| Summary: | 博士 === 臺北醫學大學 === 藥學研究所 === 95 === 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.
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