In Vitro and in Vivo Evaluation of the Anti-Inflammatory and Antithrombotic Efficacy of Natural Bioactives in Human Platelets

For the In vivo clinical study, blood samples were collected from all participants at baseline (day 0), prior to supplementation (t = 0), and again after 28 days of daily supplementation of VC or VCF. Platelet aggregation was evaluated using three agonists: platelet activating factor (PAF), ADP, and thrombin. Immediately after collection, blood samples collected in citrate containing monovette tubes were centrifuged at 194 × g for 18 minutes at 24 °C to isolate platelet-rich plasma (PRP). The remaining blood was subsequently centrifuged at 1465 × g for 20 minutes at 24 °C to obtain platelet-poor plasma (PPP). PRP and PPP were separated into distinct tubes for further analysis. For aggregometry assays, 250 μL of PRP containing a magnetic stir bar and 500 μL of PPP without stir bar were transferred to each aggregometer cuvette and placed at the appropriate positions at the aggregometer. Platelet aggregation was quantified by determining the mean EC₅₀ values, representing the concentration of each agonist required to induce 50% platelet aggregation. These values were normalized per gram of total content , vitamin C , and flavonoid content as regarding vitamin C and flavonoid supplement. The change in EC₅₀ values after 28 days of supplementation provided an indication of the supplement's modulatory effect on platelet aggregation