712 Journal of Health Science, 56(6) 712–716 (2010) — Research Letter — Safety Evaluation of Black Rice the present study suggests that kurosu from a jar does not have toxic effect and does not alter expression lev- Vinegar (Kurosu) from a Jar on els of principal drug metabolism enzymes and trans- Food-drug Interaction: 30-day porters. Ingestion Study on Expressions Key words —— black rice vinegar, cytochrome P450, of Drug Metabolism Enzymes and transporter Transporters in Rats Yoshihiko Shibayama,∗, a Masanobu Nagano,b INTRODUCTION Akira Fujii,b Miyuki Taguchi,c Yasuo Takeda,c and Katsushi Yamadac Food-drug interaction evidence has been accu- mulated; natural products can interact with drugs aEducation Research Center for Clinical Pharmacy, Faculty of by the same mechanisms as drugs. Clinically im- Pharmaceutical Sciences, Hokkaido University, Nishi 6, Kita portant interactions appear to involve effects on 12, Kita-ku, Sapporo 060–0812, Japan, bSakamoto Brewing drug metabolism via cytochrome P450 (Cyp) isoen- Co., Ltd., 21–15 Uenosnono-cho, Kagoshima 890–0052, Japan zymes, impairment of hepatic or renal function, and cClinical Pharmacy and Pharmacology, Graduate School and other possible mechanisms.1) For example, a of Medical and Dental Sciences, Kagoshima University, 8–35– healthcare supplement St. John’s Wort, interacts 1 Sakuragaoka, Kagoshima 890–8520, Japan with prescription drugs.1, 2) The family of ATP bind- (Received February 18, 2010; Accepted August 17, 2010; ing cassette (ABC) transporters, for example, p- Published online August 23, 2010) glycoprotein (P-gp), multidrug resistance protein 2 (Mrp2) and breast cancer resistance protein (Bcrp), Black rice vinegar (kurosu) made from a jar is a also has important roles in the detoxification and traditional vinegar in Japan. Kurosu has been com- excretion of xenobiotics.3) In hepatocytes, organic monly used as a healthcare supplement; however, it anion transport polypeptide 2 (Oatp2) plays impor- is not clear whether kurosu has a treatment effect tant roles in the vector transport of bile acids from on the expression of drug metabolism enzymes and blood to bile. Bile acid is a regulation factor of hep- transporters, whose expression alterations may in- atic and intestinal transporters.4) The organic anion duce food-drug interaction. Alteration of the prin- transporter (Oat) family also plays important roles cipal drug metabolism enzymes and expression fol- in the elimination of a variety of endogenous sub- lowing kurosu and moromi, the residue from black stances, xenobiotics and their metabolites from the vinegar brewing, for 30-day treatment was evaluated. body.5) Water, 0.23% acetic acid, concentrated kurosu (con- Black rice vinegar (kurosu) from a jar is a tradi- taining 0.23% acetic acid) and moromi was admin- tional vinegar in Kagoshima, Japan.6) It has been re- istered to Wister rats. The treatment did not signif- ported that kurosu improved hypertension, allergy, icantly affect the biochemical parameters of serum, hypercholesterol, carbohydrate metabolism and tu- alanine aminotransferase, alkaline phosphatase, to- mor growth.6, 7) Kurosu has been commonly used tal protein, creatinine in the serum, and body weight. as a healthcare supplement; however, it is not clear The treatment also did not affect the expression lev- whether kurosu treatment effects on the expression els of cytochrome P450 (Cyp) 1a2, Cyp2b1/2, Cyp3a1, of drug metabolism enzymes and transporters. glutathione S-transferase, p-glycoprotein, multidrug To evaluate the food-drug interaction with resistance protein 2, breast cancer resistance protein, kurosu, the authors investigated the effect of kurosu organic anion transport polypeptide 2, and organic treatment on the expression of principal drug anion transport 2 and 3 in the liver. In conclusion, metabolism enzymes and transporters. ∗To whom correspondence should be addressed: Education Re- search Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Hokkaido University, Nishi 6, Kita 12, Kita-ku, Sap- MATERIALS AND METHODS poro 060–0812, Japan. Tel. & Fax: +81-11-706-3705; E-mail: [email protected] Chemicals —— Primary antibodies for Western C 2010 The Pharmaceutical Society of Japan No. 6 713 Table 1. Primary Antibody Properties and Immunodetection Condition Molecular Clone or catalog No.HostProtein Dilution ratio size (kDa) (µg) (primary antibody) β-actin 43 SIGMA-ALDRICH, AC-74Mouse 10 10,000 Cyp1a2 58 MILLIPORE, AB10089 Rabbit 20 2,000 Cyp2b1/2 56 BIOMOL, CR 3290 Rabbit 20 1,000 Cyp3a1 58 BIOMOL, CR 3310 Rabbit 10 3,000 GSTa) 26 ROCKLAND, 3D4 Mouse 10 2,000 Mrp2 170 MONOSAN, M2III-6 Mouse 20 400 P-gp 160 Calbiochem, C219 Mouse 20 1,000 Bcrp 60 Kamiya Biomedical, bxp-21 Mouse 20 500 Oatp290Santa Cruz, sc-33610 Rabbit 20 500 Oat2 60 TransGenic,KE031 Rabbit 50 250 Oat3 130 TransGenic,KE035 Rabbit 50 250 Whole cell homogenate of the liver was separated with SDS-PAGE. The amount of protein and the dilution ratio of the primary antibodies are indicated. Clone name of mouse monoclonalantibodies or catalog number of polyclonal rabbit antibody is shown in the “Clone or catalog No.” lane. a)GST:glutathione s-transterase. blotting detection were purchased from Sigma- treated by oral gavage once a day as follows: ve- Aldrich Japan (Tokyo, Japan), Nihon Millipore hicle group, distilled water was given at a dose of K.K. (Tokyo, Japan), BIOMOL (Plymouth Meet- 10 ml/kg; acetic acid group, 1.5 ml of 0.23% acetic ing, PA, U.S.A.), ROCKLAND (Gilbertsville, PA, acid (pH 3.5) was diluted with 8.5 ml distilled wa- U.S.A.), MONOSAN (AM Uden, The Nether- ter, and the diluted solution was given at a dose lands), Calbiochem (San Diego, CA, U.S.A.), of 10 ml/kg; black vinegar group, 1.5 ml concen- Kamiya Biomedical (Seattle, WA, U.S.A.), Santa trated black vinegar solution was diluted with 8.5 ml Cruz Biotechnology (Santa Cruz, CA, U.S.A.) and distilled water, and the diluted solution was given TransGenic (Kumamoto, Japan). The clone names at a dose of 10 ml/kg; moromi group, 50 mg mo- and catalog numbers of the primary antibodies romi was suspended in 10 ml distilled water, and the are summarized Table 1. The second antibod- suspension was given at a dose of 10 ml/kg. The ies, anti-rabbit and anti-mouse IgG antibody con- rats were killed by exsanguination after blood col- jugated with horseradish peroxidase (HRP), were lection. As a positive control treatment, dexam- purchased from Nacalai Tesque (Kyoto, Japan). ethasone was intraperitoneally injected at a dose of Nitrocellulose membranes, ECL Western blotting 50 mg/kg for consecutive 4 days8, 9) and methotrex- detection system and Hyperfilm ECL were pur- ate was intraperitoneally injected once at a dose of chased from GE Healthcare U.K. Ltd. (Bucking- 150 mg/kg.10, 11) Dexamethasone-treated rats were hamshire, U.K.). Protease inhibitor cocktail tablets killed the day after 4-day treatment. Methotraxate- (CompleteR ,EDTA free) were purchased from treated rats were killed on day 4 after treatment. Roche Diagnostics GmbH (Basel, Switzerland). All Target tissues were promptly removed and stored other reagents were purchased from Wako Pure at −30◦C until Western blot analysis. Serum pre- Chemical Industries (Osaka, Japan). Concentrated pared from each rat was stored at 4◦C until analysis. black vinegar from a jar (10-fold concentrated, pH. Hemolysate samples were excluded from samples 4.41, acetic acid distilled) and moromi (residue for analysis. Animal experiments were performed from black vinegar brewing) were obtained from in accordance with the criteria of Kagoshima Uni- Sakamoto Brewing Co., Ltd. (Kagoshima, Japan). versity for the care and use of experimental animals. Concentrated black vinegar contains 0.23% acetic Assays —— Creatinine, alanine aminotransferase acid, 3.23% lactic acid and 1.99% ash. (ALT) and alkaline phosphatase (ALP) were de- Animals —— Male Wistar rats (body weight, termined with the clinical reagents for diagnosis 90 ± 5g, mean ± S.D.,Kyudo, Kumamoto, Japan) (Shino-test, Tokyo, Japan). Protein concentra- were housed under standard conditions (21 ± 2◦C, tion was determined by DC protein assay (Bio- ventilated rooms, 12 hr light/dark cycle). The ani- Rad Japan, Tokyo, Japan). Optical densities (ODs) mals were fed rat chow and allowed free access to were measured by the public domain program Im- water during the experimental period. Rats were age J (developed by the U.S. National Institutes 714 Vo l. 56 (2010) of Health and available on the Internet at http:// further incubated with 1000-fold diluted secondary rsb.info.nih.gov/ij/download.html). Individual ex- antibody conjugated with HRP. Finally, membranes posures were scanned into TIFF images with a were rinsed once for 15 minutes and four more GT9000 color scanner (Seiko Epson, Tokyo, Japan). times for 5 minutes with buffer A. For detection, Overall differences among treatments were evalu- the ECL Western blotting detection system was ated by one-way analysis of variance (ANOVA). used. Differences among groups in vehicle-, acetic acid-, black vinegar- and moromi-treated rats were evalu- ated using ANOVA. RESULTS AND DISCUSSION Western Blotting —— Whole cell homogenate was prepared from the rat liver. Tissues ob- Rats were administered water, acetic acid, black tained from individual animals of each group vinegar and moromi once a day for 30 consecutive were cryopreserved and homogenized in buffer days. It was reported that the dose of black vinegar containing 250 mM sucrose and 5 mM N-2- and moromi corresponded to the human dose and hydroxyethylpiperazine-N-2-ethanesulfonic acid that black vinegar treatment improved blood sugar (HEPES)-tris(hydroxymethyl)-aminomethane (Tris, levels in a diabetic model mice.6) The biochemi- pH 7.4) with an ultrasonic homogenizer. Protease cal parameters of serum, ALT, ALP, total protein, inhibitor tablets were dissolved in the above- creatinine were measured to estimate general liver mentioned homogenizing buffer at appropriate and kidney functions.