Food Chemistry 411 (2023) 135409

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Investigation of the difference in color enhancement effect on

cyanidin-3-O-glucoside by phenolic acids and the interaction mechanism
Yan Cao a, Baofu Zhao b, Yougui Li c, Haiyan Gao a, *, Qile Xia a, *, Zhongxiang Fang d
a
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Key Laboratory of Post-Harvest Handling of Fruits, Ministry
of Agriculture and Rural Affairs, Key Laboratory of Fruits and Vegetables Postharvest and Processing Technology Research of Zhejiang Province, Food Science Institute,
Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
b
School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an 710021, China
c
Sericultural Research Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
d
School of Agriculture and Food, The University of Melbourne, Parkville, Vic 3010, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Co-pigmentation effect of phenolic acids on cyanidin-3-O-glucoside (C3G) and the mechanisms were investi­
Cyanidin-3-O-glucoside gated. Sinapic acid (SIA), ferulic acid (FA), p-coumaric acid (p-CA) and syringic acid (SYA) significantly
Phenolic acids enhanced C3G stability (P < 0.05), whereas vanillic acid (VA) and gallic acid (GA) showed no influence (P >
Stability
0.05). Among these phenolic acids, SIA and FA had higher binding coefficient with C3G (48.83 and 43.38),
Co-pigmentation effect
reduced degradation rate constant by 40.0 ~ 50.0 %, prolonged half-life by 74.6 ~ 94.7 % at 323 K, and
Molecular docking
significantly inhibited C3G hydration reaction (pKh = 2.87 and 2.80, P < 0.05). Molecular docking revealed that
C3G and co-pigments were connected by hydrogen bond and π-π stacking interaction. Hydroxycinnamic acids of
SIA, FA and p-CA bound with ring B and ring C of C3G, while hydroxybenzoic acids of SYA, VA and GA hardly
interacted with ring C. Generally, the protection effect of hydroxycinnamic acids on C3G was better than that of
hydroxybenzoic acids, exhibiting stronger hyperchromic effect.

1. Introduction product, leading to undesirable commercial value loss consequently

Anthocyanins, the glycosylated forms of anthocyanidins, are water-
soluble pigments widely existed in plants. There is a C6-C3-C6 struc­
ture in anthocyanin, forming ring A, C and B correspondingly (Fig. 1),
and different substituents in the rings cause the diversity of anthocya­
nins in nature (Wang et al., 2022). More than 700 anthocyanins have
been reported, but the most common anthocyanins are 3-O-glycosides,
and cyanidin moiety accounts for more than 50 % of the known an­
thocyanins (Cai et al., 2022; He et al., 2012; Wang et al., 2022). In
addition, anthocyanins belong to flavonoids, which are common dietary
polyphenols in foods, with the potential health benefits of anti-
inflammation, regulating blood sugar level and lipid metabolism, neu­
roprotective activity (Garcia & Blesso, 2021; Prior & Wu, 2006; Song,
Shen, Deng, Zhang, & Zheng, 2021). Therefore, the foods rich in an­
thocyanins, such as berry beverage, are popular to consumers, and color
is one of the most important indicators for the quality of these products.
Anthocyanin degradation could result in browning or fading of the
(Chen, Du, Li, & Li, 2020).
Anthocyanins are unstable and susceptible to high temperature, ox­
ygen, pH, light and other environmental factors, and the degradation of
anthocyanins results in the loss of its attractive color and physiological
activity (Cai et al., 2022; Patras, Brunton, O’Donnell, & Tiwari, 2010).
Anthocyanins are existed predominantly as flavylium cation (AH+) in
extremely acidic environment (pH < 2.0), and they will lose electrons as
pH increases to form blue quinonoidalbase (A) (Fig. 1). Hydration re­
action can occur in the flavylium cation, and the C–O bond at position
C2 opens to form colorless carbinol pseudobase (B) or chalcone (C)
(Chen, Du, Li, & Li, 2020). Oxygen induces the formation of hydrogen
peroxide which attacks C2 site of ring C, triggering the ring-opening
reaction and anthocyanin degradation (Cai et al., 2022; Wang et al.,
2022). High temperature and dilution also result in the break of flavy­
lium cation in juice or beverage during processing (Wang & Xu, 2007).
Therefore, protecting flavylium cation is considered as an effective way
to maintain the color stability of anthocyanins. Physical embedding and

Abbreviations: C3G, Cyanidin-3-O-glucoside; SIA, Sinapic Acid; FA, Ferulic Acid; p-CA, p-Coumaric Acid; SYA, Syringic Acid; VA, Vanillic Acid; GA, Gallic Acid.
* Corresponding authors.
E-mail addresses: spsghy@163.com (H. Gao), cookxql@163.com (Q. Xia).

https://doi.org/10.1016/j.foodchem.2023.135409
Received 9 September 2022; Received in revised form 19 December 2022; Accepted 3 January 2023
Available online 5 January 2023
0308-8146/© 2023 Elsevier Ltd. All rights reserved.
Y. Cao et al.
Fig. 1. Different forms and transformation of anthocyanin in solutions.
molecular modification are used to improve the structural stability of
anthocyanins. Physical embedding with large molecules, such as
starches and proteins, is conductive to improve the biological activity,
while molecular modification by smaller molecules (i.e. polyphenolics,
amino acids, etc.) is better for maintaining the red intensity of antho­
cyanins (Bingӧl, Turkyilmaz, & Ozkan, 2022; Gong et al., 2021; Wang
et al., 2022; Zhang et al., 2018).
Co-pigmentation is a structural modification process of stabilizing
anthocyanins via interacting with other compounds (i.e., co-pigments),
which contributes largely to preserve the color stability of red wine
(Boulton, 2001; He et al., 2012). Anthocyanins and co-pigments can
form relatively stable complexes by noncovalent bond, such as Val der
Waals force, hydrogen bond and electrostatic interaction, and the
chromophore group of anthocyanins is protected from nucleophilic
attack, improving their color stability (Cai et al., 2022; He et al., 2012).
Organic acids, polysaccharides, amino acids, phenolic acids, flavonoids
and nucleotides could be used as co-pigments, but different compounds
exhibited various co-pigmentation performance. Bingӧl, Turkyilmaz, &
Ozkan (2022) found that aspartic acid could increase the color stability
of anthocyanins in strawberry juice, while proline and valine had no
effect. The polysaccharides extracted from grape could promote the
resistance of anthocyanins to high temperature and light (Fernandes
et al., 2021). In the wine model solution, flavonols (such as quercetin,
rutin) were powerful co-pigments, but flavan-3-ols (such as epicatechin,
catechin) behaved comparably poor co-pigmentation performance, and
hydroxycinnamic acids showed intermediate behavior (Álvarez, Aleix­
andre, García, Lizama, & Aleixandre-Tudó, 2008; Trouillas et al., 2016).
Phenolic acids could enhance the color stability of anthocyanins in
different levels, and it was also found that hydroxycinnamic acids
showed more effective hyperchromic effect than that of hydroxybenzoic
acids (Eiro & Heinonen, 2002; Qian, Liu, Zhao, Cai, & Jing, 2017; Zhang,
He, Zhou, Liu, & Duan, 2015). However, the reason for this phenomenon
and the interaction mechanism between anthocyanins and different
phenolic acids are still unclear.
In this study, effect of hydroxycinnamic acids and hydroxybenzoic
acids on the color stability and thermodynamic parameters of cyanidin-
3-O-glucoside (C3G) was compared. Then the binding mechanism be­
tween C3G and different phenolic acids was investigated using molec­
ular docking, and the effect of different phenolic acids on C3G at
molecular level was illustrated. In the meantime, the structural char­
acteristics of phenolic acids with better co-pigmentation effect on C3G
was elucidated. Finally, the effect of different phenolic acids on the
anthocyanin stability in black mulberry juice was evaluated, demon­
strating the practicality of improving color stability of anthocyanins.
These results would be valuable in maintaining the color stability of
anthocyanins in food industry.
2
Food Chemistry 411 (2023) 135409
2. Materials and methods
2.1. Materials
Cyanidin-3-O-glucoside (C3G), sinapic acid (SIA), ferulic acid (FA),
p-coumaric acid (p-CA), syringic acid (SYA), vanillic acid (VA), gallic
acid (GA), HPLC grade (≥98 %), were purchased from Yuanye
Biotechnology Co., ltd. (Shanghai, China). Black mulberry (“Dashi” va­
riety) was collected from the Anji planting base in Zhejiang Province,
China. The mulberry was washed, mashed and filtrated through a filter
cloth (75 μm) after harvest, and the juice was stored in plastic bottles at
− 80 ℃.
2.2. Preparation of model solutions
C3G was dissolved in citrate–phosphate buffer (pH 3.6), and the final
concentration was 0.1 mmol/L. Phenolic acid solution was prepared in
ethanol (pH 3.6, adjusted with acetic acid). Mulberry juice and phenolic
acid solution were filtrated through a 0.22 µm sterile syringe filter
before mixing, and the molar ratio of total anthocyanin and phenolic
acid was 1: 20. The mixture was kept in darkness at 30 ℃, and total
anthocyanin content and the color in/of mulberry juice were measured
after storage for 30 days.
2.3. Anthocyanin content and color difference analysis
Total anthocyanin content was determined by pH-differential
method (Chen, Du, Li, & Li, 2020). Anthocyanin preservation ratio in
black mulberry juice after storage was determined by Eq. (1). Where, C0
and C was the total anthocyanin content at day 0 and day 30
respectively.
Anthocyanin preservation ratio (%) = [(C0-C)/C0] × 100 (1)
Ultraviolet (UV) full wavelength scanning of the mixture solution
was conducted using HP845x UV–vis spectrophotometer (Palo Alto, CA,
USA) at 25 ℃. The data was recorded in the visible range of 400 ~ 700
nm with Dk = 1 nm. The color parameters (L*, a*, b*) of mulberry juice
was determined by a colorimeter (CR-400, KonicaMinolta, Japan), and
the color change (ΔEab) was calculated with Eq. (2) (Beyaz, Ozturk, &
Turker, 2010).
ΔEab = [(L*-L*0)2 + (a*-a*0)2 + (b*-b*0)2]1/2 (2)
2.4. Analysis of thermodynamic stability parameters
The mixture solution of C3G and different phenolic acids (molar
ratio = 1: 20) was incubated at 60, 70, 80, 90 ℃ for 1, 2, 3, 4, 5 h
respectively. The anthocyanin content at time t0 and t was C0 and Ct
respectively. Anthocyanin degradation followed a first-order kinetic
model, and the degradation rate constant (k) was deduced according to
Eq. (3). Then half-life (t1/2) was determined with Eq. (4). The activation
energy (Ea) represents the energy requirement when a molecule changes
from normal state to active state. Ea of anthocyanin without/with
phenolic acids was calculated by Arrhenius equation Eq. (5) (Wang &
Xu, 2007).
ln(Ct/C0) = -kt (3)
t1/2 = ln(2)/k (4)
ln(k) = -Ea/RT + C (5)
Here, R is molar gas constant (8.31 J/mol⋅K), and T is temperature
(K).
Enthalpy changes (ΔH) was used to evaluate the chemical bonding
strength of a substance in transition state. Entropy change (ΔS) describes
Y. Cao et al.
the disorder degree of reaction system, and Gibbs free energy (ΔG) in­
dicates whether a reaction is occurred spontaneously. ΔH, ΔS and ΔG
were calculated by Eqs. (6)~(8) respectively (Mercali, Jaeschke, Tes­
saro, & Marczak, 2013).
ΔH = Ea - RT (6)
ΔG = -RTln(kh/TkB) (7)
ΔS = (ΔH-ΔG)/T (8)
Here, T is the temperature (K), kB (1.3806 × 10-23 J/K) is the
Boltzmann’s constant, and h (6.6262 × 10-34 J⋅s) is the Planck’s
constant.
2.5. Apparent hydration equilibria constant measurement
C3G solution (0.1 mmol/L, pH 0.9, 1.5, 2.0, 2.5, 3.0 and 4.2) was
prepared with citrate–phosphate buffer (0.1 mol/L), and the ionic
strength was adjusted to 1.0 mol/L with NaCl. C3G and phenolic acids
were mixed at the molar ratio of 1: 20, and kept for 2 h in darkness at 25
℃ to ensure complete hydration equilibrium. Then the absorbance value
of the mixture at λ = 520 nm was recorded by spectrophotometer. The
first-order apparent degradation rate constant (K) was the sum of hy­
dration equilibrium constant (Kh) and proton transference constant (Ka),
i.e., K = Kh + Ka. K could be deduced from the straight line fitted by A0/
(A0-A) versus 10-pH in Eq. (9). The hydration reaction was predominant
in the acidic solution, so Ka was extremely small compared with Kh (Ka
≪ Kh). Hence, the hydration constant Kh could be described as K
(González-Manzano, Santos-Buelga, Dueñas, Rivas-Gonzalo, & Escri­
bano-Bailón, 2009).
A0/(A0-A) = (10-pH)/(Kh + Ka×(1-ra)) + (Kh + Ka)/(Kh + Ka×(1-ra)) (9)
In this equation, A0 is the absorbance of anthocyanin in strongly
acidic solution (1.0 mol/L hydrochloric acid aqueous solution), A is the
absorbance at a given pH, and ra is the ratio of molar absorption co­
efficients of the pigment in quinonoidal form and flavylium form.
2.6. Co-pigmentation of phenolic acids on C3G
C3G and phenolic acid were mixed at molar ratio of 1: 0, 1: 10, 1: 20,
1: 30, 1: 40, 1: 50 and 1: 60, and the mixture was kept in darkness at 25
℃ for 2 h. The absorbance at λ = 520 nm was recorded. The binding
coefficient (Kc) and the stoichiometric ratio (n) between C3G and the co-
pigments were calculated according to Eq. (10) (Zhang, He, Zhou, Liu, &
Duan, 2016).
ln[(A-A0)/A0] = ln(Kc) + nln(C0) (10)
Here, A0 is the absorbance value of C3G solution without co-
pigments, A is the absorbance value of C3G solution with co-pigments,
and C0 is the co-pigment concentration.
2.7. Analysis of interaction simulation between C3G and phenolic acids
The dispersion correction term (GD3) was introduced to analyze the
weak intermolecular interaction between molecules on the basis of
B3LYP hybrid functional (Grimme, Hansen, Brandenburg, & Bannwarth,
2016). Meanwhile, 6-31+G(d) basis set including a diffusion basis set
suitable for describing weak interaction was used (Li et al., 2019). The
geometric structures of SIA, FA, p-CA, SYA, VA and GA were optimized,
obtaining the most stable conformation of each molecule. Then the
interaction model between C3G and different phenolic acids was con­
structed on the base of the above optimized geometric structure, gaining
the stable structure of the different complexes. The interaction between
them was judged by the interaction energy with Eq. (11):
E_int = E_com - (E_A + E_B) (11)
3
Food Chemistry 411 (2023) 135409
where E_int is the interaction energy, E_com is the energy of the complex,
and E_A and E_B are the energy of two monomers respectively. The
essence of the complex stability was calculated by relevant quantum
chemical analysis methods including charge analysis and reduced den­
sity gradient analysis. The simulation and calculation were conducted
by Gaussian 16 software (Gaussian, Inc., Wallingford, CT, USA).
2.8. Statistical analysis
All the tests were performed in triplication, and the average value,
error and significance of difference with the Tukey procedure were
analyzed using Excel (Microsoft Office version 2016, Microsoft Co.,
Redmond, USA) and SPSS 21.0 (IBM Corporation, Armonk, NY, USA).
The difference was significant if P < 0.05.
3. Results and discussion
3.1. Effect of different phenolic acids on the stability of C3G
Generally, the addition of hydroxycinnamic acids (SIA, FA and p-CA)
and hydroxybenzoic acids (SYA, VA and GA) improved C3G stability
(Table 1). The effect of SIA, FA, p-CA and SYA, especially the former two,
was significantly stronger than that of VA and GA at 323 K (P < 0.05).
The degradation rate constant k decreased to 50.0 %~90.0 % of the
control level, and half-life t1/2 prolonged 13.0 %~94.7 %. The activation
energy Ea was increased significantly (P < 0.05), indicating that SIA, FA,
p-CA and SYA could promote the structure stability of C3G (Mercali,
Jaeschke, Tessaro, & Marczak, 2013). Nonetheless, there was no sig­
nificant change in the value of k, t1/2 and Ea when VA or GA was added in
C3G solution (P > 0.05). Hence hydroxycinnamic acids showed better
co-pigmentation effect on C3G than hydroxybenzoic acids, which was in
accordance with the reported research (Eiro & Heinonen, 2002; Zhang,
He, Zhou, Liu, & Duan, 2015). High temperature is an important factor
causing anthocyanin degradation, which was reflected by the significant
change of thermodynamic stability parameters of C3G at 343 K and 363
K (P < 0.05) (Table 1). On the other hand, significant increase of ΔH
(36.69 %~82.71 %, P < 0.05) was observed under the condition of
adding FA, SIA, p-CA and SYA at 323 K, but the value of ΔH in the cases
of adding VA and GA did not change significantly (P > 0.05). This
implied that FA, SIA, p-CA and SYA might modify C3G structure, and the
degradation of the modified C3G needed more energy to overcome po­
tential barrier and became active state. The increase of ΔS when
phenolic acids were added revealed that the molecules in C3G-
copigment system were reordered and the disorder degree of system
increased, leading to higher stability of the new system (Mercali,
Jaeschke, Tessaro, & Marczak, 2013). The positive value of ΔG in this
study indicated that anthocyanin degradation was not spontaneously
occurred during heating (Patras, Brunton, O’Donnell, & Tiwari, 2010;
Wang & Xu, 2007). It might be because anthocyanin self-aggregation
was happened at mild high temperature, and the polymer had better
thermal resistance than mono anthocyanin (Qian, Liu, Zhao, Cai, & Jing,
2017). From the above data, SIA and FA were the most effective co-
pigments for C3G, p-CA and SYA showed moderate hyperchromic ef­
fect on C3G, while VA and GA had no effect (Table 1).
3.2. Effect of different phenolic acids on the UV absorption spectrum of
C3G
The maximum absorption wavelength (λmax) of C3G solution was
shifted from 510 nm to 522 ~ 524 nm in the presence of SIA, FA and p-
CA, and the maximum absorbance value (Amax) was also significantly
higher than that of control (P < 0.05, Table 2). Addition of SYA, VA and
GA also brought the increase of Amax, but bathochromic shift was not as
large as the former three phenolic acids, and the λmax was 520 ~ 522 nm.
It was suggested that the polarity of solution environment was changed
when phenolic acids were added, and the complexes of C3G-copigment
Y. Cao et al. Food Chemistry 411 (2023) 135409

Table 1
Thermodynamic parameters of C3G degradation with addition of phenolic acids.
1
Group T/K k/h− t1/2 /h Ea/ kJ/moL ΔH/ kJ/moL ΔS/ J/(moL⋅K) ΔG/ kJ/moL
a c e e e
Control 323 0.010 ± 0.001 69.46 ± 0.98 61.32 ± 4.04 58.64 ± 4.04 − 161.92 ± 11.72 113.62 ± 0.31bc
343 0.044 ± 0.005a 15.13 ± 1.60a 58.47 ± 4.04e − 160.83 ± 10.93e 116.48 ± 0.30a
363 0.167 ± 0.021a 4.18 ± 0.45a 58.30 ± 4.04e − 161.44 ± 11.53e 120.24 ± 0.32a
SIA 323 0.005 ± 0.000d 135.21 ± 5.93a 98.81 ± 0.47a 96.13 ± 0.47a − 51.40 ± 1.58a 115.42 ± 0.12a
343 0.040 ± 0.007a 17.73 ± 3.40a 95.96 ± 0.47a − 52.76 ± 1.19a 116.91 ± 0.55a
363 0.279 ± 0.001a 2.42 ± 0.11a 95.80 ± 0.47a − 54.19 ± 1.40a 118.59 ± 0.14c
FA 323 0.006 ± 0.000d 121.31 ± 8.72a 88.75 ± 2.81b 85.97 ± 1.99b − 81.95 ± 5.77b 115.12 ± 0.19a
343 0.033 ± 0.001a 21.20 ± 0.79a 85.80 ± 1.99b − 83.96 ± 5.88b 117.45 ± 0.11a
363 0.266 ± 0.018a 2.61 ± 0.17a 85.64 ± 1.99b − 82.66 ± 5.31b 118.82 ± 0.20c
p-CA 323 0.008 ± 0.000c 82.64 ± 3.94b 74.94 ± 2.40c 72.26 ± 2.40c − 121.15 ± 6.50c 114.07 ± 0.43b
343 0.037 ± 0.012a 19.28 ± 4.30a 72.09 ± 2.40c − 123.00 ± 5.47c 117.13 ± 0.69a
363 0.203 ± 0.000a 3.42 ± 0.24a 71.93 ± 2.40c − 122.45 ± 6.54c 119.64 ± 0.22b
SYA 323 0.009 ± 0.001bc 78.48 ± 6.15b 68.20 ± 1.17d 65.51 ± 1.17d − 141.64 ± 3.16d 113.95 ± 0.25b
343 0.034 ± 0.005a 20.67 ± 2.45a 65.35 ± 1.17d − 143.36 ± 3.78d 117.37 ± 0.35a
363 0.187 ± 0.001a 3.72 ± 0.31a 65.18 ± 1.17d − 141.63 ± 3.83d 119.89 ± 0.24ab
VA 323 0.009 ± 0.001ab 74.27 ± 5.59bc 65.15 ± 3.67de 62.46 ± 3.67de − 150.64 ± 10.85de 113.81 ± 0.20bc
343 0.037 ± 0.002a 18.70 ± 1.20a 62.30 ± 3.67de − 151.45 ± 10.26de 117.09 ± 0.19a
363 0.180 ± 0.032a 3.84 ± 0.51a 62.13 ± 3.67de − 150.23 ± 11.14de 119.98 ± 0.40ab
GA 323 0.010 ± 0.002a 64.46 ± 5.12c 62.85 ± 2.04e 60.17 ± 2.04e − 156.54 ± 5.12e 113.41 ± 0.40c
343 0.044 ± 0.002a 16.22 ± 2.77a 60.00 ± 2.04e − 156.88 ± 7.13e 116.66 ± 0.51a
363 0.182 ± 0.011a 3.81 ± 0.17a 59.84 ± 2.04e − 156.48 ± 5.95e 119.97 ± 0.14ab

Different letters in the superscript indicate significant difference among different groups at the same temperature (P < 0.05). There is significant difference in the
parameters in the same group at different temperature (P < 0.05).

different phenolic acids on C3G stability still need further investigation.

Table 2
Effect of phenolic acids on the binding parameters, hydration equilibrium con­
stant and UV absorption of C3G *. 3.3. Interaction mechanism between C3G and phenolic acids
Group λmax/ Amax C3G:Cop Binding Hydration
nm (n) coefficient equilibrium The geometric structure of C3G and the test six phenolic acids was
(Kc) constant (pKh)
optimized (Fig. 2). It was shown that C3G molecule had a good planarity
Control 514c 0.35 ± – – 2.58 ± 0.05d due to the small dihedral angle between ring B and the benzopyrylium
0.03c
(<10 degree), and Cl atoms tended to bind near –OH to form strong
SIA 524a 0.58 ± 0.96 ± 48.83 ± 4.24a 2.87 ± 0.08a
0.05a 0.035bc hydrogen bonds (Fig. 2a) (Sinopoli, Calogero, & Bartolotta, 2019).
FA 524a 0.55 ± 0.91 ± 43.38 ± 2.80 ± 0.09ab Phenolic acid molecules contained benzene ring and conjugate structure
0.03a 0.068bc 4.90ab and showed good planarity, but the substituents were located in
p-CA 522ab 0.49 ± 0.90 ± 32.29 ± 0.66c 2.76 ± 0.03ab different positions (Fig. 2b ~ g). The interaction between C3G and the
0.02b 0.026bc
SYA 520ab 0.52 ± 0.88 ± 38.50 ± 2.71 ± 0.04bc
six phenolic acid molecules were analyzed based on the spatial structure
0.01ab 0.008b 3.34bc in Fig. 2 and the stoichiometric ratio of 1: 1. The stable configuration
VA 522b 0.47 ± 1.01 ± 37.33 ± 2.58 ± 0.07cd and interaction energy of different C3G-copigment complexes were
0.06b 0.078a 3.07bc shown in Fig. 3, where we can see that the interaction ways between
GA 520b 0.45 ± 0.94 ± 33.03 ± 2.60 ± 0.08cd
different phenolic acids and C3G were similar, but each complex had
0.01b 0.027ac 3.50bc
their own characteristics due to the distinctive spatial conformation of
*
: The molar ratio of C3G and co-pigments is 1: 20. Different letters in the phenolic acids. The larger planar structure of the molecule was favored
superscript indicate significant difference (P < 0.05). to form interaction with pigments, and the face-to-face position between
anthocyanin and co-pigment was also necessary for stronger binding
might be formed. The stoichiometric ratio of C3G and phenolic acid was force (Trouillas et al., 2016; Zhang et al., 2018). Hydroxycinnamic acids
around 1.0 (Table 2), implying that one phenolic acid molecule was (FA, SIA and p-CA) had a relatively longer sidechain which ran over the
bound to one C3G molecule (Di Meo, Sancho Garcia, Dangles, & three rings of C3G molecule. For SIA and p-CA, the benzene ring was
Trouillas, 2012; Gong et al., 2021). The larger binding coefficient above the ring C of C3G, and the long sidechain extended over the ring B,
revealed that SIA, FA, SYA and VA had better binding affinity to C3G (P but the interaction area between SIA and C3G was larger. Thus, the
< 0.05, Table 2), indicating that the binding force between these interaction energy of C3G-SIA complex was stronger (-40.0 kcal/mol),
phenolic acids and C3G was stronger (Malaj, De Simone, Quartarolo, & and the binding energy of C3G-p-CA complex was lower (-36.4 kcal/
Russo, 2013). Furthermore, the hydration equilibrium constant (pKh) of mol). The aromatic ring of FA was close to ring B of C3G, and the
C3G was increased significantly when SIA, FA, p-CA and SYA were sidechain was across ring A and ring C in C3G. The energy of C3G-FA
added (P < 0.05, Table 2), but VA and GA had no effect on pKh (P > was also at a higher level (-37.7 kcal/mol). Nevertheless, there was no
0.05). It was demonstrated that addition of SIA, FA, p-CA and SYA could long sidechain in hydroxybenzoic acids, resulting in smaller interaction
lead to the displacement of hydration equilibrium toward the formation area with C3G. The molecular plane of SYR or VA was parallel to one
of flavylium cation, causing bathochromic shift and enhancing red in­ aromatic ring of C3G (ring A or ring B), so SYR or VA and C3G could
tensity of C3G solution (Álvarez, Aleixandre, García, Lizama, & Aleix­ form stronger π-π conjugation interaction. The interaction energy of the
andre-Tudó, 2009). It was reported that benzene ring and groups of complexes of C3G-SYR and C3G-VA was − 37.7 kcal/mol and − 36.2
–OH, –OCH3, –H, –C– – C– were prone to form π electron cloud stacking kcal/mol correspondingly. For the structure of C3G-GA complex, the
and hydrogen bonds between phenolic acids and C3G (Di Meo, Sancho planes of these two molecules were unparallel, resulting in low inter­
Garcia, Dangles, & Trouillas, 2012; Gong et al., 2021; Queiroz, Alves, & action energy of C3G-GA (-29.5 kcal/mol) (Triolo et al., 2020). The
Rivelino, 2021). Therefore, phenolic acids had the potential to bind with interaction strength between phenolic acids and C3G was C3G-SIA >
C3G and form stable complexes. However, the influencing mechanism of C3G-FA = C3G-SYA > C3G-p-CA > C3G-VA > C3G-GA, and this result

4
Y. Cao et al. Food Chemistry 411 (2023) 135409

Fig. 2. The geometric structure of cyanidin-3-O-glucose and tested phenolic acids.

Fig. 3. Visualization interaction between C3G and phenolic acids based on the reduced density gradient. Note: Spherical model is phenolic acid molecule, and rod
model is C3G molecule. The color coding scheme for the interaction indicates: red for strong attractive interactions, transition region color for typical van der Waals
interaction, and blue for strong non-bonded overlap.

was consistent with the above parameter analysis results.
The interaction between phenolic acid molecules and C3G was
analyzed by the reduced density gradient method (Johnson et al., 2010;
Queiroz, Alves, & Rivelino, 2021). The main driving force forming stable
complexes was hydrogen bond and π-π stacking interaction, but the size
of interaction regions and the interaction points were different (Fig. 3).
Hydrogen bonds or charge transfer interaction were more inclined to
form between anthocyanin and co-pigment with more hydroxyl or
methoxyl groups, and the larger region of interaction indicated much
closer contact between C3G and the co-pigment (Zhang, He, Zhou, Liu,
& Duan, 2015). The conjugated double bond in the sidechain of
hydroxycinnamic acids lead to the delocalization of π electrons, and

5
Y. Cao et al. Food Chemistry 411 (2023) 135409

generated stronger π-π interaction with the aromatic rings of C3G Table 3
(Sinopoli, Calogero, & Bartolotta, 2019). In the complexes of C3G-SIA, Anthocyanin preservation ratio, the stability parameters of anthocyanins, and
C3G-FA and C3G-p-CA, hydrogen bonds (e.g., O–H ••• O and O–H color change of black mulberry juice with different phenolic acids after storage
••• Cl) were formed between the phenolic acids and ring A, ring B and for 30 days.
sugar moiety of C3G, and the π-π interaction was concentrated on ring B Group Anthocyanin Degradation rate Half-life ΔEab a*
and ring C. The flavylium cation (ring C) was surrounded by the inter­ preservation constant (k)/ (t1/2) value
ratio (%) day− 1 /day
action energy, and the steric hindrance of C2 position was increased,
especially in the complexes of C3G-SIA and C3G-FA (Fig. 3a&b). For the Control 6.23 ± 0.77e 0.0734 ± 0.007a 9.48 ± 1.10 ± 1.81
complexes of C3G-SYA and C3G-VA, π-π conjugated interaction and 0.81d 0.12a ±
0.01c
hydrogen bonds were mainly occurred on ring A or ring B of C3G, SIA 36.36 ± 1.13a 0.0299 ± 0.001e 23.22 ± 0.64 ± 2.78
leading to poor sheltering effect of ring C. Therefore, although the 0.66a 0.06d ±
binding energy of C3G-SYA and C3G-VA was the same with C3G-FA and 0.02a
C3G-p-CA respectively, the hyperchromic effect of SYA and VA was not FA 38.24 ± 2.11a 0.0280 ± 0.004e 25.11 ± 0.68 ± 2.45
3.77a 0.07d
as well as that of FA and p-CA. The ortho-trihydroxyl on the benzene ring ±
0.12b
of GA could form intramolecular hydrogen bond which negatively p-CA 34.93 ± 1.36ab 0.0308 ± 22.67 ± 0.87 ± 2.53
affected the formation of intermolecular hydrogen bonds, forming weak 0.003de 2.44a 0.07bc ±
connecting force with C3G (Bao, Huang, Xu, & Cui, 2021). Furthermore, 0.26b
the π-π interaction was not concentrated on any ring of C3G, so GA SYA 31.48 ± 2.52bc 0.0366 ± 19.01 ± 0.73 ± 2.12
0.002cd 1.17b 0.10bc
showed poor co-pigmentation effect.
±
0.11b
The structural feature of phenolic acids influenced their interaction VA 30.06 ± 2.49bc 0.0361 ± 19.23 ± 0.97 ± 2.28
with C3G. The substituents donating/accepting proton (–OH, –C– – C-, 0.001cd 0.50b 0.12ac ±
–OCH3) and the aromatic rings were necessary to form hydrogen bond 0.07b
GA 17.61 ± 2.52d 0.0551 ± 0.005b 12.66 ± 0.98 ± 1.94
and Van der Waals interaction (Triolo et al., 2020; Zhang, He, Zhou, Liu,
1.22c 0.12ac ±
& Duan, 2016). A spatial structure with geometrical flexibility was a 0.10c
critical factor affecting the structural matching degree of co-pigments
with C3G (Dimitrić Marković, Petranović, & Baranac, 2005). There­ Different letters in the superscript indicate significant difference (P < 0.05). a*
value indicates the red intensity of black mulberry juice, and ΔEab indicate that
fore, hydroxycinnamic acids with flexible sidechain were more effective
the color change magnitude of black mulberry juice.
co-pigments for C3G. Meanwhile, the size of substituents also influenced
the interaction intensity, so the larger group (such as –OCH3) could form
stronger hydrogen bond with C3G (Malaj, De Simone, Quartarolo, & %. The change of a* value revealed that phenolic acids except GA
Russo, 2013; Rein, 2005). SIA and SYA belong to bi-methoxylated significantly enhanced the red intensity of mulberry juice (P < 0.05),
phenolic acid, which exhibited stronger interaction and better co- while the a* values in the samples of adding SIA, FA and p-CA were
pigmentation effect on C3G with respect to other hydroxycinnamic higher than that in other groups. The color change (ΔEab) during storage
acids and hydroxybenzoic acids (Fig. 3). As the number of methoxyl was minimal in the samples of adding SIA and FA (P < 0.05), indicating
group decreased in the same category of phenolic acid, the interaction that SIA and FA were the most effective co-pigments to maintain the
force with C3G decreased correspondingly. For example, there was no color stability of mulberry juice among the tested phenolic acids (Beyaz,
methoxy group in GA, thus the hydrogen bond in the C3G-GA complex Öztürk, Acar, & Turker, 2011). p-CA and SYA also slightly improved the
was weak (Triolo et al., 2020). On the other hand, the degradation of anthocyanin stability in mulberry juice, but VA and GA hardly protected
anthocyanins was usually started from the break of C–O bond at posi­ the color. These results agreed with the above stability analysis and
tion C2 in the ring C due to the lower steric hindrance (Chen, Du, Li, & molecular docking between phenolic acids and C3G. Therefore,
Li, 2020; Sinopoli, Calogero, & Bartolotta, 2019). The color enhance­ hydroxycinnamic acids with more methoxyl groups (e.g., SIA and FA)
ment performance of co-pigment was dependent on their protective could be promising color enhancers for the beverage rich in
magnitude of the position C2 of anthocyanin. The stronger interaction anthocyanins.
energy around ring C of C3G in the complexes of C3G-SIA, C3G-FA and
C3G-p-CA hindered the nucleophilic attack by water, resulting in higher 4. Conclusion
structural and color stability of C3G. It was concluded that the sidechain
characteristics and substituent type in phenolic acids had close corre­ This study demonstrated that the protection effect of hydroxycin­
lation with the structural matching degree, contacting sites and inter­ namic acids on color stability of C3G was generally superior to that of
action strength with anthocyanin, affecting the co-pigmentation effect hydroxybenzoic acids. SIA, FA and p-CA bound with C3G to form stable
consequently. complexes with lower k, longer t1/2 and higher Ea, while the improve­
ment of thermodynamic parameters by SYA, VA and GA was limited.
Molecular docking simulation indicated that hydroxycinnamic acids
3.4. Application of phenolic acids in maintaining color stability of black
could interact with all three rings and sugar sidechain in C3G due to a
mulberry juice
longer sidechain, leading to higher binding force (− 36.4 ~ − 40.0 kcal/
mol). Hence, the flavylium cation was well sheltered and the nucleo­
Mulberry was a popular berry rich in anthocyanins, mainly con­
philic attack by water was restrained, which was reflected by higher
taining C3G and cyanidin-3-O-rutinoside (C3R), but the stability of C3R
hydration equilibrium constant (pKh = 2.76 ~ 2.87) and weaker hy­
was higher than that of C3G (Prior & Wu, 2006). Therefore, the co-
dration reaction. However, the binding coefficient (Kc = 33.02 ~ 38.50)
pigmentation performance of different phenolic acids on black mul­
and interaction energy (− 29.5 ~ − 37.7 kcal/mol) between hydrox­
berry juice was investigated to demonstrate the feasibility in practical
ybenzoic acids and C3G was relatively lower, and the interaction site
application. The changes of total anthocyanin content and the color of
was deviated from ring C, resulting in poor hyperchromic effect. The
mulberry juice with addition of different phenolic acids were shown in
experimental results agreed with the simulation calculation. Meanwhile,
Table 3. In general, addition of phenolic acids significantly improved the
methoxylation degree of phenolic acids had a positive relationship with
anthocyanin stability in mulberry juice during storage (P < 0.05).
their binding energy with C3G. Effect of different phenolic acids on the
Compared with the control, the anthocyanin preservation ratio
mulberry juice color stability confirmed that hydroxycinnamic acids
increased 1.83 ~ 5.14 folds, anthocyanin degradation rate constant k
(especially SIA and FA) could slow down anthocyanin degradation,
declined 24.9 %~61.9 %, and the half-life t1/2 prolonged 33.5 %~164.9

6
Y. Cao et al.
prolong the half-life of anthocyanin (2.39 ~ 2.65 folds of control), and
maintain red intensity of the juice. These results are promising to
improve the color stability of foods rich in anthocyanins in the food
industry.
CRediT authorship contribution statement
Yan Cao: Data curation, Writing – original draft, Funding acquisi­
tion. Baofu Zhao: Methodology, Investigation. Yougui Li: Investiga­
tion, Formal analysis. Haiyan Gao: Writing – review & editing. Qile
Xia: Writing – review & editing, Supervision, Funding acquisition.
Zhongxiang Fang: Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
The authors thank National Natural Science Foundation of China
(No. 31901708), Zhejiang Province Department of Agriculture and
Rural Affairs (No. 2022SNJF040), Key Laboratory of Agricultural
Products Processing of Ministry of Agriculture and Rural Affairs in
China, and Discipline Construction Project in Zhejiang Academy of
Agricultural Sciences.
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