4 5 dicaffeoylquinic acid

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).

Formic acid (>98% purity), acetonitrile (LC-MS grade), methanol, rutin, quercetin, apigenin and kaempferol were purchased from Merck (Darmstadt, Germany). apigenin 7-O-glucoside, quercetin 3-O-glucoside, kaempferol 3-O-rutinoside and luteolin were purchased from Extrasynthese (Genay Cedex, France). Cynarin, chlorogenic acid, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, luteolin 7-O-glucuronide, apigenin, 7-O-glucoside, luteolin 7-O-glucoside and diosmetin were purchased from Phytolab (Vestenbergsgreuth, Germany). Bond Elut Jr 500 mg SPE-C18 cartridge was from Agilent (Santa Clara, California, USA). A Milli-Q purification system (Millipore, Bedford, MA, USA) was used to obtain the deionised water (18.2 MΩ cm). All chemicals used for biological assays were obtained as pure commercial products from Sigma Chemical Co (St. Louis, USA) and used without further purification. Spectrophotometric determinations were obtained with an Ultrospec 2100 spectrophotometer (Biochrom Ltd, Cambridge, England) using a 1 cm length path cell.

Authentic standards of caffeic acid-derived chlorogenic acids (3-caffeoylquinic acid; 4-caffeoylquinic acid; 5-caffeoylquinic acid; 1,3-di-caffeoylquinic acid; 1,5-di-caffeoylquinic acid; 3,4-di-caffeoylquinic acid; 3,5-di-caffeoylquinic acid and 4,5-di-caffeoylquinic acid) and flavonols (quercetin-3-O-glycoside; quercetin-4-O-glycoside; quercetin-7-O-glycoside and rutin) were purchased from Phytolab (Vestenbergsgreuth, Germany). Analytical-grade acetonitrile was obtained from Romil (Cambridge, UK). Formic acid and diatomaceous earth were obtained from Sigma-Aldrich (St. Louis, MO, USA).