The volatile compositions of some Vietnamese species of Litsea have reported recently. In our previous report  , we have identified limonene (17.5%), β-caryophyllene (14.2%), bicyclogermacrene (13.1%), bicycloelemene (12.4%) and α-phellandrene (8.0%) as the major constituents of the leaf oil of L. helferi. Likewise, the oil of L. ferruginea contained large amounts of sabinene (34.5%), α-pinene (10.1%), γ-terpinene (7.8%), limonene (6.9%) and terpinen-4-ol (6.6 %) while linalool (23.4%), α-pinene (26.1%) and β-pinene (11.7%) were the compounds occurring in large proportions in the leaf oil of L. verticillata. Moreover, (E)-β-ocimene (57.4%), was the significant compound in the leaf oil of L. glutinosa. In addition, (Z)-citral (32.9% - 66.1%), sabinene (1.4% - 14.2%), limonene (7.0% - 13.6%) and linalool (1.9% - 9.5%) were the main compounds in the leaf, stem, fruits and roots oils of L. cubeba  .
Phytochemical studies on L. acutivena revealed the characterization of dehydroxymethylailanthoidol, litseakolide D-G  , acutilactone  and 4-nonacosyl-dihydrofuran-2-one  . In a previous report  , the main volatile compounds of the leaf oil of L. acutivena were found to be γ-patchoulene (11.0%), δ-cadinene (6.3%), trans-muurola-3,5-diene (5.9%) and β-selinene (5.3%). However, the components occurring in higher quantity from the twig oil were characterised to be t-cadinol (13.1%), β-selinene (9.6%), trans-β-ocimene (6.2%) and α-cadinol (7.7%).
In continuation of our extensive research on the analysis of essential oils from Vietnamese flora    , this paper reports the volatile constituents of L. acutivena leaf oil.
2. Materials and Methods
2.1. Plants Collection
The leaves of L. acutivena were collected from Pù Huống Natural Reserve, Nghệ An Province (19˚20'N 104˚50'E), Vietnam, in August 2014. Botanical identification was achieved by Dr. Nguyen Lam. A voucher specimen NTL384 was deposited at the Botany Museum, Vinh University, Vietnam.
2.2. Preparation of Plant Sample
Prior to hydrodisitillation, the leaves of L. acutivena were air-dried (17˚C, without washing with water) under laboratory shade for two weeks to reduce the moisture contents. In addition, sediments and other unwanted materials were separated from the samples. Afterwards, samples were pulverized to coarse powder using a locally made grinder.
2.3. Hydrodistillation of Essential Oil
A total of 500 g of the pulverized plant samples were used for the experiment at different time. Known weight of samples was separately and carefully introduced into a 5 L flask and distilled water was added until it covers the sample completely. Essential oils were obtained by hydrodistillation which was carried out in an all glass Clevenger-type distillation unit designed according to Vietnamese Pharmacopoeia  as described previously    . All experiments were done in triplicate. The distillation time was 3 h and conducted at normal pressure. The volatile oils distilled over water and were collected by running through the tap in the receiver arm of the apparatus into clean and previously weighed sample bottles. The oils were kept under refrigeration (4˚C) until the moment of analyses as described previously    .
2.4. Analysis of Essential Oil
The gas chromatography (GC) analysis of the essential oil was actualised using an Agilent Technologies HP 6890 Plus Gas chromatograph containing Flame Ionization Detector, fused with HP-5MS column (dimensions: 30 m × 0.25 mm; film thickness 0.25 μm). He (1 mL/min) was used as carrier gas. The inlet pressure was 6.1 kPa. The injector and detector temperatures were maintained at 250˚C and 260˚C respectively. The analysis was done by column temperature programmed starting from 40˚C (2 min hold) and ending at 220˚C (10 min hold) at 4˚C/min. The volume of the sample injected was 1.0 μL and injection was done at the split ratio of 10:1. Each analysis was performed in triplicate. The relative amounts of individual components were calculated based on the GC peak area (FID response).
For the experiment on gas-chromatograph-mass spectrometry analysis, a HP 6890N Plus gas chromatograph interfaced with a mass spectrometer HP 5973 MSD was used. The column employed was HP-5 MS (fused capillary, dimension: 30 m × 0.25 mm; film thickness 0.25 mm). The condition for the gas chromatograph was as described above. The conditions used for the Mass Spectrometry were ionization voltage of 70 eV, with mission current 40 mA. The acquisitions scan mass range of 35 - 350 amu at a sampling rate of 1.0 scan/s was maintained throughout the experiment.
2.5. Identification of Constituents of Essential Oil
The identification of constituents from the GC/MS spectra of L. acutivena was performed on the basis of retention indices (RI) determined with reference to a homologous series of n-alkanes (C4-C40), under identical experimental conditions. In some cases, co-injection with known compounds or standards (Sigma-Aldrich, St. Louis, MO, USA) under the same GC conditions was employed. The mass spectral (MS) fragmentation patterns were checked with those of other essential oils of known composition (NIST 08 Libraries)  and with those in the literature as described previously    .
3. Results and Discussion
The average yield of the essential oil was 0.16% ± 0.01% (v/w) calculated on a dry weight basis. Both oil samples were light yellow in colouration. All compounds are listed in order of their elution from the HP-5MS column (Table 1). From the results presented (Table 1) the 22 leaf oil constituents of L. acutivena were primarily monoterpenoids. Monoterpene hydrocarbons (62.0%) occurred in higher amounts with quantitative amounts of sesquiterpene hydrocarbons (17.9%) and oxygenated sesquiterpenes (11.3%). Among the monoterpenes, a-phellandrene (30.4%) and α-pinene (14.2%) were the major compounds. Previously, sesquiterpene compound predominated in the the leaf of L. acutivena from Taiwan  . Conversely, com pounds such as γ-patchoulene and trans-muurola-3,5-diene described from Taiwanese oil were not identified in the Vietnamese sample. In addition, the quantity of α-phellandrene and α-pinene in the Taiwanese oils  are lower when compared with data obtained in the present study.
Table 1. Compounds identified in the essential oil of L. acutivena.
aElution order on HP-5MS column; bRetention indices on HP-5MS column; cLiterature retention indices (NIST, 2011); dStandard deviation (SD±) were insignificant and were excluded from the Table; Sr. No. serial Number.
Monoterpene compounds were the main classes of compounds occurring in higher quantity in Vietnamese oil sample as against the sesquiterpenoids found in Taiwnaese oil  . These results demonstrate significant regional variation in the chemical composition of L. acutivena.
The chemical composition of essential oils of Litsea plants can be classified according to class of compounds they contained. Recent information showed that the compositions of the oils of L. ferruginea, L. verticillata, L. cubeba and L. glutinosa  were dominated by monoterpene compounds like the oils of L. acutivena in the present study. The monoterpenes neral and geranial  and γ-terpinene  were the main constituents of essential oils of L. cubeba from China and several other parts of the world  . Likewise the monoterpene 1,8-cineole was present in L. pungens  while L. akoensis leaf oil limonene, thymol and p-cymene with β-phellandrene and trans-β-ocimene present in the twig oil  . It could be seen that the main monoterpene components of the individual species differed from each other. The leaf oils of L. helferi had abundance of sesquiterpene compounds including β-caryophyllene, bicyclogermacrene and bicycloelemene  while γ-patchoulene, δ-cadinene and trans-muurola-3,5-diene occurred in higher amounts in L. acutivena  . The main sesquiterpene compounds identified in L. glutinosa  were β-caryophyllene and bicyclogermacrene with α-humulene and δ-cadinene making up the major components of L. nakaii  . Previously the sesquiterpenes β-eudesmol, γ-eudesmol and δ-selinene were identified in L. kostermansii  while L. acuminata  contained β-caryophyllene, τ-cadinol, α-cadinol and α-humulene. The main components of the leaf oil of L. linii were β-eudesmol, t-cadinol and α-humulene  . However, L. mushaensis leaf oil contained higher amounts of β-selinene, α-selinene and β-caryophyllene  . As usual, the identities of the major sesquiterpene components of the individual species differed from one another. Also, mixture of monoterpene and sesquiterpene compounds (trans-β-ocimene and β-selinene) characterized the essential oil of the twig of L. mushaensis  . The compositions of essential oil of L. coreana leaf was domainted mainly by mixture of sesquiterpene compounds and fatty acids namely n-decanal, (2E, 6E)-farnesol, β-eudesmol and ethyl n-dodecanoate  . It can be postulated that both intra and inter species variation could be observed in the essential oils of Litsea plants.
The paper reported the compounds identified in the essential oils of L. acutivena grown in Vietnam. The significant compounds of the oil were α-phellandrene and α-pinene. In addition, a comparative analysis of the composition of the essential oils was performed with results from the same species reported elsewhere and other species reported from Vietnam as well as Litsea plants grown in other parts of the world. The results indicated differing compositional pattern with the same species and other species.
This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number: 106.03-2018.02.
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