Use of Nuclear Magnetic Resonance Method for Detection of Rutin-Degrading Enzyme Activity in Fagopyrum esculentum and F. tartaricum.

Abstract

Using the Nuclear Magnetic Resonance (NMR) method, the detection of the activity of rutin-degrading enzyme in buckwheat was examined. Mixture of dimethylsulfoxide (DMSO) and acetate buffer (i.e., water) solvent was selected and double homogate (DHMG) decoupling technique was used in NMR measurement in order to suppress large solvent signals and to detect the signals of rutin and quercetin. NMR signals of aromatic H of the flavonoid in rutin were detected at different positions from those in quercetin. Then, the activity of the enzyme in buckwheat was examined by the NMR method. The crude extract solution of the enzyme (acetate buffer solution pH 5.0) was added to rutin in a DMSO solution, and the activity was measured by the NMR method. The enzyme activity could be detected by the appearance of the NMR signals of quercetin, which were observed in the shifted position, i.e., in a higher field than that of rutin. When the extracts from common buckwheat were added, NMR signals did not change. However, when the extracts from tartary buckwheat were added, NMR signals which shifted from that of rutin in the higher frequency region were observed. The NMR method was useful to detect the enzyme activity. Furthermore, it was confirmed that common buckwheat did not show the rutin-degrading enzyme activity, unlike tartary buck-wheat.