Chronic consumption of orange juice modifies urinary excretion of flavanone gut-derived metabolites through gut microbiota modulation.

The metabolism and absorption of citrus flavanones are intrinsically linked to the gut microbiota, creating a bidirectional relationship where these compounds influence the microbiome, and in turn, the microbiota affects their metabolism. This study evaluates the effect of acute and chronic consumption of orange juice (OJ) on the urinary excretion of gut-derived flavanone metabolites and the gut microbiota. Health volunteers ingested 500 mL of OJ for 60 days in a single-arm human intervention study. Blood and feces were collected at baseline and after 60 days, with an additional 24-hour urine collection after a single dose on day 1 and day 63. LC-MS/MS analyzed urinary flavanone metabolites, while 16S rRNA sequencing characterized gut microbiota. Total urinary hesperetin conjugates excretion significantly decreased over 60 days, while gut-derived total phenolic acids, particularly three hydroxybenzoic acids, increased. Moreover, the heterogeneity of the total amount of flavanone conjugates, initially categorizing individuals into high-, medium- and low- urinary excretor profiles, shifted towards medium-excretor, except for five individuals who remained as low-excretors. This alteration was accompanied by a decrease in intestinal ß-glucosidase activity and a shift in the relative abundance of specific genera, such as decreases in Blautia, Eubacterium hallii, Anaerostipes, and Fusicatenibacter, among which, Blautia was associated with higher urinary flavanone conjugates excretion. Conversely, an increase in Prevotella was observed. In summary, chronic OJ consumption induced transient changes in gut microbiota and altered the metabolism of citrus flavanones, leading to distinct urinary excretion profiles of flavanone metabolites.

Effects of Hesperidin Consumption on the Cardiovascular System in Pre- and Stage 1 Hypertensive Subjects: Targeted and Non-Targeted Metabolomic Approaches (CITRUS Study).

SCOPE: The aim of the present work is to determine new biomarkers of the biological effects of hesperidin in orange juice (OJ) applying a non-targeted metabolomics approach validated by targeted metabolomics analyses of compliance biomarkers. METHODS AND RESULTS: Plasma/serum and urine targeted (HPLC-MS/MS) and untargeted (1 H-NMR) metabolomics signatures are explored in a subsample with pre- and stage-1 hypertension subjects of the CITRUS study (N = 159). Volunteers received 500 mL day-1 of control drink, OJ, or hesperidin-enriched OJ (EOJ) for 12-weeks. A 6-h postprandrial study is performed at baseline. Targeted analyses reveals plasma and urine hesperetin 7-O-ß-d-glucuronide as the only metabolite differing between OJ and EOJ groups after 12-weeks consumption, and in urine is correlated with a decreased systolic blood pressure level. The non-targeted approach shows that after single dose and 12-weeks consumption of OJ and EOJ change several metabolites related with an anti-inflammatory and antioxidant actions, lower blood pressure levels and uremic toxins. CONCLUSIONS: Hesperetin 7-O-ß-d-glucuronide can be a candidate marker for distinguishing between the consumption of different hesperidin doses at 12-weeks consumption as well as a potential agent mediating blood pressure reduction. Moreover, changes in different endogenous metabolites can explain the mechanisms of action and the biological effects of hesperidin consumption.

Dissection of the potential pharmacological function of neohesperidin dihydrochalcone - a food additive - by in vivo substances profiling and network pharmacology.

Food additives are widely used in our daily life, and the side-effects caused by them have gained extensive attention around the world. Notably, constituent-oriented metabolites, in some sense, always contribute to pharmacological changes, inducing toxicity, therapeutic effects, etc. Characterization of the metabolites and their potential functions is of great importance to the practical applications. In this work, an integrated strategy by combining metabolite profiling and network pharmacology was applied to characterize the metabolic features and reveal pharmacological changes of neohesperidin dihydrochalcone (NHDC) in vivo to demonstrate its pharmacological mechanism and potential functions. As a result, a total of 19 metabolites (3 in plasma, 19 in urine, 8 in feces, 3 in heart, 5 in liver, 0 in spleen, 1 in lung, 2 in kidneys and 2 in brain) were screened and 18 of them were characterized for the first time. Phase I metabolic reactions of hydrolysis and phase II reactions of glucuronidation, sulfation, glutamylation, N-butyryl glycylation and lactylation were the main metabolic reactions of NHDC in vivo. Moreover, the results analyzed by network pharmacology revealed that, in addition to common pathways (steroid hormone biosynthesis) of NHDC, metabolites' targets were involved in pathways in cancer, ovarian steroidogenesis, proteoglycans in cancer, PI3K-Akt signaling pathway and progesterone-mediated oocyte maturation, indicating that these functional changes might result in potential novel functions or other side-effects, such as a disorder of steroid hormones. Our work provided the metabolic features and functional modifications of NHDC in vivo for the first time, and meaningful information for further pharmacological validations or potential functions is supplied.

Urinary Biomarkers for Orange Juice Consumption.

SCOPE: As orange juice belongs to one of the most consumed juices worldwide, a human study is performed to identify urinary biomarkers for the consumption of orange juice in order to differentiate between low, medium, and high intake. METHODS AND RESULTS: The 32 study participants abstained from citrus fruits, juices and products thereof, except for one portion of orange juice, for eight days. Throughout the study, spot urine samples are collected and quantitatively analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) regarding their content of several potential biomarkers for orange juice intake after enzymatic treatment with ß-glucuronidase. Proline betaine is determined as a long-term biomarker: based on its urinary excretion, orange juice consumption is traceable for at least 72 h after intake. Naringenin and hesperetin are identified as qualitative short-term biomarkers. Synephrine sulfate also showed a fast increase and decrease in a semi-quantitative approach. In the case of phloretin, no correlation between orange juice consumption and the urinary concentration is observed. CONCLUSION: Proline betaine is the most promising biomarker for orange juice consumption and allows to differentiate between low, medium, and high intake. Hesperetin and naringenin (as well as synephrine) are applicable as supporting biomarkers, whereas phloretin does not represent a reliable biomarker for orange juice consumption.

Urinary flavanone concentrations as biomarkers of dietary flavanone intakes in the European Prospective Investigation into Cancer and Nutrition (EPIC) study.

In the present study, the aim was to investigate the correlation between the acute and habitual dietary intake of flavanones, their main food sources and the concentrations of aglycones naringenin and hesperetin in 24 h urine in a European population. A 24-h dietary recall (24-HDR) and a 24-h urine sample were collected the same day from a subsample of 475 people from four different countries of the European Prospective Investigation into Cancer and Nutrition study. Acute and habitual dietary data were captured through a standardised 24-HDR and a country/centre-specific validated dietary questionnaire (DQ). The intake of dietary flavanones was estimated using the Phenol-Explorer database. Urinary flavanones (naringenin and hesperetin) were analysed using tandem MS with a previous enzymatic hydrolysis. Weak partial correlation coefficients were found between urinary flavanone concentrations and both acute and habitual dietary flavanone intakes (Rpartial = 0·14-0·17). Partial correlations were stronger between urinary excretions and acute intakes of citrus fruit and juices (Rpartial ∼ 0·6) than with habitual intakes of citrus fruit and juices (Rpartial ∼ 0·24). In conclusion, according to our results, urinary excretion of flavanones can be considered a good biomarker of acute citrus intake. However, low associations between habitual flavanone intake and urinary excretion suggest a possible inaccurate estimation of their intake or a too sporadic intake. For assessing habitual exposures, multiple urinary collections may be needed. These results show that none of the approaches tested is ideal, and the use of both DQ and biomarkers can be recommended.

A Critical Evaluation of In Vitro Hesperidin 2S Bioavailability in a Model Combining Luminal (Microbial) Digestion and Caco-2 Cell Absorption in Comparison to a Randomized Controlled Human Trial.

SCOPE: Bioavailability strongly determines polyphenol bioactivity, and is strongly influenced by food matrix, enzymatic and microbial degradation, and gastrointestinal absorption. To avoid human trials for pre-screening of polyphenol bioavailability, studies have focused on in vitro model development. Nevertheless, their predictive value for bioavailability can be questioned. METHOD AND RESULTS: We used the orange flavonoid hesperidin 2S to validate a model combining digestion in the simulator of the human intestinal microbial ecosystem (SHIME) and Caco-2 cell transport, with a human intervention study. In vitro, hesperidin was resistant to degradation in the stomach and small intestine, but was rapidly deconjugated on reaching the proximal colon. Extensive and colon-region-specific degradation to smaller phenolics was observed. Hydrocaffeic and dihydroisoferulic acid accumulated in proximal, and hydroferulic acid in distal colon. Caco-2 transport was the highest for dihydroisoferulic acid. In humans, plasma and urine hesperetin-glucuronide levels increased significantly, whereas the impact on small phenolics was limited. CONCLUSIONS: In the combined in vitro model, smaller phenolics strongly accumulated, whereas in humans, hesperetin conjugates were the main bioavailable compounds. Future in vitro model development should focus on simulating faster polyphenol absorption and elimination of smaller phenolics to improve their predictive value of in vivo polyphenol bioavailability.

Urinary metabolite profiling of flavonoids in Chinese volunteers after consumption of orange juice by UFLC-Q-TOF-MS/MS.

The metabolism of flavonoids derived from orange juice in Chinese volunteers has not been well investigated. With the ultra-fast liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UFLC-Q-TOF-MS/MS) system, orange juice-derived flavonoids, as well as metabolites contained in urine collected from healthy Chinese volunteers after consumption of 250mL orange juice, were systematically identified and quantified. Finally, a total of 9 flavonoids and 30 metabolites were detected. Obtained results revealed that flavonoids derived from orange juice underwent extensive phase II metabolism in human, mainly comprising glucuronidation and sulfation. The overall recovery of the primary flavonoid aglycones, i.e., naringenin and hesperetin, were both approximately equivalent 22% of intake, primarily occurred in 4-12h post consumption. Meanwhile, additional 35 phenolic catabolites were identified in urine collected post consumption. However, it is difficult to determine the exact amounts of phenolic catabolites derived from specific flavonoid due to the interference of diets and other flavonoids. This work would be valuable for the clarification of metabolic profiles for flavonoids in Chinese population.

Urinary excretion of Citrus flavanones and their major catabolites after consumption of fresh oranges and pasteurized orange juice: A randomized cross-over study.

SCOPE: Orange juice contains flavanones including hesperidin and narirutin, albeit at lower concentrations as compared to orange fruit. Therefore, we compared bioavailability and colonic catabolism of flavanones from orange juice to a 2.4-fold higher dose from fresh oranges. METHODS AND RESULTS: Following a randomized two-way cross-over design, 12 healthy subjects consumed a test meal comprising either fresh oranges or pasteurized orange juice, delivering 1774 and 751 µmol of total Citrus flavanones, respectively. Deglucuronidated and desulfated hesperetin, naringenin, and the flavanone catabolites 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid, 3-(3'-hydroxyphenyl)hydracrylic acid, 4-hydroxyhippuric acid, and hippuric acid were quantitated in 24-h urine by UHPLC-MS/MS. Differences in urinary hesperetin excretion were found to be nonsignificant (p = 0.5209) both after consumption of orange fruit (21.6 ± 8.0 µmol) and juice (18.3 ± 7.2 µmol). By analogy, postprandial flavanone catabolite excretions were highly similar between treatments. Excretion of 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid was inversely related to that of hesperetin, illustrating the catabolite/precursor relationship. CONCLUSION: Despite 2.4-fold higher doses, excretion of flavanones from ingested fresh orange fruit did not differ from that following orange juice consumption, possibly due to a saturation of absorption or their entrapment in the fiber-rich matrix of the fruit.

The novel voltammetric method for determination of hesperetin based on a sensitive electrochemical sensor.

A highly sensitive voltammetric sensor, based on reduced graphene oxide on SWCNTs modified glassy carbon electrode (GCE), was constructed and used for sensitive detection of hesperetin. The electrochemical behavior of hesperetin at this sensor was investigated systematically and a novel voltammetric method for determination of hesperetin was proposed. The redox characters of hesperetin was discussed in detail and a reasonable reaction mechanism was proposed also. As the analytical method, the response currents were linear relationship with the hesperetin concentrations in the range of 5.0 × 10(-8) to 3.0 × 10(-6) mol L-(1), with a detection limit of 2.0 × 10(-8) mol L(-1) (S/N=3). The method was also applied successfully to detect hesperetin in biological samples and Chinese herbal medicine Flos buddlejae with satisfactory results.

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.

Urinary Excretion of Select Dietary Polyphenol Metabolites Is Associated with a Lower Risk of Type 2 Diabetes in Proximate but Not Remote Follow-Up in a Prospective Investigation in 2 Cohorts of US Women.

BACKGROUND: Polyphenols are phytochemicals that possess antioxidant and anti-inflammatory properties and improve glucose metabolism in animal experiments, although data from prospective epidemiologic studies examining polyphenol intakes in relation to type 2 diabetes (T2D) risk are inconsistent. OBJECTIVES: We examined urinary excretion of select flavonoid and phenolic acid metabolites, as biomarkers of intake, in relation to T2D risk. METHODS: Eight polyphenol metabolites (naringenin, hesperetin, quercetin, isorhamnetin, catechin, epicatechin, caffeic acid, and ferulic acid) were quantified in spot urine samples by liquid chromatography/mass spectrometry among 1111 T2D case-control pairs selected from the Nurses' Health Study (NHS) and NHSII. RESULTS: Higher urinary excretion of hesperetin was associated with a lower T2D risk after multivariate adjustment: the OR comparing top vs. bottom quartiles was 0.68 (95% CI: 0.49, 0.96), although a linear trend was lacking (P = 0.30). The other measured polyphenols were not significantly associated with T2D risk after multivariate adjustment. However, during the early follow-up period [≤ 4.6 y (median) since urine sample collection], markers of flavanone intakes (naringenin and hesperetin) and flavonol intakes (quercetin and isorhamnetin) were significantly associated with a lower T2D risk. The ORs (95% CIs) comparing extreme quartiles were 0.61 (0.39, 0.98; P-trend: 0.03) for total flavanones and 0.55 (0.33, 0.92; P-trend: 0.04) for total flavonols (P-interaction with follow-up length: ≤ 0.04). An inverse association was also observed for caffeic acid during early follow-up only: the OR was 0.52 (95% CI: 0.32, 0.84; P-trend: 0.03). None of these markers was associated with T2D risk during later follow-up. Metabolites of flavan-3-ols and ferulic acid were not associated with T2D risk in either period. CONCLUSIONS: These results suggest that specific flavonoid subclasses, including flavanones and flavonols, as well as caffeic acid, are associated with a lower T2D risk in relatively short-term follow-up but not during longer follow-up. Substantial within-person variability of the metabolites in single spot urine samples may limit the ability to capture associations with long-term disease risk.

Orange juice (poly)phenols are highly bioavailable in humans.

BACKGROUND: We assessed the bioavailability of orange juice (poly)phenols by monitoring urinary flavanone metabolites and ring fission catabolites produced by the action of the colonic microbiota. OBJECTIVE: Our objective was to identify and quantify metabolites and catabolites excreted in urine 0-24 h after the acute ingestion of a (poly)phenol-rich orange juice by 12 volunteers. DESIGN: Twelve volunteers [6 men and 6 women; body mass index (in kg/m(2)): 23.9-37.2] consumed a low (poly)phenol diet for 2 d before first drinking 250 mL pulp-enriched orange juice, which contained 584 µmol (poly)phenols of which 537 µmol were flavanones, and after a 2-wk washout, the procedure was repeated, and a placebo drink was consumed. Urine collected for a 24-h period was analyzed qualitatively and quantitatively by using high-performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS). RESULTS: A total of 14 metabolites were identified and quantified in urine by using HPLC-MS after orange juice intake. Hesperetin-O-glucuronides, naringenin-O-glucuronides, and hesperetin-3'-O-sulfate were the main metabolites. The overall urinary excretion of flavanone metabolites corresponded to 16% of the intake of 584 µmol (poly)phenols. The GC-MS analysis revealed that 8 urinary catabolites were also excreted in significantly higher quantities after orange juice consumption. These catabolites were 3-(3'-methoxy-4'-hydroxyphenyl)propionic acid, 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid, 3-(3'-hydroxy-4'-methoxyphenyl)hydracrylic acid, 3-(3'-hydroxyphenyl)hydracrylic acid, 3'-methoxy-4'-hydroxyphenylacetic acid, hippuric acid, 3'-hydroxyhippuric acid, and 4'-hydroxyhippuric acid. These aromatic acids originated from the colonic microbiota-mediated breakdown of orange juice (poly)phenols and were excreted in amounts equivalent to 88% of (poly)phenol intake. When combined with the 16% excretion of metabolites, this percentage raised the overall urinary excretion to ∼ 100% of intake. CONCLUSIONS: When colon-derived phenolic catabolites are included with flavanone glucuronide and sulfate metabolites, orange juice (poly)phenols are much-more bioavailable than previously envisaged. In vitro and ex vivo studies on mechanisms underlying the potential protective effects of orange juice consumption should use in vivo metabolites and catabolites detected in this investigation at physiologic concentrations. The trial was registered at BioMed Central Ltd (www.controlledtrials.com) as ISRCTN04271658.

Simplified miniaturized ultrasound-assisted matrix solid phase dispersion extraction and high performance liquid chromatographic determination of seven flavonoids in citrus fruit juice and human fluid samples: hesperetin and naringenin as biomarkers.

In the present study, for the first time, a simplified miniaturized ultrasound-assisted matrix solid-phase dispersion (SM-USA-MSPD) method with a different application for liquid matrices was developed to extract different flavonoids (hesperidin, diosmin, eriocitrin, narirutin, naringin, hesperetin and naringenin) from citrus fruit juice and human fluid samples prior to their determination using high performance liquid chromatography (HPLC). Different effective parameters were studied and under the optimum conditions (including sample volume: 150µL; solid phase: silica-based C18, 200mg; eluting solvent: methanol, 500µL; pH: 4; and sonication: 6min; at room temperature), limits of detection and limits of quantification were ranged from 23.3 to 46.8ngmL(-1) and 74.8 to 141.5ngmL(-1), respectively. Once optimized, analytical performance of the method was studied in terms of linearity (0.074-198.5µgmL(-1), r(2)>0.991), accuracy (recovery=84.6-101.5%), and precision (repeatability: intra-day precision<5.9%, and inter-day precision<7.2%). At the end, SM-USA-MSPD method was successfully applied to estimate the levels of hesperetin and naringenin in plasma and urinary excretion -after ingestion of orange, grapefruit and lime juices- and the obtained results confirmed that these compounds could be used as good biomarkers of citrus fruit juice intake.

Bioavailability of dietary (poly)phenols: a study with ileostomists to discriminate between absorption in small and large intestine.

A feeding study was carried out in which six healthy ileostomists ingested a juice drink containing a diversity of dietary (poly)phenols derived from green tea, apples, grapes and citrus fruit. Ileal fluid and urine collected at intervals over the ensuing 24 h period were then analysed by HPLC-MS. Urinary excretions were compared with results obtained in an earlier study in which the juice drink was ingested by ten healthy control subjects with an intact colon. Some polyphenol components, such as (epi)catechins and (epi)gallocatechin(s), were excreted in urine in similar amounts in ileostomists and subjects with an intact colon, demonstrating that absorption took place principally in the small intestine. In the urine of ileostomists, there were reduced levels of other constituents, including hesperetin-7-O-rutinoside, 5-O-caffeoylquinic acid and dihydrochalcones, indicating their absorption in both the small and large intestine. Ileal fluid analysis revealed that even when absorption occurred in the small intestine, in subjects with a functioning colon a substantial proportion of the ingested components still pass from the small into the large intestine, where they may be either absorbed before or after catabolism by colonic bacteria.

Concentration and solubility of flavanones in orange beverages affect their bioavailability in humans.

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 bioavailability 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.

Relative bioavailability of the flavonoids quercetin, hesperetin and naringenin given simultaneously through diet.

The bioavailability and urinary excretion of three dietary flavonoids, quercetin, hesperetin and naringenin, were investigated. Ten healthy men were asked to consume a 'juice mix' containing equal amounts of the three flavonoids, and their urine and plasma samples were collected. The resulting mean plasma area under the curve (AUC)(0-48 h) and C(max) values for quercetin and hesperetin were similar, whereas the AUC(0-48 h) of naringenin and, thus, the relative bioavailability were higher after consumption of the same dose. The study consolidates a significantly lower urinary excretion of quercetin (1.5+/-1%) compared with hesperetin (14.2+/-9.1%) and naringenin (22.6+/-11.5%) and shows that this is not due to a lower bioavailability of quercetin, but rather reflects different clearance mechanisms.

Analysis of hesperetin enantiomers in human urine after ingestion of blood orange juice by using nano-liquid chromatography.

Hesperetin (HT) is a flavanone abundantly found in citrus fruits. It has been reported that HT possesses significant antioxidant, anticancer, anti-inflammatory and analgesic activities. This explains the necessity of developing new methods more powerful and sensitive for analyzing HT in biological fluids. Taking into account the chiral nature of HT, the study of the stereospecific kinetics of in vitro and in vivo metabolism and tissue distribution could be a useful tool for further understanding stereoselective biotransformations in human body. A simple nano-liquid chromatographic method for the determination of the enantiomeric composition of hesperetin in human urine was developed. Chiral separation was achieved using a 100 microm I.D. capillary, packed with phenyl-carbamate-propyl-beta-cyclodextrin stationary phase, employing a mobile phase composed by a mixture of triethylammonium acetate buffer (1%, v/v, pH 4.5) and water/methanol (30:70, v/v) at room temperature. The detection was done by using on-column UV detector at 205 nm. Calibration curves were linear in the studied concentration range from 0.25 to 25 microg/mL (r(2)>0.999). Precision assay was <4.5% and was within 3% at the limit of quantification (0.5 microg/mL). The recovery of 7-ethoxycoumarin (IS), R- and S-hesperetin was greater than 82.48%, utilizing a liquid-liquid extraction procedure. The developed method was successfully applied to the determination of hesperetin enantiomers in urine samples obtained from a male volunteer, after the ingestion of 1L of a commercial blood orange juice.

Simultaneous determination of the flavonoid aglycones diosmetin and hesperetin in human plasma and urine by a validated GC/MS method: in vivo metabolic reduction of diosmetin to hesperetin.

Diosmetin and hesperetin are the aglycones of the flavonoid glycosides diosmin and hesperidin which occur naturally in citrus fruit. A GC/MS method for the simultaneous determination of diosmetin and hesperetin in human plasma and urine has been developed and validated. The method was linear in the 2-300 ng/mL concentration range for both diosmetin and hesperetin in plasma and urine (r > 0.999). The precision of the method was better than 6.01 and 7.16% for diosmetin and hesperetin, respectively, and the accuracy was 96.76-100.40% and 95.00-105.50% for diosmetin and hesperetin, respectively. The lower limit of quantitation was found to be 2 ng/mL for both analytes in plasma and urine. Recovery of diosmetin, hesperetin and internal standard naringenin was greater than 82.5%. The method has been applied for the determination of diosmetin and hesperetin in plasma and urine samples obtained from a healthy male subject following a single oral 1000 mg dose of the flavonoid glycoside diosmin. The presence of hesperetin in plasma and urine samples indicates the metabolic reduction of diosmetin to its flavanone analogue hesperetin through reduction of the 2,3 double bond of the C-ring by the enzymes of bacteria of the intestinal microflora.

Bioavailability and metabolism of orange juice flavanones in humans: impact of a full-fat yogurt.

The bioavailability of dietary phytochemicals may be influenced by the food matrix in which they are consumed. In this study the impact of a full-fat yogurt on the bioavailability and metabolism of orange juice flavanones was investigated. Human plasma and urine were collected over a 24 h period after the consumption of 250 mL of orange juice containing a total of 168 micromol of hesperetin-7-O-rutinoside and 12 micromol of naringenin-7-O-rutinoside, with and without 150 mL of full-fat yogurt. The juice also contained 1 g of paracetamol and 5 g of lactulose. HPLC-MS(2) analysis revealed the accumulation of hesperetin-7-O-glucuronide, and an unassigned hesperetin-O-glucuronide metabolite in plasma reached a peak concentration (C(max)) of 924 +/- 224 nmol/L, 4.4 +/- 0.5 h (T(max)) after orange juice ingestion. The T(max) is indicative of absorption in the colon. When the juice was consumed with yogurt, neither the C(max) at 661 +/- 170 nmol/L nor the T(max) at 5.1 +/- 0.4 h were significantly different from those obtained with juice alone. The two hesperetin glucuronides were also excreted in urine along with a third hesperetin-O-glucuronide, two hesperetin-O-glucuronide-O-sulfates, a hesperetin-O-diglucuronide, a naringenin-O-diglucuronide, and, tentatively identified, naringenin-7-O-glucuronide and naringenin-4'-O-glucuronide. This indicates the occurrence of substantial, postabsorption, phase II metabolism prior to urinary excretion. The quantity of flavanone metabolites excreted 0-5 h after orange juice ingestion was significantly reduced by yogurt, but over the full 0-24 h urine collection period, the amounts excreted, corresponding to ca. 7.0% of intake, were not affected by the addition of yogurt to the drink. Nor did yogurt have a significant effect on gastric emptying, as determined by plasma paracetamol levels, or on the mouth to cecum transit time of the head of the meal, assessed by measurement of lactulose-derived breath hydrogen. There is also a discussion of the merits of studies of the absorption and metabolism of flavanones based on direct analysis of metabolites by HPLC-MS and the more traditional indirect approach where samples are treated with a mollusc glucuronidase/sulfatase preparation prior to HPLC analysis of the released aglycones.

Pharmacokinetics of the citrus flavanone aglycones hesperetin and naringenin after single oral administration in human subjects.

BACKGROUND AND OBJECTIVE: Hesperetin and naringenin, the aglycones of the flavanone glycosides hesperidin and naringin, occur naturally in citrus fruits. They exert interesting pharmacological properties such as antioxidant, anti-inflammatory, blood lipid and cholesterol lowering and are considered to contribute to health benefits in humans. However, no information is available on the pharmacokinetics of the citrus flavanones hesperetin and naringenin after their oral administration to humans as pure aglycones. Therefore, the objective of the present investigation was the evaluation of the pharmacokinetic parameters of hesperetin and naringenin in plasma and urine, after their single oral administration in humans in the form of solid dispersion capsules, and also to improve the absorption rate of flavanones by using aglycones rather than the naturally occurring glycosides. DESIGN: Six healthy volunteers received orally 135 mg of each compound, hesperetin and naringenin, under fasting conditions. Blood samples were collected at 14 different time points over a 12 h period. Urine was collected over 24 h, in five sequential timed intervals. Plasma and urine hesperetin and naringenin concentrations, after enzymatic hydrolysis of their conjugated forms, were measured using validated high-pressure liquid chromatography methods. Pharmacokinetic parameters for hesperetin and naringenin, such as C(max), T(max), AUC(0-t), AUC(0-infinity), CL/F, V/F, t(1/2), MRT, A(e), A(e)((0-24)), and R(max) were calculated from their plasma or urine concentrations. RESULTS: Pharmacokinetic analysis showed that both hesperetin and naringenin were rapidly absorbed and their concentrations in plasma observed 20 min after dosing and reached a peak in 4.0 and 3.5 h, respectively. The mean peak plasma concentration (C(max)) for hesperetin and naringenin were 825.78+/-410.63 ng/ml (2731.8+/-1358.4 nmol/l) and 2009.51+/-770.82 ng/ml (7386.6+/-2833.4 nmol/l), respectively and the mean AUC(0-infinity) values were 4846.20+/-1675.99 ng h/ml and 9424.52+/-2960.52 ng h/ml for hesperetin and naringenin, respectively. The elimination half-life for hesperetin was found to be 3.05+/-0.91 h and for naringenin 2.31+/-0.40 h, respectively. The mean values of the relative cumulative urinary excretion, as percentage of the administered dose, for hesperetin and naringenin, were found to be 3.26+/-0.44 and 5.81+/-0.81%, respectively. CONCLUSIONS: Oral administration of the flavanone aglycones, hesperetin and naringenin, lead to their rapid absorption as their conjugated forms. The cumulative urinary recovery data indicated low bioavailability for both flavanone aglycones, owing to extensive first-pass metabolism partly by cleavage of the C-ring by the enzymes of intestinal bacteria leading to degradation products such as phenolic acids.