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Bioavailablility of elderberry anthocyanins
Mechanisms of Ageing and Development 123 (2002) 997– 1006 www.elsevier.com/locate/mechagedev
Bioavailablility of elderberry anthocyanins Paul E. Milbury *, Guohua Cao, Ronald L. Prior, Jeffrey Blumberg Antioxidants Research Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts Uni6ersity, Room 507, 711 Washington St. Boston, MA 02111, USA
Abstract Considerable epidemiological evidence suggests a link between the consumption of diets rich in fruits and vegetables and a decreased risk of cardiovascular disease and cancers. Anthocyanins have received attention as important dietary constituents that may provide health benefits and contribute antioxidant capacity beyond that provided by essential micronutrients such as ascorbate, tocopherols, and selenium. The emergence of renewed interest by industrial countries in traditional herbal medicines and the development of ‘functional foods’ are stimulating the need for more information regarding the bioavailability and efficacy of plant polyphenols. Flavonoids represent a numerous group of secondary plant metabolites based on the structure of a pyran ring flanked by two or more phenyl rings and varying subtly in the degree of unsaturation and the pattern of hydroxylation or methylation. Flavonoids also vary in the type of sugar attached or the degree of polymerization. Anthocyanins, potent flavonoid antioxidants widely distributed in fruits, vegetables and red wines, normally occur in nature as glycosides, a form not usually considered as bioavailable. We have examined the bioavailability and pharmacokinetics of anthocyanins in humans. Anthocyanins were detected as glycosides in both plasma and urine samples. The elimination of plasma anthocyanins appeared to follow first-order kinetics and most anthocyanin compounds were excreted in urine within 4 h after feeding. The current findings appear to refute assumptions that anthocyanins are not absorbed in their unchanged glycosylated forms in humans. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Anthocyanins; Flavonoids; Antioxidant; Pharmacokinetics; Absorption; Elderly; Elderberry
1. Introduction There is great interest in determining the role of phytonutrients in promoting improved health and in reducing cancer, cardiovascular disease, and the effects of aging. It is widely believed that antioxidant phytonutrients can inhibit the propagation of free radical reactions that may ulti* Corresponding author. Tel.: +1-617-556-3095; fax: + 1617-556-3344. E-mail address: [email protected] (P.E. Milbury).
mately lead to the development of diseases, especially those which are aging related. Analysis in several laboratories shows that many fruits and vegetables have strong antioxidant capacities, and that this capacity is due primarily to non-vitamin C phytochemicals (Cao et al., 1996; Wang et al., 1996; Guo et al., 1997; Prior et al., 1998). To date the proof that dietary antioxidant phytochemicals can reduce human diseases by inhibiting in vivo oxidative processes has been elusive. There has been insufficient study that has incorporated rigorous biochemical screens of redox
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status and antioxidant uptake in human intervention trials combined with monitoring of biomarkers of oxidative stress and correlation with disease endpoints. The best studies to date in this regarding uptake involve study of flavonoid compounds in tea, cherries, and wine (Hollman et al., 1997; Wiseman et al., 1997; Wang et al., 1999a,b,c). Flavonoids are a large group of natural phenolic compounds, consisting mainly of flavonols, flavanols and anthocyanidins. Among these flavonoids, the water-soluble glycosides and acylglycosides of anthocyanins are an important group of natural antioxidants (Sichel et al., 1991; van Acker et al., 1995; Vinson et al., 1995; Tsuda et al., 1996; van Acker et al., 1996; Wang et al., 1997). While in vitro antioxidant activities of anthocyanins are superior to vitamin E (Wang et al., 1999a), little is known regarding comparable in vivo capacity or the bioavailability of these compounds. Anthocyanin variations lend to a complexity that has made these compounds, as a group, difficult to study with regard to bioavailability. Anthocyanins vary in the degree of unsaturation and the pattern of hydroxylation, methylation, and degree of polymerization, and this variation greatly influences the antioxidant capabilities of the molecules (Milbury, 2001). Tea beverages are rich sources of flavonoids, predominantly catechins. Red wines contain flavonoids that can vary in content between 1 and 3 g/l the majority of which is usually flavanols as monomers and oligomers, however, anthocyanins can comprise 20–80% of the flavonoid content (Glories, 1988). Berries represent another rich plant sources of dietary anthocyanins. Intake of anthocyanins from berry sources has been estimated to be 180– 215 mg per day among the US population (Ku¨ hnau, 1976). This figure far exceeds the estimated intake of 23 mg per day of other flavonoids such as luteolin, kaempferol, myricetin, apigenin and quercetin (Hertog et al., 1993). The most common naturally occurring anthocyanins in plants are in the 3-O-glycoside or 3,5-di-O-glycoside forms, which were considered nonabsorbable in humans. Due to this belief, study of the pharmacokinetics of dietary an-
thocyanins was neglected in humans. Recent developments in HPLC methodology to measure the anthocyanins in plasma permitted the demonstration that anthocyanins are absorbed and are bioavailable to humans (Cao and Prior, 1999). This paper presents evidence that anthocyanins are absorbed in their original glycosylated forms in human subjects. The pharmacokinetics of dietary berry anthocyanins were determined in four elderly women.
2. Materials and methods
2.1. Subjects Four women (age: 6794 years) were recruited to participate in a pharmacokinetics study. These subjects were in good health as determined by a medical history questionnaire, physical examination, and clinical laboratory test results. Eligibility criteria for inclusion included: (1) no history of cardiovascular, hepatic, gastrointestinal, or renal disease; (2) no alcoholism; (3) no supplemental vitamin and/or mineral use or antibiotic use during a 4-week period prior to the start of the study; and (4) no smoking. The study protocol was reviewed and approved by the Human Investigation Review Committee of Tufts University and the New England Medical Center. Written informed consent was obtained from each study participant.
2.2. Study design Subjects reported 48 h prior to the experimental day for admittance to the Metabolic Research Unit (MRU) at the Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging at Tufts University. The diets provided to these subjects during their residency in the MRU were designed to have no anthocyanins and to be low in other flavonoids, but provided the Recommended Dietary Allowance for protein and energy (Cao et al., 1998). All meals were prepared under the supervision of a dietitian in the MRU. The consumption of water was not limited. Other foods or beverages were not allowed during the
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residency. Subjects were fasted overnight before the day of sampling. In the morning of the sampling day, an intravenous catheter was inserted into one forearm and a 15 ml baseline sample blood sample was collected in heparinized tubes. Each subject was then given 12 g elderberry extract, dissolved in 500 ml water. The extract ‘drink’ contained 720 mg anthocyanins by analysis and was composed predominantly of cyanidin 3-sambubioside, cyanidin 3-glucoside, and maltodextrin. Elderberry was selected for this study because of its simplicity with regard to anthocyanins with cyanidin 3-sambubioside and cyanidin 3-glucoside accounting for 92.5% of the total anthocyanins detectable within the berry (Prior et al., 1998). Blood samples (15 ml) were collected again at 10, 20, 30, 45 min, 1, 2, 4, 6 and 24 h after consumption of the elderberry anthocyanins. Urine samples were collected from these subjects before the consumption of the elderberry anthocyanins and between 0– 2, 2– 4, 4– 6, 6–8, 8–12 and 12– 24 h after the consumption of the elderberry anthocyanins. Lunch and dinner were given following the blood sampling at 4 and 11 h, respectively, after consumption of the drink.
2.3. Anthocyanin analysis in plasma and urine Blood samples were centrifuged at 500× g for 10 min at 4 °C and plasma was removed for immediate treatment with TFA. Both plasma and urine samples were treated with 0.44 mol/l TFA aqueous solution (1:0.2, v/v) as previously described (24). TFA treated plasma and urine samples were stored at − 80 °C prior to anthocyanin analysis by HPLC and HPLC/MS. The plasma samples (2 ml) were spiked with malvidin 3-galactoside to a final concentration of 1 mg/ml in the sample. As malvidin 3-galactoside is not detectable among elderberry anthocyanins, it serves as an appropriate internal standard to correct for possible anthocyanin loss during sample preparation. Anthocyanins were recovered from plasma samples by solid phase extraction using an octadecylsilane solid phase extraction cartridge (Sep-Pak C18) as previously described (24). Water-soluble compounds, polar-lipids and
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neutral-lipids in the plasma samples were removed during the solid phase extraction by elution with 25 mM sodium acetate aqueous solution (pH 1.5), dichloromethane and benzene, respectively. Finally, anthocyanins were recovered for HPLC analysis by elution with methanol containing 25 mM sodium acetate (pH 1.5) or for LC/MS analysis with methanol containing 5% formic acid. For HPLC analysis, methanol phase (anthocyanin) samples were evaporated to dryness, redissolved in methanol containing 25 mM sodium acetate, and mixed with the 25 mM sodium acetate aqueous solution (1:3, v/v). After centrifuging to remove any possible undissolved materials, the samples were injected by chilled autosampler onto a HP series 1100 system utilizing binary pumps, a Zorbax SB-C18 column (4.6× 250 nm), and a diode array detector. For LC/MS analysis, the anthocyanin containing methanol phase was evaporated to dryness and redissolved in methanol containing 5% formic acid. Low-resolution MS was performed using electrospray MS (ES-MS) on an Esquire-LC Mass Spectrometer (Bruker Daltonik, Bremen, Germany), an ion trap instrument equipped with an ES interface. The mass spectrometer was operated in the positive-ion mode (ESV, 4000 V; capillary exit, 95.6 V; capillary offset, 69.8 V; skim 1, 25.8 V; dry gas, 9 l/min; temperature, 300 °C). Urine samples were extracted using Sep-Pak C18 extraction cartridges. Ten milliliter of urine was loaded on preconditioned cartridges and washed with 15 ml of 25 mM sodium acetate aqueous solution (pH 1.5). Anthocyanins were recovered with 2 ml of methanol containing 25 mM sodium acetate (pH 1.5) or 5% formic acid, and used directly for HPLC or LC/MS analysis following filtration. The recovery of anthocyanins from urine was more than 90% as assessed by the internal standard.
3. Results Before consumption of the elderberry extract ‘drink’, no anthocyanins, other than spiked malvidin 3-galactoside internal standard, were detected at 520 nm in baseline plasma samples (Fig.
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1A). Plasma samples collected after consumption of the elderberry anthocyanins demonstrate at least five additional compounds detected at 520 nm (Fig. 1B– D); Peaks 1 and 2 (in Fig. 1B– D) have the same elution time and PDA spectrum as authentic standards of cyanidin 3-sambubioside and cyanidin 3-glucoside, respectively. Authentic
standards were used in both HPLC and LC/MS to verify retention times as slightly different gradients were used for plasma and urine HPLC and LC/MS required yet another mobile phase due to detector constraints. Fragmentation patterns in LC/MS analysis (Fig. 2) confirmed peak 1 and 2 identities as cyanidin 3-sambubioside and
Fig. 1. Representative HPLC chromatograms of plasma samples collected from elderly women before (A) and 10 (B), 20 (C), and 45 min (D) after consumption of 720 mg anthocyanins. Peak 1 and 2 had the elution time and spectrum of cyanidin 3-sambubioside and cyanidin 3-glucoside, respectively. All peaks other than the internal standard (malvidin 3-galactoside) decreased after 1 h and disappeared after 24 h. A binary linear gradient method was used for the HPLC analysis. Mobile phase A was 25 mM sodium acetate aqueous solution and mobile phase B was methanol containing 25 mM sodium acetate. Both mobile phases were adjusted to pH 1.5 with trichloroacetic acid. The flow rate was 1 ml/min.
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Fig. 2. LC/MS analysis of anthocyanins in the plasma samples collected before (A) and 60 min after (B) the consumption of 720 mg anthocyanins. A binary linear gradient method was used for the HPLC analysis. Mobile phase A was 0.5% formic acid and mobile phase B was methanol. The flow rate was 0.4 ml/min. Peak 1 and 2 labeled in this LC/MS analysis corresponded to the peak 1 and 2 showed in Fig. 1(B –D).
cyanidin 3-glucoside. The molecular ion of cyanidin 3-sambubioside (m/z: 581.1), which contains one xylose and one glucose, and its fragment, cyanidin aglycone (m/z: 287.0) were detected in the MS spectrum of the suspected cyanidin 3-sambubioside in plasma (Fig. 2). The molecular ion of cyanidin 3-glucoside (m/z: 449.1) and its aglycone (m/z: 287.0) were detected in the MS spectrum of the suspected cyanidin 3-glucoside in plasma (Fig. 2). The MS spectra of anthocyanin peaks detected in plasma were also the same as those of cyanidin 3-sambubioside and cyanidin 3-glucoside extracted from elderberries. LC/MS analysis also detected cyanidin aglycone and a malvidin hexoside in the plasma sample collected after consumption of the elderberry anthocyanins, although their concentrations were low. Unexpectedly, neither glucuronates nor sulfates of these anthocyanins were detected in the plasma samples. Maximum plasma concentration (Cmax) of to-
tal anthocyanins, including cyanidin 3-sambubioside and cyanidin 3-glucoside, in the four elderly women was ascertained by absorption at 520 nm, varied from 55.3 to 168.3 nmol/l with an average of 97.4 nmol/l, which was reached within 72 min (Tmax). The elimination of plasma anthocyanins appeared to follow first-order kinetics (Fig. 3). The elimination half-life (t1/2) of plasma total anthocyanins was calculated to be 133 min (Table 1). The Cmax, Tmax, and t1/2 of plasma cyanidin 3-sambubioside were 38.9 nmol/ l, 71.3 and 168.9 min, respectively. The Cmax, Tmax, and t1/2 of plasma cyanidin 3-glucoside were 42.5 nmol/l, 65 and 97 min, respectively (Table 1). No peaks were detected at 520 nm in the urine samples collected before consumption of the elderberry anthocyanins (Fig. 4A), but at least six peaks were detected at 520 nm in the urine samples collected after consumption of the elderberry anthocyanins (Fig. 4B–D). Two main peaks de-
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that had typical anthocyanin spectra revealed that most anthocyanin-like compounds were excreted in urine during the first 4 h. The total amount of the anthocyanins excreted during 24 h after the consumption of elderberry anthocyanins was calculated to be 397.0945.1 mg (cyanidin 3-glucoside equivalents). The excretion rate of the total anthocyanins was 77.29 10.9 mg/h during the first 4 h and 13.49 1.6 mg/h during the second 4 h (Fig. 6).
4. Discussion Fig. 3. Plasma anthocyanin concentrations after the consumption of 720 mg anthocyanins. Data are presented as mean 9 S.E. of four subjects. The point at 24 h represents all three lines.
tected in the urine collected after consumption of the elderberry anthocyanins were cyanidin 3-sambubioside and cyanidin 3-glucoside, respectively. The molecular ion of cyanidin 3-sambubioside (m/z: 581.1) and its aglycone (m/z: 287.0) were detected in the MS spectrum of the suspected cyanidin 3-sambubioside in urine (Fig. 5). The molecular ion of cyanidin 3-glucoside (m/z: 449.1) and its aglycone (m/z: 287.0) were detected in the MS spectrum of the suspected cyanidin 3-glucoside in urine (Fig. 5). The MS spectra of those two suspected anthocyanin peaks detected in urine were also the same as those of cyanidin 3-sambubioside and cyanidin 3-glucoside extracted from elderberries. Several peaks in the urine samples did not show typical anthocyanin spectra and could be anthocyanin metabolites. Examining those peaks
The mechanisms underlying the absorption of anthocyanins glycosides are not known. A recent study suggests that quercetin glucosides are capable of interacting with the sodium dependent glucose transport receptors in the mucosal epithelium (Gee et al., 1998). Quercetin and anthocyanidins (aglycones of anthocyanins) share a similar basic flavonoid structure. Therefore, the demonstration of the presence of anthocyanins in their unchanged glycosylated forms may indicate the involvement of the glucose transport receptors in the absorption of these compounds in vivo. The possibility exists that sulfated and glucuronated anthocyanin conjugates were not found by the diode array detector as configured. Alternatively, these compounds may not have been extracted during sample extraction as neither sulfatase nor D-glucuronidase digestion was employed during sample preparation. Hollman et al. (1997, 1999) has determined that the bioavailability of the flavonol quercetin depends upon which form is most prevalent in consumed food. Quercetin rutinoside, for example
Table 1 Main pharmacokinetic parameters of anthocyanins in elderly women after a single oral dose of 720 mg elderberry anthocyanins
Total anthocyanins Cyanidin 3-sambubioside Cyanidin 3-glucoside
Cmax (nmol/l)
Tmax (min)
k
t1/2 (min)
97.4 9 24.5* 38.99 7.4* 42.59 4.5
71.3 9 16.6 71.39 16.6 65.0 9 20.6
0.005355 9 0.000496 0.004534 9 0.00083 0.007230 90.000504
132.6 911.2 168.9 930.6 97.3 9 7.0
The data are presented as mean 9S.E. (n= 4). *Cyanidin 3-glucoside equivalent. Cmax, maximum plasma concentration; Tmax, time to reach the maximum plasma concentration; k, terminal elimination rate constant [slope*(−2.303), the slope was derived from linear regression of the terminal portion of the log plasma concentration versus time profile]; t1/2, elimination half-life (ln 2/k).
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Fig. 4. Representative HPLC chromatograms of urine samples collected from the elderly women before (A); and 0 – 2 (B); 2– 4 (C); and 12 – 24 h (D) after the consumption of 720 mg anthocyanins. Peak 1 and 2 had the elution time and spectrum of cyanidin 3-sambubioside and cyanidin 3-glucoside, respectively. A binary linear gradient method was used for the HPLC analysis. The gradient was modified from that for plasma samples, hence a shift in retention time is observed. Retention times were verified with authentic standards. Mobile phase A was 25 mM sodium acetate aqueous solution and mobile phase B was methanol containing 25 mM sodium acetate. Both mobile phases were adjusted to pH 1.5 with trichloroacetic acid. The flow rate was 1 ml/min.
has a bioavailability only 20% that of the glucoside form. These authors suggest that quercetin rutinoside is absorbed in the colon while quercetin glucoside is absorbed in the small intestine. Onions, high in the glucoside form of quercetin, provide more bioavailable quercetin, than does tea or wine that is higher in the rutinoside form
(de Vries et al. 2001). Olthof et al. (2000) have also demonstrated that quercetin glucosides are rapidly absorbed in humans whether the glucoside is in the quercetin 3% or 4% positions and speculate, as does Aziz et al. (1998), that these molecules are absorbed via the sodium-glucose co-transporter. This theory has yet to be proven in vivo. Olthof et
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al. data indicate that 50% of consumed quercetin glucosides are absorbed in the small intestines and subsequently metabolized in the liver to compounds such as isorhamnetin with the remainder perhaps metabolized by colonic microfluora and absorbed as the aglycone or other phenolic metabolites. It is clear that very little (B 3%) of total consumed quercetin is recovered in urine. Recent in vitro studies of human intestinal Caco2 cell show a lack of quercetin glucoside absorption (Walgren et al., 2000). This prompted these investigators to reinvestigate the absorption of quercetin glucosides in ileostomy patients where they were unable to detect either quercetin 4%-monoglucoside or quercetin 3,4% diglucoside in ileostomy fluids (Walle et al., 2000). The investigators conclude that both glucosides were effectively hydrolyzed in the human small intestine to the aglycone that is then absorbed. The exact location or agent of this hydrolysis is unknown. Clearly further investigation of flavonol absorption is needed.
Animal studies of the absorption of the anthocyanin cyanidin 3-O-beta-glucoside (Tsuda et al., 1999) found that when administered orally the glucoside rapidly appeared in the plasma of rats. The aglycone was not detected in plasma, however, it was detected in the jejunum. Protocatechuic acid, a degradation product of cyanidin was found in plasma. In the liver and kidney, cyanidin 3-O-betaglucoside was metabolized to methylated cyanidin 3-O-beta-glucoside. Flavonols and anthocyanidins differ in structure in that the flavonols posses a keto structure on the pyran C ring and the anthocyanidin does not. This structural difference can have a profound influence on the resonance within the C ring and affect the nature of the oxygen within the pyran ring. More research is required to determine if this difference may have an effect on absorption. Cao et al. (1998) demonstrated 7–25% increases in serum antioxidant capacity in eight elderly women after consumption of strawberries, spinach,
Fig. 5. LC/MS analysis of anthocyanins in the urine samples collected before (A); and 0 – 2 h after (B) the consumption of 720 mg anthocyanins. A binary linear gradient method was used for the HPLC analysis. Mobile phase A was 0.5% formic acid and mobile phase B was methanol. The flow rate was 0.4 ml/min. Peak 1 and 2 labeled in this HPLC/MS analysis corresponded to the peak 1 and 2 showed in Fig. 4(B –D).
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antioxidant status. The capacity of flavonoids to act as antioxidants in vitro and the important structure–activity relationships of the antioxidant activity have been established and literature in this regard has been reviewed by Pietta (2000). The antioxidant efficacy of flavonoids in vivo is less documented. Information on the bioavailability of different flavonoid groups is limited, however, it is crucial that we understand differences that may occur if we are to sort out the potential health benefits, antioxidant or otherwise, of both flavonoids and flavonoid metabolites. In summary, this study demonstrates that anthocyanins appear to be absorbed in their unchanged glycosylated forms in humans and provides measurements of the pharmacokinetic parameters of dietary anthocyanins absorption.
Acknowledgements
Fig. 6. Excretion of total anthocyanins in urine after the consumption of 720 mg anthocyanins. (A) Excreted anthocyanins were expressed as mg; (B) excreted anthocyanins were expressed as mg per hour. Data are presented as mean 9 S.E. of four subjects.
red wine or vitamin C. Rein et al. (2000) using an 80-g bolus of semisweet chocolate, rich in the flavan-3-ol epicatechin and procyanidin oligomers demonstrated a 12-fold increase in plasma epicatechin, from 22 to 257 nmol/l by 2 h after ingestion. This rise in epicatechin was accompanied by a significant increase of 31% in plasma total antioxidant capacity and a decrease of 40% in plasma 2-thiobarbituric acid reactive substances. These authors suggest that the increases in antioxidant capacity are due to the ability of flavonoids to quench radicals, however, there is a growing belief that flavonoids may affect antioxidant capacity via multiple mechanisms. Clearly, in one form or another, flavonoids as a group are absorbed and may play a significant role in altering
Appreciation is expressed to Dr Robert M. Russell, for serving as study physician for this experiment; to Dr Gregory G. Dolnikowski for his assistance in performing LC/MS analysis; to Helen Rasmussen for her assistance in the formulation of the diets; and to the nursing staff of the Metabolic Research Unit at the HNRCA for their assistance in the care of the subjects.
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Bioavailablility of elderberry anthocyanins Paul E. Milbury *, Guohua Cao, Ronald L. Prior, Jeffrey Blumberg Antioxidants Research Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts Uni6ersity, Room 507, 711 Washington St. Boston, MA 02111, USA
Abstract Considerable epidemiological evidence suggests a link between the consumption of diets rich in fruits and vegetables and a decreased risk of cardiovascular disease and cancers. Anthocyanins have received attention as important dietary constituents that may provide health benefits and contribute antioxidant capacity beyond that provided by essential micronutrients such as ascorbate, tocopherols, and selenium. The emergence of renewed interest by industrial countries in traditional herbal medicines and the development of ‘functional foods’ are stimulating the need for more information regarding the bioavailability and efficacy of plant polyphenols. Flavonoids represent a numerous group of secondary plant metabolites based on the structure of a pyran ring flanked by two or more phenyl rings and varying subtly in the degree of unsaturation and the pattern of hydroxylation or methylation. Flavonoids also vary in the type of sugar attached or the degree of polymerization. Anthocyanins, potent flavonoid antioxidants widely distributed in fruits, vegetables and red wines, normally occur in nature as glycosides, a form not usually considered as bioavailable. We have examined the bioavailability and pharmacokinetics of anthocyanins in humans. Anthocyanins were detected as glycosides in both plasma and urine samples. The elimination of plasma anthocyanins appeared to follow first-order kinetics and most anthocyanin compounds were excreted in urine within 4 h after feeding. The current findings appear to refute assumptions that anthocyanins are not absorbed in their unchanged glycosylated forms in humans. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Anthocyanins; Flavonoids; Antioxidant; Pharmacokinetics; Absorption; Elderly; Elderberry
1. Introduction There is great interest in determining the role of phytonutrients in promoting improved health and in reducing cancer, cardiovascular disease, and the effects of aging. It is widely believed that antioxidant phytonutrients can inhibit the propagation of free radical reactions that may ulti* Corresponding author. Tel.: +1-617-556-3095; fax: + 1617-556-3344. E-mail address: [email protected] (P.E. Milbury).
mately lead to the development of diseases, especially those which are aging related. Analysis in several laboratories shows that many fruits and vegetables have strong antioxidant capacities, and that this capacity is due primarily to non-vitamin C phytochemicals (Cao et al., 1996; Wang et al., 1996; Guo et al., 1997; Prior et al., 1998). To date the proof that dietary antioxidant phytochemicals can reduce human diseases by inhibiting in vivo oxidative processes has been elusive. There has been insufficient study that has incorporated rigorous biochemical screens of redox
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status and antioxidant uptake in human intervention trials combined with monitoring of biomarkers of oxidative stress and correlation with disease endpoints. The best studies to date in this regarding uptake involve study of flavonoid compounds in tea, cherries, and wine (Hollman et al., 1997; Wiseman et al., 1997; Wang et al., 1999a,b,c). Flavonoids are a large group of natural phenolic compounds, consisting mainly of flavonols, flavanols and anthocyanidins. Among these flavonoids, the water-soluble glycosides and acylglycosides of anthocyanins are an important group of natural antioxidants (Sichel et al., 1991; van Acker et al., 1995; Vinson et al., 1995; Tsuda et al., 1996; van Acker et al., 1996; Wang et al., 1997). While in vitro antioxidant activities of anthocyanins are superior to vitamin E (Wang et al., 1999a), little is known regarding comparable in vivo capacity or the bioavailability of these compounds. Anthocyanin variations lend to a complexity that has made these compounds, as a group, difficult to study with regard to bioavailability. Anthocyanins vary in the degree of unsaturation and the pattern of hydroxylation, methylation, and degree of polymerization, and this variation greatly influences the antioxidant capabilities of the molecules (Milbury, 2001). Tea beverages are rich sources of flavonoids, predominantly catechins. Red wines contain flavonoids that can vary in content between 1 and 3 g/l the majority of which is usually flavanols as monomers and oligomers, however, anthocyanins can comprise 20–80% of the flavonoid content (Glories, 1988). Berries represent another rich plant sources of dietary anthocyanins. Intake of anthocyanins from berry sources has been estimated to be 180– 215 mg per day among the US population (Ku¨ hnau, 1976). This figure far exceeds the estimated intake of 23 mg per day of other flavonoids such as luteolin, kaempferol, myricetin, apigenin and quercetin (Hertog et al., 1993). The most common naturally occurring anthocyanins in plants are in the 3-O-glycoside or 3,5-di-O-glycoside forms, which were considered nonabsorbable in humans. Due to this belief, study of the pharmacokinetics of dietary an-
thocyanins was neglected in humans. Recent developments in HPLC methodology to measure the anthocyanins in plasma permitted the demonstration that anthocyanins are absorbed and are bioavailable to humans (Cao and Prior, 1999). This paper presents evidence that anthocyanins are absorbed in their original glycosylated forms in human subjects. The pharmacokinetics of dietary berry anthocyanins were determined in four elderly women.
2. Materials and methods
2.1. Subjects Four women (age: 6794 years) were recruited to participate in a pharmacokinetics study. These subjects were in good health as determined by a medical history questionnaire, physical examination, and clinical laboratory test results. Eligibility criteria for inclusion included: (1) no history of cardiovascular, hepatic, gastrointestinal, or renal disease; (2) no alcoholism; (3) no supplemental vitamin and/or mineral use or antibiotic use during a 4-week period prior to the start of the study; and (4) no smoking. The study protocol was reviewed and approved by the Human Investigation Review Committee of Tufts University and the New England Medical Center. Written informed consent was obtained from each study participant.
2.2. Study design Subjects reported 48 h prior to the experimental day for admittance to the Metabolic Research Unit (MRU) at the Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging at Tufts University. The diets provided to these subjects during their residency in the MRU were designed to have no anthocyanins and to be low in other flavonoids, but provided the Recommended Dietary Allowance for protein and energy (Cao et al., 1998). All meals were prepared under the supervision of a dietitian in the MRU. The consumption of water was not limited. Other foods or beverages were not allowed during the
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residency. Subjects were fasted overnight before the day of sampling. In the morning of the sampling day, an intravenous catheter was inserted into one forearm and a 15 ml baseline sample blood sample was collected in heparinized tubes. Each subject was then given 12 g elderberry extract, dissolved in 500 ml water. The extract ‘drink’ contained 720 mg anthocyanins by analysis and was composed predominantly of cyanidin 3-sambubioside, cyanidin 3-glucoside, and maltodextrin. Elderberry was selected for this study because of its simplicity with regard to anthocyanins with cyanidin 3-sambubioside and cyanidin 3-glucoside accounting for 92.5% of the total anthocyanins detectable within the berry (Prior et al., 1998). Blood samples (15 ml) were collected again at 10, 20, 30, 45 min, 1, 2, 4, 6 and 24 h after consumption of the elderberry anthocyanins. Urine samples were collected from these subjects before the consumption of the elderberry anthocyanins and between 0– 2, 2– 4, 4– 6, 6–8, 8–12 and 12– 24 h after the consumption of the elderberry anthocyanins. Lunch and dinner were given following the blood sampling at 4 and 11 h, respectively, after consumption of the drink.
2.3. Anthocyanin analysis in plasma and urine Blood samples were centrifuged at 500× g for 10 min at 4 °C and plasma was removed for immediate treatment with TFA. Both plasma and urine samples were treated with 0.44 mol/l TFA aqueous solution (1:0.2, v/v) as previously described (24). TFA treated plasma and urine samples were stored at − 80 °C prior to anthocyanin analysis by HPLC and HPLC/MS. The plasma samples (2 ml) were spiked with malvidin 3-galactoside to a final concentration of 1 mg/ml in the sample. As malvidin 3-galactoside is not detectable among elderberry anthocyanins, it serves as an appropriate internal standard to correct for possible anthocyanin loss during sample preparation. Anthocyanins were recovered from plasma samples by solid phase extraction using an octadecylsilane solid phase extraction cartridge (Sep-Pak C18) as previously described (24). Water-soluble compounds, polar-lipids and
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neutral-lipids in the plasma samples were removed during the solid phase extraction by elution with 25 mM sodium acetate aqueous solution (pH 1.5), dichloromethane and benzene, respectively. Finally, anthocyanins were recovered for HPLC analysis by elution with methanol containing 25 mM sodium acetate (pH 1.5) or for LC/MS analysis with methanol containing 5% formic acid. For HPLC analysis, methanol phase (anthocyanin) samples were evaporated to dryness, redissolved in methanol containing 25 mM sodium acetate, and mixed with the 25 mM sodium acetate aqueous solution (1:3, v/v). After centrifuging to remove any possible undissolved materials, the samples were injected by chilled autosampler onto a HP series 1100 system utilizing binary pumps, a Zorbax SB-C18 column (4.6× 250 nm), and a diode array detector. For LC/MS analysis, the anthocyanin containing methanol phase was evaporated to dryness and redissolved in methanol containing 5% formic acid. Low-resolution MS was performed using electrospray MS (ES-MS) on an Esquire-LC Mass Spectrometer (Bruker Daltonik, Bremen, Germany), an ion trap instrument equipped with an ES interface. The mass spectrometer was operated in the positive-ion mode (ESV, 4000 V; capillary exit, 95.6 V; capillary offset, 69.8 V; skim 1, 25.8 V; dry gas, 9 l/min; temperature, 300 °C). Urine samples were extracted using Sep-Pak C18 extraction cartridges. Ten milliliter of urine was loaded on preconditioned cartridges and washed with 15 ml of 25 mM sodium acetate aqueous solution (pH 1.5). Anthocyanins were recovered with 2 ml of methanol containing 25 mM sodium acetate (pH 1.5) or 5% formic acid, and used directly for HPLC or LC/MS analysis following filtration. The recovery of anthocyanins from urine was more than 90% as assessed by the internal standard.
3. Results Before consumption of the elderberry extract ‘drink’, no anthocyanins, other than spiked malvidin 3-galactoside internal standard, were detected at 520 nm in baseline plasma samples (Fig.
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1A). Plasma samples collected after consumption of the elderberry anthocyanins demonstrate at least five additional compounds detected at 520 nm (Fig. 1B– D); Peaks 1 and 2 (in Fig. 1B– D) have the same elution time and PDA spectrum as authentic standards of cyanidin 3-sambubioside and cyanidin 3-glucoside, respectively. Authentic
standards were used in both HPLC and LC/MS to verify retention times as slightly different gradients were used for plasma and urine HPLC and LC/MS required yet another mobile phase due to detector constraints. Fragmentation patterns in LC/MS analysis (Fig. 2) confirmed peak 1 and 2 identities as cyanidin 3-sambubioside and
Fig. 1. Representative HPLC chromatograms of plasma samples collected from elderly women before (A) and 10 (B), 20 (C), and 45 min (D) after consumption of 720 mg anthocyanins. Peak 1 and 2 had the elution time and spectrum of cyanidin 3-sambubioside and cyanidin 3-glucoside, respectively. All peaks other than the internal standard (malvidin 3-galactoside) decreased after 1 h and disappeared after 24 h. A binary linear gradient method was used for the HPLC analysis. Mobile phase A was 25 mM sodium acetate aqueous solution and mobile phase B was methanol containing 25 mM sodium acetate. Both mobile phases were adjusted to pH 1.5 with trichloroacetic acid. The flow rate was 1 ml/min.
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Fig. 2. LC/MS analysis of anthocyanins in the plasma samples collected before (A) and 60 min after (B) the consumption of 720 mg anthocyanins. A binary linear gradient method was used for the HPLC analysis. Mobile phase A was 0.5% formic acid and mobile phase B was methanol. The flow rate was 0.4 ml/min. Peak 1 and 2 labeled in this LC/MS analysis corresponded to the peak 1 and 2 showed in Fig. 1(B –D).
cyanidin 3-glucoside. The molecular ion of cyanidin 3-sambubioside (m/z: 581.1), which contains one xylose and one glucose, and its fragment, cyanidin aglycone (m/z: 287.0) were detected in the MS spectrum of the suspected cyanidin 3-sambubioside in plasma (Fig. 2). The molecular ion of cyanidin 3-glucoside (m/z: 449.1) and its aglycone (m/z: 287.0) were detected in the MS spectrum of the suspected cyanidin 3-glucoside in plasma (Fig. 2). The MS spectra of anthocyanin peaks detected in plasma were also the same as those of cyanidin 3-sambubioside and cyanidin 3-glucoside extracted from elderberries. LC/MS analysis also detected cyanidin aglycone and a malvidin hexoside in the plasma sample collected after consumption of the elderberry anthocyanins, although their concentrations were low. Unexpectedly, neither glucuronates nor sulfates of these anthocyanins were detected in the plasma samples. Maximum plasma concentration (Cmax) of to-
tal anthocyanins, including cyanidin 3-sambubioside and cyanidin 3-glucoside, in the four elderly women was ascertained by absorption at 520 nm, varied from 55.3 to 168.3 nmol/l with an average of 97.4 nmol/l, which was reached within 72 min (Tmax). The elimination of plasma anthocyanins appeared to follow first-order kinetics (Fig. 3). The elimination half-life (t1/2) of plasma total anthocyanins was calculated to be 133 min (Table 1). The Cmax, Tmax, and t1/2 of plasma cyanidin 3-sambubioside were 38.9 nmol/ l, 71.3 and 168.9 min, respectively. The Cmax, Tmax, and t1/2 of plasma cyanidin 3-glucoside were 42.5 nmol/l, 65 and 97 min, respectively (Table 1). No peaks were detected at 520 nm in the urine samples collected before consumption of the elderberry anthocyanins (Fig. 4A), but at least six peaks were detected at 520 nm in the urine samples collected after consumption of the elderberry anthocyanins (Fig. 4B–D). Two main peaks de-
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that had typical anthocyanin spectra revealed that most anthocyanin-like compounds were excreted in urine during the first 4 h. The total amount of the anthocyanins excreted during 24 h after the consumption of elderberry anthocyanins was calculated to be 397.0945.1 mg (cyanidin 3-glucoside equivalents). The excretion rate of the total anthocyanins was 77.29 10.9 mg/h during the first 4 h and 13.49 1.6 mg/h during the second 4 h (Fig. 6).
4. Discussion Fig. 3. Plasma anthocyanin concentrations after the consumption of 720 mg anthocyanins. Data are presented as mean 9 S.E. of four subjects. The point at 24 h represents all three lines.
tected in the urine collected after consumption of the elderberry anthocyanins were cyanidin 3-sambubioside and cyanidin 3-glucoside, respectively. The molecular ion of cyanidin 3-sambubioside (m/z: 581.1) and its aglycone (m/z: 287.0) were detected in the MS spectrum of the suspected cyanidin 3-sambubioside in urine (Fig. 5). The molecular ion of cyanidin 3-glucoside (m/z: 449.1) and its aglycone (m/z: 287.0) were detected in the MS spectrum of the suspected cyanidin 3-glucoside in urine (Fig. 5). The MS spectra of those two suspected anthocyanin peaks detected in urine were also the same as those of cyanidin 3-sambubioside and cyanidin 3-glucoside extracted from elderberries. Several peaks in the urine samples did not show typical anthocyanin spectra and could be anthocyanin metabolites. Examining those peaks
The mechanisms underlying the absorption of anthocyanins glycosides are not known. A recent study suggests that quercetin glucosides are capable of interacting with the sodium dependent glucose transport receptors in the mucosal epithelium (Gee et al., 1998). Quercetin and anthocyanidins (aglycones of anthocyanins) share a similar basic flavonoid structure. Therefore, the demonstration of the presence of anthocyanins in their unchanged glycosylated forms may indicate the involvement of the glucose transport receptors in the absorption of these compounds in vivo. The possibility exists that sulfated and glucuronated anthocyanin conjugates were not found by the diode array detector as configured. Alternatively, these compounds may not have been extracted during sample extraction as neither sulfatase nor D-glucuronidase digestion was employed during sample preparation. Hollman et al. (1997, 1999) has determined that the bioavailability of the flavonol quercetin depends upon which form is most prevalent in consumed food. Quercetin rutinoside, for example
Table 1 Main pharmacokinetic parameters of anthocyanins in elderly women after a single oral dose of 720 mg elderberry anthocyanins
Total anthocyanins Cyanidin 3-sambubioside Cyanidin 3-glucoside
Cmax (nmol/l)
Tmax (min)
k
t1/2 (min)
97.4 9 24.5* 38.99 7.4* 42.59 4.5
71.3 9 16.6 71.39 16.6 65.0 9 20.6
0.005355 9 0.000496 0.004534 9 0.00083 0.007230 90.000504
132.6 911.2 168.9 930.6 97.3 9 7.0
The data are presented as mean 9S.E. (n= 4). *Cyanidin 3-glucoside equivalent. Cmax, maximum plasma concentration; Tmax, time to reach the maximum plasma concentration; k, terminal elimination rate constant [slope*(−2.303), the slope was derived from linear regression of the terminal portion of the log plasma concentration versus time profile]; t1/2, elimination half-life (ln 2/k).
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Fig. 4. Representative HPLC chromatograms of urine samples collected from the elderly women before (A); and 0 – 2 (B); 2– 4 (C); and 12 – 24 h (D) after the consumption of 720 mg anthocyanins. Peak 1 and 2 had the elution time and spectrum of cyanidin 3-sambubioside and cyanidin 3-glucoside, respectively. A binary linear gradient method was used for the HPLC analysis. The gradient was modified from that for plasma samples, hence a shift in retention time is observed. Retention times were verified with authentic standards. Mobile phase A was 25 mM sodium acetate aqueous solution and mobile phase B was methanol containing 25 mM sodium acetate. Both mobile phases were adjusted to pH 1.5 with trichloroacetic acid. The flow rate was 1 ml/min.
has a bioavailability only 20% that of the glucoside form. These authors suggest that quercetin rutinoside is absorbed in the colon while quercetin glucoside is absorbed in the small intestine. Onions, high in the glucoside form of quercetin, provide more bioavailable quercetin, than does tea or wine that is higher in the rutinoside form
(de Vries et al. 2001). Olthof et al. (2000) have also demonstrated that quercetin glucosides are rapidly absorbed in humans whether the glucoside is in the quercetin 3% or 4% positions and speculate, as does Aziz et al. (1998), that these molecules are absorbed via the sodium-glucose co-transporter. This theory has yet to be proven in vivo. Olthof et
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al. data indicate that 50% of consumed quercetin glucosides are absorbed in the small intestines and subsequently metabolized in the liver to compounds such as isorhamnetin with the remainder perhaps metabolized by colonic microfluora and absorbed as the aglycone or other phenolic metabolites. It is clear that very little (B 3%) of total consumed quercetin is recovered in urine. Recent in vitro studies of human intestinal Caco2 cell show a lack of quercetin glucoside absorption (Walgren et al., 2000). This prompted these investigators to reinvestigate the absorption of quercetin glucosides in ileostomy patients where they were unable to detect either quercetin 4%-monoglucoside or quercetin 3,4% diglucoside in ileostomy fluids (Walle et al., 2000). The investigators conclude that both glucosides were effectively hydrolyzed in the human small intestine to the aglycone that is then absorbed. The exact location or agent of this hydrolysis is unknown. Clearly further investigation of flavonol absorption is needed.
Animal studies of the absorption of the anthocyanin cyanidin 3-O-beta-glucoside (Tsuda et al., 1999) found that when administered orally the glucoside rapidly appeared in the plasma of rats. The aglycone was not detected in plasma, however, it was detected in the jejunum. Protocatechuic acid, a degradation product of cyanidin was found in plasma. In the liver and kidney, cyanidin 3-O-betaglucoside was metabolized to methylated cyanidin 3-O-beta-glucoside. Flavonols and anthocyanidins differ in structure in that the flavonols posses a keto structure on the pyran C ring and the anthocyanidin does not. This structural difference can have a profound influence on the resonance within the C ring and affect the nature of the oxygen within the pyran ring. More research is required to determine if this difference may have an effect on absorption. Cao et al. (1998) demonstrated 7–25% increases in serum antioxidant capacity in eight elderly women after consumption of strawberries, spinach,
Fig. 5. LC/MS analysis of anthocyanins in the urine samples collected before (A); and 0 – 2 h after (B) the consumption of 720 mg anthocyanins. A binary linear gradient method was used for the HPLC analysis. Mobile phase A was 0.5% formic acid and mobile phase B was methanol. The flow rate was 0.4 ml/min. Peak 1 and 2 labeled in this HPLC/MS analysis corresponded to the peak 1 and 2 showed in Fig. 4(B –D).
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antioxidant status. The capacity of flavonoids to act as antioxidants in vitro and the important structure–activity relationships of the antioxidant activity have been established and literature in this regard has been reviewed by Pietta (2000). The antioxidant efficacy of flavonoids in vivo is less documented. Information on the bioavailability of different flavonoid groups is limited, however, it is crucial that we understand differences that may occur if we are to sort out the potential health benefits, antioxidant or otherwise, of both flavonoids and flavonoid metabolites. In summary, this study demonstrates that anthocyanins appear to be absorbed in their unchanged glycosylated forms in humans and provides measurements of the pharmacokinetic parameters of dietary anthocyanins absorption.
Acknowledgements
Fig. 6. Excretion of total anthocyanins in urine after the consumption of 720 mg anthocyanins. (A) Excreted anthocyanins were expressed as mg; (B) excreted anthocyanins were expressed as mg per hour. Data are presented as mean 9 S.E. of four subjects.
red wine or vitamin C. Rein et al. (2000) using an 80-g bolus of semisweet chocolate, rich in the flavan-3-ol epicatechin and procyanidin oligomers demonstrated a 12-fold increase in plasma epicatechin, from 22 to 257 nmol/l by 2 h after ingestion. This rise in epicatechin was accompanied by a significant increase of 31% in plasma total antioxidant capacity and a decrease of 40% in plasma 2-thiobarbituric acid reactive substances. These authors suggest that the increases in antioxidant capacity are due to the ability of flavonoids to quench radicals, however, there is a growing belief that flavonoids may affect antioxidant capacity via multiple mechanisms. Clearly, in one form or another, flavonoids as a group are absorbed and may play a significant role in altering
Appreciation is expressed to Dr Robert M. Russell, for serving as study physician for this experiment; to Dr Gregory G. Dolnikowski for his assistance in performing LC/MS analysis; to Helen Rasmussen for her assistance in the formulation of the diets; and to the nursing staff of the Metabolic Research Unit at the HNRCA for their assistance in the care of the subjects.
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