L cysteic acid

The amino acid content of olive pomace and the LMW peptides (<3 kDa) extracted from olive pomace was determined after hydrolysis in 6 N HCl plus 1% phenol in sealed Pyrex tubes (Corning, NY, USA) at 110 °C for 24 h by high-performance liquid chromatograph (HPLC) according to the Waters Pico Tag method [47 ] as described in [48 (link)], using a Waters 2695 separation module (Waters Corporation, Mildford, MA, USA). L-alpha-amino adipic acid and DL-norleucine were used as internal standards. Pre-column derivatization was carried out with phenyl isothiocyanate. The cysteine and methionine contents were determined as cysteic acid and methionine sulphone, respectively, after oxidation of samples with performic acid prior to the protein hydrolysis step, as described in [49 (link)]. For quantification, seventeen amino acid standards (Amino Acid Standard H, Thermo Fisher Scientific, Waltham, MA, USA), L-Cysteic acid, and L-Methionine sulfone (Sigma-Aldrich, St. Louis, MO, USA) were used at concentrations of 0.1, 0.2, and 0.3 mM. Tryptophan was not determined. A Millennium 32 chromatography manager system was used for gradient control and data processing. Each sample was analysed three times with two technical replicates.

The following were obtained from Sigma-Aldrich (St. Louis, MO, USA): l-Glutamate, l-cysteic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), acetonitrile, boric acid, phenobarbital, sucrose, calcium chloride, potassium chloride, magnesium chloride, DMSO, verapamil hydrochloride, carbamazepine, and 4-aminopyridine. From the Tokyo Chemical Industry (Tokyo, Japan), the following was purchased: 4-fluoro-7-nitrobenzofurazan (NBD-F). The following were provided by Cyclolab Ltd (Budapest, Hungary): β-cyclodextrin and 6-monodeoxy-6-mono(3-hydroxy) propylamino -β-cyclodextrin hydrochloride. The following were obtained from Tocris Bioscience (Bristol, UK): dl-threo-β-benzyloxyaspartic acid (dl-TBOA), tetrodotoxin, and ω-conotoxin MVIIC. The following were purchased from Reanal (Hungary, Budapest): sodium chloride and D-glucose. Fetal bovine serum was obtained from Biosera (Nuaille, France). Lidocaine hydrochloride was provided by Egis Pharmaceutical Plc. (Budapest, Hungary). Tolperisone and pregabalin were received as kind gifts from Meditop Pharmaceuticals Ltd. (Budapest, Hungary). Ultrapure water from MilliQ Direct 8 water purification system (Merck, Darmstadt, Germany) was used for all experiments. All compounds were stored and handled as described in the product information sheets.

Sulfuric acid (VI), sodium hydroxide, boric acid, formic acid, hydrochloric acid, and hydrogen peroxide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Amino acid standard solution AASS-18, amino acid standards for protein hydrolysate A 2908, L- methionine sulfone, L-cysteic acid, and L- norleucine were purchased from Sigma Aldrich (St. Louis, MO, USA). Nitric acid and heptahydrate lanthanum chloride were obtained from Merck (Darmstadt, Germany).