Industrial Crops and Products 40 (2012) 292–295

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Short communication

Chemical composition and amoebicidal activity of Piper hispidinervum

(Piperaceae) essential oil

a b b c

Ismael Pretto Sauter , Guilherme Evaldt Rossa , Aline Machado Lucas , Samuel Paulo Cibulski ,

c d a

Paulo Michel Roehe , Luiz Antônio Alves da Silva , Marilise Brittes Rott ,

b b,∗ e

Rubem Mário Figueiró Vargas , Eduardo Cassel , Gilsane Lino von Poser

a

Departamento de Microbiologia, Setor de Parasitologia, Instituto de Ciências Básicas da Saúde, UFRGS, Rua Sarmento Leite, 500, 90050-170 Porto Alegre, RS, Brazil

b

Faculdade de Engenharia, Departamento de Engenharia Química, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga, 6681 Prédio 30 – Sala 277, 90619-900 Porto

Alegre, RS, Brazil

c

Programa de Pós-Graduac¸ ão em Ciências Veterinárias, UFRGS, Av. Bento Gonc¸ alves, 9090, 91540-000 Porto Alegre, RS, Brazil

d

Pirisa Piretro–Rua Ernesto Alves, 2640, 95600-000 Taquara, RS, Brazil

e

Programa de Pós-Graduac¸ ão em Ciências Farmacêuticas, UFRGS, Av. Ipiranga, 2752, 90610-000 Porto Alegre, RS, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Acanthamoeba, a free-living protozoan widely distributed in the environment, can cause Acanthamoeba

Received 19 January 2012

keratitis, a significant ocular microbial infection. The illness can result in blindness when not prop-

Received in revised form 19 March 2012

erly treated in the initial stage. Plants of the genus Piper (Piperaceae) are used in folk medicine for

Accepted 22 March 2012

the antibacterial, antifungal and antiprotozoan properties. In this work, the chemical composition

and the amoebicidal activity of Piper hispidinervum essential oil were investigated. The leaves of the

Keywords:

fresh plant submitted to steam distillation yielded 0.95% (w/w) of essential oil that was analyzed

Acanthamoeba

by gas chromatography–mass spectrometry (GC/MS) being safrole the main component, representing

Essential oil

Keratitis 85.08% of the oil. For the assessment of the amoebicidal activity concentrations of 0.5, 0.250, 0.125 and

0.0625 mg/mL were tested. The essential oil, at the concentrations of 0.5 mg/mL, was lethal to 100% of

Piper hispidinervum

Safrole the A. polyphaga trophozoites. By the MTT assay it was verified that the essential oil was not cytotoxic to

the mammalian cells until the concentration of 0.25 mg/mL. Nevertheless, further studies are necessary

in order to verify its applicability in Acanthamoeba keratitis treatment.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction visual acuity and eventually blindness (Visvesvara and Schuster,

2008).

Acanthamoeba is an opportunistic protozoan widely distributed The treatment of Acanthamoeba keratitis includes biguanide

in the environment. This free live amoeba (AVL) has a life (polyhexamethylene biguanide or chlorhexidine digluconate)

cycle with two stages: a vegetative trophozoite and a resistant together with diamidine (propamidine isethionate or hexami-

cyst stage. Cyst form shows minimal metabolic activity and its dine). Reinfection can occur once the trophozoite can encyst under

double-walled spherical cellulose structure protects against hostile adverse conditions during the treatment (Khan, 2006). Thus, more

conditions such as antimicrobial agents and extreme tempera- effective drugs are necessary. Plants and their products can be use-

ture. Acanthamoeba is well recognized to produce serious human ful in the search for new agents and some of them have been shown

infections, including keratitis and granulomatous encephalitis amebicide effect (Topalkara et al., 2007; Ródio et al., 2008; Sauter

(Khan, 2006). Contact lenses exposed to contaminated cleaning et al., 2011).

solutions can promote the Acanthamoeba infection. The cornea Encompassing about 1000 species, the genus Piper is, together

epithelium with a trauma or hypoxia permit the parasite inva- with Peperomia, the largest and the most known of the family Piper-

sion into the stroma initiating a cytopathic effect (Clarke and aceae. The leaves of various Piper species were typically aromatic

Niederkorn, 2006; Kliescikova et al., 2011). If the infection is not or had a pungent smell affording essential oils with commercial

promptly treated, it may lead to ulceration of the cornea, loss of importance for the fragrance and pharmaceutical industries (Bizzo

et al., 2001, 2009).

Piper species are widely used in folk medicine for the antibacte-

rial, antifungal and antiprotozoan properties. These effects could be

∗ due to the presence of essential oils. In fact, the essential oils of some

Corresponding author. Tel.: +55 51 33534585; fax: +55 51 3320 3823.

E-mail address: [email protected] (E. Cassel). Piper species were investigated and antibacterial (Oyedeji et al.,

0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.03.025

I.P. Sauter et al. / Industrial Crops and Products 40 (2012) 292–295 293

2005), antifungal (Tirillini et al., 1996), antileishmanial (Monzote twice with phosphate-buffered saline buffer (PBS). The precipitate

et al., 2010) and anti-Trichomonas (Sariego et al., 2008) activities of amoebae was diluted in PYG medium to obtain a final concen-

4

were detected. tration of 2.0 × 10 trophozoites per milliliter.

Previous works performed with Piper hispidinervum essen-

tial oil report its antifungal activity against the phytopatogenic 2.5. Assessment of amoebicidal activity

fungi Bipolaris sorokiniana, Fusarium oxysporum and Colletotrichum

gloeosporioides (Zacaroni et al., 2009), the insecticidal activity The essential oil was solubilized with 1% Tween and sterile

against Tenebrio molitor larvae. The essential oil also presented water and was tested at final concentrations of 0.5, 0.250, 0.125 and

␮

insecticidal effect against T. molitor (Fazolin et al., 2007) and 0.0625 mg/mL. For the assessment of amoebicidal activity, 100 L

␮

Spodoptera frugiperda (Lima et al., 2009; Nascimento et al., 2008). of culture of A. polyphaga and 100 L of each test solution were

The aim of the present study was to analyze the essential oil inoculated into each well of a 96-well plate. The plate was incu-

◦

obtained from the aerial parts of P. hispidinervum grown in South bated at 30 C and the trophozoites counted in a Fuchs–Rosenthal

Brazil under controlled conditions and evaluate its in vitro amoe- counting chamber after 24 h. Viability was assessed using methy-

bicidal activity against Acanthamoeba polyphaga as well as the lene blue. The control used was sterile water containing 1% Tween

cytotoxic effect on mammalian cells. 20. The experiments were performed in triplicate and repeated in

three different days.

2. Materials and methods

2.6. Cytotoxicity assay

2.1. Plant material

The effect of P. hispidinervum essential oil on mammalian cell

The aerial parts of the plants were collected in spring season. viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-

Cultures were established in 2006 in the Agriculture Centre of the diphenyltetrazolium bromide (MTT) assay, as previously described

EMATER – Porto Alegre, state of Rio Grande do Sul (Southern Brazil; (Sauter et al., 2011). MTT cleavage is mediated by the mitochon-

◦  ◦ 

latitude 30 07 S; longitude 50 10 W; altitude 100 m), from seeds drial enzyme succinate dehydrogenase, and the amount of product

obtained from Pirisa Piretro Industrial Ltda – Brazil. The plants were is dependent of metabolically active cells. Vero cells (African Green

grown in rows at 80-cm spacing within the row and 1.2 m between Monkey Kidney, ATCC CCL-81) were treated with essential oil at dif-

the rows. Irrigation was applied regularly during the growing sea- ferent concentrations (0.5, 0.250, 0.125 and 0.0625 mg/mL). After

␮

son (greenhouse). The amount of water applied was equivalent of 24 h 50 L MTT reagent (Sigma Chemical Co., Saint Louis, MO, USA)

rainfall per year, 1350 mm. Biofertilizer (3.0%, w/w) was applied solution (2 mg/mL) were added to each well and incubated for a

×

four times every 6 months. In a period of 6 months the P. hispidin- further 4 h. The plates were centrifuged (1400 g for 5 min) and

␮

ervum plant reached a height of 2.5 m and produced 3.0 kg of green the untransformed MTT was removed. Then, ethanol (100 L) was

mass (aerial parts). The plants used in this study were four years added to each well and the optical density (OD) measured in an

old and pruning was carried out every 6 months. ELISA reader (Anthos 2020) at 550 nm with a 620 nm reference fil-

ter. Results were expressed as the percentage of the quotient OD of

2.2. Essential oil viable cells and OD of untreated control cells.

The essential oil was extracted from fresh leaves (ca. 0.4 kg) 2.7. Statistical analysis

without any pre-processing in a laboratory steam distillation appa-

ratus (Xavier et al., 2011). The average moisture content of the Results are expressed as percentage and analyzed by analysis of

plant was determined (Halogen Moisture Analyzer – HB43 – Met- variance and comparison of averages with the Tukey’s test. Statis-

tler Toledo) as well as the density of the oil. The experiments were tical significance was defined as p < 0.05.

performed in triplicate.

3. Results and discussion

2.3. GC and GC–MS analysis

The average density obtained for the P. hispidinervum essential

3

The oil was analyzed by gas chromatography (GC) and gas oil was 1.251 g/cm . The essential oil yield, based on fresh weight,

chromatography-mass spectrometry (GC/MS) using an Agilent was 0.953% (w/w). The standard deviation of the yield was less than

7890A gas chromatograph equipped with a mass spectrometer 0.06%.

Agilent 5975C. The experimental conditions were as previously The oil samples from triplicate extraction experiments were

described by Xavier et al. (2011). mixed in equal proportions and then analyzed. The analysis showed

The components of the oil were identified by comparison of 27 volatile compounds, representing 98.66% of the total oil. Its

their linear retention indexes (LRIs) on the column, determined chemical composition is presented in Table 1. Safrole was the major

in relation to a homologous series of n-alkanes, with those from component, representing 85.08% of the oil, followed by terpinolene

pure standards or reported in literature. Comparison of fragmenta- (5.40%).

tion patterns in the mass spectra with those stored on the GC–MS Safrole and other methylenedioxy derivatives such as apiol, dil-

databases (Adams, 2007) was also performed. lapiol and miristicin are frequently found in Piper essential oils

as the main component (Abreu et al., 2002; Ferraz et al., 2010).

2.4. A. polyphaga cultures Moreover, safrole has been previously determined as the main

component in the essential oil of P. hispidinervum: in the Amazon

The pathogenic strain of A. polyphaga (ATCC 30461) was region its concentration was greater than 90% (Fazolin et al., 2007);

obtained from the American Type Culture Collection. The axenic in plants cultivated in southern Brazil. Nascimento et al. (2008)

cultures were kept in PYG medium (2% proteose peptone, 0.2% obtained 82.5% of safrole; while on other study also carried out

◦

yeast extract, and 1.8% glucose) at a constant temperature of 30 C. with plants from Southern Brazil, the essential oil afforded safrole

The experiments were performed in accordance with Sauter et al. contents between 76.6% and 89.9% (Riva et al., 2011).

(2011). Briefly, 1 mL of the culture was centrifuged for 5 min at According to Bizzo et al. (2001) and Zacaroni et al. (2009), the

2000 rpm, the supernatant discarded, and the precipitate washed essential oil of P. hispidinervum presents economic interest because

294 I.P. Sauter et al. / Industrial Crops and Products 40 (2012) 292–295

Table 1

GC/MS of P. hispidinervum essential oil extracted by steam distillation.

t exp

Compound LRI LRI % Area

␣-Pinene 932 924 0.23

␤-Pinene 974 985 0.22

␣

-Phellandrene 1002 998 0.12

␦

-3-Carene 1008 1004 0.50

␣-Terpinene 1014 1011 0,15

␤-Phellandrene 1025 1022 0.24

(Z)-␤-Ocimene 1032 1034 0.64

(E)-␤-Ocimene 1044 1044 1.59

␥

-Terpinene 1054 1053 0.18

Terpinolene 1086 1083 5.40

Safrole 1285 1295 85.08

Fig. 1. Amoebicidal activity of P. hispidinervum essential oil presented as percentage

␣-Copaene 1374 1368 0.05

of mortality of A. polyphaga trophozoites (p < 0.05 vs control).

␤-Elemene 1389 1383 0.04

Methyl Eugenol 1403 1398 0.16

(E)-Caryophyllene 1417 1409 0.35

␣-Humulene 1452 1443 0.07

Allo-Aromandrene 1458 1447 0.09

Germacrene D 1484 1470 0.23

Bicyclogermacrene 1500 1486 1.43

Pentadecane 1500 1491 0.72

␥

-Cadinene 1522 1504 0.08

␦-Cadinene 1513 1512 0.12

Elemicin 1555 1548 0.23

Germacrene D-4-ol + Spathulenol 1547/1577 1563 0.55

Caryophyllene oxide 1582 1570 0.10

Guaiol 1600 1585 0.11

Total identified 98.66

Linear retention indexes (LRIs), relative to C9–C20 n-alkanes on DB-5MS capillary

column (Adams, 2007).

Fig. 2. Cellular viability of Vero cells front different concentrations of P. hispidin-

ervum essential oil (p < 0.05 vs control).

safrole is the raw material in the synthesis of piperonal, which

is used in the composition of perfumes, and piperonyl butoxide,

In conclusion, P. hispidinervum essential oil showed important

which is synergistic for pyrethrum-based insecticides.

amebicidal activity against trophozoites of A. polyphaga and was

Besides the industrial importance of the essential oils, plants

able to prevent their encystment. Besides that, the essential oil

of the genus Piper have been used for many centuries in the tra-

showed a little toxic effect against Vero cells. However, further

ditional medicine for different purposes and many activities have

studies are necessary in order to determine its therapeutic poten-

been revealed. For example, the essential oil from P. cernuum and

tial.

P. regnellii presented growth inhibitory activities against Staphy-

lococcus aureus and Candida albicans (Costantin et al., 2001); the

Acknowledgments

hydroalcoholic extract and neolignans from P. regnellii leaves pre-

sented strong activity against dermatophyte fungi (Koroishi et al.,

The authors would like to thank CAPES and CNPq for financial

2008); dihydrochalcones isolated from P. elongatum proved to be

support and Dr. Naveed Khan for providing A. polyphaga strain.

active against extracellular promastigotes of Leishmania braziliensis

in vitro (Hermoso et al., 2003); extracts from the fruits of P. longum,

used in traditional remedies against intestinal distress, exerted References

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