pubs.acs.org/JAFC Published on Web 05/04/2010 © 2010 American Chemical Society 6516 J. Agric. Food Chem. 2010, 58, 6516–6524 DOI:10.1021/jf100752j Concentration and Solubility of Flavanones in Orange Beverages Affect Their Bioavailability in Humans FERNANDO VALLEJO, † MAR LARROSA, † ELISA ESCUDERO, ‡ MARI ´ A P. ZAFRILLA, § BEGON ˜ A CERDA ´ , § JULIO BOZA, # MARI ´ A TERESA GARCI ´ A-CONESA, † JUAN CARLOS ESPI ´ N, † AND FRANCISCO A. TOMA ´ S-BARBERA ´ N* ,† † Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, 30100 Campus de Espinardo, Murcia, Spain, ‡ Department of Pharmacology, Faculty of Veterinary, University of Murcia, 3071 Campus de Espinardo, Murcia, Spain, # Coca Cola Services n.v., B-1070 Brussels, Belgium, and § Department of Clinic Nutrition, UCAM, 30107 Campus Los Jero´ nimos, Guadalupe, Murcia, Spain Orange juice is a very rich source of dietary flavanones. The effect of flavanone concentration and solubility of orange beverages on their bioavailability has been studied in a crossover study with 10 healthy volunteers. Five different beverages with different flavanone concentrations were evaluated. Commercial orange juices (29.2-70.3 mg of flavanones/100 mL) were compared with experimental orange beverages in which the flavanone concentration was enhanced (110.2 mg/100 mL). Hesperetin and naringenin glucuronides and sulfates were detected and quantified in plasma and urine. The study shows that the solubility of the flavanones, and particularly that of hesperidin, in the juice is a key factor for the bio- availability as flavanone excretion and the C max in plasma correlate well with the soluble flavanone concentration in the juice, whereas it has no correlation with the total flavanone intake. In addition, a large interindividual variation was observed, this being consistent for each individual after the intake of the different beverages, suggesting that flavanone bioavailability is also dependent on the occurrence of specific microbiota that is able to remove the rutinosides from the juice glycosides, which results in aglycones that are then absorbed from the gut. KEYWORDS: Citrus; flavonoids; metabolism; absorption; extracts; juice enrichment INTRODUCTION Fruits of the genus Citrus such as oranges as well as their deri- ved products (e.g., beverages, nutraceuticals) are rich in flava- nones and are a major source of these flavonoids for humans ( 1 ). Although it is difficult to determine total dietary consumption of these compounds, the daily intake of flavanones has been esti- mated to range between 25 and 65 mg on the basis of the consum- ption of a serving of orange juice ( 1 , 2 ). These figures may be increased by additional consumption of fresh peeled oranges with surrounding white tissues (albedo) ( 1 ). Numerous animal experiments and in vitro studies have shown that Citrus flavanones exhibit a wide range of biological activities, such as antioxidant, hypocholesterolemic, hypoglycemic, preven- tion of bone loss, or antitumor activities, which indicate that these compounds may exert beneficial effects against cardiovascular diseases, osteoporosis, or cancer ( 3 ). Significantly, much of the bioactivity of Citrus flavanones appears to affect blood, micro- vascular endothelial cells, and inflammation. Hesperidin, narin- gin, and naringenin have been reported to exert noticeable in vivo anti-inflammatory systemic effects in various animal models ( 4 -6 ). Recent research indicates additional potential bioactive roles of the flavanones. Thus, naringenin has been shown to protect against lung fibrosis ( 7 ), hepatotoxicity ( 8 ), and oxida- tive renal dysfunction ( 9 ), whereas naringin can prevent alterations in mitochondrial lipid peroxides ( 10 ) and reduce genotoxicity ( 11 ). The mechanisms underlying all of these activities have not yet been elucidated, but it has been reported that some flavanones and flavanone-enriched orange extracts do exert gene expression modulatory effects with specific anti-inflammatory and antifibro- tic effects ( 12 ). Despite the accumulated experimental evidence, human clinical trials are still scarce and inconclusive, with volun- teers consuming several servings of orange juice or levels of flavanones, resulting in contradictory changes in plasma levels of cholesterol or triglycerides ( 3 ). Many of the studies looking at the bioactivity of flavanones have been conducted using either the glycosides (naringin, hes- peridin, neohesperidin), which are the forms most commonly found in the Citrus fruits ( 1 ), or the hydrolyzed corresponding aglycones (naringenin, hesperetin). For these compounds to exert their beneficial effects through the diet, it is essential that they are bioavailable and can reach their target organs in a bioactive form. Two essential factors influence flavanones’ bioavailability: (i) the physical form in which they are ingested (e.g., juice, soluble extract, capsule); (ii) the transformations suffered during gastro- intestinal digestion and metabolism (phase I, phase II, and micro- biota metabolism). The flavanones are found in the orange fruit either solubilized within vesicles or in a solid form as part of the *Corresponding author (e-mail fatomas@cebas.csic.es; telephone þ34-968-396334; fax þ34-968-396213).