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Oxidation of monoterpenes in Protium heptaphyllum oleoresins
Phytochemistry xxx (2017) 1e6
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Oxidation of monoterpenes in Protium heptaphyllum oleoresins Rayane C. Albino a, Prissila C. Oliveira a, Francisco Prosdocimi b, Osman F. da Silva e, ssia M. Sakuragui d, Carolina Furtado f, Humberto R. Bizzo c, Paola E. Gama c, Ca Danilo R. de Oliveira a, * a
Faculty of Pharmacy, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, RJ, Brazil Institute of Medical Biochemistry Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, RJ, Brazil c EMBRAPA Food Technology, Rio de Janeiro 23020-470, RJ, Brazil d Institute of Biology, Department of Botany, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, RJ, Brazil e Chemistry College, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, RJ, Brazil f National Cancer Institute (INCA), Rio de Janeiro 20231-050, RJ, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 April 2016 Received in revised form 16 January 2017 Accepted 21 January 2017 Available online xxx
Protium heptaphyllum (Burseraceae) oleoresins are rich in volatile monoterpenes, exhibiting a chemical composition that can be strongly altered with time. The present work aimed to discuss the temporal change of the volatile composition of these oleoresins, and search for related supporting evidence. Samples of P. heptaphyllum oleoresin were collected separately for fresh (n ¼ 10) and aged (n ¼ 8) oleoresins, with the essential oils obtained by hydrodistillation analyzed by GC-FID and GC-MS. Fresh oleoresins were characterized by a high content of terpinolene (28.2e69.7%), whereas aged ones contained large amounts of p-cymene (18.7e43.0%) and p-cymen-8-ol (8.2e31.8%). Multivariate analyses were performed based on the yield and major essential oil components to clearly demonstrate the existence of two subsets (fresh and aged oleoresins). In addition, an analysis of the partial genome sequencing of the species was carried out, producing the largest amount of data for the genus Protium. Subsequently, were searched for nucleotide sequences responsible for the enzymes involved in the biosynthesis of monoterpenes. Two hypotheses were formulated to understand the oxidation process during aging of the oleoresins: (i) a natural chemical oxidation of terpenes and (ii) an oxidation catalyzed by enzymes produced by microorganisms associated with the plant. The results suggested that terpinolene was most likely oxidized to p-cymene, which, in turn, was oxidized into p-cymen-8-ol during natural aging of the exudate due to abiotic factors. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Protium heptaphyllum Burseraceae Terpinolene p-Cymene p-Cymen-8-ol Monoterpene oxidation Genome Multivariate analysis
1. Introduction Protium heptaphyllum (Aubl.) Marchand belongs to the Burseraceae family of trees, that account for an important part of the structure and diversity of both humid and dry forests in many parts of the tropics (Daly et al., 2012). Trees of P. heptaphyllum can reach up to 20 m in height and 60 cm in trunk diameter, often occurring in riparian semi-decidual forests (Lorenzi, 2008). Protium species are well known to produce secondary metabolites featuring different types of terpenes, with more than 100 different mono-
* Corresponding author. Universidade Federal do Rio de Janeiro, Faculty of Pharmacy, Natural Products and Foods Department, Avenue Carlos Chagas Filho, ria, RJ, Brazil. 373, Bl.A2, Sl.01, 21941-902, Fund~ ao Island, Cidade Universita E-mail addresses: [email protected], [email protected] (D.R. de Oliveira).
and sesquiterpenes characterized (Siani et al., 2004; Silva et al., 2009; Marques et al., 2010). In view of this, Protium species have been used as models to better understand the natural variation in the genes underlying monoterpene synthesis and the possible drivers of such variation (Zapata and Fine, 2013). Plants of the Protium genus store oleoresins in secretory structures of their bark until injury allows exudation (Langenheim, 2003). The nonvolatile fraction of oleoresins is rich in triterpenoids, e.g., a-amyrin and b-amyrin, while the volatile fraction is composed predominantly of monoterpenes (Rüdiger et al., 2007). Monoterpene production is catalyzed by enzymes encoded by terpene synthase genes that belong to a highly diverse TPS gene family (Bohlmann et al., 1998). The evolutionary history of monoterpene synthases (TPSb) within Protium in the context of the TPS family has been assessed by Zapata and Fine (2013), who concluded that Protium retained at least three, and possibly up to five, copies
http://dx.doi.org/10.1016/j.phytochem.2017.01.013 0031-9422/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Albino, R.C., et al., Oxidation of monoterpenes in Protium heptaphyllum oleoresins, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.013
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of TPSb genes, and suggested that this fact is associated with the monoterpene diversity expressed in the genus. Although Protium oleoresins have been studied from genetic, chemical, ecological, and evolutionary aspects, the mechanisms involved in the temporal changes of the produced chemical compounds have not been extensively discussed. The oleoresins of Protium can be characterized as fresh or aged, depending on their texture. Fresh oleoresins are known to have a soft, viscous, and malleable nature, which is partially due to the considerable amount of monoterpenes contained therein, which can exceed 20%. About three months after exudation, the oleoresins harden in the bark of the trees, becoming aged and displaying a solid and dry texture. The aged oleoresin can remain in trees for years, and its essential oil content can be reduced to less than 2% (Costa, 1975; Langenheim, 2003; Pontes et al., 2007; Da Silva et al., 2013). Despite the fact that aged oleoresins are mostly used in folk medicine, few studies comparing the composition of essential oils obtained from fresh and aged Protium oleoresins have been performed (Siani et al., 1999; Ramos et al., 2000; Pontes et al., 2007). The aim of the present work was to evaluate the temporal changes in the volatile composition of P. heptaphyllum oleoresins. Herein, essential oils from 18 samples of fresh and aged oleoresins were analyzed, and hypotheses explaining the chemical composition changes during aging were discussed. 2. Results 2.1. Chemical compositions and yields of essential oils in fresh and aged P. heptaphyllum oleoresins The essential oil yields of P. heptaphyllum oleoresins ranged from 2.1 to 20.0% (Table 1, Supplementary Material), with best yields obtained for fresh oleoresins (3.4e20.0%) and the lowest ones for aged oleoresins (2.1e6.1%). The average yield of essential oils for fresh oleoresins (12.63 ± 5.05%) was approximately four times greater than that for aged ones (3.24 ± 1.58%). The chemical compositions of the essential oils of P. heptaphyllum oleoresins are given in Table 1 (Supplementary Material), the major components being terpinolene (1) (8.8e69.7%), p-cymene (2) (4.3e43.0%), p-cymen-8-ol (3) (2.7e31.8%), a-pinene (4) (3.6e19.4%), and limonene (5) (5.8e11.6%), resembling the chemical profile observed by Lima et al. (2016). a-Terpinene (6) (0.8e10.4%) and g-terpinene (7) (0.3e4.6%) were also present in relatively high amounts in the essential oils (Fig. 1). A large quantitative difference in the content of p-cymene (2), terpinolene (1), and p-cymen-8-ol (3) monoterpenes was observed between the essential oils of fresh and aged oleoresins (Fig. 2). The essential oils obtained from fresh oleoresins (n ¼ 10) mainly contained terpinolene (1) (28.2e69.7%) and p-cymene (2) (4.3e23.3%), whereas the ones obtained from aged oleoresins (n ¼ 8) mainly contained p-cymene (2) (18.7e43.0%) and p-cymen-8-ol (3) (8.2e31.8%), followed by terpinolene (1) (8.8e20.9%). Interestingly, the content of p-cymene (2) is often high in aged essential oils (Hausen et al., 1999; Misharina and Polshkov, 2005; Turek and Stintzing, 2013), with a large reduction of terpinolene (1) content recently reported to occur in the essential oils of oregano and laurel after days of storage at 60 C (Olmedo et al., 2015). All essential oils comprised monoterpene hydrocarbons (60.8e93.0%), represented mainly by terpinolene (1) and p-cymene (2), and oxygenated monoterpenes (4.8e35.8%), mainly represented by p-cymen-8-ol (3). Notably, the essential oils of aged oleoresins contained more oxygenated monoterpenes (9.1e35.8%) and consequently less monoterpene hydrocarbons (4.5e12.4%) than those of fresh oleoresins (Table 1). Similarly to the oxygenated monoterpenes, the content of aromatic monoterpenes was also increased in the essential oils of aged oleoresins (46.3e59.6%)
Fig. 1. Major components of Protium heptaphyllum oleoresin essential oils.
compared to those of fresh oleoresins (10.0e33.2%). 2.2. Partial genome analysis A total of 34,332,626 paired-end sequencing reads were produced by partial genome analysis, totaling 8.93 billion of DNA nucleotide bases. The sequencing reads were subjected to a cleaning procedure using Trimmomatic software (Bolger et al., 2014), which discarded most sequences from the R2 dataset due to the presence of sequencing adapters or low quality. The cleaned dataset contained 4,481,402 high-quality paired-end reads and 29,845,377 unpaired reads (mainly from the R1 dataset), totaling 4.74 gigabases of genomic information. The cleaned reads were used as a query for GMAP alignment against a pre-built catalog of seven enzymes downloaded from the KEGG database, known to account for the biosynthesis of monoterpenes found in other plants. 42 sequencing reads were found with similarities to 14 K15086 (3S)-linalool synthase [EC:4.2.3.25]; 10 reads with similarities to 10 K12467 myrcene/ocimene synthase [EC:4.2.3.15]; 42 reads similar to 2 K12467 b-myrcene/(E)-b-ocimene synthase [EC:4.2.3.15]; 25 reads with similarities to the two paralogs of 1,8-cineole synthase K07385 [EC:4.2.3.108]; and 9 reads with similarities to the two paralogs of a short-chain dehydrogenase/reductase, namely 2 K15095 (þ)-neomenthol dehydrogenase [EC:1.1.1.208]. Although the above observation proves that Protium shows monoterpene metabolism, it does not clarify whether the conversion of terpinolene (1) to p-cymene (2) and p-cymen-8-ol (3) is due to its metabolism. Actually, the reported investigations of enzymes responsible for the conversion of terpinolene (1) to other compounds are scarce. The KEGG database (KEGG, 2015) presents the most comprehensive catalog of enzymes and metabolic pathways known and contains only enzymes that convert p-cymene (2) to another compound, named p-cumate. This suggests that p-cymene (2) cannot be converted to p-cymen-8-ol (3) through the action of any presently known enzyme. 2.3. Chemometric statistical analysis Prior to multivariate analysis, an analysis of variance (ANOVA) was performed to determine which conditions, if any, influence the yields and monoterpene contents. The collection time and oleoresin type (fresh or aged) were significantly (p < 0.05) important for essential oil yields, with the mean values obtained for terpinolene
Please cite this article in press as: Albino, R.C., et al., Oxidation of monoterpenes in Protium heptaphyllum oleoresins, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.013
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Fig. 2. Major components of Protium heptaphyllum oleoresin essential oils. Results shown were obtained from one subject (tree 1) and are expressed as mean ± SD of the data obtained from GC-FID (Table 1), performed in triplicate. The significance of these results is discussed in the Chemometric statistical analysis section for all subjects and variables involved.
(1), p-cymene (2), and p-cymen-8-ol (3) differing significantly among the oleoresin types and tree individuals. Principal component analysis (PCA) of the variables generated two factors (principal components) that together accounted for almost 80% of the total data variance. The use of two factors to represent all present variables was reinforced by the use of the Eigenvalues plot, which showed just two Eigenvalues exceeding unity for these data (data not shown). Factor 1 (57.35%) was found to be well correlated with most variables (Fig. A, Supplementary Material), except for a-pinene (4) and limonene (5), which were both better correlated with Factor 2 (21.43%). The conducted PCA (Fig. B, Supplementary Material) generated a graph, whose case coordinates showed the same segregation pattern seen in the earlier cluster analysis (data not shown). Fresh oleoresins were restricted to the negative values of Factor 1, and aged oleoresins to the positive ones. Another important observation was the fact that most cases with positive coordinates for Factor 2 belong to the data obtained from tree 1, demonstrating an evident and direct relationship between a-pinene (4) content and this specimen (see both plots; Figs. A and B). In addition, these results clearly show a direct relationship between aged oleoresins and two continuous variables, p-cymene (2) and p-cymen-8-ol (3) content. In the same way, fresh oleoresins were directly correlated to other four continuous variables: essential oil yields and the content of terpinolene (1), a-terpinene (6), and g-terpinene (7). 3. Discussion The compound modification seems to show dynamics as the oleoresin ages. To explain the origin of this dynamics, are suggested two hypotheses. The first and probably most likely one assumes that an abiotic chemical oxidation is responsible for the conversion of terpinolene (1) into p-cymene (2), with subsequent oxidation of p-cymene (2) into p-cymene-8-ol (3). Some relevant literature data corroborate this interpretation. pCymene (2) and p-cymen-8-ol (3) have been identified as products of terpinolene (1) oxidation, and p-cymene (2) has also been identified as a product of terpinolene (1) isomerization (Borglin et al., 1950; Matsuura et al., 1960; Mizrahi and Nigam, 1966; Comelli et al., 2005; Zhao et al., 2011). The chemical oxidation of p-cymene (2) to p-cymen-8-ol (3) has also been reported by independent groups (Bi et al., 2012; Martins et al., 1999; Matsuura et al., 1960). Furthermore, Turek and Stintzing (2011) observed a significant increase in the p-cymene (2) content accompanying the
decrease of terpinolene (1) content in pine essential oil after weeks of storage under stress conditions. The same compound dynamics was observed by Brophy et al. (1989) for tea tree essential oil and by Nguyen et al. (2009) for lemon essential oil. Terpinolene (1) has a doubly activated allylic position, which is very susceptible to oxidation (Matsuura et al., 1960; Lempers and Sheldon, 1996; Grassmann et al., 2005). It is therefore likely that terpinolene (1) undergoes isomerization into a- and g-terpinenes (6 and 7) as the first step of aromatization. The obtained a- and gterpinenes (6 and 7) are then dehydrogenated to form p-cymene (2) (Mizrahi and Nigam, 1966; Stanislaus and Yeddanapalli, 1972; Grau et al., 1999; Bueno et al., 2008; Zhao et al., 2011), in agreement with the findings here. Although the content of both compounds in the essential oils was not very high, it was significant, and similarly to terpinolene (1), their content was reduced in aged oleoresin essential oils compared to fresh ones. In the same way, according to PCA analysis, although the five compounds (terpinolene (1), a- and g-terpinenes (6 and 7), p-cymene (2), and p-cymene-8-ol (3)) were all correlated with principal component 1 (Fig. A), each of them was located at one specific quadrant (except for a- and g-terpinenes (6 and 7), which are parallel compounds in the “metabolic route”), showing a certain reverse relationship to its own direct parent compound. This result is in accordance with literature data, showing the existence of a linear transformation of terpinolene (1) to p-cymen-8-ol (3), which can be represented by a variable flux in the clockwise direction of Figure A (Supplementary Material). Notwithstanding, in order to produce a larger amount of data for P. heptaphyllum and to explore other possible explanations, was sequenced a partial genomic dataset for P. Heptaphyllum with the objective of trying to search for enzymes responsible for monoterpene metabolism. Terpinolene (1) is generated from geranyl diphosphate (0), with some monoterpene synthases known to catalyze this reaction (Huber et al., 2005; Degenhardt et al., 2009). However, there is no evidence that terpinolene (1) could be a biosynthetic precursor of p-cymene (2). The only precursor of pcymene (2) reported in literature is g-terpinene (7) (Poulose and Croteau, 1978a, 1978b; Galata et al., 2014), but enzymes responsible for this conversion remain unknown, and mentions of pcymen-8-ol (3) in biosynthetic studies was not found in the literature. The current knowledge on the biosynthesis of the main monoterpenes found in P. heptaphyllum oleoresins is summarized in Fig. 3. The dotted lines in the figure represent the putative route for the formation of p-cymene (2) and p-cymen-8-ol (3). Alternatively, the KEGG database contains enzymes responsible
Please cite this article in press as: Albino, R.C., et al., Oxidation of monoterpenes in Protium heptaphyllum oleoresins, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.013
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Fig. 3. Suggested biosynthetic routes of the major monoterpenes found in P. heptaphyllum oleoresins adapted from the scheme proposed by Keszei et al. (2010).
for the conversion of p-cymene (2) into p-cumate in bacteria from the genus Bacillus. Therefore, it is hypothesized here that this transition might also occur in microorganisms associated with the plant and the corresponding oleoresin. However, enzymes converting p-cymene (2) to p-cymene-8-ol (3) are unknown, and no evidence was found for the presence of p-cumate here. The abiotic chemical conversion of terpinolene (1) into p-cymene (2) and p-cymen-8-ol (3) is the currently most likely hypothesis. In future, a stability study might solve this question. 4. Conclusion The fresh and aged P. heptaphyllum oleoresins differ greatly from each other in terms of chemical composition. The essential oils of fresh oleoresins mainly contain terpinolene (1), while those of aged oleoresins mainly contain p-cymene (2) and p-cymen-8-ol (3). Terpinolene (1) seems to be converted into p-cymene (2) and pcymen-8-ol (3) with time. This chemical conversion is most likely due to abiotic factors, but it may also be enzymatic. Stability tests of these essential oils and monoterpene standards under stress conditions and biosynthetic studies are approaches that can help to clarify this intriguing modification occurring in P. heptaphyllum oleoresins with time. 5. Experimental 5.1. Plant material Oleoresins were periodically collected from four Protium hep~o Joa ~o da Barra (S 21 taphyllum trees located at a cattle farm in Sa 310 34500 ; W 041 040 72200 ) during the years 2013 and 2014 in the state of Rio de Janeiro, where this plant is known as amescla or
cega. The collections (c) were named as follows: c1 e May alme 2013; c2 e October 2013; c3 e February 2014; c4 e June 2014. The 18 oleoresin samples obtained were separated into fresh (10) and aged oleoresins (8), with masses ranging from 15.18 to 369.1 g (Table 1). In addition to the physical and organoleptic characteristics, the bees and other insects (e.g., the ants seen in Fig. 4) generally surrounding fresh oleoresins aid their identification. A voucher collection is deposited in the RFA herbarium at the Federal University of Rio de Janeiro under the registration number 40,868 ^nico do and in the herbarium of Instituto de Pesquisa Jardim Bota Rio de Janeiro under the number RB 598,021. The material was identified by one of the authors (Sakuragui, C.M.) based on the material type, bibliography, and herbaria materials identified by world specialists.
5.2. Essential oil distillation and analysis The oleoresins (15 g for three samples and 50 g for other samples) were hydrodistilled in a Clevenger-type apparatus for 4 h, and the obtained essential oils were stored in dark glass bottles in a freezer until used. The oils were analyzed in a gas chromatograph (Agilent 7890A) equipped with a flame ionization detector (GCFID), using H2 as a carrier gas at a flow rate of 1 mL/min and an HP5 capillary column (5% phenyl methyl silicone, 30 m 0.25 mm 0.25 mm). The injector and the detector (FID) were maintained at 250 and 280 C, respectively. A 1-mL aliquot of a 1% CH2Cl2 solution of each oil was injected with a split ratio of 1:20. The temperature was programmed to rise from 60 to 240 C at 3 C/min. Each oil was injected in triplicate, and quantitation was performed using the normalization method based on the electronic integration of the FID peak areas.
Please cite this article in press as: Albino, R.C., et al., Oxidation of monoterpenes in Protium heptaphyllum oleoresins, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.013
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Fig. 4. a. fresh oleoresin; b. aged oleoresin.
Samples were subsequently analyzed using an Agilent 6890 gas chromatograph coupled to an Agilent 5973N mass spectrometer (GC-MS). Chromatographic and injection conditions were as above, except for the carrier gas, which was He (1.0 mL/min). Constituents were identified by comparison of their mass spectra with commercial databases (Wiley), and linear retention indices calculated from the injection of a series of n-alkanes (C7 to C26) for the same column and conditions as above (Van Den Dool and Kratz, 1963) were compared to literature data (Adams, 2007).
5.5. Catalog of enzymes related to the biosynthesis of monoterpenes
5.3. Chemometric statistical analysis
The cataloged enzymes were aligned against the database of sequencing reads using the GMAP algorithm. The reads found were aligned amongst themselves for building partial gene predictions for all genes found.
Multivariate analyses were employed to determine possible relationships between the content of volatile compounds from P. heptaphyllum oleoresins and the subject (tree), oleoresin age, and collection step. PCA was employed to confirm the results obtained by prior cluster analysis and determine the number of principal components needed to represent the largest variance of the present data, describing the related variables. The following variables were selected: yield, terpinolene (1), p-cymene (2), p-cymen-8-ol (3), apinene (4), limonene (5), a-terpinene (6), and g-terpinene (7) content. The selected variables were present in all samples and had a content of 1% in at least 50% of the samples. Statistical analysis was carried out using the STATISTICA software package (Version 7.0, Stat Soft, Inc., USA). Prior to multivariate analysis, ANOVA was performed to verify if the means of the selected essential oil components differed significantly (p < 0,05) among subjects (trees), oleoresin ages, and collection times/steps.
Enzymes responsible for monoterpene synthesis were obtained via manual curation of the KEGG database (Kanehisa et al., 2010) for the monoterpene biosynthesis pathway (map00902). Other enzymes were downloaded manually. 5.6. Finding and assembling genes for enzymes in the (partial) Protium genome database
Acknowledgements We thank Getúlio Ribeiro de Alvarenga for preservating P. heptaphyllum specimens in his property and for allowing us to Luiz Rodrigues Pinto for his helpful collect samples, and Andre knowledgement about this species and for his help in the collections. This works was supported by FAPERJ (E-26/110.727/2013 e APQ1) and PIBIC/UFRJ (undergraduate scholarship). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.01.013. References
5.4. DNA extraction and next-generation sequencing P. heptaphyllum DNA was extracted from leaf tissue using the DNAeasy blood & tissue kit (Quiagen). Partial genome sequencing was performed with Illumina HiSeq using a multiplexing kit (Nextera) as 1/10th of a lane at Instituto Nacional do Cancer (INCA) in Brazil. All sequencing reads were submitted into the SRA database at the NCBI under the BioSample ID SAMN06212794 and BioProject ID PRJNA360605.
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Oxidation of aromatic monoterpenes with hydrogen peroxide catalysed by Mn(III) porphyrin complexes. J. Mol. Catal. A Chem. 137, 41e47. Matsuura, T., Saito, K., Shimakawa, Y., 1960. Oxidation of terpene compounds with tert-butyl chromate. VI. Oxidation of terpinolene. Bull. Chem. Soc. Jpn. 33 (9), 1151e1154. Misharina, T.A., Polshkov, A.N., 2005. Antioxidant properties of essential oils: autoxidation of essential oils from laurel and fennel and of their mixtures with essential oil from coriander. Appl. Biochem. Microbiol. 41, 610e618. Mizrahi, I., Nigam, I.C., 1966. Reaction gas chromatography: II. Dehydrogenation of monoterpene compounds on platinum-alumina catalyst. J. Chromatogr. A 25, 230e241. Nguyen, H., Campi, E.M., Jackson, W.R., Patti, A.F., 2009. Effect of oxidative deterioration on flavour and aroma components of lemon oil. Food Chem. 112, 388e393. Olmedo, R.H., Asensio, C.M., Grosso, N.R., 2015. Thermal stability and antioxidant activity of essential oils from aromatic plants farmed in Argentina. Ind. Crops Prod. 69, 21e28. Pontes, W.J.T., Oliveira, J.C.S., C^ amara, C.A.G., Gondim Junior, M.G.C., Oliveira, J.V., leos essenciais de folhas e Schwartz, M.O.E., 2007. Atividade acaricida dos o caro rajado (Tetranychus urticae Koch). Quím. frutos de Xylopia sericea sobre o a Nova 30, 838e841. Poulose, A.J., Croteau, R., 1978b. Gamma-terpinene synthetase: a key enzyme in the biosynthesis of aromatic monoterpenes. Arch. Biochem. Biophys. 191 (1), 400e411. Poulose, A.J., Croteau, R., 1978a. Biosynthesis of aromatic monoterpenes. Conversion of g -terpinene to p-cymene and thymol in Thymus vulgaris L. Arch. Biochem. Biophys. 187, 307e314. ~es, A.C., Ribeiro, J.E.L.S., 2000. Ramos, M.F.S., Siani, A.C., Tappin, M.R.R., Guimara Essential oils from oleoresin of Protium spp. of the Amazon region. Flavour Fragr. J. 15, 383e387. Rüdiger, A.L., Siani, A.C., Veiga-Junior, V.F., 2007. The chemistry and pharmacology of the South America genus Protium Burm. F. (Burseraceae). Pharmacogn. Rev. 1, 93e104. Siani, A.C., Garrido, I.S., Monteiro, S.S., Carvalho, E.S., Ramos, M.F.S., 2004. Protium icicariba as a resource of volatile essences. Biochem. Syst. Ecol. 32, 477e489. Siani, A.C., Ramos, M.S.F., Guimar~ aes, A.C., Susunaga, G.S., Zoghbi, M.G.B., 1999. Volatile constituents from oleoresin of Protium heptaphyllum (Aubl.) March. J. Essent. Oil Res. 11, 72e74. Silva, J.R.A., Zoghbi, M.G.B., Pinto, A.C., Godoy, R.L.O., Amaral, A.C.F., 2009. Analysis of the hexane extracts from seven oleoresins of Protium species. J. Essent. Oil Res. 21, 305e308. Stanislaus, A., Yeddanapalli, L.M., 1972. Vapor phase catalytic transformations of terpene hydrocarbons in the C10H16 series. ii. Aromatization of a-pinene over chromiaealumina. Can. J. Chem. 50 (1), 113e118. Turek, C., Stintzing, F.C., 2011. Evaluation of selected quality parameters to monitor essential oil alteration during storage. J. Food Sci. 76, 1365e1375. Turek, C., Stintzing, F.C., 2013. Stability of essential oils: a review. Food Sci. Food Saf. 12, 40e53. Van Den Dool, H., Kratz, P.D., 1963. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr. A 11, 463e471. Zapata, F., Fine, P.V.A., 2013. Diversification of the monoterpene synthase gene family (TPSb) in Protium, a highly diverse genus of tropical trees. Mol. Phylogenetics Evol. 68, 432e442. Zhao, C., He, J., Lemonidou, A.A., Li, X., Lercher, J.A., 2011. Aqueous-phase hydrodeoxygenation of bio-derived phenols to cycloalkanes. J. Catal. 280, 8e16.
Please cite this article in press as: Albino, R.C., et al., Oxidation of monoterpenes in Protium heptaphyllum oleoresins, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.013
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Oxidation of monoterpenes in Protium heptaphyllum oleoresins Rayane C. Albino a, Prissila C. Oliveira a, Francisco Prosdocimi b, Osman F. da Silva e, ssia M. Sakuragui d, Carolina Furtado f, Humberto R. Bizzo c, Paola E. Gama c, Ca Danilo R. de Oliveira a, * a
Faculty of Pharmacy, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, RJ, Brazil Institute of Medical Biochemistry Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, RJ, Brazil c EMBRAPA Food Technology, Rio de Janeiro 23020-470, RJ, Brazil d Institute of Biology, Department of Botany, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, RJ, Brazil e Chemistry College, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, RJ, Brazil f National Cancer Institute (INCA), Rio de Janeiro 20231-050, RJ, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 April 2016 Received in revised form 16 January 2017 Accepted 21 January 2017 Available online xxx
Protium heptaphyllum (Burseraceae) oleoresins are rich in volatile monoterpenes, exhibiting a chemical composition that can be strongly altered with time. The present work aimed to discuss the temporal change of the volatile composition of these oleoresins, and search for related supporting evidence. Samples of P. heptaphyllum oleoresin were collected separately for fresh (n ¼ 10) and aged (n ¼ 8) oleoresins, with the essential oils obtained by hydrodistillation analyzed by GC-FID and GC-MS. Fresh oleoresins were characterized by a high content of terpinolene (28.2e69.7%), whereas aged ones contained large amounts of p-cymene (18.7e43.0%) and p-cymen-8-ol (8.2e31.8%). Multivariate analyses were performed based on the yield and major essential oil components to clearly demonstrate the existence of two subsets (fresh and aged oleoresins). In addition, an analysis of the partial genome sequencing of the species was carried out, producing the largest amount of data for the genus Protium. Subsequently, were searched for nucleotide sequences responsible for the enzymes involved in the biosynthesis of monoterpenes. Two hypotheses were formulated to understand the oxidation process during aging of the oleoresins: (i) a natural chemical oxidation of terpenes and (ii) an oxidation catalyzed by enzymes produced by microorganisms associated with the plant. The results suggested that terpinolene was most likely oxidized to p-cymene, which, in turn, was oxidized into p-cymen-8-ol during natural aging of the exudate due to abiotic factors. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Protium heptaphyllum Burseraceae Terpinolene p-Cymene p-Cymen-8-ol Monoterpene oxidation Genome Multivariate analysis
1. Introduction Protium heptaphyllum (Aubl.) Marchand belongs to the Burseraceae family of trees, that account for an important part of the structure and diversity of both humid and dry forests in many parts of the tropics (Daly et al., 2012). Trees of P. heptaphyllum can reach up to 20 m in height and 60 cm in trunk diameter, often occurring in riparian semi-decidual forests (Lorenzi, 2008). Protium species are well known to produce secondary metabolites featuring different types of terpenes, with more than 100 different mono-
* Corresponding author. Universidade Federal do Rio de Janeiro, Faculty of Pharmacy, Natural Products and Foods Department, Avenue Carlos Chagas Filho, ria, RJ, Brazil. 373, Bl.A2, Sl.01, 21941-902, Fund~ ao Island, Cidade Universita E-mail addresses: [email protected], [email protected] (D.R. de Oliveira).
and sesquiterpenes characterized (Siani et al., 2004; Silva et al., 2009; Marques et al., 2010). In view of this, Protium species have been used as models to better understand the natural variation in the genes underlying monoterpene synthesis and the possible drivers of such variation (Zapata and Fine, 2013). Plants of the Protium genus store oleoresins in secretory structures of their bark until injury allows exudation (Langenheim, 2003). The nonvolatile fraction of oleoresins is rich in triterpenoids, e.g., a-amyrin and b-amyrin, while the volatile fraction is composed predominantly of monoterpenes (Rüdiger et al., 2007). Monoterpene production is catalyzed by enzymes encoded by terpene synthase genes that belong to a highly diverse TPS gene family (Bohlmann et al., 1998). The evolutionary history of monoterpene synthases (TPSb) within Protium in the context of the TPS family has been assessed by Zapata and Fine (2013), who concluded that Protium retained at least three, and possibly up to five, copies
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Please cite this article in press as: Albino, R.C., et al., Oxidation of monoterpenes in Protium heptaphyllum oleoresins, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.013
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of TPSb genes, and suggested that this fact is associated with the monoterpene diversity expressed in the genus. Although Protium oleoresins have been studied from genetic, chemical, ecological, and evolutionary aspects, the mechanisms involved in the temporal changes of the produced chemical compounds have not been extensively discussed. The oleoresins of Protium can be characterized as fresh or aged, depending on their texture. Fresh oleoresins are known to have a soft, viscous, and malleable nature, which is partially due to the considerable amount of monoterpenes contained therein, which can exceed 20%. About three months after exudation, the oleoresins harden in the bark of the trees, becoming aged and displaying a solid and dry texture. The aged oleoresin can remain in trees for years, and its essential oil content can be reduced to less than 2% (Costa, 1975; Langenheim, 2003; Pontes et al., 2007; Da Silva et al., 2013). Despite the fact that aged oleoresins are mostly used in folk medicine, few studies comparing the composition of essential oils obtained from fresh and aged Protium oleoresins have been performed (Siani et al., 1999; Ramos et al., 2000; Pontes et al., 2007). The aim of the present work was to evaluate the temporal changes in the volatile composition of P. heptaphyllum oleoresins. Herein, essential oils from 18 samples of fresh and aged oleoresins were analyzed, and hypotheses explaining the chemical composition changes during aging were discussed. 2. Results 2.1. Chemical compositions and yields of essential oils in fresh and aged P. heptaphyllum oleoresins The essential oil yields of P. heptaphyllum oleoresins ranged from 2.1 to 20.0% (Table 1, Supplementary Material), with best yields obtained for fresh oleoresins (3.4e20.0%) and the lowest ones for aged oleoresins (2.1e6.1%). The average yield of essential oils for fresh oleoresins (12.63 ± 5.05%) was approximately four times greater than that for aged ones (3.24 ± 1.58%). The chemical compositions of the essential oils of P. heptaphyllum oleoresins are given in Table 1 (Supplementary Material), the major components being terpinolene (1) (8.8e69.7%), p-cymene (2) (4.3e43.0%), p-cymen-8-ol (3) (2.7e31.8%), a-pinene (4) (3.6e19.4%), and limonene (5) (5.8e11.6%), resembling the chemical profile observed by Lima et al. (2016). a-Terpinene (6) (0.8e10.4%) and g-terpinene (7) (0.3e4.6%) were also present in relatively high amounts in the essential oils (Fig. 1). A large quantitative difference in the content of p-cymene (2), terpinolene (1), and p-cymen-8-ol (3) monoterpenes was observed between the essential oils of fresh and aged oleoresins (Fig. 2). The essential oils obtained from fresh oleoresins (n ¼ 10) mainly contained terpinolene (1) (28.2e69.7%) and p-cymene (2) (4.3e23.3%), whereas the ones obtained from aged oleoresins (n ¼ 8) mainly contained p-cymene (2) (18.7e43.0%) and p-cymen-8-ol (3) (8.2e31.8%), followed by terpinolene (1) (8.8e20.9%). Interestingly, the content of p-cymene (2) is often high in aged essential oils (Hausen et al., 1999; Misharina and Polshkov, 2005; Turek and Stintzing, 2013), with a large reduction of terpinolene (1) content recently reported to occur in the essential oils of oregano and laurel after days of storage at 60 C (Olmedo et al., 2015). All essential oils comprised monoterpene hydrocarbons (60.8e93.0%), represented mainly by terpinolene (1) and p-cymene (2), and oxygenated monoterpenes (4.8e35.8%), mainly represented by p-cymen-8-ol (3). Notably, the essential oils of aged oleoresins contained more oxygenated monoterpenes (9.1e35.8%) and consequently less monoterpene hydrocarbons (4.5e12.4%) than those of fresh oleoresins (Table 1). Similarly to the oxygenated monoterpenes, the content of aromatic monoterpenes was also increased in the essential oils of aged oleoresins (46.3e59.6%)
Fig. 1. Major components of Protium heptaphyllum oleoresin essential oils.
compared to those of fresh oleoresins (10.0e33.2%). 2.2. Partial genome analysis A total of 34,332,626 paired-end sequencing reads were produced by partial genome analysis, totaling 8.93 billion of DNA nucleotide bases. The sequencing reads were subjected to a cleaning procedure using Trimmomatic software (Bolger et al., 2014), which discarded most sequences from the R2 dataset due to the presence of sequencing adapters or low quality. The cleaned dataset contained 4,481,402 high-quality paired-end reads and 29,845,377 unpaired reads (mainly from the R1 dataset), totaling 4.74 gigabases of genomic information. The cleaned reads were used as a query for GMAP alignment against a pre-built catalog of seven enzymes downloaded from the KEGG database, known to account for the biosynthesis of monoterpenes found in other plants. 42 sequencing reads were found with similarities to 14 K15086 (3S)-linalool synthase [EC:4.2.3.25]; 10 reads with similarities to 10 K12467 myrcene/ocimene synthase [EC:4.2.3.15]; 42 reads similar to 2 K12467 b-myrcene/(E)-b-ocimene synthase [EC:4.2.3.15]; 25 reads with similarities to the two paralogs of 1,8-cineole synthase K07385 [EC:4.2.3.108]; and 9 reads with similarities to the two paralogs of a short-chain dehydrogenase/reductase, namely 2 K15095 (þ)-neomenthol dehydrogenase [EC:1.1.1.208]. Although the above observation proves that Protium shows monoterpene metabolism, it does not clarify whether the conversion of terpinolene (1) to p-cymene (2) and p-cymen-8-ol (3) is due to its metabolism. Actually, the reported investigations of enzymes responsible for the conversion of terpinolene (1) to other compounds are scarce. The KEGG database (KEGG, 2015) presents the most comprehensive catalog of enzymes and metabolic pathways known and contains only enzymes that convert p-cymene (2) to another compound, named p-cumate. This suggests that p-cymene (2) cannot be converted to p-cymen-8-ol (3) through the action of any presently known enzyme. 2.3. Chemometric statistical analysis Prior to multivariate analysis, an analysis of variance (ANOVA) was performed to determine which conditions, if any, influence the yields and monoterpene contents. The collection time and oleoresin type (fresh or aged) were significantly (p < 0.05) important for essential oil yields, with the mean values obtained for terpinolene
Please cite this article in press as: Albino, R.C., et al., Oxidation of monoterpenes in Protium heptaphyllum oleoresins, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.013
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Fig. 2. Major components of Protium heptaphyllum oleoresin essential oils. Results shown were obtained from one subject (tree 1) and are expressed as mean ± SD of the data obtained from GC-FID (Table 1), performed in triplicate. The significance of these results is discussed in the Chemometric statistical analysis section for all subjects and variables involved.
(1), p-cymene (2), and p-cymen-8-ol (3) differing significantly among the oleoresin types and tree individuals. Principal component analysis (PCA) of the variables generated two factors (principal components) that together accounted for almost 80% of the total data variance. The use of two factors to represent all present variables was reinforced by the use of the Eigenvalues plot, which showed just two Eigenvalues exceeding unity for these data (data not shown). Factor 1 (57.35%) was found to be well correlated with most variables (Fig. A, Supplementary Material), except for a-pinene (4) and limonene (5), which were both better correlated with Factor 2 (21.43%). The conducted PCA (Fig. B, Supplementary Material) generated a graph, whose case coordinates showed the same segregation pattern seen in the earlier cluster analysis (data not shown). Fresh oleoresins were restricted to the negative values of Factor 1, and aged oleoresins to the positive ones. Another important observation was the fact that most cases with positive coordinates for Factor 2 belong to the data obtained from tree 1, demonstrating an evident and direct relationship between a-pinene (4) content and this specimen (see both plots; Figs. A and B). In addition, these results clearly show a direct relationship between aged oleoresins and two continuous variables, p-cymene (2) and p-cymen-8-ol (3) content. In the same way, fresh oleoresins were directly correlated to other four continuous variables: essential oil yields and the content of terpinolene (1), a-terpinene (6), and g-terpinene (7). 3. Discussion The compound modification seems to show dynamics as the oleoresin ages. To explain the origin of this dynamics, are suggested two hypotheses. The first and probably most likely one assumes that an abiotic chemical oxidation is responsible for the conversion of terpinolene (1) into p-cymene (2), with subsequent oxidation of p-cymene (2) into p-cymene-8-ol (3). Some relevant literature data corroborate this interpretation. pCymene (2) and p-cymen-8-ol (3) have been identified as products of terpinolene (1) oxidation, and p-cymene (2) has also been identified as a product of terpinolene (1) isomerization (Borglin et al., 1950; Matsuura et al., 1960; Mizrahi and Nigam, 1966; Comelli et al., 2005; Zhao et al., 2011). The chemical oxidation of p-cymene (2) to p-cymen-8-ol (3) has also been reported by independent groups (Bi et al., 2012; Martins et al., 1999; Matsuura et al., 1960). Furthermore, Turek and Stintzing (2011) observed a significant increase in the p-cymene (2) content accompanying the
decrease of terpinolene (1) content in pine essential oil after weeks of storage under stress conditions. The same compound dynamics was observed by Brophy et al. (1989) for tea tree essential oil and by Nguyen et al. (2009) for lemon essential oil. Terpinolene (1) has a doubly activated allylic position, which is very susceptible to oxidation (Matsuura et al., 1960; Lempers and Sheldon, 1996; Grassmann et al., 2005). It is therefore likely that terpinolene (1) undergoes isomerization into a- and g-terpinenes (6 and 7) as the first step of aromatization. The obtained a- and gterpinenes (6 and 7) are then dehydrogenated to form p-cymene (2) (Mizrahi and Nigam, 1966; Stanislaus and Yeddanapalli, 1972; Grau et al., 1999; Bueno et al., 2008; Zhao et al., 2011), in agreement with the findings here. Although the content of both compounds in the essential oils was not very high, it was significant, and similarly to terpinolene (1), their content was reduced in aged oleoresin essential oils compared to fresh ones. In the same way, according to PCA analysis, although the five compounds (terpinolene (1), a- and g-terpinenes (6 and 7), p-cymene (2), and p-cymene-8-ol (3)) were all correlated with principal component 1 (Fig. A), each of them was located at one specific quadrant (except for a- and g-terpinenes (6 and 7), which are parallel compounds in the “metabolic route”), showing a certain reverse relationship to its own direct parent compound. This result is in accordance with literature data, showing the existence of a linear transformation of terpinolene (1) to p-cymen-8-ol (3), which can be represented by a variable flux in the clockwise direction of Figure A (Supplementary Material). Notwithstanding, in order to produce a larger amount of data for P. heptaphyllum and to explore other possible explanations, was sequenced a partial genomic dataset for P. Heptaphyllum with the objective of trying to search for enzymes responsible for monoterpene metabolism. Terpinolene (1) is generated from geranyl diphosphate (0), with some monoterpene synthases known to catalyze this reaction (Huber et al., 2005; Degenhardt et al., 2009). However, there is no evidence that terpinolene (1) could be a biosynthetic precursor of p-cymene (2). The only precursor of pcymene (2) reported in literature is g-terpinene (7) (Poulose and Croteau, 1978a, 1978b; Galata et al., 2014), but enzymes responsible for this conversion remain unknown, and mentions of pcymen-8-ol (3) in biosynthetic studies was not found in the literature. The current knowledge on the biosynthesis of the main monoterpenes found in P. heptaphyllum oleoresins is summarized in Fig. 3. The dotted lines in the figure represent the putative route for the formation of p-cymene (2) and p-cymen-8-ol (3). Alternatively, the KEGG database contains enzymes responsible
Please cite this article in press as: Albino, R.C., et al., Oxidation of monoterpenes in Protium heptaphyllum oleoresins, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.013
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Fig. 3. Suggested biosynthetic routes of the major monoterpenes found in P. heptaphyllum oleoresins adapted from the scheme proposed by Keszei et al. (2010).
for the conversion of p-cymene (2) into p-cumate in bacteria from the genus Bacillus. Therefore, it is hypothesized here that this transition might also occur in microorganisms associated with the plant and the corresponding oleoresin. However, enzymes converting p-cymene (2) to p-cymene-8-ol (3) are unknown, and no evidence was found for the presence of p-cumate here. The abiotic chemical conversion of terpinolene (1) into p-cymene (2) and p-cymen-8-ol (3) is the currently most likely hypothesis. In future, a stability study might solve this question. 4. Conclusion The fresh and aged P. heptaphyllum oleoresins differ greatly from each other in terms of chemical composition. The essential oils of fresh oleoresins mainly contain terpinolene (1), while those of aged oleoresins mainly contain p-cymene (2) and p-cymen-8-ol (3). Terpinolene (1) seems to be converted into p-cymene (2) and pcymen-8-ol (3) with time. This chemical conversion is most likely due to abiotic factors, but it may also be enzymatic. Stability tests of these essential oils and monoterpene standards under stress conditions and biosynthetic studies are approaches that can help to clarify this intriguing modification occurring in P. heptaphyllum oleoresins with time. 5. Experimental 5.1. Plant material Oleoresins were periodically collected from four Protium hep~o Joa ~o da Barra (S 21 taphyllum trees located at a cattle farm in Sa 310 34500 ; W 041 040 72200 ) during the years 2013 and 2014 in the state of Rio de Janeiro, where this plant is known as amescla or
cega. The collections (c) were named as follows: c1 e May alme 2013; c2 e October 2013; c3 e February 2014; c4 e June 2014. The 18 oleoresin samples obtained were separated into fresh (10) and aged oleoresins (8), with masses ranging from 15.18 to 369.1 g (Table 1). In addition to the physical and organoleptic characteristics, the bees and other insects (e.g., the ants seen in Fig. 4) generally surrounding fresh oleoresins aid their identification. A voucher collection is deposited in the RFA herbarium at the Federal University of Rio de Janeiro under the registration number 40,868 ^nico do and in the herbarium of Instituto de Pesquisa Jardim Bota Rio de Janeiro under the number RB 598,021. The material was identified by one of the authors (Sakuragui, C.M.) based on the material type, bibliography, and herbaria materials identified by world specialists.
5.2. Essential oil distillation and analysis The oleoresins (15 g for three samples and 50 g for other samples) were hydrodistilled in a Clevenger-type apparatus for 4 h, and the obtained essential oils were stored in dark glass bottles in a freezer until used. The oils were analyzed in a gas chromatograph (Agilent 7890A) equipped with a flame ionization detector (GCFID), using H2 as a carrier gas at a flow rate of 1 mL/min and an HP5 capillary column (5% phenyl methyl silicone, 30 m 0.25 mm 0.25 mm). The injector and the detector (FID) were maintained at 250 and 280 C, respectively. A 1-mL aliquot of a 1% CH2Cl2 solution of each oil was injected with a split ratio of 1:20. The temperature was programmed to rise from 60 to 240 C at 3 C/min. Each oil was injected in triplicate, and quantitation was performed using the normalization method based on the electronic integration of the FID peak areas.
Please cite this article in press as: Albino, R.C., et al., Oxidation of monoterpenes in Protium heptaphyllum oleoresins, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.013
R.C. Albino et al. / Phytochemistry xxx (2017) 1e6
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Fig. 4. a. fresh oleoresin; b. aged oleoresin.
Samples were subsequently analyzed using an Agilent 6890 gas chromatograph coupled to an Agilent 5973N mass spectrometer (GC-MS). Chromatographic and injection conditions were as above, except for the carrier gas, which was He (1.0 mL/min). Constituents were identified by comparison of their mass spectra with commercial databases (Wiley), and linear retention indices calculated from the injection of a series of n-alkanes (C7 to C26) for the same column and conditions as above (Van Den Dool and Kratz, 1963) were compared to literature data (Adams, 2007).
5.5. Catalog of enzymes related to the biosynthesis of monoterpenes
5.3. Chemometric statistical analysis
The cataloged enzymes were aligned against the database of sequencing reads using the GMAP algorithm. The reads found were aligned amongst themselves for building partial gene predictions for all genes found.
Multivariate analyses were employed to determine possible relationships between the content of volatile compounds from P. heptaphyllum oleoresins and the subject (tree), oleoresin age, and collection step. PCA was employed to confirm the results obtained by prior cluster analysis and determine the number of principal components needed to represent the largest variance of the present data, describing the related variables. The following variables were selected: yield, terpinolene (1), p-cymene (2), p-cymen-8-ol (3), apinene (4), limonene (5), a-terpinene (6), and g-terpinene (7) content. The selected variables were present in all samples and had a content of 1% in at least 50% of the samples. Statistical analysis was carried out using the STATISTICA software package (Version 7.0, Stat Soft, Inc., USA). Prior to multivariate analysis, ANOVA was performed to verify if the means of the selected essential oil components differed significantly (p < 0,05) among subjects (trees), oleoresin ages, and collection times/steps.
Enzymes responsible for monoterpene synthesis were obtained via manual curation of the KEGG database (Kanehisa et al., 2010) for the monoterpene biosynthesis pathway (map00902). Other enzymes were downloaded manually. 5.6. Finding and assembling genes for enzymes in the (partial) Protium genome database
Acknowledgements We thank Getúlio Ribeiro de Alvarenga for preservating P. heptaphyllum specimens in his property and for allowing us to Luiz Rodrigues Pinto for his helpful collect samples, and Andre knowledgement about this species and for his help in the collections. This works was supported by FAPERJ (E-26/110.727/2013 e APQ1) and PIBIC/UFRJ (undergraduate scholarship). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.01.013. References
5.4. DNA extraction and next-generation sequencing P. heptaphyllum DNA was extracted from leaf tissue using the DNAeasy blood & tissue kit (Quiagen). Partial genome sequencing was performed with Illumina HiSeq using a multiplexing kit (Nextera) as 1/10th of a lane at Instituto Nacional do Cancer (INCA) in Brazil. All sequencing reads were submitted into the SRA database at the NCBI under the BioSample ID SAMN06212794 and BioProject ID PRJNA360605.
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Please cite this article in press as: Albino, R.C., et al., Oxidation of monoterpenes in Protium heptaphyllum oleoresins, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.013