Yellow gentian root extract provokes concentration- and time-dependent response in peripheral blood mononuclear cells

Yellow gentian root (serial number 03970219) was purchased from the Institute of Medicinal Plant Research “Dr Josif Pančić”, Belgrade, Serbia (CAC/RCP 1-1969, rev.4-2003). Before extraction, the root was ground with a hand grinder. In order to optimise and standardise the extraction technique, ground particles were imaged after each stage of extract preparation, namely powder, lyophilisate, and dissolved lyophilisate (Figure 1), with a field emission scanning electron microscope (FESEM) with energy dispersive spectroscope (EDS) (FEI SCIOS 2, Thermo Fisher Scientific FEI Company, Tokyo, Japan) at 350x magnification and their size determined with SemAfore 5.2 software (Insinööritoimisto J. Rimppi Oy, Ojakkala, Finland).

Figure 1

The obtained aqueous extract was placed in 25 mL acrylic chambers, transferred in the freezer at -18 °C and allowed to freeze for 24 h. Pre-frozen supernatant was then vacuum freeze-dried under vacuum pressure of 400 Pa for 48 h.

Before dissolving, lyophilised powder was kept in a desiccator at 4 °C and then dissolved in 50 % ethanol to obtain the concentration of 50 mg/mL. Particle size measurements showed that lyophilised particle aggregates consisted of soluble compounds. Dissolved lyophilisate was filtered through a 0.2 μm pore diameter Minisart^® filter (Sartorius, Göttingen, Germany) in order to eliminate the remaining non-soluble particles and sterilise the extract solution. The filtrate was to determine the concentration of secoiridoids in the extract. The final extract concentrations in cell cultures were 0.5, 1, and 2 mg/mL.

Chromatographic separations of YGRE constituents were done by ACQUITY UPLC system (Malvern Panalytical Ltd, Malvern, UK) with a photodiode array (PDA) detector and a LUNA 3u, C18(2), 3 μm, 100 mm × 2 mm Phenomenex column as a stationary phase (Phenomenex, Torrance, CA, USA). All analyses were done under the gradient condition (Table 1), with a mobile phase consisting of solvent A (0.1 wt % HCOOH in water) and solvent B (0.1 wt % HCOOH in methanol) at a constant flow rate of 0.3 mL/min. Autosampler was maintained at 4 °C. 3D chromatograms were recorded in the wavelength range of 210–450 nm. The run time was 6 min, and the injection volume 3.5 μL.

Peripheral blood samples were collected in Li-heparin vacutainers (2 x 10 mL) from three healthy donors aged 20 to 40 years. All subjects signed informed consent, and the study was approved by the Ethics Committee of the Vinča Institute of Nuclear Sciences – National Institute of the Republic of Serbia.

Peripheral blood mononuclear cells (PBMC) were isolated in Ficoll™ medium (Thermo Fisher Scientific, Waltham, MA, USA) and resuspended in RPMI 1640 medium supplemented with 1 % penicillin–streptomycin and 10 % foetal bovine serum (FBS) at a concentration of 1×10⁶ viable cells/mL. The obtained cell cultures were then incubated with root extract at 37 °C for 48 and 72 h.

Trypan blue was used to evaluate cell viability as described elsewhere (17). Briefly, equal volumes of PBMC suspension and 0.4 % Trypan blue dye were mixed and applied to a haemocytometer (Cambridge Instruments Inc., Buffalo, NY, USA). Live, unstained cells were counted in four sets of 16 squares. Viable cell counts per mL represent the mean of each set, multiplied by 10⁴ and by 2 as dye dilution correction factor.

To establish cell cultures, 0.5 mL of heparinised whole blood was added in RPMI 1640 medium supplemented with 1 % penicillin–streptomycin, 10 % FBS, and phytohaemagglutinin and treated with YGRE to obtain final concentrations of 0.5, 1, and 2 mg/mL. All chemicals used for cell culture were purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). Harvesting was performed 48 and 72 h after culture initiation. Three hours before harvesting, colchicine (Sigma-Aldrich Co., St. Louis, MO, USA) was added (in the final concentration of 2.5 μg/mL) to collect metaphase cells. Cell cultures were then centrifuged and treated with hypotonic solution (0.56 % KCl) at 37 °C for 20 min. The supernatant was removed and cells fixed in Carnoy’s fixative solution (3:1 methanol-to-glacial acetic acid ratio), washed three times in fixative, and pipetted on clean microscopic slides. Slides were dehydrated with increasing concentrations of ethanol (70, 95, and 100 %), stained with 4′,6′- diamidino-2-phenylindole (DAPI)-containing Vectashield solution (Vector Laboratories Ltd, Peterborough, UK), and analysed under a Zeiss-Axioimager A1 microscope (Carl Zeiss, Jena, Germany), with the ISIS imaging software package (MetaSystems Hard & Software GmbH, Altlussheim, Germany). At least 200 complete metaphase spreads per sample were analysed, whereas the scoring criteria included determination of karyotype and indicators of chromosome damage (chromosome breaks, dicentric and ring chromosomes, acentric fragments, and radial figures) (18, 19).

The alkaline comet assay was an adaptation of the method described by Singh et al. (20). Briefly, PBMC treated for 48 h were washed in phosphate buffer saline (PBS), resuspended in 1 % low melting point agarose (LMPA) in PBS at 37 °C, and placed on microscopic slides precoated with a thin layer of 1 % normal melting point agarose (NMPA). Slides were allowed to settle at 4 °C for 10 min and then immersed in a lysis solution at 4 °C for 1 h. Electrophoresis was performed at the same temperature and voltage of 25 V (1 V/cm, 300 mA) for 20 min. Slides were then washed three times in 0.4 mol/L Tris-HCl, pH 7.5, at 4 °C, air dried, and counterstained with a DAPI-containing Vectashield solution. At least 300 cells were evaluated for each slide under a Zeiss-Axioimager A2 microscope with automated Metafer CometScan software (MetaSystems). The results are presented as the percentage of DNA in the comet tail (% DNA).

The assay was done as described by Alamdari et al. (21), with minor modifications. This method is based on two concomitant oxidation-reduction reactions in the same sample. In the first reaction, catalysed by horse radish peroxidase (HRP), chromogen 3,3’,5,5’-tetramethylbenzidine (TMB) is oxidised to a coloured cation. In the second reaction, this coloured cation is reduced by antioxidants to a colourless compound.

Standard solution was prepared by mixing different proportions (0–100 %) of 1 mmol/L H₂O₂ with 6 mmol/L uric acid in 10 mmol/L NaOH. To prepare solution of TMB cation, a mixture of 0.1 mL TMB/DMSO (6 mg/mL), 5 mL acetate buffer (0.05 mmol/L, pH 4.5) and 17.5 μL freshly made chloramine T (100 mmol/L), was mixed, shacked for 1 h at 37 ºC, and incubated in a dark place. Then 2.5 U of enzyme solution HRP was added to this mixture. TMB solution II was made of 0.1 mL TMB/DMSO (6 mg/mL) and 5 mL acetate buffer (0.05 mol/L, pH 5.6).

In each well of reaction plate, 10 μL of sample/standard/blank was mixed with 180 μL of working solution, freshly made from 0.5 mL of TMB cation and 5 mL TMB solution II that were mixed for 6 min at room temperature in the dark. Then, the plate was incubated for 12 min at 37 °C, in a dark place. The reaction was stopped by adding 40 μL of 2 mol/L HCl to each well.

The difference between these parallel reactions was measured as optical density (OD) at 450 nm wavelength, with a reference wavelength of 570 nm on absorbance microplate reader Sunrise (Tecan Group Ltd, Männedorf, Switzerland). The PAB values were calculated from the standard curve that represents the percentage of hydrogen peroxide in the standard solution and were expressed in arbitrary Hamidi-Koliakos (HK) units.

Lipid peroxidation product (LPP) assay used in this study is based on the reaction of chromogen N-methyl-2-phenylindole with products of lipid peroxidation, namely malondialdehyde (MDA) and 4-hydroxyalkenals (HNE) (22, 23).

After treatment, cells were washed and lysed in 0.9 % NaCl in freeze-thaw cycles. An aliquot of the samples was used for protein determination. The reaction mixture contained 140 μL of sample/standard/blank, 455 μL of reagent diluted in acetonitrile/methanol (3:1) to a final concentration of 10 mmol/L, and 105 μL of methanesulphonic acid containing 34 μmol/L Fe(III). It was added to microcentrifuge tubes, vortexed, and incubated at 45 °C for 60 min. Samples were centrifuged at 15000xg for 10 min to obtain a clear supernatant.

The product of this reaction is a stabile chromophore, whose OD value was measured at 586 nm on the Sunrise absorbance microplate reader (Tecan). The values are presented as nmol of LPP per mg of protein using a standard curve of 1,1,3,3-tetramethoxypropane.

All experiments were done in duplicate and repeated three times. The results are presented as means ± standard error (SE). ANOVA test and Pearson correlation coefficient were used for statistical analysis. The level of significance was set at p<0.05. All statistical analyses were run on the SPSS 10 for Windows (IBM, Armonk, NY, USA).

Analysis of FESEM photomicrograph showed that the particle size in dispersed root powder was 53.9±2.95 μm (Figure 1 a). After filtration and lyophilisation, the particles displayed laminar or plate-like structure: smaller particles formed aggregates, whose average dimension was 68.4±3.6 μm (Figure 1 b). The aggregates were mostly composed of soluble compounds, since particles observed in the lyophilisate dissolved in 50 % ethanol (concentration of 50 mg/mL) were of a much smaller dimension – 15.8±1.3 μm (Figure 1 c).

Analytical standards of selected compounds were used to identify some of the most intensive peaks in chromatogram. Detailed data are presented in the report by Valenta Šobot et al. (24). Figure 2 shows UPLC chromatograms of YGRE ethanolic solution exported from 3D chromatograms at three wavelengths, revealing a high content of gentiopicroside and high stability of other compounds during treatment.

Figure 2

Figure 3a shows results of cell viability assay. All treatments reduced cell viability to a certain extent ranging from mild (91.84 % for 0.5 mg/mL, 48 h treatment) to more noticeable (59.54 % for 2 mg/mL, 72 h treatment, p<0.001). Cell viability decreases with increasing YGRE concentration, at both exposure times. Statistically significant reduction was observed for concentrations of 1 and 2 mg/mL for both 48 (p<0.05 and p<0.01, respectively) and 72 h treatment (p<0.01 and p<0.001, respectively, Figure 3a).

Figure 3

The results of chromosome aberrations assay showed that incidence of chromosomal breaks increased with the increase of YGRE concentration after 48 h of treatment, from 0.8 % (0.5 mg/mL) to 8.11 % (2 mg/mL) (Figure 3 b). Chromosome aberrations were also analysed after 72 h to evaluate the impact of prolonged incubation on DNA damage repair. The trend of increasing frequency of breaks was noticed after 72 h treatment for concentrations of 1 and 2 mg/mL (0.93 and 5.1 %, respectively), however the percentage of breaks was reduced comparing to 48 h treatment (Figure 3). The concentration of 0.5 mg/mL did not induce chromosomal breaks after 72 h. Figure 4 shows the appearance radial figures between homologous chromosomes at 2 mg/mL of YGRE, which are indicators of double-strand break (DSB) repair.

Figure 4

As expected, the percentage of chromosome breaks inversely correlated with viability assay for both treatment times (p<0.01, r=-0.67 for 48 h treatment and p<0.01, r=-0.89 for 72 h treatment).

All tested YGRE concentrations increased the percentage of DNA in the comet tail compared to control after 48 h of treatment and the difference was significant for the two higher concentrations (p<0.001) (Figure 5). Again, as expected, the parameter percentage of DNA in the comet tail inversely correlated with cell viability (p<0.01, r=-0.797).

Figure 5

The levels of PAB and LPP were the highest (67.9 arbitrary HK units and 1.79 nmol/mg of proteins, respectively) and statistically significant for the lowest concentration of 0.5 mg/mL (p<0.001), while they dropped toward control levels at higher concentrations (Figure 6). PAB and LPP positively correlated (p<0.01, r=0.894).

Figure 6