抑制Resveratrol在培養基中降解的方法與其在生理環境降解的可能性

白藜蘆醇(Resveratrol; Res)是一種存在於葡萄皮及紅酒的多酚類，具有抗氧化、抗發炎、抗糖尿病、抗癌、心臟保護、神經保護等功效。本研究目的為(1)發展一個可以抑制Res在培養基中氧化降解的方法，以減少細胞模式研究時的干擾。(2)瞭解NaHCO3誘發Res在水溶液中降解的產物及機制。(3)得知Res在正常生理情況下，經胰液及血中NaHCO3催化是否會降解產生具有功能性的Res降解產物。我們以HPLC研究如何避免Res降解；利用methanol沉澱蛋白的方法，探討Res-FBS對於Res降解的影響；以波長265 nm分析經NaHCO3所誘發的降解產物，並且探討NaHCO3誘發降解的機轉與Res在體內是否會降解。結果發現在DMEM中額外加入≥ 5 U/mL SOD或同時加入10 % FBS及1 mM pyruvate，可以完全抑制Res在培養基降解。Res-FBS複合物的形成對抑制Res降解的能力有限。此外，HPLC的分析可以發現三個可能降解物及一個未知衍生物的波峰，而且以不同的酸性溶液調整成pH = 7.4，對於Res在NaHCO3溶液中降解會有不同的影響。另外，我們發現Res在模擬胰液或大鼠血漿皆不會降解，而模擬胰液中含有121.3 ± 2.0 U/mL的SOD活性，若將模擬胰液的SOD過濾去除後，Res則發生降解情形。本研究證實SOD具有抑制Res降解的能力，並且成功開發避免Res在培養基中降解的方法。此外，本論文發現Res的降解產物可以被偵測以及影響Res降解的因素並非只有pH值，而且我們發現Res在血漿中並不會降解，以及內源性的SOD活性可以避免Res在模擬胰液中降解，顯示可能在體內不會有Res的降解產物。

Resveratrol (Res) is one kind of polyphenols in grape skins and red wine, which has several biological benefits, for instance, anti-oxidantive, anti-inflammatory, anti-diabetic, anti-cancer, cardioprotective and neuroprotective effects. The aim of this study was to (1) develop a method that inhibits the oxidative degradation of Res in the media to reduce interference in cell model studies, (2) understand the products and mechanisms of NaHCO3 induced degradation of Res in aqueous solution, and (3) find out under normal physiological environments, whether Res will degrade through pancreatic juice and blood NaHCO3 to produce the functional degradation products of Res.We investigated how to avoid Res degradation by HPLC. The method of precipitation of protein by methanol was used to investigate the effect of Res-FBS on the degradation of Res. The NaHCO3 induced degradation products was analyzed with wavelength 265 nm, and we explored the mechanism of NaHCO3 induced Res degradation and whether Res will degrade in vivo. The results showed that the addition of ≥ 5 U/mL SOD or adding 10 % FBS and 1 mM pyruvate in DMEM completely avoided the Res degradation in media. The ability of Res-FBS complex formation for inhibiting the Res degradation was limited. In addition, the peaks of three degraded products and an unkonwn derivative were found in the HPLC chromatogram, and adjusting the pH to 7.4 with different acidic solutions had different effects on Res degradation in the NaHCO3 solution. Moreover, we found that Res did not degrade in the simulated pancreatic juice or rat plasma, and the simulated pancreatic juice contained 121.3 ± 2.0 U/mL of SOD activity. When the SOD of the simulated pancreatic juice was removed by filtration, Res will degrade. This study demonstrated that SOD had the ability of inhibiting the Res degradation and successfully developed methods for preventing the Res degradation in the culture media. In addition, this study found that the degradation products of Res could be detected by HPLC and the factors affecting the degradation of Res are not only the pH value. Furthermore, we found that Res did not degrade in the plasma, and endogenous SOD activity could prevent Res from degrading in simulated pancreatic juice, indicating that there may be no degradation products of Res in vivo.