Optimization of extraction technology from compound Huanghuai based on coagulation activity and central composite design-response surface methodology

Abstract

To establish a method for the determination of coagulation activity in vitro by using fibrinogen plate method. The extraction route of compound Huanghuai was optimized by selecting thrombin as the reference substance. The comprehensive score of extract yield and the extraction transfer rate of baicalin and rutin was set as the dependent variable, with alcohol concentration, solvent volume and extraction time as the influence factors in central composite design for quadratic fitting, and the extraction process of compound Huanghuai was optimized by using response surface methodology. The results of thrombin concentration and precipitation zone area showed a good linear relationship in 0.4-16 U•mL⁻¹, r=0.997 5. The average recovery rate was 103.8% and the RSD was 4.7 %. The circle of precipitation area of compound Huanghuai combined extract was 38.81 mm², which was bigger than that of fractionated extract. The activity on the daily amount of compound Huanghuai extract was 84.28 U; and the optimum extraction technology was as follows: alcohol concentration 40%, extracted 3 times, the liquid-solid ratio 6∶1, and extraction time 2 h. The predicted value of comprehensive score was 94.26 and the measured value was 88.34, respectively, with a relative deviation of 6.28%. The coagulation activity of the intermediate obtained by optimal extraction process was better. So the method established in this paper was simple, fast and accurate for determination of coagulation activity of compound Huanhuai, which can be also used for the screening of follow-up process and quality control.