In vitro bioactivity of leaf extract fractions and Quercetin-3-O-rhamnoside from Combretum elaeagnoides against Staphylococcus species implicated in causing bovine mastitis

Abstract

Globally, antibiotic resistance is a growing concern, motivating the search for alternatives. Bovine mastitis is an inflammatory disease of the udder caused by various microorganisms, many of which are resistant to various antibiotics, impacting the quality of dairy products and farmer income. In this study, the in vitro bioactivity of the methanol leaf extract, fractions (ethyl acetate (CeEtOAc), butanol (CeBuOH), hexane (CeHx), dichloromethane CeDCM), and water (CeAq), and a purified compound, quercetin-3-O-rhamnoside isolated from the CeEtOAc fraction of Combretum elaeagnoides Klotzsch, were investigated against six Staphylococcus aureus (S. aureus) strains isolated from clinical cases of bovine mastitis and two reference ATCC strains (S. aureus ATCC 29213 and S. epidermidis ATCC 35984). Methods used for assessing bioactivity included serial microdilution for antibacterial efficacy, crystal violet staining and p-iodonitrotetrazolium (INT) metabolic assays for anti-biofilm activity, and a microdilution assay for anti-quorum-sensing potential. The anti-inflammatory assays included 15-lipoxygenase enzyme inhibition and nitric oxide assays. Cytotoxicity screening was conducted using a tetrazolium-based colorimetric assay against bovine dermis cells. The extracts and fractions exhibited moderate to good antibacterial activity with minimum inhibitory concentration (MIC) values ranging from 0.07 to 1.04 mg/mL, with the ethyl acetate fraction being the most effective. The anti-biofilm activity of the extract, fractions, and isolated compound (quercetin-3-O-rhamnoside) varied at time zero (T0), with inhibition ranging from 3% to 100%. The CeDCM and CeEtOAc fractions exhibited the most potent anti-biofilm effects after 24 h, with inhibition ranging from 24% to 91%. The extracts and fractions exhibited significant inhibition (>50%) of biofilm within the incubation times (T0–T48), and quercetin-3-O-rhamnoside alone had >60% inhibition at 48 h. The CeEtOAc fraction had the most significant anti-quorum-sensing activity (IC50 < 0.08 mg/mL). The methanol extract and fractions exhibited significant anti-inflammatory activity, inhibiting nitric oxide production (IC50: 7–26 µg/mL). In contrast, the CeAq, CeHx, and CeDCM fractions showed the best inhibitory activity against the 15-lipoxygenase enzyme (IC50 = 3–4 µg/mL). The extracts and fractions were non-cytotoxic to bovine dermis cells (LC50 = 0.88–1 mg/mL). Combretum elaeagnoides extract and its fractions are recommended for further investigation as potential herbal treatments for the management of mastitis and its symptoms.