| Cephalotaxus mannii, the national secondary key conservation species is rare and endangered, which containing a class of harringtonines with anti-cancer activity and with very high medicinal value. Cephalotaxus is scarce resources, using of the cells to produce harringtonine can effectively solve the contradiction between supply and demand of rare medicinal resources.To get enough cell material resources for producing harringtonines, cell morphology, growth and proliferation characteristics were investigated by fluorescence staining and paraffin section technique after optimizing culture conditions (light, hormones, inoculation concentration and subculture cycle). Then, the possibility to enhance the content of total alkaloids by eliciting suspension cells was investigated and the new method to determine the content of total alkaloids were estabalished. Details are as follows:(1) The method to observe the structure of suspension cells of Cephalotaxus mannii by paraffin section was established.(2) Through the observation of the suspension cell tissue morphology and growth process, followings were found that:1. Cell proliferation mode is mainly grow small cells from the cells suspended particles surface, the small cells fall off and grow up again.2. The cells'grow condition has close relationship with the browning phenomenon. The cells, which morphological characteristics are similar and with less internal brown, grow well. While, the cells with more internal brown grow slowly.3. The level of s browning is closely related to the cell tissue's size and surface morphology. The small tissue and with scraggly surface go brown less. While, the large particles and with smooth surface, ofen go brown inside, and inhibit cell growth.(3) The optimized culture condition of Cephalotaxus mannii suspension cells is as follows:subculture every 15 days, inoculation concentration range of 0.3?0.5 g/mL, culture under far-red (1000 lx), and with NAA (15?M/L).6-BA used alone can not propitious the cells growth.(4) The new established method for determining harringtonine total alkaloids:using phosphoric acid and hydrogen peroxide to replace 98% of sulfuric acid, replacing water bath heating with microwave heating, volatile solvents methanol, eliminate the disturbance of the solvent, the best microwave heating time is 35 s, the best detection wavelength is 558nm. the standard curve made with the new testing method is Y=0.0184 X, the linear correlation coefficient r=0.9995; The detection limit is 0?20?g/mL, repeatability RSD= 0.59%; The prototype recovery=98.85%?106.86%; And the color stability within 24 h; New method is more safe, quick, accurate compared with the traditional testing methods, and it can be used as a new measurement method of tseting the harringtonine total alkaloids content of Cephalotaxus mannii.(5) The experiment also optimized the total alkaloids extraction method, and the extract efficiency of optimized extraction method can be improved by 38% compared with the traditional extraction method methods.(6) The total alkaloids content of Cephalotaxus mannii suspension cells fresh sample and the dried samples testing by the new method were 0.0041% and 0.0149%.(7) The study preliminarily explored the induced effect:use Flg22(20 nM), ethylene (400?L/L), MeJA (1mM), salicylic acid (2 mM) and copper sulfate (50?g/mL) to induced the suspension cells and leaf of Cephalotaxus mannii for 24 h and 48 h, then, detect the total alkaloids content. The result shows that these inducers cannot effectively improve the total alkaloids content in suspension cells and leaves of Cephalotaxus mannii... |
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