J. Res. Technol. Eng. 5 (1), 2024, 79-83 79 JRTE©2024 DEVELOPMENT OF AN ASSAY PROCEDURE FOR ASPARTIC PROTEASE INHIBITORY ACTIVITY AND SCREENING OF SOME MEDICINAL PLANTS IN SRI LANKA * T.S.G.S. B. Wijayarathna, S. Rajapakse Department of Molecular Biology and Biotechnology, Faculty of Science, University of Peradeniya, Sri Lanka *gayanjali90@gmail.com Received:27 Dec 2023; Revised: 28 Dec 2023; Accepted: 31 Dec 2023; Available online: 10 Jan 2024 Abstract— Aspartic acid proteases participate in many physiological processes and their activities are associated with the spreading of diseases such as Alzheimer’s disease, hypertension, cancers, and inflammatory, cardiovascular, viral , and other parasitic diseases. Aspartic proteases have become a widely spoken topic due to human immunodeficiency virus protease and malaria parasite protease that are targeted as key therapeutic intervention points in treating AIDS and malaria, respectively. But, relatively few studies have been reported on natural aspartic inhibitors. Therefore inhibition of proteases is one of the most promising approaches as therapeutic drugs for such conditions. In this case, plants have become a good source of proteinaceous and non-proteinaceous inhibitors. The present study is aimed at the identification, characterization, and partial purification of potent aspartic protease inhibitor/s from several medicinal plants in Sri Lanka. For this study plant samples were collected from Kandy district, Sri Lanka. Aqueous extracts of equal concentrations were prepared separately using fresh mature bark samples from Annona muricata, Garcinia quaesita Pierre, Bauhinia tomentosa, Caesalpinia bonduc, Crotalaria laburnifoloria, Crotalaria micans, Entada zeylanica (kosterm), Erythrina suberosa, Azadirachta indica, and Limonia acidissima L. To our knowledge to recent, there are no aspartate inhibitors identified from these plants. Aspartate inhibitory activity was determined by spectrophotometric stop rate determination method using 0.1mg ml-1 pepsin enzyme, 2.5% bovine hemoglobin substrate with plant extracts (inhibitors). The reaction was terminated by adding 5% TCA solution. The absorbance of the acid-soluble peptides was measured at 280 nm. The inhibitor was purified by ammonium sulfate precipitation and ion exchange chromatography methods. Molecular weight was estimated by dialysis. All the experiments were conducted in duplicate three times. Maximum inhibition of pepsin was shown by Garcinia quaesita Pierre while other species didn't show any significant inhibition of pepsin. Hence for further studies, Garcinia quaesita bark extract was selected. The thermal stability of the inhibitor in the crude extract was studied by incubating the extract at different temperatures and determining the remaining activity. The inhibitors were subjected to ammonium Sulphate precipitation and Ion exchange chromatography, in an attempt to purify the inhibitor/s. Molecular weight was estimated by dialysis which implies that the inhibitors comprise small and large molecules with different molecular weights ranging from less than 3.5 kDa to more than 12 kDa. Crude extract of Garcinia quaesita retained more than 50% of inhibitory activity over a wide range of temperatures (4-95 o C) for 30 minutes and also at 4 o C for one month. But the remaining inhibitory activities of the crude extract incubated at room temperature and 37 o C for one month were 20% and 10%, respectively. This suggests that inhibitor molecules are moderately thermostable. This assay procedure provided a quantitative measurement of the inhibitory activity of the inhibitor/s present in the crude bark extract. Fractions obtained from ammonium sulfate precipitation and ion exchange chromatography didn’t show inhibition towards pepsin suggesting that inhibitors could be non-proteinaceous. Further studies on purified inhibitors and necessary to characterize and elucidate the structure of the inhibitors Index Terms— Aspartic acid protease, Garcinia quaesita Pierre, Inhibitors, Pepsin