Protective effects of oleic acid and polyphenols in extra virgin olive oil on cardiovascular diseases

Yan Lu,Jun Zhao,Qiqi Xin,Rong Yuan,Yu Miao,Manli Yang,Hui Mo,Kji Chn,,Wihong Cong,

a Laboratory of Cardiovascular Diseases, Xiyuan Hospital of China Academy of Chinese Medical Sciences, Beijing 100091, China

b National Clinical Research Center for Chinese Medicine Cardiology, Beijing 100091, China

c Traditional Chinese Medicine Department, The Aff iliated Hospital of Qingdao University, Qingdao 266000, China

d Nanjing University of Chinese Medicine, Nanjing 210023, China

e Health Bureau of the Government of the Macao Special Administrative Region, Macao 999078, China

Keywords: Cardiovascular diseases Extra virgin olive oil Oleic acid Polyphenols

ABSTRACT The Mediterranean diet has long been recognized as one of the most effective ways to prevent and improve cardiovascular disease.Extra virgin olive oil (EVOO) is the typical sources of fat in the Mediterranean diet which have been shown to have noteworthy nutritional value and positive impact on human health.I t is worth noting that EVOO owes its superior nutritional value to its bioactive composition.The main component of EVOO is monounsaturated fatty acids (MUFAs) in the form of oleic acid.Oleic acid accounts for up to 70%-80% of EVOO.Secondly,EVOO contains approximately more than 30 phenolic compounds,of which HT is essential for the protection against cardiovascular diseases.In this review,we focused on the potential mechanisms of oleic acid and polyphenols combat cardiovascular diseases risk in terms of oxidative stress,inf lammation,blood pressure,endothelial function and cholesterol.This review might provide a reference for the studies on cardiovascular protective effects of EVOO.

1.Introduction

Cardiovascular diseases (CVDs),which includes heart disease,arrhythmias,coronary artery disease and various types of vascular lesions,is one of the leading causes of death worldwide for decades[1-2].The etiology of CVDs is divided into genetic and acquired causes.Acquired factors mainly include diet,atherosclerosis,hypertension,diabetes,hyperlipidemia,smoking,etc.[3].It is well known that a healthy diet is effective in suppressing risk factors such as CVDs,diabetes and metabolic syndrome,while a faulty lifestyle may increase mortality from CVDs by up to 40%[4].As a result,different dietary strategy,such as the Mediterranean diet (MD),the vegetarian diet,the Stop Hypertension diet,the ketogenic diet,have been proposed to address these issues[5].The MD advocates a high consumption of plant foods (vegetables,fruits,nuts,grains,and legumes),fat supplementation through the intake of olive oil (OO),regular intake of f ish and dairy products and small amounts of red meat and wine[6].The MD was first proposed by Ancel Keys in the 1950s after studied the diets and health conditions of seven countries[7].It was recommended to be adopted in 2015-2020 Dietary Guidelines for Americans as one of the important measures against CVDs[8-9].One of the core features of the MD that differs from other dietary patterns is its special source of fat — OO.

A 24-year follow-up study found a kind of negative association between OO consumption and CVDs prevalence.People who consumed high levels of OO (＞ 7 g/day) had a 14% reduction in the CVDs prevalence and an 18% reduction in the coronary heart disease prevalence compared to those who did not consume OO[10].A 10-year follow-up of adults without CVDs found that participants who never or intermittently consumed OO were 4.2 and 5.3 times more likely to develop CVDs than those who consumed only OO[11].In 2004,the US Federal Drug Administration authorized the possibility of labeling OO with the following statement: “Due to the MUFAs contained in OO,consumption of approximately 23 g of OO per day is beneficial for coronary heart disease”[12].All of these findings highlighted the importance of OO for CVDs.OO is divided into regular OO,virgin OO and extra virgin OO (EVOO) according to its quality.The advantage of OO is that it is rich in MUFAs and polyphenols,but this is limited to virgin OO and EVOO rather than regular OO[13].In the EVOO extraction process,malaxation has a significant impact on the quality of the product,particularly with regard to the concentration of phenolic compounds and tocopherols.This step involves the continuous kneading of the olive paste under controlled temperature and time conditions,which significantly reduces the viscosity of the olive paste,thereby facilitating subsequent separation in the decanter[14].

Table 1 Major EVOO components[15-16].

Special attention is paid to the compounds in EVOO (Table 1)[15-16].The high nutritional value of EVOO consists largely in its rich and complex chemical profile containing over 200 compounds.In particular,MUFAs in the form of oleic acid (OA) are the primary components of EVOO accounting for up to 70%-80% in EVOO[17].Other minor yet nutritious components,such as phenolic compounds,squalene and tocopherols,comprise approximately 1%-2% of EVOO content[18].Although these minor components (such as chlorophyll and carotenoids) also contribute to the beneficial effects of EVOO,the major pharmacological effects of EVOO are mainly attributed to its main components,OA and polyphenols[11,19].

Large body of evidence have demonstrated that the consumption of EVOO has cardiovascular benefits with wide range of mechanisms of actions beyond reduction of cholesterol.Understanding these mechanisms are important as it could help to implement appropriate interventions in conjunction with clinical treatment.In this review,we focused on reviewing the molecular mechanisms behind the prevention of CVDs by OA and polyphenols,the main components of EVOO,which may provide the rationale for EVOO being healthier than other types of cooking oil.

2.Mechanisms underlying the protective effects of OA on CVDs

High concentration of OA in EVOO possesses significant biological actions and is regarded as the major contributor in reducing cardiovascular risks in populations that consume MD.To clarify whether the main biological effect of EVOO is due to OA or other components,researchers fed rats with 20% EVOO,sunflower oil with high OA content or safflower oil.By comparing the proliferation of spleen lymphocytes,they concluded that the biological effect of EVOO may be mainly due to OA and less related to other components[20-21].

2.1 Lowering blood lipid level

As a MUFAs,OA accounts for about 70%-80% of EVOO[22].According to an animal study,5% dietary OA significantly reduced plasma total cholesterol (TC),very low-density lipoprotein and low-density lipoprotein (LDL) by inhibiting hepatic LDL receptor activity,plasma cholesterolestertransferprotein (CETP) activity and hepatic particulate HMG-CoA reductase activity,and therefore had anti-atherogenic properties[23].A systematic review and metaanalysis suggested that OA substitution fatty acids decreased triglyceride concentrations and increased high-density lipoprotein (HDL) levels[24].In an animal study demonstrated that an OA-rich diet reduced plasma LDL and triglyceride concentrations,and to some extent,increased HDL[25].At the same time,polyunsaturated fatty acids(PUFAs) were not found in the adipose tissue,suggesting that OA was fully utilized during blood transport and only a few of OA was utilized during adipose tissue synthesis,thus effectively lowering lipid level and incidence of CVDs[25].In a multicenter,randomized,double-blind trial,males and females at a mean age of 44 were enrolled and their fasting lipids were assessed.The results showed that a high OA oil diet (19.1% MUFAs,7% PUFAs,and 6.4%saturated fatty acids) reduced endpoint TC,LDL,apolipoprotein B(ApoB) and non-HDL cholesterol.In addition,the ratios of TC:HDL and ApoB:ApoA1 were reduced after the high OA oil diet compared to the control diet (10.5% MUFAs,10% PUFAs,and 12.3% saturated fatty acids).These data suggest that high OA oils reduce PUFAs at the expense of increasing MUFAs,resulting in an improvement in atherosclerotic lipid distribution[26].

2.2 Inhibiting postprandial reflex effects

Blood pressure is closely associated with the risk of CVDs.Elevated postprandial blood triglycerides lead to decreased arterial compliance and increased blood pressure.In addition,postprandial hypertriglyceridemia is considered to be a very important cardiovascular risk factor[27-28].Therefore,the capability of OA improving the postprandial reflex effect may be one of its major cardiovascular protective mechanisms.The hypotensive effect of EVOO is mainly attributed to OA,which may be related to the effect of lipids on cell membrane structure and physiology[29].Docking of G-protein with signaling proteins plays an important role in blood pressure control and has been implicated in the regulation of hypertension on multiple occasions.Hence,alteration in the structural property of the plasma membrane could affect blood pressure regulation.A previous clinical study showed that membrane lipid and G-protein levels were altered in hypertensive patients and the membrane concentrations of G-proteins were reduced after longterm consumption of OO.Subsequently,similar results were found in an experiment exploring the effects of VOO,triolein and OA on blood pressure in SHR rats.The above results suggest that the hypotensive mechanism of OO is triggered by OA[17].OA regulates a variety of signaling pathways that are absorbed through the intestine and transferred to the vasculature.Upon entering the cell membrane,OA enhances the non-laminar phase tendency of the cell membrane and reduces phospholipid head group accumulation.It has been reported that Gγ subunits tend to be non-lamellar while Gα subunits tend to be lamellar,and which explains the decrease in Gα protein content in cell membranes after OA intake.Furthermore,OA also promotes the secretion of vasodilators and inhibits the release of vasoconstrictors[30].

2.3 Protecting vascular endothelial function

The vascular endothelium is an important regulator of vascular tone.Impaired endothelium implies an imbalance in vasoconstrictor and vasodilator function,which is highly associated with CVDs risk factors.The vascular endothelium plays a crucial role in the control of vascular tone.Damage to the vascular endothelium means an imbalance in vasoconstrictor and diastolic function,which is highly associated with CVDs risk factors.During lipoprotein catabolism,fatty acids are deposited on the vascular endothelium[31-32].A highfat diet transiently causes vascular endothelial dysfunction,which is closely related to postprandial oxidative stress.It is well known that the atherosclerotic process is driven by a broad inflammatory response involving various inflammatory markers and mediators,such as intercellular cell adhesion molecule-1 and C-reactive protein[33].According to the results of a clinical randomized crossover trial,consuming MUFAs (e.g.OA) for more than 4 weeks was effective in avoiding postprandial vascular endothelial dysfunction caused by fat deposition and oxidative stress.Compared to diets rich in saturated fatty acids and alpha-linolenic acid,a higher bioavailability of nitrogen oxide (NOX) after consumption of MUFAs may imply a positive effect on lipids and nitric oxide synthase in the vascular endothelium and a stronger endothelium-mediated vasodilatory response.In addition,it has been proved that the OA-rich diet may alleviate oxidative stress by inhibiting the nuclear factor kappa-B(NF-κB) signaling pathway,which in turn suppresses the postprandial inflammatory response[34].OA had also been shown to inhibit LPS-induced inflammatory mediators expression and formation in murine macrophages[35].Therefore,long-term consumption of foods rich in OA,such as EVOO,could protect against postprandial vascular endothelial damage and enhance vasodilatory capacity,effectively impeding the development of CVDs such as atherosclerosis or hypertension.

2.4 Alleviating insulin resistance and atherosclerosis

Most metabolic responses to insulin depend on the activation of the insulin receptor substrate-1 (IRS1)/phosphatidylinositide 3-kinases(PI3K) pathway.A human study analyzed the effects of different fatty acids on visceral adipose tissue in non-obese and morbidly obese patients.The results showed that OA could alleviate insulin resistance by inhibiting IRS1 serine phosphorylation,inducing insulin receptor substrate-1 tyrosine phosphorylation,and activating the PI3K signaling pathway.Subsequently,this effect of OA enhanced insulin sensitivity[36].OA also reduces insulin resistance in vascular smooth muscle cells (VSMCs) by modulating NF-κB.Saturated free fatty acids induce inflammatory responses and insulin resistance,including activation of NF-κB.NF-κB not only plays a central role in insulin resistant,but also is a major signaling molecule for the development and progression of atherosclerosis.Once activated,NF-κB induces the expression of a large number of inflammatory factors,such as IL-6 and tumor necrosis factor-α (TNF-α),and promotes the expression of various genes involved in plaque instability,such as adhesion molecules,endothelial nitric oxide synthase (eNOS),monocyte chemotactic protein-1 (MCP-1),and plasminogen activator inhibitor-1(PAI-1)[37-39].VSMC apoptosis is one of the key mechanisms of atherosclerotic plaque instability or even rupture,which also accelerates thrombosis[40-41].In vitroexperiments showed that OA inhibits the proliferation of VSMCs induced by angiotensin II,palmitate or TNF-α,thereby inhibiting atherosclerotic plaque growth.Furthermore,In addition,OA attenuated TNF-α-induced PAI-1 expression in VSMCs by regulating NF-κB,so as to slow down thrombosis and fibrinolysis[42-43].

In brief,sufficient evidence proves that OA has beneficial effects on CVDs by altering the composition and fluidity of cell membranes,regulating lipid and lipoprotein concentrations,and stabilizing postprandial blood glucose and blood pressure levels.OA also improves endothelial dysfunction caused by postprandial oxidative stress and inflammatory responses,alleviates cardiovascular insulin resistance and reduces the proliferation and apoptosis of VSMCs,which ultimately stabilizes atherosclerotic arterial plaques and delays the pathological process of atherosclerosis (Fig.1)[44-48].

Fig.1 Mechanisms underlying the protective effects of OA on CVDs.CM: chylomicron.FA: free fatty acid.VLDL: very LDL.LDLR: LDL receptors.IDL: intermediate density lipoprotein.oxLDL: oxidation of LDL.HL: hepatiltriglyceridase.VSM: vascular smooth muscles.PLCβ: phospholipase Cβ.ER: endoplasmic reticulum.IP3: inositol triphosphate.CaM: calmodulin.VCAM-1: vascular cell adhesion molecule1.ILs: interleukins.AngII: angiotensin II.ICAM-1: intercellular cell adhesion molecule-1.FC: free cholesterol.ABCG1: ATP binding cassette subfamily G member 1.IR: insulin receptors.IRS: insulin receptorsubstrate.EC: endothelial cells.Red: inhibitory effect of OA.Green: activation effect of OA.

3.Mechanisms underlying the protective effects of polyphenols on CVDs

Previously,the health benefits of EVOO were always linked to the action of OA.In recent years,phenolic compounds have received more attention.Present in the hydrophilic part of EVOO,polyphenols are compounds containing multiple phenolic hydroxyl structures with one or more aromatic rings.Polyphenols are often present in free,bound or esterified form and belong to one of the largest and most ubiquitous classes of secondary metabolites in the plant kingdom[49-50].The concentration of polyphenols in EVOO fluctuates in the range of 50-800 mg/kg,depending mainly on climatic altitude factors,extraction techniques,storage time and conditions.Polyphenols represent about 3% of the total OO[51].Polyphenols have excellent antioxidant properties,which help prevent the autoxidation of EVOO and extend its shelf life[52-53].More than 30 phenolic compounds were detected in EVOO,including secoiridoids (e.g.oleuropein),phenolic alcohols (e.g.hydroxytyrosol and tyrosol),phenolic acids(e.g.ferulic and vanillic acid),flavones and lignans (Table 2)[54].Of these,hydroxytyrosol (HT) was reported to be the main biologically active component of the phenolic compounds.Many of these phenolic compounds could easily enter the bloodstream and produce therapeutic effects.After the consumption of EVOO,some of the phenolic compounds are hydrolyzed in the gastrointestinal tract,resulting in substantial amounts of free HT and tyrosol.In contrast,glycosylated secoiridoids (e.g.,oleuropein),which are not susceptible to the gastric environment,are not hydrolyzed.These unmodified molecules are accompanied by large amounts of HT and tyrosol reaching the intestine[52].Passive bidirectional transport occurring in human enterocytes membranes is now a widely acknowledged mechanism of absorption[55].All phenolic molecules that are not absorbed by the small intestine enter the colon and are degraded by intestinal microorganisms into a wide range of metabolites that are eventually absorbed or excreted.In addition,unmodified metabolites reach the large intestine and exert prebiotic-like effects to promote intestinal homeostasis,influence microbiota composition and inhibit the growth of harmful bacteria[55-57].After polyphenols undergo intense phase I and phase II metabolism,where they are hydrolysed and conjugated leading to diverse glucuronidated,methylated and sulphated forms.About 98% of HT is present in plasma and urine as glucuronide conjugated forms.The levels of HT and tyrosol increase rapidly achieving a peak concentration at approximately 1 h in human plasma and around 2 h in urine.Phenolic compounds and their metabolites were found in almost every part of the body and can also penetrate the blood-brain barrier,accumulating mainly in the brain,liver and kidney,while are detoxified by the kidney[58].

Table 2 Phenolic compound contents (mg/kg) of EVOO[54].

3.1 Inhibiting oxidative stress

The health benefits of polyphenols are strongly linked to their inhibitory effect on oxidative reaction.Dysfunction of the cardiovascular system leads to thrombosis,inflammatory cell infiltration,fatty deposits and hemodynamic instability.Oxidative stress has been regarded as a common cause for these problems,directly leading to the disruption of cardiovascular homeostasis[59].Oxidative stress is triggered when there is a disturbance in the balance between intracellular levels of reactive oxygen species (ROS) and antioxidants[60-61].Therefore,a very important means against CVDs is to inhibit ROS production and improve oxidative defenses.Zrelli et al.concluded that the antioxidant effect of HT was determined by H2O2-induced ROS production in vascular endothelial cells.Their results indicated that HT (10-50 µmol/L) prevented the increase in ROS production[62].In addition to the ability of directly scavenging ROS,heart benefits of HT were discovered to be mostly owing to its ability to decrease ROS production through modulating the oxidative stress-related pathways.Another key factor in blocking oxidative stress is the forkhead transcription factor (FOXO),which contains FOXO1,FOXO3a or FOXO3,FOXO4 and FOXO6.Adenosine-5’-monophosphate-activated protein kinase (AMPK) has been shown to directly activate FOXO3 at six sitesin vitro(Thr179,Ser399,Ser413,Ser355,Ser588,Ser626and Ser413) and at least two sites in cells (Ser626and Ser413) to promote cellular stress resistance-related gene expression[63].FOXO3a acts directly on the expression of antioxidant enzymes such as manganese superoxide dismutase,catalase,and thioredoxin,protecting cells from oxidative stress.These researchers demonstrated by the same experimental procedure as above that HT promotes a range of antioxidant genes,such as thioredoxin reductase and catalase,by triggering AMPK phosphorylation and activating the expression of FOXO3 in the nucleus[62].When AMPK is completely silenced,HT fails to activate the expression of catalase[63-65].In addition,HT (10-100 µmol/L) acting on endothelial cells and human hepatocytes (HepG2) promotes nuclear factor erythroid 2 (Nrf2)nuclear translocation through activating the PI3K/protein kinase B(AKT) pathway and extracellular regulated protein kinases 1/2 (ERK1/2)pathway,followed by activation of heme oxygenase-1 (HO-1),which inhibits oxidative stress,enhances vascular endothelial cell proliferation and promotes repair function[66].HT also inhibits the cytotoxicity produced by H2O2and enhances the antioxidant capacity of vascular endothelial cells[67-68].

3.2 Inhibiting inflammatory response

Oxidative process is often intertwined with inflammatory responses and inflammation-related pathways which contain the main targets of polyphenols[69].HT has been proved to produce the anti-inflammatory effects via the regulation of NF-κB pathway.For instant,HT effectively cuts off the activation of transcription factors such as NF-κB,I-κBα,and STAT-1α,which are key factors in regulating the inflammatory response[70-71].In order to provide a clear interpretation of the role and molecular mechanisms played by specific components of OO,Mario et al.[72]investigated the corresponding biological effects of OO phenolic extracts on human monocytes (THP-1).This experiment reported that polyphenols inhibited NF-κB activation by blocking NF-κB p65 nuclear translocation and DNA binding,and reduced oxidative stress-related genes (e.g.COX-2 and MMP9) and NF-κB mediated inflammatory factors (e.g.interleukin-1,intercellular cell adhesion molecule-1,TNF-α,MCP-1,etc.).This experimental result suggested that polyphenol extracts (7.5 μg/mL) had attenuated monocyte inflammatory potential.At the same time,the team further analyzed the functions of the components of polyphenols in their study and indicated that oleuropein (10-50 µmol/L) was one of the main substances that exerted a regulatory effect on MMP9,whereas tyrosol,HT and homovanillyl alcohol had no effect on MMP9[72].Prostaglandin E2 (PGE2) is involved in various pathogenic mechanisms,such as inflammation,increased vascular tone and platelet aggregation,all of which contribute to atherosclerotic plaque instability[73].The PGE2-cAMP signaling pathway activates matrix metalloproteinase 2 (MMP2) and MMP9.At the same time,MMP9 activation is also associated with the cyclooxygenase 2(COX-2)/PGE2 pathway[74].HT can significantly inhibit the increase of ROS production and activation of COX-2 induced by LPS stimulation.To investigate the molecular mechanisms of polyphenols on endothelial angiogenesis,human umbilical vein endothelial cells (HUVEC) were intervened by HT.The results showed that 10 µmol/L HT reduced COX-2 expression and MMP9 activity,and that this inhibition occurred at pre-translational levels.The multiple inhibitory effects of HT on MMP9,COX-2 and on other pro-inflammatory factors were mediated by interfering with the early steps of the signaling cascade,including inhibition of classical PKC activation.This study demonstrated for the first time that OO polyphenols inhibited PKC in human inflammation,which helps explain the attenuating effect of OO on redox-sensitive NF-κB-mediated activation of vascular inflammation.In peripheral monocytes,HT inhibited the expression of PGE2 and COX-2 and reduced the concentration of MMP9,but had no effect on COX-1[75].By blocking multiple signaling pathways,HT acts on numerous pro-inflammatory factors,COX-2 and MMP9,which are associated with oxidative stress,vascular permeability and tone,angiogenesis,foam cell formation and leukocyte adhesion,the key risk factors for CVDs[76].In addition,the plasma concentrations of inflammatory markers were measured in 66 participants at baseline,at 3 and 5 years later,respectively.The results demonstrated that EVOO reduced inflammatory molecules levels such as IL-5,IL-6,IL-7,IL-8,MCP-1,TNF-α,interferon γ (IFN-γ) and increased atherosclerotic stability[77].A study of 20 patients with metabolic syndrome showed that acute intake of VOO reduced the postprandial inflammatory response and the expression of proinflammatory genes,mainly by reducing the activation of NF-kB,activator protein-1 transcription factor complex AP-1,cytokines,mitogen-activated protein kinases (MAPKs),or the arachidonic acid pathway,secondary to a reduction in intestinal absorption of LPS after a high-fat meal[78].

3.3 Regulating LDL level

The well-known cholesterol theory suggests that cholesterol be the key to the formation of atherosclerosis.There are two types of lipoproteins that carry cholesterol throughout the body.The low-density lipoprotein is often referred as the ‘bad’ cholesterol which has been regarded as the primary cause of CVDs.LDL is the key to the formation of atherosclerosis,and its levels are positively correlated with CVDs’ occurrence.Once LDL is oxidized,it can be harmful to human health[79-81].Steinberg et al.[82]stated in 1989 that LDL could be prevented from going ‘bad’ if antioxidants were given appropriately to suppress oxidative stress.When LDL is oxidatively modified,it is phagocytosed by macrophages,thus evolving into cholesterol-rich lipid foam cells.Endothelial cells mediate oxidized LDL (oxLDL) via oxidized LDL receptor-1,which reduces NO generation and overexpresses adhesion molecules,accelerating the oxidative stress response in endothelial cells.Hence,foam cell formation and endothelial cell dysfunction could promote the formation of atherosclerotic plaques synergistically[83-85].Increased lipoprteinlipase (LPL) contributes to the hydrolysis of TG-rich lipoproteins such as LDL,as LPL is the main enzyme involved in the removal of TGs from the blood and has a similar role to the LDL receptors.Improved oxidative status is mostly associated with high LPL activity[86].LDL is more atherogenic when they are small and dense,because they cannot be accurately identified by LPL,can crosses the endothelial barrier and are oxidized more easily[87].Small LDL is therefore more closely associated with CVDs risk factors.Under this background,278 dyslipidemia patients were enrolled and were investigated for the effect of EVOO polyphenols on LDL concentrations in a clinical study.The results found that small LDL particles were depleted by 15.3% after polyphenol consumption.As reported,the improvement of oxidative status by OO polyphenols was associated with a decrease in small LDL particles to inhibit the increase in LDL concentration.Polyphenols also prevented LDL from causing atherosclerotic plaques,which was related to the enhanced antioxidant capacity of LDL and the reduction of small LDL particles[77].An improved oxidative status and an increased LPL expression may help explain these changes.

3.4 Regulating HDL level

HDL is the second major type of cholesterol in the body and commonly refereed as the ‘good’ cholesterol.A dose-dependent relationship was found between the increased HDL level and the EVOO polyphenol concentration[88].HDL transports cholesterol from macrophages to the liver and ultimately out of the body,known as“cholesterol reversal transport”[89-90].Another important function of HDL in the pathological process of CVDs is endothelial protection.For instance,HDL inhibits the activity of cell surface adhesion factors,thus preventing monocytes from adhering to endothelial cells,which means that HDL blocks the early process of atherosclerotic plaque formation.The “Virgin Olive Oil and HDL Functionality”(VOHF) study was reported to be the first study to evaluate the effect of polyphenols on HDL,a project that included randomized controlled trials,double-blind,crossover designs,etc.Pedret team integrated the main published results of the VOHF study from 1997 to 2017 and stated that the intake of high polyphenol VOO altered HDL subclass distribution toward larger and more mature HDL particles[88].VOO polyphenols increased HDL size and large HDL (l-HDL)quantity,and also decreased small HDL (s-HDL) number[91].Cholesterol efflux capacity is inversely associated with s-HDL particle levels and was directly associated with l-HDL,medium HDL,and HDL size[92].Similarly,the EUROLIVE and the PREDIMED studies indicated that the use of polyphenol-rich VOO contributed to the formation of larger HDL particles.These changes in HDL size and distribution were accompanied by increases cholesterol efflux capacity,lecithin-cholesterolacyltransferase (LCAT),and CETP activities,antioxidant,anti-inflammatory capacities,and vasodilatory capacity[93].The paraoxonase (PON) family,platelet-activating factor acetylhydrolase (PAF-AH) and glutathione selenoperoxidase-3(GSPx-3) are the main antioxidant enzymes in HDL.Farràs team[93]gave high polyphenol OO to 33 hypercholesterolemic patients for 3 weeks and observed that high polyphenol OO reduced PON1 expression,enhanced PON3 activity and PON1-related activity(paraoxonase and lactonase).High PON1 protein levels and low associated activities are detrimental to CVDs;conversely,increased PON3 protein levels are beneficial to CVDs[94].In recent years,PON1 activity has been proposed as a nascent marker of HDL function and risk of CVDs.It is thus clear that polyphenols are beneficial for the regulation of the PON family,which is important for HDL function enhancement and oxidative homeostasis[95].Furthermore,it was observed in the VOHF study that increased antioxidant content in HDL could provide a better antioxidant environment for apolipoprotein (ApoA-I).ApoA-I activates ATP-binding cassette subfamily A member 1 (ABCA1),a key protein that initiates HDL production and promotes cholesterol reversal,and enhances the antiatherogenic capacity of HDL.Based on this evidence,Pedret team reveal a close inter-relationship between HDL oxidation status,ApoA-I and HDL monolayer mobility,which are the three main factors associated with cholesterol efflux capacity in the VOHF study[88,96].

3.5 Protecting vascular endothelial function

The endothelium is regarded to be a layer of mononuclear cells that attaches to the inner wall of blood vessels and separates them from the blood and vascular tissue.However,this theory has evolved considerably through continuous research and it is now considered as a very complex autocrine and paracrine organ that maintains vascular tone through the interaction of endothelial cells with vascular smooth muscle[97-98].Endothelial dysfunction results in an inflammatory response,vasodilation and vasoconstriction dysfunction,altered permeability,thrombosis,vascular sclerosis,and platelet aggregation,all of which are the key elements in the formation of vascular diseases and contribute to the development of atherosclerotic plaques and the emergence of complications[99-101].Nitric oxide (NO) is crucial to the maintenance of endothelial function.Storniolo et al.[102]demonstrated for the first time that HT (10 µmol/L) may have a regulatory effect on NO/endothelin-1 (ET-1) levels in endothelial cells through anin vitrosimulated diabetes model (high glucose and elevated free fatty acids).Polyphenols achieve enhanced NO formation is due to the redox-sensitive activation of the PI3K/AKT pathway,which lead to eNOS activation subsequent to its phosphorylation on Ser1177.The same team indicated that HT might stimulate eNOS phosphorylation at the Ser1177site via p38,ERK,and AKT.In addition,they observed that high glucose and free fat acid reduced acetylcholine-induced NO bioavailability,which was significantly inhibited by HT.Polyphenols may regulate NO generation by modulating Ser1177eNOS phosphorylation and Ca2+.The above results suggest that for pathological events of endothelial dysfunction,high polyphenol EVOO may be of greater benefit than high OA seed oil without polyphenols.HT (50 µmol/L) might mediate NO production via p-eNOS in vascular endothelial cells,which was associated with HT’s inhibition to TNF-α-induced alterations in NF-κB and NF-κB inhibitor (IκB) phosphorylation[103].In addition to the effects on eNOS,HT (25-100 µmol/L) reduces inducible nitric oxide synthase (iNOS) expression in monocytes and is therefore known for its anti-inflammatory and antioxidant properties in monocytes[103].HT (10 µmol/L) had also been shown to potentially proximally activate the target,cyclic guanosine monophosphate(cGMP),resulting in an endothelium-dependent vasodilatory effect[76].

3.6 Relieving hypertension

Hypertension is one of the major risk factors for myocardial infarction and stroke,and its pathology is closely related to oxidation and inflammation[104].Polyphenols are regarded to activate nitric oxide synthase and enhance endothelial functionality[105].Moreno-Luna et al.[106]investigated the ability of EVOO polyphenols to lower blood pressure and improve endothelial function in a singleblind,randomized,crossover setting in 24 hypertensive patients.They found that polyphenols have a significant hypotensive effect by decreasing serum ADMA concentration and further increasing NO utilization.In line with that,phenolic compounds have shown to induce vasodilatation in response to transient postprandial ischemia.Many studies have pointed out that the antihypertensive mechanisms of polyphenols were strongly linked to the antioxidant,anti-inflammatory,and improved vascular endothelial functions.Additionally,Moreno-Luna et al.[106]discovered that EVOO polyphenols were more effective in severe hypertension.Therefore,EVOO polyphenols might restore the endothelial function to produce antihypertensive effect,which is more prominent in the presence of endothelial damage and hypertension[106-108].Some people claimed that diastolic and systolic blood pressures significantly decreased after the consumption of oleuropein[109].As we know,oleuropein is hydrolyzed into HT when entering the gastrointestinal tract.Therefore,it is debatable whether the antihypertensive effect comes from oleuropein or HT.Besides,HT has shown in other studies to elevate plasma nitrate and nitrite levels,which are key donors for NO formation.In addition,HT shows to be more effective than OA on inhibiting the expression of endothelin-1,a powerful contractile factor linked to elevated blood pressure.In conclusion,polyphenols’ effect on lowering blood pressure might mostly be attributed to HT[76,102].

Polyphenols present a significant protective effect on the hearts of rats with isoproterenol-induced myocardial infarction by preventing pathological ST-segment elevation,inhibiting markers of myocardial injury,and increasing acetylcholinesterase levels,improving hemodynamic levels,reducing myocardial hypertrophy and delaying cardiac remodeling[63,110].In healthy individuals,postprandial arterial stiffness is improved,and pro-atherogenic genes and inflammatory factors are downregulated in the presence of EVOO polyphenols (Fig.2)[111].

Fig.2 Mechanisms underlying the protective effects of polyphenols on CVDs.AC: adenylyl cyclone.PKC: protein kinase C.CAT: catalase.TrxR: thioredoxin reductase.LOX-1: oxidized LDL receptor 1.CAM: cell adhesion molecules.PGI2: prostacyclin.CaMK II: calcium/calmodulin-dependent protein kinase II.Red: inhibitory effect of polyphenols.Green: activation effect of polyphenols.

4.Contribution of EVOO on CVDs by intestinal microbiota regulating

The gut microbiota has drawn a great deal of attention and medical professionals are very interested in how it relates to CVDs.Under normal conditions,a complex and sophisticated ecological network is formed between these normal non-pathogenic bacteria and their human hosts,maintaining a positive symbiotic connection and significantly enhancing human health[112].The digestive system might be viewed as a large ecosystem containing numerous intestinal microbiota.The gastrointestinal tract of a healthy person is occupied by more than 500 microbial species.These microbiota and intestinal cells complement each other to regulate barrier function and stimulate the immune system to protect the body from external damages,which also have a regulatory impact on the absorption and utilization of drugs.Among the functions of the intestinal microflora,the most important one is to help the food be well decomposed and digested and the resulting metabolites be absorbed into the body to play biological roles[113].Metabolites produced by the gut microbiota enter the body’s circulation,affecting host mediators and altering the cardiovascular phenotype[114].The intestinal microbiota is complex,abundant and widely distributed,with complex ecological relationships and close metabolic communication with the host.Studies on animal and human feces have revealed that variations in dietary patterns cause changes in the composition of the intestinal flora and its metabolites.It has been proposed that dietary variations may cause nearly 60% changes in the structure of the gut flora[115].

Complex phenols are hydrolyzed in the acidic environment of the stomach to form simple phenols (e.g.,tyrosol,HT,etc.).Although most simple phenols are rapidly absorbed in the small intestine,some simple and complex phenols still reach the large intestine and are metabolized by the intestinal microbiota.A wholesale fermentation study of the human colon microbiota revealed that oleuropein was catabolized into HT,followed by oxidation into 2-(3’,4’-dihydroxyphenyl) acetic acid and eventual dehydrogenation to 2-(4’-hydroxyphenyl) acetic acid and phenylacetic acid.Tyrosol was oxidized to 2-(4’-hydroxyphenyl) acetic acid[116].Probiotics,represented byBifidobacteriumandLactobacillus,take oleuropein as a carbon source in the proliferation process.Thus,oleuropein,acting as antioxidants,also have the prebiotic effect and are used as a carbon source by probiotics such asBifidobacteriumandLactobacillusduring the proliferation phase[55].Prebiotics are mainly fermented through glycolysis,and then change the intestinal flora components,which is beneficial to the growth of probiotics such asLactobacillusandBifidobacterium[117].Probiotics can also change the environment in the gut by affecting histone deacetylation,which in turn affects intestinal epithelial permeability.This has a significant impact on lowering TC and LDL while also reducing cardiovascular risk factors.Probiotics protect the intestinal barrier function,inhibit the production of pro-inflammatory factors (TNF-α,IL-6),promote mucin production,as well as lowering LDL and TC levels[118].There was evidence thatBifidobacteriumandLactobacillusare frequently used in the treatment of hypertension,hyperlipidemia and type 2 diabetes.In addition,probiotics may also be used to address cardiovascular risk factors.According to several reports that describe the antihypertensive properties of probiotics,some of their metabolites may also have comparable antihypertensive effects by inhibiting the angiotensin-converting enzyme[119].In brief,probiotics have been found to have antioxidant and anti-inflammatory activities as well as to preserve the ecological balance of gut flora,which reduces CVDs symptoms[117].Polyphenols and bacterial survival are tightly connected.Bacteroidesreduce LPS circulation,decrease intestinal permeability,inhibit endotoxemia and inflammatory response,and have protective effects against CVDs.It was demonstrated that consuming EVOO continuously for more than 12 weeks was beneficial to survival and increased the level of theBacteroides.Polyphenols can also inhibit the growth of pathogenic bacteria,such asEscherichia coli[120].A clinical trial showed that 3-week intake of polyphenolrich OO reduced LDL levels in patients with hypercholesterolemia and such cardioprotective effect could be mediated by an increase inBifidobacteriumand an increase in polyphenols metabolites with antioxidant activity,such as catechins and HT.This study is also the first to demonstrate the potential prebiotic activity of polyphenolrich OO[121].Gut flora is influenced by diets,lifestyles,and physical exercise,which in turn affect cardiovascular health.To treat CVDs,a specific microbial therapy may offer fresh promise (Fig.3).

Fig.3 Contribution of OO on CVDs by intestinal microbiota regulating.Ole: oleuropein.Tyr: tyrosol.Green arrows: promoting effect.Red arrows: inhibitory action.

5.Conclusions

Both pre-clinical and clinical studies have shown that each of these single components within EVOO could produce cardiovascular protective effects through multiple cellular mechanisms and signaling pathways.Although contradictory results are reported by some individual studies,such discrepancies could be due to a number of factors,such as the variation in the origin and quality of the EVOO adopted,the doses or routes of administration,and difference of the experimental models.Nevertheless,current evidence clearly highlighted EVOO could modulate several common cellular mechanisms and signaling pathways to produce its cardiovascular protective effects.In general,EVOO is considered as a functional food with CVDs benefits including antioxidant,anti-inflammatory,and in improvement in lipid levels,insulin sensitivity and endothelial function,as well as antithrombotic and anti-atherosclerotic.All these effects can be attributed to the bioactive components of OO,such as polyphenols and mon-and poly-unsaturated fatty acids (mainly OA).

With the increase of knowledge about EVOO promoting health,more works are needed to further define the biological mechanisms of action of EVOO,and to better characterized the biological activities by the diverse of phenolic compounds which might be potentially beneficial to the development of new drugs in the future.Carefully designed randomized controlled clinical trials are also required to provide more evidence for the value of health promotion of EVOO in CVDs.

Conflict of interest

Weihong Cong is an editorial board member forFood ScienceandHuman Wellnessand was not involved in the editorial review or the decision to publish this article.All authors declare that there are no competing interests.

Acknowledgements

This work was supported by the CACMS Innovation Fund(CI2021A00914);the Beijing Novaprogram (Z211100002121062);the Opening Project of the Key Laboratory of Integrative Chinese and Western Medicine for the Diagnosis and Treatment of Circulatory Diseases of Zhejiang Province (2C32001) and the National Natural Science Foundation of China (82004193).