Caryophyllene

The separation was achieved on a Perkin Elmer Elite-5-MS fused-silica capillary column (30 m × 0.25 mm, film thickness 0.25 µm), using helium as carrier gas at a standard flow rate of 1.1 mL/min. The temperature of the injector was adjusted at 250 °C with an initial temperature of 50 °C, initial hold 5 min, and ramp 4.0 °C/min to 280 °C. The total running time was 62.50 min and the solvent delay was from 0 to 4.0 min. MS scan time was from 4 to 62.5 min, covering mass range 50.00 to 300.00 m/z. The chemical ingredients of the EO were characterized by comparing their mass spectra with the reference spectra in the MS Data Centre of the National Institute of Standards and Technology, and by matching their Kovats and retention indices with values reported in the literature [29 (link),30 (link)]. In addition, the EOs Kovats and retention indices with values were compared with 20% of HPLC grade of reference EOs including α-pinene, eucalyptol, caryophyllene, γ-terpinene, ocimene, endo-borneol, α-terpineol, jasmone, α-cadinene, p-menthane that were purchased from Sigma-Aldrich, Hamburg, Germany) [31 (link)].

For identification of volatiles the following analytical standards were purchased from Sigma-Aldrich (Milan, Italy): α-pinene, β-pinene, p-cymene, limonene, γ-terpinene, linalool, trans-pinocarveol, terpinen-4-ol, α-terpineol, myrtenol, nerol, carvone, geraniol, geranial, eugenol, (E)-caryophyllene, α-humulene, (E)-β-farnesene, (E)-β-ionone, (E)-nerolidol, caryophyllene oxide, n-pentacosane, n-heptacosane; (E)-Phytol was previously isolated from Onosma echioides [43 (link)]. For retention-index (RI) determination, a mixture of hydrocarbons, ranging from octane (C₈) to triacontane (C₃₀) (Supelco, Bellefonte, PA, USA) was used and run under the experimental conditions reported below. All compounds were of analytical standard grade. Analytical-grade hexane solvent was purchased from Carlo Erba (Milan, Italy); it was successively distilled through a Vigreux column before use.

Phosphate‐buffered saline (PBS), 4‐Nitrophenyl β‐D‐glucopyranoside (pNPG), 3,5‐dinitro salicylic acid (DNS), caryophyllene, bovine serum albumin (BSA), α‐tocopherol, α‐amylase, α‐glucosidae, streptokinase, aspirin, 2, 2‐diphenyl‐1‐picrylhydrazyl (DPPH), and acarbose were purchased from Sigma‐Aldrich, Singapore. All other reagents and solvents were of analytical grade.

Ethanol absolute was obtained from Carlo Erba (Milan, Italy). Acetic acid, acetonitrile, methanol, and water (high performance liquid chromatography—HPLC grade) were purchased from VWR (Milan, Italy).
α-pinene, β-pinene, linalool, β-myrcene, terpinolene, caryophyllene, humulene, and β-bisabolene, gallic acid, p-OH benzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, ferulic acid, and rosmarinic acid (from Sigma-Aldrich, Darmstadt, Germany) standards were employed. Working standard mixtures were prepared by appropriate dilution of the standards in methanol. All solutions were stored at −20 °C in the dark.
Folin–Ciocâlteu’s reagent, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH˙), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS˙⁺) were purchased from Sigma-Aldrich (Darmstadt, Germany). Sodium carbonate, potassium persulfate, potassium hexacyanoferrate(III), trichloroAcetic acid, ferric chloride, and potassium phosphate monobasic were obtained from Carlo Erba (Milan, Italy).

The authentic standards borneol, camphor, carvacrol, carvone, caryophyllene, p-cymene, estragole, eucalyptol, limonene, linalool, menthol, menthone, β-myrcene, γ-terpinene, and thymol for EO components identification as well as the control antibiotics ciprofloxacin and tioconazole were purchased from Sigma-Aldrich (Prague, Czech Republic). Hexane (Merck, Prague, Czech Republic), dimethyl sulfoxide (DMSO) (Lach-Ner, Neratovice, Czech Republic), and Tween 80 (Sigma-Aldrich, Prague, Czech Republic) were used as solvents. The plant material used for the EO distillation was purchased from commercial sources (F-DENTAL, Hodonín, Czech Republic). The EO was extracted by hydrodistillation using Clevenger type apparatus.

Terpenoid standards used in the study and their purity percentages as obtained from Sigma-Aldrich (UK) were: alloaromadendrene (-) (98%), camphene (95%), camphor (R) (95%), caryophyllene (-) (trans) (98.5%), 1,8-cineole, citral (95%), p-cymene (99%), geraniol (98%), geranyl acetate (97%), ledene (+) (95%), limonene (R) (+) (97%), linalool (±) (95%), linalyl acetate (97%), 6-methyl-5-hepten-2-one (96%), myrcene (90%), α-pinene (-) (99%), β-pinene (-) (99%), sabienene hydrate (97%), α-terpinene (85%), γ-terpinene (97%), terpinolene (95%), terpinen-4-ol (95%), α-terpineol (96%), terpinyl acetate (±) (90%) and verbenol (95%).