| Chinese water-chestnut (Eleocharis tuberosa) usually eats after peeling. However, the cutting surface easily turns yellow in a short term, which seriously affects the quality of its appearance. The etiolated tissue of fresh-cut Chinese water-chestnut was taken as the experiment material in this study. HPLC was established to explore the simultaneous determination of main etiolated substance (Naringenin and Eriodictyol) in etiolation extract of fresh-cut Chinese water-chestnut. Meanwhile, the etiolated key enzyme that were chalcone isomerase (CHI) and flavonoid 3'-hydroxylase (F3'H) were isolated,purified and characterized. Based on that, the model system in vitro about the metabolic pathways of Naringenin and Eriodictyol was established. Finally, the research would verify the metabolic pathways of naringenin and eriodictyol in vivo and clarify the mechanism of fresh-cut Chinese water-chestnut etiolation.The results were as follows:1. Naringenin and Eriodictyol in etiolation extract of fresh-cut Chinese water-chestnut were simultaneously determinated by HPLC. It showed that the optimal simultaneous determination conditions were the mobile phase composed of 0.5% (v/v) acetic acid in water (eluent A) and acetonitrile (eluent B) in a linear gradient program at a continual flow rate of 1.0 mL·min-1, the column temperature was 40 ?, the injection volume was 10 ?L,and the UV detection was performed at 280 nm, which could make naringenin and eriodictyol separate successfully. The chromatographic conditions are simple, accurate and reliable, with good reproducibility, which can eliminate the chromatography of other substances contained and interference of other substances produced.2. CHI from fresh-cut Chinese water-chestnut was isolated and purified by precipitation with ammonium sulfate (35%-80%), sequential chromatography using DE-52 and SephadexTM G-100. The purified CHI was obtained with a 12.35-fold purity, a yield of 6.86% and a specific activity of 125 U/mg·proteins. The molecular weight was estimated to approximately 14.4 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Further analysis based on enzymatic characterization indicated the optimal temperature was 45 ? as well as pH was 7.5. The CHI activity wassignificantly stimulated by Ca2+ and Cu2+, but inhibited by Zn2+, Fe2+ and Na+. A significant active effect of DTT, Triton X-100 and PVP on the enzyme activity was observed, while a negative correlation was found between the CHI activity and the chemical compounds of?-mercaptoethanol, SDS and Tween 80. In addition, EDTA, ascorbic acid, sodium ascorbate and citric acid presented no significant (p > 0.05) influence on CHI activity.Finally, the Km (2.91 mM) and Vmax (45.25 U/mL) for naringenin chalcone were obtained from the Lineweaver-Burk plot.3. The crude enzyme solution of F3'H extracted from fresh-cut Chinese water-chestnut was isolated and purified by ammonium sulfate precipitation, dialysis, and ion-exchange column chromatography on DEAE-cellulose followed by gel filtration on Sephadex G-100. After purification procedure, F3'H exhibited final purification fold of 14.01, specific activity of 478.49 U/mg·protein and yield of 6.38%, respectively. The molecular mass of F3'H was about 53.09 kDa via SDS-PAGE. Further enzymatic characterization assays showed that the purified F3'H attained maximal activity at 30 ?and pH 7.5, and had a Km and Vmax of 1.08 mmol/L and 416.67 U/ (min·mL) respectively,using naringein as the substrate. The F3'H activity was slightly inhibited by Ca2+ and citric acid, strongly inhibited by Na+. However, Fe2+, Mg2+, NADPH and ascorbic acid exhibited strongly inhibition activated on F3'H activity.4. On the basis of our previous study, to further validate the metabolic pathways in vivo of Naringenin and Eriodictyol in fresh-cut Chinese water-chestnut, the model system in vitro about this pathways was established by using Naringenin Chalcone and purified CHI and F3'H. Naringenin Chalcone was catalyzed by CHI to produce Naringenin, of which the Naringenin was catalyzed by F3'H into .Eriodictyol. The reaction productions of model system in vitro and the main etiolated substance of fresh-cut Chinese water-chestnut were analyzed by HPLC-MS and compared with the standard of Naringenin and Eriodictyol. It indicated that the main etiolated substance which were Naringenin and Eriodictyol consisted in the model system in vitro were same with fresh-cut Chinese water-chestnut, but the peak intensity of ion fragment and content of Naringenin and Eriodictyol is different. |
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