【作者】 詹勤；

【导师】 陈万生； 孙连娜；

【作者基本信息】 第二军医大学 ， 生药学， 2012， 博士

【摘要】 方剂是中药应用的最常用形式，方剂通过配伍提高了临床疗效，几千年来对人类健康发挥了积极作用。方剂配伍具有系统的中医药理论体系，严格依据君臣佐使的主要规则。引经药是能引方中诸药以达病所的使药。合理利用引经药，能提高用药的准确性，增加病所的有效药量，从而改善疗效。研究药物的“引经”作用对揭示方剂配伍机制、创新药物研制、指导临床合理用药都有重要意义。桔梗是中医药领域常用的引经药，为诸药舟楫，能载药上浮。升陷汤为临床常用的方剂，主治“大气下陷”。升陷汤由黄芪、知母、桔梗、升麻、柴胡五味中药组成，桔梗在方中作为使药，用之为向导。本课题研究目的是通过现代药物研究的方法，探讨桔梗在升陷汤中配伍作用，以期验证桔梗“引经报使”理论的科学性，从而更好的理解古人桔梗“为诸药舟楫”之说。主要研究内容包括四个方面：一、基于代谢组学技术确证桔梗在升陷汤中的配伍作用以左冠状动脉结扎诱导大鼠心肌缺血致慢性心力衰竭为模型，运用代谢组学方法结合超声心动图检查及生化指标测定，评价升陷汤全方组、升陷汤缺桔梗组及桔梗单独给药组对慢性心力衰竭大鼠的治疗作用，进而推断桔梗在升陷汤中的配伍作用。对基于超高效液相色谱/四级杆-飞行时间质谱（UPLC-Q-TOF/MS）的代谢组学技术的方法参数进行优化，开发适用于尿液代谢组和血清代谢组研究的高通量分析方法。通过建立的UPLC-Q-TOF/MS方法获得了大鼠血清样本及尿液样本的代谢指纹谱，采用主成分分析（PCA）和正交偏最小二乘判别分析法（PLS-DA）分析了各给药组之间的代谢物谱差异及升陷汤全方组不同治疗时间代谢物谱的变化趋势。结果显示，在给药结束时（第37天），PLS-DA得分图上，不同处理组能清楚的分开，升陷汤组体内的代谢物变化水平与假手术组较接近，而升陷汤缺桔梗组与假手术组相距较远；升陷汤全方组，随着治疗时间的延长，慢性心力衰竭大鼠体内代谢物水平有向假手术组回归的趋势。在给药第35天时对不同处理组的大鼠进行心脏超声检查，计算左室射血分数（EF）和左室短轴缩短率（FS）以检验左心室收缩功能。结果显示，升陷汤全方组、升陷汤缺桔梗组EF值均高于模型组（P<0.05），而桔梗单独给药组与模型组比较则无明显差异；升陷汤全方组FS值高于模型组（P<0.01），升陷汤缺桔梗组及桔梗组与模型组比较，则没有显著差异（P>0.05）。采用速率法对不同处理组的大鼠血清中乳酸脱氢酶（LDH）和肌酸激酶（CK）进行测定。结果显示，模型组血清CK和LDH均高于假手术组（P<0.01）。升陷汤全方及阳性药倍他乐克用药后，均能降低血清CK和LDH含量，与模型组比较，均有统计学意义（P<0.05）；而桔梗组、升陷汤缺桔梗组的LDH和CK值与模型组比较，均无明显差异。通过代谢组学研究结合药理指标测定，结果说明：升陷汤对大鼠慢性心力衰竭具有一定的治疗作用，而方中缺少桔梗后治疗效果不如全方，从而证实了桔梗在升陷汤中具有“引经”作用。通过代谢组学方法，鉴定出潜在的慢性心力衰竭相关的主要生物标志物，并推断出升陷汤治疗慢性心力衰竭机制可能与体内的脂质代谢过程相关。二、基于UPLC-Q-TOF/MS技术，在快速表征升陷汤化学组分的基础上，探讨桔梗的配伍对升陷汤中化学组分的影响利用UPLC-Q-TOF/MS对升陷汤化学成分进行了快速分析和鉴定，从升陷汤中鉴定了36个化合物，并归属了各化合物的单味药来源。其化学成分按照保留时间顺序依次为： neomangiferin、 caffeic acid、 mangiferin、 isomangiferin、 rutin、calycosin-7-O-β-D-glucoside、fukinolic acid、ferulic acid、isoferulic acid、timosaponin E1、timosaponin O（或timosaponin P）、timosaponin N、timosaponin B-II、ononin、2-feruloyl-piscidic acid （或2-isoferuloyl-piscidic acid）、 platycodin D、3"-O-acetylplatycodin D、2,6,4′-trihydroxy-4-methoxybenzophenone、2"-O-acetylplatycodin D、 calycosin、 timosaponin B、 astragaloside IV、anemarrhenasaponin I（或anemarrhenasaponin II）、formononetin、saikosaponin C、saikosaponin A、10-hydroxy-3,9-dimethoxy-pterocarpan （或3-hydroxy-9,10-dimethoxy-pterocarpan）、timosaponin G（或anemarrhenasaponin III）、24-O-acetylhydroshengmanol-3-O-xyloside、1α-hydroxycimigenol-3-O-β-D-galactopyranoside、astragaloside I（或isoastragaloside I）、1α-hydroxycimigenol-3-O-β-D-galactopyranoside、7,8-didehydrocimigenol-3-O-α-L-arabinopyranoside、 timosaponin A-III、24-O-acetylhydroshengmanol-3-O-xyloside、7,8-didehydrocimigenol。结果显示,升陷汤的主要成分包括黄芪皂苷、黄芪异黄酮、知母皂苷、双苯吡酮、柴胡皂苷、桔梗皂苷、黄酮类及咖啡酸衍生物等，该研究比较全面地阐明了升陷汤的化学组成。为了揭示桔梗配伍前后升陷汤中其他中药的化学成分表征区别，在升陷汤化学组分辨认和识别的基础上，比较升陷汤全方（扣除桔梗的离子信号）与缺桔梗方的UPLC-Q-TOF/MS化学指纹谱差异以及主要化学成分提取离子峰的面积差异。结果显示，桔梗配伍前后升陷汤中其它中药化学成分表征差异不显著，提示桔梗可能不是通过改变复方的化学组成而起到“引经”作用。三、建立升陷汤中主要活性成分的含量测定方法，并从药代动力学角度探讨桔梗在升陷汤中发挥引经作用的机制建立灵敏、可靠的HPLC-MS/MS分析方法，用以测定升陷汤中主要活性成分芒柄花素、毛蕊异黄酮苷、芒柄花苷、咖啡酸、异阿魏酸、芒果苷、知母皂苷E1、知母皂苷B-II和知母皂苷B的血浆药物浓度。本方法选择简便、快捷的蛋白沉淀法处理血浆样品，选择人参皂苷Re和葛根素作为内标，然后将处理后的样品分为两个部分，建立两个色谱条件，分两次测定。质谱条件均采用负离子模式下的多反应监测。此方法成功应用于升陷汤主要化学成分体内药代动力学研究，得到这些化合物在大鼠体内的药动学参数。运用所建立的方法对升陷汤全方及升陷汤缺桔梗方分别灌胃给药后，主要活性成分的药代动力学参数进行比较研究。结果表明，当升陷汤中缺少桔梗后，其主要活性成分知母皂苷E1、知母皂苷B-II、知母皂苷B和芒果苷的体内过程发生了明显改变。具体表现在，桔梗缺失后，吸收入血的活性成分未能及时向组织分布和消除，缺桔梗方组与全方组药-时曲线形状具有较大差别。充分体现了桔梗在升陷汤中的配伍意义，从药代动力学角度，说明了桔梗具有“引经”作用。四、桔梗的化学成分研究利用硅胶柱色谱、葡聚糖凝胶柱色谱、ODS-C18反相硅胶柱色谱、MCI凝胶柱色谱、半制备高效液相色谱等现代色谱分离技术，对桔梗的化学成分进行了系统分离和研究，从桔梗水提物中共分离得到46个单体化合物。运用UV、IR、ESI-MS、1D-NMR、2D-NMR等光谱和波谱分析技术及理化方法对这46个化合物进行了结构鉴定，结果如下：Rpg-1：1-pentacosanol、Rpg-2：α-spinasterol、Rpg-3：7-stigmasterol、Rpg-4：α-spinasteryl-3-O-β-D-glucoside、Rpg-5：ergosterol peroxide、Rpg-6：platycodonoidA、Rpg-7：β-amyrin、Rpg-8：Δ7-stigmastenol-3-O-β-D-glucoside、Rpg-9：platycodoniodB、Rpg-10：3-O-β-D-glucopyranosyl-polygalacic acid、Rpg-11：3-O-β-D-glucopyranosyl-platycodigenin、Rpg-12：3-O-β-D-laminaribiosyl-polygalacicacid、Rpg-13：hexyl-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside、Rpg-14：platycoside K、Rpg-15：platycoside L、Rpg-16：2"-O-acetylpolygalacin D、Rpg-17：platycodin A (2"-O-acetylplatycodin D)、Rpg-18：2"-O-acetylplatycodin D2、Rpg-19：3"-O-acetylpolygalacin D、Rpg-20：platycoside B、Rpg-21：polygalacin D、Rpg-22：platycoside C、Rpg-23：platycodin C (3"-O-acetylplatycodin D)、Rpg-24：polygalacin D2、Rpg-25：3"-O-acetylplatycodin D2、Rpg-26：hexadecanoic acid Rpg-27：platycoside H(deapio-polygalacin D3)、Rpg-28：platycodin D、Rpg-29：platycodin D2、Rpg-30：16-oxo-platycodin D、Rpg-31：platyconic acid A、Rpg-32：platycoside G3、Rpg-33：deapio platycodin D、Rpg-34：3-O-β-D-glucopyranosyl platycodigenin methyl ester、Rpg-35：platycoside P、Rpg-36：deapio-platyconic acid A lactone、Rpg-37：platycosideN、Rpg-38：2"-O-acetylplatycodin D3、Rpg-39：platycodin D3、Rpg-40：3"-O-acetylplatycodin D3、Rpg-41：deapio-platycodin D3、Rpg-42：platycoside D、Rpg-43：1Glc6-1Glc6-1Glc-platycodigenin、Rpg-44：hexitol、Rpg-45：platycoside E、Rpg-46：deapio-platycoside E。其中，三萜及其皂苷类成分35个，其它成分11个。Rpg-6、Rpg-9和Rpg-35为新化合物。Rpg-1、Rpg-5、Rpg-13和Rpg-44为首次从桔梗中分得。本研究丰富了桔梗的化学成分信息，更重要的是获得了桔梗皂苷类成分的标准品，为桔梗的全面质量控制研究及“引经”作用物质基础研究提供了必要的前提。更多还原

【Abstract】 A classical Chinese Formula is a combination of compatible Chinese herbs in fixeddosages according to classical or well-known texts of Traditional Chinese Medicine(TCM), which including one of TCM pharmacological theories---principal (Jun),adjuvant (Chen), assistant (Zuo) and guide (Shi). It has existed for over two thousandyears, and plays an important role in the medical system used in health care and treatmentof diseases. A well-designed formula tailored to the pathophysiologic state of the patient.In a formula, the “guiding drug” which could change other drug’s direction or/and location,and then made them concentrated in specially appointed direction or/and location. It ismeaningful to study the “guiding action” of the drug, which has contributed tounderstanding the TCM formula compatibility, new drug exploitation, and rational use ofdrugs in clinical practice.It is a TCM theory that Platycodi Radix acts as a “guiding drug” which assists indelivering the main herb to the organ or meridian. Platycodi Radix plays an important andspecific role in Shengxian Decoction, a famous TCM formula, which was composed ofAstragali Radix, Asphodeloides Anemarrhena, Platycodi Radix, Cimicifugae Rhizoma, andBupleuri Radix. This study established systematic method for identification of mainconstituents and determination of main active constituents in Shengxian Decoction in ratserum, and provided a scientific explanation of “guiding action” for Platycodi Radix. Thecontent includes the following three main parts:The first, myocardial ischemisa was created in rats by ligating the left anteriordescending brach of the coronary artery (LAD), resulting in chronic cardiac failure in thismodel; a metabonomics-based approach to study the anti-chronic cardiac failure activitiesbetween Shengxian Decoction and Shengxian Decoction that removed Platycodi Radix:In a rat model of chronic cardiac failure, metabonomics-based approaches were used to study the function of Shengxian Decoction (SXT), decreased Shengxian Decoction(Platycodi Radix removed, SXT-PG) and Platycodi Radix extraction. In this study, aUPLC-Q-TOF/MS-based metabolomics platform was developed for the qualitativeprofiling of urinary and serum metabonomics in rats, which were randomly assigned infive groups: Sham Surgery group, Model group, positive control group (Betaloc), PGgroup, SXT-PG group, and SXT group. Our method was successful in discriminating thedifferentially processed rats. Both the unsupervised principal component analysis (PCA)and the supervised partial least square-data analysis (PLS-DA) demonstrated strongclassification and clear trajectory patterns with regard to different groups. The PLS-DAresults (37days after oral administration) showed that most of the raw and differentiallyprocessed samples were clearly clustered in the score plot, and SXT group and ShamSurgery group had similar scores clustering together, while SXT-PG group didn’t act.Besides, this time-dependent metabolic profiling of SXT group achieved its desiredactivity and clustered near Sham Surgery group in the PLS-DA model.Echocardiography results (35days after oral administration) showed that ejectionfraction (EF) value and shortening fraction (FS) value of ventriculus sinister in SXT-PGgroup and SXT group were larger than those in Model group, which showed no significantdifference from those in PG group. In addition, EF value in SXT group were larger thanthose in Model group (P<0.01), while EF value in SXT-PG group showed no significantdifference from those in Model group and PG group (P>0.05).Serum activities of LDH and CK were determined according to the instructions ofbiochemical reagent. The velocity study results showed that LDH and CK contents inModel group were larger than those in Sham Surgery group (P<0.01), while LDH and CKcontents in SXT group and Betaloc group were smaller than those in Sham Surgery group(P<0.05). Besides, LDH and CK contents in PG group showed no significant differencefrom those in SXT-PG group.The above study verified that Shengxian Decoction (SXT group) exhibited potentanti-chronic cardiac failure activity, but the Shengxian Decoction that removed Platycodi Radix (SXT-PG group) not, which revealed the “guiding action” of the Platycodi Radix. Potential biomarkers related with chronic cardiac failure were identified, and theirmetabolic pathways were also discussed.The second, an UPLC-Q-TOF/MS method was established and validated to analyzethe chemical profiles of Shengxian Decoction and Shengxian Decoction that removedPlatycodi Radix:A rapid UPLC-Q-TOF/MS based chemical profiling method was established andvalidated to qualitative evaluation of Shengxian Decoction. Based on m/z, mass spectrumcomparison and retention time data,36compounds were unambiguously identified asneomangiferin, caffeic acid, mangiferin, isomangiferin, rutin, calycosin-7-O-β-D-glucoside,fukinolic acid, ferulic acid, isoferulic acid, timosaponin E1, timosaponin O (or timosaponinP), timosaponin N, timosaponin B-II, ononin,2-feruloyl-piscidic acid (or2-isoferuloyl-piscidic acid), platycodin D,3"-O-acetylplatycodin D、2,6,4′-trihydroxy-4-methoxybenzophenone,2"-O-acetylplatycodin D, calycosin,timosaponin B, astragaloside IV, anemarrhenasaponin I (or anemarrhenasaponin II),formononetin, saikosaponin C, saikosaponin A,10-hydroxy-3,9-dimethoxy-pterocarpan (or3-hydroxy-9,10-dimethoxy-pterocarpan), timosaponin G (or anemarrhenasaponin III),24-O-acetylhydroshengmanol-3-O-xyloside,1α-hydroxycimigenol-3-O-β-D-galactopyranoside, astragaloside I (or isoastragaloside I),1α-hydroxycimigenol-3-O-β-D-galactopyranoside,7,8-didehydrocimigenol-3-O-α-L-arabinopyranoside, timosaponin A-III,24-O-acetylhydroshengmanol-3-O-xyloside,7,8-didehydrocimigenol. This study indicatedthat astragalosides, isoflavonoids that from Radix Astragali, timosaponins, xanthones thatfrom Anemarrhena Asphodeloides, saikosaponins, platycodins, flavonoids, caffeic acidderivatives were the main compositions of Shengxian Decoction, which was a powerfultool to reveal the chemical profile of Shengxian Decoction. Besides, comparison of thedefference of chemical fingerprints between Shengxian Decoction and ShengxianDecoction that removed Platycodi Radix truly reflected the influence in the presentation ofthe chemical components of other four TCM in Shengxian Decoction by Platycodi Radix. The third, establishement of a sensitive and validated method for determination ofnine active compounds, and a comparetive study of the pharmacokinetics in rat plasmabetween SXT and SXT-PG groups:A sensitive and reliable high performance liquid chromatography-electrosprayionization-tandem mass spectrometry (HPLC-MS/MS) has been developed and validatedfor determination of9main active compounds (formononetin,calycosin-7-O-β-D-glucoside, ononin, caffeic acid, isoferulic acid, mangiferin,timosaponin E1, timosaponin B-II and timosaponin B) of Shengxian Decotion. Plasmasamples were pretreated by protein precipitation with acetonitrile containing formic acid(0.1%, v/v). Ginsenoside Re and puerarin were used as the internal standards. Then treatedsamples were devided into two parts. Two LC separation methods were established foranalyzing each part, respectly. The detection was carried out by a triple-quadrupole tandemmass spectrometer in negative ionization mode. This method was successfully applied topharmacokinetic study of the main active compounds in rats.A comparative pharmacokinetic study was carried out for the main active ingredientsusing the established method between SXT and SXT-PG. The results showed that theprocesses in vivo of mangiferin, timosaponin E1, timosaponin B-II and timosaponin Bwere changed significantly when removed PG from SXT. The shape of concentration-timecurves has a greater difference between the two groups. This may be due to thecompositions fail to distributing to the tissues timely when missing PG in SXT. Theoutcome reflected the corrigent function of Platycodi Radix in SXT at some extent.The last, the chemical investigation of Platycodi Radix:A total of46compounds including3new ones were isolated from the aqueousexaction of Platycodi Radix through various separation techniques and structurallyidentified on the basis of diverse spectroscopic methods including UV, IR, ESI-MS,1Dand2D NMR spectra.35triterpenoids and triterpenoid glycosides as well as11compoundsin other types were obtained. All compounds were listed as follows:1-pentacosanol(Rpg-1), α-spinasterol (Rpg-2),7-stigmasterol (Rpg-3), α-spinasteryl-3-O-β-D-glucoside (Rpg-4), ergosterol peroxide (Rpg-5), platycodonoid A (Rpg-6), β-amyrin (Rpg-7),Δ7-stigmastenol-3-O-β-D-glucoside (Rpg-8), platycodoniod B (Rpg-9),3-O-β-D-glucopyranosyl-polygalacic acid (Rpg-10),3-O-β-D-glucopyranosyl-platycodigenin (Rpg-11),3-O-β-D-laminaribiosyl-polygalacic acid (Rpg-12),hexyl-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (Rpg-13), platycoside K (Rpg-14),platycoside L (Rpg-15),2"-O-acetylpolygalacin D (Rpg-16), platycodinA(2"-O-acetylplatycodin D, Rpg-17),2"-O-acetylplatycodin D2(Rpg-18),3"-O-acetylpolygalacin D (Rpg-19), platycoside B (Rpg-20), polygalacin D (Rpg-21),platycoside C (Rpg-22), platycodin C(3"-O-acetylplatycodin D, Rpg-23), polygalacin D2(Rpg-24),3"-O-acetylplatycodin D2(Rpg-25), hexadecanoic acid (Rpg-26), platycosideH(deapio-polygalacin D3, Rpg-27), platycodin D (Rpg-28), platycodin D2(Rpg-29),16-oxo-platycodin D (Rpg-30), platyconic acid A (Rpg-31), platycoside G3(Rpg-32),deapio platycodin D (Rpg-33),3-O-β-D-glucopyranosyl platycodigenin methyl ester(Rpg-34), platycoside P (Rpg-35), deapio-platyconic acid A lactone (Rpg-36), platycosideN (Rpg-37),2"-O-acetylplatycodin D3(Rpg-38), platycodin D3(Rpg-39),3"-O-acetylplatycodin D3(Rpg-40), deapio-platycodin D3(Rpg-41), platycoside D(Rpg-42),6-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl-platycodigenin (Rpg-43), hexitol (Rpg-44), platycoside E (Rpg-45),deapio-platycoside E (Rpg-46). Among these constituents, Rpg-6, Rpg-9, and Rpg-35were identified as new compounds, and Rpg-1, Rpg-5, Rpg-13, and Rpg-44were reportedfrom Platycodi Radix for the first time. The platycodins yielded in this research detailedthe chemical profile of Platycodi Radix, provided standard substance for quality control ofthis herb, and made contribution for the scientific explanation of “guiding action” for thisherb.更多还原