4.2 Part two: Effects of epigallocatechin-3-gallate on mitochondrial integrity and antioxidative enzyme activity in aging process of human fibroblast (Paper II) 4.2.1 Introduction As stated in Section 1.3 the free radical theory of aging, the oxidative damages to many biological macromolecules as a result of ROS attack is probably a direct cause of cell senescence [89, 155]. And as justified in Section 3.1 Part one, mitochondrial dysfunction is probably a major underlying event in aging. Mitochondria are the main resource of intracellular ROS, and at the same time they are subjected to ROS attack themselves. A number of age-related oxidative damages have been identified in mtDNA, especially within the mtDNA control region for replication [156]. ROS also causes damage to mitochondrial membrane, resulting in a decreased mitochondrial membrane potential [146]. The impairment of mitochondria concomitantly causes even greater leakage of ROS, leading to the formation of a vicious cycle [136]. On the other hand, the antioxidant defense system is considered to involve in the scavenging of ROS and thus protect organisms against oxidative damage. Generally, the primary enzymatic antioxidant defense system, including CAT, GPx, SOD1 and SOD2, is the firstline defense to detoxify ROS. Meanwhile, components from the non-enzymatic antioxidant defense system such as vitamin C and E, co-enzyme Q10 and other small molecules and compounds which are better known as ‘nutrition supplements’, not only participate in radical scavenging directly, but also serve as essential cofactors for various enzymes that decrease oxidative stress. Researches 79 aim to delay the aging process are usually based on three principles. The first one is to control the ROS production, which can be achieved by calorie restriction [157] or manipulating the oxygen concentration [158]. The second is to reinforce the enzymatic antioxidant system in transgenic organisms or over-expression of antioxidative enzymes [80]. A third alternative is to enhance the non-enzymatic antioxidant system through pharmacological administration or dietary supplementation, so that the non-enzymatic antioxidant system could compensate for the deficiency of the enzymatic system, or both the non-enzymatic and enzymatic systems could work synergically and effectively. Epigallocatechin-3-gallate (EGCG), the main component of the green tea extract, is well-known for its radical and oxidant scavenging activity [159] as well as its chemotherapeutic properties [87, 88], whilst its anti-aging effect has little been known yet. In this study, therefore, we attempt to fill up this gap by extrapolating the anti-aging effect of EGCG on the human diploid fibroblast (HDF), a wellestablished model for cellular aging studies [160, 161]. The LC50 value of EGCG and its anti- and pro-oxidant effects are examined initially. Then in the short term study, HDF is exposed to H2O2 induced oxidative stress and pre-senescence, and in the long term study, HDF is continuously cultured till they reach replicative senescence. In both of the approaches, ROS accumulation, mitochondrial integrity and antioxidative enzyme regulation are studied in HDF in the presence or absence of EGCG. 80 4.2.2 Results Cytotoxicity of EGCG In order to determine the cytotoxicity of EGCG, both young (PDL20-30) and old (PDL>45) HDF grown in 96 well plates were treated with 0, 1, 2, 3, 6.25, 12.5, 25, 50 and 100 μM of EGCG for up to days. Cell viability result shows that for the young HDF, 50 μM EGCG significantly (p . two: Effects of epigallocatechin- 3- gallate on mitochondrial integrity and antioxidative enzyme activity in aging process of human fibroblast (Paper II) 4.2 .1 Introduction As stated in Section. cellular aging studies [16 0, 16 1]. The LC50 value of EGCG and its anti- and pro-oxidant effects are examined initially. Then in the short term study, HDF is exposed to H 2 O 2 induced oxidative. as 0, 6.25, 12 .5, 25, 50 and 10 0 μM were added into the MEM. For the first 30 min, 10 0 μM of EGCG cleared about 32 % free radicals of DPPH but its radical scavenging activity declined over time.