黄芪当归复方的物质基础及其治疗肝纤维化的实验研究
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摘要
中药色谱指纹图谱是中药(包括中药单味药与中药复方制剂)整体性的化学表征。联用仪器和色谱指纹图谱技术为阐明中药药效的物质基础研究提供了强有力的手段。本论文利用GC-MS、HPLC-DAD和HPLC-DAD-MS等联用色谱仪器和中药指纹图谱技术,结合能有效利用色谱和光谱/质谱信息的化学计量学方法,实现了对黄芪当归复方及其单味药化学组分的归属分析;考察了黄芪当归复方治疗兔血吸虫病肝纤维化的药效作用;建立了一种反相高效液相色谱方法快速测定尿羟脯氨酸的含量并讨论了其与肝纤维化程度的关系。
利用联用色谱仪器和指纹图谱技术定性分析了黄芪当归复方中挥发性和非挥发性化学成分。利用GC-MS、HPLC-DAD和HPLC-DAD-MS联用仪器得到黄芪当归复方及单味药的色谱指纹图谱,结合紫外光谱、质谱数据进行定性和归属分析,发现当归和黄芪配伍组成当黄芪当归复方后,复方中的组分绝大部分来源于各单味药的化学成分,但含量有消长变化。这些变化的化学组分有待进一步的分析研究。
研究了不同剂量黄芪当归复方对兔血吸虫病肝纤维化的药效作用。根据黄芪当归复方的益气补血功效,研究了不同剂量黄芪当归复方对兔血吸虫病肝纤维化的治疗作用。结合吡喹酮治疗兔血吸虫病肝纤维化,用B超声图像、组织病理学切片和血清学指标(透明质酸和羟脯氨酸)评估不同剂量的黄芪当归复方治疗血吸虫病肝纤维化的效果,发现血吸虫病大耳兔经黄芪当归复方治疗后肝纤维化程度降低,其血清透明质酸含量与羟脯氨酸含量也较治疗前明显降低。这一结果表明黄芪当归复方对血吸虫病肝纤维化有很好的治疗作用,且高剂量优于低剂量的治疗效果。
建立了一种新的选择性测定尿中羟脯氨酸含量的高效液相色谱(HPLC)法。本文以9-芴基甲氧基羰酰氯为衍生试剂,以3,4-脱羟脯氨酸为内标,用HPLC-DAD联用仪器快速测定尿中羟脯氨酸含量。此方法检出限为2.0μmol/L(S/N=3),回收率为97.45%。本方法具有样品处理步骤简单、准确度高、重现性好、分离时间短等优点,适合大批量样品测定。并用此方法测定了血吸虫病肝纤维化动物模型治疗前后的尿羟脯氨酸含量变化,对比肝纤维化病理学诊断结果,发现尿羟脯氨酸含量与肝纤维化程度呈一定的相关关系。
The chromatographic fingerprints of traditional Chinese medicine (TCM),including single herb and formula,represent appropriately the "chemical integrities" of TCM.Multiple constituents are usually responsible for its therapeutic effects and may work 'synergistically'.In order to address the integrity and synergistic effects of TCM,the chromatographic fingerprinting is possible to provide a powerful tool for the analysis of complex mixture of TCM,with the rapid development of hyphenated instruments and related chemometric methods.
Firstly,qualitative and quantitative analysis of chemical components in the formula of radix astragali and angelica sinensis was conducted with the help of hyphenated GC-MS,HPLC-DAD and HPLC-DAD-MS methods.Chromatographic fingerprinting is proposed as a tool for identifying and quantifying the volatile and nonvolatile components in the formula.By comparing the retention time,UV spectra,mass spectra and molecular weight of detected compounds,some components of the formula of radix astragali and angelica sinensis could be tentatively identified.
Then,the therapeutic effect against schistosomiasis hepatic fibrosis in rabbits of the formula of radix astragali and angelica sinensis was investigated in some detail.The rabbit model of hepatic fibrosis was obtained by infection of schistosoma japonicum.After being treated with high dosage of the formula of radix astragali and angelica sinensis,low dosage and no formula just normal feed for ten weeks separately,the therapeutic effect of a rabbit model of schistosomiasis hepatic fibrosis was evaluated using B-ultrasonic images,histopathological examination, hyaluronic acid and hydroxyproline in serum.The results indicated that the formula of radix astragali and angelica sinensis have a good therapeutic effect for schistosomiasis hepatic fibrosis.The statistical results showed that the degree of liver fibrosis treated with high dosage of the formula of radix astragali and angelica sinensis is lower than those of low dosage.
Finally,a determination method of urinary by reversed phase high performance liquid chromatography(RP-HPLC)hydroxyproline(HYP) was developed.After derivatization by 9-fluorenylmethyl chloroformate (FMOC),the hydroxyproline was determined by the hyphenated HPLC-DAD.The recovery of urinary hydroxyproline was 97.45%.The method developed in this work is easier to operate,accurate and rapid, and suitable for the analysis of great mass of sample.Before and after oral administration of the formula of radix astragali and angelica sinensis, the variance in concentration of urinary hydroxyproline in rabbit models, which had been treated with the formula of radix astragali and angelica sinensis,was measured in this new method.The result suggests that the concentration of urinary hydroxyproline is related with the degree of hepatic fibrosis.
利用联用色谱仪器和指纹图谱技术定性分析了黄芪当归复方中挥发性和非挥发性化学成分。利用GC-MS、HPLC-DAD和HPLC-DAD-MS联用仪器得到黄芪当归复方及单味药的色谱指纹图谱,结合紫外光谱、质谱数据进行定性和归属分析,发现当归和黄芪配伍组成当黄芪当归复方后,复方中的组分绝大部分来源于各单味药的化学成分,但含量有消长变化。这些变化的化学组分有待进一步的分析研究。
研究了不同剂量黄芪当归复方对兔血吸虫病肝纤维化的药效作用。根据黄芪当归复方的益气补血功效,研究了不同剂量黄芪当归复方对兔血吸虫病肝纤维化的治疗作用。结合吡喹酮治疗兔血吸虫病肝纤维化,用B超声图像、组织病理学切片和血清学指标(透明质酸和羟脯氨酸)评估不同剂量的黄芪当归复方治疗血吸虫病肝纤维化的效果,发现血吸虫病大耳兔经黄芪当归复方治疗后肝纤维化程度降低,其血清透明质酸含量与羟脯氨酸含量也较治疗前明显降低。这一结果表明黄芪当归复方对血吸虫病肝纤维化有很好的治疗作用,且高剂量优于低剂量的治疗效果。
建立了一种新的选择性测定尿中羟脯氨酸含量的高效液相色谱(HPLC)法。本文以9-芴基甲氧基羰酰氯为衍生试剂,以3,4-脱羟脯氨酸为内标,用HPLC-DAD联用仪器快速测定尿中羟脯氨酸含量。此方法检出限为2.0μmol/L(S/N=3),回收率为97.45%。本方法具有样品处理步骤简单、准确度高、重现性好、分离时间短等优点,适合大批量样品测定。并用此方法测定了血吸虫病肝纤维化动物模型治疗前后的尿羟脯氨酸含量变化,对比肝纤维化病理学诊断结果,发现尿羟脯氨酸含量与肝纤维化程度呈一定的相关关系。
The chromatographic fingerprints of traditional Chinese medicine (TCM),including single herb and formula,represent appropriately the "chemical integrities" of TCM.Multiple constituents are usually responsible for its therapeutic effects and may work 'synergistically'.In order to address the integrity and synergistic effects of TCM,the chromatographic fingerprinting is possible to provide a powerful tool for the analysis of complex mixture of TCM,with the rapid development of hyphenated instruments and related chemometric methods.
Firstly,qualitative and quantitative analysis of chemical components in the formula of radix astragali and angelica sinensis was conducted with the help of hyphenated GC-MS,HPLC-DAD and HPLC-DAD-MS methods.Chromatographic fingerprinting is proposed as a tool for identifying and quantifying the volatile and nonvolatile components in the formula.By comparing the retention time,UV spectra,mass spectra and molecular weight of detected compounds,some components of the formula of radix astragali and angelica sinensis could be tentatively identified.
Then,the therapeutic effect against schistosomiasis hepatic fibrosis in rabbits of the formula of radix astragali and angelica sinensis was investigated in some detail.The rabbit model of hepatic fibrosis was obtained by infection of schistosoma japonicum.After being treated with high dosage of the formula of radix astragali and angelica sinensis,low dosage and no formula just normal feed for ten weeks separately,the therapeutic effect of a rabbit model of schistosomiasis hepatic fibrosis was evaluated using B-ultrasonic images,histopathological examination, hyaluronic acid and hydroxyproline in serum.The results indicated that the formula of radix astragali and angelica sinensis have a good therapeutic effect for schistosomiasis hepatic fibrosis.The statistical results showed that the degree of liver fibrosis treated with high dosage of the formula of radix astragali and angelica sinensis is lower than those of low dosage.
Finally,a determination method of urinary by reversed phase high performance liquid chromatography(RP-HPLC)hydroxyproline(HYP) was developed.After derivatization by 9-fluorenylmethyl chloroformate (FMOC),the hydroxyproline was determined by the hyphenated HPLC-DAD.The recovery of urinary hydroxyproline was 97.45%.The method developed in this work is easier to operate,accurate and rapid, and suitable for the analysis of great mass of sample.Before and after oral administration of the formula of radix astragali and angelica sinensis, the variance in concentration of urinary hydroxyproline in rabbit models, which had been treated with the formula of radix astragali and angelica sinensis,was measured in this new method.The result suggests that the concentration of urinary hydroxyproline is related with the degree of hepatic fibrosis.
引文
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