Objective

2

To establish microbial limit test methods for

Prunella vulgaris

and

Verbenae Herba

formula granules.

Method

2

Microbial limit tests were conducted in accordance with General Chapters 1105 and 1106 of the Chinese Pharmacopoeia （2020 edition）， including suitability tests for microbial enumeration and specified microorganism tests. The total counts of molds and yeasts in both formula granules were determined using the conventional plate-pouring method （1∶10 test solution）， and the total count of aerobic bacteria was determined using the dilution method （1∶100 test solution）. For Escherichia coli detection， the volume of the culture medium was increased to 200 mL for

Verbenae Herba

formula granules， and increased to 500 mL with the addition of a neutralizing agent for

Prunella vulgaris

formula granules.

Results

2

When using the plate-pouring method with a 1∶10 test solution to determine aerobic bacteria counts in both formula granules， the recovery rates for common indicator bacteria （Staphylococcus aureus， Pseudomonas aeruginosa， Aspergillus niger， and Candida albicans） were all above 50%， meeting the requirements in Chinese Pharmacopoeia （50%~200%）. However， the recovery rates for Bacillus subtilis were 48% and 32% for

Prunella vulgaris

and

Verbenae Herba

granules， respectively， which were below the required threshold. Therefore， the conventional plate method （1∶10） is not suitable for determining the total aerobic bacteria count in these products. The recovery rates for A. niger and C. albicans （molds and yeasts） were both above 0.5， meeting the requirements， indicating that the conventional plate method （1∶10） is suitable for determining mold and yeast counts in both formulas. When the test solution was diluted to 1∶100， the plate-pouring method was used again to check aerobic bacteria counts. The recovery rates for B. subtilis were 82% an

d 88% for

Prunella vulgaris

and

Verbenae Herba

formula granules， respectively， both meeting the 50%–200% requirement， indicating that dilution to 1∶100 eliminated the antibacterial activity of the test samples against B. subtilis. It suggests that plate-pouring method with a 1∶100 test solution is suitable for determining aerobic bacteria counts in both formula granules. When using the conventional method （100 mL tryptic soy broth） for E. coli detection， both granules showed negative results in the positive control group （no growth of E. coli）， indicating inhibition. When the medium volume was increased to 200 mL， the positive control for

Verbenae Herba

formula granules showed positive results （E. coli growth）， indicating that increasing the medium volume can eliminate the antibacterial effect and is suitable for E. coli detection in this formula. However，

Prunella vulgaris

formula granules still showed negative results in the positive control group， which was indicative of inhibition， suggesting the need for further optimization. When both the culture medium volume was increased to 500 mL and a neutralizing agent was added，

Prunella vulgaris

formula granules yielded a positive result in the positive control group， indicating that this method can eliminate the antibacterial effect and is suitable for E. coli detection in this product.

Conclusion

2

The conventional plate method （1∶10） is applicable for determining mold and yeast counts in

Prunella vulgaris

and

Verbenae Herba

formula granules， while the dilution method （1∶100） is suitable for aerobic bacterial counts. Increasing the culture medium volume allows for effective detection of E. coli in

Verbenae Herba

formula granules， and combining increased volume with a neutralizing agent is effective for E. coli detection in

Prunella vulgaris

formula granules.