| Summary: | 碩士 === 國立中興大學 === 食品科學系 === 89 === 3,3”-Dihydroxyterphenyllin (3,3”-di-OH-terphenyllin) and 3- hydroxyterphenyllin (3-OH-terphenyllin), both terphenyl compounds, are antioxidative secondary metabolites produced by Aspergillus candidus. The antioxidant mechanisms of 3,3”-di-OH-terphenyllin and 3-OH- terphenyllin which prevent lipid peroxidation have not been well investigated. The objectives of this study were to investigate the antioxidant activities, safety, and cell protective effects of 3,3”-di-OH- terphenyllin and 3-OH-terphenyllin. The antioxidant properties of 3,3”- di-OH-terphenyllin and 3-OH-terphenyllin were evaluated by oxygen- radical absorbance capacity assay (ORAC assay), reducing power, free radicals scavenging effect, ferrous iron chelating effect, and the safety of which was investigated in Int 407 by trypan blue exclusion, MTT test, melondialdehyde (MDA) formation and DNA damage. Hydrogen peroxide was used as pro-oxidant to investigate the protective effects of 3,3”-di-OH-terphenyllin and 3-OH-terphenyllin against oxidative damage in Int 407 cells.
The total antioxidant capacity of 3,3”-di-OH-terphenyllin and 3-OH- terphenyllin (200 mg/mL) measured by the ORAC assay was found to be equal to 2.5 and 1.5 mM trolox, respectively. The reducing power of 3,3”-di-OH-terphenyllin and 3-OH-terphenyllin was found to be 50 and 30% to that of gallic acid. The scavenging effects of 3,3”-di-OH- terphenyllin to a,a-Diphenyl-b-picrylhydrazyl (DPPH․), 2,2’-azino- bis[3-ethylbenzthia- zoline-6-sulfonic acid ] (ABTS+․) and nitric oxide were 80, 60 and 24%, respectively, and were higher than that of 3-OH- terphenyllin (50,10 and 5 %). The superoxide anion scavenging activity of 3,3”-di-OH-terphenyllin (41%), however, was lower than that of 3-OH-terphenyllin (52%). Neither 3,3”-di-OH-terphenyllin and 3-OH- terphenyllin had any chelating ability with respect to ferrous iron. As for safety evaluation, both trypan blue exclusion and MTT test showed high viability (>94%) when Int 407 cells were treated with those two compounds for 20 h at 100 mg/mL. There was no significant increase (p >0.05) in melondialdehyde (MDA) formation or DNA damage in Int 407 after treatment under the same experimental conditions. These results suggest that the mechanisms of antioxidant activity of 3,3”-di-OH- terphenyllin and 3-OH-terphenyllin includ free radical scavenging and reducing power.
The protective effects of 3,3”-di-OH-terphenyllin and 3-OH- terphenyllin against oxidative damage in Int 407 cells induced by hydrogen peroxide were evaluated. Int 407 cells were pre-cultured with 3,3”-di-OH-terphenyllin and 3-OH-terphenyllin at 100 mg/mL for 20 h. After the samples were removed, the cells were treated with 50 mM H2O2 for 30 min, and then the oxidative damage levels were measured. The results showed that H2O2 caused lactate dehydrogenase (LDH) leakage, MDA formation and DNA damage in Int 407 to increase; however, the addition of 3,3”-di-OH-terphenyllin and 3-OH-terphenyllin significantly reduced those increasing (p<0.05). Intracellular reactive oxygen species (ROS) formation in 3,3”-di-OH-terphenyllin and 3-OH-terphenyllin pre- cultured Int 407 cells decreased (30 and 35%, respectively). The lag time of 3,3”-di-OH-terphenyllin and 3-OH-terphenyllin pre-cultured Int 407 cells during lipid peroxidation was 60 min compared with the control (0 min). The activity of glutathione peroxidase (GPx) and catalase (Cat) in 3,3”-di-OH-terphenyllin pre-cultured Int 407 cells increased 25 and 33%, respectively; however, Cat increased 30% in 3-OH- terphenyllin pre-cultured Int 407 cells. Int 407 cells pre-cultured with 3,3”-di-OH-terphenyllin and 3-OH-terphenyllin caused 36 and 21% decrease, respectively in glutathione reductase (GRd) activity . The intracellular glutathione (GSH) level was invariable (p>0.05), but the oxidized glutathione (GSSG) was increased in Int 407 cells pre-incubated with those two compounds. These findings suggest that 3,3”-di-OH-terphenyllin and 3-OH-terphenyllin have protective ability against oxidative damage in Int 407 cells, and that the protective effects might be associated with the ability to reduce MDA and ROS formation, and raise Cat activity.
Based on the results of this study, 3,3”-di-OH-terphenyllin and 3-OH-terphenyllin are potent antioxidants produced from Aspergillus candidus.
Keywords: Aspergillus candidus, 3,3”-Dihydroxyterphenyllin, 3-hydroxyterphenyllin, antioxidant, Int 407, oxidative damage; oxidative stress
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