Optimization of ultrasound-assisted extraction and development of an HPLC method for simultaneous determination of thirteen flavonoids in mulberry (Fructus Mori)

This study established an integrated methodological framework for the efficient extraction and comprehensive analysis of flavonoids from mulberry ( Morus alba L.). A high-performance liquid chromatography (HPLC) method was developed to simultaneously separate and identify 13 flavonoid components, including rutin, quercetin, kaempferol, and naringin, as well as their derivatives. Using seven representative flavonoids as standards, the ultrasonic-assisted extraction process was systematically optimized through single-factor and orthogonal experimental designs, followed by response surface methodology. The optimal extraction conditions were determined as follows: 80% ethanol (v/v), an extraction time of 60 min, a solid-liquid ratio of 1:2.5 (g mL⁻¹), and a temperature of 40℃, conducted over two cycles. This optimized protocol achieved a high total flavonoid extraction yield of 84.96%. The newly established HPLC protocol, employing a C18 column with gradient elution and dual-wavelength detection, demonstrated excellent stability and reproducibility in detecting target compounds across multiple mulberry samples. This approach features simple operation and high time efficiency. The optimized parameters suggest a profile compatible with potential scale-up, though direct evidence from pilot-scale tests and economic analysis would be required to confirm its practical industrial feasibility. Nonetheless, it provides a valuable methodological reference for the efficient extraction of natural active substances. • Response surface methodology optimized mulberry flavonoid extraction for efficiency, eco-friendliness, and scalable operability. • A novel dual-wavelength HPLC method simultaneously separated and qualified 13 flavonoid compounds. • Method reproducibility and reliability were confirmed via stability tests and statistical analysis.