Perillyl alcohol

Salvia dorisiana plant tissues were harvested in three biological replicates. The FW/DW ratio was determined by drying at 105 °C for 20 h. To measure metabolites, the plant tissues were frozen in liquid N₂, ground in a mortar and pestle while still frozen, and 1 ml of ethyl acetate, with 3 µg ml^–1 γ-terpinen-4-ol as internal standard, was added to 100 mg of frozen plant powder, vortexed for 20 s, subjected to sonication for 5 min, and centrifuged for 5 min at 3400 rpm. The supernatant was analysed by GC-MS as described below. A standard mix of 3 µg ml^–1 terpinen-4-ol, (R)-(+)-limonene (97%, Sigma Aldrich), perillyl alcohol (96%, Sigma Aldrich), (S)-(–)-perillaldehyde (Sigma Aldrich), and (S)-(–)-perillic acid (95%, Sigma Aldrich) was prepared for identification and semi-quantification. The standard mix was methylated by addition of diazomethane 1:1 and injected as well, to be able to identify and semi-quantify MePA in the samples.

Perillyl alcohol (PubChem CID: 369312; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and 3-phenylpropionic acid (referred to along this study as hydrocinnamic acid) (PubChem CID: 107; Thermo Fisher Scientific, Acros Organics, Radnor, PA, USA) were tested in this study. Chloramphenicol (PubChem CID: 5959; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and amoxicillin (PubChem CID: 33613; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) were the antibiotics selected. Stock solutions were prepared for all the compounds using DMSO (Avantor, VWR, Radnor, PA, USA). DMSO was used at 5%, and it was verified that this concentration did not affect the growth of E. coli. Stock solutions of antibiotics and phytochemicals were prepared and stored at −18 °C.

POH was formulated for intranasal delivery and the preparation supplied by the University Pharmacy according to following patents: US Patent Application 20040087651 6 May 2004, and BR Application Number PI 0107262-5 17 December 2012. Perillyl alcohol (Sigma Chem. Co., St. Louis, MI, USA) 0.3% v/v (55 mg) was administered to all patients 4 times daily by intranasal (inhalation) delivery and the protocol consisted of an initial dose of 66.7 mg/dose (totaling 266.8 mg/daily) and then escalation to a 133.4 mg/dose (totaling 533.6 mg/daily).

Human IFNα2 was purchased from PBL Assay Science (#11101) and IFNβ from Peprotech (#300-02BC). Remdesivir and ruxolitinib were obtained from Cayman Chemicals (#30354) and Cell Guidance Systems (#SM87-10), respectively. The substances cinnamic acid, hydroxy-cinnamic acid, and dihydroxy-cinammic acid/caffeic acid were purchased from Sigma (#8002350250, #8002370050, #8220290010). Perilla aldehyde and perillyl alcohol were also obtained from Sigma (#W355704 and #218391). Size exclusion and protein fractionation of components of herbal infusions were conducted by use of Amicon 100 K, 30 K, 10 K, and 3 K filters (Sigma #UFC510024, #UFC501096, #UFC503096, #Z740183-96EA). The fraction of proteins > 1 kDa was obtained by dialysis using the Mini Dialysis Kit 1 kDa (Sigma #GE80-6483–94). All kits were applied according to the manufacturer’s instructions.

Tricaprylin was kindly supplied by Croda Health Care (Edison, NJ, USA). Soy phosphatidylcholine (PC) and 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) were obtained from Avanti Polar Lipids (Alabaster, AL, USA), and propylene glycol and glycerol were purchased from Synth (São Paulo, SP, Brazil). Paclitaxel and elacridar were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Tributyrin (Tri), perillyl alcohol (PA), tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and polysorbate 80 were purchased from Sigma (St Louis, MO, USA). Other specific reagents are described along with the respective methodology. Ultrapure water was employed unless stated.

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).