| Summary: | 碩士 === 台北醫學院 === 細胞及分子生物研究所 === 87 === The damages of human tissues caused by reactive oxygen species (ROS) (some of which are free radicals) are the most critical factors to many chronic diseases. The cases, for example, reduction of the fluidity of cell membrane caused by the oxidation of unsaturated fatty acids; the coronary arteriosclerosis derived from the oxidized low density lipoprotein (oxidized LDL); moreover, cancer due to the damage of DNA, are all by reason of ROS. Presently, most of the antioxidants that have relatively strong activity and long lasting effect are mostly artificial, e.g. butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). In contrast, most of the nature antioxidants such as β-carotene and α-tocopherol, have lower activity and shorter in lasting effect than the artificial. For this reason, it is a trend and an urgent need to search for more potent and reliable antioxidants from nature sources in preventing people from suffering disease caused by ROS. Microbes are good sources in production of industrially useful secondary metabolites due to its predominance in mass production. Integrating the above-mentioned background, our study has focused in search of novel and target-specific nature antioxidants from microbial resources. For this purpose, we firstly collected a number of domestic soil samples from different areas, and resulted in isolating nearly 200 strains of actinomycetes for following research. Small-scale fermentation (60ml culture medium in a 500 ml Sakaguchi flask) was carried out followed by antioxidant screening using lipid auto-oxidation and TBARS (thiobarbituric acid reactive substances) as the detection methods. Six experimental stages were designed in our procedure, those are: (1) metabolite recovery and tested sample preparation: the metabolites were recovered by Amberlite XAD-2 absorption, followed by MeOH elution and solvent concentration; (2) antioxidant detection and strain selection: samples were quantitatively analyzed by the inhibition effects on formation of lipid peroxides (primary oxidants) and TBARS (secondary oxidants) to screen the strains able to produce antioxidants. According to the established screening methods, we chose out a strain of actinomycetes, designed as AMBL-029C; (3) antioxidant purification: the fermentation broth was recovered by a series of separation techniques including centrifugation, Amberlite XAD-2 absorption, followed by MeOH elution and a successive TLC purification. The resulting primary purified compound [temperately designed as AMBL-029C-TS (TS)] was further analyzed by HPLC to monitor its purity; (4) physical-chemical characteristics: judging from the acid-base fractionation experiments, and the pH and temperature stability tests, the compound was deduced to be a acidic compound with the properties of low polarity and highly pH and temperature stable; (5) mechanism of the antioxidant: in comparison with some other known antioxidants, TS was subjected to investigate its antioxidant mechanism, together with BHT, -tocopherol, as well as two streptomyces metabolites, homogentisic acid (HA) and -phenylpyruvic acid (-PPA), which were previously isolated as the natural antioxidants in our laboratory. The research items included: 1. Scavenging of α,α-dipheny-β-picryhydrazyl (DPPH); 2. Chelating of metal ions; 3. Scavenging of superoxide anion; In the results, HA and TS, but notβ-PPA, exhibited inhibitory activity against lipid peroxidation, and HA was comparable in potency withα-Toc. Moreover, all of HA, β-PPA and TS had the ability in scavenging superoxide anion radical (˙O2-) and DPPH, but were unable to scavenge metal ions, such as Fe+2. (6) Evaluation of the antioxidant activity in clinical uses: four experimental models were selected to evaluate the antioxidant activities of TS, HA and -PPA. These models included: (1) Rat primary hepatocyte system: to evaluate the protective ability of tested antioxidants toward cells against oxidative stress; (2) LDL oxidation system: to evaluate the ability of tested antioxidants in preventing LDL from oxidation; (3) HL-60 cells apoptosis system: to evaluate the ability of tested antioxidants in inducing and/or preventing HL-60 cells from apoptosis; (4) Telomerase inhibition system: to evaluate the ability of tested antioxidants in inhibiting telomerase activity. The results indicated that the high dose (5mM) of β-PPA exhibited more potent activity thanα-Toc in preventing LDL from being oxidized. However, HA and TS, evenβ-PPA in a low dose (1mM), didn’t show any effect against LDL oxidation. In addition, HA, β-PPA and TS could induce apoptosis of HL-60 cells. Nevertheless, as to the antioxidant activity, HA andβ-PPA could also protect cells against oxidative stress within a relatively short duration time when compared to α-Toc. TS didn’t show any effect against oxidative stress in the range of our treated doses. In the remanding experiments, all of HA, β-PPA and TS didn’t show any activity in the protection of rat primary hepatocyte cells against H2O2-induced oxidative stress, and in the inhibition of telomerase.
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