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The effect of virgin olive oil (VOO) enriched with its own polyphenols (PC) and/or thyme-phenols on the protection of oxidative DNA damage and antioxidant endogenous enzymatic system (AEES) were estimated in 33 hyperlipidemic subjects after the consumption of VOO, VOO enriched with its own PC (FVOO), or complemented with thyme PC (FVOOT). Compared to pre-intervention, 8-hydroxy-2'-deoxyguanosine (marker for DNA damage) decreased in the FVOO intervention and to a greater extent in the FVOOT with a parallel significant increase in olive and thyme phenolic biomarkers. Superoxide Dismutase (AEES enzyme) significantly increased in the FVOO intervention and to a greater extent in the FVOOT with a parallel significant increase in thyme phenolic metabolites. When comparing all three oils, FVOOT appeared to have the greatest effect in protecting against oxidative DNA damage and improving AEES. The sustained intake of a FVOOT improves DNA protection against oxidation and AEES probably due to a greater bioavailability of thyme PC in hyperlipidemic subjects.
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Virgin Olive Oil Enriched with Its Own Phenols or Complemented
with Thyme Phenols Improves DNA Protection against Oxidation and
Antioxidant Enzyme Activity in Hyperlipidemic Subjects
Marta Romeu,
Laura Rubió,
,
Vanessa Sánchez-Martos,
Olga Castañer,
§
Rafael de la Torre,
§
Rosa M. Valls,
Rosa Ras,
Δ
Anna Pedret,
Úrsula Catalán,
María del Carmen López de las Hazas,
María J. Motilva,*
,
Montserrat Fitó,
§
Rosa Solà,*
,
and Montserrat Giralt
Pharmacology Unit, Department of Basic Medical Sciences, Facultat de Medicina i Ciències de la Salut, NFOC group, Universitat
Rovira i Virgili, C/Sant Llorenç21, 43201 Reus, Spain
Food Technology Department, UTPV-XaRTA, Agrotecnio Research Center, Universitat de Lleida, C. Alcalde Rovira Roure 191,
25198 Lleida, Spain
§
Cardiovascular Risk and Nutrition Research Group, CIBER de Fisiopatología de la Obesidad y la Nutrición (CIBEROBN),
IMIM-Institut Hospital del Mar dInvestigacions Mèdiques, C. Doctor Aiguader 88, 08003 Barcelona, Spain
Unitat de Recerca en Lı́
pids i Arteriosclerosis, CIBERDEM, St. Joan de Reus University Hospital, CTNS, IISPV, Facultat de
Medicina i Ciències de la Salut, NFOC Group, Universitat Rovira i Virgili, C. Sant Llorenç21, 43201 Reus, Spain
Δ
Center for Omics Sciences, Avenida Universitat 1, 43204 Reus, Spain
*
SSupporting Information
ABSTRACT: The eects of virgin olive oil (VOO) enriched with its own phenolic compounds (PC) and/or thyme PC on the
protection against oxidative DNA damage and antioxidant endogenous enzymatic system (AEES) were estimated in 33
hyperlipidemic subjects after the consumption of VOO, VOO enriched with its own PC (FVOO), or VOO complemented with
thyme PC (FVOOT). Compared to pre-intervention, 8-hydroxy-2-deoxyguanosine (a marker for DNA damage) decreased in
the FVOO intervention and to a greater extent in the FVOOT with a parallel signicant increase in olive and thyme phenolic
metabolites. Superoxide dismutase (AEES enzyme) signicantly increased in the FVOO intervention and to a greater extent in
the FVOOT with a parallel signicant increase in thyme phenolic metabolites. When all three oils were compared, FVOOT
appeared to have the greatest eect in protecting against oxidative DNA damage and improving AEES. The sustained intake of a
FVOOT improves DNA protection against oxidation and AEES probably due to a greater bioavailability of thyme PC in
hyperlipidemic subjects.
KEYWORDS: virgin olive oil, phenol enrichment, thyme phenols, hyperlipidemia, oxidative stress, enzymatic antioxidants
INTRODUCTION
Virgin olive oil (VOO) is a typical food found in the
Mediterranean diet, and several experimental and human
studies have revealed that it has a unique phenolic composition
with relevant biological properties related to its antioxidant
capacity and also modulating gene expression.
1
The measure-
ment of the antioxidant status of biological uids is used as an
early warning sign of possible disease onset and also as an
indicator of the status of the antioxidant endogenous enzymatic
system (AEES).
2
The polyphenol content of commercial VOOs is inuenced
by multiple agronomic and technological factors. In this
context, the enrichment of VOOs with its own phenolic
compounds (PC) is an interesting strategy to increase and
standardize the daily intake of PC in the real food matrix
without increasing caloric intake. Additionally, avoring olive
oils with herbs and spices can improve their PC prole. The
leafy parts of thyme and its essential oil have been used in foods
for avor, aroma, and preservation and also in traditional
medicines. Thyme is rich in PC, for example, favonoids,
phenolic acids, and monoterpenes.
3
Thus, the enrichment of
VOOs with complementary PC from thyme was proposed as a
novel approach to investigate the combined or synergic
benecial eects of PC from dierent sources. In previous
studies, we observed that when PC from olive and thyme in a
combined extract were administered to rats, an enhanced
bioavailability of olive PC occurred in the presence of thyme
PC.
4
In agreement with these ndings, when the volunteers
from the VOHF Project (Virgin Olive oil and HDL
Functionality (VOHF): a model for tailoring functional food)
ingested VOO enriched with its own PC plus complementary
PC from thyme, an improved bioavailability of olive PC was
also observed.
5
The combination of dierent PC sources might,
therefore, be a promising approach to not only improve the
bioavailability but also provide a consequent enhancement of
their biological eects.
Received: October 13, 2015
Revised: February 17, 2016
Accepted: February 18, 2016
Article
pubs.acs.org/JAFC
© XXXX American Chemical Society ADOI: 10.1021/acs.jafc.5b04915
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Antioxidant enzymes, such as superoxide dismutase (SOD),
glutathione peroxidase (GSH-Px), and catalase (CAT), which
are of endogenous origin and constitute the rst line of
antioxidant defense, provide a real state of long-term defense
against oxidative stress. The activity of this rst line of
antioxidants may be modulated by dietary bioactive com-
pounds. Thus, PC provided by VOO can protect against
systemic oxidation, which is modulated by the main AEES.
6
The protection of body cells and molecules such as DNA,
proteins, and lipids from oxidative damage could be considered
as a benecial physiological eect. Dierent markers of
oxidative damage or repair to molecules should preferably be
determined in the same study and could be useful if appropriate
techniques are used for its analysis.
7
In this regard, mass
spectrometry determination of 8-hydroxy-2-deoxyguanosine
(oxidative damage to DNA), F2-isoprostanes (oxidative
damage to lipids), and methionine sulfoxide (oxidative damage
to proteins) is appropriate.
810
Our aim was to investigate the eect of two functional
VOOs, either enriched with their own PC (FVOO) or
complemented with thyme PC (FVOOT), on the protection
of oxidative stress, using urine and plasma oxidation biomarkers
and erythrocyte antioxidant enzymes, simultaneously with the
detection of urine, plasma, and erythrocyte phenolic metabo-
lites in hyperlipidemic subjects.
MATERIALS AND METHODS
Study Participants and Experimental Design. The VOHF-
sustained study was a randomized, double-blinded, crossover,
controlled trial with 33 hypercholesterolemic volunteers (total
cholesterol > 200 mg/dL) (19 men and 14 women), aged 3580
years. Exclusion criteria included the following: BMI > 35 kg/m2,
smokers (>7 cigarettes/week), athletes with high physical activity
(>3000 kcal/day), diabetes, multiple allergies, intestinal diseases, or
any other disease or condition that would worsen adherence to the
measurements or treatment.
Subjects were randomized to one of three orders of administration
of 25 mL/day of (i) virgin olive oil (VOO; 2.88 mg total phenols/
day), (ii) VOO enriched with its own PC (FVOO; 12.59 mg total
phenols/day), and (iii) VOO enriched with both its own PC and
thyme PC (FVOOT; 12.10 mg total phenols/day). In the randomized,
double-blinded, controlled crossover design, intervention periods were
of 3 weeks with a daily ingestion of 25 mL of raw VOO distributed
among meals and preceded by a 2 week wash-out with a common olive
oil (Figure 1). The random allocation sequence was generated by a
statistician, participant enrollment was carried out by a researcher, and
participantsassignment to interventions according to the random
sequence was done by a physician.
To avoid an excessive intake of antioxidants, such as PC, during the
clinical trial period, participants were advised to limit the consumption
of polyphenol-rich food. A 3-day dietary record was administered to
the participants before and after each intervention period to control
their habitual diet throughout the study. A set of portable containers
with the corresponding 25 mL of VOO for each day of consumption
was delivered to the participants at the beginning of each VOO
administration period. The participants were instructed to return the
containers to the center after the corresponding period to register the
amount consumed. Subjects with <80% of treatment adherence (5
full VOO or FVOO or FVOOT containers returned) were considered
noncompliant for this treatment.
Twenty-four-hour urine was collected in containers before each
visit. Urine samples were stored at 80 °C prior to use. Blood samples
were collected at fasting state. Plasma samples were obtained by
centrifugation of whole blood directly after being drawn and were
preserved at 80 °C until use. Erythrocytes were obtained by
centrifugation, washed twice with saline, and preserved at 80 °C until
use.
The VOHF study was approved by the Clinical Research Ethical
Committee of the Institut de Recerca Hospital del Mar (IMIM)
(CEIC 2009/3347/I), and the study was listed on ISRCTR.org,
ISRCTN77500181. Protocols were according to the Helsinki
Declaration and good clinical practice guidelines of the International
Conference of Harmonization (ICH GCP); the trial was conducted
according to extended CONSORT 2010 guidelines.
Sample Size and Power Analysis. The sample size of 30
individuals allows at least 80% power to detect a statistically signicant
dierence among three groups of 3 mg/dL of high-density lipoprotein
cholesterol (HDL-C) and a standard deviation of 1.9, using an
ANOVA test and assuming a dropout rate of 15% and a type I error of
0.05.
Preparation and Characterization of VOO. VOO with a low
phenolic content (80 mg total phenols/kg oil) was used as a control
condition in the intervention and as an enrichment matrix for the
preparation of the two phenol-enriched VOOs with the same amount
of PC (500 mg total phenols/kg oil) but with dierent phenolic
composition. FVOO was enriched with its own PC by adding a phenol
extract obtained from freeze-dried olive cake, and FVOOT was
enriched with its own PC (50%) and complemented with thyme PC
(50%) using a phenol extract made up of a mixture of olive cake and
dried thyme. FVOOT contained 50% of olive PC (hydroxytyrosol
derivates) and 50% thyme PC (avonoids, phenolic acids, and
monoterpenes) (Table 1). The procedure for obtaining the phenolic
extracts and enriched oils had been previously developed.
11
For the
wash-out period, a commercial common olive oil kindly provided by
Borges Mediterranean Group was used. The total phenolic content of
the VOOs was measured with the FolinCiocalteu method.
12
The
phenolic prole of the VOOs was analyzed by high-performance liquid
chromatography coupled to tandem mass spectrometry (HPLC-MS/
MS) using a previously described method.
13
Tocopherols and fatty
Figure 1. VOHF study design in human volunteers. This was a randomized, crossover, controlled trial with 30 hyperlipemic individuals comparing
the eects of three types of virgin olive oil: control (VOO), enriched with its own phenolics (FVOO), and enriched not only with its own phenolics
but also with phenolics from thyme (FVOOT). WO: wash-out.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b04915
J. Agric. Food Chem. XXXX, XXX, XXXXXX
B
acids in the VOOs were analyzed following the procedure described by
Morellóet al.,
14
and the carotenoid content was analyzed as previously
described by Criado et al.
15
Lipid Prole. Blood samples were collected at fasting state at least
10 h prior to the study, at the commencement of the study and before
and after each treatment. EDTAplasma glucose, total cholesterol
(TC), and triglyceride (TG) levels were measured using standard
enzymatic automated methods in a PENTRA-400 autoanalyzer (ABX-
Horiba Diagnostics, Montpellier, France). HDL-C was measured as
soluble HDL-C determined by an accelerator selective detergent
method (ABX-Horiba Diagnostics). Low-density lipoprotein choles-
terol (LDL-C) was calculated by the Friedewald equation whenever
TGs were <300 mg/dL.
LC-MS Oxidative Stress Markers. A 1290 UHPLC series liquid
chromatograph coupled to a 6490 QqQ/MS (Agilent Technologies,
Palo Alto, CA, USA) was used for 8-hydroxy-2-deoxyguanosine (8-
OHdG), methionine (Met), methionine sulfoxide (MetSO), and 8-iso
prostaglandin F2α(8-iso PGF2α) quantication. Ionization was
carried out by electrospray ion source (ESI), and acquisition was
done in multiple reaction monitoring (MRM) mode. ESI and MRM
conditions are summarized in Supplementary Table 1 for all of the
compounds.
Chromatographic separation in both the 8-OHdG method and Met
and MetSO methods was performed in an Acquity UPLC BEH
HILIC, 2.1 ×100 mm, 1.8 μm (Waters, Milford, MA, USA), at a ow
rate of 0.4 mL/min, using 50 mM NH4AcO in water (solvent A) and
ACN (solvent B). The elution gradient for the 8-OHdG method was
02 min, 100% B isocratic; 24 min, 80% B; 45 min, 80% B
isocratic; 57 min, 20% B; 79 min, 20% B isocratic; and 910 min,
100 B, applying a post-run of 1.5 min and injecting a sample volume of
2μL. The retention time of 8-OHdG was 4.37 min. The elution
gradient for Met and MetSO was 01 min, 95% B isocratic; 16 min,
20% B; 610 min, 20% B isocratic; and 1011 min, 95% B, with a
post-run of 1.5 min and a sample volume injection of 5 μL. Retention
times of Met and MetSO were 3.51 and 4.30 min, respectively.
For 8-OHdG quantication, an aliquot of 50 μL of freshly thawed
urine sample was mixed with 20 μL of 100 ng/mL of 8OH-2dOG-
15N5 as internal standard in ACN. After a vortex of 10 s and
centrifugation at 15000 rpm for 10 min at 4 °C, supernatant was
analyzed by liquid chromatography coupled to mass spectrometry
(LC-MS).
For Met and MetSO quantication, an aliquot of 50 μL of freshly
thawed plasma sample was mixed with 25 μLof25μg/mL of L-
methionine-13C,d3as internal standard and 150 μL of ACN/H2O50
mM NH4AcO 95:5 (v/v). After a vortex of 10 s and centrifugation at
15000 rpm for 10 min at 4 °C, the supernatant was analyzed by LC-
MS.
For 8-iso PGF2α, the chromatographic separation was carried out in
an Eclipse XDB-C18, 2.1 ×150 mm, 1.8 μm (Agilent Technologies),
at a ow rate of 0.4 mL/min, using 0.2% acetic acid in water (solvent
A) and ACN (solvent B). The elution gradient was 02 min, 0% B
isocratic; 210 min, 50% B; 1011 min, 100% B; 1314 min, 100% B
isocratic. A pos- run of 1.5 min was applied. Injected sample volume
was of 20 μL. Its retention time was 9.97 min.
For 8-iso PGF2αquantication, an aliquot of 250 μL of freshly
thawed urine sample was mixed with 20 μL of 100 ng/mL of 8iso
PSF2α-d4 as internal standard in water/methanol 2:1 (v/v) to protein
precipitation. After a vortex of 10 s, extraction was done by the
addition of 750 μL of diethyl ether, agitation for 10 min at room
temperature, and centrifugation at 4000 rpm for 10 min at 4 °C. A
volume of 700 μL of the upper organic phase was dried under a
nitrogen gas ow and resuspended in 50 μL of water/methanol 2:1 (v/
v). After vortex and centrifugation at 15000 rpm at 4 °C for 10 min,
the supernatant was analyzed by LC-MS.
In the quantication of samples, standard solutions at dierent
levels of concentration were used to obtain calibration curves, and
compounds in the samples were quantied by interpoling the analyte/
IS peak abundance ratio in these curves.
Antioxidant Enzymes in Erythrocytes. Determination of the
hemoglobin (Hb) content of lysate erythrocytes was carried out by
laser-impedance colorimetry. SOD activity in erythrocytes was
performed following McCord and Fridovich methodology
16
(Ransel
RS 125, Randox Laboratories, Crumlin, UK) and was expressed in
Table 1. Composition of the Olive Oils Used in the Study
Regarding Phenolic Compounds, Fat-Soluble
Micronutrients, and Fatty Acid Prole
a
VOO FVOO FVOOT
phenolic compounds (mg/25 mL/day)
hydroxytyrosol 0.01 ±0.00 0.21 ±0.02 0.12 ±0.00
3,4-DHPEA-AC nd 0.84 ±0.06 0.39 ±0.04
3,4-DHPEA-EDA 0.04 ±0.00 6.73 ±0.37 3.43 ±0.29
3,4-DHPEA-EA 0.26 ±0.04 0.71 ±0.06 0.36 ±0.03
total HT derivates 0.30 8.49 4.30
p-hydroxybenzoic acid nd 0.02 ±0.00 0.06 ±0.00
vanillic acid nd 0.07 ±0.00 0.13 ±0.01
caeic acid nd 0.00 ±0.00 0.06 ±0.00
rosmarinic acid nd nd 0.41 ±0.03
total phenolic acids 0.09 0.65
thymol nd nd 0.64 ±0.05
carvacrol nd nd 0.23 ±0.02
total monoterpenes 0.86
luteolin 0.04 ±0.00 0.18 ±0.02 0.21 ±0.02
apigenin 0.02 ±0.00 0.06 ±0.00 0.10 ±0.00
naringenin nd nd 0.20 ±0.02
eriodictyol nd nd 0.17 ±0.01
thymusin nd nd 1.22 ±0.09
xanthomicrol nd nd 0.53 ±0.06
7-methylsudachitin nd nd 0.53 ±0.09
total flavonoids 0.06 0.23 2.95
pinoresinol 0.05 ±0.00 0.12 ±0.00 0.10 ±0.05
acetoxipinoresinol 2.47 ±0.19 3.66 ±0.31 3.24 ±0.28
total lignans 2.52 3.78 3.34
fat-soluble micronutrients (mg/25 mL/day)
α-tocopherol 3.27 ±0.01 3.40 ±0.02 3.44 ±0.01
lutein 0.05 ±0.00 0.06 ±0.00 0.06 ±0.00
β-cryptoxanthin 0.02 ±0.00 0.03 ±0.00 0.02 ±0.00
β-carotene 0.01 ±0.00 0.02 ±0.00 0.02 ±0.00
fatty acids (relative area %)
palmitic acid 11.21 11.20 11.21
stearic acid 1.92 1.92 1.92
araquidic acid 0.36 0.36 0.36
behenic acid 0.11 0.11 0.11
total saturated 13.75 13.74 13.75
palmitoleic acid 0.70 0.70 0.69
oleic acid 76.74 76.83 76.75
gadoleic acid 0.27 0.27 0.27
total
monounsaturated
77.71 77.80 77.72
linoleic acid 7.43 7.36 7.43
timnodonic acid 0.36 0.36 0.35
linolenic acid 0.43 0.43 0.43
total
polyunsaturated
8.22 8.15 8.22
a
Values provide the individual phenolic characterization of the olive
oils expressed as means ±SD of mg phenols/25 mL oil/day.
Abbreviations: VOO, virgin olive oil; FVOO, functional virgin olive oil
enriched with its own phenolics; FVOOT, functional virgin olive oil
enriched with both its own phenolics and phenolics from thyme; 3,4-
DHPEA-AC, 4-(acetoxyethyl)-1,2-dihydroxybenzene; 3,4-DHPEA-
EDA, dialdehydic form of elenolic acid linked to hydroxytyrosol;
3,4-DHPEA-EA, oleuropein aglycone; HT, hydroxytyrosol; nd, not
determined.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b04915
J. Agric. Food Chem. XXXX, XXX, XXXXXX
C
units per gram of Hb. This method employs xanthine and xanthine
oxidase to generate superoxide radicals, which react with 2-(4-
iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a
red formazan dye. The SOD activity is then measured by the degree of
inhibition of this reaction. GSH-Px activity was measured by a
modication of the method of Paglia and Valentine
17
(Ransel RS 505,
Randox Laboratories) and expressed in units per liter. GSH-Px
catalyzes the oxidation of glutathione (GSH) by cumene hydro-
peroxide. CAT activity was measured according to the method of
Aebi
18
with slight modications. Briey, 70 mL of phosphate buer, 50
mL of erythrocyte lysate (5 mg protein/mL), and 50 mL of 1% H2O2
were added in each well of a quartz microplate (Hellma, Müllheim,
Germany). After 12 s of shaking in a plate reader (FisherScientic,
Madrid, Spain), the absorbance at 240 nm was monitored for 1 min in
15 s intervals. The nal value is expressed as units per milligram of
protein.
Analysis of Phenolic Metabolites in Urine, Plasma, and
Erythrocytes. The extraction of the phenolic metabolites from urine
and plasma samples was carried out as previously reported.
5
The PC
from erythrocyte samples were extracted with the solid phase
extraction (SPE) system using OASIS HLB 200 mg cartridges
(Waters Corp., Milford, MA, USA). The conditioning of the SPE
cartridges was done by adding sequentially 2 mL of methanol and 2
mL of Milli-Q water acidied at pH 2 with acetic acid. Extractions
were performed by loading 1 mL of washed erythrocytes, which had
previously been mixed with 3 mL of distilled water and 20 μLof
phosphoric acid at 85% to break the bonds between the proteins and
PC. The loaded cartridges were washed with 1 mL of Milli-Q water
and 1 mL of methanol at 5%. Finally, the retained PC were eluted
using 3 mL of methanol, which was evaporated to dryness and
reconstituted with 100 μL of methanol.
The phenolic metabolites in biological uids were selected on the
basis of our previous work in which olive and thyme PC intake
biomarkers were dened.
5
Thus, hydroxytyrosol sulfate (HTS; urine,
plasma, and erythrocytes) and hydroxytyrosol acetate sulfate (HTAS;
urine and plasma) were analyzed as VOO phenol metabolites.
Hydroxyphenylpropionic acid sulfate (HPPAS; urine, plasma, and
erythrocytes), thymol sulfate (TS; urine, plasma, and erythrocytes),
and p-cymene-diol glucuronide (PCymeneDG; urine) were analyzed
as thyme phenol metabolites. The analysis of the phenolic metabolites
was carried out by ultraperformance liquid chromatography (UPLC)
coupled to tandem MS (MS/MS) on the basis of the method
described by Rubióet al.
5
Animals and Experimental Procedure. Twenty Wistar rats were
obtained from Charles River Laboratories (Barcelona, Spain). They
were separated into four groups of ve rats each (four females and one
male): group 1, control diet (CON); group 2, secoiridoids (SEC);
group 3, secoiridoids combined with thyme phenols (SEC+THY); and
group 4, thyme phenols (THY). The diet preparation and character-
istics are explained in more detail in Supplementary Table 2. Rats were
fed during 21 days at a dose of 5 mg of phenolic compounds/kg rat
weight/day. SEC extract and SEC+THY were the same phenolic
extracts used for the preparation of FVOO and FVOOT, respectively,
as described previously.
3
Additionally, THY extract was used to
investigate the eect of a comparable phenolic dose exclusively from
thyme. The animal procedures were conducted in accordance with the
guidelines of the European Communities Directive 86/609/EEC
regulating animal research and approved by the local ethical committee
(CEEA-Universitat de Lleida, reference 7675).
The rats were sacriced by intracardiac puncture after isourane
anesthesia (IsoFlo, Veterinarian Esteve, Bologna, Italy). After blood
collection, the rats were perfused with an isotonic solution of sodium
chloride (NaCl) 0.9% to remove the remaining blood irrigating the
tissues, and their livers were excised. Tissue samples were stored at
80 °C and freeze-dried.
NF-κBDNA Binding Activity. NF-κB p65DNA binding was
assessed in rat hepatic tissue lysate using a Cayman kit (catalog no.
10007889). A specic double-stranded DNA sequence containing the
NF-κB response element was immobilized in the wells of a 96-well
plate. NF-κB contained in whole-cell extract from tissue binds
Figure 2. VOHF study owchart.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b04915
J. Agric. Food Chem. XXXX, XXX, XXXXXX
D
specically to the NF-κB response element and was detected by the
addition of specic primary antibody directed against NF-κB (p65).
Addition of a secondary antibody conjugated to horseradish peroxidase
(HRP) provided a sensitive colorimetric readout at 450 nm. The
activity of NF-κB p65DNA binding was represented as relative
absorbance at 450 nm/μg of protein.
Data Analysis and Statistical Procedures. Descriptive data
were expressed as the mean ±standard deviation, and postpre
intervention changes were expressed as the mean ±95% condence
interval [95% CI]. Prior to all analyses, normality of data was assessed
using ShapiroWilksWtest, and those lacking a normal distribution
were log-transformed to achieve normality. Linear regression models
were used to adjust post-intervention values for pre-intervention
values, age, and sex. Comparisons among groups were analyzed by
General Linear Models. A paired ttest was used to test the postpre
intervention period changes on oxidative biomarkers, AEES, and PC
biomarkers. Dierences were considered statistically signicant at P<
Table 2. Baseline Characteristics of the Participants in the Chronic Consumption Study
a
sequence 1 (n= 11) sequence 2 (n= 11) sequence 3 (n= 11)
gender, male/female 7/4 7/4 5/6
age, years 55.45 ±7.84 55.18 ±11.88 54.91 ±12.57
body wt, kg 84.45 ±17.74 74.60 ±18.49 74.75 ±16.80
BMI, kg/m227.85 ±4.71 26.33 ±5.29 25.63 ±3.68
SBP, mmHg 130 (106166) 128 (96151) 125 (104153)
DBP, mmHg 72 (4490) 72 (5285) 68 (52101)
glucose, mg/dL 90.91 ±10.53 93.00 ±13.33 88.55 ±11.63
total colesterol, mg/dL 218.82 ±82 231.91 ±32.70 228.36 ±42.70
LDL colesterol, mg/dL 142.45 ±25.64 152.00 ±28.45 150.80 ±34.08
HDL colesterol, mg/dL 53.39 ±9.55 52.96 ±12.82 52.78 ±11.75
triglycerides, mg/dL 115.82 ±32.49 134.36 ±60.53 126 ±86.68
a
Values are expressed as means ±SD; numbers in parentheses are the median (25th75th percentile). Sequence 1 = VOO, FVOO, and FVOOT;
sequence 2 = FVOOT, VOO, and FVOO; sequence 3 = FVOO, FVOOT, and VOO. Abbreviations: BMI, body mass index; SBP, systolic blood
pressure; DBP, diastolic blood pressure; LDL, low-density lipoprotein; HDL, high-density lipoprotein; VOO, virgin olive oil; FVOO, functional
virgin olive oil enriched with its own phenolics; FVOOT, functional virgin olive oil enriched with both its own phenolics and phenolics from thyme.
Table 3. Post-intervention Values and Changes from Baseline of Oxidation Biomarkers and Phenolic Metabolite Biomarkers in
Urine
a
VOO (n= 33) FVOO (n= 33) FVOOT (n= 33)
mean SD [95% CI]
Pvalue
compared to
pre mean SD [95% CI]
Pvalue
compared to
pre mean SD [95% CI]
Pvalue
compared to
pre
post-intervention urine HT biomarkers
HTS, μmol/24 h urine 9.6 11.3 0.660 18.0
b
21.3 0.007 12.1
c
22.4 0.350
HTAS, μmol/24 h urine 10.7 8.2 0.231 13.0
b
7.5 0.010 9.7
c
5.3 0.412
changes in urine HT biomarkers (post, pre)
HTS, μmol/24 h urine 0.8 [4.7, 3.0] 8.1 [2.4, 13.8] 3.1 [3.6, 9.7]
HTAS, μmol/24 h urine 3.9 [2.6, 10.5] 6.0 [1.6, 10.5] 2.6 [3.9, 9.1]
post-intervention urine thyme biomarkers
HPPAS, μmol/24 h urine 8.0 4.3 0.012 23.1
b
6.7 0.707 324.7
b
,
c
73.6 <0.001
TS, μmol/24 h urine 58.8 39.0 0.068 65.9 59.4 0.116 539.0
b
,
c
287.9 <0.001
PCymeneDG,
μmol/24 h urine 0.1 0.16 0.107 1.6
b
4.26 0.351 53.4
b
,
c
25.1 <0.001
changes in urine thyme biomarkers (postpre)
HPPAS, μmol/24 h urine 22.3 [39.2, 5.4] 3.4 [21.6, 14.9] 294.9 [187.6, 402.3]
TS, μmol/24 h urine 29.1 [60.4, 2.3] 21.8 [49.4, 5.8] 470.2 [291.7, 648.7]
PCymeneDG,
μmol/24 h urine
1.0 [2.2, 0.2] 0.6 [0.7, 1.8] 55.2 [35.2, 75.1]
post-intervention urine oxidation biomarkers
8-OHdG, nM 15.3 8.28 0.796 12.9
b
5.48 0.015 10.6
b
,
c
3.97 0.008
8-iso PGF2α,μg/L 0.46 0.12 0.574 0.45 0.13 0.359 0.45 0.18 0.493
changes in urine oxidation biomarkers (postpre)
8-OHdG, nM 0.4 [2.4, 3.1] 2.0 [3.7, 0.4] 4.4 [7.6, 1.2]
8-iso PGF2α,μg/L 0.03 [0.14, 0.08] 0.03 [0.09, 0.03] 0.03 [0.13, 0.06]
a
Values are means and standard deviation (SD) for post-intervention or 95% condence interval [95% CI] for changes postpre. Post-intervention
comparison between administered olive oils. Pvalue, paired ttest comparison between postintervention and preintervention. Abbreviations: VOO,
virgin olive oil; FVOO, functional virgin olive oil enriched with its own phenolics; FVOOT, functional virgin olive oil enriched with both its own
phenolics and phenolics from thyme; 8-OHdG, 8-hydroxy-2-deoxyguanosine; 8-iso PGF2α, 8-isoprostaglandin F2α; HTS, hydroxytyrosol sulfate;
HTAS, hydroxytyrosol acetate sulfate; HPPAS, hydroxyphenylpropionic acid sulfate; TS, thymol sulfate; PCymeneDG, p-cymene-diol glucuronide.
b
P< 0.05 compared to VOO.
c
P< 0.05 compared to FVOO.
Journal of Agricultural and Food Chemistry Article
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J. Agric. Food Chem. XXXX, XXX, XXXXXX
E
0.05. Data were analyzed by SPSS version 20.0 (SPSS, Inc., IBM,
Armonk, NY, USA).
RESULTS
Participants and Compliance. The study was conducted
at IMIM-Hospital del Mar Medical Research Institute
(Barcelona, Spain) from April 2012 to September 2012 with
33 enrolled participants completing the intervention period.
The participantsowchart is described in Figure 2, and a
discontinued single intervention occurred in three volunteers
due to an investigators decision. Participants had a BMI range
indicative of normal weight to overweight, and they were
normotensive and hyperlipidemic (total cholesterol > 200 mg/
dL) according to established criteria. All 33 participants had
borderline-high values of total cholesterol and LDL-C. There
were no statistically signicant dierences in baseline character-
istics of the participants among sequences 1, 2, and 3 (Table 2).
Compliance was monitored through the determination of
biomarkers of intake analyzing the phenolic metabolites in the
subjects biological uids (urine and plasma), and a successful
dietary intervention was guaranteed. No adverse side eects
were reported by participants during any of the study
treatments.
Olive Oil Characterization. Table 1 shows the chemical
characterization of VOO, FVOO, and FVOOT, including
individual PC, fat-soluble micronutrients, and fatty acids
composition. Only the phenolic composition diered among
the three VOOs as they presented the same composition
regarding fat-soluble micronutrients and fatty acids. In
comparison to VOO, FVOO was basically enriched with HT
and its derivatives, providing 8.5 mg/25 mL oil/day. FVOOT
enrichment consisted of a mixture of HT and its derivatives
(4.3 mg/25 mL oil/day), phenolic acids (0.65 mg phenols/25
mL oil/day), avonoids (2.95 mg/25 mL oil/day), and
monoterpenes (0.86 mg/25 mL oil/day). Thus, FVOOT
contained 50% of olive PC and 50% of thyme PC.
Olive and Thyme Phenolic Metabolites in Biological
Fluids. Results of the phenolic metabolites in urine and plasma
are presented in Tables 3 and 4, respectively. Apart from urine
and plasma, in the present work results of the phenolic
metabolites detected in erythrocytes are presented (Table 5).
When all three VOOs were compared, metabolites derived
from olive PC were signicantly higher in FVOO compared to
VOO and FVOOT in urine, plasma, and erythrocytes (Tables
35). With regard to the postpre-intervention changes, HTS
and HTAS signicantly increased after FVOO intervention in
urine. HTAS was also signicantly increased in plasma after
FVOO. No postpre-intervention changes in FVOOT were
observed in HT biomarkers in any biological uid. The thyme
phenolic metabolites detected in urine, plasma, and eryth-
rocytes were HPPAS, TS, and PCymeneDG (detected only in
urine). When the three interventions were compared, HPPAS
and TS levels were signicantly higher in the FVOOT group
compared to the VOO and FVOO groups in all biological
uids, and PCymeneDG was also signicantly higher in urine
(Tables 35). With regard to the postpre-intervention
changes, HPPAS, TS, and PCymeneDG signicantly increased
after the FVOOT. HPPAS appeared to be a clear erythrocyte
biomarker for thyme phenolics, as it was detected only after
FVOOT intervention (Table 5).
Eects of VOO PC Enrichment on Oxidative Stress.
The outcome measurements of urine oxidation biomarkers (8-
Table 4. Post-intervention Values and Changes from Baseline of Oxidation Biomarkers and Phenolic Metabolite Biomarkers in
Plasma
a
VOO (n= 33) FVOO (n= 33) FVOOT (n= 33)
mean SD [95% CI] Pvalue compared
to pre mean SD [95% CI] Pvalue compared
to pre mean SD [95% CI] Pvalue compared
to pre
post-intervention plasma HT biomarkers
HTS, μM 0.84 0.69 0.547 1.52
b
0.74 0.099 1.23
b
,
c
0.85 0.088
HTAS, μM 0.97 0.69 0.475 1.73
b
0.97 0.002 1.14
c
0.75 0.206
changes in plasma HT biomarkers (postpre)
HTS, μM 0.13 [0.30, 0.56] 0.75 [0.15, 1.66] 0.50 [0.08, 1.09]
HTAS, μM 0.15 [0.28, 0.59] 0.92 [0.38, 1.46] 0.39 [0.23, 1.01]
post-intervention plasma thyme biomarkers
HPPAS, μM 0.12 0.15 0.018 1.12
b
0.62 0.352 24.9
b
,
c
13.9 <0.001
TS, μM 0.84 0.26 0.002 1.61
b
0.37 0.221 26.7
b
,
c
9.5 <0.001
changes in plasma thyme biomarkers (postpre)
HPPAS, μM1.70 [3.1, 0.31] 0.56 [1.8, 0.7] 24.2 [13.6, 34.9]
TS, μM1.89 [3, 0.73] 0.78 [2.1, 0.5] 24.7 [16.3, 33.1]
post-intervention plasma oxidation biomarkers
MetSO in total
Met, % 5.4 0.58 0.033 5.6
b
0.61 0.006 5.5 0.86 0.016
changes in plasma oxidation biomarkers (postpre)
MetSO in total
Met, % 0.71 [0.06, 1.37] 0.85 [0.27, 1.43] 0.79 [0.6, 1.42]
a
Values are means and standard deviation (SD) for post-intervention or 95% condence interval [95% CI] for changes postpre. Post-intervention
comparison between administered olive oils. Pvalue, paired ttest comparison between post-intervention and pre-intervention. Abbreviations: VOO,
virgin olive oil; FVOO, functional virgin olive oil enriched with its own phenolics; FVOOT, functional virgin olive oil enriched with both its own
phenolics and phenolics from thyme; LDL, low-density lipoprotein; methionine SO, methionine sulfoxide; Met, methionine; HTS, hydroxytyrosol
sulfate; HTAS, hydroxytyrosol acetate sulfate; HPPAS, hydroxyphenylpropionic acid sulfate; TS, thymol sulfate.
b
P< 0.05 compared to VOO.
c
P<
0.05 compared to FVOO.
Journal of Agricultural and Food Chemistry Article
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J. Agric. Food Chem. XXXX, XXX, XXXXXX
F
iso PGF2αand 8-OHdG) and the postpre-intervention
changes are presented in Table 3. When the three VOO
interventions were compared, FVOOT presented lower values
of urinary 8-OHdG compared to FVOO and VOO after
intervention. In addition, urinary 8-OHdG was also signicantly
lower in FVOO than in VOO. Urinary 8-iso PGF2αdid not
dier when the three VOO interventions were compared. With
regard to the postpre-intervention changes, urinary 8-OHdG
decreased in the FVOO and to a greater extent in the FVOOT
intervention group. No postpre-intervention changes were
observed in urinary 8-iso PGF2α. The outcome measurements
of plasma percent of MetSO in total Met and the postpre-
intervention changes are shown in Table 4. There were no
dierences between groups of administered olive oils in plasma
percent of MetSO. Compared to baseline values, percent of
Table 5. Post-intervention Values and Changes from Baseline of Oxidation Biomarkers and Phenolic Metabolite Biomarkers in
Erythrocytes
a
VOO (n= 33) FVOO (n= 33) FVOOT (n= 33)
mean SD [95% CI] Pvalue
compared to pre mean SD [95% CI] Pvalue
compared to pre mean SD [95% CI] Pvalue
compared to pre
postintervention erythrocyte HT biomarkers
HTS, nM 0.16 0.67 0.436 0.64
b
0.17 0.171 1.55
b
,
c
1.28 0.167
changes in erythrocyte HT biomarkers (postpre)
HTS, nM 0.09 [0.15, 0.33] 0.44 [0.21, 1.10] 1.44 [0.65, 3.53]
postintervention erythrocyte thyme biomarkers
HPPAS, nM nd nd 28.5 13.6 0.007
TS, nM nd 1.07 1.31 0.328 10.26
c
1.92 0.006
changes in erythrocyte thyme biomarkers (postpre)
HPPAS, nM 27.2 [8, 46.3]
TS, nM 0.87 [0.93, 2.67] 10.25 [3.25, 17.3]
postintervention erythrocytes endogenous antioxidants
GSH-Px activity,
U/l 72.1 9.90 0.835 72.8
b
9.51 0.329 74.3
b
,
c
8.83 0.228
SOD activity,
U/g Hb 716.6 53.8 0.875 739
b
76.7 0.033 771
b
,
c
111.6 0.043
CAT activity,
U/mg Hb 111.7 22.9 0.142 115
b
22.3 0.308 115.2
b
23.4 0.760
changes in erythrocytes endogenous antioxidants (postpre)
GSH-Px activity,
U/l 0.17 [1.51, 1.85] 0.71 [0.73, 2.14] 2.18 [1.45, 5.82]
SOD activity,
U/g Hb 3.43 [40.7, 47.6] 26.4 [2.14, 50.7] 48.1 [1.65, 94.6]
CAT activity,
U/mg Hb
6.49 [15.28, 2.30] 3.12 [9.16, 2.93] 2.17 [16.65, 12.31]
a
Values are means and standard deviation (SD) for post-intervention or 95% condence interval [95% CI] for changes postpre. Post-intervention
comparison between administered olive oils. Pvalue, paired ttest comparison between post-intervention and pre-intervention. Abbreviations: VOO,
virgin olive oil; FVOO, functional virgin olive oil enriched with its own phenolics; FVOOT, functional virgin olive oil enriched with both its own
phenolics and phenolics from thyme; SOD, superoxide dismutase; CAT, catalase; HTS, hydroxytyrosol sulfate; HPPAS, hydroxyphenylpropionic
acid sulfate; TS, thymol sulfate; GSH-Px, glutathione peroxidase.
b
P< 0.05 compared to VOO.
c
P< 0.05 compared to FVOO.
Figure 3. Eect of phenolic compound supplementation on NF-κB activity in whole-cell extract from rat liver after 21 days of feeding at a dose of 5
mg of phenolic compounds/kg rat weight/day: control standard feed (CON), secoiridoids (SEC), secoiridoid combined with thyme phenols (SEC
+THY), and thyme phenols (THY). pvalues are with respect to CON. Values are shown as the mean ±SD.
Journal of Agricultural and Food Chemistry Article
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J. Agric. Food Chem. XXXX, XXX, XXXXXX
G
MetSO was signicantly increased in all groups (between 0.7
and 0.8%).
Eects of VOO PC Enrichment on Erythrocyte
Antioxidant Enzymes. The outcome measurements of
erythrocyte GSH-Px, SOD, and CAT activities after the three
VOO treatments and the postpre-intervention changes of
each VOO group are shown in Table 5. When the three
interventions were compared, the activities of all enzymes were
signicantly higher after the FVOOT and FVOO treatments
compared to VOO. In addition, GSH-Px and SOD were also
signicantly higher after the FVOOT treatment compared to
the FVOO (P<0.05).Withregardtothepostpre-
intervention changes, SOD activity signicantly improved
after the FVOO intervention and signicantly improved even
to a greater extent after the FVOOT one (P< 0.05). All other
measurements of antioxidant enzyme activities did not dier
between post- and pre-interventions.
Animal Experiment: NF-κBDNA Binding Activity.
Thyme supplementation in rat feed (THY) signicantly
reduced the NF-κBDNA binding activity with respect to
control (CON) (Figure 3). As shown in Figure 3, it appears
that supplementation with olive oil PC (SEC) and both thyme
and olive oil PC (SEC+THY) starts a trend to reduced activity
of NF-κB, which is established as signicant when rats are
supplemented with only thyme PC (THY).
DISCUSSION
Our study demonstrates that a sustained intake of FVOOT,
which provided the same amount of PC but dierent PC
composition of FVOO, appeared to have a greater eect against
oxidative stress in hyperlipidemic subjects. VOO presented the
highest 8-OHdG values followed by FVOO and FVOOT,
suggesting that FVOOT intervention provided major protec-
tion against oxidative DNA damage.
The antioxidant protection was also reected in the activity
of antioxidant enzymes in erythrocytes. In this sense, the SOD
activity was also increased to a greater extent after the FVOOT
than after the FVOO and VOO interventions with a parallel
increase in thyme phenolic metabolites detected in both urine
and erythrocytes after FVOOT compared to FVOO. Our data
therefore provide the rst level of evidence for an antioxidant
DNA action and antioxidant enzymatic induction through a
combination of olive and thyme PC, after a sustained
consumption of real-life doses of olive oil in hyperlipidemic
subjects.
The 8-OHdG is a major base product formed after DNA
oxidative damage and has been widely used as a DNA damage
indicator in nutritional studies.
19
Large amounts of 8-OHdG
are produced in mammalian cells, either as a byproduct of
normal oxidative metabolism or as a result of exogenous
sources of reactive oxygen species (ROS). Increased levels of 8-
OHdG in tissues represent a signal of a strong DNA-damaging
stimulus or the specicdecient DNA repair mechanism.
20
Oxidative damage to the DNA base produces a point mutation
through an AT substitution when incorporated into DNA,
causing mutagenesis and carcinogenesis.
21
In a previous study
the urinary excretion of oxidation products of guanine, the most
commonly used markers for DNA oxidation, was not modied
after 3 weeks of consumption of 25 mL of olive oil with low
(2.7 mg/kg of caeic acid equiv), medium (164 mg/kg), and
high (366 mg/kg) PC in humans.
22
In the same way, no
signicant eect was detected in urinary excretion of DNA
adducts after the consumption of phenol-rich olive oil (PC
content from 2.7 to 366 mg/kg).
23
In contrast, a decreased
amount of 8-OHdG in urine after short-term consumption, a 4
consecutive day intervention of 25 mL of three VOO, with low
(10 mg/kg of caeic acid equiv), medium (133 mg/kg), and
high (486 mg/kg) PC with a linear trend signicantly
correlated to the content of PC.
24
Similarly, a 30% reduction
of oxidative DNA damage in peripheral blood lymphocytes was
observed after substitution of all types of fat and oils habitually
consumed with the study oil (50 g/day) for two periods of 8
weeks on postmenopausal women with VOO containing high
amounts of phenols (592 mg total phenols/kg) compared to
those that consumed the lowest levels (147 mg/kg) in
postmenopausal women.
25
Our results are in accordance with
the latter two studies as a signicant decrease in urinary 8-
OHdG was observed after the sustained consumption of
phenol-enriched olive oils, FVOO and FVOOT. Despite
containing the same amount of PC, the 8-OHdG reduction
was signicantly 2-fold higher in the FVOOT compared to the
FVOO; this reduction may be attributed to the dierent PC
composition. Moreover, when compared with the VOO control
group, the 8-OHdG reduction was signicantly 10-fold higher
in the FVOOT and 5-fold higher in the FVOO.
In parallel to the oxidative DNA protection, the postpre
change values in 24 h urine of thyme phenolic biomarkers
(HPPAS, TS, and PCymeneDG) signicantly increased in
FVOOT group, which could be related to the signicant
reduction of 8-OHdG observed after the FVOOT intake. Thus,
the signicant decrease in urinary 8-OHdG after FVOOT
consumption suggests that olive and thyme PC could act
synergistically as bioactive molecules protecting against
oxidative DNA damage and improving oxidative systemic
balance as reected also in the increase of erythrocyte SOD
activity.
The postpre-intervention increase in erythrocyte SOD
activity was about 14-fold higher in the FVOOT group
compared to the VOO and 2-fold higher compared to
FVOO. These data support again that olive and thyme PC
may act synergistically as bioactive molecules improving the
erythrocyte antioxidant enzymatic system, in which SOD plays
the primary role.
26
Erythrocytes, oxygen carriers with high polyunsaturated fatty
acid content in their membranes and high cellular concen-
tration of hemoglobin, are particularly exposed to oxidative
damage. The hemoglobin released from erythrocytes is
potentially dangerous because when reacting with H2O2,itis
converted into the oxidized forms with powerful promoters of
oxidative processes.
27
For this reason, newer functional agents,
such as PC from the diet, can target oxidative stress in
erythrocytes, as a valuable way to prevent or delay the
development of organ complications.
28
In the present study, PC metabolites derived from olive or
thyme were analyzed in erythrocytes for the rst time after an
oral administration of olive oil in humans. HTS was the only
phenolic metabolite derived from olive PC detected in
erythrocytes, whereas HPPAS and TS were detected in
erythrocytes as thyme phenolic metabolites. With regard to
the postpre-intervention changes, both erythrocyte HPPAS
and TS signicantly increased after intervention in the FVOOT
group. In this regard, the parallel signicant augmentation in
the SOD activity observed after the FVOOT intake could be
attributed to the presence of these metabolites in erythrocytes.
This fact allows us to postulate that erythrocytes could be cell
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b04915
J. Agric. Food Chem. XXXX, XXX, XXXXXX
H
targets for PC and its metabolites, which could exert an
antioxidant eect in situ.
Thus, a clear parallelism appears between the modulations of
antioxidant or oxidative markers and PC metabolites observed
in urine and in erythrocytes after VOO, FVOO, or FVOOT
intervention.
To clarify the mechanistic pathways responsible for the
higher protective antioxidant eects observed after FVOOT
compared to FVOO, a parallel experiment in animals with the
same PC and similar doses administered to humans was
performed. It has been seen that hydroxytyrosol acts as an
inhibitor of NF-κB activation, leading to the inhibition of
proliferation and promotion of apoptosis in human hepatocel-
lular carcinoma cells.
29
Furthermore, inhibiting NF-κB
activation reduces ROS production and oxidative damage to
lipids and DNA.
30
In our animal experiment, results revealed
that after supplementation with olive oil PC and both thyme
and olive oil PC, a reduction trend in the activity of hepatic NF-
κB is observed, which is established as signicant when rats are
supplemented only with thyme PC. In that sense, the
suppression of the NF-κB pathway by thyme PC could be
sucient to reduce the endogenous DNA damage produced
naturally by cells. Further studies are needed to verify this
mechanistic pathway responsible for the protective antioxidant
eect observed in humans.
Considering the described results, it is surprising that percent
of MetSO in total Met was increased in all groups after
intervention. The three intervention groups have ingested oils
with dierent phenolic proles; therefore, this cannot explain
the similar increase of the MetSO observed in all groups. The
exogenous antioxidants, including PC, are considered double-
edged swordsin the cellular redox state, and several studies of
exogenous antioxidants have shown controversial results,
especially when administered at high doses.
31,32
However, in
the present study the data obtained from the three intervention
groups after a regular consumption of phenol-enriched VOO
did not go globally in this direction, despite the increase in
percent of MetSO. On the other hand, no changes of 8-iso
PGF2αwere observed in both the prepost-intervention levels
and between VOO. As we are aware of the limitations of the
use of this biomarker, we have taken into account some
important aspects to use it in a reliable manner. We tried to
prevent the ex vivo oxidation during processing and storing of
samples. In addition, the use of urine samples collected during
24 h globally reects changes in lipid peroxidation and
minimizes the possible circadian variation of 8-iso PGF2α.
One of the strengths of the present study was its design.
Randomized, controlled, clinical trials were those able to
provide the rst level of scientic evidence. The crossover
design, in which each subject acts as the corresponding control,
minimizes the intervariability. In addition, the fatty acid
composition, vitamin E content, and parental matrix of the
three olive oils were similar, the only dierences being the PC
proles and amounts.
One potential limitation of the study was that although the
trial was blinded, some participants might have identied the
type of olive oil ingested by its organoleptic characteristics.
Another limitation was the inability to assess potential synergies
and interactions among the VOOs and other diet components.
Nevertheless, the controlled diet followed throughout the trial
should have limited the scope of these interactions.
In conclusion, the sustained intake of a phenol-enriched
VOO with its own PC and complemented with thyme PC
improves DNA protection against oxidation and antioxidant
endogenous enzymatic activity probably due to a greater
bioavailability of thyme phenolic compounds in hyperlipidemic
subjects.
ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.5b04915.
ESI and MRM conditions for all of the compounds
(Supplementary Table 1) (PDF)
Diet characteristics of the animal experiment (Supple-
mentary Table 2) (PDF)
AUTHOR INFORMATION
Corresponding Authors
*(R.S.) E-mail: rosa.sola@urv.cat. Phone: +34 977 759369.
Fax: +34 977 759378. Mail: Dept. Medicina i Cirurgia,
Universitat Rovira i Virgili, Sant Llorenç21, 43201 Reus,
Spain/
*(M.J.M.) E-mail: motilva@tecal.udl.es. Phone: +34 973
702817. Fax: +34 973 702596. Mail: Dept. Tecnologia de
Alimentos, Universitat de Lleida, Alcalde Rovira Roure 191,
25198 Lleida, Spain.
Funding
This study was supported by the Ministerio de Economia y
Competitividad (AGL2012-40144-C03-02, AGL2012-40144-
C03-01, and AGL2012-40144-C03-03 projects and AGL2009-
13517-C03-01 and AGL2009-13517-C03-03 projects), CIBER-
OBN, FPI fellowship (BES-2010-040766), ISCIII, and a
Departament de Salut joint contract (CP06/00100).
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
We thank Borges Mediterranean Group for providing the
common olive oil used in the study.
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Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b04915
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... This could show a discrepancy between the in vivo and the in vitro trials, since the concentrations of the active compounds and their activities often differ between the two. Some authors reported an increase on the activities of GPx and SOD caused by dietary olive polyphenols in rats [28] and in humans [29,30]. However, the animals in our trial might have reacted differently, since we included high amounts of PUFA that increased the oxidative load in comparison to the animals and humans in other trials. ...
... In our trial, diet OLE-1 showed some protective effects on DNA measured as tail DNA % in comparison to Cont+ (p = 0.072), although the effect was not present in higher concentrations of OLE (OLE-2 and OLE-3). This partly agrees with some in vitro [32]) and in vivo [30] studies showing the protective effects of olive polyphenols against DNA damage. Interestingly, vitamin E presented prooxidative effects, as we detected higher OTM in the blood of animals fed Vit-E than in those fed Cont+, which does not comply with the previous findings of Frankič and Salobir [11]. ...
... In the present study, piglets fed Vit-E and OLE-1 had a similar excretion of 8-OHdG as piglets fed Cont−, which may suggest that the extent of DNA repair and/or damage was not altered in these groups. Contrary to our results, studies concerning DNA damage reported a reduction of 8-OHdG with dietary supplementation of olive polyphenols in humans [30]. The latter suggests that olive polyphenols might not be efficient in preventing DNA damage caused by PUFA-induced oxidative stress, or that the concentration of polyphenols might not have been sufficient to protect against DNA damage. ...
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The aim of the current study was to assess the antioxidative effects of the dietary supplementation of olive leaf extract (OLE) in different concentrations compared to those of vitamin E in piglets under conditions of dietary n-3 PUFA-induced oxidative stress. Forty-eight castrated male piglets (10.6 ± 0.99 kg) were fed the following experimental diets: Cont− (low-fat diet, no supplement), Cont+ (high linseed oil diet, no supplement), Vit-E (as Cont+, 105 IU vitamin E/day), OLE-1 (as Cont+, 3.84 mg hydroxytyrosol equivalents (HEQs)/day), OLE-2 (as Cont+, 38.4 mg HEQ/day), and OLE-3 (as Cont+, 96 mg HEQ/day). After 21 days of feeding, the experimental diets, blood and urine samples were collected to assess the extent of the oxidative stress. Results indicated that diet OLE-1 lowered the activity of gamma-glutamyl transferase, protected DNA (measured as DNA tail %) and altered urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG). Dietary vitamin E lowered the levels of urinary F2-isoprostanes, as well as of plasma malondialdehyde and γ-tocopherol, but raised the plasmatic α-tocopherol and altered the level of urinary 8-OHdG. In conclusion, only minor positive effects of dietary OLE on the oxidative stress parameters were observed. Additionally, OLE did not show concentration dependence.
... Some nutrition interventional research, including the renowned PREDIMED (prevention with Mediterranean Diet), have revealed that an elevated phenolic intake from virgin olive oil or nuts is related to a reduced incidence of metabolic syndrome, cardiovascular diseases, cancer and age-related cognitive decline [5][6][7][8]. Moreover, rich-polyphenol virgin olive oils (VOO) have been shown to improve antioxidant and anti-inflammatory effects and to reduce the proliferation of cell adhesion molecules compared with low-polyphenol VOO [9][10][11]. ...
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The purpose of this research was to improve the properties of functional edible oils with potential health promoting effects, enriched with phenolic-rich extracts obtained from pistachio and walnut (5.1 and 27.4% phenolic contents respectively), by means of emulsion and micro emulsion systems. Stable water-in-oil (W/O) emulsions were obtained employing polyglycerol polyrhizinoleate (PGPR) as emulsifier (0.5, 2% H2O in oil), despite having a whitish and opaque appearance; transparent and stable microemulsions were prepared using proper proportion (e.g., 97:3) between the oily phase and the mixture of aqueous phase and emulsifiers (3:2 lecithin-distilled monoglycerides (DMG). Total polar phenolics contents ranging between 257 and 835 mg/kg were obtained in the novel functional edible oils’ formulations, reaching higher content using walnut as compared to pistachio extracts. Antioxidant capacity determined by the 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) method increased approx. 7.5 and 1.5 times using walnut and pistachio extracts respectively. An emulsion using gallic acid and a microemulsion employing hydroxytyrosol, two well-known antioxidants, were also studied to compare antioxidant capacity of the proposed enriched oils. Furthermore, the oxidative stability of these products—very relevant to establish their commercial value—was measured under accelerated testing conditions employing the Rancimat equipment (100 °C) and performing an oven test (at 40 °C for walnut oils and 60 °C for pistachio and refined olive oils). Rancimat oxidative stability greatly increased and better results were obtained with walnut (2–3 times higher) as compared to pistachio extract enriched oils (1.5–2 times higher). On the contrary, under the oven test conditions, both the initial oxidation rate constant and the time required to reach a value of peroxide value equal to 15 (upper commercial category limit), indicated that under these assay conditions the protection against oxidation is higher using pistachio extract (2–4 times higher) than walnut’s (1.5–2 times higher). Stable emulsions and transparent microemulsions phenolic-rich nut oils (250–800 mg/kg) were therefore developed, possessing a higher oxidative stability (1.5–4 times) and DPPH antioxidant capacity (1.5–7.5 times).
... The other two trials failed to find a protective effect on DNA oxidation. Four of these studies were conducted by the same research group, and had similar experimental designs (Weinbrenner et al., 2004;Hillestrøm et al., 2006;Machowetz et al., 2007;Romeu et al., 2016). Therefore, there is very little evidence relating the intake of VOO phenolic compounds with cancer. ...
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Current research suggests that virgin olive oil (VOO) phenolics are potent preventive and therapeutic agents against metabolic diseases associated with inflammation and oxidative stress. Evidence-based medicine requires these effects be proved in randomized controlled trials (RCT), which are then assessed in meta-analyses, to ensure that the alleged health benefits really proceed in humans. The available evidence is limited to the ability of VOO phenolic compounds to protect lipoproteins from oxidation and to reduce systolic pressure in hypertensive individuals. No RCT assessing the effects of VOO phenolics on diabetes and neurodegenerative diseases have been performed, and those focused on osteoarthritis and cancer provided very scarce information. Therefore, RCT in extensive and diverse population groups, with different disorders and phenolic doses adjusted to usual VOO consumptions are necessary to achieve high quality scientific evidence before nutritional recommendations can be given to the general public.
... DNA damage can also be reversed in some cases. [13][14][15][16] In addition to intrinsic damage repair mechanisms, other protection mechanisms may also be operable. For example, DNA damage can be prevented by certain molecules, especially natural antioxidant compounds such as flavonoids, polyphenols, and carotenoids, which exist in fruits, vegetables, and medicinal plants. ...
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Relationships among the cholesterol‐lowering, antioxidant and DNA damage protection activity of four solvent extracts from the Morus alba L. (Cultivar Hongguo 2) fruits, leaves and twigs were investigated. The petroleum ether extract of fruit (FPE) was with the highest amount of total flavonoids, cholesterol‐lowering activity and better DPPH radical scavenging capacity. The ethyl acetate extract of fruit (FEE) had the highest antioxidant activity. The petroleum ether extract of the leaf (LPE), fruit (FPE) and ethyl acetate extract of twig (TEE) had strong inhibiting lipid oxidation activity and DNA damage protection potential, accompany with high total phenols and flavonoids content. It suggested that polyphenol contents, flavonoid contents or both to Hongguo 2 were positively correlated with the three biological activities, moreover, the low polarity extracts of the high inhibiting lipid oxidation activity could improve the cholesterol‐lowering activity or DNA damage protection potential through the synergism among different polyphenolic and flavonoids compounds.
... This effect was independent of the phenolic content and related to the monounsaturated fatty acid content of the olive oil [38]. In addition, a protection of DNA oxidation in hyperlipidemic subjects was observed after the intake of a VOO complemented with thyme phenolic compounds [39], and after eight weeks of supplementation with 50 g/d of an olive oil with 592 ppm of phenols in postmenopausal women [40]. Furthermore, 11.13 mg of olive's phenolic compounds have demonstrated postprandial protection against in vivo DNA oxidation in healthy males after four days of supplementation [41]. ...
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