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*
Antioxidant Research Laboratory, National Institute for Food and Nutrition Research, 546-00178 Rome, Italy; and
Cirio Ricerche, Piana di Monte Verna, Caserta, Italy
2To whom correspondence should be addressed. E-mail: bugianesi{at}inran.it.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • naringenin • tomato • bioavailability • men
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This bioflavonoid exhibits anti-estrogenic activity (1
–3
) that may be responsible for the decreased incidence of breast cancer in women consuming a large amount of phytoestrogens (4
), and could exert cholesterol-lowering properties by inhibiting cholesteryl ester synthesis (5
).
Furthermore, naringenin seems to affect different oxidative processes associated with chronic degenerative diseases. In fact, it partially deactivates the Fenton reaction (6
), restores glutathione-dependent protection against lipid peroxidation in 
-tocopherol-deficient liver microsomes (7
) and inhibits malonaldehyde production induced either by ascorbic acid in rat brain mitochondria (8
) or by autoxidation in rat brain homogenates (9
). Naringenin may modulate cytochrome P450-dependent monooxygenase, the primary enzyme involved in the metabolism of many xenobiotics such as drugs, carcinogens and environmental pollutants (10
).
Because it has been hypothesized that bioflavonoids activity in vivo is dependent on their incorporation rate into cells and on their orientation in biomembranes (11
,12
), the finding that naringenin interacts with phospholipid bilayers (9
) suggests a possible role in human physiology.
The main sources of naringenin are citrus fruits and tomato (Lycopersicum esculentum) (13
,14
). In citrus fruits, naringenin is principally present in glycosidic forms such as naringenin-7-neohesperidoside (naringin) and naringenin-7-rutinoside (narirutin), whereas in tomato, where naringenin is one of the most abundant polyphenols, it is present in the skin as aglycone. The naringenin concentration of tomato is reported to range from 0.8 to 4.2 mg/100 g whole red tomato (14
,15
). So far, no studies have been conducted to investigate the bioavailability of naringenin from tomato in humans. Given the large daily consumption of tomato and tomato-based products, especially in the Mediterranean region, we investigated naringenin bioavailability in humans after consumption of a meal containing tomato. Because bioavailability is strongly affected by food matrices, we administered cooked tomato paste to obtain results providing information on a frequent dietary habit.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All chromatographic standards, ß-glucuronidase and sulfatase were purchased from Sigma (St. Louis, MO). Methanol was obtained from Carlo Erba (Milan, Italy). Orthophosphoric acid (85%) and sodium dihydrogen orthophosphate 1-hydrate were purchased from BDH Italia (Milan, Italy). All chemicals and solvents were of analytical or HPLC grade. Distilled water was purified using a Milli-Q (18 M
) water purification system (Millipore, Milan, Italy).
Tomato paste.
Commercial tomato (Lycopersicum esculentum) paste was supplied by Cirio Ricerche (Caserta, Italy). Tomatoes from the same harvest and genotype were used to obtain the paste. The experiment was conducted 4 d after tomato paste bottling.
Study design.
Five nonsmoking healthy men gave their signed consent to study participation. The protocol complied with the Helsinki declaration as revised in 1983. The subjects were 24–38 y old, had body weights ranging from 67 to 81 kg and body mass indexes ranging from 22 to 26 kg/m2. No subjects were under medical treatment and they did not consume any dietary supplements for at least 2 wk before the study began.
During the 3 d before the study, subjects were asked to not eat citrus fruits and tomatoes, and, in general, to limit their intake of fruits and vegetables. Furthermore, daily food intakes were checked during this period to verify that dietary habits were in compliance with daily nutritional requirements.
A homogenous sauce was prepared by cooking for 10 min at 
40°C 150 g of commercial tomato paste (containing 3.8 mg of naringenin) with 30 g of corn oil. A test meal containing 70 g of pasta seasoned with the tomato sauce prepared as above described was administered to the subjects. Approximately 50 g of bread were also included to allow the consumption of all the sauce remaining in the dish. The test meal gave a dietary intake and an energy contribution (2500 kJ) suitable for a typical breakfast.
On the day of the study, the men fasted for 12 h and were subjected to blood samplings before the test meal (0 h) and 2, 4, 6, 8 and 24 h after the meal. Blood was collected into vacutainers tubes containing heparin; plasma was separated by centrifugation at 3000 x g for 10 min. Plasma samples were stored at -80°C until analysis. At 5 h after the test meal, the men consumed potatoes and water for lunch. They could choose between fried or boiled potatoes and could eat without quantity restriction. From 8 to 24 h after the meal, the men were not restricted other than to avoid foods rich in naringenin.
Two weeks later, the study described above was repeated except the same subjects received a control meal containing 70 g of pasta seasoned with 30 g of corn oil.
Sample preparation.
Three repeated extractions of food polyphenols were performed as described by Hertog et al. (16
) both with and without acid hydrolysis at 70°C to cleave glycosides.
Plasma naringenin was extracted with or without enzymatic hydrolysis of the conjugated forms. To perform the enzymatic hydrolysis, 0.5 mL of an enzyme solution containing 5.5 x 105 [SCAP]U/L of sulfatase and 1.0 x 107 U/L of ß-glucuronidase (sulfase S 3009 type H-5 from Helix Pomatia; Sigma), in 0.2 mol/L acetate buffer (pH 5) were added to 0.5 mL of plasma. The mixture was incubated at 37°C for 45 min. Immediately after incubation, 1 mL of 3 mol/L HCl: MeOH, 1:1 (v/v) was added to precipitate proteins. To measure the unconjugated form, 0.5 mL of acetate buffer (pH 5) without enzymes and 1 mL of 3 mol/L HCl: methanol, 1:1 (v/v) were added to 0.5 mL of plasma. Naringenin from both hydrolyzed and unhydrolyzed mixtures were extracted by adding 2 mL of ethylacetate, followed by vortex mixing for 3 min and sonicating for 1 min before centrifuging at 500 x g for 5 min. The extraction procedure was repeated and the two organic layers were combined and evaporated under a flow of nitrogen. The residue was dissolved into 250 µL of mobile phase (phosphate buffer, pH 2.8, and methanol, 1:1 in volume).
HPLC analysis and calibration.
The HPLC system used consisted of an ESA Model 540 refrigerated autoinjector (4°C), an ESA Model 580 solvent delivery module with two pumps, an ESA 5600 eight-channel coulometric electrode array detector and the ESA CoulArray operating software, which controlled all the equipment and carried out data processing (ESA, Chelmsford, MA). A Supelcosil LC-18 (particle size, 5 µm) column (250 x 4.6 mm) with a Perisorb Supelguard LC-18 (Supelco) were used at 30°C. Injection was performed with an autoinjector (100-µL fixed loop) and the volume injected was 30 µL. Mobile phases and elution program have been described (17
). The flow rate of the eluent was constant at 1 mL/min and the setting potentials were: 60, 120, 200, 340, 580, 620, 760 and 900 mV (vs. Pd).
Peaks were determined by retention time and by hydrodynamic voltammogram. Sample peaks were matched with standard peaks based on their retention times (± 4%) and of the accuracy the ratio between adjacent channels (± 30%).
External standards were used for calibration. A standard stock solution of naringenin (0.73 mmol/L in methanol) was stored at -20°C. The stability of the standard was checked each day by ultraviolet spectroscopy. Progressive dilutions (ranging from 0.04 to 0.73 µmol/L) of the stock solution were prepared and three injections for each standard dilution were randomly performed. The calibration graphs were obtained by a least squares linear fitting of the peak height (nA) vs. naringenin concentration.
Method validation.
To evaluate method precision and absolute recovery, we analyzed spiked samples in replicates of four on two separate occasions. Spiked samples were prepared at four concentrations ranging from 0.07 to 0.4 µmol/L. Suitable volumes of naringenin solution [0.16 mmol/L in methanol diluted 1:100 (v/v) in the mobile phase] were added to 500 µL of blank plasma. Spiked samples were processed and analyzed exactly as described above. Repeatability was estimated as the CV of the replicates.
Statistical analysis.
Results are reported as means ± SD. Statistical analysis was performed using the nonparametric Friedman ANOVA test and the Wilcoxon matched pairs test. Differences were considered significant at P 
0.05. The computer program used was Statistica for Windows, Release 4.5.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Method validation experiments gave a CV 9.0% and an absolute recovery >80% at every concentration tested (Table 1
), sufficient to accept data obtained in this experiment.
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,18
–20
). Because the naringenin content of tomato paste was analyzed without acid hydrolysis and heat treatment, the possibility that naringenin in our extracts derived from cyclization of chalconaringenin caused by the extraction procedure, as reported by other authors (21
), must be excluded. Extraction after acid hydrolysis at 70°C did not affect the naringenin peak height, confirming the absence of chalconaringenin (21
). To verify that the polyphenolic composition of tomato paste was unaffected by cooking, tomato paste samples were spiked with standard solutions and heated at 40°C for 10 min. No changes in polyphenol contents added were detected (data not shown).
Naringenin was not detected after the men consumed the control meal at any time, whereas after test meal consumption, a significant increment (P < 0.05) occurred between 0 and 2 h (Fig. 1
), followed by a significant decrement (P < 0.05) between 2 and 4 h. The peak plasma naringenin concentration (Cmax) at 2 h (Tmax) was 0.12 ± 0.03 µmol/L (Table 2
). Unconjugated naringenin was not detected in plasma samples that were not subjected to enzymatic hydrolysis. Although present in tomato paste, neither chlorogenic acid nor rutin or their derivatives, caffeic acid and quercetin, were detected in plasma samples.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Tomatoes are considered one of the most important sources of lycopene, and, so far, their potential anticancer properties have been attributed only to lycopene’s antioxidant activity (22
,23
). To our knowledge, although plant polyphenols may contribute to the health protection ascribed to fruit and vegetable consumption (24
), no studies of polyphenol absorption after tomato or tomato-based product intake have been published. In this study, the bioavailability of all the polyphenols identified in tomato paste was investigated and the suitability of our method for their quantification was verified (data not shown), but only naringenin was detectable in plasma. The test meal contained 1.08 mg of chlorogenic acid, 4.68 mg of rutin and 3.81 mg of naringenin. Although the amount of rutin ingested was the highest, neither rutin nor free quercetin was detected in plasma. Other authors (25
) found a low peak quercetin concentration (0.3 µmol/L) 9 h after ingestion of 202 mg of pure rutin. Given the small amount of rutin in the test meal, it was predicted that no quercetin peak would be detected over the limit of detection (0.08 µmol/L) if drawing at 9 h was performed.
Chlorogenic acid absorption in humans was observed in a recent study (26
) carried out by supplementation of 1 g of chlorogenic acid to healthy ileostomy subjects. Absorption, calculated as difference between the amount of supplement ingested and the amount in the ileostomy effluent, was 33%. The authors concluded that chlorogenic acid is absorbed intact and metabolized extensively in the liver. In our study, calculating 33% of chlorogenic acid ingested amount and dividing this quantity by 3 L of plasma that are normally present in a man of 70 kg, we should have detected a peak 10-fold higher (0.30 µmol/L) than our limit of detection for chlorogenic acid (0.03 µmol/L). However, this calculation is affected by a high grade of approximation because it does not take into account the balance among absorption, distribution and excretion rates. Furthermore, bioavailability is affected by bacterial degradation in the gastrointestinal tract that might cause an overestimation of absorption in the ileostomy model. Because we considered the possibility that chlorogenic acid was hydrolyzed into caffeic and quinic acids in the gastrointestinal tract, chromatograms were analyzed for caffeic acid. Although our extraction procedure and chromatography were suitable to identify caffeic acid in plasma (data not shown), it was not detected at any time, consistent with another study (26
).
The in vivo study here described showed consistent results on naringenin absorption from tomato paste. In tomato paste, only the naringenin aglycone was detected, as in other studies of the polyphenol composition of tomato (15
,18
–20
).
In a study by Erlund et al. (27
) orange or grapefruit juices, naturally rich in naringenin glycosides, were administered to humans. They found peaks of naringenin in plasma at 4.8 and 5.5 h after consumption of orange and grapefruit juices, respectively, while, in our case, the Tmax was reached after 2 h. Differences in Tmax between citrus and tomato naringenin could be because of different absorption routes. In fact, absorption of glycosidic forms probably takes place in the distal part of the small intestine, where the enzymes for breaking glycosidic linkages are present (28
,29
), whereas aglyconic forms seem to be absorbed early in the digestive tract (30
,31
).
We found a Cmax ranging from 0.07 to 0.12 µmol/L after administration of 3.81 mg of naringenin aglycone with the test meal. We did not find unconjugated naringenin in plasma samples and concluded that naringenin is largely metabolized in the liver and enters the general circulation as the conjugated form. Unfortunately, our chromatography did not permit the detection of unknown peaks that may be conjugated forms. A study in rats (32
) showed results in agreement with ours, demonstrating the absence of naringenin as the free form in plasma of rats fed naringenin.
The naringenin plasma level at baseline (T0) was undetectable, as expected, for all subjects except one. The compositional analysis of the diet consumed by this subject during the 3 d before the experiment was conducted did not reveal any anomaly to justify this incoherence, which remains unsolved.
In conclusion, our study demonstrated naringenin absorption from cooked tomato paste in men. Furthermore, because a recent study (33
) showed that lycopene administered with some polyphenols enhances its antioxidant properties, our results support the hypothesis that tomato benefits could be attributed to a positive synergistic action in vivo among lycopene and other bioavailable tomato constituents, such as naringenin, rather than to only lycopene properties.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: Cmax, highest concentration observed in plasma; MW, molecular weight; nA, peak height; Tmax, time at which the highest concentration occurs in plasma.
Manuscript received 29 April 2002. Initial review completed 30 May 2002. Revision accepted 16 July 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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