Chemical Composition, Enantiomeric Distribution, and Antifungal Activity of the Oleoresin Essential Oil of Protium amazonicum from Ecuador

Background: Protium species (Burseraceae) have been used in the treatment of various diseases and conditions such as ulcers and wounds. Methods: The essential oil from the oleoresin of Protium amazonicum was obtained by hydrodistillation and analyzed by GC-MS, GC-FID, and chiral GC-MS. P. amazonicum oleoresin oil was screened for antifungal activity against Candida albicans, Aspergillus niger, and Cryptococcus neoformans. Results: A total of 54 components representing 99.6% of the composition were identified in the oil. The essential oil was dominated by δ-3-carene (47.9%) with lesser quantities of other monoterpenoids α-pinene (4.0%), p-cymene (4.1%), limonene (5.1%), α-terpineol (5.5%) and p-cymen-8-ol (4.8%). Chiral GC-MS revealed most of the monoterpenoids to have a majority of levo enantiomers present with the exceptions of limonene and α-terpineol, which showed a dextro majority. P. amazonicum oleoresin oil showed promising activity against Cryptococcus neoformans, with MIC = 156 μg/mL. Conclusions: This account is the first reporting of both the chemical composition and enantiomeric distribution of the oleoresin essential oil of P. amazonicum from Ecuador. The oil was dominated by (−)-δ-3-carene, and this compound, along with other monoterpenoids, likely accounts for the observed antifungal activity of the oil.


Essential Oil
The oleoresin (relatively fresh, yellow, with a terpenic odor) of P. amazonicum was collected from Quito, Ecuador (0 • 14 0" S, 78 • 31 0" W, 3000 m above sea level). The tree was identified by Rafael Parducci, and a voucher specimen has been deposited in Saintoil S.A. The essential oil was obtained by hydrodistillation using a Clevenger apparatus as previously described [29] to give the essential oil.

Gas Chromatography-Mass Spectrometry (GC-MS)
The oleoresin essential oil of P. amazonicum was analyzed by GC-MS using a Shimadzu GC-MS-QP2010 Ultra (Shimadzu Corp., Columbia, MD, USA) operated in the electron impact (EI) mode (electron energy = 70 eV), with a scan range of 40-400 atomic mass units (amu), a scan rate of 3.0 scans/s, and the GC-MS Solution software (Shimadzu GC-MS-QP2010 Ultra, Columbia, MD, USA). The GC column was ZB-5MS fused silica capillary column (Phenomenex Inc., Torrance, CA, USA) (30 mL × 0.25 mm ID) with a (5% phenyl)-polymethylsiloxane stationary phase with a film thickness of 0.25 µm. The carrier gas was helium with a column head pressure of 551.6 kPa and flow rate of 1.37 mL/min. The injector temperature was 250 • C, and the ion source temperature was 200 • C. The GC oven temperature program was programmed for 50 • C initial temperature, the temperature increased at a rate of 2 • C/min to 260 • C. A 5% w/v solution of the sample in CH 2 Cl 2 was prepared and 0.1 µL was injected with a splitting mode (30:1). Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those reported in the literature [30], and stored in the MS library.

Gas Chromatography-Flame Ionization Detection
The gas chromatograph was a Shimadzu GC 2010 (Shimadzu Corp., Columbia, MD, USA) equipped with a flame ionization detector, a split/splitless injector, and autosampler AOC-20i (Shimadzu Corp., Columbia, MD, USA). The capillary column was a ZB-5MS (Phenomenex Inc., Torrance, CA, USA) with a film thickness of 0.25 µm. The column temperature was programmed, 50-250 • C at 2 • C/min, the injector temperature was 250 • C, the detector temperature was 280 • C, the carrier gas was nitrogen, and the flow rate was maintained at 1.0 mL/min. Injection mode split with a split ratio of 1:100. The injected volume was 0.3 µL of diluted oil (1:10 v/v with CH 2 Cl 2 ). The percent composition of the oleoresin essential oil was calculated from raw peak areas without standardization.

Chiral Gas Chromatography-Mass Spectrometry
Chiral analysis of the P. amazonicum oil was performed on a Shimadzu GCMS-QP2010S (Shimadzu Corp., Columbia, MD, USA) operated in the EI mode (electron energy = 70 eV), scan range = 40-400 amu, scan rate = 3.0 scans/s. GC equipped with a Restek B-Dex 325 capillary column (30 m × 0.25 mm ID × 0.25 µm film) (Restek Corp., Bellefonte, PA, USA). Oven temperature was started at 50 • C, and then gradually raised to 120 • C at 1.5 • C/min. The oven was then raised to 200 • C at 2 • C/min and held for 5 min. Helium was the carrier gas and the flow rate was maintained at 1.8 mL/min. The sample was diluted 3% w/v with CH 2 Cl 2 and then a 0.1 µL sample was injected in a split mode with a split ratio of 1:45. The enantiomers of each monoterpene were identified by comparison of retention times to authentic samples obtained from Sigma-Aldrich (Milwaukee, WI, USA).

Antifungal Screening
The broth microdilution method was performed to determine antifungal activity as previously reported [31,32]. Briefly, cultures of Candida albicans (ATCC 18804) and Cryptococcus neoformans var. neoformans (ATCC 24067) were initially grown on potato dextrose agar (PDA) plates for 72 h at 37 • C. A single colony was used to inoculate approximately 5 mL of potato dextrose broth (PDB) which was subsequently grown for an additional 24 h at 37 • C. Aspergillus niger (ATCC 16888) cultures were grown on PDA plates for 5 days at room temperature (RT, 22 • C). A. niger conidia were collected, placed in PDB, and filtered through sterile cheesecloth into fresh PDB. The absorbance of the fresh solution was read at 625 nm and adjusted accordingly with PDB to an absorbance of 0.15. Minimum inhibitory concentrations (MICs) were determined in triplicate using 96-well plates. C. albicans and C. neoformans were diluted in 3-(N-morpholino)propanesulfonic acid (MOPS) buffered Roswell Park Memorial Institute (RPMI) medium to 2000 cells/mL whereas A. niger was diluted with PDB to an OD 625 of 0.15. Initially, 50 µL of MOPS buffered RPMI was added to each well of the plate. In the first row, 50 µL of essential oil was added and mixed well, then 50 µL of this mixture was removed and then added to the medium in the next row. This serial dilution process was repeated for each row of the plate, with the removed volume from the last row being discarded. To each well was added 50 µL of cells to achieve a final volume of 100 µL. C. albicans and C. neoformans were incubated at 37 • C for 48 h. The A. niger plates were incubated at RT for 6 days. The MIC was determined from turbidity or growth on the plates in comparison to positive and negative controls. In order to verify the results, MIC determinations were carried out in nine replicates. A combination of Cyprodinil and Fludioxonil served as the positive control with MOPS buffered RPMI serving as the negative control.

Compounds
Relative
The antifungal mechanisms of activity of monoterpenoids are poorly understood. It has been suggested that these hydrophobic compounds disrupt the cytoplasmic membranes or membrane
The antifungal mechanisms of activity of monoterpenoids are poorly understood. It has been suggested that these hydrophobic compounds disrupt the cytoplasmic membranes or membrane proteins of fungal cells, leading to cytoplasmic leakage, cell lysis, and death [51]. Chirality of monoterpenoids, therefore, may not play a critical role in antimicrobial activity. Nevertheless, Kusumoto and co-workers have shown that (+)-α-pinene showed significantly better antifungal activity against Heterobasidion parviporum than (−)-α-pinene [52]. Likewise, Filipowicz et al. showed (−)-β-pinene to be slightly more active than (+)-β-pinene against Candida albicans [53], and Omran and co-workers found that (−)-limonene had better antifungal activity than (+)-limonene [54]. (+)-δ-3-Carene has shown antifungal activity against several fungal strains [47], but there are apparently no reports on antifungal activity of (−)-δ-3-carene, which is not commercially available. Overall, these findings indicate that P. amazonicum resin oil has promising potential for further antifungal consideration, in particular against C. neoformans and potentially other yeast-like fungi.

Conclusions
This is the first reported chemical analysis of the oleoresin essential oil of Protium amazonicum. The P. amazonicum resin oil collected in Ecuador was dominated by (−)-δ-3-carene and is therefore, an excellent source of this enantiomer. The abundance of this compound, along with other monoterpenoids, likely account for the observed antifungal activity of the oil. The activity against Cryptococcus neoformans and Candida albicans indicates promise against these opportunistic fungal pathogens. Additional research into this tree species and other Protium species, their chemistry and their biological activities, is needed.