Cannabichromene

The chemicals β-caryophyllene (≥98.5% purity), β-caryophyllene oxide (≥99% purity), α-humulene (≥96.0% purity), cannabidiol (CBD, ≥99.0% purity), cannabichromene (CBC, ≥99.0% purity), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT, ≥97.5% purity) were purchased from Sigma Aldrich Co (St. Louis, MO, USA), while AM281 (≥98.0% purity) and AM630 (≥98.0% purity) from Tocris bioscience (Bristol, UK). Dulbecco’s Modified Eagle’s Medium (DMEM) was provided by Aurogene (Rome, Italy).
To perform the experiments, all the solutions were prepared in the appropriate solvent, sterilized, and stored at the recommended temperature for a conservation time. Nonintoxicating phytocannabinoids (i.e., cannabidiol and cannabichromene) and caryophyllane sesquiterpenes (i.e., β-caryophyllene, β-caryophyllene oxide, and α-humulene) were dissolved in EtOH 100% v/v, while the CB1 and CB2 receptor antagonists (i.e., AM281 and AM630) in DMSO 100% v/v. EtOH and DMSO were used at a maximum concentration of 1% v/v in cell medium to avoid any cytotoxicity.

The following terpenoid standards were purchased from Supelco: α-pinene, camphene, β-pinene, myrcene, 3-carene, α-terpinene, p-cymene, limonene, γ-terpinene, fenchone, terpinolene, linalool, camphor, isoborneol, borneol, menthol, α-terpineol, β-citronellol, neryl acetate, geranyl acetate, α-cedrene, β-caryophyllene, α-humulene, cis-nerolidol, geranyl isobutyrate, caryophyllene oxide, β-eudesmol, α-bisabolol, and phytol. Cannabinoid standards were purchased from Sigma Aldrich (1 mg/mL): cannabidiolic acid (CBD-A), cannabigerolic acid (CBG-A), cannabigerol (CBG), cannabidiol (CBD), cannabinol (CBN), Δ-9-tetrahydrocannabinol (Δ-9-THC), Δ-8-tetrahydrocannabinol (Δ-8-THC), cannabichromene (CBC), Δ-9-tetrahydrocannabinolic acid (Δ-9-THC-A), and cannabichromenic acid (CBC-A). Acid standards were dissolved in acetonitrile, and nonacid standards were dissolved in methanol. Hexane, ammonium format, and acetonitrile were purchased from Sigma Aldrich, methanol was purchased from J. T. Baker and formic acid was purchased from Scharlau. Chemical structures of essential oils and cannabinoids are presented in Supplementary Materials [37 ,38 ,39 ].

Gradient grade water, acetonitrile, chloroform, methanol, and formic acid were purchased from Merck Life Science (Milano, Italy). Cannabinoid reference standards dissolved in methanol, namely, cannabidivarin (CBDV, 1 mg/mL), cannabigerol (CBG, 1 mg/mL), cannabidiol (CBD, 1 mg/mL), cannabidiolic acid (CBDA, 1 mg/mL), cannabinol (CBN, 1 mg/mL), (–)-Δ9-tetrahydrocannabinol ((–)-Δ9-THC, 1 mg/mL), Δ9-tetrahydrocannabinolic acid ((–)-Δ9-THCA, 1 mg/mL), (–)-Δ8-tetrahydrocannabinol ((–)-Δ8-THC, 1 mg/mL), and cannabichromene (CBC, 1 mg/mL) with purity ≥ 99%, were purchased from Merck Life Science (Milano, Italy). Deuterated water (D₂O) 99.97 atom% of deuterium and 3-(trimethylsilyl)-propionic-2,2,3,3-d₄ acid sodium salt (TSP) were purchased from Euriso-Top (Saclay, France). Magnesium oxide (MgO), potassium phosphate monobasic (KH₂PO₄), and potassium phosphate dibasic (K₂HPO₄) 2,2-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu reagent, gallic acid (GA), and sodium carbonate (Na₂CO₃) were purchased from Merck Life Science (Milano, Italy).

All solvents used for
UHPLC analyses were HPLC grade and were purchased from Merck Life
Science (Milan, Italy) and Carlo Erba Reagents (Milan, Italy). Cannabinoid
reference standards dissolved in methanol, namely, cannabidivarin
(CBDV, 1 mg/mL), cannabidiolic acid (CBDA, 1 mg/mL), cannabigerol
(CBG, 1 mg/mL), cannabidiol (CBD, 1 mg/mL), cannabinol (CBN, 1 mg/mL),
(−)-Δ⁹-tetrahydrocannabinol [(−)-Δ⁹-THC, 1 mg/mL], (−)-Δ⁸-tetrahydrocannabinol
[(−)-Δ⁸-THC, 1 mg/mL], cannabichromene (CBC,
1 mg/mL), and (−)-Δ⁹-tetrahydrocannabinolic
acid ((−)-Δ⁹-THCA, 1 mg/mL) with a purity
of ≥99%, were purchased from Merck Life Science (Milan, Italy).
Preparative TLC plates UV 254 nm (surface 20 × 20 cm, thickness
500 μm), used for the purification of CBC, were purchased from
Merck (Darmstadt, Germany).

Gradient grade water, acetonitrile, chloroform, methanol, and formic acid were purchased from Merck Life Science (Milano, Italy). Cannabinoid reference standards dissolved in methanol, namely, cannabidivarin (CBDV, 1 mg/mL), cannabigerol (CBG, 1 mg/mL), cannabidiol (CBD, 1 mg/mL), cannabidiolic acid (CBDA, 1 mg/mL), cannabinol (CBN, 1 mg/mL), (–)-Δ9-tetrahydrocannabinol ((–)-Δ9-THC, 1 mg/mL), Δ9-tetrahydrocannabinolic acid ((–)-Δ9-THCA, 1 mg/mL), (–)-Δ8- tetrahydrocannabinol ((–)-Δ8-THC, 1 mg/mL), and cannabichromene (CBC, 1 mg/mL) with purity ≥ 99%, were purchased from Merck Life Science (Milano, Italy). Deuterated water (D₂O) 99.97 atom% of deuterium and 3-(trimethylsilyl)-propionic-2,2,3,3-d₄ acid sodium salt (TSP) were purchased from Euriso-Top (Saclay, France). Magnesium oxide (MgO), potassium phosphate monobasic (KH₂PO₄), and potassium phosphate dibasic (K₂HPO₄) 2,2-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, gallic acid (GA), and sodium carbonate (Na₂CO₃) were purchased from Merck Life Science (Milano, Italy).

LC–MS grade chemicals (methanol, ethyl acetate, isopropanol, ammonium formate, formic acid, n-hexane, n-heptane, n-pentane) with purities in the range of 98.5–99.8% were purchased from Merck. Deionized water (18 mΩ) was obtained from an internal Milli-Q system (Millipore). P.A. 96% ethanol was purchased from Lach-Ner. Analytical standards of 17 phytocannabinoids (cannabidiol (CBD), cannabidiolic acid (CBDA), delta-8-tetrahydrocannabinol (∆⁸-THC), delta-9-tetrahydrocannabinol (∆⁹-THC), delta-9-tetrahydrocannabinolic acid (∆⁹-THCA-A), cannabigerol (CBG), cannabigerolic acid (CBGA), cannabicyclol (CBL), cannabicyclolic acid (CBLA), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), tetrahydrocannabivarin (THCV), tetrahydrocannabivarinic acid (THCVA), cannabinol (CBN), cannabinolic acid (CBNA), cannabichromene (CBC), and cannabichromenic acid (CBCA)) in purity ranges from 95 to 100% were purchased from Merck.