桑叶1-脱氧野尻霉素含量快检方法建立及其对α-葡萄糖苷酶的抑制作用

Development of rapid method for determination of 1-deoxynojirimycin in Morus alba L.leaves and its inhibitory effect onα-glucosidase

桑叶中的1-脱氧野尻霉素(1-deoxynojirimycin,DNJ)是α-葡萄糖苷酶的天然抑制剂,因此,快速检测桑叶中的DNJ含量,并探究桑叶DNJ对α-葡萄糖苷酶抑制作用尤为重要。通过建立高效液相-蒸发光检测器法(HPLC-ELSD)快速检测桑叶中的DNJ含量,并比较了6种桑叶中DNJ的含量;优化α-葡萄糖苷酶反应体系,比较6种桑叶DNJ对α-葡萄糖苷酶的抑制率,并通过抑制动力学实验探究其抑制反应类型。结果表明:6种桑叶中DNJ含量存在差异,其中,不同地域桑叶中DNJ的含量由高到低分别为沁县桑、阳城桑和韩国桑,沁县桑中DNJ含量为2.419 mg/g,不同品种桑叶中DNJ的含量由高到低分别为果桑、蛋白桑和叶桑,果桑中DNJ含量为2.694 mg/g;α-葡萄糖苷酶最优反应体系条件为蔗糖质量浓度32 mg/mL,酶用量0.4 U/mL,反应时间60 min;6种桑叶提取物对α-葡萄糖苷酶均具有抑制作用,不同品种桑叶提取物对α-葡萄糖苷酶的抑制率由高到低依次为蛋白桑、叶桑和果桑,蛋白桑提取物对α-葡萄糖苷酶的抑制率为86.0%,不同地域桑叶提取物对α-葡萄糖苷酶的抑制率由高到低依次为沁县桑、阳城桑和韩国桑,沁县桑叶提取物对α-葡萄糖苷酶的抑制率为89.0%,为6种桑叶中最高,其抑制作用类型为可逆性非竞争性抑制。综上所述,实验结果可为桑叶中DNJ的开发及有效利用提供理论依据。

1-Deoxynojirimycin(DNJ)in mulberry leaves is a natural inhibitor ofα-glucosidase,which can reduce the hydrolysis degree of disaccharides such as maltose and sucrose.In addition,DNJ can inhibit the absorption of monosaccharides in the small intestine,and effectively reduce the blood glucose level after eating.Therefore,it is particularly important to quickly detect the content(mass concentration)of DNJ and explore its inhibitory effect onα-glucosidase.In this study,a high performance liquid chromatography-evaporative light-scattering detector method(HPLC-ELSD)was developed to determine the content of DNJ in mulberry leaves,and the contents of DNJ in mulberry leaves of six varieties were compared.The reaction system ofα-glucosidase was optimized,and then the inhibition rates of DNJ in mulberry leaves of six varieties onα-glucosidase were compared,and finally the type of reaction that DNJ in mulberry leaves has inhibition onα-glucosidase was explored.The results showed that the DNJ contents in mulberry leaves of six varieties were different.The contents of DNJs among the mulberry of different regions from high to low were in order of Qinxian mulberry,Yangcheng mulberry and Korea mulberry.The content of DNJ in Qinxian mulberry leaves was 2.419 mg/g,which was significantly higher than that of mulberry leaves in the other two regions.The contents of DNJs among the mulberry of different wood species from high to low were in order of fruit mulberry,protein mulberry and leaf mulberry.The DNJ content in fruit mulberry was 2.694 mg/g,and the protein mulberry was significantly higher than the leaf mulberry.The best reaction system ofα-glucosidase was the sucrose mass concentration of 32 mg/mL,enzyme dosage of 0.4 U/mL,and reaction time of 60 min.Six kinds of extracts had inhibitory effect onα-glucosidase.The inhibitory rates of extracts among the mulberry of different regions onα-glucosidase from high to low were in order of Qinxian mulberry,Yangcheng mulberry and Korea mulberry.The inhibitory rate of extract of Qinxian was 89.0%,which was significantly higher than that of the Yangcheng mulberry and Korea mulberry.The inhibitory rate of the extract among the mulberry of different species onα-glucosidase from high to low was in order of the protein mulberry,leaf mulberry and fruit mulberry.The inhibitory rate of the extract of the protein mulberry was 86.0%,which was significantly higher than that of the leaf mulberry and fruit mulberry.This reaction was reversible non-competitive inhibition.In this study,DNJ in mulberry leaves in terms of content,inhibition rate and type ofα-glucosidase are greatly significant for improving economic benefit of mulberry in China and promoting industrial upgrading of mulberry.The experimental results provide a theoretical basis for the development and effective utilization of DNJ in mulberry leaves in the future.