Antimicrobial Constituents from Machaerium Pers.: Inhibitory Activities and Synergism of Machaeriols and Machaeridiols against Methicillin-Resistant Staphylococcus aureus, Vancomycin-Resistant Enterococcus faecium, and Permeabilized Gram-Negative Pathogens

Abstract

Two new epimeric bibenzylated monoterpenes machaerifurogerol (1a) and 5-epi-machaerifurogerol (1b), and four known isoflavonoids (+)-vestitol (2), 7-O-methylvestitol (3), (+)-medicarpin (4), and 3,8-dihydroxy-9-methoxypterocarpan (5) were isolated from Machaerium Pers. This plant was previously assigned as Machaerium multiflorum Spruce, from which machaeriols A-D (6-9) and machaeridiols A-C (10-12) were reported, and all were then re-isolated, except the minor compound 9, for a comprehensive antimicrobial activity evaluation. Structures of the isolated compounds were determined by full NMR and mass spectroscopic data. Among the isolated compounds, the mixture 10 + 11 was the most active with an MIC value of 1.25 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA) strains BAA 1696, -1708, -1717, -33591, and vancomycin-resistant Enterococcus faecium (VRE 700221) and E. faecalis (VRE 51299) and vancomycin-sensitive E. faecalis (VSE 29212). Compounds 6-8 and 10-12 were found to be more potent against MRSA 1708, and 6, 11, and 12 against VRE 700221, than the drug control ciprofloxacin and vancomycin. A combination study using an in vitro Checkerboard method was carried out for machaeriols (7 or 8) and machaeridiols (11 or 12), which exhibited a strong synergistic activity of 12 + 8 (MIC 0.156 and 0.625 µg/mL), with >32- and >8-fold reduction of MIC's, compared to 12, against MRSA 1708 and -1717, respectively. In the presence of sub-inhibitory concentrations on polymyxin B nonapeptide (PMBN), compounds 10 + 11, 11, 12, and 8 showed activity in the range of 0.5-8 µg/mL for two strains of Acinetobacter baumannii, 2-16 µg/mL against Pseudomonas aeruginosa PAO1, and 2 µg/mL against Escherichia coli NCTC 12923, but were inactive (MIC > 64 µg/mL) against the two isolates of Klebsiella pneumoniae.

Keywords: 5-epi-machaerifurogerol; MRSA; Machaerium Rimachi 12161; VRE; gram negative bacteria; isoflavonoid; machaeridiol A–C; machaerifurogerol; machaeriol A–C.

Figure 1

Structures of the isolated compounds.

Figure 2

Structure of the epimeric compound (1a) (major) and (1b) (minor) in mixture.

Figure 3

Key 2D NMR COSY, HMBC, and NOESY correlations of compound 1a.

Figure 4

Isobologram of combination of compounds 8 and 12 in MRSA 1708 (red) and 1717 (blue). The green series represents the additivity line of compounds 8 and 12 green dots represent the MIC of each compound alone; the green line represents all possible additive combinations). The red (MRSA 1708) and blue (1717) dots represent the combination of compounds 8 and 12, and show that they fell below the additivity line (the combination of compounds produces a synergistic effect beyond additivity).