Chlorogenic acid combined with lactobacillus reuteri in treating metabolic disorder related diseases and pharmaceutical composition

Abstract

The application discloses a use of a phenolic acid compound combined with probiotics in treating metabolic disorder related diseases (including obesity, diabetes, hypercholesterolemia and non-alcoholic fatty liver) and a pharmaceutical composition, and particularly relates to a use of a compound of formula I, chlorogenic acid, combined with lactobacillus reuteri in treating metabolic disorder.The chlorogenic acid combined with lactobacillus reuteri as the pharmaceutical composition can regulate host intestinal homeostasis, improve sugar and lipid metabolism and energy metabolism of the host, and has potential value in preventing and treating metabolic disorder related diseases.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a method for treating metabolic disorder-related diseases using 3-O-caffeoylquinic acid in combination with Lactobacillus reuteri, and its application in drug preparation. Background Technology

[0002] With rapid economic development, metabolic syndrome, including obesity, diabetes, and fatty liver disease, caused by unhealthy lifestyles such as high-fat, high-calorie diets and sedentary lifestyles, has become prevalent, exacerbating the burden on socioeconomic development and becoming one of the most important public health issues of the 21st century. Metabolic syndrome, clinically manifested primarily as disordered glucose and lipid metabolism, is a metabolic syndrome resulting from the complex interaction between environmental and genetic factors. Increasing evidence suggests that metabolic disorders significantly increase the incidence of cardiovascular diseases (such as hypertension, myocardial infarction, and stroke), musculoskeletal diseases (such as arthritis), central nervous system diseases (such as Alzheimer's disease and depression), and certain types of cancer (such as breast cancer, liver cancer, and colon cancer).

[0003] The gut microbiota, colonizing the mammalian gut, is an extremely complex ecosystem. It is estimated that the normal adult gut microbiota contains over 7,000 bacterial strains. The collection of the microbiota's genomes, known as the microbiome, is much larger than the human genome and typically participates in host physiological activities alongside various organs. The abundance and composition of the microbiota largely depend on the host's diet, medication, weight, and metabolic status. With rapid societal development, unhealthy dietary habits disrupt the balance of the gut microbiota, leading to imbalances in the body mass index (BMI). Compared to normal individuals, obese individuals have lower gut microbial genetic diversity, with a reduction in the number of certain Bacteroides relative to Firmicutes. High-fat diets can alter the composition of the small intestinal microbiota, such as leading to the proliferation of Staphylococcus aureus and Enterobacteriaceae. Abnormally expressed microbes can promote the production and secretion of digestive enzymes, accelerating the absorption and transport of fat in the intestine. A 2013 study published in Science found that mice transplanted with fecal microbiota from obese patients experienced increased body weight and fat mass, exhibiting a significant obesity-related metabolic phenotype. This suggests a causal relationship between gut microbiota and host metabolic diseases.

[0004] In the development of metabolic syndrome, dysbiosis disrupts host weight and blood glucose levels, and increases lipid reserves in adipose tissue. Multiple human trials have confirmed the feasibility of using probiotic supplements to reverse metabolic disorders. Lactic acid bacteria have been recognized by the FDA as safe probiotics, with several species participating in metabolic regulation, including *Lactobacillus johnsonii*, *Lactobacillus gasseri*, and *Lactococcus lactis* subsp. *milk fat*. Simultaneously, lactic acid bacteria can respond to exogenous stimuli, regulating the production of downstream microbial metabolites or metabolic enzymes, such as lactic acid, short-chain fatty acids, and bile salt hydrolases. These endogenous substances can target key obesity-related tissues and organs such as the liver, fat, and intestines. Studies have found that intermittent fasting can effectively increase the abundance of endogenous lactic acid bacteria, promote lactic acid and acetic acid secretion, and thus improve metabolic disorders. Therefore, based on the probiotic effects of lactic acid bacteria, the related microbial mechanisms, including proliferative capacity and endogenous metabolic behavior, can serve as a basis for the beneficial effects of microbial-targeted strategies in treating metabolic syndrome.

[0005] Caffeoylquinic acid (CQA) is a class of phenolic acid compounds with ester bonds, formed by the esterification reaction of caffeic acid (CA) and quinic acid. It is widely found in green coffee beans and traditional Chinese medicinal herbs such as Eucommia ulmoides, Lonicera japonica, and honeysuckle. In the 2015 edition of the Chinese Pharmacopoeia, chlorogenic acid is used as a standard for identifying the effective substances in many traditional Chinese medicines. Current research has described the effects of chlorogenic acid on improving glucose and lipid metabolism from aspects such as anti-oxidation, inhibition of liver glycogen synthesis, and delaying fat absorption. Considering that most unabsorbed chlorogenic acid remains in the intestine and interacts with the gut microbiota, it can be inferred that the gut microbiota may be an important mediator for chlorogenic acid compounds, suggesting that chlorogenic acid has potential prebiotic effects. However, there are currently no reports in the literature on the application of chlorogenic acid in treating obesity, hyperglycemia, hyperlipidemia, and fatty liver from the perspective of the interaction between chlorogenic acid and probiotics.

[0006] Chinese patent application CN111514127 A discloses the use of chlorogenic acid in the preparation of drugs for treating lung cancer brain metastases, revealing that chlorogenic acid can improve lung cancer brain metastases and overall condition and symptoms, showing unique advantages in improving quality of life and prolonging survival, and can reduce the side effects of radiotherapy and chemotherapy, demonstrating good application prospects. Chinese patent application CN111346114 A discloses the use of *Lactobacillus reuteri* in the preparation of drugs, foods, food additives, or health products for the treatment or prevention of type 2 diabetes. Chinese patent application CN114344344 A discloses the application of *Lactobacillus reuteri* in alleviating obesity induced by a high-fat diet in mice and a complex containing *Lactobacillus reuteri*, showing that combining *Lactobacillus reuteri* with extracellular polysaccharides has a potent effect. However, currently, no patents disclose combinations of chlorogenic acid and *Lactobacillus reuteri* for enhanced efficacy in treating obesity, diabetes, hypercholesterolemia, and non-alcoholic fatty liver disease. Summary of the Invention

[0007] The purpose of this invention is to provide the application of chlorogenic acid combined with Lactobacillus reuteri in the treatment of metabolic syndromes such as obesity, diabetes, hypercholesterolemia, and non-alcoholic fatty liver disease.

[0008] In a first aspect of the invention, a pharmaceutical composition is provided, characterized in that it comprises: (1) A compound of formula I as the first active ingredient, or a pharmaceutically acceptable salt, solvate, optically pure isomer, stereoisomer or combination thereof; (2) Lactobacillus reuteri as the second active ingredient; and Optional (3) pharmaceutically acceptable carrier.

[0009] In some embodiments, the Lactobacillus reuteri is a wet cell of Lactobacillus reuteri.

[0010] In some embodiments, the *Lactobacillus reuteri* is selected from the group consisting of *Lactobacillus reuteri* (…). Limosilactobacillus reuteri Strains: ATCC 23272, CCUG 33624, CIP 101887, DSM 20016, IFO 15892, JCM 1112, LMG 13557, LMG 9213, NBRC 15892, NRRL B-14171, or combinations thereof.

[0011] In some embodiments, the pharmaceutical composition is used to prevent, treat, or improve metabolic disorder-related diseases.

[0012] In some embodiments, the metabolic disorder-related disease is obesity.

[0013] In some embodiments, the prevention, treatment, or improvement refers to: reducing the fat index, reducing weight gain and / or fat content in obesity induced by a high-fat diet; and / or improving energy metabolism.

[0014] In some embodiments, the treatment of metabolic disorder-related diseases is diabetes.

[0015] In some embodiments, the prevention, treatment, or improvement refers to: improving oral glucose tolerance and / or improving insulin sensitivity.

[0016] In some embodiments, the treatment of metabolic disorder-related diseases is hypercholesterolemia.

[0017] In some embodiments, the prevention, treatment, or improvement refers to reducing the total cholesterol and / or low-density lipoprotein levels in the blood.

[0018] In some embodiments, the metabolic disorder-related disease being treated is non-alcoholic fatty liver disease.

[0019] In some embodiments, the prevention, treatment or improvement refers to: reducing liver cholesterol content, and / or reducing liver triglyceride content, and / or reducing liver lipid accumulation.

[0020] In a second aspect of the invention, a composition is provided, characterized in that the composition comprises: (1) Compound of Formula I as the first active ingredient; (I), and (2) Lactobacillus reuteri as the second active ingredient.

[0021] In a third aspect of the invention, a pharmaceutical combination is provided, characterized in that the pharmaceutical combination comprises: (1) Compound of Formula I as the first therapeutic ingredient; (I); (2) Lactobacillus reuteri as a second therapeutic component; and optional instructions for use, which describe the method of use: administering the first and second therapeutic components simultaneously or sequentially to the patient.

[0022] In another preferred embodiment, the first therapeutic ingredient is administered twice a week at a dose of 20 mg / kg to 150 mg / kg each time; The second therapeutic ingredient is administered twice a week, at a dose of 0.5 × 10⁻⁶ each time. 8 ~2×10 8 CFU was administered.

[0023] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0024] Figure 1 This is a summary of the intestinal flora data analysis results of the model mice and the chlorogenic acid-treated mice in Example 1.

[0025] Figure 2 This is a bar graph showing the in vitro proliferation-promoting effect of chlorogenic acid on the intestinal bacteria Lactobacillus reuteri in Example 1.

[0026] Figure 3 This is a bar chart showing the weight gain, food intake, and fat coefficient of each group of mice in Example 2.

[0027] Figure 4 These are the results of body temperature changes in mice in each group under 4°C cold stimulation in Example 2.

[0028] Figure 5 This is the relative expression result of thermogenic genes in the subcutaneous white fat of mice in Example 2.

[0029] Figure 6 These are the results of the glucose tolerance test and insulin sensitivity test of each group of mice in Example 3.

[0030] Figure 7 These are the results of serum total cholesterol and serum low-density lipoprotein in each group of mice in Example 4.

[0031] Figure 8 These are the results of liver triglycerides, total cholesterol, and Oil Red O staining and quantification of liver sections in each group of mice in Example 5.

[0032] Figure 9 This is the relative expression result of liver lipid synthesis-related genes in each group of mice in Example 5. Detailed Implementation

[0033] The inventors of this application, through extensive and in-depth research, discovered a combination of chlorogenic acid and *Lactobacillus reuteri* fortifying efficacy in treating obesity, hyperglycemia, hypercholesterolemia, and fatty liver. Analysis of the intestinal flora composition of mice treated with chlorogenic acid revealed that chlorogenic acid significantly upregulated the relative abundance of *Lactobacillus reuteri*. In in vitro culture, chlorogenic acid increased the abundance of *Lactobacillus reuteri*. The combined use of chlorogenic acid and *Lactobacillus reuteri* can reduce the dosage and frequency of chlorogenic acid administration while exerting a stronger metabolic-improving effect, primarily manifested in a significant reduction in body weight, blood glucose, serum cholesterol, and low-density lipoprotein in model animals, while also reducing liver triglyceride and total cholesterol levels. Therefore, chlorogenic acid, as a novel prebiotic, synergistically enhances therapeutic efficacy with the probiotic *Lactobacillus reuteri*, making it suitable for the prevention and treatment of metabolic disorders such as obesity, diabetes, hypercholesterolemia, and non-alcoholic fatty liver disease. Based on this, this invention was completed.

[0034] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art.

[0035] As used herein, the terms “chlorogenic acid” or “3-O-caffeoylquinic acid” refer to compounds as shown in formula (I).

[0036] Pharmaceutical Composition The present invention provides a pharmaceutical composition for preparing a medicine for preventing or treating a high-fat diet, comprising chlorogenic acid and Lactobacillus reuteri wet cells as active ingredients.

[0037] There are no particular limitations on the administration of the active ingredient or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0038] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0039] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0040] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0041] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0042] The main advantages of this invention are: This invention provides a safe and effective application of chlorogenic acid combined with Lactobacillus reuteri in alleviating obesity, which can reduce the fat index, reduce cholesterol and low-density lipoprotein content in high-fat diet-induced obese mice, improve liver lipid accumulation, and increase core body temperature.

[0043] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions (such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0045] Example 1: Chlorogenic acid promotes the growth of Lactobacillus reuteri. 1. Experimental Methods 1.1 Gut microbiota sequencing Total genomic DNA was extracted from the cecal contents of mice from different groups using column extraction. The genomic DNA was then analyzed using a 1% agarose gel (voltage, 100 V; time, 20 min) to evaluate DNA integrity, purity, fragment size, and concentration. 16S rRNA sequencing was then performed. a) Amplicon generation Using 10 μL of the DNA template, the target region (V3-V4 region primer 341F-805R, theoretical amplification length 460 bp) was amplified. The PCR reaction system was 30 μL, including 15 μL of high-fidelity DNA polymerase, 0.2 μmol / L forward and reverse primers, and approximately 10 μL of template DNA. The thermal cycling steps included initial denaturation at 95℃ for 3 min, 25 cycles of denaturation at 95℃ for 30 s each, annealing at 55℃ for 30 s, and extension at 72℃ for 30 s, for a total of 25 cycles. Finally, the temperature was lowered to 16℃ for 2 min. The amplified target fragment was then enriched, and specific index sequences were added to different samples to distinguish the data from each sample and create separate folders.

[0046] b) Qualitative and quantitative detection of PCR products Mix the same volume of 1× loading buffer (containing SYB green fluorescent dye) with the PCR product. First, perform electrophoresis on a 2% agarose gel to detect the brightness of bands V3-V4. Then, use a real-time PCR instrument to perform quantitative PCR, detect the effective concentration of the product, and perform accurate quantification.

[0047] c) PCR product mixing and purification procedures The PCR products were mixed at an equal density, and then the mixed PCR products were purified using a gel extraction kit.

[0048] d) Library construction and sequencing Gene libraries were constructed using a library preparation kit, involving fragmentation, end repair, A-tailing, and adapter ligation. Library quality was assessed using a fluorescence analyzer and a microarray analysis system. Finally, the libraries were sequenced on a Novaseq 6000 PE250 platform to obtain the raw sequences.

[0049] e) Data quality control The raw sequencing sequences are filtered to remove sequences containing sequencing adapters, sequences with an N ratio greater than 50%, and sequences with more than 50% of bases having a quality value lower than 19, thus obtaining high-quality sequence samples.

[0050] f) Sequence splicing and OTU alignment The PEAR sequence assembly algorithm was used to merge paired-end sequencing sequences into a single sequence based on the overlap between their beginning and end. Usearch software was used to cluster the tags of all samples, and UCLUST was used to remove chimeras. By default, sequences were clustered into Operational Taxonomic Units (OTUs) with a similarity of 97%. Then, the Closed Reference method of the QIIME platform was used to align the representative sequences of the OTUs with the database to obtain their annotation information.

[0051] g) Construction of the evolutionary tree Based on a reference database, a phylogenetic tree was constructed by aligning the representative sequences of each OTU using PyNAST. Graphlan software was then used to plot relative abundances and perform phylogenetic analysis.

[0052] h) α Diversity Analysis The alpha diversity of the bacterial community, including indices such as Shannon and Simpson, was calculated using QIIME (qiime-1.8.0) and displayed using R language.

[0053] i) Principal component analysis We used the QIIME platform to calculate and visualize the differences between bacterial communities using R language, and evaluated the differences between samples from different species using principal component analysis.

[0054] j) Taxonomic analysis Taxonomic analysis was performed on representative OTU sequences with 97% similarity using the UCLUST classification method (http: / / www.drive5.com / uclust / downloads1_2_22q.html). A redundancy analysis (RDA) model was established based on the relative abundance (log10-transformed) of each OTU, and the community composition of each sample was statistically analyzed at each taxonomic level (kingdom, phylum, class, order, family, genus, species).

[0055] 1.2 Lactobacillus reuteri ( Limosilactobacillus reuteri , L. reuteri Cultivation Prepare MRS medium (1.0 L deionized water, 66.2 g MRS), autoclave at 121℃ for 15 minutes, and use after cooling. Amplify the preserved Lactobacillus reuteri into the medium and incubate under anaerobic conditions of 5% CO2, 85% N2, and 10% H2 until the liquid becomes turbid, indicating successful amplification.

[0056] 1.3 Drug activity screening Add 5 mmol / L chlorogenic acid (CQA) to the culture system to adjust the seed culture concentration to OD. 600 The value was approximately 0.1. The culture system was placed in an anaerobic workstation at 37℃ and cultured. After 24 h, 80 μL of the bacterial solution was taken from each well, and the OD value of the bacterial solution was measured. 600 value.

[0057] 2. Experimental Results 2.1 The regulatory effect of chlorogenic acid on intestinal flora imbalance caused by a high-fat diet.

[0058] α-Diversity Analysis Results: Shannon and Simpson indices were used to assess the α-diversity of the gut microbiota in each group of mice. No significant differences were found between the model control group and the chlorogenic acid-treated group in any of the indices, suggesting that the gut microbiota regulatory behavior of chlorogenic acid does not affect intra-community diversity. Figure 1 As shown.

[0059] Principal component analysis results: Unsupervised dimensionality reduction analysis was performed on the OTU data, and linear transformations were applied to multiple variables to identify key variables, thereby revealing differences in data structure and enabling principal component analysis of the samples. Based on the PCA analysis of the gut microbiota of each group of mice, the results showed that the different group sets were significantly clustered on both sides of the two-dimensional coordinate axis, indicating significant differences in the microbiota composition between groups. Figure 1 As shown.

[0060] Species annotation and analysis results: Species annotation revealed that Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the four main phyla in the gut microbiota of this experiment. The abundance ratio of Firmicutes / Bacteroidetes in the gut microbiota of the chlorogenic acid-treated group was significantly lower than that of the model group. p<0.05), probiotics Lactobacillus reuteri ( Limosilactobacillus reuteri The abundance increased significantly. p<0.05), such as Figure 1 As shown.

[0061] The above experimental results indicate that chlorogenic acid has the effect of regulating intestinal flora, promoting the increase of the abundance of the probiotic Lactobacillus reuteri, thereby treating metabolic disorders.

[0062] 2.2 The in vitro proliferation-promoting effect of chlorogenic acid on Lactobacillus reuteri.

[0063] By administering 5 mmol / L of the compound with L. reuteri Co-incubate and measure the absorbance (OD) of the bacterial culture. 600 It was found that 5 mmol / L CQA could significantly promote L. reuteri The proliferation (P < 0.05), such as Figure 2 As shown.

[0064] The above results confirm in vitro that chlorogenic acid can specifically promote the proliferation of Lactobacillus reuteri.

[0065] Example 2: The therapeutic effect of chlorogenic acid combined with Lactobacillus reuteri on obesity induced by a high-fat diet. 1. Experiment and Methods 1.1 Experimental Materials Chlorogenic acid was purchased from Dalian Meilun Biotechnology Co., Ltd. The type strain Lactobacillus reuteri ( Limosilactobacillus reuteri , DSM 20016) was purchased from Beina Chuanglian Biotechnology Co., Ltd. Other analytical grade reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0066] 1.2 Animal Experiment Specific pathogen-free (SPF) male C57BL / 6 mice aged 6-8 weeks were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (License No. SCXK (Hu) 2018-0003). After one week of adaptive feeding, the mice were given a high-fat diet (HFD, 60% Kcal, purchased from Researchdiet, D12492), and body weight was monitored weekly.

[0067] After 4 weeks of continuous modeling, the mice were reweighed. After excluding unsuccessfully modeled animals, the model animals were randomly divided into groups. The intestinal microorganisms of the mice were cleared by adding a triple antibiotic to their drinking water for 5 days, and drug administration was started after a 3-day recovery period. During the administration period, the model group and the administration groups continued to be fed with high-fat diet. The animals were divided into: model group, chlorogenic acid administration group, Lactobacillus reuteri colonization group, and combined administration group of Lactobacillus reuteri plus chlorogenic acid (all compounds were prepared with sterile normal saline as solvent, pH was adjusted to 5.0 with NaOH, dosage: 50 mg / kg; colonization density of the strain was 10 8 CFU per mouse), and the normal group and the model group were given the same volume of blank solvent. Intragastric administration was performed twice a week for 10 consecutive times.

[0068] 1.3 Detection of Basic Indicators The food intake of mice in each group was recorded daily starting from the beginning of administration, and the body weight of mice in each group was weighed and recorded weekly. After the experiment reached the endpoint, the mice were fasted for 8 hours, and their body weight was measured according to the fasting sequence. After tissue sampling, the fat weight of the mice was weighed.

[0069] 1.4 Energy Metabolism Detection (1) Rectal temperature measurement Rectal temperature of the mice was measured using a small animal temperature maintainer, with the detection position at 4-5 cm from the anal sphincter. In the cold stimulation experiment, the mice were placed in an environment of 4°C, and their rectal temperature was measured at 0, 2, and 4 hours to draw the body temperature change curve of mice under different treatments.

[0070] (2) Adipose RNA extraction and qRT-PCR quantification 50 mg of frozen fat sample was placed in an enzyme-free centrifuge tube, 1 mL of TRIzol and two 3 mm ceramic beads were added, and the sample was homogenized in a homogenizer. First, the sample was centrifuged at 4°C and 12,000 rpm for 5 min to remove excess fat from the supernatant. Then, 200 μL of chloroform was added to the homogenate, mixed thoroughly, and extracted on ice for 5 min. Afterward, it was centrifuged at 4°C and 12,000 rpm for 15 min. The supernatant was collected, and an equal volume of isopropanol was added. The mixture was incubated at -20°C overnight. The precipitated liquid was centrifuged at 4°C and 12,000 rpm for 30 min, the supernatant was discarded, and the precipitate was washed twice with 500 μL of pre-chilled 75% ethanol. After evaporating the residual ethanol, 20 μL of DEPC water was added to the precipitate, and then RNA concentration was detected. RNA was reverse transcribed into cDNA using PrimeScript reverse transcriptase. Real-time PCR was performed using a SYBR Premix Ex Taq system on a CFX384 Touch Real-Time PCR detection system. The results were analyzed using the ΔCt method and normalized to the reference gene.

[0071] The primer sequences are as follows: 2. Experimental Results 2.1 The regulatory effect of chlorogenic acid combined with Lactobacillus reuteri on high-fat diet-induced overweight During a combined colonization process lasting 5 weeks and totaling 10 times, the weight gain in the Lactobacillus reuteri + chlorogenic acid group was effectively controlled. p<0.001, and the weight gain in the chlorogenic acid-only group was also controlled to some extent. (p<0.05), while the weight gain in the Lactobacillus reuteri colonization group alone was not significantly different from that in the model control group. At the endpoint of drug administration, the organ coefficients of subcutaneous white adipose tissue and epididymal white adipose tissue in the combined colonization group were significantly lower than those in the model control group, while neither of the two individual treatment groups showed significant differences from the model control group. Meanwhile, daily food intake data suggested that the effective weight loss of the combined colonization model was not achieved by suppressing food intake, such as... Figure 3 As shown.

[0072] 2.2 The regulatory effect of chlorogenic acid combined with Lactobacillus reuteri on energy metabolism disorders induced by a high-fat diet Mice were subjected to cold stimulation at 4°C. The results showed that the combined colonization group experienced a smaller decrease in body temperature at 4 hours, suggesting that the combined treatment of *Lactobacillus reuteri* and chlorogenic acid primarily maintains body temperature by enhancing adaptive non-shivering thermogenesis. Figure 4 As shown.

[0073] The expression levels of thermogenesis-related genes in mouse adipose tissue were detected. Molecular-level experiments showed that adipose-derived thermogenesis-related genes were significantly upregulated in subcutaneous white adipose tissue after co-colonization, for example... Ucp1 , Pgc1a and Mct4, This indicates that co-colonization can effectively enhance thermogenesis in obese hosts. For example... Figure 5 As shown.

[0074] The above experimental results indicate that chlorogenic acid combined with Lactobacillus reuteri, as a drug combination with enhanced efficacy, can treat obesity induced by a high-fat diet.

[0075] Example 3: The therapeutic effect of chlorogenic acid combined with Lactobacillus reuteri on diabetes induced by a high-fat diet. 1. Experiment and Methods 1.1 Experimental Materials Same as Example 2.

[0076] 1.2 Animal Experiments Same as Example 2.

[0077] 1.3 Detection of glucose metabolism capacity (1) Glucose tolerance test After the dosing cycle, an oral glucose tolerance test (OGTT) was performed. The test was conducted after a 16-hour fast by gavage administration of 2 g / kg glucose. Glucose loading time points were set at 0, 15, 30, 60, 90, and 120 minutes, and blood glucose concentrations in the blood samples were measured using blood glucose test strips.

[0078] (2) Insulin sensitivity test After the dosing cycle, an intraperitoneal insulin tolerance test (IPITT) was performed. The test was conducted after a 4-hour fast, with 0.75 IU / kg of insulin administered intraperitoneally. Insulin loading time points were set at 0, 15, 30, 60, 90, and 120 minutes, and blood glucose concentrations in the blood samples were measured using blood glucose test strips.

[0079] 2. Experimental Results 2.1 The regulatory effect of chlorogenic acid combined with Lactobacillus reuteri on high-fat diet-induced glucose metabolism disorders OGTT and IPITT experiments were performed on mice in different groups. The results suggested that the combined colonization model of Lactobacillus reuteri and chlorogenic acid could effectively improve the glucose metabolism level in obese mice. Figure 6 As shown.

[0080] The above experimental results indicate that chlorogenic acid combined with Lactobacillus reuteri, as a drug combination with enhanced efficacy, can treat diabetes induced by a high-fat diet.

[0081] Example 4: The therapeutic effect of chlorogenic acid combined with Lactobacillus reuteri on hypercholesterolemia induced by a high-fat diet. 1. Experiment and Methods 1.1 Experimental Materials Same as Example 2.

[0082] 1.2 Animal Experiments Same as Example 2.

[0083] 1.3 Biochemical index detection For serum markers, the levels of total cholesterol (T-CHO) and low-density lipoprotein cholesterol (LDL-C) in serum samples were measured using a kit (Nanjing Jiancheng).

[0084] 2. Experimental Results 2.1 The regulatory effect of chlorogenic acid combined with Lactobacillus reuteri on high cholesterol induced by a high-fat diet Serum lipid metabolism indicators (total cholesterol and low-density lipoprotein) were measured in each group of mice. The results showed that the combined colonization model significantly reduced the levels of total cholesterol and low-density lipoprotein in the serum of obese mice. Figure 7 As shown.

[0085] The above experimental results indicate that chlorogenic acid combined with Lactobacillus reuteri, as a drug combination with enhanced efficacy, can treat hypercholesterolemia induced by a high-fat diet.

[0086] Example 5: The therapeutic effect of chlorogenic acid combined with Lactobacillus reuteri on high-fat diet-induced non-alcoholic fatty liver disease 1. Experiment and Methods 1.1 Experimental Materials Same as Example 2.

[0087] 1.2 Animal Experiments Same as Example 2.

[0088] 1.3 Biochemical index detection To determine the lipid content in liver tissue, 20 mg of frozen liver was homogenized in 10% (w / v) 50 mmol / L Tris and 1% Triton X-100 using a sample homogenizer. After centrifugation, triglycerides and total cholesterol in the liver homogenate supernatant were quantified using a kit (Nanjing Jiancheng). The procedure was as follows: 2 μL of liver homogenate sample and corresponding standards were added to each microplate, with distilled water or liver homogenate solvent used as a blank control. Then, 200 μL of working solution was added, mixed, and incubated at 37°C for 10 min. The absorbance of each well was read at 510 nm using a microplate reader.

[0089] 1.4 Liver Histological Analysis OCT cryogenic embedding and frozen sectioning were performed on the large lobe of mouse liver under different treatments. The degree of lipid accumulation in the liver was observed by Oil Red O staining. The sections were scanned at different magnifications using a slide scanner, and Oil Red O staining was quantified using ImageJ.

[0090] 1.5 Liver RNA extraction and qRT-PCR quantification A 20 mg frozen liver sample was placed in an enzyme-free centrifuge tube, and 1 mL of TRIzol and two 3 mm ceramic beads were added. The sample was homogenized in a homogenizer. 200 μL of chloroform was added to the homogenate, and the mixture was thoroughly mixed and extracted on ice for 5 min. Then, it was centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected, and an equal volume of isopropanol was added. The mixture was incubated at -20 °C overnight to precipitate. The precipitate was centrifuged at 12,000 rpm for 30 min at 4 °C, the supernatant was discarded, and the precipitate was washed twice with 500 μL of pre-chilled 75% ethanol. After evaporating the residual ethanol, 20 μL of DEPC water was added to the precipitate, and RNA concentration was then measured. RNA was reverse transcribed into cDNA using PrimeScript reverse transcriptase. Real-time PCR was performed using a SYBR Premix Ex Taq system on a CFX384 TouchReal-Time PCR detection system. The results were analyzed using the ΔCt method and normalized to a reference gene.

[0091] The primer sequences are as follows: 2. Experimental Results 2.1 The regulatory effect of chlorogenic acid combined with Lactobacillus reuteri on high-fat diet-induced hepatic lipid accumulation The liver lipid metabolism parameters (triglycerides and total cholesterol) of mice in each group were detected. The results showed that the combined colonization model could effectively reduce the levels of liver triglycerides and total cholesterol. Meanwhile, Oil Red O staining results showed that lipid droplets in hepatocytes were significantly smaller and the Oil Red O positive area was significantly reduced in the combined colonization model. Figure 8 As shown.

[0092] 2.2 The regulatory effect of chlorogenic acid combined with Lactobacillus reuteri on high-fat diet-induced hepatic lipid synthesis Molecular-level quantitative results showed that the combined colonization model effectively downregulated the expression levels of lipid synthesis genes in the liver of metabolically disordered mice. For example... Figure 9 As shown.

[0093] The above experimental results indicate that chlorogenic acid combined with Lactobacillus reuteri, as a drug combination with enhanced efficacy, can treat non-alcoholic fatty liver disease induced by a high-fat diet.

[0094] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A pharmaceutical composition, characterized in that, include: (1) A compound of formula I as the first active ingredient, or a pharmaceutically acceptable salt thereof, or a combination thereof; （I） (2) Lactobacillus reuteri as the second active ingredient; and Optional (3) pharmaceutically acceptable carrier; The Lactobacillus reuteri strain is DSM 20016.

2. The use of the pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition is used to prepare a medicament for treating or improving metabolic disorder-related diseases, wherein the metabolic disorder-related diseases are selected from the group consisting of: obesity, diabetes, hypercholesterolemia, and non-alcoholic fatty liver disease.

3. The use as described in claim 2, characterized in that, The treatment or improvement mentioned refers to: reducing body fat index, reducing weight gain induced by a high-fat diet, and / or improving energy metabolism; The fat index is selected from the following adipose tissues: subcutaneous white adipose tissue, epididymal white adipose tissue, and brown adipose tissue; The improvement in energy metabolism includes increasing the expression levels of the following thermogenic genes in adipose tissue: Ucp1, Pgc1a, and Mct4.

4. The use as described in claim 2, characterized in that, The treatment or improvement referred to is: improving oral glucose tolerance and / or improving insulin sensitivity.

5. The use as described in claim 2, characterized in that, The metabolic disorder-related disease mentioned is hypercholesterolemia.

6. The use as described in claim 2, characterized in that, The treatment or improvement mentioned refers to: reducing the level of total cholesterol and / or low-density lipoprotein cholesterol in the blood.

7. A composition, characterized in that, The composition comprises: (1) Compound of Formula I as the first active ingredient; (I), and (2) Lactobacillus reuteri as the second active ingredient; The Lactobacillus reuteri strain is DSM 20016.

8. A drug combination, characterized in that, The drug combination includes: (1) Compound of Formula I as the first therapeutic ingredient; （I）； (2) Lactobacillus reuteri as a second therapeutic ingredient; and Optional instruction manual, which describes the method of use: administering the first therapeutic ingredient and the second therapeutic ingredient to the treatment subject simultaneously or sequentially; The Lactobacillus reuteri strain is DSM 20016.

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