两株真菌的次生代谢产物结构鉴定及生物学活性研究

发布时间：2018-03-06 21:19

本文选题：极地真菌　切入点：次级代谢产物　出处：《第二军医大学》2016年博士论文　论文类型：学位论文

【摘要】：从天然产物中筛选药物先导化合物一直是新药开发的重要思路。而真菌则一直是临床小分子药物的重要来源,临床药物中,青霉素、头孢菌素等抗生素、他汀类降血脂药物等,均产生自真菌。近些年,为了进一步获得微生物新药的多样性,科研人员不断地扩大药源真菌的来源,特别是加强来源于极端环境的真菌的活性筛选,以期从中寻找到结构新颖、活性强的代谢产物。本课题即是在这种背景下展开的。本课题的研究目的是(1)寻找极地来源的药源真菌,获取活性菌株;(2)对活性菌株进行次级代谢产物研究,重点寻找结构新颖、生物学活性强的代谢产物。为达到上述目的,我们需确定有活性的极地真菌菌株后进行规模发酵,累积活性菌株的初提物的量以供分离鉴定真菌代谢产物化合物单体;然后开展对活性真菌的系统的化学成分研究;获取化合物单体后,开展针对病死率高的恶性肿瘤和药物体内靶标蛋白的活性评价工作,以明确极地药源活性真菌的主要药效成分及作用强度。这些工作,对于系统地阐明极地真菌中药源真菌的药效成分和化学多样性,发现具有抗真菌及抗肿瘤活性的新颖化学结构有重要意义。本课题的研究方法是:首先对极地真菌样品进行小量发酵,获取初提物浸膏,对初提物浸膏展开生物学活性初筛,获取活性菌株。初筛采用纸片法进行抗菌活性实验,采用微量液基稀释法进行抗真菌活性实验以及采用MTT法进行体外细胞毒活性实验。确定活性菌株后,对活性菌株进行发酵和代谢产物分离工作,通过分析核磁共振谱、质谱图结合文献比对,确定化合物结构。再采用生物分子相互作用分析仪寻找化合物的体内靶标蛋白,再根据有结合的靶标蛋白的生物学作用进行免疫抑制活性测试。研究结果:(1)78株极地真菌的生物学活性初筛结果表明,有15株真菌表现出抑菌活性,占19.23%;D-1,Z1-1,W-1-18,S-1-10,S-1-16,S-1-19和S-1-17-Z1菌株活性较强,可分别抑制多株指示菌株的生长;40株菌株表现出稻瘟霉生长抑制作用,占到所筛选菌株的51.28%;其中,最大稀释倍数在128倍及以上的有12株,占15.38%;抗人类致病真菌筛选结果只获得1株活性菌株,即B-13;另外,有24株菌株表现出细胞毒活性,占到总筛选菌株的30.77%。抑制活性表现最突出的是S-3-88和S-3-62,在50μg/m L浓度下,S-3-88菌株初提物对MCF,He La,H446,A549,SW1990和SGC7901细胞的抑制率分别为78.79%,76.20%,50.38%,45.90%,53.18%和74.66%;同样浓度下,S-3-62菌株初提物对这6种肿瘤细胞株的抑制率分别为70.90%,69.8%,43.58%,40.64%,46.30%和35.96%。所有活性菌株中,具有2种或2种以上生物学活性的有4株菌,分别是D-1,B-13,S-1-10和S-1-16。(2)从极地Nectria sp.B-13的初提物浸膏中分离并鉴定了12个化合物的结构,分别是ilicicolin C(化合物1),LL-Z 1272ε(化合物2),deacetylchloronectrin(化合物3),dimethoxyilicicolin C(化合物4),ilicicolin D(化合物5),ilicicolin F(化合物6),ilicicolin E(化合物7),collectochlorin B(化合物8),ilicicolin A(化合物9),ilicicolin H(化合物10),3,5-二羟基甲苯(化合物11)和3,5-二羟基苯甲醇(化合物12).其中,化合物4是新化合物。(3)从极地Eutypella sp.D-1的初提物浸膏中分离并鉴定了7个化合物结构,分别是麦角甾醇(化合物13),α-亚麻酸(化合物14),脱氢松香酸(化合物15),Libertellenone B(化合物16),eutypenoid A(化合物17),eutypenoid D(化合物18)和eutypenoid E(化合物19)。其中化合物18和19是新化合物,属于海松烷型二萜。eutypenoid E的结构较为特殊,其在C-11有羰基取代,和一般的海松烷型二萜不同,属于变异的海松烷型二萜结构。(4)对化合物进行细胞毒活性研究表明,化合物6,7和9具有抑制淋巴细胞白血病Jurkat细胞增殖的作用,10μM浓度下,抑制率分别为59.27%,73.97%和71.43%。(5)生物分子相互作用分析仪研究结果表明,化合物3,7和8能与蛋白FKBP12(FK506 binding protein 12)结合,其结合常数Ka分别为7.85,220和7.47×10~4(阳性对照为8.03×10~4),化合物8的结合常数与阳性对照相当;解离常数Kd分别为1.01×10~(-5),0.026和4.17×10~(-6)(阳性对照为1.4×10~(-3));平均解离常数分别为1.28×10~(-6),1.18×10~(-4)和5.58×10~(-11)(阳性对照为1.75×10~(-8)),说明化合物8与FKBP12蛋白的亲和力高于阳性对照雷帕霉素,提示化合物8有可能具有类似于雷帕霉素的免疫抑制作用。然而,从接下来的免疫抑制活性研究中我们发现,所有测试的化合物仅有微弱的免疫抑制作用,提示化合物与蛋白FKBP12的结合并不影响B细胞和T细胞的增殖,可能与FKBP12其他的生理功能相关。化合物7的免疫抑制活性稍强,可能与其细胞毒性有关。研究结论:通过对极地真菌菌株的筛选,获得了一批活性菌株;通过对2株活性菌株代谢产物的分离鉴定,获得了19个极地真菌来源天然产物,其中有3个新化合物。生物学活性研究结果发现化合物8与FKBP12蛋白结合的亲和力高于阳性对照。以上研究提供了一批新颖结构和生物学活性的极地真菌天然产物,也发现了化合物8的体内结合蛋白,为进一步开发极地真菌的药用潜力提供了实验依据。
[Abstract]:Screening of lead compounds from natural products has been an important way for the development of new drugs. While fungi has been an important source of clinical drugs, clinical medicine, penicillin, cephalosporin antibiotics, cholesterol lowering statin drugs, were produced from fungi. In recent years, in order to obtain the microbial diversity of new drugs. Researchers continue to expand the source of medicine source fungi, especially to strengthen the source of screening in extreme environment fungal activity, in order to find novel structure, metabolism strong activity. This project is carried out in this context. The purpose of this study is to find the source of fungal medicine (1) polar sources, to obtain active strains; (2) to study the secondary metabolites of strains, focus on finding novel structure, strong biological activity of metabolites. In order to achieve the above goal, we need to determine the active Polar fungal strains after scale fermentation, extract the cumulative amount of active strains early for isolation and identification of fungal metabolite compounds; chemical constituents and to carry out the system on the activity of fungi; to obtain compound monomer, carry out the activity evaluation for high mortality rate of malignant tumor and drug target proteins work the major components and functions, clear the polar drug source strength. These fungi activity, for the system to clarify the polar active ingredients of traditional Chinese medicine source of fungal fungal and chemical diversity, findings have important implications with a novel chemical structure of antifungal and antitumor activity. The research method is: first, a small amount of fermentation the polar fungi samples, obtained initial extraction, to extract on biological activity screening, screening for obtaining active strains. The antibacterial activity experiment by slip method, Using the broth microdilution method for antifungal activity test and MTT method was used to determine the in vitro cytotoxicity experiment. The active strains, the strains were isolated and fermentation metabolites, through the analysis of NMR spectra, mass spectra with literature comparison, determine the structures of the compounds. The bio molecular interaction analyzer for compounds in vivo target protein, according to the biological function of the target protein binding of immune inhibitory activity test. Results: (1) 78 strains of biological activity of polar fungi screening results showed that 15 strains of fungi showed antibacterial activity, accounting for 19.23%; D-1, Z1-1, W-1-18, S-1-10, S-1-16, S-1-19 and S-1-17-Z1 strains strong activity, can inhibit indicator strains the strains were grown; 40 strains inhibited growth of Pyricularia oryzae, accounted for 51.28% of the strains were screened; among them, the maximum dilution times The number accounted for 15.38% in 128 times and more than 12 strains of human pathogenic fungi; resistance screening results were only obtained 1 strains, namely B-13; in addition, there were 24 strains showed cytotoxic activity, total screening strains of the 30.77%. inhibitory activity of the most outstanding performance is S-3-88 and S-3-62, in 50 g/m the concentration of L, S-3-88 strain extracts on MCF, He La, H446, A549, SW1990 and SGC7901 cell inhibition rates were 78.79%, 76.20%, 50.38%, 45.90%, 53.18% and 74.66%; the same concentration of S-3-62, inhibition of extracts of this strain at the beginning of 6 tumor cell lines were respectively 70.90%, 69.8% 43.58%, 40.64%, 46.30%, and 35.96%. of all strains, with 2 or more than 2 kinds of biological activity of 4 strains, namely D-1, B-13, S-1-10 and S-1-16. (2) separation and the structures of 12 compounds were identified in the extract from the polar Nectria sp.B-13, ilicicolin C respectively. (compound 1), LL -Z 1272 e (compound 2), deacetylchloronectrin (compound 3), dimethoxyilicicolin C (compound 4), ilicicolin D (compound 5), ilicicolin F (compound 6), ilicicolin E (compound 7), collectochlorin B (compound 8), ilicicolin A (compound 9), ilicicolin H (compound 10) 3,5-, two hydroxy toluene (compound 11) and 3,5- two hydroxy benzene methanol (compound 12). Among them, 4 compounds are new compounds. (3) separation and identification of the compounds 7 compounds in the extract from the polar Eutypella sp.D-1 at the beginning, respectively as erogostero (compound 13), alpha linolenic acid (compound 14), dehydroabietic acid (compound 15), Libertellenone B (compound 16), eutypenoid A (compound 17), eutypenoid D (compound 18) and eutypenoid E (compound 19). Compounds 18 and 19 are new compounds, which belongs to the structure of pimarane type two.Eutypenoid E from the particularity of the C-11 Substituted, and different pimarane general two terpenes, two terpene pimarane type structure belongs to variation. (4) to study cytotoxic activity compounds indicated that compounds 6,7 and 9 can inhibit the proliferation of lymphocytic leukemia Jurkat cells, 10 M concentration, the inhibition rates were 59.27%, 73.97% and 71.43%. (5) bio molecular interaction analyzer results showed that compounds 3,7 and 8 with FKBP12 (FK506 binding protein protein 12) binding, the binding constant of Ka were 7.85220 and 7.47 (8.03 * 10~4 * 10~4 as positive control), compound constant and positive control is combined with 8 Kd respectively; the dissociation constant 1.01 * 10~ (-5), 0.026 and 4.17 * 10~ (-6) (1.4 x 10~ as positive control (-3)); the average dissociation constants were 1.28 * 10~ (-6), 1.18 * 10~ (-4) and 10~ (-11) 5.58 * (1.75 * 10~ (-8) as a positive control). That compound 8 and FKBP12 protein affinity The positive control is higher than that of rapamycin, suggest that compounds 8 may have an immunosuppressive effect similar to rapamycin. However, from the immunosuppressive activity of the study we found that the immunosuppressive effect of all tested compounds only faint, with egg white FKBP12 and suggest that compounds did not affect B and T cell proliferation may be associated with the physiological function of FKBP12. The immune inhibitory activity of compound 7 slightly, which may be related to cell toxicity. Conclusion: through the screening of polar fungal strains, obtained a number of active strains; through the isolation and identification of 2 strains of metabolites, obtained 19 polar fungal natural products, of which there are 3 Study on the biological activity of new compounds. The results showed that the compounds 8 and FKBP12 protein binding affinity is higher than that of positive control. Provide a number of new nodes above research The natural products of polar fungi, which are constructed and biologically active, have also found the binding protein of compound 8, which provides experimental evidence for further developing the medicinal potential of polar fungi.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R915

【相似文献】