Herniaria

Kolodziejczyk-Czepas, Joanna, Kozachok, Solomiia, Pecio, Łukasz, Marchyshyn, Svitlana & Oleszek, Wiesław, 2021, Determination of phenolic profiles of Herniaria polygama and Herniaria incana fractions and their in vitro antioxidant and anti-inflammatory effects, Phytochemistry (112861) 190, pp. 1-21 : 18

4.7. Evaluation of antioxidant effects of the examined Herniaria fractions

4.7.1. Preparation of blood plasma samples

Blood plasma was isolated from human buffy coat units, commercially available at the Regional Centre of Blood Donation and Blood Treatment in Lodz, Poland. Experiments were approved by the committee on the Ethics of Research at the University of Lodz, Poland (Protocol No. 10/KBBN–UŁ/ I/2017). Stock solutions of the analysed fractions were dissolved in 30% DMSO (dimethyl sulfoxide) to the final concentration of DMSO of ≤0.03%, in the assayed samples. Plasma samples were pre-incubated for 15 min at 37 ◦ C with the fractions (1–50 μg/ml) or a reference compound, i.e. Trolox (5 μg/ml), and then they were exposed to ONOO (at final concentrations of 100 μM or 150 μM). Samples containing blood plasma treated with ONOO in the absence of the examined substances were also prepared. Control plasma was treated with neither the investigated plant fractions/reference antioxidant nor ONOO (but it contained 0.03% DMSO, as a vehicle for the fractions).

4.7.2. Measurements of oxidative stress biomarkers in blood plasma

Oxidative stress was induced by the exposure of blood plasma to 100 μM ONOO. Lipid peroxidation was determined based on the level of thiobarbituric acid-reactive substances (TBARS) ( Wachowicz, 1984) and lipid hydroperoxides generation (ferric-xylenol orange assay ( Gay and Gebicki, 2000)). Modifications of blood plasma proteins were analysed using 3-nitrotyrosine and thiol groups as oxidative stress biomarkers. Presence of 3-nitrotyrosine in blood plasma proteins was determined with the use of a competitive enzyme-linked immunosorbent assay (ELISA) ( Olas et al., 2004). Results from this assay were expressed as equivalents of 3-nitrotyrosine-containing standard protein (i.e. nitrated fibrinogen, 3-NT-Fg, per mg of blood plasma protein). The level of protein thiol groups was determined spectrophotometrically ( Rice-Evans et al., 1991).

4.7.3. Determination of the ferric reducing ability of blood plasma (the

FRAP assay)

Blood plasma was exposed to 150 μM ONOO in the presence or absence of the examined substances. The influence of Herniaria fractions on the non-enzymatic antioxidant capacity (NEAC) of blood plasma was established colorimetrically by measurements of the ability of the assayed samples to reduce ferric ions (Fe 3+) to ferrous ions (Fe 2+). The FRAP assay was conducted according to the previously described protocol ( Kolodziejczyk-Czepas et al., 2014).

4.7.4. Electrophoretic analysis of the peroxynitrite-induced changes in fibrinogen structure

Fibrinogen was isolated from human blood plasma based on a technique of precipitation with cold ethanol ( Doolittle et al., 1967). Isolated protein (2 mg of fibrinogen/ml, in 0.01 M Tris/HCl buffer, pH 7.4) was pre-incubated (15 min, at 37 ◦ C) with the examined Herniaria fractions (1–50 μg/ml) or the reference antioxidant, and then, exposed to peroxynitrite (100 μM). Protein samples were separated in 4–20% SDS-PAGE gradient gels (i.e. Mini-Protean TGX™ precast gels; 8 μg of protein per each lane), under reducing conditions. Electrophoresis was conducted using BioRad Mini Protean Tetra Cell equipment and BioRad reagents. Protein bands were visualized with the use of Coomassie Blue R250 dye.

4.8. COX-inhibitor screening and experiments on peripheral blood mononuclear cells (PBMCs)

Cells were isolated from human buffy coat units using density centrifugation in BioWestLymphosep medium (diatrizoic acid dihydrate – 95.281 g /l, EDTA tetrasodium salt dihydrate – 0.231 g /l, sodium hydroxide pellets – 5.860 g /l, polysaccharose 400– 57 g /l, water), according to the protocol provided by the manufacturer.

4.8.1. Determination of COX-inhibitory effects

Inhibitory effects of the examined Herniaria fractions (1–50 μg/ml) on COX-1 and -2 activities were evaluated using a commercial colorimetric kit, measuring the enzymatic activity of the peroxidase component of COXs (responsible for the oxidation of the N, N, N ′, N ′ - tetramethylp -phenylenediamine (TMPD) chromogenic substrate). Assays were carried out in triplicate, using indomethacin (1–50 μg/ml) as a reference inhibitor.

4.8.2. Determination of anti-inflammatory effects in PBMCs

Isolated PBMCs were suspended in RPMI 1640 medium (supplemented with 10% fetal calf serum and 0.1% of penicillin-streptomycin), at the density of 1.5 × 10 6 cells/ml and pre-incubated for 60 min with the examined fractions, in a laboratory CO 2 incubator. Then, PBMCs were stimulated with concanavalin A (Con A; at the final conc. of 10 μg/ ml) and cultured for 24 h (in 96-well microplates, at 37 ◦ C, with 5% of CO 2 concentration and 95% humidity). The next day, microplates were centrifuged to obtain supernatants (cell culture medium) for further analyses. Anti-inflammatory effect of the examined Herniaria fractions was evaluated based on measurements of interleukin-2 and TNF-α secretion from PBMCs. Concentrations of these cytokines in supernatants were determined with commercial ELISA kits, according to protocols provided by the manufacturer.

4.8.3. Cytotoxicity assays

For preliminary evaluation of cellular safety of the examined fractions, a short-term incubation cytotoxicity test was applied ( Kolodziejczyk-Czepas et al., 2017; Marchelak et al., 2017). PBMCs were suspended in 0.02 PBS only (1 × 106 PBMCs/ml) and incubated with the analysed Herniaria fractions (5, 25 and 50 μg/ml) to assess the possibility of direct damage to the cell membrane. This cytotoxicity assay was based on a dye excluding test, employing 0.4% Trypan blue ( Strober and Strober, 2001). Cell viability was measured in a micro-chamber automated cell counter, after 6 h of incubation (at 37 ◦ C, with gentle mixing on a rotary shaker).

Analyses based onTrypan blue exclusion were complemented with the resazurin-based cell viability assay. PBMCs (1.5 × 106 PBMCs/ml) were suspended in the RPMI-1640 medium (supplemented with 10% fetal calf serum and 0.1% of penicillin-streptomycin) and incubated with the fractions (5, 25 and 50 μg/ml) for 24 h (in 96-well microplates, at 37 ◦ C, with 5% CO 2 and 95% humidity). Then, resazurin solution was added to the final concentration of 10%. Measurements of cell viability were executed 4 h after resazurin application, using a BMG Labtech SectroStarNano microplate spectrophotometer, at λ = 600 nm (690 nm was a reference wavelength).

4.9. Statistical analysis

Data are presented as mean values ± SD; p <0.05 was assumed as statistically significant, and “n” corresponded to the number of independent experiments/blood donors. Uncertain data were excluded by the Q-Dixon test. In experiments employing blood plasma, isolated fibrinogen and PBMCs, at least two independent pre-incubations of examined substances with biological material from each donor were performed. Statistical significance of obtained results was established by the Student’ s t-test or ANOVA and Tukey’ s post-hoc test.