Synergistic Combination of Citrus Flavanones as Strong Antioxidant and COX-Inhibitor Agent

Abstract

Recently, we demonstrated that a Citrus flavanone mix (FM) shows antioxidant and anti-inflammatory activity, even after gastro-duodenal digestion (DFM). The aim of this study was to investigate the possible involvement of the cyclooxygenases (COXs) in the anti-inflammatory activity previously detected, using a human COX inhibitor screening assay, molecular modeling studies, and PGE2 release by Caco-2 cells stimulated with IL-1β and arachidonic acid. Furthermore, the ability to counteract pro-oxidative processes induced by IL-1β was evaluated by measuring four oxidative stress markers, namely, carbonylated proteins, thiobarbituric acid-reactive substances, reactive oxygen species, and reduced glutathione/oxidized glutathione ratio in Caco-2 cells. All flavonoids showed a strong inhibitory activity on COXs, confirmed by molecular modeling studies, with DFM, which showed the best and most synergistic activity on COX-2 (82.45% vs. 87.93% of nimesulide). These results were also corroborated by the cell-based assays. Indeed, DFM proves to be the most powerful anti-inflammatory and antioxidant agent reducing, synergistically and in a statistically significant manner (p < 0.05), PGE2 release than the oxidative stress markers, also with respect to the nimesulide and trolox used as reference compounds. This leads to the hypothesis that FM could be an excellent antioxidant and COX inhibitor candidate to counteract intestinal inflammation.

Keywords: COX; Caco-2 cell; Citrus flavanones; GSH/GSSG; ROS; TBARS; anti-inflammatory activity; antioxidant activity; carbonylated proteins; molecular modeling studies.

Figure 1

Docking poses of HED (green sticks), ERI (magenta sticks), NHE (orange sticks), and NER (yellow sticks) into (a) COX-1 (gray cartoons and sticks) and (b) COX-2 (cyan cartoons and sticks).

Figure 2

Protein/ligand interaction of (a) HED/COX-1; (b) HED/COX-2; (c) NHE/COX-1; (d) NHE/COX-2; (e) ERI/COX-1; (f) ERI/COX-2; (g) NER/COX-1, and (h) NER/COX-2.

Figure 3

Molecular dynamics snapshots (at 6 ns simulation time) of ERI (magenta sticks) in complex with COX-2 (cyan sticks) and of NER (yellow sticks) in complex with COX-2 (gray sticks).

Figure 4

Prostaglandin (PGE2) release upon exposure of Caco-2 monolayers to 10 mM arachidonic acid (AA) after treatment with 25 ng/mL IL-1β. CTR−, negative control treated only with 10 mM AA; CTR+, positive control treated both with 25 ng/mL IL-1β and AA; NIM, nimesulide, used as reference standard; DFM, 10 µM digested flavanone mix; NHE, 10 µM neohesperidin; ERI, 10 µM eriocitrin; HET, 10 µM hesperetin; NER, 10 µM neoeriocitrin; HED, 10 µM hesperidin. ^a p < 0.001 vs. CTR−; ^b p < 0.001 vs. CTR+; ^c p < 0.05 vs. NIM; ^d p < 0.05 vs. DFM.

Figure 5

Exposure to 25 ng/mL IL-1β causes pro-oxidant response in Caco-2 monolayers. CTR−, negative control treated only with culture medium; CTR+, positive control treated with 25 ng/mL IL-1β; TRX, 10 µM Trolox, used as reference standard; DFM, 10 µM digested flavanone mix; NHE, 10 µM neohesperidin; ERI, 10 µM eriocitrin; HET, 10 µM hesperetin; NER, 10 µM neoeriocitrin; HED, 10 µM hesperidin. ^a p < 0.001 vs. CTR−; ^b p < 0.001 vs. CTR+; ^c p < 0.05 vs. TRX; ^d p < 0.05 vs. DFM.

Figure 6

Screening of the synergistic effect of DFM (5–20 µM) at constant ratio of 2.0 by combining HED, NHE, HET, ERI, and NER, with each one at the following concentrations: 1.0–4.0 µM. The simulated lines were generated from CompuSyn by plotting CI versus Fa values. (A) ROS, (B) TBARS, (C) GSH/GSSG, (D) protein carbonyl, (E) COX-1, (F) COX-2, and (G) PGE2.