Biological activity of acetylated phenolic compounds

Article abstract of DOI:10.1021/jf0627221

In recent years an effort has been made to isolate and identify biologically active compounds that are included in the Mediterranean diet. The existence of naturally occurring acetylated phenolics, as well as studies with synthetic ones, provide evidence that acetyl groups could be correlated with their biological activity. Platelet activating factor (PAF) is implicated in atherosclerosis, whereas its inhibitors seem to play a protective role against cardiovascular disease. The aim of this study was to examine the biological activity of resveratrol and tyrosol and their acetylated derivatives as inhibitors of PAF-induced washed rabbit platelet aggregation. Acetylation of resveratrol and tyrosol was performed, and separation was achieved by HPLC. Acetylated derivatives were identified by negative mass spectrometry. The data showed that tyrosol and its monoacetylated derivatives act as PAF inhibitors, whereas diacetylated derivatives induce platelet aggregation. Resveratrol and its mono- and triacetylated derivatives exert similar inhibitory activity, whereas the diacetylated ones are more potent inhibitors. In conclusion, acetylated phenolics exert the same or even higher antithrombotic activity compared to the biological activity of the initial one.

Full text of DOI:10.1021/jf0627221

80 J. Agric. Food Chem. 2007, 55, 80−89
Biological Activity of Acetylated Phenolic Compounds
ELIZABETH FRAGOPOULOU,† TZORTZIS NOMIKOS,† HARALABOS C. KARANTONIS,†
CONSTANTINOS APOSTOLAKIS,† EMMANUEL PLIAKIS,† MARTINA SAMIOTAKI,§
GEORGE PANAYOTOU,§ AND SMARAGDI ANTONOPOULOU*,†
Department of Science of Nutrition-Dietetics, Harokopio University of Athens, 17671 Athens, Greece,
and Protein Chemistry Laboratory B.S.R.C. “Alexander Fleming”, Athens, Greece
In recent years an effort has been made to isolate and identify biologically active compounds that
are included in the Mediterranean diet. The existence of naturally occurring acetylated phenolics, as
well as studies with synthetic ones, provide evidence that acetyl groups could be correlated with
their biological activity. Platelet activating factor (PAF) is implicated in atherosclerosis, whereas its
inhibitors seem to play a protective role against cardiovascular disease. The aim of this study was to
examine the biological activity of resveratrol and tyrosol and their acetylated derivatives as inhibitors
of PAF-induced washed rabbit platelet aggregation. Acetylation of resveratrol and tyrosol was
performed, and separation was achieved by HPLC. Acetylated derivatives were identified by negative
mass spectrometry. The data showed that tyrosol and its monoacetylated derivatives act as PAF
inhibitors, whereas diacetylated derivatives induce platelet aggregation. Resveratrol and its mono-
and triacetylated derivatives exert similar inhibitory activity, whereas the diacetylated ones are more
potent inhibitors. In conclusion, acetylated phenolics exert the same or even higher antithrombotic
activity compared to the biological activity of the initial one.
KEYWORDS: HPLC separation; acetylation; platelet activating factor; electrospray mass spectrometry;
resveratrol; tyrosol
INTRODUCTION
red/white must, fish, milk, and yogurt (5-10). These compounds
had antithrombotic and anti-inflammatory actions because they
inhibited or antagonized PAF (4). Even though these molecules
have different origins, the biological activity has always been
detected in the polar lipid fraction. Specifically, these molecules
belong to phospholipids, glycolipids, phenolics, and phenolic
glycosides. It should be noted that despite the different skeletons
of these molecules, the majority of them had acetyl groups,
which seems to be essential for their biological activity.
Moreover, our data showed that the hydrolysis as well as the
addition of acetyl groups in their structures led to differentiation
of their biological action.
The existence of acetylated phenolic compounds in natural
sources is also established from other research groups. Acetyl-
ated glycosides of flavonoids (quercetin, kaempferol, apigenin,
luteolin) (11-14) and resveratrol (15, 16) have been found in
plants, triacetylated resveratrol has been found in marine
invertebrates (17), and acetylated tyrosol has been found in black
olives (18) and olive oils (19). These data provide evidence
about the existence of acetyl groups in naturally occurring
compounds.
Diet is the cornerstone of cardiovascular disease prevention.
Although Mediterranean diets are associated with a low
incidence of atherosclerotic disease, data about the specific
dietary constituents involved and the mechanisms concerning
cardioprotection are still sparse (1). Nutritional constituents that
typically occur in small quantities in foods may be responsible
for this effect (2). In recent years an effort has been made to
isolate and identify a variety of biologically active compounds
from foods included in the Mediterranean diet. Phenolic
compounds present in all plants are considered to be biologically
active constituents and have been studied extensively.
Platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-
phosphocholine, PAF) (3) is a potent inflammatory phospholipid
mediator that is implicated in the initiation of the inflammation
as well as in the process of atherogenesis (4). PAF and PAF-
like lipids are degraded in plasma by the lipoprotein-bound
enzyme, PAF acetyl hydrolase, leading to completely inactive
compounds.
In our previous studies we have isolated and characterized
bioactive lipids from vegetable oils, olive oil, red/white wine,
Recently, attempts were made to correlate biological activity
with the molecular structure. Structural modification of natural
phenolics is expected to produce analogues that may be useful
tools to study the structure-activity relationships. The synthesis
of acetylated coumarins (20), acetylated O-glycosides (21),
acetylated resveratrol (22) and its oligomers (23, 24), and
* Address correspondence to this author at 70 El. Venizelou St., Athens
17671, Greece (telephone 0030-2109549230; fax 0030-2109577050; e-mail
antonop@hua.gr).
† Harokopio University of Athens.
§ Protein Chemistry Laboratory B.S.R.C. “Alexander Fleming”.
10.1021/jf0627221 CCC: $37.00
© 2007 American Chemical Society
Published on Web 01/03/2007

Acetylated Phenolic Compounds
J. Agric. Food Chem., Vol. 55, No. 1, 2007 81
Table 1. Phenolic Determination of Tyrosol Acetylated Derivatives
before and after Mild Alkaline Hydrolysis
HPLC
tyrosol (
µ
mol)
tyrosol (µmol)
fraction
before hydrolysis
after hydrolysis
% recovery
1
2
3
4
2.95
NDa
2.95
ND
3.22
3.08
2.75
1.60
10.6
22.2
21.6
19.0
11.0
73.5
total
a Not detected.
tumor cell lines (23), and the down-regulation of NO synthase
(iNOS) in leukemia cells (24) and macrophages (25).
On the other hand, although the use of phenolic compounds
in pharmaceuticals as well as in food preparations is very
promising, limitations occur with regard to their weak solubility
and stability in a lipophilic environment. The existence of
lipophilic derivatives of flavonoids via esterification of the
hydroxyl functions with aliphatic molecules can be used as a
tool to increase their lipophilicity and therefore improve their
intestinal absorption and cell permeability of the compounds
(26).
We have previously demonstrated that acetylation of standard
sphingophospholipids as well as phosphoglycolipids resulted in
compounds with significant biological activity that also exist
in nature (27-29).
Figure 1. Typical HPLC separation of tyrosol acetylated derivatives. The
separation was made on C8 reverse phase HPLC using an isocratic elution
On the other hand, acetylation occurs as a modification of
proteins (histones and transcription factors) inside the cell. Even
though the acetyl-CoA is usually the acetyl donor for acetylation
reactions, it has also been reported that nonenzymatic transfer
of the acetyl group from aspirin to cyclooxygenase exists,
resulting in inhibition of prostaglandin synthesis (30). Recently,
evidence for the existence of an enzyme that transfers acetyl
groups from polyphenolic acetates to proteins has beenfound,
and this mechanism appears to be analogous to phosphorylation/
dephosphorylation regulation of enzyme activity (31). It seems
that the acetyl group, despite improving the cell permeability
of compounds, may also serve as a messenger group.
Resveratrol (3,4′,5-trihydroxy-trans-stilbene) is a common
phytoalexin found in a few edible materials, such as grape skins,
peanuts, and red wine, and seems to protect against oxidation,
inflammation, platelet aggregation (8), and thrombus formation
(32, review). Tyrosol is also a phenolic compound found in olive
oil and wine (especially white wine) and seems to have
antioxidant properties and anti-inflammatory actions (33, 34).
The aforementioned data support the prospect that the acetyl
group is a structural characteristic that provides significant
biological activity. The majority of these studies investigate the
activity of acetylated phenolics in cancer cells. The aim of this
study is to examine the antithrombotic activity of resveratrol
and tyrosol as well as their acetylated derivatives with respect
to platelet aggregation and more specifically their ability to act
as PAF antagonists or inhibitors.
system consisting of 40% aqueous methanol. Flow
detection was at 256 nm.
) 1 min/mL, and UV
Figure 2. Typical HPLC separation of resverartol acetylated derivatives.
The separation was made on C8 reverse phase HPLC using a gradient
elution system from 40% aqueous methanol to 60% aqueous methanol
MATERIALS AND METHODS
Materials. All reagents and chemicals were of analytical grade
supplied by Merck (Darmstadt, Germany). Resveratrol and tyrosol were
purchased from Sigma (St. Louis, MO). High-performance liquid
chromatography (HPLC) solvents were from Merck (Walkerburn,
Peebleshire, U.K.). Semisynthetic PAF (80% C-16PAF and 20%
C-18PAF) was synthesized in our laboratory as previously described
(4). Bovine serum albumin (BSA) and BN 52021 were obtained from
Sigma.
in 30 min. Flow
) 1 min/mL, and UV detection was at 256 nm.
acetylated quercetin (25) has already been reported. In all studies
the acetylated phenolics revealed the same or higher biological
activity compared with the one of the initial phenolic compound,
concerning the inhibition of lipid peroxidation (21), the inhibi-
tion of cancer cells growth (22), the cytotoxicity against human

82 J. Agric. Food Chem., Vol. 55, No. 1, 2007
Fragopoulou et al.
HPLC Separation. The reaction mixture was separated on a reverse
phase C8 HPLC column. An elution system consisting of 40% aqueous
methanol was used to separate the tyrosol reaction mixture. The
resveratrol reaction mixture separation was achieved by an elution
system consisting of a gradient from 40% aqueous methanol to 60%
aqueous methanol in 30 min and finally a hold for 5 min. The flow
rate was 1 mL/min, and the detection was at 256 nm.
Table 2. Phenolic Determination of Resveratrol Acetylated Derivatives
before and after Mild Alkaline Hydrolysis
HPLC
resveratrol (
µ
mol)
resveratrol (µmol)
fraction
before hydrolysis
after hydrolysis
% recovery
1
2
3
4
5
1.92
0.83
2.09
0.17
0.61
NDa
1.88
1.05
2.85
0.50
1.49
0.82
8.59
15.9
8.9
24.0
4.2
12.6
6.9
72.5
Biological Assay. PAF and the examined samples were dissolved in
2.5 mg of BSA/mL of saline (solution of 0.9% w/v of NaCl in water).
Various concentrations of the examined sample were added into the
aggregometer cuvette, and the aggregation induced by the sample was
studied in a Crono-Log aggregometer (3). Experiments with PAF
specific inhibitor BN 52021 0.1 mM (0.3% aqueous DMSO) were also
performed. The inhibitor was added to washed rabbit platelets 1 min
prior to the addition of the examined sample into the aggregometer
cuvette. In cross-desensitization experiments platelets were desensitized
by the addition of the examined lipid to the platelet suspension at a
concentration that caused reversible aggregation. A second stimulation
with PAF inducing the same height of aggregation as the tested
compound was performed immediately after complete disaggregation.
The platelet aggregation induced by PAF (2.5 × 10-11 M, final
concentration) was measured as PAF-induced aggregation in washed
rabbit platelets before (considered as 0% inhibition) and after the
addition of various concentrations of the examined sample. Conse-
quently, the plot of percent inhibition (ranging from 0 to 100%) versus
different concentrations of the sample is linear. From this curve, the
concentration of the sample that inhibited 50% PAF-induced aggrega-
tion is calculated. This value is defined as IC50, namely, inhibitory
concentration 50.
6
total
a Not detected.
Instrumentation. HPLC was performed on a Hewlett-Packard
(Avondale, PA) series 1100, supplied with a 100 µL loop Rheodyne (i
7725) injector. An 1100 HP UV spectrometer was used as detector.
The spectrometer was connected to a Hewlett-Packard model HP-3396A
integrator-plotter.
Separation of lipids was carried out on a 15 cm × 4.6 mm i.d. reverse
phase Zobax Eclipse XDB-C8 column (Agilent Technologies) at room
temperature.
The PAF-induced aggregation was measured in a Crono-Log
(Havertown, PA) aggregometer coupled to a Crono-Log recorder.
The electrospray ionization (ESI) mass spectrometry experiments
were performed on an Electronspray MS-LCQ-Deca (Thermo-Finni-
gan), low-flow mass spectrometer. Samples were dissolved in a small
volume of HPLC grade methanol/water (70:30, v/v). Electrospray
samples were typically introduced into the mass analyzer at a rate of
3 µL/min. Nitrogen of purity 99.99% was used as nebulizing gas and
as bath gas.
The spectrum analysis was contacted in the ion space of m/z 50-
1500, and the conditions for the highest intensity of the summits with
the lowest rupture were as follows: spray voltage, 5 kV; capillary
voltage, 7 V; capillary temperature, 275 °C; lens-entrance voltage, -54
V; lens voltage, -22 V.
Mild Alkaline Hydrolysis. This procedure was carried out according
to the method of Demopoulos et al. (3). Briefly, the sample was
dissolved in 1 mL of chloroform/methanol (1:4 v/v), and then 0.1 mL
of 1.2 N NaOH 1 N 50% methanol was added and kept for 20 min at
60 °C. The mixture was neutralized with 0.15 mL of 1 N acetic acid,
evaporated to dryness, and dissolved in ethanol.
Phenolic Determination. The phenol content was determined using
a modified method of Singleton and Rossi (35). Samples were dried
under a stream of nitrogen and dissolved in 3.5 mL of water. An amount
of 0.1 mL of Folin-Ciocalteu reagent was added, and after 3 min, 0.4
mL of 35% aqueous Na2CO3 was added. The reaction mixture was
rested for 1 h, and the intensity of the blue color was measured at 725
nm. Standards of tyrosol or resveratrol were prepared similarly.
Methods. Acetylation Reaction. The acetylated phenolics were
prepared under standard conditions using acetic anhydride. Specifically,
a mixture of tyrosol or resveratrol (2-3 mg) and acetic acid anhydride
(3 mL) was warmed at 60 °C for 45 min. The reaction mixture was
evaporated to dryness under N2 and was dissolved in ethanol. A part
of it was kept for biological assay, and the rest was subjected to HPLC
separation.
Statistical Analysis. Data were expressed as the mean value (
standard deviation (SD), and t test analysis was used for the comparison.
Figure 3. Negative ion mass spectra of tyrosol acetylated derivatives: fraction 1 (A); fraction 2 (B); fraction 4 (C).

Acetylated Phenolic Compounds
J. Agric. Food Chem., Vol. 55, No. 1, 2007 83
Figure 4. Negative ion mass spectra of resveratrol acetylated derivatives: fraction 1 (A); fraction 2 (B).
All analyses were done with the Statistical Package for Social Sciences
(SPSS, version 10.0, SPSS Inc., Chicago, IL). Differences were
considered to be statistically significant at the 5% level.
two isomers of a monoacetylated derivative and one diacetylated
derivative. To separate them, an isocratic elution system was
developed consisting of 40% aqueous methanol. A typical
profile of HPLC separation of acetylated tyrosol derivatives is
shown in Figure 1. Four fractions were selected, evaporated to
air N2, and dissolved in ethanol. Standard tyrosol on this system
is eluted at 2.8 min, corresponding to fraction 1. Under the above
conditions of acetylation, an amount of 69.8 ( 1.2% of tyrosol
is acetylated on the basis of phenolic determination, performed
on each HPLC fraction separately.
RESULTS
Acetylation and HPLC Separation of Phenolic Com-
pounds. To prepare the acetylated derivatives of tyrosol and
resveratrol, acetic acid anhydride was used. Several conditions
were tested including different quantities of acetic anhydride,
temperatures, and incubation times. Finally, 1 mL of acetic acid
anhydride was used per milligram of phenolic compound, and
the reaction was performed at 60 °C for 45 min. Initial amounts
of 2 and 2.7 mg were used for the acetylation of tyrosol and
resveratrol, respectively. After the reaction, the mixture was
evaporated to air N2 and dissolved in ethanol.
Resveratrol shares three hydroxyl groups and therefore five
acetylated derivatives were expected, specificcally, two isomers
of monoacetylated derivatives, two isomers of diacetylated
derivatives, and one triacetylated derivative. Their separation
was finally achieved by a gradient elution system from 40%
aqueous methanol to 60% aqueous methanol in 30 min. A
Two hydroxyl groups exist in the tyrosol structure, and
therefore three acetylated derivatives were expected, specifically,

84 J. Agric. Food Chem., Vol. 55, No. 1, 2007
Fragopoulou et al.
Figure 5. Negative ion mass spectra of resveratrol acetylated derivatives: fraction 4 (A); fraction 5 (B).
typical profile of HPLC separation of acetylated resveratrol
derivatives is shown in Figure 2. Six fractions were selected,
evaporated to air N2, and dissolved in ethanol. The elution time
for standard resveratrol on this system is 6.5 min, corresponding
to fraction 1. Under the above conditions of acetylation, an
amount of 78.1 ( 1.6% of resveratrol is acetylated on the basis
of phenolic determination, performed on each HPLC fraction.
Phenolic Determination. The phenolic determination re-
quires at least one free hydroxyl group on the phenolic ring
that is oxidized to give a quinoid structure. The substitution of
this type of hydroxyl group by an acetyl group results in partial
or complete inhibition of phenolic determination. For this reason,
phenolic determinations were performed in equal amounts of
all HPLC fractions including phenolic standards before and after
mild alkaline hydrolysis. Moreover, these data provide evidence
about the chemical structure of tyrosol and resveratrol acetylated
derivatives. The results of phenolic determination before and
after the mild alkaline hydrolysis of acetylated tyrosol deriva-
tives are summarized in Table 1. The data show that the
phenolic determination of fractions 2 and 4 was blocked before
the mild alkaline hydrolysis, indicating that the acetylation took
place at the phenolic hydroxyl group. Their chromatographic
behavior indicates that fraction 2 corresponds to monoacetylated
derivative and fraction 4 should be the diacetylated one.
All of the hydroxyl groups of resveratrol are on the phenolic
ring and participate in the phenolic determination. The results
of the phenolic determination of resveratrol derivatives before
and after mild alkaline hydrolysis are shown in Table 2. Only
fraction 8 gave a negative phenolic determination before mild
alkaline hydrolysis, indicating that this is the triacetate deriva-
tive.
ESI-MS. To establish the structure of phenolic derivatives,
ESI-MS of all fractions was performed. Standard tyrosol gave
only an [M - 1]- fragment at m/z 137 (data not shown). The

Acetylated Phenolic Compounds
J. Agric. Food Chem., Vol. 55, No. 1, 2007 85
Figure 6. Negative ion mass spectrum of resveratrol acetylated derivatives: fraction 6.
negative ion mass spectrum of fraction 1 gave a peak at m/z
137 (Figure 3A), indicating that this is the nonacetylated tyrosol.
Fractions 2 and 3 showed almost identical negative mass spectra
(Figure 3B). The predominant peak at m/z 179 [M - 1]-
corresponds to the molecular weight of a tyrosol molecule plus
an acetyl group, whereas the removal of the acetyl group gave
rise to the peak at m/z 137. These data are in accordance with
the results of the phenolic determination and lead to the
conclusion that fractions 2 and 3 are the monoacetylated
derivatives (Figure 3B). The negative ion ESI-MS of fraction
4 gave two peaks at m/z 137 and 179 as well as at m/z 221,
corresponding to the molecular weight of tyrosol plus two acetyl
groups. These data in combination with phenolic determination
results lead to the conclusion that fraction 4 is the diacetyl
derivative of tyrosol (Figure 3C).
Fraction 1 of resveratrol derivatives gave a negative ion mass
spectrum (Figure 4A) identical with the one of the standard
resveratrol. Specifically, the predominant peak at m/z 227
corresponds to fragment [M - 1]-. Moreover, peaks at m/z 455
and 683 correspond to fragments [2M - 1]- and [3M - 1]-,
respectively. Negative ESI-MS of fraction 2 showed an intense
peak at m/z 269 [M - 1]- corresponding to the molecular weight
of the resveratrol molecule plus an acetyl group, the peak at
m/z 227 resulting from the cleavage of the acetyl group [M -
1 - COCH3 + H]- (Figure 4B). Moreover, the peaks at m/z
539 and 497 represent the fragments [2M - 1]- and [2M - 1
- COCH3 + H]-, respectively. The above data confirm that
fraction 2 is a monoacetylated derivative of resveratrol. Negative
ESI-MS of fraction 3 gave fragments identical with the ones of
fraction 2, showing that it is also a monoacetylated derivative
(data not shown). Fractions 4 and 5 gave a predominant peak
at m/z 311 corresponding to the fragment [M - 1]- that is the
molecular weight of resveratrol plus two acetyl groups, indicat-
ing that they are the diacetylated derivatives of resveratrol
(Figure 5). Cleavage of the first and second acetyl groups gave
rise to fragments at m/z 269 [M - 1 - COCH3 + H]- and 227
[M - 1 - 2(COCH3 + H)]-. The mass spectrum of fraction 6
showed a peak at m/z 353 corresponding to fragment [M - 1]-
that is the molecular weight of resveratrol plus three acetyl
groups (Figure 6). Peaks at m/z 311, 269, and 227 arise after
the cleavage of the first [M - 1 - COCH3 + H], the second
[M - 1 - 2(COCH3 + H)]-, and the third [M - 1 - 3(COCH3
+ H)] - acetyl group, respectively. These data in combination
with the negative phenolic determination led to the conclusion
that fraction 8 is the triacetate derivative of resveratrol.
Biological Activity. The acetylated derivatives of tyrosol and
resveratrol as well as standards of tyrosol and resveratrol were
tested for their ability to induce washed rabbit platelet aggrega-
tion and/or to inhibit PAF-induced washed rabbit platelet
aggregation. The results are summarized in Tables 3 and 4,
respectively.
Data showed that tyrosol inhibits PAF-induced rabbit platelet
aggregation with an IC50 value of (21.6 ( 3.98) × 10-4 M.
The monoacetylated derivatives (fractions 2 and 3) were more
potent inhibitors of PAF-induced rabbit platelet aggregation
because IC50 values were (0.40 ( 0.08) × 10-4 and (0.27 (
0.05) × 10-4 Μ, respectively, that is, 2 orders of magnitude
more potent (p < 0.05). The diacetylated derivative (fraction
4) induced aggregation of washed rabbit platelet with an EC50
value of (0.14 ( 0.02) × 10-4 Μ. The addition of the second
acetyl group inverts the biological activity from inhibition to
aggregation. Cross-desensitization experiments and experiments
with specific PAF inhibitor were performed on this case.
Fraction 4 seems to act through the PAF pathway because it
desensitized platelets against PAF and its aggregation was fully
inhibited by the specific inhibitor of PAF, namely, BN 52021.
Resveratrol also inhibited PAF-induced rabbit platelet ag-
gregation as we have previously shown (8) with an IC50 value
(0.52 ( 0.12) × 10-4 M. The monoacetylated derivatives of
resveratrol (fractions 2 and 3) had a similar inhibitory activity
as resveratrol. More specifically, as shown in Table 4 the IC50
values of these fractions have the same order of magnitude as
that of resveratrol.
Diacetylated derivatives of resveratrol (fractions 4 and 5) also
showed inhibitory activity. The IC50 values of these fractions
of (0.08 ( 0.02) × 10-4 and (0.24 ( 0.05) × 10-4 M,
respectively, were significantly different from that of resveratrol
(p < 0.05). Even more, the IC50 value of fraction 4 is different
by 1 order of magnitude, indicating that the entrance of the
second acetyl group is capable of improving the inhibitory
activity of resveratrol.

86 J. Agric. Food Chem., Vol. 55, No. 1, 2007
Fragopoulou et al.
Table 3. Biological Activity of Tyrosol and Its Acetylated Derivativesa
a Data are expressed as mean values
± SD derived from three experiments performed on different platelet preparations.
The triacetylated derivative of resveratrol, fraction 6, gave
inhibitory activity of the same order of magnitude as resveratrol.
as well as of its monoacetylated derivatives gave predominant
peaks corresponding to their [M - H]-. The mass spectrum of
the diacetylated derivatives showed an [M - H]- fragment, but
the predominant peak was the one produced after the loss of
one acetyl group. To our knowledge, no report of the mass
spectrometry analysis of tyrosol exists, with the exception of
one publication in which no clearly distinctive molecular ions
were observed for tyrosol (36). Fractions 2 and 3 derived from
HPLC are the monoacetylated derivatives, fractions 4 and 5 are
the diacetylated derivatives, and fraction 6 is the triacetylated
derivative of resveratrol. Resveratrol as well as the mono- and
diacetylated derivatives gave predominant ions corresponding
to [M - H]-. Our results are in accordance with a previous
finding of negative ion mass spectrometry of resveratrol showing
an ion at m/z 227 (37). In addition, under our ESI-MS conditions
fragments corresponding to oligomerics of resveratrol were also
observed. Even though the ESI-MS analysis of the triacetylated
showed the [M - H]- fragment, the predominant ion was the
one produced after the loss of one acetyl group.
DISCUSSION
It is now common knowledge that the consumption of a
Mediterranean diet is protective against cardiovascular diseases
(1). Responsible for this protection are thought to be biological
active compounds that typically occur in small quantities in
foods (2). Among them, phenolic compounds are thought to be
important constituents and have been found to exert many
biological activities.
In addition, data indicate the existence of acetyl groups in
naturally occurring compounds. Acetyl groups seem to be a
structural characteristic that provides biological activity, and
therefore recently an attempt was made to synthesize acetylated
derivatives (20-25).
In this study the semisynthesis of acetylated resveratrol and
tyrosol derivatives was performed using acetic anhydride.
Acetylation reaction products were separated on HPLC. The
identification of the acetylated derivatives has been made
according to their chromatographic behavior and the phenolic
determination before and after mild alkaline hydrolysis and
mainly from negative ESI-MS of each fraction. The combination
of these data leads to the conclusion that HPLC fractions 2 and
3 are the monoacetylated derivatives and fraction 4 is the
diacetylated derivative of tyrosol. ESI-MS analysis of tyrosol
Previous studies reported that acetylated phenolics exert the
same or higher biological activity compared with the initial
phenolic compound (21-25). The majority of these studies
investigated the activity of acetylated phenolics in cancer cells.
In this study the antithrombotic activity of resveratrol and
tyrosol as well as their acetylated derivatives with respect to
platelet aggregation and more specifically their ability to act as

Acetylated Phenolic Compounds
J. Agric. Food Chem., Vol. 55, No. 1, 2007 87
Table 4. Biological Activity of Resveratrol and Its Acetylated Derivativesa
a Data are expressed as mean values
± SD derived from three experiments performed on different platelet preparations.
PAF antagonists or inhibitors was investigated. Resveratrol is
a stilbene that has been exhaustively studied, and it is known
to have many biological cardioprotective actions (34, review).
Tyrosol is a simple phenolic compound that is reported to have
antioxidant activity (33, 34).
Our results showed that tyrosol inhibited PAF-induced washed
rabbit platelet aggregation with an IC50 value of 21.6 × 10-4
M. As far as we know, there are no other data concerning
tyrosol-induced inhibition of platelet aggregation. Τhe monoacetyl-
ated derivatives were more potent inhibitors of PAF-induced
rabbit platelet aggregation, indicating that the addition of an
acetyl group on the tyrosol structure results in phenolic
compounds that are 2 orders of magnitude more potent. The
exact position of the acetyl group on the structure of tyrosol
does not seem to be crucial. The diacetylated derivative induced
washed rabbit platelet aggregation. According to these data, the
addition of two acetyl groups on the structure of tyrosol is
capable of inverting the biological activity from inhibition to
aggregation.
Resveratrol inhibited PAF-induced washed rabbit platelet
aggregation as we have previously reported (8). The monoacetyl-
ated derivatives of resveratrol had inhibitory activity similar to
that of resveratrol. Diacetylated derivatives of resveratrol also
exerted more potent inhibitory activity, indicating that the
entrance of the second acetyl group is capable of improving
the inhibitory activity of resveratrol. The triacetylated derivative
of resveratrol gave an inhibitory activity of the same order of
magnitude as resveratrol.
In conclusion, acetylation of phenolics leads to compounds
with more potent antithrombotic activity. Further investigation
is needed to elucidate the exact role of acetylated phenolics in
cardiovascular disease.

88 J. Agric. Food Chem., Vol. 55, No. 1, 2007
Fragopoulou et al.
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Received for review September 22, 2006. Revised manuscript received
November 6, 2006. Accepted November 8, 2006.
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