厚朴之酚類化合物及金奈米輔助聚合酶連鎖 反應產物之毛細管電泳分析

Abstract

本研究第一部份利用毛細管電泳進行厚朴(houpo)之兩種活性成份厚朴酚(magnolol)及和厚朴酚(honokiol)之分析，並以UV吸收及雷射誘導螢光偵測方式來進行分析。最佳化的分析條件各為：pH為9.6之45 mM與10 mM硼酸鈉(sodium tetraborate)緩衝液，分析時間各於七分鐘和三分鐘內完成，且偵測極限各可達μM及nM level。文獻指出厚朴酚會導致細胞凋亡，於是本研究培養結腸癌細胞(WiDr)觀察厚朴酚對於細胞作用之時間與濃度的關係。亦將80 μM的厚朴酚加入至細胞培養液一天後，分析培養液之成分，發現僅約25%之厚朴酚存留於培養基中。 本研究第二部份利用毛細管電泳分析金奈米粒子輔助聚合酶連鎖反應(nanogold-assisted polymerase chain reaction)之產物。以粒線體與第九凝血因子(factor IX)為PCR放大之產物，加入13 nm的金奈米粒子(gold nanoparticles, GNPs)或聚合物(poly(ethylene oxide), PEO)於聚合酶連鎖反應中，結果發現適量的加入1.0 μl的GNPs和0.5%的 PEO (8 MDa)，其複製效率(efficiency)增加。此外，改變熔融溫度(melting temperature, Tm)，亦發現加入13 nm GNPs於PCR中可提高其專一性(specificity)。

This thesis was consisted of two parts. In the first part, two active compounds, honokiol and magnolol, were analyzed by capillary electrophoresis with UV and laser-induced fluorescence detection. The optimized conditions of two detection methods were 45 mM and 10 mM tetraborate (pH 9.6), and the separations were accomplished within seven and three minutes, respectively. The limits of detection (LOD) were in μM and nM range. Furthermore, we studied the concentration effect and exposure of magnolol on WiDr cell cultrues. Also, about 25% of 80 μM magnolol remained in the cell medium after being added into cell medium. In the second part, 13-nm gold nanoparticles and poly(ethylene oxide) were added in PCR and the PCR products were analyzed by capillary electrophoresis. The amplified products were mitochondria DNA (6 kbp) and factor IX (20 kbp). The results showed that the addition of gold nanoparticles and poly(ethylene oxide) could enhance the yield and that the adjustment of the melting temperature could increase the specificity in the presence of gold nanoparitcles in PCR.