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Journal of Ethnopharmacology 133 (2011) 834–842



                                                               Contents lists available at ScienceDirect


                                                       Journal of Ethnopharmacology
                                             journal homepage: www.elsevier.com/locate/jethpharm




Taraxacum officinale Weber extracts inhibit LPS-induced oxidative stress and
nitric oxide production via the NF- B modulation in RAW 264.7 cells
Chung Mu Park a , Ji Young Park b , Kyung Hee Noh a , Jin Hyuk Shin a , Young Sun Song a,∗
a
    Department of Smart Foods and Drugs, Inje University, Obang-dong 607, Gimhae, Gyeongnam 621-749, Republic of Korea
b
    Sambang High School, Sambang-dong, Gimhae, Gyeongnam 621-910, Republic of Korea




a r t i c l e          i n f o                          a b s t r a c t

Article history:                                        Ethnopharmacological relevance: The common dandelion (Taraxacum officinale G.H. Weber ex Wiggers,
Received 21 June 2010                                   Asteraceae) has been widely used in folklore medicine to treat dyspepsia, heartburn, and spleen and liver
Received in revised form 31 October 2010                disorders.
Accepted 3 November 2010
                                                        Aim of the study: To compare the antioxidative and anti-inflammatory activities of Taraxacum officinale
Available online 11 November 2010
                                                        methanol extract (TOME) and water extract (TOWE) in lipopolysaccharide (LPS)-stimulated RAW 264.7
                                                        cells and assess their constitutional differences, including luteolin, chicoric acid, and total phenol content.
Keywords:
                                                        Materials and methods: Antioxidative enzyme activities, nitric oxide (NO) production, and inducible NO
Taraxacum officinale Weber
Asteraceae
                                                        synthase (iNOS) and nuclear factor (NF)- B expression were estimated by biochemical analysis, the Griess
Oxidative stress                                        reaction, reverse transcription-polymerase chain reaction, western hybridization, and electrophoretic
Inflammation                                             mobility shift assay. High-performance liquid chromatography and the Folin-Ciocalteau method were
Inducible nitric oxide synthase                         used to analyze functional phytochemicals and total phenol content.
Nuclear factor- B                                       Results: TOME and TOWE significantly reduced NO production with an IC50 of 79.9 and 157.5 g/mL,
                                                        respectively, without cytotoxicity. Depleted glutathione (GSH) and antioxidative enzyme activities,
                                                        including superoxide dismutase, catalase, GSH-peroxidase, and GSH-reductase, were restored by dande-
                                                        lion extracts. Both extracts inhibited LPS-stimulated iNOS gene expression and that of its transcription
                                                        factor, NF- B, in parallel with nitrite reduction. TOME showed more potent antioxidative and anti-
                                                        inflammatory capacities than TOWE, which was attributable to its high total phenol, luteolin, and chicoric
                                                        acid content.
                                                        Conclusions: These results indicate that TOME and TOWE inhibit oxidative stress and inflammatory
                                                        responses through elevated de novo synthesis of antioxidative enzymes and suppression of iNOS expres-
                                                        sion by NF- B inactivation.
                                                                                                                    © 2010 Elsevier Ireland Ltd. All rights reserved.



1. Introduction                                                                          plex with the inhibitor protein I B . In response to inflammatory
                                                                                         stimuli such as reactive oxygen species (ROS), lipopolysaccharide
    Macrophages protect the body from external intruders through                         (LPS), and cytokines, I B is phosphorylated and released from NF-
phagocytosis. During this process, macrophages produce many                                B. Activated NF- B, namely, p50 and p65 dimer, migrates to the
kinds of inflammatory mediators such as interleukin (IL)-1 , tumor                        nucleus and up-regulates inflammation-related genes such as iNOS
necrosis factor (TNF)- , nitric oxide (NO), and prostaglandins.                          and cyclooxygenase (COX)-2 (Gius et al., 1999; Allen and Tresini,
Excessively generated mediators have been implicated in various                          2000).
physiological disorders, including tumor formation, autoimmune                               The common dandelion (Taraxacum officinale G.H. Weber ex
reactions, and inflammatory diseases (Gordon, 2002). NO, which is                         Wiggers, Asteraceae) is widely used as a folklore medicinal
one of the major inflammatory mediators, is controlled by NO syn-                         plant against various disorders such as liver diseases, gallbladder
thases (NOS), with inducible NOS (iNOS) markedly up-regulated                            disorders, digestive complaints, and arthritic and rheumatic dis-
in inflammatory disorders (Kleinert et al., 2004). The expression                         eases (Racz-Kotilla et al., 1974; Bisset and Wichtl, 1994; Newall
of iNOS is regulated by transcription factor nuclear factor (NF)-                        et al., 1996). Diverse biological activities of dandelion, such as
  B, which exists ubiquitously in the cytoplasm as a heterodimer                         anti-angiogenic, anti-inflammatory, and anti-nociceptive activities
consisting of p50 and p65 as an inactive form by forming a com-                          were estimated in mice and murine macrophage cell line (Jeon
                                                                                         et al., 2008). In addition, signaling molecule for anti-inflammatory
                                                                                         activity was analyzed in RAW 264.7 cells (Koh et al., 2010). Dan-
    ∗ Corresponding author. Tel.: +82 55 320 3235; fax: +82 55 321 0691.                 delion leaf extract has been shown to exhibit a protective effect
      E-mail address: fdsnsong@inje.ac.kr (Y.S. Song).                                   against cholecystokinin octapeptide-induced acute pancreatitis

0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2010.11.015
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 extractants
liorated 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 peroxide
scavenger, because of its high polyphenol content (Hagymasi et al.,               2.3. HPLC analysis for luteolin and chicoric acid
2000). 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 followed
Lima et al., 2007). In our previous study, 4 kinds of dandelion                   by the method of Hu and Kitts (2003). Two identical Agilent
extract (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-C18
production. 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 columns
luteolin and chicoric acid may be important for the amelioration of               (4.0 mm × 3.0 mm; Phenomenex, Torrance, CA, USA) were used to
LPS-induced oxidative stress and inflammation (Park et al., 2010a).                protect the column at room temperature using a linear gradient
In 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)]. Solvent
liver injury, which indicates luteolin (including the glycosidic form)            B increased from 1 to 100% in 30 min and was kept at 100% for
and 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 nm
and 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 and
264.7 cells and investigate their underlying molecular mechanisms.                chicoric acid were used as external standards. Retention times of
In 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 content
chicoric acid.
                                                                                     Total phenol concentration was determined by the method of
2. 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]. One
2.1. 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 as
synthesis 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 calibration
phenylmethylsulfonyl fluoride were purchased from Sigma (St.                       curve.
Louis, MO, USA). Anti-mouse iNOS antibody was obtained from BD
Transduction Laboratories (Lexington, KY, USA), and anti-mouse                    2.5. Cell culture and treatment
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody
was obtained from Abcam (Cambridge, UK). All other chemicals                         The RAW 264.7 murine macrophage cell line was obtained
were of the highest commercial grade available.                                   from the American Type Culture Collection (TIB-71; Rockville,
                                                                                  MD, USA) and cultured DMEM supplemented with 10% FBS and
2.2. 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 dose
Dle-Leh-Food (Uiryeong, Korea). The scientific name of the                         of TOME was determined as 100 g/mL, since its IC50 value was
collected 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 LPS
men (CMPark 04-12-2009) was deposited in the herbarium of the                     (1 g/mL) for 18 h at 37 ◦ C in a humidified atmosphere containing
Gyeongsang 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 were
preparation, air-dried dandelion leaf powder (5 g) was mixed with                 incubated under the same conditions to keep the stimulation of
methanol (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 at
dandelion 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, both
extracts were filtered (Whatmann paper no. 4). Then, TOME was                      2.6. Nitrite production and cell viability measurement
concentrated 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 of
ery rate of TOME and TOWE was 34.2 and 36.1%, respectively. To                    NO production was measured according to the Griess reaction
prepare 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% phosphoric
they were mixed with the 100 mL of ethyl acetate. The mixtures                    acid) and 50 L of 0.1% naphthylenediamine dihydrochloride and
were shaken thoroughly. After 30 min, the separated ethyl acetate                 then incubated at room temperature for 10 min. Absorbance at
layers 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) 834–842


from 0 to 100 M, and nitrite production was determined. Cell via-              violet transilluminator. Data were quantified using the Gel Doc EQ
bility was assessed through measuring the uptake of the supravital             System (Bio-Rad Laboratories, Hercules, CA, USA). All signals were
dye neutral red by viable cells according to the procedure of Fautz            normalized to mRNA levels of the house keeping gene, GAPDH, and
et 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 (Tomy
substance production, as described by Fraga et al. (1988). GSH was             Seiko, Tokyo, Japan) and centrifuged at 13,000 × g and 4 ◦ C for
measured 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 were
nitrobenzoic 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 temperature
2.8. 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 were
itoring 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 G
that inhibited the rate of pyrogallol oxidation. Catalase activity was         secondary antibody for 1 h at room temperature. The blots were
analyzed according to the method of Aebi (1984), by following the              developed with 5-bromo-4-chloro-3-indoyl phosphate/nitroblue
decrease in absorbance of H2 O2 at 240 nm. One unit of catalase                tetrazolium color developing solution, and data were quantified
was 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 nM
of NADPH per minute. GSH-reductase (GR) activity was measured                  2.11. Assay of NF-ÄB translocation
by following the oxidation of NADPH. A unit of GR was defined as
the 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 min
2.9. 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 the
acid for 2 h, and then incubated with LPS (1 g/mL) for 18 h. Total             microspin G-25 column (Amersham biosciences, Cardiff, UK). Five
RNA 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 electrophoresis
total 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 by
washed 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 analysis
UK) to determine OD260 and OD260/280 values. Five micrograms of
total RNA were used to produce first strand cDNA using the MMLV                    All data are expressed as mean (SD). Statistical analyses were
first 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 to
taining 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 for
iNOS were 5 -GCC TTC AAC ACC AAG GTT GTC TGC A-3 (sense) and                   3. Results
5 -TCA TTG TAC TCT GAG GGC TGA CAC A-3 (anti-sense); primers
for GAPDH were 5 -CAA TGC CAA GTA TGA TGA CAT-3 (sense) and                    3.1. Total phenol, luteolin, and chicoric acid contents
5 -CCT GTT ATT ATG GGG GTC TG-3 (anti-sense). The expected
sizes of PCR products were 920 bp for iNOS and 375 bp for GAPDH.                  The total phenol content of TOME and TOWE was 0.17 ± 0.02
The amplification profile consisted of an initial denaturation at                and 0.14 ± 0.01 mg chlorogenic acid equivalent per gram of dried
94 ◦ 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 was
49 ◦ 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 between
samples. Expression of the house keeping gene, GAPDH, served as                3.2. NO production and cell viability
control. The PCR products specific for each cDNA were analyzed
by 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                                               837




Fig. 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, chicoric
acid in TOWE).


increased by 33.6 and 49.2 times, respectively, compared with the                       3.4. GSH content and antioxidative enzyme activities
untreated group. TOME and TOWE suppressed NO production with
an 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 decreased
3.3. 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 significantly
ined in LPS-stimulated RAW 264.7 cells. As shown in Fig. 3, TOME                        (p < 0.05) increased by TOME and TOWE treatment (Table 1). When
and TOWE significantly (p < 0.05) inhibited MDA concentration in                         comparing both extracts, TOME was found to increase catalase
a 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 concentrations
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 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 highest
amount of GSH activity, mostly via high induction of GR activity,                                         concentration.
though TOME strongly oxidized GSH.

                                                                                                          3.6. NF-ÄB activity
3.5. 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 of
detected in the untreated group, whereas it was highly up-                                                dandelion extracts in LPS-induced inflammation. The results
regulated 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 indicated
concentrations 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
triplicate 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 indicated
concentrations 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
triplicate 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-induced
tive 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 RAW
identified as luteolin, chicoric acid, chlorogenic acid, and chryso-                                                       264.7 cells (Choi et al., 2005; Shan et al., 2009). In this study, we
eriol (Williams et al., 1996; Hu and Kitts, 2005; Schutz et al.,                                                          analyzed both phytochemicals, luteolin and chicoric acid, due to
2005). Among these compounds, luteolin and chicoric acid play                                                             their synergistic NO inhibitory activity in LPS-stimulated murine
diverse 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 show
Chen 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 they
Chen et al., 2007). Chicoric acid, a derivative of caffeic acid, has                                                      have remarkable antioxidative activities (Kim et al., 2004; Huang

Table 1
Effects 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 ± 7.7d

Data 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 value
sharing 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 different
LPS-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 internal
control. All signals were normalized to mRNA levels of GAPDH and expressed as a
ratio. Cells were pre-incubated with and without indicated concentrations of agents
for 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 and
extracts 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. This
idative enzyme activities, such as catalase, SOD, GPx, and GR, and                       difference was attributed to the fact that TOME contained more
GSH 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 has
activities of antioxidative enzymes compared to LPS-treated con-                         been shown to be elevated in precancerous and cancerous lesions
trols. Cho et al. (2002) reported that hepatic GSH content was                           (Kundu and Surh, 2008). When macrophages are activated by
increased significantly following supplementation of dandelion leaf                       inflammatory stimuli, NF- B translocates into the nucleus and
extract in hypercholesterolemic rats. In this study, TOME and TOWE                       binds to the promoter region of inflammatory mediators. Prolonged
increased 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.


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Papadie 2

  • 1. Journal of Ethnopharmacology 133 (2011) 834–842 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Taraxacum officinale Weber extracts inhibit LPS-induced oxidative stress and nitric oxide production via the NF- B modulation in RAW 264.7 cells Chung Mu Park a , Ji Young Park b , Kyung Hee Noh a , Jin Hyuk Shin a , Young Sun Song a,∗ a Department of Smart Foods and Drugs, Inje University, Obang-dong 607, Gimhae, Gyeongnam 621-749, Republic of Korea b Sambang High School, Sambang-dong, Gimhae, Gyeongnam 621-910, Republic of Korea a r t i c l e i n f o a b s t r a c t Article history: Ethnopharmacological relevance: The common dandelion (Taraxacum officinale G.H. Weber ex Wiggers, Received 21 June 2010 Asteraceae) has been widely used in folklore medicine to treat dyspepsia, heartburn, and spleen and liver Received in revised form 31 October 2010 disorders. Accepted 3 November 2010 Aim of the study: To compare the antioxidative and anti-inflammatory activities of Taraxacum officinale Available online 11 November 2010 methanol extract (TOME) and water extract (TOWE) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and assess their constitutional differences, including luteolin, chicoric acid, and total phenol content. Keywords: Materials and methods: Antioxidative enzyme activities, nitric oxide (NO) production, and inducible NO Taraxacum officinale Weber Asteraceae synthase (iNOS) and nuclear factor (NF)- B expression were estimated by biochemical analysis, the Griess Oxidative stress reaction, reverse transcription-polymerase chain reaction, western hybridization, and electrophoretic Inflammation mobility shift assay. High-performance liquid chromatography and the Folin-Ciocalteau method were Inducible nitric oxide synthase used to analyze functional phytochemicals and total phenol content. Nuclear factor- B Results: TOME and TOWE significantly reduced NO production with an IC50 of 79.9 and 157.5 g/mL, respectively, without cytotoxicity. Depleted glutathione (GSH) and antioxidative enzyme activities, including superoxide dismutase, catalase, GSH-peroxidase, and GSH-reductase, were restored by dande- lion extracts. Both extracts inhibited LPS-stimulated iNOS gene expression and that of its transcription factor, NF- B, in parallel with nitrite reduction. TOME showed more potent antioxidative and anti- inflammatory capacities than TOWE, which was attributable to its high total phenol, luteolin, and chicoric acid content. Conclusions: These results indicate that TOME and TOWE inhibit oxidative stress and inflammatory responses through elevated de novo synthesis of antioxidative enzymes and suppression of iNOS expres- sion by NF- B inactivation. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction plex with the inhibitor protein I B . In response to inflammatory stimuli such as reactive oxygen species (ROS), lipopolysaccharide Macrophages protect the body from external intruders through (LPS), and cytokines, I B is phosphorylated and released from NF- phagocytosis. During this process, macrophages produce many B. Activated NF- B, namely, p50 and p65 dimer, migrates to the kinds of inflammatory mediators such as interleukin (IL)-1 , tumor nucleus and up-regulates inflammation-related genes such as iNOS necrosis factor (TNF)- , nitric oxide (NO), and prostaglandins. and cyclooxygenase (COX)-2 (Gius et al., 1999; Allen and Tresini, Excessively generated mediators have been implicated in various 2000). physiological disorders, including tumor formation, autoimmune The common dandelion (Taraxacum officinale G.H. Weber ex reactions, and inflammatory diseases (Gordon, 2002). NO, which is Wiggers, Asteraceae) is widely used as a folklore medicinal one of the major inflammatory mediators, is controlled by NO syn- plant against various disorders such as liver diseases, gallbladder thases (NOS), with inducible NOS (iNOS) markedly up-regulated disorders, digestive complaints, and arthritic and rheumatic dis- in inflammatory disorders (Kleinert et al., 2004). The expression eases (Racz-Kotilla et al., 1974; Bisset and Wichtl, 1994; Newall of iNOS is regulated by transcription factor nuclear factor (NF)- et al., 1996). Diverse biological activities of dandelion, such as B, which exists ubiquitously in the cytoplasm as a heterodimer anti-angiogenic, anti-inflammatory, and anti-nociceptive activities consisting of p50 and p65 as an inactive form by forming a com- were estimated in mice and murine macrophage cell line (Jeon et al., 2008). In addition, signaling molecule for anti-inflammatory activity was analyzed in RAW 264.7 cells (Koh et al., 2010). Dan- ∗ Corresponding author. Tel.: +82 55 320 3235; fax: +82 55 321 0691. delion leaf extract has been shown to exhibit a protective effect E-mail address: fdsnsong@inje.ac.kr (Y.S. Song). against cholecystokinin octapeptide-induced acute pancreatitis 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.11.015
  • 2. 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 extractants liorated 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 peroxide scavenger, because of its high polyphenol content (Hagymasi et al., 2.3. HPLC analysis for luteolin and chicoric acid 2000). 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 followed Lima et al., 2007). In our previous study, 4 kinds of dandelion by the method of Hu and Kitts (2003). Two identical Agilent extract (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-C18 production. 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 columns luteolin and chicoric acid may be important for the amelioration of (4.0 mm × 3.0 mm; Phenomenex, Torrance, CA, USA) were used to LPS-induced oxidative stress and inflammation (Park et al., 2010a). protect the column at room temperature using a linear gradient In 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)]. Solvent liver injury, which indicates luteolin (including the glycosidic form) B increased from 1 to 100% in 30 min and was kept at 100% for and 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 nm and 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 and 264.7 cells and investigate their underlying molecular mechanisms. chicoric acid were used as external standards. Retention times of In 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 content chicoric acid. Total phenol concentration was determined by the method of 2. 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]. One 2.1. 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 as synthesis 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 calibration phenylmethylsulfonyl fluoride were purchased from Sigma (St. curve. Louis, MO, USA). Anti-mouse iNOS antibody was obtained from BD Transduction Laboratories (Lexington, KY, USA), and anti-mouse 2.5. Cell culture and treatment glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Abcam (Cambridge, UK). All other chemicals The RAW 264.7 murine macrophage cell line was obtained were of the highest commercial grade available. from the American Type Culture Collection (TIB-71; Rockville, MD, USA) and cultured DMEM supplemented with 10% FBS and 2.2. 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 dose Dle-Leh-Food (Uiryeong, Korea). The scientific name of the of TOME was determined as 100 g/mL, since its IC50 value was collected 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 LPS men (CMPark 04-12-2009) was deposited in the herbarium of the (1 g/mL) for 18 h at 37 ◦ C in a humidified atmosphere containing Gyeongsang 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 were preparation, air-dried dandelion leaf powder (5 g) was mixed with incubated under the same conditions to keep the stimulation of methanol (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 at dandelion 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, both extracts were filtered (Whatmann paper no. 4). Then, TOME was 2.6. Nitrite production and cell viability measurement concentrated 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 of ery rate of TOME and TOWE was 34.2 and 36.1%, respectively. To NO production was measured according to the Griess reaction prepare 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% phosphoric they were mixed with the 100 mL of ethyl acetate. The mixtures acid) and 50 L of 0.1% naphthylenediamine dihydrochloride and were shaken thoroughly. After 30 min, the separated ethyl acetate then incubated at room temperature for 10 min. Absorbance at layers were collected and extracted in a rotary evaporator. This 550 nm was measured with a NaNO2 serial dilution standard curve
  • 3. 836 C.M. Park et al. / Journal of Ethnopharmacology 133 (2011) 834–842 from 0 to 100 M, and nitrite production was determined. Cell via- violet transilluminator. Data were quantified using the Gel Doc EQ bility was assessed through measuring the uptake of the supravital System (Bio-Rad Laboratories, Hercules, CA, USA). All signals were dye neutral red by viable cells according to the procedure of Fautz normalized to mRNA levels of the house keeping gene, GAPDH, and et 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 (Tomy substance production, as described by Fraga et al. (1988). GSH was Seiko, Tokyo, Japan) and centrifuged at 13,000 × g and 4 ◦ C for measured 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 were nitrobenzoic 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 temperature 2.8. 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 were itoring 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 G that inhibited the rate of pyrogallol oxidation. Catalase activity was secondary antibody for 1 h at room temperature. The blots were analyzed according to the method of Aebi (1984), by following the developed with 5-bromo-4-chloro-3-indoyl phosphate/nitroblue decrease in absorbance of H2 O2 at 240 nm. One unit of catalase tetrazolium color developing solution, and data were quantified was 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 nM of NADPH per minute. GSH-reductase (GR) activity was measured 2.11. Assay of NF-ÄB translocation by following the oxidation of NADPH. A unit of GR was defined as the 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 min 2.9. 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 the acid for 2 h, and then incubated with LPS (1 g/mL) for 18 h. Total microspin G-25 column (Amersham biosciences, Cardiff, UK). Five RNA 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 electrophoresis total 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 by washed 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 analysis UK) to determine OD260 and OD260/280 values. Five micrograms of total RNA were used to produce first strand cDNA using the MMLV All data are expressed as mean (SD). Statistical analyses were first 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 to taining 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 for iNOS were 5 -GCC TTC AAC ACC AAG GTT GTC TGC A-3 (sense) and 3. Results 5 -TCA TTG TAC TCT GAG GGC TGA CAC A-3 (anti-sense); primers for GAPDH were 5 -CAA TGC CAA GTA TGA TGA CAT-3 (sense) and 3.1. Total phenol, luteolin, and chicoric acid contents 5 -CCT GTT ATT ATG GGG GTC TG-3 (anti-sense). The expected sizes of PCR products were 920 bp for iNOS and 375 bp for GAPDH. The total phenol content of TOME and TOWE was 0.17 ± 0.02 The amplification profile consisted of an initial denaturation at and 0.14 ± 0.01 mg chlorogenic acid equivalent per gram of dried 94 ◦ 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 was 49 ◦ 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 between samples. Expression of the house keeping gene, GAPDH, served as 3.2. NO production and cell viability control. The PCR products specific for each cDNA were analyzed by 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
  • 4. C.M. Park et al. / Journal of Ethnopharmacology 133 (2011) 834–842 837 Fig. 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, chicoric acid in TOWE). increased by 33.6 and 49.2 times, respectively, compared with the 3.4. GSH content and antioxidative enzyme activities untreated group. TOME and TOWE suppressed NO production with an 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 decreased 3.3. 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 significantly ined in LPS-stimulated RAW 264.7 cells. As shown in Fig. 3, TOME (p < 0.05) increased by TOME and TOWE treatment (Table 1). When and TOWE significantly (p < 0.05) inhibited MDA concentration in comparing both extracts, TOME was found to increase catalase a dose-dependent manner. and SOD activities more than TOWE. However, in the GSH-related
  • 5. 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 concentrations 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 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 highest amount of GSH activity, mostly via high induction of GR activity, concentration. though TOME strongly oxidized GSH. 3.6. NF-ÄB activity 3.5. 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 of detected in the untreated group, whereas it was highly up- dandelion extracts in LPS-induced inflammation. The results regulated 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 indicated concentrations 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 triplicate experiments. Values sharing the same superscript are not significantly different at p < 0.05.
  • 6. 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 indicated concentrations 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 triplicate 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-induced tive 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 RAW identified as luteolin, chicoric acid, chlorogenic acid, and chryso- 264.7 cells (Choi et al., 2005; Shan et al., 2009). In this study, we eriol (Williams et al., 1996; Hu and Kitts, 2005; Schutz et al., analyzed both phytochemicals, luteolin and chicoric acid, due to 2005). Among these compounds, luteolin and chicoric acid play their synergistic NO inhibitory activity in LPS-stimulated murine diverse 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 show Chen 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 they Chen et al., 2007). Chicoric acid, a derivative of caffeic acid, has have remarkable antioxidative activities (Kim et al., 2004; Huang Table 1 Effects 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 ± 7.7d Data 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 value sharing same superscript is not significantly different at p < 0.05. a Lipopolysaccharide. b Superoxide dismutase. c Glutathione peroxidase. d Glutathione reductase.
  • 7. 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 different LPS-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 internal control. All signals were normalized to mRNA levels of GAPDH and expressed as a ratio. Cells were pre-incubated with and without indicated concentrations of agents for 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 and extracts 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. This idative enzyme activities, such as catalase, SOD, GPx, and GR, and difference was attributed to the fact that TOME contained more GSH 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 has activities of antioxidative enzymes compared to LPS-treated con- been shown to be elevated in precancerous and cancerous lesions trols. Cho et al. (2002) reported that hepatic GSH content was (Kundu and Surh, 2008). When macrophages are activated by increased significantly following supplementation of dandelion leaf inflammatory stimuli, NF- B translocates into the nucleus and extract in hypercholesterolemic rats. In this study, TOME and TOWE binds to the promoter region of inflammatory mediators. Prolonged increased antioxidative enzyme activities, such that these dan- up-regulation of inflammatory mediators by NF- B enhances the
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