Anthocyanins are a class of water-soluble flavonoids, which show a range of pharmacological effects, such as prevention of cardiovascular disease, obesity control and antitumour activity. Their potential antitumour effects are reported to be based on a wide variety of biological activities including antioxidant; anti-inflammation; anti-mutagenesis; induction of differentiation; inhibiting proliferation by modulating signal transduction pathways, inducing cell cycle arrest and stimulating apoptosis or autophagy of cancer cells; anti-invasion; anti-metastasis; reversing drug resistance of cancer cells and increasing their sensitivity to chemotherapy. In this review, the latest progress on the anticancer activities of anthocyanins and the underlying molecular mechanisms is summarized using data from basic research in vitro and in vivo, from clinical trials and taking into account theory and practice.

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This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc

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Effects of anthocyanins on the prevention and treatment of cancer

Oxidative cellular stress initiates Nrf2 translocation into the nucleus, thus inducing antioxidant response element (ARE)-mediated expression of Phase II enzymes involved in detoxification and antioxidant defence. We investigated whether coffee extracts (CEs) of different proveniences and selected constituents have an impact on the Nrf2/ARE pathway in human colon carcinoma cells (HT29). Assessed as increased nuclear Nrf2 protein, Nrf2 nuclear translocation was modulated by different CEs as observed by Western blot analysis. In addition to the known Nrf2 activator 5-O-caffeoylquinic acid (CGA), pyridinium derivatives like the N-methylpyridinium ion (NMP) were identified as potent activators of Nrf2 nuclear translocation and ARE-dependent gene expression of selected antioxidative Phase II enzymes in HT29. Thereby, the substitution pattern at the pyridinium core structure determined the impact on Nrf2-signalling. In contrast, trigonelline was found to interfere with Nrf2 activation, effectively suppressing the NMP-mediated induction of Nrf2/ARE-dependent gene expression. In conclusion, several coffee constituents, partly already present in the raw material as well as those generated during the roasting process, contribute to the Nrf2-translocating properties of consumer-relevant coffee. A fine tuning in the degradation/formation of activating and deactivating constituents of the Nrf2/ARE pathway during the roasting process appears to be critical for the chemopreventive properties of the final coffee product.

Coffee constituents as modulators of Nrf2 nuclear translocation and ARE (EpRE)-dependent gene expression.

Cell culture is widely used by biochemists and cell/molecular biologists, but the fluctuating (and often elevated) levels of O 2 to which cells in culture are exposed can affect many of their properties. So can the low level of antioxidants found in some cell culture media. Reagents, especially "antioxidants," added to cell culture media can react with the constituents of the media to produce H 2 O 2 and degradation products that can influence cell behavior. Several published papers describing the cellular effects of ascorbate, polyphenols, and carotenoids have, in fact, reported artifacts due to the actions of the degradation products of these "antioxidants." A greater awareness of the potential artifacts in cell culture studies is needed among the free radical/antioxidant community.

Cell culture, oxidative stress, and antioxidants: avoiding pitfalls.

To date the in vitro mechanistic bioactivity of anthocyanins has been exclusively explored using aglycones and glycoside conjugates, despite a lack of evidence establishing these as the biologically available forms. We conducted intestinal epithelial cell (Caco-2 cells) culture experiments, which indicated that after a 4 h incubation of anthocyanins in cell-free culture media (DMEM), 57% of the initial cyanidin-3-glucoside (C3G) and 96% of cyanidin had degraded. The level of degradation was not statistically different from that of cultured cell incubations, suggesting that degradation was spontaneous. Degradation products were identified as protocatechuic acid (PCA) and phloroglucinaldehyde (PGA), and were confirmed in two other buffer matrices (phosphate and Hank's buffers). In cultured cell media the degradation products PCA and PGA were metabolised to glucuronide and sulphate conjugates, as indicated by both enzyme hydrolysis (sulphatase and glucuronidase treatments) and MS (PCA and PGA m/z = 155; sulphate = 235; glucuronide = 331). These data suggest a significant proportion of intestinal metabolites of anthocyanins are likely to be conjugates of their degradation products. Future efforts to establish the biological activities of anthocyanins should therefore include the investigation of phenolic acid and aldehyde products of degradation, along with their respective metabolites.

The bioactivity of dietary anthocyanins is likely to be mediated by their degradation products

Anthocyanins degradation during storage of Hibiscus sabdariffa extract and evolution of its degradation products.

In the present study we investigated the stability of anthocyanidins under cell culture conditions and addressed the question whether degradation products might contribute to the cellular effects assigned to the parent compounds. Substantial degradation was found already after 30 min, measured by HPLC/DAD. However, the decrease of detectable anthocyanidins exceeded by far the formation of the respective phenolic acids. From the formed phenolic acids only gallic acid (GA) exhibited growth inhibitory properties. However, also GA was found to be degraded rapidly. Furthermore, the incubation with delphinidin (DEL) or GA resulted in a substantial formation of hydrogen peroxide. The suppression of hydrogen peroxide accumulation by catalase modified significantly the growth inhibitory effects of DEL and GA, indicating that hydrogen peroxide formation might generate experimental artefacts. In summary, the results show that the phenolic acids formed by the degradation of cyanidin (CY), pelargonidin (PG), peonidin (PN) and malvidin (MV) do not contribute to the growth inhibitory effect of the parent compound. The degradation of DEL generates a phenolic acid with substantial growth inhibitory properties (GA). However, taken into account the small proportion of generated GA and its lacking stability, the contribution of GA to the growth inhibitory properties of DEL might be limited.

Limited stability in cell culture medium and hydrogen peroxide formation affect the growth inhibitory properties of delphinidin and its degradation product gallic acid.

In the present study, we addressed the question whether cyanidin-3-glucoside (C3G) or complex C3G-rich blackberry extracts affect human topoisomerases with special emphasis on the contribution of the potential degradation products phloroglucinol aldehyde (PGA) and protocatechuic acid (PCA). In HT29 colon carcinoma cells a C3G-rich blackberry extract suppressed camptothecin- (CPT-) or doxorubicin- (DOX-) induced stabilization of the covalent DNA-topoisomerase intermediate, thus antagonizing the effects of these classical topoisomerase poisons on DNA integrity. As a single compound, C3G (100 μM) decreased the DNA-damaging effects of CPT as well, but did not significantly affect those induced by DOX. At the highest applied concentration (100 μM), cyanidin protected DNA from CPT- and DOX-induced damage. Earlier reports on DNA-damaging properties of cyanidin were found to result most likely from the formation of hydrogen peroxide as an artifact in the cell culture medium when the incubation was performed in the absence of catalase. The suppression of hydrogen peroxide accumulation, achieved by the addition of catalase, demonstrated that cyanidin does not exhibit DNA-damaging properties in HT29 cells (up to 100 μM). The observed effects on topoisomerase interference and DNA protection against CPT or DOX were clearly limited to the parent compound and were not observed for the potential cyanidin degradation products PGA and PCA.

Anthocyanin-rich blackberry extract suppresses the DNA-damaging properties of topoisomerase I and II poisons in colon carcinoma cells.

In the present study, the question was addressed whether anthocyanins interfere with the topoisomerase I poison irinotecan in vivo In vivo complexes of enzyme to DNA bioassay was used to detect irinotecan-induced stabilization of topoisomerase I/DNA complexes and single cell gel electrophoresis to determine DNA-strand-break induction in the colon of male Wistar rats Furthermore, analysis of anthocyanin concentrations in rat plasma and rat colon was included in the testing, demonstrating that anthocyanins reach the colon and the concentrations do not differ between rats that only received anthocyanins and the anthocyanin/irinotecan group Blackberry extract was found to significantly reduce irinotecan-mediated topoisomerase I/DNA cleavable complex formation Overall, anthocyanins did not notably increase cleavable complex formation However, a significant increase of DNA damage was shown after a single dose of irinotecan as well as the single compounds cyanidin (cy) and cyanidin-3-glucoside (cy-3-g) Furthermore, a significant reduction of irinotecan-induced DNA-strand breaks after a pretreatment with cy, cy-3-g and blackberry extract was observed Thus, the question arises whether anthocyanin-rich preparations might interfere with chemotherapy or whether, due to low systemic bioavailability, the preparations might provide protective potential in the gastrointestinal tract

Anthocyanins suppress the cleavable complex formation by irinotecan and diminish its DNA-strand-breaking activity in the colon of Wistar rats

Quercetin and its microbial degradation product 3,4-dihydroxyphenylacetic acid generate hydrogen peroxide modulating their stability under in vitro conditions.

S. Skrbek

Papers

Limited stability in cell culture medium and hydrogen peroxide formation affect the growth inhibitory properties of delphinidin and its degradation product gallic acid.

Anthocyanin-rich blackberry extract suppresses the DNA-damaging properties of topoisomerase I and II poisons in colon carcinoma cells.

Anthocyanins suppress the cleavable complex formation by irinotecan and diminish its DNA-strand-breaking activity in the colon of Wistar rats

Quercetin and its microbial degradation product 3,4-dihydroxyphenylacetic acid generate hydrogen peroxide modulating their stability under in vitro conditions.