| Aciduric microorganism are extremophiles that can survive in low pH environment. Aciduric microorganism could produce compounds with novel structures and biological activity in the secondary metabolites because of possessing special ecology, specific physiological mechanism. Fungi capable of surviving at low pH conditions have attracted much attention in recent years for their diverse secondary metabolites with a range of biological activities. But up to date, few studies have been done about secondary metabolites of aciduric microorganism, the research work are worthy of further Study."Talented strains" refer to those strains possessing large metabolic production, producing a series of new compounds with biological activity. The special acid environment where aciduric microorganism live could active silent gene and induce other specific metabolic pathway. Therefore, our study was carried out to search novel bioactive compounds from the secondary metabolites of aciduric talented strains under acid environment. Studies include the screening for aciduric talented strains, isolation and structural elucidation of the secondary metabolites, preliminary bioactivity evaluation of compounds, the impact of acid regulation for secondary metabolites of aciduric talented strains.121strains of aciduric fungi have been isolated from acid soil collected in Jianshui, Yunnan province and Yuntaishan National Geological Park from Jiaozuo, Henan, China. Using SRB methods, eighteen active aciduric strains were screened out of two121strains, on the bioassay of P388cell lines. The EtOAc extracts of these fungi were evaluated with the combinatory method of chemical screening (TLC and HPLC) and bioactive evaluation (cytotoxicities and antiviral activity). Four aciduric fungi were determined as aciduric talented strains because of their metabolic production and cytotoxicity or antiviral activities.First of all, fermentation conditions of target strains were optimizated, including medium, fermentation time and so on. After fermentation of four aciduric talented strains, the whole broths were extracted with ethyl acetate to give ferment extracts. Then the EtOAc extracts were subjected to extensive silica gel column chromatography, Sephadex LH-20and HPLC to give compounds. Sixty-seven compounds were isolated from four aciduric talented strains.18compounds were isolated from Penicillium purpurogenum JSO3-21(1-17,67);19compounds were isolated from Penicillium sclerotiorum ZZ07-5(18-36);17compounds were isolated from Penicillium purpurogenum ZZ05-4(37-53);13compounds were isolated from Penicillium griseofulvum ZZ10-35(54-66). By means of modern spectral analysis (NMR, MS, CD, UV, IR), quantum chemical calculations and chemical methods, the structures (see Fig.0-1) of sixty-two compounds were respectively determined, among them twenty-eight were new structures. Types of new compounds include azaphilones (1-4,21-27,35), benzofuranones (6-9), naphthoquinone (10), sesquiterpene derivatives (14-15), lactones (37-40), fatty acids (41-42), polyketides (55-57). In addition, the chemical structure types of compounds reported are involved in azaphilones (18-20,28-29,31-34), sesquiterpene derivatives (16-17), benzene derivatives (11-13,43-46), furan derivatives (47-50), chromone (30), terpene (35), lactams (51-52), anthraquinone (58), tetralones (59-62).The activity of compounds against influenza A virus (H1N1) was evaluated by the CPE inhibition assay. Compounds2,3,6,11,27exhibited stronge anti-H1N1activity with IC50values of61.3、64.0、85.3、58.6、25.5μM, respectively. Compound2was effective in vivo evaluation, too. Compound2could reduce lung inflammation of mice caused by influenza virus infection, play a protective role in mice. Using the agar dilution method the compounds were evaluated on anti-pathogenic microorganisms activity. In antifungal assay, compounds14-17showed moderate antifungal activity against C. albicans with MIC values of3.3,1.3,1.2and1.3μM, respectively. And compounds14and15showed moderate antimicrobial activity against E. aerogenes and P. aeruginosa with MIC values of2.4and2.6μM, and1.2and2.6μM, respectively. In order to evaluate whether the amino acid side chain attributed to the antimicrobial activity, compound17was subjected to basic hydrolysis to produce1α-hydroxyconfertifolin (17a), but no anti-C. albicans activity for17a was observed, indicating that the esterification with amino acid moiety is indispensable for the antifungal activity against C. albicans. Besides, compound9has weak antifungal activity against E. aerogenes with MIC76.7μM. Compounds were evaluated for their cytotoxicities against several cancer cell lines such as HL-60, P388, K562, K562, A549, A172, U87, MCF-7, MCF-7/Adr, MG-63by the MTT or SRB method. Compounds19-21,23-24showed moderate cytotoxicity against A172, U87, MCF-7, MCF-7/Adr, MG-63. Compound57exhibited strong cytotoxicity against HL-60cell line with inhibitory rate of89.1%.To further explore the pH and acid conditions effects on chemodiversity of secondary metabolites of aciduric talented strains, secondary metabolites from four aciduric talented strains under different culture conditions containing different pH and medium were analyzed by comparing weight, TLC, HPLC, bioactivities and compounds. The secondary metabolites contained in the extract of aciduric talented strains fermented at pH2showed a more extensive chemical diversity and better bioactivities than those at pH7. Aciduric talented strains could produce different compounds by fermented in different culture conditions. It has been reported that in general the secondary metabolites can be regulated by external pH. In some cases, the pH effects were larger than carbon and nitrogen effects, and certain silent genes can be activated by low pH. |
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