HPLC法测定桑叶1-脱氧野尻霉素(DNJ)含量的方法学研究

Methodological Study on the Determination of 1-DNJ in Mulberry Leaf with HPLC

为优化桑叶DNJ含量的测定方法,本研究采用单因素试验,分别考察了桑叶的粉碎粒度、提取溶剂、提取液pH、提取液体积、超声提取时间等因素对桑叶1-脱氧野尻霉素(1-deoxynojirimycin,DNJ)提取率的影响,在确定最优提取条件后,进一步考察该提取条件下用HPLC法测定桑叶DNJ的精密度、重复性、回收率、中间精密度等参数,最后利用该提取测定方法测定不同品种桑叶DNJ含量。结果显示,桑叶DNJ的最优提取测定方法为:桑叶粉碎过50目筛,0.2 g样品加200 mL pH=2.0缓冲液,超声提取20 min,10000 rpm离心10 min,保留上清液,精密吸取样品溶液10μL,置1.5 mL离心管中,与10μL 0.2 mol/L(pH=7)硼酸钾缓冲液在1.5 mL试管中混合,加入20μL 5 mmol/L FMOC-CL(溶解于乙腈中),迅速混匀并在室温条件下反应10 min,加10μL 0.1 mol/L甘氨酸液终止反应。该条件下用HPLC法测定同批次桑叶样品的精密度、重复性、回收率、中间精密度均良好,RSD均小于3%。用该方法测定的不同品种桑叶DNJ含量为0.8 g/kg~5.7 g/kg。以上研究结果为桑叶DNJ开发利用提供参考依据。

Mulberry leaves(mori folium)are dry leaves of Morus alba L.(family moraceae),which is a kind of medicinal and edible plant resource.The alkaloid 1-deoxynojirimycin(DNJ)is one of the main active components in mulberry leaves with hypoglycemic effect.In order to optimize the method for the determination of DNJ content in Mulberry Leaf,we conducted the single factor test to investigate the effects of factors such as grinding size,extraction solvent,pH of extraction solution,volume of extraction liquid,ultrasonic extraction time,et al for precipitation on the extraction rate of DNJ from Mulberry Leaf.Under the selected optimal extraction conditions,the precision,repeatability,recovery rate and intermediate precision of DNJ determination method by using HPLC were further investigated.Finally,the method was used to determine the content of DNJ in Mulberry Leafsamples from different varieties.The results showed that the optimal extraction and determination method of DNJ was as follows:dry grinded Mulberry Leaf powder were were sifted through 50 mesh sieve;0.2 g sample were added to 200 mL buffer with pH=2.0 and ultrasonic extraction 20 min.After centrifugation at 10000 rpm for 10 min,the supernatant was retained,10μL of sample solution was accurately absorbed,placed in a 1.5 mL centrifuge tube,mixed with 10μL of 0.2 mol/L(pH=7)potassium borate buffer in a 1.5 mL test tube,and 20μL of 5 mmol/L FMOC-Cl(dissolved in acetonitrile)was added.The mixture was quickly mixed and reacted at room temperature for 10 min.The reaction was terminated by adding 10μL of 0.1 mol/L glycine solution.Under these extraction conditions,the RSD of the precision,repeatability,recovery and intermediate precision of the same batch of Mulberry Leaf sample were all less than 3%.The DNJ content of 10 batches of Mulberry Leaf samples from different sources in Zhejiang province determined by this method was 0.8 g/kg~5.7 g/kg.Our results provide methodological basis and reference value for the determination of DNJ content of Mulberry Leaf.