Lipid-lowering and antioxidant effects of hydroxytyrosol and its triacetylated derivative recovered from olive tree leaves in cholesterol-fed rats

Article abstract of DOI:10.1021/jf072589s

This study was designed to test the lipid-lowering and antioxidative activities of triacetylated hydroxytyrosol compared with its native compound, hydroxytyrosol, purified from olive tree leaves. Wistar rats fed a standard laboratory diet or a cholesterol-rich diet for 16 weeks were used. The serum lipid levels, the thiobarbituric acid-reactive substances (TBARS) level, as an indicator of lipid peroxidation, and the activity of superoxide dismutase (SOD) as well as that of catalase (CAT) were examined. The cholesterol-rich diet induced hypercholesterolemia that was manifested in the elevation of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). Administration of hydroxytyrosol and triacetylated hydroxytyrosol (3 mg/kg of body weight) decreased the serum levels of TC, TG, and LDL-C significantly and increased the serum level of high-density lipoprotein cholesterol (HDL-C). Furthermore, the content of TBARS in liver, heart, kidney, and aorta decreased significantly when hydroxytyrosol and its triacetylated derivatives were orally administered to rats compared with those fed a cholesterol-rich diet. In addition, triacetylated hydroxytyrosol and hydroxytyrosol increased CAT and SOD activities in the liver. These results suggested that the hypolipidemic effect of triacetylated hydroxytyrosol and hydroxytyrosol might be due to their abilities to lower serum TC, TG, and LDL-C levels as well as to their antioxidant activities preventing the lipid peroxidation process.

Full text of DOI:10.1021/jf072589s

2630 J. Agric. Food Chem. 2008, 56, 2630–2636
Lipid-Lowering and Antioxidant Effects of
Hydroxytyrosol and Its Triacetylated Derivative
Recovered from Olive Tree Leaves in
Cholesterol-Fed Rats
HEDYA JEMAI,† INES FKI,† MOHAMED BOUAZIZ,† ZOUHAIER BOUALLAGUI,†
ABDELFATTAH EL FEKI,§ HIROKO ISODA,# AND SAMI SAYADI*,†
Laboratoire des Bioprocédés, Pôle d’Excellence Régional AUF, Centre de Biotechnologie de Sfax,
B.P. «K» 3038 Sfax, Tunisia; Laboratoire d’Ecophysiologie animale, Faculté des Sciences de Sfax,
B.P. 802, 3018 Sfax, Tunisia; and Graduate School of Life and Environmental Sciences, University of
Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
This study was designed to test the lipid-lowering and antioxidative activities of triacetylated
hydroxytyrosol compared with its native compound, hydroxytyrosol, purified from olive tree leaves.
Wistar rats fed a standard laboratory diet or a cholesterol-rich diet for 16 weeks were used. The
serum lipid levels, the thiobarbituric acid-reactive substances (TBARS) level, as an indicator of lipid
peroxidation, and the activity of superoxide dismutase (SOD) as well as that of catalase (CAT) were
examined. The cholesterol-rich diet induced hypercholesterolemia that was manifested in the elevation
of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C).
Administration of hydroxytyrosol and triacetylated hydroxytyrosol (3 mg/kg of body weight) decreased
the serum levels of TC, TG, and LDL-C significantly and increased the serum level of high-density
lipoprotein cholesterol (HDL-C). Furthermore, the content of TBARS in liver, heart, kidney, and aorta
decreased significantly when hydroxytyrosol and its triacetylated derivatives were orally administered
to rats compared with those fed a cholesterol-rich diet. In addition, triacetylated hydroxytyrosol and
hydroxytyrosol increased CAT and SOD activities in the liver. These results suggested that the
hypolipidemic effect of triacetylated hydroxytyrosol and hydroxytyrosol might be due to their abilities
to lower serum TC, TG, and LDL-C levels as well as to their antioxidant activities preventing the lipid
peroxidation process.
KEYWORDS: Triacetylated hydroxytyrosol; hydroxytyrosol; cholesterol-fed rat; antioxidant enzymes;
hypolipidemic; serum lipid levels
INTRODUCTION
would avidly remove LDL from the interstitium and generate
macrophage foam cells, a major cell type present within fatty
streaks and fibrous plaque (3, 4). Therefore, it has been proposed
that inhibition of the generation of the oxidative LDL-generated
foam cells and reductions in the level of triglyceride, cholesterol,
and LDL, by naturally occurring compounds, would result in
retardation of atherosclerostic lesion development. Phenolic
compounds from various sources have been reported to prevent
LDL oxidation in vitro and show marked hypolipidemic activity
in vivo, suggesting the effectiveness of polyphenols for the
prevention and treatment of atherosclerosis (5, 6).
Atherosclerosis, the principal contributor to the pathogenesis
of myocardial and cerebral infarctions, is known to be one of
the leading causes of morbidity and mortality worldwide (1).
Hyperlipidemia resulting from lipid metabolic changes is a major
cause of atherosclerosis. Hypercholesterolemia, or more specif-
ically elevated plasma low-density lipoprotein cholesterol (LDL-
C), is an important risk factor for the development and
progression of atherosclerosis (2). Moreover, it has been reported
that the oxidative modified LDL might be important in the
progression of atherosclerosis, due to the observations that
oxidized LDL is cytotoxic, chemotactic, and chemostatic.
Monocyte macrophages in an environment of oxidized LDL
Among the different phenolic compounds, particular attention
has been focused on hydroxytyrosol (7), which occurs naturally
in olive oil (8), in olive mill solid–liquid wastes from two-phase
olive oil processing (9), and in olive mill wastewaters. This
o-diphenol has been proven to be a potent scavenger of
superoxide anion and hydroxyl radical (10, 11), and it is more
active than antioxidant vitamins (12) as well as the synthetic
* Corresponding author (telephone/fax 216 74 874 452; e-mail
sami.sayadi@cbs.rnrt.tn).
† Centre de Biotechnologie de Sfax.
§ Faculté des Sciences de Sfax.
# University of Tsukuba.
10.1021/jf072589s CCC: $40.75  2008 American Chemical Society
Published on Web 04/02/2008

Hypolipidemic Effect of Triacetylated Hydroxytyrosol
J. Agric. Food Chem., Vol. 56, No. 8, 2008 2631
Table 1. Composition of the Control Diet
antioxidants (13). Clear epidemiological and biochemical evi-
dence indicates that hydroxytyrosol is endowed with significant
antithrombotic, antiatherogenic, and anti-inflammatory activi-
ties (14, 15). Recently, the hypocholesterolemic potential of
hydroxytyrosol has been demonstrated (16–18).
diet ingredient
concn (g/kg)
casein
200
3
393
154
50
DL-methionine
cornstarch
sucrose
cellulose
mineral mixa
vitamin mixb
In this respect, however, a major problem is that hydroxy-
tyrosol is chemically unstable, unless preserved dried in the
absence of air and in the dark. Therefore, the efficiency of this
molecule added in its native form to biological matrices as a
protective agent against reactive oxygen species could not be
guaranteed. On the basis of these considerations, it would be
useful to conveniently produce hydroxytyrosol in chemically
more stable derivatives able to be biochemically converted in
vivo into its original active form. Considering that the acetyl
group is a ubiquitous substrate in the biochemical processes
and that the acetylating agents are very common and manage-
able, we have planned and succeeded in preparing hydroxyty-
rosol acetyl derivatives.
35
10
a Mineral mixture contained (mg/kg of diet) the following: CaHPO4, 17200; KCl,
4000; NaCl, 4000; MgO, 420; MgSO4, 2000; Fe2O3, 120; FeSO4 ·7H2O, 200; trace
elements, 400 (MnSO4H2O, 98; CuSO4 ·5H2O, 20; ZnSO4 ·7H2O, 80; CoSO4 ·7H2O,
0.16; Kl, 0.32; sufficient starch to bring to 40 g (per kg of diet). b Vitamin mixture
contained (mg/kg of diet) the following: retinol, 12; cholecalciferol, 0.125, thiamin,
40; riboflavin, 30; pantothenic acid, 140; pyridoxine, 20; inositol, 300; cyanoco-
balamin, 0.1; menadione, 80; nicotinic acid, 200; choline, 2720; folic acid, 10;
p-aminobenzoic acid, 100; biotin, 0.6; sufficient starch to bring to 20 g (per kg of
diet).
The aim of this study is to examine the effect of the
triacetylated hydroxytyrosol on the cholesterol metabolism and
antioxidative status in hypercholesterolemic diet fed rats
compared with the parent purified hydroxytyrosol from Olea
europaea L. leaves.
white precipitation appeared (pyridine chloridrate). The formed pre-
cipitate was filtered, and the obtained solution was dried at 35 °C under
vacuum to give triacetylated hydroxytyrosol as a pale brown
residue.
Purification of Triacetylated Hydroxytyrosol. The material con-
sisted of an AKTA basic system (Biosciences-Amersham) equipped
with a UV detector and a C18-Bioscale column eluted with the same
gradient as in HPLC.
MATERIALS AND METHODS
Olive Leaf Extract Preparation. The extraction was carried out
on Chemlali olive leaves. Samples of fresh green leaves were used.
Leaves were dried and powdered for the extraction. A mixture of
methanol and water (80:20 v/v) was added to the olive leaf powder,
and the mixture was left to stand under agitation for 24 h and then was
filtered.
Acid Hydrolysis. Hydroxytyrosol-rich extract was prepared as
follows: 1 g of the olive leaf extract was dissolved in 10 mL of a MeOH/
H2O (4:1) mixture in a sealed vial. The solution was hydrolyzed at
100 °C for 1 h using 5 mL of HCl (2 M) (Prolabo, France). After 1 h,
the sample was cooled and diluted with water (10 mL) and the
hydrophobic fraction was extracted by a separatory funnel three times
with 50 mL of ethyl acetate (Prolabo, France), which was subsequently
removed by evaporation.
GC-MS Analysis. GC-MS analysis was performed with a HP model
5975B inert MSD, equipped with a capillary HP5MS column (30 m
length, 0.25 mm i.d., 0.25 mm film thickness, Agilent Technologies,
J&W Scientific Products). The carrier gas was used at 1 mL min-1
flow rate. The oven temperature program was as follows: 1 min at
100 °C, from 100 to 260 at 4 °C min-1 and 10 mn at 260 °C. OMW
samples (40 mL) were acidified at pH 2 by HCl (1 N) and extracted
with ethyl acetate (4/40 mL). The organic layer was collected and
reduced to 10 mL by rotary evaporation (37 °C) and then silylated.
For the silylation procedure, a mixture of pyridine (40 µL) and BSTFA
(200 µL) was added and vortexed in screw-cap glass tubes and
consecutively placed in a water bath at 80 °C for 45 min. From the
silylated mixture 1 µL was directly analyzed by GC-MS.
Animals and Diets. Forty male Wistar rats weighing between 150
and 170 g were purchased from the Pasteur Institute (Tunis). During
the treatment, the animals were individually housed in stainless steel
cages in a controlled room temperature at 24 °C, under a 12 h light/12
h dark cycle and with free access to food and water. The rats were
randomly divided into four experimental groups (n ) 10). Group 1
was fed a standard laboratory diet (CD) (Table 1). Group 2 was fed a
cholesterol-rich diet (HCD) (normal diet supplemented with 1%
cholesterol and 0.25% bile salts). Groups 3 and 4 received HCD with
hydroxytyrosol and triacetylated hydroxytyrosol (3 mg/kg of body
weight), respectively. Hydroxytyrosol and triacetylated hydroxytyrosol
were dissolved in drinking water. The duration of the treatment was
16 weeks. The body weight was measured every day. At the end of
the experimental period, the rats were killed by decapitation. Blood
samples were collected to determine the plasma lipid profile. The livers,
hearts, kidneys, and aortas were removed and rinsed with physiological
saline solution. All samples were stored at -80 °C until analysis.
Serum Lipids. Concentrations of total cholesterol (TC), triglycerides
(TG), LDL-C, and high-density lipoprotein cholesterol (HDL-C) in
serum were determined by enzymatic colorimetric methods using
commercial kits (Kyokuto Pharmaceuticals). The atherosclerotic index
(AI) was calculated for different groups. It is defined as the ratio of
LDL-C and HDL-C.
HPLC Analysis. A reversed-phase high-performance liquid chro-
matographic (HPLC) technique was developed to identify and quantify
the major phenolic compounds contained in the hydrolyzed extract.
For this purpose, a standard mixture solution of phenolic compounds
was analyzed. Sample concentrations were calculated on the basis of
peak areas compared to those of each of the external standards. The
HPLC chromatograph was a Shimadzu apparatus equipped with a (LC-
10ATvp) pump and a (SPD-10Avp) detector. The column was 4.6 ×
250 mm (Shim-pack, VP-ODS), and the temperature was maintained
at 40 °C. The flow rate was 0.5 mL/min. The mobile phase used was
0.1% phosphoric acid in water (A) versus 70% acetonitrile in water
(B) for a total running time of 40 min, and the gradient changed as
follows: solvent B started at 20% and increased immediately to 50%
in 30 min. After that, elution was conducted in the isocratic mode with
50% solvent B within 5 min. Finally, solvent B decreased to 20% until
the end of the running time.
Chromatographic Purification of Hydroxytyrosol. Hydrolyzed
extract (1 g) was chromatographed on a C-18 silica gel (liChroprep
RP-18; 25–40 µm) column (2.5 × 70 mm) under medium pressure.
Phenolic compound elution was carried out with the same gradient
solvent as used in the HPLC. The flow rate was adjusted to 0.3 mL/
min, and 5 mL fractions were collected. These fractions were measured
by optical density at 280 nm and the chromatogram (optical density
versus fraction number) was represented (data not shown).
Antioxidant Enzyme Activities. CAT and SOD activities were
evaluated in liver tissue. The preparation of the enzyme source fraction
was as follows. One gram of liver tissue was homogenized in 10 mL
of KCl (1.15%) and centrifuged at 7740g for 15 min. The supernatants
were removed and stored at -80 °C for analysis. The protein content
in supernatant was measured according to the method of Bradford (19)
using bovine serum albumin as standard. CAT activity was measured
Hydroxytyrosol Acetylation. One hundred milligrams of hydroxy-
tyrosol (0.65 mmol) was dissolved in diethyl ether (20 mL) and mixed
with pyridine (165 µL) in a glass vial equipped with a magnetic stirrer.
Then 1654 µL (2.3 mmol) of acetyl chloride in 10 mL of diethyl ether
was added dropwise. The mixture was stirred at 0 °C for 10 h, and a

2632 J. Agric. Food Chem., Vol. 56, No. 8, 2008
Jemai et al.
Figure 2. HPLC chromatogram at 280 nm of hydroxytyrosol after
acetylation. Peak 2 represents triacetylated hydroxytyrosol.
Table 2. Abbreviated Mass Spectra of Hydroxytyrosol and Triacetylated
Hydroxytyrosol
TMS derivatives of
hydroxytyrosol
mass spectra (m/z and % of the base peak)
370 (M+, 39), 267 (90), 193 (25), 179 (12),
73 (100)
triacetylated hydroxytyrosol 280 (M+, 5), 220 (6), 196 (3), 178 (18), 137 (10),
136 (100), 135 (5), 123 (10), 107 (2), 77 (2)
Figure 1. HPLC chromatogram at 280 nm of an olive leaf extract after
acid hydrolysis (A) and purified hydroxytyrosol (B) (peak 1).
using the method of Regoli and Principato (20). Briefly, 20 µL of the
supernatant was added to a cuvette containing 780 µL of a 50 M
potassium phosphate buffer (pH 7.4), and then the reaction was initiated
by adding 200 µL of 500 mM H2O2 to make a final volume of 1.0 mL
at 25 °C. The decomposition rate of H2O2 was measured at 240 nm for
1 min on a spectrophotometer. A molar extinction coefficient of 0.0041
mM-1 cm-1 was used to determine the CAT activity. The activity was
defined as the micromoles of H2O2 decrease per milligram of protein
per minute.
Figure 3. Effects of triacetylated hydroxytyrosol and hydroxytyrosol on
the liver/body weight ratios: 1, standard diet (CD); 2, high-cholesterol diet
(HCD); 3, HCD + hydroxytyrosol (3 mg/kg); 4, HCD + triacetylated
hydroxytyrosol (3 mg/kg). Each bar represents the mean ( SE from 10
rats. Bars with different letters differ; p < 0.05.
SOD activity was measured according to the method of Marklund
and Marklund (21). This method is based on pyrogallol oxidation by
superoxide anion (O2-) and its dismutation by SOD. Briefly, 25 µL of
the supernatant was mixed with 935 µL of a Tris-EDTA-HCl buffer
(pH 8.5) and 40 µL of 15 mM pyrogallol. The activity was measured
after 45 s at 440 nm. One unit was determined as the amount of enzyme
that inhibited the oxidation of pyrogallol by 50%. The activity was
expressed as units per milligram of protein.
Thiobarbituric Acid-Reactive Substances (TBARS) Assay. As a
marker of lipid peroxidation product, the TBARS concentration was
measured using the method of Park et al. (22). Briefly, 200 µL of a
10% (w/v) solution of the tissue homogenate was mixed with 600 µL
of distilled H2O and 200 µL of 8.1% (w/v) SDS, vortexed, and then
incubated at room temperature for 5 min. The reaction mixture was
heated at 95 °C for 1 h after the addition of 1.5 mL of 20% acetic acid
(pH 3.5) and 1.5 mL of 0.8% (w/v) TBA. After the mixture had cooled,
1.0 mL of distilled water and 5.0 mL of a butanol/pyridine (15:1)
solution were added and vortexed. This solution was centrifuged at
1935g for 15 min, and the resulting colored layer was measured at
532 nm using a malondialdehyde (MDA) standard curve.
to pure hydroxytyrosol. The purity of hydroxytyrosol was further
confirmed with HPLC analysis (Figure 1B).
To prepare the acetylated derivatives of hydroxytyrosol, acetyl
chloride was used. Several conditions were tested including
different quantities of acetyl chloride, pyridine, different tem-
peratures, and different incubation times. Three hydroxyl groups
exist in the hydroxytyrosol structure, and therefore different
acetyl derivatives were expected. Under our experimental
conditions, triacetylated derivative was obtained resulting in
96.8% initial hydroxytyrosol conversion. The acetylated raw
material was further purified. A typical HPLC profile of
triacetylated hydroxytyrosol derivative is shown in Figure 2.
The identification of hydroxytyrosol and its acetylated derivative
was confirmed by using the GC-MS apparatus (Table 2).
Body and Organ Weights. There was no significant dif-
ference in the body weight evolution in all groups throughout
the treatment (data not shown). In the same way, there were no
differences in the heart and kidney/body weight ratios. However,
the liver/body weight ratio increased in rats fed a cholesterol-
rich diet (HCD) compared with the rats fed a control diet (CD)
(Figure 3). Triacetylated hydroxytyrosol and hydroxytyrosol
decreased significantly the liver/body weight ratio compared
with those of the HCD group.
Statistical Analysis. All data presented are the mean ( SE. Statistical
differences were calculated using a one-way analysis of variance
(ANOVA), followed by Student’s test. Differences were considered to
be significant at p < 0.05.
RESULTS
Purification and Acetylation of Hydroxytyrosol. The hy-
drolysis reaction of the olive leaf extracts was realized to
produce large quantities of hydroxytyrosol. Figure 1A shows
that the hydrolysate solution was composed of a single major
phenolic compound, identified as hydroxytyrosol. The hydroly-
sate was submitted to the purification using the C-18 column
under medium pressure. The first separated peak corresponds
Serum Lipids. Serum lipid levels were measured at the end
of the experiment. After the treatment, the TC, TG, and LDL-C

Hypolipidemic Effect of Triacetylated Hydroxytyrosol
J. Agric. Food Chem., Vol. 56, No. 8, 2008 2633
Figure 4. Effects of hydroxytyrosol and triacetylated hydroxytyrosol on rat total cholesterol (TC) (A), triglycerides (TG) (B), low-density lipoprotein cholesterol (LDL-C) (C),
high-density lipoprotein cholesterol (HDL-C) (D) and atherogenic index (AI) (E) levels: 1, standard diet (CD); 2, high-cholesterol diet (HCD); 3, HCD + hydroxytyrosol (3
mg/kg); 4, HCD + triacetylated hydroxytyrosol (3 mg/kg). Each bar represents the mean ( SE from 10 rats. Bars with different letters differ; p < 0.05.
concentrations of rats fed a cholesterol-rich diet (HCD) showed
a significant increase compared with the rats fed a normal diet
(CD). However, a decrease of HDL-C concentration of rats in
the HCD group was observed (Figure 4). Rats having received
an oral administration of triacetylated hydroxytyrosol and
hydroxytyrosol had lower concentrations of TC, TG, and LDL-C
than those that received a HCD. Indeed, triacetylated hydroxy-
tyrosol and hydroxytyrosol reduced the TC, TG, and LDL-C
levels by 47, 28, and 44% and 45, 25, 42%, respectively.
Moreover, the HDL-C of rats treated with triacetylated hy-
droxytyrosol and hydroxytyrosol increased significantly com-
pared with those of rats in the HCD group (p < 0.05). The AI
was significantly reduced by orally administering phenolic
compounds (p < 0.05).
The supplementation of HCD-fed animals with triacetylated
hydroxytyrosol and hydroxytyrosol was able to restore the lipid
profile to the normal level of control group. In fact, the TC,
TG, LDL-C, and HDL-C concentrations of animals treated with
these phenolics were similar to those of the control group (p <
0.05).
Hepatic Antioxidant Enzyme Activities. The hepatic anti-
oxidant enzyme activities significantly decreased in rats fed a
cholesterol-rich diet compared to those fed a control diet (Figure
Figure 5. Effects of hydroxytyrosol and triacetylated hydroxytyrosol on
5). The decrease was significantly restored (p < 0.05) in the
CAT (A) and SOD (B) activities in liver: 1, standard diet (CD); 2, high-
presence of triacetylated hydroxytyrosol and hydroxytyrosol.
cholesterol diet (HCD); 3, HCD + hydroxytyrosol (3 mg/kg); 4, HCD +
TBARS Levels. The TBARS levels were significantly
triacetylated hydroxytyrosol (3 mg/kg). Each bar represents the mean (
increased (p < 0.05) in the liver, heart, and kidneys of the
SE from 10 rats. Bars with different letters differ; p < 0.05.
animals fed the high-cholesterol diet compared to the control
diet group. The treatment of HCD rats with triacetylated
number of previous epidemiological studies have implied a role
hydroxytyrosol and hydroxytyrosol significantly reduced the
for polyphenols in reducing the risk of coronary heart disease
TBARS concentration (Figure 6).
based on the antioxidant activity of these compounds (23, 24).
The results reported in this paper represent the first evidence
that triacetylated hydroxytyrosol, a chemically stable acetyl
DISCUSSION
Vascular disease is a prevalent disorder leading to coronary
heart disease and strokes attributed to atherosclerosis, a complex
disease process often initiated by hypercholesterolemia. A
analogue of hydroxytyrosol, is as effective as the native
compound in preventing hypercholesterolemia and oxidative
stress in cholesterol fed rats.

2634 J. Agric. Food Chem., Vol. 56, No. 8, 2008
Jemai et al.
(26), biochemical (27), or biotechnological (28) synthesis
starting from a synthetic precursor. However, because hydroxy-
tyrosol is easily oxidized, it has to be dried and preserved in
darkness in the absence of air. Therefore, the efficiency of
hydroxytyrosol added in its native form to biological matrices
as a protective agent against reactive oxygen species could not
be guaranteed. For these reasons, triacetylated hydroxytyrosol
derivative was prepared. It has been demonstrated that hydroxy-
tyrosol acetyl derivatives offer two practical advantages: (i)
increased efficiency when added to alimentary, pharmaceutical,
or cosmetic matrices as a protective agent against reactive
oxygen species (ROS) in human cells and (ii) possible exploita-
tion as a nontoxic additive to lipophilic matrices (29). Moreover,
it has been established that hydroxytyrosol acetyl derivatives
showed a high free radical scavenging capacity, preventing
protein oxidation and lipid peroxidation when cells ex vivo were
exposed to active-oxygen substances and/or free radicals. This
property makes them potentially useful in treating chronic
pathological states associated with a high generation of active
oxygen substances and/or free radicals (30). Our findings
demonstrated that triacetylated hydroxytyrosol administration
induced a protective effect against experimental atherogenesis.
Triacetylated hydroxytyrosol could be converted in vivo by
esterases into the native form, which is responsible for protecting
animals from atherosclerosis. Indeed, it was recently reported
that hydroxytyrosol acetyl derivative was as efficient as the
parent compound in protecting human cells from oxidative
stress-induced cytotoxicity, after metabolization by esterases in
the intestinal tract (31).
In the current study, the high-cholesterol diet appeared to
cause an increase of liver weights. This could be related to an
accumulation of lipids such as triglycerides and cholesterol in
the liver. In contrast to its inhibitory effect on cholesterol
biosynthesis, dietary cholesterol was shown to stimulate hepatic
fatty acid biosynthesis and the incorporation of newly synthe-
sized fatty acid to hepatic TG (32). The decrease of liver weight,
in triacetylated hydroxytyrosol and hydroxytyrosol groups, leads
us to conclude that these phenolic compounds could reduce the
accumulation of lipids in liver.
Results from the serum lipid status of the high-cholesterol-
fed rats for 16 weeks showed increased concentrations of serum
TC, TG, and LDL, whereas HDL was decreased. The elevations
in serum total TC and TG levels observed in our study on HCD
animals are in agreement with those reported in several
studies (33, 34). The high levels of LDL-C found in HCD rats
may be attributed to a down-regulation in LDL receptors by
cholesterol included in the diet (35). Treatment of HCD-fed rats
with triacetylated hydroxytyrosol and hydroxytyrosol showed
a significant decrease in TC, TG, and LDL-C concentrations
and an increase in HDL-C levels compared to the corresponding
values of HCD group. A higher content of HDL-C is very
important in humans because it is correlated with a reduced
risk of coronary heart disease (36). The increased HDL
facilitates the transport of cholesterol from the serum to the liver,
where it is catabolized and excreted from the body.
Figure 6. Effects of triacetylated hydroxytyrosol and hydroxytyrosol on
rat liver (A), heart (B), kidney (C), and aorta (D) TBARS levels: 1, standard
diet (CD); 2, high-cholesterol diet (HCD); 3, HCD + hydroxytyrosol (3
mg/kg); 4, HCD + triacetylated hydroxytyrosol (3 mg/kg). Each bar
represents the mean ( SE from 10 rats. Bars with different letters differ;
p < 0.05.
The AI, defined as the ratio of LDL-C and HDL-C, is believed
to be an important risk factor of atherosclerosis. Our data clearly
demonstrate that triacetylated hydroxytyrosol and hydroxyty-
rosol significantly decrease the ratio. It has shown that abnor-
mally high serum levels of LDL-C and low serum levels of
HDL-C are associated with an increased atherosclerosis risk
(37). Increasing the HDL-C concentrations and decreasing the
LDL-C concentrations in HCD-fed rats indicates the antiathero-
genic property of triacetylated hydroxytyrosol and hydroxytyrosol.
In this study, a relatively high amount of purified hydroxy-
tyrosol was obtained in a short time by a simple hydrolysis
reaction of Olea europaea leaf extract followed by purification
using a C-18 silica gel column. There are several methods for
the production of hydroxytyrosol, and recently several publica-
tions dealing with the production of such compound have been
proposed. Hydroxytyrosol could be recovered from olive mill
wastewaters (25) or from solid–liquid waste (10) or by chemical

Hypolipidemic Effect of Triacetylated Hydroxytyrosol
J. Agric. Food Chem., Vol. 56, No. 8, 2008 2635
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It has been reported that oxidative stress is one of the
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pathogenesis of atherosclerosis (41). This stress results from
an imbalance between the production of free radicals and the
effectiveness of the antioxidant defense system (42). Dietary
polyphenols appear to have physiological antioxidant properties,
which quench reactive oxygen and nitrogen species, thereby
potentially contributing against the pathogenesis of cardiovas-
cular disease (43). In the present study we have observed
decreased activities of antioxidant enzymes SOD and CAT in
the liver of rats fed a high-cholesterol diet as compared to those
on normal diet. Our results are in agreement with reports of
other workers which suggest that feeding a high-cholesterol diet
to experimental animals depresses their antioxidant system due
to increased lipid peroxidation and formation of free radicals
(44). The treatment of cholesterol-fed rats with triacetylated
hydroxytyrosol and hydroxytyrosol increased the SOD and CAT
activities. The increase may have been due to the activation of
both enzymes by triacetylated hydroxytyrosol and hydroxyty-
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ACKNOWLEDGMENT
We are grateful to A Gargoubi (Technician, CBS) and H.
Auissaoui (Engineer, CBS) for technical assistance.

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Received for review August 30, 2007. Revised manuscript received
January 23, 2008. Accepted January 29, 2008. This work received
financial support from the Ministry of Higher Education, Scientific
Research and Technology in Tunisia and Agence Universitaire Fran-
cophone (PER program).
JF072589S