Before each extraction, the green coffee beans were frozen in liquid nitrogen and
ground to a fine powder using an MM 400 ball mill (Retsch, Germany) for 90 s at 30 Hz. Two types of extracts were prepared for analysis of the non-volatile composition of green coffee, a methanol and a water extract. Methanol extracts were prepared by Soxhlet extraction using a Büchi extraction system B-811 (Büchi, Switzerland). Soxhlet extraction was carried out in four cycles, extracting 2 g of ground coffee powder with 100 ml of methanol, with heating set to 14 (arbitrary value), followed by a 10 min reflux washing step. The water extracts were prepared using hot water; 3 g of the green coffee powder was infused in 100 ml of water at 92 °C, stirred for 5 min and filtered using a Forskolin paper filter. All samples were prepared in triplicate. The methanol extracts of the green coffee ATM/ATR inhibitor drugs were analysed on an HPLC-MS system (Agilent 1200 HPLC with 6130 quadrupole MS). Separation was carried out on a Poroshell 120
EC-C18 2.7 μm, 2.1 x 100 mm column (Agilent) and a corresponding pre-column, with a flow rate of 0.3 ml/min and the following elution of linear gradients of the mobile phases A (water: methanol 90: 10 (v/v) with 0.1% formic acid) and B (water: methanol 5: 95 (v/v) with 0.1 formic acid): 0-1 min 10% B, 5 min 20% B, 12 min 40% B 18 min 70% B and 19 min 10% B. The injection volume was 2 μl and the post run equilibration time was 6 min. Both MS and UV/VIS detection were used. MS and comparison of retention times to standards were used
for identification purposes. Quantification of compounds was carried out by a UV/VIS detector by integrating peak areas at 325 nm for CGAs and 275 nm for caffeine. CGAs were quantified as 5-CQA equivalents. Samples for analysis and for standards were prepared Glycogen branching enzyme by pipetting 1 ml of the methanol extract or 1 ml of the solution of the standard in methanol, respectively, into 10 ml volumetric flasks, which were then filled to volume with water. Samples were filtered prior to injection using 0.45 μm PET syringe filters (Machenerey-Nagel, Germany). High-performance size-exclusion chromatography was performed based on a modification of a previously described method (Smrke, Opitz, Vovk, & Yeretzian, 2013). Two columns were used in series, first a SupermultiporePW-N 4 μm, 6 x 150 mm and secondly a SupermultiporePW-M 5 μm, 6 x 150 mm column, both from TSKGel (Tosoh Bioscience, Stuttgart, Germany). The mobile phase was a 0.1 M aqueous solution of sodium phosphate with pH adjusted to 7.0 with phosphoric acid. A flow rate of 0.4 ml/min and an injection volume of 5 μl were used. Detection was performed by UV/VIS at 210 nm, 280 nm and 325 nm. At 210 nm and 280 nm, the total high-molecular weight (HMW) fraction of the chromatogram was integrated from a retention time of 11.2 min to 24.5 min and at 325 nm the low-molecular weight (LMW) peak was integrated (Fig. 2). Water extracts were filtered using 0.