Stereochemistry and glycosidic linkages of C3-glycosylations affected the reactivity of cyanidin derivatives

Highlights

• Stereochemistry and glycosidic linkage of cyanidin-3-glycosides impacted reactivity.

• Cyanidin-3-galactoside bleached & hydrated more than cyanidin-3-glucoside.

• Cyanidin (Cy) with 1 → 6 disaccharides had higher t_1/2 than with 1 → 2 dissacharides.

• 1 → 2 disaccharides resisted hydration & bleaching more than their 1 → 6 counterparts.

• Cy with 1 → 2 & 1 → 6 trisaccharide showed greatest stability & intermediate bleaching.

Abstract

The impact of glycosylation on anthocyanin stability has largely been associated with sugar type, site, and size, with glycosyl stereochemistry being under-explored. Seven cyanidin-3-glycosides were isolated by HPLC, diluted in pH 1–9, mixed with bisulfite or ascorbic acid at pH 3, and stored for 8 weeks (25 °C, dark). Spectral changes, half-lives, and bleaching rates were determined. Cyanidin-3-galactoside was more reactive (susceptible to hydration and bleaching) than cyanidin-3-glucoside. The 1 → 2 disaccharides exhibited greater λ_vis-max (≤16 nm), resistance to hydration, and bleaching compared to 1 → 6 disaccharides.The 1 → 6 disaccharides had similar λ_vis-max (∼2 nm) to the monosaccharides but slightly improved resistance to hydration and bleaching. The tri-glycosylated anthocyanin had the greatest stability and its spectral and bleaching characteristics was intermediate to 1 → 2 and 1 → 6 disaccharides. The 1 → 2 disaccharides generally exhibited lower half-lives compared to monosaccharides; whereas, 1 → 6 disaccharides exhibited higher stability. These findings highlight the role of glycosyl assembly on anthocyanin reactivity and stability.

Introduction

The reactivity and stability of anthocyanins are known to be affected by the natural chemical composition of the pigment. Anthocyanins are composed of a basic structure of 2-phenylbenzopyrylium of flavylium salt, with varying degrees of substitution. Anthocyanidins are inherently unstable and are primarily found glycosylated in nature; the glycosyl addition is believed to support anthocyanin stability through several mechanisms such as formation of H-bonding, formation of van der Waals interactions with the chromophore while exhibiting a hydrophobic face, enhanced streric hindrance, and decreased electron delocalization (Cabrita et al., 2014, Jing et al., 2008, Zhao et al., 2014). The most common glycosylation site is C3, followed by C3 and C5, and least common are C3 with either C4 or C7 substitution. Anthocyanins are typically mono-glycosylated, but attachments as large as tri-saccharides have been reported (Andersen & Jordheim, 2010).

It is frequently reported that increasing the number of glycosyls at C3 results in increased stability of the molecules (Zhao et al., 2014). Several sources have provided evidence of the enhanced stability of 3-trisaccharide anthocyanins in comparison to 3-monosaccharides (Bonerz et al., 2007, Eiro and Heinonen, 2002, García-Viguera et al., 1998, Sui et al., 2014). Evidence regarding stability for anthocyanin 3-disaccharides, however, is generally more conflicting. For example, cyanidin-3-rutinoside (rhamnosyl(1 → 6)glucoside) was reported as being more stable to heat treatment as compared to trisaccharide derivative cyanidin-3-glucosyl(1 → 2)-rutinoside in sour cherry juice (Szalóki-Dorkó, Végvári, Ladányi, Ficzek, & Stéger-Máté, 2015) and also cyanidin-3-glucoside (Sui et al., 2014). In contrast, cyanidin-3-xylosyl(1 → 2)-rutinoside was reported as being less stable than cyanidin-3-rutinoside and cyanidin-3-sophoroside (glucosyl(1 → 2)glucoside) (Bonerz et al., 2007). In the same study mentioned by Bonerz et al. (2007), cyanidin-3-glucosyl(1 → 2)-rutinoside always exhibited superior stability to the disaccharides present in sour cherry juice even when cyanidin-3-xylosyl(1 → 2)-rutinoside did not. The differences in stability among C3 di- and tri-saccharides may be influenced by primary sugar type attached to the anthocyanidin or how subsequent sugars are bonded in the glycosylations. Susceptibility to acid hydrolysis of anthocyanin monosaccharides has been reported in the following order arabinoside > galactoside > glucoside (Ichiyanagi, Oikawa, Tateyama, & Konishi, 2001).

More recently, it was reported that how the molecules are assembled, primarily the location of glycosidic bonds, may influence the formation of pyranoanthocyanins; 1 → 6 disaccharides were reported to produce higher yields than monosaccharides and 1 → 2 disaccharides (Farr, Sigurdson, & Giusti, 2018). These findings suggest that the type of sugar attachment, their stereochemical organization around the chromophore, and thus location of glycosidic linkages within the di- or tri-saccharide all may affect anthocyanin stability and reactivity. Similarly these differences in glycosidic linkages may play roles in anthocyanin stability and reactivity; subsequent investigation could contribute further to understanding anthocyanin chemistry and ameliorate previously reported unexpected findings regarding di and tri-saccharide anthocyanin-3-glycosides.

Beyond being innately reactive, anthocyanins have been found to bleach (decrease in Chroma, increase in Lightness) in response to certain chemical stimuli. Bleaching is thought to occur as a result of nucleophilic species, such as bisulfite, hydrogen peroxide, or ascorbic acid, attacking the electrophilic sites of the aglycone. There is evidence specifically in regards to bisulfite addition suggesting these types of reactions are intitated with condensation between the reactive species and anthocyanin primarily at Carbon-4 and and less preferentially at Carbon-2 of the aglycone structure (Berké, Chèze, Vercauteren, & Deffieux, 1998). Bleaching behaviors are often investigated in regards to a plant source such as an anthocyanin mixture present in a juice, lacking a systematic comparison of the influence of the 3-substituted glycosylation. The differing number or stereochemical arrangement of glycosyl units could influence the extent and rate of bleaching of anthocyanins.

The objective of this study was to evaluate the possible effects of spatial organizations of glycosylations on the stability and reactivity of cyanidin derivatives. Significant differences in reactivity of anthocyanins bearing 1 → 2 and 1 → 6 disaccharides was hypothesized to occur with 1 → 6 anthocyanins being less stable and more prone to bleaching as suggested by pyranoanthocyanin yield as previously discovered.