【作者】 邱新建；

【导师】 陈泽奇；

【作者基本信息】 中南大学 ， 中西医结合临床， 2011， 博士

【摘要】 背景和目的：功能性消化不良是指具有上腹部疼痛、上腹部胀满、早饱、嗳气、食欲不振、恶心、呕吐等上腹部不适症状,经检查排除引起这些症状的器质性疾病的一组临床综合症。柴胡疏肝散是治疗功能性消化不良的经典古方。中药复方的治疗作用是多种成分共同作用的结果。本实验的目的：①建立快速、简单、灵敏的超高效液相色谱-光电二极管阵列(UPLC-PDA)测定方法,定量分析柴胡疏肝散汤剂中没食子酸、氧化芍药苷、芍药内酯苷、芍药苷、甘草苷、阿魏酸、柚皮芸香苷、柚皮苷、橙皮苷、新橙皮苷、水合橙皮内酯、甘草素、槲皮素、苯甲酰芍药苷、异甘草素和芒柄花素16种成分。②建立液相色谱-质谱联用(LC-MS)定性测定功能性消化不良病人口服柴胡疏肝散汤剂后血浆中成分阿魏酸和水合橙皮内酯。③研究柴胡疏肝散,阿魏酸和水合橙皮内酯对大鼠胃肠运动的影响,部分阐明柴胡疏肝散治疗功能性消化不良的药效物质基础。④建立超高效液相色谱法(UPLC-PDA)研究功能性消化不良病人口服柴胡疏肝散后血浆中阿魏酸和水合橙皮内酯的药物动力学。方法：第一章柴胡疏肝散汤剂中16种活性成分的定量分析采用UPLC-PDA法测定柴胡疏肝散汤剂中没食子酸、氧化芍药苷、芍药内酯苷、芍药苷、甘草苷、阿魏酸、柚皮芸香苷、柚皮苷、橙皮苷、新橙皮苷、水合橙皮内酯、甘草素、槲皮素、苯甲酰芍药苷、异甘草素和芒柄花素16种成分的含量,绘制各个成分的标准曲线,并对其最低检测浓度、精密度、回收率、样本稳定性和重现性进行验证。色谱柱：ACQUITY BEH C18柱(2.1 mm×50 mm,1.7μm),流动相：乙腈(A)-0.5%醋酸水(B),梯度洗脱,流速：0.5mL/min,柱温：40℃,检测波长：190-480nm。第二章阿魏酸和水合橙皮内酯在功能性消化不良病人口服柴胡疏肝散汤剂后血浆中的定性分析招募功能性消化不良病人,予一次口服柴胡疏肝散汤剂后取血。血浆经甲醇直接沉淀法去除蛋白质后,采用LC-MS法对病人血浆中阿魏酸和水合橙皮内酯进行分离分析,通过电离子喷雾(ESI)法,以选择离子反应检测(SRM)方式进行检测,阿魏酸和水合橙皮内酯离子通道m/z分别为194.8-177.0和261.1-243.0.采用Waters Xterra C18(150 mm×2.1 mm,5μm)色谱柱,流动相为10 mmol·L-1甲酸胺-乙腈(40：60,v/v),流速为0.2 mL·min-1,进样体积为20μL,柱温为40℃,样晶室温度为5℃。第三章柴胡疏肝散及其成分阿魏酸和水合橙皮内酯促胃肠动力作用研究采用大鼠在体胃排空和肠推进方法研究柴胡疏肝散,阿魏酸和水合橙皮内酯促胃肠动力作用；采用离体肠管实验研究阿魏酸和水合橙皮内酯对大鼠肠管收缩作用个影响。第四章功能性消化不良病人口服柴胡疏肝散后吸收入血的阿魏酸和水合橙皮内酯药物动力学研究用蛋白沉淀法预处理血样,UPLC法同时测定7名功能性消化不良病人口服柴胡疏肝散汤剂后13个时间点血浆样本中阿魏酸与水合橙皮内酯的血药浓度。应用3P97药动学分析软件对血药浓度-时间数据进行拟合。选用磺胺甲嗯唑为内标,阿魏酸和水合橙皮内酯检测波长分別为321nm和324nm。结果：第一章柴胡疏肝散汤剂中16种活性成分的定量分析(1)建立了一个简单、快速和灵敏的能同时检测柴胡疏肝散汤剂中16种成分的UPLC-PDA法。样品分析时间：17.5min,色谱图中各成分的色谱峰分离完全,无杂峰干扰。16种成分在其线性检测范围内显示出良好的线性回归,r2>0.9992。该方法日内、日间精密度良好,其相对标准差(RSD)波动在1.87%-5.61%之间；胡疏肝散16种成分的加样回收率良好,平均加样回收率在96.13%-102.46%之间,RSD<6.74%。柴胡疏肝散汤剂样本在24小时内稳定性好,RSD波动在0.78%～2.98%。柴胡疏肝散汤剂重现性好,RSD波动于1.94%-5.36%。(2)经UPLC-PDA方法检测后,柴胡疏肝散汤剂中没食子酸、氧化芍药苷、芍药内酯苷、芍药苷、甘草苷、阿魏酸、柚皮芸香苷、柚皮苷、橙皮苷、新橙皮苷、水合橙皮内酯、甘草素、槲皮素、苯甲酰芍药苷、异甘草素和芒柄花素16种化学成分被定量分析。第二章阿魏酸和水合橙皮内酯在功能性消化不良病人口服柴胡疏肝散汤剂后血浆中的定性分析通过研究阿魏酸和水合橙皮内酯主要色谱峰的质谱特征,功能性消化不良病人口服柴胡疏肝散汤剂后血浆中阿魏酸和水合橙皮内酯被成功检出。第三章柴胡疏肝散及其成分阿魏酸和水合橙皮内酯促胃肠动力作用研究与鼠空白对照组相比,柴胡疏肝散汤剂(20 g/kg),阿魏酸(100mg/kg)和水合橙皮内酯(14,28 mg/kg)能明显增加大鼠的胃排空率和肠推进率,P<0.05。、与大鼠空白肠相比,阿魏酸(100μmol/L)和水合橙皮内酯(10μmol/L)能明显增加大鼠空肠纵行肌和横行肌肉的收缩,P<0.05,水合橙皮内酯(30,100μmol/L)能显著增加大鼠空肠纵行肌和横行肌肉的收缩,P<0.01.第四章功能性消化不良病人口服柴胡疏肝散后吸收入血的阿魏酸和水合橙皮内酯药物动力学研究功能性消化不良患者(7名)口服柴胡疏肝散汤剂后,血浆中阿魏酸与水合橙皮内酯被检测。在色谱图上两种化合物分离完全,未出现其他物质干扰峰,保留时间分别为6.5 min和15.7min,内标磺胺甲嗯唑为8.0 min。阿魏酸与水合橙皮内酯的回收率分别为95.35%-102.56%和95.58%～102.09%,相对标准偏差(relative standard deviation, RSD)分别小于2.81%和3.86%。用空白血清加标准品绘制标准曲线,阿魏酸线性范围为10～320 ng/ml,信噪比为3时最低检测线为1.5 ng/ml,最低检测浓度为5 ng/ml;水合橙皮内酯范围为7.5～720ng/mL,最低检测线为2.5ng/mL,最低检测浓度7.5 ng/mL。阿魏酸与水合橙皮内酯在功能性消化不良病人体内的代谢过程被拟合为二室模型,阿魏酸与水合橙皮内酯分别在27.50 mmin和23.57 min时达到最大血药浓度0.199 mg/L和0.371 mg/L,阿魏酸的消除半衰期和AUC分别为131.27min和14.83μg·min/mL,水合橙皮内酯的消除半衰期和AUC分别为139.53 min和31.445μg·min/mL结论：(1)用超高效液相色谱-光电二极管阵列(UPLC-PDA)法检测柴胡疏肝散中主要有效成分快速灵敏,准确可靠、稳定且重现性好。此方法可成功用于同时检测柴胡疏肝散汤剂中16种目标成分的含量。(2)功能性消化不良病人口服柴胡疏肝散后血浆中阿魏酸和水合橙皮内酯被的LC-MS法成功分离和定性。(3)柴胡疏肝散及体内吸收成分阿魏酸和水合橙皮内酯均有明显促胃肠动力的作用,阿魏酸和水合橙皮内酯是柴胡疏肝散治疗功能性消化不良的主要物质基础。(4)采用超高效液相色谱法对功能性消化不良病人服用柴胡疏肝散后血浆中的阿魏酸和水合橙皮内酯进行同步测定。柴胡疏肝散及其促胃肠动力成分阿魏酸和水合橙皮内酯的药物动力学研究将有助于监测该方的临床应用以及理解其治疗机理。更多还原

【Abstract】 Background and objective:Functional dyspepsia is a syndrome defined by chronic or recurrent upper abdominal symptoms without any organic or biochemical abnormality. The clinical symptoms often include upper abdominal pain, upper abdominal fullness, early satiety, belching, losing of appetite, nausea, vomiting and other symptoms of upper abdominal discomfort. Chaihu-Shugan-San (CSS) is a popular traditional Chinese medicine formula to treat functional dyspepsia. It is widely accepted that the combined effect of multiple constituents of traditional Chinese medicine formula is considered to be crucial for treating diseases in clinical practice. The objective of the study are as follows:①To develop a simple, rapid and sensitive Ultra performance liquid chromatography coupled with photodiode array (UPLC-PDA) detection method for simultaneous determination of sixteen compounds in Chaihu-Shugan-San:gallic acid, oxypaeoniflora, albiflorin, paeoniflorin, liquiritin, ferulic acid (FA), narirutin, naringin, hesperidin, neohesperidin, meranzin hydrate (MH), liquiritigenin, quercimelin, benzoylpaeoniflorin, isoliquiritigenin, formononetin.②To develop a liquid chromatography-tandem mass spectrometry (LC-MS) method for the determination of FA and MH in plasmas of patients with functional dyspepsia after oral administration of Chaihu-Shugan-San decoction.③To investigated the effect of CSS, FA and MH on gastrointestinal motility in rats, partly elucidate the effective substance of CSS treating functional dyspepsia.④To develop a Ultra performance liquid churomatography coupled with photodiode array (UPLC-PDA) detection method to explore the pharmacokinetics of FA and MH in plasmas of patients with functional dyspepsia after oral administration of CSS decoction.Method:Part 1 The quantitative analysis of the sixteen active constituents in CSSThe analysis was performance on UPLC-PDA system with BEH C18 column (100×2.1 mm id,1.7μM). The mobile phase comprising acetonitrile (A) and 0.5% acetic acid (B) was used to elute the targets in gradient elution mode. Flow rate and detection wavelength were set at 0.5ml/min and 284nm. The sixteen constituents in CSS including gallic acid, oxypaeoniflorin, albiflorin, paeoniflorin, liquiritin, FA, narirutin, naringin, hesperidin, neohesperidin, MH, liquiritigenin, quercimelin, benzoylpaeoniflorin, isoliquiritigenin and formononetin were quantitative determinate, and the limit of determination (LOD), precision, recovery, sample stability and reproducibility of the sixteen constituents were examined in this study.Part 2 The qualitative analysis of FA and MH in the plasma of patients with functional dyspepsia after oral administration of CSS.The patients with functional dyspepsia were recuited and the plasmas were collected from the patients after oral administration of CSS. The plasma samples were precipitated with methanol. FA and MH were determined by LC-MS. Electrospray ionization (ESI) source was applied and operated in the positive ion mode. Selected reaction monitoring (SRM) mode with the transition of m/z 194.8-m/z 177.0 and m/z 261.1-m/z243.0 were used to detect FA and MH. Waters Xterra C18 column (150 mm×2.1 mm,5μm) was used in the experiment. The column temperature was set at 40℃.10 mmol·L-1 ammonium formate-acetonitrile (40:60, v/v) was used as mobile phase and the flow rate was 0.2mL·min-1 The sample room temperature was set at 5℃and the injection volume was 20μL.Part 3 The effects of CSS, FA and MH on promoting gastrointestinal motility.Gastric emptying (GE) and intestinal transit (IT) were used to study the effects of CSS, FA and MH on promoting gastrointestinal motility in vivo in rat. The intestinal muscle strips test in vitro was used to investigate the effects of FA and MH on Intestinal contraction.Part 4 The pharmacokinetics of FA and MH in plasmas of patients with functional dyspepsia after oral administration of CSS decoction.A total of 7 patients with functional dyspepsia were recruited in this study. The plasma samples were preprocessed by protein precipitation method. The UPLC system was used to determine the contents of FA and MH in the blood samples of 7 volunteers with functional dyspepsia, and the blood was collected through the vein at 5,10,15,30,45,60,90, 120,180,240,300,360,480 min. The dates of plasma concentration-time were fitted by using the 3P97 analytical program of pharmacokinetics. The internal standard (IS) was sulfamethoxazole. The detection wavelengths of FA and MH were set at 321 nm and 324 nm respectively.Result:Part1(1) A simple, rapid and sensitive Ultra performance liquid chromatography coupled with photodiode array (UPLC-PDA) detection method was established for the simultaneous determination of sixteen compounds in CSS. The 16 marker constituents were sufficiently separated successfully in less than 17.5 min. There were no interference peaks in the vicinity of the target compounds in the chromatograms of CSS decoction. All calibration curves showed good linear regression (r2> 0.9992) within test ranges of the sixteen targets. The relative standard deviations (RSD) of intra-and inter-day precision were from 1.87% to 5.61% indicating good precision. The average recoveries of investigated sixteen targets ranged from 96.13% to 102.46%, with RSD values<6.74%, showing good recoveries. The sample of CSS decoction has good stability in 24 h, with RSD values from 0.78% to 2.98%. The RSD of the sixteen components in the reproducibility test fluctuates between 1.94% and 5.36%, indicating good reproducibility. (2) Quantitative determination of the sixteen marker constituents in CSS decoction was made by UPLC-PDA system.Part 2According to the mass spectrometry analysis of main chromatogram peaks, FA and MH were successfully detected in the plasmas of patients with functional dyspepsia after oral administration of CSS.Part 3Compared with the control group, CSS (20g/kg), FA (100 mg/kg) and MH (14,28 mg/kg) significantly increase the rate of GE and IT in rats (P<0.05) FA (100μmol/L) and MH (10μmol/L) increased the mean amplitude of contraction in the longitudinal and circular strip compared with untreated controls (P<0.05). in addition, MH (30,100μmol/L) increased the mean amplitude of contraction in the longitudinal and circular strip compared with untreated controls (P<0.01)Part 4After oral administration of CSS, FA and MH were separated completely in the plasma of patients with functional dyspepsia and no other interfering peaks were found in the spectrum of the chromatograms. The retention times of FA and MH were 6.5 and 15.7 min, and that of sulfamethoxazole was 8,0 min. The recovery rates of FA and MH were from 95.35% to 102.56% with RSD< 2.81% and from 95.58% to102.09% with RSD<3.86%, respectively. The calibration curve for FA was linear over the concentration range from 10 to 320 ng/ml. The low detection limit of FA based on a signal-to-noise ratio of 3 was 1.5 ng/mL and the low concentration limit was 4.5 ng/ml. The calibration curve for MH was linear over the concentration range from 7.5 to 720 ng/mL. The low detection limit was 2.5 ng/mL and the low concentration limit was 7.5 ng/mL. The pharmacokinetics of FA and MH in patient plasma was fitted with a two-compartment model. Time to reach peak concentration of FA (0.199 mg/L) and MH (0.371 mg/L) was 27.50 min and 23.57 min, respectively. The elimination half-life and area under the concentration-time curve from t= 0 to the last time of FA and MH were 131.27 min and 14.835μg·min/mL and 139.53 min and 31.445μg·min/mL, respectively.Conclusion:1. A rapid, sensitive, and reliable UPLC-PDA method for the simultaneous determination of sixteen compounds in CSS was developed. The method was validated for specificity, accuracy, precision, and limits of detection and was successfully applied to determine the contents of sixteen components in CSS.2. LC-MS is a convenient, rapid and effective method to separate and quality FA and MH in plasmas of patients with functional dyspepsia after oral administration of CSS. 3. CSS and its absorbed compound FA and MH have the significant effect on promoting gastrointestinal motility in rats. FA and MH are the effective substance of CSS to treat functional dyspepsia.4. FA and MH in the plasmas of patients with functional dyspepsia were simultaneously determined by the UPLC method after oral administration of CSS. The pharmacokinetics of prokinetic CSS and its compounds are useful for monitoring CSS formulas in clinical practice and for understanding therapeutic mechanisms.更多还原