Pharmaceutical composition for treating type 2 diabetes mellitus and application thereof

Abstract

The invention discloses a pharmaceutical composition for treating type 2 diabetes mellitus and application of the pharmaceutical composition. The pharmaceutical composition for treating type 2 diabetes mellitus is prepared from atractylenolide I, catalpol, berberine, quercetin, magnolol and nobiletin according to the mass ratio of (2.5 to 7.5): (1 to 3): (5 to 20): (5 to 10): (1 to 10): (1 to 40). According to the research on the pathogenesis of type 2 diabetes mellitus and the active ingredients of traditional Chinese medicines, the active ingredients of the traditional Chinese medicines with a targeted therapeutic mechanism are screened by applying a molecular docking theory, and the composition is self-planned to be composed of various active ingredients of the traditional Chinese medicines based on the traditional Chinese medicine state target theory and clinical Pi-dan prescription application experience, and is explored in the research on the pharmacological mechanism of treating T2DM. Researches find that the pharmaceutical composition has a good T2DM treatment effect, and the action mechanism of the pharmaceutical composition is realized by improving bile acid metabolism, intestinal flora homeostasis and glucose homeostasis of liver-intestinal axes of T2DM rats.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and particularly relates to a pharmaceutical composition for treating type 2 diabetes mellitus (T2DM) and its use. Background Art

[0002] Diabetes mellitus (DM) is a metabolic disease characterized by elevated blood glucose levels caused by insulin secretion or action defects due to multiple etiologies such as genetics and environment. With the improvement of living standards and the change of lifestyle, the incidence of diabetes has risen rapidly globally. Among diabetes, T2DM (Type 2 diabetes mellitus, T2DM) accounts for more than 90% of the total number of diabetes patients, with approximately 422 million patients globally. Studies have found that hyperglycemia and insulin resistance are the main characteristics of T2DM, and a chronic inflammatory state exists throughout the progression of T2DM. These factors are all involved in the body's metabolic disorders, have an adverse impact on multiple organs such as the heart, brain, kidneys, liver, large blood vessels, and microvessels, and thus promote the occurrence and development of diseases such as diabetic cardiovascular diseases, diabetic retinopathy, and diabetic nephropathy, and increase the incidence of non-alcoholic fatty liver disease. It greatly affects the quality of life of diabetes patients. Currently, in China, more than 60% of diabetes patients have at least one chronic complication. The annual direct medical costs of diabetes patients with microvascular and macrovascular complications are 4-5 times higher than those of patients without complications. Diabetes has brought a heavy economic burden to society and has now become one of the major public health problems threatening human health.

[0003] With the development and application of sequencing technologies, the research on the microbiome and metabolic health has gradually attracted attention and achieved many beneficial results. The gut microbiota is currently considered a new and complex organ, consisting of 500 - 1000 species and 10^14 bacteria, with a quantity more than 10 times that of human cells. The genome of gut microbes is more than 100 times larger than the human nuclear genome. The gut microbiota occupies a major position in the body and is also very complex, playing a crucial role in maintaining nutrient metabolism in the body, enhancing the body's immune function, and protecting against the invasion of foreign substances, and is considered a key factor in human health. The benefits of the gut microbiota for the body mainly include helping the body digest food and absorb nutrients; protecting the intestinal barrier and resisting the invasion of pathogenic bacteria; improving the development and function of the intestinal immune system; and regulating host metabolism. Research shows that changes in the gut microbiota are closely related to the occurrence and development of metabolic diseases such as T2DM. When the gut microbiota is disordered, intestinal metabolites are abnormal, the intestinal barrier is damaged and its permeability increases, endotoxins and pro-inflammatory cytokines are produced in increased amounts, energy intake increases, insulin resistance can be induced, and further metabolic disorders and chronic inflammatory responses occur in T2DM patients. The metabolites of the gut microbiota can affect intestinal barrier function and glucose and lipid metabolism, playing an important role in the occurrence and development of metabolic diseases such as T2DM.

[0004] Bile acid (BA) can promote the digestion and absorption of lipids and vitamins in the intestine during the enterohepatic circulation, improve the body's immune function, and maintain the health of the body. The regulation of bile acids is a complex process that requires the combined action of the gut microbiota, the intestine, and the liver. BA is synthesized from cholesterol through the classical pathway and the alternative pathway. At least 17 enzymes such as cholesterol 7α-hydroxylase (CYP7A1), sterol 12α-hydroxylase (CYP8B1), and sterol-27-hydroxylase (CYP27A1) play important regulatory roles together in the process of BA synthesis.

[0005] After being synthesized from cholesterol in the liver tissue, BA is secreted into the intestinal lumen. Most of the bile acids are then reabsorbed at the terminal ileum and transported back to the liver for recycling. In hepatocytes, the classical pathway is the main pathway for BA synthesis, and CYP7A1 is the first rate-limiting enzyme in the classical pathway of BA synthesis. Cholesterol is converted into 7α-hydroxysterol under the action of CYP7A1, and then cholic acid is formed through the action of CYP8B1, synthesizing most of the BA in the bile acid pool. On the other hand, in tissues or macrophages, the alternative pathway involves the rate-limiting catalysis of cholesterol by CYP27A1 and oxysterol 7α-hydroxylase (CYP7B1) to form chenodeoxycholic acid, synthesizing a small part of the BA in the bile acid pool. Subsequently, the formed primary bile acids are activated by bile acid coenzyme A synthetase and amino acid N-acetyltransferase and conjugated with taurine or glycine to generate taurocholic acid or glycocholic acid, enhancing the water solubility of BA, reducing the damage to cell membranes, and at the same time reducing the toxicity of BA. However, some of them, after reaching the colon, change their physicochemical properties under the modification of intestinal bacteria. BA affects the composition and function of the intestinal flora. Most importantly, BA, as a key regulator of the metabolic pathway network, plays a key role in liver and intestinal gene expression and is directly involved in complex metabolic networks, triggering the physiological effects of BA by activating specific receptors expressed in different cell types, thereby regulating the metabolism of organisms and triggering cell signaling pathways to regulate glucose, lipid, and energy metabolism. BA is an effective ligand for receptors such as farnesol X receptor (FXR), fibroblast growth factor receptor 4 (FGFR4), and G protein-coupled bile acid receptor 5 (TGR5), and non-genomic effects can be produced through the action of these receptors.

[0006] After BA is synthesized in the liver, taurine or glycine is secreted into bile through the Bile Salt Export Pump (BSEP), etc. BSEP is the main transport pathway for BA. Mutations in BSEP can lead to cholestasis in the liver. In the BA-mediated FXR signaling pathway, BA is the most effective endogenous ligand for the FXR receptor, and the FXR receptor is also an inhibitor of BA synthesis in the liver, playing an important regulatory role in the BA metabolism process of the enterohepatic circulation and effectively controlling the BA content in the liver and intestine. In the liver, after activation, FXR regulates the expression of the nuclear orphan receptor Liver Related Homologue-1 (LRH-1) and hepatocyte nuclear factor 4α (HNF4α) genes by inducing the Small Heterodimer Partner (SHP), inhibits the activities of related factors, and then reduces the expression levels of CYP7A1 and CYP8B1, thereby decreasing bile acid synthesis. At the same time, hepatic FXR inhibits NTCP expression through the SHP-dependent process, reduces the uptake of bile acids from the portal circulation, enhances BSEP expression, and promotes the excretion of bile acids.

[0007] After bile acids enter the intestine, the Apical sodium-dependent bile acid transporter (ASBP) absorbs about 95% of the BAs into intestinal epithelial cells, and under the promotion of bile acid transporters, most of the BAs are actively reabsorbed back into the liver through the organic solute transporter α / β (OSTα / OSTβ) on the basolateral membrane of hepatocytes via the portal vein blood circulation system. Under the action of cholangiocytes and renal tubular cells, only about 5% of the BAs are not reabsorbed and are excreted out of the body with wastes such as feces and urine, minimizing the excretion of BAs. Among them, about 5% of the unreabsorbed BAs are still partly reabsorbed by the intestine under the action of intestinal flora and then enter the liver for effective reuse again. When returning to the liver, the reabsorbed BAs are re-secreted in the next digestive and metabolic reaction through the action of sodium taurocholate cotransporting polypeptide and organic anion transporters, thus completing the enterohepatic circulation of BAs. In the intestine, BAs stimulate the FXR receptor, activate the FXR-FGF15 / 19 signaling pathway, mediate the expression of fibroblast growth factor (FGF15 / 19) in intestinal epithelial cells, and then affect the secretion of the cell surface FGFR4 receptor (Fibroblast Growth Factor Receptors 4, FGFR4), thereby inhibiting the expression of CYP7A1, CYP8B1, etc., and finally maintaining the metabolic homeostasis of BAs in the body.

[0008] Traditional Chinese medicine classifies T2DM as "diabetes". "Suwen Qibing Lun" states: "This is the overflow of the five elements, which is called spleen disease. The five flavors enter the mouth... This is where the fat and delicious food comes from. This person must eat sweet and delicious food frequently and eat a lot of fat. Fat makes people feel hot inside, and sweet makes people feel full inside." It clearly points out that eating irregularly and eating too much fat and sweet food are the source of spleen disease, and diabetes is transformed from spleen disease; "fullness inside heat" is the core pathogenesis of T2DM. In addition, "Lingshu Wubian" mentions: "If all five internal organs are weak, they are prone to disease and diarrhea." It can be seen that congenital deficiency and weak organ function are also important internal factors for the onset of T2DM. The existing Chinese herbal compound prescriptions for the treatment of type 2 diabetes are a combination of multiple Chinese medicines. However, there are many ingredients in a single Chinese medicine, and the ingredients of the Chinese medicine after formulation are complex. The administration method is mostly Chinese medicine decoction. The active ingredients of some drugs are difficult to decoct and some ingredients are non-water-soluble. Therefore, its specific effective mechanism is still unclear and needs further analysis, resulting in the inability to more effectively target the treatment of T2DM. Summary of the invention

[0009] The purpose of the present invention is to provide a pharmaceutical composition for treating type 2 diabetes and its use. The pharmaceutical composition of the active ingredients of traditional Chinese medicine of the present invention includes atractylodes lactone I, catalpol, berberine, quercetin, magnolol, and nobiletin. The pharmacological effect is evaluated by observing the basic indicators such as blood sugar, blood lipids, insulin, and C-peptide of T2DM rats, and the histopathological morphological changes are observed to explore the mechanism of the pharmaceutical composition on the bile acid metabolism of the liver-intestinal bile axis and the intestinal flora of T2DM rats. The study found that the pharmaceutical composition has a good effect on treating T2DM, and its mechanism of action is achieved by improving the bile acid metabolism of the liver-intestinal axis of T2DM rats and the homeostasis of intestinal flora and glucose homeostasis. According to the pathogenesis of type 2 diabetes and the study of active ingredients of traditional Chinese medicine, the present invention uses the molecular docking theory to screen the active ingredients of traditional Chinese medicine with targeted treatment mechanism, and based on the theory of state target of traditional Chinese medicine and the experience of clinical application of spleen-dampening prescription, it is composed of multiple effective active ingredients of traditional Chinese medicine, and explored in the study of the pharmacological mechanism of treating T2DM.

[0010] In a first aspect, the present invention provides a pharmaceutical composition for treating type 2 diabetes, which is composed of atractylodes lactone I, catalpol, berberine, quercetin, magnolol and nobiletin in a mass ratio of (2.5-7.5):(1-3):(5-20):(5-10):(1-10):(1-40).

[0011] As an example, the pharmaceutical composition consists of atractylodes lactone I, catalpol, berberine, quercetin, magnolol and nobiletin in a mass ratio of 7:3:10:8:5:10.

[0012] Atractylodes macrocephala is a plant of the Asteraceae family. Atractylodes macrocephalaKoidz.), and atractylodes lactone I is one of the main active ingredients of lactones isolated from Atractylodes macrocephala. In the present invention, atractylodes lactone I in the pharmaceutical composition is a pure product of atractylodes lactone I.

[0013] Catalpol is an iridoid glucoside compound, which is mainly found in plants such as Rehmannia glutinosa. In the present invention, the catalpol in the pharmaceutical composition is pure catalpol.

[0014] Berberine is a type of isoquinoline alkaloid extracted and separated from plants such as the Chinese herbal medicine Coptis chinensis, and is widely distributed in the plant kingdom. In the present invention, the berberine in the pharmaceutical composition is pure berberine.

[0015] Quercetin is a flavonoid compound widely found in plants, mainly in plants such as oak, oak, eucalyptus, neem, etc. In the present invention, the quercetin in the pharmaceutical composition is pure quercetin.

[0016] Magnolia officinalis, Magnoliaceae Magnoliaceae Magnolia officinalis Magnolia officinalis Rehd.et Wils. or Magnolia officinalis Magnolia officinalis The dried bark, root bark and branch bark of Rehd. ed Wils. cheng, and magnolol are separated from magnolia bark. In the present invention, the magnolol in the pharmaceutical composition is pure magnolol.

[0017] Nobiletin is a citrus peel from the rutaceae family Citrus reticulaia A polymethoxylated flavonoid compound extracted from the peel of Blanco fruit. Nobiletin has a wide range of sources and is mainly found in parts such as orange peel, citrus peel, citrus leaves, stems, etc. In the present invention, the nobiletin in the pharmaceutical composition is pure nobiletin.

[0018] In a second aspect, the present invention provides use of any of the above-mentioned pharmaceutical compositions in the preparation of a product for treating type 2 diabetes.

[0019] In the above application, the treatment of type 2 diabetes includes improving bile acid metabolism in the liver-gut axis, improving intestinal flora homeostasis and / or improving glucose homeostasis.

[0020] In the above application, the treatment of type 2 diabetes is embodied in at least one of the following aspects: 1) reducing fasting blood glucose FBG; 2) reducing two-hour blood glucose 2hPG in the oral glucose tolerance test OGTT; 3) reducing glycosylated serum protein GSP; 4) reducing triglycerides TG and total cholesterol TC; 5) reducing insulin INS; 6) increasing C-peptide CP; 7) improving hepatocyte morphology and colon morphology; 8) improving bile acid metabolism in the liver-gut axis; 9) improving intestinal flora imbalance.

[0021] As an example, the improvement of bile acid metabolism in the liver-intestine axis is specifically manifested as increasing the protein expression of FXR and SHP in the liver, decreasing the protein expression of CYP7A1 and CYP8B1, increasing the expression of ASBT, FXR, FGF15, and FGFR4 in the colon, decreasing the expression of CYP7A1 and CYP8B1, and decreasing the mRNA expression of BSEP, FXR, LRH-1, and HNF4α in the liver.

[0022] As an example, the improvement of intestinal microbiota dysbiosis is specifically manifested as decreasing the abundance of Firmicutes, increasing the level of Bacteroidetes, and decreasing the F / B ratio.

[0023] In a third aspect, the present invention protects the use of the pharmaceutical composition according to any one of the above in the preparation of a product for improving abnormal bile acid metabolism in the liver-intestine axis.

[0024] In the above use, the product is used for treating type 2 diabetes. As an example, the improvement of bile acid metabolism is specifically manifested as increasing the protein expression of FXR and SHP in the liver, decreasing the protein expression of CYP7A1 and CYP8B1, increasing the expression of ASBT, FXR, FGF15, and FGFR4 in the colon, decreasing the expression of CYP7A1 and CYP8B1, and decreasing the mRNA expression of BSEP, FXR, LRH-1, and HNF4α in the liver.

[0025] In a fourth aspect, the present invention protects the use of the pharmaceutical composition according to any one of the above in the preparation of a product for improving intestinal microbiota dysbiosis.

[0026] In the above use, the product is used for treating type 2 diabetes. As an example, the improvement of intestinal microbiota dysbiosis is specifically manifested as decreasing the abundance of Firmicutes, increasing the level of Bacteroidetes, and decreasing the F / B ratio.

[0027] In a fifth aspect, the present invention protects a product, the active ingredient of which comprises the pharmaceutical composition according to any one of the above.

[0028] In the present invention, the product can be a drug or a health product.

[0029] In the present invention, the object of drug treatment is a human or a mammal (such as a rat, etc.). In the present invention, the doses of the pharmaceutical composition administered by gavage to the T2DM rat model are 0.215 g / kg / d and 0.43 g / kg / d. The results show that the effect of 0.43 g / kg / d is better.

[0030] Based on the pathogenesis of type 2 diabetes and the research on active ingredients of traditional Chinese medicine, this invention screened the active ingredients of traditional Chinese medicine targeting the treatment mechanism by using the molecular docking theory. Based on the theory of state-target in traditional Chinese medicine and the application experience of clinical prescriptions for spleen dampness-heat syndrome, a formula composed of various effective active ingredients of traditional Chinese medicine was self-formulated and explored in the research on the pharmacological mechanism of treating T2DM. This invention constructed a T2DM rat model by feeding with high-fat and high-sugar diet combined with intraperitoneal injection of streptozotocin, applied the compatibility combination of monomer components of traditional Chinese medicine, and intervened in the T2DM rat model. The changes in blood glucose, blood lipids, bile acid metabolism of the liver-intestinal axis, and the expression of intestinal flora in T2DM rats were observed, the regulatory effect of the formula of active ingredients of traditional Chinese medicine on bile acid metabolism of the liver-intestinal axis and the homeostasis of intestinal flora was explored, and the mechanism of preventing and treating T2DM by this formula of active ingredients of traditional Chinese medicine based on bile acid metabolism and intestinal flora was discussed.

[0031] The present invention has the following beneficial effects: The present invention uses a traditional Chinese medicine active ingredient formula as a therapeutic prescription, and the formula composition and dosage ratio are innovative. By feeding high-fat and high-sugar diets combined with intraperitoneal injection of streptozotocin to construct a T2DM rat model, the blood glucose changes of the rats are detected, and the serum insulin, C-peptide, blood lipids, etc. of the rats are detected, as well as the HE staining and glycogen staining of the liver and colon. Western blot is used to detect the related pathway proteins such as FXR, SHP, CYP7A1, and CYP8B1 in the liver, and the protein expressions of ASBT, FXR, FGF15, FGFR4, CYP7A1, and CYP8B1 in the colon are detected. RT-PCR is used to detect the expressions of related mRNAs such as BSEP, FXR, LRH-1, and HNF4α in the liver. The feces of various rats are collected for intestinal flora detection to explore the regulatory effects of the traditional Chinese medicine active ingredient formula on bile acid metabolism and intestinal flora homeostasis, and to determine the mechanism of action of this traditional Chinese medicine active ingredient formula in preventing and treating T2DM based on bile acid metabolism and intestinal flora. The research of the present invention shows that the protein expressions of FXR and SHP in the liver of the model group rats decrease, while the protein expressions of CYP7A1 and CYP8B1 increase; the mRNA expressions of BSEP, FXR, LRH-1, and HNF4α in the liver increase; the expressions of ASBT, FXR, FGF15, and FGFR4 in the colon decrease, while the expressions of CYP7A1 and CYP8B1 increase; the abundance of Firmicutes increases, the level of Bacteroidetes decreases, and the F / B ratio increases. It shows that the bile acid metabolism of the model group rats decreases and the intestinal flora is dysregulated. After treatment with the traditional Chinese medicine active ingredient formula, the protein expressions of FXR and SHP in the liver increase, while the protein expressions of CYP7A1 and CYP8B1 decrease; the mRNA expressions of BSEP, FXR, LRH-1, and HNF4α in the liver decrease; the expressions of ASBT, FXR, FGF15, and FGFR4 in the colon increase, while the expressions of CYP7A1 and CYP8B1 decrease; the abundance of Firmicutes decreases, the level of Bacteroidetes increases, and the F / B decreases. It shows that the traditional Chinese medicine active ingredient formula has the effect of improving bile acid metabolism and intestinal flora homeostasis, achieving the effect of treating T2DM. Description of the Drawings

[0032] Figure 1 It is the result diagram of HE staining and PAS staining of the liver of rats in each group in Example 1 of the present invention (×200); the result diagram of HE staining and PAS staining of the colon of rats in each group (×100); Figure 2 It is the result diagram of the protein expressions of FXR, SHP, CYP7A1, and CYP8B1 in the liver of rats in each group in Example 1 of the present invention; Figure 3 It is the result diagram of the protein expressions of ASBT, FXR, FGF15, FGFR4, CYP7A1, and CYP8B1 in the colon of rats in each group in Example 1 of the present invention; Figure 4 shows the detection results of the intestinal flora of rats in Example 1 of the present invention: the relative abundance distribution at each taxonomic level (a - Phylum, b - Class, c - Order, d - Family, e - Genus, f - Species) and the clustering heat map of species abundance at the genus level (g).

[0033] Note: Figure 1 - Each letter in Figure 4 represents the following: A. Normal group; B. Model group; C. Metformin group; D. High - dose group; E. Low - dose group. Detailed implementation manners

[0034] The present invention will be further described in detail below in combination with the specific implementation manners. The provided examples are only for clarifying the present invention and not for limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0035] The methods used in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0036] The sources of the drugs and reagents in the following examples are as follows: Atractylenolide I was purchased from Chengdu Push Biotechnology Co., Ltd., product number: PU0047-0025; Catalpol, Berberine, and Quercetin were purchased from Beijing Solarbio Science & Technology Co., Ltd., product numbers: SC8150, SB8130, and SQ8030; Magnolol and Nobiletin were purchased from Shanghai Yuanye Bio-Technology Co., Ltd., product numbers: S31396 and S31600. Metformin Hydrochloride was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: D9351. The Glycated Serum Protein (GSP) Assay Kit, Total Cholesterol (T-CHO) Test Kit, and Triglyceride (TG) Test Kit were purchased from Nanjing Jiancheng Bioengineering Institute, product numbers: A037-2-1, A111-1-1, and A110-1-1. The Rat Insulin (INS) ELISA Kit and C-P Kit were purchased from Xiamen Lunchangshuo Biotechnology Co., Ltd., product numbers: ED-30973 and ED-30079. The FXR antibody (Lot number: 25055-1-AP), CYP7A1 antibody (Lot number: 18054-1-AP), ASBT antibody (Lot number: 25245-1-AP), FGFR4 antibody (Lot number: 11098-1-AP), and HRP-labeled goat anti-mouse secondary antibody (Lot number: SA00001-1) were all purchased from Wuhan Sanying Biotechnology Co., Ltd.; the β-actin antibody (Lot number: T0022) was purchased from Affinity; the FGF15 antibody (Lot number: sc-398338) was purchased from Santa; the CYP8B1 antibody (Lot number: bs-14165R) was purchased from Bioss; the SHP antibody (Lot number: A16454) was purchased from ABclonal; the HRP-labeled goat anti-rabbit secondary antibody (Lot number: A0208) was purchased from Beyotime Biotechnology Co., Ltd.

[0037] Example 1. Animal Experiment In this example, an animal model of T2DM was constructed to experimentally verify the application of the drug combination of traditional Chinese medicine active ingredient compatibility in the treatment of T2DM, as follows: Since the liver is the most important organ for glucose regulation in the body in T2DM, and the metabolites of gut microbiota can affect intestinal barrier function, glucose and lipid metabolism, and bile acid metabolism, playing an important role in the occurrence and development of metabolic diseases such as T2DM. To further clarify the mechanism of action of the traditional Chinese medicine active ingredient formula, the protein expressions of related pathways of FXR, SHP, CYP7A1, and CYP8B1 in the liver were detected, the protein expressions of ASBT, FXR, FGF15, FGFR4, CYP7A1, and CYP8B1 in the colon were detected, and the expressions of related mRNAs such as BSEP, FXR, LRH-1, and HNF4α in the liver were detected by RT-PCR. Various rat feces were collected for gut microbiota detection. To clarify the multi-target mechanism of action of the traditional Chinese medicine active ingredient formula in preventing and treating T2DM.

[0038] Step 1: Grouping, model establishment, and administration of drugs: Prepare experimental animals. Fifty SPF healthy male SD rats were caged and fed with normal diet for 1 week for adaptation. Randomly select 8 rats as the normal group and continue to feed them with normal diet. Feed the remaining 42 rats with a high-sugar and high-fat diet (containing 20 kcal of protein, 35 kcal of carbohydrates, and 45 kcal of fat) for 4 weeks, and then intraperitoneally inject streptozotocin at a dose of 30 mg / kg. After 72 h, rats with a fasting blood glucose level at the tail tip ≥ 11.1 mmol / L were considered as the successful model. During the feeding process of model establishment, 3 rats in the model group died due to improper gavage, and 7 rats did not form the model. A total of 32 rats formed the model.

[0039] Randomly divide the rats in the successfully established model experimental group into 4 groups: control group (8 rats), model group (8 rats), low-dose group of traditional Chinese medicine active ingredient formula (8 rats), and high-dose group of traditional Chinese medicine active ingredient formula (8 rats), and start the intervention on the second day. The low-dose group of traditional Chinese medicine active ingredient formula and the high-dose group of traditional Chinese medicine active ingredient formula were fed with a high-fat diet and respectively gavaged with the low-dose traditional Chinese medicine active ingredient formula at 0.215 g / kg / day and the high-dose traditional Chinese medicine active ingredient formula at 0.43 g / kg / day. The concentration ratio of low dose to high dose was 1:2. The control group was fed with a high-fat diet and gavaged with metformin solution at a dose of 200 mg / kg / day. The model group was fed with a high-fat diet and gavaged with an equal volume of normal saline. The normal group was fed with normal diet and gavaged with an equal volume of normal saline. The intervention lasted for 8 weeks, and during this period, the hair color, mental state, etc. of the rats were observed.

[0040] The traditional Chinese medicine active ingredient formula consists of atractylenolide I, catalpol, berberine, quercetin, magnolol, and nobiletin with a mass ratio of 7:3:10:8:5:10.

[0041] For the metformin group: Rats in the metformin group were gavaged with metformin at 200 mg / kg / day. For the blank group and the model group: They were respectively gavaged with normal saline at 10 ml / kg / day.

[0042] The drug intervention lasted for 8 weeks in total, and samples were taken after 8 weeks.

[0043] Step 2: Perform detections: 1) Blood glucose detection: Fasting blood glucose was detected at 1, 3, and 5 weeks after the model was established; OGTT was detected at 2, 4, 6, and 8 weeks. Before the detection, the rats were fasted but allowed to drink water for 12 h, the bedding was changed to remove the influence of residual food, and blood was taken from the tail tip vein after disinfection with 75% alcohol, and measured with a Roche blood glucose meter.

[0044] 2) After 8 weeks of administration, 10% chloral hydrate-ethyl carbamate (1:1.5 mL / kg) was injected intraperitoneally for anesthesia, and blood was collected from the abdominal aorta, centrifuged, and serum was collected for testing of relevant indicators. ELISA kits and biochemical kits were used to test efficacy indicators such as INS, CP, and blood lipids.

[0045] 3) The liver and colon fixed with 4% paraformaldehyde were cut into appropriate sizes and placed in an embedding tank with running water overnight. The tissues were dehydrated and embedded in paraffin for 24 hours, cut into 5 μm thick sections, and the pathological morphological changes of the liver and colon were observed using HE and PAS staining.

[0046] 4) WB detection of bile acid metabolism-related indicators in the liver and colon: Total protein in the liver and colon was extracted and the protein concentration was determined. Proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with TBST containing 5% skim milk powder for two hours, and rabbit polyclonal antibodies against β-actin (1:7000), SHP (1:500), FXR (1:3000), CYP7A1 (1:3000), CYP8B1 (1:600), ASBT (1:1000), and FGFR4 (1:400); mouse monoclonal antibody against FGF15 (1:200) were added. The membranes were incubated overnight at 4°C, and the PVDF membranes were fully washed with TBST for 5 times, 5 min / time, and the corresponding HRP-labeled secondary antibodies (1:600) were diluted with TBST. The membranes were incubated on a shaker at room temperature for 2 hours, and the PVDF membranes were fully washed with TBST for 5 times, 5 min / time, and exposed by ECL chemiluminescence. The ImageJ image analysis system analyzed the grayscale values ​​of the bands and calculated the relative expression of the proteins.

[0047] 5) RT-PCR detection of liver Bsep,Fxr,Lrh-1 ,and Hnf4α RNA was extracted by Trizol method according to the mRNA expression of OD260 / OD280 ratio, and the RNA quality was estimated. The ratio between 1.8-2.0 met the experimental requirements. RT reverse transcription was performed into cDNA, and real-time fluorescence quantitative PCR was performed. The instrument's own software was used to analyze the amplification curve and melting curve to obtain the Ct value. Calculate the 2- ΔΔ The Ct value was used to determine the mRNA expression of the target gene relative to β-actin. The primer sequences for real-time fluorescence quantitative PCR are shown in Table 1.

[0048]

[0049] Step 3, comparing the test results; Statistical methods were adopted. Quantitative data were expressed as X±S, and data statistics were performed using SPSS 23.0 software. One-way ANOVA was used for comparison between groups. Bonferroni test was used for post hoc comparison when the variances were homogeneous, and Tamhane T2 test was used when the variances were heterogeneous. Taking P <0.05 as the criterion, a statistically significant difference was considered.

[0050] 1) FBG, 2hPG and body weight at 0 week: Refer to Table 2. Before treatment intervention, compared with the normal group, FBG and 2hPG in the model group, metformin group, high-dose traditional Chinese medicine active ingredient formula group, low-dose traditional Chinese medicine active ingredient formula group were significantly increased, with statistically significant differences (P<0.01); compared with the normal group, body weight in the model group, metformin group, low-dose traditional Chinese medicine active ingredient formula group, high-dose traditional Chinese medicine active ingredient formula group were significantly increased, with statistically significant differences (P<0.01).

[0051]

[0052] Note: Compared with the normal group, △ P <0.01.

[0053] 2) FBG, 2hPG and GSP after 8 weeks of drug administration: After 8 weeks of drug intervention, the results showed that FBG in the metformin group, low- and high-dose traditional Chinese medicine active ingredient formula groups was significantly lower than that in the model group (P<0.01), and the high-dose traditional Chinese medicine active ingredient formula group had the best effect; 2hPG in the metformin group, low- and high-dose traditional Chinese medicine active ingredient formula groups was significantly lower than that in the model group (P<0.01), and the metformin group had the best effect; the GSP results showed that the metformin group, low- and high-dose traditional Chinese medicine active ingredient formula groups were significantly lower than that in the model group (P<0.01), and the high-dose traditional Chinese medicine active ingredient formula group had the best effect; see Table 3 for details:

[0054] Note: Compared with the normal group, △ P<0.05, ○ P<0.01; compared with the model group, ▲ P<0.01.

[0055] 3) TG and TC after 8 weeks of drug administration: After 8 weeks of drug intervention, the TG results showed that the TG levels in the metformin group, the low-dose traditional Chinese medicine active ingredient formula group, and the high-dose traditional Chinese medicine active ingredient formula group were significantly lower than those in the model group after 8 weeks of drug intervention (P < 0.05), and the high-dose traditional Chinese medicine active ingredient formula group had the best effect; the TC results showed that the TC levels in the metformin group, the low-dose traditional Chinese medicine active ingredient formula group, and the high-dose traditional Chinese medicine active ingredient formula group were significantly lower than those in the model group after 8 weeks of drug intervention (P < 0.01), and the metformin group had the best effect. See Table 4 for details:

[0056] Note: Compared with the normal group, △ P < 0.05, ○ P < 0.01; compared with the model group, ● P < 0.05, ▲ P < 0.01.

[0057] 4) INS and C-P after 8 weeks of drug administration; The results of 8 weeks of drug intervention showed that the INS and C-P levels in the metformin group, the low- and high-dose traditional Chinese medicine active ingredient formula groups were significantly lower than those in the model group (P < 0.01), and the metformin group had the best effect. See Table 5 for details:

[0058] Note: Compared with the normal group, ○ P < 0.01; compared with the model group, ▲ P < 0.01.

[0059] 5) Effects on the pathological morphological structures of the liver and colon of rats in each group; See Figure 1 , in the normal group stained with HE, the hepatocytes were arranged radially, the cells were closely arranged, the cell nuclei were round, large and centered, the cytoplasm was rich, and the nuclear membrane was clear; in the model group, there were infiltrations of inflammatory cells in the hepatocytes, the cytoplasm was loose or even vacuolated, and a few hepatocytes showed fatty degeneration; compared with the model group, the hepatocyte morphology in the traditional Chinese medicine active ingredient formula group and the metformin group was significantly improved, the hepatocytes were closely arranged, and occasional fatty degeneration of hepatocytes was seen, the cell nuclei were round, large and centered, and the cytoplasm was rich. In the normal group stained with PAS, the glycogen-positive substances in the liver were purple-red, the cell nuclei were blue, and the glycogen was distributed plumply and evenly; in the model group, the hepatocytes were arranged disorderly, there were fat vacuoles, the glycogen-positive reaction was weak and pink, and the glycogen was distributed thinly and unevenly; compared with the model group, the PAS staining in the high-dose traditional Chinese medicine active ingredient formula group and the metformin group was relatively plump and even, and the glycogen distribution in the low-dose traditional Chinese medicine active ingredient formula group was less.

[0060] In the normal HE staining group, the mucosal structure of the colon was intact, the villi were relatively long, the crypts were arranged neatly, and the cells were arranged closely. In the model group, the mucosal structure of the colon was basically intact, the cell arrangement was relatively loose, the crypts and villi were relatively clear, and there was infiltration of a small amount of inflammatory cells. In the traditional Chinese medicine active ingredient formula group and the metformin group, the mucosal structure of the colon was more complete than that in the model group, the cell arrangement was relatively tight, the intestinal crypts and villi of the rats were relatively clear, the cell nuclei were clearly visible, and the high-dose traditional Chinese medicine active ingredient formula group was basically close to the morphological characteristics of the colon of normal rats.

[0061] In the normal PAS staining group, goblet cells and mucus-like substances in the colon were purple-red, and the distribution of mucopolysaccharides was full and uniform; in the model group, the arrangement of colon cells was disordered, the positive reaction of mucopolysaccharides was weak, and the distribution was thin and uneven; compared with the model group, the PAS staining in the traditional Chinese medicine active ingredient formula group and the metformin group was relatively saturated, and the positive reaction of mucopolysaccharides in the low-dose traditional Chinese medicine active ingredient formula group was weak.

[0062] 6) Expression of proteins related to bile acid metabolism in the liver; see Tables 6, 7 and Figure 2 、 3 , compared with the blank group, the protein expressions of FXR and SHP in the liver of rats in the model group decreased, while the protein expressions of CYP7A1 and CYP8B1 increased; the expressions of ASBT, FXR, FGF15, and FGFR4 in the colon decreased, while the expressions of CYP7A1 and CYP8B1 increased; it indicated that the bile acid metabolism in the liver of rats in the model group decreased. After treatment with the traditional Chinese medicine active ingredient formula, the protein expressions of FXR and SHP in the liver increased, while the protein expressions of CYP7A1 and CYP8B1 decreased. The expressions of ASBT, FXR, FGF15, and FGFR4 in the colon increased, while the expressions of CYP7A1 and CYP8B1 decreased.

[0063]

[0064] Note: Compared with the normal group, △ P < 0.05, ○ P < 0.01; compared with the model group, # P < 0.05, ▲ P < 0.01.

[0065]

[0066] Note: Compared with the normal group, △ P < 0.05, ○ P < 0.01; compared with the model group, # P < 0.05, ▲ P < 0.01.

[0067] 7) mRNA expression of BSEP, FXR, LRH-1 and HNF4α in the liver; see Table 8. Compared with the blank group, the mRNA expression of BSEP, FXR, LRH-1 and HNF4α in the liver of the model group decreased; after administration, compared with the model group, the mRNA expression of BSEP, FXR, LRH-1 and HNF4α in the liver increased ( P <0.01).

[0068]

[0069] Note: Compared with the normal group, △ P < 0.05, ○ P < 0.01; compared with the model group, # P < 0.05, ▲ P < 0.01.

[0070] 8) Changes in the intestinal flora of rats in each group; see Figure 4. Compared with the blank group, the abundance of Firmicutes in the model group increased, the level of Bacteroidetes decreased, and the F / B ratio (the ratio of Firmicutes to Bacteroidetes) increased. This indicates that the intestinal flora of the rats in the model group was dysregulated. After treatment with the active ingredient formula of traditional Chinese medicine, the abundance of Firmicutes decreased, the level of Bacteroidetes increased, and the F / B decreased.

[0071] Experimental conclusion: In the experimental verification of the present invention, a T2DM rat model was constructed by feeding a high-fat and high-sugar diet combined with intraperitoneal injection of streptozotocin. The changes in the body weight and blood glucose of the rats were detected, and the serum insulin, C-peptide, blood lipids, etc. of the rats were detected. HE staining and glycogen staining of the liver and colon were performed. Western blot was used to detect the expression of related pathway proteins such as FXR, SHP, CYP7A1, and CYP8B1 in the liver. The expression of proteins such as ASBT, FXR, FGF15, FGFR4, CYP7A1, and CYP8B1 in the colon was detected. RT-PCR was used to detect the expression of related mRNAs such as BSEP, FXR, LRH-1, and HNF4α in the liver. The feces of various rats were collected for intestinal flora detection to explore the regulatory effects of the formula of traditional Chinese medicine active ingredients on bile acid metabolism and intestinal flora homeostasis. The results obtained were as follows: Compared with the blank group, the blood glucose and blood lipid levels in the model group increased, and the insulin level decreased. In the model group, there was infiltration of inflammatory cells in the hepatocytes, the cytoplasm was loose or even vacuolated, and a small number of hepatocytes showed fatty degeneration. The glycogen positive reaction was weak and pink, and the glycogen was distributed thinly and unevenly. The structure of the colon mucosa in the model group was basically intact, the cells were arranged relatively loosely, the crypts and villi were relatively clear, and there was infiltration of a small amount of inflammatory cells. The mucopolysaccharide positive reaction was weak, and the distribution was thin and uneven. In the model group, the protein expressions of FXR and SHP in the liver decreased, while the protein expressions of CYP7A1 and CYP8B1 increased. The mRNA expressions of BSEP, FXR, LRH-1, and HNF4α in the liver increased. The expressions of ASBT, FXR, FGF15, and FGFR4 in the colon decreased, while the expressions of CYP7A1 and CYP8B1 increased. The abundance of Firmicutes increased, the level of Bacteroidetes decreased, and the F / B ratio increased. It was indicated that the bile acid metabolism in the liver, skeletal muscle autophagy, and intestinal flora dysregulation in the model group of rats decreased. After treatment with the formula of traditional Chinese medicine active ingredients, the blood glucose, blood lipids, insulin levels, and the morphological structures of the liver and skeletal muscle were improved. The protein expressions of FXR and SHP in the liver of each group increased, while the protein expressions of CYP7A1 and CYP8B1 decreased. The mRNA expressions of BSEP, FXR, LRH-1, and HNF4α in the liver decreased. The expressions of ASBT, FXR, FGF15, and FGFR4 in the colon increased, while the expressions of CYP7A1 and CYP8B1 decreased. The abundance of Firmicutes decreased, the level of Bacteroidetes increased, and the F / B decreased.

[0072] Conclusion: It was indicated that the formula of traditional Chinese medicine active ingredients had the effect of lowering blood glucose, and its mechanism was closely related to improving bile acid metabolism in the liver-intestinal axis and intestinal flora homeostasis, and could achieve the effect of treating T2DM.

[0073] In summary, in T2DM rats, blood glucose and blood lipid levels increase, pancreatic islet levels decrease, pathological morphology of the liver and skeletal muscle changes, bile acid metabolism in the liver and colon decreases, and intestinal flora is dysregulated; the active ingredient formula of traditional Chinese medicine has the effect of improving the disorder of glucose and lipid metabolism in T2DM rats, and its mechanism of action is closely related to improving bile acid metabolism and intestinal flora homeostasis, and can achieve the effect of treating T2DM.

[0074] In this invention, a self - formulated Pi Dan Decoction is made by adding Coptis chinensis and Scrophularia ningpoensis to Pingwei Powder. In the formula, Coptis chinensis and Scrophularia ningpoensis are the monarch drugs. Shi Jinmo often used the combination of Scrophularia ningpoensis and Coptis chinensis to treat diabetes, believing that diabetes mostly belongs to yin deficiency and internal heat. Scrophularia ningpoensis nourishes yin and promotes fluid production, while Coptis chinensis clears heat and purges fire. The combination of the two can improve the symptoms of yin deficiency and excessive fire. Pingwei Powder has the effect of drying dampness and strengthening the spleen. The active ingredients of the key drugs are selected, and the active ingredients in traditional Chinese medicine are found. Atractylenolide I, catalpol, berberine, quercetin, magnolol and nobiletin are selected according to molecular docking and literature review, and are combined and formulated to treat type 2 diabetes. Atractylenolide I can improve the intestinal flora composition of NAFLD mice, improve the intestinal mucosal barrier, reduce liver inflammation, and improve glucose uptake in C2C12 skeletal muscle cells through the AMPK and PI3K pathways; catalpol has the effects of reducing blood glucose and improving blood lipids in the treatment of diabetes; berberine can improve insulin resistance, regulate glucose metabolism, regulate blood lipid metabolism, have anti - inflammatory effects, protect pancreatic islet cells and have antioxidant effects; quercetin, magnolol and nobiletin can improve insulin sensitivity, inhibit gluconeogenesis, have antioxidant and anti - inflammatory effects, regulate intestinal flora, inhibit α - glucosidase and other effects. Each active ingredient plays a synergistic role to achieve the effect of reducing blood sugar.

[0075] The above details the present invention. For those skilled in the art, within the scope not departing from the purpose and spirit of the present invention, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. A pharmaceutical composition for treating type 2 diabetes, characterized in that: It is composed of atractylodes lactone I, catalpol, berberine, quercetin, magnolol and nobiletin in a mass ratio of (2.5-7.5):(1-3):(5-20):(5-10):(1-10):(1-40).

2. The Chinese medicine composition according to claim 1, characterized in that: The pharmaceutical composition consists of atractylodes lactone I, catalpol, berberine, quercetin, magnolol and nobiletin in a mass ratio of 7:3:10:8:5:

10.

3. Use of the pharmaceutical composition according to claim 1 or 2 in the preparation of a product for treating type 2 diabetes.

4. The use according to claim 3, characterized in that: The treatment of type 2 diabetes includes improving bile acid metabolism in the liver-gut axis, improving intestinal flora homeostasis and / or improving glucose homeostasis.

5. The use according to claim 3 or 4, characterized in that: The treatment of type 2 diabetes is embodied in at least one of the following aspects: 1) reducing fasting blood glucose FBG; 2) reducing two-hour blood glucose 2hPG in the oral glucose tolerance test OGTT; 3) reducing glycosylated serum protein GSP; 4) reducing triglycerides TG and total cholesterol TC; 5) reducing insulin INS; 6) increasing C-peptide CP; 7) improving hepatocyte morphology and colon morphology; 8) improving bile acid metabolism in the liver-gut axis; 9) improving intestinal flora imbalance.

6. Use of the pharmaceutical composition according to claim 1 or 2 in the preparation of a product for improving abnormal bile acid metabolism in the liver-gut axis.

7. The use according to claim 6, characterized in that: The product is used to treat type 2 diabetes.

8. Use of the pharmaceutical composition according to claim 1 or 2 in the preparation of a product for improving intestinal flora imbalance.

9. The use according to claim 8, characterized in that: The product is used to treat type 2 diabetes.

10. A product, characterized in that The active ingredient comprises the pharmaceutical composition according to claim 1 or 2.

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