Occurrence of serine protease inhibitory activity in the bark extract of Entada pursaetha

Abstract

Serine proteases carry out a diverse array of physiological functions and incorrect regulation of expression of serine proteases lead to many disease processes including cancer, renal diseases, neurodegenerative disorders. Serine protease inhibitors have become a target in the treatment of many such disorders leading to numerous investigations of the natural serine protease inhibitors. Members of the family Fabaceae are well known to contain serine protease inhibitors. Thus, the current study was aimed at identification and characterization of serine protease inhibitors from Entada pursaetha of the family Fabaceae. An assay procedure was developed and optimized to determine the serine protease inhibitory activity of the aqueous mature bark extract of Entada pursaetha. For the assay procedure, trypsin was used as the serine protease and casein as the substrate. Dialysis was performed by using a membrane of 14 kDa molecular weight cut off point followed by ion exchange chromatography and ammonium sulphate precipitation to purify the serine protease inhibitors in the crude extract. Thermal stability of serine protease inhibitory activity of the crude extract and partially purified sample was determined by incubating them at 37 ⁰ C and 60 ⁰ C for one month. Optimum pH for the inhibitory activity was pH 7.6 and 10% bark extract resulted in a 50.24% inhibitory activity. Dialysis with 14 kDa membrane increased the remaining inhibitory activity to 149.62%. Thus, it can be concluded that inhibitory molecules are macromolecules with molecular weight greater than 14 kDa. Thermal stability studies revealed that the inhibitor molecules are thermally stable. Partial purification of the protease inhibitors was achieved by anion exchange chromatography (pH 7.6, 8.5), cation exchange chromatography (pH 7.0, 5.5 and 5.0) and ammonium sulphate precipitation. This assay procedure provided the quantitative measurement of the inhibitory activity for the inhibitor/s present in the crude bark extract. The bark extract may contain both proteinaceous and non-proteinaceous serine protease inhibitors. Further studies on purified inhibitors are necessary in order to characterize and to elucidate the structure/s of the inhibitors.