Resveratrol As a Bioenhancer to Improve Anti-Inflammatory Activities

Article Resveratrol as a Bioenhancer to Improve Anti-Inflammatory Activities of Apigenin Jin-Ah Lee †, Sang Keun Ha †, EunJung Cho and Inwook Choi * Received: 18 August 2015 ; Accepted: 9 November 2015 ; Published: 19 November 2015 Research Group of Nutraceuticals for Metabolic Syndrome, Korea Food Research Institute, 1201-62, Anyangpangyoro, Seongnam, Gyeonggi 463-746, Korea; [email protected] (J.-A.L.); [email protected] (S.K.H.); [email protected] (E.J.C.) * Correspondence: [email protected]; Tel.: +82-31-780-9097; Fax: +82-31-709-9876 † These authors contributed equally to this work. 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: anti-inflammation; bioenhancer; hepatic metabolites of apigenin; UDP-glucuronosyltransferase (UGT) 1. Introduction Apigenin (for chemical structure see Figure1), a common bioactive flavonoid, is found in high amounts in several herbs including parsley, thyme, and peppermint. It has been reported as an important dietary flavonoid with strong anti-inflammatory activities [1]. It is extensively metabolized and mainly eliminated in humans by first-pass metabolism via glucuronidation and sulfation [2,3]. Rapid metabolism results in large amount of conjugates in the systemic circulation and cause low bioavailability [4]. Bioavailability is the rate and extent to which a therapeutically active substance enters systemic circulation and becomes available at the required site of action. Therefore, enhancement of bioavailability would be of utmost importance in order to exert health effects of flavonoids in a body. Bioenhancers improve activity and bioavailability of flavonoids in combination therapy. Many studies are increasingly showing interest toward the improvement of bioavailability of a large number of flavonoids by bioenhancers. In the 1920’s, Bose, an acknowledged author of “Pharmacographia India”, reported an enhanced anti-asthmatic effect of an Ayurevdic formula containing Adhatoda vasica when administered with long pepper [5]. Piperine, the major plant alkaloid present in P. nigrum Nutrients 2015, 7, 9650–9661; doi:10.3390/nu7115485 www.mdpi.com/journal/nutrients Nutrients 2015, 7, page–page Nutrients 2015, 7, 9650–9661 containing Adhatoda vasica when administered with long pepper [5]. Piperine, the major plant alkaloid present in P. nigrum Linn (Black pepper) and P. longum Linn (Long pepper), has Linnbioavailability (Black pepper) enhancing and P.activity longum forLinn curcumin (Long pepper),[6] and has(-)-epigallocatechin bioavailability enhancing-3-gallate (EGCG) activity [ for7]. curcuminAnother bioenhancer [6] and (-)-epigallocatechin-3-gallate is quercetin that inhibited (EGCG) the activity [7]. Another of UGT1A1 bioenhancer and UGT1A9 is quercetin [8] thatand inhibitedincreased thebioavailability activity of UGT1A1 of EGCG and in rats UGT1A9 [9]. [8] and increased bioavailability of EGCG in rats [9]. The main objective of this study is to examine resveratrol as a possible bioenhancer for improving thethe anti-inflammatoryanti-inflammatory activityactivity ofof apigenin.apigenin. To achieve this objective, hepatic metabolites of apigenin in the presence or absence of resveratrol were produced, identifiedidentified and examined for their suppressivesuppressive impact on production of pro pro-inflammatory-inflammatory markers in LPS LPS-stimulated-stimulated RAW 264.7 cells. In In addition, addition, we we tried tried to toverify verify the theresults results obtained obtained from from the cell the line cell experiment line experiment through through an animal an animalstudy. study. Figure 1.1. Structures of apigenin (A) and resveratrolresveratrol ((B).). 2. Materials Materials and and Methods Methods 2.1. Cell Culture Human hepatoma cell line, HepG2, and murine macrophage cell line, RAW 264.7, were purchased from the American Type Culture Collection (ATCC; Rockville,Rockville, MD, USA). The cells were incubated inin Dulbecco’s Dulbecco’s modified modif Eagle’sied Eagle’s medium medium (DMEM; (DMEM; Hyclone, Logan,Hyclone, UT, USA)Logan, supplemented UT, USA) withsupplemented 100 U/mL with penicillin, 100 U/mL 100 penicillin,µg/mL streptomycin100 μg/mL streptomycin (Gibco, Grand (Gibco, Island, Grand NY, Island, USA) NY, and USA) 10% fetaland ˝ bovine10% fetal serum bovine (FBS; serum Hyclone, (FBS; Hyclone, Logan, UT,Logan, USA) UT, at USA) 37 C at in 37 a humidified°C in a humidified atmosphere atmosphere of 5% COof 52%. CO2. 2.2. Pr Productionoduction of Hepatic Metabolites of Apigenin and/or Resveratrol inin HepG2HepG2 CellsCells HepG2 cells (2(2 ˆ× 106 cells/well)cells/well) were were seeded seeded in intoto 6 6 well well plates. The The cells cells were were incubated with apigenin (20 µμM)M) and/orand/or resveratrol resveratrol (0, 10, 20, and 40 μM)µM) for 10 h. The medium fro fromm HepG2 cells waswas treatedtreated withwith anan equalequalvolume volume of of methanol methanol to to precipitate precipitate protein protein and and was was centrifuged centrifuge ford for 2 min2 min at 12,000at 12,000 rpm. rpm. The supernatantsThe supernatants were usedwere to used test theto anti-inflammatorytest the anti-inflammatory effect after higheffect performance after high liquidperformance chromatograph liquid chromatograph (HPLC) analysis. (HPLC) analysis. 2.3. Determination of NO Production 4 RAW 264.7264.7 cellscells (5(5 ˆ× 104 cells/well) were were seeded seeded in intoto 96 96 well well plates plates in in phenol red red-free-free DMEM, treated withwith a standarda standard concentration concentration and hepaticand hepatic metabolites metabolites of apigenin of apigenin (20 µM) and/or (20 μM) resveratrol and/or (0,resveratrol 10, 20, and (0, 10, 40 20,µM) and for 40 30 μM) min, for and 30 min, then and incubated then incubated in the presence in the presence of LPS of (1 LPSµg/mL) (1 μg/mL) for 18 for h. Nitrite18 h. Nitrite accumulation accumulation in the in culture the culture medium medium was measured was measured as an indicator as an indicator of NO production.of NO production. Briefly, 50Briefly,µL of 5 cell0 μL culture of cell mediumculture medium was mixed was withmixed 50 withµL of 50 Griess μL of reagentGriess reagent (Sigma, (Sigma, St. Louis, St. Louis, MO, USA). MO, TheUSA). mixture The mixture was incubated was incubated at room at temperatureroom temperature for 15 for min, 15 andmin, theand absorbance the absorbance at 540 at nm540 wasnm measuredwas measured using using a microplate a microplate reader reader (BioTek (BioTek instruments, instruments, Winooski, Winooski, VT, USA). VT, Fresh USA). culture Fresh medium culture wasmedium used was as a used blank, as anda blank, the quantityand the quantity of nitrite of was nitrite determined was determined by comparison by comparison to a sodium to a sodium nitrite standardnitrite standard curve. curve. 2.4. Measurement of PGE2 and Pro-Inflammatory Cytokine Production 2.4. Measurement of PGE2 and Pro-inflammatory Cytokine Production Cells were treated as described above, and the concentration of PGE2, IL-1β, IL-6 and TNF-α Cells were treated as described above, and the concentration of PGE2, IL-1β, IL-6 and TNF-α in in the culture media were quantified using a competitive ELISA kit (PGE2, Cayman Chemical, Ann the culture media were quantified using a competitive ELISA kit (PGE2, Cayman Chemical, Ann 2 9651 Nutrients 2015, 7, 9650–9661 Arbor, MI, USA; IL-1β, IL-6 and TNF-α, R&D system, Minneapolis, MN, USA), according to the manufacturer’s instructions. The absorbance at 450 nm was measured in a microplate reader. 2.5. Preparation of Whole Cell Extracts Cells were treated with hepatic metabolites and standard of apigenin (20 µM) and/or resveratrol (0, 10, 20, and 40 µM) for 30 min, and then incubated in the presence of LPS (1 µg/mL) for 24 h. The cells were then collected by centrifugation and washed twice with phosphate-buffer saline (PBS). Whole cell extracts were prepared using RIPA buffer (Sigma, St. Louis, MO, USA) containing protease inhibitor (Roche, Mannheim, Germany). Protein concentrations were determined using a protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) according

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