New and sustainable essential oils obtained from the long-term explored cinnamomum-like Aniba canelilla

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New and sustainable essential oils obtained from the long-term explored cinnamomum-like Aniba canelilla Paula Cristina Souza Barbosa a , Karenn Silveira Fernandes a , Adriana Pellegrini Manhães b , Simone Braga Carneiro a , Paulo de Tarso Barbosa Sampaio b , Larissa Silveira Moreira Wiedemann a , Valdir Florêncio da Veiga Junior a,∗ a Department of Chemistry, Institute of Exact Sciences, Federal University of Amazonas, Av. General Rodrigo Octavio Jordão Ramos, 3000, University Campus, Japiim, CEP: 69077-000, Manaus, AM, Brazil b National Institute for Amazonian Research, Av. Efigênio Sales, 2239, 69060-001, Manaus, Amazonas, Brazil

a r t i c l e

i n f o

Article history: Received 27 September 2015 Received in revised form 9 November 2016 Accepted 13 November 2016 Available online xxx Keywords: Aniba canelilla Precious bark Multivariate analysis hierarchical cluster analysis principal component analysis

a b s t r a c t The traditional approach to extract the essential oils from precious-wood (Aniba canelilla), implies in obtaining it from the bark of the trunk of adult trees, usually resulting on the dead of the tree. In order to investigate the effect of seasonality, pruning and leaf development stages, essential oils from leaves and branches were obtained and the chemical composition analyzed by GC-FID and GC–MS. Multivariate analysis, PCA and HCA, allowed the distinction of three different types of essential oils from leaves and branches, with different chromatographic profiles. In general, 1-nitro-2-phenylethane contents were significantly higher in branches and have lower internal variation of this constituent than in leaves. The multivariate analysis also allowed the observation that seasonality and the stage of development did not influence the chemical composition of essential oils, as leaves and twigs were collected in different seasons, at different stages of development in the same area, are in a same group of similar chemical compositions, particularly characterized by the predominance of 1-nitro-2-phenylethane. Although variable, the levels of 1-nitro-2-phenylethane in leaves and twigs are comparable to the levels of 1-nitro2-phenylethane observed in the stem wood and bark of A. canelilla. Thus, the extraction of essential oil of its leaves and branches may be an alternative way to prevent the overthrow of the trunk to produce essential oils of the specie. © 2016 Published by Elsevier GmbH.

1. Introduction Aniba canelilla (HBK) Mez (syn. Aniba elliptica AC Sm., Cryptocarya canelilla Kunth) belongs to the Lauraceae family and occurs mostly in the Amazon region. This species stands out as one of the plant resources of greater economic importance due to its high value markets in the food, cosmetics and perfumes. Popularly known as bark-precious, precious-wood, precious leaf, stick-precious and false cinnamon, is easily found in street markets, herbalists and health food pharmacies throughout Brazil and is also widely used in folk medicine to treat various diseases (Maia et al., 2001). The leaves and bark are used to treat colds, fevers, nausea, headaches, injuries, various types of infection, nervous ten-

∗ Corresponding author. E-mail address: [email protected] (V.F.d. Veiga Junior).

sion, acne and other dermatitis (Lorenzi and Matos, 2008). The bark is also used in the treatment of malaria (Botsaris, 2007), Alzheimer’s disease (Madaleno, 2011) and in the treatment of problems related to the digestive system, as anti-inflammatory, nervous system stimulant, and has carminative properties (Lima et al., 2004, 2009; Lorenzi and Matos, 2008; Perazzo et al., 2009). The essential oil of A. canelilla is the main product of this species, being commercially extracted from the wood of the trunk, due to the high oil yield (Barata and Lupe, 2007), leading to the indiscriminate cutting of mature trees of reproductive age and an impact negative in the event of natural populations of the species. Similar to Aniba rosaeodora (rosewood), which was already on the list of endangered species because of this extraction process (IBAMA Ordinance No. 37-N, of April 3, 1992), A. canelilla runs the same risk extinction by predatory extraction. Regardless of plant body of which the essential oil of A. canelilla is extracted, its composition is usually rich in benzenoids, espe-

http://dx.doi.org/10.1016/j.jarmap.2016.11.002 2214-7861/© 2016 Published by Elsevier GmbH.

Please cite this article in press as: Barbosa, P.C.S., et al., New and sustainable essential oils obtained from the long-term explored cinnamomum-like Aniba canelilla. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.11.002

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cially the 1-nitro-2-phenylethane and presents monoterpenes and sesquiterpenes. The essential oils of A. canelilla have the same biological activities reported for infusions of bark, leaves and twigs. The essential oils from leaves showed cytotoxic effect and proven leishmanicidal activity (Silva et al., 2009), as well as the trunk oils that showed cytotoxic effect and antioxidant activity (Silva et al., 2007), and oils of bark exercising showed hypotensive cardiovascular activity (Lahlou et al., 2005). Several studies point to the 1-nitro-2-phenylethane as one of the substances responsible for the biological activities described for the essential oils of A. canelilla. Studies showed that 1-nitro-2-phenylethane has fungistatic (Oger et al., 1994), analgesic (Lima et al., 2009) and vasorelaxant effect (Interaminense et al., 2011, 2013). Although generally the 1-nitro-2-phenylethane is the major constituent of essential oils A. canelilla, various factors can influence the chemical composition and yield of these oils, such as plant material damage caused by mechanical or chemical stress, injury biotic, as herbivores (Figueiredo et al., 2008; Edwards et al., 1993). Environmental factors can also influence such as light and humidity (Sangwan et al., 2001), which can still be the cause of the variation in seasonality, although some studies have not found differences in chemical composition between stations (Silva et al., 2009) and may also be variations between plant organs which oil will be extracted and their stage of development (Sangwan et al., 2001). According to Silva et al. (2009), the yield of essential oils from twigs A. canelilla was higher than the yield from the trunk bark and wood. In addition, the authors observed that the yields of 1-nitro2-phenylethane did not show any difference among the stations. In contrast, another study showed no yield variations between bark, leaves and wood of the trunk and highest levels of 1-nitro2-phenylethane observed during the rainy season (Taveira et al., 2003). In this sense, the extraction of essential oils from A. canelilla from other plant organs such as leaves and branches, would be a sustainable alternative, which would facilitate field work as well as being more economically viable. In addition, different responses observed in the composition and production of essential oil from A. canelilla can be used to improve the quality and productivity of these oils in specimens from natural forests or those cultivated for marketing purposes. Therefore, the objective of this work was to study the variations in the chemical composition of essential oils from leaves and branches of A. canelilla collected before pruning (pruning crown) and after pruning (sprouting) in different seasons (wet and dry). The essential oils were analyzed by gas chromatography coupled to flame ionization detection (GC-FID) and mass spectrometry (GC–MS), and the results were evaluated by hierarchical cluster analysis (HCA) and principal components (PCA).

2. Materials and methods

(F2) and branches (G2) the bottom of the previously pruned canopy (regrowth); and they were also collected sheets (FR) and branches (GR) of the remaining crown. The minimum diameter of the collected branches was 10 cm. 2.2. Sample preparation and essential oil extraction The plant material was air dried and subsequently taken to an oven at 30 ◦ C for a week, until constant weight is obtained. Then, the already-dried samples were ground in a Wiley mill and stored in labeled plastic bags inside the freezer for preserving the properties of the essential oils until the moment of extraction of oils. Essential oils leaves and branches A. canelilla were extracted by hydrodistillation by the method of steam distillation using a modified Clevenger apparatus all-glass. They were weighed 170 g samples of leaves and branches which have been hydrodistilled in 1700 mL of water using a 3,000 mL glass flask for 3 h (until no further essential oil to be recovered) at a temperature of 100 ◦ C. After each extraction, Clevenger apparatus was washed with dichloromethane to remove the oil that was trapped in the device wall. The hydrosols (water + oil) resulting from hydrodistillation were stored in a freezer and the next day were dried with sodium sulfate (Na2 SO4 ) and anhydrous stored in amber bottles at 4 ◦ C before chromatographic analysis. 2.3. GC–FID analysis GC–FID analysis were performed with a GC 2010 system (Shimadzu Ltd., Japan) equipped with a DB-5 capillary column (30 m × 0.25 mm i.d.; film thickness 0.25 ␮m) using the following temperature program: temperature was increased from 60 ◦ C to 240 ◦ C at a rate of 3 ◦ C min−1 and held isothermally for 3 min. The injector and detector temperatures were set to 250 ◦ C and 290 ◦ C, respectively. The carrier gas was nitrogen at a flow rate of 2.0 mL min−1 ; split ratio was 1:200; the volume injected was 1.0 ␮L. The percentages of the constituents were calculated by electronic integration of the FID peak areas without response factor correction. The oils were diluted in hexane (1 mg mL−1 ) before the chromatographic determination of their composition. 2.4. GC–MS analysis GC–MS analysis were performed with a GC 2010 system (Shimadzu Ltd., Japan) equipped with a DB-5 capillary column (30 m × 0.25 mm i.d.; film thickness 0.25 ␮m). The injector and detector temperatures were set to 250 ◦ C and 300 ◦ C, respectively. The carrier gas was nitrogen at a flow rate of 1.0 mL min−1 ; split ratio was 1:100; the volume injected was 1.0 ␮L. The samples were diluted in hexane (1 mg. mL−1 ) prior to automatic injection. The ionization energy was 70 eV, with a scan time of 0.5 s and mass spectra range of were recorded from 40 to 600 m/z.

2.1. Plant material 2.5. Identification of components Leaves and branches of A. canelilla were collected from a population located at the Annual Production Unit (UPA) company Mil Woods Itacoatiara Ltda. (Precious Woods Amazon SA), in Itacoatiara, Amazonas. This forest area is located between latitudes 2◦ 43 and 3◦ 04 and 3rd 04 S and longitude 58◦ 31 and 58◦ 57 W. Two samples were taken. The first collection was carried out during the rainy season, in February 2009, resulted from the pruning done from the first fork of the trunk up to half of the total height of the canopy (50% of the canopy). Leaves were collected (F1) and branches (G1) from the bottom of the canopy, leaving 50% of the top (remaining canopy). In the second collection − held during the dry season in November 2009 − were collected sheets

The chemical composition of A. canelilla essential oils was performed using the retention time data obtained by GC-FID and mass spectra obtained by GC–MS. The retention rates were calculated using the Van der Dool Kratz equation, linking the retention times of substances present in the essential oils with the retention times of n-alkanes (homologous series of C9 -C22 ). These were co-injected with the samples, which were analyzed in sequence. As all the constituents detected were already known, the retention indices, mass spectra and fragmentation patterns are well-defined in the literature (Adams, 2007). The results obtained were compared with data from the Wiley 7.0 Spectrotech supplied by Shimadzu© .

Please cite this article in press as: Barbosa, P.C.S., et al., New and sustainable essential oils obtained from the long-term explored cinnamomum-like Aniba canelilla. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.11.002

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2.6. Statistical analysis The contents of constituents identified (chromatographic results) in the samples of essential oils of A. canelilla, constitute a multivariate data set that were interpreted by Hierarchical Cluster Analysis (HCA) and Principal Components Analysis (PCA). Preprocessing of data was carried out by normalization by logarithm, with the aim of reducing the influence of undesirable variations in the data set. With the values obtained, two data matrices were created. The first, corresponding to leaves, with 21 lines × 30 columns: 21 lines corresponded to the samples, 30 columns corresponded to the values of the constituents identified in the samples. The second, corresponding to stems, with 24 lines × 22 columns: 24 lines corresponded to the samples, 22 columns corresponded to the values of the constituents identified in the samples. 2.6.1. Hierarchical clustering analysis (HCA) Two hierarchical cluster analysis were performed, one to leaves and other to the stems. For these analyzes all variables were used. The similarity was calculated based on the Euclidean distance, using the Ward method. HCA was carried out in order to determine similarities in chemical composition between different samples from leaves and stems. The cluster analysis and dendrogram were per® formed with free software R version 2.14.0 (available at http:// www.r-project.org/). 2.6.2. Principal component analysis (PCA) Two principal component analysis were also performed: one to leaves and other to the stems. For PCA leaves, 10 variables were selected the contents of: 1-nitro-2-phenylethane, benzaldehyde, benzonitrile, benzene acetaldehyde, benzene acetonitrile, ␤-elemene, ␤-caryophyllene, ␣ and ␤-selinene, and ␦-cadinene. For PCA stems, only 4 variables were selected, the contents of: 1-nitro-2-phenylethane, benzene acetaldehyde, linalool and methyleugenol. The principal component analysis and the graphs of scores and loadings were performed with free software R version ® 2.14.0 (available at http://www.r-project.org/). 3. Results and discussion Vegetable oils are byproducts of commercial importance in the Amazon region. Despite the potential for dozens of species, their large-scale production covers only some species such as babassu, copaíba and rosewood. Sustainable production, protecting the forest, and the chemical standardization of these oils are the two main factors that reduce its value and limit its exports at larger scales. The use of sprouts leaves and pruning branches have been an alternative of sustainability for the species whose extraction by clear-cutting the trunk was the traditional use (Pinkard, 2003; Medhurst et al., 2006; Manhães et al., 2012.). For chemical patterning the output is to analyze a large number of tree samples from the same region, where issues such as climate and natural diversity (chemotypes) are minimized and effects of seasonality, age and type of soil can be analyzed. Recent studies with Copaifera multijuga analyzed the influence of seasonality, soil type and diameter at breast height (DBH) on the chemical composition of 16-resin oils copaíba collected in Ducke Forest Reserve, which allowed the differentiation of samples into two groups with different chemical compositions. According to the authors, only the type of soil would have influenced the chemical composition of copaiba oils, the difference between the sample groups was based primarily on the content of major constituents, ␤-caryophyllene and caryophyllene oxide (Barbosa et al., 2012) In this study, leaves and branches of A. canelilla were collected from different specimens of the same population in the same cli-

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mate region and soil without variation. In the first collection a pruning from the first fork of the trunk up to half of the total height of the canopy (50% of the canopy) was performed, which resulted in the collection of 7 leaf samples (F1) and 8 samples branches (G1), leaving 50% of the top of the canopy (remaining canopy) intact. In the second collection 7 leaf samples (F2) and 8 samples branches (G2) were collected at the bottom of the previously pruned canopy (regrowth); and 7 were also collected leaf samples (FR) and 8 samples branches (GR) of the remaining crown (part of the crown that was intact in the first collection). In total, 21 samples were collected from 24 samples of leaves and branches of A. canelilla. 3.1. Identification of volatile compounds in essential oils of leaves and stems of A. canelilla The chemical composition and their relative quantitation linear retention index (LRI) to the chemical constituents of essential oils extracted by hydrodistillation of the 1 st collecting sheets (F1) of the leaves of the 2nd collection (F2) and leaves the remaining canopy (FR) of A. canelilla are shown in Tables 1–3 , respectively; the same data is presented to the branches of the 1 st collection (G1) to the branches of the 2nd collection (G2) and the canopy of the remaining crown (GR) of A. canelilla in Tables 4–6, respectively. All sheets were identified 30 constituents, although the total number of detected constituent (total constituents identified unidentified +) was up to 78 components, which is between 86.62% and 99.00% of the total content of essential oils. The branches were identified 22 constituents, which has been detected up to 37 constituents, which is between 94.76% and 98.37% of the total content of essential oils of A. canelilla. Although several studies describe the chemical composition of the bark and wood of the trunk A. canelilla, usually the number of samples is small and usually do not provide representative and reliable data. Furthermore, only three studies already described the chemical composition of leaves and branches. It is common the report of three chemical classes of constituents: monoterpenes, sesquiterpenes and benzenoids. This last class is often the dominant, representing about 75% of the total composition of essential oils A. canelilla. Among benzenoids present, it is unanimous that the 1-nitro-2phenylethane is the chemical marker of this species, presenting as major constituent of essential oils of bark, leaves, branches and wood A. canelilla trunk. In the present study, the 1-nitro-2phenylethane was the major constituent of the 21 samples on 19 sheets of samples and all samples branches, although in different amounts. The levels of 1-nitro-2-phenylethane in the leaves ranged between 31.22% and 84.33% in the leaves collected during the rainy season (1 st collection), with a difference of up to 53.11% between the maximum and minimum levels of 1-nitro-2-phenylethane; 2nd collects the contents of that benzenoid ranged between 13.17% and 74.55% in the leaves of regrowth, with a difference of up to 61.38% between the maximum and minimum levels of 1-nitro-2phenylethane. At the leaves of the remaining cup, the contents of such constituent varied between 13.54% and 84.39%, with a difference of 70.85% between the maximum and minimum content. In summary, these results allowed the observation that the levels of 1nitro-2-phenylethane in the leaves are usually quite variable, with a difference of up to 70.85% between the maximum and minimum levels of constituent. A similar result was observed in six leaf samples collected in three different regions in the Serra dos Carajás/PA. Taveira et al., (2003) collected three samples of leaves in the dry season and three leaf samples in rainy season and found that the levels of 1-nitro-2-phenylethane ranged between 39.0% and 42.1% among samples collected in dry season, with a difference of up to 3.1%; while in samples collected in the rainy season the concentra-

Please cite this article in press as: Barbosa, P.C.S., et al., New and sustainable essential oils obtained from the long-term explored cinnamomum-like Aniba canelilla. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.11.002

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Table 1 Chemical composition of essential oils from A.canelilla leaves (1a collection). Compound

RI

F1 -1

F1 -3

F1 -6

F1 -7

F1 -8

F1 -9

F1 -10

␣-pinene benzaldehyde ␤-pinene benzonitrile p-cimene limonene 1,8-cineole benzene acetaldehyde Linalool benzene acetonitrile ␣-terpineol 1-nitro-2-phenylethane eugenol ␣-copaene ␤-elemene ␤-caryophyllene aromadendrene ␣-humulene germacrene D ␤-selinene ␣-selinene ␤-bisabolene ␤-sesquiphellandrene trans-calamenene ␦-cadinene trans-cadina-1,4-diene trans-nerolidol spathulenol caryophyllene oxide globulol Total

932 952 974 985 1020 1024 1026 1036 1095 1134 1186 1350 1356 1374 1389 1417 1439 1452 1484 1489 1498 1505 1521 1521 1522 1533 1561 1577 1582 1590

0.48 6.98 0.29 2.90 0.22 0.49 0.33 2.54 0.46 0.32 1.18 39.78 – 0.13 5.90 8.01 – 0.48 0.21 5.84 1.63 2.86 0.77 0.65 4.04 0.41 0.95 1.81 0.97 0.62 91.25

0.22 1.25 – 0.75 – 0.16 – 1.36 0.43 0.24 0.11 84.33 – – 1.06 4.95 – 0.40 – 0.77 0.36 0.62 0.52 – 0.26 – 0.10 0.75 0.27 0.10 99.01

0.59 1.25 – 1.05 0.17 0.46 0.13 1.89 0.41 0.29 0.52 77.37 0.10 – 2.66 4.22 0.69 0.53 0.09 1.37 0.56 – 0.75 0.08 0.49 0.15 0.25 0.96 0.48 0.24 97.75

0.09 2.54 – 0.61 – 0.08 0.09 1.82 0.27 0.40 0.60 84.31 – 0.08 0.68 1.89 – – – 2.35 0.54 0.23 0.24 0.14 0.91 0.12 0.22 0.08 0.35 0.18 98.82

0.21 2.99 – 1.31 0.10 0.20 0.08 3.63 0.38 0.23 0.21 76.00 – 0.08 1.67 3.35 – 0.09 – 2.42 0.48 0.15 0.26 0.19 0.74 0.07 0.37 0.07 0.71 0.48 96.47

0.16 4.22 0.11 1.17 0.16 0.28 0.09 1.33 0.91 0.21 0.23 31.22 0.08 0.08 2.94 16.34 0.37 1.06 0.28 8.82 2.26 1.66 0.73 1.18 6.80 0.19 1.85 1.65 1.93 1.24 89.55

0.28 2.04 – 0.89 0.13 0.28 0.18 1.54 1.00 0.29 1.07 70.97 – 0.07 2.13 7.28 0.19 0.58 – 2.40 0.66 0.49 0.36 0.18 0.87 – 0.44 0.42 0.78 0.52 96.04

Table 2 Chemical composition of essential oils from A.canelilla leaves (2a collection). Compound

RI

F2 -1

F2 -3

F2 -6

F2 -7

F2 -8

F2 -9

F2 -10

␣-pinene Benzaldehyde ␤-pinene Benzonitrile p-cimene Limonene 1,8-cineole benzene acetaldehyde Linalol benzene acetonitrile ␣-terpineol 1-nitro-2-phenylethane Eugenol ␣-copaene ␤-elemene ␤-caryophyllene Aromadendrene ␣-humulene germacrene D ␤-selinene ␣-selinene ␤-bisabolene ␤-sesquiphellandrene trans-calamenene ␦-cadinene trans-cadina-1,4-diene trans-nerolidol Spathulenol caryophyllene oxide Globulol Total

932 952 974 985 1020 1024 1026 1036 1095 1134 1186 1350 1356 1374 1389 1417 1439 1452 1484 1489 1498 1505 1521 1521 1522 1533 1561 1577 1582 1590

0.38 2.71 0.39 1.08 0.08 0.36 0.16 2.98 0.25 0.37 0.87 68.30 – 0.14 3.40 4.19 2.00 0.78 0.08 2.75 0.96 1.98 0.49 0.17 1.59 0.07 0.12 0.66 – 0.16 97.47

0.10 5.24 0.10 5.19 – 0.21 – 3.76 1.72 0.47 0.42 46.49 – 0.14 1.66 12.64 0.07 0.79 0.11 2.09 0.84 1.88 1.00 0.32 4.81 0.16 0.21 2.58 – 0.29 93.29

– 0.95 – 0.81 – 0.12 – 4.00 0.66 0.27 0.63 74.55 – 0.22 2.04 5.43 0.69 0.25 0.09 1.09 0.41 – 0.50 0.28 2.33 0.17 0.41 1.82 – 0.58 98.30

0.10 8.80 0.13 7.38 – 0.12 0.34 6.33 1.39 0.83 3.01 33.52 – 0.17 3.06 9.66 0.53 0.64 – 6.87 1.72 0.84 0.99 0.45 5.71 0.35 0.82 0.25 0.19 0.58 94.78

0.10 3.96 – 5.49 – 0.09 – 4.07 0.82 0.28 0.43 58.87 – 0.18 2.80 11.49 0.10 0.65 – 3.17 0.84 0.44 0.30 0.19 1.86 – 0.22 0.10 – 0.28 96.73

0.06 5.29 0.08 4.58 – 0.11 0.12 1.34 1.55 0.61 0.40 16.65 0.24 0.21 4.90 12.04 11.08 2.23 0.19 9.21 2.90 2.27 0.68 0.77 7.22 0.22 1.13 2.64 – 0.63 89.35

– 0.89 – 1.80 – – 0.13 0.11 5.10 0.42 2.99 13.17 – 0.17 6.60 7.36 10.10 2.35 0.25 14.98 4.86 3.37 3.42 0.78 6.44 0.78 1.18 2.85 – 0.58 90.68

tions of 1-nitro-2-phenylethane ranged between 70.6% and 95.3%, with a difference of up to 24.7%, which according to the authors, suggested influence of seasonality. However, Silva et al. (2009) reported no such variation in the essential oils of leaves collected in Manaus/AM, at different times

of the year. At that time, the essential oils in rainy season collected leaves showed 88.5% of 1-nitro-2-phenylethane, while samples collected during the dry season showed 88.9% of this constituent. Despite the observed variation, the levels of 1-nitro-2phenylethane also observed here are consistent with the results

Please cite this article in press as: Barbosa, P.C.S., et al., New and sustainable essential oils obtained from the long-term explored cinnamomum-like Aniba canelilla. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.11.002

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Table 3 Chemical composition of essential oils from A.canelilla leaves (remaining canopy). Compound

RI

FR -1

FR -3

FR -6

FR -7

FR -8

FR -9

FR -10

␣-pinene Benzaldehyde ␤-pinene Benzonitrile p-cimene Limonene 1,8-cineole benzene acetaldehyde Linalol benzene acetonitrile ␣-terpineol 1-nitro-2-phenylethane Eugenol ␣-copaene ␤-elemene ␤-caryophyllene Aromadendrene ␣-humulene germacrene D ␤-selinene ␣-selinene ␤-bisabolene ␤-sesquiphellandrene trans-calamenene ␦-cadinene trans-cadina-1,4-diene trans-nerolidol Spathulenol caryophyllene oxide Globulol Total

932 952 974 985 1020 1024 1026 1036 1095 1134 1186 1350 1356 1374 1389 1417 1439 1452 1484 1489 1498 1505 1521 1521 1522 1533 1561 1577 1582 1590

0.27 3.10 0.38 5.03 0.08 0.34 0.37 0.17 0.86 0.57 2.74 15.12 0.11 0.12 7.01 7.52 10.21 2.55 0.11 7.74 2.79 5.14 1.19 0.78 7.06 0.43 1.08 3.07 – 0.69 86.63

– 1.41 – 0.82 – 0.07 – 1.57 0.38 0.26 0.12 84.39 – 0.20 0.82 4.07 – – – 0.70 0.27 0.52 0.33 0.11 1.24 – 0.14 0.85 – 0.15 98.42

– 0.77 – 0.66 0.12 0.11 0.06 1.23 0.17 0.15 0.32 80.02 0.10 0.14 2.47 0.67 0.99 0.17 0.07 1.11 0.31 – 0.45 0.09 3.69 0.27 0.93 1.16 0.20 0.42 96.85

– 2.36 – 1.20 – 0.09 – 2.66 0.40 0.20 0.24 77.04 – 0.17 1.99 4.41 – – – 2.84 0.53 0.17 0.26 0.16 1.58 – 0.31 – 0.35 0.31 97.27

– 5.89 – 6.14 – 0.08 0.20 3.26 1.41 1.02 3.54 36.23 0.09 0.19 3.11 8.88 3.76 1.10 0.11 6.62 1.94 0.92 1.28 0.39 4.88 0.31 0.76 0.41 0.12 0.45 93.09

0.12 2.78 0.16 2.73 – 0.19 0.12 0.21 1.32 0.50 0.33 13.54 0.48 0.17 5.51 13.47 15.13 2.72 0.18 10.34 3.27 2.63 0.65 0.77 7.80 0.19 1.15 2.16 – 0.62 89.24

– 2.51 – 1.15 – 0.15 0.13 2.48 1.34 0.29 0.82 70.44 – 0.17 2.27 4.84 – 0.12 – 4.77 1.21 0.90 0.63 0.24 1.48 0.19 0.22 0.74 0.09 0.27 97.45

Table 4 Chemical composition of essential oils from A.canelilla stems (1a collection). Compound

RI

G1 -1

G1 -3

G1 -5

G1 -6

G1 -7

G1 -8

G1 -9

G1 -10

␣-pinene benzaldehyde ␤-pinene benzonitrile limonene 1,8-cineole benzene acetaldehyde linalool benzene acetonitrile 1-nitro-2-phenylethane eugenol ␣-copaene ␤-elemene methyleugenol ␤-caryophyllene ␣-humulene germacrene D ␤-selinene ␣-selinene ␤-bisabolene ␦-cadinene trans-cadina-1,4-diene Total

932 952 974 985 1024 1026 1036 1095 1134 1350 1356 1374 1389 1405 1417 1452 1484 1489 1498 1505 1522 1533

0.10 0.27 – 0.13 0.08 – 1.98 0.06 0.15 92.10 – 0.06 0.08 0.28 0.22 – 0.05 0.31 0.21 0.19 0.19 0.09 96.55

0.09 0.21 – 0.12 0.07 – 1.44 0.78 0.14 91.78 0.07 0.06 0.21 0.09 0.54 0.12 0.07 0.16 0.22 0.16 0.33 0.10 96.76

0.14 0.24 – 0.13 0.07 – 0.82 0.40 0.27 93.44 0.56 0.09 0.32 0.44 0.25 – – 0.40 0.23 0.08 0.38 0.09 98.35

0.06 0.25 – 0.12 0.06 – 1.42 0.11 0.19 93.20 1.06 0.07 0.10 0.10 0.14 – – 0.17 0.15 – 0.22 0.06 97.48

0.05 0.16 – 0.07 – – 0.84 0.24 0.14 90.83 0.85 0.07 0.26 1.82 0.19 0.06 0.08 0.36 0.21 – 0.62 0.20 97.05

– 0.23 – 0.16 – – 0.48 0.18 0.14 93.45 1.45 0.09 0.10 0.15 0.32 – – 0.43 0.18 – 0.28 0.10 97.74

0.06 0.15 – 0.09 0.06 – 1.02 0.40 0.14 90.94 0.88 0.08 0.06 0.20 0.16 0.07 0.07 0.55 0.33 0.10 0.27 0.10 95.73

– 0.20 – 0.08 – – 0.62 0.58 0.27 93.58 0.91 0.08 – 0.07 0.16 0.07 – 0.19 0.21 0.08 0.21 0.08 97.39

for leaves collected in Manaus/AM, during the dry season, which showed 71.2% of 1-nitro-2-phenylethane (Lima et al., 2004). Another study that analyzed essential oils from a mixture of fine leaves and twigs collected during the rainy season, presented content of 74.0% of 1-nitro-2-phenylethane for samples collected in Ulinópolis/PA and content of 91.8% of 1-nitro −2-phenylethane in samples collected in New Airão/AM (Silva et al., 2007), also results comparable to those obtained in this study. In the case of branches, the content of 1-nitro-2-phenylethane varied between 90.83% and 93.58% in samples of branches collected in the rainy season (1st collection), with a difference of up

to 2.75% between the maximum and minimum content; 2nd collects the contents of this constituent varied between 87.85%, 94.16% the branches of regrowth, with a difference of up to 6.3% between the maximum and minimum content; while the branches of the remaining cup, the contents of that benzenoid varied between 85.53% and 92.05%, with a difference of up to 6:52% between the maximum and minimum content. In summary, these results allowed the observation that changes in the levels of 1-nitro-2-phenylethane the branches is small compared to the variation observed in the leaves, with a maximum difference of only 6.52%. A single work studied the chemical compo-

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Table 5 Chemical composition of essential oils from A.canelilla stems (2a collection). Compound

RI

G2 -1

G2 -3

G2 -5

G2 -6

G2 -7

G2 -8

G2 -9

G2 -10

␣-pinene benzaldehyde ␤-pinene benzonitrile limonene 1,8-cineole benzene acetaldehyde linalool benzene acetonitrile 1-nitro-2-phenylethane eugenol ␣-copaene ␤-elemene methyleugenol ␤-caryophyllene ␣-humulene germacrene D ␤-selinene ␣-selinene ␤-bisabolene ␦-cadinene trans-cadina-1,4-diene Total

932 952 974 985 1024 1026 1036 1095 1134 1350 1356 1374 1389 1405 1417 1452 1484 1489 1498 1505 1522 1533

– 0.17 – 0.26 – – 0.84 0.05 0.30 92.84 0.19 0.15 0.13 0.08 0.37 0.16 – 0.53 0.26 0.44 0.17 – 96.94

– 0.19 – 0.16 – – 0.08 2.14 0.22 90.52 0.08 0.25 0.32 – 1.03 – – 0.18 0.15 0.27 0.32 – 95.91

– 0.20 – 0.22 – – 0.62 3.08 0.33 89.48 0.90 0.14 0.15 – 0.59 – – 0.38 0.22 0.17 0.15 – 96.63

0.36 0.22 0.22 0.31 0.24 0.10 1.08 0.79 0.28 87.85 0.87 0.16 0.49 0.07 0.50 – – 0.53 0.32 0.47 0.11 94.97

– 0.16 – 0.13 – – 1.22 1.23 0.26 91.64 0.46 0.20 0.28 0.26 0.66 – – 0.54 0.21 0.11 0.34 0.07 97.77

0.15 0.14 0.10 0.19 0.10 – 1.11 0.39 0.24 92.15 1.19 0.15 0.14 0.09 0.46 – – 0.50 0.18 0.10 0.15 – 97.53

– 0.10 – 0.08 – – 0.67 0.46 0.32 94.16 0.44 0.16 0.04 0.07 0.15 – – 0.41 0.20 0.12 0.09 – 97.47

0.19 0.22 0.14 0.16 – 0.07 1.03 1.99 0.27 90.90 0.38 0.15 0.40 0.08 0.28 – – 0.72 0.31 0.19 0.40 – 97.88

Table 6 Chemical composition of essential oils from A.canelilla stems (remaining canopy). Compound

RI

GR -1

GR -3

GR -5

GR -6

GR -7

GR -8

GR -9

GR -10

␣-pinene benzaldehyde ␤-pinene benzonitrile limonene 1,8-cineole benzene acetaldehyde linalool benzene acetonitrile 1-nitro-2-phenylethane eugenol ␣-copaene ␤-elemene methyleugenol ␤-caryophyllene ␣-humulene germacrene D ␤-selinene ␣-selinene ␤-bisabolene ␦-cadinene trans-cadina-1,4-diene Total

932 952 974 985 1024 1026 1036 1095 1134 1350 1356 1374 1389 1405 1417 1452 1484 1489 1498 1505 1522 1533

– 0.53 – 0.25 – – 1.15 0.30 0.15 89.12 0.11 0.22 0.15 – 0.65 0.13 0.07 0.66 0.35 0.56 0.25 0.11 94.76

– 0.19 – 0.17 – – 0.63 1.37 0.13 92.05 0.06 0.16 0.26 – 0.80 0.10 – 0.16 0.19 0.21 0.26 0.08 96.82

– 0.13 – 0.15 – – 1.06 2.33 0.18 89.92 1.42 0.15 0.10 – 0.29 0.13 – 0.26 0.21 0.15 0.20 0.06 96.74

– 0.13 – 0.15 0.09 – 1.62 0.25 0.12 90.47 1.59 0.19 0.23 – 0.12 0.12 0.07 0.27 0.24 – 0.38 0.14 96.18

– 0.12 – 0.12 – – 0.78 1.14 0.20 91.82 0.94 0.17 0.26 – 0.33 0.12 – 0.44 0.21 – 0.42 0.13 97.20

– 0.19 – 0.26 – – 1.27 0.43 0.15 91.64 1.21 0.16 0.18 – 0.56 0.13 – 0.54 0.21 0.08 0.23 0.07 97.31

– 0.14 – 0.29 0.06 – 0.92 1.10 0.22 89.48 0.96 0.16 0.08 – 0.19 0.19 – 0.73 0.39 0.24 0.26 0.09 95.50

– 0.12 – 0.16 0.09 – 1.02 2.86 0.12 85.53 0.63 0.17 0.75 – 0.16 0.28 0.11 1.00 0.55 0.34 0.82 0.22 94.93

sition of the branches A. canelilla. Lima and colleagues (2004) found that thin branches of A. canelilla collected in Manaus/AM in the dry season, showed 68.2% of 1-nitro-2-phenylethane, lower content of those found in this study. Many other works describe the content of 1-nitro-2phenylethane to the trunk bark and wood. For samples of essential oils extracted from bark with hydrodistillation, the observed levels were between 48.6% and 68.1% of 1-nitro-2-phenylethane in samples collected in dry season, and levels between 78.2% and 94.3% in samples collected in rainy season in the region Serra dos Carajás/PA (Taveira et al., 2003); 89.8% of 1-nitro-2-phenylethane to sample collected in Fatima Chimanes/Bolivia (Oger et al., 1994); 90.3% of 1-nitro-2-phenylethane to sample collected in Ulinópolis/PA (Silva et al., 2007); 52.4% of 1-nitro-2-phenylethane to sample collected in Paragominas/PA (Lahlou et al., 2005). A study that analyzed the essential oil extracted from the bark by supercritical fluid reported 71.12% of 1-nitro-2-phenylethane in a sample from the Cardoso Island/SP (Vilegas et al., 1998).

Another important benzenoid on A. canelilla essential oils is methyleugenol, which is often described with significant levels of bark and the stem wood of this species. The methyleugenol was not identified in any of the leaf samples collected in this work. This result is in agreement with the results for leaves collected in the dry season (Lima et al., 2004; Silva et al., 2009) and leaves collected in the rainy season (Silva et al., 2009), all collected in Manaus/ASilva et al. (2007) have not identified even identified methyleugenol in mixtures of leaves and twigs, although methyleugenol has already been described in leaves (Taveira et al., 2003) and twigs (Lima et al., 2004). In contrast, Taveira et al. (2003) had already described methyleugenol leaves. Concentrations ranged from 0.5% to 0.6% in leaves collected in the dry season; and 0.2% to 3.4% during the rainy season, all collected in three different regions in the Serra dos Carajás/PA. In the case of branches, methyleugenol was identified in 14 of 24 samples analyzed branches: 8 samples being branches collected in

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the rainy season (1st collection) and 6 samples collected in dry season, the branches of regrowth (2nd collection). The methyleugenol content varied between 12.07% and 1.82% in the branches collected in the rainy season (1st collection); 2nd collects the contents of this constituent varied between 0.07 and 0.26%% in the branches of regrowth. This result is in agreement with the results obtained for samples twigs collected in Manaus/AM in dry period, where the concentration of methyleugenol reached 1.1% of the total composition of the essential oil (Lima et al., 2004). According Taveira et al., (2003), the concentration of methyleugenol in samples of leaves, bark and wood trunk collected in the Serra dos Carajás/PA, show dependence on seasonality, a fact that was not observed in this study. As the 1-nitro-2-phenylethane, methyleugenol was also described in essential oils from the stem wood and bark of A. canelilla at levels quite variable. To essential oils extracted samples of shells from hydrodistillation, the observed levels were between 24.6% and 45.3% of methyleugenol in dry period collected samples, and levels between 1.0% and 14.7% in samples collected in the rainy season in the region Serra dos Carajás/PA (Taveira et al., 2003); 2.90% of methyleugenol to sample collected in Fatima Chimanes/Bolivia (Oger et al., 1994); 2.0% of methyleugenol to sample collected in Ulinópolis/PA (Silva et al., 2007); 38.6% of methyleugenol to sample collected in Paragominas/PA (Lahlou et al., 2005). A study that analyzed the essential oil extracted from the bark by supercritical fluid reported 26.9% of methyleugenol in a sample from the Cardoso Island/SP (Vilegas et al., 1998). For trunk wood samples observed methyleugenol contents were between 22.0% and 45.8% for samples collected in dry season, and levels between 10.7% and 17.7% in samples collected in the rainy season in the region of Serra dos Carajás/PA (Taveira et al., 2003); 4.3% of methyleugenol to sample collected in New Airão/AM and 25.8% for sample collected in Ulinópolis/PA (Silva et al., 2007). As described in the literature, variations of the levels of 1nitro-2-phenylethane and methyleugenol in A. canelilla oils are commonly observed in samples from different areas collected at different time extracted by diverse methods and plant organs. However, this study allowed us to observe that although variable, the levels of 1-nitro-2-phenylethane and methyleugenol in leaves and twigs are comparable to the levels observed in the stem wood and bark of A. canelilla. Thus, the essential oil extraction from its leaves and branches becomes an alternative way to prevent the overthrow of the trunk to produce essential oils that species. Although present at low concentrations, it is worth noting the presence of other benzenoids derivative of phenylalanine, which is

7

probably also the precursor of 1-nitro-2-phenylethane: benzaldehyde, benzeneacetaldehyde, benzonitrile and benzeneacetonitrile are mainly identified in the essential oils of bark and wood trunk A. canelilla and may be the final products of the degradation of phenylalanine. Therefore, it is possible that these compounds are biologically interchangeable and dependent impact extrinsic factors (Taveira et al., 2003). These four benzenoid were identified in all essential oils leaves and branches analyzed in this study, suggesting that together can also be markers of this chemical species. The knowledge of the variation of these constituents in the essential oils of A. canelilla is of paramount importance, since all of them can somehow influence the concentration of 1-nitro2-phenylethane, which has extensive commercial application in cosmetics or medicines due to its wide range of proven biological activities. The sesquiterpene germacrene D, which is an important precursor to other sesquiterpenes as cadinenes and muurolenes was identified in 12 samples of leaves and branches 7 samples. Generally, the germacrene D occurs together with ␦-cadinene, a fact that was also observed in this study: all the samples showing germacrene D also possessed ␦-cadinene in its composition. 3.2. Hierarchical cluster analysis (HCA) of GC–MS and GC-FID fingerprint of essential oils of leaves of A. canelilla The identified and quantified constituents in the essential oils of leaves of A. canelilla constitute a set of data submitted to the hierarchical clustering analysis (HCA) and principal component analysis (PCA) to the classification of 21 samples. Based on the content of each constituent built up the date array with 21 rows, corresponding to the samples, and 30 columns corresponding to the contents of the constituents identified and quantified in the leaf samples. Preprocessing of the data was done by normalization by log in order to reduce the influence of unwanted variations. The similarity between the samples was calculated using the Euclidean distance in the space of the variables, using Ward’s method. The results of HCA, performed with the purpose of verifying similarities in chemical composition between samples, were represented as a two dimensional graph, known the dendrogram (Fig. 1), through which it is possible to observe two groupings (clusters) to separate the leaves. The Cluster I includes 11 samples: 5 samples of leaves of the 1 st collection (F1-3, F1-6, F1-7, F1-8 and F1-10), 2 samples of the 2nd collection sheets (F2-1 and F2 6) Samples 4 and leaves the remaining canopy (3-Fr, Fr-6, 7-Fr and Fr-10). This grouping together the samples characterized by having high levels of 1-nitro-

Fig. 1. Dendrogram corresponding to the classification of the essential oils of essential oils of leaves of A. canelilla.

Please cite this article in press as: Barbosa, P.C.S., et al., New and sustainable essential oils obtained from the long-term explored cinnamomum-like Aniba canelilla. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.11.002

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Fig. 2. Chromatographic profile of samples essential oils of leaves of A. canelilla, cluster I.

2-phenylethane with concentrations ranging between 68.30% and 84.39% of that benzenoid. The Cluster II was divided into two sub-clusters, IIA and IIB. The sub-cluster IIA includes 4 samples: 2 samples of the 2nd collection sheets (F2-9 and F2-10) and 2 samples of leaves of the remaining crown (Fr-1 and Fr-9). This grouping brings together the samples characterized by having reduced concentration of 1-nitro2-phenylethane among the analyzed samples. Only two samples (F2-9 and Fr-1) in this group had 1-nitro-2-phenylethane as a major constituent, with levels varying between 13:17% and 16.65%. The F2-10 samples and Fr-9 presented as main constituent the ␤selinene and aromadendrene respectively; however, the content of 1-nitro-2-phenylethane these samples are similar to the levels in other samples of that benzenoid group, for which reason they are the same group. The sub-cluster IIB includes 6 samples: 2 samples of the 1 st collection sheets (F1-1 and F1-9), 3 samples of the 2nd collection sheets (F2-3, F2-7 and F2-8) and a sample leaves the remaining canopy (Rf = 8). This grouping together the samples characterized by having intermediate levels of 1-nitro-2-phenylethane with concentrations ranging between 31.22% and 58.87%. In summary, we observed that groups I and II correspond to samples with levels of 1-nitro-2-phenylethane above 60% and samples with levels of 1-nitro-2-phenylethane less than 60%, respectively. It is common that 1-nitro-2-phenylethane can be found as the main constituent of essential oils A. canelilla, although the contents of that benzenoid may undergo variations, as observed in this study. However, it is common for this variation is linked to seasonality, a fact that was not observed in this study. Although the yields 1-nitro-2-phenylethane occurred at similar ratios within each of the three groups, it was possible to observe a variation of that benzenoid levels between the three groups. In the dendrogram you can see that there is no difference between the samples of the 1 st collection, the 2nd collection and samples of the remaining cup, as it was possible to observe leaves collected at different times in the same grouping. However, two qualitatively different chromatographic profiles were observed (Figs. 2 and 3). In general, Group I samples (Fig. 2), with high levels of 1-nitro2-phenylethane showed less complex and less qualitative variation chromatographic profiles when compared to the Group II samples

(Fig. 3), with lower levels of 1-nitro-2-phenylethane, that showed the highest qualitative variation and complex chromatographic profiles. Although only 30 constituents have been identified in the leaves, the number of detected constituent (total of identified constituents unidentified +) ranged between 30 and 49 samples in the cluster I and cluster II, the number of components varied between 44 and 97. 3.3. Principal component analysis (PCA) of GC–MS and GC-FID fingerprint of essential oils of leaves of A. canelilla The PCA was performed by means of linear combinations of original 10 variables selected: 1-nitro-2-phenylethane, benzaldehyde, benzonitrile, benzene acetaldehyde, benzene acetonitrile, ␤elemene, ␤-caryophyllene, ␣-selinene, ␤-selinene and ␦-cadinene. These variables were selected because of their frequency and importance, since it was detected in all samples analyzed and leaves showed significant variations in their levels. Thus, the matrix arrangement for the principal component analysis consisted of 21 lines, corresponding to the samples, and 10 columns, corresponding to the selected variables. The PCA showed the distribution of the samples and the value of the variable. The result of PCA was represented in the form of scores and loading graphs (Figs. 4a and 4b, respectively), that is, the distribution graphs of the samples according to the significance of the variables. The charts of scores and loadings, both of dimension 1 (Dim 1) with 58.93% of the variance versus dimension 2 (Dim 2) with 24.15% of the variance, which is 83.08% of the total information. In these figures, it is possible to note that the first dimension separates the essential oils from leaves of A. canelilla with levels of 1-nitro-phenylethane above 60% and low contents of benzaldehyde, benzonitrile, ␤-elemene, ␤-caryophyllene, ␣ and ␤selinene ␦-cadinene and scores negative in value, of the oils with concentrations of 1-nitro-phenylethane below 60% and benzaldehyde contents, benzonitrile, ␤-elemene, ␤-caryophyllene, ␣ and ␤-selinene and ␦- cadinene higher than the other samples, scores positive values; Dimension 2 separates the oils with higher levels of benzaldehyde, benzonitrile, benzenoacetaldehyde, benzenoacetonitril and ␤-caryophyllene in positive scores values, oils with

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Fig. 3. Chromatographic profile of samples essential oils of leaves of A. canelilla, cluster II.

Fig. 4. Graphs of (a) scores and (b) loadings for Dim 1 vs. Dim 2, corresponding to the of the essential oils of leaves of A. canelilla.

higher ␤-elemene levels, ␣ and ␤-selinene and ␦-cadinene in negative scores. In summary, three different types of essential oils from leaves of A. canelilla were observed. The first type (cluster I) correspond to samples rich in 1-nitro-2-phenylethane with concentrations ranging between 68.30% and 84.39%, and minor amounts of benzaldehyde, benzonitrile, ␤-elemene, ␤-caryophyllene, ␣ and ␤selinene and ␦-cadinene observed between samples. The second type (sub-cluster IIA) corresponds to samples with lower levels 1-nitro-2-phenylethane, with levels varying between 13.17% and 16.65%, ␤-elemene and contents, and ␣ and ␤-selinene ␦- cadinene superior to the samples of the third type I. The Cluster (Sub Cluster IIB) with intermediate levels of 1-nitro-2-phenylethane with concentrations ranging between 31.22% and 58.87%, and levels of ␤-elemene content, ␣ and ␤-selinene and ␦-cadinene comparable to the samples Cluster IIA, and also higher than the levels of the samples Cluster I.

3.4. Hierarchical cluster analysis (HCA) of GC–MS and GC-FID fingerprint of essential oils of stems of A. canelilla The chromatographic analyzes of 24 samples of branches were also submitted to HCA and PCA for classification. Based on the levels of each compound present, determined by GC-FID and GC–MS, constructed a data matrix (24 rows x 22 columns): being 24 lines corresponding to the samples and 22 columns corresponding to the contents of the identified constituents and quantified in samples of branches. Preprocessing of the data for normalization was also performed for the log and the similarity between the samples was also calculated from the Euclidean distance, using Ward’s method. The results of the HCA the branches are represented in the following dendrogram (Fig. 5), through which you can see two groups (clusters) to separate the branches. Cluster I included 8 samples: 4 samples branches of the 2nd collection (G2-3, G2-5, G2-6 and G2-10) and 4 samples of branches

Please cite this article in press as: Barbosa, P.C.S., et al., New and sustainable essential oils obtained from the long-term explored cinnamomum-like Aniba canelilla. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.11.002

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Fig. 5. Dendrogram corresponding to the classification of the essential oils of essential oils of stems of A. canelilla.

of the remaining crown (Gr-1, Gr-5, GR 9 and Gr-10). This grouping brings together the samples characterized by having high levels of 1-nitro-2-phenylethane, but with lower levels dessebenzenóide between all samples, with levels ranging between 85.53% and 90.90%. The Cluster II was divided into two sub-clusters, IIA and IIB. The sub-cluster IIA includes 5 samples: 4 samples of branches of the 1 st collection (G1-5, G1-6, G1-8 and G2-10) and a branch of the sample remaining canopy (Gr-9). This grouping brings together the samples characterized by having the highest levels of 1-nitro2-phenylethane among all samples, with levels ranging between 93.00% and 94.16%. The sub-cluster IIB includes 11 samples: 4 samples branches of the 1 st collection (G1-1, G1-3, G1-7 G1-9 and), 3 samples branches of the 2nd collection (g2-1, and G2-7 G2-8) and 4 samples of the remaining branches canopy (Gr-3, 6-Gr, Gr Gr-7 and-8). This grouping together the samples characterized by having intermediate levels of 1-nitro-2-phenylethane with concentrations ranging between 90.47% and 92.15%. Again it was not possible to distinguish between samples of the 1st collection, the 2nd collection and samples of the remaining canopy in the dendrogram, as we observed branches collected at different times in the same grouping. It was also possible to see the homogeneity of the samples of the branches that have the same qualitative profile (Fig. 6). As the complexity of the chromatographic profile, while only 22 components have been identified in the branches, the number of detected constituent ranged between 28 and 37 samples in cluster i, and vary between 21 and 33 samples in cluster II. That is, there was almost no qualitative variation between samples branches. However, it was noticeable that the chromatographic profile is very different branches of the chromatographic profile of the leaves, which is much more complex, both qualitatively and quantitatively. Although the yields 1-nitro-2-phenylethane occurred in similar proportions between the two groups of branches, other variables allow the distinction of samples. The benzenoacetaldehyde and linalool, for example, although in small concentrations, showed variations in their levels in the various groups; while methyleugenol, commonly described as an important constituent in A. canelilla oils, was identified in only two samples of 8 samples analyzed and grouped in Cluster I, and identified 12 samples of the 16 samples analyzed and grouped in Cluster II. To assess the importance of these variables, it then performed the PCA also for samples of branches.

3.5. Principal component analysis (PCA) of GC–MS and GC-FID fingerprint of essential oils of stems of A. canelilla The PCA was performed through the fourth linear combinations of original variables selected: 1-nitro-2-phenylethane, benzenoacetaldehyde, linalool and methyleugenol. These variables were selected due to their frequency and importance − since it was detected in all samples analyzed leaves and showed large variations in their levels − in the case of the first and due to the absence/presence of the last variable in the case that It is described as one of A. canelilla oils markers. Thus, the matrix arrangement for the principal component analysis consisted of 21 lines corresponding to samples and 4 columns corresponding to the selected variables. The result is shown in the PCA scores plots and loading (Fig. 7a and b, respectively). The charts of scores and loadings, both of dimension 1 (Dim 1) with 43.37% of the variance versus dimension 2 (Dim 2) with 27.13% of the variance, which is 70.50% of the total information. In these figures, it is possible to note that the first dimension separates the essential oils of A. canelilla branches with levels of 1nitro-phenylethane above 90% and higher levels of benzaldehyde and methyleugenol in positive scores values, the oils with concentrations of nitro-1-phenylethane below 90% and higher linalool contents, in scores negative values. The second dimension separates the oils with higher levels benzenoacetaldehyde scores in positive values, oils with lower benzenoacetaldehyde levels in negative scores. In summary, three different types of essential oil from branches A. canelilla were observed. The first type (cluster I) corresponds to samples with lower levels of 1-nitro-2-phenylethane observed compared to samples Cluster II with levels varying between 85.53% and 90.90%, the Cluster I higher contents of benzenoacetaldehyde and linalool, and the absence of methyleugenol in 6 samples of 8 samples of the group. The second type (Sub-cluster IIA) corresponds to the samples with the highest levels of 1-nitro-2-phenylethane (93.00% and 94.16%), but with smaller benzenoacetaldehyde contents and linalool and presence of methyleugenol. The third type (sub-cluster IIB) has intermediate levels of 1-nitro-2-phenylethane (90.47% and 92.84%) but with high levels of benzenoacetaldehyde, low levels of linalool and methyleugenol presence of 7 samples in the 11 samples group. Several factors may influence the yield and chemical composition of essential oils, such as mechanical, chemical or biological

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Fig. 6. Chromatographic profile of samples essential oils of stems of A. canelilla.

Fig. 7. Graphs of (a) scores and (b) loadings for Dim 1 vs. Dim 2, corresponding to the of the essential oils of stems of A. canelilla.

injuries such as herbivory (Figueiredo et al., 2008; Edwards et al., 1993.). produce even more essential oil that the branches of the remaining Environmental factors such as light and moisture can also have canopies, suggesting that the branches produce more essential oil large effects on the yield and composition of essential oils in the early stage of its development. Leaves income did not show (Sangwan et al., 2001), and can be the cause of seasonal variation any difference between the different phases. However, the produc(Taveira et al., 2003). Different plant organs and their stages of tion of essential oil of regrowth of leaves was the favored canopy development are also described as possible yield of varying ratios opening, indicating that the light increases the production of essenand chemical composition (Sangwan et al., 2001) tial oil on the sheets. In addition, the essential oil yield of branches Manhães et al., (2012) studied the yield and leaf composition and was higher in the dry season, when compared with the rainy seaA. canelilla branches collected at different stages of development, son the same year; while the oil yield of the leaves did not change where we analyzed the sampling stations (dry and wet), openin the meantime. In this case, pruning would be more profitable if ing the canopy (the ground level and crown) and damage caused the branches were collected during the dry season due to higher by herbivory, and observed that the essential oils of leaves and essential oil production. The production of oil leaves and twigs was branches of A. canelilla have different incomes and that this difnot influenced by herbivores (Manhães et al., 2012). ference is more pronounced in regrowth branches. The production The chemical composition, the main components identified in of oil is higher in branches than in leaves and regrowth branches the oils A. canelilla were. The content of 1-nitro-2-phenylethane,

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eugenol and methyleugenol differ between the leaves and branches. The content of 1-nitro-2-phenylethane were significantly higher in the branches (92.7 ± 2.69%) and have lower internal variation of this constituent than in leaves (25.15 ± 52.2%), which in turn, had higher levels of eugenol and methyleugenol the branches (Manhães et al., 2012). In the present study we observed that seasonality and the stage of development of the leaves and branches did not affect the chemical composition, as leaves and twigs collected in different seasons, at different stages of development in the same area, are in a same group of similar chemical compositions, particularly characterized by the predominance of 1-nitro-2-phenylethane. It is only possible to observe that the chemical composition of leaves and branches is different and that the qualitative and quantitative chemical variation is greater in leaves than in the collected branches. 4. Conclusion Three different types of essential oils leaves and three different types of branches A. canelilla oils were distinguished. In general, the content of 1-nitro-2-phenylethane were significantly higher in branches and have lower internal variation of this constituent than in leaves. The three different types of essential oils from leaves of A. canelilla had the highest levels of 1-nitro-2-phenylethane (68.30%–84.39%), minor amounts of 1nitro-2-phenylethane (13.17%–16.65%) and intermediate levels of 1-nitro-2-phenylethane (31.22% and 58.87%). To the branches, the other three different types of essential oils were found: all with similar content for the 1-nitro-2-phenylethane, but the first type was characterized by having higher contents of benzenoacetaldehyde and linalool, and the absence of methyleugenol in 6 samples of 8 samples of the group. The second type is characterized by having the lower levels of benzenoacetaldehyde and linalool and presence of methyleugenol. Finally, the third type, characterized by the highest levels benzenoacetaldehyde, low linalool contents and the presence of methyleugenol in 7 of the 11 samples of the sample group. The multivariate analysis allowed the observation that the seasonality and the stage of development of the leaves and branches did not affect the chemical composition of the essential oils of this species. From these results, it is possible to propose that these oils can be used in the manufacture of different products with various applications, since all major constituents, present in varying levels as oil chemotype, are of great importance in the pharmaceutical and cosmetics, with different purposes. Acknowledgments The authors thank Foundation for the Amazonas State Research (FAPEAM), Higher Education Personnel Improvement Coordination (CAPES) and National Council for Scientific and Technological Development (CNPq) for financial support. References Adams, R.P., 2007. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy, Allured: Card Stream I L. Barata, L.E.S., Lupe, F., De, A., 2007. Study Composition Essential Oils Chemistry In The Amazon Aromatic Plants. Editora Unicamp, Campinas, SP.

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Please cite this article in press as: Barbosa, P.C.S., et al., New and sustainable essential oils obtained from the long-term explored cinnamomum-like Aniba canelilla. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.11.002