The Secondary Metabolites Of Endophytic Fungi Isolated From Nicotiana Tabacum L. And Their Antitumor Activities

The endophytic fungi of plants have abundant biodiversity,and they have a symbiotic relationship with the host during the co-evolutionary process.Thus the endophytic fungi can produce the same or similar secondary metabolites as the host.It has been a research focus to explore natural produces with excellent activities in the secondary metabolites of endophytic fungi.This thesis was designed to systematically investigate the richness of endophytic fungi separated from the medicinal plant Nicotiana tabacum L.in Enshi Hubei using the tissue isolation method,and to reveal their biodiversity and community structures at different tissues,developmental periods,and altitudes.We selected functional strains from the isolated and purified endophytic fungi with antitumor activity by CCK-8 assay.We had a large-scaled fermentation of the functional strain in order to separate and purify monomer compounds with antitumor activity from the secondary metabolites.The main experimental results were as follows:1.A total of 539 strains of the endophytic fungi were isolated and purified from the healthy tissues（roots,stems,and leaves）of tobacco at four developmental periods（seedling stage,resettling stage,vigorous growth stage and maturation stage）and three altitudes（600 m,1000 m,1300 m）.The isolated strains belonged to 31 genera,73 species according to morphological characteristics and molecular identification,of which the genera Aspergillus sp.and Fusarium sp.were confirmed as the dominant flora,with the relative frequency values of 22.63%and 12.99%,respectively.Meanwhile,the Shannon diversity index was 2.78.There were 13 common genera and 16 rare genera.The biodiversity of rare genera was the main difference between different tissues,developmental periods,and altitudes.2.There were 19 genera,24 genera and 18 genera separated from roots,stems,and leaves,respectively.The biodiversity of the endophytic fungi in stems was higher compared with those in roots and leaves.The difference of rare genera at roots,stems,and leaves,which had 6,5 and 1 respectively,indicated that the distribution of endophytic fungi showed specialization of tissues.3.There were 8 genera,15 genera,21 genera and 23 genera separated from seedling stage,resettling stage,vigorous growth stage and maturation stage,respectively.With the extension of developmental period,the diversity of the fungal endophytes increased gradually.The difference of rare genera at different growth periods,which had 1,4 and 9 respectively from resettling stage to maturation stage,indicated that the distribution of endophytic fungi showed specialization of developmental period.4.There were 30 genera,28 genera and 22 genera separated from 600 m,1000 m,and 1300 m,respectively.With the rising of altitude,the diversity of the fungal endophytes decreased gradually.The rare genera at 600 m and 1000 m were 2 and 1 respectively,which indicated that the distribution of endophytic fungi showed specialization of altitude.5.The crude extract of fermentation liquid from 20 endophytic fungi showed some antitumor activities to Caki-1 and HepG2 cell lines,especially to Caki-1 cell lines.The IC₅₀s of the PDB crude extract from the strain TE-1S、TE-6L、TE-14L and the rice crude extract from the strain TE-6L、TE-8L、TE-20R to Caki-1 at 48 h were all less than 40μg/mL.The IC₅₀s of the PDB crude extract and the rice crude extract from the strain F.sambucinum to Caki-1 at 48 h were 13.28μg/mL and 0.36μg/mL,respectively.6.There were 9 monomer compounds separated from the secondary metabolites of F.sambucinum,of which the compounds 1,7 and 9 showed antitumor activities to Caki-1 with the IC₅₀s at 24 h and 48 h were 18.24、3.32μg/mL,10.97、1.91μg/mL and 7.81、0.83μg/mL,respectively.The compound 9 had better antitumor activity,which was confirmed as（22E,24R）-5α,8α-epidioxyergosta-6,22-dien-3β-ol.