| Ardisia crenata is one of the well-known medicinal floral plants, and its main medicinal ingredient is bergenin. The callus of Ardisia crenata was used as the initial materials for carrying out the following studies:①research of the factors affecting the callus growth and differentiation;②optimization of extraction process on bergenin and development of the HPLC determination on the contents of bergenin of the callus;③establishment of the culture system of suspension cells and research of the kinetics in the process of cell culture;④investigation of the relationship between the growth of suspension cells and the contents of bergenin. The main results were as follows:1 The research of callus growth and differentiation in Ardisia crenataThe effects of some factors such as the medium types, carbon sources and the concentrations, nitrogen, plant growth regulators, light intensity, light quality and adding salicylic acid on the maintenance of the callus in Ardisia crenata were compared in the experiment, and the results showed that the MS medium supplemented with 0.5 mg·L-1 2,4-D was beneficial to the maintenance of callus, the best carbon source was 25 g·L-1 sucrose, the best nitrogen source was 1900 g·L-1 KNO3, the best culture condition was cultured with shading; the best addition concentration of salicylic acid was 10-5 mg·L-1, and the best addition time was the 20th day.The rate of differentiation of roots was the highest on the medium supplemented of the MS with 0.05 mg·L-1 2,4-D, 2.0 mg·L-1 KT, 100 mg·L-1 inositol, 4 % sucrose, 3 % mannitol, 5 % coconut milk,but lasted longer; under the same conditions, the effect of hormones on root differentiation from callus in Ardisia crenata was KT>6-BA>NAA; 1/2 MS medium was better than MS medium for promoting root differentiation from callus in Ardisia crenata.2 The research of extraction process, measurement and contents of brgenin of callus in Ardisia crenataThe effects of different temperature, time and the ratio of solid to solution on the extraction yield of bergenin in Ardisia crenata were investigated based on composite design of quadratic regression. The extraction technique parameters were optimized with response surface methodology, and the results showed that the optimum extraction parameters were the temperature of 80.0846℃,the time of 49.7411minutes and the ratio of solid to solution of 1:9.9966. Under those conditions, the concentration of bergenin reached the maximum with 81.3388 ug·mL-1.Determination: the HPLC mobile phase was methanol: water = 20:80 (gradient elution); detection wavelength was 275 nm. The effects of factors such as the medium types, carbon sources, nitrogen, plant growth regulators, light intensity, light quality and adding salicylic acid on the contents of bergenin of the callus in Ardisia crenata were compared in the experiment, and the results showed that the MS medium supplemented with 4 mg·L-1 2,4-D, 1.5 mg·L-1 NAA and 0.5 mg·L-1 KT was beneficial to the accumulation of bergenin of callus; 0.5 g·L-1 CH could improve the content of bergenin; the best nitrogen source was NH4NO3 with 1650 mg·L-1; the best culture condition was cultured with shading; the green light could improve the content of bergenin; adding the salicylic acid with 10-5 mg·L-1 on the 20th days could improve the content of bergenin.3 Establishment of the culture system of suspension cells and research the kinetics in the process of cell culture in Ardisia crenataThe granular callus was used as the initial material for suspension culture, and the effects of some factors such as the basal medium, hormones, culture conditions, amount of inoculation, cell concentration and carbon source on the cells growth and the content of bergenin in the suspension culture were compared in Ardisia crenata in this experiment; cell growth and the contents of bergenin in the process of suspension cell culture were measured for studying the dynamic change, and the physical and chemical properties and dynamics of cell suspensions were preliminary studied. The results showed that suspension cells grew well in the MS medium, and the cell dispersion was good; the best hormone combination was 0.5 mg·L-1 2,4-D combined with 0.05 mg·L-1KT and 0.1 mg·L-1 NAA; the proper subculture time was about 8 days and suspension cells developed well within 23℃and 25℃at 110 rpm; the best carbon source was 15 g·L-1 sucrose combined with 0.03 g·L-1 Ad; the best inoculation density of suspension cells was 0.0714 g·mL-1.On the 7th day, the cell dry weight reached the maximum, and the bergenin content also reached the maxiumum with 0.0148%. Bergenin content was positively linear correlative to cell growth at the growth stage within 7 days. Nutrients (such as nitrogen, phosphorus and carbon source) consumption of the suspension medium was relevant with the accumulation of biomass and bergenin biosynthesis.In this experiment the kinetic equation about cells growth, sugar consumption, bergenin synthesis in Ardisia crenata was set up as follows: Cell growth:Substrate consumption:Product synthesis: On the above formula,μrepresented specific growth rate,μm represented maximum specific growth rate,KS represented saturation constant of substrate, KI represented inhibition constant of substrate,Cs represented substrate concentration, qs represented specific consumption rate of substrate,YX/S represented yield coefficient of substrate consumption to cells,m represented maintain the coefficient of cells,YP/S represented yield coefficient of product to substrate consumption,qp represented specific production rate of product,α,βrepresented proportion coefficient.After verification, the model prediction fitted the experimental results at a high degree, which suggested that the model be rational and credible to some extent.4 Investigation of the relationship between the growth of suspension cells and the contents of bergenin in Ardisia crenataThe cell suspension achieved the highest biomass when the nitrate concentration increased to 60 mM and ammonium was removed, and with the initial nitrogen concentration of 40 mM at a NH4+/N03- molar ratio of 5:1,the maximum bergenin content was achieved. A two-stage flask culture system was established by altering nitrogen source supply and the results showed that the cell dry weight and the bergenin content in suspension culture cells by such a process increased by 0.3498 g and 0.0478 % respectively when compared with those of the control, reaching up to 311 % and 357 %, respectively.When the concentration of phosphate was the concentration of MS basal medium, the cell dry weight and the bergenin content in suspension culture cells by such a concentration of phosphate increased to maximum by 0.1851 g and 0.0239 %, respectively. I-,MoO42-,Cu2+,Co2+ had greater effects on the synthesis of bergenin among the eight microelements( Mn2+,Zn2+,BO33+,I-,MoO42-,Cu2+,Co2+,Fe2+). The increase of concentrations of I-,MoO42-,Cu2+,Co2+ to 5, 10, 10 and 20 times higer respectively than those of the standard MS medium promoted the synthesis of bergenin. Combined treatment of I- (25μM), MoO42-(10μM), Cu2+(1μM), Co2+(2μM) improved cell the dry weight and bergenin content to 0.2105 g and 0.0287 %, respectively, which were 168.43 % and 227.78 % increment respectively compared with those of the control.Adding the salicylic acid with 10-5 mg·L-1 on the 6th day could improve the content of bergenin.
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