Antiplatlet Effects of Standardized Tomato Extract in Hypertensive Subjects

Study design The study involved 82 high-risk hypertensive patients (44 men and 38 women), aged 28-74years, conducted between July 2015 and February 2017 in the Department of Hypertension at the University of Medical Sciences in Poznan. Seventeen patients reported resignation during the study. The permission no. 377/15 to conduct the study was published by the Ethics Committee at the Poznan University of Medical Sciences. All the patients gave informed consent for participation in the study. Patients with primary hypertension and high cardiovascular risk were randomly assigned to one of two groups. Group 1(ASA) included 33 patients who received acetylsalicylic acid at a dose of 75 mg in the morning. Group 2 (STE) included 32 patients receiving standardized tomato extract (STE) (ZAAX, Sequia, Poland) at a dose of 213 mg orally in the morning.

The patients had two visits within 4 weeks according to the scheme:

Visit 1: During the first visit, patients were qualified for the study. The patients with previously diagnosed hypertension were referred from an outpatient clinic, to the hospital ward. After admission, the patients with high cardiovascular risk were isolated and written consents were obtained.

On the basis of stage of hypertension, presence of risk factors and subclinical organ damage, diabetes coexistence, cardiovascular complications and chronic kidney disease the cardiovascular risk was estimated. Each patient with one or two risk factors and a blood pressure (BP) > 180/110 mmHg or three or more risk factors, and a BP >160/100 mmHg or with the presence of organ damage and BP >140/90 mmHg were classified as a high risk Group.There have been no changes in the concomitant treatment (lipid-lowering, anti-hypertensive and antidiabetic) during the study.

Numerous laboratory tests and imaging, such as aldosterone and ARO levels, both before and after tilting, creatinine, urea, GFR, sodium, potassium, pro BNP, TSH, FT3, FT4, and a 24-hour collection for electrolytes were collected. Abdominal ultrasound examinations, computed tomography of the abdomen, and Doppler ultrasound of the renal arteries were performed to exclude secondary causes of hypertension. Clinical BP measurements were performed three times, as well as, ambulatory blood pressure monitoring (ABPM) and echocardiography was conducted. Whole blood samples were taken and baseline platelet aggregation was determined using the VerifyNow analyzer from Accumetrics. In Group 1, two whole blood samples for VerifyNow Aspirin test were assayed. In Group 2, four whole blood samples for VerifyNow Aspirin and P2Y12 tests were assayed.

Visit 2: After 4 weeks of taking STE or ASA the patients visited the hospital for the second visit. All examinations from visit 1, including platelet aggregation inhibition assays, were repeated. Whole blood samples were collected 1-4 h after the ingestion of a morning ASA or STE dose from a peripheral vein using a 21 gauge or larger needle in a partial fill 3.2% citrate vacuum collection tube.

Standarized tomato extract characteristics STE is a water-soluble tomato concentrate, free of lycopene and fats, which contains 37 biologically active compounds of well-known structure. These bioactive compounds contain nucleosides (adenosine, cytidine, guanosine, inosine, AMP, GMP), polyphenols including flavonoids (rutin, quercetin, kemferol, luteoline, naryngenin) and phenolic acids (chlorogenic, coffee, p-coumar, ferulic).

In 2009, the European Food Safety Authority (EFSA) approved the water soluble tomato concentrate (WSTC) as a food supplement which helps to maintain normal platelet aggregation. EFSA recommended the use of STE in healthy individuals between 35 and 70. The effective daily dose was 3 g of bioactive compounds contained in syrup or 65-150 mg in powdered form.

This extract affects the inhibition of ADP-mediated aggregation, thromboxane, collagen, von Willebrand factor, thrombin and inflammatory mediators. What is more, it blocks the P2Y12 receptor for ADP and limits intracellular granular release of thromboxane, ADP, selectin P and factor 4 (PF4, platelet factor 4). Additionally, it prevents activation of αIIbβIII integrin and inhibits (GP) IIb / IIIa receptor activation. By inhibiting the P2Y12 receptor for ADP, it increases cAMP activity and this results in decreasing intracellular calcium ions concentration. Inhibition of collagen binding process with GP VI as well as von Willebrand factor with GP Ib receptor and decrease in Cβ and Cy2 phospholipase activity are also included in anti-aggregation effects of STE. These phenomena affect the shape of thrombocytha and are reversible while maintaining a level of platelet activity that allows aggregation in case of vascular injury. ZAAX can be used by people with hypersensitivity to salicylates and other non-steroidal anti-inflammatory drugs, with bronchial asthma, with active peptic ulceration.

Studies have shown that STE inhibits the activation of endothelial inflammatory processes, which prevents the development of atherosclerosis. STE reduces the production of inflammatory cytokines (TNF-α, IL-1β and IL-12) and to increases the synthesis of interleukin 10, which has anti-inflammatory properties.

VerifyNow® Tests Procedure The study protocol included measurements of platelet reactivity using a VerifyNow Tests Procedure (Accumetrics Inc., USA). This method detects platelet activity by measurement of platelet aggregation, in a blood sample, exposed to a specific agonist. This leads to an inhibition of platelet function in response to anti-platelet therapy. The VerifyNow System is a whole blood device that performs qualitative measurement of induced platelet aggregation based on the increase in light transmittance. Two types of VerifyNow test kits were used in the study: VerifyNow Aspirin Test and VerifyNow P2Y12. The Aspirin test results are reported as Aspirin Reaction Units (ARU), which are calculated as a function of the rate of aggregation. ARU values less than 550 indicate effective result of ASA, while values > 550 ARU indicate no effect of the drug. The VerifyNow P2Y12 test measures platelet function based on the ability of activated platelets to bind fibrinogen and provides information on the aggravation of platelet aggregation in reactivity units of P2Y12 receptor (PRU). It is considered that the range of 95 - 208 PRU is responsible for optimal antiplatelet activity. High PRU values after drug administration, point to the lack of expected anti-platelet effect.

Blood pressure measurements In all the patients, during each visit, BP measurements were performed three times at rest, in the supine position, in standard conditions, and using an upper arm blood pressure monitor (Omron 705IT). Ambulatory, 24-hour blood pressure measurements (ABPM) were carried out using an A\&D, 24-hour ambulatory peripheral blood pressure monitor. The frequency of measurements was every 15 minutes between 7:00 and 22:00 and every 30 minutes between 22:00 and 7:00.

Statistical analysis Statistical analyses were performed with Statistica, version 12.5. (StatSoft, USA). Since the tested data did not meet the assumption of Gaussian distribution (evaluated with Shapiro-Wilk method), the non-parametric methods were applied. The Wilcoxon signed-rank test was used for evaluation of the differences between the initial values and the values obtained after the treatment, for factors of; body weight composition, blood pressure, and platelet aggregation. To evaluate differences and correlations between the two independent groups the Mann-Whitney U test and Spearman׳s rank correlation coefficient (Rs) were used, respectively. The data presented on graphs and tables included median and interquartile ranges. A p<0.05 was considered as significant.