Chemical Composition of Essential Oils of Litsea cubeba Harvested from Its Distribution Areas in China

Litsea cubeba (Lour.) Pers. is a promising industrial crop with fruits rich in essential oils. The chemical composition of essential oils of L. cubeba (EOLC) were determined for fruits harvested from eight regions in China. The overall essential oil content, obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry (GC-MS), ranged from 3.04% to 4.56%. In total, 59 compounds were identified, the dominant components being monoterpenes (94.4–98.4%), represented mainly by neral and geranial (78.7–87.4%). D-limonene was unexpectedly a lesser constituent (0.7–5.3%) in fruits, which differed from previous reports (6.0–14.6%). Several components were only detected in certain regions and compounds such as o-cymene and eremophilene have never before been reported in EOLC. These results demonstrate significant regional variation in the chemical composition of EOLC. This investigation provides important information with regard to the bioactivity, breeding work and industrial applications of L. cubeba.

. Climate and geographic habitats of Litsea cubeba.   Of the monoterpene constituents, most were oxygenated, including 28 of the monoterpenes in oil A (97.4% of the total oil), 26 in oil B (98.4% of the oil), 30 in oil C (96.5% of the oil), 23 in oil D (97.3% of the oil), 28 in oil E (98.0% of the oil), 23 in oil F (98.4% of the oil), 20 in oil G (94.6% of the oil), and 26 in oil H (94.4% of the oil). The predominant compounds were neral and geranial, accounting for 78.7% (G) to 87.4% (E) of the total oil of L. cubeba. Note that, neral and geranial are cis-trans isomers of citral. Neral always occurred in higher quantities than geranial. The citral content in sample G was relatively low, despite a higher relative yield of oil.    The chemical compounds present in all of the oils were neral, geranial, α-pinene, β-pinene, methyl heptenone, β-myrcene, D-limonene, cineole, linalool, citronellal, verbenol, isopulegone, α-terpineol, (R)-citronellol, piperitone, geraniol, β-caryophyllene and caryophyllene oxide. D-Limonene prevailed in the third oil extracted from L. cubeba samples A, C, D, F, but was substituted for geraniol, isopulegone, citronellal and isopulegone in the oils of samples B, E, G, and H, respectively. Other constituents were only identified in samples obtained from certain regions. These unique compounds included o-cymene, camphor, selina-6-en-4-ol, α-cadinol in oil A, α-terpinene, γ-terpinolene and (Z)-β-terpineol in oil B, geranyl acetate and eremophilene in oil F, 3-carene and p-cymene in oil G, and β-bisabolene and humulene epoxide II in oil H. Only trace amounts of these unique compounds were shared among samples.

Discussion
Unexpectedly, d-limonene was present in EOLC only as a minor constituent (0.7-5.3%), which differs significantly from the findings of previous studies [20][21][22], where it was a primary component (6.0-14.6%). In addition, certain trace compounds, such as o-cymene and eremophilene had never before been found in oil obtained from L. cubeba friut. o-Cymene has, however, been found in the leaf oil of Litsea glutinosa in trace amounts of less than 0.1% [23], and eremohpilene has been reported in other Lauraceae plants [24].
As expected, both quantitative and qualitative differences in composition were observed among the essential oils, all of which contained citral as the primary ingredient. Even EOLCs from the same province exhibited significant differences in composition. Even plants gathered at the same stage of development and from within the same locality and ecological environment have shown possible differences in their essential oils [25]. EOLC composition may ultimately result from geneenvironment interactions.
EOLC is natural product, consisting primarily of terpenes with some non-terpene compounds. Terpenes were thought to be products of detoxification and overflow metabolism until several were confirmed to be repellents or attractants to other organisms. This led to the belief that terpenes play an important role in antagonistic or mutualistic interactions between organisms [26]. Synthesis-related enzyme genes of some terpenes in EOLC have been partially functionally characterized [27]. Nevertheless, we still know relatively little about their actual bioactivity. Biological activity can vary depending on the composition of the essential oil [28]. Therefore, the bioactivity of EOLC may depend on the area from which it is harvested.
Note that the current study examined the chemical compositins of L. cubeba oil taken from several provinces in China. Preliminary information does exist, however, on the chemical fingerprint of L. cubeba from those provinces and will be the focal point of a future study. The powerful tools provided by modern molecular biology, chemical biology, and analytical chemistry promise to yield a much more comprehensive view of the terpenes associated with bioactivity.

Chemicals and Reagents
All chemicals and reagents were of analytical grade. Standards were supplied by Sigma-Aldrich Company (Steinheim, Germany).

Isolation of Essential Oils
The dried vegetable matter (100 g) was subjected to hydrodistillation using a Clevenger-type apparatus to extract essential oil. The Clevenger-type apparatus consisted of a 2,000-mL flask, a vertical tube, a condenser, a measuring tube with stopcock, and a return tube. The return tube connected the bottom of the measuring tube to the vertical tube, which combined with the top of the condenser. The flask was filled with 1,500 mL of distilled water and heated by an electric heating mantle. The extraction time was 5 h, after which no more essential oil was obtained. The volatile distillate was dried over anhydrous sodium sulfate and stored at 4 °C until subsequent analysis.

GC-MS Analysis
GC-MS analysis was carried out on an Agilent 6890N gas chromatograph coupled to a mass spectrometer (Agilent 5975B, Santa Clara, CA, USA) using a fused silica capillary column (HP-5MS) coated with polydimethylsiloxane (19091 S-433) (30 m × 0.25 mm internal diameter, 0.25 μm film thickness). The column temperature was set at 50 °C for 2 min, ramped at a rate of 3 °C/min to 120 °C for 2 min, then increased to 250 °C at 15 °C/min for 5 min. Helium was used as the carrier gas at a flow rate of 1 mL/min. The sample was diluted in the ether (1:10) and a volume of 1.0 μL was injected. The injector was held at 220 °C and operated in the split mode at a ratio of 1:40 for each sample. The MS operating parameters were an ionization voltage of 70 eV, ion source temperature of 230 °C, and electron multiplier energy of 1,024 V.

Qualitative and Quantitative Analysis
Compound identifications were based on comparisons their mass spectra with the mass spectra obtained from a MS database (NIST 08) and by comparisons of the RI with values reported in the literature [29,30] or against values obtained on authentic compounds. A homologous series of n-alkanes (C 7 -C 30 ) were run under the same operating conditions as the essential oil to determine RIs. The relative amounts of individual components were calculated via peak area normalization.

Data Analysis
Yields of essential oil are expressed as mean values ±1 standard deviation. Graphs were drawn in SigmaPlot v10.0. Images were identically processed using Adobe Photoshop CS5 software.

Conclusions
EOLCs harvested from various areas of China were obtained by hydrodistillation of L. cubeba and analyzed by GC-MS. Fifty-nine components were identified. Citral was the most abundant component in all of the oils. The relative composition of EOLC varied with the region from which the sample was collected. For the first time, eremophilene and o-cymene were identified in EOLC.