| In this article, the alkaloids extraction conditions of Lycoris radiata fresh sampleswas studied; the conditions of alkaloids separation was optimized by thin-layerchromatographic （TLC）; L. radiata alkaloids were prepared by TLC; the conditions ofalkaloids separation was optimized by column chromatography; the HPLC analysismethod of alkaloids in L. radiata was established; the contents of L. radiate bulbs’alkaloids in different months were detected, as well as the alkaloids contents of differentparts in L. radiate; bud induction system was studied; the changes of alkaloids during thegrowth of the buds were studied; the effects of galantamine and tyramine on thebiosynthesis of L. radiata alkaloids were also studied. The biosynthetic pathway of L.radiata alkaloids was preliminarily studied. The following results were obtained:1. L. radiata alkaloids extraction from fresh samples. The best extraction conditionsof L. radiata alkaloids were obtained as90%ethanol, microwave power320W for60+60S, and the leaching time1h. This method could extract alkaloids in short time withless material, and is suitable for processing precious materials.2. Alkaloids analysis by thin-layer chromatographic （TLC）. The results showed thatthe best separation was CHCl3: MeOH: NH3·H2O as9:1:0.1in one-dimensional TLC.The best separation was solvent1EtOAc: MeOH: NH3·H2O as9:1:0.1and solvent2CHCl3: MeOH: NH3·H2O as9:1:0.1in two-dimensional TLC, which had a betteranalysis effect than one-dimensional TLC.15kinds of alkaloids were prepared withtwo-dimensional TLC, and these alkaloids had a high purity. This method is simple andhas a high value on the analysis of L. radiata alkaloids.3. Alkaloids separation by column chromatography. This study investigated theinitial separation and purification of the alkaloids by silica gel column chromatography.The results showed that the best conditions were bed volume80cm3, injection1.5mLconcentration100μg·mL-1, flow rate2.0BV·h-1, and eluent EtOAc: MeOH: NH3·H2Oas9:1:0.05. This method was suitable for the preliminary separation and purification oflots of alkaloids.4. The analysis of L. radiata alkaloids by high-performance liquid chromatography（HPLC）.14kind of alkaloids were separated from L. radiate sample on a ZORBAXODS-C18Reversed phase column with0.9%triethylamine aqueous solution （pH8.0adjusted with acetic acid）（A）, acetonitrile （B） and methanol （C） as mobile phase, gradient elution, flow rate1.0mL·min-1, detection wavelength234nm. This method can accuratelyanalyze a variety of alkaloids, and provide an accurate analytical method for L. radiataalkaloids research.5. The result showed that the alkaloids contents of L. radiata bulb and leaf weredifferent in different months. The contents trends of galanthamine, lycoramine andlycorine were consistent. The alkaloids contents of different parts in L. radiata were alsonot the same. The content in leaf was the most, bulb, root, flower and footstalk took turns.The research provided an experimental basis for the rational use of L. radiata resources.6. The experiment of tissue culture showed that the best medium for bud inductionwas MS+6-BA3.0mg·L-1+NAA0.2mg·L-1, and the best medium for small bulb rootingwas MS+NAA3mg·L-1. The effect of one divides into four and one divides into eight onbud induction was not significantly different.7. The changes of alkaloids during the growth of the buds and the effects ofgalantamine and tyramine on the biosynthesis of alkaloids. The results showed that thealkaloids contents of the buds and small bulbs were first increased and then decreasedduring the growth of the buds. The kind and content of alkaloids in the small bulbs weremore than in the buds. Compared with the wild bulbs and leaves, the small bulbs andbuds contained unique alkaloids. Tyramine promoted the biosynthesis of all alkaloids inL. radiata, and galantamine inhibited the biosynthesis of all alkaloid. So we presumedthat all L. radiata alkaloids had a common precursor. |
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