Novel Countercurrent Chromatography Methods And Its Application In Natural Products

Countercurrent chromatography (CCC) is a continuous liquid-liquid partition chromatography with no solid supportmatrix. Therefore, it benefits from great advantages when compared with the traditional liquid-solid separation methods and extremely useful for the separation of natural products. However, the plant extracts are extremely complex and always present a significant challenge for the standard CCC technique. In this dissertation, several novel CCC methods were developed and successfully applied in rapid and preparative separation of major phytochemicals present in complex natural extracts, exhibiting high separation efficiency and great potential for natural drug discovery.?. Using an upright preparative CCC instrument, two phenylbutenoids, (E)-4-(3', 4'-dimethoxyphenyl)but-3-enyl acetate and (E)-4-(3',4'-dimethoxyphenyl) but-1,3-diene, were separated from the rhizomes of Zingiber cassumunar with the high purity of 98.7 and 95.1%, respectively. Similarly,120 mg of oridonin at the purity of 97.8% was obtained from 200 mg of the crude sample of Rabdosia rubescens in a single-step upright CCC separation. Moreover, a comparative study of preparative isolation and purification of the phenolic compounds magnolol and honokiol from the Chinese medicinal plant Magnoliae officinalis by upright CCC and semi-preparative HPLC was also presented. Results indicated that using the present CCC approach, 1250 mg of honokiol and 520 mg of magnolol, with a purity of 98.7 and 99.5%, respectively, were obtained from 2.0 g of crude sample in a single separation. In contrast, semi-preparative HPLC allowed isolation and purification of these two phenolic compounds with significantly lower productivity and higher solvent consumption.?. An on-line heart-cutting two-dimensional CCC (2D-CCC) approach for preparative isolation and purification of three prenylflavonoids from Artocarpus altilis was presented. The upright CCC instrument was used as the first dimension. Effluent of interest was collected on-line into a 30 mL sample loop by a customized column-switching interface and introduced into a high-speed CCC instrument for the second dimension separation. With this 2D-CCC system and a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (5:5:7:3 and 5:5:6.5:3.5, v/v), which had been selected by high-speed CCC, about a 500 mg amount of the crude extract was separated, yielding three prenylflavonoids in milli-gram level with high purity. In addition, the 2D system was improved based on the use of HSCCC in the first dimension (1st-D) and UCCC in the second dimension (2nd-D). The interface was a homemade column-switching system with a 50-ml sample loop. In this way, almost the whole interested region from the first dimension was transferred on-line to the second dimension for further separation. The use of a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (1:5:1:5 and 3:5:3:5, v/v) in the two dimensions permitted the simultaneous separation of oridonin and ponicidin, making this 2D-CCC system with satisfactory resolution and peak capacity.?. A novel elution method, called back-extrusion CCC (BECCC) was developed. It used the fact that the liquid stationary phase, that contained the retained solutes, can be easily moved. Switching the column inlet and outlet ports without changing the liquid phase used as the mobile phase caused the rapid collapse of the two immiscible liquid phases inside the column, the previously stationary phase being gathered at the new column outlet. Then this previously stationary liquid phase was extruded outside the CCC column carrying the retained solutes. The back-extrusion method was tested with a standard mixture of five compounds and compared with the recently described elution-extrusion method. It was shown that the chromatographic resolution obtained during the back-extrusion step was good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. In addition, the proposed method was successfully applied in the fractionation of two complex plant extracts, exhibiting great potential for rapid analysis of complex samples, such as high-throughput natural drug discovery programs.?. The potential of CCC to fractionate complex natural extracts was studied. The association of the rational heptane-ethyl acetatemethanol-water Arizona scale with the elution-extrusion operating mode allowed designing the 2VC EECCC rapid screening protocol. Its use with an integrated three 40 mL column apparatus allows us to find the correct AZ composition able to fractionate any given natural extract in a half-day of work using less than a total volume of 1 L of combined solvents. Once the right liquid system is well-established, the scaling-up for preparative fractionation of compounds contained in the extract is straightforward. This protocol is very promising to increase the throughput of finding new active molecules from plant extracts. Comparison of the proposed CCC protocol with conventional flash chromatography methods turns to the advantage of CCC in terms of sample pretreatment, solubility problems (an important factor for sample loading), and fraction recovery.In addition, the principle of CCC technique and its recent progress was reviewed in details.