| Objective:（1）To study the applicability of microbial limit test for Liuwei Dihuang capsule,capsule shell and capsule contents,to determine the cause of the strong bacteriostasis of this variety of drugs and to establish the microbial limit test method.（2）To investigate the bacteriostatic effect of vacant gelatin capsules on bacteria and fungi by studying the applicability of counting method and control bacteria test method for vacant gelatin capsules.（3）To establish a HPLC method for the simultaneous determination of benzoic acid and its sodium salt（calculated by benzoic acid）,sorbic acid and its potassium salt（calculated by sorbic acid）in Vacant Gelatin Capsules.Methods:（1）According to the relevant requirements of the general principles1105 and 1106 of the fourth part of the Chinese Pharmacopoeia（2020）,eight batches of Liuwei Dihuang capsules were collected,and the applicability of the counting method and the control bacteria test method were tested on the capsule,the capsule contents and the capsule shell respectively.（2）The Staphylococcus aureus,Candida albicans,Escherichia coli,Pseudomonas aeruginosa and salmonella paratyphi B were selected as the test strains.According to the general principles 1105 and 1106 of the four parts of Chinese Pharmacopoeia 2020 edition,30 batches of vacant gelatin capsules were counted,and the method of adding bacteria to the plate was used to test the recovery of vacant gelatin capsules,and the test method of controlling bacteria Escherichia coli and salmonella was used to test the applicability of vacant gelatin capsules,the bacteriostatic effect of gelatin hollow capsule on bacteria and fungi was investigated.（3）The protein was removed by adding protein precipitator and C18 solid phase extraction（SPE）column,then eluted with 60%methanol water and filtered at constant volume,methanol:formic acid-ammonium acetate solution was used as mobile phase,flow rate was 1.0ml/min,detection wavelength was 230 nm,column temperature was 30℃,injection volume was 10μL.Results:（1）Two out of the eight batches of Liuwei dihuang capsules have very strong antibacterial activity against bacteria such as Staphylococcus aureus,Escherichia coli and salmonella,the total number of aerobic bacteria needed 1:1000（1 m L/dish）to eliminate its bacteriostasis.The methods of controlling Escherichia coli,salmonella and bile salt-resistant gram-negative were all the dilution method of bacteria-increasing solution,the volume of culture medium inoculated was 10 times,100 times and 20 times as much as that of conventional culture medium.The other 6 batches of capsules can be used to determine the total number of aerobic bacteria by plate method 1:50（1m L/dish）,and the control bacteria,E.coli and bile salt-resistant gram-negative bacteria can be used by conventional method,salmonella could be diluted by enrichment solution（200 m L TSB）.The bacteriostasis of 2 batches of Liuwei Dihuang capsules with strong bacteriostasis was caused by the capsule shell,and had nothing to do with the contents of the capsules.（2）The results showed that the recovery ratio of the Candida albicans of vacant gelatin capsules was in the range of 0.5～2,two batches of the test solution（1 m L/dish）were tested with plate method at a ratio of 1:10,000 to eliminate its bacteriostatic effect on bacteria,the other 28 batches were tested by plate method with1:100（1 m L/dish）to eliminate the bacteriostasis.The results of the applicability test of the control bacteria test method:among the 30 batches of vacant gelatin capsules,2 batches were examined by the dilution method（2000 m L TSB）and 28 batches were examined by the routine method（100m L TSB）Two batches（10000 m L TSB）,one batch（500 m L TSB）and the other 27 batches（100 m L TSB）were tested for salmonella.（3）Benzoic acid and sorbic acid were extracted by using precipitator and C18 solid phase extraction column,which can remove protein and some pigment effectively.The linear relationship between benzoic acid and sorbic acid was good in the concentration range of 0～54.783μg/m L and 0～55.578μg/m L,and the correlation coefficients（r）were 0.99985 and 0.99997,respectively.The detection limits of benzoic acid and sorbic acid were 3 mg/kg and 4 mg/kg,respectively,the recoveries of the samples ranged from 92.4%to 106.1%and the relative standard deviation（RSD）ranged from 0.4%to 5.5%,the relative standard deviation（RSD）was less than 3.0%.Benzoic acid and sorbic acid in30 batches of vacant gelatin capsules were determined by this method.Benzoic acid and sorbic acid were detected in 19 batches and 3 batches respectively Two batches of benzoic acid and sorbic acid were detected at the same time,and these two batches had very strong bacteriostasis to bacteria.Conclusion:（1）The established method for microbial limit test of Liuwei Dihuang capsules is scientific,effective and reasonable,and in accordance with the relevant provisions of the Chinese Pharmacopoeia（2020 edition）.The method is feasible.It was found that there was a capsule shell with strong bacteriostasis to bacteria.The strong bacteriostasis of the capsule shell affected the quality control of the capsule,so the quality control and safety of the capsule shell should be paid attention to.（2）Gelatin hollow capsule 1:10（1m L/dish）had no obvious bacteriostasis to fungi,but some had certain bacteriostasis to bacteria.The bacteriostasis intensity of gelatin hollow capsule from different manufacturers had certain difference,there are also some differences between different lot numbers of the same manufacturer.Two of the 30 batches of vacant gelatin capsules had very strong bacteriostatic effects on bacteria such as Staphylococcus aureus,Pseudomonas aeruginosa,Escherichia coli and salmonella.（3）The established method for High-performance liquid chromatography detection of benzoic acid and sorbic acid in vacant gelatin capsules is simple to operate,has good linear relationship within the curve range,and has high accuracy,precision and sensitivity,it has good repeatability and stability,and can be used for the determination of benzoic acid and its sodium salt（in benzoic acid）,sorbic acid and its potassium salt（in sorbic acid）in vacant gelatin capsules.Among the 30 batches of vacant gelatin capsules,19 batches were found to contain benzoic acid and sorbic acid,3 batches were found to contain sorbic acid,and2 batches were found to contain both benzoic acid and sorbic acid,and its Escherichia coli,salmonella and other bacteria have a very strong antibacterial effect,it is suggested that attention should be paid to the bacteriostasis of benzoic acid and its sodium salt,sorbic acid and potassium salt in vacant gelatin capsules. |
PDF Full Text Request