Optimizations Of Both Rhizoctonia Legumin1cola Culture Conditions And Swainsonine Separation Methods

Objective: Improved Rhizoctonia leguminicola growth conditions, the strain vitality andproduction of swainsonine. Search for an effective method to extract swainsonine, simplifyprocedures, increase extraction yield, in order to get more swainsonine from mycelium andculture solution of Rhizoctonia leguminicola, offering for material base to antitumor research.Method:1.Added the preparation of concentrated juice clover into the potato agarmedium. Rhizoctonia leguminicola was cultivated in potato dextrose agar medium, thetemperature was25℃and time was7days, picked the vigorous growth of sclerotia toCzapek's medium, the temperature was25℃and time was14days, then obtained theRhizoctonia leguminicola mycelium and cultures.2. The naturally dried mycelium was cut, and then put them in500ml of methanol.50℃water bath for1h,40KHz,50℃ultrasonic treatment for30min, saxhlet extraction for8-10h,abandon the hyphae. Added in200ml20ml/L glacial acetic acid, shaked well and usedisovolumetric chloroform to extract for2times, abandoned the organic phase. Put the acidsolution in the cation exchange resin, absorbed for30min, first to use deionized waterflushing, then used1mol/L ammonia flushing, collected the ammonia lotion, put it in dryingoven at60℃for drying, gained the total alkaloid, reduced pressure distillation at90℃. Thesample uses thin layer chromatography for testing.3. The cultures were condensed into extractum by volume200:1, collecting for100ml,adding in deionized water, using glacial acetic acid to adjust pH2~3,50℃water bath for1h,40KHz,50℃ultrasonic treating for30min. Use isovolumetric chloroform to extract acidsolution for10times, abandon the organic phase and oily impurities, add hydroxide to adjustpH9~10,500ml dichloromethane were added into alkali solution, then use continuousliquid/liquid extraction procedure for60~70h, concentrated organic phase, get the light yellowwax solid, it can get crude products after drying. Reduced pressure distillation at90℃. Usethin layer chromatography and gas chromatography for the sample detection.Result: The Rhizoctonia leguminicola grew vigorous on PDA medium, the medium issuitable for preserving and passaging; The Rhizoctonia leguminicola grew well on modifiedCzapek's medium, the medium were clear and with less impurity;2.3mg white crystal wasfinally obtained from50g mycelium was identified as swainsonine by means of TLC;5mgwhite crystal was finally obtained from100ml culture fluid was identified as swainsonine bymeans of TLC, GC.Conclusion: The Rhizoctonia leguminicola growth better than before when the potatoagar medium (PDA) was improved. First time to use liquid/liquid extraction procedure to extract swainsonine, simplicity of operation, extraction effect is good, and still need to befurther improved to increase the extraction yield and production.