4.1 Chemicals and materials

Ammonium thiocyanate, sodium thiosulfate pentahydrate, anhydrous sodium sulfate and iodine were purchased from Cicarelli Laboratorios (San Lorenzo, Santa Fe). AChE from Electrophorus electricus EC 3.1.1.7, Fast blue salt B (FBB), α-naphthol, α-naphthyl acetate (α-NAc), and CDCl3, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (LCMS grade) was acquired from Carlo Erba Reagents (Val de Reuil, France) and formic acid from Merck. Aluminium TLC sheets coated with Silica gel 60 F were purchased from Merck (Darmstadt, Germany). Agar was purchased from Britania (Bs. As., Argentina).

The essential oils (EOs) were purchased from EUMA (Bs. As., Argentina): Pimenta racemosa (Mill.) J. W. Moore. (PR), Thuya occidentalis L. (TO), Coriandrum sativum L. (COS), Foeniculum vulgare Mill (FV), Lavandula angustifolia Mill. (LA), Cymbopogon citratus Spreng (CyC), Litsea cubeba (Lour.) Pers. (LC), Mentha arvensis L. (MA), Origanum vulgare L. (OV), Pogostemon cablin Benth. (PC), and Salvia officinalis L. (SO).

Gas chromatography mass spectrometry (GCMS) analysis was performed on an Agilent 7890B Gas Chromatograph coupled to Agilent model 5977A Mass Spectrometer. Column: HP-5MS UI, 30 m × 0.25 mm, 0.25 μm film thick. High-resolution mass spectra (HRMS) were recorded on a Q-Exactive (Thermo Fisher Scientific) using a reported method [82].

Nuclear magnetic resonance spectra were recorded on a Bruker Avance II in CDCl3 in the presence of TMS (0.00 ppm) as the internal standard (1H at 300 MHz and 13C at 75 MHz). The assignments were made on the basis of chemical shifts (1H and 13C), 1H multiplicities and using 2D spectrums (COSY 1H-1H, HSQC and HMBC).

4.2 Preparation of chemically engineered essential oils (CEEOs)

Since the detailed exact composition of the EOs used as starting material is unknown, some average properties of EOs components were used to estimate the appropriate amounts of reagents to be used. The number of moles of reacting molecules was estimated considering 150, as the average molecular weight of EOs components, and one, as the average number of reacting groups per molecule. Finally, two mol of SCNNH4 and 0.5 mol of I2 per “estimated” mol of EO components were used in the reaction.

A suspension of EO (500 mg), SCNNH4 (507.5 mg), and I2 (423.1 mg) was stirred during 16 h at ambient temperature. Following, a solution of 15% sodium thiosulfate pentahydrate (60 mL) was added, and the reaction mixture stirred for 5 min. The organic material was then extracted with ethyl acetate (2 × 60 mL), and the organic phase dried over anhydrous sodium sulfate and the ethyl acetate from the joined fractions was removed under reduced pressure. Chemically engineered essential oil yields: PR = 92.5%, TO = 95.0%, COS = 103.9%, FV = 106.9%, LA = 104.0%, CyC = 82.7%, LC = 84.7%, MA = 89.2%, OV = 121.6%, PC = 97.9%, and SO = 92.9%.

4.3 AChE TLC and false positive autographic assays

The AChE inhibition properties of the mixtures were surveyed by TLC autography using reported protocols (Ramallo et al., 2015). Briefly, a Silica gel-TLC plate (80 cm2) was sprayed with the α-NAc: FBB (4:1) solution. Then the plate was dried under air current at room temperature. 140 mg of agar was dissolved at 80 °C in 16 mL Tris–HCl buffer (50 mM, pH 8.0), and the solution was allowed to cool down (50 °C) and 136 μL of AChE solution (132 U/mL) were added, the obtained solution was mixed by inversion and distributed over the TLC plate. After cooling and solidification, the plate was incubated at 37 °C (20 min) in the dark (in a closed and humid Petri dish).

For the AChE false positive autographic assay, a Silica gel-TLC plate (80 cm2) was sprayed with the α-NAc: FBB (4:1) solution. Then the plate was dried under air current at room temperature. 140 mg of agar was dissolved at 80 °C in 16 mL Tris–HCl buffer (50 mM, pH 8.0), then 600 of ethanolic α-naphthol solution (1.7 mg/mL) were added, the obtained solution was mixed by inversion and finally the agar-α-naphthol solution was distributed over it. The autography was analyzed immediately after it was gelled.

4.4 Fractionation of CEEO of OV

The CEEO of OV (600 mg) was fractionated by column chromatography on Silica gel with hexane: EtOAc gradient: 95:5 (5 dead volumes), 90:10 (2.5 dead volumes), 80:20 (2.5 dead volumes), 70:30 (2.5 dead volumes), 50:50 (2.5 dead volumes), 30:70) (2.5 dead volumes), and pure EtOAc (2.5 dead volumes), giving twenty two fractions. TLC-autography analysis of those fractions detected inhibition halos in four fractions: F0.2A, F0.2B, F0.4A and F0.4B.

GC–MS analysis revealed that each fraction contained one major compound each with the following m/z values: 207.1 in F0.2A, 330.2 in F0.2B, 414.2 in F0.4A, and 446.1 in F0.4B (Fig. S1–S4). The identities of these compounds were established by HRMS and NMR (1H and 13C) analyses as 5-isopropyl-2-methyl-4-thiocyanatophenol (1), 4,4′-thiobis(5-isopropyl-2-methylphenol) (2), 4-((5-(4-hydroxy-2-isopropyl-5-methylphenyl)-1,2,4-thiadiazol-3-yl)thio)-5-isopropyl-2-methylphenol (3), and 4,4′-((1,2,4-thiadiazole-3,5-diyl)bis(sulfanediyl))bis(5-isopropyl-2-methylphenol (4), respectively. The assignments for 1–4 are shown below.

4.5 Synthesis of carvarol derivatives

Standard procedure A suspension of carvacrol (250 mg), SCNNH4 (253.5 mg), and I2 (211.1 mg) was stirred during 16 h at 20 °C. Following, a solution of 15% of sodium thiosulfate pentahydrate (30 mL) was added, and the reaction mixture stirred for 5 min. The organic material was then extracted with ethyl acetate (2 × 30 mL), and the organic phase dried over anhydrous sodium sulfate. Next, ethyl acetate was removed by rotary evaporation, and the crude product was purified by column chromatography over silica gel using hexane: EtOAc gradient: 95:5 (5 dead volumes), 90:10 (2.5 dead volumes), 80:20 (2.5 dead volumes), 70:30 (2.5 dead volumes), 50:50 (2.5 dead volumes), 30:70) (2.5 dead volumes), and pure EtOAc (2.5 dead volumes).

1. Yield: 25%; 1H NMR (300 MHz, CDCl3) δ = 1.24 (6H, d, J = 6.7 Hz, CH(CH3)2), 2.22 (3H, s, Ar-CH3), 3.41 (1H, m, CH(CH3)2), 5.24 (1H, s, OH), 6.77 (1H, s, Ar), 7.41 (1H, s,Ar); 13C NMR (75 MHz, CDCl3) δ = 15.30 (CH3, Ar-CH3), 23.40 (2 CH3, CH(CH3)2), 31.34 (CH, CH(CH3)2), 111.02 (C, Ar), 112.84 (C, Ar), 113.80 (CH, Ar), 124.29 (C, SCN), 137.16 (CH, Ar), 150.64 (C, Ar), 157.18 (C, Ar); HRMS: found m/z = 206.0642, calculated m/z for C11H12OSN [M-H]− 206.0634 (0.8 mDa error).

2. Yield: 20%; 1H NMR (300 MHz, CDCl3) δ = 1.20 (12H, d, J = 6.8 Hz, 2 CH(CH3)2), 2.14 (6H, s, 2 Ar-CH3), 3.46 (1H, m, 2 CH(CH3)2), 4.67 (1H, s, 2 OH), 6.76 (1H, s, 2 Ar), 6.87 (1H, s, 2 Ar); 13CNMR (75 MHz, CDCl3) δ = 15.36 (2 CH3, Ar-CH3), 23.65 (4 CH3, CH(CH3)2), 30.42 (2 CH, CH(CH3)2), 112.70 (2 CH, Ar), 122.13 (2 C, Ar), 125.36 (2 C, Ar), 134.64 (2 CH, Ar), 148.64 (2 C, Ar), 153.56 (2 C, Ar); HRMS: found m/z = 329.1581, calculated m/z for C20H25O2S [M-H]− 329.1570 (1.1 mDa error).

3. Yield: 3%; 1H NMR (300 MHz, CDCl3) δ = 1.19 (6H, d, J = 6.7 Hz, CH(CH3)2), 1.20 (6H, d, J = 6.7 Hz, CH(CH3)2), 2.24 (3H, s, Ar-CH3), 2.26 (3H, s, Ar-CH3), 3.57 (2H, m, 2 CH(CH3)2), 4.98 (1H, s, OH), 5.14 (1H, s, OH), 6.84 (1H, s, Ar), 6.85 (1H, s, Ar), 7.49 (1H, s, Ar), 7.52 (1H, s, Ar); 13CNMR (75 MHz, CDCl3) δ = 15.08 (CH3, Ar-CH3), 15.22 (CH3, Ar-CH3), 23.76 (2 CH3, CH(CH3)2), 23.79 (2 CH3, CH(CH3)2), 29.30 (CH, CH(CH3)2), 31.06 (CH, CH(CH3)2), 112.87 (CH, Ar), 113.15 (CH, Ar), 118.12 (C, Ar), 121.32 (C, Ar), 121.81 (C, Ar), 122.36 (C, Ar), 133.21 (CH, Ar), 139.69 (CH, Ar), 147.83 (C, Ar), 152.36 (C, Ar), 155.95 (C, Ar), 156.64 (C, Ar), 172.16 (C, Ar), 187.96 (C, Ar); HRMS: found m/z = 413.1364, calculated m/z for C22H25O2N2S2 [M-H]− 413.1352 (1.2 mDa error).

4. Yield: 5%; 1H NMR (300 MHz, CDCl3) δ = 1.16 (6H, d, J = 6.7 Hz, CH(CH3)2), 1.18 (6H, d, J = 6.7 Hz, CH(CH3)2), 2.19 (3H, s, Ar-CH3), 2.22 (3H, s, Ar-CH3), 3.44 (2H, m, 2 CH(CH3)2), 5.07 (1H, s, OH), 5.33 (1H, s, OH), 6.80 (1H, s, Ar), 6.83 (1H, s, Ar), 7.42 (1H, s, Ar), 7.43 (1H, s, Ar); 13CNMR (75 MHz, CDCl3) δ = 15.15 (CH3, Ar-CH3), 15.23 (CH3, Ar-CH3), 23.75 (2 CH3, CH(CH3)2), 23.89 (2 CH3, CH(CH3)2), 31.04 (2 CH, CH(CH3)2), 113.26 (CH, Ar), 113.86 (CH, Ar), 116.99 (C, Ar), 117.49 (C, Ar), 122.39 (C, Ar), 123.77 (C, Ar), 139.38 (CH, Ar), 139.52 (CH, Ar), 152.45 (C, Ar), 152.60 (C, Ar), 156.02 (C, Ar), 157.25 (C, Ar), 172.37 (C, Ar), 194.06 (C, Ar); HRMS: found m/z = 445.1086, calculated m/z for C22H25O2N2S3 [M-H]− 445.1073 (1.3 mDa error).

4.6 Microplate assays

Inhibition percentage determinations were carried out using the method applied by Osella et al. [83]. Wells were filled in triplicate with AChE solution (in 0.1 M phosphate buffer, pH 7.5, and 13.7 µU/mL end concentration per well), Ellman’s reagent (DTNB, 5,5dithio-bis-(2-nitrobenzoic acid)), the same buffer solution (0.31 mM end concentration per well) and 10 µL of test compound in DMSO solution (0.05 µM end concentration per well). Wells containing the corresponding volume of DMSO without an inhibitor were used as references of maximum enzymatic rates, and standard drug eserine was used as the control for enzyme inhibition. The enzymatic reaction was initiated by addition of acetylthiocholine iodide (0.46 mM end concentration per well). The final volume per well was 270 µL. The plate was shaken for 2 s and the increase in absorbance at 405 nm was monitored at 37 °C for 15 min. For IC50 determination, ten serial dilutions of the compounds were prepared in DMSO, following equally spaced points on a neperian logarithm scale, starting at 2.16 mM and finishing at 0.00023 mM (end concentration per well: 80.00 to 0.00839 µM). IC50 calculated using Prism V5.01 (GraphPad Software Inc., La Jolla, CA, USA) applying a non linear regression curve fit for a log[inhibitor] vs. normalized answer model with variable slope.