Terpene Composition of Three Species of Gymnosperms from Vietnam

In the present investigation, we studied new essential oil contents and compositions of three individual plants from Cupressaceae family cultivated in Vietnam. The air-dried plants were hydrodistilled and the oils analysed by Gas chromatography (GC) and Gas chromatography couples with Mass spectrometry (GC-MS). The components were identified by comparison of their retention indices on HP-5 MS capillary column with literature reference and MS pattern with authentic library’s collection. -Pinene (36.0%), α-cedrol (18.3%) and thujopsene (5.7%) were the main constituents of Cupressus tonkiensis Silba. Monoterpenes were the quantitative significant compounds of Sabina chinensis (L.) Antoine. These are bornyl acetate (19.5%), sabinene (17.3%) and -pinene (15.8%). Moreover, the main compounds of Thuja orientalis L., were identified as -pinene (34.1%), α-cedrol (16.5%), β-caryophyllene (5.4%) and β-selinene (5.2%). The chemotaxonomy implication of these results was also discussed.


Introduction
The genus Cupressus is one of several genera within the family Cupressaceae that has the common name cypress.It is considered a polyphyletic group.Based on genetic and morphological analysis, the Cupressus genus is found in the Cupressoideae subfamily [1].As currently treated, these cypresses are native to scattered localities in mainly warm temperate regions in the Northern Hemisphere, including western North America, Central America, northwest Africa, the Middle East, the Himalayas, southern China and northern Vietnam [2].Cupressus tonkinensis Silba., is an evergreen, medium-sized tree, up to 15 -25 cm in height.Bark is grey brown with longitudinal fissures.Leaves are scaly, closely inserted on twigs.Cones are unisexual, grouped on a stalk.Male cone is subglobular.This is an endangered species in Vietnam, only found in a narrow area of the Central Region [3].Little is known about the chemical constituents and biological potential of this plant.Leaves and stems of C. tonkinensis produce monoterpene-rich oils whose composition is α-pinene (23.1%), sabinene (21.0%) and terpinen-4-ol (14.4%) in the leaf while, α-pinene (42.5%), myrcene (10.2%) and cedrol (9.0%) were identified in the stem [4].Another investigation identified sabinene (29.34%), α-pinene (25.4%), 4-terpineol (13.91%) and -terpinene (5.5%) as major compounds of its leaf oil [3].
Thuja occidentalis (syn.Platycladus orientalis (L.) Franco or Biota orientalis (L.) Endl.) is an evergreen coniferous tree, in the cypress family Cupressaceae, which is native to the northeast of the United States and the southeast of Canada, but widely cultivated as an ornamental plant.T. occidentalis has fan-like branches and scaly leaves.It is only a small tree, growing to a height of 10 -20 m.The bark is red-brown, furrowed and peels in narrow, longitudinal strips.The cones are slender, yellow-green ripening brown, 10 -15 mm broad, with 6 -8 overlapping scales.The branches may take root if the tree falls.Plants are susceptible to attacks by honey fungus [12].Plants are monoecious, male catkins being produced at the tips of branches and female cones at the base [12].The essential oil from the leaf of P. orientalis is an important natural product which is used in fragrance, air freshener, deodorizer, insectifuge, and aromatherapy.
In the present paper, we report the essential oil compositions of three gymnosperms namely Cupressus tonkinensis Silba., Sabina chinensis (L.) Antoine and Thuja occidentalis L., from Vietnam.This is part of our extensive research on the volatile composition of Vietnamese flora as they are made available [25].

Plants Collection
Leaves of C. tonkiensis were collected from Huu Lien Natural reserve, Lang Son Province, Vietnam, in August 2010.The leaves of S. chinensis and T. orientalis were collected from Nghe An Province in August 2011.Voucher specimens DND 721, DND 194 and DND 196, respectively have been deposited at the Botany Museum, Vinh University, Vietnam.Plant samples were air-dried prior to extraction.

Isolation of the Volatile Oils
0.5 kg of air-dried sample of each species was shredded and their oils obtained by hydrodistillation for 4 h at normal pressure, according to the Vietnamese Pharmacopoeia [26].The plant samples yielded a low content of essential oils: 0.20%, 0.18% and 0.20% (v/w; respectively for C. tonkiensis, S. chinensis and T. orientalis).Oil samples were light yellow colored.

Gas Chromatography (GC) Analysis
Gas chromatography (GC) analysis was performed on an Agilent Technologies HP 6890 Plus Gas chromatograph equipped with a FID and fitted with HP-Wax and HP-5MS columns (both 30 m × 0.25 mm, film thickness 0.25 m, Agilent Technology).The analytical conditions were: carrier gas H 2 (1 mL/min), injector temperature (PTV) 250˚C, detector temperature 260˚C, column temperature programmed from 40˚C (2 min hold) to 220˚C (10 min hold) at 4˚C/min.Samples were injected by splitting and the split ratio was 10:1.The volume injected was 1.0 L.Inlet pressure was 6.1 kPa.

Gas Chromatography/Mass Spectrometry Analysis/(GC-MS)
An Agilent Technologies HP 6890N Plus Chromatograph fitted with a fused silica capillary HP-5 MS column (30 m × 0.25 mm, film thickness 0.25 m) and interfaced with a mass spectrometer HP 5973 MSD was used for the GC/MS analysis, under the same conditions as those used for GC analysis.The conditions were the same as described above with He (1 mL/min) as carrier gas.The MS conditions were as follows: ionization voltage 70eV; emission current 40 mA; acquisitions scan mass range of 35 -350 amu at a sampling rate of 1.0 scan/s.

Identification of Constituents
The identification of constituents was performed on the basis of retention indices (RI) determined by co-injection with reference to a homologous series of n-alkanes under identical experimental conditions.Further identification was performed by comparison of their mass spectra with those from NIST 08 Libraries (on ChemStation HP) and Wiley 9 th Version and the home-made MS library built up from pure substances and components of known essential oils, as well as by comparison of their retention indices with literature values [27,28].

Conclusion
Quantitative and qualitative variations were observed between these oils of Cupressaceae family cultivated in Vi-etnam and elsewhere.This may be attributed to factors such as the place of collection, age and nature of the plant, climatic conditions, handling procedures etc.

Acknowledgements
Authors are grateful to Mrs. Ogunwande Musilimat for

Table 1 . Terpene composition of the oil samples.
a Elution order on HP-5MS capillary column; b Retention indices on HP-5MS capillary column; c Literature Retention indices (see Experimental); d Correct isomer not identified; -Not identified and not present in Literature; Tr, Trace amount, <0.1%; C. p = Cupressus tonkinensis; S. c = Sabina chinensis; T. o = Thuja orientalis.