| Abstract: | 白藜蘆醇(resveratrol; Res)是一種存在於葡萄皮及紅酒當中的多酚類，已知具有抗癌、抗糖尿病、抗氧化、抗發炎、抗老化、心臟保護和神經保護生理功效。本實驗室先前的研究證實Res在細胞培養基中並不穩定，其降解的程度受溶液pH值所影響，並且培養基中的HCO3-是誘發Res氧化降解的主因，此外，透過在培養基中添加超氧陰離子歧化?(superoxide dismutase; SOD)可以完全避免Res的氧化降解。然而，HCO3-誘發Res在水溶液中氧化降解的機轉，目前並不清楚？本研究假說碳酸根自由基在HCO3-誘發Res在水溶液中氧化降解的機轉中扮演重要的角色。我們將Res置入不同的pH值的NaHCO3或NaOH溶液中反應，利用electron paramagnetic resonance (EPR)檢測系統中能生成5,5'-dimethyl-1-pirroline-N-oxide (DMPO)-OH訊號的自由基，我們也利用HPLC測定Res降解的程度，並且利用亞牛磺酸氧化生成牛磺酸的方法來測定系統中是否產生碳酸根自由基。實驗結果顯示，Res不論在鹼性(pH=8.74)的NaOH或是NaHCO3水溶液中，都會有DMPO-OH訊號的產生，但若將溶調至弱鹼性(pH=7.4)及酸性(pH=6)時，僅剩下在NaHCO3水溶液中會有DMPO-OH的訊號。此外，在系統中可測得牛磺酸的生成，證明碳酸根自由基的存在，並且Res在鹼性、弱鹼性或是酸性的NaHCO3水溶液中，皆可以產生碳酸根自由基，而Res在鹼性、弱鹼性或是酸性的NaOH水溶液中，卻只有在鹼性的狀況可產生少量的羥基自由基，顯示碳酸根自由基在Res於酸性及弱鹼性的NaHCO3水溶液中降解扮演主要角色。而添加SOD抑制碳酸根和羥基自由基的生成，則可以抑制Res在NaHCO3水溶液中降解，顯示碳酸根和羥基自由基參與Res在NaHCO3水溶液中的降解。綜合上述的結果顯示碳酸根自由基在HCO3-誘發Res氧化降解扮演重要角色。
Resveratrol (Res) is a kind of polyphenol existing in both grape skins and red wine, which has several biological benefits, for instance, anti-cancer, anti-diabetes, anti-oxidative, anti-inflammatory, anti-aging, cardioprotective and neuroprotective effects. According to our previous reports, we have demonstrated that Res is unstable in culture media, the Res degradation is affected by solution pH, and HCO3- is the main cause to induce the oxidative degradation of Res. Moreover, adding superoxide dismutase (SOD) can completely avoid Res degradation in culture media. However, the mechanism of HCO3--induced oxidative degradation of Res in aqueous solution remains unclear. In the study, we hypothesized that the carbonate radical plays an important role in the mechanism of HCO3--induced Res oxidative degradation in aqueous solution. We incubated Res in NaHCO3 or NaOH solutions with different pH values to react. We used HPLC to determine the remaining level of Res after degradation and to investigate whether carbonate radicals were generated in the system by using the property of hypotaurine that can be oxidized to taurine by carbonate radicals. In addition, we also used electron paramagnetic resonance (EPR) to detect free radicals that can generate 5,5'-dimethyl-1-pirroline-N-oxide (DMPO)-OH signals. The results showed that Res would generate DMPO-OH signals in EPR no matter it is in the NaOH solution or NaHCO3 solution with pH=8.74. If we adjust the solution to weakly alkaline and acidic, the DMPO-OH signal would only produce in the NaHCO3 solution but not in the NaOH solution. In addition, the production of taurine ccould be measured in the system to deomonstrate the existence of carbonate radicals. Moreover, Res could generate carbonate radicals in the alkaline, weakly alkaline, or acidic NaHCO3 solution. However, Res can generate a small amount of hydroxyl radicals only in alkaline NaOH aqueous solution but not in weakly alkaline or acidic NaOH aqueous solution. This result indicates that carbonate radicals play a crucial role in the degradation of Res in acidic and weakly basic NaHCO3 solution. Furthermore, the addition of SOD to inhibit the formation of carbonate and hydroxyl radicals could inhibit the degradation of Res in NaHCO3 solutions, indicating that carbonate or hydroxyl radicals were involved in the degradation of Res in NaHCO3 solution. In summary, carbonate radicals play a crucial role in the mechanisms of HCO3--induced Res oxidative degradation. |
