| Compared with previous screening technology, the striking microfluidic chip analysis technology showed advantages, such as miniaturized sample pretreatment, chemical reaction, derivative, separation and detecting steps, shorter analytical time by the use of fluid and array multi passage, and a better cellular physiological and biochemical analysis of micro environment. Thus microfluidic chip became a potential drug testing platform. During the finding and screening of bioactive components in traditional Chinese medicines which had characteristics of multiple ingredients, multiple targets and combination effects, the researches to find out the active part to extract were always taken. But these methods always had shortcomings such as low throughput, low automation degree, time consuming, high cost and unitarization of indicators which might brought such bad situations like drug target not clear, drug efficacy not stable, and drug potency limited. So, a new method and technology which could synthetically evaluate the drug efficacy and compare the drug potency in order to determine the effective part of traditional Chinese medicines. In this paper, two integrated microfluidic cell chip with two different configurations were proposed and designed which were to complete the Cellular Antioxidant Activity analysis and the 5-lipoxygenase Inhibitory Activity analysis.The main research work and results are as follows:(1) An integrated microfluidic chip with arrayed micro channels which consisted of eight repeat arrayed 6×6 cell culture chamber was designed and fabricated. With this homemade microfluidic chip and test method, the antioxidant effect of various antioxidants, including free radical scavenging efficiency and capacity, can be well distinguished. All those could provide ideas for the modern drug screening and research on high content screening. In experiments, the inhibitory effect of several selected plant antioxidants, such as quercetin, rutin and kaempferol on free radicals including free radical scavenging efficiency and capacity were quantitatively detected. It was shown that the calculated IC50 of quercetin, rutin and kaempferol were 7.2 ±0.1, 52.1±0.2 and 32.6±0.1?mol/L, while the CAA units were 71.4±0.2, 74.3±0.4 and 69.9±0.1, respectively.(2) With the reaction mechanism that the 2', 7'-dichlorodihydrofluorescein(DCFH) could be oxidized to 2', 7'-dichlorofluorescin(DCF) by lipid peroxides which were metabolites of AA by 5-lipoxygenase in white blood cells, a fluorescence-based and cell-based high throughput screening assay method for in vitro screening of 5-lipoxygenase inhibitors was developed. With 2?,7?-dichlorofluorescin diacetate as a fluorescence probe, compounds including arachidonic acid, Ca2+ and ATP as agonists of 5-lipoxygenase, plenty of lipid peroxides which brought the difference in fluorescence between DCFH and DCF were generated. With monitoring of the change of fluorescence for 90 min, the inhibitory activity reflected by the reduction of fluorescent material in cells could be estimated. Caffeic acid were used as positive control which was known as 5-lipoxygenase inhibitor.(3) A homemade integrated microfluidic cell chip with 144 micro-chambers, sandwich cross channels and a size of 128mm×86mm which was suitable for the microplate reader, was proposed and designed. The integration of fluorescent labeling, washing and detection of suspension cells can be easily actualized on the integrated microfluidic cell chip. The operation parameters on chip during the screening of 5-lipoxygenase inhibitors were as follows: the White cells suspension at concentration of 106cells/m L was inoculated into micro-chambers at a fast rate of 50?L/min, and fluorescence probe 2?,7?-dichlorofluorescin diacetate with an initial concentration of 50?mol/L which was added at the rate of 20 ?L/min by dialysis exchange. Five extracts of Common Coltsfoot Flower were tested with this microchip. The 5-lipoxygenase inhibitory capacity of those extracts were: petroleum ether extracts > ethyl acetate extracts > n-butanol extracts > ethanol extracts > water extracts, while the 5-lipoxygenase inhibitory efficiency were: ethyl acetate extracts > petroleum ether extracts > n-butanol extracts > ethanol extracts > water extracts. The results kept pace with the existing results well which was taken on 96-well plates. The results showed that the chip could evaluate the activity of candidate materials in vitro effectively.(4) Based on those two systems,the general analysis and evaluation on Cellular Antioxidant Activity and the 5-lipoxygenase Inhibitory Activity of Common Coltsfoot Flower were as follows. The Cellular Antioxidant Activity analysis, Cell Cytotoxicity and the 5-lipoxygenase Inhibitory Activity analysis of the Common Coltsfoot Flower extracts were taken on the two chip respectively. The Cellular Antioxidant Activity analysis and cell cytotoxicity showed that the ethyl acetate extracts was a better antioxidant with a dose-response dependence and the IC50 was 523±71?g/m L. The n-butanol extracts(IC50=570±29?g/m L) and alcoholic extracts(IC50=1402±110?g/m L) also showed good resistance to oxidation. In the evaluation of the 5-lipoxygenase Inhibitory Activity of those extracts, the outstanding performance of the ethyl acetate extracts(IC50=5±4?g/m L,CAA under Cmax= 67±1) were particularly noticeable, too. The ethyl acetate extracts showed a better ability to inhibit 5-lipoxygenase activity than petroleum ether extracts(IC50=982±30?g/m L,CAA under Cmax= 97±2) which had a higher IC50 value. While the 5-lipoxygenase inhibitory efficiency which could more than 98%, petroleum ether extracts would be better than the ethyl acetate extracts for the CAA unit under the maximum concentration. But when the petroleum ether extracts played the biggest role in effect the activity of 5-lipoxygenase, their cytotoxicity(Cytotoxicity=1383±247?g/m L) on cells was obvious, too. In conclusion, the ethyl acetate extracts was good active site of both the oxidation resistance, and the 5-lipoxygenase inhibition. |
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