Prenylated isoflavonoids from Fabaceae against the NorA efflux pump in Staphylococcus aureus.

Overexpression of NorA efflux pumps plays a pivotal role in the multidrug-resistance mechanism in S. aureus. Here, we investigated the activities of prenylated isoflavonoids, present in the legume plant family (Fabaceae), as natural efflux pump inhibitors (EPIs) in fluoroquinolone-resistant S. aureus. We found that four prenylated isoflavonoids, namely neobavaisoflavone, glabrene, glyceollin I, and glyceollin III, showed efflux pump inhibition in the norA overexpressing S. aureus. At sub-inhibitory concentrations, neobavaisoflavone (6.25 µg/mL, 19 µM) and glabrene (12.5 µg/mL, 39 µM), showed up to 6 times more Eth accumulation in norA overexpressing S. aureus than in the control. In addition, these two compounds boosted the MIC of fluoroquinolones up to eightfold. No fluoroquinolone potentiation was observed with these isoflavonoids in the norA knockout strain, indicating NorA as the main target of these potential EPIs. In comparison to the reported NorA EPI reserpine, neobavaisoflavone showed similar potentiation of fluoroquinolone activity at 10 µM, higher Eth accumulation, and less cytotoxicity. Neobavaisoflavone and glabrene did not exhibit membrane permeabilization effects or cytotoxicity on Caco-2 cells. In conclusion, our findings suggest that the prenylated isoflavonoids neobavaisoflavone and glabrene are promising phytochemicals that could be developed as antimicrobials and resistance-modifying agents to treat fluoroquinolone-resistant S. aureus strains.

Valorisation of liquorice (Glycyrrhiza) roots: antimicrobial activity and cytotoxicity of prenylated (iso)flavonoids and chalcones from liquorice spent (G. glabra, G. inflata, and G. uralensis).

Prenylated phenolics are antimicrobials found in liquorice (Glycyrrhiza spp.). Liquorice spent is a by-product rich in prenylated phenolics obtained after water extraction of roots, and is currently not valorised. We analysed the prenylated phenolics composition of spent extracts from Glycyrrhiza glabra, G. inflata, and G. uralensis, their antimicrobial activity, cytotoxicity, and effects on Caco-2 cell viability. G. glabra, G. inflata, and G. uralensis spent extracts showed distinct phytochemical profiles. Antibacterial activity (Lactobacillus buchneri, Streptococcus mutans, and Staphylococcus aureus) of G. uralensis and G. inflata (MICs 25-250 µg mL-1) was higher than of G. glabra (MICs 75-1000 µg mL-1). Marker compounds glabridin, licochalcone A, and glycycoumarin were equally potent (MICs 12.5-25 µg mL-1). G. inflata and G. uralensis showed cytotoxicity at 500 µg mL-1, whereas G. glabra was not toxic up to 1000 µg mL-1, but showed reduced viability between 50-500 µg mL-1. Linking antibacterial activity of the liquorice spent extracts with cell viability showed that MICs against S. aureus coincide with concentrations where cell viability was not reduced, whereas for the other bacteria and yeasts MICs concurred at concentrations where cell viability was reduced. In this study we show that liquorice spent is a by-product rich in antibacterial prenylated phenolics that offers interesting oppurtunities for e.g. control of microorganisms and the discovery of novel plant-derived antimicrobials.

A targeted prenylation analysis by a combination of IT-MS and HR-MS: Identification of prenyl number, configuration, and position in different subclasses of (iso)flavonoids.

Prenylated (iso)flavonoids are potent bioactive compounds found in the Fabaceae family. Analysis and quantification of this type of phytochemicals is challenging due to their large structural diversity. In this study, the fragmentation of prenylated (iso)flavonoids was investigated using electrospray ionization ion trap mass spectrometry (ESI-IT-MSn) with fragmentation by collision induced dissociation (CID) in combination and Orbitrap-MS (ESI-FT-MS2) with fragmentation by higher energy C-trap dissociation (HCD). With this combination of IT-MSn and high resolution MS (FT-MSn), it was possible to determine the fragmentation pathways and characteristic spectral features of different subclasses of prenylated (iso)flavonoid standards, as well as characteristic fragmentations and neutral losses of different prenyl configurations. Based on our findings, a decision guideline was developed to (i) identify (iso)flavonoid backbones, (ii) annotate prenyl number, (iii) configuration, and (iv) position of unknown prenylated (iso)flavonoids, in complex plant extracts. In this guideline, structural characteristics were identified based on: (i) UV absorbance of the compound, (ii) mass-to-charge (m/z) ratio of the parent compound; (iii) ratio of relative abundances between neutral losses 42 and 56 u in MSn; (iv) retro-Diels-Alder (RDA) fragments, neutral losses 54 and 68 u, and the ratio [M+H-C4H8]+/[M+H]+. Using this guideline, 196 prenylated (iso)flavonoids were annotated in a Glycyrrhiza glabra root extract. In total, 75 skeletons were single prenylated, 104 were double prenylated, and for merely 17 skeletons prenyl number could not unambiguously be annotated. Our prenylation guideline allows rapid screening for identification of prenylated (iso)flavonoids, including prenyl number, configuration, and position, in complex plant extracts. This guideline supports research on these bioactive compounds in the areas of plant metabolomics and natural products.

Mass spectrometric characterisation of avenanthramides and enhancing their production by germination of oat (Avena sativa).

Avenanthramides are amides, with a phenylalkenoic acid (PA) and an anthranilic acid (AA) subunit, which are secondary metabolites of oat. Oat seeds were germinated, extracted, and the avenanthramides analysed by a combination of UHPLC with ion trap and high resolution ESI-MS. Typical fragmentation pathways with corresponding diagnostic fragments belonging to the PA and AA subunits were identified and summarised in a decision guideline. Based on these findings 28 unique avenanthramides were annotated in the oat seed(ling) extracts, including the new avenanthramide 6f (with a 4/5-methoxy AA subunit). Avenanthramide content increased by 25 times from seed to seedling. Avenanthramides 2p, 2c, and 2f, which are commonly described as the major avenanthramides, represented less than 20% of the total content in the seedlings. Future quantitative analyses should, therefore, include a wider range of avenanthramides to avoid underestimation of the total avenanthramide content.