Research Article Effect of Myricetin, Pyrogallol, and...

Research ArticleEffect of Myricetin Pyrogallol and Phloroglucinol onYeast Resistance to Oxidative Stress

Vanda Mendes1234 Rita Vilaccedila12 Victor de Freitas4 Pedro Moradas Ferreira123

Nuno Mateus4 and Viacutetor Costa123

1 Instituto de Investigacao e Inovacao em Saude Universidade do Porto Rua Alfredo Allen sn 4200-135 Porto Portugal2IBMC Instituto de Biologia Molecular e Celular Universidade do Porto Rua do Campo Alegre 823 4150-180 Porto Portugal3ICBAS Instituto de Ciencias Biomedicas Abel Salazar Departamento de Biologia Molecular Universidade do PortoRua de Jorge Viterbo Ferreira 228 4050-313 Porto Portugal4REQUIMTELAQV Faculdade de Ciencias da Universidade do Porto Rua do Campo Alegre sn 4169-007 Porto Portugal

Correspondence should be addressed to Vıtor Costa vcostaibmcuppt

Received 18 December 2014 Revised 30 March 2015 Accepted 30 March 2015

Academic Editor Matthew C Zimmerman

Copyright copy 2015 Vanda Mendes 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

The health beneficial effects of dietary polyphenols have been attributed to their intrinsic antioxidant activity which depends on thestructure of the compound and number of hydroxyl groups In this study the protective effects of pyrogallol phloroglucinol andmyricetin on the yeast Saccharomyces cerevisiae were investigated Pyrogallol and myricetin which have a pyrogallol structurein the B ring increased H

2O2resistance associated with a reduction in intracellular oxidation and protein carbonylation

whereas phloroglucinol did not exert protective effects The acquisition of oxidative stress resistance in cells pretreated withpyrogallol and myricetin was not associated with an induction of endogenous antioxidant defences as assessed by the analysis ofsuperoxide dismutase and catalase activitiesHowevermyricetinwhich provided greater stress resistance preventedH

2O2-induced

glutathione oxidation Moreover myricetin increased the chronological lifespan of yeast lacking the mitochondrial superoxidedismutase (Sod2p) which exhibited a premature aging phenotype and oxidative stress sensitivity These findings show that thepresence of hydroxyl groups in the ortho position of the B ring in pyrogallol and myricetin contributes to the antioxidantprotection afforded by these compounds In addition myricetin may alleviate aging-induced oxidative stress particularly whenredox homeostasis is compromised due to downregulation of endogenous defences present in mitochondria

1 Introduction

Oxidative stress is a hallmark of human disorders such ascancer and age-associated diseases [1] It results from anunbalance between the levels of reactive oxygen species(ROS) or reactive nitrogen species (RNS) and cellular antiox-idant defenses The toxicity of high levels of ROS and RNSis associated with the accumulation of damaged moleculesincluding proteins lipids and nucleic acids [1] Under normalphysiological conditions ROS are kept at low levels byantioxidant defenses such as superoxide dismutases (SOD)that catalyze the dismutation of superoxide radicals intohydrogen peroxide catalases or peroxidases that reduceH2O2into water as well as nonenzymatic defenses including

glutathione that plays critical roles in redox homeostasis

and cellular detoxification [2] In addition antioxidantsobtained in the diet such as vitamins C and E and phenoliccompounds play essential role in cellular protection [3]

Phenolic compounds are natural antioxidants present inthe humandiet through the consumption of fruits vegetablesand drinks such as juice tea coffee and wine [4 5] Struc-turally these compounds are characterized by having one ormore hydroxyl groups attached to at least one aromatic ring[4] The number and position of hydroxyl groups are impor-tant features that affect the antioxidant activity of phenoliccompounds [6] These compounds possess antiproliferativeproapoptotic and anti-inflammatory properties and theyhave been associated with the prevention of cancer andcardiovascular neurodegenerative and metabolic disorders[7 8] The protective effects of these compounds have been

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 782504 10 pageshttpdxdoiorg1011552015782504

2 Oxidative Medicine and Cellular Longevity

attributed not only to their intrinsic antioxidant activity butalso to the modulation of cell signaling pathways includingmitogen-activated protein kinase cascades which regulateoxidative stress responses [9ndash11]

Thebudding yeast Saccharomyces cerevisiaehas been usedas a eukaryoticmodel organism to characterize themolecularmechanisms underlying oxidative stress resistance and toevaluate the antioxidant potential of dietary extracts andphenolic compounds [12] We have previously reported thatquercetin the most common flavonol in the diet increasesyeast oxidative stress resistance [13] and exerts its protectiveeffects against oxidative stress by inducing the biosynthesisof trehalose a stress protectant disaccharide and the acti-vation of the cell wall integrity pathway [14] Other studieshave shown that resveratrol and catechin increase oxidativestress resistance in yeast by mechanisms associated with theactivation of catalase [15] whereas delphinidin 3-glucosideand petunidin 3-glucoside protect yeast through activationof the stress response regulators Msn2p and Msn4p [16]Moreover the sirtuin Hst3p has been implicated in oxidativestress protection afforded by a polyphenol-enriched cocoapowder [17]

Pyrogallol and phloroglucinol are simple phenols thatcontain three hydroxyl groups in the ortho- and metaposi-tion respectively of a benzene ring (Figure 1(a)) Humansare exposed to pyrogallol through ingestion of tea and coffee[18] but also from degradation of gallic acid in colon [19]Phloroglucinol is found as a monomer of phlorotannins inbrown algae which is increasing in the human diet [20]Myricetin is a naturally occurring flavonol characterized byhaving a pyrogallol structure in the B ring as well as a 4-oxo function with an unsaturated bond between the 2 and3 carbons within the C ring and the presence of hydroxylgroups at C3 and C5 [6] (Figure 1(a)) In the human dietmyricetin is commonly found in tea berries and red wine[21] In this study we investigated the effect of myricetinpyrogallol andphloroglucinol on yeast resistance to oxidativestress

2 Materials and Methods

21 Reagents All reagents and chemicals used were of ana-lytical grade Sodium or potassium phosphates riboflavinand H

2O2were purchased from Merck (Darmstadt Ger-

many) Dimethyl sulfoxide (DMSO) myricetin pyrogallolphloroglucinol and nitroblue tetrazolium were purchasedfrom Sigma (Sintra Portugal) Phenolic compounds weredissolved in DMSO at a 200mM stock concentration andstored at minus80∘C Solutions were prepared in ultrapure water(Milli-Q)

22 Yeast Strains and Growth Conditions Saccharomycescerevisiae cells (Euroscarf Germany) used in this study wereBY4741 (Mat120572 his3Δ

1 leu2Δ

0 met15Δ

0 ura3Δ

0 parental

strain) sod1Δ (BY4741 sod1ΔKanMX4) and sod2Δ (BY4741sod2ΔKanMX4) Yeast cells were grown in YPD medium[1 (wv) yeast extract 2 (wv) bactopeptone and 2(wv) glucose] or in synthetic complete (SC) drop-out

medium containing 2 (wv) glucose 067 (wv) yeastnitrogen base without amino acids supplemented with theappropriate amino acids (80mgHis Lminus1 400mg Leu Lminus1 and80mg trp Lminus1) and nucleotides (80mgUra Lminus1) Cultureswere maintained in an orbital shaker at 26∘C and 120 rpmwith a ratio of flask volumemedium volume of 5 1

23 Oxidative Stress Resistance Assays Yeast cells weregrown to the exponential phase (OD

600= 05-06) in YPD

medium pretreated with polyphenols (myricetin pyrogallolor phloroglucinol at 300 120583M) or equal volume of DMSO(vehicle) for 15min and subsequently exposed to 15mMH2O2for 1 hour Cell viability was determined by dilution

plate counts on YPDmedium containing 15 agar Colonieswere counted after growth at 26∘C for 3 days Viability wasexpressed as the percentage of colony-forming units (CFU)

24 Intracellular Oxidation The oxidant-sensitive probe2101584071015840-dichlorodihydrofluorescein (H

2DCF-DA) (Molecular

Probes) was used to measure intracellular oxidation Yeastcells grown to the exponential phase in YPD medium andpretreated with polyphenols for 15min were subsequentlyexposed to 15mMH

2O2for 1 hour in the absence or presence

of 25 120583M H2DCF-DA Cells were spun down (4000 rpm

4min) washed twice and suspended in filtered phosphate-buffered saline (PBS 137mM NaCl 27mM KCl 10mMNa2HPO4 and 18mM KH

2PO4 pH 74) Fluorescence was

measured in FL-1 channel (excitation and emission wave-length at 488 nm and 525 nm resp) in a Becton-DickinsonFACSort flow cytometer Autofluorescence was analyzed incells untreated with H

2DCF-DA Data was acquired from a

total of 10000 eventssamples BD CellQuest Pro Softwarewas used for data acquisition and FlowJo Software for dataanalysis

25 Protein Carbonylation Protein extracts were preparedin 50mM potassium phosphate buffer (pH 70) containingprotease inhibitors (Complete Mini EDTA-free ProteaseCocktail Inhibitor Tablets Roche Applied Science) by vig-orous shaking of the cell suspension in the presence of glassbeads for 5min Short pulses of 1min were used with 1minintervals on ice Protein content was estimated by the Lowrymethod with bovine serum albumin as a standard Proteincarbonylation assays were performed by slot-blot analysis aspreviously described [13] using rabbit IgG anti-dinitrophenyl(DNP) (Sigma) at a 1 5000 dilution as the primary antibodyand goat anti-rabbit IgG-peroxidase (Sigma) at 1 5000 asthe secondary antibody Immunodetection was performedby chemiluminescence with a kit from GE Healthcare (RPN2109) Quantification of bands was performed by densitome-try

26 Glutathione Levels and Enzymatic Activities All theprocedures were carried out at 4∘C Yeast cells were har-vested by centrifugation Glutathione levels were measuredby the method of Tietze [22] as described in a previouswork [13] For enzyme activities yeast extracts were pre-pared as described for the analysis of protein carbonylation

Oxidative Medicine and Cellular Longevity 3

OH

OH

OH

HO

OH

OH

HO

OH

OH

OH

OH

OH

O

O

Myricetin Pyrogallol Phloroglucinol

(a)

0

50

100

Viab

ility

()

ControlH2O2

lowast

lowast

BY4741

Myricetin Pyrogallol PhloroglucinolC(DMSO)

(b)

Figure 1 (a) Chemical structure of the polyphenolic compounds used in this work (b) Effect ofmyricetin and simple phenols (pyrogallol andphloroglucinol) on oxidative stress resistance Yeast cells were grown to the exponential phase in YPD medium pretreated with compounds(300 120583M) or equal volume of DMSO (control) for 15min and subsequently treated with 15mM H

2O2for 1 h Viability is expressed as the

percentage of the CFU Values are mean plusmn SEM of at least 3 independent assays Values were compared by one-way ANOVA Dunnettrsquosmultiple comparisons test (lowast119901 lt 005)

The activity of catalase and SOD was analyzed in situ afterseparation of proteins (50120583g) by native polyacrylamide gelelectrophoresis (PAGE) as described previously [23 24]Quantification of bands was performed by densitometry

27 Chronological Lifespan Overnight cultures in SC med-ium were diluted to OD

600= 05 and grown to the stationary

phase for 3 days (in the case of BY4741 and sod1Δ cells) orfor 1 day (in the case of sod2Δ cells) Then the compounds(300 120583Mmyricetin or pyrogallol) or DMSO (vehicle volumeidentical to compounds) were added to the cultures (day 0)These cells were kept in culture media at 26∘C and viabilitywas analyzed at indicated times by standard dilution platecounts on YPDmedium containing 15 agar Colonies werecounted after growth at 26∘C for 3 days and viability wasexpressed as the percentage in CFU relative to day 0

28 Statistical Analysis Analysis was performed in Graph-Pad Prism Data are expressed as the mean values plusmn standarderror of the mean (SEM) of at least three independent exper-iments The 005 probability level was selected as the pointof statistical significance Values of oxidative stress resistanceassays were analyzed by one-way ANOVA and compared byDunnettrsquos multiple comparisons test Intracellular ROS andprotein carbonyls were analyzed by two-way ANOVA andcompared by Sidakrsquos multiple comparisons test Statisticalanalysis of total and oxidized glutathione levels and the ratioGSSGGSHT was performed by two-way ANOVA Sidakrsquosmultiple comparisons test (lowast119901 lt 005) for comparison ofvalues between treatments in each condition (control orH2O2) and multiple 119905-tests using the Holm-Sidak method

for corrections (lowast119901 lt 005) for comparison of valuesbetween control and H

2O2for all treatments Lifespans were

compared by Studentrsquos 119905-test



100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Max

()

Max

()

Max

()

DCF fluorescence (FL-1) DCF fluorescence (FL-1) DCF fluorescence (FL-1)MyricetinMyricetin + H2O2

PyrogallolPyrogallol + H2O2

PhloroglucinolPhloroglucinol + H2O2

ControlH2O2

ControlH2O2

ControlH2O2

(a)

ControlH2O2

lowast

lowast

Myricetin Pyrogallol PhloroglucinolC(DMSO)0

5

10

15

20

ROS

leve

ls (fo

ld ch

ange

)

(b)

ControlH2O2

lowast

lowast


05

10

15

20

Carb

onyl

leve

ls

(c)

Figure 2 Effect of myricetin pyrogallol and phloroglucinol on intracellular oxidation and oxidative damage Yeast cells were grown inYPD medium to the exponential phase and pretreated with compounds (300120583M) or equal volume of DMSO (control) for 15min andsubsequently treated with 15mMH

2O2for 1 h (a) Representative histograms of intracellular ROS analyzed by flow cytometry using H

2DCF-

DA as a probe (b) Quantification of intracellular ROS expressed by mean fluorescence intensity in 10000 cells (arbitrary units) from atleast 3 independent assays (c) Quantitative analysis of protein carbonyl content was performed by densitometry using data taken from thesame membrane Proteins were derivatized with DNPH and slot-blotted into a PVDF membrane Immunodetection was performed usingan anti-DNP antibody Values are mean plusmn SEM of at least 3 independent assays Values were compared by two-way ANOVA Sidakrsquos multiplecomparisons test (lowast119901 lt 005)

3 Results

31 Myricetin and Pyrogallol Increase Hydrogen PeroxideResistance in Saccharomyces cerevisiae To assess the effect ofmyricetin pyrogallol and phloroglucinol on oxidative stressresistance exponential phase yeast cells were pretreated withthese compounds individually (300 120583M) or DMSO (control)for 15min and subsequently exposed to 15mM H

2O2for

1 hour The presence of polyphenols per se (in the absenceof H2O2) did not affect cell viability intracellular oxidation

or protein oxidation Myricetin and pyrogallol in contrastwith phloroglucinol increased cell viability from 33 (incontrol cells) to 64 and 51 respectively (Figure 1(b)) Toinvestigate if H

2O2resistance induced by these polyphenols

was correlated with a decrease in oxidative stress markersintracellular ROS levels were measured by flow cytom-etry using cells labeled with an oxidant-sensitive probe

H2DCF-DA (Figures 2(a)-2(b)) and protein oxidation was

assessed through the analysis of protein carbonyl content(Figure 2(c)) In control cells exposure to H

2O2caused a

10-fold increase in intracellular ROS and a 3-fold increasein protein carbonylation Myricetin and pyrogallol but notphloroglucinol significantly decreased H

2O2-induced intra-

cellular oxidation and protein carbonylation

32 Myricetin and Pyrogallol Do Not Affect the Activity ofSuperoxide Dismutase or Catalase To investigate if the pro-tective effect of myricetin or pyrogallol was associated withan induction of antioxidant defenses the activity of super-oxide dismutase and catalase was determined Consistentwith published data [25] SOD activity decreased 31 incontrol cells (DMSO-treated) exposed to H

2O2(Figure 3(a))

Pretreatment with the phenolic compounds did not affect


ControlH2O2


05

10

15SO

D ac

tivity

(fol

d ch

ange

)

(a)

ControlH2O2


05

10

15

lowast

lowast

lowast

lowast

lowast

mm

olG

SHT

mg

of p

rote

in

(b)

ControlH2O2


lowast

lowast lowast

000

002

004

006

008

010

mm

ol G

SSG

mg

of p

rote

in

(c)

ControlH2O2


lowastlowast

000

002

004

006

008

010

GSS

GG

SHT

(d)

Figure 3 Effect of myricetin pyrogallol and phloroglucinol on antioxidant defenses Yeast cells were grown in YPD medium to theexponential phase and pretreated with compounds (300 120583M) or equal volume of DMSO (control) for 15min and subsequently treated with15mM H

2O2for 1 h (a) SOD activity was assessed in situ after native PAGE Band intensities were measured by densitometry using data

taken from the same gel (b) GSHT levels (c) GSSG levels and (d) ratio between oxidized glutathione and total glutathione levels Values aremean plusmn SEM of at least 3 independent assays GSHT and GSSG levels were compared by two-way ANOVA Sidakrsquos multiple comparisons test(lowast119901 lt 005) and the ratio was compared by Studentrsquos 119905-test (lowast119901 lt 005)

basal SOD activity or prevent its decrease upon exposureto H2O2 Catalase is not expressed in exponential phase

cells [26] and therefore its activity was not detected incontrol cells Moreover it was not induced in cells treatedwith the tested compounds (data not shown) These resultsindicate that the increase of oxidative stress resistance in cellspretreated with myricetin or pyrogallol did not result fromthe induction of SOD and catalase

33 Myricetin Suppresses H2O2-Induced Glutathione Oxida-tion The tripeptide glutathione (GSH) is the most abun-dant low-molecular weight thiol that serves to maintain areduced intracellular environment [27] To assess the effectof myricetin pyrogallol and phloroglucinol on redox home-ostasis glutathione levels were determined in cells exposed to

H2O2(Figures 3(b)ndash3(d)) In control cells after exposure to

H2O2 total glutathione levels (GSHT) decreased 37whereas

GSSG levels increased 70 increasing the ratio betweenGSSG and GSHT Similar results were observed in cells pre-treated with phloroglucinol which is consistent with the factthat this compound did not affect oxidative stress resistanceMyricetin and pyrogallol per se (in the absence of H

2O2)

decreased GSHT levels However H2O2-induced glutathione

depletionwas lower in cells pretreatedwith these compoundscomparing with DMSO-treated cells Moreover the increasein the levels of GSSG and in the ratio GSSGGSHT inducedby H2O2was suppressed by myricetin but not by pyrogallol

This is consistent with our data showing that oxidative stressresistance in cells pretreated with myricetin was higher thanthe observed in pyrogallol pretreated cells


34 Myricetin Increases the Chronological Lifespan of sod2ΔMutant Cells Aging has been associated with an increasein intracellular oxidation and accumulation of oxidativedamage [28] Mitochondria are a major source of ROS andits dysfunction has been implicated in aging [29 30] Mito-chondria contain several antioxidant enzymes including thesuperoxide dismutases Sod1p (CuZnSOD) that is present inthe mitochondrial intermembrane space (and cytosol) andSod2p (MnSOD) located in the mitochondrial matrix Cellslacking Sod1p or Sod2p exhibit a decreased chronologicallifespan associated with the accumulation of oxidative dam-age [31] (Figures 4(a)-4(b))The protective effect of myricetinand pyrogallol against oxidative stress caused by H

2O2led

us to assess its effect on the chronological lifespan (CLS) ofparental cells and of sod1Δ and sod2Δ mutant cells Parentalcells showed a time-dependent loss of cell viability which wasnot affected by pretreatment with myricetin pyrogallol orphloroglucinol (Figure 4(a))These phenolic compounds alsodid not affect the lifespan of sod1Δ cells (data not shown)However myricetin significantly increased the CLS of sod2Δcells (Figure 4(b)) suggesting that this compound exerts aprotective effect that is particularly relevant in cells that havea decreased capacity to scavenge superoxide radicals withinthe mitochondrial matrix Consistently myricetin decreasedprotein carbonylation in aged sod2Δ cells although it had amodest effect in parental cells (Figures 4(c)ndash4(e)) In contrastpyrogallol and phloroglucinol did not extend the CLS ofsod2Δ cells (Figure 4(b))

Mitochondria play an important function during oxida-tive stress Indeed1205880 petite strains which lackmitochondrialDNA and cells deficient in electron transport chain functionare sensitive to H

2O2[32 33] A recent study showed that

H2O2increases the mitochondrial production of superoxide

radicals which have a protective effect at low concentrations[34] However high concentrations of superoxide radicalsare detrimental In agreement sod2Δ cells were sensitive toH2O2(Figure 5)We also assessed the effect of polyphenols in

sod2Δ cells exposed toH2O2The results show that pyrogallol

pretreatment slightly increased H2O2resistance of sod2Δ

cells although to levels below those observed in parental cellsIn contrast myricetin and phloroglucinol did not affect H

2O2

resistance in these mutants (Figure 5)

4 Discussion

The increased production of ROS and RNS together withthe decrease of antioxidant defenses has been implicatedin the pathogenesis of numerous diseases and aging [28]Thus a diet containing natural compounds with antioxidantproperties such as phenolic compounds may be beneficial tohuman healthThe antioxidant activity of these compounds isdetermined by structural features including the number andposition of hydroxyl groups polarity solubility and reducingpotential [35 36] In this study we used the yeast Saccha-romyces cerevisiae to assess in vivo the antioxidant capacityof the flavonol myricetin and two simple phenols pyrogalloland phloroglucinol Myricetin was the most effective inincreasing H

2O2resistance in yeast whereas phloroglucinol

had no protective effect Consistently H2O2-induced intra-

cellular oxidation and protein carbonylation decreased incells pretreated with myricetin and pyrogallol but not withphloroglucinol Pyrogallol and phloroglucinol contain threehydroxyl groups in the ortho- andmetaposition respectivelyof a benzene ringThe vicinal positions of hydroxyl groups inpyrogallol result in a lower bond dissociation energy of OndashH facilitating the donation of hydrogen to free radicals [37]In accordance with that our results show that pyrogallol incontrast with phloroglucinol increased the viability of yeastcells exposed toH

2O2 Myricetin which contains a pyrogallol

structure in the B ring provided an even higher resistanceOur results are in accordance with data demonstrating theimportance of the pyrogallol structure for the bioactivityof phenolic compounds [38] Our data is also consistentwith several reports showing a protective effect of myricetinagainst oxidative stress in mammalian cells For instancemyricetin decreases H

2O2-induced DNA damage in Caco-2

andHepG2 cells [39] and decreases tert-butyl hydroperoxide-induced protein oxidation and lipid peroxidation in erythro-cytes from T2DM patients [40]

Being redox-active compounds phenolic compounds canalso act as prooxidants and therefore induce stress responsesleading to an increase in the levels of cellular antioxidantdefenses [41 42] Our results indicate that this mechanismdoes not contribute to the protective effects of myricetinand pyrogallol in yeast since these compounds did notincrease intracellular oxidation or affect catalase and SODactivities under the conditions used in this study We havepreviously observed that hydrogen peroxide resistance inyeast incubatedwith quercetin is also not associatedwith pro-oxidant effects or modulation of antioxidant defenses [13] Incontrast other reports showed that catalase activity increasesin yeast treated with resveratrol and catechin enhancingcellular resistance to oxidative stress [15]

Glutathione is an important cellular small moleculeresponsible for the maintenance of redox homeostasis [27]The reduced form (GSH) mediates H

2O2decomposition cat-

alyzed by glutathione peroxidase [27] giving rise to oxidizedglutathione (GSSG) which is then reduced to GSH by glu-tathione reductase [43] Glutathione has also important func-tions in detoxification of toxic compounds [44] and in theprotection of proteins from oxidation through glutathiony-lation [45] Thus glutathione oxidation is a biomarker ofoxidative stress In control (DMSO-treated) cells exposure toH2O2led to an increase in GSSG levels that concomitantly

with glutathione depletion resulted in a higher GSSGGSHTratio In cells pretreated with myricetin H

2O2-induced glu-

tathione oxidation and the increase in the ratio GSSGGSHTwere suppressed which is consistent with the reduction ofintracellular oxidation Pretreatment with pyrogallol whichhad a lower protective effect comparing with myricetin didnot prevent glutathione oxidation These results suggest acorrelation between the protective effect of myricetin andmaintenance of glutathione redox status Treatmentwith bothmyricetin and pyrogallol per se led to a decrease in totalGSH levels which may result from the formation of GS-compound adducts mediated by glutathione-S-transferasesIndeed these adducts have been reported for quercetin [41


MyricetinPyrogallolPhloroglucinol

C(DMSO)

0 5 10 15 20 25

50

100

Days

Viab

ility

()

BY4741

(a)


C(DMSO)

Days

0

50

100

0 1 2 3 4

Viab

ility

()

lowast

sod2Δ

(b)

0 2 3

00

05

10

15

20

Days

C(DMSO)Myricetin

BY4741

lowast

Prot

ein

carb

onyl

s (fo

ld ch

ange

)

(c)

00

05

10

15

20

0 2 3

Days

C(DMSO)Myricetin

lowast

lowast

lowast

sod2ΔPr

otei

n ca

rbon

yls (

fold

chan

ge)

(d)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Day 0 Day 2 Day 3

BY4741

sod2Δ

(e)

Figure 4 Effect of myricetin pyrogallol and phloroglucinol on (a) BY4741 and (b) sod2Δ cells CLS Cells were grown in SC-glucose mediumto the stationary phase and treated with myricetin pyrogallol or phloroglucinol (300120583M) Viability was measured by standard dilution platecounts which were considered 100 on day 0 (first treatment day) (c d) On the indicated days the levels of protein carbonyls were analyzedduring aging of BY4741 (c) and sod2Δ (d) cells pretreated with myricetin Quantitative analysis of protein carbonyl content was performedby densitometry using data taken from the same membrane Proteins were derivatized with DNPH and slot-blotted into a PVDFmembraneImmunodetection was performed using an anti-DNP antibody A representative blot is shown in (e) Values are mean plusmn SEM of at least 3independent assays Viability values were compared by Studentrsquos 119905-test (lowast119901 lt 005) and protein carbonyl values were compared by two-wayANOVA Sidakrsquos multiple comparisons test (lowast119901 lt 005)


ControlH2O2


50

100

Viab

ility

()

lowast

sod2Δ

Figure 5 Effect of myricetin pyrogallol and phloroglucinol on theoxidative stress resistance of sod2Δ cells Yeast cells were grown tothe exponential phase in YPDmedium pretreated with compounds(300 120583M) or equal volume of DMSO (control) for 15min andsubsequently treatedwith 15mMH

2O2for 1 h Viability is expressed

as the percentage of the CFU Values are mean plusmn SEM of at least3 independent assays Values were compared by one-way ANOVADunnettrsquos multiple comparisons test (lowast119901 lt 005)

46] and glutathione-S-transferase activity may be inducedby these phenolic compounds similarly to the effects ofcoumarin [47]

High levels of ROS have been implicated in aging inyeast and higher eukaryotes [48 49] The accumulation ofoxidative damage leading to neuronal death is associatedwith age-related diseases such as Alzheimerrsquos and Parkinsondiseases [50] Therefore a diet replete in phytochemicalswith antioxidant activity characteristic of a Mediterraneandiet reduces the functional decline associated with agingand age-related disorders increasing health span [51 52]Several studies showed an increase of the lifespan of yeastcells incubated with phenolic compounds Resveratrol andphloridzin a major apple compound increase yeast replica-tive lifespan by mechanisms associated with the activationof the sirtuin Sir2p [53 54] Moreover quercetin and applepolyphenolic fractions increase yeast chronological lifespan[13 55] Here we report that although myricetin does notaffect the chronological lifespan of parental and sod1Δ cells itextends the lifespan of yeast cells lacking the mitochondrialsuperoxide dismutase which are known to exhibit a veryshort lifespan [29] In contrast pyrogallol did not extend theCLS of sod2Δ cells These results suggest that myricetin maybemore effective in protecting aged cells that have high intra-cellular ROS levels and oxidative damage especially in themitochondria In agreement with that several reports showprotective effects of myricetin in mitochondria For instance

the protective effect of L-ascorbic acid against paraquat agenerator of superoxide radicals is more pronounced insod2Δ than in parental cells [56] In addition myricetindecreased the generation of H

2O2in isolated mouse skeletal

muscle mitochondria [57] and decreased the depolarizationof the inner mitochondrial membrane potential in C6 glialcells exposed to oxygen-glucose deprivation [58] and it wasthe most efficient among other phenolic compounds in theprotection of mouse brain mitochondria against toxicityinduced by methyl mercury [59] Notably myricetin wasunable to protect sod2Δ cells against high doses of H

2O2

whereas pyrogallol slightly increased the oxidative stressresistance of these mutants It is likely that the excessiveoxidative stress in sod2Δ cells treatedwith high doses ofH

2O2

overwhelms the protective effects of these compounds

5 Conclusion

In summary our data show that myricetin and to a lesserextent pyrogallol increased yeast resistance to H

2O2 This

protective effect was correlated to a reduction in intracellularoxidation and protein carbonylation and maintenance ofGSSGGSHT ratio However changes in catalase or superox-ide dismutase activities were not associated with the protec-tive effects Furthermore myricetin attenuated the shortenedCLS of yeast cells lacking the mitochondrial superoxidedismutase (sod2Δmutants)

Abbreviations

CLS Chronological lifespanDMSO Dimethyl sulfoxideGSH Reduced glutathioneGSHT Total glutathioneGSSG Oxidized glutathioneH2DCF-DA 2101584071015840-Dichlorodihydrofluorescein diacetate

PAGE Polyacrylamide gel electrophoresisRNS Reactive nitrogen speciesROS Reactive oxygen speciesSC Synthetic completeSOD Superoxide dismutase

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Vanda Mendes was supported by a FCT (Fundacao para aCiencia e Tecnologia) Fellowship (SFRHBD784742011)

References

[1] B Halliwell and J Gutteridge ldquoCellular responses to oxidativestress adaptation damage repair senescence and deathrdquo inFree Radicals in Biology and Medicine pp 187ndash267 OxfordUniversity Press New York NY USA 4th edition 2007


[2] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[3] R A Jacob and B J Burri ldquoOxidative damage and defenserdquoTheAmerican Journal of Clinical Nutrition vol 63 no 6 pp 985Sndash990S 1996

[4] D del Rio A Rodriguez-Mateos J P E Spencer M TognoliniG Borges and A Crozier ldquoDietary (poly)phenolics in humanhealth structures bioavailability and evidence of protectiveeffects against chronic diseasesrdquo Antioxidants amp Redox Signal-ing vol 18 no 14 pp 1818ndash1892 2013

[5] D E Stevenson and R D Hurst ldquoPolyphenolic phytochemi-calsmdashjust antioxidants or much morerdquo Cellular and MolecularLife Sciences vol 64 no 22 pp 2900ndash2916 2007

[6] A Crozier I B Jaganath and M N Clifford ldquoDietary phe-nolics chemistry bioavailability and effects on healthrdquo NaturalProduct Reports vol 26 no 8 pp 1001ndash1043 2009

[7] H Sies ldquoPolyphenols and health update and perspectivesrdquoArchives of Biochemistry and Biophysics vol 501 no 1 pp 2ndash52010

[8] H E Seifried D E Anderson E I Fisher and J A Milner ldquoAreview of the interaction among dietary antioxidants and reac-tive oxygen speciesrdquo The Journal of Nutritional Biochemistryvol 18 no 9 pp 567ndash579 2007

[9] A-N T Kong R Yu C Chen S Mandlekar and T PrimianoldquoSignal transduction events elicited by natural products roleof MAPK and caspase pathways in homeostatic response andinduction of apoptosisrdquoArchives of Pharmacal Research vol 23no 1 pp 1ndash16 2000

[10] S Ramos ldquoCancer chemoprevention and chemotherapy diet-ary polyphenols and signalling pathwaysrdquo Molecular Nutritionamp Food Research vol 52 no 5 pp 507ndash526 2008

[11] Y Son Y K Cheong N H Kim H T Chung D G Kangand H O Pae ldquoMitogen-activated protein kinases and reactiveoxygen species how can ROS activate MAPK pathwaysrdquoJournal of Signal Transduction vol 2011 Article ID 792639 6pages 2011

[12] M JWu P J OrsquoDoherty H R Fernandez et al ldquoAn antioxidantscreening assay based on oxidant-induced growth arrest inSaccharomyces cerevisiaerdquo FEMS Yeast Research vol 11 no 4pp 379ndash387 2011

[13] I Belinha M A Amorim P Rodrigues et al ldquoQuercetinincreases oxidative stress resistance and longevity in Saccha-romyces cerevisiaerdquo Journal of Agricultural and Food Chemistryvol 55 no 6 pp 2446ndash2451 2007

[14] R Vilaca V Mendes M V Mendes et al ldquoQuercetin protectsSaccharomyces cerevisiae against oxidative stress by inducingtrehalose biosynthesis and the cell wall integrity pathwayrdquo PLoSONE vol 7 no 9 Article ID e45494 2012

[15] C Dani D Bonatto M Salvador M D Pereira J A P Henri-ques and E Eleutherio ldquoAntioxidant protection of resveratroland catechin in Saccharomyces cerevisiaerdquo Journal of Agricul-tural and Food Chemistry vol 56 no 11 pp 4268ndash4272 2008

[16] A Jimenez P Lisa-Santamarıa M Garcıa-Marino M TEscribano-Bailon J C Rivas-Gonzalo and J L Revuelta ldquoThebiological activity of the wine anthocyanins delphinidin andpetunidin is mediated through Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo FEMS Yeast Research vol 10 no 7 pp 858ndash869 2010

[17] P Martorell J V Forment R de Llanos et al ldquoUse of Saccharo-myces cerevisiae andCaenorhabditis elegans asmodel organisms

to study the effect of cocoa polyphenols in the resistance tooxidative stressrdquo Journal of Agricultural and Food Chemistryvol 59 no 5 pp 2077ndash2085 2011

[18] C Muller R Lang and T Hofmann ldquoQuantitative precursorstudies on di- and trihydroxybenzene formation during coffeeroasting using lsquoin beanrsquo model experiments and stable isotopedilution analysisrdquo Journal of Agricultural and Food Chemistryvol 54 no 26 pp 10086ndash10091 2006

[19] T Yasuda A Inaba M Ohmori T Endo S Kubo and KOhsawa ldquoUrinary metabolites of gallic acid in rats and theirradical-scavenging effects on 11-diphenyl-2-picrylhydrazyl rad-icalrdquo Journal of Natural Products vol 63 no 10 pp 1444ndash14462000

[20] L Montero M Herrero E Ibanez and A Cifuentes ldquoSepara-tion and characterization of phlorotannins from brown algaeCystoseira abies-marina by comprehensive two-dimensionalliquid chromatographyrdquoElectrophoresis vol 35 no 11 pp 1644ndash1651 2014

[21] M G L Hertog E J M Feskens P C H HollmanM B Katanand D Kromhout ldquoDietary antioxidant flavonoids and risk ofcoronary heart disease the Zutphen Elderly StudyrdquoThe Lancetvol 342 no 8878 pp 1007ndash1011 1993

[22] F Tietze ldquoEnzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione appli-cations to mammalian blood and other tissuesrdquo AnalyticalBiochemistry vol 27 no 3 pp 502ndash522 1969

[23] L Flohe and F Otting ldquoSuperoxide dismutase assaysrdquoMethodsin Enzymology vol 105 pp 93ndash104 1984

[24] S M Conyers and D A Kidwell ldquoChromogenic substrates forhorseradish peroxidaserdquo Analytical Biochemistry vol 192 no 1pp 207ndash211 1991

[25] D C Salo R E Pacifici S W Lin C Giulivi and K J ADavies ldquoSuperoxide dismutase undergoes proteolysis and frag-mentation following oxidative modification and inactivationrdquoThe Journal of Biological Chemistry vol 265 no 20 pp 11919ndash11927 1990

[26] T Belazzi A Wagner R Wieser et al ldquoNegative regulation oftranscription of the Saccharomyces cerevisiae catalase T (CTT1)gene by cAMP is mediated by a positive control elementrdquo TheEMBO Journal vol 10 no 3 pp 585ndash592 1991

[27] A Meister and M E Anderson ldquoGlutathionerdquo Annual Reviewof Biochemistry vol 52 pp 711ndash760 1983

[28] D F Dai Y A Chiao D J Marcinek H H Szeto and PS Rabinovitch ldquoMitochondrial oxidative stress in aging andhealthspanrdquo Longevity amp Healthspan vol 3 article 6 2014

[29] V D Longo E B Gralla and J S Valentine ldquoSuperoxidedismutase activity is essential for stationary phase survival inSaccharomyces cerevisiae mitochondrial production of toxicoxygen species in vivordquo Journal of Biological Chemistry vol 271no 21 pp 12275ndash12280 1996

[30] D V Ziegler C D Wiley and M C Velarde ldquoMitochondrialeffectors of cellular senescence beyond the free radical theoryof agingrdquo Aging Cell vol 14 no 1 pp 1ndash7 2015

[31] A B Demir and A Koc ldquoAssessment of chronological lifespandependent molecular damages in yeast lacking mitochondrialantioxidant genesrdquo Biochemical and Biophysical Research Com-munications vol 400 no 1 pp 106ndash110 2010

[32] C M Grant F H MacIver and I W Dawes ldquoMitochondrialfunction is required for resistance to oxidative stress in the yeastSaccharomyces cerevisiaerdquo FEBS Letters vol 410 no 2-3 pp219ndash222 1997


[33] G W Thorpe C S Fong N Alic V J Higgins and I WDawes ldquoCells have distinct mechanisms to maintain protec-tion against different reactive oxygen species oxidative-stress-response genesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 101 no 17 pp 6564ndash65692004

[34] G W Thorpe M Reodica M J Davies et al ldquoSuperoxideradicals have a protective role during H

2O2stressrdquo Molecular

Biology of the Cell vol 24 no 18 pp 2876ndash2884 2013[35] W Bors C Michel and K Stettmaier ldquoStructure-activity

relationships governing antioxidant capacities of plant polyphe-nolsrdquoMethods in Enzymology vol 335 pp 166ndash180 2001

[36] J C JMD SMenezes S P Kamat J A S Cavaleiro AGasparJ Garrido and F Borges ldquoSynthesis and antioxidant activityof long chain alkyl hydroxycinnamatesrdquo European Journal ofMedicinal Chemistry vol 46 no 2 pp 773ndash777 2011

[37] VThavasi L P Leong and R P A Bettens ldquoInvestigation of theinfluence of hydroxy groups on the radical scavenging ability ofpolyphenolsrdquo The Journal of Physical Chemistry A vol 110 no14 pp 4918ndash4923 2006

[38] S Mitsuhashi A Saito N Nakajima H Shima and MUbukata ldquoPyrogallol structure in polyphenols is involved inapoptosis-induction on HEK293T and K562 cellsrdquo Moleculesvol 13 no 12 pp 2998ndash3006 2008

[39] S A Aherne and N M OrsquoBrien ldquoProtection by the flavonoidsmyricetin quercetin and rutin against hydrogen peroxide-induced DNA damage in Caco-2 and Hep G2 cellsrdquo Nutritionand Cancer vol 34 no 2 pp 160ndash166 1999

[40] K B Pandey N Mishra and S I Rizvi ldquoMyricetin mayprovide protection against oxidative stress in type 2 diabeticerythrocytesrdquo Zeitschrift fur Naturforschung Teil C BiochemieBiophysik Biologie Virologie vol 64 no 9-10 pp 626ndash6302009

[41] M Kessler G Ubeaud and L Jung ldquoAnti- and pro-oxidantactivity of rutin and quercetin derivativesrdquo Journal of Pharmacyand Pharmacology vol 55 no 1 pp 131ndash142 2003

[42] V Calabrese C Cornelius A T Dinkova-Kostova et al ldquoCellu-lar stress responses hormetic phytochemicals and vitagenes inaging and longevityrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1822 no 5 pp 753ndash783 2012

[43] P Mullineaux G Creissen P Broadbent H Reynolds B Kularand A Wellburn ldquoElucidation of the role of glutathione reduc-tase using transgenic plantsrdquo Biochemical Society Transactionsvol 22 no 4 pp 931ndash936 1994

[44] M V St-Pierre S Ruetz L F Epstein P Gros and I MArias ldquoATP-dependent transport of organic anions in secretoryvesicles of Saccharomyces cerevisiaerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 91 no20 pp 9476ndash9479 1994

[45] M M Gallogly and J J Mieyal ldquoMechanisms of reversibleprotein glutathionylation in redox signaling and oxidativestressrdquo Current Opinion in Pharmacology vol 7 no 4 pp 381ndash391 2007

[46] J P E Spencer G G C Kuhnle R J Williams and C Rice-Evans ldquoIntracellular metabolism and bioactivity of quercetinand its in vivo metabolitesrdquo Biochemical Journal vol 372 no1 pp 173ndash181 2003

[47] L G Higgins and J D Hayes ldquoMechanisms of induction ofcytosolic and microsomal glutathione transferase (GST) genesby xenobiotics and pro-inflammatory agentsrdquoDrugMetabolismReviews vol 43 no 2 pp 92ndash137 2011

[48] K J Bitterman O Medvedik and D A Sinclair ldquoLongevityregulation in Saccharomyces cerevisiae linking metabolismgenome stability and heterochromatinrdquo Microbiology andMolecular Biology Reviews vol 67 no 3 pp 376ndash399 2003

[49] P Fabrizio L Battistella R Vardavas et al ldquoSuperoxide isa mediator of an altruistic aging program in SaccharomycescerevisiaerdquoThe Journal of Cell Biology vol 166 no 7 pp 1055ndash1067 2004

[50] Y-S Ho D C-H Poon T-F Chan and R C-C ChangldquoFrom small to big molecules how do we prevent and delaythe progression of age-related neurodegenerationrdquo CurrentPharmaceutical Design vol 18 no 1 pp 15ndash26 2012

[51] R N Alcalay Y Gu H Mejia-Santana L Cote K S Marderand N Scarmeas ldquoThe association betweenMediterranean dietadherence and Parkinsonrsquos diseaserdquo Movement Disorders vol27 no 6 pp 771ndash774 2012

[52] N Vassallo and C Scerri ldquoMediterranean diet and dementia oftheAlzheimer typerdquoCurrent Aging Science vol 6 no 2 pp 150ndash162 2013

[53] K THowitz K J BittermanH Y Cohen et al ldquoSmallmoleculeactivators of sirtuins extend Saccharomyces cerevisiae lifespanrdquoNature vol 425 no 6954 pp 191ndash196 2003

[54] L Xiang K Sun J Lu et al ldquoAnti-aging effects of phloridzinan apple polyphenol on yeast via the SOD and Sir2 genesrdquoBioscience Biotechnology and Biochemistry vol 75 no 5 pp854ndash858 2011

[55] V Palermo F Mattivi R Silvestri G la Regina C Falconeand C Mazzoni ldquoApple can act as anti-aging on yeast cellsrdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID491759 8 pages 2012

[56] J Saffi L Sonego Q D Varela and M Salvador ldquoAntioxidantactivity of L-ascorbic acid in wild-type and superoxide dismu-tase deficient strains of Saccharomyces cerevisiaerdquo Redox Reportvol 11 no 4 pp 179ndash184 2006

[57] G Grunz K Haas S Soukup et al ldquoStructural featuresand bioavailability of four flavonoids and their implicationsfor lifespan-extending and antioxidant actions in C elegansrdquoMechanisms of Ageing and Development vol 133 no 1 pp 1ndash102012

[58] K S Panickar andRAAnderson ldquoMechanisms underlying theprotective effects of myricetin and quercetin following oxygen-glucose deprivation-induced cell swelling and the reduction inglutamate uptake in glial cellsrdquo Neuroscience vol 183 pp 1ndash142011

[59] J L Franco T Posser F Missau et al ldquoStructure-activityrelationship of flavonoids derived frommedicinal plants in pre-venting methylmercury-induced mitochondrial dysfunctionrdquoEnvironmental Toxicology and Pharmacology vol 30 no 3 pp272ndash278 2010

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


attributed not only to their intrinsic antioxidant activity butalso to the modulation of cell signaling pathways includingmitogen-activated protein kinase cascades which regulateoxidative stress responses [9ndash11]

Thebudding yeast Saccharomyces cerevisiaehas been usedas a eukaryoticmodel organism to characterize themolecularmechanisms underlying oxidative stress resistance and toevaluate the antioxidant potential of dietary extracts andphenolic compounds [12] We have previously reported thatquercetin the most common flavonol in the diet increasesyeast oxidative stress resistance [13] and exerts its protectiveeffects against oxidative stress by inducing the biosynthesisof trehalose a stress protectant disaccharide and the acti-vation of the cell wall integrity pathway [14] Other studieshave shown that resveratrol and catechin increase oxidativestress resistance in yeast by mechanisms associated with theactivation of catalase [15] whereas delphinidin 3-glucosideand petunidin 3-glucoside protect yeast through activationof the stress response regulators Msn2p and Msn4p [16]Moreover the sirtuin Hst3p has been implicated in oxidativestress protection afforded by a polyphenol-enriched cocoapowder [17]

Pyrogallol and phloroglucinol are simple phenols thatcontain three hydroxyl groups in the ortho- and metaposi-tion respectively of a benzene ring (Figure 1(a)) Humansare exposed to pyrogallol through ingestion of tea and coffee[18] but also from degradation of gallic acid in colon [19]Phloroglucinol is found as a monomer of phlorotannins inbrown algae which is increasing in the human diet [20]Myricetin is a naturally occurring flavonol characterized byhaving a pyrogallol structure in the B ring as well as a 4-oxo function with an unsaturated bond between the 2 and3 carbons within the C ring and the presence of hydroxylgroups at C3 and C5 [6] (Figure 1(a)) In the human dietmyricetin is commonly found in tea berries and red wine[21] In this study we investigated the effect of myricetinpyrogallol andphloroglucinol on yeast resistance to oxidativestress

2 Materials and Methods

21 Reagents All reagents and chemicals used were of ana-lytical grade Sodium or potassium phosphates riboflavinand H

2O2were purchased from Merck (Darmstadt Ger-

many) Dimethyl sulfoxide (DMSO) myricetin pyrogallolphloroglucinol and nitroblue tetrazolium were purchasedfrom Sigma (Sintra Portugal) Phenolic compounds weredissolved in DMSO at a 200mM stock concentration andstored at minus80∘C Solutions were prepared in ultrapure water(Milli-Q)

22 Yeast Strains and Growth Conditions Saccharomycescerevisiae cells (Euroscarf Germany) used in this study wereBY4741 (Mat120572 his3Δ

1 leu2Δ

0 met15Δ

0 ura3Δ

0 parental

strain) sod1Δ (BY4741 sod1ΔKanMX4) and sod2Δ (BY4741sod2ΔKanMX4) Yeast cells were grown in YPD medium[1 (wv) yeast extract 2 (wv) bactopeptone and 2(wv) glucose] or in synthetic complete (SC) drop-out

medium containing 2 (wv) glucose 067 (wv) yeastnitrogen base without amino acids supplemented with theappropriate amino acids (80mgHis Lminus1 400mg Leu Lminus1 and80mg trp Lminus1) and nucleotides (80mgUra Lminus1) Cultureswere maintained in an orbital shaker at 26∘C and 120 rpmwith a ratio of flask volumemedium volume of 5 1

23 Oxidative Stress Resistance Assays Yeast cells weregrown to the exponential phase (OD

600= 05-06) in YPD

medium pretreated with polyphenols (myricetin pyrogallolor phloroglucinol at 300 120583M) or equal volume of DMSO(vehicle) for 15min and subsequently exposed to 15mMH2O2for 1 hour Cell viability was determined by dilution

plate counts on YPDmedium containing 15 agar Colonieswere counted after growth at 26∘C for 3 days Viability wasexpressed as the percentage of colony-forming units (CFU)

24 Intracellular Oxidation The oxidant-sensitive probe2101584071015840-dichlorodihydrofluorescein (H

2DCF-DA) (Molecular

Probes) was used to measure intracellular oxidation Yeastcells grown to the exponential phase in YPD medium andpretreated with polyphenols for 15min were subsequentlyexposed to 15mMH

2O2for 1 hour in the absence or presence

of 25 120583M H2DCF-DA Cells were spun down (4000 rpm

4min) washed twice and suspended in filtered phosphate-buffered saline (PBS 137mM NaCl 27mM KCl 10mMNa2HPO4 and 18mM KH

2PO4 pH 74) Fluorescence was

measured in FL-1 channel (excitation and emission wave-length at 488 nm and 525 nm resp) in a Becton-DickinsonFACSort flow cytometer Autofluorescence was analyzed incells untreated with H

2DCF-DA Data was acquired from a

total of 10000 eventssamples BD CellQuest Pro Softwarewas used for data acquisition and FlowJo Software for dataanalysis

25 Protein Carbonylation Protein extracts were preparedin 50mM potassium phosphate buffer (pH 70) containingprotease inhibitors (Complete Mini EDTA-free ProteaseCocktail Inhibitor Tablets Roche Applied Science) by vig-orous shaking of the cell suspension in the presence of glassbeads for 5min Short pulses of 1min were used with 1minintervals on ice Protein content was estimated by the Lowrymethod with bovine serum albumin as a standard Proteincarbonylation assays were performed by slot-blot analysis aspreviously described [13] using rabbit IgG anti-dinitrophenyl(DNP) (Sigma) at a 1 5000 dilution as the primary antibodyand goat anti-rabbit IgG-peroxidase (Sigma) at 1 5000 asthe secondary antibody Immunodetection was performedby chemiluminescence with a kit from GE Healthcare (RPN2109) Quantification of bands was performed by densitome-try

26 Glutathione Levels and Enzymatic Activities All theprocedures were carried out at 4∘C Yeast cells were har-vested by centrifugation Glutathione levels were measuredby the method of Tietze [22] as described in a previouswork [13] For enzyme activities yeast extracts were pre-pared as described for the analysis of protein carbonylation


OH

OH

OH

HO

OH

OH

HO

OH

OH

OH

OH

OH

O

O


(a)

0

50

100

Viab

ility

()

ControlH2O2

lowast

lowast

BY4741


(b)














100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Max

()

Max

()

Max

()




ControlH2O2

ControlH2O2

ControlH2O2

(a)

ControlH2O2

lowast

lowast


5

10

15

20

ROS

leve

ls (fo

ld ch

ange

)

(b)

ControlH2O2

lowast

lowast


05

10

15

20

Carb

onyl

leve

ls

(c)



2DCF-


3 Results


2O2for







2O2caused a


2O2-induced intra-



2O2(Figure 3(a))



ControlH2O2


05

10

15SO

D ac

tivity

(fol

d ch

ange

)

(a)

ControlH2O2


05

10

15

lowast

lowast

lowast

lowast

lowast

mm

olG

SHT

mg

of p

rote

in

(b)

ControlH2O2


lowast

lowast lowast

000

002

004

006

008

010

mm

ol G

SSG

mg

of p

rote

in

(c)

ControlH2O2


lowastlowast

000

002

004

006

008

010

GSS

GG

SHT

(d)










2O2)






2O2led









2O2


4 Discussion














2O2-induced glu-




C(DMSO)

0 5 10 15 20 25

50

100

Days

Viab

ility

()

BY4741

(a)


C(DMSO)

Days

0

50

100

0 1 2 3 4

Viab

ility

()

lowast

sod2Δ

(b)

0 2 3

00

05

10

15

20

Days

C(DMSO)Myricetin

BY4741

lowast

Prot

ein

carb

onyl

s (fo

ld ch

ange

)

(c)

00

05

10

15

20

0 2 3

Days

C(DMSO)Myricetin

lowast

lowast

lowast

sod2ΔPr

otei

n ca

rbon

yls (

fold

chan

ge)

(d)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Day 0 Day 2 Day 3

BY4741

sod2Δ

(e)



ControlH2O2


50

100

Viab

ility

()

lowast

sod2Δ









2O2


2O2


5 Conclusion


2O2 This


Abbreviations





Acknowledgment


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OH

OH

OH

HO

OH

OH

HO

OH

OH

OH

OH

OH

O

O


(a)

0

50

100

Viab

ility

()

ControlH2O2

lowast

lowast

BY4741


(b)














100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Max

()

Max

()

Max

()




ControlH2O2

ControlH2O2

ControlH2O2

(a)

ControlH2O2

lowast

lowast


5

10

15

20

ROS

leve

ls (fo

ld ch

ange

)

(b)

ControlH2O2

lowast

lowast


05

10

15

20

Carb

onyl

leve

ls

(c)



2DCF-


3 Results


2O2for







2O2caused a


2O2-induced intra-



2O2(Figure 3(a))



ControlH2O2


05

10

15SO

D ac

tivity

(fol

d ch

ange

)

(a)

ControlH2O2


05

10

15

lowast

lowast

lowast

lowast

lowast

mm

olG

SHT

mg

of p

rote

in

(b)

ControlH2O2


lowast

lowast lowast

000

002

004

006

008

010

mm

ol G

SSG

mg

of p

rote

in

(c)

ControlH2O2


lowastlowast

000

002

004

006

008

010

GSS

GG

SHT

(d)










2O2)






2O2led









2O2


4 Discussion














2O2-induced glu-




C(DMSO)

0 5 10 15 20 25

50

100

Days

Viab

ility

()

BY4741

(a)


C(DMSO)

Days

0

50

100

0 1 2 3 4

Viab

ility

()

lowast

sod2Δ

(b)

0 2 3

00

05

10

15

20

Days

C(DMSO)Myricetin

BY4741

lowast

Prot

ein

carb

onyl

s (fo

ld ch

ange

)

(c)

00

05

10

15

20

0 2 3

Days

C(DMSO)Myricetin

lowast

lowast

lowast

sod2ΔPr

otei

n ca

rbon

yls (

fold

chan

ge)

(d)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Day 0 Day 2 Day 3

BY4741

sod2Δ

(e)



ControlH2O2


50

100

Viab

ility

()

lowast

sod2Δ









2O2


2O2


5 Conclusion


2O2 This


Abbreviations





Acknowledgment


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Max

()

Max

()

Max

()




ControlH2O2

ControlH2O2

ControlH2O2

(a)

ControlH2O2

lowast

lowast


5

10

15

20

ROS

leve

ls (fo

ld ch

ange

)

(b)

ControlH2O2

lowast

lowast


05

10

15

20

Carb

onyl

leve

ls

(c)



2DCF-


3 Results


2O2for







2O2caused a


2O2-induced intra-



2O2(Figure 3(a))



ControlH2O2


05

10

15SO

D ac

tivity

(fol

d ch

ange

)

(a)

ControlH2O2


05

10

15

lowast

lowast

lowast

lowast

lowast

mm

olG

SHT

mg

of p

rote

in

(b)

ControlH2O2


lowast

lowast lowast

000

002

004

006

008

010

mm

ol G

SSG

mg

of p

rote

in

(c)

ControlH2O2


lowastlowast

000

002

004

006

008

010

GSS

GG

SHT

(d)










2O2)






2O2led









2O2


4 Discussion














2O2-induced glu-




C(DMSO)

0 5 10 15 20 25

50

100

Days

Viab

ility

()

BY4741

(a)


C(DMSO)

Days

0

50

100

0 1 2 3 4

Viab

ility

()

lowast

sod2Δ

(b)

0 2 3

00

05

10

15

20

Days

C(DMSO)Myricetin

BY4741

lowast

Prot

ein

carb

onyl

s (fo

ld ch

ange

)

(c)

00

05

10

15

20

0 2 3

Days

C(DMSO)Myricetin

lowast

lowast

lowast

sod2ΔPr

otei

n ca

rbon

yls (

fold

chan

ge)

(d)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Day 0 Day 2 Day 3

BY4741

sod2Δ

(e)



ControlH2O2


50

100

Viab

ility

()

lowast

sod2Δ









2O2


2O2


5 Conclusion


2O2 This


Abbreviations





Acknowledgment


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ControlH2O2


05

10

15SO

D ac

tivity

(fol

d ch

ange

)

(a)

ControlH2O2


05

10

15

lowast

lowast

lowast

lowast

lowast

mm

olG

SHT

mg

of p

rote

in

(b)

ControlH2O2


lowast

lowast lowast

000

002

004

006

008

010

mm

ol G

SSG

mg

of p

rote

in

(c)

ControlH2O2


lowastlowast

000

002

004

006

008

010

GSS

GG

SHT

(d)










2O2)






2O2led









2O2


4 Discussion














2O2-induced glu-




C(DMSO)

0 5 10 15 20 25

50

100

Days

Viab

ility

()

BY4741

(a)


C(DMSO)

Days

0

50

100

0 1 2 3 4

Viab

ility

()

lowast

sod2Δ

(b)

0 2 3

00

05

10

15

20

Days

C(DMSO)Myricetin

BY4741

lowast

Prot

ein

carb

onyl

s (fo

ld ch

ange

)

(c)

00

05

10

15

20

0 2 3

Days

C(DMSO)Myricetin

lowast

lowast

lowast

sod2ΔPr

otei

n ca

rbon

yls (

fold

chan

ge)

(d)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Day 0 Day 2 Day 3

BY4741

sod2Δ

(e)



ControlH2O2


50

100

Viab

ility

()

lowast

sod2Δ









2O2


2O2


5 Conclusion


2O2 This


Abbreviations





Acknowledgment


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2O2led









2O2


4 Discussion














2O2-induced glu-




C(DMSO)

0 5 10 15 20 25

50

100

Days

Viab

ility

()

BY4741

(a)


C(DMSO)

Days

0

50

100

0 1 2 3 4

Viab

ility

()

lowast

sod2Δ

(b)

0 2 3

00

05

10

15

20

Days

C(DMSO)Myricetin

BY4741

lowast

Prot

ein

carb

onyl

s (fo

ld ch

ange

)

(c)

00

05

10

15

20

0 2 3

Days

C(DMSO)Myricetin

lowast

lowast

lowast

sod2ΔPr

otei

n ca

rbon

yls (

fold

chan

ge)

(d)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Day 0 Day 2 Day 3

BY4741

sod2Δ

(e)



ControlH2O2


50

100

Viab

ility

()

lowast

sod2Δ









2O2


2O2


5 Conclusion


2O2 This


Abbreviations





Acknowledgment


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















C(DMSO)

0 5 10 15 20 25

50

100

Days

Viab

ility

()

BY4741

(a)


C(DMSO)

Days

0

50

100

0 1 2 3 4

Viab

ility

()

lowast

sod2Δ

(b)

0 2 3

00

05

10

15

20

Days

C(DMSO)Myricetin

BY4741

lowast

Prot

ein

carb

onyl

s (fo

ld ch

ange

)

(c)

00

05

10

15

20

0 2 3

Days

C(DMSO)Myricetin

lowast

lowast

lowast

sod2ΔPr

otei

n ca

rbon

yls (

fold

chan

ge)

(d)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Myr

icet

in

C(D

MSO

)

Day 0 Day 2 Day 3

BY4741

sod2Δ

(e)



ControlH2O2


50

100

Viab

ility

()

lowast

sod2Δ









2O2


2O2


5 Conclusion


2O2 This


Abbreviations





Acknowledgment


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ControlH2O2


50

100

Viab

ility

()

lowast

sod2Δ









2O2


2O2


5 Conclusion


2O2 This


Abbreviations





Acknowledgment


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





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Research Article Effect of Myricetin, Pyrogallol, and...

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