麻黄—石膏药对配伍的化学成分、药效及代谢组学研究
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摘要
研究背景
药对又称“对药”、“对子药”、“姐妹药”,由两味药成对,个别由三味以上药组成,是临床上常用的相对固定的配伍形式。药对的配伍是中药复方配伍的最简单、最基本和最常见的形式,其以一定证候特点及采用相应治法为前提,根据药物的性味归经、升降浮沉,选择性地将两味中药进行配对,其配伍蕴涵着丰富的客观规律。
本课题为麻黄类药对组成规律的基础研究(国家自然基金重点项目)的部分研究内容。麻黄-石膏药对是中医临床常用的药对之一,在很多经典方剂中都有运用,例如麻杏石甘汤、越婢汤等,现代常用于感冒、支气管肺炎、支气管哮喘等属表邪未尽,热邪壅肺等证。本课题选择经方麻杏石甘汤及其“主药对”麻黄-石膏药对为研究对象,以2味药的配比量为切入点,将麻黄-石膏药对的配伍研究分解为1)药对配伍前后的体外化学成分变化研究；2)主要药理效应(毒性)比较研究；3)药对配伍对对代谢组生物信息的干预研究；4)综合分析麻黄-石膏药对配伍、化学成分变化与药理效应改变三者间的相互关系。
研究目的
本研究以经典的寒热配伍药对麻黄-石膏为研究对象,运用现代技术手段,从化学成分变化、量效关系、药理效应改变、体内代谢途径与机制等方面对麻黄-石膏药对进行较为全面和系统的研究,通过分析麻黄-石膏药对不同比例配伍中指标性成分的含量、药效作用及代谢组学等方面的差异,可以寻求其发挥作用的最佳配伍比例,找出其量效、时效以及毒效间的关系,从而有利于更好地发挥药物的疗效,并可进一步验证、深化麻黄-石膏药对“相使”的配伍关系,据此为麻黄-石膏药对配伍的制剂研究及其临床应用,提供工作基础和实验依据。
研究方法
《伤寒论》中麻杏石甘汤组成为：“麻黄四两(去节)；杏仁五十个,去皮尖；甘草二两,炙；石膏半斤。本实验选取麻杏石甘汤中的药对麻黄—石膏(1：2)为研究对象,依据药对文献报道的配比上下成倍浮动,设计成三个配比1：1、1：2、1：4三个配伍组。
1.麻黄-石膏药对不同配伍配比的体外化学成分研究
建立测定麻黄-石膏水煎液中麻黄类生物碱的HPLC方法并进行了精密度、稳定性、加样回收率等方法学考察；采用HPLC测定麻黄、麻黄石膏药对1：1、1：2、1：4三个配伍配比水煎液中麻黄类生物碱(去甲基麻黄碱、去甲基伪麻黄碱、麻黄碱、伪麻黄碱和伪麻黄碱)的含量并比较麻黄-石膏不同比例配伍前后水煎液中5种生物碱含量的变化。
利用钙离子试剂盒测定各组水煎液中的钙离子并比较了麻黄-石膏不同比例配伍对Ca2+溶出的影响。
采用反相高效液相色谱法,选用Cosmosil C18柱(250mm×4.6mm,5μm),流动相为乙腈-0.1%磷酸水溶液(含0.1%三乙胺)进行梯度洗脱,分析并确定了麻黄-石膏不同比例配伍水煎液的“共有峰”,并研究了不同比例配伍对“共有峰”的影响。
2.麻黄石膏药对不同比例配伍的主要药理效应研究
急性毒性研究昆明小鼠,雌雄各半,分为麻黄、麻黄-石膏(1：1)、麻黄-石膏(1：2)、麻黄-石膏(1：4)5个给药组,每组分别设5个剂量组(n=10),根据预实验确定的Dn、Dm,定出相邻两组剂量比,组间比值为1：0.8。实验时将依据“低比稀释法”配制不同浓度的药液灌胃给予相应的小鼠。灌胃前禁食不禁水12小时,灌胃后观察饲养7d,观察小鼠的精神、活动、饮食、大小便、死亡等情况。记录各组动物中毒症状及死亡情况。记录实验结果,并用Bliss软件计算LD5o值和95%置信区间。
解热作用研究采用干酵母混悬液诱导雄性SD大鼠发热模型,选取体温合格大鼠,随机分为随机分为19组,即空白对照组、模型对照组、阿司匹林组、麻黄高中低剂量组、石膏高中低组、麻杏石甘汤组、麻黄石膏(1：1)高中低低剂量组、麻黄石膏(1：2)高中低剂量组、麻黄石膏(1：4)高中低组,每组6只,灌胃给药(正常对照组和模型对照组均给予等容积蒸馏水),给药后每小时测量肛温一次,连续测量三次。计算各给药组8h对大鼠体温升高抑制率(%)。利用Calcusyn统计软件(Biosoft, USA)分析麻黄-石膏两者的相互作用。
平喘作用研究采用卵蛋白诱导的哮喘大鼠模型,将60只SD大鼠随机分为正常对照组、模型组、地塞米松组、麻石1：2(高、中、低)剂量组、麻黄-石膏(1：1)组和麻黄-石膏(1：4,单味麻黄组、石膏组共10组,每组6只。观察麻黄、石膏配伍前后对大鼠引喘潜伏期、肺干湿重、EOS、WBC计数影响。
3.麻黄石膏药对不同配伍干预干酵母诱导热病症候的代谢组学研究
“发热”大鼠模型的建立及其代谢组学研究将12只雄性Wistar大鼠随机分为2组(n=6),空白对照组和模型组(背部皮下注射20%酵母混悬液10mL·kg-造模),将大鼠(正常对照组,模型组)置于代谢笼中收集6h尿液,尿液直接衍生化。采用G C-MS联用技术测定各组大鼠尿液代谢物谱。运用XCMS工具箱对代谢物谱进行峰识别、峰对齐和去噪等处理,得到由保留时间、样品号及信号强度组成的三维矩阵,将矩阵导入模式识别软件Simca-P12.0(瑞典,Umetrics AB, Umea)进行主成分分析(PCA)、偏最小二乘法判别分析(PLS-DA)。PLS-DA的结果中列出各变量的变量重要性值VIP (Variable Importance in the Projection),根据VIP大小排列,其中VIP值大于1的变量被认为是变化明显且对区分贡献较大,作为标记物分析,通过与气质工作站的数据库、对照品及查询网站进行对比分析,选择匹配度较高的内源性生物标志物作为鉴定结果。
麻黄-石膏对“发热”大鼠的代谢组学研究将雄性Wistar大鼠随机分为5组,即空白对照组、模型组、麻黄组、麻黄石膏1：2组和石膏组,每组6只。除空白组外,大鼠背部皮下注射20%干酵母(10mL/kg大鼠体重)造模,各给药组分别在造模后6h灌胃给药一次,收集给药后6h尿液,对空白组、模型组、麻黄组、麻黄石膏1：2组和石膏组的尿液代谢轮廓进行主成分分析(PCA)(?)PLS-DA分析,对其特征抽提及模式识别。选择VIP值大于1的物质作为干酵母诱导发热模型的潜在特异性生物标志物,通过与气质工作站的数据库、对照品及查询网站进行对比分析,选择匹配度较高的内源性生物标志物作为鉴定结果。分析麻黄组、麻黄石膏1：2组和石膏组生物标志物的差异及水平变化,结合现有的生物化学知识阐释麻黄、石膏药对配伍前后的调节机制。
实验结果
1.麻黄石膏药对不同比例配伍的体外化学成分研究
麻黄-石膏药对不同比例配伍对麻黄类生物碱含量的影响水煎液中5种生物碱(去甲基伪麻黄碱、去甲基麻黄碱、麻黄碱、伪麻黄碱和伪麻黄碱)含量的变化实验结果显示,除麻黄-石膏(1：4)配伍组中的去甲基麻黄碱含量比单味麻黄组降低(P<0.05),麻黄-石膏(1：1)和麻黄-石膏(1：2)配伍组各生物碱含量差异无统计学意义。麻黄-石膏(1：1)、麻黄-石膏(1：2)、麻黄-石膏(1：4)三个配伍组的水煎液中,麻黄-石膏(1：2)的总生物碱含量最高。
麻黄-石膏药对不同比例配伍对钙离子含量的影响利用析因分析麻黄、石膏不同比例配伍对Ca2+溶出量的影响：麻黄-石膏(1：1)组之间交互作用显著(F=22.222,P=0.002),通过比较发现,配伍后Ca2+溶出量则低于麻黄和石膏单煎所得Ca2+总量的加和,二者的差异具有显著性意义,说明麻黄-石膏(1：1)配伍组会抑制Ca2+的溶出。可以认为麻黄-石膏(1：1)二者之间有拮抗作用。麻黄-石膏(1：2)组之间交互作用显著(F=21.144,P=0.002),通过含量比较发现,配伍后Ca2+溶出量则低于麻黄和石膏单煎所得Ca2+总量的加和,二者的差异具有显著性意义,说明麻黄-石膏(1：2)配伍组会抑制Ca2+的溶出。麻黄-石膏(1：4)组之间交互作用显著(F=95.617,P=0.000),通过含量比较发现,配伍后Ca2+溶出量则低于麻黄和石膏单煎所得Ca2+总量的加和,二者的差异具有显著性意义,说明麻黄-石膏(1：4)配伍组会抑制Ca2+的溶出。
麻黄-石膏不同比例配伍对水煎液“共有峰”的影响不同比例麻黄-石膏的合煎液,其化学成分变化较为复杂,配伍比例不同,10个共有峰的情况变化也不相同。有的峰峰面积增大,有的降低,有的峰面积基本不保持不变。
2.麻黄石膏药对不同比例配伍的主要药理效应研究
急性毒性实验结果各比例配伍组中,在能够引起试验动物一半死亡的药物剂量下,麻黄所占的比例及其可信区间分别为单味麻黄131.67(108.23～166.81)g·kg-1,麻黄石膏(1：1)113.22(96.32～153.82)g·kg1,麻黄石膏(1：2)124.15(107.48～147.38)g.kg-1,麻黄石膏(1：4)106.05(89.14～134.02)g·kg-1。由表可知,麻黄的LDso值从大到小的顺序依次：麻黄≌麻黄石膏(1：2)>麻黄石膏(1：1)>麻黄石膏(1：4)。
解热实验结果与模型组相比,麻黄组、麻黄石膏(1：1)组、麻黄石膏(1：2)组、麻黄石膏(1：4)组均具有解热作用,且呈一定的量-效关系。各给药组8h抑制率(%)从大到小排列为：麻石1：2高剂量组(62.7)、麻杏石甘汤全方组(59.7)、麻黄高剂量组(59.0)、麻石1：2中剂量组(53.6)、麻黄中剂量组(49.4)、麻石1：1高剂量组(47.7)、麻石1：4高剂量组(47.2)、麻石1：1中剂量组(43.1)、麻石1：4中剂量组(37.1)、石膏高剂量(33.0)、麻石1：1低剂量组(30.8)、麻石1：4低剂量组(28.4)、麻石1：2低剂量组(21.7)、麻黄低剂量组(21.6)、石膏中剂量(10.9)、石膏低剂量(4.6)。通过比较麻黄石膏药对3个配比解热的作用时间与作用强度,我们发现麻黄石膏(1：2)的解热作用最佳,解热持续时间最长。麻黄-石膏(1：2)在解热方面具有协同增效作用。
平喘作用结果麻石1：2高、中剂量组均能延长引喘潜伏期(P<0.01),麻石1：2配伍组作用效果优于麻石1：1、麻石1：2组、单味麻黄、单味石膏组；麻石1：2高、中剂量组、单味麻黄组均能减少EOS数量(p<0.01)；麻石1：2高剂量组和麻石1：4组组均能减少WBC数量(p<0.01)；麻石1：2组高、中、低剂量、麻黄组、石膏组和麻石1：4组均能降低肺干湿重比值(p<0.01)；通过引喘潜伏期、肺干湿重比值、EOS和WBC计数综合分析比较,我们发现麻黄石膏(1：2)的平喘作用最佳,麻黄-石膏(1：2)其合用的效果优于同等剂量下的单味麻黄或石膏。
3.麻黄石膏药对不同比例配伍干预“发热”大鼠尿液代谢组学研究
“发热”大鼠模型的建立及其代谢组学研究通过对模型组和空白组大鼠尿液代谢物组进行PCA分析,两组大鼠的尿液样本可以在PCA的得分图上较为明显的区分开来,提示干酵母诱导的“发热”模型成功,大鼠机体代谢网络发生了明显的变化。为进一步关注干酵母引起的尿液代谢差异,找到与干酵母诱导发热相关的代谢通道的变化,实验采用PLS-DA法对模型组大鼠和正常组大鼠样本重新建模,用以鉴别造成上述分离的差异变量。结果显示,模型组和正常组在PC1上明显分离,并且该模型有较高的解释率和预测率(R2Xcum=0.878, R2Ycum=0.993, Q2Ycum=0.969)。通过上述PLS-DA法对两组大鼠样本重新建模后,根据S-Plot图(图4-6A)中离子的置信度和VIP值可知离子对分类贡献的大小,找到VIP值大于1的与干酵母诱导发热高度相关的代谢物(图4-6B),运用气质工作站的数据库(WILEY275.L及NIST05.L),结合自建的对照品物质库及查询网站(http://www.hmdb.ca/和http://metlin.scripps.edu/metabo_search_alt2.php)查找差异变量。分析结果显示,模型组和对照组共有10个物质有显著性差异,分别是：柠檬酸、酮戊二酸、3-丙酸、苯乙酸、甘氨酸、正丁胺、碳酸、丙氨酸、苯丙酮酸、苯甲酸。
麻黄、石膏及其合煎液对“发热”大鼠的代谢组学研究结果通过对空白组、模型组、麻黄组、麻黄桂枝组、石膏组大鼠尿液代谢物组进行PCA分析,5组大鼠的尿液样本可以在PCA的得分图上较为明显的区分开来,说明来大鼠给予不同处理后,其代谢物谱差异明显。不同处理组的样品之间的细微差异可以通过PLS-DA进行分析研究。
麻黄组和模型组相比,麻黄组中丙二酸、苯甲酸、丁二酸、丙氨酸、甘氨酸、缬氨酸、脯檬酸、辛二酸、尿素、马尿酸、苯丙酸、苯丙氨酸、2-羟基喹啉-羧酸含量升高(P<0.05)。
石膏处理组和模型组相比,石膏中苯甲酸、丁二酸、丙氨酸、3-丙酸、脯氨酸、辛二酸、L-谷氨酸含量升高(P<0.05)。
麻黄-石膏组与模型组相比,麻黄-石膏组中丙二酸、苯甲酸、丁二酸、甘氨酸、3-丙酸、脯氨酸、辛二酸、尿素、马尿酸、苯丙酸、苯丙氨酸、半胱氨酸含量升高(P<0.05)。
麻黄-石膏组与单味石膏相比,丙二酸、苯甲酸、甘氨酸、异亮氨酸、马尿酸、苯丙酸、1-脯氨酸含量升高(P<0.05)。
石膏组与单味麻黄相比,麻黄组中丙二酸、苯甲酸、丁二酸、3-丙酸、琥珀酸、尿素、马尿酸、苯丙酸、1-脯氨酸含量升高(P<0.05)。
对以上这些代谢物含量用单变量方差分析方法进行检验,其结果与PLS-DA的分析结果基本一致。
结论：
通过对麻黄-石膏“药对”进行化学成分、药效及代谢组学三方面的研究,确定麻黄与石膏配伍应用后,麻黄-石膏(1：2)配伍比例组在解热、平喘方面优于麻黄-石膏(1：1)、麻黄-石膏(1：4)配伍组,该最优比例与原方麻杏石甘汤中麻黄-石膏1：2相一致,从化学成分的角度,发现其增效的物质基础与增加其有效成分的含量(总生物碱以及Ca2+的溶出量)相关。推测麻黄-石膏不同配伍比例的药理作用不同,还与其通过抑制或促进某些成分的溶出,从而产生不同比例的物质群有关。代谢组学研究表明,麻黄、石膏、麻黄-石膏3个给药组均在一定程度上改善了干酵母诱导发热大鼠全身异常的代谢状态,其干预作用与调节机体的物质代谢、能量代谢相关,其中麻黄-石膏药对配伍后生物标志物的回调程度最大,表明麻黄-石膏合用比等剂量的单味麻黄或石膏效果好。从代谢组学的角度证实了麻黄-石膏配伍的合理性和优势。
Background
Herb pairs, also called drug pairs, the unique combinations of two relatively fixed herbs in clinic, are the most fundamental and the simplest form of multi-herb therapy. Without altering the basic therapeutic features of multi-herb formulae, herb pairs as the basic composition units of Chinese herbal formulae are of special clinical significance in TCM. The composing principle and manner of Chinese herb pair are based on the nature, taste, meridian distribution of the herbs and effects, which contains a wealth of objective laws. Besides, certain syndrome characteristics and the use of the appropriate treatment method as a precondition,
The meaning of our studying on the composition rules of Herb pairs:(1) Through the ways of modern technologies (for example, the material basis of the mechanism of action, and metabolic processes) to study medicine on the compatibility of Herb pairs, which play an important role in revealing the drug on the compatibility of the objective laws and scientific connotation.(2) To guide Clinical treatment, so it will benefit not only in using existing couplet medicines more efficiently, but also for creating new couplet medicines by the changes of spectrum of disease and symptom-comple.(3)The research of herb compatibility is one of the key points in revealing the compatibility law of complex prescriptions. By study of the compatibility law of Herb pairs, we will illuminate ompatibility laws of complex prescriptions. And it is also provide some basis for the reorganization of the quality and efficiency of the new prescriptions.(4) Herb pairs are vinculum of many complex prescriptions. Through the study the compatibility laws of Herb pairs, it can provide a reference for the other research containing the Herb pairs, and thus open up new avenues for compatibility rules of complex prescriptions.
Objection
Ephedra-Gypsum herb-pair are the most commonly used drug in the "cold and heat" compatibility of Traditional Chinese Medicine. The compatibility-relationship between them is classified to "xiangshi". By explore the changes in the chemical composition of ephedra-gypsum drug-pair, the dose-effect relationship, the change of the pharmacological effects and metabolic pathways and mechanisms in vivo, it will not only further to validate compatibility-relationship between ephedra and gypsum and also play a role in finding the best compatibility proportion. Furthermore, by finding out the relationship of dose-efficiency, time-efficiency and toxic-effects, thus will to bring into full play the effect of drugs, and it is also provide the basis for the clinical treament of traditional Chinese medicine, development of new drug-pairs and drugs.
Contents
To clarify the compatibility rules of Traditional Chinese herb-pair Herba Ephedrae and Gypsum with different compatibility ratio, we compared the changes in the content of active ingredients in water extracts, pharmacological effects and metabonomics. Mainly containing:
1) To clarify the chemical compatibility rules of couplet medicines Ephedr-Gypsum with different compatibility ratio, we compared the content of active ingredients in water extracts before and after different compatibility ratio of Ephedra or Gypsum. We also investigation the fingerprint peaks changes of Ephedra-Gypsum with different compatibility ratio.
2) To clarify the strength of pharmacological effects on the the target of Ephedr-Gypsum with different compatibility ratio, we compared the acute toxicity, antipyretic and anti-asthmatic effects.
3) Establishment the yeast-induced-fever rat model, collecting the urine before and after administration to process the the metabolomics study, and exploring the metabolomics variation and mechanism of Ephedr-Gypsum with different compatibility ratio Methods and materials.
Materials and Methods
The composition of ma-xin-shi-gan decoction in Treatise on Cold Pathogenic Diseases is:"Herba Ephedrae four uncia (removed knots), armeniacae semen fifty piece(decorticate), Radix Glycyrrhizae two uncia, and Gypsum eight uncia." The couplet medicines Ephedra-Gypsum (1:2) in ma-xin-shi-gan decoction were taken as study object, the compatibility ratio of Ephedra-Gypsum was duplication floating up and down basing on the ratio of the couplet medicines reported in the literature.1) Researching on chemical composition in vitro of Ephedra-Gypsum with differe-ent compatibility ratios
A high performance liquid chromatographic method (HPLC) was developed to determination the content of Norpseudoephedrine (NMP), Norephedrine (NME), Ephedrine (E), Pseudoephedrine (PE) and Methylephedrine (ME), which are the major active compounds of Herba Ephedrae.The HPLC method was validated with regard to specificity, linearity, precision, reproducibility. We compared the ephedra alkaloids content of Ephedra-Gypsum (1:1), Ephedra-Gypsum (1:2), and Ephedra-Gypsum (1:4). We also compared the contents of Ca2+in Ephedra-Gypsum (1:1), Ephedra-Gypsum (1:2), Ephedra-Gypsum (1:4), Ephedra and Gypsum water extract by calcium kit. Separations were carried out on an Cosmosil C18(250x4.6mmi.d.,5am) with a flow rate of0.7mLmin-1.The mobile phase was a mixture of H3PO4(containing0.1%triethylamine)-acetonitrile, gradient elution.
2) Study on the major pharmacological effect (major syndrome) of couplet medicines Ephedra-Gypsum with different compatibility ratio.
Acute Toxicity test:the KM mice were randomly divided into normal group, model group, Ephedra group, Ephedra-Gypsum (1:1), Ephedra-Gypsum (1:2), Ephedra-Gypsum (1:4) group, then each group was divided into five dosage group, each group had10mice, half male and half female. Dn, Dm were selected according to the preliminary experiment, the ratio between adjacent groups was1:0.8. The mice were intragastric (ig.) administrated with different dose of tested drugs, and the activities of the mice were observed the following7days, such as diet, urination and defecation, actions and even death etc. The death rates of each group were recorded. LD50and95%confidence interval were calculated with Bliss software.
Antipyretic experiment:the male rats, which had qualified temperature, were randomly divided into normal group, model group, aspirin group, Ephedra (high, middle and low)group, Ephedra-Gypsum (1:1) high, middle and low group, Ephedra-Gypsum (1:2) high, middle and low group, Ephedra-Gypsum (1:4) high, middle and low group, Gypsum (high, middle and low) group, and ma-xin-shi-gan decoction group, each of had6rats. The rats were intragastric (ig.) administrated with different dose of tested drugs. Measuring rectal temperature once per hour after administration, and measured three times. Inhibition rate (%) of each administration group is calculated8h elevated body temperature of rats.
Anti-asthmatic test Use ovalbumin-induced asthma rat model,60SD rats were randomly divided into normal control group, model group, dexamethasone group, Ephedra-Gypsum (1:2) high, middle and low group, ephedra group, Gypsum group, Ephedra-Gypsum (1:1) group and Ephedra-Gypsum (1:4) group, total10groups (n=6). The asthma incubation period, wet and dry weight of lung, EOS and WBC counts was observed.3. Metabonomics study on yeast-induced-fever rats after administration with with different compatibility ratio Ephedra-Gypsum extracts
Metabonomics study on yeast-induced-fever rats:12Wistar rats were randomly divided into normal control group and model group.The fevered rats were prepared by subcutaneous injection of20%yeast. Then6h urine of rats in each group were collected. All the urine samples were derivatized directly. Subsequently, metabolites spectra of these samples were acquired using gas chromatography-mass spectrometry (GC-MS). Identification after proeessed, the data was subjected to SIMCA-P+12.0software(Umetrics AB, Umea Sweden) for principal component analysis(PCA) and principal least squares discriminant analysis (PLS-DA). The modeling and therapeutic effects were judged by PCA visually:distinguishing effect among groups were further inspected by PLS-DA, and the correlation with classification of metabolites were evaluated according to the "variable impotance in the projection (VIP)" value. When a certain metabolite's VIP>1, it would be taken as potential metabolic marker. The differences of potential biomarkers levels in different groups were conducted with univariate statistical analysis using software of SPSS13.0, and those with significant difference were chosen as final biomarkers.
Metabonomics study on yeast-induced-fever rats after administration with with different compatibility ratio Ephedra-Gypsum extracts:30Wistar rats were randomly divided into normal control group, model group, Ephedra-Gypsum (1:2) group, Ephedra group and Gypsum group.The fevered rats were prepared by subcutaneous injection of20%yeast. Then6h urine of rats in each group were collected. All the urine samples were derivatized directly. Subsequently, metabolites spectra of these samples were acquired using gas chromatography-mass spectrometry (GC-MS). Identification after proeessed, the data was subjected to SIMCA-P+12.0software(Umetrics AB, Umea Sweden) for principal component analysis(PCA) and principal least squares discriminant analysis (PLS-DA). The modeling and therapeutic effects were judged by PCA visually:distinguishing effect among groups were further inspected by PLS-DA, and the correlation with classification of metabolites were evaluated according to the "variable impotance in the projection (VIP)" value. When a certain metabolite's VIP>1, it would be taken as potential metabolic marker. The differences of potential biomarkers levels in different groups were conducted with univariate statistical analysis using software of SPSS13.0, and those with significant difference were chosen as final biomarkers.
Results:
1Researching on chemical composition of different compatibility ratio of couplet medicines Ephedra-Gypsum
When compared to Ephedra group, the content of Norpseudoephedrine (NMP), Norephedrine (NME), Ephedrine (E), Pseudoephedrine (PE), Methylephedrine (ME), in Ephedra-Gypsum (1:1) group, Ephedra-Gypsum (1:2) group and Ephedra-Gypsum (1:4) group almost have no significance different. Except the Norephedrine (NME) was lower in Ephedra-Gypsum (1:4) group (P<0.05), compared to Ephedra group.
The results of the contents of Ca2+are as follows:The interaction of Ephedra-gypsum (1:1) was significant (F=22.222, P=0.002), the contents of Ca2+increased after compatibility, which showed Ephedra-gypsum (1:1) had synergistic effect; The interaction of ephedra-the gypsum (1:2) was significant (F=21.144, P=0.002), the contents of Ca2+increased after compatibility, which showed Ephedra-gypsum (1:2) had synergistic effect; The interaction of ephedra-the gypsum (1:2) was significant (F=95.617, P=0.000), the contents of Ca2+decreased after compatibility, which showed Ephedra-gypsum (1:4) had antagonistic action.
2Study on the major pharmacological effect (major syndrome) of Ephedra-Gypsum herb-pair with different compatibility ratio
Acute Toxicity test:the LD50of Ephedra, Ephedra-Gypsum (1:1), Ephedra-Gypsum(1:2), Ephedra-Gypsum(1:4) were131.67g·kg-1,226.43g·kg-1,372.36g·kg-1,530.25g·kg-1, respectively.95%confidence interval were108.23～166.81g·kg-1,192.64～307.64g·kg-1,322.46～442.14g·kg-1,445.68～670.11g·kg-1, respectively.
Antipyretic effect and interaction experiment:Compared with model group, Ephedra, Ephedra-Gypsum (1:1), Ephedra-Gypsum (1:2), Ephedra-Gypsum (1:4) extracts had antipyretic effect. They also showed a good dose-effect relationship. Single Gypsum with large dose had antipyretic effect, but lower doses even cause the raise of body temperature in rats.8h inhibition rate (%) were sorted: Ephedra-Gypsum1:2high-dose group (62.7), Ma-Xing-Shi-Gan Tang (59.7), Ephedra high-dose group (59.0), Ephedra-Gypsum1:2middle-dose group (53.6), Ephedra middle-dose group (49.4), Ephedra-Gypsum1:1high-dose group (47.7), Ephedra-Gypsum1:4high-dose group (47.2), Ephedra-Gypsum1:1middle-dose group (43.1), Ephedra-Gypsum1:4middle-dose group (37.1), gypsum high-dose (33.0), Ephedra-Gypsum1:1low-dose group (30.8), Ephedra-Gypsum1:4low-dose group (28.4), Ephedra-Gypsum1:2low-dose group (21.7), Ephedra low-dose group (21.6), Gypsum middle-dose group (-7.8), gypsum low-dose group (-18.3). Based on the median-effect principle, the interaction of antipyretic effect was analyzed with Calcusyn software.
Anti-asthmatic test:Ephedra-Gypsum1:2high and middle dose group could prolong the latent period of asthma (P<001), Ephedra-Gypsum (1:2) group effect was significantly better than Ephedra-Gypsum (1:1), Ephedra-Gypsum (1:4) of single ephedra, single gypsum group; Ephedra-Gypsum (1:2) high and middle dose group, single ephedra group could significantly reduce the the EOS counts in blood (p <0.01); Ephedra-Gypsum (1:2) high-dose group and Ephedra-Gypsum (1:4) group could significantly reduce the WBC counts (p<0.01); Ephedra-Gypsum (1:2) high, middle and low groups, Ephedra group, gypsum group and Ephedra-Gypsum (1:4) group can significantly reduce lung wet and dry weight ratio (p<0.01); Through analysis and comparison of the latent period of asthma, lung wet and dry weight ratio, EOS and WBC counts, we found that the antiasthmatic effects of Ephedra-Gypsum (1:2) group show better than a single herb ephedra or gypsum in equal doses.
3Metabonomics study on yeast-induced-fever rats after administration with with different compatibility ratio Ephedra-Gypsum extracts
Through the PCA analysis of the model group and blank group, results showed that the model groups and blank group distinguished well, which showed the yeast-induced-fever model success, urinary metabolites changed significantly. To further attention to the urine metabolic differences caused by the dry yeast, to find changes induced fever associated with dry yeast metabolic pathways, experimental re-modeling of the PLS-DA model rats and normal rats samples, used to identify the causethe difference variable for the above separation. The results show that the model group and the normal group on PCl clearly separated, and the model has a high interpretation rate and the predicted rate at (R2Xcum=0.878R2Ycum=0.993, Q2Ycum=0.969).By the PLS-DA method after the re-modeling of the two groups of rats sample, according to the S-Plot in confidence and VIP value that the ion size, find the classification contribution VIP value greater than1were yeast-induced-fever highly related metabolites, the use of temperament workstation database (WILEY275.L NIST05.L), combined with self-built reference standard material library and inquiries Web site(http://www.hmdb. ca/http://metlin.scripps.edu/metabo_search_al t2.php) find the difference variable.The analysis results show that compared with the model group:blank control group the contents of propylamine, glycine, malonic acid phenethyl uric acid, benzeneAcetic acid, hippuric acid significantly increased; the contents of glycine, malonic acid phenethyl uric acid, benzene, acetic acid, hippuric acid significantly decreased.
3.2Metabonomics study on yeast-induced-fever rats after administration with with normal group, model group,hedra-Gypsum(1:2) group, Ephedr group, and Gypsum group
Through the PCA analysis of the model group and blank group, results showed that the model groups, blank group, Ephedra-Gypsum(1:2) group, Ephedr group, and Gypsum group distinguished well. The subtle differences among the treatment groups will further analysis by PLS-DA.3.4Biomakers, which were obtained by PLS-DA analysis of normal group, model group, Ephedrae group, Gypsum group, Ephedra-Gypsum(1:2) group together, including organic acids, fatty acids, amino acids, amines and so on.
Model group compared to blank group, the content of Citric acid, ketoglutaric acid,3-propionic acid, phenylacetic acid, glycine, n-butylamine, carbonic acid, alanine, phenyl pyruvic acid, benzoic acid decreased (P<0.05); The results by univariate analysis are basically consistent with the results of PLS-DA analysis.
Ephedra group compared with the model group, the contents of in Ephedra group Malonic acid, benzoic acid, succinic acid, alanine, glycine, valine, proline citric acid, suberic acid, urea, hippuric acid, benzenepropanoic acid, phenylalanine,2-hydroxy-quinoline-carboxylicacid content significantly increased The results of these metabolites content by univariate analysis are basically consistent with the results of PLS-DA analysis.
Gypsum compared to model group, Alanine,3-propionic acid, proline, suberic acid, benzoic acid, succinic acid, L-glutamic acid decreased (P<0.05). The results by univariate analysis are basically consistent with the results of PLS-DA analysis.
Ephedra-Gypsum group compared with model group, Malonic acid, benzoic acid, succinic acid, glycine,3-propionic acid, proline acid, suberic acid, urea, hippuric acid, benzenepropanoic acid, phenylalanine, cysteine decreased (P<0.05). The results by univariate analysis are basically consistent with the results of PLS-DA analysis.
Ephedra-Gypsum group compared with Gypsum group, Malonic acid, benzoic acid, glycine, isoleucine, leucine, hippuric acid, phenylpropionic acid,1-proline increased (P<0.05) in Ephedra-gypsum group. The results by univariate analysis are basically consistent with the results of PLS-DA analysis.
Ephedra group compared with Gypsum group, Malonic acid, benzoic acid, succinic acid, propionic acid, succinic acid, urea, hippuric acid,3-benzenepropanoic acid,,1-prolineincreased (P<0.05) in Ephedra group. The results by univariate analysis are basically consistent with the results of PLS-DA analysis.
Conclusion:1) Results of chemical composition analysis experiment showed that Ephedra-gypsum (1:4) group significant decrease the content of methyl-ephedrine and Ca2+(P<0.05), but the other two compatibility groups has no significant change. The total contents of alkaloids and Ca2+in Ephedra-gypsum (1:2) group were higher than Ephedra-gypsum (1:1) group, which is conducive to the dissolution of the active ingredient. In the original prescription, Ma-xin-shi-gan Tang, which Ephedra:Gypsum (1:2) compatibility, the foundings provide some basis for the advantages and reasonable of this compatibility proportion(1:2), but the mechanism has yet to be studied further.2Compatibility rules of herb pairs should consider safety and efficacy.
The LD50f Ephedra-Gypsum (1:1) group, Ephedra-Gypsum (1:2) group and Ephedra-Gypsum (1:4) group were greater than the single Ephedra, that is to say, when Ephedra combined with Gypsum, the safety improved. Ephedra-Gypsum (1:1) group, Ephedra-Gypsum (1:2) group and Ephedra-Gypsum (1:4) group have a synergistic effect in antipyretic activity. Ephedra-Gypsum (1:2) is the best compatibility ratio among three. Antiasthma test results showed, among Ephedra-Gypsum (1:1) group, Ephedra-Gypsum (1:2) group and Ephedra-Gypsum (1:4) grou, Ephedra-Gypsum (1:2) group is optimal ratio.
3The metabolomics success applied for yeast-induced-fever rat model, which laid the foundation for further research on antipyretic effect of Traditional Chinese Medicines. Yeast-induced-fever will caused the Krebs cycle disorders, increased anaerobic glycolysis, amino acids excessive consumption and fatty acid metabolism disorders. Ephedra-Gypsum (1:2) group, Ephedra group and Gypsum group were taken PCA analysis together, the results showed that compared with Ephedra group and Gypsum group, the metabolic state of Ephedra-Gypsum (1:2) group was closer to the normal group, which proved that Ephedra-Gypsum (1:2) had a better intervention effect on the yeast-induced fever symptoms. PLS-DA analysis results showed that, the biomarker intervention yeast-induced fever symptoms of Ephedra-Gypsum (1:2) group were similar to normal group, which proved that Ephedra-Gypsum (1:2) group has better antipyretic effect than Ephedra or Gypsum group.
药对又称“对药”、“对子药”、“姐妹药”,由两味药成对,个别由三味以上药组成,是临床上常用的相对固定的配伍形式。药对的配伍是中药复方配伍的最简单、最基本和最常见的形式,其以一定证候特点及采用相应治法为前提,根据药物的性味归经、升降浮沉,选择性地将两味中药进行配对,其配伍蕴涵着丰富的客观规律。
本课题为麻黄类药对组成规律的基础研究(国家自然基金重点项目)的部分研究内容。麻黄-石膏药对是中医临床常用的药对之一,在很多经典方剂中都有运用,例如麻杏石甘汤、越婢汤等,现代常用于感冒、支气管肺炎、支气管哮喘等属表邪未尽,热邪壅肺等证。本课题选择经方麻杏石甘汤及其“主药对”麻黄-石膏药对为研究对象,以2味药的配比量为切入点,将麻黄-石膏药对的配伍研究分解为1)药对配伍前后的体外化学成分变化研究；2)主要药理效应(毒性)比较研究；3)药对配伍对对代谢组生物信息的干预研究；4)综合分析麻黄-石膏药对配伍、化学成分变化与药理效应改变三者间的相互关系。
研究目的
本研究以经典的寒热配伍药对麻黄-石膏为研究对象,运用现代技术手段,从化学成分变化、量效关系、药理效应改变、体内代谢途径与机制等方面对麻黄-石膏药对进行较为全面和系统的研究,通过分析麻黄-石膏药对不同比例配伍中指标性成分的含量、药效作用及代谢组学等方面的差异,可以寻求其发挥作用的最佳配伍比例,找出其量效、时效以及毒效间的关系,从而有利于更好地发挥药物的疗效,并可进一步验证、深化麻黄-石膏药对“相使”的配伍关系,据此为麻黄-石膏药对配伍的制剂研究及其临床应用,提供工作基础和实验依据。
研究方法
《伤寒论》中麻杏石甘汤组成为：“麻黄四两(去节)；杏仁五十个,去皮尖；甘草二两,炙；石膏半斤。本实验选取麻杏石甘汤中的药对麻黄—石膏(1：2)为研究对象,依据药对文献报道的配比上下成倍浮动,设计成三个配比1：1、1：2、1：4三个配伍组。
1.麻黄-石膏药对不同配伍配比的体外化学成分研究
建立测定麻黄-石膏水煎液中麻黄类生物碱的HPLC方法并进行了精密度、稳定性、加样回收率等方法学考察；采用HPLC测定麻黄、麻黄石膏药对1：1、1：2、1：4三个配伍配比水煎液中麻黄类生物碱(去甲基麻黄碱、去甲基伪麻黄碱、麻黄碱、伪麻黄碱和伪麻黄碱)的含量并比较麻黄-石膏不同比例配伍前后水煎液中5种生物碱含量的变化。
利用钙离子试剂盒测定各组水煎液中的钙离子并比较了麻黄-石膏不同比例配伍对Ca2+溶出的影响。
采用反相高效液相色谱法,选用Cosmosil C18柱(250mm×4.6mm,5μm),流动相为乙腈-0.1%磷酸水溶液(含0.1%三乙胺)进行梯度洗脱,分析并确定了麻黄-石膏不同比例配伍水煎液的“共有峰”,并研究了不同比例配伍对“共有峰”的影响。
2.麻黄石膏药对不同比例配伍的主要药理效应研究
急性毒性研究昆明小鼠,雌雄各半,分为麻黄、麻黄-石膏(1：1)、麻黄-石膏(1：2)、麻黄-石膏(1：4)5个给药组,每组分别设5个剂量组(n=10),根据预实验确定的Dn、Dm,定出相邻两组剂量比,组间比值为1：0.8。实验时将依据“低比稀释法”配制不同浓度的药液灌胃给予相应的小鼠。灌胃前禁食不禁水12小时,灌胃后观察饲养7d,观察小鼠的精神、活动、饮食、大小便、死亡等情况。记录各组动物中毒症状及死亡情况。记录实验结果,并用Bliss软件计算LD5o值和95%置信区间。
解热作用研究采用干酵母混悬液诱导雄性SD大鼠发热模型,选取体温合格大鼠,随机分为随机分为19组,即空白对照组、模型对照组、阿司匹林组、麻黄高中低剂量组、石膏高中低组、麻杏石甘汤组、麻黄石膏(1：1)高中低低剂量组、麻黄石膏(1：2)高中低剂量组、麻黄石膏(1：4)高中低组,每组6只,灌胃给药(正常对照组和模型对照组均给予等容积蒸馏水),给药后每小时测量肛温一次,连续测量三次。计算各给药组8h对大鼠体温升高抑制率(%)。利用Calcusyn统计软件(Biosoft, USA)分析麻黄-石膏两者的相互作用。
平喘作用研究采用卵蛋白诱导的哮喘大鼠模型,将60只SD大鼠随机分为正常对照组、模型组、地塞米松组、麻石1：2(高、中、低)剂量组、麻黄-石膏(1：1)组和麻黄-石膏(1：4,单味麻黄组、石膏组共10组,每组6只。观察麻黄、石膏配伍前后对大鼠引喘潜伏期、肺干湿重、EOS、WBC计数影响。
3.麻黄石膏药对不同配伍干预干酵母诱导热病症候的代谢组学研究
“发热”大鼠模型的建立及其代谢组学研究将12只雄性Wistar大鼠随机分为2组(n=6),空白对照组和模型组(背部皮下注射20%酵母混悬液10mL·kg-造模),将大鼠(正常对照组,模型组)置于代谢笼中收集6h尿液,尿液直接衍生化。采用G C-MS联用技术测定各组大鼠尿液代谢物谱。运用XCMS工具箱对代谢物谱进行峰识别、峰对齐和去噪等处理,得到由保留时间、样品号及信号强度组成的三维矩阵,将矩阵导入模式识别软件Simca-P12.0(瑞典,Umetrics AB, Umea)进行主成分分析(PCA)、偏最小二乘法判别分析(PLS-DA)。PLS-DA的结果中列出各变量的变量重要性值VIP (Variable Importance in the Projection),根据VIP大小排列,其中VIP值大于1的变量被认为是变化明显且对区分贡献较大,作为标记物分析,通过与气质工作站的数据库、对照品及查询网站进行对比分析,选择匹配度较高的内源性生物标志物作为鉴定结果。
麻黄-石膏对“发热”大鼠的代谢组学研究将雄性Wistar大鼠随机分为5组,即空白对照组、模型组、麻黄组、麻黄石膏1：2组和石膏组,每组6只。除空白组外,大鼠背部皮下注射20%干酵母(10mL/kg大鼠体重)造模,各给药组分别在造模后6h灌胃给药一次,收集给药后6h尿液,对空白组、模型组、麻黄组、麻黄石膏1：2组和石膏组的尿液代谢轮廓进行主成分分析(PCA)(?)PLS-DA分析,对其特征抽提及模式识别。选择VIP值大于1的物质作为干酵母诱导发热模型的潜在特异性生物标志物,通过与气质工作站的数据库、对照品及查询网站进行对比分析,选择匹配度较高的内源性生物标志物作为鉴定结果。分析麻黄组、麻黄石膏1：2组和石膏组生物标志物的差异及水平变化,结合现有的生物化学知识阐释麻黄、石膏药对配伍前后的调节机制。
实验结果
1.麻黄石膏药对不同比例配伍的体外化学成分研究
麻黄-石膏药对不同比例配伍对麻黄类生物碱含量的影响水煎液中5种生物碱(去甲基伪麻黄碱、去甲基麻黄碱、麻黄碱、伪麻黄碱和伪麻黄碱)含量的变化实验结果显示,除麻黄-石膏(1：4)配伍组中的去甲基麻黄碱含量比单味麻黄组降低(P<0.05),麻黄-石膏(1：1)和麻黄-石膏(1：2)配伍组各生物碱含量差异无统计学意义。麻黄-石膏(1：1)、麻黄-石膏(1：2)、麻黄-石膏(1：4)三个配伍组的水煎液中,麻黄-石膏(1：2)的总生物碱含量最高。
麻黄-石膏药对不同比例配伍对钙离子含量的影响利用析因分析麻黄、石膏不同比例配伍对Ca2+溶出量的影响：麻黄-石膏(1：1)组之间交互作用显著(F=22.222,P=0.002),通过比较发现,配伍后Ca2+溶出量则低于麻黄和石膏单煎所得Ca2+总量的加和,二者的差异具有显著性意义,说明麻黄-石膏(1：1)配伍组会抑制Ca2+的溶出。可以认为麻黄-石膏(1：1)二者之间有拮抗作用。麻黄-石膏(1：2)组之间交互作用显著(F=21.144,P=0.002),通过含量比较发现,配伍后Ca2+溶出量则低于麻黄和石膏单煎所得Ca2+总量的加和,二者的差异具有显著性意义,说明麻黄-石膏(1：2)配伍组会抑制Ca2+的溶出。麻黄-石膏(1：4)组之间交互作用显著(F=95.617,P=0.000),通过含量比较发现,配伍后Ca2+溶出量则低于麻黄和石膏单煎所得Ca2+总量的加和,二者的差异具有显著性意义,说明麻黄-石膏(1：4)配伍组会抑制Ca2+的溶出。
麻黄-石膏不同比例配伍对水煎液“共有峰”的影响不同比例麻黄-石膏的合煎液,其化学成分变化较为复杂,配伍比例不同,10个共有峰的情况变化也不相同。有的峰峰面积增大,有的降低,有的峰面积基本不保持不变。
2.麻黄石膏药对不同比例配伍的主要药理效应研究
急性毒性实验结果各比例配伍组中,在能够引起试验动物一半死亡的药物剂量下,麻黄所占的比例及其可信区间分别为单味麻黄131.67(108.23～166.81)g·kg-1,麻黄石膏(1：1)113.22(96.32～153.82)g·kg1,麻黄石膏(1：2)124.15(107.48～147.38)g.kg-1,麻黄石膏(1：4)106.05(89.14～134.02)g·kg-1。由表可知,麻黄的LDso值从大到小的顺序依次：麻黄≌麻黄石膏(1：2)>麻黄石膏(1：1)>麻黄石膏(1：4)。
解热实验结果与模型组相比,麻黄组、麻黄石膏(1：1)组、麻黄石膏(1：2)组、麻黄石膏(1：4)组均具有解热作用,且呈一定的量-效关系。各给药组8h抑制率(%)从大到小排列为：麻石1：2高剂量组(62.7)、麻杏石甘汤全方组(59.7)、麻黄高剂量组(59.0)、麻石1：2中剂量组(53.6)、麻黄中剂量组(49.4)、麻石1：1高剂量组(47.7)、麻石1：4高剂量组(47.2)、麻石1：1中剂量组(43.1)、麻石1：4中剂量组(37.1)、石膏高剂量(33.0)、麻石1：1低剂量组(30.8)、麻石1：4低剂量组(28.4)、麻石1：2低剂量组(21.7)、麻黄低剂量组(21.6)、石膏中剂量(10.9)、石膏低剂量(4.6)。通过比较麻黄石膏药对3个配比解热的作用时间与作用强度,我们发现麻黄石膏(1：2)的解热作用最佳,解热持续时间最长。麻黄-石膏(1：2)在解热方面具有协同增效作用。
平喘作用结果麻石1：2高、中剂量组均能延长引喘潜伏期(P<0.01),麻石1：2配伍组作用效果优于麻石1：1、麻石1：2组、单味麻黄、单味石膏组；麻石1：2高、中剂量组、单味麻黄组均能减少EOS数量(p<0.01)；麻石1：2高剂量组和麻石1：4组组均能减少WBC数量(p<0.01)；麻石1：2组高、中、低剂量、麻黄组、石膏组和麻石1：4组均能降低肺干湿重比值(p<0.01)；通过引喘潜伏期、肺干湿重比值、EOS和WBC计数综合分析比较,我们发现麻黄石膏(1：2)的平喘作用最佳,麻黄-石膏(1：2)其合用的效果优于同等剂量下的单味麻黄或石膏。
3.麻黄石膏药对不同比例配伍干预“发热”大鼠尿液代谢组学研究
“发热”大鼠模型的建立及其代谢组学研究通过对模型组和空白组大鼠尿液代谢物组进行PCA分析,两组大鼠的尿液样本可以在PCA的得分图上较为明显的区分开来,提示干酵母诱导的“发热”模型成功,大鼠机体代谢网络发生了明显的变化。为进一步关注干酵母引起的尿液代谢差异,找到与干酵母诱导发热相关的代谢通道的变化,实验采用PLS-DA法对模型组大鼠和正常组大鼠样本重新建模,用以鉴别造成上述分离的差异变量。结果显示,模型组和正常组在PC1上明显分离,并且该模型有较高的解释率和预测率(R2Xcum=0.878, R2Ycum=0.993, Q2Ycum=0.969)。通过上述PLS-DA法对两组大鼠样本重新建模后,根据S-Plot图(图4-6A)中离子的置信度和VIP值可知离子对分类贡献的大小,找到VIP值大于1的与干酵母诱导发热高度相关的代谢物(图4-6B),运用气质工作站的数据库(WILEY275.L及NIST05.L),结合自建的对照品物质库及查询网站(http://www.hmdb.ca/和http://metlin.scripps.edu/metabo_search_alt2.php)查找差异变量。分析结果显示,模型组和对照组共有10个物质有显著性差异,分别是：柠檬酸、酮戊二酸、3-丙酸、苯乙酸、甘氨酸、正丁胺、碳酸、丙氨酸、苯丙酮酸、苯甲酸。
麻黄、石膏及其合煎液对“发热”大鼠的代谢组学研究结果通过对空白组、模型组、麻黄组、麻黄桂枝组、石膏组大鼠尿液代谢物组进行PCA分析,5组大鼠的尿液样本可以在PCA的得分图上较为明显的区分开来,说明来大鼠给予不同处理后,其代谢物谱差异明显。不同处理组的样品之间的细微差异可以通过PLS-DA进行分析研究。
麻黄组和模型组相比,麻黄组中丙二酸、苯甲酸、丁二酸、丙氨酸、甘氨酸、缬氨酸、脯檬酸、辛二酸、尿素、马尿酸、苯丙酸、苯丙氨酸、2-羟基喹啉-羧酸含量升高(P<0.05)。
石膏处理组和模型组相比,石膏中苯甲酸、丁二酸、丙氨酸、3-丙酸、脯氨酸、辛二酸、L-谷氨酸含量升高(P<0.05)。
麻黄-石膏组与模型组相比,麻黄-石膏组中丙二酸、苯甲酸、丁二酸、甘氨酸、3-丙酸、脯氨酸、辛二酸、尿素、马尿酸、苯丙酸、苯丙氨酸、半胱氨酸含量升高(P<0.05)。
麻黄-石膏组与单味石膏相比,丙二酸、苯甲酸、甘氨酸、异亮氨酸、马尿酸、苯丙酸、1-脯氨酸含量升高(P<0.05)。
石膏组与单味麻黄相比,麻黄组中丙二酸、苯甲酸、丁二酸、3-丙酸、琥珀酸、尿素、马尿酸、苯丙酸、1-脯氨酸含量升高(P<0.05)。
对以上这些代谢物含量用单变量方差分析方法进行检验,其结果与PLS-DA的分析结果基本一致。
结论：
通过对麻黄-石膏“药对”进行化学成分、药效及代谢组学三方面的研究,确定麻黄与石膏配伍应用后,麻黄-石膏(1：2)配伍比例组在解热、平喘方面优于麻黄-石膏(1：1)、麻黄-石膏(1：4)配伍组,该最优比例与原方麻杏石甘汤中麻黄-石膏1：2相一致,从化学成分的角度,发现其增效的物质基础与增加其有效成分的含量(总生物碱以及Ca2+的溶出量)相关。推测麻黄-石膏不同配伍比例的药理作用不同,还与其通过抑制或促进某些成分的溶出,从而产生不同比例的物质群有关。代谢组学研究表明,麻黄、石膏、麻黄-石膏3个给药组均在一定程度上改善了干酵母诱导发热大鼠全身异常的代谢状态,其干预作用与调节机体的物质代谢、能量代谢相关,其中麻黄-石膏药对配伍后生物标志物的回调程度最大,表明麻黄-石膏合用比等剂量的单味麻黄或石膏效果好。从代谢组学的角度证实了麻黄-石膏配伍的合理性和优势。
Background
Herb pairs, also called drug pairs, the unique combinations of two relatively fixed herbs in clinic, are the most fundamental and the simplest form of multi-herb therapy. Without altering the basic therapeutic features of multi-herb formulae, herb pairs as the basic composition units of Chinese herbal formulae are of special clinical significance in TCM. The composing principle and manner of Chinese herb pair are based on the nature, taste, meridian distribution of the herbs and effects, which contains a wealth of objective laws. Besides, certain syndrome characteristics and the use of the appropriate treatment method as a precondition,
The meaning of our studying on the composition rules of Herb pairs:(1) Through the ways of modern technologies (for example, the material basis of the mechanism of action, and metabolic processes) to study medicine on the compatibility of Herb pairs, which play an important role in revealing the drug on the compatibility of the objective laws and scientific connotation.(2) To guide Clinical treatment, so it will benefit not only in using existing couplet medicines more efficiently, but also for creating new couplet medicines by the changes of spectrum of disease and symptom-comple.(3)The research of herb compatibility is one of the key points in revealing the compatibility law of complex prescriptions. By study of the compatibility law of Herb pairs, we will illuminate ompatibility laws of complex prescriptions. And it is also provide some basis for the reorganization of the quality and efficiency of the new prescriptions.(4) Herb pairs are vinculum of many complex prescriptions. Through the study the compatibility laws of Herb pairs, it can provide a reference for the other research containing the Herb pairs, and thus open up new avenues for compatibility rules of complex prescriptions.
Objection
Ephedra-Gypsum herb-pair are the most commonly used drug in the "cold and heat" compatibility of Traditional Chinese Medicine. The compatibility-relationship between them is classified to "xiangshi". By explore the changes in the chemical composition of ephedra-gypsum drug-pair, the dose-effect relationship, the change of the pharmacological effects and metabolic pathways and mechanisms in vivo, it will not only further to validate compatibility-relationship between ephedra and gypsum and also play a role in finding the best compatibility proportion. Furthermore, by finding out the relationship of dose-efficiency, time-efficiency and toxic-effects, thus will to bring into full play the effect of drugs, and it is also provide the basis for the clinical treament of traditional Chinese medicine, development of new drug-pairs and drugs.
Contents
To clarify the compatibility rules of Traditional Chinese herb-pair Herba Ephedrae and Gypsum with different compatibility ratio, we compared the changes in the content of active ingredients in water extracts, pharmacological effects and metabonomics. Mainly containing:
1) To clarify the chemical compatibility rules of couplet medicines Ephedr-Gypsum with different compatibility ratio, we compared the content of active ingredients in water extracts before and after different compatibility ratio of Ephedra or Gypsum. We also investigation the fingerprint peaks changes of Ephedra-Gypsum with different compatibility ratio.
2) To clarify the strength of pharmacological effects on the the target of Ephedr-Gypsum with different compatibility ratio, we compared the acute toxicity, antipyretic and anti-asthmatic effects.
3) Establishment the yeast-induced-fever rat model, collecting the urine before and after administration to process the the metabolomics study, and exploring the metabolomics variation and mechanism of Ephedr-Gypsum with different compatibility ratio Methods and materials.
Materials and Methods
The composition of ma-xin-shi-gan decoction in Treatise on Cold Pathogenic Diseases is:"Herba Ephedrae four uncia (removed knots), armeniacae semen fifty piece(decorticate), Radix Glycyrrhizae two uncia, and Gypsum eight uncia." The couplet medicines Ephedra-Gypsum (1:2) in ma-xin-shi-gan decoction were taken as study object, the compatibility ratio of Ephedra-Gypsum was duplication floating up and down basing on the ratio of the couplet medicines reported in the literature.1) Researching on chemical composition in vitro of Ephedra-Gypsum with differe-ent compatibility ratios
A high performance liquid chromatographic method (HPLC) was developed to determination the content of Norpseudoephedrine (NMP), Norephedrine (NME), Ephedrine (E), Pseudoephedrine (PE) and Methylephedrine (ME), which are the major active compounds of Herba Ephedrae.The HPLC method was validated with regard to specificity, linearity, precision, reproducibility. We compared the ephedra alkaloids content of Ephedra-Gypsum (1:1), Ephedra-Gypsum (1:2), and Ephedra-Gypsum (1:4). We also compared the contents of Ca2+in Ephedra-Gypsum (1:1), Ephedra-Gypsum (1:2), Ephedra-Gypsum (1:4), Ephedra and Gypsum water extract by calcium kit. Separations were carried out on an Cosmosil C18(250x4.6mmi.d.,5am) with a flow rate of0.7mLmin-1.The mobile phase was a mixture of H3PO4(containing0.1%triethylamine)-acetonitrile, gradient elution.
2) Study on the major pharmacological effect (major syndrome) of couplet medicines Ephedra-Gypsum with different compatibility ratio.
Acute Toxicity test:the KM mice were randomly divided into normal group, model group, Ephedra group, Ephedra-Gypsum (1:1), Ephedra-Gypsum (1:2), Ephedra-Gypsum (1:4) group, then each group was divided into five dosage group, each group had10mice, half male and half female. Dn, Dm were selected according to the preliminary experiment, the ratio between adjacent groups was1:0.8. The mice were intragastric (ig.) administrated with different dose of tested drugs, and the activities of the mice were observed the following7days, such as diet, urination and defecation, actions and even death etc. The death rates of each group were recorded. LD50and95%confidence interval were calculated with Bliss software.
Antipyretic experiment:the male rats, which had qualified temperature, were randomly divided into normal group, model group, aspirin group, Ephedra (high, middle and low)group, Ephedra-Gypsum (1:1) high, middle and low group, Ephedra-Gypsum (1:2) high, middle and low group, Ephedra-Gypsum (1:4) high, middle and low group, Gypsum (high, middle and low) group, and ma-xin-shi-gan decoction group, each of had6rats. The rats were intragastric (ig.) administrated with different dose of tested drugs. Measuring rectal temperature once per hour after administration, and measured three times. Inhibition rate (%) of each administration group is calculated8h elevated body temperature of rats.
Anti-asthmatic test Use ovalbumin-induced asthma rat model,60SD rats were randomly divided into normal control group, model group, dexamethasone group, Ephedra-Gypsum (1:2) high, middle and low group, ephedra group, Gypsum group, Ephedra-Gypsum (1:1) group and Ephedra-Gypsum (1:4) group, total10groups (n=6). The asthma incubation period, wet and dry weight of lung, EOS and WBC counts was observed.3. Metabonomics study on yeast-induced-fever rats after administration with with different compatibility ratio Ephedra-Gypsum extracts
Metabonomics study on yeast-induced-fever rats:12Wistar rats were randomly divided into normal control group and model group.The fevered rats were prepared by subcutaneous injection of20%yeast. Then6h urine of rats in each group were collected. All the urine samples were derivatized directly. Subsequently, metabolites spectra of these samples were acquired using gas chromatography-mass spectrometry (GC-MS). Identification after proeessed, the data was subjected to SIMCA-P+12.0software(Umetrics AB, Umea Sweden) for principal component analysis(PCA) and principal least squares discriminant analysis (PLS-DA). The modeling and therapeutic effects were judged by PCA visually:distinguishing effect among groups were further inspected by PLS-DA, and the correlation with classification of metabolites were evaluated according to the "variable impotance in the projection (VIP)" value. When a certain metabolite's VIP>1, it would be taken as potential metabolic marker. The differences of potential biomarkers levels in different groups were conducted with univariate statistical analysis using software of SPSS13.0, and those with significant difference were chosen as final biomarkers.
Metabonomics study on yeast-induced-fever rats after administration with with different compatibility ratio Ephedra-Gypsum extracts:30Wistar rats were randomly divided into normal control group, model group, Ephedra-Gypsum (1:2) group, Ephedra group and Gypsum group.The fevered rats were prepared by subcutaneous injection of20%yeast. Then6h urine of rats in each group were collected. All the urine samples were derivatized directly. Subsequently, metabolites spectra of these samples were acquired using gas chromatography-mass spectrometry (GC-MS). Identification after proeessed, the data was subjected to SIMCA-P+12.0software(Umetrics AB, Umea Sweden) for principal component analysis(PCA) and principal least squares discriminant analysis (PLS-DA). The modeling and therapeutic effects were judged by PCA visually:distinguishing effect among groups were further inspected by PLS-DA, and the correlation with classification of metabolites were evaluated according to the "variable impotance in the projection (VIP)" value. When a certain metabolite's VIP>1, it would be taken as potential metabolic marker. The differences of potential biomarkers levels in different groups were conducted with univariate statistical analysis using software of SPSS13.0, and those with significant difference were chosen as final biomarkers.
Results:
1Researching on chemical composition of different compatibility ratio of couplet medicines Ephedra-Gypsum
When compared to Ephedra group, the content of Norpseudoephedrine (NMP), Norephedrine (NME), Ephedrine (E), Pseudoephedrine (PE), Methylephedrine (ME), in Ephedra-Gypsum (1:1) group, Ephedra-Gypsum (1:2) group and Ephedra-Gypsum (1:4) group almost have no significance different. Except the Norephedrine (NME) was lower in Ephedra-Gypsum (1:4) group (P<0.05), compared to Ephedra group.
The results of the contents of Ca2+are as follows:The interaction of Ephedra-gypsum (1:1) was significant (F=22.222, P=0.002), the contents of Ca2+increased after compatibility, which showed Ephedra-gypsum (1:1) had synergistic effect; The interaction of ephedra-the gypsum (1:2) was significant (F=21.144, P=0.002), the contents of Ca2+increased after compatibility, which showed Ephedra-gypsum (1:2) had synergistic effect; The interaction of ephedra-the gypsum (1:2) was significant (F=95.617, P=0.000), the contents of Ca2+decreased after compatibility, which showed Ephedra-gypsum (1:4) had antagonistic action.
2Study on the major pharmacological effect (major syndrome) of Ephedra-Gypsum herb-pair with different compatibility ratio
Acute Toxicity test:the LD50of Ephedra, Ephedra-Gypsum (1:1), Ephedra-Gypsum(1:2), Ephedra-Gypsum(1:4) were131.67g·kg-1,226.43g·kg-1,372.36g·kg-1,530.25g·kg-1, respectively.95%confidence interval were108.23～166.81g·kg-1,192.64～307.64g·kg-1,322.46～442.14g·kg-1,445.68～670.11g·kg-1, respectively.
Antipyretic effect and interaction experiment:Compared with model group, Ephedra, Ephedra-Gypsum (1:1), Ephedra-Gypsum (1:2), Ephedra-Gypsum (1:4) extracts had antipyretic effect. They also showed a good dose-effect relationship. Single Gypsum with large dose had antipyretic effect, but lower doses even cause the raise of body temperature in rats.8h inhibition rate (%) were sorted: Ephedra-Gypsum1:2high-dose group (62.7), Ma-Xing-Shi-Gan Tang (59.7), Ephedra high-dose group (59.0), Ephedra-Gypsum1:2middle-dose group (53.6), Ephedra middle-dose group (49.4), Ephedra-Gypsum1:1high-dose group (47.7), Ephedra-Gypsum1:4high-dose group (47.2), Ephedra-Gypsum1:1middle-dose group (43.1), Ephedra-Gypsum1:4middle-dose group (37.1), gypsum high-dose (33.0), Ephedra-Gypsum1:1low-dose group (30.8), Ephedra-Gypsum1:4low-dose group (28.4), Ephedra-Gypsum1:2low-dose group (21.7), Ephedra low-dose group (21.6), Gypsum middle-dose group (-7.8), gypsum low-dose group (-18.3). Based on the median-effect principle, the interaction of antipyretic effect was analyzed with Calcusyn software.
Anti-asthmatic test:Ephedra-Gypsum1:2high and middle dose group could prolong the latent period of asthma (P<001), Ephedra-Gypsum (1:2) group effect was significantly better than Ephedra-Gypsum (1:1), Ephedra-Gypsum (1:4) of single ephedra, single gypsum group; Ephedra-Gypsum (1:2) high and middle dose group, single ephedra group could significantly reduce the the EOS counts in blood (p <0.01); Ephedra-Gypsum (1:2) high-dose group and Ephedra-Gypsum (1:4) group could significantly reduce the WBC counts (p<0.01); Ephedra-Gypsum (1:2) high, middle and low groups, Ephedra group, gypsum group and Ephedra-Gypsum (1:4) group can significantly reduce lung wet and dry weight ratio (p<0.01); Through analysis and comparison of the latent period of asthma, lung wet and dry weight ratio, EOS and WBC counts, we found that the antiasthmatic effects of Ephedra-Gypsum (1:2) group show better than a single herb ephedra or gypsum in equal doses.
3Metabonomics study on yeast-induced-fever rats after administration with with different compatibility ratio Ephedra-Gypsum extracts
Through the PCA analysis of the model group and blank group, results showed that the model groups and blank group distinguished well, which showed the yeast-induced-fever model success, urinary metabolites changed significantly. To further attention to the urine metabolic differences caused by the dry yeast, to find changes induced fever associated with dry yeast metabolic pathways, experimental re-modeling of the PLS-DA model rats and normal rats samples, used to identify the causethe difference variable for the above separation. The results show that the model group and the normal group on PCl clearly separated, and the model has a high interpretation rate and the predicted rate at (R2Xcum=0.878R2Ycum=0.993, Q2Ycum=0.969).By the PLS-DA method after the re-modeling of the two groups of rats sample, according to the S-Plot in confidence and VIP value that the ion size, find the classification contribution VIP value greater than1were yeast-induced-fever highly related metabolites, the use of temperament workstation database (WILEY275.L NIST05.L), combined with self-built reference standard material library and inquiries Web site(http://www.hmdb. ca/http://metlin.scripps.edu/metabo_search_al t2.php) find the difference variable.The analysis results show that compared with the model group:blank control group the contents of propylamine, glycine, malonic acid phenethyl uric acid, benzeneAcetic acid, hippuric acid significantly increased; the contents of glycine, malonic acid phenethyl uric acid, benzene, acetic acid, hippuric acid significantly decreased.
3.2Metabonomics study on yeast-induced-fever rats after administration with with normal group, model group,hedra-Gypsum(1:2) group, Ephedr group, and Gypsum group
Through the PCA analysis of the model group and blank group, results showed that the model groups, blank group, Ephedra-Gypsum(1:2) group, Ephedr group, and Gypsum group distinguished well. The subtle differences among the treatment groups will further analysis by PLS-DA.3.4Biomakers, which were obtained by PLS-DA analysis of normal group, model group, Ephedrae group, Gypsum group, Ephedra-Gypsum(1:2) group together, including organic acids, fatty acids, amino acids, amines and so on.
Model group compared to blank group, the content of Citric acid, ketoglutaric acid,3-propionic acid, phenylacetic acid, glycine, n-butylamine, carbonic acid, alanine, phenyl pyruvic acid, benzoic acid decreased (P<0.05); The results by univariate analysis are basically consistent with the results of PLS-DA analysis.
Ephedra group compared with the model group, the contents of in Ephedra group Malonic acid, benzoic acid, succinic acid, alanine, glycine, valine, proline citric acid, suberic acid, urea, hippuric acid, benzenepropanoic acid, phenylalanine,2-hydroxy-quinoline-carboxylicacid content significantly increased The results of these metabolites content by univariate analysis are basically consistent with the results of PLS-DA analysis.
Gypsum compared to model group, Alanine,3-propionic acid, proline, suberic acid, benzoic acid, succinic acid, L-glutamic acid decreased (P<0.05). The results by univariate analysis are basically consistent with the results of PLS-DA analysis.
Ephedra-Gypsum group compared with model group, Malonic acid, benzoic acid, succinic acid, glycine,3-propionic acid, proline acid, suberic acid, urea, hippuric acid, benzenepropanoic acid, phenylalanine, cysteine decreased (P<0.05). The results by univariate analysis are basically consistent with the results of PLS-DA analysis.
Ephedra-Gypsum group compared with Gypsum group, Malonic acid, benzoic acid, glycine, isoleucine, leucine, hippuric acid, phenylpropionic acid,1-proline increased (P<0.05) in Ephedra-gypsum group. The results by univariate analysis are basically consistent with the results of PLS-DA analysis.
Ephedra group compared with Gypsum group, Malonic acid, benzoic acid, succinic acid, propionic acid, succinic acid, urea, hippuric acid,3-benzenepropanoic acid,,1-prolineincreased (P<0.05) in Ephedra group. The results by univariate analysis are basically consistent with the results of PLS-DA analysis.
Conclusion:1) Results of chemical composition analysis experiment showed that Ephedra-gypsum (1:4) group significant decrease the content of methyl-ephedrine and Ca2+(P<0.05), but the other two compatibility groups has no significant change. The total contents of alkaloids and Ca2+in Ephedra-gypsum (1:2) group were higher than Ephedra-gypsum (1:1) group, which is conducive to the dissolution of the active ingredient. In the original prescription, Ma-xin-shi-gan Tang, which Ephedra:Gypsum (1:2) compatibility, the foundings provide some basis for the advantages and reasonable of this compatibility proportion(1:2), but the mechanism has yet to be studied further.2Compatibility rules of herb pairs should consider safety and efficacy.
The LD50f Ephedra-Gypsum (1:1) group, Ephedra-Gypsum (1:2) group and Ephedra-Gypsum (1:4) group were greater than the single Ephedra, that is to say, when Ephedra combined with Gypsum, the safety improved. Ephedra-Gypsum (1:1) group, Ephedra-Gypsum (1:2) group and Ephedra-Gypsum (1:4) group have a synergistic effect in antipyretic activity. Ephedra-Gypsum (1:2) is the best compatibility ratio among three. Antiasthma test results showed, among Ephedra-Gypsum (1:1) group, Ephedra-Gypsum (1:2) group and Ephedra-Gypsum (1:4) grou, Ephedra-Gypsum (1:2) group is optimal ratio.
3The metabolomics success applied for yeast-induced-fever rat model, which laid the foundation for further research on antipyretic effect of Traditional Chinese Medicines. Yeast-induced-fever will caused the Krebs cycle disorders, increased anaerobic glycolysis, amino acids excessive consumption and fatty acid metabolism disorders. Ephedra-Gypsum (1:2) group, Ephedra group and Gypsum group were taken PCA analysis together, the results showed that compared with Ephedra group and Gypsum group, the metabolic state of Ephedra-Gypsum (1:2) group was closer to the normal group, which proved that Ephedra-Gypsum (1:2) had a better intervention effect on the yeast-induced fever symptoms. PLS-DA analysis results showed that, the biomarker intervention yeast-induced fever symptoms of Ephedra-Gypsum (1:2) group were similar to normal group, which proved that Ephedra-Gypsum (1:2) group has better antipyretic effect than Ephedra or Gypsum group.
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