C.M. Park et al. / Journal of Ethnopharmacology 133 (2011) 834–842 835(Seo et al., 2005). Acute lung injury induced by LPS was ame- step was repeated three times. Harvested ethyl acetate extractantsliorated through increased antioxidative capacities and inhibited were dissolved in methyl alcohol and ﬁltered (Nylon, Whatmann,inﬂammatory cytokines production in mice (Liu et al., 2010). Dan- 0.2 m).delion leaf is also known to be an effective hydrogen peroxidescavenger, because of its high polyphenol content (Hagymasi et al., 2.3. HPLC analysis for luteolin and chicoric acid2000). Many reports have shown that polyphenols possess antiox-idative and anti-inﬂammatory activities (Velioglu et al., 1998; Luteolin and chicoric acid analyses for HPLC were followedLima et al., 2007). In our previous study, 4 kinds of dandelion by the method of Hu and Kitts (2003). Two identical Agilentextract (hot water, water, ethanol, and methanol) were applied to 1100 HPLC systems (Agilent Technologies, Palo Alto, CA, USA)LPS-induced macrophages to evaluate their antioxidative and anti- equipped with ChemStation software with a diode-array detec-inﬂammatory capacities through NO and malondialdehyde (MDA) tor was used for analysis. A reverse phase Agilent Zorbax XDB-C18production. Hot water and methanol extracts showed more potent column AAA (4.6 mm × 150 mm, 3.5 m) was used for chromato-activities than water and ethanol extracts, which indicated that graphic separation. Two Phenomenex C18 security guard columnsluteolin and chicoric acid may be important for the amelioration of (4.0 mm × 3.0 mm; Phenomenex, Torrance, CA, USA) were used toLPS-induced oxidative stress and inﬂammation (Park et al., 2010a). protect the column at room temperature using a linear gradientIn an animal model, dandelion leaf water extract showed signiﬁ- elution [solvent A = acetonitrile/0.1% phosphoric acid (25:75, v/v);cant protective effects against carbon tetrachloride (CCl4 )-induced solvent B = acetonitrile/0.1% phosphoric acid (25:75, v/v)]. Solventliver injury, which indicates luteolin (including the glycosidic form) B increased from 1 to 100% in 30 min and was kept at 100% forand polyphenol contents in dandelion leaf (Park et al., 2010b). 5 min. Samples were dissolved in the mobile phase, and 5 L was In this study, we attempted to compare the antioxidative injected. The UV–vis spectrophotometer detector was set at 350 nmand anti-inﬂammatory activities of Taraxacum ofﬁcinale methanol for free luteolin, 320 nm for chicoric acid, and 280 nm for concur-extract (TOME) and water extract (TOWE) in LPS-stimulated RAW rent monitoring. The ﬂow rate was 1.0 mL/min. Free luteolin and264.7 cells and investigate their underlying molecular mechanisms. chicoric acid were used as external standards. Retention times ofIn addition, we estimated the biological activities of both extracts free luteolin and chicoric acid are 16.38 and 13.03 min, respectively.by comparing their compositional differences regarding total phe-nol and functional phytochemical content, including luteolin and 2.4. Total phenol contentchicoric acid. Total phenol concentration was determined by the method of2. Materials and methods Bray and Thorpe (1954), with slight modiﬁcations. Both extracts were mixed in methanol/water [60:40 (v/v) with 0.3% HCl]. One2.1. Reagents hundred microliters of the samples were added to 2.0 mL of 2% Na2 CO3 . After 2 min, 100 L of 50% Folin-Ciocalteau reagent were Dulbecco’s modiﬁed Eagle Medium (DMEM), fetal bovine serum added and incubated for 30 min at room temperature. Total phenol(FBS), glutamine, TRIzol reagent, and MMLV ﬁrst strand cDNA content was measured at 750 nm and chlorogenic acid was used assynthesis kit were obtained from Invitrogen (Carlsbad, CA, USA). standard at concentrations of 0.01–0.2 mg/mL. Phenolic concentra-LPS, dimethyl sulfoxide, sodium dodecyl sulfate (SDS), NP-40, and tions were determined by comparison with the standard calibrationphenylmethylsulfonyl ﬂuoride were purchased from Sigma (St. curve.Louis, MO, USA). Anti-mouse iNOS antibody was obtained from BDTransduction Laboratories (Lexington, KY, USA), and anti-mouse 2.5. Cell culture and treatmentglyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodywas obtained from Abcam (Cambridge, UK). All other chemicals The RAW 264.7 murine macrophage cell line was obtainedwere of the highest commercial grade available. from the American Type Culture Collection (TIB-71; Rockville, MD, USA) and cultured DMEM supplemented with 10% FBS and2.2. Preparation of dandelion leaf extracts 2 mM l-glutamine. Various Korean medicinal plant extracts have been shown to inhibit LPS-induced NO production with an IC50 of Dried, powdered dandelion leaf was obtained from Min- 80 g/mL (Ryu et al., 2003). Based on this result, the highest doseDle-Leh-Food (Uiryeong, Korea). The scientiﬁc name of the of TOME was determined as 100 g/mL, since its IC50 value wascollected sample was determined by Prof. Myong Gi Chung at the about 80 g/mL. Cells were pre-incubated with and without indi-Gyeongsang National University, South Korea. A voucher speci- cated concentration extracts for 2 h, and then incubated with LPSmen (CMPark 04-12-2009) was deposited in the herbarium of the (1 g/mL) for 18 h at 37 ◦ C in a humidiﬁed atmosphere containingGyeongsang National University (GSNUC). Dandelion extracts were 5% CO2 to evaluate levels of inﬂammatory mediators and antioxida-obtained in 2 ways: methanol (TOME) and water (TOWE). For TOME tive enzyme activities. To analyze transcription factors, cells werepreparation, air-dried dandelion leaf powder (5 g) was mixed with incubated under the same conditions to keep the stimulation ofmethanol (50 mL) and heated at 85 ◦ C for 4 h. The same amount of LPS persistent, even though NF- B activation is known to peak atdandelion powder was extracted with 50 mL of distilled water and 15 min (Xiong et al., 2003).boiled at 100 ◦ C for 4 h in a double boiler. After extraction, bothextracts were ﬁltered (Whatmann paper no. 4). Then, TOME was 2.6. Nitrite production and cell viability measurementconcentrated in a rotary evaporator (Buchi, St. Gallen, Switzerland)and TOWE was lyophilized (Biotron, Bucheon, Korea). The recov- Nitrite accumulated in the culture medium as an indicator ofery rate of TOME and TOWE was 34.2 and 36.1%, respectively. To NO production was measured according to the Griess reactionprepare high-performance liquid chromatography (HPLC) samples, (D’Agostino et al., 2001). Brieﬂy, 100 L of each medium super-both extracts were dissolved in 100 mL of 10% methyl alcohol. Then, natant was mixed with 50 L of 1% sulfanilamide (in 5% phosphoricthey were mixed with the 100 mL of ethyl acetate. The mixtures acid) and 50 L of 0.1% naphthylenediamine dihydrochloride andwere shaken thoroughly. After 30 min, the separated ethyl acetate then incubated at room temperature for 10 min. Absorbance atlayers were collected and extracted in a rotary evaporator. This 550 nm was measured with a NaNO2 serial dilution standard curve
836 C.M. Park et al. / Journal of Ethnopharmacology 133 (2011) 834–842from 0 to 100 M, and nitrite production was determined. Cell via- violet transilluminator. Data were quantiﬁed using the Gel Doc EQbility was assessed through measuring the uptake of the supravital System (Bio-Rad Laboratories, Hercules, CA, USA). All signals weredye neutral red by viable cells according to the procedure of Fautz normalized to mRNA levels of the house keeping gene, GAPDH, andet al. (1991). expressed as a ratio.2.7. Lipid peroxide and glutathione (GSH) content 2.10. Western blot analysis for iNOS Lipid peroxidation was measured by thiobarbituric acid reactive The cells were disrupted with a Handy Sonic Disrupter (Tomysubstance production, as described by Fraga et al. (1988). GSH was Seiko, Tokyo, Japan) and centrifuged at 13,000 × g and 4 ◦ C formeasured by an enzymatic recycling procedure described by Tietze 20 min. Protein content was determined by the Bradford assay(1969), in which GSH is sequentially oxidized by 5,5U-dithiobis (2- (Bradford, 1976). Protein samples (50 g) from each lysate werenitrobenzoic acid) and reduced by NADPH in the presence of GSH separated on a 10% SDS-polyacrylamide gel and electrotrans-reductase. ferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Membranes were blocked for 1 h at room temperature2.8. Assay of antioxidative enzyme activities with 5% nonfat dry milk. The reactions were then incubated at 4 ◦ C overnight with a 1:1000 dilution of rabbit anti-mouse iNOS and Superoxide dismutase (SOD) activity was determined by mon- GAPDH antibodies in blocking buffer. After the membranes wereitoring the auto-oxidation of pyrogallol (Marklund and Marklund, washed, they were further incubated with a 1:1000 dilution of alka-1974). A unit of SOD activity was deﬁned as the amount of enzyme line phosphatase-conjugated goat anti-mouse immunoglobulin Gthat inhibited the rate of pyrogallol oxidation. Catalase activity was secondary antibody for 1 h at room temperature. The blots wereanalyzed according to the method of Aebi (1984), by following the developed with 5-bromo-4-chloro-3-indoyl phosphate/nitrobluedecrease in absorbance of H2 O2 at 240 nm. One unit of catalase tetrazolium color developing solution, and data were quantiﬁedwas deﬁned as the amount of enzyme that decomposes 1.0 M of using the Gel Doc EQ System (Bio-Rad, Hercules, USA). All sig-H2 O2 to H2 O and O2 per minute. GSH-peroxidase (GPx) activity was nals were normalized to protein levels of the housekeeping gene,assayed according to the method of Lawrence and Burk (1976). A GAPDH, and expressed as a ratio.unit of GPx was deﬁned as the amount of enzyme that oxidized 1 nMof NADPH per minute. GSH-reductase (GR) activity was measured 2.11. Assay of NF-ÄB translocationby following the oxidation of NADPH. A unit of GR was deﬁned asthe amount of enzyme that catalyzed a reduction of 1 nM of NADPH Nuclear protein was extracted using the method of Dignam et al.per minute. (1983), with slight modiﬁcations. Cells were lysed with buffer, vor- texed, kept on ice for 5 min, and centrifuged at 500 × g for 5 min2.9. Reverse transcription-polymerase chain reaction (RT-PCR) at 4 ◦ C. Protein concentration was determined by the Bradford assay. For the electrophoretic mobility shift assay, NF- B-speciﬁc Cells (5 × 106 cells/dish) in 100-mm dishes were pre-incubated oligonucleotide was end-labeled with [ -32 P]-ATP using T4 polynu-with and without indicated concentrations of luteolin and chicoric cleotide kinase (Promega, Madison, WI, USA) and puriﬁed using theacid for 2 h, and then incubated with LPS (1 g/mL) for 18 h. Total microspin G-25 column (Amersham biosciences, Cardiff, UK). FiveRNA was isolated using TRIzol reagent (Invitrogen) according to milligrams of nuclear protein, binding buffer, 32 P-labeled NF- B,the manufacturer’s instruction. Cells were lysed by TRIzol reagent and loading buffer were incubated for 30 min at room tempera-and transferred to the microfuge tube. Chloroform was added and ture. DNA-protein complexes were separated by electrophoresistotal RNA was collected in the aqueous phase after centrifuga- and the gels were exposed to a phosphor screen (Packard, Meri-tion. Finally, RNA was precipitated by isopropyl alcohol, and then den, CT, USA) for 2 h at −20 ◦ C, and the bands were quantitated bywashed and re-dissolved in diethyl pyrocarbonate-treated water. a phosphor imager (Packard).The concentrations of RNA samples were measured with a spec-trophotometer (Ultraspec 3000; GE Healthcare, Buckinghamshire, 2.12. Statistical analysisUK) to determine OD260 and OD260/280 values. Five micrograms oftotal RNA were used to produce ﬁrst strand cDNA using the MMLV All data are expressed as mean (SD). Statistical analyses wereﬁrst strand cDNA synthesis kit (Invitrogen). PCR (Corbett Research, performed using SPSS version 13.0 (SPSS Institute, Chicago, IL, USA).Sydney, Australia) was carried out in 50 L reaction mixture con- One-way ANOVA with Duncan’s multiple range test was used totaining the ﬁrst strand cDNA, 10× PCR buffer, 2.5 mM dNTPs, 20 pM examine differences between groups. p Values < 0.05 were consid-of each primer, and Taq. DNA polymerase (Bioneer, Korea). PCR ered signiﬁcant, unless stated otherwise.primer sequences for iNOS and GAPDH were as follows: primers foriNOS were 5 -GCC TTC AAC ACC AAG GTT GTC TGC A-3 (sense) and 3. Results5 -TCA TTG TAC TCT GAG GGC TGA CAC A-3 (anti-sense); primersfor GAPDH were 5 -CAA TGC CAA GTA TGA TGA CAT-3 (sense) and 3.1. Total phenol, luteolin, and chicoric acid contents5 -CCT GTT ATT ATG GGG GTC TG-3 (anti-sense). The expectedsizes of PCR products were 920 bp for iNOS and 375 bp for GAPDH. The total phenol content of TOME and TOWE was 0.17 ± 0.02The ampliﬁcation proﬁle consisted of an initial denaturation at and 0.14 ± 0.01 mg chlorogenic acid equivalent per gram of dried94 ◦ C for 1 min, followed by denaturation at 94 ◦ C for 2 min 30 s dandelion, respectively. The luteolin concentration of TOME and(for iNOS and GAPDH), annealing at 59 ◦ C for 2 min (iNOS) and at TOWE was 34.2 ± 0.08 and 3.53 ± 0.04 g, and chicoric acid was49 ◦ C for 2 min (GAPDH), and extension at 72 ◦ C for 2 min (for iNOS 128.6 ± 2.13 and 18.9 ± 0.07 g/g of dried dandelion, respectively.and GAPDH). Twenty seven cycles for iNOS and GAPDH resulted Chromatograms of both phytochemicals are shown in Fig. 1.in the best ampliﬁcation proﬁles to recognize differences betweensamples. Expression of the house keeping gene, GAPDH, served as 3.2. NO production and cell viabilitycontrol. The PCR products speciﬁc for each cDNA were analyzedby electrophoresis on 2% agarose gel containing ethidium bromide Fig. 2 shows the effects of TOME and TOWE on nitrite production(0.5 g/mL) at 50 V for 70 min and were visualized with an ultra induced by LPS in RAW 264.7 cells. NO concentration was sharply
C.M. Park et al. / Journal of Ethnopharmacology 133 (2011) 834–842 837Fig. 1. HPLC chromatograms of free luteolin and chicoric acid in TOME and TOWE (A, free luteolin in TOME; B, chicoric acid in TOME; C, free luteolin in TOWE; D, chicoricacid in TOWE).increased by 33.6 and 49.2 times, respectively, compared with the 3.4. GSH content and antioxidative enzyme activitiesuntreated group. TOME and TOWE suppressed NO production withan IC50 of 79.9 and 157.6 g/mL, respectively, without cytotoxicity GSH, one of the primary defense systems against oxidative(data not shown). stress, was measured to evaluate the antioxidative capacity of TOME and TOWE. Fig. 4 shows that GSH content was decreased3.3. Lipid peroxide concentration by LPS treatment and recovered signiﬁcantly (p < 0.05) by both extracts in a dose-dependent manner. In addition, antioxidative The effect of TOME and TOWE on lipid peroxide level was exam- enzymes, including catalase, SOD, GPx, and GR, were signiﬁcantlyined in LPS-stimulated RAW 264.7 cells. As shown in Fig. 3, TOME (p < 0.05) increased by TOME and TOWE treatment (Table 1). Whenand TOWE signiﬁcantly (p < 0.05) inhibited MDA concentration in comparing both extracts, TOME was found to increase catalasea dose-dependent manner. and SOD activities more than TOWE. However, in the GSH-related
838 C.M. Park et al. / Journal of Ethnopharmacology 133 (2011) 834–842 A 25 B 25 e c 20 20 c Nitrite concentration(μM) Nitrite concentration(μM) d c b b 15 15 10 10 b 5 5 a a 0 0 TOME (μg/mL) 0 0 25 50 100 TOWE (μg/mL) 0 0 25 50 100 LPS (1 μg/mL ) - + + + + LPS (1 μg/mL) - + + + +Fig. 2. Effects of TOME (Panel A) and TOWE (Panel B) on NO production in LPS-stimulated RAW 264.7 cells. Cells were pre-incubated with and without indicated concentrationsof agents for 2 h, then incubated with LPS (1 g/mL) for 18 h at 37 ◦ C in a humidiﬁed atmosphere containing 5% CO2 . Data represent the means ± S.D. of triplicate experiments.Values sharing the same superscript are not signiﬁcantly different at p < 0.05.antioxidative enzyme system, both extracts restored a similar iNOS expression signiﬁcantly (p < 0.05) only at the highestamount of GSH activity, mostly via high induction of GR activity, concentration.though TOME strongly oxidized GSH. 3.6. NF-ÄB activity3.5. iNOS gene expression level NF- B activation, an important transcription factor for inﬂam- As shown in Figs. 5 and 6, iNOS gene expression was hardly matory mediation, was measured to evaluate the effect ofdetected in the untreated group, whereas it was highly up- dandelion extracts in LPS-induced inﬂammation. The resultsregulated in the control group. TOME treatment signiﬁcantly showed that NF- B activity was hardly detected in the untreated(p < 0.05) suppressed the elevated iNOS expression in a dose- group, but signiﬁcantly (p < 0.05) increased in the LPS-treated con-dependent manner, whereas TOWE suppressed the increased trol group. Elevated NF- B activity was dose-dependently reduced A 1.2 B 0.9 c c 0.8 1 b b b 0.7 b TBARS (nmole MDA) TBARS (nmole MDA) 0.8 0.6 ab a a a 0.5 0.6 0.4 0.4 0.3 0.2 0.2 0.1 0 0 TOME (μg/mL) 0 0 25 50 100 TOWE (μg/mL) 0 0 25 50 100 LPS (1 μg/mL - + + + + LPS (1 μg/mL) - + + + +Fig. 3. Effects of TOME (Panel A) and TOWE (Panel B) on TBARS generation in LPS-stimulated RAW 264.7 cells. Cells were pre-incubated with and without indicatedconcentrations of agents for 2 h, then incubated with LPS (1 g/mL) for 18 h at 37 ◦ C in a humidiﬁed atmosphere containing 5% CO2 . Data represent the means ± S.D. oftriplicate experiments. Values sharing the same superscript are not signiﬁcantly different at p < 0.05.
C.M. Park et al. / Journal of Ethnopharmacology 133 (2011) 834–842 839 A 3.5 B 3 d c GSH concentration (pmol/2.53×107 cell) GSH concentration (pmol/2.32×107 cell) 3 c 2.5 e d b 2.5 c 2 a b 2 1.5 a 1.5 1 1 0.5 0.5 0 0 TOME (μg/ mL) 0 0 25 50 100 TOWE (μg/mL) 0 0 25 50 100 LPS (1 μg/mL) - + + + + LPS (1 μg/mL) - + + + +Fig. 4. Effects of TOME (Panel A) and TOWE (Panel B) on GSH concentration in LPS-stimulated RAW 264.7 cells. Cells were pre-incubated with and without indicatedconcentrations of agents for 2 h, then incubated with LPS (1 g/mL) for 18 h at 37 ◦ C in a humidiﬁed atmosphere containing 5% CO2 . Data represent the means ± S.D. oftriplicate experiments. Values sharing the same superscript are not signiﬁcantly different at p < 0.05.by the dandelion extracts, and evident inhibition was observed in been shown to have the greatest antioxidative activity among refer-TOME-treated cells (Fig. 7). ence compounds such as echinacoside, caffeic acid, and rosmarinic acid (Dalby-Brown et al., 2005). Besides, other phytochemicals,4. Discussion such as chlrogenic acid and chrysoeriol, from common dandelion were also reported that they exhibited anti-inﬂammatory activ- Numerous studies have attempted to isolate and evaluate bioac- ities in RAW 264.7 cells. Chlorogenic acid inhibited LPS-inducedtive compounds in dandelion, because this plant has long been used COX-2 expression through NF- B attenuation and chrysoeriol sup-as a folklore medicine; these compounds have subsequently been pressed iNOS activation via AP-1 mitigation in LPS stimulated RAWidentiﬁed as luteolin, chicoric acid, chlorogenic acid, and chryso- 264.7 cells (Choi et al., 2005; Shan et al., 2009). In this study, weeriol (Williams et al., 1996; Hu and Kitts, 2005; Schutz et al., analyzed both phytochemicals, luteolin and chicoric acid, due to2005). Among these compounds, luteolin and chicoric acid play their synergistic NO inhibitory activity in LPS-stimulated murinediverse roles as antioxidants and the prevention of inﬂammation macrophages (Supplementary Data). It has been reported that(Dalby-Brown et al., 2005; Hu and Kitts, 2005; Harris et al., 2006; plant extracts containing high total phenol concentrations showChen et al., 2007). Luteolin, a ﬂavone, has been shown to exert strong antioxidative capacity (Velioglu et al., 1998). In addition,its anti-inﬂammatory activity via NF- B and activator protein-1 phytochemicals derived from common dandelion, including lute-modulation in LPS-stimulated RAW 264.7 cells (Harris et al., 2006; olin, chicoric acid, chlorogenic acid, chrysoeriol, also reported theyChen et al., 2007). Chicoric acid, a derivative of caffeic acid, has have remarkable antioxidative activities (Kim et al., 2004; HuangTable 1Effects of TOME and TOWE on antioxidative enzyme activities in LPS-stimulated RAW 264.7 cells. Untreated TOME or TOWE ( g/mL) + LPSa (1 g/mL) 0 25 50 100 Catalase ( M/mg protein/min) TOME 0.55 ± 0.02a 0.65 ± 0.03a 2.65 ± 0.24b 2.65 ± 0.18b 3.31 ± 0.27c TOWE 0.97 ± 0.02a 1.01 ± 0.02a 1.26 ± 0.03a 2.03 ± 0.03b 2.17 ± 0.05b SODb (unit/mg protein) TOME 19.5 ± 3.4a 24.0 ± 3.8ab 33.0 ± 9.4bc 41.5 ± 2.9c 71.4 ± 4.9d TOWE 23.5 ± 1.8a 26.6 ± 1.8a 39.1 ± 4.3b 37.1 ± 2.5b 41.0 ± 2.3b GPxc (unit/mg protein) TOME 3.17 ± 0.46bc 2.70 ± 0.36ab 2.36 ± 0.12a 3.43 ± 0.21c 4.12 ± 0.20d TOWE 1.30 ± 0.14a 2.57 ± 0.05b 2.40 ± 0.13b 2.90 ± 0.08c 3.01 ± 0.24c GRd (unit/mg protein) TOME 142.8 ± 24.7c 81.7 ± 10.5ab 55.9 ± 6.60a 96.5 ± 25.2b 131.4 ± 20.7c TOWE 69.8 ± 4.4b 55.1 ± 5.4a 75.3 ± 3.5bc 81.1 ± 2.8c 100.3 ± 7.7dData represent the means ± S.D. of triplicate experiments. One-way ANOVA and Duncan’s multiple range test was used to examine the difference among groups. A valuesharing same superscript is not signiﬁcantly different at p < 0.05. a Lipopolysaccharide. b Superoxide dismutase. c Glutathione peroxidase. d Glutathione reductase.
840 C.M. Park et al. / Journal of Ethnopharmacology 133 (2011) 834–842 Fig. 6. Effects of TOME (Panel A) and TOWE (Panel B) on iNOS protein expression in LPS-stimulated RAW 264.7 cells. Panel A and B show iNOS protein expression levels by TOME and TOWE determined by Western blot analysis. GAPDH was used as an internal control. All signals were normalized to protein levels of GAPDH and expressed as a ratio. Cells were pre-incubated with and without indicated concen- trations of agents for 2 h, then incubated with LPS (1 g/mL) for 18 h at 37 ◦ C in a humidiﬁed atmosphere containing 5% CO2 . Data represent the means ± S.D. of tripli-Fig. 5. Effects of TOME (Panel A) and TOWE (Panel B) on iNOS mRNA expression in cate experiments. Values sharing the same superscript are not signiﬁcantly differentLPS-stimulated RAW 264.7 cells. Panel A and B show iNOS mRNA expression levels at p < 0.05.by TOME and TOWE determined by RT-PCR analysis. GAPDH was used as an internalcontrol. All signals were normalized to mRNA levels of GAPDH and expressed as aratio. Cells were pre-incubated with and without indicated concentrations of agentsfor 2 h, then incubated with LPS (1 g/mL) for 18 h at 37 ◦ C in a humidiﬁed atmo- delion extracts effectively removed LPS-induced oxidative stress.sphere containing 5% CO2 . Data represent the means ± S.D. of triplicate experiments. Furthermore, treatment with dandelion extracts also restored LPS-Values sharing the same superscript are not signiﬁcantly different at p < 0.05. disturbed GSH levels. The elevation in GSH and antioxidative enzyme activity may be responsible for the suppression of oxida-et al., 2009; Park et al., 2010b). Our data show that dandelion tive stress in LPS-stimulated RAW 264.7 cells. Although TOME andextracts ameliorated LPS-induced oxidative stress, as indicated by TOWE both signiﬁcantly suppressed oxidative stress, the antiox-suppressed MDA concentration, through the elevation of antiox- idative activity of TOME was stronger than that of TOWE. Thisidative enzyme activities, such as catalase, SOD, GPx, and GR, and difference was attributed to the fact that TOME contained moreGSH restoration. Living organisms contain SOD, which removes total phenols, as well as luteolin and chicoric acid than TOWE.superoxide, and are thus protected from injury caused by ROS. NO is synthesized from l-arginine by the 3 major NOS iso-Catalase mediates its function by the removal of H2 O2 generated forms, namely, neuronal NOS (nNOS), endothelial NOS (eNOS),by auto-oxidation of lipids and the oxidation of organic substances and iNOS. nNOS and eNOS are controlled by Ca2+ /calmodulin, but(Sharma et al., 1991). Our study revealed that LPS treatment signiﬁ- iNOS is up-regulated by inﬂammatory stimuli such as cytokines,cantly suppressed GPx and GR activities, but did not affect catalase IL, and bacterial endotoxin (Yu et al., 2002). Excessively gener-and SOD. However, dandelion extracts signiﬁcantly elevated the ated NO induces nitrosative and oxidative DNA damage, and hasactivities of antioxidative enzymes compared to LPS-treated con- been shown to be elevated in precancerous and cancerous lesionstrols. Cho et al. (2002) reported that hepatic GSH content was (Kundu and Surh, 2008). When macrophages are activated byincreased signiﬁcantly following supplementation of dandelion leaf inﬂammatory stimuli, NF- B translocates into the nucleus andextract in hypercholesterolemic rats. In this study, TOME and TOWE binds to the promoter region of inﬂammatory mediators. Prolongedincreased antioxidative enzyme activities, such that these dan- up-regulation of inﬂammatory mediators by NF- B enhances the
C.M. Park et al. / Journal of Ethnopharmacology 133 (2011) 834–842 841 Acknowledgement This study was supported by the 2009 Inje University research grant. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jep.2010.11.015. References Aebi, H., 1984. Catalase in vitro. Methods in Enzymology 105, 121–126. Allen, R.G., Tresini, M., 2000. Oxidative stress and gene regulation. Free Radical and Biological Medicine 28, 463–499. Bisset, N.G., Wichtl, M., 1994. Herbal Drugs and Phytopharmaceuticals: A Handbook for Practice on a Scientiﬁc Basis. CRC Press. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro- gram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Bray, H.G., Thorpe, W.V., 1954. Analysis of phenolic compounds of interest in metabolism. Methods of Biochemical Analysis 1, 27–52. 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Anti-inﬂammatory effects of chemically modiﬁed tetracyclines by the inhibition of nitric oxide and interleukin-12 synthesis in J774 cell line. International Immunopharmacology 1, 1765–1776.Fig. 7. Effects of TOME (Panel A) and TOWE (Panel B) on NF- B activity in LPS- Dalby-Brown, L., Barsett, H., Landbo, A.K., Meyer, A.S., Molgaard, P., 2005. Synergisticstimulated RAW 264.7 cells. Panel A and B show DNA binding activity of NF- B by antioxidative effects of alkamides, caffeic acid derivatives, and polysaccharideTOME and TOWE determined by EMSA analysis. Values are expressed as relative fractions from Echinacea purpurea on in vitro oxidation of human low-density lipoproteins. Journal of Agricultural and Food Chemistry 53, 9413–9423.intensity of radioactivity. Cells were pre-incubated with and without indicated con- Dignam, J.D., Lebovitz, R.M., Roeder, R.G., 1983. Accurate transcription initiation bycentrations of agents for 2 h, then incubated with LPS (1 g/mL) for 18 h at 37 ◦ C in a RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleichumidiﬁed atmosphere containing 5% CO2 . Data represent the means ± S.D. of tripli- Acids Research 11, 1475–1489.cate experiments. Values sharing the same superscript are not signiﬁcantly different Fautz, R., Husein, B., Hechenberger, C., 1991. Application of the neutral red assayat p < 0.05. (NR assay) to monolayer cultures of primary hepatocytes: rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS). Mutation Research 253, 173–179.inﬂammatory response, which is implicated in the development of Fraga, C.G., Leibovitz, B.E., Tappel, A.L., 1988. Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization andchronic diseases such as cancer and arteriosclerosis (Kang et al., comparison with homogenates and microsomes. Free Radical and Biological2000). To alleviate inﬂammation, the potential efﬁcacy of many Medicine 4, 155–161.phytochemicals as non-steroidal anti-inﬂammatory drugs has been Gius, D., Botero, A., Shah, S., Curry, H.A., 1999. Intracellular oxidation/reduction sta- tus in the regulation of transcription factors NF-kappaB and AP-1. Toxicologyexamined, and some of these compounds have been identiﬁed as Letter 106, 93–106.iNOS and COX-2 selective inhibitors (Surh et al., 2001). Therefore, Gordon, S., 2002. Pattern recognition receptors: doubling up for the innate immunethe development of nutraceutical agents, which may attenuate response. Cell 111, 927–930. Hagymasi, K., Blazovics, A., Lugasi, A., Kristo, S., Feher, J., Kery, A., 2000. In vitroinﬂammatory signaling cascades in activated macrophages, is con- antioxidant evaluation of dandelion (Taraxacum ofﬁcinale WEB.) water extracts.sidered a useful strategy for inﬂammatory diseases. In this study, Acta Alimentaria 29, 1–7.the molecular mechanisms of dandelion extract-induced NO sup- Harris, G.K., Qian, Y., Leonard, S.S., Sbarra, D.C., Shi, X., 2006. Luteolin and chrysin differentially inhibit cyclooxygenase-2 expression and scavenge reactive oxy-pression were examined. We found that iNOS gene expression and gen species but similarly inhibit prostaglandin-E2 formation in RAW 264.7 cells.NF- B activation were down-regulated following treatment with Journal of Nutrition 136, 1517–1521.dandelion extracts, which was in accordance with the observed Hu, C., Kitts, D.D., 2003. Antioxidant, prooxidant, and cytotoxic activities of solvent-suppression of NO production. In addition to the components fractionated dandelion (Taraxacum ofﬁcinale) ﬂower extracts in vitro. Journal of Agricultural and Food Chemistry 51, 301–310.we examined, other functional components of Taraxacum ofﬁci- Hu, C., Kitts, D.D., 2005. Dandelion (Taraxacum ofﬁcinale) ﬂower extract suppressesnale Weber, including polysaccharides from dandelion leaf water both reactive oxygen species and nitric oxide and prevents lipid oxidation inextract, have been shown to inhibit NF- B-mediated inﬂammatory vitro. Phytomedicine 12, 588–597. Huang, D.W., Kuo, Y.H., Lin, F.Y., Lin, Y.L., Chiang, W., 2009. Effect of Adlay (Coixsignaling pathways in CCl4 -intoxicated rat livers (Park et al., 2010c). lachryma-jobi L. var. ma-yuen Stapf) Testa and its phenolic components on Cu2+ - In conclusion, these results suggest that dandelion extracts may treated low-density lipoprotein (LDL) oxidation and lipopolysaccharide (LPS)-attenuate inﬂammatory responses and oxidative stress by reducing induced inﬂammation in RAW 264.7 macrophages. 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