2.6. Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration of methanolic extracts, essential oils and standard compounds was determined by using microdilution method. Methanolic extracts (50 μg/ml initial concentration), essential oils (10 μg/ml) from different parts of 15 Cinnamomum species and reference compounds (amoxicillin, streptomycin – 5 μg/ml initial concentration) were dissolved in dimethyl sulfoxide (5 %; Merck KGaA, Germany) for the initial stock solutions. The dilution series of methanolic extracts and essential oils were prepared in 96-well microtiter plates (Microplate Manager 4.0; Bio-Rad Laboratories, Hercules, California, USA). Each bacterial suspension containing 10⁷ CFU/ml of the bacterial cells (100 µl) were added to each well. Amoxicillin and streptomycin (Sigma-Aldrich, St. Louis, MO, USA) were used as antibacterial positive controls. Similarly, the last rows consisting of medium with selected microbes were used as negative controls. After 24 h incubation at 37 °C, the staining of viable microorganisms was conducted by mixing 20 µl resazurin solution (0.01 %) into the plates. The minimum inhibitory concentration (MIC, µg/ml) was evaluated as the lowest sample concentration, in which no microbial growth was visible. The optical densities of test samples were recorded at 655 nm and compared with blank. The MIC values of methanolic extracts of different parts of 15 Cinnamomum species were recorded in the range of 23 ± 0.01–336 ± 0.06 µg/ml against tested bacterial strains. Similarly, the MIC values of essential oils of different parts of all species were reported in between 21 ± 0.03–690 ± 0.04 µg/ml against tested bacterial species. During this study, the MIC values of standard antibacterial compounds (amoxicillin and streptomycin-18 ± 0.01–58 ± 0.06 µg/ml; positive control) were measured in different ranges. Dimethyl sulfoxide (5 %) solution was used as negative control.

The antifungal activity of methanolic extract (50 μg/ml) and essential oils (10 μg/ml) of different parts (leaf, stem bark, stem wood, root, flower, and fruit) of 15 Cinnamomum species and fluconazole (5 μg/ml) were tested against selected fungal species by using serial dilution method (A. niger, A. flavus, R. phaseoli, P. chrysogenum, and C. albicans). MIC values of tested samples were evaluated by serial dilution method using 96-well microtiter plates. Each fungal suspension containing 10⁷ CFU/ml of the fungal spores (100 µl) were added to each well. The fungal spores were cultivated in potato dextrose broth medium and stored at 4 °C during this study. The methanolic extracts and essential oils of different parts of 15 Cinnamomum species and fluconazole were dissolved in 5 % dimethyl sulfoxide solution, consisting of polysorbate-80 (0.1 %; 1 mg/ml), and added to a potato dextrose broth medium with spore inoculum. The microtiter plates were incubated in a rotary shaker (160 rpm) for 72 h at 27 °C. The minimum concentrations without conspicuous microbial growth were defined as the minimum inhibitory concentrations that completely suppressed the fungal spore growth. Similarly, C. albicans suspension was prepared according to CLSI methods (CLSI, 2002, CLSI, 2007, CLSI, 2012), where fluconazole (Sigma-Aldrich, St. Louis, MO, USA) was used as a standard antifungal agent. MIC values of methanolic extracts of different parts of all species were recorded in between 23 ± 0.01 to 444 ± 0.01 µg/ml against tested bacterial species. During this study, the MIC values of standard antifungal compound (fluconazole) were also recorded in different ranges (30 ± 0.01–76 ± 0.01 µg/ml) against selected fungal spores. Dimethyl sulfoxide (5 %) solution was used as negative control.