Interactions Between Polyphenolic Antioxidants Quercetin And Naringenin Dictate The Distinctive Redox‑related Chemical And Biological Behaviour Of Their Mixtures Part 3

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Antioxidant activity by DPPH test.

The determination of the antioxidant activity of investigated antioxidants and their mixtures was carried out by spectrophotometric assay employing DPPH radical as described previouslyl3,55. Firstly, the stock solution of DPPH radical was diluted with methanol until absorbance amounted to 0.9±0.05 at 515 nm. Secondly, antioxidants and their mixtures were diluted appropriately with 70% ethanol to achieve concentrations falling within the linear range of the assayi355. Then, the DPPH solution(1 mL) was mixed with the diluted samples（30 μL） and the absorbance was measured at 515 nm after 10 min at 37 ℃C. All reactions were carried out in 48-well plates. The absorbance measurements were performed with the aid of a TECAN Infinite M200 spectrophotometer(Tecan Group Ltd., Switzerland). The antioxidant activity of the sample was recalculated to stoichiometry coefficient ng as described previously, with modifications5. Briefly, the number of radicals scavenged by the tested samples was calculated based on the Beer-Lambert law and the molar extinction coefficient of DPPH following the measurements performed after 10 min of reaction between the antioxidant(s)solution and radical55. The value of n is calculated as a tangent of the linear relationship between the number of moles of DPPH scavenged by 1 mL of antioxidant(s) solutions within a concentration range, where the "stock" solution has the concentration"100%" and the other concentrations are a fraction of 100% as defined by the dilution factors.

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Antioxidant activity by potentiometric titration and differential pulse voltammetry.

Standard reduction potentials(E)for Q and R were measured by potentiometric titration (PT)as described elsewhere5. In short, studied compounds and the titrant(K,[Fe(CN)]) were dissolved in PBS. The concentration of purified compounds was 0.3 mg/mL, Mixtures contained 0.3 mg/mL of each compound. Measurements were carried out using JENCO 6230 N ORP-146C Micro Oxidation-Reduction equipment (USA) with the aid of an AglAgCl reference electrode (RE) and a platinum measuring electrode. PTs were performed at37±0.01°C that was mainly maintained by Ultra Thermostat (PolvScience, USA). The equal volume of titrant was added to the analyte and steady potential was read. As a result, titration curves, E=f(V..), were analyzed with the aid of SigmaPlot Version 13.0 software(Systat Software Inc., UK)by fitting the sigmoidal,5-parameters mathematical model to experimental data35. Potential at equivalence point(EP vs. RE)was read directly from this model based on the parameter a. It is equal to the volume of the titrant added at the inflection point. Finally, the values of EP versus SHE (standard reduction potential, EO) were calculated. The correction term of the potential of the RE(c) was established by titration of redox couples, FeCl.6H, O, and Na,S, O3.5H,O, characterized by known standard reduction potentials.

In turn, the antioxidant power(AOP) of studied compounds and their mixtures was measured by differential pulse voltammetry (DPV) as shown before with modifications. Briefly, measurements were carried out using the Gamry Reference 600 potentiostat (Gamry Instruments, USA)containing a three-electrode system. Glassy carbon electrode(GC,1.6 mm in diameter), platinum wire, and AgAgCl electrode (Hydromet S.C., Poland)were applied as the working (WE), the auxiliary (AE), and the reference electrode(RE), respectively13. Before experiments， the surface of the WE was polished using alumina suspension （0.05 μm particles， Buehler， USA）on micro cloth pads(MF-1040, BASi, USA)and then cleaned with distilled water and methanol. The studied compounds were diluted in DMSO and sodium phosphate buffer(pH=7.4±0.1), so the final concentration of phosphate buffer was 0.1 M in the sample. The buffer served as the supporting electrolyte. The concentration of purified compounds was 3 mM. Mixtures contained 3 mM of each compound. In order to eliminate the electrochemically reactive oxygen, the studied solutions were deoxidized by argon percolation before the measurements. DPV voltammograms for N-.N+, and mixtures: ON-and RN+were recorded in the range-0.2 to+1.3 V, while for Q and R in the range-0.2 to+0.6 V vs.RE.The potential scan rate of 0.1 V.s', pulse height of 0.05 V, and a pulse time of 0.1 s at 25±0.01 °C were set.

DPV voltammograms were analyzed by SigmaPlot Version 13.0 software (Systat Software Inc., UK). The AOP values were calculated in two steps. Calculations considered not only anodic peak potential and current(E,; I,), but also the set potential and measured current at each point of the voltammetric curve. It allowed more precise values to be obtained than those reported in our previous work3. Firstly, the parameter of antioxidant energy (AOE) was calculated according to Eq. 1:

where AwEis the surface area of the WE(equal to 0.162±0.004 cm'in our study), E is a set potential versus RE [V],is a correctional factor taking into account the presence of the liquid junction between the WE and RE (here0.103 V), I is the current measured versus the background current [A],it—is the potential sampling time [dt=0.5 s].

Secondly， AOP expressed in W cm² unit was calculated based on Eq.2∶

where t-t;—is the difference between the time of the beginning of the oxidation peak(t) and its end (t).

In order to determine Awp, cyclic voltammetry for 1.10-3M K,[Fe(CN).] solution in 0.1 M KCl was performed. This value was calculated based on Randles-Sevcik equation82 from the slope of the anodic peak current as a function of the square root of the scan rate, I, =f(pi/2). Thee was measured in the same way as in potentiometric titration. Moreover, in the present work, the thermodynamic parameter of the oxidation process was anodic peak potential corrected by the liquid junction between WE and RE (E。,=E。。+e), while kinetic parameters embraced charge transferred (Q) and anodic current (I,).

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Cell culture. In the presented study, HT29 cell line (human colon adenocarcinoma) from the ATCC was used as a model of the human intestine, The cells were maintained in McCoy's medium supplemented with antibiotics(100 U/mL streptomycins and 100 g/L penicillin) and fetal bovine serum(100 mL/L)13. The HT29 cell line was maintained at 37Cunder 5% CO, the atmosphere in a cell incubator(Heal Force)3. The cell line was employed between passages 6 and l1. Cultured cells were tested for mycoplasma contamination using Universal Mycoplasma Detection Kit from ATCC (USA).

Cytotoxicity assessment. To determine the impact of purified antioxidants(Q, N+, R, N-) and their mixtures(QN-, RN+)on HT29 cells growth, MTTtest was applied as described earlier3,5. Briefly, the exponentially growing cells were seeded in 96-well tissue culture plates (5×10 cells per well in 0.18 mL of medium)and were left to settle for 24 h at 37°Cunder 5% CO, Then, the cells were treated for 6,24 or72 h with 0.02 ml of different concentrations of the pure antioxidants or their mixtures3,5. The antioxidants and their mixtures were dissolved in ethanol (naringenin, naringin and mixtures—30% (v/v), quercetin-40%(v/v), rutin—20% (v/v)). The final concentrations of purified compounds ranged from 10 nM to 100 uM. The mixtures contained equivalent concentrations of each compound（10 nM-100 μM）. The final concentration of ethanol in culture media was 2%(v/v)in the case of rutin,3%(v/v)naringenin, naringin, and mixture as well as 4%(v/v)quercetin. After 6 and 24 h exposures, the medium was aspirated from the wells and replaced with 0.2 mL of fresh medium. The cells were incubated at 37°Cuntil 72 h of the total incubation time13,55. After72 h of incubation, to all wells 0.05 mL of MTT solution(4 g/L)was added and the cells were maintained for a further 4h at 37 ℃C13,5. Then, the medium was aspirated from wells and formazan crystals were dissolved in 0.05 mL of DMSO. The absorption of the obtained solutions was measured at 540 nm with the aid of the TECAN Infinite M200 plate reader (Tecan Group Ltd, Switzerland)15. The treatments were performed as four technical replicates. Three independent repetitions of each treatment were performed. The impact of investigated samples on HT29 cells growth was expressed as a percent of growth inhibition of cells exposed to individual antioxidants and their mixtures compared to control cells treated with the solvent only, whose growth was regarded as 100%13,55

CAA (cellular antioxidant activity) assay. CAA assay (The OxiSelect Cell Biolabs, Inc. USA) was used to evaluate the cellular antioxidant activity of compounds alone(O, N+, R, N-) and mixtures(ON-, RN+) in HT29 cells as described earlier35. The exponentially growing cells were seeded in 96-well tissue culture black plates with transparent bottoms for fluorescence measurements (3×104 cells per well in 0.2 ml of medium) and were left to settle for 24 h at 37°Cunder 5% CO,i3.55. All antioxidants were dissolved in 10% ethanol. The cells were then treated with 500 times diluted solution of 2',7'-dichlorofluorescein diacetate (0.05 mL)provided with the kit, and the same volume of different concentrations of antioxidant samples for 1, 3, or 6 h. The final concentrations of purified compounds ranged from 1 to 100 uM. The mixtures contained equivalent concentrations of each compound(1-100uM). The control cells were treated with 10%ethanol only (v/v). The final concentration of ethanol in culture media was 5%(v/v). All treatments were carried out in three technical replicates and three independent experiments were performed. Subsequent steps were carried out according to the manufacturer's recommendations(https://www.cellbiolabs.com). Calculations were performed as described earlier, Genotoxic effects. To determine the genotoxic effects exhibited by individual antioxidants (O, N+, R.N-)and their mixtures(ON-, RN+) in HT29 cells, a comet assay procedure was applied as described previously. The exponentially growing HT29 cells were seeded in 24-well tissue culture plates(10° cells per well in 1.8 mL of medium)and were allowed to settle for 24 h at 37°C under 5%CO,55, Then, the cells were treated for 24h with 0.2 mL of different concentrations of the antioxidants alone or in mixtures. The final concentrations of purified compounds ranged from 1 to 100 uM. The mixtures contained equivalent concentrations of each compound (1-100 uM). The final ethanol concentration in the culture medium was 3%(v/v). The cells used as negative controls were treated with solvent only. After treatment time, the medium was aspirated from the wells and the cells were washed with 0.5 mL PBS. The cells were then detached using 0.2 mL of trypsin solution (0.5 g/L)55 The activity of trypsin was halted by adding 1.8 mL of complete growth medium to each well. The cells were re-suspended， counted, and aliquoted into 1.5 mL tubes（30×103³cells per tube）. The cell suspension was centrifuged(100×g,5 min,4°C). The cell pellets were washed with l mL of PBS and centrifuged again(100×g,5 min, 4°C）5. Then， PBS was discarded and the cells were re-suspended in 150 μL of 0.5% LMP agarose in water pre-warmed to 42℃C and 40 μL of this mixture was placed as two spots on a microscope slide pre-coated with 1%normal melting point agarose (NMP agarose). The slides were covered with coverslips. The agarose was allowed to be set by placing the microscope slides on an ice-cold tray for at least 5 min5. Three slides with two repetitions on each were prepared for every concentration of the tested substances. After overnight lysis in a high salt alkaline buffer (2.5 M NaCl,0.1 M EDTA,0.01 M Tris,1% Triton X100,pH10), the slides were accommodated into a Bio-Rad Sub-Cell GT electrophoresis platform (UK), covered with cold electrophoresis buffer(0.3 M NaOH, 1 mM EDTA, pH 13)and chromatin was allowed to unwind for 25 min before electrophoresis. Electrophoresis was conducted at 26 V and 300 mA(0.75 V/cm) for 30 min in darkness at 4-8°C. After this step, the slides were washed firstly using PBS and then water. Subsequently, the DNA was stained with SybrGreen in TE buffer(0.1 M Trizma-Base,1 mM EDTA, pH 7.5)for 20 min~. After staining, the slides were washed with distilled water for 5 min. DNA"comets" were analyzed under a fluorescence microscope(Zeiss ImagerZ2, USA) coupled with a computerized slide scanning system(Metafer4, Germany). Comet analysis involved counting 200 consecutive nuclei per sample5. The genotoxicity of analyzed samples was expressed as the %DNA in the comet tail. Three independent replicates of each treatment were performed.

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Global DNA methylation.

For the determination of global methylation of DNA, a modified comet assay procedure was developed. Methylation sensitive comet assay was performed according to Wentzel's procedure with significant modifications. The cells were seeded in 6-well tissue culture plates(4×105 cells per well in 3.6 mL of medium) and were allowed to settle for 24 h at 37°C under 5%CO,Then, the cells were treated with 0.4 mL, of different concentrations of the tested antioxidants or their mixtures for 24 h at 37C. The final concentrations of individual compounds and solvents were the same as used for genotoxic effect assessment. After incubation time, the medium was aspirated from the wells and the cells were washed with 2 mL of PBS. The cells were detached using 0.4 mL of trypsin solution (0.5 g/L). Then,3.6 mL of medium was added to each well. The cells were re-suspended, counted, and aliquoted into 1.5 mL tubes(30×10 cells per tube). The cell suspension was centrifuged (100×g,5 min, 4°C). The cell pellets were washed with 1 mL of PBS and centrifuged again(100×g 5 min，4℃C）.PBS was discarded and the cell pellet was re-suspended in 100μL of 1%LMP agarose in water pre-warmed to 45 °C. Then，40 μL of this mixture was placed as two spots on a microscope slide pre-coated with 1%normal melting point agarose(NMP agarose)5. The slides were then covered with coverslips and left to set on an ice-cold tray for at least 5 min to solidify agarose5. Each treatment embraced a set of 3 microscope slides. After overnight lysis in a high salt alkaline buffer(2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris,1% Triton X100, pH 10), the slides were washed twice with water>5. Tightly packed chromatin was unwound by treating the nuclei with 1.5 mM proteinase K solution (0.2 mL per slide)3. The slides were covered with parafilm, placed into a plastic container lined with a damp tissue, left for 10 min at 37 °C, then washed with water3. In this way, nucleoids were prepared for digestion with restriction endonucleases(Hall and MspI). Both enzymes recognize the same restriction sequence(5'-CCGG-3') but show different sensitivity towards methylated cytosine. HpalI is presumed to digest only non-methylated sequences. MspI can cleave non-methylated sequences as well as fully methylated sequences. Thus, the relative levels of DNA methylation of the CpG sequence are reflected as the difference between the global amount of DNA within the comet tail observed with MspI digestion and HpalI digested nucleoids. To create appropriate conditions for enzymatic digestion.0.2 mL of Tango buffer diluted with molecular grade water in ratio 1:9(v/v) was applied onto each slide in the set. The slides were covered with parafilm, accommodated into a plastic container lined with a damp tissue, and left for 10 min at 37 ℃Ce. Then, the excess of the buffer was removed. Each of the three slides in the set was treated differently. Onto the first (control)slide, only 0.15 mL of diluted Tango buffer was applied. The agarose embedded nuclei on the second slide were treated with 0.15 mL of Hall enzyme （0.37 μunits）， while onto the third slide the same volume of MspI was added （0.26 μunits）. The enzyme solutions were prepared using diluted Tango buffer. The slides were covered with a parafilm and accommodated into a damp plastic container. The enzymatic digestion was carried out for 45 min at 37 °C. After digestion, the slides were twice washed with water. Further steps of the comet assay, such as electrophoresis and DNA staining, were performed as described in the section "Global DNA methylation". DNA "comets" were analyzed under a fluorescence microscope(Zeiss ImagerZ2, USA) coupled with a computerized slide scanning system (Metafer4, Germany). Comet analysis was performed with the aid of Comet score software (USA)and involved counting of 100 nuclei per sample. The mean %DNA in the comet tail was a measure of DNA fragmentation. To calculate the global DNA methylation(%CpG methylation), the following equation was used:%CpG methylation=100—Hpal/MspI×100, where Hpal/MspI is the ratio of the DNA percentage in the comet tail of nucleoid digested with HpalII and the DNA percentage in the comet tail of nucleoid digested with Mspl85. DNA damage artifacts were accounted for by subtracting the DNA percentage in the tail of the control sample from the values obtained for the samples digested with restriction enzymes. Three independent repetitions of each experiment were performed.

Microarray analysis.

Genomic analysis has been performed as shown beforei355, HT29 cells were seeded in 24-well tissue culture plates (105 cells per well in 1.8 mL of medium) and were allowed to settle for 24 h at 37°C under 5% CO, Then, the cells were treated for 24 h with 0.2 mL of different concentrations of the antioxidants alone(Q, N-)or in the mixture(QN-). The final concentrations of investigated compounds ranged from 1 to 10 uM. The mixture contained equivalent concentrations of each compound. The cells used as negative controls were treated with the solvent only. The final concentration of ethanol in culture media was 3%(v/y).Isolation of RNA. reverse transcription and real-time PCR of array consisting of 84 genes involved in antioxidant response as well as data analysis were performed as described earlier. Three independent repetitions of each treatment of cells were carried out.

Statistical analysis.

All values are expressed as mean±SD of three independent experiments unless stated otherwise. The statistical significance of determinations of antioxidant activity in a cell-free system using DPV and DPPH assay as well as in cellular models obtained by CAA test were examined by unpaired Tukey's test (p≤0.05). The results of genotoxicity and global DNA methylation analyzed by comet assays were examined by one-way ANOVA with Dunnett's post-hoc test. These statistical analyses were performed using the Prism 4.0 software package(GraphPad Software, Inc., USA). The statistical significance of changes in gene expression between samples and controls was also evaluated by unpaired Student's t-test for each gene of interest using GenGlobe Data Analysis Center(Qiagen, USA). The level of statistical significance was set atp≤0.05.

This article is extracted from www.nature.com/scientificreports