EXTRACTION, ISOLATION, CHARACTERIZATION AND NEUROPROTECTIVE ACTIVITY OF ETHANOLIC EXTRACT OF LEUCAENA LEUCOCEPHALA

Abstract

ABSTRACT This invention presents the extraction, isolation, and neuroprotective activity of ethanolic extracts from the leaves of Leucaena leucocephala. Utilizing the Soxhlet extraction method, we obtained a concentrated extract that underwent phytochemical analysis and chromatographic separation to isolate key bioactive compounds, including catechin and quercetin. Spectroscopic characterization techniques such as IR, 1H NMR, and 13C NMR were employed to confirm the structures of the isolated compounds. Pharmacological assessments in experimental rat models were conducted to evaluate the neuroprotective effects of the extract against cognitive decline associated with Alzheimer's disease. The findings revealed significant improvements in memory retention and cognitive function among treated subjects, with no signs of toxicity observed at the tested doses. This study highlights the potential of Leucaena leucocephala as a natural therapeutic agent for enhancing cognitive health and offers a promising alternative to conventional treatments for neurodegenerative diseases.

Specification

Description:FIELD OF INVENTION
The present invention relates to the field of neuropharmacology, focusing on extracting and characterizing neuroprotective agents from Leucaena leucocephala for enhancing cognitive function and treating neurodegenerative diseases like Alzheimer's disease.

BACKGROUND OF THE INVENTION
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a gradual decline in cognitive function and memory, primarily affecting the aging population and presenting significant public health challenges due to its increasing prevalence. Current treatment options, such as acetylcholinesterase inhibitors (e.g., donepezil, rivastigmine) and NMDA receptor antagonists (e.g., memantine), primarily manage symptoms without addressing the underlying mechanisms of the disease. Consequently, these therapies often come with a range of side effects, including gastrointestinal disturbances and cardiovascular issues, which can hinder patient compliance and negatively impact quality of life.

In light of these drawbacks, there has been a growing interest in exploring natural products for their potential neuroprotective properties. Leucaena leucocephala, a leguminous plant recognized for its diverse bioactive compounds like flavonoids, alkaloids, and saponins, has been traditionally utilized for various therapeutic purposes, including anti-inflammatory and antioxidant applications. However, its neuroprotective effects, particularly in enhancing cognitive function and memory retention related to Alzheimer's disease, remain under-explored.
The Conventional methods for treating Alzheimer's disease exhibit several significant drawbacks. Firstly, these synthetic medications primarily focus on symptom management rather than addressing the disease's underlying pathophysiology, resulting in limited long-term efficacy. Additionally, they are often accompanied by various side effects, including nausea, vomiting, and cardiovascular problems, which can lead to discontinuation of therapy and reduced patient adherence. The necessity for long-term use of these medications raises concerns about cumulative side effects and the need for ongoing monitoring and dosage adjustments.

Furthermore, the high cost of many synthetic neuroprotective drugs can make them inaccessible, particularly in low-income populations or regions with limited healthcare resources. Patients with Alzheimer's often take multiple medications for comorbid conditions, increasing the risk of drug-drug interactions that can complicate treatment regimens and lead to adverse effects. There is a pressing need for alternative therapies derived from natural sources that can offer safer profiles with fewer side effects. Although various plants have historical use in traditional medicine, rigorous scientific studies validating their efficacy and safety in neuroprotection remain limited, leading to skepticism regarding herbal treatments. By focusing on the extraction, isolation, characterization, and neuroprotective activity of Leucaena leucocephala, this invention aims to provide a viable alternative to conventional therapies, addressing the limitations associated with them and offering a natural, effective option for patients with Alzheimer's disease.
OBJECTS OF THE INVENTION
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
It is an object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative
An object of the present disclosure is to provide an alternative neuroprotective agent that is naturally derived, thereby minimizing potential side effects commonly associated with synthetic drugs.
Another object of the present disclosure is to provides a plant-based therapeutic agent specifically targeting memory improvement and cognitive health, which is particularly useful for neurodegenerative conditions like Alzheimer's.
Another object of the present disclosure is to create a plant-based therapeutic solution specifically targeting memory enhancement and cognitive health, particularly beneficial for individuals suffering from neurodegenerative conditions such as Alzheimer's disease.
Another object of the present disclosure is to establish a safe and effective dosage through preclinical trials, demonstrating negligible toxicity, even at high doses, thereby enhancing patient compliance.
Another object of the present disclosure is to enables a cost-effective and accessible therapeutic option due to the wide availability of Leucaena leucocephala and the relatively simple extraction and isolation techniques used.
Another object of the present disclosure is to provide a cost-effective and accessible therapeutic option, capitalizing on the wide availability of Leucaena leucocephala and employing relatively simple extraction and isolation techniques that can be easily implemented in various settings.
Another object of the present disclosure is to provide a natural formulation to the market aimed at cognitive health, providing a complementary approach to existing Alzheimer's treatments and contributing to a holistic management strategy for cognitive decline.
Another object of the present disclosure is to encourage further research into the neuroprotective effects of Leucaena leucocephala and other natural products, fostering an increased understanding of their mechanisms of action and potential therapeutic applications in neurodegenerative diseases.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY OF THE INVENTION
The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.
The present invention involves the extraction, isolation, and neuroprotective characterization of compounds from Leucaena leucocephala leaves, focusing on their potential for improving memory and cognitive function in Alzheimer's disease. Using Soxhlet extraction, ethanolic leaf extracts were subjected to chromatographic and spectroscopic techniques to isolate bioactive compounds. Compounds like catechin and quercetin were quantified via HPTLC, indicating their significant presence in the extract. Experimental testing on rats using memory assessment tasks like the Novel Object Recognition Test (NORT) and Y-Maze indicated substantial improvement in memory index and cognitive functions, supporting the extract's anti-Alzheimer's potential. Safety and toxicity studies confirmed the extract's high tolerability, making it a viable therapeutic agent. This invention introduces a potent natural formulation to the market for cognitive health, particularly targeting Alzheimer's and related cognitive disorders.
DETAILED DESCRIPTION OF THE INVENTION
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.
The present invention encompasses the extraction, isolation, and neuroprotective characterization of compounds from Leucaena leucocephala leaves, focusing on their potential for enhancing memory and cognitive function in Alzheimer's disease. Soxhlet extraction yielded an 8.36% w/w concentration of a dark brown extract, which underwent chromatographic and spectroscopic analysis to isolate bioactive compounds. Key compounds such as catechin and quercetin were quantified using High-Performance Thin-Layer Chromatography (HPTLC), confirming their significant presence. Animal testing utilizing memory assessment tasks like the Novel Object Recognition Test (NORT) and Y-Maze indicated substantial improvements in memory index and cognitive functions, supporting the extract's anti-Alzheimer's potential. Toxicity studies confirmed high tolerability of the extract, making it a viable therapeutic agent. This invention introduces a potent natural formulation to the market for cognitive health, specifically targeting Alzheimer's and related cognitive disorders.
Example 1: The present invention focuses on the extraction, isolation, characterization, and evaluation of the neuroprotective activity of ethanolic extracts from the leaves of Leucaena leucocephala. This leguminous plant is known for its rich phytochemical composition, including flavonoids, alkaloids, saponins, and phenolic compounds, which have potential therapeutic effects, particularly in cognitive health and neuroprotection.
1. Plant Material and Preparation
The leaves of Leucaena leucocephala were collected from healthy trees in a suitable location, preferably free from pollution and chemical fertilizers. The leaves were thoroughly washed to remove dirt and contaminants, then air-dried under shade to prevent degradation of sensitive phytochemicals. Once dried, the leaves were ground into a fine powder using a mechanical grinder to increase the surface area for effective extraction.
2. Extraction Process
The Soxhlet extraction method was employed to obtain the ethanolic extract from the powdered leaves. The extraction process involved the following steps:
Setup: The powdered leaves were placed in a thimble within a Soxhlet apparatus, and ethanol was used as the solvent. The Soxhlet apparatus was assembled, and heat was applied to the solvent in a round-bottom flask.
Extraction: As the ethanol boiled, it vaporized and rose into the condenser, where it cooled and returned to the thimble, extracting the soluble compounds from the leaves. This process was allowed to continue for 8 hours to ensure maximum extraction efficiency.
Evaporation: The resulting extract was collected and concentrated using a rotary evaporator at reduced pressure to remove the solvent. The final extract, characterized by its dark brown, sticky consistency, was stored in a cool, dry place for further analysis.
3. Phytochemical Analysis
The phytochemical composition of the ethanolic extract was analyzed through preliminary screening tests and chromatographic techniques.
Preliminary Screening: Qualitative analysis was conducted to identify the presence of various phytochemicals, including alkaloids, flavonoids, saponins, tannins, and phenolic compounds, which are indicative of the plant's therapeutic potential.
Chromatographic Techniques:
• Thin Layer Chromatography (TLC): TLC was performed to separate the extract components based on their polarities, allowing for the visualization of various phytochemical compounds.
• High-Performance Thin-Layer Chromatography (HPTLC): This technique was employed for precise quantification of key compounds, specifically catechin and quercetin. Calibration curves for each compound were established using known standards, and their concentrations in the extract were calculated based on regression equations.
4. Spectroscopic Characterization
The isolated compounds were characterized using advanced spectroscopic techniques, including:
Infrared (IR) Spectroscopy: The IR spectra of the extract showed characteristic absorption bands that correspond to functional groups present in flavonoids, confirming the presence of compounds such as quercetin.
Nuclear Magnetic Resonance (NMR) Spectroscopy:
1H NMR and 13C NMR: These techniques provided detailed information on the molecular structure of the isolated compounds. Quercetin was identified by its unique chemical shifts and coupling patterns, confirming its presence in the extract.
Mass Spectroscopy: Mass spectrometry was employed to determine the molecular weight and structural fragments of the isolated compounds, further validating their identity.
5. Pharmacological Evaluation
The neuroprotective effects of the ethanolic extract were evaluated using experimental models, specifically male albino rats, which were subjected to pharmacological assessments.
Experimental Design: Rats were divided into five groups (n=6 per group):
Control (saline)
Scopolamine-induced memory impairment (2 mg/kg)
Extract-treated group (100 mg/kg)
Extract-treated group (200 mg/kg)
Extract-treated group (400 mg/kg)
Treatment Protocol: The extract was administered orally for 21 consecutive days. Behavioral tests to assess memory and cognitive functions were conducted using validated paradigms.
6. Safety and Toxicity Studies
Acute toxicity studies were performed to evaluate the safety profile of the ethanolic extract. Various doses (up to 2000 mg/kg) were administered, and the rats were monitored for adverse reactions and behavioural changes.
Results: No fatalities or significant behavioural abnormalities were observed in any group, indicating the high safety margin of the extract, even at elevated doses.

Results and Advantages
The results of this invention demonstrate that the ethanolic extract of Leucaena leucocephala leaves possesses significant neuroprotective effects, evidenced by improved cognitive performance and memory retention in experimental models. The presence of bioactive compounds, particularly flavonoids like quercetin and catechin, was confirmed through rigorous analytical methods.

Advantages:
1. Natural Therapeutic Alternative: The invention provides a natural alternative for enhancing cognitive function and preventing neurodegenerative disorders, potentially beneficial for conditions such as Alzheimer's disease.
2. Safety Profile: The extract exhibits a favourable safety profile, with no observed toxicity at high doses, making it suitable for further development into therapeutic formulations.
3. Rich Phytochemical Composition: The rich array of phytochemicals present in the extract suggests potential multifaceted benefits, including antioxidant and anti-inflammatory properties, which may contribute to overall cognitive health.
4. Promising Research Foundation: This invention lays the groundwork for future studies exploring the mechanisms of action of the bioactive compounds in Leucaena leucocephala, paving the way for clinical trials and potential applications in neuropharmacology.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
, Claims:We Claim,
1. A method for producing a neuroprotective extract from Leucaena leucocephala leaves comprising the steps of:
a. Collecting fresh leaves of Leucaena leucocephala and drying them in a shaded area to reduce moisture content;
b. Grinding the dried leaves into a fine powder;
c. Subjecting the powdered leaves to Soxhlet extraction using ethanol as a solvent for a predetermined time period;
d. Concentrating the resulting extract to obtain a dark brown, sticky ethanolic extract rich in bioactive compounds including catechin and quercetin;
e. Performing preliminary phytochemical screening to confirm the presence of key phytochemicals; and
f. Characterizing the extract using chromatographic and spectroscopic techniques to confirm the identity of the bioactive compounds.
2. The method as claimed in claim 1, wherein the Soxhlet extraction is conducted at a temperature ranging from 60°C to 80°C.
3. The method as claimed in claim 1, wherein the ethanol used for extraction has a concentration of 70% to 90%.
4. The method as claimed in claim 1, wherein the phytochemical screening confirms the presence of flavonoids, alkaloids, saponins, and tannins.
5. The method as claimed in claim 1, wherein the chromatographic analysis includes Thin Layer Chromatography (TLC) and High-Performance Thin-Layer Chromatography (HPTLC) for the separation and quantification of catechin and quercetin.
6. The method as claimed in claim 1, wherein spectroscopic characterization includes infrared (IR) spectroscopy, 1H NMR, and 13C NMR for confirming the structure of the isolated compounds.
7. The method as claimed in claim 1, further comprising a safety evaluation step to assess the toxicity of the extract in animal models at specified doses.

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