Chemical Composition and Amoebicidal Activity of Piper Hispidinervum (Piperaceae) Essential
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Industrial Crops and Products 40 (2012) 292–295 Contents lists available at SciVerse ScienceDirect Industrial Crops and Products journa l homepage: www.elsevier.com/locate/indcrop 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.