Arrabidaea chica hexanic extract induces mitochondrion damage and peptidase inhibition on Leishmania...

Research ArticleArrabidaea chica Hexanic Extract Induces MitochondrionDamage and Peptidase Inhibition on Leishmania spp

Igor A Rodrigues1 Mariana M B Azevedo2 Francisco C M Chaves3 Celuta S Alviano4

Daniela S Alviano4 and Alane B Vermelho4

1 Departamento de Produtos Naturais e Alimentos Centro de Ciencias da Saude Faculdade de FarmaciaUniversidade Federal do Rio de Janeiro 219491-590 Rio de Janeiro RJ Brazil

2 Programa de Pos Graduacao Ciencias dos Alimentos Instituto de Quımica Universidade Federal do Rio de Janeiro21941-590 Rio de Janeiro RJ Brazil

3 Embrapa Amazonia Ocidental CP 319 69010-970 Manaus AM Brazil4Departamento de Microbiologia Geral Centro de Ciencias da Saude Instituto de Microbiologia Paulo de GoesUniversidade Federal do Rio de Janeiro 21941-590 Rio de Janeiro RJ Brazil

Correspondence should be addressed to Alane B Vermelho abvermelhomicroufrjbr

Received 21 February 2014 Accepted 23 March 2014 Published 9 April 2014

Academic Editor Jose Carlos Tavares Carvalho

Copyright copy 2014 Igor A Rodrigues et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Currently available leishmaniasis treatments are limited due to severe side effects Arrabidaea chica is a medicinal plant used inBrazil against several diseases In this study we investigated the effects of 5 fractions obtained from the crude hexanic extract of Achica against Leishmania amazonensis and L infantum as well as on the interaction of these parasites with host cells Promastigoteswere treated with several concentrations of the fractions obtained from A chica for determination of their minimum inhibitoryconcentration (MIC) In addition the effect of themost active fraction (B2) on parasitersquos ultrastructurewas analyzed by transmissionelectron microscopy To evaluate the inhibitory activity of B2 fraction on Leishmania peptidases parasites lysates were treated withthe inhibitory and subinhibitory concentrations of the B2 fraction The minimum inhibitory concentration of B2 fraction was 372and 186 120583gmL for L amazonensis and L infantum respectively Important ultrastructural alterations as mitochondrial swellingwith loss ofmatrix content and the presence of vesicles inside this organelle were observed in treated parasitesMoreover B2 fractionwas able to completely inhibit the peptidase activity of promastigotes at pH 55 The results presented here further support the useof A chica as an interesting source of antileishmanial agents

1 Introduction

Among individual infectious diseases leishmaniasis is inthe ninth position of the global burden of diseases Thisillness has two main clinical manifestations which are cuta-neous lesions (cutaneous leishmaniasismdashCL) and visceralimpairments (visceral leishmaniasismdashVL) [1] CL and VLrepresent a serious public health problem in 98 countries and3 territories on 5 continents where the disease can be foundAccording to World Health Organization (WHO) there aremore than 220000CL cases and 58000VL cases per year[2] In Brazil CL and VL are widespread and can be found

not only in rural areas but also in urban areas mainly due todeforestation and new settlements [3]

Despite the large number of both synthetic and naturalantileishmanial agents described in the literature only afew drugs have reached the clinical stage with approval forhuman use This fact could be partly explained by the lackof investments in drug research for poverty-related diseaseswhich includes leishmaniasis [4] The current chemotherapyfor leishmaniasis treatment still relies on the use of pen-tavalent antimonials and amphotericin B although liposo-mal amphotericin B paromomycin and miltefosine havebeen introduced for the treatment of the disease in several

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 985171 7 pageshttpdxdoiorg1011552014985171

2 BioMed Research International

countries However most of these drugs are expensivepresent toxic effects and are able to induce parasite resistance[5] Consequently the search for new and more effectiveantileishmanial agents remains crucial

Arrabidaea chica (HBK) Verlot Bignoniaceae is a scram-bling shrub native to tropical America more particularlyin the Amazon basin where it is also known as ldquoParirirdquoldquoCrajirurdquo ldquoCarajururdquo or ldquoCarajirurdquo The leaves of A chicahave been traditionally used by Brazilian Indians as a dyefor body painting in rituals and to protect the skin againstsunlight aswell as an insect repellant Chemical investigationshave been carried out since the beginning of this centuryto determine the composition of the A chica dye whichused to be commercialized as such [6] Nowadays A chicais used by the regional population as an anti-inflammatoryand astringent agent as well as a remedy for intestinal colicdiarrhea leucorrhea anemia and leukemia [7] The presentstudy aimed to evaluate the antileishmanial effects of thehexanic extract of the A chica leaves

2 Materials and Methods

21 Chemicals Resazurin RPMI 1640 medium and bovineserum albumin were purchased from Sigma Chemical CoUSA Amphotericin B was purchased from Fontoura-WyethBrazil Fetal bovine serum (FBS) was purchased from Cri-pion Biotecnologia Ltda Brazil All solvents used werespectroscopic grade from Tedia (Fairfield OH USA) Col-umn chromatographic product was purchased from Merck(Darmstadt Germany)

22 Plant Material and Acquisition of the Hexanic ExtractThe sample of A chica was kept in a germplasm bankunder the same cultivation practices at the EAFM Herbar-ium from Federal Institute of Amazonas (Manaus AM)where a voucher specimen was deposited (registry EAFM6791) Leaves of A chica were collected between 0800 and0900AM

A chica crude extract was obtained by 1 week extractionin hexane Then the extract was carefully filtered dried andstored in opaque glass vials at minus10∘C Afterwards the crudeextract was subjected to silica gel column chromatographywith an increasing gradient of polarity starting with 100 n-hexane and 100 ethyl acetate to 100 ethanol affording fivefractions (B1 B2 B3 B4 and B5)

23 Analysis of the Hexanic Extract of A chica by GC-MS The B2 fraction of the hexanic extract from ldquocrajirurdquowas analyzed by a gas chromatograph (GC) interfaced toa mass spectrometer (MS) employing the following condi-tions the oven temperature was programmed from 60∘Cto 300∘C at 10∘Cmin and helium was the carrier gas (at10mLmin) One microliter of 1 solution of the B2 fractionin dichloromethane was injected in split mode (1 50) Massspectra were obtained in an Agilent 5973N system fittedwith a low bleeding 5 phenyl95 methyl silicone (HP-5 MS 30m times 025mm times 025 120583m) fused silica capillarycolumn operating in the electronic ionization mode (EI)at 70 eV with a scan mass range of 40ndash500mz Sampling

rate was 315 scans The ion source was kept at 230∘C massanalyzer at 150∘C and transfer line at 260∘C Linear retentionindices (LRI) were measured by injection of a series of n-alkanes (C

10ndashC30) in the same column and conditions as

described above and compared with reference data Theidentification of the B2 fraction constituents was made basedon the retention indexes and by comparison of mass spectrawith computer search using NIST21 and NIST107 librariesCompound concentrationswere calculated from theGCpeakareas and they were arranged in order of GC elution

24 Parasite Strains and Cell Cultures Promastigote forms oftwo Leishmania species Leishmania (L) amazonensis (IFLABR1967PH8) and L (L) infantum (MHOMBR1974PP75)from the Leishmania Type Culture Collection of OswaldoCruz InstituteFiocruz (Rio de JaneiroRJBrazil) were usedin all experiments Parasites were axenically cultured inPBHILmediumas previously described [8] In order to assureinfectiveness of the parasites periodical infection of miceperitoneal macrophages was performed

25 Evaluation of Leishmania Inhibitory Concentrations Theassay was carried out in a 96-well microtiter plate wherethe hexanic extract from A chica was serially diluted induplicate to final test concentrations (1ndash500 120583gmL) Then50 times 10

5 promastigote forms of L amazonensis or Linfantum were harvested at the early stationary phase addedto each well and plated at 26∘C for 120 h At the end ofincubation period 25120583L of resazurin solution (5mg100mLof phosphate buffer saline pH 72) was added and the viabilityof parasites was determined in accordance with the protocolpreviously described [9] The minimal inhibitory concen-tration (MIC) was considered the lowest concentration ofthe hexanic extract that completely prevented the growthof Leishmania in vitro Alternatively 120 h-treated parasiteswere centrifuged (1000 g5min) washed twice in PBS andthen reincubated in fresh PBHIL culture medium in order toevaluate the leishmanicidal effect The lowest concentrationable to inhibit parasite growth was considered the minimalleishmanicidal concentration (MLC) The 50 inhibitoryconcentration (IC

50) was determined by logarithmic regres-

sion analysis of the data obtained as described above

26 Ultrastructure Analysis Alterations in the ultrastructureof the parasites were analyzed by transmission (TEM) elec-tron microscopy First promastigote forms of L infantumwere harvested at the early stationary phase of growthwashed twice with PBS and incubated in the presence ofa subinhibitory concentration (subMIC) of A chica hex-anic extract at 28∘C for 24 hours After the parasites werewashed twice in PBS they were fixed with glutaraldehydesolution (25 glutaraldehyde in 01M sodium cacodylatebuffer containing 35 sucrose pH 74) at 4∘C for 60minSamples of treated cells and their controls (untreated cells)were sent to Plataforma Rudolf Barth (Instituto OswaldoCruzFiocruzRJ) and processed as previously mentioned[10] The photomicrographs were obtained using an electronmicroscope Jeol JEM1011

BioMed Research International 3

Table 1 Antileishmanial activity and cytotoxic effect of the A chica hexanic extract fractions

Hexanic extract fraction L amazonensis L infantum Macrophages SIMIC (120583gmL) IC50 (120583gmL) MIC (120583gmL) IC50 (120583gmL) MCC (120583gmL)

B1 na nd na nd nd ndB2 372 318 186 147 2976 80a16b

B3 1867 1522 1867 1396 nd ndB4 368 1985 368 1797 nd ndB5 na nd na nd nd ndAmphotericin B 101 007 0625 001 146 144a234b

MIC minimum inhibitory concentration IC50 50 inhibitory concentration MCC minimum cytotoxic concentrationna not active at the highest concentration tested (500120583gmL) nd not determinedSI selective index aselective index for L amazonensis bselective index for L infantum

27 Peptidase Inhibition Assay L amazonensis and L infan-tum promastigotes (106 parasitesmL) were harvested at thelog phase washed twice by centrifugation (1500timesg5min)with PBS pH 68 and then disrupted through seven cyclesof freezing and thawing (minus80∘C37∘C) The cellular extractswere then centrifuged (12000timesg10min) and the super-natant aliquots preserved at 0∘C Peptidase (gelatinase)activity was analyzed through the protocol adapted fromCedrola et al [11] Briefly 100 120583L of the cellular lysates wasincubatedwith different concentrations of the hexanic extractin a PBS 01MpH 55 or pH 10 and gelatin 1 mixtureE64 (cysteine peptidase inhibitor) and 110-phenanthroline(metalopeptidase inhibitor) were used as positive controlsAfter the 30min incubation period at 37∘C enzymatic activ-ity was stoppedwith isopropanol and the samples were refrig-erated at 4∘C for 15min Next the samples were centrifuged(2500timesg15min) and 100 120583L supernatant was collected andthe absorbance was measured as previously described [12]One unit of gelatinase activity was defined as the amountof enzyme required to produce 1 120583g of peptides under thedescribed assay conditions

28 Peritoneal Mouse Macrophages and Cytotoxicity AssayNonelicited peritonealmacrophages from female Balbcmicewere collected in cold RPMI 1640 medium and plated in 96-well culture plates at the concentration of 105 cells100 120583LDifferent concentrations ranging from 1 to 500 120583gmL ofthe hexanic extract were added to each well and the cellswere incubated at 37∘C in 4 CO

2atmosphere for 48 h The

minimumcytotoxic concentration (MCC)was determined aspreviously described by Al-Musayeib et al using resazurinas the cellular viability indicator [13] The selective index(SI) was calculated using the MICMCC ratios The animalsused for macrophage acquisition were killed according tothe federal guidelines and institutional policies by cervicaldislocation

29 Infection of Macrophages Anti-Intracellular AmastigoteActivity and Nitric Oxide Production Peritoneal mousemacrophageswere obtained as described aboveThe infectionassays were carried out following the protocol described byPassero et al with slightmodifications [14] Briefly peritonealmacrophages (105 cells100 120583L) were plated in 96-well culture

plates and a ratio of 5 stationary phase promastigotes (Lamazonensis or L infantum) to 1macrophagewas used for theinfection procedure The parasite-macrophage interactionswere carried out in RPMI 1640 medium supplemented with10 of FBS at 35∘C for 24 hours in 4 CO

2atmosphere

After interaction assays were completed free promastigotesand nonadherent macrophages were removed by extensivewashing with PBS and the hexanic extract was added to eachwell at inhibitory and subinhibitory concentrations for Lamazonensis and L infantum After 48 hours of treatment thesupernatants from L amazonensis- and L infantum-infectedmacrophages were analyzed for their nitrite contents byGriess reaction [15] Then the plates were washed four timeswith PBS and cultures were incubated in PBHIL mediumsupplemented with 10 of FBS for 72 hours at 26∘C toevaluate the number of promastigote forms differentiatedinto the medium The number of viable promastigotes wasdetermined using a hemocytometer chamber

3 Results and Discussion

In the present study we investigated the antileishmanialeffects of the hexanic extract from A chica against two Leish-mania species the causative agents of cutaneous and visceralleishmaniasis L amazonensis and L infantum respectivelyTable 1 summarizes the inhibitory activity of five fractionsobtained from the crude hexanic extract on the growth ofthe parasites tested B2 (1 1 n-hexaneethyl acetate) was themost active fraction with MIC values of 372 and 186 120583gmLfor L amazonensis and L infantum promastigotes respec-tively Recently the antimicrobial activity of a hydroethanolicextract from A chicawas reported againstHelicobacter pyloriand Enterococcus faecalis demonstrating the potential of thisplant as a source of biologically active molecules [16] Only afew species from theArrabidaea genus have been investigatedfor their antiprotozoal activity In a study conducted byBarbosa et al the ethanol extract from A chica and fractionswere active against Trypanosoma cruzi trypomastigotes buthigh concentrations were needed to cause parasite lyses (40and 20mgmL resp) [6] Triterpenoids isolated from an Atriplinervia ethanol extract have been shown to present anti-T cruzi activity [17] However the crude ethanol extract aswell as the isolated compounds ursolic acid and oleanolicacid caused in vitro elimination of trypomastigotes at high


Table 2 Chemical characterization of the B2 fraction obtained fromthe A chica hexanic extract

RT LRI Components 15876 1968 n-Hexadecanoic acid 196117294 2114 Phytol 30517508 2143 Linoleic acid 63617575 2150 Linolenic acid methyl ester 253817777 2173 Octadecanoic acid 141019517 2363 Eicosanoic acid 13125290 3031 Vitamin E 49426244 3176 Campesterol 16026491 3206 Stigmasterol 40227135 3417 Gamma-sitosterol 1285 total 9322RT retention timeLRI linear retention indices

concentrations of 50 04 and 16mgmL respectively In thepresent study the reincubation of parasites treated at MICvalues in fresh medium revealed that those cells were nolonger able to growThus the inhibitory activity observedwasleishmanicidal for the promastigote forms of L amazonensisand L infantum (MIC values = MLC values)

Leishmania promastigotes were shown to be more sen-sitive to B2 and therefore the chemical analysis of thisfraction was carried out and the main components identifiedwere linolenic acid methyl ester (2538) n-hexadecanoicacid (1961) octadecanoic acid (1410) and gamma-sitosterol (1285) as shown in Table 2 Fatty acids havebeen reported to be active against Leishmania however theactivity of such compounds seems to be related mainly tounsaturated fatty acids rather than their saturated analogues[18] The fatty acid-rich methanol extract from Ulva lactucadisplayed antitrypanosomal and antileishmanial activitiesthrough parasite motility inhibition at low concentrations(lt100 120583gmL) In that work n-decanoic acid n-dodecanoicacid and n-hexadecanoic acid were described as the mainactive compounds [19] More recently the action mechanismof some fatty acids has been related to topoisomerase IBinhibition as demonstrated by Carballeira et al during theevaluation of the antileishmanial activity of 120572-methoxylatedfatty acids [20] Here the B2 fraction demonstrated signif-icant peptidase inhibition when tested on cellular lysatesof Leishmania (Figure 1) After incubation of the cellularlysates treated at MIC (372 and 186 120583gmL) and twice MIC(744 and 372 120583gmL) of B2 with phosphate buffer 55 pH at37∘C peptidase activity was completely inhibited for L ama-zonensis and L infantum respectively (Figure 1(a)) E64(cysteine peptidase inhibitor) was used as the positive controlcompletely inhibiting peptidase activity when incubated withLeishmania lysates under the same conditions In order toevaluate the effect of the B2 fraction on metalopeptidasescellular lysates from L amazonensis and L infantum werealso incubated with phosphate buffer 100 pH (Figure 1(b))Despite the decrease in peptidase activity the results showthat B2 was less effective against those peptidases

Naturally occurring sterols also present antileishmanialactivity In a study conducted by Pan et al sterols obtainedfrom the roots of Pentalinon andrieuxii displayed the bestinhibitory activities (IC

50) at concentrations ranging from

92 to 300 120583M and 003 to 35 120583M against promastigoteand amastigote forms of L mexicana respectively [21]The authors attributed the antileishmanial activity of thesterols tested to membrane alterations caused by cholesterolreplacement during its biosynthesis In the present studyimportant alterations on the ultrastructure of L infantumpromastigotes were also observed on B2-treated parasites(Figure 2) Parasites presented abnormal cell body shapesafter 24 h exposure to the B2 fraction at 186 120583gmL (MIC)when compared to untreated parasites (Figure 2(b)) Mito-chondrial dilatation with loss of matrix contents and Golgicomplex alterations followed by a cytoplasm vacuolizationprocess were also observed (Figure 2(c)) In addition anintense exocytic process of cytoplasmic content into theflagellar pocket was noted Mitochondrion seems to be acommon target for several natural products crude extractsor isolated compounds as has been reported by severalresearchers [10 22ndash24] The mode of action of most naturalproducts that causemitochondrial damage and parasite deathhas been attributed mainly to sterol biosynthesis inhibitionand mitochondrial membrane potential dysfunction [25ndash27] Here the L infantum mitochondrion was drasticallydamaged by the B2 fraction obtained from A chica hexanicextract as shown in Figure 2 An intense swelling of themitochondrion with the presence of vesicles was observed inthe parasites In some cases the mitochondrion membraneappears to be disrupted

The B2 fraction from theA chica hexanic extract demon-strated antileishmanial activity during the infection of peri-toneal mice macrophages After a 48-hour treatment withB2 at MIC and subMIC values the number of promastig-otes recovered in the supernatants of L amazonensis- andL infantum-infected macrophage cultures was drasticallyreduced when compared to controls (Figure 3) Accordingto Passero et al only viable amastigotes are able to differ-entiate to promastigotes under established conditions [14]Besides after treatment with 372 and 186120583gmL of the B2fraction the nitrite contents detected in the supernatant ofL amazonensis-infected macrophages was higher than thosefound on untreated cells cultures about 76 and 116 120583Mrespectively With the L infantum model of infection thenitrite content detected onMIC and subMIC-treated cultureswas about 125 and 20 120583M respectively (Figure 4) Theseresults give additional evidence of the enhancement ofmacrophage killingmechanismelicited byA chica against theintracellular form of Leishmania MoreoverA chica has beendescribed as a potent wound healing agent able to stimulatefibroblast growth and collagen synthesis at 30120583gmL (EC

50)

and 250 120583gmL respectively In vivo assays demonstratedan impressive reduction of lesion size of about 96 [28]In addition Lima de Medeiros et al reported the hepato-protective activity of the hydroethanolic extract of A chicabased on the suppression of hepatic markers such as serumglutamic oxaloacetate transaminase (GOT) and glutamicpyruvate transaminase (GPT) and decreasing levels of plasma


0

20

40

60

80

100

L amazonensisL infantum

2xMIC MIC subMIC

Pept

idas

e act

ivity

()

(a)

0

20

40

60

80

100


2xMIC MIC subMIC

Pept

idas

e act

ivity

()

(b)

Figure 1 Inhibitory activity of the B2 fraction obtained from the A chica hexanic extract on Leishmania peptidases (a) Peptidase inhibitoryactivity at pH 55 (b) peptidase inhibitory activity at pH 10 2xMIC MIC and subMIC values for L amazonensis and L infantum were 744372 and 186 120583gmL and 372 186 and 93120583gmL respectively

f

k m

L

n

(a)

fpf

k

m

(b)

fk

GL

(c)

n

m

k

(d)

m

(e)

m

fp

(f)

fpf

lowast

lowast

(g)

m

mk

G

n

(h)

Figure 2 Transmission electron microscopy of L infantum promastigotes treated with the B2 fraction from the A chica hexanic extract((a)-(b)) Thin sections of untreated promastigote forms displaying normal morphology and intracellular structures (b) Detail of themitochondrion containing the kinetoplast (k) ((c)ndash(h)) Parasites treated for 24 hours with subMIC (93120583gmL) or MIC (186120583gmL) ofthe B2 fraction showing serious cellular damage (c) Parasite treated with subMIC value of the B2 fraction presenting a dilated flagellarpocket with the presence of several vacuoles (lowast) Note the rupture of the mitochondrion membrane (arrowhead in (c)) (d) Parasite treatedwith the B2 fraction at MIC value (186 120583gmL) presenting mitochondrial swelling and some vesicles (995333) inside this organelle (details ofintramitochondrial vesicles in (e) and (f)) (g) in detail parasite flagellar pocket showing intense release of vesicles with cytoplasmic content(lowast) (h) in detail increased mitochondrial volume and Golgi complex alterations n nucleous m mitochondrion k kinetoplast f flagellumfp flagellar pocket G Golgi complex L lipid


0

10

20

30

40

50

60


Control MIC subMIC

Num

ber o

f par

asite

s (times106)

lowastlowast

Figure 3 Anti-intracellular amastigote activity of the B2 fractionfrom the A chica hexanic extract B2-treated macrophages previ-ously infected with Leishmania were incubated in fresh mediumat 28∘C for 72 hours The number of promastigote forms obtainedfrom macrophages cultures was counted using a hemocytometerchamber Each point represents the mean plusmn SE of 2 independentexperiments performed in triplicate MIC and subMIC values for Lamazonensis and L infantum were 372 and 186 120583gmL and 186 and93 120583gmL respectively Asterisks indicate that treated parasites werestatistically different (119875 lt 005) from control parasites


0

2

4

6

8

10

12

14

Control MIC subMIC

Nitr

ite (120583

M)

lowast

lowast

Figure 4 Nitric oxide synthesis by Leishmania-infected macro-phages treated with the B2 fraction After 48 hours treatment withthe B2 fraction the supernatant from L amazonensis- and L infan-tum-infected macrophages was collected and the nitrite contentdetermined through Griess reaction Each point represents themean plusmn SE of 2 independent experiments performed in triplicateMIC and subMIC values for L amazonensis and L infantum were372 and 186 120583gmL and 186 and 93 120583gmL respectively Asterisksindicate that treated parasites were statistically different (119875 lt 005)from control parasites

bilirubin [29] Considering the clinical manifestations relatedto cutaneous and visceral leishmaniasis such as skin lesionsand hepatic damage respectively A chica could represent apromising phytotherapeutic agent

In conclusion the hexanic extract from A chica espe-cially the B2 fraction possesses activity against L amazonen-sis and L infantum Fatty acids and sterols probably are themain components involved in the antileishmanial activity butfurther investigation will be necessary in order to evaluatethe isolated compounds Taken together the results presentedherein in addition to those reported in literature concerningtissue healing effects and liver protection of A chica aremotivation for further investigation of this plant using in vivomodels of Leishmania spp infection

Conflict of Interests

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

Acknowledgments

The authors thank Luciana T Zimmermann for her technicalassistance They also thank the Plataforma Rudolf Barth doInstituto Oswaldo Cruz (FiocruzRJ) for the acquisition ofthe transmission electron microscopy images This work wasfinancially supported by the Brazilian agencies MCTCNPqCAPES and FAPERJ

References

[1] M P Barrett and S L Croft ldquoManagement of trypanosomiasisand leishmaniasisrdquo British Medical Bulletin vol 104 pp 175ndash196 2012

[2] WHO ldquoLeishmaniasis epidemiology and access to medici-nesmdashan update based on the outcomes of WHO regionalmeetings literature review and expertsrsquo opinionrdquo World HealthOrganization Geneva Switzerland 2012

[3] J Alvar I D Velez C Bern et al ldquoLeishmaniasis worldwideand global estimates of its incidencerdquo PLoS ONE vol 7 no 5Article ID e35671 2012

[4] J P Ehrenberg and S K Ault ldquoNeglected diseases of neglectedpopulations thinking to reshape the determinants of health inLatin America and the Caribbeanrdquo BMC Public Health vol 5article 119 2005

[5] M den Boer D Argaw J Jannin and J Alvar ldquoLeishmaniasisimpact and treatment accessrdquo Clinical Microbiology and Infec-tion vol 17 no 10 pp 1471ndash1477 2011

[6] W L R Barbosa L D N Pinto E Quignard J M D S VieiraJ O C Silva Jr and S Albuquerque ldquoArrabidaea chica (HBK)Verlot phytochemical approach antifungal and trypanocidalactivitiesrdquo Revista Brasileira de Farmacognosia vol 18 no 4 pp544ndash548 2008

[7] J T G Siraichi D F Felipe L Z S Brambilla et al ldquoAntioxidantcapacity of the leaf extract obtained from Arrabidaea chicacultivated in Southern Brazilrdquo PLoS ONE vol 8 no 8 ArticleID e72733 2013

[8] I A Rodrigues B A da Silva A L S dos Santos A BVermelho C S Alviano and M do Socorro Santos Rosa ldquoAnew experimental culturemedium for cultivation ofLeishmania


amazonensis its efficacy for the continuous in vitro growth anddifferentiation of infective promastigote formsrdquo ParasitologyResearch vol 106 no 5 pp 1249ndash1252 2010

[9] M Rolon C Vega J A Escario and A Gomez-Barrio ldquoDevel-opment of resazurin microtiter assay for drug sensibility testingofTrypanosoma cruzi epimastigotesrdquo Parasitology Research vol99 no 2 pp 103ndash107 2006

[10] I A Rodrigues M M Azevedo F C Chaves et al ldquoIn vitrocytocidal effects of the essential oil from Croton cajucara (redsacaca) and its major constituent 7-hydroxycalamenene againstLeishmania chagasirdquo BMC Complementary and AlternativeMedicine vol 13 article 249 2013

[11] S M L Cedrola A C N de Melo A M Mazotto et alldquoKeratinases and sulfide from Bacillus subtilis SLC to recyclefeather wasterdquoWorld Journal ofMicrobiology and Biotechnologyvol 28 no 3 pp 1259ndash1269 2012

[12] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[13] NMAl-Musayeib R AMothana AMatheeussen P Cos andL Maes ldquoIn vitro antiplasmodial antileishmanial and antitry-panosomal activities of selected medicinal plants used in thetraditional Arabian Peninsular regionrdquo BMC Complementaryand Alternative Medicine vol 12 article 49 2012

[14] L F D Passero A Bonfim-Melo C E P Corbett et al ldquoAnti-leishmanial effects of purified compounds from aerial parts ofBaccharis uncinellaC DC (Asteraceae)rdquo Parasitology Researchvol 108 no 3 pp 529ndash536 2011

[15] S J Green M S Meltzer J B Hibbs Jr and C A NacyldquoActivated macrophages destroy intracellular Leishmania majoramastigotes by an L-arginine-dependent killing mechanismrdquoJournal of Immunology vol 144 no 1 pp 278ndash283 1990

[16] L Mafioleti I F da Silva Junior E M Colodel A Flach and DTMartins ldquoEvaluation of the toxicity and antimicrobial activityof hydroethanolic extract ofArrabidaea chica (Humb ampBonpl)B Verlrdquo Journal of Ethnopharmacology vol 150 no 2 pp 576ndash582 2013

[17] J P V Leite A B Oliveira J A Lombardi J D S Filho and EChiari ldquoTrypanocidal activity of triterpenes from Arrabidaeatriplinervia and derivativesrdquo Biological and PharmaceuticalBulletin vol 29 no 11 pp 2307ndash2309 2006

[18] NMCarballeiraMMCartagena C F Prada C F Rubio andR Balana-Fouce ldquoTotal synthesis and antileishmanial activityof the natural occurring acetylenic fatty acids 6-heptadecynoicacid and 6-icosynoic acidrdquo Lipids vol 44 no 10 pp 953ndash9612009

[19] L V Cunningham B H Kazan and S S Kuwahara ldquoEffectof long-chain fatty acids on some trypanosomatid flagellatesrdquoJournal of GeneralMicrobiology vol 70 no 3 pp 491ndash496 1972

[20] NM CarballeiraM Cartagena F Li et al ldquoFirst total synthesisof the (plusmn)-2-methoxy-6-heptadecynoic acid and related 2-methoxylated analogs as effective inhibitors of the leishmaniatopoisomerase IB enzymerdquo Pure and Applied Chemistry vol 84no 9 pp 1867ndash1875 2012

[21] L Pan C M Lezama-Davila A P Isaac-Marquez et alldquoSterols with antileishmanial activity isolated from the rootsof Pentalinon andrieuxiirdquo Phytochemistry vol 82 pp 128ndash1352012

[22] M D S S Rosa R R Mendonca-Filho H R Bizzo et alldquoAntileishmanial activity of a linalool-rich essential oil fromCroton cajucarardquo Antimicrobial Agents and Chemotherapy vol47 no 6 pp 1895ndash1901 2003

[23] N Sen B B Das A Ganguly et al ldquoCamptothecin inducedmitochondrial dysfunction leading to programmed cell deathin unicellular hemoflagellate Leishmania donovanirdquo Cell Deathand Differentiation vol 11 no 8 pp 924ndash936 2004

[24] M A Brenzan A O Santos C V Nakamura et al ldquoEffects of(-) mammea ABB isolated from Calophyllum brasiliense leavesand derivatives on mitochondrial membrane of Leishmaniaamazonensisrdquo Phytomedicine vol 19 no 3-4 pp 223ndash230 2012

[25] F Fonseca-Silva J D F Inacio M M Canto-Cavalheiro andE E Almeida-Amaral ldquoReactive oxygen species productionandmitochondrial dysfunction contribute to quercetin induceddeath in Leishmania amazonensisrdquo PLoS ONE vol 6 no 2Article ID e14666 2011

[26] J M Medina J C Rodrigues W de Souza G C Atellaand H Barrabin ldquoTomatidine promotes the inhibition of 24-alkylated sterol biosynthesis and mitochondrial dysfunction inLeishmania amazonensis promastigotesrdquo Parasitology vol 139no 10 pp 1253ndash1265 2012

[27] L Monzote M Garcıa J Pastor et al ldquoEssential oil fromChenopodium ambrosioides and main components activityagainst Leishmania their mitochondria and other microorgan-ismsrdquo Experimental Parasitology vol 136 pp 20ndash26 2014

[28] M P Jorge C Madjarof A L Gois Ruiz et al ldquoEvaluation ofwound healing properties of Arrabidaea chica Verlot extractrdquoJournal of Ethnopharmacology vol 118 no 3 pp 361ndash366 2008

[29] B Lima de Medeiros K dos Santos Costa J F Alves RibeiroJ O Carrera Silva Jr W L Ramos Barbosa and J C TavaresCarvalho ldquoLiver protective activity of a hydroethanolic extractof Arrabidaea chica (Humb and Bonpl) B Verl (pariri)rdquoPharmacognosy Research vol 3 no 2 pp 79ndash84 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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International Journal of

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Zoology


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GenomicsInternational Journal of


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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Stem CellsInternational



Enzyme Research



Microbiology


countries However most of these drugs are expensivepresent toxic effects and are able to induce parasite resistance[5] Consequently the search for new and more effectiveantileishmanial agents remains crucial

Arrabidaea chica (HBK) Verlot Bignoniaceae is a scram-bling shrub native to tropical America more particularlyin the Amazon basin where it is also known as ldquoParirirdquoldquoCrajirurdquo ldquoCarajururdquo or ldquoCarajirurdquo The leaves of A chicahave been traditionally used by Brazilian Indians as a dyefor body painting in rituals and to protect the skin againstsunlight aswell as an insect repellant Chemical investigationshave been carried out since the beginning of this centuryto determine the composition of the A chica dye whichused to be commercialized as such [6] Nowadays A chicais used by the regional population as an anti-inflammatoryand astringent agent as well as a remedy for intestinal colicdiarrhea leucorrhea anemia and leukemia [7] The presentstudy aimed to evaluate the antileishmanial effects of thehexanic extract of the A chica leaves

2 Materials and Methods

21 Chemicals Resazurin RPMI 1640 medium and bovineserum albumin were purchased from Sigma Chemical CoUSA Amphotericin B was purchased from Fontoura-WyethBrazil Fetal bovine serum (FBS) was purchased from Cri-pion Biotecnologia Ltda Brazil All solvents used werespectroscopic grade from Tedia (Fairfield OH USA) Col-umn chromatographic product was purchased from Merck(Darmstadt Germany)

22 Plant Material and Acquisition of the Hexanic ExtractThe sample of A chica was kept in a germplasm bankunder the same cultivation practices at the EAFM Herbar-ium from Federal Institute of Amazonas (Manaus AM)where a voucher specimen was deposited (registry EAFM6791) Leaves of A chica were collected between 0800 and0900AM

A chica crude extract was obtained by 1 week extractionin hexane Then the extract was carefully filtered dried andstored in opaque glass vials at minus10∘C Afterwards the crudeextract was subjected to silica gel column chromatographywith an increasing gradient of polarity starting with 100 n-hexane and 100 ethyl acetate to 100 ethanol affording fivefractions (B1 B2 B3 B4 and B5)

23 Analysis of the Hexanic Extract of A chica by GC-MS The B2 fraction of the hexanic extract from ldquocrajirurdquowas analyzed by a gas chromatograph (GC) interfaced toa mass spectrometer (MS) employing the following condi-tions the oven temperature was programmed from 60∘Cto 300∘C at 10∘Cmin and helium was the carrier gas (at10mLmin) One microliter of 1 solution of the B2 fractionin dichloromethane was injected in split mode (1 50) Massspectra were obtained in an Agilent 5973N system fittedwith a low bleeding 5 phenyl95 methyl silicone (HP-5 MS 30m times 025mm times 025 120583m) fused silica capillarycolumn operating in the electronic ionization mode (EI)at 70 eV with a scan mass range of 40ndash500mz Sampling

rate was 315 scans The ion source was kept at 230∘C massanalyzer at 150∘C and transfer line at 260∘C Linear retentionindices (LRI) were measured by injection of a series of n-alkanes (C

10ndashC30) in the same column and conditions as

described above and compared with reference data Theidentification of the B2 fraction constituents was made basedon the retention indexes and by comparison of mass spectrawith computer search using NIST21 and NIST107 librariesCompound concentrationswere calculated from theGCpeakareas and they were arranged in order of GC elution

24 Parasite Strains and Cell Cultures Promastigote forms oftwo Leishmania species Leishmania (L) amazonensis (IFLABR1967PH8) and L (L) infantum (MHOMBR1974PP75)from the Leishmania Type Culture Collection of OswaldoCruz InstituteFiocruz (Rio de JaneiroRJBrazil) were usedin all experiments Parasites were axenically cultured inPBHILmediumas previously described [8] In order to assureinfectiveness of the parasites periodical infection of miceperitoneal macrophages was performed

25 Evaluation of Leishmania Inhibitory Concentrations Theassay was carried out in a 96-well microtiter plate wherethe hexanic extract from A chica was serially diluted induplicate to final test concentrations (1ndash500 120583gmL) Then50 times 10

5 promastigote forms of L amazonensis or Linfantum were harvested at the early stationary phase addedto each well and plated at 26∘C for 120 h At the end ofincubation period 25120583L of resazurin solution (5mg100mLof phosphate buffer saline pH 72) was added and the viabilityof parasites was determined in accordance with the protocolpreviously described [9] The minimal inhibitory concen-tration (MIC) was considered the lowest concentration ofthe hexanic extract that completely prevented the growthof Leishmania in vitro Alternatively 120 h-treated parasiteswere centrifuged (1000 g5min) washed twice in PBS andthen reincubated in fresh PBHIL culture medium in order toevaluate the leishmanicidal effect The lowest concentrationable to inhibit parasite growth was considered the minimalleishmanicidal concentration (MLC) The 50 inhibitoryconcentration (IC

50) was determined by logarithmic regres-

sion analysis of the data obtained as described above

26 Ultrastructure Analysis Alterations in the ultrastructureof the parasites were analyzed by transmission (TEM) elec-tron microscopy First promastigote forms of L infantumwere harvested at the early stationary phase of growthwashed twice with PBS and incubated in the presence ofa subinhibitory concentration (subMIC) of A chica hex-anic extract at 28∘C for 24 hours After the parasites werewashed twice in PBS they were fixed with glutaraldehydesolution (25 glutaraldehyde in 01M sodium cacodylatebuffer containing 35 sucrose pH 74) at 4∘C for 60minSamples of treated cells and their controls (untreated cells)were sent to Plataforma Rudolf Barth (Instituto OswaldoCruzFiocruzRJ) and processed as previously mentioned[10] The photomicrographs were obtained using an electronmicroscope Jeol JEM1011













2atmosphere













50)



0

20

40

60

80

100


2xMIC MIC subMIC

Pept

idas

e act

ivity

()

(a)

0

20

40

60

80

100


2xMIC MIC subMIC

Pept

idas

e act

ivity

()

(b)


f

k m

L

n

(a)

fpf

k

m

(b)

fk

GL

(c)

n

m

k

(d)

m

(e)

m

fp

(f)

fpf

lowast

lowast

(g)

m

mk

G

n

(h)



0

10

20

30

40

50

60


Control MIC subMIC

Num

ber o

f par

asite

s (times106)

lowastlowast



0

2

4

6

8

10

12

14

Control MIC subMIC

Nitr

ite (120583

M)

lowast

lowast






Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













2atmosphere













50)



0

20

40

60

80

100


2xMIC MIC subMIC

Pept

idas

e act

ivity

()

(a)

0

20

40

60

80

100


2xMIC MIC subMIC

Pept

idas

e act

ivity

()

(b)


f

k m

L

n

(a)

fpf

k

m

(b)

fk

GL

(c)

n

m

k

(d)

m

(e)

m

fp

(f)

fpf

lowast

lowast

(g)

m

mk

G

n

(h)



0

10

20

30

40

50

60


Control MIC subMIC

Num

ber o

f par

asite

s (times106)

lowastlowast



0

2

4

6

8

10

12

14

Control MIC subMIC

Nitr

ite (120583

M)

lowast

lowast






Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










50)



0

20

40

60

80

100


2xMIC MIC subMIC

Pept

idas

e act

ivity

()

(a)

0

20

40

60

80

100


2xMIC MIC subMIC

Pept

idas

e act

ivity

()

(b)


f

k m

L

n

(a)

fpf

k

m

(b)

fk

GL

(c)

n

m

k

(d)

m

(e)

m

fp

(f)

fpf

lowast

lowast

(g)

m

mk

G

n

(h)



0

10

20

30

40

50

60


Control MIC subMIC

Num

ber o

f par

asite

s (times106)

lowastlowast



0

2

4

6

8

10

12

14

Control MIC subMIC

Nitr

ite (120583

M)

lowast

lowast






Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

20

40

60

80

100


2xMIC MIC subMIC

Pept

idas

e act

ivity

()

(a)

0

20

40

60

80

100


2xMIC MIC subMIC

Pept

idas

e act

ivity

()

(b)


f

k m

L

n

(a)

fpf

k

m

(b)

fk

GL

(c)

n

m

k

(d)

m

(e)

m

fp

(f)

fpf

lowast

lowast

(g)

m

mk

G

n

(h)



0

10

20

30

40

50

60


Control MIC subMIC

Num

ber o

f par

asite

s (times106)

lowastlowast



0

2

4

6

8

10

12

14

Control MIC subMIC

Nitr

ite (120583

M)

lowast

lowast






Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

10

20

30

40

50

60


Control MIC subMIC

Num

ber o

f par

asite

s (times106)

lowastlowast



0

2

4

6

8

10

12

14

Control MIC subMIC

Nitr

ite (120583

M)

lowast

lowast






Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Arrabidaea chica hexanic extract induces mitochondrion damage and peptidase inhibition on Leishmania...

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Transcript of Arrabidaea chica hexanic extract induces mitochondrion damage and peptidase inhibition on Leishmania...