Multitargeted Flavonoid Inhibition of the Pathogenic Bacterium Staphylococcus aureus: A Proteomic Characterization.

Growth inhibition of the pathogen Staphylococcus aureus with currently available antibiotics is problematic in part due to bacterial biofilm protection. Although recently characterized natural products, including 3',4',5-trihydroxy-6,7-dimethoxy-flavone [1], 3',4',5,6,7-pentahydroxy-flavone [2], and 5-hydroxy-4',7-dimethoxy-flavone [3], exhibit both antibiotic and biofilm inhibitory activities, the mode of action of such hydroxylated flavonoids with respect to S. aureus inhibition is yet to be characterized. Enzymatic digestion and high-resolution MS analysis of differentially expressed proteins from S. aureus with and without exposure to antibiotic flavonoids (1-3) allowed for the characterization of global protein alterations induced by metabolite treatment. A total of 56, 92, and 110 proteins were differentially expressed with bacterial exposure to 1, 2, or 3, respectively. The connectivity of the identified proteins was characterized using a search tool for the retrieval of interacting genes/proteins (STRING) with multitargeted S. aureus inhibition of energy metabolism and biosynthesis by the assayed flavonoids. Identifying the mode of action of natural products as antibacterial agents is expected to provide insight into the potential use of flavonoids alone or in combination with known therapeutic agents to effectively control S. aureus infection.

Iridoid glycoside permethylation enhances chromatographic separation and chemical ionization.

RATIONALE: While natural products isolated from medicinal plants can serve as a rich source of biologically active metabolites, mixtures of structurally related compounds of a polar nature are often difficult to chemically resolve by traditional separation techniques. Chemical derivatization to reduce metabolite polarity combined with liquid chromatography (LC) is the strategy presented here to resolve a mixture of structurally related natural product glycosides solvent extracted from the medicinal herb Teucrium polium for mass spectrometric characterization. METHODS: The partially purified plant extract was subjected to chemical derivatization and electrospray ionization mass spectrometry (ESI-MS) fragmentation pattern analysis allowed for structural characterization of iridoid and secoiridoid glycosides. Selected ions were subjected to tandem mass spectrometric (MS/MS) analysis with a relatively higher-energy collision dissociation to assist in structural elucidation. RESULTS: Permethylation replaced all protons from free hydroxyl and amino groups with methyls and resulted in increases in both hydrophobicity, for facilitated chromatographic separation, and proton affinity, for enhanced chemical ionization. Protonated and/or sodiated adducts were observed for the six compounds detected in positive-ion mode ESI-MS with a mass accuracy of less than 2 ppm. CONCLUSIONS: Permethylation combined with LC/MS analysis is shown here to be an effective chemical practice for separating and characterizing iridoid glucosinolates and is expected to be well suited for the chemical characterization of other polar natural-product mixtures of closely related compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Teucrium polium phenylethanol and iridoid glycoside characterization and flavonoid inhibition of biofilm-forming Staphylococcus aureus.

The chemical composition and biofilm regulation of 15 metabolites from Teucrium polium are reported. Compounds were isolated from a CH2Cl2-MeOH extract of the aerial parts of the plant and included iridoid and phenylethanol glycosides and a monoterpenoid, together with nine known compounds. The structures were elucidated based on standard spectroscopic (UV, (1)H and (13)C NMR), 2D NMR ((1)H-(1)H COSY, HMQC, HMBC, and NOESY), and/or LC-ESIMS/MS data analyses. Inhibition of the biofilm-forming strain Staphylococcus aureus was observed with exposure to compounds 7 and 8.

Biofilm blocking sesquiterpenes from Teucrium polium.

The chemical composition and antibacterial activity of Teucrium polium L. (Lamiaceae) were assessed; sixteen compounds were isolated from a CH2Cl2/MeOH extract of the aerial parts of the plant including four sesquiterpenes 4ß,5α-epoxy-7αH-germacr-10(14)-en-6ß-ol-1-one, 4ß,5α-epoxy-7αH-germacr-10(14)-en,1ß-hydroperoxyl,6ß-ol, 4ß,5ß-epoxy-7αH-germacr-10(14)-en,1ß-hydroperoxyl,6ß-ol and 4α,5ß-epoxy-7αH-germacr-10(14)-en,1ß-hydroperoxyl,6α-ol, together with seven known sesquiterpenes, one known iridoid glycoside, two known flavonoids, and one known phenylpropanoid glycoside. Structures were elucidated on the basis of spectroscopic (UV, (1)H and (13)C NMR) data, as well as two-dimensional NMR ((1)H-(1)H COSY, HMQC, NOESY and HMBC), and ESI-MS analysis. The relative stereochemistry of the ketone was established by X-ray crystallography, while its absolute configuration was attained by a modified Mosher's method. Antibacterial activity of the crude extract, as well as with four of the isolated metabolites, was observed with Staphylococcus aureus anti-biofilm activity in the low µMol range. Diverse sesquiterpene-skeleton structure and corresponding comprehensive enzyme capacity is discussed.