| BackgroundThis study is part of basic research on the composition of the law of Ephedra sinica herbal pairs, funded by the National Natural Science Foundation. The main line of the project is Feng, Han Biao and Shi syndromes, Ephedra sinica is taken as the major medicine, we choosed six herbal pairs which used for different accompan-ied symptoms from the Zhang Zhongjing classic prescriptions, to explore the comp-ositions of the law, and the relationship between the changes of compatibility or dos-ages and the contents of compositions and the pharmacyological effects, pharmacyo-kinetics or drug metabolism genomics, and then longitudinal analyse the internal rel-ations of six herbal pairs with prescriptions, and observe how symptoms, diagnosis and treatment of Feng, Han, Biao Shi to be affected by medicines compatibility and their dosages. These would be beneficcial to reveal the meanings and laws of the sy-stem of Traditional Chinese herbal pairs, then sum up the universal law of medicine pairs composition, and promote the development of the theory of Chinese medicine prescription. Ephedra sinica-Glycyrrhiza uralensis is one of six herbal pairs studied, which is selected from Glycyrrhiza uralensis-Ephedra sinica soup used for Feng, Han, an-d Pi Shui sydomes. Glycyrrhiza uralensis-Ephedra sinica soup is derived from "Go-lden Chamber", consists of Ephedra sinica and Glycyrrhiza uralensis.The, utility of this soup is to eliminate the water swelling, and used for the treatment of inner Shu-i, the body and face swollen, even above the waist, body fever, thirst, sweat, disadva-ntage of a urinary. The actions of Ephedra sinica are to XuanFa Wei Yang, Sanbiao QuXie, make FengXie eliminated from the outside and eliminated Lishui from the u-rine, the actions of Glycyrrhiza uralensis are to regulate ZhongQi and reconcile med-icine properties. The two combined medicines exert the actions of FaSan LiShui. In this prescription, Ephedra sinica is12g, Glycyrrhiza uralensis is6g, which is the most commonly used doses and ratio in Zhongjing prescriptions, but not set in stone, such as in the big dragon soup, Ephedra sinica-Glycyrrhiza uralensis soup and Yue pi soup, Ephedra sinica is12g, Glycyrrhiza uralensis is6g. In Ephedra sinica Ram-ulus Cinnamoml Glycyrrhiza uralensis soup, Ephedra sinica is6g, Glycyrrhiza ura-lensis is6g, which is at heavier dosage of Glycyrrhiza uralensis. In Ephedra sinica added Atractylodis Macrocephalae soup, Ephedra sinica is9g, Glycyrrhiza uralen-sis is3g."Disease are consistent to syndromes", using the different ratio of Ephedr-a sinica and Glycyrrhiza uralensis for different syndromes, is a question worth cons-idering concering the connotation of compatibility.SignificanceThe clinical efficacy of Chinese herbs depends largely on the compatibility of m-edicine. The herbal pairs, as the smallest constituent units of the Chinese herbal for-mulas and also a series of compounds in common, are simple and effective. So med-icine pairs research, as a starting point, may be beneficial to elucidate its mechanism of actions of formulas, seek the best the ratio of compatibility, and explore the basic effects, and function expansion. Carrying out research on the of compatibility law medicine pairs, is of great significance to reveal the scientific contents of formulas compatibility, mine the theory TCM compatibility, and expand clinical ideas of the treatment, as well as provide clues to the basis for in-depth study of compound com-patibility law, and the basis for the formation of the new formulas. Especially with the increasing of compatibility using active ingredients or effective parts, applying the traditional compatibility theory to guide the new form of compatibility, is of pr-actical significance to promote the new products of traditional Chinese medicine for research and innovationEphedra sinica-Glycyrrhiza uralensis is a commonly used pair in the Zhongjing prescriptions, and often used by the modern physicians. To study the pharmacologi-cal interaction effects of this pair, quantitative evaluate compatibility efficiency, seek the best ratio, and study the changes of chemical compositions in vitro and in vivo, reactions in the process of from the material basis, to find the biological markers af-fected by Ephedra sinica, Glycyrrhiza uralensis and Ephedra sinica-Glycyrrhiza uralensis by means of metabolomics, which all would be helpful to explain the compatibility of this pair gradually.ObjectivesTo reveal the compatibility connotation of Ephedra sinica-Glycyrrhiza uralens-is herbal pair from the following three levels:(1) The main aim of sudy to observe acute toxicity, anti-inflammatory effects and diuretic actions of Ephedra sinica, Glycyrrhiza uralensis and three different ratios of Ephedra sinica and Glycyrrhiza uralensis, and make a comprehensive and quantitat- ive evaluation of the interaction between Ephedra sinica and Glycyrrhiza uralensis, using calcusyn software and one-way ANOVA.(2) To analyze the changes of contents of ephedrine, pseudoephedrine, ephedrine, and methyl pseudo, ephedrine, licorice acid and liquiritin in Ephedra sinica or Glyc-yrrhiza uralensis and different ratios of Ephedra sinica-Glycyrrhiza uralensis, com-pare the links between chemical compositions and ratios, in order toelaborate the ph-armacological effects of changes.(3) To explore the effects of Ephedra sinica, Glycyrrhiza uralensis and Ephedra sinica-Glycyrrhiza uralensis administrated orally on biomarkers in urine by metabo-lomics technology, and find the biomarkers that may characterize this process of cha-nges, which would provide experimental evidence for the mechanism of interaction between Ephedra sinica and Glycyrrhiza uralensis from system and overall perspec-tive.Methods(1) The main pharmacological effects were observed by the methods of the acute toxicity in mice, acetic acid-induced capillary permeability in mice and the effect on urine volume in rats for a period of1h following oral administration of drugs. A co-mbidrug software and t test have been used to analyze the interactions of the differe-nt level in the combinations.(2) Analysis and determine the best ratio of both synergistic interaction betw-een Ephedra sinica-Glycyrrhiza uralensis. Anti-inflammatory effects of the best ratio of this pair were determined anti-inflammatory effects by utilizing the following mo-dels:xylene-induced ear edema and acetic acid-induced permeability in mice, carrag-eenin-induced edema of the hind paw, carrageenan-induced pleurisy and cotton pell-et-induced granuloma formation in rats. The anti-inflammatory mechanisms were inv- estigated by detecting the contents of prostaglandins (PGE2), tumor necrosis factor-a-lpha (TNF-a), interleukin-1β (IL-1β) in exudates, and the contents of TNF-a, IL-1β, MDA in lung tissue were also detected. HE staining was to observe the effect of Eph-edra sinica-Glycyrrhiza pair on pleurisy in rat lung tissue, and immunohistochemical detection of iNOS and ICAM-1expression in the lung tissue. The observation of Eph-edra sinica-Glycyrrhiza uralensis of PGE2in normal rat urine was to explore the di-uretic mechanism. All mean were compared with One-way ANOVA, data were statis-tically analyzed by SPSS13.0software, P<0.05was statistically significant.(3) To study the fingerprints peak changes Ephedra sinica alkaloids (ephedrine, pseudoephedrine, methyl ephedrine, methylpseudohedrine, and norpseudoephedrine and normethyl methamphetamine), using gas chromatography-liquid chromatogr-aphy (GC-MS) in Ephedra sinica and the three compatibility ratios, all mean were compared with One-way ANOVA, and liquiritin and glycyrrhizic acid in Glycyrrhiza uralensis and different ratios using high performance liquid chromatography (HPLC), then clarified the relation between differrent proportions and main medicinal compo-nents. Data were statistically analyzed by SPSS13.0software, P<0.05was statistic-ally significant.(4) Urine was collected in seven days after administration Ephedra sinica, Gly-cyrrhiza uralensis, and Ephedra sinica-Glycyrrhiza uralensis, and then centrifuged and derivatizated. Rat urine was carried out a full scan analysis of endogenous meta-bolites using GC-MS. GC-MS data were processed by the method of peak identific-ation, peak alignment, excluding the solvent peak impurity peak. Exported data (exc-el format) peak area were normalized, and then generated the two-dimensional matr-ix including sample name and a relative peak area. Metabolite spectra was analysised by principal component analysis (PCA) and partial least squares discriminant analy-sis (PLS-DA) using the software Simca-P. PCA was used for intuitive observation of changes and drug treatment of different groups. PLS-DA was used for distinction wi-thin the treated groups. The value of VIP was used for evaluation the correlation de-gree of different substances. VIP value>1was indicated as potential biomarkers, which furtherly analysised using SPSS13.0software. Those biomarkers with signify-cant difference were selected as the final biomarker. The metabolic pathways affectt-ed by Ephedra sinica, Glycyrrhiza uralensis, and Ephedra sinica-Glycyrrhiza urale-nsis were interpretated based on existing knowledge of biochemistry.Results(1) Ephedra sinica-Glycyrrhizic uralensis pair of12:3,12:6and12:12pr-oduced toxicity antagonism obviously in a dose-dependent manner associated with Glycyrrhizic uralensis. Ephedra sinica-Glycyrrhizic uralensis of12:3and12:6exerted synergy anti-inflammatory action, when the inhibition rate is25%～35%, a-nd there was significant difference compared to the control group,but12:12show-ed the obvious antagonism. Ephedra sinica-Glycyrrhizic uralensis pair12:3and12:6had close therapeutic index (TI).(2) Compared with the model group, Ephedra sinica-Glycyrrhizic uralensis pair, at at the doses of22.4,11.2g·kg-1, inhibited xylene-induced ear swelling. This pair22.4g·kg-1produced suppression against carrageenin-induced edema of the hind paw at3,5,7h after carrageenin injection,11.2,5.6g·kg-1this pair had not signi-ficant effects on edema. Ephedra sinica-Glycyrrhizic uralensis pair at the dosage of22.4g·kg-1inhibited cotton pellet-induced granuloma formation in rats. This pair inhibited inhibited the increase of the volume of exudates and total leukocytes in the exudates induced by carrageenan, as well as significantly reduced the levels of PGE2, TNF-a, IL-1β in exudates. Ephedra sinica-Glycyrrhizic uralensis pair also reduced the contents of TNF-a. IL-1β,MDA in lung tissue in carrageenan treated rats, and i-nhibited the damage to lung induced by carrageenan and the expression iNOS in the lung tissue, Ephedra sinica-Glycyrrhiza uralensis herbal pair had no significant effe-ct on ICAM-1expression. Compared with the control group, Ephedra sinica-Glyc-yrrhizic uralensis pair of22.4g·kg-1could significantly increase the urinary output and contents of K+in the rats’urine, increased PGE2excretion in urine.(3) The amount of Ephedra sinica alkaloids (ephedrine, pseudoephedrine, meth-ylph-edrine, methylpseudohedrine, and norpseudoephedrine and normethyl metham-phetamine) and glycyrrhizic acid, liquiritin, in three ratios of pair, decreased with the increase in the dosage of Glycyrrhizic uralensis. The dosage of Glycyrrhizic uralens-is was negatively correlated with the major effective compositions. The effective co-mpositions contents of Ephedra sinica-Glycyrrhizic uralensis12:12decreased m-ost significantly.(4) Compared with the control group, the biomarkers down-regulated by Glycyr-rhiza uralensis 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-Glycyrrhiza uralensis were α-ketoglutaric acid, adipic acid, aconitic acid, cit-ric acid, and cysteine, those up-regulated were alanine, high citric acid, methyl xant-hine, tyrosine and tryptophan.ConclusionThe relationship main between pharmacological interaction effects of Ephedra sinica-Glycyrrhizic uralensis and the content changes of active ingredient in diff-erent ratios is obvious. With the level of main chemical ingredients in three ratios of pair decreased, the main pharmacyological effects showed antagonism within a cer-tain range. Ephedra sinica-Glycyrrhizic uralensis drug compatibility can increase the efficiency and reduce toxicity within the appropriate dose, and the ratio of12:3and12:6showed better compatibility advantage with wider synergistic range. Ep- hedra sinica-Glycyrrhizic uralensis herbal pair (12:6) possess significant anti-inf-lammatory effects, which can be related with the inhibition of the generation of me-diators of inflammation and its antioxidation. The diuretic mechanism of action may be attributed to suppress PGE2release in urine. Ephedra sinica, Glycyrrhizic uralen-sis affected different metabolic markers. Ephedra sinica-Glycyrrhizic uralensis also affected the other three kinds of markers, in addition those affected by Ephedra sini-ca and Glycyrrhizic uralensis sololy, which suggest that there is a synergyistic actio-n between Ephedra sinica and Glycyrrhizic uralensis. |
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