| The toxiferous traditional Chinese medicine,croton has been used for thousand years in Chinese medicine. Based on theory of Chinese medicine, croton has irreplacing valuation in treatment of some stubborn disease.In recent years, since Croton alkaloids has good anti-carcinogenic to many kinds of cancer cells and tumour cells, scholars paid attention to this active part. Croton alkaloids is worth further studying. Separation and purification are always difficult problems in field of development of traditional Chinese medicine. In order to further research and utilize the Croton alkaloids,we must overcome this difficulty and know Croton alkaloids relevant information,such as chemical composition, physical characteristic, alkaloid content, and so on. This paper selected extraction and separation of Croton alkaloids and improving quality standards of croton tiglium L. as study object.Objective(1) Croton alkaloids reference substance extraction and structures identification their;(2) Extraction and purification of isoguanosine;(3) Extraction and purification of magnoflorine;(4) To establish a thin layer chromatographic(TLC) method for the Croton alkaloids;(5) To determine the content of Croton alkaloid;(6) To establish a high performance liquid chromatography(HPLC) simutaneous determination of isoguanosine and magnoflorine.Methods:(1)To establish the method for corton alkaloids extration by preliminary test to find out the dissolution character of Cotton Alkaloids. Manifold chromatographic methods were used to separate the chemical constituents of Cotton Alkaloids, and the chemical structures were identifind by spectral analyses.(2)With isoguanosine as selecting index component the best extraction and purification process and condition were confirmed.(3)With magnoflorine as selecting index component the best extractive and purification process and condition were selected.(4) With isoguanosine and magnoflorine as reference, Cotton Alkaloids were identified by TLC.(5) The content of total Alkaloids was detected from Cotton by acid dye colorimetry and compared with the isoguanosine and magnoflorine as control.(6) The contents of isoguanosine and magnoflorine were measured by HPLCResults:(1)Two compounds were isolated from croton alkaloids, one is isoguanosine, another is magnoflorine.The magnoflorine was obtained from Croton tiglium L.The magnoflorine was obtained from Croton tiglium L. for the first time.(2)Optimum extraction procedure of isoguanosine was refluxed3times with8times of20%ethanol,40min each time. Optimum purification technology of isoguanosine was as follow:D101resin was better;the concentration of sample solution was1.5g· ml’;adsorption flow rate was1.0BV/h; Ratio of crude medicinal materials weight to reisn weight was3.5.8%ethanol as eluant.(3)Optimum extraction technology of magnoflorine was refluxed2times with10times30%ethanol,40min each time. Optimum purification technology of magnoflorine was follow:D101resin was bctter;the concentration of sample solution was1.5g·ml-1;adsorption flow rate was1.5BV·h-1; Ratio of crude medicinal materials weight to reisn weight for6.4BV water,4BV8%ethanol and4BV30%ethanol were used as eluant respectively.(4) TLC identification on silicagel H plate and the proportion of chloroform:Methanol:water (6:5:1,5drops of ammonia) as the develop system was established. After launching and took out air dries and developed with iodine vapor. A same color spots in the TLC graph of sample existed at the corresponding positions compared with the controls(5) Acid dye color conditions: pH6.0buffer to8.0niL, bromothymol blue liquid3.5mL, chloroform extract with8mL, shaking out3.0minute, put it aside for at least40min for the dcterm ination of the best method, determine the wavelength for420nm.The concentration of magnoflorine showed good linearity within the range of0.0246～0.1473mg, r=0.9995, the average recovery was99.0,RSD=2.74%.(6)The contents of isoguanosine and magnoflorine were measured by HPLC. Magnoflorine have good linearity in the range of0.098～1.223μg (r=0.9997), The average recovery was99.0%,RSD=2.91%(n=6). Isoguanosine have good linearity in the range of0.604～9.06μg (r=0.9998). The average recovery was100.91%,RSD=2.39%(n=6).Conclusion:Two compounds have been isolated from croton alkaloids, one is isoguanosine, another is magnoflorine.The magnoflorine was obtained from Croton tiglium L. for the first time. To explore the optimum extraction and purification conditions of isoguanosine and magnoflorine for providing reference to preparation of pure isoguanosine and magnoflorine.Based on the determination of Croton alkaloids by TLC, method to improve different herb parts of croton TLC identification are being optimized.Based on the determination of the content of total Alkaloids was detected from Cotton by acid dye colorimetry and the contents of isoguanosine and magnoflorine were measured by HPLC, method to determine the content of croton are being optimized. |
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