New Extraction Technologies And Separation With High-speed Countercurrent Chromatography Of The Active Constituents From Licorice

Effective and efficient analytical technology for the extraction and isolation of active components in herbal plants or extracts is an important area of research in the current push towards the modernization of TCM (Traditional Chinese Medicine). In this thesis, several novel extraction techniques including microwave-assisted extraction (MAE), multistage countercurrent extraction (MCE), and high-speed counter-current chromatography (HSCCC) methods for the extraction and isolation of some important active ingredients in licorice have been developed and critically evaluated.Chapter 1 is an overview of the current research status and developing trends of TCM. It reviews important technologies for the extraction of active components from TCMs and the latest research findings on the analysis, chemistry and pharmacology of licorice.In chapter 2, MAE method was developed for the extraction of GA from licorice. The effects of important operational factors such as temperature, time and microwave power on the performance of the technique have been investigated and the optimum MAE conditions have been determined using orthogonal array design (OAD) experiments. Under the optimum conditions, the optimal solvent was selected. The results indicated that the optimum conditions of MAE are extracting for another 40min in 0.5% ammonia water after heated to 50 by microwave of 666 watt.MCE technique for the extraction of GA from licorice is discussed in detail in chapter 3. The effects of countercurrent extraction stage number, temperature, extraction time and the ratio of solvent/licorice on GA extraction yield have been investigated. The appropriate MCE conditions have been obtained fromorthogonal array design (OAD) experiments. The optimum conditions of MCE are five stages, and with an extracting time of 60min for each stage at 60. The ratio of solvent/licorice is 6:1 (mL/g). Under optimal conditions, the extraction yield of GA by MCE technique is higher than those of MAE, ultrasonic assisted extraction (USE), Soxhlet extraction (SHE) and extraction at room temperature (ERT) techniques.The history, apparatus, separation principle, solvent system of the technique, and its application in the separation of natural products, quality control, inorganic analyses and other fields are summarized in chapter 4 through the review of published references.In chapter 5, development work on the use of HSCCC technique to isolate and purify inflacoumarin A and licochalcone A from the ethanol extract of licorice is described. Primary separation was performed with a two-phase solvent system composed of hexane-chloroform-methanol-water (5:6:3:2, v/v) by eluting the lower phase at a flow rate of 1.8 mL/min and a revolution speed of 800 r/min. More refined purification step was performed with a two-phase solvent system composed of hexane-chloroform-methanol-water (1.5:6:3:2, v/v) by eluting the lower phase at a flow-rate of 1.5 mL/min and a revolution speed of 800 r/min. The purities of inflacoumarin A and licochalcone A reached 99.6% and 99.1%, respectively. In the end the structural analysis and identification of inflacoumarin A and licochalcone A by 'H- and 13C-NMR, 13C -1H -COSY, UV, FTIR and EI-MS analyses were discussed.Major conclusions and recommendations for future research are summarized in chapter 6.