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Biological activities of essential oils from six genotypes of four Ocotea species

Abstract

Essential oils from four Ocotea species collected in southern Brazil were evaluated for chemical composition using gas chromatography coupled with mass spectrometry. The primary compound identified in O. acutifolia essential oil was an unsaturated tetracyclic diterpene, phyllocladene (67.7%), followed by a sesquiterpene hydrocarbon, β-selinene (18.0%). The sesquiterpene fraction was predominant in oils from two collections of O. puberula; β-caryophyllene (25.2%) and globulol (22.6%) were the major compounds identified in collections 1 and 2, respectively.

O. silvestris essential oil contained predominantly germacrene D and bicyclogermacrene. These compounds were also predominant in essential oil from O. indecora leaves collected from shady habitats. By contrast, essential oil extracted from O. indecora grown under direct sunlight contained mainly oxygenated sesquiterpenes, such as guaiol (30.2%), α-eudesmol (27.6%), and β-eudesmol (12.7%). Chemotaxis assays showed that Ocotea essential oils had no significant inhibitory activity on leukocyte migration compared with a chemotactic stimulant (lipopolysaccharide from Escherichia coli). However, the oils exhibited antifungal activity against Candida parapsilosis, with a minimum inhibitory concentration of 500 µg/mL. To our knowledge, this is the first study to investigate the in vitro antifungal and antichemotactic activities of essential oils from Ocotea species native to southern Brazil.

Keywords:
Antifungal; Biological activity; Essential oil; Lauraceae; Ocotea

INTRODUCTION

Plants are rich natural sources of compounds capable of exerting the most diverse pharmacological actions. This property, combined with an outstanding chemical diversity, makes products of plant origin excellent sources of new drugs. Perhaps the most promising and widely researched plant secondary metabolites are essential oils, one of the largest groups of natural products and potential sources of biologically active substances. Knowledge of plant composition and secondary metabolite profiles may also assist in botanical classification, particularly for species that exhibit morphological changes as an adaptation to different environments or climates (Freire et al., 2011Freire JM, Cardoso MG, Batista LR, Andrade MA. Essential oil of Origanum majorana L., Illicium verum Hook. f. and Cinnamomum zeylanicum Blume: chemical and antimicrobial characterization. Rev Bras Plantas Med. 2011;13(2):209-214.).

In recent years, studies have reported increasing resistance to antifungal agents, and numerous efforts have been undertaken to develop alternative treatment and prophylactic strategies, such as investigations on the pharmacological activity of compounds isolated from plants (Bajpai et al., 2009Bajpai VK, Al-reza SM, Choi UK, Lee JH, Kang SC. Chemical composition, antibacterial and antioxidant activities of leaf essential oil and extracts of Metasequio aglyptostroboides Miki ex Hu. ‎ Food Chem Toxicol. 2009;47(8):1876-1883.a). Terpenoids are an example of such compounds. These volatile constituents of essential oils are associated with important biological activities, including antibacterial and antifungal properties (Reddy et al., 2017Reddy DN, Al-Rajab AJ, Sharma M, Moses MM, Reddy GR, Albratty M. Chemical constituents, in vitro antibacterial and antifungal activity of Mentha x Piperita L. (peppermint) essential oils. J King Saud Univ. doi.org/10.1016/j. jksus.2017.07.013. 2017.
https://doi.org/doi.org/10.1016/j. jksus...
; Blažeković et al., 2018Blažeković B, Yang W, Wang Y, Li C, Kindl M, Pepeljnjak S, Knežević SV. Chemical composition, antimicrobial and antioxidant activities of essential oils of Lavandula × intermedia ‘Budrovka’ and L. angustifolia cultivated in Croatia. Ind Crops Prod. 2018;123:173-182.; Danielli et al., 2018Danielli LJ, Pippi B, Duarte JÁ, Maciel AJ, Lopes W, Machado MM, Oliveira LFS, Vainstein MH, Teixeira ML, Bordignon SAL, Fuentefria AM, Apel MA. Antifungal mechanism of action of Schinus lentiscifolius Marchand essential oil and its synergistic effect in vitro with terbinafine and ciclopirox against dermatophytes. J Pharm Pharmacol. 2018;70:1216-1227.).

In addition to antimicrobial activity, several essential oils exhibit anti-inflammatory and antioxidant effects. A product possessing these properties may aid in wound healing, as infection commonly triggers an inflammatory process (Oliveira et al., 2011Oliveira PC, Paula CA, Rezende SA, Campos FT, Guimarães DAS. Anti-inflammatory activity of Lychnophora passerina, Asteraceae (Brazilian ‘’Arnica’’). J Ethnopharmacol. 2011;135(2):393-398.). By combining antifungal and anti-inflammatory actions, it is possible to limit symptoms such as itching, burning, and erythema, given that antioxidant compounds eliminate free radicals that cause tissue damage during inflammation (Havlickova, Friedrich, 2008Havlickova B, Friedrich M. The advantages of topical combination therapy in the treatment of inflammatory dermatomycoses. Mycoses. 2008;51(4):16-26.; Mastelic et al., 2008Mastelic, J, Jerkovic J, Blazevic I, Blazi MP, Borovic S, Bace II, et al. Comparative study on the antioxidant and biological activities of carvacrol, thymol and eugenol derivates. J Agric Food Chem. 2008;56(11):3989-3996.).

The family Lauraceae comprises about 50 genera, including Ocotea, about 2500-3000 species, and an extensive fossil record. This family is primarily found in tropical and subtropical regions and is well represented in the Americas, Asia, Australia, Madagascar, and rarely, in Africa (Van Der Werff, Ritcher, 1996Van Der Werff H, Ritcher HG. Toward and improved classification of Lauraceae. Ann Mo Bot Gard. 1996;83(3):409-418.; Bannister, Conran, Lee, 2012Bannister JM, Conran JG, Lee DE. Lauraceae from rainforest surrounding an early Miocene maar lake, Otago, southern New Zealand. Rev Palaeobot Palynol. 2012;178:13-34.). The genus Ocotea Aubl. is the largest in the neotropical region, consisting of circa 350 species. It is highly prevalent in the Brazilian Atlantic Forest, with 120-160 species (Baitello, 2001Baitello JB. Novas espécies de Lauraceae para a flora brasileira. Acta Bot Bras. 2001;15(3):445-450.; Baitello et al., 2003Baitello JB, Marcovino JR, Wanderley MGL, Shepherd G, Giulietti AM, Mehem TS. Lauraceae: Ocotea Aubl. In Flora fanerogâmica do Estado de São Paulo. 2003;3:179-208.).

Several studies assessed the biological activities of Ocotea essential oils. Important properties were identified, such as anti-inflammatory (Ballabeni et al., 2010Ballabeni V, Tognolini M, Giorgio C, Bertoni S, Bruni R, Barocelli E. Ocotea quixos Lam. essential oil: In vitro and in vivo investigation on its anti-inflammatory properties. Fitoterapia. 2010;81(4):289-295.; Leporatti et al., 2014Leporatti ML, Pintore G, Foddai M, Chessa M, Piana A, Petretto GL, et al. Chemical, biological, morphoanatomical and antimicrobial study of Ocotea puchury-major Mart. Nat Prod Res. 2014;28(5):294-300.), antithrombotic (Ballabeni et al., 2007Ballabeni V, Tognolini M, Bertoni S, Bruni R, Guerrini A, Rueda GM, et al. Antiplatelet and antithrombotic activities of essential oil from wild Ocotea quixos (Lam.) Kosterm. (Lauraceae) calices from Amazonian Ecuador. Pharmacol Res. 2007;55(1):23-30.), cardiovascular (Barbosa-Filho et al., 2008Barbosa-Filho JM, Cunha RM, Dias CS, Athayde-Filho PF, Silva MS, Da-Cunha EVL, et al. GC-MS Analysis and cardiovascular activity of the essential oil of Ocotea duckei. Braz J Pharmacogn. 2008;18(1):37-41.), antimicrobial (Leporatti et al., 2014Leporatti ML, Pintore G, Foddai M, Chessa M, Piana A, Petretto GL, et al. Chemical, biological, morphoanatomical and antimicrobial study of Ocotea puchury-major Mart. Nat Prod Res. 2014;28(5):294-300.,Da Silva et al., 2017Da Silva JK, Da Trindade R, Moreira EC, Maia JGS, Dosoky NS, Miller RS, et al. Chemical Diversity, Biological Activity, and Genetic. Aspects of Three Ocotea Species from the Amazon. Int J Mol Sci. 2017;18:1-15.), and cytotoxic (Da Silva et al., 2017Da Silva JK, Da Trindade R, Moreira EC, Maia JGS, Dosoky NS, Miller RS, et al. Chemical Diversity, Biological Activity, and Genetic. Aspects of Three Ocotea Species from the Amazon. Int J Mol Sci. 2017;18:1-15.). Other studies investigated associations between groups of compounds and cytotoxicity, such as Garcez et al. (2011Garcez FR, da Silva AFG, Garcez WS, Linck G, Matos MFC, Santos ECS, et al. Cytotoxic Aporphine Alkaloids from Ocotea acutifolia. Planta Med . 2011;77:383-387.), who reported that the alkaloid aporphine, isolated from O. acutifolia leaves, was cytotoxic to human cancer cell lines. Another study reported the mutagenic effects of five aporphinoid alkaloids isolated from O. acutifolia on Drosophila melanogaster wing cells (Guterres et al., 2013Guterres ZR, da Silva AFG, Garcez WS, Garcez FR, Fernandes CA, Garcez FR. Mutagenicity and recombinagenicity of Ocotea acutifolia (Lauraceae) aporphinoid alkaloids. Mutat Res. 2013;757:91-96.). It was also found that the chloroform fraction of O. puberula fruits containing the alkaloid dicentrine demonstrated antinociceptive effects (Montrucchio et al., 2012Montrucchio DP, Miguel OG, Zanin SMW, Da Silva GA, Cardozo AM, Santos ARS. Antinociceptive Effects of a Chloroform Extract and the Alkaloid Dicentrine Isolated from fruits of Ocotea puberula. Planta Med . 2012;78(14):1543- 1548.).

This study aimed to determine the chemical composition and assess the in vitro antifungal and anti- inflammatory activities of essential oils from Ocotea species occurring in southern Brazil. To our knowledge, no study has yet reported the chemical or pharmacological properties of these oils.

MATERIAL AND METHODS

Plant material

Leaves of Ocotea species were collected from native populations in southern Brazil (Table I). The plant material was identified by the botanist Dr. Sérgio L. Bordignon, and a voucher specimen of each species was deposited in the Herbarium of the Federal University of Rio Grande do Sul (ICN-UFRGS) under the following numbers: O. acutifolia, 176762; O. indecora, 192541; O. puberula, 176761; and O. silvestris, 176763.

TABLE I
Collection period, locality, and essential oil yield of different Ocotea species

Essential oil extraction

Essential oils from Ocotea species were obtained from fresh material by hydrodistillation using a Clevenger- type apparatus for 4 h, according to the procedures described in the Brazilian Pharmacopoeia (2010)Farmacopeia Brasileira, Agência Nacional de Vigilância Sanitária, Brasília, Brazil (2010).. Yield was determined as the ratio of weight to volume (w/v). Samples were stored in glass vials in a refrigerator at 4-5 °C until analysis to minimize chemical degradation.

Chemical analysis

For chemical analysis, the essential oils were diluted in ethyl ether at a ratio of 2:100 (v/v) and analyzed on a gas chromatograph coupled to a mass spectrometer (GC- MS) (Shimadzu QP5000) equipped with a Durabond DB-5 fused-silica capillary column (30 m × 0.25 mm × 0.25 μm). The injector and detector temperatures were set at 220 and 250 °C, respectively, using a column temperature program of 60-300 °C at 3 °C/min, with helium as carrier gas at a flow rate of 1 mL/min. Quantitative GC analysis was carried out using a GC-PerkinElmer Autosystem 158 XL chromatograph equipped with a Durabond DB-5 column and TotalChrom Workstation software. Injector, detector, and oven temperatures were the same as described for the GC-MS analysis. Nitrogen was used as carrier gas (1.0 mL/min) with a constant makeup flow of 40 mL/min hydrogen and 400 mL/min analytical grade air. Percentage compositions were obtained from electronic integration measurements using flame ionization detection (FID) without taking into account relative response factors.

Compound identification was based on the comparison of retention indices calculated by linear interpolation relative to retention times of a series of n-alkenes and comparison of mass spectra with those of standard samples, literature data (Adams, 2009Adams RP. Identification of Essential Oil Components by Gas Chromatogra-phy/Mass Spectrometry. Allured, IL, USA; 2009.), and National Institute of Standards and Technology (NIST) 12 and 62 spectral databases. Relative compositions were calculated by normalization of GC peak areas.

Antifungal activity

The antifungal activity of essential oils was evaluated against clinical isolates of Candida albicans (CA15), C. glabrata (CG09), C. krusei (CK03), C. parapsilosis (CP52), C. tropicalis (CT750), Cryptococcus neoformans (CN16), Microsporum canis (MC38), M. gypseum (MG01), Trichophyton mentagrophytes (TM32), and T. rubrum (TR51). All isolates are deposited in the Mycology Collection of the Federal University of Rio Grande do Sul, Porto Alegre, Brazil.

Prior to the antifungal activity assays, yeasts were cultured in Sabouraud agar containing chloramphenicol for 24 h at 35 °C and filamentous fungi were incubated at 32 °C for 5 days. A screening test was conducted with all essential oils at a fixed concentration of 500 μg/ mL. The minimum inhibitory concentration (MIC) was determined only in cases where antifungal activity was detected in the screening test. MIC values were assessed by the broth microdilution method, according to the Clinical Laboratory Standards Institute protocol (CLSI, 2008Clinical and Laboratory Standards Institute. CLSI. 2008. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Document M27-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.). Essential oil samples were tested at concentrations from 1.95 to 500 μg/mL. The MIC was defined as the lowest concentration of essential oil at which the tested microorganism did not show visible growth. Experiments were performed in quadruplicate.

Chemotaxis assay

Antichemotactic activity was evaluated according to the modified Boyden chamber method as described by Suyenaga et al. (2011Suyenaga ES, Konrath EL, Dresch RR, Apel MA, Zuanazzi JA, Chaves CG, Henriques AT. Appraisal of the antichemotactic activity of flavonoids on polymorphonuclear neutrophils. Planta Med . 2011;77(7):698-704.). Before the assay, neutrophils were treated with essential oils dissolved in Hank’s balanced salt solution (pH 7.4) at concentrations of 0.3125-5 μg/mL at 37 °C for 30 min. Samples were diluted in 1% polysorbate 80. A neutrophil suspension without antichemotactic agent and a solution of 1% polysorbate 80 were used as negative controls. Indomethacin (10 µg/ mL) was used as positive control.

RESULTS AND DISCUSSION

Chemical composition of essential oils

The essential oils of fresh leaves of four Ocotea species were analyzed in this study. Two specimens of O. indecora, O. puberula, and O. silvestris and one specimen of O. acutifolia were collected. Details on collection periods and localities are shown in Table I. No significant differences in essential oil yields were observed; however, there were important quantitative and qualitative variations in chemical composition (more specifically, terpene content) among species. Most samples were characterized by the presence of sesquiterpenes. Monoterpenes were not detected in the investigated species, except in the essential oil of one O. indecora specimen, which contained 16% monoterpene hydrocarbons, represented by α- and β-pinene (Figure 1). The chemical composition of essential oils obtained from leaves of Ocotea species is described in Table II.

FIGURE I
Terpenoid content in essential oils from four Ocotea species.

TABLE II
Percentage composition of essential oils extracted by hydrodistillation of fresh leaves of four Ocotea species

O. acutifolia essential oil, obtained in 0.2% yield, was orange in color and had a viscous consistency. Chemical analysis revealed the presence of five major compounds, accounting for about 94% of the total oil content. The oil was characterized by a predominance of an unsaturated tetracyclic diterpene, phyllocladene (67.7%), followed by a sesquiterpene hydrocarbon, β-selinene (18.0%), formed in the biosynthetic pathway of germacrene. Monoterpenes were not identified in this essential oil.

Silva et al. (2013Silva LDL, Da Silva DT, Garlet QL, Cunha MA, Mallmann CA, Baldisserotto B, et al. Anesthetic activity of Brazilian native plants in silvercat fish (Rhamdiaquelen). Neotrop Ichthyol. 2013;11(2):443-451.) analyzed essential oil of O. acutifolia leaves collected in Rio Grande do Sul and reported a total of 24 compounds, the major of which were oxygenated sesquiterpenes, such as caryophyllene oxide (56.9%), calarene epoxide (11.7%), and γ-elemene (8.2%). However, different from that observed in the current study, the authors identified the monoterpenes α-thujene, α-pinene, limonene, and linalool.

Herein, we report for the first time the presence of phyllocladene in Ocotea species. This compound was previously identified in samples of Cryptomeria japonica

D. Don (Cupressaceae) from Portugal (Moiteiro et al., 2013Moiteiro C, Esteves T, Ramalho I, Rojas R, Alvarez S, Bragança H. Essential oil characterization of two azorean Cryptomeria japonica populations and their biological evaluations. Nat Prod Commun. 2013;8(12):1785-1790.) and in the leaves, branches, and roots of Eryngium aquifolium Cav. (Apiaceae) from Spain (Palá-Paúl et al., 2010Palá-Paúl J, Usano-Alemany J, Brophy JJ, Pérez-Alonso MJ, Soria AC. Essential oil composition of the different parts of Eryngium aquifolium from Spain. Nat Prod Commun . 2010;5(5):817-821.).

Samples of O. puberula (OP) were collected from two close individuals under direct sunlight. Essential oil yield was 0.2% for both samples, and the color was light yellow. A total of 18 compounds were identified, representing about 94% and 82% of the total oil content of samples from collections 1 (OP1) and 2 (OP2), respectively. The sesquiterpene fraction was predominant in both samples. β-Caryophyllene (25.2%), (E)-nerolidol (13%), and spathulenol (11.8%) were the major compounds in OP1 essential oil, whereas globulol (22.6%), β-acoradiene (12.5%), and aromadendrene (11.5%) predominated in OP2 essential oil. No monoterpenes were identified in OP1 or OP2 essential oils. It is important to emphasize that all compounds identified in OP2 were also detected in OP1, indicating some level of similarity. Chemical differences between samples might have been due to seasonality and/or ontogenetic factors, such as plant age. The proximity of OP1 and OP2 might also have contributed to the slight variation in oil composition and the high sesquiterpene content of both samples. Quantitative variation in volatile oil components may be attributed to plant development stage, level of sunlight incidence, or presence of herbivores and pathogens, as terpenoids are related to plant defense.

In a previous study, De Araújo et al. (2001De Araújo AJ, Lordello ALL, Maia BHLNS. Análise comparativa dos óleos essenciais de folhas e galhos de Ocotea puberula (Lauraceae). Revista Visão Acadêmica. 2001;2(2):81-84.) reported the presence of 3 monoterpenes and 10 sesquiterpenes in the essential oil of O. puberula leaves, with β-caryophyllene and bicyclogermacrene as the most abundant compounds. Later, Raggi (2008Raggi L. Estudo da composição química e das atividades biológicas de óleos voláteis de espécies de Lauraceae, em diferentes épocas do ano. [Master’s dissertation]. São Paulo: Instituto de Botânica da Secretaria do Meio Ambiente, 2008.) examined the chemical composition of the leaf essential oil of three O. puberula individuals and reported a predominance of the sesquiterpene hydrocarbons β-caryophyllene, β-elemene, bicyclogermacrene, α-copaene, and α-humulene. The results of our study were similar to those of De Araújo et al. (2001)De Araújo AJ, Lordello ALL, Maia BHLNS. Análise comparativa dos óleos essenciais de folhas e galhos de Ocotea puberula (Lauraceae). Revista Visão Acadêmica. 2001;2(2):81-84..

O. silvestris essential oil was obtained in 0.2% yield from both specimens (OS1 and OS2). The oils were transparent and light yellow in color. Samples were collected from close individuals under direct sunlight. Bicyclogermacrene and germacrene D were the major compounds, accounting for 83.9% of the total oil content in OS1 and 100% in OS2. OS1 essential oil was found to contain 10 terpenes, which together accounted for about 92% of the total oil content. In this sample, germacrene D (54.1%) and bicyclogermacrene (29.8%) were the predominant compounds, and other oxygenated sesquiterpenes and sesquiterpene hydrocarbons were identified at smaller concentrations. OS2 oil contained only germacrene D and bicyclogermacrene, representing 100% of the total oil content.

O. indecora samples were collected at the same locality but from different individuals, one located under the shade of large trees (OI1) and the other under direct sunlight (OI2). The yields of leaf essential oils were 0.3 and 0.2% for OI1 and OI2, respectively. Oils exhibited a limpid and clear yellow aspect. A total of 24 components were identified in OI1 and OI2 oils, accounting for

99.2 and 95.2% of the total oil content, respectively. The sesquiterpene fraction was the most predominant in both samples. In OI1, bicyclogermacrene (40.7%), germacrene D (10.5%), and α-cadinol (10.2%) were the major components; only OI1 contained monoterpenic compounds. The oil of OI2 leaves, collected under direct sunlight, had the following oxygenated sesquiterpenes as the major compounds: guaiol (30.2%), α-eudesmol (27.6%), and β-eudesmol (12.7%).

Of note, OI2 oil had a higher content of oxygenated compounds than OI1 oil, suggesting that sunlight can directly influence essential oil composition. Germacrene D and bicyclogermacrene, detected in OI1 oil, may undergo rearrangements and diverse or nonenzymatic oxidation, affording different sesquiterpenoids, such as cadinene and guaiane or those with a eudesmane skeleton. These reactions can be stimulated by elevation of temperature (Bulow, Konig, 2000Bulow N, Konig WA. The role of germacrene D as a precursor in sesquiterpene biosynthesis: investigations of acid catalyzed, photochemically and thermally induced rearrangements. Phytochemistry. 2000;55(2):141-168.). We highlight that specimens were stored in a freezer until extraction and oils were stored at low temperatures until chemical analysis.

Another difference between the two samples was the presence of monoterpenes in OI1, collected in a shady habitat. Monoterpenes are derived from geranyl pyrophosphate, the initial precursor of monoterpenes, which, after some cyclization reactions, gives rise to structural compounds such as pinenes (α- and β-pinene), p-menthanes (limonene), myrcene, and acyclic compounds (Poulose, Croteau, 1978Poulose AJ, Croteau R. Y-terpinene to p-cymene and thymol in Thymus vulgaris L. Arch Biochem Biophys. 1978;187:307- 314.). No terpenes of this class were identified in OI2 essential oil.

Quantitative and qualitative chemical variability may be attributed to several factors. Biochemical and physiological changes occurring throughout plant development can modify the production of biologically active substances, thereby influencing the content and quality of essential oils. For this reason, oils do not have constant quality or quantity and are directly influenced by environmental factors, such as soil, light, temperature, radiation, water deficiency, and place and time of collection. Different combinations of soil (acidity or alkalinity), climate, topography, moisture, temperature, and sunlight conditions can result in differences in the chemical and physical characteristics of oils. This is supported by the fact that plants collected from areas under different light, heat, and temperature conditions may differ in development (Almeida et al., 2016Almeida LFR, Portella RO, Bufalo J, Marques MOM, Facanali R, Frei R. Non-oxygenated sesquiterpenes in the essential oil of Copaifera langsdorffii Desf. increase during the day in the dry season. Plos One. 2016;11(2):1-12.). Species that grow under light may suffer more from the action of herbivores and pathogens, as they are more exposed than plants grown under shade (Herms, Mattson, 1992Herms DA, Mattson WJ. The dilemma of plants: to grow or defend. Q Rev Biol. 1992;67(3):283-335.). Thus, we can infer that the qualitative difference in essential oil composition between OI1 and OI2 might be related to the adaptation of plants to their environment. Possibly, the high percentage of oxygenated compounds is associated with direct sunlight, suggesting that these compounds do not have such a high deterrent property.

Chemical characterization of essential oil from Dalbergia frutescens (Vell.) Britton (Fabaceae) and analysis of the influence of environmental conditions revealed that essential oil yield was strongly affected by environmental conditions, being directly proportional to temperature and inversely proportional to cloudiness and rainfall (Mendes et al., 2012Mendes CE, Casarin F, Ohland AL, Flach A, Costa LAMA, Denardin RBN, et al. Efeitos das condições ambientais sobre o teor e variabilidade dos óleos voláteis de Dalbergia frutescens (Vell.) Britton (Fabaceae). Quim Nova. 2012;35(9):1787-1793.). The concentrations of linalool, α-ionone, and β-ionone, the major compounds of essential oil extracted in the spring, were directly influenced by temperature and sunlight. It was also observed that autumn and winter specimens were characterized by the presence of β-damascenone and geranyl acetone, compounds that, at first, were not related to environmental conditions (Mendes et al., 2012Mendes CE, Casarin F, Ohland AL, Flach A, Costa LAMA, Denardin RBN, et al. Efeitos das condições ambientais sobre o teor e variabilidade dos óleos voláteis de Dalbergia frutescens (Vell.) Britton (Fabaceae). Quim Nova. 2012;35(9):1787-1793.).

Antifungal activity

Antifungal activity was assessed against yeasts and filamentous fungi by the broth microdilution method. For this assay, oils obtained from O. acutifolia, O. indecora (OI1 and OI2), O. puberula (OP2), and O. silvestris (OS1) were tested at 500 µg/mL. In the screening assay, samples were tested against four species of filamentous fungi (M. canis, M. gypseum, T. mentagrophytes, and T. rubrum) and six species of yeasts (C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, and C. neoformans). Oils did not inhibit the growth of yeasts or fungi, except that of C. parapsilosis, whose growth was inhibited by all samples.

Given this result, the MIC values of essential oils were determined against C. parapsilosis isolates. Filamentous fungal strains were not analyzed because the oils did not show inhibitory activity at the tested concentration. MIC was determined by the broth microdilution method against five strains of C. parapsilosis (RL07, RL11, RL13, RL27, and RL52). All tested essential oils showed a MIC of 500 µg/mL.

According to Burt (2004Burt S. Essential oils: their antibacterial properties and potential applications in foods- a review. Int J Food Microbiol. 2004;94(3):223-253.), essential oils comprise a large number of chemical compounds, which is why their mechanism of action involves several targets in microbial cells. It is well accepted that the lipophilicity of essential oil constituents is the main factor responsible for antimicrobial properties, given that this characteristic can facilitate the interaction of compounds with cell membrane lipids and mitochondria, increasing membrane permeability and leading to leakage of cellular contents (Cowan, 1999Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev. 1999;12(4):564-582.). Nevertheless, as argued by Deuschle et al. (2007Deuschle RAN, De Camargo T, Alves SH, Mallmann CA, Heizmann BM. Fracionamento do extrato diclorometânico de Senecio desiderabilis Vellozo e avaliação da atividade antimicrobiana. Rev Bras Farmacogn. 2007;17(2):220-223.), this might not be the sole mechanism involved in the antimicrobial activity of essential oils. For example, germacrene D, a sesquiterpene hydrocarbon, exhibited pronounced lipophilic characteristics but did not inhibit microbial growth at concentrations lower than 500 µg/ mL. This result corroborates the findings of Biavatti et al. (2001Biavatti MW, Vieira PC, Da Silva MFGF, Fernandes JB, Albuquerque S, Magalhães CMI, et al. Chemistry and bioactivity of Raulinoa echinata Cowan, an endemic Brazilian Rutaceae species. Phytomedicine. 2001;8(2): 121-124.), who observed a lack of antimicrobial activity against different microorganisms.

Raggi (2008Raggi L. Estudo da composição química e das atividades biológicas de óleos voláteis de espécies de Lauraceae, em diferentes épocas do ano. [Master’s dissertation]. São Paulo: Instituto de Botânica da Secretaria do Meio Ambiente, 2008.) determined the antifungal activity of O. puberula essential oil by bioautography on silica gel plate and observed low activity against Cladosporium cladosporioides and C. sphaerospermum. The author also assessed the antifungal activity of three O. puberula specimens (collected at different times of the year) against strains of Aspergillus niger and C. albicans. The results showed no change in antifungal activity against A. niger between individuals or seasons, with a mean inhibition percentage of 67%. Essential oils did, however, differ in their antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The author suggested that this behavior was probably due to quantitative variations in oil composition. Against S. aureus, the essential oil of leaves collected in early autumn was the most active (94.4% inhibition), whereas the essential oil of leaves sampled in early spring was the least active. Plants collected in summer and early autumn produced essential oils that afforded a 70% inhibition of P. aeruginosa.

Essential oils containing germacrene B, the precursor of germacrene D (the major constituent in some samples of the current study), demonstrated fungitoxic activity (Fach et al., 2002Fach A, Gregel B, Simionatto E, Da Silva UF, Zanatta N, Morel AF, et al. Chemical analysis and antifungal activity of the essential oil of Calea clematidea. Planta Med . 2002;68(9):836-838.). Oils containing the sesquiterpene hydrocarbon bicyclogermacrene also demonstrated antimicrobial activity (Constantin et al., 2001Constantin MB, Sartorelli P, Limberger R, Henriques AT, Steppe M, Ferreira MJP, et al. Essential oils from Piper cernuum and Piper regnellii: antimicrobial activities and analysis by CG/MS and 13C-NMR. Planta Med. 2001;67(8):771-773.; Cysne et al., 2005Cysne JB, Canuto KM, Pessoa ODL, Nunes EP, Silveira ER. Leaf essential oils of four Piper species from the State of Ceará - Northeast of Brazil. J Braz Chem Soc. 2005;16(6B):1378-1381.). The oxygenated sesquiterpene spathulenol was shown to exhibit important antibacterial properties and moderate cytotoxic activity (Santos et al., 2012Santos RF, Isobe MTC, Lalla JG, Haber LL, Marques MOM, Ming LC. Chemical composition and productivity of essential oil of Baccharis dracunculifolia DC. as affected by organic compound. Rev Bras Plant Med. 2012;14:224-234.). It is important to highlight that each component contributes differently to the biological activity of essential oils (Daferera, Ziogas, Polissioi, 2003Daferera DJ, Ziogas BN, Polissioi MG. The effectiveness of plant essential oil on the growth of Botrytis cinerea, Fusarium sp. and Clavibacter michiganensis subsp. michiganensis. Crop Prot. 2003;22(1):39-44.). Although the antimicrobial activity of an essential oil is attributed to its major compounds, the synergistic and antagonistic effects of those occurring at lower concentrations should be considered, possibly explaining a poorly pronounced biological effect.

Antichemotactic activity

The ability of essential oils to inhibit leukocyte migration was determined using the Boyden chamber method. Essential oils were tested at 0.3125-5 μg/mL. Indomethacin, used as positive control, inhibited 62.9% of leukocyte migration at the tested concentration tested (10 μg/mL). None of the samples demonstrated antichemotactic activity compared with the negative control.

Previous research showed that Ocotea essential oils exert anti-inflammatory effects. O. quixos essential oil was found to significantly inhibit lipid polysaccharide-induced cyclooxygenase-2 expression and carrageenan- induced edema in the rat paw (Ballabeni et al., 2010Ballabeni V, Tognolini M, Giorgio C, Bertoni S, Bruni R, Barocelli E. Ocotea quixos Lam. essential oil: In vitro and in vivo investigation on its anti-inflammatory properties. Fitoterapia. 2010;81(4):289-295.). Of note, the oil was characterized by the predominance of trans-cinnamaldehyde and methyl cinnamate, whose anti-inflammatory action has not yet been elucidated.

CONCLUSION

In this study, essential oils obtained from different Ocotea species showed important qualitative and quantitative variations in chemical composition, primarily in the sesquiterpene fraction. The main compounds identified were β-caryophyllene, germacrene D, bicyclogermacrene, globulol, α-eudesmol, guaiol, (E)- nerolidol, and phyllocladene. The last compound was described for the first time in the genus Ocotea. All tested essential oils exhibited antifungal activity against C. parapsilosis. These findings contribute to the knowledge of the chemical composition and biological activities of Ocotea species.

ACKNOWLEDGEMENTS

The authors are grateful to the National Institute of Science and Technology in Biodiversity and Natural Products (INCT-BioNat), the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), and the Brazilian National Council for Scientific and Technological Development (CNPq) for the financial support.

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Publication Dates

  • Publication in this collection
    20 Apr 2022
  • Date of issue
    2022

History

  • Received
    03 Oct 2018
  • Accepted
    03 Feb 2021
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br