2株海鞘共附生真菌的次生代谢产物研究

分别对采集自防城港白龙的皱瘤海鞘(Styela plicata)和北海南汅的冠瘤海鞘(Styela canopus)样品的共附生真菌Purpureocillium sp. FBZ-1和Penicillium sp. BNG-1进行研究。通过显微镜观察鉴定其形态,并通过分析生物学特征鉴定其种属。通过对两种真菌的次级代谢产物进行分离和结构鉴定,共分离鉴定28个小分子化合物,从中未发现新的化合物,主要的化合物类型是芳香胺类、生物碱类和二酮哌嗪类,分别为:lumichrome、 1,2,3,4-四氢-β-咔啉-3-羧酸、β-咔啉、环(异亮氨酸-脯氨酸)、环(亮氨酸-羟基脯氨酸)、环(色氨酸-缬氨酸)、环(亮氨酸-脯氨酸)、环(苯丙氨酸-脯氨酸)、环(异亮氨酸-丙氨酸)、环(色氨酸-亮氨酸)、苯乙胺、N-乙酰苯乙胺、吲哚乙胺、N-甲基色胺、N-(4-羟基)丁酰苯乙胺、对羟基苯乙胺、 5,7,4-三羟基异黄酮、 7,4-二羟基异黄酮、 5,7,3,4-三羟基异黄酮、邻苯二甲酸异辛酯、 2,4-N,N-dimethyl-lumichrome、 3,4-二氢-3-甲基-β-咔啉-1-酮、色氨酸、 2-羟基-3-吲哚丙酸、环(缬氨酸-脯氨酸)、环(亮氨酸-丙氨酸)、环(苯丙氨酸-5-甲基-3,4-脱氢脯氨酸)、环(色氨酸-天冬酰胺)。研究结果对利用我国丰富的海鞘及其共附生微生物资源并研究其次级代谢产物结构多样性,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,具有十分重要的理论意义和潜在的应用价值。

Secondary metabolites from two strains Purpureocillium sp. FBZ-1 and Penicillium sp. BNG-1

Recently, secondary metabolites from ascidians have become a hot spot in marine natural product research and new drug research and development, while secondary metabolites from ascidian associated microbes have not been widely used. Two strains Purpureocillium sp. FBZ-1 from S. plicata collected at Bailong of Fangchenggang and Penicillium sp. BNG-1 from S. canopus collected at Nanwan of Beihai were well cultivated and investigated. Sample solution were prepared and diluted into different concentrations(1×10-1, 1×10-2, 1×10-3), and each was spread on rose bengal medium, potato dextrose agar medium(PDA) and yeast extract peptone dextrose(YPD) medium with inverted cultivation under 27 ℃ to select colony. After cultivation(1-2 weeks), 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 suction filtration, the extraction solutions were divided into extracellular extract and intracellular extract, and tow extracts were extracted by different polar organic solvents to obtain different polar crude extracts. Microscope method and molecular biology method(18 S rDNA method which was adopted by the multiple mequence alignment with NCBI) were combined together to characterize the species of these two fungi. The results showed that these two fungi belonged to Purpureocillium sp. and Penicillium sp. respectively. To separate and purify their secondary metabolites, a variety of biological and chemical methods were performed. Macroporous resin chromatography, different gel chromatographies, silica gel plate chromatography and semipreparative high performance liquid chromatography(HPLC) 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 twenty eight small molecular compounds isolated and identified totally, belonging to aromatic amine, alkaloids, and diketopiperazines, and no new compound was found. These were lumichrome, 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid,β-carboline, cyclo(isoleucine-proline), cyclo(leucine-hydroxyproline), cyclo(tryptophan-proline), cyclo(leucine-valine), cyclo(phenylalanine-proline), cyclo(isoleucine-alanine), cyclo(tryptophan-leucine), phenylethylamine, N-acetylphenethylamine, indoleethylamine, N-methyltryptamine, N-(4-hydroxy)butylidine acylphenylamine, p-hydroxyphenylethylamine, 5,7,4-trihydroxyisoflavone, 7,4-dihydroxyisoflavone, 5,7,3,4-trihydroxyisoflavone, isophthalate ester, 2,4-N,N-dimethyl-lumichrome, 3,4-dihydro-3-methyl-β-carboline-1-ketone, tryptophane, 2-hydroxy-3-indolepropionic acid, cyclo(valine-proline), cyclo(leucine-alanine), cyclo(phenylalanine-5-methyl-3,4-dehydroproline) and cyclo(tryptophan-asparagine), respectively. The study will provide useful information for utilization and analysis of Styela resources. Meanwhile, further analysis of bioactivity of separated and purified compounds could provide positive advice in food and medicine industries. Making full use of abundant Chinese ascidians sources and their epiphytic microbial resources, exploring structural diversity of the secondary metabolites to find novel chemical substances that may be contained in natural products, and developing new antibacterial, antiviral and anti-tumor drugs with China’s independent intellectual property rights are of great theoretical significance and potential application value.