Journal: Scientific Reports
Article Title: Pyrocatechol, a component of coffee, suppresses LPS-induced inflammatory responses by inhibiting NF-κB and activating Nrf2
Figure Lengend Snippet: The R2 fraction prepared from the ethyl acetate fraction of the roasted coffee bean extract exhibits anti-inflammatory activity. ( A ) Procedure for the fractionation of the ethyl acetate fraction of coffee bean extract using the Sep Pak C18 column. ( B ) The ethyl acetate fraction of the roasted coffee bean extract ( E ) and the R1-R3 fractions were analyzed by HPLC. ( C – F ) RAW264.7 cells were pretreated with fractions R1-R3, equivalent to 5% (v/v) of coffee, for 1 h prior to the LPS stimulation (1 μg/mL). ( C ) Nitrate concentrations in culture supernatants were measured 24 h after the LPS stimulation using Griess reagent. ( D ) iNOS mRNA expression was assessed 12 h after the LPS stimulation by RT-PCR. GAPDH mRNA expression was used as an internal control. ( E ) The amounts of CCL2 in supernatants were evaluated 24 h after the LPS stimulation by ELISA. ( F ) CCL2 mRNA expression was assessed 2 h after the LPS stimulation by RT-PCR. ( G ) RAW264.7 cells were transfected with pNF-κB-Luc and pRL-TK. Transfected cells were pretreated with fractions R1-R3, equivalent to 5% (v/v) of coffee, for 1 h prior to the stimulation with LPS (1 μg/mL) for 6 h. NF-κB-dependent luciferase activity was normalized to the activity of constitutively expressed Renilla luciferase. ( H ) RAW264.7 cells were treated with fractions R1-R3, equivalent to 5% (v/v) of coffee, for 1 h. Nuclear extracts were immunoblotted with an anti-Nrf2 or anti-Lamin B antibody. The relative expression levels of Nrf2 in the nucleus are shown in the graph.
Article Snippet: To separate ethyl acetate extracts, dried ethyl acetate extracts (equivalent to 15 mL coffee) were dissolved in 15 mL of methanol/ethyl acetate (2:1), applied to a Sep-Pak C18 column (Waters, Milford, MA), and eluted by 15-mL stepwise rinses of water (R1), 20% methanol (R2), and 100% methanol (R3).
Techniques: Activity Assay, Fractionation, High Performance Liquid Chromatography, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Luciferase