Optimizing the efficiency of antioxidants in emulsions by lipophilization: Tuning interfacial concentrations

Article abstract of DOI:10.1039/c6ra18282h

Optimization of the efficiency of antioxidants, AOs, in lipid-based emulsions via chemical modifications of their reactive moieties is not always possible because of the inherent experimental difficulties and because of the regulatory status of AOs. Esterification of hydrophilic AOs may be a practical, convenient, alternative approach. Here we employed a series of caffeic acid derivatives bearing the same reactive moiety but of different hydrophobicity (alkyl chain lengths of 1 to 16 carbon atoms) to investigate the effects of hydrophobicity on the oxidative stability of stripped corn oil-in-water emulsions. AO efficiency was determined by monitoring the production of primary oxidation products (conjugated dienes) with time and a non-linear, parabolic-like, variation of their efficiency with the number of C atoms in their alkyl chain, with a maximum at the C₈ derivative, found. To rationalize the results, we also determined the distribution of the AOs between the oil, interfacial and aqueous regions of the same emulsions by employing a recently developed kinetic method that provides the partition constants of the AO between the oil-interfacial, PIO, and water-interfacial, PIW, regions of the intact emulsions. Values of both PIO and PIW range between 180-2000, suggesting that the transfer of the AOs to the interfacial region is spontaneous. The results indicate that the variations of both the percentage of AO in the interfacial region, % AO_I, of the emulsions and the AO efficiency with the number of C atoms in the AO alkyl chain parallel each other with a maxima at the C₈ derivative. The results illustrate an effective and convenient way to control lipid oxidation by modulation of the hydrophobicity (HLB) of the AOs. An increase in the alkyl chain length of the AOs promote their incorporation into the interfacial region of emulsions but only up to a critical chain length, after which a further increase makes their efficiency decrease as a consequence of the decrease in their % AO_I.

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Optimizing the efficiency of antioxidants in emulsions by
lipophilization: tuning interfacial concentrations
Received 00th January 20xx,
Accepted 00th January 20xx
Marlene Costa,a* Sonia Losada-Barreiro,a,b Fátima Paiva-Martins,a Carlos Bravo-Díazb
Optimization of the efficiency of antioxidants, AOs, in lipid-based emulsions via chemical modifications of their reactive
moieties is not always possible because of the inherent experimental difficulties and because of the regulatory status of
AOs. Esterification of hydrophilic AOs may be a practical, convenient, alternative approach. Here we employed a series of
caffeic acid derivatives bearing the same reactive moiety but of different hydrophobicity (alkyl chain lengths of 1 to 16
carbon atoms) to investigate the effects of hydrophobicity on the oxidative stability of stripped corn oil-in-water
emulsions. AO efficiency was determined by monitoring the production of primary oxidation products (conjugated dienes)
with time and a non-linear, parabolic-like, variation of their efficiency with the number of C atoms in their alkyl chain, with
a maximum at the C8 derivative, was found. To rationalize the results, we also determined the distribution of the AOs
between the oil, interfacial and aqueous regions of the same emulsions by employing a recently developed kinetic method
DOI: 10.1039/x0xx00000x
www.rsc.org/
I
that provides the partition constants of the AO between the oil-interfacial, PO , and water-interfacial, PWI, regions of the
I
I
intact emulsions. Values of both PO and PW range 180-2000, suggesting that the transfer of the AOs to the interfacial
region is spontaneous. Results indicate that the variations of both the percentage of AO in the interfacial region, %AOI, of
the emulsions and the AO efficiency with the number of C atoms in the AO alkyl chain parallel each other with a maxima at
the C8 derivative. Results illustrate an effective and convenient way to control lipid oxidation by modulation of the
hydrophobicity (HLB) of the AOs. An increase in the alkyl chain length of the AOs promote their incorporation into the
interfacial region of emulsions but only up to a critical chain length, after which a further increase makes their efficiency to
decrease as a consequence of the decrease in their %AOI.
release them under specific conditions, the control of the
concentrations of oxygen or radical initiators in the vicinity of the
Introduction
lipids and the addition of antioxidants (AOs). The latter is probably
Consumer demands for healthy foods lead to the preparation of
functional foods containing bioactive compounds such as omega-3,
6 and 9 polyunsaturated fatty acids (PUFAs), which are added as
ingredients with well-recognized positive health benefits.1-4
However, food manufacturers must face numerous challenges
during the production, transportation and storage of fortified foods
because PUFAs are extremely susceptible to oxidative degradation.
This undesirable (radical) reaction leads to the development of off-
flavors, loss of nutrients and other bioactive molecules and,
eventually, to the formation of potentially toxic compounds, making
the foods to be either unsuitable for consumption or rejected by
consumers.5, 6 It is not surprising therefore, the continuous efforts
to developing novel methods to control lipid oxidation aiming at
retarding the effects of free radicals damage in various food
products as well as in the human body. 7-11
13
the most effective, convenient and economical method.12, The
efficiency of AOs in inhibiting lipid oxidation depends on several
factors including the nature of the AO and their concentration at
the reaction site.7, 11, 14, 15 The molecular structure of phenolic AOs
(position and number of hydroxyl or other substituents on their
aromatic ring) has a considerable effect on their antioxidant
properties.3, 5 For instance, the efficiency of phenolic AOs increases
with the number of hydroxyl groups in their structure, so that the
efficiency of phenolic AOs with only one hydroxyl group (e.g., 3- or
4-hydroxybenzoic acids) is much lower than that of AOs bearing two
hydroxyl groups, e.g., caffeic (3,4-dihydroxycinnamic) or
protocatechuic (3,4-dihydroxybenzoic ) acids, and much lower than
that of phenolic AOs bearing three hydroxyl groups, e.g, gallic
(3,4,5- trihydroxybenzoic) acid. The position of the hydroxyl groups
on the aromatic ring is also relevant because strongly affects the
stability of the resulting radicals. Phenolic AOs with –OH groups in
the ortho or para positions are much more efficient those with the
–OH groups in meta. Insertion of methylene (3,4-
dihydroxyphenylacetic acid) or ethylene groups (caffeic acid)
between the phenyl ring and the carboxylic group also has
significant effects on their antioxidant activity as a consequences of
Improvement of the oxidative stability of the lipids can be achieved
by several methods including the encapsulation of the lipids to
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the different resonance stabilization and conformation of the
Journal Name
17
molecules.16,
For example, the esters of caffeic acid and
hydroxytyrosol have different reactivity because the restrictions in
the rotation of the phenyl group due to the double (caffeic) or
single (hydroxytyrosol) bond connecting the phenyl and alkyl
groups.
However, chemical modifications of the reactive moieties to
modulate their efficiency are not always possible because of the
inherent experimental difficulties and because of the regulatory
Scheme 1. Structure of the caffeic acid derivatives employed in this
status of AOS cannot be ignored when selecting antioxidants for use
18, 19
work
in particular foods.
An alternative approach to modulate the
AO efficiency is to use of potent, natural, antioxidants and modify
their hydrophobicity so that their reactive moiety is maintained but
their HLB is chemically modified by insertion of, for instance, alkyl
chains of different length.20 This strategy exploits the changes in
their relative solubility in the distinct regions of the emulsions as a
consequence of the changes in their hydrophilic-lipophilic balance
The relative importance of each contribution cannot be easily
established, and thus the structure-efficiency relationships between
need to be determined for each oil and set of homologous AOs
under the same experimental conditions. Results are relevant to the
food industry because of the economic importance of corn oil–
based products and can provide useful information for the
design/availability of new AOs radical scavenging properties.29
(HLB) of the AOs, leading to changes in their concentrations at the
22
reaction site (the interfacial region of emulsions).21,
Thus, AO
efficiency can be improved by increasing their concentrations at the
reaction site while maintaining their antioxidant properties. This
approach represents the aim of the present study in which we have
investigated the antioxidant efficiency and the distribution of a
series of n-alkyl caffeates in a model food emulsion composed of
corn oil, water and the nonionic surfactant Tween 20.
Experimental Section
Materials
Caffeic acid (Across Organics), the emulsifier Tween 20
(polyoxyethylene (20) sorbitan monolaurate, Fluka), stripped corn
oil (Across Organics, d = 0.918 g cm-3) were of the highest purity
We chose caffeic acid, CA, because CA attracts more interest than
synthetic antioxidants as a consequence of its broad spectrum of
biological activities, including antioxidative, antimicrobial and
antitumor activities.23-25 The study was performed in an attempting
to define an optimum chain length of the molecules to achieve the
maximum antioxidant efficiency by increasing the percentage the
AO in the interfacial region of the emulsions. The results reported
are the first - to our knowledge – obtained by employing stripped
corn oil emulsions and shed some light on the complex structure-
activity relationships governing the efficiency of AOs in multiphasic
systems. Previous experiments on the effects of the HLB of the
emulsifier and of AOs were carried out in different oils (octane,
soybean, olive),21, 26-28 but extrapolation to other oils, different oil-
to-water ratios or environmental conditions (pH, T, etc.) cannot be
done because the fatty composition of oils is different and the
distribution of AOs between the different phases or regions
depends on both the differences in solvation of the solutes by
solvent molecules or emulsifiers and on the capabilities of solutes
of intra- and intermolecular hydrogen bonding with solvent.
available and used as received.
Aqueous buffered (citric
acid/citrate 0.04 M, pH = 3.6) solutions were employed in the
preparation of emulsions. The chemical probe, 4-n-
hexadecylbenzenediazonium tetrafluoroborate, 16-ArN2BF4, was
prepared in high yield and purity from commercial 4-n-
hexadecylaniline (Aldrich 97%) by diazotization following
a
published method.14 2,2 Diphenyl-1-picrylhydrazyl (DPPH●) and
reagents employed in the preparation of CA esters (bezaldehyde,
Meldrum´s acid, β-alanine and pyridine) were purchased from Acros
Organics or Sigma-Aldrich and used as received. Milli-Q grade water
(conductivity < 0.1 mS cm-1) was used in all experiments. Thin layer
chromatography (TLC) analyses were performed on aluminum silica
gel sheets 60 F254 plates (Merck, Darmstadt, Germany) and spots
were detected by using a UV lamp at 254 nm after treatment with
iodine.
Scheme 2 summarizes the synthetic route employed for the
preparation of the CA esters according to published procedure.21, 30
The C1–C16 derivatives were synthesized from manomalonates
through Verley-Doebner modification of Knoevenagel condensation
with the 3,4-dihydroxybenzaldehyde. In
a
typical experiment,
mixture of the
Meldrum’s acid (0.4 M) was added to
a
corresponding alcohol (0.4 M) and 5 mL toluene in a dry round
bottom flask and stirred for 4 hours.
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volume). Each sample was vortexed for 1 min and thermostated at
37 ºC by employing an orbital Incubator Heidolph 1000 orbital
stirrer equipped with a Heidolph thermostat 1010 to control
temperature (±1 ºC). Aliquots (50 ꢀl) of each emulsion were diluted
to 10 ml with ethanol, producing a homogeneous solution, and the
Scheme 2. Synthetic route employed to prepare CA esters of
absorbance at 233 nm was measured.
different alkyl chain lengths.
O
Determining the partition constant of
in binary oil-water
PW
O
The reaction mixture was allowed to reach room temperature and
10 mL of toluene was added. The mixture was extracted with
aqueous 0.6 M Na2CO3 (3 x 30 mL). The combined aqueous solution
was neutralized with HCl 1 M, was extracted with diethyl ether (3 x
30 mL) and dried over Na2SO4. The solvent was evaporated and the
mixtures in the absence of emulsifier.The partition constants, PW
,
of CA and its esters in binary oil-water mixtures, equation 1, were
determined in binary stripped corn oil–water mixtures in the
absence of emulsifier by employing a shake-flask method.28, 33 For
each AO, a number of binary mixtures were prepared by stirring at
high speed for one minute a mixture of 4 mL of stripped corn oil
and 6 mL of aqueous buffer (0.04 M citrate buffer. pH = 3.65).
Caffeic acid and the most polar derivatives, C1 – C3, were dissolved
in the aqueous solution and the more hydrophobic derivatives, C4 -
C16, were dissolved in the corn oil.
obtained product (1g) was added to
a mixture of 3,4-
dihydroxybenzaldehyde (1g), β-alanina (37mg) and 2.5 mL pyridine
in a dry round bottom flask and stirred 24 hours at T = 60ºC. The
product was purified by flash column chromatography over silica
gel using ethyl acetate/petroleum ether as the eluent (1:1, v:v).
Final yields were > 90% for all derivatives. 1H and 13C NMR spectra
for CA and its alkyl esters agree with literature results.30
(AOO ) %AOO VW
PO =
=
W
(AOW ) %AOW VO
Methods
The final AO concentration in the total 10 mL emulsion volume was
3.5 mM in all samples. The mixtures were gently shaken for at least
1 minute and allowed to equilibrate at T = (25 ± 1) ºC for at least 24
hours. The two phases were separated by centrifugation, and the
concentrations of the AO in the aqueous and in the oil phases were
Preparation of Emulsions. Emulsions of different oil to water ratios
(4:6 and 1:9 v:v, VT = 10 mL) were prepared from stripped corn oil,
acidic water (0.04 M citrate buffer, pH 3.65) and Tween 20 as
emulsifier (0.5 - 4% w/w). Before homogenization, hydrophilic AOs
(C1 - C3) were added to the aqueous buffer and hydrophobic AOs (≥
C4) were added to the oil phase. The mixtures were stirred at high
speed at room temperature with the aid of a Polytronic PT-1600
homogenizer for 1 min and the resulting emulsions were visually
stable for at least 12 hours, a much longer time than that required
to complete the chemical reaction between 16-ArN2+ and the AOs,
which is typically < 30 min for 3-4 half-lives of reaction (see below).
determined spectrometrically (UV-Vis) at
λ = 320 nm by
O
interpolation using previously prepared calibration curves. Each PW
value is an average of duplicate or triplicate measurements. The
results are summarized in Table 1. Complete experimental details
are published elsewhere28, 34
O
I
I
AO
CA
C1
PW
PW
PO
102 kI (M-1s-1)
3.52 ± 0.3
5.95 ± 0.3
6.00 ± 0.2
5.60 ± 0.1
5.30 ± 0.3
5.31 ± 0.2
Determination of the antioxidant efficiency in emulsions:
oxidative stability. The oxidative stability of food lipids is usually
estimated by determining the extent of oxidation under a set of
standardized conditions.5 In previous works, we estimated the
efficiency of the AOs by measuring the degree of oxidation from the
production of conjugated dienes (CD) with time and here we used
the same method because it is a sensitive and reproducible (and
identical with the AOCS official standard method31 Ti-1a-64) for
following the early stages of lipid oxidation and for the sake of
0.04 ± 0.01
3.3 ± 0.3
11 ± 0.4
268 ± 10
747 ± 44
2000 ± 130
---
---
226 ± 25
188 ± 13
177 ± 25
513 ± 27
385 ± 1
C2
C3
38 ± 3
C8
651 ± 55
1200 ± 150
---
comparisons with previous results with other AOs and oils.21 In
C16
---
32
addition, we have shown in previous work22,
that monitoring
primary and secondary products under similar conditions to those
used here lead to the same conclusions. No visual phase separation
was observed in the emulsions and the small increase constant up
to sudden increase in the formation of conjugated dienes suggests
that phase separation, if any, does not have a significant effect on
the kinetics of the reaction.
Table 1. Values of the partition constant in binary oil-water
systems in the absence of emulsifier, PWO, the partition constants
I
between the oil-interfacial, PO , and water-interfacial, PWI, regions
and the rate constant in the interfacial region, kI,, for CA and alkyl
caffeates.
4:6 (v:v) oil-in-water emulsions (VT = 10 mL) were prepared as
above in the presence and absence of AOs (final AO concentration
of 0.6 mM) and transferred to screw–capped sample vials (25 mL
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Determining the partition constants by employing the
pseudophase kinetic model: Determination of kobs values for the
+
reaction between 16-ArN2 and the AOs in intact emulsions. The
+
reaction between 16-ArN2 and the CA and its esters was followed
spectrometrically by employing the dye derivatization method (azo
32, 34 14
dye formation) described in detail elsewhere.28,
The
+
methodology exploits the rapid reaction of 16-ArN2 ions with a
suitable coupling agent such as N-(1-naphthyl)ethylenediamine
dihydrochloride, NED, yielding an stable azo dye. Its absorbance
was measured spectrometrically at
λ = 572 nm after dilution with a
50:50 (v:v) BuOH:EtOH mixture that was an optically transparent,
homogeneous solution.
In a typical experiment, a freshly prepared emulsion (10 mL)
containing the required volume of AO stock solution is placed in a
continuously stirred, water-jacketed cell (T
= 25 ºC) and
thermostated for at least 15 min. Independently, 25 numbered and
stoppered test tubes were placed in a thermostated bath (T = 25 ºC)
and 2.5 mL of a 0.02 M EtOH–BuOH (50:50 v:v) solution of NED
were added to each test tube and allowed to reach thermal
equilibrium for at least 20 min. After the reaction is initiated,
aliquots (200 µL) of the reaction mixture were removed at specific
time intervals and added immediately to test tubes to quench the
reaction by initiating azo dye formation between NED and
+
unreacted 16-ArN2 . The absorbance of the azo dye at
λ = 572 nm is
+
proportional to the concentration of unreacted 16-ArN2 and the
decrease in absorbance with time was used to determine the
observed first order rate constant, kobs. Values of kobs were obtained
by fitting the absorbance vs time data to the integrated first order
rate equation by using a non-linear least squares method provided
by the GraFit 5.0.5 computer program.
+
Figure 1. Typical kinetic plots obtained for the reaction of 16-ArN2
with the C1 (A) and C16 (B) CA derivatives in 4:6 corn oil-in-water
emulsions composed of acidic water (citric/citrate buffer, 0.04 M,
+
pH = 3.65) and Tween 20 (ΦI = 0.0284), [16-ArN2 ]
≈
2.9 x 10-4 M,
[AO] = 3 x 10-3 M, T = 25 ºC.
Figure 1 shows examples of absorbance versus time plots for azo
dye formation and the fitting curves to the integrated and
+
(AOI )
linearized first-order equations for the reactions of 16-ArN2 with
I
PW
=
(2)
the C1 and C16 derivatives in 4:6 corn oil-in-water emulsions. The
excellent fits of the (A, t) data pairs to the first-order kinetic
equation demonstrate that any changes in droplet size are not
kinetically significant.14, 22, 32 The results also show that diffusion of
reactants is not rate-limiting because if it was, we would not get
good first order kinetics.35
(AOW )
(AOI )
(AOO )
PI =
(3)
O
PWI (AOI )/(AOW ) (AOO )
PI (AOI )/(AOO ) (AOW )
=
=
= PWO (4)
Kinetic analyses of data: application of the pseudophase kinetic
model to determine the interfacial rate constants and the
partition constants of the AOs between the oil-interfacial and
aqueous-interfacial regions of the emulsions. Addition of a
surfactant to produce a kinetically stable emulsion creates a new
interfacial region between the oil and water regions, and the
O
In pseudophase models emulsions are divided into three reaction
regions, the oil, interfacial and aqueous regions, Scheme 3. The
volume fraction of each region is determined by the volumes of
added oil, VO, surfactant, VI, and aqueous solution, VW, and is
defined as the ratio between the volume of the region and the total
distribution of AOs is now described by two partition constants,
I
Scheme 3, that between the aqueous and interfacial regions, PW
,
volume, e.g.,
Φ
I = VI / (VW + VI + VO).
I
and that between the oil and interfacial regions, PO , which are
defined by equations 2 and 3, respectively. Note that the value of
the ratio PWI / POI is equal to that of the partition constant between
the oil and water in the absence of surfactant, PWO, as shown in
equation 4.
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v = kobs[16ꢀArN+2T ] = k2[16−ArN2+T ][AOT ] =
= kI (16−ArN+2I )(AOI )ΦI
(5)
Equations 2 and 3 are combined with the relevant mass balance
equation to give the relationship between kobs and partition constant
values, equation 6,11 where subscripts W, O, and I stand for water, oil and
interfacial regions. The mathematical treatment can be simplified for
very hydrophilic and very hydrophobic AOs. For AOs that are oil
insoluble, i.e., that distribute between the aqueous and interfacial
I
region only the partition constant PW is needed to define its
distribution and the derivation of equation 6 simplifies to give
14
equation 7.11,
For water insoluble AOs, i.e., that distribute
between the oil and interfacial regions, only the partition constant
POI is needed and equation 6 simplifies to equation 8.
AO k PI PI
[
]
T
I
W
O
(6)
kobs
=
ΦO PWI +Φ PWI PI +ΦW PI
O
O
I
kI[AO]T PWI
ΦIPWI + ΦW
kobs =
(7)
kI[AO]T PI
O
kobs =
ΦI PI + ΦO
(8)
Scheme 3. Conceptual division of an emulsion droplet showing the
O
oil (O), interfacial (I) and aqueous (W) regions. The distribution of
I
Equations 6-8 predict that kobs values should decrease
asymptotically with increasing I. Equation 9 has the same form as
an AO between the regions is defined by the partition constants PO
I
Φ
and PW
,
Φ
stands for the volume fraction of a region and kI is the
+
the reciprocal of equation 6, where the parameters a and b are
given by equations 10 and 11, respectively. Equations 12 and 13
are the reciprocals of equations 7 and 8, respectively, and they
predict that plots of 1/kobs vs ΦI should be linear with positive
intercepts. Equations 9, 12 and 13 were employed to calculate the
rate constant for reaction between 16-ArN2 and the AO in the
interfacial region.
No visual breakdown of the emulsions was observed for at least 6
hours, a time much higher than that required to complete the
I
I
partition constants PO and PW and the rate constant kI in the
+
interfacial region as described elsewhere.11, 14
reaction between 16-ArN2 and AOs (t1/2 = 2-30 min). After bulk
mixing is complete, the distributions of reactants are determined by
their relative solubilities in the oil, interfacial and aqueous regions.
1
1
b
=
+ ΦI
a
+
(9)
16-ArN2 is located only in the interfacial region and oriented with
kobs
a
its tail in the hydrophobic core and its headgroup in the interfacial
14
region in contact with water,11,
where it reacts with the AO,
AO k PI PI 1+ Φ ΦO
[
]
(
)
T
I
W
O
W
Scheme 3. The chemical reaction does not significantly perturb the
distribution of the reactants because both reactants and emulsion
components are in dynamic equilibrium, and because the surfactant
and AO concentrations are in large excess with respect to that of
(10)
a =
PWI + ΦW ΦO PI
O
PI PI 1+ Φ ΦO
(
)
W
O
W
+
(11)
(12)
(13)
b =
−1
16-ArN2 . Thus, overall rate of the bimolecular reaction, v, in the
PI + ΦW ΦO PI
W
O
interfacial region of an emulsion, Scheme 3, depends only on the
measured (or observed) rate constant, kobs
,
and on the
concentrations of 16-ArN2 and antioxidant, AO, in the interfacial
region, equation 5, where square brackets, ], denote the
+
ΦW
1
1
=
+
ΦI
kobs kI[AOT ]PWI kI[AOT ]
[
concentration in molarity of total emulsion volume, parenthesis,
e.g., (AOI), stand for concentrations in moles per liter of interfacial
volume; kI (M-1s-1 ) is the second order interfacial rate constant and
ΦI the volume fraction of the interfacial region.
1
ΦO
1
=
+
ΦI
kobs kI[AO]TPOI kI[AO]T
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Once the partition constants are known, the distribution of the a certain time of reaction was determined as %DPPH● = 100[DPPH●
antioxidant can be determined as the percentage of the antioxidant (time = t)/ DPPH● (time = to)]. Second, the %DPPH● was plotted
in each region. Equations 14-16 were employed to calculate the against the mole AO/mol DPPH● ratio at selected times of reaction
fraction of AO in the interfacial region for AOs that distributes (5, 15, 30 and 60 min), Figure 2B. On these plots, the value of EC50
between the three regions and for those that distribute between is equal to the value the intersection of the line at %DPPH● = 50%.
the aqueous-interfacial and oil-interfacial regions, respectively. Representative plots are shown below. Each sets of experiments
Similar equations used to calculate the percentage of AO in the oil was performed in quadruplicate. The EC50 values given in Table 2
and aqueous regions were derived previously and are published are the average of these repetitions. Note that the variations in EC50
elsewhere.11, 14
times are small and virtually independent of AO chain length.
Effects of the alkyl chain length on the oxidative stability of corn
oil-in-water emulsions. The relative efficiency of CA and its esters
in 4:6 (v:v) corn oil-in-water emulsions was determined by
monitoring the formation of the primary oxidation products (
conjugated dienes) with time until a suitable end point is reached.
We run the experiments at low temperature (T = 37 ºC) to minimize
hydroperoxide side reactions (thermal decomposition) that may
difficult the interpretation of the results. Figure 3A illustrates a
typical kinetic plot obtained by monitoring the formation of
conjugated dienes, CDs, in corn oil-in-water emulsions showing the
initiation (very slow increase in the formation of CDs), propagation
(much faster increase where the scavenging antioxidant has been
consumed and does not inhibit the reaction) and termination steps
of the radical reaction. The oxidation level defined as the time
required to increase the content of conjugated dienes in 0.6% , a
time higher than that necessary to reach the propagation step both
in the presence and absence (control experiments) of AOs. All runs
were in triplicate. The uniformity of the induction period for
different AOs indicates minimal effects of phase separation on the
kinetics of oxidation because, otherwise, the values should have
shown more scatter.
100ΦIPWI PI
O
%AOI =
ΦOPWI + ΦIPWI PI + ΦWPI
(14)
(15)
(16)
O
O
100ΦIPWI
ΦIPWI + ΦW
%AOI =
%AOI =
100ΦIPI
O
ΦIPI + ΦO
O
Statistical Analysis.Duplicate or triplicate kinetic experiments were
run for 2-3 t1/2. The kobs values were within ± 7 – 9 % with typical
correlation coefficients of > 0.995. Reported partition constants in
binary oil-water systems are the average of 6 - 8 replicates. The
Dixon´s Q-test was employed in deciding whether to accept or
reject the datum before calculating the average of the set of
replicates. All oxidation experiments were
run in triplicate and SPSS 21.0 software was used for statistical
analysis by one-way analysis of variance (ANOVA, with Tukey’s HSD
multiple comparison) with the level of significance set at P < 0.05.
Data are presented as mean values ± standard deviation.
Figure 3B shows that the variation of AO efficiencies at 0.6% diene
formation goes through a maximum at C8 followed by a decrease.
The variation illustrated in Figure 3B is qualitatively similar to the
33
maxima reported by ourselves11,
in emulsions prepared with
other oils and by Laguerre and others when employing different
AOs.36-39 This behavior is known as the “cut-off effect”, indicating a
non-linear dependence of the various chemical or biological
activities of homologous series of reactants with the length of their
alkyl, reaching a maximum at a critical chain length after which their
activity decreases.
Results
Effects of AO chain length on the reactivity with radicals: DPPH•
radical scavenging activity and EC50 values for CA esters. The
procedure for determining EC50 values as a function of AO chain
length has been published in detail.21, 32 The rate of disappearance
of DPPH● from reaction with each AO was monitored at 515 nm by
using a Powerwave XS Microplate Reader (Bio-Tek Instruments, Inc)
thermostated at T = 25.0 ± 0.1 °C. The wells of a 96-well microplate
contained a methanolic solution of AO (3-20 ꢀM) and 80 ꢀM
DPPH●. The absorbance of each well was recorded at one min
intervals for a 60 min period. The absorbance of each solution was
subtracted from the blank (80 ꢀM DPPH● solution without AO).
The value of EC50 for each AO was obtained in a two-step
process. First, the DPPH● absorbance as a function of time (up to 60
minutes) was plotted for six mole AO/mole DPPH● ratios between
3-20 μL of AO, Figure 2A. The percentage of the remaining DPPH● at
6 | J. Name., 2012, 00, 1-3
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Table 2
.
EC50 (mole AO / mole DPPH●) values for CA derivatives
obtained at different reaction times with a level of significance P <
0.05. aStandard deviation (n = 4).
EC50± σa
AO
t
5
15
30
60
(min)
0.330 ±
0.005
0.329 ±
0.001
0.334 ±
0.002
0.344 ±
0.005
CA
0.308 ±
0.012
0.290 ±
0.008
0.262 ±
0.005
0.222 ±
0.010
C1
C2
0.323 ±
0.005
0.277 ±
0.005
0.263 ±
0.003
0.233 ±
0.006
0.322 ±
0.002
0.291 ±
0.006
0.260 ±
0.017
0.225 ±
0.001
C3
0.310 ±
0.006
0.293 ±
0.009
0.259 ±
0.005
0.216 ±
0.006
C4
0.315 ±
0.005
0.262 ±
0.008
0.243 ±
0.006
0.198 ±
0.007
C6
0.293 ±
0.012
0.269 ±
0.013
0.239 ±
0.005
0.199 ±
0.005
C8
0.333 ±
0.003
0.285 ±
0.003
0.254 ±
0.009
0.212 ±
0.008
C10
C12
C14
C16
0.315 ±
0.004
0.294 ±
0.007
0.266 ±
0.006
0.211 ±
0.002
0.317 ±
0.008
0.298 ±
0.001
0.257 ±
0.003
0.199 ±
0.005
Figure 2. A) Illustrative time course for the change in DPPH●
absorbance, (initial DPPH●] = 100 ꢀM) with different concentrations
of propyl caffeate (C3) expressed as mole ratios of C3 to DPPH●. [C3]
= 0.047 – 0.282 M ([C3] ∼ 5% to 25% of [mole AO/mole DPPH●). B)
Linear variation of the percentage of initial DPPH radical with the
mole ratio of AO to DPPH● at selected times of reaction. The EC50
0.314 ±
0.003
0.282 ±
0.007
0.244 ±
0.001
0.196 ±
0.006
The large variations in antioxidant efficiency in emulsions cannot be
attributed to changes in the length of the alkyl chain of the AOs
(HLB) because we previously demonstrated the independence of
the rate constant of the reaction of AOs with the free radical DPPH
(no statistically significant differences in the EC50 values were
values obtained at each time interval are listed in Table 2
.
detected
, see Table 2 H
). The DPPH● test measures the
radical/electron-transferring ability of an antioxidant versus the
stable free radical DPPH and it gives a rough index of the scavenging
activity of the AOS because it is carried out in homogeneous
solution, and the AOs bear the same reactive phenolic moiety both
in homogeneous solution and in emulsions.
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Figure 3. A) Illustrative kinetic plot for the oxidation of the corn
lipids at T = 37ºC (1% emulsifier volume fraction), showing the
initiation, propagation and termination steps. B) Variation of the
time necessary increase the percentage of conjugated dienes 0.6%
as a function of the alkyl chain length of the caffeic acid derivatives.
Thus, we hypothesized as in previous works that the observed
differential rate of the reaction with the lipidic radicals, Figure 3B
,
Figure 4. Illustrative variations of kobs with ΦI for the reaction of 16-
ArN2 with CA in 1:9 (A) and 4:6 (B) corn oil emulsions and with
can be caused by a differential partitioning of the AOS between the
different regions of the emulsion caused by the variation in their
hydrophobicity and analyzed the effects of the alkyl chain length on
their distribution within the emulsion.
+
octyl caffeate(C) in 4:6 emulsions (citric citrate (0.04 M) buffer , pH
+
3.6). [CA] = 4.4x10-3 M, [C8] = 3.5x10-3 M, [16-ArN2 ] = 2,8x10-4 M, T
= 25 ºC.
Effects of alkyl chain length on the distribution of CA and its alkyl
esters in corn oil emulsions
The distribution of AOs in emulsions is defined by the partition
I
constants between the oil-interfacial, PO , and that between the
8 | J. Name., 2012, 00, 1-3
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aqueous-interfacial, PWI, regions, equations 2 and 3, respectively. consistent with caffeic acid having the lowest solublity in water.44
I
I
For hydrophilic antioxidants such as caffeic acid, which is oil
The higher PWI values for the C1 (PW = 747) and for C2 (PW = 2000)
caffeates indicate that their solubility in water relative to the
interfacial region is reduced upon increasing their HLB. Moreover,
insoluble, only the partition constant PWI is needed to describe their
distributions, meanwhile for very hydrophobic AOs such as the C12
I
I
the PW values for C1 and C2 are much higher than the PO values,
Table 1, consistent with the expected higher solubility of C1 and C2
in corn oil compared to water.
I
or C16 alkyl derivatives only PO is needed. These simplifications are
discussed in greater detail elsewhere.34, 40, 41
I
The partition constants PO and PWI, as well as the interfacial rate
I
I
The variation of PO seems to be more complex because the PO
constant kI were determined in the intact emulsions by employing
the method described before. Figure 4 is illustrative and shows the
value for the the C8 derivative is much higher than those for the C1-
C3 and C16 derivatives. However, this complexity is more apparent
that real as shown when calculating the distribution of the AOs.
+
variations of kobs for the reaction of 16-ArN2 with CA and C8 in 1:9
(CA) and 4:6 (CA, C8) stripped corn oil/Tween 20/water emulsions.
I
The excellent fit of the (kobs , ΦI and 1/kobs , Φ) data sets, Figure 4
,
Comparisons of PW values for different AOs highlight the effect of
changing HLB. The partition constant for caffeic acid, PWI = 268, is
demonstrate that the assumptions of the pseudophase model are
met. Values of kobs decrease asymptotically and 1/kobs increase
linearly with increasing ΦI at constant AO concentration and the 6-9
significantly higher than those obtained for gallic acid (PWI = 125)34
,
consistent with caffeic acid having the lowest solublity in water.44
The higher PWI values for the C1 (PW = 747) and for C2 (PW = 2000)
I
I
fold decreases in kobs with increasing ΦI are similar to those found
28, 34
for other AOs.14,
Values of kobs decrease asymptotically on
caffeates indicate that their solubility in water relative to the
going from ΦI = 0.005 to ΦI = 0.05, consistent with the predictions interfacial region is deeply reduced upon increasing their HLB.
I
of equations 7 and 8. The straight lines in Figures 4A and
B
are plots Moreover, the PWI values for C1 and C2 are much higher than the PO
values, Table 1, consistent with the expected higher solubility of C1
of 1/kobs vs ΦI, equations 12 - 13, from where the partition
constants and the kI values displayed in Table 1 were determined.
and C2 in corn oil compared to water.
Changes in the oil to water (o:w) ratio from 1:9 to 4:6 (CA) leads to
differences in the partition constants PWI and POI less than 20%. This
low dependence of the ratio o:w on the partition constants was
previously observed for hydrophilic and hydrophobic AOs, and for
this reason we only employed 4:6 (o:w) emulsions to investigate the
distribution of AOs of moderate and high hydrophobicity.
The percentage of the antioxidant in the interfacial region of the
corn oil emulsions was calculated using equations 14-16 and the
I
O
calculated PW and PW values (Table 1). Similar expressions were
used to determine the fraction of AO in the oil and aqueous
regions. Figure 5 shows the variation of %AO with ΦI for the
different alkyl caffeates. For any given AO, the fraction of AO in the
interfacial region increases upon increasing ΦI so that at ΦI = 0.005
more than 60% of the AOs are located in the interfacial region and
the percentage increases up to 95% at ΦI = 0.04. It is worth noting
that, at any ΦI, %AOI does not correlate with the hydrophobicity of
+
kI is the rate constant of the reaction of 16-ArN2 with AOs, which
takes place in the interfacial region of the emulsion, Scheme 3. kI
values are not needed to assess the distribution of AOs, however,
their values are important because changes in kI values may detect
changes in the reaction mechanism. For example, the variation of kI the AO, following the order %C1 < %C2 < %CA ≈ %C3 < %C4 < %C6 <
with temperature provides the activation parameters for the %C16 < %C8. The most hydrophilic AOs (CA and C1) are more soluble
+
reaction between 16-ArN2 and AOs.42 Changes in kI with acidity in the aqueous region than in the oil, despite their much lower
may also allow to determining whether the reactive species is the solubility in water than that of other hydrophilic AOs such as gallic
43
anionic, dianionic or neutral form of the AO.41,
In addition, or ascorbic acids.44, 45
comparison of kI values for a number of AOs could be used as a
basis for assessing a scale of AO activity that is independent of the
AO distribution in the emulsion because the same chemical probe
However, for each antioxidant type, as the alkyl chain length
increases, its solubility in water decreases and increases in the oil
O
+
(16-ArN2 ) is used in all distribution experiments.14
region, e.g., following PW values (Table 1), which means the HLBs
of different antioxidants depend on the interactions of their polar
I
I
The positive PW and PO values in Table 1
(
ranging 177-2000) groups with water and oil as well as on the length of their alkyl
indicate that the transfer process of the AOs from the oil or chains.
aqueous to the interfacial region is spontaneous (Gibss free energy
negative) highlighting the natural tendency of these AOs to be
mainly located in the interfacial region.
I
PW values increase with increasing the alkyl chain length (i.e., the
HLB of the AO) in keeping with the hydrophobnic effect.
I
Comparisons of PW values for different AOs highlight the effect of
changing HLB. The partition constant for caffeic acid, PWI = 268, is
significantly higher than those obtained for gallic acid (PWI = 125)34
,
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Relationships between the AOs efficiency and the fraction of AOs
in the interfacial region of the emulsions. We note that the set of
AOs we employed here constitute of a group of AOS with the same
reactive moiety (catechol) but of different hydrophobicity. Because
AO efficiency depends, other things being equal, on the
concentration of AOs at the reaction site which, in emulsions, is
believed to the interfacial region, we analyzed if there is any
relationship between %AOI and their efficiency by plotting their
variations with the alkyl chain length. The non-linear, parabolic-
like, variation in the efficiency of the AOs, with a maximum at C8,
Figure 6, parallels that of %AOI, confirming that there is a direct
relationship between AO efficiency and %AOI. Such a parabolic
variation is known as the “cut-off “effect, and has been observed
for several series of AOs bearing the same reactive moieties but
different aliphatic chain lengths in oil-in-water emulsions.21, 22
Figure 6. Variations of the effects of alkyl chain length on the
percentage of AO in the interfacial region of 4:6 corn oil-in-water
emulsions (ΦI = 0.01) and the AO efficiency (measured as the time
necessary to reach an increase of 0.6% in the production of
conjugated dienes).
Figure 5. Distributions of caffeic acid derivatives between the
aqueous (A), interfacial (B) and oil (C) regions of corn oil-in-water
emulsions.
10 | J. Name., 2012, 00, 1-3
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Conclusions
Acknowledgements
We have determined the effects of the alkyl chain length of a We would like to thank Prof. Larry Romsted for
homologous series of caffeic acid derivatives on their efficiency in encouragement and extremely fruitful iscussions. Support
inhibiting the oxidation of the lipids of corn oil-in-water emulsions. from the following institutions is acknowledged: Xunta de
We also determined the partition constants of the AOs between the Galicia (10TAL314003PR), Programa Operacional Factores de
I
oil-interfacial, PO , and water-interfacial, PWI, regions. For any AO, Competitividade – COMPETE) and Fundação para a Ciência e a
both POI and PWI are >> 1, indicating all AOs have a natural tendency Tecnologia (FCT, grant UID/QUI/50006/2013 – POCI-01-0145-
I
to be located in the interfacial region. PW values increase upon FEDER-007265), Universidades de Vigo and Porto.
increasing the alkyl chain length in keeping with the increase in
I
their hydrophobicity. The variation of PO is, however, more
Notes and references
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distributions of the AOs.
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P. C. Calder, in Food enrichment with omega-3 fatty acids,
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DOI: 10.1039/C6RA18282H
TOC
Antioxidant efficiencies in emulsions can be optimized by tailoring interfacial
concentrations