Research Article Constituents of French Marigold ( …downloads.hindawi.com/journals/omcl/2016/4216285.pdfResearch Article Constituents of French Marigold ( Tagetes patula L.) Flowers

Research ArticleConstituents of French Marigold (Tagetes patula L) FlowersProtect Jurkat T-Cells against Oxidative Stress

Irakli Chkhikvishvili1 Tamar Sanikidze1 Nunu Gogia1 Maia Enukidze1

Marine Machavariani1 Nana Kipiani1 Yakov Vinokur2 and Victor Rodov2

1 Institute of Medical Biotechnology Tbilisi State Medical University 33 Vazha Pshavela Avenue 0177 Tbilisi Georgia2Department of Postharvest Science of Fresh Produce Agricultural Research Organization The Volcani CenterHaMaccabim Road 68 PO Box 15159 7528809 Rishon LeZion Israel

Correspondence should be addressed to Victor Rodov vrodovagrigovil

Received 12 January 2016 Revised 3 April 2016 Accepted 27 April 2016

Academic Editor Rodrigo Franco

Copyright copy 2016 Irakli Chkhikvishvili et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The flowers of French marigold (Tagetes patula L) are widely used in folk medicine in particular for treating inflammation-relateddisorders However cellular mechanisms of this activity demand further investigation In the present work we studied the potentialof T patula compounds to alleviate the oxidative stress in hydrogen peroxide-challenged human lymphoblastoid Jurkat T-cellsCrude extracts of marigold flowers and purified fractions containing flavonoids patuletin quercetagetin and quercetin and theirderivatives as well as the carotenoid lutein were brought in contact with Jurkat cells challenged with 25 or 50120583MH

2O2 Hydrogen

peroxide caused oxidative stress in the cells manifested as generation of superoxide and peroxyl radicals reduced viability arrestedcell cycle and enhanced apoptosis The stress was alleviated by marigold ingredients that demonstrated high radical-scavengingcapacity and enhanced the activity of antioxidant enzymes involved in neutralization of reactive oxygen species Flavonoid fractionrich in quercetin and quercetagetin showed the highest cytoprotective activity while patuletin in high dose exerted a cytotoxiceffect associated with its anticancer potential T patula compounds enhanced the production of anti-inflammatory and antioxidantinterleukin-10 (IL-10) in Jurkat cells Both direct radical-scavenging capacity and stimulation of protective cellular mechanisms canunderlay the anti-inflammatory properties of marigold flowers

1 Introduction

ThegenusTagetes (Asteraceae) is native toAmericas but someof its members (in particular T erecta and T patula) com-monly known asmarigoldswere naturalized in theOldWorld(India North Africa and Europe) as early as in 16th century[1] Moreover some researchers suggest that both speciesreached India anciently throughpre-Columbian transoceanicvoyages [2] Marigold was introduced to Georgia from Indiaand its ground dried petals became one of the most popularlocal spices [3] Both T erecta and T patula are grown inGeorgia as spice and dye plants [4] recognized for theirhealth-beneficial properties [5]

Tagetes is a multipurpose plant having ornamental ritualmedicinal anthelmintic insecticidal colorant food and for-age applications [6 7] Healing properties of Tagetes species

have been implemented by folk medicine for centuries [8]In particular flowers and entire herb of T patula (Frenchmarigold) are used for preparing ethnobotanical remediesagainst rheumatism stomach and intestinal problems kidneyand hepatic disorders fever and pneumonia [6 9] Theinfusion of T patula flowers is also implemented as eyewash[6]The efficacy of orally administeredmethanolic extracts ofT patula florets against acute and chronic inflammation wasconfirmed in experiments with animal models [10] Similarresults were obtained forT erecta (Africanmarigold) extracts[11] Furthermore a double-blind placebo-controlled clinicaltrial showed effectiveness of marigold therapy using T patulapreparations in treating human inflammation-associated dis-orders such as bunion [12]The anti-inflammatory effect of Tpatula extracts could be reproduced in animal model by oraladministration of its flavonoid constituents patuletin and

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 4216285 10 pageshttpdxdoiorg10115520164216285

2 Oxidative Medicine and Cellular Longevity

patulitrin [13] Lipophilic ingredients of marigold flowersthe carotenoid lutein and essential oil compounds were alsoreported to possess anti-inflammatory properties [14 15] Inour previous study both hydrophilic and lipophilic fractionsfrom T patula petals showed the highest radical-scavengingcapacities among all Georgian spices tested [16]

However the cellular mechanisms by which the marigoldextracts exert their anti-inflammatory effects are not fullyunderstood and demand further investigation Methanolextracts of T patula flowers as well as isolated patuletinwere reported to scavenge peroxyl and superoxide radicals inchemical systems and in human neutrophils and at the sametime to exert cytotoxic and growth inhibitory effects towardsa range of human cancer cell lines in particular HeLa cells[17] On the other hand ethanolic and ethyl acetate extracts ofmarigold flowers showed no cytotoxicity towards H460 lungcancer and theCaco-2 colon cancer cell lines in anMTT assay[18] Furthermore the MTT assay revealed a cytoprotectiveeffect of patuletin on the human lung carcinoma GLC

4cell

line challenged by cytotoxic sesquiterpene lactone helenalin[19] Activation of antioxidant enzymes rather than direct freeradical scavenging was suggested as a possible mechanismunderlying this phenomenon Mesaik et al [20] demon-strated that immunomodulatory and antiarthritic potentialof patuletin was associated with inhibited production of theproinflammatory cytokine TNF-120572 with no cytotoxic prop-erty Chew et al [21] reported that marigold-derived dietarylutein enhanced phytohemagglutinin-induced lymphocyteproliferation in mice but had no effect on interleukin-2production or lymphocyte cytotoxicity

Human lymphoblastoid T-cell Jurkat line is a popularmodel for the study of immune signaling and inflammation[22] Jurkat cells can imitate both healthy and inflammatoryT-cells in their response to oxidative metabolites such ashydrogen peroxide [23] Although H

2O2plays an impor-

tant role in antigen-dependent lymphocyte activation [24]excessive production of H

2O2induces oxidative stress and

impairs T-cell activity leading to chronic inflammation andcell death In the presence of oxygen in aqueous mediumhydrogen peroxide can produce additional cytotoxic reactiveoxygen species (ROS) such as superoxide and peroxyl radi-cals [25] To control the level of ROS cells employ antioxidantenzymes for example catalase that decomposes hydrogenperoxide and superoxide dismutase neutralizing superoxideradicals as well as low-molecular antioxidants The lattergroup includes internally produced glutathione and dietaryantioxidants such as ascorbic acid and phenolic compoundsThe function of the antioxidant system ismaintained by addi-tional enzymes such as glutathione reductase that restoresthe antioxidant capacity of oxidized glutathione Signalingfor regulation of oxidative stress and inflammatory responsesinvolve cytokines such as anti-inflammatory and antioxidantinterleukin-10 (IL-10) [26]

Theoxidative stress can interferewith normal progressionof cell growth and division arranged in a cell cycle In eukary-otes a normal cell cycle consists of four main stages G

1

during which a cell is metabolically active and continuouslygrows S phase during which DNA replication takes placeG2 during which the growth of cell continues and the cell

prepares for division and the M (mitosis) phase duringwhich the cell divides into two daughter cells each with afull copy of DNA After the M phase the cells can enter G

1

or G0 a quiescent phase When the cell detects any defects

(eg oxidative DNA damage) which necessitate halting thecell cycle inG

1 cell cycle arrest occurs Efforts to correct these

problems may slow growth and induce cell death [27]The response of Jurkat cells to H

2O2is dose-dependent

Reversible oxidative changes that could be repaired by cellularantioxidant systems occurred at a H

2O2concentration of

20120583M while the signs of apoptosis (programmed cell death)were noted at 50120583MH

2O2[28] Both apoptosis and necrosis

(a nonprogrammed cell death caused by damage) wereobserved in the Jurkat cells exposed to 100 120583M H

2O2[29]

whereas the necrosis prevailed at 500 120583MH2O2[30]

The balance between prooxidant and antioxidant repairmechanisms determining cellular survival and functioncan be affected by dietary bioactive compounds possessingradical-scavenging and anti-inflammatory activity There-fore in the present work we investigated the effects of anti-inflammatory T patula flower extract and of its purifiedfractions on the behavior of theH

2O2-challenged Jurkat cells

2 Materials and Methods

21 Plant Material The flowers were collected from theplants of a local Georgian landrace of T patula grown atexperimental plot near Tbilisi from seeds purchased from acommercial supplier The collected flowers were air-dried inthe shade at 25ndash30∘CThe dried matter was stored in a closedglass container in a cool dry place

22 Extraction and Purification ofMarigold Constituents Theisolation was performed by sequential solvent extraction ofT patula flowers A sample of 600 g of dried pulverizedplant material was extracted with 12-dichloroethane in aSoxhlet apparatus for 48 h until color loss The residue afterthe dichloroethane extraction was reextracted with ethanol(solventplant matter ratio 1 5) for isolation of compoundsof higher polarity The solvents were evaporated undervacuum at 40∘C giving dichloroethane and ethanol crudeextracts Further separation of individual compounds fromthe dichloroethane extract was performed by column chro-matography on silica gel column with chloroform-hexanesolvent system The elution of fractions from the columnwas started with hexane with further increase of chloroformcontent in the system The elution with 3 chloroform inhexane gave compound 1 Compound 2 was present in thefraction eluted from the column with 5 chloroform inhexane

The ethanolic extract was separated on a silica gel col-umn by elution with dichloroethanemethanol using thin-layer chromatography (TLC) for preliminary characteriza-tion of fractionsThe elution was started with dichloroethanewith subsequent stepwise increase of methanol content inthe system Elution with 2 3 5 7 and 10 methanolin dichloroethane produced fractions 1 2 3 4 and 5respectively Rechromatography of fraction 2 on a Sephadex

Oxidative Medicine and Cellular Longevity 3

LH-20 column with 2methanol in chloroform with furtherTLC separation produced compound 2 that was also foundin the dichloroethane extract Compound 3 was obtained byrechromatography of fraction 5 on a silica gel column elutedwith 8 methanol in chloroform and further purified on apolyamide column with elution with aqueous ethanol

The TLC separation was performed using silica gelplatesMerck (Germany) Separation of lipophilic compoundswas performed in the solvent systems of dichloroethane-methanol (9 1) and chloroform-methanol (9 1) More polarcompounds from ethanolic extracts were separated in thesolvent systems of chloroformmethanolwater (26 14 3)The chromatograms were inspected under UV light of 254and 360 nm before and after applying staining reagents forflavonoids detection Flavonoids were detected as yellowspots revealed after heating the plates sprayed with 1ethanolic solution of aluminium chloride Other compoundswere detected by spraying 20 sulfuric acid solutions Afterheating the sprayed plates to 100∘C the compounds wererevealed as spots of blue to green shades depending onspecific compounds

23 Liquid Chromatography-Mass Spectrometry (LC-MS)Analysis The samples were dissolved in HPLC-grademethanol and filtered through a Millex-HV Durapore(PVDF) membrane (022120583m) before being injected into theLC-MS instrument Mass spectral analyses were carried outusing the Ultraperformance LC-Quadruple Time of Flight(UPLC-QTOF) instrument (Waters Premier QTOF MilfordMA USA) with the UPLC column connected online to aPDA detector and then to an MS detector equipped with anelectrospray ion (ESI) source (used in ESI-positive mode)Separation was performed on a 21 times 50mm id 17 120583mUPLC BEH C18 column (Waters Acquity)

The chromatographic andMSparameters were as followsthe mobile phase consisted of 01 formic acid in water(phase A) and 01 formic acid in acetonitrile (phase B)The linear gradient program was as follows 100 to 95A over 01min 95 to 5 A over 97min held at 5 Aover 32min and then returned to the initial conditions(95 A) in 42min The flow rate was 03mL minminus1 and thecolumn was kept at 35∘C Masses of the eluted compoundswere detected with a QTOF Premier MS instrument TheUPLC-MS runs were carried out at the following settingscapillary voltage of 28 kV cone voltage of 30 eV and collisionenergy of 5 eV Argon was used as the collision gas The119898119911 range was 70 to 1000D The MS system was calibratedusing sodium formate and Leu-enkephalin was used asthe lock mass The MassLynx software version 41 (Waters)was used to control the instrument and calculate accuratemasses The compounds were identified using the molecularformulae calculated on the basis of accuratemass and isotopicpattern information and UVvisible spectra in comparisonwith authentic standards of quercetin quercetagetin andquercetagetin-7-glucoside (Extrasynthese Genay France)

24 Cell Culture and Experimental Design The humanT-cell leukemia lymphoblastoid Jurkat cells (DSMZ ACC

282) were obtained from the Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ BraunschweigGermany) The cells were grown in suspension cultureat 37∘C under 5 humidified CO

2in bioactive medium

RPMI 1640 (Gibco Grand Island NY USA) containinginactivated embryonic bovine serum (Sigma St Louis MOUSA) L-glutamine (4mM) penicillin (100UmLminus1) andstreptomycin (100UmLminus1) The experiments were carriedout at cell densities of 03 to 06 times 106 cells mLminus1 In orderto imitate the oxidative stress conditions H

2O2(Sigma)

was added to the Jurkat culture to reach the concentrationsof 25 and 50 120583M corresponding to low and intermediatestress severity respectively [28] In the unstressed controltreatment water was added to the samples instead of H

2O2

The crude marigold extracts and isolated fractions wereadded to the cultures at a rate of 2mgmLminus1 if not specifieddifferently in the text

25 Electron Paramagnetic Resonance (EPR) SpectroscopyThe effect of marigold extracts on the generation of freeradicals in H

2O2-challenged and unchallenged cells was

studied using the electron paramagnetic resonance (EPR)method EPR spectra were registered on a radiospectrometerRE 1307 (EPSI Chernogolovka Russia) Peroxyl radicals weredetected with spin-trap 120572-phenyl-tert-butylnitrone (PBNSigma) (50mM on 06 times 106 cells in 05mL medium) atroom temperature at microwave power (20mV) Superox-ide radicals were detected with a spin-trap 55-dimethyl-I-pyrrole-IV-oxide (DMPO) (Sigma) (50mM on 06 times 106 cellsin 05mLmedium) at room temperature at microwave power(20mV)

26 Cell Viability Cell viability was assayed by the MTTtest based on evaluating cellular dehydrogenase activity[31] Cell suspensions (2 times 106 cells mLminus1) were incubatedwith H

2O2and marigold preparations as described above

After the incubation period the cells were harvested bycentrifugation at 1500 g for 5 minutes washed and resus-pended in fresh medium The 8mgmLminus1 solution of 3-(45-dimethylthiazol-2)-25-diphenyltetrazolium bromide (MTT)(Sigma) in buffer (140mM NaCl 5mM HEPES pH 74)was added to the cell suspension at a rate of 30 120583L per100 120583L suspension and the mixture was incubated for 4 h at37∘C in a 5 CO

2atmosphere After this incubation the

supernatant was carefully removed and the colored formazancrystals produced from the MTT were dissolved in 100120583Lof dimethyl sulfoxide (DMSO) The absorption values of thesolutions reflecting the cellular dehydrogenase activity weremeasured at 570 nm The effects of various treatments oncellular activity and viability were expressed as percentagesof their absorption values related to those of nontreated cells

27 Cell Cycle Analysis Cell distribution into cell cycle phaseswas studied by flow cytometry using propidium iodidestaining [32] The method is based on the capacity of propid-ium iodide to intercalate with double-stranded DNA givinginformation about the DNA distribution between the cell


cycle phases The cells were fixed in 70 ethanol at 40∘b for12 hours After removal of the ethanol 100120583gmLminus1 RNAase(Sigma) was added to the cellular pellet and incubatedfor 30 minutes at room temperature After suspending thecells with propidium iodide solution they were incubatedfor 30 minutes at room temperature and analyzed by flowcytometry (excitation and emission at 488 and 617 nm resp)in accordance with the DNA content as follows (a) haploidstate-apoptotic cells (b) diploid state-cells in G

0G1phase

(c) transitional state between diploid and tetraploid-cellsin phase S (d) tetraploid state-cells in G

2M phase The

percentage of cells in each state was determinedIn addition a percentage of apoptotic cells in the pop-

ulation (apoptotic ratio) was determined by flow cytometryon the basis of mitochondrial transmembrane potential(ΔΨ) measurement using a lipophilic cation test with 331015840-dihexyloxacarbocyanine iodide (DiOC

6) [33] In order to

determine the mitochondrial potential 105 cells were incu-bated with 120 120583L of 02 120583M DiOC

6solution for 15min at

37∘C The studies were conducted using the FACSCaliburflow cytometer (Becton Dickinson Franklin Lakes NJ US)excitation and emission wavelengths for DiOC

6were 488 and

530 nm respectivelyThe structure of the Jurkat cells was investigated under

transmission electronmicroscope (TEM) Tesla BS 500 (TeslaBrno CzechRepublic) after fixationwith 25glutaraldehyde(pH 74) and toluidine blue staining

28 Antioxidant Enzymes Jurkat cell extract was preparedby centrifugation of the cell suspensions at 500 g and thenhomogenizing the cellular precipitate in a lysis buffer (pH79) that was comprised of 15mMMgCl

2 10mM KCl 1mM

dithiothreitol 1 120583gmLminus1 leupeptin 1 120583gmLminus1 aprotinin and10mM HEPES The volume of the buffer was twice thevolume of the precipitate Lysis of the cells was performed bypassing the suspension through a 27-gauge needle 10 timesThe obtained homogenate was centrifuged for 20min at10000 g The supernatant was used to determine the levels ofenzyme activity Catalase (EC 11116) activity was measuredspectrophotometrically as the decomposition of H

2O2at

240 nm [23] One unit of catalase activity was defined asthe amount of enzyme decomposing 1 120583mol of H

2O2per

minute Superoxide dismutase (SOD EC 11511) was assayedusing NADPH and phenazine methosulfate (PMS) reagentsfor the reduction of nitroblue tetrazolium salt (NBT) intoblue-colored formazan measured spectrophotometrically at560 nm [24] One unit of SOD activity was defined as theamount of enzyme oxidizing 1 nmol NADPH per minuteGlutathione reductase (GR EC 1817) activity was measuredspectrophotometrically as oxidation ofNADPHmonitored at340 nm in the presence of oxidized glutathione Glutathionereductase activity was expressed as nmol substrate oxidizedper minute The activity of the enzymes was expressed interms of units permg of protein A total proteinmicro-Lowrykit (Sigma) was used to determine the protein content

29 ORAC Assay of Nonenzymatic Radical-ScavengingActivity Dried marigold petals were extracted by stepwise

extraction with acetate buffer acetone and hexane andrepeated partition of water-soluble and water-insolubleportions as described by Vinokur and Rodov [34] Thehydrophilic wateracetone fraction was used for measuringthe oxygen radical absorbance capacity (ORAC) accordingto the procedure described by Gillespie et al [35] The assayis based on measuring the degradation of a fluorescentprobe by free radicals resulting in decline of its fluorescenceintensity The radical-scavenging efficacy of antioxidants isassessed by delay of the fluorescence decay in comparisonwith a standard antioxidant Trolox (6-hydroxy-2578-tetramethylchroman-2-carboxylic acid Sigma) Fluorescein(Sigma) 008 120583M was used as a fluorescent probe Peroxylradicals were generated by 150mM of azo-initiator 221015840-azobis(2-amidinopropane) dihydrochloride (AAPH)(Sigma) at 37∘C A SPEX fluorometer (SPEX IndustriesEdison NJ USA) was used for fluorescence measurementat excitation and emission wavelengths of 485 and 530 nmrespectively

210 Interleukin Analysis Jurkat cells were prestimulatedby incubation with 50 120583gmL phytohemagglutinin (PHA) at37∘C for 5min and cultured for 24 h with nonstimulatedJurkat cells (40 stimulated and 60 nonstimulated cells)The level of anti-inflammatory and antioxidant cytokine IL-10 was assayed using ELISA kit (BenderMedsystems ViennaAustria) and the Multiscan microplate reader (LabSystemHelsinki Finland)

211 Statistics The trials were performed in five replicationsThe statistical analysis of the obtained results includingcalculation of means and standard deviations was conductedusing the IBM SPSS Statistics programThe statistical signifi-cance of the differences between the treatment results versusnontreated control was analyzed by pairwise comparisonusing Studentrsquos 119905-test at 119875 values of le0001 le001 and le005designated as lowastlowastlowast lowastlowast and lowast respectively

3 Results and Discussion

31 Composition of T patula Extracts Several flavonoidsall belonging to the group of flavonols (Figure 1(a) (AndashF)) were identified in the marigold extracts Compound2 was identified as patuletin (6-methoxyquercetin) withprotonated molecule mass [M+H]+ at 119898119911 333 and com-pound 3 as quercetagetin (6-hydroxyquercetin) [M+H]+ at119898119911 319 Quercetagetin prevailed in fraction 5 In additionthis fraction contained quercetin glucosylated derivativesof quercetin and quercetagetin and diglucoside of querc-etagetin with protonated molecules at119898119911 303 465 481 and643 respectively Such composition of flavonoids is typicalto T patula [36] Compound 1 was identified on the basis ofits spectral characteristics as carotenoid lutein (Figure 1(b))known to be the major carotenoid in marigold flowers [37]Purified compounds lutein and patulin and the flavonoidfraction rich in quercetagetin and quercetin as well as crudemarigold extracts were used for further trials with Jurkatcells


O

O

OH

OH

OH

HO

OH

O

O

OH

OH

OH

HO

OH

HO

O

O

OH

OH

OH

HO

OH

O

O

OH

OH

HO

OH

OGlu

O

O

OH

OH

OH

OGlu

GluO

HO

O

O

OH

OH

OH

OH

GluO

HO

(A)

(E)(D)

(C)(B)

(F)

H3CO

(a)

OH

HO

H3CH3C

H3C CH3CH3CH3CH3

CH3CH3 CH3

(b)

Figure 1 The constituents of Tagetes patula (a) Flavonols (A) quercetin (B) quercetagetin (C) patuletin (D) quercetin-3-glucoside (E)quercetagetin-7-glucoside (F) quercetagetin-37-diglucoside (b) Lutein

32 Effects on Jurkat Cell Viability Addingmarigold fractionsto Jurkat cultures in the absence of exogenous H

2O2resulted

just in moderate changes in MTT test results not exceeding20 of the nontreated cells level Flavonoid fraction richin quercetagetin and quercetin caused certain increase inapparent cell viability above the control level due to theenhancement of dehydrogenase activity andor cell prolif-eration (Figure 2) On the other hand slight decline of thecell viability was observed in the presence of patuletin inagreement with the previous observation of Woerdenbag etal [19] who reported moderate to low cytotoxicity of thiscompound towards nonchallenged cells

H2O2-induced oxidative stress reduced the viability of

Jurkat cells in a dose-dependent manner This hydrogenperoxide effect was alleviated by application of variousT patula fractions The most efficient protection againstH2O2damage was rendered to the Jurkat cells by the

quercetagetinquercetin flavonoid fraction The superiorityof quercetin over other phenolic compounds in protectingJurkat cells against H

2O2-induced cell death was earlier

shown by Zhang et al [38] Purified patuletin in the concen-tration of 2mgmLminus1 showed no cytoprotective activity andeven aggravated the cytotoxic effect of H

2O2 However at

low concentration of 40120583gmLminus1 patuletin did reveal certaincytoprotective activity against 25 120583M H

2O2 increasing the

cell viability from 50 to almost 70 A similar observationwas reported by Woerdenbag et al [19] showing that mod-erate concentrations of flavonols protected the cells against

0

30

60

90

120

MTT

test

resu

lts (

of c

ontro

l)

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

Que

rcet

aget

in

quer

cetin

frac

tion

Patu

letin

004

mg

mL

Patu

letin

2

mg

mL

02550

H2O2 120583M

lowastlowastlowast

lowastlowastlowast

lowast lowastlowast lowast

lowastlowastlowast

lowastlowast

lowast

lowastlowast

lowast lowast

lowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

Figure 2 Effects of French marigold extracts and purified fractionson the results of MTT cell viability test ( of the nontreated con-trol) Error bars represent standard deviations of five replicationsValues marked with asterisks were significantly different from thenontreated control according to Studentrsquos 119905-test at119875 values ofle0001le001 and le005 designated as lowastlowastlowast lowastlowast and lowast respectively

the helenalin cytotoxicity while in higher doses patuletin(and to a much lesser extent other flavonoids studied) turned


0

1

2

3EP

R sig

nal i

nten

sity

(arb

itrar

y un

its)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M

lowastlowast lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowast

(a) Superoxide radicalO2minus

0

1

2

3

4

EPR

signa

l int

ensit

y (a

rbitr

ary

units

)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M

lowast


lowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

(b) Peroxyl radical LOO∙

Figure 3 Effects of French marigold extracts and purified fractions on the generation of superoxide (a) and peroxyl (b) radicals in Jurkatcells subjected to hydrogen peroxide-induced oxidative stress (EPR signal intensity arbitrary units) Error bars represent standard deviationsof five replications Values marked with asterisks were significantly different from the control subjected to the same H

2O2concentration

according to Studentrsquos 119905-test at 119875 values of le0001 le001 and le005 designated as lowastlowastlowast lowastlowast and lowast respectively

cytotoxic by themselves The combination of antioxidantand radical-scavenging activity of patuletin on one handwith its cytotoxic effect on the other one was described byKashif et al [17] in relation to its anticancer potential Dueto these findings patuletin was applied in further trials inconcentration of 40120583gmLminus1

33 H2O2-Induced ROS Generation and Antioxidant Activity

of T patula Extracts The addition of 25 or 50120583M of hydro-gen peroxide caused generation of superoxide and peroxylradicals in the Jurkat culture evidenced by EPR spectroscopy(Figure 3) most probably through themechanisms describedby Petlicki and Van De Ven [25] No radicals were detected inthe absence of hydrogen peroxide (data not shown) AddingT patula fractions significantly reduced the level of super-oxide and peroxyl radicals in the H

2O2-challenged Jurkat

cultures with purified patuletin being the most efficientradical scavenger

The radical-scavenging capacity of T patula extract wasconfirmed by theORACassay (Figure 4) Figure 4 exemplifiesthe protective effect of T patula extract in comparisonwith a standard antioxidant Trolox against fluorescent probedegradation caused by AAPH-generated peroxyl radicalsThe ORAC value of the dried marigold petals was calculatedas 1779 plusmn 28 120583M Trolox equivalent gminus1 comparable withpotent antioxidant spices such as paprika black pepper andcurry [39]

Figure 5 presents the effect of marigold fractions on theactivity of antioxidant enzymes superoxide dismutase (SOD)catalase and glutathione reductase (GR) in the Jurkat cellsThe activities of the three enzymes in the H

2O2-challenged

cells were enhanced by addition of patuletin fraction The

0

02

04

06

08

1

0 10 20 30 40 50

Nor

mal

ized

fluo

resc

ence

Incubation time (min)T patula extract concentration

06 120583gmL03 120583gmL003 120583gmL

0120583gmLTrolox 1120583M

Figure 4 ORAC assay representative fluorescence decay curves offluorescein in the presence of different marigold extract concentra-tions and of the standard antioxidant Trolox

most significant increase was observed with superoxide dis-mutase whose activity more than doubled in the presence ofpatuletin In addition the activity of catalase was stimulatedby lutein and quercetagetinquercetin fractions

Thus themarigold extracts could eliminate ROS and alle-viate the oxidative stress in H

2O2-challenged Jurkat cultures

through two mechanisms (a) nonenzymatic scavenging offree radicals as revealed in ORAC assay and (b) stimulatingthe activity of ROS-neutralizing antioxidant enzymes such asSOD and catalase


0

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50SO

D (u

nits

mg

prot

ein)

lowast

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H2O2

patuletinlutein flavonoidfraction

H2O2 +H2O2 +H2O2 +

(a)

0

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Cata

lase

(uni

tsm

g pr

otei

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patuletinH2O2 +

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H2O2 +

luteinH2O2 +

(b)

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(uni

tsm

g pr

otei

n)

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patuletinH2O2 +

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H2O2 +

luteinH2O2 +

(c)

Figure 5 Effect of hydrogen peroxide and French marigold extract fractions on the activity of antioxidant enzymes in Jurkat T-cells (a)Superoxide dismutase (SOD) (b) catalase (c) glutathione reductase (GR) Error bars represent standard deviations of five replications Valuesmarked with asterisks were significantly different from the nontreated control in the same series according to Studentrsquos 119905-test at 119875 values ofle001 and le005 designated as lowastlowast and lowast respectively

0

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Cell

cycle

pha

ses (

)

lowastlowast

lowast


H2O2 25120583MT patula extract

SG0G1 G2M

G0apoptosis

H2O2 +

Figure 6 Effects of hydrogen peroxide and of the ethanolic French marigold extract on the percentage of cell cycle phase distributions ofJurkat cells (results of flow cytometry of propidium iodide-stained cell populations) Values marked with asterisks were significantly differentfrom the respective cell cycle phase percentage in the nontreated control according to Studentrsquos 119905-test at119875 values of le001 and le005 designatedas lowastlowast and lowast respectively


(a) (b) (c) (d)

Figure 7 Transmission electron microscopy images of the Jurkat cells (a) normal cell morphology (b) and (c) early apoptosis stages inH2O2-challenged cells representing chromatin condensation and cytoplasmic vacuolization (d) late apoptosis stage in H

2O2-challenged cell

representing nuclear fragmentation

0

20

40

60

80

100

Apop

totic

ratio

()

H2O2 25120583MNoadditives(control)

lowastlowast

lowastlowastlowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

Figure 8 Effect of hydrogen peroxide and French marigoldfractions on the percentage of apoptotic cells (apoptotic ratio)in the Jurkat cell population (results of flow cytometry basedon mitochondrial transmembrane potential measurement usinga DiOC

6staining) Error bars represent standard deviations of

five replications Values marked with asterisks were significantlydifferent from the nontreated control according to Studentrsquos 119905-test at119875 values of le0001 and le001 designated as lowastlowastlowast and lowastlowast respectively

34 Effects on the Cell Cycle The H2O2-caused oxidative

stress changed the cell cycle phase distribution restrictingDNA replication (phase S) and increasing the relative pro-portions of G

0G1cells (the G

0G1arrest) and apoptotic cells

(Figure 6) The characteristic apoptotic changes (chromatincondensation nuclear fragmentation and cytoplasmic vac-uolization) in typicalH

2O2-exposed Jurkat cells are presented

in transmission electron microscope images (Figure 7)Adding the T patula extract to the H

2O2-challenged cells

largely normalized their cell cycle (Figure 6) Similar trendswere revealed by evaluating the percentage of apoptotic cellsin the population (the apoptotic ratio) by flow cytometry

on the basis of mitochondrial transmembrane potentialmeasured by DiOC

6test An upsurge in the apoptotic ratio

was induced by hydrogen peroxide alone while this increasewas counteracted by coadministering the T patula fractionscontaining patuletin quercetagetinquercetin or lutein (Fig-ure 8) Certain discrepancy in the proportion of apoptoticcells was evident between the two flow cytometry methodsmost probably due to the different separation criteria usedSimilarly Ozgen et al [40] reported that in the T-cellsthe DiOC

6technique gave higher estimation of apoptotic

cell population as compared to the propidium iodide-basedmethod

35 Interleukin-10 (IL-10) Production Anti-inflammatoryand antioxidant interleukin-10 (IL-10) increased in the Jurkatcells subjected toH

2O2challenge most probably as a part of a

defense mechanism against oxidative stress Similar responsewas observed in cultured human keratinocytes exposed toultraviolet irradiation [41] Flavonoid fractions of T patulafurther enhanced the IL-10 level in the H

2O2-challenged

Jurkat cells (Figure 9) similar to our previous work withSatureja hortensis [42] The IL-10 is known to inhibit theapoptotic death of T-cells presumably through upregulationof Bcl-2 [26]This mechanismmight underlay the alleviationof apoptosis observed in our study On a whole organismlevel the diet rich in phenolic antioxidants enhanced theIL-10 production in the animals enduring proinflammatoryconditions resulting in a decrease in proinflammatory fac-tors inhibited lipid peroxidation increased HDL levels andalleviated inflammation [43]

4 Conclusions

The present research has demonstrated for the first timethat both flavonoid and carotenoid constituents of Frenchmarigold (Tagetes patula L) extract can protect Jurkat cellsfrom hydrogen peroxide caused oxidative stress Both direct


0

3

6

9

12

15

H2O2Noadditives(control)

lowastlowast

lowastlowast

lowast

IL-10(p

gmLminus

1)

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

Figure 9 Effect of hydrogen peroxide and French marigold frac-tions on the level of interleukin-10 (IL-10) in the Jurkat cellsError bars represent standard deviations of five replications Valuesmarked with asterisks were significantly different from the non-treated control according to Studentrsquos 119905-test at 119875 values of le001 andle005 designated as lowastlowast and lowast respectively

radical-scavenging effects and stimulation of the cellularantioxidant enzymes and anti-inflammatory factors suchas IL-10 can be involved in these protective mechanismsThe findings are in line with the antioxidant and anti-inflammatory properties of marigold preparations used infolk medicine and confirmed in animal and human studiesAt the same time it was found that some T patula flavonoidsprimarily patuletin can exert cytotoxic effect on Jurkat cellsassociated with its anticancer potential The shift betweencytoprotective and cytotoxic activity depends on concentra-tion and chemical nature of the compound

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors are grateful to Dr Mira Weissberg (Departmentof Plant Science ARO) for running the LC-MS analysis andto Professor Karen Schaich (Department of Food ScienceRutgers University USA) for the guidance on performingthe ORAC assay The research was partially supported bythe Chief Scientist Grant 430-0593 of the Israeli Ministry ofAgriculture and Rural Development

References

[1] L Kaplan ldquoHistorical and Ethnobotanical aspects of domesti-cation in tagetesrdquo Economic Botany vol 14 no 3 pp 200ndash2021960

[2] J L Sorenson and C L Johannessen ldquoScientific evidence forpre-Columbian transoceanic voyages to and from the Amer-icasrdquo Sino-Platonic Papers 133 University of PennsylvaniaPhiladelphia Pa USA 2004

[3] M Akhalkatsi J Ekhvaia and Z Asanidze ldquoDiversity andgenetic erosion of ancient crops and wild relatives of agricul-tural cultivars for food implications for nature conservation inGeorgia (Caucasus)rdquo in Perspectives on Nature ConservationmdashPatterns Pressures and Prospects J Tiefenbacher Ed chapter 3pp 51ndash92 InTech Rijeka Croatia 2012

[4] R K Beridze P Hanelt V N Kandelaki D Mandzgaladzeand J Schultze-Motel ldquoCollecting plant-genetic resources in theGeorgian SSR (Kartli Meskheti) 1989rdquo Die Kulturpflanze vol38 no 3 pp 157ndash171 1990

[5] M Esaiashvili N Gogia T Meladze I Chkhikvishvili ASephashvili and M Gongadze ldquoThe effect of marigold extracton experimental diabetes mellitusrdquo Experimental and ClinicalMedicine vol 2011 no 1 pp 44ndash47 2011

[6] R TNeher ldquoThe ethnobotany ofTagetesrdquoEconomic Botany vol22 no 4 pp 317ndash325 1968

[7] P Vasudevan S Kashyap and S Sharma ldquoTagetes a multipur-pose plantrdquo Bioresource Technology vol 62 no 1-2 pp 29ndash351997

[8] B O de Montellano ldquoAztec medicinal herbs evaluation oftherapeutic effectivenessrdquo in Plants in Indigenous Medicine andDiet-Biobehavioral Approaches pp 113ndash127 Redgrave Publish-ing Company Bedford NY USA 1986

[9] G S Yonzone and D K N Yonzone ldquoEthnobotany of Dar-jeeling Himalaya Indiardquo in ISHS Acta Horticulturae 500 IIWOCMAP Congress Medicinal and Aromatic Plants Part 1Biological Resources Sustainable Use Conservation and Ethnob-otany vol 500 pp 209ndash213 1999

[10] Y Kasahara K Yasukawa S Kitanaka M T Khan and FJ Evans ldquoEffect of methanol extract from flower petals ofTagetes patula L on acute and chronic inflammation modelrdquoPhytotherapy Research vol 16 no 3 pp 217ndash222 2002

[11] N V Shinde K G Kanase V C Shilimkar V R Undale andAV Bhosale ldquoAntinociceptive and anti-inflammatory effects ofsolvent extracts of Tagetes erectus Linn (Asteraceae)rdquo TropicalJournal of Pharmaceutical Research vol 8 no 4 pp 325ndash3292009

[12] M T Khan ldquoThe podiatric treatment of hallux abducto valgusand its associated condition bunion with Tagetes patulardquoJournal of Pharmacy and Pharmacology vol 48 no 7 pp 768ndash770 1996

[13] K Yasukawa and Y Kasahara ldquoEffects of flavonoids fromFrench Marigold (Florets of Tagetes patula L) on acute inflam-mationmodelrdquo International Journal of Inflammation vol 2013Article ID 309493 5 pages 2013

[14] S-Y Li F K C Fung Z J Fu D Wong H H L Chanand A C Y Lo ldquoAnti-inflammatory effects of lutein in retinalischemichypoxic injury in vivo and in vitro studiesrdquo Investiga-tive Ophthalmology and Visual Science vol 53 no 10 pp 5976ndash5984 2012

[15] P Karimian G Kavoosi and Z Amirghofran ldquoAnti-oxidativeand anti-inflammatory effects of Tagetes minuta essential oilin activated macrophagesrdquo Asian Pacific Journal of TropicalBiomedicine vol 4 no 3 pp 219ndash227 2014

[16] V Rodov Y Vinokur N Gogia and I ChkhikvishvilildquoHydrophilic and lipophilic antioxidant capacities of Georgianspices formeat and their possible health implicationsrdquoGeorgianMedical News vol 179 pp 61ndash66 2010


[17] M Kashif S Bano S Naqvi et al ldquoCytotoxic and antioxidantproperties of phenolic compounds from Tagetes patula flowerrdquoPharmaceutical Biology vol 53 no 5 pp 672ndash681 2015

[18] O Vallisuta V Nukoolkarn A Mitrevej et al ldquoIn vitro studieson the cytotoxicity and elastase and tyrosinase inhibitory activi-ties ofmarigold (Tagetes erectaL) flower extractsrdquoExperimentaland Therapeutic Medicine vol 7 no 1 pp 246ndash250 2013

[19] H J Woerdenbag I Merfort T J Schmidt et al ldquoDecreasedhelenalin-induced cytotoxicity by flavonoids from Arnica asstudied in a human lung carcinoma cell linerdquo Phytomedicinevol 2 no 2 pp 127ndash132 1995

[20] M A Mesaik A Jabeen S Faizi S U Simjee and S BanoldquoPatuletin a potent anti-TNF-120572 and anti-arthritic compoundfrom Tagetes patulardquo US Patent Application 13944387 filedJuly 17 2013

[21] B P Chew MWWong and T S Wong ldquoEffects of lutein frommarigold extract on immunity and growth ofmammary tumorsinmicerdquoAnticancer Research vol 16 no 6 pp 3689ndash3694 1996

[22] R T Abraham and A Weiss ldquoJurkat T cells and developmentof the T-cell receptor signalling paradigmrdquo Nature ReviewsImmunology vol 4 no 4 pp 301ndash308 2004

[23] G Nindl N R Peterson E F Hughes L R Waite and M TJohnson ldquoEffect of hydrogen peroxide on proliferation apop-tosis and interleukin-2 production of Jurkat T cellsrdquo BiomedicalSciences Instrumentation vol 40 pp 123ndash128 2004

[24] M Reth ldquoHydrogen peroxide as second messenger in lympho-cyte activationrdquoNature Immunology vol 3 no 12 pp 1129ndash11342002

[25] J Petlicki and T G M Van De Ven ldquoThe equilibrium betweenthe oxidation of hydrogen peroxide by oxygen and the dismu-tation of peroxyl or superoxide radicals in aqueous solutions incontact with oxygenrdquo Journal of the Chemical Society - FaradayTransactions vol 94 no 18 pp 2763ndash2767 1998

[26] J J Haddad and C S Fahlman ldquoRedox- and oxidant-mediatedregulation of interleukin-10 an anti-inflammatory antioxidantcytokinerdquo Biochemical and Biophysical Research Communica-tions vol 297 no 2 pp 163ndash176 2002

[27] R S DiPaola ldquoTo arrest or not to G2-M cell-cycle arrestrdquoClinical Cancer Research vol 8 no 11 pp 3311ndash3314 2002

[28] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[29] R Chiaramonte E Bartolini P Riso et al ldquoOxidative stresssignalling in the apoptosis of Jurkat T-lymphocytesrdquo Journal ofCellular Biochemistry vol 82 no 3 pp 437ndash444 2001

[30] Y Saito K Nishio Y Ogawa et al ldquoTurning point in apop-tosisnecrosis induced by hydrogen peroxiderdquo Free RadicalResearch vol 40 no 6 pp 619ndash630 2006

[31] D M L Morgan ldquoTetrazolium (MTT) assay for cellularviability and activityrdquo in Polyamine Protocols D Morgan Edpp 179ndash184 Humana Press Totowa NJ USA 1998

[32] A Krishan ldquoRapid flow cytofluorometric analysis of mam-malian cell cycle by propidium iodide stainingrdquo The Journal ofCell Biology vol 66 no 1 pp 188ndash193 1975

[33] N Zamzami S A Susin P Marchetti et al ldquoMitochondrialcontrol of nuclear apoptosisrdquo Journal of Experimental Medicinevol 183 no 4 pp 1533ndash1544 1996

[34] Y Vinokur and V Rodov ldquoMethod for determining total(hydrophilic and lipophilic) radical-scavenging activity in thesame sample of fresh producerdquo Acta Horticulturae vol 709 pp53ndash60 2006

[35] K M Gillespie J M Chae and E A Ainsworth ldquoRapidmeasurement of total antioxidant capacity in plantsrdquo NatureProtocols vol 2 no 4 pp 867ndash870 2007

[36] P Guinot A Gargadennec G Valette A Fruchier and CAndary ldquoPrimary flavonoids inmarigold dye extraction struc-ture and involvement in the dyeing processrdquo PhytochemicalAnalysis vol 19 no 1 pp 46ndash51 2008

[37] S Bhattacharyya S Datta B Mallick P Dhar and S GhoshldquoLutein content and in vitro antioxidant activity of differentcultivars of Indian marigold flower (Tagetes patula L) extractsrdquoJournal of Agricultural and Food Chemistry vol 58 no 14 pp8259ndash8264 2010

[38] J Zhang R A Stanley A Adaim L D Melton andM A Skin-ner ldquoFree radical scavenging and cytoprotective activities ofphenolic antioxidantsrdquoMolecular Nutrition and Food Researchvol 50 no 11 pp 996ndash1005 2006

[39] D BHaytowitz and S Bhagwat ldquoUSDAdatabase for the oxygenradical absorbance capacity (ORAC) of selected foodsrdquo Release2 US Department of Agriculture 2010

[40] U Ozgen S Savasan S Buck and Y Ravindranath ldquoCom-parison of DiOC6(3) uptake and annexin V labeling for quan-tification of apoptosis in leukemia cells and non-malignant Tlymphocytes from childrenrdquo Cytometry vol 42 no 1 pp 74ndash78 2000

[41] MGrewe KGyufko and J Krutmann ldquoInterleukin-10 produc-tion by cultured human keratinocytes regulation by ultravioletB and ultraviolet A1 radiationrdquo Journal of Investigative Derma-tology vol 104 no 1 pp 3ndash6 1995

[42] I Chkhikvishvili T Sanikidze N Gogia et al ldquoRosmarinicacid-rich extracts of summer savory (Satureja hortensis L) pro-tect Jurkat T cells against oxidative stressrdquo Oxidative Medicineand Cellular Longevity vol 2013 Article ID 456253 9 pages2013

[43] M J Kim J Ohn J H Kim and H-K Kwak ldquoEffects of freeze-dried cranberry powder on serum lipids and inflammatorymarkers in lipopolysaccharide-treated rats fed an atherogenicdietrdquo Nutrition Research and Practice vol 5 no 5 pp 404ndash4112011

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


patulitrin [13] Lipophilic ingredients of marigold flowersthe carotenoid lutein and essential oil compounds were alsoreported to possess anti-inflammatory properties [14 15] Inour previous study both hydrophilic and lipophilic fractionsfrom T patula petals showed the highest radical-scavengingcapacities among all Georgian spices tested [16]

However the cellular mechanisms by which the marigoldextracts exert their anti-inflammatory effects are not fullyunderstood and demand further investigation Methanolextracts of T patula flowers as well as isolated patuletinwere reported to scavenge peroxyl and superoxide radicals inchemical systems and in human neutrophils and at the sametime to exert cytotoxic and growth inhibitory effects towardsa range of human cancer cell lines in particular HeLa cells[17] On the other hand ethanolic and ethyl acetate extracts ofmarigold flowers showed no cytotoxicity towards H460 lungcancer and theCaco-2 colon cancer cell lines in anMTT assay[18] Furthermore the MTT assay revealed a cytoprotectiveeffect of patuletin on the human lung carcinoma GLC

4cell

line challenged by cytotoxic sesquiterpene lactone helenalin[19] Activation of antioxidant enzymes rather than direct freeradical scavenging was suggested as a possible mechanismunderlying this phenomenon Mesaik et al [20] demon-strated that immunomodulatory and antiarthritic potentialof patuletin was associated with inhibited production of theproinflammatory cytokine TNF-120572 with no cytotoxic prop-erty Chew et al [21] reported that marigold-derived dietarylutein enhanced phytohemagglutinin-induced lymphocyteproliferation in mice but had no effect on interleukin-2production or lymphocyte cytotoxicity

Human lymphoblastoid T-cell Jurkat line is a popularmodel for the study of immune signaling and inflammation[22] Jurkat cells can imitate both healthy and inflammatoryT-cells in their response to oxidative metabolites such ashydrogen peroxide [23] Although H

2O2plays an impor-

tant role in antigen-dependent lymphocyte activation [24]excessive production of H

2O2induces oxidative stress and

impairs T-cell activity leading to chronic inflammation andcell death In the presence of oxygen in aqueous mediumhydrogen peroxide can produce additional cytotoxic reactiveoxygen species (ROS) such as superoxide and peroxyl radi-cals [25] To control the level of ROS cells employ antioxidantenzymes for example catalase that decomposes hydrogenperoxide and superoxide dismutase neutralizing superoxideradicals as well as low-molecular antioxidants The lattergroup includes internally produced glutathione and dietaryantioxidants such as ascorbic acid and phenolic compoundsThe function of the antioxidant system ismaintained by addi-tional enzymes such as glutathione reductase that restoresthe antioxidant capacity of oxidized glutathione Signalingfor regulation of oxidative stress and inflammatory responsesinvolve cytokines such as anti-inflammatory and antioxidantinterleukin-10 (IL-10) [26]

Theoxidative stress can interferewith normal progressionof cell growth and division arranged in a cell cycle In eukary-otes a normal cell cycle consists of four main stages G

1

during which a cell is metabolically active and continuouslygrows S phase during which DNA replication takes placeG2 during which the growth of cell continues and the cell

prepares for division and the M (mitosis) phase duringwhich the cell divides into two daughter cells each with afull copy of DNA After the M phase the cells can enter G

1

or G0 a quiescent phase When the cell detects any defects

(eg oxidative DNA damage) which necessitate halting thecell cycle inG

1 cell cycle arrest occurs Efforts to correct these

problems may slow growth and induce cell death [27]The response of Jurkat cells to H

2O2is dose-dependent

Reversible oxidative changes that could be repaired by cellularantioxidant systems occurred at a H

2O2concentration of

20120583M while the signs of apoptosis (programmed cell death)were noted at 50120583MH

2O2[28] Both apoptosis and necrosis

(a nonprogrammed cell death caused by damage) wereobserved in the Jurkat cells exposed to 100 120583M H

2O2[29]

whereas the necrosis prevailed at 500 120583MH2O2[30]

The balance between prooxidant and antioxidant repairmechanisms determining cellular survival and functioncan be affected by dietary bioactive compounds possessingradical-scavenging and anti-inflammatory activity There-fore in the present work we investigated the effects of anti-inflammatory T patula flower extract and of its purifiedfractions on the behavior of theH

2O2-challenged Jurkat cells

2 Materials and Methods

21 Plant Material The flowers were collected from theplants of a local Georgian landrace of T patula grown atexperimental plot near Tbilisi from seeds purchased from acommercial supplier The collected flowers were air-dried inthe shade at 25ndash30∘CThe dried matter was stored in a closedglass container in a cool dry place

22 Extraction and Purification ofMarigold Constituents Theisolation was performed by sequential solvent extraction ofT patula flowers A sample of 600 g of dried pulverizedplant material was extracted with 12-dichloroethane in aSoxhlet apparatus for 48 h until color loss The residue afterthe dichloroethane extraction was reextracted with ethanol(solventplant matter ratio 1 5) for isolation of compoundsof higher polarity The solvents were evaporated undervacuum at 40∘C giving dichloroethane and ethanol crudeextracts Further separation of individual compounds fromthe dichloroethane extract was performed by column chro-matography on silica gel column with chloroform-hexanesolvent system The elution of fractions from the columnwas started with hexane with further increase of chloroformcontent in the system The elution with 3 chloroform inhexane gave compound 1 Compound 2 was present in thefraction eluted from the column with 5 chloroform inhexane

The ethanolic extract was separated on a silica gel col-umn by elution with dichloroethanemethanol using thin-layer chromatography (TLC) for preliminary characteriza-tion of fractionsThe elution was started with dichloroethanewith subsequent stepwise increase of methanol content inthe system Elution with 2 3 5 7 and 10 methanolin dichloroethane produced fractions 1 2 3 4 and 5respectively Rechromatography of fraction 2 on a Sephadex










2O2(Sigma)


2O2













0G1phase


2M phase The



6) [33] In order to




6were 488 and




2 10mM KCl 1mM


2O2at


2O2per









O

O

OH

OH

OH

HO

OH

O

O

OH

OH

OH

HO

OH

HO

O

O

OH

OH

OH

HO

OH

O

O

OH

OH

HO

OH

OGlu

O

O

OH

OH

OH

OGlu

GluO

HO

O

O

OH

OH

OH

OH

GluO

HO

(A)

(E)(D)

(C)(B)

(F)

H3CO

(a)

OH

HO

H3CH3C

H3C CH3CH3CH3CH3

CH3CH3 CH3

(b)



2O2resulted







2O2 However at


2O2 increasing the


0

30

60

90

120

MTT

test

resu

lts (

of c

ontro

l)

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

Que

rcet

aget

in

quer

cetin

frac

tion

Patu

letin

004

mg

mL

Patu

letin

2

mg

mL

02550

H2O2 120583M

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowast

lowastlowast

lowast lowast

lowastlowast

lowastlowastlowast





0

1

2

3EP

R sig

nal i

nten

sity

(arb

itrar

y un

its)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M



lowastlowastlowast


lowastlowastlowast


0

1

2

3

4

EPR

signa

l int

ensit

y (a

rbitr

ary

units

)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M

lowast


lowastlowast




2O2concentration









2O2-challenged


0

02

04

06

08

1

0 10 20 30 40 50

Nor

mal

ized

fluo

resc

ence


06 120583gmL03 120583gmL003 120583gmL








0

10

20

30

40

50SO

D (u

nits

mg

prot

ein)

lowast

lowastlowast


H2O2


H2O2 +H2O2 +H2O2 +

(a)

0

10

20

30

40

50

60

Cata

lase

(uni

tsm

g pr

otei



H2O2

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(b)

0

100

200

300

400


GR

(uni

tsm

g pr

otei

n)

H2O2

lowast

lowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(c)


0

20

40

60


Cell

cycle

pha

ses (

)

lowastlowast

lowast



SG0G1 G2M

G0apoptosis

H2O2 +



(a) (b) (c) (d)


2O2-challenged cell


0

20

40

60

80

100

Apop

totic

ratio

()


lowastlowast

lowastlowastlowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +






0G1cells (the G















2O2-challenged


4 Conclusions



0

3

6

9

12

15


lowastlowast

lowastlowast

lowast

IL-10(p

gmLminus

1)

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +



Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























2O2(Sigma)


2O2













0G1phase


2M phase The



6) [33] In order to




6were 488 and




2 10mM KCl 1mM


2O2at


2O2per









O

O

OH

OH

OH

HO

OH

O

O

OH

OH

OH

HO

OH

HO

O

O

OH

OH

OH

HO

OH

O

O

OH

OH

HO

OH

OGlu

O

O

OH

OH

OH

OGlu

GluO

HO

O

O

OH

OH

OH

OH

GluO

HO

(A)

(E)(D)

(C)(B)

(F)

H3CO

(a)

OH

HO

H3CH3C

H3C CH3CH3CH3CH3

CH3CH3 CH3

(b)



2O2resulted







2O2 However at


2O2 increasing the


0

30

60

90

120

MTT

test

resu

lts (

of c

ontro

l)

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

Que

rcet

aget

in

quer

cetin

frac

tion

Patu

letin

004

mg

mL

Patu

letin

2

mg

mL

02550

H2O2 120583M

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowast

lowastlowast

lowast lowast

lowastlowast

lowastlowastlowast





0

1

2

3EP

R sig

nal i

nten

sity

(arb

itrar

y un

its)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M



lowastlowastlowast


lowastlowastlowast


0

1

2

3

4

EPR

signa

l int

ensit

y (a

rbitr

ary

units

)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M

lowast


lowastlowast




2O2concentration









2O2-challenged


0

02

04

06

08

1

0 10 20 30 40 50

Nor

mal

ized

fluo

resc

ence


06 120583gmL03 120583gmL003 120583gmL








0

10

20

30

40

50SO

D (u

nits

mg

prot

ein)

lowast

lowastlowast


H2O2


H2O2 +H2O2 +H2O2 +

(a)

0

10

20

30

40

50

60

Cata

lase

(uni

tsm

g pr

otei



H2O2

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(b)

0

100

200

300

400


GR

(uni

tsm

g pr

otei

n)

H2O2

lowast

lowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(c)


0

20

40

60


Cell

cycle

pha

ses (

)

lowastlowast

lowast



SG0G1 G2M

G0apoptosis

H2O2 +



(a) (b) (c) (d)


2O2-challenged cell


0

20

40

60

80

100

Apop

totic

ratio

()


lowastlowast

lowastlowastlowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +






0G1cells (the G















2O2-challenged


4 Conclusions



0

3

6

9

12

15


lowastlowast

lowastlowast

lowast

IL-10(p

gmLminus

1)

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +



Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0G1phase


2M phase The



6) [33] In order to




6were 488 and




2 10mM KCl 1mM


2O2at


2O2per









O

O

OH

OH

OH

HO

OH

O

O

OH

OH

OH

HO

OH

HO

O

O

OH

OH

OH

HO

OH

O

O

OH

OH

HO

OH

OGlu

O

O

OH

OH

OH

OGlu

GluO

HO

O

O

OH

OH

OH

OH

GluO

HO

(A)

(E)(D)

(C)(B)

(F)

H3CO

(a)

OH

HO

H3CH3C

H3C CH3CH3CH3CH3

CH3CH3 CH3

(b)



2O2resulted







2O2 However at


2O2 increasing the


0

30

60

90

120

MTT

test

resu

lts (

of c

ontro

l)

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

Que

rcet

aget

in

quer

cetin

frac

tion

Patu

letin

004

mg

mL

Patu

letin

2

mg

mL

02550

H2O2 120583M

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowast

lowastlowast

lowast lowast

lowastlowast

lowastlowastlowast





0

1

2

3EP

R sig

nal i

nten

sity

(arb

itrar

y un

its)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M



lowastlowastlowast


lowastlowastlowast


0

1

2

3

4

EPR

signa

l int

ensit

y (a

rbitr

ary

units

)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M

lowast


lowastlowast




2O2concentration









2O2-challenged


0

02

04

06

08

1

0 10 20 30 40 50

Nor

mal

ized

fluo

resc

ence


06 120583gmL03 120583gmL003 120583gmL








0

10

20

30

40

50SO

D (u

nits

mg

prot

ein)

lowast

lowastlowast


H2O2


H2O2 +H2O2 +H2O2 +

(a)

0

10

20

30

40

50

60

Cata

lase

(uni

tsm

g pr

otei



H2O2

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(b)

0

100

200

300

400


GR

(uni

tsm

g pr

otei

n)

H2O2

lowast

lowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(c)


0

20

40

60


Cell

cycle

pha

ses (

)

lowastlowast

lowast



SG0G1 G2M

G0apoptosis

H2O2 +



(a) (b) (c) (d)


2O2-challenged cell


0

20

40

60

80

100

Apop

totic

ratio

()


lowastlowast

lowastlowastlowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +






0G1cells (the G















2O2-challenged


4 Conclusions



0

3

6

9

12

15


lowastlowast

lowastlowast

lowast

IL-10(p

gmLminus

1)

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +



Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

O

OH

OH

OH

HO

OH

O

O

OH

OH

OH

HO

OH

HO

O

O

OH

OH

OH

HO

OH

O

O

OH

OH

HO

OH

OGlu

O

O

OH

OH

OH

OGlu

GluO

HO

O

O

OH

OH

OH

OH

GluO

HO

(A)

(E)(D)

(C)(B)

(F)

H3CO

(a)

OH

HO

H3CH3C

H3C CH3CH3CH3CH3

CH3CH3 CH3

(b)



2O2resulted







2O2 However at


2O2 increasing the


0

30

60

90

120

MTT

test

resu

lts (

of c

ontro

l)

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

Que

rcet

aget

in

quer

cetin

frac

tion

Patu

letin

004

mg

mL

Patu

letin

2

mg

mL

02550

H2O2 120583M

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowast

lowastlowast

lowast lowast

lowastlowast

lowastlowastlowast





0

1

2

3EP

R sig

nal i

nten

sity

(arb

itrar

y un

its)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M



lowastlowastlowast


lowastlowastlowast


0

1

2

3

4

EPR

signa

l int

ensit

y (a

rbitr

ary

units

)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M

lowast


lowastlowast




2O2concentration









2O2-challenged


0

02

04

06

08

1

0 10 20 30 40 50

Nor

mal

ized

fluo

resc

ence


06 120583gmL03 120583gmL003 120583gmL








0

10

20

30

40

50SO

D (u

nits

mg

prot

ein)

lowast

lowastlowast


H2O2


H2O2 +H2O2 +H2O2 +

(a)

0

10

20

30

40

50

60

Cata

lase

(uni

tsm

g pr

otei



H2O2

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(b)

0

100

200

300

400


GR

(uni

tsm

g pr

otei

n)

H2O2

lowast

lowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(c)


0

20

40

60


Cell

cycle

pha

ses (

)

lowastlowast

lowast



SG0G1 G2M

G0apoptosis

H2O2 +



(a) (b) (c) (d)


2O2-challenged cell


0

20

40

60

80

100

Apop

totic

ratio

()


lowastlowast

lowastlowastlowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +






0G1cells (the G















2O2-challenged


4 Conclusions



0

3

6

9

12

15


lowastlowast

lowastlowast

lowast

IL-10(p

gmLminus

1)

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +



Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3EP

R sig

nal i

nten

sity

(arb

itrar

y un

its)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M



lowastlowastlowast


lowastlowastlowast


0

1

2

3

4

EPR

signa

l int

ensit

y (a

rbitr

ary

units

)

Flav

onoi

dfr

actio

n

Patu

letin

No

addi

tives

(con

trol)

Chlo

rofo

rmex

trac

t

Etha

nolic

extr

act

Lute

in

2550

H2O2 120583M

lowast


lowastlowast




2O2concentration









2O2-challenged


0

02

04

06

08

1

0 10 20 30 40 50

Nor

mal

ized

fluo

resc

ence


06 120583gmL03 120583gmL003 120583gmL








0

10

20

30

40

50SO

D (u

nits

mg

prot

ein)

lowast

lowastlowast


H2O2


H2O2 +H2O2 +H2O2 +

(a)

0

10

20

30

40

50

60

Cata

lase

(uni

tsm

g pr

otei



H2O2

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(b)

0

100

200

300

400


GR

(uni

tsm

g pr

otei

n)

H2O2

lowast

lowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(c)


0

20

40

60


Cell

cycle

pha

ses (

)

lowastlowast

lowast



SG0G1 G2M

G0apoptosis

H2O2 +



(a) (b) (c) (d)


2O2-challenged cell


0

20

40

60

80

100

Apop

totic

ratio

()


lowastlowast

lowastlowastlowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +






0G1cells (the G















2O2-challenged


4 Conclusions



0

3

6

9

12

15


lowastlowast

lowastlowast

lowast

IL-10(p

gmLminus

1)

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +



Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50SO

D (u

nits

mg

prot

ein)

lowast

lowastlowast


H2O2


H2O2 +H2O2 +H2O2 +

(a)

0

10

20

30

40

50

60

Cata

lase

(uni

tsm

g pr

otei



H2O2

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(b)

0

100

200

300

400


GR

(uni

tsm

g pr

otei

n)

H2O2

lowast

lowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +

(c)


0

20

40

60


Cell

cycle

pha

ses (

)

lowastlowast

lowast



SG0G1 G2M

G0apoptosis

H2O2 +



(a) (b) (c) (d)


2O2-challenged cell


0

20

40

60

80

100

Apop

totic

ratio

()


lowastlowast

lowastlowastlowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +






0G1cells (the G















2O2-challenged


4 Conclusions



0

3

6

9

12

15


lowastlowast

lowastlowast

lowast

IL-10(p

gmLminus

1)

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +



Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c) (d)


2O2-challenged cell


0

20

40

60

80

100

Apop

totic

ratio

()


lowastlowast

lowastlowastlowast

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +






0G1cells (the G















2O2-challenged


4 Conclusions



0

3

6

9

12

15


lowastlowast

lowastlowast

lowast

IL-10(p

gmLminus

1)

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +



Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

3

6

9

12

15


lowastlowast

lowastlowast

lowast

IL-10(p

gmLminus

1)

patuletinH2O2 +

flavonoidfraction

H2O2 +

luteinH2O2 +



Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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