Bioactive Substances Of The Endophytic Fungus HCCB1621

Endophyte is a special and important group of microorganisms with taxonomic diversity, which can produce various of bioactive substances, including insecticidal, antimicrobial and antitumor compounds etc. In this research work, from a large amount of endophytes which had been screened based on several models, dozens of strains showed activities. One of which were taxonomically identified, and their secondary metabolites were studied.1280 fermentation samples were prepared from 1280 strains of endophytes. After primary and secondary screening on two models, 21 samples showed high and stable antitumor activities while 8 samples showed the strong antibiotic activities. Among them, the strain HCCB1621 was selected for further study.Using traditional taxonomic methods, electronic microscope observation, 18S rDNA, ITS sequences and phylogenetic analysis, the endophytic fungus HCCB1621 was identified as Trichoderma viride Pers.ex Fr.And it was found that the broth showed strongest anti-tumor activity after cultured 120 hours. This activity could remain stable after treatment at 60℃for 1h or at pH within 4.0 to 9.0.Through several methods of purification such as the macroporous adsorptive resins, solvent extraction, silica gel chromatography, gelatum LH-20, HPLC preparation, five compounds named as HCCB1621-1-5 were purified from HCCB1621 fermentation broth.Via mass spectrum and ¹H-NMR, ¹³C-NMR and planar NMR, the related physical and chemical characteristics were tested and their molecular weight had been ascertained.Their molecular weight are 256, 396,112, 376, 110Da respectively, and HCCB1621-1-4 were identified as 6,8,10-trimethylbenzopteridine-2,4(3H,10H)-dione, ergosta-7,9,22-trien-3-ol,pyramidin-2,4(1H,3H)-dione,7,8-dimethyl-10-tetrahydroxypentyl benzopteridine-2,4(3H,10H)-dione,which was commonly called riboflavin.The compound of HCCB1621-1 showed strong cytotoxycities against A549 and LoVo cell lines at inhibitory rate 79.85% when the concentration reaches 10μg ml^-1. The compound of HCCB1621-5 showed antibiotic activities to Micrococcus luteus sp, Blastomyces albicans sp, but its antibiotic activity to Micrococcus luteus sp. was weaker than the broad-spectrum antibiotic of ciprofloxacin.Under the same concentration, the inhibitory effect is 70.9% contrast to ciprofloxacin.