Non-clinical investigations about cytotoxic and anti-platelet activities of gamma-terpinene

Non-clinical investigations about cytotoxic and anti-platelet activities of gamma-terpinene | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Non-clinical investigations about cytotoxic and anti-platelet activities of gamma-terpinene Railson Pereira Souza, Vinícius Duarte Pimentel, Rayran Walter Ramos de Sousa, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4260336/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Gamma-terpinene (γ-TPN) is a cyclohexane monoterpene, isolated from essential oils of pharmacologically active plant species, such as tea tree ( Melaleuca alternifolia ), oregano ( Origanum vulgare ), rosemary ( Rosmarinus officinalis L.), thyme ( Thymus vulgaris Marchand) and eucalyptus ( Eucalyptus sp.). Terpenes are widely studied for their recognized pharmacological actions on the cardiovascular system, hemostasis and antioxidant actions. The objective of this study was to investigate the cytotoxic and antiplatelet activity of γ-TPN in non-clinical study models. For the in silico evaluation, the PreADMET, SwissADME and SwissTargetPrediction software were used. Molecular docking was performed using the AutoDockVina and BIOVIA Discovery Studio databases. The cytotoxicity of γ-TPN was analyzed by the MTT assay with normal murine endothelial (SVEC4-10) and fibroblast (L929) lines. Platelet aggregation was evaluated with platelet-rich (PRP) and platelet-poor (PPP) plasma from spontaneously hypertensive rats (SHR), in addition to SVEC4-10 cells pre-incubated with γ-TPN (50, 100 and 200 µM) for 24 h. In in vivo tests, SHR animals were also used, pre-treated by gavage with γ-TPN for 7 days, distributed into four groups (control, 25, 50 and 100 mg/Kg). At the end, blood samples were collected to measure nitrites using the Griess reagent. γ-TPN proved to be quite lipid-soluble (Log P = + 4.50), with a qualified profile of similarity to the drug, good bioavailability, and adequate pharmacokinetics. The monoterpene exhibited affinity mainly for the P2Y12 receptor (6.450 ± 0.232 Kcal/mol), moderate cytotoxicity for L929 (CC 50 = 333.3 µM) and SVEC 4–10 (CC 50 = 366.7 µM). The presence of γ-TPN in SVEC 4–10 cells was also able to reduce platelet aggregation by 51.57 and 44.20%, respectively, at the lowest concentrations (50 and 100 µM). It was concluded that γ-TPN has a good affinity with purinergic receptors and an effect on the reversal of platelet aggregation and oxidative stress, being promising and safe for therapeutic targets and subsequent studies in the control of thromboembolic diseases. Cyclohexane monoterpenes molecular docking purine receptors toxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cardiovascular diseases (CVDs) are a group of chronic non-communicable diseases that affect the heart and blood vessels, being responsible for 17.7 million deaths worldwide, most of them (85%) associated with the occurrence of thromboembolic events as heart attack and stroke (Benjamin et al. 2019 ; Massa et al. 2019 ; World Health Organization 2019). It is widely accepted that platelets play a fundamental role in hemostasis and blood clotting at sites of vascular injuries, representing a link between thrombosis, inflammation and atherogenesis. When blood vessels are injured, platelets are activated by the exposed subcutaneous matrix and aggregate at the site of injury to stop bleeding, causing thrombotic occlusive ischemic events (Papapanagiotou et al. 2016 ; McFadyen et al. 2018 ; Rengasamy et al. 2019 ; Olgasi et al. 2024 ). The development of drugs that inhibit platelet aggregation has been one of the main targets for the prevention and treatment of thrombotic brain and cardiovascular diseases (Xiang et al. 2019 ; Iqbal et al. 2020 ; Gerhards et al. 2021 ). Thrombosis therapy involves the use of P2Y12 purinergic receptor inhibitors, such as ticlopidine, clopidrogrel, prasugrel, cangrelor and ticagrelor (Mackman 2008 ; Hamilton 2009). However, limitating adverse effects increase the risk of intracranial hemorrhage and dyspnea (Berger 2018 ). In this context, the use of medicinal plants can be a complementary alternative (Simonetti et al. 2016 ; Sales & Alencar 2019 ; Lima et al. 2019 ; Mortada 2024 ). Medicinal plants contain essential oils (EOs), secondary metabolites of low molecular weight molecules ofteh used as drugs, aromas and fragrances, pharmaceutical products, agrochemicals, dyes and pigments, pesticides, cosmetics, food additives, among others (Alves-Silva et al. 2020 ; Çakmak et al. 2020 ; Toledo et al. 2020 ; Rocha et al. 2022 ). In the pharmacological point of view, terpenes stand out, namely gamma-terpinene (γ-terpinene, γ-TPN or 1-isopropyl-4-methylcyclohexa-1,4diene), a monoterpene found in “melaleuca” ( Melaleuca alternifolia ), “orégano” ( Origanum vulgare ), rosemary ( Rosmarinus officinalis L.), thyme ( Thymus vulgaris Marchand), tangerine ( Citrus delicious ) and eucalyptus ( Eucalyptus sp.) (Silva et al. 2015 ; Ramalho et al. 2016 ; Baldissera et al. 2016 ; Khan et al. 2020 ; Xu et al. 2020 ; Koyama & Heinbockel 2020 ; Mota 2022 ). Taking into account the biological properties of γ-TPN that have been elucidated so far, such as: antimicrobial activity (Piaru et al. 2012 ; Ramalho et al. 2016 ), in vitro antioxidant and preventive potential for chronic and cardiovascular (Choi et al. 2000 ; Li & Liu 2009 ); anti-inflammatory (Ramalho et al. 2016 ), antinociceptive (Passos et al. 2015 ) and lipid-lowering (Takahashi et al. 2003 ), the objective of this article was to investigate the cytotoxic and antiplatelet activity of this compound in study models non-clinical. Materials and methods Chemicals Gamma-terpinene (γ-TPN), sodium nitrite, Griess reagent, xylazine, adenosine diphosphate (ADP), sodium phosphate and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Fetal calf serum, DMEM, DMEM High, trypsin-EDTA, penicillin and streptomycin were purchased from Cultilab (Campinas, Brazil). Tween 80 and dimethylsulfoxide (DMSO) were obtained from Dinâmica Química Contemporânea LTDA (São Paulo, Brazil). Ketamine, ammonium oxalate and sodium thiopental were obtained from Cristalia (São Paulo, Brazil). Stock solutions were prepared with distilled water or DMSO and diluted to appropriate concentrations. γ-TPN was dissolved in DMSO for in vitro and in vivo protocols. Tween 80 (0.1% v/v) was used as eluent. All solutions were stored at 0°C. Cells and animals’ facilities Murine axillary lymph node endothelium-like cell lines (SVEC4-10) and murine fibroblasts (L-929) were maintained in DMEM and DMEM high glucose cell media, 10% fetal serum fetal bovine and 1% (w/v) penicillin/streptomycin. The culture flasks (containing 2 x 10 6 viable cells) were observed under an inverted microscope (Biosystems, USA), followed by incubation in an oven at 37°C, 95% humidity and a 5% CO 2 atmosphere (Shel Lab CO 2 Incubator, USA). Spontaneously hypertensive (SHR) female Wistar rats ( Rattus norvegicus ) weighing between 180 and 200 g were obtained from the Central Animal Facility at Universidade Federal do Piauí, Teresina, Brazil. They were kept in well-ventilated cages under standard conditions of light (12 h with alternative day and night cycles) and temperature (22 ± 1°C) and were housed with access to commercial rodent stock diet (Nutrilabor, Campinas, Brazil) and water ad libitum . All procedures were approved by the Committee on Animal Research at UFPI (#572/2019) and followed Brazilian ( Colégio Brasileiro de Experimentação Animal - COBEA) and International rules on the care and use of experimental animals (Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes). In silico evaluation of γ-TPN The 2D, 3D structures and the chemical representation with normal characters (ASCII), called SMILE (Simplified Molecular Input Line Entry Specification) of γ-TPN are shown in Fig. 1 below. These parameters were used for in silico analysis of ADMET (Absorption, distribution, metabolism, excretion/toxicity) and target predictions were extracted from the PubChem database ( https://pubchem.ncbi.nlm.nih.gov ). The molecular optimization of γ-TPN was carried out using ChemSketch software, version 14.0 and the molecular targets were prepared using Discovery Studio 20.1.0. For the prediction of physicochemical properties (lipophilicity - logP, molecular weight, polar surface area, number of hydrogen bond donors and acceptors, number of rotatable bonds and water solubility), pharmacokinetic parameters (ADMET) and similarity to drugs (Lipinski rules, CMC like, MDDR like, Leadlike and WDI like), the software PreADMET ( https://preadmet.bmdrc.kr/ ) and SwissADME ( http://www.swissadme.ch/ ) were used. Complementary data were exposed by the Bioavailability Radar and BOILED-Egg. To predict the possible macromolecular targets of γ-TPN, the SwissTargetPrediction program ( http://www.swisstargetprediction.ch/ ) was also applied. Regarding metabolic parameters, it can be checked whether the substance undergoes first pass metabolism and whether it inhibits cytochrome P450 (CYP) enzymes. The mathematical models used detected mutagenicity results, considering the Ames test and strains [ Salmonella typhimurium (TA98, TA100 and TA1535)] (Ames et al. 1975 ; Mortelmans & Zeiger 2000 ). The prediction of carcinogenicity in rodents was carried out based on data from the National Toxicology Program (NTP) and the Food and Drug Administration (FDA) (Dolabela et al. 2018 ). Regarding acute ecotoxicity, the maximum acceptable concentrations for Daphnia sp., Pimephales promelas , Oryzias latipes , as well as the inhibition of growth in algae were investigated. Additionally, the classification of cardiotoxic risk was investigated based on inhibition of the human gene related to ether-a-go-go (hERG). Analysis of docking molecular First, the molecular design and optimization of the three-dimensional structure of γ-TPN were carried out using the ACD/ChemSketch software, version 14.0, based on classical mechanics parameters (bond distance, bond angle and dihedral angle). The human P2Y12 receptor (4NTJ) was obtained from the Protein Data Bank (PDB). Only structures with a resolution below 3.0Å and obtained from Homo sapiens were considered for this study. The files obtained were imported into the Discovery Studio Visualizer software, version 21.1.0. For ligand removal, crystallographic water molecules were kept and targets were saved in (.pdb) format. Next, the .pdb files were opened in the AutoDockTools software, version 1.5.7, where polar hydrogens and Gasteiger charges were added and saved in (.pdbqt) format (Panda et al. 2020 ; Melo et al. 2022 ; He et al. 2023 ). The molecular structures of γ-TPN and conventional antiplatelet agents (Prasugrel and Ticagrelor) were obtained from the PubChem website in (.sdf) format and individually subjected to geometry optimization using the Avogadro software, version 1.2.0, using the field of MMFF94s strength, subsequently being saved in (.pdb) format. Then, the optimized ligands were also imported into the AutoDockTools software, version 1.5.7, to add polar hydrogens and Gasteiger charges. All ligands were kept flexible before being saved in (.pdbqt) format (Hanwell et al. 2012 ; Yan et al. 2022 ; Kintamani et al. 2023 ). The dimensions and coordinations in the Cartesian plane of the docking grid were automatically calculated using the AutoGrid module of AutoDock software version 4.2.6 and used to generate the configuration files. The grid volume was defined at 60x60x60 points (dimensions X, Y, Z), with a spacing of 0.375 Å, for the two targets tested. The AutoDock Vina software version 1.2.0 was used to perform the molecular docking of the ligands on each target following the scripts for flexible and hydrated docking made available by the Center of Computational Structural Biology (CCSB) at Scripps Research, with the exhaustivity parameter set at 100 races. The analysis of the bonds between targets and ligands and creation of interaction images was carried out using the BIOVIA Discovery Studio Visualizer version 21.1.0 and PyMOL version 2.5.4 software (Marzaro et al. 2014 ; Forli et al. 2016 ; Eberhardt et al. 2021 ). MTT assay Cytototoxic tests used MTT ([3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide]) according to Mosmann ( 1983 ). SVEC4-10 and L-929 (5 x 10 3 cells/well) were incubated and about twenty-four hours later, γ-TPN (0 to 734 µM) was added to each well and the plates were incubated at 37 °C in a 5% CO 2 atmosphere (Shel Lab CO 2 Incubator, USA) for 72 h. After the incubation period, 20 µL of the MTT solution (5 mg/mL) was added to each well and the plates were reincubated for 4 h. Next, formazan product was dissolved in DMSO and absorbance was read using a multiple reader (GloMax Explorer Multimode Microplate Reader, USA) at 595 nm. Design of experimental in vivo protocol SHR rats were orally treated by gavage with γ-TPN at different doses (25, 50 and 100 mg/kg/day) for seven days. The animals were randomly divided into four groups of 5 animals each as follows: Group I (control group, saline solution), Group II (25 mg/kg/day of γ-TPN), Group III (50 mg/kg/day of γ-TPN), and Group IV (100 mg/kg/day of γ-TPN). Following the treatment, the animals were anesthetized with ketamine (90 mg/kg) plus xylazine (4.5 mg/kg) for blood collection Afterwards, all mice were euthanized with sodium thiopental (150 mg/kg i.p.). Nitrite dosage To determine the nitrite content, the test use the Griess reaction (Green et al. 1981 ) in which, a total of 500 µL of Griess reagent was added to 500 µL of distilled water. In another test tube, 500 µL of Griess reagent was added to 500 µL of 10% erythrocyte homogenates (50 mM sodium phosphate buffer pH 7.4) extracted from γ-TPN-treated mice. Measurements were carried out at 560 nm and results were expressed as µM/mg of protein. Antiplatelet activity of γ-TPN Blood was collected (3–5 mL/animal) from anesthetized SHR rats through the aorta artery and by cardiac puncture (right ventricle) with plastic syringes and stored in tubes with citrate and 3.2% sodium in a ratio of 9:1 (blood: anticoagulant). The blood was centrifuged at 1650 rpm for 10 min to obtain platelet-rich plasma (PRP: 2.5 to 4.5 x 10 5 platelets/mm 3 ) and at 3000 rpm for 15 min to obtain platelet-poor plasma (PPP: < 2.0 x 10 4 platelets/mm 3 ). PPP was used as a diluent to adjust the final volume of PRP (Vinholt et al. 2017 ). Platelet counting was perfomed using a Neubauer chamber (Brecher & Cronkite 1950 ) with plasma diluted in 1% ammonium oxalate in a proportion of 1:200 (Tomasiak et al. 2004 ). The antiplatelet activity of γ-TPN was determined in citrated PRP by the turbidimetric method described by Born & Cross ( 1963 ) and monitored buy an aggregometer (Platelet Agregometer, EasyAgreg software, Benfer). Platelet aggregation was expressed as percentage of aggregation for ADP and as aggregation speed (Cattaneo 2009 ; Koltai et al. 2017 ). SVEC4-10 cells were pretreated with γ-TPN at different concentrations (50, 100 and 200 µM) for 24 h. After this period, the level of platelet aggregation was determined in a suspension (300 µL) containing pre-treated platelets (3 x 10 8 platelets/mL) and SVEC4-10 cells (1.5 x 10 3 cells/mL). First, the first cuvette was placed in the device containing 300 µL of PPP. Then, the cuvette with 270 µL of PRP was placed with 20 µL of cells pretreated with γ-TPN. After the first five minutes of recording platelet aggregation, 10µL of the aggregator ADP (10 µM) was added (Cho et al. 2017 ), followed by additional 5 min for final recording. Statistical analysis Results were expressed as mean ± standard error of the mean (S.E.M.). The binding energy values were subjected to normality analysis using the D’Agostino & Pearson test. Nonlinear regression was used to calculate average values of IC 50 (50% growth inhibition of cell proliferation). In order to determine differences between treatments, data were compared by unpaired T-test and one-way analysis of variance (ANOVA) followed by Dunnet, Tukey or Bonferroni test, considering p values < 0,05 as statistically significant (GraphPad Prism version 6.0). Results and discussion In silico parameters In silico analysis data revealed physicochemical characteristics, pharmacokinetic profile and drug similarity prediction of γ-TPN (Table 1 ). Regarding physicochemical aspects, γ-TPN has a molecular weight of 136.23 g/mol, and it was found to be quite lipid-soluble (Log P = + 4.50), moderately water-soluble (Log S=-3.45), with 1 rotary bond, without hydrogen donor and acceptor groups, sp 3 carbon fraction of 0.60, molar refractivity of 47.12, and tension in polar surface area (TSPA) of 0 Å2. γ-TPN revealed complete human intestinal absorption (100%), high permeability in Madin-Darby canine kidney cells - MDCK (244.91) and low permeability in human epithelial adenocarcinoma (Caco-2, 23.64 nm/s), besides a high degree of plasma protein binding (100%). It presented a comparative concentration ratio in the brain and plasma of 8.037, resulting in excellent penetration in the blood-brain barrier. The compound exhibited a skin permeability of -0.8857 and was not able to inhibit P-glycoprotein and CYP2C19 and CYP2C9 enzymes. On the other hand, it is a substrate of CYP3A4 (Table 1 ). Regarding the prediction of drug similarity, Table 1 showed that γ-TPN was classified as “drug-like” taking into consideration the Lipinski and World Drug Index (WDI like) rules, intermediate by the Modern Drug Data Report rule (MDDR like) and disqualified in this parameter by the Leadlike rule and the Comprehensive Medicinal Chemistry (CMC) database. Table 1 Physicochemical, pharmacokinetic properties and drug-like prediction of γ-terpinene. Property Parameter Result Physical chemistry Molecular weight (g mol − 1 ) Water solubility (Log S) Liposubility (Log P; XLog P3) Number of rotary connections Number of hydrogen bond donors Number of hydrogen bond acceptors Fraction of sp 3 carbons Molar refractivity Voltage on polar surface area (Å2) 136.23 -3.45 + 3.35; +4.50 1 0 0 0.60 47.12 0.00 Pharmacokinetics Human Intestinal Absorption (%) Permeability coefficient in Caco-2 (nm s − 1 ) Permeability coefficient in MDCK (nm s − 1 ) Binding to Plasma Proteins (%) Penetration of the Blood-Brain Barrier Skin permeability (cm 2 h − 1 ) P-glycoprotein inhibition Inhibition of CYP2C19 CYP2C9 inhibition CYP2D6 inhibition CYP2D6 substrate CYP3A4 inhibition CYP3A4 substrate 100.00 23.64 244.91 100.00 8.037 -0.8857 No Yes Yes No No No Yes Drug-likeness prediction Lipinski's Rule CMC Like Rule MDDR Like Rule LEAD Like Rule Qualified Disqualified Intermediary Disqualified WDI Like Rule Qualified There is an intrinsic correlation between the physicochemical parameters of a compound and its biological performance, which facilitates the interpretation of its pharmacokinetics, pharmacodynamics and toxicity (Wenlock and Barton 2013 ). As seen in Table 1 , in silico ADME predicts 100% human intestinal absorption (HIA) for the drug candidate (Yee 1997 ). γ-TPN has high intestinal absorption due to the oil-water partition coefficient (Log P), which guarantees elevated lipid solubility and absorption (from 70–100%) crossing membranes by passive diffusion (Hadda et al. 2013 ). It should be noted that the ideal HIA value of a drug will depend on its pharmaceutical indications and must be produced according to the therapeutical purposes (Dolabela et al. 2018 ). Anyway, the best-known rule for linking chemical cores to biological activities is the Lipinski's rule, named “rule of five”. It is based on lipid solubility (LogP) ≤ 5, Molecular weight (MW) ≤ 500, and number of H acceptors (HBA) ≤ 10 and H donors (HBD) ≤ 5 (Lipinski et al. 1997 ; Lagorce et al. 2017 ). Thus, outocomes revealed that γ-TPN met all these prerequisites. Permeability by intestinal Caco-2 cells is used for selecting drug candidates for oral administration, classifying γ-TPN as showing medium permeability through the intestinal epithelium (Yamashita et al. 2000 ). Meanwhile, γ-TPN revealed to be highly permeable through MDCK cells, a useful tool for rapid screening of cell membrane permeability and pharmacological viability (Vistoli et al. 2008 ). γ-TPN also exposed high permeability with regard to penetration of the blood-brain barrier (BBB) (Table 1 ) and is therefore classified as active on the Central Nervous System (CNS) (Zhao et al. 2001 ). This biological property is relevant for ADME studies, as it provides data about therapeutic action on the CNS, binding to plasma proteins, their disposition and efficacy, especially in cerebrovascular diseases’ conditions (Sekhar et al. 2014 ; Harika et al. 2017 ). Another essential ADME parameter is the assessment of binding to the P-glycoprotein (P-gp). This protein is part of the ATP-dependent efflux pump, acting as a physiological barrier to protect the body against toxins and xenobiotics. Then, P-gp is directly involved to the intestinal absorption, metabolism and penetration of the BBB for the majority of drugs, and its inhibition or induction can significantly alter oral bioavailability and metabolism of a drug (Pereira 2019 ). It was also provided a module whose objective is to predict the skin permeability coefficient (Kp) using a linear regression model (logKp). The skin permeability parameter reveals the ability of γ-TPN to be absorbed through the skin, anticipating possible exposure to toxins or even accidental absorption of the compound during handling. Negative value for this coefficient (Table 1 ) indicates the compound is impermeable to the skin, with a low possibility of being used intradermally (Souza et al. 2022), but probably without toxic effects upon skin exposure. Within this myriad of enzymes and transporters, the cytochrome P450 (CYP) superfamily of metabolic isoenzymes plays a crucial role (Testa & Kraemer 2007). Many drugs are targeted by these catalytical proteins, causing high rate of drug individual variability due to different degrees of CYP expression (Hollenberg 2002 ; Huang et al. 2008 ). Herein, in silico showed γ-TPN inhibits the enzymes CYP2C19 and CYP2C9 (Table 1 ). This type of activity may result in increased plasma drug concentrations, but it can induce early adverse effects (Teague et al. 1999 ; Sekhar et al. 2014 ). On the other hand, γ-TPN did not show inhibitory activity of the CYP2D6 gene, which is responsible for the oxidative metabolism of many drugs and other xenobiotics and toxins in the cellular environment. The cytochrome CYP3A4, one of the most oxidative liver enzymes, an isoform that metabolizes 50% of all drugs, it is not inhibited by the compound, acting discreetly as a substrate of this enzyme (Oprea 2000 ). Regarding genotoxic aspects (Table 2 ), γ-TPN presented a positive result for the Ames test, a mutagenicity method that uses strains of Salmonella typhimurium carrying mutations in histidine synthesis-involved genes (Prival & Zeiger 1998 ; Araki et al. 2004 ). Table 2 also displays the acute toxicity profile of γ-TPN upon aquatic organisms, with the maximum tolerable concentrations for Daphnia sp., Pimephales promelas , and Oryzias latipes of 0.23728, 0.04082, and 0.06008 mg/L, respectively. Table 2 In silico prediction of γ-terpinene toxicity. Parameter Result Ames test Carcinogenicity test (rats and mice) Acute toxicity to Daphnia sp. Acute toxicity to Pimephales promelas Acute toxicity to Oryzias latipes Inhibition of growth in algae hERG gene inhibition Mutagen Carcinogenic Acceptable C max : 0.23728 mg L − 1 Acceptable C max : 0.04082 mg L − 1 Acceptable C max : 0.06008 mg L − 1 0.02505 mg L − 1 Medium risk C max = Maximum concentration. Acute evalatuations on aquatic organisms, as well as those with Artemia salina and Danio rerio (zebrafish, family Cyprinidae), have been widely used to predict ecotoxicity of contaminants in aquatic organisms with compatible enzyme receptors (Chen et al. 2020 ; Kingcade et al. 2021 ). According to the European legislation 92/32/EEC on safety of chemical substances (Weyers et al. 2000 ), γ-TPN has no potential to cause long-term adverse effects on aquatic environments (Solubility < 1mg/L). γ-TPN presented a moderate risk for inhibition of the human gene related to ether-a-go-go (hERG). It is responsible for encoding the subunit responsible for the formation of fast-type delayed-rectification potassium channels (IKr), important in the cardiac repolarization stage. Inhibition and dysfunction of this gene causes QT prolongation and fatal ventricular arrhythmia (Lamothe et al. 2016 ; Bjerregaard 2018 ). Figure 2 demonstrates a diagram corresponding to the appropriate oral bioavailability profile of γ-TPN. The colored zone reveals itself as the appropriate physicochemical area, configuring an excellent oral bioavailability and meeting parameters of lipophilicity (LIPO), size (SIZE), polarity (POLAR), insolubility (INSOLU), unsaturations (INSATU), and flexibility (FLEX). Bioavailability Radar (Fig. 2 ) was displayed for rapid assessment of drug similarity, where six physicochemical properties are taken into consideration. To be considered a drug, the compound line under study must be fully included in the pink area (Daina et al. 2017 ). Any deviation, as observed in the result expressed by γ-TPN regarding size, represents a sub-optimal physicochemical property for bioavailability. Figure 2 also shows the BOILED-Egg analysis and points out that γ-TPN in the yellow region presents good HIA and excellent BBB penetration power. However, it was not shown to be a substrate for P-glycoprotein, due to the indication of the red dot (Pgp-). The graph called BOILED-Egg displays a correlation of TPSA with lipophilicity (LogP). It is important to emphasize that the ability to cross the BBB is only relevant when the clinical target is located in the CNS, otherwise the drug may cause side effects, such as headache, drowsiness, dizziness, partial loss of vision, among others depending on the area where chemical interaction(s) may occur. Furthermore, it is important that the drug is not a substrate for proteins such as P-gp and therefore remains at an adequate concentration at the site of action to obtain the desired effect (Borges 2018 ; Pereira 2019 ). Figure 2 predicts the possible and best targets of the molecule and cytoplasmic receptors (26.3%), G protein-coupled receptors (20%) and the CYP enzyme (12.7%) were recognized as the majority. G protein-coupled receptors, as seen in Fig. 4 , represent the second largest class of pharmacological targets of γ-TPN, a result that corroborates the proposal of this study, which is to verify the antiaggregation potential of the monoterpene, considering purinergic ADP receptors, which belong to the Gi class of GPCRs (Hollopeter et al. 2001 ). Docking molecular of γ-TPN The values of ∆G lig. of γ-TPN and RP2Y12, comparing them with conventional antiplatelet therapy drugs are also detailed ion Fig. 2 . The affinity of γ-TPN (-6.450 ± 0.232 Kcal/mol) was statistically higher than that of Prasugrel (-5.793 ± 0.223 Kcal/mol) in RP2Y12. Ticagrelor (-6.883 ± 0.276 Kcal/mol), in turn, showed higher affinity than γ-TPN and Prasugrel. Interaction energy and affinity of the ligand-target complex are inversely proportional quantities, therefore, the lower the binding energy, the greater the stability of the interaction between ligand and protein (Trott & Olson 2010 ; Zhang et al. 2021 ; Xiang et al. 2022 ). The RP2Y12 exhibits a response to the ADP signal and is involved in the inhibition of adenyl cyclase, Ca 2+ -dependent cell migration, regulation of cell morphology, and cell aggregation. RP2Y12 must be activated for ADP-induced platelet aggregation to occur, which can be prevented by a substance that impartially inhibits the receptor. RP2Y12 has a specific tissue distribution, making it a fundamental target for therapeutic intervention (Ahn et al. 2016 ). Such binding inhibits the activity of P2Y12 on platelet aggregation. Therefore, the best coupling position of the γ-TPN in the 2D and 3D structures can be seen in Fig. 3 . There is similarity in the interaction sites of γ-TPN and Prasugrel in RP2Y12, with repetition of some amino acid residues (A:LYS:80, A:PHE:104, A:TYR:105). Ticagrelor, on the other hand, interacted with RP2Y12 in a region very close, but wider to the active site, probably due to its greater molecular weight. The amino acids that interacted with both γ-TPN and Ticagrelor were A:THR:76, A:LYS:80, A:SER:101, A:PHE:104, A:TYR:105 and A:LEU:284. It is known that γ-TPN had a lower average interaction energy with RP2Y12 than Prasugrel, and is therefore capable of forming a complex with greater stability (Zhang et al. 2021 ; Xiang et al. 2022 ). The justification for greater affinity of γ-TPN with the receptor arises from higher number of hydrophobic bonds created in relation to Prasugrel. On the other hand, still according to Fig. 3 E, even though the Ticagrelor- RP2Y12 complex formed an unfavorable bond (represented by the red color), which compromises the stability of the complex, the overall number of bonds formed was higher than the other two molecules, which may justify target greater affinityies (Dhorajiwala et al. 2019 ; Bender et al. 2021 ). Similarly, Nikitina et al. ( 2022 ) evaluated the molecular fit of newly synthesized myrtenol-derived monoterpenes carrying different heteroatoms (sulfur, oxygen or nitrogen) as possible antiplatelet agents, comparing their affinities to P2Y12 with of ticagrelor. It was evident that molecular anchoring confirmed the interaction of all tested compounds with RP2Y12, suggesting that their antiaggregation properties are implemented by blocking P2Y12 function. The results of the study also show that the activity of the monoterpene is linked to the selective inhibition of platelet aggregation. In vitro cytotoxicity of γ-TPN by colorimetric assays It was essential to carry out a cytotoxic screening of a compound in order to know at what concentration it is capable of causing damage to normal cells and, thus, proceed with other experiments with concentrations that are not toxic. Gama-terpinene at concentrations of 367 and 734 µM (50 and 100 µg/mL) significantly interfered with the cell viability of SVEC4-10 and L-929, compared to the control with untreated cells (Fig. 4 ). It was not detected statistically significant viability differences on both cell lines tested at concentrations ≤ 183.50 µM (25 µg/mL). Furthermore, it was found that γ-TPN presented a CC 50 of 366.70 and 333.33 µM (49.96 and 45.41 µg/mL), respectively, for SVEC4-10 and L-929 cells. The US National Cancer Institute (NCI) classifies a compound as having high cytotoxic activity when i) CC 50 500 µg/mL (Thienthiti et al. 2017 ). According to these categorization, γ-TPN has moderate cytotoxicity on normal cells. Previous studies with γ-TPN-treated HaCaT cell lines (long-lived human keratinocytes) showed increase in cell viability after 2h of exposure (Casalle 2016 ). At any rate, terpenes, especifically, act on cells causing damage to lipids and proteins, breaking down cell walls and membranes, resulting in cell lysis. In eukaryotic cells, they also destabilize the mitochondrial membrane and damage plasma membrane proteins (Bakkali et al. 2008 ). Consequently, the number of metabolically active cells should be followed to understand cell toxic effects. Knowing that γ-TPN is a monoterpene mostly found in essential oils from plants whose anticoagulant and antithrombotic properties have already been duly proven, we seek to identify its antiplatelet action (Lamponi 2021 ). Antiplatelet effect and nitrite dosage of γ-TPN The antiplatelet activity of PRP from rats was evaluated against 10 µM ADP. Firstly, it was observed that in the presence of ADP, there was platelet aggregation, a percentage extrapolated to 100% (Fig. 5 A). In the presence of SVEC4-10, there was a significant 39.59% of reduction in platelet aggregation when compared to the negative control (ADP alone) (Fig. 5 B and 5 F). In the presence of γ-TPN 50 and 100 µM (Fig. 5 C and 5 D), there was a greater reduction in platelet aggregation promoted by ADP (51.57 and 44.27%, respectively). Meanwhile, γ-TPN 200 µM promoted a reversal of platelet antiaggregation significantly compared to the control with ADP and SVEC4-10 (Fig. 5 E and 5 F). Under in vitro conditions, the aggregation stimulated ADP to PRP triggers to its G protein-coupled receptors - P2Y1 and P2Y12 - initiating the process. ADP signaling through the RP2Y1 coupled to the Gq protein, promotes a rapid short-lasting response. It activates phospholipase C (PLC) and converts phosphatidylinositol 4,5-bisphosphate (PIP 2 ) into inositol (1,4,5)-triphosphate (IP 3 ) and diacylglycerol (DAG). DAG mobilizes intracellular Ca 2+ and, after activation of protein kinase C (PKC), these both secondary messengers induces increase of free Ca 2+ levels in the cytoplasm environment, resulting in reversible platelet aggregation (Gachet 2012 ; Fayaz & Rajanikant 2012 ; Aslam et al. 2013 ). Endothelial SVEC4-10 cells exposed to ADP (Fig. 5 B and 5 F) promoted a significant reduction in platelet aggregation, possibly justified by healthy endothelial cells expressing antiplatelet agents. Thus, when platelets encounter endothelial cells, platelet-endothelium interactions can occur for stimulating endothelial surface to produce and secrete mediators such as prostacyclin (PGI 2 ) and nitric oxide (NO) (D'amico & Villaça 2006 ; Xie et al. 2024 ). Regarding γ-TPN-pre-treated SVEC4-10 cells, it was found that the compound alter platelet anti-aggregation, since under lower concentrations (Fig. 5 C, 5 D and 5 F), it stimulates endothelial cells, probably through endothelial nitric oxide synthases (eNOS or NOS3) to produce nitric oxide (NO). It was confimed by in vivo studies, in which the dose of 100 mg/kg (91.86 ± 12.31µM) markedly increased indirect levels of nitric oxide quantified as nitrite content, which could be one of the factors responsible for inhibiting platelet aggregation (Radomski et al. 1990 ; Tran et al. 2022 ). This method was established using a calibration curve (R 2 = 0.9942) (Fig. 6 ). With NO formed, the enzyme soluble guanylyl cyclase (GCs) was activated and the second messenger cyclic guanosine monophosphate (cGMP) was produced (Förstermann & Sessa 2012 ; Wittenborn & Marletta 2021 ). After activation, the platelets themselves generated NO (Radomski et al. 1990 ; Malinski et al. 1993 ), inhibiting adhesion (Radomski et al. 1987a ; Radomski et al. 1987b ) and aggregation (Freedman et al. 1997 ). cGMP, in turn, is the agent that determines the inhibitory actions of NO on platelets and this occurs through the decrease in the concentration of intracellular Ca 2+ ([Ca 2+ ] i ) and modulation of the expression of surface receptors. The limitation of [Ca 2+ ] i occurs due to the inhibition of the release of Ca 2+ via the receptor of the dense tubular system, an increase in the rate of Ca 2+ extrusion, lower than its entry, through the extracellular environment, an increase in the activity of the Ca 2+ - ATPase of the endoplasmic reticulum, resulting in a lower amount of Ca 2+ available for platelet activation and aggregation mechanisms. Furthermore, cGMP also promotes the reduction of conformationally active GPIIb/IIIa receptors on the platelet surface, generating greater dissociation between fibrinogen and the GPIIb/IIIa receptor, producing platelet antiaggregation (Jin & Loscalzo 2010 ; Ren et al. 2024 ). Regarding purinergic receptors, γ-TPN may also be strongly involved in the inhibition of RP2Y12, due to the results found at lower concentrations (Fig. 5 C, 5 D and 5 F) and the high molecular affinity of the compound with this receptor, the which was compared with prasugrel and ticagrelor. Additionally, it is important to clarify that higher concentration of γ-TPN caused a nearly complete reversal of antiplatelet effect, suggesting γ-TPN 200 µM likely has cytotoxic action, promoting death of endothelial cells and decline in the production of antiaggregation mediators. Zhou et al. ( 2019 ) also evaluated in vitro antiplatelet activity of monoterpenes and found that suchm molecules showed moderate inhibitory activities on ADP-induced blood platelet aggregation. Aragão et al. ( 2006 ) describe a mixture of triterpenes called α- and β-amyrin, isolated from Protium heptaphyllum (Aubl) March (Burseraceae) with antiplatelet activity in a concentration-dependent manner which probably acts in a common biochemical pathway to all established agonists (ADP, collagen, arachidonic acid and AAS). Conclusions The compound γ-TPN has promising potentialities as to anti-platelet prototype, since it promoted platelet aggregation in the presence of ADP, while lower doses inhibited aggregation and weak cytotoxicity on endothelial cells and fibroblasts. These biological activities are linked to drug-likeness properties, including good bioavailability, heigh lipophilicity and intestinal absorption and capacity for crossing the blood-brain barrier. Moreiver, molecular docking analysis revealed γ-TPN as a molecule with affinity for the active site of the P2Y12 receptor (Fig. 7 ). Declarations Funding This project was partially supported by the public Brazilian agence: “Conselho Nacional de Desenvolvimento Científico e Tecnológico” [CNPq - MCTI/CNPq Nº 14/2014)]. Acknowledgments Railson Pereira Souza is grateful to “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES) (Finance code 001). Aldeídia Pereira de Oliveira also thank to the Brazilian agency “Conselho Nacional de Desenvolvimento Científico e Tecnológico” [CNPq - MCTI/CNPq Nº 14/2014)] for his personal scholarships. Thank you also to the reviewer Paulo Michel Pinheiro Ferreira, responsible for translating the article into English. Authors’ contribution statement All authors participated in the study. RPS wrote the article. RPS and RWRS performed cytotoxicity assays with MTT and nitrite dosing protocols. RPS and VDP carried out in silico and molecular docking studies. RPS, EPS and ACAS were responsible for the in vitro and in vivo antiplatelet aggregation. DD and PMPF supervised and scientifically supported the experiments. APO planned the research, managed scientific and financial support, supervised all stages and reviewed the final article. The authors further stated that all data was generated internally and that no paper mills were used. Compliance with ethical standards All procedures were approved by the Committee on Animal Research at UFPI (#572/2019) and followed Brazilian ( Colégio Brasileiro de Experimentação Animal - COBEA) and International rules on the care and use of experimental animals (Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes). Data availability statement We declare that, in case of acceptance of the manuscript for publication, we agree to transfer the right of first publication to Naunyn-Schmiedeberg's Archives of Pharmacology and the open access policy, therefore, the texts are available for anyone to read, download, copy, print, share, reuse and distribute, with due citation of the source and authorship. The datasets generated and/or analyzed during the current study are not yet publicly available, but are available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this manuscript. References Ahn YA, Lee J-Y, Park HD, Kim TH, Park MC, Choi G & Kim S (2016). Identification of a new morpholine scaffold as a P2Y12 receptor antagonist. Molecules 21:1114. https://doi.org/10.3390/molecules 21091114 Alves-Silva JM, Guerra I, Gonçalves MJ, Cavaleiro C, Cruz MT, Figueirinha A et al. (2020). Chemical composition of Crithmum maritimum L. essential oil and hydrodistillation residual water by GC-MS and HPLC-DAD-MS/MS, and their biological activities. Industrial Crops and Products 149:1-9. https://doi.org/10.1016/j.indcrop.2020.112329 Ames BN, Mccann J & Yamasaki E (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation research 31:347–364. https://doi.org/10.1016/0165-1161(75)90046-1 Aragão GF, Carneiro LM, Junior AP, Vieira LC, Bandeira PN, Lemos TL, Viana GS (2006). A possible mechanism for anxiolytic and antidepressant effects of alpha- and beta-amyrin from Protium heptaphyllum (Aubl.) March. Pharmacol Biochem Behav 85:827-34. https://doi.org/10.1016/j.pbb.2006.11.019 Araki A, Kamigaito N, Sasaki T & Matsushima T (2004). Mutagenicity of carbon tetrachloride and chloroform in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, using a gas exposure method. Environmental and molecular mutagenesis 43:128–133. https://doi.org/10.1002/em.20005 Aslam M, Sedding D, Koshty A, Santoso S, Schulz R, Hamm C, Gündüz D (2013). Nucleoside triphosphates inhibit ADP, collagen, and epinephrine-induced platelet aggregation: role of P2Y1 and P2Y12 receptors. Thrombosis Research132:548-57. https://doi.org/10.1016/j.thromres.2013.08.021 Bakkali F, Averbeck S, Averbeck D & Idaomar M (2008). Biological effects of essential oils--a review. Food and Chemical Toxicology: an International journal published for the British Industrial Biological Research Association 46:446–475. https://doi.org/10.1016/j.fct.2007.09.106 Baldissera MD, Grando TH, Souza CF, Gressler LT, Stefani LM, da Silva AS, Monteiro SG (2016). In vitro and in vivo action of terpinen-4-ol, γ-terpinene, and α-terpinene against Trypanosoma evansi. Exp Parasitol. 162:43-8. https://doi.org/10.1016/j.exppara.2016.01.004 Bender BJ, Gahbauer S, Luttens A, Lyu J, Webb CM, Stein RM, Fink EA, Balius TE, Carlsson J, Irwin JJ & Shoichet BK (2021). A practical guide to large-scale docking. Nature protocols 16:4799–4832. https://doi.org/10.1038/s41596-021-00597-z Benjamin EJ, Muntner P, Alonso A, et al. (2019). Heart disease and stroke statistics-2019 Update: a report from the American Heart Association. Circulation. 139:e56-e528. https://doi.org/10.1161/CIR.0000000000000659 Berger JS (2018). Oral antiplatelet therapy for secondary prevention of acute coronary syndrome. Am J Cardiovasc Drugs. 18:457-472. https://doi.org/10.1007/s40256-018-0291-2 Bjerregaard P (2018). Diagnosis and management of short QT syndrome. Heart rhythm 15:1261–1267. https://doi.org/10.1016/j.hrthm.2018.02.034 Born GV & Cross MJ (1963). The aggregation of blood platelets. The Journal of Physiology 168:178-95. https://doi.org/10.1113/jphysiol.1963.sp007185 Borges NHPB (2018). Análise in silico das propriedades farmacológicas e toxicológicas dos organosselênios e ensaios in vitro de atividades antibacteriana e moduladora da resistência a drogas em Staphylococcus aureus . Tese (Doutorado) - UFPB/CCS. 99 f. https://repositorio.ufpb.br/jspui/bitstream/123456789/19288/1/NathalieHelenPaesBarretoBorges_Tese.pdf Brecher G & Cronkite EP (1950). Morphology and enumeration of human blood platelets. Journal of Applied Physiology 3:365–377. https://doi.org/10.1152/jappl.1950.3.6.365 Çakmak H, Özselek Y, Turan OY et al. (2020). Whey protein isolate edible films incorporated with essential oils: antimicrobial activity and barrier properties. Polym. Degrad. Stabil. 179:109285. https://doi.org/10.1016/j.polymdegradstab.2020.109285 Casalle N (2016). Susceptibilidade de carcinoma espinocelular oral ao óleo essencial de Melaleuca alternifolia e suas principais porções solúveis. 43 f. Dissertação (Mestrado em Ciências Odontológicas), Universidade Estadual Paulista, Faculdade de Odontologia, Araraquara. http://hdl.handle.net/11449/144695 Cattaneo M. (2009). Light transmission aggregometry and ATP release for the diagnostic assessment of platelet function. Seminars in thrombosis and hemostasis 35:158–167. https://doi.org/10.1055/s-0029-1220324 Chen G, Jia Z, Wang L & Hu T. (2020). Effect of acute exposure of saxitoxin on development of zebrafish embryos ( Danio rerio ). Environmental research 185:109432. https://doi.org/10.1016/j.envres.2020.109432 Cho MS, Noh K, Haemmerle M, Li D, Park H, Hu Q, Hisamatsu T, Mitamura T, Mak SLC, Kunapuli S, Ma Q, Sood AK & Afshar-Kharghan V (2017). Role of ADP receptors on platelets in the growth of ovarian cancer. Blood 130:1235-1242. https://doi.org/10.1182/blood-2017-02-769893 Choi HS, Song HS, Ukeda H & Sawamura M (2000). Radical-scavenging activities of citrus essential oils and their components: detection using 1,1-diphenyl-2-picrylhydrazyl. Journal of Agricultural and Food Chemistry 48:4156-61. https://doi.org/10.1021/jf000227d D'Amico ÉA & Villaça PR (2006). Distúrbios da coagulação no departamento de emergência. In Emergências clínicas baseadas em evidências: disciplina de emergências clínicas. São Paulo: Editora Atheneu. https://repositorio.usp.br/item/001528493 Daina A, Michielin O & Zoete V (2017). SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific reports 7:42717. https://doi.org/10.1038/srep42717 Dhorajiwala TM, Halder ST and Samant L (2019). Comparative in silico molecular docking analysis of L-threonine-3-dehydrogenase, a protein target against african Trypanosomiasis using selected phytochemicals. Journal of Applied Biotechnology Reports 6:101-108. https://doi.org/10.29252/JABR.06.03.04 Dolabela MF, Silva ARP, Ohashi LH, Bastos MLC, Silva MCM & Vale VV (2018). Estudo in silico das atividades de triterpenos e iridoides isolados de Himatanthus articulatus (Vahl) Woodson. Revista Fitos 12:227-242. https://doi.org/10.17648/2446-4775.2018.602 Eberhardt J, Santos-Martins D, Tillack AF & Forli S (2021). AutoDock Vina 1.2.0: new docking methods, expanded force field, and python bindings. Journal of chemical information and modeling 61:3891–3898. https://doi.org/10.1021/acs.jcim.1c00203 Fayaz SM & Rajanikant GK (2012). A critical appraisal of the functional evolution of P2Y12 antagonists as antiplatelet drugs. Current Pharmaceutical Design 18:1625–1634. https://doi.org/10.2174/138161212799958558 Forli S, Huey R, Piqué M, Sanner MF, Goodsell DS & Olson AJ (2016). Docking computacional de proteína-ligante e triagem virtual de drogas com o conjunto AutoDock. Nature Protocols 11:905–919. https://doi.org/10.1038/nprot.2016.051 Förstermann U, Sessa WC (2012). Nitric oxide synthases: regulation and function. Eur Heart J. 33:829-37, 837a-837d. https://doi.org/10.1093/eurheartj/ehr304 Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, Michelson AD (1997). Nitric oxide released from activated platelets inhibits platelet recruitment. J Clin Invest. 1997 100:350-6. https://doi.org/10.1172/JCI119540 Gachet C. (2012). P2Y(12) receptors in platelets and other hematopoietic and non-hematopoietic cells. Purinergic signalling 8:609–619. https://doi.org/10.1007/s11302-012-9303-x Gerhards C, Uhlig S, Etemad M, Christodoulou F, Bieback K, Klüter H & Bugert P (2021). Expression of ADP receptor P2Y12, thromboxane A2 receptor and C-type lectin-like receptor 2 in cord blood-derived megakaryopoiesis. Platelets 32:618–625. https://doi.org/10.1080/09537104.2020.1782868 Green LC, Tannenbaum SR & Goldman P (1981). Nitrate synthesis in the germfree and conventional rat. Science 212:56–58. https://doi.org/10.1126/science.6451927 Hadda TB, Ali MA, Masand V, Gharby S, Fergoug T & Warad I (2013). Tautomeric origin of dual effects of N1-nicotinoyl-3-(4′-hydroxy- 3′-methyl phenyl)-5-[(sub)phenyl]-2-pyrazolines on bacterial and viral strains: POM analyses as new efficient bioinformatics' platform to predict and optimize bioactivity of drugs. Medicinal Chemistry Research 22 :1438-1449. https://doi.org/10.1007/s00044-012-0143-6 Hamilton JR. Protease-activated receptors as targets for antiplatelet therapy (2009). Blood Rev 23:61-5. https://doi.org/10.1016/j.blre.2008.06.002 Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E & Hutchison GR (2012). Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J Cheminformatics 4:17 (2012). https://doi.org/10.1186/1758-2946-4-17 Harika MS, Kumar R & Reddy SS. Docking studies of benzimidazole derivatives using HEX 8.0 M (2017). International Journal of Pharmaceutical Sciences and Research 8:1677–1688. https://doi.org/10.13040/IJPSR.0975-8232.8(4).1677-88 He Q, Liu C, Wang X, Rong K, Zhu M, Duan L, Zheng P & Mi Y (2023). Exploring the mechanism of curcumin in the treatment of colon cancer based on network pharmacology and molecular docking. Frontiers in pharmacology 14: 1102581. https://doi.org/10.3389/fphar.2023.1102581 Hollenberg PF (2002). Characteristics and common properties of inhibitors, inducers, and activators of CYP enzymes. Drug metabolism reviews 34:17-35. https://doi.org/10.1081/dmr-120001387 Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A & Julius D (2001). Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 409:202–207. https://doi.org/10.1038/35051599 Huang SM, Strong JM, Zhang L, Reynolds KS, Nallani S, Temple R, Abraham S, Habet SA, Baweja RK, Burckart GJ, Chung S, Colangelo P, Frucht D, Green MD, Hepp P, Karnaukhova E, Ko HS, Lee JI, Marroum PJ, Norden JM, … Lesko LJ (2008). New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process. Journal of clinical pharmacology, 48:662–670. https://doi.org/10.1177/0091270007312153 Iqbal NS, Jascur TA, Harrison SM, Edwards AB, Smith LT, Choi ES, Arevalo MK, Chen C, Zhang S, Kern AJ, Scheuerle AE, Sanchez EJ, Xing C & Baker LA (2020). Prune belly syndrome in surviving males can be caused by Hemizygous missense mutations in the X-linked Filamin A gene. BMC Med Genet 21:38. https://doi.org/10.1186/s12881-020-0973-x Jin RC, Loscalzo J (2010). Vascular nitric oxide: formation and function. J Blood Med 2010:147-162. https://doi.org/10.2147/JBM.S7000 Khan N, Jamila N, Ejaz R, Nishan U & Kim KS (2020). Volatile oil, phytochemical, and biological activities evaluation of Trachyspermum ammi seeds by chromatographic and spectroscopic methods. Analytical Letters 53:6, 984-1001. https://doi.org/10.1080/00032719.2019.1688825 Kingcade A, Ahuja N, Jefferson A, Schaffer PA, Ryschon H, Cadmus P, Garrity D & Ramsdell H (2021). Morbidity and mortality in Danio rerio and Pimephales promelas exposed to antilipidemic drug mixtures (fibrates and statins) during embryogenesis: Comprehensive assessment via ante and post-mortem endpoints. Chemosphere 263:127911. https://doi.org/10.1016/j.chemosphere.2020.127911 Kintamani E, Batubara I, Kusmana C, Tiryana T, Mirmanto E & Asoka SF (2023). Essential oil compounds of andaliman (Zanthoxylum acanthopodium DC.) Fruit varieties and their utilization as skin anti-aging using molecular docking. Life 13:754. https://doi.org/10.3390/life13030754 Koltai K, Kesmarky G, Feher G, Tibold A & Toth K (2017). Platelet aggregometry testing: molecular mechanisms, techniques and clinical implications. International Journal of Molecular Sciences 18:1803. https://doi.org/10.3390/ijms18081803 Koyama S & Heinbockel T (2020). The effects of essential oils and terpenes in relation to their routes of intake and application. International Journal of Molecular Sciences 21:1558. https://doi.org/10.3390/ijms21051558 Lagorce D, Douguet D, Miteva MA & Villoutreix BO (2017). Computational analysis of calculated physicochemical and ADMET properties of protein-protein interaction inhibitors. Scientific reports 7:46277. https://doi.org/10.1038/srep46277 Lamothe SM, Guo J, Li W, Yang T & Zhang S (2016). The human ether-a-go-go-related gene (hERG) potassium channel represents an unusual target for protease-mediated damage. Journal of Biological Chemistry 291:20387-401. https://doi.org/10.1074/jbc.M116.743138 Lamponi S (2021). Bioactive natural compounds with antiplatelet and anticoagulant activity and their potential role in the treatment of thrombotic disorders. Life (Basel) 11:1095. https://doi.org/10.3390/life11101095 Lima JF, Oliveira Neto JG, Araújo AE, Silva DA, Freitas JT (2019). Levantamento participativo e formação continuada sobre plantas medicinais cultivadas no município de Serraria-PB. Brazilian Journal of Agroecology and Sustainability 1:1-17. https://doi.org/10.52719/bjas.v1i2.2886 Li GX & Liu ZQ (2009). Unusual antioxidant behavior of alpha- and gamma-terpinene in protecting methyl linoleate, DNA, and erythrocyte. J Agric Food Chem. 57:3943-8. https://doi.org/10.1021/jf803358g Lipinski CA, Lombardo F, Dominy BW & Feeney PJ (1997). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews 23: 3-25. https://doi.org/10.1016/S0169-409X(96)00423-1 Mackman N. Triggers, targets and treatments for thrombosis (2008). Nature 451:914-8. https://doi.org/10.1038/nature06797 Malinski T, Radomski MW, Taha Z & Moncada, S. (1993). Direct electrochemical measurement of nitric oxide released from human platelets. Biochemical and Biophysical Research Communications 194:960–965. https://doi.org/10.1006/bbrc.1993.1914 Marzaro G, Ferrarese A & Chilin A (2014). Autogrid-based clustering of kinases: selection of representative conformations for docking purposes. Molecular diversity 18:611–619. https://doi.org/10.1007/s11030-014-9524-8 Massa KHC, Duarte YAO & Chiavegatto Filho ADP (2019). Análise da prevalência de doenças cardiovasculares e fatores associados em idosos, 2000-2010. Cien Saude Colet 24:105-114. https://doi.org/10.1590/1413-81232018241.02072017 Mcfadyen JD, Schaff M, Peter K (2018). Current and future antiplatelet therapies: emphasis on preserving haemostasis. Nature Reviews Cardiology 15:181–191. https://doi.org/10.1038/nrcardio.2017.206 Melo DS, Nery Neto JAO, Santos MSD, Pimentel VD, Carvalho RCV, Sousa VC, Sousa RGC, Nascimento LGD, Alves MMM, Arcanjo DDR, Sousa DP & Carvalho FAA (2022). Isopropyl gallate, a gallic acid derivative: in silico and in vitro investigation of its effects on Leishmania major . Pharmaceutics 14:2701. https://doi.org/10.3390/pharmaceutics14122701 Mortada EM (2024). Evidence-based complementary and alternative medicine in current medical practice. Cureus 16:e52041. https://doi.org/10.7759/cureus.52041 Mortelmans K & Zeiger E (2000). The Ames Salmonella /microsome mutagenicity assay. Mutation research 455: 29–60. https://doi.org/10.1016/s0027-5107(00)00064-6 Mosmann T (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods 65:55–63. https://doi.org/10.1016/0022-1759(83)90303-4 Mota AA (2022). Terpenos: métodos de extração, sua formação e aplicações. Gama, DF: UNICEPLAC, 68 p. https://dspace.uniceplac.edu.br/bitstream/123456789/1170/1/Terpenos%20-%20m%C3%A9todos%20de%20extra%C3%A7%C3%A3o%2C%20sua%20 forma%C3%A7%C3%A3o%20e%20aplica%C3%A7%C3%B5es.pdf Nikitina LE, Pavelyev RS, Gilfanov IR, Kiselev SV, Azizova ZR, Ksenofontov AA, Bocharov PS, Antina EV, Klochkov VV, Timerova AF, Rakhmatullin IZ, Ostolopovskaya OV, Khelkhal MA, Boichuk SV, Galembikova AR, Andriutsa NS, Frolova LL, Kutchin AV, Kayumov AR (2022). Unraveling the mechanism of platelet aggregation suppression by monoterpenoids. Bioengineering (Basel) 9:24. https://doi.org/10.3390/bioengineering9010024 Olgasi C, Assanelli S, Cucci A & Follenzi A (2024). Hemostasis and endothelial functionality: the double face of coagulation factors. Haematologica. https://doi.org/10.3324/haematol.2022.282272 Oprea TI (2000). Property distribution of drug-related chemical databases. Journal of computer-aided molecular design 14:251–264. https://doi.org/10.1023/a:1008130001697 Panda PK, Arul MN, Patel P, Verma SK, Luo W, Rubahn HG, Mishra YK, Suar M & Ahuja R. (2020). Structure-based drug designing and immunoinformatics approach for SARS-CoV-2. Science advances 6:eabb8097. https://doi.org/10.1126/sciadv.abb8097 Papapanagiotou A, Daskalakis G, Siasos G, Gargalionis A, Papavassiliou AG (2016). The Role of Platelets in Cardiovascular Disease: Molecular Mechanisms. Current Pharmaceutical Design 22:4493-4505. https://doi.org/10.2174/1381612822666160607064118 Passos FF, Lopes EM, de Araújo JM, de Sousa DP, Veras LM, Leite JR & Almeida FR (2015). Involvement of Cholinergic and Opioid System in γ-Terpinene-Mediated Antinociception. Evid Based Complement Alternat Med. 2015:829414. https://doi.org/10.1155/2015/829414 Pereira ALC (2019). Síntese, elucidação estrutural e estudos in silico de novos compostos 2-amino-tiofênicos imídicos candidatos a fármacos antifúngicos, antileishmanicida e antitumorais. Dissertação (Mestrado em Produtos Naturais e Sintéticos Bioativos). 164 f. João Pessoa – JP. https://repositorio.ufpb.br/jspui/handle/123456789/15841 Piaru SP, Mahmud R, Abdul Majid AM, Ismail S & Man CN (2012). Chemical composition, antioxidant and cytotoxicity activities of the essential oils of Myristica fragrans and Morinda citrifolia. J Sci Food Agric. 92:593-7. https://doi.org/10.1002/jsfa.4613 Prival MJ & Zeiger E (1998). Chemicals mutagenic in Salmonella typhimurium strain TA1535 but not in TA100. Mutation Research - Genetic Toxicology and Environmental Mutagenesis 412:251–260. https://doi.org/10.1016/s1383-5718(97)00196-4 Radomski MW, Palmer RM & Moncada S (1990). Characterization of the L-arginine: nitric oxide pathway in human platelets. British Journal of Pharmacology 101:325–328. https://doi.org/10.1111/j.1476-5381.1990.tb12709.x Radomski MW, Palmer RM & Moncada S (1987b). Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets. British Journal of Pharmacology, 92:181–187. https://doi.org/10.1111/j.1476-5381.1987.tb11310.x Radomski MW, Palmer RM & Moncada S (1987a). The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. British Journal of Pharmacology 92:639–646. https://doi.org/10.1111/j.1476-5381.1987.tb11367.x Ramalho TR, Filgueiras LR, Pacheco de Oliveira MT, Lima AL, Bezerra-Santos CR, Jancar S & Piuvezam MR (2016). Gamma-terpinene modulation of LPS-stimulated macrophages is dependent on the PGE2/IL-10 Axis. Planta Med. 82:1341-1345. https://doi.org/10.1055/s-0042-107799 Ren H, Sun Y, Li Y, Yuan X, Jiang B, Zhang W, Liu G & Lu P (2024). Potential mechanism of platelet GPIIb/IIIa and fibrinogen on retinal vein occlusion. Current Eye Research 1-11. https://doi.org/10.1080/02713683.2024.2327055 Rengasamy KRR, Khan H, Ahmad I, Lobine D, Mahomoodally F, Suroowan S, Hassan STS, Xu S, Patel S, Daglia M, Nabavi SM, Pandian SK (2019). Bioactive peptides and proteins as alternative antiplatelet drugs. Medicinal Research Reviews . 39:2153-2171. https://doi.org/10.1002/med.21579 Rocha MM, Rodrigues RDS, Guimarães PHV, et al. (2022). Potencial larvicida de óleos essenciais de plantas brasileiras contra Aedes aegypti . Pesquisa, Sociedade e Desenvolvimento 2:e53211226140. https://doi.org/10.33448/rsd-v11i2.26140 Sales AD, Alencar CMM (2019). Produção e uso de plantas medicinais como processo pedagógico de educação ambiental. Revista Gestão & Sustentabilidade Ambiental 8:468-488. https://doi.org/10.19177/rgsa.v8e42019468-488 Sekhar KC, Syed R, Golla M, Kumar M V J, Yellapu NK, Chippada AR, Chamarthi NR (2014). Novel heteroaryl phosphonicdiamides PTPs inhibitors as anti-hyperglycemic agents. Daru Journal of Pharmaceutical Sciences 22:76. https://doi.org/10.1186/s40199-014-0076-3 Silva RF, Rezende CM, Pereira JB et al. (2015). Scents from Brazilian Cerrado: chemical composition of the essential oil from Pseudobrickellia brasiliensis (Asteraceae). J Essent Oil Res 27:417–420. https://doi.org/10.1080/10412905.2015.1037021 Simonetti E, Ethur ME, Castro LC, Kauffmann C, Giacomin AC, Ledur A et al. (2016). Avaliação da atividade antimicrobiana de extratos de Eugenia anomala e Psidium salutare (Myrtaceae) frente à Escherichia coli e Listeria monocytogenes . Revista Brasileira de Plantas Medicinais 18:9-18. https://doi.org/10.1590/1983-084X/15_005 Takahashi Y, Inaba N, Kuwahara S & Kuki W (2003). Effects of γ-terpinene on lipid concentrations in serum using triton ER1330- treated rats. Bioscience, Biotechnology, and Biochemistry 67:2448-2450. http://dx.doi.org/10.1271/bbb.67.2448 Teague SJ, Davis AM, Leeson PD, Oprea T (1999). The design of leadlike combinatorial libraries. Angewandte Chemie - International Edition 38:3743–3748. https://doi.org/10.1002/(SICI)1521-3773(19991216)38:243.0.CO;2-U Thienthiti K, Tuchind P, Wongnoppavich A, et al. (2017). Cytotoxic effect of compounds isolated from Goniothalam usmarcanii Craib stem barks. Pharm. Sci. Asia . 44:86–95. https://doi.org/10.29090/psa.2017.02.086 Toledo AG, Souza JGL, Silva JPB, et al. (2020). Chemical composition, antimicrobial and antioxidant activity of the essential oil of leaves of Eugenia involucrata DC. Bioscience Journal. 36:568-577. https://doi.org/10.14393/BJ-v36n2a2020-48096 Tomasiak M, Stelmach H, Rusak T & Wysocka, J (2004). Nitric oxide and platelet energy metabolism. Acta biochimica Polonica 51:789–803. https://ojs.ptbioch.edu.pl/index.php/abp/article/view/3562/2620 Tran N, Garcia T, Aniqa M, Ali S, Ally A, Nauli SM (2022). Endothelial nitric oxide synthase (ENOS) and the cardiovascular system: in physiology and in disease states. American Journal of Biomedical Science and Research 152: 153-177. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8774925/pdf/nihms-1769792.pdf Trott O, Olson AJ (2010). AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455-61. https://doi.org/10.1002/jcc.21334 Vinholt PJ, Frederiksen H, Hvas AM, Sprogøe U & Nielsen C (2017). Measurement of platelet aggregation, independently of patient platelet count: a flow-cytometric approach. Journal of thrombosis and haemostasis 15:1191–1202. https://doi.org/10.1111/jth.13675 Vistoli G, Pedretti A & Testa B. (2008). Assessing drug-likeness--what are we missing?. Drug discovery today, 13:285–294. https://doi.org/10.1016/j.drudis.2007.11.007 Wenlock MC & Barton P. (2013). In silico physicochemical parameter predictions. Molecular pharmaceutics 10: 1224–1235. https://doi.org/10.1021/mp300537k Weyers A, Sokull-Klüttgen B, Baraibar-Fentanes J & Vollmer G (2000). Acute toxicity data: a comprehensive comparison of results of fish, Daphnia and algae tests with new substances notified in the European Union. Environmental Toxicology and Chemistry 19:1931–1933. https://doi.org/10.1002/etc.5620190731 Wittenborn EC & Marletta MA (2021). Structural Perspectives on the Mechanism of Soluble Guanylate Cyclase Activation. Int J Mol Sci 22:5439. https://doi.org/10.3390/ijms22115439 Xiang Q, Pang X, Liu Z et al. (2019). Progress in the development of antiplatelet agents: focus on the targeted molecular pathway from bench to clinic. Pharmacol Ther 203:107393. https://doi.org/10.1016/j.pharmthera.2019.107393 Xiang C, Chen C, Li X, Wu Y, Xu Q, Wen L, Xiong W, Liu Y, Zhang T, Dou C, Ding X, Hu L, Chen F, Yan Z, Liang L & Wei G (2022). Computational approach to decode the mechanism of curcuminoids against neuropathic pain. Computers in biology and medicine 147:105739. https://doi.org/10.1016/j.compbiomed.2022.105739 Xie B, Tang W, Wen S, Chen F, Yang C, Wang M, Yang Y, Liang W (2024). GDF-15 Inhibits ADP-induced human platelet aggregation through the GFRAL/RET Signaling Complex. Biomolecules 14 , 38. https://doi.org/10.3390/biom14010038 Xu L, Tsona NT, You B, Zhang Y, Wang S, Yang Z et al. (2020). NOx enhances secondary organic aerosol formation from nighttime γ-terpinene ozonolysis. Atmospheric Environment 225:1-39. https://doi.org/10.1016/j.atmosenv.2020.117375 Yamashita S, Furubayashi T, Kataoka M, Sakane T, Sezaki H & Tokuda H (2000). Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells. European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 10:195–204. https://doi.org/10.1016/s0928-0987(00)00076-2 Yan L, Zhang Z, Liu Y, Ren S, Zhu Z, Wei L, Feng J, Duan T, Sun X, Xie T & Sui X (2022). Anticancer activity of erianin: cancer-specific target prediction based on network pharmacology. Frontiers in molecular biosciences 9:862932. https://doi.org/10.3389/fmolb.2022.862932 Yee S (1997). In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth. Pharmaceutical research 14:763–766. https://doi.org/10.1023/a:1012102522787 Zhang Y, Hui J & Chen X (2021). Preprocedural ticagrelor treatment was associated with improved early reperfusion and reduced short-term heart failure in east-asian st-segment elevation myocardial infarction patients undergoing primary percutaneous coronary intervention. International Journal of General Medicine 14:1927–1938. https://doi.org/10.2147/IJGM.S307404 Zhao YH, Le J, Abraham MH, Hersey A, Eddershaw PJ, Luscombe CN, Butina D, Beck G, Sherborne B, Cooper I & Platts JA (2001). Evaluation of human intestinal absorption data and subsequent derivation of a quantitative structure-activity relationship (QSAR) with the Abraham descriptors. Journal of pharmaceutical sciences 90:749–784. https://doi.org/10.1002/jps.1031 Zhou J, Zhai JX, Zheng W, Han N, Liu Z, Lv G, Zheng X, Chang, S., Yin, J (2019). The antithrombotic activity of the active fractions from the fruits of Celastrus orbiculatus Thunb through the anti-coagulation, anti-platelet activation and anti-?brinolysis pathways. Journal of Ethnopharmacology 241:111974. https://doi.org/10.1016/j.jep.2019.111974 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 28 Apr, 2024 Reviews received at journal 27 Apr, 2024 Reviewers agreed at journal 18 Apr, 2024 Reviewers invited by journal 18 Apr, 2024 Submission checks completed at journal 17 Apr, 2024 Editor assigned by journal 17 Apr, 2024 First submitted to journal 13 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4260336","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":293508813,"identity":"732f68a0-ce51-42ce-9ef7-fe09083e6e79","order_by":0,"name":"Railson Pereira Souza","email":"","orcid":"","institution":"Federal University of Piauí","correspondingAuthor":false,"prefix":"","firstName":"Railson","middleName":"Pereira","lastName":"Souza","suffix":""},{"id":293508814,"identity":"732d632b-98c7-4542-ae9b-63a94ab4073f","order_by":1,"name":"Vinícius Duarte Pimentel","email":"","orcid":"","institution":"Federal University of Piauí","correspondingAuthor":false,"prefix":"","firstName":"Vinícius","middleName":"Duarte","lastName":"Pimentel","suffix":""},{"id":293508815,"identity":"a1618661-5d21-4260-8eaa-a2c9fe63bf40","order_by":2,"name":"Rayran Walter Ramos de Sousa","email":"","orcid":"","institution":"Federal University of Piauí","correspondingAuthor":false,"prefix":"","firstName":"Rayran","middleName":"Walter Ramos","lastName":"de Sousa","suffix":""},{"id":293508816,"identity":"3dcbdfec-7a84-4bf4-9a02-abec12199aec","order_by":3,"name":"Emerson Portela Sena","email":"","orcid":"","institution":"Federal University of Piauí","correspondingAuthor":false,"prefix":"","firstName":"Emerson","middleName":"Portela","lastName":"Sena","suffix":""},{"id":293508817,"identity":"9c02bc9a-7180-4f5c-9143-5506987bbff5","order_by":4,"name":"Alda Cássia Alves da Silva","email":"","orcid":"","institution":"Federal University of Piauí","correspondingAuthor":false,"prefix":"","firstName":"Alda","middleName":"Cássia Alves da","lastName":"Silva","suffix":""},{"id":293508818,"identity":"c03d6e4e-abf7-4e51-8ea1-b3a8caf46a66","order_by":5,"name":"Dalton Dittz","email":"","orcid":"","institution":"Federal University of Piauí","correspondingAuthor":false,"prefix":"","firstName":"Dalton","middleName":"","lastName":"Dittz","suffix":""},{"id":293508819,"identity":"27fddabf-6e14-4340-b432-d6b30f239b78","order_by":6,"name":"Paulo Michel Pinheiro Ferreira","email":"","orcid":"","institution":"Federal University of Piauí","correspondingAuthor":false,"prefix":"","firstName":"Paulo","middleName":"Michel Pinheiro","lastName":"Ferreira","suffix":""},{"id":293508820,"identity":"5d4d6efe-56f8-4f3f-83c9-b52434e77252","order_by":7,"name":"Aldeídia Pereira de Oliveira","email":"data:image/png;base64,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","orcid":"","institution":"Federal University of Piauí","correspondingAuthor":true,"prefix":"","firstName":"Aldeídia","middleName":"Pereira","lastName":"de Oliveira","suffix":""}],"badges":[],"createdAt":"2024-04-13 04:14:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4260336/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4260336/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55089800,"identity":"a1088e1e-2b82-4750-9308-48c8d6edfe25","added_by":"auto","created_at":"2024-04-22 12:13:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":83559,"visible":true,"origin":"","legend":"\u003cp\u003e2D, 3D and SMILE molecular structures of γ-terpinene\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4260336/v1/6658616ddfb1141c1f4649e3.png"},{"id":55090110,"identity":"a46181db-3020-4c5f-9f14-5e75e9cb5a7b","added_by":"auto","created_at":"2024-04-22 12:21:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":104483,"visible":true,"origin":"","legend":"\u003cp\u003ePhysicochemical \u003cem\u003ein silico \u003c/em\u003eproperties of γ-terpinene according to: A) Bioavailability radar; B) BOILED-Egg for γ-terpinene (BBB: Blood-Brain Barrier; HIA: Human Intestinal Absorption; PGP: P-glycoprotein); C) Main classes of pharmacological targets; D) Binding energy (∆G) of γ-Terpinene, Prasugrel and Ticagrelor in RP2Y12. Analysis of variance ANOVA followed by Tukey's test for unpaired samples. Values were expressed as mean ± standard deviation (SD). (*) represents statistical difference with p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4260336/v1/e6c9b4f096604c10b7f9875d.png"},{"id":55090442,"identity":"60d144ce-64c2-4bdc-8ce5-f4886d792f8c","added_by":"auto","created_at":"2024-04-22 12:29:18","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":792744,"visible":true,"origin":"","legend":"\u003cp\u003eIntermolecular interactions of γ-Terpinene (A), Prasugrel (B), and Ticagrelor (C) with RP2Y12 alone and in combination (D).\u003c/p\u003e","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4260336/v1/900b40a023e622ce5df5d63b.jpeg"},{"id":55090112,"identity":"5f968f80-397f-4b20-907c-f962defbd613","added_by":"auto","created_at":"2024-04-22 12:21:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":28400,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability and cytotoxic concentration assessed by MTT assay in SVEC4-10 (A) and L929 (B) cells after 72 h of exposure to γ-terpinene Values represent the mean ± standard deviation (SD) of three independent experiments, with ****p \u0026lt;0.001 when comparing control and γ-terpinene concentrations (11.50 to 734µM). CC50 = cytotoxic concentration, which indicates 50% inhibition; CI= confidence interval; R2= coefficient of determination. ANOVA followed by Tukey (multiple comparisons) one-way post-test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4260336/v1/aa023af2ed73a6e247cf1dc0.png"},{"id":55089801,"identity":"513480ca-4339-4f06-bf5d-1e6d64f509fd","added_by":"auto","created_at":"2024-04-22 12:13:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":222504,"visible":true,"origin":"","legend":"\u003cp\u003ePlatelet aggregation in the presence of SVEC4-10 cells treated with γ-TPN (50, 100 and 200 µM) for 24h and incubated with aggregated platelets under ADP stimulation (10 µM) The percentage of platelet aggregation as a function of time was determined using an aggregometer (A-E) and expressed as the average of maximum aggregation between different concentrations (F). ***p\u0026lt;0.001 (one-way ANOVA, Tukey post-test).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4260336/v1/a9d7b40a3382ab3f05f2897a.png"},{"id":55089804,"identity":"9dc05e2b-593f-4060-95fc-b609de139951","added_by":"auto","created_at":"2024-04-22 12:13:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":29704,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curve (A) and nitrite dosage (B) in erythrocyte concentrate from rats treated with different doses of γ-terpinene (25, 50 and 100 mg Kg\u003csup\u003e-1\u003c/sup\u003e). \u003csup\u003ea \u003c/sup\u003ecompared to the negative control. ****p\u0026lt;0.0001 (one-way ANOVA, Dunnet post-test).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4260336/v1/23c9b5660fe610a603ac38ee.png"},{"id":55089806,"identity":"2ab1a9d5-75ed-4951-b016-bd4f5f74d57d","added_by":"auto","created_at":"2024-04-22 12:13:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":521746,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e biological actions of γ-terpinene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLegend:\u003c/strong\u003e AA – arachidonic acid; AC: adenylyl cyclase; cAMP – cyclic adenosine monophosphate; ATP – adenosine triphosphate; Ca2+ - calcium ions; COX1 – Cyclooxygenase 1; DAG – diacylglycerol; eNOS – endothelial nitric oxide synthase; IP3 – inositol 1,2,4-trisphosphate; NO – Nitric oxide; PLA2 - Phospholipase A2; PLC – Phospholipase C; PI3K – phosphoinositide 3-kinase; PKA – protein kinase A; PKC - protein kinase C; PIP2: phosphatidylinositol 4,5-bisphosphate; PGI2 – prostacyclin; VASP – vasodilator-stimulated phosphoprotein; VASP-P – phosphorylated vasodilator-stimulated phosphoprotein.\u003c/p\u003e","description":"","filename":"floatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-4260336/v1/2e2ccf3e8f43f88b06efb749.png"},{"id":55090780,"identity":"5670d5fd-1ff9-4d14-8915-02bf267d381d","added_by":"auto","created_at":"2024-04-22 12:37:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1438466,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4260336/v1/35f754a9-8abc-48a3-927c-4f0643e01c09.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Non-clinical investigations about cytotoxic and anti-platelet activities of gamma-terpinene","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular diseases (CVDs) are a group of chronic non-communicable diseases that affect the heart and blood vessels, being responsible for 17.7\u0026nbsp;million deaths worldwide, most of them (85%) associated with the occurrence of thromboembolic events as heart attack and stroke (Benjamin et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Massa et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; World Health Organization 2019).\u003c/p\u003e \u003cp\u003eIt is widely accepted that platelets play a fundamental role in hemostasis and blood clotting at sites of vascular injuries, representing a link between thrombosis, inflammation and atherogenesis. When blood vessels are injured, platelets are activated by the exposed subcutaneous matrix and aggregate at the site of injury to stop bleeding, causing thrombotic occlusive ischemic events (Papapanagiotou et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; McFadyen et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rengasamy et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Olgasi et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe development of drugs that inhibit platelet aggregation has been one of the main targets for the prevention and treatment of thrombotic brain and cardiovascular diseases (Xiang et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Iqbal et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gerhards et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Thrombosis therapy involves the use of P2Y12 purinergic receptor inhibitors, such as ticlopidine, clopidrogrel, prasugrel, cangrelor and ticagrelor (Mackman \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Hamilton 2009). However, limitating adverse effects increase the risk of intracranial hemorrhage and dyspnea (Berger \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this context, the use of medicinal plants can be a complementary alternative (Simonetti et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sales \u0026amp; Alencar \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lima et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mortada \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMedicinal plants contain essential oils (EOs), secondary metabolites of low molecular weight molecules ofteh used as drugs, aromas and fragrances, pharmaceutical products, agrochemicals, dyes and pigments, pesticides, cosmetics, food additives, among others (Alves-Silva et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; \u0026Ccedil;akmak et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Toledo et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rocha et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the pharmacological point of view, terpenes stand out, namely gamma-terpinene (γ-terpinene, γ-TPN or 1-isopropyl-4-methylcyclohexa-1,4diene), a monoterpene found in \u0026ldquo;melaleuca\u0026rdquo; (\u003cem\u003eMelaleuca alternifolia\u003c/em\u003e), \u0026ldquo;or\u0026eacute;gano\u0026rdquo; (\u003cem\u003eOriganum vulgare\u003c/em\u003e), rosemary (\u003cem\u003eRosmarinus officinalis\u003c/em\u003e L.), thyme (\u003cem\u003eThymus vulgaris\u003c/em\u003e Marchand), tangerine (\u003cem\u003eCitrus delicious\u003c/em\u003e) and eucalyptus (\u003cem\u003eEucalyptus\u003c/em\u003e sp.) (Silva et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ramalho et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Baldissera et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Koyama \u0026amp; Heinbockel \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mota \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaking into account the biological properties of γ-TPN that have been elucidated so far, such as: antimicrobial activity (Piaru et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ramalho et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), in vitro antioxidant and preventive potential for chronic and cardiovascular (Choi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Li \u0026amp; Liu \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e); anti-inflammatory (Ramalho et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), antinociceptive (Passos et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and lipid-lowering (Takahashi et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), the objective of this article was to investigate the cytotoxic and antiplatelet activity of this compound in study models non-clinical.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eChemicals\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGamma-terpinene (γ-TPN), sodium nitrite, Griess reagent, xylazine, adenosine diphosphate (ADP), sodium phosphate and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Fetal calf serum, DMEM, DMEM High, trypsin-EDTA, penicillin and streptomycin were purchased from Cultilab (Campinas, Brazil). Tween 80 and dimethylsulfoxide (DMSO) were obtained from Din\u0026acirc;mica Qu\u0026iacute;mica Contempor\u0026acirc;nea LTDA (S\u0026atilde;o Paulo, Brazil). Ketamine, ammonium oxalate and sodium thiopental were obtained from Cristalia (S\u0026atilde;o Paulo, Brazil).\u003c/p\u003e \u003cp\u003eStock solutions were prepared with distilled water or DMSO and diluted to appropriate concentrations. γ-TPN was dissolved in DMSO for \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e protocols. Tween 80 (0.1% v/v) was used as eluent. All solutions were stored at 0\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCells and animals\u0026rsquo; facilities\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMurine axillary lymph node endothelium-like cell lines (SVEC4-10) and murine fibroblasts (L-929) were maintained in DMEM and DMEM high glucose cell media, 10% fetal serum fetal bovine and 1% (w/v) penicillin/streptomycin. The culture flasks (containing 2 x 10\u003csup\u003e6\u003c/sup\u003e viable cells) were observed under an inverted microscope (Biosystems, USA), followed by incubation in an oven at 37\u0026deg;C, 95% humidity and a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere (Shel Lab CO\u003csub\u003e2\u003c/sub\u003e Incubator, USA).\u003c/p\u003e \u003cp\u003eSpontaneously hypertensive (SHR) female Wistar rats (\u003cem\u003eRattus norvegicus\u003c/em\u003e) weighing between 180 and 200 g were obtained from the Central Animal Facility at Universidade Federal do Piau\u0026iacute;, Teresina, Brazil. They were kept in well-ventilated cages under standard conditions of light (12 h with alternative day and night cycles) and temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) and were housed with access to commercial rodent stock diet (Nutrilabor, Campinas, Brazil) and water \u003cem\u003ead libitum\u003c/em\u003e. All procedures were approved by the Committee on Animal Research at UFPI (#572/2019) and followed Brazilian (\u003cem\u003eCol\u0026eacute;gio Brasileiro de Experimenta\u0026ccedil;\u0026atilde;o Animal\u003c/em\u003e - COBEA) and International rules on the care and use of experimental animals (Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003eevaluation of γ-TPN\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 2D, 3D structures and the chemical representation with normal characters (ASCII), called SMILE (Simplified Molecular Input Line Entry Specification) of γ-TPN are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e below. These parameters were used for \u003cem\u003ein silico\u003c/em\u003e analysis of ADMET (Absorption, distribution, metabolism, excretion/toxicity) and target predictions were extracted from the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The molecular optimization of γ-TPN was carried out using ChemSketch software, version 14.0 and the molecular targets were prepared using Discovery Studio 20.1.0.\u003c/p\u003e\u003cp\u003eFor the prediction of physicochemical properties (lipophilicity - logP, molecular weight, polar surface area, number of hydrogen bond donors and acceptors, number of rotatable bonds and water solubility), pharmacokinetic parameters (ADMET) and similarity to drugs (Lipinski rules, CMC like, MDDR like, Leadlike and WDI like), the software PreADMET (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://preadmet.bmdrc.kr/\u003c/span\u003e\u003cspan address=\"https://preadmet.bmdrc.kr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SwissADME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissadme.ch/\u003c/span\u003e\u003cspan address=\"http://www.swissadme.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used. Complementary data were exposed by the Bioavailability Radar and BOILED-Egg. To predict the possible macromolecular targets of γ-TPN, the SwissTargetPrediction program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was also applied.\u003c/p\u003e \u003cp\u003eRegarding metabolic parameters, it can be checked whether the substance undergoes first pass metabolism and whether it inhibits cytochrome P450 (CYP) enzymes. The mathematical models used detected mutagenicity results, considering the Ames test and strains [\u003cem\u003eSalmonella typhimurium\u003c/em\u003e (TA98, TA100 and TA1535)] (Ames et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Mortelmans \u0026amp; Zeiger \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The prediction of carcinogenicity in rodents was carried out based on data from the National Toxicology Program (NTP) and the Food and Drug Administration (FDA) (Dolabela et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Regarding acute ecotoxicity, the maximum acceptable concentrations for \u003cem\u003eDaphnia\u003c/em\u003e sp., \u003cem\u003ePimephales promelas\u003c/em\u003e, \u003cem\u003eOryzias latipes\u003c/em\u003e, as well as the inhibition of growth in algae were investigated. Additionally, the classification of cardiotoxic risk was investigated based on inhibition of the human gene related to ether-a-go-go (hERG).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of docking molecular\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFirst, the molecular design and optimization of the three-dimensional structure of γ-TPN were carried out using the ACD/ChemSketch software, version 14.0, based on classical mechanics parameters (bond distance, bond angle and dihedral angle). The human P2Y12 receptor (4NTJ) was obtained from the Protein Data Bank (PDB). Only structures with a resolution below 3.0\u0026Aring; and obtained from \u003cem\u003eHomo sapiens\u003c/em\u003e were considered for this study. The files obtained were imported into the Discovery Studio Visualizer software, version 21.1.0. For ligand removal, crystallographic water molecules were kept and targets were saved in (.pdb) format. Next, the .pdb files were opened in the AutoDockTools software, version 1.5.7, where polar hydrogens and Gasteiger charges were added and saved in (.pdbqt) format (Panda et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Melo et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; He et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe molecular structures of γ-TPN and conventional antiplatelet agents (Prasugrel and Ticagrelor) were obtained from the PubChem website in (.sdf) format and individually subjected to geometry optimization using the Avogadro software, version 1.2.0, using the field of MMFF94s strength, subsequently being saved in (.pdb) format. Then, the optimized ligands were also imported into the AutoDockTools software, version 1.5.7, to add polar hydrogens and Gasteiger charges. All ligands were kept flexible before being saved in (.pdbqt) format (Hanwell et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kintamani et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe dimensions and coordinations in the Cartesian plane of the docking grid were automatically calculated using the AutoGrid module of AutoDock software version 4.2.6 and used to generate the configuration files. The grid volume was defined at 60x60x60 points (dimensions X, Y, Z), with a spacing of 0.375 \u0026Aring;, for the two targets tested. The AutoDock Vina software version 1.2.0 was used to perform the molecular docking of the ligands on each target following the scripts for flexible and hydrated docking made available by the Center of Computational Structural Biology (CCSB) at Scripps Research, with the exhaustivity parameter set at 100 races. The analysis of the bonds between targets and ligands and creation of interaction images was carried out using the BIOVIA Discovery Studio Visualizer version 21.1.0 and PyMOL version 2.5.4 software (Marzaro et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Forli et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Eberhardt et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cb\u003eMTT assay\u003c/b\u003e \u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eCytototoxic tests used MTT ([3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide]) according to Mosmann (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). SVEC4-10 and L-929 (5 x 10\u003csup\u003e3\u003c/sup\u003e cells/well) were incubated and about twenty-four hours later, γ-TPN (0 to 734 \u0026micro;M) was added to each well and the plates were incubated at 37 \u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere (Shel Lab CO\u003csub\u003e2\u003c/sub\u003e Incubator, USA) for 72 h. After the incubation period, 20 \u0026micro;L of the MTT solution (5 mg/mL) was added to each well and the plates were reincubated for 4 h. Next, formazan product was dissolved in DMSO and absorbance was read using a multiple reader (GloMax Explorer Multimode Microplate Reader, USA) at 595 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDesign of experimental\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eprotocol\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSHR rats were orally treated by gavage with γ-TPN at different doses (25, 50 and 100 mg/kg/day) for seven days. The animals were randomly divided into four groups of 5 animals each as follows: Group I (control group, saline solution), Group II (25 mg/kg/day of γ-TPN), Group III (50 mg/kg/day of γ-TPN), and Group IV (100 mg/kg/day of γ-TPN). Following the treatment, the animals were anesthetized with ketamine (90 mg/kg) plus xylazine (4.5 mg/kg) for blood collection Afterwards, all mice were euthanized with sodium thiopental (150 mg/kg i.p.).\u003c/p\u003e \u003cp\u003e \u003cb\u003eNitrite dosage\u003c/b\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo determine the nitrite content, the test use the Griess reaction (Green et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) in which, a total of 500 \u0026micro;L of Griess reagent was added to 500 \u0026micro;L of distilled water. In another test tube, 500 \u0026micro;L of Griess reagent was added to 500 \u0026micro;L of 10% erythrocyte homogenates (50 mM sodium phosphate buffer pH 7.4) extracted from γ-TPN-treated mice. Measurements were carried out at 560 nm and results were expressed as \u0026micro;M/mg of protein.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAntiplatelet activity of γ-TPN\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBlood was collected (3\u0026ndash;5 mL/animal) from anesthetized SHR rats through the aorta artery and by cardiac puncture (right ventricle) with plastic syringes and stored in tubes with citrate and 3.2% sodium in a ratio of 9:1 (blood: anticoagulant). The blood was centrifuged at 1650 rpm for 10 min to obtain platelet-rich plasma (PRP: 2.5 to 4.5 x 10\u003csup\u003e5\u003c/sup\u003e platelets/mm\u003csup\u003e3\u003c/sup\u003e) and at 3000 rpm for 15 min to obtain platelet-poor plasma (PPP: \u0026lt; 2.0 x 10\u003csup\u003e4\u003c/sup\u003e platelets/mm\u003csup\u003e3\u003c/sup\u003e). PPP was used as a diluent to adjust the final volume of PRP (Vinholt et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlatelet counting was perfomed using a Neubauer chamber (Brecher \u0026amp; Cronkite \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1950\u003c/span\u003e) with plasma diluted in 1% ammonium oxalate in a proportion of 1:200 (Tomasiak et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The antiplatelet activity of γ-TPN was determined in citrated PRP by the turbidimetric method described by Born \u0026amp; Cross (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1963\u003c/span\u003e) and monitored buy an aggregometer (Platelet Agregometer, EasyAgreg software, Benfer). Platelet aggregation was expressed as percentage of aggregation for ADP and as aggregation speed (Cattaneo \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Koltai et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSVEC4-10 cells were pretreated with γ-TPN at different concentrations (50, 100 and 200 \u0026micro;M) for 24 h. After this period, the level of platelet aggregation was determined in a suspension (300 \u0026micro;L) containing pre-treated platelets (3 x 10\u003csup\u003e8\u003c/sup\u003e platelets/mL) and SVEC4-10 cells (1.5 x 10\u003csup\u003e3\u003c/sup\u003e cells/mL). First, the first cuvette was placed in the device containing 300 \u0026micro;L of PPP. Then, the cuvette with 270 \u0026micro;L of PRP was placed with 20 \u0026micro;L of cells pretreated with γ-TPN. After the first five minutes of recording platelet aggregation, 10\u0026micro;L of the aggregator ADP (10 \u0026micro;M) was added (Cho et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), followed by additional 5 min for final recording.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eResults were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (S.E.M.). The binding energy values were subjected to normality analysis using the D\u0026rsquo;Agostino \u0026amp; Pearson test. Nonlinear regression was used to calculate average values of IC\u003csub\u003e50\u003c/sub\u003e (50% growth inhibition of cell proliferation). In order to determine differences between treatments, data were compared by unpaired T-test and one-way analysis of variance (ANOVA) followed by Dunnet, Tukey or Bonferroni test, considering p values\u0026thinsp;\u0026lt;\u0026thinsp;0,05 as statistically significant (GraphPad Prism version 6.0).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003eparameters\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn silico\u003c/em\u003e analysis data revealed physicochemical characteristics, pharmacokinetic profile and drug similarity prediction of γ-TPN (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Regarding physicochemical aspects, γ-TPN has a molecular weight of 136.23 g/mol, and it was found to be quite lipid-soluble (Log P\u0026thinsp;=\u0026thinsp;+\u0026thinsp;4.50), moderately water-soluble (Log S=-3.45), with 1 rotary bond, without hydrogen donor and acceptor groups, sp\u003csup\u003e3\u003c/sup\u003e carbon fraction of 0.60, molar refractivity of 47.12, and tension in polar surface area (TSPA) of 0 \u0026Aring;2.\u003c/p\u003e \u003cp\u003eγ-TPN revealed complete human intestinal absorption (100%), high permeability in Madin-Darby canine kidney cells - MDCK (244.91) and low permeability in human epithelial adenocarcinoma (Caco-2, 23.64 nm/s), besides a high degree of plasma protein binding (100%). It presented a comparative concentration ratio in the brain and plasma of 8.037, resulting in excellent penetration in the blood-brain barrier. The compound exhibited a skin permeability of -0.8857 and was not able to inhibit P-glycoprotein and CYP2C19 and CYP2C9 enzymes. On the other hand, it is a substrate of CYP3A4 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding the prediction of drug similarity, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showed that γ-TPN was classified as \u0026ldquo;drug-like\u0026rdquo; taking into consideration the Lipinski and World Drug Index (WDI like) rules, intermediate by the Modern Drug Data Report rule (MDDR like) and disqualified in this parameter by the Leadlike rule and the Comprehensive Medicinal Chemistry (CMC) database.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical, pharmacokinetic properties and drug-like prediction of γ-terpinene.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eProperty\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eResult\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePhysical chemistry\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eMolecular weight (g mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003eWater solubility (Log S)\u003c/p\u003e \u003cp\u003eLiposubility (Log P; XLog P3)\u003c/p\u003e \u003cp\u003eNumber of rotary connections\u003c/p\u003e \u003cp\u003eNumber of hydrogen bond donors\u003c/p\u003e \u003cp\u003eNumber of hydrogen bond acceptors\u003c/p\u003e \u003cp\u003eFraction of sp\u003csup\u003e3\u003c/sup\u003e carbons\u003c/p\u003e \u003cp\u003eMolar refractivity\u003c/p\u003e \u003cp\u003eVoltage on polar surface area (\u0026Aring;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e136.23\u003c/p\u003e \u003cp\u003e-3.45\u003c/p\u003e \u003cp\u003e+\u0026thinsp;3.35; +4.50\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e0.60\u003c/p\u003e \u003cp\u003e47.12\u003c/p\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePharmacokinetics\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eHuman Intestinal Absorption (%)\u003c/p\u003e \u003cp\u003ePermeability coefficient in Caco-2 (nm s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003ePermeability coefficient in MDCK (nm s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003eBinding to Plasma Proteins (%)\u003c/p\u003e \u003cp\u003ePenetration of the Blood-Brain Barrier\u003c/p\u003e \u003cp\u003eSkin permeability (cm\u003csup\u003e2\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003eP-glycoprotein inhibition\u003c/p\u003e \u003cp\u003eInhibition of CYP2C19\u003c/p\u003e \u003cp\u003eCYP2C9 inhibition\u003c/p\u003e \u003cp\u003eCYP2D6 inhibition\u003c/p\u003e \u003cp\u003eCYP2D6 substrate\u003c/p\u003e \u003cp\u003eCYP3A4 inhibition\u003c/p\u003e \u003cp\u003eCYP3A4 substrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003cp\u003e23.64\u003c/p\u003e \u003cp\u003e244.91\u003c/p\u003e \u003cp\u003e100.00\u003c/p\u003e \u003cp\u003e8.037\u003c/p\u003e \u003cp\u003e-0.8857\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eDrug-likeness prediction\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eLipinski's Rule\u003c/p\u003e \u003cp\u003eCMC Like Rule\u003c/p\u003e \u003cp\u003eMDDR Like Rule\u003c/p\u003e \u003cp\u003eLEAD Like Rule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eQualified\u003c/p\u003e \u003cp\u003eDisqualified\u003c/p\u003e \u003cp\u003eIntermediary\u003c/p\u003e \u003cp\u003eDisqualified\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eWDI Like Rule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eQualified\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThere is an intrinsic correlation between the physicochemical parameters of a compound and its biological performance, which facilitates the interpretation of its pharmacokinetics, pharmacodynamics and toxicity (Wenlock and Barton \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). As seen in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cem\u003ein silico\u003c/em\u003e ADME predicts 100% human intestinal absorption (HIA) for the drug candidate (Yee \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). γ-TPN has high intestinal absorption due to the oil-water partition coefficient (Log P), which guarantees elevated lipid solubility and absorption (from 70\u0026ndash;100%) crossing membranes by passive diffusion (Hadda et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It should be noted that the ideal HIA value of a drug will depend on its pharmaceutical indications and must be produced according to the therapeutical purposes (Dolabela et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnyway, the best-known rule for linking chemical cores to biological activities is the Lipinski's rule, named \u0026ldquo;rule of five\u0026rdquo;. It is based on lipid solubility (LogP)\u0026thinsp;\u0026le;\u0026thinsp;5, Molecular weight (MW)\u0026thinsp;\u0026le;\u0026thinsp;500, and number of H acceptors (HBA)\u0026thinsp;\u0026le;\u0026thinsp;10 and H donors (HBD)\u0026thinsp;\u0026le;\u0026thinsp;5 (Lipinski et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Lagorce et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thus, outocomes revealed that γ-TPN met all these prerequisites.\u003c/p\u003e \u003cp\u003ePermeability by intestinal Caco-2 cells is used for selecting drug candidates for oral administration, classifying γ-TPN as showing medium permeability through the intestinal epithelium (Yamashita et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Meanwhile, γ-TPN revealed to be highly permeable through MDCK cells, a useful tool for rapid screening of cell membrane permeability and pharmacological viability (Vistoli et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eγ-TPN also exposed high permeability with regard to penetration of the blood-brain barrier (BBB) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and is therefore classified as active on the Central Nervous System (CNS) (Zhao et al. \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). This biological property is relevant for ADME studies, as it provides data about therapeutic action on the CNS, binding to plasma proteins, their disposition and efficacy, especially in cerebrovascular diseases\u0026rsquo; conditions (Sekhar et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Harika et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother essential ADME parameter is the assessment of binding to the P-glycoprotein (P-gp). This protein is part of the ATP-dependent efflux pump, acting as a physiological barrier to protect the body against toxins and xenobiotics. Then, P-gp is directly involved to the intestinal absorption, metabolism and penetration of the BBB for the majority of drugs, and its inhibition or induction can significantly alter oral bioavailability and metabolism of a drug (Pereira \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt was also provided a module whose objective is to predict the skin permeability coefficient (Kp) using a linear regression model (logKp). The skin permeability parameter reveals the ability of γ-TPN to be absorbed through the skin, anticipating possible exposure to toxins or even accidental absorption of the compound during handling. Negative value for this coefficient (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) indicates the compound is impermeable to the skin, with a low possibility of being used intradermally (Souza et al. 2022), but probably without toxic effects upon skin exposure.\u003c/p\u003e \u003cp\u003eWithin this myriad of enzymes and transporters, the cytochrome P450 (CYP) superfamily of metabolic isoenzymes plays a crucial role (Testa \u0026amp; Kraemer 2007). Many drugs are targeted by these catalytical proteins, causing high rate of drug individual variability due to different degrees of CYP expression (Hollenberg \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Herein, \u003cem\u003ein silico\u003c/em\u003e showed γ-TPN inhibits the enzymes CYP2C19 and CYP2C9 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This type of activity may result in increased plasma drug concentrations, but it can induce early adverse effects (Teague et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Sekhar et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). On the other hand, γ-TPN did not show inhibitory activity of the CYP2D6 gene, which is responsible for the oxidative metabolism of many drugs and other xenobiotics and toxins in the cellular environment. The cytochrome CYP3A4, one of the most oxidative liver enzymes, an isoform that metabolizes 50% of all drugs, it is not inhibited by the compound, acting discreetly as a substrate of this enzyme (Oprea \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding genotoxic aspects (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), γ-TPN presented a positive result for the Ames test, a mutagenicity method that uses strains of \u003cem\u003eSalmonella typhimurium\u003c/em\u003e carrying mutations in histidine synthesis-involved genes (Prival \u0026amp; Zeiger \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Araki et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e also displays the acute toxicity profile of γ-TPN upon aquatic organisms, with the maximum tolerable concentrations for \u003cem\u003eDaphnia\u003c/em\u003e sp., \u003cem\u003ePimephales promelas\u003c/em\u003e, and \u003cem\u003eOryzias latipes\u003c/em\u003e of 0.23728, 0.04082, and 0.06008 mg/L, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e prediction of γ-terpinene toxicity.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResult\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmes test\u003c/p\u003e \u003cp\u003eCarcinogenicity test (rats and mice)\u003c/p\u003e \u003cp\u003eAcute toxicity to \u003cem\u003eDaphnia\u003c/em\u003e sp.\u003c/p\u003e \u003cp\u003eAcute toxicity to \u003cem\u003ePimephales promelas\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAcute toxicity to \u003cem\u003eOryzias latipes\u003c/em\u003e\u003c/p\u003e \u003cp\u003eInhibition of growth in algae\u003c/p\u003e \u003cp\u003ehERG gene inhibition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMutagen\u003c/p\u003e \u003cp\u003eCarcinogenic\u003c/p\u003e \u003cp\u003eAcceptable C\u003csub\u003emax\u003c/sub\u003e: 0.23728 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAcceptable C\u003csub\u003emax\u003c/sub\u003e: 0.04082 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAcceptable C\u003csub\u003emax\u003c/sub\u003e: 0.06008 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e0.02505 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMedium risk\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e= Maximum concentration.\u003c/p\u003e \u003cp\u003eAcute evalatuations on aquatic organisms, as well as those with \u003cem\u003eArtemia salina\u003c/em\u003e and \u003cem\u003eDanio rerio\u003c/em\u003e (zebrafish, family Cyprinidae), have been widely used to predict ecotoxicity of contaminants in aquatic organisms with compatible enzyme receptors (Chen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kingcade et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). According to the European legislation 92/32/EEC on safety of chemical substances (Weyers et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), γ-TPN has no potential to cause long-term adverse effects on aquatic environments (Solubility\u0026thinsp;\u0026lt;\u0026thinsp;1mg/L).\u003c/p\u003e \u003cp\u003eγ-TPN presented a moderate risk for inhibition of the human gene related to ether-a-go-go (hERG). It is responsible for encoding the subunit responsible for the formation of fast-type delayed-rectification potassium channels (IKr), important in the cardiac repolarization stage. Inhibition and dysfunction of this gene causes QT prolongation and fatal ventricular arrhythmia (Lamothe et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bjerregaard \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e demonstrates a diagram corresponding to the appropriate oral bioavailability profile of γ-TPN. The colored zone reveals itself as the appropriate physicochemical area, configuring an excellent oral bioavailability and meeting parameters of lipophilicity (LIPO), size (SIZE), polarity (POLAR), insolubility (INSOLU), unsaturations (INSATU), and flexibility (FLEX).\u003c/p\u003e \u003cp\u003eBioavailability Radar (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was displayed for rapid assessment of drug similarity, where six physicochemical properties are taken into consideration. To be considered a drug, the compound line under study must be fully included in the pink area (Daina et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Any deviation, as observed in the result expressed by γ-TPN regarding size, represents a sub-optimal physicochemical property for bioavailability.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e also shows the BOILED-Egg analysis and points out that γ-TPN in the yellow region presents good HIA and excellent BBB penetration power. However, it was not shown to be a substrate for P-glycoprotein, due to the indication of the red dot (Pgp-).\u003c/p\u003e \u003cp\u003eThe graph called BOILED-Egg displays a correlation of TPSA with lipophilicity (LogP). It is important to emphasize that the ability to cross the BBB is only relevant when the clinical target is located in the CNS, otherwise the drug may cause side effects, such as headache, drowsiness, dizziness, partial loss of vision, among others depending on the area where chemical interaction(s) may occur. Furthermore, it is important that the drug is not a substrate for proteins such as P-gp and therefore remains at an adequate concentration at the site of action to obtain the desired effect (Borges \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pereira \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e predicts the possible and best targets of the molecule and cytoplasmic receptors (26.3%), G protein-coupled receptors (20%) and the CYP enzyme (12.7%) were recognized as the majority.\u003c/p\u003e \u003cp\u003eG protein-coupled receptors, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e, represent the second largest class of pharmacological targets of γ-TPN, a result that corroborates the proposal of this study, which is to verify the antiaggregation potential of the monoterpene, considering purinergic ADP receptors, which belong to the Gi class of GPCRs (Hollopeter et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDocking molecular of γ-TPN\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe values of ∆G lig. of γ-TPN and RP2Y12, comparing them with conventional antiplatelet therapy drugs are also detailed ion Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The affinity of γ-TPN (-6.450\u0026thinsp;\u0026plusmn;\u0026thinsp;0.232 Kcal/mol) was statistically higher than that of Prasugrel (-5.793\u0026thinsp;\u0026plusmn;\u0026thinsp;0.223 Kcal/mol) in RP2Y12. Ticagrelor (-6.883\u0026thinsp;\u0026plusmn;\u0026thinsp;0.276 Kcal/mol), in turn, showed higher affinity than γ-TPN and Prasugrel. Interaction energy and affinity of the ligand-target complex are inversely proportional quantities, therefore, the lower the binding energy, the greater the stability of the interaction between ligand and protein (Trott \u0026amp; Olson \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Xiang et al. \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe RP2Y12 exhibits a response to the ADP signal and is involved in the inhibition of adenyl cyclase, Ca\u003csup\u003e2+\u003c/sup\u003e-dependent cell migration, regulation of cell morphology, and cell aggregation. RP2Y12 must be activated for ADP-induced platelet aggregation to occur, which can be prevented by a substance that impartially inhibits the receptor. RP2Y12 has a specific tissue distribution, making it a fundamental target for therapeutic intervention (Ahn et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Such binding inhibits the activity of P2Y12 on platelet aggregation. Therefore, the best coupling position of the γ-TPN in the 2D and 3D structures can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThere is similarity in the interaction sites of γ-TPN and Prasugrel in RP2Y12, with repetition of some amino acid residues (A:LYS:80, A:PHE:104, A:TYR:105). Ticagrelor, on the other hand, interacted with RP2Y12 in a region very close, but wider to the active site, probably due to its greater molecular weight. The amino acids that interacted with both γ-TPN and Ticagrelor were A:THR:76, A:LYS:80, A:SER:101, A:PHE:104, A:TYR:105 and A:LEU:284.\u003c/p\u003e \u003cp\u003eIt is known that γ-TPN had a lower average interaction energy with RP2Y12 than Prasugrel, and is therefore capable of forming a complex with greater stability (Zhang et al. \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Xiang et al. \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The justification for greater affinity of γ-TPN with the receptor arises from higher number of hydrophobic bonds created in relation to Prasugrel. On the other hand, still according to Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, even though the Ticagrelor- RP2Y12 complex formed an unfavorable bond (represented by the red color), which compromises the stability of the complex, the overall number of bonds formed was higher than the other two molecules, which may justify target greater affinityies (Dhorajiwala et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bender et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilarly, Nikitina et al. (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) evaluated the molecular fit of newly synthesized myrtenol-derived monoterpenes carrying different heteroatoms (sulfur, oxygen or nitrogen) as possible antiplatelet agents, comparing their affinities to P2Y12 with of ticagrelor. It was evident that molecular anchoring confirmed the interaction of all tested compounds with RP2Y12, suggesting that their antiaggregation properties are implemented by blocking P2Y12 function. The results of the study also show that the activity of the monoterpene is linked to the selective inhibition of platelet aggregation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecytotoxicity of γ-TPN by colorimetric assays\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIt was essential to carry out a cytotoxic screening of a compound in order to know at what concentration it is capable of causing damage to normal cells and, thus, proceed with other experiments with concentrations that are not toxic. Gama-terpinene at concentrations of 367 and 734 \u0026micro;M (50 and 100 \u0026micro;g/mL) significantly interfered with the cell viability of SVEC4-10 and L-929, compared to the control with untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It was not detected statistically significant viability differences on both cell lines tested at concentrations\u0026thinsp;\u0026le;\u0026thinsp;183.50 \u0026micro;M (25 \u0026micro;g/mL). Furthermore, it was found that γ-TPN presented a CC\u003csub\u003e50\u003c/sub\u003e of 366.70 and 333.33 \u0026micro;M (49.96 and 45.41 \u0026micro;g/mL), respectively, for SVEC4-10 and L-929 cells.\u003c/p\u003e \u003cp\u003eThe US National Cancer Institute (NCI) classifies a compound as having high cytotoxic activity when i) CC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;20 \u0026micro;g/mL; ii) moderate cytotoxic activity if CC\u003csub\u003e50\u003c/sub\u003e ranges between 21\u0026ndash;200 \u0026micro;g/mL; iii) weak cytotoxic activity if CC\u003csub\u003e50\u003c/sub\u003e is between 201\u0026ndash;500 \u0026micro;g/mL, and iv) no cytotoxic activity if CC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;500 \u0026micro;g/mL (Thienthiti et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). According to these categorization, γ-TPN has moderate cytotoxicity on normal cells. Previous studies with γ-TPN-treated HaCaT cell lines (long-lived human keratinocytes) showed increase in cell viability after 2h of exposure (Casalle \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt any rate, terpenes, especifically, act on cells causing damage to lipids and proteins, breaking down cell walls and membranes, resulting in cell lysis. In eukaryotic cells, they also destabilize the mitochondrial membrane and damage plasma membrane proteins (Bakkali et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Consequently, the number of metabolically active cells should be followed to understand cell toxic effects. Knowing that γ-TPN is a monoterpene mostly found in essential oils from plants whose anticoagulant and antithrombotic properties have already been duly proven, we seek to identify its antiplatelet action (Lamponi \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntiplatelet effect and nitrite dosage of γ-TPN\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe antiplatelet activity of PRP from rats was evaluated against 10 \u0026micro;M ADP. Firstly, it was observed that in the presence of ADP, there was platelet aggregation, a percentage extrapolated to 100% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In the presence of SVEC4-10, there was a significant 39.59% of reduction in platelet aggregation when compared to the negative control (ADP alone) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In the presence of γ-TPN 50 and 100 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), there was a greater reduction in platelet aggregation promoted by ADP (51.57 and 44.27%, respectively). Meanwhile, γ-TPN 200 \u0026micro;M promoted a reversal of platelet antiaggregation significantly compared to the control with ADP and SVEC4-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eUnder \u003cem\u003ein vitro\u003c/em\u003e conditions, the aggregation stimulated ADP to PRP triggers to its G protein-coupled receptors - P2Y1 and P2Y12 - initiating the process. ADP signaling through the RP2Y1 coupled to the Gq protein, promotes a rapid short-lasting response. It activates phospholipase C (PLC) and converts phosphatidylinositol 4,5-bisphosphate (PIP\u003csub\u003e2\u003c/sub\u003e) into inositol (1,4,5)-triphosphate (IP\u003csub\u003e3\u003c/sub\u003e) and diacylglycerol (DAG). DAG mobilizes intracellular Ca\u003csup\u003e2+\u003c/sup\u003e and, after activation of protein kinase C (PKC), these both secondary messengers induces increase of free Ca\u003csup\u003e2+\u003c/sup\u003e levels in the cytoplasm environment, resulting in reversible platelet aggregation (Gachet \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Fayaz \u0026amp; Rajanikant \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Aslam et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEndothelial SVEC4-10 cells exposed to ADP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) promoted a significant reduction in platelet aggregation, possibly justified by healthy endothelial cells expressing antiplatelet agents. Thus, when platelets encounter endothelial cells, platelet-endothelium interactions can occur for stimulating endothelial surface to produce and secrete mediators such as prostacyclin (PGI\u003csub\u003e2\u003c/sub\u003e) and nitric oxide (NO) (D'amico \u0026amp; Villa\u0026ccedil;a \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding γ-TPN-pre-treated SVEC4-10 cells, it was found that the compound alter platelet anti-aggregation, since under lower concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), it stimulates endothelial cells, probably through endothelial nitric oxide synthases (eNOS or NOS3) to produce nitric oxide (NO). It was confimed by \u003cem\u003ein vivo\u003c/em\u003e studies, in which the dose of 100 mg/kg (91.86\u0026thinsp;\u0026plusmn;\u0026thinsp;12.31\u0026micro;M) markedly increased indirect levels of nitric oxide quantified as nitrite content, which could be one of the factors responsible for inhibiting platelet aggregation (Radomski et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Tran et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This method was established using a calibration curve (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9942) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWith NO formed, the enzyme soluble guanylyl cyclase (GCs) was activated and the second messenger cyclic guanosine monophosphate (cGMP) was produced (F\u0026ouml;rstermann \u0026amp; Sessa \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wittenborn \u0026amp; Marletta \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). After activation, the platelets themselves generated NO (Radomski et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Malinski et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), inhibiting adhesion (Radomski et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e1987a\u003c/span\u003e; Radomski et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1987b\u003c/span\u003e) and aggregation (Freedman et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). cGMP, in turn, is the agent that determines the inhibitory actions of NO on platelets and this occurs through the decrease in the concentration of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e ([Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e) and modulation of the expression of surface receptors. The limitation of [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e occurs due to the inhibition of the release of Ca\u003csup\u003e2+\u003c/sup\u003e via the receptor of the dense tubular system, an increase in the rate of Ca\u003csup\u003e2+\u003c/sup\u003e extrusion, lower than its entry, through the extracellular environment, an increase in the activity of the Ca\u003csup\u003e2+\u003c/sup\u003e- ATPase of the endoplasmic reticulum, resulting in a lower amount of Ca\u003csup\u003e2+\u003c/sup\u003e available for platelet activation and aggregation mechanisms. Furthermore, cGMP also promotes the reduction of conformationally active GPIIb/IIIa receptors on the platelet surface, generating greater dissociation between fibrinogen and the GPIIb/IIIa receptor, producing platelet antiaggregation (Jin \u0026amp; Loscalzo \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ren et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding purinergic receptors, γ-TPN may also be strongly involved in the inhibition of RP2Y12, due to the results found at lower concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) and the high molecular affinity of the compound with this receptor, the which was compared with prasugrel and ticagrelor. Additionally, it is important to clarify that higher concentration of γ-TPN caused a nearly complete reversal of antiplatelet effect, suggesting γ-TPN 200 \u0026micro;M likely has cytotoxic action, promoting death of endothelial cells and decline in the production of antiaggregation mediators.\u003c/p\u003e \u003cp\u003eZhou et al. (\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) also evaluated \u003cem\u003ein vitro\u003c/em\u003e antiplatelet activity of monoterpenes and found that suchm molecules showed moderate inhibitory activities on ADP-induced blood platelet aggregation. Arag\u0026atilde;o et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) describe a mixture of triterpenes called α- and β-amyrin, isolated from \u003cem\u003eProtium heptaphyllum\u003c/em\u003e (Aubl) March (Burseraceae) with antiplatelet activity in a concentration-dependent manner which probably acts in a common biochemical pathway to all established agonists (ADP, collagen, arachidonic acid and AAS).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe compound γ-TPN has promising potentialities as to anti-platelet prototype, since it promoted platelet aggregation in the presence of ADP, while lower doses inhibited aggregation and weak cytotoxicity on endothelial cells and fibroblasts. These biological activities are linked to drug-likeness properties, including good bioavailability, heigh lipophilicity and intestinal absorption and capacity for crossing the blood-brain barrier. Moreiver, molecular docking analysis revealed γ-TPN as a molecule with affinity for the active site of the P2Y12 receptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was partially supported by the public Brazilian agence: \u0026ldquo;Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico\u0026rdquo; [CNPq - MCTI/CNPq N\u0026ordm; 14/2014)].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRailson Pereira Souza\u003csup\u003e\u0026nbsp;\u003c/sup\u003eis grateful to \u0026ldquo;Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior\u0026rdquo; (CAPES) (Finance code 001). Alde\u0026iacute;dia Pereira de Oliveira also thank to the Brazilian agency \u0026ldquo;Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico\u0026rdquo; [CNPq - MCTI/CNPq N\u0026ordm; 14/2014)] for his personal scholarships. \u0026nbsp;Thank you also to the reviewer Paulo Michel Pinheiro Ferreira, responsible for translating the article into English.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors participated in the study. RPS wrote the article. RPS and RWRS performed cytotoxicity assays with MTT and nitrite dosing protocols. RPS and VDP carried out in silico and molecular docking studies. RPS, EPS and ACAS were responsible for the in vitro and in vivo antiplatelet aggregation. DD and PMPF supervised and scientifically supported the experiments. APO planned the research, managed scientific and financial support, supervised all stages and reviewed the final article. The authors further stated that all data was generated internally and that no paper mills were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were approved by the Committee on Animal Research at UFPI (#572/2019) and followed Brazilian (\u003cem\u003eCol\u0026eacute;gio Brasileiro de Experimenta\u0026ccedil;\u0026atilde;o Animal\u003c/em\u003e - COBEA) and International rules on the care and use of experimental animals (Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that, in case of acceptance of the manuscript for publication, we agree to transfer the right of first publication to Naunyn-Schmiedeberg\u0026apos;s Archives of Pharmacology and the open access policy, therefore, the texts are available for anyone to read, download, copy, print, share, reuse and distribute, with due citation of the source and authorship. The datasets generated and/or analyzed during the current study are not yet publicly available, but are available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhn YA, Lee J-Y, Park HD, Kim TH, Park MC, Choi G \u0026amp; Kim S (2016). Identification of a new morpholine scaffold as a P2Y12 receptor antagonist. Molecules 21:1114. https://doi.org/10.3390/molecules 21091114\u003c/li\u003e\n \u003cli\u003eAlves-Silva JM, Guerra I, Gon\u0026ccedil;alves MJ, Cavaleiro C, Cruz MT, Figueirinha A et al. (2020). \u0026nbsp;Chemical composition of \u003cem\u003eCrithmum maritimum\u003c/em\u003e L. essential oil and hydrodistillation residual water by GC-MS and HPLC-DAD-MS/MS, and their biological activities. Industrial Crops and Products 149:1-9. https://doi.org/10.1016/j.indcrop.2020.112329\u003c/li\u003e\n \u003cli\u003eAmes BN, Mccann J \u0026amp; Yamasaki E (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation research 31:347\u0026ndash;364. https://doi.org/10.1016/0165-1161(75)90046-1\u003c/li\u003e\n \u003cli\u003eArag\u0026atilde;o GF, Carneiro LM, Junior AP, Vieira LC, Bandeira PN, Lemos TL, Viana GS (2006). A possible mechanism for anxiolytic and antidepressant effects of alpha- and beta-amyrin from Protium heptaphyllum (Aubl.) March. Pharmacol Biochem Behav 85:827-34. https://doi.org/10.1016/j.pbb.2006.11.019\u003c/li\u003e\n \u003cli\u003eAraki A, Kamigaito N, Sasaki T \u0026amp; Matsushima T (2004). Mutagenicity of carbon tetrachloride and chloroform in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, using a gas exposure method. Environmental and molecular mutagenesis 43:128\u0026ndash;133. https://doi.org/10.1002/em.20005\u003c/li\u003e\n \u003cli\u003eAslam M, Sedding D, Koshty A, Santoso S, Schulz R, Hamm C, G\u0026uuml;nd\u0026uuml;z D (2013). Nucleoside triphosphates inhibit ADP, collagen, and epinephrine-induced platelet aggregation: role of P2Y1 and P2Y12 receptors. Thrombosis Research132:548-57. https://doi.org/10.1016/j.thromres.2013.08.021\u003c/li\u003e\n \u003cli\u003eBakkali F, Averbeck S, Averbeck D \u0026amp; Idaomar M (2008). Biological effects of essential oils--a review. Food and Chemical Toxicology: an International journal published for the British Industrial Biological Research Association 46:446\u0026ndash;475.\u0026nbsp;https://doi.org/10.1016/j.fct.2007.09.106\u003c/li\u003e\n \u003cli\u003eBaldissera MD, Grando TH, Souza CF, Gressler LT, Stefani LM, da Silva AS, Monteiro SG (2016).\u0026nbsp;In vitro and in vivo action of terpinen-4-ol,\u0026nbsp;\u0026gamma;-terpinene, and\u0026nbsp;\u0026alpha;-terpinene against Trypanosoma evansi. Exp Parasitol. 162:43-8.\u0026nbsp;https://doi.org/10.1016/j.exppara.2016.01.004\u003c/li\u003e\n \u003cli\u003eBender BJ, Gahbauer S, Luttens A, Lyu J, Webb CM, Stein RM, Fink EA, Balius TE, Carlsson J, Irwin JJ \u0026amp; Shoichet BK (2021). A practical guide to large-scale docking. Nature protocols 16:4799\u0026ndash;4832.\u0026nbsp;https://doi.org/10.1038/s41596-021-00597-z\u003c/li\u003e\n \u003cli\u003eBenjamin EJ, Muntner P, Alonso A, et al. (2019). Heart disease and stroke statistics-2019 Update: a report from the American Heart Association. Circulation. 139:e56-e528.\u0026nbsp;https://doi.org/10.1161/CIR.0000000000000659\u003c/li\u003e\n \u003cli\u003eBerger JS (2018). Oral antiplatelet therapy for secondary prevention of acute coronary syndrome. Am J Cardiovasc Drugs. 18:457-472.\u0026nbsp;https://doi.org/10.1007/s40256-018-0291-2\u003c/li\u003e\n \u003cli\u003eBjerregaard P (2018). Diagnosis and management of short QT syndrome.\u0026nbsp;Heart rhythm\u0026nbsp;15:1261\u0026ndash;1267.\u0026nbsp;https://doi.org/10.1016/j.hrthm.2018.02.034\u003c/li\u003e\n \u003cli\u003eBorn GV\u0026nbsp;\u0026amp;\u0026nbsp;Cross MJ (1963). The aggregation of blood platelets. The Journal of Physiology 168:178-95.\u0026nbsp;https://doi.org/10.1113/jphysiol.1963.sp007185\u003c/li\u003e\n \u003cli\u003eBorges\u0026nbsp;NHPB (2018). An\u0026aacute;lise in silico das propriedades farmacol\u0026oacute;gicas e toxicol\u0026oacute;gicas dos organossel\u0026ecirc;nios e ensaios in vitro de atividades antibacteriana e moduladora da resist\u0026ecirc;ncia a drogas em \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Tese (Doutorado) - UFPB/CCS. 99 f. https://repositorio.ufpb.br/jspui/bitstream/123456789/19288/1/NathalieHelenPaesBarretoBorges_Tese.pdf\u003c/li\u003e\n \u003cli\u003eBrecher G \u0026amp; Cronkite EP (1950). Morphology and enumeration of human blood platelets. Journal of Applied Physiology 3:365\u0026ndash;377.\u0026nbsp;https://doi.org/10.1152/jappl.1950.3.6.365\u003c/li\u003e\n \u003cli\u003e\u0026Ccedil;akmak H, \u0026Ouml;zselek Y, Turan OY et al. (2020). Whey protein isolate edible films incorporated with essential oils: antimicrobial activity and barrier properties.\u0026nbsp;Polym. Degrad. Stabil. 179:109285.\u0026nbsp;https://doi.org/10.1016/j.polymdegradstab.2020.109285\u003c/li\u003e\n \u003cli\u003eCasalle N (2016). Susceptibilidade de carcinoma espinocelular oral ao \u0026oacute;leo essencial de \u003cem\u003eMelaleuca alternifolia\u003c/em\u003e e suas principais por\u0026ccedil;\u0026otilde;es sol\u0026uacute;veis. 43 f. Disserta\u0026ccedil;\u0026atilde;o (Mestrado em Ci\u0026ecirc;ncias Odontol\u0026oacute;gicas), Universidade Estadual Paulista, Faculdade de Odontologia, Araraquara.\u0026nbsp;http://hdl.handle.net/11449/144695\u003c/li\u003e\n \u003cli\u003eCattaneo M. (2009). Light transmission aggregometry and ATP release for the diagnostic assessment of platelet function. Seminars in thrombosis and hemostasis 35:158\u0026ndash;167.\u0026nbsp;https://doi.org/10.1055/s-0029-1220324\u003c/li\u003e\n \u003cli\u003eChen G, Jia Z, Wang L \u0026amp; Hu T. (2020). Effect of acute exposure of saxitoxin on development of zebrafish embryos (\u003cem\u003eDanio rerio\u003c/em\u003e). Environmental research 185:109432.\u0026nbsp;https://doi.org/10.1016/j.envres.2020.109432\u003c/li\u003e\n \u003cli\u003eCho MS, Noh K, Haemmerle M, Li D, Park H, Hu Q, Hisamatsu T, Mitamura T, Mak SLC, Kunapuli S, Ma Q, Sood AK\u0026nbsp;\u0026amp;\u0026nbsp;Afshar-Kharghan V (2017). Role of ADP receptors on platelets in the growth of ovarian cancer. Blood 130:1235-1242.\u0026nbsp;https://doi.org/10.1182/blood-2017-02-769893\u003c/li\u003e\n \u003cli\u003eChoi HS, Song HS, Ukeda H\u0026nbsp;\u0026amp;\u0026nbsp;Sawamura M (2000). Radical-scavenging activities of citrus essential oils and their components: detection using 1,1-diphenyl-2-picrylhydrazyl. Journal of Agricultural and Food Chemistry\u0026nbsp;48:4156-61.\u0026nbsp;https://doi.org/10.1021/jf000227d\u003c/li\u003e\n \u003cli\u003eD\u0026apos;Amico \u0026Eacute;A \u0026amp; Villa\u0026ccedil;a PR (2006).\u0026nbsp;Dist\u0026uacute;rbios da coagula\u0026ccedil;\u0026atilde;o no departamento de emerg\u0026ecirc;ncia. In Emerg\u0026ecirc;ncias cl\u0026iacute;nicas baseadas em evid\u0026ecirc;ncias: disciplina de emerg\u0026ecirc;ncias cl\u0026iacute;nicas. S\u0026atilde;o Paulo: Editora Atheneu.\u0026nbsp;https://repositorio.usp.br/item/001528493\u003c/li\u003e\n \u003cli\u003eDaina A, Michielin O \u0026amp; Zoete V (2017).\u0026nbsp;SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific reports 7:42717.\u0026nbsp;https://doi.org/10.1038/srep42717\u003c/li\u003e\n \u003cli\u003eDhorajiwala TM, Halder ST and Samant L (2019). Comparative in silico molecular docking analysis of L-threonine-3-dehydrogenase, a protein target against african Trypanosomiasis using selected phytochemicals.\u0026nbsp;\u003cem\u003eJournal of Applied Biotechnology Reports\u003c/em\u003e 6:101-108. https://doi.org/10.29252/JABR.06.03.04\u003c/li\u003e\n \u003cli\u003eDolabela MF, Silva ARP, Ohashi LH, Bastos MLC, Silva MCM\u0026nbsp;\u0026amp;\u0026nbsp;Vale VV (2018).\u0026nbsp;Estudo in silico das atividades de triterpenos e iridoides isolados de \u003cem\u003eHimatanthus articulatus\u003c/em\u003e (Vahl) Woodson. Revista Fitos 12:227-242.\u0026nbsp;https://doi.org/10.17648/2446-4775.2018.602\u003c/li\u003e\n \u003cli\u003eEberhardt J, Santos-Martins D, Tillack AF \u0026amp; Forli S (2021).\u0026nbsp;AutoDock Vina 1.2.0: new docking methods, expanded force field, and python bindings. Journal of chemical information and modeling 61:3891\u0026ndash;3898.\u0026nbsp;https://doi.org/10.1021/acs.jcim.1c00203\u003c/li\u003e\n \u003cli\u003eFayaz SM \u0026amp; Rajanikant GK (2012). A critical appraisal of the functional evolution of P2Y12 antagonists as antiplatelet drugs. Current Pharmaceutical Design 18:1625\u0026ndash;1634.\u0026nbsp;https://doi.org/10.2174/138161212799958558\u003c/li\u003e\n \u003cli\u003eForli S, Huey R, Piqu\u0026eacute; M,\u0026nbsp;Sanner MF, Goodsell DS\u0026nbsp;\u0026amp;\u0026nbsp;Olson\u0026nbsp;AJ\u0026nbsp;(2016).\u0026nbsp;Docking computacional de prote\u0026iacute;na-ligante e triagem virtual de drogas com o conjunto AutoDock. Nature Protocols 11:905\u0026ndash;919.\u0026nbsp;https://doi.org/10.1038/nprot.2016.051\u003c/li\u003e\n \u003cli\u003eF\u0026ouml;rstermann U, Sessa WC (2012).\u0026nbsp;Nitric oxide synthases: regulation and function. Eur Heart J. 33:829-37, 837a-837d.\u0026nbsp;https://doi.org/10.1093/eurheartj/ehr304\u003c/li\u003e\n \u003cli\u003eFreedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, Michelson AD (1997). Nitric oxide released from activated platelets inhibits platelet recruitment. J Clin Invest. 1997 100:350-6.\u0026nbsp;https://doi.org/10.1172/JCI119540\u003c/li\u003e\n \u003cli\u003eGachet C. (2012). P2Y(12) receptors in platelets and other hematopoietic and non-hematopoietic cells. Purinergic signalling 8:609\u0026ndash;619.\u0026nbsp;https://doi.org/10.1007/s11302-012-9303-x\u003c/li\u003e\n \u003cli\u003eGerhards C, Uhlig S, Etemad M, Christodoulou F, Bieback K, Kl\u0026uuml;ter H \u0026amp; Bugert P (2021).\u0026nbsp;Expression of ADP receptor P2Y12, thromboxane A2 receptor and C-type lectin-like receptor 2 in cord blood-derived megakaryopoiesis.\u0026nbsp;Platelets 32:618\u0026ndash;625.\u0026nbsp;https://doi.org/10.1080/09537104.2020.1782868\u003c/li\u003e\n \u003cli\u003eGreen LC, Tannenbaum SR \u0026amp; Goldman P (1981). Nitrate synthesis in the germfree and conventional rat. Science 212:56\u0026ndash;58.\u0026nbsp;https://doi.org/10.1126/science.6451927\u003c/li\u003e\n \u003cli\u003eHadda TB, Ali MA, Masand V, Gharby S, Fergoug T \u0026amp; Warad I (2013). Tautomeric origin of dual effects of N1-nicotinoyl-3-(4\u0026prime;-hydroxy- 3\u0026prime;-methyl phenyl)-5-[(sub)phenyl]-2-pyrazolines on bacterial and viral strains: POM analyses as new efficient bioinformatics\u0026apos; platform to predict and optimize bioactivity of drugs.\u0026nbsp;\u003cem\u003eMedicinal Chemistry Research\u003c/em\u003e \u003cem\u003e22\u003c/em\u003e:1438-1449.\u0026nbsp;https://doi.org/10.1007/s00044-012-0143-6\u003c/li\u003e\n \u003cli\u003eHamilton JR. Protease-activated receptors as targets for antiplatelet therapy (2009). Blood Rev 23:61-5.\u0026nbsp;https://doi.org/10.1016/j.blre.2008.06.002\u003c/li\u003e\n \u003cli\u003eHanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E \u0026amp; Hutchison GR (2012). Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J Cheminformatics 4:17 (2012).\u0026nbsp;https://doi.org/10.1186/1758-2946-4-17\u003c/li\u003e\n \u003cli\u003eHarika MS, Kumar R\u0026nbsp;\u0026amp;\u0026nbsp;Reddy SS. Docking studies of benzimidazole derivatives using HEX 8.0 M (2017). International Journal of Pharmaceutical Sciences and Research 8:1677\u0026ndash;1688.\u0026nbsp;https://doi.org/10.13040/IJPSR.0975-8232.8(4).1677-88\u003c/li\u003e\n \u003cli\u003eHe Q, Liu C, Wang X, Rong K, Zhu M, Duan L, Zheng P \u0026amp; Mi Y (2023). Exploring the mechanism of curcumin in the treatment of colon cancer based on network pharmacology and molecular docking. Frontiers in pharmacology 14: 1102581.\u0026nbsp;https://doi.org/10.3389/fphar.2023.1102581\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHollenberg PF (2002). Characteristics and common properties of inhibitors, inducers, and activators of CYP enzymes. Drug metabolism reviews 34:17-35.\u0026nbsp;https://doi.org/10.1081/dmr-120001387\u003c/li\u003e\n \u003cli\u003eHollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A\u0026nbsp;\u0026amp;\u0026nbsp;Julius D (2001). Identification of the platelet ADP receptor targeted by antithrombotic drugs.\u0026nbsp;Nature\u0026nbsp;409:202\u0026ndash;207.\u0026nbsp;https://doi.org/10.1038/35051599\u003c/li\u003e\n \u003cli\u003eHuang SM, Strong JM, Zhang L, Reynolds KS, Nallani S, Temple R, Abraham S, Habet SA, Baweja RK, Burckart GJ, Chung S, Colangelo P, Frucht D, Green MD, Hepp P, Karnaukhova E, Ko HS, Lee JI, Marroum PJ, Norden JM, \u0026hellip;\u0026nbsp;Lesko LJ (2008). New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process. Journal of clinical pharmacology, 48:662\u0026ndash;670.\u0026nbsp;https://doi.org/10.1177/0091270007312153\u003c/li\u003e\n \u003cli\u003eIqbal NS, Jascur TA, Harrison SM, Edwards AB, Smith LT, Choi ES, Arevalo MK, Chen C, Zhang S, Kern AJ, Scheuerle AE, Sanchez EJ, Xing C \u0026amp; Baker LA (2020). Prune belly syndrome in surviving males can be caused by Hemizygous missense mutations in the X-linked Filamin A gene. BMC Med Genet 21:38.\u0026nbsp;https://doi.org/10.1186/s12881-020-0973-x\u003c/li\u003e\n \u003cli\u003eJin RC, Loscalzo J (2010). Vascular nitric oxide: formation and function. J Blood Med 2010:147-162.\u0026nbsp;https://doi.org/10.2147/JBM.S7000\u003c/li\u003e\n \u003cli\u003eKhan N, Jamila N, Ejaz R, Nishan U \u0026amp; Kim KS (2020).\u0026nbsp;Volatile oil, phytochemical, and biological activities evaluation of \u003cem\u003eTrachyspermum ammi\u003c/em\u003e seeds by chromatographic and spectroscopic methods. Analytical Letters 53:6, 984-1001. https://doi.org/10.1080/00032719.2019.1688825\u003c/li\u003e\n \u003cli\u003eKingcade A, Ahuja N, Jefferson A, Schaffer PA, Ryschon H, Cadmus P, Garrity D \u0026amp; Ramsdell H (2021). Morbidity and mortality in Danio rerio and Pimephales promelas exposed to antilipidemic drug mixtures (fibrates and statins) during embryogenesis: Comprehensive assessment via ante and post-mortem endpoints.\u0026nbsp;Chemosphere 263:127911.\u0026nbsp;https://doi.org/10.1016/j.chemosphere.2020.127911\u003c/li\u003e\n \u003cli\u003eKintamani E, Batubara I, Kusmana C, Tiryana T, Mirmanto E\u0026nbsp;\u0026amp;\u0026nbsp;Asoka SF (2023).\u0026nbsp;Essential oil compounds of andaliman (Zanthoxylum acanthopodium DC.) Fruit varieties and their utilization as skin anti-aging using molecular docking.\u0026nbsp;\u003cem\u003eLife\u003c/em\u003e 13:754.\u0026nbsp;https://doi.org/10.3390/life13030754\u003c/li\u003e\n \u003cli\u003eKoltai K, Kesmarky G, Feher G, Tibold A\u0026nbsp;\u0026amp;\u0026nbsp;Toth K (2017). Platelet aggregometry testing: molecular mechanisms, techniques and clinical implications. International Journal of Molecular Sciences 18:1803.\u0026nbsp;https://doi.org/10.3390/ijms18081803\u003c/li\u003e\n \u003cli\u003eKoyama S\u0026nbsp;\u0026amp;\u0026nbsp;Heinbockel T (2020). The effects of essential oils and terpenes in relation to their routes of intake and application.\u0026nbsp;International Journal of Molecular Sciences\u0026nbsp;21:1558.\u0026nbsp;https://doi.org/10.3390/ijms21051558\u003c/li\u003e\n \u003cli\u003eLagorce D, Douguet D, Miteva MA \u0026amp; Villoutreix BO (2017). Computational analysis of calculated physicochemical and ADMET properties of protein-protein interaction inhibitors. Scientific reports 7:46277.\u0026nbsp;https://doi.org/10.1038/srep46277\u003c/li\u003e\n \u003cli\u003eLamothe SM, Guo J, Li W, Yang T\u0026nbsp;\u0026amp;\u0026nbsp;Zhang S (2016). The human ether-a-go-go-related gene (hERG) potassium channel represents an unusual target for protease-mediated damage. Journal of Biological Chemistry 291:20387-401.\u0026nbsp;https://doi.org/10.1074/jbc.M116.743138\u003c/li\u003e\n \u003cli\u003eLamponi S (2021). Bioactive natural compounds with antiplatelet and anticoagulant activity and their potential role in the treatment of thrombotic disorders.\u0026nbsp;Life (Basel) 11:1095.\u0026nbsp;https://doi.org/10.3390/life11101095\u003c/li\u003e\n \u003cli\u003eLima JF, Oliveira Neto JG, Ara\u0026uacute;jo AE, Silva DA, Freitas JT (2019). Levantamento participativo e forma\u0026ccedil;\u0026atilde;o continuada sobre plantas medicinais cultivadas no munic\u0026iacute;pio de Serraria-PB.\u0026nbsp;Brazilian Journal of Agroecology and Sustainability 1:1-17.\u0026nbsp;https://doi.org/10.52719/bjas.v1i2.2886\u003c/li\u003e\n \u003cli\u003eLi GX \u0026amp; Liu ZQ (2009). Unusual antioxidant behavior of alpha- and gamma-terpinene in protecting methyl linoleate, DNA, and erythrocyte. J Agric Food Chem. 57:3943-8.\u0026nbsp;https://doi.org/10.1021/jf803358g\u003c/li\u003e\n \u003cli\u003eLipinski CA, Lombardo F, Dominy BW\u0026nbsp;\u0026amp;\u0026nbsp;Feeney PJ (1997).\u0026nbsp;Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings.\u0026nbsp;Advanced Drug Delivery Reviews\u0026nbsp;23: 3-25.\u0026nbsp;https://doi.org/10.1016/S0169-409X(96)00423-1\u003c/li\u003e\n \u003cli\u003eMackman N. Triggers, targets and treatments for thrombosis (2008).\u0026nbsp;Nature 451:914-8.\u0026nbsp;https://doi.org/10.1038/nature06797\u003c/li\u003e\n \u003cli\u003eMalinski T, Radomski MW, Taha Z \u0026amp; Moncada, S. (1993).\u0026nbsp;Direct electrochemical measurement of nitric oxide released from human platelets. Biochemical and Biophysical Research Communications 194:960\u0026ndash;965.\u0026nbsp;https://doi.org/10.1006/bbrc.1993.1914\u003c/li\u003e\n \u003cli\u003eMarzaro G, Ferrarese A \u0026amp; Chilin A (2014). Autogrid-based clustering of kinases: selection of representative conformations for docking purposes.\u0026nbsp;Molecular diversity 18:611\u0026ndash;619.\u0026nbsp;https://doi.org/10.1007/s11030-014-9524-8\u003c/li\u003e\n \u003cli\u003eMassa KHC, Duarte YAO \u0026amp; Chiavegatto Filho ADP (2019). An\u0026aacute;lise da preval\u0026ecirc;ncia de doen\u0026ccedil;as cardiovasculares e fatores associados em idosos, 2000-2010.\u0026nbsp;Cien Saude Colet 24:105-114.\u0026nbsp;https://doi.org/10.1590/1413-81232018241.02072017\u003c/li\u003e\n \u003cli\u003eMcfadyen JD, Schaff M, Peter K (2018).\u0026nbsp;Current and future antiplatelet therapies: emphasis on preserving haemostasis.\u0026nbsp;Nature Reviews Cardiology\u0026nbsp;15:181\u0026ndash;191.\u0026nbsp;https://doi.org/10.1038/nrcardio.2017.206\u003c/li\u003e\n \u003cli\u003eMelo DS, Nery Neto JAO, Santos MSD, Pimentel VD, Carvalho RCV, Sousa VC, Sousa RGC, Nascimento LGD, Alves MMM, Arcanjo DDR, Sousa DP \u0026amp; Carvalho FAA (2022).\u0026nbsp;Isopropyl gallate, a gallic acid derivative: in silico and in vitro investigation of its effects on \u003cem\u003eLeishmania major\u003c/em\u003e. Pharmaceutics 14:2701.\u0026nbsp;https://doi.org/10.3390/pharmaceutics14122701\u003c/li\u003e\n \u003cli\u003eMortada EM (2024). Evidence-based complementary and alternative medicine in current medical practice.\u0026nbsp;Cureus 16:e52041.\u0026nbsp;https://doi.org/10.7759/cureus.52041\u003c/li\u003e\n \u003cli\u003eMortelmans K\u0026nbsp;\u0026amp;\u0026nbsp;Zeiger E (2000).\u0026nbsp;The Ames \u003cem\u003eSalmonella\u003c/em\u003e/microsome mutagenicity assay.\u0026nbsp;Mutation research 455: 29\u0026ndash;60.\u0026nbsp;https://doi.org/10.1016/s0027-5107(00)00064-6\u003c/li\u003e\n \u003cli\u003eMosmann T (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.\u0026nbsp;Journal of immunological methods 65:55\u0026ndash;63.\u0026nbsp;https://doi.org/10.1016/0022-1759(83)90303-4\u003c/li\u003e\n \u003cli\u003eMota AA (2022). Terpenos: m\u0026eacute;todos de extra\u0026ccedil;\u0026atilde;o, sua forma\u0026ccedil;\u0026atilde;o e aplica\u0026ccedil;\u0026otilde;es. Gama, DF: UNICEPLAC, 68 p. https://dspace.uniceplac.edu.br/bitstream/123456789/1170/1/Terpenos%20-%20m%C3%A9todos%20de%20extra%C3%A7%C3%A3o%2C%20sua%20 forma%C3%A7%C3%A3o%20e%20aplica%C3%A7%C3%B5es.pdf\u003c/li\u003e\n \u003cli\u003eNikitina LE, Pavelyev RS, Gilfanov IR, Kiselev SV, Azizova ZR, Ksenofontov AA, Bocharov PS, Antina EV, Klochkov VV, Timerova AF, Rakhmatullin IZ, Ostolopovskaya OV, Khelkhal MA, Boichuk SV, Galembikova AR, Andriutsa NS, Frolova LL, Kutchin AV, Kayumov AR (2022). Unraveling the mechanism of platelet aggregation suppression by monoterpenoids. Bioengineering (Basel) 9:24.\u0026nbsp;https://doi.org/10.3390/bioengineering9010024\u003c/li\u003e\n \u003cli\u003eOlgasi C, Assanelli S, Cucci A \u0026amp; Follenzi A (2024). Hemostasis and endothelial functionality: the double face of coagulation factors. Haematologica.\u0026nbsp;https://doi.org/10.3324/haematol.2022.282272\u003c/li\u003e\n \u003cli\u003eOprea TI (2000). Property distribution of drug-related chemical databases.\u0026nbsp;Journal of computer-aided molecular design\u0026nbsp;14:251\u0026ndash;264.\u0026nbsp;https://doi.org/10.1023/a:1008130001697\u003c/li\u003e\n \u003cli\u003ePanda PK, Arul MN, Patel P, Verma SK, Luo W, Rubahn HG, Mishra YK, Suar M \u0026amp; Ahuja R. (2020).\u0026nbsp;Structure-based drug designing and immunoinformatics approach for SARS-CoV-2. Science advances 6:eabb8097.\u0026nbsp;https://doi.org/10.1126/sciadv.abb8097\u003c/li\u003e\n \u003cli\u003ePapapanagiotou A, Daskalakis G, Siasos G, Gargalionis A, Papavassiliou AG (2016). The Role of Platelets in Cardiovascular Disease: Molecular Mechanisms.\u0026nbsp;Current Pharmaceutical Design\u0026nbsp;22:4493-4505.\u0026nbsp;https://doi.org/10.2174/1381612822666160607064118\u003c/li\u003e\n \u003cli\u003ePassos FF, Lopes EM, de Ara\u0026uacute;jo JM, de Sousa DP, Veras LM, Leite JR\u0026nbsp;\u0026amp;\u0026nbsp;Almeida FR (2015).\u0026nbsp;Involvement of Cholinergic and Opioid System in\u0026nbsp;\u0026gamma;-Terpinene-Mediated Antinociception. Evid Based Complement Alternat Med. 2015:829414.\u0026nbsp;https://doi.org/10.1155/2015/829414\u003c/li\u003e\n \u003cli\u003ePereira ALC (2019).\u0026nbsp;S\u0026iacute;ntese, elucida\u0026ccedil;\u0026atilde;o estrutural e estudos in silico de novos compostos 2-amino-tiof\u0026ecirc;nicos im\u0026iacute;dicos candidatos a f\u0026aacute;rmacos antif\u0026uacute;ngicos, antileishmanicida e antitumorais. Disserta\u0026ccedil;\u0026atilde;o (Mestrado em Produtos Naturais e Sint\u0026eacute;ticos Bioativos). 164 f. Jo\u0026atilde;o Pessoa \u0026ndash; JP.\u0026nbsp;https://repositorio.ufpb.br/jspui/handle/123456789/15841\u003c/li\u003e\n \u003cli\u003ePiaru SP, Mahmud R, Abdul Majid AM, Ismail S \u0026amp; Man CN (2012). Chemical composition, antioxidant and cytotoxicity activities of the essential oils of Myristica fragrans and Morinda citrifolia. J Sci Food Agric. 92:593-7.\u0026nbsp;https://doi.org/10.1002/jsfa.4613\u003c/li\u003e\n \u003cli\u003ePrival MJ \u0026amp; Zeiger E (1998). Chemicals mutagenic in Salmonella typhimurium strain TA1535 but not in TA100. Mutation Research -\u0026nbsp;Genetic Toxicology and Environmental Mutagenesis\u0026nbsp;412:251\u0026ndash;260.\u0026nbsp;https://doi.org/10.1016/s1383-5718(97)00196-4\u003c/li\u003e\n \u003cli\u003eRadomski MW, Palmer RM \u0026amp; Moncada S (1990).\u0026nbsp;Characterization of the L-arginine: nitric oxide pathway in human platelets.\u0026nbsp;British Journal of Pharmacology\u0026nbsp;101:325\u0026ndash;328.\u0026nbsp;https://doi.org/10.1111/j.1476-5381.1990.tb12709.x\u003c/li\u003e\n \u003cli\u003eRadomski MW, Palmer RM \u0026amp; Moncada S\u0026nbsp;(1987b).\u0026nbsp;Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets. British Journal of Pharmacology, 92:181\u0026ndash;187.\u0026nbsp;https://doi.org/10.1111/j.1476-5381.1987.tb11310.x\u003c/li\u003e\n \u003cli\u003eRadomski MW, Palmer RM \u0026amp; Moncada S (1987a). The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide.\u0026nbsp;British Journal of Pharmacology 92:639\u0026ndash;646.\u0026nbsp;https://doi.org/10.1111/j.1476-5381.1987.tb11367.x\u003c/li\u003e\n \u003cli\u003eRamalho TR, Filgueiras LR, Pacheco de Oliveira MT, Lima AL, Bezerra-Santos CR, Jancar S \u0026amp; Piuvezam MR (2016).\u0026nbsp;Gamma-terpinene modulation of LPS-stimulated macrophages is dependent on the PGE2/IL-10 Axis. Planta Med. 82:1341-1345.\u0026nbsp;https://doi.org/10.1055/s-0042-107799\u003c/li\u003e\n \u003cli\u003eRen H, Sun Y, Li Y, Yuan X, Jiang B, Zhang W, Liu G \u0026amp; Lu P (2024). Potential mechanism of platelet GPIIb/IIIa and fibrinogen on retinal vein occlusion. Current Eye Research 1-11.\u0026nbsp; https://doi.org/10.1080/02713683.2024.2327055\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eRengasamy KRR, Khan H, Ahmad I, Lobine D, Mahomoodally F, Suroowan S, Hassan STS, Xu S, Patel S, Daglia M, Nabavi SM, Pandian SK (2019). Bioactive peptides and proteins as alternative antiplatelet drugs.\u0026nbsp;\u003cem\u003eMedicinal Research Reviews\u003c/em\u003e.\u0026nbsp;39:2153-2171.\u0026nbsp;https://doi.org/10.1002/med.21579\u003c/li\u003e\n \u003cli\u003eRocha MM, Rodrigues RDS, Guimar\u0026atilde;es PHV, et al. (2022). Potencial larvicida de \u0026oacute;leos essenciais de plantas brasileiras contra \u003cem\u003eAedes aegypti\u003c/em\u003e.\u0026nbsp;Pesquisa, Sociedade e Desenvolvimento\u0026nbsp;2:e53211226140. https://doi.org/10.33448/rsd-v11i2.26140\u003c/li\u003e\n \u003cli\u003eSales AD, Alencar CMM (2019). Produ\u0026ccedil;\u0026atilde;o e uso de plantas medicinais como processo pedag\u0026oacute;gico de educa\u0026ccedil;\u0026atilde;o ambiental. Revista Gest\u0026atilde;o \u0026amp; Sustentabilidade Ambiental 8:468-488. \u0026nbsp;https://doi.org/10.19177/rgsa.v8e42019468-488\u003c/li\u003e\n \u003cli\u003eSekhar KC, Syed R, Golla M, Kumar M V J, Yellapu NK, Chippada AR, Chamarthi NR (2014).\u0026nbsp;Novel heteroaryl phosphonicdiamides PTPs inhibitors as anti-hyperglycemic agents. Daru Journal of Pharmaceutical Sciences 22:76.\u0026nbsp;https://doi.org/10.1186/s40199-014-0076-3\u003c/li\u003e\n \u003cli\u003eSilva RF, Rezende CM, Pereira JB et al.\u0026nbsp;(2015).\u0026nbsp;Scents from Brazilian Cerrado: chemical composition of the essential oil from \u003cem\u003ePseudobrickellia brasiliensis\u003c/em\u003e (Asteraceae). J Essent Oil Res 27:417\u0026ndash;420.\u0026nbsp;https://doi.org/10.1080/10412905.2015.1037021\u003c/li\u003e\n \u003cli\u003eSimonetti E, Ethur ME, Castro LC, Kauffmann C, Giacomin AC, Ledur A et al.\u0026nbsp;(2016). Avalia\u0026ccedil;\u0026atilde;o da atividade antimicrobiana de extratos de Eugenia anomala e Psidium salutare (Myrtaceae) frente \u0026agrave; \u003cem\u003eEscherichia coli\u003c/em\u003e e \u003cem\u003eListeria monocytogenes\u003c/em\u003e. Revista Brasileira de Plantas Medicinais 18:9-18.\u0026nbsp;https://doi.org/10.1590/1983-084X/15_005\u003c/li\u003e\n \u003cli\u003eTakahashi Y, Inaba N, Kuwahara S \u0026amp; Kuki W (2003).\u0026nbsp;Effects of\u0026nbsp;\u0026gamma;-terpinene on lipid concentrations in serum using triton ER1330- treated rats. Bioscience, Biotechnology, and Biochemistry 67:2448-2450. http://dx.doi.org/10.1271/bbb.67.2448\u003c/li\u003e\n \u003cli\u003eTeague SJ, Davis AM, Leeson PD, Oprea T (1999). The design of leadlike combinatorial libraries. Angewandte Chemie - International Edition 38:3743\u0026ndash;3748.\u0026nbsp;https://doi.org/10.1002/(SICI)1521-3773(19991216)38:24\u0026lt;3743::AID-ANIE3743\u0026gt;3.0.CO;2-U\u003c/li\u003e\n \u003cli\u003eThienthiti K, Tuchind P, Wongnoppavich A, et al. (2017). Cytotoxic effect of compounds isolated from\u0026nbsp;\u003cem\u003eGoniothalam usmarcanii\u0026nbsp;\u003c/em\u003eCraib stem barks. Pharm. Sci. Asia\u003cem\u003e.\u0026nbsp;\u003c/em\u003e 44:86\u0026ndash;95.\u0026nbsp;https://doi.org/10.29090/psa.2017.02.086\u003c/li\u003e\n \u003cli\u003eToledo AG, Souza JGL, Silva JPB, et al. (2020).\u0026nbsp;Chemical composition, antimicrobial and antioxidant activity of the essential oil of leaves of \u003cem\u003eEugenia involucrata\u003c/em\u003e DC. Bioscience Journal. 36:568-577.\u0026nbsp;https://doi.org/10.14393/BJ-v36n2a2020-48096\u003c/li\u003e\n \u003cli\u003eTomasiak M, Stelmach H, Rusak T \u0026amp; Wysocka, J (2004). Nitric oxide and platelet energy metabolism.\u0026nbsp;Acta biochimica Polonica 51:789\u0026ndash;803.\u0026nbsp;https://ojs.ptbioch.edu.pl/index.php/abp/article/view/3562/2620\u003c/li\u003e\n \u003cli\u003eTran N, Garcia T, Aniqa M, Ali S, Ally A, Nauli SM (2022).\u0026nbsp;Endothelial nitric oxide synthase (ENOS) and the cardiovascular system: in physiology and in disease states. American Journal of Biomedical Science and Research 152: 153-177.\u0026nbsp;https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8774925/pdf/nihms-1769792.pdf\u003c/li\u003e\n \u003cli\u003eTrott O, Olson AJ (2010). AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455-61.\u0026nbsp;https://doi.org/10.1002/jcc.21334\u003c/li\u003e\n \u003cli\u003eVinholt PJ, Frederiksen H, Hvas AM, Sprog\u0026oslash;e U \u0026amp; Nielsen C (2017). Measurement of platelet aggregation, independently of patient platelet count: a flow-cytometric approach. Journal of thrombosis and haemostasis 15:1191\u0026ndash;1202.\u0026nbsp;https://doi.org/10.1111/jth.13675\u003c/li\u003e\n \u003cli\u003eVistoli G, Pedretti A \u0026amp; Testa B. (2008).\u0026nbsp;Assessing drug-likeness--what are we missing?. Drug discovery today, 13:285\u0026ndash;294.\u0026nbsp;https://doi.org/10.1016/j.drudis.2007.11.007\u003c/li\u003e\n \u003cli\u003eWenlock MC \u0026amp; Barton P. (2013). In silico physicochemical parameter predictions. Molecular pharmaceutics 10: 1224\u0026ndash;1235.\u0026nbsp;https://doi.org/10.1021/mp300537k\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWeyers A, Sokull-Kl\u0026uuml;ttgen B, Baraibar-Fentanes J\u0026nbsp;\u0026amp;\u0026nbsp;Vollmer G (2000). Acute toxicity data: a comprehensive comparison of results of fish, \u003cem\u003eDaphnia\u003c/em\u003e and algae tests with new substances notified in the European Union. Environmental Toxicology and Chemistry 19:1931\u0026ndash;1933.\u0026nbsp;https://doi.org/10.1002/etc.5620190731\u003c/li\u003e\n \u003cli\u003eWittenborn EC\u0026nbsp;\u0026amp;\u0026nbsp;Marletta MA (2021). Structural Perspectives on the Mechanism of Soluble Guanylate Cyclase Activation. Int J Mol Sci 22:5439.\u0026nbsp;https://doi.org/10.3390/ijms22115439\u003c/li\u003e\n \u003cli\u003eXiang Q, Pang X, Liu Z et al. (2019).\u0026nbsp;Progress in the development of antiplatelet agents: focus on the targeted molecular pathway from bench to clinic.\u0026nbsp;\u003cem\u003ePharmacol Ther\u003c/em\u003e 203:107393.\u0026nbsp;https://doi.org/10.1016/j.pharmthera.2019.107393\u003c/li\u003e\n \u003cli\u003eXiang C, Chen C, Li X, Wu Y, Xu Q, Wen L, Xiong W, Liu Y, Zhang T, Dou C, Ding X, Hu L, Chen F, Yan Z, Liang L \u0026amp; Wei G (2022). Computational approach to decode the mechanism of curcuminoids against neuropathic pain. Computers in biology and medicine 147:105739.\u0026nbsp;https://doi.org/10.1016/j.compbiomed.2022.105739\u003c/li\u003e\n \u003cli\u003eXie B, Tang W, Wen S, Chen F, Yang C, Wang M, Yang Y, Liang W (2024). GDF-15 Inhibits ADP-induced human platelet aggregation through the GFRAL/RET Signaling Complex.\u0026nbsp;\u003cem\u003eBiomolecules\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003e14\u003c/em\u003e, 38.\u0026nbsp;https://doi.org/10.3390/biom14010038\u003c/li\u003e\n \u003cli\u003eXu L, Tsona NT, You B, Zhang Y, Wang S, Yang Z et al. (2020). NOx enhances secondary organic aerosol formation from nighttime \u0026gamma;-terpinene ozonolysis. Atmospheric Environment 225:1-39.\u0026nbsp;https://doi.org/10.1016/j.atmosenv.2020.117375\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eYamashita S, Furubayashi T, Kataoka M, Sakane T, Sezaki H \u0026amp; Tokuda H (2000).\u0026nbsp;Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells.\u0026nbsp;European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences\u0026nbsp;10:195\u0026ndash;204.\u0026nbsp;https://doi.org/10.1016/s0928-0987(00)00076-2\u003c/li\u003e\n \u003cli\u003eYan L, Zhang Z, Liu Y, Ren S, Zhu Z, Wei L, Feng J, Duan T, Sun X, Xie T \u0026amp; Sui X (2022).\u0026nbsp;Anticancer activity of erianin: cancer-specific target prediction based on network pharmacology. Frontiers in molecular biosciences 9:862932.\u0026nbsp;https://doi.org/10.3389/fmolb.2022.862932\u003c/li\u003e\n \u003cli\u003eYee S (1997). In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth. Pharmaceutical research 14:763\u0026ndash;766.\u0026nbsp;https://doi.org/10.1023/a:1012102522787\u003c/li\u003e\n \u003cli\u003eZhang Y, Hui J \u0026amp; Chen X (2021). Preprocedural ticagrelor treatment was associated with improved early reperfusion and reduced short-term heart failure in east-asian st-segment elevation myocardial infarction patients undergoing primary percutaneous coronary intervention. International Journal of General Medicine 14:1927\u0026ndash;1938.\u0026nbsp;https://doi.org/10.2147/IJGM.S307404\u003c/li\u003e\n \u003cli\u003eZhao YH, Le J, Abraham MH, Hersey A, Eddershaw PJ, Luscombe CN, Butina D, Beck G, Sherborne B, Cooper I \u0026amp; Platts JA (2001). Evaluation of human intestinal absorption data and subsequent derivation of a quantitative structure-activity relationship (QSAR) with the Abraham descriptors. Journal of pharmaceutical sciences 90:749\u0026ndash;784.\u0026nbsp;https://doi.org/10.1002/jps.1031\u003c/li\u003e\n \u003cli\u003eZhou\u0026nbsp;J,\u0026nbsp;Zhai\u0026nbsp;JX,\u0026nbsp;Zheng\u0026nbsp;W,\u0026nbsp;Han\u0026nbsp;N,\u0026nbsp;Liu\u0026nbsp;Z,\u0026nbsp;Lv\u0026nbsp;G,\u0026nbsp;Zheng\u0026nbsp;X, Chang,\u0026nbsp;S.,\u0026nbsp;Yin,\u0026nbsp;J (2019).\u0026nbsp;The\u0026nbsp;antithrombotic\u0026nbsp;activity\u0026nbsp;of\u0026nbsp;the\u0026nbsp;active fractions\u0026nbsp;from\u0026nbsp;the\u0026nbsp;fruits\u0026nbsp;of\u0026nbsp;\u003cem\u003eCelastrus\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eorbiculatus\u003c/em\u003e Thunb through the anti-coagulation, anti-platelet activation and anti-?brinolysis pathways. Journal of Ethnopharmacology 241:111974. https://doi.org/10.1016/j.jep.2019.111974\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cyclohexane monoterpenes, molecular docking, purine receptors, toxicity","lastPublishedDoi":"10.21203/rs.3.rs-4260336/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4260336/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGamma-terpinene (γ-TPN) is a cyclohexane monoterpene, isolated from essential oils of pharmacologically active plant species, such as tea tree (\u003cem\u003eMelaleuca alternifolia\u003c/em\u003e), oregano (\u003cem\u003eOriganum vulgare\u003c/em\u003e), rosemary (\u003cem\u003eRosmarinus officinalis\u003c/em\u003e L.), thyme (\u003cem\u003eThymus vulgaris\u003c/em\u003e Marchand) and eucalyptus (\u003cem\u003eEucalyptus\u003c/em\u003e sp.). Terpenes are widely studied for their recognized pharmacological actions on the cardiovascular system, hemostasis and antioxidant actions. The objective of this study was to investigate the cytotoxic and antiplatelet activity of γ-TPN in non-clinical study models. For the in silico evaluation, the PreADMET, SwissADME and SwissTargetPrediction software were used. Molecular docking was performed using the AutoDockVina and BIOVIA Discovery Studio databases. The cytotoxicity of γ-TPN was analyzed by the MTT assay with normal murine endothelial (SVEC4-10) and fibroblast (L929) lines. Platelet aggregation was evaluated with platelet-rich (PRP) and platelet-poor (PPP) plasma from spontaneously hypertensive rats (SHR), in addition to SVEC4-10 cells pre-incubated with γ-TPN (50, 100 and 200 \u0026micro;M) for 24 h. In in vivo tests, SHR animals were also used, pre-treated by gavage with γ-TPN for 7 days, distributed into four groups (control, 25, 50 and 100 mg/Kg). At the end, blood samples were collected to measure nitrites using the Griess reagent. γ-TPN proved to be quite lipid-soluble (Log P\u0026thinsp;=\u0026thinsp;+\u0026thinsp;4.50), with a qualified profile of similarity to the drug, good bioavailability, and adequate pharmacokinetics. The monoterpene exhibited affinity mainly for the P2Y12 receptor (6.450\u0026thinsp;\u0026plusmn;\u0026thinsp;0.232 Kcal/mol), moderate cytotoxicity for L929 (CC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;333.3 \u0026micro;M) and SVEC 4\u0026ndash;10 (CC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;366.7 \u0026micro;M). The presence of γ-TPN in SVEC 4\u0026ndash;10 cells was also able to reduce platelet aggregation by 51.57 and 44.20%, respectively, at the lowest concentrations (50 and 100 \u0026micro;M). It was concluded that γ-TPN has a good affinity with purinergic receptors and an effect on the reversal of platelet aggregation and oxidative stress, being promising and safe for therapeutic targets and subsequent studies in the control of thromboembolic diseases.\u003c/p\u003e","manuscriptTitle":"Non-clinical investigations about cytotoxic and anti-platelet activities of gamma-terpinene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-22 12:13:13","doi":"10.21203/rs.3.rs-4260336/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-28T13:03:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-27T21:58:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72673644-4093-49a2-9be7-c9342d3633b9","date":"2024-04-18T21:43:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-18T14:13:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-18T02:55:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-18T02:55:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2024-04-13T04:01:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9ae26f23-e802-4d95-8648-3ab1a9f0221e","owner":[],"postedDate":"April 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-18T17:23:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-22 12:13:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4260336","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4260336","identity":"rs-4260336","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}