摘要
本论文主要运用天然产物样品库的高通量制备技术构建一个具有相关生物活性的小型天然产物组分库。基于初步活性筛选结果,从中选取具有一定抗肿瘤作用的小秦艽(Gentiana dahurica)、白佳熊葡(Arctostaphylos pajaroensis)、刺山柑(Capparis spinosa)和具有抗抑郁效果的乌灵菌粉(Xylaria nigripes)作为研究对象；对它们的主要化学成分及生物活性进行了一定的研究：采用硅胶柱层析、微孔树脂(MCI)、Sephadex LH-20凝胶柱层析、半制备型HPLC等分离纯化手段从上述三种植物和一种真菌中共分离得到65个化合物。综合运用多种波谱和光谱技术(HR-ESIMS,1H、13C-NMR及2D NMR)、化学转化、单晶衍射及与标准品对照等手段共鉴定了46个化合物的结构(包括15个三萜、5个环烯醚萜苷、6个生物碱、5个大柱烷型降碳倍半萜及其苷、4个甾醇及其苷、4个黄酮和异黄酮、2个糖苷、1个香豆素、1个糠醛、2个有机酸,1个苯丙素类化合物)。其中新化合物3个,另39个化合物为首次从相应原材料中分离得到。生物活性筛选结果表明部分化合物具有一定的抗肿瘤细胞毒作用；本论文进而对其中五环三萜类化合物的抗肿瘤作用机制进行了初步探讨。论文最后运用高效液相色谱-质谱联用(HPLC-DAD-ESI-MS)技术对上述四种样品的中的主要化学成分进行了指纹图谱分析研究。
主要研究内容简述如下：
(1)构建小型生物活性特异性天然产物组分库
运用天然产物化学技术,主要包括快速自动硅胶柱层析(FlashMasterⅡ及FlashMaster Parallel)等对中医药临床上具有抗肿瘤、延缓脑衰老等的中(草)药进行提取、分离,构建小型天然产物样品库,并开展基于天然产物库的体外活性筛选。目前,已建成拥有106种中药的2226个Flash组分库。并采用液相色谱质谱联用技术对Flash组分库中的部分样品进行了分析。
(2)开展三种具有抗肿瘤活性的植物提取物的主要化学成分及生物功能研究
从中药小秦艽干燥根的乙醇提取物中共分离得到20个化合物,已鉴定出结构的有16个化合物,包括七个五环三萜(2.1,2.4-2.9),五个环烯醚萜苷(2.2,2.3,2.10-2.12)及四个其他类型化合物(2.13-2.16),其中2.1-2.3为新化合物,化合物2.4-2.12为首次从该植物中分离得到。体外抗MCF-7乳腺癌、SK-Hep1肝癌和U20S人骨肉瘤细胞毒性的检测结果显示,六个五环三萜类化合物(2.4-2.9)均具有中等细胞毒作用(IC50为16.7-85.2μg/mL)。本实验挑选并首次对化合物2.6抗肿瘤作用机制进行了初探。结果表明,化合物具有促进肿瘤细胞凋亡作用。此外,化合物2.7(科罗索酸)具有较好的抗钙库调控钙离子通道的作用。
熊果属植物白佳熊葡的化学成分未见有文献报道。本论文首次对该植物的化学成分及其生物活性进行研究。从该植物有机提取物(Org Extract)和甲醇提取物(MeOH Extract)中共分离纯化出16个化合物,已鉴定出结构的有12个,包括三个羽扇豆烷型五环三萜(3.1-3.3),两个齐墩果烷型五环三萜(3.4,3.8)三个乌苏烷型五环三萜(3.5-3.7),β-熊果苷(3.11)和a-D-呋喃果糖(3.12)等。这些化合物均为首次从该植物中分离得到。体外抗肿瘤细胞(胃癌细胞NUGC3、肝癌细胞SNU739、神经胶质瘤细胞SHSY-5Y和乳腺癌细胞MCF-7)毒性实验表明,两个三萜类化合物3.2和3.4具有中等细胞毒作用。
从刺山柑果实提取物中共分离纯化出化合物15个,已鉴定出结构的化合物10个,包括1个生物碱(4.1)、5个大柱烷型降碳倍半萜及其苷(4.2-4.6)、1个有机酸(4.7)、1个黄酮苷(4.8)、1个糠醛(4.9)及β-胡萝卜苷(4.10)。其中化合物4.2-4.5均为首次从刺山柑中分离得到。
(3)开展乌灵菌粉化学成分及抗抑郁活性成分分析研究
对药食两用真菌---乌灵菌粉乙酸乙酯部分进行初步分离纯化,从中分离得到几类(生物碱、吡咯衍生物、甾醇、异黄酮)单体化合物14个,鉴定出结构的有11个(5.1-5.11),这些均为首次从乌灵菌粉中分离得到。采用体外细胞筛选模型开展对乌灵菌粉粗提物及单体化合物(5.1-5.11)活性初筛发现,乌灵菌粉部分组分及化学成分能够提高脑源性神经营养因子(BDNF)的表达水平。
(4)采用液相色谱-质谱联用技术首次对上述四种天然提取物的主要化学成分进行分析研究。
对小秦艽、白佳熊葡、刺山柑及乌灵菌粉四种样品的复杂提取物分别采用高效液相色谱-质谱联用技术,正、负离子两种电离模式进行了分析。通过紫外光谱、质谱数据,结合文献对照等信息,对其所含的主要化学成分进行了初步分析鉴定,探索从复杂样品中快速、有效地寻找分析鉴定化学成分。
Natural product chemistry method, mainly consisting of automated FlashMaster chromatography and HPLC-DAD-ESI-MS systems, has been applied to the generation and analysis of a 'drug-like' natural product library---Flash fractions for drug discovery and chemical biology research. Three plants (Gentiana dahurica Fisch., Arctostaphylos pajaroensis and Capparis spinosa L.) together with the edible fungus Xylaria nigripes (K1.) Sacc., have been phytochemically investigated. A total of sixty-five naturally occurring compounds were obtained by using column chromatography (CC) over silica gel, MCI gel, Sephadex LH-20, and semi-preparative HPLC. Forty-six different compounds were characterized as fifteen triterpenoids, five iridoid glycosides, six alkaloids, five sesquiterpenoids, four steroids, four flavonoids and isoflavonoids, two glycosides, one coumarin, one furfural, two organic acids and one phenylpropanoid. Among them, three were new, and another thirty-nine were isolated for the first time from the corresponding plant. Their structures were identified on the basis of extensive spectroscopic methods (especially LR-/HR-ESIMS,1H,13C-NMR and 2D NMR), chemical transformations and single-crystal X-ray diffraction analysis. Biological tests revealed that some compounds exhibited moderate cytotoxicity, and several pentacyclic triterpenoids were further found to be the anti-cancer effective substance of G. dahurica and A. pajaroensis. The technique of high performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) has been developed for the rapid determination of complex components in the above selected crude materials.
The main research topics in the dissertation are outlined as follows:
(1) Generation and analysis of natural product library---Flash fractions
Natural product chemistry methods (mainly include automated FlashMaster chromatography and HPLC-DAD-ESI-MS) were utilized to generation and analysis of natural product library (Flash fractions) from herb medicines. Up to now, the natural product libraries consist of 2226 Flash fractions from 106 biologically selected herb medicines.
(2) Studies on chemical constituents and bioactivities of three plant materials
Sixteen compounds including seven pentacyclic triterpenoids (2.1,2.4-2.9), five iridoid glycosides (2.2,2.3,2.10-2.12) and four other compounds (2.13-2.16) were isolated from G. dahurica Fisch. Among them, three (2.1-2.3) were new, while nine compounds (2.4-2.12) were reported from this plant for the first time. Most compounds were tested for their cytotoxic effects, and compounds 2.6-2.9 were found to show moderate cytotoxicity against MCF-7 human breast cancer cells, SK-Hepl hepatocarcinoma cells, and U20S osteosarcoma. In addition, corosolic acid (2.7) could effectively block Store-Operated Calcium Entry (SOCE).
A. pajaroensis has never been phytochemically and pharmacologically investigated. In this study, twelve compounds (3.1-3.12) were isolated from the organic extract and MeOH extract of A. pajaroensis. The isolated compounds were tested against the human hepatocarcinoma SNU739 cancer cell line, the human neuroblastoma SHSY-5Y cell line, the human gastric carcinoma NUGC-3 cancer cell line and the human breast MCF-7 cancer cell line. Only compounds 3.2 and 3.4 were found to show moderate cytotoxicities.
The fruits of C. spinosa L. have been studied. One alkaloid (4.1), five sesquiterpenoids (4.2-4.6) and four other compounds (4.7-4.10) were isolated. Compounds 4.2-4.5 were obtained from this plant for the first time.
(3) Investigation on chemical constituents and anti-depression effect of X. nigripes
Eleven known compounds (5.1-5.11) were obtained and determined from X. nigripes (K1.) Sacc. The structures included spiro-alkaloids, steroids and isoflavonoids. Some crude fractions and purified compounds could enhance the expression leve of the brain derived neurophic factor (BDNF).
(4) Analysis of compounds in crude extracts using HPLC-DAD- ESI-MS technique
The HPLC-DAD-ESI-MS method was developed for the analysis of complex components of G. dahurica, A. pajaroensis and C. spinosa. The main components were primarily identified by comparison of the tR values, UV spectra and mass spectra with reported literature data.
引文
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