Trans 3 hydroxycinnamic acid

Phenolic standards such as gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillin acid, caffeic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid and trans-3-hydroxycinnamic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were obtained from Fisher (HPLC-grade, Thermo Fisher Scientific, Waltham, MA, USA). Other unmarked reagents were of an analytical grade.

HPLC was performed based on the method published by Suleiman et al. (2021) [46 (link)]. Bee bread powder was suspended in water and methanol to produce aqueous and methanol solutions, respectively, to achieve final concentrations of 100 mg/mL. The solutions were then vortexed, sonicated and followed with centrifugation at 20,111× g for 5 min prior to HPLC analysis. The samples were analyzed using a Dionex RS3000 system (Thermo Scientific, Waltham, MA, USA). The chromatic separation was achieved at 25 °C on a Zorbax SB-C18 column (3.5 µm, 4.6 mm I.D × 150 mm) (Agilent Technologies, Santa Clara, CA, USA). A binary solvent system was employed consisting of 0.1% formic acid in water as solvent A and 0.1% formic acid in methanol (40:60, v/v) as solvent B. The chromatographic analyses were conducted at a run time of 0, 20, 25, 25.1 and 30 min. The flow rate was 1.0 mL/min, and the injection volume was 20 µL. The eluted components were monitored at 340 nm. The standard substances of gallic acid, caffeic acid, mangiferin, trans-ferulic acid, 2-hydroxycinnamic acid, trans-3-hydroxycinnamic acid, quercetin, kaempferol and apigenin were purchased from Sigma (St. Louis, MO, USA) and used as reference compounds.