| Anti-cancer active ingredients of paclitaxel in Taxus, which is known as the "plant gold", has significant curative effect on the prevention and treatment of much cancer. Because of slow growth of Taxus and extremely low content of taxol can not meet the increasing market demand, and Taxus is a national level of protection of plant, the priority is the use of modern technology to obtain taxol through different channels on the premise of the protection of Taxus resource. Cell culture in the production of taxol increasingly favored by researchers because of many advantages. There are domestic and foreign relevant reports, but most of them focus on the basic theory research instead of application technology research of obtain taxol efficiently. Especially, the construction of technical system tending to industrialization is still blank. In this paper, Taxus cuspidate was chosen as raw materials to set up a cell suspension system though the induction of callus and screening of high-yielding cell lines, and the regulation factors of metabolic were used to improve production of taxol of cell suspension system. Then a technology system of efficiently obtain of taxol by cell suspension culture of Taxus cuspidate was established to laid a good foundation for scale, commercialization and industrialization production of taxol by Taxus cuspidate. And it can also provide technical support for reasonable exploitation and efficient use of medicinal plants of Changbai mountain’s wild resources with important realistic meaning and profound social influence. Research conclusions are as follows:1. Single factor experiment and experiment design of Box-Behnken were used in taxol extraction of Taxus cuspidata with ultrasonic and a method of separate and detect rapidly of taxol was set up, the results showed that:the best condition on single factor experiment is the extraction solvent of ethanol-methylene chloride (1:1), material liquid ratio of1:50, ultrasonic time of1h and ultrasonic power of200W; the correction factor K of separation and detection of taxol by TLC-UV spectrophotometry is0.009152; making material liquid ratio(Xi), ultrasonic time(X2) and ultrasonic power(X3) as the independent variable and extraction volume of taxol as the dependent variable of multifactorial experiment, response surface analysis was carried out, egression equation is obtained as follows: Y=127.78+3.86X1+6.06X2+1.65X3-3.68X2-8.23X22-5.95X32+0.57X1X2+2.05X1X3+1.80X2X3The optimum process parameters of the extraction were material liquid ratio of1:53.23, ultrasonic time of1.11h, ultrasonic power of207.88W, at this time, the amount of taxol extraction is130.576mu g/g.2. Callus induction and successive transfer culture of Taxus cuspidate were carried out, the results showed that:improved B5medium with2mg/L NAA and0.5mg/L6-B A could make the effect of callus best when young stems of Taxus cuspidata were used as explants:callus appeared on the11th days, induced rate was94.9%and the proliferation ratio of callus was1.13; the effect of callus adhered to the explant was better than that stripped from explant to subculture, the former callus had more proliferation and light browning; using7.5mg/L citric acid as an browning inhibitor could make the proliferation ratio of subculture callus to2.36and browning phenomenon was eliminated; subculture8to9times continuously could make fresh weight proliferation and taxol content steady, proliferation ratio of callus fresh weight was3.83and content of taxol was227μg/g after8times of subculture, the content of taxol was73.85%increase over that of extracted directly from the plant.3. The cultivating method of high yield cell strain of Taxus cuspidate was screened, the results showed that:no matter conditional plate culture or nursing plate culture, they both got higher plant efficiency under lower cell density, concentration of300cells/mL was best according to the growth of cell clones and appropriate plant efficiency; cells which were suspension cultured for12days used as cloning material cost the shortest time for cell clones appearing, and cells for16days’growth got the highest plant efficiency; growth rate of subculture cells on the solid medium were more steady than that in the liquid medium, but no matter which medium was used, taxol content had some fluctuation.4. Effective induction of cell suspension culture of Taxus cuspidate was carried out, the results showed that:the growth curve of suspension cells was "S" type, cell fresh weight reach the maximum of54.5g/L on the24th day; seven kinds of regulatory factor were used for cells induced, the impacts of methyl jasmonic acid, salicylic acid, phenylalanine and gibberellin on the growth and taxol production of suspension cell were more obvious; making the concentration of the four regulatory factors as independent variables, total taxol production in suspension cells as the dependent variable, the quadratic general rotary combination experimental design was carried out, regression equation is obtained as follows(a=0.10):Y=4.32000-0.12000X1+0.06667X2+0.10000X3-0.03333X4-0.40667X12-0.32167X22-0.22167X32-0.57667X42+0.02500X1X3+0.01500X2X4The best combination of regulatory factors was methyl jasmonic acid of100μmol/L, salicylic acid of20mg/L, phenylalanine of400mg/L and gibberellin of2mg/L, at this point, the taxol production up to4.32mg/L in the cells; regulatory factors were joined on the seventh day of suspension cells’growth, then taxol production of cells inside and outside was highest of4.855mg/L, and taxol release rate was10.48%; the way of continuously adding small doses regulatory factors into growth system of suspension cells was better than the way of a one-time adding, fresh weight and total taxol production increased by6.08%and11.57%respectively; the synthesis metabolic of taxol lag behind the growth of cells in growth system ofsuspension cells. |
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