HPLC-DAD analysis was conducted using the chromatograph LaChrom Elite (Hitachi, Tokyo, Japan) equipped with an L2455 DAD detector adjusted in the range of 230 to 400 nm, L2200 injector, L2130 pump, L2300 column oven, and a C18 column (150 × 4.6 mm, 5 µm, Merck, Darmstadt, Germany). Data were obtained with EZChrom Elite software, version 3.3.2 SP1 (Santa Clara, CA, USA). Analysis conditions were according to [20 (link)]. The mobile phase consisted of a gradient of 1% phosphoric acid and acetonitrile (Table 1 ) with a flow rate of 0.5 mL per minute.
Samples were obtained by diluting the extract with methanol to the concentration of 4 mg/mL followed by filtration with a 0.45 μm filter (Millex, Merck KGaA, Darmstadt, Germany). Commercial standards were used in an attempt to identify the compounds present in HEMNL by comparison of retention times and ultraviolet spectra. The standards were caffeic acid, chlorogenic acid, ferulic acid, kaempferol, gallic acid, rosmarinic acid, ellagic acid, isoquercitrin, hesperetin, quercetin, resveratrol, vitexin, isovitexin, coumarin, and myricetin acquired from Sigma-Aldrich®, hyperoside acquired from Hwi Analytik Gmbh® (Rülzheim, Germany), and rutin from Chromadex® (Los Angeles, CA, USA).
Samples were obtained by diluting the extract with methanol to the concentration of 4 mg/mL followed by filtration with a 0.45 μm filter (Millex, Merck KGaA, Darmstadt, Germany). Commercial standards were used in an attempt to identify the compounds present in HEMNL by comparison of retention times and ultraviolet spectra. The standards were caffeic acid, chlorogenic acid, ferulic acid, kaempferol, gallic acid, rosmarinic acid, ellagic acid, isoquercitrin, hesperetin, quercetin, resveratrol, vitexin, isovitexin, coumarin, and myricetin acquired from Sigma-Aldrich®, hyperoside acquired from Hwi Analytik Gmbh® (Rülzheim, Germany), and rutin from Chromadex® (Los Angeles, CA, USA).
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