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Methicillin-resistant Staphylococcus aureus (MRSA) causes mastitis in dairy cattle with serious economic and public health significance. This study developed nanoemulsions of Linum usitatissimun oil loaded with Achyrocline satureioides (macela) extract and investigated their in vitro antimicrobial activity against MRSA. Macela-nanoemulsions (NE-ML) were prepared using high-pressure homogenization (HPH) with different proportions of flaxseed oil, Tween 80 and crude extract. Four majoritarian flavonoids were identified in the macela extract: 3-O methylquercetin, achyrobichalcone, quercetin and luteolin (187.3 ± 0.1, 155.4 ± 11.6, 76.3 ± 0.1 and 30.4 ± 0.0 μg ml−1, respectively). NE-ML nanoemulsions were successfully obtained by the HPH method and showed a milky aspect with yellowish color. The mean particle size was around 200 nm with monodisperse distribution (PdI < 0.2), remaining stable for 160 days at room temperature. When analyzed on a LUMiSizer high-end dispersion analyzer, low values were found (≤0.5), indicating high stability index, mainly for NE-ML1:5 (0.2). The encapsulation efficiency of macela-nanoemulsions was greater than 94%, considering the four chemical compounds from extract. Minimum inhibitory concentration (MIC) against planktonic bacteria, inhibition of biofilm formation (MBIC), and eradication of MRSA biofilms (MBEC) were determined through in vitro tests on microplates. The MIC of NE-ML against planktonic MRSA showed values ranging from 1.2 to 10% (v/v), while blank-nanoemulsions (NE-B, without macela extract) showed values ranging from 6 to 50% (v/v). MBIC and MBEC of NE-ML were 25 and 80% (v/v), respectively. MBIC showed a mass reduction greater than 64%, and MBEC showed a mass reduction greater than 73%. Macela-nanoemulsions (NE-ML), mainly NE-ML1:5, showed high antimicrobial activity and appeared to represent a new alternative of sustainable antimicrobial product for the control of MRSA. Since this innovative nanoemulsion can impact animal health, future research should include in vitro and in vivo studies to evaluate intramammary therapy and control of MRSA infections in organic and agroecological milk production systems.

The objective of this study was to evaluate in vitro the antimicrobial activity of Brazilian propolis from Urupema, São Joaquim, and Agua Doce (Santa Catarina State) and green propolis from Minas Gerais State, and the effects of propolis on bovine mammary gland explant viability. The propolis samples differed in flavonoid content and antioxidant activity. Green propolis showed the highest content of flavonoids, followed by the sample from São Joaquim. The propolis from Urupema showed the lowest flavonoid content along with the lowest antioxidant activity. The total phenolics were similar across all studied samples. Despite phytochemical differences, the propolis samples from Minas Gerais, São Joaquim, and Urupema presented the same level of antimicrobial activity against Staphylococcus aureus strains. The reduction in S. aureus growth was, on average, 1.5 and 4 log10 times at 200 and 500 μg/mL, respectively. At concentrations of 1,000 μg/mL, all propolis reduced bacterial growth to zero. On the other hand, when the propolis were tested against strains of Escherichia coli, the samples presented weak antimicrobial activity. Mammary explants were maintained in culture for 96h without a loss in viability, demonstrating the applicability of the model in evaluating the toxicity of propolis. The origin and chemical composition of the propolis had an effect on mammary explant viability. We encountered inhibitory concentrations of 272.4, 171.8, 63.85, and 13.26 μg/mL for the propolis from Água Doce, Urupema, São Joaquim, and Mina Gerais, respectively. A clear association between greater antimicrobial activity and toxicity for mammary explants was observed. Of all propolis tested, the Urupema sample was noteworthy, as it showed antimicrobial activity at less toxic concentrations than the other samples, reducing bacterial growth to an average of 9.3 × 10(2) cfu/mL after 6h of contact using 200 μg/mL of extract. The results demonstrate the potential for Brazilian propolis in the treatment of mastitis, although effectiveness is dependent on geographical origin and concentration. The results from the mammary gland explant assays are promising for the investigation of other natural products with antimicrobial and anti-inflammatory properties that can be used in the intramammary treatment of subclinical mastitis and during dry cow therapy.

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This study describes the development of propolis nanoparticles (PNP) to treat bovine mastitis. Three PNP prepared with varying concentrations of propolis (5% and 7%, w/v) and the surfactants [poloxamer (1%, 3%, and 4%, w/v) and soy lecithin (0.25%, 0.7%, and 1%, w/v)]. PNP were characterized according to their size, polydispersity, zeta potential, pH, morphology, and physical stability. PNP were evaluated for their in vitro antimicrobial and cytotoxic effects. PNP obtained were spherical with a monodisperse distribution (polydispersity index < 0.2) and an average particle size between 181 and 201 nm. Stability studies showed that PNP were stable over 150 d. The encapsulation efficiency of total phenolic content varied between 73% and 91%. The chromatographic profile of phenolic compounds from PNP showed selective encapsulation efficiency according to the polarity of compounds. All PNP showed antimicrobial activity against Staphylococcus aureus with a minimum inhibitory concentration ranging from 156 to 310 μg mL−1. The IC50 (the concentration responsible for reduction of cellular viability by half) for epithelial cells of bovine mammary gland (MAC-T, mammary alveolar cell-T) varied from 122.2 to 268.4 μg mL−1. Results showed that PNP represent a promising nanocarrier for high concentrations of propolis extract in a stable aqueous medium, while, at the same time, presenting antimicrobial activity accompanied by moderate cytotoxicity to the MAC-T cells.

The low levels of toxicity and cytoprotective effect attributed to Achyrocline satureioides (Lam.) DC, a medicinal plant native to South America, are of interest for bovine mastitis therapy. This research paper reports the hypothesis that a nanoemulsion of macela extract (Achyrocline satureioides) exerts protective effects on bovine mammary alveolar cells -T (MAC-T) and increases the permeation of flavonoid compounds through mammary epithelium. Extract-loaded nanoemulsions (2.5 mg/ml) (NE-ML) (n = 4) were prepared using high-pressure homogenization with varying concentrations of flaxseed oil and Tween 80. Permeation and retention of free and nanoencapsulated quercetin, 3-O-methylquercetin and luteolin were performed on mammary glandular epithelium using Franz diffusion cells. The cell viability was evaluated on mammary epithelial cells (MAC-T lineage) using the MTT method (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) after exposure to loaded and blank nanoemulsions (NE-ML and NE-BL). Necrotic or apoptotic cell death was evaluated by flow cytometry after exposure to nanoemulsions (NE-ML and NE-BL). Subsequently, the cell death was assessed by previously treating MAC-T cells with NE-ML for 23 h, followed by exposure to H2O2 (2 mM) for 1 h. Higher permeation of quercetin and 3-O-methylquercetin in NE-ML was found compared to that of free extract with a final permeated amount of 50.7 ± 3.2 and 111.2 ± 0.6 μg/cm2 compared to 35.0 ± 0.6 and 48.9 ± 1.2, respectively. For NE-BL, the IC50 was at least 1.3% (v/v), while for the NE-ML, it was at least 2.6% (v/v). After exposure to NE-ML (5 and 1.2%, v/v), the percentage of apoptotic cells was reduced (±30%). For the H2O2 assay, the percentage of cells in necrosis was reduced by 40% after exposure to NE-ML1% (v/v) + H2O2 2 mM. The protective effects and increased permeation of macela nanoemulsion make this a promising new candidate for bovine mastitis therapy.

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