Study On Tissue Culture,Extraction And Separation Of Deguelin From Derris Trifoliate Lour.

Deguelin was one of the most common rotenoids in plant. In recent years, it was found that it had strong antitumor activity and potential application prospects in the prevention and treatment of lung cancer. In this paper , stems and leaves tissue from Derris Lour. were cultured by biotechnological methods, then deguelin was extracted and isolated from callus, then studied on reduction reaction of deguelin. The results were as follows:1. Optimization of tissue culture from Derris trifoliate Lour.Compared the effect of different sterilization time on tissue culture by 0.1% HgCl2 and 2% NaClO, then studied on the impact of the growth of tissue culture in different pH and the combination of concentrations hormone. The results indicated that the optimal conditions for tissue culture: explant sterilizated with 2% NaClO for 14min, pH=6.0 and the combination of concentration hormone as 2,4-D 2.0mg/ml, 6-BA 1.0mg/ml. At the same time, for the stem and leaf callus, the death rate and pollution rate were no higher than 16%, the highest rates of callus induction up to 95% and the largest quantity harvest were 3.9522g, 3.5944g.2. Optimization extraction of deguelinExtracted deguelin from callus by Ultrasonic extraction, shaking extraction and Soxhlet extraction, then compared the content of deguelin with each organs of Derris. The results showed that the extraction rate of deguelin was up to 10.31% by Ultrasonic extracted 5min. Tissued to the 60d, it had the highest content of deguelin was up to 51.40% from callus. Compared the extraction rate, the results was as follows: root> nodule> Callus> leaf> stem, the extraction rate of root was up to 9.29%, the extraction rate of callus was 8.23%.3. Optimization separation of deguelinDeguelin was separated by silica gel column chromatography (gradient elution as petroleum ether/ethyl acetate) and thin-layer chromatography, then detected by HPLC (RP-C18 column, mobile phase as methanol-water (66:34), flow rate as 0.5ml/min, column temperature as 30℃). The results showed that the content of deguelin was 96.76%, when the gradient elution of petroleum ether/ethyl acetate is 5:1. With developing solvent as acetone: chloroform: petroleum ether = 1:2:7, it could better separated the deguelin when Rf2 = 0.31, and the content was 93.33% by TLC.4. Discussion of the conditions for reduction derivativeBy HPLC analysis, the sample dissolved in methanol, then reduction reaction with NaBH4. The results showed that deguelin was completely reaction, when heated to 80℃and reaction 3h under NaOH.