Abstract
The essential oils from Aniba parviflora and the balsam from Protium rubrum, which are two woody scented Amazonian species, were analyzed by gas chromatography-mass spectrometry (GC-MS), 1H and 13C nuclear magnetic resonance (NMR). GC-MS analysis revealed the presence of unknown constituents. These compounds were then isolated, analyzed and characterized by NMR. Both substances presented distinct woody notes that contributed to the bulk woody character of the crude oils.
Keywords:
Aniba parviflora
;
Protium rubrum
; β-phellandrene; ∆4(4a)-(2R*; 8R*; 8aS*)-eremophilen-9-ol; thymol derivatives
Introduction
Brazil is one of world’s richest countries in terms of biodiversity, but it has made only a small contribution of new natural raw materials to the favors and fragrances (F&F) industry. Despite the growing number of published articles, the aromatic ingredients available from Brazilian plants in the F&F market are almost the same as 50 years ago: rosewood, copaiba and tonka beans.11 2013-2017 Flavor & Fragrance Industry Leaders, http://www.leffingwell.com/top_10.htm, accessed in September 2021.
http://www.leffingwell.com/top_10.htm...
With the aim of improving this situation, the chemical composition and potential application in the F&F market were examined for an essential oil and a balsam from the Amazon region after an evaluation by a perfumer.
The essential oil of Aniba parviflora (Meissner) Mez is produced by a species belonging to Lauraceae, a family possessing approximately 52 genera and 1900 species that are well distributed in the tropics.22 Souza-Junior, F. J. C.; Luz-Moraes, D.; Pereira, F. S.; Barros, M. A.; Fernandes, L. M. P.; Queiroz, L. Y.; Maia, C. F.; Maia, J. G. S.; Fontes-Junior, E. A.; Chem. Biodiversity 2021, 18, 10. Examples of Lauraceae essential oils that are consumed worldwide include bay (Laurus nobilis), cinnamon (Cinnamomum zeylanicum and Cinnamomum cassia), and camphor (Cinnamomum camphora). The genus Aniba is restricted to the Neotropical region, and rosewood (Aniba rosaeodora) is its most famous species.
Aniba parviflora is a small tree similar to rosewood, and it is often misclassified as a young A. rosaeodora; it is native to the central eastern Brazilian Amazon and occurs naturally near small rivers in non-fooded areas. The wounded bark is yellow in A. parviflora and reddish in rosewood, and there is a remarkable olfactory difference between them. Tw o A. parviflora experimental plantations that exist for commercial purposes can be found near the city of Belém, and these were made by mistake 15 years ago when the researchers thought they were planting rosewood. A third plantation is located between the Amazon and Tapajos rivers at the experimental campus of Amazon Federal Rural University (UFRA), and it is approximately 30 years old and occupies 5 hectares of a forest regeneration experimental station founded by FAO (Food and Agriculture Organization of United Nation). Plant material from this 30-year-old plantation was used for this study.
The other woody scent comes from Protium rubrum, known as “Cuatrec”, which is a rare Burseraceae distributed in Brazil, Peru and Colombia.33 Hopkins, M. J. G.; Rev. Saude Publica 2005, 56, 2175. The Burseraceae family has approximately 20 genera and 600 species that are well distributed in the tropics.44 Daly, D. C. B.: A Taxonomic Revision of Protium (Burseraceae) in Eastern Amazonia and Guianas; PhD thesis, City University of New York, New York, USA, 1987, available at https://www.proquest.com/docview/303462480?pq-origsite=gscholar&fromopenview=true, accessed in September 2021.
https://www.proquest.com/docview/3034624...
This family is well known for its triterpenic resins, a bark exudate, such as myrrh, olibanum and frankincense. In Brazil, this kind of resin is very common in the Amazon region and on the Bahia coast, where people use it to add perfume to their homes by burning it, or to seal boat hull cracks. Unlike other species of Protium trees, this P. rubrum has a balsam instead of a resin. Similar to Copaiba trees, the balsam is collected by utilizing a deep hole in the tree bark. This balsam does not have a traditional use and was discovered when farmers were cutting down an area for cassava cultivation in Silves, AM.
Experimental
Reagents and solvents
Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich (São Paulo, Brazil), and the solvents were of high-performance liquid chromatography (HPLC) grade (São Paulo, Brazil).
Plant material
Thin branches and leaves of A. parviflora were collected (year 2000) in the Curuá-una Experimental Field of the Wood Technology Center (CTM) of UFRA. The material (5200 kg) was collected for 3 days and was transported by river to the town of Santarem in a 24 h trip, where it was steam distilled in a 3000 L iron tank fed by an antique iron retort operating with a vapor fow of 30 kg h m3. The distillation tank could process 700 kg with each operation; therefore 3 days were required to perform the 8 necessary operations. Water came from an artesian well and went directly to the retort. The plant material was ground before undergoing distillation, and it was distilled for four hours at atmospheric pressure. The oil was treated with sodium sulfate before chromatographic analyses.
The Protium rubrum balsam was collected at the São Pedro community, Silves, in the central Amazon (year 2000). A tree was cut while an area was cleared for a cassava plantation, and the balsam was released from it in abundance. It was collected in a polyethylene bottle, brought to the city and transferred to a glass bottle. Before analysis, the balsam was treated with sodium sulfate and fltered.
Compound isolation
Sesquiterpenoid AF105 (later identified as eremophilen-9-ol, 1), which is responsible for some woody and floral notes, is present at 5% in A. parviflora essential crude oil. For isolation, 10 g of the essential oil was fractionally distilled with 10 g of paraffin (Synth, São Paulo, Brazil) to increase the thermal capacity of the system. The mixture was heated under a vacuum in an oil bath. The monoterpenes and nonoxygenated sesquiterpenes were distilled using up to 80% of the total oil. The cooled residue was then extracted 3 times with ethanol. This procedure increased the concentration of the woody scented target compound from 5 to 25%. Then, regular fractioning was performed using a silica column (Acros, São Paulo, Brazil, 0.035-0.070 mm particles with 6 nm pore diameter) followed by silica coated with 10% AgNO3 (Acros, São Paulo, Brazil). Both columns were eluted with hexane that contained increasing amounts of ethyl acetate. This procedure yielded compound 1 with 97% purity. Previous attempts without the fractionation/distillation step resulted in a contamination of oxygenated monoterpene after two chromatographic separations.
Compounds 5 and 6, isolated from P. rubrum, were obtained by single silica column fractionation.
Synthesis of the valerianol compound
To obtain the valerianol substrate, two synthetic steps were necessary.
(i) Trifluoroacetylation of valencene
Two grams of commercial Acros brand valencene (São Paulo, Brazil) were solubilized in 20 mL of hexane. The mixture was homogenized by adding 1.6 g of trifluoroacetic acid. Immediately the solution turned from colorless to violet.
The mixture remained under magnetic stirring at about 50 °C for 120 min. Due to the low yield verified by thin layer chromatography (TLC), the reaction was kept for another 24 h at room temperature. The mixture was washed with saturated sodium bicarbonate solution (NaHCO3), extracted with ethyl acetate and dried over anhydrous sodium sulfate (Na2SO4). The solvent was initially evaporated in a rotary evaporator and then in a N2 gas line. The product generated, valerianyl trifluoroacetate, was isolated by column chromatography.
(ii) Hydrolysis of valerianyl trifluoroacetate
Valerianyl trifluoroacetate (31.8 mg) and 4 mL of solvent were added as follows: 2.4 mL of tetrahydrofuran, 0.8 mL of methanol, 0.8 mL of water. After homogenization, 16.8 mg of hydrated lithium hydroxide (LiOH·H2O) was added. The mixture was kept under magnetic stirring in ambient temperature for 2 h.
It was then washed with 10% hydrochloric acid solution, extracted with ethyl acetate and dried over sodium sulfate. The solvent was removed in an analogous manner to the previous reaction. The alcohol formed, valerianol, was isolated by chromatography on column.
Gas chromatography-mass spectrometry (GC-MS) analysis
Both the A. parviflora oil and P. rubrum balsam were analyzed on a Hewlett-Packard gas chromatograph Model 6890 coupled to a Hewlett-Packard mass spectrometer (MS) Model 5973 (Agilent, São Paulo, Brazil) equipped with an HP5 column (30 m × 0.25 mm, 0.25 μm film thickness) programmed from 50 to 190 °C at 3 °C min−1 with a 5 min hold. The carrier gas was helium at 1 mL min–1; split mode injection (1:30) and an injector temperature of 200 °C were used. The MS ran in electron impact mode at 70 eV, the electron multiplier was set to 1800 V and the ion source temperature was 280 °C. Mass spectral data were acquired in the scan mode from m/z 40 to 500 a.m.u.
1H and 13C nuclear magnetic resonance (NMR) analysis
The isolated compounds underwent 1H, 13C, and 1 and 2D NMR experiments with Bruker Avance III equipment (Eisenhutweg, Germany). The spectra were acquired with a Varian INOVA-500 (B0 = 11.7 T) operating at 499.88 MHz for 1H and 125.71 MHz for 13C, using 5 mm probes for direct and indirect detections. The material was dissolved in CDCl3 at concentrations varying from 2 to 20 mg mL−1. The residual chloroform present in deuterochloroform (7.27 ppm) was used as the internal reference.
Results and Discussion
The steam distillation of A. parviflora leaves yielded 0.10% of an essential oil, which was analyzed by GC-MS (Table 1), revealing a nonidentified compound named AF105 that was present at ca. 5% with a calculated retention index of 1626.
With the chemical composition analysis of essential oil, it was possible to observe that main compounds were terpenoids β-phellandrene (15.12%), linalool (14.60%), α-phellandrene (7.98%), bicyclogermacrene (6.75%) and β-caryophyllene (6.05%), corroborating with Xavier et al.66 Xavier, J. K. A. M.; Maia, L.; Figueiredo, P. L. B.; Folador, A.; Ramos, A. R.; Andrade, E. H.; Maia, J. G. S.; Setzer, W. N.; Silva, J. K. R.; Molecules 2021, 26, 1914. and Oliveira et al.,77 Oliveira, F. P.; Rodrigues, A. C. B. C.; Lima, E. J. S. P.; Silva, V. R.; Santos, L. S.; Anunciação, T. A.; Nogueira, M. L.; Soares, M. B. P.; Dias, R. B.; Rocha, C. A. G.; Junior, S. D.; Albuquerque, P. M.; Lima, E. S.; Gonçalves, J. F. C.; Bataglion, G. A.; Costa, E. V.; Silva, F. M. A.; Koolen, H. H. F.; Bezerra, D. P.; Chem. Biodiversity 2021, 18, e2000938. who report the presence of these compounds in essential oil of A. parviflora with similar percentages.
To evaluate the contribution of AF 105, an unknown constituent, to the entire oil odor, a sniff test was set up with two columns of equal length attached to the same injector with one end linked to the mass detector and the other used for sniffing experiments. The odor of AF105 was therefore classified as having a woody-foral note with green touches.
The next step was to isolate the compound and determine its chemical structure by 1D and 2D NMR in a Varian probe (1H NMR, 13C NMR heteronuclear single quantum correlation spectroscopy (HSQC), heteronuclear multiple bond correlation spectroscopy (HMBC), correlation spectroscopy (COSY)). By comparing the 13C NMR chemical shifts with the other eremophilane,88 Ishihara, M.; Tsuneya, T.; Uneyama, K.; Phytochemistry 1993, 33, 1147., 99 Itokawa, I.; Morita, H.; Watanabe, K.; Mirashi, S.; Itaka, Y.; Chem. Pharm. Bull. 1985, 33, 1148. we could conclude that this compound is identical to Δ4(4a)-(2R*,8R*,8aS*)-eremophilen-9-ol or (2-[(2R*,8R*,8aS*)-8,8a-dimethyl-1,2,3,5,6,7,8,8a-octa-hydronaphthalen-2-yl]-propan-2-ol), 1, which was previously isolated by Itokawa et al.99 Itokawa, I.; Morita, H.; Watanabe, K.; Mirashi, S.; Itaka, Y.; Chem. Pharm. Bull. 1985, 33, 1148. (Figure 1). The carbon chemical shift differences between the jinkoheremol reported by Ishihara et al.88 Ishihara, M.; Tsuneya, T.; Uneyama, K.; Phytochemistry 1993, 33, 1147. and 1 are probably due to diastereomers of these two compounds, which also display different optical rotations (jinkoheremol55 Adams, R. P.; Identification of Essential Oils Components by Gas Cromatography/Mass Spectroscopy, 2nd ed.; Allured Publishing Coorporation: Carol Stream, 1995. ; eremophilen-9-ol7 ; isolated compound (Supplementary Information (SI) section).
Compound 1 was then sent to a perfumer for an olfactory evaluation, and its odor was classified as a floral balsamic, which was a bottom note in the entire oil odor.
The high structural similarity between eremophilen-9-ol 1 and valerianol 4, a major constituent of Valeriana officinalis essential oil,1010 Jommi, G.; Krepinsky, J.; Herout, V.; Sorm, F.; Collect. Czech. Chem. Commun. 1969, 34, 593. apud Arantes, F. A.; Hanson, J. R.; Hitchcock, P. B.; Phytochemistry 1999, 52, 1063. inspired us to produce 4 to perform an olfactory comparison between these two isomers. Therefore, valencene 2 was first derivatized to valerianyl trifluoroacetate 3 in 70% by applying Mattos et al.1111 Mattos, M. C. S.; Coelho, R. B.; Sanseverino, A. M.; Synth Commun. 2004, 34, 525. methodology, which produced valerianol 4 upon hydrolysis (Figure 2).
Valerianol 4 was characterized by GC-MS, 1H NMR and 13C NMR and compared with data in the literature.1212 Arantes, S. F.; Hanson, J. R.; Hitchcock, P. B.; Phytochemistry 1999, 52, 1063.
The olfactory comparison revealed high similarity between 1 and 4. Eremophilen-9-ol 1 has a slightly more pronounced floral character, while valerianol 4 has a more pronounced green note.
Protium rubrum
Compounds PR192 and PR210 were isolated and identified by 1H NMR and 13C NMR spectra (SI section). The suggested structures are 1-isopropenyl-2,5-dimethoxy-4-methylbenzene (5) and 1-isopropanol-2,5-dimethoxy-4-methylbenzene (6) (Figure 3).
Structure of compounds 1-isopropenyl-2,5-dimethoxy-4-methyl-benzene (5) and 1-isopropanol-2,5-dimethoxy-4-methylbenzene (6).
These molecules were also sent for an olfactory evaluation to determine their contribution to the whole oil fragrance and the olfactory difference caused by the presence of a hydroxyl group.
Compound 5 was classified as almost odorless with a very weak balsamic note, and although it is the main constituent, it has a very small contribution to the balsam fragrance. Compound 6 possesses a dry woody note that is easily identified in crude balsam. The GC-MS analysis of P. rubrum balsam is presented in Table 2.
Conclusions
The structure of compound AF105 was suggested to be 2-[(2R*,8R*,8aS*)-8,8a-dimethyl-1,2,3,5,6,7,8,8a-octa-hydronaphthalen-2-yl]-propan-2-ol based on GC-MS and 1H and 13C NMR data, which were identical to the eremophilenol previously isolated from Alpinia japonica. However, this is the first time eremophilenol 1 has been isolated from a Lauraceae.
The double bond position in 1 and in 4 did not change the olfactory characteristics of these compounds.
The same analytical tools were applied to 5 and 6, revealing that for the first time these aromatic compounds were isolated from Burseraceae. Their olfactory properties were evaluated by a perfumer, and the authors believe that both essential oils have olfactory potential to be included in the perfumer’s pallet.
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Supplementary InformationSupplementary information (1H NMR and 13C NMR spectra for compounds 1, 5, and 6) are available free of charge at http://jbcs.sbq.org.br as PDF file.
Acknowledgments
The authors would like to thank CNPq and FAPESP for support; Avive for the sample of Protium rubrum balsam; Kairos Fitoquímicos and Givaudan do Brasil for sponsoring Eduardo Mattoso’s fellowship; and Marco Carmini for support during the beginning of the work.
References
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12013-2017 Flavor & Fragrance Industry Leaders, http://www.leffingwell.com/top_10.htm, accessed in September 2021.
» http://www.leffingwell.com/top_10.htm -
2Souza-Junior, F. J. C.; Luz-Moraes, D.; Pereira, F. S.; Barros, M. A.; Fernandes, L. M. P.; Queiroz, L. Y.; Maia, C. F.; Maia, J. G. S.; Fontes-Junior, E. A.; Chem. Biodiversity 2021, 18, 10.
-
3Hopkins, M. J. G.; Rev. Saude Publica 2005, 56, 2175.
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4Daly, D. C. B.: A Taxonomic Revision of Protium (Burseraceae) in Eastern Amazonia and Guianas; PhD thesis, City University of New York, New York, USA, 1987, available at https://www.proquest.com/docview/303462480?pq-origsite=gscholar&fromopenview=true, accessed in September 2021.
» https://www.proquest.com/docview/303462480?pq-origsite=gscholar&fromopenview=true -
5Adams, R. P.; Identification of Essential Oils Components by Gas Cromatography/Mass Spectroscopy, 2nd ed.; Allured Publishing Coorporation: Carol Stream, 1995.
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6Xavier, J. K. A. M.; Maia, L.; Figueiredo, P. L. B.; Folador, A.; Ramos, A. R.; Andrade, E. H.; Maia, J. G. S.; Setzer, W. N.; Silva, J. K. R.; Molecules 2021, 26, 1914.
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7Oliveira, F. P.; Rodrigues, A. C. B. C.; Lima, E. J. S. P.; Silva, V. R.; Santos, L. S.; Anunciação, T. A.; Nogueira, M. L.; Soares, M. B. P.; Dias, R. B.; Rocha, C. A. G.; Junior, S. D.; Albuquerque, P. M.; Lima, E. S.; Gonçalves, J. F. C.; Bataglion, G. A.; Costa, E. V.; Silva, F. M. A.; Koolen, H. H. F.; Bezerra, D. P.; Chem. Biodiversity 2021, 18, e2000938.
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8Ishihara, M.; Tsuneya, T.; Uneyama, K.; Phytochemistry 1993, 33, 1147.
-
9Itokawa, I.; Morita, H.; Watanabe, K.; Mirashi, S.; Itaka, Y.; Chem. Pharm. Bull. 1985, 33, 1148.
-
10Jommi, G.; Krepinsky, J.; Herout, V.; Sorm, F.; Collect. Czech. Chem. Commun. 1969, 34, 593. apud Arantes, F. A.; Hanson, J. R.; Hitchcock, P. B.; Phytochemistry 1999, 52, 1063.
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11Mattos, M. C. S.; Coelho, R. B.; Sanseverino, A. M.; Synth Commun. 2004, 34, 525.
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12Arantes, S. F.; Hanson, J. R.; Hitchcock, P. B.; Phytochemistry 1999, 52, 1063.
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Publication Dates
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Publication in this collection
24 Jan 2022 -
Date of issue
2022
History
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Received
18 July 2021 -
Accepted
30 Sept 2021