Applicaions Of Countercurrent Chromatography (CCC) And Techniques Coupllng To The CCC On Chinese Herbal Medlcine Analyses

Countercurrent chromatography(CCC) was employed as a focal point, to carry out a supercritical fluid extraction and ultrafiltration chromatography–coupled countercurrent chromatography analyses. In this dissertation, several novel CCC methods were developed and suecessfully applied in rapid and preparative separation of major phytoehemieals present in complex extracts, exhibiting high separation efficiency and great potential for Chinese herbal medicine diseovery.1. A novel separation method of HPLC coupled with CCC was established and applied to the isolation of components from the extract of Apocynum venetum flowers. The main benefit of CCC over conventional separations is the ability to scale-up and to conduct preparative chromatography on gram scale. However, separation of the compounds by conventional CCC in a single operation is limited due to the wide range of the polarities. Two sets of solvent systems, ethyl acetate–acetonitrile–water–acetic acid and ethyl acetate–acetonitrile–water–acetic acid were used as binary solvent systems of CCC for the one–step elution. Via HPLC–HPCCC, nine compounds with a wide range of polarities were separated in one–step separation. This study demonstrates that HPLC coupled with CCC is an efficient technique to prepare pure compounds from Chinese herbal medicine.2. The main advantages of gradient SFE/CCC with two stages are as follows. ①Since SFE is very effective, and the extracts can be purified online by SFE, it can save a lot of extraction solvent. ②The second stage of SFE and the first stage of CCC separation can be simultaneously carried out, so a lot of time will be saved. ③Since SFE coupled with CCC is an online separation technique, it can promote the efficiency of extraction and isolation of chemical components, and the instrumental setup of SFE/CCC has opened up the prospects of “extraction–separation” automation in various chemistry domains. Six compounds, delphinidin–3–O–glucoside, cyanidin–3–O–glucoside, peonidin–3–O–glucoside, delphinidin, peonidin, and malvidin were extracted and on–line isolated by SFE coupled with CCC from the petals of C. sinensis. Under optimal SFE and CCC conditions, the solvent systems were optimized by a multiexponential function model. In addition, six compounds including formononetin, delphinidin, cyanidin, 5,6,4′–trihydroxy–7,8–dimethoxy flavone, 5,3′–dihydroxy–7,8–dimethoxy flavone, 5–hydroxy–6,7,8,3′,4′–pentamethoxy flavone were extracted and isolated online by SFE coupled with CCC from R. damascena. Under optimal SFE and CCC conditions, the solvent systems were optimized via multiexponential function model.3. In the present paper, xanthine oxidase(XOD), an enzyme related to gout and α–glucosidase, an enzyme related to diabetes mellitus were selected as the drug target. A method for simultaneous evaluation α–glucosidase and xanthine oxidase inhibitors activity of compounds was established. The present work demonstrated that UF–LC–MS coupled with CCC is a powerful tool for the rapid screening, characterizing, and isolating of α–glucosidase and xanthine oxidase inhibitors from C. Phellodendri extracts. In our experiment, this method was used on this plant for the first time and positive results were obtained. Three compounds(palmatine, berberine, and obaculactone) have been screened for the study of their inhibitory activities on α–glucosidase and xanthine oxidase. The obtained results are expected to be valuable for discovering α–glucosidase and xanthine oxidase inhibitors from Chinese herbal medicine and designing drugs efficiently for the prevention and treatment of DM and gout.4. Before CCC sepration, the CCC solvent systems are usually prepare 10 candidate solvent systems, and the optimal solvent system has to be selected by determining the K values of these systems. However, this screening method has a great deal of randomness and blindness, and the solvent system so obtained may not be the best solvent system. Therefore, it is necessary to apply a mathematical method to calculate the relationship between the solvent composition of the two-phase solvent system and the solvent system obtained so can be identified as the optimum solvent system in our work. This method was applied to analyzing the extract of P. japlcus and P. japonicas for the first time. The present work demonstrates that ultrafiltration liquid chromatography/mass spectrometry coupled with high–speed counter–current chromatography is a powerful tool for rapid screening, characterizing and isolating α–glucosidase inhibitors and xanthine oxidase inhibitors from P. japonicas extracts and P. japlcus extracts.