| 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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