Isolation, Identification, And Bioactivity Of Producing-taxol Endophytic Fungus

Endophytic fungus is one of the source producing active products benefited for patients during symbiosis with host plants.Endophytic fungus are also one of the source producing active products benefited for patients during symbiosis with host plants,thus,in the plant the fresh fungus secondary metabolite multiplicity as well as other merits cause the present situation which its hopeful solution partial medicine falls short of demand.To protect the resource of plants and relieve the urgent requirement for taxol,a total of 155 isolates of endophytic fungus were isolated from the Podocrapus in Cartagena territory GuiZhou province in this study.Furthermore, we separated an endophytic fungi strain producing-taxol from Podocrapus.It was resulted that the aerial part endophytic fungus is 83.87%of these 155 isolates of endophytic fungus,and the underground part is 16.13%.Moreover,these funguses were cultured amplifty to PDB,and were conservated.Thereby,the antibacterial activity of 155 strains Podocrapus endophytic fungi was tested through antimicrobial experiments in the exterior body at equal pace,the result showed that 47 strains(30.3%of the total isolated endophytic fungus) are able to produce antibacterial active constituents(inhibition zone≥0.5mm),6 strains are antibacterial activity strongly when their extract was diluted to 10~2 times,and 5 strains are antiboisis activity to experimental baeterias overall on this study.Meanwhile,the anti-tumor activity of endophytic fungi strains was detected by Methyl Thiazolyl Tetrazolium(MTT) method.It was confirmed that one strain produced Taxol by ways of thin layer chromatography(TLC) and high performance liquid chromatography(HPLC).This strain was classified by morphology together with similarity of internal transcribed spacer(ITS) sequence by Clustal W method. The activity of anti-tumor of Taxol produced from the strain was detected by Vero cells.A total of 155 endophytic fungi were isolated from the tissue of Podocrapus.The result showed that twenty-eight strains(18.1%) were able to inhibit the growth of Vero and BHK-21 cell(inhibitory ratio≥10%),and seven strains(4.52%) were detected with high activity(inhibitory ratio≥70%).It was confirmed that one of the seven strain A2 produced Taxol by ways of TLC and HPLC.Therefore,we recognized strain A2 as Endophytic fungi Producing Taxol from Podocrapus-1 (EPTP-1).EPTP-1 was classified as Aspergillus fumigates.Taxol extracted from strain EPTP-1 resulted in significant apoptosis of Vero and BHK-21 cells at concentration of 10μg/L for 24h.And the activity of anti-Vero and anti-BHK-21 growth was not significant between the purchased standard taxol and the taxol extracted from strain EPTP-1(P＞0.05,x~2).It suggested that the taxol extracted from strain EPTP-1 was similar with the standard taxol in anti-tumor activity.The distinction of taxol extracted from strain EPTP-1,furthermore,was not significant between Vero cells and BHK-21 cells(P＞0.05,x~2).It suggested also that the taxol extracted from strain EPTP-1 was activity when it treated a variety of cancer cell.The cultured hypha of the strain EPTP-1 was moved to PDB and cultured at 28℃,200rpm.The maximum yield of Taxol was 0.5578±0.0294mg in per liter liquid fungal culture on day 4,thus,we recognized the strain EPTP-1 output of Taxol was 0.56mg/L.The identified endophytic fungi,strain EPTP-1,might be a candidate for Taxol production and benefit to understand the relationship of ecology and pharmacology between endophytic fungi and the host.