Rhamnus prinoides (gesho): A source of diverse anti-biofilm activity

Ethnopharmacological relevance: Rhamnus prinoides (gesho) is an evergreen shrub from East Africa traditionally used for the treatment of illnesses including atopic dermatitis, ear, nose and throat infections, pneumonia, arthritis, brucellosis, flu, indigestion and fatigue. Aim of the study: Several of the conditions for which gesho is traditionally used are associated with communities of surface-attached microorganisms, or biofilms. We hypothesized that gesho has anti-biofilm activity. The principal aim of this study was to evaluate gesho-associated anti-biofilm activity and identify active compounds. Materials and methods: Lyophilized ethanol and aqueous extracts were prepared from dried Rhamnus prinoides stems and leaves. Biofilm inhibition was measured by crystal violet staining and subsequent viability assays were conducted on growth agar. Chemical fractionation, chemical testing, Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS) were used to isolate and identify active compounds. Results: Leaf and stem ethanol extracts significantly inhibited Staphylococcus aureus, Bacillus subtilis and Streptococcus mutans biofilm formation up to 99.9% and reduced planktonic cell growth up to 10 log units relative to untreated controls. The anti-biofilm activity of the ethanol stem extracts was due to a biocidal or bacteriostatic mechanism while bacteriostatic or anti-pathogenic mechanisms were attributed to the leaf ethanol extract. Gesho extracts showed activity against all three species tested but the treatment efficacy and mechanism were species dependent. Chemical fractionation and activity screens of the leaf ethanol extract identified ethyl 4-ethoxybenzoate and 4-hydroxy 4-methyl pentanone to be compounds with anti-biofilm activity. Ethyl 4-ethoxybenzoate activity was potentiated by DMSO. Notably, concentrations of both compounds were identified where biofilm formation was prevented without inhibition of cell growth; i.e. anti-pathogenic characteristics were evident. Conclusion: Gesho leaf ethanol extract contains chemicals with anti-biofilm and bactericidal activities. This work lends support to the traditional use of gesho for treating topical infections and warrants further investigation into Rhamnus prinoides as a source of antibacterial and anti-biofilm agents.