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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Bioavailability Is Improved by Enzymatic Modification of the Citrus Flavonoid Hesperidin in Humans: A Randomized, Double-Blind, Crossover Trial¹

Inge Lise F. Nielsen^*, Winnie S. S. Chee^*, Lea Poulsen, Elizabeth Offord-Cavin^*, Salka E. Rasmussen, Hanne Frederiksen, Marc Enslen^*, Denis Barron^*, Marie-Noelle Horcajada^** and Gary Williamson^*,2

^* Nestlé Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland; Danish Institute for Food and Veterinary Research, DK-2860 Søborg, Denmark; and ^** INRA de Clermont-Theix, Unité des Maladies Métaboliques et Micronutrients, 63122 St Genes Champanelle, France

² To whom correspondence and reprint requests should be addressed. E-mail: gary.williamson{at}rdls.nestle.com.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Hesperidin is the predominant polyphenol consumed from citrus fruits and juices. However, hesperidin is proposed to have limited bioavailability due to the rutinoside moiety attached to the flavonoid. The aim of this study was to demonstrate in human subjects that the removal of the rhamnose group to yield the corresponding flavonoid glucoside (i.e., hesperetin-7-glucoside) will improve the bioavailability of the aglycone hesperetin. Healthy volunteers (n = 16) completed the double-blind, randomized, crossover study. Subjects randomly consumed hesperetin equivalents supplied as orange juice with natural hesperidin ("low dose"), orange juice treated with hesperidinase enzyme to yield hesperetin-7-glucoside, and orange juice fortified to obtain 3 times more hesperidin than naturally present ("high dose"). The area under the curve (AUC) for total plasma hesperetin of subjects consuming hesperetin-7-glucoside juice was 2-fold higher than that of subjects consuming the "low" dose hesperidin juice [3.45 ± 1.27 vs. 1.16 ± 0.52 mmol/(L·h), respectively, P > 0.0001]. The AUC for hesperetin after consuming the hesperetin-7-glucoside juice was improved to the level of the "high" dose hesperidin juice [4.16 ± 1.50 mmol/(L·h)]. The peak plasma concentrations (C_max) of hesperetin were 4-fold higher (2.60 ± 1.07 mmol/L, P < 0.0001) after subjects consumed hesperetin-7-glucoside juice compared with those consuming "low" dose hesperidin juice (0.48 ± 0.27 mmol/L), and 1.5-fold higher than those consuming "high" dose hesperidin juice (1.05 ± 0.25 mmol/L). The corresponding T_max was much faster (0.6 ± 0.1 h, P < 0.0001) after subjects consumed hesperetin-7-glucoside juice compared with "low" dose (7.0 ± 3.0 h) and "high" dose (7.4 ± 2.0 h) hesperidin juices. The results of this study demonstrated that the bioavailability of hesperidin was modulated by enzymatic conversion to hesperetin-7-glucoside, thus changing the absorption site from the colon to the small intestine. This may affect future interventions concerning the health benefits of citrus flavonoids.

KEY WORDS: • hesperidin • hesperetin-7-glucoside • bioavailability • randomized crossover study • citrus antioxidant

Over the past decade, polyphenols, abundant in fruits and vegetables, have gained recognition for their antioxidant properties and their roles in protecting against chronic diseases such as cancer and cardiovascular diseases (1–3). Flavonoids are the largest class of polyphenols; among these, the flavanone hesperetin, found abundantly in citrus fruits, was reported to provide health benefits including antioxidant, anti-inflammatory, anticarcinogenic effects and to prevent bone loss (4,5). Consumption of hesperetin in the diet can be substantial from citrus fruits and juices; for example, in Finland, hesperetin contributed to 50% of total flavonoid intake (6).

Therefore, ensuring bioavailability of hesperetin is important to fully exploit its beneficial properties. Low plasma concentrations of hesperetin aglycone (<2 µmol/L) after ingesting 0.5–1 L of orange juice indicated limited bioavailability in human volunteers (7,8). Flavonoids are commonly present in plants conjugated to glycosides. Hesperetin is present in citrus foods as hesperetin-7-O-rutinoside (hesperidin), i.e., the aglycone is linked to glucose and rhamnose sugars at position-7 of the A ring (Fig. 1). The sugar moiety of flavonoids was proposed to be the major determinant of their absorption in humans, whereas glycosides with rhamnose are poorly absorbed compared with their aglycones and glucoside forms (9). This is clearly shown by studies investigating the bioavailability of quercetin. In rats and humans, the absorption of rutin (quercetin-3-O-glucosyl-rhamnose) is slower and less efficient compared with quercetin-4-O-glucoside and the quercetin aglycone itself (9–13). Similarly, absorption of quercetin was more rapid after ingestion of onions, which are rich in glucosides, compared with apples, which contain both glucosides and various other glycosides(14).

View larger version (12K): [in this window] [in a new window]   FIGURE 1  Chemical structure of hesperidin (hesperetin-7-O-rutinoside).

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Subjects. Healthy volunteers (n = 16; 8 men and 8 women) were recruited into the study. Subjects were 36 ± 7 y old, weighed 65.7 ± 10.5 kg, and had a BMI of 21.8 ± 1.8 kg/m². They fulfilled the following criteria: no history of and not taking medication for any gastrointestinal or metabolic disease; they did not regularly consume >2 glasses of alcohol/d or perform physical exercise lasting >45 min, >3 times/wk. The Ethical Committee of Nestlé (Lausanne, Switzerland) found the protocol to be in accordance with the Helsinki Declaration of 1975 as revised in 1983. All volunteers signed informed consent before entering the study.

    Study design. The study was a double-blind, placebo-controlled, randomized, 3-treatment crossover study. Volunteers agreed to refrain from consuming citrus fruits in any form for 3 d before each treatment and in addition refrain from ingesting tea, coffee, cola, alcohol, and whole-grain cereal or use any medication 12 h before each treatment. Compliance with these restrictions was evaluated based on a time point 0 urine sample. Subjects underwent the 3 treatments on 3 different days, separated by a washout period of at least 3 d.

After an overnight fast, the subjects arrived on each day of treatment at the metabolic unit at 0630. A urine sample was collected (time point 0), a catheter was installed in one arm, and a time point 0 blood sample was collected. Subjects then randomly consumed 1 of the 3 preparations of orange juice with a standard breakfast consisting of a croissant, 1 small plain piece of white bread, butter, and water. Subjects were given 5 mL/kg body weight of 1 of the 3 following orange juice treatments: orange juice containing natural hesperidin providing 2 mg/kg body weight hesperidin (low-dose hesperidin treatment), orange juice treated with hesperidinase enzyme to yield hesperetin-7-glucoside and providing 1.52 mg/kg body weight hesperidin (hesperetin-7-glucoside treatment), and orange juice containing deactivated hesperidinase and fortified to provide 6 mg/kg body weight hesperidin (high-dose hesperidin treatment). The low- and the high-dose levels were roughly equal to a 60-kg person ingesting 300 and 900 mL, respectively, of a standard orange juice. The 3 treatments were indistinguishable in aroma and appearance. Samples of 4.5 mL blood were collected at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 24 h after treatment. Urine was collected in 3 different fractions: 0–5, 5–10, and 10–24 h. The subjects were asked to empty their bladder at the end of each sampling interval. Around midday, 5 h after ingestion of the test products, the subjects were provided a lunch consisting of beef, rice, salad with vinaigrette dressing, and water. Water was freely available during the entire day. After collection of the 10-h blood sample, the catheter was removed and the subjects were provided with a dinner consisting of white bread, cold cut meat, and water. The volunteers were allowed to return home with the 10- to 24-h urine collection bottle, which they were asked to keep at 4°C; they came back the next morning to hand in the last urine sample, and the last blood sample was collected 24 h after ingestion of the test product. During each test period, the subjects filled in a form concerning adverse effects of the treatments and noncompliance with diet regimen and urine sample collection.

    Reagents and chemicals. Orange juice was obtained from Nestlé and Hesperidinase Amano Concentration (67000 units/g) from Amano Enzyme. Orange bioflavonoid complex (OBC 90%), containing 90% hesperidin, was obtained from Nutrafur. Vittel mineral water was obtained from Nestlé Waters. Sodium hydroxide, acetic acid (glacial) 100%, sodium acetate trihydrate and di-ammonium oxalate-monohydrate were all obtained from Merck. N,N-Dimethylformamide was obtained from Fluka. As standards, hesperidin was obtained from Fluka. Hesperetin-7-O-ß-D-glucoside was synthesized by reaction of 5,7-di-O-acetylhesperetin with 2,3,4,6-tetraacetyl-D-glucopyranosyl-(N-phenyl)-2,2,2-trifluoroacetimidate, followed by removal of all of the protective acetyl groups by refluxing with a methanolic solution of anhydrous zinc acetate. Elemental analysis of the sample accounted for C₂₂H₂₄O₁₁ + H₂O + 0.7 CH₃OH. The isotopically labeled internal standards, 3x¹³C daidzein and 3x¹³C O-desmethylangolensin (ODMA) were purchased from the School of Chemistry, University of St. Andrews, UK.

    Orange juice treatments. The treatments were as follows: 1) Orange juice with naturally occurring hesperidin: 100 mL of autoclaved hesperidinase solution (20.1 U/L) in acetic acid buffer (10 µmol/L, pH 3.5) and 4.9 mL of 1 mol/L NaOH were added to each liter of Nestlé Thailand orange juice. 2) Hesperetin-7-glucoside orange juice: 100 mL hesperidinase solution (20.1 U/L) in acetic acid buffer (10 µmol/L, pH 3.5) and 4.9 mL of 1 mol/L NaOH were added to each liter of Nestlé Thailand orange juice. 3) High dose orange juice: 100 mL autoclaved hesperidinase solution (20.1 U/L) in acetic acid buffer (10 µmol/L, pH 3.5) and 4.9 mL of 1 mol/L NaOH containing 300 g OBC 90%/Lwere added to each liter of Nestlé Thailand orange juice.

The 3 treatments were incubated for 4h at 70°C during stirring in a Stephan VM 60 incubator (Stephan Machinery), frozen at –50°C and freeze-dried in a Lyo-beta 35 (Telstar). The freeze-dried products were stored at +4°C until use (maximum 2 mo). Immediately before each treatment, the products were reconstituted to their original volume in Vittel mineral water.

    Analysis of total hesperetin aglycone in orange juice. Aliquots (1 mL) of reconstituted juice were combined with 1 mL of 0.025 mol/L ammonium oxalate pH 5, 1 mL dimethylformamide, and internal standard ¹³C daidzein (195 µmol/L final concentration). After 5 min extraction at 80°C, the mixture was centrifuged at 3000 x g for 3 min. HPLC-MS was performed on the system as previously described (16) by injection of 20 µL extract on a Zorbax SB-C18 column (4.6 x 150 mm, 3.5 µm) with a guard column from Agilent Technologies. The gradient used was slightly different: A, 0.01% aqueous formic acid (v:v); and B, acetonitrile; 0 min; 5% B (v:v), 2–10 min; 15% B (v:v), 25 min; 20% B (v:v), 26 min; 25% B (v:v), 45 min; 30% B (v:v), 50 min; 40% B (v:v), 53 min; 60% B (v:v), 55–60min; 100% B. Negative APCI mass spectra were obtained in scan-mode. Calibration curves were obtained by injection of 50, 100, 250, and 500 mg/L hesperetin and hesperetin-7-glucoside.

    Collection of plasma and urine samples. Blood samples were collected in 5-mL EDTA tubes. The tubes were stored on ice for a maximum of 15 min before centrifugation (1500 x g, 10 min, 4°C). Plasma was pipetted into 850-µL aliquots and stored at –80°C until analysis. Urine samples were collected in preweighed urine collection flasks containing 2 mL of 100 kg/L aqueous ascorbic acid solution. The flasks were stored at 4°C during the collection interval after which the total weight of the urine samples was recorded and the pH of the samples adjusted to 3.0–3.5 using 1 mol/L HCl. Urine samples were stored at –80°C until analysis. Hesperetin was quantified in the plasma and urine after hydrolysis of their conjugated forms with ß-glucuronidase and sulfatase enzymes as previously described (16).

    Determination of total hesperetin in urine samples. Hesperetin was determined in 200 µL of urine essentially as previously described (16). The ¹³C ODMA and ¹³C daidzein were used as internal standards. After enzymatic hydrolysis (16) solid-phase extraction was performed basically as described by Nielsen and Dragsted (17) on an Isolute 101 cartridge (100 mg; International Sorbent Technology). After preconditioning, and application of the sample, the cartridge was washed with 1.5 mL 5% methanol, 0.5% formic acid (v:v). The sample was eluted with 2 mL of 90% methanol, 1% formic acid, and 0.1% ascorbic acid (v:v) through a 0.2-µm filter. After complete evaporation, the residue was dissolved in 250 µL of 10% methanol and 1% formic acid (v:v) and analyzed by HPLC-MS as previously described (16).

    Determination of total hesperetin in plasma samples. Plasma samples were centrifuged for 10 min at 11,000 x g, and 100 µL of plasma supernatant was combined with 10 µL internal standard mix containing ¹³C-ODMA and ¹³C-daidzein (5 mg/L DMSO), 73 µL MilliQ water, 10 µL sulfatase and 2 µL of ß-glucuronidase and incubated for 1 h as described for the urine samples. After incubation, the pH was adjusted to 2.5 with the addition of 5 µL of 100% formic acid. Samples were stored at –20°C until HPLC-MS analysis. Before analysis, samples were thawed and centrifuged for 10 min (11,000 x g), and 100 µL was injected onto the HPLC-MS system. HPLC-MS was performed as for the urine samples, but with an electrospray (ES) interface. Conditions for the ES-MS in negative selective ion monitoring (SIM)-mode were as follows: gas temperature, 350°C; drying gas, 13.0 L/min; nebulizer pressure, 40 psig; and capillary voltage (negative), 3500 V. The following parent ions and fragment ions were included in the SIM: Hesperetin (m/z 301, 151), ¹³C-ODMA (m/z 260), and ¹³C-daidzein (m/z 256). To separate the analytes from plasma proteins a restricted access material SPS octyl (4.6 x 150 mm, 5 µm) column was used (Regis Technologies). The column temperature was 40°C. Identical mobile phases as for juice and urine analyses were used (v:v): 0–2 min; 1% B, 2.5 min; 25% B, 4 min; 30% B, 7 min; 45% B, 7.5 min; 60% B, 8–9 min; 100% B, 9.5 min; 1% B. Standard curves with correlation coefficients 0.95 between peak area and concentration were obtained by spiking blank plasma samples with 0.5, 5, 10, 20, 50, 100, 200, 500, and 1000 µg/L of analytes. The samples for calibration were hydrolyzed and otherwise treated as described for the plasma samples. The limit of quantification (S/N = 10) and detection (S/N = 3) was determined as 4 and 1 µg/L, respectively; the CV% was 13% (n = 77).

    Statistical analysis. Demographic and baseline characteristics were documented by descriptive statistics. All results are expressed as means ± SD or SEM. The primary outcome is the area under the available plasma hesperetin curve vs. time after treatment. It is calculated by the trapezoidal rule as follows:

where m_i is the i^th minute, H_i is the H^th available hesperetin value, and n is the number of minutes. Plasma bioavailability of hesperetin was from 0 to 24 h, but between 10 and 24 h, there was no blood sampling. The last value was not considered in the calculation of the AUC and the AUC _{(0–10 h)} was analyzed instead of AUC_{(0–24 h).} The secondary outcomes were C_max i.e., maximum concentration, and T_max i.e., time after treatment when reaching C_max.

For both primary and secondary outcomes, the linear mixed model was used with treatment as the fixed effect and subjects as the random effect. Both intention-to-treat (ITT) and per protocol analyses were done. The 95% CI for treatment effect were calculated. Post hoc analyses were carried out using the Bonferroni test. The rejection level in statistical tests was set at 5%. All statistical analyses were done with SAS software (version 8.2).

One subject did not complete all of the treatments due to difficulty in inserting the catheter for blood drawing during the visit for hesperetin-7-glucoside treatment. There were some measurement periods in which subjects reported intake of citrus-containing foods or beverages or took medication; these were excluded from the per protocol analyses. Overall, for ITT analysis, the number of subjects was 16 for low-dose hesperidin treatment and high-dose hesperidin treatment and 15 for hesperetin-7-glucoside treatment. For the per protocol analysis, there were 10 subjects for low-dose hesperidin treatment and high-dose hesperidin treatment and 12 subjects for hesperetin-7-glucoside treatment. Results did not differ between ITT and per protocol analysis for all variables reported below. Subjects reported no adverse events throughout the study.

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Hesperetin aglycone intake by treatment groups. Subjects ingested approximately similar quantities of total hesperetin aglycone for the low-dose hesperidin treatment (0.93 mg/kg body weight hesperetin) and hesperetin-7-glucoside treatment (1.21 mg/kg body weight hesperetin), and 3 times as much for the high-dose hesperidin treatment (2.92 mg/kg body weight hesperetin) compared with low-dose hesperidin treatment; this is in agreement with the protocol (Table 1).

View larger version (15K): [in this window] [in a new window]   FIGURE 2  Plasma concentration vs. time curve of total hesperetin in healthy humans after consumption of 3 orange juice treatments. Values are means ± SEM, n = 16.

    Total urinary hesperetin excretion. The total volume of urine produced by the subjects after each of the 3 treatments did not differ. The total hesperetin excretion over 24 h was calculated by pooling the 3 fractions of urine collected expressed as the percentage of hesperetin intake.

The relative urinary excretion of total hesperetin of the subjects was significantly higher after consuming hesperetin-7-glucoside than after consumption of low-dose hesperidin and high-dose hesperidin (Table 1). Subjects also had significantly higher urinary excretion after consuming the high-dose hesperidin juice compared with the low-dose hesperidin juice.

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Our study clearly provided evidence that hesperetin-7-glucoside is more bioavailable than hesperidin (hesperetin-7-O-rutinoside). This is as judged by the total hesperetin AUC, C_max, and urinary excretion data that were significantly higher for hesperetin-7-glucoside compared with both the low and high doses of hesperidin in orange juice. This was the first demonstration of this effect in human subjects for hesperetin-7-glucoside.

Over the past few years, understanding of the absorption and metabolism of flavonoids has increased. Flavonoid glycosides are thought to reach the small intestine intact (18) and require deglycosylation for absorption across the intestine (18,19). Our study showed that relative bioavailability was lowest with orange juice containing low-dose hesperidin, and maximum plasma concentration of hesperetin was achieved only 7 h after ingestion of the orange juice. The time to reach maximum plasma concentration was not any shorter with orange juice containing high-dose hesperidin. This is supported by the concept that flavonoids that contain rutinose groups are absorbed only in the distal part of the intestine i.e., after hydrolysis by the colonic microflora. It was demonstrated that there is no hydrolysis of rhamnosides in human intestinal tissues (20,21) but strains of bacteria, which are able to hydrolyze flavonoid glycosides bearing a rhamnosyl moiety, have been isolated from human gut microflora (22). Enterobacteria secrete - and ß-rhamnosidases to cleave the attached sugars. The released aglycone diffuses passively into the blood or alternatively is broken down into phenolic acids.

However, in our study, the cleavage of the rhamnose sugar from hesperidin in the orange juice to yield hesperetin-7-glucoside was carried out before consumption by the subjects. This rendered the glucoside molecule a suitable substrate for deglycosylation to occur at the small intestine instead of the colon. This also explained the fast attainment of peak plasma concentration in <1 h for hesperetin-7-glucoside. There are 2 hypotheses concerning why flavonoid glucosides are favored for absorption in the small intestine. First, the glucoside is hydrolyzed by lactase phlorizin hydrolase (23), and the free aglycone diffuses through the epithelial cells passively or by facilitated diffusion. The deglycosylation process is not only specific but has high capacity, hence the fast detection of maximum plasma concentration of the hesperetin aglycone. Alternatively, the glycoside molecule can be transported into the enterocyte via a sugar transporter such as SGLT1 and then deglycosylated by the ß-glucosidase enzyme present in the intestinal cells (24). Both pathways of absorption give rise to intracellular aglycones, which become conjugated to glucuronides or sulfates. Fig. 3 indicates the possible absorption pathways of hesperidin and hesperetin-7-glucoside.

View larger version (27K): [in this window] [in a new window]   FIGURE 3  Possible pathway for hesperidin and hesperetin-7-glucoside absorption showing the proposed shift in the site of absorption of hesperetin from the colon to the small intestine.

The results of this study demonstrated that the bioavailability of hesperidin was modulated by enzymatic conversion to hesperetin-7-glucoside, thus changing the absorption site from the colon to the small intestine. This may affect future interventions concerning the health benefits of citrus flavonoids.

	ACKNOWLEDGMENTS

 
We thank Corrine Hager and Martin van't Hoff for statistical advice, Sylviane Oguey-Araymon and Anny Blondel-Lubrano for performing the clinical study, Juliet Farrar for contact to the Metabolic Unit, William Sauret for data management, and Alain Fracheboud for assistance in producing the test products.

	FOOTNOTES

 
¹ I.L.N., W.S.S.C., M.E., D.B., E.O.C., and G.W. are employees of Nestlé Research Center. No other authors had any financial interest in the Nestlé Research Center, the organization sponsoring this study.

³ Abbreviations used: AUC, area under the curve; ES, electrospray; ITT, intention-to-treat; OBC, orange bioflavonoid complex; ODMA, O-desmethylangolensin; SIM, selective ion monitoring.

Manuscript received 29 September 2005. Initial review completed 18 October 2005. Revision accepted 22 November 2005.

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TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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