ReseachArticle - downloads.hindawi.comdownloads.hindawi.com/journals/bmri/2018/1942451.pdfgroup with just dsDNA in the solution. On the contrary, if an extract-test has a genoprotective

Research ArticleAnalysis of In Vitro Cyto- and Genotoxicity of BarbatimatildeoExtract on Human Keratinocytes and Fibroblasts

Neida L Pellenz1 Fernanda Barbisan 2 Veronica F Azzolin1 Thiago Duarte1

Aline Bolignon3 Moiseacutes H Mastella2 Cibele F Teixeira 1 Euler E Ribeiro4

Ivana B Macircnica da Cruz 12 andMarta M M F Duarte15

1Postgraduate Program of Pharmacology Federal University of Santa Maria Santa Maria RS Brazil2Postgraduate Program of Gerontology Federal University of Santa Maria Santa Maria RS Brazil3Phytochemical Research Laboratory Department of Industrial Pharmacy Federal University of SantaMaria SantaMaria RS Brazil4Third Age Open University University of Amazonas State Manaus AM Brazil5Brazilian Lutheran University (ULBRA) Santa Maria RS Brazil

Correspondence should be addressed to Ivana B Manica da Cruz ivanaufsmgmailcom

Received 30 April 2018 Revised 24 August 2018 Accepted 13 September 2018 Published 8 October 2018

Guest Editor Francesco Facchiano

Copyright copy 2018 Neida L Pellenz et alThis 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

Barbatimao (Stryphnodendron adstringens Mart) is a native Brazilian species used in traditional medicine and some commercialpreparations owing to its strong wound-healing activity However controversy regarding its use due to safety concerns over thepotential genotoxic effect of this plant remains In order to clarify this issue the effect of hydroalcoholic extract of barbatimao invitro on cell viability DNAdamage and induction of apoptosis in two commercial cell lines of keratinocytes (HaCaT) andfibroblasts(HFF-1) was evaluated Barbatimao stem bark hydroalcoholic extract (70 ethanol) was obtained and lyophilized for subsequentuse in all experiments The main bioactive molecules quantified by HPLC were gallic acid caffeic acid quercetin catechin andepigallocatechin gallate (EGCG) Barbatimao (0024 to 199mgmL) was found to decrease cellular mortality as compared to thecontrol group GEMO assay a noncellular DNA protocol that uses H2O2-exposed calf thymus DNA revealed not only a genotoxiceffect of barbatimao but also a potential genoprotective action against H2O2-triggered DNA fragmentationThese results indicatedthat barbatimao at concentrations of 049 and 099mgmL which are near to the levels found in commercial preparations exertedan in vitro genoprotective effect on cells by decreasing the levels of DNA oxidation quantified by 8-hydroxy-21015840-deoxyguanosine (8-OHdG) and reactive oxygen species (ROS) levels Gene and protein apoptotic markers quantified by qRT-PCR (BAXBcl-2 genes)and immunoassays (Caspases 3 and 8) respectively also indicated a decrease in apoptotic events in comparison with control cellsCollectively the results suggest that barbatimao could exert genoprotective and antiapoptotic effects on human keratinocytes andfibroblasts

1 Introduction

Many studies have described potential effectiveness of phe-nolic compounds in medicine due to their antioxidant anti-inflammatory antimicrobial and proliferative properties [12] Some of these polyphenol molecules have been describedin plants such as Stryphnodendron adstringens (Mart) aBrazilian species popularly known as ldquobarbatimaordquo withstrong wound-healing activity [3 4] Barbatimao is a nativespecies of the Savanah Biomes (Cerrado and Caatinga)where a bark extract is used by traditional communities

as a wound-healing natural product [5ndash9] Prior investiga-tions have described potential causal mechanisms associatedwith the healing efficacy of barbatimao such as antioxi-dant anti-inflammatory and antimicrobial activities [6 10ndash13]

Considering that barbatimao is effective and inexpensivethis bark extract has been included in some commercialBrazilian medicines Its inclusion as a phytotherapeutic plantis also based on complementary studies that have describedlow acute and chronic toxicity in barbatimao experimentalmodels [14ndash17]

HindawiBioMed Research InternationalVolume 2018 Article ID 1942451 11 pageshttpsdoiorg10115520181942451

2 BioMed Research International

However there are some aspects of the plant that need tobe studied for the safe use of barbatimao as a drug such asits cito- and genotoxic potential Therefore it is necessary toperform complementary investigations to evaluate the effectof barbatimao on human cellular DNA damage modulationThe present study evaluated the modulation of genotoxicityand apoptosis as an indicator of the effect of a commercialbarbatimao extract on DNA damage in human keratinocytesand dermal fibroblast commercial cell lines

2 Materials and Methods

21 Reagents Chemical reagents including acetonitrileformic acid (purity gt 98) gallic acid (purity gt 98) andcaffeic acid (purity gt 98) were purchased from Merck(Darmstadt FRM Germany) and quercetin (purity gt98) catechin (purity gt 98) rutin (purity gt 98) andkaempferol (purity gt 98) used as reference moleculeswere obtained from Sigma (Saint Louis MI United States)High-pressure liquid chromatography with a diode arraydetector (HPLC-DAD) was performed using ShimadzuProminence Auto Sampler (SIL-20A) high-pressure liquidchromatography (HPLC) system (Shimadzu Kyoto Japan)equipped with Shimadzu LC-20AT reciprocating pumpsconnected to a DGU 20A5 degasser with a CBM 20Aintegrator SPD-M20A diode array detector and LC solution122 SP1 software Materials used in cell culture werepurchased from Vitrocell Embriolife (Campinas SP Brazil)and Gibco-Life Technologies (Carlsbad CA United States)Molecular biology reagents were obtained from QIAGEN(Hilden NW Germany) Invitrogen (Carlsbad CA UnitedStates) and Bio-Rad Laboratories (Hercules CA UnitedStates)

Biochemical reagent protocols for performing the spec-trophotometry assays were obtained from Sigma-Aldrich (StLouis MI United States) The apoptotic and genotoxic mark-ersmarked by anELISA immunoassay kitwere obtained fromAbcam (CambridgeMAUnited States)The equipment usedincluded a SpectraMax i3x Multimode microplate reader(Molecular Devices Sunnyvale CA United States) andRotor-Gene Q 5plex HRM System (QIAGEN biotechnologyHilden NW Germany)

22 Good InVitroMethods Practices andExperimental DesignThe in vitro protocols performed in the present investigationare according to presumptions described in OECD Guide-lines for the Testing of Chemicals and in a draft of guidancedocument on Good In vitro Methods Practices (GIVIMP)organized by Griesinger [18] The followed standard proce-dures were used in the present study to guarantee high dataquality (1) reagents and plastics of high quality and originwere used (2) ATCC commercial lineages and standardizedconditions described by the ATCC were used (3) all experi-ments were initialized with cells at of 1 times 105 concentration(4) all experiments were carried out at controlled culturetimes identified in the results andfigures (5) experimentsconducted in 96-well plates were just in the internal wellsin order to attenuate problems related to the evaporation of

the medium which can cause large intravariation of the data(6) in the 96-well plates each treatment was repeated at leastfive times (7) all experiments were replicated at differentmoments at least three times In each replication at leastfive repetitions of each treatment were tested (8) details ofdata analysis used in GIVIMP were also followed and arepresented in statistical section of this study

General analysis performed in the present investigationis synthetized in Figure 1 Further details of assays used inthe present study will be detailed Initially to test potentialtoxicological effect of barbatimao on human cell lines ahydroalcoholic extract using bark samples of this plantcollected in Manaus Amazonas State Brazil (-310719S -600261 3∘ 61015840 2610158401015840W) and voucher specimens were depositedat the Herbarium of the Biological Sciences Course FederalUniversity of Santa Maria RS Brazil (Figure 1(A)) Mainbioactive molecules present in the barbatimao extract werequantified by HPLC-DAD analyses (Figure 1(B))

Before in vitro analyses the noncellular GEMO assay wasperformed to identify potential genotoxic andor genopro-tective barbatimaosrsquos capacity according to different concen-trations (Figure 1(C)) The principle of this assay previouslydeveloped and validated by Cadona et al [19] is to verifythe effect of some extract-test on fragmentation of double-strand (ds) DNA molecule that is concomitantly exposed toH2O2 an oxidative molecule Therefore if an extract-testhas genotoxic capacity dsDNA fragmentation triggered byH2O2 exposure will be more intense than negative controlgroup with just dsDNA in the solution On the contraryif an extract-test has a genoprotective capacity dsDNAfragmentation will be less intense than positive controlgroup with dsDNA plus H2O2 GEMO assay is performedusing a highly specific dsDNA dye (PicoGreen) as a basicreagent This dye is an ultrasensitive fluorescent reagentthat allows quantification in the solution just dsDNA butnot single DNA molecules and nucleotides Therefore highdsDNAconcentration is associated to high fluorescence levelsquantified by fluorimeter (Figure 1(C-1)) When dsDNAis fragmented by the presence of oxidant molecules suchas H2O2 fluorescence drops in comparison with negativenon-H2O2 exposed control (Figure 1(C-2)) Therefore theeffect of some extract-test on this reaction can indicate itsgenotoxic or genoprotective effect Genoprotective capacityis detected when fluorescence increases significantly in asolution containing dsDNA H2O2 and extract-test in com-parison with a solution containing just dsDNA plus H2O2On the contrary if fluorescence decreases significantly withaddition of extract-test in the same solution this resultindicates genotoxic capacity of the extract Therefore thisassay was used as preliminary indication of genoprotectiveandor genotoxic (Figure 1(C-3)) capacity of barbatimaoextract at different concentrations

All in vitro protocols were performed using two cell com-mercial lines of keratinocytes (HaCaT) and dermal fibroblasts(HFF-1) in 24 h cell cultures (Figure 1(D)) First protocoldetermined if similar concentrations tested in the GEMOassay could present some cytotoxic effects on these cell linesTherefore cell cultures were supplemented with barbatimaoat 10 different concentrations (0012 0024 0049 0099 012

BioMed Research International 3

GEMO AssayGenotoxicGenoprotective Capacity

KeratinocytesHaCaT

FibroblastsHFF-1

CitotoxicityGenotoxicity

Oxidative marker (ROS)Apoptosis induction

C-1 C-2 C-3

A

B

C D

H

H

H

H

Figure 1 General experiment design (A) Initially was obtained a hydroalcoholic extract using barbatimao bark samples (B) Main bioactivecompounds were quantified by HPLC-DA (C) An exploratory noncelular GEMO assay was conducted to determine if barbatimao at 10different concentrations could present some genotoxic or genoprotective capacity of the extract (D) The in vitro protocols were performedin two commercial human cell lines of keratinocytes (HaCaT) and dermal fibroblasts (HFF-1) Additional protocols were conducted toevaluate barbatimao effects on DNA oxidation by quantification of DNA-8-OhdG levels and by intrinsic or extrinsic apoptosis induction byquantification and comparison with negative control group of gene BAXBcl-2 ratio and CASP 3 and 8 protein levels Details of experimentaldesign and assays used in this study are presented in Methods section

024 049 099 199 and 392) From these results twobarbatimao concentrations estimated to be found in Braziliancommercial wound-healing barbatimao products describedin pharmaceutical package were used in the complementaryprotocols (049 and 099mgmL)

These protocols evaluated if barbatimao extract couldinduce cellular in vitro genotoxicity by quantification of8-deoxyguanosine (DNA-8-OhdG) levels This molecule isformed when DNA is oxidized and is considered a goodbiomarker of oxidative stress and oxidative DNA damage[20] Extensive cellular and DNA damage can trigger apop-totic events on the cells The intrinsic apoptotic events aretriggered by increase of p53 protein levels which are able todetect no-repaired DNA lesions and induce overexpressionof Bcl-2-associated X protein (BAX gene) an apoptoticregulator On the contrary p53 protein induces downreg-ulation of B-cell lymphoma 2 (Bcl-2) that plays a crucialrole in promoting cellular survival and proliferation There-fore BAXBcl-2 ratio has been used as marker of intrinsicapoptosis events (when ratio is ge 10) in a large number ofstudies such as performed by Bergandi et al [21] Uponinduction of apoptosis BAX becomes organelle membraneassociated and in particular mitochondrial membrane thatbecomes permeabilized releases cytochrome C into cytosolThe elevated cytochrome C concentration in the cytoplasmtriggers caspases (CASP) pathway (including CASP 3 and8) that regulate further apoptotic events Moreover caspasespathway can be triggered by an extrinsic pathway related

to binding of some molecules with dead receptors that arepresent in the outside of cellular membrane In this contextconcomitant quantification of BAXBcl-2 gene expressionratio and CASP 3 and 8 proteins can be considered informa-tive if some extract or product triggers apoptotic events and ifevents involve intrinsic or extrinsic apoptosis pathways Thisprotocol of apoptosis induction has been used in previousstudies performed by our research group [22 23]

As barbatimao extract can be clinically tested in severaldays a final protocol was performed to evaluate if the chroniccell culture exposure to babartimao could present elevationof reactive oxygen species (ROS) levels and DNA 8-OHdGlevels which indicates DNA damage measure in 1 3 and 5days of cultures

23 Barbatimao Extract Obtention and HPLC-DAD Proce-dures Barbatimao hydroalcoholic extract was obtained ina manner similar to that described by Betoni et al [24]Barbatimao bark was dried ground and extracted with 70ethanol at 4ndash8∘C then filtered after 48 h Filtration was doneusing Whatman No 1 paper and the solvent removed usinga rotary evaporator at reduced pressure 45∘C at 115 rpmThe resulting dry extract was obtained by lyophilization andstored at 20∘C in a sterile flask until use Quantification ofthe main bioactive molecules in the barbatimao stem barkextract was performed as previously described by Da Silva etal [25] Barbatimao hydroalcoholic extract at a concentrationof 12mgmLwas injected in amodel SIL-20A Shimadzu Auto


sampler Separations were carried out using PhenomenexC18 column (46mm times 250mm times 5120583m particle size) Themobile phase consisted of water with 1 formic acid (vv)(solvent A) and HPLC grade acetonitrile (solvent B) at aflow rate of 06mLmin and injection volume 40 120583L Thecomposition gradient was 5 solvent B reaching 15 at10min 30 solvent B at 20min 65 solvent B at 30minand 98 solvent B at 40min followed by 50min at isocraticelution until 55min At 60min the gradient reached theinitial conditions again following the method described byDa Silva et al [25] with small modifications The sample andmobile phasewere filtered through a 045 120583mmembrane filter(Millipore) and then degassed by ultrasonic bath prior touse Stock solutions of standards references were prepared inacetonitrile at a concentration range of 0030ndash0500mgmLQuantifications were carried out by integration of the peaksusing the external standard method at 254 nm for gallic acid280 nm for catechin 327 nm for caffeic acid and 366nm forquercetin rutin and kaempferol The chromatography peakswere confirmed by comparing retention time with those ofreference standards and by DAD spectra (200 to 700nm)All chromatography operations were carried out at ambienttemperature and in triplicate

24 Cell Culture Conditions and Treatments The in vitroinvestigation used two commercial cell lines immortalizedhuman keratinocytes (HaCaT) and neonatal foreskin humandermal fibroblasts (HFF-1) obtained from American TypeCulture Collection (ATCC) Cell cultures were reared in con-trolled conditions with Dulbeccorsquos modified Eagle medium(DMEM) culture medium supplemented with 15 fetalbovine serum 100 IUmL penicillin and 100120583gmL strepto-mycin Cells were maintained at 37∘C with 5 CO2 and 95humidified atmosphere for 24 h Potential cytotoxic effectsof hydroalcoholic barbatimao extract on keratinocytes andfibroblasts were tested using ten different concentrations Thecurve concentration of barbatimao analyzed here was basedon a prior study byCosta et al [15] andwas used as a reference(0012 to 392mgmL) To perform this curve barbatimaolyophilized extractwas diluted in culturemediumandfilteredusing Whatman filter paper (2 120583) to prevent the presence ofmicroorganisms in the solution

Two barbatimao concentrations near to those found incommercial preparations of barbatimao extract were usedto perform complementary analysis (049 and 099mgmL)in keratinocyte and fibroblast 24 h cell cultures In cellsexposed to these concentrations DNA oxidative damage wasevaluated by quantification of 8-hydroxy-21015840-deoxyguanosine(8-OHdG) that is considered a biomarker for oxidative DNAmarkers and has been widely used in in vivo and in vitrostudies [20] We also evaluated potential modulation ofapoptosis by barbatimao exposure via analysis of expressionof two genes Bcl-2 (B-cell lymphoma 2) and BAX (bcl-2-likeprotein 4) Bcl-2 gene is an inductor of cellular proliferationwhereas BAX is an inductor of apoptotic events GenerallyBAXBcl-2 gene expression ratio is used to detect if cellcultures are in apoptotic or proliferative processes [26] Inthis case values gt 1 indicate an apoptotic state and potentialgenotoxic effect of barbatimao on cells Quantification of two

caspase (CASP 3 and 8) protein levels was also performed toconfirm the influence of barbatimao on apoptotic processesMethods used to determine gene expression and proteinquantification involved qRT-PCR and immunoassay analysisare further described in more details below

25 GEMO Assay Before testing the potential effect ofbarbatimao on the DNA of keratinocytes and fibroblastsa GEMO assay was performed as previously described byCadona et al [19] which used Quant-IT PicoGreenDNA kit (Invitrogen Life Technologies SP Brazil) GEMOis a noncellular protocol previously described by Cadonaet al [19] that uses exposure of calf-DNA to H2O2 Thismolecule is genotoxic increasing DNA fragmentation thatis detected by PicoGreen dye If a chemical molecule in theplant extract has a genoprotective effect DNA fragmentationwill be attenuated and fluorescence in the test group willdecrease in relation to the positive control group (obtainedby exposure of calf-DNA to H2O2) A 96-well plate wasfilled with 10 120583L of calf thymus DNA (1 120583gmL plus 70120583Lof TE buffer) containing varying barbatimao concentrationsand 70120583L of H2O2 (1M) The reaction was incubated for30min After 30min PicoGreen DNA dye was added andthe fluorescence was read (excitation at 480 nmemission at520 nm) The genoprotective effect was considered presentwhen the absorbance was lower than the positive controlgroup [19]

26 Cytotoxic Assays Two assays were used to test thepotential cytotoxic effect of barbatimao on keratinocytesand fibroblasts trypan dye exclusion assay and cf-DNAPicoGreen assayThe cf-DNAPicoGreen assay is based on thepresence of double-strandedDNA fragments that are releasedwhen cells die as the cytoplasmic and nuclear membranerupture The cellular mortality rate is quantified by super-natant cell-free DNA (cf-DNA) levels using PicoGreen dyea fluorescent marker of double-stranded DNA fragmentsTherefore high cf-DNA levels indicate high mortality rate inthe cell culture [19] This assay was performed using Invit-rogen Quant-IT kit following manufacturerrsquos instructionsTen microliters of cell culture supernatants were collectedand added in a 96-well black plate together with reagentsfrom Quant-iTTM PicoGreen and diluted in Tris-EDTA(TE) buffer (10mM TrisndashHCl 1mM EDTA pH 75) Then100120583L of the PicoGreen dye diluted 1200 in TE buffer wasadded to the microplate to make a final volume of 200 120583Lper well Following incubation in the dark for 10min atroom temperature the fluorescent signal of the sampleswas measured at 480 nm excitation and 520 nm emission atroom temperature using SpectraMax M2M2e MultimodePlate Reader Molecular Devicesrsquo equipment Elevated cf-DNA levels indicated high cellular mortality

27 Reactive Oxygen Species (ROS) Assay As barbatimao isrich in antioxidant molecules ROS levels of cell cultures werequantified using a 27 dichlorofluorescein diacetate (DCFH-DA) ROS production assay DCFH-DA is hydrolysed byintracellular esterase enzymes to DCFH which is trapped


within the cell and the nonfluorescent molecule is thenoxidized with fluorescent dichlorofluorescein (DCF) usingcellular oxidants Therefore to quantify ROS levels cell cul-tures were treated with DCF-DA (10mM) for 60min at 37∘CThe fluorescence was measured at an excitation of 488 nmand an emission of 525nm and the results were obtainedas pmolemL of DCF production from 27 dichlorofluorescinin reaction with ROS molecules present in the samples [27]After the data were obtained results were converted to ofcontrol group

28 Immunoassay Tests The levels of DNA 8-OHdG wereused to quantify DNA damage and those of CASP 3 and8 were measured using a Quantikine Human Kit accordingto the manufacturerrsquos instructions All reagents and workingstandards were prepared and the excess microplate stripswere removed before adding 50120583L of the assay diluent RD1Wto each well 100120583L of standard control for the sample wasadded per well after which the well was covered with anadhesive strip and incubated for 15 h at room temperatureEach well was subsequently aspirated and washed twice fora total of three washes The antiserum of each moleculeanalyzed here was added to each well and covered witha new adhesive strip before being incubated for 30min atroom temperature The aspirationwash step was repeatedand the molecule-1 conjugate (100120583L) was added to eachwell and incubated for 30min at room temperature Theaspirationwash step was repeated before adding 100120583L ofsubstrate solution to each well followed by incubation atroom temperature for an additional 20min Finally 50 120583L ofstop solution was added to each well and the optical densitywas determined within 30min using a microplate reader setto 450 nm

29 mRNA Expression Analysis by Quantitative QT-PCRAssay Gene expressions of BAX and Bcl-2 were analyzedin cells exposed to barbatimao extract Total RNA wasextracted using Trizol following the manufacturerrsquos instruc-tions (Ludwig-Biotec RS Brazil) The extracted RNA wasmeasured by a Thermo Scientific NanoDrop 1000 Spec-trophotometer To perform reverse transcription 1 120583gmLRNA was added to the samples with 02120583L of DNAase at37∘C for 5min followed by heating at 65∘C for 10min ThecDNA was generated with 1 120583L of Iscript cDNA and 4120583L ofMix IscriptThenext steps of the reactionwere 5∘C for 10min25∘C for 5min and 85∘C for 5min with a final incubation at5∘C for 60min The qRT-PCR was performed in the Rotor-Gene Q 5plex HRM System (QIAGEN biotechnology NWGermany) using QuantiFast SYBR Green PCR Master Mixstarting with a temperature of 95∘C for 3min followed by 40cycles of 95∘C for 10 s 60∘C for 30 s and a melt curve of 60∘Cto 90∘C in 05∘C increments for 5 s The reactions of eachsample were made in triplicate using 1 120583M of each primerand with 2times QuantiFast SYBR Green PCR Master Mix thefinal volume was 20120583L The beta-actin gene sense (51015840TGT-GGATCAGCAAGCAGGAGTA31015840 ) antisense (51015840TGCGCA-AGTTAGGTTTTGTCA31015840 ) was used as a housekeeping

geneBAX gene sense (51015840CCCTTTTCTACTTTGCCAGCA-A31015840) antisense (51015840CCCGGAGGAAGTCCAATGT31015840) BCL-2 gene sense (51015840GAGGATTGTGGCCTTCTTTGAGT31015840 )antisense (51015840AGTCATCCACAGGGCGATGT31015840 ) [23]

210 Statistical Analysis Statistical tests were performedusing Graph Pad Prism Software Results of all experimentswere replicated three times with at least five repetitions ofeach treatment Data from repetitions were evaluated beforestatistical analysis and normalized by of control groupTherefore results were expressed as mean plusmn SD (standarddeviation) of the control group This procedure is broadlyused in the in vitro protocols (Antonieli et al 2017) to allowcomparison between results obtained on different days bydifferent tests and by different laboratories The upper andlower values of 2-SD range were considered outliers andexcluded of the analysis because generally these outliersgenerate relative SD gt 10 indicating presence of someexperimental imprecision Treatments were statistically com-pared by one-way analysis of variance (ANOVA) followedby Tukey post hoc test In results showed in Figures differentletters identified statistical differences (p le 005) amongtreatments

3 Results

Details of the barbatimao tree bark extract prepara-tion samples and chemical characterization are presentedin Figure 2 Barbatimao hydroalcoholic extract presentedhigher levels (mgg) of gallic acid (1248plusmn005) caffeicacid (806plusmn002) quercetin (816plusmn004) catechin (593plusmn001)and rutin (471plusmn001) (Figure 2) Low levels of kaempferoland other bioactive molecules were also detected (Fig-ure 2)

Initially potential genoprotective capacity of barbatimaoextract at different concentrationswas determined and resultsare presented in Figure 3(a) In this assay the control groupwas compounded by a solution containing isolated dsDNAplus H2O2 that caused extensive DNA fragmentation Whenbarbatimao extract was added in this solution an inversedose-dependent action on dsDNAwas observed In this casebarbatimao at lower concentration (0012mgmL) presentedhigher protective effect against H2O2 dsDNA fragmentationthan more elevated concentrations Concentrations near tobarbatimao therapeutic doses used to healing wound showedsome dsDNA protection but this effect was less intense thanthat observed in the other lower concentrations

The first in vitro protocol performed here using ker-atinocytes (Figure 3(b)) and fibroblast (Figure 3(c)) cellsevaluated potential barbatimao effect on viability of thesecells using the same range of concentrations evaluated byGEMO assay (Figure 3(a)) Barbatimao did not presenta cytotoxic effect on keratinocytes and fibroblasts whencompared to control group that did not receive any addi-tional treatment numerically represented by 0 value in thegraphic On the contrary cell culture barbatimao extractsupplementation increased viability of both cell lines whencompared to no-treated control group (0) However this


(a)

(b)

(c) (d)

(e)

(f)

Gallic ac

Catechin

EGCG

Caffeic ac

Rutin

Querceti

n

Kaempfer

ol

Bioa

ctiv

e mol

ecul

es (m

gm

L)

0

5

10

15

0

50

100

150

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40 50 min

mA

U

solv

ent

302010

1

2 34

5

6

7

0

Figure 2 Barbatimao (Stryphnodendron adstringens Mart) components and measurement of bioactive molecules (a) barbatimao tree (b)stem bark used to produce barbatimao extract (c) lyophilized barbatimao extract (d) solution of hydroalcoholic extract of barbatimao usedto treat cell cultures and (e) representative HPLC profile of barbatimao hydroalcoholic extract Gallic acid (peak 1) catechin (peak 2) EGCG(peak 3) caffeic acid (peak 4) rutin (peak 5) quercetin (peak 6) kaempferol (peak 7) (f) graphical representation of bioactive moleculequantification detected in barbatimao HPLC analysis

effect was higher in cultures supplemented with inter-mediary concentrations (Keratinocytes 0099-049mgmLfibroblasts 0099-099mgmL)

Two barbatimao concentrations (049 and 099mgmL)near to those found in commercial preparations were used toperform analysis of genotoxicity in keratinocyte and fibrob-last 24 h cell cultures In cells exposed to these concentrations

DNA damage quantified by DNA 8-OHdG levels BAXBcl-2 gene expression ratio and CASP 3 and 8 levels werequantified In both barbatimao concentrations there was asignificant decrease in ROS levels when 24 h keratinocytecultures were exposed to 049mgmL (872plusmn25 of controlgroup) and 099mgmL (743plusmn30of control group) Similarresults were also found in fibroblasts where barbatimao


0

5

10

15

Barbatimatildeo (mgmL)

A

B

C

DE

FF

GH I

00012

00490099

012024

049099

199392

DN

A P

icoG

reen

Gen

opro

tect

ive H

O

as

say (

co

ntro

l)

(a)

0

50

100

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200

Barbatimatildeo extract (mgmL)

AB B B

C C C BC BA

C

00012

00240049

0099012

024049

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392

Kera

tinoc

ytes

Cel

l via

bilit

y (

of c

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(b)

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AAB

B BBC C C C C

B AB

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00240049

0099012

024049

099199

392

Fibr

obla

sts

Cel

l via

bilit

y (

of c

ontr

ol)

(c)

Figure 3 Barbatimao preliminary assays (a) genoprotective capacity determined by GEMO noncellular assay that uses DNA PicoGreenfluorescent dye (Cadona et al 2014) This dye presents specific affinity to binding with double-strand (ds) DNA levels In GEMO assaya solution containing isolated calf dsDNA and H2O2 (1M) is produced The H2O2 trigger extensive DNA fragmentation that causesdecreasing in the fluorescence and here is considered the control group represented by 0 value Cell culture supplementation with barbatimaohydroalcoholic extract at different concentrations showed significant increase in the fluorescence indicating genoprotective effect of thisextract barbatimaorsquos viability effect on (b) keratinocytes and (c) fibroblast 24 h cell cultures measured by MTT-assay Data were analyzedby ANOVA one-way analysis of variance followed by Tukey post hoc test and all statistical tests with p le 005 were considered significantStatistical differences among treatmentswere identified by different alphabetical letters whereas same letters indicated no differences betweeneach treatment compared to the others

extract at 049mgmL reduced ROS levels to 824plusmn30 ofcontrol group and at 099mgmL reduced ROS levels to695plusmn30 of control group (p le 0001)

Barbatimao treated samples also showed a decrease inDNA damage evaluated by 8-OHdG levels (Figure 4(a)) inboth 24 h cell cultures tested Analysis of apoptotic mark-ers showed a decrease in BAXBcl-2 gene expression ratio(Figure 4(b)) Moreover barbatimao decreased CASP 3 and8 in both keratinocyte and fibroblast cell lines (Figures4(c) and 4(d)) However the genoprotective and caspaselowering effect was more pronounced in fibroblasts than inkeratinocytes

Considering that the effect of barbatimao could be tran-sient and cause subsequent triggering of an increase in ROS

levels and DNA damage in older cultures a complementaryanalysis was performed in fibroblasts in order to evaluatethe effect of barbatimao on 1 3 and 5 day cell cultures Asseen in Figure 5 barbatimao maintained its antioxidant andgenoprotective effect in cultures of several days old Howeverthis result was more pronounced in 72 h cell cultures exposedto 099mgmL concentration of barbatimao extract

4 Discussion

The present investigation evaluated potential genotoxiceffects of barbatimao a Brazilian plant used tradition-ally for wound healing Most results obtained from the


0 049 099 0 049 0990

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AB

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p lt 0001p lt 001

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-deo

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rol)

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(a)

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p lt 001A

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p lt 001


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ACB

p lt 0001A

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p lt 00001

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e 8 (

of c

ontr

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(d)

Figure 4 Comparison among genotoxic and apoptotic markers of keratinocytes and fibroblasts exposed to barbatimao hydroalcoholicextraction (a) 8-OHdG levels (b) BAXBcl-2 gene expression ratio quantified by qRT-PCR that indicates modulation of intrinsic apoptoticevents (c) caspase 3 protein levels (d) caspase 8 protein levels Data were compared by one-way analysis of variance (ANOVA) followed bya Tukey post hoc test In each marker tested here statistical differences at p le 005 among 24 h cell cultures treatments were identified bydifferent letters (A B C)

1 3 50

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( o

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(a)

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BC

p lt 0001 p lt 0001

A A

C

p lt 0001A B

C

Fibroblast cultures (days)Control049 mgmL099 mgmL

8-de

oxyg

uano

sine

( o

f con

trol

)

(b)

Figure 5 Modulation of oxidative markers on three different cell culture days of keratinocytes and fibroblasts exposed to two differenthydroalcoholic barbatimao extracts to evaluate potential chronic genotoxicity of this extract on cells (a) Reactive oxygen species (ROS)levels (b) 8-OHdG levels Data were compared by one-way analysis of variance (ANOVA) followed by a Tukey post hoc test In each markertested here statistical differences at p le 005 among 24 h cell cultures treatments were identified by different letters (A B C)


in vitro protocol using keratinocyte and fibroblasts indi-cated noncytotoxic and genoprotective effects of barbatimaoextract The genoprotective effect was estimated by a non-cellular assay quantification of 8-deoxyguanosine levelsand gene and proteins involving in apoptosis processes inboth cell lines These results could be considered relevantsince use of phytotherapeutics from traditional medicine hasbeen considered a part of healthcare by the World HealthOrganization (WHO) since 2002 Barbatimao has been usedas an astringent antihemorrhagic and antidiarrheal as wellas a treatment for scurvy genitourinary disorders and lowerairways conditions [2]

The Brazilian Official Pharmacopoeia [28] estimated thatthe wound-healing effect of barbatimao could be associatedwith the high tannin concentration in its dried bark (8) Infact most bioactive molecules quantified in the barbatimaoextract used here were previously described in the literaturesuch as gallic acid [29] and catechins [3] It is important tomention that other bioactive molecules of barbatimao suchas quercetin rutin and kaempferol have been identified andquantified in this study and have not been described in pre-vious chemical analyses of barbatimao Quercetin and rutinalso called quercetin-3-O-rutinoside are flavonoids foundin several fruits and vegetables including citrus and onionStudies on quercetin have described interesting biologicaleffects including cell protection against UV irradiation andskin regeneration in wound healing [30]

Due to its commercial use it is important to clarify thepotential genotoxic activity of barbatimao in human cellsThere are relatively few studies that evaluate potential geno-toxic effect of barbatimao leading to the controversy over thesafety of this traditional medicine Costa et al [15] evaluatedpotential micronucleus induction in mice orally treated withproanthocyanidin polymer-rich fraction (F2) of the stembark of barbatimao and did not find a genotoxic effect of thesemolecules Moreover a study involving potential somaticmutation and recombination analysis (SMART test) andchromosome damage in germ cell lines of fruit flies exposedto barbatimao bark extract did not find genotoxic effect ofthis plant [31] However when Vilar et al [32] evaluatedlyophilized solution produced from barbatimao stem barkresults indicated some cytotoxic and genotoxic effect usingSOS-Inductest in E coli prokaryotic model Therefore theauthors considered it important that further in vitro and invivo studies are performed to clarify these potentially negativeresults

In this context it is plausible to consider that triggeringhealing with barbatimao extract could cause some unde-sirable chromosomal instability associated with procarcino-genic processes Initially we obtained data from the GEMOassay that indicated the genoprotective activity of barbatimaoextract This validated assay was developed by Cadona et al[19] due to the necessity of identifying the genoprotective andgenotoxic capacity of plant extracts that are generally ana-lyzed in in vitro cell cultures which are very expensive andpresent some influence on testing response due to differentialbiochemical and molecular specifications of each cell lineTherefore results obtained from the GEMO assay are fast andinexpensive involving double-strand DNA (dsDNA) damage

caused by H2O2 exposure that was completely reverted in thepresence of barbatimaomainly in lower concentrations of theextract

Subsequent analysis confirmed no genotoxic effect ofbarbatimao by analysis of 8-OHdG levels which decreasedin the presence of this extract This molecule is considereda biomarker of oxidative stress and oxidative DNA damagefound both in physiological fluids and cells and is frequentlyused as a marker of exposure to oxidants as well as apotential marker of some chronic nontransmissible diseaseprogression [33 34] In the presence of 049 and 099mgmLbarbatimao 8-OHdG levels were shown to decrease sug-gesting that barbatimao extract could act as genoprotectivecompound

Barbatimao extract also decreased BAXBcl-2 geneexpression ratio and CASP 3 and 8 protein levels Thesemarkers are related mainly to intrinsic apoptosis triggeredvia accumulation of DNA lesions from genotoxic compoundexposure [33]

5 Conclusions

Despite methodological limitations related to in vitro studiesour data corroborate results found in rats and fruit flieswith respect to lack of genotoxicity and safety Moreoverthe results described here suggest that barbatimao couldpresent a genoprotective and antiapoptotic effect on humankeratinocytes and fibroblasts

Data Availability

The data used to support the findings of this study areincluded in the article

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

Theauthorswould like to thank theCNPq [Nos 4023252013-3 4907602013-9 3114462012-4] FAPERGS FAPEAM andCAPES for grants and fellowships

References

[1] M Działo J Mierziak U Korzun M Preisner J Szopa andA Kulma ldquoThe potential of plant phenolics in prevention andtherapy of skin disordersrdquo International Journal of MolecularSciences vol 17 no 1 pp 1ndash9 2016

[2] L M Ricardo B M Dias F L B Mugge V V Leite andM G L Brandao ldquoEvidence of traditionality of Brazilianmedicinal plantsThe case studies of Stryphnodendron adstrin-gens (Mart) Coville (barbatimao) barks and Copaifera spp(copaıba) oleoresin inwoundhealingrdquo Journal of Ethnopharma-cology vol 219 pp 319ndash336 2018

[3] J Sousa N Pedroso L Borges G A J Paula and EConceicao ldquoOptimization of Ultrasound-assisted extraction of


polyphenols tannins and epigallocatechin gallate from barksof Stryphnodendron adstringens (Mart) Coville bark extractsrdquoPharmacognosy Magazine vol 10 no 38 p 318 2014

[4] A C Isler G C Lopes M L C Cardoso J C P De Mello andL C Marques ldquoDevelopment and validation of a LC-methodfor the determination of phenols in a pharmaceutical formu-lation containing extracts from stryphnodendron adstringensrdquoQuımica Nova vol 33 no 5 pp 1126ndash1129 2010

[5] T H L Bernardo R C Sales Santos Verıssimo V Alvino et alldquoAntimicrobial Analysis of an Antiseptic Made from EthanolCrude Extracts of P granatum and E uniflora in Wistar Ratsagainst Staphylococcus aureus and Staphylococcus epidermidisrdquoTheScientificWorld Journal vol 2015 Article ID 751791 7 pages2015

[6] B F de Santana R A Voeks and L S Funch ldquoEthnomedicinalsurvey of a maroon community in Brazilrsquos Atlantic tropicalforestrdquo Journal of Ethnopharmacology vol 181 pp 37ndash49 2016

[7] S C G Pinto F G Bueno G P Panizzon et al ldquoStryphnoden-dron adstringens ClarifyingWoundHealing in Streptozotocin-Induced Diabetic Ratsrdquo Planta Medica vol 81 no 12-13 pp1090ndash1096 2015

[8] LA F SilvaM I deMouraC EDambros S L R Freitas L ASouza and M P Abreu ldquoStryphnodendron adstringens extractassociated with the hooves trimming surgical procedure for thetreatment of bovine digital dermatitisrdquo Tropical Animal Healthand Production vol 45 no 5 pp 1177ndash1181 2013

[9] L Hernandes L M da Silva Pereira F Palazzo and J CP de Mello ldquoWound-healing evaluation of ointment fromStryphnodendron adstringens (barbatimao) in rat skinrdquo Brazil-ian Journal of Pharmaceutical Sciences vol 46 no 3 pp 431ndash436 2010

[10] B O Henriques O Corrıa E P C Azevedo et al ldquoInvitro TNF- a inhibitory activity of brazilian plants and anti-inflammatory effect of Stryphnodendron adstringens in anacute arthritis modelrdquo Evidence-based Complementary andAlternative Medicine vol 2016 Article ID 9872598 15 pages2016

[11] E M R Pereira R T Gomes N R Freire E G Aguiar MD G L Brandao and V R Santos ldquoIn vitro antimicrobialactivity of Brazilian medicinal plant extracts against pathogenicmicroorganisms of interest to dentistryrdquo Planta Medica vol 77no 4 pp 401ndash404 2011

[12] A T Morey F C de Souza J P Santos et al ldquoAntifungal activ-ity of condensed tannins from stryphnodendron adstringensEffect on Candida tropicalis growth and adhesion propertiesrdquoCurrent Pharmaceutical Biotechnology vol 17 no 4 pp 365ndash375 2016

[13] G M Lanchoti Fiori A L Fachin V S Correa et alldquoAntimicrobial Activity and Rates of Tannins in Stryphnoden-dron adstringens Mart Accessions Collected in the BrazilianCerradordquo American Journal of Plant Sciences vol 04 no 11 pp2193ndash2198 2013

[14] M A Costa J C Palazzo De Mello E N Kaneshima et alldquoAcute and chronic toxicity of an aqueous fraction of the stembark of stryphnodendron adstringens (Barbatimao) in rodentsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 841580 9 pages 2013

[15] M A Costa K Ishida V Kaplum et al ldquoSafety evaluation ofproanthocyanidin polymer-rich fraction obtained from stembark of Stryphnodendron adstringens (BARBATIMO) for useas a pharmacological agentrdquo Regulatory Toxicology and Phar-macology vol 58 no 2 pp 330ndash335 2010

[16] M A Rebecca E L Ishii-Iwamoto R Grespan et al ldquoToxi-cological studies on Stryphnodendron adstringensrdquo Journal ofEthnopharmacology vol 83 no 1-2 pp 101ndash104 2002

[17] P Cintra O Malaspina and O C Bueno ldquoToxicity of bar-batimao to Apis mellifera and Scaptotrigona postica underlaboratory conditionsrdquo Journal of Apicultural Research vol 42no 1-2 pp 9ndash12 2003

[18] C Griesinger ldquoValidation of Alternative in vitro Methods toAnimal Testing Concepts Challenges Processes and ToolsrdquoAdvances in Experimental Medicine and Biology vol 1 pp 1ndash92016

[19] F C Cadona M F Manica-Cattani A K Machado et alldquoGenomodifier capacity assay A non-cell test using dsDNAmolecules to evaluate the genotoxicgenoprotective propertiesof chemical compoundsrdquo Analytical Methods vol 6 no 21 pp8559ndash8568 2014

[20] N Dabrowska and A Wiczkowski ldquoAnalytics of oxidativestress markers in the early diagnosis of oxygen DNA damagerdquoAdvances in Clinical and Experimental Medicine vol 26 no 1pp 155ndash166 2017

[21] L Bergandi E Mungo R Morone B Rolando and S Dou-blier ldquoHyperglycemia Promotes ChemoresistanceThrough theReduction of the Mitochondrial DNA Damage the BaxBcl-2 and BaxBcl-XL Ratio and the Cells in Sub-G1 Phase Dueto Antitumoral Drugs Induced-Cytotoxicity in Human ColonAdenocarcinomardquoCells vol 13 p 866 2018

[22] V F Azzolin F Barbisan L S Lenz et al ldquoEffects of Pyridostig-mine bromide on SH-SY5Y cells An in vitro neuroblastomaneurotoxicity modelrdquo Mutation Research - Genetic Toxicologyand Environmental Mutagenesis vol 8 pp 1ndash10 2018

[23] F Barbisan J De Rosso Motta A Trott et al ldquoMethotrexate-related response on human peripheral bloodmononuclear cellsmay bemodulated by the Ala16Val-SOD2 gene polymorphismrdquoPLoS ONE vol 9 no 10 Article ID e107299 2014

[24] J E C Betoni R P Mantovani L N Barbosa L C di Stasiand A Fernandes Jr ldquoSynergism between plant extract andantimicrobial drugs used on Staphylococcus aureus diseasesrdquoMemorias do Instituto Oswaldo Cruz vol 101 no 4 pp 387ndash390 2006

[25] E da Silva Brum L da Rosa Moreira A R H da Silva et alldquoTabernaemontana catharinensis ethyl acetate fraction presentsantinociceptive activity without causing toxicological effects inmicerdquo Journal of Ethnopharmacology vol 191 pp 115ndash124 2016

[26] X-C Xie Y Cao X Yang Q-H Xu W Wei and M WangldquoRelaxin Attenuates Contrast-Induced Human Proximal Tubu-lar Epithelial Cell Apoptosis by Activation of the PI3KAktSignaling Pathway InVitrordquo BioMed Research International vol2017 Article ID 2869405 7 pages 2017

[27] V F Azzolin F C Cadona A K Machado et al ldquoSuperoxide-hydrogen peroxide imbalance interferes with colorectal cancercells viability proliferation and oxaliplatin responserdquoToxicologyin Vitro vol 32 pp 8ndash15 2016

[28] Brasil Farmacopeia Brasileira vol 2 Agencia Nacional deVigilancia Sanitaria Brasılia Anvisa 546p 2010

[29] S C SantosW F Costa J P Ribeiro et al ldquoTannin compositionof barbatimao speciesrdquo Fitoterapia vol 73 no 4 pp 292ndash2992002

[30] T Hatahet M Morille A Hommoss J M Devoisselle RH Muller and S Begu ldquoQuercetin topical application fromconventional dosage forms to nanodosage formsrdquo EuropeanJournal of Pharmaceutics and Biopharmaceutics vol 108 pp 41ndash53 2016


[31] R Sarıkaya K Erciyas M I Kara U Sezer A F Erciyas and SAy ldquoEvaluation of genotoxic and antigenotoxic effects of boronby the somatic mutation and recombination test (SMART) onDrosophilardquo Drug and Chemical Toxicology vol 39 no 4 pp400ndash406 2016

[32] J B Vilar M I P DrsquoOliveira S D C Santos and L CChen ldquoCytotoxic and genotoxic investigation on barbatimao[Stryphnodendron adstringens (Mart) Coville 1910] extractrdquoBrazilian Journal of Pharmaceutical Sciences vol 46 no 4 pp687ndash694 2010

[33] J Chayapong H Madhyastha R Madhyastha et al ldquoArsenictrioxide induces ROS activity and DNA damage leading toG0G1 extension in skin fibroblasts through the ATM-ATR-associated Chk pathwayrdquo Environmental Science and PollutionResearch vol 24 no 6 pp 5316ndash5325 2017

[34] E A Prokhorova A V Zamaraev G S Kopeina B Zhiv-otovsky and I N Lavrik ldquoRole of the nucleus in apoptosisSignaling and executionrdquo Cellular and Molecular Life Sciencesvol 72 no 23 pp 4593ndash4612 2015

Medicinal ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

ToxicologyJournal of


PainResearch and TreatmentHindawiwwwhindawicom Volume 2018


Arthritis

Neurology Research International


StrokeResearch and TreatmentHindawiwwwhindawicom Volume 2018

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawiwwwhindawicom Volume 2018


Anesthesiology Research and Practice

Journal of


Pharmaceutics

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Infectious Diseases and Medical Microbiology


Canadian Journal of


Autoimmune DiseasesScientica



MEDIATORSINFLAMMATION

of

Submit your manuscripts atwwwhindawicom


However there are some aspects of the plant that need tobe studied for the safe use of barbatimao as a drug such asits cito- and genotoxic potential Therefore it is necessary toperform complementary investigations to evaluate the effectof barbatimao on human cellular DNA damage modulationThe present study evaluated the modulation of genotoxicityand apoptosis as an indicator of the effect of a commercialbarbatimao extract on DNA damage in human keratinocytesand dermal fibroblast commercial cell lines

2 Materials and Methods

21 Reagents Chemical reagents including acetonitrileformic acid (purity gt 98) gallic acid (purity gt 98) andcaffeic acid (purity gt 98) were purchased from Merck(Darmstadt FRM Germany) and quercetin (purity gt98) catechin (purity gt 98) rutin (purity gt 98) andkaempferol (purity gt 98) used as reference moleculeswere obtained from Sigma (Saint Louis MI United States)High-pressure liquid chromatography with a diode arraydetector (HPLC-DAD) was performed using ShimadzuProminence Auto Sampler (SIL-20A) high-pressure liquidchromatography (HPLC) system (Shimadzu Kyoto Japan)equipped with Shimadzu LC-20AT reciprocating pumpsconnected to a DGU 20A5 degasser with a CBM 20Aintegrator SPD-M20A diode array detector and LC solution122 SP1 software Materials used in cell culture werepurchased from Vitrocell Embriolife (Campinas SP Brazil)and Gibco-Life Technologies (Carlsbad CA United States)Molecular biology reagents were obtained from QIAGEN(Hilden NW Germany) Invitrogen (Carlsbad CA UnitedStates) and Bio-Rad Laboratories (Hercules CA UnitedStates)

Biochemical reagent protocols for performing the spec-trophotometry assays were obtained from Sigma-Aldrich (StLouis MI United States) The apoptotic and genotoxic mark-ersmarked by anELISA immunoassay kitwere obtained fromAbcam (CambridgeMAUnited States)The equipment usedincluded a SpectraMax i3x Multimode microplate reader(Molecular Devices Sunnyvale CA United States) andRotor-Gene Q 5plex HRM System (QIAGEN biotechnologyHilden NW Germany)

22 Good InVitroMethods Practices andExperimental DesignThe in vitro protocols performed in the present investigationare according to presumptions described in OECD Guide-lines for the Testing of Chemicals and in a draft of guidancedocument on Good In vitro Methods Practices (GIVIMP)organized by Griesinger [18] The followed standard proce-dures were used in the present study to guarantee high dataquality (1) reagents and plastics of high quality and originwere used (2) ATCC commercial lineages and standardizedconditions described by the ATCC were used (3) all experi-ments were initialized with cells at of 1 times 105 concentration(4) all experiments were carried out at controlled culturetimes identified in the results andfigures (5) experimentsconducted in 96-well plates were just in the internal wellsin order to attenuate problems related to the evaporation of

the medium which can cause large intravariation of the data(6) in the 96-well plates each treatment was repeated at leastfive times (7) all experiments were replicated at differentmoments at least three times In each replication at leastfive repetitions of each treatment were tested (8) details ofdata analysis used in GIVIMP were also followed and arepresented in statistical section of this study

General analysis performed in the present investigationis synthetized in Figure 1 Further details of assays used inthe present study will be detailed Initially to test potentialtoxicological effect of barbatimao on human cell lines ahydroalcoholic extract using bark samples of this plantcollected in Manaus Amazonas State Brazil (-310719S -600261 3∘ 61015840 2610158401015840W) and voucher specimens were depositedat the Herbarium of the Biological Sciences Course FederalUniversity of Santa Maria RS Brazil (Figure 1(A)) Mainbioactive molecules present in the barbatimao extract werequantified by HPLC-DAD analyses (Figure 1(B))

Before in vitro analyses the noncellular GEMO assay wasperformed to identify potential genotoxic andor genopro-tective barbatimaosrsquos capacity according to different concen-trations (Figure 1(C)) The principle of this assay previouslydeveloped and validated by Cadona et al [19] is to verifythe effect of some extract-test on fragmentation of double-strand (ds) DNA molecule that is concomitantly exposed toH2O2 an oxidative molecule Therefore if an extract-testhas genotoxic capacity dsDNA fragmentation triggered byH2O2 exposure will be more intense than negative controlgroup with just dsDNA in the solution On the contraryif an extract-test has a genoprotective capacity dsDNAfragmentation will be less intense than positive controlgroup with dsDNA plus H2O2 GEMO assay is performedusing a highly specific dsDNA dye (PicoGreen) as a basicreagent This dye is an ultrasensitive fluorescent reagentthat allows quantification in the solution just dsDNA butnot single DNA molecules and nucleotides Therefore highdsDNAconcentration is associated to high fluorescence levelsquantified by fluorimeter (Figure 1(C-1)) When dsDNAis fragmented by the presence of oxidant molecules suchas H2O2 fluorescence drops in comparison with negativenon-H2O2 exposed control (Figure 1(C-2)) Therefore theeffect of some extract-test on this reaction can indicate itsgenotoxic or genoprotective effect Genoprotective capacityis detected when fluorescence increases significantly in asolution containing dsDNA H2O2 and extract-test in com-parison with a solution containing just dsDNA plus H2O2On the contrary if fluorescence decreases significantly withaddition of extract-test in the same solution this resultindicates genotoxic capacity of the extract Therefore thisassay was used as preliminary indication of genoprotectiveandor genotoxic (Figure 1(C-3)) capacity of barbatimaoextract at different concentrations

All in vitro protocols were performed using two cell com-mercial lines of keratinocytes (HaCaT) and dermal fibroblasts(HFF-1) in 24 h cell cultures (Figure 1(D)) First protocoldetermined if similar concentrations tested in the GEMOassay could present some cytotoxic effects on these cell linesTherefore cell cultures were supplemented with barbatimaoat 10 different concentrations (0012 0024 0049 0099 012



KeratinocytesHaCaT

FibroblastsHFF-1



C-1 C-2 C-3

A

B

C D

H

H

H

H





















3 Results





(a)

(b)

(c) (d)

(e)

(f)

Gallic ac

Catechin

EGCG

Caffeic ac

Rutin

Querceti

n

Kaempfer

ol

Bioa

ctiv

e mol

ecul

es (m

gm

L)

0

5

10

15

0

50

100

150

200

40 50 min

mA

U

solv

ent

302010

1

2 34

5

6

7

0






0

5

10

15


A

B

C

DE

FF

GH I

00012

00490099

012024

049099

199392

DN

A P

icoG

reen

Gen

opro

tect

ive H

O

as

say (

co

ntro

l)

(a)

0

50

100

150

200


AB B B

C C C BC BA

C

00012

00240049

0099012

024049

099199

392

Kera

tinoc

ytes

Cel

l via

bilit

y (

of c

ontr

ol)

(b)

0

50

100

150

200

AAB

B BBC C C C C

B AB

00012

00240049

0099012

024049

099199

392

Fibr

obla

sts

Cel

l via

bilit

y (

of c

ontr

ol)

(c)






4 Discussion



0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

AB

CA

BC

p lt 0001p lt 001

DN

A d

amag

e(8

-deo

xygu

anos

ine

cont

rol)


(a)

049 099 049 09900

05

15

10 ACB

p lt 001A

CB

p lt 001


0 0

Bax

Bcl-2

Gen

e exp

ress

ion

ratio

(Bet

a-ac

tin n

orm

aliz

ed)

Barbatimao (mgmL)

(b)

0 049 099 0 049 0990

50

150

100

Barbatimao (mgmL)

A

CB

p lt 0001

AB

plt0001

C

Casp

ase 3

( o

f con

trol

)


(c)

0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

ACB

p lt 0001A

B

p lt 00001

CCa

spas

e 8 (

of c

ontr

ol)


(d)


1 3 50

50

100

150p lt 001A

B C

p lt 0001

AB

C

p lt 0001A

B C


ROS

( o

f con

trol

)


(a)

1 3 50

50

150

100A

BC

p lt 0001 p lt 0001

A A

C

p lt 0001A B

C


8-de

oxyg

uano

sine

( o

f con

trol

)

(b)










5 Conclusions


Data Availability




Acknowledgments


References












































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of




KeratinocytesHaCaT

FibroblastsHFF-1



C-1 C-2 C-3

A

B

C D

H

H

H

H





















3 Results





(a)

(b)

(c) (d)

(e)

(f)

Gallic ac

Catechin

EGCG

Caffeic ac

Rutin

Querceti

n

Kaempfer

ol

Bioa

ctiv

e mol

ecul

es (m

gm

L)

0

5

10

15

0

50

100

150

200

40 50 min

mA

U

solv

ent

302010

1

2 34

5

6

7

0






0

5

10

15


A

B

C

DE

FF

GH I

00012

00490099

012024

049099

199392

DN

A P

icoG

reen

Gen

opro

tect

ive H

O

as

say (

co

ntro

l)

(a)

0

50

100

150

200


AB B B

C C C BC BA

C

00012

00240049

0099012

024049

099199

392

Kera

tinoc

ytes

Cel

l via

bilit

y (

of c

ontr

ol)

(b)

0

50

100

150

200

AAB

B BBC C C C C

B AB

00012

00240049

0099012

024049

099199

392

Fibr

obla

sts

Cel

l via

bilit

y (

of c

ontr

ol)

(c)






4 Discussion



0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

AB

CA

BC

p lt 0001p lt 001

DN

A d

amag

e(8

-deo

xygu

anos

ine

cont

rol)


(a)

049 099 049 09900

05

15

10 ACB

p lt 001A

CB

p lt 001


0 0

Bax

Bcl-2

Gen

e exp

ress

ion

ratio

(Bet

a-ac

tin n

orm

aliz

ed)

Barbatimao (mgmL)

(b)

0 049 099 0 049 0990

50

150

100

Barbatimao (mgmL)

A

CB

p lt 0001

AB

plt0001

C

Casp

ase 3

( o

f con

trol

)


(c)

0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

ACB

p lt 0001A

B

p lt 00001

CCa

spas

e 8 (

of c

ontr

ol)


(d)


1 3 50

50

100

150p lt 001A

B C

p lt 0001

AB

C

p lt 0001A

B C


ROS

( o

f con

trol

)


(a)

1 3 50

50

150

100A

BC

p lt 0001 p lt 0001

A A

C

p lt 0001A B

C


8-de

oxyg

uano

sine

( o

f con

trol

)

(b)










5 Conclusions


Data Availability




Acknowledgments


References












































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of
















3 Results





(a)

(b)

(c) (d)

(e)

(f)

Gallic ac

Catechin

EGCG

Caffeic ac

Rutin

Querceti

n

Kaempfer

ol

Bioa

ctiv

e mol

ecul

es (m

gm

L)

0

5

10

15

0

50

100

150

200

40 50 min

mA

U

solv

ent

302010

1

2 34

5

6

7

0






0

5

10

15


A

B

C

DE

FF

GH I

00012

00490099

012024

049099

199392

DN

A P

icoG

reen

Gen

opro

tect

ive H

O

as

say (

co

ntro

l)

(a)

0

50

100

150

200


AB B B

C C C BC BA

C

00012

00240049

0099012

024049

099199

392

Kera

tinoc

ytes

Cel

l via

bilit

y (

of c

ontr

ol)

(b)

0

50

100

150

200

AAB

B BBC C C C C

B AB

00012

00240049

0099012

024049

099199

392

Fibr

obla

sts

Cel

l via

bilit

y (

of c

ontr

ol)

(c)






4 Discussion



0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

AB

CA

BC

p lt 0001p lt 001

DN

A d

amag

e(8

-deo

xygu

anos

ine

cont

rol)


(a)

049 099 049 09900

05

15

10 ACB

p lt 001A

CB

p lt 001


0 0

Bax

Bcl-2

Gen

e exp

ress

ion

ratio

(Bet

a-ac

tin n

orm

aliz

ed)

Barbatimao (mgmL)

(b)

0 049 099 0 049 0990

50

150

100

Barbatimao (mgmL)

A

CB

p lt 0001

AB

plt0001

C

Casp

ase 3

( o

f con

trol

)


(c)

0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

ACB

p lt 0001A

B

p lt 00001

CCa

spas

e 8 (

of c

ontr

ol)


(d)


1 3 50

50

100

150p lt 001A

B C

p lt 0001

AB

C

p lt 0001A

B C


ROS

( o

f con

trol

)


(a)

1 3 50

50

150

100A

BC

p lt 0001 p lt 0001

A A

C

p lt 0001A B

C


8-de

oxyg

uano

sine

( o

f con

trol

)

(b)










5 Conclusions


Data Availability




Acknowledgments


References












































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of








3 Results





(a)

(b)

(c) (d)

(e)

(f)

Gallic ac

Catechin

EGCG

Caffeic ac

Rutin

Querceti

n

Kaempfer

ol

Bioa

ctiv

e mol

ecul

es (m

gm

L)

0

5

10

15

0

50

100

150

200

40 50 min

mA

U

solv

ent

302010

1

2 34

5

6

7

0






0

5

10

15


A

B

C

DE

FF

GH I

00012

00490099

012024

049099

199392

DN

A P

icoG

reen

Gen

opro

tect

ive H

O

as

say (

co

ntro

l)

(a)

0

50

100

150

200


AB B B

C C C BC BA

C

00012

00240049

0099012

024049

099199

392

Kera

tinoc

ytes

Cel

l via

bilit

y (

of c

ontr

ol)

(b)

0

50

100

150

200

AAB

B BBC C C C C

B AB

00012

00240049

0099012

024049

099199

392

Fibr

obla

sts

Cel

l via

bilit

y (

of c

ontr

ol)

(c)






4 Discussion



0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

AB

CA

BC

p lt 0001p lt 001

DN

A d

amag

e(8

-deo

xygu

anos

ine

cont

rol)


(a)

049 099 049 09900

05

15

10 ACB

p lt 001A

CB

p lt 001


0 0

Bax

Bcl-2

Gen

e exp

ress

ion

ratio

(Bet

a-ac

tin n

orm

aliz

ed)

Barbatimao (mgmL)

(b)

0 049 099 0 049 0990

50

150

100

Barbatimao (mgmL)

A

CB

p lt 0001

AB

plt0001

C

Casp

ase 3

( o

f con

trol

)


(c)

0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

ACB

p lt 0001A

B

p lt 00001

CCa

spas

e 8 (

of c

ontr

ol)


(d)


1 3 50

50

100

150p lt 001A

B C

p lt 0001

AB

C

p lt 0001A

B C


ROS

( o

f con

trol

)


(a)

1 3 50

50

150

100A

BC

p lt 0001 p lt 0001

A A

C

p lt 0001A B

C


8-de

oxyg

uano

sine

( o

f con

trol

)

(b)










5 Conclusions


Data Availability




Acknowledgments


References












































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



(a)

(b)

(c) (d)

(e)

(f)

Gallic ac

Catechin

EGCG

Caffeic ac

Rutin

Querceti

n

Kaempfer

ol

Bioa

ctiv

e mol

ecul

es (m

gm

L)

0

5

10

15

0

50

100

150

200

40 50 min

mA

U

solv

ent

302010

1

2 34

5

6

7

0






0

5

10

15


A

B

C

DE

FF

GH I

00012

00490099

012024

049099

199392

DN

A P

icoG

reen

Gen

opro

tect

ive H

O

as

say (

co

ntro

l)

(a)

0

50

100

150

200


AB B B

C C C BC BA

C

00012

00240049

0099012

024049

099199

392

Kera

tinoc

ytes

Cel

l via

bilit

y (

of c

ontr

ol)

(b)

0

50

100

150

200

AAB

B BBC C C C C

B AB

00012

00240049

0099012

024049

099199

392

Fibr

obla

sts

Cel

l via

bilit

y (

of c

ontr

ol)

(c)






4 Discussion



0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

AB

CA

BC

p lt 0001p lt 001

DN

A d

amag

e(8

-deo

xygu

anos

ine

cont

rol)


(a)

049 099 049 09900

05

15

10 ACB

p lt 001A

CB

p lt 001


0 0

Bax

Bcl-2

Gen

e exp

ress

ion

ratio

(Bet

a-ac

tin n

orm

aliz

ed)

Barbatimao (mgmL)

(b)

0 049 099 0 049 0990

50

150

100

Barbatimao (mgmL)

A

CB

p lt 0001

AB

plt0001

C

Casp

ase 3

( o

f con

trol

)


(c)

0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

ACB

p lt 0001A

B

p lt 00001

CCa

spas

e 8 (

of c

ontr

ol)


(d)


1 3 50

50

100

150p lt 001A

B C

p lt 0001

AB

C

p lt 0001A

B C


ROS

( o

f con

trol

)


(a)

1 3 50

50

150

100A

BC

p lt 0001 p lt 0001

A A

C

p lt 0001A B

C


8-de

oxyg

uano

sine

( o

f con

trol

)

(b)










5 Conclusions


Data Availability




Acknowledgments


References












































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



0

5

10

15


A

B

C

DE

FF

GH I

00012

00490099

012024

049099

199392

DN

A P

icoG

reen

Gen

opro

tect

ive H

O

as

say (

co

ntro

l)

(a)

0

50

100

150

200


AB B B

C C C BC BA

C

00012

00240049

0099012

024049

099199

392

Kera

tinoc

ytes

Cel

l via

bilit

y (

of c

ontr

ol)

(b)

0

50

100

150

200

AAB

B BBC C C C C

B AB

00012

00240049

0099012

024049

099199

392

Fibr

obla

sts

Cel

l via

bilit

y (

of c

ontr

ol)

(c)






4 Discussion



0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

AB

CA

BC

p lt 0001p lt 001

DN

A d

amag

e(8

-deo

xygu

anos

ine

cont

rol)


(a)

049 099 049 09900

05

15

10 ACB

p lt 001A

CB

p lt 001


0 0

Bax

Bcl-2

Gen

e exp

ress

ion

ratio

(Bet

a-ac

tin n

orm

aliz

ed)

Barbatimao (mgmL)

(b)

0 049 099 0 049 0990

50

150

100

Barbatimao (mgmL)

A

CB

p lt 0001

AB

plt0001

C

Casp

ase 3

( o

f con

trol

)


(c)

0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

ACB

p lt 0001A

B

p lt 00001

CCa

spas

e 8 (

of c

ontr

ol)


(d)


1 3 50

50

100

150p lt 001A

B C

p lt 0001

AB

C

p lt 0001A

B C


ROS

( o

f con

trol

)


(a)

1 3 50

50

150

100A

BC

p lt 0001 p lt 0001

A A

C

p lt 0001A B

C


8-de

oxyg

uano

sine

( o

f con

trol

)

(b)










5 Conclusions


Data Availability




Acknowledgments


References












































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

AB

CA

BC

p lt 0001p lt 001

DN

A d

amag

e(8

-deo

xygu

anos

ine

cont

rol)


(a)

049 099 049 09900

05

15

10 ACB

p lt 001A

CB

p lt 001


0 0

Bax

Bcl-2

Gen

e exp

ress

ion

ratio

(Bet

a-ac

tin n

orm

aliz

ed)

Barbatimao (mgmL)

(b)

0 049 099 0 049 0990

50

150

100

Barbatimao (mgmL)

A

CB

p lt 0001

AB

plt0001

C

Casp

ase 3

( o

f con

trol

)


(c)

0 049 099 0 049 0990

50

100

150

Barbatimao (mgmL)

ACB

p lt 0001A

B

p lt 00001

CCa

spas

e 8 (

of c

ontr

ol)


(d)


1 3 50

50

100

150p lt 001A

B C

p lt 0001

AB

C

p lt 0001A

B C


ROS

( o

f con

trol

)


(a)

1 3 50

50

150

100A

BC

p lt 0001 p lt 0001

A A

C

p lt 0001A B

C


8-de

oxyg

uano

sine

( o

f con

trol

)

(b)










5 Conclusions


Data Availability




Acknowledgments


References












































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of










5 Conclusions


Data Availability




Acknowledgments


References












































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of













Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of








Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of


Documents

ReseachArticle - downloads.hindawi.comdownloads.hindawi.com/journals/bmri/2018/1942451.pdfgroup with just dsDNA in the solution. On the contrary, if an extract-test has a genoprotective