Study On The Main Chemical Compasition And Urine Metabolites Of Mahuang-Xingren Herb-pair

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. Couplet medicines is a bridge between single Chinese herb and compound traditional Chinese medicine, it’s the main trunk of compound traditional Chinese medicine, also the foundation of Chinese herbal medicines."The Shen Nong Ben Cao Jing" is not proposed couplet medicines directly, but there were already have a theory of "traditional Chinese medicine have yin combined yang, child and mother, brothers". Composition of couplet medicines mainly based on the seven emotions concerted application, follow theory of "monarch, minister, adjuvant and dispatcher", which has a fundamental indications of compound traditional Chinese medicine, forms a major feature of traditional Chinese medicine, has closely linked to the pathogenesis, the efficacy of single-minded, the curative effect. The study of couplet medicines both in theory and in practice are important. Use modern technology to research the aspect of composition of couplet medicines from the chemistry, pharmacology, processing to clinical application in a deeper level, It can reveal concerted effect of some couplet medicines, to explore the mechanism, seek for a dosage ratio of cooperate medicine which develop the best function, find out the relationship between its dosage-effect and time-effect, as well as toxic-effect, and take advantage of drug treatment.San-ao decoction derived from<Tai Ping Hui Ming He Ji Ju Fang>, it composed of Herba Ephedrae、Armeniacae Semen Amarum and Glycyrrhiza uralensis Fisch. In the modern clinical research, San-ao decoction has a significant effect in the aspect of curing cold, hoarseness and asthmatic, which is closely related to the material basis. 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 couplet medicines Herba Ephedrae-Armeniacae Semen Amarum as study object in San-ao decoction, and making different ratio of medicinal ingredients as the entry point, In order to evaluate rationality of concerted application of couplet medicines, as the starting point, from the change of chemical composition and some endogenous small molecules derived from the content of the asthma rat urine metabolites,(including organic acids, amino acids and fatty acids, etc.), the dynamic response revealing changes of medicinal ingredients and dosage of couplet medicines can cause the change of rules and connotation of couplet medicines. 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(1) Clarify the chemical compatibility rules of couplet medicines Herba Ephedrae and Armeniacae Semen Amarum compatibility ratio, by comparing the content of active ingredients in water extracts before and after different compatibility ratio of Herba Ephedrae and. Armeniacae Semen Amarum. (2) To evaluate the influence Of temperature and acid-base on isomerization of amygdalin in Armeniacae Semen Amarum. Further revealling the objective law and scientific connotation of concerted application of couplet medicines.(3) By comparison of the metabonomics of intervention of couplet medicines Herba Ephedrae and Armeniacae Semen Amarum on OVA sensitized asthma syndrome, to explore the scientific connotation before and after compatibility of Herba Ephedrae and Armeniacae Semen Amarum.Methods1. Date collectionLooked up articles about pharmacology, toxicology, metabonism and excretion of san-ao decoction, Herba Ephedrae, Armeniacae Semen Amarum and their active ingredients by the way of computer retrieval and manualretrieval, and making use of many kinds of databases, such as CNKI, Science Direct and PubMed.2. Researching on chemical composition in vitro of different compatibility ratio of Herba Ephedrae and Armeniacae Semen Amarum.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 Armeniacae Semen Amarum during the preparation process. The couplet medicines Herba Ephedrae and Armeniacae Semen Amarum (1:1) in san-ao decoction were taken as study object, the compatibility ratio of Herba Ephedrae and Armeniacae Semen Amarum was multiplied floating up and down basing on the ratio of the couplet medicines reported in the literature. Herba Ephedrae water extract, Armeniacae Semen Amarum water extract, Herba Ephedrae-Armeniacae Semen Amarum (1:0.5) water extract, Herba Ephedrae-Armeniacae Semen Amarum (1:1) water extract, Herba Ephedrae-Armeniacae Semen Amarum (1:2) water extract and san-ao decoction were individually prepared. The major active compounds before and after different compatibility ratio of Herba Ephedrae and Armeniacae Semen Amarum in the extracts were analyzed by HPLC individually.3. The Study of the existing form of amygdalin in Armeniacae Semen Amarum and the influence factorsTake Armeniacae Semen Amarum (which is not remove the peel) as study object, use HPLC to determine the peak area of D-amygdalin and L-amygdalin in different decocting temperature, and use this method qualitatively investigate the influence Of acid-base on isomerization of amygdalin monomer.4. Study on the intervention of different compatibility of couplet medicines Herba Ephedrae-Armeniacae Semen Amarum on OVA sensitized asthma syndrome by metabonomics.We use a method of atomizing inhalation OVA sensitized excitation to copy asthmatic rats. Before injected ovalbumin and aluminum hydroxide solution,10h urine of rats in each group were collected. Except positive drug group, the other groups collected urine of1,3,5,7d. when atomizing. All the urine samples were derivatized directly. Subsequently, metabolites spectra of these samples were acquired using GC-MS. Identification after proeessed, the data was subjected to SIMCA-P+12.0software for principal component analysis (PCA) and principal least squares discriminantanalysis (PLS-DA). Study on the intervention of different compatibility of couplet medicines Herba Ephedrae-Armeniacae Semen Amarum on OVA sensitized asthma syndrome by metabonomics. 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 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). 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).Results1Researching on chemical composition of different compatibility ratio of couplet medicines Herba Ephedrae-Armeniacae Semen AmarumUse HPLC to determine the content of amygdalin, and the content of Norephedrine (NME), Norpseudoephedrine (NMP), Ephedrine (E), Pseudoephedrine (PE) and Methylephedrine (ME) in water extracts. Compare to Herba Ephedrae decoction, when the ratio of Armeniacae Semen Amarum increased2times than Herba Ephedrae, the content of the5kinds of ephedrine in water extracts of couplet medicines were significant (P<0.05), Compare to Armeniacae Semen Amarum decoction, With a increase of Herba Ephedrae compatibility ratio, The content of amygdalin have increased in a varying degrees.2. The influence Of acid-base and temperature on isomerization of amygdalin monomer.Amygdalin in Armeniacae Semen Amarum in different temperature ranges have different isomerization performance, amygdalin monomer did not occur isomerization in acid environment, isomerization under the effect of ammonia, sodium hydroxide.3. Study on the intervention of different compatibility of couplet medicines Herba Ephedrae-Armeniacae Semen Amarum on OVA sensitized asthma syndrome by metabonomics. 3.1OVA sensitized rats can be successfully established allergic bronchial asthma model.3.2The metabonomic technique and PCA and PLS-DA analysis methods, GC-MS were utilized to study the metabolic characteristics of asthma syndrome induced by OVA and intervention effect of couplet medicine Herba Ephedrae-Armeniacae Semen Amarum. Results showed that different times of the model groups and control group distinguished well, which was classified normal physiological metabolism of OVA 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. Atomized Id group, endogenous product of the type, concentration and relative proportions of disturbance occurs, gradually away from the control group, the dynamic changes of the modeling process can be clearly seen from the PCA.3th.day atomized group, have a trend of moving toward the control group, but after4th day atomization, then gradually away from the control group, and reaches the maximum in the7th day atomized group, indicating that the metabolite profiling of this day was the most intense changes.3.3Further analysis of normal group, model group, Herba Ephedrae group, Armeniacae Semen Amarum group and Herba Ephedrae-Armeniacae Semen Amarum group of7th. day atomized group. 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. This three drug groups gradually closer to the direction of the control group, indicating that each drug group regression control group trajectory of change over time in the model; and Herba Ephedrae-Armeniacae Semen Amarum group and control group located in the fourth quadrant together, overlapping and difficult to fully distinguish. 3.4Biomakers, which were obtained by PLS-DA analysis of normal group, model group, Herba Ephedrae group, Armeniacae Semen Amarum group, Herba Ephedrae-Armeniacae Semen Amarum (1:1) 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 lower levels of glycine, pyruvic acid, malic acid, a-ketoglutaric acid, adipic acid, suberic acid, aspartic acid, citric acid, hippuric acid, phenylalanine (P<0.05), and higher levels of propylamine(P<0.05); Herba Ephedrae group display significantly lower levels of glycine, malic acid, adipic acid, suberic acid, aspartic acid, citric acid, hippuric acid, phenylalanin (P<0.05); Armeniacae Semen Amarum group display significantly lower levels of glycine, pyruvic acid, malic acid, adipic acid, hippuric acid, phenylalanine(P<0.05); Herba Ephedrae-Armeniacae Semen Amarum (1-1) group display significantly lower levels of succinic acid, glycine, pyruvic acid, malic acid, α-ketoglutaric acid, adipic acid, suberic acid, aspartic acid, citric acid, hippuric acid and phenylalanine.Conclusion:1. The results of chemical composition analysis experimental showed that after compatibility of Herba Ephedrae and Armeniacae Semen Amarum, when Armeniacae Semen Amarum compatibility proportion increases to a certain extent, the content of ephedra alkaloids declined. At the same situation, the dissolution rate of amygdalin in Herba Ephedrae-Armeniacae Semen Amarum extract significantly increased.2. Armeniacae Semen Amarum with95%ethanol extracted only get D-amygdalin while when extracted by boiling water can get both D-amygdalin and L-amygdalin. With the rise of decocting temperature results in the peak area of D-amygdalin and L-amygdalin gradually increased. Amygdalin monomer is stable in the acidic environment, alkali lead one of the factors of isomerization.3. We can see the trend of biomark from the table:(1) after drug administration, The asthmatic rats’ glycolytic pathway callbacked performance closely associated with the citric acid cycle, glycolysis energy metabolism of citric acid, succinic acid, malic acid and other endogenous metabolic intermediate content change.(2) after drug administration, The asthmatic rats’ amino acid metabolism callbacked performance closely associated with the aspartic acid, glycine, phenylalanine.(3) after drug administration,The asthmatic rats’ fatty acid metabolism callbacked performance closely associated with the pyruvic acid, adipic acid and fatty acid.4Currently, ingredients of traditional Chinese medicine is complexed, and metabolic pathways is not clear, so we still can not explain a clear correlation between the metabolic network and metabolites. As can be seen from figure3-5, couplet medicines Herba Ephedrae-Armeniacae Semen Amarum group is closer to the control group than Armeniacae Semen Amarum group and Herba Ephedrae group. Table3-2shows that Armeniacae Semen Amarum group, Herba Ephedrae group and couplet medicines Herba Ephedrae-Armeniacae Semen Amarum group improved abnormal metabolic state that OVA sensitized asthmatic rats systemic. PLS-DA analysis results showed that Herba Ephedrae-Armeniacae Semen Amarum is better.