Separation Of Active Components From Tripterygium Wilfordii

Tripterygium wilfordii Hook. f. (TWHF) is a traditional Chinese herb grown in the south of China and used for various immune and inflammatory diseases for many years. The extracts of the roots of TWHF are used widely for the treatment of a number of autoimmune disorders such as rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, and skin diseases. The main active constituents of the herb have been reported to be diterpenes, triterpenes and alkaloid's compounds. In this dissertation, a series of rapid, sensitive and accurate LC/MS analytical methods were established for the determination of triptolide, tripdiolide, wilfortrine, wilfordine, wilforgine and wilforine in extract of TWHF. The separation and isolation of triptolide, tripdiolide, wilfortrine, wilfordine, wilforgine and wilforine from TWHF is emphasized in this dissertation. The main contents of this work are as following:(1) A novel method using liquid chromatography (LC) coupled with an atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in the negative mode has been developed and validated for the rapidly simultaneous determination of triptolide and tripdiolide.(2)The first analytical procedure determination of wilfortrine-1, wilfordine-2, wilforgine-3 and wilforine-4 is described. Identity of the analytes was confirmed in a LC-MS experiment, with all compounds being clearly assignable in an atmospheric pressure chemical ionization (APCI) positive mode.(3)The sample preparation of crude triptolide from leaves of TWHF was optimized. A single-step preparative high-speed counter-current chromatography (HSCCC) method was developed for the separation and purification of triptolide from the extract of leaves of TWHF. The ratio of compositions of the solvent system composed of petroleum ether-ethyl acetate-ethanol- water were optimized. The effects of rotation speed of the multilayer coil column and flow rate of mobile phase on the retention of the stationary phase were investigated.(4)In column chromatography, the technique of separation of triptolide was researched with silca as solid phase. The effect of solvent, flow-rate of mobile phase, loading amount andconcentration on the resolution and theoretical plate height were investigated. A rate model was set up to simulate the experimental elution curve. The model fits the experimental elution curve well.(5)Separation of triptolide by solvent extraction coupled with HSCCC was developed for the enrichment and purification of tripdiolide from the multi-glycoside of Tripterygium wilfordii Hook F (GTW). The effects of the species and the volume ratio of extract solvents on the distribution coefficients of triptolide in several extraction system were studied. The purity of triptolide of the GTW was improved significantly after extraction. Then the triptolide enriched extract was subjected to HSCCC. The ratio of compositions of the solvent system for HSCCC separation were also optimized. The effects of rotation speed of the multilayer coil column and flow rate of mobile phase on the retention of the stationary phase were investigated.(6) A HSCCC method was developed for preparative isolation and purification of alkaloids from TWHF waste material. Preparative HSCCC with a two-phase solvent system composed of light petroleum-ethyl acetate-ethanol-water as the two-phase solvent system.. The ratio of compositions of the solvent system composed of light petroleum-ethyl acetate-ethanol-water were optimized. The effects of rotation speed of the multilayer coil column and flow rate of mobile phase on the retention of the stationary phase were investigated. In a single operation, four kinds of alkaloids were obtained.