Antioxidant and antiproliferative properties of methylated metabolites of anthocyanins Iva Fernandes, Filipe Marques, Victor de Freitas, Nuno Mateus ⇑ Chemistry Investigation Centre (CIQ), Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Portugal article info Article history: Received 20 March 2013 Received in revised form 2 May 2013 Accepted 8 May 2013 Available online 18 May 2013 Keywords: Anthocyanins Metabolites Methylation Antioxidant Antiproliferative abstract Anthocyanins are major flavonoids in many plant foods and have been related to health promotion. In the human organism anthocyanins are metabolised to different metabolites. One of the most important phase II reactions of flavonoids is the methylation of the catechol group. This feature is expected to have an effect on the antioxidant and antiproliferative properties of flavonoids including anthocyanins. In this work, delphinidin-3-glucoside, cyanidin-3-glucoside and petunidin-3-glucoside methylated metabolites were obtained by enzymatic hemi-synthesis. The compounds were identified as monomethylated prod- ucts by HPLC–MS and NMR. The methylated metabolites were found to still retain significant radical scavenging activity and reducing activity, suggesting that they could act as potential antioxidants in vivo. The antiproliferative activity of the metabolites in comparison with the parental anthocyanins was also evaluated in three cancer cell lines by sulforhodamine B assay. The conjugation with methyl groups decreased or did not alter the antiproliferative effect of the original anthocyanin. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Anthocyanins are the most important group of water-soluble plant pigments. These pigments are responsible for a great variety of colours of several fruits, vegetables and plants. The most com- monly known anthocyanins are based on six anthocyanidins: cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petuni- din; there are almost 600 anthocyanins reported to be isolated from plants (Andersen & Jordheim, 2005). Anthocyanins are found in the human diet usually associated with fruits (cherries, plums, strawberries, raspberries, blackberries, grapes, redcurrants and blackcurrants) but they also occur in veg- etables, roots, legumes and cereals (Markakis, 1982; Mazza & Mini- ati, 1993). In the US the daily intake of anthocyanins has been reported to be 180–215 mg/person (Kuhnau, 1976). More recently, other studies have reported a lower consumption range from around 3 to 15 (Chun, Chung, & Song, 2007; Wu et al., 2006) up to 150 mg/day (Heinonen, 2007). The antioxidant capacity of anthocyanins has been demon- strated by radical scavenging of reactive oxygen species, reducing capacity, inhibition or delaying of lipoprotein oxidation and plate- let aggregation (Azevedo et al., 2009). Over the last years, more attention has been paid to the putative anti-tumoral properties of anthocyanins and anthocyanin extracts (Faria et al., 2010; Fernandes et al., 2010). Although there are several works in the literature dealing with in vitro antioxidant and biological properties of anthocyanins, there is a lack of in vivo evidence. Extensive knowledge of the bioavail- ability and metabolism of anthocyanins is thus essential if their health effects are to be understood. Current evidence in the litera- ture poorly describes the human metabolism of anthocyanins, with no information on the biological effects of their metabolites. Anthocyanins have been proposed to be absorbed from both stom- ach (Fernandes, de Freitas, Reis, & Mateus, 2012; Passamonti, Vrhovsek, Vanzo, & Mattivi, 2003; Talavera et al., 2003) and small intestine (Talavera et al., 2004) appearing in blood circulation and urine as intact and/or conjugated (methylated, glucurono- and/or sulphoconjugated) forms (Felgines et al., 2003; Kay, Mazza, & Holub, 2005; Kay, Mazza, Holub, & Wang, 2004; Talavera et al., 2004; Wu, Cao, & Prior, 2002). However, the identification of de- rived metabolites has been limited as a result of their diversity, low concentrations in the blood and lack of commercially available standards. Unlike other flavonoids, the chemical synthesis of anthocyanins metabolites is limited, due to their low stability under the synthe- sis conditions (pH and temperature) normally applied to other flavonoid compounds to obtain metabolites (Dueñas, González- Manzano, González-Paramás, & Santos-Buelga, 2010). Nonetheless, some anthocyanin metabolites have been synthesised through an enzymatic approach (Fernandes et al., 2009). As it is fundamental to determine whether these new com- pounds are the bioactive forms responsible for some of the biolog- ical activities reported for anthocyanins, the acquisition of purified 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.05.033 ⇑ Corresponding author. Tel.: +351 220402562; fax: +351 220402659. E-mail address: nbmateus@fc.up.pt (N. Mateus). Food Chemistry 141 (2013) 2923–2933 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem