Abstract (eng)
Resistances to antibiotics and antimycotics are a worldwide rising issue and the misuse of antimicrobials created diverse multi-resistant germs. Natural sources can provide unknown substances and fungal endophytes are a relative unused origin for molecules with antimicrobial activities. Therefore, this Diploma Thesis deals with the discovery of new antimicrobials derived from fungal endophytes.
Bioactivity-guided fractionation, a method that is largely used for bioactivity screenings of new molecules, was applied within this work to fractionize fungal extracts and to isolate the compounds of interest. Dichloromethane/methanol (2:1) was used for the extraction of the fungi Ab1 (Preussia sp.), Ab11 (Nemania sp.) and Bl73 (Helotiales). High performance liquid chromatography (HPLC) was utilized as chromatography system. Analytical HPLC was used to investigate the extracts, while semi - preparative HPLC was applied for fractionation of larger amounts of the extracts. For antimicrobial activity testings of the extracts and their fractions disc diffusion assays were performed at the Department of Pharmacognosy (not part of this work). By liquid chromatography - mass spectrometry (LCMS) analysis the molecules of interest were identified and characterized through their mass within the fractions. The compounds of interest, which were determined by LCMS, were subsequently gained by subfractionation of the according fractions.
For this Diploma Thesis fungal extracts of Ab1, Ab11 and BL73 were provided for intial antibiotic activity testing. The provided extracts of Ab1 and Ab11 both inhibited B. subtilis, while Bl73 extract inhibited B. subtilis, S. cerevisiae, A. niger and F. graminearum in disc diffusion assays. The extracts, which were produced in context of this work, all showed the same antimicrobial activities, except Ab11 extract, which lost activity against B. subtilis. Consequently Ab11 extract was excluded for the rest of this work.
After analytical HPLC, Ab1 extract was fractionized by semi - preparative HPLC and some fractions showed inhibition of B. subtilis. In the subsequent work on Ab1 extract, however, problems in the dissolution of the extract occurred, which lead to the loss of the antimicrobial effect on B. subtilis and therefore the work on this extract was stopped. Following analytical HPLC of Bl73 the extract was also fractionized by semi - preparative HPLC and various fractions inhibited B. subtilis, S. cerevisiae, A. niger and F. graminearum.
In the follow up of the work on Bl73 extract the LCMS analysis of these active fractions revealed two compounds with mass [M+Na]+ 495.28 [m/z] and mass [M+Na]+ 323.06 [m/z]. These substances were supposed to be responsible for the antimicrobial effects on the test organisms. The isolation by semi - preparative HPLC of the compound with the mass [M+Na]+ 495.28 [m/z] resulted in an unknown pure substance, (which was determined by NMR spectroscopy and was not part of this work). The antibacterial effect of the substance with mass [M+Na]+ 495.28 [m/z] on B. subtilis was confirmed by a disc diffusion assay. However, the inhibition of B. subtilis was very weak both in the according fraction and pure substance. By the isolation of the substance with mass [M+Na]+ 323.06 [m/z] antifungal properties against S. cerevisiae, A. niger and F. graminearum were confirmed by disc diffusion assays. Nevertheless at the end of this work no information about the purity or the structure of the compound was available. Moreover the inhibitory effect on the fungi was not as good as the effect of the corresponding fraction.
As a conclusion by the isolation of the substance with mass [M+Na]+ 495.28 [m/z] one particular compound of Bl73 extract could be gained that inhibited B. subtilis. Subsequently to this work the structural analysis of the substance with mass [M+Na]+ 323.06 [m/z] could be done. Furthermore the analysis of other molecules in the fraction containing substances 495.28 [m/z] and 323.06 [m/z] should be done to confirm whether a synergistical effect of inhibition is present.