Synergi 4 μm hydro rp column

We analyzed each of the four phenolic extracts obtained as described in Section 2.2 in triplicate for the total phenol content (TP), major flavonoids, and antioxidant activity (AOA). For the quantification of the phenolic content, the modified Folin–Ciocalteu spectrophotometric method according to Alu’Datt et al. (2017) [21 (link)] was used. The measurements were taken using a UV–Vis spectrophotometer (V-1200 VWR, VWR, Radnor, PA, USA) and the phenolic content was expressed as mg GAE/100 g dry basis (db). The flavonoid profile of the extracts was analyzed by UHPLC (Jasco equipment, Cremella, Italy), using a Synergi 4 μm Hydro-RP column (Phenomenex, Valencia, Spain). MeOH and HPLC-grade H₂O were used as the mobile phase. The flow rate was 1 mL/min and the injection volume was 10 μL. Once the profiles were obtained, identification was performed at a wavelength of 284 nm, and for quantification, the proper patterns were used (TCI Europe N.V., Paris, France). The AOA was determined by the DPPH (1,1-diphenyl-2-pricrylhydrazyl) method [22 (link)]. The results were converted to mmol Trolox equivalent (TE)/100 g (db) using a standard curve of Trolox (Sigma-Aldrich, Steinheim am Albuch, Germany) and the same spectrophotometer described above.

The analysis of HES and NAT were carried out by using ultra-high-performance liquid chromatography (UHPLC) equipment connected to a DAD detector (Jasco equipment, Italy) and a Synergi 4 μm Hydro-RP column (Phenomenex, Madrid, Spain). Methanol and HPLC-grade water were used as the mobile phase. The flow rate was 1 mL/min, and the injection volume was 10 μL. Flavonoids were extracted by mixing the samples with 1 mL of double-distilled water and 2 mL of dimethylsulfoxide under magnetic agitation (200 rpm) for 10 min (MS-51M, Jeio Tech, Seoul, Korea) [24 (link)]. Subsequently, the solution was centrifuged at 2031× g (GYROZEN 123 GR, Daejeon, Korea) for 10 min at 4 °C. The supernatant was filtered through a 0.45 μm membrane filter for injection into the UHPLC. The flavonoid identification was performed at wavelength 284 nm, and proper patterns were used for quantification.