Figure 9
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.
