Studies On The Material Foundation And Analysis Methods Of Feixingcao And Jusanqi

As we all know, quality control is one of the key issues of the modernization for TCM. Studies on material foundation and analysis methods of TCM are the premise of quality control.Tripterospermum Chinese (Migo) H. Smith (Feixingcao), Gynura segetum (Lour.) Merr. (Jusanqi) and Shenmai injection were chosen as objects for exploring the material foundation and analysis methods by chromatography and hyphenated techniques. In this thesis, systemic studies on the effective constituents, toxic constituents and ineffective constituents of TCM were carried out. The results supplied a scientific research methodology used for quality control and reasonable application of the TCM. The main innovation and academic contributions of this dissertation are presented as follows:1. Column chromatography techniques were used to separate constituents from the 70% ethanol extract of the whole plants of T. Chinese and the structures of six compounds were elucidated by ESI-MS, TOF-MS, ¹H NMR, ¹³C NMR, HMQC, ¹H-¹HCOSY, HMBC. Four of them were xanthones which had antioxidant activity, the other two of which one belonged to new compounds were isolated from the genus Tripterospermum for the first time. 2. Based on sub-2μm (1.8μm) column, a novel chromatography method was constructed. The chromatography parameters, such as signal acquisition, column temperature, flow rate and volume of sample injection, were optimized. SST was good enough to meet the quantitative analysis of xanthones in feixingcao. The four xanthones were baseline separated within 6 min. Linearity of the method was excellent with correlation coefficients (R²) in the range of 0.9992-1.000 when the concentration of xanthones varied from 0.75 to 300μg/ml. Limit of detection was in the range of 0.1-0.3μg/ml. This method could provide high-speed separation and high sensitivity under ordinary pressure, meanwhile the consumption of mobile phase decreased.3. In this study, pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs) were extracted from the whole plant of G. segetum and analyzed by high performance liquid chromatography coupled to ion trap mass spectrometry. Identification of eluted peaks as PANOs was indicated by virtue of their MS and MSⁿ analysis, in addition to the [M+H]⁺ adduct ion, characteristically showed a significant (usually 100% abundance) dimer adduct [2M+H]⁺ that is not observed in the MS of the parent PAs. A total of 20 compounds were identified or tentatively characterized based on their mass spectra and possible biosynthetic pathways. Sixteen constituents were reported for the first time from G. segetum and tetrahydrosenecionine has not been previously reported as a natural product.4. A novel, rapid and sensitive MEKC method was developed for the separation and determination of two hepatotoxic pyrrolizidine alkaloids in G. segetum within 8 min. The optimal running buffer containing 35 mM lauric acid, 120 mM Tris and 20% methanol (v/v) was employed. Linearity of the method was excellent with correlation coefficients (R²) in the range of 0.9916-0.9939 when the concentration of PAs varied from 3.1 to 200μg/ml. Limit of detection of the two PAs was 1.0μg/ml. Compared with traditional SDS/inorganic bases system, this method provided high efficiencies, on account of its low ionic strength and therefore low conductivity. This method was suit for the quality control of Jusanqi.5. A novel, rapid and high-sensitivity CZE-MS method was developed for the simultaneous determination of hepatotoxic pyrrolizidine alkaloids and N-oxides. The optimal running buffer containing 20 mM ammonium acetate, 1% formic acid and 5% methanol (v/v) was employed. Field-amplified sample stacking with electromigration-injection (FASS-EMI) was used for the on-line concentration of the alkaloids. This online preconcentration strategy resulted in sensitivity enhancement factors 30-fold for PAs and 200-fold for PANOs. The LOD was 0.01 ng/ml for the two PAs and was 0.1 ng/ml for the two PANOs. The method is suited to determination of trace amount PAs and PANOs in TCM.6. The method of HPLC-ELSD for the analysis of contents of D-fructose, D-glucose, sucrose and maltose in Shenmai injection was established. Saccharides were separated by NH₂ column and the mobile phase was acetonitrile-water. Linearity of the method was excellent with correlation coefficients (R²) in the range of 0.9983-0.9992 when the concentration of saccharides varied from 0.025 to 1.000 mg/ml. The total saccharide in Shenmai injection was also determined using Phenylhydrate-sulfuric acid and Freeze Drying methods. Furthermore, these three methods were compared seriously.