ABSTRACT
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.
ABSTRACT
Plinia cauliflora (jaboticaba) is a native fruit tree from Brazilian rainforest widely used in popular medicine to prevent diarrhea, asthma, and infections. Studies have shown that the major therapeutic potential of jaboticaba fruits is on its peel, a rich source of anthocyanins. These secondary metabolites have well-known antioxidant and anti-inflammatory activities and have been claimed to be effective to treat diabetes, cancer, cardiovascular diseases, and stroke. This chapter describes a series of methodologies to evaluate important in vitro biological activities like cytotoxicity, proliferation, and migration of a hydroalcoholic extract of jaboticaba peel on mouse fibroblast L929 line. Assays to assess total phenolic, flavonoid, and anthocyanin contents and antioxidant activities are described as well.
Subject(s)
Anthocyanins/chemistry , Anthocyanins/pharmacology , Drug Evaluation, Preclinical/methods , Myrtaceae/chemistry , Animals , Anthocyanins/isolation & purification , Antioxidants/chemistry , Antioxidants/isolation & purification , Antioxidants/pharmacology , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Fibroblasts/cytology , Fibroblasts/drug effects , Flavonoids/chemistry , Flavonoids/isolation & purification , Flavonoids/pharmacology , Mice , Phenols/chemistry , Phenols/isolation & purification , Phenols/pharmacology , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Wound Healing/drug effectsABSTRACT
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.
