Diet of Fruits and Vegetables with Experimental Antithrombotic Effect may be Beneficial to Humans in the Prevention of Arterial Thrombotic Diseases

Prevention of arterial thrombotic disorders is a very important socioeconomic challenge. The most convenient way of prevention could be by dietary means. In testing diets for antithrombotic effect, selection of pathologically relevant models of arterial thrombosis is of crucial importance. We demonstrated that the shear-induced platelet-rich thrombosis/thrombolysis in vitro/ex vivo test, Global Thrombosis test (GTT), provided the closest approximation to the pathology of arterial thrombogenesis. We have found that specified fruit and vegetable varieties have antithrombotic activity in animal experiments using shear-induced thrombosis/thrombolysis in vitro/ex vivo and laser-induced in vivo tests and that antithrombotic activity is due to inhibition of thrombosis and/or enhancement of thrombolysis. Here we present a pilot study in a small number of volunteers on diet containing potatoes, onions and apples which were tested antithrombotic with the Global Thrombosis Test. This study shows a trend of beneficial effect of such diet in humans and encourages the organization of a large scale, well controlled crossover study to establish the real benefit of such diet in preventing atherothrombotic diseases. Ijiri Y, Ishii H, Yamamoto J (2016) Diet of Fruits and Vegetables with Experimental Antithrombotic Effect may be Beneficial to Humans in the Prevention of Arterial Thrombotic Diseases. Int J Drug Dev & Res 8: 012-016 Volume 8(3): 006-010 (2016)-07 Int J Drug Dev & Res ISSN: 0975-9344 blood with added 0.4 ml of juices from potato, onion and apple varieties was added to the GTT test tube and the antithrombotic/ thrombolytic activities were measured. Antithrombotic activity of juices prepared from potato, onion and apple varieties was measured after oral administration by the in vivo by helium neon laser-induced thrombosis test. Intake procedure of the diets over 12 weeks Both the Antithrombotic and Control groups, each of 4 males and 2 females consumed potatoes every day (mean 83 g potatoes per day), onions every day (mean 83 g onions per day) and apples with skin every day (mean 230 g per day). The study started in December and finished in March. Effects of fruits and vegetables on platelet reactivity (Occlusion Time; OT) and endogenous thrombolytic activity (Lysis Time; LT) were measured monthly with GTT. This project was approved by the Ethical Committee of Ishii Hospital and Kobe Gakuin University and was performed after informed consent from all volunteers. GTT measurement of shear-induced platelet reactivity and endogenous thrombolytic activity Blood was collected from the antecubital vein in the morning before breakfast. The first 2 ml was used for different laboratory blood tests and the next withdrawn 3.0 ml non-anticoagulated blood samples were collected and transferred into GTT test tubes. The principle and procedure of GTT measurement has been described in detail elsewhere [14,15,21,22]. In Figures 1A and 1B illustrate the embodiment and the principle of this test. Four flat segments created along the inner wall of a conical plastic tube prevent the inserted round ceramic bearing balls to occlude the lumen. When blood is added to such tube, it flows through the four narrow gaps by the balls and exits in droplets into a reservoir tube. The latter is trans-illuminated by a light emitter coupled with a sensor, to generate signal whenever a drop of blood interrupts the light path. In essence, the instrument detects the time interval (d; sec) between consecutive blood drops. Blood flows by gravity at 37°C through the narrow gaps formed between the larger upper ball and the inner wall of the tube, and the resulting high shear stress (145 dyne/cm2) activates platelets. These activated platelets remain single, because the very short transit time and high shear prevent aggregation. However, in the space distal to the large ball bearing, the low shear and turbulent flow favor large platelet aggregate formation. Furthermore, in this space between the two balls, activated platelets generate thrombin, which propagate platelet aggregation and initiates coagulation of blood. Flow then carries these fibrin-stabilized platelet aggregates into the gaps by the lower and smaller ball, where they gradually occlude the gaps and arrest the flow. Increased or decreased OT indicates lowered or enhanced platelet reactivity, respectively. At the start, blood flow is rapid and hence (d) is small. Subsequently, the flow rate gradually decreases and hence (d) increases. When the actual (d) exceeds an arbitrarily set d ≥15 sec, the instrument displays “Occlusion Time (OT)”, which is before the complete occlusion and stop of flow. OT is calculated by the instrument in seconds from the detection of the first drop of blood until d ≥15 sec. Later, the flow is completely arrested. Eventually, due to thrombolysis, flow is restored as indicated by detection of the first blood drop, and this is an indication of lysis of thrombi. Following OT, for a set time (200 sec) blood drops were not detected, because during this time the flow stops and the formed thrombi were stabilized with fibrin. Lysis Time (LT) was calculated as LT=[the time (sec) of the first drop after OT]–OT. Prolonged or shortened LT indicates inhibited or Antithrombotic diets Potato (Nishiyutaka) Onion (ONA-03) Apple (AP-13) Raw material Antithrombotic activity yes yes no Thrombolytic activity not determined not determined yes Heated material Antithrombotic activity yes yes not be done Thrombolytic activity not determined not determined not be done Non-thrombotic diets Potato Onion Apple (Cynthia) (ONA-05) (AP-12) Raw material Antithrombotic activity yes but weak no no Thrombolytic activity not determined no no Heated material Antithrombotic activity not be done not be done not be done Thrombolytic activity not be done not be done not be done Table 1: Characteristics of foods, measured by Haemostatometry or GTT (in vitro). Antithrombotic activity Potato Onion Apple (Nishiyutaka) (ONA-03) (AP-13) Raw material not be done yes yes Heated material yes yes yes Antithrombotic activity Potato Onion Apple (Cynthia) ( ONA-05) ( AP-12) Raw and heated materials not be done not be done not be done Non-thrombotic diets: Diets without the effect on thrombosis; Data cited from references 18-20 and revised. Table 2: Characteristics of diets, measured by He-Ne laser-induced thrombosis test (in vivo) Ijiri Y, Ishii H, Yamamoto J (2016) Diet of Fruits and Vegetables with Experimental Antithrombotic Effect may be Beneficial to Humans in the Prevention of Arterial Thrombotic Diseases. Int J Drug Dev & Res 8: 012-016 Volume 8(3): 006-010 (2016)-08 Int J Drug Dev & Res ISSN: 0975-9344 enhanced endogenous thrombolysis, respectively (Figure 1C). Statistical analysis Due to low number of participant, trends rather than statistically significant findings were expected. Data from GTT were analysed by repeated ANOVA (General Linear Model), followed by the multiple comparison test (Student-Newman-Keuls). Values were expressed as means ± SEM. P<0.05 was considered as the limit of significance. Analyses were performed using Statistical Package Unistat 5.6 Light (London, UK). Results and Discussion Effects of antithrombotic diet on shear-induced platelet reactivity and endogenous thrombolysis GTT tests were performed just before the start of the diet intake, at 4, 8 and 12 weeks after the start. As the present study was performed from December until March for 12 weeks, only three kinds of raw fruits and vegetables were available. Results of GTT tests are shown in Figure 2. First, the data were analysed by unpaired Student t-test. Significant difference between two groups over time was not detected. However, OT values (platelet reactivity) and LT values (endogenous thrombolytic activity) have been demonstrated to be diverse even in the same cohorts [23]. In addition, as the number of fruits and vegetables, and the number of the volunteers were small in the present study, repeated ANOVA was employed for the analysis of significance (Figure 2). No significant difference in platelet reactivity was found over time in both groups. In contrast, endogenous thrombolytic activity significantly decreased over time in the Control group, while remained unchanged in the Antithrombotic group. These findings suggest that antithrombotic foods may have prevented the increase in thrombotic status as observed in the Control group. A series of our study to find antithrombotic fruit and vegetable varieties were targeting raw materials and raw antithrombotic materials were further examined in antithrombotic activity after heat treatment. Appearance and Low shear stress with vortices Figure 1: Global Thrombosis Test (GTT). A) Embodiment of GTT with 4 gaps; B) Principle of GTT; C) A typical pattern of dropping interval over time in GTT. OT: occlusion time, index of platelet reactivity; LT: lysis time, index of thrombolytic activity. Ijiri Y, Ishii H, Yamamoto J (2016) Diet of Fruits and Vegetables with Experimental Antithrombotic Effect may be Beneficial to Humans in the Prevention of Arterial Thrombotic Diseases. Int J Drug Dev & Res 8: 012-016 Volume 8(3): 006-010 (2016)-09 Int J Drug Dev & Res ISSN: 0975-9344 0 100 200 300 400 500 before 4 8 12 0 50