Collagen and adenosine diphosphate (ADP) were purchased from Chrono-Log (Havertown, PA, USA). The human PF4 enzyme-linked immunosorbent assay (ELISA) kit, TXB2 ELISA kit, and 6-keto-PGF1α ELISA kit were purchased from Abcam (Boston, MA, USA).
2.2. Preparation of intervention supplements
Fruitflow (FF) and placebo tablets were provided by By-Health Co., Ltd (Zhuhai, Guangdong, China). Each FF tablet contained 150 mg of water-soluble tomato extract. The main biologically ingredients of FF tomato extract are flavone, adenosine, and chlorogenic acid (total > 8 mg/g). Each aspirin tablet contained 100 mg ASA (Bayer, purchased from Beijing Hospital, Beijing, China).
One hundred ninety subjects completed this clinical trial. The subjects were over 50 years old, with no medical history of serious disease, hemostatic disorders, or intolerance to ASA. The platelet aggregation rate in these subjects reached higher than 40% in ADP- and collagen-stimulated platelets. The exclusion criteria were blood pressure ≥ 160/100 mmHg, platelet count < 100 × 109/L, prothrombin time outside the normal reference range, the chronic use of medications that may affect platelet function and dietary supplements (e.g., fish oil and evening primrose oil), allergy to tomatoes or their ingredients, intolerance or allergy to ASA, contraindications for ASA, and positive fecal occult blood test. Moreover, according to the researchers’ judgment, individuals with serious hematological diseases, digestive diseases, organic diseases of the heart, liver, or kidneys, severe liver or kidney dysfunction, or metabolic, endocrine, or nervous system diseases were also excluded.
The present study was conducted according to the principles of the Declaration of Helsinki. All procedures that involved human subjects were approved by the Ethics Committee of Beijing Hospital (no. 2018BJYYEC-195-02). Written informed consent was obtained from all of the participants. The study was registered at the Chinese Clinical Trial Registry (www.clinicaltrials.gov; registration no. ChiCTR2000034647).
A flowchart of the recruitment procedure and disposition of the subjects is shown in Fig. 1.
2.4. Study design
We first used statistical software to calculate the sample size for this study. We set power to 95% and used the Cochran-Armitage test for linear trend in proportions. The results of the sample size calculation showed that the sample size for each group should be approximately 50 subjects.
The study followed a randomized placebo-controlled trial. All study activities were carried out in the Beijing Hospital, Beijing, China. The subjects were randomly assigned by a computer program to one of four groups: placebo (150 mg/day), FF (150 mg/day), ASA (100 mg/day), and FF (150 mg/day) + ASA (100 mg/day). The subjects were asked to take their respective intervention after dinner daily for 7 days. The duration of the interventions was based on O’Kennedy, who showed that the FF intervention for 7 days is sufficient to evaluate its effectiveness .
Fasting blood was collected from the participants on days 0 and 8 to analyze platelet aggregation rate and the content of thromboxane B2 (TXB2), 6-keto-prostaglandin F1α, and platelet factor 4 (PF4).
2.5. Biochemical determination
Biochemical testing was performed using a LABOSPECT 008 AS automatic biochemical analyzer (Hitachi, Tokyo, Japan).
2.6. Platelet aggregation rate measurement
Venous blood was collected from the participants in the placebo, FF, ASA, and FF + ASA groups pre-intervention and post-intervention. The blood samples were immediately mixed with 3.2% sodium citrate. The blood samples were centrifuged at 200 ⋅ g for 10 min to obtain platelet-rich plasma and then centrifuged again at 1500 ⋅ g for 10 min to obtain platelet-poor plasma. Platelet-rich plasma (300 µl) was used to analyze platelet aggregation using a Chrono-Log aggregometer. Platelet-rich plasma was set in the platelet aggregation assay channel with stirring for 1 min, and then ADP (2 µM) or collagen (2 µg/ml) was added to induce platelet aggregation. The rate of platelet aggregation was recorded using a Chrono-Log aggregometer.
2.7. Enzyme-linked immunosorbent assay
Venous blood was collected from the participants on days 0 and 8. After centrifugation at 1500 ⋅ g for 15 min, plasma was collected and stored at -80 °C. The levels of TXB2, 6-keto-PGF1α, and PF4 were determined using ELISA kits (Abcam, Boston, MA, USA) according to the manufacturer’s instructions.
Prothrombin time, fibrinogen, activated partial thromboplastin time, and thrombin time were measured using an ACL-TOP-700 coagulometer (Werfen, Barcelona, Spain).
2.9. Statistical analysis
The data are expressed as mean ± SD. All of the variables were tested for a normal distribution using the Kolmogorov-Smirnov test. Two-tailed paired Student’s t-test was used to compare platelet aggregation and TXB2, 6-keto-PGF1α, and PF4 levels between pre- and post-intervention. The Kruskal-Wallis H test was used to analyze differences between groups. The statistical analyses were performed using SPSS 25.0 software (Armonk, NY, USA). Values of p < 0.05 were considered statistically significant.