Research Article A Bio-Guided Fractionation to Assess the ...downloads.hindawi.com › journals ›...

Research ArticleA Bio-Guided Fractionation to Assess the Inhibitory Activity ofCalendula officinalis L on the NF-120581B Driven Transcription inHuman Gastric Epithelial Cells

Elisa Colombo1 Enrico Sangiovanni1 Michele DrsquoAmbrosio2

Enrica Bosisio1 Alexandru Ciocarlan2 Marco Fumagalli1 Antonio Guerriero2

Petru Harghel2 and Mario DellrsquoAgli1

1Department of Pharmacological and Biomolecular Sciences Universita degli Studi di Milano Via Balzaretti 9 20133 Milan Italy2Laboratory of Bioorganic Chemistry Department of Physics Universita degli Studi di Trento Via Sommarive 14 38123 Trento Italy

Correspondence should be addressed to Mario DellrsquoAgli mariodellagliunimiit

Received 21 May 2015 Accepted 8 September 2015

Academic Editor Kuttulebbai N S Sirajudeen

Copyright copy 2015 Elisa Colombo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Calendula officinalis L has been largely known for its topical anti-inflammatory properties however there are no experimentalevidences about its antiphlogistic effect at the gastric level To investigate whether marigold might exert an activity against gastricinflammation a CH

2Cl2extract obtained from C officinalis flowers was evaluated in vitro on the NF-120581B pathway The lipophilic

extract demonstrated a significant inhibitory effect on the NF-120581B driven transcriptionThe identification of active compounds wasconducted by a bio-guided fractionation of the extract that afforded 16 fractions Fraction J exhibited a concentration-dependentinhibitory activity on the NF-120581B driven transcription and significantly contributed to the antiphlogistic effect showed by CH

2Cl2

extract The main components of fraction J were loliolide and the fucoside acetates of 120573-eudesmol and viridiflorol HPLC analysisof fractions D and E led to the identification and isolation of triterpene esters that showed a concentration-dependent inhibitionof the NF-120581B driven transcription with faradiol-3-myristate and the corresponding aglycone being the most active compoundsThe present study provides some experimental evidences that Calendula officinalis L may exert an anti-inflammatory activity onthe gastric district by the inhibition of the NF-120581B system identifying the compounds responsible at least in part for the observedeffect

1 Introduction

Calendula officinalis L (marigold Asteraceae) is an annualherb of Mediterranean origin widely cultivated as an orna-mental plant Dried flowersrsquo heads are characterized by thepresence of steroids terpenoids free and esterified triter-penic alcohols (faradiol arnidiol and calenduladiol mainlyas myristate) phenolic acids flavonoids (quercetin rutinnarcissin isorhamnetin kaempferol) carotenoids and otherminor compounds [1] Marigold apolar extracts have beenlargely applied for external use in the treatment of skin ulcer-ations eczema and conjunctivitis [2] Several studies both invitro and in vivo demonstrated (1) the biological properties ofpreparations frommarigold flowers as antioedematous agent[3ndash5] (2) the activity in wound healing [6ndash8] and (3) their

antioxidant properties at both topical [9ndash12] and systemiclevel [13ndash15] Among secondary metabolites the flavonoidsthe carotenoids and the triterpene fatty acid esters seem tobe the major responsible for the anti-inflammatory activityof Calendula officinalis [3ndash5 16 17]

Gastritis is one of the most common inflammatory dis-eases affecting about 50 of the worldrsquos population Gastricmucosa is continuously exposed to many noxious factors andsubstances that can alter its integrity and lead to inflamma-tionAmong those factors the infection byHelicobacter pylori(H pylori) is the most relevant one H pylori is a Gram-negative pathogen that colonizes the stomach of humans andprimates and is classified as a carcinogen type I Usuallyacquired during childhood the infection can persist in thegastric district causing chronic gastritis or evolving intomore

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 727342 8 pageshttpdxdoiorg1011552015727342

2 Evidence-Based Complementary and Alternative Medicine

severe diseases such as atrophic gastritis peptic ulcer orgastric adenocarcinoma [18] As a consequence of untreatedinfections the gastric epithelial cells secrete many cytokinesand chemokines that are attracted to the mucosal layerneutrophils lymphocytes and macrophages responsible forthe maintenance of the inflammatory status Several studiesindicate that nuclear factor 120581B (NF-120581B) plays a crucial role atthe molecular level of this process [19ndash21]

Aqueous preparations from marigold flowers have beentraditionally used for the treatment of gastrointestinal dis-eases such as gastritis ulcers and colitis Two studies demon-strated the efficacy of herbal mixtures containing marigoldin the therapy of duodenal ulcers gastroduodenitis andchronic hyposecretory gastritis [22 23] and amore recent onedescribed the hypoglycemic and gastroprotective activities ofCalendula officinalis L in vivo [24] Moreover another workshowed the effect of marigold preparations in amelioratinginflammatory symptoms in an animal model of ulcerativecolitis [25] So far no one has established the clinical effective-ness of Calendula officinalis L extracts as anti-inflammatoryagent by internal use [26] Thus the aim of this work was(i) to evaluate in vitro the anti-inflammatory activity of aCH2Cl2extract from marigold flowers in a cellular model

of gastritis and (ii) to identify mixtures and compoundsresponsible for this effect by a bio-guided fractionation ofthe CH

2Cl2extract considering the NF-120581B system as the

molecular target

2 Materials and Methods

21 Reagents Dulbeccorsquos modified Eaglersquos medium(DMEM)F12 (1 1) penicillin streptomycin L-glutamineand trypsin-EDTAwere fromGibco (Life Technologies ItaliaMonza Italy) Foetal bovine serum (FBS) and disposablematerial for cell culture were purchased by Euroclone(Euroclone SpA Pero Milan Italy) Parthenolide dim-ethyl sulfoxide (DMSO) and all solvents (from Sigma-Aldrich Milan Italy) were of high-performance liq-uid chromatography purity grade (gt98) Tumor necrosisfactor alpha (TNF-120572) was from ImmunoTools (FriesoytheGermany) Human adenocarcinoma cells (AGS) werepurchased from European Collection of Cell Culture (EACCUK cat number 89090402) NF-120581B-luc plasmid was akind gift from Professor N Marx (Department of InternalMedicine II-Cardiology Ulm Germany) Britelite plus wasfrom PerkinElmer (Monza Italy)

Flash chromatography (FC) was performed by MerckKieselgel 60 (70 230mesh) Merck RP 18 LiChroprep (40ndash65 120583m) TLC by Merck Kieselgel 60 PF254 high pressure liq-uid chromatography (HPLC) by Merck Hitachi L7100 pumpL7400 UV detector D7500 integrator Rheodyne injectorHPLC column and method by Synergi Hydro column (150times 10mm 4 120583m particle size 80 A pore size PhenomenexTorrance CA USA) 50mLmin 120582 = 210 nm The eluentswere acetonitrile (ACN) (A) and ACNwater 50 50 vv (B)in method 1 the gradient was changed linearly from 0 to85 A in 45min in method 2 the eluent A was appliedin the gradient of 0 at 119905 = 0 37 at 119905 = 30 85 at119905 = 31 85 at 119905 = 45 and 100 at 119905 = 46min Optical

rotations were measured on a Bellingham + Stanley ADP 440polarimeter NMRwas performed by Bruker Avance 400 (1Hat 400MHz 13C at 100MHz) 5mm BBI probe 120575 in ppmusing residual solvent signals as internal reference (CDCl

3

= 770 CHCl3= 726 and CD

2HOD = 331) 119869 values in Hz

multiplicities and peak assignments from 1H 1HCOSY 1119869CH(HSQC) 119899119869CH (HMBC) and NOESY experiments NOESYdata are reported as correlation map(s) between protons1H harr 1H HMBC data are reported as (13C) rarr correlatedto 1HMS was performed by Bruker Esquire LCmultiple iontrap Electrospray ionization (ESI) was as follows positive ionmode capillary voltage 4000V nebulizing pressure 300 psidrying gas flow 7mLmin and temperature 300∘C Electronimpact mass spectra (EIMS HREIMS) were recorded on aKratos MS80 spectrometer with home-built data system andelectron ionization at 70 eVmz (rel)

22 Plant Material Dry flowers of Calendula officinalisL (variety Calypso Orange Florensis) were obtained andextracted as previously described [27 28] A voucher spec-imen (number 20040929) is deposited at the Laboratory ofBioorganic Chemistry (Trento Italy)

23 Cell Culture AGS cells (EACC number 89090402) weregrown in DMEMF-12 medium supplemented with 100 unitspenicillinmL 100mg streptomycinmL 2mM L-glutamineand 10 heat-inactivated foetal bovine serumThe cells wereincubated at 37∘C in humidified atmosphere with 5 CO

2

until confluence For the NF-120581B driven transcription assayAGS cells were plated at the concentration of 15 times 104cellsmL in 24-well plates with complete medium to reachabout 80 of confluence After 48 h medium was replacedwith FBS-free medium and cells were exposed for 24 h to thecompounds or fractions under study in the presence of TNF-120572 (10 ngmL) as proinflammatory stimulus To study the NF-120581B nuclear translocation cells were plated in 10mm dishes (3times 106 cellsdish) with fresh complete medium for 48 h Thenmedium was replaced with FBS-free medium containingincreasing concentrations of fractions in the presence ofTNF-120572 (10 ngmL) for 1 h We previously found that thefractions H and I are highly cytotoxic to AGS cells [28] sothese fractions were excluded from the biological assays

24 Transient Transfection and Luciferase Reporter Assay Toassess the effect of different fractions and isolated compoundson the NF-120581B driven transcription AGS cells were tran-siently transfected by the calcium-phosphate method witha plasmid containing the luciferase reporter gene under thecontrol of the E-selectin promoter with three NF-120581B bindingsites Sixteen hours after transfection cells were stimulatedwith TNF-120572 (10 ngmL) in the presence of CH

2Cl2extract

(1ndash20120583gmL) or fractions AndashP and subfractions (005ndash20120583gmL) or individual compounds (1ndash50120583M) After 24 hcells were harvested and luciferase activity was measuredusing Britelite plus reagent on a luminometer Parthenolide(final concentration 10 120583M) is a natural inhibitor of the NF-120581B driven transcription [29] it was used as positive controland showed around 50 inhibition in all the experiments

Evidence-Based Complementary and Alternative Medicine 3

reported in the present studyTheNF-120581Bdriven transcriptionactivation was expressed as a percentage relative to TNF-120572treatment alone (100)

25 NF-120581B Nuclear Translocation To clarify the effect ofmarigold fractions on the NF-120581B pathway we focused on thenuclear translocation as well AGS cells were stimulated withTNF-120572 (10 ngmL) in the presence or in the absence of theCH2Cl2extract (25ndash10 120583gmL) or fraction J (25ndash75120583gmL)

for 1 h Nuclear and cytoplasmatic extracts were obtainedwith Nuclear Extraction Kit (Cayman Chemical CompanyAnn Arbor MI USA) following manufacturer instructionsBriefly cells were first pelleted and resuspended in ice-cold hypotonic buffer Addition of detergent (NP-40 10)broke the cell membranes allowing access to the cytoplas-matic fraction while maintaining the integrity of the nuclearmembrane After separation of the cytoplasmatic fractionthe pelleted nuclei were lysed in ice-cold extraction bufferobtaining the nuclear fraction Total protein concentration ofthe nuclear extracts was determined by the Bradford method[30] 10 120583g of total nuclear extracts for each sample was usedfor the measurement of nuclear NF-120581B (p65 subunit) usinga commercial ELISA kit (Cayman Chemical Company AnnArbor MI USA)The NF-120581B nuclear quantity was expressedas a percentage relative to TNF-120572 treatment alone (100)

26 Statistical Analysis Results represented the mean plusmnsd of at least three independent experiments replicated induplicates or triplicates Statistical analysis was performedwith GraphPad Prism 60 software using one-way ANOVAanalysis of variance followed by Bonferronirsquos post-hoc testThe significance was set at 119875 lt 005

3 Results and Discussion

31 Effect of Marigold CH2Cl2Extract on the NF-120581B Pathway

Calendula officinalis L is a botanical worldwide known forits topical anti-inflammatory properties that are supportedby clinical studies Oral use of marigold infusions for thetreatment of gastrointestinal diseases such as gastritis is onlyacknowledged in traditional medicine and is not currentlysupported by clinical trials [26] Among the componentsof marigold flowers triterpene fatty acid esters present inlipophilic extracts are the main responsible for the anti-inflammatory effects [3 4 16 17] However internal useof lipophilic preparations from marigold flowers is notrecommended for their renowned toxicity In fact our grouphas recently succeeded in identifying fractions and purecompounds which occur in the lipophilic extract fromCalendula officinalis flowers and are toxic at the gastric level[28] In the sameworkwe demonstrated that the compositionof lipophilic extract was not altered by gastric digestionAccordingly two studies were performed both in vivo [5] andex vivo [31] and revealed that marigold extracts could exertthe anti-inflammatory effect even after oral administrationIn fact the consumption of extracts frommarigold flowers ledto a decrease in plasmatic proinflammatory cytokines (TNF-120572 and IL-1120573) indicating a stability of the anti-inflammatorycomponents through stomach passage [5 31]

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

+ + + + + +minus

minus minus 1 25 5 10 20

TNF-120572CH2Cl2 extract

(120583gmL)

0

20NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

40

60

80

100

120

Figure 1 Effect of CH2Cl2extract on the NF-120581B driven tran-

scription AGS cells were transiently transfected with NF-120581B-lucplasmid and treatedwith TNF-120572 in the presence of dichloromethaneextract (1ndash20 120583gmL) for 24 h The NF-120581B driven transcription wasproportional to luciferase signal measured lowast119875 lt 005 lowastlowast119875 lt 001and lowastlowastlowast119875 lt 0001

In order to investigate if apolar constituents of marigoldcould exert anti-inflammatory effects at the gastric levels viainhibition of the NF-120581B pathway the CH

2Cl2extract was

assayed in a widely used human gastric epithelial cell lineAGS cells were transfectedwith a reporter plasmid containingthe luciferase gene under the control of three NF-120581B respon-sive elements Cells were then incubated with the extract inthe presence of TNF-120572 as proinflammatory stimulus for 24 hLuciferase activity was directly proportional to activationof the NF-120581B driven transcription TNF-120572 alone increasedluciferase activity by about fivefold when compared withthe control cells Dichloromethane extract obtained fromCalendula officinalis L flower heads inhibited the NF-120581Bdriven transcription in a concentration-dependent manner(IC50of 631plusmn068 120583gmLmeanplusmn sd) exhibiting a complete

inhibition of TNF-120572-induced NF-120581B activation at 20120583gmL(Figure 1)

When NF-120581B is activated by proinflammatory stimuliit translocates into the nucleus where it promotes the tran-scription of responsive genes encoding for many mediatorsof the gastric phlogistic process To clarify if CH

2Cl2extract

could inhibit NF-120581B nuclear translocation as well AGS cellswere treated with TNF-120572 (10 ngmL) as proinflammatorystimulus in the presence of the extract (5ndash10 120583gmL) for 1 hCells were then lysed and nuclear fraction was separated thenuclear NF-120581B was quantified through a commercial ELISAkit Surprisingly CH

2Cl2extract did not exert a significant

inhibitory effect on the NF-120581B nuclear translocation thussuggesting that differentmechanisms of the NF-120581B activationcascade might be involved

32 Bio-Guided Fractionation of CH2Cl2Extract and Char-

acterization of the Fractions The identification of biologi-cally active compounds occurring in the lipophilic extractwas achieved through a bio-guided fractionation Briefly aportion of the CH

2Cl2extract was subjected to FC on silica


Table 1 Bio-guided fractionation of CH2Cl2extract and IC

50

of fractions on the NF-120581B driven transcription AGS cells weretransiently transfected with NF-120581B-luc plasmid and treated withTNF-120572 in the presence of fractions (25ndash20 120583gmL) for 24 h NF-120581Bdriven transcription was proportional to luciferase signal measuredThe inhibitory activity of fractions was considered significant ifle50 120583gmL

Fraction Hexane AcOEt IC50(mean plusmn sd)

A 95 5 ge50120583gmLB 90 10 ge50120583gmLC 80 20 ge50120583gmLD 70 30 2301 plusmn 851 120583gmLE 60 40 ge50120583gmLF 50 50 ge50120583gmLG 40 60 ge50120583gmLH 30 70 TOXICI 20 80 TOXICJ 10 90 429 plusmn 097 120583gmLK AcOEt 1959 plusmn 577 120583gmLL AcOEt 1552 plusmn 521 120583gmLM Acetone 1284 plusmn 443 120583gmLN Acetone 1159 plusmn 326 120583gmLO ActiPrOH 9 1 782 plusmn 213120583gmLP ActiPrOH 9 1 ge50120583gmL

increasing gradually the eluent polarity from hexaneEtOAc95 5 to absolute EtOAc and then washed with acetoneThis procedure afforded 16 fractions (AndashP) the triterpeneesters were contained in fractions D-E (Table 1) All thefractions were assayed on AGS cells for their cytotoxicity aspreviously described [28] Nontoxic fractions were tested fortheir biological activity on the NF-120581B driven transcription inAGS cells stimulated with TNF-120572 for 24 h

As shown in Table 1 14 fractions were assayed but only 7inhibited the NF-120581B driven transcription in a concentration-dependent manner FractionD showed only amild biologicalactivity while the most active fractions were J and Owith an IC

50of 429 plusmn 097 120583gmL and 782 plusmn 213 120583gmL

respectively Fraction J exhibited an inhibitory activity on theNF-120581B driven transcription comparable to that showed bythe CH

2Cl2extract Consequently it was also assayed on the

NF-120581Bnuclear translocation using the samemodel describedabove Fraction J (25ndash75 120583gmL) did not inhibit the NF-120581Bnuclear translocation thus confirming that both the extractand fraction J could directly prevent the activation of NF-120581Bdriven transcription Namely natural products present in theCH2Cl2extract and in fraction J could exert their inhibitory

effect on the binding of NF-120581B to DNA This mechanismof action has already been demonstrated for other naturalcompounds such as the sesquiterpene lactone parthenolide[32] and artemisinin [33 34]

321 Biological Activity of Triterpene Esters Present in Frac-tions D-E All the biologically active fractions were analysedin RP-HPLC to identify the components responsible forthe inhibitory effect on the NF-120581B driven transcription

The analysis was firstly devoted to the identification andisolation of triterpene esters present in marigold flowers thathave been previously described for their anti-inflammatoryproperties [3 4 16 17] Thus myristic esters of faradiolarnidiol and calenduladiol were identified and isolated infractions D and E by HPLC analysis as previously described[27] and then assayed for their biological activity

For the first time our results demonstrated that alltriterpenoid esters exhibited a significant and concentration-dependent inhibitory activity of the NF-120581B driven transcrip-tion (Figure 2) Triterpene alcohols are not present in CH

2Cl2

extract however they could be released after ingestion ofmarigold preparations and are thought to have a greater bio-logical activity thus we synthesized [27] and assayed themAs expected triterpene alcohols demonstrated a strongerinhibitory effect on the NF-120581B driven transcription thanthe corresponding esters (Figure 2) Faradiol and faradiol-3-myristate were themost potent compounds (IC

5030plusmn73 120583M

and 10plusmn26 120583M resp)However the relative concentrations ofthese compounds in the extract (Figure 2) revealed that othercomponents contributed to the antiphlogistic effect shown bythe extract

322 Identification and Biological Activity of CompoundsPresent in Fraction J Fraction J showed the strongestinhibition of the NF-120581B driven transcription (IC

50429 plusmn

097 120583gmL) and its action was comparable to that observedfor the lipophilic extract Thus J was separated by HPLCon RP-18 column (method 1) into two subfractions the firstcontained mainly loliolide (25 ww) the second (8 ww)consisted of a mixture of few compounds Loliolide is a bittercomponent previously described in Calendula officinalis Lflowers [35] and in other plants including Fumaria officinalisand in different marine algae [36] It was considered aphytotoxic compound and a biomarker of photooxidativealterations [36] Recently it did not exhibit any antiphlogisticeffect in a macrophage cell line stimulated with LPS [37]According to these findings loliolide revealed no activityon the NF-120581B cascade in our cellular model exhibiting a10 inhibition on the NF-120581B driven transcription at theconcentration of 5120583M (corresponding to the IC

50of fraction

J) The second subfraction inhibited the NF-120581B driven tran-scription in a concentration-dependent manner (IC

50311 plusmn

230 120583gmL) largely contributing to the effect demonstratedby fraction J Subsequently an improved analytical method(method 2) allowed the separation of fraction J into 10 HPLCpeaks which corresponded to pure compounds Those ofinterest contained the following compounds 1 (peak 1 119905

119877=

21min mg 89) 2 (peak 4 119905119877= 172min mg 34) 3

(peak 6 119905119877= 196min mg 27) The compounds 2 and 3

were peracetylated and the products were analyzed by EI-MSwithout any preliminary purification [28] The examinationof 1D- and 2D-NMR spectra as well as the MS data ofthose compounds allowed their structural characterizationloliolide 1 120573-eudesmol 11-O-120573-D-(21015840-acetyl)-fucopyranoside2 and viridiflorol 10-O-120573-D-(21015840-acetyl)-fucopyranoside 3(Figure 3) The spectroscopic data of loliolide were identicalwith those from the literature [38] This is the first report onthe isolation and structural elucidation of the natural product


ArnidiolArnidiol-3-myristate

0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1

log (120583M)

(a)

0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1

log (120583M)

CalenduladiolCalenduladiol-3-myristate

(b)

0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1

log (120583M)FaradiolFaradiol-3-myristate

Compound

Faradiol-3-myristate 169

Arnidiol-3-myristate 67

Calenduladiol-3-myristate 12

Faradiol

Arnidiol

Calenduladiol

CH2Cl2 extract

IC50 (mean plusmn sd) CH2Cl2 extract ()

mdash

mdash

mdash

mdash

631 plusmn 068120583gmL

30 plusmn 73 120583M

30 plusmn 46 120583M

35 plusmn 58 120583M

10 plusmn 26 120583M

21 plusmn 52 120583M

19 plusmn 38 120583M

(c)

Figure 2 Effect of triterpene esters and the corresponding alcohols ((a) arnidiol-3-myristate arnidiol (b) calenduladiol-3-myristatecalenduladiol (c) faradiol-3-myristate faradiol) on the NF-120581B driven transcription AGS cells were transiently transfected with NF-120581B-lucplasmid and treated with TNF-120572 in the presence of triterpene esters or alcohols (1ndash50120583M) for 24 h The table reports the IC

50values and

relative percentages (ww) of triterpene esters in lipophilic extract

2 spectral data of compound 3 has been previously reported[39] but the authors assigned the opposite configuration atC-10

Compound 2 C23H38O6 white amorphous powder

[120572]25

D +100 (c 04 CHCl3) ESI-MS (positive mode) mz

433 [M+Na]+ MS2 of [M+Na]+ mz 229 [M+Na-C15H24]+

MS3 of [M+Na]+ rarr [M+Na-C15H24]+ mz 211 [M+Na-

C15H24-H2O]+ EIMS mz 273 [M-C

15H25O]+ (28) 204

[M-C12H18O8]+∙ (20) 43 (100) HREIMS mz 2730973

(calcd for C12H17O7 2730974) 2041878 (calcd for C

15H24

2041878) 1H NMR (CDCl3 400MHz) 120575 sim149 (1H m H-

1120573) 116 (1H dd H-1120572) sim159 (2H m H-2) 229 (1H brdH-3120573) 198 (1H brq H-3120572) 172 (1H brd 119869 = 120Hz H-5)166 (1H brd 119869 = 127Hz H-6120572) 109 (1H q 119869 = 120HzH-6120573) 144 (1H tt H-7) sim157 (1H m H-8120572) sim123 (1H mH-8120573) 142 (1H brd H-9120573) sim125 (1H m H-9120572) 117 (3Hs H-12) 121 (3H s H-13) 068 (3H s H-14) 470 (1H brsH-15a) 442 (1H brs H-15b) 454 (1H d 119869 = 79Hz H-11015840)482 (1H dd 119869 = 97 79Hz H-21015840) 362 (1H brd H-31015840) 369(1H brd 119869 = 36Hz H-41015840) 361 (1H brq H-51015840) 132 (3H


O

O

OAc

OH

OH O

O

OAc

OH

OH

H

H

H

2 3114

11

2

813

12

15

106

12

3 48

6

4

14OO

HO

1

H120572

H120573 H120573

H120573

H120573

HaHb

2998400

5998400

6998400

Figure 3 Chemical structure of compounds identified in fraction J

d 119869 = 66Hz H-61015840) 210 (3H s H-210158401015840) 13C NMR (CDCl3

100MHz) 120575 414 (t C-1) 235 (t C-2) 369 (t C-3) 1508 (sC-4) 498 (d C-5) 246 (t C-6) 484 (d C-7) 222 (t C-8)419 (t C-9) 360 (s C-10) 801 (s C-11) 228 (q C-12) 250(q C-13) 164 (q C-14) 1053 (t C-15) 950 (d C-11015840) 735 (dC-21015840) 739 (d C-31015840) 721 (d C-41015840) 699 (d C-51015840) 165 (q C-61015840) 1712 (s C-110158401015840) 210 (q C-210158401015840) NOESY 14 harr 2 and 6120573 and8120573 3120573 harr 15a 15b harr 6120572 and 6120573 11015840 harr 12 and 13 HMBC (1)rarr 14 (10) rarr 14 (5) rarr 14 (7) rarr 12 and 13 (11) rarr 11015840 and12 and 13 (110158401015840) rarr 210158401015840


[120572]25

D ndash68 (c 015 CHCl3) ESI-MS (positive mode) mz


EIMSmz 494 [M]+∙ (03) 273 [M-C15H25O]+ (16) 204 [M-

C12H18O8]+∙ (71) 43 (100) HREIMS mz 4942870 (calcd

for C27H42O8 4942880) 2730975 (calcd for C

12H17O7

2730974) 2041871 (calcd for C15H24 2041878) 1H NMR

(CDCl3 400MHz) 120575 sim188 (1Hm H-1) sim160 (2Hm H-2)

sim181 (1Hm H-3120573) sim128 (1Hm H-3b) sim192 (1Hm H-4)170 (1H m H-5) 007 (1H t 119869 = 95Hz H-6) 058 (1H td119869 = 95 95 79Hz H-7) sim153 (2H m H-8) sim176 (1H mH-9120573) sim157 (1H m H-9b) 095 (3H s H-12) 100 (3H sH-13) 117 (3H s H-14) 092 (3H d 119869 = 67Hz H-15) 458(1H d 119869 = 79Hz H-11015840) 485 (1H dd 119869 = 79 95Hz H-21015840)359 (1H m H-31015840) 367 (1H brd 119869 = 4Hz H-41015840) 358 (1Hqd 119869 = 65 10Hz H-51015840) 130 (3H d 119869 = 65Hz H-61015840) 209(3H s H-210158401015840) 1HNMR (CD

3OD 400MHz) 120575 sim194 (1Hm

H-1) 171ndash155 (2H m H-2) sim183 (1H m H-3a) sim128 (1Hm H-3b) sim196 (1H m H-4) sim173 (1H m H-5) 123 (1Ht H-6) 060 (1H ddd H-7) 159 (H m H-8a) sim147 (H mH-8b) sim174 (1Hm H-9a) sim162 (1Hm H-9b) 097 (3H sH-12) 101 (3H s H-13) 118 (3H s H-14) 094 (3H d H-15)463 (1H d H-11015840) 496 (1H dd H-21015840) 364ndash360 (3H m H-31015840 and H-41015840 and H-51015840) 123 (3H d H-61015840) 205 (1Hm H-210158401015840)13C NMR (CDCl

3 100MHz) 120575 542 (d C-1) 254 (t C-2)

289 (t C-3) 384 (d C-4) 397 (d C-5) 221 (d C-6) 287 (dC-7) 182 (t C-8) 379 (t C-9) 820 (s C-10) 187 (s C-11)161 (q C-12) 286 (q C-13) 266 (q C-14) 162 (q C-15) 945(d C-11015840) 737 (d C-21015840) 739 (d C-31015840) 722 (d C-41015840) 698 (dC-51015840) 165 (q C-61015840) 1711 (s C-110158401015840) 210 (q C-210158401015840) NOESY 6harr 3b and 7 and 13 and 15 5 harr 12 11015840 harr 1 and 14 210158401015840harr 12HMBC (1) rarr 14 (9) rarr 14 (10) rarr 14 and 11015840 (6) rarr 12 and13 (7) rarr 12 and 13 (11) rarr 12 and 13 (110158401015840) rarr 21015840 and 210158401015840

We assayed the 10 peaks for their biological activity onAGS cells All compounds exhibited amild inhibitory activityon the NF-120581B driven transcription (Figure 4) thus pointingout that together they participate in the biological effect of

lowastlowast

lowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

+

TNF-1205720

20

40

60

80

100

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

120

140

2a 2b 2c 2d 3 4 5 6 71Fractions of J(5120583gmL)

+ + + + + + + + + + +minus

minus minus

Figure 4 Effect of fractions J1ndashJ7 on the NF-120581B driven transcrip-tion AGS cells were transiently transfected with NF-120581B-luc plasmidand treated with TNF-120572 in the presence of fractions (5120583gmL) for24 hThe NF-120581B driven transcription was proportional to luciferasesignal measured lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

fraction J We identified peak 1 as loliolide and peaks 4 and6 as the fucoside acetates of 120573-eudesmol and viridiflorolrespectively Similar glycosides were previously described ascomponents of Calendula persica C Mey [40] and manyadditional analogues have been recently characterized inCalendula officinalis L flowers [28] It has been shown that120573-eudesmol exerts an anti-inflammatory activity in vitro [4142] through inhibition of the NF-120581B driven transcriptionand activation at concentrations (120583molar order) similar tothat occurring in fraction J [42] These evidences are inagreement with our results obtained on fraction J and led tothe hypothesis that the presence of derivatives of 120573-eudesmolmight explain the inhibitory effect of fraction J on the NF-120581Bactivation

4 Conclusions

The present study provides some experimental evidence thatlipophilic preparations from Calendula officinalis L mayexert an anti-inflammatory activity on the gastric districtthrough the inhibition of the NF-120581B system Results obtainedthrough a bio-guided fractionation of a lipophilic extractfrom marigold flowers show for the first time that theinhibitory effect demonstrated by the extract on the NF-120581Bactivation could be ascribed to the presence of a mixture


of components (the so-called phytocomplex) among whichtriterpene esters and derivatives of 120573-eudesmol play a pivotalrole

The results described in this work could be of greatinterest for the following reasons (1) in the present studythe anti-inflammatory activity of a lipophilic extract frommarigold flowers has been proven for internal use Despitethe toxicity renowned for this type of extract now in theliterature toxic events after oral administration of marigoldpreparations are not reported (2) even if other in vivo studiesare needed to confirm the antiphlogistic activity and thesafety of this type of extract the identification of somebiologically active compounds could be the first step forthe formulation of preparations enriched with individualcomponents or extracts from marigold (as faradiol esters or120573-eudesmol derivatives) useful in the treatment or preventionof gastric inflammatory diseases

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was financially supported by MIUR and by ldquoPianodi SviluppoUNIMI-lineaBrdquoThe fellowship of ElisaColombowas partially funded by FSE Regione LombardiaThe authorsgratefully acknowledge the excellent technical assistance ofMr Sandro Gadotti

References

[1] D Arora A Rani and A Sharma ldquoA review on phytochemistryand ethnopharmacological aspects of genus Calendulardquo Phar-macognosy Reviews vol 7 no 14 pp 179ndash187 2013

[2] M Butnariu and C Z Coradini ldquoEvaluation of biologicallyactive compounds from Calendula officinalis flowers usingspectrophotometryrdquo Chemistry Central Journal vol 6 no 1article 35 2012

[3] R Della Loggia A Tubaro S Sosa H Becker S Saarand O Isaac ldquoThe role of triterpenoids in the topical anti-inflammatory activity of Calendula officinalis flowersrdquo PlantaMedica vol 60 no 6 pp 516ndash520 1994

[4] K Zitterl-Eglseer S Sosa J Jurenitsch et al ldquoAnti-oedematousactivities of the main triterpendiol esters of marigold (Calen-dula officinalis L)rdquo Journal of Ethnopharmacology vol 57 no 2pp 139ndash144 1997

[5] K C Preethi G Kuttan and R Kuttan ldquoAnti-inflammatoryactivity of flower extract of Calendula officinalis Linn and itspossible mechanism of actionrdquo Indian Journal of ExperimentalBiology vol 47 no 2 pp 113ndash120 2009

[6] K C Preethi and R Kuttan ldquoWound healing activity of flowerextract of Calendula officinalisrdquo Journal of Basic and ClinicalPhysiology and Pharmacology vol 20 no 1 pp 73ndash79 2009

[7] M Fronza B Heinzmann M Hamburger S Laufer andI Merfort ldquoDetermination of the wound healing effect ofCalendula extracts using the scratch assay with 3T3 fibroblastsrdquoJournal of Ethnopharmacology vol 126 no 3 pp 463ndash467 2009

[8] L M L Parente R D S Lino Junior L M F Tresvenzol MC Vinaud J R De Paula and N M Paulo ldquoWound healing

and anti-inflammatory effect in animal models of Calendulaofficinalis L growing in Brazilrdquo Evidence-Based Complementaryand Alternative Medicine vol 2012 Article ID 375671 7 pages2012

[9] Y M Fonseca C D Catini F T M C Vicentini A Nomizo RF Gerlach and M J V Fonseca ldquoProtective effect of Calendulaofficinalis extract against UVB-induced oxidative stress in skinevaluation of reduced glutathione levels andmatrixmetallopro-teinase secretionrdquo Journal of Ethnopharmacology vol 127 no 3pp 596ndash601 2010

[10] Y M Fonseca C D Catini F T M C Vicentini J C CardosoR L Cavalcanti De Albuquerque Junior and M J VieiraFonseca ldquoEfficacy of marigold extract-loaded formulationsagainst UV-induced oxidative stressrdquo Journal of PharmaceuticalSciences vol 100 no 6 pp 2182ndash2193 2011

[11] L H D Roveroni-Favaretto K B Lodi and J D AlmeidaldquoTopical Calendula officinalis L successfully treated exfoliativecheilitis a case reportrdquo Cases Journal vol 2 no 11 article 90772009

[12] A M Alnuqaydan C E Lenehan R R Hughes and B JSanderson ldquoExtracts from Calendula officinalis offer in vitroprotection against H

2O2induced oxidative stress cell killing of

human skin cellsrdquo Phytotherapy Research vol 29 no 1 pp 120ndash124 2015

[13] H Ozkol Y Tuluce and I Koyuncu ldquoSubacute effect ofcigarette smoke exposure in rats protection by pot marigold(Calendula officinalis L) extractrdquo Toxicology and IndustrialHealth vol 28 no 1 pp 3ndash9 2012

[14] M A Hamzawy E S El-Denshary N S Hassan F AMannaa and M A Abdel-Wahhab ldquoDietary supplementationof Calendula officinalis counteracts the oxidative stress andliver damage resulted from aflatoxinrdquo ISRN Nutrition vol 2013Article ID 538427 9 pages 2013

[15] B D Shivasharan P Nagakannan B S Thippeswamy and VP Veerapur ldquoProtective effect of Calendula officinalis L flowersagainst monosodium glutamate induced oxidative stress andexcitotoxic brain damage in ratsrdquo Indian Journal of ClinicalBiochemistry vol 28 no 3 pp 292ndash298 2013

[16] H Neukirch M DrsquoAmbrosio S Sosa G Altinier R DellaLoggia and A Guerriero ldquoImproved anti-inflammatory activ-ity of three new terpenoids derived by systematic chemicalmodifications from the abundant triterpenes of the floweryplant Calendula officinalisrdquo Chemistry and Biodiversity vol 2no 5 pp 657ndash671 2005

[17] M Ukiya T Akihisa K Yasukawa H Tokuda T Suzukiand Y Kimura ldquoAnti-inflammatory anti-tumor-promotingand cytotoxic activities of constituents of marigold (Calendulaofficinalis) flowersrdquo Journal of Natural Products vol 69 no 12pp 1692ndash1696 2006

[18] B J Egan K Holmes H J OrsquoConnor and C A OrsquoMorainldquoHelicobacter pylori gastritis the unifying concept for gastricdiseasesrdquo Helicobacter vol 12 supplement2 pp 39ndash44 2007

[19] S Keates Y S Hitti M Upton and C P Kelly ldquoHelicobacterpylori infection activates NF-120581B in gastric epithelial cellsrdquoGastroenterology vol 113 no 4 pp 1099ndash1109 1997

[20] S A Sharma M K R Tummuru M J Blaser and L D KerrldquoActivation of IL-8 gene expression by Helicobacter pylori isregulated by transcription factor nuclear factor-120581B in gastricepithelial cellsrdquo Journal of Immunology vol 160 no 5 pp 2401ndash2407 1998

[21] Y Nozawa K Nishihara RM Peek Jr et al ldquoIdentification of asignaling cascade for interleukin-8 production by Helicobacter


pylori in human gastric epithelial cellsrdquo Biochemical Pharmacol-ogy vol 64 no 1 pp 21ndash30 2002

[22] I Chakurski M Matev G Stefanov A Koichev and IAngelova ldquoTreanntment of duodenal ulcers and gastroduo-denitis with a herbal combination of Symphitum officinalisand Calendula officinalis with and without antacidsrdquo VutreshniBolesti vol 20 no 6 pp 44ndash47 1981

[23] V V Krivenko G P Potebnia and V V Loıko ldquoExperiencein treating digestive organ diseases with medicinal plantsrdquoVrachebnoe Delo no 3 pp 76ndash78 1989

[24] M Yoshikawa T Murakami A Kishi T Kageura and HMatsuda ldquoMedicinal flowers III Marigold (1) hypoglycemicgastric emptying inhibitory and gastroprotective principles andnew oleanane-type triterpene oligoglycosides calendasaponinsA B C and D from egyptian Calendula officinalisrdquo Chemicaland Pharmaceutical Bulletin vol 49 no 7 pp 863ndash870 2001

[25] D Mehrabani M Ziaei S V Hosseini et al ldquoThe effect ofCalendula officinalis in therapy of acetic acid induced ulcerativecolitis in dog as an animal modelrdquo Iranian Red Crescent MedicalJournal vol 13 no 12 pp 884ndash890 2011

[26] C Di Lorenzo M DellrsquoAgli M Badea et al ldquoPlant foodsupplements with anti-inflammatory properties a systematicreview (II)rdquo Critical Reviews in Food Science and Nutrition vol53 no 5 pp 507ndash516 2013

[27] H Neukirch M DrsquoAmbrosio J Dalla Via and A GuerrieroldquoSimultaneous quantitative determination of eight triterpenoidmonoesters from flowers of 10 varieties of Calendulla officinalisL and characterisation of a new triterpenoid monoesterrdquoPhytochemical Analysis vol 15 no 1 pp 30ndash35 2004

[28] M DrsquoAmbrosio A Ciocarlan E Colombo et al ldquoStructure andcytotoxic activity of sesquiterpene glycoside esters from Calen-dula officinalis L studies on the conformation of viridiflorolrdquoPhytochemistry vol 117 pp 1ndash9 2015

[29] M DellrsquoAgli G V Galli E Bosisio and M DrsquoAmbrosioldquoInhibition of NF-120581B and metalloproteinase-9 expression andsecretion by parthenolide derivativesrdquo Bioorganic amp MedicinalChemistry Letters vol 19 no 7 pp 1858ndash1860 2009

[30] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[31] T V Gorchakova I V Suprun I A Sobenin and A NOrekhov ldquoUse of natural products in anticytokine therapyrdquoBulletin of Experimental Biology and Medicine vol 143 no 3pp 316ndash319 2007

[32] A J Garcıa-Pineres M T Lindenmeyer and I Merfort ldquoRoleof cysteine residues of p65NF-120581B on the inhibition by thesesquiterpene lactone parthenolide andN-ethylmaleimide andon its transactivating potentialrdquo Life Sciences vol 75 no 7 pp841ndash856 2004

[33] E Aldieri D Atragene L Bergandi et al ldquoArtemisinin inhibitsinducible nitric oxide synthase and nuclear factor NF-kBactivationrdquo FEBS Letters vol 552 no 2-3 pp 141ndash144 2003

[34] C Zhu Z Xiong X Chen et al ldquoArtemisinin attenuateslipopolysaccharide-stimulated proinflammatory responses byinhibiting NF-kappaB pathway in microglia cellsrdquo PLoS ONEvol 7 no 4 Article ID e35125 2012

[35] G Willuhn and R G Westhaus ldquoLoliolide (Calendin) fromCalendula officinalisrdquo Planta Medica vol 53 no 3 article 3041987

[36] A Percot A Yalcin V Aysel H Erdugan B Dural and K CGuven ldquoLoliolide in marine algaerdquo Natural Product Researchvol 23 no 5 pp 460ndash465 2009

[37] S-Y Cheng K-J Huang S-K Wang Z-H Wen P-W Chenand C-Y Duh ldquoAntiviral and anti-inflammatory metabolitesfrom the soft coral sinularia capillosardquo Journal of NaturalProducts vol 73 no 4 pp 771ndash775 2010

[38] T Wang Z Wang L Chen S Zhang and J Lin ldquoIsolationand characterization of (6S9R) 6-hydroxy-447a-trimethyl-5677a-tetrahydro-1-benzof uran-2(4H)-one from Scutellariabarbatardquo Journal of Medicinal Plants Research vol 5 no 4 pp613ndash625 2011

[39] C Pizza and N De Tommasi ldquoSesquiterpene glycosides basedon the alloaromaden-drane skeleton from Calendula arvensisrdquoPhytochemistry vol 27 no 7 pp 2205ndash2208 1988

[40] J Jakupovic M Grenz F Bohlmann A Rustaiyan and SKoussari ldquoSesquiterpene glycosides from Calendula persicardquoPlanta Medica vol 54 no 3 pp 254ndash256 1988

[41] M B Sghaier H Harizi T Louhichi M Krifa K Ghediraand L Chekir-Ghedira ldquoAnti-inflammatory and antiulcero-genic activities of leaf extracts and sesquiterpene from Teu-crium ramosissimum (Lamiaceae)rdquo Immunopharmacology andImmunotoxicology vol 33 no 4 pp 656ndash662 2011

[42] M-J Seo S-J Kim T-H Kang et al ldquoThe regulatory mech-anism of 120573-eudesmol is through the suppression of caspase-1 activation in mast cell-mediated inflammatory responserdquoImmunopharmacology and Immunotoxicology vol 33 no 1 pp178ndash185 2011

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


severe diseases such as atrophic gastritis peptic ulcer orgastric adenocarcinoma [18] As a consequence of untreatedinfections the gastric epithelial cells secrete many cytokinesand chemokines that are attracted to the mucosal layerneutrophils lymphocytes and macrophages responsible forthe maintenance of the inflammatory status Several studiesindicate that nuclear factor 120581B (NF-120581B) plays a crucial role atthe molecular level of this process [19ndash21]

Aqueous preparations from marigold flowers have beentraditionally used for the treatment of gastrointestinal dis-eases such as gastritis ulcers and colitis Two studies demon-strated the efficacy of herbal mixtures containing marigoldin the therapy of duodenal ulcers gastroduodenitis andchronic hyposecretory gastritis [22 23] and amore recent onedescribed the hypoglycemic and gastroprotective activities ofCalendula officinalis L in vivo [24] Moreover another workshowed the effect of marigold preparations in amelioratinginflammatory symptoms in an animal model of ulcerativecolitis [25] So far no one has established the clinical effective-ness of Calendula officinalis L extracts as anti-inflammatoryagent by internal use [26] Thus the aim of this work was(i) to evaluate in vitro the anti-inflammatory activity of aCH2Cl2extract from marigold flowers in a cellular model

of gastritis and (ii) to identify mixtures and compoundsresponsible for this effect by a bio-guided fractionation ofthe CH

2Cl2extract considering the NF-120581B system as the

molecular target

2 Materials and Methods

21 Reagents Dulbeccorsquos modified Eaglersquos medium(DMEM)F12 (1 1) penicillin streptomycin L-glutamineand trypsin-EDTAwere fromGibco (Life Technologies ItaliaMonza Italy) Foetal bovine serum (FBS) and disposablematerial for cell culture were purchased by Euroclone(Euroclone SpA Pero Milan Italy) Parthenolide dim-ethyl sulfoxide (DMSO) and all solvents (from Sigma-Aldrich Milan Italy) were of high-performance liq-uid chromatography purity grade (gt98) Tumor necrosisfactor alpha (TNF-120572) was from ImmunoTools (FriesoytheGermany) Human adenocarcinoma cells (AGS) werepurchased from European Collection of Cell Culture (EACCUK cat number 89090402) NF-120581B-luc plasmid was akind gift from Professor N Marx (Department of InternalMedicine II-Cardiology Ulm Germany) Britelite plus wasfrom PerkinElmer (Monza Italy)

Flash chromatography (FC) was performed by MerckKieselgel 60 (70 230mesh) Merck RP 18 LiChroprep (40ndash65 120583m) TLC by Merck Kieselgel 60 PF254 high pressure liq-uid chromatography (HPLC) by Merck Hitachi L7100 pumpL7400 UV detector D7500 integrator Rheodyne injectorHPLC column and method by Synergi Hydro column (150times 10mm 4 120583m particle size 80 A pore size PhenomenexTorrance CA USA) 50mLmin 120582 = 210 nm The eluentswere acetonitrile (ACN) (A) and ACNwater 50 50 vv (B)in method 1 the gradient was changed linearly from 0 to85 A in 45min in method 2 the eluent A was appliedin the gradient of 0 at 119905 = 0 37 at 119905 = 30 85 at119905 = 31 85 at 119905 = 45 and 100 at 119905 = 46min Optical

rotations were measured on a Bellingham + Stanley ADP 440polarimeter NMRwas performed by Bruker Avance 400 (1Hat 400MHz 13C at 100MHz) 5mm BBI probe 120575 in ppmusing residual solvent signals as internal reference (CDCl

3

= 770 CHCl3= 726 and CD

2HOD = 331) 119869 values in Hz

multiplicities and peak assignments from 1H 1HCOSY 1119869CH(HSQC) 119899119869CH (HMBC) and NOESY experiments NOESYdata are reported as correlation map(s) between protons1H harr 1H HMBC data are reported as (13C) rarr correlatedto 1HMS was performed by Bruker Esquire LCmultiple iontrap Electrospray ionization (ESI) was as follows positive ionmode capillary voltage 4000V nebulizing pressure 300 psidrying gas flow 7mLmin and temperature 300∘C Electronimpact mass spectra (EIMS HREIMS) were recorded on aKratos MS80 spectrometer with home-built data system andelectron ionization at 70 eVmz (rel)

22 Plant Material Dry flowers of Calendula officinalisL (variety Calypso Orange Florensis) were obtained andextracted as previously described [27 28] A voucher spec-imen (number 20040929) is deposited at the Laboratory ofBioorganic Chemistry (Trento Italy)

23 Cell Culture AGS cells (EACC number 89090402) weregrown in DMEMF-12 medium supplemented with 100 unitspenicillinmL 100mg streptomycinmL 2mM L-glutamineand 10 heat-inactivated foetal bovine serumThe cells wereincubated at 37∘C in humidified atmosphere with 5 CO

2

until confluence For the NF-120581B driven transcription assayAGS cells were plated at the concentration of 15 times 104cellsmL in 24-well plates with complete medium to reachabout 80 of confluence After 48 h medium was replacedwith FBS-free medium and cells were exposed for 24 h to thecompounds or fractions under study in the presence of TNF-120572 (10 ngmL) as proinflammatory stimulus To study the NF-120581B nuclear translocation cells were plated in 10mm dishes (3times 106 cellsdish) with fresh complete medium for 48 h Thenmedium was replaced with FBS-free medium containingincreasing concentrations of fractions in the presence ofTNF-120572 (10 ngmL) for 1 h We previously found that thefractions H and I are highly cytotoxic to AGS cells [28] sothese fractions were excluded from the biological assays

24 Transient Transfection and Luciferase Reporter Assay Toassess the effect of different fractions and isolated compoundson the NF-120581B driven transcription AGS cells were tran-siently transfected by the calcium-phosphate method witha plasmid containing the luciferase reporter gene under thecontrol of the E-selectin promoter with three NF-120581B bindingsites Sixteen hours after transfection cells were stimulatedwith TNF-120572 (10 ngmL) in the presence of CH

2Cl2extract

(1ndash20120583gmL) or fractions AndashP and subfractions (005ndash20120583gmL) or individual compounds (1ndash50120583M) After 24 hcells were harvested and luciferase activity was measuredusing Britelite plus reagent on a luminometer Parthenolide(final concentration 10 120583M) is a natural inhibitor of the NF-120581B driven transcription [29] it was used as positive controland showed around 50 inhibition in all the experiments









lowast

lowastlowastlowast


+ + + + + +minus



(120583gmL)

0

20NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

40

60

80

100

120




2Cl2extract was




2Cl2extract









50














2Cl2


5030plusmn73 120583M



50429 plusmn


50of fraction


50311 plusmn


119877=

21min mg 89) 2 (peak 4 119905119877= 172min mg 34) 3





0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1

log (120583M)

(a)

0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1

log (120583M)


(b)

0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1


Compound




Faradiol

Arnidiol

Calenduladiol

CH2Cl2 extract


mdash

mdash

mdash

mdash

631 plusmn 068120583gmL

30 plusmn 73 120583M

30 plusmn 46 120583M

35 plusmn 58 120583M

10 plusmn 26 120583M

21 plusmn 52 120583M

19 plusmn 38 120583M

(c)


50values and




[120572]25





15H25O]+ (28) 204

[M-C12H18O8]+∙ (20) 43 (100) HREIMS mz 2730973


15H24




O

O

OAc

OH

OH O

O

OAc

OH

OH

H

H

H

2 3114

11

2

813

12

15

106

12

3 48

6

4

14OO

HO

1

H120572

H120573 H120573

H120573

H120573

HaHb

2998400

5998400

6998400





[120572]25



EIMSmz 494 [M]+∙ (03) 273 [M-C15H25O]+ (16) 204 [M-



12H17O7

2730974) 2041871 (calcd for C15H24 2041878) 1H NMR



3OD 400MHz) 120575 sim194 (1Hm


3 100MHz) 120575 542 (d C-1) 254 (t C-2)



lowastlowast

lowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

+

TNF-1205720

20

40

60

80

100

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

120

140


+ + + + + + + + + + +minus

minus minus



4 Conclusions







Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























lowast

lowastlowastlowast


+ + + + + +minus



(120583gmL)

0

20NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

40

60

80

100

120




2Cl2extract was




2Cl2extract









50














2Cl2


5030plusmn73 120583M



50429 plusmn


50of fraction


50311 plusmn


119877=

21min mg 89) 2 (peak 4 119905119877= 172min mg 34) 3





0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1

log (120583M)

(a)

0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1

log (120583M)


(b)

0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1


Compound




Faradiol

Arnidiol

Calenduladiol

CH2Cl2 extract


mdash

mdash

mdash

mdash

631 plusmn 068120583gmL

30 plusmn 73 120583M

30 plusmn 46 120583M

35 plusmn 58 120583M

10 plusmn 26 120583M

21 plusmn 52 120583M

19 plusmn 38 120583M

(c)


50values and




[120572]25





15H25O]+ (28) 204

[M-C12H18O8]+∙ (20) 43 (100) HREIMS mz 2730973


15H24




O

O

OAc

OH

OH O

O

OAc

OH

OH

H

H

H

2 3114

11

2

813

12

15

106

12

3 48

6

4

14OO

HO

1

H120572

H120573 H120573

H120573

H120573

HaHb

2998400

5998400

6998400





[120572]25



EIMSmz 494 [M]+∙ (03) 273 [M-C15H25O]+ (16) 204 [M-



12H17O7

2730974) 2041871 (calcd for C15H24 2041878) 1H NMR



3OD 400MHz) 120575 sim194 (1Hm


3 100MHz) 120575 542 (d C-1) 254 (t C-2)



lowastlowast

lowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

+

TNF-1205720

20

40

60

80

100

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

120

140


+ + + + + + + + + + +minus

minus minus



4 Conclusions







Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















50














2Cl2


5030plusmn73 120583M



50429 plusmn


50of fraction


50311 plusmn


119877=

21min mg 89) 2 (peak 4 119905119877= 172min mg 34) 3





0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1

log (120583M)

(a)

0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1

log (120583M)


(b)

0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1


Compound




Faradiol

Arnidiol

Calenduladiol

CH2Cl2 extract


mdash

mdash

mdash

mdash

631 plusmn 068120583gmL

30 plusmn 73 120583M

30 plusmn 46 120583M

35 plusmn 58 120583M

10 plusmn 26 120583M

21 plusmn 52 120583M

19 plusmn 38 120583M

(c)


50values and




[120572]25





15H25O]+ (28) 204

[M-C12H18O8]+∙ (20) 43 (100) HREIMS mz 2730973


15H24




O

O

OAc

OH

OH O

O

OAc

OH

OH

H

H

H

2 3114

11

2

813

12

15

106

12

3 48

6

4

14OO

HO

1

H120572

H120573 H120573

H120573

H120573

HaHb

2998400

5998400

6998400





[120572]25



EIMSmz 494 [M]+∙ (03) 273 [M-C15H25O]+ (16) 204 [M-



12H17O7

2730974) 2041871 (calcd for C15H24 2041878) 1H NMR



3OD 400MHz) 120575 sim194 (1Hm


3 100MHz) 120575 542 (d C-1) 254 (t C-2)



lowastlowast

lowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

+

TNF-1205720

20

40

60

80

100

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

120

140


+ + + + + + + + + + +minus

minus minus



4 Conclusions







Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1

log (120583M)

(a)

0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1

log (120583M)


(b)

0

25

50

75

100

125

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

0 1 2 3minus1


Compound




Faradiol

Arnidiol

Calenduladiol

CH2Cl2 extract


mdash

mdash

mdash

mdash

631 plusmn 068120583gmL

30 plusmn 73 120583M

30 plusmn 46 120583M

35 plusmn 58 120583M

10 plusmn 26 120583M

21 plusmn 52 120583M

19 plusmn 38 120583M

(c)


50values and




[120572]25





15H25O]+ (28) 204

[M-C12H18O8]+∙ (20) 43 (100) HREIMS mz 2730973


15H24




O

O

OAc

OH

OH O

O

OAc

OH

OH

H

H

H

2 3114

11

2

813

12

15

106

12

3 48

6

4

14OO

HO

1

H120572

H120573 H120573

H120573

H120573

HaHb

2998400

5998400

6998400





[120572]25



EIMSmz 494 [M]+∙ (03) 273 [M-C15H25O]+ (16) 204 [M-



12H17O7

2730974) 2041871 (calcd for C15H24 2041878) 1H NMR



3OD 400MHz) 120575 sim194 (1Hm


3 100MHz) 120575 542 (d C-1) 254 (t C-2)



lowastlowast

lowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

+

TNF-1205720

20

40

60

80

100

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

120

140


+ + + + + + + + + + +minus

minus minus



4 Conclusions







Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

O

OAc

OH

OH O

O

OAc

OH

OH

H

H

H

2 3114

11

2

813

12

15

106

12

3 48

6

4

14OO

HO

1

H120572

H120573 H120573

H120573

H120573

HaHb

2998400

5998400

6998400





[120572]25



EIMSmz 494 [M]+∙ (03) 273 [M-C15H25O]+ (16) 204 [M-



12H17O7

2730974) 2041871 (calcd for C15H24 2041878) 1H NMR



3OD 400MHz) 120575 sim194 (1Hm


3 100MHz) 120575 542 (d C-1) 254 (t C-2)



lowastlowast

lowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

+

TNF-1205720

20

40

60

80

100

NF-120581

B dr

iven

tran

scrip

tion

( v

ersu

s TN

F-120572

)

120

140


+ + + + + + + + + + +minus

minus minus



4 Conclusions







Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article A Bio-Guided Fractionation to Assess the ...downloads.hindawi.com › journals ›...

Documents

Transcript of Research Article A Bio-Guided Fractionation to Assess the ...downloads.hindawi.com › journals ›...