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Antioxidative activity of 3,3"-dihydroxyterphenyllin and 3-hydroxyterphenyllin, two metabolites from Aspergillus candidus, and their protective effects against oxidative damage in Int 407 cells
|關鍵字:||Aspergillus candidus;Aspergillus candidus;antioxidant;Int 407;oxidative damage;oxidative stress;3;3”-Dihydroxyterphenyllin;3-hydroxyterphenyllin;抗氧化劑;腸道細胞Int 407;氧化傷害;氧化壓力;3-hydroxyterphenyllin;3;3”-Dihydroxyterphenyllin||出版社:||食品科學系||摘要:||
3,3”-Dihydroxyterphenyllin (3,3”-DHT) 及3-hydroxyterphenyllin (3-HT)為Aspergillus candidus具有抗氧化性之二次代謝產物，屬三聯苯類化合物，具有延遲油脂過氧化之抗氧化特性，然而3,3”-DHT及3-HT的抗氧化機制仍不明。此外3,3”-DHT及3-HT在安全性方面的探討仍較缺乏。因此本研究以氧自由基吸收能力分析法(ORAC assay)、還原力、螯合金屬離子及清除自由基等方法，來探討其抗氧化特性以及機制；另外以trypan blue exclusion、3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tertrazolium bromide (MTT) assay、melondialdehyde (MDA)生成及DNA損傷，評估3,3”-DHT及3-HT對腸道細胞株Int 407之安全性。最後以過氧化氫誘發Int 407氧化傷害，並探討3,3”-DHT及3-HT對Int 407之保護作用及對抗氧化酵素活性之影響。
3,3”-DHT及3-HT 200 mg/mL其總抗氧化力分別為2.5及1.5 mM trolox當量。3,3”-DHT及3-HT之還原力約為沒食子酸的50 及30%。3,3”-DHT (200 mg/ml)對DPPH及ABTS+自由基的清除率分別為80 %及96 %；此外能抑制41 %的超氧陰離子生成及抑制24 %的一氧化氮生成。同濃度的3-HT清除自由基之能力則較弱，對DPPH及ABTS+自由基分別為50 %及10 %；抑制超氧陰離子生成為52 %較3,3”-DHT高，但不具清除一氧化氮之能力。3,3”-DHT及3-HT在100 mg/mL以內不會造成Int 407存活率之下降，亦不會增加MDA的生成及造成DNA損傷。由本研究結果可知3,3”-DHT及3-HT具有良好的抗氧化性，其抗氧化機制與其清除自由基能力及還原力有關。3,3”-DHT及3-HT不會影響Int 407之正常生長，亦不會傷害Int 407之細胞膜及DNA。
在保護Int 407氧化傷害方面，3,3”-DHT及3-HT (100 mg/mL)分別與Int 407培養20小時後，去除樣品加入細胞氧化傷害劑－H2O2及脂質過氧化起始劑－Fe(Ⅲ)/ADP，並以LDH滲漏率、胞內活性氧變化、MDA生成、細胞對脂質過氧化的感受度(susceptibility) 及DNA損傷，來評估樣品對Int 407的保護效果。Int 407經與3,3”-DHT及3-HT (100 mg/mL)預培養後，LDH滲漏率降低，分別達75及85%。同樣的條件下，胞內ROS的降低比率分別為30與35 %。MDA生成量分別由1.3和1.2降為0.78及0.72 nmole/mg protein。由螢光強度顯示，與3,3”-DHT或3-HT反應後的Int 407，脂質過氧化起始時間較控制組延遲60分鐘。而由彗星試驗法發現，tail moment值由22分別降低至13與14。與3,3”-DHT作用20 h後，Int 407胞內之glutathione peroxidase (GPx)及catalase (Cat)活性分別上升25%及33%，而glutathione reductase (GRd)活性下降36%。Int 407與3-HT作用後，Cat活性上升(30%)而GRd活性下降(21%)。此外兩種化合物與Int 407預培養並不會影響胞內glutathione (GSH)之含量，然而會使氧化態GSH (GSSG)含量增加。由本研究結果顯示3,3”-DHT及3-HT與Int 407預培養後，增加Int 407對抗氧化傷害之能力，推測與降低胞內ROS、減少脂質過氧化物生成及提昇胞內Cat活性有關。
綜合以上所述，由Aspergillus candidus所生產的3,3”-DHT及3-HT，具有良好的抗氧化性且其機制與還原力、清除自由機能力有關。在測試系統下，兩者對Int 407均不具細胞毒性與基因毒性。此外3,3”-DHT及3-HT預培養能降低過氧化氫誘發Int 407之氧化傷害，並能影響Int 407之抗氧化防禦狀態，使Int 407對抗過氧化氫氧化傷害之能力提昇。因此3,3”-DHT及3-HT具有開發作為微生物來源抗氧化劑之潛力。
關鍵字：Aspergillus candidus、3,3”-Dihydroxyterphenyllin、3-hydroxyterphenyllin、抗氧化劑、腸道細胞Int 407、氧化傷害、氧化壓力
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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