FIG. 4.

(A) Anti-inflammatory activity of C. sativa ethanolic extracts (C2F; 163 μg/mL), F7 at three different concentrations (an HPLC fraction of C2F at concentrations of 142, 163, and 190 μg/mL), fractions F1–F9-excluding F7 (F1–F9-exc F7) at two concentrations (HPLC fractions of C2F at concentrations of 163 and 190 μg/mL), combination of each concentration of F1–F9-excluding F7 along with each concentration of F7 on HCT 116 cells measured as level of IL-8 (ng/mL). HCT116 cells were seeded (50,000 per well) in triplicate in 500 μL growing media and incubated for 24 h at 37°C in a humidified 5% CO₂–95% air atmosphere. Cells were treated with 300 ng/mL TNFα and 50 μL of C. sativa ethanol extract of C2F or fractions for 4 h. Nontreated are the cells without TNFα and treatments. Levels of IL-8 were measured from the supernatant using a commercial kit. Values (ng/mL) were calculated relative to a TNFα-treated control. (B) Determination of HCT116 cell viability using Alamar Blue fluorescence (resazurin assay) as a function of live cell number. Cells were seeded and treated as described in (A). Next, the cells were incubated with Alamar Blue for 2 h. Relative fluorescence at the excitation/emission of 544/590 nm was measured. Values were calculated as percentage of live cells relative to the nontreated (cells without TNFα and treatments) control after reducing the autofluorescence of Alamar Blue without cells. Error bars indicate±SE (n=3). **^ΔΔCt *** Indicate data statistically significantly different in comparison with the control (TNFα-treated cells) at p≤0.001 and p≤0.0001, respectively. Levels with different letters are significantly different from all combinations of pairs by Tukey's HSD.