Screening And Preliminary Identification Of Components With Trypsin Inhibitor Activity From Traditional Chinese Herbals

Proteinase inhibitors (PI) exist in a wide range of organisms including animals, plants andmicroorganisms. These inhibitors play important roles in regulating many physiologicalprocesses by inhibiting protease activity through formation of complexes between PI andtarget protease. Trypsin inhibitor (TI), one of the most important protease inhibitors, has greatmedicinal value for its effectiveness in treatments of acute pancreatitis, emphysema,multifunctional failure, anti-shock, cardiac surgery to reduce blood loss, preventing cerebraledema and cerebral ischemia, prevention of premature and unilateral pulmonaryischemia-reperfusion injury, and other related diseases treatments. Recently, it draws eyes ofmost scientists worldwide due to its special application in HIV and cancer therapy.In this study,60kinds of chinese herbal medicine, referred to anti-virus chinese medicine,were selected to test their TI activities, and then the target herbals with high TI activity werefurther used to characterize the TI partially. The results showed that7kinds of above-listedchinese herbal medicine exerted an inhibition rate of trypsin more than15%underconcentration of100mg/mL, and Ephedra herb presented strongest inhibition of trypsin by37.95%, and Rhubarb by36.35%, and Houttuynia cordata by28.03%, and Paeoniae albaradix by23.15%, and Forsythia suspense by18.66%, and Ramulus cinnamomi by17.96%and Huguenot fern herb by17.63. And10kinds of chinese herbal medicine performed10%inhibition of trypsin under concentration of50mg/mL, and Rhubarb was32.73%, andHouttuynia cordata16.51%, and Ephedra herb14.67%. From above-mentioned data, it wasconcluded that6kinds of examed chinese herbal medicine have inhibition rate of trypsinmore than10%under both concentrations of100mg/mL and50mg/mL, respectively, theyare Ephedra herb, Rhubarb, Houttuynia cordata, Paeoniae alba radix, Forsythia suspens andAsiasarum.For the sake of high inhibition rate, Houttuynia cordata Thuhb was selected to extract TIfor further characterization. First, the optimized criteria of TI extraction procedure were45℃,30times of solvent volume, pH7.0and2h of extraction per time. Second, using bothammonium sulphate precipitation and DEAE-cellulose column chromatography techniques, the TI from Houttuynia was purified partially; it showed the peak of specific activity of TIin the part of45-60%(NH4)2SO4precipitation, and consequently was purified byDEAE-cellulose column gradigent elution. The activity of TI can be detected under thecondition of pH8.0Tris-HCL balanced solution containing240mM NaCl. The molecularweight of BCTI was determined by SDS-polyacrylamide gel electrophoresis as28KDa.