(E)-2,6,10-Trimethyldodec-8-en-2-ol: An Undescribed Sesquiterpenoid from Copaiba Oil

The use of copaiba oil has been reported since the 16th century in Amazon traditional medicine, especially as an anti-inflammatory ingredient and for wound healing. The use of copaiba oil continues today, and it is sold in various parts of the world, including the United States. Copaiba oil contains mainly sesquiterpenes, bioactive compounds that are popular for their positive effect on human health. As part of our ongoing research endeavors to identify the chemical constituents of broadly consumed herbal supplements or their adulterants, copaiba oil was investigated. In this regard, copaiba oil was subjected to repeated silica gel column chromatography to purify the compounds. As a result, one new and seven known sesquiterpenes/sesquiterpenoids were isolated and identified from the copaiba oil. The new compound was elucidated as (E)-2,6,10-trimethyldodec-8-en-2-ol. Structure elucidation was achieved by 1D- and 2D NMR and GC/Q-ToF mass spectral data analyses. The isolated chemical constituents in this study could be used as chemical markers to evaluate the safety or quality of copaiba oil.


Introduction
European settlers reported that the people of the Brazilian Amazon region used copaiba trees to treat their wounds [1]. Marcgraf and Piso were the first to describe the copaiba tree in 1638 [2]. Amazonians use copaiba oil resin as an anti-inflammatory and healing agent [3]. Copaiba oil is obtained by tapping the trunk of the Copaifera tree. Copaiba oil can be obtained from several species of Copaifera trees, but Copaifera reticulata is responsible for 70% of oil production [4,5]. Copaifera trees belong to the family Fabaceae (Leguminosae) [4]. Copaifera trees range from 0.5 to 5 m in diameter and up to 60 m in height, and can live up to 400 years [4]. Copaiba oil is an exudate secretion that results from the trees' detoxification process. The secretion acts as a defensive mechanism against ordinary predators, such as fungi and bacteria [6]. South America, particularly Brazil, has diverse Copaifera species and is considered the largest global exporter of copaiba oil.
Copaiba oil has been used in folk medicine for centuries as a wound-healing agent [7]. This use was likely inspired by animals, as the indigenous people observed injured animals rubbing their bodies on the stem of Copaifera trees [4]. The indigenous people of the Amazon region also used the Copaifera tree to treat several other conditions, such as urinary tract infections, sore throats, stomach ulcers, and infectious diseases. Copaifera trees play a vital role as an alternative remedy in the Amazon region of Brazil, and it is no surprise that phytotherapeutic and cosmetic products using copaiba oil have found their way not only into the Brazilian market but also to the international markets [1]. The main chemical constituent of copaiba oil is β-caryophyllene, which can be found in various essential oils [8]. The chemical profile of copaiba oil might be slightly different from one species to another, but in general, the main constituents are β-caryophyllene, α-humulene, α-copaene, α-bergamotene, δ-cadiene, and β-bisabolol. Some copaiba oils contain diterpene acids, such as copalic acid, clorechinic acid, and hardwickiic acid [4]. However, several factors might cause chemical composition variation in copaiba oil, such as seasonal and climatic characteristics of the environment, soil type, and composition, rainfall index, and species genetics [4,5]. In this study, copaiba oil was investigated as part of a continuing effort to isolate and identify chemical markers for use in quality studies of dietary supplements and botanical drug products under development in the United States. In this regard, eight sesquiterpenes/sesquiterpenoids (Figure 1), including one new, were isolated and characterized by analyzing their 1D and 2D NMR and GC/Q-ToF mass data.

Plant Material
The copaiba oil obtained from Copaifera officinalis L. was provided by doTERRA (Pleasant Grove, UT, USA). A reference sample # 613 was deposited in the product repository at the National Center for Natural Products Research (NCNPR), University of Mississippi.

GC/MS Analysis
For a general analysis of copaiba samples, an Agilent 7890 GC equipped with a 7693 autosampler and an Agilent 5975C quadrupole mass spectrometer was used. Separation was achieved with an Agilent DB-5MS Ultra Inert column (60 m × 0.25 mm × 0.25 µm). The inlet was held at 260 • C and was operated in the split mode with a split ratio of 50:1 and an injection volume of 1uL. The initial GC oven temperature was 80 • C; it was then heated at 3 • C/min to 125 • C, then ramped at 1 • C/min to 140 • C and held for 10 min, then heated at 3 • C/min to 170 • C, before a final ramp of 8 • C/min to 280 • C held for 10 min was used. The mass spectrometer was equipped with an electron ionization source, which was operated with an electron energy of 70 eV. The ion source, quadrupole, and transfer line temperatures were set to 230, 150, and 280 • C. Mass spectra data were recorded from 35 to 500 m/z after a 5 min solvent delay. Data were acquired utilizing Agilent MassHunter software (version B7.06.274). All of the copaiba samples were diluted in dichloromethane (0.01%, v/v), and n-dodecane (IS) was added to each sample solution at a constant concentration of 300 µg/mL.
In order to obtain the accurate mass of compound 1, analysis was performed utilizing an Agilent 7890B gas chromatographic (GC) instrument which was equipped with a RS185 PAL3 autosampler. The GC was connected to an Agilent 7250 Accurate-Mass Quadrupole Time-of-Flight (Q-ToF) mass spectrometer. The capillary column (30 m × 0.25 mm i.d.) utilized was coated with a 5% Phenyl Methyl Siloxane (J&W HP-5MS) film (0.25 µm). Helium at a constant flow rate of 1 mL/min was used as the carrier gas. The following GC oven program was utilized: 50 • C held for 1 min and then heated at a rate of 6 • C/min to 260 • C. The inlet was programmed at 260 • C in split mode. A split ratio of 50:1 with an injection volume of 0.2 uL was used for compound 1. The Q-ToF mass spectrometer was equipped with a high-emission low-energy electron ionization source which was operated with an electron energy of 70 eV and an emission current of 5.0 µA. The source, quadrupole, and transfer line temperatures were 230 • C, 150 • C, and 260 • C, respectively, during the experiment. All mass spectra data were recorded at a rate of 5 Hz from 35 to 450 m/z after a 4 min solvent delay. Data were acquired utilizing Agilent MassHunter software (version B7.06.274).

Conclusions
In this study, eight compounds were isolated from the copaiba oil. All compounds were characterized as sesquiterpenes/sesquiterpenoids. Out of them, (E)-2,6,10-trimethyldodec-8-en-2-ol (1) was found to be previously undescribed. Possible contribution of the new compound (1) to the biological activity of copaiba oil will have to be investigated in further studies.