【作者】 李翔；

【导师】 吴玉田； 柴逸峰；

【作者基本信息】 第二军医大学 ， 药物分析学， 2008， 博士

【摘要】 四逆汤是《伤寒论》的经典要方,由附子、干姜和甘草组成。四逆汤组方精良,配伍严谨。现代研究表明,四逆汤在心、脑血管疾病的预防和临床治疗中表现出独特的优势和作用特点。附子是四逆汤的君药,主要含生物碱类成分;干姜主要含挥发油成分和二苯庚烷、姜辣素类等不挥发性成分;甘草主要含三萜皂苷和黄酮类成分。本课题在组方药材淡附片、干姜、炙甘草及四逆汤HPLC-MS指纹图谱研究的基础上,采用HPLC-TOF-MS进行组方药材和四逆汤的化学成分分析研究,建立四逆汤HPLC-TOF-MS指纹图谱表征四逆汤化学物质基础。采用化学计量学方法,进行不同配比四逆汤指纹图谱与抗心肌缺血作用的评价,确定四逆汤的药效物质基础及可能的药效成分,建立了全新的中药复方药效物质基础评价方法。结合四逆汤的拆方研究,对化学计量学的研究结果进行验证。一、四逆汤化学成分的分析研究(一)四逆汤HPLC-MS指纹图谱的研究分别建立乌头碱、6-姜酚和甘草酸等3个成分的HPLC快速测定方法,并分别以淡附片、干姜和炙甘草中3个成分的含量为指标,采用正交设计的方法,优化固液比例、浸泡时间和超声时间等前处理因素,确定甲醇超声提取的最佳条件。分别建立四逆汤3个组方药材淡附片、干姜和炙甘草的HPLC-MS指纹图谱,进行方法学考察,获取组方药材甲醇提取液和水煎液的指纹图谱。在组方药材HPLC-MS指纹图谱研究的基础上,对四逆汤HPLC-MS指纹图谱的流动相体系、醋酸含量和醋酸铵浓度等色谱条件进行逐步优化,选择乙腈—2 mmol/L醋酸铵溶液(含0.01%醋酸)梯度洗脱为色谱条件。在正离子检测模式,扫描范围m/z 200～1200建立四逆汤HPLC-MS指纹图谱。以干姜挥发油的得率为指标,分别采用正交设计和均匀设计的方法对水蒸气蒸馏条件进行优化,选择加17倍量水,提取8 h为提取条件,建立干姜挥发油GC-MS指纹图谱并鉴别挥发油中的化学成分。(二)四逆汤HPLC-TOF-MS化学成分的分析研究根据组方药材中化学成分的类别,分别建立5个化学成分数据库,分别包括淡附片生物碱类、干姜二苯庚烷类和姜辣素类、炙甘草三萜皂苷类和黄酮类化学成分。通过HPLC-TOF-MS分析组方药材甲醇提取液和水煎液的化学成分,对已知化合物和未知化合物分别进行匹配和标记,表征组方药材的化学物质基础。在此基础上,通过HPLC-TOF-MS分析四逆汤的化学成分,表征四逆汤的化学物质基础。从四逆汤HPLC-TOF-MS总离子流色谱图中发现156个色谱峰,其中49个色谱峰来自淡附片,40个色谱峰来自干姜,67个色谱峰来自炙甘草,根据准确分子量、同位素分布、元素组成和保留时间可以鉴定其中19个色谱峰为已知生物碱类化合物,18个色谱峰为已知二苯庚烷类化合物,13个色谱峰为已知姜辣素类化合物,12个色谱峰为已知三萜皂苷类化合物,23个色谱峰为已知黄酮类化合物,其余色谱峰为来自各组方药材的相应未知化合物。二、四逆汤抗心肌缺血作用的研究(一)四逆汤药效物质基础的研究采用小鼠腹腔注射垂体后叶素建立急性心肌缺血模型,选择心肌组织SOD的活性为指标。在HPLC-TOF-MS化学成分指纹图谱研究的基础上,进行不同配比四逆汤的抗心肌缺血作用研究,将色谱峰面积的变化与小鼠心肌组织SOD的活性相关联,采用相关分析和多元线性回归方法确认四逆汤的药效物质基础及可能的药效成分,同时明确组方药材来源,实现从化学物质基础到药效物质基础的提升。采用相关分析的方法,确认22个化合物的色谱峰为四逆汤抗心肌缺血的可能的药效物质基础,其中11个色谱峰为已知生物碱类化合物,3个色谱峰为已知二苯庚烷类化合物,2个色谱峰为已知姜辣素类化合物,其余6个未知化合物色谱峰有4个来自淡附片,各有1个来自干姜和炙甘草。采用多元线性回归的方法,确认2个生物碱类化合物森布星B和去甲猪毛菜碱为四逆汤中可能药效成分。从药效物质基础和药效成分的分析结果中,可以发现淡附片在四逆汤抗心肌缺血作用中具有突出的作用。(二)四逆汤抗心肌缺血作用的拆方研究选择小鼠心肌组织SOD活性为指标,通过比较整方四逆汤、缺味四逆汤和组方药材之间的药效差异,以四逆汤的整体药效为参照,在中药复方的整体水平上进一步明确了淡附片在四逆汤抗心肌缺血作用中具有突出作用,同时也验证了化学计量学方法确定四逆汤药效物质基础所得结论的正确性,说明采用化学计量学方法,通过HPLC-TOF-MS化学成分指纹图谱与药效结果相结合的方式确认中药复方的药效物质基础是可行的。更多还原

【Abstract】 Sini decoction(SND) is one of the most famous and important compound in Shanghan Lun,which was composed with Radix aconite lateralis praeparata(RA), Rhizoma zingiberis(RZ) and Radix et rhizoma glycyrrhizae praeparata cum melle(RE). SND is excellent and compact,and modern study has indicated that SND has expressed distinct superiority and effect characteristic in prevention and clinical treatment of heart and cerebrovascular disease.RA is recognized as principal material containing alkaloids constituents.The chemical constituents of RZ are volatile oils and non-volatile constituents including diarylheptanoids and gingerol-related compounds,and the chemical constituents of RE are triterpene glycosides and flavonids.The HPLC-MS fingerprint chromatograms of component medicinal materials and SND were investigated and the HPLC-TOF-MS were used to analysis the chemical constituents of component medicinal materials and SND,and establish HPLC-TOF-MS fingerprint chromatograms to represent the chemical substances base of SND.THE methods of chemometrics were used to evaluate the SND of different ratio and their anti-myocardial ischemia effects.The pharmacodynamic substance base and the possible pharmaceutical effective constituents were confirmed.Combined with the decomposed recipes of SND,the results of chemometrics were validated.1 Study of chemical constituents analysis of SND(1) Study of HPLC-MS fingerprint chromatograms of SNDThe rapid HPLC methods of aconitine,6-gingerol and glycyrrhizic acid were established respectively,and the contents of three chemical constituents in RA,RZ and RE were set as index to investigate and optimize the conditions of preliminary treatment with orthogonal experimental design of solid-liquid proportion,saturate time and ultrasonic time of ultrasound with methanol.The HPLC-MS fingerprint chromatogram of three component medicinal materials were established and methodology investigation was completed,and the fingerprint chromatogram of methanol and boiling water extract were acquired,respectively.On the foundation of fingerprint chromatogram study of three component medicinal materials,the chromatographic conditions of HPLC-MS of SND,including mobile phase system,content of acetic acid and concentration of ammonium acetate were gradually optimized and the best gradient elution of HPLC-MS fingerprint chromatogram was confirmed as acetonitrile-2 mmol/L ammonium acetate solution(0.01%acetic acid) under positive mode with mass spectra range of m/z 100～1200.The yield of volatile oil of RZ was set as index,and the orthogonal design and homogeneous design was used to optimize the conditions of wet distillation.The 17 times water and extract with 8 h was selected as extract condition,and characterized the chemical constituents of volatile oil of RZ.(2) HPLC-TOF-MS analysis of chemical constituents of SNDAccording to the category of chemical constituents of three component medicinal materials including RA,RZ and RE,The chemical constituents database including alkaloids,diarylheptanoids,gingerol-related compounds,triterpene glycosides and flavonids were established.The chemical constituents of methanol and boiling water extracts of component medicinal materials was analyzed with HPLC-TOF-MS,and the known and unknown compounds were matched and marked,the chemical substances base of SND was represented.156 peaks were found from HPLC-TOF-MS total ions chromatogram of SND,in which 49 peaks from RA,40 peaks from RZ,67 peaks from RE.Depending on the accurate mass,abundance of isotopic distribution,elemental compositions and retention time,19,18,13,12 and 23 peaks were identified as known alkaloids,diarylheptanoids, gingerol-related compounds,triterpene glycosides and flavonids respectively,and other peaks were unknown compounds from three component medicinal materials.2 Study of anti-myocardial ischemia effect of SND(1) Study of pharmacodynamic substance base of SNDThe model of mice myocardial ischemia was established by intraperitoneal injection of posterior pituitary,and the activity of SOD in cardiac muscle tissue was selected as index.On the foundation of chemical constituents fingerprint chromatogram of HPLC-TOF-MS,anti-myocardial ischemia effects of SND with different ratios were investigated.The variation of peak area and activity of SOD in cardiac muscle tissue were associated,and the methods of correlation analysis and multiple linear stepwise regression were used to confirm the pharmacodynamic substance base of SND and possible pharmaceutical effective constituents.Meanwhile,the resource of component medicinal materials was identified,and the chemical substances base was upgrade to the pharmacodynamic substance base.With method of correlation analysis,23 compounds peaks were confirmed as the pharmacodynamic substance base of anti-myocardial ischemia effect of SND,11,3 and 2 peaks were identified as known alkaloids,diarylheptanoids and gingerol-related compounds respectively,and 4 peaks of unknown compounds were from RA,other 2 peaks of unknown compounds were from RZ and RE respectively.Furthermore,with the method of multiple linear stepwise regression,2 alkaloids compounds peaks,Senbusine B and Salsolinol were reckoned as possible effective chemical constituents.From the results of the pharmacodynamic substance base and effective chemical constituents,RA can be found with distinguished effect in anti-myocardial ischemia effect of SND.(2) The decomposed recipes study of anti-myocardial ischemia effect of SNDThe activity of SOD in cardiac muscle tissue was selected as index to compare the effect discrepancy of SND,incompleted SND and component medicinal materials.The whole effect of SND was set as reference,and the important effect of RA in anti-myocardial ischemia effect of SND was further identified.Meanwhile,the correctness of pharmacodynamic substance base of SND was confirmed,and the chemometrics method of confirmation of pharmacodynamic substance base of Chinese herb compound was feasible with the mode of combination of HPLC-TOF-MS chemical constituents fingerprint chromatogram and pharmacodynamic action result.更多还原