Research Article The Herbal Drug Melampyrum pratense L

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 395316 10 pageshttpdxdoiorg1011552013395316

Research ArticleThe Herbal Drug Melampyrum pratense L (Koch)Isolation and Identification of Its Bioactive CompoundsTargeting Mediators of Inflammation

S Vogl1 A G Atanasov1 M Binder2 M Bulusu2 M Zehl1 N Fakhrudin13

E H Heiss1 P Picker1 C Wawrosch1 J Saukel1 G Reznicek1 E Urban4 V Bochkov2

V M Dirsch1 and B Kopp1

1 Department of Pharmacognosy University of Vienna 14 Althanstrasse 1090 Vienna Austria2 Department of Vascular Biology andThrombosis Research Medical University of Vienna 17 Schwarzspanierstrasse1090 Vienna Austria

3 Department of Pharmaceutical Biology Faculty of Pharmacy Universitas Gadjah Mada Sekip Utara Yogyakarta 55281 Indonesia4Department of Medicinal Chemistry University of Vienna 14 Althanstrasse 1090 Vienna Austria

Correspondence should be addressed to A G Atanasov atanasatanasovunivieacat and B Kopp brigittekoppunivieacat

Received 21 November 2012 Revised 17 January 2013 Accepted 20 January 2013

Academic Editor Il-Moo Chang

Copyright copy 2013 S Vogl et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Melampyrum pratense L (Koch) is used in traditional Austrian medicine for the treatment of different inflammation-relatedconditions In thisworkwe show that the extracts ofM pratense stimulated peroxisomeproliferator-activated receptors- (PPARs-)120572and -120574 that are well recognized for their anti-inflammatory activities Furthermore the extract inhibited the activation of the pro-inflammatory transcription factor NF-120581B and induction of its target genes interleukin-8 (IL-8) and E-selectin in vitro Bioassay-guided fractionation identified several active flavonoids and iridoids including melampyroside andmussaenoside and the phenoliccompound lunularin that were identified in this species for the first timeThe flavonoids apigenin and luteolin were distinguished asthe main components accountable for the anti-inflammatory properties Apigenin and luteolin effectively inhibited tumor necrosisfactor 120572 (TNF-120572)-induced NF-120581B-mediated transactivation of a luciferase reporter gene Furthermore the two compounds dose-dependently reduced IL-8 and E-selectin protein expression after stimulation with lipopolysaccharide (LPS) or TNF-120572 in endo-thelial cells (ECs) The iridoids melampyroside and mussaenoside prevented the elevation of E-selectin in LPS-stimulated ECsLunularin was found to reduce the protein levels of the proinflammatory mediators E-selectin and IL-8 in ECs in response to LPSThese data validate the ethnomedical use ofM pratense for the treatment of inflammatory conditions and point to the constituentsaccountable for its anti-inflammatory activity

1 Introduction

Melampyrum pratense L (Koch) or common cow-wheatbelongs to the family of Orobanchaceae and is a herbaceousflowering plant In Austrian ethnomedicine the dried herbaldrug has been used externally in a pillow or as tea to treatgout and rheumatism [1] In Romanian traditional medicineMelampyrum species are used to treat rheumatic disordersand skin infections [2] In England the plant was recom-mended as decoction for kidney ailments [3] Altogether

the applications of this plant in traditional medicine stronglysuggest anti-inflammatory activity

This work therefore aims at validating the anti-inflam-matory activity ofM pratense and identifying the compoundsresponsible for this effect by means of bioassay-guided frac-tionation To this end we applied a panel of functional andtarget-oriented cell models in order to identify constituentsof M pratense that abolish TNF-120572 or LPS-induced expres-sion of proinflammatory adhesion molecules (E-selectin) orchemokines (IL-8) inhibit the nuclear factor 120581-light-chain

2 Evidence-Based Complementary and Alternative Medicine

enhancer of activated B cells (NF-120581B) pathway or activateperoxisome-proliferator-activated receptors (PPARs) thusinterfering with different steps of the complex inflammatoryprocess

Although there are various molecular mediators involvedin the development of the inflammatory response manyproinflammatory pathways converge at the point of the acti-vation of NF-120581B [4ndash6] It regulates the expression of an arrayof inflammatory response genes such as cytokines (eg TNF-120572 IL-1120573 IL-61012 and IFN-120574) chemokines (eg monocytechemotactic protein-1 (MCP-1) IL-8) enzymes producinginflammatory mediators (eg COX-2 5-lipoxygenase andinducible nitric oxide synthase) adhesion molecules (egE-selectin ICAM-1) and enzymes degrading extracellularmatrix (eg matrix metallopeptidase 9) Hence targetingdifferent steps of the NF-120581B signaling cascade is a promisingapproach to combat different inflammatory conditions [7]E-selectin belongs to the endothelial adhesion moleculesand plays an important role in recruiting leukocytes to thesite of inflammation as it recognizes and binds to sialylatedcarbohydrates present on their surface proteins Interleukin-8(IL-8) a prototype of CXC chemokines mainly acts as a neu-trophil chemoattractant its production is induced by TNF-120572LPS and IL-1 [8 9] Another ubiquitously expressed classof transcription factors that are relevant regulators of theinflammatory response is the one of the nuclear receptors[10] Recently much attention has been drawn to the PPARswhich control the expression of inflammatory and meta-bolic genes in macrophages and lymphocytes [11ndash13] PPARsare ligand-activated transcription factors exerting importantfunctions in lipid and glucose metabolism but also regulatinginflammation PPAR120572 which is highly expressed in thevascular wall skeletal muscle heart liver and kidney ismainly involved in the regulation of lipid catabolism [14]Its activation was also shown to inhibit proliferation [1516] and to modulate the inflammatory response of vascularsmoothmuscle cells (VSMCs) [17]The activation of the otherPPAR subtype studied in this work PPAR120574 increases insulinsensitivity and contributes to the regulation of genes involvedin inflammation hypertension and atherosclerosis [10 18ndash20]

The chemical composition ofM pratense is very scarcelydescribed in the literature The aucubin content was deter-mined as 511 [21] 23 phenolic compounds including 17flavonoids and 6 phenol carboxylic acids have been identifiedby two-dimensional paper chromatography [22 23] Suomiet al (2001) quantified iridoid glycosides inM pratense usingoptimal on-line combination of partial filling micellar elec-trokinetic chromatography and electrospray ionization massspectrometry (ESI-MS) and found aucubin at a concentrationof ca 100mgL and traces of catalpol [24]

In this work we performed detailed chemical analysisof M pratense and tested a number of plant constituents incell-based in vitro assays for PPAR120572 and PPAR120574 activationNF-120581B inhibition and downregulation of TNF-120572- or LPS-stimulated expression of E-selectin and IL-8 Our dataclearly demonstrate in vitro anti-inflammatory activity ofMpratense extracts and their individual components

2 Materials and Methods

21 Materials Methanol (MeOH) and acetonitrile (MeCN)were of pa grade for extraction and ofHPLC grade forHPLCanalyses (VWR Vienna Austria) Ethyl acetate (EtOAc) pachloroform GPR Rectapur (pa) dichloromethane (DCM)AnalR Normapur (pa) n-hexane pa and toluol pawere obtained from VWR (Vienna Austria) Formic acidgt 98 pa was purchased from Carl Roth (KarlsruheGermany) Reference compounds apigenin (batch number050818082968 gt99 purity) and luteolin (batch number020717205864 gt99 purity) were from Extrasynthese (LyonFrance) chrysoeriol (batch number 47792261 gt98 purity)and diosmetin (batch number 30680794 gt98 purity) werefrom Carl Roth GmbH und CoKG (Karlsruhe Germany)aucubin (batch number 3495 gt99 purity) catalpol (batchnumber 1344 gt99 purity) loganin (batch number 5970gt99 purity) and loganic acid (batch number 3518 gt99purity) were from Phytolab (Vestenbergsgreuth Germany)and mussaenosidic acid (batch number AD111810-01 81purity) was from AnalytiCon Discovery GmbH (PotsdamGermany)

For bioactivity evaluation all indicated compounds ordried extracts were reconstituted in dimethyl sulfoxide(DMSO) aliquoted and stored at minus20∘C until use

22 Plant Material The aerial parts ofMelampyrum pratensewere field collected at Neustift amWalde Vienna Austria Avoucher specimen (MP16072008) is deposited at the Depart-ment of Pharmacognosy University of Vienna Austria

23 Extraction and Fractionation 183 g of dried and pow-dered plant material was first processed with the nonpolarsolvent DCM yielding 37 g (20 yield) extract and subse-quently with the polar solvent MeOH yielding 284 g (155yield) using an accelerated solvent extractorASE200 (ThermoScientific Austria GmbH Vienna Austria) The instrumentwas equipped with 22mL stainless steel extraction cells and60mL glass collection bottles The extraction was performedat 40∘C and 150 bar using 3 extraction cycles 5min heat-uptime 2min static time 10 flush volume and 60 sec nitrogenpurge

Subsequently tannins and chlorophylls were excludedfrom polar and nonpolar extracts respectively in order toavoid possible interferences with the assays and to increasethe relative amount of the active compounds The tanninswere removed using liquid-liquid partitions between CHCl

3

and mixtures of MeOHH2O [25] leading to a loss of 95 of

the original MeOH extract yielding 14 g (49 of the MeOHextract 07 of the crude drug) Chlorophyll was removedfrom the DCM extract by liquid-liquid-partition betweenDCM and MeOH H

2O (1 1) yielding 6475mg (175 of

the DCM extract 03 of the crude drug) In detail 1 gextract was dissolved in 150mL DCM 150mL MeOH H

2O

(1 1) was added and DCM was evaporated under reducedpressure (800ndash600mbar) at 40∘C Consequently insolublechlorophyll precipitated in the methanol-water phase andcould be filtered

Evidence-Based Complementary and Alternative Medicine 3

Solid phase extraction (SPE) on 60cc Bond Elut C18

cartridges (Varian Harbor City CA USA) was used forfurther fractionation of the purified extracts The cartridgeswere prewashed with H

2O and MeOH and conditioned

with 30 MeOHH2O After the application of the extract

successive elutionwith differentmixtures ofMeOHH2Owas

performed under reduced pressure (flow rate asymp2 dropssec)to obtain three fractions of decreasing polarity (30 70and 100 methanolic SPE subfractions)

178mg of the 30 MeOHH2O SPE fraction of chlo-

rophyll-depleted DCM extract was subjected to columnchromatography (CC) on Silica Gel 60 (particle size of0063ndash0200mmMerck Darmstadt Germany)The columnwas first eluted with EtOAc MeOH H

2O 17 3 1 and 265

fractions were collected Afterwards the column was elutedwith EtOAc MeOH H

2O17 4 1 As result of the two elution

steps overall 6main combined fractionswere obtained (301ndash306) Fraction 301 (127mg) mainly contained aucubinFrom subfractions 303 (191mg 107 of original SPE frac-tion) melampyroside was isolated by SPE on C

18material

upon elution with H2O and then MeOH H

2O 1 9 and 2 8

From fraction 305 (491mg 275 of original SPE fraction)mussaenoside was purified using SPE on C

18material upon

elution with H2O and then MeOH H

2O 1 9 and 2 8

239mg of the 70 MeOHH2O SPE fraction of the chlo-

rophyll-depleted DCM extract was subjected to CC on SilicaGel 60 (particle size of 0063ndash0200mm Merck DarmstadtGermany) using a mobile phase of CHCl

3MeOH H

2O

90 35 02 and 85 8 05 from fraction 675 on Fractionswere combined to 13 main fractions (701ndash7013) Fractions704 (25mg 11 of original SPE fraction) and 706 (95mg40 of original SPE fraction) mainly consisted of lunularinFraction 705 (234mg 98 of original SPE fraction) con-tained pure lunularin (structure clarified via LC-MS andNMR)

24 HPLC Analysis An HPLC instrument from Shimadzu(Kyoto Japan) equipped with a CBM-20A system controlleraDGU-20A5membrane degasser an LC-20AD solvent deliv-ery unit an SIL-20ACHT autosampler a CTO-20AC columnoven an SPD-M20A photodiode array detector and a low-temperature evaporative light scattering detector (ELSD-LT)was used for HPLC-DADELSD experiments Data analysiswas conducted with the chromatography software LCsolu-tion Ver12 (Shimadzu) Chromatographic separation wasperformed on an Acclaim 120 C

18reversed-phase column

(150mm times 21mm id 3 120583m) equipped with an Acclaim120 C

18guard column (10mm times 21mm id 5120583m) from

Dionex (Germering Germany) at 35∘C and a flow rate of04mLminWater modified with formic acid (pH = 26) andMeCN were used as mobile phase A and B respectively Forthe methanolic extract gradient elution was performed asfollows start isocratic at 2 of B for 5min followed by 2ndash10 of B in 11min 10ndash20 of B in 24min 20ndash50 of B in20min and 50ndash95 of B in 5min For all other extractsthe following gradient was used start isocratic at 2 of Bfor 5min followed by 2ndash15 of B in 5min 15ndash21 ofB in 3min 21ndash35 of B in 27min and 35ndash95 of B in

10min The injection volume was 5 120583L for all samples TheDAD collected data from 190 to 400 nm

25 LC-MS Parameters The tentative identification of themain flavonoids and iridoid glycosides was facilitated byHPLC-DAD-MS These analyses were performed on anUltiMate 3000 RSLC-series system (Dionex) coupled to anHCT 3D quadrupole ion trap mass spectrometer equippedwith an orthogonal ESI source (Bruker Daltonics BremenGermany) HPLC separation was carried out as describedabove The eluent flow was split roughly 1 8 before the ESIion source which was operated as follows capillary voltage+35minus37 kV nebulizer 26 psi (N

2) dry gas flow 9 Lmin

(N2) and dry temperature 340∘C The mass spectrometer

was operated in an automated data-dependent acquisition(DDA) mode to obtain MS2 MS3 andMS4 spectra (collisiongas He isolation window 4 Th and fragmentation ampli-tude 10 V)

The identity of the main compounds in the extracts wasconfirmed either by the comparison of the retention timesUV- and MS119899-spectra with reference compounds (apigeninluteolin chrysoeriol diosmetin aucubin and catalpol) or byisolation and structural characterization by 1D and 2D NMRand MS (melampyroside mussaenoside and lunularin)

26 NMR Analysis All 1D (1H and 13C) and 2D (COSYNOESY HMQC and HMBC) NMR spectra were recordedon a Bruker Avance 500NMR spectrometer (Bruker BioSpinRheinstetten Germany) The respective compounds weredissolved in methanol-d4 (998 atom D) CD

3OD [120575(1H) =

331 ppm and 120575(13C) = 4900 ppm] The 1H and 13C NMRspectra were operated at 500 and 125MHz respectively 13Cand 1H NMR data were consistent with literature values formelampyroside [26 27] and mussaenoside [27] Detaileddata for melampyroside ([1S-(11205724a12057251205727a120572)]-7-[(benzoy-loxy)methyl]-14a57a-tetrahydro-5-hydroxycyclopenta[c]pyran-1-yl-120573-D-glucopyranoside) 1H-NMR (d

4-methanol

500MHz) 120575 807 (2H Bz H-2 Bz H-6) 762 (1H Bz H-4)749 (2H Bz H-3 Bz H-5) 635 (1H H-3) 586 (1H H-6)512 (1H H-4) 511 498 (2H OCH

2) 501 (1H H-1) 470

(1H H-11015840) 448 (1H H-5) 384 364 (2H H-61015840) 337 (1HH-31015840) 328 (1H H-41015840) 327 (1H H-51015840) 323 (1H H-21015840)300 (1H H-7a) 270 (CH H-4a) 13C-NMR (d

4-methanol

125MHz) 120575 16771 (C COO) 14261 (C C-7) 14179 (CHC-3) 13442 (CH Bz C-4) 13278 (CH C-6) 13132 (C BzC-1) 13064 (CH Bz C-2 Bz C-6) 12968 (CH Bz C-3Bz C-5) 10551 (CH C-4) 10017 (CH C-11015840) 9793 (CHC-1) 8288 (CH C-5) 7832 (CH C-51015840) 7797 (CH C-31015840)7491 (CH C-21015840) 7150 (CH C-41015840) 6407 (CH

2 OCH

2)

6278 (CH2 C-61015840) 4856 (CH C-7a) 4636 (CH C-4a)

Detailed data for mussaenoside ([1S-(11205724a12057271205727a120572)]-1-(120573-D-glucopyranosyloxy)-14a5677a-hexahydro-7-hydroxy-7-methyl-cyclopenta[c]pyran-4-carboxylic acid methyl ester)1H-NMR (d

4-methanol 500MHz) 120575 741 (1H H-3) 546

(1H H-1) 467 (1H H-11015840) 390 364 (2H H-61015840) 371 (3HOMe) 336 (1H H-31015840) 332 (1H H-51015840) 324 (1H H-41015840)319 (1H H-21015840) 317 (1H H-4a) 228 142 (2H H-5) 222(1H H-7a) 171 (2H H-6) 132 (3H Me at C-7) 13C-NMR


(d4-methanol 125MHz) 120575 16938 (C COO) 15204 (C C-3)

11338 (C C-4) 9981 (CH C-11015840) 9535 (CH C-1) 8051 (CC-7) 7840 (CH C-51015840) 7800 (CH C-31015840) 7474 (CH C-21015840)7171 (CH C-41015840) 6294 (CH

2 C-61015840) 5231 (CH C-7a) 5164

(CH3 OMe) 4069 (CH

2 C-6) 3204 (CH C-4a) 3073

(CH2 C-6) 2464 (CH

3 Me at C-7)The results for lunularin

(310158404-ethylenebisphenol) were as follows 1H-NMR (d4-

methanol 500MHz) 120575 704 (1H H-51015840) 697 (2H H-2 H-6)667 (2H H-3 H-5) 663 (1H H-61015840) 659 (1H H-21015840) 658(1H H-41015840) 276 (4H benzyl CH

2) 13C-NMR (d

4-methanol

125MHz) 120575 15829 (C C-31015840) 15641 (C C-4) 14482 (C C-11015840)13402 (C C-1) 13037(CH C-2 C-6) 13017 (CH C-51015840)12085 (C C-61015840) 11636 (CH C-21015840) 11599 (CH C-3 C-5)11368 (CH C-41015840) 3950 (CH

2 CH2at C-41015840) 3624 (CH

2

CH2at C-41015840)

27 Interleukin-8 and E-Selectin ELISA Telomerase reversetranscriptase technology (hTERT) immortalized humanumbilical vein endothelial cells (HUVECtert) [28] werecultured inM199medium supplementedwith 20FCS (bothfrom Sigma-Aldrich St Louis USA) cell growth supplement(PromoCell Germany) and antibiotics The experimentswere performed in sixtuplicate in 96-well plates in M199medium containing 5 serum Plant extracts were tested ata concentration of 30 120583gmL reference pure compounds at30 120583M BAY 11-7082 was used as positive control (75120583M)Subconfluent HUVECtert cells were pretreated for 30minwith the plant material or inhibitor as indicated followed bystimulation with 10 ngmL of TNF-120572 (PeproTech Rocky HillUSA) or 100 ngmL of LPS (Sigma-Aldrich St Louis USA)for 6 h E-selectin and secreted interleukin-8 were deter-mined by whole-cell ELISA or in cell culture supernatantsrespectively

IL-8 ELISA was performed using the Quantikine HumanCXCL8IL-8 Immunoassay Kit (RampD Systems MinneapolisUSA) Supernatants were transferred into 96-well plates(NALGE-NUNC Int Rochester USA) coated with capturingantibody for IL-8 and developed with the respective detec-tion antibody Peroxidase activity was assessed with TMB2-Component Microwell Peroxidase Substrate Kit (KPLGaithersburg USA) while the optical density (OD) wasmeasured with a SynergyHT Multi-Detection MicroplateReader (BioTek Instruments Winooski USA) at 450 nmusing 620 nm wavelength as reference

For measuring E-selectin cells were fixed using 01 glu-taraldehyde in PBS E-selectin was detected using 300 ngmLCD62E mouse antihuman antibody (RampD Systems Min-neapolis USA) and horseradish peroxidase-linked sheepanti-mouse IgG (GE Healthcare Waukesha USA) The samesubstrate and detection method were used as for IL-8

28 NF-120581B Transactivation Assay The transactivation of aNF-120581B-driven luciferase reporter gene was quantified inHEK293NF-120581B-luc cells (Panomics RC0014) as previouslydescribed [29] The cells were maintained at 37∘C and 5CO2cell culture incubators in Dulbeccorsquos modified Eaglersquos

medium (DMEM Lonza Basel Switzerland) supplementedwith 2mM glutamine 100 120583gmL hygromycin B 100UmL

benzylpenicillin 100 120583gmL streptomycin and 10 fetalbovine serum (FBS) On the day before the experimentthe cells were stained by incubation for 1 h in serum-freemedium supplemented with 2 120583M Cell Tracker GreenCMFDA (C2925 Invitrogen) The cells were then reseededin 96-well plates at a density of 4times 104 cellswell in phenol red-free and FBS-free DMEM overnight Cells were pretreated asindicated for 30min prior to stimulation with 2 ngmL TNF-120572 for 4 h The final concentration of DMSO in the experi-ments was 01 or lower An equal amount of DMSO wasalways tested in each experiment to assure that the solventvehicle does not influence the results After cell lysis theluminescence of the firefly luciferase and the fluorescence ofthe Cell Tracker Green CMFDA were quantified on a GeniosPro plate reader (Tecan Grodig Austria) The luciferase-derived signal from the NF-120581B reporter was normalizedby the Cell Tracker Green CMFDA-derived fluorescence toaccount for differences in the cell numberThe knownNF-120581Binhibitor parthenolide (Sigma-Aldrich Vienna Austria) wasused as a positive control

29 PPAR Luciferase Reporter Gene Assay To evaluatePPAR120572 or PPAR120574 activation transient transfection of HEK-293 cells with the respective PPAR expression plasmid anda luciferase reporter plasmid containing PPAR responseelement (PPRE) was used [30] HEK-293 cells (ATCC USA)were cultured in DMEMwith phenol red supplemented with100UmL benzylpenicillin 100120583gmL streptomycin 2mMglutamine and 10FBSThe cells were seeded in 10 cmdishesat a density of 6 times 106 cellsdish incubated for 18 h andtransfected by the calcium phosphate precipitation methodwith 4120583g of the reporter plasmid (tk-PPREx3-luc) 4120583gPPAR120572 or PPAR120574 receptor expression plasmid and 2 120583ggreen fluorescent protein plasmid (pEGFP-N1 Clontech CAUSA) as internal control After 6 h the transfected cells wereharvested and reseeded (5 times 104 cellswell) in 96-well platescontaining DMEM without phenol red supplemented with100UmL benzylpenicillin 100120583gmL streptomycin 2mMglutamine and 5 charcoal-stripped FBS The cells werefurther treated with the indicated extracts or compounds orthe solvent vehicle and incubated for 18 h The medium wasthen discarded the cells were lysed and luciferase activityand fluorescence weremeasured on a Genios Pro plate reader(Tecan Grodig Austria) The luminescence signals obtainedfrom the luciferase activity measurements were normalizedto the EGFP-derived fluorescence to account for differencesin the transfection efficiency or cell number The specificPPAR120572 agonist GW7647 (Cayman Missouri USA) and thePPAR120574 agonist pioglitazone (Molekula Ltd Shaftesbury UK)were used to verify the subtype specificity of the performedmeasurements

210 Statistical Analysis Statistical analyses were performedusing Prism Software (ver 403 GraphPad Software Inc SanDiego CA) For NF-120581B transactivation and PPAR luciferasereporter gene assay data were normalized to DMSO treatedcontrol the mean value of which was set as 10 and forE-selectin and IL-8 expression the data were analyzed as


percent inhibition in comparison to the respective LPS- orTNF-120572-treatment controls The experimental data are pre-sented as means plusmn standard error (SE) Statistical significancewas determined by ANOVA using Bonferroni post hoc test119875 values lt 005 were considered significant (lowast119875 lt 005 lowastlowast119875 lt001 lowastlowastlowast119875 lt 0001)

3 Results and Discussion

The upregulation of E-selectin and IL-8 production inHUVECtert cells was induced by two diverse stimuli theproinflammatory cytokine TNF-120572 or the bacterial prod-uct LPS TNF-120572 and LPS activate distinct but partiallyoverlapping signaling pathways implemented in acute andchronic inflammation They were used as chemically distinctinflammatory agonists interacting with different receptors toenhance the reliance of the screen Since next to endothelialcells the LPS receptor toll-like receptor 4 and the tumornecrosis factor receptor play a key role in the activation ofother cell types for example leukocytes results of the screenare likely to be relevant to other cell types as well

The investigations of extracts of Melampyrum pratenseherb with respect to downregulation of LPS- or TNF-120572-induced IL-8 and E-selectin expression PPAR activationor NF-120581B inhibition showed equal or even better resultsfor chlorophyll-depleted and detannified extracts comparedto crude DCM and MeOH extracts (Table 1) Further frac-tionation of these extracts was performed using solid phaseextraction

The SPE subfractions were further tested for their anti-inflammatory activity in vitro The strongest downregulationof IL-8 and E-selectin induced by LPS or TNF-120572was found inthe 70 SPE fraction of the chlorophyll-free DCM extract aswell as in the 30 and 70 SPE subfraction of the detannifiedMeOH extract (Table 2) Investigations regarding PPAR acti-vation and NF-120581B inhibition showed highest activities in the70 SPE subfractions (Table 2)The high activity of the 100SPE fractions of the chlorophyll-free DCM extract could beexplained by the presence of fatty acids in these fractions thatwere identified by TLC and GC-MS (data not shown) usingreference compounds [31]

HPLC analysis revealed that besides mussaenoside 4and melampyroside 5 the crude methanolic extract con-tained several other iridoid glycosides identified as catalpol1 (shoulder of 2nd peak) aucubin 2 and mussaeno-sidicloganic acid 3 (see Figure 1) The identification of thesecompoundswas donewith commercial reference compoundsvia the comparison of the UV-spectra and retention time aswell as by LC-MS analysis

On the other hand the detannifiedMeOH extract mainlycontained flavonoids of which luteolin 6 apigenin 7 chryso-eriol 8 and traces of diosmetin could be identified (Figure 2)by comparison of the UV-spectra and retention time withcommercial reference standards as well as a comparison withan MS flavonoid database [32]

The chlorophyll-free DCM extract mainly containedmussaenoside next to lunularin 9 and melampyroside(Figure 3) Mussaenoside and melampyroside were enriched

Time (min)Ligh

t sca

tterin

g re

spon

se (m

V)

0 5 10 15 20 25 30 35 40 450

500

1000AD2

1

2

3 4

5

Figure 1 HPLC-ELDS-LT chromatogram of the methanolic extractof Melampyrum pratense Analyses were performed on an Acclaim120 C

18

reversed-phase column (150mm times 21mm id 3 120583m) equip-ped with an Acclaim 120 C

18

guard column (10mm times 21mm id5 120583m) from Dionex (Germering Germany) with the followingmobile phase gradient of acetonitrile-water (A H

2

O + formic acid(pH = 25) B MeCN) 2 B (5min) 2ndash10 of B in 11min 10ndash20 of B in 24min 20ndash50 of B in 20min and 50ndash95 ofB in 5min at a flow rate of 04mLmin and a temperature set to35∘C The following compounds were identified (1) catalpol (2)aucubin (3) mussaenosidicloganic acid (4) mussaenoside and (5)melampyroside

5 10 15 20 25 30 35

0255075

100

7

6

5

8

Time (min)

Abso

rptio

n (m

AU)

254 nm 4 nm (1)

Figure 2 HPLC-DAD (detection wavelength 254 nm) chromato-gram of the detannified MeOH extract of Melampyrum pratenseThe chromatographic separationwas assessed on anAcclaim 120C

18

reversed-phase column (150mm times 21mm id 3120583m) equipped withan Acclaim 120 C

18

guard column (10mm times 21mm id 5 120583m) fromDionex (Germering Germany) using the following mobile phasegradient of acetonitrile-water (A H

2

O + formic acid (pH = 25)B MeCN) 2 B (5min) in 5min to 15 B in 3min to 21 B in27min to 35 B and in 10min to 95 B at a flow rate of 04mLminand a temperature set to 35∘C The following compounds could beidentified (5) melampyroside (6) luteolin (7) apigenin and (8)chrysoeriol

in the 30 SPE subfraction The main component of the70 SPE subfraction was lunularin The identification wasvalidated via the comparison of retention times as well asUV-spectra with those of the isolated purified referencecompounds (structure confirmed by NMR and LC-MS)

The main compounds from M pratense were testedtogether with their initial extract for PPAR activation NF-120581B inhibition and the downregulation of LPS- or TNF-120572-induced production of IL-8 and E-selectin The iridoid gly-cosides aucubin catalpol melampyroside andmussaenosidereduced E-selectin expression in response to LPS stimulation(Figure 4) The effect of iridoidglycosides was specific for E-selectin as they did not downregulate IL-8 (Figure 5) nor


Table 1 Investigation ofMelampyrumpratense extracts for agonistic activity towards PPAR120572 and PPAR120574 inhibition of TNF-120572 inducedNF-120581Bactivity and reduction of TNF-120572 or LPS induced expression of IL-8 and E-selectin in vitro

Plantmaterial Preparation PPAR120572

activationPPAR120574

activationNF-120581B

inhibitionE-selectin+TNF

IL-8+TNF

E-selectin+LPS

IL-8+LPS

Melampyrum pratenseherb

DCM extract minus minus minus + + + + ++ + + +

DCM extractchlorophyll-depleted minus minus minus ++ + + + ++ + + +

MeOH extract minus minus minus + + + + + + + + + + + +

MeOH extractdetannified minus minus + + + + + + + + + ++ + + +

DCM dichloromethane MeOH methanol Dry material from all test samples was dissolved in DMSO at a concentration of 10mgmL and tested at a finalconcentration of 10120583gmL Regarding PPARs no activation compared to control (01 DMSO) minus Regarding NF-120581B IL-8 and E-selectin inhibition in therange of 0ndash25 compared to the vehicle treated control (01DMSO) minus 25ndash50 inhibition (119875 lt 005) + 50ndash80 inhibition (119875 lt 005) ++ over 80inhibition (119875 lt 005) +++ At least three independent experiments were performed with every sample and statistical analysis was performed by ANOVA

Table 2 Results of the SPE subfractions ofMelampyrum pratense chlorophyll-depleted DCM extract and detannified MeOH extract testedon inflammatory targets in vitro

Initial extract Preparation PPAR120572activation

PPAR120574activation

NF-120581Binhibition

E-selectin+TNF

IL-8+TNF

E-selectin+LPS

IL-8+LPS

DCM extractchlorophyll-depleted

SPE 30 MeOH minus + + ++ minus ++ minus

SPE 70MeOH + + + + + + + + + + + + minus + + + + + +

SPE 100 MeOH + + + + + + + + + minus minus ++ ++

MeOH extractdetannified

SPE 30 MeOH minus + + + + ++ minus ++ + + +

SPE 70MeOH + + + + + + + + + + + + minus ++ ++

SPE 100 MeOH minus + + minus minus + + + + + +

Fractions were tested in triplicates at a concentration of 10 120583gmL for PPARs activation TNF-120572 induced NF-120581B inhibition and reduction of LPS or TNF-120572induced expression of IL-8 and E-selectin Regarding PPARs no activation compared to control (01DMSO) minus 125 to 15-fold activity compared to control(119875 lt 005) + 15 to 2-fold activity compared to control (119875 lt 005) ++ more than 2-fold of the control (119875 lt 005) +++ Regarding NF-120581B IL-8 and E-selectin inhibition in the range of 0ndash25 compared to the vehicle treated control (01DMSO) minus 25ndash50 inhibition (119875 lt 005) + 50ndash80 inhibition(119875 lt 005) ++ over 80 inhibition (119875 lt 005) +++ At least three independent experiments were performed with every sample and statistical analysis wasperformed by ANOVA

Ligh

t sca

tterin

g re

spon

se (m

V)

Time (min)5 10 15 20 25 30 35

0

100

200

300AD2

4 95

Figure 3 HPLC-ELDS-LT chromatogram of the chlorophyll-deple-ted dichloromethane extract of Melampyrum pratense Analyseswere performed on an Acclaim 120 C

18

reversed-phase column(150mm times 21mm id 3 120583m) equipped with an Acclaim 120 C

18

guard column (10mm times 21mm id 5 120583m) from Dionex (Germer-ing Germany) with the following mobile phase gradient of aceto-nitrile-water (A H

2

O + formic acid (pH = 25) B MeCN) 2 B(5min) in 5min to 15 B in 3min to 21 B in 27min to 35 Band in 10min to 95 B at a flow rate of 04mLmin and a tem-perature set to 35∘C The following compounds were identified (4)mussaenoside (5) melampyroside and (9) lunularin

activate PPARs (data not shown) or inhibit NF-120581B (Figure 6)The iridoids catalpol and aucubin can be found inmany plant

families and have already been described for M pratenseand other Melampyrum species [24 33] Jeong et al (2002)showed that aucubin inhibited the expression of TNF-120572 andIL-6 in Ag-stimulated rat basophilic leukemia- (RBL-) 2H3mast cells in a dose-dependent manner with an IC

50of 010

and 019 120583gmL respectively In that study aucubin was alsodetermined to inhibit antigen-induced nuclear translocationof the p65 subunit of NF-120581B Since activator protein-1 bindingactivity was not affected its results suggested that aucubin isa specific inhibitor of NF-120581B activation in mast cells whichmight explain its beneficial effect in the treatment of chronicinflammatory diseases [34] However in our experimentalsetup aucubin did not inhibit NF-120581B at 30 120583M

The flavonoids apigenin and luteolin on the other handshowed strong inhibition of NF-120581B (Figure 6) as well as astrong downregulation of both IL-8 and E-selectin proteins(Figures 4 and 5) Since luteolin and apigenin displayedthe highest activity on all anti-inflammatory targets dose-response studies were performed Luteolin downregulated E-selectin with an IC

50of 567120583M in TNF-120572- and an IC

50

of 510 120583M in LPS-stimulated endothelial cells IL-8 on theother hand was downregulated with an IC

50of 321 120583M and

271120583M respectively Apigenin showed significant inhibitionof both E-selectin and IL-8 An IC

50of 317 120583M in TNF-120572-

and 189 120583Min LPS-stimulated cells was observed concerning


0102030405060708090

100110

lowastlowastlowastlowastlowastlowast

lowast

lowastlowastlowast

lowastlowast

lowast


lowast

lowastlowast

lowast

OD

(450

620

nm

)

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110


lowastlowastlowastOD

(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)

Figure 4 Effect ofMelampyrum pratense extracts and their individual components on the induction of E-selectin protein after stimulationwith LPS (a) or TNF-120572 (b) Mela MeOH methanolic extract Mela det detannified methanol extract and Mela OC 30OC 70 3070methanolic SPE subfraction of the chlorophyll-depleted dichloromethane extract Extracts and fractions were tested at 30 120583gmL compoundsat 30 120583M BAY (5120583M) was used as a positive control The data are presented as mean values plusmn standard errors of sixtuplicate measurementsVery similar results were obtained in an independent experiment Stars indicate statistical significance compared to LPS or TNF-120572 activation(ANOVA) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

102030405060708090

100110

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastOD

(450

620

nm

)

0

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110120

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

OD

(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)

Figure 5 Effect of Melampyrum pratense extracts and their individual components on the induction of IL-8 protein after stimulation withLPS (a) or TNF-120572 (b)MelaMeOHmethanolic extractMela det detannifiedmethanol extract andMelaOC 30OC 70 3070methanolicSPE subfraction of the chlorophyll-depleted dichloromethane extract Extracts and fractions were tested at 30 120583gmL compounds at 30120583MBAY (5120583M)was used as a positive controlThe data are presented asmean values plusmn standard errors of sixtuplicatemeasurements Very similarresults were obtained in an independent experiment Stars indicate statistical significance compared to LPS or TNF-120572 activation (ANOVA)lowast

119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

IL-8 expression E-selectin expression was inhibited with anIC50

of 177 120583M after stimulation with TNF-120572 and 136 120583Mafter stimulation with LPS For flavonoids in general inhi-bitory effects on numerous inflammatory targets or signalingpathways in the micromolar concentration range have beenreportedTheNF-120581B inhibitory effect of apigenin and luteolinwas detected in our study by an NF-120581B-responsive luciferasereporter assay (Figure 6) and by ELISA monitoring theexpression of the NF-120581B target genes E-selectin and IL-8(Figures 4 and 5) A number of previous studies have also

demonstrated the NF-120581B inhibitory action of these twoflavonoids utilizing a number of different methods and cellmodels [35ndash41] Depending on little changes in the flavonebackbone flavonoids can play a modulating biphasic andregulatory action on immunity and inflammation [42] Stud-ies regarding the effect of flavonols (kaempferol quercetinand myricetin) and flavones (flavone chrysin apigenin lute-olin baicalein and baicalin) on TNF-120572-stimulated ICAM-1 expression which is like E-selectin a proinflammatoryadhesion molecule revealed kaempferol chrysin apigenin


0

05

1

15

2

25

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

DM

SO+

TNF-120572

Part

heno

lide 1

0 120583M

NF120581

B ac

tivat

ion

(rel

ativ

e uni

ts)

Unt

reat

ed

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

Figure 6 Influence ofMelampyrum pratense extracts (MelaMeOHmethanolic extractMela det detannifiedmethanol extract) subfractions(Mela OC 30OC 70 3070 methanolic SPE subfraction of the chlorophyll-depleted dichloromethane extract) and the respectivecompounds thereof on NF-120581B activation Extracts and fractions were tested at a concentration of 30 120583gmL compounds at 30120583Mwith TNF-120572 cotreatment in quadruplicate in three independent experiments Parthenolide (10120583M) was used as positive control Bars represent meanvalues error bars consider standard error and stars indicate significance compared to TNF-120572 induction (ANOVABonferroni) lowastlowastlowast119875 lt 0001

and luteolin as active inhibitors of ICAM-1 expression Addi-tionally apigenin and luteolin were shown to inhibit the NF-120581B signaling at the level of I120581B kinase (IKK) activity withconsequent effect on I120581B120572 degradation NF-120581B binding tothe DNA and NF-120581B transactivation activity [35] Severalflavonoids were investigated for the inhibitory effect onLPS-induced TNF-120572 production frommacrophages Positiveresults were provided for the flavones luteolin apigeninand chrysin the flavonols quercetin and myricetin theflavanonol taxifolin and the anthocyanidin cyanidin chloridein vitro Nevertheless serum TNF-120572 production in vivo wasinhibited only by luteolin or apigenin and only luteolinor quercetin inhibited 12-O-tetradecanoylphorbol-13-acetate(TPA) induced ear edema These results suggest that thestructure of luteolin is the most suitable for the oral anti-inflammatory activity and that the presence of absence ofa hydroxy group may cause a loss of efficiency [43] More-over luteolin was shown to display excellent radical scav-enging and cytoprotective properties especially when testedin complex biological systems where it can interact withother antioxidants like vitamins Nevertheless its anti-inflammatory effects at micromolar concentrations can onlybe partly explained by its antioxidant capacitiesThey includethe activation of antioxidative enzymes suppression of theNF-120581B pathway and inhibition of proinflammatory sub-stances The glycosidic form of luteolin often present inplants is cleaved in vivo and the aglycones are conjugatedand metabolized after nutritional uptake which has to beconsidered when evaluating in vitro studies [44] Besides theinhibition of the induction of inflammatory cytokines suchas TNF-120572 IL-8 IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) luteolin was found to attenuatecyclooxygenase (COX)-2 expression and rise in intracellularCa2+ levels [45] In our study the stilbene-like compoundlunularin only had an inhibitory effect on IL-8 and E-selec-tin after stimulation with LPS but not with TNF-120572 Thiscompound ismainly known from liverworts [46 47] Besides

in higher plants it was found in celery Apium graveolens[48] andMorus species [49] Activity studies have only beenconducted concerning antibacterial antifungal and antiox-idant characteristics [50] but the anti-inflammatory effectsof lunularin have not yet been reported Thus by showingits ability to downregulate E-selectin and IL-8 in endothelialcells stimulated with LPS the present study gives first insightinto a possible anti-inflammatory action of this compound

4 Conclusion

The extracts ofMelampyrum pratensewere found to decreasethe production of inflammatory proteins E-selectin and IL-8in HUVECtert cells in vitro Using bioactivity-guided frac-tionation and HPLC-ELSD-DAD-MS structure elucida-tions several iridoids and flavonoids were isolated and iden-tified as potential active principles Melampyroside mus-saenoside and lunularin were identified in M pratense forthe first time The isolated iridoid glycosides mussaenosideand melampyroside were able to downregulate E-selectinespecially after stimulation with LPS suggesting them to acton this specific pathwayTheflavonoids apigenin and luteolinon the other hand showed strong inhibition of NF-120581B aswell as strong downregulation of both IL-8 and E-selectinand thus were considered the main constituents responsiblefor the anti-inflammatory activity of the plant The stilbene-like compound lunularin only inhibited E-selectin and IL-8expression after stimulation with LPS not with TNF-120572 Addi-tionally this compound did slightly activate PPAR120574 (datanot shown) Aside from lunularin the only further PPAR120572and PPAR120574 ligands that we identified in the plant extractswere different fatty acids which represent well-known PPARactivators [31] and thus were not further investigated

Finally the present investigations contribute to the under-standing of the chemical composition in relation to theanti-inflammatory effects elicited by Melampyrum pratenseextracts


Abbreviations

ASE Accelerated solvent extractorCC Column chromatographyDCM DichloromethaneDet Detannified methanol extractDMEM Dulbeccorsquos modified Eaglersquos mediumDMSO DimethylsulfoxideEGFP Enhanced green fluorescent proteinESIMS Electrospray ionization mass spectrometryEtOAc Ethyl acetateFBS Fetal bovine serumGC-MS Gas chromatography mass spectrometryIKK I120581B kinaseIL InterleukinLPS LipopolysaccharideMeCN AcetonitrileMeOH MethanolNF-120581B Nuclear factor 120581BNMR Nuclear magnetic resonanceOC Chlorophyll-depleted dichloromethane extractPPAR Peroxisome proliferator-activated receptorPPREs PPAR response elementsSPE Solid phase extractionTLC Thin layer chromatographyTNF Tumor necrosis factor

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

S Vogl and A G Atanasov contributed equally to this work

Acknowledgments

The authors thank Professor Ronald M Evans for provid-ing the PPRE luciferase reporter construct They gratefullyacknowledge the expert technical assistance of E Geigerand J Benedics This project was supported by the AustrianScience Fund (NFN S10704-B06 NFN S10713-B13 and NFNS10704-B03B13)

References

[1] S Gerlach J Saukel and W Kubelka ldquoPflanzen in der oster-reichischen Volksmedizin Die VOLKSMED-DATENBANKrdquoScientia Pharmaceutica vol 74 supplement 1 p 36 2006

[2] CMogosan andM FMunteanu ldquoA comparative study on anti-inflammatory effect of the tinctures from Melampyrum biha-riense Kern and Melapyrum cristatum L (scrophulariaceae)rdquoFarmacia vol 56 no 4 pp 389ndash392 2008

[3] B Maloney Traditional Herbal Cures in Country Cavan Part 1vol 1 Ulster Folklife Holywood Calif USA 1st edition 1972

[4] M S Hayden and S Ghosh ldquoNF-120581B in immunobiologyrdquo CellResearch vol 21 no 2 pp 223ndash244 2011

[5] T Lawrence ldquoThe nuclear factor NF-120581B pathway in inflamma-tionrdquo Cold Spring Harbor Perspectives in Biology vol 1 no 6Article ID a001651 2009

[6] G Sethi B Sung and B B Aggarwal ldquoNuclear factor-120581Bactivation from bench to bedsiderdquo Experimental Biology andMedicine vol 233 no 1 pp 21ndash31 2008

[7] T Lawrence andC Fong ldquoThe resolution of inflammation anti-inflammatory roles for NF-120581Brdquo International Journal of Bio-chemistry and Cell Biology vol 42 no 4 pp 519ndash523 2010

[8] M Baggiolini M Uguccioni and M DrsquoApuzzo ldquoInterleukin-8and other chemokines new perspectives in inflammationrdquo FalkSymposium vol 78 pp 5ndash13 1995

[9] J-M Schroeder ldquoInterleukin 8rdquoAdvances inNeuroimmunologyvol 2 no 2 pp 109ndash124 1992

[10] C K Glass and S Ogawa ldquoCombinatorial roles of nuclearreceptors in inflammation and immunityrdquo Nature ReviewsImmunology vol 6 no 1 pp 44ndash55 2006

[11] A Chawla ldquoControl of macrophage activation and function byPPARsrdquo Circulation Research vol 106 no 10 pp 1559ndash15692010

[12] WHuang andCKGlass ldquoNuclear receptors and inflammationcontrol molecular mechanisms and pathophysiological rele-vancerdquo Arteriosclerosis Thrombosis and Vascular Biology vol30 no 8 pp 1542ndash1549 2010

[13] B P Neve J C Fruchart and B Staels ldquoRole of the peroxisomeproliferator-activated receptors (PPAR) in atherosclerosisrdquo Bio-chemical Pharmacology vol 60 no 8 pp 1245ndash1250 2000

[14] J C Fruchart ldquoPeroxisome proliferator-activated receptor-alpha (PPAR120572) at the crossroads of obesity diabetes and car-diovascular diseaserdquo Atherosclerosis vol 205 no 1 pp 1ndash82009

[15] F Gizard and D Bruemmer ldquoTranscriptional control of vascu-lar smoothmuscle cell proliferation by peroxisome proliferator-activated receptor-120574 therapeutic implications for cardiovascu-lar diseasesrdquo PPAR Research vol 2008 Article ID 429123 11pages 2008

[16] F Gizard T Nomiyama Y Zhao et al ldquoThe PPAR120572p16INK4apathway inhibits vascular smooth muscle cell proliferation byrepressing cell cycle-dependent telomerase activationrdquo Circula-tion Research vol 103 no 10 pp 1155ndash1163 2008

[17] T Okayasu A Tomizawa K Suzuki K I Manaka and Y Hat-tori ldquoPPAR120572 activators upregulate eNOS activity and inhibitcytokine-induced NF-120581B activation through AMP-activatedprotein kinase activationrdquo Life Sciences vol 82 no 15-16 pp884ndash891 2008

[18] C K Glass and K Saijo ldquoNuclear receptor transrepressionpathways that regulate inflammation in macrophages and Tcellsrdquo Nature Reviews Immunology vol 10 no 5 pp 365ndash3762010

[19] A C Li and C K Glass ldquoPPAR- and LXR-dependent pathwayscontrolling lipid metabolism and the development of athero-sclerosisrdquo Journal of Lipid Research vol 45 no 12 pp 2161ndash21732004

[20] G Pascual A L Sullivan S Ogawa et al ldquoAnti-inflammatoryand antidiabetic roles of PPAR120574rdquo Novartis Foundation Sympo-sium vol 286 pp 183ndash196 2007

[21] B Broda L Swiatek and J RUzynski ldquoDetermination of aucu-bin in some species of the plants from family ScrophulariaceaerdquoActa Poloniae Pharmaceutica vol 26 no 3 pp 255ndash258 1969

[22] E E Galishevskaya and V M Petrichenko ldquoPhenolic com-pounds from twoMelampyrum speciesrdquoPharmaceutical Chem-istry Journal vol 44 no 9 pp 497ndash500 2010

[23] E E Galishevskaya V M Petrichenko and E N SkryabinaldquoPhenolic compounds of plants of the genus MelampyrumrdquoFarmatsiya pp 25ndash29 2011


[24] J Suomi S K Wiedmer M Jussila and M-L RiekkolaldquoDetermination of iridoid glycosides by micellar electrokineticcapillary chromatography-mass spectrometry with use of thepartial filling techniquerdquo Electrophoresis vol 22 no 12 pp2580ndash2587 2001

[25] M EWall M CWani DM Brown et al ldquoEffect of tannins onscreening of plant extracts for enzyme inhibitory activity andtechniques for their removalrdquo Phytomedicine vol 3 no 3 pp281ndash285 1996

[26] R K Chaudhuri F U Afifi-Yazar O Sticher and T Winklerldquo13C NMR spectroscopy of naturally occurring iridoid gluco-sides and their acylated derivativesrdquo Tetrahedron vol 36 no 16pp 2317ndash2326 1980

[27] T Ersoz F N Yalcin D Tasdemir O Sticher and I CalisldquoIridoid and lignan glucosides from Bellardia trixago (L) AllrdquoTurkish Journal of Medical Sciences vol 28 pp 397ndash400 1998

[28] B Giessrigl G Yazici M Teichmann et al ldquoEffects of Scro-phularia extracts on tumor cell proliferation death and intrava-sation through lymphoendothelial cell barriersrdquo InternationalJournal of Oncology vol 40 no 6 pp 2063ndash2074 2012

[29] E Rozema A G Atanasov N Fakhrudin et al ldquoSelectedextracts of Chinese Herbal Medicines their effect on NF-120581BPPAR120572 and PPAR120574 and the respective bioactive compoundsrdquoEvidence-Based Complementary and Alternative Medicine vol2012 Article ID 983023 10 pages 2012

[30] MW-F Chang J Grillari C Mayrhofer et al ldquoComparison ofearly passage senescent and hTERT immortalized endothelialcellsrdquo Experimental Cell Research vol 309 no 1 pp 121ndash1362005

[31] B Desvergne andW Wahli ldquoPeroxisome proliferator-activatedreceptors nuclear control of metabolismrdquo Endocrine Reviewsvol 20 no 5 pp 649ndash688 1999

[32] M Burgert Aufbau einer MS119899 Datenbank mittels ESI-Ionenfallen-Massenspektrometrie [MS thesis] Department ofPharmacognosy University of Vienna Vienna Austria 2011

[33] J Suomi S K Wiedmer M Jussila and M L RiekkolaldquoAnalysis of eleven iridoid glycosides by micellar electrokineticcapillary chromatography (MECC) and screening of plantsamples by partial filling (MECC)-electrospray ionisation massspectrometryrdquo Journal of Chromatography A vol 970 no 1-2pp 287ndash296 2002

[34] H-J Jeong H N Koo H J Na et al ldquoInhibition of TNF-120572and IL-6 production by aucubin through blockade of NF-120581Bactivation in RBL-2H3 mast cellsrdquo Cytokine vol 18 no 5 pp252ndash259 2002

[35] C-C Chen M P Chow W C Huang Y C Lin and Y JChang ldquoFlavonoids inhibit tumor necrosis factor-120572-inducedup-regulation of intercellular adhesion molecule-1 (ICAM-1)in respiratory epithelial cells through activator protein-1 andnuclear factor-120581B structure-activity relationshipsrdquo MolecularPharmacology vol 66 no 3 pp 683ndash693 2004

[36] J-S Choi Y J Choi S H Park J S Kang and Y H KangldquoFlavones mitigate tumor necrosis factor-120572-induced adhesionmolecule upregulation in cultured human endothelial cells roleof nuclear factor-120581Brdquo Journal of Nutrition vol 134 no 5 pp1013ndash1019 2004

[37] M Comalada I Ballester E Bailon et al ldquoInhibition of pro-inflammatorymarkers in primary bonemarrow-derivedmousemacrophages by naturally occurring flavonoids analysis of thestructure-activity relationshiprdquo Biochemical Pharmacology vol72 no 8 pp 1010ndash1021 2006

[38] M Funakoshi-Tago K Nakamura K Tago T Mashinoand T Kasahara ldquoAnti-inflammatory activity of structurally

related flavonoids Apigenin Luteolin and Fisetinrdquo Interna-tional Immunopharmacology vol 11 no 9 pp 1150ndash1159 2011

[39] E-K Kim K-B Kwon M-Y Song et al ldquoFlavonoids protectagainst cytokine-induced pancreatic beta-cell damage throughsuppression of nuclear factor kappaB activationrdquo Pancreas vol35 no 4 pp e1ndashe9 2007

[40] P A Ruiz and D Haller ldquoFunctional diversity of flavonoidsin the inhibition of the proinflammatory NF-120581B IRF and Aktsignaling pathways in murine intestinal epithelial cellsrdquo Journalof Nutrition vol 136 no 3 pp 664ndash671 2006

[41] E Tahanian L A Sanchez T C Shiao R Roy and BAnnabi ldquoFlavonoids targeting of I120581B phosphorylation abro-gates carcinogen-induced MMP-9 and COX-2 expression inhuman brain endothelial cellsrdquo Drug Design Development andTherapy vol 5 pp 299ndash309 2011

[42] S Chirumbolo ldquoThe role of quercetin flavonols and flavonesin modulating inflammatory cell functionrdquo Inflammation andAllergy vol 9 no 4 pp 263ndash285 2010

[43] H Ueda C Yamazaki and M Yamazaki ldquoA hydroxyl group offlavonoids affects oral anti-inflammatory activity and inhibitionof systemic tumor necrosis factor-120572 productionrdquo BioscienceBiotechnology and Biochemistry vol 68 no 1 pp 119ndash125 2004

[44] G Seelinger I Merfort and C M Schempp ldquoAnti-oxidantanti-inflammatory and anti-allergic activities of luteolinrdquoPlanta Medica vol 74 no 14 pp 1667ndash1677 2008

[45] O-H Kang J G Choi J H Lee and D Y Kwon ldquoLuteolinisolated from the flowers of Lonicera japonica suppressesinflammatory mediator release by blocking NF-120581B andMAPKsactivation pathways inHMC-1 cellsrdquoMolecules vol 15 no 1 pp385ndash398 2010

[46] Z-Q Lu P H Fan M Ji and H X Lou ldquoTerpenoids andbisbibenzyls from Chinese liverworts Conocephalum conicumand Dumortiera hirsutardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 187ndash192 2006

[47] A Ludwiczuk I Komala A Pham J P Bianchini P Raha-rivelomanana and Y Asakawa ldquoVolatile components fromselected Tahitian liverwortsrdquoNatural Product Communicationsvol 4 no 10 pp 1387ndash1392 2009

[48] K Zhou BWu Y Zhuang L Ding Z Liu and F Qiu ldquoChemi-cal constituents of fresh celeryrdquo Zhongguo Zhongyao Zazhi vol34 no 12 pp 1512ndash1515 2009

[49] Y M Syah S A Achmad E L Ghisalberti E H Hakim LMakmur and N H Soekamto ldquoA stilbene dimer andalasin Bfrom the root trunk of Morus macrourardquo Journal of ChemicalResearch no 5 pp 339ndash340 2004

[50] F A Ramos Y Takaishi M Shirotori et al ldquoAntibacterialand antioxidant activities of quercetin oxidation products fromyellow onion (Allium cepa) skinrdquo Journal of Agricultural andFood Chemistry vol 54 no 10 pp 3551ndash3557 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


enhancer of activated B cells (NF-120581B) pathway or activateperoxisome-proliferator-activated receptors (PPARs) thusinterfering with different steps of the complex inflammatoryprocess

Although there are various molecular mediators involvedin the development of the inflammatory response manyproinflammatory pathways converge at the point of the acti-vation of NF-120581B [4ndash6] It regulates the expression of an arrayof inflammatory response genes such as cytokines (eg TNF-120572 IL-1120573 IL-61012 and IFN-120574) chemokines (eg monocytechemotactic protein-1 (MCP-1) IL-8) enzymes producinginflammatory mediators (eg COX-2 5-lipoxygenase andinducible nitric oxide synthase) adhesion molecules (egE-selectin ICAM-1) and enzymes degrading extracellularmatrix (eg matrix metallopeptidase 9) Hence targetingdifferent steps of the NF-120581B signaling cascade is a promisingapproach to combat different inflammatory conditions [7]E-selectin belongs to the endothelial adhesion moleculesand plays an important role in recruiting leukocytes to thesite of inflammation as it recognizes and binds to sialylatedcarbohydrates present on their surface proteins Interleukin-8(IL-8) a prototype of CXC chemokines mainly acts as a neu-trophil chemoattractant its production is induced by TNF-120572LPS and IL-1 [8 9] Another ubiquitously expressed classof transcription factors that are relevant regulators of theinflammatory response is the one of the nuclear receptors[10] Recently much attention has been drawn to the PPARswhich control the expression of inflammatory and meta-bolic genes in macrophages and lymphocytes [11ndash13] PPARsare ligand-activated transcription factors exerting importantfunctions in lipid and glucose metabolism but also regulatinginflammation PPAR120572 which is highly expressed in thevascular wall skeletal muscle heart liver and kidney ismainly involved in the regulation of lipid catabolism [14]Its activation was also shown to inhibit proliferation [1516] and to modulate the inflammatory response of vascularsmoothmuscle cells (VSMCs) [17]The activation of the otherPPAR subtype studied in this work PPAR120574 increases insulinsensitivity and contributes to the regulation of genes involvedin inflammation hypertension and atherosclerosis [10 18ndash20]

The chemical composition ofM pratense is very scarcelydescribed in the literature The aucubin content was deter-mined as 511 [21] 23 phenolic compounds including 17flavonoids and 6 phenol carboxylic acids have been identifiedby two-dimensional paper chromatography [22 23] Suomiet al (2001) quantified iridoid glycosides inM pratense usingoptimal on-line combination of partial filling micellar elec-trokinetic chromatography and electrospray ionization massspectrometry (ESI-MS) and found aucubin at a concentrationof ca 100mgL and traces of catalpol [24]

In this work we performed detailed chemical analysisof M pratense and tested a number of plant constituents incell-based in vitro assays for PPAR120572 and PPAR120574 activationNF-120581B inhibition and downregulation of TNF-120572- or LPS-stimulated expression of E-selectin and IL-8 Our dataclearly demonstrate in vitro anti-inflammatory activity ofMpratense extracts and their individual components

2 Materials and Methods

21 Materials Methanol (MeOH) and acetonitrile (MeCN)were of pa grade for extraction and ofHPLC grade forHPLCanalyses (VWR Vienna Austria) Ethyl acetate (EtOAc) pachloroform GPR Rectapur (pa) dichloromethane (DCM)AnalR Normapur (pa) n-hexane pa and toluol pawere obtained from VWR (Vienna Austria) Formic acidgt 98 pa was purchased from Carl Roth (KarlsruheGermany) Reference compounds apigenin (batch number050818082968 gt99 purity) and luteolin (batch number020717205864 gt99 purity) were from Extrasynthese (LyonFrance) chrysoeriol (batch number 47792261 gt98 purity)and diosmetin (batch number 30680794 gt98 purity) werefrom Carl Roth GmbH und CoKG (Karlsruhe Germany)aucubin (batch number 3495 gt99 purity) catalpol (batchnumber 1344 gt99 purity) loganin (batch number 5970gt99 purity) and loganic acid (batch number 3518 gt99purity) were from Phytolab (Vestenbergsgreuth Germany)and mussaenosidic acid (batch number AD111810-01 81purity) was from AnalytiCon Discovery GmbH (PotsdamGermany)

For bioactivity evaluation all indicated compounds ordried extracts were reconstituted in dimethyl sulfoxide(DMSO) aliquoted and stored at minus20∘C until use

22 Plant Material The aerial parts ofMelampyrum pratensewere field collected at Neustift amWalde Vienna Austria Avoucher specimen (MP16072008) is deposited at the Depart-ment of Pharmacognosy University of Vienna Austria

23 Extraction and Fractionation 183 g of dried and pow-dered plant material was first processed with the nonpolarsolvent DCM yielding 37 g (20 yield) extract and subse-quently with the polar solvent MeOH yielding 284 g (155yield) using an accelerated solvent extractorASE200 (ThermoScientific Austria GmbH Vienna Austria) The instrumentwas equipped with 22mL stainless steel extraction cells and60mL glass collection bottles The extraction was performedat 40∘C and 150 bar using 3 extraction cycles 5min heat-uptime 2min static time 10 flush volume and 60 sec nitrogenpurge

Subsequently tannins and chlorophylls were excludedfrom polar and nonpolar extracts respectively in order toavoid possible interferences with the assays and to increasethe relative amount of the active compounds The tanninswere removed using liquid-liquid partitions between CHCl

3

and mixtures of MeOHH2O [25] leading to a loss of 95 of

the original MeOH extract yielding 14 g (49 of the MeOHextract 07 of the crude drug) Chlorophyll was removedfrom the DCM extract by liquid-liquid-partition betweenDCM and MeOH H

2O (1 1) yielding 6475mg (175 of

the DCM extract 03 of the crude drug) In detail 1 gextract was dissolved in 150mL DCM 150mL MeOH H

2O

(1 1) was added and DCM was evaporated under reducedpressure (800ndash600mbar) at 40∘C Consequently insolublechlorophyll precipitated in the methanol-water phase andcould be filtered










2O 17 3 1 and 265




18material


2O 1 9 and 2 8


18material upon


2O 1 9 and 2 8



3MeOH H

2O














3OD [120575(1H) =


4-methanol


2) 501 (1H H-1) 470


4-methanol


2 OCH

2)

6278 (CH2 C-61015840) 4856 (CH C-7a) 4636 (CH C-4a)


4-methanol 500MHz) 120575 741 (1H H-3) 546





2 C-61015840) 5231 (CH C-7a) 5164

(CH3 OMe) 4069 (CH

2 C-6) 3204 (CH C-4a) 3073

(CH2 C-6) 2464 (CH




2) 13C-NMR (d

4-methanol


2 CH2at C-41015840) 3624 (CH

2

CH2at C-41015840)


















Time (min)Ligh

t sca

tterin

g re

spon

se (m

V)

0 5 10 15 20 25 30 35 40 450

500

1000AD2

1

2

3 4

5


18


18


2


5 10 15 20 25 30 35

0255075

100

7

6

5

8

Time (min)

Abso

rptio

n (m

AU)

254 nm 4 nm (1)


18


18


2










IL-8+TNF

E-selectin+LPS

IL-8+LPS











E-selectin+TNF

IL-8+TNF

E-selectin+LPS

IL-8+LPS



SPE 70MeOH + + + + + + + + + + + + minus + + + + + +




SPE 70MeOH + + + + + + + + + + + + minus ++ ++



Ligh

t sca

tterin

g re

spon

se (m

V)

Time (min)5 10 15 20 25 30 35

0

100

200

300AD2

4 95


18


18


2




50of 010




50


50of 321 120583M and


50of 317 120583M in TNF-120572-



0102030405060708090

100110


lowast

lowastlowastlowast

lowastlowast

lowast


lowast

lowastlowast

lowast

OD

(450

620

nm

)

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110



(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)


102030405060708090

100110

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast


(450

620

nm

)

0

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110120

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

OD

(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)







0

05

1

15

2

25

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

DM

SO+

TNF-120572

Part

heno

lide 1

0 120583M

NF120581

B ac

tivat

ion

(rel

ativ

e uni

ts)

Unt

reat

ed

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin




4 Conclusion




Abbreviations






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























2O 17 3 1 and 265




18material


2O 1 9 and 2 8


18material upon


2O 1 9 and 2 8



3MeOH H

2O














3OD [120575(1H) =


4-methanol


2) 501 (1H H-1) 470


4-methanol


2 OCH

2)

6278 (CH2 C-61015840) 4856 (CH C-7a) 4636 (CH C-4a)


4-methanol 500MHz) 120575 741 (1H H-3) 546





2 C-61015840) 5231 (CH C-7a) 5164

(CH3 OMe) 4069 (CH

2 C-6) 3204 (CH C-4a) 3073

(CH2 C-6) 2464 (CH




2) 13C-NMR (d

4-methanol


2 CH2at C-41015840) 3624 (CH

2

CH2at C-41015840)


















Time (min)Ligh

t sca

tterin

g re

spon

se (m

V)

0 5 10 15 20 25 30 35 40 450

500

1000AD2

1

2

3 4

5


18


18


2


5 10 15 20 25 30 35

0255075

100

7

6

5

8

Time (min)

Abso

rptio

n (m

AU)

254 nm 4 nm (1)


18


18


2










IL-8+TNF

E-selectin+LPS

IL-8+LPS











E-selectin+TNF

IL-8+TNF

E-selectin+LPS

IL-8+LPS



SPE 70MeOH + + + + + + + + + + + + minus + + + + + +




SPE 70MeOH + + + + + + + + + + + + minus ++ ++



Ligh

t sca

tterin

g re

spon

se (m

V)

Time (min)5 10 15 20 25 30 35

0

100

200

300AD2

4 95


18


18


2




50of 010




50


50of 321 120583M and


50of 317 120583M in TNF-120572-



0102030405060708090

100110


lowast

lowastlowastlowast

lowastlowast

lowast


lowast

lowastlowast

lowast

OD

(450

620

nm

)

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110



(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)


102030405060708090

100110

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast


(450

620

nm

)

0

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110120

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

OD

(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)







0

05

1

15

2

25

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

DM

SO+

TNF-120572

Part

heno

lide 1

0 120583M

NF120581

B ac

tivat

ion

(rel

ativ

e uni

ts)

Unt

reat

ed

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin




4 Conclusion




Abbreviations






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2 C-61015840) 5231 (CH C-7a) 5164

(CH3 OMe) 4069 (CH

2 C-6) 3204 (CH C-4a) 3073

(CH2 C-6) 2464 (CH




2) 13C-NMR (d

4-methanol


2 CH2at C-41015840) 3624 (CH

2

CH2at C-41015840)


















Time (min)Ligh

t sca

tterin

g re

spon

se (m

V)

0 5 10 15 20 25 30 35 40 450

500

1000AD2

1

2

3 4

5


18


18


2


5 10 15 20 25 30 35

0255075

100

7

6

5

8

Time (min)

Abso

rptio

n (m

AU)

254 nm 4 nm (1)


18


18


2










IL-8+TNF

E-selectin+LPS

IL-8+LPS











E-selectin+TNF

IL-8+TNF

E-selectin+LPS

IL-8+LPS



SPE 70MeOH + + + + + + + + + + + + minus + + + + + +




SPE 70MeOH + + + + + + + + + + + + minus ++ ++



Ligh

t sca

tterin

g re

spon

se (m

V)

Time (min)5 10 15 20 25 30 35

0

100

200

300AD2

4 95


18


18


2




50of 010




50


50of 321 120583M and


50of 317 120583M in TNF-120572-



0102030405060708090

100110


lowast

lowastlowastlowast

lowastlowast

lowast


lowast

lowastlowast

lowast

OD

(450

620

nm

)

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110



(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)


102030405060708090

100110

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast


(450

620

nm

)

0

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110120

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

OD

(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)







0

05

1

15

2

25

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

DM

SO+

TNF-120572

Part

heno

lide 1

0 120583M

NF120581

B ac

tivat

ion

(rel

ativ

e uni

ts)

Unt

reat

ed

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin




4 Conclusion




Abbreviations






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Time (min)Ligh

t sca

tterin

g re

spon

se (m

V)

0 5 10 15 20 25 30 35 40 450

500

1000AD2

1

2

3 4

5


18


18


2


5 10 15 20 25 30 35

0255075

100

7

6

5

8

Time (min)

Abso

rptio

n (m

AU)

254 nm 4 nm (1)


18


18


2










IL-8+TNF

E-selectin+LPS

IL-8+LPS











E-selectin+TNF

IL-8+TNF

E-selectin+LPS

IL-8+LPS



SPE 70MeOH + + + + + + + + + + + + minus + + + + + +




SPE 70MeOH + + + + + + + + + + + + minus ++ ++



Ligh

t sca

tterin

g re

spon

se (m

V)

Time (min)5 10 15 20 25 30 35

0

100

200

300AD2

4 95


18


18


2




50of 010




50


50of 321 120583M and


50of 317 120583M in TNF-120572-



0102030405060708090

100110


lowast

lowastlowastlowast

lowastlowast

lowast


lowast

lowastlowast

lowast

OD

(450

620

nm

)

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110



(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)


102030405060708090

100110

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast


(450

620

nm

)

0

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110120

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

OD

(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)







0

05

1

15

2

25

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

DM

SO+

TNF-120572

Part

heno

lide 1

0 120583M

NF120581

B ac

tivat

ion

(rel

ativ

e uni

ts)

Unt

reat

ed

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin




4 Conclusion




Abbreviations






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















IL-8+TNF

E-selectin+LPS

IL-8+LPS











E-selectin+TNF

IL-8+TNF

E-selectin+LPS

IL-8+LPS



SPE 70MeOH + + + + + + + + + + + + minus + + + + + +




SPE 70MeOH + + + + + + + + + + + + minus ++ ++



Ligh

t sca

tterin

g re

spon

se (m

V)

Time (min)5 10 15 20 25 30 35

0

100

200

300AD2

4 95


18


18


2




50of 010




50


50of 321 120583M and


50of 317 120583M in TNF-120572-



0102030405060708090

100110


lowast

lowastlowastlowast

lowastlowast

lowast


lowast

lowastlowast

lowast

OD

(450

620

nm

)

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110



(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)


102030405060708090

100110

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast


(450

620

nm

)

0

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110120

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

OD

(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)







0

05

1

15

2

25

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

DM

SO+

TNF-120572

Part

heno

lide 1

0 120583M

NF120581

B ac

tivat

ion

(rel

ativ

e uni

ts)

Unt

reat

ed

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin




4 Conclusion




Abbreviations






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0102030405060708090

100110


lowast

lowastlowastlowast

lowastlowast

lowast


lowast

lowastlowast

lowast

OD

(450

620

nm

)

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110



(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)


102030405060708090

100110

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast


(450

620

nm

)

0

LPS+

BAY

(5 120583M

)LP

S (1

00 n

gm

L)

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin

(a)

0102030405060708090

100110120

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

OD

(450

620

nm

)

TNF-120572

(100

ng

mL)

TNF-120572+

BAY

(5 120583M

)M

ela M

eOH

Aucu

bin

Cata

lpol

Loga

nic a

cid

Mela

det

Lute

olin

Apig

enin

Mela

OC

30M

elam

pyro

side

Mus

saen

osid

eM

ela O

C 70

Lunu

larin

(b)







0

05

1

15

2

25

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

DM

SO+

TNF-120572

Part

heno

lide 1

0 120583M

NF120581

B ac

tivat

ion

(rel

ativ

e uni

ts)

Unt

reat

ed

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin




4 Conclusion




Abbreviations






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

25

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

DM

SO+

TNF-120572

Part

heno

lide 1

0 120583M

NF120581

B ac

tivat

ion

(rel

ativ

e uni

ts)

Unt

reat

ed

Mel

a MeO

HAu

cubi

nCa

talp

olLo

gani

c aci

dM

ela d

etLu

teol

inAp

igen

inM

ela O

C 30

Mel

ampy

rosid

eM

ussa

enos

ide

Mela

OC

70Lu

nula

rin




4 Conclusion




Abbreviations






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Abbreviations






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Research Article The Herbal Drug Melampyrum pratense L