Study On The Quality Control Of Fructus Cnidii By Modern Chromatography

Fructus cnidii (Chinese name she chuang zi) is the dried ripe fruit of Cnidium monnieri (L.) Cusson, family Umbelliferae. As one of the most popular traditional Chinese medicinal herbs, it has been used for the treatments of impotence, frigidity and skin-related diseases such as suppurative dermatitis and pudendum itching.Fructus cnidii have a widely distribution in China. In different regions, it was found that the coumarins in Fructus cnidii were changeable. At present coumarins are regarded as main active components in Fructus cnidii. This paper has focused on the coumarins from Fructus cnidii, using the method of representation on chemical ingredients and biological activity for active parts by modern chromatography. The platform of RP-high performance liquid chromatography, RP-high performance liquid chromatography using a narrow-bore column,β-cyclodextrin modified micellar electrokinetic chromatography and pressurized capillary electrochromatography were established to represent the quality control of coumarins from Fructus cnidii. The results of this thesis provided the methodogical examples and technique platform for research on modernization for quality control of Fructus cnidii.1. Determination of coumarins in Fructus cnidii by RP-HPLC Established a RP-HPLC method for separation and determination of active constituents in Fructus cnidii from 12 different regions, and evaluated the quality of Fructus cnidii. DiamonsilTM C18 (150 mm×4.6 mm, 5μm) was used with a mobile phase of ammonium acetate buffer (pH 4.0)/acetonitrile (50/50 v/v) and a flow rate of 1.0 mL/min, at the detection wavelength of 320 nm. The calibration curves were linear in the range of 4-100μg/mL for bergapten, 4-100μg/mL for imperatorin, 10-300μg/mL for osthole (r = 0.9997), 4-100μg/mL for 2'-acetylangelicin, 4-100μg/mL for oroselone, 4-100μg/mL for O-acetylcolumbianetin, respectively. The average recoveries were 98.1%, 96.2%, 96.4%, 101.9%, 102.4%, 98.4% with the RSD of 4.58%, 5.73%, 3.58%, 4.89%, 5.85%, 4.51%, respectively. The method is rapid, simple and accurate, and it can be suitable for the separation and determination of coumarins in Fructus cnidii extracts, quality control and geoherbalism research of Fructus cnidii.2. A simple PR-HPLC method using a narrow-bore column for the analysis of coumarins in Fructus cnidii extractsA simple and sensitive RP-HPLC method was developed and validated for the determination of coumarins in Fructus cnidii extracts. Phenomenex C18 Luna narrow-bore column (15 cm×2.1 mm, 5μm) was used with a mobile phase of ammonium acetate buffer (pH 4.0)/acetonitrile (50/50 v/v) and a flow rate of 0.3 mL/min, at the detection wavelength of 320 nm. The method was rapid, reproducible, and accurate.3. Determination of coumarins in Fructus cnidii by micellar electrokinetic capillary chromatography (MECC)Aβ-cyclodextrin modified MECC method was established to simultaneous determine 6 coumarin constituents in Fructus cnidii. The influence of different parameters (internal standard, pH, concentration of the running buffer, concentration of SDS andβ-cyclodextrin, applied voltage) was systematically studied. Running buffer: 40 mM borate-25 mM SDS-10 mMβ-cyclodextrin (pH 9.5) containing 20% (v/v) methanol; capillary: 50.2 cm×75μm i.d., effective length 40 cm, uncoated; applied voltage: 30 kV(+)→(-); column temperature: 25℃; pressure injection: 1 psi×5 s; detection: 210 nm. The quantitative analysis results showed that the content ranges of the coumarin constituents in the raw materials from different regions of China were different. Large variations in the contents of these constituents were found.4. Separation and determination of coumarins in Fructus cnidii extracts by pressurized capillary electrochromatography A microcolumn analytical method, pressurized capillary electrochromatography (pCEC) was developed for main coumarins from Fructus cnidii. The fabrication of the frit in column was studied since column is the heart of pCEC. We used a monolithic outlet frit to displace the tradition sintered ones and proposed the application of the new packed capillary column to pCEC experiments. First, the performance of the column has been studied. Then, the coumarins from Fructus cnidii were separated on the home-made capillary column. After a thorough study of analytical parameters such as acetonitrile content of the mobile phase, the concentration and pH of the buffer, and the applied voltage, a methodology was proposed to separate and determine six coumarins of Fructus cnidii extracts in less than 15 min. The analysis methods were fully validated with satisfactory accuracy and adequate reproducibility and the results showed that the method was rapid, reproducible and accurate.