Industrial Crops & Products 125 (2018) 236–240 Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Chemical composition and antimicrobial activity of the essential oil of Lippia lasiocalycina Cham. (Verbenaceae) T Wanessa Sales de Almeidaa, , Sidney Gonçalo de Limad, Humberto Medeiros Barretob, Leila Maria de Sousa Andradeb, Lorenna Fonsecad, Candido Athayde Sobrinhoc, Ananda Rosa Beserra Santosc, Maria Christina Sanches Muratoria ⁎ a Food and Nutrition Postgraduate Program, Federal University of Piauí, CampusMinistro Petrônio Portela, 64049-550, Teresina, PI, Brazil Postgraduate Program in Pharmaceutical Sciences, Federal University of Piauí, CampusMinistro Petrônio Portela, 64049-550 Teresina, PI, Brazil c Brazilian Agricultural Research Corporation, Av. Duque de Caxias, no. 5650 CEP: 64008-780, Teresina, PI, Brazil d Laboratory of Organic Geochemistry, Postgraduate Program in Chemistry, Federal University of Piauí, Campus Ministro Petrônio Portela, 64049-550 Teresina, PI, Brazil b A R T I C LE I N FO A B S T R A C T Keywords: Piperitenone oxide Candida albicans Limonene Lippia The use of plant species of the genus Lippia in the treatment of diseases is an old practice, however, some species still needs studies. Thus, the present work aimed to characterize chemically and to evaluate the antimicrobial activity of the essential oil of Lippia lasiocalycina. The oil was extracted by hydrodistillation and the analysis of the chemical constituents done using gas chromatography coupled to the mass spectrometer. Minimum inhibitory concentrations were determined by microdilution method against Staphylococcus aureus, Escherichia coli and Candida albicans strains. The constituents present were identified (90.09%) and piperitenone oxide was defined as the major compound (57.55%) followed by limonene (20.69%). The essential oil of Lippia lasiocalycina presented activity against C. albicans strain, signaling for a potential application in the treatment of infections caused by this yeast. 1. Introduction The use of medicinal plants has been constant since the origin of mankind. (Boukhatem et al., 2014). Among the substances of natural origin, essential oils are very important both economically and scientifically, being versatile products with applicability in the most varied sectors (Bernardos et al., 2015). Essential oils are rich in bioactive substances (Medeiros et al., 2011). Its chemical composition is complex and presents a wide variety of constituents like monoterpenes, sesquiterpenes, and their derivatives such as aldehydes and phenols. This composition varies between plant species and seasons of the year. (Hajlaoui et al., 2010). In plants, the essential oils are directly related to the processes of pollination, dissemination of seeds, and in defense against attacks of herbivores as well as fungi and bacteria (Costa et al., 2015; Li et al., 2013). The Verbenaceae family has approximately 36 genera of plants and 1000 plant species distributed in pantotropical regions. Brazil is the country with the greatest diversity of taxon with 16 genera and about 290 species. The plants of this family usually present in the form of herbs, shrubs, sub-shrubs and lianas (Costa et al., 2017). Among the ⁎ genus belonging to this family, we can highlight Lippia, constituted by 200 species that exhibits a striking appearance and pleasant odor (Oliveira et al., 2006). The genus Lippia is widely used in folk medicine in gastrointestinal disorders and respiratory diseases. The infusion and the essential oil of various parts of plants is used as antifungal, antimicrobial, larvicide, and anesthetic agents (Linde et al., 2010). In recent years, we have seen the emergence of a problem that permeates the treatment of various diseases, the phenomenon of microbial resistance. This resistance has rapidly proliferated by involving Gram-positive and Gram-negative bacteria, such as Staphylococcus and Escherichia coli (Silveira et al., 2006) and opportunistic fungal species like Candida albicans (Casto and Lima, 2011). Although the advances in research on the chemical and pharmacological properties of the Lippia genus, there are still species that needs clarifying studies such as Lippia lasiocalycina. To the best of our knowledge, there have been just one literature sources reporting the preliminary study of the chemical constituents of L. lasiocalycina alcohol extract (Funari et al., 2012). No information on the biological activities or chemical composition of the essential oil is available. Therefore, the chemical characterization and determination of the Corresponding author. E-mail address: wanessa.salmeida@yahoo.com.br (W.S. de Almeida). https://doi.org/10.1016/j.indcrop.2018.09.007 Received 2 May 2018; Received in revised form 2 September 2018; Accepted 4 September 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved. Industrial Crops & Products 125 (2018) 236–240 W.S. de Almeida et al. prepared by dissolving 10.000 μg of the product in 1 mL−1 of dimethylsulfoxide (DMSO). This initial solution was then diluted in sterile distilled water to a concentration of 1024 μg mL−1. MIC of the LLEO was determined by microdilution in BHI with microbial suspensions of 105 CFU mL−1 and with LLEO at concentrations 8, 16, 32, 64, 128, 256, 512 μg mL−1. Microplates were incubated at 37 °C for 24 h. MIC was defined as the lowest concentration of the drug in which no microbial growth is observed. All experiments were done in triplicate. The control group used in this study consisted of culture medium and inoculum without the addition of essential oil. The control of the inoculum and the control of the sterility of the culture medium were made. Antifungal assays were performed by the microdilution method in SDB double concentrated with a yeast suspension of 105 CFU mL−1 and LLEO solutions ranging from 8 to 512 μg mL−1. Microplates were incubated at 37 °C for 48 h. antimicrobial activity of the essential oil of Lippia lasiocalycina was the objective of the present study. 2. Experimental 2.1. Plant material The plant material was collected at 9:00 am in the Gilbués city, Piauí State, Brazil, in April 2016. The region presents subhumid tropical climate with high temperatures and low rainfall index being located in the semi-arid region of northeastern Brazil. Plant material was made available by the Brazilian Agricultural Research Company - EMBRAPA Meio-Norte (-5.036410, -42.797898). Voucher specimen was deposited in the Embrapa Herbarium Genetic Resources and Biotechnology - CEN under the number CEN92437. 2.2. Essential oil extraction 2.6. Determination of the minimum microbicide concentration Lippia lasiocalycina essential oil (LLEO) was obtained from fresh leaves of the plant by hydrodistillation using Clevenger type distillation apparatus for approximately 3 h. The essential oils yields ranged from 0.40 to 0.52%. At the end of the process, the resulting oil was collected, dried with sodium sulphate, weighed, and stored under refrigeration. The oil was solubilized in H2CCl for gas chromatography and mass spectrometry analysis. Inhibition of the bacterial or fungal growth was confirmed transferring an aliquot of 10 μl from each well of the MIC test microplates to a Petri dish containing BHIA (or SDA) and checking cell viability after incubation at 37 °C for 24 to 48 h. Minimal microbicide concentration (MMC) was defined as the lowest concentration of the drug in which no microbial growth was observed. 2.3. Chromatography conditions 3. Results and discussion For the characterization of the chemical composition of the volatile compounds of LLEO, a gas chromatograph, coupled to the mass spectrometer (GC–MS) was used. A Shimadzu® Chromatograph, model CGMS-QP2010 SE equipped with AOC-5000 automatic injector and SLB-5 ms column (30 m x 0.25 mm x 0.25 μm) was used. The conditions for the CG-MS analysis were as follows: Helium as carrier gas at a flow rate of 1 mL min−1, a temperature of 250 °C in the injector; a temperature program, starting at 60 °C (3 min), at a rate of 3 °C / min until reach 240 °C (for 10 min); the detector temperature was 250 °C. Previously the essential oil was diluted into dichloromethane (1:10) and so 1 μL was injected. The MS conditions were triple quadrupole type of ion detector operating by electronic impact (70 eV, 45–450 Da). Identification of the essential oil components was performed by comparing their GC–MS retention indices. The spectra were considered coincident if the similarity index was equal to or greater than 90%. The Kovats index was estimated by comparison between some known compounds in the chromatogram and the compatible Kovats indices of the database records (WILEY, NIST, PHEROBASE). In the last years we have seen a change in the habits of life of the population, a change that includes a greater concern with the quality of the food ingested as well as to the safety of the industrialized products. At the same time, the industry has been looking for options to reduce the amount of synthetic chemicals in its products, signaling a global trend towards adherence to more natural means of conservation and food production (Machado et al., 2011). In this scenario, essential oils appear as a promising alternative. One of the main factors that make them interesting for the food industry is the fact that these oils are mostly used routinely in cooking or in folk medicine, making their acceptability much higher when compared with synthetic additives (Pereira et al., 2006). The volatile compounds of L. lasiocalycina essential oil were detected for the first time by GC–MS. Representing 90.09% of the essential oil fourteen constituents were identified of which the major ones, piperitenone oxide (57.55%) and limonene (20.69%), accounted for 78.24% (Table 1). The chemical composition of species of the genus Lippia has been extensively studied. Gonçalves et al. (2015) and Guimarães et al. (2014) 2.4. Microbial strains Table 1 Constituents of Lippia lasiocalycina essential oil analyzed by GC–MS. Evaluation of the antimicrobial activity of EOLL was performed against standard strains Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Candida albicans ATCC 10231, as well as, against a multidrug-resistant S. aureus strain (SA-1199B). Bacterial strains were maintained on Brain Heart Infusion Agar (BHIA, Himedia, India) slant at 4 °C, and prior to assay the cells were grown overnight at 37 °C in Brain Heart Infusion (BHI, Himedia, India). The yeast strain was maintained on Sabouraud Dextrose Agar (SDA, Himedia, India) slant at 4 °C and prior to assay the cells were grown for 24 h at 37 °C in Sabouraud Dextrose Broth (SDB, Himedia, India). 2.5. Determination of the minimum inhibitory concentration The determination of the minimum inhibitory concentration (MIC) was performed according to the microdilution method (CLSI, 2003). In this assay, 96-well microplates (12 columns and 8 lines) with flat bottom were used. A stock solution of the test product was previously RTa Compound KIb Area 3.677 7.121 8.816 10.195 10.399 10.986 11.538 19.849 21.998 22.354 24.199 25.283 31.175 31.597 Methylbenzene α-pinene Myrcene p-Cimene Limonene Trans-β-ocimene γ-Terpinene Carvone m-Thymol Tridecane Piperitenone Piperitenone oxide Bicyclogermacrene β-Bisabolene 770 939 991 1026 1031 1050 1062 1242 1290 1299 1342 1365 1494 1509 0.31 1.17 0.20 0.47 20.69 1.01 0.18 0.26 0.83 1.03 3.04 57.55 2.67 0.68 a b 237 Retention time. Kovats index. Industrial Crops & Products 125 (2018) 236–240 W.S. de Almeida et al. found carvacrol and 1,8-cineol as major components for Lippia sidoides. Costa et al. (2005) when analyzing the same species found thymol and α-phellandrene. Nogueira et al. (2007), in a study carried out in Paraná, Brazil, found citral and trans-dihydrocarvone while Silva et al. (2006) identified citral as the main component of Lippia alba. Previous researches verified that essential oil from aerial parts of L. origanoides collected in different Brazilian states showed carvacrol and thymol as major chemical components (Sarrazin et al., 2015a; Queiroz et al., 2014; Borges et al., 2012). A variation of the essential oil composition is influenced by several factors, such as place of cultivation, soil type, climate, temperature, luminosity and collection time (Alencar-Filho et al., 2017; Bitu et al., 2015; Sarrazin et al., 2015b). The chemical profile may vary both between species and interspecies (Lima et al., 2016; Morais, 2009). In addition, some constituents appear as typical of certain species while others are considered as an exception. Piperitenone oxide (PO) is a natural oxygenated monoterpene, also known as rotundifolone, used in different products such as detergents, creams, and lotions. In the food industry, it is usually used as a flavoring agent (Sotto et al., 2017). The growing market demand for exempt food products or with a reduction in the amount of synthetic chemicals used in their production and conservation has increased the industry's interest in alternatives that have better consumer acceptance (Galo et al., 2018). Thus, the presence of PO in high concentration in LLEO beckons to a new vegetable source of this substance commonly used in food flavoring, appearing as a more natural option to its synthetic analogues. Besides that, several pharmacological activities have been evidenced for PO including antiviral activity against herpesvirus type 1 (Civitelli et al., 2014), repellent action against Aedes aegypti (Tripathi et al., 2004), thevasodilatory effect in hypertensive rats (Lan et al., 2017), anticancer potential (Nakamura et al., 2014) and antiparasitic action (Teles et al., 2011). Piperitenone oxide is not a common occurring compound in the genus Lippia being. It is most common in different species of mint such as Mentha spicata and Mentha rotundifolia. Some Lippia species may exhibit piperitenone oxide as a minor component (Bozovic et al., 2015), but their presence as a major constituent is not a common finding. Among the species of the genus Lippia the PO was recently identified in Lippia pedunculosa at a concentration higher than 70% (Nascimento et al., 2017). The presence of PO as the major constituent of the LLEO (57.55%) is an unprecedented discovery, indicating that it is a good source of this compound and signalizing for a range of possible technological applications for the LLEO. Terpenes are considered the secondary metabolites most produced by plants. This class of substances is often used in the pharmaceutical and solvent industries. (Wang et al., 2016). They have a great structural diversity and can exert crucial functions for plant development, being usually involved in defense mechanisms (Srividya et al., 2015; Viegas Júnior, 2003). Limonene, the second main compound found in the LLEO, is a monocyclic monoterpene present in more than 300 plant species. It is present in two possible conformations, S-(-)-limonene and R-(+)-limonene, and both are directly related to inhibition of growth of microorganisms, especially fungi (Duetz et al., 2003). In citrus essential oils the presence of the R-(+)-limonene isomer is more common, while S-(-)-limonene is more common herbs and some plant varieties as in species of the Mentha genus (Maróstica Júnior and Pastore, 2007). In the essential oil market, limonene is one of the most exported from Brazil to European Union countries with the main purposes being the use of cleaning products. From 2005 to 2008, 427 t of limonene were exported, generating millions of dollars of revenue (Bizzo et al., 2009). Natural products from plants are considered as having a good inhibitory activity if they present MICs ≤100 μg/mL−1, a moderate inhibitory activity if they present MICs ranging from 100 to 500 μg/ mL−1, a weak inhibitory activity if they present MICs ranging from 500 to 1000 μg/mL−1, and no inhibitory activity if they present MICs > 1000 μg/mL−1 (Holetz et al., 2002). According to these Table 2 MIC and MMC of Lippia lasiocalycina essential oil on microbial strains. Strains MICa (μg/mL−1) S. aureus ATCC 25923 S. aureus SA1199-B E. coli ATCC 25922 C. albicans ATCC 10231 ≥1024 – – No activity ≥1024 – – No activity ≥1024 – – No activity 512 512 1 Fungicide a b MMCb (μg/mL−1) MMC/MIC Inhibitory Effect Minimum inhibitory concentration. Minimum microbicide concentration. criteria, the results obtained from assays to evaluate the antimicrobial activity (Table 2) showed that LLEO was inactive against both Grampositive and Gram-negative bacterial strains. On the other hand, the oil was active against the yeast strain tested (Table 2), and although this activity, it is considered as weak, its inhibitory effect was fungicide. This result indicates that LLEO is a source of phytochemicals that could be used in the prevention or treatment of mycoses in human and animal hosts, as well as, they could be used for prevention of food contamination by fungal species. The antifungal activity of the LLEO could be attributed to the presence of hydrophobic monoterpenes, such as PO and limonene, as well as, other minor components which are able to cause lipid partitioning of cell membrane and increasing its permeability to essential electrolytes (Bakkali et al., 2008; Duetz et al., 2003; Oumzil et al., 2002). Candida spp. are commensal yeasts commonly found in mucous membranes and epithelial tissues, however species of the genus Candida can lead to opportunistic fungal infections, being Candida albicans the most involved (Gullo et al., 2016; Castro and Lima, 2011). The low host immunity combined with the versatility of surviving in the most different environments and conditions is directly related to the transition from a harmless microorganism to an infectious one (Cassone, 2015; Li et al., 2015). Yeasts have several virulence factors such as the ability to adhere to tissues, the possibility of converting unicellular yeasts into filamentous forms, a hydrophobic cell wall and the production of some extracellular enzymes that help in the expression of their pathogenicity (Ishida et al., 2006; Calderoni and Fonzi, 2001). Several drugs are used in the treatment of mycotic infections. Antiseptics such as iodine tincture and gentian violet are examples of some substances very employed as antimycotics. The development of pharmacological research has enabled the discovery and use of more modern antifungal drugs such as ketoconazole, miconazole, amphotericin B, and clotrimazole (Lima et al., 2006). However, extensive use has led to resistance to treatment and difficulties in combating opportunistic fungal infections (Barbosa et al., 2016). Protein mutations that reduce the ability of drugs to interact and bind to the cell wall and the active efflux of drugs out of the cell by means of membrane-expressed transporters, and pumps are some mechanisms developed by Candida albicans to circumvent the action of major antifungal agents (Schneider and Morschhauser, 2015; Shafreen et al., 2014). Given the increasing occurrence of microorganisms that express resistance to drug therapies, alternatives should be sought to enable the treatment of these conditions efficiently. The use of plant products, such as essential oils, has been identified as one of the most promising sources, both for the discovery of new drugs and as an aid to existing ones (Guimarães et al., 2017; Oliveira et al., 2016; Lucena et al., 2015). The fact that they are a complex mixture of substances allied to action at multiple target sites makes the development of resistance to essential oils an unlikely event (Sales et al., 2014). In addition, the synergistic effect between essential oils and synthetic drugs can inhibit efflux pumps (Barreto et al., 2014) and eliminate plasmids that are involved in 238 Industrial Crops & Products 125 (2018) 236–240 W.S. de Almeida et al. antimicrobial resistance (Pereira et al., 2015). Grow Aerobically; Approved Standard M7-A6, 6rd ed. Clin. Lab. Stand. Inst., 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA. Costa, J.G.M., Rodrigues, F.F.G., Angélico, E.C., Silva, M.R., Mota, M.L., Santos, N.K.A., Cardoso, A.L.H., 2005. T.L.G. 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Nova 30, 382–387. https://doi.org/10.1590/ S0100-40422007000200027. Medeiros, R.T.S., Gonçalez, E., Felicio, R.C., Felicio, J.D., 2011. Evaluation of antifungal activity of Pittosporum undulatum L. Essential oil against Aspergillus flavus and aflatoxin production. Cienc Agrotec. 35, 71–76. https://doi.org/10.1590/S141370542011000100008. 4. Conclusion The essential oil of L. lasiocalycina presented a high concentration of piperitenone oxide and fungicide activity on strains of Candida albicans being a good source for future research aimed at the development of new drugs and as a source of obtaining flavorings used in foods. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-prof it sectors. Declarations of interest None. 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