Plants have long been considered as a valuable source of compounds beneficial for humans. Many compounds that are found in plants exhibit a biological activity such as antimicrobial, antioxidative, antiviral, antiinflammatory, antimutagenic and anticarcinogenic -. Polyphenolics in medicinal and fragrant plants, fruit and vegetables belong to such compounds. Their broad protective effect consists in a number of individual complementary actions contributing to the upbuilding and maintenance of homeostasis.
These compounds can help prevent harmful consequences of oxidative damage to biomolecules due to their biological activity and their action play important roles in prevention of various diseases such as cancer or cardiovascular and neurodegenerative diseases -. The antioxidative activity of polyphenolic compounds is given by the ability to bind reactive forms of oxygen and transition metal ions that catalyze formation of free radicals and, also, to inhibit enzymes involved in formation of free radicals -.
Vitis vinifera L. is a species of Vitis, its production is widespread throughout the world for fresh consumption and industrial processing . This plant has been used for the production of food and beverage as well as a remedy against various complaints in traditional medicine . The varied chemical composition of Vitis vinifera grapes appears to contribute to their biological potential. The grapes are rich in polyphenolic compounds such as anthocyanins, flavanols, flavonols, proanthocyanidins, stilbenes (resveratrol), and phenolic acids -. The VinOserae extract contains phenolic compounds such as oligomeric proanthocyanidins and trans-resveratrol. Phenolic compounds are important phytochemicals in grape because they possess many biological activities and health-promoting benefits -.
The above findings led us to investigate the antimutagenic and DNA protective abilities of the grape extract VinOserae from Vitis vinifera applicable as a promising supplement with effective biological potential. In this work, in order to detect a protective effect of the VinOserae extract that prevents induction of oxidative DNA damages, a molecular biology method based on detection of changes in the topological state of the pBluescript II SK(-) plasmid was used. A different electrophoretic mobility of topological DNA forms was measured by the agarose gel electrophoresis. In the plasmid DNA test, the protective effect of the extract was determined on the basis of its capability to inhibit formation of DNA strand breaks in the plasmid DNA induced by hydrogen peroxide in the presence of transition metal ions -.
The protective effect of the extract was independently evaluated with the Ames test using Salmonella typhimurium His− TA102 suitable to detect mutagenic effects of oxidative compounds. The evaluation of antimutagenic effects of the extract included detection of the ability to inhibit a mutagenic effect induced by hydrogen peroxide or tert-butyl hydroperoxide. These in vitro tests are useful to screen the antigenotoxic potential of plant extracts and to search potential bioactive compounds for possible aplication in food industry -. To the best of our knowledge this is the first report on DNA strand scission inhibitory and antimutagenic activities of the VinOserae extract.
2. Material and Methods
2.1. Chemicals and Material
The chemical reagents D-glucose-6-phosphate sodium salt, hydrogen peroxide, L-histidine, nicotinamide adenine dinucleotide phosphate and tert-butyl hydroperoxide were purchased from Sigma-Aldrich (St. Louis, USA), agarose and ethidium bromide from Serva Electrophoresis (Heidelberg, Germany). All reagents used were of analytical grade. The dry VinOserae extract was obtained from Favea, spol. s r.o. (Kopřivnice, Czech Republic). In the extract, phenolic compounds such as oligomeric proanthocyanidins and trans-resveratrol were identified.
2.2. Bacterial Strain and Plasmid DNA
Plasmid pBluescript II SK(-) DNA was isolated from the bacterial strain Escherichia coli TOP10. The isolation of plasmid pBluescript II SK(-) was carried out using columns Qiagen (QIAGEN Plasmid Mini Kit). The obtained plasmid DNA was dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and stored at −20˚C. Detection of mutagenic and antimutagenic activities of the plant extract was investigated using the auxotrophic bacterial strain Salmonella typhimurium His‒ TA102. For each experiment, a frozen stock of the tester strain culture (stored at −80˚C) was grown overnight in Oxoid nutrient broth No. 2 using the procedure described in .
2.3. Antimutagenicity Test
The Ames test standard plate incorporation procedure was used for the assessment of antimutagenicity of the VinOserae extract. In order to detect the effect of the extract on tert-butyl hydroperoxide (t-BHP) or hydrogen peroxide (H2O2), an induced mutagenicity assay was employed using the Salmonella typhimurium His‒ TA102 strain sensitive to oxidant mutagens  . A volume of 100 µl of a bacterial culture (prepared as described in ), 50 µl of the test sample (50 - 300 µg/plate), 50 µl of a mutagen (t-BHP or H2O2, 100 µg/plate) and 500 µl of phosphate buffer (0.1 mM, pH 7.4) were added to 2 ml of the top agar that was supplemented with 0.05 mM histidine. The mixture was poured on minimal agar plates that were subsequently incubated at 37˚C for 48 h and then histidine-revertant colonies on each plate were counted. The number of induced revertants was obtained by subtracting the number of spontaneous revertants from the number of revertants on the plates containing mutagen and antioxidant. The inhibition rate of mutagenicity (%) was calculated as follows: percent inhibiton (%) = [1 − (number of revertants on plates with oxidant and test compound − number of spontaneous revertants)/(number of revertants on plates with oxidant alone − number of spontaneous revertants)] × 100 . The results were analyzed with the statistical software “R”.
2.4. DNA Strand Scission Assay
The plasmid pBluescript II SK(-) DNA was used to study the protective effect of the VinOserae extract on DNA damage induced by hydroxyl radicals. The method using plasmid DNA is based on different electrophoretic mobility of topological forms of the plasmid DNA. DNA strand breaks were measured by a conversion of supercoiled (Sc) form to open circular (Oc) and linear (Lin) forms of DNA  . Plasmid DNA (0.5 µg) was incubated with various concentrations of the extract (0.02 - 0.4 µg/µl), 0.1 M Tris-HCl (pH 8.0), 0.1 mM FeSO4, and 0.3% H2O2 in an Eppendorf tube (37˚C, 30 min). The final volume of the mixture was 10 µl. DNA conformation changes were analyzed by agarose gel electrophoresis in 1xTAE buffer (1% agarose, 7 V/cm, 80 min, room temperature). The gels were stained with ethidium bromide for 30 min, followed by 30 min destaining in water. Photos of the gels were taken in UV light (transilluminator GeneGenius, SynGene, Cambridge, UK) and the bands were quantified using the software ImageJ .
3. Results and Discussion
Detection of the antimutagenic activity of the VinOserae extract was carried out using the Ames test with Salmonella typhimurium His‒ TA102. The antimutagenic effect of compounds can be evaluated on the basis of their ability to inhibit mutagen’s mutagenicity -. The indicator strain Salmonella typhimurium His‒ TA102 is used for detection of mutagens inducing substitution mutations, often in the case of compounds with oxidative effect -. In order to measure an antimutagenic effect of the extract, both H2O2 and tert-butyl hydroperoxide (t-BHP) were used at a concentration of 100 µg per plate to ensure a sufficient amount of revertants compared to the negative control  . The VinOserae extract was tested at a range of 50 - 300 µg sample per plate. The results showed that a significant antimutagenic effect of the extract was detected with both oxidative mutagens used (Figure 1). The maximal decrease in the revertant numbers was found when the
Figure 1. Antimutagenic activity of VinOserae extract in Ames test (without metabolic activation, strain TA102). Mutations were induced by t-BHP or H2O2 (100 µg/plate). The amount of spontaneous revertants is subtracted. Line ▬▬: t-BHP; line ――: H2O2; line ˗ ˗ ˗ ˗: standard error
highest concentration of the extract was used with the two mutagens. The respective percentages of the inhibition of mutagenicity with t-BHP and H2O2 when a concentration of 300 µg of the VinOserae extract per plate was used were 44.2 and 67.0% (Table 1).
Mutagenicity of the extract was examined as described in , we found no mutagenic effect irrespective of metabolic activation (not shown).
The ability of the VinOserae extract to inhibit oxidative damage of DNA was further investigated using the DNA strand scission assay where the protective effect of the extract was evaluated on the basis of its capability to inhibit formation of strand breaks in the plasmid pBluescript II SK(-) DNA. The principle of detection includes electrophoretic evaluation of changes in the topological state of the plasmid DNA. In its native form the double strand DNA of the plasmid pBluescript II SK(-) is in a compact supercoiled (Sc) conformation and has a relatively high electrophoretic mobility (Figure 2, lane 1). After a single strand break is introduced in the DNA, the supercoiled tertiary structure is impaired and results in the formation of an open circular (Oc) DNA form with a decreased electrophoretic mobility in the agarose gel. The linear (Lin) DNA form whose formation is induced by double strand breaks has a medium electrophoretic mobility between those of the supercoiled (Sc) and open circular (Oc) forms of plasmid DNA -.
When the protective effect of the VinOserae extract was tested using plasmid DNA, the sample was not dissolved in dimethylsulfoxide but in ethanol. Some studies indicate that dimethylsulfoxide effectively inhibits formation of DNA strand breaks induced by the effect of hydroxyl radicals  . Figure 2 shows how the extract affected the formation of DNA strand breaks induced by H2O2 in the presence of FeSO4 (Oc and Lin forms of DNA, lane 3). The sample was tested in a concentration range of 0.02 - 0.4 µg/µl. When the smallest dose of 0.02 µg/µl was applied, no inhibition of the formation of the open circular (Oc) and linear (Lin) DNA forms was observed (lane 4). The protective effect was observed when a dose of 0.05 µg/µl was used (lane 5). The strongest effect of inhibition of DNA strand breaks formation was found in lanes 7 - 10 when 0.15 - 0.4 µg/µl sample was applied. The protective effect measured probably reflected the presence of these compounds in the extract since the ability to inhibit damage of plasmid DNA had been proven for many polyphenolic compounds   .
Figure 3 shows results of quantitative measurement of the protective activity of the VinOserae extract capable to inhibit formation of DNA strand breaks in the plasmid pBluescript II SK(-). In the case of any extract concentration tested, a quantitative measurement of the content of supercoiled (Sc) DNA form was made using software ImageJ . The value obtained for the supercoiled DNA form was related to that of the control. In a concentration range of 0.05 - 0.4 µg/µl used an increasing rate of the protective activity of the extract was found,
Table 1. Antimutagenic effect of VinOserae extract measured with Ames test using Salmonella typhimurium His‒ TA102 with t-BHP or H2O2 as the mutagenic compounds.
aThe numbers represent a percentage (%) of mutagenicity inhibition.
Figure 2. Electrophoretic pattern of pBluescript II SK(-) plasmid where DNA strand breaks were induced by 0.3% H2O2 and 0.1 mM FeSO4 in the presence of VinOserae extract. Lane 1: control DNA; lane 2: H2O2 treatment alone; lane 3: H2O2 and FeSO4; lanes 4-10: H2O2 and FeSO4 in the presence of the extract (4: 0.02 µg/µl; 5: 0.05 µg/µl; 6: 0.1 µg/µl; 7: 0.15 µg/µl; 8: 0.2 µg/µl; 9: 0.3 µg/µl; 10: 0.4 µg/µl).
Figure 3. Analysis of supercoiled form of the plasmid pBluescript II SK(-) after DNA damage by •OH in the presence of VinOserae extract (0.02 - 0.4 µg/µl).
expressed by per cent value of the presence of supercoiled (Sc) form of the plasmid pBluescript II SK(-). The experiments showed no potential genotoxicity of the extract when applied to the plasmid pBluescript II SK(-).
Using DNA strand scission assay and the Ames test with Salmonella typhimurium His‒, an ability of the VinOserae extract from Vitis vinifera grapes to efficiently inhibit oxidative damage of DNA was documented. No potential genotoxicity of the extract was detected. The results extend the knowledge of beneficial biological effects of compounds isolated from natural raw materials and bring further evidence of the VinOserae extract to be an interesting source of compounds with protective biological effect and applicability at food industry.
The work was supported by the project CZ.1.05/2.1.00/03.0100 (IET) financed by the Structural Funds of the Europe Union and project LO1208 of the National Feasibility Programme I of the Czech Republic.