Research Article Phytochemical Constituents and Toxicity of...

Research ArticlePhytochemical Constituents and Toxicity of Duguetiafurfuracea Hydroalcoholic Extract in Drosophila melanogaster

Francisca Valeacuteria Soares de Arauacutejo Pinho12 Gustavo Felipe da Silva3

Giulianna Echeverria Macedo3 Katiane Raquel Muller3 Illana Kemmerich Martins3

Ana Paula Lausmann Ternes3 Joseacute Galberto Martins da Costa1

Margareth Linde Athayde4 Aline Augusti Boligon4 Jean Paul Kamdem25

Jeferson Luis Franco3 Irwin Rose Alencar de Menezes1 and Thaiacutes Posser3

1 Departamento de Quımica Biologica Universidade Regional do Cariri 63100-000 Crato CE Brazil2 Departamento de Quımica Programa de Pos-Graduacao em Bioquımica Toxicologica Universidade Federal de Santa Maria97105-900 Santa Maria RS Brazil

3 Centro Interdisciplinar de Pesquisa em Biotecnologia Universidade Federal do Pampa Campus Sao Gabriel97300-000 Sao Gabriel RS Brazil

4Departamento de Farmacia Industrial Laboratorio de Pesquisa em Fitoquımica Universidade Federal de Santa MariaPredio 26 Sala 1115 97105-900 Santa Maria RS Brazil

5 Departamento de Bioquımica Instituto de Ciencias Basica da Saude Universidade Federal do Rio Grande do Sul90035-003 Porto Alegre RS Brazil

Correspondence should be addressed toThaıs Posser thaisposserhotmailcom

Received 28 May 2014 Revised 16 September 2014 Accepted 27 September 2014 Published 10 November 2014

Academic Editor Wenchuan Lin

Copyright copy 2014 Francisca Valeria Soares de Araujo Pinho et al This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Duguetia furfuracea is frequently used as amedicinal plant in Brazil However studies have evidenced its cytotoxic bactericide andantitumor activities In the present studywe aimed to evaluate the potential toxicity of hydroalcoholic leaves extracts ofD furfuracea(HEDF) in a Drosophila melanogaster model Toxicity was assessed as changes in locomotor performance mitochondrial activityoxidative stressMAPKs phosphorylation and apoptosis induction after exposure toHEDF concentrations (1ndash50mgmL) for 7 daysThe phytoconstituents of the plant were screened for the presence of alkaloids tannins xanthones chalcones flavonoids auronesand phenolic acids Exposure of adult flies to HEDF caused mitochondrial dysfunction overproduction of ROS and alterations inthe activity of detoxifying enzymes GST SOD and CAT Induction of ERK phosphorylation and PARP cleavage was also observedindicating occurrence of HEDF-induced cell stress and apoptotic cell death In parallel alterations in cholinesterase activity andimpairments in negative geotaxis behavior were observed Our study draws attention to the indiscriminate use of this plant bypopulation and suggests oxidative stress as a major mechanism underlying its toxicity

1 Introduction

Theuse of plant extracts in the treatment andor prevention ofvarious diseases including cancer and cardiovascular diseasesis recognized since ancient time and some of these plantshave led to the development of an impressive number of newdrugs [1 2] The growing use of plant extracts instead ofsynthetic compounds is primary because they are generally

regarded as safe easily accessible affordable and culturallyacceptable form of health care trusted by large number ofpeople [3ndash6] Despite the beneficial effects of plant extractsthere are substantial evidences suggesting they can causecytotoxicity [7 8]Therefore evaluation of the toxic effects ofplant extracts used in folk medicine seems to be imperativesince they are generally consumed by population withoutconcerns on their toxicity [9]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 838101 11 pageshttpdxdoiorg1011552014838101

2 Evidence-Based Complementary and Alternative Medicine

Oxidative stress caused by overproduction of free rad-icals andor alterations in antioxidant defense systems isimplicated as a mechanism of toxicity of many synthetic andnatural compounds [10]The antioxidant cellular defense sys-tem including enzymatic (glutathione-S-transferase (GST)catalase (CAT) and superoxide dismutase (SOD)) andnonenzymatic (glutathione (GSH) ascorbic acid (vitaminC) 120572-tocopherol (vitamin E) and 120573-carotene (vitamin A))antioxidants act on reactive oxygen species (ROS) resultingfrom physiological or pathological processes [11] Environ-mental stressors toxic agents and natural compounds asflavonoids [12ndash14] are reported to modulate the phosphory-lation of mitogen activated protein kinases family (MAPKs)represented by ERK c-Jun NH

2-terminal kinase (JNK) and

p38MAPK kinases which participate in signaling pathwaysthat are responsible for many cellular functions such asgrowth differentiation and apoptosis

Duguetia furfuracea belonging to theAnnonaceae familyis a perennial and shrubby species found in the CentralWest Southeast and Northeast regions of Brazil [15 16] Itis popularly known as ldquoAraticum-Miudordquo ldquoAraticum-secordquoand ldquoAta bravardquo and is used in Brazilian folk medicine as anantihyperlipidemic and anorectic agent [17] Accordingly Dfurfuracea has also been reported in the treatment of rheuma-tism and renal colic [18] as an antiparasitary agent Thepowder of its seeds is used in the treatment of pediculosis [19]D furfuracea has shown to exhibit larvicidal activity againstAedes aegypti [20] and isolated alkaloids from the stembark of the plant have been reported to exhibit antitumoraltrypanocidal and leishmanicidal activities [21] Although Dfurfuracea have been used by population due its therapeuticproperties recently attention has been paid regarding thetoxicity of this plant speciesThe aqueous extract of the leavesof D furfuracea presented toxic effect in pregnant rats [22]Studies have demonstrated cytotoxic effects of the leaves ofD furfuracea in bacteria and animal models [23 24]

Phytochemical analysis of essential oil from leaves ofD furfuracea revealed the presence of sesquiterpenoids[25] and the bark of the underground stem revealed thepresence of the alkaloid (-)-duguetine 120573-N-oxide [26] inaddition flavonoids and several alkaloids [16] have been alsodescribed for aerial parts of Duguetia furfuracea Previousstudies demonstrated the at least five alkaloids isolatedfrom this plant have cytotoxic antitumoral trypanocidaland leishmanicidal activities [21] while sesquiterpenes arepotential anticancer agents [27] Flavonoids are recognizedby their beneficial and prooxidative effects depending onthe concentration and frequency of exposure presentingproperties such as anti-inflammatory diuretic antimicrobialantiviral antioxidant and proapoptotic [12]

In the present study we investigated the potential toxicityof a hydroalcoholic extract from leaves of D furfuracea(HEDF) in a fruit fly D melanogaster model The advantageof using this model is based on the fact that it raises fewethical concerns and has served as a unique and powerfulmodel to study human genetics diseases and for screeningsynthetic and natural compounds [28 29] Particularly weinvestigated the behavioral (negative geotaxis assay and

acetylcholinesterase activity) and biochemical markers ofoxidative stress and apoptotic cell death (ROS generation cellviability antioxidant enzymes p38MAPK and ERK phospho-rylation and PARP cleavage) following exposure of flies toHEDF up to seven days In addition the identification andquantification of phenolic compounds present in HEDFwerecarried out by HPLC

2 Materials and Methods

21 Materials Reduced glutathione (GSH) tetramethyleth-ylenediamine (TEMED) sodiumorthovanadate (Na

3VO4)

Quercetin (Q4951) 3-(45-dimethylthiazol-2-yl)-25-diphen-yltetrazolium bromide (MTT) Secondary antibodies(anti-rabbit IgG) horse radish peroxidase (HRP) conjugat-ed 55-dithiobis (2-nitrobenzoic acid) DTNB (D8130) acet-ylthiocholine iodide (A5751) 1-Chloro 24-dinitrobenzene(CDNB) (237329) and 2101584071015840-dichlorofluorescein diacetate(DCFH-DA) (35845) were obtained from Sigma-Aldrich(St Louis MO) The anti-phospho-p38 (Thr180Tyr182)total anti-p38 anti-phospho ERK12 (Thr202Tyr204) andanti-total-ERK12 antibodies and b-actin were purchasedfrom Cell Signaling (Beverly MA USA) Poly (ADP) ribosepolymerase (PARP) antibody was obtained from SantaCruz Biotechnology (Santa Cruz CA) SDS acrylamidebis-acrylamide chloride hybond nitrocellulose and dithio-threitol (DTT) were obtained from GE Healthcare LifeDivision Acetonitrile and formic gallic chlorogenic ellagicand caffeic acids were purchased from Merck (DarmstadtGermany) Catechin quercetin quercitrin isoquercitrinrutin and kaempferol were purchased from Sigma ChemicalCo (St Louis MO USA) High performance liquidchromatography (HPLC-DAD) was performed using HPLCsystem (Shimadzu Kyoto Japan) prominence autosampler(SIL-20A) equippedwith plunger pumps LC-20AT ShimadzuDGU connected to the integrator with 20A5 Degasser CBM20A UV-VIS detector DAD (diode) and SPD-M20A LC 122SP1 software solution All other chemicals were of analyticalgrade

22 Plant Material Leaves of D furfuracea were col-lected from Barreiro Grande Crato-Ceara (7∘2210158402810158401015840S39∘28101584042410158401015840W and altitude of 892m above sea level) Brazilin September 2011 and identified by Dr Maria Arlene Pessoada Silva A voucher specimen (n 6703) was deposited in theHerbarium Caririense Dardano de Andrade Lima (HCDAL)of the Regional University of Cariri (URCA)

23 Preparation of Crude Ethanolic Extract Fresh leaves(1050 g) of D furfuracea were washed with water thencrushed and macerated with 998 of ethanol and water(1 1 vv) for three days The suspension was filtered and thesolvent evaporated under reduced pressure and lyophilized toobtain 26113 g of crude ethanolic extract The dried extractwas then kept frozen prior to use

24 Preliminary Phytochemical Analysis Classes of sec-ondarymetabolites were screened for the presence or absence

Evidence-Based Complementary and Alternative Medicine 3

of alkaloids tannins xanthones chalcones flavonoids andaurones in HEDF using the methods previously describedby Matos [30] This is a qualitative test based on visualobservation of change in coloration or precipitate formationin addition to specific reagents

25 Identification and Quantitation of Phenolic Compoundsof HEDF by HPLC Reverse phase chromatographic analyseswere carried out under gradient conditions using C

18column

(46mm times 250mm) packed with 5120583m diameter particlesThe mobile phase was water containing 1 formic acid (A)and acetonitrile (B) and the composition gradient was 13of B until 10min and changed to obtain 20 30 5060 70 20 and 10 B at 20 30 40 50 60 70 and80min respectively [31] with slight modifications HEDFwas analyzed at a concentration of 5mgmL The presenceof ten antioxidants compounds namely gallic chlorogenicellagic and caffeic acids catechin quercetin quercitrin iso-quercitrin rutin and kaempferol were investigatedThe iden-tification of these compounds was performed by comparingtheir retention time and UV absorption spectrum with thoseof the commercial standards The flow rate was 07mLmininjection volume 40 120583L and the wavelength 254 nm for gallicacid 280 nm for catechin 325 nm for caffeic ellagic andchlorogenic acids and 365 nm for quercetin isoquercitrinquercitrin rutin and kaempferol All the samples andmobilephase were filtered through 045 120583m membrane filter (Milli-pore) and then degassed by ultrasonic bath prior to use Stocksolutions of standards references were prepared in the HPLCmobile phase at a concentration range of 0030ndash0250mgmLfor kaempferol quercetin quercitrin isoquercitrin catechinand rutin and 0030ndash0250mgmL for gallic caffeic ellagicand chlorogenic acids All chromatography operations werecarried out at ambient temperature and in triplicateThe limitof detection (LOD) and limit of quantification (LOQ) werecalculated based on the standard deviations of the responsesand the slopes using three independent analytical curves asdefined by [32] LOD and LOQ were calculated as 33 and10120590S respectively where 120590 is the standard deviation of theresponse and S is the slope of the calibration curve

26 Drosophila melanogaster Stock and Media Drosophilamelanogaster (Harwich strain) was obtained from theNational Species Stock Center Bowling Green OH USAThe flies were maintained at 25 plusmn 1∘C and 60ndash70 relativehumidityThe diet was composed of 6mL of cereal flour cornflour water and antifungal agent (Nipagin) as previouslydescribed [14]

27 Experimental ProceduremdashFlies Exposed to HEDF

271 Mortality In order to determine the doses and theduration of exposure 45 adults flies of both genders (1-to 5-day-old) per vial were exposed for 7 days to vari-ous concentrations of HEDF (0 1 2 10 20 50 100 and200mgmL)mixed to the diet Each concentration containedthree replicates The number of dead and alive flies wasrecorded daily for 7 days Based on these preliminary data

the concentrations range of 0 1 10 and 50mgmL of HEDFwere chosen with 7-day-exposure

272 Negative Geotaxis Test Locomotor activity of flieswas determined based on negative geotaxis behavior assayas previously described [33] with some modifications Theflies were exposed to HEDF as described above Followingexposure 10 flies (both genders) were sorted under a brief iceanesthesia and transferred in labeled vertical glass columnstube (15 cm length and 15 cm in diameter) After 30min ofrecovery from ice flies were gently tapped to the bottomof the tube and the number of flies that climbed up to6 cm mark of the column (ie the top) in 5 sec as well asthose that remained below the mark was counted separatelyThe procedure was repeated five times per group at 1minintervals

273 Mitochondrial Activity-MTT Reduction Assay Cellviability was determined by the mitochondrial dehydro-genase activity using the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) After exposure theviability of HEDF-treated and nontreated flies was evaluatedin the whole body of flies according to the method describedby Sudati et al [34] For this set of experiment three wholeflies of similar size were used per well The absorbance wasmeasured at 540 nm in a microplate reader (2300 EnspireMultilabel Plate Reader 2009) and the results were expressedas mean of absorbance of three determinations performed intriplicate

274 Measurement of ROS Production ROS generation wasmeasured as a general index of oxidative stress using the2101584071015840-dichlorofluorescein diacetate (DCHF-DA) a nonpolarcompound which after conversion to a polar derivative byintracellular esterase activity rapidly reacts with ROS toform the highly fluorescent compound dichlorofluorescein(DCF) [35 36] Briefly after exposure of flies to HEDF (0ndash50mgmL) the whole body of 20 flies was homogenated in1mL of 20mM Tris buffer (pH 70) and then centrifugedat 1000 rpm for 10min at 4∘C An aliquot of 20120583L of thesupernatant was incubated with 6 120583L of 10mM DCFH-DAfor 60min The formation of the fluorescent product DCFwas measured using a microplate reader (2300 MultilabelPlate reader Enspire 2009) at 488 and 525 nm excitationand emission wavelengths respectively The results wereexpressed in arbitrary units as themean of DCF fluorescence

(1) Estimation of Protein Thiol (PSH) and Nonprotein Thiol(NPSH) Protein thiols (PSH) and nonprotein (NPSH) weremeasured as previously described [37] For thiols estimation20 flies were homogenated in 300 120583L of percholic acid (PCA05M) and centrifuged at 10000timesg for 5min at 4∘C Thesupernatant was used to measure NPSH and the pellet wasresuspended in 200 120583L of TrisHCl 05M pH 80 to measurePSH

(2) Activity of Selected Enzymes (AChE CAT GST and SOD)To determine acetylcholinesterase (AchE) catalase (CAT)


glutathione-S-transferase (GST) and superoxide dismutase(SOD) activities flies homogenates were centrifuged at1000timesg for 5min at 4∘C and an aliquot of the supernatant(S1) was used for measurement of AchE [38] while the (S1)was centrifuged again at 14000timesg for 30min at 4∘C for themeasurement CAT [35] GST [36] and SOD [39]

275 Western Blotting Western blotting methodology wasperformed according to the method described [14] Fortyflies from HEDF-treated and untreated were homogenizedat 4∘C in 200120583L of buffer (pH 70) containing 50mM Tris1mMEDTA 01mMphenyl methyl sulfonyl fluoride 20mMNa3VO4 100mMsodiumfluoride and phosphatase inhibitor

cocktail The homogenates were centrifuged at 1000timesg for10min at 4∘C and the supernatants (S1) were collected Afterdetermination of the protein [40] 10 DTT was addedto the samples Then the samples were frozen at minus20∘Cfor later determination of total and phosphorylated formsof ERK 12 and phosphorylated form of p38MAPK usingspecific antibodies The immunoblots were visualized andquantified on the 400MM Pro Bruker imaging system usingECL detection reagents The density of the bands was mea-sured and expressed as percent of increase over the control(nontreated flies) 120573-actin total p38MAPK and total ERK wereused as loading controls for Western blotting experimentsProtein levels were quantified using bovine serum albuminas standard

28 Statistical Analysis Theresults are shown asmeanplusmn SEM(standard error of mean) of three independent experimentsperformed in duplicate Statistical significance was measuredby one-way analysis of variance (ANOVA) followed byDunnettrsquos or Tukeyrsquos posttest when appropriated Differencesbetween groups were considered to be significant when 119875 lt005

3 Results

31 Phytochemical Screening of HEDF The presence of alka-loids tannins flavones flavonols chantonas chauconas andauronas was identified in HEDF (data not shown)

32 Identification and Quantification of Phenolic Compoundsof HEDF by HPLC HPLC fingerprinting of HEDF revealedthe presence of the gallic acid (119905

119877= 995min peak 1)

catechin (119905119877= 1608min peak 2) chlorogenic acid (119905

119877=

2014min peak 3) caffeic acid (119905119877= 2463min peak

4) ellagic acid (119905119877= 3729min peak 5) rutin (119905

119877=

3987min peak 6) isoquercitrin (119905119877= 4493min peak

7) quercitrin (119905119877= 4815min peak 8) quercetin (119905

119877=

5107min peak 9) and kaempferol (119905119877= 6156min peak

10) They were identified by comparisons with the retentiontimes and UV spectra of the standards analyzed under thesame analytical conditions (Figure 1) Caffeic acid (3317 plusmn003mgg) rutin (2005 plusmn 001mgg) quercitrin (1907 plusmn002mgmL) and isoquercitrin (1861 plusmn 001mgmL) werethe major components present in HEDF while kaempferol

0

250 500

1 2

3

4

5

67

8

910

100

200

300

(min)

(mAU

)

0

Figure 1 High performance liquid chromatography profile ofHEDF detection UV was at 325 nm Gallic acid (peak 1) catechin(peak 2) chlorogenic acid (peak 3) caffeic acid (peak 4) ellagicacid (peak 5) rutin (peak 6) isoquercitrin (peak 7) quercitrin (peak8) quercetin (peak 9) and kaempferol (peak 10) Calibration curvefor gallic acid Y = 14286x + 13958 (119903 = 09996) catechin Y =15097x + 11893 (119903 = 09997) caffeic acid Y = 12758x + 12597(119903 = 09996) chlorogenic acid Y = 13461x + 12753 (119903 = 09992)ellagic acid Y = 13576x + 13464 (119903 = 09999) rutin Y = 12845 +13057 (119903 = 09999) quercetin Y = 13560x + 11926 (119903 = 09991)isoquercitrin Y = 12873x + 13256 (119903 = 09998) quercitrin Y =11870x + 13298 (119903 = 09993) and kaempferol Y = 14253x + 12389(119903 = 09997)

Table 1 Quantification of phenolic compounds from the HEDF

Compounds HEDF LOD LOQmgg 120583gmL 120583gmL

Gallic acid 529 plusmn 001a 052 0015 0049

Catechin 531 plusmn 001a 053 0032 0105

Chlorogenic acid 1603 plusmn 002b 160 0009 0029Caffeic acid 3317 plusmn 003

c 331 0024 0078Ellagic acid 730 plusmn 001

d 073 0013 0042Rutin 2005 plusmn 001

e 200 0027 0090Isoquercitrin 1861 plusmn 001

f 186 0008 0026Quercitrin 1907 plusmn 002

ef 180 0035 0114Quercetin 587 plusmn 001

a 058 0019 0063Kaempferol 536 plusmn 001

a 053 0026 0085Results are expressed asmeanplusmn standard deviations (SD) of three determina-tions Averages followed by different letters differ by Tukeyrsquos test at119875 lt 0001

(536 plusmn 001mgmL) catechin (531 plusmn 001mgmL) and gallicacid (529 plusmn 001mgmL) were less abundant (Table 1)

33 Effects of HEDF Exposure on Survival of Flies Ourpreliminary results showed that exposure of flies to HEDF(0ndash200mgmL) caused 78 (119875 lt 005) mortality at con-centration of 50mgmL after 7 days of exposure while 100and 200mgmL of HEDF caused 100 mortality (Figure 2)Considering the high toxicity of the higher concentrationsof the extract we decided to use the concentrations of 1 10and 50mgmL of HEDF for the evaluation of biochemicalanalyses


1 2 3 4 5 6 7

0

10

20

30

40

50

CTL

Days of exposure

Num

ber o

f dea

d fli

es (i

n a t

otal

of 4

5 fli

esg

roup

)

1mgmL2mgmL10mgmL

20mgmL50mgmL100mgmL200mgmL

lowast

lowast lowastlowast

lowastlowast

lowast

Figure 2 Mortality curve of HEDF (0ndash200mgmL) exposed fliesThe number of live and dead flies was counted daily for seven daysThe data represent the mean plusmn SEM of three separate experimentsand are expressed number of dead flies in relation to total number offlies Doses of 100 and 200mgmL were significant when comparedto control group lowast119875 lt 005

331 HEDF Exposure Induced Locomotor Deficits in D mela-nogaster Exposure of flies to 1 and 10mgmL of HEDFfor 7 days did not change the locomotion activity of flieswhen compared to control (Figure 3 119875 gt 005) Howeverflies treated with 50mgmL of HEDF remained mostly atthe bottom of the column when compared to the controlgroup (Figure 3) indicating locomotor deficit In parallel tobehavioral parameter the activity of AchE was evaluatedsince acetylcholine is themain excitatory neurotransmitter ofthe insect central nervous system [41] As shown in Figure 4the activity of AchE was significantly elevated (119875 lt 0001) inflies exposed to 1 and 10mgmL of HEDF when comparedto control flies In contrast 50mgmL of HEDF caused asignificant inhibition of AchE activity when compared tocontrol flies (Figure 4 119875 lt 005)

332 Mitochondrial Activity of HEDF Exposed Flies Themetabolic viability of cells was evaluated by assessing theMTT reducing capacity of fliesrsquo homogenates HEDF expo-sure caused a significant decrease in MTT reduction at con-centrations of 10 and 50mgmL when compared to controlflies (Figure 5) (119875 lt 005)

333 Oxidative Stress Analysis As depicted in Figure 6HEDF (50mgmL) caused significant increase of DCF flu-orescence in the homogenates of flies when compared tocontrol (119875 lt 001) Our results also revealed that only thehighest concentration of HEDF (50mgmL) exposed fliescaused a significant decrease in the levels of nonproteinthiols (NPSH) (Figure 7 119875 lt 005) However no changesin the levels of protein thiols (PSH) were observed in HEDFexposed flies when compared to control (Figure 7)

0 1 10 5000

25

50

75

100

125

TopBase

Num

ber o

f flie

s at b

ase a

nd to

p(to

tal o

f ten

flie

s)

HEDF (mgmL)

lowastlowastlowast

Figure 3 Locomotive performance in groups of 10 flies exposed toHEDF for seven days at doses of 0 1 10 and 50mgmL by negativegeotaxis testThemeasurements were repeated five times at intervalsof 1min The test was performed in triplicate (119899 = 10) and datarepresents the mean plusmn SEM of the number of flies that were in thetop and bottom Exposure of flies to 50mgmL of HEDF causedsignificant deficit in locomotive performance when compared tocontrol group (lowastlowastlowast119875 lt 00001)

lowastlowastlowast

0 1 10 500

50

100

150

200

HEDF (mgmL)

lowastlowastlowast

AchE

activ

ity (m

Um

g pr

otei

n)

Figure 4 Effect of HEDF exposed flies on the activity of AchEHEDF (1 and 10mgmL) induced alteration in the activity of AchEin HEDF-treated flies after 7 days of exposure The data representthe mean plusmn SEM of three replicates performed in duplicate lowastlowastlowast119875 lt0001 and lowast119875 lt 005 indicate significant difference when comparedto control

Considering that oxidative stress was induced by expo-sure of flies toHEDF (evidenced by increasedROS generationand decreasedNPSH levels) wemeasured the activity ofGSTSOD and CAT which are antioxidant enzymes involved inthe cell adaptive response to oxidative stress HEDF induceda marked increase in the activities of GST (Figure 8(a)) SOD(Figure 8(b)) and CAT (Figure 8(c)) at concentrations of 1and 10mgmL when compared to their respective control


000

002

004

006

008

0 1 10 50

HEDF (mgmL)

lowastlowast

lowastMTT

redu

ctio

n(a

bsor

banc

e120582540

nm)

Figure 5 MTT reduction of flies exposed to different concentra-tions of HEDF for 7 days The experiments were performed intriplicate and the bars represent an average plusmn SEM of absorbanceobtained in control group (untreated flies) and HEDF exposed flies119899 = 10 lowast119875 lt 005 versus control flies (untreated)

0 1 10 500

200

400

600

800

1000

Relat

ive fl

uore

scen

ce in

tens

ity lowastlowast

HEDF (mgmL)

Figure 6 ROS production in response to exposure to HEDF (1ndash50mgmL) flies for seven days ROS production is represented bythe intensity of fluorescence emitted by the DCF and representsthe mean plusmn SEM of three independent experiments performed intriplicate (119899 = 3) lowastlowast119875 lt 001 compared to the control group(untreated flies)

(119875 lt 0001) However at the highest concentration tested(50mgmL) there was a substantial decrease in GST activity(Figure 8(a)119875 lt 005) while CAT and SOD activities did notchange (Figures 8(b) and 8(c))

334 Phosphorylation of MAPKs and PARP Cleavage Thephosphorylation of MAPK family components was assayedafter 7 day-exposure of D melanogaster to HEDF Phospho-rylation of ERK2 was significantly increased in flies treatedwith 10mgmL of HEDF while p38MAPK was not modifiedat all the concentrations tested when compared to untreatedflies (Figures 9(b) and 9(c)) Exposure to all concentrations

0

5

10

15

20

PSHGSH

HEDF (mgmL) HEDF (mgmL)

lowastlowast

0 1 10 50 0 1 10 50

(120583M

olg

of fl

ies)

Figure 7 Levels of protein (PSH) and nonprotein thiol (NPSH) inhomogenates of flies exposed to different concentrations of HEDF(1 10 and 50mgmL) for 7 days There was a significant decreasein PSH levels at dose of 50mgmL of HEDF PSH or NPSH levelsare represented in 120583Molg of flies lowastlowast119875 lt 001 versus untreatedflies Data are expressed as mean plusmn SEM of 119899 = 4 independentexperiments performed in triplicate

of extract caused PARP cleavage (Figure 9(a)) indicating theoccurrence of apoptotic cell death

4 Discussion

Although the use of plant extracts has been reported to exert avariety of pharmacological actions by different mechanismsthere is evidence that some of them can cause toxicity [42 43]Therefore it is imperative to explore the toxicity potentialof plant extracts popularly used in folk medicine In thepresent study we investigated the potential toxicity of thehydroalcoholic extract of the leaves of D furfuracea (HEDF)in aDrosophila melanogastermodel and identified some phy-tochemicals of the plant extract Our results demonstratedthat 7 days of exposure of D melanogaster to HEDF causedtoxicity in a process involving oxidative stress alterations inthe antioxidant enzymes MAPK proteins phosphorylationand apoptotic cell death

The flies exposed to HEDF showed a significant decreasein the MTT reduction indicating that viability of fliesrsquo cellswas compromised In the same line flies that were exposedto HEDF revealed a significant increase in ROS production atconcentration of 50mgmL whereas lower concentrations ofHEDF (1 and 10mgmL) seemed to stimulate ROS generationin minor extent and improve antioxidant enzymes activitiesIn parallel PARP cleavage a general index of apoptoticcell death was observed at all concentrations analyzed Theobserved increase of antioxidant enzymes could represent anadaptive cellular response in counteracting HEDF toxicityThis apparent adaptive response was possibly induced by themild increase of ROS at the lower concentrations of HEDFleading to an increase of GST SOD and CAT activities inthe flies thus improving its antioxidant capacity The highestlevel of ROS generation in the homogenates of flies exposed to


0 1 10 500

100

200

300

HEDF (mgmL)

GST

activ

ity (m

Um

g pr

otei

n)

lowastlowast

lowastlowastlowast

(a)

0 1 10 500

5

10

15

20

HEDF (mgmL)

SOD

activ

ity (m

Um

g pr

otei

n) lowastlowastlowast

lowastlowastlowast

(b)

0 1 10 500

50

100

150

200

HEDF (mgmL)

Cata

lase

activ

ity (m

Um

g pr

otei

n)

lowastlowastlowast

lowastlowastlowast

(c)

Figure 8 Analysis of antioxidant enzymes activities after 7 days exposure of flies to various concentrations of HEDF (1ndash50mgmL) (a) GST(b) SOD and (c) CAT activities are expressed as mUmg of protein The data represent the mean plusmn SEM of three independent replicatesperformed in duplicates lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus control flies (untreated)

50mgmL may be at least in part responsible for the deple-tion of GST and CAT activities decrease in mitochondrialactivity as well as NPSH oxidation In fact oxidative stressresults from an unbalance between the production of reactivespecies and the capacity of cellular antioxidant defense toneutralize those species When this capacity is overwhelmedit leads to a diversity of cellular damages [44] A significantreduction of NPSH (ie GSH) levels was identified in fliesexposed to the 50mgmL of HEDF It is well described thatlow levels of cellularGSH results froman increase in oxidativestress associated with toxic agents (ROS inducers) [45 46] Itis interesting to note that GST SOD and CAT activities wereincreased in the flies that were exposed to the lower doses ofHEDF (1 and 10mgmL) Low to moderate level of oxidativestress has been associated with increased antioxidant defensesystem [46] In previous studies flies exposed to rotenonepresented a significant elevation of antioxidant enzymes andthis effect was related to the increased ROS generation andformation of toxic aldehydes [47] Excessive ROS generationcan cause lipid peroxidation mitochondrial dysfunction and

damage to proteins lipids and nucleic acids thereby alteringthe normal function of the cells [48]

The activity of acetylcholinesterase (AChE) was increasedin the flies exposed to 1 and 10mgmL of HEDF and wasinhibited at 50mgmL In fact acetylcholine (Ach) playsseveral functions in the nervous system acting in cognitiveprocess motivation and reward stimuli processing andthe sleep cycle The inhibition of AChE compromises thehydrolysis of the neurotransmitter ACh leading to the accu-mulation of this neurotransmitter in the synapses [49 50]Many studies with toxic agents such as rotenone paraquatand organophosphorus revealed that these agents can leadto the inhibition of AchE generating the loss of the cholin-ergic homeostasis which causes many neurochemical andneurobehavioral disturbances [51ndash53] The central nervoussystem is very susceptible to oxidative stress due to highoxygen consumption lower levels of antioxidant defenseshigh levels of polyunsaturated fatty acids (phospholipidmembrane) and high levels of iron [54] The inhibitionof AchE at 50mgmL of HEDF correlated well with the


HEDF

PARP

117 kDa

83 kDa

120573-Actin

(mgmL)Cont 1 10 50

p-P38MAPK

t-P38MAPK

p-ERK

t-ERK

(a)

lowast

0

50

100

150

ControlER

K 2

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol

1mgmL 10mgmL 50mgmL

(b)

0

50

100

150

p38

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol

Control 1mgmL 10mgmL 50mgmL

(c)

Figure 9Western blot analysis of the phosphorylated and total forms of ERK (a) and p38MAPK (b) and PARP cleavage in homogenates of fliesexposed to HEDF (1ndash50mgmL) for seven days The total content and phosphorylation of proteins were detected by specific antibodies andthe reaction was developed by the method of detection by enhanced chemiluminescence (ECL) The graph represents the quantification ofthe immunoreactive bands and means plusmn SEM of three independent experiments ( of control) (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 comparedto the control group

observed locomotor deficits induced by HEDF at the sameconcentration These results indicate that HEDF-inducedtoxicity may be associated with impairment of neurologicalfunctionsThere is evidence that enhanced activation of AchEcan cause a reduction of cholinergic neurotransmission andaffect other related functions including cell proliferation andpromote apoptosis [55 56] In the present study exposureof flies to 1 and 10mgmL of HEDF significantly increasedAchE activity Alterations in AchE activity can compromisethe normal motor activity of the flies and this was evidencedin our study by the impairment of locomotive performancein the negative geotaxis behavior assay

Mitogen-activated protein kinases (MAPKs) are a familyof proteins that participate in transduction pathways affected

by various environmental pollutants including heavy metals[14] Usually the ERK is activated by mitogenic stimuliand regulates mainly the growth and differentiation TheJNK and the p38MAPK are activated by cellular stress andinflammatory cytokines and are involved in the apoptoticprocess These proteins regulate the transmission of signalsfrom membrane to the nucleus and are well conserved fromunicellular to more complexes organisms [57 58] Studiesdemonstrated that plant derivatives such as curcumin canmodulate this pathway mediating the antitumoral propertyattributed to this compound [58 59] In our study exposureof flies to 10mgmL of HEDF significantly increased ERKphosphorylation It well known that ERK pathway is sus-ceptible to oxidative stress In addition hydrogen peroxide


metals and other cell stressors are demonstrated to stim-ulate phosphorylation of this MAPK protein [58 60] thuscontributing to activation of several downstream targets astranscriptional factors It was demonstrated that medicinalplants are able to induce gene activation dependent of thetranscriptional factor Nrf-2 via ERK [61] This transcriptionfactor is known as the main regulator of the antioxidant cellresponse and control the expression of several antioxidantenzymes [61] Here the phosphorylation of ERK occurredin parallel with an increase in antioxidant enzymes activitypossibly in response to extracellular stimuli [62] induced byHEDF exposure A possible link between the activity of theseenzymes and ERK activation in our model must be furtherinvestigated

In the present work the phytochemical prospection andHPLC analysis of the leaves of HEDF revealed the presenceof alkaloids and chalcones in addition to polyphenolic com-pounds Of particular importance chalcone and some chal-cone analogues have been reported to be toxic in zebrafishmodel [63] Additionally isolated oxoaporphine alkaloidsfrom three species of the Annonaceae family including Dfurfuracea have shown cytotoxic effect in the lineage ofHep2-cells (laryngeal carcinoma) [64] On the other hand Liet al 2014 [65] showed the potentiality of total flavonoidsfrom Arachniodes exilis to induce apoptotic cell death inhuman hepatoma HepG2 cells and cause oxidative stressSimilarly it was shown that the flavonoid quercetin causedapoptotic cell death in different human tumoral cell typesdependently of ERKphosphorylation and this effect occurredin parallel with a time dependent ROS production [12] Itwas previously demonstrated that the flavonoid quercetinwas protective against H

2O2induced toxicity only at low

concentrations while at higher concentration it inductedapoptotic cell death in hepatoma cells [66] and acting as aprooxidant agent [66 67] In accordance our data showsthat effects of D furfuracea on survival and biochemicalparameters in flies are related with the concentrations andtime of exposure

5 Conclusions

The present study demonstrated for the first time the toxicityofDuguetia furfuracea in theDrosophila melanogastermodelsystem The adverse effects of HEDF to flies were evidencedby alterations in several markers of cell stress and neurobe-havioral parameters The toxicity induced by the extract toflies may be attributed to an individual or synergistic actionof phytochemicals found in this plant over the period ofexposure Overall our results suggest that oxidative stressmay be major mechanism underlying D furfuracea inducedtoxicity in D melanogaster

Conflict of Interests

The authors declare no conflict of interests regarding thepublication of this paper

Authorsrsquo Contribution

Francisca Valeria Soares de Araujo Pinho and Gustavo Felipeda Silva contributed equally to this work

Acknowledgments

The authors acknowledge CAPES CNPq FAPERGS andFAPERGS-PRONEM for financial support Jean Paul Kam-dem particularly thanks TWAS the Alexander vonHumboldtFoundation (AvH) TWAS-CNPq and CAPES for the finan-cial support Jeferson Luis Franco Jose Galberto Martins daCosta and Margareth Linde Athayde are CNPq fellowshiprecipients

References

[1] M J Balunas and A D Kinghorn ldquoDrug discovery frommedicinal plantsrdquo Life Sciences vol 78 no 5 pp 431ndash441 2005

[2] B B Mishra and V K Tiwari ldquoNatural products an evolvingrole in future drug discoveryrdquo European Journal of MedicinalChemistry vol 46 no 10 pp 4769ndash4807 2011

[3] World Health Organization ldquoReport technical briefing ontraditional medicinerdquo in Proceedings of the 49th Regional Com-mittee Meeting WHO Regional Office for the Western PacificManila Philippines September 1998

[4] World Health Organization Consultation Meeting on Tradi-tional Medicine and Modern Medicine Harmonizing the TwoApproaches World Health Organization Geneva Switzerland1999 (document reference (WP)TMICPTM001RB98ndashS99GE32(CHN))

[5] World Health Organization Traditional Complementaryand Alternative Medicines and Therapies Working GroupOPSOMS WHO Regional Office for the AmericasPanAmerican Health Organization Washington DC USA 1999

[6] World Health Organization WHO Traditional Medicine Strat-egy 2002ndash2005 World Health Organization Geneva Switzer-land 2002

[7] N Penha-Silva C B Firmino F G de Freitas Reis et al ldquoInflu-ence of age on the stability of human erythrocyte membranesrdquoMechanisms of Ageing and Development vol 128 no 7-8 pp444ndash449 2007

[8] M V de Freitas R D C M Netto J C da Costa Huss et alldquoInfluence of aqueous crude extracts of medicinal plants on theosmotic stability of human erythrocytesrdquo Toxicology in Vitrovol 22 no 1 pp 219ndash224 2008

[9] A L Guimaraes A P Oliveira and J R G S AlmeidaldquoChallenges to implementation of Herbal Medicine in HealthSystemrdquo in Completeness and Health Epistemology Politics andPractices of Care A F Barreto Ed pp 97ndash107 University of EdUFPE Recife Brazil 2011

[10] H Kim B-S Park K-G Lee et al ldquoEffects of naturallyoccurring compounds on fibril formation and oxidative stressof 120573-amyloidrdquo Journal of Agricultural and Food Chemistry vol53 no 22 pp 8537ndash8541 2005

[11] C G Noschang R Krolow L F Pettenuzzo et al ldquoInteractionsbetween chronic stress and chronic consumption of caffeine onthe enzymatic antioxidant systemrdquoNeurochemical Research vol34 no 9 pp 1568ndash1574 2009


[12] W-M Lee M Hsiao J-L Chang et al ldquoQuercetin inducesmitochondrial-derived apoptosis via reactive oxygen species-mediated ERK activation in HL-60 leukemia cells andxenograftrdquo Archives of Toxicology 2014

[13] S J Harper and P LoGrasso ldquoSignalling for survival and deathin neurones the role of stress-activated kinases JNK and P38rdquoCellular Signalling vol 13 no 5 pp 299ndash310 2001

[14] M T Paula A P Zemolin A P Vargas et al ldquoEffects of Hg (II)exposure on MAPK phosphorylation and antioxidante systeminDmelanogasterrdquo Environmental Toxicology vol 29 no 6 pp621ndash630 2014

[15] H Lorenzi and F J A MatosMedicinal Plants in Brazil Nativeand Exotic Plantarum Institute Sao Paulo Brazil 2002

[16] C A Carollo A R Hellmann-Carollo J M de Siqueira andS Albuquerque ldquoAlkaloids and a flavonoid from aerial parts(leaves and twigs) of Duguetia furfuraceamdashannonaceaerdquo Jour-nal of the Chilean Chemical Society vol 51 no 2 pp 837ndash8412006

[17] M A B Silva L V L Melo and R V Ribeiro ldquoEthnobotanicalsurvey of plants used as anti-hyperlipidemic and the anorecticpopulation of New Xanthine-MT Brazilrdquo Revista Brasileira deFarmacognosiaBrazilian Journal of Pharmacognosy vol 20 no4 pp 540ndash562 2010

[18] V R G Rodrigues and D A Carvalho Eds Medicinal Plantsin the Field Clenched UFLA Lavras Brazil 2001

[19] M de Fatima Agra P F de Freitas and J M Barbosa-FilholdquoSynopsis of the plants known as medicinal and poisonous inNortheast of Brazilrdquo Brazilian Journal of Pharmacognosy vol17 no 1 pp 114ndash140 2007

[20] AM S Rodrigues J E de Paula N Degallier J FMolez and LS Espındola ldquoLarvicidal activity of someCerrado plant extractsagainstAedes aegyptirdquo Journal of the AmericanMosquito ControlAssociation vol 22 no 2 pp 314ndash317 2006

[21] D B da Silva E C O Tulli G C G Militao et al ldquoThe antitu-moral trypanocidal and antileishmanial activities of extract andalkaloids isolated from Duguetia furfuraceardquo Phytomedicinevol 16 no 11 pp 1059ndash1063 2009

[22] M R Toledo M T L Peres M C Vieira et al ldquoFitotoxicidadedo extrato aquoso de Duguetia furfuraceae (St Hill) B etH em ratas (Rattus novergicus)rdquo Revista Brasileira de PlantasMedicinais vol 8 no 4 pp 218ndash222 2006

[23] C R Silva P M Vieira S C Santos and L Chen-ChenldquoAssessment of Duguetia furfuracea genotoxic and cytotoxicactivity in bacteria and micerdquo Anais da Academia Brasileira deCiencias vol 84 no 1 pp 149ndash156 2012

[24] L S Coelho L P Felicio C T Miranda et al ldquoModulatoryeffects of Duguetia furfuracea (A St Hil) Benthand HookF in Drosophila melanogaster somatic and germinative cellsrdquoGenetics and Molecular Research vol 10 no 1 pp 75ndash85 2011

[25] C A Carollo A R Hellmann and J M de Siqueira ldquoSesquiter-penoids from the essential oil from leaves of Duguetia fur-furacea (Annonaceae)rdquo Biochemical Systematics and Ecologyvol 33 no 6 pp 647ndash649 2005

[26] D B da Silva E C O TulliW S Garcez E A Nascimento andJ M de Siqueira ldquoChemical constituents of the undergroundstem bark of Duguetia furfuracea (Annonaceae)rdquo Journal of theBrazilian Chemical Society vol 18 no 8 pp 1560ndash1565 2007

[27] BM Fraga ldquoNatural sesuiterpenoidsrdquoNatural Product Reportsvol 23 pp 947ndash972 2006

[28] K M Beckingham J D Armstrong M J Texada R Mun-jaal and D A Baker ldquoDrosophila melanogastermdashthe model

organism of choice for the complex biology of multi-cellularorganismsrdquoGravitational and Space Biology Bulletin vol 18 no2 pp 17ndash29 2005

[29] S-I Kim J-W Jung Y-J Ahn L L Restifo and H-W KwonldquoDrosophila as a model system for studying lifespan and neuro-protective activities of plant-derived compoundsrdquo Journal ofAsia-Pacific Entomology vol 14 no 4 pp 509ndash517 2011

[30] F J AMatos Introduction to Experimental Phytochemical UFCFortaleza Brazil 2nd edition 1997

[31] A A Boligon T F de Brum J K Frolhich A L F Froeder andM L Athayde ldquoHPLCDAD profile and determination of totalphenolics flavonoids tannins and alkaloids contents of scutiabuxifolia reissek stem barkrdquoResearch Journal of Phytochemistryvol 6 no 3 pp 84ndash91 2012

[32] A A Boligon T F Kubica D N Mario et al ldquoAntimicrobialand antiviral activity-guided fractionation from Scutia buxifoliaReissek extractsrdquoActa Physiologiae Plantarum vol 35 no 7 pp2229ndash2239 2013

[33] HCoulomand S Birman ldquoChronic exposure to rotenonemod-els sporadic Parkinsonrsquos disease in Drosophila melanogasterrdquoJournal of Neuroscience vol 24 no 48 pp 10993ndash10998 2004

[34] J H Sudati F A Vieira S S Pavin et al ldquoValeriana offic-inalis attenuates the rotenone-induced toxicity in Drosophilamelanogasterrdquo NeuroToxicology vol 37 pp 118ndash126 2013

[35] H Aebi and P Lester ldquoCatalase in vitrordquo Methods in Enzymol-ogy vol 105 pp 121ndash126 1984

[36] W B Jakoby and W H Habig ldquoGlutathione S-transferases (ratand human)rdquoMethods in Enzymology vol 77 pp 218ndash231 1981

[37] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[38] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961

[39] V A Kostyuk and A I Potapovich ldquoSuperoxide-driven oxida-tion of quercetin and a simple sensitive assay for determinationof superoxide dismutaserdquoBiochemistry International vol 19 no5 pp 1117ndash1124 1989

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

[41] Y H Kim and S H Lee ldquoWhich acetylcholinesterase functionsas the main catalytic enzyme in the Class Insectardquo InsectBiochemistry and Molecular Biology vol 43 no 1 pp 47ndash532013

[42] S R Mariz G S Cerqueira W C Araujo et al ldquoAcute toxico-logical study of the ethanol extract of aerial parts of Jatro-pha gossypiifolia L in ratsrdquo Revista Brasileira de Farmacog-nosiaBrazilian Journal of Pharmacognosy vol 16 no 3 pp 372ndash378 2006

[43] D Ntelios M Kargakis T Topalis A Drouzas and E PotolidisldquoAcute respiratory failure due to Nicotiana glauca ingestionrdquoHippokratia vol 17 no 2 pp 183ndash184 2013

[44] A L A Ferreira and L S Matsubara ldquoFree radicals conceptsdiseases and oxidative stress defense systemsrdquo Revista daAssociacao Medica Brasileira vol 43 no 1 pp 61ndash68 1997

[45] R Hosamani and S R Ramesh ldquoAttenuation of rotenone-induced mitochondrial oxidative damage and neurotoxicty inDrosophila melanogaster supplemented with creatinerdquo Neuro-chemical Research vol 35 no 9 pp 1402ndash1412 2010


[46] D Anderson ldquoAntioxidant defences against reactive oxygenspecies causing genetic and other damagerdquo Mutation Researchvol 350 no 1 pp 103ndash108 1996

[47] R Hosamani and Muralidhara ldquoNeuroprotective efficacy ofBacopa monnieri against rotenone induced oxidative stress andneurotoxicity in Drosophila melanogasterrdquo NeuroToxicologyvol 30 no 6 pp 977ndash985 2009

[48] M Valko D Leibfritz J Moncol M T D Cronin M Mazurand J Telser ldquoFree radicals and antioxidants in normal physio-logical functions and human diseaserdquoThe International Journalof Biochemistry amp Cell Biology vol 39 no 1 pp 44ndash84 2007

[49] M Calic A L Vrdoljak B Radic et al ldquoIn vitro and invivo evaluation of pyridinium oximes Mode of interactionwith acetylcholinesterase effect on tabun- and soman-poisonedmice and their cytotoxicityrdquo Toxicology vol 219 no 1ndash3 pp 85ndash96 2006

[50] D M Roberts and C K Aaron ldquoManaging acute organophos-phorus pesticide poisoningrdquo British Medical Journal vol 334no 7594 pp 629ndash634 2007

[51] P Eyer ldquoNeuropsychopathological changes by organophospho-rus compoundsmdasha reviewrdquo Human and Experimental Toxicol-ogy vol 14 no 11 pp 857ndash864 1995

[52] T A Slotkin ldquoDevelopmental cholinotoxicants nicotine andchlorpyrifosrdquo Environmental Health Perspectives vol 107 no 1pp 71ndash80 1999

[53] A P Da Silva F C Meotti A R S Santos and M FarinaldquoLactational exposure to malathion inhibits brain acetyl-cholinesterase inmicerdquoNeuroToxicology vol 27 no 6 pp 1101ndash1105 2006

[54] T A Slotkin ldquoCholinergic systems in brain development anddisruption by neurotoxicants nicotine environmental tobaccosmoke organophosphatesrdquo Toxicology and Applied Pharmacol-ogy vol 198 no 2 pp 132ndash151 2004

[55] Q H Jin H Y He Y F Shi H Lu and X J Zhang ldquoOverex-pression of acetylcholinesterase inhibited cell proliferation andpromoted apoptosis in NRK cellsrdquo Acta Pharmacologica Sinicavol 25 no 8 pp 1013ndash1021 2004

[56] R Schmatz C M Mazzanti R Spanevello et al ldquoResver-atrol prevents memory deficits and the increase in acetyl-cholinesterase activity in streptozotocin-induced diabetic ratsrdquoEuropean Journal of Pharmacology vol 610 no 1ndash3 pp 42ndash482009

[57] B E Stronach andN Perrimon ldquoStress signaling inDrosophilardquoOncogene vol 18 no 45 pp 6172ndash6182 1999

[58] Y Zhou S Zheng J Lin Q-J Zhang and A Chen ldquoTheinterruption of the PDGF and EGF signaling pathways bycurcumin stimulates gene expression of PPARgamma in ratactivated hepatic stellate cell in vitrordquo Laboratory Investigationvol 87 no 5 pp 488ndash498 2007

[59] C T Chu D J Levinthal S M Kulich E M Chalovich andD B DeFranco ldquoOxidative neuronal injury the dark side ofERK12rdquo European Journal of Biochemistry vol 271 no 11 pp2060ndash2066 2004

[60] T Posser J L Franco L Bobrovskaya R B Leal P WDickson and P R Dunkley ldquoManganese induces sustainedSer40 phosphorylation and activation of tyrosine hydroxylasein PC12 cellsrdquo Journal of Neurochemistry vol 110 no 3 pp 848ndash856 2009

[61] M Richter A F Winkel D Schummer et al ldquoPau drsquoarcoactivates Nrf2-dependent gene expression via the MEKERK-pathwayrdquoThe Journal of Toxicological Sciences vol 39 no 2 pp353ndash361 2014

[62] L A Tibbles and J R Woodgett ldquoThe stress-activated proteinkinase pathwaysrdquo Cellular and Molecular Life Sciences vol 55no 10 pp 1230ndash1254 1999

[63] Y-T Lee T-H Fong H-M Chen et al ldquoToxicity assessmentsof chalcone and some synthetic chalcone analogues in azebrafish modelrdquoMolecules vol 19 no 1 pp 641ndash650 2014

[64] D B da Silva M D F C Matos S T Nakashita et al ldquoIsolationand cytotoxicity evaluation of some oxoaporphine alkaloidsfrom Annonaceaerdquo Quimica Nova vol 30 no 8 pp 1809ndash18122007

[65] H Li J Chen C Xiong HWei C Yin and J Ruan ldquoApoptosisinduction by the total flavonoids from Arachniodes exilis inHepG2 Cells through reactive oxygen species-mediated mito-chondrial dysfunction involving MAPK activationrdquo Evidence-Based Complementary and Alternative Medicine vol 2014Article ID 906941 11 pages 2014

[66] W Watjen G Michels B Steffan et al ldquoLow concentrationsof flavonoids are protective in rat H4IIE cells whereas highconcentrations cause DNA damage and apoptosisrdquo Journal ofNutrition vol 135 no 3 pp 525ndash531 2005

[67] D Metodiewa A K Jaiswal N Cenas E Dickancaite and JSegura-Aguilar ldquoQuercetin may act as a cytotoxic prooxidantafter its metabolic activation to semiquinone and quinoidalproductrdquo Free Radical Biology and Medicine vol 26 no 1-2 pp107ndash116 1999

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


Oxidative stress caused by overproduction of free rad-icals andor alterations in antioxidant defense systems isimplicated as a mechanism of toxicity of many synthetic andnatural compounds [10]The antioxidant cellular defense sys-tem including enzymatic (glutathione-S-transferase (GST)catalase (CAT) and superoxide dismutase (SOD)) andnonenzymatic (glutathione (GSH) ascorbic acid (vitaminC) 120572-tocopherol (vitamin E) and 120573-carotene (vitamin A))antioxidants act on reactive oxygen species (ROS) resultingfrom physiological or pathological processes [11] Environ-mental stressors toxic agents and natural compounds asflavonoids [12ndash14] are reported to modulate the phosphory-lation of mitogen activated protein kinases family (MAPKs)represented by ERK c-Jun NH

2-terminal kinase (JNK) and

p38MAPK kinases which participate in signaling pathwaysthat are responsible for many cellular functions such asgrowth differentiation and apoptosis

Duguetia furfuracea belonging to theAnnonaceae familyis a perennial and shrubby species found in the CentralWest Southeast and Northeast regions of Brazil [15 16] Itis popularly known as ldquoAraticum-Miudordquo ldquoAraticum-secordquoand ldquoAta bravardquo and is used in Brazilian folk medicine as anantihyperlipidemic and anorectic agent [17] Accordingly Dfurfuracea has also been reported in the treatment of rheuma-tism and renal colic [18] as an antiparasitary agent Thepowder of its seeds is used in the treatment of pediculosis [19]D furfuracea has shown to exhibit larvicidal activity againstAedes aegypti [20] and isolated alkaloids from the stembark of the plant have been reported to exhibit antitumoraltrypanocidal and leishmanicidal activities [21] Although Dfurfuracea have been used by population due its therapeuticproperties recently attention has been paid regarding thetoxicity of this plant speciesThe aqueous extract of the leavesof D furfuracea presented toxic effect in pregnant rats [22]Studies have demonstrated cytotoxic effects of the leaves ofD furfuracea in bacteria and animal models [23 24]

Phytochemical analysis of essential oil from leaves ofD furfuracea revealed the presence of sesquiterpenoids[25] and the bark of the underground stem revealed thepresence of the alkaloid (-)-duguetine 120573-N-oxide [26] inaddition flavonoids and several alkaloids [16] have been alsodescribed for aerial parts of Duguetia furfuracea Previousstudies demonstrated the at least five alkaloids isolatedfrom this plant have cytotoxic antitumoral trypanocidaland leishmanicidal activities [21] while sesquiterpenes arepotential anticancer agents [27] Flavonoids are recognizedby their beneficial and prooxidative effects depending onthe concentration and frequency of exposure presentingproperties such as anti-inflammatory diuretic antimicrobialantiviral antioxidant and proapoptotic [12]

In the present study we investigated the potential toxicityof a hydroalcoholic extract from leaves of D furfuracea(HEDF) in a fruit fly D melanogaster model The advantageof using this model is based on the fact that it raises fewethical concerns and has served as a unique and powerfulmodel to study human genetics diseases and for screeningsynthetic and natural compounds [28 29] Particularly weinvestigated the behavioral (negative geotaxis assay and

acetylcholinesterase activity) and biochemical markers ofoxidative stress and apoptotic cell death (ROS generation cellviability antioxidant enzymes p38MAPK and ERK phospho-rylation and PARP cleavage) following exposure of flies toHEDF up to seven days In addition the identification andquantification of phenolic compounds present in HEDFwerecarried out by HPLC

2 Materials and Methods

21 Materials Reduced glutathione (GSH) tetramethyleth-ylenediamine (TEMED) sodiumorthovanadate (Na

3VO4)

Quercetin (Q4951) 3-(45-dimethylthiazol-2-yl)-25-diphen-yltetrazolium bromide (MTT) Secondary antibodies(anti-rabbit IgG) horse radish peroxidase (HRP) conjugat-ed 55-dithiobis (2-nitrobenzoic acid) DTNB (D8130) acet-ylthiocholine iodide (A5751) 1-Chloro 24-dinitrobenzene(CDNB) (237329) and 2101584071015840-dichlorofluorescein diacetate(DCFH-DA) (35845) were obtained from Sigma-Aldrich(St Louis MO) The anti-phospho-p38 (Thr180Tyr182)total anti-p38 anti-phospho ERK12 (Thr202Tyr204) andanti-total-ERK12 antibodies and b-actin were purchasedfrom Cell Signaling (Beverly MA USA) Poly (ADP) ribosepolymerase (PARP) antibody was obtained from SantaCruz Biotechnology (Santa Cruz CA) SDS acrylamidebis-acrylamide chloride hybond nitrocellulose and dithio-threitol (DTT) were obtained from GE Healthcare LifeDivision Acetonitrile and formic gallic chlorogenic ellagicand caffeic acids were purchased from Merck (DarmstadtGermany) Catechin quercetin quercitrin isoquercitrinrutin and kaempferol were purchased from Sigma ChemicalCo (St Louis MO USA) High performance liquidchromatography (HPLC-DAD) was performed using HPLCsystem (Shimadzu Kyoto Japan) prominence autosampler(SIL-20A) equippedwith plunger pumps LC-20AT ShimadzuDGU connected to the integrator with 20A5 Degasser CBM20A UV-VIS detector DAD (diode) and SPD-M20A LC 122SP1 software solution All other chemicals were of analyticalgrade

22 Plant Material Leaves of D furfuracea were col-lected from Barreiro Grande Crato-Ceara (7∘2210158402810158401015840S39∘28101584042410158401015840W and altitude of 892m above sea level) Brazilin September 2011 and identified by Dr Maria Arlene Pessoada Silva A voucher specimen (n 6703) was deposited in theHerbarium Caririense Dardano de Andrade Lima (HCDAL)of the Regional University of Cariri (URCA)

23 Preparation of Crude Ethanolic Extract Fresh leaves(1050 g) of D furfuracea were washed with water thencrushed and macerated with 998 of ethanol and water(1 1 vv) for three days The suspension was filtered and thesolvent evaporated under reduced pressure and lyophilized toobtain 26113 g of crude ethanolic extract The dried extractwas then kept frozen prior to use

24 Preliminary Phytochemical Analysis Classes of sec-ondarymetabolites were screened for the presence or absence




18column
















3 Results



119877= 995min peak 1)


119877=



119877=



119877=



0

250 500

1 2

3

4

5

67

8

910

100

200

300

(min)

(mAU

)

0








d 073 0013 0042Rutin 2005 plusmn 001









1 2 3 4 5 6 7

0

10

20

30

40

50

CTL

Days of exposure

Num

ber o

f dea

d fli

es (i

n a t

otal

of 4

5 fli

esg

roup

)

1mgmL2mgmL10mgmL


lowast

lowast lowastlowast

lowastlowast

lowast





0 1 10 5000

25

50

75

100

125

TopBase

Num

ber o

f flie

s at b

ase a

nd to

p(to

tal o

f ten

flie

s)

HEDF (mgmL)

lowastlowastlowast


lowastlowastlowast

0 1 10 500

50

100

150

200

HEDF (mgmL)

lowastlowastlowast

AchE

activ

ity (m

Um

g pr

otei

n)




000

002

004

006

008

0 1 10 50

HEDF (mgmL)

lowastlowast

lowastMTT

redu

ctio

n(a

bsor

banc

e120582540

nm)


0 1 10 500

200

400

600

800

1000

Relat

ive fl

uore

scen

ce in

tens

ity lowastlowast

HEDF (mgmL)




0

5

10

15

20

PSHGSH


lowastlowast

0 1 10 50 0 1 10 50

(120583M

olg

of fl

ies)



4 Discussion




0 1 10 500

100

200

300

HEDF (mgmL)

GST

activ

ity (m

Um

g pr

otei

n)

lowastlowast

lowastlowastlowast

(a)

0 1 10 500

5

10

15

20

HEDF (mgmL)

SOD

activ

ity (m

Um

g pr

otei


lowastlowastlowast

(b)

0 1 10 500

50

100

150

200

HEDF (mgmL)

Cata

lase

activ

ity (m

Um

g pr

otei

n)

lowastlowastlowast

lowastlowastlowast

(c)






HEDF

PARP

117 kDa

83 kDa

120573-Actin

(mgmL)Cont 1 10 50

p-P38MAPK

t-P38MAPK

p-ERK

t-ERK

(a)

lowast

0

50

100

150

ControlER

K 2

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol

1mgmL 10mgmL 50mgmL

(b)

0

50

100

150

p38

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol


(c)










5 Conclusions






Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















18column
















3 Results



119877= 995min peak 1)


119877=



119877=



119877=



0

250 500

1 2

3

4

5

67

8

910

100

200

300

(min)

(mAU

)

0








d 073 0013 0042Rutin 2005 plusmn 001









1 2 3 4 5 6 7

0

10

20

30

40

50

CTL

Days of exposure

Num

ber o

f dea

d fli

es (i

n a t

otal

of 4

5 fli

esg

roup

)

1mgmL2mgmL10mgmL


lowast

lowast lowastlowast

lowastlowast

lowast





0 1 10 5000

25

50

75

100

125

TopBase

Num

ber o

f flie

s at b

ase a

nd to

p(to

tal o

f ten

flie

s)

HEDF (mgmL)

lowastlowastlowast


lowastlowastlowast

0 1 10 500

50

100

150

200

HEDF (mgmL)

lowastlowastlowast

AchE

activ

ity (m

Um

g pr

otei

n)




000

002

004

006

008

0 1 10 50

HEDF (mgmL)

lowastlowast

lowastMTT

redu

ctio

n(a

bsor

banc

e120582540

nm)


0 1 10 500

200

400

600

800

1000

Relat

ive fl

uore

scen

ce in

tens

ity lowastlowast

HEDF (mgmL)




0

5

10

15

20

PSHGSH


lowastlowast

0 1 10 50 0 1 10 50

(120583M

olg

of fl

ies)



4 Discussion




0 1 10 500

100

200

300

HEDF (mgmL)

GST

activ

ity (m

Um

g pr

otei

n)

lowastlowast

lowastlowastlowast

(a)

0 1 10 500

5

10

15

20

HEDF (mgmL)

SOD

activ

ity (m

Um

g pr

otei


lowastlowastlowast

(b)

0 1 10 500

50

100

150

200

HEDF (mgmL)

Cata

lase

activ

ity (m

Um

g pr

otei

n)

lowastlowastlowast

lowastlowastlowast

(c)






HEDF

PARP

117 kDa

83 kDa

120573-Actin

(mgmL)Cont 1 10 50

p-P38MAPK

t-P38MAPK

p-ERK

t-ERK

(a)

lowast

0

50

100

150

ControlER

K 2

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol

1mgmL 10mgmL 50mgmL

(b)

0

50

100

150

p38

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol


(c)










5 Conclusions






Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results



119877= 995min peak 1)


119877=



119877=



119877=



0

250 500

1 2

3

4

5

67

8

910

100

200

300

(min)

(mAU

)

0








d 073 0013 0042Rutin 2005 plusmn 001









1 2 3 4 5 6 7

0

10

20

30

40

50

CTL

Days of exposure

Num

ber o

f dea

d fli

es (i

n a t

otal

of 4

5 fli

esg

roup

)

1mgmL2mgmL10mgmL


lowast

lowast lowastlowast

lowastlowast

lowast





0 1 10 5000

25

50

75

100

125

TopBase

Num

ber o

f flie

s at b

ase a

nd to

p(to

tal o

f ten

flie

s)

HEDF (mgmL)

lowastlowastlowast


lowastlowastlowast

0 1 10 500

50

100

150

200

HEDF (mgmL)

lowastlowastlowast

AchE

activ

ity (m

Um

g pr

otei

n)




000

002

004

006

008

0 1 10 50

HEDF (mgmL)

lowastlowast

lowastMTT

redu

ctio

n(a

bsor

banc

e120582540

nm)


0 1 10 500

200

400

600

800

1000

Relat

ive fl

uore

scen

ce in

tens

ity lowastlowast

HEDF (mgmL)




0

5

10

15

20

PSHGSH


lowastlowast

0 1 10 50 0 1 10 50

(120583M

olg

of fl

ies)



4 Discussion




0 1 10 500

100

200

300

HEDF (mgmL)

GST

activ

ity (m

Um

g pr

otei

n)

lowastlowast

lowastlowastlowast

(a)

0 1 10 500

5

10

15

20

HEDF (mgmL)

SOD

activ

ity (m

Um

g pr

otei


lowastlowastlowast

(b)

0 1 10 500

50

100

150

200

HEDF (mgmL)

Cata

lase

activ

ity (m

Um

g pr

otei

n)

lowastlowastlowast

lowastlowastlowast

(c)






HEDF

PARP

117 kDa

83 kDa

120573-Actin

(mgmL)Cont 1 10 50

p-P38MAPK

t-P38MAPK

p-ERK

t-ERK

(a)

lowast

0

50

100

150

ControlER

K 2

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol

1mgmL 10mgmL 50mgmL

(b)

0

50

100

150

p38

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol


(c)










5 Conclusions






Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1 2 3 4 5 6 7

0

10

20

30

40

50

CTL

Days of exposure

Num

ber o

f dea

d fli

es (i

n a t

otal

of 4

5 fli

esg

roup

)

1mgmL2mgmL10mgmL


lowast

lowast lowastlowast

lowastlowast

lowast





0 1 10 5000

25

50

75

100

125

TopBase

Num

ber o

f flie

s at b

ase a

nd to

p(to

tal o

f ten

flie

s)

HEDF (mgmL)

lowastlowastlowast


lowastlowastlowast

0 1 10 500

50

100

150

200

HEDF (mgmL)

lowastlowastlowast

AchE

activ

ity (m

Um

g pr

otei

n)




000

002

004

006

008

0 1 10 50

HEDF (mgmL)

lowastlowast

lowastMTT

redu

ctio

n(a

bsor

banc

e120582540

nm)


0 1 10 500

200

400

600

800

1000

Relat

ive fl

uore

scen

ce in

tens

ity lowastlowast

HEDF (mgmL)




0

5

10

15

20

PSHGSH


lowastlowast

0 1 10 50 0 1 10 50

(120583M

olg

of fl

ies)



4 Discussion




0 1 10 500

100

200

300

HEDF (mgmL)

GST

activ

ity (m

Um

g pr

otei

n)

lowastlowast

lowastlowastlowast

(a)

0 1 10 500

5

10

15

20

HEDF (mgmL)

SOD

activ

ity (m

Um

g pr

otei


lowastlowastlowast

(b)

0 1 10 500

50

100

150

200

HEDF (mgmL)

Cata

lase

activ

ity (m

Um

g pr

otei

n)

lowastlowastlowast

lowastlowastlowast

(c)






HEDF

PARP

117 kDa

83 kDa

120573-Actin

(mgmL)Cont 1 10 50

p-P38MAPK

t-P38MAPK

p-ERK

t-ERK

(a)

lowast

0

50

100

150

ControlER

K 2

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol

1mgmL 10mgmL 50mgmL

(b)

0

50

100

150

p38

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol


(c)










5 Conclusions






Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















000

002

004

006

008

0 1 10 50

HEDF (mgmL)

lowastlowast

lowastMTT

redu

ctio

n(a

bsor

banc

e120582540

nm)


0 1 10 500

200

400

600

800

1000

Relat

ive fl

uore

scen

ce in

tens

ity lowastlowast

HEDF (mgmL)




0

5

10

15

20

PSHGSH


lowastlowast

0 1 10 50 0 1 10 50

(120583M

olg

of fl

ies)



4 Discussion




0 1 10 500

100

200

300

HEDF (mgmL)

GST

activ

ity (m

Um

g pr

otei

n)

lowastlowast

lowastlowastlowast

(a)

0 1 10 500

5

10

15

20

HEDF (mgmL)

SOD

activ

ity (m

Um

g pr

otei


lowastlowastlowast

(b)

0 1 10 500

50

100

150

200

HEDF (mgmL)

Cata

lase

activ

ity (m

Um

g pr

otei

n)

lowastlowastlowast

lowastlowastlowast

(c)






HEDF

PARP

117 kDa

83 kDa

120573-Actin

(mgmL)Cont 1 10 50

p-P38MAPK

t-P38MAPK

p-ERK

t-ERK

(a)

lowast

0

50

100

150

ControlER

K 2

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol

1mgmL 10mgmL 50mgmL

(b)

0

50

100

150

p38

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol


(c)










5 Conclusions






Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1 10 500

100

200

300

HEDF (mgmL)

GST

activ

ity (m

Um

g pr

otei

n)

lowastlowast

lowastlowastlowast

(a)

0 1 10 500

5

10

15

20

HEDF (mgmL)

SOD

activ

ity (m

Um

g pr

otei


lowastlowastlowast

(b)

0 1 10 500

50

100

150

200

HEDF (mgmL)

Cata

lase

activ

ity (m

Um

g pr

otei

n)

lowastlowastlowast

lowastlowastlowast

(c)






HEDF

PARP

117 kDa

83 kDa

120573-Actin

(mgmL)Cont 1 10 50

p-P38MAPK

t-P38MAPK

p-ERK

t-ERK

(a)

lowast

0

50

100

150

ControlER

K 2

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol

1mgmL 10mgmL 50mgmL

(b)

0

50

100

150

p38

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol


(c)










5 Conclusions






Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HEDF

PARP

117 kDa

83 kDa

120573-Actin

(mgmL)Cont 1 10 50

p-P38MAPK

t-P38MAPK

p-ERK

t-ERK

(a)

lowast

0

50

100

150

ControlER

K 2

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol

1mgmL 10mgmL 50mgmL

(b)

0

50

100

150

p38

phos

phor

ylat

ion

(rat

io p

hosp

hoto

tal)

o

f con

trol


(c)










5 Conclusions






Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusions






Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Phytochemical Constituents and Toxicity of...

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Transcript of Research Article Phytochemical Constituents and Toxicity of...