麩胱甘肽; 過氧化酶 ; 活性染色新方法之建立及山藥塊莖儲藏性蛋白質對於心血管疾病之預 防–從調節血壓到改善血液流變之研究
中文摘要
為了能夠快速尋找麩胱甘肽過氧化酶 (Glutathione peroxidase, GSH-Px) 的活性，市售的 GSH-Px (Sigma Co., G6137) 在 7.5 % native PAGE 或是 7.5 % SDS-PAGE 電泳後浸泡在 50 mM Tris-HCl buffer (pH 7.9) 內含 13 mM 麩胱甘肽 (glutath ione) 及 0.004 % 過氧化氫 (H2O2) 中搖晃 10- 20 分鐘，再參照 Ukai 等人在 1997 年所提出對於 C-S lyase 活性染色的 方法加以修改，以 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) 及 1.6 mM phenazine met hsulfate (PMS) 來對 GSH-Px 做活性染色，可以發現在反應 10 分鐘後 GSH-Px 的活性會在膠體上以透明澄清的部分在 紫色的背景下呈現出來。接著從薑塊莖的萃取液以不同濃度之硫酸銨沈澱後 ( 30–45 %, 45–60 %, 60–75 %, 75–90% ) 的各部分重複上述方法，發現薑塊莖的萃取物有 GSH-Px 的活性。本實驗建立了這個具有快速靈敏的新方法，可以 用在動植物細胞中酵素的純化及鑑定。
另外第二部分的研究是從台農一號山藥、台農二號山藥、基隆山藥、名間山藥以及日本山藥中經由 DE-52 陰離子交 換樹脂分離出來之儲藏性蛋白質 dioscorin ，在運用 N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) 當受質時皆具有抑 制血管收縮素轉化酶 (angiotensin- converting enzyme ， ACE) 之活性，且台農一號山藥 dioscorin 之作用較為明顯，其 IC50 為 15.98 μM ( 相當於 623.8 μg) ；另進行酵素動力學分析以台農一號 dioscorin 12.8 μM 當作抑制劑，由 Micha els-Menten 方程式求得 Km 及 Km’ 各為 0.387 及 0.727 mM ，帶入公式 Ki = [I] / (Km’/Km) – 1 ，求得抑制常數 Ki 為 27.35 μM ，其 Vmax 不變 Km 變大，所以得知為競爭型抑制。在自發性高血壓鼠餵食實驗中發現一天餵食一次台農 一號山藥 dioscorin 20 及 40 mg/kg ，與對照組作比較，在第 4 小時有最高之降血壓量，收縮壓分別下降 2.4 mmHg 、 21.5 mmHg 。而且以 pepsin 水解台農一號山藥 dioscorin (PH-YSP) 40 mg/kg 其第八小時收縮壓下降了 32.8 mmHg 。 另外再以台農一號山藥 dioscorin 40 mg/kg 連續餵食 25 天，與對照組比較發現，實驗組血壓都比空白對照組低，第 九天時其收縮壓有 27.7 mmHg 之最大降低效果。
在血液流變的部分， Wistar 誘導成糖尿病三週後再餵食台農一號山藥 dioscorin 10mg/kg 三週，其紅血球的變形度指 數與沒餵食台農一號山藥 dioscorin 時比較差別不大 (p=0.854) ，但紅血球聚集指數也降低了 15.3 % (p<0.001) ，而剪 應力 = 300, 600 sec-1 時所測得的黏度比對照組時的黏度降低了 18.2 % (p=0.1008) 、 27.5 % (p<0.05) 。而 Wistar 誘導 成糖尿病八週後再餵食台農一號山藥 dioscorin 10 , 20 及 40 mg/kg 三週，其紅血球變形度指數分別提高了 7.0 % (p<0.001), 9.7 % (p<0.001) 及 11.7 % (p<0.001) 。另外在紅血球聚集度分別降低了 20.8 % (p<0.001), 42.3 % (p<0.001) 及 54.
4 % (p<0.001) ，在給予剪應力 =10, 300, 600 sec-1 時餵食 10 mg/kg 分別有 35.2 % (p= 0.201), 21.2 % (p<0.01), 28.4 % (p<0.01) 的降幅、 20 mg/kg 有 23.0 % (p=0.0579), 21.5 % (p<0.01), 23.9 % (p<0.01) 的降幅、 40 mg/kg 有 5.3 % (p<0.0 5), 15.2 % (p<0.01) , 12.5 % (p<0.01) 的降幅，比起空白對照組，有餵食台農一號山藥 dioscorin 皆有明顯改善血液流 變參數的效果。在現階段的農產保健食品當中，山藥可謂具有很大的發展，且對於未來開發成輔助調整血壓及改善 血液流變的食品，亦具有相當的潛力。
The new staining method of glutathione peroxidase and studies on the yam tuber storage protein in the protection of the cardiovascular disease – from blood pressure regulation to improvement of the hemorheology
英文摘要
To quickly screen the activity of the glutathione peroxidase (GSH-Px), from commercial bovine erythrocytes or ammonium sulfate fractionations (30- 45%, 45-60%, 60-75% and 75-90% saturations) of ginger rhizome, was detected on polyacrylamide gels after native polyacrylamide gel electrophoresi s (PAGE) or sodium dodecyl sulfate (SDS-PAGE). The gel was submerged in a 50 mM Tris-HCl buffer (pH 7.9) containing 13 mM glutathione and 0.
004 % hydrogen peroxide with gentle shaking for 10-20 min. The GSH-Px activity was stained with a solution containing 1.2 mM 3-(4,5-dimethylthia zol-2-yl)-2,5-diphenyl- tetrazolium bromide (MTT) and 1.6 mM phenazine methsulfate (PMS) for 10 min. The clear zone of GSH-Px activity on a pur ple background was found in both native and SDS-PAGE gels. This fast and sensitive method can be used in the process of enzyme purification and ch aracterization of mammalian or plant cells.
In the second part, the dioscorins, the tuber storage protein of yam from Dioscorea alata L. cv. Tainong No.1 (TN1), D. alata L. cv. Tainong No.2, D. p seudojaponica Tamamoto, D. alata L. var. purpurea and D. batatas Decne were purified from DE-52 ion-exchange chromatography. The activities of i nhibit angiotensin-converting enzyme (ACE) were shown by using N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) as substrate. And the effect was m ore observably by TN1 with the 50% inhibition (IC50) of ACE activity was 15.98 μM (equal to 623.8 μg) dioscorin. In the analysis of enzyme kinstics , using 12.8 μM TN1 dioscorin as an inhibitor, the Km and Km’ were 0.387 and 0.727 mM obtained by the equation of Michaels-Menten. The inhibiti on constant (Ki) was 27.35 μM obtain from Ki=[I]/(Km’/ Km)-1. In the test of spontaneous hypertension rat (SHR), it was also found that the systolic pressure of SHR was decreased 12.4 mmHg and 21.5 mmHg respectively after 4 hours fed on 20 and 40 mg/kg of the TN1 dioscorin. And 40 mg/kg o f the peptic hydrolyzate was fed, 8 hours later the systolic pressure of SHR was decreased 32.8 mmHg. For long-term 25-day oral administration of 40 mg/kg TN1 dioscorin SHR once a day, it was found that the SBP were lower than the control, and the ninth day the systolic pressure of SHR was maxi mum reducetion by 27.7 mmHg.
