Optimization of the Extraction Process and Preliminary Purification of Total Flavonoids from Hedysari Radix Using Response Surface Methodology

Hedysari Radix, a valuable traditional Chinese medicinal herb, is rich in flavonoid constituents with considerable potential for the development of anticancer and immunomodulatory therapeutic agents. In this work, an integrated high-efficiency technical workflow was constructed for the extraction and purification of total flavonoids from H. Radix. A fully validated HPLC quantitative assay was adopted for component quantification, and spectroscopic analyses were performed to characterize the structural profiles of purified flavonoid fractions. Single-factor experiments coupled with response-surface methodology were implemented to optimize ethanolic extraction parameters, while tandem purification utilizing HPD722 macroporous resin and polyamide resin was carried out for crude sample refinement. The HPLC method was comprehensively validated in terms of system suitability, precision and solution stability. FT-IR and NMR spectroscopic measurements were further applied to identify the chemical structures of purified flavonoid components. The validated HPLC assay exhibited favorable precision, repeatability, and 24 h solution stability. Optimized extraction conditions were determined as 75% ethanol, solid–liquid ratio of 1:16, and extraction duration of 90 min, affording a total flavonoid yield of 6.17 mg·g−1, with calycosin and formononetin contents of 90.4 μg·g−1 and 212.9 μg·g−1, respectively. After sequential purification by macroporous resin followed by polyamide resin, the purity of total flavonoids reached 47.54%, corresponding to a 77-fold enhancement relative to the crude extract. Structural characterization confirmed that the purified compounds belonged to isoflavonoid glycosides. Collectively, these findings demonstrate that the established protocol enables stable and efficient extraction and purification of flavonoids from H. Radix, and provides theoretical basis and technical reference for developing anticancer and immunomodulatory therapeutics derived from this medicinal herb.