Determination of MIC
The broth-dilution method7 was adopted to determine the minimal inhibitory concentration (MIC) of the active extract revealed by the previous well assay. The inoculum of each test bacterium was prepared by diluting the overnight culture of the bacterium in Mueller Hinton broth to a level of 1.5107 CFU/ml. Two millilitres of the extracts diluted in two-folก dilution with DMSO were added to a sterile glass tube containing 0.5 ml Mueller Hinton broth (the concentration of bacteria approximately 1.5107 CFU/ml). The tubes were incubated at 37 °C for 18–24 h. Because of the turbidity and dark colour of the extracts, 0.1 ml of the mixture in the tubes were spread onto the surface of Mueller Hinton agar. Plates were incubated at 37 °C for 18–24 h. The MIC was defined as the lowest concentration (mg/ml) of the extract resulting
in bacteria density lower than 300 colonies per plate. The test was conducted twice.