Objective: To study and establish a microbial limit test method for pyrrole antifungal preparations. Methods: Using the mixed solution containing histidine, lecithin and polysorbate 80 as diluent and washing liquid, the microbial limit of these drugs was examined by centrifugal precipitation and membrane filtration, and the methodology was verified. Results: It was verified according to the appendix of China Pharmacopoeia (2005 edition). The results showed that the recovery rate of all strains was above 70%, and the positive bacteria of the control bacteria also grew well. Conclusion: It is feasible to use the mixed solution containing histidine, lecithin and polysorbate 80 as diluent and washing liquid, and to carry out the microbial limit test of pyrrole antifungal drug preparations by centrifugal sedimentation and bacterial collection combined with membrane filtration.
[Keywords:] preparation of pyrrole antifungal drugs; Microbial limit test; Centrifugal bacteria collection method; Membrane filtration method
Pyrrole antifungal agents, such as tebuconazole nitrate and clotrimazole, have broad-spectrum antifungal activities, and have good antibacterial activities against dermatophytes, Trichophyton, Aspergillus, pigmented fungi, Cryptococcus and Candida. By interfering with the activity of cytochrome P-450, these drugs inhibit the biosynthesis of ergosterol, the main sterol in fungal cell membrane, damage fungal cell membrane, change its permeability, and lead to the leakage of important substances in cells. They can also inhibit the biosynthesis of triacylglycerol and phospholipids, inhibit the activities of oxidase and peroxidase, cause the accumulation of hydrogen peroxide in cells, and lead to the degeneration of cell ultrastructure and cell necrosis. These drugs also have a certain inhibitory effect on bacteria. According to the current domestic requirements, it is necessary to establish a microbial limit test method for pharmaceutical preparations that do not need sterilization, including antibacterial and antifungal pharmaceutical preparations. However, due to the strong antibacterial activity of these drugs, how to eliminate their antibacterial activity and make the experiment go smoothly has become an extremely important and difficult key point in establishing the method. In this paper, the author selected pyrrole (such as tebuconazole nitrate and clotrimazole) antifungal preparations which are not suitable for microbial limit inspection in China at present for experimental study. After many experiments, the composition and amount of washing liquid were investigated and optimized. Finally, referring to the European Pharmacopoeia, it is determined that the mixed solution containing histidine, lecithin and polysorbate 80 is used as diluent and washing liquid, and the centrifugal sedimentation bacteria collection method is combined with membrane filtration method. Therefore, the microbial limit test method of tebuconazole nitrate and clotrimazole pharmaceutical preparations was established and verified by methodology. The results show that this method is reasonable and feasible.
1 reagents and instruments
1. 1 reagent test
Three batches of tebuconazole nitrate vaginal cream, three batches of clotrimazole vaginal tablets from two manufacturers, three batches of compound clotrimazole cream, three batches of clotrimazole cream and three batches of miconazole acetate cream are all commercially available drugs. Nutrient broth medium, modified Martin medium, nutrient agar medium, rose sodium agar medium, bile salt lactose medium, cetyltrimethylamine bromide agar medium and mannitol sodium chloride agar medium were all purchased from China Institute for the Control of Pharmaceutical and Biological Products. Histidine, lecithin, polysorbate 80, peptone, sodium chloride, potassium dihydrogen phosphate and disodium hydrogen phosphate are all commercially available analytical pure reagents and reagents. Staphylococcus aureus [CMCC(B)26003], Bacillus subtilis [CMCC(B)6350 1], Escherichia coli [CMCC(B)44 102], Pseudomonas aeruginosa [CMCC (b)1kl. Aspergillus Niger [CMCC(F)98003], the above strains were purchased from the strain room of China Institute for the Control of Pharmaceutical and Biological Products. According to the requirements of the appendix of China Pharmacopoeia (2005 edition), the above five verified strains were made into bacterial liquid, and the bacterial content per 1 ml was 50~ 100 cfu.
1.2 instrument
Clean workbench, medical aspirator, low-speed centrifuge, etc.
2 methods and results
2. 1 pharmacopoeia appendix collection method test results
The above drugs were made into a test solution of 1∶ 10 with the common diluent sterile sodium chloride-peptone buffer recommended by China Pharmacopoeia (2005 edition), and several treatment methods to eliminate the antibacterial effect of drugs were adopted in turn. That is, culture medium dilution method (taking 0.2 ml of 1∶ 10 test solution or injecting 0.2 ml of 1∶ 100 test solution into a Petri dish), centrifugal sedimentation bacteria collection method, membrane filtration method with pH 7.0 sterile sodium chloride-peptone buffer as washing solution, and centrifugal sedimentation bacteria collection method combined with membrane filtration method. Among them, using sterile sodium chloride-peptone buffer solution with pH 7.0 as washing solution, the total washing amount reached 65438±0000ml. See table 1 for the determination results of the recovery rate of the combined test of centrifugal sedimentation, bacteria collection and membrane filtration.
2.2 Determination of recovery rate
Even if centrifugal sedimentation method combined with membrane filtration method is adopted, the recovery rate of four of the five verified strains is still zero, which shows that pyrrole antifungal drugs have strong inhibitory effect on bacteria and fungi, or membrane filtration has strong adsorption effect on these drugs, and the washing liquid commonly used at present is difficult to wash, and several treatment methods commonly used in China are not suitable for these drugs.
2.3 Comparison of the Effects of Different Formulas of Diluent and Rinse
According to China Pharmacopoeia (2005 edition) [1] and European Pharmacopoeia (5th edition) [2], the following five kinds of diluents and rinsing solutions are prepared, as shown in Table 2.
A batch of clotrimazole vaginal tablets were selected to test the above five diluents and washing liquid in turn. Centrifugal sedimentation, bacterial collection and membrane filtration were combined, and the experimental results were compared. See table 3 for details.
The above results show that it is satisfactory to use European Pharmacopoeia formula solution and lecithin as imported reagents, or to use self-made No.5 solution as diluent and washing solution to eliminate the bacteriostatic effect of drugs, but when lecithin is replaced by domestic reagents, it cannot be filtered because of the turbidity of the solution. When the dosage of lecithin and polysorbate 80 is reduced to half of that in the European Pharmacopoeia, the clarity of the solution is not affected by the quality of reagents, and the washing effect can also meet the experimental requirements. 2.4 preparation method
2.4. 1 According to the method established by the above test results, take 10 g of test sample, add the mixed solution containing sterile histidine-lecithin-polysorbate 80 (see preparation method) to 100 ml, keep the temperature at 45℃, and shake well until the test sample is evenly dispersed, and make1. The counting of bacteria, molds and yeasts is combined by centrifugal precipitation and membrane filtration. Take 50 ml of 1: 10 stock solution, centrifuge at 500 rpm for 5 minutes, take all the supernatant, add the above mixed solution to 50 ml, centrifuge at 3,000 rpm for 20 minutes, take about 5 ml of bottom bacteria collection solution, and add the above mixed solution to 50 ml, namely1:650. Take 1: 10's test solution 1 ml, add it to the above-mentioned 100 ml mixed solution, filter it all with a membrane filter, use the above-mentioned mixed solution as a washing solution, wash 100 ml for three times, and take the filter membrane, according to the law.
2.4.2 Control bacteria (such as Staphylococcus aureus and Pseudomonas aeruginosa). ) should be checked by centrifugal precipitation and membrane filtration. Take the sample solution 10 ml under the above-mentioned colony number 1: 10, add it into the above-mentioned 100 ml mixed solution, and filter it all with a membrane filter. The washing method is the same as colony counting, and the filter membrane is inoculated into the corresponding culture medium and checked according to law (Appendix J of China Pharmacopoeia, 2005).
2.4.3 preparation method of sterile histidine-lecithin-polysorbate 80 mixed solution polysorbate 80 15 g, lecithin 1.5 g, histidine 0.5 g, peptone 0.5 g, sodium chloride 2. 15 g, potassium dihydrogen phosphate 1.8 g, and dibasic phosphate.
2.5 Verification test
According to the requirements of China Pharmacopoeia (2005 edition) appendix, the established method was verified.
2.5. Verification of the counting method of1bacteria, mold and yeast: Take the above five kinds of bacteria solutions 1 ml respectively, and determine the number of live bacteria per ml in the prepared bacteria solution by plate counting method. Test control group: take 1∶ 10 test solution 1 ml, add 100 ml of the above mixed solution, filter it all with a filter membrane, wash it in the same way as above, take the filter membrane, cultivate it at a specified temperature and count it. Experimental group: Take 1: 10 test solution 1 ml, and the operation is the same as the experimental control group. Add 1 ml of the above bacteria solution (50~ 100 cfu test bacteria) to the third rinse solution, take the filter membrane, culture at the specified temperature, count and calculate the recovery rate, as shown in the table. Diluent control group: Take the above five kinds of bacteria liquid 10ml(500 ~ 1000 cfu test bacteria) respectively, and operate the same as the control group, and calculate the recovery rate, as shown in Table 4. Calculate the recovery rate (%) according to the following formula:
The above experiments show that the recovery rate of each verified bacteria meets the requirements of the appendix of Pharmacopoeia, which shows that it is feasible to count bacteria, molds and yeasts by this method.
2.5.2 Verification of test group by controlled bacteria test method: Take the above 1: 10 test solution 10 ml, add it to the above 100 ml mixed solution, filter it all with filter membrane, and add the filter membrane to the corresponding culture medium 100 ml for enrichment culture. Blank control group: take 65438±00ml of the above mixed solution, and operate the same as the diluent blank control group. Negative bacteria control group: take 10 ml test solution, add it to the above mixed solution, and filter it all with filter membrane. The cleaning method is the same as above, but appropriate negative control bacteria are added to the third cleaning solution (for example, Escherichia coli is selected as the negative control bacteria for Staphylococcus aureus inspection). Positive bacteria control group: take 10 ml test solution, add it to the above mixed solution, and filter it all with filter membrane. The washing method is the same as above, but for the third time (10 ~ 65448), appropriate bacteria (such as Staphylococcus aureus) are added to the washing solution.
All the above groups were cultured at 35 ~ 37℃ 18 ~ 24 h, respectively, and then cultured at 35 ~ 37℃18 ~ 24 h. As a result, the positive bacteria control group grew well, indicating that these drugs have no bacteriostatic effect or their bacteriostatic effect can be ignored after this treatment. No negative bacteria were detected, indicating that the method of controlling bacteria is specific and feasible.
3 discussion
This experimental study shows that pyrrole drugs have strong antibacterial effects on bacteria and fungi. For these drugs with strong antibacterial activity, we should first explore ways to eliminate their antibacterial effects, and then we can successfully check their microbial limits.
If the membrane filtration method is used to check the microbial limit of drugs, the type and dosage of diluent and washing liquid are very important, and even the applicability of the method can be determined. As a diluent and rinse for microbial limit test, it should not only have the function of dissolving drugs, but also have the function of maintaining the permeability of bacterial cell membrane, repairing damaged cells and destroying the damage of drugs to bacterial cells. Histidine is one of the important nitrogen sources for the growth of Candida albicans in the cleaning solution formula determined in this experiment. Lecithin plays an important role in the repair of cell membrane; Polysorbate 80, as a surfactant, can reduce the surface tension between the periphery of bacteria and the contact surface of culture medium, so that peripheral nutrients can enter cells faster, thus promoting the growth of bacteria and other activities faster. The combination of lecithin and polysorbate 80 can neutralize the bacteriostatic agent, and the neutralization product has little effect on bacteria and culture medium. Therefore, adding these substances into diluent and washing liquid can effectively reduce the inhibitory effect of drugs on bacteria and fungi and promote the growth of damaged bacteria and fungi.
There is a certain quality difference between some domestic reagents and imported reagents. The contents of lecithin and polysorbate 80 in the washing liquid formula recorded in the European Pharmacopoeia are relatively high. When prepared with domestic products, the solubility is not good, resulting in turbid solution. When the washing amount is large, the filtration speed of the solution is slow, which affects the washing effect and even cannot be filtered. This problem can be effectively solved by reducing the dosage of each component in the formula. It is verified that even if the dosage of each component in the formula is halved, the effect can still meet the experimental needs, and the filtration speed is appropriate, which can also reduce the experimental cost.
[References]
[1] National Pharmacopoeia Committee. China Pharmacopoeia. The second part. Beijing: Chemical Industry Press, 2005. Appendix 93.
[2] European Pharmacopoeia. The fifth edition. Appendix XVI B.A377.