Flavonoids from Taraxacum coreanum protect from radical-induced oxidative damage

The active components and protective activity of Taraxacum coreanum from oxidative stress under in vitro and cellular system using LLC-PK1 renal epithelial cells were investigated. T. coreanum was extracted with methanol (MeOH) and then fractionated into four different layers, n-hexane, trichloromethane (CHCl3), ethyl acetate (EtOAc), and n-butanol (n-BuOH). In vitro, the scavenging activities of the extract and fractions from T. coreanum and its active components, luteolin and luteoloside, on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and hydroxyl radical (·OH) were measured. In LLC-PK1 cellular model, the protective activity of the EtOAc fraction, and the active components from oxidative stress induced by 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), a generator of peroxyl radicals, were studied. Among the fractions, the EtOAc fraction and its active components, luteolin and luteoloside, exerted the strongest protective effect from DPPH and ·OH. Furthermore, the LLC-PK1 cells showed a decrease in cell viability and an increase in lipid peroxidation by the treatment of AAPH. However, the EtOAc fraction and its active components led to the significant increases in the cell viability and inhibition in lipid peroxidation. The present results indicated that T. coreanum and its active components, luteolin and luteoloside, are promising antioxidants with the protective effect from oxidative stress induced by overproduction of free radical.


INTRODUCTION
Reactive oxygen species (ROS)-induced oxidative stress leads to the modification of DNA, cellular proteins and membrane lipids; therefore, it plays a crucial role in a wide range of diseases and age-related degenerative conditions including cardiovascular diseases, inflammatory conditions and neurodegenerative diseases such as Alzheimer's disease and cancer (Bokov et al., 2004;Halliwell, 1997).This relationship has led to considerable interest in searching for antioxidants to scavenge free radicals and elevate defense activity in biological systems.Although several synthetic antioxidants have been *Corresponding author.E-mail: ejcho@pusan.ac.kr.Tel: (+82) 51-510-2837.Fax: (+82) 51-583-3648.
suggested for the prevention and treatment of diseases, some side effects and toxicities have become an issue.Natural antioxidants, because of relatively low toxicity and side effects compared with synthetic antioxidants, have attracted much attention as preventive and therapeutic agents for attenuating oxidative damage.
Plants of the genus Taraxacum also known as dandelions, are in the family of the Asteraceae and have long been used as medicinal herbs.The name is derived from the Greek words "taraxis", for inflammation, and "akeomai", for curative.Taraxacum coreanum, a native plant of Korea, grows chiefly in the Korea and China.T. coreanum has been used as diuretic drugs and antiinflammatory medicines (Ahn, 1998;Koo et al., 2004).The functions of T. coreanum are related to its phytochemical compounds including phenols and flavonoids, which are important sources of natural antioxidants for use as a dietary supplement or as the agent in pharmaceutical products.A recent study demonstrated that the extracts of T. coreanum contained higher phenolic compounds (Lee and Lee, 2008;Lee et al., 2011) and inhibited aldose reductase (Mok et al., 2011).However, the scientific results have not been sufficient to elucidate its biological effect and its bioactive compounds.Therefore, we investigated the radical scavenging effect of fractions and active compounds from T. coreanum under in vitro radical scavenging assay, and evaluated the protective activity of these components against peroxyl radical-induced oxidative damage under in a cellular system.

MATERIALS AND METHODS
The plant of T. coreanum was collected in 2007 near the West coast Express Highway, Republic of Korea.A voucher specimen (no.LEE 2007-01) was deposited at the Herbarium of Department of Integrative Plant Science, Chung-Ang University, Republic of Korea.

General instruments
The electron ionization-mass spectrometry (EI-MS) was measured with a JEOL JMS-600W (Japan) mass spectrometer, and the fast atom bombardment-mass spectrometry (FAB-MS) was measured with a JEOL JMS-AX505WA (Japan) mass spectrometer. 1H-and 13 C-NMR spectra were recorded with a Bruker AVANCE 300 NMR (Germany) spectrometer using tetramethylsilane (TMS) as an internal standard.Chemical shifts were reported in parts per million (δ), and coupling constants (J) were expressed in hertz.Evaporation was conducted using an EYELA rotary evaporator system (Japan) under reflux in vacuo.Thin layer chromatography (TLC) was conducted with Kiesel gel 60 F254 (Art.5715, Merck Co., Germany) plates (silica gel, 0.25 mm layer thickness).Recycling preparative high performance liquid chromatography (HPLC) was conducted by a JAI LC-9014 system and determination was performed by an L-6050 system pump with a UV-3702 system UV/VIS detector.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity
In a microwell plate, 100 µL of fractions and flavonoids from T. coreanum (control: 100 µL of distilled water) were added to an ethanolic solution of DPPH (60 mM) according to the method of Hatano et al. (1989).After being mixed gently and left for 30 min at room temperature, the DPPH radicals were determined using a microplate reader (model SPECTRA max 340PC, Molecular Devices, Sunnyvale, CA, U.S.A.).

Hydroxyl radical (•OH) scavenging activity
The reaction mixture contained 0.45 ml of 0.2 M sodium phosphate buffer (pH 7.0), 0.15 ml of 10 mM 2-deoxyribose, 0.15 ml of 10 mM FeSO4-EDTA, 0.15 ml of 10 mM H2O2, 0.525 ml of H2O, and 0.075 ml of sample solution.The reaction was initiated by the addition of H2O2.After incubation at 37°C for 4 h, the reaction was stopped by adding 0.75 ml of 2.8% trichloroacetic acid (TCA) and 0.75 ml of 1.0% of 2-tribarbituric acid in 50 ml of NaOH.The solution was boiled for 10 min, and then cooled on ice.Finally, the absorbance of the solution was measured at 520 nm.Hydroxyl radical scavenging activity was evaluated as the inhibition rate of 2-deoxyribose oxidation by •OH (Gutteridge, 1987).

Cell culture and treatment with peroxyl radical generator
Commercially available LLC-PK1 cells were maintained in a culture flask containing 5% FBS-supplemented DMEM/F-12 medium (pH 7.2) at 37°C in a humidified atmosphere of 5% CO2 in air.All subsequent procedures were carried out under these conditions.Cells were sub-cultured weekly with 0.05% trypsin-EDTA in PBS.After confluence had been reached, the cells were seeded into 96well plates at 10 4 cells/ml and allowed to adhere for 2 h.Next, 1.0 mM of AAPH was treated to generate LOO -, respectively.After 24 h of incubation, sample was treated in the test wells at various concentrations for 24 h (Yokozawa et al., 2003).

MTT assay
Cell viability was assessed using the MTT colorimetric assay.MTT solution (1 mg/ml) was added to each 96-well culture plate and incubated for 4 h at 37°C, and then the medium containing MTT was removed.The incorporated formazan crystals in the viable cells were solubilized with 100 ml of dimethyl sulfoxide (DMSO) and the absorbance of each well was read at 540 nm using a microplate reader.Lee et al. 5379

Measurement of thiobarbituric acid reactive substances (TBARS)
The level of lipid peroxidant released from the cultured cells was estimated as TBARS according to the methods of Yagi (1976) and Yokode et al. (1988) with slight modifications.One aliquot of medium was mixed with 1.5 ml of 0.67% TBA solution and 1.5 ml of 20% TCA, and boiled at 95 -100°C for 45 min.The mixture was cooled with water and shaken vigorously with 3.0 ml of n-butanol.
After the mixture was centrifuged at 4000 X g for 10 min, the nbutanol layer was removed, and the absorbance was measured at 520 nm on a fluorescence spectrophotometer (Model FR-550, Shimadzu, Kyoto, Japan).

Statistical analysis
All statistical analyses were assessed by SAS software (SAS Institute, Cary, NC, USA).P < 0.05 was determined as statistically significant.Measurement data (n = 6) were expressed as mean ± standard deviation.

DPPH scavenging activity
The MeOH extracts and 4 fractions from T. coreanum showed strong DPPH radical scavenging activity (Table 1).Among all fractions, the EtOAc fraction showed the strongest DPPH radical scavenging activity.Based on this result of DPPH radical, we investigated the radical scavenging activities of EtOAc fraction from T. coreanum.
The treatment of the EtOAc fraction from T. coreanum increased DPPH radical scavenging activity in a dosedependent manner (Table 2).At the 100 mg/ml concentration, 90% inhibition of DPPH radical was observed.

•OH scavenging activity
The EtOAc fraction also had the strong protective effect against •OH in a dose-dependent manner (Table 3).At the concentrations of 0.5, 2.5, 50 and 100 µg/ml, the EtOAc fraction showed 87.9, 89.9, 90.2 and 90.4% of •OH scavenging activity, respectively.

Protective activity against peroxyl radical-induced oxidative stress
The viability of LLC-PK 1 renal epithelial cells treated with AAPH was reduced to 35.4% by the treatment with 1 mM AAPH for 24 h (Figure 2).However, the treatment of the EtOAc fraction exerted protective activity against AAPHinduced cellular damage.When the EtOAc fraction was treated at the dose of 100 µg/ml, the cell viability was elevated to 80%.

Inhibition of lipid peroxidation against peroxyl radical-induced oxidative stress
AAPH led to an increase of lipid peroxidation in LLC-PK 1 renal tubular epithelial cells, whereas the EtOAc fraction significantly decreased the formation of TBARS in a concentration-dependent manner (Figure 3).Although 0.734 nmol/mg protein of TBARS was produced in cells treated only with AAPH, the treatment with the EtOAc fraction at the 100 µg/ml decreased lipid peroxidation to

Antioxidative activity of luteolin and luteoloside
The IC 50 values of the DPPH radical scavenging effects of flavonoids, luteolin and luteoloside, were 0.05 and 0.03 µg/ml, respectively (Table 3).In addition, as revealed in Figure 4, AAPH treatment reduced LLC-PK 1 cell viability to 32.2%.Treatment with both luteolin and luteoloside was able to recover the cellular damage induced by AAPH in a dose-dependent manner, and at the concentration of 10 µg/ml, the cell viability was elevated to 90.8 and 95.1%, respectively.In addition, luteolin and luteoloside also showed protective effects against lipid peroxidation induced by peroxyl radicals (Figure 5).Peroxyl radicals increased the formation of MDA from 0.50 nmol MDA/mg protein to 0.95 nmol MDA/mg protein.

DISCUSSION
Free radical-mediated oxidative stress results in a variety of pathological conditions (Bokov et al., 2004;Halliwell, 1997).Therefore, antioxidants that prevent damage caused by free radicals are considered to be worthy of study.T. coreanum is a well-known traditional herbal medicine with a long history.Until recently, only limited scientific information has been available to justify its reputed uses (Gurib-Fakim, 2006).Therefore, the present investigation was focused on the protective activity of T. coreanum and its active components, luteolin and luteoloside, from free radical-induced oxidative stress in both in vitro and cellular system.
We tested the radical scavenging activity of the MeOH extract and 4 fractions of T. coreanum against DPPH and •OH scavenging activities.DPPH is a stable free radical and has been widely used to test the ability of compounds or plant extracts to act as free radical scavengers or hydrogen donors (Lee et al., 2011;Zhu et al., 2002).Antioxidants directly react with the DPPH radical and restore it by transfer of electrons or hydrogen.Therefore, we used this system for assessing the radical scavenging activity of the MeOH extract and 4 fractions of T. coreanum.In the DPPH scavenging activity tests of the MeOH extract and 4 fractions of T. coreanum, the EtOAc fraction showed the strongest DPPH radical scavenging activity.Based on the DPPH result, we investigated the radical scavenging activities of the EtOAc fraction from T. coreanum.The treatment with the EtOAc fraction increased DPPH radical scavenging activity in a dose-dependent manner.These results suggest that the EtOAc fraction from T. coreanum includes the promising agents for scavenging of free radicals.
•OH induces various injuries to the surrounding organs and plays a vital role in some clinical disorders.Therefore, removal of •OH is the most effective defense of living body against diseases (Lin et al., 1995).In particular, among various different radicals, the •OH is an extremely  reactive and short-lived species that can attack biological molecules such as DNA, proteins, and lipids.The reactivity of •OH has been related to several human diseases such as neurodegenerative disease and diabetes.Therefore, its scavenging activity has received much attention (Halliwell and Gutteridge, 1984;Halliwell et al., 1992;Zhang et al., 1996).The EtOAc fraction showed the strongest protective effect against •OH in a dose-dependent manner.From these results, we confirmed that the EtOAc fraction from T. coreanum may contain an effective •OH scavenger capable of protecting against radical-induced oxidative damage.The present results suggest that the EtOAc fraction may play a protective role against free radical-induced oxidative stress.
Meanwhile, the reactions of free radicals in biological systems are complicated.To study these reactions, a well-designed in vitro model system is required.Thermal decompositions of free radical initiators, including peroxides, hyponitrites, and azo compounds induce oxidative stress.To generate free radicals at a known, constant and well-defined rate, thermal decomposition of free radical initiators is preferred.AAPH, one of the hydrophilic azo compounds, generates free radicals at a constant and measurable rate by its thermal decomposition without biotransformation (Terao and Niki, 1986).The free radicals generated from AAPH react with oxygen molecules rapidly to yield peroxyl radicals.The lipid peroxyl radicals in turn attack other lipid molecules to form lipid hydroperoxide and new lipid radicals.This reaction takes place repeatedly with resultant attacks upon various biological molecules, and induces physiochemical alterations and cellular damage (Miki et al., 1987).Finally, AAPH causes a diverse array of pathological changes.Therefore, AAPH-intoxication experiments are useful for evaluating biological activities of antioxidants.To investigate the antioxidative activity of the EtOAc fraction from T. coreanum in a cellular system using LLC-PK 1 renal tubular epithelial cells that are susceptible to oxidative stress, we employed such an AAPH model system.
Several reports have documented that AAPH decreased the viability of hepatic and neuron cells (Matsura et al., 1992;Rapin et al., 1998).The treatment with AAPH induces apoptosis in the cells, causing loss of viability.The present study also shows that AAPH leads to a decline in viability of LLC-PK 1 renal epithelial cells.However, our results demonstrated that the EtOAc fraction exerted a protective effect against oxidative damage by AAPH to LLC-PK 1 cells, resulting in increased cell viability in a dose dependent manner.It is well accepted that lipid peroxidation in biological systems is toxicological phenomenon, resulting in pathological consequences (Hochstein and Jain, 1981).Therefore, measurement of lipid peroxidation end products such as TBARS provides a good index of cell destruction.Our results revealed that AAPH treatment increased the formation of TBARS in LLC-PK 1 renal epithelial cells, indicating cellular damage by AAPH.However, the treatment with the EtOAc fraction also showed decrease in AAPH-induced lipid peroxidation.These results Lee et al. 5383 indicate that T. coreanum exerted protective activity from AAPH-induced cell injury and lipid peroxidation by scavenging peroxyl radicals generated from AAPH, suggesting the roles as promising antioxidants.
The present study clearly demonstrates that luteolin and luteoloside primarily are responsible for the radical scavenging effect and protective activity from oxidative damage.Several studies have demonstrated that Taraxacum officinal extracts possess antibiotic activities, anti-oxidative properties, anti-inflammatory and antitumor substances (Kimura et al., 1985;Wagner, 1989).Moreover, T. officinal contains terpenoid and sterol compounds such as taraxacin and taraxacerin, which are distributed equally in the roots, leaves and flowers.Other terpene/sterol compounds include β-amyrin, taraxasterol, taraxerol, sitosterin, stigmasterin, and phytosterin.However, the active compounds from T. coreanum with antioxidative effect have not been identified yet.We previously isolated and identified the active components from T. coreanum, luteolin and luteolin-7-glucoside (Lee et al., 2011).Our present study supported the protective role of luteolin and luteoloside from oxidative stress induced by free radicals.
In conclusion, T. coreanum exhibited radical scavenging activity in vitro and antioxidative activity against oxidative stress in a cellular model.In addition, luteolin and luteoloside isolated from T. coreanum have been shown to have antioxidative activity.Although further study on the protective mechanisms of these compounds is necessary, the present study supports the promising role of T. coreanum as a source of antioxidative compounds against free radical-induced oxidative stress.

Figure 2 .
Figure 2. Effect of the EtOAc fraction from T. coreanum on of LLC-PK1 cells treated with AAPH.Values are mean ± SD. a-c Means with the different letters are significantly different (P<0.05) by Duncan's multiple range test.

Figure 3 .
Figure 3.Effect of the EtOAc fraction from T. coreanum on lipid peroxidation of LLC-PK1 cells treated with AAPH.Values are mean ± SD. a-d Means with the different letters are significantly different (P<0.05) by Duncan's multiple range test.

Figure 4 .
Figure 4. Effect of luteolin and luteoloside from T. coreanum on viability of LLC-PK1 cells treated with AAPH.Values are mean ± SD. a-d Means with the different letters are significantly different (P<0.05) by Duncan's multiple range test.

Figure 5 .
Figure 5.Effect of luteolin and luteoloside from T. coreanum on lipid peroxidation of LLC-PK1 cells treated with AAPH.Values are mean ± SD. a-d Means with the different letters are significantly different (P<0.05) by Duncan's multiple range test.

Table 1 .
IC50 values of the MeOH extract and 4 fractions from T. coreanum against DPPH radical.Means with the different letters are significantly different (P<0.05) by Duncan's multiple range test.
bValues are mean ± SD.a-e

Table 2 .
DPPH and •OH scavenging activity of the EtOAc fraction from T. coreanum.
Values are mean ± SD.a-d Means with the different letters are significantly different (P<0.05) by Duncan's multiple range test.