Chlorogenic acid

THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1 × non-essential amino-acids at 37 °C under 5% CO₂. All products were purchased from SIGMA-Aldrich (Saint-Quentin-Fallavier, France). THP-1 cells, at a density of 0.8 × 10⁶ cells/mL in 24-wells plates, were differentiated for 3 days into macrophages by adding in the cell culture medium 20 nM of phorbol myristate acetate (SIGMA-Aldrich). Then, differentiated THP-1 cells were incubated for 24 h with 100 ng/mL of LPS added one hour before or after a pure polyphenol or tomato leaf extract. CP, Kuk_A and TDHCS were dissolved in RPMI 1640 medium and sterilized with 0.2 µM syringe filters (SIGMA-Aldrich). Thus, CP, Kuk_A and TDHCS were tested at 15, 30, 45 and 60 µM. Tomato leaf extracts were used at 50, 100 and 150 µg/mL representing, respectively, a content in chlorogenic acid of 15, 30 and 45 µg/mL. chlorogenic acid was used as standard at 15, 30 and 45 µg/mL (SIGMA-Aldrich).

HiActive^® North American wild blueberry powder was purchased from FutureCeuticals, Inc. (Momence, IL, USA). Rutin, chlorogenic acid, high performance liquid chromatography (HPLC) grade methanol, HPLC grade acetonitrile, potassium metabisulfite, formic acid, acetic acid, chlorogenic acid and Rutin were purchased from Sigma-Aldrich (St. Louis, MO, USA). A standard mixture of delphinidin, cyanidin, petunidin, peonidin, pelargonidin, and malvidin glucosides was purchased from Polyphenols (Sandnes, Norway).

1% of chlorogenic acid (Sigma-Aldrich, St. Louis, USA) was solubilized in a mixture (2/8, v/v) of PEG-400 (Cooper, Melun, France) and methanol (Sigma-Aldrich, St. Louis, USA). 20 μL of this solution were added to skin samples and incubated in a thermostated Franz cell for 24 hours. The donor compartment of the cell was covered with parafilm to prevent evaporation of the applied compounds. After 24 hours, skin samples were dried; surfaces were gently dabbed with methanol (Sigma Aldrich) using gauze prior to embedding in OTC matrix (CellPath, UK) and frozen in liquid nitrogen. The samples were then stored at −80°C until preparation of microscope slides using a cryostat. Six-micron cryo-cross-sections were observed by fluorescence microscopy (Leica DMR-Camera Leica DFC 310 FX, Nanterre, France) with a DAPI filter (excitation 350 nm and 450–490 nm emission) with few drops of Neu reagent [36 ] (1% of 2-aminoethyl-diphenylborinate) (Sigma-Aldrich, St. Louis, USA) in methanol (Sigma-Aldrich, St. Louis, USA), which potentiates the fluorescence of chlorogenic acid.

DPPH, Folin–Ciocalteu reagent, gallic acid, quercetin, chlorogenic acid, 5% carbon dioxide, DMSO and Trolox (Sigma-Aldrich, Poznan, Poland); kaemferol-3-rutinoside and kaempferol-3-O-robinoside-7-O-rhamnoside (Sigma-Aldrich, Schnelldorf, Germany); gallic acid, quercetin, Trolox, methyl nonadecanoate, MTBE, TMSH, chlorogenic acid, rosmarinic acid, t-BOOH, DCFH-DA, JC-1 dye, CCCP (Sigma-Aldrich, Poznan, Poland); methanol, ethanol, isooctane, sodium carbonate, sodium hydroxide, aluminium chloride, sodium acetate, sulfuric acid, formic acid, acetonitrile, and resazurin (POCh Gliwice, Poland).

The contents of phenolic compounds, as well as the antioxidant potential of dandelion leaves and roots, were determined for 70% methanolic plant extracts. The contents of phenolic compounds in dandelion leaves and roots were analyzed after the reaction with the Folin-Ciocalteu reagent [54 (link)]. The leaf or root methanolic extract (0.25 mL) was mixed with 0.25 mL of 25% Na₂CO₃, 0.125 mL of the twice-diluted Folin–Ciocalteu reagent (Sigma-Aldrich, diluted twice with water prior to the analysis) and 2.25 mL of water and mixed. After a 15 min incubation in a dark place, the absorbance was measured at 760 nm (JASCO V-530 UV/Vis spectrophotometer). The final results were expressed as mg of chlorogenic acid (Sigma-Aldrich) per 1 g dry weight (chlorogenic acid equivalents, mg CAE g⁻¹).