| BackgroundHerb 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.ObjectionEphedra-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.ContentsTo 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 MethodsThe 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 ratiosA 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 extractsMetabonomics 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-GypsumWhen 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 ratioAcute 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 extractsThrough 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 groupThrough 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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