4株海洋真菌和2种海洋动物次级代谢产物及其抗肿瘤活性研究

【作者】 陈立；

【导师】 顾谦群； 朱伟明；

【作者基本信息】 中国海洋大学 ， 药物化学， 2008， 博士

【摘要】 海洋生物生存环境特殊,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、抗氧化活性先导化合物,本论文采用活性追踪的方法开展了4株海洋微生物和2种海洋底栖动物活性次级代谢产物的研究工作。内容包括:不同培养条件下微生物次级代谢产物的比较和目标菌株的选择;活性成分的追踪分离;单体化合物的结构解析;单体化合物的活性初步评价。在对本实验室已研究过次级代谢产物的20株海洋活性真菌进行培养条件改变摸索研究,发现了2株真菌的粗提物仍然保持抗肿瘤活性,但TLC薄层色谱和高压液相指纹图谱显示其次级代谢产物与原菌株的明显不同,发现两株菌的代谢产物中主要成分极性明显增大以及紫外吸收明显发生改变,于是决定将其作为本论文的主要研究对象。此外,本论文还对2种具有抗肿瘤活性的海洋底栖动物和2株采自韩国沿海具有抗氧化活性的海洋真菌次级代谢产物进行了研究。对4株目标活性菌进行了大量发酵,对2种海洋动物进行了匀浆和浸提。并对发酵产物和浸提物运用萃取,薄层层析,正相、反相硅胶柱层析,LH-20凝胶柱层析,反相高压液相等化学的分离纯化手段进行活性追踪分离,从土壤青霉菌(Penicillium terrestre)的代谢产物中分离得到29个化合物单体(1-29);从匍匐曲霉(Aspergillus repens)中分离得到了7个单体化合物(30-36);从韩国沿海真菌B382中分离得到6个单体化合物(37-42);从韩国沿海真菌B523中分离得到了6个单体化合物(43-48);从哈氏刻肋海胆(Temnopleurus hardwickii)中分离得到了3个单体化合物(49-51);从星座美洲海鞘(Amaroucium constellatum)中分离得到了9个单体化合物(52-60),共计60个化合物。继而,利用理化性质和波谱学方法(IR,UV,MS,NMR,X-ray)结合化学反应的方法阐明了这60个化合物的化学结构(化合物结构参见Figure 1),其中发现新化合物20个,包括8个聚gentisyl alcohol类化合物(1-8)(首次报道该类单体的三聚物),3个gentisyl alcohol单体的衍生物(12,18,19),5个Sorbicillin类衍生物(14-17,20),1个玉米赤霉烯酮类化合物(37),3个硫酸酯铵盐类化合物(49-51)。此外还有1个化合物(13)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还涉及17个苯的衍生物(9-11,22-29,43,47-48,56-58),1个Sorbicillin类衍生物(21),3个甾醇类化合物(33,34,53),2个脑苷酯类化合物(31-32),3个生物碱类化合物(30,35-36),5个玉米赤霉烯酮类化合物(38-42),3个蒽醌类衍生物(44-46),4个核苷类衍生物(52、55、59-60),1个甘油醚类化合物(54)。利用流式细胞术结合形态学检测、MTT和SRB法,对所分得的新化合物的体外抗肿瘤活性进行了初步评价。新化合物1-8对HL-60、MOLT-4、A-549和BEL-7402四种细胞株都表现出不同程度的抑制活性,其中化合物6对四种癌细胞的活性都较强。在对6的进一步研究中发现,该化合物在10μM浓度下对酪氨酸激酶Src和KDR,抑制率分别为35.9%和31.8%。同时还发现新化合物14、15对HL-60有较弱的抑制活性(IC50分别为9.2和37.8μM),14对A-549有较弱的抑制活性(IC50为39.7μM)。此外,在以DPPH为模型的抗氧化试验中,发现聚酚类化合物(1-8)和单体(12,13)均显示较强的活性(IC50分别为4.3, 4.6, 4.4, 6.2, 5.2, 4.1, 6.3, 2.6, 8.5和9.8μM),明显强于阳性对照物维生素C(IC50为17.4μM)。综上所述,本文对4株海洋真菌和2种海洋底栖动物的次级代谢产物进行了系统研究,共分离鉴定了60个化合物的结构,包括新化合物20个并推测了其中12个新化合物可能的生合成途径;发现了一类结构新颖的具有抗肿瘤、抗氧化等多重活性gentisyl alcohol聚合体(1-8);最值得一提的是,通过对2株真菌改变培养条件所分离得到的36个化合物与原条件所分得的22个化合物无一重复。因此上述研究既为抗肿瘤新药提供了先导结构,也为今后充分发掘活性菌株次级代谢的潜力,从而获得大量结构新颖的活性次级代谢产物提供实验依据。更多还原

【Abstract】 Marine microorganisms and invertebrates continue to be an important source of bioactive secondary metabolites. A study on four strains of marine microorganisms and two invertebrates was carried out to investigate the potential anti-tumor and antioxidant compounds. Studies include comparison of microbial metabolites under different culture conditions, selecting aimed strains, fermentation studies, bioassay-guided fractionation, structural elucidation and preliminary evaluation for activities of pure compounds.After careful comparison of microbial metabolites under different culture conditions, the metabolite patterns of two fungal strains exhibited TLC and HPLC profiles very distinct from those of the original strains, such as higher polarity and different UV absorbance. However, their anti-tumor activity remained. So they were chosen as main objects of this thesis. Furthermore, two invertebrates with anti-tumor activity and two marine fungus of Korean coast with antioxidant activity were selected.Large-scale fermentations of the active strains and smash of the invertebrates were performed. Following the bioactivity, compounds were isolated and purified by using solvent extraction, silica gel column, Sephadex LH20, PHPLC and etc. From fungus Penicillium terrestre, 29 compounds (1-29) were isolated; from fungus Aspergillus repens, 7 compounds (30-36) were isolated; from fungus B382, 6 compounds (37-42) were isolated; from fungus B523, 6 compounds (43-48) were isolated; from echinus Temnopleurus hardwickii, 3 compounds (49-51) were isolated; from ascidian Amaroucium constellatum, 9 compounds (52-60) were isolated.By means of physico-chemical properties and spectral analysis (IR, UV, MS, NMR, X-ray, etc.), structures of 60 pure compounds were respectively determined, Among them there are 20 new compounds, including 8 new gentisyl alcohol polymers (1–8), 3 gentisyl alcohol derivatives (12, 18, 19), 5 sorbicillin derivatives (14-17, 20), 1 zearalenone derivative (37), 3 sulfated alkenes (49-51). Furthermore, 1 new natural product (13) was also isolated.In addition, the types of compounds are involved in benzene derivates (9-11,22-29,43,47-48,56-58), sorbicillin derivative (21), steroides (33,34,53), cerebrosides (31-32), alkaloids (30, 35-36), zearalenone derivatives (38-42), anthraquinones (44-46), nucleosides (52, 55, 59-60), glycerol derivative (54). The antitumor activity against several cancer cell lines of the new compounds was assayed by MTT, SRB and flow cytometry methods. Compounds 1-8 all showed inhibitive activity against HL-60, MOLT-4, A-549, and BEL-7402, and compound 6 showed stronger activity than others. In the continuous research, 6 showed moderate inhibitive activity against protein tyrosine kinases (Src and KDR) by ELISA (inhibitive rate 35.9% and 31.8% on concentration 10μM). In addition, compounds 14 and 15 have weak cytotoxicity against HL-60 with IC50= 9.2 and 37.8μM; 14 also has weak cytotoxicity against A-549 with IC50=39.7μM. When evaluated for their radical scavenging activity against DPPH, compounds 1–8, 12 and 13 showed moderate activity(IC50=4.3, 4.6, 4.4, 6.2, 5.2, 4.1, 6.3, 2.6, 8.5 and 9.8μM respectively. They were all stronger than the positive control (ascorbic acid, IC50=17.4μM).Summarily, this work obtained sixty compounds from four strains of marine fungus and two invertebrates. Among them, 20 compounds were identified new and the possible biogenetic pathways of 12 new compounds were given. A series of novel gentisyl alcohol polymers with antitumor and antioxidant activity was reported. The most important, none of the 36 compounds isolated from the new cultural condition was the same as the 22 compounds isolated from the original condition. Studies mentioned above provided novel structures for searching leading antitumor compounds, and gave a good example for the deep development of the active strains.更多还原