Bile acid mixture and application thereof

Abstract

The invention relates to a bile acid mixture in the field of food nutrition supplements and medicines, the bile acid mixture comprises Taurohycholic acid, Taurohycholic acid, Hyocholic acid, Taurohycholic acid, Taurohycholic acid, Taurohycholic acid, Glycholic acid, Chocholic acid, Glycholic acid, 7-Ketoithocholic acid, Glycholic acid and Lithocholic acid, and the bile acid mixture can be used for preventing and treating obesity.

Description

Technical Field

[0001] This invention relates to the fields of food nutritional supplements and pharmaceuticals, and more specifically to a mixture of bile acids and its applications. Background Technology

[0002] Globally, overweight and obesity have become the leading chronic diseases threatening public health, with obesity leading to complications such as type 2 diabetes, non-alcoholic fatty liver disease, and atherosclerosis.

[0003] Currently, the main treatments for obesity include lifestyle interventions, chemotherapy, and surgery. Lifestyle interventions primarily involve dietary control and exercise for weight loss, but they rely heavily on patient adherence, have inconsistent clinical outcomes, and are prone to weight rebound. Chemotherapy mainly uses weight-loss drugs such as orlistat, liraglutide, and smegglutide. While this method can control weight or improve metabolic indicators in the short term, it is generally expensive and accompanied by significant gastrointestinal side effects and rapid weight rebound after discontinuation. Surgical treatments such as gastric bypass and sleeve gastrectomy are only suitable for severely obese patients. These procedures are highly invasive, expensive, and prone to postoperative complications such as malnutrition and anastomotic leakage, thus limiting their applicability. Bile acids, as key signaling molecules in gut microbiota and metabolic regulation, have gradually become a research hotspot in this field. Bile acids are a class of steroidal compounds synthesized by the liver and secreted into the intestine via the bile duct. They not only promote the digestion and absorption of fats and fat-soluble vitamins, but also regulate hepatic glucose and lipid metabolism, intestinal barrier function, and microbial composition by activating signaling pathways such as farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5). However, existing research largely focuses on the physiological functions of single bile acids such as ursodeoxycholic acid (UDCA) and obeticholic acid, or on synthetic ligands targeting their receptors (such as FXR agonists and TGR5 agonists). However, single bile acids or receptor agonists often only activate specific signaling pathways and cannot fully mimic the complex physiological functions of the body's natural bile acid pool. This may lead to limited efficacy or unpredictable side effects, such as obeticholic acid-induced itching and hyperlipidemia, and the potential damage to the intestinal mucosa when high concentrations of lithocholic acid are used.

[0004] Therefore, developing a bile acid mixture with clearly defined components, significant synergistic effects, and high safety has important clinical value and application prospects for solving the treatment challenges of obesity and related metabolic diseases. Summary of the Invention

[0005] The present invention aims to provide a mixture of bile acids that can be used for the prevention and treatment of obesity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a bile acid mixture (GBP), comprising the following raw materials and their weight parts: 30-35 parts Taurohyocholic acid, 18-25 parts Taurochenodeoxycholic acid, 10-15 parts Taurodeoxycholic acid, 3-5 parts Hyocholic acid, 1-2 parts Taurohyodeoxycholic acid, 0.5-1 part Taurocholic acid, 0.3-0.5 parts Glycodeoxycholic acid, 0.1-0.2 parts Cholicacid, 0.01-0.03 parts Glycocholic acid, 0.005-0.015 parts 7-Ketolithocholic acid, 0.005-0.015 parts Glycochenodeoxycholic acid, and 0.005-0.01 parts Lithocholic acid.

[0007] Taurohyocholicacid is taurocholic acid (THCA), taurochenodeoxycholicacid is taurochenodeoxycholic acid (TCDCA), taurodeoxycholicacid is taurodeoxycholic acid (TDCA), hyocholicacid is cholic acid (HCA), taurohyodeoxycholicacid is taurochenodeoxycholic acid (THDCA), taurocholicacid is taurocholic acid (TCA), gycodeoxycholicacid is gynodeoxycholic acid (GDCA), cholic acid (CA), gycocholic acid (GCA), 7-Ketolithocholicacid is 7-ketolithocholicacid (7-KLCA), gycochenodeoxycholicacid is gynochenodeoxycholic acid (GCDCA), and lithocholicacid is lithocholic acid (LCA).

[0008] Studies have found that the bile acid mixture of this application can significantly inhibit body weight gain in a high-fat diet-induced obese mouse model, and the effect is clearly dose-dependent; moreover, the bile acid mixture can significantly reduce the weight of liver tissue and abdominal white adipose tissue (epididymal fat and inguinal fat) in mice, indicating an overall decrease in lipid accumulation. Therefore, this application seeks protection for the use of the bile acid mixture in the preparation of drugs or health foods for the prevention or treatment of obesity.

[0009] Furthermore, the prevention or treatment of obesity involves inhibiting body weight gain and / or reducing lipid accumulation.

[0010] Studies have found that the bile acid mixture of this application can effectively reduce serum TC, TG, and LDL-C levels, while ALT and AST levels decrease simultaneously, indicating that lipid accumulation-related liver injury is effectively alleviated. Therefore, this application also claims protection for the use of the bile acid mixture in the preparation of pharmaceuticals or health foods for the prevention or treatment of lipid accumulation-related liver injury.

[0011] Studies have found that the bile acid mixture of this application can effectively lower fasting blood glucose and improve insulin sensitivity. Therefore, this application also claims protection for the use of the bile acid mixture in the preparation of pharmaceuticals or health foods for lowering fasting blood glucose and / or improving insulin sensitivity.

[0012] Studies have found that the bile acid mixture of this application can effectively improve the intestinal flora structure. Therefore, this application also claims protection for the use of the bile acid mixture in the preparation of drugs or health foods for improving intestinal flora.

[0013] Furthermore, the improvement of gut microbiota refers to a decrease in the F / B ratio and an increase in the abundance of probiotics.

[0014] Studies have found that the bile acid mixture of this application can effectively regulate metabolism. Therefore, this application also claims protection for the use of the bile acid mixture in the preparation of drugs or health foods for the prevention or treatment of obesity-related metabolic disorders.

[0015] Furthermore, the prevention or treatment of obesity-related metabolic abnormalities involves reducing the inhibition of farnesoid X receptors in the intestine by a mixture of bile acids, thereby enhancing their ability to regulate systemic bile acid homeostasis, lipid metabolism, and glucose metabolism.

[0016] Studies have found that the bile acid mixture of this application can effectively inhibit fat synthesis, promote fat breakdown, and suppress chronic inflammation. Therefore, this application also claims protection for the use of the bile acid mixture in the preparation of drugs or health foods for suppressing chronic inflammation.

[0017] Beneficial effects of this invention: 1. Definite Efficacy: GBP demonstrated significant weight loss in a high-fat diet-induced obese mouse model, while simultaneously improving hepatic lipid accumulation and histopathological damage. Specifically, GBP treatment significantly reduced body weight, liver and white adipose tissue weight, and lipid levels in the liver and blood of obese mice, lowered the ALT / AST ratio, reduced fasting blood glucose and insulin levels, and improved the HOMA index. Gut microbiota analysis showed a decreased F / B ratio and an increased abundance of probiotics (such as Akkermansia, Blautia, Bacteroides, and Roseburia). Targeted bile acid metabolomics results indicated that relevant thresholds and fingerprint profiles were met; non-targeted metabolomics data further suggested that lipogenesis was inhibited, lipolysis was accelerated, and chronic inflammation was alleviated.

[0018] 2. Multiple physiological benefits: GBP significantly restores the thermogenesis function of brown adipose tissue while reducing body weight and fat cell volume, achieving multiple benefits including fat reduction, liver protection, inhibition of fat synthesis, promotion of energy expenditure, and anti-inflammation. This mechanism of action provides a new treatment strategy for obese individuals at risk of liver damage.

[0019] 3. Dual-threshold release and efficacy anchoring: This invention is the first to propose using the TβMCA / TDCA ratio as a dual anchor point for quality and efficacy (<0.3 in serum, <0.5 in intestinal contents). Through the mechanism pathway of "intestinal-preferred FXR pathway → relief of intestinal FXR antagonism → bile acid pool remodeling," it provides a causal evidence chain from fingerprint spectrum to threshold to signal axis, breaking through the traditional quality control method that relies solely on the content of a single component. This strategy anchors "quality-efficacy" through functionally verifiable dual thresholds (serum TβMCA / TDCA <0.3, preferably with added intestinal contents <0.5) and directional changes in the bile acid fingerprint spectrum, and proposes characteristic fingerprint spectra (Tα / β-MCA↓; UDCA / THDCA / T-LCA / DCA / TDCA↑; GCA / CA / CDCA / TCA↓), facilitating industrial release and consistency control.

[0020] 4. Mechanism innovation and safety advantages: Compared with single-target FXR agonists / antagonists or bile acid sequestrants, this invention defines functional compositions based on in vivo functional thresholds, focusing on the remodeling of bile acid pool structure and the "gut-first" mechanism that is not 12α-OH uplift / 12α-OH downlift, which not only reduces the risk of systemic side effects, but also improves the reproducibility of efficacy. Attached Figure Description

[0021] Figure 1 The effect of GBP on the body weight of high-fat diet-induced obese mice; where A represents the weekly change in body weight during the experiment, B represents the final body weight of the mice, and C represents the phenotype of mice in different groups; # indicates comparison with group Con.P <0.05; * indicates compared to the Veh group P <0.05.

[0022] Figure 2 The effects of GBP on liver weight, white fat weight, liver index, white fat index, and liver function in mice are shown in Figure 1. A represents the liver and epididymal fat phenotype in mice; B represents liver weight; C represents liver index; D represents epididymal fat weight; E represents epididymal fat index; F represents inguinal fat weight; G represents inguinal fat index; H represents total cholesterol in the liver; and I represents liver triglycerides. eWAT (Epididymal WAT) represents epididymal adipose tissue, and # indicates a comparison with the Con group. P <0.05; * indicates compared to the Veh group P <0.05.

[0023] Figure 3 The effect of GBP on serum biochemical parameters in mice; A represents total cholesterol, B represents triglycerides, C represents low-density lipoprotein cholesterol, D represents alanine aminotransferase (ALT), and E represents aspartate aminotransferase (AST); # indicates comparison with group Con. P <0.05; * indicates compared to the Veh group P <0.05.

[0024] Figure 4 Images of hematoxylin-eosin (HE) staining and Oil Red O staining of various tissues, scale bar = 50 μm; from top to bottom, they are: liver Oil Red O staining, liver HE staining, epididymal fat HE staining, inguinal fat HE staining, and brown fat HE staining; eWAT represents epididymal adipose tissue, iWAT represents inguinal adipose tissue, and BAT represents brown adipose tissue.

[0025] Figure 5 The effect of GBP on blood glucose and insulin levels in mice; A represents fasting blood glucose level, B represents fasting insulin level; # indicates comparison with group Con. P <0.05; * indicates compared to the Veh group P <0.05.

[0026] Figure 6 The effect of GBP on the characteristic genera and diversity of the intestinal flora in mice. (A) Venn plot; (B) PCoA plot; (C) Chao1 index; (D) Shannon index. Con, blank control group; Veh, model control group; GBP, bile acid mixture; n = 8. Note: #P<0.05 compared with blank control group; *P<0.05 compared with model control group.

[0027] Figure 7The effect of GBP on the gut microbiota of mice. (A) Relative abundance of mouse gut microbiota at the phylum level; (B) Ratio of Firmicutes / Bacteroidota in mice; (C) Relative abundance of mouse gut microbiota at the genus level; (D) Heatmap of species abundance of mouse gut microbiota at the genus level. Con, blank control group; Veh, model control group; GBP, bile acid mixture; n = 8. Note: #P<0.05 compared with the blank control group; *P<0.05 compared with the model control group.

[0028] Figure 8 Linear discriminant analysis was performed. (A) Biomarkers of differential gut microbiota between groups (LDAscore>4); (B) Evolutionary clade diagram from inside to outside corresponding to phylum to genus, node size reflects relative abundance, and color marks significantly different microbiota between groups (colorless nodes indicate no significant difference). Con, blank control group; Veh, model control group; GBP, bile acid mixture; n = 8.

[0029] Figure 9 To target bile acid serum metabolomics.

[0030] Figure 10 This is non-targeted serum metabolomics. (A) PCA diagram; (B) differential metabolite enrichment pathway diagram; (C) differential metabolite heatmap. Detailed Implementation

[0031] The following detailed description illustrates the specific implementation method: Example 1 A mixture of bile acids, comprising the raw materials and their contents and sources, is shown in Table 1 below: Table 1. Content and Suppliers of Each Bile Acid in the Bile Acid Mixture (GBP)

[0032] The above 12 bile acids were mixed evenly to obtain a bile acid mixture.

[0033] Effect verification Using the bile acid mixture from Example 1 as the test subject, a high-fat diet-induced obese mouse experiment was conducted to verify the improved effect of the bile acid mixture on lipid reduction and weight loss.

[0034] 1. Experimental Materials and Methods 1.1 Experimental Materials Bile acid mixture; 45% high-fat diet, Nantong Trofi Feed Technology Co., Ltd.; Triglyceride (TG) test kit, Total cholesterol (TC) test kit, Low-density lipoprotein cholesterol (LDL-C) test kit, High-density lipoprotein cholesterol (HDL-C) test kit, Aspartate aminotransferase (AST) test kit, Alanine aminotransferase (ALT) test kit, Glucose (GLU) test kit, Mouse insulin (INS) enzyme-linked immunosorbent assay kit, Nanjing Jiancheng Bioengineering Institute; BCA protein concentration assay kit, Shanghai Beyotime Biotechnology Co., Ltd.; UPLC-Q-Orbitrap / MS240 high-resolution mass spectrometer, Thermo; 1.2 Establishment of high-fat mouse model and experimental design Sixty healthy adult male Kunming mice, SPF grade, weighing 18–22 g, were provided by the Guangdong Provincial Center for Medical Laboratory Animals [License number: SCXK (Guangdong) 2022-0002]. The animals were housed in a barrier environment with constant temperature (21–27 °C), constant humidity (45–55 %), low noise, and a 12-hour light-dark cycle. After 10 days of adaptive feeding, they were randomly divided into 5 groups of 12 mice each (n = 12): (1) Blank control group (Con): Basal diet, intragastrically administered with an equal volume of pure water (10 mL / kg) daily.

[0035] (2) High-fat model group (Veh): High-fat diet (45% fat, 35.6% carbohydrate, 19.4% protein), intragastrically administered with an equal volume of pure water (10 mL / kg) daily.

[0036] (3) Low-dose group of bile acid mixture (GBP-L): High-fat diet + 75 mg / kg·d GBP (intragastrically administered at 10 mL / kg).

[0037] (4) Medium-dose group of bile acid mixture (GBP-M): High-fat diet + 150 mg / kg·d GBP (intragastrically administered at 10 mL / kg).

[0038] (5) High-dose group of bile acid mixture (GBP-H): High-fat diet + 300 mg / kg·d GBP (intragastrically administered at 10 mL / kg).

[0039] The experimental period was 10 weeks in total: The first 4 weeks were for model establishment, and the following 6 weeks were for drug administration intervention. All operations were approved by the Experimental Animal Ethics Committee of Zunyi Medical University (Approval number: ZMU21-2503-377), and the experimental animal ethics norms were strictly observed.

[0040] Mice were weighed and their weight recorded weekly. At the end of week 10, blood was collected by enucleation after mild anesthesia with ether. The blood samples were allowed to stand at room temperature for 2 hours, then centrifuged at 1000 ×g for 15 minutes at 4 °C to separate the serum, which was then frozen at -80 °C. Subsequently, the mice were quickly dissected, and the liver, epididymal fat, and inguinal fat were completely removed. One portion was fixed with 4% paraformaldehyde for histopathological observation; the other portion was flash-frozen in liquid nitrogen and stored at -80 °C for biochemical and lipidomics analysis.

[0041] Liver tissue was added to pre-cooled PBS at a ratio of 1:9 (w / v), homogenized using a low-temperature homogenizer, and centrifuged at 4 ℃ and 10,000 rpm for 10 min. The supernatant was collected for later use. Serum and liver homogenate supernatants were equilibrated at room temperature for 15 min, and then measured according to the kit instructions: serum (fasting blood glucose, fasting insulin, TC, TG, LDL-C, AST, ALT); liver homogenate (TC, TG).

[0042] 1.3 Measurement of body weight and food intake Observe the mice's general condition, including their mental state, diet, and coat. Weigh them weekly and record any changes in body weight. Record the amount of food consumed by the mice (feed amount - uneaten feed) and the average amount of food consumed (total food consumption of 5 mice / 5).

[0043] 1.4 Serum and Liver Biochemical Analysis Thaw mouse serum samples on ice and determine TC, TG, LDL-C, HDL-C, AST, and ALT according to the kit instructions. Homogenize 0.1g of liver tissue in 0.9mL of physiological saline and centrifuge at 2500 × g for 10 minutes at 4°C. Collect the supernatant and determine the protein concentration according to the kit instructions. Then determine TC and TG according to the kit instructions.

[0044] 1.5 Blood glucose and insulin level analysis Fasting blood glucose (GLU) levels in mouse serum were measured. Fasting serum insulin (INS) levels in mice were measured using an enzyme-linked immunosorbent assay (ELISA) according to the kit instructions. Insulin resistance index was calculated as follows: HOMA-IR = GLU × INS / 22.5; insulin sensitivity index: HOMA-IS = 22.5 / (GLU × INS); pancreatic β-cell function was calculated as: HOMA-β = (20 × INS) / (GLU - 3.5).

[0045] Mice were fasted for 12 hours, and blood was collected from their tails using a lancet. Fasting blood glucose levels were rapidly measured using a glucometer. After sacrifice, blood was collected and centrifuged to obtain serum. Fasting serum insulin levels were measured according to the insulin assay kit instructions. The HOMA-IR (Homologous Orthopaedic Molecular Weighted Index) is an indicator used to evaluate individual insulin resistance levels. It is calculated as follows: HOMA-IR = [Fasting blood glucose (mmol / L) × Fasting insulin (mIU / L)] ÷ 22.5 1.6 Histological Analysis Liver lobules and colon were fixed in 4% paraformaldehyde, while epididymal fat, inguinal fat, and brown fat were fixed in fat fixative. After gradient dehydration, the tissues were embedded in paraffin. Hematoxylin and eosin (H&E) staining was performed on the liver tissue, colon, epididymal fat, inguinal fat, and brown fat, and Oil Red O staining was performed on the liver tissue. Histopathological changes were observed under a microscope (Olympus BX50, Tokyo, Japan) and assessed using an acute pathological scoring system. Quantitative analysis was performed using ImageJ software (version 1.8.0, NIH, Bethesda, MD).

[0046] 1.7 Gut Microbiome Analysis Microbial DNA (total mass 1.2–10.0 ng) was extracted from mouse fecal samples using the CZ Soil DNA Extraction Kit (FINDROP, Guangzhou, China). DNA purity and concentration were assessed using a NanoDropOne spectrophotometer (Thermo Fisher Scientific, MA). The V3–V4 region of the 16S rRNA gene was amplified using specific primers (338F: 5′-ACTCCTACGGGAGGCAGCA-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′). PCR product concentration was determined using GeneTools analysis software (version 4.03.05.0, SynGene). PCR products were purified using the EZNA Gel Extraction Kit (Omega, USA). Libraries were prepared according to the protocol of the NEBNext® Ultra™ II DNA Library Preparation Kit for Illumina (New England Biolabs, USA). Sequencing was performed on an Illumina Nova 6000 platform (Guangdong MageGene Biotechnology Co., Ltd., Guangzhou, China). Sequences with ≥97% similarity were clustered into operational taxonomic units (OTUs) using USEARCH (https: / / www.drive5.com / usearch / ).

[0047] 1.8 Serum metabolomics analysis Serum samples were gently thawed on ice to maintain their integrity. For the preparation of each sample, 50 μL of serum was carefully mixed with 150 μL of frozen methanol:acetonitrile solution (2:1, v / v). The mixture was vortexed for 60 s to induce protein precipitation, then centrifuged at 3000 × g and 4°C for 10 min. The supernatant was collected and filtered through a 0.22 μm filter membrane for analysis.

[0048] The UPLC-Q-Orbitrap / MS240 high-resolution mass spectrometer was used. The column was a Thermo Scientific Accucore C18 (150 × 2.1 mm, 2.6 μm). The mobile phase A was 0.1% formic acid aqueous solution and B was acetonitrile solution. The gradient elution program was 0–10 min, 5%–100% B; 10–12 min, 100%–100% B; 12–13 min, 100%–5% B; 13–15 min, 5% B. The column temperature was 30 ℃, the flow rate was 0.3 mL·min⁻¹, and the injection volume was 2 µL.

[0049] A QExactive quadrupole / electrostatic field orbital trap high-resolution mass spectrometer (H-ESI) was used for analysis and detection. Electrospray ionization (ESI) was employed, with positive and negative ion switching scanning modes. The sheath gas flow rate was 30 arb; the auxiliary gas flow rate was 10 arb; the spray voltage was 3.5 kV (+) / 3.0 kV (-); the purge gas flow rate was 0 arb; the capillary temperature was 320 ℃; automatic gain control (AGC) was 200; and the s-lens RF level was 50. Full MS / dd-MS2 scanning mode was used for data acquisition. The primary scan resolution was 120,000 m / z (140–2000 m / z), and the secondary scan resolution was 30,000 m / z (120–1200 m / z). Normalized collision energies were 30%, 50%, and 150%. Dynamic exclusion was used to avoid duplicates, with a repetition count of 5.

[0050] The raw UPLC-Q-Exactive-MS data were preprocessed using ProgenesisQI-2.1 to obtain a multidimensional dataset containing detailed information such as retention time, m / z value, and normalized intensity for each ion peak. Volcano plots were then generated, saved as .csv files, and imported into SIMCA-P13.1 software for multivariate statistical analysis. Potential biomarkers were screened based on projection values ​​(VIP > 1), P < 0.05, and fold change > 2. Differential metabolic pathways were analyzed using Metaboanalyst, and enrichment and topological analyses were performed on these pathways.

[0051] 1.9 Statistical Methods Data analysis and graphing were performed using SPSS 29.0 statistical software. All results are expressed as mean ± standard deviation. One-way ANOVA and Tukey post-hoc tests were used for comparisons between groups. In the significance level labeling, different letters indicate significant differences between different groups (p < 0.05).

[0052] 2. Experimental Results 2.1 Effect of GBP on body weight of C57BL / 6 mice like Figure 1 As shown, the mouse body weight gradually increased with the increase of modeling time. Compared with mice fed a normal diet (Con), mice fed a high-fat diet (HFD) had a significantly increased body weight. P <0.05%. Notably, compared with the HFD-fed control group (Veh), all three GBP-administered groups significantly reduced the body weight of obese mice ( Figure 1 A, B, P <0.05), and shows a certain gradient trend. Figure 1 C indicates that the Veh group mice exhibited a significant obesity phenotype, being significantly more obese than the Con group. After GBP administration, the obesity phenotype in the mice lessened, and they became leaner, especially those treated with GBP at 300 mg / kg. In conclusion, GBP treatment caused a certain degree of change in the body weight of HFD mice.

[0053] 2.2 Effects of GBP on liver weight, white fat weight, liver index, white fat index and liver function in mice like Figure 2 The results of studies A and B showed that the liver weight of mice in the Veh group was significantly higher than that of mice in the Con group. P <0.05%, different doses of GBP significantly reduced liver weight in mice ( P <0.05). Regarding the liver coefficient ( Figure 2 C), the liver coefficient in mice decreased significantly after administration ( P <0.05). Figure 2 Results A, D, and E showed that, compared to the Con group, the epididymal fat weight and epididymal fat index of mice in the Veh group were significantly increased. P <0.05), while compared with the Veh group, the epididymal fat weight and epididymal fat index of GBP 300 mg / kg were significantly reduced ( P <0.05). Figure 2 FG results showed that the groin fat weight and groin fat index of the Veh group mice were significantly higher than those of the Con group (P<0.05), while the groin fat weight of the mice was significantly lower after administration of GBP 150mg / kg and 300mg / kg (P<0.05).

[0054] Hepatic total cholesterol (TC) and triglycerides (TG) are important indicators for assessing lipid metabolism status and liver function, such as... Figure 2 The levels of TC and TG in the liver of mice in the H, I, and Veh groups were significantly higher than those in the Con group. P <0.05), while administration of GBP 150 mg / kg and 300 mg / kg significantly reduced the levels of TC and TG in the liver of mice ( P <0.05). This demonstrates that GBP intervention reduced the weight of various adipose tissues in mice to some extent, thus decreasing the amount of fat in the mice.

[0055] 2.3 Effects of GBP on serum biochemical parameters in mice TC, TG, and LDL-C levels are closely related to lipid metabolism. For example... Figure 3 AC, the results showed that the levels of TC, TG, and LDL-C in the Veh group mice were significantly higher than those in the Con group ( P <0.05), while GBP administration significantly reduced serum TC, TG, and LDL-C levels in mice ( P <0.05). Among them, GBP 300mg / kg was more effective than the other two doses in reducing TC, TG, and LDL-C levels. Regarding liver injury markers, ALT and AST levels were measured in mouse serum, and the results showed ( Figure 3 D, E), long-term HFD feeding caused liver damage in mice, affecting normal liver function, as evidenced by significantly higher ALT and AST levels in the Veh group compared to the Con group ( P <0.05, different doses of GBP significantly reduced ALT and AST levels ( P The value <0.05 indicates that GBP can significantly alleviate liver damage caused by lipid accumulation. This suggests that GBP has a certain ability to regulate blood lipid levels in HFD mice.

[0056] 2.4 Results of hematoxylin-eosin (HE) staining and Oil Red O staining of various tissues like Figure 4 Oil Red O staining of liver sections showed a large number of red lipid droplets in the livers of Veh group mice, indicating that HFD caused a large accumulation of lipids in the livers of mice; after GBP administration, the area stained with Oil Red O was significantly reduced, indicating that GBP can significantly reduce lipid accumulation in the livers of mice.

[0057] HE staining of liver sections showed that in the Con group mice, the liver cells were round, plump, and neatly arranged, with the cell nuclei located in the center of the cell. In the Veh group mice, the liver showed a large number of vacuoles, localized hepatocyte ballooning, disordered cell arrangement, and pyknosis of the cell nuclei, indicating the presence of lipid accumulation in the liver. After administration of GBP 75 mg / kg and 150 mg / kg, a small number of vacuoles were still observed in localized areas of the liver tissue, but no hepatocyte ballooning was observed. After administration of GBP 300 mg / kg, the hepatocytes were neatly arranged, and the vacuoles and ballooning were significantly reduced.

[0058] HE staining of adipose tissue sections showed that the adipocytes in the Con group of epididymal adipose tissue (eWAT) and inguinal subcutaneous adipose tissue (iWAT) were morphologically regular and had uniform matrix septa; the adipocytes in the Veh group were significantly hypertrophic; after GBP administration, the adipocyte volume decreased significantly with changes in the administration concentration; the adipocyte morphology in the GBP 300 mg / kg group was close to normal, the matrix structure was intact, and no obvious pathological changes were observed.

[0059] Brown adipose tissue (BAT) showed that adipocytes in the Con group were small, with abundant small multilocular lipid droplets in the cytoplasm, centrally located nuclei, densely packed mitochondria (showing eosinophilic granular appearance in HE staining), and a rich vascular network, exhibiting typical thermogenic activity. In the Veh group, lipid droplets were abnormally enlarged (whitened), the multilocular structure transformed into a unilocular structure, mitochondrial density decreased, cytoplasmic eosinophilicity weakened, and vascular distribution sparse, suggesting impaired brown adipose tissue function. In the GBP 75 mg / kg group, the proportion of multilocular lipid droplets increased, and mitochondrial granules partially recovered. In the 150 mg / kg group, vascular density increased, and cytoplasmic eosinophilicity enhanced. In the 300 mg / kg group, the morphology and function of brown adipose tissue were close to normal, with small and uniform lipid droplets and clear mitochondrial structure.

[0060] These results indicate that GBP can effectively reduce lipid accumulation in HFD-induced obese mice in a concentration-dependent manner, with GBP 300 mg / kg being superior to the other two doses.

[0061] Effects of 2.5 GBP on blood glucose, insulin levels, and insulin-related indices in mice Existing studies have confirmed that obesity is often accompanied by high blood sugar and decreased insulin sensitivity. Figure 5 The results showed that, compared with the Con group, the fasting blood glucose level of mice in the Veh group was significantly increased (P<0.05); while after GBP intervention, the fasting blood glucose and fasting insulin levels of mice in the high-dose group were significantly lower than those in the Veh group (P<0.05), suggesting that GBP can effectively improve glucose metabolism disorder induced by high-fat diet and enhance insulin sensitivity.

[0062] 2.6 Gut microbiota analysis 2.6.1 Effects of GBP on Gut Microbiota Diversity in Mice like Figure 6 As shown in Figure A, Venn diagram analysis revealed that the number of OUTs specific to the Con, Veh, and GBP groups were 2453, 1740, and 1510, respectively, indicating that GBP intervention can alter the diversity of the gut microbiota. Figure 6 As shown in Figure B, PCoA analysis revealed a significant separation in the microbial community structure among different treatment groups, further confirming the uniqueness of the gut microbiota in each group. The Chao1 index, which estimates the number of out-of-sample (OUT) samples, reflects the richness of community species, while the Shannon index, based on species abundance, reflects both the richness and evenness of community species. Combining these two indices can analyze the α-diversity characteristics of the gut microbiota. Figure 6 As shown in CD, the Chao1 and Shannon indices of the Veh group mice were both lower than those of the Con group (P<0.05); compared with the Veh group, the Chao1 index (P>0.05) and Shannon index of the GBP group mice were lower (P<0.05). These results indicate that GBP effectively improves the richness and diversity of the gut microbiota in obese mice by altering the abundance of gut microbiota.

[0063] 2.6.2 Effects of GBP on the gut microbiota of mice at the phylum and genus levels Regarding the taxonomic composition and relative abundance of the microbial community, we further analyzed the data at the phylum and genus levels. Figure 7 A shows the top 10 bacterial groups by relative abundance at the phylum level, with the GBP group effectively reducing the relative abundance of Firmicutes and increasing the relative abundance of Actinobacteriota (Bacteroidota). Figure 7 B specifically quantified this change. Compared with the Con group, the Firmicutes / Bacteroidota value was significantly increased in the Veh group. After GBP intervention, the Firmicutes / Bacteroidota value was significantly decreased (P<0.05). Firmicutes / Bacteroidota, as an important biological marker of intestinal homeostasis, is a characteristic evaluation parameter of the gut microbiota.31 At the genus level, Figure 7 C shows the top 15 bacterial groups by relative abundance. Compared with the Veh group, the GBP group increased the relative abundance of Akkermansia, Blautia, Bacteroides, and Roseburia, while decreasing the relative abundance of Staphylococcus, Dubosiella, Ileibacterium, and Bifidobacterium. Figure 7The species abundance heatmap of D further validates the changing trends of these fungal genera.

[0064] 2.6.3 Effects of GBP on mouse gut microbiota biomarkers Linear discriminant analysis (LEfSe) was used to determine bacterial taxa that differed in abundance between groups. Figure 8 A shows the biomarkers and effect values ​​(LDAscore>4) at each classification level in different groups, with 13, 8, and 11 biomarkers selected in the Con, Veh, and GBP groups, respectively. Figure 8 B represents the hierarchical distribution of the taxonomic groups, visually demonstrating the distribution of these biomarkers at different taxonomic levels. At the genus level, the biomarkers for the Con group are g_Lachnospiraceae_NK4A136_group (LDAscore = 4.63), g_Alloprevotella (LDAscore = 4.56), g_Ileibacterium (LDAscore = 4.54), and g_Anaerostipes (LDAscore = 4.02); for the Veh group, the biomarkers are g_Dubosiella (LDAscore = 4.90) and g_Bifidobacterium (LDAscore = 4.23); and for the GBP group, the biomarkers are g_Blautia (LDAscore = 4.47) and g_Roseburia (LDAscore = 4.39). Significant differences in gut microbiota structure exist among the different groups, indicating that GBP can improve the gut microbiota balance by specifically regulating the proportion of core microbiota.

[0065] Effects of 2.7GBP on mouse serum metabolomics Bile acid targeted metabolomics data showed that GBP intervention significantly reshaped the serum bile acid composition profile. Specifically, the levels of 10 bile acid metabolites showed a significant reversal: T-β-MCA, glycocholic acid (GCA), cholic acid (CA), chenodeoxycholic acid (CDCA), and taurocholic acid (TCA) levels decreased; while ursodeoxycholic acid (UDCA), taurodeoxycholic acid (THDCA), taurolithocholic acid (T-LCA), deoxycholic acid (DCA), and taurodeoxycholic acid (TDCA) levels increased (see [link to relevant documentation]). Figure 9This change further leads to a decrease in inhibitory bile acids (such as Tα / β-MCA) and an increase in activating bile acids (such as LCA and DCA). In terms of overall composition, the proportion of 12α-OH bile acids (promoting obesity) significantly decreased, while the proportion of non-12α-OH bile acids (anti-obesity) significantly increased. Simultaneously, the serum TβMCA / TDCA ratio decreased to below 0.3, and in intestinal contents <0.5 (Table 2). This change indicates that the antagonistic state of the intestinal farnesoid X receptor (FXR) was unblocked, thereby triggering a "gut-first" regulatory mode. This shift in regulatory mode shifts the focus of bile acid signaling regulation to the intestinal region, enhancing its ability to regulate systemic bile acid homeostasis, lipid metabolism, and glucose metabolism by reducing the inhibitory state of FXR in the intestine. This may be one of the core mechanisms by which GBP improves metabolic disorders.

[0066] Table 2. Serum and intestinal T-β-MCA / TDCA ratios

[0067] Untargeted metabolomics analysis revealed that the serum metabolic profiles of mice in the GBP, Con, and Veh groups were clearly distinguishable using PCA analysis. Figure 10 A) indicates that the serum metabolic profile of the Veh group mice changed significantly compared with the Con group, which also indicates that the experimental model was successfully established. Finally, based on the projection value (VIP>1), P<0.05 and fold change>2, 106 differentially expressed metabolites in the serum were screened.

[0068] Serum differentially expressed metabolites were imported into MetaboAnalyst 6.0 for topological analysis to obtain relevant metabolic pathways. These mainly involved bile acid biosynthesis, α-linolenic acid and linoleic acid metabolism, fatty acid biosynthesis, biotin metabolism, β-oxidation of very long-chain fatty acids, glycerol ester metabolism, phospholipid synthesis, mitochondrial medium-chain saturated fatty acid β-oxidation, mitochondrial long-chain saturated fatty acid β-oxidation, phospholipid biosynthesis, starch and sucrose metabolism, fructose and mannose degradation, amino sugar metabolism, mitochondrial fatty acid elongation, galactose metabolism, porphyrin metabolism, fatty acid metabolism, steroid formation, and steroid biosynthesis—a total of 19 metabolic pathways. Figure 10 B) A total of 31 differential metabolites were involved. Among them, the p values ​​of the three pathways of bile acid biosynthesis, α-linolenic acid and linoleic acid metabolism, and fatty acid biosynthesis were < 0.05, involving a total of 15 serum differential metabolites.

[0069] In terms of lipid metabolism, GBP intervention significantly inhibited hepatic de novo lipid synthesis. Specifically, the expression of key enzymes in the fatty acid biosynthesis pathway was downregulated, and the levels of various free fatty acids, such as palmitic acid, lauric acid, myristic acid, and (R)-3-hydroxyhexadecanoic acid, were significantly reduced. Simultaneously, the glycerol metabolism pathway also showed a weakening trend, indicating that triglyceride synthesis was inhibited. These metabolic changes effectively reduced the production and accumulation of endogenous fat, lowered lipid levels in the circulatory system and liver, thereby contributing to weight loss and improving hepatic lipid metabolism disorders.

[0070] Regarding lipolysis, the GBP-treated group exhibited a significant acceleration in lipolysis. Simultaneously, mitochondrial β-oxidation was comprehensively enhanced, with a significant increase in the oxidation capacity of very long-chain, long-chain, and medium-chain fatty acids. This process was accompanied by a correction in dodecanoic acid and stearic acid levels, indicating improved fatty acid metabolic utilization efficiency. Mechanistically, GBP may promote mitochondrial biogenesis and fatty acid oxidation capacity, thereby accelerating lipolysis and energy consumption.

[0071] In terms of inflammation regulation, GBP intervention significantly improved the metabolic imbalance of alpha-linolenic acid and linoleic acid. Specifically, key polyunsaturated fatty acid metabolites such as linoleic acid, docosapentaenoic acid (22n-6), and tetracosapentaenoic acid (24:5n-3) returned to normal levels. Consequently, the production of pro-inflammatory mediators derived from Omega-6 fatty acids (such as prostaglandin E2 and leukotrienes B4) decreased, while the production of anti-inflammatory mediators derived from Omega-3 fatty acids (such as EPA and 14-hydroxyDHA) significantly increased, thereby effectively alleviating chronic low-grade inflammation.

[0072] In summary, GBP exerts a multi-target mechanism of action, including reconstructing the bile acid spectrum, activating the gut-preferred FXR signaling, inhibiting hepatic lipid synthesis, promoting lipolysis and fatty acid oxidation, and improving polyunsaturated fatty acid metabolism and inflammatory states, thereby comprehensively regulating energy metabolism and inflammatory responses. These findings not only provide a solid scientific basis for the weight-loss effect of GBP but also offer a theoretical foundation for its application in the treatment of metabolic syndrome and related diseases.

[0073] In summary, GBP demonstrated a comprehensive and systematic metabolic improvement effect in a high-fat diet-induced obese mouse model. After 10 weeks of continuous intervention, GBP significantly inhibited body weight gain in a dose-dependent manner, with the overall obesity state closest to normal levels at a dose of 300 mg / kg. Simultaneously, the absolute weight of liver and white adipose tissue, as well as the liver TC and TG content, were significantly reduced. Histological examination confirmed the effective reversal of hepatic lipid vacuolation and ballooning degeneration, the alleviation of white adipocyte hypertrophy, and the restoration of multilocular lipid droplet structure and thermogenic activity in brown adipose tissue. Serum biochemical results showed that TC, TG, and LDL-C levels continued to decrease with increasing dose, and liver injury markers ALT and AST also decreased synchronously. Furthermore, GBP significantly downregulated fasting blood glucose and insulin levels, and significantly improved insulin sensitivity. These results indicate that GBP, through a synergistic "fat reduction-liver protection-sensitivity enhancement" mechanism, provides a safe, effective, and economical potential intervention strategy for the prevention and treatment of high-fat diet-related obesity and its metabolic complications.

[0074] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A mixture of bile acids, characterized in that: The raw materials and their weight proportions are as follows: 30-35 parts Taurohyocholic acid, 18-25 parts Taurochenodeoxycholic acid, 10-15 parts Taurodeoxycholic acid, 3-5 parts Hyocholic acid, 1-2 parts Taurohyodeoxycholic acid, 0.5-1 part Taurocholic acid, 0.3-0.5 parts Glycodeoxycholic acid, 0.1-0.2 parts Cholic acid, 0.01-0.03 parts Glycocholic acid, 0.005-0.015 parts 7-Ketolithocholic acid, 0.005-0.015 parts Glycochenodeoxycholic acid, and 0.005-0.01 parts Lithocholic acid.

2. The use of the bile acid mixture of claim 1 in the preparation of a medicament or health food for the prevention or treatment of obesity.

3. The use of the bile acid mixture according to claim 2 in the preparation of a drug or health food for the prevention or treatment of obesity, characterized in that: The prevention or treatment of obesity refers to inhibiting body weight gain and / or reducing lipid accumulation.

4. The use of the bile acid mixture of claim 1 in the preparation of a medicament or health food for the prevention or treatment of lipid accumulation-related liver injury.

5. The use of the bile acid mixture of claim 1 in the preparation of a medicament or health food for lowering fasting blood glucose and / or improving insulin sensitivity.

6. The use of the bile acid mixture of claim 1 in the preparation of a drug or health food for improving intestinal flora.

7. The use of the bile acid mixture according to claim 6 in the preparation of drugs or health foods for improving intestinal flora, characterized in that: The improvement of gut microbiota refers to a decrease in the F / B ratio and an increase in the abundance of probiotics.

8. The use of the bile acid mixture of claim 1 in the preparation of a drug or health food for the prevention or treatment of obesity-related metabolic disorders.

9. The use of the bile acid mixture according to claim 8 in the preparation of drugs or health foods for the prevention or treatment of obesity-related metabolic disorders, characterized in that: The prevention or treatment of obesity-related metabolic abnormalities involves reducing the inhibition of farnesol X receptors in the intestine by a mixture of bile acids, thereby enhancing their ability to regulate systemic bile acid homeostasis, lipid metabolism, and glucose metabolism.

10. The use of the bile acid mixture of claim 1 in the preparation of a drug or health food for inhibiting chronic inflammation.

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