Study On Active Metabolites Of Endophytic Fungus From Cephalotaxus Fortunei In Qin-ba Mountain Areas

As the secondary protected rare and endangered medicinal plants, the anti-inflammatory,antitumor, antibacterial and other effects of Cephalotaxus fortunei have been recorded inancient times. while the modern people to the in-depth study on Cephalotaxus fortunei can betraced back to1971, until now it has isolated dozens of alkaloids, for example, harringtonineand homoharringtonine which are already used in clinical treatment of acute leukemia andsome solid tumors. However, for the sake of plant resources protection and ecological balance,the traditional extraction process has gradually been replaced; the method of microorganismfermentation for the production of active components with medicinal value has shown moreand more broad prospects. The endophytic fungus is a great treasure house of resources,different growth environment will exist different plant endophytic fungus and their secondarymetabolites that will provide the possibility for more valuable medicinal ingredients.The endophytic fungus this study used was isolated from roots of wild Cephalotaxusfortunei on the special geographical environment of Qin-Ba mountain areas. Antibacterial andcytotoxic activity test of fermentation broth by disk diffusion method and MTT method, thenwe chose a strain of good activity for fermentation, fermentation liquid was extracted with anequal amount of ethyl acetate three times repeatedly. The ethyl acetate solution wasevaporated under reduced pressure to give a crude extract which was separated on a silica gelcolumn. Then we selected the best activity concentration for GC-MS detection and analysisthrough activity tracking test, and found the chemical compositions of value. The mainresearch methods and results are as follows:1. Screening of endophytic fungus: fermentation liquid of10endophytic fungus isolatedfrom Cephalotaxus fortunei were tested for antibacterial and antitumor activity by diskdiffusion method and MTT method. The results showed that, all of endophytic fungi hadinhibitory effects on human promyelocytic leukemia HL-60cells and at least two indicatorbacteria. The strain of CEP18with higher activity which the inhibition rate of HL-60cellswas41.41%, the diameters of inhibition zone against Bacillus coli and Staphylococcus aureuswere22mm and20mm, the diameter of inhibition zone against Salmonella was9mm,therefore we selected the strain of CEP18as the target strain for the next experiment.2. Extraction and separation of secondary metabolites of the target strain: The culture broth (50liters) of the strain of CEP18was filtered and the filtrate was extracted with ethylacetate and evaporated under reduced pressure to give a crude extract (10.2g), which wasseparated into5fractions on a silica gel column: Fr1(105.0mg), Fr2(387.0mg), Fr3(29.0mg),Fr4(16.0mg), Fr5(22.0mg).3. The activity tracking tests of five components: the components (1.0mg/ml) were testedfor antibacterial and antitumor activity by disk diffusion method and MTT method, the resultsshowed that the component of Fr2had obviously inhibition on Staphylococcus aureus and Bacillussubtilis growth, the diameters of inhibition zone were23.0mm and26.0mm respectively.Meanwhile, it had a weaker activity on Salmonella typhl, and no inhibitory effects on Escherichiacoli and Monilia albicans. The cytotoxic activity test of five components showed that, thecomponent of Fr2showed a significant inhibitory effect on HL-60cells, with the growth inhibitoryrate of50.65%and further testing IC50value of8.721μg/ml. The component of Fr1showed aweaker activity with the growth inhibitory rate of22.51%, and the other fractions did not showsignificant cytotoxic activity. Judging from this, the compositions of antibacterial andcytotoxic activity of the ethyl acetate extract better focus on Fr2. The component of Fr2wasused for the next research.4. GC-MS detection and analysis of the component of Fr2: the component of Fr2(30.0mg) was dissolved in methanol and filtered by filter paper for GC-MS analysis. Theresult showed that27kinds of chemical compositions were retrieved and18of them wereidentified by multiple alignment which accounted for94.37%of the total, the majorconstituents were4-hydroxyphenylacetic acid（8.27%）, elaidic acid,methyl ester（10.07%）,hexadecanoic acid（7.54%）, et al. Among them, the compound of4-hydroxyphenylacetic acidwas discovered and reported for the first time in relevant researches of plants of Cephalotaxusgenus.