桑叶中1-脱氧野尻霉素的检测、提取及其生理活性研究
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摘要
桑叶中含有的1-脱氧野尻霉素(1-deoxynojirimycin, DNJ)可以抑制α-葡萄糖苷酶、葡萄糖淀粉酶的活性,具有抗病毒、减肥和增加胰岛素敏感性等多种重要生理功能。本文首先利用反相高效液相色谱建立了检测桑叶中DNJ含量的方法。利用该方法检测了132份桑树品种的DNJ含量,分析了不同纯化工艺对提高桑叶DNJ纯度的影响。利用糖尿病模型小鼠研究了DNJ的降血糖活性。
为了测定桑叶中1-脱氧野尻霉素(DNJ)含量,我们建立了以芴甲氧酰氯(FMOC-Cl)为衍化剂的柱前衍生化-反相高效液相紫外检测法。以0.05mol/LHCl为粗提桑叶粉DNJ的溶剂,芴甲氧酰氯(FMOC-Cl)为衍生化试剂,pH值8.50硼酸盐为衍生化缓冲液时,衍生化和RP-HPLC检测效果最好。反相高效液相色谱-紫外检测法检测DNJ时,流动相中乙腈：0.1%-醋酸为55：45(V／V)时色谱分离效果最好,DNJ-FMOC的峰面积与DNJ浓度呈高度正相关关系,相关系数为0.9852。
在建立了检测方法后,我们对132个不同品种的桑叶中DNJ含量进行了测定。结果显示这些品种的DNJ平均含量为0.07511%,标准差为0.028428。其中,竹场2号的DNJ含量最高,达到0.1472%,观音桑的DNJ含量最低,为0.0134073%。此外,不同叶位的桑叶的DNJ含量不同,随着桑叶发育成熟,DNJ的含量从上叶位到下叶位逐渐降低。
根据以上实验结果和桑叶价格、产地等方面的综合因素,我们确定了作为最终提取原料的桑叶材料。利用该材料,以乙醇结合超声处理所得的粗提物中,DNJ纯度仅为1.2842%。利用大孔树脂(3.3452%)、阳离子交换树脂(12.3873%)和硅胶树脂分级纯化后,纯度最终可达到19.0857%,符合食品添加剂的要求。
为了调查DNJ生理活性,我们以糖尿病小鼠模型为研究对象,将DNJ添加到饲料中喂养小鼠,并和喂养阿卡波糖及喂养普通饲料的小鼠进行对比,来评价DNJ长期食用抑制糖尿病的作用。这一我们首创的喂养方法,避免了常规灌胃方法对小鼠的伤害,保证了分析结果的可行性。结果表明：喂食高剂量DNJ(40mg/kg)40天后,小鼠血糖显著降低,与喂饲正常饲料的模型差异达到显著水平；而低剂量(20mg/kg)和中剂量(30mg/kg)组与对照组差异不显著。
1-deoxynojirimycin (DNJ) in Mulberry leaves has a variety of important physiological function, such as a-glucosidase and glucoamylase inhibition, anti-virus, body weight loss and insulin sensitivity increase. Firstly, a sensitive method of RP-HPLC-UV was established to detect DNJ contents in mulberry leaves. Using the method, we not only measured DNJ contents in 132 varieties of mulberry, but also studied various purification process of DNJ from mulberry leaves. Finally, hypoglycemic activity of DNJ was estimated in diabetic mice.
In order to determine DNJ contents in mulberry leaves, a RP-HPLC-UV method with 9-fluorenylmethyl chloroformate (FMOC-Cl) as the derived agent of pre-column was used. The results showed that the best condition of derivatization and RP-HPLC detection was 0.05mol/L HCl, FMOC-Cl and pH 8.50 borate buffer solution as DNJ extract reagent, derivatization reagent and derivatiztion buffer, respectively. The best RP-HPLC-UV mobile phase, whose DNJ-FMOC peak area and DNJ concentration are high positive correlative and the correlation coefficient is 0.9852, was acetonitrile:0.1%-acetic acid (55:45, V/V).
And then, the DNJ contents of 132 species of mulberry varieties were investigated. The results showed that the mean value was 0.07511% and standard deviation was 0.028428. The DNJ content of Zu-Chang 2 was the highest (0.14720%) and the lowest one was Guanyin-Sang (0.01341%). In addition, the DNJ contents of different site of mulberry leaves were not same. Accroding to growth degree of the mulberry leaves, the content of DNJ decreased gradually from the upper to lower sites.
Accroding to the results and other screening facters, the final mulberry leaf material was selected. Using the material and ultrasonic treatment combined with ethanol, the DNJ purity of crude extracts is 1.2842%. After a series of purification, including macroporous resin (3.3452%), cation exchange resin (12.3873%) and silica resin, the purity of DNJ reached 19.0857%, which conforms to the requirements as a food additive.
To evalue the diabetes inhibition role of long-term DNJ treatmen, diabetic mice were fed with DNJ-included dite and compared with those fed with acarbose and normal diet. After 40 days, blood glucose of high dose (40mg/kg) of DNJ mice reduced significantly, and the difference between them and mice fed with the normal diet is significant. The difference of low dose (20mg/kg) and medium dose (30mg/kg) of DNJ treatment mice group compared with control group is not significant.
为了测定桑叶中1-脱氧野尻霉素(DNJ)含量,我们建立了以芴甲氧酰氯(FMOC-Cl)为衍化剂的柱前衍生化-反相高效液相紫外检测法。以0.05mol/LHCl为粗提桑叶粉DNJ的溶剂,芴甲氧酰氯(FMOC-Cl)为衍生化试剂,pH值8.50硼酸盐为衍生化缓冲液时,衍生化和RP-HPLC检测效果最好。反相高效液相色谱-紫外检测法检测DNJ时,流动相中乙腈：0.1%-醋酸为55：45(V／V)时色谱分离效果最好,DNJ-FMOC的峰面积与DNJ浓度呈高度正相关关系,相关系数为0.9852。
在建立了检测方法后,我们对132个不同品种的桑叶中DNJ含量进行了测定。结果显示这些品种的DNJ平均含量为0.07511%,标准差为0.028428。其中,竹场2号的DNJ含量最高,达到0.1472%,观音桑的DNJ含量最低,为0.0134073%。此外,不同叶位的桑叶的DNJ含量不同,随着桑叶发育成熟,DNJ的含量从上叶位到下叶位逐渐降低。
根据以上实验结果和桑叶价格、产地等方面的综合因素,我们确定了作为最终提取原料的桑叶材料。利用该材料,以乙醇结合超声处理所得的粗提物中,DNJ纯度仅为1.2842%。利用大孔树脂(3.3452%)、阳离子交换树脂(12.3873%)和硅胶树脂分级纯化后,纯度最终可达到19.0857%,符合食品添加剂的要求。
为了调查DNJ生理活性,我们以糖尿病小鼠模型为研究对象,将DNJ添加到饲料中喂养小鼠,并和喂养阿卡波糖及喂养普通饲料的小鼠进行对比,来评价DNJ长期食用抑制糖尿病的作用。这一我们首创的喂养方法,避免了常规灌胃方法对小鼠的伤害,保证了分析结果的可行性。结果表明：喂食高剂量DNJ(40mg/kg)40天后,小鼠血糖显著降低,与喂饲正常饲料的模型差异达到显著水平；而低剂量(20mg/kg)和中剂量(30mg/kg)组与对照组差异不显著。
1-deoxynojirimycin (DNJ) in Mulberry leaves has a variety of important physiological function, such as a-glucosidase and glucoamylase inhibition, anti-virus, body weight loss and insulin sensitivity increase. Firstly, a sensitive method of RP-HPLC-UV was established to detect DNJ contents in mulberry leaves. Using the method, we not only measured DNJ contents in 132 varieties of mulberry, but also studied various purification process of DNJ from mulberry leaves. Finally, hypoglycemic activity of DNJ was estimated in diabetic mice.
In order to determine DNJ contents in mulberry leaves, a RP-HPLC-UV method with 9-fluorenylmethyl chloroformate (FMOC-Cl) as the derived agent of pre-column was used. The results showed that the best condition of derivatization and RP-HPLC detection was 0.05mol/L HCl, FMOC-Cl and pH 8.50 borate buffer solution as DNJ extract reagent, derivatization reagent and derivatiztion buffer, respectively. The best RP-HPLC-UV mobile phase, whose DNJ-FMOC peak area and DNJ concentration are high positive correlative and the correlation coefficient is 0.9852, was acetonitrile:0.1%-acetic acid (55:45, V/V).
And then, the DNJ contents of 132 species of mulberry varieties were investigated. The results showed that the mean value was 0.07511% and standard deviation was 0.028428. The DNJ content of Zu-Chang 2 was the highest (0.14720%) and the lowest one was Guanyin-Sang (0.01341%). In addition, the DNJ contents of different site of mulberry leaves were not same. Accroding to growth degree of the mulberry leaves, the content of DNJ decreased gradually from the upper to lower sites.
Accroding to the results and other screening facters, the final mulberry leaf material was selected. Using the material and ultrasonic treatment combined with ethanol, the DNJ purity of crude extracts is 1.2842%. After a series of purification, including macroporous resin (3.3452%), cation exchange resin (12.3873%) and silica resin, the purity of DNJ reached 19.0857%, which conforms to the requirements as a food additive.
To evalue the diabetes inhibition role of long-term DNJ treatmen, diabetic mice were fed with DNJ-included dite and compared with those fed with acarbose and normal diet. After 40 days, blood glucose of high dose (40mg/kg) of DNJ mice reduced significantly, and the difference between them and mice fed with the normal diet is significant. The difference of low dose (20mg/kg) and medium dose (30mg/kg) of DNJ treatment mice group compared with control group is not significant.
引文
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