Chemical Diversity, Biological Activity, and Genetic Aspects of Three Ocotea Species from the Amazon

Ocotea species present economic importance and biological activities attributed to their essential oils (EOs) and extracts. For this reason, various strategies have been developed for their conservation. The chemical compositions of the essential oils and matK DNA sequences of O. caudata, O. cujumary, and O. caniculata were subjected to comparison with data from O. floribunda, O. veraguensis, and O. whitei, previously reported. The multivariate analysis of chemical composition classified the EOs into two main clusters. Group I was characterized by the presence of α-pinene (9.8–22.5%) and β-pinene (9.7–21.3%) and it includes O. caudata, O. whitei, and O. floribunda. In group II, the oils of O. cujumary and O. caniculata showed high similarity due amounts of β-caryophyllene (22.2% and 18.9%, respectively). The EO of O. veraguensis, rich in p-cymene (19.8%), showed minor similarity among all samples. The oils displayed promising antimicrobial and cytotoxic activities against Escherichia coli (minimum inhibitory concentration (MIC) < 19.5 µg·mL−1) and MCF-7 cells (median inhibitory concentration (IC50) ≅ 65.0 µg·mL−1), respectively. The analysis of matK gene displayed a good correlation with the main class of chemical compounds present in the EOs. However, the matK gene data did not show correlation with specific compounds.


Introduction
The genus Ocotea is not a monophyletic group of the Lauraceae, which includes about 400 species occurring mostly in tropical and subtropical regions (Central and South America, the West Indies, and Africa) [1,2]. These species present alternate penninerved leaves; inflorescenses thyrsopaniculate to botryoid. Flowers are trimerous, bisexual, polygamous, or unisexual; tepals equal, rarely persistent on the rim; nine fertile stamens, the third whorl with glands; anthers with four loculos; receptacle very small and deeply tubular; in male flowers, rudimentary ovary to absent; fruit and cupule extremely variable in size and shape [1]. It is a very variable genus morphologically, being the largest genus in the Neotropics, with 170 species occurring in Brazil [3,4].
The economic importance of Ocotea species in the Amazon region has been related to numerous applications such as the use of their wood in lightweight construction and luxury furniture [5]. 2 of 15 Phytochemical studies reported the occurrence of benzylisoquinoline alkaloids, neolignans, catechins from leaves and bark of O. porosa [6,7]; aporphine alkaloids from leaves of O. macrophylla; flavonols from O. vellosiana; and eudesmane sesquiterpenoids from O. corymbosa [8][9][10]. The volatile chemical profiles of Ocotea species are characterized by high concentrations of phenylpropanoids and terpenoids (hydrocarbons or oxygenated) [11,12].
Many studies have been reported on the biological activities of Ocotea metabolites: the alkaloid reticuline isolated from extract of O. duckei showed potent central nervous system depressant action [13]. (−)-Caaverine, a noraporphine alkaloid isolated from O. lancifolia, has shown high antiprotozoal activity against Leishmania and Trypanosoma cruzi parasites [14]. The chloroform fraction obtained from an extract of fruits of O. puberula and the alkaloid dicentrine displayed antinociceptive effects [15]. The butanolides isolated from roots of O. macrocarpa showed good cytotoxic activities against the A2780 ovarian cell line [16]. The flavonoids of O. notata showed antimycobacterial activity and ability to inhibit NO production by macrophages [17]. The essential oil of O. quixos, rich in trans-cinnamaldehyde and methyl cinnamate, showed anti-inflammatory properties in vitro and in vivo models [18]. Studies on the chemical characteristics of the species O. caudata, O. cujumary, and O. caniculata are rare and important because Ocotea species are classified as threatened to extinction by the Brazilian List and the risk of extinction is increased due the reduction of genetic variability [19,20] so knowledge of the genetic diversity is necessary for our understanding of the factors that determine essential oil quantity and quality in these economically important species [21].

Antimicrobial and Cytotoxic Activities
All essential oils displayed high antimicrobial activity against E. coli (minimum inhibitory concentration (MIC) < 19.5 µg·mL −1 ). Cau-L and Cuj-L oils showed notable activity against S. epidermis and B. cereus (Table 3). It is not obvious what component(s) are responsible for the activity against E. coli. Most essential oil components show only marginal activity against this organism. The antibacterial activities against the other bacteria are consistent with the activities of the essential oil components (Table 3). On the other hand, the cytotoxic activity against MCF-7 cells did not display variation in the median inhibitory concentration (IC50) values among all samples with an average of 63.0 µg·mL −1 ( Table 3). The main compounds germacrene D, bicyclogermacrene, β-caryophyllene, αpinene, β-pinene, caryophyllene oxide, β-selinene, 7-epi-α-selinene have been reported as antimicrobial and cytotoxic [29][30][31][32][33]. The observed cytotoxicities of some of the essential oil components are consistent with the cytotoxicities of the essential oils themselves (Table 3).

Antimicrobial and Cytotoxic Activities
All essential oils displayed high antimicrobial activity against E. coli (minimum inhibitory concentration (MIC) < 19.5 µg·mL −1 ). Cau-L and Cuj-L oils showed notable activity against S. epidermis and B. cereus (Table 3). It is not obvious what component(s) are responsible for the activity against E. coli. Most essential oil components show only marginal activity against this organism. The antibacterial activities against the other bacteria are consistent with the activities of the essential oil components (Table 3). On the other hand, the cytotoxic activity against MCF-7 cells did not display variation in the median inhibitory concentration (IC 50 ) values among all samples with an average of 63.0 µg·mL −1 ( Table 3). The main compounds germacrene D, bicyclogermacrene, β-caryophyllene, α-pinene, β-pinene, caryophyllene oxide, β-selinene, 7-epi-α-selinene have been reported as antimicrobial and cytotoxic [29][30][31][32][33]. The observed cytotoxicities of some of the essential oil components are consistent with the cytotoxicities of the essential oils themselves (Table 3).

Phylogenetic Analysis
MatK from chloroplast genes has been used as a marker for the construction of plant phylogenies, due to its rapid evolution and their ubiquitous presence in plants [40]. The sequences have a size of about 1550 bp and encode the maturase K enzyme [41].
Lauraceae species used in this study formed a defined group in the phylogenetic analysis, and the results of the phylogenetic tree were robust and supported by bootstrap value (=100). Species of Calycanthaceae were used as outgroup; this corroborates with the taxonomic classification and supports the topology of the tree (Figure 3). bicyclogermacrene, α-pinene, and β-pinene against MCF-7 cells were 19.0, 30.7, and 80.2 µg/mL, respectively [24,39].

Phylogenetic Analysis
MatK from chloroplast genes has been used as a marker for the construction of plant phylogenies, due to its rapid evolution and their ubiquitous presence in plants [40]. The sequences have a size of about 1550 bp and encode the maturase K enzyme [41].
Lauraceae species used in this study formed a defined group in the phylogenetic analysis, and the results of the phylogenetic tree were robust and supported by bootstrap value (=100). Species of Calycanthaceae were used as outgroup; this corroborates with the taxonomic classification and supports the topology of the tree (Figure 3).  The genetic distances estimated for Lauraceae species were lower. The values ranged from 0 to 0.003 to Cau-L, Cuj-L, Can-L and from 0 to 0.009 to Ver-L, Flo-L and Whi-L.
The tree shows that the geographically closest species are grouped: Cau-L, Cuj-L, and Can-L were collected in Caxiuanã National Forest and Ver-L, Flo-L, and Whi-L were collected in Monteverde, Costa Rica. Although there may be differences in the composition of essential oils in plants of the same species with the same geographical location, the results of phylogenetic tree showed a good correlation with classes of chemical compounds. The species Cau-L and Can-L are rich in sesquiterpene hydrocarbons and the samples Cuj-L, Ver-L, and Whi-L showed similar amounts of monoterpene hydrocarbons. A higher difference could be observed for sample Flo-L, which had diterpenes as its main compound class. Our results support the hypothesis that matK gene is reasonably useful in phylogenetic reconstructions at high taxonomic levels (to order or family), but shows poorer reliability with lower taxonomic levels of classification (to genus or species) [42,43].  (Table 1).

Essential Oil Extraction
Leaves and branches were air-dried, pulverized, and subjected to hydrodistillation using a Clevengertype apparatus (100 g, 3 h). The essential oils were dried over anhydrous sodium sulfate, and the essential oil yields were calculated on the basis of the dry weight of plant material. The moisture contents of the samples were calculated after phase separation using a Dean-Stark trap (5 g, 60 min) using toluene as the solvent phase.

Gas Chromatographic-Mass Spectral Analysis
The volatile compositions were analyzed by gas chromatography using an Agilent 6890 GC with an HP-5 ms column, and mass spectrometry with an Agilent 5973 mass selective detector (MSD) operated in the electron impact (EI) mode with electron energy = 70 eV. The scan range was 40-400 atomic mass units (amu) and the scan rate was 3.99 scans/s. The data were processed with an Agilent ChemStation data system. The gas chromatography (GC) column was a fused silica capillary with a (5% phenyl)-polymethylsiloxane stationary phase that had a film thickness of 0.25 µm and a length of 30 m, and an internal diameter of 0.25 mm. Helium was the carrier gas with a flow rate of 1.0 mL/min and a column head pressure of 48.7 kPa. The injector temperature was 200 • C and the detector temperature was 280 • C. The GC oven temperature was programed to start with an initial temperature of 40 • C, which was held for 10 min. The temperature was then increased at a rate of 3 • C/min to 200 • C, and then increased at 2 • C/min to 220 • C. A 1-µL injection of a solution (0.2% w/v in CH 2 Cl 2 ) of the sample was performed using a splitless injection technique. The percentages of each component were based on total ion current and are reported without standardization. Individual components were identified by comparison of both their mass spectra and GC retention data with authentic compounds present in commercial libraries [44] and our own in-house library.

Antibacterial Assay
The antimicrobial activity of EOs was determined against Bacillus cereus (ATCC No. 14579), Escherichia coli (ATCC No. 10798), Pseudomonas aeruginosa (ATCC No. 27853), Staphylococcus aureus (ATCC No. 29213), and Staphylococcus epidermidis (ATCC No. 12228), using the microbroth serial dilution method as previously reported [45]. Thus, 50 µL of 1% w/v solution of the samples in dimethylsulfoxide (DMSO) was placed in the top well of 96-well microtiter plates and 50 µL of cation-adjusted Mueller Hinton broth (CAMHB) was added. The sample solutions were then serially diluted (1:1) by transferring 50 µL of sample-CAMHB mixture to the next lane and adding 50 µL fresh CAMHB to obtain a concentration range from 2500 to 12.5 µg·mL −1 . The bacteria were harvested from a fresh culture and added to each well at a concentration of approximately 1.5 × 10 8 colony forming units (CFU)·mL −1 . The plates were incubated at 37 • C for 24 h and the final minimum inhibitory concentrations (MICs) were determined as the lowest concentrations free of turbidity. The antibiotic gentamicin was used as positive control and DMSO solvent was used as negative control.

Cytotoxicity Assay
MCF-7 human mammary adenocarcinoma cells (ATCC No. HTB-22) were cultured in RPMI (Roswell Park Memorial Institute) 1640 medium supplemented with 10% fetal bovine serum (FBS), 30 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer), NaHCO 3 , and penicillin streptomycin. The cytotoxicity of the essential oils on MCF-7 cells was determined using the 96-well MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as reported previously [45]. Cells were seeded into 96-well cell culture plates at a concentration of 1.2 × 10 4 cells/well and a volume of 100 µL in each well. The plate was then labeled and incubated at 37 • C and 5% CO 2 for 48 h. After 48 h, the cells reached 70-80% confluent growth. The supernatant fluid was gently aspirated (without touching the bottom of the well to avoid removing cells) and replaced with 100 µL growth medium that contained either 1.0 or 0.5 µL of essential oil (1% in DMSO), to give final concentrations of 100 and 50 µg·mL −1 . The plate was then incubated at 37 • C and 5% CO 2 for 48 h. After 48 h, the liquid was gently aspirated from each well. In a tube, 10 mL feeding medium was mixed with 2 mL of MTT stock solution (and was protected from light). Into each well, 100 µL of the MTT solution was added and the pre-read absorbance was immediately measured spectrophotometrically at 570 nm (using a Molecular Devices Spectra Max Plus 384 microplate reader). Formazan crystals were formed over the course of 4 h at 37 • C and 5% CO 2 . After 4 h, DMSO was used to dissolve the purple crystals. The amount of MTT-formazan produced was determined spectrophotometrically at 570 nm. Growing medium, DMSO, and tingenone (100 µg·mL −1 ) served as negative, compound, and positive controls, respectively. Solutions were added to wells in eight replicates. Average absorbances, standard deviations, and percent kill ratios (% kill oil /% kill control ) were calculated. Median inhibitory concentrations (IC 50 ) were determined using the Reed-Muench method [46].

Multivariate Statistical Analysis of Chemical Composition
Hierarchical cluster analysis was carried out to organize and cluster the essential oils according to their main volatile constituents. Complete linkage and absolute correlation coefficient distance was chosen to determine similarity. Agglomerative hierarchical clustering was utilized for clustering the essential oils. The MINITAB 14.0 software was used to statistically analyze all data.

Conclusions
In this present study, the main compounds identified in the essential oils of species collected in Caxiuanã Forest were terpenoid hydrocarbons and oxygenated terpenoids, which had good antimicrobial and cytotoxic activities. The main class of chemical compounds displayed a good correlation with chloroplast DNA region (matK gene) analysis. However, the analysis of phylogenetic data and specific chemical compounds showed differences. These results suggest that matK gene was not efficient for representing this relationship.