Resveratrol as a Bioenhancer to Improve Anti-Inflammatory Activities of Apigenin

Abstract

The aim of this study was to improve the anti-inflammatory activities of apigenin through co-treatment with resveratrol as a bioenhancer of apigenin. RAW 264.7 cells pretreated with hepatic metabolites formed by the co-metabolism of apigenin and resveratrol (ARMs) in HepG2 cells were stimulated with lipopolysaccharide (LPS). ARMs prominently inhibited (p < 0.05) the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), interleukin (IL)-1β, IL-6 and TNF-α. Otherwise no such activity was observed by hepatic metabolites of apigenin alone (AMs). ARMs also effectively suppressed protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Co-administration of apigenin (50 mg/kg) and resveratrol (25 mg/kg) also showed a significant reduction of carrageenan-induced paw edema in mice (61.20% to 23.81%). Co-administration of apigenin and resveratrol led to a 2.39 fold increase in plasma apigenin levels compared to administration of apigenin alone, suggesting that co-administration of resveratrol could increase bioavailability of apigenin. When the action of resveratrol on the main apigenin metabolizing enzymes, UDP-glucuronosyltransferases (UGTs), was investigated, resveratrol mainly inhibited the formation of apigenin glucuronides by UGT1A9 in a non-competitive manner with a Ki value of 7.782 μM. These results suggested that resveratrol helps apigenin to bypass hepatic metabolism and maintain apigenin's anti-inflammatory activities in the body.

Keywords: UDP-glucuronosyltransferase (UGT); anti-inflammation; bioenhancer; hepatic metabolites of apigenin.

Figure 1

Structures of apigenin (A) and resveratrol (B).

Figure 2

Effects of ARMs on LPS-stimulated NO and PGE₂ production, and iNOS and COX-2 expressions in RAW264.7 cells. The samples of hepatic metabolites were made as described in the Materials and Methods section. Cells were treated with the standard concentration (A; 20 μM) and hepatic metabolites of apigenin (AMs; 20 μM), hepatic metabolites of apigenin (20 μM) and resveratrol (10, 20 and 40 μM) (ARMs 1:0.5, 1:1 and 1:2), and hepatic metabolites of resveratrol (40 μM) for 30 min, and then incubated in the presence of LPS (1 μg/mL) for 18 h. (A) Nitrite accumulation was measured as described in the Materials and Methods section; (B) PGE₂ production was measured as described in the Materials and Methods section. Data were presented as means ± SEM and were representative of triplicate experiments: * p < 0.05 compared to cells cultured with LPS (1 μg/mL) alone; # p < 0.05 compared to cells cultured with LPS (1 μg/mL) and AMs; (C) The expression of iNOS and COX-2 were examined by Western blot analysis.

Figure 3

Effects of ARMs on LPS-stimulated IL-1β, IL-6 and TNF-α production in RAW 264.7 cells. The samples of hepatic metabolites were made as described in the Materials and Methods section. Cells were treated with the standard concentration (A; 20 μM) and hepatic metabolites of apigenin (AMs; 20 μM), hepatic metabolites of apigenin (20 μM) and resveratrol (10, 20 and 40 μM) (ARMs 1:0.5, 1:1 and 1:2), and hepatic metabolites of resveratrol (40 μM) for 30 min, and then incubated in the presence of LPS (1 μg/mL) for 18 h. The concentration of (A) IL-1β, (B) IL-6 and (C) TNF-α in the cell culture supernatants were measured as described in the Materials and Methods section. Data were presented as means ± SEM and were representative of triplicate experiments: * p < 0.05 compared to cells cultured with LPS (1 μg/mL) alone; # p < 0.05 compared to cells cultured with LPS (1 μg/mL) and AMs.

Figure 4

Effects of co-administration of apigenin and resveratrol on carrageenan-induced paw edema in mice. (A) Percentage of carrageen-induced paw edema of control group (vehicle), apigenin administered (50 mg/kg body weight), resveratrol administered (25 mg/kg body weight), apigenin and resveratrol administered (50 and 25 mg/kg body weight), and indomethacin (10 mg/kg body weight). Data were expressed as means ± SEM, n = 5. * p < 0.05 compared to administration of apigenin; (B) HPLC chromatograms of plasma of control (up), apigenin administered (middle), and apigenin and resveratrol co-administered (down) groups. Plasma samples were collected and analyzed for apigenin and its metabolites 5 h after carrageenan injection.

Figure 5

Inhibition of resveratrol towards UGT1A9-catalyzed apigenin glucuronidation (A) and kinetic profiles for formation of apigenin glucuronide in the presence of resveratrol by recombinant UGT1A9 (B and C). Recombinant UGT1A9 (5 μg of protein) was incubated with 5 to 40 μM apigenin and 2 mM UDPGA at 37 °C for 30 to 360 min in the presence (10, 20, and 40 μM) or absence of resveratrol. Data were represented as means ± SEM and were representative of triplicate experiments.