Hydroxytyrosol an Examination of Its Potential Role in Cardiovascular Disease, Inflammation, and Longevity MATTHEW J

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Peer-reviewed scientific article Hydroxytyrosol An examination of its potential role in cardiovascular disease, inflammation, and longevity MATTHEW J. KILLEEN*, PAOLO PONTONIERE, ROBERTO CREA *Corresponding author CreAgri Inc., Hayward, California, USA

ABSTRACT: Multiple lines of converging evidence link polyphenols, particularly hydroxytyrosol, to improved clinical outcomes. In human and animal studies, hydroxytyrosol has been shown to induce beneficial effects on the cardiovascular system and exert anti-inflammatory effects. Hydroxytyrosol has also been shown to be a critical regulator of cell signalling pathways that have been implicated in longevity; in a series of experiments, hydroxytyrosol was shown to be superior to resveratrol in preventing cell death and increasing the expression of longevity proteins. In this review article we describe the experimental and clinical research that has been performed on hydroxytyrosol, and a novel hydroxytyrosol-based product, to highlight the therapeutic potential, across a broad range of disease states, of this polyphenol.

INTRODUCTION important impact on the prevention of cardiovascular disease and SCD. The Mediterranean diet has been associated with a lower The Eurolive trial was one of the first major, multicentre clinical incidence of morbidity, mortality, and improved clinical trials to provide strong evidence in support of the beneficial outcomes. A prospective study of over 22,000 people effects of olive oil polyphenols on blood cholesterol levels. In determined that strong adherence to the Mediterranean diet, this study 200 healthy male volunteers received 25 ml of olive which is rich in fruit, vegetables, and fish, was associated with oil containing either low, medium, or high concentrations of a reduced incidence of cardiovascular disease, cancer, and polyphenols once daily for three weeks. High polyphenol olive death (1). The purported clinical benefits of the oil resulted in significantly raised levels of HDL cholesterol (5).

Polyphenols Mediterranean diet have been attributed to the consumption HDL levels in study participants increased by 0.045 mmol/L of foods containing high levels of free-radical scavenging (P=0.046) (5); previously in a separate study, a 0.026 mmol/L polyphenols, particularly olives and olive oil. Of the olive increase in HDL was associated with a 2-3 percent decrease polyphenols that have been characterized, hydroxytyrosol in the risk of cardiovascular disease (30). A number of has emerged as the polyphenol with one of the greatest emerging pharmaceutical therapies, such as the potential therapeutic effects; it has the highest level of free cholesterylester transfer protein inhibitors anacetrapib and

vol 22 n 5 radical protection activity ever reported for any natural dalcetrapib, are in mid- to late-stage development for the - antioxidant compound and a variety of laboratory and treatment of dyslipidemia and the prevention of clinical studies have demonstrated its beneficial effects in a cardiovascular disease; the beneficial effects of these agents broad range of diseases. The effects of isolated polyphenols, is thought to be partly due to their ability to raise HDL levels. such as hydroxytyrosol, have been extensively documented, Therefore by similarly raising HDL, polyphenol-rich olive oil has by the Visioli laboratory (32, 34, 37, 38), in olive mill waste the potential to exert beneficial effects on the cardiovascular water (OMWW), which is one of the major by-products of olive system. oil production (35). Polyphenols have been shown to be The Eurolive clinical trial also demonstrated that high

September/October 2011 present at considerably higher concentrations in OMWW, polyphenol olive oil reduced the levels of oxidized LDL - which could act as an important source of bioactive cholesterol in healthy volunteers (5). Numerous studies have polyphenols for laboratory studies and clinical research (32). also associated oxidized LDL with the development of For example, an OMWW extract, containing hydroxytyrosol obstructive, atherosclerotic plaques within arteries (5-6). was shown to increase the plasma antioxidant activity of Oxidation of LDL results in a conformational change which industry hi-tech animals (34). In the following sections we discuss studies of permits its entrance into the arterial wall where it may hydroxytyrosol’s effects on cardiovascular disease, promote atherosclerotic processes and the subsequent OOD inflammatory disorders, and its potential role in regulating formation of plaques which impair the flow of blood to vital

gro F longevity. organs, including the heart. Such is the correlation between A oxidized LDL and atherosclerosis, measurements of LDL oxidation have been shown to be powerful predictors of THE POTENTIAL BENEFITS OF HYDROXYTYROSOL IN acute cardiovascular events in both coronary heart disease CARDIOVASCULAR DISEASE patients and in larger patient populations (7-8). Importantly virgin olive oil also contains a wide array of other Sudden cardiac death (SCD) is the leading cause of mortality minor components, in addition to polyphenols, such as α- and in the western world; in the United States, it has been γ-tocopherols, phytosterols, and flavonoids (32), all of which estimated to account 400,000 deaths per year (2-3) while in may have, in part, contributed to the effects observed in the the United Kingdom up to 70,000 deaths per years are Eurolive clinical study. attributed to SCD (4). SCD therefore represents a major public Hydroxytyrosol has been shown to play a role in health burden. Numerous human and animal studies have atherogenesis; however, conflicting findings have been shown that olive polyphenols, particularly hydroxytyrosol, can reported in different studies. For example, in one report improve blood cholesterol profiles and reduce the risk of administration of hydroxytyrosol to rabbits for one month potentially lethal thrombosis. These effects of hydroxytyrosol, resulted in a prominent reduction of the size of plaques which are described in the following sections, could have an formed in the aorta (9). Additionally, hydroxytyrosol resulted in

AgroFOOD industry hi-tech - September/October 2011 - vol 22 n 5 Polyphenols 17 , which is which , 2 , in patients with hyperlipidemia (11). In (11). hyperlipidemia with , in patients ® (a metabolite of thromboxane A thromboxane of (a metabolite 2 induce notable anti-inflammatory effects. In LPS- In effects. anti-inflammatory notable induce treatedmiceadministered the 35of mg hydroxytyrosol-based supplement or 12.5 mg of supplement acted synergistically with glucosamine toglucosamine with synergistically acted supplement glucosamine TNF-α levels were significantly reduced; determined that the OMWW hydroxytyrosol-based determined that the OMWW hydroxytyrosol-based recorded (P<0.05) (13). Further experiments also recorded (P<0.05) Further experiments also (13). recorded (P<0.05) (13). Further experiments also recorded (P<0.05) Further experiments also (13). setting with the OMWW hydroxytyrosol-based in vivo setting with the OMWW hydroxytyrosol-based in vivo supplement; significant reductions in TNF-α levels were supplement; significant reductions in TNF-α levels were supplement, LPS-treated animals were administered the administered the were animals supplement, LPS-treated supplement, LPS-treated animals were administered the administered the were animals supplement, LPS-treated To explore if anti-inflammatory effects could also be seen inseen be also could effects anti-inflammatory if explore To the the To explore if anti-inflammatory effects could also be seen inseen be also could effects anti-inflammatory if explore To responsible for platelet aggregation and the formation of of formation the and aggregation platelet for responsible occurred effects beneficial these Importantly, thromboses). within a very short time frame (4 days) (12). THE ANTI-INFLAMMATORY EFFECTS OF HYDROXYTYROSOL that shown have studies clinical and Laboratory experiments effects; anti-inflammatory exerts hydroxytyrosol supplement have performed using a hydroxytyrosol-based of inflammation and reported a reduction in biomarkers (13-14). In animproved quality of life in osteoarthritis patients lipopolysaccharideusing joint inflammation, of animal model and a (LPS) mice, the effects of pure hydroxytyrosol from OMWW onhydroxytyrosol-based supplement generated tumour necrosis factor-α (TNF-α) (a pivotal cytokine involved Whereas (13). investigated were inflammation) in supplement administration of the hydroxytyrosol-based obtained from OMWW evoked a statistically significant reduction in TNF-α levels in an LPS-treated human cell line significant any exert to failed hydroxytyrosol pure (P<0.01), Pure(13). cytokine inflammatory this of levels the on effects be ineffective in other cellhydroxytyrosol was also shown to other that imply findings such (13); inflammation of models components that are present within the hydroxytyrosol-based couldhydroxytyrosol, pure in absent are but product, OMWW these differential effects. Indeed cellular for be responsible studies measuring the antioxidant profiles of a hydroxytyrosol- hydroxytyrosol and pure OMWW based supplement from antioxidant had a greater former the that demonstrated capability (13), thus further indicating that additional components present within the OMWW product may provide supplementary benefits over pure hydroxytyrosol alone. (LPS) mice, the effects of pure hydroxytyrosol and a and a (LPS) mice, the effects of pure hydroxytyrosol from OMWW onhydroxytyrosol-based supplement generated tumour necrosis factor-α (TNF-α) (a pivotal cytokine involved Whereas (13). investigated were inflammation) in supplement administration of the hydroxytyrosol-based obtained from OMWW evoked a statistically significant reduction in TNF-α levels in an LPS-treated human cell line significant any exert to failed hydroxytyrosol pure (P<0.01), Pure(13). cytokine inflammatory this of levels the on effects be ineffective in other cellhydroxytyrosol was also shown to other that imply findings such (13); inflammation of models components that are present within the hydroxytyrosol-based couldhydroxytyrosol, pure in absent are but product, OMWW these differential effects. Indeed cellular for be responsible studies measuring the antioxidant profiles of a hydroxytyrosol- hydroxytyrosol and pure OMWW based supplement from antioxidant had a greater former the that demonstrated capability (13), thus further indicating that additional components present within the OMWW product may provide supplementary benefits over pure hydroxytyrosol alone. benefits of a novel, hydroxytyrosol-based product generated product hydroxytyrosol-based of a novel, benefits OMWW, HIDROX from this clinical study, 35 patients were assigned to twice daily oral assigned to twice study, 35 patients were this clinical for six supplement of the hydroxytyrosol-based administration significant a statistically six months of treatment, months. After cholesterol was documentedin the levels of V-LDL reduction cholesterol V-LDL (11). (P=0.001) population patient the in the highestcontains all triglyceride fat of amount of and high levels of V-LDL cholesterol cancholesterol subtypes and disease artery coronary of risk the raise significantly V-LDL cholesterol, By reducing infarction. myocardial thereducing in role important an play could hydroxytyrosol disease. risk of cardiovascular effects on aforementioned its to in addition Hydroxytyrosol, also been shown to play an importantblood lipid profiles, has study; a clinical in thrombosis of prevention the in role andinfarction myocardial of cause important an is thrombosis trial, conducted in a high risk patient SCD. A small clinical areduce could hydroxytyrosol that demonstrated population, thus highlighting its potentially thrombosis biomarker, therapeutic antithrombotic effects (12). In this study, type I a of mg 12.5-25 administered were patients diabetic Hydroxytyrosol 4 days. daily for once solution hydroxytyrosol plasma levels of produced a notable decrease in B thromboxane sulphate (39). An important clinical trial assessed the therapeutic sulphate (39). An important clinical trial assessed the therapeutic oxidation induced by copper oxidation induced by copper oxidation induced by copper oxidation induced by copper shown to effectively inhibit LDL LDL inhibit effectively to shown shown to effectively inhibit LDL LDL inhibit effectively to shown a 50 percent reduction in total cholesterol levels (9). However,(9). levels cholesterol in total reduction 50 percent a pure of that administration found study a different E deficient mice weeks to apo 10 ten for hydroxytyrosol and atherosclerotic increased total cholesterol resulted in studythat speculated latter The authors of this lesions (31). matrix and out of their original when taken phenolic products, (13); such adverse effects could induce natural environment products hydroxytyrosol of use the support may theory a occurring, naturally of complement full the containing as studied in previous reportsadditional phenolic compounds isolated, that suggests evidence Additionally, 13-14). (11, bioavailabilityreduced from a suffer polyphenols may purified that are contained within their compared to polyphenols to shown was bioavailability hydroxytyrosol’s matrix; natural oil olive of component natural a as given when higher be to olive oil or yoghurt (33). versus when it was added demonstrated that hydroxytyrosol Clinical studies have associates with following LDL oral administration the of polyphenol. In one report, 10 healthy volunteers were formulation of hydroxytyrosol and bloodadministered an oral elucidate theperformed at regular intervals to was sampling and investigate polyphenol’s pharmacokinetic profile within LDL (10). Inwhether hydroxytyrosol could be detected LDL volunteers’ in detected was hydroxytyrosol study this following oral fractions at 10 minutes and 20 minutes ain seen been also have findings Similar (10). administration virgin olive oil withstudy of 12 healthy volunteers administered varying polyphenol concentrations (6). Following oral olive oil, totaladministration of 40 ml of polyphenol-enriched fractions LDL subjects’ the in polyphenol compounds present changes these hours; 1.5 after by 57 percent increased tyrosol and hydroxytyrosol plasma with correlated directly that demonstrated also study same This (6). concentrations polyphenol-enriched olive oil LDL reduced oxidation in findings clinical these Collectively (6). volunteers healthy in hydroxytyrosol of effects beneficial potential the support the reduction of LDL oxidation. atherogenesis through been also has oleuropein hydroxytyrosol, to addition In however, if both compounds were activate SirT-1 and its effects on administered to animals an even longevity have been attributed to greater reduction in TNF-α was seen this mechanism of action (22-23). In (13). These important findings a compelling series of experiments demonstrated, for the first time, that using rat hearts, hydroxytyrosol was an OMWW hydroxytyrosol-based shown to increase the expression of supplement acted synergistically several longevity proteins, prevent with glucosamine, and that a cell death, and reduce the size of combination of both products could infarcted tissue following ischemia; in evoke prominent anti-inflammatory the majority of these experiments effects in animals. hydroxytyrosol exerted greater Hydroxytyrosol has been shown to effects than resveratrol (23). reduce inflammation in animal paws Following administration of and to also reduce levels of pain experimental compounds, including associated with inflammation. Oral hydroxytyrosol and resveratrol, for 14 administration of hydroxytyrosol to days, the expression levels of various rats with experimentally-induced SirT proteins (SirT-1, SirT-3, and SirT-4) paw inflammation induced were evaluated in excised hearts reductions in the degree of paw (23). Hydroxytyrosol led to a swelling and pain induced by paw statistically significantly increase in inflammation (15). An additional SirT-1 in cardiac tissue (P<0.05); of all study assessed the anti-inflammatory the test compounds studied, effects of olive polyphenols in including resveratrol, hydroxytyrosol animals with acute and chronic was the most effective at increasing inflammation (16). Rats were fed on the levels of this longevity protein diets of either sunflower oil, palm oil, (23). Hydroxytyrosol also evoked fish oil, or an olive polyphenol diet for significant increases in the SirT-3 and 8 weeks. After the study period, SirT- 4 longevity proteins (P<0.05); inflammatory biomarkers were the SirT-3 and SirT-4 are localized in the lowest in animals given olive mitochondria and they have been polyphenols (16). implicated in the regulation of

Polyphenols In keeping with the above animal aging and energy metabolism (23). studies of hydroxytyrosol, human In contrast, resveratrol failed to studies have also demonstrated its Table. The biological effects of hydroxytyrosol reported in evoke an increase in the expression powerful anti-inflammatory effects. A laboratory and clinical studies. of SirT-3 (23). placebo-controlled clinical study of In subsequent experiments the the OMWW hydroxytyrosol-based product in patients suffering potential cardioprotective effects of hydroxytyrosol were

vol 22 n 5 from rheumatoid arthritis revealed that the supplement evaluated in isolated rat hearts that had been subjected to 30 - reduced levels of C-reactive protein after eight weeks (14). minutes of ischemia followed by reperfusion. In control hearts C-reactive protein is an important biomarker of inflammation the size of the infarct, following the ischemic episode, was and it has been previously associated with rheumatoid arthritis, approximately 40 percent of the total heart tissue (23). cardiovascular disease, and mortality. The same clinical study Hydroxytyrosol treatment resulted in a significant reduction in also demonstrated that the supplement significantly reduced the size of the infarct to approximately 21 percent (P<0.05) (23). levels of homocysteine after eight weeks (P<0.01) (14). Hydroxytyrosol also evoked the lowest levels of cell death Homocysteine is also an important biomarker of inflammation following the ischemic episode compared to other 5 test

September/October 2011 and a number of large clinical studies have established compounds, including resveratrol (23). These important findings - homocysteine as an independent risk factor for venous demonstrate that hydroxytyrosol can increase the expression of thromboembolism, stroke, coronary heart disease, and death multiple SirT longevity proteins and potentially reduce the (17). Additionally, an eight-week placebo-controlled clinical deleterious pathophysiological effects of myocardial infarction study of the OMWW hydroxytyrosol-based supplement in through reducing the size of the infarct and preventing cell industry hi-tech patients with osteoarthritis demonstrated that it induced death. Additionally, hydroxytyrosol was shown to be more significant improvements in patient’s quality of life as measured effective than resveratrol in these studies (23). OOD by a health assessment questionnaire disability index (HAQ-DI)

gro F (14). The HAQ-DI evaluated nine categories of functional A activity; 69 percent of osteoarthritis patients receiving the CONCLUSION supplement reported a greater than 20 percent improvement in their HAQ-DI scores (14). Hydroxytyrosol, a polyphenol found at high concentrations in OMWW, is a potent natural antioxidant. Multiple experimental and clinical studies have identified a number of potential THE EFFECTS OF HYDROXYTYROSOL ON CELLULAR PATHWATS therapeutic effects of hydroxytyrosol (see Table). IMPLICATED IN LONGEVITY Hydroxytyrosol has been shown to reduce V-LDL in a clinical study (11) and have notable antithrombotic effects. The sirtuin (SirT) family of proteins, of which seven human forms Hydroxytyrosol’s anti-inflammatory effects have been have been described, have been implicated in variety of demonstrated in a number of laboratory studies; a biological processes including fatty acid metabolism and the hydroxytyrosol-based supplement reduced levels of TNF-α in extension of life span (18-19). To date the most widely an animal model of joint inflammation (13), and improved the researched SirT protein is SirT-1; among other functions, SirT-1 is quality of life in osteoarthritis patients (14). Finally, animal associated with muscle development, gene silencing, and studies have indicated that hydroxytyrosol may play a role in longevity (20-21). Resveratrol has previously been shown to the extension of life span through its effects on the

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