Preparations from selected cucurbit vegetables as antiplatelet agents

Increased blood platelet activation plays an important role in cardiovascular diseases (CVDs). Recent experiments indicate that certain fruits and vegetables, including onion, garlic, and beetroot, have anti-platelet potential and therefore may reduce the likelihood of CVDs. While vegetables from the Cucuritaceae family are known to exerting beneficial antioxidant and anti-inflammatory effects, their effects on blood platelet activation are poorly understood. Therefore, the aim of the present study was to determine the effect on platelet adhesion of preparations from selected cucurbits: pumpkin (Cucurbita pepo; fruit without seeds), zucchini (Cucurbita pepo convar. giromontina; fruit with seeds), cucumber (Cucumis sativus; fruit with seeds), white pattypan squash (Cucurbita pepo var. patisoniana; fruit without seeds) and yellow pattypan squash (Cucurbita pepo var. patisoniana, fruit without seeds). It also evaluates the activity of these preparations on enzymatic lipid peroxidation in thrombin-activated washed blood platelets by TBARS assay. The study also determines the anti-platelet properties of these five cucurbit preparations in whole blood by flow cytometry and with the total thrombus-formation analysis system (T-TAS) and evaluates the cytotoxicity of the tested preparations against platelets based on LDH activity. The results indicate that the yellow Cucurbita pepo var. patisoniana preparation demonstrated stronger anti-platelet properties than the other tested preparations, reducing the adhesion of thrombin-activated platelets to collagen/fibrinogen, and inhibiting arachidonic acid metabolism and GPIIb/IIIa expression on 10 µM ADP-activated platelets. None of the preparations was found to cause platelet lysis. Our findings provide new information on the anti-platelet activity of the tested cucurbit preparations and their potential for treating CVDs associated with platelet hyperactivity.


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| (2021) 11:22694 | https://doi.org/10.1038/s41598-021-02235-w www.nature.com/scientificreports/ in facilitating interactions with potential ligands. During its resting state, GPIIb/IIIa is bent and its "headpiece" is closed, thus reducing its affinity for physiological ligands; however, stimulation by certain stimulatory signals, such as from an inside-out signal, causes conformation changes that expose the extracellular binging domain 6,7 . Platelet activation can be stimulated by multiple pathways; however, two key routes are connected with signal transduction pathways and have their foundation in membrane glycoproteins that are solely expressed in platelets. One pathway is based on the activation of platelets through G protein-coupled receptors, leading to the release of ADP and thromboxane A 2 , resulting in an increase of cytosolic calcium concentration. This initiates specific signaling pathways, and causes further platelet activation. Alternatively, the coagulation pathway generates thrombin, a highly potent platelet activator which is needed to convert fibrinogen into fibrin to stabilize the platelet "plug". Thrombin activates blood platelets through protease activated receptors (PAR) on the platelet surface 1 . After activation, the platelets release P-selectin from α-granules to the surface. P-selectin is a ligand responsible for the interaction between platelets, leukocytes and endothelial cells, and plays a key role in linking hemostasis and inflammation 7 .
In addition, platelet activation can result in the activation of cytosolic phospholipase A 2 (cPLA 2 ), which generates arachidonic acid from phospholipid membranes. After formation, arachidonic acid is available for oxidization by cyclooxygenase (COX-1) or lipoxygenase . The results of oxidation are dependent on the type of blood cell. While oxidized arachidonic acid generates prostaglandin E 2 and leukotriene B 4 in leukocytes, it results in the production of thromboxane A 2 and 12-hydroxy-5,8,10,14-eicosatetraenoic (12-HETE) in platelets 8 . Arachidonic acid can be also oxidized by cytochrome P 450, a heme-containing enzyme found in different tissues 9 . Hemostasis is not the only function of platelets, due to their high sensitivity to different diseases states, which make them one of the most accessible markers. Platelets interact with leukocytes and endothelium cells and their reactivity for various pathogenesis states are widely dependent upon active markers, including CD36, CD41, CD42a, CD42b, CD61. Platelets are also able to release and transfer many substances, which interact with endothelium cells. They can store high amount of amyloid precursor protein and its metabolism may accumulated Aβ in the brain, leading to its vasculature through blood brain barrier. In renal diseases platelets are in the presence of toxic products in the circulation leading to the form of bleeding diatheses, which are hemorrhage and other pathological feature like thrombocytopenia and glomerular thrombosis. Moreover, blood platelets play also an important role in metastasis 10 .
The members of the Cucurbitaceae family are rich in phenolic acids, flavonoids and terpenoids, all of which show strong antioxidant activity and may influence various parts of hemostasis. Oxidative stress is a risk factor in cardiovascular disease and hemostasis disorders. The components of cucurbits have been found to have a positive effect on biomarkers of oxidative stress in plasma 11 . Indeed, our previous studies have found selected vegetables from the Cucurbitaceae family, including pumpkin, zucchini, cucumber, yellow pattypan squash and white pattypan squash, to contain various secondary metabolites with antioxidant activity 11 ; however, the influences of these vegetable preparations on the biological properties of blood platelets remain unknown. Therefore, to continue our previous research, the present study examined the anti-platelet potential of the same five cucurbit preparations, viz. pumpkin (Cucirbita pepo; fruit without seeds), zucchini (Cucurbita pepo convar. giromontina; fruit with seeds), cucumber (Cucumis sativus; fruit with seeds), white pattypan squash (Cucurbita pepo var. patisoniana; fruit without seeds) and yellow pattypan squash (Cucurbita pepo var. patisoniana, fruit without seeds) in tested washed human blood platelets and human whole blood in vitro. In addition to their anti-adhesive action, the present study also examines the activity of these preparations on enzymatic lipid peroxidation-arachidonic acid metabolism in thrombin-activated washed blood platelets based on thiobarbituric acid reactive substances (TBARS) assay. In addition, the present study also determines the anti-platelet properties of these five cucurbit preparations in whole blood using flow cytometry and a total thrombus-formation analysis system (T-TAS) and evaluates their cytotoxicity against platelets based on extracellular lactate dehydrogenase (LDH) activity. The action of cucurbit preparations was compared to commercial product-Aronox (Aronia melanocarpa berry extract with anti-platelet and antioxidant activities 12,13 .
A stock solution of commercial product-Aronox (Aronia melanocarpa berry extract, Agropharm Ltd., Poland) was prepared in H 2 O. Plant material. Obtained vegetable preparations. Five of the most well-known and easily-available types of cucurbit vegetables were selected for the study, these being pumpkin (Cucurbita pepo L., fruit without seeds); zucchini (Cucurbita pepo L. convar. Giromontina, fruit with seeds); cucumber (Cucumis sativus L., fruit with seeds); white pattypan squash (Cucurbita pepo L. var. patisoniana, fruit without seeds) and yellow pattypan squash (Cucurbita pepo L. var. patisoniana, fruit without seeds). All obtained materials were bought from organic farming in Poland 51°09′15. www.nature.com/scientificreports/ relevant institutional, national or international guideline. The entire section was previously described in Rolnik et al. 11 .
Extraction and chemical analysis of vegetable preparations. The extraction process was performed based on the following conditions: extraction solvent: 80% methanol, solvent pressure: 1500 psi, extraction cell temperature: 40 °C, extraction cycles: three, using an automatic extractor (Dionex ASE 200 Accelerated Solvent Extraction System). The extracts were dried by evaporation under reduced pressure, at 40 °C (HeidolphHei-Vap Advantage, rotary evaporator). The five preparations were purified from mostly sugars using solid phase extraction (SPE), as described previously 11 . The most diverse phytochemical profile was demonstrated by the zucchini preparation, and the least by the cucumber. Almost all identified compounds could be classified as phenylethanoids, flavonoids, glycoside lipids or fatty acids. The pumpkin and cucumber contained, inter alia, kaempferol and synaptic acid; while the other three preparations only contained phenylethanoids as glycosides.
Glycerophospholipids were identified in all cucurbit preparations. Fatty acids such as linoleic acid and octadecadienoic acid derivatives were also found in all the tested vegetables; however, the γ-linolenic acid derivative was present only in zucchini and yellow pattypan squash (Table 1) 11 .

Stock solutions of vegetable preparation.
To analyse the biological activity, stock solutions of the vegetable preparations were dissolved in 50% DMSO. The final concentration of DMSO in the samples (human plasma) was lower than 0.05% and its effects were determined in each experiment.
Blood and blood platelets. Isolation of blood platelets. Human blood was collected from healthy, medication-free volunteers in the Medical Center in Lodz; all of whom reported not smoking or consuming alcohol. The blood was collected into tubes with citrate/phosphate/dextrose/adenine (CPDA) anticoagulant. Blood platelets were separated from fresh blood through differential centrifugation, as described previously 14,15 . Following this, the platelets were suspended in Barber's buffer, in a modified Tyrode's buffer (0.14 M NaCl, 0.014 M Tris,

Effect of vegetable preparations on hemostasis parameters. Flow cytometry.
To study the effects of the cucurbit preparations on the reactivity and activation of resting and stimulated blood platelets, whole blood models were used. Firstly, whole blood was incubated with preparations from selected cucurbit vegetables for 15 min at 37 °C, and then for another 15 min at room temperature (RT) with the addition of 10 and 20 μM ADP or collagen as platelet agonists. After incubation, the tested samples were diluted tenfold in sterile PBS with Mg 2+ , and then stained with 3 μL of anti-CD61/PerCP, anti-CD62/PE, or PAC-1/FITC antibodies for 30 min at RT in the dark. Isotype controls were also prepared; these contained resting blood samples stained with 3 μL of anti-CD61/PE and isotype control antibodies marked with FITC/PEisotype. Finally, all samples were fixed with 1% CellFix for 60 min at 37 °C.
The platelets were counted using an LSR II Flow Cytometer (Becton Dickinson, San Diego, CA, USA), based on the fluorescence of 10,000 platelets (CD61/PerCP positive objects). The platelets were distinguished from other blood cells by a forward light scatter (FCS) vs. side light scatter (SSC) plot on a log/log scale (first gate) and by positive staining with monoclonal anti-CD61/PerCP antibodies (second gate). The percentages of CD62Ppositive and PAC-1-positive platelets were calculated in each sample. All results were analyzed using FlowJo _v.10.7.2 (Becton Dickinson, San Diego, CA, USA) 12,15,16,17 .

Total Thrombus Formation Analysis System (T-TAS)
. T-TAS was used to determine the thrombus formation process under flow conditions using the PL-chip microchip coated with collagen. Fresh whole blood collected on BAPA (benzylsulfonyl-d-arginyl-prolyl-4-amidinobenzylamide) was incubated with preparations from the five cucurbit samples for 30 min at 37 °C. Then the samples were transferred to the PL-chip. The results were recorded as AUC 10 i.e., Area Under the Curve 18 .
Platelet adhesion. Platelet adhesion was measured based on the activity of exoenzyme acid phosphatase in platelets. The plates were coated with 0.04 mg/mL collagen or 2 mg/mL fibrinogen. After isolation from fresh blood, the blood platelets were incubated with selected cucurbit preparations for 30 min at 37 °C. Next samples, which contain platelets and cucurbits preparations were added on plates and left to adhere for hour at 37 °C. The platelets were then dissolved with Triton X-100 and treated with the phosphatase substrate (p-nitrophenylphosphate), resulting in the formation of p-nitrophenol. The level of p-nitrophenol were measured at λ = 405 nm using a SPECTROstarNanoMicroplate Reader (96-well microtiter plates, BMG LABTECH, Germany). To achieve a color reaction in the samples, 2 M NaOH was added. All readings were taken in reference to the control sample containing only blood platelets with Barber's buffer, in a modified Tyrode's buffer, whose expression was assumed to be 100% 13,19 . Effect of vegetable preparations on parameters of damages. Activity of LDH. The cytotoxic effects of the selected Cucurbitaceae preparations on blood platelets were evaluated based on the release of lactate dehydrogenase (LDH) from the platelets. After incubation, the test samples were centrifuged for 15 min at 25 °C at 2500 rpm, and 10 μL of supernatant was transferred to a microtiter plate. The plate was then loaded with 270 μL of 0.1 M phosphate buffer and 10 μL of NADH. After a 20-min incubation at room temperature, 10 μL of pyruvate (5 mg) was added and the absorbance measured immediately afterwards. The further readings were taken at one-minute intervals over a 10-min period. Absorbance was measured at λ = 340 nm using a SPEC-TROstarNanoMicroplate Reader (BMG LABTECH, Germany) 20,21 . Effect of vegetable preparations on lipid peroxidation. The level of lipid peroxidation on the blood platelets was determined based on thiobarbituric acid reactive substances (TBARS) content. The samples were mixed with 0.37% thiobarbituric and 15% trichloroacetic acid and heated for 10 min at 100 °C in a heating block. Following this, the samples were allowed to cool and centrifuged at 10,000 rpm for 15 min at 18 °C. The absorbance of the supernatant was measured at λ = 535 nm using a SPECTROstarNanoMicroplate Reader (BMG LABTECH, Germany) 22,23 . Data analysis. Several tests were used to carry out the statistical analysis. All the values were expressed as mean ± SD. First the results were checked for normality with the Kolmogorow-Smirnow test, and the equality of variance was determined with Levine's test. Statistically significant differences were identified using an ANOVA test (assuming a significance level of p < 0.05), followed by either Tukey's multiple comparisons test or the Kruskal-Wallis test as appropriate.

Effect of vegetable preparations on parameters of damages.
To determine the toxic effect of all the tested cucurbit preparations on human blood platelets, the level of extracellular LDH activity was measured.
The results indicate no significant difference in blood platelet viability after exposure to the used plant preparations at 5 and 50 µg/mL compared to control, however, these changes were not statistically significant (Fig. 1).

Effect of vegetable preparations on platelet adhesion. The anti-adhesive properties of five plant
preparations were studied in vitro using washed blood platelets. The results were presented as percent of level of adhesion for control samples. The obtained results showed the level of adhesion of resting platelets to collagen was significantly inhibited after pre-incubation with four tested preparations: pumpkin (50 µg/mL), zucchini (50 µg/mL), cucumber (5 and 50 µg/mL), and pattypan squash (white) (50 µg/mL) ( Fig. 2A).
For the ADP-stimulated platelets, all of the tested plant preparations (5 and 50 µg/mL) have not anti-adhesive properties (Fig. 3B). Fig. 4, all the tested plant preparations (5 and 50 µg/mL) altered the level of lipid peroxidation in platelets stimulated by thrombin. Most significantly, the pattypan squash (yellow) preparation (50 µg/mL) demonstrated 85% inhibition relative to the positive control.

Effect of vegetable preparations on lipid peroxidation. As shown in
Effect of vegetable preparations on the hemostasis parameters in whole blood. The samples treated with five plant preparations demonstrated different levels of blood platelet activation (measured by flow cytometry) compared with untreated whole blood control samples (Figs. 5 and 6). Only three preparations demonstrated clear changes at the highest tested concentration (50 µg/mL): the cucumber, the pattypan squash (white), and pattypan squash (yellow) preparations significantly reduced PAC-1 binding in platelets activated by 10 µM ADP (Fig. 5B).
The effects of all five tested plant preparations (at the highest tested concentration) on chosen blood platelet activation parameters are compared with aronia berry extract in Table 2. Of all preparations, the yellow pattypan squash preparation demonstrated the strongest anti-platelet properties, reducing the adhesion of thrombinactivated platelets to collagen/fibrinogen, and inhibiting arachidonic acid metabolism and GPIIb/IIIa expression on 10 µM ADP-activated platelets. In addition, aronia berry extract (50 µg/mL) decreased PAC-1 binding in

Discussion
A fundamental part of the primary and secondary treatment of atherosclerotic thrombotic disease is the use of drugs affecting platelet function. Although several such drugs are available in the clinical armory, aspirin and clopidogrel have the most favorable profile of currently-used drugs and are the most widely-studied. Aspirin plays a crucial role in preventing thromboembolic complications from atherosclerotic disease. It is believed to prevent platelet activation by permanently inactivating key platelet enzymes. In contrast, clopidogrel offers slightly better effectiveness regarding the secondary prevention of vascular events. While it has no direct antiplatelet activity of its own, it stimulates metabolites, like free thiols group to bind to P 2 Y 12 platelet receptors to form disulfide bridges with extracellular cysteine residues, leading to irreversible inhibition of ADP-induced platelet aggregation, because the P2Y 1 receptor plays an significant role in ADP -induced activation of platelets 24 .
In recent years the antiplatelet drugs are mostly related to role of mechanism of thrombus formation, which is exclusively expressed on blood platelet. Due to this important pharmacological research directions for treating In the graphs, the adhesion is expressed as a percentage of the control sample (platelets without plant preparation). Results are given as mean ± SD (n = 5). Kruskal-Wallis test: *p < 0.05, **p < 0.01, compared with control (i.e. not treated with plant preparation). There wasn't any statistically significant between effect of 5 and 50 µg/mL (p > 0.05). The baseline spectral reading (absorbance) for plant preparations range between 0.00075 and 0.0095.  25 . The greatest adverse effect for antiplatelet drugs is the increased risk of bleeding, which is associated with an elevated risk of thrombosis. Antiplatelet therapy should inhibit platelet function during periods of high thrombotic risk. In addition, to avoid the risk of recurrence of ischemic events after premature cessation of medication or non-compliance, patients often require long-term antiplatelet therapy 24 . The Cucurbitaceae family includes a range of phytochemicals, such as saponins and cardiac glycosides, which are known to influence on the cardiovascular system and are often used in the therapy of heart disease. Saponins are able to coagulate blood and reduce bleeding, and the cucurbitacins can exert an anti-atherosclerotic effect due to their ability to inhibit lipid peroxidation products, like malonaldehyde 26,27 . Cucurbitacin B has a protective effect against cardiac hypertrophy by increase autophagy among cardiomyocytes; while hypertrophy In the graphs, the adhesion is expressed as a percentage of the control sample (platelets without plant preparation). Results are given as mean ± SD (n = 5). Kruskal-Wallis test: *p < 0.05, **p < 0.01, ***p < 0.001, compared with control (i.e. not treated with plant preparation). There wasn't any statistically significant between effect of 5 and 50 µg/mL. There wasn't any statistically significant between effect of 5 and 50 µg/mL (p > 0.05). The baseline spectral reading (absorbance) for plant preparations range between 0.0025 and 0.0055. www.nature.com/scientificreports/ is a dynamic and adaptive process in physiological conditions, it can lead to pressure or volume overload, often resulting in heart failure if prolonged 28 .

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The leaves and seeds of Momoridace balsamina represent a ready source of glycosides, which can be used to treat cardiac diseases by intensifying the force of heart contraction, based on its influence on calcium release. It has also been found that zucchini can also help alleviate symptoms related with heart diseases, such as high cholesterol 27 . Other research indicates that ethanolic extract of Lagenaria siceraria (Cucurbitaceae) fruit inhibits ADP-induced platelet aggregation in vitro, and increases bleeding time and plasma re-calcification time in mice 29 . Other experiments have found cucumber sap extract to inhibit blood platelet aggregation induced by different agonists (ADP, collagen, and epinephrine) in platelet-rich plasma and to decrease plasma re-calcification time and prothrombin time 30 . In addition, a recent study by Sanzana et al. found that various pumpkin seed extracts (aqueous, ethanolic and methanolic extract) have anti-platelet properties in vitro 31 ; the extracts inhibited platelet aggregation stimulated by ADP, collagen and thrombin receptor activator peptide 6 (TRAP-6) in vitro, and reduced P-selectin expression and GPIIb/IIIa activation on blood platelets stimulated by TRAP-6 31 .
Our present findings, and those of our previous studies 11 , suggest that the selected cucurbit preparations offer promise as candidates for reducing blood platelet activation. It is worth noting that the present study used a combination of flow cytometry and T-TAS to study platelet activation in its natural environment, i.e. after blood collection and incubation with plant preparations, and that is the first paper to examine the anti-platelet potential of the five tested cucurbit preparations in two in vitro models: one based on washed blood platelets and the other with whole blood. An important, and novel, finding is that three tested cucurbit preparations: the yellow pattypan squash, white pattypan squash, and cucumber, influenced blood platelet activation, as indicted by flow cytometry. In addition, four cucurbit preparations (pumpkin, cucumber, pattypan squash (white), and pattypan squash (yellow)) changed AUC 10 values measured by T-TAS. The differences in anti-platelet potential observed between samples may be explained by their different chemical profiles. An important consideration is that the concentration of plant preparation (≤ 50 µg/mL) used in the study corresponds with physiological concentrations of phenolic compounds after oral administration 32 .
Our results indicate that the yellow pattypan squash preparation appears to demonstrate the best anti-platelet properties of the tested cucurbits. The preparation was found to inhibit adhesion of thrombin-activated platelets to collagen and fibrinogen. It also significantly inhibited GPIIb/GPIIIa activation in ADP-activated platelets; it is likely that this inhibition may be responsible for the anti-adhesive potential of this preparation. This plant preparation was found to demonstrate anti-platelet potential measured by T-TAS. Again, its strong antiplatelet activity may well be correlated with its chemical composition. Preparations from pattypan squash are include a range of phenolic derivates, many of which show anti-platelet activity. One of the main compounds found in pattypan squash was phenylpropanoid glycoside, known to demonstrate anti-platelet activity 33  www.nature.com/scientificreports/ plasma in vitro. This extract contains a high level of phenylpropanoid glycoside, and a certain amount of benzoic acid derivates 33 , both of which were identified in pattypan squash (Table 1), with the benzoic acid derivative in the pattypan squash demonstrating a strong anti-aggregation effect 33 . The batch variability of plant matter is connected with the quantitative differences in chemical composition, due to the various plant cultivation methods and soil quality. www.nature.com/scientificreports/ Pattypan squash also contains various diterpenoids, which are also known to demonstrate anti-platelet activity ( Table 1). For example, Thisoda et al. 34 found diterpenoids from Andrographi spaniculate extract to demonstrate anti-aggregatory effects of in vitro and to significantly inhibit thrombin-induced platelet aggegation 34 . However, further studies are needed to precisely identify the compound responsible for anti-platelet activity in pattypan squash. It may well be the case that this effect is derived from the synergistic action of multiple compounds together. Nevertheless, our results provide new information on the anti-platelet activity of the tested cucurbit preparations, especially the yellow pattypan squash preparation, and their possible use in CVDs associated with platelet hyperactivity.

Pattypan squash (yellow) Aronia berry extract
Adhesion of thrombinactivated platelet to collagen Inhibition of this process (anti-platelet potential)

No effect
Inhibition of this process (anti-platelet potential)

No effect
Inhibition of this process (anti-platelet potential) Inhibition of this process (anti-platelet potential) Adhesion of thrombinactivated platelet to fibrinogen Inhibition of this process (anti-platelet potential) Inhibition of this process (anti-platelet potential) No effect Inhibition of this process (anti-platelet potential) Inhibition of this process (anti-platelet potential) License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.