Cox ovine human inhibitor screening assay kit

The anti-inflammatory activity of the samples was examined using a COX (ovine/human) Inhibitor Screening Assay Kit (Cayman Chemical). All samples were examined according to the instruction provided by the producer. Primary solutions of examined extracts were dissolved in 80% methanol. Measurements were performed in triplicate for each sample and averaged. At least five dilutions of each extract and the positive control were examined to plot a dose-response curve and determine the half maximal inhibitory concentration (IC₅₀). All measurements were made in triplicate and averaged.

The evaluation of the inhibition of COX-1 and COX-2 by CDXs was conducted using the commercially available COX (ovine/human) Inhibitor Screening Assay Kit (Cayman Chemical, Michigan, MI, USA). Briefly, the assay implies an enzymatic immunoassay based on the competition between prostaglandins (PGs) and a PG-acetylcholinesterase (AChE) conjugate (PG tracer), which is then evaluated by the addition of Ellman’s reagent. All the solutions required for the experiment were prepared according to the manufacturer’s instructions. Each compound was analysed in two independent days, in triplicate. CDX working solutions were prepared in DMSO to a final concentration of 20 µM. Indomethacin (1 µM) was used as positive control. Absorbance measurements at 412 nm were performed in a Synergy HT microplate reader (Bio-Tek Instruments, Winooski, VT, USA) operated with Gen5 software (Bio-Tek Instruments).

In vitro anti-inflammatory activity of CPL was determined using the COX (ovine/human) Inhibitor Screening Assay Kit (Cayman, city, MI, USA) following the manufacturer’s instructions. This assay directly measures the amount of prostaglandin 2α (PGF2α) produced by stannous chloride reduction of COX-derived PGH2. Briefly, 10 µL of a polysaccharide sample (0.25 to 5mg/mL, in 5% DMSO) were added to a reaction mixture containing 160μL of assay buffer (0.1 M Tris-HCL, pH-8 with 5 mM EDTA and 2 mM phenol), 10μL of heme, and 10μL of the enzyme (either ovine COX-1 or human recombinant COX-2). After 10-minute incubation at 37 °C, 10 µL of arachidonic acid were added. The reaction mixtures were mixed and incubated for 2 min at 37 °C. Then, 30 µL of a saturated SnCl₂ solution in 1 M HCl were added to stop COX catalysis. The mixtures were vortexed and incubated for 5 min at room temperature. The prostanoid product was quantified by the enzyme immunosorbent assay (ELISA) using a specific antiserum. The blank contained 10 µl of 5% DMSO instead of the sample and 10 µL of the inactivated enzyme instead of its active form. Aspirin (acetylsalicylic acid, 1mM) was used as a positive control for inhibition of COX-1 and COX-2. The percent COX inhibition was calculated as follows:

where ASP = absorbance of the inhibitor sample and AIAS = absorbance of the 100% initial activity sample.

The extracts of E. japonicum were examined for cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitory activity using a COX (ovine/human) Inhibitor Screening Assay Kit (Cayman Chemical, MI, USA) according to the protocol of the manufacturer. The extracts were tested at different concentrations (20–100 µg/mL). Indomethacin (1 µg/mL) was used as a positive control.

The extracts of Cephalaria species were tested for cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitory activity using a COX (ovine/human) Inhibitor Screening Assay Kit (Cayman Chemical, MI, USA) according to the protocol of the manufacturer. The stock solution of the studied extracts were dissolved in ethanol, and their final concentration was 50 and 100 µg/mL. Indomethacin was used as a positive control for inhibition of COX-1 and COX-2.

COX (ovine/human) inhibitor screening assay kit (Cayman Chemical, Ann Arbor, MI, USA) was used according to the manufacturer’s instructions for measuring COX-1 and COX-2 inhibition rates. Briefly, COX reactions were performed. Background tubes were prepared for COX-1 and COX-2 in boiling water for three minutes. The inactivated enzymes were used to generate the background values. Then, 160 µL reaction buffer, 10 µL of heme, and 10 µL of COX-1 or COX-2 were added to reaction tubes for COX-1 or COX-2 100% initial activity tubes and inhibitor tubes. Next, 10 µL of inhibitors was added to inhibitor tubes, whilst 10 µL of vehicle was added to 100% initial activity tubes and background tubes. Celecoxib and indomethacin were administered at 0.01–10 µM concentrations, whereas compound 2e was applied at 10–100 µM concentrations. Then, these tubes were incubated for 10 min at 37 °C. Reaction was initiated by adding 10 µL of arachidonic acid to all the reaction tubes, mixed, and incubated for two minutes at 37 °C. Then 30 µL of the saturated stannous chloride solution was added to each tube. The prostaglandins were quantified by ELISA. The plate was read at 405 nm in an ELISA reader (BioTek Instruments Synergy HTX S1 LFA).

Cell culture reagents were purchased from Euroclone (Milan, Italy). COX (ovine/human) inhibitor screening assay kit (Catalog No. 560131, Cayman Chemicals, Ann Arbor, MI, USA) was purchased from Sigma-Aldrich, Burlington, MA, USA. Mofezolac was prepared in our laboratory [29 ]. The other reagents were purchased from Sigma-Aldrich, Burlington, MA, USA.