Applicability of diffusion methods required by the pharmacopoeias for testing antibacterial activity of natural compounds.

The diffusion methods required by various pharmacopoeias were investigated in comparison to the bioautographic TLC assay in regard to the applicability and the statistical evaluation for testing the antibacterial activity of natural substances. Naringenin as an example for a weakly effective lipophilic natural compound and tetracycline as standard were used. Data were analysed by linear regression and ANOVA. The comparison of the disk diffusion method, the hole-plate diffusion method, and the bioautographic TLC assay showed that nature and amount of substance influence to a great extent the selection of the test method. The evaluation of parallelism demanded by the pharmacopoeias may provoke problems in comparing natural substances with strongly effective antibiotics. The simultaneous influence of different variables was examined with the help of ANOVA. The bioautographic TLC assay showed for spotted compounds a comparable reproducibility and accuracy to the diffusion methods mentioned above.