Research On The Antimicrobial Active Constituents Of Fibrous Root Of Bletilla Striata(Thunb.) Reichb.f.and Antibacterial Mechanism

Objective:To extract and separate the constituents of fibrous root of Bletilla striata,and to study the in vitro antibacterial activity and mechanism.Methods:(1)The chemical constituents were separated and purified by various chromatographic procedures,e.g.silica gel,AB-8 macropore absorption chromatography,Sephadex LH-20,and Semi-preparative HPLC.The chemical structures of the compounds were identified on the basis of HPLC with known compounds.(2)The antibacterial activity of the constituents were determined by disc diffusion method and antibacterial spectrum.The MIC and MBC were determined by the double dilution method,the time-sterilizing curve was drawn with the method of colony,the bacterial morphological change was observed under a microscope.(3)FtsZ protein was expressed successfully using E.coli and purified by Ni affinity column chromatography and G-50 column chromatography.(4)BSA method was used to determine protein concentration,malachite green method GTP enzyme activity and the FtsZ protein aggregation method of 90 fluorescent scattering analysis.Results:(1)15 compounds were isolated from 95%ethanol extract of fibrous root of Bletilla striata,including 12 liposoluble components.and 3water-soluble components.Compared with HPLC chromatograms of the compounds which were identified in the laboratory before,13 of them are known compounds and the purity was above 90%.Two compounds are unknown and need further identification.(2)The compounds were germicidal effect on gram-positive was better than that on gram-negative,the N-butanol extraction and separation of part of compound antibacterial effect no good ethyl acetate extraction and separation of the compounds.Of the isolated compounds,the compound 3 showed the most potent activity against all tested Gram-positive bacteria with MICs of 2μg/ml.Furthermore,the compound 3 were found to induce filamentation in Bacillus subtilis,suggesting FtsZ may be the target of these antibiotic compounds.(3)FtsZ(Mycobacterium tuberculosis)was overexpressed and purified from Escherichia coli BL21 strain.The SDS-PAGE analysis indicated that the purity of the protein was more than 90%.(4)FtsZ mycobacterium tuberculosis protein was obtained through column chromatography,with His tag protease activity is without His tag protease activity about three times.The GTPase activity of FtsZ and Light scattering assays indicated that compound 3 increased the polymerization of FtsZ,suggesting FtsZ may be the target of these antibiotic compounds.Conclusion:The ethylacetate extract of fibrous roots of Bletilla striata has broad-spectrum antibacterial activity.The antibacterial effect of gram positive bacteria is better than that of gram-negative bacteria and the phenanthrenes have good antibacterial activity.Antibacterial mechanism may be concerned with the FtsZ protein function.In this paper,the constituents of fibrous roots of Bletilla striata were isolated,the antimicrobial activity and mechanism were studied and disscussed.It provides experimental evidences for the utilization of Bletilla striata and it also deserves further study.