Primary Isolation Swainsonine From Rhizoctonia Leguminicola Culture Solution

Subject:The purpose is seeking a new source of swainsonine ,searching for a batch of swainsonie gaining approach ,lowering SW anti-tumor clinical application cost,realize SW officinal value,offering new pharmic base to therapy and research for human and animal tumour disease. Method:1. Inoculating Rhizoctonia leguminicola on PDA in a stated time ,making it holding hearty vitality.2. High yield strains were cultured on Czapek′s medium and Rhizoctonia leguminicola culture solution was obtained 3.Pretreating culture solution with flocculation,centrifugal,sedimentation,adsorption method and so on .4. Using five different methods to gropeing for SW isolation.â‘ Taking 500ml pretreated culture solution of strains 10.2411, adjust to pH 10 with 2mol/L ammonia ,extract 3 times with butanol and combine butanol, adjust to pH 7 with 0.5 mol/L vitriol, iterative surge, placed,leave water layer , callback butano , water layer was adjusted to pH 10 with 2mol/L ammonia , vaporized at 50℃,that is the total alkaloids. It decompression sublime at 90℃,SW was obtained.â‘¡Taking 500ml pretreated culture solution of strains 10.2411, braise to ointment at 50℃,extracted with ethanol(Soxhlet 24h), callback ethanol,the residues braise, adjust to pH 10 with 2mol/L ammonia , airing, distill 5 times using ammoniac chloroform without water, filtrate infusibility matter, braise, crude SW was obtained.â‘¢Taking 500ml pretreated culture solution of strains 3.2871A, adjust to pH 10, shaken (3×) with H₂O∶CH₂Cl₂ (v/v) , the water extract was adjusted to pH 10 and again extracted (3×) with H₂O∶CH₂Cl₂ (v/v). The water layer was adjusted to pH 7 and absorbed (30 m in) on a strong cationexchange resin (H + ) , eluted with 1mol/L NH₄OH , and the filtrate was dried. The residue was adjusted to pH 7 and lyohilized to obtain a light brown powder,crude SW was obtained.â‘£Taking 500ml pretreated culture solution of strains 3.2888, adjust to pH 2 with 2mol/L hydrochloric acid, join fresh ammonium reinecke,ammonium tetrathiocyana todiammine chromate saturated solution , gather deposit and dissolve it in the acetone, filtrate infusibility matter, join silver sulphate saturated solution to filtrate, braise filtrate ,it may obtain total alkaloids hydrochloric salt, adjust to pH 10 with 2mol/L ammonia, airing and gained powder 3.467g,applied it to a silica gel column,eluting with different proportion ethyl acetate and methanol just like10:1,5∶1,3∶1,2∶1,1∶1,1∶4,collected 30ml ⁵0ml as one sample,total is 85 bottles,monitored with UV analyst,incorporated the same fraction,monitored by TLC, collate with standardized sample of swainsonine , incorporated the part that dyed with Ehrlich's reagent and all the volumes of Rf and dot are close, freeze-dry and gained crude matter 0.88g.It decompression sublime at 90℃,SW was obtained.⑤Taking 500ml pretreated culture solution of strains 3.2888, adjust to pH 7, applied it to colophony, enrichment 5 times,placed 12h, eluted with water using 2 chapiter,collected it, in succession eluted with ethanol using 2 Chapiter, collected it, at last collected 2 chapiter ethanol, decompression concentrate separately, monitored by TLC and it indicate that alkaloids are mainly lying in ethanol elution, incorporated it and decompression concentrate callback ethanol, freeze-dry the residue and gained crude SW. Result: Gaining some crude SW by primary isolation method. Brief summary: SW is a kind of alkaloids which has greatly water-solubiliting,culture solution is a water circumstance,it brings many difficulties to isolation , losing through extraction and applied to silica are big ,it should be perfected in future.