journal of food and drug analysis 23 (2015) 516e528 Available online at www.sciencedirect.com ScienceDirect journal homepage: www.jfda-online.com Original Article Effects of aqueous extract of Ruta graveolens and its ingredients on cytochrome P450, uridine diphosphate (UDP)-glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-quinone oxidoreductase in mice * Yune-Fang Ueng a,b,c, , Chien-Chih Chen d,e,f, Yu-Ling Huang d,g, I-Jung Lee h, Chul-Ho Yun i, Yu-Hsuan Chen a, Chiung-Chiao Huang a a Division of Basic Chinese Medicine, National Research Institute of Chinese Medicine, Taipei, Taiwan b Department and Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan c Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan d Division of Chinese Medicinal Chemistry, National Research Institute of Chinese Medicine, Taipei, Taiwan e Department of Biotechnology, Hungkuang University, Taichung County, Taiwan f Department of Nursing, Hungkuang University, Taichung County, Taiwan g Department of Cosmetic Science, Chang Gung University of Science and Technology, Taoyuan County, Taiwan h Division of Chinese Materia Medica Development, National Research Institute of Chinese Medicine, Taipei, Taiwan i School of Biological Sciences and Technology, Chonnam National University, Gwangju, South Korea article info abstract Article history: Ruta graveolens (the common rue) has been used for various therapeutic purposes, including Received 15 October 2014 relief of rheumatism and treatment of circulatory disorder. To elucidate the effects of rue on Received in revised form main drug-metabolizing enzymes, effects of an aqueous extract of the aerial part of rue and 29 January 2015 its ingredients on cytochrome P450 (P450/CYP), uridine diphosphate (UDP)- Accepted 24 March 2015 glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate) Available online 21 May 2015 (NAD(P)H):quinone oxidoreductase were studied in C57BL/6JNarl mice. Oral administration of rue extract to males increased hepatic Cyp1a and Cyp2b activities in a dose-dependent Keywords: manner. Under a 7-day treatment regimen, rue extract (0.5 g/kg) induced hepatic Cyp1a and cytochrome P450 Cyp2b activities and protein levels in males and females. This treatment increased hepatic furanocoumarins UDP-glucuronosyltransferase activity only in males. However, NAD(P)H:quinone oxidore- Ruta graveolens ductase activity remained unchanged. Based on the contents of rutin and furanocoumarins * Corresponding author. Division of Basic Chinese Medicine, National Research Institute of Chinese Medicine, 155-1, Li-Nong Street, Section 2, Taipei 112, Taiwan. E-mail address: [email protected] (Y.-F. Ueng). http://dx.doi.org/10.1016/j.jfda.2015.03.005 1021-9498/Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved. journal of food and drug analysis 23 (2015) 516e528 517 rutin of mouse dose of rue extract, rutin increased hepatic Cyp1a activity and the mixture of UDP-glucuronosyltransferase furanocoumarins (Fmix) increased Cyp2b activities in males. The mixture of rutin and Fmix increased Cyp1a and Cyp2b activities. These results revealed that rutin and Fmix contributed at least in part to the P450 induction by rue. Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved. can be linked to the phototoxicity and antifertility of rue [10]. 1. Introduction The flavonoid rutin (Fig. 1) has been identified as a water- soluble glycoside in rue [11]. In the evaluation of the utero- Drug-metabolizing enzymes play a crucial role in the tonic activities of rue fractionations, rutin was found in the biotransformation of xenobiotics, including drugs and chemi- active fraction and had the most potent uterotonic effect [11].It cal carcinogens, as well as physiological substances such as has been suggested that furanocoumarins and rutin play steroids. In Phase I metabolism, microsomal cytochrome P450 important roles in the pharmacological/toxicological effects of (P450/CYP)-dependent monooxygenase is the primary enzyme rue. system that is responsible for oxidations. This monooxygenase In the modulation of drug-metabolizing enzymes, intra- system participates in the oxidation of a variety of endogenous peritoneal administration of a single dose of 25 mg/kg 8-MOP and exogenous substrates, such as testosterone, methadone, to rats induced hepatic CYP2B after 24 hours and 48 hours [3]. and codeine [1]. P450 catalyzes the oxidations by transfer of two Chalepensin activated the mouse constitutive androstane e electrons through NADPH P450 reductase (CPR), and the sec- receptor (CAR) and induced Cyp2b9/10 in male C57BL/6JNarl ond electron can also be transferred by cytochrome b5.Enzymes mice after 1 week of treatment [4]. By contrast, some of the of the P450 superfamily exhibit broad substrate specificity and furanocoumarins in rue, such as 5-MOP, 8-MOP, and chale- functional diversity. Therefore, selective substrates have been pensin, are known potent mechanism-based inhibitors of used as chemical probes for characterizing functional changes CYP2A and CYP3A in vitro and/or in vivo [5,12]. Rutin is catab- in individual P450 isoforms [1]. In the nomenclature of P450 olized by human intestinal/fecal bacteria to form deglycosy- isoforms, lowercase letters are used only for mouse P450, such lated products including leucocyanidin and quercetin [13]. as Cyp1a2 (http://drnelson.uthsc.edu/cytochromeP450.html). Quercetin induced CYP1A1 expression in HepG2 cells, In Phase II metabolic reactions, uridine diphosphate (UDP)- glucuronosyltransferase (UGT)-catalyzed glucuronidation is quantitatively the most important conjugative reaction and OH appears to be the main metabolic pathway of a variety of drugs, such as acetaminophen. Reduced nicotinamide adenine dinu- HO O cleotide (phosphate) (NAD(P)H):quinone oxidoreductase (NQO) OH OH catalyzes further oxidation of quinone substrates, including the HO OH antineoplastic agent mitomycin C [2]. Functions of drug- O O O metabolizing enzymes can be modulated by several natural OH O products, such as psoralen derivatives [3e5]. Alterations of drug-metabolizing enzymes may result not only in herbedrug H3C O HO interaction, but also in changes in physiological functions of HO endogenous substrates or in carcinogenicity of xenobiotics. Rutin OH Ruta graveolens L. (Yuin Siang), commonly known as rue, has been used in America, Asia, and Europe for the treatment of CH3 heart disease, wounds, and rheumatism [6,7]. In Taiwan, the O aerial part of rue has been reputedly used as a folk medicine for the treatment of circulatory disorders. In Europe, this plant has also been used as an abortifacient and emmenagogue [6]. Aqueous extract of rue was found to reduce human sperm O O O O O O motility [8]. According to the 53-day spermatogenic cycle in rats, Psoralen 5-MOP a study of 60-day treatment of rue was designed and the changes in the rue-treated group were compared to those in the vehicle control group [9]. Oral administration of rue aqueous extract at 0.5 g/kg/d to male adult rats for 60 days decreased O O their serum testosterone level, suppressed sperm motility and O O O O number of mounts, and prolonged ejaculatory latency. Types of O Chalepensin chemical compounds identified in rue include coumarins, fla- H3C 8-MOP vonoids, lignans, and quinoline alkaloids [10]. Furanocoumar- ins, such as 8-methoxypsoralen (MOP) and chalepensin (Fig. 1), Fig. 1 e Structures of rutin and furanocoumarins identified represent one group of biologically active rue constituents and in the rue aqueous extract. MOP ¼ methoxypsoralen. 518 journal of food and drug analysis 23 (2015) 516e528 whereas rutin did not [14]. To a lesser extent, quercetin was collected, filtered through a filter paper (ADVANTEC; Toyo elevated the UGT mRNA level. These reports indicated that Roshi Kaisha, Ltd, Tokyo, Japan) and lyophilized. A total of rutin and furanocoumarins may affect drug-metabolizing 20.54 g of extract was obtained from 1 kg of rue. To determine enzymes. However, the effect of rue remained unclear. In the rutin and furanocoumarin contents, rue aqueous extract this study, to elucidate the effects of rue on hepatic and renal (100 mg) was extracted twice using 2 mL of methanol. After drug-metabolizing enzymes, an aqueous extract of rue was centrifugation and filtration through a 0.45-mm filter, the prepared and the contents of rutin and furanocoumarins in filtrate was subjected to HPLC analysis. An Agilent 1100 HPLC the extract were determined. The effects of oral administra- system (Agilent Technology, Santa Clara, CA, USA) equipped tion of rue on P450 isoforms, UGT, and NQO were studied. with a photodiode array detector and a C18 column (Cosmosil Based on the contents of the rue extract, the effects of rutin 5C18-AR-II, 4.6 Â 250 mm2,5mm; Nacalai Tesque, Kyoto, Japan) and the mixture of furanocoumarins (Fmix) were studied to was used. Ingredients of the rue extract were separated using reveal their contribution to the enzyme induction by rue. acetonitrile/methanol (1:1) (A) and 10% methanol (B), with stepwise changes of linear gradients: 0 minute, 100% B; 45 minutes, 50% A and 50% B; 50 minutes, 50% A and 50% B; 60 2. Materials and methods minutes, 100% A; 62 minutes, 100% B; 70 minutes, 100% B. Rutin and furanocoumarins were detected by measuring the 2.1. Chemicals and antibodies absorbance at 246 nm. In addition to the retention