Equivalent component group of Gegen Qinlian decoction for improving type 2 diabetes mellitus and application of equivalent component group

Abstract

The invention discloses an equivalent component group of Gegen Qinlian decoction for improving type 2 diabetes mellitus and application of the equivalent component group. The invention provides an equivalent component group for treating type 2 diabetes mellitus by using a Gegen Qinlian decoction. The equivalent component group consists of 13, 19 or 27 chemical components. A zebra fish diabetes model and a high fat diet combined streptozotocin induced type 2 diabetes mouse model prove that the equivalent component group consisting of 13, 19 or 27 chemical components has an obvious treatment effect on type 2 diabetes, the treatment effect is not remarkably different from that of the original formula of the Gegen Qinlian decoction, and the excellent equivalence is shown. Therefore, the composition provided by the invention can be used as an equivalent component group of the Gegen Qinlian decoction for treating the type 2 diabetes mellitus, and is further used for preparing a medicine for equivalently replacing the Gegen Qinlian decoction for improving the type 2 diabetes mellitus, or is used for preparing a composition for controlling the quality of the Gegen Qinlian decoction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to the discovery and application of an equivalent component group of a classic prescription of traditional Chinese medicine, in particular to an equivalent component group of Gegen Qinlian Decoction for improving type 2 diabetes and application thereof. BACKGROUND

[0002] Diabetes is divided into type 1 diabetes, type 2 diabetes and gestational diabetes, and the most common type in clinical is type 2 diabetes with decreased insulin sensitivity and defective function of islet beta cells. The pathogenesis of type 2 diabetes is extremely complex, and is related to genetic factors, lifestyle, obesity, insulin resistance, islet beta cell dysfunction, chronic inflammation, intestinal flora imbalance and other internal and external factors, and various factors are causally related and jointly cause the disease.

[0003] Gegen Qinlian Decoction is a compound preparation composed of four traditional Chinese medicines, namely, radix puerariae, radix scrophulariae, rhizoma coptidis and honey-fried licorice, and has the effects of relieving superficies and clearing interior, clearing heat and stopping diarrhea, and is often used in the treatment of various diseases caused by damp-heat accumulation in clinical practice. It has good clinical application value in the treatment of acute enteritis, bacterial dysentery, intestinal typhoid, gastrointestinal cold, non-alcoholic fatty liver disease, type 2 diabetes and other metabolic diseases. Although Gegen Qinlian Decoction has definite efficacy, due to the complexity and diversity of its prescription, the key scientific problem of which components jointly exert the efficacy, i.e., the “pharmacodynamic component group”, has been difficult to clarify.

[0004] The “equivalent component group” is a research hypothesis for the effective component group of traditional Chinese medicine, that is, although there are numerous components in traditional Chinese medicine, not all components exert the efficacy for a specific syndrome / efficacy, and there naturally exists a group of components with clear composition, definite content ratio and equivalent efficacy to the original traditional Chinese medicine, which interact with the key nodes / targets / pathways of the body and exert the efficacy in an overall coordinated manner. For a complex traditional Chinese medicine compound preparation like Gegen Qinlian Decoction, a thinking mode of organically linking “component- efficacy-syndrome” is established, which provides a basis for establishing a quality control mode of traditional Chinese medicine with dose-effect correlation. At the same time, based on equivalent substitution, the component group of the composition with clear components and easy-to-control quality can be used for equivalent substitution of the clinical treatment of traditional Chinese medicine. This has important significance for the modernization and internationalization of traditional Chinese medicine.

[0005] At present, among the hundreds of chemical components contained in Gegen Qinlian Decoction, it is not clear whether there is a combination of certain components and which combination of components can equivalent replace the effect of Gegen Qinlian Decoction for improving type 2 diabetes. Based on this research and discovery, the present application is proposed. SUMMARY

[0006] The present application aims to overcome the deficiencies of the prior art and provide an equivalent component group of Gegen Qinlian Decoction for improving type 2 diabetes and application thereof.

[0007] The above-mentioned object of the present application is achieved by the following technical solutions.

[0008] An equivalent composition representing the efficacy of Gegen Qinlian Decoction in treating type 2 diabetes mellitus is composed of 13 compounds: puerarin, genistein, 3'-hydroxypuerarin, formononetin, daidzin, daidzein, wogonin, wogonoside, baicalein, glycyrrhizic acid, glycyrrhizin charlton A, berberine hydrochloride and palmatine hydrochloride; and the content ratio of each compound is the same as that in Gegen Qinlian Decoction.

[0009] An equivalent composition representing the efficacy of Gegen Qinlian Decoction in treating type 2 diabetes mellitus is composed of 19 compounds: genistein, puerarin, daidzein, baicalein, wogonin, wogonoside, baicalin, berberine hydrochloride, chrysin, formononetin, glycyrrhetic acid, hedygripin A, isofraxidin, glycyrrhizin charlton A, palmatine hydrochloride, 3'-methoxypuerarin, hydrochloride of coptisine, mufangchin and daidzin; and the content ratio of each compound is the same as that in Gegen Qinlian Decoction.

[0010] An equivalent composition representing the efficacy of Gegen Qinlian Decoction in treating type 2 diabetes mellitus is composed of 27 compounds: genistein, puerarin, daidzein, baicalein, wogonin, wogonoside, baicalin, berberine hydrochloride, chrysin, formononetin, glycyrrhetic acid, hedygripin A, isofraxidin, glycyrrhizin charlton A, palmatine hydrochloride, 3'-methoxypuerarin, hydrochloride of coptisine, mufangchin, daidzin, glycyrrhizic acid, glycyrrhizin saponin G2, glycyrrhetol, magnoflorine, 3'-hydroxypuerarin, hydrochloride of epiberberine, afrapiamine and puerarin-6"-O-xyloside; and the content ratio of each compound is the same as that in Gegen Qinlian Decoction.

[0011] The above-mentioned equivalent composition is used for preparing a drug for replacing Gegen Qinlian Decoction in treating type 2 diabetes mellitus.

[0012] Preferably, the drug takes the equivalent composition as active ingredient, and is prepared into a pharmaceutically acceptable dosage form by a pharmaceutically acceptable carrier or excipient.

[0013] More preferably, the carrier or excipient is solid, liquid or semi-solid.

[0014] More preferably, the dosage form includes tablets, capsules, pills and injections.

[0015] The above-mentioned equivalent composition is used for preparing a composition for controlling the quality of Gegen Qinlian Decoction, which contains standard samples of each compound in the equivalent composition, and the quality of Gegen Qinlian Decoction is based on the efficacy of Gegen Qinlian Decoction in treating type 2 diabetes mellitus.

[0016] Advantages:

[0017] The application provides an equivalent component group of Gegen Qinlian Decoction for treating type 2 diabetes, which is composed of 13, 19 or 27 chemical components. Zebrafish diabetes model and type 2 diabetes mouse model induced by high-fat diet combined with streptozotocin prove that the equivalent component group composed of the 13, 19 or 27 chemical components has obvious therapeutic effect on type 2 diabetes, and the therapeutic effect has no significant difference compared with the original Gegen Qinlian Decoction, which shows excellent equivalence.

[0018] The application provides a certain reference for elucidating the chemical basis of the medicinal effect of Gegen Qinlian Decoction, and promotes the research on the medicinal material basis and quality standard of Gegen Qinlian Decoction, in particular: (1) the combination of the 13, 19 or 27 components can be prepared into a drug composition for treating type 2 diabetes as an equivalent substitute for Gegen Qinlian Decoction; (2) the combination of the 13, 19 or 27 components can be prepared into a marker composition for controlling the quality of Gegen Qinlian Decoction, so as to realize the stability, repeatability and controllability of the chemical components in monitoring the stability, repeatability and controllability of Gegen Qinlian Decoction in improving the activity of type 2 diabetes. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The figure is a Venn diagram of the action target of the components of Gegen Qinlian Decoction and the action target of type 2 diabetes;

[0020] Figure 2 The figure is a protein-protein interaction (PPI) network;

[0021] Figure 3 The figure is a network relationship diagram of “traditional Chinese medicine-component-disease target”;

[0022] Figure 4 The figure is the top 10 entries obtained by GO enrichment of 147 key targets;

[0023] Figure 5 The figure is the top 20 signal pathways obtained by KEGG pathway enrichment of 147 key targets;

[0024] Figure 6 The figure is the influence of Gegen Qinlian Decoction and candidate component groups I-IV on zebrafish glucose level and lipid accumulation;

[0025] Figure 7 The figure is the influence of Gegen Qinlian Decoction and equivalent component group IV on the body weight and fasting blood glucose of T2DM mice;

[0026] Figure 8 The figure is the influence of Gegen Qinlian Decoction and equivalent component group IV on the OGTT and ITT of T2DM mice;

[0027] Figure 9Effects of Gegen Qinlian Decoction and equivalent component group IV on pancreas of T2DM mice;

[0028] Figure 10 Effects of Gegen Qinlian Decoction and equivalent component group IV on liver of T2DM mice;

[0029] Figure 11 Effects of Gegen Qinlian Decoction and equivalent component group IV on fat of T2DM mice. DETAILED DESCRIPTION

[0030] The substantial content of the present application will be specifically introduced below in combination with examples, but the protection scope of the present application is not limited by this.

[0031] I. Experimental materials, reagents and animals

[0032] The experimental materials, reagents and animals used in the present application are all commercially available products.

[0033] The information of the original medicinal materials of Gegen Qinlian Decoction is shown in Table 1.

[0034] Table 1 Information of original medicinal materials of Gegen Qinlian Decoction

[0035] The information of the standard samples of some chemical components in Gegen Qinlian Decoction is shown in Table 2.

[0036] Table 2 Information of standard samples of chemical components in Gegen Qinlian Decoction

[0037]

[0038]

[0039]

[0040] Acetonitrile and methanol (MS grade) were purchased from Merck Chemical Technology (Shanghai) Co., Ltd., D(+)-Dextrose (lot number: S11022-100g) was purchased from Shanghai Yuan Ye Biological Technology Co., Ltd., Dimethyl sulfoxide (lot number: G75927B) and anhydrous ethanol (lot number: G73537AD) were purchased from Shanghai Titan Scientific Co., Ltd., Sodium chloride (lot number: 1000582) was purchased from Xilong Scientific Co., Ltd., Anhydrous calcium chloride (lot number: C0050540223) was purchased from Nanjing Chemical Reagent Co., Ltd., Potassium chloride (lot number: 10016308) and anhydrous magnesium sulfate (lot number: D10203) were purchased from Shanghai Lingfeng Chemical Co., Ltd., Streptozotocin (lot number: S0130-1G) and sodium carboxymethyl cellulose (lot number: C8621) were purchased from Beijing Solabio Science and Technology Co., Ltd., Insulin (lot number: BS901-25mg) was purchased from Beijing Lanjiekai Technology Co., Ltd., Glucose reagent kit (glucose oxidase method), ALT, AST, TG, LDL-C, HDL-C, total cholesterol, insulin, etc. determination kit were purchased from Nanjing Jiancheng Biological Engineering Research Institute Co., Ltd., Oil red (lot number: O104972-25g) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and Fengnian shrimps were purchased from Tianjin Fengnian Aquaculture Co., Ltd.

[0041] Wild AB line zebrafish were purchased from Nanjing Yaoshunyu Biological Technology Co., Ltd. The water temperature was set to 28±1℃, and the light cycle was 14h light / 10h dark (lights on at 8am, lights off at 22pm) to ensure regular spawning of adult fish in the zebrafish recirculating water culture system. The system water was kept circulating to ensure adequate oxygen, and the water level was maintained by daily replenishment of the water lost by evaporation. The conductivity was adjusted to 400-500μs·cm-1 by sodium chloride, and the pH was adjusted to 6.8-7.2 by sodium bicarbonate and sodium chloride. -1 The Fengnian shrimp eggs were incubated under light for more than 24 hours (incubation ratio: 96g sea salt, 12g Fengnian shrimp eggs in 6L pure water), and the bottom shrimp eggs were washed clean with pure water, and fed twice a day. Male and female fish were separated during the experiment. Two hours after feeding at night, the fish were placed in the spawning tank at a ratio of 1:1 or 1:2, and separated by a partition. At the beginning of the light period the next morning, the partition was removed, and after the spawning was completed, the fertilized eggs were collected, impurities were removed, and transferred to a 28.5℃ light incubator (light cycle 14h / 10h) for culture. Unfertilized eggs and dead eggs were removed twice a day, and the culture water was changed.

[0042] The mouse strain is C57BL / 6J, SPF level, 6-8 weeks old, and weighs 20-22 g, purchased from Vantianlihua Experimental Animal Technology Co., Ltd. (Beijing, China). The animals were housed in the Animal Experimental Center of China Pharmaceutical University, and the feeding environment strictly followed the experimental animal feeding standards. The animals were allowed to eat and drink freely, and the environment temperature was about 25℃, and the relative humidity was about 50%. Before the experiment, the animals were adaptively fed for one week. The animal experiments strictly followed the "Regulations on the Management of Experimental Animals" and "Jiangsu Provincial Management Measures for Experimental Animals". All animal experiments were approved by the Animal Ethics Committee of China Pharmaceutical University (Ethical Number: 2024-11-028).

[0043] II. Experimental methods

[0044] 1. Quantitative analysis of the chemical components of Gegen Qinlian Decoction

[0045] 1.1 Quantitative analysis of the chemical components of Gegen Qinlian Decoction based on UHPLC-QQQ MS

[0046] Preparation of Gegen Qinlian Decoction samples: Accurately weigh 80 g of Gegen decoction pieces, 30 g of Huangqin decoction pieces, 30 g of Huanglian decoction pieces, and 20 g of Zhigancao decoction pieces, respectively, and place them in an electric sand pot. Add 1280 mL (equivalent to 8 times the total amount of medicinal materials) of water. Soak the Gegen decoction pieces for 30 minutes, then start boiling with a strong fire, and then switch to a gentle fire for 30 minutes. Then add Huangqin decoction pieces, Huanglian decoction pieces, and Zhigancao decoction pieces, and continue to heat to boiling with a strong fire, then switch to a gentle fire for 1 hour, and filter. Then add 1280 mL (equivalent to 8 times the total amount of medicinal materials) of water to the residue, heat to boiling with a strong fire, then switch to a gentle fire for 1 hour, and filter. Combine the filtrates obtained from the two filtrations. Concentrate by rotary evaporation at 60°C and 60 rpm, then vacuum freeze-dry. Grind the freeze-dried sample with a mortar and mix it evenly, then store it in a -20°C refrigerator for subsequent use.

[0047] Preparation of the test solution: Take about 0.1 g of the freeze-dried powder of Gegen Qinlian Decoction prepared above, accurately weigh it, and place it in a conical flask with a stopper. Accurately add 10 mL of 75% methanol, weigh it, ultrasonically extract it (40 kHz, 500 W) for 20 minutes, cool it, re-weigh it, make up the weight loss with 75% methanol, centrifuge it at 13000 rpm for 10 minutes, take 10 μL of the supernatant, dilute it to 1 mL with 75% methanol, and add the internal standard solution, as the test solution.

[0048] Preparation of the control solution: Weigh each control substance accurately, add methanol to dissolve and dilute the concentration to about 1 mg / mL of the control substance mother liquor, then take an appropriate amount of each control substance mother liquor, mix it to prepare a mixed solution containing about 1 μg / mL of the control substance, and shake it to obtain the mixed control substance solution.

[0049] Chromatographic conditions: The analytical instrument was a Shimadzu LC-30A ultra-high performance liquid chromatograph. Chromatographic separation was performed on an Agilent ZORBAX SB-Aq column (2.1 x 100 mm, 1.8 μm) with a pre-column filter for protection. Mobile phase: A phase was 0.1% formic acid water (v / v), B phase was acetonitrile; flow rate: 0.3 mL / min; column temperature: 35 °C; injection volume: 1 μL; Gradient elution 1 was used for detecting flavonoids and saponins under negative ion mode, and gradient elution 2 was used for detecting alkaloids under positive ion mode. Gradient elution 1: 0-22 min, 10-32% B; 1-2 min, 22-35% B; 2-9 min, 32-50% B; 35-36 min, 50-95% B; 36-40 min, 95% B. Gradient elution 2: 0-15 min, 20-25% B.

[0050] Mass spectrometric conditions: The mass spectrometric instrument was an AB SCIEX Qtrap 5500 triple quadrupole and linear ion trap technology hybrid mass spectrometer. Ion source parameters: nebulizer gas flow rate (N2): 3 L / min; drying gas flow rate (N2): 10.0 L / min; interface temperature: 300 °C; desolvation tube temperature: 250 °C; quantification was performed using the Multiple Reaction Monitoring (MRM) data acquisition mode; positive and negative ion modes were scanned and collected separately.

[0051] Data analysis: The results were reviewed and data were processed using Analyst software.

[0052] 1.2 Methodology validation

[0053] Standard curve, limit of quantification and limit of detection: The control substance mother liquor of each compound was diluted into a series of gradient concentrations, and an equal amount of internal standard was added. The injection amount of each mixed control substance solution was 1 μL. The peak area ratio of each test compound to the internal standard compound was fitted to a linear regression equation, and the standard curve of each component was obtained accordingly. The mixed control substance solution was further diluted with solvent and analyzed. The concentration with a signal-to-noise ratio of 10 was defined as the limit of quantification, and the concentration with a signal-to-noise ratio of 3 was defined as the limit of detection.

[0054] Intra-day precision: Three different concentrations of mixed control substance solutions at high, medium and low positions in the concentration gradient were selected. The injection amount of each concentration of mixed control substance solution was 1 μL, and the injection was performed 3 times within a day. The peak area ratio of each test compound to the internal standard compound was recorded, and the relative standard deviation (RSD) between 3 injections was calculated.

[0055] Day precision: select three different concentrations of mixed control solution in high, medium and low position in the concentration gradient, continuously for three days, 3 times a day for each concentration of mixed control solution, the injection amount is 1 μL, record the peak area ratio of each test compound and internal standard compound, calculate the RSD between 9 needles.

[0056] Repeatability: take gegen qinlian decoction freeze-dried powder, respectively, 6 parallel preparation of test solution, the injection amount is 1 μL, record the peak area ratio of each test compound and internal standard compound, calculate the RSD between 6 needles.

[0057] Stability: test solution was analyzed at 0h, 2h, 4h, 8h, 12h, 24h, respectively, record the peak area ratio of each test compound and internal standard compound, calculate the RSD between 6 needles.

[0058] Accuracy: the recovery experiment was used to investigate. Take gegen qinlian decoction freeze-dried powder, respectively, 6 parallel preparation of test solution, the injection amount is 1 μL. Record the peak area ratio of each test compound and internal standard compound, into the standard curve to calculate the content of each test compound. According to the formula, the recovery rate was calculated, the RSD between 6 needles was calculated. Recovery % = (actual measured content-sample content) / added control amount x 100%.

[0059] 1.3 content determination

[0060] According to the established analysis method, the content of the ingredients in the prepared gegen qinlian decoction freeze-dried powder was determined, the injection amount was 1 μL, the peak area ratio of each test compound and internal standard compound was recorded, and the content of each compound was calculated by standard curve.

[0061] 2, based on network pharmacology screening of potential components of gegen qinlian decoction to improve type 2 diabetes

[0062] 2.1 screening of chemical components of gegen qinlian decoction and the action target of disease

[0063] To predict the potential targets of 60 chemical components, we introduced these components into multiple authoritative databases, including PubMed (https: / / pubmed.ncbi.nlm.nih.gov / ) for literature retrieval, PharmMapper (http: / / www.lilab-ecust.cn / pharmmapper / ) for drug-target prediction, SwissTargetPrediction (http: / / www.swisstargetprediction.ch / ) for structure-based target prediction, and TCMSP (https: / / tcmspw.com / tcmsp.php) for traditional Chinese medicine component analysis. Then, we used the Uniprot database (https: / / www.uniprot.org / ) to standardize the gene names of the targets associated with diabetes for the compounds, thereby constructing a standardized target dataset for the compounds and diabetes. Based on the VENNY 2.1 online tool (https: / / bioinfogp.cnb.csic.es / tools / venny / ), we drew a Venn Diagram of the compound targets and diabetes targets to obtain the potential action targets of Gegen Qinlian Decoction in the treatment of diabetes.

[0064] 2.2 Protein-protein interaction (PPI) network construction and key target analysis

[0065] We used the STRING 11.0 database to conduct in-depth analysis of the protein-protein interaction (PPI) information of the 60 chemical components and common targets of diabetes, setting the species to "Homo Sapiens" and keeping other parameters at their default settings. Based on the analysis results, we used Cytoscape 3.8.1 software to construct the PPI network of Gegen Qinlian Decoction for the treatment of diabetes. Subsequently, we used the NetworkAnalyzer toolkit built into Cytoscape to evaluate the topological isomerization parameters of the PPI network, including degree, betweenness centrality, closeness centrality, and other parameters.

[0066] 2.3 Compound-diabetes target network construction

[0067] First, we organized and imported the data information of the 60 compounds in Gegen Qinlian Decoction and their corresponding diabetes targets into an Excel table, and further used Cytoscape 3.8.1 software to conduct detailed visualization processing of the compound-diabetes target interaction information of Gegen Qinlian Decoction.

[0068] 2.4 KEGG pathway analysis and GO biological function annotation

[0069] To further explore the potential mechanism of Gegen Qinlian Decoction in treating diabetes, the previously screened compound target genes were first introduced into the DAVID 6.8 (https: / / david.ncifcrf.gov / ) database one by one. On this basis, systematic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and GO (Gene Ontology) enrichment analysis were performed. Bubble charts and bar charts were generated using R-Studio software to more intuitively display the pathways and biological functions involved in treating diabetes.

[0070] 2.5 Calculation of Network topology score (NTS) value

[0071] Firstly, the degree, betweenness centrality, closeness centrality and neighborhood connectivity parameters obtained through network pharmacology analysis were normalized to ensure the comparability of each index in numerical value. Subsequently, the professional statistical software SIMCA 14.0 was used to perform Principal Component Analysis (PCA) on the four normalized parameters. Through the dimension reduction processing of PCA, a comprehensive index, Network Topology Score (NTS), was calculated and obtained to comprehensively evaluate the pharmacological characteristics of the network.

[0072] 3. Proposal of candidate component group

[0073] According to the content of each component in Gegen Qinlian Decoction and its network topology score in network pharmacology, the candidate component group was designed.

[0074] 4. Capture and preparation of equivalent component group and residual component group based on preparation liquid phase

[0075] A preparative Shimadzu Prominence LC-20AP liquid chromatography system was used with an Agilent Zorbax SB-C18 semi-preparative column (9.4 x 250 mm, 5 μm). The mobile phase was 0.1% formic acid in water (pH 3.4 adjusted with ammonia) (A) and acetonitrile (B). The gradient elution program was 0-17 min, 15-25% B; 17-40 min, 25-37% B; 40-64 min, 37-60% B; 64-65 min, 60-95% B; 65-80 min, 95-95% B at a flow rate of 2 mL / min. The column temperature was set at 30 °C and the detection wavelength was 280 nm. Candidate BECCs were prepared by real-time manual injection after injecting 25 μL of a 70% (v / v) methanol solution of GQD at a concentration of 8 mg / mL. The collection program was as follows: 3'-hydroxy puerarin, 9.15-10.30 min; puerarin, 13.75-15.00 min; daidzin, 21.40-22.50 min; wogonoside, 41.25-42.00 min; daidzein, 45.85-47.00 min; glycyrrhizic acid, 51.50-53.50 min; palmatine hydrochloride, 54.30-55.75 min; berberine hydrochloride, 57.20-59.25 min; genistein, 59.50-60.50 min; baicalein, 60.60-61.70 min; formononetin, 66.25-67.00 min; wogonin, 72.25-73.00 min; glyyrrhetol, 77.75-79.00 min. The remaining fraction was collected in addition to the above 13 components. The solvent was removed using a Speedvac evaporator (Genevac EZ-2plus, Genevac Technologies, UK). Then, an appropriate amount of dimethyl sulfoxide (DMSO) was added to reconstitute the solution to prepare a stock solution at a concentration of 20 mg / mL, which was stored in a refrigerator at -20 °C.

[0076] 5. Discovery of the equivalent component group of Gegen Qinlian Decoction for improving type 2 diabetes based on the zebrafish diabetes model

[0077] 5.1 Solution preparation

[0078] Preparation of E3 medium (60x): weigh 17.532 g of sodium chloride, 0.7695 g of potassium chloride, 2.1978 mg of calcium chloride, and 2.3833 g of magnesium sulfate into 1 L of ultrapure water. The preparation ratio of embryo culture water is: 1 L of pure water plus 16.6 mL of E3 medium (60x).

[0079] Preparation of glucose: weigh an appropriate amount of glucose, accurately weigh, dissolve in embryo culture water to prepare 3.0% glucose E3 medium, and use it immediately after preparation.

[0080] Oil red dye preparation: 0.5 g of oil red dry powder was weighed, dissolved in a small amount of isopropyl alcohol, and then isopropyl alcohol was added to 100 mL, and stored at 4°C in the dark as a stock solution. The oil red stock solution and deionized water were diluted according to 3:2, and then used after filtration.

[0081] Gegen Qinlian decoction preparation: Gegen Qinlian decoction lyophilized powder was accurately weighed, dissolved in DMSO to 100 mg / mL as GQD mother liquor, and then diluted with embryo culture water to 200, 100, 80, 40, 20 and 10 μg / mL for use.

[0082] Preparation of candidate component group solution: According to the quantitative analysis results of Gegen Qinlian decoction, the content of chemical components in Gegen Qinlian decoction was calculated, accurately weighed, dissolved in DMSO to form a mother liquor, and then mixed into each candidate component group solution according to the design. When used, it was diluted with E3 culture solution to the required concentration.

[0083] 5.2 Construction of zebrafish diabetes model and animal grouping

[0084] Zebrafish larvae that developed normally after 2 days of fertilization (2dpf) were selected and placed in a six-well plate, with 30 larvae per well. The experiment was divided into control group, model group, Gegen Qinlian decoction group, candidate component group I, candidate component group II, candidate component group III and candidate component group IV group. When dosing, the embryo culture water in the six-well plate was removed, 3 mL of embryo culture water was added to the control group, 3.0% glucose culture solution was given to the model group, and 3.0% glucose culture solution mixed with 20 μg / mL Gegen Qinlian decoction was given to the Gegen Qinlian decoction group, with a total volume of 3 mL. The candidate component group was given 3.0% glucose culture solution mixed with standard solution prepared according to the quantitative results of Gegen Qinlian decoction (the dosages of each chemical component in the candidate component group were the same as those in the Gegen Qinlian decoction group, and the same below), with a total volume of 3 mL.

[0085] 5.3 Evaluation of glucose levels in zebrafish

[0086] Ten zebrafish from each group were taken in a 1.5 mL centrifuge tube, the culture solution was removed, and the appropriate PBS was used to wash 4 times. After the last washing, the PBS was discarded, and the tube was placed in the 4°C refrigerator overnight with the opening, and the solution was evaporated. 20 μL PBS was added and ground with a tissue grinder, centrifuged, and the supernatant was taken and determined for glucose content using a glucose kit.

[0087] 5.4 Evaluation of lipid levels in zebrafish

[0088] Each group of zebra fish was fixed with paraformaldehyde for 2 h, washed with PBS twice, and then stained with oil red solution for 1 h in the dark. The solution was eluted with 70% ethanol until the signal was clear. Ten zebra fish from each group were fixed on slides with 3% methyl cellulose. The zebra fish were kept in a lateral recumbent position. The staining of each group of zebra fish was observed and photographed using a body microscope. The photographs were processed and analyzed using ImageJ software.

[0089] 6. Evaluation of the activity of the Gegen Qinlian Decoction equivalent component group in improving type 2 diabetes mellitus based on a mouse model of T2DM

[0090] 6.1 Solution preparation

[0091] STZ injection solution preparation: Citric acid was weighed and accurately measured, and then dissolved and diluted in pure water to obtain a 0.1% citric acid aqueous solution. Sodium citrate was weighed and accurately measured, and then dissolved and diluted in pure water to obtain a 3% sodium citrate aqueous solution. The citric acid aqueous solution and the sodium citrate aqueous solution were mixed at a ratio of 1:1, filtered with a 0.22 μm filter membrane, and stored at 4°C for later use. STZ was weighed and accurately measured in the dark, and then dissolved and diluted in the citric acid-sodium citrate buffer to obtain a 10 mg / mL STZ injection solution, which was used in the dark.

[0092] Carboxymethylcellulose sodium solution: Carboxymethylcellulose sodium was weighed and accurately measured, and then dissolved and diluted in pure water to obtain a 0.5% carboxymethylcellulose sodium aqueous solution, which was stored at 4°C for later use.

[0093] Gegen Qinlian Decoction: Gegen Qinlian Decoction lyophilized powder was weighed and accurately measured, and then dissolved and diluted in the carboxymethylcellulose sodium solution to obtain a 0.18 g / mL (low dose) and a 0.36 g / mL (high dose) solution, which was stored at 4°C for later use.

[0094] Gegen Qinlian Decoction candidate equivalent component group IV: The reference substances in this section were weighed and accurately measured, and then mixed and dissolved and diluted in the carboxymethylcellulose sodium solution to obtain a solution with a concentration equivalent to that of the low-dose Gegen Qinlian Decoction (0.18 g / mL) and the high-dose Gegen Qinlian Decoction (0.36 g / mL), which was stored at 4°C for later use.

[0095] 6.2 Establishment of a mouse model of T2DM and experimental grouping

[0096] 5-week-old male C57BL / 6J mice were fed for one week, among which, the blank group mice were normally fed, and the rest were all replaced with high-fat feed (fatty energy supply accounted for 60%) for continuous feeding for 4 weeks. The C57BL / 6J mice were divided into cages according to the body weight, and the mice were fasted for 16 h. The mice were injected with STZ through intraperitoneal injection, and the dosage was 40 mg / kg. The blood glucose level was determined after continuous injection for 5 days. The male C57BL / 6J mice with successful modeling were randomly divided into 6 groups, 10 in each group. The random grouping conditions were as follows: model group (normal saline), positive drug group (200 mg / kg Metformin), Gegen Qinlian Decoction low-dose group (1.8 g / kg GQD), Gegen Qinlian Decoction high-dose group (3.6 g / kg Gegen Qinlian Decoction), candidate component group low-dose group (the dosage was equivalent to that of the GQD low-dose group), and candidate component group high-dose group (the dosage was equivalent to that of the GQD high-dose group), and the blank group was given the same amount of normal saline. Continuous gavage administration was performed for 6 weeks.

[0097] 6.3 Determination of fasting blood glucose level of mice

[0098] The mice were changed into clean cages and fasted for 16 h. During the fasting period, the mice were kept normal drinking water. The mice were taken out of the cages, gently placed on the iron grid, and the tail ends of about 1-2 mm were cut off with scissors. The mouse tail was gently squeezed to make the blood concentrate into a drop. After the first drop of blood was discarded, the fasting blood glucose was determined with a blood glucose meter, and the blood glucose determination value was read.

[0099] 6.4 Oral glucose tolerance test (OGTT)

[0100] Glucose solution preparation: an appropriate amount of glucose was accurately weighed, added with pure water to dissolve and dilute into a glucose solution with a concentration of 0.1 g / mL, and stored at 4°C for standby use.

[0101] The mice were transferred to clean cages, and the oral glucose tolerance test was performed after fasting for 16 h. During the fasting period, the mice were kept normal drinking water. The tail ends of about 1-2 mm of the mice were cut off with scissors, and the mouse tail was gently squeezed to make the blood concentrate into a drop. After the first drop of blood was discarded, the fasting blood glucose was determined with a blood glucose meter, and the value was recorded as the blood glucose value at 0 min. Then, the mice were given a dose of 0.5 g / kg of glucose solution by gavage. After the gavage was completed, the timing was immediately started. The blood glucose value of each mouse was determined at 15 min, 30 min, 60 min, 90 min and 120 min after gavage, and was recorded. The time was taken as the X axis, and the blood glucose value was taken as the Y axis, to draw a line graph. The Area Under the Curve (AUC) was calculated using GraphPad software.

[0102] 6.5 Insulin Tolerance Test (ITT)

[0103] Insulin solution preparation: about 1 mg of insulin powder was weighed and added to pure water in an ice water bath, and then ultrasonically dissolved and diluted to obtain an insulin solution with a concentration of 1.0 mg / mL. 40 μL of the insulin solution was taken and diluted with 21.56 mL of normal saline to obtain an insulin solution with a concentration of 0.05 U / mL, which was stored at 4°C for later use.

[0104] The mice were changed into clean cages and fasted. After fasting for 4 h, the insulin tolerance test was performed. During the fasting period, the mice were allowed to drink water normally. The tail tip of the mouse was cut off about 1-2 mm with scissors, and the mouse tail was gently squeezed to make the blood concentrate into a drop. After discarding the first drop of blood, the blood glucose was measured using a blood glucose meter, and the value was recorded as the blood glucose value at 0 min. Then, the mouse was injected intraperitoneally with an insulin solution at a dose of 0.5 U / kg. After the injection was completed, timing was immediately started. The blood glucose value of each mouse was measured at 15 min, 30 min, 60 min, 90 min, and 120 min after the injection, and was recorded. A line graph was plotted with time as the X axis and the blood glucose value as the Y axis. The area under the curve (AUC) was calculated using GraphPad software.

[0105] 6.6 Measurement of serum biochemical indicators

[0106] The blood of each group of mice was collected by enucleation, and the blood was naturally precipitated for 4 h. The serum was separated by centrifugation at 3000 rpm for 10 min. The supernatant (serum) was aliquoted and stored at -80°C for later use. The contents of AST, ALT, TG, T-CHO, LDL-C, HDL-C, and insulin were determined according to the kit instructions, and the contents in the serum were calculated.

[0107] 6.7 Pathological section detection

[0108] The pancreas, liver, and adipose tissue of each group of mice were taken and immersed in 4% paraformaldehyde fixing solution, and then sent to Wuhan Sevier Biological Technology Co., Ltd. for pathological section preparation.

[0109] H&E staining of liver: after the section was deparaffinized to water, hematoxylin staining was performed for 8-15 min, and then washed with water. The cells were differentiated with 1% hydrochloric acid alcohol for several seconds to 30 seconds, and then washed with water until the cell nucleus was blue. Then, eosin staining was performed for 1-3 min, and the floating color was removed by washing with water. Finally, the section was dehydrated with 80% and 95% ethanol and anhydrous ethanol, transparentized with xylene, and sealed with neutral resin.

[0110] Liver oil red staining: The frozen sections of liver tissue were washed with distilled water, immersed in 60% ethanol, and then placed in oil red O staining solution (oil red O stock solution mixed with distilled water 3:2) for 10-15 minutes in the dark. Then, the sections were differentiated with 60% ethanol, washed with distilled water for 3 times, and the cell nuclei were counterstained with Mayer's hematoxylin for 1-2 minutes, differentiated with 1% hydrochloric acid, rinsed, dried, and mounted with glycerol gelatin.

[0111] Pancreas H&E staining: Similar to the liver, the cell nuclei were stained blue-violet with hematoxylin, and the cytoplasm was stained red with eosin. The staining was completed by dehydration, transparency, and fixation steps.

[0112] Fat H&E staining: After deparaffination of the sections to water, hematoxylin staining was performed for 1 minute, followed by eosin staining after water washing, and then 70% ethanol differentiation, and finally dehydration, transparency, and fixation.

[0113] 7. Data analysis and statistics

[0114] All mouse activity data were expressed as mean ± standard error of the mean (SEM). Statistical analysis of the data was performed using Graph Pad Prism 9.0. One-way ANOVA and Tukey's multiple comparison test were used for comparison between groups, and p<0.05 indicated that the difference was statistically significant.

[0115] 8. Equivalence evaluation

[0116] In order to facilitate the evaluation of biological activity equivalence, all biological assay results were converted into efficacy values according to the following equation:

[0117]

[0118] Activity drug : Determined value of biological activity index of the administration group (Metformin, GQD and BECC);

[0119] Activity model : Determined value of biological activity index of the model group;

[0120] Activity control : Determined value of biological activity index of the control group.

[0121] Biological activity equivalence was evaluated by calculating the 90% confidence interval of the ratio of the efficacy of the candidate BECC and GQD (two one-sided t-tests). Efficacy data usually need to meet normal distribution or lognormal distribution. When the data do not meet the normal distribution, the efficacy can be taken as the logarithm for statistical analysis:

[0122]

[0123] The 90% confidence interval of relative efficacy can be calculated by the following formula, if the 90% confidence interval of relative efficacy is in the range of 70-143% compared with GQD, the candidate BECC is considered to be bioequivalent active ingredient group with GQD.

[0124]

[0125] df = n BECC + n GQD - 2

[0126] III. Experimental results

[0127] 1. Quantitative analysis of the chemical components of Gegen Qinlian Decoction

[0128] Based on the previous qualitative characterization of the chemical components of Gegen Qinlian Decoction by the research group, high-content or active components accurately identified in the literature were selected, and a total of 60 representative compounds were selected for quantitative analysis.

[0129] UHPLC-QTRAP MS method validation: 60 analytes showed good linearity (r 2 was 0.940-0.999), the recovery rate was 84%-125% and RSD <10%, the intra-day and inter-day precision, repeatability, and sample stability within 24 hours were good (RSD <10%), meeting the requirements of quantitative analysis methods. The quantitative results showed that the contents of puerarin, wogonin, baicalein, and berberine hydrochloride in Gegen Qinlian Decoction freeze-dried powder were relatively high, ranging from 2.0% to 12.0%. The results showed that the established quantitative method was accurate, reproducible, and reliable, and could simultaneously determine the 60 selected compounds in Gegen Qinlian Decoction.

[0130] 2. Screening of target points for Gegen Qinlian Decoction in the treatment of type 2 diabetes

[0131] The chemical components of Gegen Qinlian Decoction were input into SwissTargetPrediction, PharmMapper, and TCMSP databases, and a total of 445 target points of the chemical components of Gegen Qinlian Decoction were retrieved. A total of 465 diabetes-related target points were obtained from PharmGKB, CTD, TTD, and DrugBank databases. VENNY 2.1 was used to construct the Venn diagram of the chemical components of Gegen Qinlian Decoction and the target points of diabetes Figure 1 ), and a total of 147 intersection target points were obtained, which were the potential target points of Gegen Qinlian Decoction in the treatment of diabetes.

[0132] 3. Regulatory network analysis of Gegen Qinlian Decoction in the treatment of diabetes

[0133] The 147 target points of Gegen Qinlian Decoction for treating diabetes were input into the STRING database to construct the PPI network of common target points, and the target points with a "combine score ≥ 0.7" were screened. Based on Cytoscape 3.8.1 software, the PPI network diagram of chemical components and common target points of diabetes was established ( Figure 2 ), which contained 135 nodes and 902 edges. Genes with a degree > 20 were selected as key target points in the protein-protein interaction network, and 37 key target points were obtained. Among them, the degree values of target points such as serine / threonine-protein kinase AKT (AKT1), transcription factor AP-1 (JUN), tumor necrosis factor (TNF), interleukin 6 (IL6), and mitogen-activated protein kinase 3 (MAPK3) were larger, and the interaction frequency was high, indicating that these target points played a significant role in the PPI network, suggesting that these genes may be key target points for the treatment of diabetes.

[0134] 4. Construction of "Traditional Chinese Medicine-Component-Disease Target" Network

[0135] The 60 chemical components of Gegen Qinlian Decoction and the 135 common target points for treating diabetes were input into Cytoscape 3.8.1 software to draw the visual network of chemical components of Gegen Qinlian Decoction and target points of diabetes ( Figure 3 ). The compound-disease target network of Gegen Qinlian Decoction for treating diabetes contained 199 nodes (4 medicinal components, 60 chemical components, and 135 target points) and 835 edges. Network Analyzer was used for network analysis, and GG3 (Genistein), HQ6 (Apigenin), GG13 (Daidzein), GG1 (Puerarin), HQ1 (Wogonin), HQ5 (Baicalein), and HQ8 (Chrysin) had a degree value greater than 20. These compounds were closely related to the target points for treating diabetes, suggesting that these compounds were key pharmacodynamic components of Gegen Qinlian Decoction for treating diabetes. At the same time, 16 genes (CA2, ESR1, TTR, GSTP1, and PTGS2) with a degree ≥ 15 were screened in the network, indicating that these genes were key target points for Gegen Qinlian Decoction in treating diabetes.

[0136] 5. KEGG signaling pathway and GO enrichment analysis

[0137] GO and KEGG pathway enrichment analysis of 135 intersection targets of Gegen Qinlian Decoction in treating diabetes were constructed by DAVID online database. The results of GO enrichment analysis of intersection targets were shown in Figure 4 . The results were as follows: 793 biological processes (BP) were obtained, mainly related to drug / alcohol response, heterotypic stimulus, positive / negative regulation of cell aging and apoptosis process, and positive regulation of nitric oxide biosynthesis process; 92 cell compositions (CC) were obtained, mainly related to cell membrane, mitochondria, cytoplasm, and plasma membrane functions; 142 molecular functions (MF) were obtained, mainly related to protein binding, enzyme binding, heme binding, oxidoreductase binding, and protein kinase binding. Further KEGG pathway analysis of targets of Gegen Qinlian Decoction in treating diabetes was performed, and 175 significantly related signaling pathways (P<0.05) Figure 5 ) were obtained. These signaling pathways were mainly endocrine resistance pathway, apoptosis signaling pathway, TNF signaling pathway, and non-alcoholic fatty liver pathway. KEGG pathway analysis showed that Gegen Qinlian Decoction mainly exerted anti-diabetic activity through regulating multiple pathways such as apoptosis, immune regulation, and lipid regulation.

[0138] 6. Proposal of GQD candidate component group

[0139] The importance of GQD candidate components was evaluated considering both NTS and content. First, SIMCA 14.0 was used for principal component analysis to obtain the loading matrix of four topological parameters: degree, betweenness centrality, closeness centrality, and neighborhood connectivity. Then, the NTS value of each compound was calculated and sorted, which reflected the topological importance of the compound target in the GQD regulatory network. The calculation of topological parameters and NTS values of each quantitative component was shown in Table 3. Based on the quantitative results of multiple components, components with NTS greater than 0.5 or content greater than 5.0% were selected as candidate component group I, components with NTS greater than 0.25 or content greater than 1.0% were selected as candidate component group II, and components with NTS greater than 0.2 or content greater than 0.3% were selected as candidate component group III. The detailed information of each candidate component group was shown in Tables 4-7.

[0140] Table 3 Calculation of topological parameters and NTS values of each quantitative component

[0141]

[0142]

[0143]

[0144] Table 4 Compound combinations of candidate component group I

[0145]

[0146] Table 5 Compound combinations of candidate component group II

[0147]

[0148] Table 6 Compound combinations of candidate component group III

[0149]

[0150]

[0151] Table 7 Compound combinations of candidate component group IV

[0152]

[0153] 7. Discovery of equivalent component groups of Gegen Qinlian Decoction for improving type 2 diabetes based on zebrafish diabetes model

[0154] The 3% glucose-induced zebrafish diabetes model was established, and Gegen Qinlian Decoction and candidate component groups I-IV were administered. It was found that Gegen Qinlian Decoction and candidate component groups I-IV could significantly reduce the glucose level and lipid accumulation of 3% glucose-induced zebrafish (Figures 2A, 2B). The results showed that Gegen Qinlian Decoction and the four candidate component groups could improve type 2 diabetes. Figure 6

[0155] For bioactivity equivalence evaluation, the bioassay results were converted into efficacy values, and the bioactivity equivalence between the candidate component groups and Gegen Qinlian Decoction was evaluated by calculating the 90% confidence interval (CI). Compared with the original prescription, if the 90% CI of the relative efficacy is within 70-143%, it is considered that the candidate component group is equivalent to Gegen Qinlian Decoction. Table 8 lists the bioactivity equivalence evaluation results of the four candidate component groups. Among them, the 90% CI of the relative efficacy of candidate component groups II, III and IV for zebrafish type 2 diabetes is within 70-143%, which can be considered equivalent to Gegen Qinlian Decoction. After comprehensive consideration, candidate component group IV (a total of 13 components) was finally selected as the equivalent component group of Gegen Qinlian Decoction for subsequent experiments.

[0156] Table 8 Bioactivity equivalence evaluation results of candidate component groups in zebrafish model

[0157]

[0158] 8. Pharmacodynamic evaluation of Gegen Qinlian Decoction and equivalent component groups for improving type 2 diabetes based on zebrafish diabetes model

[0159] ​The efficacy of the drug in a zebrafish model was validated by preparing candidate component group IV for liquid-phase capture and the remaining component groups. In vivo assessment of the bioactivity equivalence of glucose levels in zebrafish showed that both Ge Gen Qin Lian Tang and the equivalent component group (BECC being the prepared liquid-phase capture component group, and MRCs being the control compound component group) significantly reduced glucose levels, while the remaining component groups did not significantly reduce glucose levels. Figure 6 Table 9 shows the bioactivity equivalence evaluation results of candidate component group IV in the zebrafish diabetes model. Oil Red staining results indicate that ( Figure 6 In the zebrafish model group (GQD), compared with the control group, lipid accumulation was significantly increased. After treatment with Ge Gen Qin Lian Tang and the equivalent component group (BECC being the preparative liquid-phase capture component group, and MRCs being the control standard blended component group), the lipid accumulation area decreased to varying degrees, while the lipid accumulation area in the remaining groups showed almost no significant reduction. According to the bioactivity equivalence evaluation analysis, the equivalent component group and GQD within the 90% confidence interval were comparable in reducing glucose levels and improving lipid accumulation in zebrafish.

[0160] Table 9. Bioactivity equivalence evaluation results of candidate component group IV in the zebrafish diabetes model.

[0161]

[0162] 9. Efficacy evaluation of Gegen Qinlian Decoction and its equivalent components in improving type 2 diabetes based on a T2DM mouse model

[0163] A type 2 diabetes mouse model was established by inducing a high-fat diet with streptozotocin (STZ). Compared with the control group, the fasting blood glucose of the model group mice was significantly increased. Then, the mice were administered Ge Gen Qin Lian Tang and its equivalent components by gavage. The efficacy and equivalence of Ge Gen Qin Lian Tang and its equivalent components in treating T2DM were preliminarily evaluated using key indicators such as oral glucose tolerance test (OGTT), insulin tolerance test (ITT), fasting blood glucose (FBG) levels, and lipid accumulation. The evaluation results are shown in Table 10. The results showed that the equivalent components had similar activity to Ge Gen Qin Lian Tang and had no significant effect on the body weight of the mice. Figure 7 (A, B), but it can effectively improve blood glucose levels in T2DM mice ( Figure 7 (C, D) Improve glucose tolerance ( Figure 8 (A, B) and insulin sensitivity ( Figure 8 (C, D) have potential therapeutic activity for type 2 diabetes mellitus. Compared with the control group, the serum insulin level of mice in the model group was significantly increased, while Gegen Qinlian Decoction and its equivalent components could reduce the serum insulin level of mice in the model group to varying degrees. Figure 9The histological observation found that the islet volume of the model mice was significantly reduced, and the boundary was blurred, while the Gegen Qinlian Decoction and the equivalent component group can significantly improve the atrophy of the islet of the diabetic mice (Fig. 2A, B). Figure 9 In addition, the Gegen Qinlian Decoction and the equivalent component group can significantly reduce the organ index of the liver (Fig. 2C). Figure 10 In addition, the Gegen Qinlian Decoction and the equivalent component group can significantly reduce the organ index of the liver (Fig. 2C). Figure 10 The histological observation found that the islet volume of the model mice was significantly reduced, and the boundary was blurred, while the Gegen Qinlian Decoction and the equivalent component group can significantly improve the atrophy of the islet of the diabetic mice (Fig. 2A, B). Figure 10 In addition, the Gegen Qinlian Decoction and the equivalent component group can significantly reduce the organ index of the liver (Fig. 2C). Figure 11 In addition, the Gegen Qinlian Decoction and the equivalent component group can significantly reduce the organ index of the liver (Fig. 2C). Figure 11 In addition, the Gegen Qinlian Decoction and the equivalent component group can significantly reduce the organ index of the liver (Fig. 2C). Figure 11 In addition, the Gegen Qinlian Decoction and the equivalent component group can significantly reduce the organ index of the liver (Fig. 2C).

[0164] Table 10 Bioactivity equivalence evaluation results of the candidate component group in the T2DM mouse model

[0165]

[0166]

[0167] As can be seen from the above, the present application provides an equivalent component group of Gegen Qinlian Decoction for treating type 2 diabetes, which is composed of 13, 19 or 27 chemical components. The zebrafish diabetes model and the type 2 diabetes mouse model induced by high-fat diet combined with streptozotocin prove that the equivalent component group composed of 13, 19 or 27 chemical components has obvious therapeutic effect on type 2 diabetes, and the treatment effect has no significant difference compared with the original prescription of Gegen Qinlian Decoction, which shows excellent equivalence.

[0168] The present application provides certain reference for elucidating the chemical basis of the medicinal effect of Gegen Qinlian Decoction, and promotes the research on the medicinal material basis and quality standard of Gegen Qinlian Decoction, specifically: (1) the combination of the 13, 19 or 27 components can be prepared into a drug composition equivalent to Gegen Qinlian Decoction for treating type 2 diabetes; (2) the combination of the 13, 19 or 27 components can be prepared into a marker composition for controlling the quality of Gegen Qinlian Decoction, so as to realize the stability, repeatability and controllability of the activity of Gegen Qinlian Decoction for improving type 2 diabetes.

[0169] The above examples serve to specifically introduce the essential content of the present application, but those skilled in the art should know that the protection scope of the present application should not be limited to the specific examples.

Claims

1. An equivalent composition representing the therapeutic efficacy of Gegen Qinlian Decoction for type 2 diabetes mellitus, characterized in that, Composed of the following 13 compounds Composition: Puerarin, genistein, 3'-hydroxypuerarin, gentiopicrin, daidzein, daidzeinogen, baicalin, baicalin, baicalin, glycyrrhizic acid, glycyrrhizin chalcone A, berberine hydrochloride and palmatine hydrochloride; and the content ratio of each compound is the same as its content ratio in Ge Gen Qin Lian Tang.

2. An equivalent composition representing the therapeutic efficacy of Ge Gen Qin Lian Tang in treating type 2 diabetes, characterized in that, It is composed of the following 19 compounds: genistein, puerarin, daidzein, baicalin, wogonin, wogonin, baicalin, berberine hydrochloride, stigmosiderin, gentioside, glycyrrhetinic acid, stigmosiderin A, isoglycyrrhizin, glycyrrhizin chalcone A, palmatine hydrochloride, 3'-methoxypuerarin, berberine hydrochloride, mefenoxam, and daidzein; and the content ratio of each compound is the same as its content ratio in Ge Gen Qin Lian Tang.

3. An equivalent composition representing the therapeutic efficacy of Ge Gen Qin Lian Tang in treating type 2 diabetes, characterized in that, It is composed of the following 27 compounds: genistein, puerarin, daidzein, baicalin, wogonin, wogonin, baicalin, berberine hydrochloride, salicumin, gentiopicrin, glycyrrhetinic acid, sennain A, isoglycyrrhizin, glycyrrhizin chalcone A, palmatine hydrochloride, 3'-methoxypuerarin, berberine hydrochloride, mefenoxam, daidzein, glycyrrhizic acid, glycyrrhizin G2, glycyrrhizin, magnoflorine, 3'-hydroxypuerarin, epiberberine hydrochloride, tetrandrine, and puerarin-6"-O-xyloside; and the content ratio of each compound is the same as its content ratio in Ge Gen Qin Lian Tang.

4. The use of the equivalent composition according to claim 1, 2 or 3 in the preparation of an equivalent alternative to Ge Gen Qin Lian Tang for the treatment of type 2 diabetes.

5. The use according to claim 4, characterized in that: The drug uses the equivalent composition as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using a pharmaceutically acceptable carrier or excipient.

6. The use according to claim 5, characterized in that: The carrier or auxiliary material is a solid, liquid or semi-solid.

7. The use according to claim 5, characterized in that: The dosage forms include tablets, capsules, pills, and injections.

8. Use of the equivalent composition of claim 1, 2 or 3 for preparing a composition for controlling the quality of Gegen Qinlian Decoction, the composition containing standards of each compound in the equivalent composition, wherein the quality of Gegen Qinlian Decoction is based on the efficacy of Gegen Qinlian Decoction in treating type 2 diabetes.

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