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中药马齿苋有效成分提制分析及其质量控制新方法研究
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摘要
中药质量的优劣不但直接影响预防和治疗疾病的效果,而且密切关系到人们的健康与生命安全。为了保证用药的安全、合理和有效,在中药的研究、生产、保管、供应及临床使用过程中都应进行严格的分析检验,全面控制中药的质量。将现代分析方法和技术应用于中药的研究开发和生产过程中以保障中药质量提高中药疗效和加快中药现代化和国际化进程,是中药现代化面临的主要课题和重要任务。本研究以中药马齿苋为研究对象,以现代新兴样品预处理技术(微波辅助提取技术、分子印迹技术和固相萃取技术等)为工具,从马齿苋中提取黄酮类化合物,研究了各种因素对提取效率的影响。另外,本研究还建立了马齿苋药材色谱和光谱指纹图谱质量控制标准,并对马齿苋药材中的以山奈酚为代表的黄酮类化合物的样品前处理方法进行了相关研究,为合理利用马齿苋药材提供了相关参照。具体内容如下:
     1.中药马齿苋中总黄酮的提制分析
     以中药马齿苋为研究对象,研究马齿苋中总黄酮的最佳提取方法。研究比较了五种提取马齿苋中总黄酮的方法,包括:微波辅助提取法、超声提取法、热浸回流提取法、索氏提取法和室温冷浸提取法。通过单因素实验及正交实验对微波辅助提取条件进行优化,以总黄酮得率为指标,考察了提取溶剂、提取时间、提取温度和提取固液比等因素对实验结果的影响。采用分光光度法对马齿苋中总黄酮进行了测定,以NaNO2-Al(NO3)3-NaOH显色体系,在碱性条件下,利用其与黄酮类化合物在可见光区具有特征吸收,以芦丁为对照测定其吸光度。实验结果表明,马齿苋中总黄酮的最佳提取工艺条件为:70%的乙醇,料液比1:50,提取温度为50℃,微波辅助提取9 min。总黄酮测定的线性回归方程为A=-0.00171+11.58482C,线性相关系数为0.9999,平均回收率为102.6%,RSD=1.13%(n=5),用所拟方法对8组马齿苋样品进行了测定,其总黄酮含量分别为7.16,7.10,9.38,6.82,6.78,11.36,5.12和1.76 mg/g。
     2.中药马齿苋GC/MS指纹图谱和红外光谱指纹图谱的建立
     研究采用气相色谱质谱联用技术和红外光谱技术建立了中药马齿苋GC/MS指纹图谱和红外光谱指纹图谱并进行了比较,结合相似度分析和聚类分析对指纹图谱的实用性进行了分析,鉴别了马齿苋挥发油的主要成分和共有成分。研究结果表明,通过建立色谱质谱指纹图谱可以得到其主要成分信息及各成分含量间的关系,而红外光谱指纹图谱得到的是所有化合物的叠加信息。GC/MS指纹图谱和IR指纹图谱聚类分析的结果是相似的,色谱质谱指纹图谱可以用于其主要成分的快速分离鉴定,二阶导数红外光谱可以明显提高其红外光谱指纹图谱的分辨率。
     3.微波合成分子印迹微球并结合固相萃取法分析中药中的山奈酚
     将微波加热的方法研究应用于山奈酚分子印迹聚合物微球的合成中,聚合的时间大大缩短,而且所得聚合物微球的形状均匀,其静态吸附性能实验结果和选择性能实验结果均优于常规加热方法合成的分子印迹聚合物微球。实验所得分子印迹微球直径分布在6-9μm,平均直径为8μm,微波加热得到的分子印迹微球的印迹效率为5.0,而常规加热方法得到的分子印迹微球的印迹效率为4.2;选用印迹效率较高的微波合成的分子印迹微球作为固相萃取的吸附剂进行固相萃取性能研究,结果表明,山奈酚分子印迹微球作为固相萃取吸附剂对山奈酚表现出了良好的萃取性能,且具有较高的洗脱率;将自制的山奈酚分子印迹微球固相萃取柱用于中药马齿苋和山奈中的山奈酚的分析,回收率分别为90.2%和88.0%,RSD为1.21%和1.18%,结果满意。
The quality of Chinese herds has an important role on the effectiveness of prevention and treatment of disease and closely related to people's health and safety. In order to ensure the safety, rational and effective of drugs the testing should be rigorous operated and comprehensive quality of traditional Chinese medicine should be controlled in the process of preparation, production, storage, supply and clinical research of traditional Chinese medicine. It is the main issues and important tasks of the modernization of Chinese medicine that modern analysis methods and techniques be used in Chinese medicine research and production process to improve the quality of Chinese medicine and speed up the modernization and internationalization of Chinese medicine. The noval types of sample pretreatment techniques (microwave-assisted extraction approaches, molecular imprinting technique and solid-phase microextraction) have been used for extraction of flavonoids from Portulaca oleracea L. In this work, the parameters which influence the extraction efficiencies in each technique had been investigated. Additionally, the quality control standard of Portulaca oleracea L. was established by the techniques of chromatographic and spectroscopic techniques, which was used to control the quality of Portulaca oleracea L. And then sample pre-treatment methods of the flavonoids, such as kaempferol, from Portulaca oleracea L. was studied, which can provide reference informations for the rational use of Portulaca oleracea L. The concrete contents are as follows:
     1. Analysis of Flavonoids in Portulaca oleracea L. by UV-Vis Spectrophotometry with Comparative Study on Different Extraction Technologies
     Using Portulaca oleracea L. as the research object and the extraction method of total flavonoids in Portulaca oleracea L. was studied. Five extraction technologies of flavonoids from Portulaca oleracea L. were investigated and compared, including microwave-assisted extraction, ultrasonic extraction, reflux extraction, Soxhlet extraction, and marinated extraction. The conditions of microwave assisted extraction were optimized by single factor experiments and orthogonal test, and the extraction solvent, extraction time, extraction temperature and extraction of solid to liquid ratio was investigated. Quantification was performed by means of UV-Vis spectrophotometry with chromogenic system of NaN02-Al (NO3)3-NaOH. In alkaline conditions the chromogenic system has a characteristic absorption in the visible region and could be determined. The results showed that microwave assisted extraction were most suitable for the extraction of flavonoids from Portulaca oleracea L. because of its high effect and short extraction time. The found optimum extraction conditions were that the ethanol concentration was 70%(v/v), solid-liquid ratio was 1:50, extracting temperature was 50℃and irradiation time was 9 min. Under the optimum conditions, the linear regression equation was A=-0.00171+11.58482C, the calibration curve for the analyte was linear with the correlation coefficients was greater than 0.9999. The average recovery was 102.6%, and its RSD was 1.13%(n=5). Eight types of Portulaca oleracea L. according to different habits were investigated. The total content of flavonoids was 7.16,7.10,9.38,6.82, 6.78,11.36,5.12, and 1.76 mg/g, respectively.
     2. Identification of Portulaca oleracea L. from different sources using GC/MS and FT-IR spectroscopy
     A fingerprinting approach was developed by means of gas chromatography/mass spectrometry (GC/MS) and IR spectroscopy for quality control of Portulaca oleracea L., and the similarity evaluation and hierarchical cluster analysis were performed to evaluate the similarity and variation of these samples. Major components of volatile oil and characteristic peaks in the common pattern were identified. The results show that the information of the compounds and the information of the relative content of the compounds could be obtained through chromatography mass spectrometry analysis and The IR spectrum shows a total overlap of each absorption spectrum of all components. The cluster analysis results of GC/MS and IR spectra were similar. GC-MS fingerprint can be used for the rapid separation and identification of major components and the second derivative spectrum can enhance the apparent resolution of IR spectrum.
     3. Development and Characterization of Molecular Imprinted Polymer for the Selective Detection of Kaempferol in Traditional Chinese Medicines
     Microwave heating was applied to the preparation of kaempferol molecular imprinted polymer microspheres, the term of the polymerization was dramatically shortened by using microwave heating and the results of morphology observation, static absorption performance and selectivity performance of the MIP microspheres were all superior to the MIPs prepared by conventional heating. The MIP microsphere were demonstrated with a narrow diameter distribution (6-9μm) and with a spherical shape and the imprinting efficiency of the MIP microspheres prepared by microwave heating was 5.0 and by conventional heating was 4.2. Based on the imprinting effect, the MIPs prepared by microwave was used as the sorbent of solid phase extraction and the properties of the resultant extraction cartridge shows a good extraction performance of kaempferol. The MIPs coupled with solid phase extraction was used for extracting kaempferol in Portulaca oleracea L. and Alpinia officinarum, the recoveries were 90.2% and 88.0% and RSD were 1.21% and 1.18%. The results indicated that the MIPs can be favorably used for the extraction of the kaempferol in traditional Chinese medicines.
引文
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