摘要
海绵的体表及体内积聚了大量的微生物,这些微生物的次级代谢产物化学结构新颖、种类繁多,是新药先导化合物的重要来源。为了寻找新的活性先导化合物,本论文采用了活性筛选和现代化学筛选相结合的方法,选取了2株海绵来源具有抗肿瘤和抗炎活性真菌的次级代谢产物进行系统研究。
从海南琼海及三亚附近海域的27个海绵样品中分离得到了208株微生物。采用SRB法,以小鼠白血病P388肿瘤细胞为筛选模型进行体外抗肿瘤活性的一级、二级筛选,并结合HPLC指纹图谱及薄层色谱进行化学筛选,得到具有较强细胞毒活性的菌株15株。对15株活性菌株同时进行抗炎活性筛选,其中2株具有强抗炎活性的海绵真菌,经ITS序列进行测序比对后,鉴定为附球菌属Epicoccum sp. JJY-40和链格孢属Alternaria sp. JJY-32,因此本文选择这2株活性菌株作为研究目标。
在考察了2株目标菌株的培养条件后,对目标活性菌进行了大量发酵。对发酵产物运用萃取,薄层色谱,正相、反相硅胶柱色谱,LH-20凝胶柱色谱,反相高压液相等化学的分离纯化手段,从附球菌属Epicoccum sp. JJY-40的次级代谢产物中分离得到18个单体化合物(1-15,29,30,31);从链格孢属Alternaria sp. JJY-32的次级代谢产物中分离得到16个单体化合物(16-28,32,33,34),共34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(乙酰化反应等)阐述了其中28个化合物(1-28)的化学结构(化合物结构及名称参见Table 1),其中新化合物12个。新化合物结构类型包括:萜类衍生物(18-25,27,28),Aucubinine B的羟基化衍生物(8)以及吡喃衍生物(15)。其中,新化合物18-25为系列具有新颖侧链的萜类衍生物,此类结构以前未曾报道,新化合物28为一罕见的杂螺环萜类衍生物。其它已知化合物分别为4个苯的衍生物(1-4)、4个二酮哌嗪(6,9-11)、1个糖苷(12)、1个核苷(13)、3个生物碱(5,7,14)、1个甾醇类化合物(16)以及2个Tricycloalternarene类萜类衍生物(17,26)。
先天性免疫是机体抗感染的第一防线,而巨噬细胞在其中发挥了关键性的作用,RAW264.7细胞可以很好的模拟体内的巨噬细胞系统,LPS可以通过刺激细胞因子的分泌而激活巨噬细胞,从而诱导炎症反应,因此实验利用LPS刺激下的RAW264.7细胞作为细胞模型,采用ELISA,RT-PCR,Western Blot和免疫荧光染色的筛选方法,对部分单体化合物的体外免疫调节活性进行了初步评价,发现化合物15和17可以明显的抑制LPS诱导的TNF-α和NO的分泌以及mRNA基因的表达,同时这些化合物亦可以明显的抑制LPS诱导的iNOS以及COX-2的表达,说明化合物15和17具有很强的免疫炎症抑制作用。此为化合物15和17抗炎活性的首次报道。剩余新化合物的构型和抗炎、抗肿瘤活性正在评价过程中。
综上,本文从海绵样品中共分离筛选出15株细胞毒活性菌株, 2株强抗炎活性菌株;阐明2株真菌中28个次级代谢产物的结构,其中12个新化合物,发现1个罕见的杂螺环萜类衍生物;首次发现化合物15和17具有显著的抗炎活性。上述研究为天然产物化学提供了新的结构类型,为抗炎药物的研究提供了活性模板化合物,并为从海绵来源真菌中活性次级代谢产物的研究提供了基础资料。
Sponge-associated microorganism is a rich source of secondary metabolites with diverse structures and biological activities. A study is carried out to investigate the bioactive lead compounds derived from sponge-associated microorganism. This thesis describes the study of the secondary metabolites produced by two selected sponge-associated fungi with anti-tumor and anti-inflammatory activities.
Two hundred and eight fungal strains were isolated from twenty seven sponge samples which were collected from the seashore of Qiong hai and Sanya in Hainan Province. Fifteen active strains were picked out based on the cytotoxic assay using the P388 cell line by the SRB method and on the HPLC and TLC chemical screening. Among them, two strains which were identified as Epicoccum sp. JJY-40 and Alternaria sp. JJY-32 by ITS sequencing also exhibited high anti-inflammatory activities. So they were selected for further study of their secondary metabolites.
Large-scale fermentations of the two bioactive strains were performed to obtain the active extracts. By means of chromatography over silica gel column, Sephadex LH20, preparative TLC and semi-preparative HPLC, eighteen (1-15,29,30,31) and sixteen compounds (16-28,32,33,34) were isolated from the sponge-associated fungi JJY-40 and JJY-32, respectively.
Basing on their physico-chemical properties and spectral data (IR, UV, MS, NMR, etc.), twenty eight structures were elucidated as 5-methylbenzene-1,3-diol(1), 1,3-Dihydroxy-4,5-dimethylbenzene (2), 2,4-dihydroxyl-5,6-dimethyl- benzoic acid (3), 2,4-dihydroxyl-6-methyl-benzoic acid (4), 3-indolylacetic acid methyl ester (5), cyclo-(Tyr-Pro)(6),N-acetyltyramine(7),3-hydroxy-7-methyl-6,7-dihydrocyclop-enta [c]pyridine-5-one (8), cyclo-(Pro-Leu) (9), cyclo-(Pro-Ile) (10),cyclo-(Val-Pro)(11), isomaltol-glucoside(12), Inosine(13), flazin(14), D8646-2-6’(15), Ergosterol (16), ACTG-toxins H (17), Dicycloalternarene 1b(18), Dicycloalternarene 2b(19), Dicycloalternarene 3a(20), Dicycloalternarene 3b(21), Dicycloalternarene 4b(22), Dicycloalternarene 4a(23), Phencycloalternarene 1(24), Phencycloalternarene 2(25), Tricycloalternarene 6a(26) , Tricycloalternarene 12a(27) and Tricycloalternarene 13a(28). Among the twelve new compounds, there were ten terpenoid derivatives (18-25,27,28), a hydroxy derivative of Aucubinine B (8) and a pyran derivative (15). New compounds 18-25 are a series of terpenoid derivatives with novel side-chain which have not been reported previously. New compound 28 is a rare spiro-oxindole terpenoid derivative. The known compounds were identified as four benzol derivatives (1-4), four cyclic dipeptides (6,9-11), a glucoside (12), a nucleoside (13), three alkaloids (5 , 7 , 14), a sterol (16) and two tricycloalternarene-type terpenoid derivatives (17,26), respectively.
Macrophages play a crucial role in innate immunity which is the body's first line of defense against infection and LPS can activate macrophages to induce inflammatory response by stimulating the secretion of cytokines. So we used LPS- stimulated RAW264.7 cells which can simulate macrophage system in vivo perfectly as cell model to evaluate immunomodulatory activity of some compounds in vitro by ELISA, RT-PCR, Western Blot and Immunofluorescent staining screening methods. The results indicated that compounds 15 and 17 could inhibit secretion and mRNA expression of LPS-induced TNF-αand NO. They also inhibited LPS-induced iNOS and COX-2 expression obviously. The high anti-inflammatory activities of such compounds have not been reported yet. The anti-inflammatory and anti-tumor activities of the rest new compounds are being evaluated.
In a word, fifteen active sponge–associated fungal strains with cytotoxic activities were obtained by combinatorial screening. Among them two strains also exhibited high anti-inflammatory activities. Twenty eight compounds were obtained from the two aimed fungi. Among them twelve compounds were new including a rare spiro-oxindole terpenoid derivative. Compounds 15 and 17 were first found to show high anti-inflammatory activities. This study provides novel structure types for natural product chemistry and bioactive template compounds for exploring new anti-inflammatory drugs. It also proves that the method we used is effective in looking for new bioactive secondary metabolites from sponge-associated fungi.
引文
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