Use of a pharmaceutical composition of a phenolic acid compound and probiotics

Abstract

The application discloses a use of a pharmaceutical composition of a phenolic acid compound and probiotics, and particularly relates to a combination of chlorogenic acid and lactobacillus reuteri. The combination of chlorogenic acid and lactobacillus reuteri can improve glycolipid metabolic disorder, improve cognitive dysfunction, emotional disorder and delay aging, and can be used for preparing a medicine for treating metabolic disorder combined with neuropsychiatric diseases.

Description

Technical Field

[0001] This invention belongs to the fields of medicine, health products, and food, specifically involving the use of 3-O-caffeoylquinic acid in combination with Lactobacillus reuteri for the prevention and treatment of metabolic disorders and related cognitive impairment, mood disorders, and aging, as well as its application in the preparation of drugs, health products, and food. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease that seriously affects 6% of people aged 65 and older. AD patients experience memory loss and cognitive impairment, accompanied by neuropsychiatric symptoms such as anxiety and depression that accelerate cognitive decline. Previous studies have confirmed that disorders of glucose and lipid metabolism damage the nervous system, leading to microvascular lesions and neuronal damage in the brain, thereby exacerbating cognitive dysfunction. This is closely related to oxidative stress, inflammation, and mitochondrial dysfunction.

[0003] Mounting evidence suggests that targeting the gut-microbe axis for metabolic regulation plays a crucial role in improving brain function. As the second largest genome in the human body, the gut microbiota's role in metabolic disorders and related cognitive impairments has been increasingly revealed. Xylo-oligosaccharides and *Lactobacillus paracasei* HII01, used alone or in combination, effectively restored cognitive impairment in obesity-related insulin resistance rats, improved hippocampal plasticity and brain mitochondrial function, and reduced microglia activation via the gut-brain axis. *Akkermansia muciniphila* significantly reduced weight gain induced by a high-fat diet, improved insulin resistance, delayed amyloid plaque deposition in the brains of AD model mice, and improved spatial learning and memory impairment. *Lactobacillus paracasei* improved cognitive impairment in high-fat-induced obese mice by affecting insulin signaling and neuroinflammatory pathways. These findings demonstrate the feasibility and effectiveness of targeting the gut-microbe axis to improve brain function. There is a need to develop drugs to treat metabolic disorders combined with neuropsychiatric diseases. Summary of the Invention

[0004] The purpose of this invention is to provide the application of chlorogenic acid combined with Lactobacillus reuteri in the treatment of metabolic disorders and related cognitive impairments, mood disorders, and anti-aging drugs.

[0005] A first aspect of the present invention provides the use of a composition for preparing a medicament for preventing, treating, or improving metabolic disorders complicated with neuropsychiatric diseases or for delaying aging, wherein the composition comprises:

[0006] First active component: a compound of formula I, a pharmaceutically acceptable salt thereof, or a solvation thereof; and

[0007]

[0008] The second active component: Limosilactobacillus reuteri.

[0009] In another preferred embodiment, the Lactobacillus reuteri is a wet cell of Lactobacillus reuteri.

[0010] In another preferred embodiment, the Lactobacillus reuteri strain is selected from the group consisting of: ATCC23272, CCUG 33624, CIP 101887, DSM 20016, IFO 15892, JCM 1112, LMG 13557, LMG 9213, NBRC 15892, NRRL B-14171, or a combination thereof.

[0011] In another preferred embodiment, the metabolic disorder is a glucose metabolism disorder or a lipid metabolism disorder.

[0012] In another preferred embodiment, the metabolic disorder refers to hyperlipoproteinemia, hypertriglyceridemia, hypercholesterolemia, diabetes, fatty liver, obesity, or arteriosclerotic cardiovascular and cerebrovascular diseases.

[0013] In another preferred embodiment, the neuropsychiatric disease is a cognitive impairment or a mood disorder.

[0014] In another preferred embodiment, the mood disorder is anxiety disorder, depression, or bipolar disorder.

[0015] In another preferred embodiment, the composition prevents, treats, or improves metabolic disorders complicated with neuropsychiatric diseases by:

[0016] (1) Slow down weight gain;

[0017] (2) Lower blood sugar levels;

[0018] (3) Regulate lipid metabolism, reduce fat content, lower the fat / lean meat ratio, and lower blood lipid levels;

[0019] (4) Increase the abundance of probiotics and reduce the abundance of pathogenic bacteria;

[0020] (5) Increases cell density in the hippocampal subcortex and increases neuronal survival;

[0021] (6) Reduce the expression of glial fibrillary acidic protein, reduce the proliferation of astrocytes, and reduce damage to the nervous system;

[0022] (7) Increase the exploration of new objects;

[0023] (8) Improve cognitive index;

[0024] (9) Increase the alternation percentage;

[0025] (10) Increase the dwell time of the open arm.

[0026] According to the present invention, chlorogenic acid combined with Lactobacillus reuteri as a pharmaceutical composition can improve cognitive dysfunction, mood disorders and delay aging by regulating host metabolic homeostasis, and has potential value in the prevention and treatment of metabolic disorders combined with neuropsychiatric diseases (including glucose and lipid metabolism disorders and related cognitive dysfunction, mood disorders and aging).

[0027] In another preferred embodiment, the treatment for preventing, treating, or improving metabolic disorders combined with neuropsychiatric diseases is cognitive impairment.

[0028] In another preferred embodiment, the prevention, treatment, or improvement refers to: improving oral glucose tolerance and / or reducing blood glucose and lipid levels, thereby improving glucose and lipid metabolism disorders; improving cognitive index and spontaneous alternation rate, thereby delaying the onset of cognitive impairment.

[0029] In another preferred embodiment, the metabolic disorder combined with neuropsychiatric illness is an emotional disorder such as anxiety or depression.

[0030] In another preferred embodiment, the prevention, treatment, or improvement refers to: increasing the dwell time of the open arm and improving mood disorders such as anxiety and depression.

[0031] In another preferred embodiment, the prevention, treatment or improvement refers to: prolonging the body's lifespan and anti-aging.

[0032] In another preferred embodiment, the drug is administered orally, by injection, inhalation, or via a cavity.

[0033] In another preferred embodiment, the dosage form of the drug is selected from: injections, capsules, tablets, granules, sprays, gels, sustained-release preparations, oral liquids, pellets, and nano-preparations.

[0034] In another preferred embodiment, the medicament further includes pharmaceutically acceptable excipients selected from: solvents, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavorings, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, antioxidants, preservatives, carbohydrates, fats, vitamins, amino acids, trace elements, or proteins, or one or more of these.

[0035] In another preferred embodiment, the first active component and the second active component are administered to the treatment subject simultaneously or sequentially.

[0036] In another preferred embodiment, the first active component is administered twice a week at a dose of 20 mg / kg to 150 mg / kg each time;

[0037] The second active ingredient is administered twice a week at a dose of 0.5 × 10⁻⁶ each time. 8 ~2×10 9 CFU was administered.

[0038] In another preferred embodiment, the first active component is administered twice a week at a dose of 25 mg / kg to 75 mg / kg each time;

[0039] The second active ingredient is administered twice a week at a dose of 0.5 × 10⁻⁶ each time. 8 ~1.5×10 8 CFU was administered.

[0040] In another preferred embodiment, the first active component is administered twice a week at a dose of 25 mg / kg to 75 mg / kg each time.

[0041] In another preferred embodiment, the second active ingredient is administered twice a week at a dose of 0.5 × 10⁻⁶ each time. 8 ~1.5×10 8 CFU was administered.

[0042] In another preferred embodiment, the first active component is administered twice a week at a dose of 30 mg / kg to 60 mg / kg each time.

[0043] In another preferred embodiment, the second active ingredient is administered twice a week at a dose of 0.6 × 10⁻⁶ each time. 8 ~1.2×10 8 CFU was administered.

[0044] In another preferred embodiment, the first active component is administered at a dose of 50 mg / kg twice a week.

[0045] The second active ingredient is administered twice a week at a dose of 1×10. 8 CFU was administered.

[0046] This invention provides a method for preventing and treating metabolic disorders combined with neuropsychiatric diseases, by administering a first active component and a second active component to the recipient.

[0047] This invention provides a method for delaying aging by administering a first active component and a second active component to a desired subject. In another preferred embodiment, the desired subject is *C. elegans*.

[0048] The present invention also provides a composition for the prevention and treatment of metabolic disorders combined with neuropsychiatric diseases, comprising the above-mentioned first active component and second active component.

[0049] The present invention also provides an anti-aging composition comprising a first active component and a second active component.

[0050] The combined use of chlorogenic acid and *Lactobacillus reuteri* in this invention demonstrated beneficial regulatory effects on metabolic indicators such as body weight, body composition, blood glucose, and blood lipids in a high-fat diet-induced 5×FAD mouse model. This combined administration enhanced the mice's exploratory drive, improved their environmental awareness and spatial memory, and alleviated cognitive impairment; it also effectively improved mood disorders such as anxiety and depression. Furthermore, this drug combination increased the survival rate of *C. elegans*, suggesting an anti-aging effect.

[0051] 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

[0052] Figure 1 This is a bar chart showing the weight gain and food intake of mice in each group in Example 1.

[0053] Figure 2 These are the results of fasting blood glucose, random blood glucose, and glucose tolerance tests in each group of mice in Example 1.

[0054] Figure 3 These are the results of body composition analysis, serum triglycerides, and cholesterol in each group of mice in Example 1.

[0055] Figure 4 This is a summary of the data analysis results of the intestinal flora of mice in each group in Example 1.

[0056] Figure 5 These are the trajectory diagrams of mice in each group during the new object recognition experiment in Example 2.

[0057] Figure 6 This is the statistical result of the cognitive index of the new object recognition experiment in Example 2.

[0058] Figure 7 This is the result of the Y-maze experiment in Example 2.

[0059] Figure 8 This is the histological evaluation result from Example 2.

[0060] Figure 9 This is the result of the elevated cross maze experiment in Example 3.

[0061] Figure 10These are the growth curves of nematodes under different treatments in Example 4.

[0062] In the figures, ns: no significant difference, *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0063] The inventors of this application, through extensive and in-depth research, discovered a pharmaceutical composition with preventive and therapeutic effects on metabolic disorders combined with neuropsychiatric diseases (including glucose and lipid metabolism disorders and related cognitive impairment, mood disorders, and aging). The combined use of chlorogenic acid and *Lactobacillus reuteri* effectively improves metabolic disorders and related cognitive impairment, mood disorders, and delays aging, primarily manifested in slowed weight gain, reduced fat content, fat / lean ratio, and blood glucose and lipid levels; significantly increased cognitive index and spontaneous alternation rate in mice; significantly increased time spent in the open arm of the elevated cruciate maze; and prolonged survival time in nematodes. Therefore, chlorogenic acid, as a novel prebiotic, can synergistically enhance therapeutic efficacy with the probiotic *Lactobacillus reuteri*, and can be used to prevent and treat metabolic disorders and related cognitive impairment, mood disorders, and delay aging. Based on this, this invention was completed.

[0064] the term

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art.

[0066] As used herein, the terms “chlorogenic acid” or “3-O-caffeoylquinic acid” refer to compounds represented by formula (I).

[0067]

[0068] Pharmaceutical Composition

[0069] This invention provides a pharmaceutical composition for the prevention or treatment of metabolic disorders complicated with neuropsychiatric diseases (including disorders of glucose and lipid metabolism and related cognitive impairment, mood disorders, and aging), comprising chlorogenic acid and Lactobacillus reuteri, particularly wet cells of Lactobacillus reuteri.

[0070] There are no particular limitations on the administration of the active ingredients or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0071] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] Main advantages of the invention

[0076] This invention provides a safe and effective application of chlorogenic acid combined with Lactobacillus reuteri in alleviating metabolic disorders and related cognitive impairment, mood disorders, and delaying aging. This drug combination can improve glucose and lipid metabolism disorders induced by a high-fat diet in mice; increase cognitive index and alternation percentage, improve cognitive impairment; increase open arm dwell time, improve mood disorders; and improve the survival rate of nematodes, thus having an anti-aging effect.

[0077] 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.

[0078] 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.

[0079] Example 1

[0080] Chlorogenic acid combined with Lactobacillus reuteri improves metabolic disorders and gut microbiota dysbiosis in obese AD mice.

[0081] 1. Experiment and Methods

[0082] 1.1 Experimental Materials

[0083] Chlorogenic acid was purchased from Dalian Meilun Biotechnology Co., Ltd., and the type strain *Limosilactobacillus reuteri* (DSM 20016) was purchased from Beina Chuanglian Biotechnology Co., Ltd. Other analytical grade reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0084] 1.2 Animal Experiments

[0085] 5×FAD mice, 23 weeks old, were used for breeding. All mice were housed, modeled, administered drugs, and observed in the barrier animal facility of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, at a temperature of 22±2℃ and a relative humidity of 60±10%. All animal studies were approved by the Experimental Ethics Committee of the Shanghai Institute of Materia Medica (IACUC No.: 2023-XC-102).

[0086] After 8 weeks of continuous high-fat diet modeling, the patients were re-randomized into the model group and the treatment group.

[0087] Specifically, the animals were grouped (7 mice per group) as follows:

[0088] Normal diet control group (given a normal diet),

[0089] High-fat diet model group (fed a high-fat diet),

[0090] High-fat diet chlorogenic acid + Lactobacillus reuteri colonization group (under high-fat diet model, 50 mg / kg chlorogenic acid, 1×10 8 CFU (Chemical Fusarium oxysporum) and chlorogenic acid were prepared using sterile physiological saline as a solvent, and the pH was adjusted to 5.0 with NaOH.

[0091] Both the control and model groups were given the same volume of sterile saline. Mice were administered the drug-eluting dose twice a week via gavage, with a gavage volume of 5 mL / kg, for 8 consecutive weeks.

[0092] 1.3 Basic Indicator Testing

[0093] Food intake was recorded for each group of mice for one week starting from the start of drug administration, and body weight was measured and recorded weekly. At the end of the experiment, body composition (body fat and lean meat content) of the mice was determined using a body composition analyzer.

[0094] 1.4 Detection of glucose and lipid metabolism indicators

[0095] (1) Random blood glucose and fasting blood glucose

[0096] Random blood glucose levels were measured in mice after the drug administration period ended; fasting blood glucose levels were measured in mice after they had been starved for 8 hours.

[0097] (2) Glucose tolerance test

[0098] 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 concentration in the blood samples was measured using blood glucose test strips.

[0099] (3) Biochemical index detection

[0100] For blood lipid indicators, the levels of triglycerides (TG) and total cholesterol (T-CHO) in serum samples were measured using a kit (Nanjing Jiancheng).

[0101] 1.5 Gut microbiota sequencing

[0102] a) Extraction and PCR amplification of genomic DNA

[0103] Genomic DNA was extracted from the samples using the CTAB method. The purity and concentration of the DNA were then determined by agarose gel electrophoresis. An appropriate amount of sample DNA was transferred to a centrifuge tube and diluted with sterile water to 1 ng / μL. Using the diluted genomic DNA as a template, specific primers with barcodes (from New England Biolabs) were selected based on the chosen sequencing region. High-Fidelity PCR Master Mix with GC Buffer and high-efficiency, high-fidelity enzymes are used for PCR to ensure amplification efficiency and accuracy.

[0104] b) Mixing and purification of PCR products

[0105] PCR products were detected by electrophoresis using a 2% agarose gel. Qualified PCR products were purified by magnetic beads, quantified using enzyme-linked immunosorbent assay (ELISA), and mixed in equal volumes according to the PCR product concentration. After thorough mixing, the PCR products were detected by electrophoresis using a 2% agarose gel. The mixed PCR products were then purified using a gel extraction kit.

[0106] c) Library construction and sequencing

[0107] use The DNA PCR-Free Sample Preparation Kit was used to construct the library. The constructed library was then quantified using Qubit and Q-PCR. Once the library was deemed acceptable, it was processed using a NovaSeq6000.

[0108] d) OTU clustering and species annotation

[0109] The Uparse algorithm (Uparse v7.0.1001, http: / / www.drive5.com / uparse / ) was used to cluster all effective tags of all samples. By default, sequences were clustered into OTUs (Operational Taxonomic Units) with a 97% identity. Representative sequences of OTUs were selected based on the algorithm's principles, choosing the most frequent sequence among the OTUs. Species annotation was performed on the OTU sequences using the Mothur method and the SSUrRNA database of SILVA138.1 (http: / / www.arb-silva.de / ) (with a threshold of 0.8-1).

[0110] e) α Diversity Analysis

[0111] 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.

[0112] f) Principal coordinate analysis

[0113] The differences between bacterial communities were calculated using the QIIME platform and visualized using R language. Principal coordinate analysis was performed based on weighted unifrac distance and unweighted unifrac distance.

[0114] g) Taxonomic analysis

[0115] 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).

[0116] 2. Experimental Results

[0117] 2.1 Regulatory effect of chlorogenic acid combined with Lactobacillus reuteri on metabolic disorders in obese AD mice

[0118] like Figure 1 As shown, chlorogenic acid and Lactobacillus reuteri colonization effectively slowed weight gain in mice on a high-fat diet without affecting food intake.

[0119] like Figure 2 As shown, the drug combination significantly reduced blood glucose levels, and the OGTT test results suggest that the combined colonization of chlorogenic acid and Lactobacillus reuteri can effectively improve glucose metabolism in obese mice.

[0120] like Figure 3 As shown, this drug combination can regulate lipid metabolism, reduce fat content, lower the fat / lean meat ratio, and reduce blood lipid levels. The results suggest that the combined colonization of chlorogenic acid and Lactobacillus reuteri can effectively improve lipid metabolism in obese mice.

[0121] The above experimental results indicate that chlorogenic acid combined with Lactobacillus reuteri, as a drug combination with fortifying effects, can improve glucose and lipid metabolism disorders induced by a high-fat diet.

[0122] 2.2 The regulatory effect of chlorogenic acid combined with Lactobacillus reuteri on intestinal flora dysbiosis in obese AD mice

[0123] like Figure 4 As shown, principal coordinate analysis was performed on the gut microbiota of mice in each group. The results showed that the normal diet group and the high-fat diet group were significantly clustered on both sides of the two-dimensional coordinate axis, indicating that there were significant differences in the composition of the microbiota among different diet groups. The species composition structure also changed after colonization by chlorogenic acid and Lactobacillus reuteri.

[0124] Species annotation revealed that Firmicutes, Bacteroidetes, Verrucomicrobia, and Actinobacteria were the four main phyla-level bacterial groups in the gut microbiota colonized by chlorogenic acid and Lactobacillus reuteri in this experiment.

[0125] Correlation analysis revealed that *Lactobacillus reuteri* can increase the abundance of probiotics such as *Alistipes indistinctus*, *Lactobacillus intestinalis*, and *Bacteroides acidifaciens*, while decreasing the abundance of pathogenic bacteria *Clostridium sp.*.

[0126] The above experimental results indicate that chlorogenic acid and Lactobacillus reuteri colonization can increase the abundance of probiotics and reduce the abundance of pathogenic bacteria, which is beneficial for regulating intestinal flora imbalance induced by a high-fat diet, thereby treating metabolic disorders.

[0127] Example 2

[0128] Chlorogenic acid combined with Lactobacillus reuteri improves cognitive impairment in obese AD mice.

[0129] 1. Experiment and Methods

[0130] 1.1 Experimental Materials

[0131] Same as Example 1.

[0132] 1.2 Animal Experiments

[0133] Same as Example 1.

[0134] 1.3 Cognitive Function Assessment

[0135] (1) New Object Recognition Experiment

[0136] Before the experiment, the mice were placed in the experimental room for 1-2 hours to acclimatize. Before the test, the mice were placed in an empty test box (25cm×25cm×25cm) to familiarize themselves with the environment for 8 minutes. At the start of the experiment, the mice were allowed to explore for 5 minutes. After 1 hour, the testing phase began, and one of the old objects was replaced with a new object for 5 minutes. The activity of each mouse was captured and recorded using a camera, and the data was analyzed using ANY-maze animal behavior analysis software. The Recognition Index (RI) was calculated using the formula: RI = (New object / (New object + Old object)) × 100%.

[0137] (2) Y-maze experiment

[0138] Before the experiment, the experimental animals were moved into the laboratory 1-2 hours in advance. During the 8-minute test, each mouse was placed in the middle of the three arms and allowed to explore the maze freely. The order and number of times the mouse entered each arm of the Y-maze were recorded. Entering an arm was defined as entering one arm when all four of the mouse's limbs entered the arm.

[0139] Alternation percentage = Number of times the three arms are entered sequentially / Maximum number of possible alternations (total number of arms entered - 1) × 100%.

[0140] (3) Brain histological analysis

[0141] Brain tissues from the three groups of mice were placed in paraformaldehyde fixative and sent to Seville Bioservices for paraffin embedding, sectioning, and hematoxylin and eosin staining (H&E).

[0142] Immunofluorescence (IF) staining was used to observe the deposition of amyloid-β-protein (Aβ) and the activation of astrocytes. Brain slices were dewaxed for antigen retrieval, then blocked with 5% bovine serum albumin at room temperature for 1 hour, followed by overnight incubation with Aβ (1:400) primary antibody at 4°C. The slices were then incubated with AlexaFluor@488 secondary antibody (1:400) at room temperature for 1 hour. To observe astrocyte proliferation, after antigen retrieval and blocking, the slices were incubated overnight with GFAP (1:200) primary antibody at 4°C, followed by incubation with AlexaFluor@488 secondary antibody (1:400) at room temperature for 1 hour. Finally, the cell nuclei were stained with DAPI, and photographs were taken using a Zeiss 900 inverted laser confocal microscope.

[0143] 2. Experimental Results

[0144] The effect of chlorogenic acid combined with Lactobacillus reuteri on improving cognitive impairment in obese AD mice

[0145] like Figure 5 As shown, the results of the novel object recognition experiment indicate that after a high-fat diet with chlorogenic acid and colonization by Lactobacillus reuteri, mice increased their exploration of novel objects.

[0146] like Figure 6 As shown, a high-fat diet significantly reduced the cognitive index, suggesting that a high-fat diet impairs the cognitive function of mice. The cognitive index significantly increased after colonization with chlorogenic acid and Lactobacillus reuteri.

[0147] like Figure 7 As shown, the Y-maze experiment results indicated that the percentage of alternation in mice on a high-fat diet was significantly reduced, suggesting that a high-fat diet impairs the spatial memory ability of mice. The percentage of alternation was significantly increased after colonization with chlorogenic acid and Lactobacillus reuteri.

[0148] like Figure 8 As shown, H&E staining results indicated that under a high-fat diet, cell density in the hippocampal subventral region decreased, and neuronal survival was reduced. After combined administration of chlorogenic acid and *Lactobacillus reuteri*, cell density was significantly improved, effectively enhancing neuronal survival. IF results showed no significant difference in Aβ protein deposition among the groups. Astrocyte proliferation is associated with central nervous system damage and is considered crucial in the pathogenesis of neurodegenerative diseases. Its marker is glial fibrillary acidic protein (GFAP). Increased GFAP expression in the hippocampus of mice under a high-fat diet suggested neuronal damage and astrocyte proliferation. GFAP expression decreased after combined administration of chlorogenic acid and *Lactobacillus reuteri*.

[0149] The above experimental results indicate that chlorogenic acid combined with Lactobacillus reuteri, as a drug combination with enhancing effects, is beneficial in increasing the survival of neurons in mice under a high-fat diet, alleviating the proliferation of astrocytes, reducing nervous system damage, and improving cognitive dysfunction related to metabolic disorders.

[0150] Example 3

[0151] Chlorogenic acid combined with Lactobacillus reuteri improves mood disorders in obese AD mice.

[0152] 1. Experiment and Methods

[0153] 1.1 Experimental Materials

[0154] Same as Example 1.

[0155] 1.2 Animal Experiments

[0156] Same as Example 1.

[0157] 1.3 Emotional Level Assessment

[0158] Elevated cross maze experiment: Mice were placed in the environment for acclimatization 1-2 hours before the experiment. When the experiment officially started, the mice were placed in the central area of ​​the elevated cross maze, facing the open arms. The mice were allowed to freely explore the elevated maze for 5 minutes. The time the mice spent in the open arms was recorded using a camera and Super-maze software.

[0159] 2. Experimental Results

[0160] The effect of chlorogenic acid combined with Lactobacillus reuteri on improving mood disorders in obese AD mice

[0161] like Figure 9 As shown, the elevated cross maze test results indicate that mice on a high-fat diet spend less time in the open arm, while chlorogenic acid and Lactobacillus reuteri colonization increase the time mice spend in the open arm and have an ameliorative effect on mood disorders in obese AD mice.

[0162] The above results indicate that chlorogenic acid combined with Lactobacillus reuteri, as a drug combination with enhancing effects, can improve mood disorders such as anxiety and depression in mice on a high-fat diet.

[0163] Example 4

[0164] Chlorogenic acid combined with Lactobacillus reuteri improves the survival rate of Caenorhabditis elegans.

[0165] 1. Experiment and Methods

[0166] The nematode lifespan experiment was conducted at 20°C. Synchronized L1-stage *C. elegans* were seeded onto Nematode growth media (NGM) and allowed to grow to the L4 stage. On day 0, approximately 100 L4 nematodes from each plate were transferred to NGM plates. All plates were supplemented with 100 μm of 5-fluorodeoxyuridine (FUdR) to inhibit progeny production. At the L4 stage, the nematodes were transferred to NGM plates containing FUdR to spend the remainder of their lives.

[0167] The experimental groups are as follows:

[0168] Control group: Nematodes were cultured using standard nematode culture medium (NGM), prepared as follows: per 1000 mL of medium, 18–25 g agar, 2.7 g beef peptone, 0.55 g Tris-base, 0.27 g Tris-HCl, and 2 g NaCl, autoclaved at 121°C for 20 min. The culture temperature was 20°C. The control group culture medium contained sterile deionized water.

[0169] Chlorogenic acid group: NGM was prepared in the same way as the control group. After adding chlorogenic acid, NGM culture medium was prepared with a final concentration of 10 mg / ml chlorogenic acid and the pH was adjusted to 7.0.

[0170] Lactobacillus reuteri group: 1×10⁻⁶ samples were plated on NGM medium. 8 CFU Lactobacillus reuteri

[0171] Chlorogenic acid + Lactobacillus reuteri group: Chlorogenic acid was administered to NGM medium to a final concentration of 10 mg / ml. The pH was adjusted to 7.0, and 1×10⁻⁶ samples were spread. 8 CFU Lactobacillus reuteri

[0172] Record the survival status of the nematodes until all of them die.

[0173] 2. Experimental Results

[0174] Anti-aging effects of chlorogenic acid combined with Lactobacillus reuteri

[0175] like Figure 10 As shown, the survival rate of nematodes in the chlorogenic acid + Lactobacillus reuteri group was higher than that in the control group, while there were no statistically significant differences in the other groups.

[0176] The above experimental results indicate that chlorogenic acid combined with Lactobacillus reuteri, as a drug combination with enhanced efficacy, can prolong the lifespan of nematodes and play an anti-aging role.

[0177] 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. Use of a composition, characterized in that, This is used to prepare a medicament for the prevention, treatment, or improvement of metabolic disorders complicated with neuropsychiatric diseases or for delaying aging, wherein the composition comprises: First active component: a compound of formula I, a pharmaceutically acceptable salt thereof, or a solvation thereof; and The second active component: Limosilactobacillus reuteri.

2. The use as described in claim 1, characterized in that, The Lactobacillus reuteri mentioned is a wet cell of Lactobacillus reuteri.

3. The use as described in claim 1, characterized in that: The Lactobacillus reuteri strains mentioned are selected from the following groups: ATCC 23272, CCUG 33624, CIP 101887, DSM 20016, IFO 15892, JCM 1112, LMG13557, LMG 9213, NBRC 15892, NRRL B-14171, or combinations thereof.

4. The use as described in claim 1, characterized in that, The metabolic disorder mentioned refers to either a disorder of carbohydrate metabolism or a disorder of lipid metabolism.

5. The use as described in claim 1, characterized in that, The metabolic disorders mentioned refer to hyperlipoproteinemia, hypertriglyceridemia, hypercholesterolemia, diabetes, fatty liver, obesity, or arteriosclerotic cardiovascular and cerebrovascular diseases.

6. The use as described in claim 1, characterized in that, The neuropsychiatric disorders mentioned are cognitive impairments or mood disorders.

7. The use as described in claim 6, characterized in that, The mood disorder referred to is anxiety disorder, depression, or mania.

8. The use as described in claim 1, characterized in that, The composition described herein can prevent, treat, or improve metabolic disorders complicated with neuropsychiatric illnesses by means of: (1) Slow down weight gain; (2) Lower blood sugar levels; (3) Regulate lipid metabolism, reduce fat content, lower the fat / lean meat ratio, and lower blood lipid levels; (4) Increase the abundance of probiotics and reduce the abundance of pathogenic bacteria; (5) Increases cell density in the hippocampal subcortex and increases neuronal survival; (6) Reduce the expression of glial fibrillary acidic protein, reduce the proliferation of astrocytes, and reduce damage to the nervous system; (7) Increase the exploration of new objects; (8) Improve cognitive index; (9) Increase the alternation percentage; (10) Increase the dwell time of the open arm.

9. The use as described in claim 1, characterized in that, The drug is administered orally, by injection, inhalation, or via cavity.

10. The use as described in claim 1, characterized in that, The dosage form of the drug is selected from: injections, capsules, tablets, granules, sprays, gels, sustained-release preparations, oral liquids, drop pills, and nano-preparations.

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