Griess reagent

n-Hexane, chloroform, and methanol (analytical grade) were purchased from Carlo Erba Reagenti (Milan, Italy). β-Pinene, limonene, sabinene, myrcene, γ-terpinene, linalool, neral, linalyl acetate, geranial, geranyl acetate, and β-caryophyllene were supplied by Fluka (Milan, Italy). 2,2′-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were from Sigma-Aldrich (Milan, Italy). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, streptomycin, and penicillin were acquired from Lonza (Verviers, Belgium). Lipopolysaccharides (LPS, E. coli O111:B4) was obtained from Sigma-Aldrich (Schnelldorf, Germany). Griess reagents and nitrite standard were purchased from Cayman Chemical (Ann Arbor, MI, USA).

To ensure the safety of TP, cytotoxicity assay was performed using Cytotoxicity LDH Assay Kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Cells were cultured in culture medium with or without TP at indicated concentrations (10–1,000 µg/mL), or FFA solely (0–500 µM), or combined, cultured for 24 h followed by LDH assay. Measurement of nitrite in medium was used as an indicator of NO production. Culture supernatants were collected and nitrite, the stable reaction product generated from NO with molecular oxygen, was measured using Griess reagent (Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer’s instructions. PGE₂ production was measured with ELISA according to the manufacturer’s instructions (Cayman Chemical Co.).

The production of NO was measured indirectly via its stable metabolite nitrite using the Griess reagent, as per manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI, USA). The conditioned growth media from the MIO-M1 cell cultures were added, in sequence, to a 96-well plate. Both the Griess reagent R1 and Griess reagent R2 were then added to the 96-well plate. The 96-well plates were allowed to develop for ten minutes at RT, and the absorbance was measured using a plate reader at 540 nm. The nitrite concentration was calculated via comparison to a standard reference.

To measure the NO production, nitrite concentration in the culture supernatant was determined using Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride in 5% H3PO4) (Cayman Chemical, Ann Arbor, Michigan, USA). 100 μl of cell culture supernatant and 100 μl Griess reagent were mixed and incubated for 10 min, to color development. The absorption was estimated at 540 nm, using a Glomax Multireader spectrophotometer (Promega, Madison, WI, USA). Nitrite standard (Cayman Chemical, Ann Arbor, Michigan, USA) was used to generate a standard curve for quantification. Results were obtained from three independent experiment measurements.

The effect of 17-βE in solution and loaded in the CREKA-peptide nanoemulsion system on NO production from cultured ECs was quantified as total oxidative NO products (nitrate/nitrite) with a colorimetric assay kit based on the Griess reagent (Cayman Chemical). For the experiment, ECs were plated in T25 flasks at a density of 50,000 cells per flask. The cells were treated with17-βE loaded solution, the CREKA-peptide modified blank nanoemulsion, or the 17-βE loaded CREKA-peptide loaded nanoemulsion system for 24 hours. One T25 flask was used for each treatment group. After treatment, the cells were washed and trypsinized. The cell pellet was collected by centrifugation, re-suspended in PBS, and centrifuged at 30,000 rpm for 30 minutes. Sixty μl of the cellular extract was used for the assay and the nitrate/nitrite levels were measured using the assay kit as per the manufacturer’s instructions. The remaining cellular extract was treated with lysis buffer and protein concentration was determined by the BCA assay. The final nitrate + nitrite concentration was reported as pmol of nitrate/nitrite per μg of total cellular protein.