Chlorogenic acid cga

Chemicals and reagents were obtained from the following commercial sources:
2',7'-dichlorodihydrofluorescein diacetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), RPMI 1600 and an antibiotic solution containing 10,000 units of penicillin, 10 mg of streptomycin and 25 μg of amphotericin B per mL were purchased from Thermo Fisher Scientific, Gibco (Grand Island, NY, USA). BlockAce was purchased from DS Pharma Biomedical (Japan), Biotin-HABP (biotinylated linker protein hyaluronan, 0.25mg/mL) was purchased from Hokudo Co. (Japan), Anti-Human Collagen type 1 antibody was purchased from Rockland Immunochemicals (US), Anti-human MMP-1, anti-IgG/HRP, and Streptavidin-HRP were purchased from R&D Systems (Japan), Can Get Signal was purchased from Toyobo Co. (Japan), EZ West blue dye was purchased from Atto Corp. (Japan), Chlorogenic acid (CGA) was purchased from Cayman Chemical (US). Fluorescein-5-thiosemicarbazide and sodium hyaluronate, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The bicinchoninic acid (BCA) protein assay reagent kit was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

Samples (2 g) were macerated with 5 mL of extraction solvent (9:1 methanol: formic acid, v/v) and incubated overnight at 3 °C, without stirring. The obtained suspension was centrifuged at 3500× g for 15 min (Rolco, CM4080, Ciudad de Buenos Aires, Argentina) and the supernatant was recovered. Three independent extractions (one from each of three plastic boxes containing the carrot samples) were performed for each treatment, accession, and sampling time. Total phenolic content was determined using the Folin–Ciocalteu reagent, according to Singleton and Rossi [37 ] with minimal modifications. Briefly, 50 µL-aliquots of the extracts were mixed with 250 μL of Folin–Ciocalteu reagent, 1000 μL of Na₂CO₃ (20% w/v), and 3700 μL of distillated water. The absorbance at 765 nm was measured in a spectrophotometer (T60 UV/VIS, PG Instruments Ltd., Wibtoft, Lutterworth, UK) after 60 min of reaction. A commercial standard of chlorogenic acid (CGA) (Cayman Chemical, Buenos Aires, Argentina) was used as a reference to estimate TP levels, and results were expressed as CGA equivalents in mg kg⁻¹ of fresh weight (fw). For each extract, three determinations were performed (i.e., three technical replicates).