Characterization and anti-staphylococcal activity of the essential oil from Turnera subulata

Characterization and anti-staphylococcal activity of the essential oil from Turnera subulata Sm. The present study is pioneer in analyzing the chemical composition of the essential oil from aerial parts of Turnera subulata Sm. and evaluates their antibacterial activity against a panel of drug-resistant strains of Staphylococcus aureus. The aerial parts were kiln-dried and then powdered in mechanical mill, and was subjected to hydrodistillation in a Clevenger-type apparatus. The components were analyzed using a mass spectrometrycoupled gas chromatography (GC-MS), and the identification of substances was performed by comparison of the mass spectra obtained with the mass spectra of the database of the GC-MS and retention indices . The essential oil was evaluated using the method of broth dilution at concentrations ranging from 3.125μg/mL to 3200μg/mL. It was possible to identify 45 substances (92.1%) of the essential oils, and the major components were trans-caryophyllene (6.7%), citronellol (5.6%), sphatulenol (5.3%), α-cadinol (4.3%), n-tricosano (4.3%), geraniol (4.1%) and transgeranilacetone (3.7%), n-pentacosano (3.5%), globulol (3.4%), caryophyllene oxide (3.2%). The essential oil of T. subulata Sm. showed effective antibacterial activity for the various strains of S. aureus tested, with the MIC values between 25 μg/mL and 1600 μg/mL. The study of the essential oil of Turnera subulata Sm. showed that it consists of a complex mixture of several classes of compounds having the sesquiterpenes as major constituents followed by monoterpenes, and showed an antibacterial activity significant front the strains tested.

Palavras-chave: Turnera subulata, CG-EM, Turneraceae, óleo essencial, atividade antiestafilocócica ABSTRACT: Characterization and anti-staphylococcal activity of the essential oil from Turnera subulata Sm.The present study is pioneer in analyzing the chemical composition of the essential oil from aerial parts of Turnera subulata Sm. and evaluates their antibacterial activity against a panel of drug-resistant strains of Staphylococcus aureus.The aerial parts were kiln-dried and then powdered in mechanical mill, and was subjected to hydrodistillation in a Clevenger-type apparatus.The components were analyzed using a mass spectrometrycoupled gas chromatography (GC-MS), and the identification of substances was performed by comparison of the mass spectra obtained with the mass spectra of the database of the GC-MS and retention indices .The essential oil was evaluated using the method of broth dilution at concentrations ranging from 3.125µg/mL to 3200μg/mL.It was possible to identify 45 substances (92.1%) of the essential oils, and the major components were trans-caryophyllene (6.7%), citronellol (5.6%), sphatulenol (5.3%), α-cadinol (4.3%), n-tricosano (4.3%), geraniol (4.1%) and transgeranilacetone (3.7%), n-pentacosano (3.5%), globulol (3.4%), caryophyllene oxide (3.2%).The essential oil of T. subulata Sm. showed effective antibacterial activity for the various strains of S. aureus tested, with the MIC values between 25 μg/mL and 1600 μg/mL.The study of the essential oil of Turnera subulata Sm. showed that it consists of a complex mixture of several classes of compounds having the sesquiterpenes as major constituents followed by monoterpenes, and showed an antibacterial activity significant front the strains tested.

INTRODUCTION
Essential oils are volatile elements contained in many vegetable organs, and are related to several functions necessary for the plant survival, also has great importance in the defense against pathogens (Siqui et al., 2000).Researchers have been interested in biologically active compounds isolated from plant species for the elimination of pathogenic microorganisms because of the resistance that microorganisms have built against antibiotics (Essawi, 2000).
The bacterial resistance to multiple antibiotics has increased throughout the decades, making conventional antibiotics impotent against the bacterial resistance, such as Staphylococcus aureus (lowy, 2003), which culminates in the necessity for new drugs discovery.The essential oils have been used in folk medicine for decades as antimicrobial (Fisher and Phillips, 2008).Thus, natural products of plant origin are a rich source of new antimicrobials (Lewis & Ausubel, 2006).
Turnera subulata Sm, popularly known as "chanana" or "flor-do-Guarujá", is a member of the family Turneraceae (Gonzalez and Arbo, 2005), which consists of 10 genera and about 200 species that have distribution from North to South America, and in Africa, including Madagascar and the Mascarene Islands (Arbo, 2007).
In Brazil the family Turneraceae is represented by two genera, Piriqueta and Turnera, being this the most representative in the country having about 140 species (Arbo, 2008).According to Arbo (2007), Brazil holds the top number of species, the highest number of endemics being found in the mountains of the states of Bahia, Goiás and Minas Gerais.The genus is characterized by the presence of terpenoids, like mono and sesquiterpenes, flavonoids, steroids, benzenoids, alkaloids and lipids (Barbosa et al., 2007).
The roots of Turnera chamaedrifolia Cambess.and Turnera subulata Sm. are sold in street markets of the State of Paraíba and used in folk medicine (in the form of tea) in the treatment of amenorrhea, dysmenorrhea and as an abortifacient (Barbosa et al., 2007).
Previous studies have defined the many constituents of the essential oil of several species of Turnera, including T. diffusa, T. aphrodisiaca and T. brasiliensis (Kumar et al., 2006;Maia et al., 2007).
It was observed that the phytochemical studies have mainly focused on the characterization of the extracts, with few reports on the composition of essential oils.The present study is pioneer in analyzing the chemical composition of the essential oil from aerial parts of Turnera subulata Sm.The essential oil was also evaluated for antibacterial activity against a panel of drug-resistant strains of Staphylococcus aureus.

Experimental
Plant material: The aerial parts of Turnera subulata Sm. was collected in the University campus, in João Pessoa, the coastal region of Paraíba, Brazil, in August 2008, and indentified by one of the authors (M.F.A.).A voucher specimen (Agra & Barbosa 6273) was deposited at the herbarium Prof. Lauro Pires Xavier (JPB), Federal University of Paraíba.
The aerial parts were kiln-dried, with air circulating system, at 40°C for a period of 48 hours, and then powdered in mechanical mill.427.7 g of plant material was subjected to hydrodistillation in a Clevenger-type apparatus for 3 hours.For the analysis, 1μL of the essential oil was diluted in 1mL of ethyl acetate (chromatographic degree).

Analysis of the essential oils:
The components of the essential oil were analyzed using a gas chromatograph coupled to mass spectroscopy (GC-MS, Shimadzu QP-5000, Japão) equipped with a capillary column OV-5 (Ohio Valley Specialty Chemical, Inc.; 30.0m x 0.25mm x 0.25m).The column temperature was programmed from 60°C to 280°C, at a rate of 3°C/min.The temperatures of the injector and detector were 220°C and 230°C, respectively.Helium was used as the carrier gas, at a flow rate of 1mL/min.The injection of the oil was manually, 1μL the solution, with split of 1:10.The mass spectra were obtained with electron impact (EI) of 70 eV.
Identification and quantification the components of the oil were identified by comparison of their retention indices (determined in relation to an homologous series of n-alkanes) with those of pure standards or as reported in the literature (Adams, 1995).Comparision of fragmentation patterns in the MS with those stored on the CG-MS databases (NIST 62) was also performed.The percentages of each component were reported as raw percentages without standardization.
Antibacterial assay: The stock solution of the essential oil was prepared in DMSO which at its highest final concentration after dilution in the broth (4%) caused no inhibition of bacterial growth.The minimum inhibitory concentration (MIC) of the essential oil was determined in BHI by the microdilution assay (CSLI, 2005) using a suspension of ca. 10 5 cfu/mL and an essential oil concentration range of 3200 -3.125 µg/mL (two-fold serial dilutions).The MIC, determined in duplicate, is defined as the lowest concentration at which no growth is observed.

RESULTS AND DISCUSSION
Through the analysis of the essential oil of Turnera subulata in GC-MS it was possible to identify 45 substances , representing 92.1% of the total essential oil (Table 1).
It can be verified that this essential oil is composed of a complex mixture of several kinds of substances, comprising an alcohol, five monoterpenes, twenty-three sesquiterpenes, eight hydrocarbons, four fatty acids, one ester, two ketones and one aldehyde.
A study with Turnera ulmifolia showed that its essential oil had excellent antifungal activity against dermatophyte species, inhibiting 100% of the strains tested (Lima et al., 2000).MIC values of the essential oil of Turnera subulata Sm against strains of S aureus are presented in Table 2. Essential oils are potential sources of new antimicrobial compounds especially against bacterial pathogens (Prabuseenivasan et al., 2006).
An important characteristic of the essential oil is its hydrophobicity, which allows them to change the lipids in the cell membrane of bacteria, destructuring and making them more permeable to ions and other molecules that may lead to cell death (Sikkema et al., 1994).

Table 1 .
Chemical composition (%) of the essential oil of Turnera subulata Sm.

Table 2 .
Anti-staphylococcal activity of the essential oil of Turnera subulata Sm