Identification of Ellagic Acid Rhamnoside as a Bioactive Component of a Complex Botanical Extract with Anti-biofilm Activity

Abstract

Staphylococcus aureus is a leading cause of hospital-acquired infections. It is listed among the top "serious threats" to human health in the USA, due in large part to rising rates of resistance. Many S. aureus infections are recalcitrant to antibiotic therapy due to their ability to form a biofilm, which acts not only as a physical barrier to antibiotics and the immune system, but results in differences in metabolism that further restricts antibiotic efficacy. Development of a modular strategy to synthesize a library of phenolic glycosides allowed for bioactivity testing and identification of anti-biofilm compounds within an extract of the elmleaf blackberry (Rubus ulmifolius). Two ellagic acid (EA) derivatives, EA xyloside and EA rhamnoside, have been identified as components of the Rubus extract. In addition, EA rhamnoside has been identified as an inhibitor of biofilm formation, with activity comparable to the complex extract 220D-F2 (composed of a mixture of EA glycosides), and confirmed by confocal laser scanning microscopy analyses.

Keywords: Rubus ulmifolius; Staphylococcus aureus; biofilm; ellagic acid; natural products.

FIGURE 1

Synthesis of phenol and catechol glycosides. (a) BF₃-OEt₂, CH₂Cl₂, glycosyl donor, room temperature, 10-43%; (b) Et₃N, MeOH, rt, 99%.

FIGURE 2

Synthesis of ellagic acid glycosides. (a) TBSCl, Im, DMAP, CH₂Cl₂/DMF, 50°C, 36 h, 71%; (b) TASF, CH₂Cl₂, room temperature, 10 min; then glycosyl donor, Bu₄NI (xyloside only), reflux, 48 h, 13–15%. (c) (1) K₂CO₃, DMF/H₂O; (2) K₂CO₃, MeOH/H₂O, 86–92%.

FIGURE 3

Identification of ellagic acid glycosides in the 220D-F2 extract. (A) Total UV-visible absorbance (210–500 nm) chromatogram. (B) FTMS base peak chromatogram. (C) FTMS chromatogram filter for ellagic acid rhamnoside m/z range (447–448). (D) FTMS chromatogram filter for ellagic acid xyloside m/z range (433–434). Comparison of fragmentation patterns from compounds identified in 220D-F2 extract and synthetic standards can be found in Supplementary Figure S1.

FIGURE 4

Impact on biofilm formation as assessed by confocal laser scanning microscopy. Biofilm inhibition assays were undertaken with UAMS-1 and its isogenic sarA mutant (UAMS-929) as a control. An orthogonal view is included to illustrate overall biofilm architecture at a magnification of 10×.