Ronald L. Prior, Ph.D. and Xianli Wu, Ph.D., USDA, ARS, Arkansas Children’s Nutrition Center, Little Rock, AR 72202
Anthocyanins are water-soluble glycosides and acylglycosides of anthocyanidins. They are widely distributed in foods of plant origin, especially in fruits with dark red and blue colors. Anthocyanins are strong antioxidants in vitro. Whether anthocyanins are effective antioxidants in vivo is still an open question primarily because of the relatively low apparent absorption of anthocyanins compared to other phenolic compounds. In order to understand potential mechanisms whereby anthocyanins produce health benefits, an understanding of their bioavailability and metabolism is critical. Although there have been several recent studies of the apparent absorption of anthocyanins, much remains to be learned about the metabolism of anthocyanins and what might account for some rather large differences in metabolites and urinary excretion rates observed.
Berry Anthocyanin Composition: The absorption/metabolism of five different berries (Marionberry, MB; Black Raspberry, BR; Black Currant, BC; Chokeberry, CB; Elderberry, EB) have been studied in a weanling pig model (6-10 kg BW). Freeze dried berry powders were given by stomach tube to study the absorption and metabolism of anthocyanins (ACNs). Four major ACNs were found in MB: cyanidin-3-glucoside (Cy-3-glc, 78%), cyanidin-3-rutinoside (Cy-3-rut, 20%), pelargonidin-3-glucoside (Pg-3-glc, 0.4%) and one unknown acylated cyanidin based ACN (UACy, 1.5%). There were 5 major ACN’s in BR (Cy-3-rut, 57.7%; cyanidin-3-sambubioside-5-rhamnoside (Cy-3-sam-5RH), 27.7%; Cy-3-glc, 9.5%; cyanidin-3-sambubioside (Cy-3-sam), 2.9%; and pelargonidin-3-rutinoside (Pg-3-rut), 2.2%). Four major ACNs were present in BC (delphinidin-3-rutinoside (Dp-3-rut), 44.8%; delphinidin-3-glucoside (Dp-3-glc), 25.1%; Cy-3-rut, 23.5%; and Cy-3-glc, 6.6%) and four in CB (cyanidin-3-galactoside (Cy-3-gal), 65.6%; cyanidin-3-arabinoside (Cy-3-arab), 28.3%; Cy-3-glc 2.5%; and cyanidin-3-xyloside (Cy-3-xyl), 3.7%). Three major ACNs were present in EB: Cy-3-glc, 53.8%; Cy-3-sam, 39.7%; and cyanidin-3-sambubioside-5-glucoside (Cy-3-sam-5glc), 6.0%.
Anthocyanins in the Gastrointestinal (GI) Tract of the Pig Following Black Raspberry Consumption: Freeze dried BR powder has a very high in vitro antioxidant capacity (ORAC: 901 μmol TE/g, dry weight). Five weaningling pigs (16.3±5.8 kg) were fed BR powder. After 4 hours, the pigs were killed and the contents in five segements of the GI tract (small intestine, cecum, proximal ileum, terminal ileum and large intestine) were collected for the analysis of anthocyanins and antioxidant capacity. Total recovery of antioxidant capacity (ORAC) was 43.6±6.4% which was similar to that of total ACNs which was 41.7±10.9% (Mean±SD, % of dose). However, recovery of individual anthocyanins was quite different. Recovery of Cy-3-sam, Pg-3-rut, Cy-3-sam5RH, Cy-3-rut and Cy-3-glc was 78.2±17.2, 50.0±8.0, 46.4±3.5, 43.7±6.4 and 1.7±1.0% (Mean±SEM, % of dose), respectively. Cy-3-glc showed a very low recovery in the GI tract. Since we saw a relatively high amount of Cy-3-sam, cleavage of rhamnose from Cy-3-sam5RH seems to happen in GI tract. Increased recovery in the GI tract indicates an apparent decreased absorption and/or decreased degradation within the GI tract.
Absorption/Metabolism of Anthocyanins: In the urine from the berry fed pigs, all of the original major ACNs from each berry were present in the urine in the intact glycosylated form. Methylated froms (either 3' or 4' position of the flavylium ring) were observed in the urine for all of the original anthocyanins in these berries except delphinidin and pelaragonidin. The cyanidin-monoglucuronide metabolite as well as the mixed conjugates (methylated + glucuronidated forms) likewise was observed following consumption of all berries. Cyanidin-3-glucoside-monoglucuronide and the mixed conjugate was only observed following the consumption of MB or EB, which is likely due to the low amounts of Cy-3-glc consumed from BR, BC or CB.
In pigs given MB, eleven metabolites were identified and quantified. Total recovery of the four original ACNs plus their related metabolites was over 6 times greater for Pg-3-glc than for Cy-3-glc or Cy-3-rutin. The plasma concentration/dose ratio of Cy-3-rutin was significantly higher than that of Cy-3-glc. Most of the Cy-3-glc (65.3% of dose) and Pg-3-glc (91.3% of dose) were excreted in the form of metabolites, whereas only 8.1% of the Cy-3-rut was excreted in a metabolized form. Urinary recovery of an unidentified cyanidin based acylated anthocyanin was lower than that of nonacylated anthocyanins.
In urine samples from pigs fed BR, besides the 5 major parent compounds (Cy-3-glc, Cy-3-sam5RH, Cy-3-rut and Pg-3-rut), 7 of 11 metabolites were identified. They were cyanidin monoglucuroinide, (Iso)Peonidin-3-glucoside, (Iso)Peonidin-3 monoglucuronide, and Pg monoglucuronide.
A total of 7 metabolites from BC and 14 from CB were identified in urine of pigs fed the respective berries. Dp ACNs from BC were found to have lower urine recoveries compared to Cy ACNs. Dp ACNs were present in urine as their original form, whereas a high proportion of Cy ACNs in urine were in metabolized forms. Recoveries (% of dose) of rutinosides of Dp and Cy were higher than the recovery of the glucoside forms in the urine. In CB fed pig, urine concentrations of cyanidin monoglucuronide were more than 2-fold higher than that of Cy-3-glc, suggesting that the formation of glucuronide was not directly from glucoside but that glucuronidation occurred following cleavage of the sugar from the aglycone.
Conclusions: ACN metabolism in the pig appears similar to that observed in the human. ACNs with complex glycosylation patterns remained at higher concentrations for a longer period of time in the GI tract. Intestinal cells may be exposed to ACN concentrations as high as 800 μM. Major parent ACNs from all berries and from 7-14 different metabolites were identified in pig urine after berry feeding. Methylation and glucuronidation were the two major pathways in the metabolism of ACNs. Delphinidin (Dp) ACNs were found to have lower urinary recoveries compared to cyanidin (Cy) or pelargonidin (Pg) and were present in urine in their original intact form, whereas a high proportion of Cy and Pg ACNs in urine were metabolized to methylated and/or glucuronide forms. Different ACN aglycones and sugar moieties significantly influence the absorption/metabolism of ACNs in vivo. Metabolically, anthocyanins (ACNs) appear to be quite different from other flavonoids with much lower quantities excreted in the urine relative to the amount consumed.
References:
(1) Wu, X.; Prior, R. L. Anthocyanidin and glycoside moieties alter anthocyanin absorption and metabolism following a meal of berries in the weanling pig. J. Nutr. 2005, (Submitted).
(2) Wu, X.; Pittman, H. E. I.; Prior, R. L. Fate of Black Raspberry (BR) Anthocyanin (ACN) and Antioxidant Capacity in Contents of the Gastrointestinal (GI) Tract of Weanling Pigs. J. Nutr. 2005, 154, (In preparation).
(3) Wu, X.; Prior, R. L. Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/MS in Common Foods in the United States: Fruits and Berries. J. Agric. Food Chem. 2005, 53, 2589-2599.
(4) Cho, M. J.; Howard, L. R.; Prior, R. L.; Clark, J. R. Flavonol glycosides and antioxidant capacity of various blackberry and blueberry genotypes determined by high-performance liquid chromatography/mass spectrometry. J. Sci. Food Agric. 2005, In press.
(5) Wu, X.; Gu, L.; Prior, R. L.; McKay, S. Characterization of anthocyanins and proanthocyanins in some cultivars of Ribes, Aronia and Sambucus and their antioxidant capacity. J. Agric. Food Chem. 2004, 52, 7846-7856.
(6) Wu, X.; Pittman, H. E.; Prior, R. L. Pelargonidin Is Absorbed and Metabolized Differently than Cyanidin after Marionberry Consumption in Pigs. J. Nutr. 2004, 134, 2603-2610.
(7) Prior, R. L.; Joseph, J. Berries and Fruits in Cancer Chemoprevention. In Phytopharmaceuticals in cancer chemoprevention; D. Bagchi and H. G. Preuss, Eds.; CRC Press: Boca Raton, 2004; pp 465-479.
(8) Prior, R. L. Absorption and metabolism of anthocyanins: Potential Health Effects. In Phytochemicals: Mechanisms of Action; M. Meskin; W. R. Bidlack; A. J. Davies; D. S. Lewis and R. K. Randolph, Eds.; CRC Press: Boca Raton, FL, 2004; pp 1-19.
(9) Wu, X.; Cao, G.; Prior, R. L. Absorption and metabolism of anthocyanins in human subjects following consumption of elderberry or blueberry. J. Nutr. 2002, 132, 1865-1871.
(10) Cao, G.; Muccitelli, H. U.; Sanchez-Moreno, C.; Prior, R. L. Anthocyanins are absorbed in glycated forms in elderly women: a pharmacokinetic study. Am. J. Clin. Nutr. 2001, 73, 920-926.
(11) Cao, G.; Prior, R. L. Anthocyanins are detected in human plasma after oral administration of an elderberry extract. Clinical Chemistry 1999, 45, 574-576.