| The resources of sugarcane and lotus are very abundant.However,the study of the active ingredients of discarded sugarcane and lotus plants is not deep,resulting in a waste of large numbers of natural plant resources.The pH-zone-refining CCC is an efficient liquid-liquid chromatography.Compared with conventional high speed countercurrent chromatography(HSCCC),it offers ten-fold larger sample loading capacity and yields in highly concentrated compounds.So,in this thesis,pH-zone-refining CCC was used to separate phenolic acids from sugarcane rinds.Ionic liquids(ILs)are a group of unique properties with green extraction solvents.This is not only because ILs have a low vapor pressure,good thermal stability,but also because they can be combined with water and organic solvent to form a two-phase system.They can be used as solvents for separation.So,in the thesis,ILs and pH-zone-refining CCC were combined to isolate alkaloids from lotus.The main research results were as follows:1.A high efficient method for separation and purification of phenolic acids from the sugarcane rinds based on pH-zone-refining CCC technique was established.The separation was performed with a solvent system of methyl tert-butyl ether/acetonitrile/water at a volume ratio of 4:1:5,where 5 mM trifluoroacetic acid was added to upper phase as a retainer and 3 mM NH4OH added to lower phase as an eluter.As a result,three phenolic acids including 4.8 mg of caffeic acid,12.9 mg of ferulic acid and 65.2 mg of p-coumaric acid were successfully purified in one run from 1.00 g crude extract with the purities of 93.4%,94.6%and 98.8%,respectively.2.The antioxidant activity of phenolic acids from the sugarcane rinds was studied.Total antioxidant capacity and 2,2-diphenyl-l-picrylhydrazyl(DPPH)radical assay were used to evaluate antioxidant activities.The separated individual phenolic acid had higher antioxidant activity than their mixture.3.The extraction method of crude extract of lotus plants was optimized.The results showed that the IC50of DPPH free radicals was 110.5 ?g/mL,which indicated that the dichloromethane extraction showed a certain ability to remove DPPH free radicals,and is rich in alkaloids.A rapid method of DPPH-HPLC was established,and six alkaloids with different degrees of antioxidant activity were identified from lotus plants.4.A general ionic liquid pH-zone-refining CCC method for isolation of six alkaloids from the whole lotus plant was established.The conditions of ionic liquid pH-zone-refining CCC,involving types of ionic liquids and the content of ionic liquids,were optimized to improve extraction efficiency.The established general method has been respectively applied to the crude samples of lotus leaves and lotus plumules.Finally,the separation of six alkaloids was n-hexane-ethyl acetate-methanol-water-[C4mim][PF6]at a volume ratio of 5:2:2:8:0.1,where 10 mM TEA was as a retainer,3 mM HC1 was as an eluter.As a result,a total of 37.3 mg of liensinine,57.7 mg of isoliensinine and 179.9 mg of neferine were successfully purified in one run from 1.00 g crude extract of lotus plumule with the purities of 93.2%,96.5%and 98.8%,respectively.Amount of 45.6 mg N-nornuciferine,21.6 mg nuciferine and 11.7 mg roemerine were obtained in one step separation from 1.05g crude extract of lotus leaves,with the purity of 96.9%,95.6%and 91.33%,respectively.5.Nuciferine and neferine,which content were the highest in lotus leaves and plumules,were large-scale prepared by pH-zone-refining CCC and developed as national standards.Their definite values were determined by eight laboratories as 98.56 ± 0.15%and 98.31±0.10%,respectively. |
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