| Background and SignificanceCouplet 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.ObjectionClarify 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.Method1. 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 informationResearching 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).Result1. 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 macrocephalaAcute 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 MetabonomicsCompared 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.Conclusion1.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. |
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