Discovery And Purification Of A Non-competitive Trypsin Inhibitor From Sunflower Seeds

Protease inhibitors, as a kind of physiological active substances, play important role in the fields, such as anti-infectivity, anti-virus, anti-tumor, and prevention of premature delivery. In the recent years, the trypsin inhibitors which competitively inhibit serine proteases, i.e. aprotinin, urinary trypsin inhibitor, and soybean protease inhibitor, etc. were widely used. A variety of human serine proteases share the active center with almost identical primary structure and special structure; therefore, the normal functions of other serine proteases are usually changed when the trypsin competitive inhibitors are used. It was the typical example that State Food and Drug Administration of China decided to suspend aprotinin injection in the sale and use. Consequently, it is necessary to search the new trypsin inhibitors.The powder of dried sunflower seeds which planted in Jilin Province was defatted by using petroleum ether reflux, and then was extracted by 0.001mol/L HCl. The supernatant was microfiltrated and ultrafiltrated to 4 fractions, which were Fraction1 (F1 , microfiltration: > 0.2μm), Fraction 2 (F2, microfiltration: < 0.2μm ~ ultrafiltration: 6kDa), Fraction 3 (F3, ultrafiltration: 6kDa ~ ultrafiltration: 3kDa), and Fraction 4 (F4, ultrafiltration: <3kDa). Based on gelatin plate method, F3 with trypsin inhibitory activity (the inhibition activity ratio is 14.21) was determined, and it was further confirmed by using azocasein method. 4 fractions (F3 -Ⅰ, F3 -Ⅱ, F3 -Ⅲ, F3-Ⅳ) were isolated and collected by using ion exchange chromatography. F3-Ⅲ, as interest ingredient, was quantitatively determined (inhibition activity, 8719.35U/mg). 4 peaks shown in RP-HPLC for F3-Ⅲindicates that it is not the non-pure component and needs further purification. The F3-Ⅲinhibitor type which was conducted by Lineweaver-Burk mapping shows that it is non-competitive trypsin inhibitor.Taken together, a new non-competitive inhibitor which molecular weight ranges from 3K to 6KDa was discovered and isolated. Based on the gelatin plate method, casein protein assay and BAPNA assay, an evaluation technology portfolio of trypsin inhibition effectiveness was established. For a new trypsin inhibitor of research and development, development and application lay the foundation.