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Objective: The effect of ethanol to material ratio on ethanol precipitation and sediment morphology of Shuanghuanglian preparation (SHLP) were researched, and the feasibility of comprehensive evaluation of ethanol precipitation sediment fractal dimension was discussed. Methods: For the SHLP ethanol precipitation process, HPLC and gravimetric analysis were used to investigate the effects of ethanol to material ratio on the overall retention of the index components and the liquid content of the filter cake. The direct observation, atomic force microscope observation and "area-perimeter" calculation method were used to investigate the influence of ethanol to material ratio on the apparent morphology, micro-morphology and fractal dimension of the precipitate. At the same time, Pearson correlation analysis method was used to investigate the correlation between the fractal dimension and the above evaluation index of ethanol precipitation effect. Results: With the increase of the ethanol to material ratio, the comprehensive retention of the index components and the fractal dimension of the ethanol precipitation precipitates was gradually increased, while the liquid content of the filter cake was gradually decreased. Therefore, the fractal dimension of sediment was positively correlated with the comprehensive retention rate of index components, but negatively correlated with the liquid content of the filter cake. Conclusion: The fractal dimension of ethanol precipitation sediment can be used as comprehensive evaluation index for reflecting the ethanol precipitation effect in the SHLP ethanol precipitation process, which can provide a reference for the visualized fine control research of the subsequent SHLP ethanol precipitation process.
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OBJECTIVE：To optim ize the water extraction-ethanol precipitation technology of polysaccharide from Litchi chinensis seed，and to evaluate its hypoglycemic activity in vitro . METHODS ：The content of polysaccharides was determined by phenol-sulfuric acid colorimetry ，and the extraction rate of polysaccharides was calculated. Single factor test and response surface methodology were used to optimize the water extraction technology with the ratio of material to liquid ，extraction times and extraction time as factors ，and the extraction rate of polysaccharide as index. Single factor test was used to screen the concentration volume fraction of water extract and ethanol precipitation Using acarbose as contro l，4-nitrophenol-α-D-glucopyranoside method was used to investigate in vitro inhibitory activity of polysaccharide from L. chinensis seed to α-glucosidase. RESULTS ：The optimal technology was the ratio of material to liquid 1∶19 （g/mL），decocting for 3 times，1 h for each time ，concentrating the water extract to 40％ of original volume ，and adding ethanol to 80％ volume fraction. After deproteinization by Sevage method ，the crude polysaccharide of L. chinensis seed was obtained. The results of 3 times of validation tests showed that ，extraction rates of polysaccharide were 7.61％，7.89％，7.99％，average extraction rate was 7.83％（RSD＝2.52％，n＝3）. The contents of polysaccharide were 55.57％，55.83％ and 56.66％，average content was 56.02％（RSD＝1.81％，n＝3）. The inhibitory activity of the polysaccharide from L. chinensis seed to α-glucosidase were increased as concentration ；its IC 50 was 0.056 mg/mL，which was lower than positive control acarbose （0.196 mg/mL）. CONCLUSIONS：The optimal water extraction-ethanol precipitation technology of polysaccharide from L. chinensis seed is stable and feasible. The polysaccharide from L. chinensis seed show significant in vitro inhibitory effect on α-glucosidase，which is better than that of acarbose.
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OBJECTIVE：To optimize the water extraction and ethanol precipitation technology of Zhuang medicine Baijin granules. METHODS ：HPLC method was adopted to determine the content of bergenin in Baijin granules extract. The extraction routes of Baijin granules （water decoction ，70％ ethanol reflux extraction ，water decoction combined with 70％ ethanol reflux extraction）was screened primarily with the yield of extract and the experiment of reducing uric acid of mice. The orthogonal test was adopted to optimize water extraction technology of Baijin granules with water multiple ，extraction time and extraction times as factors，taking the extraction yield and the bergenin content as index ，then the validation test was carried out. The orthogonal test was adopted to optimize the alcohol precipitation process of Baijin granules including the relative density of medicinal materials ， alcohol content and the alcohol precipitation time ，then the validation test was carried out. By the experiment of reducing uric acid ， the effects of medicinal materials extract of Baijin granules extract were compared before and after ethanol precipitation. RESLUTS：Established method for content determination of bergenin with linearity range of 0.007 2-0.288 mg/mL，had good precision，reproducibility，stability and accuracy. The initially chosen extraction process of Baijin granules was water decoction extraction. The optimal water extraction technology was soaked for 0.5 h，then decocted for 3 times with 14-fold water （mL/g）and 1.0 h each time. The optimal ethanol precipitation process was to concentrate the water extract to a relative density of 1.0 g/mL with alcohol content of liquid at 60％ and precipitated for 12 h. Validation tests showed that RSDs of extract yield and beragenin content were all lower than 2％（n＝3）. The experiment of pharmacodynamics showed that water extract （before ethanol precipitation ）and water extract after alcohol precipitation could significantly decrease the level of uric acid in hyperuricemia model mice （P＜0.01）. There was no statistical significance in the reduction of uric acid between 2 groups（P＞0.05）. CONCLUSIONS ：The optimized water extraction technology can obtain good extract yield and bergenin content ，and combined with ethanol precipitation technology for removing excess impurities would not affect the pharmacodynamics. The water extraction and ethanol precipitation technology is feasible，and can be used for extracting the medicinal materials of Baijin granules and its edulcoration.
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Objective: Critical process parameters (CPPs) identification is an important step of the implementation of quality by design (QbD) concept. There are many CPP identification methods, such as risk analysis method, sensitivity analysis method, multiple linear regression method, standard partial regression coefficient (SPRC) method, and so on. The SPRC method can consider multiple process critical quality attributes (CQAs) simultaneously, but the determination of CPP number is subjective. Therefore, new CPP identification method is still required. Methods: The manufacturing process of Astragali Radix extract, which contained water reflux extraction, concentration, and ethanol precipitation, was used as an example. First, the multiple process CQAs were determined to be the yield of pigment, dry matter, sugars, and active ingredients. Second, the potential CPPs were determined by a knowledge organization method. Plackett-Burman designed experiments were then performed. A weighted determination coefficient (Rw2) method was presented to identify CPPs. In this method, the importance of different CQAs was considered. Process parameters were removed one-by-one according to their importance index. The decrease in Rw2 was used to characterize the importance of the removed parameter. If the decrease of Rw2 was less than a preset threshold, the removed parameter was not a CPP. Results: During the manufacturing process of Astragali Radix extract, the potential CPPs determined by the knowledge organization method were water consumption, reflux extraction time, extraction frequency, ethanol content, ethanol consumption, and concentration endpoint. Reflux extraction time, the first ethanol consumption, the second ethanol consumption, and the second ethanol precipitation refrigeration temperature were found to be CPPs using the weighted determination coefficient method with the threshold of 10%. Conclusion: Using the weighted determination coefficient method, CPPs can be determined with all the CQAs considered based on their importance. The determination of CPP number is more objective compared with the SPRC method.
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Objective: To establish a method for the determination of fractal dimension of ethanol precipitation flocs in two kinds of root medicinal herbs (Astragali Radix and Isatidis Radix). Method: The ethanol-precipitated flocs of Astragali Radix and Isatidis Radix were taken as the research objects. Firstly, the original microscopic images of the ethanol precipitation flocs were binarized by Ostu algorithm. Secondly, the influence of sampling volume, dilution multiple, image magnification ratio and number of image acquisition sites on fractal dimension was investigated using perimeter-area method. Result: The ethanol precipitation flocs of Astragali Radix and Isatidis Radix had fractal characteristics. At the same time, the stable and reliable fractal dimension of the flocs could be obtained when the sampling volume was 600-800 μL, the sample was diluted to 5-20 times, the microscopic image was enlarged to 200 times or 400 times, and four different image acquisition sites were selected. Conclusion: The established method is stable and feasible, which can provide reference for the fractal theory of ethanol precipitation flocs of root medicinal herbs.
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OBJECTIVE: To study the immunoregulatory effect of Dendrobium officinale polysaccharide (DOP) obtained by step ethanol precipitation on macrophages and explore its action mechanisms. METHODS: Step ethanol precipitation method was used to get DOPs of different molecular weights, MTT method was used to detect the protective effect of DOP on macrophages induced by LPS, neutral red method was used to detect the phagocytosis activity of macrophages, ELISA method was used to detect the contents of TNF-α, IL-6, and TGF-β1, and Griess method was used to detect the NO release quantity. RESULTS: The yield of DOP was the largest when the ethanol concentration was 75%. DOP could promote the proliferation and phagocytosis activity of RAW264. 7 cells, reduce the release of TNF-α, IL-6 and NO, and promote the secretion of TGF-β1 and the proportion of M2 significantly (P < 0.01). CONCLUSION: Dendrobium officinale polysaccharide has immune regulation effect on RAW264. 7 cells, and the mechanism may be related to the promotion of cell phagocytosis activity, inhibition of inflammatory factor release, promotion of TGF-β1 secretion, and increase of the proportion of M2.
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Objective: To evaluate the optimal separation and purification processes of water extract, the analytic techniques of particle size analysis, powder fluidity testing, and scanning electronic microscope were adopted to compare the influence factor of different separation and purification techniques on microscopic preparation characteristic of intermediate product prepared from water extract of traditional Chinese medicine (TCM) formula purification. Methods: Taking Gubi Granules (GG) which have established the production technology and quality standards in the previous study as an example, three common techniques used for water extract of TCM formula purification, including ethanol precipitation, column chromatogram of macroporous resin, and membrane separation, were applied to preparing the intermediate of GG. Results: Through the comprehensive analysis of fluidity, adhesive property, compressibility, permeability, particle microstructure and particle size distribution, it was found that membrane separation could obtain intermediates with better performance, which was conducive to the subsequent granulation, tabletting, and other process smoothly. The operating conditions for liquid concentration were 0.05 g crude drug/mL, 30 ℃ liquid temperature, 0.15 MPa pressure and 5 m/s flow rate. Conclusion: The different spray dried powders varied greatly. The membrane separation method of 0.2 μm Al2O3 ceramic was selected as the optimal process for separating and purifying of water extract of GG by analyzing the influencing factors of the pharmaceutical properties.
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The present study was designed to determine the relationships between the performance of ethanol precipitation and seven process parameters in the ethanol precipitation process of Re Du Ning Injections, including concentrate density, concentrate temperature, ethanol content, flow rate and stir rate in the addition of ethanol, precipitation time, and precipitation temperature. Under the experimental and simulated production conditions, a series of precipitated resultants were prepared by changing these variables one by one, and then examined by HPLC fingerprint analyses. Different from the traditional evaluation model based on single or a few constituents, the fingerprint data of every parameter fluctuation test was processed with Principal Component Analysis (PCA) to comprehensively assess the performance of ethanol precipitation. Our results showed that concentrate density, ethanol content, and precipitation time were the most important parameters that influence the recovery of active compounds in precipitation resultants. The present study would provide some reference for pharmaceutical scientists engaged in research on pharmaceutical process optimization and help pharmaceutical enterprises adapt a scientific and reasonable cost-effective approach to ensure the batch-to-batch quality consistency of the final products.
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Chemical Precipitation , Chromatography, High Pressure Liquid , Methods , Drugs, Chinese Herbal , Chemistry , Ethanol , Injections , Principal Component AnalysisABSTRACT
Continuous pharmaceutical manufacturing is one of the development directions in international pharmaceutical technology. In this study, a continuous mixing technology of ethanol and concentrated extract in the ethanol precipitation of Salvia miltiorrhiza was realized by using a membrane dispersion method. The effects of ethanol flowrate, concentrated extract flowrate, and flowrate ratio on ethanol precipitation results were investigated. With the increase of the flowrates of ethanol and concentrated extract, retention rate of active phenolic acids components was increased, and the total solid removal rate was decreased. The purity of active components in supernatants was mainly affected by the ratio of ethanol flowrate and concentrated extract flowrate. The mixing efficiency of adding ethanol under continuous flow mixing mode in this study was comparable to that of industrial ethanol precipitation. Continuous adding ethanol by using a membrane dispersion mixer is a promising technology with many advantages such as easy enlargement, large production per unit volume, and easy control.
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To determine the process parameters of optimal water-extraction and ethanol precipitation method for Xuanbi'antong (XBF) extract, which is a clinically experience formula for coronary disease. Orthogonal test L9(34) was conducted for the study of XBF water-extraction and ethanol precipitation process. Extractum, salvianolic acid B, rhizoma coptidis alkaloid, paeoniflorin, puerarin, ginsenoside Rb₁, ginsenosides and echinacoside were selected as marker components and multi-index comprehensive weighted score was used to select and verify optimal water-extraction and ethanol precipitation process. The optimal extraction process was as follows： XBF was added with 10 times distilled water, decocted for half an hour for 3 times. The best ethanol-precipitation process was established where the ethanol was added up to 70% and precipitated for 24 hours in 1.12 extract density (20 ℃). The optimized water-extraction and ethanol precipitation method is stable and reliable, and can provide reference for further development and utilization of the formula.
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Background Thermostable DNA polymerase (Taq Pol ?) from Thermus aquaticus has been widely used in PCR, which was usually extracted with Pluthero's method. The method used ammonium sulfate to precipitate the enzyme, and it saved effort and money but not time. Moreover, we found that 30-40% activity of Taq Pol I was lost at the ammonium sulfate precipitation step, and the product contained a small amount of DNA. Results We provided a novel, simplified and low-cost method to purify the Taq Pol ? after overproduction of the enzyme in Escherichia coli, which used ethanol instead of ammonium sulfate to precipitate the enzyme. The precipitate can be directly dissolved in the storage buffer without dialysis. In addition, DNA and RNA contamination was removed with DNase I and RNase A before precipitation, and the extraction procedure was optimized. Our improvements increase recovery rate and specific activity of the enzyme, and save labor, time, and cost. Conclusions Our method uses ethanol, DNase I, and RNase A to purify the Taq Pol ?, and simplifies the operation, and increases the enzyme recovery rate and quality.
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Taq Polymerase/isolation & purification , Taq Polymerase/genetics , Ethanol/chemistry , Chemical Precipitation , Polymerase Chain ReactionABSTRACT
Objective To compare the refining effects between ethanol precipitation and chitosan flocculation methods on extracting solutions of Shuyu compound granules.Methods Orthogonal experiment was utilized to examine the influence of liquid concentration, alcohol concentration and standing time on refining of Shuyu compound granules extracting solution by ethanol precipitation method, and compare the impact of ethanol precipitation and chitosan flocculation methods on the contents of solids and paeoniflorin.Results The optimized condition of ethanol precipitation method was 0.8 g/mL solution concentration (crude drug) with 20% ethanol standing for 6 h. Both ethanol precipitation method and chitosan flocculation method reduced the extraction ratio of Shuyu compound granules decoction, but chitosan flocculation method caused a great loss of paeoniflorin.Conclusion Low concentration ethanol precipitation method can be used for the purification of Shuyu compound granules. Whether chitosan flocculation method is suitable requires further study.
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Objective:To optimize the ethanol precipitation technique for Fufang Shenqi soft capsules. Methods: An orthogonal design was used to optimize the technique with the relative density of the concentrated solution, ethanol concentration, standing time, temperature of ethanol precipitation as the influencing factors and the yield of dry extract and the content of total polysaccharides as the indices. Results:The best ethanol precipitation technique was as follows:the relative density of the concentrated solution was 1. 10, 95% ethanol was used to obtain 60% ethanol concentration, and the standing time was 48 h under the temperature of 10-30℃. Con-clusion:The optimized ethanol precipitation technique for Fufang Shenqi soft capsules is simple and practicable, and suitable for prac-tical production.
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OBJECTIVE:To optimize the ethanol precipitation condition for Shaoshi hujing capsules.METHODS:The content of Paeoniflorin in ethanol precipitated solution was determined by HPLC.Effects of three factors-relative density of water decoction before decoction,concentration of ethanol and time of reaction on the content of paeoniflorin in the ethanol precipitation were investigated by orthogonal design.RESULTS:The relative density of water decoction before decoction had a significant impact on ethanol precipitation(P
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Objective To develop recovery prediction models for danshensu,salvianolic acid B,and salvianolic acid D in danshen ethanol precipitation.Methods Recorded the operation parameters of 15 normal batches of danshen ethanol precipitation in one Chinese medicine plant and measured five active component contents in danshen concentrated liquid and danshen ethanol precipitated liquid,the Stepwise-MLR recovery prediction models of dashensu,salvianolic acid B,and salvianolic acid D were developed based on an approach which combined the process parameters and the active component contents in danshen concentrated liquid.The importance of variables in the prediction models was analyzed later.Results The coefficient correlations of dashensu,salvianolic acid B,and salvianolic acid D recovery prediction models were all above 0.95.Conclusion The models developed for active component recovery prediction show a good predictability.Models can be used to help manufacturers to understand the ethanol precipitation process and improve the quality control ability of danshen ethanol precipitation.
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AIM: To test the effect of ethanol precipitation on the purifying technology of chlorogenic acid (CGA). METHODS: Using HPLC, the effect of the extraction of CGA from the crude Flos Lonicerae Japonica powder with three concentrations of ethanol was compared. RESULTS: Ethanol with concentration of 60% was optimal. CGA content was raised from 5.5% to 37.72% by use of ethanol precipitation and extraction in combination. CONCLUSION: The ethanol precipitation can get rid of the impurity in the crude powder of Flos Lonicerae Japonica effectively and the yield of CGA can reach 90.16%.