Study On Detection, Extraction, Separation And Purification Of 1-Deoxynojirimycin In Mulberry Leaves

1-deoxynojirimycin(DNJ) is a unique polyhydroxy alkaloids with a strong hypoglycemic activity in mulberry leaves. It is the main active components of hypoglycemic in mulberry leaves, which can be used for the treatment of diabetes.The modern pharmacology have showed that DNJ also has the functions of lowering blood lipid, antiviral, anti-tumor and losing weight and so on. It has important application value in the clinical treatment of diabetes and molecular biology research.In order to obtain high purity DNJ samples, the article mainly studies on qualitative and quantitative detection, extraction process,separation and purification technology of DNJ in mulberry leaves, which can provide theoretical basis for the development and application of DNJ in future.1.The conditions of DNJ qualitative analysis was determined. We had compared several developing reagent and chromogenic reagent that were used for the qualitative detection to alkaloids, and detected the DNJ through thin-layer chromatography method. Experimental results showed that when n-butanol: acetic acid:water(4:1:1) as developing agent, chloro-o-tolidine as chromogenic agent,unfold effect of DNJ in the thin layer plate was the best. The separation effect of the method was good, and DNJ spot was roundness and clear. It can be used for rapid qualitative analysis and detection of DNJ.2.Because DNJ is a small molecular piperidine alkaloid that, very strong, a structure is similar to monosaccharides with stronger polarity and good water solubility, so the C18 column is not suitable for detecting it. With amino column as chromatographic column, this experiment had established a high performance liquid chromatographic(HPLC) method by UV-detector detection after precolume dervatization with 9-fluorenylmethyl chlorformate(FMOC-Cl). The method showed a good linear relationship in the range of 5~50μg/mL, and the linear correlation coefficient of standard curve was 0.9897. According to the results of stability,precision, repeatability and recovery test, we know that the method was stable and reliable to be used as an effective method to detect the content of DNJ in mulberryleaves.3.Extraction process of the ultrasonic-surfactant extraction technology of DNJ in mulberry leaves was optimized through the single factor experiment and response surface design, and the optimum conditions were obtained as follows: ultrasonic time was 32 min, the liquid ratio of 10mL/g, extraction temperature was 73℃, the concentration of surfactant was 2.1%, under the conditions, the extraction amount of DNJ was 0.996mg/g, the yield was 83%. In the best conditions, the extraction efficiency of DNJ in mulberry leaves by reflux extraction, ultrasonic extraction and ultrasonic-surfactant extraction method were compared.4.In this experiment, the crude extraction extracted from mulberry leaves were isolated and purified by cationic exchange resin(D72, H+), anion exchange resin(D296, OH-) and neutral alumina column chromatography. At last, we obtained high purity DNJ after the detection and analysis by HPLC and HPLC-MS.