The Journal of Nutrition

Nutrition and Disease

An Extra-Virgin Olive Oil Rich in Polyphenolic

Compounds Has Antioxidant Effects in
Of1 Mice1,2
Mara-Jesus Oliveras-Lopez,3 Genoveva Berna,3 Everardo M. Carneiro,4 Herminia Lopez-Garca de la Serrana,5
Franz Martn,3* and M. Carmen Lopez5
3
Department of Molecular Biology and Biochemistry Engineering, Andalusian Center of Molecular Biology and Regenerative Medicine,
CIBERDEM, University of Pablo de Olavide of Seville, 41092 Seville, Spain; 4Department of Physiology and Biophysic, Institute
of Biology, State University of Campinas, CP-13.083-970 Campinas, Brazil; and 5Department of Nutrition and Food Science,
Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, 18071 Granada, Spain

Abstract
are because of its antioxidant content. We studied the antioxidant activity and mechanisms of an extra-virgin OO that is
rich in phenolics on pancreatic islets and liver in control mice (CTL) fed a nonpurified diet and in mice supplemented with 50
mL/d sunflower oil (SO) or 50 mL/d extra-virgin OO for 4 d. Plasma hydroxytyrosol concentration was determined by HPLCdiode array detector. Plasma antioxidant capacity, enzymatic activities, and lipid peroxidation were measured by
spectrophotometry. Islet function was studied by measuring insulin release. Islet cell gene expression was examined
using quantitative RT-PCR. The plasma hydroxytyrosol concentration was greater in OO mice than in CTL or SO mice (P ,
0.05) and was greater in SO mice than in CTL mice. The ratio of reduced:oxidized glutathione and the antioxidant capacity
in plasma was greater in OO mice than in CTL or SO mice (P , 0.05) and higher in SO mice than in CTL mice. Glucosestimulated insulin secretion was greater in OO mice than in CTL or SO mice (P , 0.05) and was also higher in SO mice than
in CTL mice. Protection against liver cell and b cell membrane lipid peroxidation was greater in OO mice than in CTL or SO
mice (P , 0.05) and was greater in SO mice than in CTL mice. Catalase (CAT) expression in the islet of Langerhans was
higher in OO mice than in CTL mice and SO mice (P , 0.05). The CAT and glutathione peroxidase 1 activities in the islet of
Langerhans were 25% greater in OO mice than in CTL mice and higher than in SO mice (P , 0.05) and they were greater in
SO mice than in CTL mice. These results indicate that, in metabolic tissues, protection by extra-virgin OO against oxidative
stress occurs primarily through a direct antioxidant effect as well as through an indirect mechanism that involves greater
expression and activity of certain enzymes with antioxidant activities. J. Nutr. 138: 10741078, 2008.

Introduction
Extra-virgin olive oil (OO)6 is becoming more important in daily
diets due to its beneficial effects on human health. Phenolic
compounds, oleic acid, and tocopherols are thought to be related
to some of these beneficial effects (1). Among these antioxidants
from extra-virgin OO, phenolic compounds have received the
most attention. Oleuropein derivatives, especially hydroxytyrosol, have been shown to have protective effects against
markers associated with the atherogenic process (13) and to
have an antioxidant capacity higher than that of other known
1

Supported by Instituto de Salud Carlos III (RCMN C03/08; ISCIII-RETIC

RD06/0015; CIBERDEM) and Center of National Research-Q-Brazil-2000-01/03-5.
Author disclosures: M.-J. Oliveras-Lopez, G. Berna, E. M. Carneiro, H. LopezGarca de la Serrana, F. Martn, and M. C. Lopez, no conflicts of interest.
6
Abbreviations used: CAT, catalase; CTL, control; GSSG, oxidized gluthatione;
GTpx, glutathione peroxidase; GSH, reduced glutathione; KRB, Krebs ringer
buffer; MDA, malondialdehyde; OO, olive oil; PGK1, Phosphoglycerate kinase 1;
SO, sunflower oil; SOD, superoxide dismutase; TAC, total antioxidant capacity.
* To whom correspondence should be addressed. E-mail: fmarber@upo.es.
2

1074

antioxidants such as vitamins E and C (4). Recent studies (5)

now show, however, opposite results with hydroxytyrosol
administration in the atherosclerotic process.
Previous articles (6) have shown that seed oils, with low levels
of polyphenols and a greater concentration of vitamin E, exhibited a smaller antioxidant capacity than the extracts obtained
from extra-virgin OO.
Due to the possible health benefits derived from the consumption of polyphenols, the study of their possible mechanisms
of protection is becoming a matter of great importance. In
humans, several studies have investigated the bioavailability of
polyphenols from extra-virgin OO after diet supplementation
(1). Reported results with humans and animals show that the
antioxidant activity of hydroxytyrosol is preserved after ingestion (7,8).
The studies of total antioxidant capacity (TAC) attempted to
evaluate general levels of antioxidants in plasma (9), finding
decreased TAC in animals exposed to oxidative stress. Tissues
with low levels of the reduced form of glutathione (GSH) are

0022-3166/08 $8.00 2008 American Society for Nutrition.

Manuscript received 26 July 2007. Initial review completed 13 August 2007. Revision accepted 20 March 2008.

Downloaded from jn.nutrition.org by guest on September 20, 2016

Extra-virgin olive oil (OO) is becoming more important in daily diets due to its beneficial effects on health, most of which

Materials and Methods

Mice and diets. Twelve to 16-wk-old male OF1 mice purchased from
Charles River were used throughout the experiments. The mice were
allowed free access to food and water and were maintained on a 12-h
light-dark cycle at 24C and constant humidity. Mice were fed with a
standard nonpurified diet containing 145 g/kg protein, 35 g/kg fat, 3.2
kcal/g (13.4 kJ/g), and 45 g/kg fiber (Teklad Global 14% Protein Rodent
Maintenance Diet 2014, Harlan Interfauna Iberica S.A.). Three groups
of mice with the same mean body weights (3133 g) were established: 1)
control (CTL) group (n 21), mice fed a standard rodent nonpurified
diet; 2) sunflower oil (SO) group (n 46), mice fed with standard rodent
nonpurified diet plus SO for 4 d; and 3) OO group (n 49), fed a
standard rodent nonpurified diet plus extra-virgin OO for 4 d. Mice were
dosed daily (at 09001000) with 50 mL of oil. The extra-virgin OO used
had a high total phenol concentration (.1500 mg/kg) with an elevated
hydroxytyrosol concentration (10 mg/g), as determined by Mateos et al.
(17). The SO used had very low levels of total polyphenols (6,18). Mice
received the oil by oral feeding using a sounding line connected to a
syringe to control the dosage employed. Extra-virgin OO or SO supplementation did not modify food or water intake (data not shown). The
review boards of animal ethics at our institutes (Andalusian Center of
Molecular Biology and Regenerative Medicine of Seville and Institute of
Biology, State University of Campinas) approved this study. We followed
the requirements regarding the protection of animals used for experimental and other scientific purposes (council directive of 24 November
1986; 86/609/EEC).
Sample collection and processing. Twenty-four hours after the 4th
oil dose, mice were anesthetized with 50 mg/kg of sodium pentobarbital,
and 1 mL of blood was obtained by intracardiac puncture. Plasma was
separated by centrifugation at 1000 3 g; 12 min t 4C in nonsiliconecoated tubes with sodium heparin. All samples were stored at 280C
until analysis.
Plasma hydroxytyrosol determination. We performed polyphenol
plasma extraction and quantitative plasma hydroxytyrosol determination as reported by Ruiz-Gutierrez et al. (19). All analytical determinations were performed in triplicate. The plasma hydroxytyrosol
concentration was determined in CTL, OO, and SO mice after 3 and 4 d
of supplementation with 50 mL/d and 100 mL/d of oil.

Measurements of plasma antioxidant capacity. Antioxidant capacity and the GSH:GSSG relationship were measured by colorimetric
assays using the Antioxidant Potential assay kit (Deltaclon) and the
GSH/GSSG-412 assay kit (Oxis International), respectively. Regarding
the GSH:GSSG measurements, the plasma was immediately frozen at
280C until the determination day to maintain the redox state of the
GSH. All analytical determinations were conducted in triplicate.
Insulin measurements. Insulin measurements were performed as
previously described (20). Briefly, fresh collagenase-isolated islets were
incubated for 30 min at 37C in fresh modified Krebs ringer buffer (KRB)
containing 115 mmol/L NaCl, 10 mmol/L NaHCO3, 5 mmol/L KCl,
2 mmol/L NaH2PO4, 1.1 mmol/L MgCl2, 25 mmol/L HEPES acid, and
2.56 mmol/L CaCl2 and supplemented with 5.6 mmol/L glucose and 1%
bovine serum albumin (Sigma), pH 7.4. Then, one-half of the islets were
incubated for 30 min at 37C in the same KRB buffer plus 10 mmol/L
hydrogen peroxide, whereas the rest were incubated in the same KRB
buffer alone. Finally, islets were incubated in groups of 4 in 1 mL of KRB
with 1% bovine serum albumin plus the different glucose concentrations
(3 and 22.2 mmol/L) for 60 min at 37C. Insulin content and secretion
were measured as previously described (20). In addition, 15 mL aliquots
of the same samples were stored at 280C for protein determination by
Bradford assay. Insulin was assayed by radioimmunoassay using a kit
from Diagnostic Products. Standard curves and experimental points
were conducted in triplicate.
Lipid peroxidation determination. Fresh collagenase-isolated islets and
50 mg of disaggregated liver were incubated for 30 min at 37C in KRB
buffer plus 10 mmol/L hydrogen peroxide. Afterwards, both tissues were
washed with PBS, sonicated at 4C in lysis buffer [50 mmol/L Tris-HCl
(pH 7.5), 0.9% NaCl, 1 mmol/L EDTA, 100 mmol/L phenylmethylsulphonyl fluoride, 1 mmol/L leupeptin, 1 g/L pepstatin, and 100 g/L
aprotinin], and centrifuged at 20,800 3 g; 15 min at 4C to obtain
supernatants. In addition, 15-mL aliquots of the same samples were stored
at 280C for protein determination by Bradford assay. Lipid peroxidation
was assessed using an assay based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole, with MDA and 4-hydroxyalkenals at
45C, as described by Esterbauer and Cheeseman (21).
Quantitative PCR. RNA was extracted with a specific RNA protocol.
RNA was prepared using Trizol (Invitrogen) reagent according to the
instructions, and 2 mg of total RNA was reverse transcribed using
TaqMan Universal PCR Master mix (Applied Biosystems). The purity of
the RNA extracted was measured in a spectrophotometer at 260 nm. The
cDNA was amplified by quantitative real-time PCR using an ABI prism
7700 Sequence Detection system (Applied Biosystems). Quantitative
real-time PCR were performed in triplicate using 5 mL of cDNA, 10 mL
of TaqMan Universal PCR Master mix 23, and 1 mL of mix 203 of
probe and primers in a final volume of 20 mL. Primers and probes for
genes were from Applied Biosystems Assay on-Demand Gene expression
products: Catalase (Mn 00437992_m1), Glutathione peroxidase 1 (Mm
00656767_g1), Superoxide dismutase 2 (Mm 00449726_m1), and
Phosphoglycerate kinase 1 (PGK1) (Mm00435617_m1) as a housekeeping gene. We followed the Applied Biosystems protocol by selecting
primers with introns located between 2 exons of each gene to avoid
potential amplification of contaminating genomic DNA. Amplifications
were normalized to PGK1 and the quantification of gene expression was
performed using the DDCt calculation, where Ct was the threshold cycle.
The amount of the target gene was normalized to PGK1 relative to the
calibrator (islet from CTL group) using the equation 22DDCt.
Measurement of antioxidant enzyme activities. The activities of
SOD, CAT, and GTPX were measured by spectrophotometric assay kits
from Cayman Chemicals. Antioxidant enzyme activities were measured
in freshly isolated islets incubated for 60 min at 37C in the presence of
5.6 mmol/L glucose. Islets were homogenized in cold buffer containing
50 mmol/L potassium phosphate and 1 mmol/L EDTA at pH 7.4. Total
SOD activity was measured by utilization of a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. CAT activity was measured using the peroxidatic function of
Antioxidant effects of extra-virgin olive oil

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much more sensitive to external oxidative injuries (10), as cells

exposed to higher oxidation exhibit accumulation of oxidized
glutathione (GSSG).
Pancreatic islets have a higher risk of developing oxidative
stress (11) than other tissues. In this regard, a protective effect of
polyphenol-rich diets has been studied previously in diabetes
(12). In addition, a positive effect of monounsaturated fat-rich
diets on insulin sensitivity has been shown in both healthy (13)
and diabetic (14) subjects.
Malondialdehyde (MDA) accumulation results from membrane peroxidation (15). Therefore, quantification of MDA accumulation provides insight into the integrity of lipid membranes.
Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GTPX) are considered some of the more important antioxidant defenses of the organism against the production
of free radicals (10). The low level of expression of these antioxidant enzymes has been considered the main reason for the
susceptibility of the b cell to oxidative stress (16); therefore, it
was predicted that increasing the expression of these enzymes
would impart a beneficial effect of antioxidant protection.
In the present study, some of the mechanisms by which polyphenols from extra-virgin OO impart protective and beneficial
effects as antioxidants were proposed. The in vivo biological
effects of a diet rich in extra-virgin OO phenolics, especially
hydroxytyrosol, were studied in mice.

CAT. The GTPX activity was measured indirectly by a coupled reaction

with glutathione reductase. All analytical determinations were conducted in triplicate.

TABLE 2

Lipid peroxidation in liver and pancreatic islets in

OF1 mice fed different oils for 4 d1

Hydrogen peroxide treatment

Statistical analysis. Results are expressed as means 6 SEM. Differences in means were tested for significance by 1-way ANOVA and by the
following post hoc tests when necessary for multiple comparisons:
Bonferroni, Fisher least significant difference tests, and the Tukey-Kramer
test when the group size was the same. Analyses were performed using
the SPSS statistical software package (version 15.0). Differences were
considered significant for P , 0.05.

Results

TABLE 1

The relationship between GSH:GSSG and plasma

antioxidant potential in OF1 mice fed different oils
for 4 d1

GSH:GSSG
Potential antioxidant, mmol/L
copper-reducing equivalents

SO

OO

3.73 6 0.31c
17.42 6 2.97c

4.12 6 0.28b
22.62 6 3.65b

5.39 6 0.48a
36.91 6 4.57a

Values are means 6 SEM, n 6 (potential antioxidant) or 10 (GSH:GSSG). Means in

a row without a common letter differ, P , 0.05.

1076

Oliveras-Lopez et al.

OO

mmol MDAL21mg protein21

19.68 6 2.39c 11.98 6 1.56b
8.32 6 0.89a
31.81 6 3.5c
21.97 6 2.98b 14.62 6 1.33a

Liver
Pancreatic islets
1

Values are means 6 SEM, n 4. Means in a row without a common letter differ,
P , 0.05.

SO group was higher than that of the OO group (P , 0.05) in

both tissues (Table 2).
In islets of Langerhans from mice supplemented with extravirgin OO, CAT expression was 98% greater than in the CTL
group (P , 0.05) (Table 3). SO supplementation, however,
elicited a 40% decrease relative to the CTL group. By contrast,
SO and OO groups showed a decrease of 22% in SOD 2
compared with the CTL group, with no significant differences
(Table 3). Neither SO nor extra-virgin OO supplementation
modified GTPX 1 expression compared with the CTL group
(Table 3).
Though SO and extra-virgin OO supplementation did not
modify SOD 2 activity compared with the CTL group (Table 4),
CAT and GTPX 1 had greater activity in the OO group (P ,
0.05) compared with the CTL and SO groups (Table 4).

Discussion
To establish the relationship between polyphenols from foods
and their biological effects related to disease prevention, it is
essential to understand the factors influencing the absorption
of these compounds (22). Mechanisms of absorption, bioavailability, and distribution of hydroxytyrosol in human tissues are
currently being studied by several authors (8,23), because this
compound is actually considered one of the polyphenols with the
highest antioxidant activity (7,8). Bioavailability assays performed
with animals and humans reveal that phenolics from extra-virgin
OO are absorbed in a proportion of 5560% (24), but most of
these tests are generally achieved with polyphenol amounts higher
than those usually seen in the common daily diet.
In this article, we tested different periods of time and different
oil doses (up to 100 mL) and found the same levels of plasma
hydroxytyrosol content after 3 d of oil administration. Thus, our
results reveal the first day at which we observed a significant
increase in the hydroxytyrosol levels. We have shown that with
the consumption of an extra-virgin OO with a high phenolic
content (0.494 mg of hydroxytyrosol), the plasma levels for this
compound increases up to 10-fold greater. Although this
compound has been shown to be absorbed in a dose-dependant
manner by mammals (7), we revealed that increased oil ingestion

TABLE 3

Antioxidant enzyme gene expression in pancreatic

islets in OF1 mice fed different oils for 4 d1

mRNA relative concentration

CTL

SO

SO

OO
-fold of CTL

CAT
SOD-2
GTPX
1

0.6 6 0.1
0.78 6 0.1
1.16 6 0.26

1.98 6 0.6a
0.77 6 0.12
0.85 6 0.25

Values are means 6 SEM, n 4. Means in a row without a common letter differ,
P , 0.05.

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Mice from the OO and SO groups reached their maximum

plasma hydroxytyrosol levels after 3 d of supplementation with
50 mL/d of oil. The plasma hydroxytyrosol concentration was
greater in OO mice than in CTL or SO mice (P , 0.05) and was
greater in SO mice than in CTL mice for all the doses and time
points assayed. There were, however, no significant differences
within each group. After 4 d of 50 mL/d oil supplementation, the
plasma hydroxytyrosol concentration in OO mice (2.3 6 0.19
mmol/L) was 10-fold higher than that of the CTL group (0.21 6
0.06 mmol/L) and 0.9-fold higher than in the SO group (1.21 6
0.09 mmol/L) (P , 0.05). The hydroxytyrosol concentration
from the SO group, however, was 4.7-fold higher than that of the
CTL group (P , 0.05). The plasma GSH:GSSG ratio was greater
in the OO group than in the SO group (P , 0.05) and CTL
group (P , 0.05) (Table 1). In addition, plasma antioxidant
capacity differed in mice from the OO group compared with the
SO (P , 0.05) and CTL groups (P , 0.05). The CTL and SO
groups did not differ (Table 1).
Extra virgin OO or SO supplementation did not modify basal
(3 mmol/L) or glucose-stimulated (22 mmol/L) insulin secretion.
The CTL group did not differ from the OO or SO groups in
glucose-stimulated insulin secretion. In contrast, after 10 mmol/L
hydrogen peroxide incubation, islets from extra-virgin OOsupplemented mice had a higher (P , 0.05) glucose-stimulated
insulin secretion (0.1 6 0.006 pmolmg protein21h21) by 22
mmol/L glucose compared with the CTL group (0.05 6 0.007
pmolmg protein21h21) and the SO group (0.08 6 0.007
pmolmg protein21h21). SO supplementation, however, resulted
in an improvement (P , 0.05) of 22 mmol/L glucose-stimulated
insulin secretion compared with CTL mice.
Oil supplementation protected against cell membrane lipid
peroxidation. Liver tissue from the SO and OO groups incubated
with 10 mmol/L hydrogen peroxide displayed significant differences in MDA production compared with the CTL group (n 4)
(Table 2). Pancreatic islets from the SO and OO groups incubated with 10 mmol/L hydrogen peroxide displayed the same
significant difference in MDA production compared with the
CTL group (n 4). In addition, the MDA concentration in the

CTL

TABLE 4

Antioxidant enzymes activity in pancreatic islets in

OF1 mice fed different oils for 4 d1

Antioxidant enzyme activity

CAT
SOD-2
GTPX4.3

CTL

SO

OO

mmolmin21 mg protein21
6.2 6 0.2
6.4 6 0.3b
7.8 6 0.2a
115 6 12
103 6 10
107 6 13
4.3 6 0.2b
4.6 6 0.3b
5.6 6 0.3a
b

Values are means 6 SEM, n 4. Means in a row without a common letter differ,
P , 0.05.

Acknowledgments
We thank N. Illera and M. P. Navarro for their skilled technical
assistance and the American Journal Experts Association
English Language Science & Medical Research Editing and
Proofreading (www.journalexperts.com) for its English revision. We thank A. M. Sanchez and M. J. Porras for their
statistical expertise.

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and more days of oil administration do not lead to further

increases in the plasma levels.
The results of our study suggest that the differences among
the 3 mice groups studied were a result of the total phenol
content of their respective diet. In this sense, our results are in
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to hydrogen peroxide. The importance of this approach is enhanced if we consider that diabetic patients have higher oxidative stress (12), which is also a common mechanism involved
in diabetic complications. In addition, hyperglycemia causes enhanced free radical concentration (11), so islets of Langerhans
are exposed to higher levels of oxidation products. The results
from our study show that, in this tissue, supplementation of mice
with extra-virgin OO potentially has a direct protective effect
against oxidation.
It is important to investigate the relationship between free
radical production and membrane damage combined with the
subsequent production of toxic products from peroxidation.
Most of the studies regarding MDA formation describe the
mechanisms involved in the in vitro degradation of proteins,
DNA, RNA, and other biomolecules (15). Previous articles
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polyphenolic antioxidants from tea, were added to cell cultures
exposed to hydrogen peroxide (10). This study, however,
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against cell lipid peroxidation. We performed in vitro assays of 2
metabolic tissues easily subjected to oxidative stress, confirming
that the group fed with extra-virgin OO was protected over a
longer duration.

An increase in expression of antioxidant enzymes is an

important defense against oxidative stress (10). Previous studies
report that overexpression of CAT, SOD, or GTPX reduces
oxidative damage (29,30). In all cases, damage had previously
been induced in cell cultures. More recently, pure antioxidant
compounds have also been tested in diverse in vitro and in vivo
(31) assays and have been shown to promote gene expression of
these antioxidant enzymes.
Still, few articles have reported (32,33) the influence of diet
supplementation on antioxidant enzyme expression. This is a
novel approach, in which gene expression from antioxidant
enzymes was measured in healthy mice fed extra-virgin OO as a
part of their diet. Pancreatic islets from assays had not
previously been exposed to an oxidizing agent and the supplementation of mice with extra-virgin OO was performed in doses
lower than that in current articles. The CAT enzyme exhibited a
higher response in mRNA synthesis modulated by extra-virgin
OO ingestion. The 3 studied enzymes operate in a coordinated
system, so antioxidant protection would not involve the overexpression of all of them (34). In addition, the enzymatic activity
of CAT and GTPX 1 increased significantly in the OO group.
Thus, we suggest that extra-virgin OO probably also has an
indirect effect against oxidative stress by modulating gene
expression and enzyme activity, which enhances enzymatic
antioxidant defenses.
Our results show that extra-virgin OO can have high nutritional value. These findings indicate that a moderate daily ingestion of extra virgin OO for 3 d reduces oxidative stress in the
pancreas, so the use of this oil could have advantages for use in
enteral formulas. These findings could have significant consequences for human health and thus deserve further investigation.

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