The results were expressed as CE50 ± sd, where CE50 represents the sample concentration required to obtain half the ABTS + radical scavenging activity and sd is the calculated standard deviation. The chromatographic analyses were conducted on a Shimadzu (Kyoto, Japan) high-performance
liquid chromatograph (HPLC). The chromatograph was equipped with an automatic Rheodyne 7125i injector with a 20-μL loop and a diode array detector. The columns used were a Shimadzu LC-18 column (25 cm × 4.6 mm from Supelco, Bellefonte, PA), a Rexchrom LC-18 column (15 cm × 4.6 mm; Supelco) and a Shimadzu pre-column C-18 ODS. For the analysis of the phenolic acids, the elution system was composed of 5% formic acid (solvent A) and MeOH (solvent see more B). The elution conditions
were: 0.01–15 min 20–30% B, 15–20 min 30% B, 20–30 min 30–40% B and www.selleckchem.com/products/AZD2281(Olaparib).html 40–50 min 100% B, at a flow rate of 1.0 mL min−1. For the monitoring, the wavelengths of 254 and 290 nm were employed. For the determination of flavonoids, the elution system used 1% formic acid (solvent A) and MeOH (solvent B) for the mobile phase. The elution conditions were as follows: 0.01–3 min 40% B, 5–15 min 45% B, 17–25 min 50% B, 27–35 min 55% B and 40 min 40% B. For the monitoring, the wavelength of 320 nm was utilised. The flow rate of the mobile phase was 1 mL min−1, and the oven temperature of the column was fixed at 35 °C. The identification of phenolic compounds was based on the retention times, the UV-spectra and chromatographic comparison (co-injection) with authentic markers (Silva et al., 2013). Based on compounds previously found in honeys, the phenolic standards selected for comparison were as follows:
apigenin, isorhamnetin, kaempferol, luteolin, myricetin, quercetin, tricetin, taxifolin, naringenin, ferulic acid, 3-hydroxy-4-methoxycinnamic acid, caffeic acid, p-coumaric acid, cinnamic acid, sinapic acid, 4-methoxycinnamic acid, chlorogenic acid, 3,4,5-trihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, syringic acid, (trans–trans)-abscisic acid, (cis–trans)-abscisic acid, salicylic acid, catechol, gallic acid and vanillic acid. Strains of Staphylococcus aureus: ATCC 25923, S. epidermidis ATCC ADP ribosylation factor 12228, Pseudomonas aeruginosa and Escherichia coli were kept in Müller–Hinton agar (bacteria) at 4 °C, and strains of Candida albicans ATCC 6645, C. tropicalis ATCC 13803 and C. krusei LM 13 were kept in Sabouraud Dextrose Agar at 35 °C. All the strains were obtained from the Pharmaceutical Sciences Department, São Paulo University, Adolfo Lutz Institute, Brazil. For the evaluation of the antimicrobial activity, the EtOAc fractions were solubilised in 10% dimethyl sulfoxide (DMSO) and were tested at concentrations ranging from 0.032 to 1.024 mg mL−1. For each microorganism, a suspension at 106 CFU mL−1 (0.5 McFarland Scale) was prepared.