Study On Secondary Metobolites Of Endophyte F127 With High Activity From Cephalotaxus Hainanensis Li.

Endophytes, microorganisms that reside in the tissues of living plants, are potential important sources of novel natural products for exploitation in medicine,agriculture, and industry. Currently, the related research, to find the novel and bioactive metabolites, and /or the products associated with the host plant from medicinal plant species, has been worldwide of concern. In this research, we deeply studied the strain F127 (Phomopsis sp.) with strong antimicrobial and antitumor activtiy, which was screened from the endophytes of Cephalotaxus hainanensis Li. by our previous work. The crude extract from the optimized fermention method and its crude fraction was separated in order , based on the mutli-bioactive evaluation, to elucidate the highly active compounds, and possibly discover the compounds with novel structure or novel bioactivity.In this study, the F127 was first fermented by different methods in liquid or solid,and determined to take solid fermentation with rice medium to further cultured the strain by large scale, through the comparison of their metabolites of yields, chemical compositions, antimicrobial activities and antitumor activities; secondly, the chemical compositions and the antitumor acitivities of the crude extract from the rice solid fermentation were stressingly measured as well as the antimicrobial activities and antioxidant activities done; then, the crude extract were isolated and purified , during which the multi-bioactivities of the crude fraction were tracked and discussed, to further separate the bioactive ones; finally the compounds were elucidated by NMR and their anti-tumor activities, antimicrobial activities, and antioxidant activities, were evaluated in vitro. The main results are as follows:(1) F127 was cultured by different fermentation methods with different mediums,among which the yield of metabolite by rice solid fermentation was the highest, the chemical composition spectrum of metabolite by rice solid fermentation was the most abundant, and the better antimicrobial activity and stronger leukemia cytotoxic activity were also observed in it. Therefore, the strain F127 was determined to take solid fermentation with rice medium in large scale.(2) The crude extract, weighed 520.5g, by solid fermentation with 15.0kg rice and ethylacetate extraction was obtained. Seven categories of substances, phenols,alkaloids, flavonoids, saponins, steroids, anthraquinones and volatile oil,were positively detected in the crude extract by tube qualitative test, stating that the substances of metabolite are plentiful. Substantially cytotoxic activitities of the crude extract against K562, NB4, HL-60 cells were reavealed with reaching IC50 value of 1.92ug / mL, 10.29 ug/mL and 17.12 ug/mL, distinctively. In the antimicrobial activity test, the crude extract was found to have significant inhibitions against Escherichia coli , Bacillus subtilis , Streptococcus agalactiae , Sclerotinia sclerotiorum, Fusarium oxysporum f.sp. Cubense in seven tested strains. Results of antioxidiant-activity was showed with 51.54mg / g, 21.39mg / g, 0.642, 90.42% and 85.32%, which were observed from the total phenolics and total flavonoids in the crude extract, the total abosorbance and the clearance rates of ABTS � and DPPH �respectively, displaying that the crude extract is with good antioxidiant activity.(3) The crude fractions, F1-F6, were obatained by column chromatography from the crude extract by rice solid fermentation. The bioactive crude fractions, F2, F3, and F4 were further separated by CC, ODS, TLC and HPLC techniques. And the 10 single compounds were identified on the basis of NMR, which are ergosterol (1),oblongolide S (2), 4-hydroxybenzaldehyde (3), (R) -6-Hydroxymellein (4),phomopsolidone B (5),oblongolide A1 (6),phomopsolide B (7),oblongolide B1(8),oblongolide T (9),and penixanacid A (10). Compound (6) and compound (8) are new compounds without any report. Besides compound (10) belongs to xanthone ,which was isolated from Phomopsis endophytic fungi for the first time. And 4 of 10 belongs to oblongolides, which are the known compounds from the gensus Phomopsis endophyte.(4) The antitumor activites , antimicrobial activites and antioxidiant activites,were in vitro evaluated by MTT, MIC and DPPH method, respectively.The results indicated that new compound oblongolide A1 showed a certain antioxidant activity with IC50 of 386.61 ?g mL, and good inhibitory against Staphylococcus aureus with the MIC of 62.5?g / mL; and new compound oblongolide B1 was not only found to have strong resistance against Staphylococcus aureus and Escherichia coli, both with the MIC value of 62.5?g / mL, but also resistant to K562, NB4, HL- 60 with good cytotoxic activity especially for HL-60 cell with IC50 values of 31.03?g / mL. Besides,the remaining nine substances showed one or more biological activities, except for oblongolide S .Among them, 4-hydroxybenzaldehyde and (R) -6-Hydroxymellein both showed good antioxidiant activity with the IC50 of 70.96?g/mL and 104.01?g/mL;ergosterol, phomopsolidone B and penixanacid A displayed strong inhibitory against Streptococcus agalactiae from tilapia, with the MIC values of 1.95?g / mL,7.81?g / mL and 3.91?g/mL; ergosterol, phomopsolide B and penixanacid A exhibited substantial cytotoxicities against K562, NB4 and HL-60 in which phomopsolide B was strongest with IC50 of 7.62?g/mL, 1.44?g/mL and 2.78?g/mL.In conclusion, the secondary metabolites of Phomoposis sp. F127 are aboundant and multi-bioactive. And some compounds showed significant bioactivities, and some compounds were with new structure. Thus, F127 is worthy studying further to provide the scientific basis for developing the potential drug resoures from the endophytes of Cephalotaxus hainanensis Li. finally.