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Enzymatic synthesis, structural characterization

Cite this: Food Funct., 2016, 7, 2754
and antioxidant capacity assessment of a new
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lipophilic malvidin-3-glucosideoleic acid conjugate

Luis Cruz,* Iva Fernandes, Marta Guimares, Victor de Freitas and Nuno Mateus

Received 1st April 2016,
Accepted 12th May 2016
DOI: 10.1039/c6fo00466k
www.rsc.org/foodfunction
The chemical modication of anthocyanins (water-soluble pigments) into more lipophilic compounds is
very important to expand their application in the food, medical and cosmetic industries. In this work, the
synthesis of a pure malvidin-3-glucosideoleic acid ester derivative was achieved by enzymatic catalysis.
This approach allowed us to synthesize a novel compound, malvidin-3-O-(6-oleoyl)glucoside (Mv3glc
OA), which was structurally characterized by mass spectrometry and for the rst time by NMR spectro-
scopy. The enzymatic reaction revealed to be regioselective giving only one ester product. Antioxidant
features of the malvidin-3-glucoside lipophilic derivative by means of DPPH, FRAP and lipid peroxidation
assays were assessed, which conrmed that the structural modication of the genuine malvidin-3-gluco-
side into a more lipophilic compound did not compromise its antioxidant potential and protected more
eectively a lipidic substrate from oxidation, which is an important insight for future technological
applications.

Introduction stabilize anthocyanins in order to increase their application

Anthocyanins are the largest group of polyphenolic pigments
in the plant kingdom present in many plant foods and pro-
cessed beverages such as red wine and red fruit juices.1
Research on the application of anthocyanins and their deriva-
tives has attracted great interest over the last few years in the
food, nutraceutical, cosmetic and pharmaceutical industries,
due to their antioxidant and biological properties yielding
health benefits.2,3 The eectiveness of anthocyanins depends
on preserving their stability, bioactivity and bioavailability.
However, anthocyanins are quite unstable and susceptible to
degradation. Their stability is aected by pH, temperature,
light, metals, enzymes, oxygen, ascorbic acid, sugars and their
degradation products, proteins, and sulfur dioxide.46 Antho-
cyanins are hydrophilic water-soluble pigments while sparingly
soluble in more lipophilic media, which may compromise
their eective application in lipophilic systems, such as fats,
oils, lipid-based food or cosmetic formulas and emulsions, as
well as biological environments. Considering this, new ways to
REQUIMTE/LAQV, Departamento de Qumica e Bioqumica, Faculdade de Cincias,
Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal.
E-mail: luis.cruz@fc.up.pt; Fax: +351 220402658; Tel: +351 220402558
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6fo00466k
potential have been extensively used namely microencapsula-
tion of anthocyanins through dierent techniques such as
spray drying and freeze drying, among others.7,8 However,
after release from their capsule by an external stimulus the
compounds become unstable. A solution to improve the hydro-
phobic nature of anthocyanins consists of the esterification of
the hydroxyl groups with fatty acids. Studies that have been
performed include chemical and enzymatic acylation to
improve flavonoid properties.914 Some synthesized flavan-3-ol
fatty acid derivatives have exhibited greater anticancer, anti-
angiogenesis, and antioxidant activities in diverse lipid systems
than their native forms.15,16 These results suggest that these
derivatives have a higher application potential in dierent
matrices, namely in the food, cosmetic and pharmaceutical
industries, among others.17
In contrast to other flavonoids, the colour displayed by
anthocyanin pigments is much more appealing and attractive
for consumers and represents an added-value for cosmetic and
food companies in the development of products enriched with
these compounds. However, there are only few investigations
on the acylation of anthocyanin molecules because the chem-
istry involving flavylium compounds is not easy due to their
positive charge in the flavylium form, low solubility in organic
solvents and instability under neutral and basic conditions.
To the best of our knowledge, there is only one work in the

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Food & Function
literature that reported the enzymatic esterification of antho-
cyanins from jaboticaba fruits,18 but the full structural charac-
terization of the anthocyanin derivatives was not determined.
In recent years, some studies have been dedicated to
the synthesis and chemical derivatization of modified
anthocyanins.1921
The aim of this work was to perform a selective acylation of
malvidin-3-glucoside (Mv3glc) from an anthocyanin-rich red
wine extract by enzymatic catalysis with oleic acid as an acyl
donor using dierent experimental conditions. Furthermore, a
Mv3glcOA derivative was purified, and characterized by NMR
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and its antioxidant capacity was assessed.
Materials and methods
Reagents
TSK Toyopearl gel HW-40(S) was purchased from Tosoh
(Tokyo, Japan). Gel RP-18 (4063 m) LiChroprep was provided
by Merck (Darmstadt, Germany). Oleic acid and linoleic acid
were purchased from Sigma-Aldrich (Madrid, Spain). Lipase
acrylic resin from Candida antarctica (5000 U g1, recombi-
nant, expressed in Aspergillus niger) was purchased from
Sigma-Aldrich (Madrid, Spain). Molecular sieves 4 were
purchased from Sigma-Aldrich (Madrid, Spain). TPTZ (2,4,6-
tris(2-pyridyl)-s-triazine), DPPH (2,2-diphenyl-1-picrylhydrazyl),
AAPH (2,2-azobis(2-amidinopropane) dihydrochloride), -caro-
tene and 1,1,3,3-tetraethoxypropane were purchased from
Sigma-Aldrich (Madrid, Spain).
Preparation of the Mv3glc-rich extract
50 litres of red wine were concentrated in a nanofiltration
system. Evaporation of ethanol was then performed and sugars
were removed by C18 reverse gel column chromatography, fol-
lowed by lyophilization. 5 grams of lyophilized red wine was
extracted with ethyl acetate to remove non-anthocyanin
organic compounds. The aqueous layer was then added in a
Buchner funnel loaded with TSK Toyopearl gel HW-40(S) and
the anthocyanin 3-monoglucosides were eluted with 10%
aqueous methanol acidified with 2% HCl. The methanol was
removed by evaporation and the aqueous phase was concen-
trated and purified by elution in a C18 gel. After freeze-drying,
the anthocyanin extract was obtained as a red solid (720 mg).
Enzymatic synthesis and purification of the Mv3glcoleic
acid conjugate
To a Schlenk tube loaded with activated 4 molecular sieves
(100 g L1), the Mv3glc extract (9.47 mol) and anhydrous
2-methyl-2-butanol (2.5 mL) were added. To this solution, the
respective fatty acid was added (100 equivalents). The reaction
started with the addition of the lipase (20 g L1). The reaction
was stirred at 60 C under an argon atmosphere for 48h. The
reaction progression was followed by ESI-MS and HPLC-DAD.
The reaction was stopped by filtration of the molecular
sieves and lipase and by solvent evaporation under vacuum.
The residue was redissolved in acidulated methanol and some
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impurities were extracted with heptane. The methanol fraction
was evaporated, ethyl acetate and water were added and the
Mv3glcoleic acid ester was separated from the starting
material (soluble in the aqueous phase). After the ethyl acetate
evaporation, the residue was dissolved in 85% methanol/water
and the isolation of the Mv3glcoleic acid conjugate was per-
formed by semi-preparative HPLC. Then the compound was
purified by column chromatography loaded with TSK Toyo-
pearl HW-40 (S) gel (300 mm 16 mm i.d.). Methanol was
removed under vacuum, and protected from light at room
temperature. The pigments were freeze-dried and stored at
18 C under an argon atmosphere until use. The compound
was further characterized by LC-DAD/ESI-MS and its structure
and purity were assessed by NMR analyses.
HPLC
The enzymatic reactions were analyzed by HPLC (Merck-
Hitachi L-7100) on a 150 mm 2.1 mm i.d. reversed-phase C8
column (Merck, Darmstadt, Germany) thermostated at 25 C;
detection was carried out at 520 nm using a diode array detec-
tor (Merck-Hitachi L-7450A). Solvents were (A) water/formic
acid 9 : 1 (v : v) and (B) acetonitrile/formic acid 9 : 1 (v : v), with
the following gradient: 020% B over 5 min, 20100% over
10 min and isocratic 100% B for 15 min at a flow rate of
0.4 mL min1. The sample injection volume was 20 L. The
chromatographic column was washed with 100% B for 10 min
and then stabilized with the initial conditions for another
10 min.
For the analysis of anthocyanins from the wine extract a
reversed-phase C18 column (150 mm 4.6 mm i.d.) was used.
Solvents were (A) water/formic acid 9 : 1 (v : v) and (B) aceto-
nitrile, with the following gradient: 1035% B over 50 min at a
flow rate of 0.5 mL min1.
The semi-preparative HPLC system (Elite LaChrom) was
composed of an L-2130 quaternary pump, a manual injector
with a loop of 500 L and an L-2420 UV-vis detector. The
stationary phase was composed of a reversed-phase C8 column
(Merck, Darmstadt, Germany) (150 mm 2.1 mm i.d.) and the
eluents were (A) water/formic acid 9 : 1 (v : v) and (B) aceto-
nitrile/formic acid 9 : 1 (v : v), with the following gradient:
020% B over 5 min, 20100% over 10 min and isocratic 100%
B for 15 min at a flow rate of 0.4 mL min1. The detection
wavelength was set to 520 nm.
LC-DAD/ESI-MS
LC-DAD/ESI-MS analyses were performed on a Finnigan Sur-
veyor series liquid chromatograph equipped with a Finnigan
LCQ DECA XP MAX (Finnigan Corp., San Jose, Calif., USA)
mass detector and an atmospheric pressure ionization (API)
source using an electrospray ionization (ESI) interface. The
samples were analyzed on reversed-phase columns (150 mm
2.1 mm, 5 m, C8 and 150 mm 4.6 mm, 5 m, C18) thermo-
stated at 25 C using the same solvents, gradients, injection
volume and flow rate mentioned above for HPLC analyses.
Double-online detection was carried out by using a photodiode
spectrophotometer and mass spectrometer. The vaporizer and
Food Funct., 2016, 7, 27542762 | 2755

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the capillary voltages were 5 kV and 4 V, respectively. The capil-
lary temperature was set at 325 C. Nitrogen was used both as
sheath and auxiliary gas at flow rates of 90 and 25, respectively
(in arbitrary units). Spectra were recorded in positive and nega-
tive ion modes between m/z 250 and 1500. The mass spectro-
meter was programmed to perform a series of three scans: a
full mass (MS), a zoom scan of the most intense ion in the
first scan (MS2), and a MS-MS of the most intense ion using a
relative collision energy of 30 and 60 (MS3).
NMR
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1
H NMR (600.13 MHz) and 13C NMR (125.77 MHz) spectra
were recorded in DMSO/TFA (9 : 1) on a Bruker-Avance 600
spectrometer at 303 K and with TMS as an internal standard
(chemical shifts () in parts per million, coupling constants ( J)
in hertz). 1H chemical shifts were assigned using the 2D NMR
(COSY) experiment while 13C resonances were assigned using
2D NMR techniques (gHMBC and gHSQC). The delay for the
long range C/H coupling constant was optimized to 7 Hz.
Radical scavenging activity (DPPH)
Following the method described in the literature (Bondet,
Brand-Williams, & Berset, 1997) with some modifications,
radical scavenging activity was determined using DPPH as the
free radical. In a 96-well micro plate, 30 L of antioxidant
(1000 M dissolved in methanol/dimethylsulfoxide) was added
to 270 L of radical DPPH (60 M in methanol). The reaction
was carried out at 25 C, and the decrease in absorbance was
measured at 515 nm, at 0 and 20 min in a microplate reader
(Biotek Instruments Inc., Powerwave XS, Winooski, USA). The
antiradical activity was expressed in micromolar Trolox
equivalents, determined using a calibration curve of Trolox
(2.550 M).
Ferric reducing antioxidant power (FRAP)
The FRAP method was developed to measure the ferric redu-
cing ability of plasma at low pH (Benzie & Strain, 1996). An
intense blue color is formed when the ferric (Fe3+)-tripyridyl-
triazine complex is reduced to the ferrous (Fe2+) complex. In a
96-well micro-plate, 30 L of antioxidant (1000 M dissolved in
methanol/dimethylsulfoxide) was added to 270 L of FRAP
reagent [(10 volumes of 300 mM acetate buer, pH 3.6 + 1
volume of 10 mM TPTZ in 40 mM HCl + 1 volume of 20 mM
FeCl3), diluted to one-third with acetate buer and preheated
at 37 C]. The blank assay was performed using 270 L of the
FRAP reagent and 30 L of methanol. The reaction was per-
formed at 37 C, and the absorbance was measured at 0 and
4 min at 593 nm in a microplate reader (Biotek Instruments
Inc., Powerwave XS, Winooski, USA). The reducing power was
expressed in micromolar Trolox equivalents determined using
a calibration curve of Trolox (2.550 M).
Lipid peroxidation assays
Lipid peroxidation was evaluated using a linoleic acid model
constituted of 345 L of linoleic acid, 5 mL of ethanol, 4 mL of
potassium phosphate buer 50 mM ( pH 7), and 66.7 mg of
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AAPH, and the volume was adjusted to 10 mL with phosphate
buer. The samples containing the antioxidants (Mv3glc and
the Mv3glcoleic acid conjugate (Mv3glcOA)) were prepared
by addition of 50 L of a stock solution in methanol (2 mM). A
control containing no added sample represents 100% lipid
peroxidation. The samples and the control were left to react at
37 C over 5 days. From the mixtures 50 L was taken, and
added ethyl acetate (500 L), water (500 L), and 1,1,3,3-tetra-
ethoxypropane (0.83 M) as the internal standard. The organic
phase was extracted, dried over anhydrous sodium sulphate,
filtered (0.2 m filters) and analyzed by GC-MS. Quantification
of two oxidation products, caprylic acid and 2,4-decadienal, in
the samples and the control was performed from the respect-
ive standard curves. All the results were expressed as percent
antioxidant activity (%AO), defined as: %AO = (caprylic acid
value of the control caprylic acid value of the sample)/
caprylic acid value of the control 100.
The eect of Mv3glc and the respective Mv3glcOA conju-
gate on inhibition of lipid peroxidation was also determined
in the -carotene linoleate system according to the method
described in the literature.22 A variation of the above method,
in the absence of Tween 40, was also performed which
included the following: 400 L of -carotene (0.1 mg mL1)
and 4.4 L of linoleic acid were mixed and chloroform was
evaporated. To the flask were added 500 L of methanol and
4.5 mL of water and the solution was shaken vigorously. The
compounds (25 L, final concentration 200 M) and 225 L of
-carotene solution were added to a 96-well plate. The control
consisted of 25 L of methanol and the blank was performed
in the absence of linoleic acid. The experiments were per-
formed in quadruplicate. The samples were subjected to
thermal oxidation over 90 minutes at 50 C in a microplate
reader. The zero time absorbance was read at 470 nm immedi-
ately after the emulsion was added and after 90 minutes of
incubation. Inhibition of lipid peroxidation was calculated
according to the following equation: % inhibition = [1 (A0
At)/(A0 At)] 100, where A0 is the absorbance of the sample
at zero time, At is the absorbance of the sample after incu-
bation (90 min) at 50 C, A0 is the absorbance of the control at
zero time and At is the absorbance of the control after incu-
bation (90 min) at 50 C.
GC-MS
The samples were analyzed in a GC-MS system Thermo Scienti-
fic Trace 1300 equipped with an automatic liquid sampler (AI
1310 Thermo Scientific) and coupled to an ISQ Single Quadru-
pole mass detector. The GC was fitted with an apolar column
TG-5MS Thermo Scientific (60 m, 0.25 mm i.d., 0.25 m film
thickness) and helium was used as the carrier gas at 1.5 mL
min1. Injection was carried out in splitless mode with an
injection volume of 1 L and an injector temperature of
280 C. The column temperature was initially 90 C, then
increased to 150 C at 15 C min1, to 170 C at 5 C min1
and finally to 280 C at 20 C min1 and kept at this tempera-
ture for 5 min (total time 19 min). The solvent delay was
5 min. The detector was operated in electron impact ionization
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mode (electron energy 70 eV), scanning the 301000 m/z range.
The temperatures of the ion source and the interface were 230
and 280 C, respectively. Identification of the fatty acid deriva-
tives was based on the retention time of standard compounds
and with the assistance of the NIST 11 mass spectral library.
Results/discussion
Anthocyanin wine extract
The red wine extract was analyzed by HPLC-DAD and LC-DAD/
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enzyme-catalyzed esterifications is the water content in the
organic medium as it alters the thermodynamic equilibrium
of the reaction towards hydrolysis (excess of water) or synthesis
(adequate amount). Bearing this in mind, as the enzymatic
reaction releases water to the medium, the yield of the reaction
could be improved by adding activated molecular sieves 4 .
On the other hand, when the water content is too low, the
enzyme activity may be impaired.24,25 In the reactions
performed the variable conditions were the amount of lipase
and molecular sieves 4 , the oleic acid/anthocyanin molar
ratio, the concentration and the time of reaction. The highest

ESI-MS in the positive ion mode and confirmed that Mv3glc
represents the main anthocyanin present. The other minor
peaks correspond to the following anthocyanins: delphinidin-
3-glucoside (Dp3glc), petunidin-3-glucoside (Pt3glc), peonidin-
3-glucoside (Peo3glc) and the corresponding acetyl derivative
of Mv3glc (Mv3actglc).
Acylation reaction
Several attempts for the acylation of the Mv3glc-rich wine
extract with oleic acid catalyzed by lipase B were performed
(Scheme 1 and Table 1). All reactions were performed in an-
hydrous 2-methyl-2-butanol and at 60 C as frequently reported
in the literature.12 The solvent and the temperature chosen
allowed the best enzyme activity, good solubility of substrates
and high conversion yields. The enzyme used (lipase B) was in
the immobilized form which improves its stability, facilitates
product isolation and enables enzyme reuse.23 Another crucial
factor that aects the performance and regioselectivity of the
conversion yield was obtained for reaction 1 (Table 1).
The progress of the reactions was followed by HPLC-DAD
and the formation of a new peak around 16 min was detected
(Fig. 1). The residue was analyzed by LC-DAD/ESI-MS in posi-
tive ion mode and its identification was performed bearing
their molecular ions and related fragments (Fig. 2). The new
peak was identified as corresponding to a Mv3glc molecule
attached to an oleic acid moiety according to its full MS spec-
trum, [M]+ at m/z 757. In addition, the Mv3glcoleic acid con-
jugate revealed an MS2 fragment, [M 426]+ at m/z 331, which
is in agreement with the Mv3glc aglycone structure (loss of the
glucose residue attached to the oleic acid group). This means
that acylation of Mv3glc occurred preferentially in the OH
groups of the glucose moiety instead of any hydroxyl groups
from the flavylium core. The third fragmentation corresponds
mainly to the losses of methyl groups of ring B, [M 426
16]+ at m/z 315 and [M 426 32]+ at m/z 299. Smaller peaks
corresponding to other anthocyanins were also detected

Scheme 1 The reaction scheme of enzymatic esterication of the malvidin-3-glucoside-rich wine extract.

Table 1 Reaction conditions of enzymatic lipophilization of the Mv3glc extract with oleic acid

Extract Oleic acid Volume Lipase Molecular sieves 4 Conversion

Reaction (mg) (eq.) (mL) (g L1) (g L1) t (h) (%)

1 5 100 2.5 20 100 48 21.2

2 5 100 2.5 10 100 25 5.6
3 5 100 2.5 40 100 25 2.1
4 5 100 2.5 20 200 23 4.8
5 5 200 2.5 20 100 23 3.2
6 28 100 10 20 100 45 1.9

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Fig. 1 (a) HPLC chromatogram of the enzymatic esterication after 48 h (reaction 1). (b) UVvis spectra of Mv3glc and the Mv3glcOA conjugate.
Fig. 2 MS spectra of the Mv3glcOA conjugate (full MS, MS2, and MS3) and respective fragmentation patterns recorded in positive ion mode.
namely Pt3glc ([M]+ at m/z 743, [M 426]+ at m/z 317, [M
426 15]+ at m/z 302) and Peo3glcoleic acid conjugates ([M]+
at m/z 727).
The novel compound maintained the attractive colour of
the native Mv3glc. However, the native max revealed a hypo-
chromic and bathochromic shift and the maximum absorption
is now located in the UV region (max 283 nm) (Fig. 1b).
NMR
The new compound was purified by semi-preparative HPLC and
Toyopearl gel column chromatography, and thereafter stru-
cturally characterized by NMR spectroscopy. The structure of
the Mv3glcoleic acid conjugate was fully confirmed by 1H and
13
C NMR assignment using 1D and 2D NMR techniques (COSY,
HSQC and HMBC) in DMSO/TFA 9 : 1 (Table 2).
The enzymatic reaction yielded only a monoester and the
regioselectivity of the reaction was constant, showing that acy-
lation always took place on the primary hydroxyl group present
on the glycoside moiety of the molecule (C6-OH). The location
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of the acylated site has been well documented in previous studies
for a range of acyl donors.11,12,2628 This was confirmed by com-
paring the downfield and upfield shifts between the Mv3glcoleic
acid ester and the reported catechinMvglc dimer adduct in the
same solvent.29 13C NMR spectra showed that the acylation on the
6-OH position led to a downfield of the C-6 signal by approxi-
mately 3 ppm (60.9 to 63.9 ppm) due to the resonance eect
towards the carbonyl of the newly generated ester.
Antioxidant features
Some antioxidant features of the novel Mv3glc derivative
obtained as well as its precursor were assessed by means of
in vitro techniques, namely DPPH, FRAP and lipid peroxidation.
DPPH and FRAP. The antiradical capacity and reducing
power of the Mv3glcOA conjugate was revealed to have a
statistically little decrease compared to the native Mv3glc prob-
ably due to the increase of the structural complexity of the new
compounds arising from the long carbon chain of oleic acid
attached to glucose (Fig. 3). Overall, the introduction of an
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Table 2 1H and 13
C NMR data of the Mv3glcOA conjugate in DMSO/ lations would be facilitated because of the higher solubility
TFA (9 : 1)a and stability of these compounds.
Lipid peroxidation. In order to evaluate the antioxidant
capacity of the synthesized Mv3glcOA adduct in a more lipo-
philic system, a lipid peroxidation assay was performed using
linoleic acid as the lipidic substrate. The extent of the reaction
was quantified by measuring the area of the two products
resulting from linoleic acid oxidation, caprylic acid and 2,4-
decadienal.31 The chromatograms in Fig. 4 represent three of
the tested samples after 5 days of incubation, namely the reac-
tion control (absence of antioxidant), Mv3glcOA (antioxidant
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and lipidic substract) and Mv3glcOA blank (absence of lipidic

substract).
No peaks were observed in the blank sample as expected,
which confirms the stability of the conjugate, as no free oleic
acid or oleic acid degradation products were observed. On the
other hand, in the control sample, containing only linoleic
acid, the presence of linoleic acid degradation products was
Position (1H); J (Hz) (13C) HMBC HSQC
high (caprylic acid and 2,4-decadienal isomers). In the sample
2C 161.4 H-4C, H-2,6 containing Mv3glcOA, the formation of these products was
B lower, which confirmed the protection of the lipidic substrate
3C 144.0 H-4C
4C 8.85; s 135.2 H-4C from peroxidation.
4aA 112.3 H-6A The results of lipid peroxidation were expressed in function
5A 158.3 H-4C of the area of caprylic acid and 2,4-decadienal isomers (Fig. 5).
6A 6.73; s 102.7 H-6A
7A 158.0 H-6A, H-8A Considering the quantification by caprylic acid no statistical
8A 7.06; s 94.9 H-8A dierences were observed between the control, native Mv3glc
8aA 156.3 H-4C and Mv3glcOA conjugate. However, for 2,4-decadienal
1B 118.4 H-2,6B
2,6B 7.95; s 109.6 H-2,6B isomers it was possible to observe the statistical dierences
3B 145.0 H-2,6B between the control and the samples containing Mv3glc and
4B 148.3 H-2,6B the Mv3glcOA conjugate. Indeed, these results confirm once
5B 145.0 H-2,6B
OCH3 3.91; s 56.6 CH3 again that the introduction of oleic acid in the native Mv3glc
did not aect its antioxidant capacity.
Glucose The antioxidant capacity of the synthesized Mv3glcOA
1 5.43; d, 7.25 101.9 H-1
2 3.47; m 73.7 H-2 adduct was also assessed by using a dierent method, the
3 3.41; m 76.5 H-3 -carotene bleaching assay. Two dierent approaches were con-
4 3.21; m 70.3 H-4 ducted in the presence and in the absence of a surfactant
5 3.77; m 74.6 H-5
6a 4.32; m 63.9 H-6a (Tween 40) in the system (Fig. 6).
6b 4.09; m 63.9 H-6b In the presence of Tween 40, the antioxidant eect of
Mv3glc and Mv3glcOA was superimposed, which reflects that
Oleic acid
1 174.6 H-2, H-3 the lipophilic residue of the new conjugate does not aect the
910 CHvCH 5.3; m 129.8 H-9, H-10 antioxidant capacity of the anthocyanic precursor.
2 CH2 2.15; t, 7.4 33.8 H-2 Conversely, in the absence of Tween 40, no oxidation of -car-
8/11 (CH2)2 1.96; m 26.8 H-8, H-11
3 CH2 1.46; q 24.6 H-3 otene was observed in the control and in the presence of Mv3glc
47/1217 1.171.32; 29.1 H-4H-7/H12 OA. In this system, Mv3glc appears to act as a pro-oxidant being
(CH2)10 m H17 responsible for the oxidation of the -carotene, which was
18 CH3 0.83; t, 6.9 14.0 H-18
detected by the decrease of the absorbance intensity at 470 nm.
a
Key: s, singlet; m, multiplet; d, doublet; t, triplet; q, quintuplet. In the presence of Tween 40, Mv3glc and the Mv3glcOA
conjugate have the same solubilization in the oil-in-water
emulsion which explains the same antioxidant activity of both
compounds. In the system without Tween 40, the Mv3glcOA
oleic acid group on the Mv3glc molecule did not fully compro- conjugate showed a higher protection of lipidic substrate oxi-
mise its antioxidant activity. Besides, these derivatives are dation than Mv3glc which could be attributed to the amphi-
more liposoluble which would facilitate their transport to cell philic behaviour of the new Mv3glcOA conjugate possibly
targets, increasing their bioavailability and consequently their acting as a surfactant of the system (Fig. 6).
positive biological eects.15,16,30 Furthermore, their techno- This result is quite interesting since the introduction of the
logical application in foods and in cosmetic lipophilic formu- oleic acid in the anthocyanin moiety not only maintains the

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Fig. 3 Radical scavenging activity (DPPH) and ferric reducing antioxidant power (FRAP) of 100 M Mv3glc and the Mv3glcOA conjugate. Columns
represent mean standard deviation. Columns with the same letter do not dier statistically (*p < 0.05).

Fig. 4 Chromatogram representation of control, Mv3glcOA and Mv3glcOA blank samples after the lipid peroxidation reaction.

Fig. 5 Quantication of caprylic acid and 2,4-decadienal isomers in the

control, Mv3glc and Mv3glcOA samples by GC-MS. All reactions were
performed in triplicate. No oxidation products were detected in the
compound blank samples. *Signicantly dierent from control P < 0.05.

antioxidant capacity of the native molecule (evidenced by

this and the other antioxidant tests), but also reduces the
pro-oxidant activity of the anthocyanin, possibly by the more
stabilized structure, unable to form radicals (quinones). Pro-
oxidant activity is thought to be directly proportional to the
total number of hydroxyl groups in a flavonoid molecule.32
Multiple hydroxyl groups, especially in the B-ring, significantly Fig. 6 -Carotene bleaching assays of 200 M of Mv3glc and the
increased the production of hydroxyl radicals in the Fenton Mv3glcOA conjugate. All reactions were performed in quadruplicate.
reaction.32 Although all the anthocyanin monoglucosides (a) With Tween 40 and (b) without Tween 40.

2760 | Food Funct., 2016, 7, 27542762 This journal is The Royal Society of Chemistry 2016

Food & Function
including Mv3glc showed pro-oxidant activity of human LDL
even at 2.5 M.33
Conclusions
This work reports the lipophilization of malvidin-3-glucoside
with oleic acid by enzymatic catalysis esterification. This pro-
cedure proceeded selectively yielding only one ester derivative.
To the best of our knowledge, this is the first time that struc-
tural modification of a pure natural anthocyanin with a fatty
Published on 13 May 2016. Downloaded by FAC DE QUIMICA on 17/04/2017 20:06:30.
acid by an enzymatic approach is described as well as its full
NMR structural characterization. The novel compound pre-
served its attractive chromatic features (red-violet colour) as
well as its antioxidant activity. These structural transform-
ations of anthocyanins to more lipophilic derivatives have the
main advantage of increasing or improving their technological
application potential in lipophilic systems such as fats, oils,
lipid-based food or cosmetic formulations. In the future, it is
intended to optimize the yields of the enzymatic synthesis in
order to obtain significant amounts of novel compounds to
proceed to the next stages which will include transepithelial
studies using cell barrier models, biological studies, design of
formulations for incorporating the compounds and stability
tests.
Acknowledgements
The authors thank Dr Zlia Azevedo for the MS analysis and
Dr Mariana Andrade for the NMR analysis. This research was
supported by a research project grant (PTDC/AGR-TEC/3078/
2014) with financial support from FCT/MEC through national
funds and co-financed by FEDER, under the Partnership
Agreement PT2020 (UID/QUI/50006/2013 POCI/01/0145/
FEDER/007265). Lus Cruz and Iva Fernandes gratefully
acknowledge the Post.Doc.Grants from FCT (SFRH/BPD/72652/
2010 and SFRH/BPD/86173/2012, respectively).
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This journal is The Royal Society of Chemistry 2016