Assessment of the Antioxidant Activity of Silybum marianum Seed Extract and Its Protective Effect against DNA Oxidation , Protein Damage and Lipid Peroxidation

Silybum marianum (L.) Gaertn. (Asteraceae), also known as milk thistle, is an important herbal medicine. It is grown primarily for the extraction of its active component (silymarin) from edible seeds, which has been used to treat liver disorders. The liver-protecting ability of silymarin is due to the antioxidant and free radical scavenging properties. Silybum marianum seeds contain more than seven fl avonolignans. Many studies have reported the biological evaluation of silymarin, such as in cancer chemoprevention and hepatoprotection (1,2). Silymarin has the ability to scavenge free radicals, it has been found to increase the production of glutathione in hepatocytes and the activity of superoxide dismutase in erythrocytes (3). In one animal study it has been shown to increase intracel lular glutathione level by as much as 50 % (3).


Introduction
Silybum marianum (L.) Gaertn.(Asteraceae), also known as milk thistle, is an important herbal medicine.It is grown primarily for the extraction of its active component (silymarin) from edible seeds, which has been used to treat liver disorders.The liver-protecting ability of silymarin is due to the antioxidant and free radical scavenging properties.Silybum marianum seeds contain more than seven fl avonolignans.Many studies have reported the biological evaluation of silymarin, such as in cancer chemoprevention and hepatoprotection (1,2).Silymarin has the ability to scavenge free radicals, it has been found to in-crease the production of glutathione in hepatocytes and the activity of superoxide dismutase in erythrocytes (3).
In one animal study it has been shown to increase intracel lular glutathione level by as much as 50 % (3).
Flavonolignans showed radical scavenging properties and protective eff ects against the damage of lipid membranes (4) and low-density lipoproteins (5).The antioxidant eff ect is due to the modulation of pathways such as cell growth, apoptosis and diff erentiation (6).
Reactive oxygen species (superoxide radical, hydro xyl radical (˙OH) and lipid peroxide (LOO˙) radicals) may cause many diseases such as cancer and Alzheimer's (7).
They are generated in human body as a by-product of the normal biochemical reactions and increased exposure to the environmental stress (8).Oxidation of fatt y acids in the cell membranes can disrupt their fl uidity and permeability and damage macromolecules, such as DNA, RNA, protein and other cellular components (9).Silymarin supports the normal fl uidity of cell membrane by interacting with its components (10).
Many publications show the inhibitory eff ects of plant extracts or their components against protein oxidation and DNA damage caused by reactive oxygen species (11)(12)(13).To our knowledge, this is the fi rst study that has investigated the inhibition ability of DNA damage, protein oxidation and lipid peroxidation by ethanol extract of S. marianum seeds in vitro.

Seed sample
Cultivated Silybum marianum seeds were harvested in the autumn of 2012 from the fi eld of the Department of Field Crops, Faculty of Agriculture, University of Dicle, Diyarbakır, Turkey.The extract was prepared from dried S. marianum seeds, which were ground into fi ne powder using blender (model 7011S; Waring, Atlanta, GA, USA).

Extraction procedure
Silybum marianum seeds were ground into fi ne powder with blender to a diameter of 0.4 mm (14).Silymarin was extracted in a two-step process in which powdered seeds were fi rst defatt ed.In order to defat the seeds, about 10 g of fi nely powdered samples were weighed and extracted with petroleum ether (370 mL) for 4 h and then with ethanol (350 mL) for 8 h in a Soxhlet apparatus (Sigma-Aldrich).Ethanol solution was evaporated at a temperature not exceeding 40 °C and ethanol extract of seeds (1.08 g) was obtained as soft yellow powder.

Determination of total phenolic content
The phenolic content of milk thistle seed extract was measured with the Folin-Ciocalteau method (15).A mass concentration of 0.5 mg/mL of stock solution of the extract was prepared in ethanol.Then, 40 μL of the sample and the control were diluted with 1160 μL of water and 200 μL of 2 M Folin-Ciocalteu agent.The absorbance was determined at λ=765 nm.A standard gallic acid solution (50-400 μg/mL) was used to construct the calibration curve.The phenolic content was expressed in μg of gallic acid equivalents (GAE).The equation below was used to calculate the concentration of phenolic compounds:

Determination of total fl avonoid content
The fl avonoid content was determined by the previously described method (16).The stock solution of 0.5 mg/mL of milk thistle seeds was prepared in ethanol.In 10-mL test tube, 1 mL of extract, 0.1 mL of 10 % Al(NO 3 ) 3 , 0.1 mL of 1 M CH 3 COOK and 3.8 mL of methanol were mixed.The reaction mixture was incubated at 25 o C for 40 min.The absorbance was measured at λ=415 nm.The standard curve for total fl avonoids was made using quercetin standard solution (1-25 μg/mL).The equation below was used to measure the concentration of fl avonoid compounds expressed as quercetin equivalents (QE):

DPPH radical scavenging activity assay
The 1,1-diphenyl-2-picryl-hydrazil (DPPH) radical scavenging activity of S. marianum seeds was studied using previously reported procedure (17).A volume of 3 mL of S. marianum seed ethanol extract solutions of diff erent concentrations (5,10,25,50, 100 and 150 μg/mL) was mixed with 1 mL of 0.1 mM DPPH.The mixture was kept at room temperature for 30 min.Then the absorbance was measured at λ=517 nm with a Cary 100 Bio UV/Vis spectrophotometer (Varian Inc., Palo Alto, CA, USA).The following equation was used to measure the radical scavenging activity:

Reducing power assay
The reducing power of the S. marianum seeds was eva luated using the procedure described previously (18), with some modifications.Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used as positive controls.In a test tube, 1 mL of sample (5-150 μg/ mL), 2.5 mL of 0.2 M phosphate buff er (PBS; pH=6.6) and 2.5 mL of 1 % K 3 Fe(CN) 6 solution were mixed.
Aft er 20 min of incubation at 50 o C, 2.5 mL of 10 % trichloroacetic acid were added to each mixture, and then centrifuged at 1500×g for 10 min.A supernatant volume of 2.5 mL was taken and mixed with 2.5 mL of deionized water containing 0.5 mL of 0.1 % iron(III) chloride.Aft er 10 min of incubation at room temperature the absorbance A was measured at λ=700 nm.

Rat liver homogenate preparation
The rat liver homogenate (10 % by mass per volume) was prepared according to a previously reported method (19).For this study, albino Wistar rats (mass of (150±25) g) were used.Permission was obtained from Dicle Universi-ty Ethical Committ ee (IAEC; Diyarbakır, Turkey).The animals were housed in polypropylene cages at controlled temperature ((22±3) o C).Standard formula and water were given to the animals ad libitum.The liver was removed and perfused with 120 mM potassium chloride and 50 mM phosphate buff er, pH=7.4.The ratio of liver mass per volume of solution was 1:10.In order to obtain the pellet, the samples were centrifugated at 700×g at 4 °C.
The following equation was used to calculate the percentage of lipid peroxidation inhibition:

DNA damage protection capacity
DNA protection capacity of milk thistle seed extract against oxidation was determined according to Kızıl (21) using plasmid DNA.The band intensity for the forms I and II of plasmid DNA was quantifi ed using Quantity One v. 4.5.2soft ware (Bio-Rad, Hercules, CA, USA).Genejet miniprep kit (Thermo Fisher Scientifi c, Inc, Waltham, MA, USA) was employed for the isolation of plasmid DNA.The following equation was used to calculate the inhibition of the DNA cleavage: where S m+a is the percentage of the remaining supercoiled DNA aft er treatment with a mix consisting of buff er, DNA, H 2 O 2 and UV, and the agent (Silybum marianum extract), S c is the percentage of the remaining supercoiled DNA in the control (untreated plasmid), and S m is the percentage of the remaining supercoiled DNA with the mix, but without the agent.

Inhibition of protein carbonyl formation
The eff ect of milk thistle seed extract on protein oxidation was determined using a modifi ed method of Wang et al. (22).Bovine serum albumin (BSA; 4 mg/mL) was treated with iron(III) chloride (50 μM), hydrogen peroxide (1 mM) and ascorbic acid (100 μM) in potassium phosphate buff er (20 mM, pH=7.4) in the presence of seed extract (10-1000 μg/mL) at 37 °C for 30 min and then the absorbance was measured at λ=370 nm.The inhibition of protein carbonyl formation was calculated as follows (21):

SDS-PAGE analysis
The inhibitory eff ect of the ethanol extract of S. marianum seeds on protein damage was also determined by SDS-PAGE (12).The gel was run at a maximum voltage in running buff er containing 25 mM Tris, pH=8.3, 190 mM glycine and 0.1 % sodium dodecyl sulphate (SDS).The current of 25 mA was used per gel.Coomassie Brillant Blue R-250 (0.15 %) was used for staining of the gel, which was scanned with Gel Doc XR System (Bio-Rad).

Statistical analysis
In order to determine the diff erences between the groups, one-way ANOVA was used.Results are presented as the mean value±standard deviation of three replicates.

Phenolic and fl avonoid content of S. marianum seed extract
Total phenolic content (expressed as GAE) of ethanol extract of milk thistle seeds was determined to be (620.0±4.93)μg/g.In addition to this, the fl avonoid content (expressed as QE) was found to be (39.32±0.11)μg/g.These values show that the ethanol extract of milk thistle seed has good antioxidant activity.Besides that, hydroxyl groups present in the compounds isolated from the milk thistle extract have good radical scavenging activity.Pereira et al. (23) described the content of phenolics and fl avonoids in milk thistle.In their study, milk thistle plant was used as dry material for infusion and pill preparation.The phenolic and fl avonoid contents (expressed as GAE and catechin equivalent, respectively) were found to be (23.26±0.22)mg/g and (6.95±0.23)mg/g in infusions, and (20.92±0.45)and (3.88±0.13)mg/g in dietary supplements, respectively (23).On the other hand, Tupe et al. (24) reported phenolic content (as GAE) of methanolic plant extract of milk thistle of (18.33±0.16) mg/g.The high contents of phenolics and fl avonoids obtained in infusions, dietary supplements and methanolic milk thistle plant extract may depend on the part of the plant used.Phenolics and fl avonoids are very important plant components due to their radical scavenging properties (25,26).It is well known that fl avonoids have anticarcinogenic activity, antiallergenic, antiviral, antiageing and anti-infl ammatory properties.

DPPH radical scavenging activity
The S. marianum extract showed a strong DPPH radical scavenging ability in a concentration-dependent manner (Fig. 1).The order of radical scavenging activities was found to be as follows: S. marianum>BHT>BHA and was 92.0±5.2, 88.0±5.7 and 87.8±2.1 at 150 μg/mL.The antioxidant (DPPH scavenging) activity of S. marianum essential components and diff erent tissues was also reported by Hadaruga and Hadaruga (27).There was no signifi cant diff erence between the DPPH radical scavenging activity of S. marianum seed extract and both standards.

Reducing power assay
It was found that S. marianum has a benefi cal eff ect against acute and chronic liver failure.Silibinin, a major active constituent of silymarin, was found to have antitumour properties in hepatocarcinoma cell and animal models (28).The tested samples at the concentrations of 5, 10, 25, 50, 100, and 150 μg/mL exhibited a dose-dependent reducing power activity.Reducing power of 100 μg/mL of milk thistle seed extract measured as A 700 nm was determined to be 0.11±0.00.On the other hand, reducing powers of BHA and BHT at 100 μg/mL measured as A 700 nm were determined to be 0.61±0.03and 0.34±0.08,respectively.The reducing power of S. marianum seed ethanol extract is shown in Fig. 2 with BHT and BHA as positive controls.
There are reports on antioxidant activity and reducing power of milk thistle infusions, dietary supplements and syrups (23,28).Reducing power of infusions, dietary suplements and syrups (measured as A 700 nm ) was found to be 1.73±0.03,1.10±0.02,and 0.05±0.0,respectively (23,28).Tupe et al. (24) have investigated 19 frequently used medicinal plants and concluded that the strongest antioxi-dant activity was observed in Terminalia chebula since it had high phenolic content, outstanding reducing power and highest radical scavenging activity.

Lipid peroxidation
The compound antioxidant activity depends on their capacity to delay the oxidation by inhibiting reactive oxygen species (29).As shown in Fig. 3, thiobarbituric acid reactive substances (TBARS), which are formed during lipid oxidation, were measured in rat liver homogenate and it was found that the S. marianum extract moderately repressed TBARS formation.At the concentration of 250 μg/mL of S. marianum seed extract, 26.14 % of TBARS formation was inhibited.BHT inhibited 31.40 % at the same concentration.There were no signifi cant diff erences between the activity of S. marianum seed extract and BHT.The inhibitory activity of Mangifera indica fractions against lipid peroxidation was studied by Badmus et al. (30), who found that the ethyl acetate fraction had the highest inhibitory activity against lipid peroxidation in brain homogenate (66.3 %), followed by methanol (61.6 %), chloroform (46.5 %) and aqueous (39.5 %) fractions.High percentage of lipid peroxidation inhibition of 89.1 % was observed in aqueous fraction of liver homogenate, followed by ethyl acetate (71.6 %), methanol (59.0 %) and chloroform (48.5 %) fractions.

DNA damage protection
The band intensity for the forms I and II of plasmid DNA was quantifi ed (Fig. 4a).Fig. 4b shows plasmid DNA aft er UV-induced photolysis of H 2 O 2 (2.5 mM) in the presence or absence of the S. marianum etanol extract (in the concentration of 100, 500, 750 or 1000 μg/mL).The supercoiled DNA was converted to form I DNA by UV light (Fig. 4b, lane 4).Formation of form I DNA was suppresed in the presence of the extract (Fig. 4b, lanes 6-9) and the amount of form II of DNA was increased.The inhibitory activity of the ethanol extract of cultivated milk thistle seed against oxidative DNA damage was found to be 4.72, 41.08, 66.95 and 87.80 % at 100, 500, 750 and 1000 μg/mL, respectively.DNA damage was suppressed with the increased concentration of the extract.The ethanol extract of milk thistle seeds showed a concentration-dependent free radical scavenging activity (Fig. 4a).The protective eff ect of the extract against DNA damage may be att ributed to the presence of fl avonoid and phenolic compounds, such as silymarin and silibinin, which can prevent the production of reactive oxygen species (ROS) by making complexes with cations such as copper and iron that participate in hydroxyl radical formation (5).

Inhibition of protein carbonyl formation by S. marianum seed extract
The eff ect of milk thistle seed extract on BSA hydroxyl radical-mediated oxidation is shown in Fig. 5. BHT and S. marianum seed extract at 50, 100, 250, 500 and 1000 μg/mL decreased the free radical-induced protein damage caus ed by protein carbonyl group formation, induced by Fenton reaction system.The inhibitory eff ect of BHT at 50, 100, 250, 500 and 1000 μg/mL was found to be 12.56, 37.33, 38.16, 40.43 and 45.40 %, respectively, and of S. marianum seed extract at 50, 100, 250, 500 and 1000 μg/mL was found to be 9. 16, 9.46, 11.36, 21.10, 42.75 and 86.27 %, respectively.The inhibition of protein oxidation by S. maria num seed extract may be due to its high radical scavenging activity.Inhibitory eff ects of methanolic extract of Feronia limonia pericarp on in vitro protein glycoxidation was studied by Kilari et al. (31).The study demonstrated that F. limonia extract was effi cient in preventing oxidative protein damage, including protein carbonyl formation during glycoxidation.They proposed that the antioxidant eff ect of F. limonia might be due to the presence of volatile fl avours and free fatt y acids in the pericarp, which are involved in the inhibitory mechanisms of protein carbonyl formation.

SDS-PAGE
The diff erent concentrations of BHT and S. marianum seed extract showed inhibitory activity on BSA oxidation induced by Fenton reaction system as measured with SDS-PAGE.

Conclusions
The present work investigated the in vitro antioxidative and oxidative DNA, protein and lipid damage protective eff ects of milk thistle (Silybum marianum) seed ethanol extract.The extract was found to have protective eff ect against DNA, protein and lipid oxidation induced by hydroxyl radical.The results obtained in this study show that ethanol extract of seeds can be used as a dietary supplement and it can protect DNA from damage.The inhibitory activity of seed extract against hydroxyl radical-induced DNA, protein and lipid damage may be mainly responsible for the cancer chemoprevention and hepatoprotection eff ects.The presence of silymarin could account for the increased antioxidant activity and enhanced DNA, protein and lipid damage protection capacity of the extract.Protection against the DNA, protein and lipid oxidation is due to phenolic and fl avonoid compounds that scavenge the free radicals.These fi ndings also imply that the presence of antioxidants can decrease the occurrence of degenerative disorders in which free-radical damage may be involed.

Confl ict of interest
Authors state that they have no confl ict of interest regarding this submission.

Fig. 4 .Fig. 6 .Fig. 5 .
Fig. 4. The quantifi ed band intensity of the scDNA (form I) and ocDNA (form II) (a), and (b) electrophoretic patt ern of pBluescript M13+DNA aft er UV photolysis of H 2 O 2 in the presence or absence of ethanol extract of Silybum marianum seeds.Reaction vials contained 200 ng of supercoiled DNA (31.53nM) in distilled water, pH=7

Fig. 7 .
Fig. 7. Protection against bovine serum albumin (BSA) oxidative damage by ethanol extract of Silybum marianum seeds: a) the electrophoretic patt ern of BSA, and b) densitometric analysis of the corresponding band intensity.BSA was oxidised by Fenton reaction system (Fe 3+ /H 2 O 2 /ascorbic acid).Each bar represents the mean value±S.D. (N=3).Mean values with diff erent lett ers diff er signifi cantly (p<0.05), while those with the same lett ers are not signifi cantly diff erent at p>0.05 compared with Fenton reaction system