Antimicrobial activity and protective effect of Geranium thunbergii against oxidative DNA damage via antioxidant effect

The G. thunbergii was purchased from Omniherb of Gyeongsan (Gyeongbuk province, Korea). The samples were pulverized to 80 mesh size using Sung Chang Machine (ACM10INCH, Namyangju, Korea) and kept at -20℃ until assayed. Ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu’s phenol reagent, sodium carbonate, dimethyl sulfoxide (DMSO), potassium ferricyanide, butylated hydroxy anisole (BHA), trolox, trichloroacetic acid, ferric chloride and potassium persulfate were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2,2-Azino-bis (3-ethyl benzthiazoline- 6-sulfonic acid) (ABTS) was purchased from Wacko chemical (Tokyo, Japan). All other reagents and chemicals used in this study were analytical grade.

The dried sample (100 g) was extracted with 1.0 L of 95% methanol overnight at room temperature for 2 days. The methanol extract was then filtered through filter paper Advantec 5C (Advantec, Toyo Roshi Kaisha Ltd., Tokyo, Japan) and by vacuum rotary evaporator (BÜCHI Rotavapor R-210, Flawil, Switzerland). The methanol extract was partitioned with different solvents (H; n-hexane C; chloroform E; ethyl acetate B; butanol, W; water). Five fractions were completely evaporated and dried and then their yields were calculated. The yield (g/dry) of solvent-partitioned fractions was hexane fraction (1.21 g), chloroform fraction (0.36 g), ethyl acetate fraction (0.58 g), butanol fraction (1.52 g), and water fraction (4.15 g), respectively. The solvent-partitioned fractions were stored at -20℃ prior to use.

The TPC of the extract was measured using the Folin-Denis method (19). 0.5 mL of Folin Ciocalteu’s phenol reagent was added to 0.5 mL sample and the reaction mixture incubated for 3 min. After this, 0.5 mL of Na₂CO₃ (10%, w/v) was added and further incubated at room temperature (25℃) for 60 min. Absorbance of the reaction mixture was then measured at 760 nm using spectrophotometer (Jasco, Tokyo, Japan). TPC was expressed in terms of mg gallic acid equivalents (GAE)/g.

The antioxidant activity of G. thunbergii was measured using DPPH radical scavenging assay (20). Fifty microliters of the sample was added to 100 μL of 0.2 mM DPPH solvent, vortex-mixed, and allowed to stand at room temperature for 10 min. Absorbance was measured at 517 nm. All determinations were carried out in triplicate. BHA was used as a positive control and the capability of scavenging DPPH radicals was calculated using the following equation:

${\text{Radical scavenging activity=[1-(A}}_{\text{sample}}{\text{/A}}_{\text{control}}\text{)]×100}$

ABTS radical cation decolorization assay was performed using the Re method (21). 7.0 mM 2,2-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) was added to 2.45 mM potassium-persulfate and the mixture was incubated at room temperature in the dark for 24 h. Absorbance of the ABTS radical cation solution was measured at 734 nm using ethanol. Fifty microliters of the sample was added to 100 μL ABTS radical cation solution and the reaction mixture was incubated at room temperature for 10 min. Its absorbance was measured at 734 nm. All determinations were carried out in triplicate. Trolox was used as a positive control and the capability scavenging ABTS radicals was calculated using the following equation:

${\text{Radical scavenging activity=[1-(A}}_{\text{sample}}{\text{/A}}_{\text{control}}\text{)]×100}$

Reducing power assay was performed using the Oyaizu method (22). One microliters of the sample was added to 1 mL of potassium ferricyanide (1%, w/v) and the reaction mixture was incubated in water bath at 50℃ for 20 min. And then 1 mL of trichloroacetic acid (10%, w/v) was added. 1 mL of this mixture was added with 1 mL of distilled water and 0.1 mL of ferric chloride (0.1%, w/v). Absorbance was measured at 700 nm.

Conversion of the supercoiled form of plasmid DNA to the open-circular and linear forms has been used as index of DNA damage. Reaction mixtures (20 μL) contained 5 μL of ΦΧ-174 RF I plasmid DNA, 10 μL of varying concentrations of the ethyl acetate fraction, 5 μL of 1 mM FeSO₄ or 1 mM hydrogen peroxide were incubated at 37℃ for 30 min. After 30 min, 5 μL of a solution containing 50% glycerol (v/v), 40 mM EDTA and 0.05% bromophenol blue was added to stop the reaction. The mixtures were electrophoresed on 1% agarose gel (w/v). DNA in the gel was visualized and photographed under ultraviolet light after ethidium bromide staining.

To determine antimicrobial activity, Bacillus subtilis (KCTC 1666), Bacillus cereus (KCTC 1012), Staphylococcus epidemidis (KCTC 1917), Staphylococcus aureus (KCTC 1916), Listeria monocytogenes (KCTC 3569), Escherichia coli (KCTC2643) were used. These microorganisms were kept frozen at -80℃ in broth containing glycerol (40%, v/v). These cultures were grown in liquid and solid media at optimal temperature of microorganisms, respectively. All media were purchased from Difco Co. (Sparks, MD, USA).

Antimicrobial activity of solvent-partitioned fraction was performed using the paper disc method (15). The fractions were dissolved in DMSO. The culture of each strain was inoculated with colony on agar plate, and incubated at 37℃ for 24 h and the 100 μL of each strain culture was spread on agar plate. The sample 40 μL added to the paper disk (Advantec, Toyo Roshi Kaisha, Tokyo, JAPAN) was placed on the inoculated plate. The plates were incubated at 37℃ for 24 h and diameter of the inhibition zones was measured. All analyses were conducted in triplicate for each extract.

The MIC of ethyl acetate fraction from G. thunbergii methanol extract was measured using paper disc method. The concentrations of ethyl acetate fraction were from 0.025 to 1.6 mg/mL. All analyses were conducted under same conditions as paper disc method, and MIC was defined as the lowest concentration of ethyl acetate fraction required for inhibition of bacteria. All analyses were conducted in triplicate for each extract.

Analysis of major phenolic compounds in G. thunbergii ethyl acetate fraction was conducted using high performance liquid chromatography (Alliance, Waters, USA). An Eclipse plus C₁₈ column (Agilent Technologies Ltd., 4.6×250 mm, particle size 5 μm) was used to analyze gallic acid and ellagic acid. The mobile phases were 0.1% formic acid (v/v) in 10% acetonitrile (solvent A) and, 0.1% formic acid (v/v) in 90% acetonitrile (solvent B). All reagents used were HPLC grade. The gradient followed the order: 0-6 min 0% B, 6-31 min 10-90% B, 31-41 min 20-80%, 41-45 min 50-50% B, 45-50 min 0% B. The separated phenolic compounds were detected with photodiode array detector at 280 nm.

All analyses were conducted in triplicate. Statistical comparisons were carried out using SPSS 18.0 statistical software (SPSS, Chicago, IL, USA) and significance was determined by one-way ANOVA followed by Duncan multiple range test for multiple comparisons. Means with different letters in groups one significantly different (p<0.05).

In the present study, G. thunbergii was extracted twice with methanol at room temperature, followed by progressive partitioning in different solvents. The yield of solventpartitioned fractions followed the order: water fraction > butanol fraction > n-hexane fraction > ethyl acetate fraction > chloroform fraction and was found to be 49.52%, 18.16%,14.51%, 6.94%, and 4.35%, respectively. TPC has been used to evaluate the function of various materials as a measure of biological activity (8). TPC of solventpartitioned fractions from G. thunbergii methanol extract are shown in Table 1. Also, five fractions had high phenolic content, which ranged from 83.72 to 604.28 mg/g with the ethyl acetate fraction being highest 604.28 mg/g. In addition, the butanol fraction (465.65 mg/g) had higher TPC than the hexane, chloroform, and water fractions. Lee et al. (15) also reported TPC of G. thunbergii ethanol extract (96.51 mg/g) and investigated its correlation with antioxidant potential using radical scavenging assays. The phenolic compounds in herbs, plants and seaweeds have been regarded as antioxidants owing to their involvement in oxidationreduction reactions and their important role in various functions such as antimicrobial, DNA damage inhibition and anti-inflammatory activities (23). Several studies have shown that high amounts of TPC in the sample extract may correlate with their antioxidant activities (24). Zeng et al. (6) showed that the antioxidant activity of Rhizoma homalomenae extract according to total polyphenolic content increases as also shown by this study. The correlation values were estimated as DPPH/ABTS radical scavenging activity and reducing power with correlation coefficients of r²=0.9804, r²=0.9828, and r²=0.9875, respectively (data not shown).

The DPPH assay provides basic information on the antiradical reaction of numerous extracts because of theformation of stable free radicals and follows a radical-radical interaction to form a stable molecule (25). The DPPH scavenging activity of G. thunbergii solventpartitioned fractions significantly increased in a dose dependent manner at concentrations from 25 to 200 μg/mL. The DPPH scavenging activity of G. thunbergii solventpartitioned fractions at 50 μg/mL concentration was as following order ethyl acetate fraction > butanol fraction > chloroform fraction > water fraction > n-hexane fraction, with the following values 80.88%, 73.48%, 26.24%, 13.76%, and 13.43%, respectively. The DPPH radical scavenging activities of the positive controls (butylated hydroxyanisole and ascorbic acid) were 27.02% and 96.52%, respectively (Table 1).

ABTS assay is a commonly used method to evaluate the antioxidant capacity of various plants, seaweeds, and other sources. The ABTS radical scavenging activity of G. thunbergii solvent-partitioned fractions significantly increased in a dose-dependent manner at concentrations from 25 to 200 μg/mL. The ABTS radical scavenging activity of G. thunbergii solvent-partitioned fractions at 50 μg/mL concentration was following the order: ethyl acetate > butanol > chloroform > water > n-hexane with values of 80.12%, 70.72%, 21.36%, 15.54%, and 6.63%, respectively. The ABTS radical scavenging activities of the positive controls (butylated hydroxyanisole and ascorbic acid) were 87.70% and 99.05%, respectively (Table 1).

Also the reducing power assay is used to screen for antioxidant in plants, foods, and various fields (26,27). The reducing power of G. thunbergii solvent-partitioned fractions at 50 μg/mL concentration showed following the order: ethyl acetate fraction > butanol fraction > chloroform fraction > water fraction > n-hexane fraction with values of 0.28, 0.23, 0.13, 0.09, and 0.08, respectively. The reducing powers of the positive controls butylated hydroxyanisole and ascorbic acid, were 0.36 and 0.34, respectively (Fig. 1). According to their report, G. thunbergii ethanol extract at 1,000 μg/mL concentration had DPPH radical scavenging activity of 56.79%, TPC (96.51 mg/g) G. thunbergii ethanol extract of was higher than that of other plant extracts (24,28).

Fig. 1. Reducing power ability of Geranium thunbergii solventpar titioned fractions. Butylated hydroxyl anisole and ascorbic acid were used as positive controls. Data represent the means±SD of three determinations. Means with different letters on the bar are significantly different at p<0.05.

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Plants are known for having various biological activities such as antioxidant, anti-inflammatory, anti-obesity, and antimicrobial effects owing to the presence of diverse phenolic compounds (18,29). Kim et al. (18) reported the antioxidant activity and cell toxicity of pressurized liquid extracts of 20 selected plant species. The extracts of these species had high total phenolic content, antioxidant activities, and inhibitory activity against nitric oxide production in lipopolysaccharideinduced RAW 264.7 macrophages. In addition, Singh et al. (29) demonstrated the antioxidant and antimicrobial activities of Trigonella foenum-gracum, and its protective effect against oxidative DNA damage. Thus, various plants including G. thunbergii can be utilized as a good source of antioxidant in food industry and medicine.

Figure 2 shows gel electrophoretogram demonstrating the protective effect of G. thunbergii ethyl acetate fraction against cleavage of plasmid DNA by ferrous ion-induced oxidative damage. Addition of ferrous ion in the absence of GTEF converted the supercoiled form of DNA into open-circular form. DNA was not detected after the addition of ferrous ion in absence of GTEF. Recently, the protective effect of plant extracts with antioxidant activity has been reported. Cheng et al. (30) have reported the protective effect of extract of Crataegus pinnatifida pollen against DNA damage response to oxidative stress. According to the results, C. pinnatifida pollen showed antioxidant activity, protective effect against DNA damage, and their main phenolic compound was examined. Further, Abbas et al. (31) showed the phenolic profile, antioxidant potential, and protective effect on DNA of sugarcane (Saccharum officinarum), and their results showed that these cultivars can be used not only as accessible source of natural antioxidants but also as an ingredient of functional food for the control of degenerative diseases. Thus, various plants including G. thunbergii may be useful as ingredients for prevention of oxidative damagemediated diseases.

Fig. 2. Protective effect of G. thunbergii ethyl acetate fraction against DNA cleavage induced by FeCl₂ (A) and hydroxyl radical (B) using phiX-174RFI plasmid DNA. Control was treated with nothing, and another control was treated with FeCl₂ and hydroxyl radical pre-reaction without G. thunbergii ethyl acetate fraction. Sample lane was treated with varying concentrations of the G. thunbergii ethyl acetate fraction (50, 100, and 200 μg/mL).

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Table 2 shows the antimicrobial activities of all fractions. The ethyl acetate fraction showed remarkable antimicrobial activity with diameter of inhibition zones ranging from 13.33 to 15.67 mm. In addition, the MIC values of the ethyl acetate fraction ranged from 0.2 to 0.8 mg/mL. Previous reports on studies with G. thunbergii and other plant extracts had shown similar antimicrobial activities. Lee et al. (15) reported the antimicrobial activity of four herbal ethanol extracts. Among the samples, G. thunbergii ethanol extract had good antimicrobial activity against L. monocytogenes, S. aureus, B. cereus, and E. coli and their activities remained relatively high heat treatment. Shen et al. (32) demonstrated the antimicrobial effect of blueberry (Vaccinium corymbosm L.) extracts against L. monocytogenes and Salmonella enteritidis and showed four active phenolic products in blueberry extract. Quercetin was ineffective at 8 μg/mL, but ellagic acid, quercetin, and quercetin-3-galctoside at 200 μg/mL, showed significant (p<0.05) inhibitory effect when compared to the control. These antimicrobial activities have been attributed to phenolic compounds and flavonoids.

To analyze the contents of phenolic compounds in G. thunbergii ethyl acetate fraction, we compared it with standard substances (gallic acid, chlorogenic acid, caffeic acid, ellagic acid, myricetin, icariin, quercetin, and kampferol). From HPLC analysis, ellagic acid and gallic acid were detected in the ethyl acetate fraction and their contents were 55.14 and 5.42 mg/g, respectively (Fig. 3). Other reports have indicated significant roles for these and other phenolic and flavonoid compounds with the antioxidant, neuroprotective, and anticarcinogenic effects of plant extracts. Choi et al. (16) reported α-glucosidase inhibitory effect of active compounds isolated from G. thunbergii ethyl acetate fraction and confirmed p-hydroxybenzoic acid, geraniin, and ellagic acid to be the active components. Liu et al. (17) also reported inhibitory activity against IL-6 production of flavonoids and phenolic compounds from G. thunbergii and confirmed kampferol-3-rhamnoside, protocatechuic acid, and gallic acid to be the active components. Xiong et al. (33) reported that gallic acid was expressed as 7.99 mg/g in flower. They demonstrated that gallic acid and chlorogenic acid have attracted considerable attention for their antioxidant activity, neuroprotective effects, and anticarcinogenic properties.

Fig. 3. HPLC chromatogram of ellagic acid and gallic acid in G. thunbergii ethyl acetate fraction. The separated phenolic compounds were detected with a photodiode array detector at 280 nm.

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