摘要
一、番荔枝内生真菌Periconia sp. F-31中新颖结构化学成分研究
Periconia sp. F-31为本实验室前期从刺果番荔枝中分离得到的一株具有较强体外细胞毒活性的内生真菌,菌种鉴定结果表明,其可能为黑团孢霉属的一个新种(Periconia sp.)。本论文以寻找新颖结构的活性PKS-NRPS杂合物为重点,对其次生代谢产物进行了系统的研究。
紧密结合HPLC-UV-MS"分析技术指导跟踪分离,采用正相硅胶柱色谱、SephadexLH-20凝胶柱色谱、正/反相半制备HPLC等多种分离手段进行快速定向分离纯化,并通过理化性质、多种波谱技术(IR, UV, MS、1D NMR,2DNMR)、CD、单晶X-ray、化学方法等手段鉴定化合物的结构。从Periconia sp. F-31中共分离得到29个化合物,新化合物24个(P1-P24), PKS-NRPS杂合物16个(P1-P16),包括8个来源于PKS-NRPS杂合途径的具有新颖结构骨架的细胞松弛素类化合物(P1-P8)和2个具6/6/5含氧三环系新颖结构骨架的PKS-NRPS杂合物(P9-P10)。其中8个细胞松弛素类化合物又包含三类骨架结构：第一类为9/6/5三环系的细胞松弛素,第二类为其衍生的5/6/6/5四环系和5/5/6/6/5五环系的细胞松弛素,第三类为7/6/5三环系的细胞松弛素。在确定新化合物的绝对构型过程中,除了常用波谱手段外,还运用了化学衍生法、X-ray、CD[包括Rh2(OCOCF3)4-鳌合CD、Mo2(OAC)4-鳌合CD]等方法。另外,对新化合物的生物合成途径进行了合理的推测。
对部分化合物进行了体外抗肿瘤活性评价和抗病毒(HIV-1)活性评价。抗肿瘤筛选结果显示,化合物P1和P2对HCT-8、BGC-823、Bel-7402三种肿瘤细胞株显示良好的抑制活性。上述新颖结构骨架PKS-NRPS杂合物的发现不仅为药物先导物提供了结构多样化的化合物分子,还为这些PKS-NRPS杂合物生物合成途径的进一步研究奠了基础。
二、丹参细胞培养物中脂溶性化学成分研究
丹参为我国传统中药,是最常用的活血化瘀中药之一,现代药理学研究表明,丹参有扩张冠状动脉、增加冠状动脉血流量、减慢心率、改善心肌缺氧、改善微循环、抗肿瘤、抗菌消炎等多种药理活性。临床广泛用于治疗心脑血管疾病。丹参的脂溶性成分是活血化瘀的有效成分之一,主要用于治疗冠心病。丹参中化学成分已有较多文献报道,而其细胞培养物中化学成分则少有研究。为明确丹参细胞培养物中脂溶性化学成分,寻找新的活性天然产物。我们对丹参细胞培养物80%乙醇提取物的石油醚部位进行了系统的化学成分研究。
采用正相硅胶柱色谱、Sephadex LH-20凝胶柱色谱、制备薄层、正／反相半制备HPLC等手段进行分离纯化,根据理化性质及波谱数据鉴定化合物的结构,共分离鉴定了50个化合物,其中8个新化合物(M1-M8),包括6个含嗯唑环的芳香化松香烷二萜类化合物(M1-M6)和2个降碳二萜类化合物(M7-M8),并首次通过单晶X衍射法和圆二色谱(CD)技术确定了含嗯唑环的芳香化松香烷二萜类化合物中手性碳的绝对构型。另外,对含噁唑环的芳香化松香烷二萜的生物合成途径进行了合理的推测。
对分离得到的化合物进行了抗肿瘤、抗病毒、神经保护等多种药理活性筛选。在抗肿瘤活性测试中,化合物M19-M21, M31-M33具有良好的体外抗肿瘤活性。在抗病毒活性筛选中,发现多个化合物具有很强的抗HIV-1活性,并且发现其在低浓度和高浓度下对HIV-1的抑制活性不呈浓度依赖性,其中2个化合物在0.1μM浓度下抑制率可达40-50%。在小胶质细胞释放炎症因子的抑制模型上,化合物M19-M23,M31,M33在10μM浓度下对小胶质细胞释放的炎症因子的抑制率可达100%,显示出很强的神经保护活性。以上结果表明,植物细胞培养物不仅可以产生原植物中已发现的活性成分,还可以产生新颖结构活性天然产物,为药物先导物的发现提供了新的来源。
1. Chemical constituents from endophytic fungus Periconia sp. F-31isolated from Annona muricata
Periconia sp. F-31was an endophytic fungus isolated from the medicinal plant Annona muricata, which might be a new species according to the identification by using morphological and molecular (ITS1-5.8S-ITS2rDNA sequence) analyses, the crude extract of which displayed potent cytotoxicity against several human cancer cell lines.
As part of our search for novel bio-active PKS-NRPS metabolites from Periconia sp. F-31, the EtOAc extract of the filtrate and mycelia of this strain were systematically investigated. Under the guidance of HPLC-UV-MS" analysis, twenty-nine compounds were isolated by using various chromatographic techinques, and their structures were elucidated by extensive spectroscopic data analysis. Especially, the absolute configurations were determined by analyzing the single-crystal X-ray diffraction data, CD [including Rh2(OCOCF3)4-induced CD^Mo2(OAC)4-induced CD] spectroscopic data. Twenty-four (P1-P24) of them are new compounds, including sixteen PKS-NRPS metabolites (P1-P16), these PKS-NRPS metabolites contain eight novel cytochalasans (P1-P8) and two PKS-NRPS metabolites with an unique6/6/5tricyclic ring system (P9-P10). The cytochalasan contain three type structures:the first type of which possesses novel9/6/5tricyclic ring system, the second type has unique5/6/6/5and5/5/6/6/5carbocyclic ring system which might be derived from the former, the third type displays unusual7/6/5tricyclic ring system. The plausible biosynthetic pathways of new compounds were also proposed.
The in vitro bio-activities of part of compounds were evaluated for the cytotoxicity against five cancer cell lines and antiviral activity. Comopunds PI and P2showed selective and significant cytotoxicity against HCT-8, BGC-823, and Bel-7402cancer cell lines. The discovery of novel PKS-NRPS metabolites with cytotoxicities not only provide the diverse molecules for lead compounds, but also are useful for further biosynthesis investigation of these significant PKS-NRPS metabolites.
2. Liposoluble chemical constituents from cell cultures of Salvia miltiorrhiza
Salvia miltiorrhiza Bunge (Labiatae), which is well-known as the traditional Chinese medicine "Danshen", has been used for the treatment of menstrual disorders, menorrhagia, menostasis, insomnia and cardiovascular diseases, particularly angina pectoris and myocardial infarction. The liposoluble chemical constituents of S. miltiorrhiza were confirmed as effective constituents, which were mainly used for the treatment of coronary heart disease. Numerous studies of chemical constituents of S. miltiorrhiza plants have been reported, however, the chemical constituents of the cell cultures of S. miltiorrhiza have hardly reported till now. As part of our search for bioactive compounds, the petroleum ether fraction of80%EtOH extract of S. miltiorrhiza cell cultures were systematically investigated, which led to the isolation of fifty compounds including eight new compounds (M1-M8). Their structures with absolute configuration were elucidated by detailed analyses of spectroscopic data (IR, MS, NMR, UV), CD, and single-crystal X-ray diffraction. The new compounds included six new oxazole-containing diterpenoids (M1-M6) and two norditerpenoids (M7-M8). Especially, the single-crystal X-ray diffraction experiment was the first time for confirming the oxazole ring and the absolute configuration of this type diterpenoids. The plausible biosynthetic pathway of aromatic oxazole-containing diterpenoids was also proposed.
The isolated compounds were evaluated for their cytotoxicities, antiviral activities, neuroprotective activities. Compounds M19-M21, M31-M33showed selected and significant activity against several cancer cell lines. Several compounds displayed potent activity against HIV-1replication. It's interesting that the values of inhibitory activities of these compounds are not concentration-dependent, the values of inhibitory activities of2compounds in0.1μM were in the range of40-50%. Compounds M19-M23, M31, M33inhibited lipopolysaccharide (LPS)-induced pro-inflammatory factors in BV2-macrophages with the inhibition ratio of100%at10μM. The results imply that plant cell cultures can not only produce known bioactive compounds, but also yield new bioactive metabolites, which offer alternative and effective sources of lead compounds.
引文
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