Oxygen radical absorbance capacity

The Oxygen Radical Absorbance Capacity (ORAC) technique developed by Ou et al. was used, with a few modifications detailed by Denev et al.^{[20, 21]} This method assesses an in vitro antioxidants ability to neutralize peroxide radicals. The method is based on inhibiting fluorescein fluorescence decline during oxidation in the presence of an in vitro antioxidant. As a peroxide radical generator, the thermal breakdown of 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH) was used. The results are given in terms of mol Trolox equivalents per gram of extract (TE/g). FLUOstar OPTIMA fluorimeter (BMG LABTECH, Offenburg, Germany) was used for the measurements. The excitation wavelength was 485 nm, and the emission wavelength was 520 nm.

The Hydroxyl Radical Averting Capacity (HORAC) method developed by Ou et al.^[22] assesses an in vitro antioxidant’s ability to form complexes in Fenton-like reaction caused by the interaction of Co (II) with H₂O₂. The results are given in terms of mol gallic acid equivalents per gram of extract (GAE/g). FLUOstar OPTIMA fluorimeter (BMG LABTECH, Offenburg, Germany) was used for the measurements. The excitation wavelength was 485 nm, and the emission wavelength was 520 nm.

The electrochemical technique was used for evaluating the antioxidant activity in vitro.^[23] The methodology of the experiment consists in recording voltamperograms of cathodic electroreduction of oxygen with the “Analyst AOA” (RU.C.31.113.A N28715) linked to a PC. The in vitro antioxidant activity of the investigated samples was calculated using the kinetic criteria K (in micromoles per liter/minute), which indicates the quantity of reactive oxygen species in time and is represented as a percentage of the Trolox kinetic criterion via the formula:

АOA = K_sample / K_Trolox

Animals

All experiments are in agreement with the approval of the Ethics Committee of Medical University of Plovdiv (as of protocol No. 01-2/10.04.2020) and the approval of the Bulgarian Food Safety Agency (as of protocol No. 258), based on the position of the Ethic Committee, Bulgarian Food Safety Agency No. 174 from October 8, 2019.

Rats were raised in the vivarium of the Medical University of Plovdiv and housed under standard conditions: 20°–22°C, 12-hour light/dark cycle, with free access to food and water.

Carvacrol and rosmarinic acid (RA) were bought from Sigma-Aldrich (St. Louis, Missouri, USA). Carvacrol was dissolved in olive oil and RA was dissolved in saline. Both solutions were administered orally via stomach gavage (1 ml/100 g b.w.).

The dry extract of Satureja montana (SME) was prepared by methodology of Vesselino EOOD, Kazanlak, Bulgaria via methanol-aqueous (70:30) extraction, followed by spray drying at 40°C until complete evaporation of both solvents. The extract was dissolved in saline and applied orally via stomach gavage in the volume of 0.5 ml/100 g b.w. (for a dose of 250 mg/kg b.w.) and 1 ml/100 g b.w. (for a dose of 500 mg/kg b.w.).

Acute stress model

For investigation of the impact of dry SME on the serum concentration of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in acute cold stress model, 56 male 8-week-old Wistar rats with average bodyweight 110 g (range 100-120 g) were used. The rodents were randomly divided into 7 groups (n=8). One of the groups was sham control, which was only gavaged without administration of substances. The rest groups were treated daily for 14 days orally via stomach gavage with saline and olive oil – 1 ml/100 g b.w. (positive controls), dry SME at doses of 250 mg/kg b.w. and 500 mg/kg b.w., carvacrol 500 mg/kg b.w., and RA 15 mg/kg b.w. – experimental groups.

On day 15 from the beginning of the experiment, rodents from both positive controls and all experimental groups were exposed to the stress factor, −4°C for 60 minutes in the refrigerator. Animals were placed in plastic boxes 20×20 cm, and allowed to move freely into the box. At the same time, there were four animals in the refrigerator, each of it in a single box. Boxes were cleaned with 70% alcohol after each animal. The sham group was not exposed to the stress factors.

Fifteen minutes after removing the rodents from the refrigerator, they were decapitated under narcosis with ether and 2 ml blood was collected. The blood was centrifuged and 1 ml of serum was separated. The investigation of serum levels of IL-1β, IL-6, and TNF-α was performed with ELISA method and Ebioscience kits (ThermoFisher Scientific, USA).

To investigate the effect of dry SME on the serum pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in a chronic unpredictable mild stress model, 56 male, 8-week-old Wistar rats with average bodyweight of 110 g (100-120 g) were used. They were randomly divided in 7 groups (n=8), which were similar to these, described above in acute stress model. From the first day of the experiment, animals from both positive controls and all experimental groups were exposed to mild stress stimuli – food or water deprivation for 24 hours, placing an empty water bottle for 1 hour, tilting of home cage (45° for 3 hours), leaving of light for 24 hours, soiling the bedding (200 ml, 25°C water per 100 g bedding material), predator sounds (two rounds of 20 minutes). Stress factors were applied 60 minutes after the daily treatment. Each stressor was used once a week and the order was changed every week of the experiment to avoid habituation to them. The sham group was not exposed to the stress stimuli.

On day 60, one hour after the exposure to the stressor, the rodents were decapitated under narcosis with ether and 2 ml of blood was collected. This blood was centrifuged and 1 ml of serum was separated. The investigation of serum levels of IL-1β, IL-6, and TNF-α was performed as described above in the section of acute stress model.

Diluted rat serums (1:2) for IL-1, IL-6, and TNF, as well as internal controls and test standards, were dripped on solid phase with monoclonal antibodies against the respective cytokine for quantitative cytokine testing. A peroxidase conjugate (second anti-species antibody) is added after incubation and washing to generate a cytokine complex. A second wash is used to eliminate any unbound conjugate. A color response occurs when a chromogenic substrate is introduced to the enzyme, indicating the presence of cytokines. Absorption is evaluated colorimetrically on a TECAN ELISA reader at 450 nm and 620 nm and is proportional to cytokine concentration. A standard curve is used to determine the concentration of each cytokine in pg/ml.

The results were analyzed statistically using one-way ANOVA and LSD post hoc tests with IBM SPSS 19.0 software. Results were expressed as arithmetic means (X̅), standard error of the mean (±SEM for cytokine levels) ,and standard deviation (±SD for the antioxidant activity). A p-value ≤0.05 was considered statistically significant. For each one-way ANOVA test F statistics and p value is given. Statistical significance between groups, which is found with post hoc analyses, is presented as a p-value.

The dry SME showed in vitro antioxidant activity in all used methods. The results are presented in Table 1.

Acute stress model

Acute cold stress significantly increased the serum concentrations of TNF-α and IL-6 in the positive controls, treated with olive oil (p=0.041 and p<0.001, respectively). In the positive saline control, a statistically significant increase was found only in IL-6 serum concentrations (p=0.003). The same cytokine was found to be statistically higher in the groups treated with dry SME 500 mg/kg b.w., RA, and carvacrol compared with sham control (p=0.028, p<0.001, and p<0.001, respectively). TNF-α was statistically increased in the groups that received RA and carvacrol when compared to the sham control (p=0.003, p=0.015, respectively).

None of all tested substances had a significant impact on lowering the serum cytokine levels compared to corresponding positive control (p>0.05 for all measurements). The results are presented on Fig. 1A–B.

The dry SME at a dose at 250 mg/kg b.w. decreased significantly the serum concentration of IL-6 and TNF-α compared to RA (p=0.011 and p=0.036). When the same dose of dry SME was compared with the carvacrol treated group, a significant impact was found only on the IL-6 levels (p=0.019). The higher dose of Satureja montana dry extract did not show significant anti-inflammatory effect compared to RA and carvacrol. The results are presented in Fig. 2.

Chronic stress significantly increased serum concentrations of IL-6 and TNF-α in the positive olive oil control group (p=0.001 and p=0.006, respectively) as well as IL-6 in the positive saline control group (p=0.015). The chronic stress had no impact on serum levels of IL-1β.

The dry SME decreased non-significantly the serum concentrations of IL-6 and TNF-α compared to the positive saline control (p>0.05 for all measurements). RA increased statistically significantly IL-6 compared to the sham control (p=0.013) and showed non-significant anti-inflammatory effect compared to the positive saline control (p>0.05). Carvacrol decreased significantly serum concentrations of IL-6 and TNF-α compared to the olive oil control (p=0.011 and p=0.004, respectively). The results are presented in Fig. 3.

Both doses of the dry SME 250 mg/kg b.w. and 500 mg/kg b.w. did not show significant effect on serum concentrations of IL-6 and TNF-α when compared with the RA or the carvacrol treated groups (p>0.05 for all measurements). Visualization of results is not shown.