974 Biol. Pharm. Bull. 27(7) 974—977 (2004) Vol. 27, No. 7 Screening of Furanocoumarin Derivatives in Citrus Fruits by Enzyme- Linked Immunosorbent Assay Tetsuya SAITA,* Hiroshi FUJITO, and Masato MORI Department of Pharmacy, Saga University Hospital; 5–1–1 Nabeshima, Saga 849–8501, Japan. Received January 30, 2004; accepted March 16, 2004 This paper reports a sensitive and specific enzyme-linked immunosorbent assay for screening of fura- -nocoumarin derivatives as cytochrome P450 3A4 inhibitors in citrus fruits. Anti-6؅,7؅-dihydroxybergamottin an -tibody was obtained by immunizing rabbits with 6؅,7؅-dihydroxybergamottin conjugated with bovine serum al -bumin using the N-succinimidyl ester method. An enzyme marker was similarly prepared by coupling 6؅,7؅-dihy droxybergamottin with b-D-galactosidase. The enzyme-linked immunosorbent assay is capable of detecting as lit- tle as 800 pg/ml of 6؅,7؅-dihydroxybergamottin and 4 ng/ml of bergamottin. Cross-reactivity data showed that the antibody well recognizes both the furanocoumarin and 6,7-dihydroxy-3,7-dimethyloct-2-enyloxy moieties of the -6؅,7؅-dihydroxybergamottin, and is thus specific to the structure of furanocoumarin derivatives containing ger anyloxy side chain as the cytochrome P450 3A4 inhibitors in grapefruit juice. The antibody was, therefore, used .for screening a large number of citrus fruits for furanocoumarin derivatives such as 6؅,7؅-dihydroxybergamottin Fifteen citrus fruits were examined and significant reactivity was observed in 8 of these: red pummelo, sweetie, melogold, banpeiyu pummelo, hassaku, sour orange, lime and natsudaidai. This enzyme-linked immunosorbent assay may be a powerful tool for screening for furanocoumarin derivatives as cytochrome P450 3A4 inhibitors in grapefruit juice. Key words furanocoumarin derivative; enzyme-linked immunosorbent assay; screening; citrus fruit; grapefruit juice Grapefruit juice (GFJ) has been reported to increase the Besides the Rutaceae family (like grapefruit), many plants oral availability of many clinically important drugs.1—5) The of other families such as Umbelliferae, Leguminosae and interaction is caused by the inhibition of their first pass me- Moraceae also contain furanocoumarin derivatives.11) Many tabolisms mainly catalyzed by an intestinal cytochrome P450 of these plants are used as common vegetables12) or tradi- (CYP) 3A4.6,7) Recently, six furanocoumarin derivatives, tional medicines.13) Thus it is possible that furanocoumarin bergamottin, 6Ј,7Ј-epoxybergamottin, 6Ј,7Ј-dihydroxyberg- derivatives contained in these vegetables or crude drugs also amottin (DHB) and three furanocoumarin dimers (Fig. 1) change the pharmacokinetics of a drug. These findings re- were isolated from GFJ as inhibitors of CYP3A4 and are quire us to develop a simple screening method for the fura- now suggested to be clinically active constituents.8—10) nocoumarin derivatives. Their determination in plants has usually been made by thin layer or column chromatography, however, it is very difficult to find these derivatives by chro- matography alone. Therefore, we wanted to develop a simple and specific enzyme-linked immunosorbent assay (ELISA) for screening of furanocoumarin derivatives in plants. The methodology for the antibody production, the labeling of furanocoumarin derivative with b-D-galactosidase (b-Gal) to act as a tracer, the characterization of antibody specificity, and the technique developed for the screening of fura- nocoumarin derivatives in citrus fruits by ELISA is described here. MATERIALS AND METHODS Reagents Bergamottin and DHB were purchased from Ultrafine, Ltd. (Manchester, England). b-Gal (EC 3.2.1.23) from Escherichia coli and 4-methylumbelliferyl-b-D-galac- topyranoside were obtained from Boehringer Mannheim (Mannheim, Germany). All other reagents and solvents were of the highest grade commercially available. Samples of Citrus Fruits Citrus fruits were obtained from local commercial sources and information on them is shown in Table 1. All fruits were squeezed and filtered, and the juices were stored at Ϫ20 °C until needed for the study. The fruit juice samples were diluted 50, 250 and 1250 times with 0.25 M phosphate buffer (pH 7.4) containing 10 mM eth- Fig. 1. Chemical Structures of Furanocoumarin Derivatives in GFJ ylenediaminetetraacetate, 0.1% bovine serum albumin (BSA) * To whom correspondence should be addressed. e-mail: [email protected] © 2004 Pharmaceutical Society of Japan July 2004 975 Table1. Species and Origin of Citrus Fruits Used in the Present Study Citrus fruit Species Origin Grapefruit (white) C. paradisi MACF. California U.S.A. Red pummelo C. grandis OSBECK California U.S.A. Sweetie C. grandis OSBECKϫC. paradisi MACF. Israel Melogold C. grandis OSBECKϫC. paradisi MACF. California U.S.A. Banpeiyu pummelo C. grandis OSBECK Kumamoto Japan Hassaku C. hassaku HORT. ex TANAKA Saga Japan Sour orange C. aurantium L. Saga Japan Lime C. aurantifolia SWINGLE Mexico Natsudaidai C. natsudaidai HAYATA Kumamoto Japan Navel orange C. sinensis L. OSBECK var. brasiliensis TANAKA Australia Sweet orange C. sinensis OSBECK Australia Yuzu C. junos SIEB. ex TANAKA Miyazaki Japan Iyokan C. iyo HORT. ex TANAKA Ehime Japan Satsuma mandarin C. unshu MARCOVITCH Fukuoka Japan Ponkan C. reticulata BLANCO Oita Japan Dekopon C. reticulata BLANCOϫ(C. unshu MARCOVITCHϫC. sinensis L. OSBECK) Oita Japan and 0.1% NaN3 (buffer A), then used to screen fura- female rabbits were each given multiple subcutaneous injec- nocoumarin derivatives using ELISA. tions over sites along both sides of their backs. Booster injec- Preparation of the Immunogen A solution of DHB tions were then given three times at biweekly intervals, using (4.5 mg, 12 mmol) and succinic anhydride (12 mg, 120 mmol) one-half the amount of the dose of the first immunization. in pyridine (300 ml) was stirred overnight at 60 °C. After re- The rabbits were bled from an ear vein 10 weeks after immu- moving pyridine by passing nitrogen through the reaction nization began. The sera (10 ml) were separated by centrifu- mixture, 1 ml of ethyl acetate and 1 ml of H2O were added to gation and heated at 55 °C for 30 min. Fractions of IgG were the residue, and the mixture was shaken vigorously. The separated from the sera with 50% saturated ammonium sul- ethyl acetate layer was washed with saturated sodium chlo- fate and chromatographed on a column of DEAE-Sephacel ride, dried over anhydrous sodium sulfate, and evaporated to (2.1ϫ23 cm) using 17.5 mM phosphate buffer (pH 6.8) as an give DHB hemisuccinate (3.4 mg) as a white solid. The re- eluant.14) The fraction passed through the column was sulting DHB hemisuccinate was used without further purifi- lyophilized and used as anti-DHB IgG for ELISA. cation for preparing the conjugates with BSA and b-Gal, re- Enzyme Labeling DHB was labeled by binding to b- spectively, as the immunogen and the tracer in the ELISA. Gal, essentially by the same principle as used for the prepara- The yield of DHB hemisuccinate was tentatively estimated to tion of the DHB immunogen. In brief, a solution of b-Gal be 60% according to HPLC measurements of the quantity of (156 mg, 0.28 nmol) in 1 ml of 0.1 M phosphate buffer (pH nonreacted DHB. 7.0) was mixed with a solution of succinimidyl DHB 1-Ethyl-3,3-dimethylaminopropyl-carbodiimide hydrochlo- hemisuccinate (approximately 0.2 mg, 0.35 mmol) in 50 ml of ride (EDPC) (3 mg, 15.6 mmol) and N-hydroxysuccinimide DMF and incubated at room temperature for 1 h. The mixture (1.8 mg, 15.6 mmol) were added to a solution of DHB was chromatographed on a Sepharose 6B column (2.0ϫ40 hemisuccinate (approximately 3.4 mg, 7.2 mmol) in 95% diox- cm) using 20 mM phosphate buffer (pH 7.0) containing 0.1 M ane (0.5 ml), and the resulting solution was allowed to stand NaCl, 1 mM MgCl2, 0.1% BSA, and 0.1% NaN3 (buffer B) to at room temperature for 2 h. After the addition of H2O, the remove any remaining small molecules. Four-milliliter frac- resulting mixture was extracted with ethyl acetate. The ethyl tions were collected, and fractions 15 to 17, representing the acetate layer was dried over anhydrous sodium sulfate, and main peak showing enzyme activity, were combined and evaporated to give succinimidyl DHB hemisuccinate as a used as a label in the ELISA. white solid. A solution of BSA (10 mg, 0.15 mmol) in 0.1 M ELISA Method ELISA is based on the principle of phosphate buffer (pH 7.0, 1 ml) was mixed with a solution of competition between enzyme-labeled and unlabeled drugs succinimidyl DHB hemisuccinate (approximately 3.8 mg, for an immobilized antibody, followed by measurement of 6.7 mmol) in N,N-dimethylformamide (DMF, 300 ml) and in- the marker enzyme activity of the immunocomplex bound to cubated at room temperature for 2 h. The reaction mixture the solid phase. Briefly, the wells in microtiter plates (Nunc F was chromatographed on a Sephadex G-100 column (2.8ϫ Immunoplates I, Nunc, Reskilde, Denmark) were coated by 42 cm) with an eluant of 0.1 M phosphate buffer (pH 7.0) loading 150 ml of anti-DHB IgG (0.5 mg/ml) in 10 mM containing 3 M urea. Then the purified conjugate was exam- Tris–HCl buffer (pH 8.5) containing 10 mM NaCl and 10 mM ined spectrophotometrically and estimated to contain approx- NaN3 and allowed to stand for 1 h at 37 °C. After the plates imately 3.1 molecules of DHB per BSA molecule, assuming had been washed twice with buffer A, they were incubated the molar extinction coefficients of DHB to be 5300 at with 200 ml of 10 mM Tris–HCl buffer (pH 8.5) containing 280 nm and 14300 at 310 nm, and those of BSA to be 43600 10 mM NaCl, 10 mM NaN3 and 2% BSA for 20 min at 37 °C at 280 nm.