Cardiovascular diseases (CVDs) are associated with high mortality, which is mainly due to a relative increase in the unhealthy lifestyle and the aging population.
Epidemiological studies have shown that diets rich in fruits and vegetables may prevent from CVDs. This protective effect might be related to their bioactive compounds, which has been described for fresh and processed tomatoes ( Solanum lycopersicum ) providing a cardioprotective effect through antioxidant (1) and antiplatelet activities (2) and reduction of blood lipid levels (3). The consumption of tomato products was shown to reduce the oxidative stress induced by postprandial lipidemia and associated inflammatory response (4). Patients suffering from atherosclerosis showed a significantly lower level of serum lycopene.
Pomace is a byproduct of industrial processing of tomatoes into paste and canned products. It mainly consists, of seeds and the peel. However, apart from lycopene, pomace still contains other valuable compounds exerting complementary biological activities. Therefore, the evaluation of its qualitative and quantitative compositions regarding its utilization as a possible functional ingredient is of particular interest.
Platelets and atherogenesis: Platelet anti-aggregation activity and endothelial protection from tomatoes (Solanum lycopersicum L.) - 2012.
Antioxidant and Antiplatelet Activities in Extracts from Green and Fully Ripe Tomato Fruits (Solanum lycopersicum) and Pomace from Industrial Tomato Processing - 2013.
Platelet anti-aggregation activity of bioactive compounds from tomatoes
It has been observed that the tomato has platelet anti-aggregation activity in vitro and in vivo by inhibiting platelet aggregation induced by ADP and collagen (5). The various platelet anti-aggregation activity levels observed in different varieties of tomatoes can be explained by the existence of one or more bioactive compounds or different concentrations of the same compound. The platelet anti-aggregation activity of aqueous and methanol extracts of tomatoes in vitro were similar. Both types of extract showed inhibition of platelet aggregation (30-40%) at 1 mg/ml induced by ADP. When collagen was used as agonist, inhibition was lower, whereas the use of arachidonic acid and peptide receptor activator of thrombin showed no inhibitory effect. The experimental results obtained by Fuentes et al (2) indicate that aqueous and methanol extracts resuspended in 0.9% saline exhibit a pH of 4.5; when resuspended in more acidic (pH 2.0) and basic (pH 10.0) suspensions, they maintained their inhibitory activity of maximum platelet aggregation. As we know that carotenoids are unstable at pH extremes, this finding may exclude the possibility that these antioxidant compounds exhibit platelet anti-aggregation activity. In addition, the platelet anti-aggregation activity is inversely related to tomato ripening and the increase in the concentration of lycopene. In the study by Fuentes et al (2), aqueous and methanol extracts under various temperatures (22, 60 and 100 ̊C) maintained their platelet anti-aggregation activity, indicating that the active compounds with platelet anti-aggregation activity present in the two extracts were not affected by heat treatment.
There are a wide range of bioactive compounds in tomatoes with platelet anti-aggregation activity. Based on the results, the bioactive compounds of tomatoes have thermal and acid-base stability, are devoid of lycopene and have low molecular weight (<1000 Da).
Bioassay-Guided Isolation and HPLC Determination of Bioactive Compound That Relate to the Antiplatelet Activity (Adhesion, Secretion, and Aggregation) from Solanum lycopersicum - 2012.
The results of platelet aggregation induced by the agonists ADP, collagen, TRAP-6, and arachidonic acid, respectively, with added extracts from green and fully mature tomatoes are presented in Table 1. Both extracts from green ripe and fully mature tomatoes inhibited platelet aggregation induced by ADP and collagen, respectively, but to a different extent. For the extract obtained from different tissues of red-ripe tomatoes, inhibition of platelet aggregation induced by ADP compared to control (𝑃 < 0.05) was in the following order: myxotesta of the seeds (65 ± 2%) > pulp (41 ± 4%) > peels (40 ± 3%). For the extracts from green ripe tomatoes, relative inhibition of platelet aggregation induced by ADP was even greater amounting to 51 ± 5% for the extract from seed myxotesta than for the pulp extract (44 ± 8%, 𝑃 < 0.05) and peels (1±1%, 𝑃 > 0.05). Despite their different ripeness stage, the relative inhibition of platelet aggregation did not differ significantly (𝑃 > 0.05) for the pulp extracts from green and red tomatoes amounting to 44 ± 2 and 41 ± 3%, respectively. Inhibition of platelet aggregation induced by collagen compared to negative control was in the following order (𝑃 < 0.05): extract from seed myxotesta of red ripe tomatoes (43 ± 4%) > peels (21 ± 3%) > pulp (19 ± 2%), while the extract from green tomato pulp only showed 18 ± 1% inhibition of platelet aggregation (𝑃 < 0.05). Extracts from the pulp and seed myxotesta of red ripe tomatoes displayed a net lag time of 118 ± 1 and 205 ± 1 s, respectively (𝑃 < 0.05), in the platelet aggregation assay induced by arachidonic acid, reaching the maximum aggregation rate of >80% at 360 s.
Each of the pomace extracts exerted a potent inhibition of platelet aggregation induced by ADP, collagen, TRAP-6 and arachidonic acid, respectively (Table 2). Considering the different agonists tested in this study, the inhibition of platelet aggregation by the aqueous pomace extract was in the following order: collagen (36 ± 2%) > ADP (35 ± 3%) > TRAP-6 (22 ± 4%) > arachidonic acid (19 ± 2%) as compared to control (𝑃 < 0.05). When testing the aqueous extract of seed mucilage, inhibition of platelet aggregation was in the following order: collagen (80 ± 2%) > ADP (53 ± 4%) > TRAP-6 (35 ± 3%) > arachidonic acid (30 ± 4%) relative to the negative control (𝑃 < 0.05). Finally, the petroleum ether extract of seed mucilage inhibited platelet aggregation in the following order: collagen (80 ± 3%) > arachidonic acid (76 ± 2%) > TRAP-6 (74 ± 4%) > ADP (69 ± 3%) compared to negative control (𝑃 < 0.05).
Characteristics of Tomato Pulp
The approximate chemical composition of tomato pulp showed moisture 94 ± 2%, protein 12 ± 0.1%, fat 3 ± 0.1%, ash 15 ± 0.1%, carbohydrate 63 ± 0.4%, and crude fiber 7 ± 0.2%. Eighteen grams (18g) (0.3%w/w yield) of a yellow aqueous extract were obtained from 6kg of tomato pulp. Such extract showed the highest yield over ethyl acetate extract (0.05% w/w yield) and petroleum ether extract (1.4 × 10−3% w/w yield). When comparing different types of extracts, differences in their antioxidant potential were significant. At a concentration of 1000 μg/mL, ethyl acetate extract (87 ± 2%) was superior to ether petroleum (6 ± 2%, P < 0.05) and aqueous extract (15 ± 5%, P < 0.05).
Isolation of Antiplatelet Compound
To advance in the search for a bioactive compound with antiplatelet activity, the aqueous extract was subjected to repeated permeation. The fractions were monitored and two subfractions were identified (subfractions A and B). Since platelet aggregation induced by ADP was completely inhibited by subfraction B at 1mg/mL further purification was carried out (Figure 1 (a)).
The subfraction B was applied on semipreparative TLC. Under UV light (254 nm) three bands (BA, BB, and BC) were observed and removed, and extracted with methanol. Then each band was filtered and evaporated under vacuo. Since platelet aggregation induced by ADP was completely inhibited by BC at 1 mg/mL, further identification of compound was carried out.
Identification of the Antiplatelet Compound
The BC band was identified as adenosine according to the UV spectrum (λmax = 221 and 261nm). The structure was confirmed by NMR spectroscopy.
Effect of Aqueous Extract and Adenosine on Platelet Secretion and Aggregation
The results of platelet aggregation induced by the agonist ADP with extract, fraction, and adenosine (band C) are presented in Figure 1 (a). After the liquid-liquid separation, the inhibition of platelet aggregation induced by ADP in aqueous extract increased to 54 ± 8% (P < 0.05). As well as inhibiting the platelet aggregation, the aqueous extract inhibited the platelet secretion in 50 ± 5%. The inhibited ADP-induced platelet aggregation of adenosine was concentration dependent (2.3–457μM), in which a concentration of 4.6 μM inhibited 50 ± 12% platelet aggregation (P < 0.05). At the same concentration, it completely inhibited platelet secretion.
The role of tomato in CVD
The development and progression of CVD lies in the interactive processes of atherosclerotic lesions and thrombus formation, an interaction established primarily by platelet-endothelial binding (6). The activation of vascular endothelium occurs early in the development of atherosclerosis, where the inflammatory component, present in all phases of atherosclerosis, is a vascular response to guard against cardiovascular risk factors (i.e., hypertension, diabetes, smoking and obesity). This inflammatory process generates a microenvironment characterized by oxidative stress and cell damage. The process triggers a loss of endothelial function through a decrease in the bioavailability of nitric oxide and the physiological mechanisms of cardiovascular protection that are derived from it.
The role of platelets in arterial thrombosis is well known (7). When there is a damaged atheromatous plaque, platelets adhere, secrete their contents, and then attach to it. This activation causes a redistribution of anionic phospholipids, creating a negatively charged surface which, in addition to the synthesis and expression of tissue factor, favors the consecutive formation of protein complexes in coagulation and fibrin and the consolidation of the thrombus.
Epidemiological studies have demonstrated the cardiovascular protective role of a healthy diet (8). In this context, the beneficial effects of fruits and vegetables (F&V) may be related to the bioactive compounds found therein, which explains the increasing amount of attention in research on phytochemicals in the prevention of CVD. In addition to their nutritional value, tomatoes ( Solanum lycopersicum L. ), fresh or processed, have been found to provide a cardioprotective effect at both the endothelial and platelet levels.
Platelets are not only involved in the inflammatory complications of the atheromatous lesion but are also involved in the initiation and progression of atherosclerotic plaque. Accordingly, platelets act as a bridge between the inflammatory processes characteristic of atherosclerosis and thrombosis.
Union of activated platelets to ECs in the normal state.
Endothelium in the normal state plays a fundamental role in regulating the hemostatic balance through various mechanisms of antiplatelets, anticoagulants and fibrinolysis, which are regulated by the secretion of NO and prostacyclin.
In vitro studies have demonstrated platelet adhesion to ECs in the normal state. Platelet adhesion occurs because it is activated in circulation. By contrast, in other in vivo studies, the binding occurs under shear conditions. Once activated, platelets may adhere to ECs and promote local vascular inflammation through inflammatory mediators such as the secretion of chemokines, which disrupt normal functioning of the endothelium. For example, platelets store and express CD40L (inflammatory modulator) on their surface, releasing the protein into the environment once they are activated. CD40L also induces the expression and release of metalloproteinases, which degrade extracellular matrix proteins that are exposed to the circulation when damage occurs at the endothelial level. In addition to the release of sCD40, IL-1β, which promotes increased IL-6 and IL-8 and the expression of cell adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the EC, is also released. This leads to the recruitment of leukocytes to the site from which the injury (i.e., damaged endothelium) originated. The leukocyte-platelet interaction causes a wide range of responses in the innate and adaptive immune systems, giving the platelet a new function as regulator of the immune system, which contributes to the pathogenesis of an inflammatory response.
Moreover, animal models have shown a high level of P-selectin expression and endothelial growth factor (VEGF) in atherosclerotic plaques with involvement in the progression of CVD. Platelet factor 4 (PF-4) and platelet-derived growth factor (PDGF) molecules released from activated platelets cause chemotaxis of monocytes and other leukocytes on the EC, and promote the retention of low density lipoprotein (LDL) and oxidized LDL in the subendothelium. In patients with CVD, the presence of oxidized LDL increase in chemoattractant capacity. It has been shown that PF-4 have heterophile actions, and that the latter must undergo structural modifications in order to amplify its effects on monocytes. PDGF stimulates smooth muscle cell proliferation, causing hyperplasia of the intima layer of the arterial wall, thus acting as an amplifier of the inflammatory response.
The initiation and development of CVD is marked by platelet-endothelial interaction. This interaction promotes the expression of adhesion molecules on the endothelium and the recruitment of inflammatory cells, and stimulates the activation of circulating platelets. In the prevention of CVD, the consumption of F&V is crucial. At the level of primary prevention, tomato consumption promotes cardiovascular health through its role in platelet anti-aggregation activity and its endothelium-protective effects. Platelet antiaggregation activity is regulated by one or more bioactive compounds that act on ADP and collagen receptors.
The antiplatelet activity is specific for adenosine, so that, it is possible to establish the degree of such activity in the different types of extracts. The extracts of ripe tomato fruits and their processing by-products due to their adenosine content may be used as functional ingredients adding antiplatelet activities to processed foods which may be supportive in the primary prevention of CVD.
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