Extraction, Isolation And Activities Of Antioxidant And Tyrosinase Inhibition Of The Main Active Compounds In Silymarin

Silymarin, derived from the milk thistle plant Silybum marianum, has been used widely for centuries for the protection of the liver from toxic substances. And has been widely used in medicine, health products, cosmetics and feed industry fields for its other activities. Silybin is the principal active component of silymarin, which is a mixture of silybin A and silybin B. The main factors on the extraction of active ingredients in silymarin from the seeds of milk thistle were investigated and the mathematical model on enzyme-assisted extraction of the silybin was developed. The separation and purification techniques of taxifolin, silychristin, silydianin, silybin A, silybin B, isosilybin B and isosilybin A were developed. The activities of antioxidant and tyrosinase inhibition of the main active compounds in silymarin were determined. The main contents and results were shown in the following:(1) A simple and rapid chromatography method for the quality control of silymarin in plant extract and diary supplement. Moreover, comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency and sensitivity. The run time for all the analytes in silymarin was about 9min in the UPLC system, while the run time for all the analytes from silymarin was about 45 min in our HPLC system. The UPLC method allowed shortening the analysis time up to 5-fold compared to that on our HPLC method and in the literature. LOD of the UPLC method was lower twice than that of our HPLC method.(2) A preliminary two-level orthogonal array design (OAD) was used to choose the significant factors that affect the emzymolysis and a Box-Behnken design was used to further optimize the important variables, such as enzyme incubation temperature (EIT), the pH of enzyme solution (PES) and the size of seeds (SS). The optimal conditions for enzymolysis were:EIT (40℃), PES (4.5) and SS (700 mm). Maximum yield of silybin (24.60 mg/g defatted seeds) was achieved.Compared with conventional reflux extraction, ultrasonic and microwave extraction, the silybin yield was higher 136%,50.92%and 43.02%; purity was higher 106.7%,111%, and 139.1%.(3) A response Surface Method was used to investigate the main operating parameters of the pre-LC for the separation of the silybin A and silybin B from silybin. Optimum operating conditions were:flow 8mL/min, injection volume of 3mL, mobile phase ratio of 40:60 (v/v). The first time, injection volume was important factor in the separation of insoluble compounds to obtain satisfing purity of the products and the higher yield.Four new compounds were obtained from the co-products of the silybin refined process by pre-LC. Preliminary structural identifications were performed by MS-MS and CD spectra. (4) The systematic selection of the series of CDs and optimization of the two-phase solvent system for the chiral separation of silybin using a two-level orthogonal array design (OAD) were tried on an analytical HSCCC from the silymarin extracts. Using the optimum solvent system, the purities of silybin B and isosilybin A from silymarin by preparative HSCCC used hydroxypropyl-B-cyclodextrin as the chiral selector were over 95%.A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of silychristin, silydianin and taxifolin in the co-products of the silybin refined process from the silymarin was successfully established. The results demonstrated that HSCCC was a more powerful technique to separate of these compounds in a large scale. Application of HSCCC in the separation of taxifolin, silychristin and silydianin would increase the revenues of the silybin refined process.(5) Activities of antioxidant and tyrosinase inhibition of silymarin by different extraction methods from the seeds of Silybum marianum were determined. Using statistical tools, the correlations among the levels of tyrosinase inhibition, free radicals scaveing levels, total flavonoid content and the contents of seven major active compounds in different silymarin extracts were also studied.The activities anti-DPPH scavenging and tyrosinase inhibition of silymarin, silybin and its monomers in vitro were systematically compared. The results were the same with that of the correlation analysis. The results showed that the activities of two enantiomers of silybin were significantly different. The DPPH scavenging activity of silybin A and silybin B were slightly higher than the silibin. The activity of tyrosinase inhibition of silybin B was over 4 times of quercetin and 9 times of silymarin, repectively. The activity of tyrosinase inhibition of the enantiomers of silybin was significant different. The activity of tyrosinase inhibition of silybin B was twice of silybin, while the activity of of tyrosinase inhibition of silybin A was slightly less than one times of silybin. Silybin B could be used as a new natural tyrosinase inhibitor.