in vivo seeds against amastigote stage of...

Research ArticleIn Vivo Antiprotozoal Activity of the Chloroform Extract fromCarica papaya Seeds against Amastigote Stage of Trypanosomacruzi during Indeterminate and Chronic Phase of Infection

Matilde Jimenez-Coello1 Karla Y Acosta-Viana1 Antonio Ortega-Pacheco2

Salud Perez-Gutierrez3 and Eugenia Guzman-Marin1

1 Laboratorio de Biologıa Celular CIR ldquoDr Hideyo Noguchirdquo CA Biomedicina de Enfermedades Infecciosas y ParasitariasUniversidad Autonoma de Yucatan Avenida Itzaes No 490 x 59 Centro 97000 Merida YUC Mexico

2Departamento de Salud Animal y Medicina Preventiva CA Salud Animal Facultad de Medicina Veterinaria y ZootecniaUniversidad Autonoma de Yucatan Carretera Merida-Xmatkuil Km 155 Carr Merida-Xmatkuil AP 4-116 Merida YUC Mexico

3 Universidad Autonoma Metropolitana-Xochimilco Calzada del Hueso No 1100 AP 23-181 04960 Mexico DF Mexico

Correspondence should be addressed to Eugenia Guzman-Marin gmarinuadymx

Received 11 June 2014 Accepted 27 July 2014 Published 8 September 2014

Academic Editor Rainer W Bussmann

Copyright copy 2014 Matilde Jimenez-Coello et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

In order to evaluate the antiprotozoal activity of the chloroform extract of Carica papaya seeds during the subacute and chronicphase of infection of Trypanosoma cruzi doses of 50 and 75mgkg were evaluated during the subacute phase including a mixtureof their main components (oleic palmitic and stearic acids) Subsequently doses of 50 and 75mgkg in mice during the chronicphase of infection (100 dpi) were also evaluated It was found that chloroform extract was able to reduce the amastigote nestsnumbers during the subacute phase in 555 and 697 (P gt 005) as well as in 5645 in animals treated with the mixture offatty acids Moreover the experimental groups treated with 50 and 75mgkg during the chronic phase of the infection showed asignificant reduction of 468 and 5313 respectively (P lt 005) It is recommended to carry out more studies to determine if higherdoses of chloroformic extract or its administration in combination with other antichagasic drugs allows a better response over theintracellular stage of T cruzi in infected animal models and determine if the chloroform extract of C papaya could be consideredas an alternative for treatment during the indeterminate and chronic phase of the infection

1 Introduction

American trypanosomiasis (AT) also known as Chagasrsquodisease is a neglected infectious disease caused by thehemoflagellate protozoa Trypanosoma cruzi (T cruzi) Thepresence of the parasite is widely distributed in the Americancontinent from the south of the United States of NorthAmerica to Argentina AT is endemic throughout muchof Mexico Central America and South America where anestimated 8 million people are infected [1]

Currently the treatment protocols of Chagasrsquo disease arebased on the use of two drugs nifurtimox and benznidazoleBoth drugs are considered inefficient not only because of thenarrow therapeutic range but also because of the associatedtoxicity Natural products are considered an important source

of biologically active compounds against various infectiousorganisms [2]

The Carica papaya (Linn) (C papaya) commonly knownas papaya is a fruit crop cultivated in tropical and subtropicalregions and well-known for its nutritional benefits andmedicinal applications [3] It is widely distributed in the southof Mexico including the Caribbean and grows at an altituderange of 10 to 1600m above sea level The antimicrobialantifungal larvicidal [4] insecticidal [5] and antiprotozoalproperties of C papaya against Trichomona vaginalis [6] andT cruzi [7] have been previously reported

Particularly the antiprotozoal activity of the crude chlo-roformic seed extract ofC papayawas observed from in vitroas in vivo studies during the acute phase of the infection [7]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 458263 7 pageshttpdxdoiorg1011552014458263

2 Evidence-Based Complementary and Alternative Medicine

The chemical composition of chloroform seed extracts of Cpapaya has been previously reported by GC-MS with oleic(4597) palmitic (241) and stearic (852) acids beingthe more abundant components [5] The chloroform extractofC papayahas no demonstrated toxicitywhen administeredto different animal species (mice rabbits dogs andmonkeys)for periods of 30 60 120 or 300 days It has been reported thatthe oral route administration of this extract does not inducesignificant changes in hematological values of treated animalsshowing any signs of toxicity caused by the administration ofthe extract [8ndash12]

In the present study the in vivo antiprotozoal activityof crude C papaya seeds (extract and a mixture of maincomponents) against T cruzi intracellular replicative stage(amastigote) was evaluated during the subacute and chronicphases of the disease

2 Materials and Methods

21 Plant Material and Crude Extract Elaboration Caricapapaya seeds from ripe fruits were collected from EscarcegaCampeche State Mexico through July to September of 2011The plant was authenticated by Dr Salvador Flores-Guidoand a voucher (10284) was deposited at the herbarium ofUniversidad Autonoma de Yucatan (UADY) Seeds weredried at room temperature under shadow conditions Freshseeds from ripe fruits were shade-dried for 15 days and latercoarsely powdered In a 1 L bottom flask fitted with a refluxcondenser 100 g of dry ground papaya seed and 500mL ofchloroform were heated for 4 h cooled to room temperatureand filtered The solvent was dried under a vacuum in arotary evaporator and then dried in a vacuum oven at roomtemperature for 12 h [5]

22 Parasites Trypomastigotes of T cruzi strain H4 wereused for intraperitoneal (IP) inoculation Parasites for inoc-ulation were obtained from the blood of previously infectedmice from where the strain is maintainedThe selected strain(H4) was isolated in the Yucatan Peninsula area from ahuman clinical case and it has been described as a highlyvirulent strain [13] capable of producing a mortality rate of50 inmice after 30 days of inoculation Also the amastigotesof H4 strain have shown tropism to cardiac tissue and in aminor proportion of parasites invade skeletal muscle [14]

23 Animals In order to evaluate the antiprotozoal activityof the chloroform extract and their main components duringthe subacute phase of AT a total of 40 female BALBc micewere infected with an IP inoculation of 5 times 102 trypomastig-otes (in 200120583L of saline solution) Also to evaluate theantiprotozoal activity of the extract during the chronic phaseof theAT another 32 femalemicewere inoculatedwith 1times102trypomastigotesMice included in the assayswere 8weeks oldat day of inoculation

The case definition for a subacute phase of Chagas diseasewas mice negative to T cruzi at a parasitological test (Stroutmethod or thick smear) inoculated at least 45 days beforethe evaluation and with the presence of positive IgG The

definition for chronic phase was mice with a negative T cruziparasitological test (Stroutmethod or thick smear) inoculatedat least 100 days before the evaluation and with the presenceof positive IgG

For the subacute phase evaluation mice after 45-daypostinfection (dpi) began to receive the extract during 15 daysevery 24 hours The chloroform extract of C papaya wasadministrated per os (PO) at the doses of 50 and 75mgKg(119899 = 8 in each group) Before administration the extract wasmixed with phosphate buffered saline (PBS 137mM NaCl27mM KCl 43mM Na

2HPO4 and 14mM KH

2PO4 pH

74)One group was treated with a mixture of the main fatty

acids present in the extract (palmitic oleic and stearic acids)at a dose of 100mgKg (119899 = 8) The proportion of eachcompound was estimated as a function of the percentage thateach one of them is usually found in the crude chloroformextracts of C papaya [5] The main compounds from thechloroform extract of C papaya tested in this research wereobtained froma commercial presentation of every one (SigmaAldrich codes oleic acid O1008 palmitic acid P0500 andstearic acid 605581)

For the chronic phase bioassay mice after 100 dpi beganto receive the chloroform crude extract (50 and 75mgkg)during 15 days PO every 24 hours (119899 = 8 in each experimentalcondition)

For each bioassay two control groupswere considered Asa negative control (119899 = 8) a group of infected mice receivedonly 50 120583L orally out of the vehicle (PBS) whilst a positivecontrol group including infected mice (119899 = 8) was treatedorally with allopurinol (Sigma Aldrich A8003) (85mgkg)diluted in 50 120583L of PBS every day during 15 days

24 Evaluation of the In Vivo Antiprotozoal Activity against Tcruzi during the Subacute and Chronic Phase of the DiseaseTo determine the antiprotozoal activity against the intra-cellular amastigote form of T cruzi cardiac tissue samplesfrom treated and untreated mice were collected and fixedin formaldehyde (10) Afterwards the paraffin-embeddedtissue sectionswere stainedwith hematoxylin-eosin (HE) andexamined under a light microscope Four nonconsecutiveslides from the heart of each mouse were also examined ina blinded mode

The number of amastigote nests was quantified in 100zones for each heart All procedures were conducted inaccordance with the internationally accepted principles forlaboratory animal use and care [15]

In animals treated during subacute phase the para-site burden was evaluated by absolute quantification withQuantitative Polymerase Chain Reaction (qPCR) Tissues(25mg) were subjected to proteinase K lysis and totalDNA was isolated by chromatography columns using thecommercial kit DNeasy Blood and Tissue (QIAGEN cat no69 506) Total DNA (100 ng) was used as the template ina real-time PCR (as described above) with oligonucleotidesspecific for a sequence encoding for satDNA of T cruzi(TCZF 51015840-GCTCTTGCCCACAMGGGTGC-31015840 TCZR 51015840-CCAAGCAGCGGATAGTTCAGG-31015840) [16]

Evidence-Based Complementary and Alternative Medicine 3

The amplification was conducted under the followingcycling conditions after 15min of denaturation at 95∘C PCRamplification was carried out for 50 cycles (95∘C for 10 s55∘C for 15 s and 72∘C for 10 s) Fluorescence data collectionwas performed at 72∘C at the end of each cycle Afterquantification a melt curve was made with 74ndash85∘C raisingby 05∘C each step and waiting for 4 seconds afterwardsacquiring on green channel Melting temperature (119879

119898) of the

amplicon was 81∘C Each 96-well reaction plate contained thestandard curve and two negative controls Negative controlsconsisted of a reaction with T cruzi-specific primers withoutDNA or DNA extracted from the cardiac tissue of healthymice Each DNA sample was carried out in triplicate andT cruzi load was estimated by the absolute quantificationmethod CT values for the T cruzi-specific signal werenormalized to GAPDH gene DNA levels

25 Statistical Analyses Data are expressed as means plusmnstandard deviations (SD) and were derived from at leasttriplicate observations per sample (eight animals per group)Results were analyzed for significant differences by usinganalysis-of-variance procedures followed by Tukeyrsquos multiplecomparison tests The level of significance was accepted as119875 lt 005

3 Results and Discussion

The evaluation of the extract during the subacute phase withdoses of 50 and 75mgkg and using 100mgkg of a mixtureof the major fatty acid components (stearic palmitic andoleic acids) showed a good antiprotozoal activity againstamastigote forms of T cruzi

A reduced number of amastigote nests were observed inthe cardiac tissue of infected mice during the subacute phaseof the disease with the doses of extract evaluated (50 and75mgkg) showing 555 and 697 respectively (119875 gt 005)comparedwith no treated group (negative control) (Figure 1)The mixture of the major fatty acid components exhibited anantiprotozoal activity of 5645 similar to the crude extractevaluated doses and the mice group treated with allopurinol(positive control)

In agreement with the results recorded with histologicaltechniques when the parasite burden was measured byqPCR a similar antiprotozoal effect in treated animals wasobserved where a reduction of the parasite burdenwas 84428286 and 6864 (in comparison with the negative controlgroup) for doses 50 and 75mgkg of crude chloroform extractand 100mgkg of the compound mixture respectively (119875 gt005) However a total elimination of the DNA of the parasitewas not observed in any of the doses tested (Figure 2)

During the examination of histological sections fromthe heart a significant degeneration with severe diffusecoagulated necrosis in the cardiomyocyte cells was observedas well in the negative as in positive control group (Figures3(a) and 3(b)) In the tissue of mice treated with 50mg kgdose of crude extract ofC papaya a degeneration with severediffuse coagulated necrosis with a lower histiocytic infiltratewas recorded (Figure 3(c)) Mice treated with a 75mgkg

0 5 10 15 20 25 30 35 40

Number of amastigote nests in 100 fields

Negative control(PBS)

Positive controlallopurinol(85mgkg)

C papaya extract(50mgkg)


Compound mixture(100mgkg) 1187

825

1212

1275

2725

Figure 1 Effect of chloroform extract of seeds from C papaya onthe number of amastigote nests observed in cardiac tissue frommice BALBc infected with trypomastigotes of T cruzi and treatedwith 45 dpi at doses 50 and 75mgkg of chloroform crude extractand 100mgkg of the mixture of the fatty acids previously identifiedin the crude extract (119899 = 8 in each evaluated group) PBS andallopurinol (85mgKg) were used as negative and positive controlrespectively (119899 = 8 in each control group)

0

10

20

30

40

50

60

70

80

301

387 4698 51679443Tc

satD

NA

Neg

ativ

e con

trol (

PBS)

Parasite load

Posit

ive c

ontro

l (al

lopu

rinol

85

mg

kg)

C p

apay

ase

ed ex

trac

t(50

mg

kg)

C p

apay

ase

ed ex

trac

t(75

mg

kg)

Com

poun

d m

ixtu

re(100

mg

kg)

(eminus10)

Figure 2 Effect of chloroform extract of seeds from C papaya onthe parasite burden (measured by qPCR) inDNAcardiac tissue frommice BALBc infected with trypomastigotes of T cruzi and treatedwith 45 dpi at doses 50 and 75mgkg of chloroform crude extractand 100mgkg of themixture of the fatty acids identified in the crudeextract PBS and allopurinol (85mgKg) were used as negative andpositive control respectively (119899 = 8 in each control group)

showed similar lesions and multifocal fibrosis (Figure 3(d))Mice treated with the mixture of fatty acids showed necrosiswith severe diffuse coagulated necrosis histiocytic lym-phocytic and moderated plasmacytic multifocal infiltration(Figure 3(e))

After the analysis of the results the most promisingtreatment option was chosen for the evaluation of theantiprotozoal activity during the chronic phase This assaycould bemore complicated due to the long period (more than100 days) required to reach the chronic phase and for ethicalrecommendations and just the more efficient chloroformcrude extract doses were assayed

Results from monitoring of the doses of 50 and 75mgkgof the extract during the chronic phase confirmed the


(a) (b)

(c) (d) (e)

Figure 3 Microphotographs of cardiac tissue from BALBc (40x HE) during subacute phase of Chagasrsquo disease Untreated animals (negativecontrol) (a) treated with allopurinol (positive control 85mgkg) (b) treated with seed CE of C papaya (50mgkg) (c) treated with seedCE of C papaya (75mgkg) (d) and treated with the combination of FA (100mgkg) (CE = chloroform extract FA = fatty acids and arrowsindicate the presence of amastigote nests)

antiprotozoal activity of the evaluated extract Inmice treatedwith the extract (50 as 75mgkg) a significantly lower parasiteamount (119875 lt 005) was observed projecting an 468 and5313 reduction in amastigote nests number compared tonegative control group during chronic phase of the infectionOnly the higher evaluated dose (75mgkg) showed statisti-cally difference from positive control (119875 lt 005) (Figure 4)

The use of C papaya has been recurrent in the traditionalmedicine having a significant number of reports due to theirproperties for the treatment such as skin ulcers [17] andantiprotozoal activity against Leishmania amazonensis [18]Trichomonas vaginalis [6] and Plasmodium falciparum (Pfalciparum) [19]The antiprotozoal activity of the chloroformcrude extract of C papaya during the subacute and chronicphase of Chagasrsquo disease is demonstrated in this study inconcordance with previous reports [7]

Currently the existing treatment for Chagasrsquo disease islimited to only two drugs [2] being both toxic andwith severeside effects Natural products are an alternative source in thesearch for new drugs against T cruzi Also the few treatmentoptions tend to be subscribed during the acute phase of thedisease therefore in the indeterminate and chronic stage

of the diseases there is an urgent need for alternatives oftreatment

During the chronic phase of the disease patients havedifferent systemic pathologies predominantly at the cardio-vascular and digestive level and drugs used with adverseeffects after consumption involve more risks than a potentialbenefit Therefore it is important to search for alternativeand effective options during the chronic phase (intracellularamastigote form) and no harmful to the infected individuals

There are just few studies describing the antiprotozoalactivity of natural products against amastigote form of Tcruzi and most of those investigations have been conductedunder in vitro conditions [20ndash22] In the performed bioas-says the in vivo antiprotozoal activity of chloroform extractof C papaya seeds during the subacute and chronic phaseof Chagasrsquo disease was evaluated During these stages of thedisease it is not feasible to determine the presence of parasitesin the blood circulation (trypomastigote forms) and the onlyway to measure the antiprotozoal activity is to record theamount of the replicative intracellular form of amastigoteparticularly in the cardiac tissue because the H4 strain hadpreviously shown high tropism there


0 5 10 15 20



Positive control(allopurinol85mgkg)


C papaya extract(75mgkg) 6

68

92

128

lowast

lowastlowastlowast

Figure 4 Effect of chloroform extract of seeds from C papaya overthe number of amastigote nests observed in cardiac tissue frommiceBALBc infected with trypomastigotes of T cruzi and treated with100 dpi at doses 50 and 75mgdg of chloroform crude extract (119899 =8 in each evaluated group) PBS and allopurinol (85mgKg) wereused as negative and positive control respectively (119899 = 8 in eachcontrol group) (lowast119875 lt 005 versus negative control lowastlowast119875 lt 005 versuspositive control)

Some natural products have been described as activecompounds against the amastigote stage of T cruzi such asthe ethyl acetate extract of Piper jericoense in which one ofits fractions (F4) showed significant activity against T cruziamastigotes yielding an IC50 of 56 120583gmL and its selectivityindex was 224 times higher than that of benznidazoleThis fraction was reported as not cytotoxic mutagenicor genotoxic [20] Another compound with antiprotozoalactivity against the amastigote stage in vitro is the ter-penoid hypnophilin purified from Lentinus strigosus whichdemonstrated a significant inhibitory activity over T cruzitripanotion reductase [22]

Finally another relevant report from natural productsagainst T cruzi was the one described by Veiga-Santoset al [21] reporting piperovatine and piperlonguminineas compounds isolated from Piper ovatum which showedantiprotozoal activity against the T cruzi amastigote stageThese compounds were able to cause severe alterations inthe plasma membrane and cytoplasm of the parasite butshow lower toxicity in mammalian cells The C papayaextract has not shown toxicity against mammal cells butdue to the antiprotozoal activity shown by the evaluatedC papaya seeds crude extract it would be important todetermine the possible targets in the parasite of this extractThe identification of differential proteins expressed betweenexposed and not exposed parasites to the evaluated extractallows for identifying the possible targets or metabolic routesinvolved in the antiprotozoal activity from the evaluatedextract against T cruzi

The composition of the chloroform seed extract of Cpapaya used in this study was previously determined by GC-MS and the oleic (4597) palmitic (241) and stearic(852) acids were the main components [4] In otherresearch conducted under in vivo conditions but with ananimalmodel infected with Plasmodium spp the effect of theC18 fatty oleic elaidic and linoleic acids on malaria parasiteswas tested These fatty acids inhibited the parasitaemiain mice infected with Plasmodium vinckei petteri or with

Plasmodiumyoelii nigeriensis in a 4-day suppressive test Aftervarious experiments this group concluded that fatty acids didnot act at mitochondrial level of pyrimidine synthesis [23]

In another in vivo report the antiprotozoal activity of Cpapaya (crude aqueous extract obtained from leaves) againstPlasmodium berghei has been reported The combinationsof artesunic acid and crude aqueous extract of C papayaalso prolonged the survival time of the infected mice andincreased the recovery rate compared to artesunic acidalone [24] On the other hand Pohl et al [25] emphasizedthat fatty acids and their derivatives hold great potential asenvironmentally friendly antifungal agents or leads for novelantifungal drugs and these facts could be partially sharedwith trypanosomatidae because some similar metabolicmechanisms are also present in these parasites

For the case of C papaya crude extract it possesses aliquid consistency and it reaches a concentration around1 120583g120583L for that reason it is not feasible to evaluate higherdoses in mice models It is necessary to develop bioassaysin other animal models (ie dogs) and to evaluate theantiprotozoal activity against T cruzi at higher doses aswell as to evaluate the combination of different doses of theextract with specific antiprotozoal drugs (ie benznidazol) todeterminewhether any of these therapeutic options can resultin a better response allowing the removal of the parasite

The search for active compounds to identify new treat-ment alternatives for subacute and chronic phases of thedisease is relevant whether these new options may be usefulto be used alone or in combination with other antiprotozoaldrugs Because AT is a neglected disease and pharmaceuticalcompanies are not interested in searching for new drugsthere is a strong need to treat patients especially during thesubacute and chronic phases of the disease

Although administration of the chloroform extract ofC papaya seeds in the present study did not allow thecomplete elimination of the parasite during the subacute andchronic phases of the disease these assays demonstrate asignificantly positive effect in treated animals particularlyduring the chronic phase of the disease suggesting that betterresults could be obtained for antiprotozoal activity against theamastigote stadium when administered in combination withother antichagasic drugs

The reduction of the parasite load during these phases ofthe disease may improve the prognosis and life expectancyof infected patients For that reason AT should be treatedas a parasitic disease with the primary objective on reducingthe parasite load in order to improve the effectiveness ofthe immune response and to reduce disease progression tocardiomyopathy [26]

4 Conclusions

The antiprotozoal bioactivity of extract obtained from theseeds of C papaya has been shown to be effective againstamastigotes of T cruzi during subacute and chronic phaseof infection in an animal model However it is necessary toevaluate higher doses if the assessed extract could have a bet-ter antiprotozoal activity when administered in combination


with another drug which is currently used in the treatment ofpatients diagnosed with AT

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

Thanks are due to Dr Leonardo Guillermo-Cordero (CCBAUADY) for his support in the interpretation of histopathol-ogy findings from the tested tissues and to the stu-dents J Raymundo Llanes-Cocom Luis Angulo-Cancheand Maria Jose Valdez for their enthusiastic collabora-tion during the experimental and laboratory work Theauthors gratefully thank FOMIX-CAMPECHE-CONACYT(CAMP-2009-C01-126514) BASICA-CONACYT (BASICA-CONACYT-2009-129903) and APOYOS COMPLEMEN-TARIOS GRANTS (CONACYT-INFRA2013-1015-204848)for the financial support to the research

References

[1] Centers for disease Control Prevention CDC ldquoParasitesmdashAmerican Trypanosomiasis (also known as Chagas Disease)rdquo2013 httpwwwcdcgovparasiteschagasgen infodetailedhtml

[2] G E Garcıa Linares E L Ravaschino and J B RodriguezldquoProgresses in the field of drug design to combat tropicalprotozoan parasitic diseasesrdquoCurrent Medicinal Chemistry vol13 no 3 pp 335ndash360 2006

[3] R Ming S Hou Y Feng et al ldquoThe draft genome of thetransgenic tropical fruit tree papaya (Carica papaya Linnaeus)rdquoNature vol 452 no 7190 pp 991ndash996 2008

[4] P J Wabo N J D Ngankam B C F Bilong and M MpoameldquoA comparative study of the ovicidal and larvicidal activities ofaqueous and ethanolic extracts of pawpaw seeds Carica papaya(Caricaceae) on Heligmosomoides bakerirdquo Asian Pacific Journalof Tropical Medicine vol 4 no 6 pp 447ndash450 2011

[5] S Perez-Gutierrez M A Zavala-Sanchez M M Gonzalez-Chavez N C Cardenas-Ortega and M A Ramos-LopezldquoBioactivity of Carica papaya (Caricaceae) against Spodopterafrugiperda (Lepidoptera Noctuidae)rdquo Molecules vol 16 pp7502ndash7509 2011

[6] F Calzada L Yepez-Mulia and A Tapia-Contreras ldquoEffectof Mexican medicinal plant used to treat trichomoniasis onTrichomonas vaginalis trophozoitesrdquo Journal of Ethnopharma-cology vol 113 no 2 pp 248ndash251 2007

[7] M Jimenez-Coello E Guzman-Marın A Ortega-Pacheco SPerez-Gutierrez and K Y Acosta-Viana ldquoAssessment of theanti-protozoal activity of crude Carica papaya seed extractagainst Trypanosoma cruzirdquoMolecules vol 18 pp 12621ndash126322013

[8] R J Verma D Nambiar andN J Chinoy ldquoToxicological effectsof Carica papaya seed extract on spermatozoa of micerdquo Journalof Applied Toxicology vol 26 no 6 pp 533ndash535 2006

[9] N K Lohiya N Pathak P K Mishra and B ManivannanldquoContraceptive evaluation and toxicological study of aqueousextract of the seeds of Carica papaya in male rabbitsrdquo Journal ofEthnopharmacology vol 70 no 1 pp 17ndash27 2000

[10] N K Lohiya N Pathak P K Mishra and B ManivannanldquoReversible contraception with chloroform extract of Caricapapaya Linn seeds in male rabbitsrdquo Reproductive Toxicologyvol 13 no 1 pp 59ndash66 1999

[11] A Ortega-Pacheco M Jimenez- Coello E Gutierrez-Blancoet al ldquoEffects of chloroformic extracts from washed andunwashed papaya seeds (Carica papaya) on the sperm concen-tration of dogsrdquo Reproduction in Domestic Animals vol 45 pp1126ndash1129 2010

[12] N K Lohiya B Manivannan P K Mishra et al ldquoChloroformextract of Carica papaya seeds induces long-term reversibleazoospermia in langur monkeyrdquo Asian Journal of Andrologyvol 4 no 1 pp 17ndash26 2002

[13] E Dumonteil J Escobedo-Ortegon N Reyes-Rodriguez AArjona-Torres and M J Ramirez-Sierra ldquoImmunotherapy ofTrypanosoma cruzi infection with DNA vaccines in micerdquoInfection and Immunity vol 72 no 1 pp 46ndash53 2004

[14] M A Barrera-Perez M E Rodriguez-Felix E Guzman-Marinand J ZavalaVelazquez ldquoBiological behaviour of three strains ofTrypanosoma cruzi from Yucatan Mexicordquo Revista Biomedicavol 12 pp 224ndash230 2001

[15] NORMA Oficial Mexicana NOM-062-ZOO-1999 specificac-iones tecnicas para la produccion cuidado y uso de los anim-ales de laboratorio Tecnicas para la Produccion Cuidado y Usode los Animales de Laboratorio 2014 httpwwwordenjuri-dicogobmxPublicacionesCDs2007CDAgropecuariapdf58NOMpdf

[16] A G Schijman M Bisio L Orellana et al ldquoInternational studyto evaluate PCR methods for detection of Trypanosoma cruziDNA in blood samples from Chagas disease patientsrdquo PLoSNeglected Tropical Diseases vol 5 no 1 article e931 2011

[17] G Odonne F Berger D Stien P Grenand and G BourdyldquoTreatment of leishmaniasis in the Oyapock basin (FrenchGuiana) A KAP survey and analysis of the evolution ofphytotherapy knowledge amongst Wayapi Indiansrdquo Journal ofEthnopharmacology vol 137 no 3 pp 1228ndash1239 2011

[18] V Celine P Adriana D Eric et al ldquoMedicinal plants from theYanesha (Peru) evaluation of the leishmanicidal and antimalar-ial activity of selected extractsrdquo Journal of Ethnopharmacologyvol 123 no 3 pp 413ndash422 2009

[19] V Venkatesalu N Gopalan C R Pillai et al ldquoIn vitro anti-plasmodial activity of some traditionally used medicinal plantsagainst Plasmodium falciparumrdquo Parasitology Research vol 111no 1 pp 497ndash501 2012

[20] A L Hamedt I C Ortiz P A Garcıa-Huertas et al ldquoCytotoxicmutagenic and genotoxic evaluation of crude extracts andfractions from Piper jericoense with trypanocidal actionrdquo ActaTropica vol 131 pp 92ndash97 2014

[21] P Veiga-Santos V C Desoti N Miranda et al ldquoThe nat-ural compounds piperovatine and piperlonguminine induceautophagic cell death on Trypanosoma cruzirdquo Acta Tropica vol125 no 3 pp 349ndash356 2013

[22] E M Souza-Fagundes B B Cota L H Rosa et al ldquoIn vitroactivity of hypnophilin from Lentinus strigosus a potentialprototype for chagas disease and leishmaniasis chemotherapyrdquoBrazilian Journal of Medical and Biological Research vol 43 no11 pp 1054ndash1061 2010

[23] M Krugliak E Deharo G Shalmiev M Sauvain C Morettiand H Ginsburg ldquoAntimalarial effects of c18 fatty acids onPlasmodium falciparum in culture and on Plasmodium vinckeipetteri and Plasmodium yoelii nigeriensisin vivordquo ExperimentalParasitology vol 81 no 1 pp 97ndash105 1995


[24] L O Onaku A A Attama V C Okore A Y Tijani A ANgene and C O Esimone ldquoAntagonistic antimalarial prop-erties of pawpaw leaf aqueous extract in combination withartesunic acid in Plasmodium berghei-infected micerdquo Journal ofVector Borne Diseases vol 48 no 2 pp 96ndash100 2011

[25] H C Pohl J L F Kock and V S Thibane ldquoAntifungal freefatty acids a reviewrdquo in Science against Microbial PathogensCommunicating Current Research and Technological AdvancesA Mendez-Vilas Ed vol 3 of Microbiology Series pp 61ndash71Formatex 2011

[26] S Sosa-Estani R Viotti and E L Segura ldquoTherapy diagnosisand prognosis of chronic Chagas disease insight gained inArgentinardquo Memorias do Instituto Oswaldo Cruz vol 104 no1 pp 167ndash180 2009

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


The chemical composition of chloroform seed extracts of Cpapaya has been previously reported by GC-MS with oleic(4597) palmitic (241) and stearic (852) acids beingthe more abundant components [5] The chloroform extractofC papayahas no demonstrated toxicitywhen administeredto different animal species (mice rabbits dogs andmonkeys)for periods of 30 60 120 or 300 days It has been reported thatthe oral route administration of this extract does not inducesignificant changes in hematological values of treated animalsshowing any signs of toxicity caused by the administration ofthe extract [8ndash12]

In the present study the in vivo antiprotozoal activityof crude C papaya seeds (extract and a mixture of maincomponents) against T cruzi intracellular replicative stage(amastigote) was evaluated during the subacute and chronicphases of the disease

2 Materials and Methods

21 Plant Material and Crude Extract Elaboration Caricapapaya seeds from ripe fruits were collected from EscarcegaCampeche State Mexico through July to September of 2011The plant was authenticated by Dr Salvador Flores-Guidoand a voucher (10284) was deposited at the herbarium ofUniversidad Autonoma de Yucatan (UADY) Seeds weredried at room temperature under shadow conditions Freshseeds from ripe fruits were shade-dried for 15 days and latercoarsely powdered In a 1 L bottom flask fitted with a refluxcondenser 100 g of dry ground papaya seed and 500mL ofchloroform were heated for 4 h cooled to room temperatureand filtered The solvent was dried under a vacuum in arotary evaporator and then dried in a vacuum oven at roomtemperature for 12 h [5]

22 Parasites Trypomastigotes of T cruzi strain H4 wereused for intraperitoneal (IP) inoculation Parasites for inoc-ulation were obtained from the blood of previously infectedmice from where the strain is maintainedThe selected strain(H4) was isolated in the Yucatan Peninsula area from ahuman clinical case and it has been described as a highlyvirulent strain [13] capable of producing a mortality rate of50 inmice after 30 days of inoculation Also the amastigotesof H4 strain have shown tropism to cardiac tissue and in aminor proportion of parasites invade skeletal muscle [14]

23 Animals In order to evaluate the antiprotozoal activityof the chloroform extract and their main components duringthe subacute phase of AT a total of 40 female BALBc micewere infected with an IP inoculation of 5 times 102 trypomastig-otes (in 200120583L of saline solution) Also to evaluate theantiprotozoal activity of the extract during the chronic phaseof theAT another 32 femalemicewere inoculatedwith 1times102trypomastigotesMice included in the assayswere 8weeks oldat day of inoculation

The case definition for a subacute phase of Chagas diseasewas mice negative to T cruzi at a parasitological test (Stroutmethod or thick smear) inoculated at least 45 days beforethe evaluation and with the presence of positive IgG The

definition for chronic phase was mice with a negative T cruziparasitological test (Stroutmethod or thick smear) inoculatedat least 100 days before the evaluation and with the presenceof positive IgG

For the subacute phase evaluation mice after 45-daypostinfection (dpi) began to receive the extract during 15 daysevery 24 hours The chloroform extract of C papaya wasadministrated per os (PO) at the doses of 50 and 75mgKg(119899 = 8 in each group) Before administration the extract wasmixed with phosphate buffered saline (PBS 137mM NaCl27mM KCl 43mM Na

2HPO4 and 14mM KH

2PO4 pH

74)One group was treated with a mixture of the main fatty

acids present in the extract (palmitic oleic and stearic acids)at a dose of 100mgKg (119899 = 8) The proportion of eachcompound was estimated as a function of the percentage thateach one of them is usually found in the crude chloroformextracts of C papaya [5] The main compounds from thechloroform extract of C papaya tested in this research wereobtained froma commercial presentation of every one (SigmaAldrich codes oleic acid O1008 palmitic acid P0500 andstearic acid 605581)

For the chronic phase bioassay mice after 100 dpi beganto receive the chloroform crude extract (50 and 75mgkg)during 15 days PO every 24 hours (119899 = 8 in each experimentalcondition)

For each bioassay two control groupswere considered Asa negative control (119899 = 8) a group of infected mice receivedonly 50 120583L orally out of the vehicle (PBS) whilst a positivecontrol group including infected mice (119899 = 8) was treatedorally with allopurinol (Sigma Aldrich A8003) (85mgkg)diluted in 50 120583L of PBS every day during 15 days

24 Evaluation of the In Vivo Antiprotozoal Activity against Tcruzi during the Subacute and Chronic Phase of the DiseaseTo determine the antiprotozoal activity against the intra-cellular amastigote form of T cruzi cardiac tissue samplesfrom treated and untreated mice were collected and fixedin formaldehyde (10) Afterwards the paraffin-embeddedtissue sectionswere stainedwith hematoxylin-eosin (HE) andexamined under a light microscope Four nonconsecutiveslides from the heart of each mouse were also examined ina blinded mode

The number of amastigote nests was quantified in 100zones for each heart All procedures were conducted inaccordance with the internationally accepted principles forlaboratory animal use and care [15]

In animals treated during subacute phase the para-site burden was evaluated by absolute quantification withQuantitative Polymerase Chain Reaction (qPCR) Tissues(25mg) were subjected to proteinase K lysis and totalDNA was isolated by chromatography columns using thecommercial kit DNeasy Blood and Tissue (QIAGEN cat no69 506) Total DNA (100 ng) was used as the template ina real-time PCR (as described above) with oligonucleotidesspecific for a sequence encoding for satDNA of T cruzi(TCZF 51015840-GCTCTTGCCCACAMGGGTGC-31015840 TCZR 51015840-CCAAGCAGCGGATAGTTCAGG-31015840) [16]



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in vivo seeds against amastigote stage of...

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