Studies On Isolation And Identification Of Antifungal Components Of Artemisia Hedinii And Their Antifungal Mechanism

Artemisia hedinii（A.hedinii）was a angiosperm,and the crude extracts of A.hedinii had antifungal activity.Using A.hedinii as test material,Alternaria alternat（A.alternat）and Trichoderma viride as test strains,the extraction method of antifungal substance of A.hedinii and the antifungal activity of the crude extracts of A.hedinii were studied,and the antifungal substances were isolated and purified,Analysis of antimicrobial substances,and antimicrobial substances antifungal mechanism to explore.1.By comparing the bacteriostasis experiment results with the extraction rate,we get the suitable method of extracting A.hedinii.The dried stems and leaves of A.hedinii were crushed by a crusher and then sieved through a sieve with a hole diameter of 0.154mm（100 meshes）,mixed with petroleum ether（class II）at a ratio of material to liquid of1:15,and subjected to ultrasonic assisted constant temperature magnetic stirring extraction,the extract was dissolved in ethanol,ethanol was filtered to obtain a solution,evaporated to dry A.hedinii coarse extract.The virulence test results of the crude extract of Artemisia odorats on fungi showed that the EC₅₀0 of A.alternat mycelia EC₅₀=1.25mg/mL,and the EC₅₀0 of Trichoderma viride mycelium 2.51 mg/mL,which was the germination of A.alternat EC₅₀=5.04 mg/mL.It shows that the crude extract of A.hedinii had antifungal activity.2.The extraction of A.hedinii were rough and further isolated by chromatographic separation technology,and the antifungal activity of the component was evaluated by the the spore germination method.Furthermore,The composition of antimicrobial components was analyzed by high performance liquid chromatography-mass spectrometry（LC-MS）.The results showed that 13 kinds of components（Fr.1^Fr.13）were rough isolated from the extraction of A.hedinii,in which the component Fr.5showed the best antifungal activity.Four kinds of components（Fr.5.1^Fr.5.4）were obtained when the component Fr.5 was further separated.The EC₅₀0 of A.hedinii crude extraction on A.alternate spores was 5.04 mg/mL,while the component Fr.5.2 was 0.57mg/mL.The LC-MS results showed that the component of component Fr.5.2 mainly contained deoxy double hydroxyl artemether（17%）,deoxy dihydroartemisinin（43.65%）and deoxyartemisinin（16.15%）.The content of Dihydroartemisinin（DHA）of component Fr.5.2 was calculated as 40.95%by the standard substance of DHA.The EC₅₀of DHA on A.alternate was 2.70 mg/mL,indicating that the major antifungal matter in component Fr.5.2 was artemisinin derivatives.3.The Minimum inhibitory concentration（MIC）and Minimum bactericidal concentration（MBC）,the integrity of cell membrane,the leakage of intracellular substances,Scanning Electron Microscopy（SEM）observation of bacteria and their spores were determinated to study the antifungal mechanism of A.hedinii antifungal component Fr.5.2 against A.alternate,as DHA the positive control.The results showed that the MIC and MBC of aganist A.alternate were 1.25 mg/mL and 2.5 mg/mL,respectively,while the MIC and MBC were 5 mg/mL and 10 mg/mL of DHA aganist A.alternate.The cell integrity of A.alternate decreased and the intracellular nucleic acid macromolecules losed with the time extension in 0-3 h as the component Fr.5.2 and DHA concentration of 0.5MIC.SEM observation shows that the cell membrane of mycelium was small,concave and hollow and the surface of the spores was also sunken,A.hedinii antifungal component Fr.5.2 and DHA can inhibit the synthesis of ergosterol and lead to its content decreasing by29.77%,28.99%,respectively,comparing to the control group.The number of spores increased,however,the germination rate decreased.These results indicate that artemisinin substances show antifungal effect on fungi,and can cause the synthesis of fungal ergosterol inhibited,the cell membrane destroyed,the metabolism of fungal cells hindered and fungal spores germinated.