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Studies on the Fingerprint Analysis and Quality Control of Fructus Ligustri Lucidum and Its Preparation

Author: DingXue
Tutor: ShiLiFu
School: Second Military Medical University
Course: Pharmaceutical Analysis
Keywords: Fingerprinting Chinese medicine Ligustrum Tonic Capsule HPLC UPLC-ESI-TOF/MS
CLC: R284
Type: Master's thesis
Year: 2009
Downloads: 301
Quote: 0
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Abstract


Ligustrum lucidum for the traditional beneficial traditional Chinese medicines, has been used for thousands of years of history, has the basis of a wide range of clinical applications. To develop, improve, and the establishment of a modern Chinese medicine quality standards evaluation system, with internationally recognized standards so that Chinese medicine is one of the strategic objectives of the modernization of Chinese medicine. Characterization of compounds with a representative of traditional Chinese medicine pharmacodynamic substances based group fingerprints to comprehensive evaluation of the quality of TCM, in line with the characteristics of the chemical composition of traditional Chinese medicine theory and traditional Chinese medicine, can reflect more fully the original appearance of the traditional Chinese medicine quality, and is internationally recognized. In this paper, combined with several modern detection techniques, such as HPLC / UV and UPLC-ESI-TOF/MS Ligustrum lucidum establish fingerprints of herbs and its compound preparation, separation and identification of the active ingredient, Chemoinformatics combination and pharmacodynamics, and evaluate the quality of research. For the first part of the Ligustrum herbs fat-soluble ingredients and the extraction and separation of water-soluble components: Extract Ligustrum different parts of the group, and retain different parts of the active ingredient. Methods: To compare the 100%, 75%, 50% ethanol solution of different proportions of the different extraction methods, ultrasonic extraction and maceration for extraction; the extracted samples were then layered extraction in accordance with the selection of different polarity difference extractant; suitable conditions for the HPLC Determination of Total peak area and number, determine the appropriate pre-treatment program. Results: establishing a suitable method of extraction of the fat-soluble components and water-soluble components: extracted two times with 75% ethanol solution of ultrasound, every 30min; extractant hierarchical extraction and then chloroform and ethyl acetate, respectively, to shake off extractant, namely to get the liposoluble constituents and water-soluble components. HPLC fingerprint of the second part of Ligustrum HPLC fingerprint Objective: To establish the HPLC analysis method, Ligustrum herbs, to evaluate the quality of different origin Ligustrum herbs. Methods: A high-performance liquid chromatography to Platisil (Platinum) C18 ODS (250 × 4.6 mm, 5 μm) column, mobile phase, water and acetonitrile, gradient elution of fat-soluble ingredients detection wavelength of 240nm, elution procedures : t :0-15-23-33-80min, A% (the proportion of acetonitrile): 15% -30% -40% -90% -100%; water soluble component, the detection wavelength was 220nm, elution program: t :0-25-35-40-60min, A% the acetonitrile proportion: 20% -30% -60% -90% -100%, the flow rate are 1.0ml · min-1, column temperature was 25 ° C. Results: The chromatogram obtained using a gradient elution of each peak separation is better to fingerprint requirements. Reliable analytical methods to provide for better control Ligustrum intrinsic quality. 10 Origin of similarity differences, the water-soluble components of different origin Ligustrum herbs fingerprint similarity is above 0.85, while the fat-soluble ingredients fingerprint similarity in the 0.45 to 0.93 range, indicating a different origin of the female between Sadako herbs, there is a big difference, but the difference is mainly reflected in the fat-soluble ingredients. The use of cluster analysis to prove that the origin is an important factor to affect the of Ligustrum quality differences. The quality is very important to the protection of traditional Chinese medicine, Jiangsu Province GAP base Ligustrum herbs with higher levels of active ingredients, and the implementation of GAP system in our country. The third part Ligustrum the medicinal chemistry ingredients UPLC-ESI-TOF/MS qualitative Objective: to use UPLC-ESI-TOF/MS qualitative analysis Ligustrum different parts of the main chemical components of Chinese herbal medicines. : The ESI-TOF/MS quality detector and UPLC C18 column (100 × 2.1mm, 1.7 μm) and MASS LYNX system. Optimization of nitrogen as the atomizing cone gas; electrospray ionization positive ion mode; flight the tube detection mode V-shaped; capillary voltage 3.2kv; cone voltage 35v; the extraction cone Hole 1.0v; source temperature 120 ° C; desolvation air temperature of 350 ° C; reverse cone gas flow 50l / h; off the solvent gas 600 l / h; collision gas flow rate 0.5mL/min; detector 1700v; scanning time 0.45s; scanning time interval 0.05s; quality Hebi Fan Wai: 50-1000m / z; forms of data collection center ionization. Results: find the molecular weight of 456.6825 and 456.7134 of the compounds, oleanolic acid and ursolic by the structures were confirmed, preliminary derivation cracking mechanism of oleanolic acid and ursolic possible to enrich pentacyclic triterpenoids ESI -MSn data, provides an effective mass spectrometric methods for the identification of other pentacyclic triterpenoids. The fourth part of the compound preparation Zhenqifuzheng capsule HPLC fingerprint of purpose: To establish an HPLC analysis method to establish the fingerprint of of Ligustrum lucidum preparations Zhenqifuzheng capsules, and evaluate its quality. Method: Tonic Capsule filtration, dissolved in methanol sample concentration of 0.1mg/ml. High performance liquid chromatography of Platisil (Platinum) C18 ODS (250 × 4.6mm, 5μm) column with a mobile phase of water and acetonitrile, gradient elution program: t :0-12-35-40 -50min, A% the acetonitrile proportion: 6% -20% -40% -60% -100% of the wavelength of 236nm, flow rate of 1.0 ml min-1, column temperature was 25 ° C. Results: The chromatogram obtained using a gradient elution of peak separation is better, the main peak separation is greater than 1.5, to achieve the requirements of fingerprints. 10 batches Tonic Capsule sample similarity greater than 0.85, to prove that the production process is stable and reliable. Established fingerprint analysis method was validated by the method, operational, precision, reproducibility, stability, Tonic Capsule can be used for quality control.

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