5 cqa

Commercial standards (purity＞95％) of tocopherols (α-, β-, γand δ-) , fatty acid methyl ester mix (37-FAME mix, Supelco) , phenolic compounds (gallic acid, 5-CQA, di-OHphenylacetic acid, caffeic acid, 2,4-di-OH-benzoic acid, hydroxycinnamic acid, rosmarinic acid, and naringenin) and phytosterols (β-sitosterol, stigmasterol, and cholestanol) were acquired from Sigma-Aldrich Chemical Co. (São Paulo, Brazil) . Anhydrous reagent alcohol (90％ ethanol, 5％ methanol, 5％ 2-propanol; v/v) was used in SC-CO 2 extractions and referred to simply as alcohol throughout this work. All solvents were HPLC grade (Tedia ® , Rio de Janeiro, Brazil) . The high purity CO 2 (purity 99.99％) used for supercritical extraction was purchased from AltaTec LTDA (Rio de Janeiro, Brazil) . Ultrapure water (Milli-Q system, Millipore, Bedford, MA, USA) was used throughout the experiments.

For sugars analysis were used 1-methylimidazole (C 4 H 6 N 2 , ≥99%, Sigma-Aldrich), 2-deoxy-D-glucose (C 6 H 12 O 5 , ≥99%, Sigma-Aldrich), ammonium hydroxide solution (NH 4 OH, 25%, Sigma-Aldrich), acetic anhydride (C 4 H 6 O 3 , ≥99%, Carlo Erba Reagents), acetic acid glacial (C 2 H 4 O 2 , ≥99%, Carlo Erba Reagents), dichloromethane (CH 2 Cl 2 , 99.8%, Fischer Scientific), dimethyl sulfoxide ((CH 3 ) 2 SO, 99.7%, Fischer Scientific), hydrochloric acid (HCl, 37%, Sigma-Aldrich), iodomethane (CH 3 I, ≥99%, Sigma-Aldrich), sodium borodeuteride (NaBD 4 , >90%, Sigma-Aldrich), sodium borohydride (NaBH 4 , >95%, Fischer Scientific), sodium hydroxide (NaOH, 98%, José Manuel Gomes dos Santos), sulfuric acid (H 2 SO 4 , 98%, Biochem Chemopharma) and trifluoroacetic acid (C 2 HF 3 O 2 , 99%, Alfa Aeasar). For lipids analysis was used n-hexane (C 6 H 14, 95%, Fischer Scientific). For caffeine/5-CQA determinations Milli-Q water, formic acid (Honeywell) and methanol (Fischer Scientific) were HPLC-grade reagents and as standards were used 5-CQA (C 16 H 18 O 9 , ≥95%, Sigma-Aldrich) and caffeine (C 8 H 10 N 4 O 2 , ≥99%, Sigma-Aldrich). For foam properties experiments were used citric acid (C 6 H 8 O 7 , 99.5%, Honeywell Fluka) and sodium bicarbonate (NaHCO 3 , ≥99.7%, Sigma-Aldrich).

The CGA and placebo test beverages were prepared to be indistinguishable on the basis of appearance and flavour, and were canned (100 ml). All CGA isomers are presented according to the IUPAC nomenclature (9) . The test beverage contained 0 or 600 mg of CGA, which consisted of CQA (68 %), FQA (14 %) and diCQA (19 %) (Table 1). CGA were prepared from green coffee beans using hot water extraction followed by spray drying and girding. After activated carbon filtration, caffeine was not detected (<0•5 mM). The CGA composition in test beverages was measured by HPLC with a UV detector (325 nm). All peaks of the isomers were confirmed by LC-MS (34) . The standard, 5-CQA, was purchased from Sigma, and other standards, 3-CQA, 4-CQA, 3-FQA, 4-FQA, 5-FQA, 3,4-diCQA, diCQA and 3,5-diCQA, were obtained by repeated fractionations of green coffee bean extract (purity >96 %, confirmed by LC-UV detection and 1 H-NMR) using preparative chromatography with an octa decyl silyl (ODS) column (34) . Both beverages were caffeine-free, and the energy contents were 29 kJ/100 ml (7 kcal/100 ml) and 8 kJ/100 ml (2 kcal/100 ml) in the CGA and placebo beverages, respectively. Table 1. Chlorogenic acid (CGA) compositions of the test beverages (mg/100 ml)
CON, control; CQA, caffeoylquinic acid; FQA, feruloylquinic acid; diCQA, dicaffeoylquinic acid.

Standards of 5-O-caffeoylquinic acid (chlorogenic acid; 5-CQA), 4-O-caffeoylquinic acid (cryptochlorogenic acid; 4-CQA) and 3-O-caffeoylquinic acid (neochlorogenic acid; 3-CQA) were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). All standards were at least 95% in purity. HPLC grade methanol was obtained from Fisher Scientific (Ottawa, ON, Canada). The water used throughout this study was from a Millipore Milli-Q water system (Millipore Corp., Milford, MA, USA). Commercial apple and pear juice concentrates were from China and the United States, respectively.
Stock solutions of the caffeoylquinic acids at a concentration of 1.0 mM were prepared by dissolving the standards in 50% (v:v) aqueous methanol. The standards were then syringe filtered (nylon, 0.2 μm pore size, 13 mm diameter; Chromatographic Specialties, Brockville, ON, Canada) and stored at -20°C until use.

Green coffee (Coffea arabica L. from Colombia) and yerba mate (Ilex paraguariensis L.) were purchased in a local supermarket in Madrid (Spain). 3,5-DCQA was acquired from PhytoLab (Vestenbergsgreuth, Germany). Caffeine was obtained from Fluka (Madrid, Spain). 5-CQA, CA, FA, theobromine, DHCA, DHFA were obtained from Sigma-Aldrich (Madrid, Spain). Phycoerythrin (PE)-conjugated mouse anti-human CD61 (CD61-PE), allophycocyanin (APC)-conjugated mouse anti-human P-selectin (CD62-APC), PE-conjugated mouse IgG1κ, APC-conjugated mouse IgG1κ, AF488-conjugated mouse IgG1κ, sodium chloride (NaCl) and FACS Flow sheath fluid were acquired from BD Biosciences (Oxford, UK). AF488-conjugated fibrinogen from human plasma was obtained from Fisher Scientific (Loughborough, UK). Adenosine 5`-diphosphate (ADP), phosphate-buffered saline (PBS), dimethylsulfoxide (DMSO), potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO 4 -7H 2 O), 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), phorbol 12-myristate 13-acetate (PMA) and quercetin were acquired from Sigma-Aldrich (Dorset, UK). All other chemicals were of analytical grade.