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Inhibitory effects of kaurenoic acid from Aralia continentalis on LPS-induced inflammatory response in RAW264.7 macrophages.



Aralia continentalis (Arafiaceae)

Kaurenoic acid

Anti-inflammatory activity



This study investigates the anti-inflammatory effects of a diterpenoid, kaurenoic acid, isolated from the root of Aralia continentalis (Avaliaceae). To determine its anti-inflammatory effects, LPS-induced RAW264.7 macrophages were treated with different concentrations of kaurenoic acid and carrageenan-induced paw edema mice model was used in vivo. Kaurenoic acid (en/-kaur-16-en-19-oic acid) dose-dependently inhibited nitric oxide (NO) production, prostaglandin [E.sub.2] (PG[E.sub.2]) release, cydooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression at micromolar concentrations in LPS-induced RAW264.7 macrophages with I[C.sub.50] (the half maximal inhibitory concentration) values of 51.73 [+ or -]2.42[micro]M and 106.09 [+ or -]0.27[micro]M in NO production and PG[E.sub.2] release, respectively. Kaurenoic acid also dose-dependently inhibited LPS-induced activation of NF-kB as assayed by elec-trophorectic mobility shift assay (EMSA) and it almost abolished NF-kB DNA binding affinity at 100[micro]M. Furthermore, the in vivo antiinflammatory effect of kaurenoic acid was examined in a carrageenan-induced paw edema model. Eight ICR mice in each group were injected with carragcenan and observed hourly, compared with the control group. Kaurenoic acid dose-dependently reduced paw swelling up to 34.4% at 5h after induction, demonstrating inhibition in an acute inflammation model. Taken together, our data suggest that kaurenoic acid, a major diterpenoid from the root of A continentalis shows antiinflammatory activity and the inhibition of iNOS and COX-2 expression might be one of the mechanisms responsible for its anti-inflammatory properties.

[c] 2010 Elsevier GmbH. All rights reserved.


Inflammation responses are regulated by many inflammatory mediators, including nitric oxide (NO), prostaglandin [E.sub.2] (PC[E.sub.2]), NF-[kappa]B, etc. When the regulation of the inflammatory response is broken down, acute or chronic inflammatory diseases such as arthritis, inflammatory bowel diseases and asthma can occur. Many of these diseases are incurable, but treatments are available and they usually involve non-steroidal anti-inflammatory drugs (NSAlDs). Coxibs, a class of NSAIDs, have been widely used for the treatment of acute and chronic inflammation. However, long-term treatment may cause cardiovascular complications such as myocardial infarctions and hypertension, causing the worldwide withdrawal of rofecoxib from use (Rainsford 2005).

The roots of Aralia continentalis (Araliaceae) have been widely used in oriental countries for analgesia, headache, rheumatism, lumbago, and lameness (Kim and Kang 1998). Its extract shows hepatotoxicity (Hwang et al. 2009) and also has anti-inflammatory effect in vivo (Park etal. 2005; Han etal. 1983,1985; Limetal. 2009). In addition, this plant and its components were reported to exhibit cytotoxicity and COX-2 inhibition against tumor cell lines (Lee et al. 2006) and anticancer activities (Seo et al. 2007). Kaurenoic acid is a diterpenoid and one of the major biologically active components in the roots of A continentalis. For instance, kaurenoic acid shows anti-tumor (Cavalcanti et al. 2006), vasorelaxant (Ambrosio et al. 2004), and antibacterial (Gil et al. 2006) activities and the capacity to kill human sperm (Valencia et al. 1986). Furthermore, kaurenoic acid was also reported to possess analgesic (Okuyama et al. 1991) and antimicrobial activities (Porto et al. 2009). Recently, several kaurane-type diterpenes have caused inhibitory effects towards protein tyrosine phosphatase IB (Na et al. 2006; Kim et al. 2006) and cholinesterase (Ertas et al. 2009), and anti-HIV activities (Wu et al. 1996). Based on earlier investigations of Han et al., who demonstrated an anti-inflammatory effect of kaurenoic acid in the paw edema model, we aimed at elucidating the mode of action of the anti-inflammatory activity of kaurenoic acid on the cellular level. Therefore, kaurenoic acid was treated on LPS-stimulated RAW264.7 macrophages to determine its inhibitory effects on the production of NO and PGE2, the enzymes iNOS and COX-2, producing these pro-inflammatory mediators, and the activation of NF-[kappa]B pathway.


Materials and methods

Plant material

The root of A continentalis was purchased at a local market (Korea), and authenticated by Prof. J.H. Lee (Dongguk University, Korea). A voucher specimen (no. 20080320) was deposited in the laboratory of Prof.J.S. Choi. (Pukyong National University, Korea)

Isolation of kaurenoic acid

The roots of A. continentalis (12 kg) were refluxed with methanol (MeOH) for 3 h (3 x 20 l).The total filtrate was then concentrated to dryness in vacuo at 40[degrees]C in order to render the crude MeOH extract. This extract was suspended in distilled [H.sub.2]0 and successively partitioned with n-hexane, methylene chloride (C[H.sub.2][Cl.sub.2]), ethyl acetate (EtOAc) and n-butanol (n-BuOH) to yield 360g n-hexane, 40.9g C[H.sub.2] [Cl.sub.2], 104.5 g EtOAc, and 81.3 g n-BuOH fractions and 207.1 g [H.sub.2]O residue. Repeated chromatography of the n-hexane fraction over a Si gel column with an n-hexane:EtOAc solvent system (10:1 to 0:1 gradient), followedbyanEtOAc:MeOHsolventsystem(10:l to 10:3 gradient), resulted in 15 sub-fractions (HF01-HF15). A portion of HFO3 (62.8 g) was successively loaded onto an RP-18 column with 100% MeOH and 100% C[H.sub.3]CN to obtain 2.5g kaurenoic acid. Kaurenoic acid was isolated and identified by spectroscopic methods, including [sup.1.H] (400 MHz) and [sup.13.C] NMR (100 MHz), and by comparison with published data (Shibata et al. 1967; Yahara et al. 1974; Mihashi et al. 1969; Herz et al. 1983; Dang et al. 2005). The structure of the compound is shown in Fig. 1.

Animals, chemicals and reagents

Male 1CR mice (Samtako, Korea), 4-5 weeks old and weighing 25-30g, were housed in a pathogen-free barrier zone of the Seoul National University Animal Laboratory, according to procedures outlined in the Guide for the Care and Use of Laboratory Animals and the procedures for the experiments were permitted by the Institutional Animal Care and Use Committee, Seoul National University, Korea. Thirty-two animals were acclimated for at least one week, caged eight per group, and were fed a diet of animal chow and water ad lib. The animals were housed at 23 [+ or -] 0.5[degrees]C with 10% humidity in a 12-h light-dark cycle. Unless otherwise indicated, ail chemicals were purchased from Sigma-Aldrich Co.

Cell culture

RAW264.7 murine macrophages were obtained from American Type Culture Collection (Manassas, VA, USA). These cells were maintained at sub-confluence in a 95% air and 5% C [O.sub.2] humidified atmosphere at 37[degrees]C. DMEM medium supplemented with 10% fetal bovine serum (FBS)( lOOU/ml penicillin and 100[micro]g/ml streptomycin was used for routine subculturing and all in vitro experiments.

Cell viability

The cytotoxicity of kaurenoic acid was evaluated by Cell Counting Kit (CCK-8) purchased from Dojindo Laboratories (Japan). Briefly, RAW264.7 cells were plated at a density of 1 x [10.sup.4] per well in a 96-well plate and incubated at 37[degrees]C for 24 h. The cells were treated with various concentrations of kaurenoic acid for 3h as previously described (Ahn et al. 2005). Water-soluble tetrazolium salt, WST8-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disu;fophenyl)-2H-tetrazolium, monosodium salt] in CCK-8 produces a water-soluble forrnazan dye upon reduction, and the resulting color was assayed at 450 nm using a EMax[R]microplate reader (Molecular Devices, CA, USA).

Nitric oxide determination

The nitrite concentration in the medium was measured by Griess reagent as an indicator of nitric oxide production as previously described (Shin et al. 2008). In brief, 1 x [10.sup.5] RAW264.7 cells were plated in 24-well plates, incubated for 24 h and pre-treated with the indicated concentrations of kaurenoic acid, or a vehicle for another 2h, then challenged with LPS (1[micro]g/ml) for an additional 18h. To quantify the nitrite concentration, standard nitrite solutions were prepared, and the absorbances of the solutions were determined with an EMax[R] microplate reader (Molecular Devices, CA, USA) at 540 nm. For this experiment, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) was used as a positive control.

Semi-quantitative reverse transcriptase (RT)-PCR

To perform a semi-quantitative RT-PCR, 1[micro]g of isolated total RNA (easy-BLUE[TM], iNtRON Biotechnology, Korea) was reverse-transcribed into cDNA with an RT PreMix Kit (iNtRON Biotechnology, Korea), according to the manufacturer's instructions. In brief, cDNA synthesis was carried out by PCR (Genius FGEN05TD, Teche, England) under the condition of 45[degrees]C for 1 h and 95[degrees]C for 5 min. Then, the PCR mixture consisted of cDNA 2[micro]l, 10x PCR buffer 2[micro]l, dNTPs 2[micro]l, Taq DNA polymerase 0.2[micro]l, forward/reverse primer 0.2[micro]and 0.1% diethyl pyrocarbonate water for final volume 20[micro]l were amplified as follows: denaturation at 95[degrees]C for 5 min and 94[degrees]C for 30 s, annealing at 50[degrees]C for 30 s, extension at 74[degrees]C for 1 min (35 cycles) and final extension at 72[degrees]C for 10 min. The primers for COX-2 amplification were sense 5'-GGA GAG ACT ATC AAG ATA GTG ATC-3' and antisense 5'-ATG GTC ACT AGA CTT TTA CAG CTC-3'. The primers for iNOS amplification were sense 5'-CCC TTC CGA AGTTTC TGG CAG C-3' and antisense 5'-GGC TGT CAG AGC CTC GTG GCTT-3'.The primers for GAPDH were sense 5'-GCT CGG AGT CAA CGG ATT TGG TCG-3' and antisense 5'-CTT CCG ACG CCT GCT TCA CCA C-3'. The amplified cDNA products were separated by 2% agarose gel electrophoresis and stained with ethid-ium bromide. The gels were viewed using Doc-It LS Image Analysis software (UVP Inc., CA, USA) and quantified using UN-SCAN-IT[TM] gel 6.1 software (Silk Scientific Corp., UT, USA). The PCR products were normalized to the amount of GAPDH for each band.

Western immunoblot analysis

RAW264.7 macrophages were pre-treated with the indicated concentrations of kaurenoic acid or vehicle for 2 h and stimulated with 1[micro]g/ml LPS for 18 h to detect ([beta]-actin, COX-2 and iNOS. Ten micrograms of total protein extracts was separated by 8% SDS-PAGE, electro-transferred to nitrocellulose membranes (Whatman GmbH, Germany), blotted with each primary antibody (1:1000) and the corresponding secondary antibody (1:5000) (Santa Cruz, CA, USA), and detected with WEST-SAVE Up[TM] luminol-based ECL reagent (ABFrontier, Korea). The COX-2 primary antibody (goat polyclonal IgG, sc-1745)and its corresponding secondary antibody (mouse anti-goat IgG-HRP, sc-2354) were used. The iNOS primary antibody (rabbit polyclonal IgG, sc-8310) and its corresponding secondary antibody (goat anti-rabbit IgG-HRP, sc-2004) were used. The [beta]-actin primary antibody (mouse monoclonal IgG, sc-47778) and the corresponding secondary antibody (goat anti-mouse IgG-HRP, sc-2005) were also used. All antibodies were obtained from Santa Cruz (CA, USA). The target bands were quantified using UN-SCAN-1[TM] gel 6.1 software (Silk Scientific Corp., UT, USA),

Determination of PG[E.sub.2] production

PG[E.sub.2] produced from activated macrophages was quantified using an enzyme immunoassay (EIA) kit for PG[E.sub.2] (Cayman Chemical, MI, USA) according to the manufacturer's instructions. Briefly, RAW264.7 macrophages were pre-treated with several concentrations of kaurenoic acid or vehicle for 2 h and then activated by 1[micro]g/ml LPS to express COX-2 for an additional 18 h. These media were diluted 2 times with distilled water and transferred to a PG[E.sub.2] antibody-coated 96-well culture plate in the EIA kit and treated according to manufacturer's instruction. The PG[E.sub.2] produced in the specimen was quantified to determine COX-2 expression using a PG[E.sub.2] standard curve. Absorbance at 405 nm was recorded using an EMax[R] microplate reader (Molecular Devices, CA, USA). For comparison, 50[micro]M of celecoxib (Celebrex[R], Pfizer) was used as a positive control.

Etectrophoretic mobility shift assay (EMSA)

In order to check activation of NF-[kappa]B, electrophoretic mobility shift assay (EMSA) was performed as previously described with some modifications (Anand et al. 2008). Briefly, RAW264.7 macrophages were pre-incubated with the indicated concentrations of vehicle, kaurenoic acid or the positive control, parthenolide for 2h and then, 1[micro]g/ml LPS was added for 1 h, and nuclear extracts prepared from LPS-treated cells were incubated with a (32) P-end-labeled 45-mer double-stranded NF-kB oligonucleotide sequence from the HIV long terminal repeat (5'-TGTTACMGGGACnTCCGCTGGGGACnTCCAGGGAGGCGTGG-3' (NF-kB binding sites are bolded)) for 30min at 37[degrees]C, and the DNA-protein complexes were separated from the free oligonucleotide on 6% native polyacrylamide gels. Specificity was determined using competition with an unlabeled oligonucleotide. The gels were dried and visualized using a BAS-1500 apparatus (Fuji, Japan).

Carrageenan-induced paw edema test in mice

Paw edema was induced by subcutaneous injection of 0.05 ml of 1% carrageenan into the right hind paw of mice as previously described (Shin et al. 2008). Control animals received identical treatments but with the vehicle, which, in this study, was 10% Tween 80 (10 ml/kg, i.p.) in saline. Thirty minutes prior to carrageenan administration, the animals received an i.p. injection of saline, dexamethasone (lOmg/kg) or kaurenoic acid (10 or 50 mg/kg), and the paw thickness was measured using a dial thickness gauge (No. 2046F, Mitutoyo, Japan) before and every hour after edema induction for 5 h. The percent increase of paw thickness was calculated based on the volume difference between the two paws with and without carrageenan injection.s


Data analysis

The results were expressed as mean [+ or -] standard deviation (S.D.). Analysis of variance (ANOVA) with Dunnett's t-test was used for the statistical analysis of multiple comparisons of data. p-Values less than 0.05 were considered statistically significant. [*p <0.05; **p < 0.01;***p < 0.001],


Cell viability

The cytotoxic effect of kaurenoic acid on RAW264.7 cells was measured by CCK-8, and no cytotoxic effect was observed with up to 100[micro]M of kaurenoic acid (Fig. 2). Therefore, sample treatments between 10 and 100[micro]M were used in the subsequent experiments.

Inhibition of LPS-induced NO production by kaurenoic acid in RAW264.7 macrophages

We investigated the inhibitory effect of kaurenoic acid on LPS-induced NO production in RAW264.7 macrophages. Pretreat-ment with kaurenoic acid inhibited LPS-induced NO production in a concentration-dependent manner, which corresponds to the half maximal inhibitory concentration (I[C.sub.50]) at 51.73 [+ or -]2.42[micro]M (Fig. 3A).

Inhibition of LPS-induced PG[E.sub.2] secretion by kaurenoic acid on RAW264.7 macrophages

The PG[E.sub.2] produced in the specimen was quantified to determine COX-2 expression using a PG[E.sub.2] standard curve. Kaurenoic acid decreased PGE2 expression in a dose-dependent manner (Fig. 3B).

Inhibition of LPS-induced protein and mRNA expression ofiNOS and COX-2 by kaurenoic acid

Fig. 4A shows that kaurenoic acid significantly reduced the protein levels of both COX-2 and iNOS. The target bands were quantified by [beta]-actin expression. In considering mRNA expression, kaurenoic acid also dosedependently decreased iNOS mRNA expression, and did effectively suppress COX-2 mRNA expression (Fig.4B).


Inhibition of LPS-induced NF-[kappa]B nuclear protein DNA binding activity by kaurenoic acid

Since NF-[kappa]B, a transcription factor, which is activated by inflammatory stimuli such as LPS, its activation is an essential stage in gene expression including iNOS and COX-2 in macrophages. LPS increased the binding activity of nuclear extracts to the NF-[kappa]B DNA sequence, kaurenoic acid almost completely abolished the NF-[kappa]B DNA binding activity at 100[micro]M (Fig. 5).

Inhibitory effect on carrageenan-induced paw edema in mice by kaurenoic acid

In order to evaluate the in vivo anti-inflammatory effect of kaurenoic acid, a carrageenan-induced paw edema test was conducted. As shown in Fig. 6, maximal edema formation was observed 1 h after 1% carrageenan injection, and treatment with kaurenoic acid showed inhibitory effects on paw swelling in a dose-dependent manner, compared with the control group. Paw swelling was reduced 21.5% at 5h after the induction at lOmg/kg treatment group, and decreased up to 34.4% in 50mg/kg group. Blood samples were collected immediately after in vivo tests to measure GOT (glutamic oxaloacetic transminase), GPT (glutamic pyruvic transminase) and creatine levels. Both kaurenoic acid-treated animals showed similar levels with those of normal controls except dex-amethasone (data not shown).



Macrophage activation is important to the progression of multiple diseases through the release of inflammatory mediators. Lipopolysaccharide (LPS)-induced RAW264.7 macrophages are widely used in vitro, because LPS is a pathogen that triggers toll-like receptor 4 (TLR4J and activates various inflammatory signals (Denlinger et at 1996). NF-kB is a transcription factor that plays a critical role in inflammatory and immune responses. It is present in the cytoplasm, binding to the inhibitory protein I[kappa]B in un-stimulated cells. When the cells are exposed to the stimulants such as LPS, IkB is phosphorylated and liberates NF-[kappa]B, resulting in NF-kB translocation into the nucleus. Nuclear NF-[kappa]B then binds to the promoters of pro-inflammatory mediators, resulting in the induction of their gene expression (Gilroy et al. 2004).



Here, we have elucidated that kaurenoic acid significantly inhibits inflammatory mediators in LPS-stimulated RAW264.7 macrophages, which are associated with many acute and chronic inflammatory diseases. A nitrite assay revealed that kaurenoic acid inhibited the production of NO, and through an ELISA assay, it was found to reduce PG[E.sub.2] secretion in a concentration-dependent manner. Moreover, kaurenoic acid decreased the expression of iNOS and COX-2 both on protein and mRNA levels. The inhibition of iNOS and COX-2 protein by kaurenoic acid paralleled the LPS-induced decrease of NO and PGE2. NF-[kappa]B-DNA binding affinity was also diminished by kaurenoic acid treatment, which supports that this compound inhibits COX-2 and iNOS induction via NF-[kappa]B inhibition. In addition, we confirmed the anti-inflammatory activity of kaurenoic acid in an in vivo carrageenan-induced paw edema model. Previous reports have indicated the anti-inflammatory potential of kaurenoic acid in acetic acid-induced colitis (Paiva et al. 2002), as well as the therapeutic effect of A continentalis extracts in a collagenase-induced arthritis rabbit model (Park et al. 2009). Han et al. demonstrated that the anti-inflammatory activity of kaurenoic acid in carrageenan-induced edema test in rat hind paw (Han et al. 1985). He and his co-workers reported that administration s.c. showed a lower anti-inflammatory activity than doing p.o. We decided to reduce the concentration of kaurenoic acid, which are 10 and 50mg/kg on Lp. administration in an in vivo carrageenan-induced paw edema model. By comparison to controls, kaurenoic acid effectively reduced carrageenan-induced paw swelling. Considering both the in vitro and in vivo results, it appears that kaurenoic acid might be viable anti-inflammatory candidate in the clinical field. Blood samples were collected immediately to measure GOT, GPT and creatine levels after in vivo tests. Kaurenoic acid-treated animals showed similar levels with those of normal controls, except in the case of dexamethasone (data not shown). Even though the dexamethasone-treated group showed the significant effect in paw swelling, dexamethasone caused liver damage based on the blood test

Recently, several research groups reported that kaurane type diterpenes have shown anti-inflammatory activity regarding the inhibition of NF-[kappa]B activation (Castrillo et al. 2001; Park et al. 2007; Aquila et al. 2009). Since kaurenoic acid possesses antiinflammatory effects without harmful influences based on our findings, we suggest that kaurane diterpenes are potential candidates for anti-inflammation without concerning liver damage In conclusion, the present study demonstrated that kaurenoic acid isolated from A. continentalis display antiinflammatory effects both in vitro and in vivo, kaurenoic acid inhibited LPS-induced NO, PG[E.sub.2], iNOS and COX-2 expression by blocking DNA binding, in macrophages. In addition, this diterpenoid effectively inhibited carrageenan-induced paw edema by suppressing PG[E.sub.2] production in vivo. Taken together, we suggest that kaurenoic acid could be a potential drug source for the medication of inflammatory disorders such as colitis, rheumatoid arthritis, asthma and gastritis.


This work was supported by a grant from the Korea Food and Drug Administration (2007) for Studies on the Identification of the Efficacy of Biologically Active Components from Oriental Herbal Medicines and was also supported in part by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST) (No. 20090083533).

0944-7113/$ - see front matter[c] 2010 EtsevierGmbH. All rights reserved. doi: 10.1016/j.phymed.2010.11.010


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doi:l0.1016/j.phymed.20l0.l 1.010

Ran Joo Choi (a), Eun Myoung Shin (a), Hyun Ah Jungbt Jae Sue Choi (b), Yeong Shik Kima (a), *

(a) Natural Products Research Institute, College of Pharmacy, Seoul National University, 599 Gwanangno, Gwanak-gu, Seoul 151-742, Republic of Korea

(b) Division of Food Science and Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea

* Corresponding author. Tel.: +82 2 880 2479; fax: +82 2 765 4768. E-mail address: (Y.S. Kim).
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Title Annotation:lipopolysaccharides
Author:Choi, Ran Joo; Shin, Eun Myoung; Jung, Hyun Ah; Choi, Jae Sue; Kima, Yeong Shik
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9SOUT
Date:Jun 15, 2011
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