflammatory cytokine in the hippocampus homogenate samples was estimated by utilizing commercially available ELISA kits as per their protocol. FIGURE 7 shows effect of test formulations on TNF-a activity in the brain of Amyloid-ß treated rats. Data presented as mean ± SEM; n = 6. **** P <0.0001, ** P < 0.05, ns: non- significant versus corresponding value in the disease control group.
Estimation of IL-6 :- Higher dose of the composition shows significant reduction in the pro-inflammatory cytokine IL-6. FIGURE 8 shows effect of test formulations on IL-6 activity in the brain of Amyloid-ß treated rats. Data presented as mean ± SEM; n = 6. **** P <0.0001, **** P < 0.001, ** P < 0.05. ns: non- significant versus corresponding value in the disease control group.
Estimation of AChE: The level of acetylcholinesterase in the hippocampus homogenate samples was estimated as per the kit protocol. FIGURE 9 Effect of test formulations on Acetylcholinesterase activity in the brain of Amyloid-ß treated rats. Data presented as mean ± SEM; n = 6. **** P <0.0001, * P < 0.05, ns: non-significant versus corresponding value in the disease control group.
Conclusion of the studies : The Polyherbal formulation is used for tablet oral dosage forms. The results of the study showed a significant neuroprotective effect at mid-dose and high dose of the tablet formulations were evaluated in Aß 1- 42 induced AD animal model. The tablet (high dose) significantly attenuated oxidative stress (MDA) and pro-inflammatory cytokines levels while increasing the defensive anti-oxidant enzyme (GSH, SOD and catalase) levels when compared with the disease control group. Interestingly, the maximum efficacy was observed in the case of a high dose of the tablet.
Likewise, the levels of pro-inflammatory cytokines TNF-a & IL-6 significantly reduced when administered with the different doses of the formulations i.e mid and high dose of the tablet formulation. The results showed maximum efficacy at high doses of tablet formulation.
Moreover, the level of acetylcholinesterase enzymes was also found to be significantly reduced in the treated groups. The maximum efficacy in reducing the level of acetylcholinesterase was observed in the groups administered with a high dose of the tablet formulation. The treatment groups did not show any changes in the phosphorylated tau protein when compared with disease group but the pathological biomarkers significantly attenuated at high dose of tablet. The assessment of the behavioural89 parameters also showed significant improvements in cognitive functions when compared with the disease control group using MWM test.
The experiment showed a significant result in favour of the neuroprotective effect when the data obtained from the control and treated groups that were compared with the diseased group. The data obtained from the histopathological studies also showed that the brain sections of the high-dose of tablet group show numerous healthy neurons, which appeared spherical/ oval shapes with clear cytoplasm. The result indicates that test formulations are effective in imparting neuroprotection against the amyloid-ß induced AD. However, maximum efficacy was provided by the high dose of the tablet.

Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed present invention are illustrated by way of example and appropriate reference to accompanying drawings. Those skilled in the art to which the present invention pertains may make modifications resulting in other embodiments employing principles of the present invention without departing from its spirit or characteristics, particularly upon considering the foregoing teachings. Accordingly, the described embodiments are to be considered in all respects only as illustrative, and not restrictive, and the scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description or drawings. Consequently, while the present invention has been described regarding particular embodiments, modifications of structure, sequence, materials and the like apparent to those skilled in the art still fall within the scope of the invention as claimed by the applicant.
,CLAIMS:1. A composition for use in prevention and management of the neurodegenerative disorder, the said composition comprising:
a) Bacoside A3 in the range of 0.5083 mg to 1.5248 mg;
b) Diosgenin in the range of 0.0549mg to 0.1646 mg;
c) Quercetin in the range of 0.0103mg to 0.0308mg;
d) Vitamin C in the range of 0.0439mg to 0.1316mg;
e) Gallic Acid in the range of 0.0914mg to 0.2742mg;
f) Omega 3 fatty acids in the range of 0.0134mg - 0.0402mg;
g) Luteolin in the range of 0.0019 mg to 0.0057mg;
h) Beta-sitosterol in the range of 0.0081mg to 0.0242mg;
i) Folic Acid in the range of 0.0119 mg to 0.0357mg; and
a pharmaceutically acceptable carrier and/or diluent;
Wherein, the constituents (a-i) are the active compounds separated chromatographically from the hydro-alcoholic extracts.
2. The hydro-alcoholic extracts claimed in claim 1, wherein the hydro-alcoholic extracts are selected from Dioscorea bulbifera, Hippophae rhamnoides, and Bacopa monnieri.
3. The composition claimed in claim 1, wherein the composition is a synergistic combination of the said active components having a definite action in restoring a neuromodulatory activity.
4. The composition claimed in claims 1, wherein said neurodegenerative disease or disorder is amnesia, learning and memory deficits, amyotrophic lateral sclerosis, circumscribed brain atrophy, corticobasal degeneration, degeneration of nervous system due to alcohol, unspecified degenerative disease of the nervous system, degenerative syndrome, diabetic polyneuropathy, epilepsy, Friedreich's ataxia, frontotemporal dementia and Parkinsonism of Chromosome, Huntington's disease, mild cognitive impairment, Morbus Pick, non-classified degenerative diseases or disorders of the nervous system, progressive isolated aphasia, progressive supranuclear palsy, non-classified senile degeneration of the brain, spinocerebellar ataxia/degenerations, mental retardation, spinal cord injury, forgetfulness, and Parkinson's disease.
5. The composition claimed in claim 1, wherein, the active Phyto-constituents Bacoside-A3, Beta-Sitosterol, Luteolin, and Quercetin are extracted from the dry leaf extract of Bacopa Monnieri; Diosgenin, Quercetin, Gallic Acid, and Vitamin-C are the target phytochemicals extracted from the dry leaf extract of Dioscorea Bulbifera; Omega-3 Fatty acid, Vitamin-C, Quercetin, and Folic Acid are extracted from the dry leaf extract of Hippophae Rhamnoides.
6. The method for the preparation of the composition used in the management of the neurodegenerative disorder, the method comprises of:
Step 1:
iii. a dry extract of Dioscorea Bulbifera, Bacopa Monnieri, and Hippophae Rhamnoides are sieve through no.30#, and
iv. Sift Microcrystalline Cellulose Plain and Maize Starch through Sieve No. 30#;
Step 2: Mixing and Granulation
iv. the sifted raw material is collected in an RMG and is mixed for 15 minutes at the slow speed of 40 RPM, resulting in a blended mixture,
v. isopropyl Alcohol and PVPK-30 is added in a container along with continuous stirring to get a clear solution, and
vi. the clear solution is added slowly in the above-blended mixture in RMG to make granules;
Step 3: Drying
iii. the above-wet granules are loaded in FBD and are air-dried for 10 to 12 minutes, and
iv. the granules are dried in FBD at inlet temperature 50°C ± 5°C for approximately 30 minutes to achieve the LOD of between 2% to 3% w/w (by IR/halogen moisture analyzer at 105 °C in auto mode);
Step 4: Dry Screening
v. Check the Integrity of the Screen,
vi. Sift the dried granules through 16# mesh and collect in doubled polythene lined container,
vii. the oversized granules are passed through a 2.0 mm screen with the help of multi mill, and after milling the granules are passed through 16 # mesh, and
viii. Collect the said granules in IPC/HDPE container, weigh and label it;
Step 5: Blending & Lubrication
vii. the sifted granules are loaded in an Octagonal Blender,
viii. Sift Low-Substituted Hydroxypropyl Cellulose LH-11 through sieve No. 20# and then load in Octagonal Blender,
ix. Sift Crospovidone through Mesh No. 40# and load in Octagonal Blender and run the blender for 20 minutes at 7 RPM,
x. Sift Colloidal Anhydrous Silica and Magnesium Stearate through 40# mesh. Add above materials into Octagonal blender and mix for 3 minutes at 7 RPM,
xi. Unload the lubricated granules in double polythene lined container, and
xii. Collect the said granules in IPC/HDPE container;
Step 6: Compression
iii. Compress the granules by using 20.9 mm X 9.9 mm punches on Compression Machine, and
iv. Maintain the temperature between 25ºC ± 2ºC and relative humidity 45% ± 5% RH; and
Step 7: Coating
Seal Coating:
vi. Take Isopropyl Alcohol and add Hydroxy Propyl Methyl Cellulose 15 cps with continuous stirring,
vii. Add Methylene Dichloride to the above solution of step under continuous mixing,
viii. Stir continuously for 45 minutes until homogenous dispersion is obtained,
ix. Filter the coating solution through 100# filter cloth, and
x. Load uncoated de-dusted solid dosage forms into Auto Coater and warm the solid dosage forms for 10 min at 70°C ± 5°C then start coating of solid dosage forms,
Film Coating:
v. Take Isopropyl alcohol and add Novomix 80021 Red with continuous stirring,
vi. Add Methylene Dichloride to the above solution of step under continuous mixing,
vii. Filter the coating solution through 100# filter cloth then start Colour coating, and
viii. Sampling: Take a sample from the final Coated & solid dosage form send it for analysis to the lab.
7. The composition claimed in claims 1 to 5, wherein said composition comprises at least one further pharmaceutically active ingredient selected from the group consisting of minerals, vitamins, salt, fillers, binders, and combinations thereof.