| Summary: | 碩士 === 台北醫學院 === 醫學研究所 === 89 === Antioxidants have at present been recognized to play two major roles in industrial applications: (1) healthy foods and food additives; (2) clinically useful supplements for the prevention of chronic diseases, such as cancers and cardiovascular diseases. Due to the social demands and the close relationship between antioxidants and the physiological function of our bodies, the objective of our research was thus aimed at new antioxidant developments in order to improve the safety and usefulness of them. Actinomycetes have been known to be a good source for the development of biologically active compounds, we therefore set these microbes as the screening targets in this experiments.
Beginning to our job, we firstly collected a number of domestic soil samples from different areas, and isolated nearly 200 strains of actinomycetes. The isolates were then cultured and fermented by conventional process in order to screen antioxidant-producing strains. The samples were recovered from the microbial metabolites followed by EtOAc extraction and then evaporation to dryness in vacuo. After that, they were subjected to a series of tests in their antioxidative activity. The activity of the prepared samples screened by measurement of their DPPH radical scavenging ability, activity in the inhibition of thiobarbituric acid reactive substances (TBARS) formation, and the potency of reducing power. With above-mentioned categories, we successfully chose out a highly antioxidant producing strain, named AMBL-019C. AMBL-019C was furthermore confirmed to be a strain of Streptomyces sp. in accordance with its taxonomical characteristics. We then lavishly fermented AMBL-019C in YMD + 0.1% Tween80 medium and meanwhile, extracted its metabolites with EtOAc. In the further steps of purification, we used acid-base fractionation combined with antioxidative activity analysis to pick up the neutral phase, an active part of the fraction collected from the crude material. After that, TLC was applied to sample analysis and resulted in obtaining the Rf value of active metabolites. The active part was collected and subsequently subjected to column chromatography ( Hexane：EtOAc 96：4) for further large-scale purification and conducted to gernerate a pale yellow crystal, designed as AMBL-019C-TS (TS). In order to confirm the purity of the target compound, HPLC was further performed and TS was consequently as well as detected at retention time of 7.30~7.70 min with high purity. TS was soluble in methanol and less polar solvents but insoluble in water. The integrate results of mass spectroscopy, Ultraviolet-visible spectroscopy, 1H-NMR, 13C-NMR and 1H-1H 2D NMR to TS showed that it was a benzoic and keto group containing compound with molecule weight of 236. As for antioxidative activity, TS showed an remarkable inhibitory activity against LDL oxidation in a dose-dependent manner over a concentration ranging from 2.0 to 10.0 mM. Since oxidative modification of LDL has proved to play a crucial role in atherosclerosis and coronary artery disease (CAD), our experiments implied the potential of TS in the prevention of CAD, though supplementary animal model has not yet been established. As regards the anti-cellular oxidative activity test of Balb-3T3 cells, TS also showed protective effect to the cells, however, no cytotoxicity was observed. Therefore, further study of TS on the antioxidative activity in cellular and physiological level as well as its mechanism and applications in human will be a research topic with value in the future.
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