| Dahuang Gancao Decoction is the classic diarrhea vomiting prescription,which was firstly introduced in the "Golden Chamber" It contains only two herbs,rhubarb and licorice.The application of rhubarb-licorice herb pair already appeared in "Treatise on Febrile Diseases".With experience application for thousands of years,rhubarb-licorice has been included in the category of herb pairs,used as a classic diarrhea drug pair.Based on the literature research on the Dahuang Gancao Decoction,rhubarb-licorice herb pair and the early researches of our research group,this paper mainly sduty on quantity-effect relationship of rhubarb in constipation mouse and the harmonic effect of liquorice on rhubarb as well as the material basis and mechanismThis thesis is divided into four chapters.The research contents and results of each chapter are as follows:Chapter 1 Literature Research of Rhubarb-Licorice Herb pair Section 1 Analysis of Chinese Traditional Medicine Application Data containing Rhubarb-Licorice Herb pairIt was found that there are 26,185 prescriptions containing licorice and 9713 prescriptions containing rhubarb in the database of traditional Chinese medicine prescriptions.We calculated the statistical information of rhubarb and licorice used in Chinese herbal formulas by computer programming and keyword mapping.Frequency statistics,network and associated rules were used in data mining on the application information of Chinese herbal formulas of rhubarb and licorice.The results showed that licorice was widely used in formulas,accounting for 27.1%of all formulas.The most commonly used ratio of licorice and other traditional Chinese medicines is 1:1.It was commonly used to treat medical diseases.The main efficacys of these prescriptions are relieving pain,clearing heat,dampness,relieving cough and asthma.Rhubarb is often used in combination with astragalus,peony,angelica,gardenia,and licorice.When rhubarb and licorice are used together,the common compatibility ratio is 1:1,accounting for 37.85 percent of the total amount of prescriptions.The majority the weight of licorice is less than or equal to the weight of rhubarb.When the ratio is different,the order of formulas' efficacy is also different,mainly containing relieving pain,clearing heat,detoxification,relieving phlegm,cough and diarrhea,etc.Section 2 Progress in Chemical and Biological Research of Chinese Herbal RhubarbIn rhubarb-licorice herb pair,rhubarb plays a major role of diarrhea,while licorice is used to reconcile rhubarb drug properties.Therefore,we reviewed the research progress of chemical and biological of rhubarb in this part.Section 3:Progress of Research on Chemical Composition and Compatibility of Dahuang Licorice DecoctionIn this section,we reviewed the relevant literature of Dahuang Gancao Decoction and rhubarb-licorice herb pair in recent years,including its material basis,compatibility mechanism,and clinical application.Chapter 2 Dahuang Gancao Decoction and its dose-effect relationshipSection 1 evaluation of the purgative effect of Dahuang Gancao Decoction on constipation mouseIn this section,loperamide hydrochloride was used to replicate the constipation model in mouse.Lactulose was given as a positive drug,and rhubarb,licorice,Dahuang Gancao Decoction were used to treat the constipation mouse.The effects of rhubarb and Dahuang Gancao Decoction on constipation mouse were investigated with mouse defecation characteristics,intestinal propulsion rate,organ coefficient,blood biochemical indicators,and pathological sections.It was found that rhubarb had a significant diarrhea effect at the 4 times higher dose in the pharmacopoeia.Combination with licorice can slow the diarrhea effect of rhubarb.We also found that rhubarb had a damaging effect on liver and colon in constipation mouse.Compatibility with licorice can reduce the damage to liver and colon of rhubarb.There was no evidence shown rhubarb had damage to the kidney and resulted water and salt balance disorders.Section II:The Quantitative-Efficacy Relationship of Dahuang Gancao Decoction on Constipation MouseIn this section,we set control group,model group,positive drug group,rhubarb 0.5-fold group,Rhubarb 0.5-fold group,rhubarb 2-fold group,Dahuang Gancao Decoction 2-fold group,rhubarb 8-fold group,and Dahuang Gancao Decoction 8-fold group.To investigate the dose-effect relationship of rhubarb diarrhea effect and the harming effect of licorice,we admitted mouse with loperamide to make constipation models.The study found that rhubarb had good diarrhea effect at dose of 0.5 times in pharmacopoeia.Rhubarb has a purgative effect in a dose-dependent manner,but high-dose rhubarb has liver and colon toxicity in constipation mouse.It can increase the ALT and AST of mouse,vacuolar degeneration of hepatocytes,and thicken the murine colon muscle.Compared with the rhubarb group,the defecation volume,fecal water content and the intestinal propulsion rate decreased,as well as the time of oral and anus transport were increased in the Dahuang Gancao Decoction group.The combination of licorice can reduce the level of ALT and AST in mouse,alleviate the thickening of murine colon muscle layer and reduce the vacuolation of liver cells caused by rhubarb.Licorice eases the diarrhea effect of rhubarb and reduces the liver and colonic toxicity of rhubarb to constipation mouse.The harmonization of licorice to rhubarb decreases with the increase of rhubarb dose.Chapter 3 Research on the Material Basis of Dahuang Gancao Decoction Section 1 Study on the dissolution changes of chemical constituents in Rhubarb-Licorice in vitroThis section aims to study the changes of chemical composition of rhubarb and licorice before and after compatibility in vitro.We selected 9 rhubarb-licorice compatibility ratios 4:0,4:1,4:2,4:3,4:4,3:4,2:4,1:4,0:4 to detect.Simultaneous determination of 17 Chemical Components(rhein,emodin,aloe-emodin,chrysophanol,emodin,rhein-8-O-?-D-glucoside,emodin-8-O-?-D-glucoside,sennoside A,sennoside B,glycyrrhizic acid,glycyrrhetinic acid,glycyrrhizin,isoliquiritigenin,glycyrrhizin,isoliquiritigenin,licorice chalcones,light glyphosate)in rhubarb and licorice by UHPLC-TQ-MS/MS.It was found that licorice promoted the dissolution of eight active ingredients of rhubarb.However,rhubarb had two effects on the dissolution of components in licorice.Rhubarb promoted the dissolution of glycyrrhizic acid,glycyrrhetinic acid,isoliquiritigenin,and glycyrrhizin,while inhibitted the dissolution of glycyrrhizin,isoliquiritigenin and licorice chalcone.Section 2 Pharmacokinetics and Tissue Distribution Changes of Dahuang Gancao Decoction in Normal and Constipated MouseThis section aims to study the pharmacokinetics and tissue distribution differences of Dahuang Gancao Decoction in the control and constipation mouse.A UHPLC-TQ-MS/MS method was established to simultaneously determine rRhein,emodin,aloe-emodin.rhein-8-O-?-D-glucose,sennoside A,glycyrrhizic acid,and glycyrrhizin in Dahuang Gancao Decoction in the control and model mouse.The results showed that the main chemical constituents of Dahuang Gancao Decoction in model mouse in the plasma and liver were higher than in the control group,and the changes in the kidney and colon were not completely the same.In general,the amount of rhein,emodin,aloe-emodin,rhein-8-O-?-D-glucose,sennoside A.glycyrrhizic acid,and glycyrrhizin absorbed in constipation mouse increased.The active ingredients of rhubarb in the liver and colon of constipated mouse are also higher than in control mouse.Chapter 4 Study on the mechanism of diarrhea effect of Dahuang Gancao Decoction based on intestinal bacteria and metabonomics study Section 2 Effect of Dahuang Gancao Decoction on the Diversity of Intestinal Bacteria inConstipation MouseIn this section,the second-generation high-throughput sequencing method was used to determine the differences of intestinal microflora between mouse in the control group,the model group,the positive drug group,the rhubarb group,the licorice group,and the Dahuang Gancao Decoction group.547 OUT sequences were found in all samples.The level of intestinal bacterial diversity in the rhubarb group and the Dahuang Gancao Decoction group were lower than that in the other four groups,and the glycyrrhizae can ease the richness and evenness decrease of the intestinal bacteria in constipation mouse caused by rhubarb.There were 208 OUT sequences in the intestinal flora of mouse in each group.At the level of the phylum,Streptomyces,Bacteroidetes,and Proteobacteria were the most microorganisms.At the level of the genus,Mercaprum accounted for 32.02%,was the highest proportion of the species belonging to the these mouse,and Bacteroides accounted for 21.14%,following Mercaprum.In the PLS-DA plot,the intestinal flora of the Rhubarb group showed a significant left shift.Compatibility with licorice can ease the constipation caused by rhubarb in constipation mouse intestinal bacteria.Licorice has a harmonizing effect.From the analysis of the differences in intestinal microflora structure between groups,we found the differences in intestinal microflora structure between the control group and the model group was small,while the differences between the model group and the rhubarb group were greater,9 kinds of intestinal bacteria with significant differences.Compared the Dahuang Gancao Decoction group with the model group,there were significant differences among 8 Intestinal bacteria.Compared the Dahuang Gancao Decoction group with rhubarb group,3 intestinal bacteria had significant differences.Compatibility with licorice can reduce the rhizosphere increasing caused by rhubarb in constipation mouse,increase the number of lactobacillus,regulate the imbalance of intestinal microflora,maintain the stability of intestinal flora,reduce the toxicity of rhubarb.Section 2 Study on the Potenterating Mechanism of Dahuang Gancao Decoction Based on MetabolomicsIn this section,we set up control group,model group,rhubarb group,licorice group,and Dahuang Gancao Decoction group to reseach potenterating mechanism based on metabolomics.A metabolomic study was conducted on the plasma of mouse.Thirteen endogenous differential metabolites were found.13 metabolic pathways were obtained from these differential metabolites,inclouding glycerophospholipid metabolism,glyoxylate and dicarboxylic acid metabolism,citric acid cycle(TCA cycle),alanine,aspartic acid and glutamate metabolism,pantothenate and CoA biosynthesis,pyrimidine metabolism,pyruvate metabolism and so on.Rhubarb and Dahuang Gancao Decoction can reverse the changes of endogenous markers in mouse caused by constipation.Rhubarb and Dahuang Gancao Decoction may be used to treat constipation by regulating the above metabolic pathways,while licorice has a moderating effect on the reversal of rhubarb. |
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