In Vitro Inhibition of the Transcription Factor NF-κB and Cyclooxygenase by Bamboo...

* Correspand PhytocB-9000 GhE-mail: Ar

PHYTOTHERAPY RESEARCHPhytother. Res. (2013)Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/ptr.4978

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In Vitro Inhibition of the Transcription FactorNF-kB and Cyclooxygenase by Bamboo Extracts

Laura Van Hoyweghen,1* Karolien De Bosscher,2,3 Guy Haegeman,2 Dieter Deforce1and Arne Heyerick11Laboratory of Pharmacognosy and Phytochemistry, Faculty of Pharmaceutical Sciences, Ghent University, B-9000, Ghent, Belgium2Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST), Department of Physiology, Ghent University,B-9000, Ghent, Belgium3Cytokine receptor Laboratory, Department of Medical Protein Research, VIB, and Department of Biochemistry, Faculty of Medicineand Health Sciences, Ghent University, A. Baertsoenkaai 39000, Ghent, Belgium

Several bamboo species have been used in traditional medicine for the treatment of inflammatory conditions.The present study evaluates the in vitro anti-inflammatory properties of the traditionally used bamboo speciesPhyllostachys nigra (Lodd.) Munro and Sasa veitchii (Carr.) Rehder to explore their future researchopportunities and therapeutic potential as anti-inflammatory agents. The extracts were evaluated for theirpotential inhibitory activity at the level of NF-kB-induced gene expression and suppression of cyclooxygenase(COX)-1 and COX-2 enzyme activities, representative pharmacological targets for the anti-inflammatory actionof glucocorticoids and non-steroidal anti-inflammatory drugs, respectively. The activity of P. nigra (Lodd.)Munro and S. veitchii (Carr.) Rehder was compared with bamboo species without traditional anti-inflammatoryindications. High-performance liquid chromatography with diode-array detection and liquid chromatography–tandemmass spectrometry analyses were performed to phytochemically characterize the extracts. P. nigra (Lodd.) Munroleaf extract potently inhibited NF-kB-induced gene expression, while S. veitchii (Carr.) Rehder leaf extract exerteda selective COX-2 inhibition. The crude extracts consistently showed a more potent bioactivity than the solid phaseextraction fractions. P. nigra (Lodd.) Munro and S. veitchii (Carr.) Rehder both exert anti-inflammatory properties,but act via a different molecular mechanism. Copyright © 2013 John Wiley & Sons, Ltd.

Keywords: Bamboo leaves; traditional medicine; cyclooxygenase; NF-kB; LC-MS/MS.

Abbreviations: COX, cyclooxygenase; GC, glucocorticoids; IkBa, inhibitory protein kappa B a; NF-kB, nuclear factor-kappa B; TNF,tumor necrosis factor

INTRODUCTION

Inflammation can be considered as a response of tissuesto injury by many stimuli such as microbial invasion,physical injury and ultraviolet (UV) irradiation. Themainfunction of inflammation is to resolve infection and torepair the damage in order to achieve homeostasis.Chronic inflammation occurs when the production ofcytokines is disturbed, thereby amplifying and perpetuatingthe inflammatory state (Barton, 2008).Currently, the most commonly used drugs in the

symptomatic treatment of inflammation are glucocorti-coids (GCs) and non-steroidal anti-inflammatory drugs(NSAIDs). However, upon long-term use, these drugsare associated with unwanted side effects. For GCs,adverse effects such as skin atrophy, impaired woundhealing, diabetes, osteoporosis, increased susceptibilityto infections and growth suppression are observed(McDonough et al., 2008). Long-term administration ofNSAIDs unavoidably leads to side effects such asgastro-intestinal damage, kidney disorders, inhibition

ondence to: Dr. Arne Heyerick, Laboratory of Pharmacognosyhemistry, Faculty of Pharmaceutical Sciences, Ghent University,ent, [email protected]

© 2013 John Wiley & Sons, Ltd.

of platelet aggregation and bronchoconstriction, due totheir non-selective inhibition of cyclooxygenase (COX)-enzymes (Dannhardt and Kiefer, 2001). Therefore,scientists are still exploring alternatives with an improvedbenefit-to-risk ratio. In this respect, information derivedfrom traditional medicine can be a valuable approachfor the discovery of novel anti-inflammatory agents.

GCs exert their activity by interference with thetranscription factors NF-kB and activator protein (AP)-1.A range of immunoregulatory genes coding for cytokines,cytokine receptors, chemotactic proteins, or adhesionmolecules contain NF-kB and/or AP-1 sites in theirpromoters or regulatory regions (De Bosscher et al., 2003).

The mechanism of action of NSAIDs is based on thereversible inhibition of COX by competing with thesubstrate arachidonic acid (AA). COX is the rate-limitingenzyme that catalyzes the initial step ofAA transformationinto prostaglandins (PGs), thromboxanes (TXA2) andprostacyclin (PGI2) (Vane and Botting, 1996). PGE2 isthe most important PG which mediates the typicalsymptoms of inflammation: redness, heat development,swelling, pain and a gradual loss of function (DannhardtandKiefer, 2001). Agents that inhibit COX-2 while sparingCOX-1 exert an anti-inflammatory effect without the sideeffects associated with COX-1 inhibition (Dannhardt andKiefer, 2001). However, long-term use of the selectiveCOX-2 inhibitors ‘coxibs’ have also been linked with

Received 3 August 2012Revised 06 February 2013

Accepted 14 February 2013

L. VAN HOYWEGHEN ET AL.

severe cardiovascular adverse side effects associated withtheir long-term use (Baron et al., 2008), emphasizing theneed for alternative approaches even more.Phyllostachys nigra (Lodd.) Munro and Sasa veitchii

(Carr.) Rehder are two bamboo species with atraditional medicinal use in Asian countries. The leavesof Phyllostachys nigra (Lodd.) Munro have been usedfor their antipyretic and diuretic properties (Duke andAyensu, 1985). Sasa veitchii (Carr.) Rehder leaves havebeen used to treat burns and diuretic problems(Akamatsu, 1970). Shirotake et al. (2009) reportthat the leaves of Sasa veitchii (Carr.) Rehder are usedas a treatment for halitosis and body odor, stomatitis,gingivitis, bedsores, burns, hemorrhoids, cuts andpurulent wounds.In this study, we investigated the effect of the leaf

extracts of P. nigra (Lodd.) Munro and S. veitchii onNF-kB-induced gene expression and on COX-1and COX-2 enzyme activities, as key targets in thepathway leading towards inflammation. To emphasizethe importance of gathering prior information derivedfrom traditional medicine, we compared their anti-inflammatory activity with 10 other bamboo species whichdo not have any known anti-inflammatory properties. Thespecies Arundinaria gigantea (Walter) Muhl., Dinochloascandens (Blume ex Nees) Kuntze and Pseudosasajaponica (Steud.) Makino have reported traditional uses(Stuart, 1911; Sturtevant, 1955; Moerman, 1998; BPI,2005), but not related to inflammation. Bambusa balcooaRoxb., Dendrocalamus hamiltonii Nees & Arn. exMunro, Fargesia robusta T.P. Yi ‘Pingwu’, Fargesiarufa T.P. Yi ‘Green Panda’, Guadua amplexifoliaJ. Presl, Phyllostachys humilis and Pleioblastus variegatus(Siebold ex Miq.) Makino have, to our knowledge, nohistory of traditional medicinal applications.The current study provides substantial insight into the

therapeutic potential of bamboo and will probably spurfuture research and application opportunities ofbamboo species as anti-inflammatory agents.

Table 1. Extraction yield of leaves (% DW)

A. gigantea 16B. balcooa 21D. hamiltonii 20D. scandens 22F. robusta ’Pingwu’ 18F. rufa ’Green Panda’ 18G. amplexifolia 21P. variegatus 21P. humilis 16P. nigra 24P. japonica 20S. veitchii 19

MATERIALS AND METHODS

Plant collection and extraction. The bamboos werein vitro propagated and further grown outdoors or ingreenhouses. These plants were kindly provided byOprins plant NV. A voucher specimen of each specieswas deposited in the Herbarium of the Ghent UniversityBotanical Garden, Faculty of Sciences, Ghent University.Each species was provided with an accessionnumber: 2009–1565G (Fargesia robusta T.P. Yi ‘Pingwu’),2009–1552G (Phyllostachys nigra (Lodd.) Munro), 2009–1557G(Sasaveitchii (Carr.)Rehder),2009–1551G(Dendrocalamushamiltonii Nees & Arn. ex Munro), 2009–1547G(Dinochloa scandens (Blume ex Nees) Kuntze), 2009–1549G (Guadua amplexifolia J. Presl), 2009–1556G(Phyllostachys humilis), 2009–1563G (Pleioblastus variegatus(Siebold ex Miq.) Makino), 2009–1555G (Pseudosasajaponica (Steud.) Makino), 2009–1564G (Fargesia rufa T.P.Yi ‘Green Panda’), 2010–2642G (Arundinaria gigantea(Walter)Muhl.), and 2010–2641G (Bambusa balcooaRoxb.).Freeze-dried leaves were milled and successively

extracted three times with MeOH/H2O (1/1, v/v) at40 �C in a sonication bath (Bandelin sonorex) for

Copyright © 2013 John Wiley & Sons, Ltd.

30min. The solvent was then removed under reducedpressure, and the extract was freeze-dried. Theextraction yield was calculated as a percentage of dryweight (Table 1.).

High-performance liquid chromatography (HPLC)analysis.HPLC solvents were purchased from Biosolve(Valkenswaard, The Netherlands). The chromatographicprofiles of the extracts were determined by HPLC withdiode-array detection (DAD) using a Waters 2695Alliance separations module and 996 photodiode arraydetector (Waters, Milford, MA) in combination with aphenomenex Luna 5m C18(2) column (i.d.: 4.6mm,length: 250mm). The UV detection range was set from200 to 800nm. The mobile phase consisted of 0.025%HCOOH in H2O (solvent A), 0.025% HCOOH inCH3CN (solvent B) and 0.025% HCOOH in MeOH(solvent C). The elution program was: 0–3min – isocraticat 85%A (7.5%B andC); 3–8min. – linear gradient from85 to 76% A; 8–11min. – isocratic at 76%A; 11–18min –linear gradient from 76 to 66% A; 18–28min. – lineargradient from 66 to 56% A; 28–36min. - linear gradientfrom 56 to 19% A; 36–42min. from 19 to 5% A;42–50min. – isocratic at 5% A; 50–57min. – lineargradient from 5 to 85% A; 57–60min. – isocratic at 85%A. The flow rate was 0.7ml/min., injection volume 10mland the column oven temperature was set at 35 �C.Extracts were diluted with H2O and/or MeOH to aconcentration of 10mg/ml prior to analysis. Liquidchromatography–mass spectrometry (LC-MS) analysiswas performed using a Waters 2695 Alliance separationsmodule coupled to a Q-TOF micro (Waters) massspectrometer. Chromatographic separationswere obtainedusing the same column and the samemobile phase but with0.1% HCOOH in H2O (solventA) and 0.1% HCOOH inMeOH/CH3CN (1/1) (solvent B) as mobile phases. Theflow rate was 0.7ml/min with a split ratio of ¼, and theinjection volume was 10 mL. MS data were acquired inthe positive and negative mode. A cone voltage of 25Vand a capillary voltage of 2.5kVwere used in both positiveand negative mode. The desolvation temperature was setto 300 �C, and the source temperature to 120 �C. MSand MS/MS spectra were acquired over an m/z range of100–800. The MCP detector potential was set to 2200Vboth for positive and negative mode.

Solid phase extraction (SPE). Freeze-dried crudeextract was dissolved in H2O/MeOH (9/1; 0.025%HCOOH) and applied to an activated and conditioned

Phytother. Res. (2013)

Figure 1. Inhibition of COX enzymes by indometacin (54 nM) andS. veitchii leaf extract. * p ≤0.05; **p ≤0.01; ***p ≤0.001.(Inhibition percentage and statistical values are expressed in relation tosolvent control). Values are the means (+ SD) of three measurementsof one sample.

BAMBOO EXTRACTS INHIBIT NF-kB AND CYCLOOXYGENASE

SPE column (5000mg/20ml, C18, GracePureTM,Alltech Associates, Lokeren, Belgium). Elution with100% H2O (20ml), 30% MeOH (30ml), 60% MeOHin H2O (30ml) and 100% MeOH (40ml) yielded fourfractions with compounds of different polarity. Theeluentia were acidified with 0.025% HCOOH. Eachfraction was dried under nitrogen or freeze-dried anddissolved in dimethyl sulfoxide (DMSO) or H2O forfurther dilution in the assays.

COX inhibitor screening assay. This assay wasperformed using a commercial COX inhibitor screeningassay kit (Cayman Chemical, kit N� 560131, Ann Arbor,MI, USA), according to the manufacturer’s protocol.All extracts were initially dissolved in DMSO or H2O(50mg/ml) and further diluted in reaction buffer (finalconcentration of 0.5% solvent). The same solventconcentration was used in the control wells. The crudeextracts (200 mg/ml) were incubated with either COX-1or COX-2 enzymes for 5min. at 37 �C. The reactionwas initiated by addition of AA and incubated for2min. at 37 �C. Then the reaction was stopped by adding1M HCl. The PGH2 produced in the COX reactionswas converted to the more stable PGF2a by the additionof a saturated solution of stannous chloride (SnCl2). TheCOX enzyme activity was than measured based onthe amount of PGF2a generated in the reaction tubeand detected by enzyme immunoassay. Absorptionmeasurements were registered on a Tecan microplate(Tecan, Austria) reader with Magellan™ software.Indometacin was used as positive control (at 54 nM) andwas bought from Sigma Aldrich (Bornem, Belgium).

Cell culture and reagents. Mouse fibrosarcoma L929sAcells stably transfected with a recombinant NF-kB-responsive promoter, derived from the IL-6 promoter,coupled to a luciferase reporter gene, p(IL6-kB)3-50hu.IL6P-luc+ (Plaisance et al., 1997) and the pPGKbGeobpAexpression vector, constitutively expressing b-Galactosidase,were available at the Faculty of Sciences, Laboratory ofEukaryotic Gene Expression and Signal Transduction,Ghent University. The cells were maintained in DMEM(Gibco, Invitrogen, CA, USA) supplemented with 5% fetalcalf serum and 5% newborn calf serum, 100 U/ml penicillinand 0.1mg/ml streptomycin. Recombinant murine TNFwas produced in the same laboratory. Dexamethasone(DEX) was purchased from Sigma Chemical (St. Louis,MO, USA) and used as a positive control in the reportergene assays. The luciferase assay reagent comprised270mM coenzyme A, 470mM luciferin (both from Sigma),and 530mMadenosine triphosphate (BoehringerMannheim,Germany) in a reaction buffer containing 20mM tricine,1mM (MgCO3)4.Mg(OH)2, 2.7mMMgSO4, 0.1mMEDTAand 33.3mM dithiothreitol (all from Sigma). Galactosidaseactivity was quantified by a Tropix Galactostar kit (AppliedBiosystems, Lennik, Belgium). We verified that the highestsolvent concentration (not exceeding 0.5%) of the testcompounds and extracts did not interfere with any of theassays. Each experiment was performed at least in triplicate.

Reporter gene assay. Cells were seeded into a 24-wellplate (120000 cells/well) and allowed to adhere for24 h. After 24 h cells were incubated with crude extractsor SPE fractions at 100, 250, 500, 750 and 1000mg/mldissolved in DMSO or H2O at a maximum concentration


of 0.5%; the solvent concentration that was also usedin the control wells. For homoorientin and orientin, aconcentration range of 0–50mM was used. The cellswere subsequently incubated for 45min, after whichTNF was added.

After the appropriate inductions (5h), cells werewashedwith phosphate-buffered saline (PBSA), lysed in lysisbuffer (Tropix, Bedford, MA, USA) and frozen overnight.Thawed cell lysates were transferred to two black 96-wellplates, and samples were assayed for their reporter geneactivity according to the manufacturer’s instructions(Promega, Madison, WI, USA). Light emission wasmeasured in a luminescencemicroplate counter (VictorX3;Perkin Elmer, Turku, Finland). Luciferase activities,expressed in arbitrary light units, were corrected for theprotein concentration in the sample, by normalization tothe constitutively co-expressed b-galactosidase levels.Βeta-galactosidase protein levels were quantified usingthe chemiluminescent reporter assay of a Galactostar kit(Applied Biosystems, Lennik, Belgium).

Statistical analysis. The data on NF-kB and COXinhibition was statistically analyzed using one-wayanalysis of variance and Bonferroni post-hoc test. Thevalues with p≤0.05 were considered statistically significant.

RESULTS

Indometacin (54 nM) was used as a positive control inthe COX assays. Although not significant at the testedconcentration, indometacin tended to have a higherselectivity towards COX-2 (Fig. 1). Of the 12 investigatedspecies, only S. veitchii (Carr.) Rehder extract was able toinhibit the conversion of AA to PGH2 at the testedconcentration (200mg/ml) (Fig. 1). More specifically,S. veitchii (Carr.) Rehder showed a five times strongerinhibition ofCOX-2 compared toCOX-1 (p< 0.001) (Fig. 1).

HPLC-DAD and LC-MS/MS of the 12 extractsrevealed the presence of phenolic compounds asmajor compounds, as discussed in previous work (VanHoyweghen et al., 2012).

Fractionation of the total extract of S. veitchii (Carr.)Rehder (269.9mg) with SPE resulted in four fractionscontaining compounds of different polarity: 100% H2O



(177.8mg), 30% MeOH (22.9mg), 60% MeOH in H2O(40.6mg) and 100%MeOH (12.7mg). The fractions wereevaluated in the COX inhibitory screening assay at aconcentration corresponding to their relative part in the200mg/ml total extract: 140mg/ml (100% H2O), 18mg/ml(30% MeOH), 32 mg/ml (60% MeOH), 10 mg/ml(100% MeOH).Both H2O and MeOH fractions exerted a significant

inhibition of COX enzyme activities (Fig. 2). The H2Ofraction showed a 2.2 times stronger activity towardsCOX-2 compared to COX-1 (p< 0.001), while nosignificant difference was observed for the MeOHfraction. There was no significant inhibition using the30% and 60% MeOH fractions.HPLC-DAD and LC-MS/MS analysis of the H2O

fraction revealed the presence of quinic acid andphenolic acids (Table 2).HPLC-DAD and LC-MS/MS analysis of the

MeOH fraction showed the presence of tricin andoxygenated fatty acids as major compounds (Table 2).The oxygenated fatty acids (oxylipins) present werecharacterized as oxidation products of linoleic acid, bycomparison with literature data (Dufour and Loonis,2005; Oliw et al., 2006; Levandi et al., 2009; Pussa et al.,2009; Aghofack-Nguemezi et al., 2011) (Table 2).We investigated the influence of bamboo leaf extracts

on NF-kB-induced gene expression in TNF-stimulatedmouse fibrosarcoma cells. DEX was used as positivecontrol (Fig. 3). As shown in Fig. 4, Phyllostachys nigra(Lodd.) Munro, Fargesia robusta T.P. Yi ‘Pingwu’,Fargesia rufa T.P. Yi ‘Green Panda’ and Pseudosasajaponica (Steud.) Makino significantly inhibited NF-kB-driven luciferase reporter gene expression. The otherspecies showed no or no significant inhibition at 1mg/ml(data not shown).The IC50 values of P. nigra (Lodd.) Munro, F. robusta

‘Pingwu’, F. rufa ‘Green Panda’ and P. japonica leafextract are 0.89, 0.81, 0.81 and 0.61mg/ml, respectively.Results from a previous study (Van Hoyweghen et al.,

2012) showed that these extracts contain high amountsof flavone conjugates and phenolic acids.As traditionally used species with an inhibitory

activity on the transcriptional activity of NF-kB, thetotal extract of P. nigra (Lodd.) Munro (261.1mg) wassubjected to SPE to yield a 100% H2O (141.6mg),30% MeOH (17.8mg), 60% MeOH (36.0mg) in H2Oand a 100% MeOH (43.2mg) fraction, which wereevaluated for their inhibitory activity on NF-kB-drivengene expression.Both the 60%MeOH and 100%MeOH SPE fractions

of P. nigra (Lodd.) Munro extract showed a reduced

Figure 2. COX inhibitory activity of the SPE fractions of S. veitchii.* p ≤0.05; ***p ≤0.001. (Inhibition percentage and p-valuesare expressed in relation to solvent control). Values are the means(+ SD) of three measurements of one sample.


NF-kB-dependent luciferase reporter gene expression,with IC50 values of 1.34mg/ml and 1.06mg/ml, respec-tively (Fig. 5 A and B). Thus, the total extract was morepotent than either SPE fractions.

LC-MS/MS and HPLC-DAD analysis of the 60%MeOH fraction revealed the presence of luteolin andapigenin glycosides, together with phenolic acids asmain compounds. More specifically, the presence ofhomoorientin, orientin and isovitexin, previously identifiedin P. nigra (Lodd.) Munro (Van Hoyweghen et al., 2012),could be demonstrated in the 60% MeOH fraction.

Chlorogenic acid was identified as one of the cinnamicacid derivatives present. This was confirmed throughco-elution with an authentic standard.

We also demonstrated the presence of salicylic acid inthe 60% MeOH fraction, which was identified throughco-elution with an authentic standard (Table 2).

LC-MS/MS and HPLC-DAD analysis of the 100%MeOH fraction revealed the presence of homoorientin,luteolin, tricin and an oxylipin (Table 2). This wasconfirmed through spiking with standards.

The bioactive extracts P. japonica and F. robusta‘Pingwu’ were also subjected to SPE fractionation tolocate bioactive compounds. None of the SPE fractionsof P. japonica showed inhibition of NF-kB induced geneexpression (data not shown). Similarly, there was noinhibition observed for any of the SPE fractions ofF. robusta ‘Pingwu’.

DISCUSSION

The traditionally used species, S. veitchii (Carr.)Rehder and P. nigra (Lodd.) Munro showed both anti-inflammatory activity, but act via a different molecularmechanism. S. veitchii (Carr.) Rehder showed directinhibition of COX enzyme activities. P. nigra (Lodd.)Munro demonstrated anti-inflammatory activity viasuppression of NF-kB-induced gene expression. Otherspecies suppressing the activity of this transcription factorwere,F. rufa ‘Green Panda’, F. robusta ‘Pingwu’ and P. japonica.

Using SPE, bio-active compounds could be located.In the case of S. veitchii (Carr.) Rehder, both the100% H2O and the 100% MeOH fractions contributedto the bio-activity of the total extract. The 100% H2Ofraction showed a two times stronger inhibition ofCOX-2 than COX-1, while no selectivity was seen forthe 100% MeOH fraction. Therefore, the 100% H2Ofraction seems to be responsible for the observedactivity of the total extract. This fraction contains quinicacid and phenolic acids as main compounds.

HPLC analysis revealed the presence of tricin andoxylipins in the MeOH fraction of S. veitchii (Carr.)Rehder. Tricin has been reported to have COXinhibitory activities (Cai et al., 2005). The oxylipinsdetected in S. veitchii (Carr.) Rehder were tentativelyidentified as oxidized derivatives of linoleic acid. Similarcompounds have been found in wheat (Triticumaestivum) varieties (Poaceae) (Levandi et al., 2009).Linoleic acid has been shown to exhibit selectiveCOX-2 inhibition (Sato et al., 2007). In a study ofFujimoto et al. (2008), a hydroperoxy adduct of linoleicacid was able to inhibit both COX-1 and COX-2.Further studies will clarify if these compounds areresponsible for the observed activity.


Table2.

UVan

dMS/MSda

taof

theiden

tified

compo

unds

inthebioa

ctivefractio

nsof

S.veitchii(C

arr.)

Reh

deran

dP.

nigra(L

odd.)Mun

ro

ES-Q

TOF

t R(m

in)

UV

l max(nm)

[M+

H]-/[M

+H]+

Mainfrag

men

tsAssignm

ent

Rem

arks

S.ve

itch

ii_H2O

3.7

/341/

179,161,11

9,89

caffeo

ylhe

xose

Rivera-Pas

tran

aet

al.,2010[24]

4.8

/191/

173,129,11

1,87

quinic

acid

Bas

toset

al.,2007[25]

8.6

310

315/

153,109

dihy

drob

enzo

icac

idhe

xose

Enge

lset

al.,2012[26]

S.ve

itch

ii_MeO

H37.1

269,346

329/

314,299,271,227

tricin

Van

How

eghe

net

al.,2012[18]

38.1

/327/

229,211

,183,171

oxo-DHODEa

Leva

ndie

tal.,2009[21];Pus

saet

al.,2009[19]

39.2

/329/

329,229,211

,171

THODEb

Agh

ofac

k-Ngu

emez

ietal.,2011

[22];

Pus

saet

al.,2009[19]

42.5

/355/

311

,293,211

,171

oxylipin

Dufou

ran

dLo

onis,2005[23];Oliw

etal.,2006[20];

Pus

saet

al.,2009[19]

P.nigra_6

0%

MeO

H12.8

304(sh),326

353/

353,191,96

chloroge

nicac

idUV,MSan

dstan

dard

19.1

257(sh),269,349

/449

383,353,329,299

homoo

rien

tin

UV,MSan

dstan

dard

19.7

255,268,344

/449

431,413,395,383

orientin

UV,MSan

dstan

dard

22.4

271,329

/433

415,337,313,283

isov

itex

inUV,MSan

dstan

dard

31.0

238.303

137/

137,93

salic

ylic

acid

UV,MSan

dstan

dard

P.nigra_M

eOH

34.5

253,267,349

285/

285,175,151,133

luteolin

UV,MSan

dstan

dard

37.1

269,346

329/

314,299,271,227

tricin

NMR,UV,MSan

dliteratureda

ta38.1

/327/

229,211

,183,171

oxo-DHODE

Leva

ndie

tal.,2009[21];Püs

saet

al.,2009[19]

aOxo

-DHODE:

oxo-dihy

drox

y-oc

tade

ceno

icac

id.

bTHODE:

trihyd

roxy

-octad

ecen

oicac

id.


Copyright © 2013 John Wiley & Sons, Ltd. Phytother. Res. (2013)

Figure 4. Inhibitory activity of P. nigra (A) F. robusta ‘Pingwu’ (B), F. rufa ‘sion. * p ≤0.05; ** p ≤0.01; ***p ≤0.001 (p-values are expressed in rmeans (+ SD) of three measurements of one sample.

Figure 5. Inhibitory activity of the 60% MeOH (A) and 100%MeOH fracare expressed in relation to negative control). NC=negative control. Va

Figure 3. Inhibition of NF-kB-induced transcription by the syntheticglucocorticoid dexamethasone (1mM). NC=negative control(solvent EtOH); Dex=dexamethasone. ***p ≤0.001(p-valuesare expressed in relation to negative control). Values are the means(+ SD) of three measurements of one sample.



Bioactivity-guided fractionation of P. nigra (Lodd.)Munro showed that the 60% MeOH and the 100%MeOH fractions were able to inhibit NF-kB inducedgene expression. These fractions had a low relative part,13.8 and 16.5% (by mass), respectively, in the totalextract. Therefore, the total anti-inflammatory activityof 127 mg/ml 60% MeOH fraction and 155 mg/ml 100%MeOH fraction was found lower than the activity ofthe total extract at 0.91mg/ml. It seems that theobserved anti-inflammatory activity of P. nigra (Lodd.)Munro is the result of a combined effect of differentphytochemicals.

Several compounds identified in the 60%MeOH fraction, including isovitexin, chlorogenic acidand salicylic acid, have been previously shown byother authors to inhibit the transcriptional activity of

Green Panda’ (C) and P. japonica (D) on NF-kB-induced gene expres-elation to negative control). NC=negative control. Values are the

tion (B) on NF-kB-induced gene expression.***p<0.001 (p-valueslues are the means (+ SD) of three measurements of one sample.



NF-kB (Kopp and Ghosh, 1994; Lin et al., 2005; Shanet al., 2009). It is likely that these compoundscontribute to the observed activity. This has to beconfirmed by future studies.Luteolin was identified in theMeOH fraction. In a study

of Kim et al. (2003), it showed significant suppression of thetranscriptional activity of NF-kB at a concentration.The crude extracts of F. robusta ‘Pingwu’, F. rufa ‘Green

Panda’ and P. japonica showed also significant suppressionof NF-kB induced gene expression. In correspondencewith P. nigra, (Lodd.) Munro, a decrease in bioactivitywas seen upon fractionation of the extracts.


Acknowledgements

The authors cordially thank Ms. Debby Bracke for excellent technicalassistance with cell culture and reporter gene assays. LVH is gratefulto the IWT-Vlaanderen (Institute for the Promotion of Innovationby Science and Technology in Flanders, Brussels, Belgium) forproviding pre-doctoral grants. KDB is a postdoctoral fellow at theFWO-Vlaanderen.

Conflict of Interest

The authors have declared that there is no conflict of interest.

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