Optimized Pretreatment and Preparative Chromatography for Vitexin Purification from Mung Bean Seed Coats

This research focuses on the valorization of mung bean hull, a major agro-industrial byproduct, through the development of an optimized process for the high-purity isolation of the bioactive flavonoid, vitexin. Vitexin is recognized for its significant nutraceutical properties, including anticancer, antioxidant, and anti-inflammatory activities, making its high-purity isolation crucial for pharmaceutical and functional food applications. The study emphasized the importance of pretreatment methods to enhance extraction efficiency and downstream purification. Raw material preparation involved controlled grinding and sieving, followed by the selection of a particle size fraction (80-150 mesh) for subsequent pretreatment. This fraction was subjected to a crucial washing step with ethanol to effectively remove residual starch particles. This ethanol washing minimized the loss of vitexin (unlike deionized water washing) and was vital for preventing column clogging and back pressure issues during subsequent chromatographic purification. The optimal solvent extraction condition was determined to be a 50% ethanol/deionized water mixture, which yielded the highest vitexin concentration of 24.29 mg/ga result consistent with tuning the solvent polarity to an optimal dielectric constant (∼45.4) and the effective removal of the starch matrix. For the high-purity purification of vitexin, a Reverse Phase Preparative Liquid Chromatography (Prep-LC) system was utilized. The key operating parametersmobile phase composition and flow ratewere systematically optimized. An initial mobile phase composition of 20:80 acetonitrile:deionized water resulted in poor separation. Fine-tuning the composition to 15:85 acetonitrile:deionized water was critical, achieving the complete baseline separation of vitexin and its isomer, iso-vitexin. The flow rate of 3 mL/min proved to be the optimal compromise, effectively reducing the separation time by approximately 2-fold while maintaining the ability to produce high-purity vitexin (purity ≥ 90%). This study establishes a process-oriented and experimentally validated strategy for producing high-purity vitexin from mung bean hull, integrating effective starch removal and optimized Prep-LC parameters. The identified optimal conditionsextraction with 50% ethanol and purification using Prep-LC with 15:85 acetonitrile:deionized water at 3 mL/minprovide practical operational guidelines that may inform future scale-up studies and process development for vitexin production.