Based On Chinese Herbal Medicine Extract 1 - Deoxy-d-xylulose 5 - Phosphate Restore Heterogeneous Enzyme Inhibitor Screening

The increasing incidence of bacterial drug resistance has stimulated the development of strategies for new antibiotics screening. Up to now, more than 40,000 terpenes have been isolated from natural sources, which constitute one of the biggest families of the natural products. Two pathways for the biosyntehsis of this kind of compounds have been found and established, namely the classical mevalonate (MVA) pathway and non-mevalonate (MEP) pathway. The latter one was set up as a new biosynthetic pathway of terpenoids in the end of last century and most human pathogenic bacteria rely their sources of terpenoids, while the human beings and animals use only MVA pathway. Therefore, every enzyme of this novel pathway can be employed as ideal targets to screen antibiotics. Protein 1-deoxy-D-xylulose 5-phosphate reductoisomerase (IspC or DXR) is the rate-limiting enzyme of MEP pathway and therefore, it has been used as a target to screen new antibiotics. We have rich resources of medicinal plants in our country and it has been shown by a plenty of clinical and pharmacological studies that lots of Chinese herbal medicines possess good anti-microbial activity. Thus, Chinese herbal medicines based antibiotic screening using ispC protein as a target will certainly lead to the discovery of new antimicrobial agents. In this thesis, the essential oils of several medicinal plants which show excellent anti-microbial activity were selected to screen inhibitors of ispC protein and the results indicated that eugenol isolated from the essential oils of Eugenia caryophyllata Thunb. Display medium inhibition on the target.Firstly, the volatile components were extracted from a dozen of antimicrobial Chinese herbal medicines and then separated by column chromatography. The different fractions were used for the further study.Secondly, we did a study of the reaction system which was used for quickly detecting IspC activity. The results were that:the concentration of Tris-HCl (pH 7.4), NADPH, DXP, Mg2+, and IspC protein 100mM, 0.1mM, 1mM,5mM and 0.2mg/ml respectively. Besides, the suitable reaction tempreture was 30℃, and the reaction time was 50min. Then, we screened the segments of various volatile oils from different Chinese herbal medicines with antimicrobial activity according to the above-mentioned results. Consequently we found that the sixth fraction of the volatile oil of cloves exhibits medium inhibitory activity on IspC. After further purification of this fraction and spectroscopic elucidation, the structure of the active compound was identified as eugenol. Its IspC inhibitory activity was being further studied.Finally, we made some simple structural modification on eugenol, and investigated the IspC inhibitory activity of the derivatives. Unfortunately, no derivative with better IspC inhibitory activity than eugenol wae found. More structural modification of the lead was being carried out in the lab.