Asian Pac J Trop Med 2014; 7(Suppl 1): S327-S331 S327

Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine

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Document heading doi: 10.1016/S1995-7645(14)60254-6 Anti-inflammatory activity of methyl ferulate isolated from Lour

1 2 3 Nguyen Thi Mai Phuong *, Trinh Tat Cuong , Dang Ngoc Quang 1Institute of Biotechnology, Academy of Science and Technology, Hanoi, Vietnam 2Key Laboratory for Enzyme and Protein Technology, Hanoi University of Science, Vietnam 3Faculty of Chemistry, Hanoi National University of Education, Vietnam

ARTICLE INFO ABSTRACT

Article history: Objective: ( ) TStemonao evaluate tuberosa the anti-inflammatoryS. tuberosa activity of methyl ferulate MF isolated from Received 16 May 2014 the roots of ( ) Lour () in lipopolysaccharide activated Received in revised form 23 May, 2nd revised form 15 Jun, 3rd revised form 25 Jun 2014 macrophageMethods: cells. S. tuberosa Accepted 3 Aug 2014 Methanol extracts of a root powder of were prepared for isolation of Available online 27 Jun 2014 a potential anti-inflammatory agent using ultrasound extraction combined with repeated chromatography on silica gel. After the quantitative analyses, anti-inflammatory activity of the Keywords: isolated compound was evaluated by measurement of cytokine release, NO generation, expression Stemona tuberosa of cyclooxygenase-2 and phosphorylation of mitogen activated protein kinases including p38 and c-Results:Jun NH2-terminal kinase using quantitative kits and Western blotting with specific antibodies. Inflammation The isolation process yielded a potential anti-inflammatory compound with a purity Toll-like 4 receptor level of 99% determined by high performance liquid chromatography. The compound was Macrophage MF MF identified as by using nuclear magnetic resonance. strongly inhibitedα γ the release of pro-inflammatory cytokines from macrophages, including IL-6, TNF , IFN , yet it did not affect the anti-inflammatory cytokine IL-10. Phosphorylation of p38 and c-Jun NH2-terminal kinase were clearly reduced in MF-treated macrophages stimulated with lipopolysaccharide. cyclooxygenase-2 expression and NO generation by macrophages were also suppressed when the Conclusions:cells were treated with MF. The data suggested that MF is a possible inhibitor of the mitogen activated phosphor kinase pathwayS. andtuberosa could be a potential anti-inflammatory agent isolated for the first time in medicinal .

1. Introduction activity of this plant required further investigation. In this Stemona tuberosa S. tuberosa study, we aimed to examine the anti-inflammatory activity ( ) Lour (Stemonaceae), known and potential mechanisms of action in lipopolysaccharide as Bach Bo in Vietnam, is a hairless, perennial, herbaceous (LPS)-activated macrophageS. tuberosa cells for methyl ferulate (MF) twiner which can grow up to 4-10 m long. Its rootsS. tuberosa form a isolated from roots of . fascicle of many thick, fleshy shoots. The root of 2. Materials and methods has been used in Vietnamese traditional medicine for its antitussive and anti-ectoparasitic activities moisten 2.1. Plant material the lungs and stop cough, as well as kill parasites[1]. The chemical constituents of the . tuberosa are alkaloids, stilbenoides S. tuberosa and tocopherols[2,3]. Recently, extracts have been The roots of and eleven other traditional attracting new interest for their multi-biological functions medicinal plants for screening test were collected in including anti-tuberculotic, antifungal, demulcent, and Quang Binh, Thanh Hoa, Phu Tho, Hoa Binh provinces anti-cancer activities[1,2]. However, the anti-inflammatory and identified by the Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, *Corresponding author: Nguyen Thi Mai Phuong, Institute of Biotechnology, Vietnam Hanoi. Academy of Science and Technology, Hanoi, Vietnam. Tel: +84 917500965 2.2. Chemicals Fax: +84 438360853 E-mail: [email protected] Foundation Project: Supported by Vietnam Academy of Science and Technology LPS S A (S L MO) (VAST) Grant No. (VAST03.02/12-13). was purchased from igma- ldrich t. ouis, . Nguyen Thi Mai Phuong et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S327-S331 S328 α γ γ ELISA kits for IL-6, IL-10, TNF , IFN were purchased from IL10, and IFN- secreted by cell culture were measured by BD Pharmingen (Franklin Lakes, NJ). Antibodies p38 and ELISA reagents (BD Pharmingen). phospho-(Thr180/Tyr182)-p38, c-Jun NH2-terminal kinase 2.7. Western blotting (JNK) and p-JNK were purchased from Cell Signalling Technology (Beverly, MA). The anti-cyclooxygenase-2 (COX-2) antibody was purchased from Abfrontier (Korea). The The treated BMDMs were processed for analysis by Western NO detection kit was purchased from iNtRON Biotechnology blotting[4]. Briefly, BMDMs were treated with the test agent for ( ) 60 48 Korea . All chemicals used were of analytical grade. minµ in Corning Costar well plate before stimulation with 2.3. Extraction and isolation LPS (1 g/mL). The total cell lysates obtained by using lysis buffer NP 40% (Life Technologies) were then separated by 10% S. tuberosa sodium dodecyl sulfate polyacrylamide gel electrophoresis The air-dried and powdered roots of (2.0 and transferred overnight to nitrocellulose membranes, kg) were extracted five times with 75% methanol (5伊10 L) (AmershamTM HybondTM-ECL) and the nonspecific binding in ultrasound extracting system (Elma, ) at room of antibodies was blocked with 3% nonfat dried milk in temperature and filtered. The combined extract was phosphate-buffered saline. Membranes were probed with concentrated to dryness by evaporating in vacuo to give a p38, p-p38, JNK, and p-JNK antibodies (1:1 000 dilution in residue (18 g), which was used for antiflammatory screening Tris-buffered saline, pH 7.4) for 2 h at room temperature. assays. The residue was chromatographed on silica gel After three washes with phosphate-buffered saline, blots (200-300 mesh) column chromatography, eluting with an were developed by a chemiluminescence assay (ECL; ether:acetone gradient (9:1) to give 10 fractions E 1-10. The Amersham-Pharmacia). further separation of fraction E4 (0.82 g) by silica gel column 2.8. Measurement of NO content chromatography eluted with a hexane:ethyl acetate gradient ( ) ( ) ( ) 2:1 , yielded ten fractions H1-10 . Compound 1 85 mg was 6 obtained from fraction H5 by preparative high performance Raw 246.7 cells at density of 10 were seeded in Corning liquid chromatography (HPLC) (hexane-EtOAc, 2/1; flow rate Costar 48 well plate for 16 h before treatment. The cells were 1 ) 60 1 mL/min . µtreated with the test agent for min and activated with 2.4. Cell culture g/mL of LPS. The culture supernatants were obtained by centrifuge and concentrations of NO were assessed by Griess reaction using a NO detection kit (iNtRON Biotechnology, Primary bone marrow derived-macrophages (BMDMs) were Korea). isolated from the bone marrow of mice, which were reviewed 2.9. Determination of COX-2 expression and approved by the animal care unit of National Institute of Hygiene and Epidemiology,- Vietnam. The cells were differentiated for 5 7 d in macrophage colony-stimulating RAW 246.7 cells were treated with test agent in Corning ( CSF) 48 60 factor M-’ -containing ’media. The medium contains Costar µ well plate for min before stimulation with Dulbecco s modified Eagle s medium (DMEM, Gibco-BRL, LPS (1 g/mL). The total cell– lysates were then analyzed Gaithersburg, MD) with 10% L929 cell-conditioned medium with Western blot using anti COX-2 antibody (Santa Cruz (as a source of M-CSF), 10% heat-inactivated fetal bovine Biotechnology Inc., Santa Cruz, CA). The antibodies were (FBS) ( BRL) 1 50 1 1 000 serum Gibco- µ , mmol/L sodium pyruvate,5 diluted at ratio of : . The membranes were developed by 50 5 10 (ECL ) IUβ/mL penicillin, g/mL streptomycin and 伊 mol/ a chemiluminescence assay ; Amersham-Pharmacia . L -mercaptoethanol, sodium pyruvate, non-essential 2.10. Statistical analyses (100 ) aminoµ acids, penicillin G IU/mL , and streptomycin (100 g/mL). Mouse macrophage cell line, RAW264.7 (American Type Culture Collection; ATCC) was maintained For statistical analysis, data obtained from independent in complete medium (DMEM with 10% FBS), sodium pyruvate, experiments are’ tpresented as mean依SD and were analyzed (100 ) ANOVA non-essential aminoµ acids, penicillin G IU/mL , and by the Student s -test with for multipleP comparisons. streptomycin (100 g/mL). Differences were considered significant at <0.05. 2.5. Cell viability assay 3. Results

Cell viability assessment was performed using a Cell 3.1. Isolation of potential anti-inflammatory agent(s) from Counting Kit-8 (CCK-8, Dojindo’ Laboratories, Kumamoto, S. tuberosa extract ) 10 µJapan as per the manufacturer s instructions. Briefly, L of CCK-8 solution was added and incubated with treated cells for 60 min in Corning Costar 48 well plate and then Macrophages and lymphocytes produce powerful pro- 450 6 1 8 absorbance was measured at nm. Values from each inflammatoryα mediators, including IL , IL , IL , and treatment were calculated as a percent relative to the TNF , one of the most important mediators that regulates untreated control (100% survival). biochemical changes and symptomatic pathophysiological 2.6. ELISA responses in the human body. In later stages e.g.of sepsis, anti-inflammatory mediators are produced ( IL10), leading to an abatement in the production of many of the BMDMs were treated in Corning Costar 48 well plate pro-inflammatory mediators[5]. Macrophages activated 3 4 LPS as indicated in the Figures and . The cell-cultureα by bacterial induce antibody production and local supernatants were analyzed for levels of cytokines TNF , IL6, inflammation, which includes the release of pro- Nguyen Thi Mai Phuong et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S327-S331 S329 Table 1 inflammatory cytokines, chemokines, and inflammatory 1 13 mediators[5]. Therefore, macrophage cell lines are routinely The spectroscopic data of MF (CDCl3) about compound of H NMR and C NMR. Position Compound 1 used to evaluate the anti-inflammatory activities of crude 1 13 extracts. in vitro H NMR (J, Hz) C NMR In this study, assaying LPS-activated macrophages , 1 167.7 we screened for a potential anti-inflammatory extract from 2 6.28 d, J=16.0 115.2 12 3 7 62 J 16 0 145 0 traditional medicinalα plants of Vietnam by measurement . d, = . . of IL6 and TNF production when LPS-induced macrophage 4 126.9 5 7 03 109 4 cells were treatedS. tuberose with these plant extracts. We found that . brd s . 6 148.0 a methanolic α extract strongly inhibited pro- inflammatory IL6 and TNF release while it did not affect the 7 146.8 release of the anti-inflammatory cytokine IL10, suggesting 8 6.92 d, J=8.5 114.7 9 7 07 J 1 0 8 0 123 0 that the extract possesses potential anti-inflammatoryS. tuberosa . dd, = . , . . properties (data not shown). Based on this result, 1-OMe 3.80 s 51.6 was selected for isolation of the desired compound. The 6-OMe 3.93 s 55.9 isolation process was based on the bioassay-guided 7-OH 5.90 s chromatographic separations. Compound 1 (85 mg) in the 3.2. Effect of MF on cell viability form of a yellow-brown oil was received by a preparative chromatography system after two repeated silica gel column chromatography steps showed a purity of up to 99% by To evaluate the anti-inflammatory activity of MF, we HPLC (NMR) . The obtained nuclear magnetic resonance examined the cytotoxic effects of MF and a dexamethasoneµ spectroscopic data indicated that compound 1 is MF with a (Dex) control on BMDMs. BMDMs were treated with MF (25 g/ molecular fomular of C11H13O4 (Figure 1), which displays the mL) or Dex (100 nmol/L) for various times, and the percentage UV (M OH) 236 322 IR CCK 8 following spectral-1 data: e _max nm: , m/z; of surviving cells was counted using - cell viability ( ) EIMS KBr _max cm : 3424, 2903, 2649, 25491 , 174413, 1670; : assay. As shown in Figure 2, no significant difference + 208 [M] , 177, 145, 137, 131, 89, 77, 51; H and C NMR (Table 1). was observed for MF or Dex-inducedP cell viability at the Ferulic acid and some derivatives, especially ethyl indicated times, up to 48 h ( >0.05). ferulate, have been reported to be effective antioxidant, 120

anti-microbial, anti-inflammatory, hepatoprotective, ) % neuroprotective, anticarcinogenic, anti-diabetic, anti- ( 90 cholesterolemic, UV-protective and radioprotective compounds[6,7]. However, anti-inflammatory activity of MF 60

derivative has not been well documented. ell viability

C 30

1 COOCH3 0 0 6 18 48 Time (h) MF Dex Figure 2. No effect on cell viability after treatment of BMDMs with MF or Dex. 6 BMDMs were seeded in Corning Costar 48 well plate at a density of 1伊10 cells/ 2 µ H well. After 3-4 d, cells were treated with MF (25 g/mL) for the times indicated. Cell viability was assessed after incubation for indicated times in the presence of CCK-8. Data are presented as the mean依SD of three independent 3 H experiments.

3.3. Inhibition of MF against cytokine production by LPS activated macrophages

4 0 5 Anti-inflammatoryµ properties of MF concentrations , , 5 10 25 L 9 , and g/m were confirmed by examination of their 6 effectγ on αpro-inflammatory cytokine production of IL- , IFN TNF 10 , , and post-anti-inflammatory cytokine IL- γ. ( 3) 6 INF The resultsα Figure indicated that the release of IL , , 8 TNF BMDM IL10 6 and was strongly inhibited in s while P was unaffected compared to untreated control cells ( <0.05). 7 These data indicate- that MF could be a good candidate for 3 10 OCH useµ as an antiµ inflammatory agent. At a concentration of g/mL (45 mol/L), release of all the tested cytokines in the treated BMDMs were suppressed by at least 50%. 3.4. MF inhibits phosphorylation by p38 and JNK in MAPK OH pathway Figure 1. S. tuberosa. Chemical structure of methyl ferulate isolated from Mitogen activated protein kinases (MAPK) are a group Nguyen Thi Mai Phuong et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S327-S331 S330 of signaling molecules that also appear to play important to prostaglandin, resulting in pain and inflammation[10]. roles in inflammatory processes. At least three MAP kinase Inhibitors of COX-2 have profound anti-inflammatory cascades are well described including, extracellular signal effects[10]. We also examined COX-2 expression in MF- and regulated kinase, JNK and p38[8,9]. These have also been treated and LPS-stimulated BMDMs, as COX-2 is an important reported to differentially activate depending on the stimuli inflammatory mediator that is induced by various stimuli, present and cell type affected[9]. As shown in Figure 4, the including LPS and cytokines[9]. As shown in Figure 6, MF 38 JNK COX 2 activation of p and was significantly reduced µin the clearlyµ suppressed - expression at a concentration of RAW 264 7 25 25 MF-treated . cells at a concentration of g/mL g/mL while the inhibitory effectµ seems to not be very (compared to the control). profound at a concentration of 10 g/mL. 16 000 16 000 A B 16 12 000 12 000 (pg/mL)

(pg/mL)

8000 α 8 000 6

) 12 IL L

TNF 4 000 4 000

0 0 g/m µ ( 8 0 5 0 10 0 25 0 D . µ . . ex 0 5.0 µ 10.0 25.0 Dex MF ( L) MF ( L) g/m g/m NO 15 000 15 000 C D 4 10 000 10 000 (pg/mL) (pg/mL)

γ 5000 10 5000 IL 0 IFN µ µ Control LPS 10.0 g/mL 25.0 g/mL 0 0 Figure 5. 0 5 0 10 0 25 0 0 5 0 10 0 25 0 . µ . . Dex . µ . . Dex Inhibition of MF against NO generation in BMDMs. MF ( g/mL) MF ( g/mL) 6 Figure 3. α γ Raw 246.7 cells at density of 10 were seeded in Corning Costar 48 well plate Effect of MF on cytokines TNF , IL6, IFN and IL10 produced by for 16 h before treatment. The cells were treated with MF at concentrations of macrophages. µ 10 and 25 ug/mL for 60 min and activated with 1 ug/mL of LPS for another 1.5 BMDMs were treated with MF at concentrations of 0, 5, 10, and 25 g/mL in h. The culture supernatants were harvested by centrifuge and concentrations vehicle DMSO 0.1% in 60 min before stimulation with LPS. Supernatants were µ of NO were determined using a NO detection kit. Data are presented as the harvested 18 h after stimulation with 1 g/mL LPS to induce inflammation. γ α mean依SD of three independent experiments. Concentrations of IL-10, IFN , IL-6, and TNF in the culture supernatants were determined by ELISA. Dexamethasone (Dex) was used as positive control. 0 LPS MF1 MF2 Data are presented as the mean依SD of three independent experiments. COX-2 -LPS LPS MF+LPS -LPS LPS MF+LPS p-p38 p-pJNK

β p38 pJNK -Actin Figure 4. Inhibition of MF against p38 and JNK phosphorylation in BMDMs. µ Figure 6. BMDMs were treated with MF at concentrations of 25 g/mL in vehicle Inhibition of MF against COX-2 expression in BMDMs. µ µ DMSO 0.1% for 60 min before stimulation with LPS (1 g/mL). The cells then RAW 246.7 cells were treated with MF at concentrations of 10 g/mL (MF1) µ were then analyzed with Western blot using p38, p-p38, JNK, and p-JNK and 25 g/mL (MF2) in vehicle DMSO 0.1% in Corning Costar 48 well plate for µ antibodies. 60 min before stimulation with LPS (1 g/mL). The cells were then analyzed 3.5. Inhibition of MF against NO formation in BMDMs with Western blot using anti COX-2 antibody.

4. Discussion Reactive oxygen species (ROS), such as H2O2, NO, superoxide anions, and hydroxyl radicals (OH) are often important intracellular signaling molecules in pathways In the treatment of inflammation, non-steroid anti- involved in cell proliferation, stress responses and rheumatic drugs, such as acetylsalicylic acid, more apoptosis[5,9]. The inhibitory effects of MF on activation of commonly known as aspirin or ibuprofen, have been popular MAPK (phosphorylation of p38 and JNK), which was mentioned choices. However, many of these drugs cause potential in section 3.4, suggested that p38 and JNK could be the risks and side effects, such as stomach and cardiovascular important signals involved in the MF-induced inhibition problems[11,12]. Therefore, novel anti-inflammatory, of NO generation. Therefore, we investigated whether MF especially natural agents are badly needed. Plants are rich inhibits LPS-induced NO generation in BMDMs. The RAW264.7 in naturally-occurring bioactive compounds. This is an ideal cells were treated with MF and inflammation was induced by bio-resource to develop new therapeutic agents[13-15]. In an using LPS. The data (Figure 5) indicated that LPS-induced NO attempt to find novel anti-inflammatory agentsS. fromtuberosa plants generation was inhibited in a dose dependent manner withµ of Vietnam, we were screened and selected , a about 20% and 50% inhibition at concentrations 10 and 25 g/ traditional medicinal plant. We found the MF compound, mL MF, respectively. and even though it is not a new compound, this is the first 3.6. Inhibition of MF against COX-2 expression time that it has been isolated from this plant andin vitro shown to possess anti-inflammatory activity when tested . First, MF strongly inhibited MAPK phosphorylation induced COX-2 is primarily present at sites of inflammation by LPS, which affected two important signal pathways p38 and plays a role in the conversion of arachidonic acid and JNK. The most well-known pathway involved in LPS- Nguyen Thi Mai Phuong et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S327-S331 S331 Planta Med 72 induced pro-inflammatory responses in macrophages activities of stemona alkaloids. 2006; (2): 99-113. [2] M urthy K, Rama S, Reddy MC, Kondamudi R, Pullaiah T. is the MAPK pathway, which is involved intracellular Stemona tuberosa — [8,16] Micropropagation of Lour. an endangered signaling cascades . Intense efforts have been made to Indian J and rare medicinal plant in Eastern Ghats of India. develop and evaluate compounds that target components Biotechnol 12 of these pathways[9,16]. The most extensive activity in 2013; : 420-424. MAPK JNK 38 [3] S chinnerl J, Brem B, But PP, Vajrodaya S, Hofer O, Greger H. inhibitor development has found withα p and p , [17] Pyrrolo-and pyridoazepine alkaloids as chemical markers in which regulate the production of TNF and IL1 . p38 Stemona Phytochemistry 68 inhibitors are expected to inhibit not only the production species. 2007; (10): 1417-1427. of pro-inflammatory cytokines, but also their actions, [4] Y ang CS, Ko SR, Cho BG, Shin DM, Yuk JM, Li S, et al. The thereby interrupting the vicious cycle that often occurs in ginsenoside metabolite compound K, a novel agonist of [5,9] glucocorticoid receptor, induces tolerance to endotoxin-induced inflammatory and immune-responsive diseases . Our data J Cell Mol Med 12 suggest that MF is a potential inhibitor of the MAPK pathway. lethal shock. 2008; : 1739-1753. Second, several studies have demonstrated that activation [5] A ldridge C, Razzak A, Babcock TA, Helton WS, Espat NJ. MAPK Lipopolysaccharide-stimulated RAW 264.7 macrophage inducible of plays a role in the regulationκ of inflammationκ by [8,17] nitric oxide synthase and nitric oxide production is decreased affecting the activation of NF- B . Moreover, NF- B plays J Surg Res 149 an important role in the regulation of inflammatory genes, by an omega-3 fatty acid lipid emulsion. 2008; (2): ( NOS) COX 2 296-302. such as, αinducible nitric oxide synthetaseκ i , - , and TNF- [9,10,17]. In our study, NF- B expression was also [6] P aiva LBD, Goldbeck R, Santos WDD, Squina FM. Ferulic acid BMDM 50 105 and derivatives: molecules with potential application in the evaluated in κ s using specific antibodies to the p / Braz J Pharm Sci 49 pharmaceutical field. 2013; (3): 395-411. subunitκ of NF- B. We found an only minor suppression of [7] S ultana R. Ferulic acid ethyl ester as a potential therapy in NF- B expression byµ MF in the Western blot image at a Biochim Biophys Acta 1822 concentration of 25 g/mL (data not shown). However, the neurodegenerative disorders. 2012; (5): inhibitory effect of MF on NO generation and COX-2 at the 748-752. [8] E nglish JM, Cobb MH. Pharmacological inhibitors of MAPK same concentrationκ suggests etthat al MF could still be affecting Trends Pharmacol Sci 23 the NF- B pathway. Islam . demonstrated that anti- pathways. 2002; : 40-45. inflammatory activity of ethyl ferulate, another derivative of [9] P ark HJ, Lee HJ, Choi MS, Son DJ, Song HS, Song MJ, et al. JNK ROS pathway is involved in the inhibition of inflammatory target gene ferulate acid comes from the inhibition ofκ production J Inflamm expression and NF-kappaB activation by melittin. and the significant suppressionκ of the NF- B activity when (Lond) 5 the translocation of NF- B p65 in LPS-stimulated RAW 2008; : 7. 264.7 macrophages was measured[18].This suggests that an [10] L ee S, Shin S, Kim H, Han S, Kim K, Kwon J, et al. Anti- inflammatory function of arctiin by inhibiting COX-2 expression additional study would be warranted to furtherκ investigate J Inflamm (Lond) 8 via NF-kB pathways. 2011; : 16. this, perhaps by employing the use of an NF- B reporter. Br [11] B arnes PJ. Glucocorticosteroids: current and future directions. Thus, it is possible thatκ there could be cross-talk between J Pharmacol 163 - 2011; (1): 29 43. the MAPK and the NF- B signals that may be important foret Cell [12] D inarello CA. Anti-inflammatory agents: present and future. al.relaying the biological effect of MF. Moreover, Minutoli 140 - demonstrated that during the development of testicular 2010; (6): 935 950. JNK 38 [13] C hao WW, Lin BF. Isolation and identification of bioactive ischemia-reperfusionκ injury, the and p signalset al. were Andrographis paniculata Chin Med [19] compounds in (Chuanxinlian). abolished when NF- B was disruptedκ . Sethi also 5 found that TNF-induced NF- B activation was stopped in 2010; : 17. [14] M ahesh G, Ramkanth S, Mohamed Saleem TS. Anti-inflammatory cells deleted of the MKK4 gene encoding activates both JNK - Int J Rev [20] drugs from medicinal plants a comprehensive review. and p38 MAPK . Life Sci 1 2011 (1) 1 10 In summary, the present study suggests MF is a potential ; : í - . [15] M ayer AM, Rodr guez AD, Taglialatela-Scafati O, Fusetani N. inflammatory compoundS. tuberosa isolated for the first time from - medicinal plant grown in Vietnam. Marine pharmacology in 2009 2011: marine compounds with antibacterial, antidiabetic, antifungal, anti-inflammatory, Conflict of interest statement antiprotozoal, antituberculosis, and antiviral activities; affecting the immune and nervous systems, and other miscellaneous Mar Drugs 11 - mechanisms of action. 2013; (7): 2510 2573. Annu [16] J aneway CA Jr, Medzhitov R. Innate immune recognition. We declare that we have no conflict of interest. Rev Immunol 20 2002; : 197-216. [17] S eo MB, Lee SK, Jeon YJ, Im JS. Inhibition of p65 nuclear Acknowledgements Toxicol Res 27 translocation by Baicalein. 2011; (2): 71-76. [18] I slam MS, Yoshida H, Matsuki N, Ono K, Nagasaka R, Ushio H, This study was accomplished with support from the Project et al. Antioxidant, free radical-scavenging, and NF-kappaB- ( inhibitory activities of phytosteryl ferulates: structure-activity of the Vietnam Academy of Science and Technology Grant J Pharmacol Sci 111 No VAST 03.02/12-13). 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