Anhydrous sodium sulphate

All standards of fatty acid methyl esters were purchased from Sigma (St. Louis, MO, USA). Methanol, n-hexane, petroleum ether, chloroform, potassium iodide, Folin–Ciocalteu reagent, sodium carbonate, starch, concentrated sulphuric, nitric and hydrochloric acid (37%) were purchased from Kemika (Zagreb, Croatia). Anhydrous sodium sulphate and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) were purchased from Sigma (St. Louis, MO, USA).
Sodium and potassium hydroxides in the form of pellets, chloroform and analytical standards (dihydrocholesterol, campesterol, stigmasterol, β-sitosterol, eicosanol, docosanol, tricosanol, tetracosanol, pentacosanol, hexacosanol, heptacosanol, octacosanol, squalene and (±)-α-tocopherol) were purchased from Sigma (St. Louis, MO, USA). Ethanol and a silylating mixture according to Sweeley were purchased from Fluka (Buchs, Switzerland).
Hydroxytyrosol, tyrosol, oleuropein, lutein and apigenin were purchased from Extrasynthese (Genay, France). Caffeic acid and gallic acid were purchased from Sigma (St. Louis, MO, USA). Standard stock solutions of each compound were 1 mg/mL in Methanol.
All mineral standards were purchased from Merck (Darmstadt, Germany) and diluted to the appropriate obtained concentration.
All standards stored at +4 °C in amber vials and in a dark place until analysis.

Potassium hydroxide (1 M), sodium hydroxide (1 M), sodium bicarbonate (50 mM), anhydrous sodium sulphate, sulphuric acid (3 M), and 2,3,3,3-d4-alanine were purchased from Sigma-Aldrich (St. Louis, USA). Cambridge Isotope Laboratories (Andover, USA) supplied the three other internal standards; 2,2,4,4-d4-citric acid, 2,3,4,5,6-d5-phenylalanine, and 3,3-d2-tyrosine. Methanol (Optima grade) was bought from Adamas-beta® (Shanghai, China); chloroform from Chongqing Xinan Chemical Reagent Company (Chongqing, China); pyridine from Merck (Darmstadt, Germany); and methyl chloroformate (MCF) from Shandong Huayang Pesticide Chemical Industry Group (Shandong, China).

Mature seeds of P. sylvestris were harvested from the Ludeștii de Jos, Hunedoara County (Coordinates: 45°43′5″ N 23°10′21″ E) in August 2018. After the identification, a voucher specimen (VSNH.BUASTM-122) was deposited in the Herbarium of the Faculty of Agronomy, Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I of Romania” from Timisoara. The seeds were dried under natural conditions (protected from sunlight and natural ventilated). The P. sylvestris essential oil (PEO) was obtained through steam distillation, according to the method previously described [35 ]. After decantation, the EO was dried over anhydrous sodium sulphate (Sigma, Germany) and stored in sealed amber vials at −18 °C.

Methanol and dichloromethane from analytical HPLC grade, anhydrous sodium sulphate, 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Butyrylcholinesterase from equinum serum, Acetylcholinesterase from Electrophorus electricus, phosphate buffered saline, Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid), Acetylthiocoline iodide, donepezil hydrochloride, were purchased from Sigma-Aldrich (San Luis, MO, USA).The standard aliphatic hydrocarbons were purchased from ChemService (West Chester, PA, USA). Helium was purchased from INDURA (Guayaquil, Ecuador). All chemicals were of analytical grade and used without further purifications.

Approximately 0.5 g of meconium sample was employed, then pulverized with 8 g of diatomaceous earth and placed into a 66 mL stainless steel extraction cell with a little anhydrous sodium sulphate (Sigma Aldrich, Steinheim, Germany) before being placed in the accelerated solvent extraction system (ASE 350, Dionex, Sunnyvale, CA, USA). The samples were subjected to extraction using n-hexane and dichloromethane (1:1) as a solvent. The extraction was performed at 100 °C under a pressure of 1500 psi for 5 min and three static cycles with a static time of 5 min. A flush volume equivalent to 90% of the extraction cell capacity was used. Following extraction, pressurized nitrogen was used to purge the extract for 100 s. The extract was then filtered and transferred to a Turbovap tube, with anhydrous sodium sulphate added beforehand to remove any water present.

All solvents (acetone, chloroform, ethyl acetate and deuterated chloroform CDCl₃) used were of analytical standards and purchased from Sigma–Aldrich (Johannesburg, South Africa). Anhydrous sodium sulphate purchased from Sigma Aldrich. Rotary evaporator Büchi Rotavapor R-210 with Büchi Heating Bath B-491 was from Büchi Labotechnik, Meierseggstrasse 40, Postfach 9230 Flawil, Switzerland. NMR spectrometer Bruker AvanceTM III HD 400 MHz spectrometer, Topspin 3.5 pls and SampleXPress autosampler were from Bruker BioSpin, Rheinstetten, Germany.
Software materials include MetaboloAnalyst 4.0 designed by McGill University, Parasitology Building, 21,111 Lakeshore Road Ste. Anne de Bellevue, QC, Canada (http://www.metaboanalyst.ca) used for the multivariate analyses. MestReNova 14 software was from Mestrelab Research S.L., Santiago de Compostela, Spain, and used for the analysis of NMR chemical shifts.