發(fā)布時(shí)間：2018-03-06 21:19

  本文選題：極地真菌　切入點(diǎn)：次級(jí)代謝產(chǎn)物　出處：《第二軍醫(yī)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：從天然產(chǎn)物中篩選藥物先導(dǎo)化合物一直是新藥開發(fā)的重要思路。而真菌則一直是臨床小分子藥物的重要來(lái)源,臨床藥物中,青霉素、頭孢菌素等抗生素、他汀類降血脂藥物等,均產(chǎn)生自真菌。近些年,為了進(jìn)一步獲得微生物新藥的多樣性,科研人員不斷地?cái)U(kuò)大藥源真菌的來(lái)源,特別是加強(qiáng)來(lái)源于極端環(huán)境的真菌的活性篩選,以期從中尋找到結(jié)構(gòu)新穎、活性強(qiáng)的代謝產(chǎn)物。本課題即是在這種背景下展開的。本課題的研究目的是(1)尋找極地來(lái)源的藥源真菌,獲取活性菌株;(2)對(duì)活性菌株進(jìn)行次級(jí)代謝產(chǎn)物研究,重點(diǎn)尋找結(jié)構(gòu)新穎、生物學(xué)活性強(qiáng)的代謝產(chǎn)物。為達(dá)到上述目的,我們需確定有活性的極地真菌菌株后進(jìn)行規(guī)模發(fā)酵,累積活性菌株的初提物的量以供分離鑒定真菌代謝產(chǎn)物化合物單體;然后開展對(duì)活性真菌的系統(tǒng)的化學(xué)成分研究;獲取化合物單體后,開展針對(duì)病死率高的惡性腫瘤和藥物體內(nèi)靶標(biāo)蛋白的活性評(píng)價(jià)工作,以明確極地藥源活性真菌的主要藥效成分及作用強(qiáng)度。這些工作,對(duì)于系統(tǒng)地闡明極地真菌中藥源真菌的藥效成分和化學(xué)多樣性,發(fā)現(xiàn)具有抗真菌及抗腫瘤活性的新穎化學(xué)結(jié)構(gòu)有重要意義。本課題的研究方法是:首先對(duì)極地真菌樣品進(jìn)行小量發(fā)酵,獲取初提物浸膏,對(duì)初提物浸膏展開生物學(xué)活性初篩,獲取活性菌株。初篩采用紙片法進(jìn)行抗菌活性實(shí)驗(yàn),采用微量液基稀釋法進(jìn)行抗真菌活性實(shí)驗(yàn)以及采用MTT法進(jìn)行體外細(xì)胞毒活性實(shí)驗(yàn)。確定活性菌株后,對(duì)活性菌株進(jìn)行發(fā)酵和代謝產(chǎn)物分離工作,通過(guò)分析核磁共振譜、質(zhì)譜圖結(jié)合文獻(xiàn)比對(duì),確定化合物結(jié)構(gòu)。再采用生物分子相互作用分析儀尋找化合物的體內(nèi)靶標(biāo)蛋白,再根據(jù)有結(jié)合的靶標(biāo)蛋白的生物學(xué)作用進(jìn)行免疫抑制活性測(cè)試。研究結(jié)果:(1)78株極地真菌的生物學(xué)活性初篩結(jié)果表明,有15株真菌表現(xiàn)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菌株活性較強(qiáng),可分別抑制多株指示菌株的生長(zhǎng);40株菌株表現(xiàn)出稻瘟霉生長(zhǎng)抑制作用,占到所篩選菌株的51.28%;其中,最大稀釋倍數(shù)在128倍及以上的有12株,占15.38%;抗人類致病真菌篩選結(jié)果只獲得1株活性菌株,即B-13;另外,有24株菌株表現(xiàn)出細(xì)胞毒活性,占到總篩選菌株的30.77%。抑制活性表現(xiàn)最突出的是S-3-88和S-3-62,在50μg/m L濃度下,S-3-88菌株初提物對(duì)MCF,He La,H446,A549,SW1990和SGC7901細(xì)胞的抑制率分別為78.79%,76.20%,50.38%,45.90%,53.18%和74.66%;同樣濃度下,S-3-62菌株初提物對(duì)這6種腫瘤細(xì)胞株的抑制率分別為70.90%,69.8%,43.58%,40.64%,46.30%和35.96%。所有活性菌株中,具有2種或2種以上生物學(xué)活性的有4株菌,分別是D-1,B-13,S-1-10和S-1-16。(2)從極地Nectria sp.B-13的初提物浸膏中分離并鑒定了12個(gè)化合物的結(jié)構(gòu),分別是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),3,5-二羥基甲苯(化合物11)和3,5-二羥基苯甲醇(化合物12).其中,化合物4是新化合物。(3)從極地Eutypella sp.D-1的初提物浸膏中分離并鑒定了7個(gè)化合物結(jié)構(gòu),分別是麥角甾醇(化合物13),α-亞麻酸(化合物14),脫氫松香酸(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,屬于海松烷型二萜。eutypenoid E的結(jié)構(gòu)較為特殊,其在C-11有羰基取代,和一般的海松烷型二萜不同,屬于變異的海松烷型二萜結(jié)構(gòu)。(4)對(duì)化合物進(jìn)行細(xì)胞毒活性研究表明,化合物6,7和9具有抑制淋巴細(xì)胞白血病Jurkat細(xì)胞增殖的作用,10μM濃度下,抑制率分別為59.27%,73.97%和71.43%。(5)生物分子相互作用分析儀研究結(jié)果表明,化合物3,7和8能與蛋白FKBP12(FK506 binding protein 12)結(jié)合,其結(jié)合常數(shù)Ka分別為7.85,220和7.47×10~4(陽(yáng)性對(duì)照為8.03×10~4),化合物8的結(jié)合常數(shù)與陽(yáng)性對(duì)照相當(dāng);解離常數(shù)Kd分別為1.01×10~(-5),0.026和4.17×10~(-6)(陽(yáng)性對(duì)照為1.4×10~(-3));平均解離常數(shù)分別為1.28×10~(-6),1.18×10~(-4)和5.58×10~(-11)(陽(yáng)性對(duì)照為1.75×10~(-8)),說(shuō)明化合物8與FKBP12蛋白的親和力高于陽(yáng)性對(duì)照雷帕霉素,提示化合物8有可能具有類似于雷帕霉素的免疫抑制作用。然而,從接下來(lái)的免疫抑制活性研究中我們發(fā)現(xiàn),所有測(cè)試的化合物僅有微弱的免疫抑制作用,提示化合物與蛋白FKBP12的結(jié)合并不影響B(tài)細(xì)胞和T細(xì)胞的增殖,可能與FKBP12其他的生理功能相關(guān)�；衔�7的免疫抑制活性稍強(qiáng),可能與其細(xì)胞毒性有關(guān)。研究結(jié)論:通過(guò)對(duì)極地真菌菌株的篩選,獲得了一批活性菌株;通過(guò)對(duì)2株活性菌株代謝產(chǎn)物的分離鑒定,獲得了19個(gè)極地真菌來(lái)源天然產(chǎn)物,其中有3個(gè)新化合物。生物學(xué)活性研究結(jié)果發(fā)現(xiàn)化合物8與FKBP12蛋白結(jié)合的親和力高于陽(yáng)性對(duì)照。以上研究提供了一批新穎結(jié)構(gòu)和生物學(xué)活性的極地真菌天然產(chǎn)物,也發(fā)現(xiàn)了化合物8的體內(nèi)結(jié)合蛋白,為進(jìn)一步開發(fā)極地真菌的藥用潛力提供了實(shí)驗(yàn)依據(jù)。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R915

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5 秦藝e，

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