| Tripterygium wilfordii Hook.f. is Celastraceae Tripterygium perennial climbing vine contains many kinds of bioactive components, such as triptolide and alkaloids, it has a long application history in China on Chinese herbal medicine and insecticidal field. This research is to study the effects of different macroporous resins and different addition amounts of macroporous resin as well as concentrations of macro elements in H culture medium on growth of adventitious root of Tripterygium wilfordii Hook.f. and the content of triptolide,wilforgine and wilforine. The optimal conditions of 10 L airlift bioreactor culture system on inoculum size, culture duration, inoculum density and aeration rate were selected. The feasibility and necessity of scale-up culture of adventitious root of Tripterygium wilfordii Hook.f. was discussed. The main results were given as follows:1. The selection of macroporous resins type and addition amount:In the 5 kinds of macroporous resins of XAD-2, XAD-7, HP-20, ADS-8 and ADS-F8,the XAD-7 on the addition of 5% is the best macroporous resin on Tripterygium wilfordii Hook.f. culture. The growth of adventitious root dry weight reached 1.61 g as 1.16 times to CK. The content of triptolide, wilforgine and wilforine in culture medium were 2.57 times,35.77 times and 3.31 times to CK, respectively. The total content of 3 secondary metabolites reached 0.57 mg/flask, 1.08 mg/flask and 130.47 μg/flask as 2.04 times, 8.60 times and 1.66 times to CK, respectively.2. Optimization of concentrations of macro elements and and NO3-/NH4+ in H culture medium:Using H medium as the basic medium to culture Tripterygium wilfordii Hook.f.adventitious root as material to study the effects of different concentrations of macro elements and NO3-/NH4+ on the growth of adventitious root and content of triptolide,wilforgine and wilforine. Concentrations nitrogen, phosphorus and kalium were 2 times to H culture medium standard concentration, the concentration of calcium and magnesium were2.5 times and 1.5 times to the standard concentration while NO3-/NH4+ equaled to 9:1, thegrowth of adventitious root dry weight reached the highest value. The value of NO3-/NH4+equaled to 7:3 was benefit to the production of 3 secondary metabolites. Reapectively, 3.5times concentration of nitrogen and calcium, 1.5 times concentration of phosphorus and kalium, 2.5 times concentration of magnesium were conductive to the accumulation of triptolide; 1.5 times concentration of nitrogen, 3times concentration of calcium, 2 times concentration of phosphorus and kalium, 3.5 times concentration of magnesium were conductive to the accumulation of wilforgine; 3 times concentration of nitrogen and magnesium, 3.5 times concentration of calcium, 1.5 times of phosphorus and kalium were conductive to the accumulation of wilforine.3. Optimization of 10 L airlift bioreactor culture conditions:The optimal conditions of 10 L airlift bioreactor culture system were as follows: 4-5 cm as the inoculum size, 120 g FW/bioreactor as the inoculum density, 1.0 vvm as the aeration rate, 30 d as the culture duration. In these conditions, the growth of adventitious root and the content of 3 secondary metabolites reached the highest value, the total yields were 36.21 g,24.19 mg, 46.56 mg and 6.28 mg, respectively. With the scale-up of adventitious root culture,the production of adventitious root dry weight was 6.04 g/L, only half to 250 mL flask while the decrease on production capacity of triptolide and wilforgine were 9.84% and 4.55%(the production capacity of wilforine increased 28.5%). Large-scale industrialized culture could produce adventitious root rapidly with no effects of season and place to produce secondary metabolites, it could be an effective way to solve the problems on growth and reproduction of Tripterygium wilfordii Hook.f. Using bioreactors to large-scale culture Tripterygium wilfordii Hook.f. adventitious root to produce high value secondary metabolites had a very considerable prosect and has a high feasibility. |
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