Oxidative formation and structural characterisation of new α-pyranone (lactone) compounds of non-oxonium nature originated from fruit anthocyanins

Article abstract of DOI:10.1016/j.foodchem.2011.01.069

A new group of pyranoanthocyanin-derived polyphenolic compounds was recently found to occur in aged wines and named as oxovitisins, the spectrum of which displayed only a pronounced broad absorption peak around 370 nm in the UV region, being quite differe

Full text of DOI:10.1016/j.foodchem.2011.01.069

Food Chemistry 127 (2011) 984–992
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Oxidative formation and structural characterisation of new
a-pyranone
(lactone) compounds of non-oxonium nature originated from fruit anthocyanins
Jingren He a,b, Artur M.S. Silva c, Nuno Mateus a, Victor de Freitas a,
⇑
a Centro de Investigação em Química, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal
b School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, PMB1, Glen Osmond, 5069 SA, Australia
c Departamento de Química & QOPNA, Universidade de Aveiro, 3810-193 Aveiro, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A new group of pyranoanthocyanin-derived polyphenolic compounds was recently found to occur in aged
wines and named as oxovitisins, the spectrum of which displayed only a pronounced broad absorption
peak around 370 nm in the UV region, being quite different from that of anthocyanin-like chromenylium
pigments which present maximum absorption in the visible wavelength ranges. The possibilities and
reaction conditions for the oxidative transformation of anthocyanin type flavylium cations into new type
of stable pyranone structures of non-oxonium nature was studied through two-step reactions using
anthocyanins obtained from different fruit extracts. The irreversible change of pyranoflavyliums to the
neutral pyranone compounds by hydration and further oxidation reactions in the second step were
strongly related to pH. The reaction took place only in mildly acidic solutions with the most favourable
pH range 4.0–5.5, as the hemiacetal formation by the nucleophilic attack of water could be hindered at
higher or lower pH. The reaction rate increased significantly with increasing temperature. The new non-
oxonium compounds resulting from malvidin-3-coumaroylglucoside and cyanidin-3-rutinoside were
structurally characterised by MS and NMR spectroscopy and shown similarly to possess the common
Received 2 September 2010
Received in revised form 13 December 2010
Accepted 19 January 2011
Available online 25 January 2011
Keywords:
Red fruit anthocyanins
Reaction pathway
Non-oxonium derivatives
Oxidative transformation
Structural characterisation
Spectral features
a
-pyranone (lactone) ring between C-4 and the hydroxy group at C-5 of the anthocyanin core, which con-
fers them with unique spectrum characteristics. The interest in new anthocyanin derivatives of non-oxo-
nium nature with additional pyranone (lactone) ring will go beyond wine chemistry and bring
expectations concerning their use in the food and pharmaceutical industry due to their spectral and
structural similarity to flavones as well as their naturally occurring nature.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
for the relatively higher stability of pyranoanthocyanins compared
to that of the original anthocyanins (Bakker & Timberlake, 1997;
Anthocyanins constitute a major group of polyphenolic natural
pigments widely distributed in the plant kingdom and are respon-
sible for a variety of colours in flowers and fruits. These pigments
are relatively unstable and may undergo several chemical reactions
in anthocyanin-rich foodstuffs and beverages like red wine, leading
to the formation of more stable condensation products (Jurd, 1969;
Liao, Cai, & Haslam, 1992; Somers, 1971; Timberlake, 1980). One of
the most interesting group of reaction products is pyranoanthocy-
anins, possessing an additional pyran ring structure (Fig. 1) be-
tween C-4 and the hydroxyl group at C-5 of the anthocyanin core
(Bakker & Timberlake, 1997; Francia-Aricha, Guerra, Rivas-Gonz-
alo, & Santos-Buelga, 1997; Fulcrand, Benabdeljalil, Rigaud, Chey-
nier, & Moutounet, 1998; Fulcrand, Cameiro dos Santos, Sarni-
Machado, Cheynier, & Moutounet, 1996; He, Santos-Buelga, Silva,
Mateus, & de Freitas, 2006), which is thought to be responsible
Cameira dos Santos, Brillouet, Cheynier, & Moutounet, 1996; Fulc-
rand et al., 1998).
Amongst pyranoanthocyanins bearing different moieties of
small molecular reactants, one of the most important groups
was carboxy-pyranoanthocyanins (A-type vitisins), which re-
sulted from the cycloaddition reaction of pyruvic acid with antho-
cyanins (Bakker et al., 1997; Fulcrand et al., 1998). Recently, a
new class of pyranoanthocyanin dimers that present an outstand-
ing turquoise blue colour was identified in aged Port wine and
lees and shown to be formed by reactions of carboxy-pyranoanth-
ocyanin with methyl-pyranoanthocyanin (Oliveira et al., 2010).
More recently, another family of pyranoanthocyanin derived neu-
tral pigments was also found to occur in aged old Port wines and
named as oxovitisins (He, Oliveira, Silva, Mateus, & de Freitas,
2010). All these newly formed pigments point to a new pathway
involving chemical transformations of pyranoanthocyanins into
new types of structures contributing to the colour changes during
wine ageing.
⇑
Corresponding author. Tel./fax: +351 220402658.
E-mail address: vfreitas@fc.up.pt (V. de Freitas).
0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.01.069

J. He et al. / Food Chemistry 127 (2011) 984–992
985
OCH3
R1
OH
OH
R1
OH
R2
HO
O
HO
O
OCH3
R2
HO
O
O-Gluc
O-Gluc
O
O
O-Gluc
O
OH
R3
Pyranoanthocyanins
Anthocyanins
Oxovitisin A
Fig. 1. Chemical structures of anthocyanins, pyranoanthocyanins and a new non-oxonium derivative detected in wine. R1, R2 = H, OH, or OCH3; R3 = H, COOH, CH3, phenol or
flavanol; Gluc, glucose.
The interest in anthocyanin derivatives goes beyond wine
chemistry. The formation and occurrence of vitisins with different
aglycons have been extensively studied during the last decades in
table wine, Port wine or by-product like grape pomace, and fruit
and vegetable juices (Anderson, Fossen, Torskangerpoll, Fossen, &
Hauge, 2004; Fossen, & Anderson, 2003; Hillebrand, Schwarz, &
Winterhalter, 2004; Mateus, Silva, Vercauteren, & De Freitas,
anthocyanins were firstly converted into respective carboxy-pyr-
anoanthocyanins by reactions with pyruvic acid (molar ratio pyru-
vic acid/anthocyanin of 50:1) in water (pH 2.6, 35 °C) during 5 day
according to the method of Oliveira et al. (2006). The resulting car-
boxy-pyranoanthocyanins recovered were dissolved in 20% aque-
ous ethanol in amber-glass jars with screw-cap. The solutions
were then adjusted to different pH values from 1.0 to 7.0 and en-
riched with air/oxygen through mildly fortified oxygenation peri-
odically (2 min treatment every week). After treatment, the jars
were closed and sealed with a septum and the solutions were incu-
bated at 35 °C. The formation of new compounds was monitored
periodically (every 3 days) by high performance liquid chromatog-
raphy with diode array detection (HPLC-DAD).
The effect of temperature on the reaction was studied by incu-
bation of solutions at ambient temperature (25 °C), 50 and 70 °C,
respectively. For the comparison of oxidising conditions, another
two sets of sample solutions at pH 3.8 without periodical oxygen-
ation treatment or in the presence of additional 5 ppm hydrogen
peroxide were also carried out.
2001; Rentzsch, Quast, Hillebrand, Mehnert,
& Winterhalter,
2007; Schwarz, Wary, & Winterhalter, 2004). For the newly discov-
ered neutral wine pigment (not flavylium cations) which contains
an additional pyran-2-one or lactone ring onto the anthocyanin
molecules (Fig. 1), its spectral and structural similarities to flav-
ones as well as its non-oxonium nature bring expectations con-
cerning the use of these natural compounds in the food and
pharmaceutical industry. Bearing this in mind, the aim of this work
was to study the possibilities and reaction conditions for the oxida-
tive formation of non-oxonium anthocyanin-derivatives through
two-step chemical transformation of genuine anthocyanins ob-
tained from different red fruits. The resulting new polyphenolic
compounds were prepared and characterised by NMR and mass
spectrometry so as to better understand their spectral characteris-
tics and non-oxonium chemical nature as well as their oxidative
formation mechanism, which could also help to explain their pos-
sible occurrence during storage and processing of anthocyanin-rich
foodstuffs and beverages.
2.4. Preparation and purification of anthocyanin-derived pyranone
compounds
Under the optimised reaction conditions, when the newly
formed pyranone-anthocyanin derivatives detected at 370 nm
reached its maximum concentration the reaction was stopped
and the major compounds were purified. Each reaction mixture
of different anthocyanin origin was applied directly onto a TSK
Toyopearl gel HW-40(s) column chromatography. The fraction
containing the derived pigment eluted with 40% aqueous methanol
was collected and then submitted to semi-preparative HPLC using
the below indicated reversed-phase C18 column using an injection
2. Materials and methods
2.1. Reagents
TSK Toyopearl gel HW-40(s) was purchased from Tosoh (Tokyo,
Japan); pyruvic acid (d = 1.267; 97%) was purchased from Sigma–
Aldrich (Madrid, Spain). All solvents were HPLC grade.
volume of 500 ll and the same gradient programme. The isolated
compound was concentrated under vacuum and then submitted
to further purification with distiled methanol, which consisted of
a final elution on silica gel 100 C18-reversed phase using a vacuum
filtration system.
2.2. Anthocyanin extracts preparation
A total of 1 kg of sweet cherries (Prunus avium) and red grapes
(Vitis vinifera) was extracted with a solution of 50% aqueous etha-
nol (pH 1.5, acidified with HCl) for 1 day at room temperature. The
anthocyanin extracts were purified by a TSK Toyopearl gel column
(250 ꢀ 16 mm i.d.) chromatography to obtain the major anthocya-
nin malvidin-3-O-coumaroyl-glucoside 1 from grapes and cyani-
din-3-O-rutinoside from cherries 2 according to the procedures
described previously (Pissarra, Mateus, Rivas-Gonzalo, Santos-Bue-
lga, & de Freitas, 2003).
2.5. Monitoring of reaction products
2.5.1. HPLC-DAD analysis
A Knauer K-1001 HPLC with a 250 ꢀ 4.6 mm i.d. reversed-phase
C18 column (Merck, Darmstadt, Germany) was used to monitor the
reactions. The detection was carried out using a Knauer K-2800
diode array detector. An HPLC pump Knauer K-1001 was used to-
gether with a Knauer K-3800 autosampler. The solvents were A:
H2O/HCOOH (9:1), and B: HCOOH/H2O/CH3CN (1:1:8). The gradi-
ent consisted of 15–35% B over 70 min, 35–80% B over 5 min, and
then isocratic for 10 min at a flow rate of 1.0 ml/min. The column
was then stabilised with the initial conditions for another 10 min.
2.3. Oxidative formation of anthocyanin-derived pyranone compounds
of non-oxonium nature
The formation of non-oxonium pyranone derivatives was
achieved through two-step chemical reactions. The two original

986
J. He et al. / Food Chemistry 127 (2011) 984–992
2.5.2. HPLC-electrospray ionisation mass spectrometry (ESI-MS)
analysis
aglycons and different types of glycosyl and acyl moiety. The origi-
nal anthocyanins were firstly converted into respective pyranoan-
For the LC–MS analysis was used a Finnigan Surveyor series li-
thocyanins 3 (4) by reactions with pyruvic acid under the
quid chromatograph, equipped with a 150 ꢀ 4.6 mm i.d., 5
l
m
conditions described previously (Fulcrand et al., 1998; Oliveira
et al., 2006). The occurrence of carboxy-pyranoanthocyanins with
different aglycons has been extensively studied over recent years
in wine and fruit juices (Hillebrand et al., 2004; Mateus et al.,
2001; Rentzsch et al., 2007; Schwarz et al., 2004), as well as in
fresh fruits and vegetables (Anderson et al., 2004; Fossen & Ander-
son, 2003) and are believed to result from reactions of original
anthocyanins with pyruvic acid, a ubiquitous intermediate of
metabolism like aerobic glycolysis and anaerobic fermentations
during storage and processing.
When fruit anthocyanins were transformed into carboxy-pyr-
anomalvidin-3-O-coumaroyl-glucoside 3 and carboxy-pyranocy-
anidin-3-O-rutinoside 4, the progressive changes in the
concentration of pyranoanthocyanins and the appearance of new
derivatives were monitored by HPLC-DAD/MS during further incu-
bation of the solutions under oxidative conditions. Fig. 3A and B
showed the formation of new products after 20 days of further
incubation of the reaction solutions at pH 3.8, typical pH for many
fruit juices and wine. A significant increase in the intensity of the
new peaks 5 and 6, concurrent with the decrease of the pyrano-
anthocyanin peaks 3 and 4, respectively, was observed in the UV
region of the HPLC chromatograms.
LicroCARTÒ reversed-phase C18 column thermostated at 35 °C.
The mass detection was carried out by a Finnigan LCQ DECA XP
MAX (Finnigan Corp., San José, CA) mass detector with an API
(Atmospheric Pressure Ionisation) source of ionisation and an ESI
(ElectroSpray Ionisation) interface. Solvents were A: aqueous
0.1% trifluoroacetic acid, and B: acetonitrile, establishing the HPLC
gradient as reported elsewhere (He et al., 2006). The capillary volt-
age and temperature were 4 V and 190 °C, respectively. Spectra
were recorded in positive ion mode between m/z 120 and 1500.
The mass spectrometer was programmed to do a series of three
scans: a full mass, a zoom scan of the most intense ion in the first
scan, and a MS–MS of the most intense ion, using relative collision
energy of 30 and 60 V.
2.6. NMR analysis
1H NMR (500.13 MHz) and 13C NMR (125.77 MHz) spectra were
measured in CD3OD/TFA (98:2) and CDCl3 on a Bruker-Advance
500 spectrometer at 303 K with TMS as internal standard. 1H
chemical shifts were assigned using 1D and 2D 1H NMR (gCOSY
and NOESY). 13C resonances were assigned using 2D NMR tech-
niques (gHMBC and gHSQC) (Bax & Subramanian, 1986; Bax &
Summers, 1986). The delay for the long-range C/H coupling con-
stant was optimised to 7 Hz.
The mass spectra of newly formed compounds 5 and 6 were ob-
tained by LC-ESI/MS in the positive ion mode. The MS data of peak
5 showed a quasi molecular ion [M+H]+ at m/z 679 and a major
fragment ion [M+H-308]+ in MS2 at m/z 371 (Fig. 4A and B), which
could correspond to the loss of a coumaroylglucoside moiety orig-
inated from the parent anthocyanin. Whilst the minor fragment
ion [M+H-146]+ in MS2 at m/z 533 could result from the loss of
the coumaroyl moiety. The mass of peak 5 is 29 amu less than
the precursor pyranomalvidin-3-coumaroylglucoside 3 and is con-
sistent with the molecular structure where an oxo-group replaced
the carboxyl group in the C-10 position of pyran ring D to give rise
3. Results and discussion
3.1. Reaction pathway for the oxidative formation of pyranone
compounds derived from fruits anthocyanins
Anthocyanins obtained from different red fruit extracts were
used to produce the respective new derivatives of non-oxonium
nature with additional pyranone ring (Fig. 2). Two anthocyanins
malvidin-3-O-coumaroylglucoside 1 and cyanidin-3-O-rutinoside
2 were chosen aiming to obtain the anthocyanins with different
to an a
-pyranone (lactone) ring, as shown in Fig. 2. The MS3 spec-
trum of the aglycon ion at m/z 371 in MS2 revealed the presence of
a major fragment ion at m/z 343 (Fig. 4C), corresponding to the
further loss of the C-10 carbonyl (C@O) group (equal to 28 amu),
R1
R1
R1
OH
OH
R2
OH
R2
HO
O
HO
O
HO
O
pH 2.6 / 35°C
R2
HO
O-R3
O-R3
O-R3
O
OH
O
COOH
COOH
1 and 2
COOH
3 and 4
O
H
H
R2=OCH3
R1=OCH3
OH
1, 3, 5:
R1
R1
O
2'"
HO
OH
O
β
2"
4"
6"
3'"
4'"
3"
5"
OH
R2
R3=
O
OH
R2
3'
2'
1'
4'
5'
1'"
6'"
1"
α
OH
8
HO
O
5'"
8a
4a
HO
O
R1=OH R2=H
OH
4"
2
6'
7
-CO ,
OH
O
O
2, 4, 6:
2
2"
3"
O
R3= HO
5
4
6
3
-2H+, -2e-
6"
O-R3
5"
O-R3
1"
HO
O
9
O
1'"
10
2'"
3'"
O
O
HO COOH
5 and 6
5'"
H3C
OH
4'"
HO
Fig. 2. Reaction pathway for the formation of non-oxonium pyranone compounds 5 and 6 derived from anthocyanins 1 and 2, respectively.

J. He et al. / Food Chemistry 127 (2011) 984–992
987
5
A
370 nm
510 nm
3
5
10
15
20
25
30
35
40
45
Retention time (min)
B
6
370 nm
4
510 nm
5
10
15
20
25
30
35
40
45
Retention time (min)
Fig. 3. HPLC chromatograms recorded at 510 and 370 nm showing the formation of anthocyanin-derived new pyranone compounds 5 and 6 during further oxidative
incubation of the reaction solutions containing pyranoanthocyanins 3 and 4.
characteristic fragmentation pattern of the
a
-pyranone ring as ob-
(Mateus, Oliveira, Pissarra, & de Freitas, 2003; Romero & Bakker,
1999). The optimised reaction conditions in the second step for
the further transformation of pyranoanthocyanins were explored
at different temperatures and various pH values so as to better
understand the oxidative formation mechanism of new pyranone
derivatives. HPLC-DAD analysis showed that the production of
new pyranone-anthocyanins was very slow at room temperature
as presented in Fig. 5 based on the study of malvidin-3-O-cou-
maroylglucoside, but increased significantly when the temperature
was raised to 50 °C. At the higher temperature of 70 °C, the appear-
ance of pyranone-anthocyanins was faster but a lower amount of
derivatives was observed probably resulting from a higher degra-
dation. Hence, the following reactions were performed at 50 °C
due to the relatively higher stability and reaction rate of
pyranoanthocyanins.
The pH dependency of the transformation reactions was also
investigated (Fig. 6). In very acidic medium (pH < 2), the reaction
did not take place during the incubation period. With the increase
of pH to higher values in the range 3.0–6.0 the reaction rate was
improved significantly. However, at a pH above 6, the open chal-
cone form of pyranoanthocyanins was expected to occur and
therefore decreased the amount of new derivatives. The favourable
served for oxovitisin A and the coumarins (He et al., 2010; Xie,
Zhao, Zhou, Fan, & Wu, 2010). Additionally, fragmentation of the
aglycon ion also produced other fragment ions at m/z 338, 327,
310 and 282, the same phenomena observed by Hayasaka and
Asenstorfer (2002) who found this fragmentation pathway to be
typical for malvidin-derived compounds. All these results indi-
cated that the newly formed peak 5 in Fig. 3A could correspond
to the non-oxonium derivative of malvidin-3-O-coumaroylgluco-
side with additional pyranone ring, as proposed compound 5 in
Fig. 2. The quasi molecular ion [M+H]+ at m/z 635 of the peak 6
in Fig. 3B and its similar MS/MS fragmentation pattern (data not
shown) are consistent with the proposed cyanidin-3-O-rutinoside
derivative 6 in Fig. 2. Therefore the original fruit anthocyanins
could be converted into respective new derivatives with pyranone
ring by two-step reactions through the proposed pathway in Fig. 2.
3.2. Effect of reaction conditions on the oxidative transformation of
pyranoanthocyanins
Factors influencing the formation of pyranoanthocyanins from
anthocyanins and pyruvic acid have been studied previously

988
J. He et al. / Food Chemistry 127 (2011) 984–992
679.13
2400000
2200000
A
OCH3
OH
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
HO
O
OCH3
OH
O
O HO
OH
O
O
O
OH
O
701.13
836.80
400
600
800
1000
1200
1400
m/z
371.19
16000
15000
14000
13000
12000
11000
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
B
532.59
200
250
300
350
400
450
500
550
600
650
m/z
343.14
6000
5500
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
C
311.27
355.58
338.22
327.17
0
100
150
200
250
300
350
m/z
Fig. 4. LC/ESI/MS analysis of the newly formed peak 5 in Fig. 3 originated from malvidin-3-coumaroylglucoside: A, full mass spectrum (quasi molecular ion); B, MS2 spectrum,
and C, MS3 spectrum of the major ion fragment in the last spectrum.
pH for the formation of new pyranone-anthocyanins ranged be-
tween 4.0 and 5.5. The rate of formation under other oxidative con-
ditions was also studied. The reaction in the presence of hydrogen
peroxide proceeded slightly faster, but at the same time a higher
degradation of pyranoanthocyanin precursors was also observed,
which considerably lowered the reaction yields to less than 10%.

J. He et al. / Food Chemistry 127 (2011) 984–992
989
3.3. Structural analysis of anthocyanin-derived new pyranone
compounds
30
25
20
15
10
5
50°C
70°C
25°C
The structure of newly formed pyranone derivatives 5 and 6
originated from malvidin-3-O-coumaroylglucoside and cyanidin-
3-O-rutinoside respectively was elucidated by 1H and 13C NMR
analysis using 1D and 2D techniques (Table 1).
For compound 5, the protons H-6 and H-8 of ring A were as-
signed to two meta-coupled doublets (J = 2.0 Hz) at 6.44 and
6.41 ppm, respectively. Protons H-20, 60 and those of the methoxyl
groups at ring B were situated at 7.36 and 3.85 ppm, respectively.
Proton H-9 was assigned to the singlet at 5.97 ppm. Concerning the
glucosyl moiety, the b-anomeric proton H-1’’ was attributed to a
doublet (J = 7.7 Hz) at 4.77 ppm, all other protons were situated
in the 3.28–3.52 ppm region, except for the last two protons H-
6a00 and H-6b00 that were assigned at 4.22 and 4.15 ppm, respec-
0
0
10
20
30
40
50
tively. With respect to the coumaroyl group, protons H-
a and H-
Reaction time (days)
b
were assigned to two doublets (J = 15.8 Hz) at 7.38 and
6.05 ppm, respectively. Protons H-2000, 6000 and H-3000, 5000 were read-
ily attributed to two doublets (J = 8.6 Hz) at 7.31 and 6.80 ppm,
respectively.
Fig. 5. Influence of temperature on the rate of formation of new pyranone
compound 5.
The carbon resonances were attributed using two dimensional
techniques (HSQC and HMBC) (Fig. 7). Carbons C-6, C-8, C-9, C-
20,60 and OCH3 were assigned to 99.2, 99.8, 92.7, 108.2 and
57.1 ppm, respectively through their direct 1H–13C correlations
with protons H-6, H-8, H-9, H-20,60 and H-OCH3. Carbons C-7 and
C-4a were assigned to 163.8 and 102.7 ppm, respectively by their
correlations in the HMBC spectrum with protons H-6 and H-8. Car-
bon C-5 and C-8a were attributed to 155.4 and 151.9 ppm, respec-
tively by their long distance correlations with proton H-6 and H-8,
respectively. Carbons C-2, C-10, C-30, 50 and C-40 were attributed to
153.2, 122.4, 148.5 and 139.8 ppm, respectively, by their long dis-
tance correlation with protons H-20, 60. Carbon C-3 was assigned to
132.3 ppm through its correlations in the HMBC spectrum with
proton H-9 and the anomeric proton H-100. Carbon C-10 was attrib-
uted at 165.5 ppm by its long distance correlation with proton H-9.
The higher chemical shift observed for this carbon, comparing with
other pyranoanthocyanins described in literature (He et al., 2006;
Mateus et al., 2001; Oliveira, de Freitas, & Mateus, 2009), can be
due to the presence of an electronegative group, such as an oxo
group, attached to this carbon. In fact, the ESI/MS fragmentation
of the aglycon by elimination of a carbonyl (C@O) group in the
Without the periodical oxygenation treatment, the formation of
new pyranone compounds was negligible during the incubation
period and could not be detected under the present analysis
conditions.
The transformation of anthocyanins into a-pyranone-anthocya-
nins via pyranoanthocyanins apparently involves the hydration
reactions of the pyranoflavylium cations followed by hemiacetal
formation as shown in Fig. 2. Subsequent decarboxylation and fur-
ther oxidation of the pyran moiety bearing an a-hydroxyl substitu-
ent to the aromatic lactone or pyran-2-one structure led to the
formation of the final product, a stabilised neutral (non-flavylium
cation) pyranone-anthocyanin derivative. The hydration reaction
by the nucleophilic attack of water should be the key step for the
irreversible change of chromenylium pigments to the neutral pyr-
anone compounds, which also explained the negative reaction at
pH below 2 as observed in Fig. 6, where hemiacetal formation by
the nucleophilic attack of water was hindered in very acidic
medium.
35
30
25
20
15
10
5
0
1
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
pH
Fig. 6. Influence of pH on the rate of formation of new pyranone compound 5.

990
J. He et al. / Food Chemistry 127 (2011) 984–992
Table 1
assigned at 115.3, 147.1, 131.5, 117.3 ppm, respectively by their di-
rect 1H–13C correlations with protons H- , H-b, H-2000,6000 and H-
3000,5000, respectively. Carbon C-CO2 was attributed at 169.2 ppm
1H and 13C chemical shifts of compound 5 (pyranone-malvidin-3-coumaroylgluco-
side) and 6 (pyranone-cyanidin-3-rutinoside), determined in CD3OD/TFA (98:2)a.
a
Pigment
Position
5
6
by its long range correlation with protons H-
a and H-b. Carbons
C-1000 and C-4000 were attributed by their correlations in the HMBC
spectrum with protons H-2000, 6000 and H-3000, 5000 at 127.1 and
161.4, respectively.
d
1Y (ppm); J (Hz)
d
13C
d
1Y (ppm); J (Hz)
d
13C
Pyranone-anthocyanidin moiety
2
3
4
4a
5
6
7
–
–
–
–
153.2
132.3
n.a.
102.7
155.4
99.2
–
–
–
–
–
154.1
132.2
102.8
102.8
152.3
98.7
The structure of newly formed pyranone compound 6 derived
from cherry anthocyanin was also confirmed by NMR analysis (Ta-
ble 1), similar to the analysis obtained for 5. The major differences
concerned the ring B and the sugar moiety that in this case is rutin-
ose which is composed by a molecule of glucose linked to a mole-
–
6.44; d, 2.0
6.55; d, 2.0
–
163.8
99.8
–
163.6
99.6
cule of rhamnose by an a
-(1,6) linkage. The protons H-20, H-50 and
8
8a
9
6.41; d, 2.0
6.51; d, 2.0
–
151.9
92.7
–
155.5
94.0
60 of ring B were attributed at 7.66, 6.87 and 7.44 ppm as doublet
(J = 2.1 Hz), doublet (J = 8.4 Hz) and double doublet (J = 8.4,
2.1 Hz), respectively. The glucose b-anomeric proton was assigned
to the doublet (J = 7.7 Hz) at 4.63 ppm. All of the other glucosyl
protons were situated in the 3.53–3.80 ppm region, except for
the last two protons H-6a00 and H-6b00 that were assigned at 3.93
and 3.79 ppm, respectively. This suggests that the rhamnosyl moi-
ety is linked to C-6 of the glucosyl moiety. The anomeric proton of
the rhamnosyl moiety corresponds to the signal at 4.44 ppm with a
5.97; s
6.16; s
10
–
–
165.5
122.4
108.2
148.5
139.8
148.5
108.2
57.1
–
–
166.0
123.0
118.2
145.6
149.2
117.2
123.5
–
10
20
7.36; s
–
–
–
7.36; s
3.85; s
7.66; d, 2.1
–
–
6.87; d, 8.4
7.44; dd, 8.4/2.1
–
30
40
50
60
30, 50 – OMe
Glucose moiety
100
4.77; d, 7.7
3.52; dd, 9.2/7.7
3.42; t, 9.1
3.28; t, 9.1
3.46; m
103.7
75.9
76.5
72.0
78.5
64.9
64.9
4.63; d, 7.7
3.77⁄
102.8
78.2
72.3
69.0
83.3
62.4
62.4
small coupling constant (J = 1.6 Hz), suggesting an
a configuration
200
of the rhamnose molecule. Protons of the rhamnosyl moiety were
situated in the 3.16–3.66 ppm region. The methyl group of the
rhamnose was assigned to a doublet (J = 6.2 Hz) at 1.13 ppm. All
carbons of the rutinosyl moiety were assigned through direct
1H–13C correlations in the HSQC spectrum.
300
3.53⁄
400
3.75⁄
3.80–3.82; m
3.93; dd, 11.8/2.2
3.79⁄
500
6a00
6b00
4.22; dd, 11.8/2.3
⁄
4.15;
Coumaroyl group
R1CO2R2
–
169.2
115.3
147.1
127.1
131.5
117.3
161.4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3.4. UV–Vis spectrophotometry
CH@CH CO2R
7.38; d, 15.8
6.05; d, 15.8
–
7.31; d, 8.6
6.80; d, 8.6
–
a
CHb@CHCO2R
1000
E
The pyranone structure and non-oxonium nature of the newly
formed compounds confer them with unique spectral features. In-
deed, the UV–Vis spectrum of new derivatives displayed only a
pronounced broad peak at 370 nm for compound 6 derived from
cyanidin-3-O-rutinoside (Fig. 8A). For compound 5 derived from
malvidin-3-O-coumaroylglucoside the UV–Vis spectrum revealed
a maximum absorption peak at 316 nm and also a broad absorp-
tion band at 373 nm (Fig. 8B). Except for the absorption at
316 nm resulting from the coumaroyl group of the molecules,
the two new anthocyanin-derivatives presented similar spectra
characteristics, a broad absorption peak only in the UV region
around 370 nm, being quite different from that of original fruit
anthocyanins and all other anthocyanin-derived chromenylium
pigments which present maximum absorption in visible wave-
length ranges, as shown in Fig. 8C, thus coinciding with its non-
flavylium cation nature of the molecular structures. The small
spectral difference (3 nm) between the two neutral pyranone
aglycons of compounds 5 and 6 lies in the substitutive groups of
2000,6000
3000,5000
E
E
4000
E
Rhamnosyl moiety
1000
2000
3000
4000
5000
CH3
–
–
–
–
–
–
–
–
–
–
–
–
4.44; d, 1.6
3.66; dd, 3.3/1.6
3.38⁄
102.5
72.2
69.8
69.0
73.9
17.9
3.16⁄
3.26⁄
1.13; d, 6.2
Key: s, singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet; ⁄, unre-
solved; n.a., not assigned.
a
MS3 spectrum (Fig. 4) also supports the structure of a pyranone-
anthocyanin molecule, which possesses an oxo group at C-10. With
respect to the carbons of the glucose molecule, they were assigned
by their 1H–13C direct correlation in the HSQC spectrum. The Car-
bons C-
a
, C-b, C-2000,6000 and C-3000,5000 of coumaroyl group were also
OCH3
H
OH
H
A
O
B
HO
H
O
OCH3
H
C
H
H
OH
OH
O
H
O
OH
O
D
E
O
OH
H
H
H
H
O
H
Fig. 7. The important long range 1H–13C correlations observed in the HMBC spectrum of pyranone-malvidin-3-coumaroylglucoside 5.

J. He et al. / Food Chemistry 127 (2011) 984–992
991
750
600
450
300
150
0
316
A
373
220
270
320
370
420
470
520
570
620
670
720
wavelength (nm)
750
600
450
300
150
0
370
B
220
270
320
370
420
470
520
570
620
670
720
wavelength (nm)
900
800
700
600
500
400
300
200
100
0
anthocyanin 1
C
pyranoanthocyanin 3
anthocyanin 2
pyranoanthocyanin 4
360
410
460
510
560
610
660
710
wavelength (nm)
Fig. 8. UV–Vis spectrum of anthocyanin-derived new pyranone compounds 5 (A) and 6 (B) of non-oxonium nature; and UV–Vis spectra of genuine anthocyanins and
anthocyanin-derived chromenylium pyranoanthocyanins (C).
ring B, which contains two methoxy groups in the former, indicat-
ing the origin of fruit anthocyanin aglycons has minimal influence
on the characteristic kmax of the new non-oxonium derivatives
which possess commonly an additional a-pyranone (lactone) ring
between C-4 and the hydroxyl group at C-5 of the anthocyanin
core.

992
J. He et al. / Food Chemistry 127 (2011) 984–992
Cameira dos Santos, P. J., Brillouet, J. M., Cheynier, V., & Moutounet, M. (1996).
4. Conclusions
Detection and partial characterisation of new anthocyanins derived pigments in
wine. Journal of the Science of Food and Agriculture, 70, 204–208.
The results obtained in this study show that anthocyanins ob-
tained from different fruit extracts may be readily transformed into
the respective new pyranone derivatives of non-oxonium nature
through two-step chemical reactions in mildly acidic solutions
(pH 3.0–6.0) like in fermented beverages. The irreversible change
of chromenylium pyranoanthocyanins to the neutral pyranone
compounds in the second step by hydration reactions and further
oxidation are strongly related to pH, as the hemiacetal formation
by the nucleophilic attack of water could be hindered at higher
and lower pH. Additionally, the increase of temperature improves
the reaction rate significantly.
The implications of the reaction pathway studied for the trans-
formation of anthocyanin type flavylium cations into new type of
more stable non-oxonium structures via pyranoanthocyanins are
significant. As the occurrence of pyranoanthocyanins with differ-
ent aglycons has been confirmed over recent years in wine, fer-
mented and stored cherry juices and other processed foodstuffs,
as well as in some fresh fruits and vegetables, the oxidative forma-
tion of new pyranone compounds could thus be expected to take
place during processing and years of storage or ageing, as the oxo-
vitisin A of this family discovered in aged wine (He et al., 2010),
highlighting their relevance to anthocyanin reactivity and wine
colour evolution during winemaking and aging. On the other hand,
the interest in new anthocyanin derivatives of non-oxonium nat-
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chemistry as their spectral and structural similarity to flavones
as well as their naturally occurring nature open new perspectives
for potential applications of such compounds in the food and phar-
maceutical industry. Nevertheless, further studies will be per-
formed especially regarding their physicochemical properties and
biological activities.
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Acknowledgements
Oliveira, J., Fernandes, V., Miranda, C., Santos-Buelga, C., Silva, A., de Freitas, V., et al.
(2006). Color properties of four cyanidin–pyruvic acid adducts. Journal of
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Reaction between malvidin 3-glucoside and (+)catechin in model solutions
containing different aldehydes. Journal of Food Science, 68, 476–481.
The authors thank Dr. Zélia Azevedo for the LC-DAD-MS analy-
sis. This research was supported by a grant from FCT (Fundação
para a Ciência e a Tecnologia – PTDC/AGR-ALI/65503/2006, PTDC/
AGR-ALI/67579/2006) from Portugal and by FEDER funding.
Rentzsch, M., Quast, P., Hillebrand, S., Mehnert, J.,
& Winterhalter, P. (2007).
Isolation and identification of 5-carboxypyranoanthocyanins in beverages from
cherry (Prunus cerasus L.). Innovative Food Science and Emerging Technologies, 8,
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