New Natural Inhibitors Of Cathepsins, The Extraction Of Flavonoids From Herba Epimdii And The Inhibition Assay

As the main members of cysteine proteases family, more than 20 kinds of cathepsins had been found up to the present, and 11 of them are existed in human body. They involved in many kinds of diseases as osteoporosis, cancer and rheumatoid arthritis. Among them, Cathepsin K is the most important enzyme in the bone resorption; it plays the key role in the process of bone remodeling process,and therefore is becoming a more and more popular target in the study of the osteoporosis.With the low toxicity, high oral bioavailability and credible effects, the natural medication earns special attentions recently. Considering the long history of using and researching natural medication in China, we decide to screen the natural inhibitors with high selectivity and good inhibition of Cathepsin k in Chinese traditional medicine.Two significances of present study could be achieved: Firstly, during the process of purification and screening inhibitor, we have chance to found new medicines for the concerning diseases; Secondly, the relationship between Chinese traditional medicine and the new target of the osteoporosis,Cathepsin K, could be established by understanding some obscure mechanism of the traditional medicine at the molecule level. Based on such backgrounds, two parts of work are carried out in the present study.Part I, the isolation of flavonoids from the Herba Epimdii: After the extraction and several times going through of silica gel column chromatography, two compounds, Icariin and Sempervirenoside A, have been isolated from the crude extraction of the Herba Epimdii, and the identification of them have also been performed.Part II, the Inhibition AssayIn order to search for new natural inhibitors against Cathepsin K, we performed the inhibition assays of natural extractions to cathepsin K, B, L, S. The IC50 value of Icariin,Sempervirenoside A,IcarisideⅡ, Epimedoside A,Sagittatoside B,Epimedin B and Ikarisoside C to the four cathepsins have been test, respectively. The results indicate that all they are good inhibitors of the cathepsins, and most of them show selectivity toward Cathepsin K and Icariin is the best inhibitor of Cathepsin K among the seven flavonoids, and its IC50 value is 46μM.As the main component of Herba Epimdii is metabolized as IcarisideⅡin the human body, we choose IcarisideⅡfor the further investigation of inhibition mechanism, which includes both the test of Kinetic inhibition constant (Ki) valve and the type assay of the inhibition. Lineweaver-Burk plot is employed and the results show a Ki valve of 10μM for IcarisideⅡto cathepsin K and a noncompetitive mixed inhibition.The mechanism of Herba Epimdii in curing the osteoporosis is still in obscure. The present study will play a key role in the bone remodeling and such results will give a new explanation at the molecule level of the mechanism.