摘要
复方配伍理论是中医理论的重要组成部分。然而,中药自身组成的复杂性和临床用药的辩证灵活性使得复方配伍理论很难用现代科学技术作出清晰表达,因此,如何科学阐明复方配伍理论一直是中医药理论研究中的热点和难点。“君-使”药物是复方中的一组核心对药,符合中药“七情”中的“相使”关系,研究这种重要的配伍关系,发现体内效应物质对于进一步认识中药引经的实质和丰富并发展复方配伍理论有着重要意义,同时也能为高效创新药物的研发提供新的途径。本论文选择“广枣-降香”作为研究对象,系统地研究了降香对君药广枣中的效应物质在正常家兔体内药代动力学过程和组织分布特征的影响。全文共分为四章,主要研究内容如下:
1.建立了兔含药血浆和组织中没食子酸(GA)和原儿茶酸(PA)的高效液相色谱分析新方法,结果显示:没食子酸和原儿茶酸在0.04-2.0 mg·L~(-1)范围内具有良好的线性关系,低、中、高三个浓度的方法回收率、精密度和稳定性等指标均符合美国FDA生物样品的测定要求,可以作为广枣体内效应物质研究的方法,为君.使对药体内动力学和组织分布研究奠定了方法学基础。
2.采用上述方法研究了君药广枣中没食子酸和原儿茶酸在正常家兔体内的药代动力学过程;并研究了使药降香对没食子酸和原儿茶酸药代动力学过程的影响。研究发现与降香配伍后,可促进没食子酸、原儿茶酸由血液向组织中分布,提高了生物利用度,提示降香有可能增强君药广枣在治疗心血管类疾病方面的疗效,为分析降香增效.导向作用,以及科学阐明复方配伍理论提供了依据。
3.研究了广枣与降香配伍前后,没食子酸和原儿茶酸在正常家兔体内的组织分布特征变化,结果表明:没食子酸的分布特征由C_肾>C_脑>C_脾>C_心>C_肝>C_肺转变为C_肾>C_脑>C_肝>C_心>C_脾>C_肺;原儿茶酸的分布特征由C_肾>C_心>C_脾>C_肝>C_肺>C_脑转变为C_肾>C_脾>C_肝>C_心>C_肺>C_脑。结果印证了中医理论在广枣归经与降香引经方面的理论论述,为中药归经、引经等理论的现代研究提供了新思路。
4.结合代谢组学的研究方法,用主成分分析法对获得的色谱数据进行了分析,并结合药时曲线的变化规律,确定了4个未知成分为“广枣-降香”的体内效应物质。进一步通过高效液相色谱-质谱联用技术(HPLC-MS)对其进行了定性分析,推测其中2个成分的分子离子峰分别为:m/z 179.2和m/z 3 18.5,建立了“君-使”对药体内效应物质研究的新模式,为创新药物的开发提供了新途径。
The theory of prescription compatibility is an important part of the theory of traditional Chinese medicine (TCM). But, for the complex composition and clinical application with philosophic thoughts, it's very difficult to express the mechanism of prescription compatibility by modern science and technology. Therefore, how to express the principle of prescription compatibility according to modern science and technology is a hotspot and a tough problem all the time. Monarch-guide compatible herbal medicine is the core of TCM. An important approach to enrich the theories of Traditional Chinese Medicine and develop highly effective new drugs is to find the key compatibility relationship through the studies of efficacious prescriptions and find their bioactive substances. Under the guidance of the theories of Traditional Chinese Medicine, the present research chose "Guangzao - Dalbergia" to systematically study the mechanism of monarch-guide herbal medicine in the body through metabolomics. The influence of guide herb (Dalbergia,JX) was revealed through investigating the pharmacokinetical (PK) changes of bioactive substances in monarch herb(Guangzao,GZ); The main contents are as follows:
1. A new high performance liquid chromatography (HPLC) method for analysis of the gallic acid (GA) and protocatechuic acid (PA) of Guangzao in plasma and tissue is established. The results showed that the linear range of GA and PA is 0.04~2.0 mg·L~(-1). The relative recovery and absolute recovery of low, medium and high concentration, relative standard deviations (RSD) of precision and stability experiments are consistent with the requirements of biological sample analysis according to FDA.
2. After intragastric administration of GZ and GZ-JX, the PK parameters of GA and PA in plasma and tissue of normal rabbits. The results indicated that dalbergia could change the pharmacokinetics of GA, also could increase the biological availability of PA. Further, it might indicate that the the dalbergia enhance curative effect of GZ in the cardiovascular diseases, which will provide evidences of dalbergia's synergism and explaination of the theory of prescription compatibility by modem science and technology.
3. Tissue distribution of GA and PA is investigated to study the influence of synergism between SM and BC. The results show that the concentration sequence of GA in SM group is kidney >brain>spleen>heart>liver>lung. However, it convert to kidney> brain >liver> heart>spleen>lung under the influence of BC. The results show that the concentration sequence of PA in SM group is kidney >heart >spleen >liver>lung>brain, and it convert to kidney>spleen >liver> heart >lung > brain under the influence of BC, verifying the TCM theory on meridian distribution of SM and guidance distribution of BC.
4. The mass data of HPLC is statistically processed by metabonomic ,with SPSS method and considering the change of concentration-time curves, 4 unkown substances were ascertained to be the bioactive substances of GZ-JX. An initial qualitative analysis on them was carried out by HPLC -MS, then the fragment ions corresponded with 2 unknown bioactive substances were supposed to be: m/z179.2 and m/z318.5. A new mode to investigate the bioactive substances of monarch-guide compatible herb was established, which could provide new pathway to develop highly effective new drugs.
引文
[1] 周斌,张铁军,高文远,等.中药复方配伍规律及药效物质基础研究进展[J].中草药.2006,37(11):1601-1606
[2] 李冀,李晓琳.论药对与方剂配伍的关系[J].中医药信息,2005,22(1):30-31
[3] 孙洋,陈婷,徐强.从药对的角度考察复方配伍规津[J].世界科学技术.中医药现代化,2004,6(1):17-2l
[4] 刘海燕,谢建中.复方丹参注射液及丹参注射液与其他药物的配伍[J].云南医药,2005,26(5):479-480
[5] 叶建红.对药远志石菖蒲的药理作用和临床运用体会[J].现代中西医结合杂志,2002,11(22),2256-2258
[6] 王淑英,陈奕芝,魏刚,等.石菖蒲与降香配伍对高脂血症大鼠脑组织内皮素和降钙素基因相关肽的影响[C].第八次全国中西医结合虚证与老年医学学术研讨会论文集.2005,7:96-97
[7] 刘嵌.黄芪、丹参及其配伍对“血瘀”大鼠血液流变学影响[J].甘肃中医,2006,19(10):32-34
[8] 王洪.威灵仙桃仁临床配伍举隅[J].中医杂志,2006,47(7):490-491
[9] 商洪才,高秀梅,郭利平,等.丹参三七组方配比优选及信息综合评价[J].药品评价,2005,2(3):209-211
[10] 张玉杰,杨洁,邹晓翠.黄连肉桂药对配伍对黄连总碱大鼠小肠吸收的影响[J].中国中医杂志,2007,32(15):1521-1524
[11] Li SL, Li P, Sheng LH, et al.Live cell extraction and HPLC-MS analysis for predicting bioactive components of traditional Chinese medicines[J]. Journal of Pharmaceutical and Biomedical Analysis,2006,41(23):576-581
[12] 黄熙,陈可冀.“证治药动学”新假说的理论与实践[J].中医杂志,1997,38(12):745-747
[13] 贺丰,罗佳波,陈飞龙,等.GC-MS法研究麻黄汤中麻黄碱、伪麻黄碱的人体过程[J].中药新药与临床药理,2004,(5):336-338
[14] 贺丰,罗佳波.臣佐使药对麻黄汤君药麻黄中伪麻黄碱的人体药代动力学的影响[J].中国中药杂志,2005,30(18):1454-1456
[15] 贺丰,罗佳波.臣佐使药对麻黄汤君药麻黄中麻黄碱的人体药代动力学的影响[J].中草药,2005,36(9):1313-1316
[16] 张壮,刘楠,陈可冀,等.川芎赤芍配伍比例对阿魏酸在麻醉犬体内药代动力学的影响[J].中国实验方剂学杂志,2005,11(3):28-31
[17] 郑晓晖,赵欣,房敏峰,等.复方丹参方中使药降香对君药丹参药代动力学的影响[J].西安交通大学学报(医学版),2007,28(2):170-173
[18] 张丽,陈林,王世祥,等.活血化瘀方中使药郁金对君药丹参药代动力学的影响研究[J].中成药,2007,29(8):1225-1227
[19] 庸一梅,李仲谨,王世祥,等.檀香对丹参药代动力学的影响研究[J].中国药科大学学报,2007,38(2):149-152
[20] 杜力军,邢东明,刘美风,等.脑血管药CBN注射剂药代动力学研究——对植物药药代动力学研究方法学的探讨[J].世界科学技术-中医药现代化(基础研究),2003,5(1):48-52
[21] Nicholson J K, Connelly J,Lindon J C, et al. Metabonomics: a platform for studying drug toxicity and gene function [J]. Nat Rev Drug Dis, 2002,1(2):153-161
[22] Reo N V. NMR-based metabolomics [J]. Drug Chem Toxicol, 2002, 25 (4): 375-382
[23] Jun Yang, Guowang Xu, Yufang Zheng, et al. Strategy for metabonomics research based on high-performance liquid chromatography and liquid chromatography coupled with tandem mass spectrometry [J]. Journal of Chromatography A,2005,1084 (8): 214-221
[24] 刘昌孝,代谢组学的发展与药物研究开发[J].中草药,2006,37(增刊):2-7
[25] Scaraffia PY,Zhang QF, Wysocki VH, et al.Analysis of whole body ammonia metabolism in Aedes aegypti using [~(15)N]-labeled compounds and mass spectrometry [J]. Insect Biochemistry and Molecular Biology, 2006,36(8):614-622
[26] Ameenah GF. Medicinal plants: Traditions of yesterday and drugs of tomorrow [J]. Molecular Aspects of Medicine, 2006,27:1-93
[27] Y Feng, ZH Wu, XZ Zhou, et al. Knowledge discovery in traditional Chinese medicine: State of the art and perspectives [J]. Artificial Intelligence in Medicine, 2006, 38 (3): 219-236
[28] Aardema MJ, MacacGregor JT. Toxicology and genetic toxicology in the new era of "toxicogenomics": impact of "-omics" technologies [J]. Mutation Research, 2002,499 (1):13-25
[29] 李林,王建农,任建勋,等.气虚血瘀证大鼠尿液的核磁共振谱代谢组学[J].科学通报,2007,52(15):1758-1762
[30] 黄玉荣,魏广力,龙红,等.钩藤多动合剂的药效作用及用代谢物组学方法研究其生化机制[J].中草药,2005,36(3):398-402
[31] 蒋宁,周文霞,张永祥.应用代谢组学方法研究比较六味及八味地黄汤的作用机理[J].中药药理与临床,2007,23(5):45
[32] 谢跃生,潘桂湘,高秀梅,等.高效液相色谱技术在代谢组学研究中的应用[J].分析化学(评述与发展),2006,34(11):1644-1648
[33] Pham-Tuan H, Kaskavelis L, Daykin CA, et al. Method development in high-performance liquid chromatography for high-throughput profiling and metabonomic studies of biofluid samples [J].Journal of Chromatography B, 2003,789(2):283-301
[34] Williams RE, Lenz EM, Evans JA, et al. A combined ~1HNMR and HPLC-MS-based metabonomic study of urine from obese (fa/fa) Zucker and normal Wistar-derived rats [J]. Journal of Pharmaceutical and Biomedical Analysis, 2005, 38 (3): 465-471
[35] 骆泽宇.核磁共振-质谱联用技术在药学领域的应用进展[J].中国医院药学杂志,2007,27(6):807-809
[36] Sandvoss M, Bardsley B, Beck TL, et al. HPLC-SPE-NMR in pharmaceutical development:capabilities and applications [J]. Magnetic resonance in chemistry, 2005,43 (9):762-770
[37] Nicholson J K, Lindon J C, Holmes E. "Metabonomics": understanding the metabolic responses of living systems to pathophysiological stimuli via multi variate statistical analysis of biological NMR spectroscopic data [J]. Xenobiotica, 1999,29 (11):1181-1189
[38] 宋剑南.辩证论治的整体调节与中药整体效应分子组[J].世界科学技术-中医药现代化(思路与方法),2006,8(3):54-61
[39] Nobeli I, Thornton JMA. Bbioinformatician's view of the metabolome[J]. Bioessays, 2006, 28 (5): 534-545
[40] Marielle Lintinga, Jacqueline JM, Patrick JF, et al. Nonlinear Principal Components Analysis:Introduction and Application [J]. Psychological Methods, 2007,12(3):336-358
[41] Peter Forlay-Frick, Jeno Fekete, Karoly Heberger. Classification and replacement test of HPLC systems using principal component analysis [J]. Analytica Chimica Acta, 2005, 536(1-2):71-81
[42] 冉小容,司圣柱,梁琼麟,等.LC/MS/MS结合主成分分析(PCA)用于鼠尿中灯盏花素代谢组学的研究[C],第三届全国微全分析系统学术会议论文集,2005,1:213-214
[43] 王喜军,孙文军,孙晖,等.CCL_4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[J].世界科学技术-中医药现代化(科技论坛),2006,8(6):101-106
[44] Peiyuan Yin, Xinjie Zhao, Qiurong Li, et al. Metabonomics Study of Intestinal Fistulas Based on
[45] 朱建平,袁冰.明代方剂配伍理论初探[J].中国医药学报,2004,19(5):261-265
[46] 于友华.方剂配伍理论的系统科学思想[J].中国中医基础学杂志,2004,10(8):63-64
[47] 王阶,郭丽丽,杨戈,等.方剂配伍理论研究方法及研究前景[J].世界科学技术-中医药现代化,2006,8:1-5
[48] 张琦,张大方.中药复方物质基础的研究[J].吉林中医药,2007,27(2):54-55
[49] 李镤.速效救心丸及其新对药-川芎、降香[J],北京中医,2006,25(9):575-576
[50] 卫生部国家药典委员会,中华人民共和国药典(一部)[M].北京:化学工业出版社,2005:29-30
[51] 邰文泉,白玉下.蒙药广枣的提取工艺和化学成分研究新进展[J].内蒙古民族大学学报(自然科学版),2006,21(1):65-66
[52] 白勇,爱民,谢军,等.广枣总黄酮的研究概况[J].内蒙古医学院学报,2005,27(5):135-137
[53] 施润菊,戴云,郑晓晖等.广枣水提物的HPLC-ESI-MS~n分析[J].中药材,2007,30(3):294-297
[54] 樊海燕,赛音等.广枣中黄酮类化合物提取工艺研究[J].中草药,2004,35(3):253-255
[55] 刘莲英,杨瑞瑞,李维凤,等.广枣中没食子酸的含量测定[J].西北药学杂志,2002,17(5):204-205
[56] 邓科君,张艺,等.反相高效液相色谱法同时测定藏药广枣中没食子酸和原儿茶酸的含量[J].色谱,2006,24(6):652-653
[57] 尹海波,何静,王冰,等.东北八种老鹳草没食子酸含量比较[J].中药材,2007,30(6):637-638
[58] 韩莹,熊立志,杨春娟,等.高效液相色谱法测定大鼠血浆中的原儿茶酸[J].2007,25(2):207-210 .
[59] Siranoush Shahrzad, Kazumasa Aoyagi, Antje Winter, et al.Pharmacokinetics of gallic acid and its relative bioavailability from tea in healthy humans [J]. Human Nutrition and Metabolism-Research Communication, 2001,131:1207-1210
[60] Siranoush Shahrzad, Irmgard Bitsch. Determination of gallic acid and its metabolites in human plasma and urine by high-performance liquid chromatography [J]. Journal of Chromatography B,1998, 705: 87-95
[61] Guan S, Jiang B, Bao YM,et al. Protocatechuic acid suppresses MPP+- induced mitochondrial dysfunction and apoptotic cell death in PC12 cells [J]. Food and Chemical Toxicology Food Chem Toxic, 2006,44:1659-1666
[62] 连珠,张承忠,李冲,等.蒙药广枣化学成分的研究[J].中药材,2003,26:23-24
[63] 杨秀伟.基于体内过程的中药有效成分和有效效应物质的发现策略[J].中国中药杂志,2007,32(5):365-370
[64] 田金洲,王永炎,徐意,等.血瘀证动物模型的种类、评价与研究[J].北京中医药大学学报,2006,29(6):396-400