research article dna protection against oxidative damage...

Research ArticleDNA Protection against Oxidative Damage Usingthe Hydroalcoholic Extract of Garcinia mangostana andAlpha-Mangostin

Ronaldo Carvalho-Silva1 Alanna Cibelle Fernandes Pereira1

Ruacutebens Prince dos Santos Alves123 Temenouga N Guecheva4 Joatildeo A P Henriques4

Martin Brendel1 Cristina Pungartnik1 and Fabriacutecio Rios-Santos25

1Laboratorio de Biologia de Fungos Centro de Biotecnologia Universidade Estadual de Santa Cruz (UESC) Rodovia Jorge Amadokm 16 45662900 Ilheus BA Brazil2Laboratorio de Farmacogenomica e Epidemiologia Molecular Universidade Estadual de Santa Cruz (UESC) Rodovia Jorge Amadokm 16 45662900 Ilheus BA Brazil3Laboratorio de Desenvolvimento de Vacinas Instituto de Ciencias Biomedicas Departamento de MicrobiologiaUniversidade de Sao Paulo Sao Paulo SP Brazil4Genotox-Royal Universidade Federal do Rio Grande do Sul (UFRGS) Avenida Bento Goncalves 9500Setor 4-Pred 43421S11391501970 Porto Alegre RS Brazil5Laboratorio de Imunofarmacologia Universidade Federal de Mato Grosso (UFMT) Avenida Fernando Correa da Costa n∘ 2367Boa Esperanca 78060900 Cuiaba MT Brazil

Correspondence should be addressed to Cristina Pungartnik cpungartnikyahoocombr

Received 27 October 2015 Accepted 31 January 2016

Academic Editor Rainer W Bussmann

Copyright copy 2016 Ronaldo Carvalho-Silva et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Garcinia mangostana popularly known as ldquomangosteen fruitrdquo originates from Southeast Asia and came to Brazil about 80years ago where it mainly grows in the states of Para and Bahia Although mangosteen or its extracts have been used forages in Asian folk medicine data on its potential genotoxicity is missing We therefore evaluated genotoxicitymutagenicity ofhydroethanolic mangosteen extract [HEGM 10 to 640 120583gmL] in established test assays (Comet assay micronucleus test andSalmonellamicrosome test) In the Comet assay HEGM-exposed human leukocytes showed no DNA damage No significantHEGM-induced mutation in TA98 and TA100 strains of Salmonella typhimurium (with or without metabolic activation) wasobserved and HEGM-exposed human lymphocytes had no increase of micronuclei However HEGM suggested exposureconcentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeast Saccharomyces cerevisiaeHEGM preloading effectively protected against H

2

O2

-induced DNA damage in leukocytes (Comet assay) Preloading of yeast withHEGM for up to 4 h significantly protected the cells from lethality of chronic H

2

O2

-exposure as expressed in better survivalAbsence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substancescontained in it may hold promise for pharmaceutical or nutraceutical application

1 Introduction

Despite many biological activities attributed to medicinalplants and their pharmacological properties a minor contri-bution exists in regard to their safety and toxicity in humans[1] The indiscriminate use of medicinal plants or naturalproducts in traditional medicine or its self-administration in

combination with prescribed drugs could therefore causeserious therapeutic problems that is leading to unknowndeleterious effects by the plant product or severe adverse drugcross effects respectively [2 3] It is worthwhile to mentionthat apart from acute toxicity of plant effects for examplecarcinogenic potential are more difficult to evaluate as it willbemanifested only after significant time after initial exposure

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 3430405 8 pageshttpdxdoiorg10115520163430405

2 Evidence-Based Complementary and Alternative Medicine

Thus it is not only important to describe the pharmacologicalproperties of a medicinal plant but also to establish its safetyfor human consumption

The fruit of Garcinia mangostana L a tree originatingfrom Southern Asia is widely consumed in Brazil particu-larly in its northwest region as it has not only an excellenttaste but also is supposed to have a range of medicinal prop-erties that is in treating constipation diarrhea intestinal dis-orders skin diseases and cancer [4 5] In factGmangostanaextract was shown to have antitumor anti-inflammatoryantiallergy antibacterial antifungal antiviral and antimalar-ial activity [5] Most biological properties of G mangostanaextractsmay be related to xanthonesmainly isolated from thepericarp whole fruit and bark or from leaves Phytochemicalanalysis of G mangostana revealed a significant variety ofxanthones mainly alpha- beta- and gamma-mangostinsgarcinone E 8-deoxygartanin and gartanin [5]While chem-ical studies of these compounds have progressed only limiteddata is available on their cytotoxicity either when applied asa mixture contained in G mangostana extracts or as one ofits purified chemical compounds

In vivo toxicity studies established a lethal dose at1000mgkg for female BALBc mice and suggested a suitabledose for short-term studies of less than 200mgkg [6]A few cytotoxicity studies are available mouse xenographanticancer testing showed xanthones to inhibit neoplasticcell reproduction [7] anticolon cancer effect was investigatedon HCT116 human colorectal carcinoma cells includingcytotoxicity apoptosis antitumorigenicity and effect on cellsignaling pathways dose dependent killing of HCT116 cellsexposed to mangosteen xanthone extract 120572-mangostin and120574-mangostin gave IC

50values of 65 plusmn 10 120583gmL 51 plusmn

02 120583gmL and 72 plusmn 04 120583gmL respectively finally 120572-mangostin treatment induced mitochondria-mediated toxi-city and apoptosis in human breast cancer line MDA-MB231[8]

G mangostana may contain various compounds whichwhen extracted could have beneficial biological activities andthus could be a source for pharmaceutical and nutraceuticalproducts similar to those already found in Teucrium ramo-sissimum [9] Copaifera langsdorffii [10] andMoringa oleifera[11] Unfortunately data on genotoxicity of crude extract aswell as substances extracted from mangosteen pericarp isnot available This leaves a gap in our knowledge on theanticancer potential ofmangosteen extract and onmangostinsafety practiced in self-medication in traditional medicine[5]

In this work we therefore evaluated the genotoxi-citymutagenicity and the antigenotoxic potential of thehydroethanolic extract of G mangostana [HEGM] in acombination of biological tests that is micronucleus Cometassay Ames test and antioxidant activity in the yeast Saccha-romyces cerevisiae

2 Materials and Methods

21 Preparation of Extract Fruits of Garcinia mangostanaL were collected at Una Bahia Brazil After cleaning andwashing the pericarp of fruits (approximately 20 g) was

broken and macerated for 24 h with 1 L of ethanol (70)The supernatant was vacuum-filtered (filter paper) and con-centrated by rotary evaporation until complete withdrawalof ethanol followed by freezing and lyophilization to obtainthe hydroethanolic extract of G mangostana [HEGM] In allexperiments HEGM was dissolved with DMSO (7mM)

All media and chemicals used are SIGMA (Sigma-Aldrich Inc St LouisMOUSA) or VETEC (Vetec QuimicaFina Ltda RJ Brazil) or as otherwise stated

22 HEGM Cytotoxicity Human blood cell viability testby trypan blue exclusion was used to determine adequateconcentrations of HEGM Bloodwas collected from a healthyindividual Samples of 20 120583L of whole bloodmL of RPMI1640 medium contained HEGM at doses ranging from 10to 1280120583gmL Cell viability was measured by collectingHEGM-exposed samples after 1 2 and 4 h of incubation at37∘C and mixing 15 120583L of each cell suspension with 15 120583L oftrypan blue (4) in a microcentrifuge tube Cell suspensionwas analyzed by light microscopy in a Neubauer chamberViable cells were unstained while dead cells appeared stainedblue HEGM doses used in this study allowed cell viabil-ity gt90 Total 2100 cells were analyzed throughout theexperiment Through the line equation of the IC

50was IC

50

1853 120583gmL

23 HEGM Genotoxic Activity

231 Comet Assay Nucleated cells from peripheral bloodwere obtained from 8mL of human peripheral blood of amale age 26 and nonsmoking healthy individual Blood wastransferred to tubes containing anticoagulant and centrifugedfor 10min at 1000 g The buffy coat was collected along withred blood cells and serum Cells were then transferred to asterile microcentrifuge tube and stored protected from lightat room temperature until the start of microcultivations

The Comet assay was performed based on the protocolproposed by Tice et al [12] Briefly a cell suspension of 20120583Lof cell culture in 1mL of RPMI 1640 was exposed to 20120583L ofHEGM (final concentration of 10 to 640 120583gmL 1 h 37∘C)DMSO as negative control (7mM 1 h 37∘C) or hydrogenperoxide as positive control (H

2O2 1 mM 1 h 37∘C) 15 120583L

of that cell suspension was mixed with 95120583L of low-meltingagarose in a microcentrifuge tube The cell suspension wastransferred to an agarose precoated glass slide covered witha glass cover slip (22mm times 66mm) and immediately placedin a refrigerator (4∘C) for 5min to allow complete agarosesolidification Slides were prepared in duplicate Cover slipswere then removed and the slides immersed in cold (4∘C)lysing solution (25M NaCl 100mM Na

2EDTA 10mM Tris

1 (vv) Triton X-100 and 10 DMSO pH 10) protectedfrom light for at least 8 h DNA was allowed to unwind for20min in freshly prepared alkaline electrophoresis buffer(1mMNa

2EDTA 03N NaOH pH 13) prior to electrophore-

sis Horizontal electrophoresis was performed at 25Vcm for20min at 4∘C All steps were performed under yellow lightto minimize additional DNA damage The slides were thenplaced vertically and gently washed 3x for 5min with neu-tralizing buffer (04M Tris-HCl buffer pH 75)

Evidence-Based Complementary and Alternative Medicine 3

Slideswere dried at 37∘C for 15 h and then fixed for 10min(15 trichloroacetic acid 5 zinc sulphate heptahydrate and5 glycerol) washed 3 times with distilled water After 24 hfixation the slides were stained with silver nitrate (1) ina rotatory shaker for 20min at 37∘C Slides were washed 3times with distilled water neutralized with acetic acid (1)for 5min and again washed 3x with distilled waterThe slideswere then dried at room temperature and properly storeduntil analysis

Two persons analyzed each sample twice by opticalmicroscopy (100-fold magnification) In each of the twoslides 100 Comets were analyzed and classified as 0 1 2 3and 4 according to the size of their tail type [12] The indexof genetic damage at each HEGM concentration was deter-mined by summing the number of multiplications of Cometswith their respective class and the percentage of damage ineach treatment

Bars in the Comet assay represent mean and SD of threeindependent experiments (values obtained from an averageof 100 cells per pool) ANOVA-Tukey was conducted tocompare means bars represent mean plusmn SD as calculated bythe GraphPad Prismreg program (GraphPad Software Inc SanDiego CA)

232 SalmonellaMicrosome Mutagenicity Assay (Ames Test)Salmonella typhimurium strains TA98 and TA100were kindlyprovided by B M Ames (University of California BerkeleyCA USA) Mutagenicity was assayed by the preincubationprocedure The S9 metabolic activation mixture (S9 mix)was prepared according to Maron and Ames [13] Briefly100 120583L of bacterial test cultures (1-2 times 109 cellsmL) wasincubated for 20min at 37∘C with different amounts of Gmangostana extract in the presence or absence of S9 mixwithout shaking Subsequently 2mL of soft agar (06 agar05 NaCl 50120583M histidine and 50 120583M biotin pH 7446∘C) were added to the test tube and poured immediatelyonto a plate of minimal agar (15 agar Vogel-Bonner Emedium containing 2 glucose) The concentration rangeof HEGM extract was determined in experiments using Styphimurium strain TA100 with and without metabolizationand cytotoxicity was observed at concentrations higher than20120583gplate in the absence of S9 mix and 500 120583gplate in thepresence of S9 mix In the mutagenicity assay the dose rangebetween 100 and 500 120583gplate was used in the presence of S9mix and a concentration range between 4 and 20 120583gplate wasapplied in the absence of S9 mix Aflatoxin B1 (1 120583gplate)was used as positive control for all strains in the presence ofmetabolic activation (+S9 mix) In the absence of metabolicactivation 4-nitroquinoline-oxide (4-NQO 05120583gplate)wasused for strain TA98 and sodium azide (1 120583gplate) for strainTA100 Plates were incubated in the dark at 37∘C for 48 hbefore counting the revertant colonies

The results were analyzed by the Salmonella StatisticAssay (Environmental Monitoring System Laboratory EPASoftware Version 23 April 1988) A test substance wasconsidered mutagenic when significant dose response andANOVA variance were observed and the increase in themean number of revertants on test plates was at least twofoldhigher than that observed in the negative control

233 Micronucleus Test (MN) The modified cytokinesisblocked method of Fenech and Morley [14] was used todetermine the frequency of MN Briefly 1mL of whole bloodwas mixed with 9mL of RPM1 1640 10 fetal bovine serumpenicillin (100 unitsmL) and streptomycin (100 120583gmL)Phytohemagglutinin was added at a concentration of 2to stimulate lymphocyte division Cells were exposed toconcentrations of HEGM (160 to 640 120583gmL) and incubatedin 5CO

2 at 37∘C for 72 hVincristine (1 nM) and incubation

in 5 CO2at 37∘C for 72 h were used as positive control

whereas DMSO (7mM) and incubation in 5 CO2at 37∘C

for 72 h were the negative control At 44 h cytochalasin Bwas added at a concentration of 3120583gmL At 72 h cells werecollected by centrifugation at 1000 rpm for 10min and thentreatedwith 1of sodiumcitrate hypotonic solution for 5minat room temperature Treatment with methanolacetic acid(3 1 vv) was repeated twice and the cells were collected onmicroscope slides Slideswere coded stainedwith 5Giemsafor 5min and scored according to the criteria described [14]The mean MN frequency was calculated for each individualfrom 1000 binucleated cells Error bars represent standarddeviation as calculated by the GraphPad Prism program(GraphPad Software Inc San Diego CA) Values repre-sented in the graph are the average of three experiments plusmn SDfollowed by ANOVA-Tukeyrsquos analyses to compare means

24 Antigenotoxic Activity Using Comet Assay Antigenotoxicactivity wasmeasuredwith theComet assay [12] using humanperipheral blood cells (leukocytes) Cells were preloadedwith HEGM (160 320 640120583gmL) for 1 to 4 h collected bycentrifugation and twice washed with saline solution beforebeing re-suspended in RPMI 1640 medium and exposed for5min to H

2O2(1mM) at 37∘C DMSO (SIGMA 7mM up

to 4 h) was used as negative control Alternatively the sameprocedure was done with cells preloaded with 120572-mangostin125 120583gmL (M3824 SIGMA) for 1 to 4 h centrifuged washedand resuspended as above but exposed to H

2O2(1 05 or

025mM) at 37∘C Ethanol (SIGMA 2 up to 4 h) was usedas negative control due to better solubility of the chemicalError bars represent standard deviation as calculated by theGraphPad Prism program (GraphPad Software Inc SanDiego CA) Values represented in the graph are the average ofthree experiments plusmn SD followed by ANOVA-Tukeyrsquos analy-ses to compare means

25 Antioxidant Activity Using Saccharomyces cerevisiaeAntioxidant activity of HEGM was assessed using thesurvival of WT Saccharomyces cerevisiae strain BY4742[EUROSCARFMAT120572 his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0] initiallyexposed to HEGM (preloaded) and then plated on agarmedium containingH

2O2(4mM) Yeastmedia and solutions

were prepared according to [15] Complete medium (YPD)was used for routine growth of yeast cells Stationary phase(STAT) cultures with 2 times 108 cellsmL were obtained afterinoculation of an isolated colony into liquid YPD and 72 hincubation at 30∘C with aeration by shaking Five mL ofcell suspension was centrifuged and the collected cells werewashed 3x and resuspended in 2mLNaCl (09) normalizedto 107 cellsmL From this suspension 100 120583L of cells was


exposed to different HEGM concentrations (0 to 640120583gmL)for 0 to 4 h Suspensions were properly diluted and plated onYPD with and without H

2O2(4mM) Plates were incubated

at 30∘C for 72 h and colony numbers counted Graphs weregenerated using the GraphPad Prism program (GraphPadSoftware Incorporation SanDiego CAUSA) error bars rep-resent the standard deviations of at least 3 independent exper-iments The measurement of the effect of HEGM using thepercentage survival versus dose with a logarithmic ordinatewas performedusing a typical inactivation kinetics curve [16]where the rate of decrease of the number of active single yeastcells with respect to dose is proportional to the number ofactive cells remaining at that dose level When killing followssingle hit kinetics the inactivation curve is exponential andthe graph in the semilog plot will yield a linear curve witha negative slope This presentation allows a rapid estimationof the dose reduction factor [17] Statistical analysis betweenparallel experiments was performed using the standard devi-ation

3 Results and Discussion

Many plants or their extracts used in traditional medicinehave not been scientifically validated for their effectivenessand there is even less information available about the poten-tial risks that their use might pose to public health [18] Afterall some plants used as food or for therapeutic purposes havebeen proven to contain substances with cytotoxic genotoxicor mutagenic activity [1 3 6 8 18 19]

The pericarp of mangosteen has been used to treatconstipation diarrhea intestinal disorders and skin diseases[5 17 20] This plant is native from Southeast Asia where theconcentrate of the peel of the mangosteen is used to mainlyfight infections and stomach ailments [4 5] Tests performedwith different extractions of mangosteen pericarp (includinghydroethanolic) suggested the existence of compounds withanalgesic [21] antioxidant [22] and anti-inflammatory activ-ity [23] as well as the ability to induce apoptosis in cancercells [8] amongst other activities However up to date noresearch has been reported investigating possible genotoxicmutagenic activity of HEGM the hydroethanolic extract ofmangosteen pericarp

31 Genotoxic Activity The genotoxic activity of HEGM wasevaluated using the Comet assay (Figure 1) micronucleustest (Figure 2) and Salmonellamicrosome assay (Table 1)HEGM was considered not mutagenic in the three testsystems as there was no statistically significant differencebetween untreated cells [negative control] and cells exposedto different concentrations of HEGM in the Comet assay(Figure 1) there was no statistical significant differencebetween the negative control and the HEGM-exposed strainsin the Salmonellamicrosome assay either with or with-out metabolization (Table 1) Finally whereas vincristine-exposed cells exhibited a significant increase in micronucleusfrequency all other treatments that is negative control andexposure to HEGM did not (Figure 2)

The Comet assay has been used to evaluate genotoxicityof extracts from Polyalthia longifolia [24] Cedrela odorata

DMSO (mM)

lowastlowastlowast

H2O2 (mM)10 20 40 80 160 320 640 17

HEGM (120583gmL)

0

25

50

75

100

DN

A d

amag

e (

)

Figure 1 Genotoxic activity measured by Comet assay of humanleukocytes HEGM treated cells (final concentration of 10 to640 120583gmL 1 h 37∘C) DMSO (negative control 7mM 1 h 37∘C)or H2

O2

(positive control 1mM 1 h 37∘C) Error bars representmean plusmn SD of 3 independent experiments The analysis of variancenonparametric (ANOVA) and Tukeyrsquos test used to compare meansDMSO-induced DNA damage (basal) was subtracted from HEMG-induced DNA damage lowastlowastlowast119901 lt 0001

DMSO (mM) Vincristine (nM)160 320 640 1070

HEGM (120583gmL)

0

5

10

15

20

25

30M

icro

nucle

us1

000

cells

lowastlowastlowast

Figure 2 Micronuclei frequency per 1000 human lymphocytesexposed to HEGM (160 to 640120583gmL 5 CO

2

37∘C for 72 h)vincristine (1 nM 5CO

2

37∘C for 72 h positive control) orDMSO(7mM 5 CO

2

37∘C for 72 h negative control) The analysis ofvariance nonparametric (ANOVA) and Tukey test used to comparemeans lowastlowastlowast119901 lt 0001

L and Juglans regia L [25] and Acacia aroma [26] anddemonstrated their safety On the other hand a survey of barkextract ofNauclea traditionally used in African countries forthe treatment of fever [27] diarrhea and malaria showed ahigh rate of genotoxicity in the Comet assay suggesting thatingestion of this extract is a health risk These findings pointto the importance of genotoxic risk assessment [28] that isto determine whether plant extracts contain substances thatmay damage the genetic material and thus might be putativecarcinogens endangering the health of users [1 29]The sameholds true for extracts of Polyscias filicifolia S [30] Camelliaoleifera A [31] and Inula viscosa [32] which yielded positiveresults in the micronucleus test that is a bioindicator ofmutagenicity

The concentration range of HEGM extract was deter-mined in experiments using S typhimurium strain TA100


Table 1 Induction of his+ revertants in S typhimurium strains by Garcinia mangostana extract with and without metabolic activation (S9mix)

S typhimurium strains

Substance Concentration (120583gplate) TA100 TA98Revplatea MIb Revplatea MIb

Without metabolic activation (minusS9)

NCc extract

mdash 953 plusmn 199 mdash 300 plusmn 52 mdash4 903 plusmn 101 095 280 plusmn 27 0938 1020 plusmn 166 107 203 plusmn 46 06812 993 plusmn 503 104 290 plusmn 87 09716 950 plusmn 195 100 283 plusmn 68 09420 813 plusmn 571 085 197 plusmn 90 066

PCd 05 (4NQO)1 (NaN3

) 4797 plusmn 571lowastlowastlowast 503 3627 plusmn 345lowastlowastlowast 1209With metabolic activation (+S9)

NCc extract


PCd 1 (AFB1

) 2963 plusmn 627lowastlowast 328 5697 plusmn 157lowastlowastlowast 1155aNumber of revertantsplate mean of three independent experiments plusmn SD bMI mutagenic index (number of his+ induced in the samplenumber ofspontaneous his+ in the negative control) cNC negative control DMSO (10120583L) used as solvent for the extract dPC positive control (minusS9) sodium azide toTA100 4-NQO to TA98 (+S9) aflatoxin B

1 lowastlowastData significantly different in relation to the negative control 119901 lt 001 lowastlowastlowast119901 lt 0001

with and without metabolization and cytotoxicity wasobserved at concentrations higher than 20120583gplate in theabsence of S9 mix and 500120583gplate in the presence of S9 mix(data not shown) In the mutagenicity assay the dose rangebetween 100 and 500 120583gplate was used in the presence of S9mix and a concentration range between 4 and 20 120583gplate wasapplied in the absence of S9 mix (Table 1) The extract wasnot mutagenic in strain TA98 (detects frameshiftmutation inthe DNA target ndashC-G-C-G-C-G-C-G) regardless of absenceor presence of metabolic activation Also no mutagenicitywas detected in strain TA100 (detects base pair substitutions(leucine [GAG] by proline [GGG])) in the absence or pres-ence of metabolic activation This method was already usedto screen the extracts of Parthenium hysterophorus L [33]Peperomia pellucida L Eichhornia crassipes S and Colocasiaesculenta S [11] where no mutagenicity at the evaluated con-centrations was found Also the Salmonellamicrosome assayhas been used to evaluate the biosafety of extracts of Parthe-nium hysterophorus L [33] Peperomia pellucida L Eichhorniacrassipes S and Colocasia esculenta [11] no mutagenicity wasobserved at evaluated concentrations by this effectivemethodfor the detection of genotoxicity

32 Antigenotoxic Activity We also used the Comet assayto assess the antigenotoxic activity of HEGM (Figure 3) andfound it to be effective against DNA damage induced byH2O2in doses up to 320120583gmL (1 2 and 4 h exposure)

Comparing preloading of HEGM up to 4 hours of exposurewhen doubling the dose from 160 to 320 120583gmL we foundincreased DNA-induced damage protection observable asreduced amount of detected DNA damage However at

640 120583gmL results were almost the same when compared tothose at 320120583gmL indicating that the lower concentrationofHEGMalready achievedmaximumprotection Also whencomparing 2 to 4 hours of HEGM preloading results ofDNA-induced damage protection were similar Protection ofcells may be due to potential antioxidants present in HEGM[17 18] that are sufficient to neutralize DNAdamage inducingH2O2-derived free radicals [31] thus preventing DNA-strand

breaks detected by Comet assay Internal controls validatedthat there was no significant increase in DNA damage at anyexposure time and concentration of HEGM preloading whencompared to the negative control (DMSO) However therewas a significant increase when comparing positive (H

2O2)

to negative control (DMSO)Indeed it was shown that extracts of Portulaca oleracea

L [31] Gymnema montanum [32] and olive leaf [33] wereeffective to protect against DNA damage induced by H

2O2-

generated oxidative stress similarly Gentiana asclepiadeaextracts showed protection against oxidative damage causedby H2O2[34] and leaf extract of Dendrobium speciosum con-

tains compounds protecting against 4NQO-induced DNAdamage [35] The discovery of substances able to protect thegenetic material from genotoxic agents is important as it maylead to products or procedures that may aid in the preventionof genetic damage-induced diseases such as cancer [7 8]

According to HPLC measurements the HEGM extractused in this study contained 967 120572-mangostin (cf Sup-plementary Material 1 (see Supplementary Material avail-able online at httpdxdoiorg10115520163430405)) whichmeans that there is 6144 120583g of 120572-mangostin (or 161 120583M)in 640 120583g of HEGM In order to test whether 120572-mangostin


lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60D

NA

dam

age (

)

(a)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(b)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(c)

Figure 3 Antigenotoxic activity asmeasured by amount ofDNAdamage as detected by theComet assayHEGMpreloaded human leukocytes(160 320 and 640 120583gmL up to 4 h) all following cells were treated with H

2

O2

(1mM 5min at 37∘C) (a b and c) The cells were preloadedto HEGM concentrations for 1 2 and 4 h respectively followed by exposure to H

2

O2

for 5 minutes For positive control cells were treatedonly with H

2

O2

(first column)The analysis of variance nonparametric (ANOVA) and Tukey test used to compare means thus evaluating thereduction in DNA damage lowastlowastlowast119901 lt 0001

was responsible for DNA antigenotoxicity pure 120572-mangostinisolated from pericarp of Garcinia mangostana (SIGMA) wasused (Figure 4) Results demonstrated that a concentration of125 120583M 120572-mangostin protectedDNA fromdamage caused byH2O2in a dose dependent manner Higher doses led to cellu-

lar cytotoxicity (data not shown)Thus the results of Figure 4suggest that some components present in the pericarp ofthe Garcinia mangostana L especially 120572-mangostin protectagainst H

2O2-induced DNA damage

33 Anti-Oxidant Activity Theantioxidant activity ofHEGMwas shown in vitro using the DPPH assay and results weresimilar to those related by Da Silva and coworkers [22] (Sup-plementary Material 2) We then tested HEGM for its in vivoantioxidant activity using S cerevisiae preloaded withHEGM(from 160 to 640 120583gmL) and then plating these cells onH2O2-containing agar (Figure 5) After one hour of HEGM

pretreatment there was already an increase in survival at

HEGM exposure doses of 320 and 640 120583gmL and aftertwo hours before loading there was a further significantincrease in survival of yeast as compared to the one-hourtreatment and the negative control Yeast cells preloaded with320 120583gmL HEGM for two hours reached about 60 survivalandwhen again doubling theHEGMdose about 80 survivalwas achieved (Figure 5)

Doubling the time of exposure in preloading did notenhance protective antioxidant capacity Taken togetherthese results suggest that HEGM contains at least one com-pound with antioxidant activity that when taken up by theyeast cells can neutralize the oxidant effects of H

2O2 most

probably that of free radicals thus alleviating cellular oxida-tive stress Preloading yeast cells with HEGM for two andfour hours yielded better protection against H

2O2-induced

oxidative stress than shorter exposure times Our result isin agreement with published data that suggest the presenceof compounds with antioxidant activity in the pericarp ofGarcinia mangostana L [22]


H2O2

(mM)

(a)

(b)

(c)

(d)

10 125 125 125 210 05 025

H2O2

0

10

20

30

40

50

60

DN

A d

amag

e (

)

Ethanol

Figure 4 Antigenotoxicity of alpha-mangostin measured by theamount of DNA damage detected by the Comet assay Humanleukocytes were treated (preloaded) with 125 120583M alpha-mangostinfor 4 h and then exposed for 5 minutes to different concentrations ofH2

O2

(1 05 and 025mM) at 37∘C (a) Positive control 1mMH2

O2

(b c and d) H

2

O2

exposure of 1 05 and 025mM respectivelyethanol as negative control (a) versus (b c and d) 119901 lt 0001 (c)versus (d) (119901 lt 05)

1

10

100

Surv

ival

()

1 2 3 40Time of HEGM preloading (h)

Figure 5 Survival of WT strain of S cerevisiae to chronic H2

O2

(4mM) induced oxidative stress Cells preloaded (up to 4 h) witheither with saline (◻) or with HEGM at exposure concentrations of160120583gmL (998771) 320 120583gmL (e) 640 120583gmL (X)

4 Conclusions

In summary the hydroethanolic extract of G mangostanashowed no genotoxicitymutagenicity at exposure concentra-tions up to 640 120583gmL but was effective as an antioxidant foryeast and effective in protecting DNA against damage fromfree radicals produced by H

2O2

The absence of genotoxicity together with the shownantioxidant in vivo properties makes application or ingestionof extracts of G mangostana safe and even beneficial tohumans as incidence of oxidative stress-induced geneticdamage may be lowered Contributing to prevention of DNAdamage-caused disease by neutralizing free radicals derived

by either cellular metabolism or external agents mangosteenextract or isolated active compounds thereof may thus holdpromise for pharmaceutical or nutraceutical applications

Conflict of Interests

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

Acknowledgments

This research was supported by grants from CNPq RonaldoCarvalho-Silva held held a CAPES Master Fellowship (Pro-grama de Pos-Graduacao em Genetica e Biologia Molecular)and Alanna Cibelle Fernandes Pereira held a CNPq DoctoralFellowship (Programa de Pos-Graduacao em Biologia eBiotecnologia deMicrorganismos) Rubens Prince dos SantosAlves held a CNPq Scientific Initiation Fellowship MartinBrendel is Visiting Scientist at UESC and held a FAPESBFellowship Research is supported by financial support fromCNPq and CAPESPVE

References

[1] R W Bussmanna G Malcab A Glenna et al ldquoToxicity ofmedicinal plants used in traditional medicine in NorthernPerurdquo Journal of Ethnopharmacology vol 137 no 1 pp 121ndash1402011

[2] H Nordeng K Bayne G C Havnen and B S PaulsenldquoUse of herbal drugs during pregnancy among 600 Norwegianwomen in relation to concurrent use of conventional drugs andpregnancy outcomerdquoComplementaryTherapies inClinical Prac-tice vol 17 no 3 pp 147ndash151 2011

[3] M F D Amorim M F F M Diniz M S T Araujo et al ldquoThecontrovertible role of kava (Piper methysticum G Foster) ananxiolytic herb on toxic hepatitisrdquo Brazilian Journal of Phar-macognosy vol 17 no 3 pp 448ndash454 2007

[4] Y Akao Y Nakagawa M Iinuma and Y Nozawa ldquoAnti-cancereffects of xanthones from pericarps of mangosteenrdquo Interna-tional Journal of Molecular Sciences vol 9 no 3 pp 355ndash3702008

[5] J Pedraza-Chaverri N Cardenas-Rodrıguez M Orozco-Ibarra and J M Perez-Rojas ldquoMedicinal properties of man-gosteen (Garcinia mangostana)rdquo Food and Chemical Toxicologyvol 46 no 10 pp 3227ndash3239 2008

[6] N Kosem K Ichikawa H Utsumi and P Moongkarndi ldquoInvivo toxicity and antitumor activity of mangosteen extractrdquoJournal of Natural Medicines vol 67 no 2 pp 255ndash263 2013

[7] A F A Aisha K M Abu-Salah Z Ismail and A Majid ldquoInvitro and in vivo anti-colon cancer effects of Garcinia man-gostana xanthones extractrdquo BMC Complementary and Alterna-tive Medicine vol 12 article 104 2012

[8] H Kurose M-A Shibata M Iinuma and Y Otsuki ldquoAlter-ations in cell cycle and induction of apoptotic cell death in breastcancer cells treated with 120572-mangostin extracted from mangos-teen pericarprdquo Journal of Biomedicine and Biotechnology vol2012 Article ID 672428 9 pages 2012

[9] M Ben Sghaier J Boubaker I Skandrani et al ldquoAntimutagenicantigenotoxic and antioxidant activities of phenolic-enriched


extracts from Teucrium ramosissimum combination with theirphytochemical compositionrdquo Environmental Toxicology andPharmacology vol 31 no 1 pp 220ndash232 2011

[10] B C Cavalcanti L V Costa-LotufoM OMoraes et al ldquoGeno-toxicity evaluation of kaurenoic acid a bioactive diterpenoidpresent in Copaiba oilrdquo Food and Chemical Toxicology vol 44no 3 pp 388ndash392 2006

[11] B N Singh B R Singh R L Singh et al ldquoOxidative DNAdam-age protective activity antioxidant and anti-quorum sensingpotentials of Moringa oleiferardquo Food and Chemical Toxicologyvol 47 no 6 pp 1109ndash1116 2009

[12] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicology test-ingrdquo Environmental and Molecular Mutagenesis vol 35 no 3pp 206ndash221 2000

[13] D M Maron and B N Ames ldquoRevised methods for theSalmonella mutagenicity testrdquo Mutation ResearchEnvironmen-tal Mutagenesis and Related Subjects vol 113 no 3-4 pp 173ndash215 1983

[14] M Fenech and A A Morley ldquoMeasurement of micronuclei inlymphocytesrdquo Mutation Research vol 147 no 1-2 pp 29ndash361985

[15] D Burke D Dawson and T StearnsMethods in Yeast GeneticsCold Spring Harbour Laboratory Course Manual Cold SpringHarbor Laboratory Press New York NY USA 2000

[16] F I Biso C M Rodrigues D Rinaldo et al ldquoAssessment ofDNA damage induced by extracts fractions and isolated com-pounds of Davilla nitida and Davilla elliptica (Dilleniaceae)rdquoMutation Research vol 702 no 1 pp 92ndash99 2010

[17] DObolskiy I Pischel N Siriwatanametanon andMHeinrichldquoGarciniamangostanaL a phytochemical and pharmacologicalreviewrdquo Phytotherapy Research vol 23 no 8 pp 1047ndash10652009

[18] J Pedraza-Chaverrı L M Reyes-Fermın E G Nolasco-Amayaet al ldquoROS scavenging capacity and neuroprotective effect of120572-mangostin against 3-nitropropionic acid in cerebellar granuleneuronsrdquo Experimental and Toxicologic Pathology vol 61 no 5pp 491ndash501 2009

[19] J Cui W Hu Z Cai et al ldquoNew medicinal properties ofmangostins Analgesic activity and pharmacological character-ization of active ingredients from the fruit hull of Garciniamangostana Lrdquo Pharmacology Biochemistry and Behavior vol95 no 2 pp 166ndash172 2010

[20] S Tewtrakul C Wattanapiromsakul and W MahabusarakamldquoEffects of compounds from Garcinia mangostana on inflam-matory mediators in RAW2647 macrophage cellsrdquo Journal ofEthnopharmacology vol 121 no 3 pp 379ndash382 2009

[21] C J da Silva J K Bastos and C S Takahashi ldquoEvaluation ofthe genotoxic and cytotoxic effects of crude extracts of Cordiaecalyculata andEchinodorus grandiflorusrdquo Journal of Ethnophar-macology vol 127 no 2 pp 445ndash450 2010

[22] R N Da Silva V F S Kahl S M Da Silva et al ldquoAntigenotox-icity and antioxidant activity of Acerola fruit (Malpighia glabraL) at two stages of ripenessrdquo Plant Foods for Human Nutritionvol 66 no 2 pp 129ndash135 2011

[23] A Al-Zubairi P Ismail C P Pei and A Rahmat ldquoGenotoxiceffect of Catha edulis (khat) crude extract after sub-chronicadministration in ratsrdquo Environmental Toxicology and Pharma-cology vol 25 no 3 pp 298ndash303 2008

[24] S L Jothy Y Chen J R Kanwar and S Sasidharan ldquoEvaluationof the genotoxic potential against H

2

O2

-radical-mediated DNAdamage and acute oral toxicity of standardized extract ofPolyalthia longifolia leafrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 925380 13 pages2013

[25] D C Almonte-Flores N Paniagua-Castro G Escalona-Cardoso and M Rosales-Castro ldquoPharmacological and geno-toxic properties of polyphenolic extracts of Cedrela odorata Land Juglans regia L barks in rodentsrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2015 Article ID 1873468 pages 2015

[26] M Ouedraogo F L Da A Fabre et al ldquoEvaluation of the bron-chorelaxant genotoxic and antigenotoxic effects ofCassia alataLrdquo Evidence-Based Complementary and Alternative Medicinevol 2013 Article ID 162651 11 pages 2013

[27] B Lu XWu YDong J Gong andY Zhang ldquoMutagenicity andsafety evaluation of ethanolic extract of Prunus mumerdquo Journalof Food Science vol 74 no 9 pp T82ndashT88 2009

[28] A Ramos R Rivero M C Victoria A Visozo J Pilotoand A Garcıa ldquoAssessment of mutagenicity in Partheniumhysterophorus Lrdquo Journal of Ethnopharmacology vol 77 no 1pp 25ndash30 2001

[29] C Ciganda and A Laborde ldquoHerbal infusions used for inducedabortionrdquo Journal of Toxicology Clinical Toxicology vol 41 no3 pp 235ndash239 2003

[30] A Thepouyporn K Kwanbunjan S Pooudong and S Chang-bumrung ldquoMutagenicity study of weeds and common plantsused in traditional medicine and for animal feedrdquoThe SoutheastAsian Journal of Tropical Medicine and Public Health vol 37 no3 pp 195ndash202 2006

[31] J Behravan F Mosafa N Soudmand E Taghiabadi B MRazavi and G Karimi ldquoProtective effects of aqueous andethanolic extracts of Portulaca oleracea L aerial parts on H

2

O2

-induced DNA damage in lymphocytes by Comet assayrdquo Journalof Acupuncture and Meridian Studies vol 4 no 3 pp 193ndash1972011

[32] K M Ramkumar L Sankar C Manjula et al ldquoAntigenotoxicpotential of Gymnema montanum leaves on DNA damage inhuman peripheral blood lymphocytes and HL-60 cell linerdquoEnvironmental and Molecular Mutagenesis vol 51 no 4 pp285ndash293 2010

[33] L Seddik TM Bah A AouesM Slimani andM BenderdourldquoElucidation of mechanisms underlying the protective effects ofolive leaf extract against lead-induced neurotoxicity in WistarratsrdquoThe Journal of Toxicological Sciences vol 36 no 6 pp 797ndash809 2011

[34] AHudecova KHasplova EMiadokova et al ldquoGentiana ascle-piadea protects human cells against oxidation DNA lesionsrdquoCell Biochemistry and Function vol 30 no 2 pp 101ndash107 2012

[35] M Moretti L Cossignani F Messina et al ldquoAntigenotoxiceffect composition and antioxidant activity of Dendrobiumspeciosumrdquo Food Chemistry vol 140 no 4 pp 660ndash665 2013

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


Thus it is not only important to describe the pharmacologicalproperties of a medicinal plant but also to establish its safetyfor human consumption

The fruit of Garcinia mangostana L a tree originatingfrom Southern Asia is widely consumed in Brazil particu-larly in its northwest region as it has not only an excellenttaste but also is supposed to have a range of medicinal prop-erties that is in treating constipation diarrhea intestinal dis-orders skin diseases and cancer [4 5] In factGmangostanaextract was shown to have antitumor anti-inflammatoryantiallergy antibacterial antifungal antiviral and antimalar-ial activity [5] Most biological properties of G mangostanaextractsmay be related to xanthonesmainly isolated from thepericarp whole fruit and bark or from leaves Phytochemicalanalysis of G mangostana revealed a significant variety ofxanthones mainly alpha- beta- and gamma-mangostinsgarcinone E 8-deoxygartanin and gartanin [5]While chem-ical studies of these compounds have progressed only limiteddata is available on their cytotoxicity either when applied asa mixture contained in G mangostana extracts or as one ofits purified chemical compounds

In vivo toxicity studies established a lethal dose at1000mgkg for female BALBc mice and suggested a suitabledose for short-term studies of less than 200mgkg [6]A few cytotoxicity studies are available mouse xenographanticancer testing showed xanthones to inhibit neoplasticcell reproduction [7] anticolon cancer effect was investigatedon HCT116 human colorectal carcinoma cells includingcytotoxicity apoptosis antitumorigenicity and effect on cellsignaling pathways dose dependent killing of HCT116 cellsexposed to mangosteen xanthone extract 120572-mangostin and120574-mangostin gave IC

50values of 65 plusmn 10 120583gmL 51 plusmn

02 120583gmL and 72 plusmn 04 120583gmL respectively finally 120572-mangostin treatment induced mitochondria-mediated toxi-city and apoptosis in human breast cancer line MDA-MB231[8]

G mangostana may contain various compounds whichwhen extracted could have beneficial biological activities andthus could be a source for pharmaceutical and nutraceuticalproducts similar to those already found in Teucrium ramo-sissimum [9] Copaifera langsdorffii [10] andMoringa oleifera[11] Unfortunately data on genotoxicity of crude extract aswell as substances extracted from mangosteen pericarp isnot available This leaves a gap in our knowledge on theanticancer potential ofmangosteen extract and onmangostinsafety practiced in self-medication in traditional medicine[5]

In this work we therefore evaluated the genotoxi-citymutagenicity and the antigenotoxic potential of thehydroethanolic extract of G mangostana [HEGM] in acombination of biological tests that is micronucleus Cometassay Ames test and antioxidant activity in the yeast Saccha-romyces cerevisiae

2 Materials and Methods

21 Preparation of Extract Fruits of Garcinia mangostanaL were collected at Una Bahia Brazil After cleaning andwashing the pericarp of fruits (approximately 20 g) was

broken and macerated for 24 h with 1 L of ethanol (70)The supernatant was vacuum-filtered (filter paper) and con-centrated by rotary evaporation until complete withdrawalof ethanol followed by freezing and lyophilization to obtainthe hydroethanolic extract of G mangostana [HEGM] In allexperiments HEGM was dissolved with DMSO (7mM)

All media and chemicals used are SIGMA (Sigma-Aldrich Inc St LouisMOUSA) or VETEC (Vetec QuimicaFina Ltda RJ Brazil) or as otherwise stated

22 HEGM Cytotoxicity Human blood cell viability testby trypan blue exclusion was used to determine adequateconcentrations of HEGM Bloodwas collected from a healthyindividual Samples of 20 120583L of whole bloodmL of RPMI1640 medium contained HEGM at doses ranging from 10to 1280120583gmL Cell viability was measured by collectingHEGM-exposed samples after 1 2 and 4 h of incubation at37∘C and mixing 15 120583L of each cell suspension with 15 120583L oftrypan blue (4) in a microcentrifuge tube Cell suspensionwas analyzed by light microscopy in a Neubauer chamberViable cells were unstained while dead cells appeared stainedblue HEGM doses used in this study allowed cell viabil-ity gt90 Total 2100 cells were analyzed throughout theexperiment Through the line equation of the IC

50was IC

50

1853 120583gmL

23 HEGM Genotoxic Activity

231 Comet Assay Nucleated cells from peripheral bloodwere obtained from 8mL of human peripheral blood of amale age 26 and nonsmoking healthy individual Blood wastransferred to tubes containing anticoagulant and centrifugedfor 10min at 1000 g The buffy coat was collected along withred blood cells and serum Cells were then transferred to asterile microcentrifuge tube and stored protected from lightat room temperature until the start of microcultivations

The Comet assay was performed based on the protocolproposed by Tice et al [12] Briefly a cell suspension of 20120583Lof cell culture in 1mL of RPMI 1640 was exposed to 20120583L ofHEGM (final concentration of 10 to 640 120583gmL 1 h 37∘C)DMSO as negative control (7mM 1 h 37∘C) or hydrogenperoxide as positive control (H

2O2 1 mM 1 h 37∘C) 15 120583L

of that cell suspension was mixed with 95120583L of low-meltingagarose in a microcentrifuge tube The cell suspension wastransferred to an agarose precoated glass slide covered witha glass cover slip (22mm times 66mm) and immediately placedin a refrigerator (4∘C) for 5min to allow complete agarosesolidification Slides were prepared in duplicate Cover slipswere then removed and the slides immersed in cold (4∘C)lysing solution (25M NaCl 100mM Na

2EDTA 10mM Tris

1 (vv) Triton X-100 and 10 DMSO pH 10) protectedfrom light for at least 8 h DNA was allowed to unwind for20min in freshly prepared alkaline electrophoresis buffer(1mMNa

2EDTA 03N NaOH pH 13) prior to electrophore-

sis Horizontal electrophoresis was performed at 25Vcm for20min at 4∘C All steps were performed under yellow lightto minimize additional DNA damage The slides were thenplaced vertically and gently washed 3x for 5min with neu-tralizing buffer (04M Tris-HCl buffer pH 75)















2O2(1 05 or














DMSO (mM)

lowastlowastlowast

H2O2 (mM)10 20 40 80 160 320 640 17

HEGM (120583gmL)

0

25

50

75

100

DN

A d

amag

e (

)


O2



HEGM (120583gmL)

0

5

10

15

20

25

30M

icro

nucle

us1

000

cells

lowastlowastlowast


2


2


2









NCc extract




NCc extract


PCd 1 (AFB1








2O2)



2O2-





lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60D

NA

dam

age (

)

(a)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(b)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(c)


2

O2


2

O2


2

O2









2O2 most


2O2-induced



H2O2

(mM)

(a)

(b)

(c)

(d)

10 125 125 125 210 05 025

H2O2

0

10

20

30

40

50

60

DN

A d

amag

e (

)

Ethanol


O2


O2

(b c and d) H

2

O2


1

10

100

Surv

ival

()



O2


4 Conclusions


2O2





Acknowledgments


References



























2

O2









2

O2











of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























2O2(1 05 or














DMSO (mM)

lowastlowastlowast

H2O2 (mM)10 20 40 80 160 320 640 17

HEGM (120583gmL)

0

25

50

75

100

DN

A d

amag

e (

)


O2



HEGM (120583gmL)

0

5

10

15

20

25

30M

icro

nucle

us1

000

cells

lowastlowastlowast


2


2


2









NCc extract




NCc extract


PCd 1 (AFB1








2O2)



2O2-





lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60D

NA

dam

age (

)

(a)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(b)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(c)


2

O2


2

O2


2

O2









2O2 most


2O2-induced



H2O2

(mM)

(a)

(b)

(c)

(d)

10 125 125 125 210 05 025

H2O2

0

10

20

30

40

50

60

DN

A d

amag

e (

)

Ethanol


O2


O2

(b c and d) H

2

O2


1

10

100

Surv

ival

()



O2


4 Conclusions


2O2





Acknowledgments


References



























2

O2









2

O2











of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























DMSO (mM)

lowastlowastlowast

H2O2 (mM)10 20 40 80 160 320 640 17

HEGM (120583gmL)

0

25

50

75

100

DN

A d

amag

e (

)


O2



HEGM (120583gmL)

0

5

10

15

20

25

30M

icro

nucle

us1

000

cells

lowastlowastlowast


2


2


2









NCc extract




NCc extract


PCd 1 (AFB1








2O2)



2O2-





lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60D

NA

dam

age (

)

(a)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(b)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(c)


2

O2


2

O2


2

O2









2O2 most


2O2-induced



H2O2

(mM)

(a)

(b)

(c)

(d)

10 125 125 125 210 05 025

H2O2

0

10

20

30

40

50

60

DN

A d

amag

e (

)

Ethanol


O2


O2

(b c and d) H

2

O2


1

10

100

Surv

ival

()



O2


4 Conclusions


2O2





Acknowledgments


References



























2

O2









2

O2











of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















NCc extract




NCc extract


PCd 1 (AFB1








2O2)



2O2-





lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60D

NA

dam

age (

)

(a)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(b)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(c)


2

O2


2

O2


2

O2









2O2 most


2O2-induced



H2O2

(mM)

(a)

(b)

(c)

(d)

10 125 125 125 210 05 025

H2O2

0

10

20

30

40

50

60

DN

A d

amag

e (

)

Ethanol


O2


O2

(b c and d) H

2

O2


1

10

100

Surv

ival

()



O2


4 Conclusions


2O2





Acknowledgments


References



























2

O2









2

O2











of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60D

NA

dam

age (

)

(a)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(b)

lowastlowastlowast

H2O2 (1mM)

160 320 640

HEGM (120583gmL)

0

10

20

30

40

50

60

DN

A d

amag

e (

)

(c)


2

O2


2

O2


2

O2









2O2 most


2O2-induced



H2O2

(mM)

(a)

(b)

(c)

(d)

10 125 125 125 210 05 025

H2O2

0

10

20

30

40

50

60

DN

A d

amag

e (

)

Ethanol


O2


O2

(b c and d) H

2

O2


1

10

100

Surv

ival

()



O2


4 Conclusions


2O2





Acknowledgments


References



























2

O2









2

O2











of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















H2O2

(mM)

(a)

(b)

(c)

(d)

10 125 125 125 210 05 025

H2O2

0

10

20

30

40

50

60

DN

A d

amag

e (

)

Ethanol


O2


O2

(b c and d) H

2

O2


1

10

100

Surv

ival

()



O2


4 Conclusions


2O2





Acknowledgments


References



























2

O2









2

O2











of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































2

O2









2

O2











of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article dna protection against oxidative damage...

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