Article
J. Agric. Food Chem., Vol. 57, No. 12, 2009 5277
conditions is associated with the stoichiometric formation of
phenolic acid constituents. As the observed rates of anthocyani-
din degradation to phenolic acids were rapid, it is likely that these
compounds are present at far higher concentrations than antho-
cyanidins/anthocyanins in processed foods, the gut, and the
systemic circulation. Although it is likely that pH and tempera-
ture are the chief contributors to anthocyanin degradation under
biological conditions, the extent to which other physiological
forces, such as protein interactions and enzymatic or microbial
transformation, contribute to anthocyanin degradation is yet to
be fully elucidated.
Phloroglucinol aldehyde represented the A-ring constituent of
anthocyanins in the present investigation; however, it is possible
that phloroglucinol aldehyde could also be derived from other
sources in the diet, as this A-ring constituent is common to many
other flavonoids. Further study is, however, required to fully
establish the extent to which phloroglucinol aldehyde formation
is derived from the degradation of anthocyanins and other
flavonoids within the diet. An apparent lack of appreciation for
the rate and extent of anthocyanin degradation under physiolo-
gical conditions may explain the almost complete absence of data
aimed at quantifying anthocyanin breakdown products in human
samples (tissue, urine, and plasma) and the current lack of reports
concerned with the biological activities of these compounds and
their impact, as dietary constituents on human health. We
contend that anthocyanins may be regarded as “pro-drugs” for
the delivery of bioactive phenolic intermediates, and this should
be a focus for future research.
In this report, we provide evidence to show that anthocyanins
degrade at a faster rate in buffered solution than in water.
However, the rate of end stage degradation product (phenolic
acids) formation was relatively slower in buffered solution than in
water. These observations imply that the buffered environment
confers a greater stability to the pH-dependent anthocyanin
isoforms (including chalcone, hemiketal, and R-diketone inter-
mediate structures). However, it is possible that slight differences
in sample pH could account for some of the differences in
anthocyanin stability as observed in the present investigation.
As biological samples (i.e., serum, urine, and cell culture media)
possess a degree of buffering agents, the degradation character-
istics of anthocyanins in these matrices should be considered in
light of the current finding, where anthocyanins may demonstrate
differential degradation rates in other matrices such as foods,
which will be dependent on their individual buffering character-
istics.
anthocyanin structure contribute to their experimental recovery.
Consequently, these factors should be considered when analyzing
clinical or biological samples (such as cell culture medium, serum/
plasma, or urine) to avoid the underestimation of anthocyanin
bioavailability.
While some studies have evaluated the storage stability of
anthocyanins in various food matrices (26-28), no studies have
formally reported their storage stability under clinically relevant
conditions. Thus, this study has shown that anthocyanins are
stable when stored at -80 °C and epH 2, while anthocyanidins
are relatively less stable. This indicates that the poor recovery of
anthocyanins following clinical feeding trials is not likely to be the
result of degradation in stored samples but the result of degrada-
tion in vivo or during initial sample processing.
In conclusion, this report demonstrates that anthocyanins are
rapidly degraded to their phenolic acid and aldehyde constituents
under simulated (in vitro) physiological conditions and that
increased anthocyanin B-ring hydroxylation is associated with a
decrease in stability; these observations have major implications
for the design and interpretation of dietary intervention studies
utilizing anthocyanins. Furthermore, anthocyanin losses during
sample preparation were accounted for by the stoichiometric
appearance of their phenolic acid degradation products, particu-
larly during SPE. Hence, we suggest that the degradation of
anthocyanins and the representative formation of their phenolic
acid constituents be of important consideration when investigat-
ing anthocyanins within clinical and laboratory settings and that
these degradation products be targeted in future bioavailability
and bioactivity studies to establish their true occurrence and
influence on human health and disease.
ACKNOWLEDGMENT
We thank Shika Saha and Paul Needs at the Institute of Food
Research (Norwich) for their advice on analytical methodology.
We also thank GlaxoSmithKline plc. (United Kingdom) for their
support of this study.
LITERATURE CITED
(1) Mink, P. J.; Scrafford, C. G.; Barraj, L. M.; Harnack, L.; Hong, C.
P.; Nettleton, J. A.; Jacobs, D. R. Jr. Flavonoid intake and
cardiovascular disease mortality: A prospective study in postmeno-
pausal women. Am. J. Clin. Nutr. 2007, 85, 895–909.
(2) Xia, M.; Ling, W.; Zhu, H.; Wang, Q.; Ma, J.; Hou, M.; Tang, Z.; Li,
L.; Ye, Q. Anthocyanin prevents CD40-activated proinflammatory
signaling in endothelial cells by regulating cholesterol distribution.
Arterioscler., Thromb., Vasc. Biol. 2007, 27, 519–524.
The data presented here show that B-ring hydroxylation
significantly affected the recovery of anthocyanins following
routine SPE. This observation suggests that an underestimation
of anthocyanin levels in clinically derived samples is possible
when employing SPE. Furthermore, we observed that the dis-
appearance of anthocyanins was accompanied by the stoichio-
metric formation of their respective phenolic acid breakdown
products, demonstrating that degradation rather than poor
recovery was the foremost cause of anthocyanin loss following
SPE, particularly in the case of cyanidin-3-glucoside. This high-
lights the importance of establishing method accuracy and pre-
cision when estimating anthocyanin levels in clinical studies. Our
data suggested that the degradation of anthocyanins observed
during SPE may be the result of sample evaporation. We provide
direct evidence that evaporation of SPE eluents to dryness
contributes significantly to loss of anthocyanins and that recov-
eries can be substantially improved by partially evaporating SPE
eluents. This may be the result of acid concentration and eventual
hydrolysis in non-neutralized samples during post-SPE evapora-
tion. Thus, it is clear that both sample preparation technique and
(3) Xia, X.; Ling, W.; Ma, J.; Xia, M.; Hou, M.; Wang, Q.; Zhu, H.;
Tang, Z. An anthocyanin-rich extract from black rice enhances
atherosclerotic plaque stabilization in apolipoprotein E-deficient
mice. J. Nutr. 2006, 136, 2220–2225.
(4) Feng, R.; Ni, H. M.; Wang, S. Y.; Tourkova, I. L.; Shurin, M. R.;
Harada, H.; Yin, X. M. Cyanidin-3-rutinoside, a natural polyphenol
antioxidant, selectively kills leukemic cells by induction of oxidative
stress. J. Biol. Chem. 2007, 282, 13468–13476.
(5) Rossi, M.; Garavello, W.; Talamini, R.; Negri, E.; Bosetti, C.; Dal
Maso, L.; Lagiou, P.; Tavani, A.; Polesel, J.; Barzan, L.; Ramazzotti,
V.; Franceschi, S.; La Vecchia, C. Flavonoids and the risk of oral and
pharyngeal cancer: A case-control study from Italy. Cancer Epide-
miol. Biomarkers Prev. 2007, 16, 1621–1625.
(6) Tsuda, T.; Horio, F.; Uchida, K.; Aoki, H.; Osawa, T. Dietary
cyanidin 3-O-β-D-glucoside-rich purple corn color prevents obesity
and ameliorates hyperglycemia in mice. J. Nutr. 2003, 133, 2125–2130.
(7) Markakis, P. Anthocyanins and their stability in foods. CRC Crit.
Rev. Food Technol. 1974, 4, 437–456.
(8) Fleschhut, J.; Kratzer, F.; Rechkemmer, G.; Kulling, S. E. Stability
and biotransformation of various dietary anthocyanins in vitro. Eur.
J. Nutr. 2006, 45, 7–18.