Keywords

Taxus chinensis var. mairei; seed; chemical components; GC-MS; composition analysis.

Introduction

Taxus chinensis 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 (Li et 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 (Sonia et al., 2011, Elavarasi et al., 2012, Zhao et al., 2007, Ozols, 1995, Shao et 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 protect T. chinensis v. 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 taxol active ingredient of T chinensis v. 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 a1., 2002a, 2002b, 2002c, Li, et a1., 2003, Kingston et al., 1993, Kumaran et al., 2010). At present, the taxanes and non-taxanes compounds were isolated from T. mairei (Bergstralh et al., 2006, Yang et al., 2011, Yang et al., 2012). The polysaccharide compounds of this species also can improve immunity and protect the liver and other organs (Li et al., 2007). T. chinensis v. 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. mairei spermoderm and endosperm were identified by GC-MS to provide reference for further development and utilization of this species.

Materials and Methods

Experimental Materials

T. chinensis v. mairei fruits consist of scarlet or green cuplike arils. Fruits were collected from 20-40 years old trees in an mixed broad-leaved forest in valleys and slopes at 400-500m above sea level in 2011, Xiushui County Jiujiang City, Jiangxi Province. Arils and empty seeds were 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.

Experimental Methods

Sample Extraction and Separation

Spermoderm and endosperm (including embryos) of T. chinensis v. 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. chinensis v. mairei spermoderm and endosperm were crudely isolated with system solvent (Fig.1, Zu et 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. mairei spermoderm 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 ( 30 m ×0. 25 mm; 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 (Zu et al., 2010).

Results

The peaks identified by GC-MS showed that there were a variety of organic compounds in T. chinensis v. mairei spermoderm and endosperm (Fig. 2). These compounds were mainly organic acids, benzene and ester- based. 24 kinds of organic compounds were identified in T. chinensis v. mairei spermoderm. In addition, there were 4 indefinite and 9 unknown substances in spermoderm as shown in the table 1. Petroleum ether phase of spermoderm included (Z) -6 - octadecenoic acid (32.53%), unknown 1 (31.28%), erucic acid (19.26%), and hexadecanoic acid (7.14%); ether phase mainly included 9-18 carbon acid (43.27%), hexadecanoic acid (15.05%), erucic acid (11.12%), oleic acid (10.11%), ether (6.46%), octadecanoic acid (5.16%); ethyl acetate phase of spermoderm mainly included 9 - octadecadienoic acid (44.15%), hexadecanoic acid (35.85%), octadecanoic acid (8.06%), 3 - phenyl -2 - acrylic acid (6.60%); methanol phase of spermoderm mainly included erucic acid (65.09%) 9 - 18 carbon acid (7.83%), unknown 4 (4.03%), oil acid (3.79%), and so on.

37 organic compounds were identified in T. chinensis v. mairei endosperm, which included 5 types unknown chemicals. Endosperm petroleum ether phase mainly included organic compounds, (E) -9 - octadecenoic acid (15.32%), unknown 5 (12.31%), unknown 6 (6.69%), unknown 7 (14.10%), 9 - octadecadienoic acid (6.40%), erucic acid (6.38%); ether phase mainly included erucic acid (72.25%), oleic acid (12.42%),cis-9 ,10-epoxyoctadecanoic acid (8.88%); ethyl acetate phase mainly included (Z, Z) -9,12 - 18 oleic acid (45.70%), 3 - phenyl -2 - acrylic acid (10.48%), 20 acid (10.31%), oleic acid (8.13%), erucic acid (7.21%), hexadecanoic acid (4.55%); methanol phase mainly included diethyl phthalate (38.79%) , (2,3) - dihydro--1,1,3 - trimethyl -3 - phenyl-1H-indene (22.42%), 1,2,3,4 - tetracarboxylic benzene (8.50%), 2 , 4 - 2-phenyl -4 -methyl--2 (E) - pentene (5.22%), as shown in table 1.

---

---

---

---

Conclusion And Discussion

56 peaks of T. mairei spermoderm 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. chinensis v. 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 acids existed in petroleum ether, ethyl ether phase, ethyl acetate phase of endosperm and methanol phase of spermoderm.

In recent years, researchers have made deep study of T. chinensis v. mairei, but reports on artificial propagation and cultivation of T. mairei research were rare(Shi et al., 2010, Yu et 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. chinensis v. mairei were seriously affected by its low seed germination rate(Yang et al., 2012). As a rare plant species, T. chinensis v. mairei should be vigorously cultivated on precondition of ensuring the quality of medicines, so that their medicinal value can be sustainable and developed.

Acknowledgements

The work reported here has been funded by Projects from Jiangxi Province Natural Science Foundation of China (20114BAB214007, 20122BAB214025).

References

1. Bergstralh, D.T., Ting, J.P. 2006. Microtubule stabilizing agents: their molecular signaling consequences and the potential for enhancement by drug combination. Cancer Treat. Rev, 32, 166-179.
2. Elavarasi A, Rathna G S, Kalaiselvam M. 2012. Taxol producing mangrove endophytic fungi Fusarium oxysporum from Rhizophora annamalayana. Asian Pacific Journal of Tropical Biomedicine, S1081- S1085.
3. Kingston, D C., Molinero A A., Rimoldi, J. M. et al.. 1993. Progress in the chemistry of organic natural products studies. Vienna: Springer, 61, 1-206.
4. Kumaran R S. Kim H J, Hur B K. 2010. Taxol promising fungal endophyte, Pestalotiopsis species isolated from Taxus cuspidate. Journal of Bioscience and Bioengineering, 110(5),541-546.
5. Li C, Yuan Y J, Wu J C, et a1.. 2003. A structured kinetic model for suspension of Taxus chinensis var. mairei induced by an oligosaccharide from Frusarium oxysporum Biotechnology Letters, 25, l335-1343.
6. Li C F, Liu Y, Dong M, Shi Q W. 2007. Advances in studies on chemical constituents in Taxus chinensis var. mairei. Chin Tradit Herb Drugs,38:1121-1132.
7. Li C F, Huo C H, Zhang M L, et al.. 2008. Chemistry of Chinese yew, Taxus chinensis var. mairei. Biochemical Systematics and Ecology, 36:266-282.
8. Ozols R F. 1995. USA update on pacilitaxl in ovarian cancer. Ann Med., 27, 127-128.
9. Shao N, Wang S M, Yao C, et al.. 2012. Sequential versus concurrent anthracyclines and taxanes as adjuvant chemotherapy of early breast cancer: A meta-analysis of phase III randomized control trials. The Breast, 21:389-393.
10. Shi G L, Bai B, Lu C H. 2010. Seed rain and seed bank of Chinese yew (Taxus chinensis var. mairei) population in Tianmu Mountain. Acta Ecologica Sinica 30, 276-279.
11. Sonia M, Rosa M C, Mohammad H M, et al. 2011. Production of the anticancer drug taxol in Taxus baccata suspension cultures: A review. Process Biochemistry 46,23-34.
12. Yang C, Wang J S, Kong L Y. 2011. Chemical Constituents from the Needles of Taxus canadensis. Chinese Journal of Natural Medicines, 9(2), 0188-0190.
13. Yang W X, Zhao Z G, Wang L H, et al.. 2012. Control of hypertension in rats using volatile components of leaves of Taxus chinensis var. mairei. Journal of Ethnopharmacology, 141,309-313.
14. Han B R, Huang Y G, Li X G, et. al.. 2000. Six kinds germination inhibitors in American ginseng pulp. Special Wild Economic Animal and Plant Research, 1, 13-17.
15. Yuan Y J, Li C, Hu Z D, et al.. 2002a. A double oxidative burst for taxol production in suspension cultures of Taxus chinensis var. mairei induced by oligosaccharide from Fusarium oxysprum. Enzyme and Microbial Technology, 30, 774-778.
16. Yuan Y J, Li C, Hu Z D, et al.. 2002b. Fungal elicitor-induced cell apoptosis in suspension cultures of Taxus chinensis vat. mairei for Taxol production. Process Biochemistry, 38, 193-198.
17. Yuan Y J, Li C, Wu J C, et al. 2002c. A model for signal transduction in suspension cultures of Taxus chinensis var. mairei induced by an oligosaccharide from Fusarium oxysporum. Biotechnology Letters, 24, 407-412.
18. Yu J H, Wang Y B, Qian H. 2012a. Polyprenols from the needles of Taxus chinensis var. mairei. Fitoterapia, 83,831–837.
19. Yu J H, Wang Y B, Qian H, et al.. 2012b. Polyprenols from Taxus chinensis var. mairei prevent the development of CCl4-induced liver fibrosis in rats. Journal of Ethnopharmacology 142,151-160.
20. Zhao D, Qin K, Cao C M, et al.. 2007. Effect of the compounds from Taxus chinensis var. mairei and T. cuspidata on the proliferation of human breast cancer cells. Natural Product Research andDevelopment, 4, 635-638.
21. Zu Y G, Pang H H, Yu J H, et al.. 2010. Responses in the morphology, physiology and biochemistry of Taxus chinensis var. mairei grown under supplementary UV-B radiation. Journal of Photochemistry and Photobiology B: Biology 98,152-158.