Carveol

Mouse monoclonal anti-HO1 (SC-136960), rabbit polyclonal anti Nrf2 (SC-722), mouse monoclonal anti-p-JNK (SC-6254), mouse monoclonal anti-COX-2 (SC-514489), mouse monoclonal anti-p-NFκB (SC-271908), mouse monoclonal anti-Bcl2 (SC-7382), mouse monoclonal anti-caspase-3 (SC-56053), ABC Elite kit (SC-2018), Ultra Cruz mounting media (SC-516212), mouse anti-rabbit IgG-R (SC-2492), and 3,3’-diaminobenzidine peroxidase (DAB) (SC-216567) were purchased from Santa Cruz Biotechnology. The horseradish peroxidase-conjugated secondary antibodies were obtained from Abcam (ab-6789, ab-6721). p-NF-κB Elisa kit (Cat # SU-B28069) and Nrf2 Elisa kit (cat. no. SU-B30429) were purchased from (Shanghai Yuchun Biotechnology, China). HO-1 Elisa kit (cat. No. E-EL-R0488), and TNF-α Elisa kit (cat. No. E-EL-R0019) were got from Elabscience. PBS tablets and proteinase K were obtained from (MP Bio, United States). Formaldehyde, hydrogen peroxide (H₂O₂), reduced glutathione (GSH), trichloroacetic acid (TCA), 1-chlor-2,4-dinitrobenzene (CDNP), N-(1-naphthyl)ethylenediamine dihydrochloride, 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), and carveol (catalog No: 192384 a mixture of isomers, with 97% purity) was purchased from (Sigma-Aldrich, United States). All-trans retinoic acid (ATRA) of the highest analytical grade (99% HPLC) was purchased from the local pharmaceutical industry (GSK).

Carveol and carvone were obtained from Sigma-Aldrich (Portugal) and citronellol and citronellal were obtained from Acros Organics (USA). Each compound (the compounds were weighed out leading to μg/mL units) was tested at various concentrations in the range of 0.066 to 3000 μg/mL in dimethyl sulfoxide (DMSO, 100%) (Sigma-Aldrich). The solutions of EOs components in DMSO did not exceed 10% (v/v) of the final volume of cell suspensions. Cell suspensions with DMSO (10%, v/v) and cell suspension without EOs components were used as controls. To assess the mode of action, each EOs component was tested at MIC concentration. All tests were performed in triplicate with three repeats.

MeSA (99.5%) and n-hexane (≥98%) were purchased from Kaitong Chemical Reagents Co., Ltd., Tianjin, China. p-Cymene, Carveol and m-Diethylbenzenel (≥98%) were purchased from Sigma Aldrich, Germany.

The antifungal activity of eleven EOs and eight commercial terpenes was evaluated in vitro. EOs were distilled from five plants of different L. origanoides chemotypes (Codes 2206, 0008, 0010, 0018, and 0019), the L. alba citral chemotype (Code 0046), L. micromera (Code 0020), V. curassavica (Code 0042), P. marginatum (Code 0024), A. cf. popayanensis (Code 0034), and P. cablin (Code 0049). The terpenes tested were limonene (97%), carvacrol (98%), thymol (98.5%), p-cymene (99%), perillyl alcohol (96%), carveol, mixture cis and trans (≥95%), verbenone (≥99%), and trans-β-caryophyllene (98.5%) (Sigma-Aldrich, St. Louis, MO, USA). A stock solution of each sample was prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA).

LiChrolut EN cartridges (500 mg, 6 mL) were obtained from Merck (Darmstadt, Germany). All chemicals were of analytical reagent grade unless otherwise stated, and water was obtained from a Milli-Q purification system (MilliporeSigma, Burlington, MA, USA). Folin-Ciocalteu reagent, sodium carbonate, sodium acetate, potassium chloride, sodium chloride, sucrose, methanol (HPLC grade), ethanol (HPLC grade), dichloromethane (HPLC grade) and gallic acid were purchased from SCR^® (Shanghai, China). Eucalyptol (99%), linalool (≥95%), (−)-myrtenol (95%), carveol (97%, mixture of isomers), borneol, (≥99.0%, sum of enantiomers, GC), terpinolene (≥94.0%), β-citronellol (95%), geraniol (98%), ethyl-2-methylbutyrate (99%), ethyl-3-methylbutyrate (98%), (Z)-3-hexenol (98%), (E)-2-hexenol (96%), benzyl alcohol (≥99%), phenylethyl alcohol (≥99%), cuminic alcohol (97%), methyl butanoate (99%), isobutyl acetate (99%), ethyl butanoate (99%), isoamyl acetate (≥99%), methyl benzoate (99%), ethyl benzoate (≥99%), diethyl succinate (99%), methyl salicylate (≥99%), ethyl octanoate (≥99%), ethyl decanoate (98%), methyl vanillate (99%), decanoic acid (≥98.0%), benzaldehyde (≥99%), p-cresol (99%), 4-vinylguaiacol (≥98.0%), vanillin (99%), (E)-asarone (98%), (Z)-asarone (70%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All volatile standards were prepared by dilution with HPLC grade methanol.