Phytochemical and Antibacterial Evaluation of Essential Oils from Ottonia Martiana Miq . ( Piperaceae )

Três óleos essenciais extraídos das folhas, frutos e raízes de Ottonia martiana Miq. (Piperaceae), espécie comum da floresta Atlântica brasileira, e conhecida popularmente por “anestésia”, foram analisados por CG-EM e submetidos a um ensaio antibacteriano bioautográfico frente a Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Pseudomonas aerogenes (ATCC 27853) e Escherichia coli (ATCC 25922). Setenta e sete compostos foram identificados e submetidos a uma análise comparativa, a qual revelou uma variabilidade no teor dos componentes majoritários desses óleos (espatulenol, óxido de cariofileno, (E)-nerolidol, viridiflorol, β-cariofileno, δ cadineno e aloaromadendreno). A presença de zonas de inibição de crescimento bacteriano nos bioautogramas analisados (Rfs 0,29 e 0,34) revelou o potencial antibacteriano dos óleos analisados frente às bactérias Gram-positivas testadas e permitiu identificar alguns dos componentes bioativos.


Introduction
Actually, research of healthy habits for world population has promoted a progressive increase on production and consumption of natural products, as the case of essential oils from plants, source of bioactive substances. 1 Chemical investigation of Piperaceae essential oils has revealed the presence of monoterpenes, sesquiterpenes and arylpropanoids that have shown interesting biological properties including psychotropic, antimicrobial, antioxidant and cytotoxic effects stimulating studies on plants within this family. 2mong Piperaceae species of medicinal interest, there is Ottonia martiana Miq, a characteristic shrub of the Brazilian Rain Forest 3,4 and known as "anestésia" for the natives of the Paraná coast due to its anesthetically action on the oral mucous. 5,6][9] No chemical data on essential oil of O. martiana has been found, but two papers discussing on essential oils constitution of other species from genus Ottonia have been previously published. 10,11his work relates a phytochemical investigation on essential oils extracted from leaves, fruits and roots of O. martiana and their antibacterial activity.

General
All reagents were of analytical grade.Column chromatography and TLC were performed on silica gel 60 F 254 (Merck).Mass spectra was determined with a Varian 3800 Series GC-EIMS (Varian SATURN 2000), 70 eV, capillary column CP-SIL PONA CB (100 m × 0.25 mm I.D. × 0.25 μm film thickness) with helium as the carrier gas at a flow rate of 1.6 mL min -1 ; the temperature program was 140 °C (10 min) and increased at a rate of 5 °C min -1 to 230 °C, standing 25 min at this temperature; injection in the split mode (1:200) at an injector, temperature of 200 °C; detector temperature at 200 °C; injection volume, 0.5 μL.][12] The NIST (National Institute for Standard Technology -62.235 compounds) was used for comparison of mass spectra.The retention indices were calculated for all volatile constituents using a homologous series (C 9 to C 20 ) recorded under the same operating conditions.Retention indices (RI) have been obtained according to the method of Van den Dool. 13The quantitative data were obtained by electronic integration of the GC-FID peak areas.
The bacterial strains were provided by the Laboratory of Basic Pathology-Universidade Federal do Paraná.Tests were carried out in duplicate with strains of Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Pseudomonas aerogenes (ATCC 27853) and Escherichia coli (ATCC 25922).New cultures of each strain were standardized in sterile saline solution following 0.5 MacFarland scale.Miller Hinton Agar (Difco Laboratories) was used for culture medium.

Extraction of the oils
Essential oils of leaves (188.4 g) and fruits (30.0 g) from O. martiana were obtained by hydrodistillation for four hours in a modified Clevenger-type apparatus, after cooling to 0 o C.
The essential oil of roots was obtained in previously phytochemical investigation.The air-dried and powdered roots of O. martiana (470.0 g) were extracted with 95% ethanol in a Soxhlet for 5 h at hot stage (470 g / 3 L).The obtained filtered material, after removal of the ethanol under reduced pressure was partitioned with 250 mL of hexane, dichloromethane, ethyl acetate and methanol, respectively.The fraction hexane (4.0 g) obtained after evaporation of the solvent under vacuum at 40 ºC, was submitted to column cromatography on a column of silica gel and developed with solvent mixtures of increasing polarity at 5% (hexane, hexane/ethyl acetate, ethyl acetate/ methanol and methanol), yielding 0.1 mL of the colorless essential oil (0.02%) correspondent to the grouped subfractions 5-13.

Antimicrobial activity assay
The bioautography method, 14 adapted from Hostettmann 15 was based on the application of 3 μL of the test oils on a TLC plate.The samples were first applied on a TLC plate GF 254 (2.5 × 5.0 cm) and developed with toluene/EtOAc (97:3), transferred to a Petri dish, covered with Miller Hinton Agar containing triphenyltetrazolium chloride (TTC -1%) and inoculated with bacterial strains previously standardized.Inhibition zones after 24 h of incubation (37 ºC) indicated the presence of active compounds.Chloramphenicol (Newprov -3 μL) was used as positive control of growth inhibition.
MIC tests were carried out according to Eloff, 16 using a tissue culture testplate (96 wells).The stock solutions of the oils were diluted and transferred into the first well (100 μL), and serial dilutions were performed so that concentrations in the range of 1 -0.001 mg mL -1 were obtained.Chloramphenicol (Merck) was used as the reference antibiotic control.The inoculum was added to all wells and the plates were incubated at 37 o C during 24 h.Antimicrobial activity was detected by adding 20 μL of 1% TTC aqueous solution.MIC was defined as the lowest concentration of oil that inhibited visible growth.
Phytochemical and Antibacterial Evaluation of Essential Oils J. Braz.Chem.Soc.
A total of 77 compounds were identified, accounting for 68.22 to 78.87% of the constituents.The identification of the constituents was performed by computer library search, retention indices (RI) and visual interpretation of the mass spectra. 12Results obtained for the qualitative and quantitative analysis of these essential oils are shown in Table 1.
Several compounds of the oils remained unidentified.The comparative analysis between oils showed variability in chemical constitution related to content of their principal components in different organs (Figure 1).
Roots and aerial parts (leaves and fruits) of O. martiana were collected in February 2003, in Paraná State, Southern region of Brazil.The botanist Dr. Gerdt Hatschbach of the Museu Botânico Municipal -Prefeitura de Curitiba, Paraná (MBM) identified the plant.Voucher specimens are deposited at the Herbarium of the MBM under number 259057.

Figure 1 .
Figure 1.Chromatograms of GC-MS analysis of essential oils from the Ottonia martiana.

Table 2 .
Antibacterial activity of essential oils from Ottonia martiana on four bacterial species American Type Culture Collection); + = inhibition zone; -= no inhibition zone.