The Secondary Metabolites And Antimicrobial And Antitumor Activities Of Endophytical Fungi From Cephalotaxus Hainanensis Li

The study selected medicinal plants Cephalotaxus hainanensis Li as research material. Total of183strains of endophytic fungi were isolated from leaves and bark from Hainan Tropical Botanical Garden, Jianfengling Nature Reserve and Bawangling Nature Reserve,which contained3bacteria such as Escherichia coli ATCC25922, Staphylococcu aureus ATCC51650, and Bacillus subtilis strains ATCC6633,as well as4plant pathogen such as Rhizoctonia solani ZLL101, Sclerotinia sclerotiorum XJS01, Fusarium oxysporum f.sp. cubense RKY102and Fusarium oxysporum f.sp.cubense FJAT-370and an animal pathogen, tilapia source agalactiae Streptococcus agalactiae HYXY08. For the eight test strains, their antimicrobial activities were evaluated to screen the active strains. And24screened active strains had the antimicrobial activities, further screening on the active strains’metabolite showed that there were11higher active strains from27strains.The traditional fungal morphological identification and18s rDNA-ITS sequence analytical method were adopted to identify11higher active endophytic fungi strains Al: Phomopsis destruens, A9:Penicillium citrinum, A21:Diaporthe vexans, A31:Penicillium soppii, B6:Nemania diffusa, B14:Diaporthe actinidiae, B21:Corynespora cassiicola, C7a: Phomopsis phyllanthicola, E2:Leptostroma sp., F52:Neonectria macroconidialis, F127: Phomopsis eucommicola.Crude extracts from metabolites of the11high antibacterial activity strains were screened for their antitumor activity, and results showed that each strain crude extracts had inhibitory effect on at least one tumor cell, while9of11strains crude extracts had inhibitory effect on more than two tumor cells. The ICso alue of F127strains on3tumor cells are below32μg/mL, and F127strains also showed strong antitumor activity on the leukemia cell K562and leukemia cells NB4, whose IC50alue were8.07μg/mL and16.68μg/mL, respectively. In consideration of the antimicrobial activity, antitumor activity and chemical composition analysis, F127was selected as target strain.Cephalotaxus hainanensis Li endophytic fungi F127(Phomopsis eucommicola), which had the high activity of antimicrobial and antitumor activity, were cultured by extensive fermentation.15.6g crude extracts from metabolites were obtained from F127. The methods of Silica gel column chromatography, thin-layer preparation and recrystallization were adopted to carry out separation and purification on the fermentation crude extracts from metabolites. Five compounds were obtained and they were oblongolides t, sorbicillin ((2E,4E)-1-(2,4-dihydroxy-3,5-dimethylphenyl)-,2,4-Hexadien-1-one), dibutyl phthalate, Phomopsolide B and6,8-dihydroxy-3-methyl-3,4-dihydroiso-cou marin, respectively. Throμgh chemical analysis and spectroscopic data analysis, the structures of the five compounds were ultimately determined.Five compounds were tested for their antitumor activities to five tumor cells. The results showed that only sorbicillin and dibutyl phthalate did not have antitumor activity, but compound6,8-dihydroxy-3-methyl-3,4-dihydroisocoumarin had some antitumor activity on NB4and hepatoma cells (HepG-2), and the IC50values were87.08μg/mL and96.57μg/mL, respectively. Compound oblongolides T had a better inhibitory activity on K562, NB4and HL-60leukemia cells, and their IC50values were51.82μg/mL,54.25μg/mL and29.31μg/mL. Compound Phomopsolide B had inhibitory effect on five kinds of tumor cells, and it had warm inhibitory effect on the HepG-2and colon cancer cells LoVo, and the IC50values were92.94μg/mL and87.34ug/mL. However, Phomopsolide B had very strong inhibitory effect on the leukemia cells K562, NB4and HL-60, and the IC50values were3.35μg/mL,0.014μg/mL and0.16μg/mL.