Raymond Brouillard

The mechanism of co-pigmentation of anthocyanins in aqueous solutions

Abstract Factors affecting co-pigmentation of anthocyanins with chlorogenic acid in aqueous solutions were studied. The anthocyanins used were cyanidin 3,5-diglucoside, malvidin 3,5-diglucoside, cyanidin 3-glucoside and malvidin 3-glucoside. The variables studied were concentration of anthocyanin, concentration of chlorogenic acid, pH, temperature, and solvent. On the basis of visible absorption spectrometric measurements, a mechanism for the co-pigmentation/complexation of anthocyanins with chlorogenic acid is established. This mechanism involves formation of 1:1 pigment-co-pigment complexes forced together by hydrophobic interactions. Indeed, the extent of the pigment-co-pigment association is under the control of the unique hydrogen-bonded molecular structure of liquid water, and is affected by pH, temperature and composition of the medium.

Recent developments in the stabilization of anthocyanins in food products

Abstract The most recent findings on the influence of chemical structure, pH and copigments on colour stabilization of anthocyanin-containing media are reviewed. The pH is shown to be the most important factor affecting the colour of anthocyanins, but various means of stabilization are given. The discussion on copigmentation is based mainly on results of studies of this phenomenon in flowers. It is evident from the list of known copigments, however, that some of these compounds are commonly found in foods and there is sufficient evidence to show that they do play a role in the colour stabilization of food products. Most interesting is the recent discovery of acylated anthocyanins, which retain a stable colour in slightly acidic or neutral solutions. This discovery may prove to be of particular importance to the technology of foods, since with new and better sources of these compounds and with better understanding of their physicochemical properties they may find application in the colouring of food products.

The in vivo expression of anthocyanin colour in plants

Abstract During the last two decades both model experiments and in vivo investigations have given a deeper insight into our understanding of the organization of anthocyanins in living tissues. The main factors known to affect the colour of anthocyanin containing media are: the chemical nature of an anthocyanin, its concentration, the co-occurrence of several pigments, the pH and the presence of copigments. Under conditions as similar as possible to the conditions in nature, low concentrated aqueous solutions of the most common anthocyanins are characterized by the existence of several chemical equilibria: a fast acid and base catalysed prototropic tautomerism; a hydration of the pyrylium ring and a ring-chain prototropic tautomerism leading to opening of the pyranol ring. In their natural states, anthocyanins must be protected against nucleophilic reagents, especially water which is the main constituent of the vacuole. Nucleophilic addition of water to the flavylium cation results in colour loss. Copigmentation is probably the most efficient mean of protecting the flavylium cation against these compounds. Until now, the only suitable spectroscopic technique for anthocyanins in vivo studies was UV-visible absorption spectroscopy. Very recently, resonance Raman spectroscopy was successfully applied to both aqueous anthocyanin solutions and to living tissues containing these pigments.

Anthocyanin intramolecular copigment effect

Five structurally coherent anthocyanins, extracted from red-purple cultivars of Pharbitis nil, have been investigated for their pigmentation properties in slightly acidic aqueous solution and at 25°. They all possess the same aglycone (pelargonidin) and the same glycosyl moieties (sophorosyl at C-3 and glucosyl at C-5 of the aglycone). The reference compound is not acylated, while two members are monocaffeylated, the fourth and the fifth in the series being di- and tricaffeylated, respectively. It is shown that caffeylation of the sophorosyl group has a profound effect on some properties of pelargonidin, e.g. it reduces both the rate and the extent of the pelargonidin hydration step. Our kinetic and equilibrium data strongly suggest the existence of an interaction taking place between the pelargonidin chromophore and the caffeyl group(s), the glycosyl unit playing the role of a spacer. This highlights the function of glycosyl residues in the complex anthocyanins found during the past decade.

Anthocyanin molecular interactions: the first step in the formation of new pigments during wine aging?

Abstract The changes in red wine colour on aging are still a challenge. From recent work involving model experiments, it has been concluded that high quality red wine storage conditions (long period of time, permanent contact with wood, low pH values, low temperature and aqueous environment), are all in favour of good molecular associations between the wine anthocyanins and the aromatic planar colourless molecules present in such a medium. In this article, factors which affect anthocyanin non-covalent interactions will be reviewed. They include pigment and copigment concentrations and structures, pH, solvent and temperature. The complex of a pigment with one of its copigments may be the starting point of a covalent linkage between the molecules. This could explain changes in colour and in anthocyanin reactivity observed during the aging of red wines, for instance.

Coupling reactions between flavylium ions and catechin

Abstract In order to model natural polymeric pigments present in old red wines, new covalent adducts have been synthesized upon condensation of synthetic flavylium ions (models of anthocyanins) with catechin (model of tannins) in the presence and in the absence of acetaldehyde. These new pigments have been investigated by 1D and 2D NMR, HPLC, FAB-mass and UV-visible spectroscopies and molecular modelling. The two flavylium salts used in this work (3,4′-dimethoxy-7-hydroxyflavylium chloride and 5,7-dihydroxy-3,4′-dimethoxyflavylium chloride) display quite different reactivities toward catechin. The electronic donating effect of the catechin moiety and the formation of noncovalent dimers in acidic aqueous or methanolic solution should be mainly responsible for the improved stability of the flavylium chromophore in the new pigments.

Resveratrol analog (Z)-3,5,4′-trimethoxystilbene is a potent anti-mitotic drug inhibiting tubulin polymerization

Resveratrol (3,5,4′‐trihydroxystilbene) a natural polyphenol present in medicinal plants, grapes and wines, has potent chemopreventive properties on intestinal carcinogenesis. A methylated derivative (Z‐3,5,4′‐trimethoxystilbene: R3) was synthesized. R3 at 0.3 μM exerted a 80% growth inhibition of human colon cancer Caco‐2 cells and arrested growth completely at 0.4 μM (R3 was 100‐fold more active than resveratrol). The cis conformation of R3 was also 100‐fold more potent than the trans isomer. R3 (0.3 μM) caused cell cycle arrest at the G2/M phase transition. The drug inhibited tubulin polymerization in a dose‐dependent manner (IC50= 4 μM), and it reduced also by 2‐fold ornithine decarboxylase and s‐adenosylmethionine decarboxylase activities. This caused the depletion of the polyamines, putrescine and spermidine, which are growth factors for cancer cells. R3 inhibited partially colchicine binding to its binding site on tubulin, indicating that R3 either partially overlaps with colchicine binding or that R3 binds to a specific site of tubulin that is not identical with the colchicine binding site modifying colchicine binding by allosteric influences. The resveratrol derivative (Z)‐3,5,4′‐trimethoxystilbene (R3) is an interesting anti‐mitotic drug that exerts cytotoxic effects by depleting the intracellular pool of polyamines and by altering microtubule polymerization. Such a drug may be useful for the treatment of neoplastic diseases.

Characterization of compounds obtained by chemical oxidation of caffeic acid in acidic conditions

Sodium periodate oxidation of caffeic acid was investigated in aqueous solutions with pH ranging from 2 to 7. Products formed from quinone evolution were monitored by HPLC. It was found that they were affected both qualitatively and quantitatively by the conditions of the reaction. The rate and the yield of the reaction increased with the pH. In particular, two products obtained at pH values lower than 4.6 approximately, were analysed and isolated by reverse phase HPLC. By using 1H and 13C NMR and mass spectrometries, these compounds were shown to be two stereoisomers of 2,5-(3′,4′-dihydroxyphenyl)tetrahydrofuran 3,4-dicarboxylic acid.

New aspects of anthocyanin complexation. Intramolecular copigmentation as a means for colour loss

Two series of structurally related anthocyanins, extracted from the blue flowers of Evolvulus pilosus cv. Blue Daze and from the blue-purple flowers of Eichhornia crassipes, exhibit remarkable colour stabilities in aqueous solution at mildly acidic pH values. All the pigments possess the same chromophore (delphinidin), but a different pattern of glycosylation and acylation. Moreover, one of the pigments has an apigenin 7-glucoside molecule (a flavone) attached to the glycosidic chain by two ester bonds with malonic acid, instead of an aromatic acid and is the only known anthocyanin with such a structure. All the molecules studied, except one which has only a 3-gentiobioside (a disaccharide) as substituent, denote an effect of reduction in the hydration constant when compared with the parent delphinidin 3-glucoside or 3,5-diglucoside molecules, which supports the existence of intramolecular hydrophobic interactions between the chromophoric skeleton and the acyl or flavonoid groups. The role played by the sugar units on the hydrophobicity/hydrophilicity of the pigments is also discussed.

New features of intramolecular copigmentation by acylated anthocyanins

Abstract Three series of structurally related anthocyanins, extracted from the red–purpleflowers of Dendrobium Pramot, xLaeliocattleya cv. Mini Purple, Bletilla striata and Phalaenopsis all belonging to the Orchidaceae family and another series extracted from the pinkflowers of Senecio cruentus (Compositae) allowed the confirmation of the existence of strongintramolecular copigmentation effects. These interactions confer stability to the coloured formsof the molecules, in a wide range of slightly acidic to neutral aqueous media. Moreover, theexistence of structural relationships among the four series stressed the different influencesexerted by the diverse substituent groups. The existence of a malonylglucoside attached toposition 3 of all but three of the molecules put forward a new role for the malonyl residue, in thisparticular position.