| Objective:Phenolic acids are the active ingredients of many Chinese medicinal herbs and are difficult to separate and prepare due to their low content and similar structures.In this thesis,the four Chinese medicinal herbs of Lonicerae japonicae Flos,Xanthii Fructus,Salvia yunnanensis Radix and Echinacea purpurea L.Moench are taken as examples to carry out a study for the preparative separation and purification methods of different types of phenolic acids using pH-zone-refining counter-current chromatography(pH-ZRCCC).The aim is to explore and summarize the common separation method and efficient preparation mode of phenolic acids using pH-ZRCCC,provide technical support for the preparative separation of standard materials of phenolic acids,and provide scientific basis for the quality standards formulation of traditional Chinese medicine containing phenolic acids and the study of pharmacological activities.Method:The K values determination of the target compounds is used to select and optimize the two-phase solvent system for pH-ZRCCC separation.The adjustment of concentration of the retainer and eluter in the solvent system is used to achieve rapid elution of large amount of samples;pH-ZRCCC combined with semi-preparative high performance liquid chromatography(semi-pre-HPLC)is used for the enrichment and preparative separation of low-content phenolic acids.The analysis and purity determinations for samples are carried out by high performance liquid chromatography(HPLC).And the chemical structures of purified compounds are identified by Mass Spectrometry(MS)and Nuclear Magnetic Resonance Spectroscopy(NMR).Results:Using the two-phase solvent system composed of ethyl acetate(EtOAc)-n-butanol(n-BuOH)-acetonitrile(ACN)-water(H2O)(3:1:1:5,v/v/v/v)with trifluoroacetic acid(TFA,10 mM)added in the upper phase and ammonium hydroxide(NH3·H2O,10 mM)added in the lower phase,four phenolic acids were separated by pH-ZRCCC from Lonicerae japonicae Flos and were identified as 3-caffeoylquinic acid(chlorogenic acid,1),3,4-dicaffeoylquinic acid(isochlorogenic acid B,2),4,5-dicaffeoylquinic acid(isochlorogenic acid C,3)and 3,5-dicaffeoylquinic acid(isochlorogenic acid A,4)with the purities all above 96%;using EtOAc-ACN-H2O(4:1:5,v/v/v)solvent system(TFA(10 mM)was added in the upper phase and NH3·H2O(10 mM)was added in the lower phase)and EtOAc-H2O(1:1,v/v)(TFA(10 mM)was added in the upper phase and NH3·H2O(20 mM)was added in the lower phase),seven phenolic acids were isolated by pH-ZRCCC combined with semi-pre-HPLC from Xanthii Fructus and were identified as 3-caffeoylquinic acid(chlorogenic acid,1),1,5-dicaffeoylquinic acid(5),4,5-dicaffeoylquinic acid(isochlorogenic acid C,3),3,5-dicaffeoylquinic acid(isochlorogenic acid A,4),caffeic acid(6),4-caffeoylquinic acid(cryptochlorogenic acid,7)and 5-caffeoylquinic acid(neochlorogenic acid,8),the purities of all compounds were higher than 93%;using Pet-EtOAc-ACN-H2O(1.5:2.5:1:5,v/v/v/v)(TFA(10 mM)was added in the upper phase and NH3·H2O(30 mM)was added in the lower phase)and methyl tert-butyl ether(TBME)-H2O(1:1,v/v)(TFA(5 mM)was added in the upper phase and NH3·H2O(60 mM)was added in the lower phase)solvent system,five compounds containing four phenolic acids were obtained by pH-ZRCCC from Salvia yunnanensis Radix and were identified as rosmarinic acid(9),caffeic acid(6),protocatechualdehyde(10),salvianolic acid B(11),and lithospermic acid(12),and the purities were all above 95%;using solvent systems composed of EtOAc-ACN-H2O(4:1:5,v/v/v)(TFA(10 mM)was added in the upper phase and NH3·H2O(30 mM)was added in the lower phase),EtOAc-H2O(1:1,v/v)and EtOAc-n-BuOH-ACN-H2O(3:1:1:5,v/v/v/v)(TFA(10 mM)was added in the upper phase and NH3·H2O(10 mM)was added in the lower phase),eight phenolic acids were isolated by pH-ZRCCC from Echinacea purpurea L.Moench and were identified as caffeoyl tartaric acid(13),feruloyl tartaric acid(14),coumaroyl tartaric acid(15),iso-feruloyl tartaric acid(16),caffeoyl tartaric acid methyl ester(17),2,3-O-dicaffeoyl tartaric acid(chicoric acid,18),ferulic acid(19)and caffeic acid methyl ester(20)with the purities all above 94%.Conclusion:pH-ZRCCC is characterized by its large sample injection,short separation time,low solvent consumption and high sample recovery.Compared with traditional column chromatography,it has higher separation efficiency and reproducibility,and is more suitable for preparation of phenolic acids from Chinese medicinal herbs.Through the separation and purification of phenolic acids from the four Chinese medicinal herbs,the selection method of the pH-ZRCCC solvent system for separating phenolic acids are summarized:for high-polarity phenolic acids separation,EtOAc-H2O(1:1,v/v)can be applied as the basic two-phase solvent system,and for low-polarity phenolic acids,Pet-EtOAc-H2O(1:4:5,v/v/v)or TBME-H2O(1:1,v/v)solvent system,of which TBME-H2O(1:1,v/v)can also be used for phenolic acids with poor resolution,then a certain propotion of "bridge solvent" such as n-BuOH and ACN can be added to the solvent system according to the polarities and the resolution factors of target compounds,and finally adjust the concentration of retainer(TFA)and eluter(NH3·H2O)in the solvent system to the optimize the retention time of sample.Combined with the above selection method of solvent system,the sample with simple composition only needs one pH-ZRCCC separation to complete the rapid separation and purification of all components;for separation of samples with large difference of polarity and retention time or containing low content of compounds,the separation strategy of "segmentation-enrichment first,then separation" can be applied to improve the separation efficiency;if necessary,other separation and purification technologies such as semi-pre-HPLC can be used to refine the trace components in the sample.In this study,the separation rules for separating phenolic acids using pH-ZRCCC from Chinese medicinal herbs also provide a useful reference for the efficient separation and preparation of other types of compounds. |
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