Based On The Establishment And Application HUVECM And Chromatography-mass Spectrometry Drug Screening Model

It is a common practice to separate and purify compounds from the traditional Chinese medicine (TCM) before their chemical characteristics, biological activity and toxicity are studied. Only those available will be given further consideration for animal test and enduringness evaluation. It is beyond argument that this kind of screening for natural compounds from medicinal plants is very important, yet it does not often comply with the philosophy for the administration and study on TCMs. The therapeutic performance of TCM and its compound formulas is supposed to be a result of multiple compounds upon multiple targets under synergistic action, so we are well advised to deem the compounds in TCM and its compound formulas as a whole rather than many individuals, i.e. it is more of a study on the rule for TCM administration experience than a mere compounds concern. At the advantage of GCMS, LCMS techniques and special affinity for active receptors on HUVECM (Human Umbilical Vein Endothelial Cell Membrane), we establish several screening models for active compounds in TCM, with an aim to screen potential TAI (Tumor angiogenesis inhibitor). The applications of the established screening models, as part of the contribution to the sreening methods for TCM, are carried out in the essential oil and alcohol extract of Chinese herb Ligusticum chuanxiong Hort., alcohol extract of Chinese herb Aconitum taipeicum Hand.-Mazz.and alcohol extract of Tie Bang Chui, respectively.The main contents of this dissertation are as follows:Part1:Development of an online method for determining ATPase bioactivity and its application to the study on activity of acceptors on HUVECM.Experiment results reveals that the bioactivity of ATPase decreases with increasing duration time in vitro in buffer solution, while the ATPase on HUVECM remains bioactive over a certain time (at least12h). It indicates the rule about variation of the acceptor activity. Thus HUVECM is eligible for pertinent medicine discovery.Part2:The establishment and application.of the screening model at the advantage of combination of active acceptors on HUVECM with GCMS techniques.HUVECM at different activity levels is incubated with multiple-compound extract of TCM, the content of individual volatile compound in the extract is determined before and after the incubation. With probing the variation of the their content, those potentially active are preliminarily screened.1. The application to the preliminary screening for potentially active compounds in the essential oil of Chinese herb Ligusticum chuanxiong Hort.According to the experimental results, there are at least28compounds which show specific affinity for active acceptors on HUVECM, they are:terpineol, terpinen, citral, thujopsene, di-epi-.alpha.-cedrene, gamma.-muurolene, chamigrene, patchoulene, cuparene, delta.-cadinene, alpha.-bergamotene, alpha.-muurolene, alpha.-calacorene, asarone, squalene, salicylic acid methyl ester, sabinene, alpha.-limonene, eugenol, alpha.-cedrene, caryophyllene, humulene, alpha.-curcumen, butylidenephthalide, neocnidilide, ligustilide, dibutylphthalate. Many of the compounds screened out have been reported to have special pharmacological activities, though our conclusion needs further confirmation with more facts.2. The applicaton to the preliminary screening for potentially active compounds in alcohol extract of Chinese herb Aconitum taipeicum Hand.-Mazz.and alcohol extract of Tie Bang Chui, respectively.Experimental results reveal that there are only a few volatile compounds in the alcohol extracts that show specific affinity, with nonacosane and one unidentified compound for Aconitum taipeicum Hand.-Mazz, and tricosane, tetracosane and acetic acid-octadecyl ester for Tie Bang Chui.Part3:The establishment of HUVEC-CMCSP based LC-GCMS screening model and its application to the alcohol extract of Chinese herb Ligusticum chuanxiong Hort.A mini-chromatographic column based on HUVEC-CMCSP (Cell membrane chromatographic stationary phase) is prepared for a special chromatographic column, with which an online LC system combined with GCMS is established for screening pontentially active compounds in alcohol extract of Chinese herb Ligusticum chuanxiong Hort. There are at least4compounds that show specific affinity for active acceptors on HUVECM, they are vanillin, ligustrazine, tetramethylpyrazine and one unidentified compound, repectively. Both ligustrazine and tetramethylpyrazine show unilateral synergistic action on vanillin, and no apparent additivity is observed between the two actions. Ligustrazine is found to show unilateral synergistic action on both vanillin and tetramethylpyrazine, it is likely that there are still some other unknown compounds in the extract that show synergistic action on both vanillin and tetramethylpyrazine. Nothing was found showing unilateral synergistic action on ligustrazine.Part4:The establishment and application of HUVEC-CMCSP-based LC-MS2screening system for potentially active compounds.1. Free fatty acids profile analysis of alcohol extracts of Aconitum taipeicum Hand.-Mazz. and of Tie Bang Chui with GCMSThe main free fatty acids in the extracts of Aconitum taipeicum Hand.-Mazz. and of Tie Bang Chui are identified and quantified, respectively, with a GCMS method. Experimental results show both Chinese herbs are all abundant in6kinds of free fatty acids, linoleic acid (13.89±0.36mg/g), palmitic acid (12.47±0.32mg/g) and linolenic acid (5.34±0.12mg/g) top the content list of alcohol extracts of Aconitum taipeicum Hand.-Mazz., while for the same top three of Tie Bang Chui, their contents are8.49±0.22mg/g,7.16±0.18mg/g,6.19±0.16mg/g, respectively. The unsaturated free fatty acids account for70%of the total content of free fatty acids.2. The application of HUVEC-CMCSP-based LC-MS2screening system to alcohol extracts of Aconitum taipeicum Hand.-Mazz. and of Tie Bang Chui.LC-MS2screening system for potentially active compounds is established based on the special and high performance of the chromatographic column that is filled with HUVEC-CMCSP, and its application to water-soluble compounds in both alcohol extracts of Aconitum taipeicum Hand.-Mazz. and of Tie Bang Chui. are carried out. Experimental results show that there seems to be a similarity of both components and binding behaviors on acceptors between the two Chinese herbs. Some compounds with specific affinity for active acceptors on HUVECM are found and identified, they include senkirkine,3-Acetylbeiwutine, daunorubicin, esorubicin,8-O-ole-benzoyldeoxyaconine and/or8-O-lino-benzoylmesaconine, etc. Some compounds screened out is reported to have special pharmacological activities or severe toxicity, this screening system has showed its difference from the traditional ones, though it still requires further improvement and perfection.