In vitro activity: Azalomycin-B possesses an antibacterial activity against Gram-positive bacteria. The minimum inhibitory concentration (MIC) of Azalomycin-B against Staphylococcus aureus (SG 511, 285 and 503) is 1.52 μM. The MIC of Azalomycin-B against Streptococcus pyogenes is 0.76 μM and 1.52 μM for strains 306A, and 77A, respectively. The MIC of Azalomycin-B against S. faecium A is 3.05 μM. In vitro, Azalomycin-B shows an antibiotic activity as a rumen fermentation efficiency enhancer and also inhibits lactic acid production in the rumen fluid with IC5O of 2.14 μM. Azalomycin-B inhibits P-type ATPases such as the P-type, K+-dependent ATPase from Escherichia coli, without affecting F-type and V-type ATPases at all. Azalomycin-B shows the potent cytotoxic effect on L929 mouse fibroblast cells, K562 human leukemia cells and HeLa cell cultures with IC50 of 0.29 μM, 0.19 μM and 0.29 μg/mL, respectively. Moreover, Azalomycin-B also produces the cytotoxicity in MRC-5 cells with IC50 of 0.85 μM.

Cell Assay: The adherent mouse fibroblast cell line L-929 is cultured in Eagle's MEM with 0.35 mg/mL sodium bicarbonate, 100 units/mL penicillin/100 μg/mL streptomycin, 10 mM HEPES, and 10% heat-inactivated FBS at 37 °C in culture flasks. The adherent cells are harvested at the logarithmic growth phase after trypsination using 0.05% trypsin in phosphate-buffered saline (PBS) containing 0.02% EDTA. The nonadherent human leukemia cell line K-562, is cultured in RPMI 1640 medium, supplemented with 100 units/mL penicillin/100 μg/mL streptomycin and 10% FBS in culture flasks. L-929 and K-562 cells are inoculated in 0.1 mL culture medium, containing NaHCO3 without HEPES, per well of the 96-well microplates. The plates are previously prepared with dilutions of the Azalomycin-B in 0.1 mL medium. The microplates are kept for 72 hour at 37 °C in a humidified atmosphere containing 5% CO2. The adherent human cell line HeLa is cultured in MEM Eagle with 100 units/mL penicillin/100 μg/mL streptomycin, 10% FBS, and 2 mM l-glutamine in vented culture flasks. The adherent cells are harvested during the logarithmic growth phase after trypsination with 0.4% trypsin in PBS containing 0.02% EDTA. These cells are seeded with in 0.1 mL culture medium per well of the 96-well microplates. HeLa cells are preincubated 48 hours without Azalomycin-B. The dilutions of Azalomycin-B are carried out on the monolayer of HeLa cells after preincubation time. After incubation, the monolayer of the adherent L-929 and HeLa cells are fixed by glutaraldehyde and stained with a 0.05% solution of methylene blue for 15 minutes. After washing the stain is eluted by 0.2 mL of 0.33 N HCl in the wells. The optical densities are measured at 630 nm in a Dynatech MR 7000 microplate reader. After incubation, the K-562 cells are analyzed using an electronic cell analyzer system CASY 1 and software CASYSTAT for determination of IC50 values. The IC50 values are determined by integrated software CASYSTAT.

J Antibiot (Tokyo). 1990 Nov;43(11):1431-40; J Antibiot (Tokyo). 1993 Feb;46(2):350-2; Biochemistry. 1993 Apr 20;32(15):3902-6.