Screening Of The Active Components From Traditional Chinese Medicine Using Online Chromatography System

With the development and modernization of traditional Chinese medicines (TCMs), screening active components in complex systes have introduced a key resolution to explore new drugs in TCMs. However, one TCM herb may contain up to hundreds or even thousands of different constituents, but only a few compounds are responsible for the pharmaceutical or toxic effects. With the interference of other components in TCMs, it is extremely difficult to screen the bioactive ingredients rapidly and efficiently.By far, a variety of strategies have been developed to separate and analyze the constituents of TCM for identification of bioactive compounds and quality control, including screening with animal models, cell bioassays, enzyme/receptor models and traditional chromatographies. The establishment and application of these models play a positive role for screening of active components. However, these methods suffer from some shortcomings such as weak selectivity and lack of the information of screened components of TCMs. So it is often time-consuming and inefficient to separate lead compound from TCMs following with the bioassay guidance, resulting from the poor affinity and selectivity of conventional procedures.This paper mainly focuses on the research of construction and application of some biochromatographic models, and aims to establish some new methods for screening bioactive components. The paper contains up to six chapters. The author’s main contributions are as follows: 1. In this paper we have, for the first time, established a lipid raft stationary phase chromatography (LRSC) to develop a high throughput screening of potential therapeutic compounds for cancer treatment. The stable lipid rafts abundant with one receptor tyrosine kinase, tropomyosin-related kinase A (TrkA) obtained from U251cells were constructed to prepare the stationary phase. An unrelated TrkA inhibitor (gemcitabine) and TrkA targeted anti-tumor drugs (lestaurtinib and gefitinib) were analyzed on LRSC to evaluate the specific affinity of the column. Using lestaurtinib as a model, the retention behavior was investigated and the thermodynamic parameters of transfer from mobile phase to stationary phase were discussed. The results showed that the ideal resolution of lestaurtinib on lipid raft chromatography could be achieved with eligible partical size (10～50μm) of stationary phase at a flow rate of0.2mL/min at37℃. The standard enthalpy change (AH0) and entropy change (ΔS) in chromatographic system were determined by Van’t Hoff relationship (lnk-1/T). The binding constants, changes of enthalpy, entropy and Gibbs free energy were obtained as negative value by using lestaurtinib as a model. The retention mechanism of solutes on the prepared stationary phase involves synergistic interaction of hydrogen bonding and van der Waals force.2. The established LRSC was further applied for the screening of potential therapeutic compounds in cancer treatment using Chinese gallnut as a model. The chromatographic column manifested desirable affinity when the ether fraction of Chinese gallnut was injected into LRSC. The established LRSC system can recognize target components through affinity interactions between the components and TrkA signaling pathway on the lipid rafts. The methyl thiazolyl tetrazolium assay confirmed the anti-tumor effect of the screened ether fraction of Chinese gallnut and further proved the selectivity of LRSC on TrkA-targeted drugs. The mode of the newly constructed LRSC was identified as an efficient approach to screen anti-tumor components in TCMs.3. In this study,2,3-dehydrosilymarin, a compound exhibiting remarkable antiradical/antioxidant activity, was synthesized from silymarin for the first time. The structures of its main components were verified by ultra-performance liquid chromatography/mass spectrometry (UPLC-MS) and other spectral analysis. The solubility, radical scavenging capacity and liver protecting activity of2,3-dehydrosilymarin were studied with silybin, dehydrosilybin and silymarin employed as controls. The antioxidant activities of silymarin and its derivatives were measured by determining their reducing activity, scavenging DPPH-free radical, superoxide radical, hydroxyl radical and the Fe2+concentration by reduction experiment. In addition, a rapid screening method, online high-performance liquid chromatography/1,1-dipheny1-2-picrylhydrazyl (DPPH-HPLC) system, was developed for identifying individual antioxidants. Both in-vitro and in-vivo results markedly proved that dehydrosilymarin has decent aqueous solubility and remarkable antiradical/antioxidation capacity. Moreover,2,3-dehydrosilybin and2,3-dehydrosilychristin were identified as the two major active compounds comprising2,3-dehydrosilymarin. Our results suggest that2,3-dehydrosilymarin may be a promising and potent alternative for inhibition of free radical and prevention of oxidation. The established on-line HPLC radical scavenging activity measurement makes it possible to directly identify active constituents in complex matrices.4. An HPLC method and a single wavelength TLC-scanning method were used to determinate the concertration of chlorogenic acid of Herba Artemisiae scopariae extracts. Their free radical scavenging activities were measured with offline UV-2,2-diphenyl-l-picrylhydrazyl (DPPH·) method. The chromatograms were obtained after spraying with DPPH-solution and the peak areas of the spots on TLC were calculated to evaluate the antioxidant capacity of Herba Artemisiae scopariae extracts. The contents of chlorogenic acid determined by TLC-bioautography were consistent with the routine method, and the total integral value measured by bioautography was significantly higher than the single peak area of chlorogenic acid. The results indicated that Herba Artemisiae scopariae had many antioxidant active ingredients including chlorogenic acid. With DPPH as color developing reagent and dual wavelength scanning, TLC-bioautography can be used for fast selecting and evaluating the antioxidant capacity of traditional Chinese medicines.