【作者】 曲丽萍；

【导师】 吴玉田； 范国荣；

【作者基本信息】 第二军医大学 ， 药物分析学， 2010， 博士

【摘要】 针对中药复杂体系定性定量分析与药效机制研究方面面临的瓶颈问题,本课题以辛夷挥发油为研究对象,应用气相色谱-质谱联用分析手段,结合化学计量学方法及代谢组学研究技术,对辛夷挥发油的组成、药代动力学、药效学、作用机制等进行了全面、系统的研究,为揭示辛夷挥发油药效物质基础提供了依据,对于含有辛夷的中药复方的药效学研究亦具有一定的借鉴意义。具体研究内容如下:1.气相色谱中目标色谱峰保留时间的预测先是基于恒温及一阶程序升温条件下目标色谱峰的保留时间,对一阶程序升温条件下另一升温速率的该目标色谱峰保留时间进行预测。结果表明,预测值和测量值的相对偏差较大;然后,在一阶程序升温条件下,用两个不同升温速率下目标色谱峰的保留时间来预测另一速率下的保留时间,预测值与实测值较为接近,只有个别组分相对偏差大于1%,其它相对偏差较小。比之在恒温下对目标色谱峰保留时间的预测值来说,相对偏差明显减小。基于实际需要,建立了可用于二阶及多阶程序升温的目标色谱峰保留时间预测的方法。先以标准对照品为研究对象,预测正十六酸和二十九烷在二阶程序升温条件下的保留时间。结果显示,预测值与实测值的相对偏差小于0.2%。然后以辛夷挥发油成分为考察目标,相对偏差小于0.15%,预测结果满意。并对一阶和二阶程序升温条件下保留时间预测相对偏差进行了比较。结果显示,二阶条件下的预测值和真实值更为接近。该预测方法可用来优化气相色谱条件,节省分析时间,提高分析效率,在实际工作中具有较好的应用价值。2.化学计量学分辨技术和联用色谱技术用于辛夷挥发油的复杂成分分析通过GC-MS联用,结合直观推导式演进特征投影法(HELP)对辛夷挥发油的成分进行了定性和定量分析。以重叠色谱峰簇A和B为例,在解析过程中,先后使用FSMWEFA,ELPG和HELP方法,对重叠色谱峰进行了解析。解析结果显示:峰簇A是含有三组分的峰簇,峰簇A中的三个化合物分别为o-Cymene,D-Limonene和Eucalyptol,相对含量分别为2.27%,4.52%,14.20%;峰簇B经FSMWEFA法解析是4组分体系,但2D-ELPG显示为3组分体系,为进一步确定体系组分数,采用3D-ELPG方法进行确认后,峰簇B包括四个化合物,解析结果和NIST数据库比对后,确认分别是Carotol,τ-Cadinol,τ-Muurolol和1á-Cadin-4-en-10-ol,相对含量分别是0.13%,1.41%,1.01%,0.41%。采用此策略对辛夷挥发油进行解析后,化合物由原来直接由NIST检索的65个增加至80个。在本研究中,我们充分利用了3D-ELPG的信息,解析结果更为准确可靠。可以看出,GC-MS联用结合HELP方法可成功的用于分析辛夷挥发油成分,也充分说明化学计量学在分析复杂体系中具有潜在的优势,可提高组分定性和定量的准确性。3.辛夷挥发油成分GC-MS指纹图谱的建立通过多元曲线分辨-偏最小二乘法(MCR-ALS)对12批辛夷挥发油的重叠色谱峰进行了批量解析,增加了定性定量的准确性;确定了12批样品中辛夷挥发油的共有组分及含量,确定了辛夷挥发油中最主要的化合物,包括桉油精,异合金欢醇,α-水芹烯,崁烯,柠檬烯,α-杜松醇,α-芳樟醇,石竹烯,邻聚伞花素等。采用n强峰、共有峰率及变异率对指纹图谱进行了评价,建立的方法稳定、可靠,较为全面的反映了辛夷挥发油的化学成分,为辛夷药材质量标准奠定了基础。4.辛夷挥发油中主要活性成分1,8-桉叶素的药代动力学初步研究以1,8-桉叶素为研究对象,采用液-液萃取法提取血浆样品,建立了以α-蒎烯为内标的气相色谱法检测血浆样品中1,8-桉叶素浓度的方法。色谱分离条件为毛细管柱:SPBTM-1,0.25 mm×30 m×0.25μm;FID检测器;载气:氮气,流速1.0 mL/min;程序升温条件:起始柱温50°C,保持2 min,再以5°C /min的速率升温至100°C,保持2 min。采用本文建立的生物样品预处理方法和气相色谱法测定样品中1,8-桉叶素的含量,生物样品预处理方法回收率高,色谱分离选择性好。本法的准确度、精密度、专属性和定量线性范围均达到体内药物分析的要求。线性相关系数为0.999,日内精密度范围是3.75~5.74%,日间精密度范围是2.41~5.58%。用上述建立的气相色谱法测定血浆中1,8-桉叶素的浓度,根据所得的血药浓度-时间曲线,采用非房室模型推算药物动力学参数。结果表明,SD大鼠口服1,8-桉叶素后体内血药浓度消除较慢,个体差异较大。经非房室模型法估算的SD大鼠口服给药后达峰时间为2h,达峰浓度为36.223μg/ml,药时曲线末端相消除半衰期为4.811h,平均滞留时间为7.923h,AUC0~12和AUC0~∞分别为227.09μg·h/ml,280.06μg·h/ml。5.辛夷挥发油抗炎作用的代谢组学初步研究首先建立角叉菜胶致大鼠急性炎症模型,然后从药效学角度证明辛夷挥发油具有较好的抗炎活性。然后采用GC-MS分析正常组、模型组及给药组的三种不同状态下大鼠的血浆代谢谱,采用NIST数据库对不同组别大鼠血浆中的内源性代谢物进行鉴定,使用多元模式识别方法分析大鼠不同组间的代谢物谱差异。在正常组和模型组的得分矩阵图中,两组的样本点完全分离,说明致炎后大鼠正常生理代谢被干扰,从机体生理内源性代谢物变化的层面可以认为炎症模型造模成功。在正常组、模型组及给药组的得分矩阵图中,三组的样本点也达到了分离,而对照组和给药组在第二主成分上毗邻,这表明给药组样本有向对照组移动的趋势。从三组的载荷矩阵图中,可以得到对分类贡献较大的生物标记物。数据结果表明,模型组中尿素、戊二胺、甘氨酸及乳酸的含量升高,且尿素和尸胺对分类贡献最大,可认为是角叉菜胶致炎对大鼠机体生理内源性代谢产生扰动的生物标记物。给药组中尿素、戊二胺、甘氨酸及乳酸的含量下降,回到正常水平,而软脂酸、亚油酸、油酸、硬脂酸、花生四烯酸、胆固醇的含量升高,且高于正常组,可认为是给药后的生物标记物。通过文献调研表明,这些标记物的改变和其抗炎活性存在一定的关系。这些脂肪酸及胆固醇可作为肝x受体的激活剂,并启动肝x受体信号通路从而发挥抗炎活性。更多还原

【Abstract】 Based on the problems faced by complex system of Chinese medicine qualitative and quantitative analysis and the research of pharmacodynamics mechanism, in the present paper, Flos magnoliae volatile oil was chosen as the research subject, and gas chromatography-mass spectrometry was used as analytical tools and chemometric resolution methods and metabonomics research techniques were carried out to conduct a comprehensive and systematic study. This research provides the basis for pharmacological activity of Flos Magnoliae volatile oil, and gives a certain amount of reference and help for pharmacodynamic study of Chinese medicine formulation containing Flos Magnoliae.Some studies are as follows,1. Retention time prediction of target chromatographic peaks in gas chromatography conditionWhen one non-isothermal run and another isothermal run were used to predict the retention time in capillary gas chromatography, the relative error is large; when two one-step programmed runs were used, the relative error is small. In the case, the relatively larger error could be due to the relatively larger difference of the sample in partition between under the non-isothermal condition and under the isothermal condition.Based on real needs, a modified method was proposed for retention time prediction in two-step temperature-programmed gas chromatography, based on the closed-form integral method which was formerly put forward to predict the retention time in one-step gas chromatography. The calculation process was simplified and the prediction error was reduced in the proposed method. The modified method was validated through predicting the retention time of two reference standards, n-hexadecanoic acid and nonacosane using two-step temperature-programmed gas chromatography, and the relative errors of prediction were less than 0.2%. Then the proposed method was applied to predict the retention time of Flos Magnoliae volatile oil, and satisfactory results were obtained as well. The relative errors of prediction were less than 0.15%. The results indicate that our modified method is effective for retention time prediction in the two-step temperature-programmed gas chromatography, and the prediction error could be reduced using the proposed method. The prediction method can be used to optimize the gas chromatographic conditions, save analysis time and improve the analysis efficiency. Thus, there are good application field in practical work.2. Determination of the volatile oil of Magnolia biondii Pamp by GC-MS combined with Chemometric TechniquesAn optimized temperature-programmed gas chromatography-mass spectrometry system combined with chemometric methods was firstly applied to analyze the volatile components of M. biondii Pamp (a kind of Flos Magnoliae). The peak purity of two-way data was controlled by fixed size moving window evolving factor analysis, two dimensional-evolving latent projection graph and three dimensional-evolving latent projection graph. Then the overlapped peak clusters were resolved using heuristic evolving latent projection. We choose peak cluster A and B as examples to illustrate the data analysis process. The results show that the components co-eluted in peak cluster A is o-cymene, D-limonene and eucalyptol. Their relative contents are 2.27%, 4.52% and 14.20%, respectively; the components co-eluted in peak cluster B is Carotol,τ-Cadinol,τ-Muurolol and 1á-Cadin-4-en-10-ol. Their relative contents are 0.13%, 1.41%, 1.01% and 0.41%, respectively. A total of 65 components were identified using similarity searches between mass spectra and MS database. This number was extended to 80 components with the help of chemometric techniques.In this study, we make full use of the 3D-ELPG information, and more accurate and reliable analytical results can be acquired. The research indicates GC-MS combined with chemometric techniques can be successfully used to analyze the volatile oil of Magnolia biondii Pamp, and the CRM method in analysis of complex systems have the potential advantages. The accuracy of qualitative and quantitative analysis can be improved. The results also prove that the reported approach is powerful for the analysis of complex herbal samples. 3. Comparative study on fingerprints of Flos Magnoliae volatile oil by GC-MS combined with MCR-ALSTwelve different sources of Flos Magnoliae were analyzed and compared with each other. The overlapped chromatographic peaks of 12 batches of Flos Magnoliae volatile oils were resolved through MCR-ALS method, and the accuracy of qualitative and quantitative was increased. It is the first time to apply MCR-ALS method to resolve the chromatographic peaks in fingerprint research, and it reduces the burden of qualitative analysis as well as the subjectivity. Among the components were determined, there were 34 components coexisting in all samples. Eucalyptol, (E, E)-Farnesol,α-phellandrene, Camphene, D-Limonene,α-Cadinol,α-Linalool, Caryophyllene and o-cymene are the main components in Flos Magnoliae volatile oil. N strong peaks, Common and Variant peak ratios were used to evaluate their fingerprint. The results showed a fair consistency in their GC-MS fingerprint.4. Quantitative analysis and preliminary study on pharmacokinetics of 1, 8-cineole in rats plasmaA gas chromatography method has been developed for the determination of 1,8-cineole in rat plasma usingα-pinene as internal standard. Liquid-liquid extraction was used as the sample pretreatment mode. Separation was obtained on a 30 m×0.25 mm i.d. capillary column coated with 0.25μm film SPBTM-1. Nitrogen was used as carrier gas at a 1.0 mL/min flow rate. To acquire good separation, the column temperature was initially maintained at 50°C for 2 min, and then increased from 50 to 100°C at a rate of 5°C/min, and held for 2 min. The chromatographic system used in this research provided good separation of compounds without interfering peaks from endogenous substances. The calibration curve was liner range with correction coefficients above 0.999. The precision of intra-day and inter-day were evaluated by analysis of variance with the result of 3.75~5.74% and 2.41~5.58%, respectively. The method has been successfully used to support the pharmacokinetics study of 1,8-cineole.Following a single oral administration with 400mg/kg to rats, the blood samples were collected at different time after dosing. All collected blood samples were centrifuged to obtain plasma and the concentration of 1,8-cineole in plasma were determined by gas chromatography method described as above. Pharmacokinetics parameter calculations were carried out using non-compartmental analysis method. It showed from the plasma concentration-time data that the concentrations of 1,8-cineole in rat plasma increased and then reduced slowly after oral administration and the individual difference of rats was evident. The peak plasma concentration was 36.223μg/ml at 2h. The estimated elimination half-time and Mean residence time in rats were 4.811h and 7.923h, respectively. AUC0~12 and AUC0~∞were 227.09μg·h/ml and 280.06μg·h/ml, respectively.These results indicated that 1,8-cineole has slow oral absorption and its biological half-time period is relative longer. If suitable pharmaceutical dosage forms could be used, its bioavailability would be higher. In addition, the relevant research suggests that 1,8-cineole has a strong permeability, it can be developed for the transdermal formulations as local anti-inflammatory or antibacterial drug.5. Intervention effect of Volatile Oil of Magnolia biondii Pamp (VOMbP) on rat model of acute inflammation: A plasma metabonomics study based on gas chromatography-mass spectrometryIn the present study, GC-MS-based metabonomics was applied to investigate the metabolic profiles of rats with acute inflammation induced by carrageenan, and the intervention effects of VOMbP on acute inflammation were evaluated as well. The endogenous metabolites in rat plasma were identified by NIST library, and the metabolic patterns were investigated using principal component analysis. The PCA scores plot showed that the inflammation group was apparently separated to the control group, indicating that the two groups had completely different metabolic profiles. When PCA was applied to treat the data of the control, inflammation and VOMbP groups, however, it can be seen that the three groups were apparently apart from each other, whereas the VOMbP group and the control group were adjacent at the same side on PC2. This indicated that the metabolic profile of VOMbP pretreated group was totally different from that of the model group and was showing a trend to return to the control group.In the corresponding loadings plot based on the GC-MS data of three groups, the metabolites which are the furthest from zero could be considered as marker metabolites that contribute most strongly to the classification of the three groups. In inflammation group, levels of urea, cadaverine, glycine and lactic acid increased, whereas the other metabolites had reduced levels. After VOMbP pretreatment, however, it can be seen that the levels of urea, cadaverine, glycine and lactic acid were reduced and the levels of the other metabolites were elevated comparing to those in the inflammation group. Among them, six metabolites, namely hexadecanoic acid, linoleic acid, oleic acid, stearic acid, arachidonic acid and cholesterol, may be mostly related to the anti-inflammatory activity of VOMbP may be involved in the anti-inflammatory activation of VOMbP, these metabolites may be regarded to act as LXRs activators to hinder the release of the inflammatory mediators and inhibit the inflammatory response. Therefore, VOMbP may exert its effectiveness on anti-inflammatory activation by involving the LXRs signaling pathway.更多还原