Extraction process and method validation for bioactive compounds from Citrus reticulata cv. Chachiensis: Application of response surface methodology and HPLC–DAD

Abstract Citrus reticulata cv. Chachiensis, a traditional Chinese herb, has extensive medicinal and edible effects. 3?,4?,5,6,7,8-Hexamethoxyflavone (HM) 5,6,7,8,4?-pentamethoxyflavone (PM) are main bioactive compounds in which have been reported to possess various biological properties. In this study, supercritical CO 2 extraction (SCE) high-speed countercurrent chromatography (HSCCC) were utilized prepare HM PM from Chachiensis. The contents of target determined by high-performance liquid method with diode-array detection (HPLC-DAD), was validated using the following parameters: linearity, sensitivity, repeatability, stability, precision accuracy. SCE conditions optimized response surface methodology central composite design. Obtained optimum temperature 37.9 °C, pressure 26.3 MPa, modifier volume 81.0 mL. Under above conditions, recoveries 92.52 ± 0.83 96.36 0.43%, respectively. most appropriate solvent system for HSCCC selected as n -hexane/ethyl acetate/methanol/water (1:0.8:1:1.2, v/v). fractions detected HPLC-DAD, chromatography-mass spectrometry (LC-MS) nuclear magnetic resonance spectroscopy ( 1 H NMR 13 C NMR). results indicated that successfully applied obtain high purities