Screening of furanocoumarin derivatives in citrus fruits by enzyme-linked immunosorbent assay

Abstract

This paper reports a sensitive and specific enzyme-linked immunosorbent assay for screening of furanocoumarin derivatives as cytochrome P450 3A4 inhibitors in citrus fruits. Anti-6′,7′-dihydroxybergamottin antibody was obtained by immunizing rabbits with 6′,7′- dihydroxybergamottin conjugated with bovine serum albumin using the N-succinimidyl ester method. An enzyme marker was similarly prepared by coupling 6′,7′-dihydroxybergamottin with β-D-galactosidase. The enzyme-linked immunosorbent assay is capable of detecting as little 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 geranyloxy 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. © 2004 Pharmaceutical Society of Japan.