Optimisation of enzyme assisted extraction of silybin from the seeds of Silybum marianum by Box–Behnken experimental design

Introduction – Silybin, a standardised extract of flavanolignans from the seeds of Silybum marianum, has been used for centuries as a natural remedy in the treatment of hepatitis and cirrhosis. The higher yield of silybin by using more efficient extraction technique is of particular interest in the herbal products manufacture. Objective – To systematically investigate the important factors of enzyme‐assisted extraction of flavanolignans from the seeds of Silybum marianum to enhance the extraction yield of silybin. Methodology – The important factors of enzyme‐assisted extraction were optimised by employing Box–Behnken design with the aid of the orthogonal array design (OAD) OA₈ (2⁷). The effects of enzyme incubation temperature (EIT), the pH of enzyme solution (PES) and the size of seeds (SS) on the yield of silybin were visualised as three‐dimensional response surface and contour plots. Results – The predictive yield was 24.6 mg/g defatted seeds under the optimum enzymolysis conditions (EIT 40°C, PES 4.5 and SS 7003 μm). The coefficient of the model was r² > 0.97 (n = 15). The actual yield of silybin was 24.81 ± 1.93 mg/g defatted seeds, higher by 138 and 123.6% than that from ethanol extraction in this study and in the previous literature, respectively. IR spectra and HPLC of the extracts by EAE were in agreement with those from ethanol extraction. SEM and TEM pictures of defatted seeds by variant extractions demonstrate that the extraction of silybin depends on the destruction of cell walls. Conclusion – The results suggest that EAE is a promising alternative for the extraction of silybin by the use of traditional ethanol extraction. Copyright © 2009 John Wiley & Sons, Ltd.