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Effect of temperature on acidity and hydration equilibrium constants of delphinidin-3-O- and cyanidin-3-O-sambubioside calculated from uni- and multi-wavelength spectroscopic data Kévin Vidot, Nawel Achir, Christian Mertz, André Mundombe Sinela, Nadirah Rawat, Alexia Prades, Olivier Dangles, Hélène Fulcrand, and Manuel Dornier J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00701 • Publication Date (Web): 28 Apr 2016 Downloaded from http://pubs.acs.org on May 3, 2016
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Journal of Agricultural and Food Chemistry
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Effect of temperature on acidity and hydration equilibrium constants of
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delphinidin-3-O- and cyanidin-3-O-sambubioside calculated from uni- and
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multi-wavelength spectroscopic data
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Kévin Vidot1, Nawel Achir2*, Christian Mertz1, André Sinéla1, Nadirah Rawat1, Alexia Prades1,
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Olivier Dangles3, Hélène Fulcrand4, Manuel Dornier2
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1 CIRAD, UMR 95 Qualisud, TA B-95/16, F-34398 Montpellier Cedex 5, France
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2 Montpellier SupAgro, UMR 95 Qualisud, TA B-95/16, F-34398 Montpellier Cedex 5, France
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3 UMR 408, University of Avignon, INRA, Safety and Quality of Plant Products, 84000
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Avignon, France
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4 INRA-IPV, Unité de Recherche des Polymères et des Techniques Physicochimiques, 2 place
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Viala, 34060 Montpellier, France
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*
[email protected] 16
1101, avenue Agropolis
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34093 Montpellier Cedex 5
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FRANCE
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Tel: +33 (0) 4 67 87 40 89
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ABSTRACT
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Delphinidin-3-O-sambubioside and cyanidin-3-O-sambubioside are the main anthocyanins of
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Hibiscus sabdariffa calyces, traditionally used to make a bright red beverage by decoction in
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water. At natural pH, these anthocyanins are mainly in their flavylium form (red) in
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equilibrium with the quinonoid base (purple) and the hemiketal (colorless). For the first
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time, their acidity and hydration equilibrium constants were obtained from a pH-jump
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method followed by UV-VIS spectroscopy as a function of temperature from 4 to 37°C.
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Equilibrium constant determination was also performed by multivariate curve resolution
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(MCR). Acidity and hydration constants of cyanidin-3-O-sambubioside at 25°C were 4.12x10-5
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and 7.74x10-4 respectively and were significantly higher for delphinidin-3-O-sambubioside
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(4.95x10-5 and 1.21x10-3 respectively). MCR enabled the obtaining of concentration and
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spectrum of each form but led to overestimated values for the equilibrium constants.
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However, both methods showed that formations of the quinonoid base and hemiketal were
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endothermic reactions. Equilibrium constants of anthocyanins in the hibiscus extract showed
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comparable values as for the isolated anthocyanins.
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Keywords: Anthocyanin, cyanidin-3-O-sambubioside, delphinidin-3-O-sambubioside, pH-
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jump, MCR, UV-vis, equilibrium constant, Hibiscus sabdariffa extract.
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Calyces of Hibiscus sabdariffa are traditionally used to prepare a red beverage called Bissap
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by decoction in water. This strong red coloration is due to an important concentration of 40
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mg/g of two main anthocyanins present in the calyces: delphinidin-3-O-sambubioside (Del-
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3Sb) and cyanidin-3-O-sambubioside (Cya-3Sb) 1. One problem during the storage of the
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beverage is the instability of anthocyanins leading to an undesirable color change 2. Indeed,
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the flavylium structure of anthocyanin, which is responsible for the red color, can undergo
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several structural modifications 3. The flavylium cation (AH+) is in equilibrium with two main
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forms, which are the quinonoid base (A) and the hemiketal (B) (Fig. 1). Depending on the pH,
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the quinonoid base (a mixture of tautomers with the 7-keto tautomer making the major
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contribution) results from a very fast proton loss from one acidic OH group (at C4’, C5 or
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preferentially at C7) while the hemiketal is formed by the much slower addition of a water
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molecule to the electrophilic C2 center with concomitant proton loss. Hemiketal B is in fast
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cycle – chain equilibrium with minor concentrations of cis-chalcone (Cc), itself in very slow
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equilibrium with the corresponding trans-chalcone (Ct). These equilibria are characterized by
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the acidity and overall hydration constants Ka and K’h (Fig. 1): Ka = (H+)(A)/(AH+), K’h = (H+)[(B)
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+ (Cc) + (Ct)]/(AH+) = Kh[1 + Kt(1 + Ki)]. These different structures have very distinct spectral
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properties as A is purple, B colorless and the chalcones pale yellow.
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Upon heating, irradiation or metabolism, anthocyanins are also known to go through several
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irreversible degradation pathways depending on pH
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enzymatic hydrolysis of the glycosidic bond at C3-OH, the chalcone forms undergo fast
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irreversible C-ring cleavage. Therefore, the quantification of the different anthocyanin forms
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as a function of pH and temperature, and thus the preliminary determination of the acidity
4–7
. In particular, after chemical or
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and hydration constants at different temperatures, are important steps for studying the
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stability of anthocyanins and rationalizing the observed color changes. The equilibrium
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constants may vary firstly as a function of the anthocyanin structure: B-ring substitution,
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extent of glycosidation (e.g., 3-glycoside vs. 3,5-diglycosides), type of sugar residues (Table
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1). To our knowledge, the equilibrium constants of delphinidin-3-O-sambubioside and
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cyanidin-3-O-sambubioside have not been estimated yet.
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Table 1. Hydration equilibrium constants of some anthocyanins 8–10.
pK’h
K’h (x10-3)
Cyanidin-3-O-xylosyl-galactoside
3.13 (± 0.02)
0.74
Cyanidin-3-O-glucoside
3.01 (± 0.04)
0.98
Malvidin-3-O-glucoside
2.60 (± 0.01)
2.51
Malvidin-3,5-O-diglucoside
1.85 (± 0.02)
14.13
Anthocyanins
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Generally, anthocyanin equilibrium constants are determined by a pH-jump method which
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allows the monitoring of the hydration kinetics as a function of pH 9 or by simple titration on
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fully equilibrated solutions 10. The former method allows the determination of both Ka and
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K’h while the latter only provides Ka + K’h. The color changes are monitored by absorbance
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measurement at a single wavelength, usually at 520 nm
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recorded the absorbance values at all wavelengths of the UV-visible spectra and used
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Journal of Agricultural and Food Chemistry
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multivariate curve resolution (MCR) to obtain the concentration of each form in equilibrium
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after deconvolution of the spectra
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pure anthocyanins and compared to the values obtained with the usual single wavelength
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method.
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In this context, the present study reports on the determination of the acidity and hydration
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equilibrium constants of Del-3Sb and Cya-3Sb as a function of temperature mimicking
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different storage conditions, using the usual single wavelength method and the alternative
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MCR method. The estimation of these equilibrium constants is also attempted directly in the
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Hibiscus sabdariffa extract in order to analyze the effect of the natural matrix on the
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distribution of the anthocyanin forms.
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. However, this technique has never been tested on
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MATERIAL AND METHODS
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Solution preparation
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Pure anthocyanin solutions. Commercial cyanidin-3-O-sambubioside chloride and
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delphinidin-3-O-sambubioside chloride (Extrasynthese, France) were dissolved separately in
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malic acid solution (0.1M - pH 2.2) to obtain anthocyanin solutions at 1.2x10-4M. All
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solutions were equilibrated at the temperature of experiments one hour before use.
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Extract of Hibiscus sabdariffa. In natural medium, apparent anthocyanin equilibrium
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constants are known to be sensitive to any compound susceptible to interact in a
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discriminate fashion with the colored and colorless forms of anthocyanins. Hence, apparent
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hydration and acidity constants were also evaluated in the Hibiscus sabdariffa extract.
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Hibiscus sabdariffa extract was obtained by soaking calyx powder in water at a ratio 1/9
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(g/g) during 30 min and by filtration. Previous experimental characterization of the extract
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showed that the pH equaled 2.2 with malic acid as main organic acid. The main anthocyanins
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are delphinidin-3-O-sambubioside (70%) and cyanidin-3-O-sambubioside (30%) 1. A 1/10
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dilution of the extract in malic acid (Sigma-Aldrich, France) solution (0.1M - pH 2.2) was
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made before UV-VIS measurements.
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Sodium hydroxide solutions: Twelve sodium hydroxide (Sigma-Aldrich, France) solutions of
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concentration ranging from 1.0 x 10-2 to 1.2 x 10-1 M were prepared, and depending on
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working temperature, only ten solutions were chosen for the experiment (4°C, 15°C and
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25°C: [NaOH] = 1.0 x 10-2 to 1.0 x 10-1M ; for 30°C and 37°C: [NaOH] = 3.0 x 10-2 to 1.2 x 10-2
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M).
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pH-jump experiments
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The pH jump method was carried out according to the literature (Brouillard and Delaporte
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1977). The equilibrium was perturbed by a sudden change in H+ concentration and the
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absorbance from 240 to 800 nm (each 0.5 nm) was recorded as a function of time with a
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diode-array spectrophotometer (Specord S600 Analytik Jena, Germany) thermostated by a
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F250 cryostat (Julabo, Germany). To do so, 1 mL of equilibrated anthocyanin solution was
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put in a quartz cell (d = 1 cm) equipped with a stirring magnet. Then, 1 mL of NaOH solution
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of concentration ranging from 1.0 x 10-2 to 1.2 x 10-1 M was quickly added to the cell, and the
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spectra were immediately recorded every second over 1 min or 1.5 min, until reaching the
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hydration equilibrium. The final pH value was carefully measured with an Inolab 7110 (WTW,
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Germany) pH-meter combined to a SenTix®Mic-D (WTW, Germany) electrode and ranged
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from 2.5 to 4.5. This set of 10 kinetic experiments was performed for both pure
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anthocyanins in solution, Cya-3Sb and Del-3Sb, at five different temperatures 4°C, 15°C,
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25°C, 30°C and 37°C. This experiment was also repeated on the anthocyanin extract of
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hibiscus at 25°C. Three replicates were done for each temperature.
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The spectra of the solution in which the anthocyanin is exclusively under the flavylium form
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(pH
