GC-MS Analysis of Chemical Components of Taxus Chinensis Var. Mairei Seeds

Taxuschinensis var.mairei is a rare species. It is distributed within the Yangtze River basin, the Henan Nanling Mountains, and several mountains and valleys in Shaanxi, Gansu, and also in the Taiwan Province in China (Liet al., 2007). It has attracted wide attention because of the significant anticancer activity of taxol present in bark, twigs, leaves and other parts of the species(Soniaet al., 2011,Elavarasiet al., 2012, Zhaoet al., 2007, Ozols, 1995, Shaoet al., 2012, Li et al., 2008).The species has faced huge harvesting pressure in recent years and populations have been decimated. Thus, researchers have been seeking ways to protectT. chinensisv. mairei populations within a framework of forest resource management and genetic conservation. In addition, seeds of this species have a combination of morpho-physiological deep dormancy with underdeveloped, dormant embryos, which need grow to a certain length before seed dormancy could be broken. Its natural reproduction is low, resulting in an endangered existing state of this species. The taxolactive ingredient of T chinensisv. mairei.is reported to have significant inhibitive effects on ovarian cancer, breast, lung, stomach, colon, melanoma, leukemia, bladder cancer, and central nervous system tumors(Yuan,et al.,2002a, 2002b, 2002c, Li,et a1.,2003, Kingstonet al., 1993, Kumaranet al., 2010). At present, the taxanes and non-taxanes compounds were isolated from T. mairei(Bergstralhet al., 2006, Yanget al., 2011, Yanget al., 2012). The polysaccharide compounds of this species also can improve immunity and protect the liver and other organs(Liet al.,2007). T. chinensisv.mairei was listed as one of China's class key protected wild plants in 1999. In this paper, extracts of petroleum ether, ethyl ether, ethyl acetate and methanol of T. maireis per moderm and endosperm were identified by GC-MSto provide reference for further development and utilization of this species.


Introduction
Taxuschinensis var.mairei is a rare species. It is distributed within the Yangtze River basin, the Henan Nanling Mountains, and several mountains and valleys in Shaanxi, Gansu, and also in the Taiwan Province in China (Liet al., 2007). It has attracted wide attention because of the significant anticancer activity of taxol present in bark, twigs, leaves and other parts of the species (Soniaet al., 2011,Elavarasiet al., 2012, Zhaoet al., 2007, Ozols, 1995, Shaoet al., 2012, Li et al., 2008.The species has faced huge harvesting pressure in recent years and populations have been decimated. Thus, researchers have been seeking ways to protectT. chinensisv. mairei populations within a framework of forest resource management and genetic conservation. In addition, seeds of this species have a combination of morpho-physiological deep dormancy with underdeveloped, dormant embryos, which need grow to a certain length before seed dormancy could be broken. Its natu-ral reproduction is low, resulting in an endangered existing state of this species. The taxolactive ingredient of T chinensisv. mairei.is reported to have significant inhibitive effects on ovarian cancer, breast, lung, stomach, colon, melanoma, leukemia, bladder cancer, and central nervous system tumors (Yuan,et al.,2002a, 2002b, 2002c, Li,et a1.,2003, Kingstonet al., 1993, Kumaranet al., 2010. At present, the taxanes and non-taxanes compounds were isolated from T. mairei (Bergstralhet al., 2006, Yanget al., 2011, Yanget al., 2012. The polysaccharide compounds of this species also can improve immunity and protect the liver and other organs (Liet al.,2007). T. chinensisv.mairei was listed as one of China's class key protected wild plants in 1999. In this paper, extracts of petroleum ether, ethyl ether, ethyl acetate and methanol of T. maireis per moderm and endosperm were identified by GC-MSto provide reference for further development and utilization of this species.

Experimental materials
T. chinensisv.mairei fruits consist of scarlet or green cuplike arils. Fruits were collected from 20-40 years old trees in anmixed broad-leaved forest in valleys and slopes at 400-500m above sea level in 2011, Xiushui County Jiujiang City, Jiangxi Province. Arils and empty seedswere floated off after fruits were collected and macerated in water. The natural dried seeds were sealed into polyethylene bags and stored in the refrigerator (4°C). The TGW of seeds was 65.048g mairei seeds were separated and weighed. Spermoderm and endosperm (1200g) were separately placed into two 1000 ml beaker after they were grind with a mill. Afterwards the beakers were filled with methanol (80%) and closed for extraction at 0 to 4 °C. The extract was then filtered by Büchner funnel at intervals of 24h. The collected filtrate was re-extracted with 80% methanol. The process was repeated several times until the extract turned pale, and all extracts were mixed. The methanol extracts of T. chinensisv.mairei spermoderm and endosperm were crudely isolated with system solvent (Fig.1, Zuet al. 2010). The crude extracts were separated into four groups: petroleum ether phase, ether phase, ethyl acetate phase and methanol phase. And then, these organic phases of spermoderm and endosperm were placed in a Rotary Evaporator RE-3000 concentrates to evaporate. Finally, the organic phases were concentrated to 200ml and placed at 5 °C.

Identification of extractions of T. chinensis v. maireis permoderm and endosperm
Each above 100 ml concentrated extract was collected and evaporated to be concentrated dry in vacuum on a rotary evaporator. These dry matters were respectively washed by the same organic solvent to obtain 3ml samples. The samples were identified by GC-MS in the Forest Products Chemistry, Analysis and Testing Center of Chinese Academy of Forestry.
Analysis was carried out on a GC-MS instrument (Antigone Lun 6890N/5973N, USA) equipped with a 1NNOWAX quartz capillary column (30m×0.25mm; film thickness 0.25 μm) with 50 °C-190°C column temperature; programmed temperature was 5°C · min -1 ; Helium was used as a carrier gas; gasification temperature was 280°C. MS conditions were: The ionization mode was EI and the ionization energy was 70eV; source temperature was 200°C; collection of current was 300μA; emission current was 1mA; instrument separation rate was 600; quality range was 10-500. Finally, spectrums of all components were checked by the inventory signal of computer-controlled and matched with the standard spectrum(Zuet al.,2010).

Results
The peaksidentified by GC-MS showed that there were a variety of organic compounds in T. chinensisv. maireispermoderm and endosperm (Fig. 2). These compounds were mainly organic acids,

Conclusion and Discussion
56 peaks of T. maireispermoderm components and 79 peaks of endosperm were obtained by GC-MS. 37 chemicals were detected by computer on-line information retrieval, 24 of which compounds were identified (64.86%), and there were other 4 indefinite kinds (10.81%) and 9 unknown kinds (24.32%). 35 compounds with content more than 1%, accounted for 94.59%of the total extracted compounds. 32 peaks were obtained in petroleum ether extractions, 7 of which were identified (21.88%), and their content were more than 1%. 36 peaks were obtained in methanol extractions, 14 of which were identified (38.89%), and 13 compounds content more than 1%, accounting for 92.86% of the total of all compounds. 34 peaks of ethyl acetate extractions were obtained and of which 10 compounds (29.41%) were identified, 13 of which compounds content were more than 1%, accounting for 92.86% of the total of all compounds. 33 peaks of ether extracts were obtained, 11 of which compounds (33.33%) were identified, 2 kinds of components had indefinite substances (6%). Their relative content of components was identified with the peak area normalization method.The results also showed more kinds of organic compounds were extracted from T. chinensisv. mairei in endosperm than those in spermoderm on the same treatment condition. The same organic compounds might exist in the different organic phase of spermoderm and endosperm, such as erucic acid also existed in petroleum ether phase, ether phase, methanol phase of spermoderm, and petroleum ether phase, ether phase and ethyl acetate phase of endosperm phase. 16   In recent years, researchers have made deep study of T. chinensisv. mairei, but reports on artificial propagation and cultivation of T. mairei research were rare (Shiet al., 2010, Yuet al., 2012a, 2012b. Because research on suitable germination conditions was rudimentary, identifying chemical components and its effects on seed germination is important for protection and cultivation of this species. The development and utilization of T. chinensisv. maire were seriously affected by its low seed germination rate (Yang et al., 2012). As a rare plant species,T. chinensisv. mairei should be vigorously cultivated on precondition of ensuring the quality of medicines, so that their medicinal value can be sustainable and developed.