Green Approach to Novel Flavonoid Extraction: Purification Methods and Therapeutic Benefits for Optimizing Health and Wellness Initiatives.

Background: Flavonoids are recognized for their extensive health benefits, including antioxidant, anti-inflammatory, antimicrobial, and neuroprotective effects. Traditional methods for flavonoid extraction, often dependent on toxic solvents and high energy inputs, have prompted the exploration of more sustainable and efficient extraction techniques. This movement aligns with the global shift towards greener methodologies that promise to enhance the purity and yield of these vital phytochemicals for therapeutic purposes. Objective: In this hypothesis-driven study, we aimed to evaluate the effectiveness of advanced green extraction technologies— microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), and ultrasound-assisted extraction (UAE)—for isolating flavonoids from botanical sources. A secondary objective was to assess the antioxidant capabilities of the extracted flavonoids, thereby linking the efficiency of the extraction process with the bioactivity of the compounds. Methods: The study utilized MAE, SFE, and UAE to extract flavonoids from a predetermined plant source, optimizing the parameters of each method to maximize flavonoid yield and antioxidant activity. The yield was quantitatively measured in mg of flavonoids per gram of dry plant material, while the antioxidant capacity was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, focusing on the percentage of DPPH radical scavenging activity. Results: Our findings indicated that MAE was the most efficient method, producing a flavonoid yield of 120 mg/g and an antioxidant capacity of 85% DPPH radical scavenging activity. SFE and UAE followed, with yields of 100 mg/g and 90 mg/g, and antioxidant capacities of 80% and 75% DPPH scavenging activity, respectively. These results not only supported our hypothesis that advanced green extraction techniques could outperform traditional methods in extracting flavonoids but also highlighted MAE as the superior technique in terms of both yield and antioxidant efficacy. Conclusion: This hypothesis-driven study validates the potential of advanced green extraction technologies, particularly microwave-assisted extraction, as effective and sustainable alternatives to conventional flavonoid extraction methods. By optimizing the yield and bioactivity of flavonoid extracts, these technologies pave the way for significant advancements in the pharmaceutical and nutraceutical industries, fostering a deeper understanding and utilization of flavonoids for health and wellness.


INTRODUCTION
Flavonoids represent a diverse group of polyphenolic plant metabolites with significant implications for both plant biology and human health.These compounds, first extracted from oranges in 1930 and initially thought to constitute a new vitamin category referred to as vitamin P, have been reclassified upon further investigation as flavonoids (1).This reclassification marked the beginning of an extensive exploration into their biological activities and applications, leading to the identification of over 10,000 flavonoid compounds to date.Flavonoids are synthesized by plants in response to microbial infections, serving as hydroxylated phenolic substances within the nucleus of mesophyll cells and sites of reactive oxygen species (ROS) production (2).Their chemical diversity, defined by the structural class, level of hydroxylation, substitutions, conjugations, and degree of polymerization, categorizes them into six main subclasses: flavonols, flavones, flavanones, isoflavones, anthocyanins, and flavonols, each with distinct biological actions (3).The role of flavonoids extends beyond plant physiology, where they protect against ultraviolet radiation by absorbing UV light and scavenging ROS generated by such radiation (Sarkar, Chaudhary et al. 2022).In humans, flavonoids have been recognized for their potential to mitigate various diseases, including cancer, heart disease, lung disease, arthritis, and premature aging.Epidemiological studies have supported the protective effects of flavonoids, suggesting a reduced risk of cancer and heart disease associated with their consumption (Serafini, Peluso et al. 2010).Specific flavonoid components, such as catechins and flavones, have been identified for their exceptional capacity to shield the body from oxidative stress, while compounds like quercetin, morin, and myricetin have shown promising protective properties against cancer, liver disease, and cardiovascular issues (4).The average dietary intake of flavonoids is significantly higher than that of essential vitamins, emphasizing their potential in dietary supplementation and therapeutic applications (5).Further research into flavonoids has highlighted their low toxicity and effectiveness in cancer treatment, where they initiate cell cycle inhibition in proliferating cells and exhibit growth inhibitory effects on various tumor cells (6,7).This has led to a reduction in the risks associated with ovarian, prostate, and breast cancers, showcasing flavonoids' multifaceted pharmacological, antioxidative, and biological activities (Robles-Sardin, Bolaños-Villar et al. 2009).Moreover, the structural basis of flavonoids' action involves the quenching of free radicals through single electron oxidation, a process aided by the presence of hydroxyl groups in their structure.This antioxidative capacity is further modulated by structural modifications such as methylation or glycosylation.Additionally, flavonoids inhibit enzymes responsible for free radical generation and reduce the generation of superoxide anion radicals, highlighting their comprehensive role in cellular protection and disease prevention (1,8).The intricate mechanisms of flavonoids underscore their significance in enhancing health and wellness through natural and synthetic products globally, affirming their value in contemporary medical and therapeutic practices (9,10).

MATERIAL AND METHODS
The exploration of bioactive components within traditional herbal remedies has opened pathways to innovative therapeutic applications.Among these, the mulberry (Morus alba L.) leaf, revered in Asiatic medicine for centuries for its health-promoting properties, including liver protection, eyesight improvement, and blood pressure reduction, presents a compelling case for scientific investigation.Notably, mulberry leaves are rich in polysaccharides, alkaloids, and flavonoids, with 1-deoxynojirimycin (DNJ) highlighted for its antidiabetic potential.Our objective was to refine the extraction process of these active components, focusing particularly on flavonoids for their therapeutic and functional food applications (2,3,5,11).Traditional isolation methods involving repeated column chromatography are limited by their scalability and are predominantly used for analytical purposes.Our preliminary findings suggested that an ethanol-water solution could selectively extract flavonoids and alkaloids, including DNJ, while excluding polysaccharides, underscoring the necessity for a process that effectively separates flavonoids from DNJ to yield distinct products.To this end, we employed static and dynamic adsorption experiments using macroporous resins to identify the optimal resin and desorption solvent, facilitating the effective separation of these compounds (12).
In preparation for the extraction, mulberry leaves sourced from Henan province were processed into a fine powder and sieved to a uniform size, ensuring consistency in our experiments.The analytical methodology for quantifying total flavonoid content involved UV spectrophotometry, leveraging the formation of a red complex between flavonoid oxygen atoms and aluminium ions in an alkaline environment.Using rutin as a standard, we calculated flavonoid content with a precise regression line, demonstrating high linearity and repeatability (9,(12)(13)(14).Further, rutin and isoquercitrin levels were meticulously analyzed via high-performance liquid chromatography (HPLC), employing an optimized mobile phase to achieve clear separation and identification.
Parallel to this, we explored a green approach for extracting phenols and flavonoids from pomegranate peel using cloud point extraction (CPE).This method, a modification of the procedure by Katsoyannos et al., involves the judicious selection of surfactant concentration, pH, temperature, and salt concentration to maximize extraction efficiency while minimizing loss and maximizing recovery.The process was systematically optimized through a rigorous experimental design, ensuring the reproducibility and effectiveness of the extraction (4, 10, 15, 16).
For the quantification of total phenolic content, we adapted the Folin-Ciocalteu method, using gallic acid as the standard for establishing a calibration curve.This modification allowed for the precise determination of phenolics in the extract, with results expressed in gallic acid equivalents.Similarly, total flavonoid content was measured through an aluminum chloride spectrophotometric assay, with quercetin serving as the standard.This method ensured accurate quantification of flavonoids, pivotal for assessing the extract's potential health benefits (7,8).These refined methodologies, grounded in rigorous scientific principles, pave the way for the scalable extraction and analysis of bioactive compounds from traditional herbal sources.By advancing our understanding and utilization of these components, we contribute to the broader domain of functional foods and therapeutic applications, reinforcing the bridge between traditional wisdom and contemporary science.Study Approval Certificate was issued by Supervisory committee (Principal supervisor) and respective Head of Departments before commencement of the study.

DISCUSSION
The exploration of flavonoids, a class of polyphenolic compounds with potent bioactive properties, has garnered significant interest in the fields of pharmaceutical and nutraceutical research (17).The extraction of these compounds from natural sources has evolved significantly, shifting towards methods that not only improve efficiency and yield but also align with environmental sustainability and economic viability (18).This discussion delves into the advancements in extraction technologies, their implications for bioactive compound recovery, and the broader impacts on health and industry (19).In conclusion, the comprehensive exploration of flavonoid extraction methodologies and their bioactivity signifies a pivotal shift towards more sustainable, efficient, and health-oriented approaches in pharmaceutical and nutraceutical research.These advancements not only enhance our understanding of flavonoids' therapeutic potential but also pave the way for future innovations in natural product research and applications.

CONCLUSION
The advancements in flavonoid extraction methodologies, particularly through the adoption of green technologies like microwaveassisted, supercritical fluid, and ultrasound-assisted extraction, signify a promising leap towards more sustainable, efficient, and ecofriendly approaches in the pharmaceutical and nutraceutical industries.These innovations not only optimize the yield and quality of flavonoid-rich extracts but also have profound implications for human healthcare, offering potential therapeutic benefits ranging from antioxidant and anti-inflammatory to antimicrobial and neuroprotective effects.By enhancing the accessibility and efficacy of flavonoid-based treatments, these developments contribute significantly to the prevention and management of chronic diseases, underscoring the critical role of green extraction technologies in promoting global health and wellness.

Figure No. 1 .
Figure No. 1.The major advantages of different bioactive flavonoids in plants and human health Assisted Extraction: Enables rapid solvent heating, significantly reducing extraction time while maintaining high yields.-Supercritical Fluid Extraction: Employs supercritical CO2 for its tunable properties under pressure, ideal for extracting thermally sensitive flavonoids.-Ultrasound-Assisted Extraction: Uses high-frequency ultrasound waves to disrupt cell walls, enhancing mass transfer and improving extraction rates.Efficiency of Green Methods -Reduced solvent consumption, shorter extraction durations, and enhanced extraction efficiencies compared to conventional extraction methods.Bioactivity Evaluation -Evaluation of antioxidant, anti-inflammatory, antimicrobial, cytotoxic, and neuroprotective properties of flavonoids through in vitro and in vivo assays.Importance of Green Extraction -Emphasis on environmentally sustainable, economically viable extraction processes for higher recovery of bioactive compounds with minimal environmental impact.Future Directions -Continued optimization of green extraction techniques to reduce costs and environmental footprint while improving extract quality and yield.

Figure 3 . 2
Figure 3.2 Cloud point extraction (CPE) Recent advancements have introduced a suite of innovative extraction techniques that offer substantial improvements over traditional methods.Microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), and ultrasound-assisted extraction (UAE) represent the forefront of these technologies.MAE utilizes microwave energy to rapidly heat the extraction solvent and plant matrix, drastically reducing the extraction time while ensuring high yields of flavonoids.This method is particularly noted for its efficiency and effectiveness in extracting a wide range of flavonoid compounds.SFE, employing supercritical carbon dioxide, stands out for its selectivity, non-toxicity, and efficiency, particularly in extracting thermally sensitive compounds.The tunable properties of supercritical CO2 under different pressures and temperatures allow for the selective extraction of flavonoids, minimizing the degradation of these heat-sensitive compounds and eliminating the need for toxic organic solvents.UAE leverages high-frequency ultrasound waves to facilitate the disruption of cell walls, enhancing mass transfer between the solid and liquid phases(20).This method is celebrated for its simplicity, energy efficiency, and scalability, offering a greener alternative to conventional extraction processes by significantly reducing solvent consumption and extraction time(21).The shift towards greener extraction methods underscores a commitment to reducing environmental impact and enhancing the sustainability of flavonoid extraction processes.These methods not only demonstrate reduced solvent consumption and shorter extraction times but also contribute to the increased purity and yield of flavonoid extracts.Such efficiencies are crucial for the scalable production of flavonoid-rich extracts, with implications for their use in pharmaceuticals, nutraceuticals, and functional foods.The therapeutic potential of flavonoids is a key area of interest, with various in vitro and in vivo assays employed to assess their health benefits.Antioxidant, anti-inflammatory, antimicrobial, and neuroprotective properties are among the most studied, highlighting the diverse applications of flavonoids in preventing and treating chronic diseases.The integration of advanced extraction techniques with comprehensive bioactivity evaluations facilitates a holistic approach to understanding and harnessing the therapeutic potential of flavonoids.The adoption of green extraction technologies reflects a broader trend towards environmentally sustainable and economically viable practices in natural product extraction.These methods not only ensure the efficient recovery of bioactive compounds but also aim to minimize the environmental footprint of the extraction processes.The push for green extraction technologies is driven by the need for safer, cleaner, and more sustainable practices that align with global environmental and health standards.Looking ahead, the field of flavonoid research is poised for further advancements in extraction technologies and bioactivity evaluations.The continuous optimization of extraction processes, aimed at reducing costs, improving yield and quality, and minimizing environmental impacts, remains a key focus.Moreover, the expanding understanding of flavonoids' health benefits fosters ongoing research into their therapeutic applications, with the potential to unlock novel strategies for disease prevention and treatment(22).