摘要
海洋真菌作为海洋微生物中的重要一员,其次级代谢产物化学结构新颖、种类繁多,是新活性先导化合物的重要来源。为了寻找抗肿瘤活性先导化合物,本论文采用活性追踪的方法开展了1株南极海泥来源的真菌和2株来源于不同海域的海藻真菌中次级代谢产物抗肿瘤活性次级代谢产物研究工作。
从6个海藻样品中分离得到了21株真菌,采用海虾生物致死法和MCF7、SF-268、NCI-H460细胞为模型的筛选模型,以细胞周期抑制、细胞凋亡诱导以及坏死性细胞毒为活性指标,结合化学筛选,获得具有活性且次级代谢产物丰富的活性菌株9株。
采用正相和反相硅胶柱色谱、Sephadex LH-20凝胶柱色谱、半制备反相高压液相色谱等分离方法,从真菌PR19N-1(Penicillium sp.)中分离得到20个化合物(1-20),从黄曲霉c-f-3(Aspergillus flavus)中分离得到18个化合物(21 - 38),从真菌v-3-1中分离得到了6个化合物(39 -44),共计44个化合物。采用现代波谱技术(UV、IR、NMR、MS、CD),并结合其理化性质,阐明了40个化合物的化学结构(化合物结构参见Fig. 1);其中新化合物14个,包括11个艾里莫芬烷型倍半萜(1-6,8-12),利用NOESY谱、偶合常数确定其相对构型,并根据该类化合物的可能生合成途径,结合旋光值,推测了该类化合物部分结构的绝对构型;1个新的二酮哌嗪类化合物(21),利用NOE差谱确定了化合物的相对构型,并通过氨基酸手性分析柱确定了化合物的绝对构型;2个2-吡喃酮类化合物(24、25)。此外还有2个化合物(13、28)为首次从天然来源中分离得到。
另外发现的其他已知化合物的结构类型主要有甾类(32、33、40、41),倍半萜类(7,14-16),苯的简单衍生物(17、18、19、26),生物碱类(20、22、23、27、28), 2-吡喃酮衍生物(29、30)等。
利用MTT法、SRB法以及流式细胞术结合形态学检测的方法,对分离获得的单体化合物的抗肿瘤活性进行了初步评价。结果表明:新化合物1对A549、HL-60细胞显示出较强的细胞毒活性;新化合物8对A549显示较强的活性,对HL-60细胞显示中等强度的活性;新化合物10对两种肿瘤细胞都有微弱的活性;新化合物9对A549显示微弱的活性;新化合物11对HL-60细胞显示较弱的活性;新化合物21对HL-60细胞显示微弱的细胞毒活性;化合物28对A549、HL-60及MOLT-4三种肿瘤细胞都显示出一定的抑制活性。
运用cAMP检测方法对新化合物24、25的GPR12受体结合活性进行了初步评价,结果显示化合物24能促进GPR12-CHO、GPR12-HEK293细胞中的cAMP含量增加,且呈一定的剂量依赖关系。
综上所述,本文采用组合筛选的方法获得9株海洋活性真菌,并对3株海洋真菌的次级代谢产物的活性部位进行了系统研究,共分离鉴定了40个化合物,其中新化合物14个。其中具有细胞增殖抑制活性的新化合物6个,具有GPR12受体激动活性的新化合物1个。上述研究结果为寻找药物的先导化合物提供了新的化学结构类型,并为海洋真菌药用资源的研究提供了重要的基础。
As an important member of marine microorganism, marine fungus have been considered an important source of bioactive leading compounds, due to their unique living environment. A study on three strains of marine fungal strains was carried out to investigate the potential anti-tumor compounds.
By combinatory use of brine shrimp bioassay testing, cytometric bioassay against MCF7、SF-268、NCI-H460 cell line, and chemical analysis, 9 bioactive strains have been screened from 21 strains isolated from six marine samples.
Monitoring by bioassay-guided isolation, the active fraction is chromatographed on silica gel, Sephadex LH-20 and prepared TLC, and preparative reversed phase HPLC, respectively, forty-two compounds were isolated from the three active strains. Twenty compounds (1-20) were isolated from fungus PR19N-1 (Penicillium sp.), eighteen compounds (21-38)) from fungus c-f-3 (Aspergillus flavus), six compounds (39-44) from fungus v-3-1.
By means of modern spectral analysis (UV, IR, NMR, MS, CD) and physico-chemical properties, the structures (see Fig. 1) of forty pure compounds were respectively determined. Among them there are fourteen new compounds, including eleven eremophilane-type sesquiterpenes (1-6,8-12), which were deduced the relative structure by NOESY spectrum and the coupling constants, and some of which were proposed the absolute stereochemistry by analysis their biosynthesis route; one new diketopiperazine (21),which was deduced the relative structure by NOE and determined the absolute stereochemistry by chiral HPLC analysis; two new 2-pyrons (24, 25). Additionally, two compounds (13,28) were isolated from nature source for the first time.
In addition, the chemical structure types of compounds are mainly involved in Steroides (32、33、40、41), sesquiterpenes (7,14-16), Benzene derivates (17、18、19、26), Alkaloids (20、22、23、27、28), 2-Pyrone (29、30), etc.
These compounds were evaluated for their cytotoxicities against several cancer cell lines such as HL-60, A549 etc, by the MTT or SRB method. The results indicated that new compounds 1 and 8 showed stronge cytotoxicities on A549 cell lines; new compounds 9 and 10 showed moderate cytotoxicities on A549 cell lines; new compounds 8, 10, 11 showed a certain cytotoxicities on HL-60 cell lines; new compound 21 showed slightly cytotoxicities on HL-60 cell lines; and compound 28 showed cytotoxicities on the three cell lines of A549、HL-60及MOLT-4.
New compounds 24, 25 were tested the GPR12 receptor binding activity by cAMP assay. The results showed that 24 could promote a small but significant cAMP increase in a dose-dependent manner on both GPR12-CHO cells and GPR12-HEK293 cells, while no significant difference on their vector controls.
Summarily, this work obtained nine important active strains from six marine alga samples, and forty compounds from three active strains. Among them, fourteen compounds were identified new. Six new compounds with antitumor activities in vitro were discovered, and one new compound with GPR12 receptor binding activity. Studies mentioned above provided novel structures for searching leading compounds and strain resources of great value for further study and development of marine fungus.
引文
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