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Abstract [Objectives] This study was conducted to establish a method for microbial limit test of Compound Gangbangui Capsule. [Methods] According to the Chinese Pharmacopoei 2015 edition of the four general rules， the microbiological limits of non-sterile products were verified. [Results] Compound Gangbangui Capsule has a strong inhibitory effect on S. aureus， and the antimicrobial activity was significantly eliminated after increasing the diluent （1∶20）； and the recoveries were in the range of 0.5-2.0 when the total quantities of aerobic microbes were determined by dilution method （1∶20）. When the total quantities of mould and yeast were determined by conventional method （1∶20）， the recoveries were from 0.5 to 2.0， and Escherichia coli can be detected in experimental groups by the test solution dilution method. [Conclusions] With the plate method， the total quantities of aerobic bacteria and the quantities of mould and yeast were examined by increasing diluent and conventional method， respectively， and control bacteria， E. coli was examined by conventional method. This method has been proved effective for microbial limit test of Compound Gangbangui Capsule， and can effectively control the quality of the preparation. The method is accurate and reliable.
Key words Compound Gangbangui Capsule； Microbial limit test method； Dilution method
"Compound Gangbangui Capsule" is a traditional Chinese medicine prescription preparation prepared by Gynecology Department， The Second Affiliated Hospital of Guiyang College of Traditional Chinese Medicine. The medicine is prepared from seven herbal materials， i.e.， Polygonum perfoliatum L.， Salvia miltiorrhiza， Paeonia lactiflora Pall.， Cyperus rotundus L.， Bupleurum chinense， prepared Rheum officinale and Polygonum cuspidatum. It has the effects of promoting blood circulation to remove blood stasis， clearing away heat and toxic materials， and removing flood retention， and could be used for treating pelvic infection and chronic pelvic inflammatory disease. The preparation is used in Gynecology Department， The Second Affiliated Hospital of Guiyang College of Traditional Chinese Medicine from 2004 until now， exhibiting very significant clinical efficacy.
In order to improve the quality control standard of the hospital preparation， according to Chinese Pharmacopoei 2015 edition of the four general rules， methodological validation was performed to the applicability test of microbial limit test methods for the preparation， and the microbial limit test method of the preparation was established. Materials
Tested materials
Compound Gangbangui Capsule （lot number： 20160901， 20160902， 20160903）， The Second Affiliated Hospital of Guiyang College of Traditional Chinese Medicine）， specification： 0.5 g per capsule， 36 capsules per bottle.
Instruments
High-pressure sterilizer； purification operation room； clean bench； biosafety cabinet； oscillator； electro-heating thermostatic water bath； electro-heating standing-temperature cultivator.
Medium
The used media included sterile sodium chloride-peptone buffer solution （pH 7.0）， trypticase soy agar medium， trypticase soy broth medium， Sabouraud dextrose agar medium， MacConkey broth medium and Maconkey agar medium. The media were all produced by Qingdao High-tech Industrial Park Haibo Biotechnology Co.， Ltd.
Strains
The strains were all of the third generation， including Pseudomonas aeruginosa[CMCC（B）10104]， Staphylococcus aureus[CMCC（B）26003]， Bacillus subtilis[CMCC（B）63501]， Candida albicans[CMCC（F）98001]， Aspergillus niger[CMCC（F）98003]， and Escherichia coli[CMCC（B）44102]. All the strains were provided by National Institutes for Food and Drug Control.
Methods and Results
The experiment was carried out in a bioclean room attained the criteria of 104-grade cleanliness with unidirectional flow of air attained the criteria of 102-grade cleanliness， satisfying the requirements by "microbial limit test" in the Chinese Pharmacopoei 2015 edition of the four general rules[1].
Preparation method of microbial liquid
（1） Each of S. aureus， P. aeruginosa， B. subtilis， and E. coli was cultured on trypticase soy broth medium at 34 ℃ for 24 h， and the culture was diluted with sterile sodium chloride-peptone buffer solution with a pH of 7.0 into a bacterial suspension with bacterial concentration not higher than 10 000 cfu/ml.
（2） C. albicans was cultured in glucose at 24 ℃ for 72 h， and the culture was diluted with sterile sodium chloride-peptone buffer solution with a pH of 7.0 into a bacterial suspension with bacterial concentration not higher than 10 000 cfu/ml.
（3） A. niger was cultured on Sabouraud dextrose agar medium at 24 ℃ for 7 d， and the slant culture was added with 0.9% sterile sodium chloride solution containing 0.05% Tween 80， to wash off the Aspergillus spores， which were sucked to a sterile test tube. The fungal suspension was diluted with 0.9% sterile sodium chloride solution containing 0.05% Tween 80 to a fungal suspension with spore concentration not higher than 10 000 cfu/ml. Preparation of testing sample
A certain amount of Compound Gangbangui Capsule （10 g） was added with sterile sodium chloride-peptone buffer solution （pH 7.0） to 100 ml， and the solution was oscillated in a water bath at 45 ℃， obtaining a 1∶10 testing solution after well mixing for later use. In addition， the 1∶10 testing solution was diluted with sterile sodium chloride-peptone buffer solution （pH 7.0） to a 1∶20 testing solution.
Total quantities of aerobic microbes
A certain amount of the 1∶10 and 1∶20 testing solutions （10 ml） were added into sterilized test tubes， respectively， and then， 0.1 ml of corresponding testing fungi （S. aureus， P. aeruginosa， B. subtilis， A. niger and C. albicans） were added， respectively， when ensuring the final concentration not higher than 100 cfu/ml. After mixing well， 1 ml of corresponding fungal liquid was added into a plate， and two plates were prepared for each strain. Into each plate， 15-20 ml of melted trypticase soy agar medium at a temperature not higher than 45 ℃ were poured， followed by shaking and solidification. The strains were cultured in a refrigerator at 34 ℃ for 5 d under inverted state. The number of colony was determined， and meanwhile， the numbers of colony in the fungal liquid group and the control group of the testing solution were also determined by the same method. The recovery of each strain was calculated according to following formula： Recovery=（Average number of colony in the experimental group-Average number of colony in the control group of testing solution）/Average number of colony in the control group of fungal liquid. The results are shown in Table 1.
The results showed that when performing aerobic microbes counting of this product by conventional method （1∶10 testing solution）， the recoveries of S. aureus were all smaller than 0.5. Therefore， conventional method is not suitable for the aerobic bacteria counting of this product. Verification was performed by increasing dilution times （1∶20）， and the recoveries of above five kinds of microbes were in the range of 0.5-2.0.
Total quantities of mould and yeast
A certain amount of the 1∶10 and 1∶20 testing solution （10 ml） were added into sterilized test tubes， respectively， and then 0.1 ml of corresponding testing fungi （C. albicans and A. niger） were added， respectively， when ensuring the final concentration not higher than 100 cfu/ml. After mixing well， 1 ml of corresponding fungal liquid was added into a plate， and two plates were prepared for each strain. Into each plate， 15-20 ml of melted Sabouraud dextrose agar medium at a temperature not higher than 45 ℃ were poured， followed by shaking and solidification. The strains were cultured in a refrigerator at 24 ℃ for 5 d under inverted state. The number of colony was determined， and meanwhile， the numbers of colony in the fungal liquid group and the control group of the testing solution were also determined by the same method. The recovery of each strain was calculated according to following formula： Recovery=（Average number of colony in the experimental group-Average number of colony in the control group of testing solution）/Average number of colony in the control group of fungal liquid. The results are shown in Table 2. The results showed that when performing mould and yeast counting of this product by conventional method （1∶10 testing solution）， the recoveries of C. albicans and A. niger were all in the range of 0.5-2.0.
Control microbe examination
Method validation was performed according to "control microbe examination method" in the Chinese Pharmacopoei 2015 edition of the four general rules.
E. coli
（1） The experimental group： At first， 10 ml of the 1∶10 testing solution was inoculated to 100 ml of trypticase soy broth medium， and two groups were prepared in parallel. Then， 0.1 ml of prepared E. coli were added into one group， and the other groups was added with 0.1 ml of S. aureus suspension. After mixing well， the bacteria were cultured at 34 ℃ for 18-24 h. Next， 1 ml of each culture was inoculated to 100 ml of MacConkey broth medium and then cultured at 44 ℃ for 48 h. Each MacConkey liquid culture was streak-inoculated onto a Maconkey agar medium plate， and cultured at 34 ℃ for 3 d.
（2） The testing sample group： Similarly， 10 ml of the 1∶10 testing solution was inoculated to 100 ml of trypticase soy broth medium. The culture condition and the examination method were the same as the experimental group.
（3） The negative group： As negative control， 10 ml of sterile sodium chloride-peptone buffer solution （pH 7.0） was added into 100 ml of trypticase soy broth medium. The culture condition and the examination method were the same as the experimental group.
According to above method， there were colonies growing on Maconkey agar medium plate， i.e.， the experimental group was examined to be positive， and the colony on the plate was isolated. Pure culture and Gram stain microscopy demonstrated that the bacteria were E. coli. The testing sample group and the negative control group showed no colony， and were thus both negative. Therefore， the control bacteria examination of the product could be performed according to conventional method. The results are shown in Table 3.
Discussion
In this study， method validation tests were carried out by the method of diluting testing solution. The data showed that during the aerobic microbe counting test， Compound Gangbangui Capsule contained antibiotic ingredients and showed a stronger inhibitory effect on S. aureus， the recovery of which did not reached 0.5-2.0. Therefore， dilution method should be adopted， and finally， for the 1∶20 testing solution， the recoveries of aerobic microbes were all in the range of 0.5-2.0. During the mould and yeast counting test， the recoveries of C. albicans and A. niger were both in the range of 0.5-2.0， and they thus could be examined by conventional method. The results of control microbe examination of Compound Gangbangui Capsule showed that the positive microbes grew well in the experimental group， indicating that the product has no inhibitory effect on E. coli. Corresponding control microbe tests were carried out by conventional method according to Chinese Pharmacopeia of the four general rules （2015 edition）， and the test results are feasible. Chinese herba preparations have the characteristics of diverse and complicated chemical components， and the antimicrobial effect of any component might affect the growth of strains used in microbial limit test， thereby affecting the accuracy of the test method. Therefore， in this study， the problem whether there are antimicrobial components in the preparation was discussed at first， and validation of the test method was then performed under the premise of excluding the interference of antimicrobial components.
It could be seen from the validation results that by the method of increasing diluent （1∶20 testing solution）， the three batches of Compound Gangbangui Capsule showed the recoveries of S. aureus， P. aeruginosa， B. subtilis， C. albicans and A. niger all in the range of 0.5-2.0， and by conventional plate method （1∶10 testing solution）， the three batches of Compound Gangbangui Capsule showed the recoveries of mould and yeast quantities of A. niger and C. albicans all in the range of 0.5-2.0. The product only has an antimicrobial effect on S. aureus. When performing control microbe examination and validation of examination method by conventional method， the results showed that the positive microbes grew well， and the product has no inhibitory effect on E. coli.
The methods for eliminating the antimicrobial effect of tested products recorded in "Chinese Pharmacopeia" （2015 edition） include increasing diluent or medium volume， adding suitable neutralizer or inactivator， performing membrane filtration or combining above method[2]. Therefore， testing solutions of different dilution times were prepared to perform validation， and the results showed that the tested product has stronger inhibitory effect on S. aureus， and counting validation of the total quantities of aerobic bacteria by increasing diluent （1∶20） and mould and yeast by conventional method was performed. The results showed that the recovery of each specific strain satisfied the pharmacopeia requirements. The method is accurate and reliable， and could be used for the quality control of Tongmai Tangyanming Capsule. Compared with "Chinese Pharmacopeia" （2010 edition）， the microbial limit test was revised a lot in the 2015 edition[3-4]： the microbial counting was changed to the total quantities of aerobic bacteria and the total quantity of yeast， and the medium was changed to wide-spectrum trypticase soy broth medium and Sabouraud dextrose agar medium； and the content and requirements of the recovery test were also revised correspondingly. The methods tend to gear to international conventions. In this paper， study and validation were carried out according to the pharmacopeia requirements， which will provide reference for the microbial limit test of such pharmaceuticals.
References
[1] Chinese Pharmacopoeia Commission. Chinese Pharmacopeia （four part， 2015 edition）[S]. Beijing： China Medical Science Press， 2015： 1105-1107.
[2] Chinese Pharmacopoeia Commission. Chinese Pharmacopeia （four part， 2015 edition）[S]. Beijing： China Medical Science Press， 2015： 142.
[3] JING RX， ZHANG GL. The revision of clean environment and plate counting medium for microbial limit test in the Chinese Pharmacopoeia[J]. China Health Industry， 2015（5）： 184-185.
[4] LI CE， JIANG YF. Comparing the microbial limit test methods of Chinese Pharmacopoeia 2010 edition and 2015 edition for microbes determination in four pharmaceuticals[J]. Drug Standards of China， 2015， 16（2）： 86-90.
Key words Compound Gangbangui Capsule； Microbial limit test method； Dilution method
"Compound Gangbangui Capsule" is a traditional Chinese medicine prescription preparation prepared by Gynecology Department， The Second Affiliated Hospital of Guiyang College of Traditional Chinese Medicine. The medicine is prepared from seven herbal materials， i.e.， Polygonum perfoliatum L.， Salvia miltiorrhiza， Paeonia lactiflora Pall.， Cyperus rotundus L.， Bupleurum chinense， prepared Rheum officinale and Polygonum cuspidatum. It has the effects of promoting blood circulation to remove blood stasis， clearing away heat and toxic materials， and removing flood retention， and could be used for treating pelvic infection and chronic pelvic inflammatory disease. The preparation is used in Gynecology Department， The Second Affiliated Hospital of Guiyang College of Traditional Chinese Medicine from 2004 until now， exhibiting very significant clinical efficacy.
In order to improve the quality control standard of the hospital preparation， according to Chinese Pharmacopoei 2015 edition of the four general rules， methodological validation was performed to the applicability test of microbial limit test methods for the preparation， and the microbial limit test method of the preparation was established. Materials
Tested materials
Compound Gangbangui Capsule （lot number： 20160901， 20160902， 20160903）， The Second Affiliated Hospital of Guiyang College of Traditional Chinese Medicine）， specification： 0.5 g per capsule， 36 capsules per bottle.
Instruments
High-pressure sterilizer； purification operation room； clean bench； biosafety cabinet； oscillator； electro-heating thermostatic water bath； electro-heating standing-temperature cultivator.
Medium
The used media included sterile sodium chloride-peptone buffer solution （pH 7.0）， trypticase soy agar medium， trypticase soy broth medium， Sabouraud dextrose agar medium， MacConkey broth medium and Maconkey agar medium. The media were all produced by Qingdao High-tech Industrial Park Haibo Biotechnology Co.， Ltd.
Strains
The strains were all of the third generation， including Pseudomonas aeruginosa[CMCC（B）10104]， Staphylococcus aureus[CMCC（B）26003]， Bacillus subtilis[CMCC（B）63501]， Candida albicans[CMCC（F）98001]， Aspergillus niger[CMCC（F）98003]， and Escherichia coli[CMCC（B）44102]. All the strains were provided by National Institutes for Food and Drug Control.
Methods and Results
The experiment was carried out in a bioclean room attained the criteria of 104-grade cleanliness with unidirectional flow of air attained the criteria of 102-grade cleanliness， satisfying the requirements by "microbial limit test" in the Chinese Pharmacopoei 2015 edition of the four general rules[1].
Preparation method of microbial liquid
（1） Each of S. aureus， P. aeruginosa， B. subtilis， and E. coli was cultured on trypticase soy broth medium at 34 ℃ for 24 h， and the culture was diluted with sterile sodium chloride-peptone buffer solution with a pH of 7.0 into a bacterial suspension with bacterial concentration not higher than 10 000 cfu/ml.
（2） C. albicans was cultured in glucose at 24 ℃ for 72 h， and the culture was diluted with sterile sodium chloride-peptone buffer solution with a pH of 7.0 into a bacterial suspension with bacterial concentration not higher than 10 000 cfu/ml.
（3） A. niger was cultured on Sabouraud dextrose agar medium at 24 ℃ for 7 d， and the slant culture was added with 0.9% sterile sodium chloride solution containing 0.05% Tween 80， to wash off the Aspergillus spores， which were sucked to a sterile test tube. The fungal suspension was diluted with 0.9% sterile sodium chloride solution containing 0.05% Tween 80 to a fungal suspension with spore concentration not higher than 10 000 cfu/ml. Preparation of testing sample
A certain amount of Compound Gangbangui Capsule （10 g） was added with sterile sodium chloride-peptone buffer solution （pH 7.0） to 100 ml， and the solution was oscillated in a water bath at 45 ℃， obtaining a 1∶10 testing solution after well mixing for later use. In addition， the 1∶10 testing solution was diluted with sterile sodium chloride-peptone buffer solution （pH 7.0） to a 1∶20 testing solution.
Total quantities of aerobic microbes
A certain amount of the 1∶10 and 1∶20 testing solutions （10 ml） were added into sterilized test tubes， respectively， and then， 0.1 ml of corresponding testing fungi （S. aureus， P. aeruginosa， B. subtilis， A. niger and C. albicans） were added， respectively， when ensuring the final concentration not higher than 100 cfu/ml. After mixing well， 1 ml of corresponding fungal liquid was added into a plate， and two plates were prepared for each strain. Into each plate， 15-20 ml of melted trypticase soy agar medium at a temperature not higher than 45 ℃ were poured， followed by shaking and solidification. The strains were cultured in a refrigerator at 34 ℃ for 5 d under inverted state. The number of colony was determined， and meanwhile， the numbers of colony in the fungal liquid group and the control group of the testing solution were also determined by the same method. The recovery of each strain was calculated according to following formula： Recovery=（Average number of colony in the experimental group-Average number of colony in the control group of testing solution）/Average number of colony in the control group of fungal liquid. The results are shown in Table 1.
The results showed that when performing aerobic microbes counting of this product by conventional method （1∶10 testing solution）， the recoveries of S. aureus were all smaller than 0.5. Therefore， conventional method is not suitable for the aerobic bacteria counting of this product. Verification was performed by increasing dilution times （1∶20）， and the recoveries of above five kinds of microbes were in the range of 0.5-2.0.
Total quantities of mould and yeast
A certain amount of the 1∶10 and 1∶20 testing solution （10 ml） were added into sterilized test tubes， respectively， and then 0.1 ml of corresponding testing fungi （C. albicans and A. niger） were added， respectively， when ensuring the final concentration not higher than 100 cfu/ml. After mixing well， 1 ml of corresponding fungal liquid was added into a plate， and two plates were prepared for each strain. Into each plate， 15-20 ml of melted Sabouraud dextrose agar medium at a temperature not higher than 45 ℃ were poured， followed by shaking and solidification. The strains were cultured in a refrigerator at 24 ℃ for 5 d under inverted state. The number of colony was determined， and meanwhile， the numbers of colony in the fungal liquid group and the control group of the testing solution were also determined by the same method. The recovery of each strain was calculated according to following formula： Recovery=（Average number of colony in the experimental group-Average number of colony in the control group of testing solution）/Average number of colony in the control group of fungal liquid. The results are shown in Table 2. The results showed that when performing mould and yeast counting of this product by conventional method （1∶10 testing solution）， the recoveries of C. albicans and A. niger were all in the range of 0.5-2.0.
Control microbe examination
Method validation was performed according to "control microbe examination method" in the Chinese Pharmacopoei 2015 edition of the four general rules.
E. coli
（1） The experimental group： At first， 10 ml of the 1∶10 testing solution was inoculated to 100 ml of trypticase soy broth medium， and two groups were prepared in parallel. Then， 0.1 ml of prepared E. coli were added into one group， and the other groups was added with 0.1 ml of S. aureus suspension. After mixing well， the bacteria were cultured at 34 ℃ for 18-24 h. Next， 1 ml of each culture was inoculated to 100 ml of MacConkey broth medium and then cultured at 44 ℃ for 48 h. Each MacConkey liquid culture was streak-inoculated onto a Maconkey agar medium plate， and cultured at 34 ℃ for 3 d.
（2） The testing sample group： Similarly， 10 ml of the 1∶10 testing solution was inoculated to 100 ml of trypticase soy broth medium. The culture condition and the examination method were the same as the experimental group.
（3） The negative group： As negative control， 10 ml of sterile sodium chloride-peptone buffer solution （pH 7.0） was added into 100 ml of trypticase soy broth medium. The culture condition and the examination method were the same as the experimental group.
According to above method， there were colonies growing on Maconkey agar medium plate， i.e.， the experimental group was examined to be positive， and the colony on the plate was isolated. Pure culture and Gram stain microscopy demonstrated that the bacteria were E. coli. The testing sample group and the negative control group showed no colony， and were thus both negative. Therefore， the control bacteria examination of the product could be performed according to conventional method. The results are shown in Table 3.
Discussion
In this study， method validation tests were carried out by the method of diluting testing solution. The data showed that during the aerobic microbe counting test， Compound Gangbangui Capsule contained antibiotic ingredients and showed a stronger inhibitory effect on S. aureus， the recovery of which did not reached 0.5-2.0. Therefore， dilution method should be adopted， and finally， for the 1∶20 testing solution， the recoveries of aerobic microbes were all in the range of 0.5-2.0. During the mould and yeast counting test， the recoveries of C. albicans and A. niger were both in the range of 0.5-2.0， and they thus could be examined by conventional method. The results of control microbe examination of Compound Gangbangui Capsule showed that the positive microbes grew well in the experimental group， indicating that the product has no inhibitory effect on E. coli. Corresponding control microbe tests were carried out by conventional method according to Chinese Pharmacopeia of the four general rules （2015 edition）， and the test results are feasible. Chinese herba preparations have the characteristics of diverse and complicated chemical components， and the antimicrobial effect of any component might affect the growth of strains used in microbial limit test， thereby affecting the accuracy of the test method. Therefore， in this study， the problem whether there are antimicrobial components in the preparation was discussed at first， and validation of the test method was then performed under the premise of excluding the interference of antimicrobial components.
It could be seen from the validation results that by the method of increasing diluent （1∶20 testing solution）， the three batches of Compound Gangbangui Capsule showed the recoveries of S. aureus， P. aeruginosa， B. subtilis， C. albicans and A. niger all in the range of 0.5-2.0， and by conventional plate method （1∶10 testing solution）， the three batches of Compound Gangbangui Capsule showed the recoveries of mould and yeast quantities of A. niger and C. albicans all in the range of 0.5-2.0. The product only has an antimicrobial effect on S. aureus. When performing control microbe examination and validation of examination method by conventional method， the results showed that the positive microbes grew well， and the product has no inhibitory effect on E. coli.
The methods for eliminating the antimicrobial effect of tested products recorded in "Chinese Pharmacopeia" （2015 edition） include increasing diluent or medium volume， adding suitable neutralizer or inactivator， performing membrane filtration or combining above method[2]. Therefore， testing solutions of different dilution times were prepared to perform validation， and the results showed that the tested product has stronger inhibitory effect on S. aureus， and counting validation of the total quantities of aerobic bacteria by increasing diluent （1∶20） and mould and yeast by conventional method was performed. The results showed that the recovery of each specific strain satisfied the pharmacopeia requirements. The method is accurate and reliable， and could be used for the quality control of Tongmai Tangyanming Capsule. Compared with "Chinese Pharmacopeia" （2010 edition）， the microbial limit test was revised a lot in the 2015 edition[3-4]： the microbial counting was changed to the total quantities of aerobic bacteria and the total quantity of yeast， and the medium was changed to wide-spectrum trypticase soy broth medium and Sabouraud dextrose agar medium； and the content and requirements of the recovery test were also revised correspondingly. The methods tend to gear to international conventions. In this paper， study and validation were carried out according to the pharmacopeia requirements， which will provide reference for the microbial limit test of such pharmaceuticals.
References
[1] Chinese Pharmacopoeia Commission. Chinese Pharmacopeia （four part， 2015 edition）[S]. Beijing： China Medical Science Press， 2015： 1105-1107.
[2] Chinese Pharmacopoeia Commission. Chinese Pharmacopeia （four part， 2015 edition）[S]. Beijing： China Medical Science Press， 2015： 142.
[3] JING RX， ZHANG GL. The revision of clean environment and plate counting medium for microbial limit test in the Chinese Pharmacopoeia[J]. China Health Industry， 2015（5）： 184-185.
[4] LI CE， JIANG YF. Comparing the microbial limit test methods of Chinese Pharmacopoeia 2010 edition and 2015 edition for microbes determination in four pharmaceuticals[J]. Drug Standards of China， 2015， 16（2）： 86-90.