Kaempferol 3 o β rutinoside

All solvents and reagents used for analysis were HPLC- and analytical-grade. Gallic acid (≥98.0%), 3,4-dihydroxybenzoic acid (≥97.0%), 4-hydroxybenzoic acid (≥99.0%), 2,4-dihydroxybenzoic acid (≥97.0%)), vanillic acid (≥97.0%), caffeic acid (≥98.0%), syringic acid (≥95.0%), p-coumaric acid (≥98.0%), ferulic acid (≥99.0%), sinapinic acid (≥98.0%), rutin (≥95.0%) quercetin 3-β-d-glucoside (≥99.0%), benzoic acid (≥99.5%), kaempferol 3-O-β–rutinoside (≥98.0%), quercetin (≥95.0%), cinnamic acid (≥99.0%), and kaempferol (≥97.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Stock standard solutions were prepared by dissolving approximately 1 mg of each substance in a 1 mL aliquot of methanol (1000 μg/mL). A mixed standard solution containing 10 μg/mL was subsequently prepared in methanol and stored at 4 °C prior to use.

An ultra performance liquid chromatography (UPLC) system (Waters, Milford, MA, USA) equipped with a binary solvent delivery pump, auto-sampler, and a photodiode array (PDA) detector was used to identify individual polyphenols, as described previously [22 (link)]. The UPLC settings used in this study are summarized in Table 1. The mobile phase consisted of a binary solvent system comprising water (Solvent A) and acetonitrile (Solvent B) supplemented with 0.1% formic acid. A total of 17 phenolic compounds such as, gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, rutin, quercetin 3-β-D-glucoside, benzoic acid, kaempferol 3-O-β-rutinoside, quercetin, cinnamic acid, and kaempferol were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as standards. For each standard compound, a methanol solution was prepared, resulting in a final concentration of 1000 ng/mL. A mixture of all standard compounds was then utilized to establish the calibration curve. The polyphenol content of each sample was calculated using the method described previously [22 (link)] with minor modification.