Acetylsalicylic acid asa

Freeze-dried crude venom (150 mg) was solubilized in 0.05 M ammonium bicarbonate pH 8.0 and subjected to ion exchange chromatography. The fraction corresponding to MjTX-II was obtained by a gradient of 0.05 to 0.5 M ammonium bicarbonate pH 8.0, as described by Soares and colleagues^{71 (link)}. For contaminant removal, this fraction was subjected to reversed-phase chromatography, with a gradient of 0–66.5% acetonitrile (in 0.1% trifluoroacetic acid) in a C18 column (Shimadzu). Acetylsalicylic acid (ASA) and rosmarinic acid (RA) were purchased from Sigma-Aldrich, St. Louis, Missouri, USA.

Collagen was purchased from Chrono-log Corporation (Havertown, PA, USA). The 2-MeSADP, thrombin, daphnetin, apyrase (type V), prostaglandin E₁ (PGE₁), sodium citrate, and acetylsalicylic acid (ASA) were bought from Sigma (St. Louis, MO, USA). Anti-phospho-cPLA₂ (Ser505), anti-cPLA₂, anti-phospho-ERK (Thr202/Tyr204), anti-ERK, Anti-phospho-AKT (Ser473), and anti-AKT antibodies were from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-labeled secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TxB₂ ELISA kit was from Enzo Life Sciences (Exeter, UK). All additional chemicals were of reagent grade.

Kraft lignin was obtained from Sigma-Aldrich (Steinheim, Germany). Poly (vinyl alcohol) (APV) M_w = 72000, 98% degree of hydrolysis, benzyl alcohol, styrene (St), and ethylene glycol dimethylacrylate (EGDMA) were obtained from Merck (Darmstadt, Germany). α,α’-Azoiso-bis-butyronitrile (AIBN) was obtained from FLUKA (Buchs, Switzerland). Toluene (TOL), decan-1-ol, tetrahydrofuran (THF), acetone (Ace), chloroform (TCM), methanol (MeOH), and acetonitrile (ACN), were obtained from Avantor Performance Materials Poland S.A. (Avantor, Gliwice Poland). Purified water came from Millipore (UMCS Lublin, Poland). The sulphuric acid, nitric acid, phosphorous acid, silver nitrite, and caffeine came from Merck (Darmstadt, Germany). The acetylsalicylic acid (ASA) was from Sigma-Aldrich (Darmstadt, Germany).

All solvents were with chromatographic grade (Tedia, Brazil). Acetylsalicylic acid (ASA), carrageenan, dexamethasone, citral and limonene were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Formalin was purchased from Merck (Germany). Morphine sulfate was kindly provided by Cristália (São Paulo, Brazil). Essential oils (EO) were dissolved in oil (Sigma-Aldrich, USA) to prepare a stock solution at 100 mg/ml. They were administered by oral gavage, at doses of 10 to 100 mg/kg 60 min prior to experiments. Morphine (5 mg/kg, p.o.), ASA (100 mg/kg, p.o.) and dexamethasone (1.5 mg/kg, i.p.) were used as reference drugs. All drugs were diluted in phosphate buffer saline (PBS) just before use. The control group was composed by vehicle (PBS with the same amount of oil used in the highest dose). The final concentration of oil did not exceed 0.5%, and had no effect per se. The doses of ASA and morphine were chosen based on previous experiments performed by our group and caused a 50% reduction on each protocol (IC₅₀).

C57BL/6J mice (Envigo Rms Spain Sl., Sant Feliu de Codines, Spain) were kept under controlled environmental conditions (relative humidity: 45–65%; temperature: 20–24 °C) with a 12 h light/dark cycle and free access to chow and water. At the time of the study, mice were 8–12 weeks old. Equivalent numbers of males and females were included. According to the oral drug administration protocol, mice were immobilized and Idelalisib, 20 mg/kg or vehicle (DMSO; Sigma-Aldrich, Madrid, Spain), was introduced directly into the stomach with an oral gavage feeding tube. One hour after the treatment, tail bleeding and arterial thrombosis assays were performed. A separate set of animals treated with other established antiplatelet drugs were also used as positive controls. Acetyl salicylic acid (ASA; Sigma-Aldrich, Madrid, Spain) was dissolved in water at 1 mg/mL and given by a single intraperitoneally injection of 100 mg/kg in mice 2–4 h before the experiment, as previously reported [33 (link)]. Moreover, two doses of clopidogrel (2 mg/kg) were also given to both animal groups by oral gavage 24 h and 2 h before the experiment.