Halogenated Terpenes and a C-15-Acetogenin from the Marine Red Alga Laurencia saitoi

Seven parguerane diterpenes: 15-bromo-2,7,19-triacetoxyparguer-9(11)-en-16-ol (1), 15-bromo-2,7,16,19-tetraacetoxyparguer-9(11)-ene (2), 15-bromo-2,19-diacetoxyparguer-9(11)-en-7,16-diol (3), 15-bromo-2,16,19-triacetoxyparguer-9(11)-en-7-ol (4), 15bromo-2,16-diacetoxyparguer-9(11)-en-7-ol (5), 15-bromoparguer-9(11)-en-16-ol (6), 15-bromoparguer-7-en-16-ol (7), two polyether triterpenes: thyrsiferol (8) and thyrsiferyl 23-acetate (9), and one C15-acetogenin, neolaurallene (10), were isolated from a sample of marine red alga Laurencia saitoi collected off the coast of Yantai. Their structures were established by detailed NMR spectroscopic analysis and comparison with literature data.

Benzaldehyde derivatives from Eurotium rubrum, an endophytic fungus derived from the mangrove plant Hibiscus tiliaceus

Four new (1-4) and seven known (5-11) benzaldehyde derivatives were characterized from the liquid fermentation cultures of Eurotium rubrum, an endophytic fungus that was isolated from the inner tissue of stems of the mangrove plant Hibiscus tiliaceus. The structures of these compounds were determined by extensive analysis of their spectroscopic data. Among these metabolites, compound 1, which was named as eurotirumin, possesses a new carbon skeleton with a cyclopentabenzopyran ring system.

Natural Anthraquinone Derivatives from a Marine Mangrove Plant-Derived Endophytic Fungus Eurotium rubrum: Structural Elucidation and DPPH Radical Scavenging Activity

There is considerable interest in the isolation of potent radical scavenging compounds from natural resources to treat diseases involving oxidative stress. In this report, four new fungal metabolites including one new bisdihydroanthracenone derivative (1, eurorubrin), two new seco-anthraquinone derivatives [3, 2-O-methyl-9-dehydroxyeurotinone and 4, 2-O-methyl4-O-(alpha-D-ribofuranosyl)-9-dehydroxyeurotinone], and one new anthraquinone glycoside [6,3-O-(alpha-D-ribofuranosyl)questin], were isolated and identified from Eurotium rubrum, an endophytic fungal strain that was isolated from the inner tissue of the stem of the marine mangrove plant Hibiscus tiliaceus. In addition, three known compounds including asperflavin (2), 2-O-methyleurotinone (5), and questin (7) were also isolated and identified. Their structures were elucidated on the basis of spectroscopic analysis. All of the isolated compounds were evaluated for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity.

Chemical constituents of marine medicinal mangrove plant Sonneratia caseolaris

Twenty-four compounds including eight steroids (1-8), nine triterpenoids (9-16, 24), three flavonoids (20-22), and four benzenecarboxylic derivatives (17-19, 23) were isolated and identified from stems and twigs of medicinal mangrove plant Sonneratia caseolaris. The structures of the isolated compounds were determined by extensive analysis of their spectroscopic data. Among these metabolites, compounds 1, 4-20 and 22-24 were isolated and identified for the first time from S. caseolaris. In the in vitro cytotoxic assay against SMMC-7721 human hepatoma cells, compound 21 (3',4',5,7-tetrahydroxyflavone) exhibited significant activity with IC50 2.8 mu g/mL, while oleanolic acid (14), 3,3'-di-O-methyl ether ellagic acid (18), and 3,3',4-O-tri-O-methyl ether ellagic acid (19) showed weak activity. None of these compounds displayed significant antibacterial activites.

Hymeniacidon perleve associated bioactive bacterium Pseudomonas sp NJ6-3-1

Among marine bacteria isolated from the cytotoxic sponge Hymeniacidon perleve, one strain NJ6-3-1 classified as Pseudomonas sp. showed both cytotoxic and antimicrobial activities. Fatty acid analysis indicated that the bacterial strain consists mainly of C16:1, C16:0, C18:1, C18:0, C15:0, C14:0. One unusual 9,10-cyclopropane-C17:0 fatty acid and C26:0 also constitute major components, as well as the existence of squalene, the precursor of triterpenoids. The major metabolites in the culture broth were identified as alkaloids, including diketopiperazines and indole compounds, namely 3,6-diisopropylpiperazine-2,5-dione, 3-benzyl-3-isopropylpiperazine-2,5-dione, 3,6-bis-(2-methylpropyl)-piperazine-2,5-dione, indole-3-carboxaldehyde, indole-3-carboxylic acid methyl ester, indole-3-ethanol, and quinazoline-2,4-dione.

Molecular biotechnology of marine algae in China

Molecular biotechnology of marine algae is referred to as the biotechnology on the identification, modification, production and utilization of marine algal molecules. It involves not only the manipulation of macromolecules such as DNA, RNA and proteins, but also deals with low molecular weight compounds such as secondary metabolites. In the last decade, molecular systematic researches to investigate the relationship and to examine the evolutionary divergence among Chinese marine algae have been carried out by Chinese scientists. For example, RAPD has been widely used in several laboratories to elucidate genetic variations of the reds, such as Porphyra, Gracilaria, Grateloupia and the greens such as Ulva and Enteromorpha. Some important data have been obtained. The study on molecular genetic markers for strain improvement is now in progress. In 1990s, genetic engineering of economic seaweeds such as Laminaria, Undaria, Porphyra, Gracilaria and Grateloupia has been studied in China. For Laminaria japonica, the successfully cultivated kelp in China, a model transformation system has been set up based on the application of plant genetic techniques and knowledge of the algal life history. Progress has been made recently in incorporating a vaccine gene into kelp genome. Evidence has been provided showing the expression of gene products as detectable vaccines. In the present paper, the progress of molecular biotechnological studies of marine algae in China, especially researches on elucidating and manipulating nucleic acids of marine algae, are reviewed.

Integrated cultivation of the red alga Kappaphycus alvarezii and the pearl oyster Pinctada martensi

The effect of simultaneously cultivating the pearl oyster Pinctada martensi and the red alga Kappaphycus alvarezii on growth rates of both species was investigated in laboratory and field studies conducted from December 1993 to June 1995. The two study sites were in subtidal areas 100 km apart off the east coast of Hainan Island, China. Pearl oysters were cultivated in the center of an algal farm and red alga was cultivated in the center of the pearl oyster farm. These field experiments showed higher growth rates of both P. martensi and K. alvarezii in a co-culture system than in a monospecies culture system. Laboratory studies showed that the algae removed nitrogenous wastes released by pearl oysters. Algae treated with pearl oyster wastes grew much faster than those without oyster wastes. Algae treated with the seawater to which NH4Cl, NaNO3 and NaNO2 were added grew at the same rate as those treated with natural seawater containing oyster nitrogenous wastes, suggesting that enhanced growth of algae in the co-culture system was largely due to nitrogenous metabolites of the pearl oysters. In the co-culture, growth of pearl oysters was positively influenced by the presence of rapidly growing algae but when seawater temperature decreased below 20 degrees C, the algae grew slowly and there was no measurable benefit of mixed culture to either algae or pearl oyster.

Antifeedant, antibacterial, and antilarval compounds from the South China Sea seagrass Enhalus acoroides

We investigated chemical constituents and the antifeedant, antibacterial, and antilarval activities of EtOH (ethanol) extracts of the South China Sea seagrass Enhalus acoroides. Eleven pure compounds including four flavonoids and five steroids were obtained. Among these compounds, three flavonoids were antifeedant against second-instar larvae of Spodoptera litura, two flavonoids had antibacterial activity towards several marine bacteria, and one flavonoid showed strong antilarval activity against Bugula neritina larvae. This is the first description of isolation and bioactivity of secondary metabolites from E. acoroides.

New sphingolipids with a previously unreported 9-Methyl-C-20-sphingosine moiety from a marine algous endophytic fungus Aspergillus niger EN-13

Asperamides A (1) and B (2), a sphingolipid and their corresponding glycosphingolipid possessing a hitherto unreported 9-methyl-C-20-sphingosine moiety, were characterized from the culture extract of Aspergillus niger EN-13, an endophytic fungus isolated from marine brown alga Colpomenia sinuosa. The structures were elucidated by spectroscopic and chemical methods as (2S,2'R,3R,3'E,4E,8E)-N-(2'-hydroxy-3'-hexadecenoyl)-9-methyl-4,8-icosadien-1,3-diol (1) and 1-O-beta-D-glucopyranosyl-(2S,2'R,3R,3'E,4E,8E)-N-(2'-hydroxy-3'-hexadecenoyl)-9-methyl-4,8-icosadien-1,3-diol (2). In the antifungal assay, asperamide A (1) displayed moderate activity against Candida albicans.

Two anthraquinone compounds from a marine actinomycete isolate M097 isolated from Jiaozhou Bay

Two anthraquinone compounds were isolated from the culture broth of a marine actinomycete isolate M097. The structures were elucidated as Aloesaponarin II and 1,6-dihydroxy-8-hydroxymethyl-anthraquinone by detailed interpretation of their spectra. It is the first time that the latter has ever been reported as a secondary metabolite from a wild-type strain. The results showed that the actinomycete isolate M097 could be a promising material for studying the biosynthetic pathway of polyketides and the production of novel recombinant polyketides.

Investigations of the characteristics and mode of action of an algalytic bacterium isolated from Tai Lake

An algalytic bacterium provisionally designated as TL1 was isolated from Tai Lake, a large freshwater lake in the Yangtze Delta plain on the border of the Jiangsu and Zhejiang provinces and close to Wuxi city in the People's Republic of China. Strain TL1 was identified as Achromobacter sp. based on its biophysical and biochemical properties and the analysis of its 16S rRNA sequence. Microcystis aeruginosa, which is the most common toxic cyanobacterium in eutrophic freshwater, could be decomposed by strain TL1. The results showed that after inoculation with the algalytic bacterium, the content of chlorophyll-a, maximum PSII quantum yield, and maximum electron transport rates of the alga decreased sharply. At first, the algal cells enhanced the activities of some antioxidative enzymes, but subsequently, the activities of antioxidative enzymes fell sharply once damage of the algal cells was achieved. The filtrate from strain TL1 culture suspension, after autoclaving and treatments with proteinase K, strongly inhibited algal growth, indicating that the lytic metabolites were extracellular and thermostable, not a protein.

Triterpenoids from the mangrove plant Hibiscus tiliaceus

Three new triterpenoids with the rarely occurring nigrum skeleton, namely (20E)-22-hydroxynigrum-20-en-3-one (1), 21 beta-hydroxynigrum-22(29)-en-3-one (2), and 21 alpha-hydroxynigrum-22(29)-en-3-one (3), were isolated from the mangrove plant Hibiscus tiliaceus. Additionally, five known triterpenoids including friedelin (4),12-oleanen-3 beta-ol (5), 3 beta-hydroxy-12-oleanen-28-oic acid (6),20(29)-lupen-3 beta,28-diol (7). and cucurbita-5,23-dien-3 beta,25-diol (8) were also isolated and identified. The latter structures were elucidated by a detailed NMR and MS analyses, as well as by comparison with reported literature data.

海洋红藻多管藻和松节藻的化学成分及抗氧化活性研究

海洋生物具有产生丰富多样的次生代谢产物的能力，其中红藻门松节藻科海藻卤代次生代谢产物以其结构新颖、生物活性独特引起了天然产物化学家的重视。 本论文对海洋红藻多管藻和松节藻进行了化学成分研究，综合利用各种色谱学方法 (硅胶柱层析、反相硅胶柱层析、凝胶Sephadex LH-20柱层析、半制备高效液相色谱以及重结晶等) 和现代波谱学技术 (IR、UV、EI-MS、FAB-MS、HR-ESI-MS、CD、1H-NMR、13C-NMR、DEPT、1H-1H COSY、HSQC、HMBC等)，共分离鉴定了100个化合物，发现25个新化合物。 从多管藻中分离鉴定38个化合物 (24个溴酚化合物)，其中7个新化合物 (均为溴酚化合物)，包括1个菲并呋喃结构溴酚 (P1), 2个二氢菲结构溴酚 (P2, P3)，1个含 5,7-dihydrodibenzo[c,e]oxepine 结构溴酚 (P4)和3个简单溴酚 (P5, P6, P7)。P1 (urceolatin) 属首例报道的具有菲并呋喃结构的天然产物，从该种中分离的化合物P12 和 P13 可能是其生源合成的前体。P2和P3为第二例报道的具有二氢菲结构的溴酚化合物。 从松节藻中分离并鉴定了62 个化合物，其中18 个为溴酚类新化合物，44 个为已知化合物。化合物具有多变的取代基团，包括2 个脲基吡咯烷酮溴酚化合物 (R1, R2), 4 个γ-脲基丁酸溴酚化合物 (R3-R6)，5 个酰胺溴酚化合物 (R7, R8, R9, R13, R14)，1 个溴酚砜化合物 (R12), 1 个Xanthene 溴酚化合物 (R10)和5 个简单溴酚化合物 (R11, R15, R16, R17, R18)。R1、R2 是首例报道的含有脲基吡咯烷酮片段的天然产物，R10 为首次报道的溴代Xanthene 类天然产物。 对分离到的化合物进行了清除DPPH 和ABTS两种自由基活性的筛选。结果发现溴酚类天然产物具有显著的DPPH自由基清除活性，其中R3 的IC50 仅为3.3 μM, 其活性强度约为阳性对照BHT (IC50 为82.1 μM) 的24倍。另外，溴酚类天然产物对ABTS自由基有较强的清除活性，R2 的TEAC（Trolox efficency activity capacity）值为5.2 mM，约为阳性对照 (ascorbic acid, 1.02 mM) 的 5 倍。初步的构效关系研究发现，稠环分子、多羟基和邻位甲氧基等结构特点能有效增强DPPH 自由基清除活性；特殊取代基如脲基、吡咯烷酮等含有氮原子的基团，能有效增强ABTS 自由基清除活性，多羟基、溴代等结构特点也使其活性有所增强。 本研究结果丰富了海藻卤代化合物的结构类型，为多管藻和松节藻的合理利用提供了一定的科学依据。

从胶州湾海洋放线菌中发现的新型星形孢菌素等7种新结构化合物

本课题组自1999年以来，将培养条件优化、生物活性跟踪及化学跟踪技术应用到胶州湾海洋放线菌的次级代谢产物研究中，发现了一批具有生物活性的新结构化合物。 本研究从青岛胶州湾分离出各类海洋微生物423株，包括海洋放线菌287株，海洋细菌116株，海洋真菌20株，对胶州湾海洋微生物的资源和分布规律有了基本的认识。对其中海洋放线菌的次级代谢产物进行了活性和化学筛选，获得了它们对八种病原微生物的抑制活性数据。发现胶州湾海洋放线菌对至少一种受试微生物具有中等以上拮抗能力的比例为50%。 从三株海洋放线菌的发酵粗提物中鉴定出7个新结构化合物和31个已知化合物。具体是：①从菌株M518中鉴定出21个化合物，包括3个新结构天然化合物：N-Foramidostaurosporine，5,7-Dihydroxy-5,6,7,8-tetrahydro-1H-azocin-2-one，Selina-4(14),7(11)-diene-8,9-diol和1个新结构衍生物Trimer 3。其中化合物5,7-Dihydroxy-5,6,7,8-tetrahydro-1H-azocin-2-one为新骨架类型的天然化合物。② 从菌株M491 中鉴定出10个化合物，包括3个新结构化合物10α-14-Dihydroxyamorph-4-en-3-one，10α,11-Dihydroxyamorph-4-ene，5α,10α,11-Triydroxyamorphan-3-one； 并首次从微生物中分离得到已知化合物10α-Hydroxyamorph-4-en-3-one；③从菌株M311中鉴定出7个已知化合物。 发现天然化合物5,7-Dihydroxy-5,6,7,8-tetrahydro-1H-azocin-2-one很容易发生重排反应，分离出了两个重排反应产物，并得出该化合物可能的重排反应过程及三聚体形成的方式。 生物活性实验结果表明新型星形孢菌素N-Formamidostaurosporine具有很强的抗肿瘤活性：对于37株人类肿瘤细胞的平均IC50，IC70和IC90分别为0.016µg/ml，0.171µg/ml和2.352µg/ml。对37株肿瘤细胞株的抗肿瘤选择性为27%。还首次发现Staurosporine类化合物具有抑制微藻生长的活性。 对产生新化合物的菌株M518 和M491 进行了形态、生理生化及分子鉴定，提交两株菌的16S rDNA 序列到GenBank（DQ184649 和DQ184648）。结合形态、生理生化特征及系统 发育树分析表明：菌株M518 属于链霉菌属，并可能属于该属中的粉红孢类群；菌株M491属于链霉菌属，并可能属于该属中的青色类群。

两种海洋生物次生代谢产物的研究

本论文对多指软珊瑚（Sinularia numerosa）和鼠尾藻（Sargassum thunbergii）两种海洋生物的化学成分进行了研究，对从其中分离得到的大部分化合物进行了初步的生物活性筛选。 多指软珊瑚采自海南三亚，样品经冷冻干燥并粉碎后用丙酮浸泡提取得到提取物；鼠尾藻采自山东青岛沿岸，样品干燥后用乙醇提取，将提取物先后用石油醚、乙酸乙酯和正丁醇萃取，得到石油醚相、乙酸乙酯相和正丁醇相提取物。 采用常规的硅胶柱层析、反相硅胶柱层析、凝胶Sephadex LH-20柱层析、制备薄层层析、重结晶以及半制备HPLC等手段分离得到38个化合物。利用各种现代波谱技术（IR、UV、MS、HR-MS、1D-NMR、2D-NMR等），确定了其中27个化合物的结构。其中，从多指软珊瑚中得到30个化合物，鉴定了其中得19个化合物，包括2个新的西松烷二萜类化合物（化合物SN1，命名为Numerolides A；化合物SN2，命名为Numerolides B）、1个新的甾醇类化合物（化合物SN3，命名为Gorgost-3β, 7β-diol）以及15个首次从该种中报道的化合物。从鼠尾藻中分离鉴定了8个化合物，包括植醇、岩藻甾醇、2个单萜类化合物、1个环戊烯酮类化合物及3个不饱和长链化合物，这些化合物皆为首次从该种中报道。对大部分化合物作了针对人肝癌细胞株SMMC-7721体外细胞毒活性和抗菌活性筛选，所测样品均未显示针对SMMC-7721的细胞毒活性，而化合物SN15具有中等强度的抑制金黄色葡萄球菌和大肠杆菌的活性，化合物SN19有促进金黄色葡萄球菌生长的活性。