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Editor-in-Chief: John M. PezzutoPharm Biol, Early Online: 1–9

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.914231

ORIGINAL ARTICLE

Essential oil of Psidium cattleianum leaves: Antioxidant andantifungal activity

Micheli R. Castro1, Francine N. Victoria1,2, Daniela H. Oliveira1, Raquel G. Jacob1, Lucielli Savegnago2, andDiego Alves1

1Laboratorio de Sıntese Organica Limpa, CCQFA, Universidade Federal de Pelotas, Pelotas-RS, Brazil and 2Centro de Desenvolvimento Tecnologico,

Unidade Biotecnologia, Universidade Federal de Pelotas, Grupo de Pesquisa em Neurobiotecnologia, Pelotas-RS, Brazil

Abstract

Context: Psidium cattleianum Sabine (Myrtacea) is rich in vitamin C and phenolic compounds,including epicatechin and gallic acid as the main components.Objective: To evaluate the antifungal and antioxidant capacity in vitro of the essential oil ofaraca (EOA). The acute toxicity of the EOA also was evaluated in mice.Materials and methods: The leaves of the P. cattleianum were extracted by steam distillation.The antioxidant capacity was evaluated by in vitro tests [1,1-diphenyl-2-picryl-hydrazyl (DPPH),2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), ferric ion reducing antioxidant power(FRAP), linoleic acid oxidation, thiobarbituric acid reactive species (TBARS)], and ex vivo analysis[TBARS, d-aminulevunilate dehydratase (d-Ala-D) and catalase activity, non-protein thiols(NPSH), and ascorbic acid levels]. The toxicity was studied in mice by a single oraladministration of EOA; and the antifungal activity was performed with five strains of fungi.Results: The EOA exhibited antioxidant activity in the FRAP assay and reduced lipidperoxidation in the cortex (Imax¼ 32.90 ± 2.62%), hippocampus (IC50¼ 48.00 ± 3.00 mg/ml andImax¼ 32.90 ± 2.62%), and cerebellum (Imax¼ 45.40 ± 14.04%) of mice. Acute administration ofthe EOA by the oral route did not cause toxicological effects in mice (LD504500mg/ml). TheEOA also showed antifungal activity through of the determination minimum inhibitoryconcentration (MIC) values ranging from 41.67 ± 18.04 to 166.70 ± 72.17 mg/ml for tested strains.Conclusion: The results of present study indicate that EOA possess antioxidant properties,antifungal and not cause toxicity at tested doses.

Keywords

Araca, oxidative stress, toxicology

History

Received 20 January 2014Revised 28 March 2014Accepted 8 April 2014Published online 24 November 2014

Introduction

Psidium cattleianum Sabine (Myrtacea) is known as straw-

berry guava or araca. The fruits have white pulp and a tart

taste, and are rich in vitamin C (Lapcik et al., 2005; Luximon-

Ramma et al., 2003; Pino et al., 2001) and contain a large

amount of phenolic compounds including epicatechin and

gallic acid as the main components (Medina et al., 2011).

Natural antioxidants are in high demand for bio-

pharmaceuticals application in several fields of study.

In fact, intensive research has been performed for the

extraction, characterization, and utilization of natural prod-

ucts/antioxidants, like essential oils (EOs) and plant extracts,

which may serve as potent candidates in combating the

oxidative-related pathologies (Ozen et al., 2011).

In this sense, EOs are aromatic oily liquids obtained from

plant material, like flowers, buds, seeds, leaves, twigs, bark,

herbs, wood, fruits, and roots (Bakkali et al., 2008). The

percentage of the chemical components of EOs varies among

species and plants parts. These components are chemically

derived from terpenes and their oxygenated derivatives

(Solorzano-Santos & Miranda-Novales, 2012). EO possesses

many pharmacological effects described, such as antioxidant

(Victoria et al., 2012; Wang et al., 2013), antimicrobial (Singh

et al., 2013; Stefanakis et al., 2013), and antinociceptive

(Amorim et al., 2009).

In the context of natural products biological potential, the

antioxidant potential of native fruits from the southern of Rio

Grande do Sul, Brazil, such as ‘‘araca’’ (Psidium cattleianum)

have been studied (Alvarenga et al., 2013; Pereira et al.,

2013).

Araca, with yellow or red berries, has a nice balance

between soluble solids and acidity, and ripens in Brazil in late

summer between February and May (Drehmer & Amarante,

2008). Preliminary exploratory studies suggested high anti-

oxidant activity and high phenolic content differing among

araca genotypes (Medina et al., 2011). The few investigations

of araca suggest nutritional and functional potential

Correspondence: Lucielli Savegnago, Centro de DesenvolvimentoTecnologico, Unidade Biotecnologia, Universidade Federal de Pelotas,Grupo de Pesquisa em Neurobiotecnologia, Pelotas-RS, Brazil; DiegoAlves, Laboratorio de Sıntese Organica Limpa, CCQFA, UniversidadeFederal de Pelotas, CEP 96010-900, Pelotas-RS, Brazil. E-mail:diego.alves@ufpel.edu.br

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(Galho et al., 2007). Although traditionally appreciated for its

sensory attributes and expected functional properties, araca is

still poorly characterized, and limited scientific information is

available about the fruit.

Few studies were performed with the leaves of araca.

Alvarenga et al. (2013) evaluated the in vivo analgesic and

toxicity of a hydroalcoholic extract from P. cattleianum, the

results demonstrated that extract demonstrated strong anal-

gesic activity and no toxicity in cultures of LLC-MK2

mammalian fibroblast cells. Aqueous extract of P. cattleia-

num leaves reduced enamel demineralization, acidogenic

potential, microorganism viability, and extracellular polysac-

charide production (Brighenti et al., 2012), and a chloroform

leaf extract activated specific intracellular death-related

pathways, leading to caspase-3 activation and induction of

apoptosis in SNU-16 cells (Moona et al., 2012).

Taken together the potential of EOs and araca, and the

experience of our research group in the search for new

biologically active compounds, the aim of this study was to

evaluate the chemical composition of EO from the leaves of

araca (EOA), the antioxidant effects and analyze whether it

caused acute toxicity when orally administered to mice.

Additionally, studies were performed to evaluate the anti-

fungal activity of EOA against fungal strains.

Materials and methods

Animals

Adult male Swiss mice (25–35 g) were used. The mice were

kept in separate animal rooms on a 12 h light/dark cycle at a

temperature of 22 ± 2 �C and with free access to food and

water. The mice were treated according to the guidelines of

the Committee on Care and Use of Experimental Animal

Resources of the Federal University of Pelotas, Pelotas, Brazil

(CEEA 2762).

Plant material and EO material

The leaves of the P. cattleianum red plant were collected from

a research orchard (germplasm collection of Embrapa Clima

Temperado, Pelotas, RS, Brazil) in February and March 2012

in the morning (voucher specimen PEL no. 26.320). EOA was

extracted by steam distillation. The fresh leaves (600 g) were

cut with scissors into small pieces and put in a 2000 ml

reaction flask. After 3 h of extraction, the organic phase was

separated and dried over Na2SO4. The distillations were

performed in triplicate to determine the yield. The chemical

composition of the crude EO was evaluated using gas

chromatography coupled with mass spectrometry (GC–MS).

Analysis of the EOA

The identification and determination of the major chemical

constituent ratio of EOA were performed by GC–MS. The oil

was dissolved in hexane, and the injected sample volume was

1.0ml. A Shimadzu GC–MS QP2010 (Tokyo, Japan) and a

polyethylene glycol (Carbowax), model Rtx-Wax (RESTEC)

(30 m� 0.25 mm i.d., film thickness 0.25mm) capillary

column were used for the analysis. The temperature was

first held at 40 �C, and then raised to 250 �C (10 min, 20� C/

min). The carrier gas was helium at a flow rate of 3 ml/min.

The components of the oil were identified based on the

comparison of their retention indices and mass spectra with

the fragmentation patterns from computer matching with the

NIST/EPA/NIH/2005 library.

Determination of in vitro antioxidant activity of EOA

The EOA was diluted in dimethyl sulfoxide (DMSO) for the

in vitro studies. The DPPH radical is a stable free radical

commonly used as a substrate to evaluate in vitro antioxidant

activity. Radical-scavenging activity was determined by the

reaction of the stable DPPH radical with the compounds, in

accordance with the method with some modifications

(Choi et al., 2002). Different concentrations of EOA (10–

500 mg/ml) were mixed with a methanol solution containing

the DPPH radical. The mixture was incubated for 30 min at

30 �C, and the absorbance was measured at 517 nm. The

values are expressed as the percentage of inhibition of DPPH

absorbance (% inhibition) in relation to the control values

without the EO.

ABTS radical scavenging activity was determined accord-

ing to the method described by Re et al. (1999) with some

modifications. ABTSS+ was produced by react the

ABTS stock solution with 2.45 mM potassium persulfate

(final concentration) and allowing the mixture to stand in the

dark at room temperature for 12–16 h before use. For the

study of samples, the ABTSS+ stock solution was diluted

with phosphate-buffered saline (5 mM, pH 7.4) to an absorb-

ance of 0.70 at 0.73 nm. Different concentrations of EOA

(10–50 mg/ml) were mixed with ABTSS+ solution, and

the mixture was left to stand for 30 min at 30 �C; the

absorbance was measured at 734 nm. The values are

expressed as the percentage of inhibition of ABTSS+

absorbance (% inhibition) in relation to the control values

without compound.

The ferric ion (Fe3+) reducing antioxidant power (FRAP)

method was used to measure the reducing capacity of EOA.

The FRAP assay was carried out as described by Stratil et al.

(2006). The FRAP reagent was prepared by mixing 38 mM

anhydrous sodium acetate in distilled water (pH 3.8), 20 mM

FeCl3�6H2O in distilled water, and 10 mM 2,4,6-

tri(2-pyridyl)-S-triazine (TPTZ) in 40 mM HCl in proportions

of 10:1:1. This reagent was freshly prepared before each

experiment. Different concentrations of EOA and FRAP

reagent were added to each sample, and the mixture was

incubated at 37 �C for 40 min in the dark. The absorbance of

the resulting solution was measured at 593 nm by a

spectrophotometer.

The capacity of EOA to reduce lipid peroxidation was

evaluated by three methods: linoleic acid oxidation, lipid

peroxidation induced by sodium nitroprusside (SNP) in

regions of mouse brain (cortex, hippocampus, and cerebel-

lum), and basal lipid peroxidation in liver, kidney, and brain

of mice.

The linoleic acid peroxidation assay was based on the

reaction mixture contained linoleic acid (48.8 mM), Tris-HCl

(100 mM, pH 7.5), SNP (100 mM), and a varying concentra-

tions of EOA (10–500 mg/ml). The reaction was initiated

by the addition of SNP, incubated for 30 min at 37 �C,

and terminated by the addition of trichloroacetic acid

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(TCA, 5.5%). An aliquot of 250 mL of thiobarbituric acid

(TBA, 1%) in 50 mM NaOH was added to 250 of the reaction

mixture, followed by heating (90 �C) for 10 min, after this

time, the organic phase was extracted with 500 mL of butanol

(PA) and centrifuged at 3500g for 10 min and the absorbance

of thiobarbituric acid-reacting substances (TBARS) in the

supernatant was read at 532 nm and converted into the

percentage oxidation (Choi et al., 2002).

To evaluate the effect of EOA in lipid peroxidation in

tissue of mice, the animals were euthanized, and the hepatic,

renal, and cerebral tissues were rapidly removed and placed

on ice and the brain regions were dissected. All tissues were

kept chilled and homogenized in 50 mM Tris-HCl at

pH 7.4 (liver and kidney (1/10) and brain and brain regions

(1/5), weight/volume [w/v]). The homogenate was centrifuged

for 10 min at 2400 rpm to yield a pellet that was discarded and

a low-speed supernatant (S1) for each tissue, which was

used for SNP-induced lipid peroxidation and basal lipid

peroxidation.

Lipid peroxidation in the cortex, hippocampus, and

cerebellum was performed by the formation of TBARS

during an acid-heating reaction as previously described by

Ohkawa et al. (1979). A 20 ml aliquot of the cortex and

cerebellum (S1) and a 10 ml aliquot of the hippocampus S1

were pre-incubated in the presence of EOA (1–500 mg/ml)

with and without SNP (100 mM) for 1 h at 37 �C. The mixture

was then incubated with sodium dodecyl sulfate (SDS, 8.1%),

TBA (0.8%), and acetic acid/HCl (pH 3.4) at 95 �C for 2 h.

Malondialdehyde (MDA) was used as a biomarker of fatty-

acid peroxidation. The absorbance of the sample was

measured at 532 nm, and the results are expressed as

percentage of lipid peroxidation.

To extend the studies about the antioxidant potential of the

EOA, its effect on the in vitro basal lipid peroxidation on

liver, kidney, and brain was evaluated according to the

methodology of Ohkawa et al. (1979). An aliquot of 200 ml of

S1 was pre-incubated for 1 h at 37 �C with different concen-

trations of EOA (10–500 mg/ml) or vehicle (DMSO). The

mixture was then incubated with SDS (8.1%), TBA (0.8%),

and acetic acid/HCl (pH 3.4) at 95 �C for 2 h. MDA was used

as a biomarker of lipid peroxidation and the results are

expressed as % of lipid peroxidation.

In vivo experiments

Acute toxicity

To investigate the potential acute toxicity caused by the EOA,

the mice received a single oral dose of the EOA (100, 200,

and 500 mg/kg) or a vehicle (10 ml/kg of canola oil). After

administration, the animals were observed for up to 72 h

(at the interval of 24 h) to determine the lethal dose (LD50).

Body weight gain was recorded as a sign of general

toxicity. After 72 h of exposure, the mice were euthanized

by cervical displacement, and the brain, liver, and kidney

were removed, homogenized (liver and kidney 1/10 v/v and

brain 1/5 v/v), and centrifuged. The S1 was separated and used

for analysis of TBARS, ascorbic acid, and non-protein thiols

(NPSH) levels as well as the (d-aminulevunilate dehydratase)

(d-Ala-D) and catalase activities (see section Analysis of

the EOA).

Ex vivo analysis

Among the lipid peroxidation products used for antioxidant

assays, MDA has been most widely used to evaluate the

antioxidant activity in lipid peroxidation systems. In this

work, MDA formation was used as a marker of lipid

peroxidation according to the method reported by Ohkawa

et al. (1979).

The d-Ala-D was assayed according to the method reported

by Sassa (1982). d-Ala-D is a sulfhydryl-containing enzyme

and numerous metals and other compounds that oxidized

sulfhydryl groups modified its activity.

Catalase (CAT) activity, an enzymatic antioxidant defense,

was assayed spectrophotometrically by the method of Aebi

(1984), which involves monitoring the disappearance of H2O2

in the homogenate at 240 nm. The enzymatic activity was

expressed in units of U CAT/mg protein. Vitamin C, a non-

enzymatic antioxidant defense, determination was performed

as described by Jacques-Silva et al. (2001) with some

modifications. NPSH levels were analyzed by the method of

Ellman (1959).

Protein determination

The protein content of the S1 was measured according to the

method reported by Lowry et al. (1951) using bovine serum

albumin (BSA) as a standard.

Antifungal activity

The antifungal activity of EOA was tested with five

fungal strains: Candida albicans, Candida parapsilosis,

Candida guilhermondii, Candida lipolytica, and

Trichosporon asahii that were obtained from the

Department of Microbiology at the Federal University of

Pelotas. Fungal strains were maintained on potato dextrose

agar (PDA) and subcultured (1% inoculum) in potato dextrose

broth at 35 �C for at least 2–4 d before being used in the

screening assays.

The minimum inhibitory concentration (MIC) of EOA was

determined according to the method of NCCLS (2002).

EOA was tested in concentrations ranging from 500 to

0.85 mg/ml, and the assays were repeated at least three times

in their entirety to confirm the results. The MIC was recorded

as the lowest concentration of EOA that inhibited the fungal

growth.

Statistical analysis

Experimental results were given as the mean ± standard

error (SE) to show variations among groups.

Statistical analyses were performed using one-way ANOVA

followed by the Newman–Keuls multiple comparison test

when appropriate. All in vitro tests were performed at least

three times in duplicate. For the in vivo assays, five to eight

animals were used per group. The IC50 values (concentration

of sample required to inhibit 50% of the effect) were

calculated from the graph of the inhibition percentage

versus the EOA concentration. Differences were considered

statistically significant at a probability of less than 5%

(p50.05).

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Results

Chemical composition of EOA

This is the first time that the chemical composition of

EOA of Rio Grande do Sul, Brazil, was investigated,

according to our knowledge. The GC/MS analysis permitted

the identification of 97.5% of the compounds presents in

EOA, with a prevalence of oxygenated sesquiterpenes

(88.13%) followed by non-oxygenated sesquiterpenes

(9.09%) (Figure 1). The major compound found was

isocaryophillene (59.62%).

In vitro assay

As shown in Figure 2(a), EOA exhibited ferric-reducing

ability at concentrations equal and higher than 100 mg/ml.

EOA did not demonstrated effect in the DPPH (electron or

hydrogen-based transfer antioxidant test) and in the ABTS test

(ET-based antioxidant test) (data not shown).

As shown in Figure 2(b), EOA effectively inhibits

SNP-linoleic acid peroxidation at a concentration equal and

higher than 50 mM. EOA presented a value of IC50 of

56.41 ± 5.41 mg/ml and a maximum inhibition of 61.59%.

Figure 3(a) showed that EOA decreased lipid peroxidation

per se at concentrations ranging from of 100 to 500 mg/ml in

the liver, with an IC50 value of 48.00 ± 3.00 mg/ml and Imax of

65.10 ± 11.71%. In the kidney (Figure 3b), EOA decreased

lipid peroxidation at concentrations of 100 and 500 mg/ml

with an Imax value of 32.90 ± 2.62%. In addition, EOA

declined lipid peroxidation in the brain and presented an Imax

of 45.40 ± 14.40%.

Figure 4 shows the effect of EOA on SNP-induced lipid

peroxidation on brain regions. Data demonstrated that SNP

significantly induced lipid peroxidation on the brain struc-

tures. EOA were effective on reducing the SNP-induced lipid

peroxidation at concentrations equal or greater than 10 mg/ml

in hippocampus, cortex and cerebellum. In the cortex, EOA

presented an IC50 value of 8.33 ± 4.04 mg/ml and a maximal

inhibition (Imax) of 80.90 ± 4.00% (4b), in the hippocampus

EOA showed an Imax of 45.87 ± 10.20% (4 a) and in the

cerebellum 28.00 ± 4.40% (Figure 4c).

In vivo assay

Acute toxicity

The oral administration of EOA at doses of 100, 200, and

500 mg/kg did not cause death of any animal. The LD50 value

obtained for the EOA possibly is greater than 500 mg/kg

given that lethality was not observed at this dose.

O

OH HO H

H

HO

α-caryophyllene(6.42%)

naphtalene(1.71%)

azulene(0.96%)

caryophylleneoxide(18.16%)

cadinol(4.63%)

naphtalenol(3.65%)

epiglobulol(2.07%)

isocaryophyllene(59.62%)

A B0

20

40

60

80

100

% o

f com

poun

ds

Figure 1. Chemical composition of the EOA and % of sesquiterpenes oxygenated (A) and not oxygenated sesquiterpenes (B).

4 M. R. Castro et al. Pharm Biol, Early Online: 1–9

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The administration of EOA at these doses did not cause

significant reduction in the body weight when compared with

the control group (data not shown).

Ex vivo analysis

The levels of TBARS after oral exposure of mice to EOA

were significantly reduced in the liver, kidney at doses 200

and 500 mg/kg, and in the brain at dose of 500 mg/kg

(Figure 5). These data suggest that the EOA does not cause

any oxidative stress in mice tissue after acute treatment,

although it reduces the levels of TBARS in mice liver, kidney,

and brain.

d-Ala-D is a sulfhydryl-containing enzyme that is inhibited

by a variety of sulfhydryl reagents (Folmer et al., 2005).

In this study, the EOA did not alter the enzyme activity in

liver, kidney and brain of mice (data not shown).

C 10 50 100 5000

200

400

600

[μg/ml]

(c)

*

[MD

A]/g

of t

issu

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Imax = 32.90 ± 2.62 %

C 10 50 100 5000

200

400

600

[μg/ml]

**

(b)

[MD

A]/g

of t

issu

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IC50 = 48.00 ± 3.00 μg/mlImax = 65.10 ± 11.71 %

Imax = 45.4 ± 14.04 %

C 10 50 100 5000

100

200

300

400

500

600

[μg/ml]

(a)

**

[MD

A]/g

of t

issu

e

Figure 3. Effect of EOA on lipid peroxidation: (a) liver, (b) kidney, and (c) brain. The values are expressed in nmol MDA/g tissue. Data are presentedas mean ± SE (n¼ 5). Asterisks represent a significant effect as compared with the control group (C). *p50.05 by the Student–Newman–Keuls test forpost-hoc comparison.

C 10 50 100 5000.0

0.2

0.4

0.6

0.8

1.0

**

[μg/ml]

(a)

Abs

C 10 50 100 5000

20

40

60

80

100 I

*

**

[μg/ml]

(b) IC50=56.41 μg/ml% Imax = 61.59

% o

xida

tion

Figure 2. Antioxidant activities of the EOA: (a) ferric ion reducing antioxidant power (FRAP). Each value is expressed as the mean ± SE (n¼ 3).Asterisks represent significant *p50.05 when compared with the respective control without essential oil. The values are expressed in absorbance; themean value of absorbance (593 nm) of the control is 0.099 ± 0.004. (b) On the linoleic acid, SNP-induced lipid peroxidation assay. Data are presented asthe mean ± S.E (n¼ 3). The values are expressed as induced percent. The IC50 indicates the necessary concentration to inhibit 50% of lipidperoxidation. The Imax is the maximal inhibition offered by the EO. Asterisks represent significant *p50.05 compared with the induced for SNP-induced sample (100% lipid peroxidation). ANOVA followed by the Newman–Keuls multiple comparison test when appropriate.

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C 100 200 5000

500

1000

1500

2000

2500

* *

[mg/kg]

(a)

[MD

A]/

g o

f tis

sue

C 100 200 5000

500

1000

1500

2000

2500

**

[mg/kg]

(b)

[MD

A]/

g o

f tis

sue

C 100 200 5000

500

1000

1500

2000

2500

*

[mg/kg]

(c)

[MD

A]/

g o

f tis

sue

Figure 5. Effect of EOA on lipid peroxidation ex vivo: (a) liver, (b) kidney, and (c) brain. The values are expressed in nmol MDA/g tissue.Data are presented as mean ± SE (n¼ 5). Asterisks represent a significant effect as compared with the control group (C). *p50.05 by theStudent–Newman–Keuls test for post-hoc comparison.

C I 10 50 100 5000

500

1000

1500

2000

2500

* * *

(a)

#

[μg/ml]

[μg/ml]

[μg/ml]

[MD

A]/

g o

f tis

sue

C I 1 10 50 100 5000

500

1000

1500

2000

2500

**

**** **

(b)

#

[MD

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f tis

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C I 1 10 50 100 5000

500

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2500

* * **

(c)

#

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A]/

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f tis

sue

Imax = 45.87 ± % 02.01 IC50 = 8.33 ± 4.04 μg/mlImax = 80.90 ± 4.00 %

Imax = 28.00 ± 4.4 %

Figure 4. Effect of EOA on lipid peroxidation induced by SNP structures brain homogenates: (a) hipocamppus, (b) cortex, and (c) cerebellum.The values are expressed in nmol MDA/g tissue. Data are presented as mean ± SE (n¼ 5). Asterisks represent a significant effect when compared withinduced *p50.05, compared with the SNP-induced sample (100% lipid peroxidation) and # p50.01 compared with the control group (C) by theStudent–Newman–Keuls test for post-hoc comparison.

6 M. R. Castro et al. Pharm Biol, Early Online: 1–9

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CAT activity was not altered in the liver, kidney, or brain

of mice treated with EOA at any of the tested doses when

compared with the control group. Also, the administration of

EOA did not modified vitamin C levels and NPSH in the liver

and kidney of mice (Table 1).

Antifungal activity

In our study of antifungal activity, EOA presented MIC values

ranging from 41.67 ± 18.04 to 166.70 ± 72.17 mg/ml for the

tested strains (Table 2). To our knowledge, this is the first

report describing the antifungal activity of EOA.

Discussion

Due to the undesirable effects of synthetic compounds, there

has been growing interest in the investigation of natural

products for the discovery of active compounds with

antimicrobial and antioxidant properties that do not have

any negative effects on the human health; one of these natural

additives are the EOs.

In this study, EOA presented an antioxidant effect in the

FRAP, TBARS in vitro (SNP-induced linoleic oxidation,

basal lipid peroxidation in liver, kidney and brain,

SNP-induced brain regions oxidation), and TBARS ex vivo.

Also, EOA did not present any sign of toxicity and did not

alter important oxidative parameters, like d-Ala-D and

catalase activities and vitamin C and NPSH levels.

Furthermore, EOA inhibited significantly the growth of

different clinically important fungal strains.

The chemical compositional analysis of EOA presents the

predominance of non-oxygenated sesquiterpenes and the

major compound found was isocaryophillene (59.62%).

Chalannavar et al. (2013) studied the chemical composition

of the EO of P. cattleianum of South Africa by hydrodistilla-

tion, the analysis revealed caryophyllene oxide as the major

compound, and a predominance of non-oxygenated sesqui-

terpenes (36.78%), corroborating with our findings.

There was a great variability in the chemical composition

of EOs. Such variability depends on several factors including

local climatic and environmental conditions, soil variations,

season, geographical location, geology, the stage of the

vegetative cycle, part of the plant, and the method used to

obtain the EO (Viuda-Martos et al., 2008).

The terpenoid compounds present in the chemical com-

position of EOs are closely associated with their antioxidant

function, mainly due to their redox properties exerted by

various possible mechanisms: free radical-scavenging

activity, hydrogen donors, transition metal chelating activity,

and/or singlet oxygen quenching capacity (Liyana-Pathirana

& Shahidi, 2006). However, the mechanism set in motion by

the antioxidant activity of these compounds is still not clearly

understood. According to Amensour et al. (2009), the

antioxidant activity of EO is believed to be mainly due to

their redox properties, which play an important role in

adsorbing and neutralizing free radicals, quenching singlet

and triplet oxygen, or decomposing peroxides.

In agreement with our results, EOA presented an antioxi-

dant effect in the FRAP assay. The FRAP assay measures the

ability of antioxidant to reduce Fe(III)–triazine complex to

Fe(II)–complex (the respective lower valence state). Studies

have demonstrated that the reducing power of natural plant

extracts and EOs might be strongly correlated with their

antioxidant activities (Gulcin et al., 2012; Stratil et al., 2006;

Victoria et al., 2012).

Other important findings of our work are the effect of EOA

in the lipid peroxidation in the assays of SNP-induce linoleic

acid oxidation, in the SNP-induced lipid peroxidation in brain

structures, reduced the basal lipid peroxidation in the liver,

kidney, and brain, and when orally administered, reduced the

levels of MDA in liver, kidney, and brain of mice. Excessive

production of free radicals can generate a lipid peroxidation

chain reaction, which are responsible for many pathological

disorders (Castro et al., 2009).

According to Choi et al. (2002), the inhibition of linoleic

acid peroxidation by natural products is effective because of

the presence of various phytochemicals, such as phenolic

compounds, amino acids, ascorbic acid, tocopherols, and

pigments that might contribute to some antioxidant activity

singly or in combination.

Recently our research group demonstrated the effect

of another EO of the Myrtaceae family, the EO of

Eugenia uniflora L. leaves, in lipid peroxidation (Victoria

Table 1. Effect of acute treatment of EOA on catalase activity and ascorbic acid and NPSH levels.

Catalase (U CAT/mg protein) Ascorbic acid (mg AA/g tissue) NPSH (nmol NPSH/g tissue)

Dose (mg/kg) Liver Kidney Brain Liver Kidney Liver Kidney

0 (control) 0.87 ± 0.85 2.21 ± 0.41 0.25 ± 0.03 746.70 ± 2.20 734.30 ± 1.62 17 669.00 ± 1064.00 9122.00 ± 281.50100 0.88 ± 0.25 1.38 ± 0.47 0.24 ± 0.04 747.70 ± 2.40 733.10 ± 1.59 17 549.00 ± 694.50 9300.00 ± 174.00200 0.84 ± 0.30 1.91 ± 0.29 0.22 ± 0.03 747.30 ± 2.70 735.80 ± 1.89 18 191.00 ± 898.50 9004.00 ± 554.70500 0.70 ± 0.17 2.30 ± 0.60 0.23 ± 0.03 744.00 ± 2.20 736.10 ± 2.36 20 830.00 ± 900.50 10 251.00 ± 300.20

The values were analyzed by a one-way ANOVA, each value is expressed as the mean ± SE (n¼ 5).

Table 2. Evaluation of the antifungal activity of EOA against fungalstrains.

Fungal strainMinimum inhibitory concentration (mg/ml)

EOA Fluconazole

C. lipolytica 125.00 ± 00.00 62.50 ± 00.00C. parapsilosis 104.20 ± 36.08 52.10 ± 18.00C. guilhermondi 125.00 ± 00.00 62.50 ± 00.00C. albicans 166.70 ± 72.17* 62.40 ± 00.00T. asahi 41.67 ± 18.04 83.30 ± 36.10

The values were analyzed by a one-way ANOVA followed by theNewman–Keuls multiple comparison test, each value is expressed asthe mean ± SE. The tests were performed three times in duplicate.Asterisks represent significant effects (*p50.01) compared withfluconazole, for each strain.

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et al., 2013). Since the chemical composition of both EOs

(P. cattleianum and E. uniflora) revealed sesquiterpenes as

major compounds, we can infer that the presence of these

compounds can be related with the effect of EOs in lipid

peroxidation. Sesquiterpenes are formed from the assembly of

three isoprene units (C15). The extension of the chain

increases the number of cyclizations which allows a great

variety of structures (Bakkali et al., 2008).

In order to evaluate the possible toxic effect of EOA, mice

were orally treated with different doses of EOA. The results

demonstrated that the EOA did not present any signal of

toxicity. The tissues of animals were used to evaluate the

effect of EOA in some oxidative parameters, like TBARS,

CAT activity, and ascorbic acid levels. According to the

results, the levels of MDA were significantly reduced in liver,

kidney, and brain. MDA, one of the products of lipid

peroxidation, is malondialdehyde, which can be determined

by measuring the amount of TBA reactive species (Reznick &

Packer, 1994). According to many authors, lipid peroxidation

is involved in neurodegenerative disorders like Huntington

disease, amyotrophic lateral sclerosis, and Alzheimer disease.

The activity of catalase, an enzymatic defense, and the

levels of ascorbic acid and NPSH, a non-enzymatic defense,

are considered markers of oxidative stress, and a decrease in

its content might indicate an increase in oxidative stress.

In this work, the activity of catalase and the levels of ascorbic

acid were not changed by the administration of EOA, based

on this information it is possible suggest that EOA, orally

administered did not induced oxidative stress. Indeed, taken

together these data with results of lipid peroxidation, EOA

reduced the oxidative stress in mice at the tested doses.

Plants are rich in a wide variety of secondary metabolites,

such as phenols, terpenoids, sesquiterpenes, hydrocarbons,

flavonoids, tannins, acids, alcohols, aldehydes, and alkaloids,

which have been found to have antimicrobial properties in

in vitro studies. In addition, minor components play an

important part for antimicrobial activity, possibly by

producing a synergistic effect between other components.

Especially EOs, the aromatic oily liquid, are responsible for

antimicrobial activities of medicinal plants, which is effective

against microbial deterioration. Supercritical fluid extracts,

methanol, and ethanol extracts, and water extracts are also

utilized, but generally less effective (Skrovankova et al.,

2012).

EOA presents interesting effect in the inhibition of

important clinical fungal strains like T. asahii, C. para-

psilosis, C. albicans, C. lipolytica, and C. guilhermondi.

There are several studies that show that coriander, celery, and

sweet basil EO have an inhibitory effect on the growth of

yeasts (Matasyoh et al., 2008). Although the EOs showed

antimicrobial activity, the reason behind this capacity is not

well documented. This antimicrobial activity could be caused

by the major compounds of the EOs or due to a synergistic

effect between the major compounds and the minor ones

(Carovic-Stanko et al., 2010).

Possible mechanisms by which mycelial growth may be

reduced or totally inhibited have been proposed. Thus, Lucini

et al. (2006) indicated mycelial growth inhibition is caused by

the monoterpenes present in the EOs. These components

could increase the concentration of lipidic peroxides such as

hydroxyl, alkoxyl, and alkoperoxyl radicals, which leads to

cell death. According to Sharma and Tripathi (2006), the EOs

would act on the hypha of the mycelium, provoking exit of

components from the cytoplasm, the loss of rigidity, and

integrity of the hypha cell wall, resulting in its collapse and

death of the mycelium.

Conclusion

The EOA showed antioxidant activity and presented a LD50

higher than 500 mg/kg in mice. EOA demonstrated anti-

microbial activity against fungal strains. Thus, based on the

promising results shown in this work, other pharmacological

studies will be performed with this EO.

Declaration of interest

All authors declare that there are no conflicts of interest. L.

S., D. S. A., and R. G. J. are recipients of CNPq fellowships.

This work was supported by CNPq (Grants 472644/2010-6),

CAPES (23038.008258/2011-93), and FAPERGS (PRONEX

10/0027-4, PqG 1012043).

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