桔梗在升陷汤中引经作用及其化学成分研究
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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。
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.
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。
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.
引文
[1]王阶,王永炎,杨戈.中药方剂配伍理论研究方法和模式[J].中国中药杂志,2005,30(1):6-11.
[2]金在久.桔梗的化学成分及药理和临床研究进展[J].时珍国医国药,2007:18(2):506-508.
[3]李然,刘立萍,李海波.舟楫之剂论桔梗[J].中医药信息,2011,28(6):6-7.
[4]石含秀,贾波.桔梗“载药上行”的含义探析[J].内蒙古中医药,2008,(8):42-43.
[5]刘萍,古今,冯建涌.天王补心丸全方及方中缺桔梗对小鼠镇静催眠作用的影响[J].中国药物应用与监测杂志,2004,(2):51-53.
[6]李外,周忻,刘萍.天王补心丸中桔梗载药上行作用的初步研究[J].中国中医药信息杂志,2007,14(9):26-27.
[7]李海静,高月,刘萍.天王补心丸全方及全方缺桔梗对少寐大鼠脑神经递质的影响[J].中国中药杂志,2009,4(2):217-223.
[8]刘萍,邹鹏,李外等.指纹图谱技术考察桔梗在天王补心丸中的配伍意义[J].中国中药杂志,200,4(2):156-160.
[9]李英伦,卢胜明,简沫.桔梗“引经”对罗红霉素肺药浓度的影响[J].中兽医医药杂志,2005,2(3):3-6.
[10]李英伦,蒋智钢,何晓俐.桔梗对左氧氟沙星在健康鸡体内药动力学影响的研究[J].畜牧兽医学报,2006,37(2):183-186.
[11]李英伦,蒋智纲,何晓俐.桔梗的“引经”作用对左旋氧氟沙星在鸡体内药物分布的影响[J].中国兽医学报,2006,26(5):541-543.
[12]刘萍,魏玲.桔梗对卡马西平家兔体内血药浓度的影响[J].中国医院用药评价与分析,2008,8(5):366-369.
[13]张锡纯.医学衷中参西录[M].上册.河北：河北人民出版社.1974,31.
[14]余云昶,史载祥,姜良铎.升解通瘀汤对大鼠心脏缺血再灌注损伤的心肌保护作用[J].陕西中医,2008,29(6):747-749.
[15]康红钰,张福华,刘喜民等.升陷汤对大鼠急性心肌缺血作用机制的探讨[J].中国医院药学杂志,2007,2(5):617-619.
[16]张智伟,黄晶,赵瑞等.升陷汤治疗心肺疾病临床研究进展[J].光明中医,2007,22(8):60-62.
[17] Plumb RS, Stumpf CL, Granger JH, et al. Use of liquid chromatography/time-of-flight-massspectrometry and multivariate statistical analysis shows promise for the detection of drugmetabolites in biological fluids [J].Rapid Commun Mass Spectrum,2003,17(26):2632-2638.
[18] DunnW B, ElliD I. Metabolomics: Current analytical platforms and method[J]. Trends Anal Chem,2005,24(4):285-294.
[19] Theodoridis, G., H. G. Gika, et al.. LC-MS-based methodology for global metabolite profiling inmetabonomics/metabolomics. TrAC Trends in Analytical Chemistry,2008,27(3):251-260.
[20]王喜军,刘莲,孙晖,孙文军,吕海涛.乙醇诱导大鼠肝损伤的代谢组学和茵陈蒿汤的干预研究.中国药理学通报,200,24(04):452-457
[21]王喜军,孙文军,孙晖,吕海涛,邹迪新,吴泽明,王萍,刘莲,吴修红. CCL4诱导大鼠肝损伤模型的代谢组学及茵陈蒿汤的干预作用研究.世界科学技术-中医药现代化,2006,8(06):101-106.
[22] Liang, X., X. Chen, et al. Metabonomic study of Chinese medicine Shuanglong Formula as aneffective treatment for myocardial infarction in rats. Journal of Proteome Research,2011
[23]阿基业.代谢组学数据处理方法——主成分分析.中国临床药理学与治疗学,2010,15(5):481-489.
[24]卢红梅,梁逸曾.代谢组学分析技术及数据处理技术.分析测试学报,2008,27(3):325-332.
[25]王喜军.基于药物代谢组学的中药及方剂中组分间协同增效作用.中国天然药物,2009,7(2):90-94.
[26]王喜军,张伯礼.基于药物代谢组学的方剂配伍规律及配伍科学价值揭示.中国中药杂志,2010,35(10):1346-1348.
[27]李然,刘立萍,李海波.舟楫之剂论桔梗.中医药信息,2011,28(6):6-7.
[28]刘萍,古今,冯建涌.天王补心丸全方及方中缺桔梗对小鼠镇静催眠作用的影响[J].中国药物应用与监测,2004,(02).
[29]李海静,高月,刘萍.天王补心丸全方及全方缺桔梗对少寐大鼠脑神经递质的影响[J].中国中药杂志,2009,(02).
[30]刘萍,邹鹏,李外,孙艳,张洁,余河水,赵阳,马百平.指纹图谱技术考察桔梗在天王补心丸中的配伍意义[J].中国中药杂志,2009,(02).
[31]王长春.议张锡纯之“大气下陷证”.中医研究,2006,19(6):8-9.
[32]马术明.慢性充血性心力衰竭的辨证论治[J].吉林中医药,2004,24(2):6-8.
[33]刘静,贾庭英.益气强心汤治疗充血性心力衰竭临床观察[J].吉林中医药,2005,25(8):14.
[34]吴素琴.生脉、黄芪注射液联用治疗充血性心力衰竭疗效分析[J].吉林中医药,2004,24(7):6-7.
[35]张持.中医对慢性充血性心力衰竭的认识及治疗进展[J].吉林中医药,2006,26(11):71-73.
[36]赵惠,李七一.充血性心力衰竭的中医研究概况[J].吉林中医药,2004,24(9):56-58.
[37]李昌繁,江时森.心力衰竭动物模型的研究进展.医学研究生学报,2007,20(5):532-534.
[38]戴闺柱. β受体阻滞剂治疗慢性心力衰竭-从禁忌证到常规治疗.中华心血管病杂志,2002,30(6):381-383.
[39]周永昌,郭万学.超声医学[M].北京:科学技术文献出版社,2004:680.
[40]陈翔,邓乐勇.强心力胶囊治疗充血性心力衰竭彩色多普勒超声心动图检测指标分析[J].湖南中医学院学报,2006,26(1):44.
[41] WAYBRIGHT TJ,VAN QN,MUSCHIK GM, et al. LC-MS in metabonomics: Optimization ofexperimental conditions for the analysis of metabolites in human urine[J]. J Liq Chromatogr RelatTechnol,2006,29(17-20):2475-2497.
[42] ZHAO Xinjie, ZHANG Yi, MENG Xianli, et al. Effect of a traditional Chinese medicinepreparation Xindi soft capsule on rat model of acute blood stasis: a urinary metabonomics studybased on liquid chromatography mass spectrometry[J]. J Chromatogr B,2008,873(2):151-158.
[43] GIRI S, KRAUSZ K W, IDLE J R, et al. The metabolomics of (+/-) arecoline oxide in the mouseand its formation by human flavin containing monooxygenases[J]. Biochem Pharmacol,2007,73(4):561-573.
[44] WONG MCY,LEEA WTK,WONG JSY, et al. An approach towards method development foruntargeted urinary metabolite profiling in metabonomic research using UPLC/Q-TOF-MS[J]. JChromatogr B,2008,871(2):341-348.
[45] XIE Baogang, GONG Tao, GAO Rong, et al. Development of rat urinary HPLC-UV profiling formetabonomic study on Liuwei Dihuang Pills[J]. J Pharm Biomed Anal,2009,49(2):492-497.
[46] LAURIDSEN M,HANSEN S H,JAROSZEWSKI J W, et al. Human urine as test material in1H-NMR based metabonomics: recommendations for sample preparation and storage[J]. AnalChem,2007,79(3):1181-1186.
[47] BRUCE SJ,TAVAZZI I,PARISOD V, et al. Investigation of human blood plasma samplepreparation for performing metabolomics using ultrahigh performance liquid chromatography/massspectrometry[J]. Anal Chem,2009,81(9):3285-3296.
[48] MICHOPOULOS F,LAI L,GIKA H, et al. UPLC-MS based analysis of human plasma formetabonomics using solvent precipitation or solid phase extraction[J]. J Proteome Res,2009,8(4):2114-2121.
[49]王咏梅,殷仁富,吴宗贵，梅长林，顾书华.左旋肉碱洽疗急性病毒性心肌炎的临床疗效分析.中华心血管病杂志,1999,20(6):405-407.
[50] Listenberger L L, Schaffer JE. Mechanisms of Lipoapoptosis*1: Implications for Human HeartDisease. Trends in cardiovascular medicine,2002,12(3):134-138.
[51]杨建峰,魏经汉.慢性心力衰竭患者血清IL-1β, TNF-α水平及临床意义.实用全科医学,2007,5(11):954-955.
[52]孙蕾,王会信,周廷冲.鞘磷脂与细胞生长、凋亡调控.军事医学科学院院刊,1998,22(3):233-235.
[53]齐炼文,周建良,郝海平,李会军,闻晓东,陈君,杨中林,李萍,王广基.基于中医药特点的中药体内外药效物质组生物／化学集成表征新方法.中国药科大学学报,2010,41(3):195-200.
[54]潘瑞乐,陈迪华,沈连刚,斯建勇.高效液相色谱法测定中药升麻中阿魏酸和异阿魏酸的含量.药物分析杂志,2000,20(6):396-398
[55] Li, W., Y. Sun, et al. Identification of caffeic acid derivatives in Actea racemosa (Cimicifugaracemosa, black cohosh) by liquid chromatography/tandem mass spectrometry. Rapidcommunications in mass spectrometry,2003,17(9):978-982.
[56] Ma, C., L. Wang, et al. Identification of major xanthones and steroidal saponins in rat urine byliquid chromatography–atmospheric pressure chemical ionization mass spectrometry technologyfollowing oral administration of Rhizoma Anemarrhenae decoction. BiomedicalChromatography,2008,22(10):1066-1083.
[57] Zhiyun, M., X. Suixu, et al. New saponins from Anemarrhena asphodeloides Bge. Chinese Journalof Medicinal Chemistry,1999,9(4):294-298.
[58] Zhang, Y. Y., Q. Wang, et al. Characterization and determination of the major constituents inBelamcandae Rhizoma by HPLC-DAD-ESI-MSn. Journal of Pharmaceutical and BiomedicalAnalysis,2011,56(2011)304-314.
[59]中国科学中国植物志编辑委员会.中国植物志.北京：科学出版社,73(2):77.
[60]国家药典委员会，2010版中国药典一部，北京：化学工业出版社，2010：259.
[61]付文卫,窦德强,等.桔梗的化学成分和生物活性研究进展.沈阳药科大学学报,2006,23(3):184-191.
[62] Gao ZZ, Xia YF, Yao XJ, Dai Y, Wang Q. A new triterpenoid saponin from Gleditisia sinensis andstructure-activity relationships of inhibitory effects on lipopolysaccharide-induced nitric oxideproduction. Nat Prod Res.2008,22(4)320-332.
[63] Yoshikawa M, Murakami T, Yashiro K, et al．Kotalanol, a potent glycosidase inhibitor withthiosugar sulfonium sulfate structure, from antidiabetic Ayurvedic medicine salaciareticulata[J]．Chemical&Pharmaceutical Bulletin,1998,46(8):1339-1340．
[64]赵晓亚,孙汉董,等.冷水七根茎的化学成分研究[J].中国中药杂志,2005,30(008):584-586.
[65]王文永,赵仕丞,刘东新.山海螺化学成分研究[J].中药材,2011,34(4)553-555.
[66]姜勇,刘蕾,屠鹏飞.远志的化学成分研究Ⅲ.中国天然药物,2003,1(3):142-145
[67]解翠华,阮丽君,夏焕章,陈代杰,葛梅.植物内生菌HCCB00167产物的发酵、分离和结构鉴定[J].沈阳药科大学学报,2007,24(4):245-248.
[68]龚小见,周欣,赵超,等.马兰中的三萜类成分[J].中国中药杂志,2010,35(3):327-330．
[69]任启生,余雄英,等.山海螺化学成分研究.中草药,2005,36(12):1773-1775.
[70]任凤霞,张爱军,赵毅民.鹿蹄草化学成分研究Ⅱ[J].解放军药学学报,2008,24(4):301-304．
[71]付文卫,侯文彬,窦德强,华会明,桂毛华,付锐,陈英杰,裴月湖.桔梗中远志酸型皂苷的化学研究,药学学报,2006,41(4):358-360
[72]李凌军,刘振华,等.桔梗的化学成分研究.中国中药杂志，2006，31(18):1506-1509
[73] Fu, W. W., N. Shimizu, et al. Five new triterpenoid saponins from the roots of Platycodongrandiflorum. Chemical and pharmaceutical bulletin，2006，54(4):557-560.
[74] NIKAIDO, T., K. KOIKE, et al. Triterpenoid saponins from root of Platycodon grandiflorum.生薬學雜誌，1998，52(1):54-59.
[75] Li, W., L. Xiang, et al. A new triterpenoid saponin from the roots of Platycodon grandiflorum.Chinese Chemical Letters,2007,18(3):306-308.
[76] Choi, Y. H., D. S. Yoo, et al. Platyconic acid A, a genuine triterpenoid saponin from the roots ofPlatycodon grandiflorum. Molecules,2008,13(11):2871-2879.
[77] He, Z., C. Qiao, et al. New triterpenoid saponins from the roots of Platycodon grandiflorum.Tetrahedron,2005,61(8):2211-2215.
[78] Fu, W. W., D. Q. Dou, et al. Studies on the chemical constituents from the roots of Platycodongrandiflorum. Journal of natural medicines,2006,60(1):68-72.
[79] Ishii, H., K. Tori, et al. Saponins from roots of Platycodon grandiflorum. Part1. Structure ofprosapogenins. J. Chem. Soc., Perkin Trans.,1981,1(0):1928-1933.
[80] Li, W., W. Zhang, et al. Platycoside N: A New Oleanane-Type Triterpenoid Saponin from the Rootsof Platycodon grandiflorum. Molecules,2010,15(12):8702-8708.
[81] Nikaido, T., K. Koike, et al. Two new triterpenoid saponins from Platycodon grandiflorum.Chemical and pharmaceutical bulletin,1999,47(6):903-904.
[82] Fu, W. W., D. Q. Dou, et al. Triterpenoid saponins from Platycodon grandiflurum. Journal of Asiannatural products research,2007,9(1):35-40.
[2]金在久.桔梗的化学成分及药理和临床研究进展[J].时珍国医国药,2007:18(2):506-508.
[3]李然,刘立萍,李海波.舟楫之剂论桔梗[J].中医药信息,2011,28(6):6-7.
[4]石含秀,贾波.桔梗“载药上行”的含义探析[J].内蒙古中医药,2008,(8):42-43.
[5]刘萍,古今,冯建涌.天王补心丸全方及方中缺桔梗对小鼠镇静催眠作用的影响[J].中国药物应用与监测杂志,2004,(2):51-53.
[6]李外,周忻,刘萍.天王补心丸中桔梗载药上行作用的初步研究[J].中国中医药信息杂志,2007,14(9):26-27.
[7]李海静,高月,刘萍.天王补心丸全方及全方缺桔梗对少寐大鼠脑神经递质的影响[J].中国中药杂志,2009,4(2):217-223.
[8]刘萍,邹鹏,李外等.指纹图谱技术考察桔梗在天王补心丸中的配伍意义[J].中国中药杂志,200,4(2):156-160.
[9]李英伦,卢胜明,简沫.桔梗“引经”对罗红霉素肺药浓度的影响[J].中兽医医药杂志,2005,2(3):3-6.
[10]李英伦,蒋智钢,何晓俐.桔梗对左氧氟沙星在健康鸡体内药动力学影响的研究[J].畜牧兽医学报,2006,37(2):183-186.
[11]李英伦,蒋智纲,何晓俐.桔梗的“引经”作用对左旋氧氟沙星在鸡体内药物分布的影响[J].中国兽医学报,2006,26(5):541-543.
[12]刘萍,魏玲.桔梗对卡马西平家兔体内血药浓度的影响[J].中国医院用药评价与分析,2008,8(5):366-369.
[13]张锡纯.医学衷中参西录[M].上册.河北：河北人民出版社.1974,31.
[14]余云昶,史载祥,姜良铎.升解通瘀汤对大鼠心脏缺血再灌注损伤的心肌保护作用[J].陕西中医,2008,29(6):747-749.
[15]康红钰,张福华,刘喜民等.升陷汤对大鼠急性心肌缺血作用机制的探讨[J].中国医院药学杂志,2007,2(5):617-619.
[16]张智伟,黄晶,赵瑞等.升陷汤治疗心肺疾病临床研究进展[J].光明中医,2007,22(8):60-62.
[17] Plumb RS, Stumpf CL, Granger JH, et al. Use of liquid chromatography/time-of-flight-massspectrometry and multivariate statistical analysis shows promise for the detection of drugmetabolites in biological fluids [J].Rapid Commun Mass Spectrum,2003,17(26):2632-2638.
[18] DunnW B, ElliD I. Metabolomics: Current analytical platforms and method[J]. Trends Anal Chem,2005,24(4):285-294.
[19] Theodoridis, G., H. G. Gika, et al.. LC-MS-based methodology for global metabolite profiling inmetabonomics/metabolomics. TrAC Trends in Analytical Chemistry,2008,27(3):251-260.
[20]王喜军,刘莲,孙晖,孙文军,吕海涛.乙醇诱导大鼠肝损伤的代谢组学和茵陈蒿汤的干预研究.中国药理学通报,200,24(04):452-457
[21]王喜军,孙文军,孙晖,吕海涛,邹迪新,吴泽明,王萍,刘莲,吴修红. CCL4诱导大鼠肝损伤模型的代谢组学及茵陈蒿汤的干预作用研究.世界科学技术-中医药现代化,2006,8(06):101-106.
[22] Liang, X., X. Chen, et al. Metabonomic study of Chinese medicine Shuanglong Formula as aneffective treatment for myocardial infarction in rats. Journal of Proteome Research,2011
[23]阿基业.代谢组学数据处理方法——主成分分析.中国临床药理学与治疗学,2010,15(5):481-489.
[24]卢红梅,梁逸曾.代谢组学分析技术及数据处理技术.分析测试学报,2008,27(3):325-332.
[25]王喜军.基于药物代谢组学的中药及方剂中组分间协同增效作用.中国天然药物,2009,7(2):90-94.
[26]王喜军,张伯礼.基于药物代谢组学的方剂配伍规律及配伍科学价值揭示.中国中药杂志,2010,35(10):1346-1348.
[27]李然,刘立萍,李海波.舟楫之剂论桔梗.中医药信息,2011,28(6):6-7.
[28]刘萍,古今,冯建涌.天王补心丸全方及方中缺桔梗对小鼠镇静催眠作用的影响[J].中国药物应用与监测,2004,(02).
[29]李海静,高月,刘萍.天王补心丸全方及全方缺桔梗对少寐大鼠脑神经递质的影响[J].中国中药杂志,2009,(02).
[30]刘萍,邹鹏,李外,孙艳,张洁,余河水,赵阳,马百平.指纹图谱技术考察桔梗在天王补心丸中的配伍意义[J].中国中药杂志,2009,(02).
[31]王长春.议张锡纯之“大气下陷证”.中医研究,2006,19(6):8-9.
[32]马术明.慢性充血性心力衰竭的辨证论治[J].吉林中医药,2004,24(2):6-8.
[33]刘静,贾庭英.益气强心汤治疗充血性心力衰竭临床观察[J].吉林中医药,2005,25(8):14.
[34]吴素琴.生脉、黄芪注射液联用治疗充血性心力衰竭疗效分析[J].吉林中医药,2004,24(7):6-7.
[35]张持.中医对慢性充血性心力衰竭的认识及治疗进展[J].吉林中医药,2006,26(11):71-73.
[36]赵惠,李七一.充血性心力衰竭的中医研究概况[J].吉林中医药,2004,24(9):56-58.
[37]李昌繁,江时森.心力衰竭动物模型的研究进展.医学研究生学报,2007,20(5):532-534.
[38]戴闺柱. β受体阻滞剂治疗慢性心力衰竭-从禁忌证到常规治疗.中华心血管病杂志,2002,30(6):381-383.
[39]周永昌,郭万学.超声医学[M].北京:科学技术文献出版社,2004:680.
[40]陈翔,邓乐勇.强心力胶囊治疗充血性心力衰竭彩色多普勒超声心动图检测指标分析[J].湖南中医学院学报,2006,26(1):44.
[41] WAYBRIGHT TJ,VAN QN,MUSCHIK GM, et al. LC-MS in metabonomics: Optimization ofexperimental conditions for the analysis of metabolites in human urine[J]. J Liq Chromatogr RelatTechnol,2006,29(17-20):2475-2497.
[42] ZHAO Xinjie, ZHANG Yi, MENG Xianli, et al. Effect of a traditional Chinese medicinepreparation Xindi soft capsule on rat model of acute blood stasis: a urinary metabonomics studybased on liquid chromatography mass spectrometry[J]. J Chromatogr B,2008,873(2):151-158.
[43] GIRI S, KRAUSZ K W, IDLE J R, et al. The metabolomics of (+/-) arecoline oxide in the mouseand its formation by human flavin containing monooxygenases[J]. Biochem Pharmacol,2007,73(4):561-573.
[44] WONG MCY,LEEA WTK,WONG JSY, et al. An approach towards method development foruntargeted urinary metabolite profiling in metabonomic research using UPLC/Q-TOF-MS[J]. JChromatogr B,2008,871(2):341-348.
[45] XIE Baogang, GONG Tao, GAO Rong, et al. Development of rat urinary HPLC-UV profiling formetabonomic study on Liuwei Dihuang Pills[J]. J Pharm Biomed Anal,2009,49(2):492-497.
[46] LAURIDSEN M,HANSEN S H,JAROSZEWSKI J W, et al. Human urine as test material in1H-NMR based metabonomics: recommendations for sample preparation and storage[J]. AnalChem,2007,79(3):1181-1186.
[47] BRUCE SJ,TAVAZZI I,PARISOD V, et al. Investigation of human blood plasma samplepreparation for performing metabolomics using ultrahigh performance liquid chromatography/massspectrometry[J]. Anal Chem,2009,81(9):3285-3296.
[48] MICHOPOULOS F,LAI L,GIKA H, et al. UPLC-MS based analysis of human plasma formetabonomics using solvent precipitation or solid phase extraction[J]. J Proteome Res,2009,8(4):2114-2121.
[49]王咏梅,殷仁富,吴宗贵，梅长林，顾书华.左旋肉碱洽疗急性病毒性心肌炎的临床疗效分析.中华心血管病杂志,1999,20(6):405-407.
[50] Listenberger L L, Schaffer JE. Mechanisms of Lipoapoptosis*1: Implications for Human HeartDisease. Trends in cardiovascular medicine,2002,12(3):134-138.
[51]杨建峰,魏经汉.慢性心力衰竭患者血清IL-1β, TNF-α水平及临床意义.实用全科医学,2007,5(11):954-955.
[52]孙蕾,王会信,周廷冲.鞘磷脂与细胞生长、凋亡调控.军事医学科学院院刊,1998,22(3):233-235.
[53]齐炼文,周建良,郝海平,李会军,闻晓东,陈君,杨中林,李萍,王广基.基于中医药特点的中药体内外药效物质组生物／化学集成表征新方法.中国药科大学学报,2010,41(3):195-200.
[54]潘瑞乐,陈迪华,沈连刚,斯建勇.高效液相色谱法测定中药升麻中阿魏酸和异阿魏酸的含量.药物分析杂志,2000,20(6):396-398
[55] Li, W., Y. Sun, et al. Identification of caffeic acid derivatives in Actea racemosa (Cimicifugaracemosa, black cohosh) by liquid chromatography/tandem mass spectrometry. Rapidcommunications in mass spectrometry,2003,17(9):978-982.
[56] Ma, C., L. Wang, et al. Identification of major xanthones and steroidal saponins in rat urine byliquid chromatography–atmospheric pressure chemical ionization mass spectrometry technologyfollowing oral administration of Rhizoma Anemarrhenae decoction. BiomedicalChromatography,2008,22(10):1066-1083.
[57] Zhiyun, M., X. Suixu, et al. New saponins from Anemarrhena asphodeloides Bge. Chinese Journalof Medicinal Chemistry,1999,9(4):294-298.
[58] Zhang, Y. Y., Q. Wang, et al. Characterization and determination of the major constituents inBelamcandae Rhizoma by HPLC-DAD-ESI-MSn. Journal of Pharmaceutical and BiomedicalAnalysis,2011,56(2011)304-314.
[59]中国科学中国植物志编辑委员会.中国植物志.北京：科学出版社,73(2):77.
[60]国家药典委员会，2010版中国药典一部，北京：化学工业出版社，2010：259.
[61]付文卫,窦德强,等.桔梗的化学成分和生物活性研究进展.沈阳药科大学学报,2006,23(3):184-191.
[62] Gao ZZ, Xia YF, Yao XJ, Dai Y, Wang Q. A new triterpenoid saponin from Gleditisia sinensis andstructure-activity relationships of inhibitory effects on lipopolysaccharide-induced nitric oxideproduction. Nat Prod Res.2008,22(4)320-332.
[63] Yoshikawa M, Murakami T, Yashiro K, et al．Kotalanol, a potent glycosidase inhibitor withthiosugar sulfonium sulfate structure, from antidiabetic Ayurvedic medicine salaciareticulata[J]．Chemical&Pharmaceutical Bulletin,1998,46(8):1339-1340．
[64]赵晓亚,孙汉董,等.冷水七根茎的化学成分研究[J].中国中药杂志,2005,30(008):584-586.
[65]王文永,赵仕丞,刘东新.山海螺化学成分研究[J].中药材,2011,34(4)553-555.
[66]姜勇,刘蕾,屠鹏飞.远志的化学成分研究Ⅲ.中国天然药物,2003,1(3):142-145
[67]解翠华,阮丽君,夏焕章,陈代杰,葛梅.植物内生菌HCCB00167产物的发酵、分离和结构鉴定[J].沈阳药科大学学报,2007,24(4):245-248.
[68]龚小见,周欣,赵超,等.马兰中的三萜类成分[J].中国中药杂志,2010,35(3):327-330．
[69]任启生,余雄英,等.山海螺化学成分研究.中草药,2005,36(12):1773-1775.
[70]任凤霞,张爱军,赵毅民.鹿蹄草化学成分研究Ⅱ[J].解放军药学学报,2008,24(4):301-304．
[71]付文卫,侯文彬,窦德强,华会明,桂毛华,付锐,陈英杰,裴月湖.桔梗中远志酸型皂苷的化学研究,药学学报,2006,41(4):358-360
[72]李凌军,刘振华,等.桔梗的化学成分研究.中国中药杂志，2006，31(18):1506-1509
[73] Fu, W. W., N. Shimizu, et al. Five new triterpenoid saponins from the roots of Platycodongrandiflorum. Chemical and pharmaceutical bulletin，2006，54(4):557-560.
[74] NIKAIDO, T., K. KOIKE, et al. Triterpenoid saponins from root of Platycodon grandiflorum.生薬學雜誌，1998，52(1):54-59.
[75] Li, W., L. Xiang, et al. A new triterpenoid saponin from the roots of Platycodon grandiflorum.Chinese Chemical Letters,2007,18(3):306-308.
[76] Choi, Y. H., D. S. Yoo, et al. Platyconic acid A, a genuine triterpenoid saponin from the roots ofPlatycodon grandiflorum. Molecules,2008,13(11):2871-2879.
[77] He, Z., C. Qiao, et al. New triterpenoid saponins from the roots of Platycodon grandiflorum.Tetrahedron,2005,61(8):2211-2215.
[78] Fu, W. W., D. Q. Dou, et al. Studies on the chemical constituents from the roots of Platycodongrandiflorum. Journal of natural medicines,2006,60(1):68-72.
[79] Ishii, H., K. Tori, et al. Saponins from roots of Platycodon grandiflorum. Part1. Structure ofprosapogenins. J. Chem. Soc., Perkin Trans.,1981,1(0):1928-1933.
[80] Li, W., W. Zhang, et al. Platycoside N: A New Oleanane-Type Triterpenoid Saponin from the Rootsof Platycodon grandiflorum. Molecules,2010,15(12):8702-8708.
[81] Nikaido, T., K. Koike, et al. Two new triterpenoid saponins from Platycodon grandiflorum.Chemical and pharmaceutical bulletin,1999,47(6):903-904.
[82] Fu, W. W., D. Q. Dou, et al. Triterpenoid saponins from Platycodon grandiflurum. Journal of Asiannatural products research,2007,9(1):35-40.