摘要
天然产物有效成分是其发挥药效作用的物质基础,对评价中药材的质量、制定中药质量控制标准、优化制剂工艺、揭示中药作用机制、实现中药现代化具有重要意义。
天然产物成分复杂,传统的成分分离-活性筛选模式虽已阐明多种天然产物活性成分,但是这样的分离分析周期长,而且经常会随着逐步分离纯化有效部位的活性越来越弱,最后可能得不到有活性的单体成分,特别是结构不稳定的微量活性成分。因此建立快速分离分析复杂体系中活性化合物的方法势在必行。在本论文中,建立了一系列以高效液相色谱和高速逆流色谱为核心的方法,实现了天然产物复杂体系中活性成分的快速筛选、分析以及快速靶向分离制备研究。
研究内容主要包括以下几个部分：
(1)将HPLC-DPPH-MS/MS联用技术用于快速筛选和识别蒙古蒲公英中抗氧化活性成分。从蒙古蒲公英的醇提物中分析和鉴定了32个具有清除自由基活性的化合物：16个黄酮类衍生物,10个苯丙素类衍生物以及6个羟基苯甲酸类化合物,其中化合物9(isoetin-7-O-β-D-gluco-pyranosyl-2'-O-α-L-arabinopyranoside)、10(isoetin-7-O-β-D-glucopyranosyl-2'-O-α-D-glucopyranoside)和30(mongolicumin A)为新化合物,并首次使用逆流色谱导向分离制备三个新化合物。
(2)建立DPPH/HPLC筛选技术,从中华卷柏乙酸乙酯部位中筛选出8个抗氧化活性成分。采用正己烷-乙酸乙酯-甲醇-水(8:8:9:7, v/v/v/v)体系首次对8个目标化合物进行HSCCC同时制备分离,其中芹菜素和4'-O-甲基-罗伯茨双黄酮为首次从中华卷柏中分离得到。
(3)同时建立DPPH/HPLC离线筛选技术,HPLC-DPPH在线筛选技术和HPLC/DPPH离线筛选技术,比较三种活性筛法方法在快速识别葛花提取液中抗氧化活性成分的差异,三种方法均无需对复杂体系进行预处理,但是DPPH/HPLC技术比其他两种方法具备较高的灵敏度和分辨率且对HPLC中色谱峰的分辨率要求不高。根据活性筛选结果,使用梯度洗脱、固定相推出、循环洗脱等多种模式的HSCCC技术导向制备18个不同极性不同含量的目标异黄酮类活性化合物,其中10个化合物(1个新化合物)为首次从葛花中分离得到。
(4)采用离心超滤/HPLC方法快速筛选出杜仲中与BSA有相互作用的6个活性成分,并使用HSCCC技术导向分离制备6个目标活性化合物。通过荧光分光光度法测定了6个目标化合物以及混合物体系与BSA的相互作用,阐明了活性成分间的相互作用机理：京尼平苷酸单独存在时与BSA无相互作用,而当复杂体系中有绿原酸等化合物存在时,京尼平苷酸与绿原酸等化合物之间发生氢键作用生成京尼平苷酸-绿原酸复合物,从而发生化合物之间的相互作用,使得绿原酸等化合物与BSA的结合浓度降低,而京尼平苷酸表现为一定的结合作用。
Active constituents in natural products are the basis of their pharmacology. They are very important in the evaluation of the quality of the herbs, the establishment of the standard for quality control, the optimization of the preparative technology, the research of the action mechanism and the modernization of traditional Chinese medicine.
Natural products have many active compounds with different polarities and concentrations. Although many active compounds have been isolated from natural products by the conventional activity-guided fractionation, it is true that the method is a time-consuming, labor intensive and expensive process, and often leads to loss of activity during the isolation and purification procedures due to dilution effects or decomposition. In order to avoid the above-mentioned problems, simple, rapid and effective methods to screen and purify potential antioxidants from complex plant extract are essential. A series of methods based on high performance liquid chromatography (HPLC) and high-speed counter-current chromatography (HSCCC) were developed for screening, analysis and target isolation of active compounds from complex mixtures of natural products.
The main contents and results are as follows:
(1) The method based on the HPLC on line scavenging DPPH radical activity is utilized for the rapid screening of radical scavengers from Taraxacum monlogicum extracts. Thirty-two compounds with scavenging radical activity including sixteen flavonoid derivatives, ten phenylpropanoid derivatives and six benzoic acid derivatives have been screened from Taraxacum mongolicum. Compounds9(isoetin-7-O-β-D-glucopyranosyl-2'-O-α-L-arabinopyranoside),10(isoetin-7-O-β-D-glucopyranosyl-2'-O-α-D-glucopyranoside) and30(mongolicumin A) were new compounds, which were finally isolated and purified by HSCCC
(2) DPPH spiking test through HPLC (DPPH-HPLC) was first used to screen antioxidants from Selaginella sinensis, and eight active compounds were screened. Then under the target-guidance of DPPH-HPLC experiment, two flavonoids and six biflavonoids were preparative separated by HSCCC method using n-hexane-ethyl acetate-methanol-water (8:8:9:7, v/v/v/v) as the solvent system. This is the first report on simultaneous separation of eight antioxidant compounds from S. sinensis by HSCCC, moreover, apigenin and4'-O-methyl-robustaflavone were first identified from this plant.
(3) Three main methods based on HPLC analysis combined with DPPH assay, DPPH spiking-HPLC analysis, on-line post-column HPLC-DPPH analysis and HPLC-based DPPH activity profiling, were developed and comparatively studied for rapid screening antioxidants from an ethyl acetate fraction of Pueraria lobata flower. The results indicated that all the three methods could achieve similar information with regard to antioxidants, without the need of preparative isolation techniques. However, there were differences in instrumental set-up, sensitivity and efficiency. Eighteen antioxidants were tentatively screened and identified form P. lobata flower, which were then systematically isolated by HSCCC using several elution modes, such as classical elution, stepwise elution, extrusion elution and recycling elution. Among them, ten compounds including one new compound were first isolated from P. lobata flower.
(4) A centrifugal ultrafiltration-HPLC method was developed to screen and identify bioactive compounds binding with BSA in E. ulmoides leaves. Six active compounds were then preparative isolated by HSCCC. The interaction between active compounds and BSA were investigated by quenching the intrinsic BSA fluorescence. The quenching process of geniposidic acid was easily affected in the presence of other active compounds. The formation of geniposidic acid-chlorogenic acid complex could increase the binding affinity of geniposidic acid with BSA, however, the increased steric hindrance of complex may make phenylpropanoid or flavonoid dissociated from BSA, and then decreased their affinities.
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