微生物来源的Aurora—B激酶抑制剂的分离、结构鉴定和抗肿瘤活性研究

目的从微生物代谢产物中分离和纯化Aurora—B激酶抑制剂并检测其抗肿瘤活性。方法以野生型酵母菌Y300和ipZl-321温度敏感型突变株为模式菌跟踪活性组分;从阳性放线菌107A-01038发酵产物中分离纯化活性化合物并进行结构鉴定;体外酶学实验验证阳性化合物对Aurora—B激酶的抑制活性;MTT法检测阳性化合物对肿瘤细胞增殖的抑制活性;AnnexinV-FITC／PI双染法检测活性化合物对肿瘤细胞早期凋亡的诱导作用。结果从阳性放线菌107A-01038发酵产物中得到活性化合物酒渣碱甲酯（flazinmethylester）,酒渣碱甲酯对咖11．321突变株具有特异性抑制作用,其在野生型酵母菌株Y300和突变株ipl1—321的Ic50分别为48μmol／L和24μmol／L;体外酶学实验证实其对Aurora—B激酶具有抑制作用,其IC50为10μmol／L（ATP=50gmol／L）：酒渣碱甲酯对HepG2、A549、Hela肿瘤细胞均具有杀伤活性,IC50分别为13、11和10μmol／L。并能够诱导Hela细胞发生早期凋亡。结论得到-个微生物来源的具有抗肿瘤活性的Aurora—B激酶抑制剂-酒渣碱甲酯。     

具有血管内皮细胞生长因子受体-2酪氨酸激酶抑制作用的链霉菌次生代谢产物2754R的研究

目的分离鉴定链霉菌I06A-02754发酵液中具血管内皮生长因子受体-2酪氨酸激酶(VEGFR2-CD)抑制活性的强极性次生代谢产物。方法采用大孔吸附树脂、阴离子交换树脂、MPLC、HPLC等分离手段对次生代谢产物进行分离纯化;通过UV、IR、HR-ESI质谱、1D-NMR和2D-NMR对其结构进行鉴定,以ELISA法检测其次生代谢产物对VEGFR2-CD的抑制活性;以MTT法检测化合物对肿瘤细胞的抑制活性。结果从发酵液的水溶性部分分离得到一个极性较大的胡桃霉素类次生代谢产物——2754R;其化学结构与胡桃霉素D一致,对VEGFR2-CD表现出一定的抑制活性;MTT实验显示化合物2754R对HepG2细胞、MCF-7细胞和BEL-7402细胞没有明显的抑制活性(IC50>10μmol/L)。结论化合物2754R是具有VEGFR2-CD活性的胡桃霉素类次生代谢产物。     

丰肉结海绵相关链霉菌LS298活性代谢产物研究

目的对我国南海丰肉结海绵相关链霉菌LS298的活性代谢产物进行研究。方法采用硅胶柱、凝胶柱及HPLC等色谱方法对LS298发酵产物进行分离纯化;通过核磁共振、质谱等波谱分析手段对分离得到的化合物进行结构鉴定;以滤纸片扩散法及MTT法分别检测其抗菌和抗肿瘤活性。结果分离得到12个化合物。分别为尿嘧啶核苷、2'-脱氧尿嘧啶核苷、邻苯二甲酸正丁二酯、邻苯二甲酸二(2-乙基己)酯、3-甲酰胺-吲哚、环(脯氨酸-缬氨酸)、环(脯氨酸-苯丙氨酸)、环(脯氨酸-酪氨酸)、环(脯氨酸-亮氨酸)、meleagrin、5-hydroxyectoine和echinomycin。其中,3-甲酰胺-吲哚系首次从微生物中分离得到;meleagrin系首次从放线菌中分离得到。初步的药理研究表明,化合物echinomycin不仅具有较强的抗菌活性,亦具有很强的体外抗肿瘤活性。结论化合物echinomycin是链霉菌LS298的主要抗菌和抗肿瘤活性成分之一。     

以结核分枝杆菌H_(37)Rv莽草酸脱氢酶为靶点的高通量筛选模型的建立及应用

目的建立以结核分枝杆菌莽草酸脱氢酶为靶点的新型抗结核药物高通量筛选模型;用此模型筛选莽草酸脱氢酶抑制剂;进一步评价化合物对莽草酸脱氢酶活性的影响。方法表达并纯化结核分枝杆菌H37Rv莽草酸脱氢酶;利用还原型辅酶II(NADPH)在溶液中的光吸收,测定酶的活性,构建了该酶抑制剂的高通量筛选模型;用Z′因子法评价该模型的可靠性,并对5万余个化合物进行筛选;测定了各抑制剂的IC50并对抑制剂6186050的酶抑制动力学进行了研究;用菌液稀释法评价了抑制剂对某些临床分离菌株包括耐药菌株的影响。结果得到了重组莽草酸脱氢酶;测得比活力为20987U/mg,所建的莽草酸脱氢酶高通量筛选模型Z′因子为0.76,符合高通量筛选的要求;对5万余个化合物进行筛选得到9个抑制率较高的化合物;抑制剂6186050为竞争性可逆抑制剂;抑制剂6230384和6186050对海分枝杆菌的最低抑菌浓度(MIC)都是32μg/ml。结论建立了稳定性好、灵敏度较高的结核分枝杆菌莽草酸脱氢酶抑制剂高通量药物筛选模型,应用该模型筛选得到的抑制剂可能具有抑菌活性。     

刺五加内生放线菌Streptomyces sp. CWJ-256次生代谢产物研究

目的研究刺五加植物内生放线菌CWJ-256次级代谢产物及其生物活性。方法采用16S rRNA基因同源进化分析对菌株CWJ-256进行初步分类鉴定,采用抗菌活性追踪方式,对CWJ-256发酵液通过硅胶柱层析、凝胶柱层析、薄层色谱显色以及高效液相色谱等方法对目标化合物分离纯化,利用核磁共振波谱法和质谱法鉴定化合物的结构。采用MTT法测试其抗肿瘤细胞增殖活性。基于Gluc报告系统对化合物3的体外抗流感病毒药效学进行初步评价。结果菌种鉴定显示Streptomycessp.CWJ-256为生黑孢链霉菌Streptomyces melanosporofaciens;从该菌株的发酵代谢产物中分离得到3个活性化合物,经结构鉴定为efomycin G(化合物1)、11,11'-O,O-dimethylelaiophylin(化合物2)、elaiophylin(化合物3);抗肿瘤细胞增殖活性评价显示,化合物1和3对体外培养的人乳腺癌细胞MDA-MB-231具有增殖抑制作用(IC50分别为4.385和2.118μmol/L)。体外抗流感病毒药效学评价显示,化合物3对流感病毒A/WSN/33(H1N1)具有一定抑制效果。结论刺五加内生放线菌Streptomycessp.CWJ-256可产生多个洋橄榄叶素类次生代谢产物,显示出抗细胞增殖、抗病毒等多种活性。     

丰肉结海绵相关真菌产黄青霉HLS111菌株活性代谢产物研究

目的对我国南海水域丰肉结海绵相关真菌产黄青霉菌HLS111的活性代谢产物进行研究。方法采用HPLC-DAD技术与活性筛选相结合的方法,通过硅胶柱、凝胶柱及HPLC等色谱方法对HLS111发酵产物进行分离纯化;通过核磁共振、质谱等波谱分析手段对分离得到的化合物进行结构鉴定;并对单体化合物进行抗肿瘤、抗HIV、抗炎活性测定。结果分离得到12个化合物。其中2个黑麦酮酸类化合物:黑麦酮酸F(1)和D(2);4个生物碱类化合物:环(L-色氨酸-L-苯丙氨酸)(3)、citreoindole(4)、meleagrin(5)、脑苷脂B(6);4个甾体类化合物:麦角甾醇(7)、(22E,24R)-5α,8α-过氧化麦角甾-6,22-烯-3β-醇(8)、globosterol(9)、β-谷甾醇(10);1个三萜类化合物:24-亚甲基环木菠萝烷醇-反式阿魏酸(11);1个蒽醌类化合物:大黄素(12)。对化合物4的氢谱、碳谱数据进行了归属。初步的药理研究表明,黑麦酮酸类化合物表现出较强的抗肿瘤活性,化合物citreoindole表现出一定的抗HIV及抗炎活性。结论海绵相关真菌产黄青霉HLS111菌株体现了次生代谢产物化学结构的多样性;黑麦酮酸类化合物为产黄青霉HLS111的主要抗肿瘤活性成分。     

罗布泊来源胀果甘草根际枯草芽孢杆菌GA21次生代谢产物结构与活性初步研究

目的分离鉴定枯草芽孢杆菌GA21发酵液中次生代谢产物并对其活性进行初步研究。方法采用TLC、HPLC等分离手段对次生代谢产物进行分离;通过HR-ESI质谱、1D-NMR和2D-NMR对其结构进行鉴定;以琼脂平板法检测其次生代谢产物对金黄色葡萄球菌、大肠杆菌、稻瘟菌、白色念珠菌的拮抗活性。结果分离得到两个异香豆素类次生代谢产物:GA21-20和GA21-26;其中GA21-20的化学结构与Amicoumacin B一致,但无抗细菌及真菌活性;GA21-26有抗真菌活性,但无抗细菌活性,分子量为435.20890,分子式为C21H29O7N3,是一新的3,4-二氢异香豆素类抗生素。GA21-26在2.5μg/纸片对白色念珠菌即可显示抑制活性,在80μg/纸片对水稻稻瘟菌显示抑制活性,表明GA21-26在拮抗医学条件致病真菌方面有潜在用途。结论枯草芽孢杆菌GA21能产生一系列3,4-二氢异香豆素类抗生素,其次级代谢产物值得进一步研究。     

天然海水高盐条件对海绵相关细菌抗菌活性及次级代谢产物的影响

海洋微生物生活在高盐、高压、低温、低光照、寡营养、弱碱性的海洋环境中,因而形成了独特的生理特征和代谢机制,可产生与陆生微生物结构与活性迥然不同的次级代谢产物,如生物碱、萜类、环肽类和聚酮类等[1-2],成为海洋药物的重要来源之一。从海洋放线菌中分离得到的salinisporamide A 已经完成了 I 期临床研究,用于治疗多发性骨髓瘤[3]。以从海洋真菌 Aspergillus sp. CNC-139中获得的化合物为模板合成的 plinabulin（NPI-2358）已经进入II 期临床研究,用于治疗非小细胞肺癌[4]。来自于卡纳里水域深海沉积物中的链霉菌 NTK937产生的 caboxamycin是一种新型的苯并噁唑抗生素,显示出较强的抗菌活性,对革兰阳性菌枯草芽孢杆菌（Bacillus subtilis）IC50仅为8μmol/L[5]。海洋是一个特殊的生态系统,据文献报道,通过模拟海洋环境的拟生态培养,可以诱导海洋真菌产生新的活性代谢产物[6-9]。同时有研究表明,高盐条件造成的极端环境（如高渗透压和营养剥夺等）可以激活生物体内的沉默基因或次级代谢产物合成酶,进而使海洋等来源的耐盐真菌产生新的活性化合物[10-11]。另据文献报道,培养基盐度对海洋真菌的活性物质具有显著影响,其生物活性和活性物质的种类和产量可能有较大的差别[12-13]。但是,应用天然海水培养基探讨海洋来源细菌的抗菌活性、次级代谢产物的 HPLC化学指纹的报道较少,本实验室曾研究报道天然海水对海绵相关细菌的抗菌活性有影响[14]。另有文献对细菌 HPLC 化学指纹研究进行了报道[15-17],此外,有文献报道天然海水可影响细菌的生长[18-19]。本文以海绵相关细菌为主要研究对象,初步开展了相关探索,以期获得具有良好抗菌活性且次级代谢产物丰富的菌株。     

链霉菌CPCC 200510产生的大黄酚及其类似物的鉴定

目的链霉菌具有丰富次级代谢产物合成能力,对链霉菌CPCC 200510产生的色素类次级代谢产物进行研究。方法对该菌株的次级代谢产物进行乙酸乙酯提取、ODS色谱柱分离、半制备HPLC分离纯化、NMR结构解析和生物活性检测。结果得到6个黄色化合物,分别为1,8-二羟基-3-甲基蒽醌(大黄酚,1)、2-乙酰-1,8-二羟基-3-甲基蒽醌(2)、2-乙酰-1,8-二羟基-3-羧甲基蒽醌(3)、1,2,8-三羟基-3-甲基蒽醌(4)、1-(7-乙酰-8-羟基苯并[de]色烯-2-基)-2-丙酮(5)和1-(8-羟基-2-甲基苯并[de]色烯-7-基)乙酮(6),为一组生物合成相关的芳香聚酮类化合物。其中,1～3为链霉菌CPCC 200510产生的主要组分。生物活性测定表明,1～3具有抗肿瘤细胞或抗病毒活性。结论链霉菌CPCC200510有望作为大黄酚类化合物的微生物产生菌。     

链霉菌CPCC 203577产生的napyradiomycins的分离与鉴定

目的对一株具有抗菌活性的链霉菌CPCC 203577产生的次级代谢产物进行系统研究。方法采用ISP2琼脂平板发酵培养链霉菌CPCC 203577,乙酸乙酯提取发酵培养物,获得粗提物;粗提物经反相色谱柱、凝胶色谱柱、制备型TLC和半制备HPLC等分离纯化获得目标化合物纯品;MS和NMR等确定化合物结构;琼脂稀释法测定抗菌活性。结果从链霉菌CPCC 203577中分离鉴定了含萘醌并吡喃母核的3个已知化合物,3-chloro-6,8-dihydroxy-8-α-lapachone(1)、16-dechloro-16-hydroxynapyradiomycin C2(2)和napyradiomycin A2(3)。化合物1具有较强的抗革兰氏阳性细菌活性,最低抑菌浓度(MIC)值为4~8μg/ml;化合物2和3没有抗菌活性。结论链霉菌CPCC 203577具有丰富的次级代谢产物合成能力,产生napyradiomycins类化合物。     

以蛋白激酶A为靶点的抗结核药物高通量筛选模型的建立和应用

目的 建立以蛋白激酶 A 为靶点的抗结核药物高通量筛选模型,应用该模型筛选具有特异性酶活抑制活性的微生物发酵液粗提物样品。 方法 以结核分枝杆菌 H37Rv 基因组 DNA 为模板,扩增目的基因片段 pknA,构建表达载体 pET43.1a-pknA,在大肠杆菌中克隆表达了重组 MTB PknA 蛋白;采用三步级联的反应方法,利用还原型烟酰胺腺嘌呤二核苷酸到氧化型烟酰胺腺嘌呤二核苷酸这一反应最大吸光值波长的变化,建立和优化蛋白激酶 A 抑制剂高通量药物筛选模型。 结果 成功构建了表达载体 pET43.1a-pknA;建立了稳定灵敏,可用于靶向结核分枝杆菌蛋白激酶 A 的抗结核药物高通量筛选模型;利用该模型对4000个微生物发酵液粗提物样品进行筛选,最终得到21个抑制蛋白激酶 A 活性的阳性样品,阳性率0.53%;以耻垢分枝杆菌和海分枝杆菌为检定菌,平板纸片法检测阳性样品的抗分枝杆菌活性,然后对阳性样品的细胞毒性和酶活抑制特异性进行评价后,最终得到8个阳性样品,其中 I10AA-02916、I09AA-02717、I09AB-02729、I08AB-00801这4个阳性样品酶活抑制特异性、抗菌活性均较好,且细胞毒性较低。 结论 建立了高稳定性的以蛋白激酶 A 为靶点的抗结核药物高通量筛选模型,应用该模型所得到的发酵液阳性样品值得进一步研究。     

The synergistic immunosuppressive potential of cyclosporin metabolite combinations.

Out of the 29 cyclosporin (CS) metabolites defined so far seven representatives were isolated from the bile of liver grafted patients, purified by HPLC and characterized by FAB-MS and/or 1H-NMR. These were used to determine the growth inhibitory effects on concanavalin A stimulated rat lymphocytes (LN). Metabolites diluted in culture medium at concentrations re-checked by HPLC at the respective assay time were added and proliferation determined by [3H]-thymidine incorporation after 48 h. A 50% growth inhibition of LN by single metabolites (AM) was achieved at the following concentrations (mg/l): CS: 0.023; primary metabolites AM1: 0.11; AM1c: 0.65; AM9: 1.05; secondary metabolites AM19: 1.02; AM4N9: 1.02; H355: 1.85; AM1A: 4.5. Although all metabolites were immunosuppressive at higher concentrations in vitro on a single metabolite level, only AM1 with 20% of the activity of native CS seemed to play a role in vivo. However, when we tested the antiproliferative effects of double or triple metabolite combinations, we found a strong synergism not only of primary metabolites, but even with combinations including secondary metabolites. The concentration of the participating metabolites necessary to decrease LN growth by 50% was far below the trough levels observed in vivo. Finally, to mimic to some extent the in vivo situation we determined the interaction of native CS with single metabolites or double combinations. In contrast to the clear synergism in the absence of CS the combinations of metabolites with native CS resulted in an additive growth inhibition. These results indicate an immunosuppressive potential of all metabolites tested and a clear synergism of metabolites in the absence of CS. Although up to double metabolite combinations did only additively enhance CS induced immunosuppression, the combination of 29 metabolites occurring in vivo might have significant immunosuppressive effects in situations where CS levels drop below active concentrations.     

Lignoren, a new sesquiterpenoid metabolite from Trichoderma lignorum HKI 0257

The new compound lignoren (1) was isolated from Trichoderma lignorum HKI 0257 by chromatographic methods. This metabolite has a santalane-like structure, which was elucidated by mass spectrometric and NMR spectroscopic investigations. Lignoren (1) shows a moderate narrow-spectrum of antibacterial and antifungal activity.     

Esters of 3,4-dihydroxybenzoic acid, highly effective inhibitors of the sn-glycerol-3-phosphate oxidase of Trypanosoma brucei brucei

Alkyl esters of 3,4-dihydroxybenzoic acid are inhibitors of the sn-glycerol-3-phosphate oxidase system of Trypanosoma brucei brucei in vitro and have significant trypanocidal activity in vivo when combined with glycerol. While the parent acid has little inhibitory activity in vitro, the esters are highly active with activity increasing as the chain length of the esterifying alcohol increases. The n-dodecyl ester was more than 400 times as active as salicylhydroxamic acid and 15 times more active than the corresponding p-n-alkyloxybenzhydroxamic acid, one of the most active sn-glycerol-3-phosphate oxidase inhibitors previously reported. When combined with glycerol (to block an alternative pathway of glycolysis) and tested in vitro against intact parasites, this ester was 100 times more effective than salicylhydroxamic acid and 10 times more effective than p-n-dodecyloxybenzhydroxamic acid. It was also active against T. b. brucei in mice when combined with glycerol whereas the latter compound was not. Esters of 3,4,5-trihydroxybenzoic acid (gallic acid) were also highly active while those of 2,3-dihydroxybenzoic acid were much less inhibitory and those of 2,5-dihydroxybenzoic acid were inactive. A related compound, 2',4',5'-trihydroxybutyrophenone, was also active as predicted by its structure but was too toxic to be of interest as a drug candidate.     

Immunostimulatory activities of a decapeptide derived from Alcaligenes faecalis FY-3 to crucian carp

A strain was isolated from a soil sample collected from Weihe river in Shaanxi province (108°03'E 34°14'N), which was identified as Alcaligenes faecalis by 16S rRNA analysis. A compound M showing potent immune activity was isolated from secondary metabolites of the strain through bioassay-guided isolation techniques. The structure of the compound M was elucidated using FT-IR, EI-MS, 1H NMR and 13C NMR spectra and identified as cyclo-(L-Pro-Gly)5 which was first time reported as a natural product. We evaluated the immune effects of the cyclo-(L-Pro-Gly)5 on the basis of serum lysozyme activity, bacterial agglutination titre assay, superoxide anion production and phagocytic activity assay, and they were found to be significantly increased by cyclo-(L-Pro-Gly)5. The effects of cyclo-(L-Pro-Gly)5 on immune-related gene expression were further investigated. The outcomes of real-time quantitative polymerase chain reaction (RQ-PCR) proved that the transcribing level of interleukin 6β (IL-6β) and inducible nitric oxide synthase 1β (iNOS-1β) mRNA in the blood leucocytes have been augmented by cyclo-(L-Pro-Gly)5. The challenge experiment showed that crucian carp injected the cyclo-(L-Pro-Gly)5 had significantly (P < 0.05) lower cumulative mortality (13.0%) compared with the control (45.4%) after infection with live Aeromonas hydrophila. These results suggested that cyclo-(L-Pro-Gly)5 is a possible immunostimulant and may strengthen the immune response and protect the heath status of crucian carp against A. hydrophila.     

Mutagenicity of benzo(a)pyrene metabolites generated on the isolated perfused lung following particulate exposure

The isolated perfused rabbit lung (IPL) is being used to study the effects of particulate exposure on the pulmonary metabolism of benzo(a)pyrene (BaP). Pasturealla-free New Zealand white rabbits were treated intraperitoneally with BaP prior to kill. The isolated lungs were then administered either 14C-labeled BaP alone or BaP plus Fe2O3 or fly ash by intratracheal injection. Rates of appearance of BaP metabolites in the perfusing blood were determined. The extent of metabolism, distribution of metabolites, and types of metabolites produced were quantified for various lung tissue types by high-performance liquid chromatography and liquid scintillation spectrometry. Procedures were developed to apply the Salmonella/microsome test in the assay of mutagenicity of lung tissue and blood extracts as an indicator of their biologic activity. With few exceptions, blood extracts from IPL receiving BaP only were not mutagenic. Lung, trachea-bronchi, and macrophage extracts, by contrast, were mutagenic. A part of this activity could be attributed to BaP metabolites rather than to parent compound remaining in extracts. When lungs were exposed to Fe2O3 or to fly ash, only macrophage extracts were consistently mutagenic. This activity was due to significant amounts of unmetabolized BaP.     

Purification and biological evaluation of the metabolites produced by Streptomyces sp. TK-VL_333

An Actinobacterium strain isolated from laterite soils of the Guntur region was identified as Streptomyces sp. TK-VL_333 by 16S rRNA analysis. Cultural, morphological and physiological characteristics of the strain were recorded. The secondary metabolites produced by the strain cultured on galactose–tyrosine broth were extracted and concentrated followed by defatting of the crude extract with cyclohexane to afford polar and non-polar residues. Purification of the two residues by column chromatography led to isolation of five polar and one non-polar fraction. Bioactivity- guided fractions were rechromatographed on a silica gel column to obtain four compounds, namely 1H-indole-3-carboxylic acid, 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde, 4-(4-hydroxyphenoxy) butan-2-one and acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester from three active polar fractions and 8-methyl decanoic acid from one non-polar fraction. The structure of the compounds was elucidated on the basis of FT-IR, mass and NMR spectroscopy. The antimicrobial activity of the bioactive compounds produced by the strain was tested against the bacteria and fungi and expressed in terms of minimum inhibitory concentration. Antifungal activity of indole-3-carboxylic acid was further evaluated under in vitro and in vivo conditions. This is the first report of 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde, 4-(4-hydroxyphenoxy) butan-2-one, acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester and 8-methyl decanoic acid from the genus Streptomyces.     

A new p-terphenyl derivative from fruiting bodies of the basidiomycete Sarcodon laevigatum

A new metabolite with p -terphenyl core, named sarcodan (1), together with three known p -terphenyl metabolites (3, 4, 5) represented as Fig. 1, was isolated from the fruiting bodies of the basidiomycete Sarcodon laevigatum . The structures of these compounds were elucidated by spectroscopic and chemical methods.     

Microbial synthesis of metabolites with antihypertensive activity: aspects of fermentation derived inhibitors of angiotensin-converting enzyme (ACE)

In this review the microbial angiotensin-converting enzyme inhibitors are described. Especially from the microbiological point of view the characteristics of these metabolites are given, e.g. occurrence, fermentation physiology and specificity. Besides these data, the structure, assays and some isolation problems are summarised. Apart from ACE inhibition the different biological activities of these secondary metabolites are discussed.