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. 2017 Jul 1;2(1):167–182. doi: 10.1089/can.2017.0027

FIG. 6.

FIG. 6.

(A) Anti-inflammatory activity of C. sativa F7 at three different concentrations (an HPLC fraction of C2F at concentrations of 190 μg/mL), fractions F1–F9-excluding F7 (F1–F9-exc F7; HPLC fractions of C2F at concentrations of 163 μg/mL), combination of F1–F9-excluding F7 along with F7 measured as level of IL-8 on HCT116 cells, with and without antagonists to CB1, CB2, and GPR55 receptors (antagonists at a concentration of 20 μM, CB1, CB1 receptor antagonist rimonabant; CB2, CB2 receptor antagonist SR144528; GPR55, GPR55 antagonist CID16020046). 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 fractions for 4 h. Treatments with F7, F1–F9, and combination of fractions without antagonists served as a positive control (Con). 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 control. (B) Determination of HCT116 cell viability using Alamar Blue fluorescence (resazurin assay) as a function of cytotoxic effect. 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 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≤0.01 and p≤0.0001, respectively. Levels with different letters are significantly different from all combinations of pairs by Tukey's HSD. CB1, cannabinoid receptor type 1; CB2, cannabinoid receptor type 2.