to get a first hint of the 
 activation of nrf2 by c. odorata , a crude methanol 
 extract of the leaves was tested in an nrf2-dependent are - driven luciferase 
 reporter gene assay . significant induction of luciferase was observed 
 at a concentration of 30 g / ml ( figure s1a , supporting information ) . the extract was further fractionated 
 by liquid  liquid extraction with solvents of increasing polarity . the diethyl ether fraction elicited potent concentration - dependent 
 activation of nrf2 ( figure s1b , supporting information ) , comparable to the extent of the positive control , 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide 
 ( cddo - im , 100 nm , for structure refer to figure 
 s1 ) , and was therefore chosen 
 for further bioassay - guided fractionation . out of the most active 
 fractions , these compounds were identified by means of mass spectrometry 
 and 1d- and 2d - nmr spectroscopy as well as by comparison of their 
 physical and spectroscopic data with those of reference values reported 
 in the literature , as cystosiphonin , scutellarein 
 tetramethyl ether , 6-methoxyhesperetin , hesperetin , naringenin , acacetin , 6-methoxyacacetin , salvigenin , ombuin , kaempferol 4-methyl ether , betuletol , kaempferol , aromadendrin 7-methyl ether , 4-hydroxybenzoic acid , and 
 five phytoprostanes . the latter included chromomoric acid c - i ( 1 ) , chromomoric acid c - iv ( 2 ) , ( 8z)-chromomoic 
 acid g ( 3 ) , ( 8e)-chromomoric acid g ( 4 ) , and ( 9s,13r)-12-oxophytodienoic 
 acid ( 5 ) . in contrast 
 to several reports on nrf2 activation by flavonoids , none of the flavonoids isolated from c. odoratum exerted nrf2 activation in the reporter gene assay used ( data not 
 shown ) . possibly , the concentrations tested , 10 to 30 m , were 
 too low to activate nrf2 under the assay conditions utilized . phytoprostanes 
 ( prostaglandin - like fatty acids ) , first identified from chromolaena 
 morrii(29 ) and some other chromolaena species , are bioactive 
 plant lipids and , from the chemical point of view , nonenzymatic radical 
 initiated peroxidation products of -linoleic acid . among the five phytoprostanes ( 15 ) isolated , compounds 4 and 5 have previously been found in the chloroform extract of c. odorata , while the others 
 ( 13 ) were identified in this plant 
 for the first time . of these , compound 1 ( chromomoric 
 acid c - i ) , which made up 0.014% w / w of the dried plant material ( supporting information , figure s2 ) , showed a 
 promising activation of nrf2 at 10 m . despite its structural 
 similarity with the other isolated phytoprostanes , only 1 was capable of markedly activating nrf2-driven gene expression ( figure 1a ) . testing 1 at different concentrations 
 revealed that already 5.2 m suffices to elicit a 2-fold activation 
 of nrf2-driven luciferase expression compared to vehicle control cells . compound 1 shows high structural similarity to the human 
 15-deoxy--prostaglandin j2 , a known nrf2 activator , which launches a concentration - dependent activation 
 of nrf2 highly comparable to 1 ( figure 1b ) . therefore , it is conceivable that 1 undergoes 
 an electrophilic attack of cysteine residues of keap1 and thereby 
 activates nrf2 signaling as shown for 15-deoxy--prostaglandin j2 ( pgj2 ) . thiol reactivity of 1 is underlined by a weaker nrf2 
 activation when the compound is added to an excess of extracellular 
 glutathione ( figure 1b ) . 15-deoxy--prostaglandin j2 also interferes with proinflammatory 
 nf-b signaling , at least in part via electrophilic attack and 
 inactivation of proteins within this pathway . by analogy , 1 inhibited the nf-b activity in 
 the respective luciferase reporter gene assay with an ic50 of 6.9 m , whereas the other isolated phytoprostanes were 
 obviously less active ( figure 1c ) . these data 
 suggest that 1 possesses optimized structural features , 
 i.e. , an exocyclic trans - configured double bond (  ) conjugated to the carbonyl group , favoring a putative 
 electrophilic attack of intracellular cysteine residues , a hypothesis 
 deserving further investigation and confirmation in the future . ( a ) cho - are luc cells were treated 
 with dmso ( 0.1% , negative control , neg ) , 100 nm cddo - im ( positive 
 control , pos ) , and 10 m of the isolated phytoprostanes ( 15 ) for 18 h , as indicated . luciferase 
 expression was assessed , normalized to the cell count , and expressed 
 as a fold induction of the negative dmso control . the bar graph depicts 
 compiled data of three independent experiments ( means + sd , * * p < 0.01 , anova ) . ( b ) cho - are luc cells were treated 
 with dmso ( 0.1% , negative control , neg ) , 100 nm cddo - im ( positive 
 control , pos ) , and different concentrations of 1 and 
 15-deoxy--prostaglandin j2 ( pgj2 ) , respectively . when indicated , the medium was supplemented with 
 2 mm glutathione ( gsh ) prior to addition of 1 then , 
 18 h later luciferase expression was assessed , normalized to the cell 
 count , and expressed as a fold induction of the negative dmso control . the bar graph depicts the compiled data of three independent experiments 
 ( means + sd , * p < 0.05 * * p < 
 0.01 , anova ) . ( c ) after staining with cell tracker green cmfda ( 2 
 m ) for 1 h , hek293/nf-b - luc cells were seeded in 96-well 
 plates ( 4  10 cells / well ) overnight and pretreated 
 for 30 min with the indicated concentration of the phytoprostanes 15 , solvent vehicle ( 0.1% dmso ) , and 
 5 m parthenolide [ as positive control for nf-b inhibition 
 ( pos ) ] . afterward , the cells were incubated for 4 h and lysed for 
 luminescence ( nf-b signal ) and fluorescence ( cell count ) determination . the data represent means + sd ( n = 3 ; * p < 0.05 , * * * p < 0.001 , vs vehicle control ; 
 anova / bonferroni ) . in the next step of this study , the newly identified nrf2 activator 1 was tested to see 
 if it could activate endogenous nrf2-dependent promoters ( in contrast 
 to the artificial simplified are - luciferase promoter in the reporter 
 gene assay ) . for this purpose , mouse embryonic fibroblasts were treated 
 with different concentrations of 1 , and the expression 
 of ho-1 was examined by immunoblot analysis . ho-1 is a cytoprotective 
 nrf2-dependent target gene and degrades heme to carbon monoxide , biliverdin , 
 and ferrous iron ( fe ) . a strong and concentration - dependent 
 induction of ho-1 was observed in wild - type mouse embryonic fibroblasts , 
 which was less evident in nrf2/ isogenic cells , clearly 
 demonstrating nrf2 dependency ( figure 2a ) . the induction of ho-1 in nrf2/ cells by 10 m 1 suggests that the compound at this concentration also activates 
 transcription factors other than nrf2 that are involved in the induction 
 of ho-1 , such as ppar or hypoxia - inducible factor 1. compound 1 induces ho-1 in a nrf2-dependent manner 
 and inhibits vsmc proliferation via ho-1 induction . wt and isogenic 
 nrf2/ mouse embryonic fibroblasts ( a ) as well as vsmc 
 ( b ) were treated with 1 at the indicated concentration 
 for 18 h before total cell lysates were subjected to immunoblot analysis 
 for ho-1 and actin as loading control . the numbers below the blots 
 indicate the compiled densitometric analysis of ho-1/actin [ referred 
 to the vehicle ( 0.1% dmso ) control , which is set at 1 ] of all performed 
 experiments . ( c ) quiescent vsmc were treated with 1 at 
 the indicated concentration for 30 min and then stimulated with 20 
 ng / ml pdgf for 48 h. proliferation was assessed based on resazurin 
 conversion as described in the experimental section . the bar graph depicts compiled data of three independent experiments 
 ( means + sd , * p < 0.05 ; * * * p < 
 0.001 , anova ) . ( d ) quiescent vsmc were treated with 1 at the indicated concentration for 48 h before their release of 
 ldh as readout for cell membrane disintegrity and cytotoxicity was 
 determined . ( e ) antiproliferative activity of 5 m 1 in the absence and presence of 10 m tin protoporphyrin ix 
 ( ho-1 inhibitor ) was assessed as in ( c ) . the bar graph depicts compiled 
 data of three independent experiments ( means + sd , * * * p < 0.001 , anova ) . vascular smooth muscle cells ( vsmc ) usually 
 contract and dilate blood vessels to facilitate circulation . under 
 atherosclerotic conditions , however , vsmc omit their contractile phenotype 
 and start to migrate and proliferate , leading to narrowing and occlusion 
 of the vessel . inhibition of vsmc proliferation is considered as a 
 valid approach in the prevention of atherosclerotic events . notably , 
 the nrf2ho-1 axis has been linked to an antiproliferative 
 influence on vsmc in many studies . this and the considerable 
 nrf2-dependent induction of ho-1 by 1 in mouse embryonic 
 fibroblasts prompted the testing of compound 1 also in 
 primary vsmc . as seen in figure 2b , 3 m 
 of this substance markedly induced ho-1 in vsmc . moreover , 1 was able to inhibit vsmc proliferation triggered by platelet - derived 
 growth factor ( pdgf ) , the most potent mitogen for vsmc in the vasculature , 
 without eliciting signs of cytotoxicity , as evident by the absent 
 release of lactate dehydrogenase ( ldh ) ( figure 2c , d ) . the ho-1 inhibitor tin protoporphyrin ix 
 abolished the antiproliferative effect of 1 , demonstrating 
 causality between ho-1 induction and inhibition of proliferation ( figure 2e ) . overall , phytoprostane 1 was 
 identified for the first time as an nrf2-activating principle of c. odorata leaves . nrf2 activation by 1 may 
 add to and/or synergize with the bioactivities of other constituents 
 of c. odorata and finally contribute its share to 
 the use of this plant in traditional medicine . pure compound 1 induced ho-1 , an endogenous nrf2 target gene , in mouse embryonic 
 fibroblasts and vsmc . notably , phytoprostanes are signals 
 of oxidative stress in plants and trigger an increased stress resistance 
 by induction of the plant detoxification machinery . this picture is highly reminiscent of the nrf2-mediated 
 detoxification response in mammalian cells upon exposure to 15-deoxy--prostaglandin j2 , a prostaglandin found under conditions 
 of inflammation and redox stress . the homology between the plant and 
 the mammalian stress response is underlined by the fact that phytoprostanes 
 can activate the stress - sensing mammalian transcription factor nrf2 . this suggests the existence of a common molecular antistress language 
 in plant and mammalian cells using a comparable vocabulary that may 
 be exploited to boost a deficient mammalian detoxification capacity 
 by appropriate plant metabolites , as exemplified with 1 in activated vsmc in this study . the optical rotations 
 were determined with a perkin - elmer 341 polarimeter ( wellesley , ma , 
 usa ) at 20 c . 1d- and 2d - nmr experiments were recorded on a 
 bruker drx 300 ( bruker biospin rheinstetten , germany ) or bruker advance 
 ii 600 nmr spectrometer ; nmr solvents : meoh - d4/cdcl3/dmso - d6/with 
 0.03% tms ( eurisotop gif - sur - yvette , france ) , which was used as internal 
 standard . lc analyses were carried out using an hp 1050 system ( agilent , 
 waldbronn , germany ) equipped with autosampler , dad , and column thermostat . separations were performed on a phenomenex aqua 125a ( 4.6 mm ( i.d . ) 
  250 mm , 5 m ) and a merck ( vwr , darmstadt , germany ) lichrocart 
 4 - 4 guard column with lichrospher 100 rp18 ( 5 m ) packing . a 
 mobile phase consisting of 0.5% fa + 1% 1-buoh + 1% thf in h2o ( v / v ) ( solvent a ) and meoh ( solvent b ) was employed with gradient 
 elution ( 0 min , 55:45 ( a : b ) ; 50 min , 20:80 ; 51 min , 2:98 ; 60 min , 
 2:98 ) . the detection wavelength was 280 nm , and the thermostat was 
 set at 45 c . the injection volume was 10 l ; the flow 
 rate was 0.5 ml / min . esims were obtained on an esquire 3000plus mass 
 spectrometer ( bruker daltonics , bremen , germany ) , using the following 
 parameters : alternating mode ; spray voltage , 4.5 kv , 350 c ; 
 dry gas , 10.0 l / min ; nebulizer 30 psi ; full scan mode , m / z 1001500 . fast centrifugal partition 
 chromatography ( fcpc ) was carried out on apparatus ( kromaton , france ) 
 equipped with a gilson 302/803c pump system model 302 ( villiers - la - bel , 
 france ) . column chromatography was performed with sephadex lh-20 ( pharmacia 
 biotech ab , stockholm , sweden ) and silica gel 60 ( 0.0400.063 
 mm ; merck , vwr , darmstadt , germany ) as stationary phases . tlc was 
 carried out on silica gel 60 f254 plates ( vwr , darmstadt , germany ) . 
 semipreparative hplc was performed with a dionex ultimate 3000-preaprative 
 hplc system with chromeleon software . a heto powerdry 6000 freeze - dryer 
 was used for water - containing fractions . primary rat vsmc were 
 purchased from lonza ( braine - lalleud , 
 belgium ) . chinese hamster ovary cells ( cho - k1 ) were obtained from 
 lgc ( wesel , germany ) , and the stable cho - are - luc clones thereof were 
 established in our laboratory as described previously . wild - type and isogenic nrf2/ 
 mouse embryonic fibroblasts were kindly provided by dr . the ho-1 inhibitor tin protoporphyrin ix 
 dichloride was from enzo life sciences ( lausen , switzerland ) , and 
 all other chemicals were obtained from sigma - aldrich ( vienna , austria ) . the anti - ho-1 antibody was from stressgene ( via enzo , lausen , switzerland ) , 
 the anti - actin antibody was from mpbio ( eschwege , germany ) , and the 
 secondary horseradish - peroxidase - coupled antibodies came from cell 
 signaling ( heidelberg , germany ) . all solvents used for isolation 
 were purchased from vwr international 
 ( darmstadt , germany ) . ultrapure water was produced by a sartorius arium 611 uv 
 water purification system ( gttingen , germany ) . the leaves of chromolaena odorata were collected 
 in chua chan mountains , dongnai , vietnam , in may 
 2011 and identified by prof . tran hung ( department of pharmacognosy , 
 faculty of pharmacy , university of medicine and pharmacy of hochiminh 
 city , vietnam ) . a voucher specimen ( dn108 ) is stored at the department 
 of pharmacognosy , faculty of pharmacy , university of medicine and 
 pharmacy of hochiminh city . extraction 
 was carried out 
 with 8 kg of the milled and air - dried leaves , which were percolated 
 with 96 l of meoh at room temperature . the solution obtained was evaporated 
 to dryness at 35 c , yielding 1356.1 g of crude extract . liquid 
 extraction ; 210 g of crude extract was suspended in 1.0 l of water 
 and extracted with n - hexane ( 500 ml  6 ) , diethyl 
 ether ( 500 ml  6 ) , and ethyl acetate ( 500 ml  4 ) followed 
 by n - butanol ( 500 ml  6 ) . each of the combined 
 organic layers as well as the aqueous layer was evaporated to dryness , 
 affording n - hexane ( 32.0 g ) , diethyl ether ( 53.2 
 g ) , ethyl acetate ( 4.5 g ) , n - butanol ( 20.4 g ) , and 
 water ( 89.1 g ) fractions . the obtained diethyl ether fraction , 
 which showed the most promising pharmacological effects , was subjected 
 to bioassay - guided isolation . an aliquot ( 45.0 g ) of this fraction 
 was subjected to silica gel cc ( petroleum ether etoac , 10:0 
 to 4:6 , v / v ) , to obtain 15 fractions ( a1 to a15 ) . fraction a11 ( 1.7 
 g ) , which showed the most potent activity in the nrf2 assay , was applied 
 to fcpc ( n - heptane  etoac  meoh h2o , 3:5:5:3 , lower phase : mobile phase ) to afford 16 fractions 
 ( a11 - 1 to a11 - 16 ) . the insoluble part of a11 in the fcpc solvent system 
 was separated by sephadex cc ( meoh ) to obtain acacetin ( 17.4 mg ) and 
 ombuin ( 13.6 mg ) . among the 16 subfractions of a11 , fractions a11 - 6 
 to a11 - 9 , a11 - 15 , and a11 - 16 exhibited nrf2 activation . fractions 
 a11 - 7 , a11 - 8 , and a11 - 9 were purified by sephadex cc ( meoh ) to afford 
 cystosiphonin ( 14.4 mg ) , scutellarein tetramethyl ester ( 20.5 mg ) , 
 6-methoxyacacetin ( 15.6 mg ) , and salvigenin ( 5.4 mg ) . fraction a11 - 15 
 was further chromatographed on a sephadex column ( meoh , followed by 
 ch2cl2acetone ; 85:15 , v / v ) to yield 
 6-methoxyhesperetin ( 10.3 mg ) , hesperetin ( 16.4 mg ) , naringenin ( 20.9 
 mg ) , kaempferol ( 7.0 mg ) , and aromadendrin 7-methyl ether ( 54.8 mg ) . fraction a11 - 16 was separated by sephadex cc ( meoh ) to yield 4-hydroxybenzoic 
 acid ( 5.6 mg ) . the subfractions of a11 containing fatty acid 
 derivatives showed 
 the most potent activities in the nrf2 assay ; however , when separated 
 from the flavonoid constituents , these fractions were found to be 
 unstable when stored at room temperature . therefore , further isolation 
 was conducted with fraction a10 , which contained the identical fatty 
 acid components when analyzed by hplc . the first part , a10-f ( 1.7 g ) , was worked up with the same 
 work flow as applied to fraction a11 , using fcpc ( n - heptane  etoac  meoh h2o , 5:3:3:3 , 
 lower phase : mobile phase ) to provide 11 fractions . fractions a10-f3 
 and a10-f4 were purified by sephadex cc ( meoh ) to yield fatty acid - containing 
 fractions : a10-f3 - 2 , a10-f4 - 2 , a10-f5 - 1 . fraction a10-f3 - 2 ( 136.5 
 mg ) was rechromatographed by silica gel cc ( ch2cl2etoac , 10:0 to 94:6 , v / v ) , followed by sephadex cc ( ch2cl2acetone , 85:15 , v / v ) to obtain compound 5 ( 15.5 mg ) . fraction a10-f3 - 2 - 4 was applied to semipreparative 
 hplc ( phenomenex aqua 5 m c18 125  ; 250  
 10.0 mm , isocratic mecn + 0.02% tfa , 68:32 ; 
 flow rate 2.0 ml / min ) to afford compounds 3 ( 3.2 mg ) 
 and 4 ( 6.1 mg ) . fraction a10-f5 - 1 ( 44.3 mg ) was purified 
 by silica gel cc ( ch2cl2etoac , 10:0 
 to 94:6 , v / v ) to yield compound 1 ( 8.8 mg ) . subfractions 
 a10-f5 - 9 and a10-f4 - 6 were combined ( 8.7 mg ) and separated by semipreparative 
 hplc ( x - terra prep c18 125  ; 100  7.8 mm , isocratic 
 meoh  water , 75:25 , v / v ; flow rate 1.0 ml / min ) , to afford beturetol 
 ( 4.6 mg ) , which was present only in a small amount in the active fraction 
 a11 - 9 . the second part of fraction a10 ( a10-s , 1.21 g ) was separated 
 by sephadex cc , to obtain a fraction containing fatty acid derivatives 
 ( a10-s2 , 90.2 mg ) and kaempferol 4-methyl ether ( 15.8 mg ) , 
 which was also present in active fraction a11 - 9 . fractions a10-f4 - 2 
 and a10-s2 were combined ( 328 mg ) and applied to silica gel cc ( ch2cl2etoac , 10:0 to 94:6 , v / v ) to produce 
 nine fractions and compound 1 ( 23 mg ) . fraction a10-s2 - 6 
 was purified by semipreparative hplc ( phenomenex aqua 5 m c18 125  ; 250  10.0 mm , isocratic meoh  h2o + 0.02% tfa , 80:20 , flow rate 2.0 ml / min ) to yield compound 2 ( 7.2 mg ) . cho - are - luc and mouse 
 embryonic fibroblasts 
 were maintained in dulbecco s modified essential medium ( dmem ) 
 supplemented with 10% fetal calf serum and 2 mm glutamine and , for 
 cho - are luc , additionally with 4 g / ml puromycin . hek293/nf-b - luc cells ( panomics , rc0014 ) 
 were maintained in dmem with 100 g / ml hygromycin b , 100 u / ml 
 benzylpenicillin , 100 g / ml streptomycin , 2 mm glutamine , and 
 10% serum . the 
 are- and nf-b - dependent 
 luciferase assays were performed as described previously . cddo - im ( 100 nm ) served as positive control in the are - dependent 
 luciferase assay and parthenolide ( 5 m ) in the nf-b 
 luciferase assay . equal concentrations of the solvent vehicle ( 0.1% 
 dmso ) were present in all treatment groups and served as negative 
 control in both assays . cell tracker green cmfda ( invitrogen ) staining 
 was used to monitor the cell viability . for quantification of nf-b activities hek293/nf-b - luc 
 cells were pretreated for 30 min as indicated and afterward stimulated 
 with 2 ng / ml tnf- for 4 h. the cells were lysed with a luciferase 
 lysis buffer ( promega ; e1531 ) , and afterward luminescence and fluorescence 
 were quantified on a genios pro plate reader ( tecan , grdig , 
 austria ) . cells ( mef or vsmc ) were seeded 
 onto six - well plates ( ( 34 )  10 cells / well ) . the next day , they were treated with dmso ( 0.1% ) or 1 at the indicated concentrations for 18 h. then , cells were lysed 
 and protein extracts were subjected to sds - page electrophoresis and 
 immunoblot analysis , as described previously . cell proliferation was assessed 
 based on metabolic conversion of resazurin to fluorescent resorufin 
 that correlates with the cell number , as described previously . briefly , serum - starved cells were pretreated 
 for 30 min with 0.1% dmso , 1 , or tin protoporphyrin ix 
 as indicated and subsequently stimulated for 48 h with pdgf ( 20 ng / ml ) . then , cells were washed with phosphate - buffered saline and incubated 
 in serum - free medium containing 10 g / ml resazurin for 45 min . samples were measured by monitoring the increase in fluorescence at 
 a wavelength of 590 nm using an excitation wavelength of 535 nm in 
 a 96-well plate reader ( tecan genios pro ) . as a readout for potential 
 cytotoxicity , membrane integrity was assessed and the amount of ldh 
 released from vsmc determined after a 48 h treatment with 1 . for this , promega s cytotox nonradioactive cytotoxicity 
 assay was used according to the manufacturer s instructions . data are presented as means and standard 
 deviation ( sd ) from three independent experiments unless stated otherwise . statistical significance was determined by anova and dunnett s 
 or bonferroni s post - test using graphpad prism software . results 
 with p <
activation of nuclear factor - erythroid 
 2-related factor 2 ( nrf2 ) 
 contributes to several beneficial bioactivities of natural products , including induction of an increased 
 cellular stress resistance and prevention or resolution of inflammation . 
 in this study , the potential of a crude leaf extract of chromolaena 
 odorata , traditionally used against inflammation and skin 
 lesions , 
 was examined for nrf2 activation . 
 guided by an nrf2-dependent 
 luciferase reporter gene assay , the phytoprostane chromomoric acid 
 c - i ( 1 ) was identified as a potent nrf2 activator from c. odorata with a cd ( concentration doubling the response 
 of vehicle - treated cells ) of 5.2 m . when tested at 110 
 m , 
 1 was able to induce the endogenous nrf2 target 
 gene heme oxygenase 1 ( ho-1 ) in fibroblasts 
 . between 2 and 5 m , 
 compound 1 induced ho-1 in vascular smooth muscle cells 
 ( vsmc ) and inhibited their proliferation in a ho-1-dependent manner , 
 without eliciting signs of cytotoxicity .