In Vivo Evaluation of Anti-inflammatory and Antithrombotic Health Promoting Properties of Extra Virgin Olive Oil Versus Classic Table Olive Oil in Healthy Subjects

Introduction: Thrombo-inflammatory manifestations are key pathophysiological mechanisms of cardiovascular diseases and are directly related to platelet activation and aggregation. Extra virgin olive oil, a key component of the Mediterranean diet, is rich in natural bioactives (e.g. unsaturated fatty acids, polar lipids, phenolic compounds, carotenoids and other pigments), which have been proposed to possess not only antioxidant activities, but mainly strong anti-inflammatory and antiplatelet properties, which contribute to the reduction of cardiovascular risk. However, to our knowledge, nutritional and biochemical evaluation of these properties through an in vivo intervention study has not been thoroughly conducted, especially for the cardioprotective benefits of consuming such an extra virgin olive oil against the main thrombo-inflammatory pathways, and compared to plain classic table olive oil.

Purpose: The purpose of this study was the qualitative characterization of a sample of extra virgin olive oil and the biochemical investigation of the effect of its daily consumption on platelet activation and on the mechanisms related to thrombo-inflammation compared to the effects observed by a similar consumption of classic table olive oil.

Materials and Methods: An in vivo dietary intervention study, randomised and single blinded (the volunteers did not know what type of olive oil they consumed) was performed on four arms:

In the 1st arm, 18 healthy volunteers consumed the nutritionally recommended daily dose (30 mL) of extra virgin olive oil daily for 28 days.

In the 2nd arm, 18 other healthy volunteers consumed the nutritionally recommended daily dose (30 mL) of plain classic table olive oil daily for 28 days.

In the 3rd arm, 9 healthy volunteers initially consumed the nutritionally recommended daily dose (30 mL) of extra virgin olive oil daily for 28 days and after a month washout period they then re-consumed the nutritionally recommended daily dose (30 mL) of plain classic table olive oil daily for another period of 28 days, in a crossover single blinded randomised design In the 4th arm, 9 healthy volunteers initially consumed the nutritionally recommended daily dose (30 mL) of plain classic table olive oil daily for 28 days and after a month washout period they then re-consumed the nutritionally recommended daily dose (30 mL) of extra virgin olive oil daily for another period of 28 days, in a crossover single blinded randomised design Blood samples were taken from all volunteers in all these 4 arms, on days 0 and 28 in each one of these interventions and platelet sensitization by various agonists was assessed just after blood sampling. Blood Plasma was stored at -20oC for further analysis Specifically the anti-inflammatory effects were expressed as the Effective Concentrations that induce 50% of platelet aggregation (EC50 values) by platelet-activating factor (PAF), the antithrombotic effects as the EC50 for Thrombin by using Thrombin Receptor Active Peptide (TRAP) and the antiplatelet effects as the EC50 values for ADP.

• Olive Oil 1 (Other), also known as Dietary Intervention, Extra virgin olive oil (EVOO) consumption, Single Blinded, Randomised: 1st arm: 18 healthy volunteers consumed 30 mL of extra virgin olive oil (EVOO) daily for 28 days. Blood samples and biochemical assays were conducted just before (day 0) and after (day 28) this dietary intervention
• Olive oil (placebo) 1 (Other), also known as randomised, single blinded, plain classic table olive oil (CTOO) consumption, dietary intervention: 2nd arm, 18 other healthy volunteers consumed 30 mL of plain classic table olive oil (CTOO) daily for 28 days.
• Olive Oil 2 (Other), also known as single blinded, randomised, crossover, dietary intervention: 3rd arm, 9 healthy volunteers initially consumed 30 mL of EVOO daily for 28 days and after a washout period of 1 month they then re-consumed 30 mL of CTOO daily for 28 days, in a crossover single blinded randomized design with the 3rd arm. Blood sampling and Biochemical Assays were conducted just before (day 0) and just after (day 28) each of these interventions
• Olive oil (placebo) 2 (Other), also known as Dietary Intervnention, Crossover, Single Blinded, Randomised: 4th arm, 9 healthy volunteers initially consumed 30 mL of CTOO daily for 28 days and after a washout period of 1 month they then re-consumed 30 mL of EVOO daily for 28 days, in a crossover single blinded randomized design with the 3rd arm. Blood sampling and Biochemical Assays were conducted just before (day 0) and just after (day 28) each of these interventions

Primary outcomes

• Effective Concentration of 50% of platelet aggregation (EC50 value) for PAF [Blood sampling and platelet sensitization by PAF is assessed just before (day 0) and just after (day 28) of each intervention in platelets and plasma of blood samples from all volunteers in all these 4 arms]: 1\. The anti-inflammatory effects of the intervention assessed in platelets and plasma of blood samples from all volunteers are expressed as the Effective Concentrations that induce 50% of platelet aggregation (EC50 values) induced by platelet-activating factor (PAF), measured in nM concentration of PAF that can cause 50% of platelet aggregation.
• Effective Concentration of 50% of platelet aggregation (EC50 value) for Thrombin [Blood sampling and platelet sensitization by TRAP is assessed just before (day 0) and just after (day 28) of each intervention in platelets and plasma of blood samples from all volunteers in all these 4 arms]: The antithrombotic effects of the intervention assessed in platelets and plasma of blood samples from all volunteers are expressed as the Effective Concentration that induce 50% of platelet aggregation (EC50 values) induced by the Thrombin Pathway, measured in μM concentration of Thrombin Receptor Active Peptide (TRAP) that can cause 50% of platelet aggregation
• Effective Concentration of 50% of platelet aggregation (EC50 value) for ADP [Blood sampling and platelet sensitization by ADP is assessed just before (day 0) and just after (day 28) of each intervention in platelets and plasma of blood samples from all volunteers in all these 4 arms]: The antiplatelet effects of the intervention assessed in platelets and plasma of blood samples from all volunteers are expressed as the Effective Concentration that induce 50% of platelet aggregation (EC50 values) induced by the Adenosine 5' Diphosphate (ADP, measured in μM concentration of ADP that can cause 50% of platelet aggregation.

Sex: All, accepts healthy volunteers

Inclusion Criteria:

* Not to have a pathological condition related to platelet and leukocyte activity
* Not to have a chronic pathological condition
* To have a Normal Body Mass Index (BMI) and waist circumference
* Not to take medications/supplements or any kind of other intervention that have anti-inflammatory and/or antithrombotic and/or antioxidant effects

Exclusion Criteria:

* To have a pathological condition related to platelet and leukocyte activity
* To have a chronic pathological condition
* Not to have a Normal Body Mass Index (BMI) and waist circumference
* To take medications/supplements/nutraceuticals or any kind of other intervention that have anti-inflammatory and/or antithrombotic and/or antioxidant effects

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• Alexandros Tsoupras, Principal investigator, Democritus University of Thrace