Artepillin c

Brazilian green propolis (EPP-AF^®) extract and gel were evaluated using an HPLC/DAD system (Shimadzu apparatus equipped with a CBM-20 A controller, a LC-20AT quaternary pump, an SPD-M 20 A diode-array detector, and Shimadzu LC solution software, version 1.21 SP1) coupled to a Shimadzu Shim-Pack CLC-ODS column (4.6 mm × 250 mm, 5 µm particle diameter, 100 Å pore diameter). The mobile phase consisted of methanol (HPLC grade) and a water-formic acid solution (0.1% v/v), pH 2.7 (A). The method consisted of a linear gradient of 20%–95% methanol over a period of 77 min at a flow rate of 0.8 mL/min. Detection was set at 275 nm, in accordance with a previously published protocol (Berretta et al., 2012 (link)). Samples were diluted in 5 mL of methanol in 10 mL volumetric flasks, subjected to sonication for 10 min and filled to volume with Milli-Q water. All samples were filtered through a 0.45 µm filter before analysis. The chemical references used were caffeic acid (Sigma-Aldrich, L: SLBZ6416), p-coumaric acid (Sigma-Aldrich, L: 091M119V), 3,5 dicaffeoylquinic acid (Phytolab, L. 3215), 4,5–dicaffeoylquinic acid (Phytolab, L. 9943), galangin (Sigma-Aldrich: BCCG2648), artepillin C (Phytolab, L: 111674647), as well as aromadendrin-4′-O-methyl ether, drupanin and baccharin previously isolated by De Sousa et al. (2007) (link).

The following isolated compounds were purchased: caffeic acid (Sigma-Aldrich, L: SLBZ6416), p-Coumaric acid (Sigma-Aldrich, L: 091M119V), and artepillin C (PhytoLab, L: 111674647). Baccharin was isolated and identified according to De Sousa et al. [16 (link)].