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

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.


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., 2011).
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., 2009a). 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., 2017;Blažeković et al., 2018;Danielli et al., 2018).

Biological activities of essential oils from six genotypes of four Ocotea species
Page 2/11 Braz. J. Pharm. Sci. 2022;58: e181097 Michele A. Rambo, Krissie D. Soares, Letícia J. Danielli, Daiane F. D. Lana, Sérgio A. L. Bordignon, Alexandre M. Fuentefria, Miriam A. Apel 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., 2011). 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, 2008;Mastelic et al., 2008).
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, 1996;Bannister, Conran, Lee, 2012). 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, 2001;Baitello et al., 2003).
Several studies assessed the biological activities of Ocotea essential oils. Important properties were identified, such as anti-inflammatory (Ballabeni et al., 2010;Leporatti et al., 2014), antithrombotic (Ballabeni et al., 2007), cardiovascular (Barbosa-Filho et al., 2008), antimicrobial (Leporatti et al., 2014, Da Silva et al., 2017, and cytotoxic (Da Silva et al., 2017). Other studies investigated associations between groups of compounds and cytotoxicity, such as Garcez et al. (2011), 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., 2013). It was also found that the chloroform fraction of O. puberula fruits containing the alkaloid dicentrine demonstrated antinociceptive effects (Montrucchio et al., 2012).
This study aimed to determine the chemical composition and assess the in vitro antifungal and antiinflammatory 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.

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.  (CLSI, 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. (2011). 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.

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 Essential oil extraction Essential oils from Ocotea species were obtained from fresh material by hydrodistillation using a Clevengertype apparatus for 4 h, according to the procedures described in the Brazilian Pharmacopoeia (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, 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  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.   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. (2001) 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 (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).
O. silvestris essential oil was obtained in 0.2% yield from both specimens (OS1 and OS2). The oils 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. (2013) 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., 2013) and in the leaves, branches, and roots of Eryngium aquifolium Cav. (Apiaceae) from Spain (Palá-Paúl et al., 2010).
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 Page 7/11 Biological activities of essential oils from six genotypes of four Ocotea species 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, 2000). 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, 1978). 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., 2016). 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, 1992). 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., 2012). 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., 2012). Michele A. Rambo, Krissie D. Soares, Letícia J. Danielli, Daiane F. D. Lana, Sérgio A. L. Bordignon, Alexandre M. Fuentefria, Miriam A. Apel

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 (2004), 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, 1999). Nevertheless, as argued by Deuschle et al. (2007), 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. (2001), who observed a lack of antimicrobial activity against different microorganisms. Raggi (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., 2002). Oils containing the sesquiterpene hydrocarbon bicyclogermacrene also demonstrated antimicrobial activity (Constantin et al., 2001;Cysne et al., 2005). The oxygenated sesquiterpene spathulenol was shown to exhibit important antibacterial properties and moderate cytotoxic activity . It is important to highlight that each component contributes differently to the biological activity of essential oils (Daferera, Ziogas, Polissioi, 2003). 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-Page 9/11 Biological activities of essential oils from six genotypes of four Ocotea species induced cyclooxygenase-2 expression and carrageenaninduced edema in the rat paw (Ballabeni et al., 2010). 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.