Chemical composition , antimicrobial and anti-acetylcholinesterase activities of essential oil from Lantana camara ( Verbenaceae ) flowers

Post-Graduate in Biodiversity and Biotechnology Program, Bionorte, State Coordination of Roraima, Federal University of Roraima UFRR, Campus Cauamé, BR 174, Km 12, District Monte Cristo, CEP 69310-250, Boa Vista-RR-Brazil. Post-Graduate in Chemistry Program, Center for Research and Post-Graduate Studies in Science and Technology, NPPGCT, UFRR, Av Capitão Ene Garcez, n. 2413, Campus Paricarana, CEP 69310-000, Boa Vista-RR-Brazil. Embrapa Brazilian Agricultural Research Corporation. Rodovia 174, Km 8, Industrial District, CEP 69301-970, Boa Vista-RR-Brazil. Institute of Exact Sciences, Department of Chemistry, Federal University of Minas Gerais, UFMG, Av Antonio Carlos, n. 6627, Pampulha, CEP 31270-901, Belo Horizonte-MG-Brazil. Chromatography Laboratory, Institute of Exact Sciences, Department of Chemistry, UFMG, Belo Horizonte-MG-Brazil.


INTRODUCTION
Lantana camara L. (Verbenaceae) is a shrub with distribution in tropical, subtropical and temperate regions, being considered a weed difficult to control.However, L camara has ornamental uses, and is reported to improve soil quality for agriculture as well as possessing insecticide, antifungal, and herbicide activities.In folk medicine, this species is known by its sudorific and antipyretic activities, action on bronco-lung problems, rheumatism and against scabies (Patel, 2011;Passos et al., 2009;Lorenzi, 2008;Kohli et al., 2006;Lorenzi, 2002).The essential oil of its flowers and leaves possess a variety of chemical compounds with leishmanicidal, antimicrobial, anticancer, antiulcer, anti-inflammatory activities, among others.However, in high doses, these species can be toxic to some animals (Oyourou et al., 2013;Machado et al., 2012;Montanari et al., 2011;Sousa et al., 2011;Costa et al., 2009;Sharma and Kumar, 2009).
The objectives of this work was to analyze the chemical constitution of essential oil obtained from dried flowers of L. camara collected in Boa Vista, Roraima, and to evaluate its bioactivities on the acetylcolinesterase inhibition, and on the pathogenic microorganisms Escherichia coli, Salmonella typhimurium (Gram-negative bacteria), Staphylococcus aureus and Streptococcus sanguinis (Gram-positive bacteria), an yeast (Candida albicans) and the filamentous fungi Aspergillusflavus and Fusariumproliferatum.

Plant material and essential oil extraction
The flowers of L. camara were collected in the Cauamé Campus of Federal University of Roraima (UFRR) in Boa Vista, Roraima, Brazil.The plant material was identified by José Ferreira Ramos (Instituto Nacional de Pesqusias da Amazonia, INPA), and a voucher specimen (268126) was deposited at the INPA Herbarium.
The flowers were dried at room temperature and 100 g of the sample were used to obtain the essential oil by hydrodistillation using a Clevenger type apparatus.The essential oil was dried over anhydrous sodium sulphate and stored at -20°C before analysis (Rubiolo et al., 2010;Sefidkon, 2002).

Gas chromatography/mass spectrometry analysis
A GCMS-QP2010 ULTRA (Shimadzu) was used.Column: Rxi-1MS dos Santos et al. 923 30 m × 0.25 mm × 0.25 microns (Restek).Column Temp: 70°C (2 min), 5°C min -1 to 250°C.Injector: 250°C Split (1:20), GC-MS interface at 250°C.MS detector (electron impact at 70 eV) temperature was 250°C.Carrier gas: helium at 1.5 ml min -1 .Vol injection: 1 μl.Essential oil was diluted at 0.1% in chloroform.Data acquisition software used was GC-MS Solution (Shimadzu) together with NIST11 library.Identification of peaks was made by comparison of the mass spectra obtained by GC-MS spectra with the NIST11 library and also by comparing the Kovats indices calculated by GC-FID and literature data.

Antibacterial and yeast assay
E. coli (ATCC 25922), S. tiphymurium (ATCC 14028), S. aureus (ATCC 25923) and S. sanguinis (ATCC 49456) bacteria and C. albicans (ATCC 18804) yeast were used in the assay.Concentrations assayed were 500, 250, 125, 62.5, 31.25, 15.6, and 3.9 µg ml -1 (Zacchino and Gupta, 2007).Samples were weighed and dissolved in DMSO to 50 mg ml -1 .40 µl of this solution was added to a flask containing 960 µl of BHI (Brain Heart Infusion) broth (working solution).A pre-inoculum was prepared in which the bacteria and the yeast, stored under refrigeration, were transferred with a platinum loop to test tubes containing 3 ml of freshly made BHI broth.The tubes were incubated at 37°C for 18 h.Then, the pre-inoculum (500 µl) was transferred to tubes containing 4.5 ml of sterile distilled water.The tubes were homogenized and the concentration adjusted to 0.5 of McFarland turbidity standard (10 8 CFU ml -1 ), thereby obtaining the inocula used in the bioassays.
Assays were performed in 96-microwell plates in duplicate.100 µL of BHI broth was added to each well.In the first well, 100 µl of working solution were also added.The solution was homogenized and 100 µl transferred to the next well and so on until the last well, from where 100 µl was discarded.Then, 100 µl of microorganism inocula were added to wells.Eight different concentrations of each sample were tested.A positive control devoid of the working solution allowed us to examine microorganism growth.A negative control, which lacked the inoculum permitted us to discount the colour coming from the working solution.A control plate containing 100 µl of BHI culture medium and 100 µl of sterile distilled water were added to the experiment as a control of BHI broth sterility.
Another control was also prepared, containing the standard antibiotics Ampicillin (antibacterial), miconazole and nystatin (antifungals) to observe the activity of these antibiotics over the microorganisms.Microorganism growth was measured in ELISA plate reader (492 nm) immediately after ending the experiment (0 h).They were incubated at 37°C and read again after 24 h of experiments, ending the test.Results were calculated as percentual inhibition using the formula: % inhibition = 100 -AC -AC × 100AH -AM AC = absorbance of the sample; AC = absorbance of control sample; AH = absorbance of microorganisms in the control control and AM = absorbance of culture medium control.

Filamentous fungi assay
Filamentous fungi used in this test were A. flavus (CCT 4952) and *Corresponding author.E-mail: ricardocs.br@gmail.com.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License F. proliferatum (CML 3287).DMSO was used for sample preparation and the concentration of sample in the assay was 250 mg mL -1 .Sabouraud broth was used for fungal growth.A spore suspension at a concentration of 5 × 10 -5 spores ml -1 was used after spores counting on a Neubauer chamber.The sample incubation time was 48 h after which absorbance was read at 490 nm on a microtitre plate reader.Data were processed using the Outlier method, Grubbs test with 95% significance level.The percentage of inhibition was calculated by using the formula: % inhibition = 100 -AC -AC × 100AH -AM AC = absorbance of the sample; AC = absorbance of control sample; AH = absorbance of microorganisms in the control control and AM = absorbance of culture medium control.
The concentrations of compounds detected in the essential oil of L. camara flowers showed to be influenced by the collection spot.For instance, in the oil obtained from a L. camara voucher collected in India (Khan et al., 2002), the major compounds identified were β-elemene (14.5%), germacrene D (10.6%), α-copaene (10.7%), αcadinene (7.2%), β-caryophyllene (7.0%) and γ-elemene (6.8%); on the other hand, the essential oil obtained from L. camara flowers grown in Iran showed sabinene (16.5%), β-caryophyllene (14.0%), 1,8-cineole (10.0%), bicyclogermacrene (8.1%) and α-humulene (6.0%) as major compounds (Sefidkon, 2002).El Baroty et al. (2014) report the essential oil chemical composition of L. camara flowers obtained in Cairo, Egypt, where the majority chemicals differ from those presented in Table 1 among other differences.The yield differences, variations in chemical composition and their respective yields can be result of a number of biotic and abiotic factors (Figueiredo et al., 2008).The variations in the chemical composition can lead to different bioactive effects against diseases and microorganisms responsible for causing various pathologies.L. camara essential oil was assayed for acetylcholinesterase inhibition and, as the result, a good inhibition was detected reaching 77.15% of inhibition.
Neurodegenerative syndromes like Alzheimer's disease have been causing great concern worldwide.This disease causes difficulties in language, memory, emotional behaviour, personality and cognitive abilities (Singh et al., 2013).The World Health Organization (WHO), presents alarming data for Alzheimer's disease.Since it was estimated in 2010 about 35.6 million people were already suffering with this illness, with projections that this value would be tripled by 2050, with approximately 115.4 million people directly affected by Alzheimer's disease (WHO, 2012).Plants components have been extensively screened for their potential for acetylcholinesterase inhibition, since many plants can be used for the treatment of neurodegenerative diseases (Mukherjee et al., 2007).According to the classification for acetylcholinesterase reducing potential of crude extracts, weak inhibitors present inhibitory value below 30%; moderate inhibitors present 30 to 50% inhibition, and potent inhibitors show over 50% of inhibition enzyme (Vinutha et al., 2007).
Considering this, essential oil of L. camara dried flowers stands out as a potent inhibitor of AChE enzyme.This inhibitory potential may have been highlighted by the synergism of the several chemical constituents present.It has been found that the inhibitory potential can be associated to synergistic compounds.As an example, interactions between 1,8-cineole/α-pinene and 1,8cineole/caryophyllene oxide are reported to improve reduction of acetylcholinesterase activity; the same effect can be also caused by miscellaneous compounds (Singh et al., 2013;Savalev et al., 2003).Therapies involving essential oils can be accomplished in several ways, the  Bacteria 500 μg ml -1 (%) 250 μg ml -1 (%) 125 μg ml -1 (%) 31.25 μg ml -1 (%) 3.91 μg ml -1 (%) most common being aromatherapy.It was possible to observe improvement in the cognitive function of patients with Alzheimer's through aromatherapy (Savalev et al., 2003).Essential oils act in the central nervous system with excellent results in improving the living conditions and treatment of several diseases, especially neurodegenerative diseases like Alzheimer's and Parkinson (Dobetsberger and Buchbauer, 2011;Jimbo et al., 2009).Filamentous fungi A. flavus and F. proliferatum were inhibited by L. camera essential oil (44.52 and 28.97%, respectively).These microorganisms affect humans and crops causing a lot of damage, especially economic losses in various parts of the world (Mulè et al., 2004;Howard, 2002).Gram (+) and gram (-) bacteria (Table 2) and the yeast were inhibited in the assay showing activity Relative absorbance Time for the essential oil from L. camara flowers.The values ranged from 3.91 μg ml -1 to 500 μg ml -1 , potentially some satisfactory results in this bioassay.Ampicillin was used as a control, a standard clinical use of antibiotics, was very efficient in the concentrations shown.Growth of the pathogenic yeast, C. albicans, was inhibited more than 90% at all concentrations.It is noteworthy that there was 95% of C. albicans inhibition at 15.6 μg/ml, while the positive controls, miconazole and nystatin where less active at the same concentration (92 and 91%, respectively).
Pathogenicity of this yeast is usually associated to low immunity in decurrence of aging, infection or therapies, which can lead to the development of candidiasis.This infection affects the skin, oral cavity, esophagus, gastrointestinal tract, vagina and the human vascular system (Calderone and Fonzi, 2001).The above results reveal the potential of this essential oil against pathogenic microorganisms, broadening the biological importance of the species, already known for its actions as antipyretic, antimutagenic, and insecticide, among others (Naz and Bano, 2013;Seth et al., 2012;Zandi-Sohani et al., 2012;Kurade et al., 2010;Sharma and Kumar, 2009;Sonibare and Effiong, 2008;Verma and Verma, 2006;Barre et al., 2005;Deena and Thoppil, 2000;Siddiqui et al., 1995).

CONCLUSION
Despite L. camara is considered a weed, it has several biological benefits, being a promising source of a natural and powerful oil that acts as an AChE inhibitor.This essential oil may be a possibility for the development of new medicines to treat neurodegenerative diseases.Besides, it has efficacy against pathogenic microorganisms that affect humans, therefore it is used as an alternative antibiotic is also suggested.

Figure 1 .
Figure 1.Chromatogram of the essential oil from L. camara flowers.

Table 1 .
Percentage composition of the L. camara flowers essential oil.

Table 2 .
Inhibition of gram (+) and gram (-) bacteria by the essential oil from L. camara flowers.