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       【关键词】  Callus;,,Cell,suspension,cultures;,,Ligusticum,chuanxiong,Hort.;,,Gas,chromatography-mass,spectromet
       (1. Institute of Medicinal Plant Development, Peking Union Medical College, Chinese Academy of Medical Sciences, Malianwa North Road 151#, Beijing 100094, China; 2.Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Xian Nong Tan Street 1#, Beijing 100050,  China; 3. Department of Chemical Engineering, Tsinghua University, Beijing 100084, China)
       Abstract:ObjectiveTo analyze the components in callus and cell suspension cultures of Ligusticum chuanxiong Hort. by gas chromatography-mass spectrometry (GC-MS). Methods Initiate the callus and cell suspension cultures of Ligusticum chuanxiong Hort.. Collect the callus and three sequent generations cell suspension cultures, and the components in callus and cell suspension cultures were analyzed by gas chromatography-mass spectrometry. ResultsThe callus and cell suspension cultures of Ligusticum chuanxiong Hort. contained similar constituenents, but the components in callus and cell suspension cultures were quite different from those of the plant, and the main bioactive constituentes of the plant were not found in callus and cell suspension cultures. ConclusionThe characteristics of callus and cell suspension cultures were different from those of the plant.
       Key words:Callus;  Cell suspension cultures;  Ligusticum chuanxiong Hort.;  Gas chromatography-mass spectrometry
       Ligusticum chuanxiong Hort. is a well-known traditional Chinese medicine (TCM) for thousands of years with haemodynamic and analgesic effects［1］. This herb is commonly used for the treatment of migraine and various cardiovascular diseases, such as angina pectoris and ischemic stroke［2,3］. The plant is mainly cultured in Sichuan province, China.
Higher plants are recognised as important sources of biochemicals, used as drugs, pesticides, flavourings and fragrances. These substances have been extracted from naturally grown whole plants. However, this approach involves large-scale crop cultivation,difficult to cultivate,a long cultivation period or a low metabolite yield. Plant cell culture provides an alternative approach. It can be produced under controlled and reproducible conditions, independent of geographical and climatic factors. From an engineering perspective, cell suspension culture has more immediate potential for industrial application than plant tissue or organ cultures, due to the extensive body of expertise which has been amassed for the treatment of submerged microbial cultures［4］. Accordingly, cell suspension cultures for commercial use have been studied recently. These include secondary metabolite production for drugs, flavorings, fragrances, micropropagation, the production of somatic embryos.  In this paper, we first report the analysis of the components in callus and cell suspension cultures of Ligusticum chuanxiong Hort. by gas chromatography-mass spectrometry.
       　　1  Experiment
       1.1  InstrumentationSamples were analyzed on a Finnigan TRACE DSQTM Single Quadrupole GC-MS (Thermo Electron Corporation) operated in EI mode and equipped with a DB-5 fused silica capillary column (30m × 0.25mm i.d., 0.25μm film thickness, J & W Scientific). The oven temperature was programmed from 50 ℃ (held for 1min) at 10℃/min to 200℃ then at 5℃/min to 300℃ (held for 30min). The injector temperature was 300℃. Splitless injection mode was used, and the injection volume was 0.2 μl. Helium was used as the carrier gas at a flow rate of 1.0 ml/min. Mass spectrometric conditions: ionization voltage, 70 ev; ion source temperature, 200℃; interface temperature, 300℃; scan range, 33-1 000 amu. The used MS library was NIST.
       1.2  Tissue cultureCallus cultures of Ligusticum chuanxiong Hort. were derived from leaf explants which were collected from the Medicinal Plant Garden of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and were authenticated by Professor Zhao Zhang (Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, China). The callus cultures were grown on MS medium (Murashige and Skoog, 1962) supplemented with 30g/L sucrose, 8g/L agar, and 1.0mg/L 2,4-dichlorophenoxyacetic acid. The pH of media was adjusted to 5.8 using 1N HCl or 1N NaOH prior to autoclaving. One hundred milliliters of medium were dispensed into 250ml Erlenmeyer flasks and autoclaved at 121℃ for 20min. Callus was grown in the dark at 25℃ and subcultured every 35 days by transferring about 5g (fresh weight) of callus to 100 ml of fresh medium in 250ml Erlenmeyer flasks.
       Media for initiation of suspension cultures consisted of MS media supplemented as above except without agar. One hundred and fifty milliliters of medium were dispensed into 500 ml Erlenmeyer flasks and autoclaved at 121 ℃ for 20 min. For initiation of suspension cultures, approximate 25 g (fresh weight) of 1 year old callus were transferred to each flask and placed on a rotary shaker at 130 rpm without light. Flasks were incubated for 16 days at 25 ℃ and subcultured by inoculating 50 ml of the 16 days old culture into 100 ml of fresh medium. Suspension cultures were subcultured every 16 days.
       1.3  Sample preparationSamples Chx-1 to Chx-5 were suspension cultures, suspension subcultures, the second suspension subcultures, callus cultures, and leaves of Ligusticum chuanxiong Hort. , respectively. Each sample was dried at 60℃ and then ground to powder. The powder (0.5 g) was extracted with 50ml methanol under sonication for 30 minutes. The mixture was filtered and the filtrate was evaporated to dryness under vacuum at 60℃. The residue was redissolved in 1ml of methanol for GC-MS analysis.
       　　2  Results and discussion
       Table 1 shows the detected constituents in suspension cultures, suspension subcultures, the second suspension subcultures, callus cultures, and leaves of Ligusticum chuanxiong Hort.. The components are listed in order of their retention time. Identification of the constituents was performed by comparison of the mass spectra of authentic compounds in NIST Library.
       Many components in suspension cultures, suspension subcultures, the second suspension subcultures, and callus cultures of Ligusticum chuanxiong Hort. were identical, including propanoic acid, 2-oxo-, methyl ester; pyrazine, methyl-; 2-methoxy-4-vinylphenol; hexadecane; dodecyl acrylate; octadecane; 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester; hexadecanoic acid, methyl ester; 9,12-octadecadienoic acid, methyl ester, (E,E)-; docosane; isopropyl stearate; tricosane; tetracosane. However, the chemical components in leaves of Ligusticum chuanxiong Hort. were quite different from those in suspension cultures, suspension subcultures, the second suspension subcultures, and callus cultures of Ligusticum chuanxiong Hort.. Leaves of Ligusticum chuanxiong Hort. mainly contained large amount of volatile oils, and neocnidilide (46.25%), trans-caryophyllene (12.89%) and 3-butylphthalide (1.81%) were the main components, but suspension cultures, suspension subcultures, the second suspension subcultures, and callus cultures of Ligusticum chuanxiong Hort. contained few volatile oils. Neocnidilide and 3-butylphthalide are known as the main bioactive constituentes of Ligusticum chuanxiong Hort.. They were found in leaves of Ligusticum chuanxiong Hort., but not found in suspension cultures, suspension subcultures, the second suspension subcultures, and callus cultures of Ligusticum chuanxiong Hort..
 
　　In conclusion, cell suspension cultures, suspension subcultures, and the second suspension subcultures of Ligusticum chuanxiong Hort. contained similar constituents. Callus and cell suspension cultures of Ligusticum chuanxiong Hort. had many same components. However, the components in callus and cell suspension cultures of Ligusticum chuanxiong Hort. were quite different from those of the plant. They were seldom the same. Callus and cell suspension cultures seldom contained the main bioactive constitutes of the plant. These results show that the characteristics of callus and cell suspension cultures are different from those of the plant.Table 1  The components in leaves, callus and cell
suspension cultures of Ligusticum chuanxiong Hort.Compound     Molecular
weight  Area (%)
       　　References:
       ［1］Pharmacopoeia of the People"s Republic of China［M］. vol 1. Beijing: Chemical Industry Press, 2000: 37.
       ［2］Ju J, Du J. Treatment of migraine with modified chuanxiong powder―a report of 30 cases ［J］. J Tradit Chin Med, 1995, 15 (3): 183.
       ［3］ Chen K J, Chen K. Ischemic stroke treated with Ligusticum chuanxiong［J］. Chin Med J (Engl), 1992, 105 (10): 870.
       ［4］Kieran P M, MacLoughlin P F, Malone D M. Plant cell suspension cultures: some engineering considerations ［J］. J Biotechnol, 1997, 59 (1-2): 39.
       Auehor：ZHAN Yulian(1973)，female(Han)，doceor, in Pharmacognosy.
Correskonding auehor：LIU Dehua，male, krofessor.