| Pueraria Lobata (Willd.) Ohwi is a traditional Chinese medicine, which contains puerarin, daidzein and many kinds of flavonoids.Pueraria Lobata distribute in the southern, central and northern parts of Shanxi. However, it was still blank in the development and utilization of the Pueraria Lobata germplasm resources so far.In this study, we analysised and compared the puerarin, daidzin and total flavonoids of Pueraria lobata from ten areas of Shanxi based on the optimization of the extraction, determination conditions. At the same time, we established the stable suspension cells culture system of Pueraria Lobata, and investigated the effect of physical, chemical factors, elicitors and precursors on the growth of suspension cells and the synthesis of total flavonoids. This research provided the experimental evidence and a new way for the industrialisation of medicinal components of Pueraria lobata.Our experiment results showed that:Several factors which influence the extraction of puerarin, daidzein and total flavonids were optimized. Ultimately we decided the extraction conditions of total flavonoids of Pueraria Lobata from Shanxi were as follows:the ratio of material to liquid 1:10, the extraction temperature 70℃, 70% ethanol ultrasonic treatment for 30 min.Puerarin,daidzin extraction conditions were as follows:30% methanol,50℃, ultrasonic treatment for 30 mm.The conditions which influence the chromatographic separation of puerarin, daidzin was optimized. Ultimately we decided the mobile phase were methanol(A) and water(B), gradient elution program as follow:0-9 min, 25%～32% A,9～24 min,32% A,24～30 min,32%～25% A and 30～35 min, 25% A.The contents of puerarin, daidzin and total flavonoids of Pueraria lobata from ten areas of Shanxi were determined. The results show that:the content of main effective components of Pueraria lobata from ten ares of Shanxi Province were significant difference. The content of puerarin were 1.46%～4.48%, daidzin were 0.11%～0.62% and total flavonoids were 11.19%～26.14%. Puerarin and total flavonoids had positive correlation with latitude.The disinfection methods of Pueraria lobata explants of different regions and growth years were selected. The results showed that:using 75% alcohol treatment for 30 s and then 0.1% or 0.2% HgCl2 treatment for 8-10 min could achieve the ideal disinfection effect for Pueraria lobata explants.Effects of basic medium, exogenous plant hormone, photoperiod, and subculture times for the growth of callus and the biosynthesis of total flavonoids of Pueraria lobata.were compared. The results showed that:the optimum induction conditions for the callus of Pueraria lobata was MS+2.0 mg/L 2,4-D+1.0 mg/L 6-BA+0.5 mg/L NAA+0.3 mg/L KT. The optimum photoperiod for the growth of callus and biosynthesis of total flavonoids of Pueraria lobata was 16 h light and 8 h dark. At the end of the stable period of callus growth (26 d), the content of total flavonoids was the highest. After 3 times subculture, the content of total flavonoids in Pueraria lobata callus achieve the highest.The influence factors on the growth of suspension cells and the biosynthesis of total flavonoids of Pueraria lobata. were studied. The results showed that:the optimum culture conditions for the growth of Pueraria lobata suspension cell was B5+1.0 mg/L 2,4-D+1.0 mg/L 6-BA+1.0 mg/L NAA+0.5 mg/L KT+30 g/L sucrose, the initial inoculation was 0.5 g, pH 5.3. The optimum culture conditions of the biosynthesis of total flavonoids of Pueraria lobata was B5+2.0 mg/L 2,4-D+1.0 mg/L 6-BA+1.0 mg/L NAA +0.2 mg/L KT+40 g/L sucrose, pH 5.8.The influence of the biological elicitors-phenylalanine, tyrosine and abiotic elicitors-Cu2+, Ag+ for the growth of cell and the biosynthesis of total flavonoids of Pueraria lobata. was studied. The results shows that:adding 0.5 mmol/L phenylalanine in cell exponential growth stage could improve the accumulation of total flavonoids. Adding 0.1 mg/L Ag+ can not only promote the growth of Pueraria lobata cell but also promote the accumulation of flavonoids. Adding 0.05～1 mg/L Cu2+,0.1～0.6 mmol/L tyrosine in culture medium inhibited the growth of Pueraria lobata cell and the accumulation of total flavonoids... |
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