Cannabidiol

(1) Terpene profiling. Terpenoid analyses (DigiPath Laboratories) were carried out on an Agilent 7890B GC/7697A Headspace/5977A mass spectrophotometer with a DB-624UI and Agilent 5181–8818 split/splitless liner. Injector port temperature was 250°C with a transfer line, valve oven and needle temperature of 180°C. Carrier gas was helium at a flow of 33.0 cm/s. The MS detector was set to scan with a range from 50 to 300 m/z. The instrument was controlled by Agilent Masshunter quantitative analysis (Vers. B.08.00 Build 8.0.593.0). Certified reference standards were from Restek (Bellefonte, PA) with Masshunter library confirmation.
(2) Strain-inspired mixture: Cannabidivarin (CBDV), Cannabichromene (CBC), Cannabidiol (CBD), Cannabidiolic Acid (CBDA), Cannabigerol (CBG), Cannabigerolic Acid (CBGA), Cannabinol (CBN), alpha-Bisabolol, alpha-Humulene, alpha-Pinene, beta-Caryophyllene, beta-Myrcene, (+)-beta-Pinene, Camphene, Limonene, Linalool, Nerolidol. (3) Cannabinoid mixture: Cannabidivarin (CBDV), Cannabichromene (CBC), Cannabidiol (CBD), Cannabidiolic Acid (CBDA), Cannabigerol (CBG), Cannabigerolic Acid (CBGA), Cannabinol (CBN). (4) Terpene mixture: alpha-Bisabolol, alpha-Humulene, alpha-Pinene, beta-Caryophyllene, beta-Myrcene, (+)-beta-Pinene, Camphene, Limonene, Linalool, Nerolidol.

Standard solution of cannabidiol (CBD) was purchased from Restek (Bellefonte, PA, USA). Solvents ethanol, methanol and hexane: ethyl acetate were purchased from Sintorgan (Buenos Aires, Argentina), and used as received.
For in vitro studies, cannabidiol (CBD) was solubilized in dimethyl sulfoxide (DMSO) at a concentration of 15 mg/mL. Subsequently, it was introduced into the culture medium, yielding final concentrations of 50, 10, 5, and 1 µg/mL. Importantly, the quantity of DMSO introduced into the culture medium did not exceed 3 µL/mL.
For in vivo studies, a suspension of albendazole (ABZ) at a concentration of 5.25 mg/mL was formulated using pharmaceutical-grade ABZ (Parafarm, Buenos Aires, Argentina). This formulation involved dispersing pure ABZ in distilled and deionized water (pH = 7.0), subjecting it to overnight shaking, and subsequently, to sonication for 30 min. Concurrently, cannabidiol (CBD) was dissolved in sesame oil (Nutra sem, Buenos Aires, Argentina) at a drug concentration of 4 mg/mL through a one-hour sonication process.

The cannabinoid standards (at a concentration of 1 mg/mL in methanol) used in this study included cannabidiol (CBD, Restek catalog no. 34011), cannabigerol (CBG, Restek catalog no. 34091), tetrahydrocannabivarin (THCV, Restek catalog no. 34100), cannabinol (CBN, Restek catalog no. 34010),a and delta-9-tetrahydrocannabidiol (Δ-9 THC, Restek catalog no. 34067). Inverse agonists (IA) to CB1 and CB2 used included AM251 (ab120088; Abcam) and SR144528 (ab146185; Abcam), respectively. The transient receptor potential ankyrin subtype 1 protein (TRPA1) blocker used was HC-030031 (ab120554; Abcam). Transient receptor potential vanilloid receptor 1 (TRPV1) and 2 (TRPV2) antagonists were SB-366791 (ab141772-5-B Abcam) and Tranilast (1098/10 Abcam), respectively. All IAs were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. Doxorubicin (D1515; Sigma Aldrich, St. Louis, MO, USA) served as a positive control in concentrations of 0.5 µg/mL on A172 cells and 50 µg/mL on U87. Temozolomid (TMZ, T2577; Sigma Aldrich, St. Louis, MO, USA) was tested as a positive control. Analytical grade methanol was used according to the indicated concentration of the treatment. Ultra-pure deionized water (MS grade) was used as received without further purification.