Polyphenols in Alcoholic Beverages Activating Constitutive Androstane Receptor CAR
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Biosci. Biotechnol. Biochem., 75 (8), 1635–1637, 2011 Communication Polyphenols in Alcoholic Beverages Activating Constitutive Androstane Receptor CAR Ruiqing YAO,1 Akihito YASUOKA,2 Asuka KAMEI,3 Yoshinori KITAGAWA,4 Tomohiro ROGI,4 y Norifumi TAIEISHI,4 Nobuo TSURUOKA,4 Yoshionobu KISO,4 Takumi MISAKA,1 and Keiko ABE1;3; 1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2Department of Biological Engineering, Maebashi Institute of Technology, 460-1 Kamisadori-machi, Maebashi, Gunma 371-0816, Japan 3Kanagawa Academy of Science and Technology, 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan 4Institute for Health Care Science, Suntory Wellness Ltd., 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan Received June 8, 2011; Accepted June 27, 2011; Online Publication, August 7, 2011 [doi:10.1271/bbb.110444] The constitutive androstane receptor CAR is a Among the 29 polyphenols examined, chrysin (5,7-OH xenosensing nuclear receptor that can be activated by flavone) and galangin (3,5,7-OH flavone) were found to natural polyphenols such as flavonoids and catechins. elicit activities as strongly as known artificial CAR We examined alcoholic beverage phytochemicals for activators. This incidence was also confirmed in vivo their ability to activate CAR. HepG2 cells were trans- using CAR KO mice, suggesting the possibility that fected with CAR expression vector and its reporter food-derived polyphenols can regulate CAR, alleviating gene, and then treated with trans-resveratrol, ellagic metabolic syndrome. The objective of the present study acid, -caryophyllene, myrcene, and xanthohumol. A was to extend the spectrum of known CAR respondents luciferase assay revealed that ellagic acid and trans- to ingredients of alcoholic beverages. Identification of resveratrol activated both human and mouse CAR. the novel CAR activators resveratrol and ellagic acid Since CAR regulates many genes involved in energy should shed light on the mechanisms of the beneficial metabolism, the possibility exists that these polyphenols effects of congeners in alcoholic beverages. would reduce the risk of certain alcohol-induced The structural formulae of the congeners investigated metabolic disorders with the help of CAR. in this study are depicted in Fig. 1. trans-resveratrol is one of the stilbenes in the peel of the grape variety used Key words: polyphenol; resveratrol; ellagic acid; in red wine production. Ellagic acid is generated from constitutive androstane receptor; alcoholic hydrolysable tannins in the oak barrel during charring beverage and aging process. Beer contains phenolic compounds derived partly from hops (30%) and partly from barley Alcoholic beverages contain a variety of phytochem- (70%). -Caryophyllene, myrcene, and xanthohumol are icals originating from fruit peels (wine), wooden barrels congeners of hop origin. Pure chemicals, ellagic acid (whisky), or additives (beer and liqueur). These non- (Fluka Biochemika, Switzerland, 45140), trans-resvera- alcoholic compounds are called congeners of alcoholic trol (Sigma, USA, R5010-500MG), -caryophyllene beverages. Constituents of the congeners are potent anti- (Wako, Japan, 329-53072), and myrcene (Tokyo oxidants evoking certain physiological activities that can Chemical Industry, Japan, M0235) were used, except reduce the risk of metabolic disorders associated with that xanthohumol was supplied as hop extract containing chronic alcohol consumption.1–3) Particularly, resvera- 85% xanthohumol (Hopsteiner, Germany, Xantho-flav- trol is well studied in the context of epigenetic extract). These congeners were subjected to luciferase regulation of energy metabolism.4,5) The constitutive assay with known CAR activators, as described below androstane receptor (CAR, NR1I3) belongs to the NR1I (CITCO and TCPOBOP, Sigma). subfamily of nuclear receptors with its relative, pre- To detect CAR activation, we used an HepG2 cell- gnene X receptor (PXR, NR1I2). While it is known as a based assay system with the CAR expression vector and drug responsive nuclear receptor that makes possible the the CYP2B6 PBREM reporter.10) Because the endoge- detoxification of xenobiotics through regulation of the nous activity of human CAR in HepG2 cells is low, the genes involved in this process,6) recent studies have PBREM reporter activity in this system depends revealed expanded roles in regulating the genes related basically on the activation of exogenously introduced to energy metabolism, ameliorating fat accumulation CAR.11) The cells were transfected with mouse or and insulin resistance.7,8) We have screened food- human CAR expression vector, a reporter plasmid derived flavonoids and related polyphenols for their (PBREM-TK-pGL3), and a control reporter plasmid ability to activate CAR in a cell-based assay system.9) (phRL-TK) prior to 48 h of treatment with 0.1% DMSO y To whom correspondence should be addressed. Fax: +81-3-5841-8006; E-mail: [email protected] Abbreviations: CAR, constitutive androstane receptor; PBREM, phenobarbital responsive enhancer module 1636 R. YAO et al. O 3.0 trans-resveratrol Ellagic acid A Human CAR OH O OH * 2.5 * HO OH 2.0 HO * 1.5 OH HO O O 1.0 β-Caryophyllene 0.5 Xanthohumol Relative Luciferase activity Luciferase Relative 0.0 5 20 5 20 5 20 5 20 5 20 0.25 (µM) H C 2 OH H H CITCO HO OH Myrcene Ellagic acid Resveratrol Xanthohumol Myrcene 0.1 % DMSO -Callyophyllene β O OCH3 6.0 B Mouse CAR * Cl Cl Cl 5.0 4.0 N Cl O TCPOBOP CITCO 3.0 2.0 * O * 1.0 N activity Luciferase Relative N O 0.0 N 520520520520520 2 (µM) Cl Cl Cl S N Myrcene TCPOBOP Ellagic acid Fig. 1. Structures of the Chemicals Used in This Study. Resveratrol 0.1 % DMSO Xanthohumol trans-resveratrol, the effective component of polyphenols in red -Callyophyllene wine; ellagic acid, a derivative of tannin solubilized from wooden β barrels; -caryophyllene, myrcene, and xanthohumol: constituents Fig. 2. Response Profile of CAR to Congeners of Alcoholic of hop and thus of beer. TCPOBOP: 1,4-Bis[2-(3,5-dichloropyridy- Beverages. loxy)]benzene and CITCO: 6-(4-chlorophenyl)-imidazo[2,1-b]thia- HepG2 cells were transfected with the CAR expression vector, a zole-5-carbaldehyde as activators of mouse and human CAR luciferase reporter for CAR activation, and an internal control respectively. reporter, and treated with these chemicals for 48 h. The concen- tration of xanthohumol was estimated from the amount in the hop as vehicle or with the various test compounds in the extract (85%). The effect of each compound on reporter activity was medium at the indicated concentrations. The cell lysates represented as a relative value compared to the response to the vehicle solution, and was then normalized by the response to were assayed for luciferase activity. Among the com- positive control drugs (CITCO and TCPOBOP, which induced 2.5- pounds described above, 20 mM ellagic acid and 5 mM fold and 5-fold increases respectively). The data are representative trans-resveratrol exhibited significant increases in of three independent experiments in triplicate. Significant increases reporter activity over basal activity (Fig. 2A and B). In in luciferase activity versus DMSO controls are asterisked (Welch’s the case of human CAR, the values were 1.63-fold for t-test, p < 0:05, n ¼ 3). Twenty mM ellagic acid and 5 mM resveratrol activated both human CAR (A) mouse CAR (B). 20 mM ellagic acid and 2.23-fold for 5 mM trans-resver- atrol, close to that of 5 mM galangin (1.90-fold) observed in a previous study.9) Similarly, mouse CAR exhibited since they undergo glyco- or sulfo-conjugation, which activation by 20 mM ellagic acid (1.47-fold) and by 5 mM decreases their accessibility to inner-cellular compo- trans-resveratrol (1.69-fold), approximating the extent nents.13,14) Previous studies using human volunteers due to 5 mM galangin (1.74-fold). When the cells were have indicated that a dose of 40 mg ellagic acid results in treated with various doses of these polyphenols, various the occurrence of 200 mg/L (0.6 mM) of pure ellagic acid response curves were obtained (Fig. 3A and B). In the in the plasma,15) while it was 25 mg versus 7 mg/L case of both human and mouse CAR, trans-resveratrol (0.03 mM) in the case of resveratrol.16) Others have caused a maximum response at 5 mM, with decreased reported relatively low penetration ratios.17–19) These responses at 10 mM and 20 mM. No change in cell shape concentrations are 200 to 300-fold smaller than the or no decrease in control reporter activity was observed effective concentrations found in our assay. Whether at these concentrations. This is in contrast with the these polyphenols can activate CAR in vivo, especially results obtained for chrysin, which showed both of these in the liver where they are metabolized concomitantly phenomena.9) As for ellagic acid, both human and with ethanol, is an issue for the future. mouse CAR exhibited significant responses only at The ameliorating effects of ellagic acid and resvera- 20 mM, indicating that its agonistic activity is less than trol on alcohol-induced metabolic disorders have been that of trans-resveratrol. investigated basically as to their anti-oxidative activity The quantities of these polyphenols in alcoholic and their effects on specific proteins. Several enzymatic drinks varies depending on the materials used;1,2,12) they systems for alcohol metabolism have been implicated as range from 5 to 50 mg per L, whisky or brandy in the sources of reactive oxygen species (ROS).20) The radi- case of ellagic acid, and from 1 to 15 mg per L of red cal-scavenging activities of ellagic acid and resveratrol wine in the case of resveratrol. There are issues can antagonize the adverse effects of these ROS.21) concerning the bioavailability of these polyphenols, Moreover, resveratrol has been reported to activate key Polyphenols Activating CAR 1637 A 3.0 Culture, Sports, Science, and Technology of Japan Human CAR (22380072 to T.