Elsevier

Phytochemistry

Volume 68, Issue 9, May 2007, Pages 1285-1294
Phytochemistry

Anthocyanins from red cabbage – stability to simulated gastrointestinal digestion

https://doi.org/10.1016/j.phytochem.2007.02.004Get rights and content

Abstract

Anthocyanins were the main polyphenol components in extracts of fresh and pickled red cabbage. The composition of anthocyanins in red cabbage was studied using liquid chromatography mass–spectrometry. Eleven major peaks absorbing at 520 nm were discerned, which represented 18 different anthocyanin structures. Another five minor anthocyanin components could be identified by searching at their respective m/z values but only in anthocyanin-enriched concentrates produced by sorption to solid phase extraction matrices. The predominant anthocyanins were constructed of cyanidin-3-diglucoside-5-glucoside “cores” which were non-acylated, mono-acylated or di-acylated with p-coumaric, caffeic, ferulic and sinapic acids. Pelargonidin-3-glucoside and novel forms of cyanidin-3-O-triglucoside-5-O-glucoside di-acylated with hydroxycinnamic acids were also detected in extracts of raw red cabbage, commercially pickled red cabbage and anthocyanin-enriched concentrates.

The stability of the anthocyanins to simulated gastrointestinal digestion was assessed. The anthocyanins were effectively stable in the acidic gastric digestion conditions but the total recovery after simulated pancreatic digestion was around 25% compared to around 100% recovery of phenol content. As anthocyanins make up the majority of red cabbage polyphenols, this suggested that anthocyanins broke down to form new phenolic components. The recovery of the individual anthocyanins was monitored by LC–MSn. All of the anthocyanins were reduced in content after pancreatic digestion but acylated forms were notably more stable than non-acylated forms. There was also a relationship between the type of acylated hydroxycinnamic acid and stability to pancreatic digestion.

Graphical abstract

The stability of red cabbage anthocyanins was assessed by an in vitro procedure that simulates human gastrointestinal digestion. Anthocyanins acylated with hydroxycinnamic acids were more stable than the unacylated forms with a marked difference in stability between different acylated hydroxycinnamic acids.

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Introduction

Increased anthocyanin content has been a breeding target for the genetic improvement of blackcurrant (Brennan, 1996) and raspberry (Graham et al., 2004) germplasm for both cosmetic reasons and potential health benefits. Anthocyanins are responsible for the red, purple and blue hues of plant fruits, flowers and leaves (Strack and Wray, 1993). Dietary consumption, mainly from red fruits, certain vegetables (such as red cabbage) and red wine (Wu et al., 2006) can reach 200 mg/day. Anthocyanins have a range of biological activities that may produce health benefits; examples range from inhibition of DNA damage in cancer cells in vitro (Hou, 2003), inhibition of digestive enzymes (McDougall and Stewart, 2006), induction of insulin production in isolated pancreatic cells (Jayaprakasam et al., 2005), reduction in inflammatory responses (Tall et al., 2004) to protection against age-related decline in brain function (Lau et al., 2006).

Eating anthocyanin-rich fruits, extracts or pure anthocyanins may prevent or suppress disease states in vivo (Ramirez-Tortosa et al., 2001, Mazza et al., 2002). Oral intake of anthocyanins increased antioxidant status of the serum (Serafini et al., 1998, Ramirez-Tortosa et al., 2001, Mazza et al., 2002, Talavera et al., 2006) but this was usually accompanied by very low uptake of anthocyanins into the serum (≪1% of dose) (Lapidot et al., 1998, Bub et al., 2001, Frank et al., 2003, Talavera et al., 2006). The apparent low bioavailability of anthocyanins casts doubt on their ability to cause their proposed beneficial effects throughout the body.

It has become clear from studies on simulated human gastrointestinal digestion that anthocyanins, whilst stable in the acidic conditions of the stomach, are less stable at the elevated pH of the small intestine (Perez-Vicente et al., 2002, Gil-Izquierdo et al., 2002, McDougall et al., 2005a, McDougall et al., 2005b). Red cabbage (Brassica oleracea L.) is a useful food colouring (Giusti and Wrolstad, 2003, Stintzing and Carle, 2004) because red cabbage anthocyanins are stable over a broader pH range than anthocyanins from (say) blackcurrants, which only retain colour at pH < 4.0 (Markakis, 1982). Therefore, red cabbage anthocyanins are used as colours for foods with neutral pH and are natural alternatives to synthetic blue colourings (Bridle and Timberlake, 1997).

The major anthocyanins of red cabbage are based on a core of cyanidin-3-O-diglucoside-5-O-glucoside (Fig. 1), which can be non-acylated, mono-acylated or di-acylated with p-coumaric, caffeic, ferulic and sinapic acids (Tanchev and Timberlake, 1969, Idaka et al., 1987a, Idaka et al., 1987b, Giusti et al., 1999, Wu and Prior, 2005). Anthocyanins exist in equilibrium of four molecular species; the coloured basic flavylium cation and three secondary structures; the quinoidal bases, the carbinol pseudobase and the chalcone pseudobase forms. At pH 2 or below, the flavylium cation form predominates but as the pH is raised towards 7 the colourless chalcone pseudobase begins to dominate. Chalcone formation is also favoured by elevated temperatures and prolonged exposure may enhance degradation between the B and C rings (Fig. 1) resulting in the destruction of the anthocyanin chromophore (Strack and Wray, 1993, Clifford, 2000). The unusual pH stability of the colour of red cabbage anthocyanins is thought to be due to the presence of these acyl groups which “hinder the hydrolysis of the red flavylium cationic form to the colourless carbinol base, allowing preferential formation of the blue quinoidal bases” (Bridle and Timberlake, 1997). Glycosylation at positions 3 and 5 shifts the colour towards the blue and the stability of colour may also be influenced by intramolecular co-pigmentation (Maulien-Aubert et al., 2001).

In this study, we assess if the known pH stability of red cabbage anthocyanins influences their stability under simulated gastrointestinal (GIT) digestion. The relative stability of the anthocyanins under GIT conditions will determine the pool size for whatever active mechanisms are present in the stomach (Passamonti et al., 2003) or the small intestine (Gee et al., 1998) to transport anthocyanins into the blood stream. Information on the relationship between the structure and gastrointestinal stability of anthocyanins will be fed-back into traditional or marker-assisted breeding programs to facilitate the generation of fruit with enhanced health benefits.

Section snippets

Extraction procedures

Fresh red cabbage and pickled red cabbage was purchased from a local supermarket. The raw cabbage (500 g) was chopped into small pieces and added to 1 l of ice-cold 0.5% (v/v) acetic acid in water. The material was homogenised in a Waring Blender then filtered through coarse then fine glass sinters. The pickled red cabbage was drained then extracted in the same manner.

Portions of the red cabbage extracts were adjusted to 0.5% (v/v) formic acid prior to application to C18 solid phase extraction

Results and discussion

The red cabbage extract contained a high content of anthocyanins (137.5 ± 2.9 mg/100 g) which after concentration by sorption to C18 solid phase extraction (SPE) units gave an anthocyanin/total phenol ratio of ∼1.00. HPLC analysis of the red cabbage extract yielded a number of peaks that absorbed at 520 nm (Fig. 2a). The HPLC profiles at 280 nm and 520 nm (Fig. 2a and b) are very similar which confirms that anthocyanins make up the majority of red cabbage polyphenols. The pickled red cabbage gave a

Acknowledgements

We thank Professor Howard Davies for his support, Alison Emslie, Paul Neave and Pauline Smith for their help. SCRI is grateful for financial support from the Scottish Executive Environment and Rural Affairs Department.

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    Present address: Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

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