| National Ministry of Health in China recognizes mulberry leaves as both medicinal and edible plants.Mulberry leaf refers to the leaves of the genus Moraceae.It contains various active ingredients such as alkaloids and flavonoids.1-Deoxynojirimycin(DNJ)is one of them.DNJ is a natural glycosidase inhibitor,has a strong hypoglycemic function,thus it is often used in the treatment of diabetes.In this paper,the subcritical water extraction method is use to extract DNJ from mulberry leaves.The pre-column derivatization and HPLC were used for the qualitative and quantitative determination,and high performance liquid chromatography-tandem quadrupole mass spectrometry(HPLC-MS/MS)was used as a kind of qualitative detection.The purified DNJ was obtained by purifying the extract with macroporous resin and cation exchange resin.The purpose of this study is to provide a high-efficiency,high-yield process for the extraction and purification of DNJ from mulberry leaves.In addition,it also provides a theoretical reference for the comprehensive utilization of mulberry leaves and industrial extraction and purification of DNJ.The experimental results are as follows:(1)In the DNJ analysis and test,the moisture content of mulberry leaves is 7.24%.Mulberry leaf extract was precolumn derivatized with fluorenylmethyloxycarbonyl chloride(FMOC-Cl)and determined by HPLC.The peak time appeared at 3.7-3.9 min.After this method,the RSD of stability is 0.53%,the RSD of reproducibility is 0.74%,and the RSD of precision is 0.39%,all less than 2%,so the detection method is great.Qualitative detection by HPLC-MS/MS confirms that the extracted mulberry extract contains DNJ.The peak time of its liquid phase was observed at 3.18 min.In the positive ion source,m/z:386.20 refers to the molecular ion peak of[M+H]+,m/z:368.20 was corresponded to[M+H-H2O]+,m/z:190.08 was origin from[M+H-C14H12O]+.(2)In the DNJ extraction,the extraction rate of DNJ was seted as the crucial index.Compared with the hot water extraction and ultrasound-assisted extraction the performance of subcritical water extraction was pleasing,having the highest yield without pollution form toxic solvent.Single-factor tests was used to explored the effect of different process parameters on the extraction rate of DNJ in mulberry leaves.When the extraction yield is relatively high,the values of the parameters are as follows:temperature is 130?,time is 2h,ratio of solid to liquid is 1:20 g/mL.Based on this,using response surface method to optimize,the optimum extraction conditions for extraction of DNJ are 2.5h,132?,and 125 g/mL.Under this condition the predicted value of DNJ extraction rate is 84.79 mg/100g.The order of influence of each process parameter is:extraction temperature>material-to-liquid ratio>extraction time.Based on the above results,a verification experiment is conducted,its extraction rate of DNJ is 82.57 mg/100g.(3)In the DNJ purification process,AB-8 resin was selected as the macroporous resin.The purification were conducted using 60%ethanol solution as the eluent under neutral pH(pH=7)to get a best result.The adsorption rate is 37.2%and the desorption rate is 84.8%,in addition,the purity is 3.1%,which is increased by 2.9 times;In the 732 type cationic resin purification experiment,the extract is eluted with a 0.5 mol/L aqueous ammonia solution,pH=4 is the best state.The adsorption rate is 31.3%,and the desorption rate is 78.1%,besides the purity is 4.1%,which increased by 3.8 times;The dynamic adsorption and desorption of AB-8 resin is similar to that of 732 resin,and it flows through the resin column at a flow rate of 2BV/h.When AB-8 resin was combined with 732 resin,the purity of DNJ reached to 12.4%,and the purity increased by 11.5 times.The purification result under this condition is the best. |
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