| ObjectiveOptimization of Mulberry hypoglycemic component extraction, purification and clarification molding process to determine the optimum preparation and ensure he actual operation of the process is stable and feasible, achieving cost saving, in addition. To establish Sangyuan Zhike capsule quality standards, we through the DNJ, mulberry leaf total polysaccharide and flavonoids content are determined, which is used to control the capsule quality, in order to lay the foundation for new drug application.Methods1. Extraction process of mulberry leaves hypoglycemic componentsOn the basis of single factor experiment, mulberry leaf alkaloid extraction rate as index, through the Box-Behnken response surface analysis method of extraction time, extraction temperature, ethanol volume fraction, and solid-liquid ratio and extraction times and solvent pH of mulberry leaf total alkaloid extraction yield, the effect of using the Design Expert 8.0.5 software to analyze the test data, to determine the optimum extraction process. Then mulberry leaf powder residue in water, extraction time, extraction temperature, solid-liquid ratio and extraction times of single factor experiment, select the significant factor, mulberry total polysaccharides and flavonoids extraction rate index analysis and optimization response surface methodology to determine the optimum extraction technology of mulberry polysaccharides and flavonoids.2. Clarification process of compound mulberry hypoglycemic ingredient extract with ZTC1+1Ⅱ natural clarifying agentThe remaining rate of mulberry leaf alkaloid, polysaccharides, flavonoids and the solids removal rate were used as indexes, optimized by single factor experiment to study the effect of some factors, such as the order and dosage of clarifying agents, the concentration and the temperature of the extracting solution, whisking time, whisking speed and holding time, response surface methodology was selected to decide the best clarification process.3. Mulberry leaf hypoglycemic components molding process researchFirst of all, according to the oral dose, to determine the granulating method, choose the appropriate drying process; Then it does not affect the determination of polysaccharide as the principle, screening pharmaceutical excipients; Next, according to the capsule quality inspection, hygroscopicity and flowability results to determine the amount of materials; Finally, grain yield as the dependent variable, material proportion, wetting agent ethanol concentration and dosage for the independent variables, using orthogonal experiment design, granulation process conditions, to determine the Sangyuan Zhike capsule best material proportion of granulating process, ethanol concentration and dosage.4. The quality standard of Sangyuan Zhike capsuleUsing thin layer chromatography (TLC) to the Sangyuan Zhike capsule material qualitative identification of mulberry leaves, with the control mulberry leaf and Rutin reference as the control. By high performance liquid tandem mass spectrometry (HPLC-MS/MS) to Sangyuan Zhike capsule main active ingredient deoxynojirimycin (DNJ) quantitative determination, to make preparation of DNJ content limits. HPLC method was developed for the determination of DNJ with AgilentHC-C18 column (4.6mm×50mm,5μm), water as the mobile phase A, acetonitrile as mobile phase B, A:B=10:90 (V:V), a flow rate of 0.4mL/min, column temperature was room temperature. MS conditions: electrospray ionization, capillary voltage 4000V, the positive ion mode, drying temperature 350℃, drying gas flow rate 10L/min, atomizing air pressure 20psi, scanning the multiple reaction monitoring (MRM), dwell time 200, fragmentation voltage 75, collision energy 18eV, used for quantitative analysis of ion pair at 164.1/69.0. Ultraviolet-visible spectrophotometer method was developed to quantitative determination of mulberry leaves total polysaccharides and flavones, to make preparation of mulberry leaves total polysaccharides and flavones content limits.Results1. The optimized condition for extracting alkaloid from mulberry leaf was heated to reflux extraction, extraction temperature 80℃, extraction time 120min, ethanol concentration 65%, pH 5, solid-liquid ratio 1:12, extraction 1, mulberry leaf alkaloid extraction rate 0.356%; Mulberry leaves residue add water to mulberry leaves total polysaccharides and total flavonoids extraction, the best extraction technology is heating reflux, extraction temperature 82℃, extracting time150min, solid-liquid ratio 1:10, extraction times two, mulberry leaf total polysaccharides and total flavonoids extraction yield of 8.161%and 2.589%respectively. Actual value and the predicted deviation rate were 2.068%,2.346%and 1.293%.2.The optimal process:the order of the ZTC1+1Ⅱ natural clarifying agents was part B after part A, the dosage of clarifiers B and A were 10% and 5% volume of the extract, the extract solution was 0.17g/mL, the temperature was 76℃, the whisking speed was 120r/min, the whisking time was 10 min,80℃ holding 30 min, and standing time was 12 h, the remaining rate of mulberry leaf alkaloid, polysaccharides and flavonoids were 92.5%,90.2% and 91.1%, the solids removal rate was 26.5%.3.Vacuum drying, dry extract granulating method, dry extract and mixed excipient (microcrystalline cellulose:of dihydrate calcium sulphate=2:1) to 10:1.5, wetting agent was ethanol concentration 95%, the dosage was 14% (14 ml/100 g dry extract powder), drying temperature was 60℃, dried for 10 min. Under these conditions, an average particle density was 0.7464 g/mL, using the 0 type capsule shell, each capsule is about 0.5g.4. Determined by LC method of mulberry leaf flavonoids in finished pharmaceutical products and HPLC-MS/MS determination of the content of DNJ, formulated the content limits of DNJ was not less than 14.3386mg/g, mulberry total polysaccharides content was not less than 612.1930 mg/g, mulberry leaf total flavonoid content was not less than 88.5849mg/g.ConclusionThrough the Box-Behnken response surface analysis to investigate the extraction time, extraction temperature, extraction times, and solid-liquid ratio and extraction solvent pH influence on mulberry leaf alkaloid extraction yield, with mulberry leaf alkaloid, polysaccharides and flavonoids extraction rate as the index, we can determine the best value of each extraction factors. The result indicated that the established model had good fitting performance, the extraction process was stable and reliable, and then the method could be used to predict the extraction yield of alkaloids, polysaccharides and flavonoids from mulberry leaves. Sangyuan Zhike capsule used orthogonal test to select the best granulating molding process, results of verification test also showed that was a reliable process. The established quality standards were stable and feasible that can be used for pre-clinical trials. These experimental data provided a basis for a new drug application of Chinese medicine. |
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