Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 279726 10 pageshttpdxdoiorg1011552013279726

Research ArticleEugenia uniflora L Essential Oil as a PotentialAnti-Leishmania Agent Effects on Leishmania amazonensisand Possible Mechanisms of Action

Klinger Antonio da Franca Rodrigues1 Layane Valeacuteria Amorim1

Jamylla Mirck Guerra de Oliveira1 Clarice Noleto Dias2 Denise Fernandes CoutinhoMoraes2 Eloisa Helena de Aguiar Andrade3 Jose Guilherme Soares Maia3

Sabrina Maria Portela Carneiro1 and Fernando Aeacutecio de Amorim Carvalho1

1 Medicinal Plants Research Center Federal University of Piauı 64049-550 Teresina PI Brazil2 Laboratory of Pharmacognosy II Department of Pharmacy Federal University of Maranhao65085-580 Sao Luıs MA Brazil

3 Postgraduate Program in Chemistry Federal University of Para 66075-900 Belem PA Brazil

Correspondence should be addressed to Klinger Antonio da Franca Rodrigues klingerantoniogmailcom

Received 13 October 2012 Revised 14 December 2012 Accepted 17 January 2013

Academic Editor Liliana V Muschietti

Copyright copy 2013 Klinger Antonio da Franca Rodrigues et al This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Eugenia uniflora L is amember of theMyrtaceae family and is commonly known as Brazilian cherry tree In this study we evaluatedthe chemical composition of Eugenia uniflora L essential oil (EuEO) by using gas chromatography-mass spectrometry (GC-MS)and assessed its anti-Leishmania activityWe also explored the potential mechanisms of action and cytotoxicity of EuEOThirty-twocompounds were identified which constituted 9265 of the total oil compositionThemost abundant components were sesquiter-penes (9192) with curzerene (473) 120574-elemene (1425) and trans-120573-elemenone (104) being the major constituents Thebioactivity shown by EuEO against promastigotes (IC

50 304 120583gsdotmLminus1) and amastigotes (IC

50 192 120583gsdotmLminus1) suggested significant

anti-Leishmania activity In the cytotoxicity determination EuEO was 20 times more toxic to amastigotes than to macrophagesHemolytic activity was 6322 at the highest concentration tested (400120583gsdotmLminus1) however there appeared to be no toxicity at50 120583gsdotmLminus1 While the data show that EuEO activity is not mediated by nitric oxide production they do suggest that macrophageactivationmay be involved in EuEO anti-Leishmania activity as evidenced by increases in both the phagocytic capacity and the lyso-somal activityMore studies are needed to determine in vivo activity aswell as additionalmechanisms of the anti-Leishmania activity

1 Introduction

Protozoan parasites of the genus Leishmania are responsiblefor a spectrum of diseases collectively known as leishmania-sis which affects the skin mucous membranes and internalorgans Over 12 million cases of leishmaniasis have beenreported worldwide with 1 to 2 million new cases beingreported annually [1 2] Increase in the incidence of leishma-niasis is associated with urban development deforestationenvironmental changes and increased migration to areaswhere the disease is endemic [3] Despite its epidemiological

importance treatment is still performed with chemothera-peutic drugs that are delivered parenterally require medicalsupervision and have many side effects [4 5]

The need to identify new anti-Leishmania compoundsthat are more effective and less toxic than conventional drugshas motivated research of substances derived from plantspecies In this context essential oils containing a group ofsecondary metabolites consisting mainly of monoterpenessesquiterpenes and phenylpropanoids have demonstratedproven anti-Leishmania activity in vivo and in vitro on pro-mastigote andor amastigote forms of Leishmania Included

2 Evidence-Based Complementary and Alternative Medicine

among these are Croton cajucara [6] Ocimum gratissimum[7] Copaifera cearensis [8] Chenopodium ambrosioides [9]Cymbopogon citratus [10 11] and Lippia sidoides [12] Thesestudies have shown that essential oils can be a promisingsource of new drugs with anti-Leishmania activity

Eugenia uniflora L commonly known as ldquopitangueirardquoor Brazilian cherry tree is a species belonging to the familyMyrtaceae which is native to South America and commonin regions with tropical and subtropical climate [13] InBrazil it is used in the treatment of digestive disorders [14]and is thought to have anti-inflammatory and antirheumaticactivities [15] It is an aromatic species and its essential oilhas pharmacological properties that are well characterizedin the literature as antioxidant and antimicrobial [16] Euniflora has known antihypertensive [17] antitumor [18]and antinociceptive properties [19] and it shows good per-formance against microorganisms demonstrating antiviralantifungal [20] anti-Trichomonas gallinae [21] and anti-Trypanosoma cruzi properties [22]

Considering the potential pharmacological benefits ofE uniflora and the increasing interest in the discovery ofessential oils with anti-Leishmania activity the aim of thisstudy was to investigate the anti-Leishmania activity of leafessential oil from this species the chemical composition ofthe oil its cytotoxicity and possible mechanisms of action

2 Materials and Methods

21 Chemicals Dimethyl sulfoxide (DMSO 99) anhy-drous sodium sulfate glacial acetic acid ethanol formal-dehyde sodium chloride calcium acetate zymosan andneutral red were purchased fromMerck Chemical Company(Germany) The 119899-alkane (C

8ndashC20) homologous series

Schneiderrsquos medium RPMI 1640 medium fetal bovineserum (FBS) MTT (3-(45-dimethylthiazol-2-yl)25-diphen-yltetrazolium bromide) Griess reagent (1 sulfanilamidein H3PO410 (vv) in Milli-Q water) and the antibiotics

penicillin and streptomycin were purchased from SigmaChemical (St Louis MO USA) The antibiotic amphotericinB (90) was purchased from Cristalia (Sao Paulo SP Brazil)

22 Plant Material E uniflora leaves were collected (January2010) from a mature tree in the flowering stage in Sao Luıs(2∘30101584045310158401015840S and 44∘1810158401110158401015840W) in the state of Maranhao innortheast BrazilThe samples were collected from a cultivatedplant and its fruits have red color A voucher specimen(no 0998SLS017213) was deposited at the Herbarium ldquoAticoSeabrardquo of the Federal University of Maranhao and plantidentification was confirmed by botanists of the HerbariumldquoMurca Piresrdquo from Museu Paraense Emılio Goeldi BelemPA Brazil

23 Extraction of the Essential Oil Theplantmaterial was air-dried for 7 days cut into small pieces and subjected to hydro-distillation using a Clevenger-type apparatus (300 g 3 h) toobtain a sesquiterpene-rich essential oil Once collected theessential oil was dried over anhydrous sodium sulfate filteredand weighed and then the oil yield was calculated in terms of (ww) Its percentage content was estimated based on the

plant dry weight by calculating the water content by using amoisture analyzer prior to distillation EuEO was then storedin a dark flask and refrigerated (at +5∘C) until use

24 Gas Chromatography-Mass Spectrometry Analysis of theEssential Oil EuEO was analyzed using a THERMO DSQ IIGC-MS instrument (Thermo Fisher Scientific Austin TXUSA) under the following conditions DB-5ms (30m times025mm id 025120583m film thickness) fused silica capillarycolumn with the following temperature program 60∘Csubsequently increased by 3∘Cmin up to 240∘C injector tem-perature 250∘C carrier gas helium (high purity) adjusted toa linear velocity of 32 cms (measured at 100∘C)The injectiontype was splitless The oil sample was diluted 1 100 in hexanesolution and the volume injected was 01 120583L The split flowwas adjusted to yield a 2 1000 ratio and the septum sweepwas constant at 10mLmin All mass spectra were acquired inelectron impact (EI)modewith an ionization voltage of 70 eVover amass scan range of 35ndash450 amuThe temperature of theion source and connection parts was 200∘C

25 Identification and Quantification of Constituents Thequantitative data regarding the volatile constituents wereobtained by peak-area normalization using a FOCUS GCFID operated under conditions similar to those used in theGC-MS assay except for the carrier gas which was nitrogenThe retention index was calculated for all the volatile con-stituents by using an 119899-alkane (C

8ndashC20) homologous series

When possible individual components were identified bycoinjection with authentic standards Otherwise the peakassignment was performed by comparison of bothmass spec-trum and GC retention data by using authentic compoundspreviously analyzed and stored in our private library as wellas with the aid of commercial libraries containing retentionindices and mass spectra of volatile compounds commonlyfound in essential oils [23 24] Percentage (relative) of theidentified compounds was computed from the GC peak area

26 Parasites andMice Leishmania (Leishmania) amazonen-sis (IFLABR67PH8) was used for the determination ofthe anti-Leishmania activity Parasites were grown in sup-plemented Schneiderrsquos medium (10 heat-inactivated fetalbovine serum (FBS) 100UsdotmLminus1 penicillin and 100120583gmLstreptomycin at 26∘C) Murine macrophages were collectedfrom the peritoneal cavities of male and female BALBcmice (4-5 weeks old) fromMedicinal Plants Research Center(NPPMCCSUFPI) located at Teresina PI Brazil Themacrophages were maintained at a controlled temperature(24 plusmn 1∘C) and light conditions (12 h lightdark cycle) Allprotocols were approved by the Animal Research EthicsCommittee (CEEAPI no 0012012)

27 Anti-Leishmania Activity Assay Promastigotes in thelogarithmic growth phase were seeded in 96-well cell cultureplates at 1 times 106 Leishmania per well Then essential oilwas added to the wells in serial dilutions of 400 200 10050 25 125 625 and 312 120583gsdotmLminus1 The plate was kept at26∘C in a biological oxygen demand (BOD) incubator and

Evidence-Based Complementary and Alternative Medicine 3

Leishmania was observed and counted by using a Neubauerhemocytometer after 24 48 and 72 h to monitor growth andviability [25] Assays were performed in triplicate and wererepeated 3 times on different days

28 Cytotoxicity Determination Cytotoxicity of EuEO wasassessed using the MTT test In a 96-well plate 100 120583Lof supplemented RPMI 1640 medium and about 1 times 105macrophages were added per well They were then incubatedat 37∘C in 5 of CO

2for 2 h to allow cell adhesion After this

time 2 washes with supplemented RPMI 1640 medium wereperformed to remove cells that did not adhere SubsequentlyEuEO was added in triplicate after being previously dilutedin supplemented RPMI 1640 medium to a final volume of100 120583L for each well at the tested concentrations (100 50 25125 625 and 312 120583gsdotmLminus1) Cells were then incubated for48 h At the end of the incubation 10 120583L of MTT diluted inPBS was added at a final concentration of 5mgsdotmLminus1 (10 ofvolume ie 10 120583L for each 100 120583L well) and was incubatedfor an additional 4 h at 37∘C in 5 CO

2 The supernatant

was then discarded and 100120583L of DMSO was added toall wells The plate was then stirred for about 30min atroom temperature to complete formazan dissolution Finallyspectrophotometric reading was conducted at 550 nm in anELISA plate reader [26]

29 Hemolytic Activity The hemolytic activity was investi-gated by incubating 20 120583L of serially diluted essential oil inphosphate-buffered saline (PBS 400 200 100 50 25 125625 and 312 120583gsdotmLminus1) with 80 120583L of a suspension of 5 redblood cells (human O+) for 1 h at 37∘C in assay tubes Thereaction was slowed by adding 200120583L of PBS and then thesuspension was centrifuged at 1000 g for 10min Cell lysiswas then measured spectrophotometrically (540 nm) Theabsence of hemolysis (blank control) or total hemolysis (pos-itive control) was determined by replacing the essential oilsolution with an equal volume of PBS orMilli-Q sterile waterrespectivelyThe results were determined by the percentage ofhemolysis compared to the positive control (100hemolysis)and the experiments were performed in triplicate [27]

210 Treatment of Infected Macrophages Macrophages werecollected in 24-well culture plates at a concentration of 2 times105 cells500 120583L in RPMI 1640 containing sterile 13mm

round coverslips They were then incubated at 37∘C in 5of CO

2for 2 h to allow cell adhesion After this time the

medium was replaced with 500 120583L of supplemented RPMI1640 and incubated for 4 h The medium was then aspiratedand a new medium containing promastigotes (in the station-ary phase) at a ratio of 10 promastigotes to 1 macrophage wasadded to each well After 4 h of incubation in 5CO

2at 37∘C

the medium was aspirated to remove free promastigotes andthe test samples were added at nontoxic concentrations to themacrophages (312 156 and 078 120583gsdotmLminus1) This preparationwas then incubated for 48 h after which the coverslips wereremoved fixed in methanol and stained with Giemsa Foreach coverslip 100 cells were evaluated and both the number

of infected macrophages and the amount of parasites permacrophage were counted [26]

211 Lysosomal Activity Measurement of the lysosomal activ-ity was carried out according to the method of Grando etal [28] Peritoneal macrophages were plated and incubatedwith EuEO in serial dilutions of 100 50 25 125 625 and312 120583gsdotmLminus1 After 4 h of incubation at 37∘C in 5CO

2 10 120583L

of neutral red solution was added and incubated for 30minOnce this time had elapsed the supernatant was discardedthe wells were washed with 09 saline at 37∘C and 100mL ofextraction solution was added (glacial acetic acid 1 vv andethanol 50 vv dissolved in bidistilled water) to solubilizethe neutral red inside the lysosomal secretion vesicles After30min on aKline shaker (model AK 0506) the plate was readat 550 nm by using an ELISA plate reader

212 Phagocytosis Test Peritoneal macrophages were platedand incubated with EuEO in serial dilutions of 100 50 25125 625 and 312 120583gsdotmLminus1 After 48 h of incubation at 37∘Cin 5 CO

2 10 120583L of zymosan color solution was added and

incubated for 30min at 37∘C After this 100mL of Bakerrsquosfixative (formaldehyde 4 vv sodium chloride 2wv andcalcium acetate 1wv in distilled water) was added to stopthe phagocytosis process Thirty minutes later the plate waswashed with 09 saline in order to remove zymosan thatwas not phagocytosed by macrophages The supernatantwas removed and added to 100mL of extraction solutionAfter solubilization in a Kline shaker the absorbances weremeasured at 550 nm by using an ELISA plate reader [28]

213 Nitric Oxide (NO) Production In 96-well plates 2 times105 macrophages were added per well and incubated at

37∘C in 5 CO2for 4 h to allow cell adhesion A new

medium containing promastigotes (in the stationary phase)at a ratio of 10 promastigotes per macrophage was addedto half of the wells The essential oil was added after beingpreviously diluted in a culture medium containing differentconcentrations (100 50 25 125 625 and 312 120583gsdotmLminus1) inthe absence or presence of L amazonensis and was thenincubated again at 37∘C in 5CO

2for 24 h After this period

the cell culture supernatant was collected and transferred toanother plate for measurement of nitrite A standard curvewas prepared with 150 120583M sodium nitrite in Milli-Q water atvarying concentrations of 1 5 10 25 50 75 100 and 150 120583Mdiluted in a culture medium At the time of dosing 100 120583Lof either the samples or the solutions prepared for obtainingthe standard curve was mixed with an equal volume of Griessreagent The analysis was performed using an ELISA platereader at 550 nm [28]

214 Statistical Analysis All assays were performed in trip-licate and in 3 independent experiments The half-maximalinhibitory concentration (IC

50) values and 50 cytotoxicity

concentration (CC50) values with 95 confidence intervals

were calculated using a probit regressionmodel and Studentrsquos119905-test Analysis of variance (ANOVA) followed by a Bonfer-roni test were performed taking a 119875 value of lt005 as theminimum level required for statistical significance

4 Evidence-Based Complementary and Alternative Medicine

3 Results

31 Analysis of the Essential Oil The yield of EuEO was03 Thirty-two components were identified in this oil byGC-MS constituting 9265 of the total mixture EuEO wasshown to be rich in oxygenated sesquiterpenes (6255)and sesquiterpene hydrocarbons (2937) Curzerenewas themajor constituent (473) followed by 120574-elemene (1425)and trans-120573-elemenone (104) The chemical constituentsare listed in Table 1

32 Anti-Leishmania Activity Assay The inhibitory profile ofEuEO on L amazonensis promastigotes showed a significantconcentration-dependent decrease (119875 lt 005) in parasiteviability with 100 inhibition of promastigote growth atconcentrations of 400 200 and 100120583gsdotmLminus1 (Figure 1) TheIC50

was 175120583gsdotmLminus1 at 72 h of exposure (Table 2) Incultures treated with EuEO we used an optical microscopeto observe morphological changes in the promastigotes suchas cells with rounded or completely spherical shapes as wellas cellular debris in contrast to the spindle forms presentin the control (data not shown) Amphotericin B was usedas a positive control and was tested at a concentration of2120583gsdotmLminus1 The highest inhibitory effect of amphotericin Bwas observed after 24 h

33 Cytotoxicity and Hemolysis Assay The cytotoxicity ofEuEO for murine peritoneal macrophages and erythrocytesis shown in Figures 2 and 3 respectively and in Table 2The EO from E uniflora significantly altered (119875 lt 005)macrophage viability at a concentration of 625 120583gsdotmLminus1 andwas able to reduce 50 of the macrophage viability (CC

50)

at 453120583gsdotmLminus1 The cytotoxicity of EO from E uniflora forhuman blood type O+ erythrocytes was 6322 at the highestconcentration tested (400120583gsdotmLminus1) however it appearedto be nontoxic at concentrations less than 50 120583gsdotmLminus1(Figure 3)

34 Treatment of Infected Macrophages Figures 4 and 5show the results of EuEO treatment of macrophages infectedwith L amazonensis The values obtained revealed a signifi-cant and concentration-dependent reduction in macrophageinfection at 48 h with all the 3 EuEO concentrations studiedThe reduction of infection was 27 4467 and 5067at 078 156 and 312 120583gsdotmLminus1 respectively EuEO alsodecreased the number of amastigotes per infected cell in aconcentration-dependentmanner (Figure 5)The IC

50at 48 h

of exposure to EuEOwas 192120583gsdotmLminus1 (Table 2)These valuesand the CC

50values for macrophages were used to calculate

the selectivity index that is useful for assessing the safety levelof EuEO in mammalian cells (Table 2) This index representsthe relationship between CC

50and IC

50for amastigotes

35 Lysosomal Activity and Phagocytosis Assay Lysosomalactivity was assessed based on the retention of neutral redin macrophage lysosomes and was determined colorimet-rically The lysosomal activity of macrophages treated withEuEO showed significant reduction at concentrations of

Table 1 Chemical composition and retention indices of the con-stituents of Eugenia uniflora essential oil

Constituentsa RRI Ad

Calb Litc

Myrcene 989 988 019Limonene 1026 1029 018Linalool 1095 1095 016(119885)-3-Hexenyl butyrate 1186 1184 010Cuminaldehyde 1240 1238 010120575-Elemene 1335 1338 132120572-Cubebene 1349 1345 010120573-Elemene 1390 1389 551Sativene 1394 1390 006(E)-Caryophyllene 1417 1417 433trans-120572-Bergamotene 1437 1432 005120574-Elemene 1434 1434 1425Aromadendrene 1440 1439 007120572-Humulene 1455 1452 021Alloaromadendrene 1462 1458 011120573-Chamigrene 1475 1476 038Germacrene D 1482 1484 119120573-Selinene 1487 1489 070Curzerene 1498 1499 473120574-Cadinene 1513 1513 012120575-Cadinene 1523 1522 028Selina-37-(11)-diene 1546 1545 024Germacrene B 1558 1559 045Spathulenol 1578 1577 017Caryophyllene oxide 1581 1582 013Globulol 1585 1590 025Viridiflorol 1590 1592 008trans-120573-Elemenone 1597 1601 104Atractilona 1655 1657 238Germacrone 1694 1693 151Eudesm-7(11)-en-4-ol 1700 1700 033Monoterpene hydrocarbons 037Oxygenated monoterpenes 036Sesquiterpene hydrocarbons 2937Oxygenated sesquiterpenes 6255Total 9265aCompounds listed in order of elution on the DB-5ms column bRelativeretention indices (RRIs) experimentally determined against n-alkanes byusing theDB-5ms column cRRIs reported in the literature [23 24] dContentexpressed as percentages obtained by integration of the GC peak area

100 and 50 120583gsdotmLminus1 (Figure 6(a)) due to cytotoxicity (CC50

453120583gsdotmLminus1) However we observed a statistically signif-icant increase in lysosomal activity at concentrations from125 to 312 120583gsdotmLminus1 compared to control Another parameterreflecting macrophage activation was the phagocytosis ofstained zymosan when the cells were exposed to an externalstimulus The phagocytic ability of macrophages incubatedwith EuEO is shown in Figure 6(b) There was a significantincrease in phagocytosis of zymosan particles in the wells

Evidence-Based Complementary and Alternative Medicine 5

0

20

40

60

80

100

400Amph 200 100 50 25 125 625 312

Gro

wth

inhi

bitio

n (

)

lowast

24 h48 h72 h

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Figure 1 Effect of Eugenia uniflora essential oil (400 200 10050 25 125 625 and 312 120583gsdotmLminus1) or amphotericin B (Amph)(2120583gsdotmLminus1) on Leishmania amazonensis promastigotes Cultures oflog-phase promastigotes (1 times 106) were incubated at 26∘C for 2448 and 72 h in different essential oil concentrations Data representthe mean percentage of growth inhibition plusmn standard error of 3experiments carried out in triplicate lowast119875 lt 005

312 625 125 25 50 100

0102030405060708090

100

Cel

l via

bilit

y (

)

lowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 2 Cytotoxicity of Eugenia uniflora essential oil on theviability ofmurine peritonealmacrophages Peritonealmacrophageswere seeded at 1 times 105well in 96-well microplates and incubated for48 h in the presence of E uniflora L essential oil at concentrationsof 100 50 25 125 625 and 312 120583gsdotmLminus1 Viability was determinedwith 3-(45-dimethylthiazol-2-yl)25-diphenyltetrazolium bromide(MTT) and the optical density was determined at 540 nmData rep-resent the mean parasite density plusmn standard error of 3 experimentscarried out in triplicate lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

treated with EuEO at concentrations ranging from 25 to312 120583gsdotmLminus1

36 NO Production NO production was determined indi-rectly by measuring nitrite produced by macrophages treatedwith EuEO and stimulated (or not stimulated) by L amazo-nensis promastigotes Macrophages treated with EuEO but

01020304050607080

4002001005025125625312

Hem

olys

is (

)

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Figure 3 Hemolytic activity of Eugenia uniflora essential oil in a 4suspension of human O+ red blood cells after 1 h of incubation

not stimulated by Leishmania showed significant reductionin NO production at concentrations of 100 and 50120583gsdotmLminus1(Figure 7(a)) while macrophages stimulated by the parasiteshowed increased NO at a concentration of 312120583gsdotmLminus1albeit without statistical significance (119875 gt 005) (Figure 7(b))

4 Discussion

The need to identify new compounds with anti-Leishmaniaproperties that are more effective and less toxic than con-ventional drugs has motivated research on natural productsisolated from plant species These substances predominantlyconsist of alkaloids terpenes flavonoids benzopyrans phe-nolics and sesquiterpene lactones and have been identifiedin plant species with documented anti-Leishmania activity[29 30] The chemical composition of EuEO obtained in thisstudy revealed sesquiterpenes to be the dominant chemicalclass corroborating the findings of many previous studies[16 31ndash33] Despite the prevalence of sesquiterpenes thespecies has different chemotypes with different major con-stituents being observed in different studies The specimenanalyzed in this study had curzerene as themajor constituentwhich again corroborated previous reported findings [3234] According to Adio [35] germacrone is a heat-sensitivecompound that may degrade to trans-120573-elemenone duringGC analysis This may be the explanation of the presence oftrans-120573-elemenone among the major compounds of EuEOSome germacrone products have shown pharmacologicalactivity as antitumor [35]

According to Costa et al [20] the composition of theessential oil from E uniflora leaves might be influenced byfruit colorsThe plants with red fruit colors have curzerene asthe major component as in our studied plant In Maranhaowhere the samples were collected the majority of plants ofE uniflora have red fruit color [32] Selina-137(11)-trien-8-one [36] germacrene B [16 33] and 120572- and 120573-selinene [37]have been found to be the major constituents of other Euniflora chemotypes Genetic variability geographical andenvironmental conditions and the method of environmentalmanagement directly influence the yield and composition of

6 Evidence-Based Complementary and Alternative Medicine

Table 2 Anti-Leishmania and cytotoxic effects of Eugenia uniflora essential oil

Macrophage Promastigote AmastigoteCC50 (120583gsdotmLminus1) IC50 (120583gsdotmLminus1)

SIIC50 (120583gsdotmLminus1) SI

48 h 24 h 48 h 72 h 48 hEuEO 453 plusmn 245 696 plusmn 102 304 plusmn 075 175 plusmn 053 149a 192 plusmn 08 2359baSIpro (selectivity index) = CC50 macrophagesIC50 promastigote forms (48 h)bSIama (selectivity index) = CC50 macrophagesIC50 amastigote forms

078 156 312

0

20

40

60

80

100

27

44675067

Mac

roph

ages

infe

ctio

n (

)

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 4 Effect of Eugenia uniflora essential oil on macrophageinfection after 48 h of exposure Peritoneal macrophage cells wereinfected with promastigotes of Leishmania amazonensis and thentreated with 312 156 or 078 120583gsdotmLminus1 of Eugenia uniflora essentialoil Data represent the mean parasite density plusmn standard error of 3experiments carried out in triplicate lowastlowastlowast119875 lt 0001

078 156 312

0

2

4

6

8

10lowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Am

astig

otes

mac

roph

age (n

)

lowastlowastlowast

lowastlowastlowast

Figure 5 Effect of Eugenia uniflora essential oil on the survival ofLeishmania amazonensis amastigotes internalized in macrophagesLeishmania amazonensis-infected mouse peritoneal macrophageswere treated with 312 156 or 078 120583gsdotmLminus1 of Eugenia unifloraessential oil After 48 h of incubation amastigote survival wasassessed Data represent the mean parasite density plusmn standard errorof 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001

essential oils [38] It is the first report of essential oil analyzedfrom E uniflora collected in Sao Luıs MA Brazil and thiscould be an excellent source of natural product with interestfor pharmaceutical industry

The present study was conducted to evaluate the anti-Leishmania activity of EuEO on promastigotes and amasti-gotes of L amazonensis its potential mechanisms of macro-phage activation and its cytotoxicity inmammalian cellsTheEuEO showed significant concentration-dependent activityagainst promastigotes of L amazonensis with an IC

50of

175120583gsdotmLminus1 Previous studies have suggested that theseclasses of natural products may have potential anti-Leishmania activity 120573-Caryophyllene (a sesquiterpenehydrocarbon) and nerolidol (an oxygenated sesquiterpene)are examples of terpenic substances with well-characterizedanti-Leishmania activity possibly associated with inhibitionof cellular isoprenoid biosynthesis [8 39]

It has been shown that the lipophilic components ofessential oils may affect layers of polysaccharides fatty acidsand phospholipids in plasma membranes of promastigotes ofLeishmania spp This then leads to cell lysis and release ofmacromolecules [40] In the cytoplasm these substances candisrupt the specific metabolic pathways of lipids and proteinsor stimulate depolarization of mitochondrial membraneswhich can lead to cell necrosis or apoptosis [41 42]

Amphotericin B is a drug of choice for the treatment ofdifferent forms of leishmaniasis Because of this it was usedas a positive control We observed a significant inhibitoryeffect on promastigote growth after 24 h of exposure toamphotericin B but leading to an increase in viable pro-mastigote forms at 48 and 72 h Similar results were observedin previous studies [43] Some Leishmania strains have shownmechanisms of resistance to amphotericin B This may hashappenedwith the strain used in this studyThe reproductionof resistant parasites may have led to a higher cell viabilityafter 48 and 72 h [44 45]

Experimental models using amastigotes internalized intomacrophages can produce results that more closely approxi-mate those obtained using animal models since these formsare responsible for the clinicalmanifestations of leishmaniasis[46] Amastigotes can be found in parasitophorous vacuolesof infected macrophages therefore a therapeutic drug willonly be effective against Leishmania spp if it can crossthe host cell membrane and act on the amastigotes insidethe vacuole Thus assays of activity against intracellularamastigotes are the most effective way to relate the in vitroactivity of a possible substance with its in vivo effectiveness[47] The inhibition of amastigote growth was even greater at

Evidence-Based Complementary and Alternative Medicine 7

C 100 50 25 125 625 312

005

115

225

335

445

5N

eutr

al re

d up

take

ABS

lowast

lowastlowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(a)

C 100 50 25 125 625 312

0

005

01

015

02

025

Phag

ocyt

osis

ABS

lowast lowast lowastlowast lowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

(b)

Figure 6 The influence of Eugenia uniflora essential oil on the lysosomal activity (a) and phagocytic activity (b) of peritoneal macrophagesat concentrations of 100 50 25 125 625 and 312 120583gsdotmLminus1 Data represent the mean density plusmn standard error of 3 experiments carried outin triplicate lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 C control ABS absorbance

0010203040506070809

1

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119871119890119894119904ℎ119898119886119899119894119886 119886119898119886119911119900119899119890119899119904119894119904

0010203040506070809

1

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(b)

Figure 7 Production of nitric oxide (NO) Murine macrophages (2 times 105) were treated with Eugenia uniflora essential oil (100 50 25 125625 and 312 120583gsdotmLminus1) in the absence (a) or presence of Leishmania amazonensis (b) over 24 h Data represent the mean density plusmn standarderror of 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001 C control ABS absorbance

48 h after exposure to oil with an IC50of 192 120583gmL whereas

the IC50

for promastigotes was 304120583gsdotmLminus1 for the sameduration of exposure Previous studies demonstrated greateractivity against amastigotes versus promastigotes of essentialoils of Artemisia absinthium and Satureja punctata [48]

Mechanisms of anti-Leishmania action on Leishmaniaspp amastigotes have also been described for essentialoils and their terpene compounds Monoterpenes such aslinalool isolated from leaves of Croton cajucara (Euphorbia-ceae) significantly increased NO production inmacrophagesinfected with L amazonensis and acts directly on the parasiteas evidenced by mitochondrial swelling and changes in thekinetoplast and the organization of nuclear chromatin[6] In this study the production of NO was determined bymeasuring nitriteThe results frommacrophages treated with

EuEO revealed that EuEO does not induce NO productionwhich demonstrates that its promising anti-Leishmaniaactivity does not occur by this route

Natural products that are able to activate macrophagesand are thus biological response modifiers have beenextensively studied [49 50] In this context the EuEOdemonstrated potential in the activation of macrophagesby increasing phagocytic capacity and lysosomal activityimmunomodulatory activities possibly involved in its anti-Leishmania activity Phagocytosis and the lysosomal systemare critical to macrophage function because of their roles ininternalization degradation and eventually presentation ofpeptides derived from antigens required for host defense Inaddition antigen presentation occurs through phagocytosisand endosomallysosomal targeting systems [51ndash53]

8 Evidence-Based Complementary and Alternative Medicine

Due to the need for anti-Leishmania substances that aremore selective for the parasite and less toxic to host cellsand since EuEO showed low IC

50values it was important to

investigate the cytotoxic activity of EuEO againstmammaliancells EuEO showed significant toxicity in macrophages witha CC50

of 453120583gsdotmLminus1 but when compared with its IC50

value in amastigotes this indicates a secure selectivity indexabove 20 The literature recommends that for amastigotesinternalized in macrophages this index must be demon-strated as near to or greater than 20 [54] The investigationof the toxic action of new substances with anti-Leishmaniaactivity onmacrophages is important since they are themaincells of the vertebrate host parasitized by Leishmania spp[55 56] Furthermore various other cytotoxic assays can beconducted using animal and human cells to determine thesafety of a test substance leading to a future treatment invivo Tests performed with erythrocytes enable assessment ofa drugrsquos potential to cause injury to the plasma membrane ofa cell either by forming pores or by total rupture leadingto cellular damage or changes in membrane permeability[57] This is a model used for the preliminary study of theprotective and toxic effects of substances being a possibleindicator of this type of damage to cells in vivo [58ndash60]The results obtained in this test with EuEO revealed lowtoxicity to erythrocytes since EuEO showed 0 hemolysis atconcentrations of 50120583gsdotmLminus1 and lower

5 Conclusions

In conclusion the data in this study show that the essentialoil of leaves from E uniflora (EuEO) characterized bysesquiterpenes has anti-Leishmania activity in both stagesof L amazonensis This study also demonstrated that EuEOactivity is not mediated by NO production On the otherhand activation of macrophages may be involved in themechanisms of EuEO anti-Leishmania activity as evidencedby increases in both phagocytic capacity and the lysosomalcompartment Further investigation is needed to determineadditional mechanisms involved in the anti-Leishmaniaactivity of this plant species as well as in vivo studies inmodels of leishmaniasis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors are grateful to the CAPES and FAPEMA forfinancial grants through the concession of scholarships andfinancial support

References

[1] S Kaur H Sachdeva S Dhuria M Sharma and T Kaur ldquoAn-tileishmanial effect of cisplatin against murine visceral leishma-niasisrdquo Parasitology International vol 59 no 1 pp 62ndash69 2010

[2] S AzzouzMMaache R G Garcia andA Osuna ldquoLeishmani-cidal activity of edelfosine miltefosine and ilmofosinerdquo Basic

and Clinical Pharmacology and Toxicology vol 96 no 1 pp 60ndash65 2005

[3] J A Patza T K Graczykb N Gellera and A Y Vittorc ldquoEffectsof environmental change on emerging parasitic diseasesrdquo Inter-national Journal of Parasitology vol 30 no 12-13 pp 1295ndash13042000

[4] B H Alizadeh A Foroumadi S K Ardestani F Poorrajaband A Shafiee ldquoLeishmanicidal evaluation of novel syntheticchromenesrdquo Archiv der Pharmazie vol 341 no 12 pp 787ndash7932008

[5] RW Ashford ldquoThe leishmaniases as emerging and reemergingzoonosesrdquo International Journal for Parasitology vol 30 no 12-13 pp 1269ndash1281 2000

[6] M S S Rosa R R Mendonca-Filho H R Bizzo et al ldquoAntil-eishmanial activity of a linalool-rich essential oil from CrotoncajucarardquoAntimicrobial Agents Chemotherapy vol 47 no 6 pp1895ndash1901 2003

[7] T Ueda-Nakamura R R Mendonca-Filho J A Morgado-Dıazet al ldquoAntileishmanial activity of eugenol-rich essential oil fromOcimum gratissimumrdquo Parasitology International vol 55 no 2pp 55ndash59 2006

[8] A O Santos T Ueda-Nakamura B P Dias Filho V F VeigaJunior A C Pinto and C V Nakamura ldquoEffect of Braziliancopaiba oils on Leishmania amazonensisrdquo Journal of Ethnophar-macology vol 120 no 2 pp 204ndash208 2008

[9] LMonzote AMMontalvo S Almanonni R Scull MMiran-da and J Abreu ldquoActivity of the essential oil fromChenopodiumambrosioides grown in Cuba against Leishmania amazonensisrdquoChemotherapy vol 52 no 3 pp 130ndash136 2006

[10] MMachado P Pires A M Dinis et al ldquoMonoterpenic aldehy-des as potential anti-Leishmania agents activity ofCymbopogoncitratus and citral on L infantum L tropica and L majorrdquoExperimental Parasitology vol 130 no 3 pp 223ndash231 2012

[11] M R Santin A O Dos Santos C V Nakamura B P Dias FilhoI C P Ferreira and T Ueda-Nakamura ldquoIn vitro activity of theessential oil of Cymbopogon citratus and its major component(citral) on Leishmania amazonensisrdquo Parasitology Research vol105 no 6 pp 1489ndash1496 2009

[12] V C S Oliveira D M S Moura J A D Lopes P P DeAndrade N H Da Silva and R C B Q Figueiredo ldquoEffectsof essential oils from Cymbopogon citratus (DC) Stapf Lippiasidoides Cham and Ocimum gratissimum L on growth andultrastructure of Leishmania chagasi promastigotesrdquo Parasitol-ogy Research vol 104 no 5 pp 1053ndash1059 2009

[13] A E Consolini and M G Sarubbio ldquoPharmacological effectsof Eugenia uniflora (Myrtaceae) aqueous crude extract on ratrsquosheartrdquo Journal of Ethnopharmacology vol 81 no 1 pp 57ndash632002

[14] C B Alice V M F Vargas G A A B Silva et al ldquoScreening ofplants used in south Brazilian folk medicinerdquo Journal of Ethno-pharmacology vol 35 no 2 pp 165ndash171 1991

[15] P Weyerstahl H Marschall-Weyerstahl C Christiansen B OOguntimein and A O Adeoye ldquoVolatile constituents of Euge-nia uniflora leaf oilrdquo Planta medica vol 54 no 6 pp 546ndash5491988

[16] F N Victoria E J Lenardao L Savegnago et al ldquoEssential oil ofthe leaves of Eugenia uniflora L antioxidant and antimicrobialpropertiesrdquo Food and Chemical Toxicology vol 50 no 8 pp2668ndash2674 2012

Evidence-Based Complementary and Alternative Medicine 9

[17] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[18] I A Ogunwande N O Olawore O Ekundayo T M Walker JM Schmidt andWN Setzer ldquoStudies on the essential oils com-position antibacterial and cytotoxicity of Eugenia uniflora LrdquoInternational Journal of Aromatherapy vol 15 no 3 pp 147ndash1522005

[19] A C L Amorim C K F Lima A M C Hovell A L PMiranda and C M Rezende ldquoAntinociceptive and hypother-mic evaluation of the leaf essential oil and isolated terpenoidsfrom Eugenia uniflora L (Brazilian Pitanga)rdquo Phytomedicinevol 16 no 10 pp 923ndash928 2009

[20] D P Costa E G A Filho LM A Silva et al ldquoInfluence of fruitbiotypes on the chemical composition and antifungal activityof the essential oils of Eugenia uniflora leavesrdquo Journal of theBrazilian Chemical Society vol 21 no 5 pp 851ndash858 2010

[21] G F Ibikunle A C Adebajo F G Famuyiwa A J Aladesanmiand C O Adewunmi ldquoIn-vitro evaluation of anti-trichomonalactivities ofEugenia uniflora leafrdquoAfrican Journal of TraditionalComplementary and AlternativeMedicines vol 8 no 2 pp 170ndash176 2011

[22] K K Santos S R Tintino C E Souza et al ldquoAnti-Trypanosomacruzi and cytotoxic activities of Eugenia uniflora Lrdquo Experimen-tal Parasitology vol 131 no 1 pp 130ndash132 2012

[23] NIST 05 Mass Spectral Library (NISTEPANIH) NationalInstitute of Standards and Technology Gaithersburg Md USA2005

[24] R P Adams Identification of Essential Oil Components By GasChromatographyMass Spectrometry Allured Carol Stream IllUSA 4th edition 2007

[25] S M P Carneiro F A A Carvalho L C L R Santana A PL Sousa andM H Chaves ldquoThe cytotoxic and antileishmanialactivity of extracts and fractions of leaves and fruits of Azad-irachta indica (A Juss)rdquo Biological Research vol 45 no 2 pp111ndash116 2012

[26] M D G F deMedeiros A C da Silva A M D G L Cito et alldquoIn vitro antileishmanial activity and cytotoxicity of essential oilfrom Lippia sidoides Chamrdquo Parasitology International vol 60no 3 pp 237ndash241 2011

[27] S E Lofgren L C Miletti M Steindel E Bachere and M ABarracco ldquoTrypanocidal and leishmanicidal activities of differ-ent antimicrobial peptides (AMPs) isolated from aquatic ani-malsrdquo Experimental Parasitology vol 118 no 2 pp 197ndash2022008

[28] F C C Grando C A Felıcio A Twardowschy et al ldquoModula-tion of peritoneal macrophage activity by the saturation state ofthe fatty acid moiety of phosphatidylcholinerdquo Brazilian Journalof Medical and Biological Research vol 42 no 7 pp 599ndash6052009

[29] MM Iwu J E Jackson and BG Schuster ldquoMedicinal plants inthe fight against leishmaniasisrdquo Parasitology Today vol 10 no2 pp 65ndash68 1994

[30] L G Rocha J R G S Almeida R O Macedo and J M Bar-bosa-Filho ldquoA review of natural products with antileishmanialactivityrdquo Phytomedicine vol 12 no 6-7 pp 514ndash535 2005

[31] SMDeMorais A A CraveiroM I LMachado JW Alencarand F J A Matos ldquoVolatile constituents of Eugenia uniflora leafoil from northeastern Brazilrdquo Journal of Essential Oil Researchvol 8 no 4 pp 449ndash451 1996

[32] J G S Maia E H A Andrade M H L Da Silva and M G BZoghbi ldquoA new chemotype of Eugenia uniflora L from NorthBrazilrdquo Journal of Essential Oil Research vol 11 no 6 pp 727ndash729 1999

[33] J H G Lago E D Souza B Mariane et al ldquoChemical and bio-logical evaluation of essential oils from two species of myrtace-aemdashEugenia uniflora L and Plinia trunciflora (O Berg) KauselrdquoMolecules vol 16 no 12 pp 9826ndash9837 2011

[34] R Chang S A L de Morais D R Napolitano K C DuarteV B Guzman and E A do Nascimento ldquoA new approach forquantifying furanodiene and curzerene A case study on theessential oils of Eugenia uniflora (pitangueira) leavesrdquo BrazilianJournal of Pharmacognosy vol 21 no 3 pp 392ndash396 2011

[35] A M Adio ldquoGermacrenes A-E and related compounds ther-mal photochemical and acid induced transannular cycliza-tionsrdquo Tetrahedron vol 65 no 8 pp 1533ndash1552 2009

[36] D P Costa S C Santos J C Seraphin and PH Ferri ldquoSeasonalvariability of essential oils of Eugenia uniflora leavesrdquo Journal ofthe BrazilianChemical Society vol 20 no 7 pp 1287ndash1293 2009

[37] A T HenriquesM E Sobral A D Caudaro E E S SchapovalV L Bassani and G Lamaty ldquoAromatic plants from BrazilII The chemical composition of some Eugenia essential oilsrdquoJournal of Essential Oil Research vol 5 no 5 pp 501ndash505 1993

[38] B Teixeira A Marques C Ramos et al ldquoEuropean pennyroyal(Mentha pulegium) from portugal chemical composition ofessential oil and antioxidant and antimicrobial properties ofextracts and essential oilrdquo Industrial Crops and Products vol 36no 1 pp 81ndash87 2012

[39] D C Arruda F L DrsquoAlexandri A M Katzin and S RB Uliana ldquoAntileishmanial activity of the terpene nerolidolrdquoAntimicrobial Agents andChemotherapy vol 49 no 5 pp 1679ndash1687 2005

[40] RDi Pasqua G Betts NHoskinsM Edwards D Ercolini andG Mauriello ldquoMembrane toxicity of antimicrobial compoundsfrom essential oilsrdquo Journal of Agricultural and Food Chemistryvol 55 no 12 pp 4863ndash4870 2007

[41] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[42] J S Armstrong ldquoMitochondrial membrane permeabilizationthe sine qua non for cell deathrdquo BioEssays vol 28 no 3 pp253ndash260 2006

[43] A L Silva C M Adade F M Shoyama et al ldquoIn vitro leish-manicidal activity of N-dodecyl-1 2-ethanediaminerdquo Biomedi-cine and Pharmacotherapy vol 66 no 3 pp 180ndash186 2012

[44] B Purkait A Kumar N Nandi et al ldquoMechanism of ampho-tericin B resistance in clinical isolates of Leishmania donovanirdquoAntimicrobial Agents and Chemotherapy vol 56 no 2 pp 1031ndash1141 2012

[45] M Vermeersch R I Da Luz K Tote J P Timmermans P Cosand L Maes ldquoIn vitro susceptibilities of Leishmania donovanipromastigote and amastigote stages to antileishmanial referencedrugs practical relevance of stage-specific differencesrdquo Antimi-crobial Agents and Chemotherapy vol 53 no 9 pp 3855ndash38592009

[46] C Di Giorgio F Delmas V Akhmedjanova et al ldquoIn vitroantileishmanial activity of diphyllin isolated fromHaplophyllumbucharicumrdquo Planta Medica vol 71 no 4 pp 366ndash369 2005

10 Evidence-Based Complementary and Alternative Medicine

[47] H Martinez-Rojano J Mancilla-Ramirez L Quinonez-Diazand N Galindo-Sevilla ldquoActivity of hydroxyurea against Leish-mania mexicanardquo Antimicrobial Agents and Chemotherapy vol52 no 10 pp 3642ndash3647 2008

[48] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[49] A H Klimp E G E De Vries G L Scherphof and T DaemenldquoA potential role of macrophage activation in the treatment ofcancerrdquo Critical Reviews in OncologyHematology vol 44 no 2pp 143ndash161 2002

[50] M R Piemonte and D F Buchi ldquoAnalysis of IL-12 IFN-120574 and TNF-120572 production a5b1 integrins and actin filamentsdistribution in peritoneal mouse macrophages treated withhomeopathic medicamentrdquo Journal of Submicroscopic Cytologyand Pathology vol 33 no 1 pp 255ndash263 2002

[51] F Niedergang and P Chavrier ldquoSignaling and membranedynamics during phagocytosis Many roads lead to the pha-gos(R)omerdquo Current Opinion in Cell Biology vol 16 no 4 pp422ndash428 2004

[52] W L Lee R E Harrison and S Grinstein ldquoPhagocytosis byneutrophilsrdquo Microbes and Infection vol 5 no 14 pp 1299ndash1306 2003

[53] S J Green M S Meltzer J B Hibbs and C A Nacy ldquoActivatedmacrophages destroy intracellular Leishmania major amastig-otes by an L-arginine-dependent killing mechanismrdquo Journal ofImmunology vol 144 no 1 pp 278ndash283 1990

[54] E Osorio G J Arango N Jimenez et al ldquoAntiprotozoal andcytotoxic activities in vitro of Colombian Annonaceaerdquo Journalof Ethnopharmacology vol 111 no 3 pp 630ndash635 2007

[55] S Kamhawi ldquoPhlebotomine sand flies and Leishmania para-sites friends or foesrdquo Trends in Parasitology vol 22 no 9 pp439ndash445 2006

[56] P Tripathi V Singh and S Naik ldquoImmune response to leish-mania paradox rather than paradigmrdquo FEMS Immunology andMedical Microbiology vol 51 no 2 pp 229ndash242 2007

[57] L V Costa-Lotufo G M A Cunha P A M Farias et al ldquoThecytotoxic and embryotoxic effects of kaurenoic acid a diterpeneisolated fromCopaifera langsdorffii oleo-resinrdquoToxicon vol 40no 8 pp 1231ndash1234 2002

[58] R M Aparicio M J Garcıa-Celma M Pilar Vinardell and MMitjans ldquoIn vitro studies of the hemolytic activity ofmicroemul-sions in human erythrocytesrdquo Journal of Pharmaceutical andBiomedical Analysis vol 39 no 5 pp 1063ndash1067 2005

[59] L A Lexis R G Fassett and J S Coombes ldquo120572-tocopherol and120572-lipoic acid enhance the erythrocyte antioxidant defence incyclosporine A-treated ratsrdquo Basic and Clinical Pharmacologyand Toxicology vol 98 no 1 pp 68ndash73 2006

[60] J R Munoz-Castaneda J Muntane M C Munoz I BujalanceP Montilla and I Tunez ldquoEstradiol and catecholestrogens pro-tect against adriamycininduced oxidative stress in erythrocytesof ovariectomized ratsrdquo Toxicology Letters vol 160 no 3 pp196ndash203 2006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

among these are Croton cajucara [6] Ocimum gratissimum[7] Copaifera cearensis [8] Chenopodium ambrosioides [9]Cymbopogon citratus [10 11] and Lippia sidoides [12] Thesestudies have shown that essential oils can be a promisingsource of new drugs with anti-Leishmania activity

Eugenia uniflora L commonly known as ldquopitangueirardquoor Brazilian cherry tree is a species belonging to the familyMyrtaceae which is native to South America and commonin regions with tropical and subtropical climate [13] InBrazil it is used in the treatment of digestive disorders [14]and is thought to have anti-inflammatory and antirheumaticactivities [15] It is an aromatic species and its essential oilhas pharmacological properties that are well characterizedin the literature as antioxidant and antimicrobial [16] Euniflora has known antihypertensive [17] antitumor [18]and antinociceptive properties [19] and it shows good per-formance against microorganisms demonstrating antiviralantifungal [20] anti-Trichomonas gallinae [21] and anti-Trypanosoma cruzi properties [22]

Considering the potential pharmacological benefits ofE uniflora and the increasing interest in the discovery ofessential oils with anti-Leishmania activity the aim of thisstudy was to investigate the anti-Leishmania activity of leafessential oil from this species the chemical composition ofthe oil its cytotoxicity and possible mechanisms of action

2 Materials and Methods

21 Chemicals Dimethyl sulfoxide (DMSO 99) anhy-drous sodium sulfate glacial acetic acid ethanol formal-dehyde sodium chloride calcium acetate zymosan andneutral red were purchased fromMerck Chemical Company(Germany) The 119899-alkane (C

8ndashC20) homologous series

Schneiderrsquos medium RPMI 1640 medium fetal bovineserum (FBS) MTT (3-(45-dimethylthiazol-2-yl)25-diphen-yltetrazolium bromide) Griess reagent (1 sulfanilamidein H3PO410 (vv) in Milli-Q water) and the antibiotics

penicillin and streptomycin were purchased from SigmaChemical (St Louis MO USA) The antibiotic amphotericinB (90) was purchased from Cristalia (Sao Paulo SP Brazil)

22 Plant Material E uniflora leaves were collected (January2010) from a mature tree in the flowering stage in Sao Luıs(2∘30101584045310158401015840S and 44∘1810158401110158401015840W) in the state of Maranhao innortheast BrazilThe samples were collected from a cultivatedplant and its fruits have red color A voucher specimen(no 0998SLS017213) was deposited at the Herbarium ldquoAticoSeabrardquo of the Federal University of Maranhao and plantidentification was confirmed by botanists of the HerbariumldquoMurca Piresrdquo from Museu Paraense Emılio Goeldi BelemPA Brazil

23 Extraction of the Essential Oil Theplantmaterial was air-dried for 7 days cut into small pieces and subjected to hydro-distillation using a Clevenger-type apparatus (300 g 3 h) toobtain a sesquiterpene-rich essential oil Once collected theessential oil was dried over anhydrous sodium sulfate filteredand weighed and then the oil yield was calculated in terms of (ww) Its percentage content was estimated based on the

plant dry weight by calculating the water content by using amoisture analyzer prior to distillation EuEO was then storedin a dark flask and refrigerated (at +5∘C) until use

24 Gas Chromatography-Mass Spectrometry Analysis of theEssential Oil EuEO was analyzed using a THERMO DSQ IIGC-MS instrument (Thermo Fisher Scientific Austin TXUSA) under the following conditions DB-5ms (30m times025mm id 025120583m film thickness) fused silica capillarycolumn with the following temperature program 60∘Csubsequently increased by 3∘Cmin up to 240∘C injector tem-perature 250∘C carrier gas helium (high purity) adjusted toa linear velocity of 32 cms (measured at 100∘C)The injectiontype was splitless The oil sample was diluted 1 100 in hexanesolution and the volume injected was 01 120583L The split flowwas adjusted to yield a 2 1000 ratio and the septum sweepwas constant at 10mLmin All mass spectra were acquired inelectron impact (EI)modewith an ionization voltage of 70 eVover amass scan range of 35ndash450 amuThe temperature of theion source and connection parts was 200∘C

25 Identification and Quantification of Constituents Thequantitative data regarding the volatile constituents wereobtained by peak-area normalization using a FOCUS GCFID operated under conditions similar to those used in theGC-MS assay except for the carrier gas which was nitrogenThe retention index was calculated for all the volatile con-stituents by using an 119899-alkane (C

8ndashC20) homologous series

When possible individual components were identified bycoinjection with authentic standards Otherwise the peakassignment was performed by comparison of bothmass spec-trum and GC retention data by using authentic compoundspreviously analyzed and stored in our private library as wellas with the aid of commercial libraries containing retentionindices and mass spectra of volatile compounds commonlyfound in essential oils [23 24] Percentage (relative) of theidentified compounds was computed from the GC peak area

26 Parasites andMice Leishmania (Leishmania) amazonen-sis (IFLABR67PH8) was used for the determination ofthe anti-Leishmania activity Parasites were grown in sup-plemented Schneiderrsquos medium (10 heat-inactivated fetalbovine serum (FBS) 100UsdotmLminus1 penicillin and 100120583gmLstreptomycin at 26∘C) Murine macrophages were collectedfrom the peritoneal cavities of male and female BALBcmice (4-5 weeks old) fromMedicinal Plants Research Center(NPPMCCSUFPI) located at Teresina PI Brazil Themacrophages were maintained at a controlled temperature(24 plusmn 1∘C) and light conditions (12 h lightdark cycle) Allprotocols were approved by the Animal Research EthicsCommittee (CEEAPI no 0012012)

27 Anti-Leishmania Activity Assay Promastigotes in thelogarithmic growth phase were seeded in 96-well cell cultureplates at 1 times 106 Leishmania per well Then essential oilwas added to the wells in serial dilutions of 400 200 10050 25 125 625 and 312 120583gsdotmLminus1 The plate was kept at26∘C in a biological oxygen demand (BOD) incubator and

Evidence-Based Complementary and Alternative Medicine 3

Leishmania was observed and counted by using a Neubauerhemocytometer after 24 48 and 72 h to monitor growth andviability [25] Assays were performed in triplicate and wererepeated 3 times on different days

28 Cytotoxicity Determination Cytotoxicity of EuEO wasassessed using the MTT test In a 96-well plate 100 120583Lof supplemented RPMI 1640 medium and about 1 times 105macrophages were added per well They were then incubatedat 37∘C in 5 of CO

2for 2 h to allow cell adhesion After this

time 2 washes with supplemented RPMI 1640 medium wereperformed to remove cells that did not adhere SubsequentlyEuEO was added in triplicate after being previously dilutedin supplemented RPMI 1640 medium to a final volume of100 120583L for each well at the tested concentrations (100 50 25125 625 and 312 120583gsdotmLminus1) Cells were then incubated for48 h At the end of the incubation 10 120583L of MTT diluted inPBS was added at a final concentration of 5mgsdotmLminus1 (10 ofvolume ie 10 120583L for each 100 120583L well) and was incubatedfor an additional 4 h at 37∘C in 5 CO

2 The supernatant

was then discarded and 100120583L of DMSO was added toall wells The plate was then stirred for about 30min atroom temperature to complete formazan dissolution Finallyspectrophotometric reading was conducted at 550 nm in anELISA plate reader [26]

29 Hemolytic Activity The hemolytic activity was investi-gated by incubating 20 120583L of serially diluted essential oil inphosphate-buffered saline (PBS 400 200 100 50 25 125625 and 312 120583gsdotmLminus1) with 80 120583L of a suspension of 5 redblood cells (human O+) for 1 h at 37∘C in assay tubes Thereaction was slowed by adding 200120583L of PBS and then thesuspension was centrifuged at 1000 g for 10min Cell lysiswas then measured spectrophotometrically (540 nm) Theabsence of hemolysis (blank control) or total hemolysis (pos-itive control) was determined by replacing the essential oilsolution with an equal volume of PBS orMilli-Q sterile waterrespectivelyThe results were determined by the percentage ofhemolysis compared to the positive control (100hemolysis)and the experiments were performed in triplicate [27]

210 Treatment of Infected Macrophages Macrophages werecollected in 24-well culture plates at a concentration of 2 times105 cells500 120583L in RPMI 1640 containing sterile 13mm

round coverslips They were then incubated at 37∘C in 5of CO

2for 2 h to allow cell adhesion After this time the

medium was replaced with 500 120583L of supplemented RPMI1640 and incubated for 4 h The medium was then aspiratedand a new medium containing promastigotes (in the station-ary phase) at a ratio of 10 promastigotes to 1 macrophage wasadded to each well After 4 h of incubation in 5CO

2at 37∘C

the medium was aspirated to remove free promastigotes andthe test samples were added at nontoxic concentrations to themacrophages (312 156 and 078 120583gsdotmLminus1) This preparationwas then incubated for 48 h after which the coverslips wereremoved fixed in methanol and stained with Giemsa Foreach coverslip 100 cells were evaluated and both the number

of infected macrophages and the amount of parasites permacrophage were counted [26]

211 Lysosomal Activity Measurement of the lysosomal activ-ity was carried out according to the method of Grando etal [28] Peritoneal macrophages were plated and incubatedwith EuEO in serial dilutions of 100 50 25 125 625 and312 120583gsdotmLminus1 After 4 h of incubation at 37∘C in 5CO

2 10 120583L

of neutral red solution was added and incubated for 30minOnce this time had elapsed the supernatant was discardedthe wells were washed with 09 saline at 37∘C and 100mL ofextraction solution was added (glacial acetic acid 1 vv andethanol 50 vv dissolved in bidistilled water) to solubilizethe neutral red inside the lysosomal secretion vesicles After30min on aKline shaker (model AK 0506) the plate was readat 550 nm by using an ELISA plate reader

212 Phagocytosis Test Peritoneal macrophages were platedand incubated with EuEO in serial dilutions of 100 50 25125 625 and 312 120583gsdotmLminus1 After 48 h of incubation at 37∘Cin 5 CO

2 10 120583L of zymosan color solution was added and

incubated for 30min at 37∘C After this 100mL of Bakerrsquosfixative (formaldehyde 4 vv sodium chloride 2wv andcalcium acetate 1wv in distilled water) was added to stopthe phagocytosis process Thirty minutes later the plate waswashed with 09 saline in order to remove zymosan thatwas not phagocytosed by macrophages The supernatantwas removed and added to 100mL of extraction solutionAfter solubilization in a Kline shaker the absorbances weremeasured at 550 nm by using an ELISA plate reader [28]

213 Nitric Oxide (NO) Production In 96-well plates 2 times105 macrophages were added per well and incubated at

37∘C in 5 CO2for 4 h to allow cell adhesion A new

medium containing promastigotes (in the stationary phase)at a ratio of 10 promastigotes per macrophage was addedto half of the wells The essential oil was added after beingpreviously diluted in a culture medium containing differentconcentrations (100 50 25 125 625 and 312 120583gsdotmLminus1) inthe absence or presence of L amazonensis and was thenincubated again at 37∘C in 5CO

2for 24 h After this period

the cell culture supernatant was collected and transferred toanother plate for measurement of nitrite A standard curvewas prepared with 150 120583M sodium nitrite in Milli-Q water atvarying concentrations of 1 5 10 25 50 75 100 and 150 120583Mdiluted in a culture medium At the time of dosing 100 120583Lof either the samples or the solutions prepared for obtainingthe standard curve was mixed with an equal volume of Griessreagent The analysis was performed using an ELISA platereader at 550 nm [28]

214 Statistical Analysis All assays were performed in trip-licate and in 3 independent experiments The half-maximalinhibitory concentration (IC

50) values and 50 cytotoxicity

concentration (CC50) values with 95 confidence intervals

were calculated using a probit regressionmodel and Studentrsquos119905-test Analysis of variance (ANOVA) followed by a Bonfer-roni test were performed taking a 119875 value of lt005 as theminimum level required for statistical significance

4 Evidence-Based Complementary and Alternative Medicine

3 Results

31 Analysis of the Essential Oil The yield of EuEO was03 Thirty-two components were identified in this oil byGC-MS constituting 9265 of the total mixture EuEO wasshown to be rich in oxygenated sesquiterpenes (6255)and sesquiterpene hydrocarbons (2937) Curzerenewas themajor constituent (473) followed by 120574-elemene (1425)and trans-120573-elemenone (104) The chemical constituentsare listed in Table 1

32 Anti-Leishmania Activity Assay The inhibitory profile ofEuEO on L amazonensis promastigotes showed a significantconcentration-dependent decrease (119875 lt 005) in parasiteviability with 100 inhibition of promastigote growth atconcentrations of 400 200 and 100120583gsdotmLminus1 (Figure 1) TheIC50

was 175120583gsdotmLminus1 at 72 h of exposure (Table 2) Incultures treated with EuEO we used an optical microscopeto observe morphological changes in the promastigotes suchas cells with rounded or completely spherical shapes as wellas cellular debris in contrast to the spindle forms presentin the control (data not shown) Amphotericin B was usedas a positive control and was tested at a concentration of2120583gsdotmLminus1 The highest inhibitory effect of amphotericin Bwas observed after 24 h

33 Cytotoxicity and Hemolysis Assay The cytotoxicity ofEuEO for murine peritoneal macrophages and erythrocytesis shown in Figures 2 and 3 respectively and in Table 2The EO from E uniflora significantly altered (119875 lt 005)macrophage viability at a concentration of 625 120583gsdotmLminus1 andwas able to reduce 50 of the macrophage viability (CC

50)

at 453120583gsdotmLminus1 The cytotoxicity of EO from E uniflora forhuman blood type O+ erythrocytes was 6322 at the highestconcentration tested (400120583gsdotmLminus1) however it appearedto be nontoxic at concentrations less than 50 120583gsdotmLminus1(Figure 3)

34 Treatment of Infected Macrophages Figures 4 and 5show the results of EuEO treatment of macrophages infectedwith L amazonensis The values obtained revealed a signifi-cant and concentration-dependent reduction in macrophageinfection at 48 h with all the 3 EuEO concentrations studiedThe reduction of infection was 27 4467 and 5067at 078 156 and 312 120583gsdotmLminus1 respectively EuEO alsodecreased the number of amastigotes per infected cell in aconcentration-dependentmanner (Figure 5)The IC

50at 48 h

of exposure to EuEOwas 192120583gsdotmLminus1 (Table 2)These valuesand the CC

50values for macrophages were used to calculate

the selectivity index that is useful for assessing the safety levelof EuEO in mammalian cells (Table 2) This index representsthe relationship between CC

50and IC

50for amastigotes

35 Lysosomal Activity and Phagocytosis Assay Lysosomalactivity was assessed based on the retention of neutral redin macrophage lysosomes and was determined colorimet-rically The lysosomal activity of macrophages treated withEuEO showed significant reduction at concentrations of

Table 1 Chemical composition and retention indices of the con-stituents of Eugenia uniflora essential oil

Constituentsa RRI Ad

Calb Litc

Myrcene 989 988 019Limonene 1026 1029 018Linalool 1095 1095 016(119885)-3-Hexenyl butyrate 1186 1184 010Cuminaldehyde 1240 1238 010120575-Elemene 1335 1338 132120572-Cubebene 1349 1345 010120573-Elemene 1390 1389 551Sativene 1394 1390 006(E)-Caryophyllene 1417 1417 433trans-120572-Bergamotene 1437 1432 005120574-Elemene 1434 1434 1425Aromadendrene 1440 1439 007120572-Humulene 1455 1452 021Alloaromadendrene 1462 1458 011120573-Chamigrene 1475 1476 038Germacrene D 1482 1484 119120573-Selinene 1487 1489 070Curzerene 1498 1499 473120574-Cadinene 1513 1513 012120575-Cadinene 1523 1522 028Selina-37-(11)-diene 1546 1545 024Germacrene B 1558 1559 045Spathulenol 1578 1577 017Caryophyllene oxide 1581 1582 013Globulol 1585 1590 025Viridiflorol 1590 1592 008trans-120573-Elemenone 1597 1601 104Atractilona 1655 1657 238Germacrone 1694 1693 151Eudesm-7(11)-en-4-ol 1700 1700 033Monoterpene hydrocarbons 037Oxygenated monoterpenes 036Sesquiterpene hydrocarbons 2937Oxygenated sesquiterpenes 6255Total 9265aCompounds listed in order of elution on the DB-5ms column bRelativeretention indices (RRIs) experimentally determined against n-alkanes byusing theDB-5ms column cRRIs reported in the literature [23 24] dContentexpressed as percentages obtained by integration of the GC peak area

100 and 50 120583gsdotmLminus1 (Figure 6(a)) due to cytotoxicity (CC50

453120583gsdotmLminus1) However we observed a statistically signif-icant increase in lysosomal activity at concentrations from125 to 312 120583gsdotmLminus1 compared to control Another parameterreflecting macrophage activation was the phagocytosis ofstained zymosan when the cells were exposed to an externalstimulus The phagocytic ability of macrophages incubatedwith EuEO is shown in Figure 6(b) There was a significantincrease in phagocytosis of zymosan particles in the wells

Evidence-Based Complementary and Alternative Medicine 5

0

20

40

60

80

100

400Amph 200 100 50 25 125 625 312

Gro

wth

inhi

bitio

n (

)

lowast

24 h48 h72 h

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Figure 1 Effect of Eugenia uniflora essential oil (400 200 10050 25 125 625 and 312 120583gsdotmLminus1) or amphotericin B (Amph)(2120583gsdotmLminus1) on Leishmania amazonensis promastigotes Cultures oflog-phase promastigotes (1 times 106) were incubated at 26∘C for 2448 and 72 h in different essential oil concentrations Data representthe mean percentage of growth inhibition plusmn standard error of 3experiments carried out in triplicate lowast119875 lt 005

312 625 125 25 50 100

0102030405060708090

100

Cel

l via

bilit

y (

)

lowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 2 Cytotoxicity of Eugenia uniflora essential oil on theviability ofmurine peritonealmacrophages Peritonealmacrophageswere seeded at 1 times 105well in 96-well microplates and incubated for48 h in the presence of E uniflora L essential oil at concentrationsof 100 50 25 125 625 and 312 120583gsdotmLminus1 Viability was determinedwith 3-(45-dimethylthiazol-2-yl)25-diphenyltetrazolium bromide(MTT) and the optical density was determined at 540 nmData rep-resent the mean parasite density plusmn standard error of 3 experimentscarried out in triplicate lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

treated with EuEO at concentrations ranging from 25 to312 120583gsdotmLminus1

36 NO Production NO production was determined indi-rectly by measuring nitrite produced by macrophages treatedwith EuEO and stimulated (or not stimulated) by L amazo-nensis promastigotes Macrophages treated with EuEO but

01020304050607080

4002001005025125625312

Hem

olys

is (

)

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Figure 3 Hemolytic activity of Eugenia uniflora essential oil in a 4suspension of human O+ red blood cells after 1 h of incubation

not stimulated by Leishmania showed significant reductionin NO production at concentrations of 100 and 50120583gsdotmLminus1(Figure 7(a)) while macrophages stimulated by the parasiteshowed increased NO at a concentration of 312120583gsdotmLminus1albeit without statistical significance (119875 gt 005) (Figure 7(b))

4 Discussion

The need to identify new compounds with anti-Leishmaniaproperties that are more effective and less toxic than con-ventional drugs has motivated research on natural productsisolated from plant species These substances predominantlyconsist of alkaloids terpenes flavonoids benzopyrans phe-nolics and sesquiterpene lactones and have been identifiedin plant species with documented anti-Leishmania activity[29 30] The chemical composition of EuEO obtained in thisstudy revealed sesquiterpenes to be the dominant chemicalclass corroborating the findings of many previous studies[16 31ndash33] Despite the prevalence of sesquiterpenes thespecies has different chemotypes with different major con-stituents being observed in different studies The specimenanalyzed in this study had curzerene as themajor constituentwhich again corroborated previous reported findings [3234] According to Adio [35] germacrone is a heat-sensitivecompound that may degrade to trans-120573-elemenone duringGC analysis This may be the explanation of the presence oftrans-120573-elemenone among the major compounds of EuEOSome germacrone products have shown pharmacologicalactivity as antitumor [35]

According to Costa et al [20] the composition of theessential oil from E uniflora leaves might be influenced byfruit colorsThe plants with red fruit colors have curzerene asthe major component as in our studied plant In Maranhaowhere the samples were collected the majority of plants ofE uniflora have red fruit color [32] Selina-137(11)-trien-8-one [36] germacrene B [16 33] and 120572- and 120573-selinene [37]have been found to be the major constituents of other Euniflora chemotypes Genetic variability geographical andenvironmental conditions and the method of environmentalmanagement directly influence the yield and composition of

6 Evidence-Based Complementary and Alternative Medicine

Table 2 Anti-Leishmania and cytotoxic effects of Eugenia uniflora essential oil

Macrophage Promastigote AmastigoteCC50 (120583gsdotmLminus1) IC50 (120583gsdotmLminus1)

SIIC50 (120583gsdotmLminus1) SI

48 h 24 h 48 h 72 h 48 hEuEO 453 plusmn 245 696 plusmn 102 304 plusmn 075 175 plusmn 053 149a 192 plusmn 08 2359baSIpro (selectivity index) = CC50 macrophagesIC50 promastigote forms (48 h)bSIama (selectivity index) = CC50 macrophagesIC50 amastigote forms

078 156 312

0

20

40

60

80

100

27

44675067

Mac

roph

ages

infe

ctio

n (

)

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 4 Effect of Eugenia uniflora essential oil on macrophageinfection after 48 h of exposure Peritoneal macrophage cells wereinfected with promastigotes of Leishmania amazonensis and thentreated with 312 156 or 078 120583gsdotmLminus1 of Eugenia uniflora essentialoil Data represent the mean parasite density plusmn standard error of 3experiments carried out in triplicate lowastlowastlowast119875 lt 0001

078 156 312

0

2

4

6

8

10lowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Am

astig

otes

mac

roph

age (n

)

lowastlowastlowast

lowastlowastlowast

Figure 5 Effect of Eugenia uniflora essential oil on the survival ofLeishmania amazonensis amastigotes internalized in macrophagesLeishmania amazonensis-infected mouse peritoneal macrophageswere treated with 312 156 or 078 120583gsdotmLminus1 of Eugenia unifloraessential oil After 48 h of incubation amastigote survival wasassessed Data represent the mean parasite density plusmn standard errorof 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001

essential oils [38] It is the first report of essential oil analyzedfrom E uniflora collected in Sao Luıs MA Brazil and thiscould be an excellent source of natural product with interestfor pharmaceutical industry

The present study was conducted to evaluate the anti-Leishmania activity of EuEO on promastigotes and amasti-gotes of L amazonensis its potential mechanisms of macro-phage activation and its cytotoxicity inmammalian cellsTheEuEO showed significant concentration-dependent activityagainst promastigotes of L amazonensis with an IC

50of

175120583gsdotmLminus1 Previous studies have suggested that theseclasses of natural products may have potential anti-Leishmania activity 120573-Caryophyllene (a sesquiterpenehydrocarbon) and nerolidol (an oxygenated sesquiterpene)are examples of terpenic substances with well-characterizedanti-Leishmania activity possibly associated with inhibitionof cellular isoprenoid biosynthesis [8 39]

It has been shown that the lipophilic components ofessential oils may affect layers of polysaccharides fatty acidsand phospholipids in plasma membranes of promastigotes ofLeishmania spp This then leads to cell lysis and release ofmacromolecules [40] In the cytoplasm these substances candisrupt the specific metabolic pathways of lipids and proteinsor stimulate depolarization of mitochondrial membraneswhich can lead to cell necrosis or apoptosis [41 42]

Amphotericin B is a drug of choice for the treatment ofdifferent forms of leishmaniasis Because of this it was usedas a positive control We observed a significant inhibitoryeffect on promastigote growth after 24 h of exposure toamphotericin B but leading to an increase in viable pro-mastigote forms at 48 and 72 h Similar results were observedin previous studies [43] Some Leishmania strains have shownmechanisms of resistance to amphotericin B This may hashappenedwith the strain used in this studyThe reproductionof resistant parasites may have led to a higher cell viabilityafter 48 and 72 h [44 45]

Experimental models using amastigotes internalized intomacrophages can produce results that more closely approxi-mate those obtained using animal models since these formsare responsible for the clinicalmanifestations of leishmaniasis[46] Amastigotes can be found in parasitophorous vacuolesof infected macrophages therefore a therapeutic drug willonly be effective against Leishmania spp if it can crossthe host cell membrane and act on the amastigotes insidethe vacuole Thus assays of activity against intracellularamastigotes are the most effective way to relate the in vitroactivity of a possible substance with its in vivo effectiveness[47] The inhibition of amastigote growth was even greater at

Evidence-Based Complementary and Alternative Medicine 7

C 100 50 25 125 625 312

005

115

225

335

445

5N

eutr

al re

d up

take

ABS

lowast

lowastlowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(a)

C 100 50 25 125 625 312

0

005

01

015

02

025

Phag

ocyt

osis

ABS

lowast lowast lowastlowast lowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

(b)

Figure 6 The influence of Eugenia uniflora essential oil on the lysosomal activity (a) and phagocytic activity (b) of peritoneal macrophagesat concentrations of 100 50 25 125 625 and 312 120583gsdotmLminus1 Data represent the mean density plusmn standard error of 3 experiments carried outin triplicate lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 C control ABS absorbance

0010203040506070809

1

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119871119890119894119904ℎ119898119886119899119894119886 119886119898119886119911119900119899119890119899119904119894119904

0010203040506070809

1

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(b)

Figure 7 Production of nitric oxide (NO) Murine macrophages (2 times 105) were treated with Eugenia uniflora essential oil (100 50 25 125625 and 312 120583gsdotmLminus1) in the absence (a) or presence of Leishmania amazonensis (b) over 24 h Data represent the mean density plusmn standarderror of 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001 C control ABS absorbance

48 h after exposure to oil with an IC50of 192 120583gmL whereas

the IC50

for promastigotes was 304120583gsdotmLminus1 for the sameduration of exposure Previous studies demonstrated greateractivity against amastigotes versus promastigotes of essentialoils of Artemisia absinthium and Satureja punctata [48]

Mechanisms of anti-Leishmania action on Leishmaniaspp amastigotes have also been described for essentialoils and their terpene compounds Monoterpenes such aslinalool isolated from leaves of Croton cajucara (Euphorbia-ceae) significantly increased NO production inmacrophagesinfected with L amazonensis and acts directly on the parasiteas evidenced by mitochondrial swelling and changes in thekinetoplast and the organization of nuclear chromatin[6] In this study the production of NO was determined bymeasuring nitriteThe results frommacrophages treated with

EuEO revealed that EuEO does not induce NO productionwhich demonstrates that its promising anti-Leishmaniaactivity does not occur by this route

Natural products that are able to activate macrophagesand are thus biological response modifiers have beenextensively studied [49 50] In this context the EuEOdemonstrated potential in the activation of macrophagesby increasing phagocytic capacity and lysosomal activityimmunomodulatory activities possibly involved in its anti-Leishmania activity Phagocytosis and the lysosomal systemare critical to macrophage function because of their roles ininternalization degradation and eventually presentation ofpeptides derived from antigens required for host defense Inaddition antigen presentation occurs through phagocytosisand endosomallysosomal targeting systems [51ndash53]

8 Evidence-Based Complementary and Alternative Medicine

Due to the need for anti-Leishmania substances that aremore selective for the parasite and less toxic to host cellsand since EuEO showed low IC

50values it was important to

investigate the cytotoxic activity of EuEO againstmammaliancells EuEO showed significant toxicity in macrophages witha CC50

of 453120583gsdotmLminus1 but when compared with its IC50

value in amastigotes this indicates a secure selectivity indexabove 20 The literature recommends that for amastigotesinternalized in macrophages this index must be demon-strated as near to or greater than 20 [54] The investigationof the toxic action of new substances with anti-Leishmaniaactivity onmacrophages is important since they are themaincells of the vertebrate host parasitized by Leishmania spp[55 56] Furthermore various other cytotoxic assays can beconducted using animal and human cells to determine thesafety of a test substance leading to a future treatment invivo Tests performed with erythrocytes enable assessment ofa drugrsquos potential to cause injury to the plasma membrane ofa cell either by forming pores or by total rupture leadingto cellular damage or changes in membrane permeability[57] This is a model used for the preliminary study of theprotective and toxic effects of substances being a possibleindicator of this type of damage to cells in vivo [58ndash60]The results obtained in this test with EuEO revealed lowtoxicity to erythrocytes since EuEO showed 0 hemolysis atconcentrations of 50120583gsdotmLminus1 and lower

5 Conclusions

In conclusion the data in this study show that the essentialoil of leaves from E uniflora (EuEO) characterized bysesquiterpenes has anti-Leishmania activity in both stagesof L amazonensis This study also demonstrated that EuEOactivity is not mediated by NO production On the otherhand activation of macrophages may be involved in themechanisms of EuEO anti-Leishmania activity as evidencedby increases in both phagocytic capacity and the lysosomalcompartment Further investigation is needed to determineadditional mechanisms involved in the anti-Leishmaniaactivity of this plant species as well as in vivo studies inmodels of leishmaniasis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors are grateful to the CAPES and FAPEMA forfinancial grants through the concession of scholarships andfinancial support

References

[1] S Kaur H Sachdeva S Dhuria M Sharma and T Kaur ldquoAn-tileishmanial effect of cisplatin against murine visceral leishma-niasisrdquo Parasitology International vol 59 no 1 pp 62ndash69 2010

[2] S AzzouzMMaache R G Garcia andA Osuna ldquoLeishmani-cidal activity of edelfosine miltefosine and ilmofosinerdquo Basic

and Clinical Pharmacology and Toxicology vol 96 no 1 pp 60ndash65 2005

[3] J A Patza T K Graczykb N Gellera and A Y Vittorc ldquoEffectsof environmental change on emerging parasitic diseasesrdquo Inter-national Journal of Parasitology vol 30 no 12-13 pp 1295ndash13042000

[4] B H Alizadeh A Foroumadi S K Ardestani F Poorrajaband A Shafiee ldquoLeishmanicidal evaluation of novel syntheticchromenesrdquo Archiv der Pharmazie vol 341 no 12 pp 787ndash7932008

[5] RW Ashford ldquoThe leishmaniases as emerging and reemergingzoonosesrdquo International Journal for Parasitology vol 30 no 12-13 pp 1269ndash1281 2000

[6] M S S Rosa R R Mendonca-Filho H R Bizzo et al ldquoAntil-eishmanial activity of a linalool-rich essential oil from CrotoncajucarardquoAntimicrobial Agents Chemotherapy vol 47 no 6 pp1895ndash1901 2003

[7] T Ueda-Nakamura R R Mendonca-Filho J A Morgado-Dıazet al ldquoAntileishmanial activity of eugenol-rich essential oil fromOcimum gratissimumrdquo Parasitology International vol 55 no 2pp 55ndash59 2006

[8] A O Santos T Ueda-Nakamura B P Dias Filho V F VeigaJunior A C Pinto and C V Nakamura ldquoEffect of Braziliancopaiba oils on Leishmania amazonensisrdquo Journal of Ethnophar-macology vol 120 no 2 pp 204ndash208 2008

[9] LMonzote AMMontalvo S Almanonni R Scull MMiran-da and J Abreu ldquoActivity of the essential oil fromChenopodiumambrosioides grown in Cuba against Leishmania amazonensisrdquoChemotherapy vol 52 no 3 pp 130ndash136 2006

[10] MMachado P Pires A M Dinis et al ldquoMonoterpenic aldehy-des as potential anti-Leishmania agents activity ofCymbopogoncitratus and citral on L infantum L tropica and L majorrdquoExperimental Parasitology vol 130 no 3 pp 223ndash231 2012

[11] M R Santin A O Dos Santos C V Nakamura B P Dias FilhoI C P Ferreira and T Ueda-Nakamura ldquoIn vitro activity of theessential oil of Cymbopogon citratus and its major component(citral) on Leishmania amazonensisrdquo Parasitology Research vol105 no 6 pp 1489ndash1496 2009

[12] V C S Oliveira D M S Moura J A D Lopes P P DeAndrade N H Da Silva and R C B Q Figueiredo ldquoEffectsof essential oils from Cymbopogon citratus (DC) Stapf Lippiasidoides Cham and Ocimum gratissimum L on growth andultrastructure of Leishmania chagasi promastigotesrdquo Parasitol-ogy Research vol 104 no 5 pp 1053ndash1059 2009

[13] A E Consolini and M G Sarubbio ldquoPharmacological effectsof Eugenia uniflora (Myrtaceae) aqueous crude extract on ratrsquosheartrdquo Journal of Ethnopharmacology vol 81 no 1 pp 57ndash632002

[14] C B Alice V M F Vargas G A A B Silva et al ldquoScreening ofplants used in south Brazilian folk medicinerdquo Journal of Ethno-pharmacology vol 35 no 2 pp 165ndash171 1991

[15] P Weyerstahl H Marschall-Weyerstahl C Christiansen B OOguntimein and A O Adeoye ldquoVolatile constituents of Euge-nia uniflora leaf oilrdquo Planta medica vol 54 no 6 pp 546ndash5491988

[16] F N Victoria E J Lenardao L Savegnago et al ldquoEssential oil ofthe leaves of Eugenia uniflora L antioxidant and antimicrobialpropertiesrdquo Food and Chemical Toxicology vol 50 no 8 pp2668ndash2674 2012

Evidence-Based Complementary and Alternative Medicine 9

[17] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[18] I A Ogunwande N O Olawore O Ekundayo T M Walker JM Schmidt andWN Setzer ldquoStudies on the essential oils com-position antibacterial and cytotoxicity of Eugenia uniflora LrdquoInternational Journal of Aromatherapy vol 15 no 3 pp 147ndash1522005

[19] A C L Amorim C K F Lima A M C Hovell A L PMiranda and C M Rezende ldquoAntinociceptive and hypother-mic evaluation of the leaf essential oil and isolated terpenoidsfrom Eugenia uniflora L (Brazilian Pitanga)rdquo Phytomedicinevol 16 no 10 pp 923ndash928 2009

[20] D P Costa E G A Filho LM A Silva et al ldquoInfluence of fruitbiotypes on the chemical composition and antifungal activityof the essential oils of Eugenia uniflora leavesrdquo Journal of theBrazilian Chemical Society vol 21 no 5 pp 851ndash858 2010

[21] G F Ibikunle A C Adebajo F G Famuyiwa A J Aladesanmiand C O Adewunmi ldquoIn-vitro evaluation of anti-trichomonalactivities ofEugenia uniflora leafrdquoAfrican Journal of TraditionalComplementary and AlternativeMedicines vol 8 no 2 pp 170ndash176 2011

[22] K K Santos S R Tintino C E Souza et al ldquoAnti-Trypanosomacruzi and cytotoxic activities of Eugenia uniflora Lrdquo Experimen-tal Parasitology vol 131 no 1 pp 130ndash132 2012

[23] NIST 05 Mass Spectral Library (NISTEPANIH) NationalInstitute of Standards and Technology Gaithersburg Md USA2005

[24] R P Adams Identification of Essential Oil Components By GasChromatographyMass Spectrometry Allured Carol Stream IllUSA 4th edition 2007

[25] S M P Carneiro F A A Carvalho L C L R Santana A PL Sousa andM H Chaves ldquoThe cytotoxic and antileishmanialactivity of extracts and fractions of leaves and fruits of Azad-irachta indica (A Juss)rdquo Biological Research vol 45 no 2 pp111ndash116 2012

[26] M D G F deMedeiros A C da Silva A M D G L Cito et alldquoIn vitro antileishmanial activity and cytotoxicity of essential oilfrom Lippia sidoides Chamrdquo Parasitology International vol 60no 3 pp 237ndash241 2011

[27] S E Lofgren L C Miletti M Steindel E Bachere and M ABarracco ldquoTrypanocidal and leishmanicidal activities of differ-ent antimicrobial peptides (AMPs) isolated from aquatic ani-malsrdquo Experimental Parasitology vol 118 no 2 pp 197ndash2022008

[28] F C C Grando C A Felıcio A Twardowschy et al ldquoModula-tion of peritoneal macrophage activity by the saturation state ofthe fatty acid moiety of phosphatidylcholinerdquo Brazilian Journalof Medical and Biological Research vol 42 no 7 pp 599ndash6052009

[29] MM Iwu J E Jackson and BG Schuster ldquoMedicinal plants inthe fight against leishmaniasisrdquo Parasitology Today vol 10 no2 pp 65ndash68 1994

[30] L G Rocha J R G S Almeida R O Macedo and J M Bar-bosa-Filho ldquoA review of natural products with antileishmanialactivityrdquo Phytomedicine vol 12 no 6-7 pp 514ndash535 2005

[31] SMDeMorais A A CraveiroM I LMachado JW Alencarand F J A Matos ldquoVolatile constituents of Eugenia uniflora leafoil from northeastern Brazilrdquo Journal of Essential Oil Researchvol 8 no 4 pp 449ndash451 1996

[32] J G S Maia E H A Andrade M H L Da Silva and M G BZoghbi ldquoA new chemotype of Eugenia uniflora L from NorthBrazilrdquo Journal of Essential Oil Research vol 11 no 6 pp 727ndash729 1999

[33] J H G Lago E D Souza B Mariane et al ldquoChemical and bio-logical evaluation of essential oils from two species of myrtace-aemdashEugenia uniflora L and Plinia trunciflora (O Berg) KauselrdquoMolecules vol 16 no 12 pp 9826ndash9837 2011

[34] R Chang S A L de Morais D R Napolitano K C DuarteV B Guzman and E A do Nascimento ldquoA new approach forquantifying furanodiene and curzerene A case study on theessential oils of Eugenia uniflora (pitangueira) leavesrdquo BrazilianJournal of Pharmacognosy vol 21 no 3 pp 392ndash396 2011

[35] A M Adio ldquoGermacrenes A-E and related compounds ther-mal photochemical and acid induced transannular cycliza-tionsrdquo Tetrahedron vol 65 no 8 pp 1533ndash1552 2009

[36] D P Costa S C Santos J C Seraphin and PH Ferri ldquoSeasonalvariability of essential oils of Eugenia uniflora leavesrdquo Journal ofthe BrazilianChemical Society vol 20 no 7 pp 1287ndash1293 2009

[37] A T HenriquesM E Sobral A D Caudaro E E S SchapovalV L Bassani and G Lamaty ldquoAromatic plants from BrazilII The chemical composition of some Eugenia essential oilsrdquoJournal of Essential Oil Research vol 5 no 5 pp 501ndash505 1993

[38] B Teixeira A Marques C Ramos et al ldquoEuropean pennyroyal(Mentha pulegium) from portugal chemical composition ofessential oil and antioxidant and antimicrobial properties ofextracts and essential oilrdquo Industrial Crops and Products vol 36no 1 pp 81ndash87 2012

[39] D C Arruda F L DrsquoAlexandri A M Katzin and S RB Uliana ldquoAntileishmanial activity of the terpene nerolidolrdquoAntimicrobial Agents andChemotherapy vol 49 no 5 pp 1679ndash1687 2005

[40] RDi Pasqua G Betts NHoskinsM Edwards D Ercolini andG Mauriello ldquoMembrane toxicity of antimicrobial compoundsfrom essential oilsrdquo Journal of Agricultural and Food Chemistryvol 55 no 12 pp 4863ndash4870 2007

[41] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[42] J S Armstrong ldquoMitochondrial membrane permeabilizationthe sine qua non for cell deathrdquo BioEssays vol 28 no 3 pp253ndash260 2006

[43] A L Silva C M Adade F M Shoyama et al ldquoIn vitro leish-manicidal activity of N-dodecyl-1 2-ethanediaminerdquo Biomedi-cine and Pharmacotherapy vol 66 no 3 pp 180ndash186 2012

[44] B Purkait A Kumar N Nandi et al ldquoMechanism of ampho-tericin B resistance in clinical isolates of Leishmania donovanirdquoAntimicrobial Agents and Chemotherapy vol 56 no 2 pp 1031ndash1141 2012

[45] M Vermeersch R I Da Luz K Tote J P Timmermans P Cosand L Maes ldquoIn vitro susceptibilities of Leishmania donovanipromastigote and amastigote stages to antileishmanial referencedrugs practical relevance of stage-specific differencesrdquo Antimi-crobial Agents and Chemotherapy vol 53 no 9 pp 3855ndash38592009

[46] C Di Giorgio F Delmas V Akhmedjanova et al ldquoIn vitroantileishmanial activity of diphyllin isolated fromHaplophyllumbucharicumrdquo Planta Medica vol 71 no 4 pp 366ndash369 2005

10 Evidence-Based Complementary and Alternative Medicine

[47] H Martinez-Rojano J Mancilla-Ramirez L Quinonez-Diazand N Galindo-Sevilla ldquoActivity of hydroxyurea against Leish-mania mexicanardquo Antimicrobial Agents and Chemotherapy vol52 no 10 pp 3642ndash3647 2008

[48] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[49] A H Klimp E G E De Vries G L Scherphof and T DaemenldquoA potential role of macrophage activation in the treatment ofcancerrdquo Critical Reviews in OncologyHematology vol 44 no 2pp 143ndash161 2002

[50] M R Piemonte and D F Buchi ldquoAnalysis of IL-12 IFN-120574 and TNF-120572 production a5b1 integrins and actin filamentsdistribution in peritoneal mouse macrophages treated withhomeopathic medicamentrdquo Journal of Submicroscopic Cytologyand Pathology vol 33 no 1 pp 255ndash263 2002

[51] F Niedergang and P Chavrier ldquoSignaling and membranedynamics during phagocytosis Many roads lead to the pha-gos(R)omerdquo Current Opinion in Cell Biology vol 16 no 4 pp422ndash428 2004

[52] W L Lee R E Harrison and S Grinstein ldquoPhagocytosis byneutrophilsrdquo Microbes and Infection vol 5 no 14 pp 1299ndash1306 2003

[53] S J Green M S Meltzer J B Hibbs and C A Nacy ldquoActivatedmacrophages destroy intracellular Leishmania major amastig-otes by an L-arginine-dependent killing mechanismrdquo Journal ofImmunology vol 144 no 1 pp 278ndash283 1990

[54] E Osorio G J Arango N Jimenez et al ldquoAntiprotozoal andcytotoxic activities in vitro of Colombian Annonaceaerdquo Journalof Ethnopharmacology vol 111 no 3 pp 630ndash635 2007

[55] S Kamhawi ldquoPhlebotomine sand flies and Leishmania para-sites friends or foesrdquo Trends in Parasitology vol 22 no 9 pp439ndash445 2006

[56] P Tripathi V Singh and S Naik ldquoImmune response to leish-mania paradox rather than paradigmrdquo FEMS Immunology andMedical Microbiology vol 51 no 2 pp 229ndash242 2007

[57] L V Costa-Lotufo G M A Cunha P A M Farias et al ldquoThecytotoxic and embryotoxic effects of kaurenoic acid a diterpeneisolated fromCopaifera langsdorffii oleo-resinrdquoToxicon vol 40no 8 pp 1231ndash1234 2002

[58] R M Aparicio M J Garcıa-Celma M Pilar Vinardell and MMitjans ldquoIn vitro studies of the hemolytic activity ofmicroemul-sions in human erythrocytesrdquo Journal of Pharmaceutical andBiomedical Analysis vol 39 no 5 pp 1063ndash1067 2005

[59] L A Lexis R G Fassett and J S Coombes ldquo120572-tocopherol and120572-lipoic acid enhance the erythrocyte antioxidant defence incyclosporine A-treated ratsrdquo Basic and Clinical Pharmacologyand Toxicology vol 98 no 1 pp 68ndash73 2006

[60] J R Munoz-Castaneda J Muntane M C Munoz I BujalanceP Montilla and I Tunez ldquoEstradiol and catecholestrogens pro-tect against adriamycininduced oxidative stress in erythrocytesof ovariectomized ratsrdquo Toxicology Letters vol 160 no 3 pp196ndash203 2006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

Leishmania was observed and counted by using a Neubauerhemocytometer after 24 48 and 72 h to monitor growth andviability [25] Assays were performed in triplicate and wererepeated 3 times on different days

28 Cytotoxicity Determination Cytotoxicity of EuEO wasassessed using the MTT test In a 96-well plate 100 120583Lof supplemented RPMI 1640 medium and about 1 times 105macrophages were added per well They were then incubatedat 37∘C in 5 of CO

2for 2 h to allow cell adhesion After this

time 2 washes with supplemented RPMI 1640 medium wereperformed to remove cells that did not adhere SubsequentlyEuEO was added in triplicate after being previously dilutedin supplemented RPMI 1640 medium to a final volume of100 120583L for each well at the tested concentrations (100 50 25125 625 and 312 120583gsdotmLminus1) Cells were then incubated for48 h At the end of the incubation 10 120583L of MTT diluted inPBS was added at a final concentration of 5mgsdotmLminus1 (10 ofvolume ie 10 120583L for each 100 120583L well) and was incubatedfor an additional 4 h at 37∘C in 5 CO

2 The supernatant

was then discarded and 100120583L of DMSO was added toall wells The plate was then stirred for about 30min atroom temperature to complete formazan dissolution Finallyspectrophotometric reading was conducted at 550 nm in anELISA plate reader [26]

29 Hemolytic Activity The hemolytic activity was investi-gated by incubating 20 120583L of serially diluted essential oil inphosphate-buffered saline (PBS 400 200 100 50 25 125625 and 312 120583gsdotmLminus1) with 80 120583L of a suspension of 5 redblood cells (human O+) for 1 h at 37∘C in assay tubes Thereaction was slowed by adding 200120583L of PBS and then thesuspension was centrifuged at 1000 g for 10min Cell lysiswas then measured spectrophotometrically (540 nm) Theabsence of hemolysis (blank control) or total hemolysis (pos-itive control) was determined by replacing the essential oilsolution with an equal volume of PBS orMilli-Q sterile waterrespectivelyThe results were determined by the percentage ofhemolysis compared to the positive control (100hemolysis)and the experiments were performed in triplicate [27]

210 Treatment of Infected Macrophages Macrophages werecollected in 24-well culture plates at a concentration of 2 times105 cells500 120583L in RPMI 1640 containing sterile 13mm

round coverslips They were then incubated at 37∘C in 5of CO

2for 2 h to allow cell adhesion After this time the

medium was replaced with 500 120583L of supplemented RPMI1640 and incubated for 4 h The medium was then aspiratedand a new medium containing promastigotes (in the station-ary phase) at a ratio of 10 promastigotes to 1 macrophage wasadded to each well After 4 h of incubation in 5CO

2at 37∘C

the medium was aspirated to remove free promastigotes andthe test samples were added at nontoxic concentrations to themacrophages (312 156 and 078 120583gsdotmLminus1) This preparationwas then incubated for 48 h after which the coverslips wereremoved fixed in methanol and stained with Giemsa Foreach coverslip 100 cells were evaluated and both the number

of infected macrophages and the amount of parasites permacrophage were counted [26]

211 Lysosomal Activity Measurement of the lysosomal activ-ity was carried out according to the method of Grando etal [28] Peritoneal macrophages were plated and incubatedwith EuEO in serial dilutions of 100 50 25 125 625 and312 120583gsdotmLminus1 After 4 h of incubation at 37∘C in 5CO

2 10 120583L

of neutral red solution was added and incubated for 30minOnce this time had elapsed the supernatant was discardedthe wells were washed with 09 saline at 37∘C and 100mL ofextraction solution was added (glacial acetic acid 1 vv andethanol 50 vv dissolved in bidistilled water) to solubilizethe neutral red inside the lysosomal secretion vesicles After30min on aKline shaker (model AK 0506) the plate was readat 550 nm by using an ELISA plate reader

212 Phagocytosis Test Peritoneal macrophages were platedand incubated with EuEO in serial dilutions of 100 50 25125 625 and 312 120583gsdotmLminus1 After 48 h of incubation at 37∘Cin 5 CO

2 10 120583L of zymosan color solution was added and

incubated for 30min at 37∘C After this 100mL of Bakerrsquosfixative (formaldehyde 4 vv sodium chloride 2wv andcalcium acetate 1wv in distilled water) was added to stopthe phagocytosis process Thirty minutes later the plate waswashed with 09 saline in order to remove zymosan thatwas not phagocytosed by macrophages The supernatantwas removed and added to 100mL of extraction solutionAfter solubilization in a Kline shaker the absorbances weremeasured at 550 nm by using an ELISA plate reader [28]

213 Nitric Oxide (NO) Production In 96-well plates 2 times105 macrophages were added per well and incubated at

37∘C in 5 CO2for 4 h to allow cell adhesion A new

medium containing promastigotes (in the stationary phase)at a ratio of 10 promastigotes per macrophage was addedto half of the wells The essential oil was added after beingpreviously diluted in a culture medium containing differentconcentrations (100 50 25 125 625 and 312 120583gsdotmLminus1) inthe absence or presence of L amazonensis and was thenincubated again at 37∘C in 5CO

2for 24 h After this period

the cell culture supernatant was collected and transferred toanother plate for measurement of nitrite A standard curvewas prepared with 150 120583M sodium nitrite in Milli-Q water atvarying concentrations of 1 5 10 25 50 75 100 and 150 120583Mdiluted in a culture medium At the time of dosing 100 120583Lof either the samples or the solutions prepared for obtainingthe standard curve was mixed with an equal volume of Griessreagent The analysis was performed using an ELISA platereader at 550 nm [28]

214 Statistical Analysis All assays were performed in trip-licate and in 3 independent experiments The half-maximalinhibitory concentration (IC

50) values and 50 cytotoxicity

concentration (CC50) values with 95 confidence intervals

were calculated using a probit regressionmodel and Studentrsquos119905-test Analysis of variance (ANOVA) followed by a Bonfer-roni test were performed taking a 119875 value of lt005 as theminimum level required for statistical significance

4 Evidence-Based Complementary and Alternative Medicine

3 Results

31 Analysis of the Essential Oil The yield of EuEO was03 Thirty-two components were identified in this oil byGC-MS constituting 9265 of the total mixture EuEO wasshown to be rich in oxygenated sesquiterpenes (6255)and sesquiterpene hydrocarbons (2937) Curzerenewas themajor constituent (473) followed by 120574-elemene (1425)and trans-120573-elemenone (104) The chemical constituentsare listed in Table 1

32 Anti-Leishmania Activity Assay The inhibitory profile ofEuEO on L amazonensis promastigotes showed a significantconcentration-dependent decrease (119875 lt 005) in parasiteviability with 100 inhibition of promastigote growth atconcentrations of 400 200 and 100120583gsdotmLminus1 (Figure 1) TheIC50

was 175120583gsdotmLminus1 at 72 h of exposure (Table 2) Incultures treated with EuEO we used an optical microscopeto observe morphological changes in the promastigotes suchas cells with rounded or completely spherical shapes as wellas cellular debris in contrast to the spindle forms presentin the control (data not shown) Amphotericin B was usedas a positive control and was tested at a concentration of2120583gsdotmLminus1 The highest inhibitory effect of amphotericin Bwas observed after 24 h

33 Cytotoxicity and Hemolysis Assay The cytotoxicity ofEuEO for murine peritoneal macrophages and erythrocytesis shown in Figures 2 and 3 respectively and in Table 2The EO from E uniflora significantly altered (119875 lt 005)macrophage viability at a concentration of 625 120583gsdotmLminus1 andwas able to reduce 50 of the macrophage viability (CC

50)

at 453120583gsdotmLminus1 The cytotoxicity of EO from E uniflora forhuman blood type O+ erythrocytes was 6322 at the highestconcentration tested (400120583gsdotmLminus1) however it appearedto be nontoxic at concentrations less than 50 120583gsdotmLminus1(Figure 3)

34 Treatment of Infected Macrophages Figures 4 and 5show the results of EuEO treatment of macrophages infectedwith L amazonensis The values obtained revealed a signifi-cant and concentration-dependent reduction in macrophageinfection at 48 h with all the 3 EuEO concentrations studiedThe reduction of infection was 27 4467 and 5067at 078 156 and 312 120583gsdotmLminus1 respectively EuEO alsodecreased the number of amastigotes per infected cell in aconcentration-dependentmanner (Figure 5)The IC

50at 48 h

of exposure to EuEOwas 192120583gsdotmLminus1 (Table 2)These valuesand the CC

50values for macrophages were used to calculate

the selectivity index that is useful for assessing the safety levelof EuEO in mammalian cells (Table 2) This index representsthe relationship between CC

50and IC

50for amastigotes

35 Lysosomal Activity and Phagocytosis Assay Lysosomalactivity was assessed based on the retention of neutral redin macrophage lysosomes and was determined colorimet-rically The lysosomal activity of macrophages treated withEuEO showed significant reduction at concentrations of

Table 1 Chemical composition and retention indices of the con-stituents of Eugenia uniflora essential oil

Constituentsa RRI Ad

Calb Litc

Myrcene 989 988 019Limonene 1026 1029 018Linalool 1095 1095 016(119885)-3-Hexenyl butyrate 1186 1184 010Cuminaldehyde 1240 1238 010120575-Elemene 1335 1338 132120572-Cubebene 1349 1345 010120573-Elemene 1390 1389 551Sativene 1394 1390 006(E)-Caryophyllene 1417 1417 433trans-120572-Bergamotene 1437 1432 005120574-Elemene 1434 1434 1425Aromadendrene 1440 1439 007120572-Humulene 1455 1452 021Alloaromadendrene 1462 1458 011120573-Chamigrene 1475 1476 038Germacrene D 1482 1484 119120573-Selinene 1487 1489 070Curzerene 1498 1499 473120574-Cadinene 1513 1513 012120575-Cadinene 1523 1522 028Selina-37-(11)-diene 1546 1545 024Germacrene B 1558 1559 045Spathulenol 1578 1577 017Caryophyllene oxide 1581 1582 013Globulol 1585 1590 025Viridiflorol 1590 1592 008trans-120573-Elemenone 1597 1601 104Atractilona 1655 1657 238Germacrone 1694 1693 151Eudesm-7(11)-en-4-ol 1700 1700 033Monoterpene hydrocarbons 037Oxygenated monoterpenes 036Sesquiterpene hydrocarbons 2937Oxygenated sesquiterpenes 6255Total 9265aCompounds listed in order of elution on the DB-5ms column bRelativeretention indices (RRIs) experimentally determined against n-alkanes byusing theDB-5ms column cRRIs reported in the literature [23 24] dContentexpressed as percentages obtained by integration of the GC peak area

100 and 50 120583gsdotmLminus1 (Figure 6(a)) due to cytotoxicity (CC50

453120583gsdotmLminus1) However we observed a statistically signif-icant increase in lysosomal activity at concentrations from125 to 312 120583gsdotmLminus1 compared to control Another parameterreflecting macrophage activation was the phagocytosis ofstained zymosan when the cells were exposed to an externalstimulus The phagocytic ability of macrophages incubatedwith EuEO is shown in Figure 6(b) There was a significantincrease in phagocytosis of zymosan particles in the wells

Evidence-Based Complementary and Alternative Medicine 5

0

20

40

60

80

100

400Amph 200 100 50 25 125 625 312

Gro

wth

inhi

bitio

n (

)

lowast

24 h48 h72 h

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Figure 1 Effect of Eugenia uniflora essential oil (400 200 10050 25 125 625 and 312 120583gsdotmLminus1) or amphotericin B (Amph)(2120583gsdotmLminus1) on Leishmania amazonensis promastigotes Cultures oflog-phase promastigotes (1 times 106) were incubated at 26∘C for 2448 and 72 h in different essential oil concentrations Data representthe mean percentage of growth inhibition plusmn standard error of 3experiments carried out in triplicate lowast119875 lt 005

312 625 125 25 50 100

0102030405060708090

100

Cel

l via

bilit

y (

)

lowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 2 Cytotoxicity of Eugenia uniflora essential oil on theviability ofmurine peritonealmacrophages Peritonealmacrophageswere seeded at 1 times 105well in 96-well microplates and incubated for48 h in the presence of E uniflora L essential oil at concentrationsof 100 50 25 125 625 and 312 120583gsdotmLminus1 Viability was determinedwith 3-(45-dimethylthiazol-2-yl)25-diphenyltetrazolium bromide(MTT) and the optical density was determined at 540 nmData rep-resent the mean parasite density plusmn standard error of 3 experimentscarried out in triplicate lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

treated with EuEO at concentrations ranging from 25 to312 120583gsdotmLminus1

36 NO Production NO production was determined indi-rectly by measuring nitrite produced by macrophages treatedwith EuEO and stimulated (or not stimulated) by L amazo-nensis promastigotes Macrophages treated with EuEO but

01020304050607080

4002001005025125625312

Hem

olys

is (

)

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Figure 3 Hemolytic activity of Eugenia uniflora essential oil in a 4suspension of human O+ red blood cells after 1 h of incubation

not stimulated by Leishmania showed significant reductionin NO production at concentrations of 100 and 50120583gsdotmLminus1(Figure 7(a)) while macrophages stimulated by the parasiteshowed increased NO at a concentration of 312120583gsdotmLminus1albeit without statistical significance (119875 gt 005) (Figure 7(b))

4 Discussion

The need to identify new compounds with anti-Leishmaniaproperties that are more effective and less toxic than con-ventional drugs has motivated research on natural productsisolated from plant species These substances predominantlyconsist of alkaloids terpenes flavonoids benzopyrans phe-nolics and sesquiterpene lactones and have been identifiedin plant species with documented anti-Leishmania activity[29 30] The chemical composition of EuEO obtained in thisstudy revealed sesquiterpenes to be the dominant chemicalclass corroborating the findings of many previous studies[16 31ndash33] Despite the prevalence of sesquiterpenes thespecies has different chemotypes with different major con-stituents being observed in different studies The specimenanalyzed in this study had curzerene as themajor constituentwhich again corroborated previous reported findings [3234] According to Adio [35] germacrone is a heat-sensitivecompound that may degrade to trans-120573-elemenone duringGC analysis This may be the explanation of the presence oftrans-120573-elemenone among the major compounds of EuEOSome germacrone products have shown pharmacologicalactivity as antitumor [35]

According to Costa et al [20] the composition of theessential oil from E uniflora leaves might be influenced byfruit colorsThe plants with red fruit colors have curzerene asthe major component as in our studied plant In Maranhaowhere the samples were collected the majority of plants ofE uniflora have red fruit color [32] Selina-137(11)-trien-8-one [36] germacrene B [16 33] and 120572- and 120573-selinene [37]have been found to be the major constituents of other Euniflora chemotypes Genetic variability geographical andenvironmental conditions and the method of environmentalmanagement directly influence the yield and composition of

6 Evidence-Based Complementary and Alternative Medicine

Table 2 Anti-Leishmania and cytotoxic effects of Eugenia uniflora essential oil

Macrophage Promastigote AmastigoteCC50 (120583gsdotmLminus1) IC50 (120583gsdotmLminus1)

SIIC50 (120583gsdotmLminus1) SI

48 h 24 h 48 h 72 h 48 hEuEO 453 plusmn 245 696 plusmn 102 304 plusmn 075 175 plusmn 053 149a 192 plusmn 08 2359baSIpro (selectivity index) = CC50 macrophagesIC50 promastigote forms (48 h)bSIama (selectivity index) = CC50 macrophagesIC50 amastigote forms

078 156 312

0

20

40

60

80

100

27

44675067

Mac

roph

ages

infe

ctio

n (

)

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 4 Effect of Eugenia uniflora essential oil on macrophageinfection after 48 h of exposure Peritoneal macrophage cells wereinfected with promastigotes of Leishmania amazonensis and thentreated with 312 156 or 078 120583gsdotmLminus1 of Eugenia uniflora essentialoil Data represent the mean parasite density plusmn standard error of 3experiments carried out in triplicate lowastlowastlowast119875 lt 0001

078 156 312

0

2

4

6

8

10lowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Am

astig

otes

mac

roph

age (n

)

lowastlowastlowast

lowastlowastlowast

Figure 5 Effect of Eugenia uniflora essential oil on the survival ofLeishmania amazonensis amastigotes internalized in macrophagesLeishmania amazonensis-infected mouse peritoneal macrophageswere treated with 312 156 or 078 120583gsdotmLminus1 of Eugenia unifloraessential oil After 48 h of incubation amastigote survival wasassessed Data represent the mean parasite density plusmn standard errorof 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001

essential oils [38] It is the first report of essential oil analyzedfrom E uniflora collected in Sao Luıs MA Brazil and thiscould be an excellent source of natural product with interestfor pharmaceutical industry

The present study was conducted to evaluate the anti-Leishmania activity of EuEO on promastigotes and amasti-gotes of L amazonensis its potential mechanisms of macro-phage activation and its cytotoxicity inmammalian cellsTheEuEO showed significant concentration-dependent activityagainst promastigotes of L amazonensis with an IC

50of

175120583gsdotmLminus1 Previous studies have suggested that theseclasses of natural products may have potential anti-Leishmania activity 120573-Caryophyllene (a sesquiterpenehydrocarbon) and nerolidol (an oxygenated sesquiterpene)are examples of terpenic substances with well-characterizedanti-Leishmania activity possibly associated with inhibitionof cellular isoprenoid biosynthesis [8 39]

It has been shown that the lipophilic components ofessential oils may affect layers of polysaccharides fatty acidsand phospholipids in plasma membranes of promastigotes ofLeishmania spp This then leads to cell lysis and release ofmacromolecules [40] In the cytoplasm these substances candisrupt the specific metabolic pathways of lipids and proteinsor stimulate depolarization of mitochondrial membraneswhich can lead to cell necrosis or apoptosis [41 42]

Amphotericin B is a drug of choice for the treatment ofdifferent forms of leishmaniasis Because of this it was usedas a positive control We observed a significant inhibitoryeffect on promastigote growth after 24 h of exposure toamphotericin B but leading to an increase in viable pro-mastigote forms at 48 and 72 h Similar results were observedin previous studies [43] Some Leishmania strains have shownmechanisms of resistance to amphotericin B This may hashappenedwith the strain used in this studyThe reproductionof resistant parasites may have led to a higher cell viabilityafter 48 and 72 h [44 45]

Experimental models using amastigotes internalized intomacrophages can produce results that more closely approxi-mate those obtained using animal models since these formsare responsible for the clinicalmanifestations of leishmaniasis[46] Amastigotes can be found in parasitophorous vacuolesof infected macrophages therefore a therapeutic drug willonly be effective against Leishmania spp if it can crossthe host cell membrane and act on the amastigotes insidethe vacuole Thus assays of activity against intracellularamastigotes are the most effective way to relate the in vitroactivity of a possible substance with its in vivo effectiveness[47] The inhibition of amastigote growth was even greater at

Evidence-Based Complementary and Alternative Medicine 7

C 100 50 25 125 625 312

005

115

225

335

445

5N

eutr

al re

d up

take

ABS

lowast

lowastlowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(a)

C 100 50 25 125 625 312

0

005

01

015

02

025

Phag

ocyt

osis

ABS

lowast lowast lowastlowast lowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

(b)

Figure 6 The influence of Eugenia uniflora essential oil on the lysosomal activity (a) and phagocytic activity (b) of peritoneal macrophagesat concentrations of 100 50 25 125 625 and 312 120583gsdotmLminus1 Data represent the mean density plusmn standard error of 3 experiments carried outin triplicate lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 C control ABS absorbance

0010203040506070809

1

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119871119890119894119904ℎ119898119886119899119894119886 119886119898119886119911119900119899119890119899119904119894119904

0010203040506070809

1

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(b)

Figure 7 Production of nitric oxide (NO) Murine macrophages (2 times 105) were treated with Eugenia uniflora essential oil (100 50 25 125625 and 312 120583gsdotmLminus1) in the absence (a) or presence of Leishmania amazonensis (b) over 24 h Data represent the mean density plusmn standarderror of 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001 C control ABS absorbance

48 h after exposure to oil with an IC50of 192 120583gmL whereas

the IC50

for promastigotes was 304120583gsdotmLminus1 for the sameduration of exposure Previous studies demonstrated greateractivity against amastigotes versus promastigotes of essentialoils of Artemisia absinthium and Satureja punctata [48]

Mechanisms of anti-Leishmania action on Leishmaniaspp amastigotes have also been described for essentialoils and their terpene compounds Monoterpenes such aslinalool isolated from leaves of Croton cajucara (Euphorbia-ceae) significantly increased NO production inmacrophagesinfected with L amazonensis and acts directly on the parasiteas evidenced by mitochondrial swelling and changes in thekinetoplast and the organization of nuclear chromatin[6] In this study the production of NO was determined bymeasuring nitriteThe results frommacrophages treated with

EuEO revealed that EuEO does not induce NO productionwhich demonstrates that its promising anti-Leishmaniaactivity does not occur by this route

Natural products that are able to activate macrophagesand are thus biological response modifiers have beenextensively studied [49 50] In this context the EuEOdemonstrated potential in the activation of macrophagesby increasing phagocytic capacity and lysosomal activityimmunomodulatory activities possibly involved in its anti-Leishmania activity Phagocytosis and the lysosomal systemare critical to macrophage function because of their roles ininternalization degradation and eventually presentation ofpeptides derived from antigens required for host defense Inaddition antigen presentation occurs through phagocytosisand endosomallysosomal targeting systems [51ndash53]

8 Evidence-Based Complementary and Alternative Medicine

Due to the need for anti-Leishmania substances that aremore selective for the parasite and less toxic to host cellsand since EuEO showed low IC

50values it was important to

investigate the cytotoxic activity of EuEO againstmammaliancells EuEO showed significant toxicity in macrophages witha CC50

of 453120583gsdotmLminus1 but when compared with its IC50

value in amastigotes this indicates a secure selectivity indexabove 20 The literature recommends that for amastigotesinternalized in macrophages this index must be demon-strated as near to or greater than 20 [54] The investigationof the toxic action of new substances with anti-Leishmaniaactivity onmacrophages is important since they are themaincells of the vertebrate host parasitized by Leishmania spp[55 56] Furthermore various other cytotoxic assays can beconducted using animal and human cells to determine thesafety of a test substance leading to a future treatment invivo Tests performed with erythrocytes enable assessment ofa drugrsquos potential to cause injury to the plasma membrane ofa cell either by forming pores or by total rupture leadingto cellular damage or changes in membrane permeability[57] This is a model used for the preliminary study of theprotective and toxic effects of substances being a possibleindicator of this type of damage to cells in vivo [58ndash60]The results obtained in this test with EuEO revealed lowtoxicity to erythrocytes since EuEO showed 0 hemolysis atconcentrations of 50120583gsdotmLminus1 and lower

5 Conclusions

In conclusion the data in this study show that the essentialoil of leaves from E uniflora (EuEO) characterized bysesquiterpenes has anti-Leishmania activity in both stagesof L amazonensis This study also demonstrated that EuEOactivity is not mediated by NO production On the otherhand activation of macrophages may be involved in themechanisms of EuEO anti-Leishmania activity as evidencedby increases in both phagocytic capacity and the lysosomalcompartment Further investigation is needed to determineadditional mechanisms involved in the anti-Leishmaniaactivity of this plant species as well as in vivo studies inmodels of leishmaniasis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors are grateful to the CAPES and FAPEMA forfinancial grants through the concession of scholarships andfinancial support

References

[1] S Kaur H Sachdeva S Dhuria M Sharma and T Kaur ldquoAn-tileishmanial effect of cisplatin against murine visceral leishma-niasisrdquo Parasitology International vol 59 no 1 pp 62ndash69 2010

[2] S AzzouzMMaache R G Garcia andA Osuna ldquoLeishmani-cidal activity of edelfosine miltefosine and ilmofosinerdquo Basic

and Clinical Pharmacology and Toxicology vol 96 no 1 pp 60ndash65 2005

[3] J A Patza T K Graczykb N Gellera and A Y Vittorc ldquoEffectsof environmental change on emerging parasitic diseasesrdquo Inter-national Journal of Parasitology vol 30 no 12-13 pp 1295ndash13042000

[4] B H Alizadeh A Foroumadi S K Ardestani F Poorrajaband A Shafiee ldquoLeishmanicidal evaluation of novel syntheticchromenesrdquo Archiv der Pharmazie vol 341 no 12 pp 787ndash7932008

[5] RW Ashford ldquoThe leishmaniases as emerging and reemergingzoonosesrdquo International Journal for Parasitology vol 30 no 12-13 pp 1269ndash1281 2000

[6] M S S Rosa R R Mendonca-Filho H R Bizzo et al ldquoAntil-eishmanial activity of a linalool-rich essential oil from CrotoncajucarardquoAntimicrobial Agents Chemotherapy vol 47 no 6 pp1895ndash1901 2003

[7] T Ueda-Nakamura R R Mendonca-Filho J A Morgado-Dıazet al ldquoAntileishmanial activity of eugenol-rich essential oil fromOcimum gratissimumrdquo Parasitology International vol 55 no 2pp 55ndash59 2006

[8] A O Santos T Ueda-Nakamura B P Dias Filho V F VeigaJunior A C Pinto and C V Nakamura ldquoEffect of Braziliancopaiba oils on Leishmania amazonensisrdquo Journal of Ethnophar-macology vol 120 no 2 pp 204ndash208 2008

[9] LMonzote AMMontalvo S Almanonni R Scull MMiran-da and J Abreu ldquoActivity of the essential oil fromChenopodiumambrosioides grown in Cuba against Leishmania amazonensisrdquoChemotherapy vol 52 no 3 pp 130ndash136 2006

[10] MMachado P Pires A M Dinis et al ldquoMonoterpenic aldehy-des as potential anti-Leishmania agents activity ofCymbopogoncitratus and citral on L infantum L tropica and L majorrdquoExperimental Parasitology vol 130 no 3 pp 223ndash231 2012

[11] M R Santin A O Dos Santos C V Nakamura B P Dias FilhoI C P Ferreira and T Ueda-Nakamura ldquoIn vitro activity of theessential oil of Cymbopogon citratus and its major component(citral) on Leishmania amazonensisrdquo Parasitology Research vol105 no 6 pp 1489ndash1496 2009

[12] V C S Oliveira D M S Moura J A D Lopes P P DeAndrade N H Da Silva and R C B Q Figueiredo ldquoEffectsof essential oils from Cymbopogon citratus (DC) Stapf Lippiasidoides Cham and Ocimum gratissimum L on growth andultrastructure of Leishmania chagasi promastigotesrdquo Parasitol-ogy Research vol 104 no 5 pp 1053ndash1059 2009

[13] A E Consolini and M G Sarubbio ldquoPharmacological effectsof Eugenia uniflora (Myrtaceae) aqueous crude extract on ratrsquosheartrdquo Journal of Ethnopharmacology vol 81 no 1 pp 57ndash632002

[14] C B Alice V M F Vargas G A A B Silva et al ldquoScreening ofplants used in south Brazilian folk medicinerdquo Journal of Ethno-pharmacology vol 35 no 2 pp 165ndash171 1991

[15] P Weyerstahl H Marschall-Weyerstahl C Christiansen B OOguntimein and A O Adeoye ldquoVolatile constituents of Euge-nia uniflora leaf oilrdquo Planta medica vol 54 no 6 pp 546ndash5491988

[16] F N Victoria E J Lenardao L Savegnago et al ldquoEssential oil ofthe leaves of Eugenia uniflora L antioxidant and antimicrobialpropertiesrdquo Food and Chemical Toxicology vol 50 no 8 pp2668ndash2674 2012

Evidence-Based Complementary and Alternative Medicine 9

[17] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[18] I A Ogunwande N O Olawore O Ekundayo T M Walker JM Schmidt andWN Setzer ldquoStudies on the essential oils com-position antibacterial and cytotoxicity of Eugenia uniflora LrdquoInternational Journal of Aromatherapy vol 15 no 3 pp 147ndash1522005

[19] A C L Amorim C K F Lima A M C Hovell A L PMiranda and C M Rezende ldquoAntinociceptive and hypother-mic evaluation of the leaf essential oil and isolated terpenoidsfrom Eugenia uniflora L (Brazilian Pitanga)rdquo Phytomedicinevol 16 no 10 pp 923ndash928 2009

[20] D P Costa E G A Filho LM A Silva et al ldquoInfluence of fruitbiotypes on the chemical composition and antifungal activityof the essential oils of Eugenia uniflora leavesrdquo Journal of theBrazilian Chemical Society vol 21 no 5 pp 851ndash858 2010

[21] G F Ibikunle A C Adebajo F G Famuyiwa A J Aladesanmiand C O Adewunmi ldquoIn-vitro evaluation of anti-trichomonalactivities ofEugenia uniflora leafrdquoAfrican Journal of TraditionalComplementary and AlternativeMedicines vol 8 no 2 pp 170ndash176 2011

[22] K K Santos S R Tintino C E Souza et al ldquoAnti-Trypanosomacruzi and cytotoxic activities of Eugenia uniflora Lrdquo Experimen-tal Parasitology vol 131 no 1 pp 130ndash132 2012

[23] NIST 05 Mass Spectral Library (NISTEPANIH) NationalInstitute of Standards and Technology Gaithersburg Md USA2005

[24] R P Adams Identification of Essential Oil Components By GasChromatographyMass Spectrometry Allured Carol Stream IllUSA 4th edition 2007

[25] S M P Carneiro F A A Carvalho L C L R Santana A PL Sousa andM H Chaves ldquoThe cytotoxic and antileishmanialactivity of extracts and fractions of leaves and fruits of Azad-irachta indica (A Juss)rdquo Biological Research vol 45 no 2 pp111ndash116 2012

[26] M D G F deMedeiros A C da Silva A M D G L Cito et alldquoIn vitro antileishmanial activity and cytotoxicity of essential oilfrom Lippia sidoides Chamrdquo Parasitology International vol 60no 3 pp 237ndash241 2011

[27] S E Lofgren L C Miletti M Steindel E Bachere and M ABarracco ldquoTrypanocidal and leishmanicidal activities of differ-ent antimicrobial peptides (AMPs) isolated from aquatic ani-malsrdquo Experimental Parasitology vol 118 no 2 pp 197ndash2022008

[28] F C C Grando C A Felıcio A Twardowschy et al ldquoModula-tion of peritoneal macrophage activity by the saturation state ofthe fatty acid moiety of phosphatidylcholinerdquo Brazilian Journalof Medical and Biological Research vol 42 no 7 pp 599ndash6052009

[29] MM Iwu J E Jackson and BG Schuster ldquoMedicinal plants inthe fight against leishmaniasisrdquo Parasitology Today vol 10 no2 pp 65ndash68 1994

[30] L G Rocha J R G S Almeida R O Macedo and J M Bar-bosa-Filho ldquoA review of natural products with antileishmanialactivityrdquo Phytomedicine vol 12 no 6-7 pp 514ndash535 2005

[31] SMDeMorais A A CraveiroM I LMachado JW Alencarand F J A Matos ldquoVolatile constituents of Eugenia uniflora leafoil from northeastern Brazilrdquo Journal of Essential Oil Researchvol 8 no 4 pp 449ndash451 1996

[32] J G S Maia E H A Andrade M H L Da Silva and M G BZoghbi ldquoA new chemotype of Eugenia uniflora L from NorthBrazilrdquo Journal of Essential Oil Research vol 11 no 6 pp 727ndash729 1999

[33] J H G Lago E D Souza B Mariane et al ldquoChemical and bio-logical evaluation of essential oils from two species of myrtace-aemdashEugenia uniflora L and Plinia trunciflora (O Berg) KauselrdquoMolecules vol 16 no 12 pp 9826ndash9837 2011

[34] R Chang S A L de Morais D R Napolitano K C DuarteV B Guzman and E A do Nascimento ldquoA new approach forquantifying furanodiene and curzerene A case study on theessential oils of Eugenia uniflora (pitangueira) leavesrdquo BrazilianJournal of Pharmacognosy vol 21 no 3 pp 392ndash396 2011

[35] A M Adio ldquoGermacrenes A-E and related compounds ther-mal photochemical and acid induced transannular cycliza-tionsrdquo Tetrahedron vol 65 no 8 pp 1533ndash1552 2009

[36] D P Costa S C Santos J C Seraphin and PH Ferri ldquoSeasonalvariability of essential oils of Eugenia uniflora leavesrdquo Journal ofthe BrazilianChemical Society vol 20 no 7 pp 1287ndash1293 2009

[37] A T HenriquesM E Sobral A D Caudaro E E S SchapovalV L Bassani and G Lamaty ldquoAromatic plants from BrazilII The chemical composition of some Eugenia essential oilsrdquoJournal of Essential Oil Research vol 5 no 5 pp 501ndash505 1993

[38] B Teixeira A Marques C Ramos et al ldquoEuropean pennyroyal(Mentha pulegium) from portugal chemical composition ofessential oil and antioxidant and antimicrobial properties ofextracts and essential oilrdquo Industrial Crops and Products vol 36no 1 pp 81ndash87 2012

[39] D C Arruda F L DrsquoAlexandri A M Katzin and S RB Uliana ldquoAntileishmanial activity of the terpene nerolidolrdquoAntimicrobial Agents andChemotherapy vol 49 no 5 pp 1679ndash1687 2005

[40] RDi Pasqua G Betts NHoskinsM Edwards D Ercolini andG Mauriello ldquoMembrane toxicity of antimicrobial compoundsfrom essential oilsrdquo Journal of Agricultural and Food Chemistryvol 55 no 12 pp 4863ndash4870 2007

[41] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[42] J S Armstrong ldquoMitochondrial membrane permeabilizationthe sine qua non for cell deathrdquo BioEssays vol 28 no 3 pp253ndash260 2006

[43] A L Silva C M Adade F M Shoyama et al ldquoIn vitro leish-manicidal activity of N-dodecyl-1 2-ethanediaminerdquo Biomedi-cine and Pharmacotherapy vol 66 no 3 pp 180ndash186 2012

[44] B Purkait A Kumar N Nandi et al ldquoMechanism of ampho-tericin B resistance in clinical isolates of Leishmania donovanirdquoAntimicrobial Agents and Chemotherapy vol 56 no 2 pp 1031ndash1141 2012

[45] M Vermeersch R I Da Luz K Tote J P Timmermans P Cosand L Maes ldquoIn vitro susceptibilities of Leishmania donovanipromastigote and amastigote stages to antileishmanial referencedrugs practical relevance of stage-specific differencesrdquo Antimi-crobial Agents and Chemotherapy vol 53 no 9 pp 3855ndash38592009

[46] C Di Giorgio F Delmas V Akhmedjanova et al ldquoIn vitroantileishmanial activity of diphyllin isolated fromHaplophyllumbucharicumrdquo Planta Medica vol 71 no 4 pp 366ndash369 2005

10 Evidence-Based Complementary and Alternative Medicine

[47] H Martinez-Rojano J Mancilla-Ramirez L Quinonez-Diazand N Galindo-Sevilla ldquoActivity of hydroxyurea against Leish-mania mexicanardquo Antimicrobial Agents and Chemotherapy vol52 no 10 pp 3642ndash3647 2008

[48] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[49] A H Klimp E G E De Vries G L Scherphof and T DaemenldquoA potential role of macrophage activation in the treatment ofcancerrdquo Critical Reviews in OncologyHematology vol 44 no 2pp 143ndash161 2002

[50] M R Piemonte and D F Buchi ldquoAnalysis of IL-12 IFN-120574 and TNF-120572 production a5b1 integrins and actin filamentsdistribution in peritoneal mouse macrophages treated withhomeopathic medicamentrdquo Journal of Submicroscopic Cytologyand Pathology vol 33 no 1 pp 255ndash263 2002

[51] F Niedergang and P Chavrier ldquoSignaling and membranedynamics during phagocytosis Many roads lead to the pha-gos(R)omerdquo Current Opinion in Cell Biology vol 16 no 4 pp422ndash428 2004

[52] W L Lee R E Harrison and S Grinstein ldquoPhagocytosis byneutrophilsrdquo Microbes and Infection vol 5 no 14 pp 1299ndash1306 2003

[53] S J Green M S Meltzer J B Hibbs and C A Nacy ldquoActivatedmacrophages destroy intracellular Leishmania major amastig-otes by an L-arginine-dependent killing mechanismrdquo Journal ofImmunology vol 144 no 1 pp 278ndash283 1990

[54] E Osorio G J Arango N Jimenez et al ldquoAntiprotozoal andcytotoxic activities in vitro of Colombian Annonaceaerdquo Journalof Ethnopharmacology vol 111 no 3 pp 630ndash635 2007

[55] S Kamhawi ldquoPhlebotomine sand flies and Leishmania para-sites friends or foesrdquo Trends in Parasitology vol 22 no 9 pp439ndash445 2006

[56] P Tripathi V Singh and S Naik ldquoImmune response to leish-mania paradox rather than paradigmrdquo FEMS Immunology andMedical Microbiology vol 51 no 2 pp 229ndash242 2007

[57] L V Costa-Lotufo G M A Cunha P A M Farias et al ldquoThecytotoxic and embryotoxic effects of kaurenoic acid a diterpeneisolated fromCopaifera langsdorffii oleo-resinrdquoToxicon vol 40no 8 pp 1231ndash1234 2002

[58] R M Aparicio M J Garcıa-Celma M Pilar Vinardell and MMitjans ldquoIn vitro studies of the hemolytic activity ofmicroemul-sions in human erythrocytesrdquo Journal of Pharmaceutical andBiomedical Analysis vol 39 no 5 pp 1063ndash1067 2005

[59] L A Lexis R G Fassett and J S Coombes ldquo120572-tocopherol and120572-lipoic acid enhance the erythrocyte antioxidant defence incyclosporine A-treated ratsrdquo Basic and Clinical Pharmacologyand Toxicology vol 98 no 1 pp 68ndash73 2006

[60] J R Munoz-Castaneda J Muntane M C Munoz I BujalanceP Montilla and I Tunez ldquoEstradiol and catecholestrogens pro-tect against adriamycininduced oxidative stress in erythrocytesof ovariectomized ratsrdquo Toxicology Letters vol 160 no 3 pp196ndash203 2006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

3 Results

31 Analysis of the Essential Oil The yield of EuEO was03 Thirty-two components were identified in this oil byGC-MS constituting 9265 of the total mixture EuEO wasshown to be rich in oxygenated sesquiterpenes (6255)and sesquiterpene hydrocarbons (2937) Curzerenewas themajor constituent (473) followed by 120574-elemene (1425)and trans-120573-elemenone (104) The chemical constituentsare listed in Table 1

32 Anti-Leishmania Activity Assay The inhibitory profile ofEuEO on L amazonensis promastigotes showed a significantconcentration-dependent decrease (119875 lt 005) in parasiteviability with 100 inhibition of promastigote growth atconcentrations of 400 200 and 100120583gsdotmLminus1 (Figure 1) TheIC50

was 175120583gsdotmLminus1 at 72 h of exposure (Table 2) Incultures treated with EuEO we used an optical microscopeto observe morphological changes in the promastigotes suchas cells with rounded or completely spherical shapes as wellas cellular debris in contrast to the spindle forms presentin the control (data not shown) Amphotericin B was usedas a positive control and was tested at a concentration of2120583gsdotmLminus1 The highest inhibitory effect of amphotericin Bwas observed after 24 h

33 Cytotoxicity and Hemolysis Assay The cytotoxicity ofEuEO for murine peritoneal macrophages and erythrocytesis shown in Figures 2 and 3 respectively and in Table 2The EO from E uniflora significantly altered (119875 lt 005)macrophage viability at a concentration of 625 120583gsdotmLminus1 andwas able to reduce 50 of the macrophage viability (CC

50)

at 453120583gsdotmLminus1 The cytotoxicity of EO from E uniflora forhuman blood type O+ erythrocytes was 6322 at the highestconcentration tested (400120583gsdotmLminus1) however it appearedto be nontoxic at concentrations less than 50 120583gsdotmLminus1(Figure 3)

34 Treatment of Infected Macrophages Figures 4 and 5show the results of EuEO treatment of macrophages infectedwith L amazonensis The values obtained revealed a signifi-cant and concentration-dependent reduction in macrophageinfection at 48 h with all the 3 EuEO concentrations studiedThe reduction of infection was 27 4467 and 5067at 078 156 and 312 120583gsdotmLminus1 respectively EuEO alsodecreased the number of amastigotes per infected cell in aconcentration-dependentmanner (Figure 5)The IC

50at 48 h

of exposure to EuEOwas 192120583gsdotmLminus1 (Table 2)These valuesand the CC

50values for macrophages were used to calculate

the selectivity index that is useful for assessing the safety levelof EuEO in mammalian cells (Table 2) This index representsthe relationship between CC

50and IC

50for amastigotes

35 Lysosomal Activity and Phagocytosis Assay Lysosomalactivity was assessed based on the retention of neutral redin macrophage lysosomes and was determined colorimet-rically The lysosomal activity of macrophages treated withEuEO showed significant reduction at concentrations of

Table 1 Chemical composition and retention indices of the con-stituents of Eugenia uniflora essential oil

Constituentsa RRI Ad

Calb Litc

Myrcene 989 988 019Limonene 1026 1029 018Linalool 1095 1095 016(119885)-3-Hexenyl butyrate 1186 1184 010Cuminaldehyde 1240 1238 010120575-Elemene 1335 1338 132120572-Cubebene 1349 1345 010120573-Elemene 1390 1389 551Sativene 1394 1390 006(E)-Caryophyllene 1417 1417 433trans-120572-Bergamotene 1437 1432 005120574-Elemene 1434 1434 1425Aromadendrene 1440 1439 007120572-Humulene 1455 1452 021Alloaromadendrene 1462 1458 011120573-Chamigrene 1475 1476 038Germacrene D 1482 1484 119120573-Selinene 1487 1489 070Curzerene 1498 1499 473120574-Cadinene 1513 1513 012120575-Cadinene 1523 1522 028Selina-37-(11)-diene 1546 1545 024Germacrene B 1558 1559 045Spathulenol 1578 1577 017Caryophyllene oxide 1581 1582 013Globulol 1585 1590 025Viridiflorol 1590 1592 008trans-120573-Elemenone 1597 1601 104Atractilona 1655 1657 238Germacrone 1694 1693 151Eudesm-7(11)-en-4-ol 1700 1700 033Monoterpene hydrocarbons 037Oxygenated monoterpenes 036Sesquiterpene hydrocarbons 2937Oxygenated sesquiterpenes 6255Total 9265aCompounds listed in order of elution on the DB-5ms column bRelativeretention indices (RRIs) experimentally determined against n-alkanes byusing theDB-5ms column cRRIs reported in the literature [23 24] dContentexpressed as percentages obtained by integration of the GC peak area

100 and 50 120583gsdotmLminus1 (Figure 6(a)) due to cytotoxicity (CC50

453120583gsdotmLminus1) However we observed a statistically signif-icant increase in lysosomal activity at concentrations from125 to 312 120583gsdotmLminus1 compared to control Another parameterreflecting macrophage activation was the phagocytosis ofstained zymosan when the cells were exposed to an externalstimulus The phagocytic ability of macrophages incubatedwith EuEO is shown in Figure 6(b) There was a significantincrease in phagocytosis of zymosan particles in the wells

Evidence-Based Complementary and Alternative Medicine 5

0

20

40

60

80

100

400Amph 200 100 50 25 125 625 312

Gro

wth

inhi

bitio

n (

)

lowast

24 h48 h72 h

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Figure 1 Effect of Eugenia uniflora essential oil (400 200 10050 25 125 625 and 312 120583gsdotmLminus1) or amphotericin B (Amph)(2120583gsdotmLminus1) on Leishmania amazonensis promastigotes Cultures oflog-phase promastigotes (1 times 106) were incubated at 26∘C for 2448 and 72 h in different essential oil concentrations Data representthe mean percentage of growth inhibition plusmn standard error of 3experiments carried out in triplicate lowast119875 lt 005

312 625 125 25 50 100

0102030405060708090

100

Cel

l via

bilit

y (

)

lowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 2 Cytotoxicity of Eugenia uniflora essential oil on theviability ofmurine peritonealmacrophages Peritonealmacrophageswere seeded at 1 times 105well in 96-well microplates and incubated for48 h in the presence of E uniflora L essential oil at concentrationsof 100 50 25 125 625 and 312 120583gsdotmLminus1 Viability was determinedwith 3-(45-dimethylthiazol-2-yl)25-diphenyltetrazolium bromide(MTT) and the optical density was determined at 540 nmData rep-resent the mean parasite density plusmn standard error of 3 experimentscarried out in triplicate lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

treated with EuEO at concentrations ranging from 25 to312 120583gsdotmLminus1

36 NO Production NO production was determined indi-rectly by measuring nitrite produced by macrophages treatedwith EuEO and stimulated (or not stimulated) by L amazo-nensis promastigotes Macrophages treated with EuEO but

01020304050607080

4002001005025125625312

Hem

olys

is (

)

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Figure 3 Hemolytic activity of Eugenia uniflora essential oil in a 4suspension of human O+ red blood cells after 1 h of incubation

not stimulated by Leishmania showed significant reductionin NO production at concentrations of 100 and 50120583gsdotmLminus1(Figure 7(a)) while macrophages stimulated by the parasiteshowed increased NO at a concentration of 312120583gsdotmLminus1albeit without statistical significance (119875 gt 005) (Figure 7(b))

4 Discussion

The need to identify new compounds with anti-Leishmaniaproperties that are more effective and less toxic than con-ventional drugs has motivated research on natural productsisolated from plant species These substances predominantlyconsist of alkaloids terpenes flavonoids benzopyrans phe-nolics and sesquiterpene lactones and have been identifiedin plant species with documented anti-Leishmania activity[29 30] The chemical composition of EuEO obtained in thisstudy revealed sesquiterpenes to be the dominant chemicalclass corroborating the findings of many previous studies[16 31ndash33] Despite the prevalence of sesquiterpenes thespecies has different chemotypes with different major con-stituents being observed in different studies The specimenanalyzed in this study had curzerene as themajor constituentwhich again corroborated previous reported findings [3234] According to Adio [35] germacrone is a heat-sensitivecompound that may degrade to trans-120573-elemenone duringGC analysis This may be the explanation of the presence oftrans-120573-elemenone among the major compounds of EuEOSome germacrone products have shown pharmacologicalactivity as antitumor [35]

According to Costa et al [20] the composition of theessential oil from E uniflora leaves might be influenced byfruit colorsThe plants with red fruit colors have curzerene asthe major component as in our studied plant In Maranhaowhere the samples were collected the majority of plants ofE uniflora have red fruit color [32] Selina-137(11)-trien-8-one [36] germacrene B [16 33] and 120572- and 120573-selinene [37]have been found to be the major constituents of other Euniflora chemotypes Genetic variability geographical andenvironmental conditions and the method of environmentalmanagement directly influence the yield and composition of

6 Evidence-Based Complementary and Alternative Medicine

Table 2 Anti-Leishmania and cytotoxic effects of Eugenia uniflora essential oil

Macrophage Promastigote AmastigoteCC50 (120583gsdotmLminus1) IC50 (120583gsdotmLminus1)

SIIC50 (120583gsdotmLminus1) SI

48 h 24 h 48 h 72 h 48 hEuEO 453 plusmn 245 696 plusmn 102 304 plusmn 075 175 plusmn 053 149a 192 plusmn 08 2359baSIpro (selectivity index) = CC50 macrophagesIC50 promastigote forms (48 h)bSIama (selectivity index) = CC50 macrophagesIC50 amastigote forms

078 156 312

0

20

40

60

80

100

27

44675067

Mac

roph

ages

infe

ctio

n (

)

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 4 Effect of Eugenia uniflora essential oil on macrophageinfection after 48 h of exposure Peritoneal macrophage cells wereinfected with promastigotes of Leishmania amazonensis and thentreated with 312 156 or 078 120583gsdotmLminus1 of Eugenia uniflora essentialoil Data represent the mean parasite density plusmn standard error of 3experiments carried out in triplicate lowastlowastlowast119875 lt 0001

078 156 312

0

2

4

6

8

10lowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Am

astig

otes

mac

roph

age (n

)

lowastlowastlowast

lowastlowastlowast

Figure 5 Effect of Eugenia uniflora essential oil on the survival ofLeishmania amazonensis amastigotes internalized in macrophagesLeishmania amazonensis-infected mouse peritoneal macrophageswere treated with 312 156 or 078 120583gsdotmLminus1 of Eugenia unifloraessential oil After 48 h of incubation amastigote survival wasassessed Data represent the mean parasite density plusmn standard errorof 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001

essential oils [38] It is the first report of essential oil analyzedfrom E uniflora collected in Sao Luıs MA Brazil and thiscould be an excellent source of natural product with interestfor pharmaceutical industry

The present study was conducted to evaluate the anti-Leishmania activity of EuEO on promastigotes and amasti-gotes of L amazonensis its potential mechanisms of macro-phage activation and its cytotoxicity inmammalian cellsTheEuEO showed significant concentration-dependent activityagainst promastigotes of L amazonensis with an IC

50of

175120583gsdotmLminus1 Previous studies have suggested that theseclasses of natural products may have potential anti-Leishmania activity 120573-Caryophyllene (a sesquiterpenehydrocarbon) and nerolidol (an oxygenated sesquiterpene)are examples of terpenic substances with well-characterizedanti-Leishmania activity possibly associated with inhibitionof cellular isoprenoid biosynthesis [8 39]

It has been shown that the lipophilic components ofessential oils may affect layers of polysaccharides fatty acidsand phospholipids in plasma membranes of promastigotes ofLeishmania spp This then leads to cell lysis and release ofmacromolecules [40] In the cytoplasm these substances candisrupt the specific metabolic pathways of lipids and proteinsor stimulate depolarization of mitochondrial membraneswhich can lead to cell necrosis or apoptosis [41 42]

Amphotericin B is a drug of choice for the treatment ofdifferent forms of leishmaniasis Because of this it was usedas a positive control We observed a significant inhibitoryeffect on promastigote growth after 24 h of exposure toamphotericin B but leading to an increase in viable pro-mastigote forms at 48 and 72 h Similar results were observedin previous studies [43] Some Leishmania strains have shownmechanisms of resistance to amphotericin B This may hashappenedwith the strain used in this studyThe reproductionof resistant parasites may have led to a higher cell viabilityafter 48 and 72 h [44 45]

Experimental models using amastigotes internalized intomacrophages can produce results that more closely approxi-mate those obtained using animal models since these formsare responsible for the clinicalmanifestations of leishmaniasis[46] Amastigotes can be found in parasitophorous vacuolesof infected macrophages therefore a therapeutic drug willonly be effective against Leishmania spp if it can crossthe host cell membrane and act on the amastigotes insidethe vacuole Thus assays of activity against intracellularamastigotes are the most effective way to relate the in vitroactivity of a possible substance with its in vivo effectiveness[47] The inhibition of amastigote growth was even greater at

Evidence-Based Complementary and Alternative Medicine 7

C 100 50 25 125 625 312

005

115

225

335

445

5N

eutr

al re

d up

take

ABS

lowast

lowastlowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(a)

C 100 50 25 125 625 312

0

005

01

015

02

025

Phag

ocyt

osis

ABS

lowast lowast lowastlowast lowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

(b)

Figure 6 The influence of Eugenia uniflora essential oil on the lysosomal activity (a) and phagocytic activity (b) of peritoneal macrophagesat concentrations of 100 50 25 125 625 and 312 120583gsdotmLminus1 Data represent the mean density plusmn standard error of 3 experiments carried outin triplicate lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 C control ABS absorbance

0010203040506070809

1

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119871119890119894119904ℎ119898119886119899119894119886 119886119898119886119911119900119899119890119899119904119894119904

0010203040506070809

1

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(b)

Figure 7 Production of nitric oxide (NO) Murine macrophages (2 times 105) were treated with Eugenia uniflora essential oil (100 50 25 125625 and 312 120583gsdotmLminus1) in the absence (a) or presence of Leishmania amazonensis (b) over 24 h Data represent the mean density plusmn standarderror of 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001 C control ABS absorbance

48 h after exposure to oil with an IC50of 192 120583gmL whereas

the IC50

for promastigotes was 304120583gsdotmLminus1 for the sameduration of exposure Previous studies demonstrated greateractivity against amastigotes versus promastigotes of essentialoils of Artemisia absinthium and Satureja punctata [48]

Mechanisms of anti-Leishmania action on Leishmaniaspp amastigotes have also been described for essentialoils and their terpene compounds Monoterpenes such aslinalool isolated from leaves of Croton cajucara (Euphorbia-ceae) significantly increased NO production inmacrophagesinfected with L amazonensis and acts directly on the parasiteas evidenced by mitochondrial swelling and changes in thekinetoplast and the organization of nuclear chromatin[6] In this study the production of NO was determined bymeasuring nitriteThe results frommacrophages treated with

EuEO revealed that EuEO does not induce NO productionwhich demonstrates that its promising anti-Leishmaniaactivity does not occur by this route

Natural products that are able to activate macrophagesand are thus biological response modifiers have beenextensively studied [49 50] In this context the EuEOdemonstrated potential in the activation of macrophagesby increasing phagocytic capacity and lysosomal activityimmunomodulatory activities possibly involved in its anti-Leishmania activity Phagocytosis and the lysosomal systemare critical to macrophage function because of their roles ininternalization degradation and eventually presentation ofpeptides derived from antigens required for host defense Inaddition antigen presentation occurs through phagocytosisand endosomallysosomal targeting systems [51ndash53]

8 Evidence-Based Complementary and Alternative Medicine

Due to the need for anti-Leishmania substances that aremore selective for the parasite and less toxic to host cellsand since EuEO showed low IC

50values it was important to

investigate the cytotoxic activity of EuEO againstmammaliancells EuEO showed significant toxicity in macrophages witha CC50

of 453120583gsdotmLminus1 but when compared with its IC50

value in amastigotes this indicates a secure selectivity indexabove 20 The literature recommends that for amastigotesinternalized in macrophages this index must be demon-strated as near to or greater than 20 [54] The investigationof the toxic action of new substances with anti-Leishmaniaactivity onmacrophages is important since they are themaincells of the vertebrate host parasitized by Leishmania spp[55 56] Furthermore various other cytotoxic assays can beconducted using animal and human cells to determine thesafety of a test substance leading to a future treatment invivo Tests performed with erythrocytes enable assessment ofa drugrsquos potential to cause injury to the plasma membrane ofa cell either by forming pores or by total rupture leadingto cellular damage or changes in membrane permeability[57] This is a model used for the preliminary study of theprotective and toxic effects of substances being a possibleindicator of this type of damage to cells in vivo [58ndash60]The results obtained in this test with EuEO revealed lowtoxicity to erythrocytes since EuEO showed 0 hemolysis atconcentrations of 50120583gsdotmLminus1 and lower

5 Conclusions

In conclusion the data in this study show that the essentialoil of leaves from E uniflora (EuEO) characterized bysesquiterpenes has anti-Leishmania activity in both stagesof L amazonensis This study also demonstrated that EuEOactivity is not mediated by NO production On the otherhand activation of macrophages may be involved in themechanisms of EuEO anti-Leishmania activity as evidencedby increases in both phagocytic capacity and the lysosomalcompartment Further investigation is needed to determineadditional mechanisms involved in the anti-Leishmaniaactivity of this plant species as well as in vivo studies inmodels of leishmaniasis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors are grateful to the CAPES and FAPEMA forfinancial grants through the concession of scholarships andfinancial support

References

[1] S Kaur H Sachdeva S Dhuria M Sharma and T Kaur ldquoAn-tileishmanial effect of cisplatin against murine visceral leishma-niasisrdquo Parasitology International vol 59 no 1 pp 62ndash69 2010

[2] S AzzouzMMaache R G Garcia andA Osuna ldquoLeishmani-cidal activity of edelfosine miltefosine and ilmofosinerdquo Basic

and Clinical Pharmacology and Toxicology vol 96 no 1 pp 60ndash65 2005

[3] J A Patza T K Graczykb N Gellera and A Y Vittorc ldquoEffectsof environmental change on emerging parasitic diseasesrdquo Inter-national Journal of Parasitology vol 30 no 12-13 pp 1295ndash13042000

[4] B H Alizadeh A Foroumadi S K Ardestani F Poorrajaband A Shafiee ldquoLeishmanicidal evaluation of novel syntheticchromenesrdquo Archiv der Pharmazie vol 341 no 12 pp 787ndash7932008

[5] RW Ashford ldquoThe leishmaniases as emerging and reemergingzoonosesrdquo International Journal for Parasitology vol 30 no 12-13 pp 1269ndash1281 2000

[6] M S S Rosa R R Mendonca-Filho H R Bizzo et al ldquoAntil-eishmanial activity of a linalool-rich essential oil from CrotoncajucarardquoAntimicrobial Agents Chemotherapy vol 47 no 6 pp1895ndash1901 2003

[7] T Ueda-Nakamura R R Mendonca-Filho J A Morgado-Dıazet al ldquoAntileishmanial activity of eugenol-rich essential oil fromOcimum gratissimumrdquo Parasitology International vol 55 no 2pp 55ndash59 2006

[8] A O Santos T Ueda-Nakamura B P Dias Filho V F VeigaJunior A C Pinto and C V Nakamura ldquoEffect of Braziliancopaiba oils on Leishmania amazonensisrdquo Journal of Ethnophar-macology vol 120 no 2 pp 204ndash208 2008

[9] LMonzote AMMontalvo S Almanonni R Scull MMiran-da and J Abreu ldquoActivity of the essential oil fromChenopodiumambrosioides grown in Cuba against Leishmania amazonensisrdquoChemotherapy vol 52 no 3 pp 130ndash136 2006

[10] MMachado P Pires A M Dinis et al ldquoMonoterpenic aldehy-des as potential anti-Leishmania agents activity ofCymbopogoncitratus and citral on L infantum L tropica and L majorrdquoExperimental Parasitology vol 130 no 3 pp 223ndash231 2012

[11] M R Santin A O Dos Santos C V Nakamura B P Dias FilhoI C P Ferreira and T Ueda-Nakamura ldquoIn vitro activity of theessential oil of Cymbopogon citratus and its major component(citral) on Leishmania amazonensisrdquo Parasitology Research vol105 no 6 pp 1489ndash1496 2009

[12] V C S Oliveira D M S Moura J A D Lopes P P DeAndrade N H Da Silva and R C B Q Figueiredo ldquoEffectsof essential oils from Cymbopogon citratus (DC) Stapf Lippiasidoides Cham and Ocimum gratissimum L on growth andultrastructure of Leishmania chagasi promastigotesrdquo Parasitol-ogy Research vol 104 no 5 pp 1053ndash1059 2009

[13] A E Consolini and M G Sarubbio ldquoPharmacological effectsof Eugenia uniflora (Myrtaceae) aqueous crude extract on ratrsquosheartrdquo Journal of Ethnopharmacology vol 81 no 1 pp 57ndash632002

[14] C B Alice V M F Vargas G A A B Silva et al ldquoScreening ofplants used in south Brazilian folk medicinerdquo Journal of Ethno-pharmacology vol 35 no 2 pp 165ndash171 1991

[15] P Weyerstahl H Marschall-Weyerstahl C Christiansen B OOguntimein and A O Adeoye ldquoVolatile constituents of Euge-nia uniflora leaf oilrdquo Planta medica vol 54 no 6 pp 546ndash5491988

[16] F N Victoria E J Lenardao L Savegnago et al ldquoEssential oil ofthe leaves of Eugenia uniflora L antioxidant and antimicrobialpropertiesrdquo Food and Chemical Toxicology vol 50 no 8 pp2668ndash2674 2012

Evidence-Based Complementary and Alternative Medicine 9

[17] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[18] I A Ogunwande N O Olawore O Ekundayo T M Walker JM Schmidt andWN Setzer ldquoStudies on the essential oils com-position antibacterial and cytotoxicity of Eugenia uniflora LrdquoInternational Journal of Aromatherapy vol 15 no 3 pp 147ndash1522005

[19] A C L Amorim C K F Lima A M C Hovell A L PMiranda and C M Rezende ldquoAntinociceptive and hypother-mic evaluation of the leaf essential oil and isolated terpenoidsfrom Eugenia uniflora L (Brazilian Pitanga)rdquo Phytomedicinevol 16 no 10 pp 923ndash928 2009

[20] D P Costa E G A Filho LM A Silva et al ldquoInfluence of fruitbiotypes on the chemical composition and antifungal activityof the essential oils of Eugenia uniflora leavesrdquo Journal of theBrazilian Chemical Society vol 21 no 5 pp 851ndash858 2010

[21] G F Ibikunle A C Adebajo F G Famuyiwa A J Aladesanmiand C O Adewunmi ldquoIn-vitro evaluation of anti-trichomonalactivities ofEugenia uniflora leafrdquoAfrican Journal of TraditionalComplementary and AlternativeMedicines vol 8 no 2 pp 170ndash176 2011

[22] K K Santos S R Tintino C E Souza et al ldquoAnti-Trypanosomacruzi and cytotoxic activities of Eugenia uniflora Lrdquo Experimen-tal Parasitology vol 131 no 1 pp 130ndash132 2012

[23] NIST 05 Mass Spectral Library (NISTEPANIH) NationalInstitute of Standards and Technology Gaithersburg Md USA2005

[24] R P Adams Identification of Essential Oil Components By GasChromatographyMass Spectrometry Allured Carol Stream IllUSA 4th edition 2007

[25] S M P Carneiro F A A Carvalho L C L R Santana A PL Sousa andM H Chaves ldquoThe cytotoxic and antileishmanialactivity of extracts and fractions of leaves and fruits of Azad-irachta indica (A Juss)rdquo Biological Research vol 45 no 2 pp111ndash116 2012

[26] M D G F deMedeiros A C da Silva A M D G L Cito et alldquoIn vitro antileishmanial activity and cytotoxicity of essential oilfrom Lippia sidoides Chamrdquo Parasitology International vol 60no 3 pp 237ndash241 2011

[27] S E Lofgren L C Miletti M Steindel E Bachere and M ABarracco ldquoTrypanocidal and leishmanicidal activities of differ-ent antimicrobial peptides (AMPs) isolated from aquatic ani-malsrdquo Experimental Parasitology vol 118 no 2 pp 197ndash2022008

[28] F C C Grando C A Felıcio A Twardowschy et al ldquoModula-tion of peritoneal macrophage activity by the saturation state ofthe fatty acid moiety of phosphatidylcholinerdquo Brazilian Journalof Medical and Biological Research vol 42 no 7 pp 599ndash6052009

[29] MM Iwu J E Jackson and BG Schuster ldquoMedicinal plants inthe fight against leishmaniasisrdquo Parasitology Today vol 10 no2 pp 65ndash68 1994

[30] L G Rocha J R G S Almeida R O Macedo and J M Bar-bosa-Filho ldquoA review of natural products with antileishmanialactivityrdquo Phytomedicine vol 12 no 6-7 pp 514ndash535 2005

[31] SMDeMorais A A CraveiroM I LMachado JW Alencarand F J A Matos ldquoVolatile constituents of Eugenia uniflora leafoil from northeastern Brazilrdquo Journal of Essential Oil Researchvol 8 no 4 pp 449ndash451 1996

[32] J G S Maia E H A Andrade M H L Da Silva and M G BZoghbi ldquoA new chemotype of Eugenia uniflora L from NorthBrazilrdquo Journal of Essential Oil Research vol 11 no 6 pp 727ndash729 1999

[33] J H G Lago E D Souza B Mariane et al ldquoChemical and bio-logical evaluation of essential oils from two species of myrtace-aemdashEugenia uniflora L and Plinia trunciflora (O Berg) KauselrdquoMolecules vol 16 no 12 pp 9826ndash9837 2011

[34] R Chang S A L de Morais D R Napolitano K C DuarteV B Guzman and E A do Nascimento ldquoA new approach forquantifying furanodiene and curzerene A case study on theessential oils of Eugenia uniflora (pitangueira) leavesrdquo BrazilianJournal of Pharmacognosy vol 21 no 3 pp 392ndash396 2011

[35] A M Adio ldquoGermacrenes A-E and related compounds ther-mal photochemical and acid induced transannular cycliza-tionsrdquo Tetrahedron vol 65 no 8 pp 1533ndash1552 2009

[36] D P Costa S C Santos J C Seraphin and PH Ferri ldquoSeasonalvariability of essential oils of Eugenia uniflora leavesrdquo Journal ofthe BrazilianChemical Society vol 20 no 7 pp 1287ndash1293 2009

[37] A T HenriquesM E Sobral A D Caudaro E E S SchapovalV L Bassani and G Lamaty ldquoAromatic plants from BrazilII The chemical composition of some Eugenia essential oilsrdquoJournal of Essential Oil Research vol 5 no 5 pp 501ndash505 1993

[38] B Teixeira A Marques C Ramos et al ldquoEuropean pennyroyal(Mentha pulegium) from portugal chemical composition ofessential oil and antioxidant and antimicrobial properties ofextracts and essential oilrdquo Industrial Crops and Products vol 36no 1 pp 81ndash87 2012

[39] D C Arruda F L DrsquoAlexandri A M Katzin and S RB Uliana ldquoAntileishmanial activity of the terpene nerolidolrdquoAntimicrobial Agents andChemotherapy vol 49 no 5 pp 1679ndash1687 2005

[40] RDi Pasqua G Betts NHoskinsM Edwards D Ercolini andG Mauriello ldquoMembrane toxicity of antimicrobial compoundsfrom essential oilsrdquo Journal of Agricultural and Food Chemistryvol 55 no 12 pp 4863ndash4870 2007

[41] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[42] J S Armstrong ldquoMitochondrial membrane permeabilizationthe sine qua non for cell deathrdquo BioEssays vol 28 no 3 pp253ndash260 2006

[43] A L Silva C M Adade F M Shoyama et al ldquoIn vitro leish-manicidal activity of N-dodecyl-1 2-ethanediaminerdquo Biomedi-cine and Pharmacotherapy vol 66 no 3 pp 180ndash186 2012

[44] B Purkait A Kumar N Nandi et al ldquoMechanism of ampho-tericin B resistance in clinical isolates of Leishmania donovanirdquoAntimicrobial Agents and Chemotherapy vol 56 no 2 pp 1031ndash1141 2012

[45] M Vermeersch R I Da Luz K Tote J P Timmermans P Cosand L Maes ldquoIn vitro susceptibilities of Leishmania donovanipromastigote and amastigote stages to antileishmanial referencedrugs practical relevance of stage-specific differencesrdquo Antimi-crobial Agents and Chemotherapy vol 53 no 9 pp 3855ndash38592009

[46] C Di Giorgio F Delmas V Akhmedjanova et al ldquoIn vitroantileishmanial activity of diphyllin isolated fromHaplophyllumbucharicumrdquo Planta Medica vol 71 no 4 pp 366ndash369 2005

10 Evidence-Based Complementary and Alternative Medicine

[47] H Martinez-Rojano J Mancilla-Ramirez L Quinonez-Diazand N Galindo-Sevilla ldquoActivity of hydroxyurea against Leish-mania mexicanardquo Antimicrobial Agents and Chemotherapy vol52 no 10 pp 3642ndash3647 2008

[48] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[49] A H Klimp E G E De Vries G L Scherphof and T DaemenldquoA potential role of macrophage activation in the treatment ofcancerrdquo Critical Reviews in OncologyHematology vol 44 no 2pp 143ndash161 2002

[50] M R Piemonte and D F Buchi ldquoAnalysis of IL-12 IFN-120574 and TNF-120572 production a5b1 integrins and actin filamentsdistribution in peritoneal mouse macrophages treated withhomeopathic medicamentrdquo Journal of Submicroscopic Cytologyand Pathology vol 33 no 1 pp 255ndash263 2002

[51] F Niedergang and P Chavrier ldquoSignaling and membranedynamics during phagocytosis Many roads lead to the pha-gos(R)omerdquo Current Opinion in Cell Biology vol 16 no 4 pp422ndash428 2004

[52] W L Lee R E Harrison and S Grinstein ldquoPhagocytosis byneutrophilsrdquo Microbes and Infection vol 5 no 14 pp 1299ndash1306 2003

[53] S J Green M S Meltzer J B Hibbs and C A Nacy ldquoActivatedmacrophages destroy intracellular Leishmania major amastig-otes by an L-arginine-dependent killing mechanismrdquo Journal ofImmunology vol 144 no 1 pp 278ndash283 1990

[54] E Osorio G J Arango N Jimenez et al ldquoAntiprotozoal andcytotoxic activities in vitro of Colombian Annonaceaerdquo Journalof Ethnopharmacology vol 111 no 3 pp 630ndash635 2007

[55] S Kamhawi ldquoPhlebotomine sand flies and Leishmania para-sites friends or foesrdquo Trends in Parasitology vol 22 no 9 pp439ndash445 2006

[56] P Tripathi V Singh and S Naik ldquoImmune response to leish-mania paradox rather than paradigmrdquo FEMS Immunology andMedical Microbiology vol 51 no 2 pp 229ndash242 2007

[57] L V Costa-Lotufo G M A Cunha P A M Farias et al ldquoThecytotoxic and embryotoxic effects of kaurenoic acid a diterpeneisolated fromCopaifera langsdorffii oleo-resinrdquoToxicon vol 40no 8 pp 1231ndash1234 2002

[58] R M Aparicio M J Garcıa-Celma M Pilar Vinardell and MMitjans ldquoIn vitro studies of the hemolytic activity ofmicroemul-sions in human erythrocytesrdquo Journal of Pharmaceutical andBiomedical Analysis vol 39 no 5 pp 1063ndash1067 2005

[59] L A Lexis R G Fassett and J S Coombes ldquo120572-tocopherol and120572-lipoic acid enhance the erythrocyte antioxidant defence incyclosporine A-treated ratsrdquo Basic and Clinical Pharmacologyand Toxicology vol 98 no 1 pp 68ndash73 2006

[60] J R Munoz-Castaneda J Muntane M C Munoz I BujalanceP Montilla and I Tunez ldquoEstradiol and catecholestrogens pro-tect against adriamycininduced oxidative stress in erythrocytesof ovariectomized ratsrdquo Toxicology Letters vol 160 no 3 pp196ndash203 2006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

0

20

40

60

80

100

400Amph 200 100 50 25 125 625 312

Gro

wth

inhi

bitio

n (

)

lowast

24 h48 h72 h

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Figure 1 Effect of Eugenia uniflora essential oil (400 200 10050 25 125 625 and 312 120583gsdotmLminus1) or amphotericin B (Amph)(2120583gsdotmLminus1) on Leishmania amazonensis promastigotes Cultures oflog-phase promastigotes (1 times 106) were incubated at 26∘C for 2448 and 72 h in different essential oil concentrations Data representthe mean percentage of growth inhibition plusmn standard error of 3experiments carried out in triplicate lowast119875 lt 005

312 625 125 25 50 100

0102030405060708090

100

Cel

l via

bilit

y (

)

lowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 2 Cytotoxicity of Eugenia uniflora essential oil on theviability ofmurine peritonealmacrophages Peritonealmacrophageswere seeded at 1 times 105well in 96-well microplates and incubated for48 h in the presence of E uniflora L essential oil at concentrationsof 100 50 25 125 625 and 312 120583gsdotmLminus1 Viability was determinedwith 3-(45-dimethylthiazol-2-yl)25-diphenyltetrazolium bromide(MTT) and the optical density was determined at 540 nmData rep-resent the mean parasite density plusmn standard error of 3 experimentscarried out in triplicate lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

treated with EuEO at concentrations ranging from 25 to312 120583gsdotmLminus1

36 NO Production NO production was determined indi-rectly by measuring nitrite produced by macrophages treatedwith EuEO and stimulated (or not stimulated) by L amazo-nensis promastigotes Macrophages treated with EuEO but

01020304050607080

4002001005025125625312

Hem

olys

is (

)

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Figure 3 Hemolytic activity of Eugenia uniflora essential oil in a 4suspension of human O+ red blood cells after 1 h of incubation

not stimulated by Leishmania showed significant reductionin NO production at concentrations of 100 and 50120583gsdotmLminus1(Figure 7(a)) while macrophages stimulated by the parasiteshowed increased NO at a concentration of 312120583gsdotmLminus1albeit without statistical significance (119875 gt 005) (Figure 7(b))

4 Discussion

The need to identify new compounds with anti-Leishmaniaproperties that are more effective and less toxic than con-ventional drugs has motivated research on natural productsisolated from plant species These substances predominantlyconsist of alkaloids terpenes flavonoids benzopyrans phe-nolics and sesquiterpene lactones and have been identifiedin plant species with documented anti-Leishmania activity[29 30] The chemical composition of EuEO obtained in thisstudy revealed sesquiterpenes to be the dominant chemicalclass corroborating the findings of many previous studies[16 31ndash33] Despite the prevalence of sesquiterpenes thespecies has different chemotypes with different major con-stituents being observed in different studies The specimenanalyzed in this study had curzerene as themajor constituentwhich again corroborated previous reported findings [3234] According to Adio [35] germacrone is a heat-sensitivecompound that may degrade to trans-120573-elemenone duringGC analysis This may be the explanation of the presence oftrans-120573-elemenone among the major compounds of EuEOSome germacrone products have shown pharmacologicalactivity as antitumor [35]

According to Costa et al [20] the composition of theessential oil from E uniflora leaves might be influenced byfruit colorsThe plants with red fruit colors have curzerene asthe major component as in our studied plant In Maranhaowhere the samples were collected the majority of plants ofE uniflora have red fruit color [32] Selina-137(11)-trien-8-one [36] germacrene B [16 33] and 120572- and 120573-selinene [37]have been found to be the major constituents of other Euniflora chemotypes Genetic variability geographical andenvironmental conditions and the method of environmentalmanagement directly influence the yield and composition of

6 Evidence-Based Complementary and Alternative Medicine

Table 2 Anti-Leishmania and cytotoxic effects of Eugenia uniflora essential oil

Macrophage Promastigote AmastigoteCC50 (120583gsdotmLminus1) IC50 (120583gsdotmLminus1)

SIIC50 (120583gsdotmLminus1) SI

48 h 24 h 48 h 72 h 48 hEuEO 453 plusmn 245 696 plusmn 102 304 plusmn 075 175 plusmn 053 149a 192 plusmn 08 2359baSIpro (selectivity index) = CC50 macrophagesIC50 promastigote forms (48 h)bSIama (selectivity index) = CC50 macrophagesIC50 amastigote forms

078 156 312

0

20

40

60

80

100

27

44675067

Mac

roph

ages

infe

ctio

n (

)

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 4 Effect of Eugenia uniflora essential oil on macrophageinfection after 48 h of exposure Peritoneal macrophage cells wereinfected with promastigotes of Leishmania amazonensis and thentreated with 312 156 or 078 120583gsdotmLminus1 of Eugenia uniflora essentialoil Data represent the mean parasite density plusmn standard error of 3experiments carried out in triplicate lowastlowastlowast119875 lt 0001

078 156 312

0

2

4

6

8

10lowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Am

astig

otes

mac

roph

age (n

)

lowastlowastlowast

lowastlowastlowast

Figure 5 Effect of Eugenia uniflora essential oil on the survival ofLeishmania amazonensis amastigotes internalized in macrophagesLeishmania amazonensis-infected mouse peritoneal macrophageswere treated with 312 156 or 078 120583gsdotmLminus1 of Eugenia unifloraessential oil After 48 h of incubation amastigote survival wasassessed Data represent the mean parasite density plusmn standard errorof 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001

essential oils [38] It is the first report of essential oil analyzedfrom E uniflora collected in Sao Luıs MA Brazil and thiscould be an excellent source of natural product with interestfor pharmaceutical industry

The present study was conducted to evaluate the anti-Leishmania activity of EuEO on promastigotes and amasti-gotes of L amazonensis its potential mechanisms of macro-phage activation and its cytotoxicity inmammalian cellsTheEuEO showed significant concentration-dependent activityagainst promastigotes of L amazonensis with an IC

50of

175120583gsdotmLminus1 Previous studies have suggested that theseclasses of natural products may have potential anti-Leishmania activity 120573-Caryophyllene (a sesquiterpenehydrocarbon) and nerolidol (an oxygenated sesquiterpene)are examples of terpenic substances with well-characterizedanti-Leishmania activity possibly associated with inhibitionof cellular isoprenoid biosynthesis [8 39]

It has been shown that the lipophilic components ofessential oils may affect layers of polysaccharides fatty acidsand phospholipids in plasma membranes of promastigotes ofLeishmania spp This then leads to cell lysis and release ofmacromolecules [40] In the cytoplasm these substances candisrupt the specific metabolic pathways of lipids and proteinsor stimulate depolarization of mitochondrial membraneswhich can lead to cell necrosis or apoptosis [41 42]

Amphotericin B is a drug of choice for the treatment ofdifferent forms of leishmaniasis Because of this it was usedas a positive control We observed a significant inhibitoryeffect on promastigote growth after 24 h of exposure toamphotericin B but leading to an increase in viable pro-mastigote forms at 48 and 72 h Similar results were observedin previous studies [43] Some Leishmania strains have shownmechanisms of resistance to amphotericin B This may hashappenedwith the strain used in this studyThe reproductionof resistant parasites may have led to a higher cell viabilityafter 48 and 72 h [44 45]

Experimental models using amastigotes internalized intomacrophages can produce results that more closely approxi-mate those obtained using animal models since these formsare responsible for the clinicalmanifestations of leishmaniasis[46] Amastigotes can be found in parasitophorous vacuolesof infected macrophages therefore a therapeutic drug willonly be effective against Leishmania spp if it can crossthe host cell membrane and act on the amastigotes insidethe vacuole Thus assays of activity against intracellularamastigotes are the most effective way to relate the in vitroactivity of a possible substance with its in vivo effectiveness[47] The inhibition of amastigote growth was even greater at

Evidence-Based Complementary and Alternative Medicine 7

C 100 50 25 125 625 312

005

115

225

335

445

5N

eutr

al re

d up

take

ABS

lowast

lowastlowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(a)

C 100 50 25 125 625 312

0

005

01

015

02

025

Phag

ocyt

osis

ABS

lowast lowast lowastlowast lowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

(b)

Figure 6 The influence of Eugenia uniflora essential oil on the lysosomal activity (a) and phagocytic activity (b) of peritoneal macrophagesat concentrations of 100 50 25 125 625 and 312 120583gsdotmLminus1 Data represent the mean density plusmn standard error of 3 experiments carried outin triplicate lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 C control ABS absorbance

0010203040506070809

1

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119871119890119894119904ℎ119898119886119899119894119886 119886119898119886119911119900119899119890119899119904119894119904

0010203040506070809

1

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(b)

Figure 7 Production of nitric oxide (NO) Murine macrophages (2 times 105) were treated with Eugenia uniflora essential oil (100 50 25 125625 and 312 120583gsdotmLminus1) in the absence (a) or presence of Leishmania amazonensis (b) over 24 h Data represent the mean density plusmn standarderror of 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001 C control ABS absorbance

48 h after exposure to oil with an IC50of 192 120583gmL whereas

the IC50

for promastigotes was 304120583gsdotmLminus1 for the sameduration of exposure Previous studies demonstrated greateractivity against amastigotes versus promastigotes of essentialoils of Artemisia absinthium and Satureja punctata [48]

Mechanisms of anti-Leishmania action on Leishmaniaspp amastigotes have also been described for essentialoils and their terpene compounds Monoterpenes such aslinalool isolated from leaves of Croton cajucara (Euphorbia-ceae) significantly increased NO production inmacrophagesinfected with L amazonensis and acts directly on the parasiteas evidenced by mitochondrial swelling and changes in thekinetoplast and the organization of nuclear chromatin[6] In this study the production of NO was determined bymeasuring nitriteThe results frommacrophages treated with

EuEO revealed that EuEO does not induce NO productionwhich demonstrates that its promising anti-Leishmaniaactivity does not occur by this route

Natural products that are able to activate macrophagesand are thus biological response modifiers have beenextensively studied [49 50] In this context the EuEOdemonstrated potential in the activation of macrophagesby increasing phagocytic capacity and lysosomal activityimmunomodulatory activities possibly involved in its anti-Leishmania activity Phagocytosis and the lysosomal systemare critical to macrophage function because of their roles ininternalization degradation and eventually presentation ofpeptides derived from antigens required for host defense Inaddition antigen presentation occurs through phagocytosisand endosomallysosomal targeting systems [51ndash53]

8 Evidence-Based Complementary and Alternative Medicine

Due to the need for anti-Leishmania substances that aremore selective for the parasite and less toxic to host cellsand since EuEO showed low IC

50values it was important to

investigate the cytotoxic activity of EuEO againstmammaliancells EuEO showed significant toxicity in macrophages witha CC50

of 453120583gsdotmLminus1 but when compared with its IC50

value in amastigotes this indicates a secure selectivity indexabove 20 The literature recommends that for amastigotesinternalized in macrophages this index must be demon-strated as near to or greater than 20 [54] The investigationof the toxic action of new substances with anti-Leishmaniaactivity onmacrophages is important since they are themaincells of the vertebrate host parasitized by Leishmania spp[55 56] Furthermore various other cytotoxic assays can beconducted using animal and human cells to determine thesafety of a test substance leading to a future treatment invivo Tests performed with erythrocytes enable assessment ofa drugrsquos potential to cause injury to the plasma membrane ofa cell either by forming pores or by total rupture leadingto cellular damage or changes in membrane permeability[57] This is a model used for the preliminary study of theprotective and toxic effects of substances being a possibleindicator of this type of damage to cells in vivo [58ndash60]The results obtained in this test with EuEO revealed lowtoxicity to erythrocytes since EuEO showed 0 hemolysis atconcentrations of 50120583gsdotmLminus1 and lower

5 Conclusions

In conclusion the data in this study show that the essentialoil of leaves from E uniflora (EuEO) characterized bysesquiterpenes has anti-Leishmania activity in both stagesof L amazonensis This study also demonstrated that EuEOactivity is not mediated by NO production On the otherhand activation of macrophages may be involved in themechanisms of EuEO anti-Leishmania activity as evidencedby increases in both phagocytic capacity and the lysosomalcompartment Further investigation is needed to determineadditional mechanisms involved in the anti-Leishmaniaactivity of this plant species as well as in vivo studies inmodels of leishmaniasis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors are grateful to the CAPES and FAPEMA forfinancial grants through the concession of scholarships andfinancial support

References

[1] S Kaur H Sachdeva S Dhuria M Sharma and T Kaur ldquoAn-tileishmanial effect of cisplatin against murine visceral leishma-niasisrdquo Parasitology International vol 59 no 1 pp 62ndash69 2010

[2] S AzzouzMMaache R G Garcia andA Osuna ldquoLeishmani-cidal activity of edelfosine miltefosine and ilmofosinerdquo Basic

and Clinical Pharmacology and Toxicology vol 96 no 1 pp 60ndash65 2005

[3] J A Patza T K Graczykb N Gellera and A Y Vittorc ldquoEffectsof environmental change on emerging parasitic diseasesrdquo Inter-national Journal of Parasitology vol 30 no 12-13 pp 1295ndash13042000

[4] B H Alizadeh A Foroumadi S K Ardestani F Poorrajaband A Shafiee ldquoLeishmanicidal evaluation of novel syntheticchromenesrdquo Archiv der Pharmazie vol 341 no 12 pp 787ndash7932008

[5] RW Ashford ldquoThe leishmaniases as emerging and reemergingzoonosesrdquo International Journal for Parasitology vol 30 no 12-13 pp 1269ndash1281 2000

[6] M S S Rosa R R Mendonca-Filho H R Bizzo et al ldquoAntil-eishmanial activity of a linalool-rich essential oil from CrotoncajucarardquoAntimicrobial Agents Chemotherapy vol 47 no 6 pp1895ndash1901 2003

[7] T Ueda-Nakamura R R Mendonca-Filho J A Morgado-Dıazet al ldquoAntileishmanial activity of eugenol-rich essential oil fromOcimum gratissimumrdquo Parasitology International vol 55 no 2pp 55ndash59 2006

[8] A O Santos T Ueda-Nakamura B P Dias Filho V F VeigaJunior A C Pinto and C V Nakamura ldquoEffect of Braziliancopaiba oils on Leishmania amazonensisrdquo Journal of Ethnophar-macology vol 120 no 2 pp 204ndash208 2008

[9] LMonzote AMMontalvo S Almanonni R Scull MMiran-da and J Abreu ldquoActivity of the essential oil fromChenopodiumambrosioides grown in Cuba against Leishmania amazonensisrdquoChemotherapy vol 52 no 3 pp 130ndash136 2006

[10] MMachado P Pires A M Dinis et al ldquoMonoterpenic aldehy-des as potential anti-Leishmania agents activity ofCymbopogoncitratus and citral on L infantum L tropica and L majorrdquoExperimental Parasitology vol 130 no 3 pp 223ndash231 2012

[11] M R Santin A O Dos Santos C V Nakamura B P Dias FilhoI C P Ferreira and T Ueda-Nakamura ldquoIn vitro activity of theessential oil of Cymbopogon citratus and its major component(citral) on Leishmania amazonensisrdquo Parasitology Research vol105 no 6 pp 1489ndash1496 2009

[12] V C S Oliveira D M S Moura J A D Lopes P P DeAndrade N H Da Silva and R C B Q Figueiredo ldquoEffectsof essential oils from Cymbopogon citratus (DC) Stapf Lippiasidoides Cham and Ocimum gratissimum L on growth andultrastructure of Leishmania chagasi promastigotesrdquo Parasitol-ogy Research vol 104 no 5 pp 1053ndash1059 2009

[13] A E Consolini and M G Sarubbio ldquoPharmacological effectsof Eugenia uniflora (Myrtaceae) aqueous crude extract on ratrsquosheartrdquo Journal of Ethnopharmacology vol 81 no 1 pp 57ndash632002

[14] C B Alice V M F Vargas G A A B Silva et al ldquoScreening ofplants used in south Brazilian folk medicinerdquo Journal of Ethno-pharmacology vol 35 no 2 pp 165ndash171 1991

[15] P Weyerstahl H Marschall-Weyerstahl C Christiansen B OOguntimein and A O Adeoye ldquoVolatile constituents of Euge-nia uniflora leaf oilrdquo Planta medica vol 54 no 6 pp 546ndash5491988

[16] F N Victoria E J Lenardao L Savegnago et al ldquoEssential oil ofthe leaves of Eugenia uniflora L antioxidant and antimicrobialpropertiesrdquo Food and Chemical Toxicology vol 50 no 8 pp2668ndash2674 2012

Evidence-Based Complementary and Alternative Medicine 9

[17] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[18] I A Ogunwande N O Olawore O Ekundayo T M Walker JM Schmidt andWN Setzer ldquoStudies on the essential oils com-position antibacterial and cytotoxicity of Eugenia uniflora LrdquoInternational Journal of Aromatherapy vol 15 no 3 pp 147ndash1522005

[19] A C L Amorim C K F Lima A M C Hovell A L PMiranda and C M Rezende ldquoAntinociceptive and hypother-mic evaluation of the leaf essential oil and isolated terpenoidsfrom Eugenia uniflora L (Brazilian Pitanga)rdquo Phytomedicinevol 16 no 10 pp 923ndash928 2009

[20] D P Costa E G A Filho LM A Silva et al ldquoInfluence of fruitbiotypes on the chemical composition and antifungal activityof the essential oils of Eugenia uniflora leavesrdquo Journal of theBrazilian Chemical Society vol 21 no 5 pp 851ndash858 2010

[21] G F Ibikunle A C Adebajo F G Famuyiwa A J Aladesanmiand C O Adewunmi ldquoIn-vitro evaluation of anti-trichomonalactivities ofEugenia uniflora leafrdquoAfrican Journal of TraditionalComplementary and AlternativeMedicines vol 8 no 2 pp 170ndash176 2011

[22] K K Santos S R Tintino C E Souza et al ldquoAnti-Trypanosomacruzi and cytotoxic activities of Eugenia uniflora Lrdquo Experimen-tal Parasitology vol 131 no 1 pp 130ndash132 2012

[23] NIST 05 Mass Spectral Library (NISTEPANIH) NationalInstitute of Standards and Technology Gaithersburg Md USA2005

[24] R P Adams Identification of Essential Oil Components By GasChromatographyMass Spectrometry Allured Carol Stream IllUSA 4th edition 2007

[25] S M P Carneiro F A A Carvalho L C L R Santana A PL Sousa andM H Chaves ldquoThe cytotoxic and antileishmanialactivity of extracts and fractions of leaves and fruits of Azad-irachta indica (A Juss)rdquo Biological Research vol 45 no 2 pp111ndash116 2012

[26] M D G F deMedeiros A C da Silva A M D G L Cito et alldquoIn vitro antileishmanial activity and cytotoxicity of essential oilfrom Lippia sidoides Chamrdquo Parasitology International vol 60no 3 pp 237ndash241 2011

[27] S E Lofgren L C Miletti M Steindel E Bachere and M ABarracco ldquoTrypanocidal and leishmanicidal activities of differ-ent antimicrobial peptides (AMPs) isolated from aquatic ani-malsrdquo Experimental Parasitology vol 118 no 2 pp 197ndash2022008

[28] F C C Grando C A Felıcio A Twardowschy et al ldquoModula-tion of peritoneal macrophage activity by the saturation state ofthe fatty acid moiety of phosphatidylcholinerdquo Brazilian Journalof Medical and Biological Research vol 42 no 7 pp 599ndash6052009

[29] MM Iwu J E Jackson and BG Schuster ldquoMedicinal plants inthe fight against leishmaniasisrdquo Parasitology Today vol 10 no2 pp 65ndash68 1994

[30] L G Rocha J R G S Almeida R O Macedo and J M Bar-bosa-Filho ldquoA review of natural products with antileishmanialactivityrdquo Phytomedicine vol 12 no 6-7 pp 514ndash535 2005

[31] SMDeMorais A A CraveiroM I LMachado JW Alencarand F J A Matos ldquoVolatile constituents of Eugenia uniflora leafoil from northeastern Brazilrdquo Journal of Essential Oil Researchvol 8 no 4 pp 449ndash451 1996

[32] J G S Maia E H A Andrade M H L Da Silva and M G BZoghbi ldquoA new chemotype of Eugenia uniflora L from NorthBrazilrdquo Journal of Essential Oil Research vol 11 no 6 pp 727ndash729 1999

[33] J H G Lago E D Souza B Mariane et al ldquoChemical and bio-logical evaluation of essential oils from two species of myrtace-aemdashEugenia uniflora L and Plinia trunciflora (O Berg) KauselrdquoMolecules vol 16 no 12 pp 9826ndash9837 2011

[34] R Chang S A L de Morais D R Napolitano K C DuarteV B Guzman and E A do Nascimento ldquoA new approach forquantifying furanodiene and curzerene A case study on theessential oils of Eugenia uniflora (pitangueira) leavesrdquo BrazilianJournal of Pharmacognosy vol 21 no 3 pp 392ndash396 2011

[35] A M Adio ldquoGermacrenes A-E and related compounds ther-mal photochemical and acid induced transannular cycliza-tionsrdquo Tetrahedron vol 65 no 8 pp 1533ndash1552 2009

[36] D P Costa S C Santos J C Seraphin and PH Ferri ldquoSeasonalvariability of essential oils of Eugenia uniflora leavesrdquo Journal ofthe BrazilianChemical Society vol 20 no 7 pp 1287ndash1293 2009

[37] A T HenriquesM E Sobral A D Caudaro E E S SchapovalV L Bassani and G Lamaty ldquoAromatic plants from BrazilII The chemical composition of some Eugenia essential oilsrdquoJournal of Essential Oil Research vol 5 no 5 pp 501ndash505 1993

[38] B Teixeira A Marques C Ramos et al ldquoEuropean pennyroyal(Mentha pulegium) from portugal chemical composition ofessential oil and antioxidant and antimicrobial properties ofextracts and essential oilrdquo Industrial Crops and Products vol 36no 1 pp 81ndash87 2012

[39] D C Arruda F L DrsquoAlexandri A M Katzin and S RB Uliana ldquoAntileishmanial activity of the terpene nerolidolrdquoAntimicrobial Agents andChemotherapy vol 49 no 5 pp 1679ndash1687 2005

[40] RDi Pasqua G Betts NHoskinsM Edwards D Ercolini andG Mauriello ldquoMembrane toxicity of antimicrobial compoundsfrom essential oilsrdquo Journal of Agricultural and Food Chemistryvol 55 no 12 pp 4863ndash4870 2007

[41] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[42] J S Armstrong ldquoMitochondrial membrane permeabilizationthe sine qua non for cell deathrdquo BioEssays vol 28 no 3 pp253ndash260 2006

[43] A L Silva C M Adade F M Shoyama et al ldquoIn vitro leish-manicidal activity of N-dodecyl-1 2-ethanediaminerdquo Biomedi-cine and Pharmacotherapy vol 66 no 3 pp 180ndash186 2012

[44] B Purkait A Kumar N Nandi et al ldquoMechanism of ampho-tericin B resistance in clinical isolates of Leishmania donovanirdquoAntimicrobial Agents and Chemotherapy vol 56 no 2 pp 1031ndash1141 2012

[45] M Vermeersch R I Da Luz K Tote J P Timmermans P Cosand L Maes ldquoIn vitro susceptibilities of Leishmania donovanipromastigote and amastigote stages to antileishmanial referencedrugs practical relevance of stage-specific differencesrdquo Antimi-crobial Agents and Chemotherapy vol 53 no 9 pp 3855ndash38592009

[46] C Di Giorgio F Delmas V Akhmedjanova et al ldquoIn vitroantileishmanial activity of diphyllin isolated fromHaplophyllumbucharicumrdquo Planta Medica vol 71 no 4 pp 366ndash369 2005

10 Evidence-Based Complementary and Alternative Medicine

[47] H Martinez-Rojano J Mancilla-Ramirez L Quinonez-Diazand N Galindo-Sevilla ldquoActivity of hydroxyurea against Leish-mania mexicanardquo Antimicrobial Agents and Chemotherapy vol52 no 10 pp 3642ndash3647 2008

[48] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[49] A H Klimp E G E De Vries G L Scherphof and T DaemenldquoA potential role of macrophage activation in the treatment ofcancerrdquo Critical Reviews in OncologyHematology vol 44 no 2pp 143ndash161 2002

[50] M R Piemonte and D F Buchi ldquoAnalysis of IL-12 IFN-120574 and TNF-120572 production a5b1 integrins and actin filamentsdistribution in peritoneal mouse macrophages treated withhomeopathic medicamentrdquo Journal of Submicroscopic Cytologyand Pathology vol 33 no 1 pp 255ndash263 2002

[51] F Niedergang and P Chavrier ldquoSignaling and membranedynamics during phagocytosis Many roads lead to the pha-gos(R)omerdquo Current Opinion in Cell Biology vol 16 no 4 pp422ndash428 2004

[52] W L Lee R E Harrison and S Grinstein ldquoPhagocytosis byneutrophilsrdquo Microbes and Infection vol 5 no 14 pp 1299ndash1306 2003

[53] S J Green M S Meltzer J B Hibbs and C A Nacy ldquoActivatedmacrophages destroy intracellular Leishmania major amastig-otes by an L-arginine-dependent killing mechanismrdquo Journal ofImmunology vol 144 no 1 pp 278ndash283 1990

[54] E Osorio G J Arango N Jimenez et al ldquoAntiprotozoal andcytotoxic activities in vitro of Colombian Annonaceaerdquo Journalof Ethnopharmacology vol 111 no 3 pp 630ndash635 2007

[55] S Kamhawi ldquoPhlebotomine sand flies and Leishmania para-sites friends or foesrdquo Trends in Parasitology vol 22 no 9 pp439ndash445 2006

[56] P Tripathi V Singh and S Naik ldquoImmune response to leish-mania paradox rather than paradigmrdquo FEMS Immunology andMedical Microbiology vol 51 no 2 pp 229ndash242 2007

[57] L V Costa-Lotufo G M A Cunha P A M Farias et al ldquoThecytotoxic and embryotoxic effects of kaurenoic acid a diterpeneisolated fromCopaifera langsdorffii oleo-resinrdquoToxicon vol 40no 8 pp 1231ndash1234 2002

[58] R M Aparicio M J Garcıa-Celma M Pilar Vinardell and MMitjans ldquoIn vitro studies of the hemolytic activity ofmicroemul-sions in human erythrocytesrdquo Journal of Pharmaceutical andBiomedical Analysis vol 39 no 5 pp 1063ndash1067 2005

[59] L A Lexis R G Fassett and J S Coombes ldquo120572-tocopherol and120572-lipoic acid enhance the erythrocyte antioxidant defence incyclosporine A-treated ratsrdquo Basic and Clinical Pharmacologyand Toxicology vol 98 no 1 pp 68ndash73 2006

[60] J R Munoz-Castaneda J Muntane M C Munoz I BujalanceP Montilla and I Tunez ldquoEstradiol and catecholestrogens pro-tect against adriamycininduced oxidative stress in erythrocytesof ovariectomized ratsrdquo Toxicology Letters vol 160 no 3 pp196ndash203 2006

Submit your manuscripts athttpwwwhindawicom

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MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Table 2 Anti-Leishmania and cytotoxic effects of Eugenia uniflora essential oil

Macrophage Promastigote AmastigoteCC50 (120583gsdotmLminus1) IC50 (120583gsdotmLminus1)

SIIC50 (120583gsdotmLminus1) SI

48 h 24 h 48 h 72 h 48 hEuEO 453 plusmn 245 696 plusmn 102 304 plusmn 075 175 plusmn 053 149a 192 plusmn 08 2359baSIpro (selectivity index) = CC50 macrophagesIC50 promastigote forms (48 h)bSIama (selectivity index) = CC50 macrophagesIC50 amastigote forms

078 156 312

0

20

40

60

80

100

27

44675067

Mac

roph

ages

infe

ctio

n (

)

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 4 Effect of Eugenia uniflora essential oil on macrophageinfection after 48 h of exposure Peritoneal macrophage cells wereinfected with promastigotes of Leishmania amazonensis and thentreated with 312 156 or 078 120583gsdotmLminus1 of Eugenia uniflora essentialoil Data represent the mean parasite density plusmn standard error of 3experiments carried out in triplicate lowastlowastlowast119875 lt 0001

078 156 312

0

2

4

6

8

10lowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

Am

astig

otes

mac

roph

age (n

)

lowastlowastlowast

lowastlowastlowast

Figure 5 Effect of Eugenia uniflora essential oil on the survival ofLeishmania amazonensis amastigotes internalized in macrophagesLeishmania amazonensis-infected mouse peritoneal macrophageswere treated with 312 156 or 078 120583gsdotmLminus1 of Eugenia unifloraessential oil After 48 h of incubation amastigote survival wasassessed Data represent the mean parasite density plusmn standard errorof 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001

essential oils [38] It is the first report of essential oil analyzedfrom E uniflora collected in Sao Luıs MA Brazil and thiscould be an excellent source of natural product with interestfor pharmaceutical industry

The present study was conducted to evaluate the anti-Leishmania activity of EuEO on promastigotes and amasti-gotes of L amazonensis its potential mechanisms of macro-phage activation and its cytotoxicity inmammalian cellsTheEuEO showed significant concentration-dependent activityagainst promastigotes of L amazonensis with an IC

50of

175120583gsdotmLminus1 Previous studies have suggested that theseclasses of natural products may have potential anti-Leishmania activity 120573-Caryophyllene (a sesquiterpenehydrocarbon) and nerolidol (an oxygenated sesquiterpene)are examples of terpenic substances with well-characterizedanti-Leishmania activity possibly associated with inhibitionof cellular isoprenoid biosynthesis [8 39]

It has been shown that the lipophilic components ofessential oils may affect layers of polysaccharides fatty acidsand phospholipids in plasma membranes of promastigotes ofLeishmania spp This then leads to cell lysis and release ofmacromolecules [40] In the cytoplasm these substances candisrupt the specific metabolic pathways of lipids and proteinsor stimulate depolarization of mitochondrial membraneswhich can lead to cell necrosis or apoptosis [41 42]

Amphotericin B is a drug of choice for the treatment ofdifferent forms of leishmaniasis Because of this it was usedas a positive control We observed a significant inhibitoryeffect on promastigote growth after 24 h of exposure toamphotericin B but leading to an increase in viable pro-mastigote forms at 48 and 72 h Similar results were observedin previous studies [43] Some Leishmania strains have shownmechanisms of resistance to amphotericin B This may hashappenedwith the strain used in this studyThe reproductionof resistant parasites may have led to a higher cell viabilityafter 48 and 72 h [44 45]

Experimental models using amastigotes internalized intomacrophages can produce results that more closely approxi-mate those obtained using animal models since these formsare responsible for the clinicalmanifestations of leishmaniasis[46] Amastigotes can be found in parasitophorous vacuolesof infected macrophages therefore a therapeutic drug willonly be effective against Leishmania spp if it can crossthe host cell membrane and act on the amastigotes insidethe vacuole Thus assays of activity against intracellularamastigotes are the most effective way to relate the in vitroactivity of a possible substance with its in vivo effectiveness[47] The inhibition of amastigote growth was even greater at

Evidence-Based Complementary and Alternative Medicine 7

C 100 50 25 125 625 312

005

115

225

335

445

5N

eutr

al re

d up

take

ABS

lowast

lowastlowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(a)

C 100 50 25 125 625 312

0

005

01

015

02

025

Phag

ocyt

osis

ABS

lowast lowast lowastlowast lowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

(b)

Figure 6 The influence of Eugenia uniflora essential oil on the lysosomal activity (a) and phagocytic activity (b) of peritoneal macrophagesat concentrations of 100 50 25 125 625 and 312 120583gsdotmLminus1 Data represent the mean density plusmn standard error of 3 experiments carried outin triplicate lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 C control ABS absorbance

0010203040506070809

1

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119871119890119894119904ℎ119898119886119899119894119886 119886119898119886119911119900119899119890119899119904119894119904

0010203040506070809

1

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(b)

Figure 7 Production of nitric oxide (NO) Murine macrophages (2 times 105) were treated with Eugenia uniflora essential oil (100 50 25 125625 and 312 120583gsdotmLminus1) in the absence (a) or presence of Leishmania amazonensis (b) over 24 h Data represent the mean density plusmn standarderror of 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001 C control ABS absorbance

48 h after exposure to oil with an IC50of 192 120583gmL whereas

the IC50

for promastigotes was 304120583gsdotmLminus1 for the sameduration of exposure Previous studies demonstrated greateractivity against amastigotes versus promastigotes of essentialoils of Artemisia absinthium and Satureja punctata [48]

Mechanisms of anti-Leishmania action on Leishmaniaspp amastigotes have also been described for essentialoils and their terpene compounds Monoterpenes such aslinalool isolated from leaves of Croton cajucara (Euphorbia-ceae) significantly increased NO production inmacrophagesinfected with L amazonensis and acts directly on the parasiteas evidenced by mitochondrial swelling and changes in thekinetoplast and the organization of nuclear chromatin[6] In this study the production of NO was determined bymeasuring nitriteThe results frommacrophages treated with

EuEO revealed that EuEO does not induce NO productionwhich demonstrates that its promising anti-Leishmaniaactivity does not occur by this route

Natural products that are able to activate macrophagesand are thus biological response modifiers have beenextensively studied [49 50] In this context the EuEOdemonstrated potential in the activation of macrophagesby increasing phagocytic capacity and lysosomal activityimmunomodulatory activities possibly involved in its anti-Leishmania activity Phagocytosis and the lysosomal systemare critical to macrophage function because of their roles ininternalization degradation and eventually presentation ofpeptides derived from antigens required for host defense Inaddition antigen presentation occurs through phagocytosisand endosomallysosomal targeting systems [51ndash53]

8 Evidence-Based Complementary and Alternative Medicine

Due to the need for anti-Leishmania substances that aremore selective for the parasite and less toxic to host cellsand since EuEO showed low IC

50values it was important to

investigate the cytotoxic activity of EuEO againstmammaliancells EuEO showed significant toxicity in macrophages witha CC50

of 453120583gsdotmLminus1 but when compared with its IC50

value in amastigotes this indicates a secure selectivity indexabove 20 The literature recommends that for amastigotesinternalized in macrophages this index must be demon-strated as near to or greater than 20 [54] The investigationof the toxic action of new substances with anti-Leishmaniaactivity onmacrophages is important since they are themaincells of the vertebrate host parasitized by Leishmania spp[55 56] Furthermore various other cytotoxic assays can beconducted using animal and human cells to determine thesafety of a test substance leading to a future treatment invivo Tests performed with erythrocytes enable assessment ofa drugrsquos potential to cause injury to the plasma membrane ofa cell either by forming pores or by total rupture leadingto cellular damage or changes in membrane permeability[57] This is a model used for the preliminary study of theprotective and toxic effects of substances being a possibleindicator of this type of damage to cells in vivo [58ndash60]The results obtained in this test with EuEO revealed lowtoxicity to erythrocytes since EuEO showed 0 hemolysis atconcentrations of 50120583gsdotmLminus1 and lower

5 Conclusions

In conclusion the data in this study show that the essentialoil of leaves from E uniflora (EuEO) characterized bysesquiterpenes has anti-Leishmania activity in both stagesof L amazonensis This study also demonstrated that EuEOactivity is not mediated by NO production On the otherhand activation of macrophages may be involved in themechanisms of EuEO anti-Leishmania activity as evidencedby increases in both phagocytic capacity and the lysosomalcompartment Further investigation is needed to determineadditional mechanisms involved in the anti-Leishmaniaactivity of this plant species as well as in vivo studies inmodels of leishmaniasis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors are grateful to the CAPES and FAPEMA forfinancial grants through the concession of scholarships andfinancial support

References

[1] S Kaur H Sachdeva S Dhuria M Sharma and T Kaur ldquoAn-tileishmanial effect of cisplatin against murine visceral leishma-niasisrdquo Parasitology International vol 59 no 1 pp 62ndash69 2010

[2] S AzzouzMMaache R G Garcia andA Osuna ldquoLeishmani-cidal activity of edelfosine miltefosine and ilmofosinerdquo Basic

and Clinical Pharmacology and Toxicology vol 96 no 1 pp 60ndash65 2005

[3] J A Patza T K Graczykb N Gellera and A Y Vittorc ldquoEffectsof environmental change on emerging parasitic diseasesrdquo Inter-national Journal of Parasitology vol 30 no 12-13 pp 1295ndash13042000

[4] B H Alizadeh A Foroumadi S K Ardestani F Poorrajaband A Shafiee ldquoLeishmanicidal evaluation of novel syntheticchromenesrdquo Archiv der Pharmazie vol 341 no 12 pp 787ndash7932008

[5] RW Ashford ldquoThe leishmaniases as emerging and reemergingzoonosesrdquo International Journal for Parasitology vol 30 no 12-13 pp 1269ndash1281 2000

[6] M S S Rosa R R Mendonca-Filho H R Bizzo et al ldquoAntil-eishmanial activity of a linalool-rich essential oil from CrotoncajucarardquoAntimicrobial Agents Chemotherapy vol 47 no 6 pp1895ndash1901 2003

[7] T Ueda-Nakamura R R Mendonca-Filho J A Morgado-Dıazet al ldquoAntileishmanial activity of eugenol-rich essential oil fromOcimum gratissimumrdquo Parasitology International vol 55 no 2pp 55ndash59 2006

[8] A O Santos T Ueda-Nakamura B P Dias Filho V F VeigaJunior A C Pinto and C V Nakamura ldquoEffect of Braziliancopaiba oils on Leishmania amazonensisrdquo Journal of Ethnophar-macology vol 120 no 2 pp 204ndash208 2008

[9] LMonzote AMMontalvo S Almanonni R Scull MMiran-da and J Abreu ldquoActivity of the essential oil fromChenopodiumambrosioides grown in Cuba against Leishmania amazonensisrdquoChemotherapy vol 52 no 3 pp 130ndash136 2006

[10] MMachado P Pires A M Dinis et al ldquoMonoterpenic aldehy-des as potential anti-Leishmania agents activity ofCymbopogoncitratus and citral on L infantum L tropica and L majorrdquoExperimental Parasitology vol 130 no 3 pp 223ndash231 2012

[11] M R Santin A O Dos Santos C V Nakamura B P Dias FilhoI C P Ferreira and T Ueda-Nakamura ldquoIn vitro activity of theessential oil of Cymbopogon citratus and its major component(citral) on Leishmania amazonensisrdquo Parasitology Research vol105 no 6 pp 1489ndash1496 2009

[12] V C S Oliveira D M S Moura J A D Lopes P P DeAndrade N H Da Silva and R C B Q Figueiredo ldquoEffectsof essential oils from Cymbopogon citratus (DC) Stapf Lippiasidoides Cham and Ocimum gratissimum L on growth andultrastructure of Leishmania chagasi promastigotesrdquo Parasitol-ogy Research vol 104 no 5 pp 1053ndash1059 2009

[13] A E Consolini and M G Sarubbio ldquoPharmacological effectsof Eugenia uniflora (Myrtaceae) aqueous crude extract on ratrsquosheartrdquo Journal of Ethnopharmacology vol 81 no 1 pp 57ndash632002

[14] C B Alice V M F Vargas G A A B Silva et al ldquoScreening ofplants used in south Brazilian folk medicinerdquo Journal of Ethno-pharmacology vol 35 no 2 pp 165ndash171 1991

[15] P Weyerstahl H Marschall-Weyerstahl C Christiansen B OOguntimein and A O Adeoye ldquoVolatile constituents of Euge-nia uniflora leaf oilrdquo Planta medica vol 54 no 6 pp 546ndash5491988

[16] F N Victoria E J Lenardao L Savegnago et al ldquoEssential oil ofthe leaves of Eugenia uniflora L antioxidant and antimicrobialpropertiesrdquo Food and Chemical Toxicology vol 50 no 8 pp2668ndash2674 2012

Evidence-Based Complementary and Alternative Medicine 9

[17] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[18] I A Ogunwande N O Olawore O Ekundayo T M Walker JM Schmidt andWN Setzer ldquoStudies on the essential oils com-position antibacterial and cytotoxicity of Eugenia uniflora LrdquoInternational Journal of Aromatherapy vol 15 no 3 pp 147ndash1522005

[19] A C L Amorim C K F Lima A M C Hovell A L PMiranda and C M Rezende ldquoAntinociceptive and hypother-mic evaluation of the leaf essential oil and isolated terpenoidsfrom Eugenia uniflora L (Brazilian Pitanga)rdquo Phytomedicinevol 16 no 10 pp 923ndash928 2009

[20] D P Costa E G A Filho LM A Silva et al ldquoInfluence of fruitbiotypes on the chemical composition and antifungal activityof the essential oils of Eugenia uniflora leavesrdquo Journal of theBrazilian Chemical Society vol 21 no 5 pp 851ndash858 2010

[21] G F Ibikunle A C Adebajo F G Famuyiwa A J Aladesanmiand C O Adewunmi ldquoIn-vitro evaluation of anti-trichomonalactivities ofEugenia uniflora leafrdquoAfrican Journal of TraditionalComplementary and AlternativeMedicines vol 8 no 2 pp 170ndash176 2011

[22] K K Santos S R Tintino C E Souza et al ldquoAnti-Trypanosomacruzi and cytotoxic activities of Eugenia uniflora Lrdquo Experimen-tal Parasitology vol 131 no 1 pp 130ndash132 2012

[23] NIST 05 Mass Spectral Library (NISTEPANIH) NationalInstitute of Standards and Technology Gaithersburg Md USA2005

[24] R P Adams Identification of Essential Oil Components By GasChromatographyMass Spectrometry Allured Carol Stream IllUSA 4th edition 2007

[25] S M P Carneiro F A A Carvalho L C L R Santana A PL Sousa andM H Chaves ldquoThe cytotoxic and antileishmanialactivity of extracts and fractions of leaves and fruits of Azad-irachta indica (A Juss)rdquo Biological Research vol 45 no 2 pp111ndash116 2012

[26] M D G F deMedeiros A C da Silva A M D G L Cito et alldquoIn vitro antileishmanial activity and cytotoxicity of essential oilfrom Lippia sidoides Chamrdquo Parasitology International vol 60no 3 pp 237ndash241 2011

[27] S E Lofgren L C Miletti M Steindel E Bachere and M ABarracco ldquoTrypanocidal and leishmanicidal activities of differ-ent antimicrobial peptides (AMPs) isolated from aquatic ani-malsrdquo Experimental Parasitology vol 118 no 2 pp 197ndash2022008

[28] F C C Grando C A Felıcio A Twardowschy et al ldquoModula-tion of peritoneal macrophage activity by the saturation state ofthe fatty acid moiety of phosphatidylcholinerdquo Brazilian Journalof Medical and Biological Research vol 42 no 7 pp 599ndash6052009

[29] MM Iwu J E Jackson and BG Schuster ldquoMedicinal plants inthe fight against leishmaniasisrdquo Parasitology Today vol 10 no2 pp 65ndash68 1994

[30] L G Rocha J R G S Almeida R O Macedo and J M Bar-bosa-Filho ldquoA review of natural products with antileishmanialactivityrdquo Phytomedicine vol 12 no 6-7 pp 514ndash535 2005

[31] SMDeMorais A A CraveiroM I LMachado JW Alencarand F J A Matos ldquoVolatile constituents of Eugenia uniflora leafoil from northeastern Brazilrdquo Journal of Essential Oil Researchvol 8 no 4 pp 449ndash451 1996

[32] J G S Maia E H A Andrade M H L Da Silva and M G BZoghbi ldquoA new chemotype of Eugenia uniflora L from NorthBrazilrdquo Journal of Essential Oil Research vol 11 no 6 pp 727ndash729 1999

[33] J H G Lago E D Souza B Mariane et al ldquoChemical and bio-logical evaluation of essential oils from two species of myrtace-aemdashEugenia uniflora L and Plinia trunciflora (O Berg) KauselrdquoMolecules vol 16 no 12 pp 9826ndash9837 2011

[34] R Chang S A L de Morais D R Napolitano K C DuarteV B Guzman and E A do Nascimento ldquoA new approach forquantifying furanodiene and curzerene A case study on theessential oils of Eugenia uniflora (pitangueira) leavesrdquo BrazilianJournal of Pharmacognosy vol 21 no 3 pp 392ndash396 2011

[35] A M Adio ldquoGermacrenes A-E and related compounds ther-mal photochemical and acid induced transannular cycliza-tionsrdquo Tetrahedron vol 65 no 8 pp 1533ndash1552 2009

[36] D P Costa S C Santos J C Seraphin and PH Ferri ldquoSeasonalvariability of essential oils of Eugenia uniflora leavesrdquo Journal ofthe BrazilianChemical Society vol 20 no 7 pp 1287ndash1293 2009

[37] A T HenriquesM E Sobral A D Caudaro E E S SchapovalV L Bassani and G Lamaty ldquoAromatic plants from BrazilII The chemical composition of some Eugenia essential oilsrdquoJournal of Essential Oil Research vol 5 no 5 pp 501ndash505 1993

[38] B Teixeira A Marques C Ramos et al ldquoEuropean pennyroyal(Mentha pulegium) from portugal chemical composition ofessential oil and antioxidant and antimicrobial properties ofextracts and essential oilrdquo Industrial Crops and Products vol 36no 1 pp 81ndash87 2012

[39] D C Arruda F L DrsquoAlexandri A M Katzin and S RB Uliana ldquoAntileishmanial activity of the terpene nerolidolrdquoAntimicrobial Agents andChemotherapy vol 49 no 5 pp 1679ndash1687 2005

[40] RDi Pasqua G Betts NHoskinsM Edwards D Ercolini andG Mauriello ldquoMembrane toxicity of antimicrobial compoundsfrom essential oilsrdquo Journal of Agricultural and Food Chemistryvol 55 no 12 pp 4863ndash4870 2007

[41] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[42] J S Armstrong ldquoMitochondrial membrane permeabilizationthe sine qua non for cell deathrdquo BioEssays vol 28 no 3 pp253ndash260 2006

[43] A L Silva C M Adade F M Shoyama et al ldquoIn vitro leish-manicidal activity of N-dodecyl-1 2-ethanediaminerdquo Biomedi-cine and Pharmacotherapy vol 66 no 3 pp 180ndash186 2012

[44] B Purkait A Kumar N Nandi et al ldquoMechanism of ampho-tericin B resistance in clinical isolates of Leishmania donovanirdquoAntimicrobial Agents and Chemotherapy vol 56 no 2 pp 1031ndash1141 2012

[45] M Vermeersch R I Da Luz K Tote J P Timmermans P Cosand L Maes ldquoIn vitro susceptibilities of Leishmania donovanipromastigote and amastigote stages to antileishmanial referencedrugs practical relevance of stage-specific differencesrdquo Antimi-crobial Agents and Chemotherapy vol 53 no 9 pp 3855ndash38592009

[46] C Di Giorgio F Delmas V Akhmedjanova et al ldquoIn vitroantileishmanial activity of diphyllin isolated fromHaplophyllumbucharicumrdquo Planta Medica vol 71 no 4 pp 366ndash369 2005

10 Evidence-Based Complementary and Alternative Medicine

[47] H Martinez-Rojano J Mancilla-Ramirez L Quinonez-Diazand N Galindo-Sevilla ldquoActivity of hydroxyurea against Leish-mania mexicanardquo Antimicrobial Agents and Chemotherapy vol52 no 10 pp 3642ndash3647 2008

[48] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[49] A H Klimp E G E De Vries G L Scherphof and T DaemenldquoA potential role of macrophage activation in the treatment ofcancerrdquo Critical Reviews in OncologyHematology vol 44 no 2pp 143ndash161 2002

[50] M R Piemonte and D F Buchi ldquoAnalysis of IL-12 IFN-120574 and TNF-120572 production a5b1 integrins and actin filamentsdistribution in peritoneal mouse macrophages treated withhomeopathic medicamentrdquo Journal of Submicroscopic Cytologyand Pathology vol 33 no 1 pp 255ndash263 2002

[51] F Niedergang and P Chavrier ldquoSignaling and membranedynamics during phagocytosis Many roads lead to the pha-gos(R)omerdquo Current Opinion in Cell Biology vol 16 no 4 pp422ndash428 2004

[52] W L Lee R E Harrison and S Grinstein ldquoPhagocytosis byneutrophilsrdquo Microbes and Infection vol 5 no 14 pp 1299ndash1306 2003

[53] S J Green M S Meltzer J B Hibbs and C A Nacy ldquoActivatedmacrophages destroy intracellular Leishmania major amastig-otes by an L-arginine-dependent killing mechanismrdquo Journal ofImmunology vol 144 no 1 pp 278ndash283 1990

[54] E Osorio G J Arango N Jimenez et al ldquoAntiprotozoal andcytotoxic activities in vitro of Colombian Annonaceaerdquo Journalof Ethnopharmacology vol 111 no 3 pp 630ndash635 2007

[55] S Kamhawi ldquoPhlebotomine sand flies and Leishmania para-sites friends or foesrdquo Trends in Parasitology vol 22 no 9 pp439ndash445 2006

[56] P Tripathi V Singh and S Naik ldquoImmune response to leish-mania paradox rather than paradigmrdquo FEMS Immunology andMedical Microbiology vol 51 no 2 pp 229ndash242 2007

[57] L V Costa-Lotufo G M A Cunha P A M Farias et al ldquoThecytotoxic and embryotoxic effects of kaurenoic acid a diterpeneisolated fromCopaifera langsdorffii oleo-resinrdquoToxicon vol 40no 8 pp 1231ndash1234 2002

[58] R M Aparicio M J Garcıa-Celma M Pilar Vinardell and MMitjans ldquoIn vitro studies of the hemolytic activity ofmicroemul-sions in human erythrocytesrdquo Journal of Pharmaceutical andBiomedical Analysis vol 39 no 5 pp 1063ndash1067 2005

[59] L A Lexis R G Fassett and J S Coombes ldquo120572-tocopherol and120572-lipoic acid enhance the erythrocyte antioxidant defence incyclosporine A-treated ratsrdquo Basic and Clinical Pharmacologyand Toxicology vol 98 no 1 pp 68ndash73 2006

[60] J R Munoz-Castaneda J Muntane M C Munoz I BujalanceP Montilla and I Tunez ldquoEstradiol and catecholestrogens pro-tect against adriamycininduced oxidative stress in erythrocytesof ovariectomized ratsrdquo Toxicology Letters vol 160 no 3 pp196ndash203 2006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

C 100 50 25 125 625 312

005

115

225

335

445

5N

eutr

al re

d up

take

ABS

lowast

lowastlowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(a)

C 100 50 25 125 625 312

0

005

01

015

02

025

Phag

ocyt

osis

ABS

lowast lowast lowastlowast lowastlowast

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

(b)

Figure 6 The influence of Eugenia uniflora essential oil on the lysosomal activity (a) and phagocytic activity (b) of peritoneal macrophagesat concentrations of 100 50 25 125 625 and 312 120583gsdotmLminus1 Data represent the mean density plusmn standard error of 3 experiments carried outin triplicate lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 C control ABS absorbance

0010203040506070809

1

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

C LPS 50 25 125 625 312100

Nitr

ite p

rodu

ctio

n A

BS

119871119890119894119904ℎ119898119886119899119894119886 119886119898119886119911119900119899119890119899119904119894119904

0010203040506070809

1

119864119906119892119890119899119894119886 119906119899119894119891119897119900119903119886 (120583gmiddotmLminus1)

lowastlowastlowast

(b)

Figure 7 Production of nitric oxide (NO) Murine macrophages (2 times 105) were treated with Eugenia uniflora essential oil (100 50 25 125625 and 312 120583gsdotmLminus1) in the absence (a) or presence of Leishmania amazonensis (b) over 24 h Data represent the mean density plusmn standarderror of 3 experiments carried out in triplicate lowastlowastlowast119875 lt 0001 C control ABS absorbance

48 h after exposure to oil with an IC50of 192 120583gmL whereas

the IC50

for promastigotes was 304120583gsdotmLminus1 for the sameduration of exposure Previous studies demonstrated greateractivity against amastigotes versus promastigotes of essentialoils of Artemisia absinthium and Satureja punctata [48]

Mechanisms of anti-Leishmania action on Leishmaniaspp amastigotes have also been described for essentialoils and their terpene compounds Monoterpenes such aslinalool isolated from leaves of Croton cajucara (Euphorbia-ceae) significantly increased NO production inmacrophagesinfected with L amazonensis and acts directly on the parasiteas evidenced by mitochondrial swelling and changes in thekinetoplast and the organization of nuclear chromatin[6] In this study the production of NO was determined bymeasuring nitriteThe results frommacrophages treated with

EuEO revealed that EuEO does not induce NO productionwhich demonstrates that its promising anti-Leishmaniaactivity does not occur by this route

Natural products that are able to activate macrophagesand are thus biological response modifiers have beenextensively studied [49 50] In this context the EuEOdemonstrated potential in the activation of macrophagesby increasing phagocytic capacity and lysosomal activityimmunomodulatory activities possibly involved in its anti-Leishmania activity Phagocytosis and the lysosomal systemare critical to macrophage function because of their roles ininternalization degradation and eventually presentation ofpeptides derived from antigens required for host defense Inaddition antigen presentation occurs through phagocytosisand endosomallysosomal targeting systems [51ndash53]

8 Evidence-Based Complementary and Alternative Medicine

Due to the need for anti-Leishmania substances that aremore selective for the parasite and less toxic to host cellsand since EuEO showed low IC

50values it was important to

investigate the cytotoxic activity of EuEO againstmammaliancells EuEO showed significant toxicity in macrophages witha CC50

of 453120583gsdotmLminus1 but when compared with its IC50

value in amastigotes this indicates a secure selectivity indexabove 20 The literature recommends that for amastigotesinternalized in macrophages this index must be demon-strated as near to or greater than 20 [54] The investigationof the toxic action of new substances with anti-Leishmaniaactivity onmacrophages is important since they are themaincells of the vertebrate host parasitized by Leishmania spp[55 56] Furthermore various other cytotoxic assays can beconducted using animal and human cells to determine thesafety of a test substance leading to a future treatment invivo Tests performed with erythrocytes enable assessment ofa drugrsquos potential to cause injury to the plasma membrane ofa cell either by forming pores or by total rupture leadingto cellular damage or changes in membrane permeability[57] This is a model used for the preliminary study of theprotective and toxic effects of substances being a possibleindicator of this type of damage to cells in vivo [58ndash60]The results obtained in this test with EuEO revealed lowtoxicity to erythrocytes since EuEO showed 0 hemolysis atconcentrations of 50120583gsdotmLminus1 and lower

5 Conclusions

In conclusion the data in this study show that the essentialoil of leaves from E uniflora (EuEO) characterized bysesquiterpenes has anti-Leishmania activity in both stagesof L amazonensis This study also demonstrated that EuEOactivity is not mediated by NO production On the otherhand activation of macrophages may be involved in themechanisms of EuEO anti-Leishmania activity as evidencedby increases in both phagocytic capacity and the lysosomalcompartment Further investigation is needed to determineadditional mechanisms involved in the anti-Leishmaniaactivity of this plant species as well as in vivo studies inmodels of leishmaniasis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors are grateful to the CAPES and FAPEMA forfinancial grants through the concession of scholarships andfinancial support

References

[1] S Kaur H Sachdeva S Dhuria M Sharma and T Kaur ldquoAn-tileishmanial effect of cisplatin against murine visceral leishma-niasisrdquo Parasitology International vol 59 no 1 pp 62ndash69 2010

[2] S AzzouzMMaache R G Garcia andA Osuna ldquoLeishmani-cidal activity of edelfosine miltefosine and ilmofosinerdquo Basic

and Clinical Pharmacology and Toxicology vol 96 no 1 pp 60ndash65 2005

[3] J A Patza T K Graczykb N Gellera and A Y Vittorc ldquoEffectsof environmental change on emerging parasitic diseasesrdquo Inter-national Journal of Parasitology vol 30 no 12-13 pp 1295ndash13042000

[4] B H Alizadeh A Foroumadi S K Ardestani F Poorrajaband A Shafiee ldquoLeishmanicidal evaluation of novel syntheticchromenesrdquo Archiv der Pharmazie vol 341 no 12 pp 787ndash7932008

[5] RW Ashford ldquoThe leishmaniases as emerging and reemergingzoonosesrdquo International Journal for Parasitology vol 30 no 12-13 pp 1269ndash1281 2000

[6] M S S Rosa R R Mendonca-Filho H R Bizzo et al ldquoAntil-eishmanial activity of a linalool-rich essential oil from CrotoncajucarardquoAntimicrobial Agents Chemotherapy vol 47 no 6 pp1895ndash1901 2003

[7] T Ueda-Nakamura R R Mendonca-Filho J A Morgado-Dıazet al ldquoAntileishmanial activity of eugenol-rich essential oil fromOcimum gratissimumrdquo Parasitology International vol 55 no 2pp 55ndash59 2006

[8] A O Santos T Ueda-Nakamura B P Dias Filho V F VeigaJunior A C Pinto and C V Nakamura ldquoEffect of Braziliancopaiba oils on Leishmania amazonensisrdquo Journal of Ethnophar-macology vol 120 no 2 pp 204ndash208 2008

[9] LMonzote AMMontalvo S Almanonni R Scull MMiran-da and J Abreu ldquoActivity of the essential oil fromChenopodiumambrosioides grown in Cuba against Leishmania amazonensisrdquoChemotherapy vol 52 no 3 pp 130ndash136 2006

[10] MMachado P Pires A M Dinis et al ldquoMonoterpenic aldehy-des as potential anti-Leishmania agents activity ofCymbopogoncitratus and citral on L infantum L tropica and L majorrdquoExperimental Parasitology vol 130 no 3 pp 223ndash231 2012

[11] M R Santin A O Dos Santos C V Nakamura B P Dias FilhoI C P Ferreira and T Ueda-Nakamura ldquoIn vitro activity of theessential oil of Cymbopogon citratus and its major component(citral) on Leishmania amazonensisrdquo Parasitology Research vol105 no 6 pp 1489ndash1496 2009

[12] V C S Oliveira D M S Moura J A D Lopes P P DeAndrade N H Da Silva and R C B Q Figueiredo ldquoEffectsof essential oils from Cymbopogon citratus (DC) Stapf Lippiasidoides Cham and Ocimum gratissimum L on growth andultrastructure of Leishmania chagasi promastigotesrdquo Parasitol-ogy Research vol 104 no 5 pp 1053ndash1059 2009

[13] A E Consolini and M G Sarubbio ldquoPharmacological effectsof Eugenia uniflora (Myrtaceae) aqueous crude extract on ratrsquosheartrdquo Journal of Ethnopharmacology vol 81 no 1 pp 57ndash632002

[14] C B Alice V M F Vargas G A A B Silva et al ldquoScreening ofplants used in south Brazilian folk medicinerdquo Journal of Ethno-pharmacology vol 35 no 2 pp 165ndash171 1991

[15] P Weyerstahl H Marschall-Weyerstahl C Christiansen B OOguntimein and A O Adeoye ldquoVolatile constituents of Euge-nia uniflora leaf oilrdquo Planta medica vol 54 no 6 pp 546ndash5491988

[16] F N Victoria E J Lenardao L Savegnago et al ldquoEssential oil ofthe leaves of Eugenia uniflora L antioxidant and antimicrobialpropertiesrdquo Food and Chemical Toxicology vol 50 no 8 pp2668ndash2674 2012

Evidence-Based Complementary and Alternative Medicine 9

[17] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[18] I A Ogunwande N O Olawore O Ekundayo T M Walker JM Schmidt andWN Setzer ldquoStudies on the essential oils com-position antibacterial and cytotoxicity of Eugenia uniflora LrdquoInternational Journal of Aromatherapy vol 15 no 3 pp 147ndash1522005

[19] A C L Amorim C K F Lima A M C Hovell A L PMiranda and C M Rezende ldquoAntinociceptive and hypother-mic evaluation of the leaf essential oil and isolated terpenoidsfrom Eugenia uniflora L (Brazilian Pitanga)rdquo Phytomedicinevol 16 no 10 pp 923ndash928 2009

[20] D P Costa E G A Filho LM A Silva et al ldquoInfluence of fruitbiotypes on the chemical composition and antifungal activityof the essential oils of Eugenia uniflora leavesrdquo Journal of theBrazilian Chemical Society vol 21 no 5 pp 851ndash858 2010

[21] G F Ibikunle A C Adebajo F G Famuyiwa A J Aladesanmiand C O Adewunmi ldquoIn-vitro evaluation of anti-trichomonalactivities ofEugenia uniflora leafrdquoAfrican Journal of TraditionalComplementary and AlternativeMedicines vol 8 no 2 pp 170ndash176 2011

[22] K K Santos S R Tintino C E Souza et al ldquoAnti-Trypanosomacruzi and cytotoxic activities of Eugenia uniflora Lrdquo Experimen-tal Parasitology vol 131 no 1 pp 130ndash132 2012

[23] NIST 05 Mass Spectral Library (NISTEPANIH) NationalInstitute of Standards and Technology Gaithersburg Md USA2005

[24] R P Adams Identification of Essential Oil Components By GasChromatographyMass Spectrometry Allured Carol Stream IllUSA 4th edition 2007

[25] S M P Carneiro F A A Carvalho L C L R Santana A PL Sousa andM H Chaves ldquoThe cytotoxic and antileishmanialactivity of extracts and fractions of leaves and fruits of Azad-irachta indica (A Juss)rdquo Biological Research vol 45 no 2 pp111ndash116 2012

[26] M D G F deMedeiros A C da Silva A M D G L Cito et alldquoIn vitro antileishmanial activity and cytotoxicity of essential oilfrom Lippia sidoides Chamrdquo Parasitology International vol 60no 3 pp 237ndash241 2011

[27] S E Lofgren L C Miletti M Steindel E Bachere and M ABarracco ldquoTrypanocidal and leishmanicidal activities of differ-ent antimicrobial peptides (AMPs) isolated from aquatic ani-malsrdquo Experimental Parasitology vol 118 no 2 pp 197ndash2022008

[28] F C C Grando C A Felıcio A Twardowschy et al ldquoModula-tion of peritoneal macrophage activity by the saturation state ofthe fatty acid moiety of phosphatidylcholinerdquo Brazilian Journalof Medical and Biological Research vol 42 no 7 pp 599ndash6052009

[29] MM Iwu J E Jackson and BG Schuster ldquoMedicinal plants inthe fight against leishmaniasisrdquo Parasitology Today vol 10 no2 pp 65ndash68 1994

[30] L G Rocha J R G S Almeida R O Macedo and J M Bar-bosa-Filho ldquoA review of natural products with antileishmanialactivityrdquo Phytomedicine vol 12 no 6-7 pp 514ndash535 2005

[31] SMDeMorais A A CraveiroM I LMachado JW Alencarand F J A Matos ldquoVolatile constituents of Eugenia uniflora leafoil from northeastern Brazilrdquo Journal of Essential Oil Researchvol 8 no 4 pp 449ndash451 1996

[32] J G S Maia E H A Andrade M H L Da Silva and M G BZoghbi ldquoA new chemotype of Eugenia uniflora L from NorthBrazilrdquo Journal of Essential Oil Research vol 11 no 6 pp 727ndash729 1999

[33] J H G Lago E D Souza B Mariane et al ldquoChemical and bio-logical evaluation of essential oils from two species of myrtace-aemdashEugenia uniflora L and Plinia trunciflora (O Berg) KauselrdquoMolecules vol 16 no 12 pp 9826ndash9837 2011

[34] R Chang S A L de Morais D R Napolitano K C DuarteV B Guzman and E A do Nascimento ldquoA new approach forquantifying furanodiene and curzerene A case study on theessential oils of Eugenia uniflora (pitangueira) leavesrdquo BrazilianJournal of Pharmacognosy vol 21 no 3 pp 392ndash396 2011

[35] A M Adio ldquoGermacrenes A-E and related compounds ther-mal photochemical and acid induced transannular cycliza-tionsrdquo Tetrahedron vol 65 no 8 pp 1533ndash1552 2009

[36] D P Costa S C Santos J C Seraphin and PH Ferri ldquoSeasonalvariability of essential oils of Eugenia uniflora leavesrdquo Journal ofthe BrazilianChemical Society vol 20 no 7 pp 1287ndash1293 2009

[37] A T HenriquesM E Sobral A D Caudaro E E S SchapovalV L Bassani and G Lamaty ldquoAromatic plants from BrazilII The chemical composition of some Eugenia essential oilsrdquoJournal of Essential Oil Research vol 5 no 5 pp 501ndash505 1993

[38] B Teixeira A Marques C Ramos et al ldquoEuropean pennyroyal(Mentha pulegium) from portugal chemical composition ofessential oil and antioxidant and antimicrobial properties ofextracts and essential oilrdquo Industrial Crops and Products vol 36no 1 pp 81ndash87 2012

[39] D C Arruda F L DrsquoAlexandri A M Katzin and S RB Uliana ldquoAntileishmanial activity of the terpene nerolidolrdquoAntimicrobial Agents andChemotherapy vol 49 no 5 pp 1679ndash1687 2005

[40] RDi Pasqua G Betts NHoskinsM Edwards D Ercolini andG Mauriello ldquoMembrane toxicity of antimicrobial compoundsfrom essential oilsrdquo Journal of Agricultural and Food Chemistryvol 55 no 12 pp 4863ndash4870 2007

[41] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[42] J S Armstrong ldquoMitochondrial membrane permeabilizationthe sine qua non for cell deathrdquo BioEssays vol 28 no 3 pp253ndash260 2006

[43] A L Silva C M Adade F M Shoyama et al ldquoIn vitro leish-manicidal activity of N-dodecyl-1 2-ethanediaminerdquo Biomedi-cine and Pharmacotherapy vol 66 no 3 pp 180ndash186 2012

[44] B Purkait A Kumar N Nandi et al ldquoMechanism of ampho-tericin B resistance in clinical isolates of Leishmania donovanirdquoAntimicrobial Agents and Chemotherapy vol 56 no 2 pp 1031ndash1141 2012

[45] M Vermeersch R I Da Luz K Tote J P Timmermans P Cosand L Maes ldquoIn vitro susceptibilities of Leishmania donovanipromastigote and amastigote stages to antileishmanial referencedrugs practical relevance of stage-specific differencesrdquo Antimi-crobial Agents and Chemotherapy vol 53 no 9 pp 3855ndash38592009

[46] C Di Giorgio F Delmas V Akhmedjanova et al ldquoIn vitroantileishmanial activity of diphyllin isolated fromHaplophyllumbucharicumrdquo Planta Medica vol 71 no 4 pp 366ndash369 2005

10 Evidence-Based Complementary and Alternative Medicine

[47] H Martinez-Rojano J Mancilla-Ramirez L Quinonez-Diazand N Galindo-Sevilla ldquoActivity of hydroxyurea against Leish-mania mexicanardquo Antimicrobial Agents and Chemotherapy vol52 no 10 pp 3642ndash3647 2008

[48] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[49] A H Klimp E G E De Vries G L Scherphof and T DaemenldquoA potential role of macrophage activation in the treatment ofcancerrdquo Critical Reviews in OncologyHematology vol 44 no 2pp 143ndash161 2002

[50] M R Piemonte and D F Buchi ldquoAnalysis of IL-12 IFN-120574 and TNF-120572 production a5b1 integrins and actin filamentsdistribution in peritoneal mouse macrophages treated withhomeopathic medicamentrdquo Journal of Submicroscopic Cytologyand Pathology vol 33 no 1 pp 255ndash263 2002

[51] F Niedergang and P Chavrier ldquoSignaling and membranedynamics during phagocytosis Many roads lead to the pha-gos(R)omerdquo Current Opinion in Cell Biology vol 16 no 4 pp422ndash428 2004

[52] W L Lee R E Harrison and S Grinstein ldquoPhagocytosis byneutrophilsrdquo Microbes and Infection vol 5 no 14 pp 1299ndash1306 2003

[53] S J Green M S Meltzer J B Hibbs and C A Nacy ldquoActivatedmacrophages destroy intracellular Leishmania major amastig-otes by an L-arginine-dependent killing mechanismrdquo Journal ofImmunology vol 144 no 1 pp 278ndash283 1990

[54] E Osorio G J Arango N Jimenez et al ldquoAntiprotozoal andcytotoxic activities in vitro of Colombian Annonaceaerdquo Journalof Ethnopharmacology vol 111 no 3 pp 630ndash635 2007

[55] S Kamhawi ldquoPhlebotomine sand flies and Leishmania para-sites friends or foesrdquo Trends in Parasitology vol 22 no 9 pp439ndash445 2006

[56] P Tripathi V Singh and S Naik ldquoImmune response to leish-mania paradox rather than paradigmrdquo FEMS Immunology andMedical Microbiology vol 51 no 2 pp 229ndash242 2007

[57] L V Costa-Lotufo G M A Cunha P A M Farias et al ldquoThecytotoxic and embryotoxic effects of kaurenoic acid a diterpeneisolated fromCopaifera langsdorffii oleo-resinrdquoToxicon vol 40no 8 pp 1231ndash1234 2002

[58] R M Aparicio M J Garcıa-Celma M Pilar Vinardell and MMitjans ldquoIn vitro studies of the hemolytic activity ofmicroemul-sions in human erythrocytesrdquo Journal of Pharmaceutical andBiomedical Analysis vol 39 no 5 pp 1063ndash1067 2005

[59] L A Lexis R G Fassett and J S Coombes ldquo120572-tocopherol and120572-lipoic acid enhance the erythrocyte antioxidant defence incyclosporine A-treated ratsrdquo Basic and Clinical Pharmacologyand Toxicology vol 98 no 1 pp 68ndash73 2006

[60] J R Munoz-Castaneda J Muntane M C Munoz I BujalanceP Montilla and I Tunez ldquoEstradiol and catecholestrogens pro-tect against adriamycininduced oxidative stress in erythrocytesof ovariectomized ratsrdquo Toxicology Letters vol 160 no 3 pp196ndash203 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

Due to the need for anti-Leishmania substances that aremore selective for the parasite and less toxic to host cellsand since EuEO showed low IC

50values it was important to

investigate the cytotoxic activity of EuEO againstmammaliancells EuEO showed significant toxicity in macrophages witha CC50

of 453120583gsdotmLminus1 but when compared with its IC50

value in amastigotes this indicates a secure selectivity indexabove 20 The literature recommends that for amastigotesinternalized in macrophages this index must be demon-strated as near to or greater than 20 [54] The investigationof the toxic action of new substances with anti-Leishmaniaactivity onmacrophages is important since they are themaincells of the vertebrate host parasitized by Leishmania spp[55 56] Furthermore various other cytotoxic assays can beconducted using animal and human cells to determine thesafety of a test substance leading to a future treatment invivo Tests performed with erythrocytes enable assessment ofa drugrsquos potential to cause injury to the plasma membrane ofa cell either by forming pores or by total rupture leadingto cellular damage or changes in membrane permeability[57] This is a model used for the preliminary study of theprotective and toxic effects of substances being a possibleindicator of this type of damage to cells in vivo [58ndash60]The results obtained in this test with EuEO revealed lowtoxicity to erythrocytes since EuEO showed 0 hemolysis atconcentrations of 50120583gsdotmLminus1 and lower

5 Conclusions

In conclusion the data in this study show that the essentialoil of leaves from E uniflora (EuEO) characterized bysesquiterpenes has anti-Leishmania activity in both stagesof L amazonensis This study also demonstrated that EuEOactivity is not mediated by NO production On the otherhand activation of macrophages may be involved in themechanisms of EuEO anti-Leishmania activity as evidencedby increases in both phagocytic capacity and the lysosomalcompartment Further investigation is needed to determineadditional mechanisms involved in the anti-Leishmaniaactivity of this plant species as well as in vivo studies inmodels of leishmaniasis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors are grateful to the CAPES and FAPEMA forfinancial grants through the concession of scholarships andfinancial support

References

[1] S Kaur H Sachdeva S Dhuria M Sharma and T Kaur ldquoAn-tileishmanial effect of cisplatin against murine visceral leishma-niasisrdquo Parasitology International vol 59 no 1 pp 62ndash69 2010

[2] S AzzouzMMaache R G Garcia andA Osuna ldquoLeishmani-cidal activity of edelfosine miltefosine and ilmofosinerdquo Basic

and Clinical Pharmacology and Toxicology vol 96 no 1 pp 60ndash65 2005

[3] J A Patza T K Graczykb N Gellera and A Y Vittorc ldquoEffectsof environmental change on emerging parasitic diseasesrdquo Inter-national Journal of Parasitology vol 30 no 12-13 pp 1295ndash13042000

[4] B H Alizadeh A Foroumadi S K Ardestani F Poorrajaband A Shafiee ldquoLeishmanicidal evaluation of novel syntheticchromenesrdquo Archiv der Pharmazie vol 341 no 12 pp 787ndash7932008

[5] RW Ashford ldquoThe leishmaniases as emerging and reemergingzoonosesrdquo International Journal for Parasitology vol 30 no 12-13 pp 1269ndash1281 2000

[6] M S S Rosa R R Mendonca-Filho H R Bizzo et al ldquoAntil-eishmanial activity of a linalool-rich essential oil from CrotoncajucarardquoAntimicrobial Agents Chemotherapy vol 47 no 6 pp1895ndash1901 2003

[7] T Ueda-Nakamura R R Mendonca-Filho J A Morgado-Dıazet al ldquoAntileishmanial activity of eugenol-rich essential oil fromOcimum gratissimumrdquo Parasitology International vol 55 no 2pp 55ndash59 2006

[8] A O Santos T Ueda-Nakamura B P Dias Filho V F VeigaJunior A C Pinto and C V Nakamura ldquoEffect of Braziliancopaiba oils on Leishmania amazonensisrdquo Journal of Ethnophar-macology vol 120 no 2 pp 204ndash208 2008

[9] LMonzote AMMontalvo S Almanonni R Scull MMiran-da and J Abreu ldquoActivity of the essential oil fromChenopodiumambrosioides grown in Cuba against Leishmania amazonensisrdquoChemotherapy vol 52 no 3 pp 130ndash136 2006

[10] MMachado P Pires A M Dinis et al ldquoMonoterpenic aldehy-des as potential anti-Leishmania agents activity ofCymbopogoncitratus and citral on L infantum L tropica and L majorrdquoExperimental Parasitology vol 130 no 3 pp 223ndash231 2012

[11] M R Santin A O Dos Santos C V Nakamura B P Dias FilhoI C P Ferreira and T Ueda-Nakamura ldquoIn vitro activity of theessential oil of Cymbopogon citratus and its major component(citral) on Leishmania amazonensisrdquo Parasitology Research vol105 no 6 pp 1489ndash1496 2009

[12] V C S Oliveira D M S Moura J A D Lopes P P DeAndrade N H Da Silva and R C B Q Figueiredo ldquoEffectsof essential oils from Cymbopogon citratus (DC) Stapf Lippiasidoides Cham and Ocimum gratissimum L on growth andultrastructure of Leishmania chagasi promastigotesrdquo Parasitol-ogy Research vol 104 no 5 pp 1053ndash1059 2009

[13] A E Consolini and M G Sarubbio ldquoPharmacological effectsof Eugenia uniflora (Myrtaceae) aqueous crude extract on ratrsquosheartrdquo Journal of Ethnopharmacology vol 81 no 1 pp 57ndash632002

[14] C B Alice V M F Vargas G A A B Silva et al ldquoScreening ofplants used in south Brazilian folk medicinerdquo Journal of Ethno-pharmacology vol 35 no 2 pp 165ndash171 1991

[15] P Weyerstahl H Marschall-Weyerstahl C Christiansen B OOguntimein and A O Adeoye ldquoVolatile constituents of Euge-nia uniflora leaf oilrdquo Planta medica vol 54 no 6 pp 546ndash5491988

[16] F N Victoria E J Lenardao L Savegnago et al ldquoEssential oil ofthe leaves of Eugenia uniflora L antioxidant and antimicrobialpropertiesrdquo Food and Chemical Toxicology vol 50 no 8 pp2668ndash2674 2012

Evidence-Based Complementary and Alternative Medicine 9

[17] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[18] I A Ogunwande N O Olawore O Ekundayo T M Walker JM Schmidt andWN Setzer ldquoStudies on the essential oils com-position antibacterial and cytotoxicity of Eugenia uniflora LrdquoInternational Journal of Aromatherapy vol 15 no 3 pp 147ndash1522005

[19] A C L Amorim C K F Lima A M C Hovell A L PMiranda and C M Rezende ldquoAntinociceptive and hypother-mic evaluation of the leaf essential oil and isolated terpenoidsfrom Eugenia uniflora L (Brazilian Pitanga)rdquo Phytomedicinevol 16 no 10 pp 923ndash928 2009

[20] D P Costa E G A Filho LM A Silva et al ldquoInfluence of fruitbiotypes on the chemical composition and antifungal activityof the essential oils of Eugenia uniflora leavesrdquo Journal of theBrazilian Chemical Society vol 21 no 5 pp 851ndash858 2010

[21] G F Ibikunle A C Adebajo F G Famuyiwa A J Aladesanmiand C O Adewunmi ldquoIn-vitro evaluation of anti-trichomonalactivities ofEugenia uniflora leafrdquoAfrican Journal of TraditionalComplementary and AlternativeMedicines vol 8 no 2 pp 170ndash176 2011

[22] K K Santos S R Tintino C E Souza et al ldquoAnti-Trypanosomacruzi and cytotoxic activities of Eugenia uniflora Lrdquo Experimen-tal Parasitology vol 131 no 1 pp 130ndash132 2012

[23] NIST 05 Mass Spectral Library (NISTEPANIH) NationalInstitute of Standards and Technology Gaithersburg Md USA2005

[24] R P Adams Identification of Essential Oil Components By GasChromatographyMass Spectrometry Allured Carol Stream IllUSA 4th edition 2007

[25] S M P Carneiro F A A Carvalho L C L R Santana A PL Sousa andM H Chaves ldquoThe cytotoxic and antileishmanialactivity of extracts and fractions of leaves and fruits of Azad-irachta indica (A Juss)rdquo Biological Research vol 45 no 2 pp111ndash116 2012

[26] M D G F deMedeiros A C da Silva A M D G L Cito et alldquoIn vitro antileishmanial activity and cytotoxicity of essential oilfrom Lippia sidoides Chamrdquo Parasitology International vol 60no 3 pp 237ndash241 2011

[27] S E Lofgren L C Miletti M Steindel E Bachere and M ABarracco ldquoTrypanocidal and leishmanicidal activities of differ-ent antimicrobial peptides (AMPs) isolated from aquatic ani-malsrdquo Experimental Parasitology vol 118 no 2 pp 197ndash2022008

[28] F C C Grando C A Felıcio A Twardowschy et al ldquoModula-tion of peritoneal macrophage activity by the saturation state ofthe fatty acid moiety of phosphatidylcholinerdquo Brazilian Journalof Medical and Biological Research vol 42 no 7 pp 599ndash6052009

[29] MM Iwu J E Jackson and BG Schuster ldquoMedicinal plants inthe fight against leishmaniasisrdquo Parasitology Today vol 10 no2 pp 65ndash68 1994

[30] L G Rocha J R G S Almeida R O Macedo and J M Bar-bosa-Filho ldquoA review of natural products with antileishmanialactivityrdquo Phytomedicine vol 12 no 6-7 pp 514ndash535 2005

[31] SMDeMorais A A CraveiroM I LMachado JW Alencarand F J A Matos ldquoVolatile constituents of Eugenia uniflora leafoil from northeastern Brazilrdquo Journal of Essential Oil Researchvol 8 no 4 pp 449ndash451 1996

[32] J G S Maia E H A Andrade M H L Da Silva and M G BZoghbi ldquoA new chemotype of Eugenia uniflora L from NorthBrazilrdquo Journal of Essential Oil Research vol 11 no 6 pp 727ndash729 1999

[33] J H G Lago E D Souza B Mariane et al ldquoChemical and bio-logical evaluation of essential oils from two species of myrtace-aemdashEugenia uniflora L and Plinia trunciflora (O Berg) KauselrdquoMolecules vol 16 no 12 pp 9826ndash9837 2011

[34] R Chang S A L de Morais D R Napolitano K C DuarteV B Guzman and E A do Nascimento ldquoA new approach forquantifying furanodiene and curzerene A case study on theessential oils of Eugenia uniflora (pitangueira) leavesrdquo BrazilianJournal of Pharmacognosy vol 21 no 3 pp 392ndash396 2011

[35] A M Adio ldquoGermacrenes A-E and related compounds ther-mal photochemical and acid induced transannular cycliza-tionsrdquo Tetrahedron vol 65 no 8 pp 1533ndash1552 2009

[36] D P Costa S C Santos J C Seraphin and PH Ferri ldquoSeasonalvariability of essential oils of Eugenia uniflora leavesrdquo Journal ofthe BrazilianChemical Society vol 20 no 7 pp 1287ndash1293 2009

[37] A T HenriquesM E Sobral A D Caudaro E E S SchapovalV L Bassani and G Lamaty ldquoAromatic plants from BrazilII The chemical composition of some Eugenia essential oilsrdquoJournal of Essential Oil Research vol 5 no 5 pp 501ndash505 1993

[38] B Teixeira A Marques C Ramos et al ldquoEuropean pennyroyal(Mentha pulegium) from portugal chemical composition ofessential oil and antioxidant and antimicrobial properties ofextracts and essential oilrdquo Industrial Crops and Products vol 36no 1 pp 81ndash87 2012

[39] D C Arruda F L DrsquoAlexandri A M Katzin and S RB Uliana ldquoAntileishmanial activity of the terpene nerolidolrdquoAntimicrobial Agents andChemotherapy vol 49 no 5 pp 1679ndash1687 2005

[40] RDi Pasqua G Betts NHoskinsM Edwards D Ercolini andG Mauriello ldquoMembrane toxicity of antimicrobial compoundsfrom essential oilsrdquo Journal of Agricultural and Food Chemistryvol 55 no 12 pp 4863ndash4870 2007

[41] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[42] J S Armstrong ldquoMitochondrial membrane permeabilizationthe sine qua non for cell deathrdquo BioEssays vol 28 no 3 pp253ndash260 2006

[43] A L Silva C M Adade F M Shoyama et al ldquoIn vitro leish-manicidal activity of N-dodecyl-1 2-ethanediaminerdquo Biomedi-cine and Pharmacotherapy vol 66 no 3 pp 180ndash186 2012

[44] B Purkait A Kumar N Nandi et al ldquoMechanism of ampho-tericin B resistance in clinical isolates of Leishmania donovanirdquoAntimicrobial Agents and Chemotherapy vol 56 no 2 pp 1031ndash1141 2012

[45] M Vermeersch R I Da Luz K Tote J P Timmermans P Cosand L Maes ldquoIn vitro susceptibilities of Leishmania donovanipromastigote and amastigote stages to antileishmanial referencedrugs practical relevance of stage-specific differencesrdquo Antimi-crobial Agents and Chemotherapy vol 53 no 9 pp 3855ndash38592009

[46] C Di Giorgio F Delmas V Akhmedjanova et al ldquoIn vitroantileishmanial activity of diphyllin isolated fromHaplophyllumbucharicumrdquo Planta Medica vol 71 no 4 pp 366ndash369 2005

10 Evidence-Based Complementary and Alternative Medicine

[47] H Martinez-Rojano J Mancilla-Ramirez L Quinonez-Diazand N Galindo-Sevilla ldquoActivity of hydroxyurea against Leish-mania mexicanardquo Antimicrobial Agents and Chemotherapy vol52 no 10 pp 3642ndash3647 2008

[48] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[49] A H Klimp E G E De Vries G L Scherphof and T DaemenldquoA potential role of macrophage activation in the treatment ofcancerrdquo Critical Reviews in OncologyHematology vol 44 no 2pp 143ndash161 2002

[50] M R Piemonte and D F Buchi ldquoAnalysis of IL-12 IFN-120574 and TNF-120572 production a5b1 integrins and actin filamentsdistribution in peritoneal mouse macrophages treated withhomeopathic medicamentrdquo Journal of Submicroscopic Cytologyand Pathology vol 33 no 1 pp 255ndash263 2002

[51] F Niedergang and P Chavrier ldquoSignaling and membranedynamics during phagocytosis Many roads lead to the pha-gos(R)omerdquo Current Opinion in Cell Biology vol 16 no 4 pp422ndash428 2004

[52] W L Lee R E Harrison and S Grinstein ldquoPhagocytosis byneutrophilsrdquo Microbes and Infection vol 5 no 14 pp 1299ndash1306 2003

[53] S J Green M S Meltzer J B Hibbs and C A Nacy ldquoActivatedmacrophages destroy intracellular Leishmania major amastig-otes by an L-arginine-dependent killing mechanismrdquo Journal ofImmunology vol 144 no 1 pp 278ndash283 1990

[54] E Osorio G J Arango N Jimenez et al ldquoAntiprotozoal andcytotoxic activities in vitro of Colombian Annonaceaerdquo Journalof Ethnopharmacology vol 111 no 3 pp 630ndash635 2007

[55] S Kamhawi ldquoPhlebotomine sand flies and Leishmania para-sites friends or foesrdquo Trends in Parasitology vol 22 no 9 pp439ndash445 2006

[56] P Tripathi V Singh and S Naik ldquoImmune response to leish-mania paradox rather than paradigmrdquo FEMS Immunology andMedical Microbiology vol 51 no 2 pp 229ndash242 2007

[57] L V Costa-Lotufo G M A Cunha P A M Farias et al ldquoThecytotoxic and embryotoxic effects of kaurenoic acid a diterpeneisolated fromCopaifera langsdorffii oleo-resinrdquoToxicon vol 40no 8 pp 1231ndash1234 2002

[58] R M Aparicio M J Garcıa-Celma M Pilar Vinardell and MMitjans ldquoIn vitro studies of the hemolytic activity ofmicroemul-sions in human erythrocytesrdquo Journal of Pharmaceutical andBiomedical Analysis vol 39 no 5 pp 1063ndash1067 2005

[59] L A Lexis R G Fassett and J S Coombes ldquo120572-tocopherol and120572-lipoic acid enhance the erythrocyte antioxidant defence incyclosporine A-treated ratsrdquo Basic and Clinical Pharmacologyand Toxicology vol 98 no 1 pp 68ndash73 2006

[60] J R Munoz-Castaneda J Muntane M C Munoz I BujalanceP Montilla and I Tunez ldquoEstradiol and catecholestrogens pro-tect against adriamycininduced oxidative stress in erythrocytesof ovariectomized ratsrdquo Toxicology Letters vol 160 no 3 pp196ndash203 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

[17] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[18] I A Ogunwande N O Olawore O Ekundayo T M Walker JM Schmidt andWN Setzer ldquoStudies on the essential oils com-position antibacterial and cytotoxicity of Eugenia uniflora LrdquoInternational Journal of Aromatherapy vol 15 no 3 pp 147ndash1522005

[19] A C L Amorim C K F Lima A M C Hovell A L PMiranda and C M Rezende ldquoAntinociceptive and hypother-mic evaluation of the leaf essential oil and isolated terpenoidsfrom Eugenia uniflora L (Brazilian Pitanga)rdquo Phytomedicinevol 16 no 10 pp 923ndash928 2009

[20] D P Costa E G A Filho LM A Silva et al ldquoInfluence of fruitbiotypes on the chemical composition and antifungal activityof the essential oils of Eugenia uniflora leavesrdquo Journal of theBrazilian Chemical Society vol 21 no 5 pp 851ndash858 2010

[21] G F Ibikunle A C Adebajo F G Famuyiwa A J Aladesanmiand C O Adewunmi ldquoIn-vitro evaluation of anti-trichomonalactivities ofEugenia uniflora leafrdquoAfrican Journal of TraditionalComplementary and AlternativeMedicines vol 8 no 2 pp 170ndash176 2011

[22] K K Santos S R Tintino C E Souza et al ldquoAnti-Trypanosomacruzi and cytotoxic activities of Eugenia uniflora Lrdquo Experimen-tal Parasitology vol 131 no 1 pp 130ndash132 2012

[23] NIST 05 Mass Spectral Library (NISTEPANIH) NationalInstitute of Standards and Technology Gaithersburg Md USA2005

[24] R P Adams Identification of Essential Oil Components By GasChromatographyMass Spectrometry Allured Carol Stream IllUSA 4th edition 2007

[25] S M P Carneiro F A A Carvalho L C L R Santana A PL Sousa andM H Chaves ldquoThe cytotoxic and antileishmanialactivity of extracts and fractions of leaves and fruits of Azad-irachta indica (A Juss)rdquo Biological Research vol 45 no 2 pp111ndash116 2012

[26] M D G F deMedeiros A C da Silva A M D G L Cito et alldquoIn vitro antileishmanial activity and cytotoxicity of essential oilfrom Lippia sidoides Chamrdquo Parasitology International vol 60no 3 pp 237ndash241 2011

[27] S E Lofgren L C Miletti M Steindel E Bachere and M ABarracco ldquoTrypanocidal and leishmanicidal activities of differ-ent antimicrobial peptides (AMPs) isolated from aquatic ani-malsrdquo Experimental Parasitology vol 118 no 2 pp 197ndash2022008

[28] F C C Grando C A Felıcio A Twardowschy et al ldquoModula-tion of peritoneal macrophage activity by the saturation state ofthe fatty acid moiety of phosphatidylcholinerdquo Brazilian Journalof Medical and Biological Research vol 42 no 7 pp 599ndash6052009

[29] MM Iwu J E Jackson and BG Schuster ldquoMedicinal plants inthe fight against leishmaniasisrdquo Parasitology Today vol 10 no2 pp 65ndash68 1994

[30] L G Rocha J R G S Almeida R O Macedo and J M Bar-bosa-Filho ldquoA review of natural products with antileishmanialactivityrdquo Phytomedicine vol 12 no 6-7 pp 514ndash535 2005

[31] SMDeMorais A A CraveiroM I LMachado JW Alencarand F J A Matos ldquoVolatile constituents of Eugenia uniflora leafoil from northeastern Brazilrdquo Journal of Essential Oil Researchvol 8 no 4 pp 449ndash451 1996

[32] J G S Maia E H A Andrade M H L Da Silva and M G BZoghbi ldquoA new chemotype of Eugenia uniflora L from NorthBrazilrdquo Journal of Essential Oil Research vol 11 no 6 pp 727ndash729 1999

[33] J H G Lago E D Souza B Mariane et al ldquoChemical and bio-logical evaluation of essential oils from two species of myrtace-aemdashEugenia uniflora L and Plinia trunciflora (O Berg) KauselrdquoMolecules vol 16 no 12 pp 9826ndash9837 2011

[34] R Chang S A L de Morais D R Napolitano K C DuarteV B Guzman and E A do Nascimento ldquoA new approach forquantifying furanodiene and curzerene A case study on theessential oils of Eugenia uniflora (pitangueira) leavesrdquo BrazilianJournal of Pharmacognosy vol 21 no 3 pp 392ndash396 2011

[35] A M Adio ldquoGermacrenes A-E and related compounds ther-mal photochemical and acid induced transannular cycliza-tionsrdquo Tetrahedron vol 65 no 8 pp 1533ndash1552 2009

[36] D P Costa S C Santos J C Seraphin and PH Ferri ldquoSeasonalvariability of essential oils of Eugenia uniflora leavesrdquo Journal ofthe BrazilianChemical Society vol 20 no 7 pp 1287ndash1293 2009

[37] A T HenriquesM E Sobral A D Caudaro E E S SchapovalV L Bassani and G Lamaty ldquoAromatic plants from BrazilII The chemical composition of some Eugenia essential oilsrdquoJournal of Essential Oil Research vol 5 no 5 pp 501ndash505 1993

[38] B Teixeira A Marques C Ramos et al ldquoEuropean pennyroyal(Mentha pulegium) from portugal chemical composition ofessential oil and antioxidant and antimicrobial properties ofextracts and essential oilrdquo Industrial Crops and Products vol 36no 1 pp 81ndash87 2012

[39] D C Arruda F L DrsquoAlexandri A M Katzin and S RB Uliana ldquoAntileishmanial activity of the terpene nerolidolrdquoAntimicrobial Agents andChemotherapy vol 49 no 5 pp 1679ndash1687 2005

[40] RDi Pasqua G Betts NHoskinsM Edwards D Ercolini andG Mauriello ldquoMembrane toxicity of antimicrobial compoundsfrom essential oilsrdquo Journal of Agricultural and Food Chemistryvol 55 no 12 pp 4863ndash4870 2007

[41] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[42] J S Armstrong ldquoMitochondrial membrane permeabilizationthe sine qua non for cell deathrdquo BioEssays vol 28 no 3 pp253ndash260 2006

[43] A L Silva C M Adade F M Shoyama et al ldquoIn vitro leish-manicidal activity of N-dodecyl-1 2-ethanediaminerdquo Biomedi-cine and Pharmacotherapy vol 66 no 3 pp 180ndash186 2012

[44] B Purkait A Kumar N Nandi et al ldquoMechanism of ampho-tericin B resistance in clinical isolates of Leishmania donovanirdquoAntimicrobial Agents and Chemotherapy vol 56 no 2 pp 1031ndash1141 2012

[45] M Vermeersch R I Da Luz K Tote J P Timmermans P Cosand L Maes ldquoIn vitro susceptibilities of Leishmania donovanipromastigote and amastigote stages to antileishmanial referencedrugs practical relevance of stage-specific differencesrdquo Antimi-crobial Agents and Chemotherapy vol 53 no 9 pp 3855ndash38592009

[46] C Di Giorgio F Delmas V Akhmedjanova et al ldquoIn vitroantileishmanial activity of diphyllin isolated fromHaplophyllumbucharicumrdquo Planta Medica vol 71 no 4 pp 366ndash369 2005

10 Evidence-Based Complementary and Alternative Medicine

[47] H Martinez-Rojano J Mancilla-Ramirez L Quinonez-Diazand N Galindo-Sevilla ldquoActivity of hydroxyurea against Leish-mania mexicanardquo Antimicrobial Agents and Chemotherapy vol52 no 10 pp 3642ndash3647 2008

[48] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[49] A H Klimp E G E De Vries G L Scherphof and T DaemenldquoA potential role of macrophage activation in the treatment ofcancerrdquo Critical Reviews in OncologyHematology vol 44 no 2pp 143ndash161 2002

[50] M R Piemonte and D F Buchi ldquoAnalysis of IL-12 IFN-120574 and TNF-120572 production a5b1 integrins and actin filamentsdistribution in peritoneal mouse macrophages treated withhomeopathic medicamentrdquo Journal of Submicroscopic Cytologyand Pathology vol 33 no 1 pp 255ndash263 2002

[51] F Niedergang and P Chavrier ldquoSignaling and membranedynamics during phagocytosis Many roads lead to the pha-gos(R)omerdquo Current Opinion in Cell Biology vol 16 no 4 pp422ndash428 2004

[52] W L Lee R E Harrison and S Grinstein ldquoPhagocytosis byneutrophilsrdquo Microbes and Infection vol 5 no 14 pp 1299ndash1306 2003

[53] S J Green M S Meltzer J B Hibbs and C A Nacy ldquoActivatedmacrophages destroy intracellular Leishmania major amastig-otes by an L-arginine-dependent killing mechanismrdquo Journal ofImmunology vol 144 no 1 pp 278ndash283 1990

[54] E Osorio G J Arango N Jimenez et al ldquoAntiprotozoal andcytotoxic activities in vitro of Colombian Annonaceaerdquo Journalof Ethnopharmacology vol 111 no 3 pp 630ndash635 2007

[55] S Kamhawi ldquoPhlebotomine sand flies and Leishmania para-sites friends or foesrdquo Trends in Parasitology vol 22 no 9 pp439ndash445 2006

[56] P Tripathi V Singh and S Naik ldquoImmune response to leish-mania paradox rather than paradigmrdquo FEMS Immunology andMedical Microbiology vol 51 no 2 pp 229ndash242 2007

[57] L V Costa-Lotufo G M A Cunha P A M Farias et al ldquoThecytotoxic and embryotoxic effects of kaurenoic acid a diterpeneisolated fromCopaifera langsdorffii oleo-resinrdquoToxicon vol 40no 8 pp 1231ndash1234 2002

[58] R M Aparicio M J Garcıa-Celma M Pilar Vinardell and MMitjans ldquoIn vitro studies of the hemolytic activity ofmicroemul-sions in human erythrocytesrdquo Journal of Pharmaceutical andBiomedical Analysis vol 39 no 5 pp 1063ndash1067 2005

[59] L A Lexis R G Fassett and J S Coombes ldquo120572-tocopherol and120572-lipoic acid enhance the erythrocyte antioxidant defence incyclosporine A-treated ratsrdquo Basic and Clinical Pharmacologyand Toxicology vol 98 no 1 pp 68ndash73 2006

[60] J R Munoz-Castaneda J Muntane M C Munoz I BujalanceP Montilla and I Tunez ldquoEstradiol and catecholestrogens pro-tect against adriamycininduced oxidative stress in erythrocytesof ovariectomized ratsrdquo Toxicology Letters vol 160 no 3 pp196ndash203 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

[47] H Martinez-Rojano J Mancilla-Ramirez L Quinonez-Diazand N Galindo-Sevilla ldquoActivity of hydroxyurea against Leish-mania mexicanardquo Antimicrobial Agents and Chemotherapy vol52 no 10 pp 3642ndash3647 2008

[48] Y Tariku A Hymete A Hailu and J Rohloff ldquoEssential-oilcomposition antileishmanial and toxicity study of Artemisiaabyssinica and Satureja punctata ssp punctata from EthiopiardquoChemistry and Biodiversity vol 7 no 4 pp 1009ndash1018 2010

[49] A H Klimp E G E De Vries G L Scherphof and T DaemenldquoA potential role of macrophage activation in the treatment ofcancerrdquo Critical Reviews in OncologyHematology vol 44 no 2pp 143ndash161 2002

[50] M R Piemonte and D F Buchi ldquoAnalysis of IL-12 IFN-120574 and TNF-120572 production a5b1 integrins and actin filamentsdistribution in peritoneal mouse macrophages treated withhomeopathic medicamentrdquo Journal of Submicroscopic Cytologyand Pathology vol 33 no 1 pp 255ndash263 2002

[51] F Niedergang and P Chavrier ldquoSignaling and membranedynamics during phagocytosis Many roads lead to the pha-gos(R)omerdquo Current Opinion in Cell Biology vol 16 no 4 pp422ndash428 2004

[52] W L Lee R E Harrison and S Grinstein ldquoPhagocytosis byneutrophilsrdquo Microbes and Infection vol 5 no 14 pp 1299ndash1306 2003

[53] S J Green M S Meltzer J B Hibbs and C A Nacy ldquoActivatedmacrophages destroy intracellular Leishmania major amastig-otes by an L-arginine-dependent killing mechanismrdquo Journal ofImmunology vol 144 no 1 pp 278ndash283 1990

[54] E Osorio G J Arango N Jimenez et al ldquoAntiprotozoal andcytotoxic activities in vitro of Colombian Annonaceaerdquo Journalof Ethnopharmacology vol 111 no 3 pp 630ndash635 2007

[55] S Kamhawi ldquoPhlebotomine sand flies and Leishmania para-sites friends or foesrdquo Trends in Parasitology vol 22 no 9 pp439ndash445 2006

[56] P Tripathi V Singh and S Naik ldquoImmune response to leish-mania paradox rather than paradigmrdquo FEMS Immunology andMedical Microbiology vol 51 no 2 pp 229ndash242 2007

[57] L V Costa-Lotufo G M A Cunha P A M Farias et al ldquoThecytotoxic and embryotoxic effects of kaurenoic acid a diterpeneisolated fromCopaifera langsdorffii oleo-resinrdquoToxicon vol 40no 8 pp 1231ndash1234 2002

[58] R M Aparicio M J Garcıa-Celma M Pilar Vinardell and MMitjans ldquoIn vitro studies of the hemolytic activity ofmicroemul-sions in human erythrocytesrdquo Journal of Pharmaceutical andBiomedical Analysis vol 39 no 5 pp 1063ndash1067 2005

[59] L A Lexis R G Fassett and J S Coombes ldquo120572-tocopherol and120572-lipoic acid enhance the erythrocyte antioxidant defence incyclosporine A-treated ratsrdquo Basic and Clinical Pharmacologyand Toxicology vol 98 no 1 pp 68ndash73 2006

[60] J R Munoz-Castaneda J Muntane M C Munoz I BujalanceP Montilla and I Tunez ldquoEstradiol and catecholestrogens pro-tect against adriamycininduced oxidative stress in erythrocytesof ovariectomized ratsrdquo Toxicology Letters vol 160 no 3 pp196ndash203 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom