Study On The Optimization Of Mulberry Leaves With High Alkaloid Content And The Extraction Technology Of Total Alkaloid

Objective: By the establishment of a method for the determination of1-deoxynojirimycin in mulberry leaves, and the investigation of the methodology, the thesis measuresthe content of1-deoxynojirimycin in different varieties, different seasons and different positions of mulberry leaves, and screens out the highest content of1-deoxynojirimycin in mulberry varieties, the best picking season and leaf position. At the same time, the thesis optimizes the extraction of mulberry total alkaloid, carries out the study on quality control and LC-MS analysis of its extract. The thesis provides theoretical bases for the research on the preparation technology of the total alkaloid extracted from mulberry leaves, the establishment of the quality control criterion.Methods:Using method (HPLC-ELSD), column: WELCH XB-NH2(4.6×250mm,5μm), mobile phase acetonitrile: water solution (86:14), column temperature:40℃, injection volume:20μL, flow rate:1mL-min-1, ELSD drift tube temperature:45℃, the flow rate of carrier gas chromatographic separation conditions for2.5L-min-1. The95%confidence interval overlap method to compare and analyze the different leaf13mulberry varieties, different seasons1-deoxynojirimycin content. Single factor study of reflux method, dipping method, ultrasonic immersion method of three kinds of extraction methods, and then to the reflux method the best screening of two extraction solvents, methanol and ethanol as the extraction solvent of single factor, the optimum extraction method that total alkaloids from mulberry leaf for ethanol reflux extraction, based on the L9(34) the orthogonal experiment, the total alkaloids and1-deoxynojirimycin content as the index, the influence factors on the reflux extraction(extraction time,solid-liquid ratio,the concentration of ethanol)optimization. Using liquid chromatography-mass spectrometry method for the analysis of the components of mulberry leaves extract, chromatographic column of WELCH XB-NH2column (4.6×250mm,5μm); mobile phase acetonitrile:water solution (86:14), column temperature:40℃, injection volume:20μL, flow rate:1mL-min-1. Mass spectrometric conditions were as follows:dry gas flow rate:10L, min-1, gas flow rate:45psi, temperature:345℃heating module, positive ion mode detection, full scan mass range:100～1500m/z.Results(1)The thesis establishes a simple, fast and accurate method for the determination of the content of1-deoxynojirimycin. Results:the linear range of1-deoxynojirimycin is2.5-20μg; regression equation and correlation coefficient was:y=402.8x-122.1, r=0.9994, the test solution was stable in10hours. Precision of the instrument is good, and RSD is0.865%. Results of repeatability (n=5):the average content of1-deoxynojirimycin (RSD) in the same batch of mulberry leaves is1.100mg/g(2.1%); recovery rate (RSD) was98.17%(1.77%). The contents of three batches of samples were1.152mg/g(1.58%),1.044mg/g (1.23%), and1.120mg/g (1.04%) respectively.(2) Of15kinds of Mulberry (66mulberry samples), the varieties with higher content of1-deoxynojirimycin were screened out, namely, Li24xmiao33, Zao Shengsang No.1, Sha2×Lun109, Yu71-1. Through the analysis of the data, the content of the autumn leaves was significantly higher than that in spring leaves, and the content in middle leaves and lower leaves was apparently higher than that in the upper leaves.(3) Comparing different extraction methods and different extraction solvent, ethanol reflux extraction was considered as the best extraction method. The extraction techniques of total alkaloids in mulberry leaves and1-deoxynojirimycin were optimized by orthogonal method. With8times amount of70%ethanol reflux extraction of mulberry powder1hour. The average content (RSD) of total alkaloid in the verification test is5.217mg/g(1.117%), and the average content (RSD) of1-deoxynojirimycin is1.170mg/g(2.33%).(4) Using UPLC-QTOF to analyze the components in mulberry leaves, two kinds of possible compounds were speculated, namely, Nigerose (Sakebiose), another is not retrieved.Conclusions(1)The thesis establishes a method for determination of the content of DNJ in mulberry leaves, which provides the basis for the setting of quality standard.(2) Optimal Mulberry variety, Li24xmiao33, Zao Shengsang No.1, Sha2×Lun109, Yu71-1was filtered out.(3) The study screened the extraction technology of total alkaloids from mulberry leaves. The technology, which is reasonable, stable, feasible, and enviroument-friendly, is suitable for industrialized production.(4) Through LC-MS analysis, two kinds of possible compounds were listed, which lays foundations for further exploration of unknown component.