| 1-deoxynojirimycin (DNJ) is a kind of piperidine alkaloid in mulberry leaves, chemically named 3,4,5-trihydroxy-2-hydroxymethyltetrahydropyridens, C6H13NO4 with molecular formula,and 163 with molecular weight. As the inhibitor ofα-glucosidase, DNJ functions by decreasing blood glucose, resisting virus and resisting tumor metastasis. Besides, DNJ is also a effective inhibitor ofα-glucoamylase.In this study, 1-deoxynojirimycin in mulberry leaves was determined by Capillary Column Gas Chromatography, utilizing the resemblance between DNJ and carbohydrate. comparing the difference in DNJ content among 110 varieties of mulberry leaves, cultivated by Shaanxi Provincial Institute of Sericulture Silk, before and after the frosting. The results showed that capillary column gas chromatography is acceptable to determine the DNJ content in mulberry leaves, certain differences existed in the DNJ content among mulberry leaves collected at different time.Influence of the different solvents and different extraction methods on DNJ extraction in mulberry leaves was tested in this study, by selecting different reagent to extract DNJ such as methanol, distilled water and pyridine, different extraction methods such as shocks, heating and ultrasonic extraction. Finally, the best conditions to determine DNJ by capillary column gas chromatography was confirmed: samples was treated by ultrasonic wave for 30 minutes with Pyridine as extraction reagent. Meanwhile, the influence of derivatization condition on it was tested, such as the derivatization reagent, time and temperature. Finally,the best derivatization condition was confirmed, using acetic glycoside as acetylation reagent, hydroxylamine hydrochloride as the catalyst, acetylating for 30 minutes in the 90℃water. The influence of different color spectrum condition, containing the kind and temperature of chromatographic column , the flow rate of carriering gas and the way of introduction etc. on the DNJ determination, also was detected.Finally,the condition of color spectrum was confirmed: using OV-1701chromatographic column, initial temperature 285℃,after continuous 5 minutes rising the temperature to 295℃at the rate of 10℃per min, then lasting another 25 minutes, after that, decreasing the temperature to 285℃at the rate of 20℃per min; the temperature of detection port is 310℃, the flow rate of hydrogen is 47mL per min; the sample size is 1μL; using flame ion detector(FID); using Shimadzu GCsolution Gas Chromatograph as the software for chromatographic analysis.The content of DNJ was calculated by standard curve method, which can simplify repetitive operation and avoid systematic errors. After experiment and calculation, equation of standard curve was calculated as y=36435x+17225, R2=0.9921, the minimum detectable concentration is 0.01μg/mL. The average recovery of the recovery rate test of added sample is 96.11%, RSD=3.24% (n=5). The result of repeatability test is RSD=2.10% (n=5). The result of stability test showed that the standards and samples are basically stable during 24 hours, the RSD is 1.00% and 2.56%(n=5)respectively.In this study, the DNJ content of 110 varieties of mulberry leaves, cultivated by Shaanxi Provincial Institute of Sericulture Silk, was determinated by capillary column gas chromatography, comparing the DNJ content in different varieties of mulberry leaves before and after the frosting, the result showed that DNJ existed in all the 110 varieties of mulberry leaves. The DNJ content in mulberry leaves collected in the late September (before frosting) is 0.059%～0.535%.By contrast, The DNJ content in mulberry leaves collected in the early November(after frosting ) is 0.001%～0.180%. The influence of frosting on DNJ content in mulberry leaves is obvious, the DNJ content in mulberry leaves from the majority of Mulberry decreases after frosting.
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