Comparative Study on the Stability and Antioxidant Activity of Six Pyranoanthocyanins Based on Malvidin-3-glucoside

Article abstract of DOI:10.1021/acs.jafc.9b06734

Pyranoanthocyanins are the important color and functional compounds in red wine. Six common kinds of pyranoanthocyanins were synthesized through the reaction of malvidin-3-O-glucoside (Mv-3-gluc) with acetone, pyruvic acid, p-cumaric acid, caffeic acid, ferulic acid, and sinapid acid, respectively, and their pH (1.0-11.0), SO₂ (0-250 ppm), and thermo (50-98 °C) stabilities and antioxidant activities were comparatively studied. Results showed that the six pyranoanthocyanins all exhibited higher pH and SO₂ color stability than Mv-3-gluc, especially vitisin-A with a carboxy group on the D ring. The six pyranoanthocyanins also showed much more thermostability than Mv-3-gluc, especially methylpyranomv-3-gluc. The degradation kinetics of Mv-3-gluc and its derivatives all fitted to a first-order reaction. Moreover, pinotin-A with the o-dihydroxyl group on the E ring presented the strongest antioxidant capability, as evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric ion reducing power (FRAP) assays. Additionally, the increase in the number of rings is beneficial to the improvement of the DPPH radical scavenging ability of anthocyanins.

Full text of DOI:10.1021/acs.jafc.9b06734

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Functional Structure/Activity Relationships
Comparative study on the stability and antioxidant activity
of six pyranoanthocyanins based Malvidin-3-glucoside
Jianxia Sun, Xinghua Li, Haixia Luo, Lijun Ding, Xinwei Jiang, Xusheng Li, Rui Jiao, and Weibin Bai
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b06734 • Publication Date (Web): 04 Feb 2020
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Journal of Agricultural and Food Chemistry
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Comparative study on the stability and antioxidant activity of six
pyranoanthocyanins based Malvidin-3-glucoside
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Jianxia Suna,1, Xinghua Li a,b,1, Haixia Luo a,1, Lijun Ding a, Xinwei Jiang c, Xusheng Li c, Rui Jiao
c, Weibin Bai*,c
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a School of Chemical Engineering and Light Industry, Guangdong University of Technology,
Guangzhou, PR China
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b Baxter Healthcare (Guangzhou) Co., Ltd, Guangzhou, PR China
c Department of Food Science and Engineering, Institute of Food Safety and Nutrition, Guangdong
Engineering Technology Center of Food Safety Molecular Rapid Detection, Jinan University,
Guangzhou, PR China
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ABSTRACT
Pyranoanthocyanins are the important color and functional compounds in red wine.
Six common kinds of pyranoanthocyanins were synthesized through the reaction of
malvidin-3-O-glucoside (Mv-3-gluc) with acetone, pyruvic acid, p-cumaric acid,
caffeic acid, ferulic acid and sinapid acid, respectively, and their pH (1.0-11.0), SO2 (0-
250 ppm) and thermo (50-98 °C) stability and antioxidant activity were comparatively
studied. Results showed that six pyranoanthocyanins all exhibited higher pH and SO2
color stability than Mv-3-gluc, especially Vitisin-A with a carboxy group on D ring.
Six pyranoanthocyanins also showed much more thermo-stability than Mv-3-gluc,
especially Methylpyranomv-3-gluc. The degradation kinetics of Mv-3-gluc and its
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derivatives all fitted to a first order reaction. Moreover, Pinotin-A with o-dihydroxyl
group on E ring presented the strongest antioxidant capability evaluated by DPPH,
ABTS and FRAP assays. Additionally, the increase in the number of rings is beneficial
to the improvement of DPPH radical scavenging ability of anthocyanins.
KEYWORDS: anthocyanin, pyranoanthocyanins, pH stability, anti-SO2 bleaching,
thermo-stability, antioxidant activity
INTRODUCTION
Anthocyanins belong to flavonoids and play a major role in wine quality. The type
and concentration of anthocyanin could effectively affect the sensible characteristics of
wine, especially color and astringency (1). The sense of red wine is changed in the
vinification process as anthocyanins react with other organic chemicals continuously.
Color is an important perceptible parameter of wine during aging (2). At the initial stage
of wine making, monomeric anthocyanins largely contribute to total color (3-4). After
that, the intramolecular of anthocyanins themselves or the intermolecular interaction of
anthocyanins with other phenolic compounds occur and generate various new
anthocyanin-derived pigments (1, 5-7).
Pyranoanthocyanins are a group of anthocyanin-derived pigments (8-9), which were
firstly discovered in red wine by Santos et al. (10) in 1996，using UV/Vis (ultraviolet–
visible spectroscopy) and ESI-LC-MS (electrospray ionization-liquid chromatograph-
mass spectrometer) spectra analysis. Thereafter, Fulcrand et al. (11) confirmed the final
structure of this new pigment by mass spectrometric and NMR (nuclear magnetic
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resonance). They reported these new pigments were formed by reaction between
anthocyanins and other phenolic compounds in red wine. The general structure of
pyranoanthocyanin is a result of cycloaddition between the hydroxy group at C-5 and
C-4 of the anthocyanin pyranic ring with an ethylenic bond of another substances (12).
This new pyranic ring may directly accompany with various substituents at C-10 site,
thereby generating the diversity of pyranoanthocyanins. In recent years,
hydroxyphenyl-pyranoanthocyanins, vitisins, vinylflavanol-pyranoanthocyanins,
portisins and rosacyanin B were the most common type of pyranoanthocyanins to be
detected and studied (13).
It has been fully elucidated that anthocyanins have strong antioxidant, hypoglycemic,
anti-tumor and anti-inflammatory activities (14-15). However, monomeric
anthocyanins are highly unstable and susceptible to light, oxygen, temperature, pH,
structure and concentration of themselves, resulting in degradation (16).
Pyranoanthocyanins, as a class of derivatives of anthocyanins, presented greater
stability than its precursor. Schwarz and Winterhalter (17) reported Pinotin-A was a lot
less susceptible to pH shifts and maintained original color within the scope of wide pH-
range. Moreover, Sun et al. presented that the methylpyranocyanidin-3-O-glucoside
condensated by cyanidin-3-O-glucoside and acetone presented better stability in
heating, pH variation and bisulfite bleaching than its precursor (18).
Due to the crucial contribution of anthocyanins to wine color as well as its bioactivity,
advancing the knowledge about structural characteristics, stability and bioactivity of
anthocyanins and their derivatives like pyranoanthocyanins is of great importance.
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However, relevant research focusing on the comparison of anthocyanins with
pyranoanthocyanins is infrequent and lacks rigorous systematics. Therefore, this study
comparatively researched the pH, SO2 and thermal stability of Mv-3-gluc and six
synthetic pyranoanthocyanins. In addition, the antioxidant activity of the Mv-3-gluc
and its six derivatives were also evaluated by the 2,2-diphenyl-1-picryhydrazyl (DPPH)
radical, 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical test and
ferric ion reducing power (FRAP) assay. This study selected six common
pyranoanthocyanins in red wine, aiming to elucidate the effects of different substituent
groups at C-10 on anthocyanin stability and antioxidant activity. Furthermore, this
study is expected to provide a theoretical basis for screening and modification of
anthocyanin structures, and expand their applications in food, health products and
cosmetics as a natural pigment.
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MATERIAL AND METHODS
Materials. Mv-3-gluc was extracted from grapes (Vitis labruscana Kyoho) and then
isolated and purified by the methods described by Sun et al. (19) until a final purity of
85.8 % at 280 nm and 90.2 % at 520 nm detected by HPLC (high-performance liquid
chromatography). The purified Mv-3-gluc was further lyophilized into powder and then
stored at -20 °C until use.
Methylpyranomv-3-gluc was synthesized with Mv-3-gluc and acetone followed the
methods described by He et al (20) with some modifications. Mv-3-gluc and acetone
(for HPLC, Shanghai Aladdin Bio-Chem Technology Co., LTD) with a molar ratio of
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1:50 in 50 mL phosphate buffer solutions (pH 2.6), the reaction was monitored at 30 °C
in the dark for 30 days.
The synthesis of Vitisin-A was referred to the method of Oliveira (21) with some
improvements. Mv-3-gluc and pyruvic acid (purity > 98 %, Shanghai Aladdin Bio-
Chem Technology Co., LTD) were incubated in 50 mL phosphate buffer solutions (pH
2.6) at 35 °C in the dark for 21 days (Mv-3-gluc and pyruvic acid of molar ratio of
1:500).
Mv-3-gluc-4-vinylphenol, Pinotin-A, Mv-3-gluc-4-vinylguaiacol and Mv-3-gluc-4-
vinylsyringol were synthesized according to the method of Schwarz (22). Four different
cinnamic acids (p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid (purity >
98 %), Shanghai Aladdin Bio-Chem Technology Co., LTD) were dissolved in the
buffer solution (pH 3.2), and then reacted with 27 mL of 400 mg/L Mv-3-gluc for every
3.0 mL of cinnamic acid (400 mg/L). The reaction liquid was incubated at a 30 ℃
water bath with a shaker (100 r/min) in the dark. Samples were analyzed in regular
intervals, and the pyranoanthocyanins finally were collected after the Mv-3-gluc was
completely consumed. Structures of six pyranoanthocyanins and Mv-3-gluc are shown
in Scheme 1.
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Separation and Purification. The synthetic pyranoanthocyanins were separated by
solid-phase extraction (SPE). The column (Sep-Pak C18, 3 cc, Waters, Milford, MA)
was previously activated with methanol, followed by 0.01% HCl aqueous solution. The
Mv-3-gluc and pyranoanthocyanins were absorbed onto the column, after which
impurities were washed by the acidified water (0.1 M HCl). Then Mv-3-gluc and the
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derivatives were eluted with 0.1 M HCl aqueous solution in methanol, then evaporated
to dryness by pressure blowing concentrator (RE-52AA, Shanghai Zhenjie
experimental equipment Co., LTD) at 37℃. The pyranoanthocyanins dry extracts were
dissolved with the 10% aqueous formic acid. The pyranoanthocyanins were purified
several times using a venusil ASB-C18 column (25 cm×2.0 cm; i.d.; 5 mm, Agela, USA)
by HPLC equipped with DAD detector (diode array detector).
HPLC/DAD experiments were performed by using a Waters HPLC/DAD System
(Waters Alliance e2695 HPLC system with Waters 2998 Photodiode Array Detector
(PDA)) at a constant temperature of 35 ℃ and a flow rate of 1.0 mL/min. The elution
solvents were A: H2O/CH3COOH (v/v, 9/1), and B: mixed solution of
HCOOH/H2O/CH3CN (v/v/v, 0.05/1.95/8). A linear gradient from 20 % to 85 % of B
in 70 min. Methylpyranomv-3-gluc was detected at the wavelength of 280 nm, 478 nm
and 520 nm, other pyranoanthocyanins were detected at 280 nm, 510 nm and 520 nm.
The purity, structure of each pyranoanthocyanin and its maximum absorption
wavelength (λmax) in UV are shown in Supplementary Table 1. The final purity of six
pyranoanthocyanins is all above 97% evaluated by the ratio of target peak area to total
peak area at 280 nm. After purification, samples were evaporated and freeze-dried into
powders, and stored at -20 ℃ until use.
UPLC-DAD/ESI-MS Analysis. The identification of pyranoanthocyanin was
conducted on a Waters Acquity TM Ultra-Performance LC system (Waters, Milford
MA, US), equipped with a DAD and ESI-MS/MS system coupled with a Waters data-
processing station. The mass spectra data were processed with the MassLynx software
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(version 4.1, Micro mass, Manchester, UK). 7.5 microliter purified sample with the
concentration of 1.0 mg/L was injected after filtration with 0.22 μm hydrophilic PTFE
millipore filter on a reversed-phase column Agilent Eclipse Plus C18 (2.1×100 mm, 1.8
μm) with a column temperature of 35 ℃. The UPLC conditions were: 0.1% formic acid
water (phase A), acetonitrile (phase B), the flow rate was 0.5 mL/min. The linear
gradient for phase B was 0 min, 5 %; 9 min, 60 %; 10 min, 95 %; 12 min, 95 %. The
column was washed with 95% of phase B for 1 min and then stabilized with the initial
conditions for another 2 min. The detection wavelength was consistent with the
purification procedure. For identification, ESI-MS/MS was adapted from the method
of Sun (23) with slight modifications: positive ion mode; dry gas, N2, 650 L/h; drying
gas flow temperature, 450 ℃; source temperature, 110 ℃; capillary, 3000 V; cone
voltage, 30 V; cone orifice flow, 50 L/h; scan range, 100-1000 m/z. The mass spectrum
data are shown in Supplementary Table 1.
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NMR Analysis (1H and 13C NMR). 1H NMR (400 MHz) and 13C NMR (100 MHZ)
spectra were performed with CD3OD containing tetramethylsilane as internal standard
on a Bruker AVANCE III 400 MHz Superconducting Fourier NMR system (AVANCE
III HD 400). All data are collected and shown in Supplementary Table 1.
pH Stability. Mv-3-gluc, Methylpyranomv-3-gluc, Vitisin-A, Mv-3-gluc-4-
vinylphenol, Pinotin-A, Mv-3-gluc-4-vinylguaiacol and Mv-3-gluc-4-vinylsyringol
were prepared in sodium dihydrogen phosphate - sodium hydrogen phosphate buffer
solutions (both 0.1 mM) with different pH values in a range between 1.0 and 11.0. All
pigment solutions were balanced under the natural light for 15 min, and then the color
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was observed, and the visible spectrogram was also recorded, using a 10 mm pathlength
cell in a UV-1800PC MAPADA spectrophotometer (MAPADA Instruments, Shanghai,
China).
SO2 Bleaching Stability. To compare the stability of anti-bleaching by SO2 among
these pigments, each pigment was dissolved in solutions of sodium dihydrogen
phosphate and sodium hydrogen phosphate at pH 2.5 (0.01 mM), and different aqueous
solution of sodium bisulfite (5.0 mg·mL-1) were added to this solution to achieve SO2
concentrations in the range between 0 and 200 ppm (24). All pigment solutions were
balanced for 15 min under the natural light, and then their visible spectroscopic
absorbance curves were recorded in a UV-1800PC MAPADA spectrophotometer with
a 1 nm sampling interval using a 10 mm path-length cell. The bleaching constant (KSO2
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was calculated from the slope of the graphic of the color intensity curve of absorbance
(at the maximum absorbance of each pigment) as a function of the concentration of SO2
(24).
Thermal Stability. Mv-3-gluc and six pyranoanthocyanins were dissolved in 10 mL
aqueous solution at pH 1.0 to a final concentration of 0.1 mM. 2 mL of each sample
were heated in a thermostatic water bath (HH-1, Botai, Shanghai, China) for 5 h at
different temperatures ranging from 50 °C to 98 °C. The absorption value of each
sample was measured every hour.
The first-order reaction rate constants (k) and half-lives (t1/2) were calculated by the
Eqs (1) and (2):
ln (Ct/C0) = – k × t
(1)
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t1/2 = – ln (0.5 × k)
(2)
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where: C0 – initial anthocyanin content; Ct – anthocyanin content after t hour heating
at a given temperature
Antioxidant Activity. Antioxidant capacity was determined by DPPH free radical
scavenging ability, ABTS free radical scavenging ability performed with Trolox
equivalent antioxidant capacity (TEAC) and ferric reducing antioxidant power (FRAP)
according to Azevedo (25), Rivero-Pérez (26) and Muselík (27) with some
modifications.
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DPPH· Method. This method is based on the reduction of free radical DPPH· (2,2-
diphenyl-1-picrylhydrazyl). Briefly, 3 μL of different samples in methanolic solution
with the same concentration (10 μM) was added to 297 μL of DPPH· methanolic
solution (60 μM) in the pore plate. After reaction for 20 min of avoiding light at 25 °C,
the absorbance at 515 nm was measured by MULTISKAN GO multimode reader
(Thermo Scientific). The reference cuvette contained methanol. All results were
expressed in μM of Trolox, using the relevant calibration curve. The Trolox (6-
Hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) standard curve was detected
with the same condition as described above. 3 μL of different concentrations of Trolox
between 5 and 100 μM in the solution of 0.5% TFA (trifluoroacetic acid) and methanol
(v/v, 7/3) reacted with 297 μL of DPPH· methanolic solution.
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ABTS Method. The ABTS radical cation could receive a maximum absorption at
734 nm. Reduction of the radical cation of ABTS would cause decoloration of the
solution. The radical was generated after 7 mM solution of ABTS in water reacted with
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2.45 mM potassium persulphate for 16 h in the dark at 20 °C. The assay was made up
with 260 μL ABTS radical cation solutions and 40 μL of different samples with the
same concentration of 10 μM. The samples were tested with a multimode reader at the
wavelength of 734 nm after being balanced in the dark for 6 min. All results were
expressed in μM of Trolox, using the relevant calibration curve. The Trolox standard
curve among 5 to 100 μM was drawn with the same condition as described above. The
preparation of Trolox solution was the same as the method of DPPH.
FRAP Method. This method is based on the increased absorbance at the wavelength
of 593 nm since the reduction of ferric 2,4,6-tripyridyl-s-triazine (Fe3+ - TPTZ) to a
deep blue colored product (Fe2+ - TPTZ) in the presence of reductant. 40 μL of different
samples with the same concentration of 10 μM were separately filled into the pore plate
with 260 μL TPTZ solution (mixed with 25 mL of 0.3 M acetate buffer, 2.5 mL of 10
mM tripyridyltriazine solution prepared in HCl (40 mM) and 5 mL of 20 mM ferric
chloride solution (FeCl3). The reactive mixture was incubated at 37 °C for 30 min and
then measured at 593 nm. All results were expressed in μM of Trolox, using the relevant
calibration curve. The Trolox standard curve between 5 and 100 μM was plotted with
the same condition as described above. The preparation of Trolox solution was
performed by using the same method as DPPH. The standard curves of three methods
were linear between 5 and 100 μM Trolox. The results are expressed as μmol TE L−1
sample.
Statistical Analysis. All experiments were conducted in triplicates. Statistical
analysis of the ANOVA (using Tukey’s procedure) was conducted using the Origin,
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version 9.1 (OriginLab Corp., Northampton, Massachusetts, USA). Significant
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difference was statistically considered at the level of p < 0.05.
RESULTS AND DISCUSSION
Pyranoanthocyanins Stability. The spectral properties of six pyranoanthocyanins
and Mv-3-gluc in relation to pH, bisulfite decoloration and heat were characterized. In
all cases, these pyranoanthocyanins showed greater stability than its precursor, Mv-3-
gluc.
pH Stability. The color changes of six pigments toward different pH values were
shown in Figure 1. In acid solution, Mv-3-gluc shows a red color, Methylpyranomv-3-
gluc presents a yellow color, and the other pyranoanthocyanins share the orange-red
color. Due to the special chemical structure, monomeric anthocyanin is very sensitive
to pH change, and present different colors in different pH buffer solutions. The color of
Mv-3-gluc almost disappears above pH 3.6, and then turns blue above pH 9.0. While
the color of other six pyranoanthocyanins remained stable as the pH change, especially
Vitisin-A that was synthesized by Mv-3-gluc and pyruvic acid, no obvious tonal
changes visible to the naked eye was found below pH 9.0. For other
pyranoanthocyanins, there are also no obvious tonal changes visible to the naked eye
below 4.5. Above pH 4.5 or higher, their color gradually changes from yellow via pink
to the final light yellow, orange-red via mauve to purple, orange-red to brown, orange-
red to deep purple, orange-red to deep purple, and orange-red via light green to violet,
for Methylpyranomv-3-gluc, Mv-3-gluc-4-vinylphenol, Pinotin-A, Mv-3-gluc-4-
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vinylguaiacol and Mv-3-gluc-4-vinylsyringol with the gradual increase of pH,
respectively.
For monomeric anthocyanins, at pH below 2.0, the flavylium cation of anthocyanin
is the primary species and contributes to purple and red color. Furthermore, colorless
species of carbinol pseudobase and chalcone can be observed at the pH range from 3.0
to 6.0. When the pH value is above 7.0, monomeric anthocyanin pigments are
deprotonated to form an ionized quinonoidal base (28-29). However, the color of
pyranoanthocyanins was still stabilized and strong even at higher pH values (3.6 and
4.5) (Figure 1). The substitution at C-4 of anthocyanins, especially the formation of a
pyranoid ring stabilizes the pigment molecule, by preventing the hydration of the
pyranoanthocyanin to a colorless carbinol base or hemiacetal form (30). Oliveira (31)
put forward that the pH change will contribute to occurrence of proton transfer reaction,
but not the hydration in pyranoanthocyanins. Pyranoanthocyanins are present in their
pyranoflavylium cation form at acidic pH conditions. When the pH increases,
pyranoanthocyanins are deprotonated and lead to the formation of the quinoidal neutral
base and the anionic base. These equilibria have been discovered at a great diversity of
pyranoanthocyanins with UV-Visible spectroscopy and NMR techniques (32-35).
Above results showed that pyranoanthocyanins can maintain stable color over a
considerable wide range. The usual pH is between 3.0 and 4.0 when anthocyanin
applied in the production of fruit juice, wine and fruit vinegar as a natural pigment.
Within this pH range, pyranoanthocyanins have no obvious tonal changes visible to the
naked eye. The chemical structure is also one of the main contributors to influencing
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the color of anthocyanins themselves. Figure 2 shows the visible spectrum variation of
Mv-3-gluc and six malvidin-based pyranoanthocyanins at different pH values ranging
from 1.0 to 11.0. When compared to Mv-3-gluc, the λmax of pyranoanthocyanins
molecules presented a hypsochromic shift and the λmax presented a lower value at pH
below 2.5. However, λmax of all the pyranoanthocyanins had a greatly steady value
during the increase of pH in acid buffer solution (pH 2.5-5.5), while absorbance of Mv-
3-gluc dropped very quickly as the pH increased and finally transferred to the colorless
pseudobase species forms at pH 3.6 (Figure 2). Mazza and Miniati (36) also mentioned
that monomeric anthocyanins easily lost about 80% of their color intensity towards the
pH value from 1.0 to 5.0, while pyranoanthocyanins do not display an apparent change
in color intensity. The higher stability can be explained by the protective effect of the
new pyran ring against the nucleophilic attack of water, to hinder the formation of the
carbinol base (13). This new pyranic ring D may have multifarious substituents
combined directly to C-10. This structural feature has been considered as the foundation
of the higher color intensity and stability of pyranoanthocyanins in a wider pH range
compared with their anthocyanin precursors (37).
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However, the stability of pyranoanthocyanins in aqueous solution is also partially
determined by the different substituent groups. There is a relationship existed between
the structural characterization of pyranoanthocyanins and the ionization constants. The
electronic delocalization could affect equilibrium forms of pyranoanthocyanins (34).
According to Oliveira (38), Vitisin-A is present in its carboxy-pyranoflavylium form,
and performs a red color at heavily acidic conditions When the pH value increased to
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3.0, a low amplitude hypsochromic shift occurred in the λmax to lower values as the
deprotonation happened at the carboxyl group present at C-10 of the pyrano ring and
then leads to the formation of the carboxypyranoflavylium zwitterion. With further
increase of pH, the color intensity of Vitisin-A reduced, accompanied by a
bathochromic shift of the λmax. This result attributed to the deprotonating at the 7-OH
finally leads to the formation of anionic quinoidal base. For higher pH values above
5.46, a third deprotonation occurs (4’-OH) leads to the formation of the dianionic
quinoidal base. The three ionization constants of Vitisin-A (pKa1 = 1.09 ± 0.09, pKa2 =
4.93 ± 0.02, pKa3 = 9.14 ± 0.05 and pKa1 = 1.18 ± 0.07, pKa2 = 4.42 ± 0.01, pKa3 =
7.78 ± 0.02 obtain by NMR and UV–Visible spectroscopy, respectively) were similar
with the value of Vitisin-B (pKa1 ≤ 0 .68, pKa2 = 4.40 ± 0.08, pKa3 = 7.45 ± 0.09 and
pKa1 ≤ 0 .63, pKa2 = 4.34 ± 0.02, pKa3 = 7.34 ± 0.03 obtain by NMR and UV–Visible
spectroscopy), which is the simplest vitisin-type pigment and has no substituent group
on the second pyran ring. The exception is the pKa1 of Vitisin-A is effectively higher
than the value of Vitisin-B, and it is due to that the carboxyl group presented at C-10
can stabilize the pyranoflavylium form.
In the case of the Methylpyranomv-3-gluc, methyl group on the new pyran ring, there
are also four equilibrium forms that appear in water with different pH values: The
Methylpyranomv-3-gluc cation, the neutral quinoidal base, the respective anionic
quinoidal base, and a dianionic base unprotonated at the methyl group. In the light of
the NMR data, deprotonation happened fast at the 7-OH of methylpyranoflavylium
cation and led to the generation of neutral quinoidal base. As the pH value increased to
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11.78, the neutral quinoidal base deprotonated at the 4’-OH and gave rise to anionic
quinoidal base. The pKa of Methylpyranomv-3-gluc were pKa1 = 5.17 ± 0.03, pKa2 =
8.85 ± 0.08 and pKa1 = 4.57 ± 0.07, pKa2 = 8.23 ± 0.04 obtained by NMR and UV-
visible spectroscopies, respectively. The pKa1 and pKa2 of the Methylpyranomv-3-gluc
was higher than the value of Vitisin-B (32). This result might be attributed to the methyl
group present in the Methylpyranomv-3-gluc, which stabilizes the quinoidal base form
and further reduces their ability to lose the second proton. Besides, the third slower
deprotonation found by NMR spectra occurred at the methyl group when the pH value
was above 12.3, and the Methylpyranomv-3-gluc was transformed to syringetin-3-O-
glucoside (35). Above results indicated that the carboxyl group and methyl group
present at C-10 could stabilize the pyranoflavylium cation form of pyranoanthocyanins.
As for Pinotin-A, an obvious bathochromic shift and a significant increase in
maximum absorption occurred with the increase of pH. A similar result for the
pyranoanthocyanin-catechols that have the same catechol structure on E ring with
Pinotin-A was reported by Oliveira (31). The pyranoanthocyanin-catechol initially
released the 7-OH proton and subsequently 4′-OH proton, and finally the hydroxyl
proton at catechol at the range of pH between 3 and 12. The pKa of the
pyranoanthocyanin-catechol was 4.20 ± 0.06, 7.84 ± 0.05 and 10.28 ± 0.07 for
above equilibrium reactions (33). The first deprotonation and the second deprotonation
process of Pinotin-A were consistent with the second deprotonation and the third
deprotonation process of Vitisin B respectively, which lack catechol group at C-10.
However, the pKa1 and pKa2 of Pinotin-A were higher than the pKa2 and pKa3 of Vitisin
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B (31). It can be concluded that the catechol group affected the ionization equilibrium
of Pinotin-A.
SO2 Stability. The impact of SO2 on the color stability of six pyranoanthocyanins
compared with Mv-3-gluc was also studied (Figure 3). Results showed that
pyranoanthocyanins have stronger resistance to SO2 bleaching (concentration from 0 to
250 ppm) than Mv-3-gluc. He et al. (24)and Oliveira et al. (39) showed that
pyranoanthocyanin pigments (pyranoanthocyanin-flavanols and Portisin) were
effectively resistant to bleaching by SO2 at the aqueous solution of different
concentrations of sodium bisulfite, and suggested that the improved stability against
bisulfite bleaching of three oligomeric pigments could be attributed to the C-4
substituted structure of pyranoanthocyanins. Bakker et al. (40) also compared the
bleaching stability of vitisin-type pyranoanthocyanins and Mv-3-gluc. Their results
showed that aqueous solution of vitisin-A at pH 3.85 was not bleached by the addition
of up to 250 ppm SO2 and that the maximum absorbance remained unchanged after 48
h storage. Moreover, the aqueous solution of Vitisin-B at pH 3.80 was partially
bleached by SO2 but was still much more resistant than Mv-3-gluc, which was almost
completely decolorized under the same SO2 concentration. It is well documented that
Mv-3-gluc is easily bleached by SO2, and reversibly generates colorless products. This
is because the nucleophilic addition of anionic bisulfite occurs at the positively charged
C-2 and C-4 position of the anthocyanin structure. The C-4 position of anthocyanin is
easier to be attacked due to its lower steric hindrance with respect to the C-2 position
(41). For six pyranoanthocyanins, C-4 is occupied by the formation of a new pyran ring,
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preventing the nucleophilic attack by SO2 in that position. Additionally, slight
decoloration of these pyranoanthocyanins could be attributed to nucleophilic attack of
SO2 at the less favored C-2 position of the adducts (21). The bleaching constant
determined by plotting the color intensity at the λmax of each pigment as the function of
SO2 concentration (Table 1) showed that all six pyranoanthocyanins are more
efficiently resistant to bleaching by SO2. The order of resistance is Vitisin-A > Mv-3-
gluc-4-vinylguaiacol > Methylpyranomv-3-gluc > Pinotin-A > Mv-3-gluc-4-
vinysyringol > Mv-3-gluc-4-vinylphenol > Mv-3-gluc. Vitisin-A shows the strongest
resistance, more than 30 times than Mv-3-gluc.
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Thermo-Stability. The degradation of six pyranoanthocyanins was also studied at
different temperatures ranging from 50 °C to 98 °C. It showed that all the six
pyranoanthocyanins presented greater stability than Mv-3-gluc at the same heating
conditions (Table 2). When the color value of Mv-3-gluc at λmax fell to 67.3% after
heating at 98 °C for 1 h, the preservation of other six pyranoanthocyanins remained
more than 80%. Moreover, the residual rate of Mv-3-gluc dropped to 16.8% for 5 h
after continuous heating at 98 °C, the six pyranoanthocyanins still had a relatively high
preservation over 55%. Among six pyranoanthocyanins, the stability of
Methlypranomv-3-gluc against heating was higher than others. The logarithm of the
pigment contents ln (Ct/C0) was plotted as a function of time in this study (Figure 4).
The linear relationship indicated that the degradation of these pigments by heating
followed first order reaction kinetic. Related kinetics parameters characterized by the
half-life time (t1/2) and reaction rate constant (k) are given in Supplementary Table 2.
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The degradation amplitude of pigments increased with increasing temperature and
thermal treatment time. The thermal degradation reaction rate constants (k) of
anthocyanin have also been studied in some previous references. A study conducted by
Kırca et al. (42) demonstrated that the degradation kinetics of monomeric anthocyanins
fitted a first-order reaction with reaction rate constants (k) of 1.15×10-5 - 8.30×10-5 s-1
over the temperature range of 70 - 90 °C. Sui et al. (43) reported the degradation rate
constants for cyanidin-3-glucoside were 8.99 × 10-4 s-1 and 0.120 s-1 obtained at pH
2.2, 100 °C and pH 6.0, 165 °C, respectively, whereas those for cyanidin-3-rutinoside
were 5.33 × 10-4 s-1 at pH 2.2, 100 °C and 7.39 × 10-2 s-1 at pH 5.0, 165 °C, respectively,
which also followed a first-order reaction. With increasing heating temperature from
50 °C to 98 °C, the Mv-3-gluc and six pyranoanthocyanins degradation amplitude
increased, and their stability decreased. In the same treatment condition, the half-lives
(t1/2) of pyranoanthocyanins were larger than the value of their precursor Mv-3-gluc. It
is indicated that pyranoanthocyanins possessed higher thermo-stability than Mv-3-gluc,
especially in the higher temperature and longer treatment time. Among six
pyranoanthocyanins, the Methylpyranomv-3-gluc showed the greatest stability against
thermo treatment, followed by four hydroxyphenyl-pyranoanthocyanins. And among
four hydroxyphenyl-pyranoanthocyanins, Mv-3-gluc-4-vinylsyringol and Mv-3-gluc-
4-vinylguaiacol presented better thermo-stability than the other two hydroxyphenyl-
pyranoanthocyanins associated with one or two methoxy groups on E ring. Moreover,
Mv-3-gluc-4-vinylsyringol with two methoxy substituents are more stable than Mv-3-
gluc-4-vinylguaiacol with one methoxy substituent, reasoning a positive effect of
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methoxyl on thermo-stability of pyranoanthocyanins. Besides, the hydroxyl group on
E ring could also have a positive contribution to pyranoanthocyanin thermo-stability.
Compared to Mv-3-gluc-4-vinylphenol, the Pinotin-A with hydroxyl group on E ring
possessed greater thermo-stability. Based on above data, the thermo-stability among six
pyranoanthocyanins followed this order: Methylpyranomv-3-gluc > Mv-3-gluc-4-
vinylsyringol > Mv-3-gluc-4-vinylguaiacol > Pinotin-A > Mv-3-gluc-4-vinylphenol >
Vitisin-A. Liu et al. (44) studied the thermal degradation kinetics of Cyanidin-3-
glucoside, Cyanidin-3-(6´´-malonyl)-glucoside and Cyanidin-3-glucoside-derived
pyranoanthocyanins from blood orange, all of three pigments followed the first order
reaction kinetics during heating at the temperature of 70, 80 and 90 °C. The activation
energy (Ea) values for Cyanidin-3-glucoside, Cyanidin-3-(6´´-malonyl)-glucoside and
Cyanidin-3-glucoside-derived pyranoanthocyanin were 75.4, 79.5, and 81.7 kJ mol-1
respectively, which meant that Cyanidin-3-glucoside-derived pyranoanthocyanin had
the highest stability, followed by cyanidin-3-(6´´-malonyl)-glucoside and cyanidin-3-
glucoside. This result revealed that the pyranoanthocyanins showed greater thermo-
stability than their precursors.
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Degradation of anthocyanins is primarily caused by oxidation, cleavage of covalent
bonds or enhanced oxidation reactions during thermal processing (45). While it is
believed up till now that anthocyanins are degraded through two pathways to end up
with chalcone and coumaric acid glycosides or aldehyde and benzoic acid derivatives
(46-48). However, knowledge of the underlying degradation mechanism is scarce. Both
two pathways of degradation are happened associated with the loss of B ring of
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anthocyanins. However, the higher stability of pyranoanthocyanins against heating may
be caused by the stronger steric hindrance to prevent the attack from water molecules
and some oxidation substances. The synthesis of monomeric anthocyanin with phenols
can generate a new ring accompanied by intramolecular copigmentation. The
intramolecular copigmentation can produce higher steric-hindrance, which is stronger
and effective in stabilizing anthocyanins than intermolecular copigmentation (44, 49).
Antioxidant Activity. In this study, the antioxidant activities of six
pyranoanthocyanins and their precursor Mv-3-gluc were evaluated by DPPH, ABTS
and FRAP methods (Figure 5). As we know, multiple reaction characteristics and
mechanisms are likely involved, no single assay will accurately reflect all antioxidant
activities of the pyranoanthocyanins. Thus, three different assays including DPPH,
ABTS and FRAP methods were used to fully evaluate a comprehensive profile of
antioxidant capacity of six pyranoanthocyanins.
The ABTS radical cation is reduced in the existence of hydrogen atom-donating
antioxidants and sensitive to hydroxylated aromatic compounds (50). DPPH is one of
stable organic nitrogen radicals, and is processed in the presence of electron-transfer
reactions and hydrogen-donating (51). FRAP assay specially takes advantage of
electron-transfer reactions. The difference of substituent group on D ring of
anthocyanins may causes the different electron-transfer reactions and H atom donation
capability, which could lead to the different results for three methods.
Results showed that the antioxidant activities of pyranoanthocyanins with different
substituent groups at C-10 were different. Overall, no matter which method was used
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in this study, Pinotin-A with o-dihydroxyl group on
E
ring always
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significantly presented the strongest antioxidant capability among these seven pigments
(p<0.05). Caffeic acid and its derivatives have better antioxidant activity than its related
hydroxycinnamic acid compounds (52-53). Pinotin-A belongs to the derivative from
caffeic acid and malvidin-3-glucoside (54), and caffeic acid retains its two OH groups
next to each other. All in all, Pinotin-A displayed the strongest antioxidant capability
among these seven pigments (p<0.05).
DPPH radical scavenging activity is widely used for quickly assessing the
antioxidant ability of polyphenols. In the DPPH assays, the H atom is transferred from
an antioxidant to DPPH, both Mv-3-gluc and other six pyranoanthocyanins were tested
at the same concentration of 10 μM. Among six pyranoanthocyanins, Pinotin-A, Mv-3-
gluc-4-vinylsyringol, Mv-3-gluc-4-vinylsyringol, Mv-3-gluc-4-vinylguaiacol and
Vitisin-A have significantly stronger DPPH scavenging ability than their precursor Mv-
3-gluc (Figure 5 a). However, Methylpyranomv-3-gluc was found to have a DPPH
scavenging ability that is similar to Mv-3-gluc. Above result shows that most of
pyranoanthocyanins possessing one or two extra ring like D ring or E ring, exhibit
stronger DPPH radical scavenging activity than basic anthocyanins, especially the four
hydroxyphenyl-pyranoanthocyanins with two extra rings in one compound. Moreover,
the Pinotin-A with o-dihydroxyl group on the E ring showed the strongest DPPH radical
scavenging ability. This experimental phenomenon is accordance with other studies.
Goupy et al. (55) suggested that pyranoanthocyanins derived from Mv-3-gluc with
higher H-donating activities could reduce more DPPH radicals than Mv-3-gluc.
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Azevedo et al. (25) also indicated that most of pyranoanthocyanins they studied
presented higher antioxidant potential than that of malvidin-3-glucoside. The
pyranoanthocyanin-catechols and pyranoanthocyanin-catechins that possess E ring
displayed a two-fold or above higher anti-radical capacity than malvidin-3-glucoside,
and vitisin-like pigments (Vitisin-A and Vitisin-B) were also found to display a higher
anti-radical capacity than their parent anthocyanin. The outstanding scavenging DPPH
radical ability of pyranoanthocyanin-catechols and pyranoanthocyanin-catechins is
likely related to the presence of an additional o-dihydroxyl group in their structures
combined with the extended conjugation of π electrons.
Ability of scavenging ABTS radical is also another frequently-used method for anti-
oxidative activity evaluation of polyphenols. All of six pyranoanthocyanins exhibited
higher TEAC value than Mv-3-gluc. Pinotin-A showed the highest activity among the
analytes, it is more than twice value than its precursor Mv-3-gluc (mean value was
80.71 and 33.47 μmol of TE L−1 respectively). The value of Methylpyranomv-3-gluc,
Mv-3-gluc-4-vinylphenol, Mv-3-gluc-4-vinylsyringol, Vitisin-A and Mv-3-gluc-4-
vinylguaiacol was 71.69, 70.44, 63.47, 55.39 and 50.13 μmol of TE L−1, severally. This
demonstrated that the new-derived pigments originated from Mv-3-gluc presented
stronger scavenging ABTS radical capacity than Mv-3-gluc at the same concentration.
To assess the influence of structure on the potential reducing capacity of Mv-3-gluc
and its six derivatives, FRAP method was adopted in this study (Figure 5 c). When the
reducing capability value of Mv-3-gluc was 17.00 μmol of TE L−1, the reducing
capacities of the individual pyranoanthocyanins were in the range of 5.83 to 92.44 μmol
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of TE L−1. Pinotin-A also showed the highest potential antioxidant capacity compared
to other pigments. Mv-3-gluc-4-vinylguaiacol and Mv-3-gluc-4-vinylsyringol with
similar values also presented greater reducing capability than Mv-3-gluc. However, the
antioxidant ability of Vitisin-A and Mv-3-gluc-4-vinlphonel were obtained lower value
than its precursor. Muselík et al. also compared the antioxidant activities between
delphinidin-3-monoglucoside, malvidin-3-monoglucoside and their pyruvic acid
adducts by FRAP assay, both two pyranoanthocyanins caused a significant decrease in
antioxidant activity in aqueous phase assays (27). Tománková (56) also assessed the
influence of structure on the potential antioxidant capacity between Mv-3-gluc and
Vitisin-A by FRAP assay and found that the antioxidant capability of Vitisin-A was 42
% less than Mv-3-gluc. According to García-Alonso et al., the decrease of reducing
capacity of Vitisin-A may be related with the intervention of an additional C3O2 group
at positions of C-4 and C-5 and the lack of the 5-hydroxyl group in the molecule,
contributing to the antioxidant activity together with the 7-hydroxyl group (57).
The molecular basis for the antioxidant properties of polyphenols is mainly revealed
in three different pathways: (I) a hydrogen atom transfer, (II) a single-electron transfer,
and (III) the chelation of metals (58-61). The first two pathways are described by Eqs
(3) and (4) respectively:
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ArOH + R• →ArO• + RH
ArOH + R• →ArOH•+ + R-
(3)
(4)
In recent years, the antioxidant capability of pyranoanthocyanins has been studied
computationally. Researchers have applied DFT/B3LYP method to evaluate the
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antioxidant radical scavenging capacity of pyranoanthocyanins. Both H atom donation
mechanism and single-electron mechanism were investigated, and the bond
dissociation energy (BDE) was determined in the gas phase medium and liquid medium.
The computational results showed that pigments with lower BDE values favor both
hydrogen abstraction and electron-transfer reaction (25, 62).
Among the pyranoanthocyanins studied by Leopoldini (62), the lowest value of BDE
was the Pt-3-Glu-VP (petunidin-3-glucoside-4-vinylphenol) and Pinotin-A that
possessed the o-dihydroxy functionality at rings B and rings E, respectively. Besides,
Pt-3-Glu-VP with an additional E ring on its structure showed a lower BDE value than
Pt-3-Glu-PA (peonidin-3-glucoside-pyruvic acid), which further confirmed the positive
effect of extra ring formation. It is possible because the increase of conjugation and
delocalization effect reduces the BDE value.
SUPPORTING INFORMATION
1
The details of maximal wavelength and H chemical shifts of Mv-3-gluc and six
pyranoanthocyanins are shown in Supplementary Table 1, and thermal degradation
parameters of six pyranoanthocyanins and Mv-3-gluc are shown in Supplementary
Table 2.
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China
(NSFC, No. 31771983 and No. 31871816). The authors also thank the Science and
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Technology Program of Guangzhou (No.201903010082 and No. 201704020050).
Besides, we thank Teus Dorresteijn for linguistic assistance during the preparation of
this manuscript.
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AUTHOR CONTRIBUTIONS
1These authors contributed equally to this work.
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