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

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

林建良(Chien-Liang Lin)

指導教授 ： 侯文琪

共同指導教授 ： 林耀輝 ； 劉得任

臺北醫學大學/藥學院/藥學研究所/博士(2007年)

https://doi.org/10.6831/TMU.2007.00013

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(glutathione)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 methsulfate (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Michaels-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<0.05), 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。

關鍵字

GSH-Px ； dioscorea

並列摘要

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 electrophoresis (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-dimethylthiazol-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 purple 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 characterization 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. pseudojaponica Tamamoto, D. alata L. var. purpurea and D. batatas Decne were purified from DE-52 ion-exchange chromatography. The activities of inhibit angiotensin-converting enzyme (ACE) were shown by using N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) as substrate. And the effect was more 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 inhibition 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 of 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 maximum reducetion by 27.7 mmHg.
And in the haemorrheological properties, after diabetes mellitus (DM) was induced by streptocozocin, 3 weeks later the Wistar rats were fed with TN1 dioscorin 10 mg/kg for 3 weeks. The blood deformation index was no difference (p=0.854) compared with the control. But the red blood aggregation index was lower 15.3 % (p<0.001), and the blood viscosity was lower by 18.2 % (p=0.1008) (γ=300), 27.5 % (p<0.05) (γ=600). After 8 weeks DM induced, the Wistar rats were fed with TN1 dioscorin 10, 20 and 40 mg/kg for 3 weeks. The red blood deformation index was elevated 7.0 % (p<0.001), 9.7 % (p<0.001) and 11.7 % (p<0.001) respectively. The blood aggregation index was lower 20.8 % (p<0.001), 42.3 % (p<0.001) and 54.4 % (p<0.001) respectively. In the blood viscosity test, when shear rate 10, 300, 600 sec-1 was given, the Wistars fed on 10 mg/kg TN1 dioscorin was lower by 35.2 % (p= 0.201), 21.2 % (p<0.01), 28.4 % (p<0.01) respectively. The Wistars fed on 20 mg/kg TN1 dioscorin was lower by 23.0 % (p=0.0579), 21.5 % (p<0.01), 23.9 % (p<0.01) respectively, and the Wistars fed on 40 mg/kg TN1 dioscorin was lower by 5.3 % (p<0.05), 15.2 % (p<0.01), 12.5 % (p<0.01) respectively. Accroding to the above, it was found that the Wistars fed on TN1 dioscorin significantly improve the blood hemorheology. In the health-care food propellent infancy, dioscorea is more potencial to design as a complementary alternative medicine to adjust blood pressure and improve the blood hemorheology.

並列關鍵字

麩胱甘肽過氧化酶山藥

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