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林业科学 ›› 2015, Vol. 51 ›› Issue (2): 112-120.doi: 10.11707/j.1001-7488.20150214

• 论文与研究报告 • 上一篇    下一篇

桑枝1-脱氧野尻霉素的提取及其含量与α-葡萄糖苷酶抑制活性的相关性分析

刘超1, 史正琴1, 向伟1, 黄先智1, 徐立1, 兰俊2   

  1. 1. 西南大学生物技术学院 重庆 400715;
    2. 厦门绿洲环保产业股份有限公司 厦门 361004
  • 收稿日期:2014-04-23 修回日期:2014-05-22 出版日期:2015-02-25 发布日期:2015-03-11
  • 通讯作者: 徐立
  • 基金资助:

    中央高校基本科研业务费专项基金项目(XDJK2013C053); 国家蚕桑产业技术体系专项(CARS-22-ZJ0503)。

1-Deoxynojirimycin Extraction from Ramulus Mori and Relationship Analysis between Its Content in Extracts and α-Glucosidase Inhibitory Activity

Liu Chao1, Shi Zhengqin1, Xiang Wei1, Huang Xianzhi1, Xu Li1, Lan Jun2   

  1. 1. College of Biotechnology, Southwest University Chongqing 400715;
    2. Oasis Sources Environmental Protection Industry Ltd. Xiamen 361004
  • Received:2014-04-23 Revised:2014-05-22 Online:2015-02-25 Published:2015-03-11

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摘要：

【目的】 1-脱氧野尻霉素(DNJ)是一种降血糖功效较好的天然活性产物,在桑树中的含量相对较高。对超声-微波联合辅助提取桑枝中DNJ的工艺进行优化,以获得较高提取率; 同时,分析桑枝水提物中DNJ含量与α-葡萄糖苷酶抑制活性的相关性,以获得其中DNJ对α-葡萄糖苷酶抑制活性的贡献程度。这可能为桑枝中DNJ的提取、分离及桑枝α-葡萄糖苷酶抑制剂的极性分布等研究提供一定的依据。【方法】 利用9-芴甲氧羰酰氯对DNJ进行衍生化,弥补其本身不具有紫外吸收特性的缺点,利用高效液相色谱在254 nm波长下进行定性定量检测; 单因素试验分别求得微波处理时间、超声波处理时间和液料比的优化范围,以响应曲面法对超声-微波联合辅助提取DNJ三因素的平方和、均方、P值、R2等参数进行优化,获得二次多项回归方程和相应的最优提取条件; 在此基础上利用系统溶剂法萃取浓缩的水提物,计算石油醚、乙酸乙酯、正丁醇和水4种极性部分的α-葡萄糖苷酶抑制活性(通过酶标仪在415 nm波长处测定α-葡萄糖苷酶对4-硝基苯-α-D吡喃葡萄糖苷的分解率来计算)与DNJ的含量,并对二者之间的关系进行分析。【结果】 用0.05 moL·L-1 的盐酸溶液提取,微波处理9.60 min、超声波处理30.89 min和液料比46.47 mL·g-1时达到最优条件,此时DNJ的产率为0.205%,结果检验值与优化值的拟合率达99.27%,相对于对照组增长了10.86%; 正丁醇部分DNJ的含量最高,乙酸乙酯次之,水部分仍含有少许,石油醚部分为零,说明正丁醇的萃取效果较好; 正丁醇部分的抑制曲线趋势与乙酸乙酯部分相近,水部分与石油醚部分相近,且各萃取部分之间的IC50都差异显著,结合DNJ的含量分布,说明桑枝中具有α-葡萄糖苷酶抑制活性的物质间有一定的极性差异; 根据各萃取部分中DNJ的含量,从其半抑制浓度中换算出DNJ的浓度,即为"其中DNJ的浓度",正丁醇部分"其中DNJ的浓度"与DNJ标准品的IC50差异不显著,说明此部分α-葡萄糖苷酶抑制活性物质几乎尽是DNJ; 水部分虽含有少量的DNJ,但其活性却相对更小,可能其中含有具备提高α-葡萄糖苷酶活性的多糖等物质,或一些糖类等黏性物质影响了DNJ与酶的结合; 乙酸乙酯部分"其中DNJ的浓度"比DNJ标准品的IC50小,可能是由于此部分含有个别黄酮类等对DNJ具有活性增强/协同作用,或其中含有一些游离生物碱、香豆素或黄酮苷元等物质具备相似活性; 石油醚部分不含DNJ,却有一定的α-葡萄糖苷酶抑制活性,说明桑枝中含有一些极性小的脂溶性化合物具备α-葡萄糖苷酶抑制活性。【结论】 微波处理初步粉碎的新鲜桑枝,既有干燥作用又有辅助提取效果,而后用超声波辅助浸提可提高DNJ的提取效率; 桑枝中主要的α-葡萄糖苷酶抑制是DNJ,除此外还可能含有一些极性相对较小的活性成分。这些结果有利于桑枝DNJ及其他降血糖活性成分的进一步研究。

关键词: 1-脱氧野尻霉素, 超声-微波联合辅助提取法, 响应曲面法, α-葡萄糖苷酶抑制活性, 桑枝

Abstract:

【Objective】 1-Deoxynojirimycin (DNJ) is one of the important natural active substances with good hypoglycemic effect. Mulberry has a relatively high DNJ content. To obtain high extraction rate from ramulus mori, the microwave-ultrasonic assisted technology was optimized. Meanwhile, to get DNJ contribution degree to α-glucosidase inhibitory activity in ramulus mori water extract, the relationship between DNJ content and α-glucosidase inhibitory activity was analyzed. The present study would provide a basis for DNJ extraction, separation, and polar distribution of α-glucosidase inhibitors. 【Method】9-Fluorenylmethyl chloroformate was used as DNJ derivatization reagent to make up for the shortcomings of DNJ that does not hold the UV absorption characteristic. Then, quantitative and qualitative analysises of DNJ had been done at a wavelength of 254 nm by high performance liquid chromatography. The optimal dimension of ultrasonic processing time, microwave processing time and solvent-to-sample ratio were obtained by single factor experiment. The optimized values including sum of squares, mean square, p-value of three-factor of face-centered cube design, regression equation and corresponding optimal extraction condition were gotten by response surface methodology. Based on the results, systematic solvent method was used to extract concentrated extracts. α-Glucosidase inhibitory activity (through the detection of decomposition rate of α-glucosidase to 4-nitrophenyl-α-D-glucopyranoside at a wavelength of 415 nm by microplate reader) and DNJ content were calculated. The relationship between them was also analyzed in four parts extracted by petroleum ether, ethyl acetate, n-butyl alcoho and water in turn. 【Result】Microwave processing time 9.60 min, ultrasonic processing time 30.89 min and solvent-to-sample ratio 46.47 mL·g-1 were found to be the optimal condition for extraction with 0.05 mol·L-1 HCl. The yield was 0.205%. The fitting rate between test value and the optimal value was 99.27%. The test yield of DNJ increased by 10.86% comparing with that of the control group. The DNJ content in n-butyl alcohol part was the highest. The second was found in the ethyl acetate part, and the third was found in the water part. Petroleum ether part had no DNJ. It informed that the extraction effect of n-butyl alcohol was the best. The inhibition curve trend of n-butyl alcohol part was close to ethyl acetate part, and that of water part and petroleum ether part was similar. The 4 parts had apparent difference in IC50 value. It suggested that some differences in polarity existed among α-glucosidase inhibitors in ramulus mori combined with the content distribution of DNJ. DNJ concentration, which was called "DNJ content at this concentration", was calculated from 50% inhibiting concentration based on DNJ content of each extraction part. "DNJ content at this concentration" of n-butyl alcohol part and DNJ IC50 had no apparent difference. It indicated that the α-glucosidase inhibitors of n-butyl alcohol part was almost entirely DNJ. A small amount of DNJ was in water part, but its activity was relatively smaller. It may be due to some components like polysaccharide with the ability of improving α-glucosidase inhibitory activity were possible or some sticky substances that affected the combination of DNJ and enzyme. "DNJ content at this concentration" of ethyl acetate part was smaller than DNJ IC50. This may be because some components like flavone which had enhancement/synergistic function with DNJ or it was possible that some components like alkaloids, coumarins and flavone glycoside etc. had similar activity with DNJ. Petroleum ether part had no DNJ, but had a certain activity. It provided that some other hypoglycemic active compounds with less polarity and lower activity than DNJ were existed in ramulus mori extracts. 【Conclusion】Crushed fresh ramulus mori was treated by microwave, which had both drying effect and assistant extraction effect. Then, extraction was done under ultrasonic conditions. The operation could improve the extraction efficiency of DNJ. The main α-glucosidase inhibitor was DNJ, but there were also some other compounds with less polarity and lower activity than DNJ in ramulus mori. The results are conducive for the further study of DNJ extraction and the composition of hypoglycemic activity compounds in mulberry.

Key words: 1-Deoxynojirimycin, ultrasonic-microwave assisted extraction, response surface methodology, α-glucosidase inhibitory activity, ramulus mori

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