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Olive oil (OO) is the primary source of fat in the Mediterranean diet and has been associated with longevity and a lower incidence of chronic diseases, particularly CHD. Cardioprotective effects of OO consumption have been widely related with improved lipoprotein profile, endothelial function and inflammation, linked to health claims of oleic acid and phenolic content of OO. With CVD being a leading cause of death worldwide, a review of the potential mechanisms underpinning the impact of OO in the prevention of disease is warranted. The current body of evidence relies on mechanistic studies involving animal and cell-based models, epidemiological studies of OO intake and risk factor, small- and large-scale human interventions, and the emerging use of novel biomarker techniques associated with disease risk. Although model systems are important for mechanistic research nutrition, methodologies and experimental designs with strong translational value are still lacking. The present review critically appraises the available evidence to date, with particular focus on emerging novel biomarkers for disease risk assessment. New perspectives on OO research are outlined, especially those with scope to clarify key mechanisms by which OO consumption exerts health benefits. The use of urinary proteomic biomarkers, as highly specific disease biomarkers, is highlighted towards a higher translational approach involving OO in nutritional recommendations.
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Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
New perspectives on bioactivity of olive oil evidence from
animal models, human interventions and the use of urinary
proteomic biomarkers
By S. Silva1,2,3, E. Combet4, M.E. Figueira3, T. Koeck5, W. Mullen6 and M.R. Bronze1,2,3, 1iBET,
Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
2 ITQB, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de
Lisboa, Av. da República, 2780-157 Oeiras, Portugal 3Pharmacy Faculty, University of Lisbon,
Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal, 4Human Nutrition, School of Medicine,
University of Glasgow, Glasgow G31 2ER, UK 5 Mosaiques diagnostics GmbH, Mellendorfer
Strabe 7-9, 30625, Hannover, Germany, and 6Institute of Cardiovascular and Medical Sciences,
University of Glasgow, Glasgow G12 8QQ, UK
This is a pre peer review manuscript of the full paper
published in Proceedings of the Nutrition 08/2015; 74(3):268-
281. DOI:10.1017/S0029665115002323
Corresponding author:
M. R. Bronze, Pharmacy Faculty, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa,
Portugal; telephone number (+351) 21 794 64 00 Ext. 14329; Fax number (+351) 217946470
Short title: New perspectives on bioactivity of olive oil
Keywords: olive oil, phenolics, coronary artery disease, inflammation, proteomic
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
Olive oil (OO) is the primary source of fat in the Mediterranean diet and has been associated
with longevity and a lower incidence of chronic diseases, particularly coronary heart disease.
Cardioprotective effects of OO consumption have been widely related with improved lipoprotein
profile, endothelial function and inflammation, linked to health claims of oleic acid and phenolic
content of OO. With cardiovascular disease being a leading cause of death worldwide, a review
of the potential mechanisms underpinning the impact of OO in the prevention of disease is
The current body of evidence relies on mechanistic studies involving animal and cell-based
models, epidemiological studies of OO intake and risk factor, small and large scale human
interventions, and the emerging use of novel biomarker techniques associated with disease risk.
This review critically appraises the available evidence to date, with particular focus on emerging
novel biomarkers for disease risk assessment. New perspectives on OO research are outlined,
especially those with scope to clarify key mechanisms by which OO consumption exerts health
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
1. Relevance of the Mediterranean diet and olive oil to health
The olive tree, Olea europaea L., is one of the oldest agricultural tree crops and provides
diversified products for human consumption such as table olives and olive oil (OO)(1). The
analytical parameters to ascertain OO quality and classify OOs are defined by European Union
(EU) regulations(2). Oils obtained only by mechanical extraction are virgin olive oils (VOOs) and
further quality assessment can lead to a classification as extra virgin olive oil (EVOO)(3).
OO is the primary source of fat in the Mediterranean diet and has been associated with longevity
and a lower incidence of chronic diseases, particularly coronary heart disease (CHD)(4-7). OO
consumption is also associated with decreased rates of cancer, diabetes and neurodegenerative
diseases(8) as well as body weight reduction and obesity prevention(9, 10). The epidemiological
evidence underpinning the relevance of the Mediterranean diet to health is strong with over
seventeen studies including 2300 volunteers confirming that a Mediterranean diet decreases
inflammation and improves endothelial function(11), and a meta-analysis of thirty-two cohort
studies (> 800,000 subjects) indicating that there is an inverse correlation between OO intake and
coronary heart disease(12).
Olive oil bioactive components
The major components of OO are glycerols (saponifiable fraction) which represent more than
98% of of the total oil weight and are mainly triglyceride esters of oleic acid (55 to 83%),
palmitic acid (7.5 to 20%), linoleic acid (3.5 to 21%) and other fatty acids such as stearic acid
(0.5 to 5%)(13). Minor components (the unsaponifiable fraction) include aliphatic and triterpenic
alcohols, sterols, hydrocarbons as squalene, volatile compounds, tocopherols, carotenes,
chlorophyll and phenolic compounds(13-15).
Special attention has been given to the phenolic compounds only found in VOO and EVOO. The
agronomic and technological aspects of OO production have an impact on the concentration of
phenolic compounds, as does the pedoclimatic conditions and agronomic techniques
(e.g.: irrigation)(4, 14). The main classes of phenolic compounds present in VOO are phenolic
acids, phenolic alcohols (hydroxytyrosol and tyrosol), flavonoids, lignans and secoiridoids,
Table 1.
Oleuropein and ligstroside, the most significant secoiridoids in Olea europaea L., are esters of
elenolic acid glucoside with hydroxytyrosol and tyrosol, respectively. During the mechanical
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
extraction of the oil, fruit endogenous β-glucosidases(14, 16) are released leading to the secoiridoid
aglycones formation, accounting for more than 50% of the phenolic content of the oil(17, 18). The
most abundant secoiridoids of VOO are the oleuropein and ligstroside aglycons and dialdehydic
forms of deacetoxy of oleuropein and ligstroside aglycons(14) also named oleacein and
oleocanthal, respectively(19).
Phenolic compounds bioavailability and bioactivity
Once OO has been ingested, it produces a micellar solution composed of a lipid and an aqueous
phase. Chemical hydrolysis of secoiridoids can take place in the acidic medium of the
stomach(20) or in alkaline conditions in the small intestine(21, 22) leading to an increase of free
phenolic alcohols released into the aqueous phase. As a result OO phenolic compounds are
further absorbed in the small intestine(23). Measuring the bioavailability of these compounds in
plasma and urine reveals that OO phenolics undergo a conjugation process of methylation,
glucuronidation and to a lesser extent sulfation indicating that there is phase 2 metabolism
involved during the absorption of these compounds.
When assessing the chemical and in vitro biological antioxidant activities of these compounds, it
is the glucuronides conjugates of hydroxytyrosol and tyrosol that must be assessed. These were
tested in the range 0.0110 μM against the radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). None
of the glucuronides displayed significant antioxidant activities at the concentrations tested,
whereas the parent aglycones did display antioxidant activity at these concentrations(24). This
conflicts with the results of others(25) with differences attributed to the fact that in one study
reference standard material(24) was used and in the other the glucuronide conjugates were
extracted from urine samples(25), and likely contained inpurities that had antioxidant activity.
Hydroxytyrosol metabolites might act as "sinks" of hydroxytyrosol that could be locally released
in the cells after enzymatic hydrolysis(26), therby explaining the proposed hydroxytyrosol
biological effects observed in vivo. Moreover, in situ deconjugation of hydroxytyrosol
metabolites (into their free form) in red blood cells was observed in rats after oral administration
of an OO phenolic extract obtained from olive cake (1.5 g/kg body weight, equivalent to 34.4 mg
of hydroxytyrosol and derivatives), highlighting a potential protective mechanism against cell
oxidative damage(27).
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
Secoiridoids that are not absorbed in the small intestine are degraded by the colonic microbiota
with oleuropein producing hydroxytyrosol as the major product (20). In vitro colonic metabolism
was evaluated on tyrosol, hydroxytyrosol, hydroxytyrosol acetate and oleuropein showing an
increase in phenolic acids, stability of hydroxytyrosol and tyrosol and degradation of
hydroxytyrosol acetate and oleuropein mainly to hydroxytyrosol(28). In order to evaluate OO
phenolic metabolites produced from colonic fermentation, faecal samples were analysed before
and after mid-term consumption of phenol-rich OO(28). A significant increase in hydroxytyrosol
concentration (p < 0.05) was observed after phenol-rich OO intake. Although absorption of OO
phenolic compounds mainly occurr in the small intestine a small proportion of hydroxytyrosol
and its derivatives still pass into the large intestine(23). This highlights the need to study the
impact of OO phenolics in the colon, either with gut microbiota interaction or local activity due
to its antioxidant and anti-inflammatory properties.
2. Olive oil and inflammation
Inflammation involves a complex cascade of events partly related with the production of an
excess of free radicals due to internal or environmental stress(29). The inflammation process
triggers signaling molecules such as nuclear factor-kappa-B (NF-kB), which up-regulates the
production of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-α)(30) inducible
NO synthase (iNOS), cyclooxygenase-2 (COX-2), and interleukin-1beta (IL-1β)(29).
A number of phenolic compounds present in OO have anti- inflammatory properties, including
oleocanthal, a secoiridoid (dose-dependent inhibition of COX-1 and COX-2 activities, similar to
the anti-inflammatory drug ibuprofen(31)). However, to achieve comparable effect to the
recommended daily dose of ibuprofen, 500 g of EVOO would need to be consumed(32, 33) making
the dose/effect relationship outwith any (acute) inflammatory benefits due to typical OO
Chronic inflammation
Rheumatoid arthritis (RA) is a major inflammatory, autoimmune, disease characterized by
chronic joint inflammation(34, 35). Hydroxytyrosol has been studied for its anti-inflammatory
effects in a RA animal model. We reported that it provided beneficial effects in the evolution of
the disease(36), with 0.5 and 5 mg/kg doses in rats, after gavage administration, using refined OO
as vehicle (human-equivalent of 4.9 and 49 mg/day, respectively, for a 60 kg adult), Figure 1.
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
Significant effects, on paw edema reduction, were observed for a human-equivalent dose of
49 mg/day, a dose 10 times higher than the approved Food and Drug Administration and
European Food Safety Authority (EFSA) dose for phenolic compounds in relation to protection
of lipid oxidation(37). The same hydroxytyrosol dose was effective on colitis, another chronic
inflammatory disease(38). This dose would only be achievable through nutraceutical
supplementation of OO with hydroxytyrosol, and the use of this functional food on a daily basis.
To further evaluate the anti-inflammatory mechanisms involved with hydroxytyrosol, we studied
COX-2 and iNOS expression(36). The treatment at 5 mg/kg dose significantly decreased
histological damage, COX-2 and iNOS expression (p<0.001 vs. positive control), markedly
reduced the degree of bone resorption, soft tissue swelling and osteophyte formation, improving
articular function in treated animals. Moreover at the same dose there was a significant decrease
(p<0.005 vs. positive control and refined OO) in TNF-α serum levels. These results are in line
with others that reported benefits on RA, in animal models, after oral administration of an EVOO
extract(39), intraperitoneal administration of oleuropein aglycone(40) or polyphenol supplemented
VOOs diets(41). The reports highlight effects on RA of OO phenolic compounds either
administered as isolated compounds or as an extract. However, doses comparison between
animal studies have to to take in consideration not only differences in species (rats vs. mice) but
also routes of administration. Compared to intraperitoneal administration, an oral dose has an
extra pass through the liver with consequent metabolism through the first-pass effect.
Acute inflammation
Acute inflammation has been commonly induced using carrageenan in animals in order to
evaluate the effects of non steroid anti-inflammatory drugs (NSAIDs)(42). We studied the effect
of hydroxytyrosol-supplemented OO on acute inflammation, induced by carrageenan in rats, at
0.5 and 5 mg/kg(36) dose, after gavage administration which occurred 30 min before the challenge
with carrageenan. Both doses significantly reduced paw edema (p<0.001 vs. positive control)
with the lowest effective dose being achievable through OO daily intake. Previous studies in
rats(43) also showed inhibition of carrageenan - acute inflammation of an aqueous hydroxytyrosol
formulation (HT-20, 22% hydroxytyrosol), and significant effects were obtained at a 22 mg/kg
hydroxytyrosol dose. Differences in dose effect might be related to the administration vehicle
with ROO or OO being better vehicles than water.
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
Vehicles of administration of OO phenolic compounds on animal and human studies
Using OO as the intake vehicle can promote absorption of hydroxytyrosol: the corresponding
bioavailability of hydroxytyrosol in rats for aqueous and OO solutions were reported as 75 and
99%(44), respectively. When a supplement containing hydroxytyrosol as a single oral
dose (2.5 mg/kg) was fed to humans, the bioavailability was below 10%(45), while previous
studies showed higher bioavailability for hydroxytyrosol supplementation in lipid vehicles(46).
The addition of hydroxytyrosol to low fat yogurt and administered to humans was also associated
with a lower excretion of hydroxytyrosol when compared with OO(46). As OO phenolic
compounds are mainly absorbed in the small intestine(23) the increase of hydroxytyrosol
bioavailability, in OO, might be related to the rate of gastric emptying(45) and slow release of
hydroxytyrosol from the oil matrix(45, 47). The presence of other antioxidants in OO might prevent
breakdown of hydroxytyrosol before absorption in the gastrointestinal tract(44).
Although there are a number of anti-inflammatory effects for OO phenolic compounds, most
cannot be achieved via normal dietary exposure to OO. This has led to development of enriched
products with natural OO phenolic compounds. OO by-products such as olive mill wastewater(48)
and olive pomace(49, 50) are potential sources of natural bioactives which could be used to
supplement OO. The development of new OO products such as pomace OO or refined OO
enriched in natural bioactives opens new perspectives in the field.
3. Cardioprotection of olive oil
Most of the interventional studies focusing on the benefit of VOO intake on cardiovascular
disease have investigated the effect of phenolic compounds on the prevention of oxidation of
low-density (LDL) and high-density (HDL) lipoproteins(51-58), two risk markers of cardiovascular
disease. A number of trials have also focused on cardioprotection against inflammation(59)
mainly on antioxidant activity and inflammatory mediators.
Impact of olive oil constituents on lipoproteins and atherosclerosis
Fat content
LDL particles carry about two-thirds of plasma cholesterol and can infiltrate the arterial wall
attracting macrophages, smooth muscle cells, and endothelial cells(60) thus driving atherosclerosis.
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
LDL particle size is influenced by type and amount of dietary fat consumed(61): Low-fat diets
lead to a decrease in the size of LDL particles compared to high-fat diets(62). The type of fat
ingested is also important: LDL particles are larger with high-monounsaturated fatty acid diets
(such as those based on OO), compared to diets with a high polyunsaturated fatty acids intake,
where LDL particles are smaller(63). LDL particle size is especially relevant, since small size
particle are more prone to oxidation and can better enter into the arterial wall when compared
with larger LDL particles(64). Conversely, HDL particles are antiatherogenic, as their primarily
role is to deliver cholesterol to the liver to be metabolized and excreted or reused. HDL may also
be able to dislodge cholesterol molecules from atheromas in arterial walls(60). It has been
reported in patients with peripheral vascular disease(65, 66), that LDL particles are less susceptible
to oxidation when the diet is enriched in VOO monounsaturated fatty acids, compared to the
polyunsaturated fatty acids of sunflower oil enriched diets. Moreover when compared to
saturated fatty acids intake, OO oleic acid reduces the level of LDL-cholesterol(57, 58).
The health benefits associated with monounsaturated fat content in OO were recognised by the
United States Food and Drug Administration (FDA) in 2004, highligthing “the benefits on the
risk of coronary heart disease of eating about two tablespoons (23 g) of OO daily”(67). Health
benefits were related with a decrease of total and LDL cholesterol in serum(67), diet improvement
of endothelial dysfunction(68), coagulation activity(69) and reduced LDL susceptibility to
Phenolic content
Antioxidants that can prevent lipid peroxidation, such as phenolic compounds, could play an
important role in preventing oxidative modification of LDL(4), with the oxidative process an
initiating factor for atherosclerotic plaques(70). Once monocytes differentiate in macrophages on
the endothelium they scavenge oxidized LDL (ox-LDL), then becoming foam cells, leading to
plaque formation(5).
The Effect of Olive Oil on Oxidative Damage in European Populations (EUROLIVE) study was
a cross-over fat replacement intervention(52), using OOs with different phenolic content in
healthy male volunteers. Its findings led to the current EFSA recommendation (Opinion of the
Scientific Committee/Scientific Panel, EFSA Journal(37, 71, 72)). A linear increase in HDL-
cholesterol levels after 3 weeks was observed after low-, medium-, and high-polyphenol OO
consumption: mean change from preintervention, 0.02 mmol/L (95% CI, 0.00 to 0.05 mmol/L),
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
0.03 mmol/L (95% CI, 0.00 to 0.05 mmol/L) , and 0.04 mmol/L (95% CI, 0.02 to 0.06 mmol/L),
respectively. Total cholesterol:HDL cholesterol ratio decreased linearly with the phenolic
content of the OO. Triglyceride levels decreased by an average of 0.05 mmol/L for all OOs(52).
Mean changes from preintervention for ox-LDL levels were 1.21 U/L (95% CI, -0.8 to 3.6 U/L),
-1.48 U/L(95% CI, -3.6 to 0.6 U/L) and -3.21 U/L (95% CI, -5.1 to -0.8 U/L) for the low-,
medium-, and high-polyphenol OO, respectively, showing a dose-dependent relation with VOO
phenolic content(52). The EFSA confirmed a cause effect relationship between consumption of
OO phenolics (standardized by the content of hydroxytyrosol and its derivatives) and protection
of LDL cholesterol particles against oxidative damage. To support the EFSA health claim, 5 mg
of hydroxytyrosol and its derivatives should be consumed daily in 20 g OO(37), but
concentrations in some OOs may be too low to achieve this target in the context of a balanced
diet. Moreover, the EFSA Panel commented study design limitations as most human
interventions with OO have been conducted in more homogeneous male populations(71) and not
in general population.
The contribution of OO phenolics toward cardiovascular health benefits has been challenged
with inconsistent results reported for ex vivo resistance of LDL to oxidation(73, 74). Seven human
intervention studies with OO were compared for impact of phenolics on ox-LDL, with no effect
seen in five of them(73), possibly explained by artifacts generated during LDL isolation.
Since the approval of the EFSA claim, both terminology and analytical methodology supporting
the dose calculation of hydroxytyrosol and derivatives have been appraised. Mastralexi et al.(75)
commented on the weaknesses of the claim terminology namely the term “olive oil polyphenols”
is not entirely clear and accurate as “olive oil” is a generic term for the type of oil, and the basic
structure of OO phenolic compounds do not coincide with a “polyphenolic” structure;
accordingly “virgin olive oil bioactive phenols” is a more appropriate term. Others also
commented about the lack of robust and reliable methods for quantifying phenolic compounds in
OO. A simple and robust method for routine analysis of hydroxytyrosol and tyrosol was
proposed(75, 76) based on hydrolysis of the polar fraction of OO. This was followed by
development and validation of a 1H NMR method enabling direct measurement of tyrosol and
hydroxytyrosol derivatives, as well as oleocanthal and oleacein in OO, overcoming analytical
issues such as chromatographic peak broadening(19).
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
Cardioprotective mechanisms of oleic acid
OO intake has been related with a decrease on blood pressure with oleic acid regarded as being a
major contributor to this effect, as evidenced in animal models(77). Chronic oral administration of
VOO (rich in oleic acid), triolein (a triacylglyceride with three oleic acid moieties) or oleic acid
over 14 days significantly reduced systolic blood pressure in rats (-26 ± 4 for VOO and -21 ± 3
mm Hg for triolein, p<0.001, and -17 ± 1.9 mm Hg for oleic acid p<0.05) when compared to the
control group that received water. Similarly acute (2 h) treatments with either VOO or triolein
also significantly reduced systolic blood pressure when compared to the control group
(-20 ± 0 mm Hg, p < 0.001, and -14 ± 2 mm Hg, respectively, p < 0.05) with oleic acid again
significantly reducing systolic blood pressure (-13.0 ± 0.3 mm Hg; p < 0.001). In contrast,
chronic treatment with the trans-monounsaturated fatty acid elaidic (18:1n-9) or the saturated
fatty acid stearic acid (18:0) did not significantly affect blood pressure. Results show that
saturation and cis/trans double bond arrangement are implicated with the cardioprotective effect
of the long chain fatty acid in this animal model at high dose levels(77). Similar significant results
were obtained after VOO and oleic acid intake in an animal model of hypertension using
spontaneously hypertensive rats(77).
The molecular mechanisms were evaluated by measuring signaling proteins involved in the
control of blood pressure in the aorta. OO intake increases oleic acid levels in membranes, which
regulate membrane lipid structure and impact on G protein-mediated signaling, causing a
reduction in blood pressure(78). Unlike its analogues elaidic and stearic acid, oleic acid, due to its
cis-18:1n-9 structure, regulates cellular membrane lipid structure and the α2 receptor system
involved in the control of blood pressure (α2A/D - adrenoreceptor/G protein/adenylyl cyclase-
cAMP/PKA) as demonstrated in vitro(78) and in vivo(77). Oleic acid can also contribute to heart
health via intramyocardial triglyceride turnover(79), which is reduced in pressure-overloaded
failing hearts. In this situation oleate (derivative of oleic acid) upregulated triglyceride dynamics
when compared to palmitate (derivative of palmitic acid and major saturated fatty acid of palm
oil). This result underscores the importance of the intracellular lipid storage type on nuclear
receptor signaling and contractility(79) in diseased hearts.
An important driver of vasorelaxation is nitric oxide, a free radical which readily reacts with fats
and proteins. Nitro-fatty acids are mediators of cardiovascular signaling actions(80) as these
compounds relax blood vessels, attenuate platelet activation, and reduce inflammation(81, 82).
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
Both oleic acid and linoleic acid are unsaturated fatty acids that after reaction with nitrite may
form nitro-fatty acids. Nitro-oleic acid mediated antihypertensive signaling actions were shown
in a mouse model(83). The mechanism was attributed to the inhibition of soluble epoxide
hydrolase by nitro-fatty acids, thus lowering blood pressure in an angiotensin II-induced
hypertension(83). It is however unclear how the extent of nitrite in the human diet may contribute
to nitration of dietary fat, and the physiological relevance of this finding.
Role of phenolic compounds on endothelium protection
Oxidative stress and reactive oxygen species (ROS) have been implicated in endothelial damage,
progression to atherosclerosis, injury in sustained myocardial infarction and ischemia
reperfusion(70, 84-86). Monocytes and macrophages are critical cells that are involved in
atherosclerosis. These cells produce proinflammatory cytokines, such as IL-, TNF-α and
C-reactive protein (CRP), which induce the expression of adhesion molecules like intercellular
adhesion molecule-1 (ICAM-1), vascular-cell adhesion molecule-1 (VCAM-1), and E-selectin(87).
Meanwhile, oxidative stress through ROS production promotes the expression of the adhesion
molecules on the endothelium(88).
Expression of adhesion molecules attracts circulating monocytes inducing their adherence to the
endothelium. OO phenolic compounds have been shown to act on endothelium protection as
evidenced in in vitro assays with typical OO phenolic compounds and less on in vivo circulating
metabolites. OO phenolic extract, oleuropein aglycone or homovanillic alcohol (metabolite of
hydroxytyrosol) had inhibitory effects on VCAM-1, ICAM-1 and E-selectin surface expression
in human umbilical vascular endothelial cells, using TNF-α as pro-inflammatory stimulus(89).
Endothelium dysfunction refers to an impairment of endothelium-dependent vasorelaxation
caused by a loss of NO bioactivity in the vessel wall. In animal models with rats oral
hydroxytyrosol administration was tested on NO production and platetet function(90). Results
showed that hydroxytyrosol administration (100 mg/kg/day) increased vascular NO production
by up to 34.2% (p < 0.01) and inhibited platelet aggregation for 50% inhibitory dose of
48.25 mg/day for hydroxytyrosol (p<0.01) when compared to control group (treated with
isotonic saline solution). Animal dose translation to humans allowed us to conclude that the
effective hydroxytyrosol doses tested would be above the expected intake through OO daily. The
reported benefits would only be achievable through nutraceutical supplementation.
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
Endothelium repair: matrix metalloproteinases and olive oil
Matrix metalloproteinases (MMPs) play a role in endothelium repair. Macrophages resident in
human and experimental atherosclerosis co-localize with and release active MMPs including the
gelatinase MMP-9, which is specialized in the digestion of basement membrane collagens and
elastin, and is implicated in atherogenesis, unstable coronary syndromes, and in aortic
aneurysms(91). Accumulating evidence points to the MMPs as major molecular mediators of
arterial diseases(91). Collagens, types 1 and 3, are the main proteins in arterial walls being also
present in the thickened intima of atherosclerotic lesions(92, 93). Fragments of collagens found in
urine are present as a result of proteolytic activity in arterial walls and other vascular structures.
Collagen type 1 or 3 fragments were up-regulated in urine in coronary artery disease (CAD)
patients(94). Increase in collagen degradation is related with an increase on collagenases
circulation, such as MMP-9, as shown in patients with CAD(95).
In an in vitro study hydroxytyrosol (1-10 μM) reduced MMP-9 (IC50 = 10 μmol/L, p< 0.05) and
COX-2 induction in activated human monocytes, with phorbol myristate acetate (PMA)(96).
These effects were mediated by inhibition of transcription factor NF-κB and protein kinase C
(PKC) α and PKCβ1 activation(96). Results are in line with previous in vitro reports that showed
inhibition of MMP-9 on endothelial cells by OO phenolics namely hydroxytyrosol in PMA
induced cells(97), and oleuropein aglycone in TNF-α induced cells by acting on NF-κB(88). No
hydroxytyrosol activity on MMP-9 was found in TNF-α induced cells(88).
The discriminatory polypetides that increase in CAD includes collagen type 1 and 3 fragments
with a C-terminal GxPGP motif (98). Increase on these polypeptides would come from a protease
decrease activity possibly related with chemical change of the substrate (e.g.: oxidative damage)
thus inhibiting it acting at a specific site, or a decrease in circulating levels by lack of enzyme
activation. MMP-2 is secreted in an inactive form (pro MMP-2) and several factors can promote
its activation such as plasmin(99) and thrombin(100). Other mechanisms that involve proteinases or
oxidative stress can also activate MMP-2(101). Therefore antioxidants, as phenolic compounds,
might have a role on MMP-2 activation and published data indicate phenolic compounds from
red wine(102) and green tea(103) as acting on prevention of thrombin-induced activation of MMP-2
in vascular smooth cells.
We evaluated the impact of a 6-week OO supplementation in healthy adults on urinary proteomic
biomarkers of coronary artery disease (CAD) in a randomized, parallel, controlled, double-blind
Silva et al 2015 New perspectives on bioactivity of olive oil
Manuscript published in Proceedings of Nutrition Society, DOI:10.1017/S0029665115002323
study(104). This study was the first to describe the significant impact of daily OO supplementation
on highly specific disease biomarkers for CAD. Analysis of urinary proteomic profiles at
baseline and endpoint enabled the identification of 12 sequenced peptides that were significantly
regulated toward healthy scoring. Eight of them included four collagen α-1(I) chain, one α-2 (1)
chain, one α-2(V) chain, and one α-2(VI) chain fragments. Changes in circulating concentrations
of collagenases may mediate these changes in the urinary fingerprint. Therefore with more data
or in future intervention studies with OO it would be interesting to link urinary fragments to the
proteases involved in their generation. This predictive analysis would enable looking at the
peptide cleavage sites studying the MMPs up or downregulated with OO intervention.
The majority of studies of dietary intake of proposed bioactive foods asses the activities of these
foods based on the major risk factors of cardiovascular disease. However marker such as
lipoprotein profile, blood pressure, endothelial function, inflammation and oxidative stress have
no direct link to the disease itself but are merely associated with it. There is a great need for more
biomarkers that appear as a direct result of the disease itself(57, 61).
Silva et al 2015 New perspectives on bioactivity of olive oil
4. Proteomics biomarkers as a mechanistic approach to explain olive oil health effects
The systems biology approach (encompassing genomics, transcriptomics, proteomics and
metabolomics using urine, blood or saliva) could provide a greater understanding of disease
development, treatment efficacy and evaluation of the influence of food bioactive
compounds (33, 105). There is a need for biomarkers of practical value for clinical intervention,
allowing disease risk prediction and more importantly early diagnosis. Accuracy, reproducibility,
availability, feasibility of implementation into the clinical settings, sensitivity and specificity are
additional characteristics to be fulfilled, and panels of biomarkers are gaining acceptance instead
of individual molecules(106), as single biomarkers are often not available and lack the ability to
adequately describe complex diseases(107). Candidate biomarkers should be carefully validated in
a wide and different cohort of samples from those used in the discovery phase as often
overfitting of the biomarker model has occurred(108) .
The proteome, corresponding to a set of expressed proteins, informs the current “status” of an
organism, constantly changing according to endogenous and exogenous factors(107). Proteins are
widely used in different clinical tests for both diagnosis and prognosis of diseases and to follow
their evolutions(92). They can be used to measure the extent of inflammation, calcification, and
the development of plaques on the arteries. Understanding what causes plaque rupture is of great
importance. As previously mentioned, MMPs could have a key role in this process(109). The
discovery of proteomic biomarkers may be useful in understanding the molecular mechanisms
involved in the onset and progression of other vascular diseases(110). Plasma, serum and urine are
the most commonly used biological matrices in cardiovascular research, due to their perceived
clinical relevance as a source of potential biomarkers(92). However proteomic studies have also
been carried out on vascular tissues (arteries), artery layers, cells looking at proteomes and
secretomes, exosomes, lipoproteins, and metabolites(92). Although sampling the tissue may seem
an obvious method there are a number of difficulties, especially where the need for a biopsy
would be required(111). Recent advances in extraction processes and LC-MS/MS analysis has
allowed the quantitative analysis of tissue samples in vascular research to be carried
out(112, 113).
Urine, as a sample source is now recognized as the source of choice for proteomic biomarker
investigations. It has a number of advantages such as being noninvasive and can be collected by
untrained personnel. Urine is produced by renal filtration of the plasma and approximately 70%
Silva et al 2015 New perspectives on bioactivity of olive oil
of proteins in the normal human urinary proteome are of kidney origin, whereas the remaining
30% are derived from plasma proteins (114, 115). It has high stability due to absence of proteolytic
agents and the low dynamic range of analyte concentration facilitates the detection and
quantification of peptides(107, 116).
Using capillary electrophoresis coupled with mass spectrometry (CE-MS)(117) urinary biomarker
classifiers for the diagnosis of diseases like chronic kidney disease(118), acute kidney injury(119),
stroke(120), and coronary artery diseases(98), were already identified, allowing classification of
case versus control groups with good accuracy(121).
Urinary peptides and protein fragments are the end products of proteolytic processes. The
different pattern of urinary excretion of peptides when comparing controls and disease patients
might indicate their role in the pathophysiology of disease. Therefore changes in the normal
urine "fingerprint" (e.g.: presence of collagen fragments) can be used as biomarkers of disease.
Besides collagens, common blood proteins (e.g., alpha-1-antitrypsin, hemoglobin, serum
albumin, and fibrinogen), and uromodulin were also identified(122) in urine which provides
additional proof of the suitability of this sample source for proteomic biomarker studies out with
the kidney and urinary tract. Collagens are the most abundant peptides sequenced so far in the
CAD biomarker (66% of all peptides)(98), with atherosclerosis associated with an increased
synthesis of several extracellular matrix components, including collagen types 1 and 3, elastin,
and several proteoglycans(123). Changes in the circulating levels of collagenases may mediate
these changes in peptides represented in the fingerprint, as reported in coronary
atherosclerosis(94), and chronic kidney disease(122).
The progress in urinary proteomics and the use of multiple biomarker classifiers opens the
possibility of establishing new tools adapted to different clinical needs(124), enabling direct
monitoring of disease overcoming limitations of indirect measurements.
Proteomic in vitro studies on olive oil phenolic compounds
Proteomics has been applied in a number of studies of OO phenolic compounds on
cardiovascular health using animal and in vitro studies. The in vitro effects of alperujo extract, an
OO production waste product containing phenolic compounds present in olive fruits, were
studied on platelet aggregation and changes in the platelet proteome(125). Nine proteins were
differentially regulated by the alperujo extract upon platelet aggregation underlying the anti-
Silva et al 2015 New perspectives on bioactivity of olive oil
platelet effects of the extract. However, like a number of previously mentioned in vitro studies,
the effective concentrations (40-500 mg/L) were far above the physiologically concentrations
achievable by dietary intake.
The effects of EVOOs, with low and high in phenolic content, were evaluated in the hepatic
proteome in Apoe-/- mice that spontaneously develop atherosclerosis(126). For 10 weeks the mice
were fed with a high fat high cholesterol diet supplemented with 0.15% (w/w) cholesterol and
either 20% (w/w) low phenolic EVOO or 20% (w/w) high phenolic EVOO versus a control
group fed with 0.15% (w/w) cholesterol and 20% (w/w) palm oil. Within this work a range of
hepatic antioxidant enzymes differentially regulated by OO(126) were identified. The authors
concluded that the up-regulation of a large array of antioxidant enzymes might explain anti-
atherogenic mechanisms of EVOOs(126). Again the dose level was above what could be achieved
through dietary intake and translation from an animal model to human has also to be considered.
Urinary proteomics biomarkers, olive oil and cardiovascular disease
Atherosclerosis is a process of chronic inflammation, characterized by the accumulation of
lipids, cells, and fibrous elements in medium and large arteries(92). The extent of
inflammation, proteolysis, calcification, and neovascularization influences the development of
advanced lesions (atheroma plaques) on the arteries(92).
Classical risk factors in atherosclerosis (hypertension, LDL-cholesterol, C-reactive protein,
aging, smoking, male gender, among others) do not actually measure disease initiation or
progression. As such, they cannot be used directly to identify individuals who have developed
atherosclerosis and prevent a fatal event(92, 127). Other, more recent markers that indicate changes
in vascular structure can still only be detected once cardiovascular disease has progressed to an
advanced stage where drug or surgical intervention is required(128).
The analysis of urine samples from diseased and healthy individuals has been used to establish a
database of naturally occurring urinary peptides, making a basis for the definition and validation
of biomarkers for diagnosis/prognosis/monitoring of a wide range of diseases using proteomic
biomarker patterns(122), such as CAD(94), emphasizing that non-invasive proteomics analysis
could become a valuable addition to assess cardiovascular disease alongside to other biomarkers
which are indicators of cardiovascular risk.
Silva et al 2015 New perspectives on bioactivity of olive oil
The first time that urinary proteomics was applied to assess cardiovascular health improvements
of OO consumption in humans, was in a randomized, parallel, controlled, double-blind study
designed to evaluate the impact of a 6 week OO supplementation in healthy adults on urinary
proteomic biomarkers of CAD(104). The impact of the supplementation with OO was also studied
on urinary proteomic biomarkers of chronic kidney disease (CKD), and diabetes.
The increase or decrease in the concentration of the peptides in the biomarker determines the
scoring value of each disease biomarker. The CAD proteomic biomarker developed for clinical
diagnosis produces a CAD scoring system from 1 (CAD case) to -1 (healthy artery). A scoring of
disease absence, presence and severity is provided, based on the concentration of a group (panel)
of urinary peptides measured by CE-MS, allowing monitoring of progression and/or effect of
treatment(129, 130). In this study, self-reported healthy participants were randomly allocated to
supplementation with a daily dose of OO either low or high in phenolic compounds. For 6
weeks, they consumed a daily dose of 20 mL OO (not heated or cooked) as a supplement (no
specific time during the day, single intake, equivalent to 6 mg of hydroxytyrosol and derivatives
for the high phenolic OO), in line with the EFSA and FDA recommendations.The impact of
supplementation with OO was evaluated on urinary proteomic biomarkers of CAD with
biomarkers being measured at baseline and 3 and 6 weeks. Consumption of both OOs
significantly improved the proteomic CAD score at endpoint compared with baseline, moving
the CAD biomarker pattern in a healthy profile direction, Table 2. No differences were observed
for CKD or diabetes proteomic biomarkers, Table 2.
In a placebo-controlled intervention, Irbesartan (angiotensin II receptor antagonist used for the
treatment of hypertension) taken at 300 mg per day over 2 years in hypertensive type 2 diabetes
patients, using the CAD 238 biomarker panel, led to a 0.35 point reduction in the CAD score for
the drug-controlled group(98), which saw a significant reduction in incidents of CAD in this
group. In the nutritional intervention(104) the CAD score change in the intervention was
significant for both OOs tested, using the same CAD 238 biomarker, leading to a similar degree
of change as observed for irbersartan over a 6 week period. This evidence highlights the
importance of the CAD biomarker as a tool for nutrition and health intervention studies. This
type of urinary biomarker enabled the measurement of health effects induced by a change in diet
that could not be detected by monitoring the conventional risk markers of CAD such as plasma
triacylglycerols, oxidized LDL, and LDL cholesterol. The overall change in CAD score in a
Silva et al 2015 New perspectives on bioactivity of olive oil
short period of time is more likely due to OO major components, such as fatty acids. However
the role of other OO minor components other than phenolic compounds should also be taken into
account. Squalene, a polyunsaturated triterpene which makes up 6075% of the unsaponifiable
fraction of OO(131), reduced atherosclerotic lesion size in male mice(132) and further investigation
is needed to clarify its role on cardiovascular disease.
Our results emphasize further the potential role of nutrition in the prevention or delay of
cardiovascular disease and offer new perspectives on OO applications. These results are highly
translatable to guidelines for nutritional recommendations. The biomarkers were originally
developed to detect early signs of diseases in clinical setting and to inform clinician as to the
effectiveness of treatment. However, the technology also provides a sensitive tool for the
assessment of potential bioactive foods in cardiovascular health, chronic kidney disease and
diabetes, with a range of additional tests under development. Further testing of reportedly
bioactive foods can now be carried out which will allow better nutritional health advice to be
advanced and could also lead to better food labeling so that the public can make informed
choices on their food purchases.
Silva et al 2015 New perspectives on bioactivity of olive oil
5. Exploring olive oil health benefits: perspectives 1
Although strong evidence from heritability is related with cardiovascular disease many forms of heart 2
disease are not genome associated(133). The epigenome is a possible link between genetics and 3
environment(133) which includes impact of food components/diet. Omics techniques (genomics, 4
transcriptomics, proteomics, epigenomics, metabolomics) have the potential, when integrated, to paint 5
a comprehensive picture of the contribution of diet toward the modulation of disease risk(141). Some 6
trials have shown the impact of OO on down-regulation of atherosclerosis-related genes(134, 135). The 7
effect of Mediterranean Diet was studied on urinary metabolome(136) and related to compounds of the 8
metabolism of carbohydrates, creatine, creatinine, amino acids, lipids and microbial cometabolites. 9
Phenolic compounds can interact with cellular signaling cascades regulating the activity of 10
transcription factors with impact on gene expression. For instance, phenolic compounds have shown to 11
affect the expression of microRNAs (miRNA)(137). miRNAs are small, noncoding RNAs implicated in 12
the regulation of gene expression that control both physiological and pathological processes, influenced 13
by external factors as diet components(138). Most of the studies reported in this field are in vitro and 14
more in vivo studies are needed to clarify miRNA targets of dietary phenolic compounds(138). 15
Interactions between genes and the bioactive components present in OO studied by nutrigenomics may 16
help to explain its health benefits(139). In this sense, besides their antioxidant and anti-inflammatory 17
capacities, OO phenolic compounds are able to modify gene expression coding in a protective mode for 18
proteins participating in the cellular mechanisms involved in oxidative stress resistance, inflammation 19
or lipid metabolism amongst others(140). 20
Glycation, a non-enzymatic reaction between reducing sugars and proteins, is a proteome wide 21
phenomenon, mainly observed in diabetes due to hyperglycemia(141), but also relevant to end organ 22
damage, disease pathogenesis and aging(142) and OO phenolic compounds have been reported as potent 23
inhibitors of the formation of advanced glycation end products(143). Our human intervention trial with 24
OO low or high in phenolics did not find a significant impact on plasma fructosamine levels(104). A key 25
factor may be the duration of the study (6 weeks) not being sufficient to detect changes in protein 26
modifications such as glycation, and may also be partly related to the quantity and quality of phenolic 27
compounds, which exert differential antioxidant and antiglycative activities depending on 28
structure(4, 144). Further studies should proceed in order to clarify anti-glycation properties of OO 29
phenolic compounds, given that glycation is a key driver for tissue damage and is present in all non-30
communicable disease scenarios. 31
Silva et al 2015 New perspectives on bioactivity of olive oil
6. Final Remarks 33
The reported health benefits of the Mediterranean diet and OO consumption may allow the 34
implementation of primary prevention programs of cardiovascular disease, based on nutritional 35
interventions. These interventions would have a particular relevance in non-regular OO consumers like 36
the Northern European populations. 37
Human intervention trials focusing on new outcomes related with proteomics and nutrigenomics are 38
needed to better clarify pathways/mechanisms by which oleic acid, phenolic compounds from OO or 39
even other components act on cardiovascular disease risk factors and affect the proteome. 40
7. Financial Support 42
QREN project Azeite+ Global nº 12228 and Ordem dos Farmacêuticos (Lisbon, Portugal). 43
8. Conflict of Interest 45
Conflict of interest: Thomas Koeck is employed at Mosaiques Diagnostics, the company that 46
developed the urinary proteomics for CE-MS technology for clinical application. No other authors 47
declare a conflict of interest. 48
Silva et al 2015 New perspectives on bioactivity of olive oil
Table 1 Main classes of phenolic compounds in virgin olive oil
Phenolic acids
Phenolic alcohols
Caffeic acid
(+) - Pinoresinol
Oleuropein aglycone
Ligstroside aglycone
Dialdehydic form of deacetoxy
oleuropein (3,4-DHPEA-EDA)
Silva et al 2015 New perspectives on bioactivity of olive oil
Table 2 Changes in scores of CAD, CKD and diabetes proteomic biomarkers at baseline, middle (3-weeks) and
end of intervention (6 weeks)1
Low phenolic olive oil (n = 34)
High phenolic olive oil (n = 28)
Changes relative to baseline
Changes relative tobaseline
CAD proteomic biomarker
-0.5 ± 0.2
-0.6 ± 0.4
3 weeks
-0.7 ± 0.3
-0.2 ± 0.3 (-0.3, -0.1)
-0.7 ± 0.3
-0.1 ± 0.4 (-0.3, 0.0)
6 weeks
-0.8 ± 0.3
-0.3 ± 0.2 (-0.4, -0.2)**
-0.8 ± 0.3
-0.2 ± 0.3 (-0.4, -0.1)*
CKD proteomic biomarker
-0.4 ± 0.2
-0.4 ± 0.3
3 weeks
-0.4 ± 0.2
0.0 ± 0.3 (-0.1, 0.1)
-0.4 ± 0.3
0.1 ± 0.3 (0.0, 0.2)
6 weeks
-0.4 ± 0.2
0.0 ± 0.3 (0.0, 0.1)
-0.4 ± 0.2
0.0 ± 0.3 (-0.1, 0.1)
Diabetes proteomic biomarker
1.3 ± 0.3
1.3 ± 0.3
3 weeks
1.3 ± 0.4
0.1 ± 0.4 (-0.1, 0.2)
1.3 ± 0.3
-0.1 ± 0.4 (-0.2, 0.1)
6 weeks
1.4 ± 0.4
0.1 ± 0.4 (0.0, 0.2)
1.2 ± 0.3
0.0 ± 0.4 (-0.2, 0.1)
1Values are means ± SDs; 95% CIs in parentheses. A repeated-measures ANOVA test was used with statistical significance at p < 0.05. ***Compared with
corresponding baseline value: *p < 0.005, **p < 0.001. There were no significant differences in changes between groups. CAD, coronary artery disease; CKD,
chronic kidney disease (adapted from Silva et al.(104)).
Silva et al 2015 New perspectives on bioactivity of olive oil
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Figure 1 Chronic inflammation model and impact on rats paw edema (ANOVA, * p < 0.001
vs. postive control Rheumatoid Arthritis, + p < 0.01 vs. Refined Olive Oil; OHTYR =
hydroxytyrosol) (adapted from Silva et al.(36))
... The study results showed that an oral hydroxytyrosol administration of 100 mg/kg/day increased vascular nitric oxide release by up to 34.2% (p < 0.01) and inhibited platelet aggregation for 50% at an inhibitory dose of 48.25 mg/day (p<0.01), compared to the control group (106). However, animal dose translation to humans permitted the assumption that the 350 effective hydroxytyrosol doses tested would be above the expected daily intake of olive oil (106). ...
... compared to the control group (106). However, animal dose translation to humans permitted the assumption that the 350 effective hydroxytyrosol doses tested would be above the expected daily intake of olive oil (106). ...
... Relying on these findings, it is possible to suggest that compounds of olive oil help to balance increased oxidative stress and the impaired antioxidant defense that affects endothelial function contributing to the atherosclerotic disease progression. However, a number of anti-oxidant effects of phenolic compounds cannot be realized by the normal dietary exposure to olive oil (106). ...
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Existing evidence supports the significant role of oxidative stress in the endothelial injury, and there is a direct link between increased oxidative stress, and the development of endothelial dysfunction. Endothelial dysfunction precedes the development of atherosclerosis and subsequent cardiovascular disease (CVD). The overproduction of reactive oxygen species facilitates the processes, such as oxidative modification of low-density lipoproteins and phospholipids, reduction in the NOS-derived nitric oxide, and the functional disruption of high-density lipids that are profoundly involved in atherogenesis, inflammation, and thrombus formation in vascular cells. Thus, under oxidative stress conditions, endothelial dysfunction was found to be associated with the following endothelial alterations: reduced nitric oxide bioavailability, increased anticoagulant properties, increased platelet aggregation, increased expression of adhesion molecules, chemokines, and cytokines. In this review, we summarized the evidence indicating that endothelial damage triggered by oxidation can be diminished or reversed by the compounds of olive oil, a readily available antioxidant food source. Olive oil bioactive compounds exhibited a potent capability to attenuate oxidative stress and improve endothelial function through their anti-inflammatory, anti-oxidant, and anti-thrombotic properties, therefore reducing the risk and progression of atherosclerosis. Also, their molecular mechanisms of action were explored to establish the potential preventive and/or therapeutic alternatives to the pharmacological remedies available.
... Oleic acid (C18:1) is the most famous MUFA abundant in olive oil and is known to decrease the risk of coronary artery disease compared to saturated fatty acids. 17 A previous study revealed the strong inverse association between LCMUFA levels in red blood cells and CVD even after adjusting for the de novo synthesized MUFAs (palmitoleic acid; C16:1 and C18:1), indicating the possible atheroprotective effects of LCMUFAs. 18 We have previously shown that fish oil-derived LCMUFAs attenuate atherosclerosis development in ApoE À/À and LDLR À/À mice. ...
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Fish oil-derived long-chain monounsaturated fatty acids (LCMUFAs) with a carbon chain length longer than 18 units ameliorate cardiovascular risk in mice. In this study, we investigated whether LCMUFAs could improve endothelial functions in mice and humans. In a double-blind, randomized, placebo-controlled, parallel-group, multi-center study, healthy subjects were randomly assigned to either an LCMUFA oil (saury oil) or a control oil (olive and tuna oils) group. Sixty subjects were enrolled and administrated each oil for 4 weeks. For the animal study, ApoE−/− mice were fed a Western diet supplemented with 3% of either gadoleic acid (C20:1) or cetoleic acid (C22:1) for 12 weeks. Participants from the LCMUFA group showed improvements in endothelial function and a lower trimethylamine-N-oxide level, which is a predictor of coronary artery disease. C20:1 and C22:1 oils significantly improved atherosclerotic lesions and plasma levels of several inflammatory cytokines, including IL-6 and TNF-α. These beneficial effects were consistent with an improvement in the gut microbiota environment, as evident from the decreased ratio of Firmicutes/Bacteroidetes, increase in the abundance of Akkermansia, and upregulation of short-chain fatty acid (SCFA)-induced glucagon-like peptide-1 (GLP-1) expression and serum GLP-1 level. These data suggest that LCMUFAs alter the microbiota environment that stimulate the production of SCFAs, resulting in the induction of GLP-1 secretion. Fish oil-derived long-chain monounsaturated fatty acids might thus help to protect against cardiovascular disease.
... These proinflammatory markers have been previously found altered in GDM placentas and are difficult to modulate even with a good metabolic control. 3,5 Also, the ability of the EVOO-enriched diet to prevent MMPs overactivity in the placenta and the umbilical cord blood observed in GDM patients is supported by previous studies addressing the effect of diets added with EVOO as negative regulators of MMPs in placentas from diabetic rats and in different human diseases 15,38 and provides evidence of the capacity of the benefits of the EVOO-enriched diet to reach the foetal compartment. ...
Aims To address the effect of a diet enriched in extra virgin olive oil (EVOO) on maternal metabolic parameters and placental proinflammatory markers in Gestational diabetes mellitus (GDM) patients. Methods Pregnant women at 24‐28 weeks of gestation were enrolled: 33 GDM patients which were randomly assigned or not to the EVOO‐enriched group and 17 healthy controls. Metabolic parameters were determined. Peroxisome proliferator activated receptor (PPAR) γ and PPARα protein expression, expression of microRNA (miR)‐130a and miR‐518d (which respectively target these PPAR isoforms) and levels of proinflammatory markers were evaluated in term placentas. Matrix metalloproteinases (MMPs) activity was evaluated in term placentas and umbilical cord blood. Results GDM patients that received the EVOO‐enriched diet showed reduced pregnancy weight gain (GDM‐EVOO:10.3±0.9, GDM:14.2±1.4, P= 0.03) and reduced triglyceridemia (GDM‐EVOO:231±14, GDM:292±21, P= 0.02) compared to the non‐EVOO‐enriched GDM group. In GDM placentas, the EVOO‐enriched diet did not regulate PPARγ protein expression or miR‐130a expression, but prevented the reduced PPARα protein expression (P =0.02 vs GDM) and the increased miR‐518d expression (P =0.009 vs GDM). Increased proinflammatory markers (interleukin‐1β, tumor necrosis factor‐α and nitric oxide overproduction) in GDM placentas were prevented by the EVOO‐enriched diet (respectively P =0.001, P =0.001 and P =0.01 vs GDM). MMPs overactivity was prevented in placenta and umbilical cord blood in the EVOO‐enriched GDM group (MMP‐9: respectively P =0.01 and P =0.001 vs GDM). Conclusions A diet enriched in EVOO in GDM patients reduced maternal triglyceridemia and weight gain and has anti‐inflammatory properties in placenta and umbilical cord blood, possibly mediated by the regulation of PPAR pathways. This article is protected by copyright. All rights reserved.
... These proinflammatory markers have been previously found altered in GDM placentas and are difficult to modulate even with a good metabolic control. 3,5 Also, the ability of the EVOO-enriched diet to prevent MMPs overactivity in the placenta and the umbilical cord blood observed in GDM patients is supported by previous studies addressing the effect of diets added with EVOO as negative regulators of MMPs in placentas from diabetic rats and in different human diseases 15,38 and provides evidence of the capacity of the benefits of the EVOO-enriched diet to reach the foetal compartment. ...
... 5,6 There is a growing body of evidence which indicates that OA is beneficial to blood pressure control and that it helps to slow the progress of cancer and inflammatory diseases. [7][8][9][10] However, the role of OA in inflammatory responses remains controversial. Indeed, a number of studies have reported the anti-inflammatory of OA; 11 however, other studies have contradicted these claims. ...
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Aim: This paper reports on the incorporation of oleic acid (OA) within nanostructured lipid carriers (OA-NLC) to improve the anti-inflammatory effects in the presence of albumin. Materials and methods: NLCs produced via hot high-shear homogenization/ultrasonication were characterized in terms of particle size, zeta potential, and toxicity. We examined the effects of OA-NLC on neutrophil activities. Dermatologic therapeutic potential was also elucidated by using a murine model of leukotriene B4-induced skin inflammation. Results: In the presence of albumin, OA-NLC but not free OA inhibited superoxide generation and elastase release. Topical administration of OA-NLC alleviated neutrophil infiltration and severity of skin inflammation. Conclusion: OA incorporated within NLC can overcome the interference of albumin, which would undermine the anti-inflammatory effects of OA. OA-NLC has potential therapeutic effects in topical ointments.
... It has been shown that the urinary metabolome can be affected by the MD [37], with variations in the compounds of the metabolism of carbohydrates, creatine, creatinine, amino acids, lipids and the cometabolites of bowel microbes. An appropriate quantity of EVOO can influence the urinary metabolome [38]. It could therefore be interesting to evaluate how the MD, with a suitable quantity of EVOO, can influence the urinary metabolome of nursing mothers and the health of breastfed children. ...
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The new base of the pyramid that represents the Mediterranean Diet (MD) includes a balanced lifestyle, healthy cooking methods, traditional, local and eco-friendly products, conviviality, physical activity with an adequate amount of rest, as well as caloric restriction and food frugality. Moreover, it has been confirmed that the main source of MD fat is Extra Virgin Olive Oil (EVOO). EVOO is considered a key feature of the healthy properties of the MD, due to its fatty acid, vitamin and polyphenol composition. However, these components need to be bioavailable to allow EVOO to exert its nutraceutical properties, which include antioxidant, anti-inflammatory, anti-cancer, antimicrobial, antiviral and hypoglycemic properties, as well as protective effects on the heart and brain, and during pregnancy and breast feeding. The main phenolic components responsible for the nutraceutical properties of EVOO are hydroxytyrosol, tyrosol and oleuropein. The adopted oil production and extraction technologies, such as extraction at low oxidative stress, determine the final polyphenol content in virgin olive oil. Limited information on the epigenetic effects of olive polyphenols is presently available, although the epigenetic effects of many other plant polyphenols have been well documented. In this context, it has been found that, if mothers consume an adequate amount of olive oil during pregnancy, their children will be exposed to a lower risk of wheezing in the first period of their lives. In addition, EVOO, because of its oleochantal content, a natural anti-inflammatory substance, may have an effect on many inflammatory diseases, even in the early period of life.
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Background Gibberellic acid (GA3) is a plant growth regulator used to improve the quality of crops but its residues in food causes many hazardous effects. In contrast, olive oil has registered several health benefits including antioxidant, anti-inflammatory, and anti-cancer. Thus, the present study suggests the use of olive oil as a natural food source to counteract the GA3 toxicity during mice development. In a preliminary experiment, 18 mature females were classified into control and GA3-treated subgroups with ascending doses of GA3 (55, 110, 240, 480, 960 mg/kg B.W.) for 2 weeks. In the main experiment, 20 pregnant females at the 7th day of gestation were divided into four groups: G1 is control, G2 treated orally with GA3 (55 mg/kg), G3 treated with olive oil, and G4 treated with GA3-olive oil. The pregnant females were dissected at prenatal stages at E14 and E18 of gestation. Results The high doses of GA3 in the preliminary experiment showed decrease of uterine folds, reduction of carbohydrates content and TNFR2 expression of the uterine glands, degeneration of the ovarian follicles, blood vessels congestion, and altered TNFR2 expression in oocyte membrane as compared with the control. In the second experiment, GA3-treated embryo at E14 and E18 revealed histopathological changes and altered TNFR2 immunostaining in the developing liver, kidney, and skin tissues. Treatment of GA3 with olive oil improves the negative effects induced by GA3. Conclusion The study concluded that a supplementation rich diet with olive oil creates a protective effect against gibberellic acid-induced embryotoxicity during pregnancy.
Coronary heart disease (CHD) is the leading cause of death worldwide. Dietary inclusion of monounsaturated fatty acids (MUFA) such as olive oil (OO), can reduce CHD risk. Tea seed oil (TSO) from Camellia oleifera grown in Thailand, with a MUFA content similar to OO, may be an alternative to OO when a higher cooking smoke point is desired. The lipid profiles, liver histology and serum chemistries of hamsters fed high‐fat diets (TSO, OO, grape seed oil or butter; 14% by weight) were analyzed. After three weeks of feeding, TSO and OO groups had similar plasma low‐density lipoprotein cholesterol (LDL‐C), very‐low‐density lipoprotein cholesterol (VLDL‐C), triacylglycerols (TAG), high‐density lipoprotein cholesterol (HDL‐C), and total cholesterol (TC) levels. Moreover, TC/HDL‐C and LDL‐C/HDL‐ C ratios were also comparable. Similar to the OO group, the TSO group had significantly lower plasma LDL‐C, VLDL‐C, TAG, TC, and a lower TC/ HDL‐C ratio as compared to the butter group. Some minor liver pathological lesions commonly found in rodents fed high‐fat diets were observed. Thai TSO may be a healthy option for cooking, and clinical studies are warranted. Practical applications: With its high smoking point, TSO is attractive to Asia consumers for their styles of cuisine. As it is produced locally, its price is cheaper than imported OO. This study demonstrates TSO's health benefits in lowering lipid profiles similar to OO. Based on this fact, this study supports Thai TSO cultivation in northern Thailand as a means of increasing rural villagers' income.
This chapter reviews the relationship between the amount and type of dietary fat and the incidence of major chronic diseases of Western countries, with a particular emphasis on the role of olive oil and monounsaturated fat. It provides an overview of literature related to examination of diet and in particular dietary fats and several aspects of health status, including coronary disease, several cancers, and obesity. Furthermore, the chapter examines the evidence regarding possible health impacts of using olive oil with its unique functional compounds as the primary source of fat, along with other foods considered part of a traditional Mediterranean diet. There was strong support for the hypothesis that dietary fat intake was an important cause of breast cancer. This was based mostly on older international correlation studies, as well as rodent studies in which animals fed high-fat diets ad libitum developed a greater incidence of mammary tumors.
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The aim of the present meta-analysis of cohort studies was to focus on monounsaturated fat (MUFA) and cardiovascular disease, cardiovascular mortality as well as all-cause mortality, and to distinguish between the different dietary sources of MUFA. Literature search was performed using the electronic databases PUBMED, and EMBASE until June 2nd, 2014. Study specific risk ratios and hazard ratios were pooled using a inverse variance random effect model. Thirty-two cohort studies (42 reports) including 841,211 subjects met the objectives and were included. The comparison of the top versus bottom third of the distribution of a combination of MUFA (of both plant and animal origin), olive oil, oleic acid, and MUFA:SFA ratio in each study resulted in a significant risk reduction for: all-cause mortality (RR: 0.89, 95% CI 0.83, 0.96, p = 0.001; I2 = 64%), cardiovascular mortality (RR: 0.88, 95% CI 0.80, 0.96, p = 0.004; I2 = 50%), cardiovascular events (RR: 0.91, 95% CI 0.86, 0.96, p = 0.001; I2 = 58%), and stroke (RR: 0.83, 95% CI 0.71, 0.97, p = 0.02; I2 = 70%). Following subgroup analyses, significant associations could only be found between higher intakes of olive oil and reduced risk of all-cause mortality, cardiovascular events, and stroke, respectively. The MUFA subgroup analyses did not reveal any significant risk reduction. The results indicate an overall risk reduction of all-cause mortality (11%), cardiovascular mortality (12%), cardiovascular events (9%), and stroke (17%) when comparing the top versus bottom third of MUFA, olive oil, oleic acid, and MUFA:SFA ratio. MUFA of mixed animal and vegetable sources per se did not yield any significant effects on these outcome parameters. However, only olive oil seems to be associated with reduced risk. Further research is necessary to evaluate specific sources of MUFA (i.e. plant vs. animal) and cardiovascular risk.
Lipid and lipoprotein responses to reduced dietary fat intake were investigated in relation to differences in distribution of low-density-lipoprotein (LDL) subclasses among 105 healthy men consuming high-fat (46% fat) and low-fat (24% fat) diets in random order for 6 wk each. With high-fat diets, 87 subjects had predominantly large, buoyant LDL (pattern A), whereas the remainder had primarily smaller, denser LDL (pattern B). With low-fat diets, 36 men changed from pattern A to B. Compared with the 51 men with pattern A with both diets (stable A group), men in the stable B group (n = 18) had significantly greater reductions in plasma LDL cholesterol, apolipoprotein B, and mass of mid-sized (LDL II) and small (LDL III) LDL subfractions. In both the stable A and change groups, there was a shift in LDL particle mass from larger, lipid-enriched (LDL I and II) to smaller, lipid-depleted (LDL III and IV) subfractions, suggestive of change in LDL composition with minimal change in particle number, and consistent with the observation of reduced plasma LDL cholesterol without reduced apolipoprotein B. Stable B subjects had significantly greater increases in the largest very-low-density-lipoprotein subfraction with the low-fat diet than the stable A group, and also had greater decreases in the high-density-lipoprotein (HDL) subclass HDL3 but smaller reductions in HDL2. Genetic and environmental factors influencing LDL subclass distributions thus may also contribute substantially to interindividual variation in plasma lipoprotein response to a low-fat diet.
Olive oil is the major edible vegetable oil of the Mediterranean countries and Portugal. It is also, perhaps, the oldest reported crop in history. The olive tree is ca­ pable of existing in a harsh climate on poor soils, and trees 500 years old still bear fruit. The oil itself is much prized for its flavor and aroma. The highest-quality oils are obtained, without solvent extraction, from fresh and healthy fruits. Although the subtle sensory characteristics of olive oil account for its popularity, despite a high market price, increasing interest has been given to its nutritional properties, which are believed to play a large role in the so-called "Mediterranean Diet. " In this book, we provide a wealth of detail about the analysis and properties of olives and their oil. After an introduction to olive oil and to technological aspects, we include a section on biochemistry because, of course, the unique properties of the oil are based on the biochemistry of the olive fruit. This applies not only to the main constituents-the various triacylglycerols-but also to minor sensory components that are derived largely from the lipoxygenase catabolic pathway. Following are chapters that deal with the analysis of olive oil from the standpoint of general methodology, and later chapters describe detailed techniques. The sophisticated analytical methods have to be evaluated by the use of math­ ematical procedures for characterization.
Background: Virgin olive oils are richer in phenolic content than refined olive oil. Small, randomized, crossover, controlled trials on the antioxidant effect of phenolic compounds from real-life daily doses of olive oil in humans have yielded conflicting results. Little information is available on the effect of the phenolic compounds of olive oil on plasma lipid levels. No international study with a large sample size has been done. Objective: To evaluate whether the phenolic content of olive oil further benefits plasma lipid levels and lipid oxidative damage compared with monounsaturated acid content. Design: Randomized, crossover, controlled trial. Setting: 6 research centers from 5 European countries. Participants: 200 healthy male volunteers. Measurements: Glucose levels, plasma lipid levels, oxidative damage to lipid levels, and endogenous and exogenous antioxidants at baseline and before and after each intervention. Intervention: In a crossover study, participants were randomly assigned to 3 sequences of daily administration of 25 mL of 3 olive oils. Olive oils had low (2.7 mg/kg of olive oil), medium (164 mg/kg), or high (366 mg/kg) phenolic content but were otherwise similar. Intervention periods were 3 weeks preceded by 2-week washout periods. Results: A linear increase in high-density lipoprotein (HDL) cholesterol levels was observed for low-, medium-, and high-polyphenol olive oil: mean change, 0.025 mmol/L (95% Cl, 0.003 to 0.05 mmol/L), 0.032 mmol/L (Cl, 0.005 to 0.05 mmol/L), and 0.045 mmol/L (Cl, 0.02 to 0.06 mmol/L), respectively. Total cholesterol-HDL cholesterol ratio decreased linearly with the phenolic content of the olive oil. Triglyceride levels decreased by an average of 0.05 mmol/L for all olive oils. Oxidative stress markers decreased linearly with increasing phenolic content. Mean changes for oxidized low-density lipoprotein levels were 1.21 U/L (Cl, -0.8 to 3.6 U/L), -1.48 U/L (-3.6 to 0.6 U/L), and -3.21 U/L (-5.1 to -0.8 U/L) for the low-, medium-, and high-polyphenol olive oil, respectively. Limitations: The olive oil may have interacted with other dietary components, participants' dietary intake was self-reported, and the intervention periods were short. Conclusions: Olive oil is more than a monounsaturated fat. Its phenolic content can also provide benefits for plasma lipid levels and oxidative damage.
Background: Olive oil (OO) consumption is associated with cardiovascular disease prevention because of both its oleic acid and phenolic contents. The capacity of OO phenolics to protect against low-density lipoprotein (LDL) oxidation is the basis for a health claim by the European Food Safety Authority. Proteomic biomarkers enable an early, presymptomatic diagnosis of disease, which makes them important and effective, but understudied, tools for primary prevention. Objective: We evaluated the impact of supplementation with OO, either low or high in phenolics, on urinary proteomic biomarkers of coronary artery disease (CAD), chronic kidney disease (CKD), and diabetes. Design: Self-reported healthy participants (n = 69) were randomly allocated (stratified block random assignment) according to age and body mass index to supplementation with a daily 20-mL dose of OO either low or high in phenolics (18 compared with 286 mg caffeic acid equivalents per kg, respectively) for 6 wk. Urinary proteomic biomarkers were measured at baseline and 3 and 6 wk alongside blood lipids, the antioxidant capacity, and glycation markers. Results: The consumption of both OOs improved the proteomic CAD score at endpoint compared with baseline (mean improvement: -0.3 for low-phenolic OO and -0.2 for high-phenolic OO; P < 0.01) but not CKD or diabetes proteomic biomarkers. However, there was no difference between groups for changes in proteomic biomarkers or any secondary outcomes including plasma triacylglycerols, oxidized LDL, and LDL cholesterol. Conclusion: In comparison with low-phenolic OO, supplementation for 6 wk with high-phenolic OO does not lead to an improvement in cardiovascular health markers in a healthy cohort.
The Mediterranean diet (MD) is considered a dietary pattern with beneficial effects on human health. The aim of this study was to assess the effect of an MD on urinary metabolome by comparing subjects at 1 and 3 years of follow-up, after an MD supplemented with either extra-virgin olive oil (MD+EVOO) or nuts (MD+Nuts), to those on advice to follow a control low-fat diet (LFD). Ninety-eight non-diabetic volunteers were evaluated, using metabolomic approaches, corresponding to MD+EVOO (n=41), MD+Nuts (n=27) or LFD (n=30) groups. The 1H-NMR urinary profiles were examined at baseline, after 1 and 3 years of follow-up. Multivariate data analysis (OSC-PLS-DA and HCA) methods were used to identify the potential biomarker discriminating groups, exhibiting a urinary metabolome separation between MD groups against baseline and LFD. Results revealed that the most prominent hallmarks concerning MD groups were related to the metabolism of carbohydrates (3-hydroxybutyrate, citrate, cis-aconitate), creatine, creatinine, amino acids (proline, N-acetylglutamine, glycine, branched-chain amino acids and derived metabolites), lipids(oleic and suberic acids), and microbial co-metabolites (phenylacetylglutamine, p-cresol). Otherwise, hippurate, trimethylamine-N-oxide, histidine and derivates (methylhistidines, carnosine, anserine) and xanthosine were predominant after LFD. The application of NMR-based metabolomics enabled the classification of individuals regarding their dietary pattern and highlights the potential of this approach for evaluating changes in the urinary metabolome at different time points of follow-up in response to specific dietary interventions.
The consumption of extra virgin olive oil (EVOO) in Mediterranean countries has shown beneficial effects. A wide range of evidence indicates that phenolic compounds present in EVOO are endowed with anti-inflammatory properties. In this work, we evaluated the effects of EVOO-polyphenol extract (PE) in a model of rheumatoid arthritis, the collagen-induced arthritis model in mice. On day 0, DBA-1/J mice were immunized with bovine type II collagen. On day 21, mice received a booster injection. PE (100 and 200 mg/kg) was orally administered once a day from days 29 to 41 to arthritic mice. We have demonstrated that PE decreases joint edema, cell migration, cartilage degradation and bone erosion. PE significantly reduced the levels of proinflammatory cytokines and prostaglandin E2 in the joint as well as the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1. Our data indicate that PE inhibits c-Jun N-terminal kinase, p38 and signal transducer and activator of transcription-3. In addition, PE decreases nuclear factor κB translocation leading to the down-regulation of the arthritic process. These results support the interest of natural diet components in the development of therapeutic products for arthritic conditions.