All the subjects were recruited from the medical examination center of the first affiliated hospital of Sun Yat-sen University and community health center through flyers, medical record reviews, or clinicians’ recommendations in Guangzhou, Guangdong, China. This study was approved by the Ethics Committee of Sun Yat-sen University (No. 2016036), and was conducted in strict accordance with the principles of the Declaration of Helsinki. This trial was registered at Chinese clinical trial registry as ChiCTR-POR-17012927. All participants signed written informed consent forms prior to enrollment.

The inclusion criteria were: (1) men or women aged 40–65 years; (2) without serious vascular or hematological diseases; and (3) normal laboratory tests for hematuria, liver and kidney function, blood glucose, and lipids. The exclusion criteria were: (1) with the history of hypertension, infectious disease, hemostatic disorders, diabetes mellitus, and CVDs; (2) agents use that could influence platelet function; (3) lactating or pregnant women; and (4) allergic to tomatoes and their ingredients.

This was a randomized crossover-designed controlled trial. First, a total of 105 eligible subjects were stratified by gender and then randomly divided into two group 1 or group 2 by random numbers generated by SPSS v22.0 (SPSS Inc., Chicago, IL, United States). Referring to other previous randomized controlled trials which focused on the effects of 4-week supplementation for other nutritional supplements rich in phytochemical (20–23), we took 4 weeks as the duration of WTE supplementation in this study. Individuals in group 1 took one WTE tablet (150 mg/day) and individuals in group 2 take one placebo tablet daily for 4 weeks, followed by a 2-week washout period. Then, the two groups switched groups and continued to take WTE (150 mg/day) or placebo tablets for another 4 weeks (Figure 1). During the 10-week trial period, all subjects were instructed to maintain their usual diet and lifestyle, but to refrain from tomatoes and tomato products. The 24-h dietary recall data were recorded on 3 consecutive days, and International Physical Activity Questionnaire (IPAQ) scores were collected via face-to-face interviews at baseline and after intervention (24, 25).

A technician who did not participate in the experiments, data collection or analysis was responsible for randomization, and the management of the packaged supplements. Participants, investigators, and laboratory technicians were blinded to the treatment assignments.

Use PASS software (version 11.0, NCSS Inc.) to estimate the sample size required for the study based on the conventional assumption of a two-tailed α level of 0.05 and β level of 0.10. According to the previous study (15), the change rates of platelet aggregation in the intervention group and the control group were −9.7 and −3.1% after the intervention of WTE. Based on a two-tailed α level of 0.05 and β level of 0.10, 42 participants in each group were needed. Considering a 10% loss to follow-up rate, 47 subjects are needed in each group at least.

Water-soluble tomato extract tablets and placebo tablets were provided by DSM (Netherlands) and By-health (China). The WTE tablets contain 150 mg WTE (Fruitflow) which comprises nucleosides, polyphenols, and flavonoids purified from fresh tomatoes (L. esculentum) by using solid-phase extraction, as described in previous study (14) (refer to Supplementary Table 1 for the ingredients of the WTE tablets). The placebo tablets contained only maltodextrin. The weight, appearance, taste, and packaging of WTE tablets and placebo tablets were same.

Body weight, height, waist circumference (WC), hip circumference (HC), heart rate (HR), and blood pressure (BP) were measured according to standard protocols, as previously described (24, 25).

Fasting blood samples were obtained at 8–9 a.m. from each subject’s antecubital vein with a 21-gauge needle at the beginning and at weeks 4, 6, and 10 of the trial. Whole human blood was collected into a Vacutainer tube containing 3.8% sodium citrate (1/9, v/v) via venipuncture. The blood sample was then centrifuged at 1,700 × g for 15 min at 4°C to obtain plasma, and the blood sample with sodium citrate was then centrifuged at 200 × g for 10 min at 22°C to obtain platelet-rich plasma (PRP) (26). PRP was immediately used to assay platelet aggregation and activation or other related parameters. Plasma samples were stored at −80°C until subsequent analyses.

Fasting blood samples were subjected to LDL-C, HDL-C, serum TG, serum TC, fasting blood glucose (FBG), urea, uric acid (UA), creatinine, alanine aminotransferase, total protein, albumin, and globulin. Lipid profile analyses were performed on the Cobas c311 automated assay analyzer (c311, Roche Diagnostics, Switzerland). Enzymatic methods were used to determine the concentrations of HDL-C, LDL-C, TC, and TG. Other biochemical analyses included FBG, urea, UA, creatinine, alanine aminotransferase, total protein, albumin, and globulin were measured using the Cobas c311 automated assay analyzer. Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin clotting time (TT), and plasma fibrinogen (Fib) estimations were performed on a Sysmex5100 system (Siemens Healthineers, Malvern, PA, United States).

The platelet granule contents including platelet factor 4 (PF4), β-thromboglobulin (β-TG), and thromboxane B2 (TXB₂) in plasma were tested using an enzyme linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions. The PF4 and β-TG in plasma were detected via the double antibody sandwich method of ELISA (Abcam, United Kingdom). TXB₂ is the stable product of the non-enzymatic hydration of thromboxane A2 (TXA₂), thus the production of TXA₂ in vivo is typically monitored by measurement of TXB₂ (27). Therefore, as previous studies (28–31), we chose the R&D’s TXB₂ competitive ELISA kit (R&D Systems, United States) to detect TXB₂ for reflecting the production of TXA₂. The SPARK^® multimode microplate reader (Tecan, Switzerland) was used to record ODs at 450 nm.

In the clinical trial, PRP (5 × 10⁶ platelets/mL) were labeled with FITC-conjugated anti-human CD62P antibody or PAC-1 antibody for 20 min and followed the stimulation of ADP or collagen, samples were then fixed with 1% paraformaldehyde and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, CA, United States), and data were analyzed with CytExpert 2.0 (Beckman Coulter, CA, United States), as previously described (32, 33).

Briefly, 500 μL of fresh PRP from citrated blood was incubated in a transparent cuvette at 37°C for 5 min. Then the cuvette was transferred into the detection hole of Chronolog aggregometer (Chrono-Log Corp., PA, United States) and stimulated by 5 μmol/L ADP or 2 μg/mL collagen. At least 500 μL of PRP need to be used for platelet aggregation test at a time, which means that a lot of whole blood needs to be collected. This is not in line with the principles of ethics. In addition, previous acute studies in Britain have found that WTE significantly inhibited collagen- and ADP-induced platelet aggregation (14, 15). Therefore, we measured platelet aggregation stimulated by collagen and ADP. Platelet aggregation was evaluated on a Chronolog aggregometer in PRP (3.0 × 10⁸ platelets/mL) at 37°C with a sample stir speed of 1,000 rpm, and the maximum-reversible platelet aggregation was monitored and recorded for at least 6 min, as previously described (33, 34).

SPSS 22.0 software was used for statistical analysis. In the clinical study, the data are expressed as the mean ± standard error of the mean (SEM) unless otherwise stated. For categorical variables, analysis was performed using the chi-square test. The changes after 4-week WTE supplementation or placebo supplementation were calculated as values after 4-week intervention (at 4 or 10 weeks) deducted from the values at baseline (at 0 week or after 2-week washout period). For continuous variables, variables were checked for normal distribution and were assessed by Kruskal–Wallis test if they were not normally distributed. The comparability of the two groups at baseline were assessed by one-way analysis of variance (ANOVA).

The effects of treatments were evaluated using one-way ANOVA, comparisons of each index at baseline (at 0 week or after 2-week washout period) and after 4-week intervention (at 4 or 10 weeks) between two groups were conducted using the Student’s t-test for unpaired data. The mean changes of each index between two groups after 4-weeks intervention were compared using the Student’s t-test for unpaired data. To explore the reversibility of 4-week supplementation of WTE, one-way ANOVA were used to assess changes between two groups at baseline, 4, 6, and 10 weeks. Stratified analyses were performed with ANOVA to compare the changes of platelet function after 4-week treatment stratified by gender. Differences were considered statistically significant at P < 0.05.

All funders of the present study (namely National Natural Science Foundation of China, Shenzhen Science, Technology, and Innovation Commission, By-health Research Foundation and Scientific Research Foundation of Chinese Nutrition Society) only provided financial support and did not participate in the design, data collection, data analysis, interpretation of the clinical trial, and the writing of this manuscript.

A total of 105 individuals completed the trial and were included in the analysis (Figure 1). The characteristics of 105 individuals are shown in Table 1. At baseline, 105 individuals were comparable in terms of age, sex, height, BMI, WC, HC, HR, SBP, DBP, lipid profile, and routine blood test (Table 1 and Supplementary Table 2). After intervention, there was no significant difference between two groups in terms mentioned above (Table 1 and Supplementary Table 2). In addition, there was no significant difference between two groups in daily physical activity status or the intake of energy, total protein, carbohydrates, lipids, dietary fiber, and vitamins at baseline and after intervention (Supplementary Table 3).

At baseline, there were no significant differences in platelet activation and aggregation between two groups. Subjects consumed 150 mg of WTE or placebo tablet daily for 4 weeks. After 4-week intervention, 150 mg/day of WTE significantly reduced the percentage of ADP- or collagen-induced activated GPIIbIIIa positive platelets, with baseline levels of 51.8 ± 1.4 or 21.3 ± 1.5% and post-intervention levels of 45.6 ± 1.8 or 17.5 ± 1.2%, respectively (P < 0.05, Table 2). Four weeks of 150 mg/day WTE also significantly decreased the percentage of ADP- or collagen-induced P-selectin positive platelets, with baseline values of 51.2 ± 1.0 or 32.3 ± 1.0% and post-intervention values of 44.3 ± 1.1 or 25.7 ± 0.9% (P < 0.05, Table 2). Similarly, 150 mg/day WTE resulted in significant reductions in platelet aggregation stimulated by ADP or collagen, with baseline values of 72.3 ± 1.4 and 81.1 ± 1.2% and post-intervention values of 61.5 ± 1.6 and 77.2 ± 0.9% after 4-week supplementation (P < 0.05, Table 2). After 4-week intervention, placebo did not significantly change platelet activation and aggregation in Chinese healthy middle-aged and older individuals (P > 0.05, Table 2). In addition, there were significant differences between 150 mg/day WTE group and the placebo group in terms of platelet activation and aggregation after 4-week intervention (P < 0.05, Table 2).

The percentage of ADP-induced activated GPIIbIIIa positive platelets from baseline to 4 weeks significantly decreased in the 150 mg/day WTE (−6.2 ± 2.0%, P < 0.05, Table 2). The percentage of collagen-induced activated GPIIbIIIa positive platelets from baseline to 4 weeks also significantly decreased in the 150 mg/day WTE groups (−3.8 ± 2.0%, P < 0.05), compared with the placebo group (3.0 ± 1.9%, Table 2). Similarly, the change in ADP- or collagen-induced platelet aggregation from baseline to 4 weeks in the 150 mg/day WTE group (−10.8 ± 1.8 or −3.9 ± 1.5%) was significantly different from the change in the placebo group (−1.6 ± 2.1 or 1.5 ± 1.1%, P < 0.05) (Table 2). Besides, there were significant differences between 150 mg/day WTE group (−6.9 ± 1.5 or −6.6 ± 1.3%) and the placebo group (4.8 ± 1.4 or −0.8 ± 1.2%) in the percentage of ADP- or collagen-induced P-selectin positive platelets (P < 0.05, Table 2). Stratified analyses indicated that WTE could significantly inhibit platelet activation and aggregation in whether male or female, and WTE might have more robust inhibition on platelet activation and aggregation among females (Supplementary Tables 4, 5).

One hundred and five individuals were comparable between two groups in terms of platelet granule secretion at baseline (P > 0.05, Table 3). Compared with baseline values, the 4 weeks of placebo supplementation did not significantly alter any of the variables (P > 0.05, Table 3). However, a 4-week intervention of 150 mg WTE per day can significantly reduce plasma β-TG from baseline value of 495.8 ± 19.1 ng/mL to post-intervention value of 366.1 ± 19.2 ng/mL (P < 0.05, Table 3). Four weeks of WTE at 150 mg/day also significantly decreased plasma PF4, with baseline value of 397.3 ± 24.5 ng/mL and post-intervention value of 276.7 ± 27.1 ng/mL (P < 0.05, Table 3). Similarly, 150 mg/day WTE resulted in significant reductions in TXB₂, with baseline value of 160.5 ± 4.5 ng/mL and post-intervention value of 118.5 ± 6.8 ng/mL after 4-week supplementation (P < 0.05, Table 3).

After 4-week intervention, there were significant differences between 150 mg/day WTE group and the placebo group plasma β-TG, PF4 and TXB₂ (P < 0.05, Table 3). The plasma β-TG from baseline to 4 weeks significantly decreased (−129.7 ± 27.5 ng/mL) in the 150 mg/day WTE group (Table 3). The decrease in plasma PF4 from baseline to 4 weeks in the 150 mg/day WTE group (−120.6 ± 33.2 ng/mL) was significantly different from the change in the placebo group (37.0 ± 40.3 ng/mL, P < 0.05), as shown in Table 3. In addition, compared with the placebo group (−1.0 ± 2.4 ng/mL), the level of plasma TXB₂ from baseline to 4 weeks was significantly reduced in the 150 mg/day WTE group (−42.0 ± 4.0 ng/mL, P < 0.05), as shown in Table 3. Stratified analyses indicated that WTE could significantly inhibit platelet granule secretion in whether male or female, and WTE might have more robust inhibition on platelet granule secretion among females (Supplementary Tables 4, 5).

After 4-week intervention, 150 mg/day WTE can significantly reduce platelet activation, aggregation, and granule contents, compared with placebo (P < 0.05, Tables 2, 3). To evaluate the reversibility of anti-platelet effect of WTE, we further performed a 2-week washout period for all volunteers after 4-week intervention. Interestingly, after a 2-week washout period, there was no significant difference in the percentage of ADP- or collagen-induced activated GPIIbIIIa positive platelets, the percentage of ADP- or collagen-induced P-selectin positive platelets, platelet aggregation stimulated by ADP or collagen, plasma β-TG and TXB₂ between 150 mg/day WTE group and placebo group (P > 0.05, Supplementary Tables 6, 7). These results showed that effects of 4-week WTE supplement on platelet activation, aggregation, and granule contents are reversible.

Considering the anti-platelet ability of WTE, we carefully determined whether the clotting pathways were affected by WTE alongside anti-platelet effects. We therefore examined the coagulation function of subjects and found that 4-week intervention of WTE had no any adverse effects on PT, PT-INR, PT-R, APTT, TT, and Fib in Chinese healthy middle-aged and older individuals (P > 0.05, Table 4). Similarly, there was also no significant difference between two groups in urea, UA, creatinine, alanine aminotransferase, total protein, albumin, and globulin at baseline and after intervention, and these results suggested that 4-week intervention of WTE had no significant effect on liver and kidney function in Chinese healthy middle-aged and older individuals (P > 0.05, Table 1). In addition, no adverse events were reported during the intervention period.

There are some strengths and limitations in our present study. The double-blind, randomized, placebo-controlled, and crossover trial design is the major strength of this study. Different from the previous trials only focused on the effects of single-dose WTE supplementation after 3 or 7 h in Britain, our present crossover trial up to 4-week WTE supplementation can be more conducive to guiding the application of WTE in Chinese. Another strength is that we tested platelet activation and aggregation at baseline and during follow-up, which are required to be detected within two hours right after blood collection, to determine the effect and reversibility of WTE on platelet in Chinese healthy middle-aged and older individuals. To monitor their dietary habits and physical activity, we maintained their usual dietary intake and physical activities via 24-h dietary recall data on 3 consecutive days and an IPAQ during the trial. It may be another strength of our study. Meanwhile, some limitations also existed in the study. The findings of our study which focused on Chinese healthy middle-aged and older individuals just indicate a potential benefit for primary prevention of CVDs in Chinese healthy middle-aged and older individuals, and the primary or secondary prevention of WTE on individuals with metabolic diseases merits further clinical study. As in most other randomized controlled trials using the maximum-reversible platelet aggregation as main outcome (51–53), our present study only focused on the effect of WTE on the maximum-reversible platelet aggregation, without evaluating its effect on the maximum irreversible aggregation rate. This may be another limitation of our study.