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*
Laboratoire de Pharmacognosie, Faculté de Pharmacie, 63001 Clermont-Ferrand, France and
Laboratoire des Maladies Métaboliques et des Micronutriments, Institut National de la Recherche Agronomique de Clermont-Ferrand/Theix, 63122 Saint-Genès Champanelle, France
1To whom correspondence should be addressed. E-mail: catherine.felgines{at}u-clermont1.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.26% of the ingested amount, whereas that of malvidin 3-glucoside was 0.67%. This result suggested that structure of the aglycone moiety of anthocyanins could play an important role in their metabolism. Low amounts of glucosides as well as of cyanidin were recovered in cecal contents. This could result from adaptation of microflora to anthocyanin degradation. Overall, these data indicate that blackberry anthocyanins are excreted in urine as intact and methylated glucoside forms and that their bioavailability is very low compared with other flavonoids.
KEY WORDS: • blackberry • cyanidin 3-glucoside • malvidin 3-glucoside • bioavailability • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The daily intake of anthocyanins in humans has been estimated to be as much as 180–215 mg/d in the United States (2
) due to their widespread distribution and occurrence in fruits and vegetables. This value is much higher than the intake of other flavonoids such as flavones and flavonols in the Dutch diet (23 mg/d) (3
). Consumption of anthocyanins through the intake of red wine or bilberry extract has been shown to be associated with a lower risk of coronary heart disease and improvement of visual function (4
,5
). Several studies have reported beneficial effects of anthocyanins in the treatment of various microcirculation diseases resulting from capillary fragility (6
,7
). Anthocyanins may also prevent cholesterol-induced atherosclerosis (8
) and inhibit platelet aggregation (4
). Other biological properties such as anti-inflammatory (9
) and anticarcinogenic (10
) activities have also been described for anthocyanins. The positive effects of these pigments could be related to their potent antioxidant activity demonstrated in various in vitro studies (9
,11
,12
).
In view of these multiple biological effects, the bioavailability of anthocyanins is an important issue. A few studies have been performed using mixtures extracted from plant sources, such as Vaccinium myrtillus anthocyanosides (13
) and wine anthocyanins (14
). Moreover, recent studies have evaluated quantitative absorption of anthocyanins using various purified molecules administered by gavage to rats (15
–17
). However, to date there are no available data on the bioavailability of red fruit anthocyanins after adaptation of rats to a supplemented diet. Thus, the aim of this work was to evaluate absorption and metabolism of anthocyanins in rats adapted for 8 d to an anthocyanin-enriched diet. This study was carried out with blackberries as anthocyanin source because these fruits are characterized by one major pigment, cyanidin 3-glucoside (1
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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ß-glucuronidase type VII-A from Escherichia coli was purchased from Sigma Chemical (Saint-Quentin-Fallavier, France). All other chemicals were purchased from Extrasynthèse (Genay, France). Deep-freezed blackberries were from a deep-freezed food product supplier (Szymczak-Nadreau, Romagnat, France).
Animals and diets.
Thirty-six male Wistar rats (Iffa-Credo, L’Arbresle, France) weighing 170 g were housed two per cage in temperature-controlled rooms (22°C), with a dark period from 0800 to 2000 h and access to food from 0800 to 1600 h. They were fed a semipurified control diet for 7 d (see detailed composition in Table 1
). They were then randomly divided into two groups, individually housed in metabolic cages fitted with urine/feces separators, and received for 8 d (25 g/d) either the control diet (control rats, n = 18) or the control diet supplemented with 200 g/kg blackberry powder plus 20 g/kg citric acid (anthocyanin-fed rats, n = 18). Blackberry powder was obtained from frozen blackberries that were lyophilized, pulverized and then sieved to eliminate seeds.
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Sampling procedure.
Rats were killed at 3, 6 and 24 h after the beginning of the last experimental meal after being anesthetized with sodium pentobarbital (40 mg/kg body). Blood was withdrawn from the abdominal aorta into heparinized tubes. Plasma was acidified with 10 mmol/L acetic acid. Urine present in the bladder was collected and acidified with 10 mmol/L acetic acid. Urine from rats killed 24 h after the beginning of the last meal was collected during the last 24 h into tubes containing 1 mL of 3 mol/L HCl. Cecal contents were drained by finger pressure into microfuge tubes and immediately frozen. All samples were rapidly frozen and stored at -20°C until analysis.
HPLC analysis.
To quantify anthocyanins administered to rats, blackberry powder (0.3 g) was treated for 30 min under agitation with 95 mL of 0.12 mol/L HCl in methanol. After filtration, the volume of solution was adjusted to 100 mL, and this solution was 10-fold-diluted with 0.12 mol/L HCl before HPLC analysis. This latter dilution (20 µL) was injected in a 150- x 4.6-mm Hypersil C18–5-µm column (Interchim, Montluçon, France). A photodiode array detector (991; Waters, Milford, MA) and an UV-visible detector (785A; Perkin Elmer, Courtab
uf, France) at 520 nm were used for identification and quantification of anthocyanins, respectively. Elution was performed using water-H3PO4 (99:1) as solvent A and acetonitrile as solvent B at a flow rate of 1.0 mL/min. Analysis were carried out with linear gradient conditions from 100% A to 90% A for 10 min and then to 75% A for 30 min. To attempt to identify aglycone moiety of anthocyanins, anthocyanins were extracted from blackberry powder for 30 min under agitation in 0.12 mol/L HCl. After filtration, anthocyanins were submitted to acid hydrolysis in 0.6 mol/L HCl for 2 h at 100°C. The hydrolysis product was then analyzed as previously described.
Anthocyanins exist as four different structures in equilibrium (1
). The proportion of each structure depends on pH. In acidic conditions (pH below 2), anthocyanins exist primarily in the form of flavylium-colored cation detectable at 520 nm (1
). Urine collected in the bladder was acidified with 10% 12 mol/L HCl for at least 1 h before analysis to give maximal yield of the colored flavylium cations. Then, 20 µL of this mixture was injected into HPLC column and eluted as previously described. Each sample was analyzed twice. To search for possible glucuroconjugates, urine samples were acidified to pH 4.9 with 0.1 volume of 0.58 mol/L acetic acid solution and incubated for 4 h at 37°C with 106 U/L ß-glucuronidase (from E. coli). Samples were treated by adding 2.8 volumes of acetone and the resulting mixtures were centrifuged for 5 min at 12,000 x g at room temperature. Supernatants were evaporated under a nitrogen stream to initial volume of urine and then acidified and analyzed as previously described.
To determine 24-h urine excretion of anthocyanins, 24-h urine samples collected into HCl were centrifuged for 5 min and the supernatants (20 µL) were injected and analyzed as previously described. Collection of urine onto HCl allowed regeneration of colored structure of anthocyanins as urine fell into the tube and, thus, increased their stability.
Plasma was treated with one volume of 0.61 mol/L trichloroacetic acid to precipitate proteins and then centrifuged for 5 min at 12,000 x g at room temperature. The supernatant (20 µL) was analyzed with the same HPLC system and elution conditions as for urine.
Cecal contents were extracted with 9 volumes of water/acetone (1/1) containing 500 mmol/L HCl, briefly sonicated and centrifuged for 5 min at 12,000 x g at room temperature. Supernatants were analyzed for anthocyanin content as previously described.
Data analysis.
Values are given as means ± SEM and, when appropriate, significance of differences between values was determined by the Kruskal-Wallis test followed by Dunn’s multiple comparisons test (GraphPad, Instat, San Diego, CA). Differences of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) with retention times as follows: peak 1, 20.47 min; peak 2, 25.05 min; and peak 3, 28.12 min. Peaks 1 and 2 were identified as cyanidin 3-glucoside and malvidin 3-glucoside, respectively, by comparison with the authentic compounds based on the retention time in the HPLC analysis and UV-visible spectrum, and by spiking with individual compounds. Peak 3 (called peak X) presented an anthocyanin UV-visible spectrum but could not have been identified by comparison with commercially available anthocyanins. Moreover, acid hydrolysis liberated, as expected, a large amount of cyanidin and a lower amount of malvidin, but no additional aglycone was detected. Therefore, peak X might be a glycoside of cyanidin or malvidin. Concentrations of cyanidin 3-glucoside and malvidin 3-glucoside in the anthocyanin-supplemented diet were 6.1 mmol/kg and 0.12 mmol/kg, respectively. The amount of food actually ingested during the last meal (of the 25-g diet provided daily to each rat) was monitored (Table 2
).
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. Whereas no peak was observed in urine from control rats (upper pannel), several peaks were present in urine from anthocyanin-fed rats killed 3 and 6 h after the beginning of the meal (lower panel). These peaks were not observed in urine collected from rats killed 24 h after the beginning of the meal (data not shown). Peaks 1 and 2 were identified as cyanidin 3-glucoside and malvidin 3-glucoside, respectively. Peak 4 was identified as peonidin 3-glucoside by comparison of the UV-visible spectrum and retention time and by spiking samples with commercially available standard. No aglycone was detected in urine. Treatment of urine with ß-glucuronidase type VII-A did not alter the HPLC profile. The urinary excretions of cyanidin 3-glucoside, peonidin 3-glucoside and malvidin 3-glucoside over a 24-h period were 190 ± 21 nmol/24 h, 91.6 ± 9.5 nmol/24 h, and 14.8 ± 1.6 nmol/24 h, respectively.
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The presence of anthocyanins was also studied in the cecal contents. Cyanidin 3-glucoside, malvidin 3-glucoside and the unidentified compound (peak X) were present in cecal contents of anthocyanin-fed rats (Fig. 3
). A low amount of cyanidin was also observed. Cecal anthocyanin content increased between 3 and 6 h after the beginning of the meal and then had decreased by 24 h (Fig. 4
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,19
). Blackberries used in this study contained, as expected, a large amount of cyanidin 3-glucoside. Moreover, small proportions of malvidin 3-glucoside (1/50 of cyanidin 3-glucoside content) and of an unidentified pigment were detected. The presence of a similar pigment that eluted after malvidin 3-glucoside has been described in some blackberry cultivars, but the investigators did not identify it (20
,21
).
Neither qualitative nor quantitative data on urinary excretion of anthocyanin metabolites in rats have been reported. This study reports for the first time information on urinary excretion of anthocyanins after adaptation of rats to an anthocyanin-containing diet. As reported in recent human studies (17
,19
,22
), anthocyanins were recovered in urine as the intact forms, whereas neither anthocyanidin (aglycone) nor glucurono conjugates were detected. As suggested (16
), the flavylium cation structure of anthocyanins seems to impart resistance against enzymic conversion into conjugates. Peonidin 3-glucoside, which was detected in urine, could result from hepatic methylation at the 3' hydroxyl moiety position of cyanidin 3-glucoside by catechol-O-methyltransferase. Previous studies conducted in cyanidin 3-glucoside-fed rats have reported a methylated metabolite in liver, but they did not detect it in plasma (15
,16
). Miyazawa et al. (16
) have suggested that methylated metabolites found in liver may be excreted directly into bile. However, our results clearly indicated that these metabolites were excreted in urine in substantial quantities.
The percentages of cyanidin 3-glucoside and malvidin 3-glucoside recovered in urine (calculated as the ratio of anthocyanin excreted to anthocyanin ingested) were 0.17% and 0.67%, respectively. Considering that peonidin 3-glucoside found in urine probably results from cyanidin 3-glucoside methylation, its excretion could be linked to cyanidin 3-glucoside ingestion. So, 0.09% of ingested cyanidin 3-glucoside was eliminated in urine as peonidin 3-glucoside. We, thus, can consider that urinary recovery of cyanidin 3-glucoside as either the intact or methylated form was 0.26% of the ingested amount. Human studies also reported very low urinary recoveries of anthocyanins (from 0.05 to 0.11% of the dose ingested) (17
,19
,22
). The discrepancy between total cyanidin 3-glucoside (0.26%) and malvidin 3-glucoside (0.67%) bioavailability suggests that the structure of the aglycone moiety of anthocyanins could play an important role in absorption and metabolism.
Recent studies carried out in rats fed a single bolus of anthocyanins by gavage have indicated that anthocyanin 3-glucosides were rapidly absorbed and had a relatively short half-life (15
–17
). In our study, we did not detect any anthocyanin in plasma at any time of sampling. Indeed, food consumption was spread over a period of 8 h and anthocyanins were slowly ingested. Therefore, they did not accumulate in plasma and their plasma levels were probably too low to be detectable by UV-visible HPLC analysis. Moreover, in the bladder, anthocyanins were present at similar levels 3 and 6 h after the beginning of the meal but they were no longer detectable 24 h later. This result confirms their rapid absorption and elimination.
Anthocyanin stability is highly influenced by pH and glycosylation also increases stability of the pigments (1
). Moreover, we have observed that after incubation of control urine or plasma with aglycones, followed by acidification to regenerate flavylium cation structure, aglycones were not recovered in their intact form (data not shown). Therefore, it was not surprising to not detect aglycones in biological fluids. Anthocyanin 3-glucosides would be partly hydrolyzed by intestinal ß-glucosidase and would, thus, liberate aglycones rapidly transformed into unknown metabolites. Some of the absorbed anthocyanins was likely metabolized to some noncolored forms that we did not detect under present conditions.
Examination of cecal content anthocyanin revealed the presence of low amounts of glucosides and cyanidin. Unlike previous studies of flavanone-fed rats in which no glucoside was found in the cecal contents after naringenin 7-glucoside feeding (23
), anthocyanin glucosides were recovered in cecum. This result underlines once again that anthocyanin metabolism is not similar to that of other flavonoids. As previously suggested (16
), the presence of the cation group in anthocyanins would render them more stable than other flavonoids against bacterial hydrolysis. Moreover, rats were adapted to the anthocyanin diet. The recovery of low amounts of blackberry pigments could result from adaptation of microflora to anthocyanin degradation. Indeed, we have previously shown in rats adapted to flavanone-enriched diets that cecal microflora have adapted and, thus, hydrolyze the flavanones that reach the large intestine into aglycones and phenolic acids (23
). The high instability of anthocyanidins at physiological pH (6–6.5) was probably responsible for the low aglycone content in the cecum and aglycones likely were rapidly transformed to noncolored forms and/or degradation products.
In conclusion, this study indicated that blackberry anthocyanins were excreted in urine as intact and methylated glycoside forms and that their bioavailability was very low compared to other flavonoids (23
,24
). However, red fruits such as blackberries also contained phenolic acids that have antioxidant properties and that are efficiently absorbed (25
,26
).
Manuscript received 3 December 2001. Initial review completed 14 January 2002. Revision accepted 26 February 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Mazza, G. & Miniati, E. (1993) Mazza, G. Miniati, E. eds. Anthocyanins in fruits, vegetables, and grains :1-28 CRC Press Boca Raton, FL. .
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Cao, G., Muccitelli, H. U., Sanchez-Moreno, C. & Prior, R. L. (2001) Anthocyanins are absorbed in glycated forms in elderly women: a pharmacokinetic study. Am. J. Clin. Nutr. 73:920-926.
20. Dugo, P., Mondello, L., Errante, G., Zappia, G. & Dugo, G. (2001) Identification of anthocyanins in berries by narrow-bore high-performance liquid chromatography with electrospray ionization detection. J. Agric. Food Chem. 49:3987-3992.[Medline]
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