Camphor

Pseudomonas aeruginosa (ATCC 27,853), Escherichia coli (ATCC 25,922), Staphylococcus aureus (ATCC 25,923), and Listeria monocytogenes (ATCC 7644) were acquired from the Pasteur Institute of Iran. Camphor, thymol, tween 80, Muler hinton broth, and Muler hinton agar were purchased from Merck Chemicals (Germany). Carboxymethylcellulose (CMC) was purchased from Sigma-Aldrich (USA).

The activity on the epimastigote forms of T. cruzi was evaluated on cultures in a LIT medium supplemented with 10% heat-inactivated (FCS). The parasites in the logarithmic growth phase (8–10 × 10⁶ epimastigote/mL) were distributed in 96-well flat-bottom plates (90 µL of culture/well). Essential oils and compounds (1,8-cineole and camphor from Sigma-Aldrich Quimica SL, Madrid, Spain) were tested in triplicate at several concentrations (EOs at 800, 400, 200, and 100 µg/mL; compounds at 100, 10, and 1 µg/mL) for 72 h. Nifurtimox (Bayer AG, Monheim am Rhein, Germany) was used as the positive control, and the parasite viability was analyzed by a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay method [7 (link)]. Data were analyzed with Statgraphics statistical analysis software (Centurion XVIII, Statgraphics Technologies, Inc., The Plains, VA, USA) using one-way analysis of variance (ANOVA) and least significant difference (LSD) test (p < 0.05) analyses.

Polydimethylsiloxane (PDMS) tapes (4 × 15 × 0.2 mm, ca. 33 mg) were placed on different areas of the adaxial and abaxial leaf lamina of S. littoralis-attacked and of control leaves. A glass coverslip was placed just above the DC-STE tape in order to exclude PDMS – air interaction. The quantitation of the collected PVs was obtained by an external standard at known concentration levels, being difficult to calculate an analyte recovery rate with DC-STE applied to in vivo plant matrices (unlike it was done in [37 (link)] with standards). Sampling was carried out in triplicate in the positions on the leaf shown in Figure 1, for the times reported above (2, 6, 24 h). Camphor (Sigma-Aldrich, Milan, Italy) was used as internal standard (I.S.) and was sorbed onto the tapes as proposed by Wang et al. [70 (link)] for Solid Phase Micro Extraction. Preliminary analysis with tapes with and without Camphor I.S. were carried out to verify any possible interference of Camphor with lima bean PV production (Additional file 4). After sampling, the PDMS tapes were placed in thermal desorption tubes, stored in sealed vials, and submitted to automatic thermal desorption (see below). Sorption tapes were provided by the Research Institute for Chromatography (Kortrijk-Belgium).

Flowers and leaves from ylang ylang were frozen in liquid nitrogen and ground to a powder with a pre-chilled mortar and pestle. About 500mg of powder was dissolved in 500 µl of ethyl acetate (Fisher Scientific) including 1 µl (10mg ml^–1) of camphor (Sigma-Aldrich) as an internal standard. The slush was vortexed and incubated on a horizontal shaker at 50rpm for 2h. After centrifugation of the mixture at 13 000g for 10min, the resulting ethyl acetate upper layer extract was transferred to a clean Eppendorf tube and mixed with 300mg of anhydrous Na₂SO₄ (Sigma-Aldrich) to remove water. Following the second centrifugation, the extract was transferred into a 2ml glass vial for gas chromatography/mass spectrometry (GC-MS) analysis (Agilent Technologies).

The new complexes were synthesized under nitrogen using Schlenk and vacuum techniques unless stated otherwise. camphor ligands (OC₁₀H₁₄NY: Y = NH₂, OH, C₆H₅, C₆H₄NH₂−4, C₆H₄CH₃−4, and C₆H₄OH−3) were prepared according to reported procedures [24 (link)]. Silver salts, camphor, camphorsulfonic acid, the amines, and hydrazine were purchased from Sigma Aldrich. Acetonitrile (PA grade) was purchased from Carlo Erba and was purified by conventional techniques [25 ,26 ] and distilled before use. The FTIR spectra were obtained from KBr pellets using a JASCO FT/IR 4100 spectrometer. The NMR spectra (¹H, ¹³C, DEPT, HSQC, and HMBC) were obtained from CD₃CN, CD₂Cl₂, CDCl₃, or DMSO solutions using Bruker Avance II+ (300 or 400 MHz) spectrometers. The NMR chemical shifts are referred to TMS (δ = 0 ppm).

All kinds of cell culture-related agents were purchased from Life Technologies Inc (Grand Island, NY, USA). β-Caryophyllene, camphor, 1,8-cineole, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 3,3’-dihexyloxacarbocyanine iodide (DiOC6), propidium iodide (PI), bisacrylamide, sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), RNase A, and other chemicals were purchased from Sigma Chemical Co. (St.Louis, MO, USA). N,N,N’,N’-tetramethyl-ethylenediamine dihydrochloride (TEMED) were purchased from Bio-Rad Laboratories (Portland, ME, USA). The following antibodies for caspase-3, Bcl-2, Bcl-X_L, Bad, CDK2, CDK4, CDK6, Cyclin D1, Cyclin E, p21^CIP1/WAF1, p27^KIP1, RB, and β-actin were purchased from Santa Cruz Biotechnology Inc. (Santa cruz, CA, USA). p-RB antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The antibodies for caspase-8 and caspase-9 were purchased from BD Biosciences, Pharmingen (San Diego, CA, USA). z-VAD-fmk was purchased from Calbiochem (Bad Soden, Germany).

The standard
solution of tigecycline (1 × 10^–3 mol L^–1) was prepared by dissolving an appropriate weighed
amount of the active substance (USP, China) in 100 mL of doubly distilled
water. The deep eutectic solvents were prepared by mixing thymol (Sigma-Aldrich,
India) and camphor (Sigma-Aldrich, China), thymol and decanoic acid
(Sigma-Aldrich, Malaysia), dodecanoic acid (Sigma-Aldrich, Malaysia)
and menthol (Sigma-Aldrich, Germany), and dodecanoic acid and dodecanol
(Sigma-Aldrich, USA) in a suitable mass ratio and stirring this mixtures
at 40 °C until formation of the clear liquids.
Acetonitrile
(purity HPLC), water, methanol, and 98% formic acid (purity LC-MS)
were obtained from Honeywell (USA) and Merck (Germany). Trichloroacetic
acid, 35–38% (w/w) hydrochloric acid, and sodium hydroxide
were supplied from POCH SA (Poland). Stock solutions of trichloroacetic
acid (0.7 mol L^–1), hydrochloric acid (0.1 mol L^–1), formic acid (0.1% v/v), and sodium hydroxide (0.1
mol L^–1) were prepared by dissolving appropriate
amounts in 500 mL of doubly distilled water.