Chemical composition and antibacterial activity of Piper lenticellosum C . D . C essential oil collected in Ecuador

1 Facultad de Ciencias Naturales, Universidad de Guayaquil, Guayaquil, Ecuador. Facultad de Farmacia y Bioanálisis. Universidad de Los Andes, Mérida, Venezuela. 2 Facultad de Farmacia y Bioanálisis. Universidad de Los Andes, Mérida, Venezuela. 3 Herbario GUAY, Universidad de Guayaquil, Facultad de Ciencias Naturales, Guayaquil, Ecuador. 4 Departamento de Química. Universidad Técnica Particular de Loja, Ecuador.


INTRODUCTION
The genus Piper L. (Piperaceae) comprises more than 700 species widely distributed in the tropical and subtropical regions of the world (Jaramillo and Manaos, 2001); and is known for its economic importance due at its aromatic and medicinal properties (Ravindran, 2000).Particularly, in Ecuador 275 Piper species have been reported, 75 of which are endemic to the country (Callejas, 1999;Quijano et al., 2006).Traditionally, Piper species have been used in Latin America as analgesics in pain management, toothache and wound treatment (Parmar et al., 1997).In Ecuador, some of this species have been used in folk medicine to treat numerous diseases as bronchitis, dysmenorrheal, anti-diarrheal, anti-parasitical, renal diseases and the leaves bath against several indispositions (Grandtner and Chevrette, 2013).The essential oils of Piper species have been recognized for showing insecticidal, bactericidal, larvicidal, molluscicidal, cytotoxic, anticholinesterasic and leishmanicidal activities (da Silva et al., 2014;Morales et al., 2013).The chemical composition of essential oil of some Piper species have been analyzed showing as main compounds phenylpropanoids such as safrole, dillapiol and myristicin and terpenes such as limonene, βcaryophyllene, spathulenol, (E)-nerolidol, -bicyclogermacrene and cadinol (Maia et al., 2009;Moura do Carmo et al., 2012).Piper lenticellosum C. DC., has been cited as a synonym of P. carpunya Ruiz & Pav. (Jorgensen & León, 1999), it is known in Ecuador as "guaviduca", and has been used in traditional medicine as anti-inflammatory, anti-ulcer, anti-diarrheal and anti-parasitical remedy as well as a treatment for skin irritations (Diaz and Dorado, 1986).
Despite the wide numbers of studies on chemical composition from Piper species, a single report on essential oil of Piper carpunya from Peruvian Amazon is available (Vargas et al., 2004).Anti-secretory, anti-inflammatory and anti-Helicobacter pylori activities such as antioxidant activities of the ethanolic extract of P. carpunya have also been studied.
These activities have been associated with the presence of flavonoids isolated from leaves of this species such as vitexin, isovitexin, rhamnopyranosylvitexin, isoembigenin also the phytosterols and phytol triterpenes (De las Heras et al., 1998;Quílez et al., 2010).In our continuing interest for the evaluation of biological proprieties and chemical composition of the medicinal plant that grows spontaneously in the Coast of Ecuador, the chemical composition and antibacterial activity of the essential oil of Piper lenticellosum C.D.C has been studied.

Plant Materials
The leaves and spikes of Piper lenticellosum C. DC. were collected in July 2015 at Matilde Esther locality, Guayas Province, Ecuador.Botanical identification was carried out by Ricardo Callejas (HUA), and a voucher specimen (code MER01) has been deposited at the GUAY Herbarium, Faculty of Natural Science, University of Guayaquil, Ecuador.

Essential oils Isolation
Fresh leaves and spikes (300 g) were subjected to hydrodistillation for 4 h, using a Clevenger-type apparatus.The oils (6.2 mL, 2.06 %) were dried over anhydrous sodium sulfate and stored in sealed vials at 4°C in the dark until analyzed and tested.

Gas chromatography (GC/FID)
The analyses of the chemical composition of the essential oil were performed on Agilent gas chromatograph (model 6890 N series) equipped with a flame ionization detector (FID) using a non-polar DB-5MS (5% phenyl-methylpolyxilosane) 30 m x 0.25 mm, thickness 0.25 μm capillary column (Agilent 122-5532).An automatic injector (series 7683) in split mode was used.The sample, 1 L of solution (1/100, v/v, essential oil/dicloromethane), was injected with a split ratio of 1:50.The initial oven temperature was held at 50 °C for 3 minutes, then it was heated to 210 °C with a ramp of 2.5 °C/min, and the temperature was maintained for 3 min until the end.The injector and detector temperatures were 210 °C and 250 °C, respectively.Helium was used as a carried gas at 0.9 mL/min in constant flow mode.The retention index was determined based on the retention times of the standard hydrocarbons TPH-6RPM of CHEM SERVICE C9-C24, which were injected after the oils under the same conditions.

Gas chromatography-mass spectrometry (GC-MS)
The GC-MS analyses were performed using an Agilent gas chromatograph coupled to a mass spectrometer detector (model Agilent series 5973 inlet).The spectrometer was operated at 70 eV, electron multiplier 1600 eV, scan rate: 2 scan/second and mass range: 40-350 m/z.This was provided with a data system MSD-Chemstation D.01.00 SP1.The GC equipped with a DB-5MS 5% phenyl-methylpolysiloxane capillary column (30 m x 0.25 mm x 0.25 μm).The ion source temperature was 250°C.The constituents of the essential oil were identified by comparison of their mass spectra with reference spectra in the computer library (Wiley) and also by comparing their retention indices, with those authentic compounds or data in the literature (Adams, 2007;Joulain and Konig, 1998).The quantitative data were obtained electronically from FID area percentage without the use of correction factor.

Antimicrobial method
The antimicrobial activity was carried out according to the disc diffusion assay described by Velasco et al., 2007.The strains were maintained in agar at room temperature.Each bacterial inoculum (2.5 mL) was incubated in Müeller-Hinton broth at 37ºC for 18 hours.The bacterial inoculum was diluted in sterile 0.85% saline to obtain turbidity visually comparable to a McFarland Nº 0.5 standard (10 6-8 CFU/mL).The minimal inhibitory concentration (MIC) was determined only with microorganisms that displayed inhibitory zones.MIC was determined by dilution of the essential oil in dimethyl sulphoxide (DMSO) pipetting 10 μL of each dilution onto a filter paper disc.Dilutions of the oil within a concentration range of 20-980 μL/mL were also carried out.MIC was defined as the lowest concentration that inhibited visible bacterial growth (CLSI, 2016).A negative control was also included in the test using a filter paper disc saturated with DMSO (10L) to check possible activity of this solvent against the assayed bacteria.The experiments were repeated at least twice.

RESULTS AND DISCUSSION
The essential oil was analyzed by Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS).All components (28, representing 98.78 % of the total oil) were characterized by comparison of each MS with the Wiley GC/MS library data also from its retention index (RI).A list of the identified components, along with their percentage of the total oil, is given in Table 1.
Additionally, this clear difference between the two essentials oils might be due to climatic conditions.It is well documented that essential oils yield vary considerably depending on the time of the year of plant collection and is also influenced by environmental conditions at the time of harvesting (Juliani et al., 2002).However, this species are widely used in traditional medicine in Ecuador, Colombia and Perú (Grandtner and Chevrette, 2013).
Antibacterial activity of Piper lenticellosum essential oil was screened against international reference bacterial strains and results obtained are summarized in Table 2.The essential oil showed a significantly inhibition of the growth of the bacterial strains producing a zone of inhibition ranging from 7 to 17 mm with MIC values ranging from 100 to 900 L/mL.These results showed that the essential oil was most active against S. aureus, E. coli and K. pneumoniae with MIC values ranging 100, 200 and 300 L/mL, respectively.The antibacterial activity of the essential oil of P. lenticellosum could be due to the presence of piperitone, the major component of the oil (33.97%).The antibacterial properties of the piperitone have been reported (Mahboubi and Haghi, 2008) thus as the capacity as nitrofurantoin resistance modulating agent (Shahverdi et al., 2015).Additionally, other Piper species have showed antibacterial activity against different Gram positive and Gram negative bacteria related at presence to monoterpens as -pinene and -pinene (Morales et al., 2013); alkylbenzenes as safrole (Guerrini et al., 2009) that are also present in the essential oil of P. lenticellosum.To the best of our knowledge, this is the first report on the antibacterial activity of the essential oils P. lenticellosum from Ecuador.With these results, we hope to contribute to the study of species of the genus Piper from Ecuador, of which many are used since ancient times by the Ecuadorian population.

CONCLUSION
In the present research, several differences were observed in the composition of P. lenticellosum essential oil comparing to Piper carpunya (Vargas et al., 2004), this might attributed to geographical environment, seasonality, physiological age of the plant, harvesting time, among other conditions.The essential oil showed a broad spectrum against both Gram positive and Gram negative bacteria attributed to piperitone, mainly.These results may explain the use of these species in the traditional medicine as anti-diarrheal, bronchitis and skin conditions.It must also consider that these apparent differences in the chemical composition may be due to P. lenticellosum should be treated as a related but separate species and not as a synonym for P. carpunya.Phylogenetic and molecular studies are recommended for both species.

Table 2 :
Antibacterial activity of the essential oil of Piper lenticellosum C.D.C.