0.45 μm ptfe filter

One gram of ground dried leaves (knife mill after oven at 60 °C with forced air circulation) was extracted by maceration in ultrasonic bath (Quimis, Diadema, Brazil) at room temperature in 10 mL ethanol:water (7:3, v/v) for 30 min. The obtained ACHE was filtered in a 0.45 μm PTFE filter (Merck Millipore, Darmstadt, Germany) before analyzed in a High-Performance Liquid Chromatograph coupled to Diode-Array Detector (HPLC-DAD). Carajurin was isolated and characterized [16 (link)]. The determined purity was 98% by HPLC-DAD [16 (link)].

Quantification of anthocyanidins was performed using carajurin as the external standard. Since there are no standards available for all anthocyanidins, their content was expressed in milligrams of carajurin by grams of dry extract. Stock solution of the isolated carajurin (98%, chromatographic determined) was prepared as 200 μg/mL in acetonitrile:methanol (both HPLC grade, Tedia) mixture (75:25; v/v) in volumetric flasks. Six concentrations of work solutions were done on the day for analytical curve preparation (20; 40; 60; 100; 150 and 200 μg/mL) (Figure S7). The solutions were filtered in a 0.45 μm PTFE filter (Merck Millipore, Darmstadt, Germany) before analysis by HPLC-DAD-UV. Injections of 10 μL were performed in triplicate to obtain the analytical curve from the areas corresponding to the peaks of carajurin. Analytical curve (20–200 μg/mL) of the standard was constructed based on the UV-Vis signal at 480 nm to better selectivity: carajurin content (μg/mL) = (Abs (mAu) + 661228)/48694; R² = 0.9993). Carajurin and other anthocyanidins amounts (mg/g of dry extract) were calculated and expressed as carajurin content.