麻黄类药对组成规律的基础研究—麻黄—甘草药对（I）
|
摘要
研究背景
本文为国家自然科学基金重点项目-麻黄类药对组成规律的基础研究的部分研究内容。该项目以风寒表实证为主线,以麻黄为主药,从张仲景经典名方中选择针对主证为风寒表实证但不同兼证的麻黄汤及其类方中的六个药对进行组成规律研究,探讨药味、药量的改变与化学成分、药理效应、药代动力学和药物代谢组学之间的相互关系；同时纵向分析六个药对组合与风寒表实证遣方用药的内在联系,探讨药对变化的内涵和组成规律,促进中医药学遣方用药理论的发展。
麻黄-甘草药对为上述六个药对之一,亦为《金匮要略》所载用于表寒兼皮水的甘草麻黄汤,有利水消肿之功,主治身体面目悉肿,腰以上肿甚,身微热,口不渴,无汗,小便不利者。方中麻黄宣发卫阳,散表祛邪,使风邪水气从外而解,又可使在里之水从小便而利；甘草和调中气、调和药性。两药合用,辛甘发散以利水,健脾和中以制水。本方中麻黄12g、甘草6g,该剂量及配比在张仲景麻黄、甘草并用的方剂中最为常用,但并不是一成不变的,如在大青龙汤、麻黄杏仁甘草石膏汤、越婢汤中,麻黄用12g,甘草用6g；在麻黄附子甘草汤中,麻黄用6g、而甘草为6g,甘草用量较重；在麻黄加术汤中,麻黄用9g、而甘草为3g。“病皆与方相应”,麻黄-甘草药对针对不同主治证候采用不同的配伍比例,这也是分析其配伍内涵探讨的问题。
本文运用现代药理学方法来研究麻黄和甘草药理效应的相互作用,定量评价药对是否存在配伍增效或配伍减毒,寻求最佳配比；同时研究其在体外过程中发生各种物理化学反应后主要化学成分的变化,从物质基础寻找量变到质变的根本所在；用代谢组学的方法观察麻黄和甘草药对配伍对生物标志物的影响,从代谢组学层面表征该药对的配伍内涵。
研究意义
复方临床疗效的发挥,很大程度上取决于中药的配伍,而药对作为中药配伍应用最为常见的形式,它构成简单、疗效确切,且具备复方基本主治功能,是中药复方的最小组成单位,同时也是系列复方的共同点；药对反映复方配伍的特殊规律与内在联系。因此,以药对研究为切入点,阐明其配伍机制,寻求发挥最佳作用的配伍比例,有利于探索与发掘药对的基本功用与功用扩展。开展药对配伍规律研究,能进一步揭示药对配伍的客观规律与科学内涵,促进中药配伍应用理论的进步；能有效地指导临床遣方用药,既利于更好运用已有的药对,亦利于针对疾病谱的变化、病证的发展而创制新药对；特别是在新药研究中以有效成分或有效部位配伍已越来越多的今天,以传统配伍的普遍规律指导新的配伍形式,对促进中药新产品研创亦具有现实意义。
研究目的
本文从以下三个层面揭示麻黄-甘草药对配伍内涵：
(1)观察麻黄-甘草药对不同配比的急性毒性、抗炎、利尿三个方面的药理效应,应用中效原理进行相互作用统计分析,定量评价麻黄和甘草主要药理效应的相互作用,以判断药物是否增效减毒。
(2)对不同配比水煎液麻黄类生物碱(麻黄碱、伪麻黄碱、去甲基麻黄碱、去甲基伪麻黄碱和甲基麻黄碱)和甘草酸、甘草苷含量进行研究,比较不同配比对这些化学成分含量的影响,以期从物质基础的变化来阐述药理效应变化的内因。
(3)运用代谢组学技术探讨大鼠灌胃麻黄、目草和麻黄-甘草药对后对尿液中生物标志物的影响,寻找表征这一变化过程的生物标志物,从系统与整体层面探讨麻黄配伍甘草的相互作用机制。
研究方法
(1)主要药理效应变化采用小鼠半数致死量(LD50)测定来评价麻黄、甘草及麻黄-甘草药对三个配比的急性毒性；以醋酸致小鼠毛细血管通透性增加实验观察抗炎作用；以对正常大鼠的排尿量的影响来观察利尿作用,应用calcusyn软件或单因素方差分析(One-way ANOVA)评价麻黄和甘草的相互作用。
(2)根据麻黄-甘草的相互作用关系确定两者协同作用的最佳配比,利用该配比进行二甲苯致小鼠耳肿胀、角叉菜胶致大鼠足肿胀、角叉菜胶致大鼠胸膜炎三个急性炎症实验和大鼠棉球肉芽肿亚急性炎症实验,进一步明确麻黄-甘草药对的抗炎作用；利用角叉菜胶致大鼠胸膜炎实验观察麻黄-甘草药对对胸膜炎大鼠HE染色肺组织的病理学影响；免疫组化检测iNOS和ICAM-1在肺组织的表达情况；测定胸膜炎大鼠胸腔渗出液前列腺素E2(PGE2)、肿瘤坏死因子-α (TNF-α)和白细胞介素-1p(IL-1β)含量以及肺组织TNF-α IL-1β和丙二醛(MDA)含量来探讨抗炎机制。观察麻黄-甘草药对对正常大鼠尿液中PGE2的影响,探讨利尿作用机制。所有均数比较用One-way ANOVA,数据由SPSS13.0软件进行统计学处理,以P<0.05为差异有统计学意义。
(3)采用气质-液质(GC-MS)联用考察麻黄水煎液及麻黄-甘草药对不同配比共煎液中麻黄类生物碱(麻黄碱、伪麻黄碱、去甲基麻黄碱、去甲基伪麻黄碱和甲基麻黄碱)指纹图谱峰的变化情况,所有均数比较用One-way ANOVA;采用高效液相色谱法(HPLC)考察甘草水煎液及麻黄-甘草药对不同配比共煎液中甘草苷和甘草酸的指纹图谱峰的变化情况；以明确不同配比对主要药效成分的影响,均数比较采用析因设计资料的方差分析。所有数据由SPSS13.0软件进行统计学处理,以P<0.05为差异有统计学意义。
(4)麻黄水煎液、甘草水煎液、麻黄-甘草药对水煎液给大鼠灌胃,于第7天收集尿液,尿液离心后进行衍生化；采用GC-MS对大鼠尿液中代谢物进行全扫描分析。将所得的GC-MS数据进行峰识别、峰对齐、扣除溶剂峰、杂质峰的处理。导出的数据进行峰面积归一化处理,生成包括样品名称、归一化峰面积的二维矩阵。利用Simca-P软件对大鼠的尿液代谢物谱进行主成分分析(PCA)和偏最小二乘法判别分析(PLS-DA), PCA直观观察不同组的差异；PLS-DA进一步考察组间的区分程度,并根据代谢物的投影重要性(VIP)评价不同物质与分类的相关性程度,以VIP值>1的代谢物作为潜在生物标志物。运用SPSS13.0软件对潜在生物标志物进行统计分析,选择差异有统计学意义的物质作为最终的生物标志物。通过与空白对照组尿液的代谢物谱比较,研究麻黄水煎液、甘草水煎液和麻黄-甘草药对(12：6)水煎液对大鼠内源性代谢标志物的影响；结合现有的生物化学知识阐释麻黄、甘草和麻黄-甘草药对所影响的代谢途径。
主要研究结果
(1)麻黄-甘草药对(12：3)、(12：6)和(12：12)均表现出毒性拮抗作用,且拮抗作用与甘草用量有明显剂量依赖关系。麻黄-甘草药对(12：3)配伍,当抑制率在25%～70%,抗炎协同作用明显,且与空白对照组比较,抗炎作用差异有统计学意义；抑制率高于70%时表现出拮抗作用。(12：6)抗炎抑制率在25%～70%协同作用明显,且与空白对照组比较,抗炎作用差异性有统计学意义。(12：12)有明显抗炎拮抗作用。麻黄-甘草药对(12：3)和(12：6),治疗指数(TI)接近。
(2)与模型对照组比较,麻黄-甘草药对(12：6)(22.4,11.2g·kg-1)2个剂量组均能抑制二甲苯所致的小鼠耳肿胀(P<0.05)；22.4g·kg-1能抑制角叉菜胶注射后5h,7h的大鼠足肿胀(P<0.05),11.2,5.6g·kg-1对大鼠足肿胀度没有抑制作用(P>0.05)；22.4g·kg-1能抑制大鼠棉球肉芽肿形成(P<0.05)；麻黄-甘草药对22.4g·kg-1能减轻角叉菜胶对肺组织的病理损伤、抑制iNOS在肺支气管上皮细胞的表达(P<0.05)、对ICAM-1的表达没有显著影响(P>0.05)；药对能抑制角叉菜胶诱导的胸膜炎大鼠胸腔液渗出P<0.05)、抑制渗出液白细胞数量增多以及渗出液PGE2、TNF-α和IL-1β含量升高、抑制肺组织TNF-α、IL-1β和MDA含量升高(所有P<0.05);麻黄-甘草药对能增加正常大鼠排尿量及尿液中K+的排泄(P<0.05)；增加尿液中PGE2排泄(P<0.05)。
(3)麻黄-甘草药对配伍,随着甘草用量的增加,麻黄类生物碱(麻黄碱、伪麻黄碱、去甲基麻黄碱、去甲基伪麻黄碱和甲基麻黄碱)和甘草酸、甘草苷含量均下降；甘草用量与7个成分的含量呈负相关,麻黄-甘草药对(12：12)各成分下降最明显。
(4)与空白对照组比较,甘草组存在差异的生物标志物有：α-酮戊二酸、己二酸、柠檬酸、氨基马尿酸、甲基黄嘌呤、色氨酸,水平显著下调的有：α-酮戊二酸、己二酸、柠檬酸；生物标志水平显著上调的有氨基马尿酸、甲基黄嘌呤、色氨酸。麻黄组存在差异上调的生物标志物：丙氨酸、色氨酸。麻黄-甘草药对组与空白对照组比较,存在差异的生物标志物有：α-酮戊二酸、己二酸、乌头酸、柠檬酸、丙氨酸、半胱氨酸、甲基黄嘌呤、酪氨酸、色氨酸；水平显著下调的有α-酮戊二酸、己二酸、乌头酸、柠檬酸、半胱氨酸；显著上调的有丙氨酸、甲基黄嘌呤、酪氨酸、色氨酸。
结论
麻黄-甘草药对主要药理效应的相互关系与两者配伍后主要有效成分含量的改变有相关性,合煎后主要化学成分群含量降低,主要药理效应会在一定范围内表现出拮抗作用；麻黄-甘草药对配伍,在合适的剂量和配比范围内,能增效减毒；(12：3)和(12：6)协同范围较宽,呈现配伍优势。麻黄-甘草药对有抗炎作用,抗炎机制可能与影响炎症介质产生和抗氧化作用有关；利尿作用机制与促进尿液PGE2排泄有关。麻黄、甘草影响的代谢标志物不同,麻黄-甘草药对除了影响两者单独作用的代谢标志物外,还影响了另外3种标志物,从系统的层面表明麻黄和甘草存在协同作用。
Background
This study is part of basic research on the composition of the law of Ephedra sinica herbal pairs, funded by the National Natural Science Foundation. The main line of the project is Feng, Han Biao and Shi syndromes, Ephedra sinica is taken as the major medicine, we choosed six herbal pairs which used for different accompan-ied symptoms from the Zhang Zhongjing classic prescriptions, to explore the comp-ositions of the law, and the relationship between the changes of compatibility or dos-ages and the contents of compositions and the pharmacyological effects, pharmacyo-kinetics or drug metabolism genomics, and then longitudinal analyse the internal rel-ations of six herbal pairs with prescriptions, and observe how symptoms, diagnosis and treatment of Feng, Han, Biao Shi to be affected by medicines compatibility and their dosages. These would be beneficcial to reveal the meanings and laws of the sy-stem of Traditional Chinese herbal pairs, then sum up the universal law of medicine pairs composition, and promote the development of the theory of Chinese medicine prescription.
Ephedra sinica-Glycyrrhiza uralensis is one of six herbal pairs studied, which is selected from Glycyrrhiza uralensis-Ephedra sinica soup used for Feng, Han, an-d Pi Shui sydomes. Glycyrrhiza uralensis-Ephedra sinica soup is derived from "Go-lden Chamber", consists of Ephedra sinica and Glycyrrhiza uralensis.The, utility of this soup is to eliminate the water swelling, and used for the treatment of inner Shu-i, the body and face swollen, even above the waist, body fever, thirst, sweat, disadva-ntage of a urinary. The actions of Ephedra sinica are to XuanFa Wei Yang, Sanbiao QuXie, make FengXie eliminated from the outside and eliminated Lishui from the u-rine, the actions of Glycyrrhiza uralensis are to regulate ZhongQi and reconcile med-icine properties. The two combined medicines exert the actions of FaSan LiShui. In this prescription, Ephedra sinica is12g, Glycyrrhiza uralensis is6g, which is the most commonly used doses and ratio in Zhongjing prescriptions, but not set in stone, such as in the big dragon soup, Ephedra sinica-Glycyrrhiza uralensis soup and Yue pi soup, Ephedra sinica is12g, Glycyrrhiza uralensis is6g. In Ephedra sinica Ram-ulus Cinnamoml Glycyrrhiza uralensis soup, Ephedra sinica is6g, Glycyrrhiza ura-lensis is6g, which is at heavier dosage of Glycyrrhiza uralensis. In Ephedra sinica added Atractylodis Macrocephalae soup, Ephedra sinica is9g, Glycyrrhiza uralen-sis is3g."Disease are consistent to syndromes", using the different ratio of Ephedr-a sinica and Glycyrrhiza uralensis for different syndromes, is a question worth cons-idering concering the connotation of compatibility.
Significance
The clinical efficacy of Chinese herbs depends largely on the compatibility of m-edicine. The herbal pairs, as the smallest constituent units of the Chinese herbal for-mulas and also a series of compounds in common, are simple and effective. So med-icine pairs research, as a starting point, may be beneficial to elucidate its mechanism of actions of formulas, seek the best the ratio of compatibility, and explore the basic effects, and function expansion. Carrying out research on the of compatibility law medicine pairs, is of great significance to reveal the scientific contents of formulas compatibility, mine the theory TCM compatibility, and expand clinical ideas of the treatment, as well as provide clues to the basis for in-depth study of compound com-patibility law, and the basis for the formation of the new formulas. Especially with the increasing of compatibility using active ingredients or effective parts, applying the traditional compatibility theory to guide the new form of compatibility, is of pr-actical significance to promote the new products of traditional Chinese medicine for research and innovation
Ephedra sinica-Glycyrrhiza uralensis is a commonly used pair in the Zhongjing prescriptions, and often used by the modern physicians. To study the pharmacologi-cal interaction effects of this pair, quantitative evaluate compatibility efficiency, seek the best ratio, and study the changes of chemical compositions in vitro and in vivo, reactions in the process of from the material basis, to find the biological markers af-fected by Ephedra sinica, Glycyrrhiza uralensis and Ephedra sinica-Glycyrrhiza uralensis by means of metabolomics, which all would be helpful to explain the compatibility of this pair gradually.
Objectives
To reveal the compatibility connotation of Ephedra sinica-Glycyrrhiza uralens-is herbal pair from the following three levels:
(1) The main aim of sudy to observe acute toxicity, anti-inflammatory effects and diuretic actions of Ephedra sinica, Glycyrrhiza uralensis and three different ratios of Ephedra sinica and Glycyrrhiza uralensis, and make a comprehensive and quantitat- ive evaluation of the interaction between Ephedra sinica and Glycyrrhiza uralensis, using calcusyn software and one-way ANOVA.
(2) To analyze the changes of contents of ephedrine, pseudoephedrine, ephedrine, and methyl pseudo, ephedrine, licorice acid and liquiritin in Ephedra sinica or Glyc-yrrhiza uralensis and different ratios of Ephedra sinica-Glycyrrhiza uralensis, com-pare the links between chemical compositions and ratios, in order toelaborate the ph-armacological effects of changes.
(3) To explore the effects of Ephedra sinica, Glycyrrhiza uralensis and Ephedra sinica-Glycyrrhiza uralensis administrated orally on biomarkers in urine by metabo-lomics technology, and find the biomarkers that may characterize this process of cha-nges, which would provide experimental evidence for the mechanism of interaction between Ephedra sinica and Glycyrrhiza uralensis from system and overall perspec-tive.
Methods
(1) The main pharmacological effects were observed by the methods of the acute toxicity in mice, acetic acid-induced capillary permeability in mice and the effect on urine volume in rats for a period of1h following oral administration of drugs. A co-mbidrug software and t test have been used to analyze the interactions of the differe-nt level in the combinations.
(2) Analysis and determine the best ratio of both synergistic interaction betw-een Ephedra sinica-Glycyrrhiza uralensis. Anti-inflammatory effects of the best ratio of this pair were determined anti-inflammatory effects by utilizing the following mo-dels:xylene-induced ear edema and acetic acid-induced permeability in mice, carrag-eenin-induced edema of the hind paw, carrageenan-induced pleurisy and cotton pell-et-induced granuloma formation in rats. The anti-inflammatory mechanisms were inv- estigated by detecting the contents of prostaglandins (PGE2), tumor necrosis factor-a-lpha (TNF-a), interleukin-1β (IL-1β) in exudates, and the contents of TNF-a, IL-1β, MDA in lung tissue were also detected. HE staining was to observe the effect of Eph-edra sinica-Glycyrrhiza pair on pleurisy in rat lung tissue, and immunohistochemical detection of iNOS and ICAM-1expression in the lung tissue. The observation of Eph-edra sinica-Glycyrrhiza uralensis of PGE2in normal rat urine was to explore the di-uretic mechanism. All mean were compared with One-way ANOVA, data were statis-tically analyzed by SPSS13.0software, P<0.05was statistically significant.
(3) To study the fingerprints peak changes Ephedra sinica alkaloids (ephedrine, pseudoephedrine, methyl ephedrine, methylpseudohedrine, and norpseudoephedrine and normethyl methamphetamine), using gas chromatography-liquid chromatogr-aphy (GC-MS) in Ephedra sinica and the three compatibility ratios, all mean were compared with One-way ANOVA, and liquiritin and glycyrrhizic acid in Glycyrrhiza uralensis and different ratios using high performance liquid chromatography (HPLC), then clarified the relation between differrent proportions and main medicinal compo-nents. Data were statistically analyzed by SPSS13.0software, P<0.05was statistic-ally significant.
(4) Urine was collected in seven days after administration Ephedra sinica, Gly-cyrrhiza uralensis, and Ephedra sinica-Glycyrrhiza uralensis, and then centrifuged and derivatizated. Rat urine was carried out a full scan analysis of endogenous meta-bolites using GC-MS. GC-MS data were processed by the method of peak identific-ation, peak alignment, excluding the solvent peak impurity peak. Exported data (exc-el format) peak area were normalized, and then generated the two-dimensional matr-ix including sample name and a relative peak area. Metabolite spectra was analysised by principal component analysis (PCA) and partial least squares discriminant analy-sis (PLS-DA) using the software Simca-P. PCA was used for intuitive observation of changes and drug treatment of different groups. PLS-DA was used for distinction wi-thin the treated groups. The value of VIP was used for evaluation the correlation de-gree of different substances. VIP value>1was indicated as potential biomarkers, which furtherly analysised using SPSS13.0software. Those biomarkers with signify-cant difference were selected as the final biomarker. The metabolic pathways affectt-ed by Ephedra sinica, Glycyrrhiza uralensis, and Ephedra sinica-Glycyrrhiza urale-nsis were interpretated based on existing knowledge of biochemistry.
Results
(1) Ephedra sinica-Glycyrrhizic uralensis pair of12:3,12:6and12:12pr-oduced toxicity antagonism obviously in a dose-dependent manner associated with Glycyrrhizic uralensis. Ephedra sinica-Glycyrrhizic uralensis of12:3and12:6exerted synergy anti-inflammatory action, when the inhibition rate is25%～35%, a-nd there was significant difference compared to the control group,but12:12show-ed the obvious antagonism. Ephedra sinica-Glycyrrhizic uralensis pair12:3and12:6had close therapeutic index (TI).
(2) Compared with the model group, Ephedra sinica-Glycyrrhizic uralensis pair, at at the doses of22.4,11.2g·kg-1, inhibited xylene-induced ear swelling. This pair22.4g·kg-1produced suppression against carrageenin-induced edema of the hind paw at3,5,7h after carrageenin injection,11.2,5.6g·kg-1this pair had not signi-ficant effects on edema. Ephedra sinica-Glycyrrhizic uralensis pair at the dosage of22.4g·kg-1inhibited cotton pellet-induced granuloma formation in rats. This pair inhibited inhibited the increase of the volume of exudates and total leukocytes in the exudates induced by carrageenan, as well as significantly reduced the levels of PGE2, TNF-a, IL-1β in exudates. Ephedra sinica-Glycyrrhizic uralensis pair also reduced the contents of TNF-a. IL-1β,MDA in lung tissue in carrageenan treated rats, and i-nhibited the damage to lung induced by carrageenan and the expression iNOS in the lung tissue, Ephedra sinica-Glycyrrhiza uralensis herbal pair had no significant effe-ct on ICAM-1expression. Compared with the control group, Ephedra sinica-Glyc-yrrhizic uralensis pair of22.4g·kg-1could significantly increase the urinary output and contents of K+in the rats'urine, increased PGE2excretion in urine.
(3) The amount of Ephedra sinica alkaloids (ephedrine, pseudoephedrine, meth-ylph-edrine, methylpseudohedrine, and norpseudoephedrine and normethyl metham-phetamine) and glycyrrhizic acid, liquiritin, in three ratios of pair, decreased with the increase in the dosage of Glycyrrhizic uralensis. The dosage of Glycyrrhizic uralens-is was negatively correlated with the major effective compositions. The effective co-mpositions contents of Ephedra sinica-Glycyrrhizic uralensis12:12decreased m-ost significantly.
(4) Compared with the control group, the biomarkers down-regulated by Glycyr-rhiza uralensis were α-ketoglutarate, adipic acid and citric acid; those up-regulated were aminohippuric acid, methyl xanthine andtryptophan, biomarkers up-regulated by Ephedra sinica were alanine and tryptophan, those down-regulated by Ephedra sinica-Glycyrrhiza uralensis were α-ketoglutaric acid, adipic acid, aconitic acid, cit-ric acid, and cysteine, those up-regulated were alanine, high citric acid, methyl xant-hine, tyrosine and tryptophan.
Conclusion
The relationship main between pharmacological interaction effects of Ephedra sinica-Glycyrrhizic uralensis and the content changes of active ingredient in diff-erent ratios is obvious. With the level of main chemical ingredients in three ratios of pair decreased, the main pharmacyological effects showed antagonism within a cer-tain range. Ephedra sinica-Glycyrrhizic uralensis drug compatibility can increase the efficiency and reduce toxicity within the appropriate dose, and the ratio of12:3and12:6showed better compatibility advantage with wider synergistic range. Ep- hedra sinica-Glycyrrhizic uralensis herbal pair (12:6) possess significant anti-inf-lammatory effects, which can be related with the inhibition of the generation of me-diators of inflammation and its antioxidation. The diuretic mechanism of action may be attributed to suppress PGE2release in urine. Ephedra sinica, Glycyrrhizic uralen-sis affected different metabolic markers. Ephedra sinica-Glycyrrhizic uralensis also affected the other three kinds of markers, in addition those affected by Ephedra sini-ca and Glycyrrhizic uralensis sololy, which suggest that there is a synergyistic actio-n between Ephedra sinica and Glycyrrhizic uralensis.
本文为国家自然科学基金重点项目-麻黄类药对组成规律的基础研究的部分研究内容。该项目以风寒表实证为主线,以麻黄为主药,从张仲景经典名方中选择针对主证为风寒表实证但不同兼证的麻黄汤及其类方中的六个药对进行组成规律研究,探讨药味、药量的改变与化学成分、药理效应、药代动力学和药物代谢组学之间的相互关系；同时纵向分析六个药对组合与风寒表实证遣方用药的内在联系,探讨药对变化的内涵和组成规律,促进中医药学遣方用药理论的发展。
麻黄-甘草药对为上述六个药对之一,亦为《金匮要略》所载用于表寒兼皮水的甘草麻黄汤,有利水消肿之功,主治身体面目悉肿,腰以上肿甚,身微热,口不渴,无汗,小便不利者。方中麻黄宣发卫阳,散表祛邪,使风邪水气从外而解,又可使在里之水从小便而利；甘草和调中气、调和药性。两药合用,辛甘发散以利水,健脾和中以制水。本方中麻黄12g、甘草6g,该剂量及配比在张仲景麻黄、甘草并用的方剂中最为常用,但并不是一成不变的,如在大青龙汤、麻黄杏仁甘草石膏汤、越婢汤中,麻黄用12g,甘草用6g；在麻黄附子甘草汤中,麻黄用6g、而甘草为6g,甘草用量较重；在麻黄加术汤中,麻黄用9g、而甘草为3g。“病皆与方相应”,麻黄-甘草药对针对不同主治证候采用不同的配伍比例,这也是分析其配伍内涵探讨的问题。
本文运用现代药理学方法来研究麻黄和甘草药理效应的相互作用,定量评价药对是否存在配伍增效或配伍减毒,寻求最佳配比；同时研究其在体外过程中发生各种物理化学反应后主要化学成分的变化,从物质基础寻找量变到质变的根本所在；用代谢组学的方法观察麻黄和甘草药对配伍对生物标志物的影响,从代谢组学层面表征该药对的配伍内涵。
研究意义
复方临床疗效的发挥,很大程度上取决于中药的配伍,而药对作为中药配伍应用最为常见的形式,它构成简单、疗效确切,且具备复方基本主治功能,是中药复方的最小组成单位,同时也是系列复方的共同点；药对反映复方配伍的特殊规律与内在联系。因此,以药对研究为切入点,阐明其配伍机制,寻求发挥最佳作用的配伍比例,有利于探索与发掘药对的基本功用与功用扩展。开展药对配伍规律研究,能进一步揭示药对配伍的客观规律与科学内涵,促进中药配伍应用理论的进步；能有效地指导临床遣方用药,既利于更好运用已有的药对,亦利于针对疾病谱的变化、病证的发展而创制新药对；特别是在新药研究中以有效成分或有效部位配伍已越来越多的今天,以传统配伍的普遍规律指导新的配伍形式,对促进中药新产品研创亦具有现实意义。
研究目的
本文从以下三个层面揭示麻黄-甘草药对配伍内涵：
(1)观察麻黄-甘草药对不同配比的急性毒性、抗炎、利尿三个方面的药理效应,应用中效原理进行相互作用统计分析,定量评价麻黄和甘草主要药理效应的相互作用,以判断药物是否增效减毒。
(2)对不同配比水煎液麻黄类生物碱(麻黄碱、伪麻黄碱、去甲基麻黄碱、去甲基伪麻黄碱和甲基麻黄碱)和甘草酸、甘草苷含量进行研究,比较不同配比对这些化学成分含量的影响,以期从物质基础的变化来阐述药理效应变化的内因。
(3)运用代谢组学技术探讨大鼠灌胃麻黄、目草和麻黄-甘草药对后对尿液中生物标志物的影响,寻找表征这一变化过程的生物标志物,从系统与整体层面探讨麻黄配伍甘草的相互作用机制。
研究方法
(1)主要药理效应变化采用小鼠半数致死量(LD50)测定来评价麻黄、甘草及麻黄-甘草药对三个配比的急性毒性；以醋酸致小鼠毛细血管通透性增加实验观察抗炎作用；以对正常大鼠的排尿量的影响来观察利尿作用,应用calcusyn软件或单因素方差分析(One-way ANOVA)评价麻黄和甘草的相互作用。
(2)根据麻黄-甘草的相互作用关系确定两者协同作用的最佳配比,利用该配比进行二甲苯致小鼠耳肿胀、角叉菜胶致大鼠足肿胀、角叉菜胶致大鼠胸膜炎三个急性炎症实验和大鼠棉球肉芽肿亚急性炎症实验,进一步明确麻黄-甘草药对的抗炎作用；利用角叉菜胶致大鼠胸膜炎实验观察麻黄-甘草药对对胸膜炎大鼠HE染色肺组织的病理学影响；免疫组化检测iNOS和ICAM-1在肺组织的表达情况；测定胸膜炎大鼠胸腔渗出液前列腺素E2(PGE2)、肿瘤坏死因子-α (TNF-α)和白细胞介素-1p(IL-1β)含量以及肺组织TNF-α IL-1β和丙二醛(MDA)含量来探讨抗炎机制。观察麻黄-甘草药对对正常大鼠尿液中PGE2的影响,探讨利尿作用机制。所有均数比较用One-way ANOVA,数据由SPSS13.0软件进行统计学处理,以P<0.05为差异有统计学意义。
(3)采用气质-液质(GC-MS)联用考察麻黄水煎液及麻黄-甘草药对不同配比共煎液中麻黄类生物碱(麻黄碱、伪麻黄碱、去甲基麻黄碱、去甲基伪麻黄碱和甲基麻黄碱)指纹图谱峰的变化情况,所有均数比较用One-way ANOVA;采用高效液相色谱法(HPLC)考察甘草水煎液及麻黄-甘草药对不同配比共煎液中甘草苷和甘草酸的指纹图谱峰的变化情况；以明确不同配比对主要药效成分的影响,均数比较采用析因设计资料的方差分析。所有数据由SPSS13.0软件进行统计学处理,以P<0.05为差异有统计学意义。
(4)麻黄水煎液、甘草水煎液、麻黄-甘草药对水煎液给大鼠灌胃,于第7天收集尿液,尿液离心后进行衍生化；采用GC-MS对大鼠尿液中代谢物进行全扫描分析。将所得的GC-MS数据进行峰识别、峰对齐、扣除溶剂峰、杂质峰的处理。导出的数据进行峰面积归一化处理,生成包括样品名称、归一化峰面积的二维矩阵。利用Simca-P软件对大鼠的尿液代谢物谱进行主成分分析(PCA)和偏最小二乘法判别分析(PLS-DA), PCA直观观察不同组的差异；PLS-DA进一步考察组间的区分程度,并根据代谢物的投影重要性(VIP)评价不同物质与分类的相关性程度,以VIP值>1的代谢物作为潜在生物标志物。运用SPSS13.0软件对潜在生物标志物进行统计分析,选择差异有统计学意义的物质作为最终的生物标志物。通过与空白对照组尿液的代谢物谱比较,研究麻黄水煎液、甘草水煎液和麻黄-甘草药对(12：6)水煎液对大鼠内源性代谢标志物的影响；结合现有的生物化学知识阐释麻黄、甘草和麻黄-甘草药对所影响的代谢途径。
主要研究结果
(1)麻黄-甘草药对(12：3)、(12：6)和(12：12)均表现出毒性拮抗作用,且拮抗作用与甘草用量有明显剂量依赖关系。麻黄-甘草药对(12：3)配伍,当抑制率在25%～70%,抗炎协同作用明显,且与空白对照组比较,抗炎作用差异有统计学意义；抑制率高于70%时表现出拮抗作用。(12：6)抗炎抑制率在25%～70%协同作用明显,且与空白对照组比较,抗炎作用差异性有统计学意义。(12：12)有明显抗炎拮抗作用。麻黄-甘草药对(12：3)和(12：6),治疗指数(TI)接近。
(2)与模型对照组比较,麻黄-甘草药对(12：6)(22.4,11.2g·kg-1)2个剂量组均能抑制二甲苯所致的小鼠耳肿胀(P<0.05)；22.4g·kg-1能抑制角叉菜胶注射后5h,7h的大鼠足肿胀(P<0.05),11.2,5.6g·kg-1对大鼠足肿胀度没有抑制作用(P>0.05)；22.4g·kg-1能抑制大鼠棉球肉芽肿形成(P<0.05)；麻黄-甘草药对22.4g·kg-1能减轻角叉菜胶对肺组织的病理损伤、抑制iNOS在肺支气管上皮细胞的表达(P<0.05)、对ICAM-1的表达没有显著影响(P>0.05)；药对能抑制角叉菜胶诱导的胸膜炎大鼠胸腔液渗出P<0.05)、抑制渗出液白细胞数量增多以及渗出液PGE2、TNF-α和IL-1β含量升高、抑制肺组织TNF-α、IL-1β和MDA含量升高(所有P<0.05);麻黄-甘草药对能增加正常大鼠排尿量及尿液中K+的排泄(P<0.05)；增加尿液中PGE2排泄(P<0.05)。
(3)麻黄-甘草药对配伍,随着甘草用量的增加,麻黄类生物碱(麻黄碱、伪麻黄碱、去甲基麻黄碱、去甲基伪麻黄碱和甲基麻黄碱)和甘草酸、甘草苷含量均下降；甘草用量与7个成分的含量呈负相关,麻黄-甘草药对(12：12)各成分下降最明显。
(4)与空白对照组比较,甘草组存在差异的生物标志物有：α-酮戊二酸、己二酸、柠檬酸、氨基马尿酸、甲基黄嘌呤、色氨酸,水平显著下调的有：α-酮戊二酸、己二酸、柠檬酸；生物标志水平显著上调的有氨基马尿酸、甲基黄嘌呤、色氨酸。麻黄组存在差异上调的生物标志物：丙氨酸、色氨酸。麻黄-甘草药对组与空白对照组比较,存在差异的生物标志物有：α-酮戊二酸、己二酸、乌头酸、柠檬酸、丙氨酸、半胱氨酸、甲基黄嘌呤、酪氨酸、色氨酸；水平显著下调的有α-酮戊二酸、己二酸、乌头酸、柠檬酸、半胱氨酸；显著上调的有丙氨酸、甲基黄嘌呤、酪氨酸、色氨酸。
结论
麻黄-甘草药对主要药理效应的相互关系与两者配伍后主要有效成分含量的改变有相关性,合煎后主要化学成分群含量降低,主要药理效应会在一定范围内表现出拮抗作用；麻黄-甘草药对配伍,在合适的剂量和配比范围内,能增效减毒；(12：3)和(12：6)协同范围较宽,呈现配伍优势。麻黄-甘草药对有抗炎作用,抗炎机制可能与影响炎症介质产生和抗氧化作用有关；利尿作用机制与促进尿液PGE2排泄有关。麻黄、甘草影响的代谢标志物不同,麻黄-甘草药对除了影响两者单独作用的代谢标志物外,还影响了另外3种标志物,从系统的层面表明麻黄和甘草存在协同作用。
Background
This study is part of basic research on the composition of the law of Ephedra sinica herbal pairs, funded by the National Natural Science Foundation. The main line of the project is Feng, Han Biao and Shi syndromes, Ephedra sinica is taken as the major medicine, we choosed six herbal pairs which used for different accompan-ied symptoms from the Zhang Zhongjing classic prescriptions, to explore the comp-ositions of the law, and the relationship between the changes of compatibility or dos-ages and the contents of compositions and the pharmacyological effects, pharmacyo-kinetics or drug metabolism genomics, and then longitudinal analyse the internal rel-ations of six herbal pairs with prescriptions, and observe how symptoms, diagnosis and treatment of Feng, Han, Biao Shi to be affected by medicines compatibility and their dosages. These would be beneficcial to reveal the meanings and laws of the sy-stem of Traditional Chinese herbal pairs, then sum up the universal law of medicine pairs composition, and promote the development of the theory of Chinese medicine prescription.
Ephedra sinica-Glycyrrhiza uralensis is one of six herbal pairs studied, which is selected from Glycyrrhiza uralensis-Ephedra sinica soup used for Feng, Han, an-d Pi Shui sydomes. Glycyrrhiza uralensis-Ephedra sinica soup is derived from "Go-lden Chamber", consists of Ephedra sinica and Glycyrrhiza uralensis.The, utility of this soup is to eliminate the water swelling, and used for the treatment of inner Shu-i, the body and face swollen, even above the waist, body fever, thirst, sweat, disadva-ntage of a urinary. The actions of Ephedra sinica are to XuanFa Wei Yang, Sanbiao QuXie, make FengXie eliminated from the outside and eliminated Lishui from the u-rine, the actions of Glycyrrhiza uralensis are to regulate ZhongQi and reconcile med-icine properties. The two combined medicines exert the actions of FaSan LiShui. In this prescription, Ephedra sinica is12g, Glycyrrhiza uralensis is6g, which is the most commonly used doses and ratio in Zhongjing prescriptions, but not set in stone, such as in the big dragon soup, Ephedra sinica-Glycyrrhiza uralensis soup and Yue pi soup, Ephedra sinica is12g, Glycyrrhiza uralensis is6g. In Ephedra sinica Ram-ulus Cinnamoml Glycyrrhiza uralensis soup, Ephedra sinica is6g, Glycyrrhiza ura-lensis is6g, which is at heavier dosage of Glycyrrhiza uralensis. In Ephedra sinica added Atractylodis Macrocephalae soup, Ephedra sinica is9g, Glycyrrhiza uralen-sis is3g."Disease are consistent to syndromes", using the different ratio of Ephedr-a sinica and Glycyrrhiza uralensis for different syndromes, is a question worth cons-idering concering the connotation of compatibility.
Significance
The clinical efficacy of Chinese herbs depends largely on the compatibility of m-edicine. The herbal pairs, as the smallest constituent units of the Chinese herbal for-mulas and also a series of compounds in common, are simple and effective. So med-icine pairs research, as a starting point, may be beneficial to elucidate its mechanism of actions of formulas, seek the best the ratio of compatibility, and explore the basic effects, and function expansion. Carrying out research on the of compatibility law medicine pairs, is of great significance to reveal the scientific contents of formulas compatibility, mine the theory TCM compatibility, and expand clinical ideas of the treatment, as well as provide clues to the basis for in-depth study of compound com-patibility law, and the basis for the formation of the new formulas. Especially with the increasing of compatibility using active ingredients or effective parts, applying the traditional compatibility theory to guide the new form of compatibility, is of pr-actical significance to promote the new products of traditional Chinese medicine for research and innovation
Ephedra sinica-Glycyrrhiza uralensis is a commonly used pair in the Zhongjing prescriptions, and often used by the modern physicians. To study the pharmacologi-cal interaction effects of this pair, quantitative evaluate compatibility efficiency, seek the best ratio, and study the changes of chemical compositions in vitro and in vivo, reactions in the process of from the material basis, to find the biological markers af-fected by Ephedra sinica, Glycyrrhiza uralensis and Ephedra sinica-Glycyrrhiza uralensis by means of metabolomics, which all would be helpful to explain the compatibility of this pair gradually.
Objectives
To reveal the compatibility connotation of Ephedra sinica-Glycyrrhiza uralens-is herbal pair from the following three levels:
(1) The main aim of sudy to observe acute toxicity, anti-inflammatory effects and diuretic actions of Ephedra sinica, Glycyrrhiza uralensis and three different ratios of Ephedra sinica and Glycyrrhiza uralensis, and make a comprehensive and quantitat- ive evaluation of the interaction between Ephedra sinica and Glycyrrhiza uralensis, using calcusyn software and one-way ANOVA.
(2) To analyze the changes of contents of ephedrine, pseudoephedrine, ephedrine, and methyl pseudo, ephedrine, licorice acid and liquiritin in Ephedra sinica or Glyc-yrrhiza uralensis and different ratios of Ephedra sinica-Glycyrrhiza uralensis, com-pare the links between chemical compositions and ratios, in order toelaborate the ph-armacological effects of changes.
(3) To explore the effects of Ephedra sinica, Glycyrrhiza uralensis and Ephedra sinica-Glycyrrhiza uralensis administrated orally on biomarkers in urine by metabo-lomics technology, and find the biomarkers that may characterize this process of cha-nges, which would provide experimental evidence for the mechanism of interaction between Ephedra sinica and Glycyrrhiza uralensis from system and overall perspec-tive.
Methods
(1) The main pharmacological effects were observed by the methods of the acute toxicity in mice, acetic acid-induced capillary permeability in mice and the effect on urine volume in rats for a period of1h following oral administration of drugs. A co-mbidrug software and t test have been used to analyze the interactions of the differe-nt level in the combinations.
(2) Analysis and determine the best ratio of both synergistic interaction betw-een Ephedra sinica-Glycyrrhiza uralensis. Anti-inflammatory effects of the best ratio of this pair were determined anti-inflammatory effects by utilizing the following mo-dels:xylene-induced ear edema and acetic acid-induced permeability in mice, carrag-eenin-induced edema of the hind paw, carrageenan-induced pleurisy and cotton pell-et-induced granuloma formation in rats. The anti-inflammatory mechanisms were inv- estigated by detecting the contents of prostaglandins (PGE2), tumor necrosis factor-a-lpha (TNF-a), interleukin-1β (IL-1β) in exudates, and the contents of TNF-a, IL-1β, MDA in lung tissue were also detected. HE staining was to observe the effect of Eph-edra sinica-Glycyrrhiza pair on pleurisy in rat lung tissue, and immunohistochemical detection of iNOS and ICAM-1expression in the lung tissue. The observation of Eph-edra sinica-Glycyrrhiza uralensis of PGE2in normal rat urine was to explore the di-uretic mechanism. All mean were compared with One-way ANOVA, data were statis-tically analyzed by SPSS13.0software, P<0.05was statistically significant.
(3) To study the fingerprints peak changes Ephedra sinica alkaloids (ephedrine, pseudoephedrine, methyl ephedrine, methylpseudohedrine, and norpseudoephedrine and normethyl methamphetamine), using gas chromatography-liquid chromatogr-aphy (GC-MS) in Ephedra sinica and the three compatibility ratios, all mean were compared with One-way ANOVA, and liquiritin and glycyrrhizic acid in Glycyrrhiza uralensis and different ratios using high performance liquid chromatography (HPLC), then clarified the relation between differrent proportions and main medicinal compo-nents. Data were statistically analyzed by SPSS13.0software, P<0.05was statistic-ally significant.
(4) Urine was collected in seven days after administration Ephedra sinica, Gly-cyrrhiza uralensis, and Ephedra sinica-Glycyrrhiza uralensis, and then centrifuged and derivatizated. Rat urine was carried out a full scan analysis of endogenous meta-bolites using GC-MS. GC-MS data were processed by the method of peak identific-ation, peak alignment, excluding the solvent peak impurity peak. Exported data (exc-el format) peak area were normalized, and then generated the two-dimensional matr-ix including sample name and a relative peak area. Metabolite spectra was analysised by principal component analysis (PCA) and partial least squares discriminant analy-sis (PLS-DA) using the software Simca-P. PCA was used for intuitive observation of changes and drug treatment of different groups. PLS-DA was used for distinction wi-thin the treated groups. The value of VIP was used for evaluation the correlation de-gree of different substances. VIP value>1was indicated as potential biomarkers, which furtherly analysised using SPSS13.0software. Those biomarkers with signify-cant difference were selected as the final biomarker. The metabolic pathways affectt-ed by Ephedra sinica, Glycyrrhiza uralensis, and Ephedra sinica-Glycyrrhiza urale-nsis were interpretated based on existing knowledge of biochemistry.
Results
(1) Ephedra sinica-Glycyrrhizic uralensis pair of12:3,12:6and12:12pr-oduced toxicity antagonism obviously in a dose-dependent manner associated with Glycyrrhizic uralensis. Ephedra sinica-Glycyrrhizic uralensis of12:3and12:6exerted synergy anti-inflammatory action, when the inhibition rate is25%～35%, a-nd there was significant difference compared to the control group,but12:12show-ed the obvious antagonism. Ephedra sinica-Glycyrrhizic uralensis pair12:3and12:6had close therapeutic index (TI).
(2) Compared with the model group, Ephedra sinica-Glycyrrhizic uralensis pair, at at the doses of22.4,11.2g·kg-1, inhibited xylene-induced ear swelling. This pair22.4g·kg-1produced suppression against carrageenin-induced edema of the hind paw at3,5,7h after carrageenin injection,11.2,5.6g·kg-1this pair had not signi-ficant effects on edema. Ephedra sinica-Glycyrrhizic uralensis pair at the dosage of22.4g·kg-1inhibited cotton pellet-induced granuloma formation in rats. This pair inhibited inhibited the increase of the volume of exudates and total leukocytes in the exudates induced by carrageenan, as well as significantly reduced the levels of PGE2, TNF-a, IL-1β in exudates. Ephedra sinica-Glycyrrhizic uralensis pair also reduced the contents of TNF-a. IL-1β,MDA in lung tissue in carrageenan treated rats, and i-nhibited the damage to lung induced by carrageenan and the expression iNOS in the lung tissue, Ephedra sinica-Glycyrrhiza uralensis herbal pair had no significant effe-ct on ICAM-1expression. Compared with the control group, Ephedra sinica-Glyc-yrrhizic uralensis pair of22.4g·kg-1could significantly increase the urinary output and contents of K+in the rats'urine, increased PGE2excretion in urine.
(3) The amount of Ephedra sinica alkaloids (ephedrine, pseudoephedrine, meth-ylph-edrine, methylpseudohedrine, and norpseudoephedrine and normethyl metham-phetamine) and glycyrrhizic acid, liquiritin, in three ratios of pair, decreased with the increase in the dosage of Glycyrrhizic uralensis. The dosage of Glycyrrhizic uralens-is was negatively correlated with the major effective compositions. The effective co-mpositions contents of Ephedra sinica-Glycyrrhizic uralensis12:12decreased m-ost significantly.
(4) Compared with the control group, the biomarkers down-regulated by Glycyr-rhiza uralensis were α-ketoglutarate, adipic acid and citric acid; those up-regulated were aminohippuric acid, methyl xanthine andtryptophan, biomarkers up-regulated by Ephedra sinica were alanine and tryptophan, those down-regulated by Ephedra sinica-Glycyrrhiza uralensis were α-ketoglutaric acid, adipic acid, aconitic acid, cit-ric acid, and cysteine, those up-regulated were alanine, high citric acid, methyl xant-hine, tyrosine and tryptophan.
Conclusion
The relationship main between pharmacological interaction effects of Ephedra sinica-Glycyrrhizic uralensis and the content changes of active ingredient in diff-erent ratios is obvious. With the level of main chemical ingredients in three ratios of pair decreased, the main pharmacyological effects showed antagonism within a cer-tain range. Ephedra sinica-Glycyrrhizic uralensis drug compatibility can increase the efficiency and reduce toxicity within the appropriate dose, and the ratio of12:3and12:6showed better compatibility advantage with wider synergistic range. Ep- hedra sinica-Glycyrrhizic uralensis herbal pair (12:6) possess significant anti-inf-lammatory effects, which can be related with the inhibition of the generation of me-diators of inflammation and its antioxidation. The diuretic mechanism of action may be attributed to suppress PGE2release in urine. Ephedra sinica, Glycyrrhizic uralen-sis affected different metabolic markers. Ephedra sinica-Glycyrrhizic uralensis also affected the other three kinds of markers, in addition those affected by Ephedra sini-ca and Glycyrrhizic uralensis sololy, which suggest that there is a synergyistic actio-n between Ephedra sinica and Glycyrrhizic uralensis.
引文
1.刘阳.《金匮要略》中麻黄的配伍用药尺度探讨[J].中医药临床杂志,2006,18(5)：427-428.
2.孙冰,邓家刚.施今墨对药应用平性药配伍的规律探讨[J].山东中医药大学学报,2009,33(6)：460-463.
3.耿建国,王代娣.《伤寒论》药对配伍规律与特点[J].江苏中医,2000,21(9)：6-7.
4.吕景山.施今墨药对[M].第3版,北京：人民军医出版社,2005：57-60.
5.陈维华.药对论[M].合肥：安徽科学技术出版社,1989.
6.苏庆英.中医临床常用对药配伍[M].北京：人民卫生出版社,2010.
7.邢袁玲.《伤寒论》中四气五味及特殊药对探析[J].中国医药导报,2009,6(13)：111-113.
8.刘萍,王平,刘松林,等.基于药对探讨中药复方配伍规律的思考[J].中华中医药学刊,2010,28(9)：1833-1835.
9.杨敏,田岩.仲景药对配伍特点浅析[J].辽宁中医学院学报,2005,7(6)：560-561.
10.杨巨成.“药对”在中医方剂配伍中的重要性[J].中国民间疗法,2010,18(1)：55.
11.高晓山.中药药性论[M].北京：人民出版社,1992：167-177.
12.张宇燕,杨洁红.附子甘草配伍对乌头碱、甘草酸、甘草苷的动态影响[J].中国药学杂志,2009,44(1)：11-14.
13.越皓,皮子凤,宋凤瑞,等.附子不同配伍药对中生物碱成分的电喷雾质谱分析[J].药学学报,2007,42(2)：201-205.
14.涂瑶生,刘法锦,孙冬梅,等.黄连、吴茱萸配伍药对的HPLC指纹图谱研究[J].中成药,2011,33(1)：5-9.
15.毛幼儿,周桂芬.大黄与芒硝药对不同比例配伍对蒽醌苷含量的影响[J].江西中医药,2011,42(338)：60-61.
16.史琪荣,于少云,孙晓迪,等.黄连干姜药对对功能性消化不良大鼠胃排空和血清胃泌素的影响[J].中国药学杂志,2011,46(13)：988-992.
17.夏伯候,王智民,林丽美,等.银翘药对的药效学研究进展[J].中国实验方剂学杂志, 2009,15(3)：80-82.
18.王敏,刘彬,齐云,等.甘草皂苷对桔梗皂苷急性毒性的影响研究[J].中国实验方剂学杂志,2008,14(1)：59-61.
19.梁日欣,黄璐琦,刘菊福,等.药对川芎和赤芍对高脂血症大鼠降脂、抗氧化及血管内皮功能的实验观察[J].中国实验方剂学杂志,2002,8(1)：34.
20.郡顺琴,凤良元,黄德武,等.枳术丸对胃排空肠推进作用的影响[J].中成药,1996,18(4)：30-31.
21.襄萍,徐朝晖,战光绪,等.对药麻黄地龙配比及平喘作用机制的研究[J].中国中药杂志,2006,31(3)：236-239.
22.柳润辉,苏娟,徐希科,等.药对“丹参-黄芪”有效组分的最佳配伍配比研究[J].中国药学杂志,2006,41(11)：815-817.
23.杨祖贻,裴瑾,刘荣敏,等.当归肉桂配伍后阿魏酸生物利用度的研究[J].中国中药杂志,2006,31(12)：1012-1015.
24.王静,袁子民,张振秋.黄连、吴茱萸药对中盐酸小檗碱在大鼠体内的药代动力学研究[J].药物分析杂志,2009,29(5)：755-759.
25.王文萍,曹琦琛,高晶晶,等.六一散配伍规律的药动学研究[J].中国实验方剂学杂志,2009,15(12)：70-72.
26.何薇,荆雷,刘树民,等.黄连解毒汤中君-使药对黄连-栀子干预2,4-二硝基苯酚热病证候模型的代谢组学[J].中国实验方剂学杂志,2011,17(20)：173-178.
27.薛蕾.袁金声教授常用药对举隅[J].辽宁中医药大学学报,2012,14(2)：140-141.
28.王冠华,严志林.王少华治疗咳喘常用对药探微[J].辽宁中医药大学学报,2011,13(2)：11-12.
29.李杭,王荣欣.治疗糖尿病肾病常用药对举隅[J].光明中医,2012,27(2)：385-386
30.王咏梅,马红,刘苏中.方剂配伍的模糊数学特性研究[J].中国实验方剂学杂志,2000,6(6)：59-60.
31.李振岳,周怡.伤寒病复方药对配伍规律的关联规则分析[J].医学信息,2009,22(5)：591-593.
32.赵蔡斌,周鲁,付超,等.中药复方的模糊分析[J].中国实验方剂学杂志,2003,9(2)：62-63.
33.黄洁,马荣华,闻晓东,等.桃仁-红花药对不同配比的应用数据分析[J].中国新药杂志,2012,21(4)：407-411.
34. Loewe S, Muischnek H. Effect of combinations:mathematical basis of the problem[J]. Arch Exp PatholPharmacol,1926,114 (2):313-326.
35. Gebharto F. Commentson the isobolemethod foranalysis of drug interactions[J]. Pain,1992, 51(2):381.
36.郑青山.复方药物设计与药物相互作用定量分析方法学[c].第四届定量药理研究方法学研讨会,2010.
37. Tallarida RJ. Drug synergism:its detection and applications[J]. J Pharmacol Exp Ther, 2001,298(3):865-872.
38. Lorenzo JI, Sanchez-Marin P. Comments on "Isobolographic analysis for combinations of a full and partial agonist:curved isoboles[J]. J Pharmacol Exp Ther,2006,316(1):476-478.
39. Grabovsky Y, Tallarida RJ. Isobolographic analysis for combinations of a full and partial ag-Onist:curved isoboles[J]. J Pharmacol Exp Ther,2004,310(3):981-986.
40. Lelas S, Rowlett JK, Spealman RD. Isobolographic analysis of chlordiazepoxide and triazo-lamcombinations in squirrel monkeys discriminating triazolam[J]. Psychopharmacology (Berl).2001,158(2):181-189.
41. Adams JU, Tallarida RJ, Geller EB, et al. Isobolographic superadditivity between delta and mu opioid agonists in the rat depends on the ratio of compounds, the mu agonist and the an-algesic assay used[J]. J Pharmacol Exp Ther,1993,266(3):1261-1267.
42. Tallarida RJ. The interaction index:a measure of drug synergism[J]. Pain,2002,98(1/2): 163-168.
43. Chang TT, Gulati SC, Chou TC, et al. Synergistic effect of 4-hydroperoxycyclophosphami-de and etoposide on a human promyelocytic leukemia cell line (HL-60) demonstrated by c- omputer analysis[J]. Cancer Res,1985,45(6):2434-2439.
44. Chou TC. The mass-action law based algorithm for cost-effective approach for cancer drug discovery and development[J]. Am J Cancer Res,2011,1(7):925-954.
45. Chou TC. The mass-action law based algorithms for quantitative econo-green bio-research [J]. Integr Biol (Camb).2011,3(5):548-559.
46. Marvania B, Lee PC, Chaniyara R, etal. Design, synthesis and antitumor evaluation of phe-nyl N-mustard-quinazoline conjugates[J]. Bioorg Med Chem,2011,19(6):1987-1998.
47. Chou TC. Drug combination studies and their synergy quantification using the Chou-Talala-y method[J]. Cancer Res,2010,70(2):440-446.
48. Vathsala A, Chou TC, Kahan BD. Analysis of the interactions of immunosuppressive drugs with cyclosporine in inhibiting DNA proliferation[J]. Transplantation,1990,49(2):463-472.
49. Chou TC. Assessment of synergistic and antagonistic effects of chemotherapeutic agents in vitro[J]. Contrib Gynecol Obstet,1994,19:91-107.
50. Chou TC, Tan QH, Sirotnak FM. Quantitation of the synergistic interaction of edatrexate and cisplatin in vitro[J]. Cancer Chemother Pharmacol,1993,31(4):259-264.
51. Ogawa S, Kamijima T, Miyamoto Y, et al. A new attempt to solve the scale-up problem for granulation using response surface methodology [J]. J Pharm Sci,1994,83(3):439-443.
52. Greco WR, Park HS, Rustum YM. Application of a new approach for the quantitation of drug synergism to the combination of cisdiamminedi chloroplatinum and 1-beta- D-ara-binofuranosylcytosine[J]. Cancer Res,1990,50(17):5318-5327.
53. Lee SI. Drug interaction:focusing on response surface models[J]. Korean J Anesthesiol, 2010,58(5):421-434.
54. Ramakrishnan R, Jusko WJ. Interactions of aspirin and salicylic acid with prednisolone for inhibition of lymphocyte proliferation[J]. Int Immunopharmacol,2001,1(11):2035-2042.
55. Creco WR. Park HS. Rustum YM. Application of a new approach for the quantitation of drug synergism to the combination of cisdiaminedichloroplatinum and 1-D-arabinofura-Nosyl- cytosine[J]. Cancer Res,1990,50:5318-5327.
56.张玉霞,陈素,刘向明.分析药物相互作用的方法在中药研究中的应用[J].上海生物医学工程杂志,2005,26(3)：156-159.
57. Hu Y, Li DM, Han XD. Analysis of combined effects of nonylphenol and Monobutyl phthalate on rat Sertoli cells applying two mathematical models[J]. Food Chem Toxicol, 2012,50(3-4):457-463.
58. Stergiopoulou T, Meletiadis J, Sein T, et al. Synergistic interaction of the triple combination of amphotericin B, ciprofloxacin, and polymorphonuclear neutrophils against Aspergillus fumigates[J]. Antimicrob Agents Chemother,2011,55(12):5923-5929.
59. Boucher AN, Tam VH. Mathematical formulation of additivity for antimicrobial agents[J]. Diagn Microbiol Infect Dis,2006,55(4):319-325.
60. Dressler V, Muller G, Suhnel J. CombiTool-a new computer program for analyzing comb-ination experiments with biologically active agents[J]. Comput Biomed Res,1999, 32(2):145-160.
61. Tam VH, Schilling AN, Lewis RE, et al. Novel approach to characterization of combined pharmacodynamic effects of antimicrobial agents[J]. Antimicrob Agents Chemother,2004, 48(11):4315-4321.
62. Lee JJ, Kong M, Ayers GD, et al. Interaction index and different methods for determining drug interaction in combination therapy [J]. J Biopharm Stat,2007,17:461-480.
63.芦娜,刘振佳,闫征,等.三种药物相互作用数学模型分析SAHA与三氧化二砷联合用药的细胞毒作用[J].药学学报,2010,45(5)：601-607.
64.金正均.合并用药中的相加[J].中国药理学报,1980,1(2)：70-76.
65. Veldstrah. Syneergism and potentiation with special reference to the combination of structu-ral analogues [J]. Pharmacol Rev,1956,8(3):339-387.
66.周元晏,王宏镛,唐朝广,等.衡量合并用药效应得两个新公式-兼论Burgi公式及金式修正公式[J].中国药理学报,1984,5：217-221.
67.顾兵,王心如.联合作用特征的评价[J].中国工业医学杂志,2000,13(1)：55-58.
68.郑青山,孙瑞元.药物相互作用动力学分析方法及其CoDrug软件[J].中国临床药理学与治疗学,2002,7(1)：81.
69. Miaskowski C, Sutters KA, Taiwo YO, et al. Antinociceptive and motor effects of delta/mu and kappa/mu combinations of intrathecal opioid agonists[J]. Pain,1992,49(1):137-144.
70. Miaskowski C, Sutters KA, Taiwo YO, et al. Comparison of the antinociceptive and motor effects of intrathecal opioid agonists in the rat[J]. Brain Res,1991,553(1):105-109.
71. Franck LS, Miaskowski C, Putris J, et al. Dissociation of antinociceptive and motor effects of supraspinal opioid agonists in the rat[J]. Brain Res,1991,563(1-2):123-126.
72. Weckwerth W. Metabolomics in systems biology[J]. Annu Rev Plant Biol,2003,54:669-689.
73. Nicholson JK, Wilson ID. Opinion:understanding 'global' systems biology:metabonom-ics and the continuum of metabolism[J]. Nat Rev Drug Discov,2003,2(8):668-676.
74. Fiehn O. Metabolomics-the link between genotypes and phenotypes[J]. Plant Mol Biol, 2002,48(1-2):155-171.
75. Dunn WB, Bailey NJ, Johnson HE. Measuring the metabolome:current analytical techno-logies[J]. Analyst,2005,130(5):606-625.
76. Zhang A, Sun H, Wang P, et al. Modern analytical techniques in metabolomics analysis[J]. Analyst,2012,137(2):293-300.
77.李仪奎.中药药理实验方法学[M].第二版.上海：上海科学技术出版社,2006：1001-1007.
78. Whittle BA.The use of changes in capillary permeability in mice to distinguish between narcotic and nonnarcotic analgesics[J]. Br J Pharmacol Chemother,1964,22:246-253.
79. Lahlo S, Tahraoui A, Israili Z, et al. Diuretic activity of the aqueous extracts of Carum carviand Tanacetum vulgare in normal rats[J]. J Ethnopharmacol,2007,110(3):458-463.
80. Romay C, Ledon N, Gonzalez R. Further studies on anti-inflammatory activity of phyco- cyanin in some animal models of inflammation[J]. Inflamm Res,1998,47(8):334-338
81. Winter CA, Risley EA, Nuss GW. Carrageenan-induced edema in hind paw of rat as an ass-ay for anti-inflammatory drugs[J]. P Soc Exp Biol Med,1962,111:544-547.
82. Bianchine JR, Ead NR. The effect of 5-hydroxytryptamine on the cotton pellet local inflam-matory response in the rat[J]. J Exp Med,1967,125(3):501-510.
83. Ghiara P, Bartalini M, Tagliabue A, et al. Anti-inflammatory activity of IFN-beta in carrag-eenan-induced pleurisy in the mouse [J]. Clin Exp Immunol,1986,66(3):606-614.
84.朱全红,陈飞龙,白霜,等GC-MS法测定尿液中麻黄汤代谢产物麻黄类生物碱的浓度[J].药物分析杂志,2005,25(9)：1030-1034.
85.国家药典委员会.中华人民共和国药典[S].一部.北京：中国医药科技出版社,2010：80-81.
86. Benton HP, Want EJ, Ebbels TM. Correction of mass calibration gaps in liquid chromatog-raphy-mass spectrometry metabolomics data[J]. Bioinformatics,2010,26(19):2488-2489.
87. Xu Y, Heilier JF, Madalinski G, et al. Evaluation of accurate mass and relative isotopic ab-undance measurements in the LTQ-orbitrap mass spectrometer for further metabolomics database building[J]. Anal Chem,2010,82(13):5490-5501.
88. Okada T, Afendi FM, Altaf-Ul-Amin M, et al. Metabolomics of medicinal plants:the importance of multivariate analysis of analytical chemistry data[J]. Curr Comput Aided Drug Des,2010,6(3):179-196.
89. Pittler MH, Schmidt K, Ernst E. Adverse events of herbal food supplements for body weig-ht reduction:systematic review [J]. Obes Rev,2005,6(2):93.
90. Powell T, Hsu FF, Turk J, et al. Ma-huang strikes again:ephedrine nephrolithiasis [J]. Am J Kidney Dis,1998,32(1):153.
91. Dunnick JK, Kissling G, Gerken DK, et al. Cardiotoxicity of Ma Huang/caffeine or ephed-rine/caffeine in a rodent model system [J]. Toxicol Pathol,2007,35(5):657.
92.孙晓英,张振军,边学峰,等.伪麻黄碱对大鼠致畸作用研究[J].癌变畸变突变,2005, 18(1)：68-70.
93.潘国华,孙晓如.国外对麻黄及其制剂的安全性评价[J].药物警戒,2007,4(2)：111-117.
94.贺敬波.《伤寒论》中的甘草运用规律探讨[J].现代中西医结合杂志,2011,20(3)：342-343.
95.张胜.《伤寒论》中甘草遣用探析[J].湖北中医,2001,23(1)：39-40.
96.杨建,颜红.从《金匮要略》中探讨甘草的使用方略[J].辽宁中医药大学学报,2011,13(4)：124-125.
97.候家玉.中药药理学[M].北京：中国中医药出版社,2002：28.
98.宋建平,韩丽,石强华.治水不必禁甘草[J].河南中医,1992,12(5)：213.
99. Wu TY, Khor TO, Saw CL, et al. Anti-inflammatory/Anti-oxidative stress activities and di-fferential regulation of Nrf2-mediated genes by non-polar fractions of tea Chrysanthemum zawadskii and licorice Glycyrrhiza uralensis uralensis[J]. AAPS J,2011,13(1):1-13.
100. Shin EM, Zhou HY, Guo LY, et al. Anti-inflammatory effects of glycyrol isolated from G1-ycyrrhiza uralensisuralensis in LPS-stimulated RAW264.7 macrophages[J]. Int Immunoph-armacol,2008,8(11):1524-1532.
101. Chandrasekaran CV, Deepak HB, Thiyagarajan P, et al. Dual inhibitory effect of Glycyrrhi-za uralensis glabra (GutGardTM) on COX and LOX products[J]. Phytomedicine,2011, 18(4):278-284.
102.陈长勋.中药药理学[M].上海：上海科学技术出版社,2006：164.
103. Crunkhorn P, Meacock SC. Mediators of the inflammation induced in the rat paw by carra-geenin[J]. Br J Pharmacol,1971,42:392-402.
104. Di Rosa M, Giroud JP, Willoughby DA. Studies of mediators of the acute inflammatory r-esponse induced in rats in different sites by carrageenan and turpentine[J]. J Patho,1971, 104:15-29.
105. Di Rosa M, Willoughby DA. Screens for anti-inflammatory drugs[J]. J Pharm Pharmacol, 1971,23:297-298.
106. Iwata M, Suzuki S, Asai Y, et al. Involvement of nitric oxide in a rat model of carrageenan-induced pleurisy[J]. Mediators of Inflammation,2010; 2010:682879.
107. Oka M, Tachibana M, Noda K, et al. Relevance of anti-reactive oxygen species activity to anti-inflammatory activity of components of eviprostat, a phytotherapeutic agent for benig-n prostatic hyperplasia[J]. Phytomedicine,2007,14:465-472.
108. Toriyabe M, Omote K, Kawamata T, et al. Contribution of interaction between nitric oxide and cyclo-oxygenases to the production of prostaglandins in carrageenan-induced inflamm-ation[J]. Anesthesiology,2004,101:983-990.
109. Kobayashi Y. The regulatory role of nitric oxide in proinflammatory cytokine expression during the induction and resolution of inflammation[J]. J Leukoc Biol,2010,88(6):1157-1162.
110. Long EO. ICAM-1:getting a grip on leukocyte adhesion[J]. J Immunol,2011,186(9): 5021-5023.
111. Mazzon E, Esposito E, Di Paola R, et al. Effect of tumour necrosis factor-a receptor 1 gen-etic deletion on carrageenan-induced acute inflammation:a comparison with etanercept [J]. Clin Exp Immunol,2008,153(1):136.
112.俞振良,卞如濂.甘草煎剂对大鼠下丘脑—腺垂体—肾上腺皮质轴的影响[J].浙江医科大学学报,1985,14(1)：5-7.
113.汉方临床.1980,27(9)：570.连策安译,程华校.
114. Dunn MJ, Hood VL. Prostaglandins and the kidney[J]. Am J Physiol,1977,233(3):169-184.
115. Weber PC, Scherer B, Siess W, et al. Formation and action of prostaglandins in the kidne-y[J]. Journal of Molecular Medicine,1979,57(19):1021-1029.
116. Scherer B, Weber PC. Time-dependent changes in prostaglandin excretion in response to frusemide in man[J]. Clin Sci (Lond),1979,56:77-81.
117. Halushka PV, Margolius HS, Allen H, et al. Urinary excretion of prostaglandin E like mat- erial and kallikrein:effects of furosemide[J]. Prostaglandins,1979,18(3):359-368.
118.孟翔宇,皮子凤,宋凤瑞,等.麻黄-甘草药对配伍前后主要药效成分及抗炎活性的变化[J].应用化学,2009,26(7)：801-805.
119.裴香萍,裴妙荣,段秀俊.酸碱对药麻黄与甘草在麻杏石甘汤中的配伍化学研究[J].2009,34(19)：2466-2468.
120.吴昭晖,罗佳波,谭晓梅.配伍对麻黄汤中甘草HPLC指纹图谱的影响[J].中国中药杂志,2006,31(2)：209-212.
121.王术玲,贾薇,曾建波,等.黄连-厚朴不同配伍对生物碱成分溶出的影响[J].中药新药与临床药理,2011,22(1)：91-93.
122.崔景朝,赵自明.中药配方颗粒研究进展(Ⅱ)-中药单煎与合煎对比研究概况[J].中国实验方剂学杂志,2011,17(4)：240-245.
123.孙晓英,张振军,边学峰,等.伪麻黄碱对大鼠致畸作用研究[J].2005,18(1)：68-70.
124.段丽萍.甘草药对分析[J].疾病监测与控制杂志,2009,3(2)：92-93.
125. Mudiam MK, Jain R, Dua VK, et al. Application of ethyl chloroformate derivatization for solid-phase microextraction-gas chromatography-mass spectrometric determination of bisp-henol-A in water and milk samples[J]. Anal Bioanal Chem,2011,401(5):1695-1701.
126. Tao X, Liu Y, Wang Y, GC-MS, et al. with ethyl chloroformate derivatization for compreh-ensive analysis of metabolites in serum and its application to human uremia[J]. Anal Bioa-nal Chem,2008,391(8):2881-2889.
127. Qiu Y, Su M, Liu Y, et al.Application of ethyl chloroformate derivatization for gas chroma-tography-mass spectrometry based metabonomic profiling[J]. Anal Chim Acta,2007,583(2) :277-283.
128. Smith CA, Want EJ, O'Maille G, et al. XCMS:processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification[J]. Anal Chem,2006,78(3):779-87.
129.王惠文,吴载斌,孟洁.偏最小二乘回归的线性与非线性方法[M],北京：国防工业出版社,2006：293-296.
2.孙冰,邓家刚.施今墨对药应用平性药配伍的规律探讨[J].山东中医药大学学报,2009,33(6)：460-463.
3.耿建国,王代娣.《伤寒论》药对配伍规律与特点[J].江苏中医,2000,21(9)：6-7.
4.吕景山.施今墨药对[M].第3版,北京：人民军医出版社,2005：57-60.
5.陈维华.药对论[M].合肥：安徽科学技术出版社,1989.
6.苏庆英.中医临床常用对药配伍[M].北京：人民卫生出版社,2010.
7.邢袁玲.《伤寒论》中四气五味及特殊药对探析[J].中国医药导报,2009,6(13)：111-113.
8.刘萍,王平,刘松林,等.基于药对探讨中药复方配伍规律的思考[J].中华中医药学刊,2010,28(9)：1833-1835.
9.杨敏,田岩.仲景药对配伍特点浅析[J].辽宁中医学院学报,2005,7(6)：560-561.
10.杨巨成.“药对”在中医方剂配伍中的重要性[J].中国民间疗法,2010,18(1)：55.
11.高晓山.中药药性论[M].北京：人民出版社,1992：167-177.
12.张宇燕,杨洁红.附子甘草配伍对乌头碱、甘草酸、甘草苷的动态影响[J].中国药学杂志,2009,44(1)：11-14.
13.越皓,皮子凤,宋凤瑞,等.附子不同配伍药对中生物碱成分的电喷雾质谱分析[J].药学学报,2007,42(2)：201-205.
14.涂瑶生,刘法锦,孙冬梅,等.黄连、吴茱萸配伍药对的HPLC指纹图谱研究[J].中成药,2011,33(1)：5-9.
15.毛幼儿,周桂芬.大黄与芒硝药对不同比例配伍对蒽醌苷含量的影响[J].江西中医药,2011,42(338)：60-61.
16.史琪荣,于少云,孙晓迪,等.黄连干姜药对对功能性消化不良大鼠胃排空和血清胃泌素的影响[J].中国药学杂志,2011,46(13)：988-992.
17.夏伯候,王智民,林丽美,等.银翘药对的药效学研究进展[J].中国实验方剂学杂志, 2009,15(3)：80-82.
18.王敏,刘彬,齐云,等.甘草皂苷对桔梗皂苷急性毒性的影响研究[J].中国实验方剂学杂志,2008,14(1)：59-61.
19.梁日欣,黄璐琦,刘菊福,等.药对川芎和赤芍对高脂血症大鼠降脂、抗氧化及血管内皮功能的实验观察[J].中国实验方剂学杂志,2002,8(1)：34.
20.郡顺琴,凤良元,黄德武,等.枳术丸对胃排空肠推进作用的影响[J].中成药,1996,18(4)：30-31.
21.襄萍,徐朝晖,战光绪,等.对药麻黄地龙配比及平喘作用机制的研究[J].中国中药杂志,2006,31(3)：236-239.
22.柳润辉,苏娟,徐希科,等.药对“丹参-黄芪”有效组分的最佳配伍配比研究[J].中国药学杂志,2006,41(11)：815-817.
23.杨祖贻,裴瑾,刘荣敏,等.当归肉桂配伍后阿魏酸生物利用度的研究[J].中国中药杂志,2006,31(12)：1012-1015.
24.王静,袁子民,张振秋.黄连、吴茱萸药对中盐酸小檗碱在大鼠体内的药代动力学研究[J].药物分析杂志,2009,29(5)：755-759.
25.王文萍,曹琦琛,高晶晶,等.六一散配伍规律的药动学研究[J].中国实验方剂学杂志,2009,15(12)：70-72.
26.何薇,荆雷,刘树民,等.黄连解毒汤中君-使药对黄连-栀子干预2,4-二硝基苯酚热病证候模型的代谢组学[J].中国实验方剂学杂志,2011,17(20)：173-178.
27.薛蕾.袁金声教授常用药对举隅[J].辽宁中医药大学学报,2012,14(2)：140-141.
28.王冠华,严志林.王少华治疗咳喘常用对药探微[J].辽宁中医药大学学报,2011,13(2)：11-12.
29.李杭,王荣欣.治疗糖尿病肾病常用药对举隅[J].光明中医,2012,27(2)：385-386
30.王咏梅,马红,刘苏中.方剂配伍的模糊数学特性研究[J].中国实验方剂学杂志,2000,6(6)：59-60.
31.李振岳,周怡.伤寒病复方药对配伍规律的关联规则分析[J].医学信息,2009,22(5)：591-593.
32.赵蔡斌,周鲁,付超,等.中药复方的模糊分析[J].中国实验方剂学杂志,2003,9(2)：62-63.
33.黄洁,马荣华,闻晓东,等.桃仁-红花药对不同配比的应用数据分析[J].中国新药杂志,2012,21(4)：407-411.
34. Loewe S, Muischnek H. Effect of combinations:mathematical basis of the problem[J]. Arch Exp PatholPharmacol,1926,114 (2):313-326.
35. Gebharto F. Commentson the isobolemethod foranalysis of drug interactions[J]. Pain,1992, 51(2):381.
36.郑青山.复方药物设计与药物相互作用定量分析方法学[c].第四届定量药理研究方法学研讨会,2010.
37. Tallarida RJ. Drug synergism:its detection and applications[J]. J Pharmacol Exp Ther, 2001,298(3):865-872.
38. Lorenzo JI, Sanchez-Marin P. Comments on "Isobolographic analysis for combinations of a full and partial agonist:curved isoboles[J]. J Pharmacol Exp Ther,2006,316(1):476-478.
39. Grabovsky Y, Tallarida RJ. Isobolographic analysis for combinations of a full and partial ag-Onist:curved isoboles[J]. J Pharmacol Exp Ther,2004,310(3):981-986.
40. Lelas S, Rowlett JK, Spealman RD. Isobolographic analysis of chlordiazepoxide and triazo-lamcombinations in squirrel monkeys discriminating triazolam[J]. Psychopharmacology (Berl).2001,158(2):181-189.
41. Adams JU, Tallarida RJ, Geller EB, et al. Isobolographic superadditivity between delta and mu opioid agonists in the rat depends on the ratio of compounds, the mu agonist and the an-algesic assay used[J]. J Pharmacol Exp Ther,1993,266(3):1261-1267.
42. Tallarida RJ. The interaction index:a measure of drug synergism[J]. Pain,2002,98(1/2): 163-168.
43. Chang TT, Gulati SC, Chou TC, et al. Synergistic effect of 4-hydroperoxycyclophosphami-de and etoposide on a human promyelocytic leukemia cell line (HL-60) demonstrated by c- omputer analysis[J]. Cancer Res,1985,45(6):2434-2439.
44. Chou TC. The mass-action law based algorithm for cost-effective approach for cancer drug discovery and development[J]. Am J Cancer Res,2011,1(7):925-954.
45. Chou TC. The mass-action law based algorithms for quantitative econo-green bio-research [J]. Integr Biol (Camb).2011,3(5):548-559.
46. Marvania B, Lee PC, Chaniyara R, etal. Design, synthesis and antitumor evaluation of phe-nyl N-mustard-quinazoline conjugates[J]. Bioorg Med Chem,2011,19(6):1987-1998.
47. Chou TC. Drug combination studies and their synergy quantification using the Chou-Talala-y method[J]. Cancer Res,2010,70(2):440-446.
48. Vathsala A, Chou TC, Kahan BD. Analysis of the interactions of immunosuppressive drugs with cyclosporine in inhibiting DNA proliferation[J]. Transplantation,1990,49(2):463-472.
49. Chou TC. Assessment of synergistic and antagonistic effects of chemotherapeutic agents in vitro[J]. Contrib Gynecol Obstet,1994,19:91-107.
50. Chou TC, Tan QH, Sirotnak FM. Quantitation of the synergistic interaction of edatrexate and cisplatin in vitro[J]. Cancer Chemother Pharmacol,1993,31(4):259-264.
51. Ogawa S, Kamijima T, Miyamoto Y, et al. A new attempt to solve the scale-up problem for granulation using response surface methodology [J]. J Pharm Sci,1994,83(3):439-443.
52. Greco WR, Park HS, Rustum YM. Application of a new approach for the quantitation of drug synergism to the combination of cisdiamminedi chloroplatinum and 1-beta- D-ara-binofuranosylcytosine[J]. Cancer Res,1990,50(17):5318-5327.
53. Lee SI. Drug interaction:focusing on response surface models[J]. Korean J Anesthesiol, 2010,58(5):421-434.
54. Ramakrishnan R, Jusko WJ. Interactions of aspirin and salicylic acid with prednisolone for inhibition of lymphocyte proliferation[J]. Int Immunopharmacol,2001,1(11):2035-2042.
55. Creco WR. Park HS. Rustum YM. Application of a new approach for the quantitation of drug synergism to the combination of cisdiaminedichloroplatinum and 1-D-arabinofura-Nosyl- cytosine[J]. Cancer Res,1990,50:5318-5327.
56.张玉霞,陈素,刘向明.分析药物相互作用的方法在中药研究中的应用[J].上海生物医学工程杂志,2005,26(3)：156-159.
57. Hu Y, Li DM, Han XD. Analysis of combined effects of nonylphenol and Monobutyl phthalate on rat Sertoli cells applying two mathematical models[J]. Food Chem Toxicol, 2012,50(3-4):457-463.
58. Stergiopoulou T, Meletiadis J, Sein T, et al. Synergistic interaction of the triple combination of amphotericin B, ciprofloxacin, and polymorphonuclear neutrophils against Aspergillus fumigates[J]. Antimicrob Agents Chemother,2011,55(12):5923-5929.
59. Boucher AN, Tam VH. Mathematical formulation of additivity for antimicrobial agents[J]. Diagn Microbiol Infect Dis,2006,55(4):319-325.
60. Dressler V, Muller G, Suhnel J. CombiTool-a new computer program for analyzing comb-ination experiments with biologically active agents[J]. Comput Biomed Res,1999, 32(2):145-160.
61. Tam VH, Schilling AN, Lewis RE, et al. Novel approach to characterization of combined pharmacodynamic effects of antimicrobial agents[J]. Antimicrob Agents Chemother,2004, 48(11):4315-4321.
62. Lee JJ, Kong M, Ayers GD, et al. Interaction index and different methods for determining drug interaction in combination therapy [J]. J Biopharm Stat,2007,17:461-480.
63.芦娜,刘振佳,闫征,等.三种药物相互作用数学模型分析SAHA与三氧化二砷联合用药的细胞毒作用[J].药学学报,2010,45(5)：601-607.
64.金正均.合并用药中的相加[J].中国药理学报,1980,1(2)：70-76.
65. Veldstrah. Syneergism and potentiation with special reference to the combination of structu-ral analogues [J]. Pharmacol Rev,1956,8(3):339-387.
66.周元晏,王宏镛,唐朝广,等.衡量合并用药效应得两个新公式-兼论Burgi公式及金式修正公式[J].中国药理学报,1984,5：217-221.
67.顾兵,王心如.联合作用特征的评价[J].中国工业医学杂志,2000,13(1)：55-58.
68.郑青山,孙瑞元.药物相互作用动力学分析方法及其CoDrug软件[J].中国临床药理学与治疗学,2002,7(1)：81.
69. Miaskowski C, Sutters KA, Taiwo YO, et al. Antinociceptive and motor effects of delta/mu and kappa/mu combinations of intrathecal opioid agonists[J]. Pain,1992,49(1):137-144.
70. Miaskowski C, Sutters KA, Taiwo YO, et al. Comparison of the antinociceptive and motor effects of intrathecal opioid agonists in the rat[J]. Brain Res,1991,553(1):105-109.
71. Franck LS, Miaskowski C, Putris J, et al. Dissociation of antinociceptive and motor effects of supraspinal opioid agonists in the rat[J]. Brain Res,1991,563(1-2):123-126.
72. Weckwerth W. Metabolomics in systems biology[J]. Annu Rev Plant Biol,2003,54:669-689.
73. Nicholson JK, Wilson ID. Opinion:understanding 'global' systems biology:metabonom-ics and the continuum of metabolism[J]. Nat Rev Drug Discov,2003,2(8):668-676.
74. Fiehn O. Metabolomics-the link between genotypes and phenotypes[J]. Plant Mol Biol, 2002,48(1-2):155-171.
75. Dunn WB, Bailey NJ, Johnson HE. Measuring the metabolome:current analytical techno-logies[J]. Analyst,2005,130(5):606-625.
76. Zhang A, Sun H, Wang P, et al. Modern analytical techniques in metabolomics analysis[J]. Analyst,2012,137(2):293-300.
77.李仪奎.中药药理实验方法学[M].第二版.上海：上海科学技术出版社,2006：1001-1007.
78. Whittle BA.The use of changes in capillary permeability in mice to distinguish between narcotic and nonnarcotic analgesics[J]. Br J Pharmacol Chemother,1964,22:246-253.
79. Lahlo S, Tahraoui A, Israili Z, et al. Diuretic activity of the aqueous extracts of Carum carviand Tanacetum vulgare in normal rats[J]. J Ethnopharmacol,2007,110(3):458-463.
80. Romay C, Ledon N, Gonzalez R. Further studies on anti-inflammatory activity of phyco- cyanin in some animal models of inflammation[J]. Inflamm Res,1998,47(8):334-338
81. Winter CA, Risley EA, Nuss GW. Carrageenan-induced edema in hind paw of rat as an ass-ay for anti-inflammatory drugs[J]. P Soc Exp Biol Med,1962,111:544-547.
82. Bianchine JR, Ead NR. The effect of 5-hydroxytryptamine on the cotton pellet local inflam-matory response in the rat[J]. J Exp Med,1967,125(3):501-510.
83. Ghiara P, Bartalini M, Tagliabue A, et al. Anti-inflammatory activity of IFN-beta in carrag-eenan-induced pleurisy in the mouse [J]. Clin Exp Immunol,1986,66(3):606-614.
84.朱全红,陈飞龙,白霜,等GC-MS法测定尿液中麻黄汤代谢产物麻黄类生物碱的浓度[J].药物分析杂志,2005,25(9)：1030-1034.
85.国家药典委员会.中华人民共和国药典[S].一部.北京：中国医药科技出版社,2010：80-81.
86. Benton HP, Want EJ, Ebbels TM. Correction of mass calibration gaps in liquid chromatog-raphy-mass spectrometry metabolomics data[J]. Bioinformatics,2010,26(19):2488-2489.
87. Xu Y, Heilier JF, Madalinski G, et al. Evaluation of accurate mass and relative isotopic ab-undance measurements in the LTQ-orbitrap mass spectrometer for further metabolomics database building[J]. Anal Chem,2010,82(13):5490-5501.
88. Okada T, Afendi FM, Altaf-Ul-Amin M, et al. Metabolomics of medicinal plants:the importance of multivariate analysis of analytical chemistry data[J]. Curr Comput Aided Drug Des,2010,6(3):179-196.
89. Pittler MH, Schmidt K, Ernst E. Adverse events of herbal food supplements for body weig-ht reduction:systematic review [J]. Obes Rev,2005,6(2):93.
90. Powell T, Hsu FF, Turk J, et al. Ma-huang strikes again:ephedrine nephrolithiasis [J]. Am J Kidney Dis,1998,32(1):153.
91. Dunnick JK, Kissling G, Gerken DK, et al. Cardiotoxicity of Ma Huang/caffeine or ephed-rine/caffeine in a rodent model system [J]. Toxicol Pathol,2007,35(5):657.
92.孙晓英,张振军,边学峰,等.伪麻黄碱对大鼠致畸作用研究[J].癌变畸变突变,2005, 18(1)：68-70.
93.潘国华,孙晓如.国外对麻黄及其制剂的安全性评价[J].药物警戒,2007,4(2)：111-117.
94.贺敬波.《伤寒论》中的甘草运用规律探讨[J].现代中西医结合杂志,2011,20(3)：342-343.
95.张胜.《伤寒论》中甘草遣用探析[J].湖北中医,2001,23(1)：39-40.
96.杨建,颜红.从《金匮要略》中探讨甘草的使用方略[J].辽宁中医药大学学报,2011,13(4)：124-125.
97.候家玉.中药药理学[M].北京：中国中医药出版社,2002：28.
98.宋建平,韩丽,石强华.治水不必禁甘草[J].河南中医,1992,12(5)：213.
99. Wu TY, Khor TO, Saw CL, et al. Anti-inflammatory/Anti-oxidative stress activities and di-fferential regulation of Nrf2-mediated genes by non-polar fractions of tea Chrysanthemum zawadskii and licorice Glycyrrhiza uralensis uralensis[J]. AAPS J,2011,13(1):1-13.
100. Shin EM, Zhou HY, Guo LY, et al. Anti-inflammatory effects of glycyrol isolated from G1-ycyrrhiza uralensisuralensis in LPS-stimulated RAW264.7 macrophages[J]. Int Immunoph-armacol,2008,8(11):1524-1532.
101. Chandrasekaran CV, Deepak HB, Thiyagarajan P, et al. Dual inhibitory effect of Glycyrrhi-za uralensis glabra (GutGardTM) on COX and LOX products[J]. Phytomedicine,2011, 18(4):278-284.
102.陈长勋.中药药理学[M].上海：上海科学技术出版社,2006：164.
103. Crunkhorn P, Meacock SC. Mediators of the inflammation induced in the rat paw by carra-geenin[J]. Br J Pharmacol,1971,42:392-402.
104. Di Rosa M, Giroud JP, Willoughby DA. Studies of mediators of the acute inflammatory r-esponse induced in rats in different sites by carrageenan and turpentine[J]. J Patho,1971, 104:15-29.
105. Di Rosa M, Willoughby DA. Screens for anti-inflammatory drugs[J]. J Pharm Pharmacol, 1971,23:297-298.
106. Iwata M, Suzuki S, Asai Y, et al. Involvement of nitric oxide in a rat model of carrageenan-induced pleurisy[J]. Mediators of Inflammation,2010; 2010:682879.
107. Oka M, Tachibana M, Noda K, et al. Relevance of anti-reactive oxygen species activity to anti-inflammatory activity of components of eviprostat, a phytotherapeutic agent for benig-n prostatic hyperplasia[J]. Phytomedicine,2007,14:465-472.
108. Toriyabe M, Omote K, Kawamata T, et al. Contribution of interaction between nitric oxide and cyclo-oxygenases to the production of prostaglandins in carrageenan-induced inflamm-ation[J]. Anesthesiology,2004,101:983-990.
109. Kobayashi Y. The regulatory role of nitric oxide in proinflammatory cytokine expression during the induction and resolution of inflammation[J]. J Leukoc Biol,2010,88(6):1157-1162.
110. Long EO. ICAM-1:getting a grip on leukocyte adhesion[J]. J Immunol,2011,186(9): 5021-5023.
111. Mazzon E, Esposito E, Di Paola R, et al. Effect of tumour necrosis factor-a receptor 1 gen-etic deletion on carrageenan-induced acute inflammation:a comparison with etanercept [J]. Clin Exp Immunol,2008,153(1):136.
112.俞振良,卞如濂.甘草煎剂对大鼠下丘脑—腺垂体—肾上腺皮质轴的影响[J].浙江医科大学学报,1985,14(1)：5-7.
113.汉方临床.1980,27(9)：570.连策安译,程华校.
114. Dunn MJ, Hood VL. Prostaglandins and the kidney[J]. Am J Physiol,1977,233(3):169-184.
115. Weber PC, Scherer B, Siess W, et al. Formation and action of prostaglandins in the kidne-y[J]. Journal of Molecular Medicine,1979,57(19):1021-1029.
116. Scherer B, Weber PC. Time-dependent changes in prostaglandin excretion in response to frusemide in man[J]. Clin Sci (Lond),1979,56:77-81.
117. Halushka PV, Margolius HS, Allen H, et al. Urinary excretion of prostaglandin E like mat- erial and kallikrein:effects of furosemide[J]. Prostaglandins,1979,18(3):359-368.
118.孟翔宇,皮子凤,宋凤瑞,等.麻黄-甘草药对配伍前后主要药效成分及抗炎活性的变化[J].应用化学,2009,26(7)：801-805.
119.裴香萍,裴妙荣,段秀俊.酸碱对药麻黄与甘草在麻杏石甘汤中的配伍化学研究[J].2009,34(19)：2466-2468.
120.吴昭晖,罗佳波,谭晓梅.配伍对麻黄汤中甘草HPLC指纹图谱的影响[J].中国中药杂志,2006,31(2)：209-212.
121.王术玲,贾薇,曾建波,等.黄连-厚朴不同配伍对生物碱成分溶出的影响[J].中药新药与临床药理,2011,22(1)：91-93.
122.崔景朝,赵自明.中药配方颗粒研究进展(Ⅱ)-中药单煎与合煎对比研究概况[J].中国实验方剂学杂志,2011,17(4)：240-245.
123.孙晓英,张振军,边学峰,等.伪麻黄碱对大鼠致畸作用研究[J].2005,18(1)：68-70.
124.段丽萍.甘草药对分析[J].疾病监测与控制杂志,2009,3(2)：92-93.
125. Mudiam MK, Jain R, Dua VK, et al. Application of ethyl chloroformate derivatization for solid-phase microextraction-gas chromatography-mass spectrometric determination of bisp-henol-A in water and milk samples[J]. Anal Bioanal Chem,2011,401(5):1695-1701.
126. Tao X, Liu Y, Wang Y, GC-MS, et al. with ethyl chloroformate derivatization for compreh-ensive analysis of metabolites in serum and its application to human uremia[J]. Anal Bioa-nal Chem,2008,391(8):2881-2889.
127. Qiu Y, Su M, Liu Y, et al.Application of ethyl chloroformate derivatization for gas chroma-tography-mass spectrometry based metabonomic profiling[J]. Anal Chim Acta,2007,583(2) :277-283.
128. Smith CA, Want EJ, O'Maille G, et al. XCMS:processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification[J]. Anal Chem,2006,78(3):779-87.
129.王惠文,吴载斌,孟洁.偏最小二乘回归的线性与非线性方法[M],北京：国防工业出版社,2006：293-296.