NEUROPROTECTIVE COMPOSITION COMPRISING BETANIN FOR TREATMENT OF DEMENTIA AND COGNITIVE DISORDERS

Abstract

ABSTRACT A neuroprotective composition comprising Betanin for the treatment of dementia and cognitive disorders is disclosed. Betanin exhibits neuroprotective properties, improving cognitive function by reducing oxidative stress, modulating cholinergic activity, and potentially activating the Nrf2 pathway. In experimental studies with scopolamine-induced memory impairment in mice, Betanin significantly improved memory performance, decreased acetylcholinesterase (AChE) activity, reduced malondialdehyde (MDA) levels, and enhanced antioxidant enzymes. The composition offers an alternative treatment for neurodegenerative diseases by leveraging the natural neuroprotective effects of Betanin.

Specification

Description:NEUROPROTECTIVE COMPOSITION COMPRISING BETANIN FOR TREATMENT OF DEMENTIA AND COGNITIVE DISORDERS

FIELD OF THE INVENTION
The present invention pertains to the fields of pharmacology and neuroprotection, specifically focusing on the use of Betanin (C24H26N2O13), a naturally derived compound, in a pharmaceutical composition for treating neurodegenerative diseases, including dementia and cognitive impairments. The invention leverages the neuroprotective, antioxidative, and anti-cholinesterase properties of Betanin, aimed at reducing cognitive decline through multiple pathways in the brain.

BACKGROUND OF THE INVENTION
Dementia and neurodegenerative diseases are increasingly prevalent as life expectancy rises globally. Dementia, including Alzheimer's disease, is characterized by progressive cognitive decline, memory loss, and eventual loss of functionality in daily activities. While the precise mechanisms of dementia vary across subtypes, common features include oxidative stress, cholinergic dysfunction, neuroinflammation, and the accumulation of amyloid-beta plaques or tau tangles in the brain. Among these, oxidative stress plays a significant role, causing cellular damage and contributing to neuronal death. The cholinergic system, responsible for transmitting signals critical for memory and cognition, is also compromised in dementia, which further exacerbates cognitive deficits.
Currently, the standard pharmacological treatments for dementia involve the use of cholinesterase inhibitors, such as donepezil, which function by enhancing cholinergic neurotransmission. However, these treatments are largely symptomatic and fail to address the underlying neurodegeneration. Additionally, cholinesterase inhibitors are associated with side effects, including gastrointestinal issues, bradycardia, and potential drug interactions. Consequently, there is a growing interest in alternative approaches, especially those that target neuroprotective pathways to slow or prevent the progression of neurodegenerative diseases.
The Nrf2 (Nuclear factor erythroid 2-related factor 2) pathway has emerged as a promising therapeutic target for neurodegenerative diseases. Nrf2 is a transcription factor that regulates the expression of antioxidant proteins, protecting cells from oxidative damage. Activation of the Nrf2 pathway results in the upregulation of various antioxidant response elements (AREs), which include enzymes like catalase, superoxide dismutase, and glutathione reductase. These enzymes mitigate oxidative stress by neutralizing free radicals and reactive oxygen species (ROS), thus reducing cellular damage and supporting neuronal survival.
Betanin, a natural pigment found in beetroot, is known for its potent antioxidative properties. Research has shown that Betanin can scavenge free radicals, reduce lipid peroxidation, and inhibit acetylcholinesterase (AChE) activity. These effects suggest Betanin as a potential candidate for neuroprotection, particularly in conditions where oxidative stress and cholinergic dysfunction are predominant factors. The exact mechanisms by which Betanin confers neuroprotection remain to be fully elucidated. however, preliminary studies indicate that Betanin may activate the Nrf2 pathway, enhancing the brain's antioxidative capacity.
The present invention provides a novel neuroprotective composition containing Betanin for the treatment of dementia. The composition offers a comprehensive therapeutic approach by simultaneously modulating cholinergic activity, reducing oxidative stress, and potentially activating the Nrf2 pathway. The present invention addresses the limitations of currently existing treatments by providing a natural alternative that can improve cognitive function without the adverse effects associated with synthetic drugs.

OBJECTIVE OF THE INVENTION
The objective of the present invention is to provide a neuroprotective composition comprising Betanin, intended to mitigate cognitive impairment and enhance memory function in subjects with neurodegenerative conditions such as dementia or Alzheimer's disease. Specifically, the invention aims to utilize the antioxidant and cholinergic-enhancing properties of Betanin to reduce oxidative stress, decrease acetylcholinesterase (AChE) activity, and upregulate neuroprotective pathways, including the Nrf2 pathway, thereby offering a therapeutic option that addresses the underlying oxidative damage associated with cognitive decline.
The present invention further seeks to improve the safety and efficacy of treatments for cognitive disorders by providing a composition with minimal side effects that can be administered alone or in combination with other neuroprotective agents.

SUMMARY OF THE INVENTION
The invention provides a pharmaceutical composition containing Betanin as the primary active ingredient, formulated to treat cognitive impairments associated with dementia and other neurodegenerative diseases.
The composition of the present invention, when administered to subjects with dementia, significantly enhances memory performance, reduces oxidative stress markers, and lowers acetylcholinesterase activity. The method of administration is through oral dosages, suitable for human and animal use.
An efficacy of the composition may be demonstrated in preclinical models of dementia. In such models, Swiss albino mice may be used to assess the neuroprotective effects of Betanin. Dementia may be induced using scopolamine, a cholinergic antagonist known to impair memory, thus providing a model for cognitive deficits. Betanin may be administered orally to the subject mice, with behavioural and biochemical assessments conducted over a period of two weeks.
In an embodiment of the present invention, the results of such models indicate that Betanin effectively improved memory performance and reduced oxidative damage in brain tissues, highlighting its potential as a therapeutic agent for neurodegenerative conditions.


BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying figures illustrate several embodiments of the disclosure and, together with the description, serve to explain the principles of the disclosure. One of ordinary skill in the art readily recognizes that the embodiments illustrated in the figures are merely exemplary, and are not intended to limit the scope of the present disclosure.
FIG. 1 illustrates chemical structure of Betanin (C24H26N2O13) used in an embodiment of the present disclosure.
FIG. 2 illustrates an exemplary Diagrammatic representation of Hypothesis of an embodiment of the present disclosure.
FIG. 3 illustrates an exemplary Diagrammatic representation of a plan of work of an embodiment of the present disclosure.
FIG. 4 discloses (A) Effect of betanin on Escape latency by using Morris Water Maze, (B) Effect of betanin on Time spent in Target Quadrant by using Morris Water Maze, and (C) Effect of betanin on Spontaneous alteration behaviour by using Y maze.
FIG. 5 illustrates betanin decreased MDA level in Scopolamine induced dementia in subject.
FIG. 6 illustrates betanin decreased Catalase level in Scopolamine induced dementia in subject.
FIG. 7 illustrates betanin increased reduced Glutathione level in Scopolamine induced dementia in subject.
FIG. 8 illustrates betanin decreased AChE level in Scopolamine induced dementia in subject.
Further areas of applicability of the present disclosure will become apparent from the complete description provided hereinafter.
It should be understood that the complete description of exemplary embodiments is intended for illustration purposes only and is, therefore, not intended to necessarily limit the scope of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
The present invention is more particularly described in the following present specification that is intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Various embodiments of the present disclosure are now described in detail. Referring to the drawings, like numbers, if any, indicate like components throughout the views. 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 and throughout the claims that follow, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. Moreover, titles or subtitles may be used in the specification for the convenience of a reader, which shall have no influence on the scope of the present disclosure. Additionally, some terms used in this specification are more specifically defined below.
The terms used in this specification generally have their ordinary meanings in the art, within the context of the present disclosure, and in the specific context where each term is used. Certain terms that are used to describe the present disclosure are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the disclosure.
For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term are the same, in the same context, whether or not it is highlighted. It will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms.
The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the disclosure or of any exemplified term. Likewise, the disclosure is not limited to various embodiments given in this specification.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the case of conflict, the present document, including definitions will control.
As used herein, "around", "about" or "approximately" shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term "around", "about" or "approximately" can be inferred if not expressly stated.
As used herein, "plurality" means two or more.
As used herein, the terms "comprising," "including," "carrying," "having," "containing," "involving," and the like are to be understood to be open-ended, i.e., to mean including but not limited to.
As used herein, the phrase at least one of A, B, and C should be construed to mean a logical (A or B or C), using a non-exclusive logical OR. It should be understood that one or more steps within a method may be executed in a different order (or concurrently) without altering the principles of the present disclosure.
The present invention relates to a neuroprotective composition containing Betanin (C24H26N2O13) for the treatment and management of cognitive impairments. The following sections detail the experimental setup, findings, and potential therapeutic applications of the composition.
Experimental Methodology
In the present invention, for an investigation of Betanin's neuroprotective potential, adult Swiss albino mice, aged 18 to 24 months and weighing 20-25 grams, may be selected as the experimental subjects. Mice of either sex may be used to eliminate potential gender-specific effects. These experimental subjects may be obtained from the Central Animal House at the Institute of Pharmaceutical Research, GLA University for housing and quarantining small laboratory animals like rats, mice, guinea pigs, and rabbits, Mathura, and kept under controlled conditions.
For the present invention, the experimental subjects resided in polypropylene cages lined with husk bedding within the institutional animal facility, maintaining ambient conditions of 24 ± 2°C temperature, 45-55% relative humidity, and a 12-hour light/dark cycle. Standardized pellet food, provided by Lipton India, Ltd., Mumbai, and water were accessible ad libitum.
The experimental procedures may be conducted in strict adherence to guidelines set by the Institutional Animal Ethics Committee (IAEC) under approval number 1260/PO/Re/S/09/CPCSEA/IAEC/GLAIPR, following the principles of laboratory animal care as outlined by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the National Research Council's Guide for the Care and Use of Laboratory Animals.
The experimental subjects may be divided into a plurality of groups, preferably six, each receiving different treatments to evaluate the effects of Betanin on cognitive performance and oxidative stress. The groups may include a normal control group, a scopolamine-induced memory impairment control group, and several Betanin-treated groups, in addition to a standard drug group receiving donepezil and an Nrf2 inhibitor group treated with ML385.
Further, each group may have uniform population, preferably 6 subjects per group, kept for acclimatization to laboratory conditions for at least five days, and may be classified as:
1. Vehicle Control Group: Subjects in this group may neither subject to Scopolamine nor Betanin but receive the vehicle solution.
2. Diseased Group (Scopolamine Only): This group receives Scopolamine (1 mg/kg; i.p.) for seven consecutive days to induce cognitive impairment, without further treatment.
3. Betanin Group (Only Betanin Treatment): Subjects in this group are administered with Betanin (50 mg/kg; i.p.) daily but are not exposed to Scopolamine-induced dementia.
4. Standard Control Group (Scopolamine + Donepezil): Serving as the positive control, subjects of this group receive Scopolamine (1 mg/kg; i.p.) to induce dementia and are treated with Donepezil (5 mg/kg; per oral).
5. Treatment Group (Scopolamine + Betanin): Subject in this group received both Scopolamine (1 mg/kg; i.p.) and Betanin (50 mg/kg; i.p.) to assess the therapeutic effects of Betanin on dementia.
6. Mechanistic Group (Scopolamine + Betanin + ML385): To elucidate the role of Nrf2 in Betanin's neuroprotective action, this group may be treated with Scopolamine, Betanin, and the Nrf2 inhibitor ML385 (30 mg/kg; per oral).
For all groups receiving Scopolamine, the agent is administered daily for seven days. The treatment period extends for 14 days, with having Betanin, Donepezil, and ML385 administered 30 minutes before Scopolamine in their respective groups.
Betanin may be administered at a dose of 50 mg/kg, delivered orally to the mice once daily for 14 days. Scopolamine, known for its memory-impairing effects due to cholinergic system inhibition, may be administered intraperitoneally (i.p.) at a dose of 1 mg/kg to induce cognitive deficits. To evaluate the role of the Nrf2 pathway, ML385, a specific inhibitor of Nrf2, may be given to one group to ascertain whether the observed neuroprotective effects of Betanin are mediated through Nrf2 activation. This protocol is maintained to evaluate the full neuroprotective potential of Betanin over the duration of the experimentation.
In another embodiment, Betanin, the active compound of the present invention, may be administered at a dosage of 50 mg/kg body weight of the experimental subject and prepared freshly before each administration. Scopolamine, a cholinergic antagonist inducing dementia-like symptoms, may be administered intraperitoneally at a dose of 1 mg/kg body weight to simulate cognitive impairment. Donepezil, a standard anti-dementia drug, may be administered orally at 5 mg/kg body weight, serving as the positive control in the present invention. The Nrf2 pathway inhibitor ML385 may be given orally at a dose of 30 mg/kg to assess the mechanistic involvement of Nrf2 in Betanin's neuroprotective action.

Behavioural Assessments
On the 14th day from the date of drug administration, behavioural tests may be conducted to assess the cognitive functions of the subjects. The Morris Water Maze (MWM) and/or Y-maze tests may be employed, both widely used to evaluate spatial memory and cognitive function.
Y-maze Test: On day 14, all animals may be subjected to the Y-maze test to assess working memory. The Y-maze test measures spatial memory based on the animal's ability to explore new arms, indicating the capacity for short-term memory retention. The apparatus comprised three arms, labelled A, B, and C each measuring 40 cm in length, 10 cm in width, and 20 cm in height, and arranged at a 120° angle to one another.
Subjects may be placed in the maze positioned at the end of a single arm and allowed to explore freely for 5-10 minutes. Spontaneous alternation may be calculated as the percentage of triads (sequential entries into each arm without repetition) divided by the total possible alternations, thereby providing an index of working memory.
Morris Water Maze (MWM) Test: Following the Y-maze, mice may be subjected to the MWM test to evaluate spatial learning and memory. The MWM consists of a circular pool, preferably having a 132 cm diameter and a 60 cm height, filled with opaque water up to at least middle of the height and a partially submerged escape platform that remains in a fixed location throughout the test. The temperature of the water may be maintained at 25.2 degrees. In another embodiment, a non-toxic paint may be used to improve water clarity. Further, the circular pool may be divided in a plurality of quadrants each having a partially submerged escape platform. Preferably, the circular pool may be divided in four quadrants.
The subjects underwent training trials to locate the hidden platform using spatial cues around the room. Escape latency (time taken to find the escape platform) may be recorded to measure cognitive impairment and the effect of Betanin on memory enhancement. Reduced escape latency in treated groups compared to the Scopolamine-only group served as an indicator of Betanin's efficacy.
In another embodiment, dementia-like cognitive impairment in mice may be induced using Scopolamine as the dementia model compound. Scopolamine may be administered intraperitoneally at a concentration of 1 mg/kg body weight per day for seven consecutive days. The procedure is based on established protocols, allowing for effective induction of dementia symptoms in mice, as reflected in cognitive tests such as the Morris Water Maze and Y-maze tasks.

Biochemical Assessments
After concluding the behavioural assessments, the subjects may be euthanized for biochemical analyses. Brain tissues may be homogenized to measure oxidative stress markers and enzyme activities, including malondialdehyde (MDA), acetylcholinesterase (AChE), glutathione reductase, and catalase.
• Malondialdehyde (MDA): MDA is a byproduct of lipid peroxidation and serves as a marker for oxidative stress. Elevated MDA levels indicate increased oxidative damage in brain tissues. Scopolamine-treated mice demonstrated significantly higher MDA levels, while Betanin administration reduced these levels to near-normal values, suggesting Betanin's antioxidative potential.
• Acetylcholinesterase (AChE): AChE is an enzyme that breaks down acetylcholine, a neurotransmitter involved in memory and learning. Increased AChE activity is associated with cognitive impairment, as it reduces acetylcholine levels. Betanin treatment led to a reduction in AChE activity, indicating enhanced cholinergic function.
• Glutathione Reductase and Catalase: These are key antioxidant enzymes regulated by the Nrf2 pathway. Glutathione reductase plays a crucial role in maintaining cellular redox balance, while catalase neutralizes hydrogen peroxide, preventing cellular damage. Betanin-treated mice showed increased activities of these enzymes, supporting the hypothesis that Betanin may activate the Nrf2 pathway, enhancing antioxidant defenses in the brain.
Each biochemical parameter may be measured using established spectrophotometric techniques, ensuring reliable and reproducible data to support the neuroprotective claims of Betanin.
Mechanisms of Action
The data as illustrated in figures of the present specification suggest that Betanin's neuroprotective effects are multifactorial, involving cholinergic modulation, reduction of oxidative stress, and potential activation of the Nrf2 pathway. The reduction in MDA levels and AChE activity suggests that Betanin can alleviate oxidative stress and improve cholinergic neurotransmission, both of which are critical in preserving cognitive function.
Furthermore, the observed increase in glutathione reductase and catalase activities in Betanin-treated mice implies an Nrf2-mediated response. Given the involvement of the Nrf2 pathway in regulating endogenous antioxidants, Betanin may function by activating this pathway, which in turn upregulates the brain's intrinsic antioxidant systems.
The inventive neuroprotective composition of the present invention is significantly potent as an alternative or adjunctive therapy for patients suffering from dementia or other neurodegenerative conditions. By simultaneously targeting oxidative stress and cholinergic dysfunction, the Betanin-based composition provides a multifaceted approach to neuroprotection. Its natural origin also minimizes the risk of adverse effects, making it a suitable option for long-term management of cognitive impairment.
The Betanin composition can be administered in various forms, including capsules, tablets, or liquid suspensions, and tailored to the patient's needs based on age, weight, and disease severity. The therapeutic dosage can range from 10 mg/kg to 100 mg/kg, with a standard administration of 50 mg/kg once or twice daily for optimal neuroprotective effects.

Formulation Development
The pharmaceutical composition may be developed in various dosage forms to ensure versatility and patient compliance. The dosage forms may include:
• Oral Dosage Forms: Capsules, tablets, and liquid formulations are the most common. Each form can be tailored for specific release profiles, allowing for immediate or sustained release of Betanin. The selection of excipients is crucial in these formulations to enhance bioavailability and stability.
• Topical Formulations: Although the primary focus is on cognitive functions, exploring the use of Betanin in topical applications for neuroinflammatory skin conditions may broaden its therapeutic scope.
• Injectable Formulations: For more immediate therapeutic effects, particularly in acute settings, injectable forms of Betanin could be developed. This would require rigorous studies to evaluate the pharmacokinetics and pharmacodynamics of Betanin in different routes of administration.
The embodiments described hereinabove are exemplary of the present invention. The disclosure may enable those skilled in the art to make and use embodiments having alternative elements that likewise correspond to the elements of the invention. The intended scope of the invention may thus include other embodiments that do not differ or that insubstantially differ from the literal language of the invention. However, the scope of the present invention is accordingly defined as set forth in the present complete specification.
, Claims:WE CLAIM:
1. A neuroprotective composition, the composition comprising Betanin in a therapeutically effective amount and a pharmaceutically acceptable carrier, wherein the composition is formulated to enhance cognitive function by reducing oxidative stress and increasing cholinergic activity.

2. The composition as claimed in claim 1, wherein the Betanin is present in a concentration of about 50 mg/kg, effective to decrease Acetylcholinesterase (AChE) activity and malondialdehyde (MDA) levels, and to increase levels of glutathione and catalase in brain tissue.

3. The composition as claimed in claim 1, wherein the composition is further configured to activate an Nrf2 pathway, thereby enhancing neuroprotection and mitigating oxidative stress.

4. The composition as claimed in claim 1, wherein the composition includes an additional acetylcholinesterase inhibitor, selected to enhance the cholinergic activity and neuroprotective effects of Betanin.

5. The composition as claimed in claim 1, wherein the composition further comprises adjunctive antioxidants or neuroprotective agents to provide synergistic neuroprotective effects.

6. The composition as claimed in claim 1, wherein the composition is formulated for oral or parenteral administration and is intended for use in treating cognitive impairments associated with neurodegenerative conditions, including dementia and Alzheimer's disease.

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