Dimethylsulfoxide (DMSO), bovine serum albumin (BSA), thiobarbituric acid (TBA), fibrinogen were acquired from Sigma-Aldrich (St. Louis, MO., USA). All other reagents were purchased from commercial suppliers, including POCH (Poland), Chempur (Poland), Chrono-log (Poland), and Kselmed (Poland). Ultrapure water was prepared in-house using a Milli-Q water purification system (Millipore, Milford, MA, USA).
A stock solution of commercial product – Aronox (Aronia melanocarpa berry extract, Agropharm Ltd., Poland) was prepared in H2O.
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°09'15.0"N 21°59'47.1"E, in 2019. The samples were shredded, frozen and freeze dried (CHRIST Gamma 2-16 LSC Freeze Dryer, Osterode am Harz, Germany), and stored in the Department of Biochemistry and Crop Quality of the Institute in Puławy, Poland. The plants were identified by Katarzyna Adamczyk: the owner of a private farm. The sample voucher for this material has been deposited in the Institute's collection under the deposit number 42/2019/IUNG, 43/2019/IUNG, 44/2019/IUNG, 45/2019/IUNG, and 46/2019/IUNG, respectively. All plant studies involved in the research were carried out in accordance with relevant institutional, national or international guideline. The entire section was previously described in Rolnik et al.10.
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 previously10.
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.
Both identified phenylethanoid glycosides, zizybeoside I and forsythoside E (isomer I), were present in zucchini. Of the identified phenolic acids, the sinapic acid hexoside was found in pumpkin and the salicylic acid O-glycoside in cucumber. Both pattypan squashes contain diterpenoids: cinncassiol A in the white pattypan and adenostemmoic acid C in the yellow pattypan. Among the flavonoids,7-methylquercetin-3-galactoside-6''-rhamnoside-3'''-rhamnoside, quercetin-3-O-rutinoside(rutin),isorhamnetin 3-O-rutinoside (narcissin) andhesperetin 7-O-(2'',6''-di-O-α-rhamnopyranosyl)-β-glucopyranoside, were identified.
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 (Tab. 1)10.
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 detached from fresh blood through differential centrifugation, as described previously11,12. Following this, the platelets were suspended in Barber’s buffer, in a modified Tyrode’s buffer (pH 7.4). The amount of platelets used for the test reached 1.5–2.0 × 108/mL and were measured using a UV-Visible Helios α spectrophotometer at 800 nm. For each experiment, blood or blood platelets were incubated for 30 minutes, at 37°C with vegetable preparations at final concentrations of 5 and 50 μg/mL.
Confirmation by human participants
All experiments were approved by the University of Lodz Committee for Research on Human Subjects and carried out under permission number 8/KBBN-UŁ/III/2018.
We confirm that all experiments were performed in accordance with relevant guidelines and regulations. All donors were informed about the purpose of the study and gave their informed consent to participate.
Effect of vegetable preparations on hemostasis parameters
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 minutes at 37°C, and then for another 15 minutes 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 10-fold in sterile PBS with Mg2+, and then stained with 3 μL of anti-CD61/PerCP, anti-CD62/PE, or PAC- 1/FITC antibodies for 30 minutes 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 minutes at 37°C.
The platelets were counted using an LSR II Flow Cytometer (Becton Dickinson, San Diego, CA, USA), based on the fluorescence of 5,000 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 CD62P-positive 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)13,14,15.
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 minutes at 37°C. Then the samples were transferred to the PL-chip. The results were recorded as AUC10 i.e., Area Under the Curve16.
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 plates were incubated with selected cucurbit preparations for 30 minutes 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, 2M 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 (0.14 M NaCl, 0.014 M Tris, 10 mM glucose; pH 7.4), whose expression was assumed to be 100%17,18.
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 0.1M 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-minute period. Absorbance was measured at λ=340 nm using a SPECTROstarNanoMicroplate Reader (BMG LABTECH, Germany)19.
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 minutes 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)20, 21.
Several tests were used to carry out the statistical analysis. All the values were expressed as mean±SE. 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.