Gastrointestinal absorption and metabolism of hesperetin-7-O-rutinoside and hesperetin-7-O-glucoside in healthy humans.

SCOPE: Hesperetin-7-O-rutinoside (hesperidin) reduces blood pressure in healthy volunteers but its intestinal absorption and metabolism are not fully understood. Therefore, we aimed to determine sites of absorption and metabolism of dietary flavanone glycosides in humans. METHODS AND RESULTS: Using a single-blind, randomized crossover design, we perfused equimolar amounts of hesperetin-7-O-rutinoside and hesperetin-7-O-glucoside directly into the proximal jejunum of healthy volunteers. We assessed the appearance of metabolites in the perfusate, blood and urine, to determine the sites of metabolism and excretion, and compared this to oral administration. The glucoside was rapidly hydrolyzed by brush border enzymes without any contribution from pancreatic, stomach, or other secreted enzymes, or from bacterial enzymes. Only ∼3% of the dose was recovered intact in the perfusate, indicating high absorption. A proportion was effluxed directly back into the perfused segment mainly in the form of hesperetin-3'-O-sulfate. In contrast, very little hydrolysis or absorption of hesperetin-7-O-rutinoside was observed with ∼80% recovered in the perfusate, no hesperetin metabolites were detected in blood and only traces were excreted in urine. CONCLUSION: The data elucidate the pathways of metabolism of dietary hesperidin in vivo and will facilitate better design of mechanistic studies both in vivo and in vitro.

Consumption of both low and high (-)-epicatechin apple puree attenuates platelet reactivity and increases plasma concentrations of nitric oxide metabolites: a randomized controlled trial.

We hypothesised that consumption of flavanol-containing apple puree would modulate platelet activity and increase nitric oxide metabolite status, and that high flavanol apple puree would exert a greater effect than low flavanol apple puree. 25 subjects consumed 230 g of apple puree containing 25 and 100mg epicatechin (low and high flavanol apple puree, respectively) and aspirin (75 mg) in random order. Measurements were made at baseline, acutely after treatment (2, 6 and 24 h), and after 14 d of treatment. Low flavanol apple puree significantly attenuated ADP and epinephrine-induced integrin-ß3 expression 2 h and 6 h after consumption and ADP and epinephrine-induced P-selectin expression within 2h of consumption. High flavanol apple puree attenuated epinephrine and ADP-induced integrin-ß3 expression after 2 and 6h. ADP and epinephrine-induced integrin-ß3 expression was significantly attenuated 2, 6 and 24 h after consumption of aspirin, whilst 14 d aspirin consumption attenuated collagen-induced P-selectin expression only. The plasma total nitric oxide metabolite conc. was significantly increased 6h after consumption of both low and high flavanol apple purees. In conclusion, consumption of apple purees containing ⩾25 or 100 mg flavanols transiently attenuated ex vivo integrin-ß3 and P-selectin expression and increased plasma nitric oxide metabolite conc. in healthy subjects, but the effect was not enhanced for the high flavanol apple puree.

Intestinal absorption, metabolism, and excretion of (-)-epicatechin in healthy humans assessed by using an intestinal perfusion technique.

BACKGROUND: (-)-Epicatechin is a dietary flavonoid present in many foods that affects vascular function, but its action is limited by incomplete absorption, conjugation, and metabolism. Factors that influence this activity may be attributed to instability in the gastrointestinal lumen, low permeability across the intestinal wall, or active efflux from enterocytes and extensive conjugation. OBJECTIVE: With the use of a multilumen perfusion catheter, we investigated the jejunal absorption, systemic availability, metabolism, and intestinal, biliary, and urinary excretion of (-)-epicatechin in humans. DESIGN: In a single-center, randomized, open, controlled study in 8 healthy volunteers, 50 mg purified (-)-epicatechin was perfused into an isolated jejunal segment together with antipyrine as a marker for absorption. (-)-Epicatechin and conjugates were measured in intestinal perfusates, bile, plasma, and urine. RESULTS: Forty-six percent of the dose was recovered in the perfusate either as unchanged (-)-epicatechin (22 mg) or conjugates (0.8 mg); with stability taken into account, this result indicates that ∼46% of the dose had apparently been absorbed. The conjugates were predominantly sulfates, which indicated conjugation by sulfotransferases followed by efflux from the enterocytes. In contrast, epicatechin glucuronides were dominant in plasma, bile, and urine. CONCLUSIONS: Almost one-half of the (-)-epicatechin is apparently absorbed in the jejunum but with substantial interindividual differences in the extent of absorption. The data suggest that the nature and substitution position of (-)-epicatechin conjugation are major determinants of the metabolic fate in the body, influencing whether the compound is effluxed into the lumen or absorbed into the blood and subsequently excreted.

Bioavailability of bioactive food compounds: a challenging journey to bioefficacy.

Bioavailability is a key step in ensuring bioefficacy of bioactive food compounds or oral drugs. Bioavailability is a complex process involving several different stages: liberation, absorption, distribution, metabolism and elimination phases (LADME). Bioactive food compounds, whether derived from various plant or animal sources, need to be bioavailable in order to exert any beneficial effects. Through a better understanding of the digestive fate of bioactive food compounds we can impact the promotion of health and improvement of performance. Many varying factors affect bioavailability, such as bioaccessibility, food matrix effect, transporters, molecular structures and metabolizing enzymes. Bioefficacy may be improved through enhanced bioavailability. Therefore, several technologies have been developed to improve the bioavailability of xenobiotics, including structural modifications, nanotechnology and colloidal systems. Due to the complex nature of food bioactive compounds and also to the different mechanisms of absorption of hydrophilic and lipophilic bioactive compounds, unravelling the bioavailability of food constituents is challenging. Among the food sources discussed during this review, coffee, tea, citrus fruit and fish oil were included as sources of food bioactive compounds (e.g. (poly)phenols and polyunsaturated fatty acids (PUFAs)) since they are examples of important ingredients for the food industry. Although there are many studies reporting on bioavailability and bioefficacy of these bioactive food components, understanding their interactions, metabolism and mechanism of action still requires extensive work. This review focuses on some of the major factors affecting the bioavailability of the aforementioned bioactive food compounds.

Epicatechin B-ring conjugates: first enantioselective synthesis and evidence for their occurrence in human biological fluids.

Herein, the first enantioselective total synthesis of a number of biologically relevant (-)-epicatechin conjugates is described. The success of this synthesis relied on (i) optimized conditions for the stereospecific cyclization step leading to the catechin C ring; on (ii) efficient conjugation reactions; and on (iii) optimized deprotection sequences. These standard compounds have been subsequently used to elucidate for the first time the pattern of (-)-epicatechin conjugates present in four different human biological fluids following (-)-epicatechin absorption.

UPLC-MS/MS quantification of total hesperetin and hesperetin enantiomers in biological matrices.

Hesperidin (hesperetin-7-O-rutinoside), a flavonoid affecting vascular function, is abundant in citrus fruits and derived products such as juices. After oral administration, hesperidin is hydrolyzed by the colonic microbiota producing hesperetin-7-O-glucoside, the glucoside group is further cleaved and the resulting hesperetin is absorbed and metabolized. Flavanones have a chiral carbon generating (R)- and (S)-enantiomers, with potentially different biological activities. A rapid UPLC-MS/MS method for the analysis of (R)- and (S)-hesperetin enantiomers in human plasma and urine was developed and validated. Biological matrices were incubated with ß-glucuronidase/sulfatase, and hesperetin was isolated by solid-phase extraction using 96-well plate mixed-mode cartridges having reversed-phase and anion-exchange functionalities. Racemic hesperetin was analyzed with a UPLC HSS T3 reversed phase column and hesperetin enantiomers with a HPLC Chiralpak IA-3 column using H(2)O with 0.1% CHOOH as solvent A and acetonitrile with 0.1% CHOOH as solvent B. The method was linear between 50 and 5000nM for racemic hesperetin in plasma and between 25 and 2500nM for (S)- and (R)-hesperetin in plasma. Linearity was achieved between 100 and 10,000nM for racemic hesperetin in urine and between 50 and 5000nM for (S)- and (R)-hesperetin in urine. Values of repeatability and intermediate reproducibility for racemic hesperetin and enantiomers in plasma and urine were below 15% of deviation in general, and maximum 20% for the lowest concentrations. In addition, the method was applied for the quantification of total hesperetin and of hesperetin enantiomers in human plasma and urine samples, obtained after oral ingestion of purified hesperetin-7-O-glucoside. In conclusion, the developed and validated method was sensitive, accurate and precise for the quantification of enantiomers of hesperetin in biological fluids.

Stereoselective conjugation, transport and bioactivity of s- and R-hesperetin enantiomers in vitro.

The flavanone hesperetin ((+/-)-4'-methoxy-3',5,7-trihydroxyflavanone) is the aglycone of hesperidin, which is the major flavonoid present in sweet oranges. Hesperetin contains a chiral C-atom and so can exist as an S- and R-enantiomer, however, in nature 2S-hesperidin and its S-hesperetin aglycone are predominant. The present study reports a chiral HPLC method to separate S- and R-hesperetin on an analytical and semipreparative scale. This allowed characterization of the stereoselective differences in metabolism and transport in the intestine and activity in a selected bioassay of the separated hesperetin enantiomers in in vitro model systems: (1) with human small intestinal fractions containing UDP-glucuronosyl transferases (UGTs) or sulfotransferases (SULTs); (2) with Caco-2 cell monolayers as a model for the intestinal transport barrier; (3) with mouse Hepa-1c1c7 cells transfected with human EpRE-controlled luciferase to test induction of EpRE-mediated gene expression. The results obtained indicate some significant differences in the metabolism and transport characteristics and bioactivity between S- and R-hesperetin, however, these differences are relatively small. This indicates that for these end points, including intestinal metabolism and transport and EpRE-mediated gene induction, experiments performed with racemic hesperetin may adequately reflect what can be expected for the naturally occurring S-enantiomer. This is an important finding since at present hesperetin is only commercially available as a racemic mixture, while it exists in nature mainly as an S-enantiomer.

Metabolism and transport of the citrus flavonoid hesperetin in Caco-2 cell monolayers.

Metabolism and transport from intestinal cells back into the lumen by ATP-binding cassette (ABC) transporters is believed to limit the bioavailability of flavonoids. We studied metabolism and transport of the citrus flavonoid hesperetin, the aglycone of hesperidin, using a two-compartment transwell Caco-2 cell monolayer system, simulating the intestinal barrier. The role of apically located ABC transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance protein 2 (ABCC2), and breast cancer resistance protein (BCRP/ ABCG2) in the efflux of hesperetin and its metabolites was studied by coadministration of compounds known to inhibit several classes of ABC transporters, including cyclosporin A, GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide], Ko143 [3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester], MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), and PSC-833 (Valspodar). Apically applied hesperetin (10 microM) was metabolized into hesperetin 7-O-glucuronide and hesperetin 7-O-sulfate, identified using high-performance liquid chromatographydiode array detector (DAD), ultraperformance liquid chromatography-DAD-tandem mass spectrometry, and authentic standards, which were transported predominantly to the apical side of the Caco-2 cell monolayer (1.12 cm(2)), at average (S.D.) rates of 14.3 (3.7) and 2.1 (0.8) pmol/min/monolayer, respectively. Hesperetin aglycone also permeated to the basolateral side, and this process was unaffected by the inhibitors used, possibly implying a passive diffusion process. Inhibition studies, however, showed that efflux of hesperetin conjugates to the apical side involved active transport, which from the pattern of inhibition appeared to involve mainly BCRP. Upon inhibition by the BCRP inhibitor Ko143 (5 micro M), the apical efflux of hesperetin conjugates was 1.9-fold reduced (p

Stability and enhancement of berry juice color.

Attractive color is one of the main sensory characteristics of fruit and berry products. Unfortunately, the color of red juices is unstable and easily susceptible to degradation, leading to a dull and weak juice color. This study was designed to investigate the color stability and copigmentation of four different berry juices enhanced by phenolic acids and commercial color enhancers. Phenolic acid enrichment improved and stabilized the color of the berry juices during storage. The commercial color enhancers immediately produced an intensive color to the juices, which, however, was not very stable. The color enhancement was intensive in strawberry and raspberry juices and effective in lingonberry and cranberry juices. Sinapic acid induced the strongest color in strawberry juice. Ferulic and sinapic acids improved raspberry juice color equally. Rosmarinic acid enhanced the color of lingonberry and cranberry juices the most. The addition of the simple cinnamic acids produced novel peaks to the end of the high-performance liquid chromatography chromatogram, indicating a formation of new compounds. It can be assumed that sinapic and ferulic acids formed new intramolecular copigmentation compounds with berry anthocyanins whereas rosmarinic acid stabilized anthocyanins intermolecularly.