Figure 8
The ethyl acetate fraction prepared from the extract of roasted coffee beans exhibits anti-inflammatory activity. (A) Procedure for the fractionation of coffee extract using organic solvents. (B) The extract of roasted coffee beans and each fraction were analyzed by HPLC. (C-F) RAW264.7 cells were pretreated with coffee extract (5% (v/v) (Cf) and each fraction equivalent to 5% (v/v) of coffee (H: hexane fraction, E: ethyl acetate fraction, B: butanol fraction, W: water fraction) 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 evaluated 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. The expression of GAPDH mRNA was used as an internal control. (G) RAW264.7 cells were transfected with pNF-κB-Luc and pRL-TK. Transfected cells were pretreated with coffee extract (5% (v/v)) and each fraction equivalent to 5% (v/v) of coffee for 1 h prior to LPS stimulation 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 coffee extract (5% (v/v)) and each fraction 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.
