2 hydroxycinnamic acid

Gallic acid, (+)-catechin, pyrocatechol, chlorogenic acid, 2,5-dihydroxybenzoic acid, 4-hydroxybenzoic acid, (−)-epicatechin, caffeic acid, syringic acid, vanillin, taxifolin, sinapic acid, p-coumaric acid, ferulic acid, rosmarinic acid, 2-hydroxycinnamic acid, pinoresinol, quercetin, luteolin, and apigenin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vanillic acid, 3-hydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, apigenin 7-glucoside, luteolin 7-glucoside, hesperidin, eriodictyol, and kaempferol were obtained from Fluka (St. Louis, MO, USA). Finally, verbascoside, protocatechuic acid, and hyperoside were purchased from HWI Analytik (Ruelzheim, RP, Germany). Methanol and formic acid of HPLC grade were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Hesse, Germany), respectively. Ultra-pure water (18 mΩ) was obtained using a Millipore Milli-Q Plus water treatment system (MILLIPORE CORPORATION, Bedford, MA, USA).

Gallic acid, (+)-catechin, pyrocatechol, chlorogenic acid, 2,5-dihydroxybenzoic acid, 4-hydroxybenzoic acid, (−)-epicatechin, caffeic acid, syringic acid, vanillin, taxifolin, sinapic acid, p-coumaric acid, ferulic acid, rosmarinic acid, 2-hydroxycinnamic acid, pinoresinol, quercetin, luteolin, and apigenin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vanillic acid, 3-hydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, apigenin 7-glucoside, luteolin 7-glucoside, hesperidin, eriodictyol, and kaempferol were obtained from Fluka (St. Louis, MO, USA). Finally, verbascoside, protocatechuic acid, and hyperoside were purchased from HWI Analytik (Ruelzheim, Germany). Methanol and formic acid of HPLC grade were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany), respectively. Ultra-pure water (18 mΩ) was obtained from a Milli-Q water purification system (Millipore Co., Ltd.)
Ethyl acetate and methanol were obtained from Carlo Erba Reagents (Milan, Italy). Ultra-pure water was obtained using a Millipore Milli-Q Plus water treatment system (Millipore Bedford Corp., Bedford, MA). All chemicals were of analytical grade.

Gallic acid, (+)-catechin, pyrocatechol, chlorogenic acid, 2,5-dihydroxybenzoic acid, 4-hydroxybenzoic acid, (−)-epicatechin, caffeic acid, syringic acid, vanillin, taxifolin, sinapic acid, p-coumaric acid, ferulic acid, rosmarinic acid, 2-hydroxycinnamic acid, pinoresinol, quercetin, luteolin, and apigenin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vanillic acid, 3-hydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, apigenin 7-glucoside, luteolin 7-glucoside, hesperidin, eriodictyol, and kaempferol were obtained from Fluka (St. Louis, MO, USA). Verbascoside, protocatechuic acid, and hyperoside were purchased from HWI Analytik (Ruelzheim, Germany).

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.

Luminol was dissolved in DMSO and then diluted in a 50 mM borate buffer (pH 9). Phenol (Sigma, 33517), 2-hydroxycinnamic acid (Sigma, H22809-5G), p-coumaric acid (Sigma, C9228-1G), and 1-(4-hydroxyphenyl)imidazole (HPI) were dissolved in DMSO and then diluted in 30% DMSO in PBS to the indicated concentrations. The measurements were done in a white opaque 96-well plate in PBS at a final reaction volume of 100 μL per well. The final recombinant APEX2 concentration was 1.5 nM. Measurements were done in the Pherastar (BMG) plate reader.

2,2′-Azobis (2-amidinopropane) dihydrochloride (AAPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), apigenin, caffeic acid, (+)-catechin, p-coumaric acid, 3,4-dihydroxy-L-phenylalanin, 2,5-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 2,4-dihydroxycinnamic acid, 3,4-dihydroxyhydrocinnamic acid, formic acid, gallic acid, 4-hydroxyphenylacetic acid, 2-hydroxycinnamic acid, 4,4′-methylenediphenol, potassium chloride, potassium persulfate, potassium phosphate monobasic, quercetin, resveratrol, rosmarinic acid, sinapic acid, syringic acid, sodium hydrogen phosphate, sodium chloride, taxifolin, and Trolox were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Methanol was purchased from J. T. Baker Co. (Phillipsburg, NJ, USA) for sample preparation and analysis. Ultrapure water used in this study was produced using a Milli-Q water purification system (Millipore Co., Bedford, MA, USA).
The aerial parts of L. meyenii were gathered from Lima in 2015 and verified by Paul H. Gonzales Arce (P.H.G.A.). The dried samples (L-2015-A30), extract (L-2015-A30E), and separated compounds (L-2015-A30C1-7) were placed at the Center for Efficacy Assessment and Development of Functional Foods and Drugs at Hallym University.

To determine cell viability, in the exponential phase of the growth cells were seeded at 5 × 10⁴/mL in 96-well plates in medium supplemented with 1% glutamine, 0.5% penicillin/streptomycin, and 5% fetal bovine serum. After seeding (24 h), cells were treated, in quadruplicate, with increasing concentrations of phytochemicals (1–500 μM) and incubated for 24 and 48 h. Ascorbic acid, 20-hydroxyecdysone, ferulic acid, 2-hydroxycinnamic acid, p-coumaric acid, β-carotene, and lutein were from Sigma-Aldrich Company Ltd. (Milan, Italy) and resuspended in dimethylsulfoxide. The cytotoxicity assay was performed by adding a small amount of the CellTiter 96R AQueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA) directly to culture wells, incubating for 4 h and then recording the absorbance at 450 nm with a 96-well plate reader (MULTISKAN EX, Thermo Electron Corporation, Vantaa, Finland). The percentage of cell growth is calculated as: