Naphthylethylenediamine dihydrochloride

For HPLC analysis, chromatographic-grade methanol was used (VWR, Vienna, Austria). A Millipore purifier (Millipore, Burlington, MA, USA) was used to obtain water for HPLC. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphoric acid, potassium chloride, quercetin, hispiduline, ascorbic acid, ibuprofen, ketoprofen, sodium chloride, hydrogen peroxide, trypsin, Tris-HCl buffer, sodium nitroprusside, sodium salicylate, ferrous sulfate, sulfanilamide, naphthylethylenediamine dihydrochloride and perchloric acid were purchased from Sigma-Aldrich, Taufkirchen, Germany. Human albumin 20%—BB, 200 g/L was ordered from BB-NCIPD Ltd., Sofia, Bulgaria.

Copaifera multijuga oleoresin (CMO) was collected from the Reserva Florestal Ducke, at the Instituto Nacional de Pesquisas da Amazônia (INPA) during the summer of 2006, where this is the only species of Copaifera available. Exsiccate was properly deposited at the INPA herbarium. β-CD, HP-β-CD, albumin, Coomassie brilliant blue G-250, hexadecyltrimethylammonium bromide, naphthyl ethylenediamine dihydrochloride, o-dianisidine, sodium nitrite, sulfanilamide, Tween 80 and λ-carrageenan were purchased from Sigma-Aldrich^® (St. Louis, MO, USA). β-caryophyllene [trans-(−)-caryophyllene] standard was purchased from ChromaDex^® (Irvine, CA, USA). Hydrogen peroxide was purchased from Merck (Darmstadt, Germany). All experiments were carried out using purified water (<1.3 μS) obtained by reverse osmosis. All reagents were of analytical grade. Phosphate buffered saline (PBS) was used in this study and was prepared with the following constituents: 137 mM NaCl, 3 mM KCl, 1.5 mM KH₂PO₄ and 10 mM Na₂HPO₄, pH 7.4.

RAW 264.7 cells were plated in 96-well plates at a density of 3 × 10⁴ cells/well for a 24 h incubation at 37 °C. Cells were either exposed to the compounds or N^G-methyl-L-arginine acetate salt (L-NMMA) at concentrations of 5, 10, 25, 50, and 100 μM for 1 h at 37 °C. Thereafter, they were incubated with 1 μg/mL LPS for 24 h at 37 °C. The supernatant was mixed with an equal volume of Griess reagent containing 0.2% naphthylethylenediamine dihydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and 2% sulfanilamide (Sigma-Aldrich) in 5% phosphoric acid (Sigma-Aldrich) and incubated for 40 min at 37 °C. The absorbance was then measured at 540 nm using a PowerWave XS microplate reader.

Nitric oxide (NO) secreted by cells into the cell culture was determined using the Griess reaction. Cell culture supernatant in 96-well plates (Nunc, Roskilde, Denmark) was mixed with an equal volume of Griess reagent made up of 1% m/v sulphanilamide (Sigma-Aldrich, Germany), 0.01% m/v naphthyl ethylenediamine dihydrochloride (Sigma-Aldrich), and 2.5% phosphoric acid. The mixture was allowed to react for 15 min at room temperature. The colour developed was measured at 540 nm using a microplate reader (Multiskan Ex, Thermo Electron Corporation). The concentration of NO was determined from a standard curve generated using 1.56–100 μM sodium nitrite (Sigma-Aldrich, Germany).

All chemicals and solvents used were of analytical grade. Rutin, gallic acid, Folin–Ciocalteu reagent, methanol, N-N-dimethyl ρ-nitrosoamine, histidine, naphthylethylenediaminedihydrochloride, sodium nitroprusside, 2 deoxy D-ribose were obtained from Sigma Aldrich, St. Louis, MO, USA through Analytical Instrument Pvt Ltd., Colombo, Sri Lanka.

Dimethyl sulfoxide (DMSO) and lipopolysaccharide (LPS), dexamethasone, formic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hyclone Laboratories Inc. Fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid (EDTA), and penicillin–streptomycin were purchased from Gibco Industries Inc. High performance liquid chromatography (HPLC)-grade water and acetonitrile were obtained from Fisher Scientific Korea Ltd. DMSO-d₆ and methanol-d₄ were purchased from Cambridge Isotope Laboratories Inc. The Griess reagent for the measurement of NO production was generated from 1% sulfanilamide (Sigma-Aldrich), 0.1% naphthylethylenediamine dihydrochloride (Sigma-Aldrich), and 2% phosphoric acid (Wako Pure Chemical Industries). The water soluble tetrazolium (WST) kit (EZ-cytox) for the cell viability assay was purchased from Daeil Lab Service Co. NMR spectra were recorded on a Bruker Avance DPX 300 and Bruker Avance III 600. The ESI-MS spectra were measured with an Agilent 6530 Q-TOF mass spectrometer (Agilent, Santa Clara, USA).

Peritoneal macrophages were treated with Lactobacillus strains for 10 h and challenged or not with C. albicans (yeast-to-macrophage ratio: 3:1). Culture supernatants of macrophages were incubated with equal volumes of Griess reagent, containing 1% sulfanilamide (Sigma) and 0.1% naphthylethylenediamine dihydrochloride (Sigma) in 2.5% phosphoric acid. After 30 min at room temperature, the absorbance was read at 550 nm and concentration was determined by comparison with standard solutions of sodium nitrite prepared in the same culture media. Each assay was conducted in triplicates.