# Anthocyanin-rich extracts: Susceptibility to color loss by hydration and thermal degradation, influence of metal ions and endogenous copigments

Sabrina Idir, Sabiha Achat, Luis Cruz, Olivier Dangles

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Sabrina Idir, Sabiha Achat, Luis Cruz, Olivier Dangles. Anthocyanin-rich extracts: Susceptibility to color loss by hydration and thermal degradation, influence of metal ions and endogenous copigments. Food Chemistry, 2025, 481, 10.1016/j.foodchem.2025.144004. hal-05037627

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# Anthocyanin-rich extracts: Susceptibility to color loss by hydration and thermal degradation, influence of metal ions and endogenous copigments

Sabrina Idir<sup>a</sup>, Sabiha Achat<sup>a,*</sup>, Luis Cruz<sup>b</sup>, Olivier Dangles<sup>c,*</sup>

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

### Keywords:

- Eggplant

- Pomegranate

- Blood orange

- Anthocyanin

- Copigment

- Aluminum

## ABSTRACT

Three anthocyanin-rich extracts from eggplant peel (EPP), pomegranate juice (PGJ), and blood orange juice (BOJ) were prepared and studied for their phenolic composition and coloring properties. BOJ and EPP anthocyanins (ACNs) were more resistant to water addition (reversible color loss) than PGJ ACNs, in agreement with differences in ACN glycosylation patterns and in the extracts' content in potent copigments. All extracts formed Al³⁺ complexes at pH 5 with concomitant reversion of the hydration equilibria and efficient color regeneration. EPP formed blue Al³⁺ complexes, while BOJ and PGJ formed purple complexes. Monitoring color loss and pigment degradation during thermal treatment at 50 °C showed that BOJ was the most stable extract, while EPP degraded rapidly, possibly because of the autoxidation of its caffeoyl derivatives. Both color loss and pigment degradation were substantially reduced upon Al³⁺-ACN binding, with BOJ again coming up as the most stable extract.

## 1. Introduction

Anthocyanins (ACNs), a class of polyphenolic compounds, are very common water-soluble pigments widely distributed in nature (flowers, vegetables and fruits) and known for their bright red-orange to violet-blue hue palette (Dangles, 2024). Anthocyanins also possess health-promoting properties (Krga & Milenkovic, 2019) and a high potential of food industry applications, as natural food additives, nutraceuticals and healthy food ingredients. Thus, ACNs are widely regarded as alternatives to synthetic colorings (Rodriguez-Amaya, 2016).

Anthocyanins are O-glycosides of a tricyclic polyphenolic chromophore called anthocyanidin, which is usually represented by its structure under highly acidic conditions ($pH < 2$), i.e. the flavylium cation. The variety of ACN structures results from the B-ring substitution (OH and OMe groups), from the position, number and structure of the glycosyl groups, and the possible acylation of the latter by aliphatic and phenolic acids (Dangles, 2024). Additional complexity arises from the structural transformations of the flavylium ion when the pH increases (Fig. 1). Thus, ACNs can be viewed as a mixture of colored forms (flavylium and quinonoid bases) in fast proton transfer equilibrium, and of colorless forms in slower equilibrium (hemiketal, cis- and trans-chalcones) resulting from reversible water addition to the flavylium ion and

subsequent tautomerization and isomerization steps (Cruz et al., 2022; Dangles & Fenger, 2018; Pina, 2014). Under the mildly acidic conditions typical of plant and food, the thermodynamically more stable colorless forms usually predominate. Hence, color-protecting mechanisms operate in nature (Trouillas et al., 2016). Indeed, any mechanism favoring the colored forms over the colorless forms, or locking the colored forms under the quinonoid structure, which does not undergo water addition, can be considered protective. The most important example of the first mechanism is copigmentation, i.e. the formation of π-stacking complexes between ACNs (through the flat polarizable chromophore of the colored forms) and colorless phenols (copigments). Metal binding is an example of the second mechanism. It requires an ortho-dihydroxy substitution on the B-ring (catechol) (Trouillas et al., 2016). These mechanisms not only enhance color by limiting water addition to the flavylium ion (reversible color loss), but also generally protect ACNs against thermal and photochemical degradation (irreversible color loss) (Oancea, 2021).

The rich physical chemistry of ACNs has been extensively investigated with pure pigments (Cruz et al., 2022; Trouillas et al., 2016), which are difficult to extract and purify in substantial amounts. Extension of these works to anthocyanin-rich extracts is highly desirable, as a) only extracts (not pure ACNs) are amenable to industrial development

<footnote><sup>a</sup> Laboratory of Biomathematics, Biochemistry, Biophysics and Scientometry, Faculty of Natural and Life Sciences, University A. Mira of Bejaia, Bejaia 06000, Algeria</footnote>

<footnote><sup>b</sup> REQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal</footnote>

<footnote><sup>c</sup> Research Unit SQPOV, Avignon University, INRAE, 84000 Avignon, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: sabiha.achat@univ-bejaia.dz (S. Achat), olivier.dangles@univ-avignon.fr (O. Dangles).</footnote>
<figure><img src="image_3.png" /><figcaption>Fig. 1. Structural transformations of the flavylium ion (e.g., cyanidin 3-O-glucoside) in mildly acidic solution.</figcaption></figure>

for obvious economic reasons, b) extracts may contain other compo-
nents, especially colorless phenols, contributing to color stability by
acting as copigments, antioxidants, UV-filters....

So far, color studies involving extracts rich in ACNs and copigments
are typically limited to reporting extract composition and global spectral
or colorimetric parameters, as well as apparent first-order rate constants
of color loss, without distinguishing the reversible and irreversible
components (Müller-Maatsch et al., 2016; Oancea, 2021; Pereira et al.,
2024). In this work, our hypothesis is that the rigorous physicochemical
models developed from pure ACNs can be adapted to anthocyanin-rich
extracts, thus applying to the pool of ACNs in interactions with the
endogenous copigments (instead of individual ACNs). This approach is
thought to provide valuable insights into the color stabilization mech-
anisms operating within the extracts. Thus, semi-purified ACN-rich ex-
tracts from pomegranate juice (PGJ), blood orange juice (BOJ) and
eggplant peel (EPP) were investigated for their composition and color
properties. The equilibrium constants of the ACN multistate were
determined and compared to those of pure anthocyanins in the litera-
ture, so as to outline the possible influence of the plant matrix on the
hydration equilibrium (reversible color loss) governing the partition of
ACNs between colored and colorless forms. As metal - ACN binding
 selective to the colored forms), is a powerful way to restore color from
 an equilibrated mixture of colored and colorless forms, this capacity was
 quantitively assessed with the three extracts at the relevant food pH of 5.
 Finally, the thermal stability of the extracts under moderate heating at
 pH 5 was also studied and the results analyzed in terms of percentage
 values of color loss (reversible + irreversible) and pigment loss (irre-
versible). Overall, from these common easy-to-prepare extracts, this is a
 systematic mapping of ACN-rich extracts that is proposed, so as to

collect useful parameters to help select the most promising ACN sources
for further development as food colors.

## 2. Materials & methods

2.1. *Plant materials*

Three plant materials, eggplant (Solanum melongena L.), blood or-
ange (Citrus sinensis (L.) Osbeck), and pomegranate (Punica granatum)
were collected from different localities of Bejaia (Algeria), then washed
by distilled water. Fresh eggplants were purchased from local market in
March 2020 and peeled using a sharp knife. The whole peel (EPP) was
dried at 40 °C into a ventilated oven. Blood orange juice (BOJ) was
collected and either directly used for the analyses, or freeze-dried after
mixing with the pulp in a blender. The dried sample was ground in a
grinder, sieved to particle size <250 nm, then stored in a deep-freezer at
-18 °C for subsequent analyses. The arils from pomegranate were me-
chanically extracted by squeezing the arils. The juice (PGJ) obtained was
centrifuged during 30 min at 9000 rpm at 4 °C.

## 2.2. *Sample preparation*

2.2.1. *Anthocyanin extraction*

Five grams of the powdered samples (eggplant peel and blood or-
ange) were extracted with 50 mL of MeOH/H₂O (7:3) containing 0.1 %
HCl (pH 1.6) in an amber flask. The mixtures were kept in an ultrasonic
bath (42 kHz) for 50 min at 50 °C, then centrifuged at 4600 rpm for 15
min. The supernatants were collected and the remaining residues re-
extracted using the same procedure. The collected extracts were
gathered, then filtered prior to their analysis for total anthocyanin and total polyphenol contents. For purification, MeOH was evaporated under reduced pressure at 40 °C and the remaining aqueous solutions were diluted to a known volume with 0.01 % aqueous HCl (v/v).

<table><caption>**Table 1**
HPLC-DAD-MS data and putative identification of anthocyanins in the different extracts.</caption><thead><tr><th>Peak</th><th>t<sub>R</sub><br>(min)</th><th>λ<sub>max</sub><br>(nm)</th><th>Full<br>MS<br>(m/z)</th><th>MS<sup>2</sup><br>(m/z)</th><th>MS<sup>3</sup>(m/z)</th><th>Structure</th></tr><tr><td colspan="7">a)</td></tr></thead><tbody><tr><td colspan="7"><strong>Eggplant peel</strong></td></tr><tr><td>1</td><td>17.3</td><td>524,<br>276</td><td>773</td><td>611,<br>303,<br>465</td><td>611 → 303,<br>465</td><td>Delphinidin-3-O-<br>rutinoside-5-O-<br>galactoside</td></tr><tr><td>2</td><td>24.3</td><td>520,<br>280</td><td>773</td><td></td><td></td><td>Delphinidin-3-O-<br>rutinoside-5-O-<br>glucoside</td></tr><tr><td>3</td><td>29.6</td><td>524,<br>276</td><td>465,<br>303</td><td></td><td></td><td>Delphinidin-3-O-<br>glucoside</td></tr><tr><td>4</td><td>31.0</td><td>524,<br>276<br>(344)</td><td>611</td><td>303,<br>465</td><td>303 → 257,<br>229, 285</td><td>Delphinidin-3-O-<br>rutinoside*</td></tr><tr><td colspan="7"><strong>Pomegranate juice</strong></td></tr><tr><td>1</td><td>19.6</td><td>520</td><td>627,<br>465,<br>303</td><td></td><td></td><td>Delphinidin-3,5-di-<br>O-glucoside</td></tr><tr><td>2</td><td>24.5</td><td>512,<br>276</td><td>611</td><td>449,<br>287</td><td>449 → 287</td><td>Cyanidin-3,5-di-O-<br>glucoside*</td></tr><tr><td>3</td><td>29.3</td><td>516,<br>272</td><td>465,<br>303</td><td></td><td></td><td>Delphinidin-3-O-<br>glucoside</td></tr><tr><td>4</td><td>34.5</td><td>516,<br>280</td><td>449</td><td>287</td><td>287 → 213,<br>231, 259,<br>241, 189,<br>175, 137,<br>269, 149</td><td>Cyanidin-3-O-<br>glucoside*</td></tr><tr><td>5</td><td>39.7</td><td>504</td><td>433,<br>271</td><td></td><td></td><td>Pelargonidin-3-O-<br>glucoside</td></tr><tr><td colspan="7"><strong>Blood orange juice</strong></td></tr><tr><td>1</td><td>29.5</td><td>528</td><td>465,<br>303</td><td></td><td></td><td>Delphinidin-3-O-<br>glucoside</td></tr><tr><td>2</td><td>31.2</td><td>520</td><td>611,<br>287</td><td></td><td></td><td>Cyanidin-3-O-<br>sophoroside</td></tr><tr><td>3</td><td>32.3</td><td>516</td><td>449,<br>287</td><td></td><td></td><td>Cyanidin-3-O-<br>galactoside</td></tr><tr><td>4</td><td>34.7</td><td>516,<br>280</td><td>449</td><td>287</td><td>287 → 213,<br>231, 259,<br>241, 189,<br>175, 137,<br>269, 149,<br>203</td><td>Cyanidin-3-O-<br>glucoside*</td></tr><tr><td>5</td><td>42.9</td><td>520</td><td>625,<br>301,<br>463</td><td></td><td></td><td>Peonidin-3,5-di-O-<br>glucoside</td></tr><tr><td>6</td><td>46.3</td><td>512</td><td>593,<br>287</td><td></td><td></td><td>Cyanidin-3-O-<br>dioxalylglucoside</td></tr><tr><td>7</td><td>47.6</td><td>516,<br>280</td><td>535</td><td>287</td><td>287 → 213,<br>231, 259,<br>241, 189,<br>175, 137,<br>269, 149</td><td>Cyanidin-3-O-(6'<br>-malonyl)glucoside*</td></tr><tr><td>8</td><td>50.6</td><td>520,<br>280</td><td>593,<br>287</td><td></td><td></td><td>Cyanidin-3-O-<br>dioxaloylglucoside</td></tr><tr><td>9</td><td>55.2</td><td>520</td><td>549,<br>301</td><td></td><td></td><td>Peonidin-3-O-(6'<br>-malonyl)glucoside</td></tr><tr><td>10</td><td>57.6</td><td>520</td><td>679,<br>287</td><td></td><td></td><td>Cyanidin-3-O-<br>(malonyl, dioxalyl)<br>glucoside</td></tr></tbody></table>

a) Positive ESI, MS fragments are ranked according to decreasing intensity. * major pigment(s).

### 2.2.2. Anthocyanin purification

The aqueous extracts of eggplant and blood orange (see 2.2.1) and the pomegranate juice (pH 3.4) were purified using a Sep-Pak C18 cartridge previously activated with HPLC-grade MeOH followed by 0.01 % aqueous HCl. ACNs and other polyphenols were adsorbed onto the column, whereas sugars, acids, and other water-soluble compounds were removed by washing the column with 18 mL of 0.01 % aqueous HCl. The column was further washed with 18 mL of EtOAc to partially remove colorless non-ACN phenols. ACNs from the extract were collected by washing the column with 0.01 % HCl in MeOH. The ACN-rich extract was dried under reduced pressure at 30 °C.

## 2.3. Characterization of the extracts

The experimental conditions for the following analyses are reported in the Appendix as Supplementary information: total anthocyanin and total polyphenol contents in crude extracts, phenolic composition in purified extracts by HPLC-DAD and HPLC-DAD-ESI-MS.

## 2.4. Thermodynamics of water addition and proton transfer

A universal buffer (2.25 mL of H<sub>3</sub>PO<sub>4</sub> (85 % w/w) + 7.0 g of citric acid, H<sub>2</sub>O + 3.54 g of boric acid + 343 mL of 1 M NaOH, completed to 1 L by H<sub>2</sub>O) was used for all experiments.

The reversible transformations of ACNs in mildly acidic solution (pH 1–6) are recalled on Fig. 1. They consist a) in a fast proton transfer between the red flavium ion (AH<sup>+</sup>) and the purple neutral base (A, mixture of tautomers), and b) in water addition to AH<sup>+</sup>, yielding hemiketal B, in fast tautomeric equilibrium with cis-chalcone Cc, itself slowly isomerizing into trans-chalcone Ct. The last three species are all colorless. Color loss by hydration was studied by UV-visible spectroscopy using the pH jump technique (Pina, 2014). Concentrated stock solutions of extracts (9.15, 10.0 and 16.67 mg/mL for the, pomegranate, eggplant and blood orange extracts, resp.) were prepared in 0.1 M HCl (pH 1, 100 % AH<sup>+</sup>). At time zero of the kinetic experiments, a small volume of stock solution was rapidly added to a volume of universal buffer (dilution by a factor 1:90, final pH ranging from 2.5 to 6) placed in the quartz cell of a UV-visible spectrophotometer (Shimadzu UV-1800) and spectra were recorded from 250 to 800 nm until the hydration equilibrium (a mixture of colored and colorless forms) was reached.

The initial and final absorbance values (A<sub>0</sub>, A<sub>f</sub>) at the flavium's λ<sub>max</sub>, as well as the apparent first-order rate constant of hydration (k<sub>obs</sub>). From the pH dependence of k<sub>obs</sub> (Pina, 2014), the rate constants of hydration / tautomerization (k<sub>h</sub>, AH<sup>+</sup> → B + Cc) and the reverse reaction (k<sub>h</sub>, B + Cc → AH<sup>+</sup>), as well as the acidity constant (K<sub>a</sub>, AH<sup>+</sup>-A equilibrium) can be estimated using Eq. 1.

$$k_{\text{obs}} = \frac{[H^+]}{[H^+] + K_a} k_h + k_{-h} [H^+] \quad (1)$$

From the pH dependence of A<sub>f</sub> (Pina, 2014), acidity constant K<sub>a</sub>, connecting the flavium ion and all neutral forms except Ct, can be determined. The curve-fitting was carried out using Eq. 2.

$$A_f = A_0 \frac{1 + r_a 10^{(pH - pK_a)}}{1 + 10^{(pH - pK_a)}} \quad (2)$$

With A<sub>0</sub> = absorbance of a solution of pure flavium ion at λ<sub>max</sub>, K<sub>a</sub> = [H<sup>+</sup>]/[A<sup>+</sup>]/[B<sup>+</sup>]/[Cc]/[AH<sup>+</sup>], K<sub>a</sub> = [H<sup>+</sup>]/[A<sup>+</sup>]/[AH<sup>+</sup>], r<sub>a</sub> = ε(A)/ε(AH<sup>+</sup>) at the flavium's λ<sub>max</sub>.

When the spectral measurements are repeated a few hours later, full equilibrium is reached (including Ct), and the corresponding global acidity constant is K<sub>a</sub> = [H<sup>+</sup>]/[A<sup>+</sup>]/[B<sup>+</sup>]/[Cc]/[Ct]/[AH<sup>+</sup>].

The pK<sub>a</sub> value can be determined from Eq. 2 with A<sub>eq</sub> (absorbance at complete equilibrium) replacing A<sub>f</sub>, pK<sub>a</sub> and replacing pK<sub>a</sub>. The percentage of residual color was also evaluated as RC% = 100 × A<sub>eq</sub>/A<sub>0</sub>. Finally, thermodynamic constants of hydration at pseudo-equilibrium
and full equilibrium can be deduced from the following relationships:

$$ 
\begin{aligned}
K_h &= \frac{k_h}{k_{-h}} = [H^+] \frac{[B] + [Cc]}{[AH^+]} = K_a - K_a, \\
K'_h &= [H^+] \frac{[B] + [Cc] + [Ct]}{[AH^+]} = K'_a - K_a
\end{aligned}
$$

## 2.5. Metal binding experiment

Metal-ACN binding experiment was performed according a previously reported method (Fenger, Roux, et al., 2021) with some modifications. To 2.67 mL of a pH 5 buffer solution (0.01 M acetate), 30 µL of ACN stock solution were rapidly added and placed in the spectrometer cell. After a few minutes (pseudo-equilibrium), 30 µL of a fresh solution of Al³⁺ (in 0.1 M HCl) were added. The metal/ACN ratios in solution were 0.5, 1 and 2 equivalents. Spectra were recorded every 0.5 s over 5 min. The binding was relatively fast and accompanied by the development of a large visible band in the range 550–600 nm characteristic of the metal-ACN complex.

At t = 0 (Al³⁺ addition), A = Aeq (pseudo-equilibrium). Then, A(t) at λmax of complex increases to a final value A<sub>f</sub> (complexation equilibrium). The percentage of color regeneration is expressed as 100xA<sub>f</sub>/A<sub>eq</sub> at λmax of the complex.

## 2.6. Thermal degradation

Thermal degradation was carried out at pH 5 and 50 °C in a thermostated water bath according to a previously reported method (Fenger et al., 2019) with some modifications. Concentrated solutions of extracts (19.1 mg/mL, 32.8 mg/mL and 36.1 mg/mL for the pomegranate, eggplant and blood orange extracts, resp.) were prepared in 0.1 M HCl. At time zero, aliquots of 0.5 mL were added to 20 mL of buffer solution (10 mM acetate, pH 5), with or without added metal ion, and placed in a ventilated oven at 50 °C under magnetic stirring. Two daily samplings were performed. Samples were cooled down and their UV-vis spectra recorded, giving A(t, pH5). Then, samples were acidified to pH 1 by 6 M HCl (negligible dilution), and stored in the dark at room temperature till the next day in order to convert all the non-degraded ACN forms (colored and colorless) into the flavylium cation for titration. The corresponding absorbance is noted A(t, pH1). Control experiments (no degradation) were also performed. To 2 mL acetate buffer, 50 µL of stock solution were added and the absorbance at λmax, A(0, pH5), immediately recorded (mostly, a solution of neutral base). The same experiments were repeated with 2 mL 0.1 M HCl to obtain A(0, pH1). The fractions of residual color and residual pigments were calculated as X<sub>color</sub> = A(t, pH5) / A(0, pH5) and X<sub>pigment</sub> = A(t, pH1) / A(0, pH1). Both were plotted vs. time.

All absorbance values were approximately corrected for light scattering (which gradually took place after prolonged heating, t > 48 h), by subtracting the absorbance at 800 nm.

# 3. Results & discussion

## 3.1. Anthocyanin and total polyphenol contents in extracts

The TPC and TAC of the three crude extracts (before fractionation on C18 silica gel) are reported in Table 1 supplementary. For comparison, similar high TAC and TPC in eggplant peel (Dranca & Oroian, 2017), pomegranate juice (Catania et al., 2020; Legua et al., 2012) and blood orange (Forner-Giner et al., 2023; Legua et al., 2022) were also reported in the literature.

After purification on C18 silica gel, the ACN content in the extracts was strongly enhanced, up to a factor ca. 100 for BOJ (Table 1 supplementary).

## 3.2. Identification of major pigments and copigments

After fractionation on C18 silica gel, ACNs and other polyphenols

<table><caption>**Table 2**

HPLC-DAD-MS data and putative identification of copigments in the extracts.</caption><thead><tr><th>Peak</th><th>t<sub>R</sub><br>(min)</th><th>λ<sub>max</sub><br>(nm)</th><th>Full<br>MS<br>(m/z)</th><th>MS<sup>2</sup><br>(m/z)</th><th>MS<sup>3</sup><br>(m/z)</th><th>Structure</th></tr></thead><tbody><tr><td colspan="7">Eggplant peel a)</td></tr><tr><td>1</td><td>37.3</td><td>280</td><td>474</td><td>222,<br>457,<br>165</td><td>222<br>→<br>165</td><td>N<sup>1</sup>,N<sup>8</sup>-Di-<br>dihydrocaffeoylspermidine</td></tr><tr><td>2</td><td>40.6</td><td>288,<br>320</td><td>472</td><td>220,<br>310,<br>455,<br>163</td><td>220<br>→<br>163</td><td>N<sup>1</sup>-Caffeoyl-N<sup>8</sup><br>-dihydrocaffeoylspermidine</td></tr><tr><td>3</td><td>42.7</td><td>288,<br>320</td><td>472</td><td>222,<br>310,<br>293,<br>455,<br>165</td><td>222<br>→<br>165</td><td>N<sup>1</sup>-Dihydrocaffeoyl-N<sup>8</sup>-<br>caffeoylspermidine</td></tr><tr><td>4</td><td>45.8</td><td>296,<br>320</td><td>470</td><td>308,<br>220,<br>453,<br>163,<br>291,<br>234</td><td>308<br>→<br>291,<br>220,<br>234</td><td>N<sup>1</sup>,N<sup>8</sup>-Dicaffeoylspermidine</td></tr><tr><td colspan="7">Pomegranate juice b)</td></tr><tr><td>1</td><td>24.8</td><td>264</td><td>783</td><td>765,<br>774,<br>689,<br>613</td><td>765<br>→<br>597,<br>613,<br>301,<br>275,<br>533,<br>427,<br>229</td><td>Pedunculagin I</td></tr><tr><td colspan="7">Blood orange juice b)</td></tr><tr><td>1</td><td>42.0</td><td>272,<br>336</td><td>593</td><td>473,<br>503,<br>353,<br>575,<br>383</td><td>473<br>→<br>353,<br>383</td><td>Apigenin-6,8-di-C-<br>glucoside</td></tr><tr><td>2</td><td>45.5</td><td>284</td><td>777*</td><td>433,<br>741,<br>271,<br>579</td><td>433<br>→<br>271</td><td>Naringenin-7-O-rutinoside-<br>4'-O-glucoside</td></tr><tr><td>3</td><td>66.6</td><td>284</td><td>579</td><td>271</td><td>271<br>→<br>151,<br>177,<br>165</td><td>Naringenin-7-O-<br>neohesperidoside</td></tr><tr><td>4</td><td>72.8</td><td>284</td><td>609</td><td>301</td><td>301<br>→<br>286,<br>283,<br>242,<br>257,<br>199,<br>125</td><td>Hesperetin-7-O-<br>neohesperidoside</td></tr></tbody></table>

a) Positive ESI, b) negative ESI. MS fragments are ranked according to decreasing intensity. * Undefined adduct (+36)

were identified using HPLC-DAD-MS by comparison with the literature. The corresponding data are presented in Tables 1 and 2 (see also Fig. 1 supplementary to Fig. 4 supplementary).

### 3.2.1. Eggplant peel

Based on comparison of retention time and LC-MS fragmentation pattern with published data (Kacjan Maršić et al., 2014; Zhang et al., 2020), four anthocyanins were identified in eggplant peel (Fig. 1 supplementary, Table 1). Peaks 3 and 4 were identified as delphinidin-3-O-glucoside (D3G) and delphinidin 3-O-rutinoside (D3R). Peaks 1 and 2 had the same molecular ion (m/z 773). Mass fragmentation indicated
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Spectral variations of anthocyanin-rich extracts after several direct pH jump in the range 2–6 at pseudo-equilibrium (**left panel**) and at equilibrium (**right panel**). **Top:** eggplant peel. **Middle:** pomegranate juice. **Down:** blood orange juice.</figcaption></figure>

that peak 1 is a delphinidin diglycoside bearing one hexose and one rutinose. Despite the absence of fragments for peak 2, both peaks were tentatively identified as delphinidin 3-O-rutinoside-5-O-galactoside (peak 1) and delphinidin 3-O-rutinoside-5-O-glucoside (peak 2) from their retention times. Delphinidin 3-O-rutinoside was the predominant ACN in eggplant peel with a peak area of 84.6 % of total ACNs identified.

The copigments identified in eggplant peel extracts were diamide derivatives of triamine spermidine following acylation by caffeic and dihydrocaffeic acids (Table 2, Fig. 2 supplementary). The main mass fragmentations observed consisted in the cleavage of a) the C→N bond

of caffeoylamides with release of caffeoyl- and dihydrocaffeoyl-spermidines (m/z 308 & 310, resp.), while the dihydro homologs were not cleaved, and b) the central amine group with release of caffeoyl- and dihydrocaffeoyl-allylamines (m/z 220 & 222, resp.), in agreement with the literature (Sun et al., 2015). Asymmetry in the latter cleavage allowed discriminating the two regioisomers (peaks 2 and 3 in Table 2).

### 3.2.2. Pomegranate juice

The ACN profile consists in mono- and di-O-glucosides of delphinidin and cyanidin, and in pelargonidin 3-O-glucoside (Table 1, Fig. 1
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** pH dependence of the apparent rate constant of water addition after pH jumps from pH 2 in aqueous solutions of anthocyanin-rich extracts. **Left:** eggplant peel. **Middle:** pomegranate juice. **Right:** blood orange juice.</figcaption></figure>

supplementary) in agreement with the literature (Mena et al., 2012). The most abundant anthocyanin is cyanidin 3-O-glucoside (C3G, peak 4), representing 42 % of the total ACN peak area, followed by cyanidin 3,5-O-diglucoside (C3,5DG, peak 2, 38 %). Minor ACNs include delphinidin 3,5-O-diglucoside (6.9 %, peak 1), delphinidin 3-O-glucoside (7.9 %, peak 3), and pelargonidin 3-O-glucoside (5.1 %, peak 5).

Ellagitannins are complex polyphenols (hydrolysable tannins) based on a d-glucose core acylated by galloyl and hexahydroxydiphenoyl (HHDP) units. They are recognized as major phenolic compounds in pomegranate juice (Díaz-Mula et al., 2019). In our pomegranate juice extract, ellagitannin 1 was detected with a molecular ion at *m/z* 783 (Table 2, Fig. 3 supplementary) consistent with a d-glucose core acylated by 2 HHDP moieties. Its fragments at *m/z* 301 is characteristic of ellagic acid released after cleavage of a HHDP moiety (Fischer et al., 2011); Ellagitannin 1 was thus identified to pedunculagin I.

### 3.2.3. Blood orange juice

The extract is the most diversified in terms of ACN distribution with ten compounds identified (Table 1, Fig. 1 supplementary), seven cyanidin derivatives (peaks 2, 3, 4, 6, 7, 8, and 10), two peonidin (peaks 5 and 9) and one delphinidin derivatives (peaks 1). Compared to the other extracts, blood orange juice is specific in its content in acylated anthocyanins. For instance, peak 7, one of the most abundant BOJ ACNs, showed a molecular ion at *m/z* 535 and a fragment at *m/z* 287 (cyanidin aglycone) corresponding to the loss of a malonylglucoside unit. Although minor ACNs, peaks 6, 8 and 10 (molecular ions at *m/z* 593, 593 and 679, resp. + a common fragment at *m/z* 287), are all cyanidin 3-O-dioxalylglucosides with an additional malonyl residue for peak 10, remarkable for its triacylated single Glc moiety. By contrast, peak 9 bears a unique malonyl residue and was identified to peonidin 3-O-(6'-malonyl)glucoside. Cebadera-Miranda et al. (2019) reported a similar ACN composition in blood orange juice. The major BOJ ACNs are cyanidin 3-O-glucoside (C3G, peak 4, 57.1 %) and cyanidin 3-O-(6'-malonyl)glucoside (C3MG, peak 7, 28.0 %) (Table 1, Fig. 1 supplementary), in agreement with the literature (Hillebrand et al., 2004; Morales et al., 2021).

Four flavonoid copigments could be identified in BOJ (Table 2, Fig. 4 supplementary) by comparing with the literature (Barreca et al., 2011). Compound 1 was identified as a diglucoside of the flavone apigenin. The absence of the typical fragmentation of O-glycoside (loss of the glycosyl unit) points to a di-C-glucoside with a complex fragmentation pattern, combining the loss of fragments at *m/z* 18 (-H₂O), 90, 120, 210, and 240. The last four fragmentations are consistent with the loss of (CH₂O)₃ and (CH₂O)₄ fragments, or a combination of them, from the C-glycosyl units. Compound 1 was thus identified as apigenin 6,8-di-C-glucoside (vicenin-2). Compounds 2–4 were identified as O-glycosides of flavanones naringenin and hesperetin. The fragmentation of compound 2 (molecular ion at *m/z* 741) clearly showed the sequential loss of glycosyl units, i.e.

-Glc (*m/z* 579), -Rha (*m/z* 433), -Glc (*m/z* 271), typical of naringenin-7-O-rutinoside-4'-O-glucoside.

### 3.3. Reversible color loss in the extracts

Several pH jump experiments were conducted and the spectral changes in the visible region were monitored over time. By fitting the decay of the final visible absorbance as a function of pH, the pK'a value (pseudo-equilibrium connecting the flavylium ion and all neutral forms, except the *trans*-chalcone) was determined. Repeating the titration after a sufficiently long period to ensure complete equilibrium afforded the pK'a value (including the *trans*-chalcone) (Fig. 2, Table 3). As expected, the trend observed is pK'a < pK'a, but the differences are very small. It can thus be stated that the mole fraction of Cₜ at equilibrium is low (< 15 %).

The pK'a values can be compared with those of the literature for the main ACNs in the extract (Leydet et al., 2012; Mazza & Brouillard, 1987), i.e. delphinidin 3-O-glucoside for EPP (assuming close behavior to delphinidin 3-O-rutinoside, the actual major EPP ACN), cyanidin 3-O-glucoside and cyanidin 3,5-O-diglucoside for PGJ, and cyanidin 3-O-glucoside for BOJ (assuming close behavior to cyanidin 3-O-(6'-malonyl)glucoside, the other major BOJ ACN).

From Table 3, it is clear that the global acidity constant pK'a mostly reflects the overall formation of the colorless forms (pK'a > > Kₐ, pK'a ≈ pK'h). and is thus a good measure of the sensitivity of a given extract to color loss through hydration. The pK'a values indicate that the color of the BOJ and EPP extracts is more stable than that of PGJ, which is fully consistent with PGJ containing a high proportion of anthocyanidin 3,5-O-diglucoside, known to be much more prone to hydration than their 3-O-glucoside counterpart (a likely consequence of the electron-withdrawing effect of the additional Glc, which increases the electrophilic character of the flavylium ion) (Seco et al., 2024). This effect is obvious from the pK'a difference between cyanidin 3,5-O-diglucoside and cyanidin 3-O-glucoside (Table 3). It is confirmed by the values of the hydration rate constant kₕ, the one of PGJ being higher by a factor 2–3 than those of BOJ and EPP. While it is satisfying to assign PGJ a pK'a value that is intermediate between those of its two main ACNs, EPP and BOJ display higher pK'a values than that of the main pure ACN they contain. This is a manifestation of intermolecular copigmentation within the extracts. Indeed, since hydroxycinnamic acids are recognized as potent ACN copigments (Trouillas et al., 2016), N,N-dicaffeoylspermidine amides are likely to interact rather strongly with the EPP anthocyanins and thus contribute to color stability by limiting hydration. This is also true for the flavones and flavanones of BOJ, but probably much less so for PGJ because ellagitannins are rather poor copigments (Trouillas et al., 2016).

Because of the competing thermodynamically favored hydration reaction, pKₐ values are typically harder to accurately assess, requiring
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Left: molar fractions of flavylium ion (red), quinonoid base (blue) and the pool of colorless forms (black) as a function of pH. Right: residual color of the equilibrated solutions. Final ACN concentration in the solutions: 0.10, 0.11 and 0.18 mg/mL for the pomegranate, eggplant and blood orange extracts, resp. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

to monitor the apparent rate constant of hydration as a function of pH (Fig. 3). However, the trend observed is that the extracts $pK_a$'s tend to be higher than that of the main pure ACNs. This again could reflect copigmentation. Indeed, phenolic copigments usually interact more strongly with the flavylium ion than with the neutral base (Trouillas et al., 2016), thus broadening the pH range of flavylium predominance.

From the $pK'_a$ and $pK_a$ values, the speciation diagrams for the colored forms $AH^+$ and A, and the complete pool of colorless forms B +

$Cc + Ct$ were plotted for each extract over the pH range 1–6 (Fig. 4). They show that above pH 3, PGJ solutions are almost colorless, while EPP, and even more so BOJ, solutions still retain part of their color. This is confirmed by their visual appearance.

### 3.4. Metal binding

As the main ACNs in the EPP, PGJ and BOJ extracts are cyanidin or
<caption>**Table 3**</caption>

Kinetic and thermodynamic data for the anthocyanin-rich extracts and a selection of pure anthocyanins for comparison. Major ACNs in extracts: D3R (EPP), C3G + C3,5diG (1:1) (PGJ), C3G + C3MG (BOJ).

<table><thead><tr><th></th><th>EPP</th><th>PGJ</th><th>BOJ</th><th>D3G<sup>‡</sup></th><th>C3G<sup>‡</sup></th><th>C3,5diG<sup>*</sup></th></tr></thead><tbody><tr><td>pK<sub>a</sub> 1)</td><td>4.0 (±<br>0.1)</td><td>3.7 (±<br>0.2)</td><td>4.4 (±<br>0.1)</td><td>3.8</td><td>3.8</td><td>3.38</td></tr><tr><td>pK<sup>h</sup> 2)</td><td>2.75 (±<br>0.01)<sup>a</sup></td><td>2.41 (±<br>0.02)<sup>b</sup></td><td>2.85 (±<br>0.04)<sup>a</sup></td><td>2.6</td><td>2.5</td><td>2.08</td></tr><tr><td>pK<sup>h</sup> 3)</td><td>2.76 (±<br>0.01)<sup>b</sup></td><td>2.47 (±<br>0.06)<sup>c</sup></td><td>2.93 (±<br>0.02)<sup>a</sup></td><td>–</td><td>–</td><td>–</td></tr><tr><td>pK<sup>h</sup> 4)</td><td>2.78</td><td>2.43</td><td>2.86</td><td>2.6</td><td>2.5</td><td>2.10</td></tr><tr><td>pK<sup>h</sup> 5)</td><td>2.79<br>(2.73)</td><td>2.50<br>(2.16)</td><td>2.94<br>(3.13)</td><td>(2.5)</td><td>(2.5)</td><td>(2.23)</td></tr><tr><td>k<sub>h</sub> (s<sup>-1</sup>)</td><td>0.11 (±<br>0.01)</td><td>0.25 (±<br>0.05)</td><td>0.08 (±<br>0.01)</td><td>0.09</td><td>0.11</td><td>–</td></tr><tr><td>k<sub>h</sub> (M<sup>-1</sup><br>s<sup>-1</sup>)</td><td>58 (± 4)</td><td>36 (± 19)</td><td>106 (± 8)</td><td>30</td><td>31</td><td>–</td></tr></tbody></table>

1) From the pH dependence of the hydration reaction, 2) titration at equilibrium, 3) titration at pseudo-equilibrium, 4) $K^{\prime h} = K^{\prime a} - K_a$, 5) $K^{\prime h} = K^{\prime a} - K_a$, values between parentheses: $K^{\prime h} = k_h/k_{\prime h}$. Values with letters are the means of two determinations with standard deviations. Different letters in the same line refer to significantly different data (p < 0.05). Other standard deviations are from the curve fitting experiments. * From Mazza and Brouillard (1987). ‡ From Leydet et al. (2012).

delphinidin derivatives, they are expected to bind hard metal ions via their B-ring. Under mildly acidic conditions, this property is selective to the colored forms, as their B-ring protons are much more acidic than those of the colorless forms (Yang et al., 2024), and thus more easily displaced by the incoming metal ion. Hence, the hydration equilibrium is reversed to the profit of the colored forms and hyperchromism is observed. Within their metal complexes, ACNs adopt quinonoid structures that are resistant to water addition and also express distinct purple – blue colors (Fenger, Sigurdson, et al., 2021; Trouillas et al., 2016). Overall, metal – ACN binding is a powerful mechanism to both stabilize and diversify ACN colors.

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Solutions of anthocyanin-rich extracts. (A) Extracts at the hydration equilibrium (pH 5). (B) Extracts after Al³⁺ binding (pH 5). (C) Extracts supplemented with Al³⁺ after thermal treatment (50 °C for 24 h, pH 5). (D) The same after acidification to pH 1.</figcaption></figure>

When Al³⁺ was added to equilibrated solutions of extract at pH 5, rapid bathochromic and hyperchromic shifts were observed (Fig. 5 supplementary). This is evidence that the colored forms selectively bind Al³⁺, thus reversing the hydration equilibrium, and that other extract's components, which could potentially compete with ACNs for Al³⁺, do not prevent metal – ACN binding. While the Al³⁺-induced bathochromic shifts (compared to free flavylium ion) were approximately the same (ca. 50 nm) for the three extracts, the hyperchromic shifts (expressed as color regenerated CR) varied with EPP and BOJ showing much higher CR values than PGJ (Table 4). The low CR for PGJ can be attributed to the presence of ellagitannins that probably compete with ACNs for Al³⁺. By contrast, the N,N-dicaffeoylspermidine amides of EPP and BOJ flavonoids probably have less affinity for Al³⁺. The highest percentage of color regenerated was achieved at a metal/ACN molar ratio of 2 in the three extracts studied. The strong influence of Al³⁺ on the color expressed in EPP is evident (Fig. 5), as delphinidin glycosides (with their additional OH group in the B-ring), once bound to Al³⁺, provide a nice blue color, while the Al³⁺ complexes of cyanidin glycosides in PGJ and BOJ remain purple.

### 3.5. Thermal degradation

The stability of the anthocyanin-rich extracts was evaluated at pH 5 and 50 °C. Under such conditions, a fast color loss occurred during the first few minutes, mainly due to hydration and subsequent C-ring opening with accumulation of the *trans*-chalcone. Beyond ca. 30 min, the color loss was much slower and mainly assigned to degradation by a combination of autoxidation and hydrolysis. The residual color intensity followed the following ranking: BOJ > PGJ > EPP. Over longer heating periods (> 20 h), the absorbance measurements gradually became blurred by browning and light scattering featuring precipitation, especially with EPP and PGJ, as evidenced by their higher browning index at 24 h (Table 5).

No such problems occurred after acidification of samples to pH 1, so as to convert all residual ACN forms to the flavylium ion for the
<caption>**Table 4**</caption>

The potential of Al³⁺ binding for color regeneration from extracts at the hydration equilibrium. A₀: maximal visible absorbance before hydration (t = 0), Aeq: maximal visible absorbance at the hydration equilibrium, Af: maximal visible absorbance after metal binding, RC: residual color at the hydration equilibrium, CR: color regenerated after metal binding. For EPP, A₀ and Aeq are measured at 548 nm, while Af is measured at 572 nm. For PGJ and BOJ, A₀ and Aeq are measured at 536 nm, while Af is measured at 562 nm.

<table><thead><tr><th>Extract</th><th>A<sub>0</sub></th><th>A<sub>eq</sub></th><th>RC (%) a)</th><th>Al<sup>3+</sup> (equiv.)</th><th>A<sub>f</sub></th><th>CR (%) b)</th></tr></thead><tbody><tr><td rowspan="5">EPP</td><td>0.523 ± 0.035</td><td>0.111 ± 0.009</td><td>19.2 ± 1.0</td><td rowspan="2">2</td><td>0.759 ± 0.052</td><td>145.1 ± 0.4</td></tr><tr><td>0.514 ± 0.016</td><td>0.113 ± 0.001</td><td>22.0 ± 0.8</td><td>0.657 ± 0.007</td><td>127.9 ± 2.6</td></tr><tr><td>0.501 ± 0.002</td><td>0.119 ± 0.001</td><td>23.3 ± 0.2</td><td rowspan="2">0.5</td><td>0.657 ± 0.003</td><td>127.8 ± 0.2</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">PGJ</td><td>0.297 ± 0.006</td><td>0.049 ± 0.006</td><td>16.7 ± 0.4</td><td rowspan="2">2</td><td>0.322 ± 0.029</td><td>108.9 ± 6.8</td></tr><tr><td>0.304 ± 0.010</td><td>0.056 ± 0.000</td><td>18.4 ± 0.7</td><td>0.270 ± 0.005</td><td>89.1 ± 2.2</td></tr><tr><td>0.304 ± 0.002</td><td>0.059 ± 0.003</td><td>19.2 ± 1.0</td><td rowspan="2">0.5</td><td>0.261 ± 0.007</td><td>85.8 ± 1.6</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">BOJ</td><td>0.475 ± 0.058</td><td>0.160 ± 0.006</td><td>34.1 ± 5.6</td><td rowspan="2">2</td><td>0.669 ± 0.031</td><td>141.4 ± 10.9</td></tr><tr><td>0.471 ± 0.006</td><td>0.165 ± 0.006</td><td>35.1 ± 0.8</td><td>0.596 ± 0.014</td><td>125.1 ± 2.8</td></tr><tr><td>0.521 ± 0.004</td><td>0.189 ± 0.005</td><td>36.4 ± 1.4</td><td rowspan="2">0.5</td><td>0.627 ± 0.004</td><td>120.5 ± 0.3</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

a) % RC = 100Aeq/A₀, b) % CR = 100Aeq/A₀. CR values higher than 100 % simply reflect εmax(complex) > εmax(free form).

<caption>**Table 5**</caption>

Color loss and pigment loss in solutions of anthocyanin-rich extracts with or without supplementation with Al³⁺ (2 equiv.) after 24 h at pH 5 and 50 °C.

<table><thead><tr><th>No added metal ion</th><th>X<sub>color</sub></th><th>% Color loss</th><th>X<sub>pigment</sub></th><th>% Pigment loss</th><th>BI a)</th></tr></thead><tbody><tr><td rowspan="2">EPP</td><td>0.32 ± 0.02</td><td>68 ± 2</td><td>0.45 ± 0.02</td><td>55 ± 2</td><td>1.9 ± 0.10</td></tr><tr><td>0.41 ± 0.02</td><td>59 ± 2</td><td>0.76 ± 0.05</td><td>24 ± 5</td><td>2.13 ± 0.03</td></tr><tr><td rowspan="2">PGJ</td><td>0.57 ± 0.05</td><td>43 ± 5</td><td>0.73 ± 0.02</td><td>27 ± 2</td><td>1.00 ± 0.03</td></tr><tr><td>0.50 ± 0.01</td><td>50 ± 1</td><td>0.57 ± 0.01</td><td>43 ± 1</td><td>-</td></tr><tr><td rowspan="2">BOJ</td><td>0.59 ± 0.01</td><td>41 ± 1</td><td>0.59 ± 0.01</td><td>41 ± 1</td><td>-</td></tr><tr><td>0.67 ± 0.02</td><td>33 ± 2</td><td>0.84 ± 0.01</td><td>16 ± 1</td><td>-</td></tr></tbody></table>

a) Browning index, defined as A(420 nm)/A(λmax visible).

assessment of the total pigment loss (Fenger et al., 2019), which was then plotted vs. time to assess the pigments' half-life (Fig. 6). Blood orange juice emerged as the most stable one, boasting a half-life (t₁/₂) of ca. 90 h. Pomegranate juice followed with t₁/₂ = 60 h, while eggplant peel showed the lowest stability with t₁/₂ = 18 h.

Previous studies have shown that the thermal degradation of purified and non-purified anthocyanins is faster when temperature and pH increase (Nayak et al., 2011; Patras et al., 2010). For example, the half-life of purified C3G at pH 3 is ca. 126, 81 and 66 min at 50, 70 and 90 °C, respectively. However, it is greatly prolonged in the presence of different phenolic copigments in large excess (100 equiv.): 321, 258, and 209 min for ferulic acid, 279, 204, and 150 min for dopamine, and 326, 296, and 261 min for (+)-catechin (Kanha et al., 2019). In our extracts, the favorable effect of intermolecular copigmentation is also manifested. For instance, BOJ, the most stable extract, contain flavonoids, especially

<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Color loss (●), pigment loss (■), and browning index (▲) in anthocyanin-rich extracts at pH 5 and 50 °C as a function of time: — (EPP), --- (PGJ), ... (BOJ). Error bars represent the standard deviation calculated from duplicate measurements.</figcaption></figure>

flavones, whose flat tricyclic nuclei are particularly suitable to develop strong π-stacking interactions with ACNs (Trouillas et al., 2016). Under acidic conditions, the *trans*-chalcone is the most vulnerable ACN form (Cruz et al., 2022; Sadilova et al., 2007). Indeed, the rate-limiting hydrolysis of its 3-O-glycosyl group releases an electron-rich enol group highly sensitive to autoxidation. Hence, strong π-stacking interactions between flavonoids and the ACN colored forms are expected to limit water addition (as evidenced by the significantly higher pK'ₐ value for BOJ, compared to its main ACN C3G, Table 3) and subsequent *trans*-chalcone accumulation, thereby also limiting thermal degradation. Cao et al. (2009) studied the thermal degradation of cyanidin 3-(6''-malonyl) glucoside (C3MG) and cyanidin 3-glucoside (C3G), the two main BOJ ACNs, at pH 3.5. During the first hours of heating, HPLC-MS analysis revealed that C3MG was hydrolyzed to C3G, which tended to accumulate, suggesting that its degradation was slower than its formation from C3MG.

Unlike flavones and flavonols, ellagitannins are only modest copigments and their impact on the thermal stability of PGJ ACNs may be negligible. Moreover, PGJ is rich in C3,5DG and anthocyanidin 3,5-O-diglucosides, which are known to be less thermally stable than their 3-O-glucoside homologs, a likely consequence of the former being more sensitive to water addition than the latter (Seco et al., 2024). Indeed, at pH 3.5, the colorless species were the first intermediates in the degradation pathway (Kanha et al., 2019). In addition, bulky ACNs (e.g., di- and triglucosides) form less stable copigmentation complexes than monoglucosides (Trouillas et al., 2016).

The faster degradation of EPP anthocyanins might reflect their higher sensitivity to autoxidation due to the presence of caffeoyl derivatives in the extract. Indeed, although the caffeoyl residues of acylated ACNs are effective in slowing down color loss by hydration (intramolecular copigmentation), their redox activity can, on the other hand, accelerate the thermal degradation (Fenger, Roux, et al., 2021). This was confirmed by adding caffeic acid to ACN solutions before heating. In this study, new pigments were detected resulting from the coupling between the nucleophilic A-ring of anthocyanins and the electrophilic o-quinone derived from the caffeoyl residues or free caffeic acid by autoxidation.

Precipitation occurred in the PGJ and BOJ extracts supplemented with 0.5 or 1 equiv. of Al³⁺. Hence, the influence of metal binding was quantified with 2 equiv. of Al³⁺. Overall, color stability during heating
at pH 5 was significantly improved, especially with PGJ and BOJ (Table 5, Fig. 6). This improvement is largely due to the quinonoid structure of the metal complexes, which does not undergo water addition (Denish et al., 2021; Fenger, Sigurdson, et al., 2021). However, with PGJ and EPP, the extent of pigment loss remains high, meaning that the metal complexes remain vulnerable to autoxidation. With BOJ, the metal complexes are more resistant to degradation and the relatively modest color loss observed is thus largely due to the gradual dissociation of the metal complexes with subsequent hydration of the free ACNs.

Overall, among the three extracts studied, the anthocyanins from blood orange juice appear most promising as food colorings, being less sensitive to water addition and capable of forming stable metal complexes.

## 4. Conclusions

In this work, extracts of common plant food were easily prepared, then enriched in anthocyanins and extensively characterized: phenolic composition, susceptibility to hydration (fast reversible color loss) and thermal degradation (slow irreversible color loss), capacity for metal binding as an efficient way of color diversification and stabilization. The corresponding protocols are both simple and rigorous, taking into account the rich physical chemistry of anthocyanins. By comparison with data on pure ACNs, they also provide an insight into the role possibly played by the other phenolic compounds in the extracts. Overall, this approach may prove valuable as a first step in the systematic search for natural food colorings. In a second step, testing the extracts under an extended range of experimental conditions (e.g., pH, temperature, time periods) will clearly be necessary to go closer to real conditions of food processing and storage.

## CRediT authorship contribution statement

**Sabrina Idrir:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization.
**Sabiha Achat:** Writing – review & editing, Supervision, Methodology, Investigation. **Luis Cruz:** Writing – review & editing, Supervision, Methodology. **Olivier Dangles:** Writing – review & editing, Supervision, Methodology, Conceptualization.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: CRUZ Luis reports financial support was provided by Fundação para a Ciência e a Tecnologia. CRUZ Luis reports a relationship with Fundação para a Ciência e a Tecnologia that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors gratefully acknowledge Prs Khodir Madani (Bejaia University), Lila Boulekbache-Makhlouf (Bejaia University), and Victor de Freitas (Porto University) for their support. Luís Cruz acknowledges Fundação para a Ciência e a Tecnologia (FCT) for his research contract (2023.07923.CEECIND).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2025.144004.

## Data availability

Data will be made available on request.

## References

Barreca, D., Bellocco, E., Caristi, C., Leuzzi, U., & Gattuso, G. (2011). Distribution of C- and O-glycosyl flavonoids, (3-hydroxy-3-methylglutaryl)glycosyl flavanones and furocoumarins in *Citrus aurantium* L. juice. *Food Chemistry*, **124**, 576-582. https://doi.org/10.1016/j.foodchem.2010.06.076

Cao, S., Liu, L., Pan, S., Lu, Q., & Xu, X. (2009). A comparison of two determination methods for studying degradation kinetics of the major anthocyanins from blood Orange. *Journal of Agricultural and Food Chemistry*, **57**, 245-249. https://doi.org/10.1021/jf8021964

Catania, P., Comparetti, A., De Pasquale, C., Morello, G., & Vallone, M. (2020). Effects of the extraction technology on pomegranate juice quality. *Agronomy*, **10**, 1483. https://doi.org/10.3390/agronomy10101483

Cebadera-Miranda, L., Domínguez, L., Dias, M. I., Barros, L., Ferreira, I. C. F. R., Igual, M., ... Cámara, M. (2019). Sanguinello and Tarocco (*Citrus sinensis* [L. Osbeck]: Bioactive compounds and colour appearance of blood oranges. *Food Chemistry*, **270**, 395-402. https://doi.org/10.1016/j.foodchem.2018.07.094

Cruz, L., Basílio, N., Mateus, N., de Freitas, V., & Pina, F. (2022). Natural and synthetic flavylium-based dyes: The chemistry behind the color. *Chemical Reviews*, **122**, 1416-1481. https://doi.org/10.1021/acs.chemrev.1c00399

Dangles, O. (2024). Anthocyanins as natural food colorings: The chemistry behind and challenges still ahead. *Journal of Agricultural and Food Chemistry*, **72**, 12356-12372. https://doi.org/10.1021/acs.jafc.4c01050

Dangles, O., & Fenger, J.-A. (2018). The chemical reactivity of anthocyanins and its consequences in food science and nutrition. *Molecules*, **23**, 1970. https://doi.org/10.3390/molecules23081970

Denish, P. R., Fenger, J.-A., Powers, R., Sigurdson, G. T., Grisanti, L., Guggenheim, K. G., ... Robbins, R. J. (2021). Discovery of a natural cyan blue : A unique food-sourced anthocyanin could replace synthetic brilliant blue. *Science Advances*, **7**, Article eabe7871. https://doi.org/10.1126/sciadv.abe7871

Díaz-Mula, H. M., Tomás-Barberán, F. A., & García-Villalba, R. (2019). Pomegranate fruit and juice (cv. Mollar), rich in ellagitannins and anthocyanins, also provide a significant content of a wide range of Proanthocyanidans. *Journal of Agricultural and Food Chemistry*, **67**, 9160-9167. https://doi.org/10.1021/acs.jafc.8b07155

Dranca, F., & Oroian, M. (2017). Total monomeric anthocyanin, total phenolic content and antioxidant activity of extracts from eggplant (*Solanum melongena* L.) peel using ultrasonic treatments: Eggplant antioxidants extraction. *Journal of Food Process Engineering*, **40**, Article e12312. https://doi.org/10.1111/jfpe.12312

Fenger, J.-A., Moloney, M., Robbins, R. J., Collins, T. M., & Dangles, O. (2019). The influence of acylation, metal binding and natural antioxidants on the thermal stability of red cabbage anthocyanins in neutral solution. *Food & Function*, **10**, 6740-6751. https://doi.org/10.1039/C9FO01884K

Fenger, J.-A., Roux, H., Robbins, R. J., Collins, T. M., & Dangles, O. (2021). The influence of phenolic acyl groups on the color of purple sweet potato anthocyanins and their metal complexes. *Dyes and Pigments*, **185**, Article 108792. https://doi.org/10.1016/j.dyeip.2020.108792

Fenger, J.-A., Sigurdson, G. T., Robbins, R. J., Collins, T. M., Giusti, M. M., & Dangles, O. (2021). Aclylated anthocyanins from red cabbage and purple sweet potato can bind metal ions and produce stable blue colors. *International Journal of Molecular Sciences*, **22**, 4551. https://doi.org/10.3390/ijms22094551

Fischer, U. A., Carle, R., & Kammerer, D. R. (2011). Identification and quantification of phenolic compounds from pomegranate (*Punica granatum* L.) peel, mesocarp, aril and differently produced juices by HPLC-DAD-ESI/MSn. *Food Chemistry*, **127**, 807-821. https://doi.org/10.1016/j.foodchem.2010.12.156

Forner-Giner, M.A., Ballesta-de los Santos, M., Melgarejo, P., Martínez-Nicolás, J. J., Melián-Navarro, A., Rufz-Canales, A., ... Legua, P. (2023). Fruit quality and primary and secondary metabolites content in eight varieties of blood oranges. *Agronomy*, **13**, 1037. https://doi.org/10.3390/agronomy13041037

Hillebrand, S., Schwarz, M., & Winterhalter, P. (2004). Characterization of anthocyanins and pyranoanthocyanins from blood orange [*Citrus sinensis* (L.) Osbeck] juice. *Journal of Agricultural and Food Chemistry*, **52**, 7331-7338. https://doi.org/10.1021/jf0487957

Kacjan Marsić, N., Mikulic-Petkovsek, M., & Štampar, F. (2014). Grafting influences phenolic profile and carpometric traits of fruits of greenhouse-grown eggplant (*Solanum melongena* L.). *Journal of Agricultural and Food Chemistry*, **62**, 10504-10514. https://doi.org/10.1021/jf503338m

Kanha, N., Surawang, S., Pitchakarn, P., Regenstein, J. M., & Laakuldilok, T. (2019). Copigmentation of cyanidin 3-O-glucoside with phenolics : Thermodynamic data and thermal stability. *Food Bioscience*, **30**, Article 100419. https://doi.org/10.1016/j.fbio.2019.100419

Krga, I., & Milenkovic, D. (2019). Anthocyanins: From sources and bioavailability to cardiovascular-health benefits and molecular mechanisms of action. *Journal of Agricultural and Food Chemistry*, **67**, 1771-1783. https://doi.org/10.1021/acs.jafc.8b06737

Legua, P., Melgarejo, P., Martínez, J. J., Martínez, R., & Hernández, F. (2012). Evaluation of Spanish pomegranate juices: Organic acids, sugars, and anthocyanins. *International Journal of Food Properties*, **15**, 481-494. https://doi.org/10.1080/10942912010.491931

Legua, P., Modica, G., Porras, I., Conesa, A., & Continela, A. (2022). Bioactive compounds, antioxidant activity and fruit quality evaluation of eleven blood orange
cultivars. *Journal of the Science of Food and Agriculture*, **102**, 2960-2971. https://doi.org/10.1002/jsfa.11636

Leydet, Y., Gavara, R., Petrov, V., Diniz, A. M., Jorge Parola, A., Lima, J. C., & Pina, F. (2012). The effect of self-aggregation on the determination of the kinetic and thermodynamic constants of the network of chemical reactions in 3-glucoside anthocyanins. *Phytochemistry*, **83**, 125-135. https://doi.org/10.1016/j.phytochem.2012.06.022

Mazza, G., & Brouillard, R. (1987). Color stability and structural transformations of cyanidin 3,5-diglucoside and four 3-deoxyanthocyanins in aqueous solutions. *Journal of Agricultural and Food Chemistry*, **35**, 422-426. https://doi.org/10.1021/jf00075a034

Mena, P., Calani, L., Dall'Asta, C., Galaverna, G., García-Viguera, C., Bruni, R., ... Del Rio, D. (2012). Rapid and comprehensive evaluation of (poly)phenolic compounds in pomegranate (*Punica granatum* L.) juice by UHPLC-MSn. *Molecules*, **17**, 14821-14840. https://doi.org/10.3390/molecules171214821

Morales, J., Bermejo, A., Navarro, P., Forner-Giner, M.À., & Salvador, A. (2021). Rootstock effect on fruit quality, anthocyanins, sugars, hydroxycinnamic acids and flavanones content during the harvest of blood oranges 'Moro' and 'Tarocco Rosso' grown in Spain. *Food Chemistry*, **342**, Article 128305. https://doi.org/10.1016/j.foodchem.2020.128305

Müller-Maatsch, J., Bechtold, L., Schweiggert, R. M., & Carle, R. (2016). Co-pigmentation of pelargonidin derivatives in strawberry and red radish model solutions by the addition of phenolic fractions from mango peels. *Food Chemistry*, **213**, 625-634. https://doi.org/10.1016/j.foodchem.2016.06.097

Nayak, B., Berrios, J. D. J., Powers, J. R., & Tang, J. (2011). Thermal degradation of anthocyanins from purple potato (cv. purple majesty) and impact on antioxidant capacity. *Journal of Agricultural and Food Chemistry*, **59**, 11040-11049. https://doi.org/10.1021/jf201923a

Oancea, S. (2021). A review of the current knowledge of thermal stability of anthocyanins and approaches to their stabilization to heat. *Antioxidants*, **10**, 1337. https://doi.org/10.3390/antiox10091337

Patras, A., Brunton, A., Nigel, P., O'Donnell, C., & Tiwari, B. K. (2010). Effect of thermal processing on anthocyanin stability in foods; mechanisms and kinetics of degradation. *Trends in Food Science & Technology*, **21**, 3-11. https://doi.org/10.1016/j.tifs.2009.07.004

Pereira, A. R., Fernandes, V. C., Delerue-Matos, C., de Freitas, F., Mateus, N., & Oliveira, J. (2024). Exploring acylated anthocyanin-based extracts as a natural alternative to synthetic food dyes: Stability and application insights. *Food Chemistry*, **461**, Article 140945. https://doi.org/10.1016/j.foodchem.2024.140945

Pina, F. (2014). Chemical applications of anthocyanins and related compounds. A source of bioinspiration. *Journal of Agricultural and Food Chemistry*, **62**, 6885-6897. https://doi.org/10.1021/jf404869m

Rodriguez-Amaya, D. B. (2016). Natural food pigments and colorants. *Current Opinion in Food Science*, **7**, 20-26. https://doi.org/10.1016/j.cofs.2015.08.004

Sadilova, E., Carle, R., & Stintzing, F. C. (2007). Thermal degradation of anthocyanins and its impact on color and in vitro antioxidant capacity. *Molecular Nutrition & Food Research*, **51**, 1461-1471. https://doi.org/10.1002/mnfr.200700179

Seco, A., Pereira, A. R., Camuenho, A., Oliveira, J., Dias, R., Brás, N. F., ... Pina, F. (2024). Comparing the chemistry of Malvidin-3-O-glucoside and malvidin-3,5-O-diglucoside networks: A holistic approach to the acidic and basic paradigms with implications in biological studies. *Journal of Agricultural and Food Chemistry*, **72**, 7497-7510. https://doi.org/10.1021/acs.jafc.4c00552

Sun, J., Song, Y.-L., Zhang, J., Huang, Z., Huo, H.-X., Zheng, J., ... Tu, P.-F. (2015). Characterization and quantitative analysis of phenylpropanoid amides in eggplant (*Solanum melongena* L.) by high performance liquid chromatography coupled with diode Array detection and hybrid ion trap time-of-flight mass spectrometry. *Journal of Agricultural and Food Chemistry*, **63**, 3426-3436. https://doi.org/10.1021/acs.jafc.5b00023

Trouillas, P., Sancho-García, J. C., De Freitas, V., Gierschner, J., Otyepka, M., & Dangles, O. (2016). Stabilizing and modulating color by copigmentation: Insights from theory and experiment. *Chemical Reviews*, **116**, 4937-4982. https://doi.org/10.1021/acs.chemrev.5b00507

Yang, P., Basflio, N., Liao, X., Xu, Z., Dangles, O., & Pina, F. (2024). The influence of acylation by hydroxycinnamic acids on the reversible and irreversible processes of anthocyanins in acidic to basic aqueous solution. *Journal of Agricultural and Food Chemistry*, **72**, 25955-25971. https://doi.org/10.1021/acs.jafc.4c06847

Zhang, Y., Deng, Z., Li, H., Zheng, L., Liu, R., & Zhang, B. (2020). Degradation kinetics of anthocyanins from purple eggplant in a fortified food model system during microwave and frying treatments. *Journal of Agricultural and Food Chemistry*, **68**, 11817-11828. https://doi.org/10.1021/acs.jafc.0c05224