Effect of the flavonoid hesperidin on glucose and fructose transport, sucrase activity and glycaemic response to orange juice in a crossover trial on healthy volunteers.

Although polyphenols inhibit glucose absorption and transport in vitro, it is uncertain whether this activity is sufficient to attenuate glycaemic response in vivo. We examined this using orange juice, which contains high levels of hesperidin. We first used a combination of in vitro assays to evaluate the potential effect of hesperidin and other orange juice components on intestinal sugar absorption and then tested whether this translated to an effect in healthy volunteers. Hesperidin attenuated transfer of 14C-labelled glucose across differentiated Caco-2/TC7 cell monolayers. The involvement of the sugar transporter GLUT2 was demonstrated by experiments carried out in the absence of Na to exclude the contribution of sodium-glucose linked transporter 1 and further explored by the use of Xenopus laevis oocytes expressing human GLUT2 or GLUT5. Fructose transport was also affected by hesperidin partly by inhibition of GLUT5, while hesperidin, even at high concentration, did not inhibit rat intestinal sucrase activity. We conducted three separate crossover interventions, each on ten healthy volunteers using orange juice with different amounts of added hesperidin and water. The biggest difference in postprandial blood glucose between orange juice and control, containing equivalent amounts of glucose, fructose, sucrose, citric acid and ascorbate, was when the juice was diluted (ΔC max=-0·5 mm, P=0·0146). The effect was less pronounced when the juice was given at regular strength. Our data indicate that hesperidin can modulate postprandial glycaemic response of orange juice by partial inhibition of intestinal GLUT, but this depends on sugar and hesperidin concentrations.

Effect of Low-Furanocoumarin Hybrid Grapefruit Juice Consumption on Midazolam Pharmacokinetics.

The objectives of this study were to investigate the effect of grapefruit juice low in furanocoumarins on CYP3A activity and to summarize previous findings of enzyme inhibition measured by the metabolism of midazolam after intake of grapefruit juice. Twelve healthy volunteers participated in a prospective, randomized, double-blinded, 3-way crossover clinical study to determine the effect of regular grapefruit juice (RGJ) and a novel, low-furanocoumarin hybrid grapefruit juice (HGJ) on the metabolism of oral midazolam, used as a probe for in vivo CYP3A activity, compared with water as a control. The RGJ was 100% hand-squeezed "Hudson" grapefruit juice, and the HGJ contained low amounts of furanocoumarin constituents. The point estimates (90% confidence intervals) for the RGJ/water midazolam AUC geometric mean ratio was 122% (107-140). The point estimate for the HGJ/water midazolam AUC ratio was within the 80% to 125% bioequivalence range, indicating an absence of interaction. This finding also prompted a systematic review of available evidence on the pharmacokinetic alteration of midazolam by grapefruit juice. Although most studies demonstrated alteration in midazolam pharmacokinetics supporting inhibition of CYP3A activity as a likely mechanism, the cohorts included in these studies and the extent of the pharmacokinetic interaction varied widely. The current study indicated grapefruit juice-drug interaction varies substantially based on patient characteristics and/or grapefruit juice product-related factors, including the amount of furanocoumarin constituents present in the juice.

Identification of genes associated with low furanocoumarin content in grapefruit.

Some furanocoumarins in grapefruit (Citrus paradisi) are associated with the so-called grapefruit juice effect. Previous phytochemical quantification and genetic analysis suggested that the synthesis of these furanocoumarins may be controlled by a single gene in the pathway. In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis of fruit tissues was performed to identify the candidate gene(s) likely associated with low furanocoumarin content in grapefruit. Fifteen tentative differentially expressed fragments were cloned through the cDNA-AFLP analysis of the grapefruit variety Foster and its spontaneous low-furanocoumarin mutant Low Acid Foster. Sequence analysis revealed a cDNA-AFLP fragment, Contig 6, was homologous to a substrate-proved psoralen synthase gene, CYP71A22, and was part of citrus unigenes Cit.3003 and Csi.1332, and predicted genes Ciclev10004717m in mandarin and orange1.1g041507m in sweet orange. The two predicted genes contained the highly conserved motifs at one of the substrate recognition sites of CYP71A22. Digital gene expression profile showed the unigenes were expressed only in fruit and seed. Quantitative real-time PCR also proved Contig 6 was down-regulated in Low Acid Foster. These results showed the differentially expressed Contig 6 was related to the reduced furanocoumarin levels in the mutant. The identified fragment, homologs, unigenes, and genes may facilitate further furanocoumarin genetic study and grapefruit variety improvement.

Effect of blueberry juice on clearance of buspirone and flurbiprofen in human volunteers.

AIM: The present study evaluated the possibility of drug interactions involving blueberry juice (BBJ) and substrate drugs whose clearance is dependent on cytochromes P4503A (CYP3A) and P4502C9 (CYP2C9). METHODS: A 50:50 mixture of lowbush and highbush BBJ was evaluated in vitro as an inhibitor of CYP3A activity (hydroxylation of triazolam and dealkylation of buspirone) and of CYP2C9 activity (flurbiprofen hydroxylation) using human liver microsomes. In clinical studies, clearance of oral buspirone and oral flurbiprofen was studied in healthy volunteers with and without co-treatment with BBJ. RESULTS: BBJ inhibited CYP3A and CYP2C9 activity in vitro, with 50% inhibitory concentrations (IC50 ) of less than 2%, but without evidence of mechanism-based (irreversible) inhibition. Grapefruit juice (GFJ) also inhibited CYP3A activity, but inhibitory potency was increased by pre-incubation, consistent with mechanism-based inhibition. In clinical studies, GFJ significantly increased area under the plasma concentration-time curve (AUC) for the CYP3A substrate buspirone. The geometric mean ratio (GMR = AUC with GFJ divided by AUC with water) was 2.12. In contrast, the effect of BBJ (GMR = 1.39) was not significant. In the study of flurbiprofen (CYP2C9 substrate), the positive control inhibitor fluconazole significantly increased flurbiprofen AUC (GMR = 1.71), but BBJ had no significant effect (GMR = 1.03). CONCLUSION: The increased buspirone AUC associated with BBJ is quantitatively small and could have occurred by chance. BBJ has no effect on flurbiprofen AUC. The studies provide no evidence for concern about clinically important pharmacokinetic drug interactions of BBJ with substrate drugs metabolized by CYP3A or CYP2C9.

Mechanism-based inhibition of human cytochrome P450-3A activity by grapefruit hybrids having low furanocoumarin content.

A citrus breeding program aimed at developing low furanocoumarin (FC) grapefruit cultivars provided 40 grapefruit juice (GFJ) samples containing variable concentrations of FC derivatives, established as being mechanism-based (irreversible) inhibitors of human CYP3A isoforms. The principal inhibitory FCs were identified as 6',7'-dihydroxybergamottin, along with a series of dimeric compounds (spiroesters) having high inhibitory potency. A random subset of the GFJ samples (n = 25) were tested as CYP3A inhibitors using an in vitro model based on human liver microsomal metabolism of the index substrate triazolam. The reciprocal values of in vitro 50% inhibitory concentrations (IC(50)) were highly correlated with concentrations of inhibitory FCs in the GFJ samples (r(2) = 0.96). However the correlations were driven mainly by a few samples having high FC content and high reciprocal IC(50) (corresponding to low IC(50)). Among the rest of the samples, the relationship was less robust. Further study is needed to determine how low the FC content needs to be (or how high the IC(50) needs to be) to assure minimal risk of clinical interactions involving GFJ and CYP3A substrate drugs.

The effect of grapefruit juice on drug disposition.

INTRODUCTION: Since their initial discovery in 1989, grapefruit juice (GFJ)-drug interactions have received extensive interest from the scientific, medical, regulatory and lay communities. Although knowledge regarding the effects of GFJ on drug disposition continues to expand, the list of drugs studied in the clinical setting remains relatively limited. AREAS COVERED: This article reviews the in vitro effects of GFJ and its constituents on the activity of CYP enzymes, organic anion-transporting polypeptides (OATPs), P-glycoprotein, esterases and sulfotransferases. The translational applicability of the in vitro findings to the clinical setting is discussed for each drug metabolizing enzyme and transporter. Reported AUC ratios for available GFJ-drug interaction studies are also provided. Relevant investigations were identified by searching the PubMed electronic database from 1989 to 2010. EXPERT OPINION: GFJ increases the bioavailability of some orally administered drugs that are metabolized by CYP3A and normally undergo extensive presystemic extraction. In addition, GFJ can decrease the oral absorption of a few drugs that rely on OATPs in the gastrointestinal tract for their uptake. The number of drugs shown to interact with GFJ in vitro is far greater than the number of clinically relevant GFJ-drug interactions. For the majority of patients, complete avoidance of GFJ is unwarranted.

Effect of maturity, processing, and storage on the furanocoumarin composition of grapefruit and grapefruit juice.

Since the early 1990's, grapefruit juice has been implicated in drug interaction with various furanocoumarins (FCs) now associated with the effect. Although FCs are present in various fruits and vegetables, it is their presence in grapefruit that has attracted the most attention. Studies have shown that FCs in grapefruit juice can vary significantly and from multiple causes. Most of all, FCs are stress-induced molecules, their levels affected by many factors ranging from UV exposure to insect infestation. There are also varietal and seasonal factors. In this study, juice processing and storage parameters were investigated. Prolonged fruit storage prior to processing and most steps involved in juice processing had little influence on the levels of 6',7'-dihydroxybergamottin (DHB), paradisin C, or bergamottin. However, products that were hot filled or stored at room temperature had lower amounts of DHB and paradisin C and higher amounts of bergaptol compared to juices that were not hot filled and stored at refrigerated temperatures. Both DHB and paradisin C are potent CYP3A4 inhibitors, while bergaptol is a very weak inhibitor. Bergamottin amounts decreased to a lesser extent. Therefore, grapefruit juice products that were hot filled or have been stored at room temperature for an extended period of time will have a reduced drug interaction potential.

New furanocoumarins detected from grapefruit juice retentate.

Grapefruit (Citrus paradisi Macf.) furanocoumarins and related compounds have been shown to interact with the enterocyte cytochrome P450, CYP3A4, and as a result they affect the bioavailibility of certain drugs. Only a few grapefruit furanocoumarins have been identified so far. In this study, grapefruit juice retentate, rich in furanocoumarins, was extracted and then separated by flash chromatography for the examination of new compounds. Finally, nine new furanocoumarins were detected in different fractions according to their UV spectra and mass spectrometric properties by LC-MS (liquid chromatography mass spectrometry) and tentatively designated as FC 338, FC 420, FC 524, FC 530, FC 540, FC 546, FC 552, FC 570 and FC 614.