In our study, we wanted to test whether the pre-incubation of lymphocytes with the A. rusticana extract or flavonoids can decrease the hydrogen peroxide-induced DNA damage. We used the hydrogen peroxide challenge assay which is a method used widely to detect the antigenotoxic potential of various plant extracts. It enables one to assess the capacity of plant extracts and their components to protect DNA against DNA oxidation in human cells [31]. First we tried to find non-genotoxic concentrations of the extract and flavonoids.

We evaluated a wide range of concentrations of the extract and flavonoids using the comet assay. For A. rusticana extract, a range of five concentrations from 0.0025 to 2.5 mg·mL⁻¹ was tested. Three concentrations of the aqueous extract from A. rusticana (0.0025 mg·mL⁻¹; 0.025 mg·mL⁻¹; 0.25 mg·mL⁻¹) did not exhibit any genotoxic activity and could be considered as non-genotoxic. Two higher concentrations of the extract from A. rusticana (0.5 mg·mL⁻¹; 2.5 mg·mL⁻¹) showed a low genotoxic activity (p < 0.05) (Figure 2). For further experiments we chose the concentration 0.0025 mg·mL⁻¹.

While searching for non-genotoxic concentrations of flavonoids, we evaluated a range of eleven concentrations from 5 to 1,500 μg·mL⁻¹. At first we tested a range from 250 to 1,500 μg·mL⁻¹. We did not find a non-genotoxic concentration because all the concentrations exhibited a low or moderate DNA damage (from p < 0.05 to p < 0.001) (data not shown).

Higher concentrations of kaempferol (from 500 to 1500 μg·mL⁻¹) showed some DNA damage comparable to the same concentrations of quercetin (from p < 0.05 to p < 0.001). Our results are in agreement with the results obtained by other authors who used these flavonoids in vitro and in vivo [32]. Therefore, we applied lower concentrations of flavonoids in the range from 5 to 100 μg·mL⁻¹.

Kaempferol concentrations in the range from 5 to 100 μg·mL⁻¹ exerted a non-genotoxic effect (Figure 3A). The three lowest concentrations of quercetin (from 5 to 25 μg·mL⁻¹) were not genotoxic either. Two higher concentrations (50 μg·mL⁻¹; 100 μg·mL⁻¹) showed a low genotoxic effect (Figure 3B). Based on these results we chose a non-genotoxic concentration 5 μg·mL⁻¹ of kaempferol and quercetin for further experiments.

After the selection of non-genotoxic concentrations of A. rusticana extract and flavonoids, we tested whether the pre-treatment of human lymphocytes challenged with hydrogen peroxide has the ability to protect human DNA. Firstly, non-genotoxic concentration of the aqueous extract from A. rusticana (0.0025 mg·mL⁻¹) was tested (Figure 4).

Lymphocytes without treatment (DNA damage was 6.6%) were used as the negative control. Lymphocytes treated only with hydrogen peroxide were used as the positive control (DNA damage was 78%). Lymphocytes incubated with non-genotoxic concentration of A. rusticana extract caused only 10.6% of DNA damage. After the pre-incubation of lymphocytes with non-genotoxic concentration prior to hydrogen peroxide exposure, the DNA damage reached only 35.75% (Figure 4). This result proves that non-genotoxic concentration (0.0025 mg·mL⁻¹) of A. rusticana extract has the ability to decrease DNA damage induced by hydrogen peroxide. We detected a reduction from 78% to 35.75% compared to the positive control (lymphocytes treated with hydrogen peroxide only). We obtained very similar results to the ones obtained after the pre-incubation of lymphocytes and HEK 293 cells with the extract from Gentiana asclepiadea [33].

We finally tested the non-genotoxic concentrations of flavonoids—5 μg·mL⁻¹. For the negative control, we used lymphocytes incubated in PBS (DNA damage was 6.4%). Lymphocytes treated with hydrogen peroxide were used as the positive control (DNA damage was 83.25%). Non-genotoxic concentration of kaempferol induced higher DNA damage (15%) than the quercetin one (9.6%). After the pre-incubation with kaempferol prior to hydrogen peroxide treatment, we found out a decrease of DNA damage from 83.25% to 19.4%. After the pre-incubation with quercetin prior to hydrogen peroxide exposure, the percentage of DNA damage significantly decreased from 83.25% to 16.2% (Figure 5A,B). Both flavonoids significantly reduced hydrogen peroxide-induced DNA damage.

In the present study, we demonstrated that the aqueous extract from A. rusticana and the flavonoids kaempferol and quercetin prevented the induction of single-strand DNA breaks. We propose that the extract from A. rusticana and flavonoids might be considered as desmutagens. Desmutagens are defined as agents being able to suppress mutations by decreasing levels of DNA lesions (including single-strand breaks as primary DNA lesions) through various mechanisms. They can suppress mutations by decreasing levels of DNA lesions via their antioxidant (scavenging, transient metals chelating) properties, or by preventing or decreasing the conversion of pro-mutagens to ultimate mutagens. They have also the ability to degrade/detoxificate mutagens or induce enzymes that can detoxificate mutagen prior to reaching DNA [34,35].

In our preliminary experiments scavenging/antioxidant activities of the extract and both flavonoids were assessed (unpublished data), so that we could hypothesize that the underlying mechanism of H₂O₂genotoxicity reduction might be a result of the antioxidant activity of A. rusticana and the tested flavonoids. It is necessary to realize that the combinations of various flavonoids usually promote antagonistic effects. Kaempferol and quercetin are unique in their synergistic antioxidant activity [30]. We propose that this synergistic antioxidant activity can effectively contribute to the overall antigenotoxic effect of A. rusticana extract. We obtained similar results when comparing the effect of lymphocytes pre-incubation with methanolic extracts from A. rusticana and Gentiana asclepiadea. All of them had the ability to modulate DNA damage induced by hydrogen peroxide in the case of pre-incubation and acted as desmutagens [36]. We also came to the conclusion that methanolic extracts from A. rusticana, G. asclepiadea had a lower modulating effect than the aqueous extract from A. rusticana presented in this study. Differences between these results might be due to the fact that different extracts obtained from the same plant (aqueous or methanolic) may differ not only in the quantity of their components, but even in their chemical composition. As the antimutagenic activity of natural compounds often correlates with the antioxidant activities [37], we could consider that the molecular mechanisms underlying their antimutagenic effect might be explained by their antioxidant potential (attributed to the free radicals capture).

Our results correlate with the study in which kaempferol or quercetin pre-treated HepG2 cells were exposed to a genotoxic agent – benzo[a]pyrene [38]. The results from this study demonstrated that the pre-incubation with flavonoids decreased DNA damage. Their unique structure and varied pharmacological activities may bring new possibilities for a discovery of drugs with a new mechanism of action [39].

The air-dried plant material (roots) weighing about 70 g was cut into small pieces and then extracted to 150 mL of water at 65 °C. This procedure was repeated five times. The hot solution of the extract was then filtered and concentrated using a vacuum evaporator. The final aqueous extract from the roots of A. rusticana was kept in the dark at 4 °C until tested, and then diluted in 1× PBS.

Both flavonoids, kaempferol and quercetin, were purchased from Sigma-Aldrich (Bratislava, Slovakia). They were dissolved in DMSO solution (1%) and kept in the dark at 4 °C until tested.

Lymphocytes were obtained from peripheral blood using the finger prick method just prior to use. Blood (40–50 μL) was taken and added to phosphate buffer solution (1 mL, 1× PBS, pH 7.5), mixed and left on ice up to 30 min. Then we underlayed it with Histopaque 1077 (Sigma, 100 μL) and spinned at 180 ×g for 5 min at 4 °C. Lymphocytes (100 μL) were retrieved from just above the boundary between the phosphate buffer and Histopaque, pipetted into new Eppendorf tubes with 1 mL of PBS and spinned again at 180 ×g for 5 min at 4 °C. Supernatant was removed and the lymphocytes were used for the subsequent analyses.

The comet assay was based on the method of Collins et al. [40]. Briefly, prior to the assay we prepared various concentrations of the aqueous extract from A. rusticana roots and of flavonoids. Lymphocytes placed on cold-resistant microscope slides were incubated with various concentrations of A. rusticana extract or flavonoids and covered for 30 min in wet room at 37 °C. Two samples were used as negative controls (1× PBS; 1% DMSO). Another sample was immediately treated with hydrogen peroxide (35 μM) for 5 min at 4 °C, and served as the positive control. All samples were placed in a lysis solution (pH 10.0) for 1 h at 4 °C to remove cellular membrane and cytoplasm while leaving nucleoids. After lysis, the samples were placed to electrophoretic tank with alkaline solution (pH 13.0) for 20 min at 4 °C for DNA unwinding. After unwinding, the electrophoresis was carried out under the following conditions: 30 min, 25 V, 260–320 mA at 4 °C. The samples were neutralised for 7 min in PBS (pH 7.5) and then 7 min in deionized water at 4 °C. Nucleoids were analysed by at 100× magnification using an Olympus BX51 fluorescence microscope equipped with a U-MNU2 filter and captured by the Olympus U-CMAD3 Color View Soft Imaging System. Images were analysed with the image analysis software CometScore™ (TriTec Corporation, San Diego, CA, USA). Comets were classified into five categories: 0 representing undamaged cells (comets with no or barely detectable tails) and 1–4 representing increasing relative tails intensities. Summing the scores (0–4) of 100 comets gives an overall score between 0 and 400 arbitrary units. The percentage of the DNA damage was subsequently evaluated with the image analysis software CometScoreTM (TriTec Corporation).

Isolated human lymphocytes were pre-incubated in the wet room at 37 °C in the dark with non-genotoxic concentrations of the extract from A. rusticana or flavonoids. After the pre-incubation lymphocytes were washed in the phosphate buffer (1× PBS, pH 7.5), incubated in H₂O₂ (35 μM) for 5 min at 4 °C and washed again in the phosphate buffer. Cells used for the positive control were immediately treated with in H₂O₂ for 5 min at 4 °C. Afterwards, the lymphocytes were submitted to the comet assay.

The results represent the mean of three experiments ± standard deviation. The significance of differences between means was evaluated by the Student’s t-test: * p < 0.05; ** p < 0.01; *** p < 0.001.