Preparation and use of tangerine peel polysaccharide and composition thereof

Abstract

Preparation and use of a tangerine peel polysaccharide and a composition thereof. Provided is use of a Citri reticulatae pericarpium crude polysaccharide and a characteristic component PCRCPI thereof in alleviating nerve injury, depression-like behaviors, and metabolism-related mental diseases; they can effectively ameliorate depression-like behaviors caused by HFD diet, restore the levels of neurotransmitters DA and 5-HT, and upregulate neuronal cell activity biomarkers DCX and Neun.

Description

Preparation and application of a tangerine peel polysaccharide and its composition Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to the preparation and application of a tangerine peel polysaccharide and its composition. Background Technology

[0002] Depression is often comorbid with cardiovascular disease and metabolic disorders, and metabolic-associated depression is considered a subtype of depression (metabolic depression, MD). Extensive clinical and animal research evidence suggests that excessive and prolonged high-fat diets (HFD) can lead to obesity and systemic insulin resistance, resulting in cognitive and / or emotional dysfunction and a depressive phenotype.

[0003] Metabolic disorders and complications caused by high blood pressure dysbiosis (HFD) are often associated with gut microbiota dysbiosis. In clinical trials and preclinical animal studies, targeted regulation of the composition and structure of gut microbiota through diet, probiotics, and fecal microbiota transplantation has effectively alleviated HFD-induced clinical symptoms. Dietary fiber is considered a potential candidate for gut microbiota-guided drug development, as its utilization by gut microbiota depends on their encoded genes and carbohydrate enzymes. Traditional Chinese medicine polysaccharides, due to their diverse sources, sugar units, and molecular structures, can promote the growth of different beneficial bacteria, thus holding great promise for gut microbiota-guided drug development. Therefore, in-depth discovery of polysaccharide components that specifically interact with specific bacterial groups and their deep application in the prevention and treatment of metabolic syndrome (MD) can provide new ideas and technical support for clinical MD intervention and new drug development. Summary of the Invention

[0004] This application aims to at least solve one of the technical problems existing in the prior art. To this end, this application proposes an application of tangerine peel polysaccharide.

[0005] This application also proposes a tangerine peel polysaccharide.

[0006] This application also proposes a method for preparing the above-mentioned tangerine peel polysaccharide.

[0007] This application also proposes a composition containing the above-mentioned tangerine peel polysaccharide.

[0008] This application also proposes applications of the above-described compositions.

[0009] According to the first aspect of this application, the use of tangerine peel polysaccharide in any one of the following (1)-(13) is proposed:

[0010] (1) Use in the preparation of products for the prevention and / or treatment of depression; (2) Use in the preparation of products for the prevention and / or treatment of neurological damage; (3) Use in the preparation of products for reducing weight gain; (4) Use in the preparation of products for regulating gut microbiota; (5) Use in the preparation of products for increasing plasma DA content; (6) Use in the preparation of products for increasing plasma 5-HT content; (7) Use in the preparation of products for increasing DCX expression; (8) Use in the preparation of products for increasing Neun expression; (9) Use in the preparation of products for decreasing Iba-1 expression; (10) Use in the preparation of products for inhibiting HFD-induced microglial activation; (11) Use in the preparation of products for increasing the expression of metabolites of retrograde endocannabinoid signaling; (12) Use in the preparation of products for activating oxidative phosphorylation signaling pathways; or (13) Use in the preparation of products for increasing the expression levels of genes related to oxidative phosphorylation signaling pathways.

[0011] In some embodiments of this application, the depression is cognitive and / or emotional dysfunction, neurological damage, and / or metabolic-related depression.

[0012] In some embodiments of this application, the metabolites of the retrograde endocannabinoid signaling include 2-arachidonic acid glycerol (2-AG) and γ-aminobutyric acid (GABA).

[0013] In some embodiments of this application, genes associated with the oxidative phosphorylation signaling pathway include at least one of COX6c, mt-Nd3, NDUFs5, ATP5e, NDUFb8, SDHB, ATP5e, UQCR11, and UQCRh.

[0014] In some embodiments of this application, the tangerine peel polysaccharide is prepared by the following method: a tangerine peel sample is taken, and subjected to defatting, water extraction, alcohol precipitation, decolorization, protein removal with Sevag reagent, and dialysis to obtain tangerine peel polysaccharide.

[0015] In some embodiments of this application, a step of fractionating and separating the tangerine peel polysaccharide is also included, the step of which is as follows: eluting the tangerine peel polysaccharide through a DEAE cellulose column, the elution reagent including a NaCl solution with a concentration of 0.1M to 0.6M.

[0016] In some embodiments of this application, the elution reagents used are, in sequence, 0.15M to 0.24M NaCl solution, 0.25M to 0.35M NaCl solution, and 0.45M to 0.55M NaCl solution.

[0017] In some embodiments of this application, the elution reagents used are, in sequence, 0.2M NaCl solution, 0.3M NaCl solution, and 0.5M NaCl solution.

[0018] In some embodiments of this application, the elution reagent also includes water.

[0019] In some embodiments of this application, the elution flow rate is 1-3 mL / min.

[0020] In some embodiments of this application, the elution flow rate is 1.5 mL / min.

[0021] In some embodiments of this application, the monosaccharides in the tangerine peel polysaccharide obtained by graded separation contain rhamnose, arabinose, galactose, glucose, and galacturonic acid; the molar ratio of rhamnose, arabinose, galactose, glucose, and galacturonic acid is (1-3):(9-13):(4-6):(1-3):(75-85).

[0022] In some embodiments of this application, the molar ratio of rhamnose, arabinose, galactose, glucose, and galacturonic acid is 2.4:11.1:5.1:1.7:79.7.

[0023] In some embodiments of this application, the tangerine peel includes those derived from tea branch mandarin oranges (Guangdong tangerine peel), Wenzhou mandarin oranges (Zhejiang tangerine peel), early tangerines (Zhejiang tangerine peel), Fujian tangerines (Jiangxi tangerine peel), Sichuan tangerines (Sichuan tangerine peel), Da Hong Pao tangerines (Sichuan tangerine peel), Nanfeng tangerines (Jiangxi tangerine peel), etc.

[0024] In some embodiments of this application, the mass fraction of the tangerine peel polysaccharide in the product is from 0.01% to 100%.

[0025] According to some preferred embodiments of this application, the mass fraction of the tangerine peel polysaccharide in the product is 0.05% to 95%.

[0026] According to some preferred embodiments of this application, the mass fraction of the tangerine peel polysaccharide in the product is 0.05% to 50%.

[0027] According to a second aspect of this application, a tangerine peel polysaccharide is proposed, wherein the monosaccharide in the tangerine peel polysaccharide contains rhamnose, arabinose, galactose, glucose, and galacturonic acid; the molar ratio of the rhamnose, arabinose, galactose, glucose, and galacturonic acid is (1-3):(9-13):(4-6):(1-3):(75-85).

[0028] In some embodiments of this application, the molar ratio of rhamnose, arabinose, galactose, glucose, and galacturonic acid is 2.4:11.1:5.1:1.7:79.7.

[0029] In some embodiments of this application, the average molecular weight of the tangerine peel polysaccharide is 48.85 kDa.

[0030] According to a third aspect of this application, a method for preparing the above-mentioned tangerine peel polysaccharide is provided, the method comprising the following steps:

[0031] (1) Take a sample of dried tangerine peel, and after defatting, water extraction, alcohol precipitation, decolorization, protein removal with Sevag reagent, and dialysis, obtain crude polysaccharide of dried tangerine peel;

[0032] (2) The crude polysaccharide of tangerine peel is eluted through a DEAE cellulose column to obtain tangerine peel polysaccharide; the elution reagents include NaCl solution with a concentration of 0.1M to 0.6M.

[0033] In some embodiments of this application, the elution reagents used are, in sequence, 0.15M to 0.24M NaCl solution, 0.25M to 0.35M NaCl solution, and 0.45M to 0.55M NaCl solution.

[0034] In some embodiments of this application, the elution reagents used are, in sequence, 0.2M NaCl solution, 0.3M NaCl solution, and 0.5M NaCl solution.

[0035] In some embodiments of this application, the elution reagent also includes water.

[0036] In some embodiments of this application, the elution flow rate is 1-3 mL / min.

[0037] In some embodiments of this application, the elution flow rate is 1.5 mL / min.

[0038] According to a fourth aspect of this application, a composition is provided containing the above-mentioned tangerine peel polysaccharide.

[0039] In some embodiments of this application, the composition further comprises lactobacillus.

[0040] In some embodiments of this application, the lactobacillus includes one of Lactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus murineis.

[0041] According to the fifth aspect of this application, the use of the above composition in any one of the following (1)-(13) is proposed:

[0042] (1) Use in the preparation of products for the prevention and / or treatment of depression; (2) Use in the preparation of products for the prevention and / or treatment of neurological damage; (3) Use in the preparation of products for reducing weight gain; (4) Use in the preparation of products for regulating gut microbiota; (5) Use in the preparation of products for increasing plasma DA content; (6) Use in the preparation of products for increasing plasma 5-HT content; (7) Use in the preparation of products for increasing DCX expression; (8) Use in the preparation of products for increasing Neun expression; (9) Use in the preparation of products for decreasing Iba-1 expression; (10) Use in the preparation of products for inhibiting HFD-induced microglial activation; (11) Use in the preparation of products for increasing the expression of metabolites of retrograde endocannabinoid signaling; (12) Use in the preparation of products for activating oxidative phosphorylation signaling pathways; or (13) Use in the preparation of products for increasing the expression levels of genes related to oxidative phosphorylation signaling pathways.

[0043] In some embodiments of this application, the depression is cognitive and / or emotional dysfunction, neurological damage, and / or metabolic-related depression.

[0044] In some embodiments of this application, the metabolites of the retrograde endocannabinoid signaling include 2-arachidonic acid glycerol (2-AG) and γ-aminobutyric acid (GABA).

[0045] In some embodiments of this application, genes associated with the oxidative phosphorylation signaling pathway include at least one of COX6c, mt-Nd3, NDUFs5, ATP5e, NDUFb8, SDHB, ATP5e, UQCR11, and UQCRh.

[0046] In some embodiments of this application, the amount of tangerine peel polysaccharide in the composition is 40-100 mg / kg / d.

[0047] In some embodiments of this application, the amount of tangerine peel polysaccharide in the composition is 60 mg / kg / d.

[0048] In some embodiments of this application, the amount of lactobacillus in the composition is 3 × 10⁻⁶. 9 Up to 7×10 9 CFU / d.

[0049] In some embodiments of this application, the amount of lactobacillus in the composition is 5 × 10⁻⁶. 9 CFU / d.

[0050] In some embodiments of this application, the product includes pharmaceuticals, health products, or functional foods.

[0051] In some embodiments of this application, the pharmaceutical product further includes pharmaceutically acceptable excipients.

[0052] In some embodiments of this application, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, sweeteners, and flavorings.

[0053] In some embodiments of this application, the excipient includes water.

[0054] In some embodiments of this application, the filler includes at least one of starch and sucrose.

[0055] In some embodiments of this application, the adhesive includes at least one of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.

[0056] In some embodiments of this application, the wetting agent includes glycerin.

[0057] In some embodiments of this application, the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate.

[0058] In some embodiments of this application, the absorption enhancer includes a quaternary ammonium compound.

[0059] In some embodiments of this application, the surfactant includes hexadecyl alcohol.

[0060] In some embodiments of this application, the adsorbent carrier includes at least one of kaolin and soap clay.

[0061] In some embodiments of this application, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0062] In some embodiments of this application, the dosage form of the drug is a solid, semi-solid, or liquid, and may be an aqueous solution, a non-aqueous solution, or a suspension.

[0063] In some embodiments of this application, the formulations are tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, drop pills, dry extracts, injections or infusions, transdermal preparations, and transdermal microneedles.

[0064] In some embodiments of this application, the administration method of the drug can be a conventional administration method in the art, including but not limited to injection or oral administration.

[0065] In some embodiments of this application, the injection administration can be via intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.

[0066] According to some embodiments of this application, at least the following beneficial effects are achieved: This application provides the application of crude polysaccharide of dried tangerine peel and its characteristic component PCRCPI in alleviating nerve damage and depressive behavior. Through the crude polysaccharide of dried tangerine peel or its characteristic component PCRCPI of this application, depressive-like behavior induced by HFD diet can be effectively improved, the reduction of DA and 5-HT caused by long-term HFD can be restored, and the RNA expression of DCX and Neun in neuronal cells can be increased and the activation of microglia can be inhibited, thus protecting HFD mice from nerve damage. This provides a new possibility for the preparation of new drugs for alleviating nerve damage and depressive behavior.

[0067] Meanwhile, this application also shows that the combined use of PCRCPI and Lactobacillus can further improve depressive-like behavior induced by HFD diet, increase the content of DA and 5-HT in HFD mice, increase the relative expression of DCX and Neun, and reduce the expression of Iba-I. This can be effectively used to alleviate nerve damage and depressive behavior, and to prepare a drug to alleviate nerve damage and depressive behavior. Attached Figure Description

[0068] The present application will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0069] Figure 1 shows the preparation and characterization results of PCR-CPI in the test examples of this application. In this figure, A is a flowchart of the extraction and purification of PCR-CPI; B is the elution curve of DEAE-52; and C is the elution curve of Sephadex G-100.

[0070] Figure 2 shows the ultraviolet spectrum of PCR-CPI in the test example of this application;

[0071] Figure 3 shows the high-performance gel permeation chromatography (HPGPC) chromatogram of PCRCPI in the test example of this application, to show its molecular weight distribution;

[0072] Figure 4 shows the FT-IR spectrum in the test example of this application;

[0073] Figure 5 shows the results of the detection of monosaccharide composition of PCRCPI identified by gas chromatography-mass spectrometry (GC-MS) in the test examples of this application. The black lines represent the graph of standard monosaccharides, and the red lines represent the graph of PCRCPI.

[0074] Figure 6 is a lgMw-RT standard curve of the molecular weight measurement of PCRCPI in the test example of this application;

[0075] Figure 7 shows the detection results of the differential improvement of HFD-induced depressive-like behavior in mice by the characteristic components of tangerine peel polysaccharide (PCRCP) in the test examples of this application. Among them, A is a schematic diagram of the animal experiment process; B is a graph of mouse body weight change; C is a graph of TST detection results; D is a graph of FST detection results; E is a graph of SPT detection results; F is a graph of plasma DA content detection results; G is a graph of plasma 5-HT content detection results; H is a graph of relative expression level of DCX in mouse hippocampus tissue; I is a graph of relative expression level of Neun in mouse hippocampus tissue; J is a graph of relative expression level of Iba-1 in mouse hippocampus tissue; K is a representative graph of HE and Nissl staining in the dentate gyrus (DG) region of the hippocampus. "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001.

[0076] Figure 8 shows the fecal metabolite profile of mice whose depressive-like behavior was significantly reshaped by PCR-CPI treatment in the test cases of this application; where A is the principal component analysis (PCA) plot; B is the volcano plot of differential metabolites, with screening criteria: P<0.05, FC≥2; and C is the expression calorimetry of differential metabolites.

[0077] Figure 9 is a pathway enrichment map of differential metabolites in the fecal metabolite profile of mice whose depressive-like behavior was significantly reshaped by PCRCPI treatment in the test cases of this application, based on KEGG term analysis.

[0078] Figure 10 is a heatmap of the expression levels of endocannabinoid pathway metabolites in the test examples of this application;

[0079] Figure 11 shows the fecal metabolite profile of mice whose depressive-like behavior was significantly reshaped by PCRCPI treatment in the test cases of this application. In the figure, A is the relative abundance of 2-AG in the PCRCPI group and the HFD group, and B is the relative abundance of GABA in the PCRCPI group and the HFD group. "****" indicates P<0.0001.

[0080] Figure 12 shows the fecal metabolite profile of mice whose depressive-like behavior was significantly reshaped by PCRCPI treatment in the test examples of this application. In the figure, A is the correlation analysis result of the relative abundance of 2-AG and SPT behavioral index; B is the correlation analysis result of the relative abundance of 2-AG and FST behavioral index; and C is the correlation analysis result of the relative abundance of 2-AG and TST behavioral index.

[0081] Figure 13 shows the detection results of the role of lactic acid bacteria in improving high-fat diet-induced depressive-like behavior in mice treated with PCR-CPI in the test cases of this application. Among them, A is a schematic diagram of the animal experiment process; B is a graph of mouse weight change; C is a graph of FST detection results; D is a graph of TST detection results; E is a representative graph of HE and Nissl staining in the dentate gyrus (DG) region of the hippocampus in the FMT group and SFF group; F is a schematic diagram of the fecal microbiota transplantation animal experiment; G is a graph of mouse weight change; H is a graph of SPT detection results; I is a graph of TST detection results; J is a graph of FST detection results; K is a representative graph of HE and Nissl staining in the dentate gyrus (DG) region of the hippocampus. "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, "****" indicates P<0.0001, and "ns" indicates no significant difference.

[0082] Figure 14 shows the detection results of the differential enhancement of the antidepressant effect of PCR-CPI by lactic acid bacteria in the test examples of this application; where A is a schematic diagram of the animal experiment process; B is a graph of mouse weight change; C is a graph of SPT detection results; D is a graph of TST detection results; E is a graph of FST detection results; F is a representative graph of H&E and Nissl staining in the dentate gyrus (DG) region of the hippocampus; G is a graph of plasma DA content detection results; H is a graph of plasma 5-HT content detection results; I is a graph of relative expression level of DCX in mouse hippocampus tissue; G is a graph of relative expression level of Neun in mouse hippocampus tissue; K is a graph of relative expression level of Iba-1 in mouse hippocampus tissue; "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001.

[0083] Figure 15 shows the results of gene expression profiling of hippocampal tissue analyzed by RNA sequencing in the test examples of this application. In the figure, A is the principal component analysis (PCA) plot; B is the volcano plot of differential metabolites. Screening criteria: P<0.05, FC≥1.5.

[0084] Figure 16 is a KOG functional enrichment map of the gene expression profile of hippocampal tissue analyzed by RNA sequencing in the test example of this application.

[0085] Figure 17 shows the results of gene expression profiling analysis of hippocampal tissue by RNA sequencing in the test examples of this application. In this figure, A is a pathway enrichment map of differentially expressed genes based on KEGG analysis; B is a heatmap of gene expression related to the oxidative phosphorylation signaling pathway.

[0086] Figure 18 shows the results of gene expression profile analysis of hippocampal tissue by RNA sequencing in the test example of this application. In this figure, A is the GSEA enrichment analysis of genes related to the retrograde endocannabinoid pathway, and B is the GSEA enrichment analysis of genes related to the oxidative phosphorylation signaling pathway.

[0087] Figure 19 shows the results of gene expression profile analysis of hippocampal tissue by RNA sequencing in the test examples of this application. In the figure, A is the Spearman correlation analysis results between genes of the oxidative phosphorylation signaling pathway and depression-related indicators; B is the detection results of relative mRNA expression levels of genes related to the oxidative phosphorylation signaling pathway; and C is the molecular docking results of 2-AG and mt-ND3.

[0088] Figure 20 shows the KEGG functional enrichment analysis results of gene expression profile analysis of hippocampal tissue analyzed by RNA sequencing in the test example of this application.

[0089] Figure 21 is a heatmap analysis of the enrichment of the retrograde endocannabinoid signaling pathway in the test examples of this application;

[0090] Figure 22 shows the expression level and fold change of mt-ND3 in the oxidative phosphorylation signaling pathway in the test examples of this application. Detailed Implementation

[0091] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0092] Example 1

[0093] This embodiment provides a method for preparing crude polysaccharide (PCRCP) from dried tangerine peel, including the following steps:

[0094] (1) Powdering: Select five-year-old dried Guangchenpi (the original place of origin of Guangchenpi sample is Xinhui District, Jiangmen City), dry it, grind it into powder by a pulverizer, collect it after passing through a 40-mesh sieve, and store it in a dry environment at room temperature.

[0095] (2) Degreasing: The dried tangerine peel powder obtained in step (1) was added to anhydrous ethanol at a material-to-liquid ratio of 1g:10mL for degreasing treatment. The treatment conditions were set as reflux at 80℃ for 1.5h, and repeated twice. After degreasing, the filter residue was collected and dried for later use.

[0096] (3) Water extraction: Take the filter residue obtained in step (2) and add pure water at a material-to-liquid ratio of 1g:10mL. Set the extraction conditions to 100℃ and reflux in a water bath for 6 hours. Use a Buchner funnel to filter the extract through 0.45μm filter paper. After filtrate, centrifuge at 8000r / min for 30min and collect the supernatant.

[0097] (4) Alcohol precipitation: The supernatant obtained in step (3) is mixed with 4 times the volume of 95wt% ethanol, and the mixture is precipitated overnight in a refrigerator at 4°C. After precipitation, the mixture is filtered and the precipitate is retained. The precipitate is dried to obtain tangerine peel crude polysaccharide powder.

[0098] (5) Decolorization: The polysaccharide powder obtained in step (4) was washed with diethyl ether and acetone respectively. After filtering the precipitate, the organic solvent was evaporated. The precipitate was dissolved in pure water at a mass ratio of 1:100. Activated carbon was added at a mass ratio of 1:2 and stirred for 2 hours. The mixture was then placed in a refrigerator at 4°C and left to stand overnight for decolorization. The mixture was centrifuged at 8000 r / min for 30 min and the decolorized solution was collected.

[0099] (6) Protein Removal: The supernatant prepared in step (5) is subjected to protein removal treatment. Sevag reagent is prepared by mixing chloroform and n-butanol solutions at a volume ratio of 4:1. The decolorizing solution and Sevag reagent are mixed in a separatory funnel at a volume ratio of 5:1, shaken thoroughly for 10-15 min, allowed to stand for separation, and the lower layer of denatured protein precipitate is discarded. The operation is repeated until no denatured protein is formed. The upper layer of crude polysaccharide solution is collected.

[0100] (7) Dialysis and freeze-drying: The crude polysaccharide solution deproteinized in step (6) is dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. The dialysate is collected and freeze-dried in a freeze dryer to obtain the crude polysaccharide of dried tangerine peel.

[0101] All animal experiments were ethically approved by the Animal Ethics Committee of the International Institute for Health and Wellness Innovation (Jiangmen, China). All animal-related experimental protocols were conducted in accordance with the institute's "Guidelines for the Care and Use of Laboratory Animals".

[0102] Male C57BL / 6J mice, SPF grade, 8 weeks old, weighing 20g, were purchased from Zhuhai Beston Biotechnology Co., Ltd. They were housed in an SPF-grade animal facility with standard facilities, a 12-hour light / dark cycle, and were acclimatized for 1 week.

[0103] Example 2: A tangerine peel polysaccharide for alleviating nerve damage and depressive-like behavior

[0104] This embodiment provides a tangerine peel polysaccharide for alleviating nerve damage and depressive-like behavior. The preparation flowchart is shown in Figure 1A. The specific method is as follows: 2g of crude polysaccharide is dissolved in 30mL of water and centrifuged at 12000rpm for 10 minutes to remove insoluble components. The sample is eluted using a DEAE-52 cellulose column (4cm). Water and different concentrations of NaCl solution (0.2, 0.3, 0.5M) are used sequentially at a flow rate of 1.5mL / min. Specifically, the crude polysaccharide solution is washed with 0.2M NaCl solution to obtain characteristic component PCRCP-I (0.2M), then the remaining polysaccharide is washed with 0.3M NaCl solution to obtain characteristic component PCRCP-II (0.3M), and finally the remaining polysaccharide is washed with 0.5M NaCl solution to obtain characteristic component PCRCP-III (0.5M). The elution curve is determined using the phenol-sulfuric acid colorimetric method (Figure 1B).

[0105] The PCRCP-I fraction was purified using a Sephadex G-100 column (1.6adex G-), with ultrapure water as the eluent and a flow rate of 4.0 mL / 12 min. The elution curve was determined by the phenol-sulfuric acid method (Figure C in Figure 1), and the main peak was collected to obtain the purified tangerine peel polysaccharide PCRCP-I fraction.

[0106] Example 3: A composition for alleviating nerve damage and depressive-like behavior

[0107] This embodiment provides a composition for alleviating nerve damage and depressive-like behavior. The composition contains the PCRCP-I component prepared in Example 2 and *Lactobacillus reuteri* LR-G100, wherein the concentration of *Lactobacillus reuteri* LR-G100 can be 5 × 10⁻⁶. 9 CFU / mL.

[0108] Example 4: A composition for alleviating nerve damage and depressive-like behavior

[0109] This embodiment provides a composition for alleviating nerve damage and depressive-like behavior. The composition contains the PCRCP-I component prepared in Example 2 and Lactobacillus johnsonii LJ-G55, wherein the concentration of Lactobacillus johnsonii LJ-G55 can be 5 × 10⁻⁶. 9 CFU / mL.

[0110] Example 5: A composition for alleviating nerve damage and depressive-like behavior

[0111] This embodiment provides a composition for alleviating nerve damage and depressive-like behavior. The composition contains the PCRCP-I component prepared in Example 2 and *Lactobacillus murineis*, with the concentration of *Lactobacillus murineis* being 5 × 10⁻⁶. 9 CFU / mL.

[0112] Test case

[0113] 1. Isolation, purification, and structural characterization of PCR-CPI

[0114] (1) Ultraviolet-Visible Spectroscopy Analysis

[0115] Two mg of the purified PCRCPI from Example 2 was dissolved in 4 mL of deionized water to prepare a polysaccharide solution with a concentration of 0.5 mg / mL. The solution was scanned at room temperature using a UV-Vis spectrophotometer (Shimadzu UV-2600) in the wavelength range of 190–400 nm.

[0116] The purified PCR-CPI was analyzed by UV-Vis spectroscopy, and the results are shown in Figure 2. As can be seen from the figure, no obvious absorption peaks were found in PCR-CPI at wavelengths of 260 nm and 280 nm, indicating that the purified PCR-CPI contained negligible amounts of nucleic acids and proteins.

[0117] (2) High-performance gel permeation chromatography (HPGPC) analysis

[0118] Purity and molecular weight of PCR CPI were determined using high-performance gel permeation chromatography (HPGPC).

[0119] The purity and molecular weight of the purified PCR-CPI obtained in Example 2 were determined using an LC-10AT instrument (Shimadzu, Japan) equipped with an RI-20A refractive index detector and a BRT 105-103101 (8×300 mm) gel filter column. The HPGPC experimental conditions were as follows: 0.05 M NaCl solution at pH 1.55 was used as the mobile phase, the flow rate was 0.8 mL / min, and the column temperature was 40 °C.

[0120] Sample preparation: Accurately weigh 5 mg of the purified PCR-CPI sample obtained in Example 2, dissolve it in 1 mL of NaCl solution to prepare a 5 mg / mL solution. After sonication for 10 min, centrifuge the solution at 12000 rpm for 10 min to collect the supernatant, pass it through a 0.22 μm hydrophilic microporous membrane, and perform sample analysis according to the established HPGPC experimental conditions.

[0121] The detection results, shown in Figure 3, reveal a single symmetrical peak, confirming the high purity of the sample. This result indicates consistency with the results obtained from Sephadex G-100 gel chromatography and UV analysis (Figures 2 and 3). Comparison with the molecular weight standard curve (Figure 6) shows that the average molecular weight of PCRCPI is 44.15 kDa.

[0122] (3) Fourier transform infrared spectroscopy (FT-IR) analysis of PCR-CPI

[0123] Accurately weigh 2 mg of the purified PCR-CPI obtained in Example 2 and mix it with 200 mg of KBr powder (spectrally pure). Then, use a tablet press to compress the mixture into granules. Spectroscopy was performed using Fourier transform infrared spectroscopy at 450–4000 cm⁻¹. 2 The infrared spectrum of PCRCPI was obtained by scanning within the range.

[0124] The FT-IR spectrum of PCRCPI is shown in Figure 4, where the peak wavelength at 3399 cm⁻¹ is [missing information]. -1 The broad and strong characteristic peak at 2933 cm⁻¹ corresponds to the stretching vibration of the OH group in the sugar molecule. -1 and 1444cm -1 The two additional absorption peaks at 1242 cm⁻¹ are attributed to the stretching and deformation vibrations of CH in the methyl group, respectively. -1 The absorption peak at 1623 cm⁻¹ further confirms the presence of CH bonds. Furthermore, the absorption peak at 1623 cm⁻¹... -1 and 1749cm -1 The distinct peaks observed at 1200-1000 cm⁻¹ are attributed to the stretching bands of the carboxylate ion (COO⁻) and the methylated carbonyl group (COO-CH₃), respectively, thus confirming the presence of uronic acid in the sample. -1 The absorption bands within this range represent stretching vibrations of CO bonds, including COH and COC bonds, indicating the presence of either COC or COH bonds. At 638 cm⁻¹... -1 The absorption peak at that point corresponds to the out-of-plane vibration of OH.

[0125] (4) Monosaccharide composition analysis of PCR-CPI

[0126] The monosaccharide composition of the purified PCRCPI obtained in Example 2 was analyzed by ion chromatography (IC). The specific steps are as follows:

[0127] 5 mg of sample was introduced into 2 mL of 3 M trifluoroacetic acid (TFA) and hydrolyzed at 120 °C for 3 h. After hydrolysis, the resulting hydrolysis product was evaporated under a nitrogen stream and then resuspended in 5 mL of ultrapure water. A 50 μL aliquot of this solution was diluted with 950 μL of deionized water and centrifuged at 12000 rpm for 5 min. The resulting supernatant (along the monosaccharide solution mixture) was analyzed by ion chromatography equipped with an electrochemical detector. (Dionex Carbopac) TM Monosaccharide separation was performed on a PA 20 column (3 × 150 mm) using a mobile phase consisting of A: H₂O; mL B: 15 mM NaOH; and C: 15 mM NaOH and 100 mM NaAc. The flow rate was maintained at 0.3 mL / min, the injection volume was 25 μL, and the column temperature was kept constant at 30 °C.

[0128] The results are shown in Figure 5. As can be seen from the figure, the homogeneous polysaccharide is composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc) and galacturonic acid (GalA), with molar percentages (mol%) of 2.4%, 11.1%, 5.1%, 1.7%, and 79.7%, respectively.

[0129] 2. Differential characteristics of tangerine peel polysaccharide (PCRCP) components improve HFD-induced depressive-like behavior in mice.

[0130] This test case verified the effects of different PCRCP components on improving HFD-induced depressive-like behavior in mice. The specific verification protocol is shown below.

[0131] (1) Establishment of a mouse model of depression induced by HFD through intervention of different components of PCRCP

[0132] SD mice were randomly divided into 6 groups of 8 mice each. The mice were administered either a normal diet or a high-fat diet, or received PCRCP, PCRCPII, PCRCPIII, or PCRCPIII via gavage for 18 weeks, respectively. The specific grouping and administration are shown in Figure 7A, as detailed below:

[0133] Chow group: water / normal feed (10% fat, 3.5 kcal / g);

[0134] HFD group: water / high-fat diet (45% fat, 16.8 kcal / g);

[0135] PCRCP group: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / kg / d PCRCP;

[0136] PCRCPI group: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / kg / d PCRCPI;

[0137] PCRCPII group: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / kg / d PCRCPII;

[0138] PCRCPIII group: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / Kg / d PCRCPIII.

[0139] (2) Index Evaluation Experiment

[0140] 1) Dissection and tissue sampling

[0141] The body weight and food intake of mice in different groups were recorded weekly. After the experiment, fresh feces, plasma, hippocampus, and brain of the mice were collected and flash-frozen in liquid nitrogen or fixed in paraformaldehyde for subsequent analysis.

[0142] 2) Behavioral testing

[0143] Sugar Preference Test (SPT): Each mouse in each group was individually housed in a cage equipped with two drinking bottles, one containing a 1% sucrose solution and the other containing control water. To avoid bias, the positions of the two bottles were switched every 12 hours. After acclimatization, water intake was restricted for 6 hours before the behavioral test. The weights of the drinking water bottle and the 1% sucrose water bottle were measured before the experiment (W1, S1) and 24 hours later (W2, S2). Sucrose consumption was calculated using the following formula: Sucrose Preference Index (%) = (S1-S2) / ((W1-W2)+(S1-S2))×100%.

[0144] Forced swimming test (FST): The test mice were placed in a transparent plexiglass cylinder (30cm high, 20cm in diameter, 20cm deep) filled with water (water temperature: 25±1℃) for 6 minutes, and the duration of static floating in the last 4 minutes was recorded.

[0145] Tail Suspension Test (TST): Mice were individually hung upside down on a horizontal bar with tape for 6 minutes. The time spent with the limbs completely or almost motionless for 4 minutes after the test was recorded.

[0146] 3) Detection of plasma dopamine (DA) and serotonin (5-HT).

[0147] Blood was collected from mice in EP tubes and allowed to stand for 2 hours. The tubes were then centrifuged at 3000g for 15 minutes at 4°C. The supernatant was collected and stored at -80°C. The concentrations of DA and 5-HT in mouse plasma were determined using an ELISA kit according to the manufacturer's instructions.

[0148] 4) HE staining

[0149] ① Dewaxing of paraffin sections: The sample was dehydrated and embedded in paraffin, with a section thickness of 5 μm. The sections were then immersed in xylene I-II for 10 min each, followed by immersion in anhydrous ethanol I-II for 5 min each. The samples were then dewaxed by sequentially immersing them in 90% ethanol, 70% ethanol, and pure water for 2 min each, and were ready for use.

[0150] ② HE staining: Stain with hematoxylin for 5 minutes, rinse with water for 3-5 seconds, then stain with eosin for 30 seconds. Immediately rinse with chromogenic solution 2-3 times, air dry, and mount with neutral resin. Take pictures using an Olympus BX41 microscope.

[0151] 5) Nissl staining

[0152] ① Dewaxing of paraffin sections: The sample was dehydrated and embedded in paraffin, with a section thickness of 5 μm. The sections were then immersed in xylene I-II for 10 min each, followed by immersion in anhydrous ethanol I-II for 5 min each. The samples were then dewaxed by sequentially immersing them in 90% ethanol, 70% ethanol, and pure water for 2 min each, and were ready for use.

[0153] ②Nissl staining: Add Nissl staining solution to the tissue section and stain for about 5 minutes. Rinse with 95% ethanol for 5 seconds, wash twice with 70% ethanol, dehydrate with 95% ethanol for 2 minutes, clear with xylene for 5 minutes, mount with neutral resin, and observe the staining results under an upright fluorescence microscope.

[0154] 6) Real-time quantitative PCR (q-PCR) analysis

[0155] ①Hippocampal RNA extraction (Trizol extraction method)

[0156] Mouse hippocampal tissue was placed in a 1.5 mL centrifuge tube, and 0.3 mL of Trizol was added. The tissue was then homogenized thoroughly using a tissue homogenizer. Another 1 mL of Trizol was added to bring the total volume to 1.3 mL. The mixture was incubated on ice for approximately 5 minutes, followed by centrifugation at 4°C and 12,000 rpm for 15 minutes to separate the tissue lysate. Approximately 1 mL of the supernatant was transferred to a new centrifuge tube, and 0.2 mL of chloroform was added at a volume ratio of 5:2. The mixture was vigorously shaken for 30 seconds, incubated on ice for 2 minutes, and then centrifuged again at 4°C and 12,000 rpm for 15 minutes.

[0157] Transfer approximately 0.5 mL of the supernatant from centrifugation to a centrifuge tube containing an equal volume of isopropanol, mix gently, and then incubate at -20°C for 8 hours to allow the RNA to precipitate completely. After incubation, collect the precipitate by centrifugation at 4°C and 12,000 rpm for 10 minutes, and discard the supernatant. Next, add 75% ethanol (prepared with DEPC water) to the precipitate, gently shake manually to ensure thorough washing, and then centrifuge again at 4°C and 12,000 rpm for 5 minutes.

[0158] After discarding the ethanol, allow the precipitate to air dry for about 5 minutes, being careful to avoid excessive drying which would make it difficult to dissolve. Finally, dissolve the RNA precipitate in an appropriate amount of DEPC water and use an Agilent 2100 bioanalyzer to check the quality and purity of the RNA to ensure that the experimental product is suitable for subsequent analysis.

[0159] ②Reverse transcription reaction

[0160] The RNA obtained above was reverse transcribed using the K1622 RevertAid First Strand cDNA Synthesis Kit. The specific system is shown in Table 1 below, and the reaction procedure is shown in Table 2.

[0161] Table 1

[0162] Table 2 PCR Procedure

[0163] ③ Real-time quantitative PCR

[0164] The cDNA obtained from the above reverse transcription was processed using RR420A TB. Premix Ex Taq TM The (Tli RNaseH Plus) kit was used for qPCR assay. The reaction system is shown in Table 3, the reaction procedure is shown in Table 4, and the primer sequences used are shown in Table 5.

[0165] Table 3

[0166] Table 4

[0167] Table 5 PCR amplification primer sequences

[0168] The test results are shown in Figure 7. The proposed method constructs an HFD-induced depression model.

[0169] As can be seen from Figure 7B, HFD feeding significantly increased the body weight of mice compared to conventionally fed mice.

[0170] As can be seen from the CE plot in Figure 7, HFD feeding induced an increase in immobility time in FST and TST and a decrease in sucrose preference in mice, which was significantly reversed after treatment with PCRCP and PCRCPPI. PCRCPII and PCRCPIII could reduce immobility time in FST to some extent, but immobility time in TST was induced to increase, while PCRCPIII decreased sucrose preference. This indicates that the PCRCP components alleviated HFD-induced depressive-like behavior in mice in a structure-dependent manner.

[0171] As can be seen from the FG plot in Figure 7, the plasma levels of DA and 5-HT in HFD mice are reduced. When treated with PCR-CPI, these changes in neurotransmitters are reversed, especially DA, which can reach levels comparable to those in normal mice.

[0172] As can be seen from the HI plot in Figure 7, compared with untreated mice, PCRCPI significantly restored the mRNA expression of DCX (a marker of immature neurons) and Neun (a marker of mature neurons) in hippocampal neurons from HFD-fed mice after 18 weeks.

[0173] As can be seen from Figure 7, PPCRCPI significantly increased the mRNA expression level of Iba-1, a classic marker of microglia, which means that PPCRCPI can inhibit HFD-induced microglia activation.

[0174] As shown in Figure 7 (K plot), HE staining and Nissl staining revealed changes in hippocampal neurons in each group of mice. In the HFD group, the pyramidal cells in the DG region of the hippocampus showed significant neuronal loss, atrophy, reduced Nissl bodies, unclear stratification, and irregular arrangement. PCR-CPI supplementation effectively protected hippocampal neurons from HFD-induced damage. These results indicate that HFD can induce depressive-like behavior and cause neurological damage in mice, while PCR-CPI can improve depressive-like behavior and reverse neurological damage.

[0175] The above experiments indicate that PCRCPI may be a key component of tangerine peel polysaccharides in exerting their antidepressant effects. PCRCPI can more effectively alleviate depressive-like behaviors in HFD mice, specifically manifested as an increased saccharide preference index in the SPT experiment and less immobility time in the TST and FST experiments. PCRCPI can also restore the reduction in DA and 5-HT caused by long-term HFD, and increase the RNA expression levels of DCX and Neu in neurons and inhibit microglial activation. Meanwhile, HE staining and Nissl staining show that PCRCPI can effectively protect HFD mice from neurological damage.

[0176] 3. Fecal metabolomics analysis

[0177] To investigate whether the protective effect of PCR-CPI on HFD-induced depression in mice is related to changes in metabolomics, this test case used feces from mice in the PCR-CPI-treated group that underwent 18 weeks of intervention to improve HFD-induced depressive-like behavior. Non-targeted metabolomics analysis was performed using GC-TOF / MS. The specific analysis steps are as follows:

[0178] 50 mg of feces was homogenized with 600 μL of ultrapure water and centrifuged at 13,000 rpm for 10 min at 4 °C. Residues were extracted with 600 μL of ice-cold methanol. 80 μL of the supernatant was collected and precipitated with 80 μL of methanol containing chloro-d-phenylalanine (100 ng / mL) and ketoprofen (10 ng / mL), then centrifuged at 13,000 rpm for 10 min at 4 °C. A total of 100 μL of supernatant was prepared from each sample for analysis. Metabolites were separated and detected using an Acquity I-Class PLUS ultra-high performance liquid chromatography (UPLC) system (Waters, USA), connected in series with a Xevo G2-XS QTOF high-resolution mass spectrometer (Thermo Fisher Scientific, USA). Chromatographic separation was achieved on a Waters Acquity UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm). Pathway enrichment analysis of differentially metabolites was performed using the KEGG database.

[0179] The results are shown in Figures 8-12. As can be seen from the AC plot in Figure 8, metabolomics revealed that PCR-CPI can significantly alter the content of fecal metabolites in HFD mice.

[0180] As shown in Figure 9, KEGG enrichment analysis revealed that differentially metabolites in the feces of PCR-CPI-treated mice were mainly enriched in the retrograde endocannabinoid signaling pathway, synaptic vesicle circulation, and GABAergic synapses. Changes in these metabolic signaling pathways are commonly associated with depression in rodents.

[0181] As can be seen from Figures 10 and 11, compared with HFD mice, PCRCPI significantly upregulated the expression of beneficial metabolites of retrograde endocannabinoid signaling, including 2-arachidonic acid glycerol (2-AG) and γ-aminobutyric acid (GABA).

[0182] As shown in Figure 12, Spearman correlation analysis was used to investigate the correlation between key indicators of depressive-like behavior and differential metabolite changes. The concentration of 2-AG in feces was positively correlated with the sucrose preference index in SPT and negatively correlated with immobility time in TST and FST, suggesting that the PCR-enriched metabolite 2-AG may be an active metabolite that helps improve the depressive phenotype.

[0183] In summary, these results indicate that PCRCPI can significantly modulate gut microbial metabolites in HFD-fed mice exhibiting depressive-like behavior.

[0184] 4. The ameliorative effect of gut microbiota and lactic acid bacteria on HFD-induced depressive-like behavior

[0185] To further investigate whether the antidepressant effect of PCRCPI is mediated by gut microbiota, feces from mice treated with PCRCPI (a group of mice with HFD-induced depressive-like behavior) that showed differential improvement in characteristic components of 2-tangerine peel polysaccharide (PCRCP) were transferred to HFD-fed mice with or without gut microbiota for 15 weeks (animal experimental procedure is shown in Figure A of Figure 13).

[0186] The experimental protocol was consistent with that of 2. The difference in characteristic components of tangerine peel polysaccharide (PCRCP) improved HFD-induced depressive-like behavior in mice.

[0187] The experimental results are shown in Figure 13. As can be seen from the BE plot in Figure 13, the fecal microbiota from mice treated with PCRCPI can improve the development of HFD-induced depression.

[0188] As shown in the FJ plot in Figure 13, PCRCP can significantly enrich *Lactobacillus* species that induce HFD-induced obesity in mice. Different live *Lactobacillus* strains, including *Lactobacillus reuteri* LR-G100 (LR), *Lactobacillus johnsonii* LJ-G55 (LJ), and *Lactobacillus murineis* (LM), were colonized in HFD-fed mice for 10 weeks to investigate their potential antidepressant effects. Oral administration of *Lactobacillus* strains significantly reduced weight gain and improved depressive-like behaviors in HFD-fed mice, such as increased sucrose preference and reduced immobility time in FST and TST.

[0189] As can be seen from the K plot in Figure 13, histological examination with HE and Nissl staining showed that the supplementation of Lactobacillus strains significantly reduced neuronal damage caused by HFD.

[0190] In summary, this evidence clearly suggests that the mitigation of HFD-induced depressive-like behavior in mice by PCRCPI may be related to the normalization of gut microbiota composition, particularly the restoration of lactobacilli.

[0191] 5. Differences in lactic acid bacteria enhanced the antidepressant effect of PCRCPI.

[0192] To further investigate how the interaction between PCRCPI and different Lactobacillus strains affects depressive-like behavior, 5 × 10⁶ cells were colonized in HFD mice. 9 The study investigated the use of live LR, LJ, and LM bacteria at CFU / d and PCR-CPI at 60 mg / kg / day for 10 weeks (method flowchart shown in Figure A of Figure 14).

[0193] Construction of animal models:

[0194] Male C57BL / 6J mice, SPF grade, 7-8 weeks old, 20±2g; after being housed normally in the animal room for 1 week, the animals were randomly divided into 9 groups of 11 each; all animals were fed either a normal diet (Chow, 13% fat for energy) or HFD (45% fat for energy).

[0195] Chow: Water / regular feed (13% fat, 3.5 kcal / g);

[0196] HFD: Water / high-fat diet (45% fat, 16.8 kcal / g);

[0197] PCRCPI: Water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / kg / d PCRCPI;

[0198] LR: Water / high-fat diet (45% fat, 16.8 kcal / g) / 5×10 9 CFU / d LR;

[0199] LJ: Water / high-fat feed (45% fat, 16.8 kcal / g) / 5×10 9 CFU / d LJ;

[0200] LM: Water / high-fat feed (45% fat, 16.8 kcal / g) / 5×10 9 CFU / d LM;

[0201] LR-PCRCPI: Water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / kg / d PCRCPI / 5 × 10 9 CFU / d LR;

[0202] LJ-PCRCPI: Water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / kg / d PCRCPI / 5 × 10 9 CFU / d LJ;

[0203] LM-PCRCPI: Water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / kg / d PCRCPI / 5 × 10 9 CFU / d LM.

[0204] The experimental protocol was consistent with that of 2. The difference in characteristic components of tangerine peel polysaccharide (PCRCP) improved HFD-induced depressive-like behavior in mice.

[0205] The results are shown in Figure 14. The BE plot in Figure 14 shows that the combination of *Lactobacillus* and *PCRCPI* significantly reduced mouse body weight gain and significantly improved depressive-like behavior. Specifically, immobility time was significantly reduced in both TST and FST, while sucrose preference was significantly increased.

[0206] As can be seen from Figure F in Figure 14, histological analysis with HE and Nissl staining showed that simultaneous administration of Lactobacillus and PCRCPI alleviated HFD-induced neurological damage in mice, with the most significant effect observed in mice receiving LM and PCRCPI.

[0207] As can be seen from the G and H plots in Figure 14, LM combined with PCRCPI treatment also significantly restored the reduction in DA and 5-HT levels caused by HFD.

[0208] Meanwhile, as can be seen from the IK plot in Figure 14, LM combined with PCRCPI treatment also significantly increased the mRNA expression of DCX and Neun in the hippocampus of HFD-fed mice and decreased the expression of Iba-1, indicating that the combined treatment effectively alleviated HFD-fed-induced depressive-like behavior and relieved neurological damage.

[0209] Therefore, the specific interaction between Lactobacillus and PCRCPI plays an important role in reducing neuronal damage and alleviating depressive-like behavior in HFD-fed mice.

[0210] 6. LM combined with PCR-CPI alters hippocampal gene signal transduction.

[0211] To further confirm the antidepressant effect and elucidate the potential mechanism by which the combination of LM and PCRCPI improves depressive-like behavior in HFD-fed mice, we performed unbiased RNA sequencing (RNA-seq) on the hippocampus of HFD mice and mice in the LM-PCRCPI treatment group after 10 weeks of intervention to analyze gene changes.

[0212] Transcriptomic sequencing (RNA-seq) and analysis were performed as follows: Total RNA was extracted from mouse hippocampal tissue using TRIZOL reagents (Invitrogen, Grand Island, NY, USA). RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and RNA quantity was assessed using an Agilent Bioanalyzer 2100 system (Agilent Technologies, San Diego, CA, USA). The library was sequenced on the Illumina NovaSeq platform (Beijing Biomarker Technology Co., Ltd., Beijing, China), generating 150 bp paired-end reads. Differential expression analysis between the two groups was performed using P.adj < 0.05 and |FC| ≥ 1.5 as criteria, and the results were analyzed and visualized using the BMKCloud platform (www.biocloud.net). The results are shown in Figures 15-22.

[0213] As shown in Figures A and B of Figure 15, LM-PCRCPI treatment significantly altered gene expression in the hippocampus of HFD-fed mice. Compared with untreated mice, 641 genes were upregulated and 544 genes were downregulated in LM-PCRCPI-treated mice (P<0.05; |FC|≥1.5).

[0214] As can be seen from Figures 16 and 20, the KOG and KEGG functional enrichment analyses show that the differentially expressed genes are mainly enriched in functional categories such as signal transduction mechanisms, energy production and conversion, and transcription.

[0215] As shown in Figure 17A, KEGG enrichment analysis revealed that these differentially expressed genes were mainly enriched in the ribosome, oxidative phosphorylation (OXPHOS), thermogenic, mitogen-activated protein kinase (MAPK) signaling pathway, and retrograde endocannabinoid signaling pathway. Among these, oxidative phosphorylation, thermogenic, Parkinson's disease, and retrograde endocannabinoid signaling pathways have been identified as potential candidates for promoting targeted prevention and developing personalized treatment strategies.

[0216] As can be seen from Figure 17B and Figure 18B, LM-PCRCPI treatment significantly activated the oxidative phosphorylation signaling pathway (OXPHOS), a key process in energy metabolism and ATP production. Damage to this pathway can lead to energy metabolism disorders, thereby promoting the development of depression.

[0217] As can be seen from Figure 18A and Figure 21, gene mutations in the retrograde endocannabinoid signaling pathway are also considered to be potential factors in the pathogenesis of major depressive disorder.

[0218] As shown in Figure 19B, q-PCR verification revealed that LM-PCRCPI treatment significantly increased the expression of mt-Nd3, NDUFs5, ATP5e, etc. in the hippocampus OXPHOS of mice fed HFD.

[0219] As can be seen from Figure A in Figure 19, Spearman correlation analysis shows that the mt-Nd3, ATP5e, and COX6c genes are positively correlated with the severity of depression-related indicators.

[0220] As can be seen from Figure 19C and Figure 22, mt-Nd3 has the highest expression level and a high fold change among differentially expressed genes in the OXPHOS pathway, and there is a strong molecular binding interaction between 2-AG and mt-Nd3. This suggests that enriching endogenous 2-AG by LM-PCRCPI may be a potential candidate substance for treating depressive-like behavior in mice by regulating mt-Nd3.

[0221] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this application. Furthermore, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

Claims

1. Application of tangerine peel polysaccharides in any of the following (1)-(13): (1) Use in the preparation of products for the prevention and / or treatment of depression; (2) Use in the preparation of products for the prevention and / or treatment of nerve damage; (3) Application in the preparation of products that reduce weight gain; (4) Application in the preparation of products that regulate intestinal flora; (5) Application in the preparation of products that increase the DA content in plasma; (6) Application in the preparation of products that increase the 5-HT content in plasma; (7) Application in the preparation of products that enhance DCX expression; (8) Application in the preparation of products that enhance Neun expression; (9) Application in the preparation of products that reduce Iba-1 expression; (10) Application in the preparation of products that inhibit HFD-induced microglial cell activation; (11) Application in the preparation of metabolites that enhance retrograde endocannabinoid signaling; (12) Application in the preparation of products that activate the oxidative phosphorylation signaling pathway; or (13) Application in the preparation of products that enhance the expression levels of genes related to the oxidative phosphorylation signaling pathway.

2. The application according to claim 1, characterized in that, The metabolites of the retrograde endocannabinoid signaling include 2-arachidonic acid glycerol and γ-aminobutyric acid; And / or, the genes associated with the oxidative phosphorylation signaling pathway include at least one of COX6c, mt-Nd3, NDUFs5, ATP5e, NDUFb8, SDHB, ATP5e, UQCR11, and UQCRh.

3. The application according to claim 1, characterized in that, The mass fraction of the tangerine peel polysaccharide in the product is 0.01% to 100%.

4. A tangerine peel polysaccharide, characterized in that, The monosaccharides in the tangerine peel polysaccharide contain rhamnose, arabinose, galactose, glucose, and galacturonic acid; the molar ratio of rhamnose, arabinose, galactose, glucose, and galacturonic acid is (1-3):(9-13):(4-6):(1-3):(75-85).

5. The tangerine peel polysaccharide according to claim 4, characterized in that, The average molecular weight of the tangerine peel polysaccharide is 48.85 kDa.

6. A method for preparing the tangerine peel polysaccharide according to claim 4 or 5, characterized in that, The preparation method includes the following steps: (1) Take a sample of dried tangerine peel, and after defatting, water extraction, alcohol precipitation, decolorization, protein removal with Sevag reagent, and dialysis, obtain crude polysaccharide of dried tangerine peel; (2) The crude polysaccharide of tangerine peel is eluted through a DEAE cellulose column to obtain tangerine peel polysaccharide; the elution reagents include NaCl solution with a concentration of 0.1M to 0.6M.

7. A composition, characterized in that, The composition contains the tangerine peel polysaccharide as described in claim 4 or 5.

8. The composition according to claim 7, characterized in that, The composition also includes lactobacillus; Preferably, the lactobacillus includes one of Lactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus murineis.

9. Use of the composition according to claim 7 or 8 in any one of the following (1)-(13): (1) Use in the preparation of products for the prevention and / or treatment of depression; (2) Use in the preparation of products for the prevention and / or treatment of nerve damage; (3) Application in the preparation of products that reduce weight gain; (4) Application in the preparation of products that regulate intestinal flora; (5) Application in the preparation of products that increase the DA content in plasma; (6) Application in the preparation of products that increase the 5-HT content in plasma; (7) Application in the preparation of products that enhance DCX expression; (8) Application in the preparation of products that enhance Neun expression; (9) Application in the preparation of products that reduce Iba-1 expression; (10) Application in the preparation of products that inhibit HFD-induced microglial cell activation; (11) Application in the preparation of metabolites that enhance retrograde endocannabinoid signaling; (12) Application in the preparation of products that activate the oxidative phosphorylation signaling pathway; or (13) Application in the preparation of products that enhance the expression levels of genes related to the oxidative phosphorylation signaling pathway.

10. The application according to claim 9, characterized in that, The products include pharmaceuticals, health products, or functional foods; Preferably, the drug further includes pharmaceutically acceptable excipients; More preferably, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, sweeteners, and flavorings; Preferably, the dosage form of the drug is tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, drop pills, dry extracts, injections or infusions, transdermal preparations, or transdermal microneedles. Preferably, the method of administration of the drug includes injection or oral administration.

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