毛细管柱气相色谱法测定桑叶中1-脱氧野尻霉素含量的研究
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摘要
1-脱氧野尻霉素(1-deoxynojirimycin)是桑叶中的一种哌啶生物碱,其化学名称是3,4,5-三羟基- 2-羟甲基四氢吡啶,分子式为C6H13NO4,分子量为163。DNJ作为α-葡萄糖苷酶的抑制剂,具有降血糖、抗病毒、抗肿瘤转移等作用。另外,DNJ还是α-葡萄糖淀粉酶的有效抑止剂。
本试验利用DNJ与糖类的相似性,按照常用的用气相色谱法测定糖类物质进行的衍生化方式,将DNJ的羟基进行乙酰化反应,探索用毛细管柱气相色谱法测定桑叶中DNJ的含量,并比较了陕西省蚕桑丝绸研究所品种桑园中种植的110个桑品种不同时期采集叶片中DNJ含量的高低。结果表明,用毛细管柱气相色谱法测定桑叶中的DNJ含量是可取的,不同品种不同采集时间的桑叶样品中DNJ的含量有一定差别。
本实验考察了不同溶剂和不同提取方式对桑叶中DNJ提取的影响,选用甲醇、蒸馏水、吡啶等不同试剂对DNJ进行提取,提取方式分别尝试了震荡、加热、超声提取。最终确定用吡啶作为提取试剂在超声波条件下处理30分钟为毛细管气相色谱法测定DNJ的最佳提取条件。同时考察了衍生化条件对该试验的影响,考察内容包括衍生化试剂、衍生化时间、衍生化温度等内容。最终确定在用醋酸苷作为乙酰化试剂,盐酸羟胺作为催化剂,在水浴90℃条件下乙酰化30分钟为最佳衍生化条件。
本试验还考察了不同色谱条件对DNJ测定的影响,考察内容包括色谱柱选择、柱温、载气流量进样方式等。最终确定色谱条件为:色谱柱OV-1701,初温285℃×5min,以10℃/min上升至295℃,维持25min,以20℃/min下降至285℃;汽化室温度310℃;检测口温度310℃;氢气流量47mL/min;进样量1μL,火焰离子检测器(FID),色谱分析软件采用GCsolution岛津气相色谱工作站。
本试验采用标准曲线法计算DNJ含量,该法具有简化重复操作,避免系统误差等优点。试验及计算后得到标准曲线方程y=36435x+17225,R2=0.9921。最低检测浓度为0.01μg/mL。加样回收率试验结果平均回收率为96.11%,RSD=3.24%(n=5)。重复性试验结果RSD=2.10%(n=5)。稳定性试验结果表明:标样及样品在24小时内基本稳定,RSD分别为1.00%和2.56%(n=5)。
用所建立的毛细管柱气相色谱法对陕西省蚕桑丝绸研究所品种桑园中110个桑品种桑叶中DNJ含量进行了测定。并比较了不同品种在霜前和霜后DNJ含量的高低,结果显示,110种桑叶中均含有DNJ。其9月底(霜前)含量在0.059%～0.535%之间,11月初(霜后)含量在0.001%～0.180%之间,同时表明下霜对桑叶中DNJ含量有较大影响,绝大多数桑叶中DNJ含量在霜后降低。
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.
本试验利用DNJ与糖类的相似性,按照常用的用气相色谱法测定糖类物质进行的衍生化方式,将DNJ的羟基进行乙酰化反应,探索用毛细管柱气相色谱法测定桑叶中DNJ的含量,并比较了陕西省蚕桑丝绸研究所品种桑园中种植的110个桑品种不同时期采集叶片中DNJ含量的高低。结果表明,用毛细管柱气相色谱法测定桑叶中的DNJ含量是可取的,不同品种不同采集时间的桑叶样品中DNJ的含量有一定差别。
本实验考察了不同溶剂和不同提取方式对桑叶中DNJ提取的影响,选用甲醇、蒸馏水、吡啶等不同试剂对DNJ进行提取,提取方式分别尝试了震荡、加热、超声提取。最终确定用吡啶作为提取试剂在超声波条件下处理30分钟为毛细管气相色谱法测定DNJ的最佳提取条件。同时考察了衍生化条件对该试验的影响,考察内容包括衍生化试剂、衍生化时间、衍生化温度等内容。最终确定在用醋酸苷作为乙酰化试剂,盐酸羟胺作为催化剂,在水浴90℃条件下乙酰化30分钟为最佳衍生化条件。
本试验还考察了不同色谱条件对DNJ测定的影响,考察内容包括色谱柱选择、柱温、载气流量进样方式等。最终确定色谱条件为:色谱柱OV-1701,初温285℃×5min,以10℃/min上升至295℃,维持25min,以20℃/min下降至285℃;汽化室温度310℃;检测口温度310℃;氢气流量47mL/min;进样量1μL,火焰离子检测器(FID),色谱分析软件采用GCsolution岛津气相色谱工作站。
本试验采用标准曲线法计算DNJ含量,该法具有简化重复操作,避免系统误差等优点。试验及计算后得到标准曲线方程y=36435x+17225,R2=0.9921。最低检测浓度为0.01μg/mL。加样回收率试验结果平均回收率为96.11%,RSD=3.24%(n=5)。重复性试验结果RSD=2.10%(n=5)。稳定性试验结果表明:标样及样品在24小时内基本稳定,RSD分别为1.00%和2.56%(n=5)。
用所建立的毛细管柱气相色谱法对陕西省蚕桑丝绸研究所品种桑园中110个桑品种桑叶中DNJ含量进行了测定。并比较了不同品种在霜前和霜后DNJ含量的高低,结果显示,110种桑叶中均含有DNJ。其9月底(霜前)含量在0.059%～0.535%之间,11月初(霜后)含量在0.001%～0.180%之间,同时表明下霜对桑叶中DNJ含量有较大影响,绝大多数桑叶中DNJ含量在霜后降低。
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.
引文
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