In the current trial, we included the participants with the age over 50 years old, with no medical history of serious disease, hemostatic disorders, or intolerance to ASA. The platelet aggregation rate in these participants reached higher than 50% 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. Eligible participants were recruited from February to December 2019. Participants undergone clinical examination on days 0 and 8. A flowchart of the recruitment procedure and disposition of the participants is shown in Fig. 1.
2.2. Sample size calculation
On the basis of the sample size formula suggested for randomized clinical trials and by considering type I error of 5% (α = 0.05), type II error of 10% (β = 0.10, power =90%), and allowing for a 20 % drop-out rate, we determined a sample size of 40 individuals in each group. To get a more confident result, we intended to recruit 100 participants for each group. However, we temporarily terminated the trial after the recruitment of 50 participants in each group because of COVID-19 crisis.
2.3. Ethical consideration
The present study was conducted according to the principles of the Declaration of Helsinki. All procedures that involved human participants 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 retrospectively registered at the Chinese Clinical Trial Registry (www.clinicaltrials.gov; registration no. ChiCTR2000034647).
2.4. Study supplements and materials
Fruitflow®(FF) and placebo were provided by By-Health Co., Ltd (Zhuhai, Guangdong, China) in tablet formats. Each FF tablet contained 150 mg of water-soluble tomato extract. The main biological ingredients of FF are flavone, adenosine, and chlorogenic acid (total > 8 mg/g). Each aspirin tablet contained 100 mg ASA (Bayer, purchased from Beijing Hospital, Beijing, China). The packages of FF and placebo tablets were same in the terms of color, appearance, and taste and were given to the participants at the study baseline.
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).
All study activities were carried out in the Beijing Hospital, Beijing, China. The study followed a randomized placebo-controlled trial. The participants were randomly allocated in a manner of 1:1:1:1 to four groups: placebo (150 mg/day), FF (150 mg/day), ASA (100 mg/day), and FF (150 mg/day) + ASA (100 mg/day). Simple randomization sequence was created using SPSS 25.0 (Armonk, NY, USA) statistical software. The participants in the placebo group did not know the aim of the study. The participants were asked to take their respective intervention after dinner daily for 7 days. The participants were asked to record their intervention form every day and to ensure their adherence in the present study. 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 . The primary outcomes were post-supplement changes in platelet aggregation response to agonist, changes in TXB2, 6-keto-prostaglandin F1α(6-keto-PGF1α) and platelet factor 4 (PF4) generation by platelets. Secondary outcomes included post-supplement changes in plasma clotting times and fecal occult blood test. Outcome variables were assessed at the study baseline and end of the trial (days 0 and 8). Fasting blood from participants was collected by a second researcher who was unaware of assignment of the groups.
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. Assessment of TXB2, 6-keto-PGF1α and PF4
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.
2.8. Biochemical determination
Biochemical testing was performed using a LABOSPECT 008 AS automatic biochemical analyzer (Hitachi, Tokyo, Japan).
2.9. Coagulometry and Fecal occult blood test
Prothrombin time, fibrinogen, activated partial thromboplastin time, and thrombin time were measured using an ACL-TOP-700 coagulometer (Werfen, Barcelona, Spain). Fecal occult blood testing using tetramethylbenzidine (Wan Hua Pu Man Biological Engineering Co., Ltd., Beijing, China).
2.10. Assessment of other variables
Required information on age, gender, having a history of other diseases, medication, body mass index (BMI), and supplement use was collected using a researcher-made questionnaire. BMI was calculated as weight in kilograms divided by height in square meters (kg/m2). Height was measured in a standing position without shoes. Weight was determined with minimal clothing and without shoes by an analog scale.
2.11. 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.