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  • Citrus plants are of great interest because


    Citrus plants are of great interest because they contain large amounts of flavonoids, and citrus fruits and their juices are consumed in large quantities around the world [18]. The flavonoid contents and profiles of citrus juices vary greatly with the type of fruit. It has been determined that citrus juices from lemon, grapefruit, bergamot, and orange citrus fruits contain at least 20 flavonoids, including diosmin, naringin, and its aglycone, naringenin [19], [20]. In studies on CYP450 activity, various experimental models, ranging from cellular fractions to human studies, were used. Most used are the animal or human liver microsomal CYP450-containing systems or S9 fractions. Microsomal fractions are rich sources of many drug-metabolizing enzymes; therefore they are widely used in in vitro studies to investigate the metabolic fate of xenobiotics. Fish microsomal fractions were also used in drug metabolism studies, but the investigations primarily centered around the presence of pharmaceutically active compounds in aquatic environments. There is little information on how flavones can affect fish metabolic pathways. A major challenge is to find the optimal animal species to serve as a model for human flavonoid–drug interactions because there are differences in primary structure, function, expression, and catalytic activities of drug-metabolizing vitamin d3 sources [21]. Little effort has been made toward comparing the effects of dietary flavonoids on drug metabolizing enzymes in differing species. Thus, the objective of this study was to investigate the effects of diosmin (3′,5,7-trihydroxy-4′-methoxyflavone 7-rutinoside), naringin, and naringenin on CYP3A-mediated 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase activity (BFCOD) in hepatic microsomes from four species.
    Materials and methods
    Results Neither diosmin nor naringin inhibited BFCOD activity in any species (Fig. 1, Fig. 2). However, diosmin did not significantly reduce BFCOD activity in fish hepatic microsomes. In rainbow trout, the activity of BFCOD decreased 60% in the presence of the highest diosmin concentration (Fig. 1). After including the pre-incubation step, BFCOD activity in human hepatic microsomes was reduced in the presence of naringin by 21% (Fig. 2). However, because this did not reach 50% at the highest concentrations of naringin or diosmin, no further analyses of these two flavonoids were performed. Naringenin inhibited BFCOD in the liver microsomes of all four species (Fig. 3). The remaining BFCOD activity at the highest concentrations of naringenin ranged from 43% to 37% in humans, pigs, and mice, and from 6.9% to 9.7% in fish. The degree of inhibition was similar in all species. Inclusion of a pre-incubation step did not enhance the degree of inhibition. Michaelis–Menten kinetic analyses indicated that naringenin competitively inhibited BFCOD activities in humans, pigs, and mice, which yielded Ki values of 24.6, 15.6, and 19.6μM, respectively, and non-competitively inhibited it in fish, which yielded a Ki value of 7.0μM (Fig. 4 and Table 1). The BFCOD reaction in microsomes from all species exhibited Michaelis–Menten kinetics and were fit best with single-enzyme kinetics. Values for Vmax were similar in humans and pigs. Fish Vmax was significantly lower than those for human, pig, and mouse (by 40-, 33-, and 21-fold, respectively). Mouse Vmax was significantly lower than those for human and pig (1.83- and 1.5-fold, respectively). Mouse Km was significantly lower than those for human, pig, and fish (Table 1).
    Discussion Many constituents of plant-based foods, including some flavonoids, have been found to be potent modulators of CYP450 activity and thus can interact with drug metabolism. The most reliable information about such interactions can be obtained from human volunteer studies. However, due to ethical constrains, such studies are restricted. Moreover, it is not possible to perform in vivo human studies on a large number of the compounds simultaneously. In the early phase of interaction studies, in vitro human microsomes are an attractive tool because they contain a full complement of CYP450 enzymes and allow studies of interactions with a large number of bioactive compounds. However, high costs and limited materials from the same human donors impede their use in the first screening phase. Therefore, there is an increasing need for methods that can be used to study interactions between bioactive compounds and CYP450s. Various liver-derived in vitro models in animals have been used. Pigs and laboratory mice are often recognized as model organisms of choice for studying human metabolic pathways because they share a number of physiologic characteristics with humans [26], [27]. Because fish evolution allows conservation of several human drug targets [28], we also tested fish as a model organism for studying the inhibitory potency of diosmin, naringin, and naringenin on CYP3A-mediated BFCOD activity.