Chemical Diversity and Biological Activities of Essential Oils from Licaria, Nectrandra and Ocotea Species (Lauraceae) with Occurrence in Brazilian Biomes

Lauraceae species are known as excellent essential oil (EO) producers, and their taxa are distributed throughout the territory of Brazil. This study presents a systematic review of chemical composition, seasonal studies, occurrence of chemical profiles, and biological activities to EOs of species of Licaria, Nectandra, and Ocotea genera collected in different Brazilian biomes. Based on our survey, 39 species were studied, with a total of 86 oils extracted from seeds, leaves, stem barks, and twigs. The most representative geographic area in specimens was the Atlantic Forest (14 spp., 30 samples) followed by the Amazon (13 spp., 30 samples), Cerrado (6 spp., 14 samples), Pampa (4 spp., 10 samples), and Caatinga (2 spp., 2 samples) forests. The majority of compound classes identified in the oils were sesquiterpene hydrocarbons and oxygenated sesquiterpenoids. Among them, β-caryophyllene, germacrene D, bicyclogermacrene, caryophyllene oxide, α-bisabolol, and bicyclogermacrenal were the main constituents. Additionally, large amounts of phenylpropanoids and monoterpenes such as safrole, 6-methoxyelemicin, apiole, limonene, α-pinene, β-pinene, 1,8-cineole, and camphor were reported. Nectandra megatopomica showed considerable variation with the occurrence of fourteen chemical profiles according to seasonality and collection site. Several biological activities have been attributed to these oils, especially cytotoxic, antibacterial, antioxidant and antifungal potential, among other pharmacological applications.


Introduction
Lauraceae is one of the most primitive angiosperm families. It belongs to the subclass Magnoliidae and order Laurales [1]. Lauraceae species have the reputation of being difficult to identify because several collections are sterile or fruiting but lack the floral characters needed for identification [2]. This family of flowering species is widely distributed in regions of tropical and subtropical climates with more than 2500 species [3].
Brazil contains six areas of biomes: Amazon, Atlantic Forrest, Cerrado, Caatinga, Pantanal, and Pampa. The Amazon biome covers 49.3% of the Brazilian territory and has an extension of

Distribution of Main Compound Classes in Essential Oil Samples
In this section, the oils were classified based on the percentage of the most abundant chemical compound class. Thus, the oils were found to be rich in monoterpene hydrocarbons, sesquiterpene hydrocarbons, oxygenated sesquiterpenoids, phenylpropanoids, and benzenoids. Some EO displayed the main compounds that belonged to different classes than the majority in the oils. For example, the oil of Ocotea bicolor Vattimo-Gil collected in Curitiba (PR, Brazil) exhibited a predominance of sesquiterpene hydrocarbons (48.77%). However, the phenylpropanoid dillapiole (15.2%) in combination with δ-cadinene (20.0%), α-cubebene (6.5%), and α-copaene (5.1%) were the main compounds. The distribution of compound classes, according to its respective biome, can be visualized in Figure 2.

Oils Rich in Sesquiterpene Hydrocarbons
As a class, the sesquiterpene hydrocarbons are very well represented in Lauraceae essential oils, especially the caryophyllane, humulane, germacrane, and selinane skeletons.
Compounds with the germacrane skeleton, such as bicyclogermacrene (33.4%) and germacrene D (16.8%), were predominant in the oil of N. megapotamica collected in Barracão (RS, Brasil). The content of sesquiterpene hydrocarbons was 79.60% [31]. In another study, the chemical composition during the different maturation stages of N. megapotamica collected in Santa Maria (RS) was evaluated. The oils showed a content of sesquiterpene hydrocarbons of 59.75% and 49.97% in young and adult plants, respectively. The main compounds identified were bicyclogermacrene (46.47%, 34.56%) and germacrene D (9.61%, 9.2%) [32]. In addition, bicyclogermacrene (28.44%) and germacrene A (7.34%) were the main compounds in the oil of leaves of Nectandra leucantha Nees & Mart collected in Cubatão (SP). The total of sesquiterpene hydrocarbons in this sample was 58.78% [33].

Oils Rich in Benzenoids
The chemical composition of the oil from the leaves of Licaria canella (Meissn.) Kosterm collected in Manaus (AM) showed a predominance of benzyl benzoate (71.35%) [56].

Occurrence of Different Chemical Profiles
The chemical composition varies among specimens of the same species of Licaria, Nectandra and Ocotea; the oils and the combination were characterized by their chemical profiles, which are based on the concentrations of the major components. These different chemical profiles may be associated with respect to ecological and geographical condition, age of plant and time of harvesting [24,32,50].

Seasonal Variation in the Volatile Constituents
Several studies on Lauraceae species have shown that changes in the chemical composition and yield of EO can be affected by humidity, temperature, seasonality, luminosity, photoperiod, geographic variations, plant age, tissue collected and phenologic stages [24,47,50]. The variations in the chemical composition in the oil from the leaves presented in this study are illustrated in Figure 3.
The seasonality and phenological aspects influenced in the EO production of N. megapotamica can probably be attributed to morphological parameters such as alterations in the leaves and metabolites due to environmental adaptation (pollinator attraction, seed dispersers, defense against herbivory and pathogens). Juvenile and mature leaves of N. megapotamica were collected in the city Morro do Elefante (Santa Maria, RS, Brasil) during the different seasons. Leaves collected in the spring, the season that includes flowering, fruiting, and foliation, displayed the higher EO yield with a percentage of 0.59% and 0.30% in juvenile and mature leaves, respectively. The range of leaf oil yield was lower (0.21-0.28%) in the autumn, the period in which the plant is in vegetative and reproductive rest, and of abscission of the vegetal organs for the winter [24].
The chemical composition and yield of EOs of leaves, fruits, and inflorescences of O. lancifolia collected in the district of Santo Antão (Santa Maria, RS) were evaluated according to climate changes during a year. Oxygenated sesquiterpenoids were predominant during all periods in the leaves (79.2%), inflorescences (81.3%), and in the fruits (69.1%). A variation of chemical composition and oil yield was observed in the samples collected between August and November and in the period from May to July. These periods are related to ripening and attack by pathogens in plants [50]. A higher yield from the leaf EOs was observed in the spring (1.03%) and the summer (0.96%) in contrast to those obtained in the winter (0.56%) and autumn (0.6%). The lowest EO production per month was observed in May (0.27%) and July (<0.1%). Caryophyllene oxide (46.4-36.4%), bicyclogermacrene (7.8-6.1%), and allo-himachalol (8.0-5.7%) were the main compounds, except in May and July, which presented β-chenopodiol (20.9%, 17.4%), (Z)-nerolidyl acetate (9.3%, 8.7%) and kaurene (11.9%, 17.1%) [50].
The oil yield from the leaves N. grandiflora and different tissues of O. odorifera showed significant variation according to seasonality. The collection sites for the samples were Jaguari (RS) and Viçosa (MG), respectively [59,60]. N. grandiflora displayed higher EO production during the spring (0.75%) and the lower yield in the winter (0.39%) [59]. Regarding O. odorifera oils, the higher EO production was observed in the summer for leaves (0.86%) and during the spring for twigs (0.9%) and bark (1.37%) [60]. These studies did not report information on EO chemical composition, however.

Biological Activities
All of the studies on biological activities of EOs of Licaria, Nectandra, and Ocotea species collected in Brazil corresponded to a total of 60 oils. Among them, six samples had no chemical composition reported. Several oils presented more than one specific activity, and the most frequent were cytotoxic, antibacterial, antioxidant, and antifungal activities. The percentages of the reported bioactivities and details of biological assays are present in Figure 4 and Table 2, respectively.

Antibacterial Activity
The antibacterial activity of several species was evaluated by the disc diffusion method. The leaf EO of O. odorifera collected from Marcelino Ramos (RS) were tested against seventeen bacterial strains: Enterococcus faecalis, Micrococcus luteus, Sarcina sp., Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans (Gram-positive) and Acinetobacter sp., Aeromonas sp., Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Salmonella choleraesuis, Serratia marcescens, Shigella flexneri and Yersinia enterocolitica (Gram-negative). The oil was tested at volumes varying from 5.0 to 20.0 µL where chloramphenicol (30.0 µg) was used as positive control. EO major components were safrole (40.23%), camphor (34.35%) and limonene (7.42%). In general, a higher potential was observed for Gram-negative (8.40-15.40 mm) than for Gram-positive bacteria (7.90-11.80 mm). Unfortunately, minimum inhibitory concentrations (MIC) were not determined [54] Furthermore, leaves of L. puchury-major from Borba (AM) were tested against Pseudomonas aeruginosa, E. coli, Streptococcus agalactiae and S. aureus. This species of EO was composed mainly of safrole (39.40%), 1,8-cineole (28.00%) and sabinene (8.50%). The plant exhibited antibacterial activity against S. agalactiae and S. aureus with zones of inhibition of 12.0 and 13.0 mm, respectively. No MIC values and standard were reported [52]. The leaf EO of O. nonata was tested against five bacteria strains (Staphylococcus aureus, S. epidermidis, Enterococcus faecalis and E. coli). Moderate activity was observed against S. aureus with inhibition zones of 12.0 mm, and against S. epidermidis and E. faecalis with inhibition halos of 10 mm. EO composition, MIC values and standard were not reported [42].

Antifungal Activity
The leaf EO of N. lanceolata was mainly composed of β-caryophyllene (32.50%), bicyclogermacrene (27.80%) and spathulenol (11.80%). On the other hand, N. megapotamica was represented by bicyclogermacrene (33.40%), germacrene D (16.80%) and limonene (14.10%). Both species were collected in Barracão (RS) and had moderate activity against the dermatophytes Trichophyton rubrum, T. mentagrophytes, Microsporum canis and M. gypseum (MIC 250-500µg/mL). The assays were performed by the microdilution method, and terbinafine was applied as reference standard (MIC 0.004-0.016µg/mL). In addition, a combination of each oil with ciclopirox was evaluated regards its synergistic effect. The interaction was defined quantitatively as a fractional inhibitory concentration (FIC). The synergism was indicated when FIC values were below 0.5. The results indicated that the N. lanceolata EO with ciclopirox had a synergistic effect (FICI 0.375) for T. rubrum (TRU43) and M. canis (MCA29), which means that the concentration of the active antifungal agent can be reduced when in combination with the EO [31].
Different concentrations of EOs of leaves of N. grandiflora from Jaguari (RS) were tested on the growth of Pycnoporus sanguineus (white-rot fungus) and Gloeophyllum trabeum (brown-rot fungus). The oil was dominated by dehydrofukinone (26.85%), valencene (6.89%) and kaurene (6.03%). The oil exhibited a LC 50 (Lethal concentration is the amount of the oil required to kills 50% of the larvae) of 0.39 µL/mL against the fungus G. trabeum and a LC 50 of 1.22 µL/mL against P. sanguineus. The bioactivity can be explained by the presence of the major compound dehydrofukinone. In a parallel experiment, this compound was isolated and had its antifungal activity evaluated. It showed mycelial inhibition ranging from 76.06% and 79.45% in comparison to the pure EO with 80.56%. The assay was performed by the radial growth technique, but no reference standard was reported [45].
The antifungal effect of leaves of Ocotea species from Borba (AM) was also evaluated by the disc diffusion method. L. puchury-major showed strong inhibitory effect against some fungi species frequently found in hospitals and potentially responsible for opportunistic mycoses such as Rhodotorula spp., Candida albicans, Fusarium spp., Alternaria spp. and mixed molds with zones of inhibition varying from 31.0 to 37.30 mm. The highest effect was found for Aspergillus fumigatus with a halo of 64.30 mm diameter. The EO composition and MIC values were not reported. The authors used 6-mm sterile paper disks containing 15 µL of each EO. Zones of inhibition ≥20 mm were considered strongly inhibitory [62]. A different specimen of L. puchury-major had its activity evaluated. The EO was mainly composed of safrole (39.40%), 1,8-cineole (28.0%) and sabinene (8.50%). Pure oil indicated strong antifungal potential (29.0 and 40.0 mm) against two yeast species (Rhodotorula sp. and Candida sp.) and a mixture of molds. A paper disc without oil was used as negative control. MIC values and reference standards were not mentioned in the manuscript [52].

Reduction of Motor and Anesthetic Activity
The leaf EO of O. acutifolia from São Francisco de Assis (RS) was mainly composed of caryophyllene oxide (56.90%), calarene epoxide (11.74%), and τ-elemene (8.17%). Anesthesia induction and recovery was evaluated in silver catfish (Rhamdia quelen) in six stages: light and deep sedation, partial and total loss of equilibrium, deep anesthesia and medullar collapse. Anesthesia was reached with 300-900 µL/L (13-18 min) of oil, and recovery time was greater than 30 min. In addition, blood glucose levels were evaluated since they are a common indicator of stress response. The EO of O. acutifolia (150 µL/L) promoted an increase in blood glucose level. The long induction and recovery times can likely be attributed to the hydrophobic characteristics of the EO [49].

Chemical Composition-Geographic Distribution Correlation
A multivariate statistical analysis was performed in order to find chemical markers according to geographic occurrence of Lauraceae species. The total percentage of compound classes (monoterpene hydrocarbons (MH), oxygenated monoterpenoids (OM), sesquiterpene hydrocarbons (SH), oxygenated sesquiterpenoids (OS) and phenylpropanoids (PP) for each of the leaf oils was used as variables. The data matrix was standardized by subtracting the mean from individual value of each compound and then subtracted it by the standard deviation. The values were submitted to Hierarchical Cluster Analysis (HCA) the Euclidian distance and complete linkage and absolute correlation coefficient distance were selected as a measure of similarity using the Minitab software (free 390 version, Minitab Inc., State College, PA, USA) ( Figure 5).
Based on the dendrogram obtained by HCA, the oils from the leaves of Lauraceae species were classified into three main clusters. Cluster I was composed of 12 samples collected in the biomes Amazon and Cerrado divided into two subgroups, which presented a similarity level of 46.9%. The subgroup I-1, the samples displayed a higher average of sesquiterpene hydrocarbons (52.1%) and phenylpropanoids (29.3%) and a similarity of 92.1%. On the other hand, the oils of subgroup I-2 showed a similarity of 87.8%, and the average of their main compounds were of 39.8%, 30.4%, and 20.9% to sesquiterpene hydrocarbons, phenylpropanoids, and monoterpene hydrocarbons, respectively. Cluster II presented a similarity of 20.7%, and it was composed of 10 samples collected in the biomes Atlantic Forest and Amazon classified into two subgroups. The main classes presented in the subgroup II-1 were sesquiterpene (72.1%) and monoterpene (16.5%) hydrocarbons and only sesquiterpene hydrocarbons (75.8%) to subgroup II-2. These subgroups displayed a similarity level of 84.4% and 83.4%, respectively.
Cluster II included 29 samples collected in the biomes Atlantic Forest, Amazon, Pampa, and Cerrado with the higher similarity level (55.0%) subdivided into three subgroups. The subgroup III-1 was composed of 10 samples collected in Atlantic Forest and Amazon with a similarity of 84.4%. These oils displayed a higher chemical diversity of the main compounds. The predominant classes were sesquiterpene (35.8%) and monoterpene hydrocarbons (13.0%), oxygenated sesquiterpenoids (21.3%) and monoterpenoids (13.3%), and phenylpropanoids (12.5%). Subgroup III-2 included nine samples rich in sesquiterpene hydrocarbons (57.2%) and oxygenated sesquiterpenoids (35.5%) with a similarity of 99.5% among samples collected in Atlantic Forest and Cerrado. Finally, the subgroup III-3 was formed by ten samples collected in Atlantic Forrest and Pampa biomes and displayed a similarity of 91.9%. These samples displayed a high average of concentrations of oxygenated sesquiterpenoids (47.3%) and sesquiterpene hydrocarbons (36.4%). In summary, sesquiterpene hydrocarbons were present in all oils extracted from the leaves collected in Brazilian biomes. However, some compound classes were able to discriminate the Lauraceae oils based on their site collection. Samples collected in the Amazon and Cerrado showed high amounts of sesquiterpene hydrocarbons and phenylpropanoids. However, these biomes displayed other chemical profiles. Chemical markers of the Pampa biome were oxygenated sesquiterpenoids followed by sesquiterpene hydrocarbons. Samples from the Amazon and Atlantic Forest showed high contents of sesquiterpene and monoterpene hydrocarbons.

Conclusions
The genera Licaria, Nectandra, and Ocotea have shown high biodiversity in the territorial extension of Brazil, corresponding about 50% of the Lauraceae species in the country. However, studies focused on their essential oils (EOs) represent only 15% of the total species. According to our bibliographic research, species from the Licaria genus were collected only in the Amazon biome, and the Cerrado biome displayed the exclusive occurrence of Nectandra species. The essential oils displayed a broad chemical diversity with generally higher amounts of sesquiterpenes, as well as considerable contents of phenylpropanoids, and monoterpenes. Sesquiterpenes were present in all oils extracted from the leaves and its combination with other compound classes could discriminate some chemical markers to species collected, especially from Amazon, Cerrado and Pampa biomes. Various species showed the occurrence of two or more chemical profiles according to its site collection or seasonality, and the EO of Nectandra megatopomica was the most studied. The EOs displayed several biological activities, especially as cytotoxic and antimicrobial agents against fungi and bacteria. The results of this review suggest the high economic potential of these essential oils as new agents in the pharmaceutical, cosmetic, and food industries.

Conflicts of Interest:
The authors declare no conflicts of interest.