Determination of anti-inflammatory activities

The callus cultures were also investigated for anti-inflammatory activities via employing sPLA2, 15-LOX, COX1 and COX2 assays by following the protocol reported by Usman et al.⁹⁴ with slight modification.

15-LOX Inhibition Assay

The inhibitory activity in the extracts towards 15-LOX was also performed by the kit method (760700, Cayman Chem. Co., Interchim, Montluçon, France) according to following the manufacturer’s guidelines.

The amount of hydroperoxides accumulated thought the lipo-oxygenation reaction was calculated via the kit using standard filtered soybean 15-lipooxygenase in Tris–HCl bu_er (10 mM) at pH 7.4. The microplate reader (BioTek ELX800; BioTek Instruments, Colmar, France) was employed to measure the absorbance at 940 nm. The 5 µM Thioetheramide-PC was used as sPLA2 inhibitor. The extraction volume in equal amount was applied as blank.

sPLA2 inhibition assay

The kit method (10004883, Cayman Chem. Co, Interchim, Montluçon, France) was followed with manufacturer instructions to measure inhibition of phospholipase A2 (sPLA2) enzyme. The Diheptanoyl thio-PC was used as substrate and thiotheramide-PC was applied as positive control inhibitor. While. Nordihydroguaiaretic acid (100 _M) was used as 15-LOX inhibitor during the assay. The extraction volume in equal amount was applied as blank. The absorbance was recorded via microplate reader (BioTek ELX800; BioTek Instruments, Colmar, France) at 420 nm.

The % inhibition was figured out with the following formula;

where the inhibition is expressed as enzyme activity with adding inhibitor; IA as 100% activity of enzyme in the absence of inhibitor.

COX-1 and COX-2 inhibition assay

The COX1 (ovine) and COX2 (human) assay kits were utilized by following the manufacturer guidelines (701050; Cayman Chem; Co, Interchim, Montluçon; France). The 10 µM ibuprofen as positive control and Arachidonic acid (1.1 mM) was employed as substrate. In order to estimate the activity, COX peroxidase component kit employed and Synergy II plate (BioTek Instruments, Colmar, France) was used to demonstrate the oxidized tetramethyl-p-phenylenediamine, Wurster’s blue (C10H16N2). The microplate reader (BioTek ELX800; BioTek Instruments, Colmar, France) was used to measure the absorbance at 5 nm for 5 min. The extraction volume in equal amount was applied as blank.