| Objective: (1) To develop a new method for large-scale preparation of natural chemicals by combination of macroporous resin column chromato- graphy (MRCC) and high-speed counter-current chromatography (HSCCC). (2) To separate and purify proteins from rat liver and Momordica charantia by HSCCC using reverse micelles solvent systems.Method: (1) HSCCC and MRCC were applied for large-scale preparative separation of three flavonoids from the flower of Daphne genkwa, a famous Chinese medicinal herb. In the study, nine kinds of resins were investigated by adsorption and desorption tests, and a suitable resin was selected for the first cleaning-up. Then, the best separation strategy was developed by optimizing the conditions of HSCCC. Finaly, the obtained compounds were detected by HPLC and identified by MS and UV compared with the standards. (2-1) The rats used in this study were treated with phenobarbial to induce the microsomal monooxygenase system. Then, the livers were quickly removed. After lots of purificating steps, the crude protein of P450 was obtained. Then, a reverse micelle solvent system coupled with HSCCC was used to separate P450 proteins, of which the stationary phase was reverse micelle phase and the mobile phase was Tris-HCl buffer. At last, the product was detected by SDS-PAGE and UV. (2-2) After the crude proteins were extracted from M. charantia by alkali solution, the reverse micelle solvent system and other conditions were optimized to separate proteins from the crude sample by HSCCC. And three proteins were obtained by one-step HSCCC process, and then the products were identified by SDS-PAGE and MALDI-TOF/TOF-MS/MS. Furthermore, the anti-tumor activities of the separatd proteins were also investigated.Result: (1) Nine kinds of resins were investigated and D101 macropo- rous resin was selected for the first cleaning-up, in which 40% aqueous ethanol was used to remove the undesired constituents and 90% aqueous ethanol was used to elute the targets. The fraction of 90% aqueous ethanol was directly subjected to the preparative HSCCC using the solvent system composed of n-hexane–ethyl acetate–methanol–water (4: 5: 4: 5, v/v/v/v). The compounds apigemin (823 mg), 3-hydroxyl-genkwanin (842 mg) and genkwanin (998 mg) with the purities of 98.79, 97.71 and 93.53%, respectively, were purificated from 3 g crude extract only in one HSCCC run. (2-1) P450 protein with 100% electrophoresis pure was obtained under the optimized HSCCC conditions. In the separation, 0.05 mol/L AOT/ isooctane was used as the stationary phase, and Tris-HCl buffer adjusted by HCl and KCl to afford suitable pH value and ionic strength was used as the mobile phase. But unfortunately, the P450 protein was almost denaturated after the HSCCC separation detected by UV spectrum. (2-2) Three proteins with 100% electrophoresis purities were successfully separated by HSCCC using reverse micelle solvent systems (mobile phase A: 0.05 mol/L Tris-HCl, pH=7.0, 0.05 mol/L KCl; mobile phase B: 0.05 mol/L Tris-HCl, pH=10.0, 0.5 mol/L KCl). And one of them was proved to have the anti-cancer activity to SGC-7901 with IC50 0.116 mg/mL at 48 h treatment. Then, it was initially identified to be a new protein with 36 KD molecular mass, pI 8.8 and 343 amino acids.Conclusion: (1) Two techniques, MRCC and prep-HSCCC, were combinated for large-scale preparation of three flavonoids from the flower of D. genkwa. The advantage of this method was friendly to the environment, and it was simple and easily for operation, which was suitable for the industrial production. (2) The successful application of HSCCC coupled with reverse micelle solvent system in protein separation indicated that HSCCC can be employed for separation and purification of biomacromolecules. But how to avoid liver protein denaturation during HSCCC procedure still needs to be solved. |
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