Enzymatic Synthesis of Tyrosol-Based Phenolipids: Characterization and Effect of Alkyl Chain Unsaturation on the Antioxidant Activities in Bulk Oil and Oil-in-Water Emulsion

Article abstract of DOI:10.1007/s11746-015-2775-4

Oxidative stability of lipids is one of the most important parameters affecting their quality. Lipase-catalyzed lipophilic tyrosyl esters with an equivalent carbon alkyl chain but different degrees of unsaturation (C18:0 to C18:4n3) were prepared, charact

Full text of DOI:10.1007/s11746-015-2775-4

J Am Oil Chem Soc (2016) 93:329–337
DOI 10.1007/s11746-015-2775-4
ORIGINAL PAPER
Enzymatic Synthesis of Tyrosol‑Based Phenolipids:
Characterization and Effect of Alkyl Chain Unsaturation on the
Antioxidant Activities in Bulk Oil and Oil‑in‑Water Emulsion
Garima Pande1 · Casimir C. Akoh1
Received: 27 April 2015 / Revised: 4 December 2015 / Accepted: 8 December 2015 / Published online: 30 December 2015
© AOCS 2015
Abstract Oxidative stability of lipids is one of the most
important parameters affecting their quality. Lipase-cat-
alyzed lipophilic tyrosyl esters with an equivalent carbon
alkyl chain but different degrees of unsaturation (C18:0 to
C18:4n3) were prepared, characterized, and used as anti-
oxidants. Free fatty acids and fatty acid ethyl esters (sub-
strate molar ratio tyrosol: acyl donor, 1:10) were used as
acyl donors and immobilized lipase from Candida ant-
arctica was the biocatalyst (10 %). The phenolipids were
Keywords Phenolipids · Tyrosol · Antioxidant ·
Structured lipids · Infant formula
Introduction
Food lipids are a significant functional and healthful con-
stituent of various food products. They affect the chemi-
cal and physical properties of food such as texture and
flavor and also have a major effect on human health. How-
ever, most oils and fats are susceptible to oxidation which
leads to the formation of off-flavors and potentially harm-
ful products [1]. Oxidation in food affects its flavor, nutri-
tional quality, and functional attributes. Antioxidants are
widely used in the manufacture, packaging, and storage of
lipid-containing foods. Much interest has developed dur-
ing the last few decades in naturally occurring antioxidants
because of the adverse attention received by synthetic anti-
oxidants, and also the worldwide trend to avoid or mini-
mize the use of artificial food additives [2].
Phenolic compounds are secondary metabolites pro-
duced by plants and reported to exhibit several health
effects [3]. However, due to their highly polar nature their
use in lipid-based food products is limited. “Phenolipids”
are lipophilized phenolic derivatives produced with an aim
to improve the hydrophobicity of the phenolic moiety and
its efficiency in lipid-based systems. These are amphiphi-
lic in nature as they can be used in both lipid and oil-in-
water emulsion systems. A number of structural factors can
affect the antioxidative activity of the phenolipids such as
the number of phenolic hydroxyl group, presence of pri-
mary hydroxyl group, and nature of the alkyl chain [4].
Phenolipids can be prepared by either esterification of phe-
nolic acids (–COOH) with fatty alcohols [5] or grafting a
fatty acid to the phenolic alcohol (–OH) [1]. The reactions
1
isolated and characterized using ESI–MS, H-NMR, and
13C-NMR. Peroxide value (PV) and para-anisidine value
(p-AV) were measured to evaluate their antioxidant activi-
ties in bulk oil structured lipid (SL) and in an oil-in-water
emulsion (SL-based infant formula). No significant differ-
ence was found in yield and reaction time between the two
types of acyl donors. However, as the unsaturation of the
fatty acids increased the reaction time also increased. In
SL, tyrosyl esters exhibited lower antioxidant activity than
tyrosol whereas the addition of an alkyl chain enhanced the
antioxidant efficiency of tyrosol in infant formula. Tyrosyl
oleate was the most efficient antioxidant in the emulsion
system followed by tyrosyl stearate and tyrosyl linoleate.
These results suggest that the synthesized phenolipids may
be used as potential antioxidants in lipid-based products.
Electronic supplementary material The online version of this
article (doi:10.1007/s11746-015-2775-4) contains supplementary
material, which is available to authorized users.
* Casimir C. Akoh
cakoh@uga.edu
1
Department of Food Science and Technology, University
of Georgia, Athens, GA 30602-2610, USA
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J Am Oil Chem Soc (2016) 93:329–337
employed can be esterification with free acids or transes-
terification with methyl or ethyl esters.
Materials and Methods
Tyrosol and hydroxytyrosol are two characteristic phe-
nolic compounds present in olive oil. Their free forms as
well as their secoroid derivatives constitutes around 30 %,
and other conjugated forms such as oleuropein and lig-
stroside aglycones represent almost half of the total phe-
nolic content of a virgin olive oil [6]. Olive oil phenolics
have beneficial biological effects [7] and also contribute to
the high stability of olive and other oils [8]. Several stud-
ies have been done on the synthesis of phenolipids from
hydroxytyrosol and tyrosol [9–16]. These studies have
focused on the effect of increasing chain length/lipophilic-
ity on the antioxidant or antimicrobial [13] activities of the
phenolipids. In this paper, we used the same chain length
but increasing degree of unsaturation (C18:0 to C18:4).
Trujillo et al. [9] reported that esterification does not affect
the antioxidant activity of hydroxytyrosol in lipid matri-
ces however antioxidant capacities of the lipophilic esters
were enhanced in a biological model. A number of stud-
ies have reported the existence of a critical chain length or
cut-off effect of the synthesized lipophilic phenolic esters
to achieve maximum antioxidant capacity [12, 13, 17–19].
The effect of chain length on the antioxidant ability of ros-
marinate esters [17] and chlorogenate esters [18] on tung
oil based emulsion using conjugated autoxidizable triene
assays, showed the nonlinear behavior and cut-off theory
of the esters. The polar paradox hypothesis explains that
hydrophilic antioxidants are more efficient in bulk oils
whereas lipophilic antioxidants work better in emulsions
and micelles. Recent studies have shown that not all anti-
oxidants follow this hypothesis in oil and emulsion systems
[20]. Most studies have focused on the effect of varying
chain lengths whereas a few have shown the difference
of behavior between saturated and unsaturated long chain
ester derivatives. In this paper we aimed at synthesizing
lipophilic tyrosyl esters with increasing degree of unsatura-
tion but with the same chain length. To this end, we evalu-
ated both free fatty acids and fatty acid ethyl esters as acyl
donors. The antioxidant efficiency of the synthesized esters
were tested in bulk oil structured lipid (SL) and in an oil-
in-water emulsion (SL-based infant formula) using perox-
ide value (PV) and para-anisidine value (p-AV). In a previ-
ous study by our group [21], it was observed that during
SL synthesis and purification the phenolics of the substrate
oil were lost in free and/or esterified form. Therefore, add-
ing them back as phenolipids can help in SL stability. With
the growing trend of clean label and natural ingredients,
use of antioxidants derived from indigenous components of
olive oil in olive oil-based SL can be a potential use of such
phenolipids.
Materials
Tyrosol was obtained from Oakwood Products Inc. (West
Columbia, SC, USA). Stearic, oleic, linoleic, α-linolenic
acids, and their ethyl esters (≥98 %) were purchased
from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
Stearidonic acid and its ethyl ester (>98 %) were pur-
chased from Cayman Chemical Company (Ann Arbor,
MI, USA). The immobilized enzyme Novozym® 435
(Candida antarctica lipase, non-specific, specific activ-
ity 10,000 PLU/g: PLU = Propyl Laurate Unit) was pur-
chased from Novozymes North America Inc. (Franklinton,
NC, USA). Tetrahydrofuran (THF, ≥99 %, anhydrous,
inhibitor-free) was purchased from Sigma-Aldrich Chemi-
cal Co. Other solvents and chemicals were from Fisher
Scientific (Norcross, GA, USA) and Sigma-Aldrich Chem-
ical Co.
Preparation of Phenolipids
Phenolipid synthesis was performed in screw-cap test
tubes. First, 0.1 g of tyrosol was reacted with various
acyl donors (C18:0–C18:4, free and ethyl esters) in a
1:10 substrate molar ratio (tyrosol: acyl donor). Then,
5 mL THF was added and the substrates were thoroughly
mixed. Novozym 435 lipase was added at 10 % total sub-
strate weight. Molecular sieves (3 Å, 4–8 mesh) were
also added (5 % by weight) to the test tubes to eliminate
any moisture formed during the reactions. The tubes were
flushed with nitrogen and the reactions were carried out
at 50 °C for 24 h under constant stirring. Samples were
analyzed at 0, 1, 4, 12, and 24 h for reaction completion.
Reactions were monitored by analyzing tyrosol using a
high-performance liquid chromatograph (HPLC; Agi-
lent Technologies 1260 Infinity, Santa Clara, CA, USA)
equipped with a Sedex 85 evaporative light scattering
detector (ELSD) at 70 °C and 3.8 bar and UV–DAD at
240 and 260 nm. A Beckman Ultrasphere® C18 column,
5 μm, 4.6 × 250 mm was used with the temperature set
at 30 °C. The injection volume was 20 μL. The mobile
phase at a flow rate of 1 mL/min consisted of methanol/
acetonitrile/water (95:3:2, by volume) [22]. The reac-
tion was stopped and the mix was filtered through anhy-
drous sodium sulfate column to remove the enzymes.
Pure tyrosyl esters were obtained using silica solid phase
extraction (SPE) by eluting with hexane/tert-butyl methyl
ether (8:1–10:1) [10]. The samples were dried under
nitrogen and reconstituted in 2 mL methanol and stored
in −20 °C until further analyses.
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331
Characterization of Phenolipids
samples were prepared for 7, 14, and 21 days analyses.
Control samples (containing no added antioxidants) were
also prepared. The PV and p-AV were determined accord-
ing to AOCS Official Method Cd 8b-90 and AOCS Offi-
cial Method Cd18-90, respectively [24]. Total oxidation
(TOTOX) value was calculated as (2 × PV) + p-AV [25].
For electrospray ionization mass spectrometry (ESI–MS)
analysis, the sample (10 µL) was dissolved in 990 µL of
methanol–isopropyl alcohol–propyl alcohol 16:3:3 mix-
ture (infusion buffer). ESI–MS analysis was performed by
direct infusion (DI) on an LCQ mass-spectrometer (Thermo
Fisher Scientific Inc. Waltham, MA, USA) equipped with
electrospray ion source. The samples were infused directly
into the instrument at a constant flow rate of 5 µL/min. The
capillary temperature was set at 275 °C and MS analysis
was performed in the negative ion mode in m/z range of
100–1500. A full FT-MS spectrum was collected at 30,000
resolution, and for MS/MS analysis, the collision energy
was set at 35 eV. For NMR sample preparation, methanol
was removed by evaporation under N2-stream and the sam-
Statistical Analysis
All reactions and analyses were performed in duplicate and
averages reported. Statistical analysis was performed with
the SAS software package (SAS Institute, Cary, NC, USA).
Duncan’s multiple-range test was performed to determine
the significant difference (P ≤ 0.05) between the tyrosyl
esters.
1
ples were dissolved in CDCl3 (deutero-chloroform). H-
and 13C-NMR spectra were recorded on a Bruker 600 MHz
spectrometer (Bruker BioSpin Corporation, Billerica, MA,
USA) equipped with a 5-mm probe at 25 °C, in CDCl3.
Chemical shifts were referenced to residual methanol peak
(δH/δC 3.35/48.8 ppm).
Results and Discussion
Preparation and Characterization of Phenolipids
The lipophilic esters of tyrosol were prepared with two
types of acyl donors, namely, free forms and ethyl esters
of stearic, oleic, linoleic, linolenic, and stearidonic acids.
The reactions were monitored for tyrosol disappearance
at regular intervals. However, complete disappearance of
tyrosol was not obtained during the monitored reaction
time. Therefore, the reactions were stopped when a stable
level of tyrosol was achieved. There was no significant dif-
ference found between the two types of acylating agents
in terms of yield and reaction time. Previous studies have
reported that water formed during esterification reaction
can lower the yield as it facilitates hydrolysis of formed
ester [13]. However, in this study molecular sieves were
added to absorb any water formed during esterification
reactions. Reaction time increased from stearic acid (6 h)
to stearidonic acid (22 h). For oleic, linoleic, and linolenic
acids the reactions were carried out for 10, 12, and 17 h,
respectively. The yields of the tyrosyl esters (measured
gravimetrically after SPE) were 71.6 2.2a, 69.2 1.8a,
64.8 1.3b, 60.4 2.6c, and 54.7 2.1d % for stearate,
oleate, linoleate, linolenate, and stearidonate esters, respec-
tively. Saturated fats have a straight chain-like structure
which allows them to stack tightly and have more inter-
molecular interactions. Unsaturated fatty acids are not lin-
ear and the double bonded carbons results in a bent chain
structure which may cause steric hindrance and decrease
close intermolecular interactions with tyrosol. Similar
results were reported by Zhong and Shahidi while synthe-
sizing epigallocatechin gallate esters with stearic (yield
56.9 %), eicosapentaenoic (yield 42.7 %), and docosahex-
aenoic (yield 30.7 %) acids [26]. In another study, higher
yield and shorter reaction time was achieved using ethyl
Oxidation Study
The peroxide value (PV) and para-anisidine value (p-AV)
were measured to evaluate their antioxidant activities in
bulk oil (SL) and in an oil-in-water emulsion, o/w (SL-
based infant formula, IF). The SL used as the lipid phase in
the infant formula was prepared using a one-pot synthesis
design using tripalmitin, extra virgin olive oil, and free fatty
acids of arachidonic and docosahexaenoic single cell oils in
the ratio of 0.5:1:0.5 [21]. Both enzymes Novozym 435 and
Lipozyme TL IM lipases were added simultaneously and
the reaction was carried out for 24 h at 60 °C [21]. After the
reaction, the extra FFA were removed and the purified SL
was used for oxidation study and infant formula prepara-
tion. Infant formula was prepared as described by Zou and
Akoh [23]. Briefly, high heat nonfat dry milk, [alpha]-lac-
talbumin enriched whey protein concentrate, lactose, and
carrageenan were fully dissolved in water and lecithin was
dispersed in SL. The oil phase was added to the aqueous
phase while stirring at 60 °C and micronutrient premix was
added. Polytron high-speed batch disperser (Kinematica,
Inc., Bohemia, NY, USA) Polytron high-speed batch dis-
perser (Kinematica, Inc., Bohemia, NY, USA) at setting 5
for 2 min and EmulsiFlex-C5 high-pressure homogenizer
(Avestin, Inc., Ottawa, Canada) at 5000–7000 psi (34.47–
48.26 MPa) were used to prepare the emulsion. After pH
adjustment, the resulting emulsion was sterilized in an
autoclave at 121 °C for 6 min 100 ppm of the tyrosyl esters
were added to the samples and stored for 21 days at 45 °C
in the dark in a shaking water bath at 200 rpm. Different
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J Am Oil Chem Soc (2016) 93:329–337
O
Fig. 1 Reaction scheme for
synthesizing lipophilic tyrosol
esters. Reactions were carried
out in tetrahydrofuran with
Novozym 435 lipase as biocata-
lyst at 50 °C for 24 h
OH
O
R
O
+
H2O
+
R
OH
OH
OH
Tyrosol
R=C17H35, stearate
R=C17H33, oleate
Tyrosyl ester
Fatty acid
O
R=C17H31, linoleate
R=C17H29, linolenate
R=C17H27, stearidonate
OH
O
R
O
CH3CH2OH
+
R
OCH2CH3
+
OH
OH
Tyrosol
Tyrosyl ester
Fatty acid ethyl ester
Fig. 2 Representative ESI–MS spectrum of phenolipid product A (tyrosyl stearate). For additional spectra, refer to the Supplementary Material
esters than free fatty acids. However, as the length of the
fatty acids increased from stearic to docosahexaenoic acid,
yield decreased significantly [27].
aqueous samples with minimized fragmentation leaving
mostly unaltered species. A representative ESI–MS spec-
trum is shown in Fig. 2. Full ESI–MS spectra can be found
in the Supplementary Material. For sample A, the most
dominant peak in the ESI–MS spectrum was the dimer
[M2−H]− located at 807 m/z. Phenolipid molecular ion
[M−H]− and fatty acid molecular ion [M−H]− were iden-
tified at 403 and 283 m/z, respectively, confirming the sam-
ple to be tyrosyl stearate. In sample B, tyrosyl ester molec-
ular ion [M−H]− was identified at 401 m/z and fatty acid
molecular ion [M−H]− at 281 m/z, identifying the sample
as tyrosyl oleate. Similarly, the analyzed and calculated m/z
for tyrosyl ester molecular ions [M−H]− for samples C, D,
and E were in accordance for tyrosyl linoleate (anal. 399,
The reaction schemes are shown in Fig. 1. Tyrosol
[4-(2-hydroxyethyl)phenol, C8H10O2 MW 138.16 g/mol]
reacts with different fatty acids (C18:0–C18:4n3) to form
tyrosyl esters, namely tyrosyl stearate (C26H44O3 MW
404.62 g/mol), tyrosyl oleate (C26H42O3 MW 402.61 g/
mol), tyrosyl linoleate (C26H40O3 MW 400.59 g/mol),
tyrosyl linolenate (C26H38O3 MW 398.58 g/mol), and
tyrosyl stearidonate (C26H36O3 MW 396.56 g/mol). The
prepared esters (samples A–E) were identified as specific
tyrosol fatty acid esters by negative ion ESI–MS. The
ESI–MS allows acquisition of mass spectra directly from
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J Am Oil Chem Soc (2016) 93:329–337
333
Table 1 1H- and 13C-NMR chemical shifts (δ, ppm) assignments for lipophilic tyrosol esters
Sample b (CH2)
1H
c (CH2)
1H
d (CH2)
1H
e (Allylic)
f (Olefinic)
g (Bis allylic) h (t-CH3)
Compound
13C
13C
13C
1H
13C
1H
13C
1H
13C
1H
13C
A
B
C
D
E
2.30 34.1 1.61 25.0 1.28 29.4
–
–
–
–
–
–
–
–
0.89 13.7 Tyrosyl stearate
0.89 13.7 Tyrosyl oleate
2.31 34.2 1.62 24.7 1.30 29.1 2.02 26.8 5.32 129.5
2.29 34.2 1.62 24.7 1.33 29.1 2.05 26.8 5.34 129.6 2.78 25.2 0.89 13.5 Tyrosyl linoleate
2.30 34.4 1.62 24.9 1.33 28.9 2.06 27.0 5.35 127.6 2.82 25.3 0.97 13.7 Tyrosyl linolenate
2.32 33.5 1.63 24.0 1.37 28.4 2.08 26.6 5.26 128.5 2.76 25.3 0.98 13.1 Tyrosyl stearidonate
Fig. 3 Representative proton NMR spectrum of phenolipid product A (tyrosyl stearate). For additional spectra, refer to Supplementary Material
calc. 399.59) tyrosyl linolenate (anal. 397, calc. 397.58),
and tyrosyl stearidonate (anal. 395, calc. 395.56). The ran-
dom peaks detected in full MS spectra can be [M−Ty]−
and fatty acid dimers.
Material). For proton labels and assignments the structure
for each sample is inserted on respective sample’s proton
spectrum. The numbers on the peaks represent chemical
shift values. The proton NMR chemical shift is affected by
proximity to electronegative atoms and unsaturated groups
and the carbon NMR chemical shift is influenced by elec-
tronegative and steric effects. For 1H-NMR, the proton
chemical shift assignments (δ, ppm) for free tyrosol were
3.73 (1′), 2.73 (2′), 7.04 (4′, 8′), and 6.73 (5′, 7′) and for
tyrosyl residue the chemical shifts were 4.21 (1′), 2.83 (2′),
1H-NMR and 13C-NMR analyses were used to confirm
the structures from the results obtained from negative ion
ESI–MS method. The results of chemical shift (δ, ppm)
assignments for the tyrosyl esters are given in Table 1
and a representative proton NMR spectrum is shown in
Fig. 3 (complete spectra can be found in Supplementary
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J Am Oil Chem Soc (2016) 93:329–337
7.04 (4′, 8′), and 6.73 (5′, 7′). A downward chemical shift
was observed for 1′ (∆δ 0.48) and 2′ (∆δ 0.10) protons. In
case of 13C-NMR, the proton chemical shift assignments (δ,
ppm) for free tyrosol were 63.5 (1′), 37.6 (2′), 129.4 (3′),
129.9 (4′, 8′), 114.7 (5′, 7′), and 155.3 (6′) and for tyrosyl
residue the chemical shifts were 64.9 (1′), 34.3 (2′), 129.4
(3′), 129.9 (4′, 8′), 114.7 (5′, 7′), and 155.3 (6′). A larger
downward chemical shift was observed for 1′ (∆δ 1.4)
and an upward shift was observed for 2′ (∆δ 3.3) protons.
The difference in shifts is because acylation on a hydroxyl
group results in a downward chemical shift at the O-acyl
carbon (1′ in tyrosyl residue) and an upward chemical shift
at the adjacent carbon (2′ in tyrosyl residue) [28]. Similar
decreasing intensity of methylene (d, CH2) proton signals
in the proton spectra of C, D, and E, indicated progressing
amount of double bonds in Samples C through E (c-tyrosyl
linoleate, d-tyrosyl linolenate, e-tyrosyl stearidonate).
These results are similar to those reported by Mateos et al.
[10]. However, free tyrosol was observed in samples A and
E but not in samples B, C, and D. Although the reactions
were monitored for tyrosol disappearance but complete
disappearance was not achieved and the reactions were
stopped when a stable level of tyrosol was observed. There-
fore, the samples may contain some tyrosol especially sam-
ple E because stearidonic acid due to steric hindrance had
the longest reaction time and the lowest yield.
1
trends in chemical shifts for H-NMR and 13C-NMR were
reported by Zhong and Shahidi [26]. No chemical shift was
detected in the aromatic ring protons. No allylic (e), bis-
allylic (g) or olefinic protons (f) were detected in Samples
A, confirming that the sample is tyrosyl stearate. No bis-
allylic protons (g) were detected in Samples B but the pres-
ence of olefinic proton (f) signals indicated that sample B
is tyrosyl oleate. Increasing intensity of bis-allylic (g), and
Oxidation Stability
The antioxidant effectiveness/efficiency of the synthesized
tyrosyl esters were tested in bulk oil (SL) and an o/w emul-
sion (SL-based infant formula, IF). The major fatty acids
in the SL were oleic (38.08 mol%), palmitic (35.23 mol%),
arachidonic (6.23 mol%), and docosahexaenoic acids
Fig. 4 Effect of lipophilic tyro-
sol esters on the oxidative sta-
bility of a bulk oil as structured
lipid (SL) and b SL-based infant
formula o/w emulsion measured
by peroxide value over time at
45 °C Data points represent the
average of duplicate samples
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J Am Oil Chem Soc (2016) 93:329–337
335
Fig. 5 Effect of lipophilic
tyrosol esters on the oxidative
stability of a structured lipid
(SL) and b SL-based infant
formula emulsion measured by
p-anisidine value over time at
45 °C. Data points represent the
average of duplicate samples
(3.71 mol%) [21]. PV (expressed as mequiv/kg) of the
SL and emulsion are shown in Fig. 4a, b, respectively. PV
measures the amount of iodine formed by the reaction of
the peroxides with the iodine ion. PV is a measurement of
the initial/primary products of oxidation. p-AV (expressed
as absorbance/mL) of the SL and infant formula emulsion
are shown in Fig. 5a, b, respectively. p-AV determines the
amount of aldehydes or decay products of the hydroper-
oxides by reaction of these compounds with p-anisidine.
It is a measure of the secondary oxidation products. The
TOTOX value is an evaluation of both primary and second-
ary oxidation products used to estimate oxidative deteriora-
tion of food lipids.
In SL, tyrosyl esters exhibited lower antioxidant effec-
tiveness than tyrosol whereas the addition of an alkyl
chain enhanced the antioxidant efficiency of tyrosol in
infant formula. The PV and p-AV of tyrosol for SL after
21 days of storage were 4.9 0.8 and 5.1 0.9 mequiv/
kg, respectively. Tyrosol being the most polar was effective
in bulk oil system following the polar paradox hypothesis.
However, there was no specific order based on polarity for
the antioxidant efficiency of the tyrosyl esters. The results
indicate that the antioxidant efficiency of a compound is
not based only on its polarity as stated in the polar para-
dox hypothesis but also depends on other factors such as
the medium and concentration. The critical site of oxida-
tion in bulk oil is not the air-oil interface rather the asso-
ciation colloids formed with traces of water and surface
active molecules such as phospholipids [20]. In another
study comparing lipophilic tyrosyl and hydroxytyrosol
esters, higher antioxidant capacity was found in hydroxyty-
rosol due to its o-diphenolic structure [10]. In lipid matrix,
lower antioxidant activity was reported for the esters than
the parent phenol. However, in FRAP and ABTS assays,
hydroxytyrosol esters were more efficient than hydroxy-
tyrosol but tyrosol esters were less active than free tyro-
sol. They also found no significant effect of chain length
on antioxidant activity [10]. Similarly, for oil-in-water
emulsion (infant formula), a nonlinear trend was observed
for the lipophilic tyrosol esters. After 21 days of stor-
age, tyrosyl oleate had the lowest PV (4.3
0.3) and p-
AV (6.1
0.8) in the emulsion system. Based on the
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total oxidation value (TOTOX) the order of antioxidant
efficiency in SL was tyrosol (14.8
0.2a), tyrosyl lino-
lenate (20.8 1.1b), tyrosyl oleate (22.6 0.7bc), tyrosyl
linoleate (24.1 0.9c), tyrosyl stearidonate (26.0 1.3d),
tyrosyl stearate (30.6
For the emulsion system, the order was tyrosyl oleate
(14.7 0.8a), tyrosyl stearate (17.6 0.9b), tyrosyl
1.7e), and control (46.4
2.2f).
linoleate (21.1
tyrosyl stearidonate (24.8
1.5c), tyrosyl linolenate (24.7
0.6d),
1.4e),
1.1d), tyrosol (31.9
and control (43.1
2.1f). These results show that the
most efficient antioxidant in the bulk oil was tyrosol, and
in oil-in-water emulsion, tyrosyl oleate was the most effi-
cient antioxidant. Tyrosyl stearidonate was less efficient
than tyrosyl oleate and linoleate. This trend may probably
be due to complex structure of stearidonate moiety which
may cause steric hindrance and hinder the mobility and
positioning of the phenolipid on the desired interface in the
matrix. It has been postulated that the structure and size of
an antioxidant may affect its mobility, location (internaliza-
tion or interfacial position), and stability (self-aggregation)
which in turn determine its antioxidant efficiency in a sys-
tem [29].
11. Lucas R, Comelles F, Alcántara D, Maldonado OS, Curcuroze
M, Parra JL, Morales JC (2010) Surface-active properties of
lipophilic antioxidants tyrosol and hydroxytyrosol fatty acid
esters: a potential explanation for the nonlinear hypothesis of
the antioxidant activity in oil-in-water emulsions. J Agric Food
Chem 58:8021–8026
12. Medina I, Lois S, Alcántara D, Lucas R, Morales JC (2009)
Effect of lipophilization of hydroxytyrosol on its antioxidant
activity in fish oils and fish oil-in-water emulsions. J Agric Food
Chem 57:9773–9779
13. Aissa I, Sghair RM, Bouaziz M, Laouini D, Sayadi S, Gargouri
Y (2012) Synthesis of lipophilic tyrosyl esters derivatives and
assessment of their antimicrobial and antileishmania activities.
Lipids Health Dis 11:1–8
14. Tofani D, Balducci V, Gasperi T, Incerpi S, Gambacorta A (2010)
Fatty acid hydroxytyrosyl esters: structure/antioxidant activity
relationship by ABTS and in cell-culture DCF assays. J Agric
Food Chem 58:5292–5299
15. Bouallagui Z, Bouaziz M, Lassoued S, Engasser JM, Ghoul M,
Sayadi S (2011) Hydroxytyrosol acyl esters: biosynthesis and
activities. Appl Biochem Biotechnol 163:592–599
16. Bernini R, Crisante F, Barontini M, Tofani D, Balducci V, Gam-
bacorta A (2012) Synthesis and structure/antioxidant activity
relationship of novel catecholic antioxidants structurally ana-
logues to hydroxytyrosol and its lipophilic esters. J Agric Food
Chem 60:7408–7416
17. Laguerre M, López Giraldo LJ, Lecomte J, Figueroa-Espinoza
MC, Baréa B, Weiss J, Decker EA, Villeneuve P (2010) Rela-
tionship between hydrophobicity and antioxidant ability of “phe-
nolipids” in emulsion: a parabolic effect of the chain length of
rosmarinate esters. J Agric Food Chem 58:2869–2876
18. Laguerre M, López Giraldo LJ, Lecomte J, Figueroa-Espinoza
MC, Baréa B, Weiss J, Decker EA, Villeneuve P (2009) Chain
length affects antioxidant properties of chlorogenate esters in
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Conclusion
In summary, several lipophilic tyrosyl esters with increasing
unsaturation levels were prepared in this study. The oxidation
study results from bulk oil and oil-in-water emulsion sug-
gest that the synthesized phenolipids may be used as poten-
tial antioxidants in lipid-based products. Addition of an acyl
moiety increased the antioxidant activity of tyrosol in the
emulsion system. In this study increasing the level of unsatu-
ration in the acyl moiety was not directly proportional to its
antioxidant efficiency. Therefore, it is important to determine
the best and type of acyl group when synthesizing phenolip-
ids for use as effective antioxidants. Structure modification
of tyrosol to increase its lipophilicity may improve its anti-
oxidant effectiveness but further investigations are required
to assess the biological activity of these phenolipids.
Acknowledgments This research was supported in part by the
Department of Energy grant “Plant and Microbial Complex Carbo-
hydrates” (DE-FG02-93ER20097) to Parastoo Azadi at the Complex
Carbohydrate Research Center and the Food Science Research at the
Department of Food Science, University of Georgia.
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