8
Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 826370, 7 pages http://dx.doi.org/10.1155/2013/826370 Research Article Remarkable Anti-Trichomonas vaginalis Activity of Plants Traditionally Used by the Mbyá-Guarani Indigenous Group in Brazil Clara Lia Costa Brandelli, 1 Patrícia de Brum Vieira, 1 Alexandre José Macedo, 2 and Tiana Tasca 1 1 Faculdade de Farm´ acia, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil 2 Faculdade de Farm´ acia e Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil Correspondence should be addressed to Tiana Tasca; [email protected] Received 16 April 2013; Accepted 4 June 2013 Academic Editor: Abhay R. Satoskar Copyright © 2013 Clara Lia Costa Brandelli et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Trichomonas vaginalis, a flagellate protozoan, is the causative agent of trichomonosis, the most common nonviral sexually trans- mitted disease worldwide. Taking into account the increased prevalence of metronidazole-resistant isolates, alternative drugs are essential for the successful treatment. Natural products are the source of most new drugs, and popular wisdom about the use of medicinal plants is a powerful tool in this search. In this study, the activity of 10 medicinal plants extensively used in daily life by Mby´ a-Guarani indigenous group was evaluated against seven different T. vaginalis isolates. Among the aqueous extracts tested, Verbena sp. (Guachu ka’a in Mby´ a-Guarani language) and Campomanesia xanthocarpa (Guavira in Mby´ a-Guarani language) showed the highest activity against T. vaginalis with MIC value of 4.0 mg/mL reaching 100% of efficacy against the parasite. e kinetic growth assays showed that the extracts promoted complete growth abolishment aſter 4 h of incubation. In addition, the extracts tested did not promote a significant hemolytic activity against human erythrocytes. Our results show for the first time the potential activity of Verbena sp. and C. xanthocarpa against T. vaginalis. In addition, this study demonstrates that indigenous knowledge is an important source of new prototype antiprotozoal agents. 1. Introduction Trichomonas vaginalis parasitizes the urogenital human tract causing trichomonosis, the most prevalent non-viral sexually transmitted disease worldwide, being responsible for 248 mil- lion new cases annually [1]. Aſter colonization, the parasite causes vaginitis, urethritis, and prostatitis [2]. Moreover, the pathogen has been associated with serious consequences as adverse pregnancy outcomes and preterm birth [3], infertility [4], predisposition to cervical cancer [5], and pelvic inflam- matory disease [6]. Importantly, trichomonosis acts as a cofactor in human immunodeficiency virus (HIV) transmis- sion and acquisition [7, 8]. In spite of impact of this infection, the therapeutic arsenal is restricting, and only metronidazole and tinidazole, both 5-nitroimidazole drugs, are approved by the FDA for trichomonosis treatment [9]. Although the cure rate is high, treatment failure can be observed and the resis- tance of T. vaginalis isolates is the main reason of treatment failure [10]. In this sense, it is undoubtedly necessary that the investigation for new alternatives for the treatment of trichomonosis and two strategies can be followed: (i) the search for new therapeutic targets, which is essential to rational development of new antiparasitic agents and (ii) the investigation of new anti-T. vaginalis compounds structurally distinct from 5-nitroimidazoles and, consequently, acting by different mechanism as demonstrated by our group [11, 12]. In this context, natural products, especially medicinal plants, are immeasurable as a potent source of bioactive molecules. Since ancient times, people use plants to treat common infectious diseases, and some of these traditional

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Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 826370 7 pageshttpdxdoiorg1011552013826370

Research ArticleRemarkable Anti-Trichomonas vaginalis Activity ofPlants Traditionally Used by the Mbyaacute-Guarani IndigenousGroup in Brazil

Clara Lia Costa Brandelli1 Patriacutecia de Brum Vieira1

Alexandre Joseacute Macedo2 and Tiana Tasca1

1 Faculdade de Farmacia Universidade Federal do Rio Grande do Sul Avenida Ipiranga 2752 90610-000 Porto Alegre RS Brazil2 Faculdade de Farmacia e Centro de Biotecnologia Universidade Federal do Rio Grande do Sul Avenida Ipiranga 275290610-000 Porto Alegre RS Brazil

Correspondence should be addressed to Tiana Tasca tianatascaufrgsbr

Received 16 April 2013 Accepted 4 June 2013

Academic Editor Abhay R Satoskar

Copyright copy 2013 Clara Lia Costa Brandelli et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Trichomonas vaginalis a flagellate protozoan is the causative agent of trichomonosis the most common nonviral sexually trans-mitted disease worldwide Taking into account the increased prevalence of metronidazole-resistant isolates alternative drugs areessential for the successful treatment Natural products are the source of most new drugs and popular wisdom about the useof medicinal plants is a powerful tool in this search In this study the activity of 10 medicinal plants extensively used in dailylife by Mbya-Guarani indigenous group was evaluated against seven different T vaginalis isolates Among the aqueous extractstestedVerbena sp (Guachu karsquoa inMbya-Guarani language) andCampomanesia xanthocarpa (Guavira inMbya-Guarani language)showed the highest activity against T vaginalis with MIC value of 40mgmL reaching 100 of efficacy against the parasite Thekinetic growth assays showed that the extracts promoted complete growth abolishment after 4 h of incubation In addition theextracts tested did not promote a significant hemolytic activity against human erythrocytes Our results show for the first timethe potential activity of Verbena sp and C xanthocarpa against T vaginalis In addition this study demonstrates that indigenousknowledge is an important source of new prototype antiprotozoal agents

1 Introduction

Trichomonas vaginalis parasitizes the urogenital human tractcausing trichomonosis the most prevalent non-viral sexuallytransmitted diseaseworldwide being responsible for 248mil-lion new cases annually [1] After colonization the parasitecauses vaginitis urethritis and prostatitis [2] Moreover thepathogen has been associated with serious consequences asadverse pregnancy outcomes and preterm birth [3] infertility[4] predisposition to cervical cancer [5] and pelvic inflam-matory disease [6] Importantly trichomonosis acts as acofactor in human immunodeficiency virus (HIV) transmis-sion and acquisition [7 8] In spite of impact of this infectionthe therapeutic arsenal is restricting and only metronidazoleand tinidazole both 5-nitroimidazole drugs are approved by

the FDA for trichomonosis treatment [9] Although the curerate is high treatment failure can be observed and the resis-tance of T vaginalis isolates is the main reason of treatmentfailure [10] In this sense it is undoubtedly necessary thatthe investigation for new alternatives for the treatment oftrichomonosis and two strategies can be followed (i) thesearch for new therapeutic targets which is essential torational development of new antiparasitic agents and (ii) theinvestigation of new anti-T vaginalis compounds structurallydistinct from 5-nitroimidazoles and consequently acting bydifferent mechanism as demonstrated by our group [11 12]

In this context natural products especially medicinalplants are immeasurable as a potent source of bioactivemolecules Since ancient times people use plants to treatcommon infectious diseases and some of these traditional

2 BioMed Research International

medicines are still integrated as part of the cure of diversepathologies [13] In addition the search based on ethnophar-macological information rescues the immense empiricalknowledge of plants utilization [14 15] In this contextregarding natural environment and vast wealth of geneticresources Brazil is a country of great interest to ethnophar-macology [16]

Indigenous people have a wealth of biodiversity informa-tion and know how to capture and use natural resources [17]Despite of the fact that several drugs have been discoveredthrough traditional cures and folk knowledge in Brazil thereare 122 indigenous ethnic groups but only 30 of them wereinvestigated with regard to ethnobotanical aspects [18 19]Therefore the study of plants used by indigenous medicineis important to interconnect traditional medicine and bioticenvironment preserving the indigenous ancient knowledge

Parasitic protozoa remain a major threat to human andanimal health and there are few effective drugs for thetreatment of many protozoal diseases Taking into accountthe indigenous ethnopharmacology as a contributor to thesystematic screening of plants with antiprotozoal activity thisstudy evaluated the anti-T vaginalis activity of the mostimportant medicinal plants used in Mbya-Guarani indige-nous medicine located in South of Brazil

2 Materials and Methods

21 Parasite Culture Conditions In this study seven isolatesof T vaginalis were used 30236 and 30238 from AmericanType Culture Collection (ATCC) TV-LACM1 TV-LACM2and TV-LACM3 (fresh clinical isolates from female patients)TV-LACH1 andTV-LACH2 (fresh clinical isolates frommalepatients) all from Laboratorio de Analises Clınicas e Tox-icologicas Faculdade de Farmacia UFRGS Brazil (projectwith ethical approval by UFRGS Ethical Research Commit-tee number 18923) Trichomonads were axenically culturedin trypticase-yeast extract-maltose (TYM)medium (pH 60)supplemented with 10 heat-inactivated bovine serum andincubated at 37∘C [20]

22 PlantMaterial Plantmaterialwas collected from indige-nous tribe Mbya-Guarani located at the Lomba do PinheiroPorto Alegre RS Brazil (30∘061015840476210158401015840 S and 51∘071015840378510158401015840W)inMarch 2011This village is denominated Tekoa Anhetengua(in EnglishTrueVillage) and 200 people live in the area gath-ered at 40 familiesThe women of the village have the knowl-edge of medicinal plants used by the entire population andconducted the collection of specimens Ten plants routinelyused in indigenous medicine for infectious diseases werecollected leaves of Aloe arborescensMill (ICN 173371) aerialparts of Bidens pilosa L (ICN 167397) aerial parts ofRhipsalisbaccifera (ICN 167402) barks of Luehea divaricata (ICN167403) roots of Trichilia sp (ICN 173126) leaves of Campo-manesia xanthocarpa O Berg (ICN 167401) leaves of Coixlacryma-jobi Lin (ICN 167396) leaves of Citrus limonium(ICN 167399) leaves of Citrus reticulata (ICN 173127) andleaves of Verbena sp (ICN 167394) The voucher specimenswere deposited at the herbarium of the Universidade Federaldo Rio Grande do Sul (ICN)

23 Preparation of Plant Extracts The fresh plants extractswere prepared conforming traditional use by indigenous peo-ple by decoction at 60∘C for 60min [1 10 (w v)] Aqueousextracts were freeze dried and the work solution was pre-pared at 80mgmL in ultrapure water sterilized by filtration(022 120583m) and stored at minus20∘C

24 Anti-Trichomonas vaginalis Assay Ten aqueous extractswere screened in vitro for activity against T vaginalis Theassay was performed in 96 microtiter plates at final concen-tration of 40mgmL in the wells The inhibitory minimumconcentration (MIC)was determined using an eightfold dilu-tion (40ndash0031mgmL) and the viability of the trophozoiteswas determined according to a fluorimetricmethod [21] Twocontrols were performed negative control only with tropho-zoites and a positive control with 80120583M metronidazole(Sigma Chemical Co St Louis MO USA)The experimentswere performed at least in three independent experimentsin triplicate The results were expressed as the percentage ofviable trophozoites compared to untreated parasites

25 Growth Effect of Aqueous Extracts In order to investigatethe effect of the active aqueous extracts on T vaginalisgrowth a kinetic growth curve was performed using theATCC 30236 isolate The parasites initial density of 20 times105 trophozoitesmL was incubated with the extracts at MICvalue in TYMmediumThe trophozoites were counted usinga hemocytometer during 72 h The results were expressed astrophozoite number (105mL) For microscopic analysis thecontrols and treated parasites were centrifuged for 10min at2000 rpm to concentrate the organisms after 4 h of incuba-tion and the preparation was observed in light microscope(magnified times1000) by trypan blue dye exclusion

26 Hemolytic Assay This assay was performed according tothe method used by Rocha et al [22] Blood type O positivefrom healthy human volunteers was collected with Alseverrsquossolution (1 1) and centrifuged at 2000 rpm for 5min Theerythrocyte fraction was washed three times with PBS 1x (pH70) and resuspended to give 1 red cell suspension (vv)Aqueous extracts concentrations were chosen according tominimum inhibitory concentration (MIC) determined in thesusceptibility assayThe erythrocytes were incubated with theextracts at 37∘C under agitation for 1 h and then were cen-trifuged at 3000 rpm for 5minThe absorbance of the super-natant was measured at 540 nm The experiment was per-formed in triplicate and the percentage of hemolysis causedby each sample tested was calculated in comparison to 100hemolytic activity of saponin from Quillaja saponaria [23]

3 Results and Discussion

This study evaluated the anti-T vaginalis activity of aqueousextracts of 10 medicinal plants used in medicine systemMbya-Guarani Importantly among the plants tested wefound two aqueous extracts with a potent anti-T vaginalis invitro activity The promising extracts are of Verbena sp andCampomanesia xanthocarpa The extract from Verbena sp

BioMed Research International 3

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(a)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(b)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(c)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(d)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(e)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(f)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(g)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(h)

Figure 1 Continued

4 BioMed Research International

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(i)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(j)

Figure 1 Effect of selected Mbya-Guarani medicinal plants at 40mgmL on different Trichomonas vaginalis isolates (a) Verbena sp (b)Campomanesia xanthocarpa (c) Bidens pilosa (d) Luehea divaricata (e) Coix lacryma-jobi (f) Trichilia sp (g) Citrus limonium (h) Aloearborescens (i) Citrus reticulata (j) Rhipsalis baccifera Data represent means plusmn standard deviation

Table 1 Hemolytic activity of the aqueous extracts that showedthe best anti-Trichomonas vaginalis activity and respective MICdetermined by susceptibility assay

Aqueous extractVerbena sp Campomanesia xanthocarpa

MIC (mgmL) 40 40Hemolysis () plusmn SD 65 plusmn 002 76 plusmn 001

at 40mgmL demonstrated the optimal anti-T vaginalisactivity inducing complete cytotoxicity (100 of parasitesdeath) upon all distinct trichomonads isolates (Figure 1(a))The leaves extract of Campomanesia xanthocarpa at the sameconcentration also showed a very high anti-T vaginalis activ-ity reducing the parasite viability up to 96 (Figure 1(b))

Other aqueous extracts displayed a variety of resultsThe aqueous extract obtained from Bidens pilosa totallyabolished the trophozoites viability only of the 30236 isolate(Figure 1(c)) The extract of the barks of Luehea divaricatainduced a reduction ofT vaginalis viability differently in eachtrichomonads isolate tested (Figure 1(d)) The leaves of Coixlacryma-jobi induced on average 75 of viability reductionon T vaginalis isolates however for the TV-LACH2 isolatethe reduction was only 40 (Figure 1(e)) The fresh clinicalisolates from male patients (TV-LACH1 and TV-LACH2)presented higher susceptibility than the isolates from femalepatients (ATCC and fresh clinical) when the root extract ofTrichilia sp was tested (Figure 1(f)) Extracts of leaves fromCitrus limonium reduced about 40 the viability of isolates(Figure 1(g)) The extracts from Aloe arborescens and Citrusreticulata decreased the viability of 50 of at least one isolate(Figures 1(h) and 1(i)) while Rhipsalis baccifera reducedonly 30 the viability of 30236 and TV-LACM2 isolates(Figure 1(j))

In comparison to the other extracts the extracts fromVer-bena sp and C xanthocarpa demonstrated the most promis-ing activity against T vaginalis and the minimum inhibitoryconcentration was determined for the 30236 isolate (Table 1)

Time (hours)0 12 24 36 48 60 72

0

5

10

15

20

Control

Time (hours)0 1 2 3 4 5 6

0

1

2

3

4

5

Verbena spCampomanesia xanthocarpa

Viab

le tr

opho

zoite

(105

mL)

Viab

le tr

opho

zoite

(105

mL)

Figure 2 Kinetic growth curve of Trichomonas vaginalis 30236 iso-late after treatment with aqueous extracts ofVerbena sp andCampo-manesia xanthocarpa at 40mgmL The trophozoites growth wascompletely inhibited by the extracts in 4 hours of incubation Datarepresent means plusmn standard deviation of at least three experimentsall in triplicate

In order to investigate the effect of the most active extracton parasite growth and viability a kinetic growth curve wasperformed The control exhibited the classical growth peakafter 24 h of incubation however the organisms treated withextracts of Verbena sp and C xanthocarpa after 4 h of incu-bation evoked a complete growth abolishment (Figure 2) andshowed a characteristic blue color by trypan blue dye exclu-sion indicating cell death (Figure 3)

In order to investigate a possible effect of the C xantho-carpa and Verbena sp extracts on parasites membrane andalso to have preliminary data on cytotoxicity against mam-malian cells hemolytic activity of these extracts was con-ducted The Verbena sp and C xanthocarpa extracts weretested at MIC values and demonstrated 65 and 76 erythro-cytes lysis respectively (Table 1) These results demonstratedthat both extracts at MIC values did not promote a signif-icant hemolytic activity indicating that the mechanism of

BioMed Research International 5

(a) Control (b) Verbena sp

(c) Campomanesia xanthocarpaFigure 3 Microscopic analysis of parasites morphology in light microscope (magnified times1000) by trypan blue dye exclusion (a) Control(b) after 4 hours of treatment with aqueous extract of Verbena sp (c) after 4 hours of treatment with aqueous extract of Campomanesiaxanthocarpa

action responsible for the parasite viability reduction proba-bly does not involve parasitic membrane disruption More-over and importantly these findings suggest that both ex-tracts are not toxic to mammalian cells since they did notlyse human erythrocytes

This study contributes to rescue the indigenous knowl-edge about medicinal plants The indigenous Mbya-Guaraniuse Verbena sp (Guachu karsquoa in Mbya-Guarani language)as tea for relief of symptoms of infectious diseases such asfeverThis is in agreement of the literature that relatesVerbenasp infusion as sedative analgesic to inflammatory disordersskin burns amenorrhea depression and gastric diseases [24ndash26] Moreover this species has a variety of chemical con-stituents as flavonoid iridoid glycosides phenylpropanoidglycoside sterols triterpenes and glycoconjugate and thebiological activity may be partially attributed to them [24]In addition this tribe utilizes the leaves of C xanthocarpa(Guavira in Mbya-Guarani language) for treatment of diar-rhea stomach pain and worms This plant is traditionallyused in the south of Brazil as depurative antidiarrhoeiccleanser and antirheumatic and to decrease the bloodcholesterol [27] and these properties may be attributedto tannins saponins and flavonoids (quercetin myricetinquercitrin and rutin) compounds present in the leaves of Cxanthocarpa [28]

The rescue ethnopharmacology of historical uses of plantsis recognized as valuable for bioprospection since they affordthe rationale for selection and research of medicinal plants[15]This study describes the first report on the antiprotozoalactivity of Verbena sp and C xanthocarpa In addition otherspecies of the families Verbenaceae and Myrtaceae were

tested against T vaginalis however without success [29 30]Several biodiversities and traditional knowledge of differentpopulations have guided studies to search new prototypesanti-T vaginalis Recently the potential of plants traditionallyused from Caatinga against T vaginalis was reported [31]Calzada et al [29] demonstrated that plants used for urogen-ital tract disorders in Mexican traditional medicine showedeffect against this protozoan Amaryllidaceae species usedagainst venereal diseases in SouthAfrica also showed promis-ing activity against T vaginalis [11 32]

Considering the impact of trichomonosis in public healthand the emergent number of resistantT vaginalis isolates it isnecessary alternatives for the treatment of this infection Nat-ural products are a promising source of active molecules andthe ethnopharmacology approach rescues the knowledge ofpopulation of medicinal plants This wisdom combined withchemical and pharmacological studies presents a preciousvalue in the bioprospection of innovates safe and accessibledrugsThis pioneer study demonstrated relevant results aboutanti-T vaginalis activity of the Verbena sp and C xantho-carpa plants traditionally used by indigenous populationMbya-Guarani for infectious diseases Despite of showingstrong anti-T vaginalis activity this report rescued the knowl-edge of indigenous people avoiding the miscarriage of thiswisdom

Authorsrsquo Contribution

Clara Lia Costa Brandelli and Patrıcia de Brum Vieira con-tributed equally to this work

6 BioMed Research International

Acknowledgments

This work was supported by the NANOBIOTEC-Brazil pro-gram from CAPES and FAPERGS The authors thank Chief-tain Jose Cirilo Pires Morinico (tribe Mbya-Guarani) formaking possible the accomplishment of this study and for thevaluable information Alexandre Jose Macedo and TianaTasca thank CNPq for researcher fellowships

References

[1] WHO Global Incidence and Prevalence of Selected Curable Sex-ually Transmitted Infections-2008 World Health OrganizationGeneva Switzerland 2012 httpappswhointirisbitstream106657518119789241503839 engpdf

[2] D Petrin K Delgaty R Bhatt and G Garber ldquoClinicaland microbiological aspects of Trichomonas vaginalisrdquo ClinicalMicrobiology Reviews vol 11 no 2 pp 300ndash317 1998

[3] M A Klebanoff J C Carey J C Hauth et al ldquoFailure of met-ronidazole to prevent preterm delivery among pregnant womenwith asymptomatic Trichomonas vaginalis infectionrdquo The NewEngland Journal of Medicine vol 345 no 7 pp 487ndash493 2001

[4] F Grodstein M B Goldman and D W Cramer ldquoRelation oftubal infertility to history of sexually transmitted diseasesrdquoAmerican Journal of Epidemiology vol 137 no 5 pp 577ndash5841993

[5] M Viikki E Pukkala P Nieminen and M Hakama ldquoGynae-cological infections as risk determinants of subsequent cervicalneoplasiardquo Acta Oncologica vol 39 no 1 pp 71ndash75 2000

[6] T L Cherpes H C Wiesenfeld M A Melan et al ldquoTheassociations between pelvic inflammatory diseaseTrichomonasvaginalis infection and positive herpes simplex virus type 2serologyrdquo Sexually Transmitted Diseases vol 33 no 12 pp 747ndash752 2006

[7] F Sorvillo L Smith P Kerndt and L Ash ldquoTrichomonas vagi-nalis HIV and African-Americansrdquo Emerging Infectious Dis-eases vol 7 no 6 pp 927ndash932 2001

[8] B van der Pol C Kwok B Pierre-Louis et al ldquoTrichomonasvaginalis infection and human immunodeficiency virus acqui-sition in African womenrdquo Journal of Infectious Diseases vol 197no 4 pp 548ndash554 2008

[9] V Ali and T Nozaki ldquoCurrent therapeutics their problemsand sulfur-containing-amino-acid metabolism as a novel targetagainst infections by ldquoamitochondriaterdquo protozoan parasitesrdquoClinical Microbiology Reviews vol 20 no 1 pp 164ndash187 2007

[10] J R Schwebke and F J Barrientes ldquoPrevalence of Trichomonasvaginalis isolates with resistance to metronidazole and tinida-zolerdquo Antimicrobial Agents and Chemotherapy vol 50 no 12pp 4209ndash4210 2006

[11] R B Giordani P B Vieira MWeizenmann et al ldquoCandimine-induced cell death of the amitochondriate parasiteTrichomonasvaginalisrdquo Journal of Natural Products vol 73 no 12 pp 2019ndash2023 2010

[12] R B Giordani P B Vieira M Weizenmann et al ldquoLycorineinduces cell death in the amitochondriate parasiteTrichomonasvaginalis via an alternative non-apoptotic death pathwayrdquoPhytochemistry vol 72 no 7 pp 645ndash650 2011

[13] J L Rıos and M C Recio ldquoMedicinal plants and antimicrobialactivityrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 80ndash84 2005

[14] MHeinrich ldquoEthnobotany and natural products the search fornewmolecules new treatments of old diseases or a better under-standing of indigenous culturesrdquo Current Topics in MedicinalChemistry vol 3 no 2 pp 141ndash154 2003

[15] C L C Brandelli R B Giordani A J Macedo G A de Carliand T Tasca ldquoIndigenous traditional medicine plants for thetreatment of diarrheardquo inNature Helps How Plants and OtherOrganisms Contribute to Solve Health Problems pp 1ndash18Springer Berlin Germany 1st edition 2011

[16] E Elisabetsky and L Wannmacher ldquoThe status of ethnophar-macology in Brazilrdquo Journal of Ethnopharmacology vol 38 no2-3 pp 137ndash143 1993

[17] F L H Rego A J Brand and R B Costa ldquoGenetic resourcesbiodiversity traditional knowledge Guarani Kaiowa and localdevelopmentrdquo Interacoes vol 11 no 1 pp 55ndash69 2010

[18] D F Coutinho L M A Travassos and F M M AmaralldquoEstudo etnobotanico de plantas medicinais utilizadas emcomunidades indıgenas no estado doMaranhaomdashBrasilrdquoVisaoAcademica vol 3 no 1 pp 7ndash12 2002

[19] A H Gilani and A-U Rahman ldquoTrends in ethnopharmacol-ogyrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 43ndash492005

[20] L S Diamond ldquoThe establishment of various Trichomonas ofanimals andman in axenic culturesrdquoThe Journal of Parasitologyvol 43 no 4 pp 488ndash490 1957

[21] M Duarte R B Giordani G A de Carli J A ZuanazziA J Macedo and T Tasca ldquoA quantitative resazurin assay todeterminate the viability of Trichomonas vaginalis and the cyto-toxicity of organic solvents and surfactant agentsrdquo ExperimentalParasitology vol 123 no 2 pp 195ndash198 2009

[22] T D Rocha P B Vieira S C B Gnoatto T Tasca and G Gos-mann ldquoAnti-Trichomonas vaginalis activity of saponins fromQuillaja Passiflora and Ilex speciesrdquo Parasitology Research vol110 no 6 pp 2551ndash2556 2012

[23] Y SunH TongM Li Y Li S Guan and J Liu ldquoImmunologicaladjuvant effect of Japanese ginseng saponins (JGS) on specificantibody and cellular response to ovalbumin and its haemolyticactivitiesrdquo Vaccine vol 26 no 47 pp 5911ndash5917 2008

[24] M I Calvo ldquoAnti-inflammatory and analgesic activity of thetopical preparation of Verbena officinalis Lrdquo Journal of Ethno-pharmacology vol 107 no 3 pp 380ndash382 2006

[25] S-W Lai M-S Yu W-H Yuen and R C-C Chang ldquoNovelneuroprotective effects of the aqueous extracts from Verbenaofficinalis LinnrdquoNeuropharmacology vol 50 no 6 pp 641ndash6502006

[26] E Speroni R Cervellati S Costa et al ldquoEffects of differentialextraction of Verbena officinalis on rat models of inflammationcicatrization and gastric damagerdquo Planta Medica vol 73 no 3pp 227ndash235 2007

[27] M W Biavatti C Farias F Curtius et al ldquoPreliminary studieson Campomanesia xanthocarpa (Berg) and Cuphea carthage-nensis (Jacq) JF Macbr aqueous extract weight control andbiochemical parametersrdquo Journal of Ethnopharmacology vol93 no 2-3 pp 385ndash389 2004

[28] B E O Markman E M Bacchi and E T M Kato ldquoAntiul-cerogenic effects of Campomanesia xanthocarpardquo Journal ofEthnopharmacology vol 94 no 1 pp 55ndash57 2004

[29] F Calzada L Yepez-Mulia and A Tapia-Contreras ldquoEffect ofMexican medicinal plant used to treat trichomoniasis on Tri-chomonas vaginalis trophozoitesrdquo Journal of Ethnopharmacol-ogy vol 113 no 2 pp 248ndash251 2007

BioMed Research International 7

[30] J Desrivot J Waikedre P Cabalion et al ldquoAntiparasitic activityof some New Caledonian medicinal plantsrdquo Journal of Ethno-pharmacology vol 112 no 1 pp 7ndash12 2007

[31] A P Frasson O dos Santos M Duarte et al ldquoFirst report ofanti-Trichomonas vaginalis activity of the medicinal plant Poly-gala decumbens from the Brazilian semi-arid region CaatingardquoParasitology Research vol 110 no 6 pp 2581ndash2587 2012

[32] P B Vieira R B Giordani G A de Carli J A S Zuanazzi andT Tasca ldquoScreening and bioguided fractionation of Amarylli-daceae species with anti- Trichomonas vaginalis activityrdquo PlantaMedica vol 77 no 10 pp 1054ndash1059 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 BioMed Research International

medicines are still integrated as part of the cure of diversepathologies [13] In addition the search based on ethnophar-macological information rescues the immense empiricalknowledge of plants utilization [14 15] In this contextregarding natural environment and vast wealth of geneticresources Brazil is a country of great interest to ethnophar-macology [16]

Indigenous people have a wealth of biodiversity informa-tion and know how to capture and use natural resources [17]Despite of the fact that several drugs have been discoveredthrough traditional cures and folk knowledge in Brazil thereare 122 indigenous ethnic groups but only 30 of them wereinvestigated with regard to ethnobotanical aspects [18 19]Therefore the study of plants used by indigenous medicineis important to interconnect traditional medicine and bioticenvironment preserving the indigenous ancient knowledge

Parasitic protozoa remain a major threat to human andanimal health and there are few effective drugs for thetreatment of many protozoal diseases Taking into accountthe indigenous ethnopharmacology as a contributor to thesystematic screening of plants with antiprotozoal activity thisstudy evaluated the anti-T vaginalis activity of the mostimportant medicinal plants used in Mbya-Guarani indige-nous medicine located in South of Brazil

2 Materials and Methods

21 Parasite Culture Conditions In this study seven isolatesof T vaginalis were used 30236 and 30238 from AmericanType Culture Collection (ATCC) TV-LACM1 TV-LACM2and TV-LACM3 (fresh clinical isolates from female patients)TV-LACH1 andTV-LACH2 (fresh clinical isolates frommalepatients) all from Laboratorio de Analises Clınicas e Tox-icologicas Faculdade de Farmacia UFRGS Brazil (projectwith ethical approval by UFRGS Ethical Research Commit-tee number 18923) Trichomonads were axenically culturedin trypticase-yeast extract-maltose (TYM)medium (pH 60)supplemented with 10 heat-inactivated bovine serum andincubated at 37∘C [20]

22 PlantMaterial Plantmaterialwas collected from indige-nous tribe Mbya-Guarani located at the Lomba do PinheiroPorto Alegre RS Brazil (30∘061015840476210158401015840 S and 51∘071015840378510158401015840W)inMarch 2011This village is denominated Tekoa Anhetengua(in EnglishTrueVillage) and 200 people live in the area gath-ered at 40 familiesThe women of the village have the knowl-edge of medicinal plants used by the entire population andconducted the collection of specimens Ten plants routinelyused in indigenous medicine for infectious diseases werecollected leaves of Aloe arborescensMill (ICN 173371) aerialparts of Bidens pilosa L (ICN 167397) aerial parts ofRhipsalisbaccifera (ICN 167402) barks of Luehea divaricata (ICN167403) roots of Trichilia sp (ICN 173126) leaves of Campo-manesia xanthocarpa O Berg (ICN 167401) leaves of Coixlacryma-jobi Lin (ICN 167396) leaves of Citrus limonium(ICN 167399) leaves of Citrus reticulata (ICN 173127) andleaves of Verbena sp (ICN 167394) The voucher specimenswere deposited at the herbarium of the Universidade Federaldo Rio Grande do Sul (ICN)

23 Preparation of Plant Extracts The fresh plants extractswere prepared conforming traditional use by indigenous peo-ple by decoction at 60∘C for 60min [1 10 (w v)] Aqueousextracts were freeze dried and the work solution was pre-pared at 80mgmL in ultrapure water sterilized by filtration(022 120583m) and stored at minus20∘C

24 Anti-Trichomonas vaginalis Assay Ten aqueous extractswere screened in vitro for activity against T vaginalis Theassay was performed in 96 microtiter plates at final concen-tration of 40mgmL in the wells The inhibitory minimumconcentration (MIC)was determined using an eightfold dilu-tion (40ndash0031mgmL) and the viability of the trophozoiteswas determined according to a fluorimetricmethod [21] Twocontrols were performed negative control only with tropho-zoites and a positive control with 80120583M metronidazole(Sigma Chemical Co St Louis MO USA)The experimentswere performed at least in three independent experimentsin triplicate The results were expressed as the percentage ofviable trophozoites compared to untreated parasites

25 Growth Effect of Aqueous Extracts In order to investigatethe effect of the active aqueous extracts on T vaginalisgrowth a kinetic growth curve was performed using theATCC 30236 isolate The parasites initial density of 20 times105 trophozoitesmL was incubated with the extracts at MICvalue in TYMmediumThe trophozoites were counted usinga hemocytometer during 72 h The results were expressed astrophozoite number (105mL) For microscopic analysis thecontrols and treated parasites were centrifuged for 10min at2000 rpm to concentrate the organisms after 4 h of incuba-tion and the preparation was observed in light microscope(magnified times1000) by trypan blue dye exclusion

26 Hemolytic Assay This assay was performed according tothe method used by Rocha et al [22] Blood type O positivefrom healthy human volunteers was collected with Alseverrsquossolution (1 1) and centrifuged at 2000 rpm for 5min Theerythrocyte fraction was washed three times with PBS 1x (pH70) and resuspended to give 1 red cell suspension (vv)Aqueous extracts concentrations were chosen according tominimum inhibitory concentration (MIC) determined in thesusceptibility assayThe erythrocytes were incubated with theextracts at 37∘C under agitation for 1 h and then were cen-trifuged at 3000 rpm for 5minThe absorbance of the super-natant was measured at 540 nm The experiment was per-formed in triplicate and the percentage of hemolysis causedby each sample tested was calculated in comparison to 100hemolytic activity of saponin from Quillaja saponaria [23]

3 Results and Discussion

This study evaluated the anti-T vaginalis activity of aqueousextracts of 10 medicinal plants used in medicine systemMbya-Guarani Importantly among the plants tested wefound two aqueous extracts with a potent anti-T vaginalis invitro activity The promising extracts are of Verbena sp andCampomanesia xanthocarpa The extract from Verbena sp

BioMed Research International 3

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(a)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(b)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(c)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(d)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(e)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(f)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(g)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(h)

Figure 1 Continued

4 BioMed Research International

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(i)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(j)

Figure 1 Effect of selected Mbya-Guarani medicinal plants at 40mgmL on different Trichomonas vaginalis isolates (a) Verbena sp (b)Campomanesia xanthocarpa (c) Bidens pilosa (d) Luehea divaricata (e) Coix lacryma-jobi (f) Trichilia sp (g) Citrus limonium (h) Aloearborescens (i) Citrus reticulata (j) Rhipsalis baccifera Data represent means plusmn standard deviation

Table 1 Hemolytic activity of the aqueous extracts that showedthe best anti-Trichomonas vaginalis activity and respective MICdetermined by susceptibility assay

Aqueous extractVerbena sp Campomanesia xanthocarpa

MIC (mgmL) 40 40Hemolysis () plusmn SD 65 plusmn 002 76 plusmn 001

at 40mgmL demonstrated the optimal anti-T vaginalisactivity inducing complete cytotoxicity (100 of parasitesdeath) upon all distinct trichomonads isolates (Figure 1(a))The leaves extract of Campomanesia xanthocarpa at the sameconcentration also showed a very high anti-T vaginalis activ-ity reducing the parasite viability up to 96 (Figure 1(b))

Other aqueous extracts displayed a variety of resultsThe aqueous extract obtained from Bidens pilosa totallyabolished the trophozoites viability only of the 30236 isolate(Figure 1(c)) The extract of the barks of Luehea divaricatainduced a reduction ofT vaginalis viability differently in eachtrichomonads isolate tested (Figure 1(d)) The leaves of Coixlacryma-jobi induced on average 75 of viability reductionon T vaginalis isolates however for the TV-LACH2 isolatethe reduction was only 40 (Figure 1(e)) The fresh clinicalisolates from male patients (TV-LACH1 and TV-LACH2)presented higher susceptibility than the isolates from femalepatients (ATCC and fresh clinical) when the root extract ofTrichilia sp was tested (Figure 1(f)) Extracts of leaves fromCitrus limonium reduced about 40 the viability of isolates(Figure 1(g)) The extracts from Aloe arborescens and Citrusreticulata decreased the viability of 50 of at least one isolate(Figures 1(h) and 1(i)) while Rhipsalis baccifera reducedonly 30 the viability of 30236 and TV-LACM2 isolates(Figure 1(j))

In comparison to the other extracts the extracts fromVer-bena sp and C xanthocarpa demonstrated the most promis-ing activity against T vaginalis and the minimum inhibitoryconcentration was determined for the 30236 isolate (Table 1)

Time (hours)0 12 24 36 48 60 72

0

5

10

15

20

Control

Time (hours)0 1 2 3 4 5 6

0

1

2

3

4

5

Verbena spCampomanesia xanthocarpa

Viab

le tr

opho

zoite

(105

mL)

Viab

le tr

opho

zoite

(105

mL)

Figure 2 Kinetic growth curve of Trichomonas vaginalis 30236 iso-late after treatment with aqueous extracts ofVerbena sp andCampo-manesia xanthocarpa at 40mgmL The trophozoites growth wascompletely inhibited by the extracts in 4 hours of incubation Datarepresent means plusmn standard deviation of at least three experimentsall in triplicate

In order to investigate the effect of the most active extracton parasite growth and viability a kinetic growth curve wasperformed The control exhibited the classical growth peakafter 24 h of incubation however the organisms treated withextracts of Verbena sp and C xanthocarpa after 4 h of incu-bation evoked a complete growth abolishment (Figure 2) andshowed a characteristic blue color by trypan blue dye exclu-sion indicating cell death (Figure 3)

In order to investigate a possible effect of the C xantho-carpa and Verbena sp extracts on parasites membrane andalso to have preliminary data on cytotoxicity against mam-malian cells hemolytic activity of these extracts was con-ducted The Verbena sp and C xanthocarpa extracts weretested at MIC values and demonstrated 65 and 76 erythro-cytes lysis respectively (Table 1) These results demonstratedthat both extracts at MIC values did not promote a signif-icant hemolytic activity indicating that the mechanism of

BioMed Research International 5

(a) Control (b) Verbena sp

(c) Campomanesia xanthocarpaFigure 3 Microscopic analysis of parasites morphology in light microscope (magnified times1000) by trypan blue dye exclusion (a) Control(b) after 4 hours of treatment with aqueous extract of Verbena sp (c) after 4 hours of treatment with aqueous extract of Campomanesiaxanthocarpa

action responsible for the parasite viability reduction proba-bly does not involve parasitic membrane disruption More-over and importantly these findings suggest that both ex-tracts are not toxic to mammalian cells since they did notlyse human erythrocytes

This study contributes to rescue the indigenous knowl-edge about medicinal plants The indigenous Mbya-Guaraniuse Verbena sp (Guachu karsquoa in Mbya-Guarani language)as tea for relief of symptoms of infectious diseases such asfeverThis is in agreement of the literature that relatesVerbenasp infusion as sedative analgesic to inflammatory disordersskin burns amenorrhea depression and gastric diseases [24ndash26] Moreover this species has a variety of chemical con-stituents as flavonoid iridoid glycosides phenylpropanoidglycoside sterols triterpenes and glycoconjugate and thebiological activity may be partially attributed to them [24]In addition this tribe utilizes the leaves of C xanthocarpa(Guavira in Mbya-Guarani language) for treatment of diar-rhea stomach pain and worms This plant is traditionallyused in the south of Brazil as depurative antidiarrhoeiccleanser and antirheumatic and to decrease the bloodcholesterol [27] and these properties may be attributedto tannins saponins and flavonoids (quercetin myricetinquercitrin and rutin) compounds present in the leaves of Cxanthocarpa [28]

The rescue ethnopharmacology of historical uses of plantsis recognized as valuable for bioprospection since they affordthe rationale for selection and research of medicinal plants[15]This study describes the first report on the antiprotozoalactivity of Verbena sp and C xanthocarpa In addition otherspecies of the families Verbenaceae and Myrtaceae were

tested against T vaginalis however without success [29 30]Several biodiversities and traditional knowledge of differentpopulations have guided studies to search new prototypesanti-T vaginalis Recently the potential of plants traditionallyused from Caatinga against T vaginalis was reported [31]Calzada et al [29] demonstrated that plants used for urogen-ital tract disorders in Mexican traditional medicine showedeffect against this protozoan Amaryllidaceae species usedagainst venereal diseases in SouthAfrica also showed promis-ing activity against T vaginalis [11 32]

Considering the impact of trichomonosis in public healthand the emergent number of resistantT vaginalis isolates it isnecessary alternatives for the treatment of this infection Nat-ural products are a promising source of active molecules andthe ethnopharmacology approach rescues the knowledge ofpopulation of medicinal plants This wisdom combined withchemical and pharmacological studies presents a preciousvalue in the bioprospection of innovates safe and accessibledrugsThis pioneer study demonstrated relevant results aboutanti-T vaginalis activity of the Verbena sp and C xantho-carpa plants traditionally used by indigenous populationMbya-Guarani for infectious diseases Despite of showingstrong anti-T vaginalis activity this report rescued the knowl-edge of indigenous people avoiding the miscarriage of thiswisdom

Authorsrsquo Contribution

Clara Lia Costa Brandelli and Patrıcia de Brum Vieira con-tributed equally to this work

6 BioMed Research International

Acknowledgments

This work was supported by the NANOBIOTEC-Brazil pro-gram from CAPES and FAPERGS The authors thank Chief-tain Jose Cirilo Pires Morinico (tribe Mbya-Guarani) formaking possible the accomplishment of this study and for thevaluable information Alexandre Jose Macedo and TianaTasca thank CNPq for researcher fellowships

References

[1] WHO Global Incidence and Prevalence of Selected Curable Sex-ually Transmitted Infections-2008 World Health OrganizationGeneva Switzerland 2012 httpappswhointirisbitstream106657518119789241503839 engpdf

[2] D Petrin K Delgaty R Bhatt and G Garber ldquoClinicaland microbiological aspects of Trichomonas vaginalisrdquo ClinicalMicrobiology Reviews vol 11 no 2 pp 300ndash317 1998

[3] M A Klebanoff J C Carey J C Hauth et al ldquoFailure of met-ronidazole to prevent preterm delivery among pregnant womenwith asymptomatic Trichomonas vaginalis infectionrdquo The NewEngland Journal of Medicine vol 345 no 7 pp 487ndash493 2001

[4] F Grodstein M B Goldman and D W Cramer ldquoRelation oftubal infertility to history of sexually transmitted diseasesrdquoAmerican Journal of Epidemiology vol 137 no 5 pp 577ndash5841993

[5] M Viikki E Pukkala P Nieminen and M Hakama ldquoGynae-cological infections as risk determinants of subsequent cervicalneoplasiardquo Acta Oncologica vol 39 no 1 pp 71ndash75 2000

[6] T L Cherpes H C Wiesenfeld M A Melan et al ldquoTheassociations between pelvic inflammatory diseaseTrichomonasvaginalis infection and positive herpes simplex virus type 2serologyrdquo Sexually Transmitted Diseases vol 33 no 12 pp 747ndash752 2006

[7] F Sorvillo L Smith P Kerndt and L Ash ldquoTrichomonas vagi-nalis HIV and African-Americansrdquo Emerging Infectious Dis-eases vol 7 no 6 pp 927ndash932 2001

[8] B van der Pol C Kwok B Pierre-Louis et al ldquoTrichomonasvaginalis infection and human immunodeficiency virus acqui-sition in African womenrdquo Journal of Infectious Diseases vol 197no 4 pp 548ndash554 2008

[9] V Ali and T Nozaki ldquoCurrent therapeutics their problemsand sulfur-containing-amino-acid metabolism as a novel targetagainst infections by ldquoamitochondriaterdquo protozoan parasitesrdquoClinical Microbiology Reviews vol 20 no 1 pp 164ndash187 2007

[10] J R Schwebke and F J Barrientes ldquoPrevalence of Trichomonasvaginalis isolates with resistance to metronidazole and tinida-zolerdquo Antimicrobial Agents and Chemotherapy vol 50 no 12pp 4209ndash4210 2006

[11] R B Giordani P B Vieira MWeizenmann et al ldquoCandimine-induced cell death of the amitochondriate parasiteTrichomonasvaginalisrdquo Journal of Natural Products vol 73 no 12 pp 2019ndash2023 2010

[12] R B Giordani P B Vieira M Weizenmann et al ldquoLycorineinduces cell death in the amitochondriate parasiteTrichomonasvaginalis via an alternative non-apoptotic death pathwayrdquoPhytochemistry vol 72 no 7 pp 645ndash650 2011

[13] J L Rıos and M C Recio ldquoMedicinal plants and antimicrobialactivityrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 80ndash84 2005

[14] MHeinrich ldquoEthnobotany and natural products the search fornewmolecules new treatments of old diseases or a better under-standing of indigenous culturesrdquo Current Topics in MedicinalChemistry vol 3 no 2 pp 141ndash154 2003

[15] C L C Brandelli R B Giordani A J Macedo G A de Carliand T Tasca ldquoIndigenous traditional medicine plants for thetreatment of diarrheardquo inNature Helps How Plants and OtherOrganisms Contribute to Solve Health Problems pp 1ndash18Springer Berlin Germany 1st edition 2011

[16] E Elisabetsky and L Wannmacher ldquoThe status of ethnophar-macology in Brazilrdquo Journal of Ethnopharmacology vol 38 no2-3 pp 137ndash143 1993

[17] F L H Rego A J Brand and R B Costa ldquoGenetic resourcesbiodiversity traditional knowledge Guarani Kaiowa and localdevelopmentrdquo Interacoes vol 11 no 1 pp 55ndash69 2010

[18] D F Coutinho L M A Travassos and F M M AmaralldquoEstudo etnobotanico de plantas medicinais utilizadas emcomunidades indıgenas no estado doMaranhaomdashBrasilrdquoVisaoAcademica vol 3 no 1 pp 7ndash12 2002

[19] A H Gilani and A-U Rahman ldquoTrends in ethnopharmacol-ogyrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 43ndash492005

[20] L S Diamond ldquoThe establishment of various Trichomonas ofanimals andman in axenic culturesrdquoThe Journal of Parasitologyvol 43 no 4 pp 488ndash490 1957

[21] M Duarte R B Giordani G A de Carli J A ZuanazziA J Macedo and T Tasca ldquoA quantitative resazurin assay todeterminate the viability of Trichomonas vaginalis and the cyto-toxicity of organic solvents and surfactant agentsrdquo ExperimentalParasitology vol 123 no 2 pp 195ndash198 2009

[22] T D Rocha P B Vieira S C B Gnoatto T Tasca and G Gos-mann ldquoAnti-Trichomonas vaginalis activity of saponins fromQuillaja Passiflora and Ilex speciesrdquo Parasitology Research vol110 no 6 pp 2551ndash2556 2012

[23] Y SunH TongM Li Y Li S Guan and J Liu ldquoImmunologicaladjuvant effect of Japanese ginseng saponins (JGS) on specificantibody and cellular response to ovalbumin and its haemolyticactivitiesrdquo Vaccine vol 26 no 47 pp 5911ndash5917 2008

[24] M I Calvo ldquoAnti-inflammatory and analgesic activity of thetopical preparation of Verbena officinalis Lrdquo Journal of Ethno-pharmacology vol 107 no 3 pp 380ndash382 2006

[25] S-W Lai M-S Yu W-H Yuen and R C-C Chang ldquoNovelneuroprotective effects of the aqueous extracts from Verbenaofficinalis LinnrdquoNeuropharmacology vol 50 no 6 pp 641ndash6502006

[26] E Speroni R Cervellati S Costa et al ldquoEffects of differentialextraction of Verbena officinalis on rat models of inflammationcicatrization and gastric damagerdquo Planta Medica vol 73 no 3pp 227ndash235 2007

[27] M W Biavatti C Farias F Curtius et al ldquoPreliminary studieson Campomanesia xanthocarpa (Berg) and Cuphea carthage-nensis (Jacq) JF Macbr aqueous extract weight control andbiochemical parametersrdquo Journal of Ethnopharmacology vol93 no 2-3 pp 385ndash389 2004

[28] B E O Markman E M Bacchi and E T M Kato ldquoAntiul-cerogenic effects of Campomanesia xanthocarpardquo Journal ofEthnopharmacology vol 94 no 1 pp 55ndash57 2004

[29] F Calzada L Yepez-Mulia and A Tapia-Contreras ldquoEffect ofMexican medicinal plant used to treat trichomoniasis on Tri-chomonas vaginalis trophozoitesrdquo Journal of Ethnopharmacol-ogy vol 113 no 2 pp 248ndash251 2007

BioMed Research International 7

[30] J Desrivot J Waikedre P Cabalion et al ldquoAntiparasitic activityof some New Caledonian medicinal plantsrdquo Journal of Ethno-pharmacology vol 112 no 1 pp 7ndash12 2007

[31] A P Frasson O dos Santos M Duarte et al ldquoFirst report ofanti-Trichomonas vaginalis activity of the medicinal plant Poly-gala decumbens from the Brazilian semi-arid region CaatingardquoParasitology Research vol 110 no 6 pp 2581ndash2587 2012

[32] P B Vieira R B Giordani G A de Carli J A S Zuanazzi andT Tasca ldquoScreening and bioguided fractionation of Amarylli-daceae species with anti- Trichomonas vaginalis activityrdquo PlantaMedica vol 77 no 10 pp 1054ndash1059 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(a)

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hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(b)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(c)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(d)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(e)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(f)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(g)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(h)

Figure 1 Continued

4 BioMed Research International

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(i)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(j)

Figure 1 Effect of selected Mbya-Guarani medicinal plants at 40mgmL on different Trichomonas vaginalis isolates (a) Verbena sp (b)Campomanesia xanthocarpa (c) Bidens pilosa (d) Luehea divaricata (e) Coix lacryma-jobi (f) Trichilia sp (g) Citrus limonium (h) Aloearborescens (i) Citrus reticulata (j) Rhipsalis baccifera Data represent means plusmn standard deviation

Table 1 Hemolytic activity of the aqueous extracts that showedthe best anti-Trichomonas vaginalis activity and respective MICdetermined by susceptibility assay

Aqueous extractVerbena sp Campomanesia xanthocarpa

MIC (mgmL) 40 40Hemolysis () plusmn SD 65 plusmn 002 76 plusmn 001

at 40mgmL demonstrated the optimal anti-T vaginalisactivity inducing complete cytotoxicity (100 of parasitesdeath) upon all distinct trichomonads isolates (Figure 1(a))The leaves extract of Campomanesia xanthocarpa at the sameconcentration also showed a very high anti-T vaginalis activ-ity reducing the parasite viability up to 96 (Figure 1(b))

Other aqueous extracts displayed a variety of resultsThe aqueous extract obtained from Bidens pilosa totallyabolished the trophozoites viability only of the 30236 isolate(Figure 1(c)) The extract of the barks of Luehea divaricatainduced a reduction ofT vaginalis viability differently in eachtrichomonads isolate tested (Figure 1(d)) The leaves of Coixlacryma-jobi induced on average 75 of viability reductionon T vaginalis isolates however for the TV-LACH2 isolatethe reduction was only 40 (Figure 1(e)) The fresh clinicalisolates from male patients (TV-LACH1 and TV-LACH2)presented higher susceptibility than the isolates from femalepatients (ATCC and fresh clinical) when the root extract ofTrichilia sp was tested (Figure 1(f)) Extracts of leaves fromCitrus limonium reduced about 40 the viability of isolates(Figure 1(g)) The extracts from Aloe arborescens and Citrusreticulata decreased the viability of 50 of at least one isolate(Figures 1(h) and 1(i)) while Rhipsalis baccifera reducedonly 30 the viability of 30236 and TV-LACM2 isolates(Figure 1(j))

In comparison to the other extracts the extracts fromVer-bena sp and C xanthocarpa demonstrated the most promis-ing activity against T vaginalis and the minimum inhibitoryconcentration was determined for the 30236 isolate (Table 1)

Time (hours)0 12 24 36 48 60 72

0

5

10

15

20

Control

Time (hours)0 1 2 3 4 5 6

0

1

2

3

4

5

Verbena spCampomanesia xanthocarpa

Viab

le tr

opho

zoite

(105

mL)

Viab

le tr

opho

zoite

(105

mL)

Figure 2 Kinetic growth curve of Trichomonas vaginalis 30236 iso-late after treatment with aqueous extracts ofVerbena sp andCampo-manesia xanthocarpa at 40mgmL The trophozoites growth wascompletely inhibited by the extracts in 4 hours of incubation Datarepresent means plusmn standard deviation of at least three experimentsall in triplicate

In order to investigate the effect of the most active extracton parasite growth and viability a kinetic growth curve wasperformed The control exhibited the classical growth peakafter 24 h of incubation however the organisms treated withextracts of Verbena sp and C xanthocarpa after 4 h of incu-bation evoked a complete growth abolishment (Figure 2) andshowed a characteristic blue color by trypan blue dye exclu-sion indicating cell death (Figure 3)

In order to investigate a possible effect of the C xantho-carpa and Verbena sp extracts on parasites membrane andalso to have preliminary data on cytotoxicity against mam-malian cells hemolytic activity of these extracts was con-ducted The Verbena sp and C xanthocarpa extracts weretested at MIC values and demonstrated 65 and 76 erythro-cytes lysis respectively (Table 1) These results demonstratedthat both extracts at MIC values did not promote a signif-icant hemolytic activity indicating that the mechanism of

BioMed Research International 5

(a) Control (b) Verbena sp

(c) Campomanesia xanthocarpaFigure 3 Microscopic analysis of parasites morphology in light microscope (magnified times1000) by trypan blue dye exclusion (a) Control(b) after 4 hours of treatment with aqueous extract of Verbena sp (c) after 4 hours of treatment with aqueous extract of Campomanesiaxanthocarpa

action responsible for the parasite viability reduction proba-bly does not involve parasitic membrane disruption More-over and importantly these findings suggest that both ex-tracts are not toxic to mammalian cells since they did notlyse human erythrocytes

This study contributes to rescue the indigenous knowl-edge about medicinal plants The indigenous Mbya-Guaraniuse Verbena sp (Guachu karsquoa in Mbya-Guarani language)as tea for relief of symptoms of infectious diseases such asfeverThis is in agreement of the literature that relatesVerbenasp infusion as sedative analgesic to inflammatory disordersskin burns amenorrhea depression and gastric diseases [24ndash26] Moreover this species has a variety of chemical con-stituents as flavonoid iridoid glycosides phenylpropanoidglycoside sterols triterpenes and glycoconjugate and thebiological activity may be partially attributed to them [24]In addition this tribe utilizes the leaves of C xanthocarpa(Guavira in Mbya-Guarani language) for treatment of diar-rhea stomach pain and worms This plant is traditionallyused in the south of Brazil as depurative antidiarrhoeiccleanser and antirheumatic and to decrease the bloodcholesterol [27] and these properties may be attributedto tannins saponins and flavonoids (quercetin myricetinquercitrin and rutin) compounds present in the leaves of Cxanthocarpa [28]

The rescue ethnopharmacology of historical uses of plantsis recognized as valuable for bioprospection since they affordthe rationale for selection and research of medicinal plants[15]This study describes the first report on the antiprotozoalactivity of Verbena sp and C xanthocarpa In addition otherspecies of the families Verbenaceae and Myrtaceae were

tested against T vaginalis however without success [29 30]Several biodiversities and traditional knowledge of differentpopulations have guided studies to search new prototypesanti-T vaginalis Recently the potential of plants traditionallyused from Caatinga against T vaginalis was reported [31]Calzada et al [29] demonstrated that plants used for urogen-ital tract disorders in Mexican traditional medicine showedeffect against this protozoan Amaryllidaceae species usedagainst venereal diseases in SouthAfrica also showed promis-ing activity against T vaginalis [11 32]

Considering the impact of trichomonosis in public healthand the emergent number of resistantT vaginalis isolates it isnecessary alternatives for the treatment of this infection Nat-ural products are a promising source of active molecules andthe ethnopharmacology approach rescues the knowledge ofpopulation of medicinal plants This wisdom combined withchemical and pharmacological studies presents a preciousvalue in the bioprospection of innovates safe and accessibledrugsThis pioneer study demonstrated relevant results aboutanti-T vaginalis activity of the Verbena sp and C xantho-carpa plants traditionally used by indigenous populationMbya-Guarani for infectious diseases Despite of showingstrong anti-T vaginalis activity this report rescued the knowl-edge of indigenous people avoiding the miscarriage of thiswisdom

Authorsrsquo Contribution

Clara Lia Costa Brandelli and Patrıcia de Brum Vieira con-tributed equally to this work

6 BioMed Research International

Acknowledgments

This work was supported by the NANOBIOTEC-Brazil pro-gram from CAPES and FAPERGS The authors thank Chief-tain Jose Cirilo Pires Morinico (tribe Mbya-Guarani) formaking possible the accomplishment of this study and for thevaluable information Alexandre Jose Macedo and TianaTasca thank CNPq for researcher fellowships

References

[1] WHO Global Incidence and Prevalence of Selected Curable Sex-ually Transmitted Infections-2008 World Health OrganizationGeneva Switzerland 2012 httpappswhointirisbitstream106657518119789241503839 engpdf

[2] D Petrin K Delgaty R Bhatt and G Garber ldquoClinicaland microbiological aspects of Trichomonas vaginalisrdquo ClinicalMicrobiology Reviews vol 11 no 2 pp 300ndash317 1998

[3] M A Klebanoff J C Carey J C Hauth et al ldquoFailure of met-ronidazole to prevent preterm delivery among pregnant womenwith asymptomatic Trichomonas vaginalis infectionrdquo The NewEngland Journal of Medicine vol 345 no 7 pp 487ndash493 2001

[4] F Grodstein M B Goldman and D W Cramer ldquoRelation oftubal infertility to history of sexually transmitted diseasesrdquoAmerican Journal of Epidemiology vol 137 no 5 pp 577ndash5841993

[5] M Viikki E Pukkala P Nieminen and M Hakama ldquoGynae-cological infections as risk determinants of subsequent cervicalneoplasiardquo Acta Oncologica vol 39 no 1 pp 71ndash75 2000

[6] T L Cherpes H C Wiesenfeld M A Melan et al ldquoTheassociations between pelvic inflammatory diseaseTrichomonasvaginalis infection and positive herpes simplex virus type 2serologyrdquo Sexually Transmitted Diseases vol 33 no 12 pp 747ndash752 2006

[7] F Sorvillo L Smith P Kerndt and L Ash ldquoTrichomonas vagi-nalis HIV and African-Americansrdquo Emerging Infectious Dis-eases vol 7 no 6 pp 927ndash932 2001

[8] B van der Pol C Kwok B Pierre-Louis et al ldquoTrichomonasvaginalis infection and human immunodeficiency virus acqui-sition in African womenrdquo Journal of Infectious Diseases vol 197no 4 pp 548ndash554 2008

[9] V Ali and T Nozaki ldquoCurrent therapeutics their problemsand sulfur-containing-amino-acid metabolism as a novel targetagainst infections by ldquoamitochondriaterdquo protozoan parasitesrdquoClinical Microbiology Reviews vol 20 no 1 pp 164ndash187 2007

[10] J R Schwebke and F J Barrientes ldquoPrevalence of Trichomonasvaginalis isolates with resistance to metronidazole and tinida-zolerdquo Antimicrobial Agents and Chemotherapy vol 50 no 12pp 4209ndash4210 2006

[11] R B Giordani P B Vieira MWeizenmann et al ldquoCandimine-induced cell death of the amitochondriate parasiteTrichomonasvaginalisrdquo Journal of Natural Products vol 73 no 12 pp 2019ndash2023 2010

[12] R B Giordani P B Vieira M Weizenmann et al ldquoLycorineinduces cell death in the amitochondriate parasiteTrichomonasvaginalis via an alternative non-apoptotic death pathwayrdquoPhytochemistry vol 72 no 7 pp 645ndash650 2011

[13] J L Rıos and M C Recio ldquoMedicinal plants and antimicrobialactivityrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 80ndash84 2005

[14] MHeinrich ldquoEthnobotany and natural products the search fornewmolecules new treatments of old diseases or a better under-standing of indigenous culturesrdquo Current Topics in MedicinalChemistry vol 3 no 2 pp 141ndash154 2003

[15] C L C Brandelli R B Giordani A J Macedo G A de Carliand T Tasca ldquoIndigenous traditional medicine plants for thetreatment of diarrheardquo inNature Helps How Plants and OtherOrganisms Contribute to Solve Health Problems pp 1ndash18Springer Berlin Germany 1st edition 2011

[16] E Elisabetsky and L Wannmacher ldquoThe status of ethnophar-macology in Brazilrdquo Journal of Ethnopharmacology vol 38 no2-3 pp 137ndash143 1993

[17] F L H Rego A J Brand and R B Costa ldquoGenetic resourcesbiodiversity traditional knowledge Guarani Kaiowa and localdevelopmentrdquo Interacoes vol 11 no 1 pp 55ndash69 2010

[18] D F Coutinho L M A Travassos and F M M AmaralldquoEstudo etnobotanico de plantas medicinais utilizadas emcomunidades indıgenas no estado doMaranhaomdashBrasilrdquoVisaoAcademica vol 3 no 1 pp 7ndash12 2002

[19] A H Gilani and A-U Rahman ldquoTrends in ethnopharmacol-ogyrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 43ndash492005

[20] L S Diamond ldquoThe establishment of various Trichomonas ofanimals andman in axenic culturesrdquoThe Journal of Parasitologyvol 43 no 4 pp 488ndash490 1957

[21] M Duarte R B Giordani G A de Carli J A ZuanazziA J Macedo and T Tasca ldquoA quantitative resazurin assay todeterminate the viability of Trichomonas vaginalis and the cyto-toxicity of organic solvents and surfactant agentsrdquo ExperimentalParasitology vol 123 no 2 pp 195ndash198 2009

[22] T D Rocha P B Vieira S C B Gnoatto T Tasca and G Gos-mann ldquoAnti-Trichomonas vaginalis activity of saponins fromQuillaja Passiflora and Ilex speciesrdquo Parasitology Research vol110 no 6 pp 2551ndash2556 2012

[23] Y SunH TongM Li Y Li S Guan and J Liu ldquoImmunologicaladjuvant effect of Japanese ginseng saponins (JGS) on specificantibody and cellular response to ovalbumin and its haemolyticactivitiesrdquo Vaccine vol 26 no 47 pp 5911ndash5917 2008

[24] M I Calvo ldquoAnti-inflammatory and analgesic activity of thetopical preparation of Verbena officinalis Lrdquo Journal of Ethno-pharmacology vol 107 no 3 pp 380ndash382 2006

[25] S-W Lai M-S Yu W-H Yuen and R C-C Chang ldquoNovelneuroprotective effects of the aqueous extracts from Verbenaofficinalis LinnrdquoNeuropharmacology vol 50 no 6 pp 641ndash6502006

[26] E Speroni R Cervellati S Costa et al ldquoEffects of differentialextraction of Verbena officinalis on rat models of inflammationcicatrization and gastric damagerdquo Planta Medica vol 73 no 3pp 227ndash235 2007

[27] M W Biavatti C Farias F Curtius et al ldquoPreliminary studieson Campomanesia xanthocarpa (Berg) and Cuphea carthage-nensis (Jacq) JF Macbr aqueous extract weight control andbiochemical parametersrdquo Journal of Ethnopharmacology vol93 no 2-3 pp 385ndash389 2004

[28] B E O Markman E M Bacchi and E T M Kato ldquoAntiul-cerogenic effects of Campomanesia xanthocarpardquo Journal ofEthnopharmacology vol 94 no 1 pp 55ndash57 2004

[29] F Calzada L Yepez-Mulia and A Tapia-Contreras ldquoEffect ofMexican medicinal plant used to treat trichomoniasis on Tri-chomonas vaginalis trophozoitesrdquo Journal of Ethnopharmacol-ogy vol 113 no 2 pp 248ndash251 2007

BioMed Research International 7

[30] J Desrivot J Waikedre P Cabalion et al ldquoAntiparasitic activityof some New Caledonian medicinal plantsrdquo Journal of Ethno-pharmacology vol 112 no 1 pp 7ndash12 2007

[31] A P Frasson O dos Santos M Duarte et al ldquoFirst report ofanti-Trichomonas vaginalis activity of the medicinal plant Poly-gala decumbens from the Brazilian semi-arid region CaatingardquoParasitology Research vol 110 no 6 pp 2581ndash2587 2012

[32] P B Vieira R B Giordani G A de Carli J A S Zuanazzi andT Tasca ldquoScreening and bioguided fractionation of Amarylli-daceae species with anti- Trichomonas vaginalis activityrdquo PlantaMedica vol 77 no 10 pp 1054ndash1059 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(i)

Trop

hozo

ite v

iabi

lity

()

0

20

40

60

80

100

120

Con

trol

3023

6

3023

8

TV-L

ACM

1

TV-L

ACM

2

TV-L

ACM

3

TV-L

ACH

1

TV-L

ACH

2

(j)

Figure 1 Effect of selected Mbya-Guarani medicinal plants at 40mgmL on different Trichomonas vaginalis isolates (a) Verbena sp (b)Campomanesia xanthocarpa (c) Bidens pilosa (d) Luehea divaricata (e) Coix lacryma-jobi (f) Trichilia sp (g) Citrus limonium (h) Aloearborescens (i) Citrus reticulata (j) Rhipsalis baccifera Data represent means plusmn standard deviation

Table 1 Hemolytic activity of the aqueous extracts that showedthe best anti-Trichomonas vaginalis activity and respective MICdetermined by susceptibility assay

Aqueous extractVerbena sp Campomanesia xanthocarpa

MIC (mgmL) 40 40Hemolysis () plusmn SD 65 plusmn 002 76 plusmn 001

at 40mgmL demonstrated the optimal anti-T vaginalisactivity inducing complete cytotoxicity (100 of parasitesdeath) upon all distinct trichomonads isolates (Figure 1(a))The leaves extract of Campomanesia xanthocarpa at the sameconcentration also showed a very high anti-T vaginalis activ-ity reducing the parasite viability up to 96 (Figure 1(b))

Other aqueous extracts displayed a variety of resultsThe aqueous extract obtained from Bidens pilosa totallyabolished the trophozoites viability only of the 30236 isolate(Figure 1(c)) The extract of the barks of Luehea divaricatainduced a reduction ofT vaginalis viability differently in eachtrichomonads isolate tested (Figure 1(d)) The leaves of Coixlacryma-jobi induced on average 75 of viability reductionon T vaginalis isolates however for the TV-LACH2 isolatethe reduction was only 40 (Figure 1(e)) The fresh clinicalisolates from male patients (TV-LACH1 and TV-LACH2)presented higher susceptibility than the isolates from femalepatients (ATCC and fresh clinical) when the root extract ofTrichilia sp was tested (Figure 1(f)) Extracts of leaves fromCitrus limonium reduced about 40 the viability of isolates(Figure 1(g)) The extracts from Aloe arborescens and Citrusreticulata decreased the viability of 50 of at least one isolate(Figures 1(h) and 1(i)) while Rhipsalis baccifera reducedonly 30 the viability of 30236 and TV-LACM2 isolates(Figure 1(j))

In comparison to the other extracts the extracts fromVer-bena sp and C xanthocarpa demonstrated the most promis-ing activity against T vaginalis and the minimum inhibitoryconcentration was determined for the 30236 isolate (Table 1)

Time (hours)0 12 24 36 48 60 72

0

5

10

15

20

Control

Time (hours)0 1 2 3 4 5 6

0

1

2

3

4

5

Verbena spCampomanesia xanthocarpa

Viab

le tr

opho

zoite

(105

mL)

Viab

le tr

opho

zoite

(105

mL)

Figure 2 Kinetic growth curve of Trichomonas vaginalis 30236 iso-late after treatment with aqueous extracts ofVerbena sp andCampo-manesia xanthocarpa at 40mgmL The trophozoites growth wascompletely inhibited by the extracts in 4 hours of incubation Datarepresent means plusmn standard deviation of at least three experimentsall in triplicate

In order to investigate the effect of the most active extracton parasite growth and viability a kinetic growth curve wasperformed The control exhibited the classical growth peakafter 24 h of incubation however the organisms treated withextracts of Verbena sp and C xanthocarpa after 4 h of incu-bation evoked a complete growth abolishment (Figure 2) andshowed a characteristic blue color by trypan blue dye exclu-sion indicating cell death (Figure 3)

In order to investigate a possible effect of the C xantho-carpa and Verbena sp extracts on parasites membrane andalso to have preliminary data on cytotoxicity against mam-malian cells hemolytic activity of these extracts was con-ducted The Verbena sp and C xanthocarpa extracts weretested at MIC values and demonstrated 65 and 76 erythro-cytes lysis respectively (Table 1) These results demonstratedthat both extracts at MIC values did not promote a signif-icant hemolytic activity indicating that the mechanism of

BioMed Research International 5

(a) Control (b) Verbena sp

(c) Campomanesia xanthocarpaFigure 3 Microscopic analysis of parasites morphology in light microscope (magnified times1000) by trypan blue dye exclusion (a) Control(b) after 4 hours of treatment with aqueous extract of Verbena sp (c) after 4 hours of treatment with aqueous extract of Campomanesiaxanthocarpa

action responsible for the parasite viability reduction proba-bly does not involve parasitic membrane disruption More-over and importantly these findings suggest that both ex-tracts are not toxic to mammalian cells since they did notlyse human erythrocytes

This study contributes to rescue the indigenous knowl-edge about medicinal plants The indigenous Mbya-Guaraniuse Verbena sp (Guachu karsquoa in Mbya-Guarani language)as tea for relief of symptoms of infectious diseases such asfeverThis is in agreement of the literature that relatesVerbenasp infusion as sedative analgesic to inflammatory disordersskin burns amenorrhea depression and gastric diseases [24ndash26] Moreover this species has a variety of chemical con-stituents as flavonoid iridoid glycosides phenylpropanoidglycoside sterols triterpenes and glycoconjugate and thebiological activity may be partially attributed to them [24]In addition this tribe utilizes the leaves of C xanthocarpa(Guavira in Mbya-Guarani language) for treatment of diar-rhea stomach pain and worms This plant is traditionallyused in the south of Brazil as depurative antidiarrhoeiccleanser and antirheumatic and to decrease the bloodcholesterol [27] and these properties may be attributedto tannins saponins and flavonoids (quercetin myricetinquercitrin and rutin) compounds present in the leaves of Cxanthocarpa [28]

The rescue ethnopharmacology of historical uses of plantsis recognized as valuable for bioprospection since they affordthe rationale for selection and research of medicinal plants[15]This study describes the first report on the antiprotozoalactivity of Verbena sp and C xanthocarpa In addition otherspecies of the families Verbenaceae and Myrtaceae were

tested against T vaginalis however without success [29 30]Several biodiversities and traditional knowledge of differentpopulations have guided studies to search new prototypesanti-T vaginalis Recently the potential of plants traditionallyused from Caatinga against T vaginalis was reported [31]Calzada et al [29] demonstrated that plants used for urogen-ital tract disorders in Mexican traditional medicine showedeffect against this protozoan Amaryllidaceae species usedagainst venereal diseases in SouthAfrica also showed promis-ing activity against T vaginalis [11 32]

Considering the impact of trichomonosis in public healthand the emergent number of resistantT vaginalis isolates it isnecessary alternatives for the treatment of this infection Nat-ural products are a promising source of active molecules andthe ethnopharmacology approach rescues the knowledge ofpopulation of medicinal plants This wisdom combined withchemical and pharmacological studies presents a preciousvalue in the bioprospection of innovates safe and accessibledrugsThis pioneer study demonstrated relevant results aboutanti-T vaginalis activity of the Verbena sp and C xantho-carpa plants traditionally used by indigenous populationMbya-Guarani for infectious diseases Despite of showingstrong anti-T vaginalis activity this report rescued the knowl-edge of indigenous people avoiding the miscarriage of thiswisdom

Authorsrsquo Contribution

Clara Lia Costa Brandelli and Patrıcia de Brum Vieira con-tributed equally to this work

6 BioMed Research International

Acknowledgments

This work was supported by the NANOBIOTEC-Brazil pro-gram from CAPES and FAPERGS The authors thank Chief-tain Jose Cirilo Pires Morinico (tribe Mbya-Guarani) formaking possible the accomplishment of this study and for thevaluable information Alexandre Jose Macedo and TianaTasca thank CNPq for researcher fellowships

References

[1] WHO Global Incidence and Prevalence of Selected Curable Sex-ually Transmitted Infections-2008 World Health OrganizationGeneva Switzerland 2012 httpappswhointirisbitstream106657518119789241503839 engpdf

[2] D Petrin K Delgaty R Bhatt and G Garber ldquoClinicaland microbiological aspects of Trichomonas vaginalisrdquo ClinicalMicrobiology Reviews vol 11 no 2 pp 300ndash317 1998

[3] M A Klebanoff J C Carey J C Hauth et al ldquoFailure of met-ronidazole to prevent preterm delivery among pregnant womenwith asymptomatic Trichomonas vaginalis infectionrdquo The NewEngland Journal of Medicine vol 345 no 7 pp 487ndash493 2001

[4] F Grodstein M B Goldman and D W Cramer ldquoRelation oftubal infertility to history of sexually transmitted diseasesrdquoAmerican Journal of Epidemiology vol 137 no 5 pp 577ndash5841993

[5] M Viikki E Pukkala P Nieminen and M Hakama ldquoGynae-cological infections as risk determinants of subsequent cervicalneoplasiardquo Acta Oncologica vol 39 no 1 pp 71ndash75 2000

[6] T L Cherpes H C Wiesenfeld M A Melan et al ldquoTheassociations between pelvic inflammatory diseaseTrichomonasvaginalis infection and positive herpes simplex virus type 2serologyrdquo Sexually Transmitted Diseases vol 33 no 12 pp 747ndash752 2006

[7] F Sorvillo L Smith P Kerndt and L Ash ldquoTrichomonas vagi-nalis HIV and African-Americansrdquo Emerging Infectious Dis-eases vol 7 no 6 pp 927ndash932 2001

[8] B van der Pol C Kwok B Pierre-Louis et al ldquoTrichomonasvaginalis infection and human immunodeficiency virus acqui-sition in African womenrdquo Journal of Infectious Diseases vol 197no 4 pp 548ndash554 2008

[9] V Ali and T Nozaki ldquoCurrent therapeutics their problemsand sulfur-containing-amino-acid metabolism as a novel targetagainst infections by ldquoamitochondriaterdquo protozoan parasitesrdquoClinical Microbiology Reviews vol 20 no 1 pp 164ndash187 2007

[10] J R Schwebke and F J Barrientes ldquoPrevalence of Trichomonasvaginalis isolates with resistance to metronidazole and tinida-zolerdquo Antimicrobial Agents and Chemotherapy vol 50 no 12pp 4209ndash4210 2006

[11] R B Giordani P B Vieira MWeizenmann et al ldquoCandimine-induced cell death of the amitochondriate parasiteTrichomonasvaginalisrdquo Journal of Natural Products vol 73 no 12 pp 2019ndash2023 2010

[12] R B Giordani P B Vieira M Weizenmann et al ldquoLycorineinduces cell death in the amitochondriate parasiteTrichomonasvaginalis via an alternative non-apoptotic death pathwayrdquoPhytochemistry vol 72 no 7 pp 645ndash650 2011

[13] J L Rıos and M C Recio ldquoMedicinal plants and antimicrobialactivityrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 80ndash84 2005

[14] MHeinrich ldquoEthnobotany and natural products the search fornewmolecules new treatments of old diseases or a better under-standing of indigenous culturesrdquo Current Topics in MedicinalChemistry vol 3 no 2 pp 141ndash154 2003

[15] C L C Brandelli R B Giordani A J Macedo G A de Carliand T Tasca ldquoIndigenous traditional medicine plants for thetreatment of diarrheardquo inNature Helps How Plants and OtherOrganisms Contribute to Solve Health Problems pp 1ndash18Springer Berlin Germany 1st edition 2011

[16] E Elisabetsky and L Wannmacher ldquoThe status of ethnophar-macology in Brazilrdquo Journal of Ethnopharmacology vol 38 no2-3 pp 137ndash143 1993

[17] F L H Rego A J Brand and R B Costa ldquoGenetic resourcesbiodiversity traditional knowledge Guarani Kaiowa and localdevelopmentrdquo Interacoes vol 11 no 1 pp 55ndash69 2010

[18] D F Coutinho L M A Travassos and F M M AmaralldquoEstudo etnobotanico de plantas medicinais utilizadas emcomunidades indıgenas no estado doMaranhaomdashBrasilrdquoVisaoAcademica vol 3 no 1 pp 7ndash12 2002

[19] A H Gilani and A-U Rahman ldquoTrends in ethnopharmacol-ogyrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 43ndash492005

[20] L S Diamond ldquoThe establishment of various Trichomonas ofanimals andman in axenic culturesrdquoThe Journal of Parasitologyvol 43 no 4 pp 488ndash490 1957

[21] M Duarte R B Giordani G A de Carli J A ZuanazziA J Macedo and T Tasca ldquoA quantitative resazurin assay todeterminate the viability of Trichomonas vaginalis and the cyto-toxicity of organic solvents and surfactant agentsrdquo ExperimentalParasitology vol 123 no 2 pp 195ndash198 2009

[22] T D Rocha P B Vieira S C B Gnoatto T Tasca and G Gos-mann ldquoAnti-Trichomonas vaginalis activity of saponins fromQuillaja Passiflora and Ilex speciesrdquo Parasitology Research vol110 no 6 pp 2551ndash2556 2012

[23] Y SunH TongM Li Y Li S Guan and J Liu ldquoImmunologicaladjuvant effect of Japanese ginseng saponins (JGS) on specificantibody and cellular response to ovalbumin and its haemolyticactivitiesrdquo Vaccine vol 26 no 47 pp 5911ndash5917 2008

[24] M I Calvo ldquoAnti-inflammatory and analgesic activity of thetopical preparation of Verbena officinalis Lrdquo Journal of Ethno-pharmacology vol 107 no 3 pp 380ndash382 2006

[25] S-W Lai M-S Yu W-H Yuen and R C-C Chang ldquoNovelneuroprotective effects of the aqueous extracts from Verbenaofficinalis LinnrdquoNeuropharmacology vol 50 no 6 pp 641ndash6502006

[26] E Speroni R Cervellati S Costa et al ldquoEffects of differentialextraction of Verbena officinalis on rat models of inflammationcicatrization and gastric damagerdquo Planta Medica vol 73 no 3pp 227ndash235 2007

[27] M W Biavatti C Farias F Curtius et al ldquoPreliminary studieson Campomanesia xanthocarpa (Berg) and Cuphea carthage-nensis (Jacq) JF Macbr aqueous extract weight control andbiochemical parametersrdquo Journal of Ethnopharmacology vol93 no 2-3 pp 385ndash389 2004

[28] B E O Markman E M Bacchi and E T M Kato ldquoAntiul-cerogenic effects of Campomanesia xanthocarpardquo Journal ofEthnopharmacology vol 94 no 1 pp 55ndash57 2004

[29] F Calzada L Yepez-Mulia and A Tapia-Contreras ldquoEffect ofMexican medicinal plant used to treat trichomoniasis on Tri-chomonas vaginalis trophozoitesrdquo Journal of Ethnopharmacol-ogy vol 113 no 2 pp 248ndash251 2007

BioMed Research International 7

[30] J Desrivot J Waikedre P Cabalion et al ldquoAntiparasitic activityof some New Caledonian medicinal plantsrdquo Journal of Ethno-pharmacology vol 112 no 1 pp 7ndash12 2007

[31] A P Frasson O dos Santos M Duarte et al ldquoFirst report ofanti-Trichomonas vaginalis activity of the medicinal plant Poly-gala decumbens from the Brazilian semi-arid region CaatingardquoParasitology Research vol 110 no 6 pp 2581ndash2587 2012

[32] P B Vieira R B Giordani G A de Carli J A S Zuanazzi andT Tasca ldquoScreening and bioguided fractionation of Amarylli-daceae species with anti- Trichomonas vaginalis activityrdquo PlantaMedica vol 77 no 10 pp 1054ndash1059 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

(a) Control (b) Verbena sp

(c) Campomanesia xanthocarpaFigure 3 Microscopic analysis of parasites morphology in light microscope (magnified times1000) by trypan blue dye exclusion (a) Control(b) after 4 hours of treatment with aqueous extract of Verbena sp (c) after 4 hours of treatment with aqueous extract of Campomanesiaxanthocarpa

action responsible for the parasite viability reduction proba-bly does not involve parasitic membrane disruption More-over and importantly these findings suggest that both ex-tracts are not toxic to mammalian cells since they did notlyse human erythrocytes

This study contributes to rescue the indigenous knowl-edge about medicinal plants The indigenous Mbya-Guaraniuse Verbena sp (Guachu karsquoa in Mbya-Guarani language)as tea for relief of symptoms of infectious diseases such asfeverThis is in agreement of the literature that relatesVerbenasp infusion as sedative analgesic to inflammatory disordersskin burns amenorrhea depression and gastric diseases [24ndash26] Moreover this species has a variety of chemical con-stituents as flavonoid iridoid glycosides phenylpropanoidglycoside sterols triterpenes and glycoconjugate and thebiological activity may be partially attributed to them [24]In addition this tribe utilizes the leaves of C xanthocarpa(Guavira in Mbya-Guarani language) for treatment of diar-rhea stomach pain and worms This plant is traditionallyused in the south of Brazil as depurative antidiarrhoeiccleanser and antirheumatic and to decrease the bloodcholesterol [27] and these properties may be attributedto tannins saponins and flavonoids (quercetin myricetinquercitrin and rutin) compounds present in the leaves of Cxanthocarpa [28]

The rescue ethnopharmacology of historical uses of plantsis recognized as valuable for bioprospection since they affordthe rationale for selection and research of medicinal plants[15]This study describes the first report on the antiprotozoalactivity of Verbena sp and C xanthocarpa In addition otherspecies of the families Verbenaceae and Myrtaceae were

tested against T vaginalis however without success [29 30]Several biodiversities and traditional knowledge of differentpopulations have guided studies to search new prototypesanti-T vaginalis Recently the potential of plants traditionallyused from Caatinga against T vaginalis was reported [31]Calzada et al [29] demonstrated that plants used for urogen-ital tract disorders in Mexican traditional medicine showedeffect against this protozoan Amaryllidaceae species usedagainst venereal diseases in SouthAfrica also showed promis-ing activity against T vaginalis [11 32]

Considering the impact of trichomonosis in public healthand the emergent number of resistantT vaginalis isolates it isnecessary alternatives for the treatment of this infection Nat-ural products are a promising source of active molecules andthe ethnopharmacology approach rescues the knowledge ofpopulation of medicinal plants This wisdom combined withchemical and pharmacological studies presents a preciousvalue in the bioprospection of innovates safe and accessibledrugsThis pioneer study demonstrated relevant results aboutanti-T vaginalis activity of the Verbena sp and C xantho-carpa plants traditionally used by indigenous populationMbya-Guarani for infectious diseases Despite of showingstrong anti-T vaginalis activity this report rescued the knowl-edge of indigenous people avoiding the miscarriage of thiswisdom

Authorsrsquo Contribution

Clara Lia Costa Brandelli and Patrıcia de Brum Vieira con-tributed equally to this work

6 BioMed Research International

Acknowledgments

This work was supported by the NANOBIOTEC-Brazil pro-gram from CAPES and FAPERGS The authors thank Chief-tain Jose Cirilo Pires Morinico (tribe Mbya-Guarani) formaking possible the accomplishment of this study and for thevaluable information Alexandre Jose Macedo and TianaTasca thank CNPq for researcher fellowships

References

[1] WHO Global Incidence and Prevalence of Selected Curable Sex-ually Transmitted Infections-2008 World Health OrganizationGeneva Switzerland 2012 httpappswhointirisbitstream106657518119789241503839 engpdf

[2] D Petrin K Delgaty R Bhatt and G Garber ldquoClinicaland microbiological aspects of Trichomonas vaginalisrdquo ClinicalMicrobiology Reviews vol 11 no 2 pp 300ndash317 1998

[3] M A Klebanoff J C Carey J C Hauth et al ldquoFailure of met-ronidazole to prevent preterm delivery among pregnant womenwith asymptomatic Trichomonas vaginalis infectionrdquo The NewEngland Journal of Medicine vol 345 no 7 pp 487ndash493 2001

[4] F Grodstein M B Goldman and D W Cramer ldquoRelation oftubal infertility to history of sexually transmitted diseasesrdquoAmerican Journal of Epidemiology vol 137 no 5 pp 577ndash5841993

[5] M Viikki E Pukkala P Nieminen and M Hakama ldquoGynae-cological infections as risk determinants of subsequent cervicalneoplasiardquo Acta Oncologica vol 39 no 1 pp 71ndash75 2000

[6] T L Cherpes H C Wiesenfeld M A Melan et al ldquoTheassociations between pelvic inflammatory diseaseTrichomonasvaginalis infection and positive herpes simplex virus type 2serologyrdquo Sexually Transmitted Diseases vol 33 no 12 pp 747ndash752 2006

[7] F Sorvillo L Smith P Kerndt and L Ash ldquoTrichomonas vagi-nalis HIV and African-Americansrdquo Emerging Infectious Dis-eases vol 7 no 6 pp 927ndash932 2001

[8] B van der Pol C Kwok B Pierre-Louis et al ldquoTrichomonasvaginalis infection and human immunodeficiency virus acqui-sition in African womenrdquo Journal of Infectious Diseases vol 197no 4 pp 548ndash554 2008

[9] V Ali and T Nozaki ldquoCurrent therapeutics their problemsand sulfur-containing-amino-acid metabolism as a novel targetagainst infections by ldquoamitochondriaterdquo protozoan parasitesrdquoClinical Microbiology Reviews vol 20 no 1 pp 164ndash187 2007

[10] J R Schwebke and F J Barrientes ldquoPrevalence of Trichomonasvaginalis isolates with resistance to metronidazole and tinida-zolerdquo Antimicrobial Agents and Chemotherapy vol 50 no 12pp 4209ndash4210 2006

[11] R B Giordani P B Vieira MWeizenmann et al ldquoCandimine-induced cell death of the amitochondriate parasiteTrichomonasvaginalisrdquo Journal of Natural Products vol 73 no 12 pp 2019ndash2023 2010

[12] R B Giordani P B Vieira M Weizenmann et al ldquoLycorineinduces cell death in the amitochondriate parasiteTrichomonasvaginalis via an alternative non-apoptotic death pathwayrdquoPhytochemistry vol 72 no 7 pp 645ndash650 2011

[13] J L Rıos and M C Recio ldquoMedicinal plants and antimicrobialactivityrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 80ndash84 2005

[14] MHeinrich ldquoEthnobotany and natural products the search fornewmolecules new treatments of old diseases or a better under-standing of indigenous culturesrdquo Current Topics in MedicinalChemistry vol 3 no 2 pp 141ndash154 2003

[15] C L C Brandelli R B Giordani A J Macedo G A de Carliand T Tasca ldquoIndigenous traditional medicine plants for thetreatment of diarrheardquo inNature Helps How Plants and OtherOrganisms Contribute to Solve Health Problems pp 1ndash18Springer Berlin Germany 1st edition 2011

[16] E Elisabetsky and L Wannmacher ldquoThe status of ethnophar-macology in Brazilrdquo Journal of Ethnopharmacology vol 38 no2-3 pp 137ndash143 1993

[17] F L H Rego A J Brand and R B Costa ldquoGenetic resourcesbiodiversity traditional knowledge Guarani Kaiowa and localdevelopmentrdquo Interacoes vol 11 no 1 pp 55ndash69 2010

[18] D F Coutinho L M A Travassos and F M M AmaralldquoEstudo etnobotanico de plantas medicinais utilizadas emcomunidades indıgenas no estado doMaranhaomdashBrasilrdquoVisaoAcademica vol 3 no 1 pp 7ndash12 2002

[19] A H Gilani and A-U Rahman ldquoTrends in ethnopharmacol-ogyrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 43ndash492005

[20] L S Diamond ldquoThe establishment of various Trichomonas ofanimals andman in axenic culturesrdquoThe Journal of Parasitologyvol 43 no 4 pp 488ndash490 1957

[21] M Duarte R B Giordani G A de Carli J A ZuanazziA J Macedo and T Tasca ldquoA quantitative resazurin assay todeterminate the viability of Trichomonas vaginalis and the cyto-toxicity of organic solvents and surfactant agentsrdquo ExperimentalParasitology vol 123 no 2 pp 195ndash198 2009

[22] T D Rocha P B Vieira S C B Gnoatto T Tasca and G Gos-mann ldquoAnti-Trichomonas vaginalis activity of saponins fromQuillaja Passiflora and Ilex speciesrdquo Parasitology Research vol110 no 6 pp 2551ndash2556 2012

[23] Y SunH TongM Li Y Li S Guan and J Liu ldquoImmunologicaladjuvant effect of Japanese ginseng saponins (JGS) on specificantibody and cellular response to ovalbumin and its haemolyticactivitiesrdquo Vaccine vol 26 no 47 pp 5911ndash5917 2008

[24] M I Calvo ldquoAnti-inflammatory and analgesic activity of thetopical preparation of Verbena officinalis Lrdquo Journal of Ethno-pharmacology vol 107 no 3 pp 380ndash382 2006

[25] S-W Lai M-S Yu W-H Yuen and R C-C Chang ldquoNovelneuroprotective effects of the aqueous extracts from Verbenaofficinalis LinnrdquoNeuropharmacology vol 50 no 6 pp 641ndash6502006

[26] E Speroni R Cervellati S Costa et al ldquoEffects of differentialextraction of Verbena officinalis on rat models of inflammationcicatrization and gastric damagerdquo Planta Medica vol 73 no 3pp 227ndash235 2007

[27] M W Biavatti C Farias F Curtius et al ldquoPreliminary studieson Campomanesia xanthocarpa (Berg) and Cuphea carthage-nensis (Jacq) JF Macbr aqueous extract weight control andbiochemical parametersrdquo Journal of Ethnopharmacology vol93 no 2-3 pp 385ndash389 2004

[28] B E O Markman E M Bacchi and E T M Kato ldquoAntiul-cerogenic effects of Campomanesia xanthocarpardquo Journal ofEthnopharmacology vol 94 no 1 pp 55ndash57 2004

[29] F Calzada L Yepez-Mulia and A Tapia-Contreras ldquoEffect ofMexican medicinal plant used to treat trichomoniasis on Tri-chomonas vaginalis trophozoitesrdquo Journal of Ethnopharmacol-ogy vol 113 no 2 pp 248ndash251 2007

BioMed Research International 7

[30] J Desrivot J Waikedre P Cabalion et al ldquoAntiparasitic activityof some New Caledonian medicinal plantsrdquo Journal of Ethno-pharmacology vol 112 no 1 pp 7ndash12 2007

[31] A P Frasson O dos Santos M Duarte et al ldquoFirst report ofanti-Trichomonas vaginalis activity of the medicinal plant Poly-gala decumbens from the Brazilian semi-arid region CaatingardquoParasitology Research vol 110 no 6 pp 2581ndash2587 2012

[32] P B Vieira R B Giordani G A de Carli J A S Zuanazzi andT Tasca ldquoScreening and bioguided fractionation of Amarylli-daceae species with anti- Trichomonas vaginalis activityrdquo PlantaMedica vol 77 no 10 pp 1054ndash1059 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

Acknowledgments

This work was supported by the NANOBIOTEC-Brazil pro-gram from CAPES and FAPERGS The authors thank Chief-tain Jose Cirilo Pires Morinico (tribe Mbya-Guarani) formaking possible the accomplishment of this study and for thevaluable information Alexandre Jose Macedo and TianaTasca thank CNPq for researcher fellowships

References

[1] WHO Global Incidence and Prevalence of Selected Curable Sex-ually Transmitted Infections-2008 World Health OrganizationGeneva Switzerland 2012 httpappswhointirisbitstream106657518119789241503839 engpdf

[2] D Petrin K Delgaty R Bhatt and G Garber ldquoClinicaland microbiological aspects of Trichomonas vaginalisrdquo ClinicalMicrobiology Reviews vol 11 no 2 pp 300ndash317 1998

[3] M A Klebanoff J C Carey J C Hauth et al ldquoFailure of met-ronidazole to prevent preterm delivery among pregnant womenwith asymptomatic Trichomonas vaginalis infectionrdquo The NewEngland Journal of Medicine vol 345 no 7 pp 487ndash493 2001

[4] F Grodstein M B Goldman and D W Cramer ldquoRelation oftubal infertility to history of sexually transmitted diseasesrdquoAmerican Journal of Epidemiology vol 137 no 5 pp 577ndash5841993

[5] M Viikki E Pukkala P Nieminen and M Hakama ldquoGynae-cological infections as risk determinants of subsequent cervicalneoplasiardquo Acta Oncologica vol 39 no 1 pp 71ndash75 2000

[6] T L Cherpes H C Wiesenfeld M A Melan et al ldquoTheassociations between pelvic inflammatory diseaseTrichomonasvaginalis infection and positive herpes simplex virus type 2serologyrdquo Sexually Transmitted Diseases vol 33 no 12 pp 747ndash752 2006

[7] F Sorvillo L Smith P Kerndt and L Ash ldquoTrichomonas vagi-nalis HIV and African-Americansrdquo Emerging Infectious Dis-eases vol 7 no 6 pp 927ndash932 2001

[8] B van der Pol C Kwok B Pierre-Louis et al ldquoTrichomonasvaginalis infection and human immunodeficiency virus acqui-sition in African womenrdquo Journal of Infectious Diseases vol 197no 4 pp 548ndash554 2008

[9] V Ali and T Nozaki ldquoCurrent therapeutics their problemsand sulfur-containing-amino-acid metabolism as a novel targetagainst infections by ldquoamitochondriaterdquo protozoan parasitesrdquoClinical Microbiology Reviews vol 20 no 1 pp 164ndash187 2007

[10] J R Schwebke and F J Barrientes ldquoPrevalence of Trichomonasvaginalis isolates with resistance to metronidazole and tinida-zolerdquo Antimicrobial Agents and Chemotherapy vol 50 no 12pp 4209ndash4210 2006

[11] R B Giordani P B Vieira MWeizenmann et al ldquoCandimine-induced cell death of the amitochondriate parasiteTrichomonasvaginalisrdquo Journal of Natural Products vol 73 no 12 pp 2019ndash2023 2010

[12] R B Giordani P B Vieira M Weizenmann et al ldquoLycorineinduces cell death in the amitochondriate parasiteTrichomonasvaginalis via an alternative non-apoptotic death pathwayrdquoPhytochemistry vol 72 no 7 pp 645ndash650 2011

[13] J L Rıos and M C Recio ldquoMedicinal plants and antimicrobialactivityrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 80ndash84 2005

[14] MHeinrich ldquoEthnobotany and natural products the search fornewmolecules new treatments of old diseases or a better under-standing of indigenous culturesrdquo Current Topics in MedicinalChemistry vol 3 no 2 pp 141ndash154 2003

[15] C L C Brandelli R B Giordani A J Macedo G A de Carliand T Tasca ldquoIndigenous traditional medicine plants for thetreatment of diarrheardquo inNature Helps How Plants and OtherOrganisms Contribute to Solve Health Problems pp 1ndash18Springer Berlin Germany 1st edition 2011

[16] E Elisabetsky and L Wannmacher ldquoThe status of ethnophar-macology in Brazilrdquo Journal of Ethnopharmacology vol 38 no2-3 pp 137ndash143 1993

[17] F L H Rego A J Brand and R B Costa ldquoGenetic resourcesbiodiversity traditional knowledge Guarani Kaiowa and localdevelopmentrdquo Interacoes vol 11 no 1 pp 55ndash69 2010

[18] D F Coutinho L M A Travassos and F M M AmaralldquoEstudo etnobotanico de plantas medicinais utilizadas emcomunidades indıgenas no estado doMaranhaomdashBrasilrdquoVisaoAcademica vol 3 no 1 pp 7ndash12 2002

[19] A H Gilani and A-U Rahman ldquoTrends in ethnopharmacol-ogyrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 43ndash492005

[20] L S Diamond ldquoThe establishment of various Trichomonas ofanimals andman in axenic culturesrdquoThe Journal of Parasitologyvol 43 no 4 pp 488ndash490 1957

[21] M Duarte R B Giordani G A de Carli J A ZuanazziA J Macedo and T Tasca ldquoA quantitative resazurin assay todeterminate the viability of Trichomonas vaginalis and the cyto-toxicity of organic solvents and surfactant agentsrdquo ExperimentalParasitology vol 123 no 2 pp 195ndash198 2009

[22] T D Rocha P B Vieira S C B Gnoatto T Tasca and G Gos-mann ldquoAnti-Trichomonas vaginalis activity of saponins fromQuillaja Passiflora and Ilex speciesrdquo Parasitology Research vol110 no 6 pp 2551ndash2556 2012

[23] Y SunH TongM Li Y Li S Guan and J Liu ldquoImmunologicaladjuvant effect of Japanese ginseng saponins (JGS) on specificantibody and cellular response to ovalbumin and its haemolyticactivitiesrdquo Vaccine vol 26 no 47 pp 5911ndash5917 2008

[24] M I Calvo ldquoAnti-inflammatory and analgesic activity of thetopical preparation of Verbena officinalis Lrdquo Journal of Ethno-pharmacology vol 107 no 3 pp 380ndash382 2006

[25] S-W Lai M-S Yu W-H Yuen and R C-C Chang ldquoNovelneuroprotective effects of the aqueous extracts from Verbenaofficinalis LinnrdquoNeuropharmacology vol 50 no 6 pp 641ndash6502006

[26] E Speroni R Cervellati S Costa et al ldquoEffects of differentialextraction of Verbena officinalis on rat models of inflammationcicatrization and gastric damagerdquo Planta Medica vol 73 no 3pp 227ndash235 2007

[27] M W Biavatti C Farias F Curtius et al ldquoPreliminary studieson Campomanesia xanthocarpa (Berg) and Cuphea carthage-nensis (Jacq) JF Macbr aqueous extract weight control andbiochemical parametersrdquo Journal of Ethnopharmacology vol93 no 2-3 pp 385ndash389 2004

[28] B E O Markman E M Bacchi and E T M Kato ldquoAntiul-cerogenic effects of Campomanesia xanthocarpardquo Journal ofEthnopharmacology vol 94 no 1 pp 55ndash57 2004

[29] F Calzada L Yepez-Mulia and A Tapia-Contreras ldquoEffect ofMexican medicinal plant used to treat trichomoniasis on Tri-chomonas vaginalis trophozoitesrdquo Journal of Ethnopharmacol-ogy vol 113 no 2 pp 248ndash251 2007

BioMed Research International 7

[30] J Desrivot J Waikedre P Cabalion et al ldquoAntiparasitic activityof some New Caledonian medicinal plantsrdquo Journal of Ethno-pharmacology vol 112 no 1 pp 7ndash12 2007

[31] A P Frasson O dos Santos M Duarte et al ldquoFirst report ofanti-Trichomonas vaginalis activity of the medicinal plant Poly-gala decumbens from the Brazilian semi-arid region CaatingardquoParasitology Research vol 110 no 6 pp 2581ndash2587 2012

[32] P B Vieira R B Giordani G A de Carli J A S Zuanazzi andT Tasca ldquoScreening and bioguided fractionation of Amarylli-daceae species with anti- Trichomonas vaginalis activityrdquo PlantaMedica vol 77 no 10 pp 1054ndash1059 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

[30] J Desrivot J Waikedre P Cabalion et al ldquoAntiparasitic activityof some New Caledonian medicinal plantsrdquo Journal of Ethno-pharmacology vol 112 no 1 pp 7ndash12 2007

[31] A P Frasson O dos Santos M Duarte et al ldquoFirst report ofanti-Trichomonas vaginalis activity of the medicinal plant Poly-gala decumbens from the Brazilian semi-arid region CaatingardquoParasitology Research vol 110 no 6 pp 2581ndash2587 2012

[32] P B Vieira R B Giordani G A de Carli J A S Zuanazzi andT Tasca ldquoScreening and bioguided fractionation of Amarylli-daceae species with anti- Trichomonas vaginalis activityrdquo PlantaMedica vol 77 no 10 pp 1054ndash1059 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology