Abstract: The aim of the present study was to optimize the continuous ultrasonic extraction process for isoflavone aglycones including genistein, daidzein and glycitein produced by Streptomyces roseolus (ATCC 31047) based on Plackett-Burman design and orthogonal array design. Results indicated that the order of three positive factors affecting extraction efficiency from strong to weak was solvent composition (ethanol aqueous solution), ultrasonic treatment time and eluent volume. However, extraction temperature and eluent flow rate exhibited a negative impact on extraction efficiency. Ultrasonic treatment time of 7 min and solvent composition of 70% ethanol concentration were found optimal, and the extraction rate of total isoflavone aglycones reached up to (98.7 ± 1.23)% under the optimal conditions. The application of solid-phase extraction (SPE) could effectively reduce the interference of impurity substances during the quantitative analysis of the above three isoflavone aglycones so that the detection limit of the developed SPE-HPLC method could reach 10-3μg/mL. The detection procedure was less than 10 min and the recovery rate was between 96.2% and 103.4% with a RSD of less than 2.34%. Therefore, this method can meet the requirements for high-efficiency extraction and rapid quantitative analysis of isoflavone aglycones produced by microbial fermentation.  

Key words: isoflavone aglycones, continuous ultrasonic extraction, solid-phase extraction, microbial fermentation