槐米和五味子的指纹图谱及部分有效成分与血清白蛋白的相互作用研究
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摘要
本文针对现行的中药指纹图谱偏重于中药化学成分的种类和相对含量的研究,缺少与中药药效间的相关性研究的问题,在建立具有整体性、模糊性、可量化等特点的指纹图谱的基础上,引入特征参数logPow,使建立的指纹图谱具有测定疏水参数的功能,突破中药化学指纹图谱的概念,拓展其应用领域。
本文对中药指纹图谱的研究概况进行了综述,然后对以下三个命题进行了研究:
其一,选取超声提取法作为槐米组分的提取方法,以槐米电泳谱图的峰数为指标,确定了电泳运行介质为:40 mmol/L硼砂(pH 9.0),运行电压为20 kV,建立了槐米的毛细管电泳指纹图谱,确定了5个共有峰,方法的精密度、稳定性和重现性,符合建立指纹图谱的要求。对产自广西、贵州、河南、江苏、四川五地的槐米中所含的芦丁分别进行了定量的测定,通过比较得到贵州槐米中芦丁含量最高,达28.34%。同时,还采用三种算法对以上五个产地的槐米指纹图谱进行了相似性评价。通过计算得到五个产地的槐米之间具有良好的相似性。
其二,以槐米中的有效成分芦丁和槲皮素为配体分子,采用荧光光谱法研究了BSA-rutin,BSA-quercetin,HSA-rutin和HSA-quercetin四个体系的相互作用,分别得到了四个体系的结合常数和结合位点数。并采用同步荧光的方法研究了这四个相互作用体系中色氨酸和酪氨酸残基的荧光变化。
其三,采用MEEKC技术,以五味子为研究对象,确定了在微乳液组成为50 mmol/L SDS、80 mmol/L正庚烷、900 mmol/L正丁醇和20 mmol/L硼砂,pH值9.0的条件下五味子甲醇提取物的分离效果最佳,并建立了中药五味子的具有脂水分配系数测定功能的指纹图谱,获得了五味子提取物中13种化合物的疏水参数,方法的精密度、稳定性和重现性符合建立指纹图谱的要求。
Traditional Chinese medicine fingerprint (TCM-FP) is one of the methods to evaluate the quality of TCM by specialization and similarity of fingerprint with the integrative overview. TCM-FP, as one of the effective means of quality control for TCM, has a great development in recent years. However, the actual state of TCM-FP emphasis on the chemical compounds of extracts and their relative contents, lack of the research on the curative effect of TCM. Based on the establishment of the fingerprint which is integrity, fuzziness and qualitified, the study established the functional fingerprint by introduce characteristic parameters, to make a great breakthrough in the concept and application of TCM-FP.
The research projects and their results are:
(1) The fingerprint spectrum of the Flos Sophorae Buds was established with methanol as the extraction solvent in high performance capillary electrophoresis with applied voltage of 20 kV and a buffer concentration of 40 mmol/L borate (pH 9.0). The fingerprint had good precision, stability and reproducibility. The RSD of relative migration time of the co-peak was below 1.3%. The RSD of the peak area above 10% of the total peak area was below 6.0%. These parameters meet the requirements for the setting-up of fingerprints of TCM. Took the different sources of Flos sophorae Buds (Guizhou, Jiangsu, Guangxi, Henan, Sichuan) as samples, the content of rutin was examined, and the similarity of their TCM-FP was evaluated by 3 different methods.
(2) Rutin and quercetin, as the effective components of Flos Sophorae Buds, were selected to interact with bovine/human serum albumin. The four interaction systems were investigated by fluorescence spectroscopic method. The four interaction systems were also investigated by synchronous fluorescence spectroscopy.
(3) The optimized electrophoretic electrolyte of microemulsion electrokinetic chromatography (MEEKC) was composed of a 50 mmol/L SDS, 80mmol/L n-heptane, 900 mmol/L n-butanol, 20 mmol/L borate solution (pH 9.0). Following the principle and method on the measurement of oil-water partition coefficients by MEEKC, a functional TCM-FP of Fructus Schisandrae Chinensis was established. The fingerprint had good precision, stability and reproducibility. The values of logPow of 13 components of Fructus Schisandrae Chinensis were examined by this method.
本文对中药指纹图谱的研究概况进行了综述,然后对以下三个命题进行了研究:
其一,选取超声提取法作为槐米组分的提取方法,以槐米电泳谱图的峰数为指标,确定了电泳运行介质为:40 mmol/L硼砂(pH 9.0),运行电压为20 kV,建立了槐米的毛细管电泳指纹图谱,确定了5个共有峰,方法的精密度、稳定性和重现性,符合建立指纹图谱的要求。对产自广西、贵州、河南、江苏、四川五地的槐米中所含的芦丁分别进行了定量的测定,通过比较得到贵州槐米中芦丁含量最高,达28.34%。同时,还采用三种算法对以上五个产地的槐米指纹图谱进行了相似性评价。通过计算得到五个产地的槐米之间具有良好的相似性。
其二,以槐米中的有效成分芦丁和槲皮素为配体分子,采用荧光光谱法研究了BSA-rutin,BSA-quercetin,HSA-rutin和HSA-quercetin四个体系的相互作用,分别得到了四个体系的结合常数和结合位点数。并采用同步荧光的方法研究了这四个相互作用体系中色氨酸和酪氨酸残基的荧光变化。
其三,采用MEEKC技术,以五味子为研究对象,确定了在微乳液组成为50 mmol/L SDS、80 mmol/L正庚烷、900 mmol/L正丁醇和20 mmol/L硼砂,pH值9.0的条件下五味子甲醇提取物的分离效果最佳,并建立了中药五味子的具有脂水分配系数测定功能的指纹图谱,获得了五味子提取物中13种化合物的疏水参数,方法的精密度、稳定性和重现性符合建立指纹图谱的要求。
Traditional Chinese medicine fingerprint (TCM-FP) is one of the methods to evaluate the quality of TCM by specialization and similarity of fingerprint with the integrative overview. TCM-FP, as one of the effective means of quality control for TCM, has a great development in recent years. However, the actual state of TCM-FP emphasis on the chemical compounds of extracts and their relative contents, lack of the research on the curative effect of TCM. Based on the establishment of the fingerprint which is integrity, fuzziness and qualitified, the study established the functional fingerprint by introduce characteristic parameters, to make a great breakthrough in the concept and application of TCM-FP.
The research projects and their results are:
(1) The fingerprint spectrum of the Flos Sophorae Buds was established with methanol as the extraction solvent in high performance capillary electrophoresis with applied voltage of 20 kV and a buffer concentration of 40 mmol/L borate (pH 9.0). The fingerprint had good precision, stability and reproducibility. The RSD of relative migration time of the co-peak was below 1.3%. The RSD of the peak area above 10% of the total peak area was below 6.0%. These parameters meet the requirements for the setting-up of fingerprints of TCM. Took the different sources of Flos sophorae Buds (Guizhou, Jiangsu, Guangxi, Henan, Sichuan) as samples, the content of rutin was examined, and the similarity of their TCM-FP was evaluated by 3 different methods.
(2) Rutin and quercetin, as the effective components of Flos Sophorae Buds, were selected to interact with bovine/human serum albumin. The four interaction systems were investigated by fluorescence spectroscopic method. The four interaction systems were also investigated by synchronous fluorescence spectroscopy.
(3) The optimized electrophoretic electrolyte of microemulsion electrokinetic chromatography (MEEKC) was composed of a 50 mmol/L SDS, 80mmol/L n-heptane, 900 mmol/L n-butanol, 20 mmol/L borate solution (pH 9.0). Following the principle and method on the measurement of oil-water partition coefficients by MEEKC, a functional TCM-FP of Fructus Schisandrae Chinensis was established. The fingerprint had good precision, stability and reproducibility. The values of logPow of 13 components of Fructus Schisandrae Chinensis were examined by this method.
引文
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