Despite the indications of their biological activity, there is up to now only sparse information on the in vivo bioavailability and bioactivity. Moreover, biokinetic data of their absorption, distribution, metabolism, and elimination in human beings are lacking so far but are a precondition for the assessment of their efficacy in the organism. Additionally, it is controversially debated whether alcohol may affect the bioavailability and renal excretion of this group of plant phenolics.

Venous blood samples were taken initially (predose as baseline) and at 0.25, 0.50, 1.0, 1.5, 2, and 3 hours after intake of juice or wine, respectively. Urine samples were additionally collected predose and quantitatively in hourly intervals over a period of 7 hours. The study design was approved by the Ethical Commission of the University of Giessen, Germany.

In plasma and urine, the anthocyanin content was analyzed by the usual HPLC methods [4, 5, 6, 7]. In brief description, after extraction from plasma by using an ODS solid phase extraction cartridge and elution with 5mL 0.44M trifluoruacetic acid (TFA) in methanol, the anthocyanins were separated using a Prontosil Eurobond RP-18 column protected by a LiChrospher 100 RP-18 guard column and isocratic elution with water/acetonitrile/formic acid (81/10/9, pH 1.6) as mobile phase. Single substances were detected at 520nm with a photodiode array detector and a UV-VIS detector. Identity of the separated substances was ensured by comparing their retention time and UV-VIS spectra with those of commercially available standards. The detection limits of the analyzed anthocyanins were between 0.65ng/mL (cyanidin-3-glucoside) and 5.2ng/mL (malvidin-3-glucoside) in all matrices. For calibration (average r² value 0.998), blank urine and plasma samples were spiked with known concentrations of standard solutions.

The chromatographic conditions were identical for beverages, plasma, and urine. Juice and red wine samples were diluted 1 : 50 with HPLC mobile phase and centrifuged before injection into the HPLC column. The analyzed anthocyanin profile in grape juice differed slightly from that in wine in a lower content of glucosides of petunidin (42 versus 91μg/mL) and malvidin (327 versus 461μg/mL) and a higher glucoside content of delphinidin (124 versus 95μg/mL) and peonidin (208 versus 45μg/mL). In predose plasma and urine samples of the volunteers, the anthocyanin levels were below the detection limit.

Total polyphenolics were determined according to [8] by a modification of the Folin-Ciocalteau method. Conjugated polyphenols were hydrolyzed with 1M hydrochloric acid (HCl) and polyphenol-lipid links broken with 2M NaOH in 75% methanol followed by precipitation of plasma proteins with 0.75M metaphosphoric acid. The polyphenols were, after centrifugation, extracted from the supernatant with acetone/water (1/1) and assayed with the Folin-Ciocalteau reagent. Results were given as milligram gallic acid equivalents (GAE) per liter. The anthocyanin glucoside values in plasma and urine were based for the calculation of the pharmacocinetics/biokinetics, such as the maximal concentration c_max, its peaking time t_max, the area under the curve AUC, and the elimination half-life t_1/2 [4]. Noncompartmental pharmacokinetic evaluation according to standard methods was performed [9] by using the WinNonlin Professional software (version 3.3, Pharsight Co, Mountain View, Calif). The extent and the relative bioavailability (wine versus juice) was tested for equivalence by calculating the 90% confidence interval (CI) on the basis of a one-sample t test of the log-transformed intraindividual differences in dose-normalized data. Distributional assumptions were confirmed by the Shapiro-Wilk test prior to performing the t test, which was based on the assumption of log-normally distributed intraindividual differences. Significant differences were accepted with P values less than or equal to .05.

The plasma profile of the absorbed substances was also reflected in the urinary excretion pattern of the anthocyanin glucosides (Table 3). The maximal excretion rate R_max could be detected between 1.5 and 2.5 hours after intake. This is consistent with observations of Miyazawa et al [5] and Matsumoto et al [11]. Despite the high variation also seen with the grape juice anthocyanin excretion pattern, differences between both beverages in the excreted total amount were statistically significant, reaching 0.23% of the administered dose after grape juice and 0.18% after wine ingestion with petunidin and peonidin glucoside as the highest rates and cyanidin glucoside as the lowest-percentage excretion rate in both cases. It should however be mentioned that the cumulative excreted amounts do not correspond exactly to the absorption rate. Biliary secretion, for example, may also contribute to the elimination process of some substances.

The content of total polyphenolics in plasma increased after intake of both beverages with a c_max after about 30 minutes (Figure 1). In contrast to plasmatic anthocyanins, the c_max value after grape juice intake significantly exceeded the analogous value after wine intake, indicating that flavonoids other than anthocyanins may be better absorbed. It should be noted that this increase occurred on the basis of a relatively high GAE fasting level that may be accounted for by other reducing substances (SH-compounds, ascorbic and uric acids, etc) reacting also with the modified Folin-Ciocalteau method as was described by other authors [8].

A first proof that anthocyanins appear in plasma as intact glucosides after oral ingestion had been furnished by Tsuda et al in animals [13]. In the meantime, it could be demonstrated by our group and other authors that besides monoglucosides, higher condensed glycosylated forms in fruit juices were also incorporated from the intestine into the blood of rats and humans and are excreted in urine as such [4, 5, 6, 11, 12, 14, 16, 17, 18, 19, 20].

The generally low plasma and urine concentrations in this study suggest that the small absorbed anthocyanin dose is subjected to a rapid metabolism. Also a decompostion of anthocyanins cannot be excluded, since Tsuda et al [13] detected protocatechuic acid in the plasma of rats fed cyanidin-3-glucoside but in a 100-fold higher dose per kg body weight than in the present study. As the percentage of the excreted cyanidin glucoside in the present study was by far lower than for the other anthocyanins, this may indicate that cyanidin in vivo serves as precursor and is methylated to peonidin as was already proved in rats [13] (Table 3, Xe (0–7)). The excreted total anthocyanin amount is in contrast to the values of Lapidot et al [21] who assessed a more than 10-fold excretion rate after red wine intake with a comparable anthocyanin dose but within 12 hours. Bub et al [20], on the other hand, found less than 0.03% of the ingested dose in the urine.

Even though the plasma biokinetics revealed no statistical differences between the beverages, the analogous values in the urinary excretion (Table 3, R_max, Ae (0–7) and Xe (0–7)) suggest a higher bioavailability of anthocyanins from red grape juice compared to red wine. Perhaps the observation period was relatively short to recognize bioequivalence in plasma kinetics, too. Nevertheless, it has also been considered that due to absorption on plasma proteins, the analytical recovery rate of anthocyanins may be lowered. Murkovic et al [14] calculated, from spiked plasma samples, a recovery rate of elderberry anthocyanins to only 20%.

The identical elimination rates (t_1/2) from plasma corresponding with a (nonstatistical) tendency to lower c_max- and higher t_max-values after red wine intake lead to the conclusion that either the anthocyanin absorption from red wine was inhibited or the intestinal uptake from red grape juice was enhanced (Table 2). Up to now, there are contradictory findings about the effect of alcohol on the gastrointestinal function and absorption of flavonoids. Levanon et al [22] suggested that white wine may inhibit the postprandial contractions and thus delay the absorption of sugars and glucosides. Bub et al [20] found no difference in the absorption of malvidin-3-glucoside between red wine and dealcoholized red wine, but found a delayed bioavailability after red grape juice intake. The latter finding is in contrast to our results. Gee et al [23] and Hollman [24] could demonstrate that quercetin glucosides may interact with the intestinal glucose transporter SGLT-1, resulting in an elevated intestinal uptake. More recently, it has been shown that the intestinal lactase-phlorizin hydrolase (LPH) in the apical membrane of intestine epithelial cells and the cytosolic β-glucosidase seems to play an important role in the absorption and metabolism of flavonoids [25, 26]. Considering the sugar content of the administered red grape juice and the similar structural features to quercetin, it is conceivable that the anthocyanins are absorbed via cotransport with the intestinal brush border SGLT-1, elevating thus the anthocyanin uptake compared to red wine [27].