Antioxidant activity of hydroxytyrosol acetate compared with that of other olive oil polyphenols

Article abstract of DOI:10.1021/jf000537w

Hydroxytyrosol acetate was synthesized, and the antioxidant activity of this olive oil component was assessed in comparison with that of other olive oil components, namely hydroxytyrosol, oleuropein, 3,4-DHPEA-EA, and α-tocopherol in bulk oil and oil-in-water emulsions. The activity of the compounds was also assessed by scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. Hydroxytyrosol acetate had a weaker DPPH radical scavenging activity than hydroxytyrosol, oleuropein, or 3,4-DHPEA-EA but it had a radical scavenging activity similar to that of α-tocopherol. In oil, the antioxidant activity of hydroxytyrosol acetate was much higher than that of α-tocopherol or oleuropein, but in an emulsion 3,4-DHPEA-EA and α-tocopherol were more effective as antioxidants than hydroxytyrosol acetate. The antioxidant activity of hydroxytyrosol acetate was rather similar to that of hydroxytyrosol in oil and emulsions despite the difference in DPPH radical scavenging activity.

Full text of DOI:10.1021/jf000537w

2480
J. Agric. Food Chem. 2001, 49, 2480−2485
An tioxid a n t Activity of Hyd r oxytyr osol Aceta te Com p a r ed w ith Th a t
of Oth er Olive Oil P olyp h en ols
Michael H. Gordon,*,† Fa´tima Paiva-Martins,‡ and Miguel Almeida‡
Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading,
Whiteknights P.O. Box 226, Reading RG6 6AP, UK, and Departamento de Qu´ımica, Faculdade de Cieˆncias,
Universidade do Porto, Rua do Campo Alegre, number 687, 4169-007 Porto, Portugal
Hydroxytyrosol acetate was synthesized, and the antioxidant activity of this olive oil component
was assessed in comparison with that of other olive oil components, namely hydroxytyrosol,
oleuropein, 3,4-DHPEA-EA, and R-tocopherol in bulk oil and oil-in-water emulsions. The activity of
the compounds was also assessed by scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals.
Hydroxytyrosol acetate had a weaker DPPH radical scavenging activity than hydroxytyrosol,
oleuropein, or 3,4-DHPEA-EA but it had a radical scavenging activity similar to that of R-tocopherol.
In oil, the antioxidant activity of hydroxytyrosol acetate was much higher than that of R-tocopherol
or oleuropein, but in an emulsion 3,4-DHPEA-EA and R-tocopherol were more effective as
antioxidants than hydroxytyrosol acetate. The antioxidant activity of hydroxytyrosol acetate was
rather similar to that of hydroxytyrosol in oil and emulsions despite the difference in DPPH radical
scavenging activity.
Keyw or d s: Olive oil; hydroxytyrosol; 3,4-DHPEA-EA; oleuropein; hydroxytyrosol acetate; R-toco-
pherol; emulsion; antioxidants
INTRODUCTION
From comparisons with a limited number of phenolic
constituents, it has been claimed that hydroxytyrosol
is the most active antioxidant compound in virgin olive
oil, although the concentration of free hydroxytyrosol
is sometimes low (7-10). Oleuropein, a hydroxytyrosol
derivative, which occurs in large amounts in olive leaves
and olive fruits (up to 14% of the dry weight in unripe
olives), has shown a weak antioxidant activity in olive
oil (7, 9 ,10). Both hydroxytyrosol and oleuropein have
been shown to be scavengers of superoxide anions, and
inhibitors of the respiratory burst of neutrophils and
hypochlorous acid-derived radicals, but hydroxytyrosol
was more effective than oleuropein (1). Both compounds
also scavenged hydroxyl radicals, but in this case
oleuropein showed greater activity (11).
Virgin and extra virgin olive oils are among the very
few oils consumed without refining, and which conse-
quently contain phenolic compounds besides toco-
pherols, which may play a role in the health benefits of
the oil. Epidemiological evidence shows that the Medi-
terranean diet is associated with lower incidence of both
coronary heart disease (CHD) and certain tumors (1,
2). Olive oil, which is rich in monounsaturated, and low
in saturated, fatty acids is the major fat component of
this diet. The oxidative stability of low-density lipopro-
teins (LDL) isolated from animals fed virgin olive oil is
increased in comparison with a control comprising a
triglyceride preparation with similar fatty acid composi-
tion, and this increased stability is attributable to the
minor components in the oil (3, 4). In recent years there
has been much interest in the effects of antioxidants in
retarding oxidative modification of LDL, which is be-
lieved to be a key step in the initiation of atherosclerosis
(5).
Olive oil hydrophilic extracts contain a large number
of phenolic compounds including phenyl-alcohols, such
as 3,4-dihydroxyphenylethanol (3,4-DHPEA or hydroxy-
tyrosol) and p-hydroxyphenylethanol (p-HPEA or tyro-
sol), as well as phenyl-acids. Oleosidic forms of 3,4-
DHPEA, in particular the dialdehydic form of elenolic
acid linked to 3,4-DHPEA (3,4-DHPEA-EDA), an isomer
of oleuropein aglycon (3,4-DHPEA-EA), and the dialde-
hydic form of elenolic acid linked to p-HPEA (p-HPEA-
EDA) have been identified as the major secoiridoid
compounds of virgin olive oil (6).
Studies with the Rancimat method at 120 °C showed
that the oxidative stability of olive oil at elevated
temperatures mainly correlated with the concentration
of hydroxytyrosol and its oleosidic forms. 3,4-DHPEA-
EA and 3,4-DHPEA-EDA showed similar antioxidant
activity to hydroxytyrosol under these conditions (12,
13). These compounds also inhibited copper sulfate-
induced LDL oxidation (14).
Recently another hydroxytyrosol derivative, hydroxy-
tyrosol acetate (3,4-dihydroxyphenylethyl acetate) has
been found in olive oil (15) (Figure 1). The aim of this
research has been to investigate the antioxidant activity
of this compound in comparison with that of the phenolic
extract from olive oil and the pure components hydroxy-
tyrosol, oleuropein, 3,4-DHPEA-EA, and R-tocopherol in
bulk oil and oil-in-water emulsions. The activity of the
single compounds and the phenolic extract were also
assessed by scavenging of diphenylpicrylhydrazyl (DPPH)
radicals.
* To whom correspondence should be addressed. Tel: 44-
1189-316723. Fax: 44-1189-310080. E-mail: m.h.gordon@
reading.ac.uk.
† The University of Reading.
‡ Universidade do Porto.
10.1021/jf000537w CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/04/2001

Antioxidant Activity of Hydroxytyrosol Acetate
J. Agric. Food Chem., Vol. 49, No. 5, 2001 2481
F igu r e 1. Structures of olive oil phenolics.
MATERIALS AND METHODS
concentrations were tested. Phenolic compound solution (0.1
mL) was added to 3.5 mL of a 0.06 mM methanol DPPH radical
solution. The decrease in absorbance was determined at 515
nm at 0, 5, and every 10 min until 250 min. The exact initial
DPPH radical concentration (CDPPH) in the reaction was
calculated from a calibration curve with the equation
Ma ter ia ls. Phenolic extract was obtained from commercial
virgin olive oil following the procedure described by Montedoro
et al. (6) and quantified by the Folin-Ciocalteu reaction,
expressed as caffeic acid equivalent. All calculations were
made by assuming a mean relative molecular mass of the
phenolic extract of 300. Hydroxytyrosol was synthesized from
3,4-dihydroxyphenylacetic acid (Sigma-Aldrich Quimica-S. A.,
Madrid, Spain) according to the procedure of Baraldi et al. (16).
Oleuropein was purchased from Extrasynthese (Genay, France)
or extracted from olive leaves according to the procedure of
Garibaldi et al. (17). 3,4 -DHPEA-EA was obtained from
oleuropein by enzymatic reaction using â-glycosidase (Fluka,
Buchs, Switzerland) according to the procedure of Limirioli
et al. (18). R-Tocopherol, trolox, tween 20, and soybean
phosphatidylcholine were obtained from Sigma. Sodium ac-
etate, acetic acid, sodium hydrogen phosphate, sodium phos-
phate, and silica gel (130-230 mesh) were supplied by Merck
(Merck Farma e Quimica, S. A., Lisbon, Portugal). LiAlH4,
benzyl bromide, dicyclohexylcarbodiimide (DCC), p-toluene-
sulfonic acid (TSA), Pd-C 10%, 1,4-cyclohexadiene, 1-octanol,
and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were supplied by
Sigma-Aldrich. All other reagents were analytical grade or
purer.
Y ) 0.1108x + 0.0001 (r2 ) 0.999)
as determined by linear regression. For each compound tested,
the reaction kinetics were plotted, and from these graphs the
percentage of DPPH radical remaining at several times was
determined. The values were transferred onto another graph
showing the percentage of residual DPPH radical as a function
of the molar ratio of phenolic compound to DPPH radical.
Antiradical activity was defined as the relative concentration
of antioxidant required to lower the initial DPPH concentration
by 50% [EC50 (mol/L phenolic compound per unit DPPH
concentration)].
Mea su r em en t of P a r tition Coefficien t (log P ). A solu-
tion (0.3 mM) of each compound in 1-octanol was kept at 60
°C for an hour. An UV spectrum was then run, and the value
of absorbance at the maximum was measured (A0). Equal
volumes of organic solution and acetate buffer (0.1 M, pH 5.5)
or phosphate buffer (0.1M, pH 7.4) were vortexed (2500 rpm)
for 1 min. The UV spectrum of the organic layer was deter-
mined after 30 min (Ax). The partition coefficient (log P) was
calculated according to the relationship, P ) Ax/(A0 - Ax ). A
solution of n-octanol saturated with water was used as the
blank.
Bu lk Oil Sa m p les. Olive oil (10 g), stripped of natural
tocopherols and phenols, with each additive at a final concen-
tration of 0.3 mmol/kg or 0.6 mmol/kg was stored in a 10-cm
Petri dish.
Em u lsion Sa m p les. 30% Oil-in-water emulsions (33 g)
were prepared in 100-mL Erlenmeyer flasks. The emulsions
were made by mixing 10 g of olive oil, stripped of natural
tocopherols and phenols, containing each additive at a final
concentration of 0.3 mmol‚kg-1, 0.66 g of tween 20 as emulsi-
fier, and 22.3 g of acetate buffer (0.1 M, pH 5.5), 3N-
morpholinopropanesulfonic acid (MOPS) or phosphate buffer
(0.1 M, pH 7.4). The mixture was sonicated for 10 min in an
ice bath.
Oxidation Exper im en ts. Bulk Oil and Emulsions. Samples
were oxidized in the dark at 60 °C. Each sample was studied
in triplicate. Isolation of oil from emulsions for analysis was
by freezing, thawing, and centrifugation. Progress of oxidation
was monitored by determination of the peroxide value (PV)
(AOCS Official Method Cd 8-53) or conjugated dienes (CD)
(AOCS Official Method Ti 1a-64) and p-anisidine value (AV)
(AOCS Official Method Cd 18-90).
Sta tistica l An a lysis. Statistical analysis to determine
significant differences in antioxidant activity involved plotting
PV or CD against time to determine times to certain values
and then applying ANOVA one-way with Tukey’s HSD mul-
tiple comparison to determine differences significant at the
5% level.
Olive oil stripped of natural tocopherols and phenols was
prepared from commercial virgin olive oil by washing with 0.5
M NaOH (Merck) solution and passing twice through an
aluminum oxide column (Merck). Complete removal of toco-
pherols was confirmed by HPLC, according to IUPAC Method
2.432.
P r ep a r a tion of Hyd r oxytyr osol Aceta te. The phenolic
groups of hydroxytyrosol were protected by the reaction with
benzyl bromide according to the method of Kanai et al. (19).
The 2-[(3,4-dibenzyloxyphenyl]ethanol was purified by eluting
through a silica gel column with chloroform/ethyl acetate (7:
3) (v/v). The yield of this reaction was 80%. This compound
(0.8 g) and acetic acid (0.14 mL) were dissolved in pyridine (3
mL); DCC (0.6 g) and TSA (0.02 g) were then added. After the
mixture was stirred at room temperature for 72 h, acetic acid
was added and the mixture was filtered. Chloroform was added
and the organic layer was washed with hydrochloric acid (5
M), water, sodium carbonate solution (0.5 M), and water. The
chloroform phase was evaporated in a vacuum rotary evapora-
tor at 35-40 °C and the mixture was purified by eluting
through a silica gel column with dichloromethane/light petro-
leum 40-60 °C (1:1) (v/v). 2-[(3,4-Dibenzyloxyphenyl]ethyl
acetate was obtained, and the yield of this reaction was 40%.
The benzyloxy groups from this compound were removed by
catalytic hydrogenation with a palladium-on-carbon catalyst
(10%) and 1,4-cyclohexadiene, according to the method of Felix
et al. (20). The hydroxytyrosol acetate obtained was purified
by eluting through a silica gel column with ethyl acetate/light
petroleum 40-60 °C (1:1) (v/v). The yield of this reaction was
75%. NMR (methanol-d) δΗ 6.69-6.49 (m, 3H, ArH), 4.91 (br
s, 2H, OH), 4.16 (t, J ) 7.1 Hz, 2H, CH2-O), 2.74 (t, J ) 7.1
Hz, CH2-Ph), 1.99 (s, 3H, CH3); δC 173.0 (CO), 146.2 and
144.9 (ArC-OH), 130.7 (ArC-CH2), 121.1, 117.0, and 116.3
(ArC-H), 66.6 (CH2-O), 35.4 (Ph-CH2), 20.8 (CH3).
RESULTS AND DISCUSSION
Deter m in a tion of Ra d ica l Sca ven gin g Activity. 1,1-
Diphenyl-2-picrylhydrazyl (DPPH•) radical was used as a
stable radical (21). For each phenolic compound, several
DP P H Sca ven gin g Test. The radical scavenging
activity, assessed by the antioxidant concentration

2482 J. Agric. Food Chem., Vol. 49, No. 5, 2001
Gordon et al.
Ta ble 1. DP P H Ra d ica l-Sca ven gin g Effects of P h en olic Com p ou n d s a fter Va r iou s Rea ction Tim es (15 m in , 60 m in , a n d
250 m in )a
time 15 minb
time 60 minb
time 250 minb
no. of
reduced DPPH
no. of
reduced DPPH
no. of
reduced DPPH
c
c
c
compound
EC50
EC50
EC50
phenolic extract
hydroxytyrosol acetate
R-tocopherol
0.40a (( 0.01)
0.26b (( 0.01)
0.25b (( 0.01)
0.24b (( 0.01)
0.22c (( 0.01)
0.19d (( 0.01)
0.12e (( 0.0)
1.3
2.0
2.0
2.1
2.2
2.7
4.3
0.36a (( 0.01)
0.22b (( 0.01)
0.25c (( 0.01)
0.24c (( 0.02)
0.18d (( 0.01)
0.13e (( 0.01)
0.10f (( 0.01)
1.4
2.2
2.0
2.1
2.6
3.6
4.7
0.26a (( 0.01)
0.14b (( 0.01)
0.24a,c (( 0.01)
0.24c,a (( 0.02)
0.12b (( 0.01)
0.099d (( 0.004)
0.062e (( 0.002)
1.5
3.6
2.1
2.1
4.0
5.1
8.0
trolox
oleuropein
hydroxytyrosol
3,4-DHPEA-EA
a
b
Superscripts within a row indicate samples that were significantly different (p < 0.05). nd, not determined. Mean (standard deviation
in parentheses) of four determinations. c EC50 expressed as mol of antioxidant/mol of DPPH radical.
Ta ble 2. Tim e for Str ip p ed Olive Oil Sa m p les Con ta in in g
Ad d itives (0.3 m m ol‚k g-1) to Rea ch a P er oxid e Va lu e of
50 Meq‚k g-1 a t 60 °C
compound
time (days)a
control
oleuropein
R-tocopherol
3,4-DHPEA-EA
phenolic extract
hydroxytyrosol acetate
hydroxytyrosol
2.36 (( 0.37)a
8.51 (( 0.33)b
20.24 (( 0.18)c
64.38 (( 1.45)d
66.03 (( 0.79)d,e
66.54 (( 0.78)e,d
70.45 (( 0.13)f
a
Mean (standard deviation in parentheses) of triplicate stored
samples. Superscripts within a column indicate samples that were
significantly different (p < 0.05).
required for 50% reduction in DPPH radical concentra-
tion in 15 min (EC50), decreased in the following order:
3,4-DHPEA-EA . hydroxytyrosol > oleuropein > hy-
droxytyrosol acetate, R-tocopherol, trolox > phenolic
extract (Table 1). After 15 min, the reaction of DPPH
radical with R-tocopherol and trolox (the water-soluble
analogue of R-tocopherol) was nearly at the steady state.
Hydroxytyrosol acetate, the other pure phenolics, and
the phenolic extract were similar to tocopherol and
trolox in reacting rapidly with the DPPH radical, but
they also showed slower reactions with the radical, with
hydroxytyrosol acetate showing a 46% reduction in EC50
between 15 and 250 min. For an antioxidant to be
effective in interrupting the propagation step of lipid
oxidation, it must react much faster with lipid radicals
than linoleic acid does, despite being present at much
lower concentrations than the polyunsaturated fatty
acid in an oil, and hence the EC50 value at 15 min, which
is a measure of more rapid reactions with DPPH
radicals, is probably more relevant than the slower
reactions. The EC50 value at 15 min for hydroxytyrosol
acetate (0.26 mol AH/mol DPPH) was significantly
greater than for hydroxytyrosol and oleuropein (0.19
and 0.22 mol AH/mol DPPH). The acetate group of the
ester may hinder the scavenging effect of the hydroxyl
groups by intra- or intermolecular hydrogen bonding.
Oxid a tive Sta bility of Bu lk Olive Oil Con ta in in g
Olive Oil P h en olics. The antioxidant effects of pure
olive oil phenolic compounds in olive oil, stripped of
natural phenolic compounds and tocopherols, were
assessed by the peroxide value (PV) to determine
primary oxidation products and by the p-anisidine value
determination (AV) to determine secondary oxidation
products. On the basis of the time to a PV of 50
meq‚kg-1, antioxidant efficiency for 0.3 mmol‚kg-1 ad-
dition decreased in the following order: hydroxytyrosol
> hydroxytyrosol acetate, phenolic extract, 3,4-DHPEA-
EA . R-tocopherol > oleuropein > control (Figure 2a,
Table 2). The AV determinations confirmed the order
F igu r e 2. Effect of pure olive oil phenolics and olive oil
phenolic extract at 0.3 mmol‚kg-1 on the oxidation of olive oil
triacylglycerols stored at 60 °C, assessed by (a) the peroxide
value and (b) the anisidine value.
Ta ble 3. P a r tition Coefficien t of P h en olic Com p ou n d s
Log Pa
compound
pH 5.5
pH 7.4
hydroxytyrosol
DHPEA-EA
hydroxytyrosol acetate
oleuropein
phenolic extract
R-tocopherol
0.03 (( 0.01)
1.13 (( 0.08)
0.82 (( 0.01)
0.17 (( 0.01)
0.78 (( 0.03)
1.37 (( 0.04)
0.02 (( 0.01)
1.16 (( 0.08)
0.82 (( 0.05)
0.13 (( 0)
nd
1.38 (( 0.01)
a
Mean (standard deviation in parentheses) of triplicate deter-
minations.
of antioxidant activity (Figure 2b). The polarity of
antioxidants was assessed by the partitioning between
octanol and water (Table 3). Comparing the order of
activity as antioxidants in oil with the DPPH radical

Antioxidant Activity of Hydroxytyrosol Acetate
J. Agric. Food Chem., Vol. 49, No. 5, 2001 2483
F igu r e 4. Effect of pure olive oil phenolics at 0.3 mmol‚kg-1
on the oxidation of stripped olive oil triacylglycerols-in-water
emulsions made with MOPS buffer at pH 7.4, stored at 60 °C,
assessed by (a) the conjugated diene content and (b) the
anisidine value.
F igu r e 3. Effect of pure olive oil phenolics and olive oil
phenolic extract at 0.3 mmol‚kg-1 on the oxidation of stripped
olive oil triacylglycerols-in-water emulsions at pH 5.5, stored
at 60 °C, assessed by (a) the conjugated diene content and (b)
the anisidine value.
of van der Waals interactions between triglyceride
molecules and the ester side chain. However, even if
hydroxytyrosol acetate and hydroxytyrosol had similar
radical scavenging activity, the polar paradox would
suggest that there would be a marked difference in
antioxidant activity in oil but the effect is rather small.
Oleuropein, which is a glycoside, is less effective than
predicted by the polar paradox in less polar media as
previously observed for other glycosides (24).
scavenging activity, it is clear that hydroxytyrosol
acetate is only slightly less effective as an antioxidant
in oil than the more polar antioxidant hydroxytyrosol
despite being considerably less polar and having re-
duced radical scavenging activity assessed by the DPPH
scavenging test. According to the polar paradox, more
polar antioxidants are more effective in less polar media
(22, 23). The good antioxidant activity of hydroxytyrosol
acetate in oil suggests that any inter- or intramolecular
hydrogen bonding between the ester group of this
molecule and the active hydroxyl groups, which con-
tributes to reduced radical scavenging activity in the
DPPH test, does not occur in oil, presumably because
Oxid a tive Sta bility of Olive-Oil-in -Wa ter Em u l-
sion s con ta in in g Olive Oil P h en olics. Antioxidant
effects were studied at 60 °C in oil-in-water emulsions
prepared from olive oil stripped of natural phenolic
compounds and tocopherols. The emulsions were physi-
cally stable during all the experiments. Oxidation was
more rapid in emulsions (Figures 3 and 4) than in bulk
oil (Figure 2). The oxidative stability of the emulsions
Ta ble 4. Tim e for Str ip p ed Olive Oil-in -Wa ter Em u lsion Sa m p les to Rea ch a Con ju ga ted Dien e Con ten t of 0.4 %
time (days) to CD ) 0.4%a)
pH 5.5
pH 7.4
compound
control
hydroxytyrosol
hydroxytyrosol acetate
oleuropein
phenolic extract
DHPEA-EA
R-tocopherol
acetate buffer
phosphate buffer
MOPS buffer
3.79 ((0.08)a
5.99 ((0.25)b
7.78 ((0.29)c
7.94 ((0.17)c
8.38 ((0.45)c
13.40 ((0.59)d
14.14 ((0.27)d
3.03 ((0)a
6.57 ((0.04)b
7.84 ((0.28)c
7.02 ((0)b
nd
6.80 ((0.50)a
16.97 ((0.52)b
17.65 ((0.50)b
20.48 ((0.99)c
nd
8.13 ((0.17)c
12.04 ((0.33)d
22.58 ((0.81)d
21.21 ((0.24)c,d
a
Mean (standard deviation in parentheses) of triplicate stored samples. Superscripts within a row indicate samples that were significantly
different (p < 0.05). nd, not determined.

2484 J. Agric. Food Chem., Vol. 49, No. 5, 2001
Gordon et al.
(3) Scaccini, C.; Nardini, M.; D’Aquino, M.; Gentili, V.;
Felice, M. D.; Tomassi, G. Effect of dietary oils on lipid
peroxidation and antioxidant parameters of rat plasma
and lipoprotein fractions. J . Lipid Res. 1992, 33, 627-
633.
(4) Wiseman, S. A.; Mathot, J . N. N. J .; De Fouw, N. J .;
Tijburg, L. B. M. Dietary non-tocopherol antioxidants
present in extra virgin olive oil increase the resistance
of low-density lipoproteins to oxidation in rabbits.
Atherosclerosis 1996, 120, 15-23.
(5) Steinberg, D.; Parthasarathy, S.; Carew, T. E.; Khoo, J .
C.; Witzum, J . L. Beyond cholosterol. Modifications of
low-density lipoprotein that increases its atherogenicity.
N. Engl. J . Med. 1989, 320, 915-924.
(6) Montedoro, G. F.; Servili, M.; Baldioli, M.; Selvaggini,
R.; Miniati, E.; Maccchioni, A. Simple and Hydrolyzable
Compounds in Virgin Olive Oil. 3. Spectroscopic Char-
acterizations of the Secoiridoid Derivatives. J . Agric.
Food Chem. 1993, 41, 2228-2234.
(7) Chimi, H.; Sadik, A.; Le Tutour, B.; Rahmani, M.
Contribuyion a` l’e´tude comparative des pouvoirs anti-
oxidants dans l’huile d’olive du tyrosol, de l’hydroxytyro-
sol, de l’acide cafe´ique, de l’oleuropeine et du B. H. T.
Rev. Fr. Corps Gras 1988, 35, 339-344.
(8) Chimi, H.; Cillard, J .; Cillard, P.; Rahmani, M. Cine´tique
du de´gradation des acides gras et compose´s phe´noliques
en solution micellaire. Rev. Fr. Corps Gras 1991, 38,
225-231.
(9) Papadopoulos, G.; Boskou, D., Antioxidant Effect of
Natural Phenols on Olive Oil. J . Am. Oil Chem. Soc.
1991, 68, 669-671.
(10) Tsimidou, M.; Papadopoulos, G.; Boskou, D. Phenolic
Compounds and Stability of Virgin Olive Oil - Part I.
Food Chem. 1992, 45, 141-144.
(11) Chimi, H.; Cillard, J .; Cillard, P.; Rahmani, M. Peroxyl
and Hydroxyl Radical Scavenging Activity of Some
Natural Phenolic Antioxidants. J . Am. Oil Chem. Soc.
1991, 68, 307-312.
(12) Servili, M.; Baldioli, M.; Miniati, E.; Montedoro, G. F.
Antioxidant activity of new phenolic compounds ex-
tracted from virgin olive oil and their interaction with
R-tocopherol and â-carotene. Riv. Ital. Sostanze Grasse
1996, 73, 55-59.
(13) Baldioli, M.; Servili, M.; Perreti, G.; Montedoro, G. F.
Antioxidant Activity of Tocopherols and Phenolic Com-
pounds of Virgin Olive Oil. J . Am. Oil Chem. Soc. 1996,
73, 1589-1593.
(14) Visioli, F.; Bellomo, G.; Montedoro, G. F.; Galli, C. Low-
density lipoprotein oxidation is inhibited in vitro by olive
oil constituents. Atherosclerosis 1995, 117, 25-32.
(15) Brenes, M.; Garc´ıa, A.; Garc´ıa, P.; Rios, J . J .; Garrido,
A. Phenolic Compounds in Spanish Olive Oils. J . Agric.
Food Chem. 1999, 47, 3535-3540.
(16) Baraldi, P. G.; Simoni, D.; Manfredini, S.; Menziani, E.
Preparation of 3,4-Dihydroxy-1-benzeneethanol: A Re-
investigation. Liebigs Ann. Chem. 1983, 684-686.
(17) Garibaldi, P.; J ommi, G.; Verotta, L. Secoiridoids From
Olea europaea. Phytochemistry 1986, 865-869.
(18) Limiroli, R.; Consonni, R.; Ranalli, A.; Bianchi, G.; Zetta,
L. 1H NMR Study of Phenolics in Vegetation Water of
Three Cultivars of Olea europaea: Similarities and
Differences. J . Agric. Food Chem. 1996, 44, 2040-2048.
(19) Kanai, F.; Isshiki, K.; Umezawa, Y.; Morishima, H.;
Naganawa, H.; Takita, T.; Takeuchi, T.; Umezawa, H.
Vanoxonin, a New Inhibitor of Thymidylate Synthetase.
II. Structure Determination and Total Synthesis. J .
Antibiot. 1985, 38, 31-38.
(20) Felix, A. M.; Heimer, E. P.; Lambros, T. J .; Tzougraki,
C.; Meienhofer, J . Rapid Removal of Protecting Groups
from Peptides by Catalytic Transfer Hydrogenation with
1,4-Cyclohexadiene. J . Org. Chem. 1978, 43, 4194-4196.
(21) Brand-Williams, W.; Cuvelier, M. E., Berset, C. Use of
a Free Radical Method to Evaluate Antioxidant Activity.
Lebensm.-Wiss. Technol. 1995, 28, 25-30.
containing added phenolic compounds was tested at pH
5.5 (acetate buffer, 0.1 M) and at pH 7.4 (MOPS buffer,
0.1M).
As shown in Figures 3 and 4, the order of antioxidant
activity in emulsions was different from that in bulk
oil. On the basis of the time to a conjugated diene
content of 0.4%, the order of antioxidant activity at pH
5.5 (Table 4) was R-tocopherol, 3,4-DHPEA-EA > phe-
nolic extract, oleuropein, hydroxytyrosol acetate > hy-
droxytyrosol > control; whereas at pH 7.4 in MOPS
buffer, the order of antioxidant activity was 3,4-DHPEA-
EA, R-tocopherol, oleuropein > hydroxytyrosol acetate,
hydroxytyrosol . control. The p-anisidine value deter-
minations confirmed the order of activity (Figures 3 and
4). Hydroxytyrosol acetate was more effective than
hydroxytyrosol in an emulsion at pH 5.5 (Figure 3) but
there was no significant difference between the two
antioxidants in MOPS buffer at pH 7.4 (Figure 4). The
effects of the additives at pH 7.4 were confirmed by
storing a set of samples with the additives in emulsions
buffered with a phosphate buffer (Table 4). Oxidation
in phosphate buffer was faster than in MOPS buffer,
and differences in the order of activity were apparent.
Hydroxytyrosol acetate was significantly more active
than oleuropein and hydroxytyrosol in phosphate buffer.
The differences in the rates of oxidation between the
two buffers suggests that MOPS may have an antioxi-
dant action. The order of antioxidant activity at pH 5.5
and at pH 7.4 in phosphate buffer was similar, although
the differences between DHPEA-EA and R-tocopherol,
and between hydroxytyrosol acetate and oleuropein
were only significant at pH 7.4. There is no change in
partition coefficient with a change in pH (Table 3), and
consequently the explanation for the change in relative
activity with pH must lie elsewhere. Hydroxytyrosol
acetate is less effective than hydroxytyrosol as a radical
scavenger (Table 1), but it is less polar and this helps
to make it more effective as an antioxidant in an
emulsion. The antioxidant activity in an emulsion
depends on partitioning of components between hydro-
philic and hydrophobic phases, the complex interfacial
effects at the oil-water interfaces, the radical-scaveng-
ing properties, and stability of the antioxidants. Oleu-
ropein has a greater radical-scavenging activity than
hydroxytyrosol acetate, and consequently would be a
better antioxidant in the absence of other effects.
However, oleuropein is the more polar of the two
molecules and this reduces its activity in an emulsion
due to the polar paradox. The net effect is that the two
compounds are similar in antioxidant activity in an
emulsion.
In conclusion, this study has demonstrated that
hydroxytyrosol acetate has a weaker DPPH radical
scavenging activity than hydroxytyrosol, but the two
compounds are similar in antioxidant activity with
hydroxytyrosol acetate being slightly less effective in oil,
but slightly more effective in an emulsion at pH 5.5 and
pH 7.4. DHPEA-EA is slightly less effective than hy-
droxytyrosol acetate in oil but is the most effective
hydroxytyrosol derivative in an emulsion.
LITERATURE CITED
(1) Visioli, F.; Bellomo, G.; Galli, C. Free Radical-Scaveng-
ing Properties of Olive Oil Polyphenols. Biochem. Bio-
phys. Res. Commun. 1998, 247, 60-64.
(2) Visioli, F.; Galli, C. Olive Oil Phenols and Their Poten-
tial Effects on Human Health. J . Agric. Food Chem.
1998, 46, 4292-4296.

Antioxidant Activity of Hydroxytyrosol Acetate
J. Agric. Food Chem., Vol. 49, No. 5, 2001 2485
(22) Porter, W. L. Paradoxical Behaviour of Antioxidants in
Food and Biological Systems. Toxicol. Ind. Health 1993,
9, 93-122.
(23) Frankel, E. N.; Huang, S. W.; Kanner, J .; German, J .
B. Interfacial Phenomena in the Evaluation of Antioxi-
dants in Bulk Oils versus Emulsions. J . Agric. Food
Chem. 1994, 42, 1054-1059.
Received for review April 28, 2000. Revised manuscript
received February 12, 2001. Accepted February 12, 2001. The
authors acknowledge the Fundac¸a˜o Caloust Gulbenkian, As-
sociac¸a˜o Luso-Britaˆnica do Porto, and Centro de Investigac¸a˜o
em Qu´ımica da Faculdade de Cieˆncias do Porto for financial
support.
(24) Hopia, A.; Heinonen, M. Antioxidant Activity of Flavonol
Aglycones and their Glycosides in Methyl Linoleate. J .
Am. Oil Chem. Soc. 1999, 76, 139-144.
J F000537W