麻黄—白术药对主要化学成分、药效及尿液代谢物研究
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摘要
研究背景与意义
药对,又称对药,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。药对组成形式主要是在七情配伍的基础上,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,选择性地将两味或两味以上的中药进行配对,其配伍蕴涵着丰富的客观规律。药对的配伍规律是基于中医药理论体系,能体现疾病证型、病机及药物特点而配合应用的药物间的联系,它是历代医家的经验总结,并经历了从感性认识到理性认识的飞跃,具有普遍性、重复性的特点。
我们进行药对组成规律研究的意义：(1)进一步揭示药对配伍的客观规律与科学内涵,阐明药对配伍的物质基础与作用机理,在更广的范围、更深的层次展现中医治疗疾病的科学原理。(2)指导临床遣方用药,既能更有效地运用已有的药对,亦利于针对疾病谱的变化、病证的发展而创制新药对。(3)随着药对的作用机理与物质基础更加明确,在新药研究中以有效成分或有效部位配伍已越来越多,中西药配伍亦不鲜见,传统配伍的普遍规律同样可以指导新的配伍形式,对中药新产品制剂处方研创具有现实意义。
本药对组成规律研究遵循中医药的“理法方药”诊疗理论,选择麻黄加术汤及麻黄-白术药对为研究对象,按性味的“七情合和”配伍,以药味配比量为切入点,将药对配伍研究分解为：(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)药对配伍对代谢组生物信息干预的研究。(4)运用解析多变量复杂关系的数理统计方法进行分析。通过麻黄-白术药对配伍、化学成分变化、药理效应改变、体内代谢途径与机制几者间的内在联系,揭示药对的组成规律,据此为中医临床遣方用药、创制新药对及开发新药提供理论和实验依据。
目的
通过比较麻黄与白术不同配伍配比对水煎液中有效成分含量的影响,探求麻黄与白术药对的化学变化规律。以期从物质基础的变化来阐述药理效应变化的内因。
通过比较麻黄与白术不同配伍配比对急性毒性、发汗作用、镇痛作用、利尿作用的影响,定量评价麻黄和白术配伍后主要药理效应的变化,以判断药物是否增效减毒。
通过运用代谢组学技术探讨大鼠灌胃麻黄、白术和麻黄—白术药对配伍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白术的相互作用机制。
方法
1.麻黄-白术药对不同配伍配比的体外化学成分研究分析在制备过程(体外)中化学成分数和量的变化,探讨麻黄、白术主要效应成分在制备过程中的理化变化和相互影响。
采用气质联用(GC-MS)考察麻黄水煎液及麻黄—白术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)含量的变化情况；采用高效液相色谱法(HPLC)考察白术水煎液及麻黄—白术药对不同配比共煎液中白术内酯Ⅰ、白术内酯Ⅱ、白术内酯Ⅲ的指纹图谱峰的变化情况。比较麻黄与白术不同比例配伍前后水煎液中有效成分含量的变化。2.麻黄-白术药对不同配比的主要药理效应(主证)比较研究以毒副作用、发汗作用、镇痛作用、利尿作用、药物相互作用等相关指标,从药理效应相互关系方面分析药对配伍(偶合)对靶标效应强度的规律。
急性毒性实验及相互作用研究采用昆明小鼠,按照麻黄、白术、麻黄白术(3：2)、麻黄白术(3：4)、麻黄白术(3：8)分为5个给药组,采用小鼠半数致死量(LD50)测定来评价麻黄、白术及麻黄—白术药对三个配比的急性毒性,无法测得LD50值的,采用测定其最大耐受量(MTD)的方法以获取其安全性信息。
发汗药效实验研究采用SD大鼠,通过比较大鼠腋窝部和足趾部皮肤汗腺,确定了取材部位,建立了定量评价发汗强度的方法。采用汗腺导管面积为指标考察了麻黄-白术药对配伍对大鼠的发汗作用。
镇痛药效实验研究采用昆明小鼠,运用热板镇痛和光辐射热甩尾镇痛法,考察麻黄、白术、麻黄-白术药对及麻黄加术汤全方的镇痛作用；依据中效原理(Chou-Talalay联合指数法),应用Calcusyn统计软件(Biosoft,USA)分析,绘制单味药及二药配伍使用对小鼠痛阈的影响曲线,确定二药的效应(fa)—合用指数(CI)的关系,根据曲线定量评价麻黄与白术镇痛的相互作用
利尿药效实验研究采用SD大鼠,运用水负荷排尿实验-代谢笼法,考察麻黄、白术、麻黄-白术药对大鼠的利尿作用；采用昆明小鼠,运用滤纸称重法,考察麻黄、白术、麻黄-白术药对小鼠的利尿作用,探索药对配伍对于利尿作用的影响。
3.麻黄-白术药对配伍的代谢组学研究选用SD大鼠40只,雄性,随机分为4组,即空白对照组、麻黄组、白术组和麻黄白术3：4组,每组10只。生理盐水、麻黄水煎液、白术水煎液、麻黄—白术药对水煎液给大鼠灌胃,每天收集8h尿液,尿液离心后衍生化。采用GC-MS联用技术测定各组大鼠尿液代谢物谱。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,生成二维矩阵。将矩阵导入模式识别软件Simca-P12.0进行主成分分析(PCA)、偏最小二乘法判别分析(PLS-DA),分析麻黄组、白术组、麻黄-白术药对组生物标志物的差异及水平变化。
4.统计学处理
运用SPSS16.0软件进行统计分析。计量资料以均数±标准差(x±s)表示,两组均数比较采用独立样本t检验；多组均数比较采用单向方差分析(One-way ANOVA),方差齐性采用LSD法；方差不齐,Welch法校正后采用Games-Howell法多重比较。所有检验水平a=0.05(双侧)。
结果
1.麻黄-白术药对不同配伍配比的体外化学成分研究
与麻黄单煎液相比,麻黄白术配伍后,随着白术的配伍比例增大,水煎液中去甲基伪麻黄碱、去甲基麻黄碱、麻黄碱、伪麻黄碱、甲基麻黄碱含量均有所下降。
与白术单煎液相比,麻黄白术配伍后,水煎液中白术内酯Ⅰ、白术内酯Ⅱ、白术内酯Ⅲ均有不同程度的降低。
2.麻黄-白术药对不同配伍配比主要药理效应(主证)比较研究
急性毒性实验麻黄LDso值为79.098g·kg-1,白术和三个药对配伍组均无法测得LD5o值,均测得其最大耐受量(MTD),白术三个配伍组及全方配伍均对麻黄的毒性具有拮抗作用。
发汗药效实验模型动物的选择,以大鼠为优,取材部位选取足趾部皮肤汗腺,取材时间为给药后1h。麻黄有较好的发汗作用,白术不明显,麻黄白术配伍后,对于麻黄的发汗效力有拮抗作用,其中以麻黄白术(3：4)组影响最为显著。
镇痛药效实验与空白组相比,各给药组均不同程度的具有提高小鼠痛阈值,抑制小鼠甩尾的作用,说明单味麻黄、单味白术及麻黄白术配伍后均具有镇痛作用,在相同剂量时,效应强弱顺序为麻黄白术(3：4)组>白术组>麻黄组；与单味药相比,麻黄白术配伍后镇痛作用有所增强,说明麻黄、白术配伍后,镇痛作用为协同作用。
利尿药效实验大鼠的代谢笼法及小鼠的滤纸称重法实验结果均表明,麻黄—白术药对配伍能增加正常大鼠及正常小鼠排尿量,与单味药材相比,药对配伍使其利尿效应增强。
3.代谢组学研究
与空白对照组比较,白术组存在差异的生物标志物有：正丙胺、α-酮戊二酸、己二酸、柠檬酸、马尿酸、色氨酸、甲基黄嘌呤、酪氨酸；麻黄组与空白对照组存在差异的生物标志物有：正丙胺、马尿酸、丙氨酸、色氨酸；麻黄白术(3：4)组与空白对照组存在差异的生物标志物有：正丙胺、α-酮戊二酸、己二酸、柠檬酸、马尿酸、半胱氨酸、乌头酸、丙氨酸、氨基马尿酸、甲基黄嘌呤、酪氨酸、色氨酸。与空白对照组比较,生物标志物水平显著下调的有：正丙胺、α-酮戊二酸、己二酸、柠檬酸、马尿酸；生物标志水平显著上调的有：半胱氨酸、乌头酸、丙氨酸、氨基马尿酸、甲基黄嘌呤、酪氨酸、色氨酸。
结论
1化学成分分析实验结果表明,麻黄与白术配伍后,麻黄中各有效成分的溶出率明显减少,其溶出率与白术在药对中所占的比例有关,比例越大,麻黄碱类成分溶出率越小；药对配伍后,白术中各有效成分的溶出率与白术单煎液相比也明显减少,麻黄白术配伍后对彼此有效成分的溶出有一定的抑制作用。
2药对配伍使用多起到增效减毒的作用。结合急性毒性实验、发汗实验、镇痛实验、利尿实验等研究结果,对麻黄-白术药对不同配伍配比进行分析。麻黄白术配伍使用后,对麻黄的毒性有拮抗作用,发汗作用有拮抗作用,镇痛作用有协同作用,利尿作用有协同作用。其中麻黄白术(3：4)配伍的减毒增效作用十分明显。这与经方麻黄加术汤中麻黄和白术的配伍比例相吻合。
3药对配伍对大鼠尿液代谢标志物的影响
麻黄、白术单味药影响的代谢标志物不同,麻黄-白术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,可能是二者在配伍使用时药理效应发生变化的影响因素。
Background and Significance
Couplet medicines, also called herbal pairs, which is made of two herbas, individually made of three drugs or more, is a relatively fixed concerted application form in clinic. Composition of couplet medicines mainly based on the seven emotions concerted application, on the premise of several syndrome characteristics and the use of the appropriate method of treatment, according to the nature and flavour, ascending and descending of Chinese medicinal herbs, selective pairing of two quantity unit of traditional Chinese medicinal herbs, concerted application of which contains a wealth of objective rules. The concerted application rules of couplet medicines are based on the theoretical system of traditional Chinese medicine and pharmacy, which to reflect the link between drugs with the application in disease syndrome type, pathogenesis and characteristics of drugs, it is the experience of ancient physicians, goes through a leap from perceptual to rational knowledge, universally and reproducible.
The meaning of our studying on the composition rules of couplet medicines:(1) further revealling the objective law and scientific connotation of concerted application of couplet medicines, clarifying its material basis and action mechanism, demonstrate the scientific principles of treatment of traditional Chinese medicine in a broader context, a deeper level;(2) guiding clinical prescription, conducing in using existing couplet medicines more efficiently, and for new couplet medicines aiming at changes in the spectrum of disease and syndromes development;(3) with the action mechanism and material basis of couplet medicines are more explicit, it's being increasingly in new drug research that making concerted application with the active principle, available composition or available fraction, even concerted application of Chinese material medica and Western medicine are common, the rules of the traditional concerted application can also guide the new form of concerted application, which also has practical significance for the research and innovation of new product of traditional Chinese medicine formulations.
The researching of composition rules of this couplet medicines was followed the clinics theory of traditional Chinese medicine and pharmacy called "principle-method-recipe-medicines", classical prescription ma-huang-jia-zhu decoction and Herba Ephedrae-Atractylodes macrocephala were selected as study object, and were paired accoding to seven emotions harmony and integration of nature and flavor, making different ratio of medicinal ingredients as the entry point, the research on compability of couplet medicines was decomposed to:(1) study on the changes of chemical compositions in vitro before and after compatibility of couplet medicines.(2) study on the main pharmacological effects (toxicity) comparison.(3) study on the compatibility of couplet medicines by metabinomics.(4) analysis by using analytical the complex relationship of multi-variable mathematical statistical methods. By studied the intrinsic link among the compatibility, changes of chemical compositions, pharmacological effects, and the in vivo metabolic pathways and mechanisms of couplet medicines Herba Ephedrae-Atractylodes macrocephala, we revealled the composition rules of couplet medicines and take it as a theoretical and experimental basis for clinical prescription of traditional Chinese medicine, development of new couplet medicines and drugs.
Objection
Clarify the chemical compatibility rules of couplet medicines Ephedrae and Atractylodes macrocephala compatibility ratio, by comparing the content of active ingredients in water extracts before and after different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala. View to explain the internal cause of pharmacological effect variation from the perspective of material basis variation.
Quantitative evaluating the variation of main pharmacological effect after compatibility of Herba Ephedrae and Atractylodes macrocephala, in order to judging whether effect-enhancing and toxicity-reducing or not, by comparing the effect of the acute toxicity, diaphoretic function, analgesia and diuretic effect.
To explore the effects of Herba Ephedrae, Atractylodes macrocephala and Herba Ephedrae-Atractylodes macrocephala administrated orallym on biomarkers in urine by metabo-lomics technology, and find the biomarkers that may characterize this process of changes, which would provide testal evidence for the mechanism of interaction between Herba Ephedrae and Atractylodes macrocephala from system and overall perspective.
Method
1. Researching on chemical composition in vitro of different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala Analyzed the content and quantity changes of chemical compositions during the preparation process (in vitro). Investigating the physical and chemical changes of major active ingredients of Herba Ephedrae and Atractylodes macrocephala during the preparation process.
Examined the content changes of Ephedra alkaloids(Norpseudoephedrine (NMP),Norephedrine(NME),Ephedrine(E),Pseudoephedrine(PE)and Methylephedrine (ME)) by GC-MS, which is in water extract of Herba Ephedrae and different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala; Examined the Fingerprint peak changes of Atractylenolide I, Atractylenolide II, Atractylodes lactone III by HPLC, which is in water extract of Atractylodes macrocephala and different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala. To comparing the change of the active ingredient content before and after different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala.
2. Researching on the major pharmacological effect (major syndrome) of couplet medicines Herba Ephedrae-Atractylodes macrocephala on the different compatibility ratio. With the side effects, diaphoretic function, analgesia and diuretic effect, interactions as related indicators, the rules of intensity that couplet medicines compatibility (coupling) effect on the target were analyzed by the relationship of pharmacologic effects of different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala.
Acute Toxicity test The kunming mice were randomly divided into5groups, which are Herba Ephedrae group, Atractylodes macrocephala group, Herba Ephedrae-Atractylodes macrocephala(3:2) group, Herba Ephedrae-Atractylodes macrocephala (3:4) group and Ephedrae-Atractylodes macrocephala (3:8) group.(LD50) was used to evaluate the acute toxicit of Ephedra, Atractylodes and couplet medicines of Herba Ephedrae and Atractylodes macrocephala. if LD50can not be measured, the method of determining MTD was used to obtain security information
Researching on pharmacodynamic test of diaphoretic function Using normal SD rats, by comparing rats axillary and toe sweat gland, having determined the material positions, to establish a method of quantitative evaluation of sweating strength. Using incidence of sweat duct area and sweat glands vacuoles to examined diaphoretic function of couplet medicines of Herba Ephedrae-Atractylodes macrocephala.
Researching on pharmacodynamic test of analgesia effect Observe and study the analgesia effect of Herba Ephedrae, Atractylodes macrocephala, couplet medicines of Herba Ephedrae-Atractylodes macrocephala, Ma-huang-jia-zhu decoction, by using hot-plate analgesia and light radiant heat tail-flick analgesia with Kunming mice; according to the median-effect principle (Chou-Talalay index method), applying Calcusyn statistical software to dispose, to draw the curve of single herb and drug compatibility on the pain threshold, determining the relationship of two drugs effects-combination index (CI), according to the curve to quantitative evaluating the interaction of Analgesia effect of Herba Ephedrae and Atractylodes macrocephala.
Researching on pharmacodynamic test of diuretic effect Useing the water load urination tests-metabolic cages method with normal SD rats, to examin diuretic effect of Herba Ephedrae, Atractylodes macrocephala, Couplet medicines of Herba Ephedrae-Atractylodes macrocephala; useing filter paper weighing method with Kunming mice, to examin diuretic effect of Herba Ephedrae, Atractylodes macrocephala, Couplet medicines of Herba Ephedrae-Atractylodes macrocephala, in order to explore the influence on diuretic effect after compatibility of medicines.
3. Researching on water extracts of couplet medicines Herba Ephedrae-Atractylodes macrocephala by metabonomics40male SD rats, were randomly divided into four groups, which are the control group, Herba Ephedra group, Atractylodes macrocephala group and Herba Ephedra-Atractylodes macrocephala (3:4)group, n=10. The saline and decoction of Herba Ephedrae, Atractylodes macrocephala, Couplet medicines of Herba Ephedrae-Atractylodes macrocephala were made to rats intragastricly.8h urine of rats in each group were collected everyday. 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, analyzing the significant differences and change of biomarker in different groups.
4. Statistical analysis The differences of samples in different groups were conducted with univariate statistical analysis using software of SPSS16.0. The measurement data was presented as (x±s). Two groups means were compared with independent-samples T test; multiple groups means were compared with One-Way ANOVA and the pairwise comparison was peformed with LSD test if the variance of the data was homogeneous, otherwise with Games-Howell method. All significant levels were0.05(two tailed).
Result
1. Researching on chemical composition of different compatibility ratio of couplet medicines Herba Ephedrae-Atractylodes macrocephala Compared to single ephedra decoction, the content of five Herba Ephedra alkaloids were reduced in varying degrees as proportion of Atractylodes macrocephala increased after Herba Ephedra-Atractylodes macrocephala matched. As the content of Atractylodes macrocephala increased, the content of demethylation pseudoephedrine, ephedrine, ephedrine, pseudoephedrine, ephedrine were declined. Compared to single Atractylodes macrocephala decoction, the content of atractylenolide Ⅰ, atractylenolide Ⅱ, atractylenolide Ⅲ were reduced in varying degrees.
2. Comparative researching on main pharmacological effects of different compatibility ratio of couplet medicines Ephedrae-Atractylodes macrocephala
Acute toxicity test:The LD50of Herba Ephedrae were79.098g·kg-1, the LD50of Atractylodes macrocephala and three drug compatibility groups can not be measured and measured MTD instead. Three Atractylodes macrocephala compatibility groups and the full compatibility group have a antagonism influence on toxicity of Herba Ephedra.
Sweating pharmacodynamic test:The choice of model animals, rats is fitter. Selecting toe sweat gland is fitter, sampling time is1h after administration. Herba Ephedra has a good effect of weating, Atractylodes macrocephala is not obvious. Couplet medicines of Herba Ephedrae and Atractylodes macrocephala has a antagonism influence on weating of Herba Ephedra and Herba Ephedra-Atractylodes macrocephala (3:4) group is the most significant.
Analgesic efficacy test:Compared to control group, each group has a effect of improving pain threshold and inhibiting drift, suggesting each group has analgesic effect. At the same dose, the effect was in the order as follows:Herba Ephedra-Atractylodes macrocephala (3:4) group> the Atractylodes macrocephala group> Herba Ephedra group; compared to single herb, analgesic effect is enhanced after Herba Ephedra-Atractylodes macrocephala matched, suggesting analgesic effect play a synergistic role.
Diuretic efficacy test:The results of metabolic cages and mice weighing method of filter pape test suggesting urine output was increased after Herba Ephedra-Atractylodes macrocephala matched. Compared to the single herb, diuretic effect has enhanced after Herba Ephedr-Atractylodes macrocephala matched.
3. Researching on Metabonomics
Compared with the control group, the biomarkers down-regulated by Atracty-lodes macrocephala 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-Atractylodes macrocephala were α-ketoglutaric acid, adipic acid, aconitic acid, citric acid, and cysteine, those up-regulated were alanine, high citric acid, methyl xant-hine, tyrosine and tryptophan.
Conclusion
1.The results of chemical composition analysis test show that the dissolution rate of the active ingredient is significant reduced after Herba Ephedra-Atractylodes macrocephala matched. The greater is the proportion, The lower is the dissolution rate; compared to single Atractylodes macrocephala decoction, the dissolution rate of the active ingredient has significant reduced after Herba Ephedra-Atractylodes macrocephala matched. There is an antagonism influence on the dissolution of each active ingredient after Herba Ephedra-Atractylodes macrocephala matched.
2. There is an influence on effect-enhancing and toxicity-reducing after Herba Ephedra-Atractylodes macrocephala matched. Combine to the results of acute toxicity test, sweating test, analgesic and diuretic test, an analysis of different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala has made. There is an antagonism influence on toxicity and sweating, a synergistic influence on analgesic test and diuretic test after Herba Ephedra-Atractylodes macrocephala matched. Couplet medicines of Herba Ephedrae and Atractylodes macrocephala (3:4) group has a significant effect of effect-enhancing and toxicity-reducing. The proportion of Herba Ephedra and Atractylodes macrocephala which has a significant effect is identical to classical prescription of Ma-huang-jia-zhu decoction.
3. The influence on urine metabolic markers which after Herba Ephedra-Atractylodes macrocephala matched are different between Herba Ephedra and Atractylodes macrocephala. Couplet medicines of Herba Ephedra and Atractylodes macrocephala not only have an influence on both each metabolic markers but also have an influence on another3metabolic markers. It may related to the changes of pharmacological effect when both of two used together.
药对,又称对药,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。药对组成形式主要是在七情配伍的基础上,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,选择性地将两味或两味以上的中药进行配对,其配伍蕴涵着丰富的客观规律。药对的配伍规律是基于中医药理论体系,能体现疾病证型、病机及药物特点而配合应用的药物间的联系,它是历代医家的经验总结,并经历了从感性认识到理性认识的飞跃,具有普遍性、重复性的特点。
我们进行药对组成规律研究的意义：(1)进一步揭示药对配伍的客观规律与科学内涵,阐明药对配伍的物质基础与作用机理,在更广的范围、更深的层次展现中医治疗疾病的科学原理。(2)指导临床遣方用药,既能更有效地运用已有的药对,亦利于针对疾病谱的变化、病证的发展而创制新药对。(3)随着药对的作用机理与物质基础更加明确,在新药研究中以有效成分或有效部位配伍已越来越多,中西药配伍亦不鲜见,传统配伍的普遍规律同样可以指导新的配伍形式,对中药新产品制剂处方研创具有现实意义。
本药对组成规律研究遵循中医药的“理法方药”诊疗理论,选择麻黄加术汤及麻黄-白术药对为研究对象,按性味的“七情合和”配伍,以药味配比量为切入点,将药对配伍研究分解为：(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)药对配伍对代谢组生物信息干预的研究。(4)运用解析多变量复杂关系的数理统计方法进行分析。通过麻黄-白术药对配伍、化学成分变化、药理效应改变、体内代谢途径与机制几者间的内在联系,揭示药对的组成规律,据此为中医临床遣方用药、创制新药对及开发新药提供理论和实验依据。
目的
通过比较麻黄与白术不同配伍配比对水煎液中有效成分含量的影响,探求麻黄与白术药对的化学变化规律。以期从物质基础的变化来阐述药理效应变化的内因。
通过比较麻黄与白术不同配伍配比对急性毒性、发汗作用、镇痛作用、利尿作用的影响,定量评价麻黄和白术配伍后主要药理效应的变化,以判断药物是否增效减毒。
通过运用代谢组学技术探讨大鼠灌胃麻黄、白术和麻黄—白术药对配伍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白术的相互作用机制。
方法
1.麻黄-白术药对不同配伍配比的体外化学成分研究分析在制备过程(体外)中化学成分数和量的变化,探讨麻黄、白术主要效应成分在制备过程中的理化变化和相互影响。
采用气质联用(GC-MS)考察麻黄水煎液及麻黄—白术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)含量的变化情况；采用高效液相色谱法(HPLC)考察白术水煎液及麻黄—白术药对不同配比共煎液中白术内酯Ⅰ、白术内酯Ⅱ、白术内酯Ⅲ的指纹图谱峰的变化情况。比较麻黄与白术不同比例配伍前后水煎液中有效成分含量的变化。2.麻黄-白术药对不同配比的主要药理效应(主证)比较研究以毒副作用、发汗作用、镇痛作用、利尿作用、药物相互作用等相关指标,从药理效应相互关系方面分析药对配伍(偶合)对靶标效应强度的规律。
急性毒性实验及相互作用研究采用昆明小鼠,按照麻黄、白术、麻黄白术(3：2)、麻黄白术(3：4)、麻黄白术(3：8)分为5个给药组,采用小鼠半数致死量(LD50)测定来评价麻黄、白术及麻黄—白术药对三个配比的急性毒性,无法测得LD50值的,采用测定其最大耐受量(MTD)的方法以获取其安全性信息。
发汗药效实验研究采用SD大鼠,通过比较大鼠腋窝部和足趾部皮肤汗腺,确定了取材部位,建立了定量评价发汗强度的方法。采用汗腺导管面积为指标考察了麻黄-白术药对配伍对大鼠的发汗作用。
镇痛药效实验研究采用昆明小鼠,运用热板镇痛和光辐射热甩尾镇痛法,考察麻黄、白术、麻黄-白术药对及麻黄加术汤全方的镇痛作用；依据中效原理(Chou-Talalay联合指数法),应用Calcusyn统计软件(Biosoft,USA)分析,绘制单味药及二药配伍使用对小鼠痛阈的影响曲线,确定二药的效应(fa)—合用指数(CI)的关系,根据曲线定量评价麻黄与白术镇痛的相互作用
利尿药效实验研究采用SD大鼠,运用水负荷排尿实验-代谢笼法,考察麻黄、白术、麻黄-白术药对大鼠的利尿作用；采用昆明小鼠,运用滤纸称重法,考察麻黄、白术、麻黄-白术药对小鼠的利尿作用,探索药对配伍对于利尿作用的影响。
3.麻黄-白术药对配伍的代谢组学研究选用SD大鼠40只,雄性,随机分为4组,即空白对照组、麻黄组、白术组和麻黄白术3：4组,每组10只。生理盐水、麻黄水煎液、白术水煎液、麻黄—白术药对水煎液给大鼠灌胃,每天收集8h尿液,尿液离心后衍生化。采用GC-MS联用技术测定各组大鼠尿液代谢物谱。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,生成二维矩阵。将矩阵导入模式识别软件Simca-P12.0进行主成分分析(PCA)、偏最小二乘法判别分析(PLS-DA),分析麻黄组、白术组、麻黄-白术药对组生物标志物的差异及水平变化。
4.统计学处理
运用SPSS16.0软件进行统计分析。计量资料以均数±标准差(x±s)表示,两组均数比较采用独立样本t检验；多组均数比较采用单向方差分析(One-way ANOVA),方差齐性采用LSD法；方差不齐,Welch法校正后采用Games-Howell法多重比较。所有检验水平a=0.05(双侧)。
结果
1.麻黄-白术药对不同配伍配比的体外化学成分研究
与麻黄单煎液相比,麻黄白术配伍后,随着白术的配伍比例增大,水煎液中去甲基伪麻黄碱、去甲基麻黄碱、麻黄碱、伪麻黄碱、甲基麻黄碱含量均有所下降。
与白术单煎液相比,麻黄白术配伍后,水煎液中白术内酯Ⅰ、白术内酯Ⅱ、白术内酯Ⅲ均有不同程度的降低。
2.麻黄-白术药对不同配伍配比主要药理效应(主证)比较研究
急性毒性实验麻黄LDso值为79.098g·kg-1,白术和三个药对配伍组均无法测得LD5o值,均测得其最大耐受量(MTD),白术三个配伍组及全方配伍均对麻黄的毒性具有拮抗作用。
发汗药效实验模型动物的选择,以大鼠为优,取材部位选取足趾部皮肤汗腺,取材时间为给药后1h。麻黄有较好的发汗作用,白术不明显,麻黄白术配伍后,对于麻黄的发汗效力有拮抗作用,其中以麻黄白术(3：4)组影响最为显著。
镇痛药效实验与空白组相比,各给药组均不同程度的具有提高小鼠痛阈值,抑制小鼠甩尾的作用,说明单味麻黄、单味白术及麻黄白术配伍后均具有镇痛作用,在相同剂量时,效应强弱顺序为麻黄白术(3：4)组>白术组>麻黄组；与单味药相比,麻黄白术配伍后镇痛作用有所增强,说明麻黄、白术配伍后,镇痛作用为协同作用。
利尿药效实验大鼠的代谢笼法及小鼠的滤纸称重法实验结果均表明,麻黄—白术药对配伍能增加正常大鼠及正常小鼠排尿量,与单味药材相比,药对配伍使其利尿效应增强。
3.代谢组学研究
与空白对照组比较,白术组存在差异的生物标志物有：正丙胺、α-酮戊二酸、己二酸、柠檬酸、马尿酸、色氨酸、甲基黄嘌呤、酪氨酸；麻黄组与空白对照组存在差异的生物标志物有：正丙胺、马尿酸、丙氨酸、色氨酸；麻黄白术(3：4)组与空白对照组存在差异的生物标志物有：正丙胺、α-酮戊二酸、己二酸、柠檬酸、马尿酸、半胱氨酸、乌头酸、丙氨酸、氨基马尿酸、甲基黄嘌呤、酪氨酸、色氨酸。与空白对照组比较,生物标志物水平显著下调的有：正丙胺、α-酮戊二酸、己二酸、柠檬酸、马尿酸；生物标志水平显著上调的有：半胱氨酸、乌头酸、丙氨酸、氨基马尿酸、甲基黄嘌呤、酪氨酸、色氨酸。
结论
1化学成分分析实验结果表明,麻黄与白术配伍后,麻黄中各有效成分的溶出率明显减少,其溶出率与白术在药对中所占的比例有关,比例越大,麻黄碱类成分溶出率越小；药对配伍后,白术中各有效成分的溶出率与白术单煎液相比也明显减少,麻黄白术配伍后对彼此有效成分的溶出有一定的抑制作用。
2药对配伍使用多起到增效减毒的作用。结合急性毒性实验、发汗实验、镇痛实验、利尿实验等研究结果,对麻黄-白术药对不同配伍配比进行分析。麻黄白术配伍使用后,对麻黄的毒性有拮抗作用,发汗作用有拮抗作用,镇痛作用有协同作用,利尿作用有协同作用。其中麻黄白术(3：4)配伍的减毒增效作用十分明显。这与经方麻黄加术汤中麻黄和白术的配伍比例相吻合。
3药对配伍对大鼠尿液代谢标志物的影响
麻黄、白术单味药影响的代谢标志物不同,麻黄-白术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,可能是二者在配伍使用时药理效应发生变化的影响因素。
Background and Significance
Couplet medicines, also called herbal pairs, which is made of two herbas, individually made of three drugs or more, is a relatively fixed concerted application form in clinic. Composition of couplet medicines mainly based on the seven emotions concerted application, on the premise of several syndrome characteristics and the use of the appropriate method of treatment, according to the nature and flavour, ascending and descending of Chinese medicinal herbs, selective pairing of two quantity unit of traditional Chinese medicinal herbs, concerted application of which contains a wealth of objective rules. The concerted application rules of couplet medicines are based on the theoretical system of traditional Chinese medicine and pharmacy, which to reflect the link between drugs with the application in disease syndrome type, pathogenesis and characteristics of drugs, it is the experience of ancient physicians, goes through a leap from perceptual to rational knowledge, universally and reproducible.
The meaning of our studying on the composition rules of couplet medicines:(1) further revealling the objective law and scientific connotation of concerted application of couplet medicines, clarifying its material basis and action mechanism, demonstrate the scientific principles of treatment of traditional Chinese medicine in a broader context, a deeper level;(2) guiding clinical prescription, conducing in using existing couplet medicines more efficiently, and for new couplet medicines aiming at changes in the spectrum of disease and syndromes development;(3) with the action mechanism and material basis of couplet medicines are more explicit, it's being increasingly in new drug research that making concerted application with the active principle, available composition or available fraction, even concerted application of Chinese material medica and Western medicine are common, the rules of the traditional concerted application can also guide the new form of concerted application, which also has practical significance for the research and innovation of new product of traditional Chinese medicine formulations.
The researching of composition rules of this couplet medicines was followed the clinics theory of traditional Chinese medicine and pharmacy called "principle-method-recipe-medicines", classical prescription ma-huang-jia-zhu decoction and Herba Ephedrae-Atractylodes macrocephala were selected as study object, and were paired accoding to seven emotions harmony and integration of nature and flavor, making different ratio of medicinal ingredients as the entry point, the research on compability of couplet medicines was decomposed to:(1) study on the changes of chemical compositions in vitro before and after compatibility of couplet medicines.(2) study on the main pharmacological effects (toxicity) comparison.(3) study on the compatibility of couplet medicines by metabinomics.(4) analysis by using analytical the complex relationship of multi-variable mathematical statistical methods. By studied the intrinsic link among the compatibility, changes of chemical compositions, pharmacological effects, and the in vivo metabolic pathways and mechanisms of couplet medicines Herba Ephedrae-Atractylodes macrocephala, we revealled the composition rules of couplet medicines and take it as a theoretical and experimental basis for clinical prescription of traditional Chinese medicine, development of new couplet medicines and drugs.
Objection
Clarify the chemical compatibility rules of couplet medicines Ephedrae and Atractylodes macrocephala compatibility ratio, by comparing the content of active ingredients in water extracts before and after different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala. View to explain the internal cause of pharmacological effect variation from the perspective of material basis variation.
Quantitative evaluating the variation of main pharmacological effect after compatibility of Herba Ephedrae and Atractylodes macrocephala, in order to judging whether effect-enhancing and toxicity-reducing or not, by comparing the effect of the acute toxicity, diaphoretic function, analgesia and diuretic effect.
To explore the effects of Herba Ephedrae, Atractylodes macrocephala and Herba Ephedrae-Atractylodes macrocephala administrated orallym on biomarkers in urine by metabo-lomics technology, and find the biomarkers that may characterize this process of changes, which would provide testal evidence for the mechanism of interaction between Herba Ephedrae and Atractylodes macrocephala from system and overall perspective.
Method
1. Researching on chemical composition in vitro of different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala Analyzed the content and quantity changes of chemical compositions during the preparation process (in vitro). Investigating the physical and chemical changes of major active ingredients of Herba Ephedrae and Atractylodes macrocephala during the preparation process.
Examined the content changes of Ephedra alkaloids(Norpseudoephedrine (NMP),Norephedrine(NME),Ephedrine(E),Pseudoephedrine(PE)and Methylephedrine (ME)) by GC-MS, which is in water extract of Herba Ephedrae and different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala; Examined the Fingerprint peak changes of Atractylenolide I, Atractylenolide II, Atractylodes lactone III by HPLC, which is in water extract of Atractylodes macrocephala and different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala. To comparing the change of the active ingredient content before and after different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala.
2. Researching on the major pharmacological effect (major syndrome) of couplet medicines Herba Ephedrae-Atractylodes macrocephala on the different compatibility ratio. With the side effects, diaphoretic function, analgesia and diuretic effect, interactions as related indicators, the rules of intensity that couplet medicines compatibility (coupling) effect on the target were analyzed by the relationship of pharmacologic effects of different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala.
Acute Toxicity test The kunming mice were randomly divided into5groups, which are Herba Ephedrae group, Atractylodes macrocephala group, Herba Ephedrae-Atractylodes macrocephala(3:2) group, Herba Ephedrae-Atractylodes macrocephala (3:4) group and Ephedrae-Atractylodes macrocephala (3:8) group.(LD50) was used to evaluate the acute toxicit of Ephedra, Atractylodes and couplet medicines of Herba Ephedrae and Atractylodes macrocephala. if LD50can not be measured, the method of determining MTD was used to obtain security information
Researching on pharmacodynamic test of diaphoretic function Using normal SD rats, by comparing rats axillary and toe sweat gland, having determined the material positions, to establish a method of quantitative evaluation of sweating strength. Using incidence of sweat duct area and sweat glands vacuoles to examined diaphoretic function of couplet medicines of Herba Ephedrae-Atractylodes macrocephala.
Researching on pharmacodynamic test of analgesia effect Observe and study the analgesia effect of Herba Ephedrae, Atractylodes macrocephala, couplet medicines of Herba Ephedrae-Atractylodes macrocephala, Ma-huang-jia-zhu decoction, by using hot-plate analgesia and light radiant heat tail-flick analgesia with Kunming mice; according to the median-effect principle (Chou-Talalay index method), applying Calcusyn statistical software to dispose, to draw the curve of single herb and drug compatibility on the pain threshold, determining the relationship of two drugs effects-combination index (CI), according to the curve to quantitative evaluating the interaction of Analgesia effect of Herba Ephedrae and Atractylodes macrocephala.
Researching on pharmacodynamic test of diuretic effect Useing the water load urination tests-metabolic cages method with normal SD rats, to examin diuretic effect of Herba Ephedrae, Atractylodes macrocephala, Couplet medicines of Herba Ephedrae-Atractylodes macrocephala; useing filter paper weighing method with Kunming mice, to examin diuretic effect of Herba Ephedrae, Atractylodes macrocephala, Couplet medicines of Herba Ephedrae-Atractylodes macrocephala, in order to explore the influence on diuretic effect after compatibility of medicines.
3. Researching on water extracts of couplet medicines Herba Ephedrae-Atractylodes macrocephala by metabonomics40male SD rats, were randomly divided into four groups, which are the control group, Herba Ephedra group, Atractylodes macrocephala group and Herba Ephedra-Atractylodes macrocephala (3:4)group, n=10. The saline and decoction of Herba Ephedrae, Atractylodes macrocephala, Couplet medicines of Herba Ephedrae-Atractylodes macrocephala were made to rats intragastricly.8h urine of rats in each group were collected everyday. 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, analyzing the significant differences and change of biomarker in different groups.
4. Statistical analysis The differences of samples in different groups were conducted with univariate statistical analysis using software of SPSS16.0. The measurement data was presented as (x±s). Two groups means were compared with independent-samples T test; multiple groups means were compared with One-Way ANOVA and the pairwise comparison was peformed with LSD test if the variance of the data was homogeneous, otherwise with Games-Howell method. All significant levels were0.05(two tailed).
Result
1. Researching on chemical composition of different compatibility ratio of couplet medicines Herba Ephedrae-Atractylodes macrocephala Compared to single ephedra decoction, the content of five Herba Ephedra alkaloids were reduced in varying degrees as proportion of Atractylodes macrocephala increased after Herba Ephedra-Atractylodes macrocephala matched. As the content of Atractylodes macrocephala increased, the content of demethylation pseudoephedrine, ephedrine, ephedrine, pseudoephedrine, ephedrine were declined. Compared to single Atractylodes macrocephala decoction, the content of atractylenolide Ⅰ, atractylenolide Ⅱ, atractylenolide Ⅲ were reduced in varying degrees.
2. Comparative researching on main pharmacological effects of different compatibility ratio of couplet medicines Ephedrae-Atractylodes macrocephala
Acute toxicity test:The LD50of Herba Ephedrae were79.098g·kg-1, the LD50of Atractylodes macrocephala and three drug compatibility groups can not be measured and measured MTD instead. Three Atractylodes macrocephala compatibility groups and the full compatibility group have a antagonism influence on toxicity of Herba Ephedra.
Sweating pharmacodynamic test:The choice of model animals, rats is fitter. Selecting toe sweat gland is fitter, sampling time is1h after administration. Herba Ephedra has a good effect of weating, Atractylodes macrocephala is not obvious. Couplet medicines of Herba Ephedrae and Atractylodes macrocephala has a antagonism influence on weating of Herba Ephedra and Herba Ephedra-Atractylodes macrocephala (3:4) group is the most significant.
Analgesic efficacy test:Compared to control group, each group has a effect of improving pain threshold and inhibiting drift, suggesting each group has analgesic effect. At the same dose, the effect was in the order as follows:Herba Ephedra-Atractylodes macrocephala (3:4) group> the Atractylodes macrocephala group> Herba Ephedra group; compared to single herb, analgesic effect is enhanced after Herba Ephedra-Atractylodes macrocephala matched, suggesting analgesic effect play a synergistic role.
Diuretic efficacy test:The results of metabolic cages and mice weighing method of filter pape test suggesting urine output was increased after Herba Ephedra-Atractylodes macrocephala matched. Compared to the single herb, diuretic effect has enhanced after Herba Ephedr-Atractylodes macrocephala matched.
3. Researching on Metabonomics
Compared with the control group, the biomarkers down-regulated by Atracty-lodes macrocephala 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-Atractylodes macrocephala were α-ketoglutaric acid, adipic acid, aconitic acid, citric acid, and cysteine, those up-regulated were alanine, high citric acid, methyl xant-hine, tyrosine and tryptophan.
Conclusion
1.The results of chemical composition analysis test show that the dissolution rate of the active ingredient is significant reduced after Herba Ephedra-Atractylodes macrocephala matched. The greater is the proportion, The lower is the dissolution rate; compared to single Atractylodes macrocephala decoction, the dissolution rate of the active ingredient has significant reduced after Herba Ephedra-Atractylodes macrocephala matched. There is an antagonism influence on the dissolution of each active ingredient after Herba Ephedra-Atractylodes macrocephala matched.
2. There is an influence on effect-enhancing and toxicity-reducing after Herba Ephedra-Atractylodes macrocephala matched. Combine to the results of acute toxicity test, sweating test, analgesic and diuretic test, an analysis of different compatibility ratio of Herba Ephedrae and Atractylodes macrocephala has made. There is an antagonism influence on toxicity and sweating, a synergistic influence on analgesic test and diuretic test after Herba Ephedra-Atractylodes macrocephala matched. Couplet medicines of Herba Ephedrae and Atractylodes macrocephala (3:4) group has a significant effect of effect-enhancing and toxicity-reducing. The proportion of Herba Ephedra and Atractylodes macrocephala which has a significant effect is identical to classical prescription of Ma-huang-jia-zhu decoction.
3. The influence on urine metabolic markers which after Herba Ephedra-Atractylodes macrocephala matched are different between Herba Ephedra and Atractylodes macrocephala. Couplet medicines of Herba Ephedra and Atractylodes macrocephala not only have an influence on both each metabolic markers but also have an influence on another3metabolic markers. It may related to the changes of pharmacological effect when both of two used together.
引文
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