| Background and SignificanceCouplet medicines, also called drug pairs, which is made of two drugs, 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", taking wind-cold superficies excess syndrome of Treatise on Cold Pathogenic Diseases as main line, classical prescription mahuang decoction and its "sovereign couplet medicines" Herba Ephedrae-Ramulus Cinnamomi 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-Ramulus Cinnamomi, 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 Herba Ephedrae and Ramulus Cinnamomi compatibility ratio, by comparing the content of active ingredients in water extracts before and after different compatibility ratio of Herba Ephedrae and Ramulus Cinnamomi.Clarify the relationship between the chemical composition and pharmacodynamic of couplet medicines Herba Ephedrae and Ramulus Cinnamomi, by comparing the acute toxicity and antipyretic effects before and after different compatibility ratio of Herba Ephedrae and Ramulus Cinnamomi.Clarify the relevance between the drug properties and the effectiveness, and preliminarily reveal the scientific connotation of property-effect relationship of Chinese tranditional medicine, by studying on the metabonomics of intervention of couplet medicines Herba Ephedrae and Ramulus Cinnamomi on dry yeast-induced fever syndrome.By studying the intrinsic link of Herba Ephedrae-Ramulus Cinnamomi among compatibility of couplets medicines, changes in chemical composition, changes in pharmacological effects, pathways and mechanisms of the in vivo metabolic, to clarify the material basis and mechanism of the compatibility of couplets medicines.Methods and materials1. Looked up articles about pharmacology, toxicology, biotransformation, metabonism and excretion of mahuang decoction, Herba Ephedrae, Ramulus Cinnamomi and their active ingredients by the way of computer retrieval and manual retrieval, and making use of many kinds of databases, such as CNKI, PubMed, Science Direct, SpringerLINK, CA, Toxnet.2. Researching on chemical composition in vitro of different compatibility ratio of Herba Ephedrae and Ramulus Cinnamomi. 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 Ramulus Cinnamomi during the preparation process.The composition of mahuang decoction in Treatise on Cold Pathogenic Diseases s is:"Herba Ephedrae three liang (removed knots), Ramulus Cinnamomi two liang (barked), Radix Glycyrrhizae one liang (roasted), seventy Semen Armeniacae Amarum(barked)." The couplet medicines Herba Ephedrae and Ramulus Cinnamomi (3:2) in mahuang decoction were taken as study object, the compatibility ratio of Herba Ephedrae and Ramulus Cinnamomi was multiplied floating up and down basing on the ratio of the couplet medicines reported in the literature. Herba Ephedrae water extract, Ramulus Cinnamomi water extract, Herba Ephedrae-Ramulus Cinnamomi (3:1) water extract, Herba Ephedrae-Ramulus Cinnamomi (3:2) water extract, Herba Ephedrae-Ramulus Cinnamomi (3:4) water extract and mahuang decoction were individually prepared. The content of Norpseudoephedrine (NMP), Norephedrine (NME), Ephedrine (E), Pseudoephedrine (PE) and Methylephedrine (ME), which are the major active compounds of Herba Ephedrae, were analyzed by gas chromatography-mass spectrometry (GC-MS). The content of coumarin, cinnamic alcohol and cinnamaldehyde, which are the major active compounds of Ramulus Cinnamomi in the extracts were analyzed by high performance liquid chromatography (HPLC).3. Study on the major pharmacological effect (major syndrome) of couplet medicines Herba Ephedrae-Ramulus Cinnamomi on the different compatibility ratio. According to the modern and relevant research results, with the side effects, antipyretic effects, 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 Herba Ephedrae, Ramulus Cinnamomi, couplet medicines Herba Ephedrae-Ramulus Cinnamomi and mahuang decoction. Used regression to analysis the results of chemical composition, acute toxicity and antipyretic effects, in order to clarify the material basis of Herba Ephedrae-Ramulus Cinnamomi.Acute Toxicity experiment:The kunming mice were randomly divided into normal group, model group, Herba Ephedrae group, Ramulus Cinnamomi group, Herba Ephedrae-Ramulus Cinnamomi (3:1) group, Herba Ephedrae-Ramulus Cinnamomi (3:2) group and Herba Ephedrae-Ramulus Cinnamomi (3:4) group, then each group was divided into five dosage group, each of which had10mice, half male and half female. Dn, Dm were selected according to the preliminary experiment, then set dose between adjacent groups, 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, diarrhea, death, hair, etc. The death rates of each group were recorded. LD50and confidence interval of95%of above mentioned tested drugs were calculated with Calcusyn software.Interaction of acute toxicity experiment:Based on the median-effect principle, the experimental data from acute toxicity experiment were analyzed with Calcusyn software. As a CI equal to1indicated additivity; a CI less than1indicated synergy; and a CI greater than1indicated antagonism.Antipyretic action experiment:The male rats, which had qualified temperature, were randomly divided into normal group, model group, aspirin group, analgin group, Herba Ephedrae group, Ramulus Cinnamomi (3) group, Ramulus Cinnamomi (6) group, Ramulus Cinnamomi (12) group, Herba Ephedrae-Ramulus Cinnamomi (3:1) group, Herba Ephedrae-Ramulus Cinnamomi (3:2)group, Herba Ephedrae-Ramulus Cinnamomi (3:4) group and mahuang decoction, each of which had8rats. The rats were intragastric (ig.) administrated with different dose of tested drugs. The rectal temperature (Tr) of rats was measured once each hour after dosaging. The most increased temperature (ΔT/℃) and3h inhibition rate were calculated.Interaction of antipyretic action experiment:Based on the median-effect principle, the experimental data from antipyretic action experiment were analyzed with Calcusyn software. As a CI equal to1indicated additivity; a CI less than1indicated synergy; and a CI greater than1indicated antagonism.Regression analysis study on the compatibility of chemical composition, acute toxicity and antipyretic effect:According to the results of chemical composition, acute toxicity and antipyretic effect, the use of SPSS16.0software, multiple regression analysis of the active ingredient content of the decoction and mortality rate,3h ΔT(℃) after dosage, inhibitory rate.Antipyretic mechanism experiment:The male rats, which had qualified temperature, were randomly divided into normal group, model group, aspirin group, analgin group, Herba Ephedrae-Ramulus Cinnamomi (3:2) low dosage group, Herba Ephedrae-Ramulus Cinnamomi (3:2) middle dosage group, Herba Ephedrae-Ramulus Cinnamomi (3:2) high dosage group, the content of PGF2and cAMP in hypothalamus of rats were determined by radioimmunoassay.4. Study on water extracts of couplet medicines Herba Ephedrae-Ramulus Cinnamomi by compatibility of different ratio on dry yeast-induced fever syndrome by metabonomicsAccording to the main symptoms of wind-cold superficies excess syndrome, febris response in rats was chosen. The male Wistar rats were randomly divided into normal group, model group, Herba Ephedrae group, Ramulus Cinnamomi group and Herba Ephedrae-Ramulus Cinnamomi group, with6rats in each group. Before induced by dry yeast,8h urine of rats in each group were collected, then the model group, Herba Ephedrae group, Ramulus Cinnamomi group and Herba Ephedrae-Ramulus Cinnamomi group of rats were prepared by back subcutaneous injection of20%dry yeast suspension,0～6h、6～10h、10～18h、18～24h、24～30h、30～36h urine of rats in each groups 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 processed, 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 SPSS16.0, and those with significant difference were chosen as final biomarkers.5. Statistial analysisThe 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). Results:1Researching on chemical composition of different compatibility ratio of couplet medicines Herba Ephedrae-Ramulus CinnamomiMethodology study was established respectively based on the gas chromatography-mass spectrometry (GC-MS) analysis method and HPLC analysis method. The separation degree of Norpseudoephedrine (NMP), Norephedrine (NME), Ephedrine (E), Pseudoephedrine (PE), Methylephedrine (ME), coumarin, cinnamic alcohol and cinnamaldehyde was above1.5with no impurity peak interference. The methods were proved accurate, with good specificity and reproducibility, and high sensitivity.The content of Norpseudoephedrine (NMP), Norephedrine (NME), Ephedrine (E), Pseudoephedrine (PE), Methylephedrine (ME), coumarin, cinnamic alcohol and cinnamaldehyde in water extracts of couplet medicines Herba Ephedrae-Ramulus Cinnamomi with different ratio were lower than that of single decoction.The content of Norpseudoephedrine (NMP), Norephedrine (NME), Ephedrine (E), Pseudoephedrine (PE), Methylephedrine (ME) from high to low:Herba Ephedra water extract, Herba Ephedrae-Ramulus Cinnamomi(3:1) water extract, Herba Ephedrae-Ramulus Cinnamomi(3:2) water extract, mahuang decoction and Herba Ephedrae-Ramulus Cinnamomi(3:4) water extract.2Study on the major pharmacological effect (major syndrome) of couplet medicines Herba Ephedrae-Ramulus Cinnamomi on the different compatibility ratioAcute Toxicity test:the LD50of Herba Ephedrae, Ramulus Cinnamomi, Herba Ephedrae-Ramulus Cinnamomi (3:1), Herba Ephedrae-Ramulus Cinnamomi (3:2), Herba Ephedrae-Ramulus Cinnamomi (3:4) were140.708g·kg-1,137.988g·kg-1,140.774g·kg-1,234.379g·kg-1,223.342g·kg-1, respectively.95%confidence interval were120.230～166.225g·kg-1,117.902～163.006g·kg-1,119.448～164.385g·kg-1,200.100～276.305g·kg-1,188.312～261.246g·kg-1, respectively. The acute toxicity of couplet medicines Herba Ephedrae-Ramulus Cinnamomi with different ratio were lower than that of single decoction. The acute toxicity of Herba Ephedrae-Ramulus Cinnamomi (3:2) was lower than Herba Ephedrae-Ramulus Cinnamomi (3:1) and Herba Ephedrae-Ramulus Cinnamomi (3:4).Interaction of acute toxicity experiment:The results showed that interaction of acute toxicity of couplet medicines Herba Ephedrae-Ramulus Cinnamomi with different ratio were drug antagonism. The interaction of acute toxicity from high to low:Herba Ephedrae-Ramulus Cinnamomi (3:1), Herba Ephedrae-Ramulus Cinnamomi (3:2), Herba Ephedrae-Ramulus Cinnamomi (3:4). With the the compatibility percentage of Ramulus Cinnamomi increased, the acute toxicity of antagonism enhanced.Antipyretic effect experiment:Compared with model group, all the treatment groups had obvious antipyretic effect. Compared with the Herba Ephedra group and Ramulus Cinnamomi group, antipyretic effects of the three ratio groups of Herba Ephedrae-Ramulus Cinnamomi were strengthened. Among the three ratio groups of Herba Ephedrae-Ramulus Cinnamomi, the effect of Herba Ephedrae-Ramulus Cinnamomi (3:2) was strongest of them.Interaction of antipyretic effect experiment:based on the median-effect principle, the interaction of antipyretic effect was analyzed with Calcusyn software. Herba Ephedrae, Ramulus Cinnamomi and Herba Ephedrae-Ramulus Cinnamomi (3:2) groups showed a good dose-effect relationship. Compared with model group, all of five dosage of Herba Ephedrae-Ramulus Cinnamomi (3:2) groups can significantly inhibit the elevated body temperature of rats (P<0.01). At the same dose, the effect of comparative sequence:the Herba Ephedrae-Ramulus Cinnamomi group (3:2)> Ramulus Cinnamomi group> Herba Ephedrae group. Regression analysis study on the compatibility of chemical composition, acute toxicity and antipyretic effect:Ephedrine, Coumarin and Cinnamaldehyde was introduced into the regression equation, the regression equation was as follows:Mortality rate=-0.105+0.588E+0.165GPQ3hΔT(℃)=1.953-12.213GPQ+29.196XDSInhibitory rate (%)=26.596+459.205GPQ-1097.774XD SEphedrine and cinnamaldehyde, the active ingredients which were introduced to the regression equation had a significant effect (P<0.05) on the death rate.Coumarin and cinnamaldehyde on the inhibitory rate had a significant effect (P<0.05).Antipyretic mechanism experiment:Compared with model group, each treatment group showed remarkable decreased the contents of PGE2and cAMP in the hypothalamus of febrile rats. The results showed that Herba Ephedrae-Ramulus Cinnamomi(3:2) group might be related with decreasing the levels of PGE2and cAMP in the hypothalamus3Study on water extracts of couplet medicines Herba Ephedrae-Ramulus Cinnamomi by compatibility of different ratio on dry yeast-induced fever syndrome by metabonomicsThe metabonomic technique and PCA and PLS-DA analysis methods, GC-MS were utilized to study the metabolic characteristics of fever syndrome induced by dry yeast and intervention effect of couplet medicine Herba Ephedrae-Ramulus Cinnamomi. In score plots urinary metabolites changed significantly after modelling and divided into two categories, which displaying the similarities and differences between the metabolic networks of two groups from microscopic view in vivo. Results showed that different times of the model groups and blank group distinguished well, which was classified normal physiological metabolism of dry yeast-induced rats were interference. Metabolites of different model groups had undergone certain changes, different times of the model group to deviate from the normal group was significantly different.0～6h model group, moving away from the blank group, the classification trend was becoming increasingly obvious, the maximum in the18～24h model group, indicating that the metabolite profiling of18～24h model group was the most intense changes;30～36h model group, after modeling, endogenous productthe type, concentration and relative proportions of the location of the gradual pullback to the blank group, but ultimately failed and the blank group overlap, indicating that the material metabolism of the model group showed a certain degree of self-regulation trend observed within the time limit, but could not be restored to their normal state. Metabolites of experimental group clustered at the route of models recuperating after the intervention of pairmedicine Herba Ephedrae-Ramulus Cinnamomi, and it indicated that couplet medicines Herba Ephedrae-Ramulus Cinnamomi accelerated the recuperation of fever syndrome model and made them develop in the direction of health.3.3Biomakers, which were obtained by PLS-DA analysis of normal group, model group, Herba Ephedrae group, Ramulus Cinnamomi group, Herba Ephedrae-Ramulus Cinnamomi (3:2) group, respectively, including organic acids, fatty acids, amino acids, amines and so on.Urinary biomarkers and their changes in different drug-tested groups:compared to model group, normal group display significantly higher levels of succinic acid, suberic acid, citric acid, methionine, hippuric acid, nicotinamide and isoleucine(P<0.05), and lower levels of propylamine, pyruvic acid, valine, benzamide, phenylacetic acid, phenylalanine, isobutyric acid, tyrosine, tryptophan and isonicotinoyl amides (P<0.05); Herba Ephedrae group display significantly higher levels of succinic acid, leucine, tyramine, methionine, hippuric acid, uric acid phenethyl, spermine, isoleucine and cyclohexyl propionate (P<0.05), and lower levels of propylamine, valine, benzamide, pimelic acid, pyroglutamic acid, phenylacetic acid, adipic acid, azelaic acid, isobutyric acid, dihydroxyphenylacetic acid, tyrosine,5-hydroxytryptamine, tryptophan, isonicotinoyl amides (P<0.05); Ramulus Cinnamomi group display significantly higher levels of succinic acid, a-ketoglutaric acid, serine, suberic acid, methionine, hippuric acid, the acetophenone uric acid, spermine and isoleucin (P<0.05), and lower levels of propylamine, pyruvate, valine, benzamide, phenylacetic acid, leucine, lauric acid, azelaic acid, phenylalanine, isobutyric acid, glutathione, tyrosine, tryptophan and isonicotinoyl amide(.P<0.05); Herba Ephedrae-Ramulus Cinnamomi (3:2) group display significantly higher levels of succinic acid, suberic acid, citric acid, hippuric acid and cysteine (P<0.05), and lower levels of propylamine, pyruvate, valine, phenylacetic acid, lauric acid, phenylalanine,2-toluene-ethyl acetate, glutathione, tyrosine, tryptophan and isonicotinoyl amides (P<0.05).3.4Biomakers, which were obtained by PLS-DA analysis of normal group, model group, Herba Ephedrae group, Ramulus Cinnamomi group, Herba Ephedrae-Ramulus Cinnamomi (3:2) group together, including organic acids, fatty acids, amino acids, amines and so on.Urinary biomarkers and their changes in different drug-tested groups:compared to model group, normal group display significantly higher levels of citric acid, hippuric acid and isoleucine(P<0.05), and lower levels of propylamine, pyruvic acid, benzamide, phenylacetic acid, leucine, lauric acid, phenylalanine, isobutyric acid, tyrosine, tryptophan and isonicotinoyl amides (P<0.05); Herba Ephedrae group display significantly higher levels of hippuric acid (P<0.05), and lower levels of valine, benzamide, phenylacetic acid, isobutyric acid, tyrosine, tryptophan and isonicotinoyl amides(P<0.05); Ramulus Cinnamomi group display significantly higher levels of hippuric acid and isoleucine (P<0.05), and lower levels of glutaric acid, valine, benzamide, phenylacetic acid, lauric acid, phenylalanine, isobutyric acid, tyrosine, tryptophan and isonicotinoyl amides (P<0.05); Herba Ephedrae-Ramulus Cinnamomi (3:2) group display significantly higher levels of citric acid and hippuric acid(P<0.05), and lower levels of propylamine, pyruvic acid, benzamide, phenylacetic acid, leucine, lauric acid, suberic acid, phenylalanine, isobutyric acid, tyrosine, tryptophan and isonicotinoyl amides (P<0.05).Conclusion:1The results from chemical composition analysis experimental showed that after compatibility of Herba Ephedrae and Ramulus Cinnamomi, compared with Herba Ephedrae, the dissolution rate of effective components in Herba Ephedrae-Ramulus Cinnamomi extract significantly reduced. The dissolution rate of effective components was related to the proportion of Ramulus Cinnamomi, the greater of Ramulus Cinnamomi, the lower of dissolution rate. At the same situation, compared with Ramulus Cinnamomi, the dissolution rate of effective components in Herba Ephedrae-Ramulus Cinnamomi extract significantly reduced. Dissolution of each other’s active ingredients in compatibility of Herba Ephedrae-Ramulus Cinnamomi was a certain inhibition.2Compatibility rules of couplet medicines should be safety and efficacy. Combined the data from acute toxicity experiment and its interaction studies with the data of of antipyretic experiment and its interaction studies, compared with the single drug, the combinations in the three ratios (3:1,3:2,3:4) of Herba Ephedrae-Ramulus Cinnamomi showed a synergistic trend in antipyretic effect and showed an antagonism in acute toxicity. Furthermore, the compatibility of Herba Ephedrae-Ramulus Cinnamomi (3:2) was the best ratio among the three ratios. This was consistent with the compatibility ratio of Herba Ephedrae-Ramulus Cinnamomi of mahuang decection. Inferred by regression analysis, ephedrine and cinnamaldehyde may be the toxicity material basis of couplet medicines Herba Ephedrae-Ramulus Cinnamomi, and coumarin and cinnamaldehyde may be the material basis of antipyretic effect of couplet medicines Herba Ephedrae-Ramulus Cinnamomi.Combined with chemical analysis results, it suggested that the effective substance of couplet medicines Herba Ephedrae-Ramulus Cinnamomi based not only on the kinds of chemical composition, and also on the content of chemical compositions, which was corresponding to the multi-component, multi-target, multi-channel and multi-link of mechanism in Chinese herbal compound.318～24h normal group, model group, Herba Ephedrae group, Ramulus Cinnamomi group, Herba Ephedrae-Ramulus Cinnamomi group were taken PCA analysis together, the results showed that compared with Herba Ephedrae group and Ramulus Cinnamomi group, the metabolic state of Herba Ephedrae-Ramulus Cinnamomi group was closer to the normal group, which proved that Herba Ephedrae-Ramulus Cinnamomi had a better intervention effect on the dry yeast-induced fever symptoms. PLS-DA analysis results showed that, the biomarker intervention dry yeast-induced fever symptoms of Herba Ephedrae-Ramulus Cinnamomi group were similar to normal group, which proved that antipyretic effect of Herba Ephedrae-Ramulus Cinnamomi was better. |
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