Abstract
Conifers are well represented in Vietnam where a new pine species has been recently discovered in Son La province: Pinus cernua, synonym P. armandii ssp. xuannhaensis. The compositions of needle, cone, and branch oils have been investigated by gas chromatography (retention index), gas chromatography-mass spectrometry, and 13C nuclear magnetic resonance. Myrcene (47.0%) was the main component of needle oil, followed by β-pinene (28.4%) and α-pinene (12.5%). Branch oil also contained myrcene (32.8%), α-pinene (17.9%), β-pinene (9.8%), and a high content of limonene (20.0%). Finally, cone oil displayed α-pinene (44.1%) beside myrcene (11.5%), β-pinene (8.1%), and limonene (5.8%).
Conifers are well represented in Vietnam with 33 species and 2 subspecies belonging to 5 families, including Pinaceae that constitutes the largest family. 1 Among Vietnamese Pinus species, the well-known Pinus dalatensis Ferré (Vietnamese white pine) endemic to Indochina and Pinus krempfii that bears flattened needles and is present in tropical rainforests of southern Vietnam 2 as well as Pinus kwangtungensis Chun ex Tsiang that produces timber of good quality or Pinus latteri Mason growing in primary mixed forests, periodically inundated swamp forests, or woodlands on foot of mountains could be cited. 1
Pinus cernua L. K. Phan ex Aver., K. S. Nguyen and T. H. Nguyen (Pinaceae, synonym Pinus armandii ssp. xuannhaensis L.K. Phan; Vietnamese name: Thông xuân nha) is a new pine species recently discovered in Northern Vietnam, from Xuan Nha Nature Reserve, on the slopes of Pha Luong mountainous range, Moc Chau district, Son La province, along the border Laos. Mature individuals occurred with a diameter of 30-75 cm and 25-30 m in height at an altitude around 1000 m above the sea level. Botanical characteristics were given in detail by Phan Ke Loc et al. 1
The genetic variation was investigated on the basis of 15 ISSR primers and data indicated low genetic variability for all the populations. 3 Analysis of molecular variance divided the samples into 2 main groups. 4 The subspecies xuannhaensis, considered endemic to Vietnam, is assessed as globally critically endangered following the accepted IUCN Red List Categories. 5
However, Businský reported that “the pine recently referred to as P. armandii, was identified as the true P. fenzeliana, currently known only from China.” 6 Later, the same author concluded that “P. cernua is relegated to a synonym of P. fenzeliana.” 7 Whatever, the history of discovery and taxonomy of this pine species is perfectly developed in a recent paper by Averyanov et al. 8 Concerning identification of phytochemicals, a unique paper reported on the chemical composition of essential oil (EO) isolated from aerial parts (needles and branches) of P. armandi subsp. xuannhaensis. 9 Unfortunately, in that paper, which also reported the EO composition of two other species of Vietnamese pines, 2 columns have been advisedly inversed in the table describing the results (personal communication of the authors). In short, monoterpene hydrocarbons, β-pinene (27.3%), α-pinene (18.0%), limonene (16.1%), and myrcene (10.4%) were the main components of this oil sample.
Therefore, we believed that, in combination with phenotypic and genetic parameters, the chemical composition of EO isolated from various parts of the tree could bring useful information about the species of the pine growing wild in Son La Province, Vietnam. In continuation of our work on the characterization of Vietnamese conifers (Cupressus tonkinensis, Fokienia hodginsii, Xanthocyparis vietnamensis) through the composition of their EOs 10-12 the aim of the present paper was to get more insight on the composition of EOs isolated from various organs of the recently discovered Pinus cernua (P. armandi subsp. xuannhaensis): needle oil, cone oil, and branch oil.
Needles, cones, and branches have been harvested on one adult tree. EOs have been isolated at laboratory scale using a Clevenger-type apparatus, and yields accounted for 0.22%, 0.39%, and 0.21%, respectively (v/w on dry weight basis). The 3 oil samples have been submitted to gas chromatography (GC) (in combination with retention indices (RIs) on two columns of different polarity), gas chromatography mass spectrometry (GC-MS), and 13C nuclear magnetic resonance (NMR) following a method developed at University of Corsica. 13-15 In total, 41 compounds have been identified, accounting for 99.0%, 97.2%, and 96.7 %, respectively, of the whole composition (Table 1). The compositions of Pinus cernua needle, cone, and branch oils are dominated by monoterpene hydrocarbons, particularly, α-pinene, β-pinene, myrcene, and limonene.
Chemical Compositions of Vietnamese Pinus Cernua Needle, Cone, and Branch Oils.
MS, mass spectrometry; NMR, nuclear magnetic resonance; Tr, traces; RIa, RIp, retention indices on apolar and polar columns.
Order of elution and percentages of components are given on apolar column (BP1). NMR: identification by 13C NMR at least in one oil sample.
Myrcene (47.0%) constituted almost half of the needle oil composition, followed by β-pinene (28.4%) and α-pinene (12.5%). Branch oil contained also myrcene (32.8%), α-pinene (17.9%), and β-pinene (9.8%) and it was characterized by a high content of limonene (20.0%). Finally, cone oil displayed α-pinene (44.1%) beside myrcene (11.5%), β-pinene (8.1%), and limonene (5.8%). Camphene (5.6%) and (E)-β-ocimene (3.6%) were more abundant than in the 2 first oil samples. Oxygenated monoterpenes were poorly represented except α-terpineol (up to 1.5%). Various sesquiterpene hydrocarbons have been identified at appreciable content, particularly (E)-β-caryophyllene (6.1% in cone oil) and δ-cadinene (3.1% in branch oil). A few oxygenated sesquiterpenes were present in cone oil at low content (0.4%-1.0%), with α-cadinol being the major one. In branch oil, 2 diterpene hydrocarbons, sclarene (0.3%) and camphorene (0.4%), have been suggested by MS matching against commercial computerized libraries. Camphorene, scarcely found in EOs, has been confirmed by 13C NMR, according to published data. Moreover, the carbon chemical shifts fitted perfectly with those of meta-camphorene previously identified in EOs from Boswelia species. 16
In conclusion, the 3 investigated oil samples isolated from needles, cones, and branches of the pine species recently discovered in Northern Vietnam and named P. cernua (synonym Pinus armandii ssp. xuannhaensis) belong to the monoterpene hydrocarbon-rich oils family, with 4 components that emerge: α-pinene (12.6%-44.1%), β-pinene (8.1%-28.4%), myrcene (11.5%-47.0%), and limonene (4.6%-22.0%). Although P. armandii Franch has not been observed in Vietnam, 1 it could be noted that the cone oil of Chinese origin contained mainly α-pinene (20.9%) and limonene (15.8%). 17 In contrast, the composition of needle oil was dominated by γ-muurolene (40.7%) and (E)-β-caryophyllene (36.3%). 18
Experimental
Plant Material and Essential Oil Isolation
Needles, cones, and branches from a P. cernua mature tree have been collected in Xuan Nha Nature Reserve, Moc Chau district, Son La province, Vietnam (Figure 1) on November 2017. The scientific name was identified by Tran Huy Thai at the Institute of Ecology and Biological Resources (IEBR), VAST. A voucher specimen (NT-P.14) was deposited at IEBR. They have been separately hydrodistilled on a Clevenger-type apparatus for 2.5, 3 and 4 hours, respectively.
Map of Vietnam. Son La province in red. Xuan Nha Nature Reserve is green.
Analytical GC
GC analyses were performed on a Perkin Elmer Clarus 500 gas chromatograph (FID) equipped with 2 fused silica gel capillary columns (50 m, 22 mm id, film thickness 0.25 µm), BP-1 (polydimethylsiloxane) and BP-20 (polyethylene glycol). The oven temperature was programmed from 60 to 220°C at 2°C/min and then held isothermal at 220°C for 20 minutes, with injector temperature 250°C, detector temperature 250°C, carrier gas hydrogen (1.0 mL/min), and split 1/60. The relative proportions of the oil constituents were expressed as percentages obtained by peak area normalization, without using correcting factors. RIs were determined relative to the retention times of a series of n-alkanes with linear interpolation (‘Target Compounds” software of Perkin Elmer).
GC-MS Analysis
The EOs were analyzed with a Perkin Elmer TurboMass detector (quadrupole), directly coupled to a Perkin Elmer Autosystem XL, equipped with a fused silica gel capillary column (50 m, 0.22 mm id, film thickness 0.25 µm), (BP-1 polydimethylsiloxane). Carrier gas, helium at 0.8 mL/min; split, 1/75; injection volume, 0.5 µL; injector temperature, 250°C; oven temperature programmed from 60 to 220°C at 2°C/min and then held isothermal (20 min); ion source temperature, 250°C; energy ionization, 70 eV; electron ionization mass spectra were acquired over the mass range 40-400 Da.
13C NMR Analysis
13C NMR analyses were performed on a Bruker AVANCE 400 Fourier Transform spectrometer operating at 100.623 MHz for 13C, equipped with a 5 mm probe, in CDCl3, with all shifts referred to internal tetramethylsilane. 13C NMR spectra were recorded with the following parameters: pulse width, 4 microseconds (flip angle 45°); acquisition time, 2.73 seconds for 128 K data table with a spectral width of 22 000 Hz (220 ppm); CPD mode decoupling; digital resolution 0.183 Hz/pt. The number of accumulated scans was 3 000 for each sample (around 40 mg of oil in 0.5 mL of CDCl3). Exponential line broadening multiplication (1.0 Hz) of the free induction decay was applied before Fourier transformation.
Identification of Components
Identification of the components was based (i) on comparison of their GC RIs on polar and apolar columns, determined relative to the retention times of a series of n-alkanes with linear interpolation (‘Target Compounds” software of Perkin Elmer), with those of authentic compounds or literature data (components 40 and 41); (ii) on computer matching against laboratory-made and commercial mass spectral libraries; 19-21 and (iii) on comparison of the signals in the 13C NMR spectra of EOs with those of reference spectra compiled in the laboratory spectral library, with the help of a laboratory-made software. 13-15 In the investigated samples, individual components were identified by NMR at contents as low as 0.3%.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by Nafosted (Project 106.06.2018.13).