Interactions between α-tocopherol and rosmarinic acid and its alkyl esters in emulsions: Synergistic, additive, or antagonistic effect?

Article abstract of DOI:10.1021/jf302673j

Many antioxidants can interact to produce synergistic interactions that can more effectively inhibit lipid oxidation in foods. Esterification of rosmarinic acid produces a variety of compounds with different antioxidant activity due to differences in polarity and thus differences in partitioning in oil, water, and interfacial regions of oil-in-water emulsions (O/W). Therefore, rosmarinic acid and rosmarinate esters provide an interesting tool to study the ability of antioxidant to interact in O/W emulsions. In O/W emulsions, rosmarinic acid (R0) exhibited the strongest synergistic interaction with α-tocopherol while butyl (R4) and dodecyl (R12) rosmarinate esters exhibited small synergistic interaction and eicosyl rosmarinate esters (R20) exhibited slightly antagonistic interaction. Fluorescence quenching and electron paramagnetic resonance (EPR) studies showed that water-soluble rosmarinic acid (R0) exhibited more interactions with α-tocopherol than any of the tested esters (R4, R12, R20). This was also confirmed in O/W emulsions where R0 altered the formation of α-tocopherol quinone and α-tocopherol increased the formation of caffeic acid from R0. This formation of caffeic acid was proposed to be responsible for the synergistic activity of R0 and α-tocopherol since the formation of an additional antioxidant could further increase the oxidative stability of the emulsion.

Full text of DOI:10.1021/jf302673j

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Article
Interactions between #-tocopherol and rosmarinic acid and its alkyl
esters in emulsions: synergistic, additive or antagonistic effect?
Atikorn Panya, Ketinun Kittipongpittaya, Mickaël Laguerre, Christelle Bayrasy,
Jerome Lecomte, Pierre Villeneuve, D. Julian McClements, and Eric A. Decker
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 18 Sep 2012
Downloaded from http://pubs.acs.org on September 26, 2012
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Interactions between αꢀtocopherol and rosmarinic acid and its alkyl esters
in emulsions: synergistic, additive or antagonistic effect?
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Atikorn Panya1, Ketinun Kittipongpittaya1, Mickaël Laguerre2, Christelle Bayrasy2, Jérôme
Lecomte2, Pierre Villeneuve2, D. Julian McClements1 AND Eric A. Decker1*
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1Department of Food Science, University of Massachusetts, Chenoweth Laboratory, 100
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Holdsworth Way, Amherst, MA 01003, USA; CIRAD, Dept PERSYST, UMR IATE, Fꢀ
34398 Montpellier 5, France
*Corresponding Author
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Running Title: Interactions between rosmarinate esters and αꢀtocopherol in oilꢀinꢀwater
emulsions
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ABSTRACT
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Many antioxidants can interact to produce synergistic interactions that can more
effectively inhibit lipid oxidation in foods. Esterification of rosmarinic acid produces a variety
of compounds with different antioxidant activity due to differences in polarity and thus
differences in partitioning in oil, water, and interfacial regions of oilꢀinꢀwater emulsions
(O/W). Therefore, rosmarinic acid and rosmarinate esters provide an interesting tool to study
the ability of antioxidant to interact in O/W emulsions. In O/W emulsions, rosmarinic acid
(R0) exhibited the strongest synergistic interaction with αꢀtocopherol while butyl (R4) and
dodecyl (R12) rosmarinate esters exhibited small synergistic interaction and eicosyl
rosmarinate esters (R20) exhibited slightly antagonistic interaction. Fluorescence quenching
and electron paramagnetic resonance (EPR) studies showed that water soluble rosmarinic acid
(R0) exhibited more interactions with αꢀtocopherol than any of the tested esters (R4, R12,
R20). This was also confirmed in O/W emulsions where R0 altered the formation of αꢀ
tocopherol quinone and αꢀtocopherol increased the formation of caffeic acid from R0. This
formation of caffeic acid was proposed to be responsible of the synergistic activity of R0 and
αꢀtocopherol since the formation of an additional antioxidant could further increase the
oxidative stability of the emulsion.
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INTRODUCTION
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Under current limitations of approved antioxidants for food applications, it is often
challenging for food scientists to maintain the oxidative stability of processed foods. To try to
solve this problem, several strategies have been attempted to improve antioxidant
performance. One interesting strategy is to use combinations of antioxidants to produce
synergistic interactions via free radical transfer mechanisms. For example, the regeneration of
oxidized αꢀtocopherol by ascorbic acid, flavonoids, carotenoids, phospholipids, amino acids
and peptides has been reported (1-7). These regeneration reactions have been postulated to
produce synergistic antioxidant interactions.
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Several models to study interactions between antioxidants combinations include the
oxygen radical absorbance capacity (ORAC) (8, 9), 2,2ꢀdiphenylꢀ1ꢀpicrylhydrazyl (DPPH)
radical scavenging (10-14), ferric reducing antioxidant power (FRAP) (12), and homogeneous
solutions of peroxidizing methyl linoleate (15). Unfortunately, these models often produce
inconsistent results. It was reported that the combinations between αꢀtocopherol and
flavonoids exhibit synergistic, additive, and antagonistic effects in the ORAC model (8, 9).
However, Hiramoto and coworkers (14) reported that synergistic antioxidant interactions were
observed only when αꢀtocopherol was combined with ascorbic acid, but not with other water
soluble antioxidants in the DPPH model.
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The free radical scavenging properties of antioxidants in homogenous model systems
(e.g. DPPH and ORAC) might not correlate to foods since foods are heterogeneous (16) and
thus may have physical attributes not encountered in homogenous systems that can impact
antioxidant interactions. For example, the existence of both lipid and aqueous phases would
affect antioxidant partitioning and thus interactions between oil and water soluble antioxidants
at the oilꢀwater interface. Fukazawa and coworkers reported that interactions between αꢀ
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tocopherol and ascorbic acid in liposomal membranes are influenced by physical barriers and
surface charge of the membranes (17). Currently, there are no systematic methods to be able
to predict how combinations of antioxidants can inhibit oxidation in real food systems in a
synergistic, additive, or antagonistic manner.
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In this research, we hypothesized that antioxidants partitioning at different locations in
oilꢀinꢀwater (O/W) emulsions may influence the ability of antioxidants to interact. Rosmarinic
acid and its alkyl esters are excellent tools to study interactions with αꢀtocopherol because
their distributions and locations in O/W emulsions can be varied without impacting on their
reactive hydroxyl groups (18). In the current study, several methods were utilized to study the
interactions between αꢀtocopherol and rosmarinic acid or its alkyl esters. Electrochemical
properties of these phenolic compounds were investigated by cyclic voltammetry (CV) to
explain thermodynamic reactions between antioxidants. Direct observations of interactions
between rosmarinates, rosmarinic acid, and αꢀtocopherol were observed by a fluorescence
quenching technique. The efficiency of tocopheroxyl radical regeneration by rosmarinic acid
and rosmarinate esters was studied by electron paramagnetic resonance (EPR) in
homogeneous (ethanol) and heterogeneous (Tween 20 micelles) systems. Finally, the sparing
effects of antioxidant interactions between rosmarinate esters, rosmarinic acid, and αꢀ
tocopherol during the oxidation of O/W emulsions were investigated by determining the rates
of antioxidant decomposition by highꢀperformance liquid chromatography (HPLC). Through
these studies, important mechanistic information can be obtained to better predict when
synergistic, additive, or antagonistic antioxidant interactions can occur in O/W emulsions.
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MATERIALS AND METHODS
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Chemicals and Materials
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Soybean oil was purchased from a local grocery market in Amherst, MA.
Ethylenediaminetetraacetic acid (EDTA) disodium salt was purchased from Chempure ™
Ultra (Houston, Texas). Acetic acid, acetonitrile, methanol, hydrochloric acid were obtained
from Fisher Scientific (Pittsburgh, PA). Chelex® 100, rosmarinic acid, caffeic acid, 2,2ꢀ
diphenylꢀ1ꢀpicrylhydrazyl (DPPH), FeSO4, Tween 20™ (Mw ≈1228), BaCl2, Bu4 NPF6,
phosphoric acid, sodium phosphate monoꢀ and dibasic, 2,2,6,6ꢀtetramethylpiperidinoxyl
(TEMPO) radical were purchased from SigmaꢀAldrich (St. Louis, MO). αꢀTocopherol
quinone was purchased from Tokyo chemical industry (Tokyo, Japan). Doubleꢀdistilled and
deionized water was used for the preparation of all solutions.
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Synthesis of Rosmarinate Esters
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The chemoenzymatic esterification of rosmarinic acid to obtain rosmarinate esters was
carried out following the procedure described by Lecomte and coworkers (18). Briefly, the
chemical esterification of rosmarinic acid (56 ꢁmol) was conducted in sealed brown flasks
each containing 5 mL of alcohol (methanol, 123.4 mmol; nꢀbutanol, 54.6 mmol; nꢀoctanol,
31.9 mmol; nꢀdodecanol, 22.5 mmol; nꢀhexadecanol, 17.0 mmol; nꢀoctadecanol, 15.1 mmol
or nꢀeicosanol, 13.6 mmol). The reaction mixtures were stirred (orbital shaker, 250 rpm, 55ꢀ
70 oC) prior to the addition of the catalyst; the strongly acidic sulfonic resin Amberlite IRꢀ
120H (5% w/w, total weight of both substrates) that had been previously dried at 110 oC for
48 h. The water generated during the reaction was removed by adding 3 A°, 4ꢀ8 mesh
molecular sieves (40 mg/mL, Aldrich, St. Louis,MO) to the medium. Samples (20 ꢁL) were
regularly withdrawn from the reaction medium and then mixed with 980 ꢁL of methanol,
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filtered (0.45 ꢁm syringe filter MillexꢀFH, Millipore Corp., Bedford, MA), and analyzed by
reverse phase HPLC with UV detection at 328 nm (18). After complete (4ꢀ21 days)
conversion of rosmarinic acid into the corresponding ester, the latter was purified in a twoꢀ
step procedure. First, a liquidꢀliquid extraction using hexane and acetonitrile was performed
to remove the excess alcohol. Then, the remaining traces of the alcohol and rosmarinic acid
were eliminated by flash chromatography on a CombiFlash Companion system (Teledyne
Isco Inc., Lincoln, NE). Separation was carried out on a silica column using an elution
gradient of hexane and ether (20ꢀ100% in 35 min). The yield of purified esters, obtained as
pale yellow to yellow amorphous powders, was calculated from calibration curves previously
established with pure compounds. Pure esters and rosmarinic acid were then fully
characterized by ESIꢀMS, 1H NMR, and 13C NMR as previously described by Lecomte et al.
(18).
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Emulsion Preparation
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Stripped soybean oil was prepared according to the method of Waraho et al. (19). The
effectiveness of stripping was monitored by measuring the removal of tocopherols by HPLC
(20). No tocopherol could be detected in the stripped oils. Oilꢀinꢀwater (O/W) emulsions
were prepared using 1.0% (wt) stripped soybean oil in a 10 mM phosphate buffer solution
(pH 7.0). Tween 20 was used as an emulsifier at a 1:10 emulsifier/oil ratio. Stripped soybean
oil, Tween 20, and phosphate buffer were added to a beaker, and a coarse emulsion was made
by blending with a handꢀheld homogenizer (M133/1281ꢀ0, Biospec Products, Inc.,
Bartlesville, OK) for 2 min. The coarse emulsion was then homogenized with a microfluidizer
(Microfluidics, Newton, MA) at a pressure of 9 kbar for three passes.
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After the O/W emulsion was prepared, rosmarinic acid and its esters with various
chain lengths (4, 8, 12, 18, and 20 carbons) in methanol were added to the emulsion at a final
concentration of 30 ꢁM and stirred for 1 h at room temperature. Samples with methanol but
without antioxidant were used as control samples. The emulsions (0.5 mL) were transferred
into 10 mL GC vials and sealed with (tetrafluoroethylene) butyl rubber septa, and then stored
at 25 oC in the dark. Three vials of each treatment were taken every day to determine lipid
hydroperoxides and hexanal formation.
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In some experiments, when needed, emulsions were washed to remove aqueous phase
surfactants as previously described by Faraji and coworkers (21) with some modifications. In
short, emulsions were centrifuged at 38,518 g (17,000 rpm) for 1 h at 4 oC using a Fiberlite®
F40Lꢀ8 x 100 rotor with a highꢀspeed centrifuge (Thermo scientific WX Ultra 80, Asheville,
NC). After the centrifugation, the bottom layer (phosphate buffer) was carefully removed
using a needle and syringe, and then the same volume of the fresh phosphate buffer was used
to reꢀdisperse the creamed emulsion droplet layer by vortexing. This washing procedure was
performed a total of three times. The lipid content of the final washed emulsion was
determined by the modified Bligh and Dyer method (22) and then phosphate buffer was used
to adjusted the lipid content back to 1% (w/w). In some experiments, Tween 20 was added
back into the washed emulsions (0, 0.1, 0.5, 1.0, and 2.5%; w/w) so that a known amount of
surfactant would be in the continuous phase.
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Measurements of particle size of emulsions
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The size of the emulsion droplets were measured by a dynamic light scattering
(Zetasizer NanoꢀZS, Model ZEN3600, Malvern Instruments, Worchester, U.K.), and
expressed as zꢀaverage mean diameter. Samples were diluted 50 times with the same buffer as
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the emulsion, mixed, and immediately measured by transferring the solution into 3 mL plastic
cuvettes for determining the size. Measurements were performed on three replicates and
repeated 3 times on each samples at room temperature. The emulsion droplets size ranged
from 173.3 ± 11.7 nm and there was no significant change in droplet size of each emulsion
over the course of study (data not shown). In addition, there was no visual observation of
creaming during storage in all treatments
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Measurements of Lipid Hydroperoxides
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Lipid hydroperoxide formation in emulsion solutions was determined according to the
method described by Panya and coworkers (23) with some modifications. Emulsion solutions
(0.2 mL) were mixed with 1.5 mL of isooctane/2ꢀpropanol (3:1 v/v) and vortexed (10 s, three
times). After centrifugation at 1000 g for 2 min, 30 ꢁL of the organic solvent phase was
mixed with 1.5 mL of methanol/1ꢀbutanol (2:1). Hydroperoxide detection was started by the
addition of 7.5 ꢁL of 3.94 M ammonium thiocyanate and 7.5 ꢁL of ferrous iron solution
(prepared by adding equal amounts of 0.132 M BaCl2 and 0.144 M FeSO4). After 20 min of
incubation at room temperature, the absorbance was measured at 510 nm using a UVꢀVis
spectrophotometer (Genesys 20, Thermo Spectronic). Hydroperoxide concentrations were
determined using a standard curve prepared from hydrogen peroxide.
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Measurements of Hexanal
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Headspace hexanal was determined according to the method described by Panya and
coworkers (23) with some modification using a Shimadzu GCꢀ2014 gas chromatograph (GC)
equipped with an AOCꢀ5000 autoinjector (Shimadzu, Tokyo, Japan). A 50/30 ꢁm
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divinylbenzene/carboxen/polydimethylsiloxane (DVB/Carboxen/PDMS) stable flex solid
phase microextraction (SPME) fiber (Supelco, Bellefonte, PA) was inserted through the vial
septum and exposed to the sample headspace for 8 min at 55 oC. The SPME fiber was
desorbed at 250 oC for 3 min in the GC detector at a split ratio of 1:7. The chromatographic
separation of volatile aldehydes was performed on a fusedꢀsilica capillary column (30 m x
0.32 mm i.d. x 1 ꢁm) coated with 100% poly(dimethylsiloxane) (Equityꢀ1, Supelco). The
temperatures of the oven, injector, and flame ionization detector were 65, 250, and 250 oC,
respectively. Sample run time was 10 min. Concentrations were calculated by using a
standard curve made from the above emulsions containing known hexanal concentrations and
200 ꢂM EDTA.
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Calculation of antioxidant interaction indexes of rosmarinate esters and αꢀtocopherol
combinations
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Interaction indexes of rosmarinate esters with αꢀtocopherol were calculated based on
the oxidation lag times of lipid hydroperoxides and hexanal formation. Lag times were
determined as the first data point that was statistically (p≤0.05) greater than time zero.
Briefly, the oxidation lag times of individual antioxidants were used to estimate the expected
oxidation lag times of its combination. Interaction indexes were calculated from the ratio
between the obtained oxidation lag times of the combination and the expected oxidation lag
time of the combination with the following equation:
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Where, A and B represent αꢀtocopherol and rosmarinates, respectively. Controls of A,
B and A+B represent the lag time of individuals and combinations without adding
antioxidants. Interaction indexes were expressed as synergistic (> 1), additive (≈ 1) and
antagonistic (< 1) antioxidant effects
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Determination of Antioxidant Partitioning
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Determination of the physical location of rosmarinic acid and its alkyl esters in the
emulsions were performed according to the procedure described by Panya and coworkers
(23). EDTA (200 ꢀM) was added to regular emulsions and washed O/W emulsions with
added surfactants (0, 0.1, 0.5, 1.0, and 2.5%; w/w) to minimize oxidation during analysis.
Rosmarinic acid and its alkyl esters in methanol were added to the emulsion at a final
concentration of 100 µM followed by stirring at room temperature for 1 h. The emulsions
were centrifuged at 162,102 g (46,000 rpm) for 1 h at 4 oC using a PTI F65Lꢀ6x13.5 rotor
with a highꢀspeed centrifuge (Thermo Scientific WX Ultra 80, Asheville, NC). The
continuous phase was carefully collected with a pipette and the amount of aqueous phase
rosmarinic acid esters was determined directly by HPLC using a modified method described
by Lecomte and coworkers (18). Briefly, HPLC determination of rosmarinic acid and its alkyl
esters was carried out with a Hypersil gold C18 reversed phase column (250 mm x 4.6 mm, 5
µm) equipped with a Hypersil gold guard column (10 mm x 4 mm, 5 µm) (Thermo Scientific,
USA) using a LCꢀ10ATvp HPLC system (Shimadzu, USA). Peak integration was performed
using Shimadzu EZstart (Version 7.2). Gradient elution was performed using methanol and 3
mM phosphoric acid at 1 mL/min at 40 oC (column temperature), in linear gradients from
0/100 (v/v) to 100/0 (v/v) for 5 min, then 100/0 (v/v) for 10 min, back to 0/100 (v/v) for 5 min
and hold at 0/100 (v/v) for 5 min. Rosmarinic acid and its alkyl esters [(R4 (4 carbons) – R20
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(20 carbons)] were detected with a photodiode array detector (SPDꢀM10Avp, Shimadzu,
USA) at 328 nm. αꢀTocopherol was detected at 295 nm. The concentrations of rosmarinic
acid esters and αꢀtocopherol were calculated using a standard curve made from each
antioxidant dissolved in methanol.
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Determination of Antioxidant Decomposition during Oxidation Studies
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Determination of the decomposition of αꢀtocopherol and rosmarinic acid and its alkyl
esters in the emulsions during storage were performed according to the procedure described
by Panya and coworkers (23). A 200 ꢂL sample of O/W emulsions was transferred into 1.5
mL eppendorf tubes containing 50 ꢂL of 200 ꢂM EDTA to inhibit further lipid oxidation.
Samples were frozen at ꢀ80 oC until freezing drying. The freezing drying condition was
operated at ꢀ10 oC for 16 h, then increased the temperature to 5 oC for 4 h. Dried emulsions
were stored at ꢀ80 oC until analysis. Antioxidants in dried emulsions were extracted
immediately prior to analysis by adding 200 ꢂL of methanol. The mixtures were vortexed for
2 min, sonicated in ultrasonic bath for 2 min, and then centrifuged at 1000 g for 5 min.
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The clear methanolic solutions were carefully collected with a pipette and antioxidant
concentrations were determined directly by HPLC using a modified method described by
Fujimoto and Masuda (24). Briefly, HPLC determination of rosmarinic acid and its alkyl
esters was carried out with a Hypersil gold C18 reversed phase column (250 mm x 4.6 mm, 5
ꢂm) equipped with a Hypersil gold guard column (10 mm x 4 mm, 5 ꢂm) (Thermo scientific,
USA) using a LCꢀ10ATvp HPLC system (Shimadzu, USA). Peak integration was performed
using Shimadzu EZstart (Version 7.2). Gradient elution was performed using acetonitrile and
1 % acetic acid at 1 mL/min in linear gradients from 5/95 (v/v) to 100/0 (v/v) for 40 min, then
100/0 (v/v) for 10 min and then back to 5/95 (v/v) for 5 min. Rosmarinic acid and dodecyl
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rosmarinate ester (12 carbons) were detected with a photodiode array detector (SPDꢀM10Avp,
Shimadzu, USA) at 328 nm. The oxidation products of rosmarinic acid and its dodecyl ester
were detected at 280 and 328 nm. αꢀTocopherol and αꢀtocopheryl quinone were detected at
295 and 265 nm, respectively. The concentrations of antioxidants (rosmarinic acid and its
ester, αꢀtocopherol, and αꢀtocopheryl quinone) were calculated using a standard curve made
from the standard antioxidants dissolved in methanol.
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FrontꢀFace Fluorescence Quenching Measurements
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Frontꢀface fluorescence quenching between αꢀtocopherol and rosmarinic acid, and its
alkyl esters in O/W emulsions was determined by steadyꢀstate emission measurements
recorded with a PTI spectrofluorometer (PTI, London, Ontario, Canada). Stripped soybean oil
(1 %; w/w) emulsions were prepared with 0.1 % (w/w) Tween 20 in 10 mM phosphate buffer
(pH 7) with 200 ꢂM EDTA to minimize oxidation. A final concentration of 100 ꢂM of αꢀ
tocopherol was added to the O/W emulsions from the stock solution of αꢀtocopherol in
methanol. The mixtures were stirred at room temperature for 1 hour. Then, rosmarinate esters
in methanol were transferred into the emulsions containing αꢀtocopherol at concentrations of
0, 5, 10, 25, 50, and 100 ꢂM.
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After vortexing for 2 min, the final emulsions (1.5 mL) were transferred into triangular
suprasil cuvettes. The samples were held at 30 oC and stirred with a 3 mm magnetic stirring
bar (Fisher scientific, USA). Emission was observed at 90o to the incident beam, that is, 22.5o
with respect to the illuminated cell surface. The emission of αꢀtocopherol was measured at
320 nm using an excitation wavelength of 295 nm. Spectral bandwidth for both excitation and
emission slits was 2.0 nm, integration time was 1 s, and the wavelength increment was 2.5
nm. The intensity of the spectra (I) of αꢀtocopherol after addition of the rosmarinate
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derivatives was determined as the emission signal intensity (counts per second) measured by
means of a photomultiplier. The intensity ratio (Io/I), where Io is the fluorescence intensity of
αꢀtocopherol, was plotted versus the concentrations of rosmarinate esters. The slope of this
line was used to determine the quenching constant of the different rosmarinate esters.
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Cyclic Voltammetry (CV) Measurements
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Cyclic voltammetry was performed according to the method described by Wilson and
coworkers (25). Rosmarinic acid and its alkyl esters (2 mM) were freshly dissolved in
acetonitrile and evaluated with a BASi Model Cꢀ3 cell stand using a planar 1 mm diameter
glassy carbon working electrode, Ag/AgCl reference electrode, and a Pt wire auxiliary
electrode. Samples were scanned at 100 mV sꢀ1 at 25 oC in acetonitrile with 0.5 M Bu4 NPF6
as a salt bridge in 50 mM phosphate buffer, pH 7 or in 1% Tween 20 in 50 mM phosphate
buffer, pH 7.
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Measurement of αꢀTocopherol Regeneration by Electron Paramagnetic Resonance
Spectroscopy
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The efficiency of rosmarinic acid and its alkyl esters to regenerate αꢀtocopherol from
αꢀtocopheroxyl radicals was determined by electron paramagnetic resonance (EPR)
spectroscopy in two different systems: a homogeneous ethanolic solution and a heterogeneous
Tween 20 micelle solution. The experiment procedure was adapted from the method
described by Pazos and coworkers (26) with some modifications.
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For experiments in homogeneous environments, stock solutions of αꢀtocopherol (2
mM), DPPH radical (0.5 mM), and rosmarinates were prepared freshly with N2 saturated
ethanol. In heterogeneous environments, a stock solution of αꢀtocopherol (2 mM) was
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prepared in 100 mM Tween 20 in N2 saturated 50 mM phosphate buffer solution at pH 7. Two
mL of the stock solution of αꢀtocopherol (ethanolic or Tween 20 solutions) was transferred
into a 4 mL vial which was purged with N2. Then, 50 ꢁL of the DPPH solution was
transferred into the stock solution of αꢀtocopherol, and mixed immediately at room
temperature. After reacting for 20 sec to form αꢀtocopheroxyl radicals, 50 ꢂL of the stock
solutions of rosmarinic acid and its alkyl esters were added and mixed. The final
concentrations of αꢀtocopherol and DPPH were 1.9 and 0.01 mM, respectively. The final
concentrations rosmarinic acid and rosmarinates were ranged from 2.5 to 20 ꢂM. All
solutions were transferred into an EPR spectrometer via a 5 mL syringe. EPR spectra were
recorded 1 min after the reaction with DPPH.
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The experiments were performed using a Bruker ELEXSYS Eꢀ500 EPR spectrometer
(Bruker, Germany) equipped with an Xꢀband microwave bridge and an ER 4122ꢀSHQE high
sensitivity single cavity. Samples were injected into the cavity through with AquaꢀX flowꢀ
through cell. EPR parameters were at the following setting: microwave power, 10 dB; sweep
width, 100 G; sweep time, 20.9 s; modulation amplitude, 3 G; time constant, 81.92 ms;
receiver gain, 80 dB. All samples were handled under N2 sealed environment at room
temperature.
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The Mn (II) marker attached with the AquaꢀXꢀflow cell was used to determine the
relative signal intensity of the αꢀtocopheroxyl radical (the peakꢀtoꢀpeak ratio between αꢀ
tocopheroxyl radical and the marker). Concentrations of αꢀtocopheroxyl radical were
quantitated by comparing the doubleꢀintegrated areas of αꢀtocopheroxyl radical to known
concentration of TEMPO radical. The integration of the signal was performed by using
Bruker Xepr software. Efficiencies of the regeneration of αꢀtocopherol by each rosmarinates
were estimated from the slopes of αꢀtocopheroxyl radical reduction at various concentrations
of rosmarinates.
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Statistical Analysis
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All analyses were performed on triplicate samples. Oxidation lag phases were defined
as the first data point significantly greater than the 0 time value. In all cases, comparisons of
the means were performed using Duncan’s multipleꢀrange tests. A significance level of
p<0.05 was defined as being statistically different. All calculations were performed using
SPSS17 (http://www.spss.com; SPSS Inc., Chicago, IL).
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RESULTS AND DISCUSSION
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Antioxidant Activity of Rosmarinic Acid and its Esters and αꢀTocopherol Combinations
in Stripped Soybean O/W Emulsions
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The ability of rosmarinic acid and rosmarinate esters and αꢀtocopherol to
synergistically inhibit lipid oxidation in the O/W emulsions was tested with rosmarinic acid
(R0) and its different esters (R4, R12, and R20). This system was used since the different
forms of rosmarinate could primarily exist in the aqueous phase, the interfacial layer, or the r
emulsion droplet core and thus could interact differently with αꢀtocopherol (T) which would
mainly associate with the oilꢀwater interface and/or the emulsions droplet core. Lipid
hydroperoxide and hexanal formation in the O/W emulsions were determined during storage
at 25 oC in the dark. All forms of rosmarinic acid and its esters (30 ꢂM) were able to inhibit
the formation of lipid hydroperoxides and hexanal compared to the control (Figure 1). For
example, the R4 was slightly better than R12 at increasing hexanal lag times and R0 was
slightly better than R20 which is the worst antioxidant. This antioxidant efficiency order
(R4>R12>>R0>R20) confirms the cutꢀoff effect we already observed in O/W emulsion with
rosmarinate derivatives (27, 28) and chlorogenate alkyl esters (29).
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All the combinations of rosmarinic acid and its esters (30 ꢂM) with αꢀtocopherol (30
ꢂM) exhibited better antioxidant activity compared to the individual compounds. This was not
entirely unexpected since the total antioxidant concentrations were higher. As shown in
Figure 2, it was noted that the combinations of αꢀtocopherol and butyl rosmarinate ester (Tꢀ
R4), and αꢀtocopherol and dodecyl rosmarinate ester (TꢀR12) exhibited similar increases in
the lag phase of lipid hydroperoxides and hexanal formation as the sum of the individual
antioxidants (Figure 1). Surprisingly, the combination of αꢀtocopherol and rosmarinic acid (Tꢀ
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R0) showed significant increases in the lag phase of lipid hydroperoxides and hexanal
formation compared to the sum of antioxidants analyzed individually (synergistic effect). The
lag times of hexanal formation and lipid hydroperoxides for the TꢀR0 combination ended up
being similar to the lag times of the TꢀR4 and TꢀR12 combinations. In this case, the strong
synergistic effect between αꢀtocopherol and R0 compensates for the much lower antioxidant
activity of R0 taken individually compared to R4 and R12. Finally, it clearly appears from
Figure 2 that the combination between R20 and αꢀtocopherol produces an antagonistic effect.
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To quantitate the effects of the antioxidant combinations, the lag times for the
formation of lipid hydroperoxides and hexanal for both individual and combined antioxidants
were used to calculate the interaction index. As illustrated in Figure 3, R0 had the strongest
antioxidant synergy with αꢀtocopherol producing interaction indexes for lipid hydroperoxides
and hexanal formation of 4 to 5, while the interaction indexes of αꢀtocopherol and R4 or R12
were approximately 1.5 to 2 meaning that the synergistic effects were smaller. In contrast,
R20 showed significant antagonistic effect with αꢀtocopherol exhibiting an interaction index
of 0.3 to 0.6.
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Partitioning of Rosmarinic Acid, Rosmarinate Esters, and αꢀTocopherol in O/W
Emulsions
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A factor that could be important for the observed variations in synergistic antioxidant
interactions would be differences in the physical location of rosmarinic acid and rosmarinate
esters which would impact their ability to interact with αꢀtocopherol. The aqueous phase of
O/W emulsions was collected to determine whether the antioxidants partitioned into the
aqueous or emulsion droplet phases (interface + droplet core). Results showed that rosmarinic
acid (R0) had the lowest association with emulsion droplets, partitioning at approximately 90
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% in aqueous phase, while butyl (R4) and dodecyl (R12) rosmarinate esters and αꢀtocopherol
were highly associated with the emulsion droplets indicated by low concentrations (2ꢀ9%) in
aqueous phase (Figure 4). The antioxidant partitioning of R4, R12 and R20 were consistent
with our previous report (28). There was slight precipitation observed in the continuous phase
of emulsions containing R20. According to our previous study, R20 may form poorly soluble
selfꢀassembled aggregates and/or coꢀmicelles with Tween 20 in the aqueous phase (27, 28).
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Interactions between Antioxidants in O/W Emulsions as Determined by FrontꢀFace
Fluorescence Quenching Measurements
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Just because antioxidants partition into the continuous phase or the emulsion droplet
does not mean they will interact to produce synergistic or antagonist interactions. Direct
observations of the rosmarinic acid or rosmarinate esters interacting with αꢀtocopherol in O/W
emulsions can be determined by quenching of αꢀtocopherol fluorescence by the rosmarinates.
Although the exact quenching mechanisms between these antioxidants have not been
reported, one potential mechanism might be Förster resonance energy transfer (FRET)
because the emission wavelength of αꢀtocopherol (325 nm) overlaps with the excitation
wavelength (323 nm) of the rosmarinate esters and rosmarinic acid. Therefore, closer the
proximity between αꢀtocopherol and the rosmarinates or rosmarinic acid would be expected to
produce greater quenching.
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In the O/W emulsions, the fluorescence intensity of αꢀtocopherol was decreased in the
presence of butyl and dodecyl rosmarinates and rosmarinic acid. As illustrated in Figure 5, R0
was more effective at quenching the αꢀtocopherol fluorescence in O/W emulsion compared to
the rosmarinate esters (R4 to R12) as shown by R0’s higher quenching constant. Results
suggest that the more water soluble rosmarinic acid (R0) was able to interact with αꢀ
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tocopherol on the emulsion droplet surface. The lower the quenching constants of rosmarinate
alkyl esters suggest that slow lateral diffusion on the droplet surface would limit their ability
to interact with αꢀtocopherol. This slowing down of the rosmarinate diffusivity may be due to
hydrophobic interactions between their alkyl chains and the aliphatic tails of surfactants and
lipids at the interface and/or in the droplet interior.
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When O/W emulsions are produced, excess surfactant that is not absorbed onto the
emulsion droplet surface partitions into the aqueous phase and forms micelles. These micelles
can alter the partitioning of antioxidants into the continuous phase by solubilizing the
antioxidants into the micelles. To eliminate the influence of surfactant micelles, the excess
surfactants in O/W emulsions were removed by a washing process (28). Removal of the
surfactant micelles decreased the ability of all the rosmarinic acid derivatives to quench the
fluorescence of αꢀtocopherol with the exception of the R4 ester. This decrease was most
dramatic for rosmarinic acid (R0) such that its quenching constant became similar to the
rosmarinic acid esters (R8, R12). This suggests that the removal of the surfactant micelles
decreased the partitioning of αꢀtocopherol into the aqueous phase which decreased the ability
of αꢀtocopherol and waterꢀsoluble rosmarinic acid to interact. The αꢀtocopherol quenching
constants of the other rosmarinate esters also decreased in washed emulsions, suggesting that
the Tween 20 micelles also decreased their ability to interact with αꢀtocopherol. Since all of
the antioxidants can partly partition into the interface, the decrease in interfacial area caused
by the removal of the micelles would be expected to decrease the partitioning of the
antioxidants in the interface of the emulsion droplets and micelles and thus decrease
antioxidant interactions especially if some of the antioxidant was forced into the interior of
the emulsion droplet.
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Electrochemical Properties of Rosmarinic Acid, Rosmarinate Esters and αꢀTocopherol
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One possible reason for the observed variations in antioxidant activity for
combinations of rosmarinic acid esters and αꢀtocopherol could be due to the regeneration of
one antioxidant by the other. The hierarchy of antioxidant regeneration by electron transfer
can be estimated by their oxidationꢀreduction potentials. Generally, an antioxidant with lower
reduction potential is thermodynamically preferred to give electrons to an antioxidant with
higher reduction potential (30). Cyclic voltammetry can be a useful method for studying the
reduction potential of antioxidant compounds (31-35). In this study, cyclic voltammetry was
performed in order to measure the electrochemical potential of αꢀtocopherol and also to
investigate the influence of esterification on the electrochemical properties of rosmarinic acid
in various solutions.
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Results showed that cyclic voltammograms of the rosmarinate esters and αꢀtocopherol
exhibited one anodic and one cathodic peak (data not shown) in acetonitrile. This is also true
for other phenolic compounds (36, 37). As shown in Table 1, all the rosmarinate esters
showed a similar oxidation peak (EPaox) at approximately 1115ꢀ1137 mV (vs. Ag/AgCl) in
acetonitrile solution which was significantly higher than αꢀtocopherol (634.9 mV).
Electrochemical parameters of the rosmarinate esters did not show the nonꢀlinear behaviors
that were observed when their activity was tested with the DPPH assay or in lipid oxidation
studies (27-29, 38).
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Oxidation peak potentials of rosmarinic acid and αꢀtocopherol were further studied in
50 mM phosphate buffer, pH 7.0 and 1% Tween 20 in 50 mM phosphate buffer solutions.
Trolox was used instead of αꢀtocopherol in these experiments due to solubility limitations of
αꢀtocopherol in aqueous solutions. Results showed that rosmarinic acid reduction potential
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decreased in the phosphate buffer, however, rosmarinic acid still had higher reduction
potentials than Trolox. This was also true in the presence of Tween 20 micelles where the
rosmarinic acid reduction potential decreased compared to acetonitrile but was still greater
than αꢀtocopherol. These results suggested that αꢀtocopherol is thermodynamically preferred
to donate electrons to rosmarinic acid.
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Regeneration Efficiency of the Rosmarinic Acid Esters to Reduce αꢀTocopheroxyl
Radical in Homogeneous and Heterogeneous Systems
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The ability of rosmarinic acid and its esters and αꢀtocopherol to regenerate each
other’s radicals can be determined by electron paramagnetic resonance (EPR) technique. To
determine these interactions, antioxidant radicals were produced by exposing the antioxidants
to DPPH radicals. However, rosmarinic acid and its esters were not able to produce stable
radicals that could be observed by electron paramagnetic resonance (EPR) using this method,
so the following studies focused on the fate of αꢀtocopherol radicals in the presence of
rosmarinic acid and its esters. Experiments were performed in both ethanol and 100 mM
Tween 20 solutions in order to observe the ability of the different alkyl chain lengths of
rosmarinic acid to impact the efficiency of αꢀtocopherol regeneration in homogenous and
heterogeneous systems. Overall, the efficiency of rosmarinic acid and its esters to reduce αꢀ
tocopherol radical was very low as predicted by electron reduction potential (Table 1). The
range of αꢀtocopherol radical regeneration efficiencies by rosmarinic acid and its esters
ranged from 0.08 to 0.55 moles αꢀtocopherol radicals reduced/ mole of phenolics (Figure 6).
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As shown in Figure 6, esterification of rosmarinic acid increased electronꢀdonating
ability towards αꢀtocopheroxyl radicals compared to rosmarinic acid in ethanolic solutions.
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Esterification has also been observed to increase the DPPH scavenging activity of rosmarinic
acid esters (18, 28) and chlorogenic acid esters (39) in methanolic solution. Lecomte and
coworkers (18) reported that dodecyl rosmarinate (R12) had the greatest DPPH scavenging
activity of all the esters tested (4ꢀ20 carbons), while LopezꢀGiraldo and coworkers (2009)
showed that butyl and octyl chlorogenate esters had higher DPPH scavenging activity than
chlorogenic acid itself and its esters with alkyl chains longer than 12 carbons. In this study,
R4 had the highest ability to regenerate the αꢀtocopheroxyl radical scavenging efficiency
compared to other esters in ethanol solution.
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To investigate the influence of physical structures on αꢀtocopherol radical
regeneration by the rosmarinates, measurements were also performed in 50 mM phosphate
buffer solution with surfactant micelles produced from 100 mM Tween 20, pH 7.0. The ratio
of scavenging regeneration efficiency in Tween 20 versus ethanol for R0 and R4 increased by
381 and 214 %, respectively while R12 and R20 where essentially the same in ethanol and
Tween 20. The results for R0 are similar to those observed for R0 quenching of fluorescence
again suggesting that the high partitioning of R0 in the aqueous phase allowed it to interact
with αꢀtocopherol radicals at the Tween 20 micelle interface. The fact that R4 was effective at
interacting with αꢀtocopheroxyl radicals but did not alter αꢀtocopherol fluorescence could be
due to the lack of emulsion droplets in the EPR study. The presence of emulsion droplets in
the fluorescence study could result in R4 partitioning in the emulsion droplet in a manner
where it did not readily interact with αꢀtocopherol whereas R4 would interact with αꢀ
tocopheroxyl radicals in surfactant micelles.
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Depletion of αꢀTocopherol, Rosmarinic Acid, and its alkyl Esters during oxidation of
O/W emulsion
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Studies on αꢀtocopherol fluorescence quenching indicated that of all the tested
polyphenols, R0 interacted with αꢀtocopherol more than the rosmarinate esters. This suggests
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that the ability of αꢀtocopherol to greatly increase the antioxidant activity of R0 could be due
to their molecular interactions. To further investigate the potential interaction between the
rosmarinates and αꢀtocopherol, decomposition of the antioxidants was determined during
storage and compared to formation of the lipid oxidation product, hexanal in the O/W
emulsion.
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The decomposition of R0, R12 and αꢀtocopherol analyzed individually in the O/W
emulsion and subsequent hexanal formation are shown in Figure 7AꢀC. Results showed that
αꢀtocopherol concentrations decreased in a linear fashion (Figure 7A). αꢀTocopherol
concentrations were approximately 8ꢀ12 µM when the lag phase of hexanal formation ended.
The concentration of R0 and R12 also decreased in a linear fashion during storage of the O/W
emulsions (Figure 7Band C). However, both R0 and R12 were completely depleted prior to
formation of hexanal.
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Depletion of R0, R12, and αꢀtocopherol during the storage of the O/W emulsions
when the antioxidants were added in combination is shown in Figure 8 and 9. In this study, αꢀ
tocopherol concentration was constant at 30 ꢀM while R0 and R12 concentrations ranged
from 15 to 60 ꢀM. As was previously observed in Figures 3, the combination of αꢀtocopherol
and R0 produced synergistic antioxidant activity, while αꢀtocopherol and R12 showed an
additive effect.
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The decomposition disappearance of R0 and R12 was very similar in the presence of
αꢀtocopherol (Figure 8A and 9 A). For example, the time at which approximately 50% of R0
and R12 was lost was about 9 days. The similarity of R0 and R12 depletion in the presence of
αꢀtocopherol suggests that the increase in the antioxidant activity of R0 by αꢀtocopherol was
not due to αꢀtocopherol regenerating R0 and keeping R0 concentrations higher. Conversely,
αꢀtocopherol depletion was much faster in the presence of R0 than R12 (Figure 8B and Figure
9B). For example at 30 days of storage, αꢀtocopherol concentrations were less than 5 ꢀM in
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the presence of R0 (60 ꢂM) compared to 11 ꢀM in the presence of R12 (60 ꢂM). In addition,
the lag times of tocopheryl quinone (TQ) formation were different between R0 and R12
samples (Figure 8B and 9B). For example, tocopheryl quinone was detected in the R0
samples when R0 was almost depleted whereas tocopheryl quinone was detected much earlier
during storage in the presence of R12 with the lag phase for TQ formation being independent
of R12 concentration.
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The observation that R0 and αꢀtocopherol samples increased the lag phase for TQ
formation and improved their oxidative stability similar to R12 and αꢀtocopherol samples
suggests that the two antioxidants were interacting. Even though the concentrations of αꢀ
tocopherol in the combination with R0 were lower than that observed in R12 and αꢀtocopherol
samples, antioxidant activity was greater. This suggests that other antioxidative compounds
might exist in the emulsions since it has been reported that some antioxidants can produce
other antioxidative compounds via their oxidative degradation (24, 40).
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The major oxidation products generated from R0 and R12 were analyzed to get a
better understanding of the dynamics of the antioxidant mechanisms in the O/W emulsions.
Only one major breakdown product of R0 was observed during storage while two major
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breakdown products were observed for R12. From LCꢀMS analysis, the products at HPLC
retention times of 10.5 (in R0 and R12) and 35.9 (in R12) min are caffeic acid [m/z = 179.2
(MꢀH)ꢀ] and a dodecyl rosmarinate quinone formed on the 2ꢀoxyphenylpropanyl moiety [m/z
= 525.06 (MꢀH)ꢀ]. The structures of major antioxidant oxidation products were showed in
Figure 10. The quinone was previously reported to be the major oxidation product of
rosmarinic acid as determined in 2,2ꢀazobis(isobutyronitrile)(AIBN)ꢀinduced oxidation of
ethyl linoleate and DPPH model systems(24).
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However, unlike the above single phase model systems using ethyl linoleate and
alcohols, our results indicated that the antioxidant product of R0 in O/W emulsions was
mainly caffeic acid (Figure 11). This could be because the R0 quinone was unstable in O/W
emulsions and was fragmented to caffeic acid. It was reported that rosmarinic acid can be
decomposed into caffeic acid via a McLafferty rearrangement (γꢀH rearrangement with βꢀ
cleavage in the electron ionization) in mass spectrometry analysis (40). Caffeic acid has also
been found to be one of the metabolites of rosmarinic acid in rats (41). Hydrolysis of
rosmarinic acid by esterases into caffeic acid and 3,4ꢀdihydroxyphenyllactic acid in vitro was
reported, however the hydrolysis was not observed in a gastrointestinal model with lipase and
pancreatic enzymes (42).
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In our conditions, however, hydrolysis is unlikely. Instead, an oxidation breakdown is
possible on the tertiary carbon (circled carbon on Figure 10) which represents, by far, the
most oxidizable carbon due to the three electronꢀwithdrawing effects exerted by the ester, the
acid, and the quinone groups. Such position could be oxidized twice which would provoke the
scission of the ester bond to produce caffeic acid (Figure 10).
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In both the absence and presence of αꢀtocopherol, caffeic acid was produced from
both R0 and R12 (Figure 11). More caffeic acid was produced from R0 in the presence of αꢀ
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tocopherol (Figure 11A) suggesting that αꢀtocopherol could be involved in the formation of
caffeic acid. The same was true with the case of R12, however, the production of caffeic acid
by αꢀtocopherol was found to a much lower (Figure 11B).
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In all treatments, caffeic acid was detected at the beginning of the oxidation process
and then decreased at the end of the oxidation lag times (Figure 11A and B). Except for R0
alone, the hexanal lag times ended before all caffeic acid was depleted. Caffeic acid has the
ability to scavenge free radicals (46) and Chen and Ho (47) reported that caffeic acid had
better antioxidant activity in O/W emulsions than rosmarinic acid and αꢀtocopherol. In this
study, the degradation of caffeic acid prior to the end of the lag phase also suggests that it is
being preferentially oxidized prior to the fatty acids and thus is acting as an antioxidant.
Therefore, the formation of caffeic acid from R0 in the presence of αꢀtocopherol could
explain why this combination had much better antioxidant activity that the individual
antioxidants since the caffeic acid would provide an additional antioxidant to slow down
oxidation (Figure 10A). In addition to the formation of caffeic acid might be driven by the
proximity between R0 and αꢀtocopherol, it might be enhanced by πꢀπ aromatic interactions
between phenolic compounds. It was suggested that the formation of a stable complex
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between antioxidant due to πꢀπ stacking between the aromatic rings of phenolic compounds
may influence on the overall electron donating capacity, resulting synergistic effects (43-45).
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A quinone of dodecyl rosmarinate ester was observed with R12 but not R0 suggesting
that the esterification of rosmarinic acid with an alkyl chain can increase the stability of the
quinone (Figure 11C) and can thus defavor the formation of caffeic acid. Steric hindrance
exerted by the dodecyl chain of the R12 quinone may slow the rosmarinic acid oxidation
reactions which produces caffeic acid. Therefore, unlike the extremely reactive R0 quinone
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which is rapidly converted into caffeic acid, the R12 quinone may exist long enough to be
detected.
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If we consider that αꢀtocopherol promote the breakdown of the quinone into caffeic
acid, the lower concentration of caffeic acid with R12 compared to R0 could also be due to
R12 reacting less with αꢀtocopherol than R0 as shown by the fluorescence and EPR data
(Figures 5 and 6) thus decreasing the conversion of the quinone to caffeic acid.
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Fluorescence quenching results demonstrates that R12 interacts in a much lesser extent
than R0 with αꢀtocopherol. In absence of bimolecular complex, consequently, the R12
quinone may be too far from the hydroxyl radicals generated by αꢀtocopherol. In these
conditions, αꢀtocopherol does not promote any conversion of R12 quinone into caffeic acid,
which is the observation made on Figure 11B. Furthermore, this could explain why R12 does
not affect the depletion rate of αꢀtocopherol during the first days (Figure 9B).
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In conclusion, R0 was physically able to interact with αꢀtocopherol in surfactant
micelles and O/W emulsions. We hypothesize that the observed synergistic antioxidant
activity of the combination of R0 and αꢀtocopherol were not due to the regeneration of αꢀ
tocopherol by rosmarinic acid due to the thermodynamic infeasibility of this reaction and the
fact that αꢀtocopherol degradation rates in O/W emulsions were not decreased by rosmarinic
acid. In addition, the regeneration of the rosmarinate radical by αꢀtocopherol was also
unlikely since this reaction was slow and αꢀtocopherol did not alter R0 degradation rates.
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Instead αꢀtocopherol and R0 interactions produced an increase in antioxidant activity by
promoting the conversion of rosmarinic acid into caffeic acid thus providing a third molecule
that could inhibit lipid oxidation and increased the oxidative stability of the O/W emulsion. In
contrast, R12 in combination with αꢀtocopherol did not produce synergistic antioxidant
activity and did not significantly increase its conversion into caffeic acid during lipid
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oxidation in O/W emulsions. This could be because R12 did not interact strongly with αꢀ
tocopherol as determined by fluorescence. Overall, physical interaction between αꢀtocopherol
and rosmarinic acid or its alkyl esters seems to be an important factor in their ability to inhibit
lipid oxidation. Further research is needed to determine if physical interactions are important
in determining the antioxidant activity of combinations of other antioxidants.
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ACKNOWLEDGMENTS
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I would like to express my thanks to Prof. Dr. Paul M. Lahti at the chemistry department,
UMass Amherst for being a member of my committee. I appreciated for his donating valuable
times to teach and suggest me on developing EPR techniques used in this research.
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BIBLIOGRAPHY
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