Efficacy of three weed species against bovine mastitis-causing bacteria, and their anti-inflammatory, antioxidant and cytotoxic activities

• Leonotis leonurus, L. nepetifolia, Tithonia rotundifolia are traditional medicines. • Extracts were active against mastitis-causing Staphylococcus aureus. • Some extracts had excellent anti-inflammatory activity and low cytotoxicity. • Antibiofilm and quorum quenching properties added value to selected extracts. • Treatments for bovine mastitis are being developed from bioactive plants. Bovine mastitis is a significant infectious disease in the dairy industry, and it tends to re-occur owing to antibiotic resistance. Management of the condition using complementary treatments could reduce the economic burden caused by the disease. Different extracts (methanol, ethanol, acetone, dichloromethane: methanol, hot and cold water) of three species commonly growing as weeds in South Africa ( Leonotis leonurus, L. nepetifolia and Tithonia rotundifolia ) were investigated. The plants are reported to be used traditionally to treat breast swelling, inflammation, abscesses, wounds, pains, fever, and muscular cramps. The antibacterial, antibiofilm, quorum quenching, anti-inflammatory, antioxidant activity and cytotoxicity of the different extracts were investigated. The antibacterial activity against six Staphylococcus aureus species isolated from clinical cases of bovine mastitis, and two Staphylococcus reference strains ( S. aureus ATCC 29213 and S. epidermidis ATCC 35984) was tested. Quorum quenching activity using Chromobacterium violaceum as a model was conducted. At the same time, antibiofilm assays were carried out using crystal violet and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2 H -tetrazolium (INT) assays to determine the antiadhesion and biofilm inhibitory activity, as well as the viability of bacterial cells after treatment with the plant extracts. The antioxidant activity was investigated using in vitro chemical assays. Inhibition of the 15-lipoxygenase enzyme (15-LOX) and nitric oxide (NO) release from activated macrophages was used to determine the anti-inflammatory potential of the extracts. The cytotoxicity of the extracts was assessed via a tetrazolium-based colorimetric reduction assay against Vero African green monkey kidney and bovine dermis cell lines. The extracts showed moderate to good inhibition of bacterial growth with minimum inhibitory concentration (MIC) values ranging between 0.02 and 2.5 mg/mL. The extracts of Leonotis leonurus had the best anti-adhesion and biofilm inhibitory activity against the pathogens. They inhibited the metabolic activity of the bacterial cells at different times, with the most active resulting in 100 % inhibition. Eight extracts had excellent anti-quorum sensing ability with violacein inhibition ≥50 % at all concentrations (IC 50 = 0.08 to 2.5 mg/mL). Most of the extracts had excellent activity against the 15-LOX enzyme compared with the positive controls, as well as good to moderate NO inhibition. The extracts were less toxic to the bovine dermis cells than to the Vero cells. The antibacterial effects, little to no cytotoxicity at low concentrations and the promising antibiofilm and quorum quenching results in this study provide motivation for further investigation of the extracts as potential candidates for development of a novel therapy for the management of pathogenic traits in organisms implicated in causing mastitis.