两株南海真菌Aspergillus aculeatus 1-P1和Paraconiothyrium cyclothyrioides 1-I2的次级代谢产物研究

Secondary metabolites from the South China Sea fungi Aspergillus aculeatus 1-P1 and Paraconiothyrium cyclothyrioides 1-I2

为进一步研究我国南海真菌活性次级代谢产物,对从中国南海表层海水中获得的2株真菌Aspergillus aculeatus 1-P1和Paraconiothyrium cyclothyrioides 1-I2进行鉴定和分析,通过ITS序列分析并构建系统发育树来鉴定菌株,综合运用硅胶柱层析、Sephadex LH-20凝胶柱层析及半制备高效液相等色谱学方法对其发酵产物进行分离纯化,获得17个化合物,化合物结构类型包括三萜(1~2)、甾醇(3~4,12)、二酮哌嗪(5~7)、甘油酯(14~17)等。研究结果有助于为我国南海丰富的海洋微生物资源及其次级代谢产物结构多样性研究提供参考。此外,对发现其中可能含有的结构新颖的化学物质以开发抗菌、抗病毒及抗肿瘤新药等也具有十分重要的理论意义和潜在的应用价值。

The South China Sea and its adjacent waters are potential sources for providing new bioactive substances and compound strains.This study mainly focused on the identification and chemical constituents from two fungi in the surface waters of the South China Sea.Sample solution were prepared and diluted into different concentrations,and was spread on rose bengal medium,potato dextrose agar(PDA)medium and yeast extract peptone dextrose(YPD)medium with inverted cultivation under 27℃to select colony.After cultivation,the big colonies were selected according to the color,form of the fungi.The final fungi strains were inoculated on agar slant culture medium and stored under 4℃.The well-stored fungi were then amplified using YPD medium with high temperature setrilization.Through suctionfiltration,the extraction solutions were divided into extracellular extract and intracellular extract,and two extracts were extracted by different polar organic solvents to obtain different polar crude extracts.Microscope method and molecular biology method(18S rDNA method was adopted by the multiple sequence alignment with NCBI)were combined together to characterize the species of these two fungi,and they were identified as Aspergillus aculeatus and Paraconiothyrium cyclothyrioides,respectively.A variety of biological and chemical methods were performed to separate and purify their secondary metabolites.Macroporous resin chromatography,silica gel plate chromatography and semipreparative high performance liquid chromatography were taken turns to achieve the separation and purification of various compounds.By high performance liquid chromatography time of flight mass spectrometry(HPLC-TOF-MS),nuclear magnetic resonance spectrum(NMR)(1H-NMR and 13C-NMR),and other physicochemical properties,there were 17 small molecular compounds isolated and identified totally,including two triterpenoids(1-2),named kudzusaponin SA4 and soyasapogenol,three sterols(3-4,12),named 24-ethylcholesta-7,22-diene-3β,5α,6β-triol,(22E,24R)-ergosta-5,7,22-trien-3β-ol,and(22E)-5α,8α-Epidioxyergosta-6,22-dien-3β-ol,three diketopiperazines(5-7),named cyclo-(4-OH-Phe-Leu),cyclo-(Phe-Pro),and cyclo-(L-Pro-L-Tyr),four glycerides(14-17),named methyl linoleate,1-O-(9Z,12Z-octadecadienoyl)glycerol,(8E,12Z)-10-Hydroxy-8,12-octadecadienoic acid,2-linoleoylglycerol,two flavenoids(10-11),named 7,4′-dihydroxy isoflavones,5,7,4-trihydroxy isoflavones,and other types of compounds named indole ethylamine(8),hydroxyphenethylamine(9),and uridine(13).The fermentation conditions of fungi will be further optimized,and different marine fungal culture media will be used to try suitable metabolic control methodslater.The scale of fermentation will be enlarged and trace ingredients will be enriched in order to obtain more complex structural novel compounds.The results are of great theoretical significance and potential to study the rich diversity of marine microbial resources and the structure diversity of secondary metabolites in the South China Sea,and to discover new chemical entities with antibacterial,antiviral and antitumor activities.