Response surface methodology (RSM), based on a central composite design, was used to determine the best liquid-to-raw material ratio (10:3-15 mL/g), extraction time (1-3 h), and ethanol concentration (50%-100%) for maximum content of α-asarone from Perilla frutescens (PF) extract. Experimental values of α-asarone were 9.51-46.36 mg/g; the results fitted a second-order quadratic polynomial model and correlated with the proposed model (R2 > 0.9354). The best conditions were obtained with extraction time of 1.76 h, liquid-to-raw material ratio of 10:13.5 mL/g, and ethanol concentration of 90.37%. Under these conditions, the model predicted extraction content of 40.56 mg/g, while experimental PF content of α-asarone was 43.84 mg/g dried plant. Optimized conditions determined for maximum content of α-asarone were similar to the experimental range. Experimental values agreed with those predicted, thus validating and indicating suitability of both the model and the RSM approach for optimizing extraction conditions. In addition, a reliable, reproducible and accurate method for the quantitative determination of α-asarone by High Performance Liquid Chromatography (HPLC) analysis was developed with limit of detection (LOD), limit of quantitation (LOQ) values of 0.10 and 0.29 µg/mL and excellent linearity (R2 > 0.9999).
Keywords: HPLC; Perilla frutescens L.; optimization; response surface methodology; validation; α-asarone.