| Objective:Ultrafiltration membrane separation is a kind of technology that can separate the big and small molecules in the solution, based on the principle of membrane screening. The extraction and refining of traditional Chinese medicine injection is to eliminate the big molecule impurity and pyrogen, in order to preserve the effective ingredient, and ensure the safety and effectiveness of clinical medication. The study is based on Qingkailing injection, utilizing the ultrafiltration technology in the refining and elimination of traditional Chinese medicine injection, in order to improve the quality, ensure the safety of medication, save the production cost, and shorten the production period.Methods:The original producing process of Qingkailing injection is as follows: The baicalin, hyodeoxycholic acid, and cholic acid are directly mixed in the injection. Cornu Bubali, Concha Margaritifera, Radix Isatidis, Fructus Gardenia, and Flos lonicera japonica are extracted and refined, mixed as 6-blended solution with alcohol precipitation,and then 8-blended solution with activated carbon, the injection is finished.In the study, through analysis of the impurity to be eliminated and the effective ingredient to be preserved in every step of the production of the Qingkailing injection, The process of membrane separation is defined; The right membrane material, aperture, and module type are found; The secondary membrane filtering mode is set up; The ultrafiltration technology in the step of 6-blended solution refining and pyrogen elimination are utilized, and the quality and effectiveness of the ultrafiltration product are evaluated.1. Utilizing ultrafiltration in the step of 6-blended solution refining:①In the ultrafiltration of 6-blended solution, the polyacrylonitrile membranes with aperture of 50KD, 100KD and 150KD are used. We analyze the impact of membrane aperture and the operation pressure on the transferring rate of total nitrogen, geniposide, cholic acid, and the impact on the membrane flux and the decreasing rate of the solid content. The change of ingredients before and after ultrafiltration is also studied.②The guideline is chosen as the recovering rate of the membrane flux, then sodium hydroxide and sodium hypochlorite are chosen as the chemical cleaning, and the ultrafiltration membrane is cleaned through several different methods. After that, the best process of cleaning the polluted membrane is found. Finally, the optimum condition of ultrafiltration for 6-blended solution and cleaning process are determined.2. Utilizing ultrafiltration in the step of pyrogen elimination:①By the limulus amebocyte lysate (LAL), we evaluate the effect of membranes with different interception precision on the eliminating of the bacterium endotoxin. Then, we choose polysulfone composite membrane of 30KD in the step of pyrogen elimination. In the ultrafiltration of Qingkailing injection with pyrogen, we use this kind of membrane, and analyze the pyrogen by the pyrogen detection method to validate the effectiveness of ultrafiltration membrane in the step of pyrogen elimination.②Through the three-factor three-level orthogonal experiment and by the index of averaged membrane flux and effective baicalin ingredient, we build up the parameters of the membrane separation in the pyrogen elimination, and also evaluate these parameters through experiments. We further analyze the relation between the pressure difference and the membrane flux, in order to avoid the concentration polarization.③By the index of the recovering rate of membrane flux and the bacterium endotoxin, we analyze the cleaning effect of different methods with sodium hydroxide solution on the elimination of the bacterium endotoxin, and finally determine the optimum cleaning process of the pyrogen eliminating membrane.3. Evaluating the quality of the ultrafiltration product:①According to the quality criteria of the Qingkailing injection, We mensurate the total nitrogen, baicalin, cholic acid and geniposide. On the basis of these results, we mensurate the content of chlorogenic acid, and establish the measuring method of the active ingredient of Radix Isatidis (guanosine and adenosine) in the injection, and also evaluate the result through methodology.②We establish the fingerprint detection method and evaluate it through methodology. Compared with the fingerprint of the Qingkailing injection produced by the original process, we carry out similarity evaluation of the fingerprint of the sample injection after ultrafiltration.③According to the pharmacopoeia, we examine the protein, tannin, resin, oxalate, kalium ion, etc. And also, we evaluate the effect of ultrafiltration on the elimination of impurity. On the basis of these results, we also explore the possibility of measuring the content of protein in the sample injection after ultrafiltration by the method of Bradford and polyacryamide gel electrophoresis, which can serve as some foundation of further evaluation of the effect of ultrafiltration on the elimination of big molecule protein.4. Evaluating the effectiveness of the ultrafiltration product: We choose some important pharmacodynamic indexes of Qingkailing injection, such as antipyretic and anti-inflammation, and compare these indexes with those of the product produced by the original process.Results:1. Utilizing ultrafiltration in the step of 6-blended solution refining:①The ultrafiltration membrane is polyacrylamide hollow fiber, with aperture of 50KD. Under the pressure of 0.10MPa, the effective transferring rates of total nitrogen, cholic acid and geniposide are 99.36%, 99.99% and 98.62%, respectively. The decreasing rate of the solid content is 0.80%, and the averaged membrane flux is 174Lm-2h-1.②The ultrafiltration membrane is cleaned with sodium hydroxide or sodium hypochlorite solution for 30 minutes, which can improve the recovering rate of the membrane flux to the level of 98% or above.2. Utilizing ultrafiltration in the step of pyrogen elimination:①Polysulfone composite membranes of 10KD, 30KD and 50KD can all eliminate the bacterium endotoxin, among which, the effectiveness of elimination of 10KD and 30KD can be affirmed. The effectiveness of 30KD polysulfone composite membrane on the elimination of pyrogen can be affirmed.②The optimum parameters of the process is as follows: The pressure is within the scope of 0.10Mpa to 0.14MPa; the rotation speed of the pump is 250r/m; and the temperature of the medicine solution is 40℃. Under these conditions, the ultrafiltration flux is the maximum, and the production efficiency can also reach the highest level.③The effect of the cleaning of the membrane can be optimum if we wash it again and again for 45 minutes, after being marinated in the 2% sodium hydroxide solution.3. Evaluating the quality of the ultrafiltration product:①The contents of the effective ingredients of the ultrafiltration product are all within the qualified scope, and compared with the product by the original process, there is no obvious difference.②The result of the similarity evaluation of the fingerprint s of the injection after ultrafiltration and the injection produced by the original process is R=0.992-0.999.③The measurement result of related substances manifests that ultrafiltration can effectively eliminate the protein, tannin, resin, oxalate, kalium ion, and some other impurities.④The examination of pyrogen and unconventional toxicity can also measure up.4. Evaluating the effectiveness of the ultrafiltration product:①We compare the Qingkailing injection after ultrafiltration with the model group, and find that the product after ultrafiltration can notably restrain the rabbit fever caused by Lipopiysaecharides.②The injection after ultrafiltration can also restrain carrageenin-induced inflammation in rat metatarsus and cotton ball granuloma to some extent, which shows notable difference compared with the model group. The ultrafiltration injection shows stronger antipyretic and anti-inflammatory effects than that produce by the original process.5. Improvement of the economic benefit by ultrafiltration membrane separation technique:①Assumed that the annual output is 20 million pieces, and each piece is 10ml, then 1 million Yuan can be saved on the alcohol cost.②The production period can be shortened by 5 days, from originally 7 days to 2.Conclusion:This study determines the process of applying ultrafiltration technology to the production of Qingkailing injection; establishes the method of membrane screening; and confirms the feasibility and superiority of the process after comparing the quality, effectiveness and economic benefit of the ultrafiltration product with that produced by the original process. |
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