Preparation and application of pericarpium citri reticulatae polysaccharide and composition thereof

Abstract

The invention discloses preparation and application of pericarpium citri reticulatae polysaccharide and a composition thereof. The invention provides application of citrus chachiensis hortorum crude polysaccharide and a characteristic component PCRCPI of the citrus chachiensis hortorum crude polysaccharide in relieving nerve injury, depression-like behaviors and metabolism-related mental diseases. According to the scheme, the citrus chachiensis hortorum crude polysaccharide or the characteristic component PCRCPI of the citrus chachiensis hortorum crude polysaccharide can effectively improve depression-like behaviors caused by HFD diet, recover the level of neurotransmitters DA and 5-HT, up-regulate neuronal cell active biomarkers DCX and Neun, protect HFD mice from nerve injury, improve the immunity of the mice, improve the immunity of the mice and improve the immunity of the mice. Effective candidates and technical supports are provided for preparing new drugs for relieving nerve injury, resisting depression and resisting spirit.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the preparation and application of tangerine peel polysaccharide and a composition thereof. Background Art

[0002] Depression is often comorbid with cardiovascular disease and metabolic disorders, and metabolic-related depression is considered a subtype of depression (metabolic depression, MD). A large amount of clinical and animal research evidence shows that excessive and long-term intake of a high-fat diet (HFD) can cause obesity and systemic insulin resistance, leading to cognitive and / or emotional dysfunction and a depressive phenotype.

[0003] Metabolic disorders and complications caused by HFD are often related to intestinal flora imbalance. In clinical trials and animal preclinical studies, targeted regulation of the composition and structure of intestinal microorganisms through diet, probiotics and fecal microbiota transplantation can effectively alleviate HFD-induced related clinical symptoms. Dietary fiber is considered to be a microbiota-directed drug development, which is attributed to the fact that the utilization of dietary fiber by intestinal microorganisms depends on its encoded genes and carbohydrate enzymes. Traditional Chinese medicine polysaccharides can promote the growth of different beneficial bacteria due to different sources, sugar units and molecular structures, and therefore have great application prospects in microbiota-directed drugs. Therefore, in-depth discovery of polysaccharide components that can specifically interact with specific flora, in-depth application in the prevention and treatment of MD, and development of a new and highly effective drug against metabolic syndrome and complications can provide new ideas and technical support for clinical MD intervention and new drug development. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides an application of tangerine peel polysaccharide.

[0005] The invention also provides tangerine peel polysaccharide.

[0006] The invention also provides a method for preparing the tangerine peel polysaccharide.

[0007] The invention also provides a composition containing the tangerine peel polysaccharide.

[0008] The present invention also provides the application of the composition.

[0009] According to the first aspect of the present invention, 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 preventing and / or treating depression;

[0011] (2) Use in the preparation of products for preventing and / or treating nerve damage;

[0012] (3) Use in the preparation of products for reducing weight gain;

[0013] (4) Application in the preparation of products for regulating intestinal flora;

[0014] (5) Application in the preparation of products for increasing DA content in plasma;

[0015] (6) Use in the preparation of products for increasing the 5-HT content in plasma;

[0016] (7) Application in the preparation of products for increasing DCX expression;

[0017] (8) Application in the preparation of products for improving Neun expression;

[0018] (9) Use in the preparation of products that reduce Iba-1 expression;

[0019] (10) Use in the preparation of products for inhibiting HFD-induced microglial activation;

[0020] (11) Use in the preparation of expression of metabolites that enhance retrograde endocannabinoid signaling;

[0021] (12) Application in the preparation of products for activating oxidative phosphorylation signaling pathways;

[0022] (13) Application in the preparation of products for increasing the expression level of genes related to the oxidative phosphorylation pathway.

[0023] In some embodiments of the present invention, the depression is depression associated with cognitive and / or affective dysfunction, neural damage and / or metabolism.

[0024] In some embodiments of the invention, the metabolites of retrograde endocannabinoid signaling include 2-arachidonoylglycerol (2-AG) and gamma-aminobutyric acid (GABA).

[0025] In some embodiments of the present invention, genes related to the oxidative phosphorylation pathway include at least one of COX6c, mt-Nd3, NDUFs5, ATP5e, NDUFb8, SDHB, ATP5e, UQCR11, and UQCRh.

[0026] In some embodiments of the present invention, the tangerine peel polysaccharide is prepared by the following method: taking a tangerine peel sample, defatting, water extraction, alcohol precipitation, decolorization, deproteinization with Sevag reagent, and dialysis to obtain tangerine peel polysaccharide.

[0027] In some embodiments of the present invention, the method further comprises the step of fractionating the tangerine peel polysaccharide, the step being as follows: eluting the tangerine peel polysaccharide through a DEAE cellulose column, wherein the reagent used for elution comprises a NaCl solution with a concentration of 0.1-0.6 M.

[0028] In some embodiments of the present invention, the reagents used for elution are 0.15-0.24 M NaCl solution, 0.25-0.35 M NaCl solution, and 0.45-0.55 M NaCl solution, respectively.

[0029] In some embodiments of the present invention, the reagents used for elution are 0.2M NaCl solution, 0.3M NaCl solution, and 0.5M NaCl solution, respectively.

[0030] In some embodiments of the present invention, the reagent used for elution also includes water.

[0031] In some embodiments of the present invention, the elution flow rate is 1-3 mL / min.

[0032] In some embodiments of the present invention, the flow rate of the elution is 1.5 mL / min.

[0033] In some embodiments of the present invention, the monosaccharides in the tangerine peel polysaccharide obtained by the fractionation 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).

[0034] In some embodiments of the present invention, the molar ratio of rhamnose, arabinose, galactose, glucose and galacturonic acid is 2.4:11.1:5.1:1.7:79.7.

[0035] In some embodiments of the present invention, the tangerine peel includes tangerine peel from tea branch tangerine (Guang tangerine peel), Wenzhou mandarin orange (Zhe tangerine peel), early tangerine (Zhe tangerine peel), lucky tangerine (Jian tangerine peel), Sichuan tangerine (Sichuan tangerine peel), Dahongpao (Sichuan tangerine peel), Nanfeng tangerine (Gan tangerine peel), etc.

[0036] In some embodiments of the present invention, the mass fraction of the tangerine peel polysaccharide in the product is 0.01% to 100%.

[0037] According to some preferred embodiments of the present invention, the mass fraction of the tangerine peel polysaccharide in the product is 0.05% to 95%.

[0038] According to some preferred embodiments of the present invention, the mass fraction of the tangerine peel polysaccharide in the product is 0.05% to 50%.

[0039] According to a second aspect of the present invention, a tangerine peel polysaccharide is proposed, wherein the monosaccharides in the tangerine peel polysaccharide contain 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).

[0040] In some embodiments of the present invention, the molar ratio of rhamnose, arabinose, galactose, glucose and galacturonic acid is 2.4:11.1:5.1:1.7:79.7.

[0041] In some embodiments of the present invention, the average molecular weight of the tangerine peel polysaccharide is 48.85 kDa.

[0042] According to a third aspect of the present invention, a method for preparing the above-mentioned tangerine peel polysaccharide is provided, and the preparation method comprises the following steps:

[0043] (1) taking a dried tangerine peel sample, and subjecting it to defatting, water extraction, alcohol precipitation, decolorization, Sevag reagent protein removal, and dialysis to obtain a dried tangerine peel crude polysaccharide;

[0044] (2) The crude tangerine peel polysaccharide is eluted through a DEAE cellulose column to obtain tangerine peel polysaccharide; the reagent used for the elution includes a NaCl solution with a concentration of 0.1-0.6M.

[0045] In some embodiments of the present invention, the reagents used for elution are 0.15-0.24 M NaCl solution, 0.25-0.35 M NaCl solution, and 0.45-0.55 M NaCl solution, respectively.

[0046] In some embodiments of the present invention, the reagents used for elution are 0.2M NaCl solution, 0.3M NaCl solution, and 0.5M NaCl solution, respectively.

[0047] In some embodiments of the present invention, the reagent used for elution also includes water.

[0048] In some embodiments of the present invention, the elution flow rate is 1-3 mL / min.

[0049] In some embodiments of the present invention, the flow rate of the elution is 1.5 mL / min.

[0050] According to a fourth aspect of the present invention, a composition is provided, comprising the above-mentioned tangerine peel polysaccharide.

[0051] In some embodiments of the invention, the composition further comprises Lactobacillus.

[0052] In some embodiments of the present invention, the lactobacillus comprises one of Lactobacillus reuteri, Lactobacillus johnsonii and Lactobacillus murinus.

[0053] According to a fifth aspect of the present invention, the use of the above composition in any one of the following (1)-(13) is proposed:

[0054] (1) Use in the preparation of products for preventing and / or treating depression;

[0055] (2) Use in the preparation of products for preventing and / or treating nerve damage;

[0056] (3) Use in the preparation of products for reducing weight gain;

[0057] (4) Application in the preparation of products for regulating intestinal flora;

[0058] (5) Application in the preparation of products for increasing DA content in plasma;

[0059] (6) Use in the preparation of products for increasing the 5-HT content in plasma;

[0060] (7) Application in the preparation of products for increasing DCX expression;

[0061] (8) Application in the preparation of products for improving Neun expression;

[0062] (9) Use in the preparation of products that reduce Iba-1 expression;

[0063] (10) Use in the preparation of products for inhibiting HFD-induced microglial activation;

[0064] (11) Use in the preparation of expression of metabolites that enhance retrograde endocannabinoid signaling;

[0065] (12) Application in the preparation of products for activating oxidative phosphorylation signaling pathways;

[0066] (13) Application in the preparation of products for increasing the expression level of genes related to the oxidative phosphorylation pathway.

[0067] In some embodiments of the present invention, the depression is depression associated with cognitive and / or affective dysfunction, neural damage and / or metabolism.

[0068] In some embodiments of the invention, the metabolites of retrograde endocannabinoid signaling include 2-arachidonoylglycerol (2-AG) and gamma-aminobutyric acid (GABA).

[0069] In some embodiments of the present invention, genes related to the oxidative phosphorylation pathway include at least one of COX6c, mt-Nd3, NDUFs5, ATP5e, NDUFb8, SDHB, ATP5e, UQCR11, and UQCRh.

[0070] In some embodiments of the present invention, the dosage of tangerine peel polysaccharide in the composition is 40-100 mg / kg / d.

[0071] In some embodiments of the present invention, the dosage of tangerine peel polysaccharide in the composition is 60 mg / kg / d.

[0072] In some embodiments of the present invention, the amount of Lactobacillus in the composition is 3×10 9 -7×10 9 CFU / d.

[0073] In some embodiments of the present invention, the amount of Lactobacillus in the composition is 5×10 9 CFU / d.

[0074] In some embodiments of the present invention, the product includes a medicine, a health product or a functional food.

[0075] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0076] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener and a flavoring agent.

[0077] In some embodiments of the invention, the excipient comprises water.

[0078] In some embodiments of the present invention, the filler comprises at least one of starch and sucrose.

[0079] In some embodiments of the present invention, the binder includes at least one of a cellulose derivative, alginate, gelatin and polyvinyl pyrrolidone.

[0080] In some embodiments of the invention, the humectant comprises glycerin.

[0081] In some embodiments of the present invention, the disintegrant comprises at least one of agar, calcium carbonate and sodium bicarbonate.

[0082] In some embodiments of the invention, the absorption enhancer comprises a quaternary ammonium compound.

[0083] In some embodiments of the invention, the surfactant comprises cetyl alcohol.

[0084] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

[0085] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.

[0086] In some embodiments of the present invention, the dosage form of the drug is in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension.

[0087] In some embodiments of the present invention, the formulation is in the form of tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, pellets, dry extracts, injections or infusions, transdermal agents, or transdermal microneedles.

[0088] In some embodiments of the present invention, the drug may be administered by any conventional method in the art, including but not limited to injection or oral administration.

[0089] In some embodiments of the present invention, the injection administration can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

[0090] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention provides an application of crude polysaccharide of dried orange peel and its characteristic component PCRCPI in alleviating neural injury and depressive behavior; the crude polysaccharide of dried orange peel or its characteristic component PCRCPI of the present invention can effectively improve the depressive-like behavior caused by HFD diet, restore the reduction of DA and 5-HT caused by long-term HFD, increase the RNA expression of DCX and Neun in neuronal cells and inhibit the activation of microglia, protect HFD mice from neural injury, and provide new possibilities for preparing new drugs for alleviating neural injury and depressive behavior.

[0091] At the same time, the present invention also combines PCRCPI with lactobacillus, which can further improve the depressive-like behavior caused by the HFD diet, increase the DA and 5-HT contents of HFD mice, increase the relative expression of DCX and Neun, and reduce the expression of Iba-I, which can be effectively used to alleviate nerve damage and depressive behavior and prepare drugs for alleviating nerve damage and depressive behavior. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0093] Figure 1The figure is a result diagram of the preparation and characterization of PCRCPI in the test example of the present invention, wherein A is a flowchart of the extraction and purification of PCRCPI; B is an elution curve diagram of DEAE-52; C is an elution curve diagram of Sephadex G-100;

[0094] Figure 2 It is the ultraviolet spectrum of PCRCPI in the test example of the present invention;

[0095] Figure 3 The high performance gel permeation chromatography (HPGPC) chromatogram of PCRCPI in the test example of the present invention is used to show its molecular weight distribution;

[0096] Figure 4 FT-IR spectrum in the test example of the present invention;

[0097] Figure 5 This is a graph showing the monosaccharide composition detection result of PCRCPI identified by gas chromatography-mass spectrometry (GC-MS) in the test example of the present invention, wherein the black line shows the graph of the standard monosaccharide and the red line shows the graph of PCRCPI;

[0098] Figure 6 lgMw-RT standard curve diagram of the molecular weight measurement of PCRCPI in the test example of the present invention;

[0099] Figure 7 It is a test result diagram of the characteristic components of tangerine peel polysaccharide (PCRCP) in the test example of the present invention that differentially improve the depressive-like behavior of mice induced by HFD, wherein A is a schematic diagram of the animal experiment process; B is a graph of mouse weight change; C is a graph of TST test results; D is a graph of FST test results; E is a graph of SPT test results; F is a graph of DA content in plasma, and G is a graph of 5-HT content in plasma; H is a graph of the relative expression of DCX in mouse hippocampal tissue; I is a graph of the relative expression of Neun in mouse hippocampal tissue; J is a graph of the relative expression of Iba-1 in mouse hippocampal tissue and Iba-1; 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;

[0100] Figure 8 The results of the test in the present invention show that PCRCPI treatment significantly reshaped the fecal metabolite profile of mice with depressive-like behavior; A is a principal component analysis (PCA) diagram; B is a volcano diagram of differential metabolites, screening criteria: P<0.05, FC≥2; C is a heat map of differential metabolite expression;

[0101] Fig. 9The PCRCPI treatment in the test example of the present invention significantly reshaped the fecal metabolite profile of mice with depressive-like behavior, and analyzed the pathway enrichment map of differential metabolites based on KEGG terms;

[0102] Fig.10 is a heat map of the expression of metabolites in the endocannabinoid pathway in the test example of the present invention;

[0103] Fig.11 The results of the test show that PCRCPI treatment significantly reshaped the fecal metabolite profile of mice with depressive-like behavior in the test example of the present invention, wherein A is the relative abundance of 2-AG in the PCRCPI group and the HFD group, B is the relative abundance of GABA in the PCRCPI group and the HFD group, and "****" indicates P<0.0001;

[0104] Fig.12 The results of the test show that PCRCPI treatment significantly reshaped the fecal metabolite profile of mice with depressive-like behavior in the test example of the present invention, wherein A is a correlation analysis result diagram of the relative abundance of 2-AG and the SPT behavioral index; B is a correlation analysis result diagram of the relative abundance of 2-AG and the FST behavioral index; C is a correlation analysis result diagram of the relative abundance of 2-AG and the TST behavioral index;

[0105] Fig.13 It is a test result diagram of the effect of lactic acid bacteria in the test example of the present invention on improving the depressive-like behavior induced by a high-fat diet in mice treated with PCRCPI, wherein A is a schematic diagram of the animal experiment process; B is a graph of changes in mouse weight; C is a graph of FST test results; D is a graph of TST test results; E is a representative graph of HE and Nissl staining of the dentate gyrus (DG) region of the hippocampus in the FMT group and the SFF group; F is a schematic diagram of the fecal microbiota transplantation animal experiment; G is a graph of changes in mouse weight; H is a graph of SPT test results; I is a graph of TST test results; J is a graph of FST test results; K is a representative graph of HE and Nissl staining of 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;

[0106] Fig.14It is a test result diagram of the lactic acid bacteria differentially enhancing the antidepressant effect of PCRCPI in the test example of the present invention; wherein, A is a schematic diagram of the animal experiment process; B is a graph of mouse weight change; C is a graph of SPT test results; D is a graph of TST test results; E is a graph of FST test 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 DA content in plasma, and H is a graph of 5-HT content in plasma; I is a graph of the relative expression of DCX in mouse hippocampal tissue; G is a graph of the relative expression of Neun in mouse hippocampal tissue; K is a graph of the relative expression of Iba-1 in mouse hippocampal tissue and Iba-1; "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001;

[0107] Fig.15 The results of the gene expression profile analysis of hippocampal tissue by RNA sequencing in the test example of the present invention are shown in Figure 1, wherein A is a principal component analysis (PCA) diagram; B is a volcano diagram of differential metabolites, and the screening criteria are: P<0.05, FC≥1.5;

[0108] Fig.16 It is a KOG function enrichment map of the gene expression profile of hippocampal tissue analyzed by RNA sequencing in the test example of the present invention;

[0109] Fig.17 : It is a test result diagram of the gene expression profile of hippocampal tissue analyzed by RNA sequencing in the test example of the present invention, wherein A is a pathway enrichment diagram of differentially expressed genes based on KEGG analysis; B is a heat map of the expression of genes related to the oxidative phosphorylation pathway;

[0110] Fig.18 : It is a test result diagram of the gene expression profile of hippocampal tissue analyzed by RNA sequencing in the test example of the present invention, wherein A is a GSEA enrichment analysis diagram of genes related to the retrograde endocannabinoid pathway, and B is a GSEA enrichment analysis diagram of genes related to the oxidative phosphorylation pathway;

[0111] Fig.19 : It is a test result diagram of the gene expression profile of hippocampal tissue analyzed by RNA sequencing in the test example of the present invention, wherein A is a Spearman correlation analysis result diagram between oxidative phosphorylation pathway genes and depression-related indicators; B is a test result diagram of the relative mRNA expression level of genes related to the oxidative phosphorylation pathway; C is a molecular docking result diagram of 2-AG and mt-ND3;

[0112] Fig. 20 This is a KEGG function enrichment analysis result diagram of the gene expression profile analysis of hippocampal tissue by RNA sequencing in the test example of the present invention;

[0113] Fig.21 This is a heat map analysis diagram of the enrichment of the retrograde endocannabinoid signaling pathway in the test example of the present invention;

[0114] Fig. 22 This is a graph showing the expression level and difference multiple detection results of mt-ND3 in the oxidative phosphorylation signaling pathway in the test example of the present invention. DETAILED DESCRIPTION

[0115] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0116] Preparation method of tangerine peel crude polysaccharide (PCRCP):

[0117] (1) Flour making:

[0118] Five-year-old dried Guang tangerine peel (the origin of the Guang tangerine peel samples was Xinhui District, Jiangmen City) was selected, dried, crushed into powder by a grinder, collected after passing through a 40-mesh sieve, and stored in a dry environment at room temperature.

[0119] (2) Degreasing:

[0120] The dried tangerine peel powder obtained in step (1) was added with anhydrous ethanol at a solid-liquid ratio of 1 g:10 mL for degreasing treatment, and the treatment conditions were set to be heated under reflux at 80° C. for 1.5 h, and repeated twice. After degreasing, the filter residue was collected and dried for later use.

[0121] (3) Water extraction:

[0122] Take the filter residue obtained in step (2) and add pure water at a solid-liquid ratio of 1g:10mL. The extraction conditions are set to 100°C and reflux in a water bath for 6 hours. The extract is filtered using a Buchner funnel and 0.45μm filter paper. The filtrate is then centrifuged at 8000r / min for 30min and the supernatant is collected.

[0123] (4) Alcohol precipitation:

[0124] The supernatant obtained in step (3) is mixed with 4 times the volume of 95wt% ethanol, and after alcohol precipitation in a refrigerator at 4°C overnight, the mixture is filtered and the precipitate is retained. The precipitate is dried to obtain tangerine peel crude polysaccharide powder.

[0125] (5) Decolorization:

[0126] The polysaccharide powder obtained in step (4) was washed with ether and acetone respectively, the precipitate was filtered and 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, stirred for 2 hours, and then placed in a refrigerator at 4°C for overnight decolorization. The mixed solution was centrifuged at 8000r / min for 30 minutes, and the decolorized solution was collected.

[0127] (6) Protein removal:

[0128] The supernatant prepared in step (5) is subjected to protein removal treatment. Chloroform and n-butanol solution are mixed in a volume ratio of 4:1 to prepare Sevag reagent. The decolorizing solution and Sevag reagent are mixed in a volume ratio of 5:1 in a separatory funnel, and fully shaken for 10-15 minutes. After standing and stratification, the denatured protein precipitate in the lower layer is discarded, and the operation is repeated until no denatured protein is formed. The upper layer of crude polysaccharide solution is collected.

[0129] (7) Dialysis and freeze-drying:

[0130] The deproteinized crude polysaccharide solution in step (6) is dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 8000-14000Da, and the dialysate is collected and freeze-dried using a freeze dryer to obtain tangerine peel crude polysaccharide.

[0131] All animal experiments were approved by the Animal Experiment Ethics Committee of the International Institute for Healthcare Innovation (Jiangmen, China). All animal-related experimental protocols were performed in accordance with the Guide for the Care and Use of Laboratory Animals of our institution.

[0132] Male C57BL / 6J mice, SPF grade, 8 weeks old, weighing 20 kg / m, were purchased from Zhuhai Baishitong Biotechnology Co., Ltd. They were housed in an SFP grade animal room with standard facilities, a light / dark cycle of 12 h, and adapted for 1 week.

[0133] Example 1: Tangerine peel polysaccharide for alleviating nerve damage and depressive-like behavior

[0134] This embodiment provides a tangerine peel polysaccharide for alleviating nerve damage and depressive-like behavior. The preparation flow chart is as follows: Figure 1As shown in Figure A, the specific method is as follows: the crude polysaccharide (2 g) is dissolved in water (30 mL) and centrifuged at 12000 rpm for 10 minutes to remove the insoluble components. The sample is eluted using a DEAE-52 cellulose column (4 cm (52)), and water and different concentrations of NaCl solutions (0.2, 0.3, 0.5 M) are used in turn to elute at a flow rate of 1.5 mL / min. Specifically, the crude polysaccharide solution is washed with a 0.2 M NaCl solution to obtain a characteristic component PCRCP-I (0.2 M), and then the remaining polysaccharide is washed with a 0.3 M NaCl solution to obtain a characteristic component PCRCP-II (0.3 M), and then the remaining polysaccharide is washed with a 0.5 M NaCl solution to obtain a characteristic component PCRCP-III (0.5 M). The elution curve is determined by the phenol-sulfuric acid colorimetric method (as shown in Figure 1). Figure 1 (as shown in Figure B).

[0135] The PCRCP-I component was purified using a Sephadex G-100 column (1.6 adex G-) with ultrapure water as the eluent at a flow rate of 4.0 mL / 12 min. The elution curve was determined by the phenol-sulfuric acid method (e.g. Figure 1 (as shown in Figure C in the figure) and the main peak was collected to obtain the purified tangerine peel polysaccharide PCRCP-I component.

[0136] Example 2 A composition for alleviating nerve damage and depressive-like behavior

[0137] This embodiment provides a composition for alleviating nerve damage and depressive-like behavior, wherein the composition contains the PCRCP-I component prepared in Example 1 and Lactobacillus reuteri LR-G100, wherein the concentration of Lactobacillus reuteri LR-G100 can be 5×10 9 CFU / mL.

[0138] Example 3 A composition for alleviating nerve damage and depressive-like behavior

[0139] This embodiment provides a composition for alleviating nerve damage and depressive-like behavior, wherein the composition contains the PCRCP-I component prepared in Example 1 and Lactobacillus johnsonii LJ-G55, and the concentration of Lactobacillus johnsonii LJ-G55 can be 5×10 9 CFU / mL.

[0140] Example 4 A composition for alleviating nerve damage and depressive-like behavior

[0141] This embodiment provides a composition for alleviating nerve damage and depressive-like behavior, wherein the composition contains the PCRCP-I component prepared in Example 1 and Lactobacillus murinus, and the concentration of Lactobacillus murinus can be 5×10 9 CFU / mL.

[0142] Test Case

[0143] 1. Isolation, purification and structural characterization of PCR-CPI

[0144] (1) UV-Vis spectroscopy analysis

[0145] 2 mg of the purified PCRCPI obtained in Example 1 was dissolved in 4 mL of deionized water to prepare a polysaccharide solution with a concentration of 0.5 mg / mL. At room temperature, a UV-visible spectrophotometer (model: Shimadzu UV-2600) was used to scan in the wavelength range of 190-400 nm.

[0146] The purified PCR CPI was analyzed by UV-visible spectroscopy. Figure 2 As shown in the figure, it can be seen that no obvious absorption peaks were found in PCRCPI at wavelengths of 260 nm and 280 nm, indicating that the purified PCRCPI contained negligible nucleic acids and proteins.

[0147] (2) High performance gel permeation chromatography (HPGPC) analysis

[0148] High performance gel permeation chromatography (HPGPC) was used to determine the purity and molecular weight of PCR CPI.

[0149] The purity and molecular weight of the PCRCPI purified in Example 1 were determined on an LC-10AT instrument (Shimadzu, Japan) equipped with a RI-20A refractive index detector and a BRT 105-103101 (8×300 mm) gel filtration column. The HPGPC test conditions were as follows: a 0.05 M NaCl solution with a pH of 1.55 was used as the mobile phase, the flow rate was 0.8 mL / min, and the column temperature was 40°C.

[0150] Sample treatment: 5 mg of the purified PCRCPI sample obtained in Example 1 was accurately weighed and dissolved in 1 mL of NaCl solution to prepare a 5 mg / mL solution. After ultrasonic treatment for 10 min, the solution was centrifuged at 12000 rpm for 10 min to collect the supernatant, passed through a 0.22 μm hydrophilic microporous membrane, and analyzed according to the established HPGPC test conditions.

[0151] Test results such as Figure 3As shown, a single symmetrical peak was shown, confirming the high purity of the sample. This result indicates that the results obtained from Sephadex G-100 gel chromatography and UV analysis are consistent ( Figure 2 and 3 Compared with the molecular weight standard curve ( Figure 6 ) were compared, the average molecular weight of PCRCPI was 44.15 kDa.

[0152] (3) Fourier transform infrared spectroscopy (FT-IR) analysis of PCRCPI

[0153] 2 mg of the purified PCRCPI obtained in Example 1 was accurately weighed and mixed with 200 mg of KBr powder (spectrally pure). The mixture was then pressed into pellets using a tablet press. The Fourier transform infrared spectroscopy was performed at 450-4000 cm 2 The infrared spectrum of PCRCPI was obtained by scanning in the range.

[0154] The FT-IR spectrum of PCRCPI is shown in Figure 4 As shown in the figure, at 3399cm -1 The broad and strong characteristic peak at 2933cm -1 and 1444cm -1 The two additional absorption peaks at 1242 cm -1 The absorption peak at 1623 cm further confirms the existence of CH bond. -1 and 1749cm -1 The obvious peaks observed at 1200-1000cm-1 are attributed to the stretching bands of carboxylate ions (COO-) and methylated carbonyl groups (COO-CH3), confirming the presence of uronic acid in the sample. -1 The absorption bands in the range represent the stretching vibration of CO bonds, including COH and COC bonds, indicating the presence of COC or COH bonds. -1 The absorption peak at corresponds to the out-of-plane vibration of OH.

[0155] (4) Analysis of monosaccharide composition of PCRCPI

[0156] The monosaccharide composition of the PCRCPI purified in Example 1 was analyzed by ion chromatography (IC), and the specific steps were as follows:

[0157] 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 hydrolyzate was evaporated under a stream of nitrogen and then resuspended in 5 mL of ultrapure water. A 50 μL aliquot of the solution was diluted with 950 μL of deionized water and centrifuged at 12000 rpm for 5 min. The resulting supernatant (along with the monosaccharide solution mixture) was analyzed using ion chromatography equipped with an electrochemical detector. The Dionex Carbopac TM Monosaccharides were separated on a PA 20 column (3×150 mm) using a mobile phase consisting of A: H2O; mLB: 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.

[0158] The results are as follows Figure 5 As shown in the figure, it can be seen that the homogeneous polysaccharide is composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc) and galacturonic acid (GalA), and the molar percentages (mol%) are 2.4%: 11.1%: 5.1%: 1.7%: 79.7%, respectively.

[0159] 2. Characteristic components of tangerine peel polysaccharide (PCRCP) differentially improve HFD-induced depressive-like behavior in mice

[0160] This test example verifies the effects of different PCRCP components on improving HFD-induced depressive-like behavior in mice. The specific verification scheme is shown below.

[0161] (1) Establishment of a HFD-induced depression model in mice using different components of PCRCP

[0162] SD mice were randomly divided into 6 groups, 8 in each group, and the SD mice were given normal diet or high-fat diet and intragastric administration of 60 mg / kg / d PCRCP, PCRCPI, PCRCPII and PCRCPIII for 18 weeks. Figure 7 As shown in Figure A, the details are as follows:

[0163] Chow: water / normal feed (10% fat, 3.5 kcal / g);

[0164] HFD: water / high-fat diet (45% fat, 16.8 kcal / g);

[0165] PCRCP: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / Kg / d PCRCP;

[0166] PCRCPI: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / Kg / d PCRCPI;

[0167] PCRCPII: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / Kg / d PCRCPII;

[0168] PCRCPIII: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / Kg / d PCRCPIII.

[0169] (2) Index evaluation experiment

[0170] 1) Anatomical sampling

[0171] The body weight and food intake of mice in different groups were recorded weekly. After the experiment, fresh feces, plasma, hippocampus, and brain of mice were collected and quickly frozen in liquid nitrogen or fixed in paraformaldehyde for subsequent analysis.

[0172] 2) Behavioral testing

[0173] Sugar water preference test (SPT): Each mouse in each group was housed individually in a cage equipped with two drinking bottles, one containing 1% sucrose solution and the other containing control water. To avoid bias, the positions of the two bottles were exchanged every 12 hours. After habituation, mice were restricted from drinking water 6 hours before behavioral testing. The weights of the drinking water bottles and 1% sucrose water bottles 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%.

[0174] Forced swimming test (FST): The test mice were placed in a transparent plexiglass cylinder (30 cm high, 20 cm diameter, 20 cm water depth) filled with water (water temperature: 25±1°C) for 6 minutes, and the duration of static floating in the last 4 minutes was recorded.

[0175] Tail suspension test (TST): The mice were individually hung upside down on a horizontal bar with tape for 6 minutes, and the time during which the limbs were completely or almost motionless was recorded for the next 4 minutes.

[0176] 3) Detection of plasma dopamine (DA) and serotonin (5-HT).

[0177] Blood was collected from mice in EP tubes, and after standing for 2 hours, the blood was centrifuged at 3000 g for 15 minutes at 4°C. The supernatant was collected and frozen at -80°C. The concentrations of DA and 5-HT in mouse plasma were determined using ELISA kits according to the manufacturer's instructions.

[0178] 4) HE staining

[0179] ① Dewaxing of paraffin sections: Dehydrate the samples and embed them in paraffin, with a section thickness of 5 μm. Soak them in xylene I-II for 10 min, then soak them in anhydrous ethanol I-II for 5 min, then soak the samples in 90% ethanol, 70% ethanol and pure water for 2 min in turn for dewaxing and set aside.

[0180] ②HE staining: stain with hematoxylin staining solution for 5 minutes, wash with water for 3-5 seconds, then stain with eosin staining solution for 30 seconds, immediately rinse with color enhancement solution 2-3 times, dry naturally, and seal with neutral gum. Use Olympus BX41 microscope for photography.

[0181] 5) Nissl dyeing

[0182] ① Dewaxing of paraffin sections: Dehydrate the samples and embed them in paraffin, with a section thickness of 5 μm. Soak them in xylene I-II for 10 min, then soak them in anhydrous ethanol I-II for 5 min, then soak the samples in 90% ethanol, 70% ethanol and pure water for 2 min in turn for dewaxing and set aside.

[0183] ②Nissl staining: Add Nissl staining solution to the tissue section, stain for about 5 minutes, rinse with 95% ethanol for 5 seconds, wash twice with 70% ethanol, dehydrate with 95% ethanol for 2 minutes, transparentize with xylene for 5 minutes, seal with neutral gum, and observe the staining results under an upright fluorescence microscope.

[0184] 6) Real-time fluorescence quantitative PCR (q-PCR) analysis

[0185] ① Hippocampal RNA extraction (Trizol extraction method)

[0186] Place the mouse hippocampal tissue in a 1.5 mL centrifuge tube, add 0.3 ml Trizol, and use a tissue grinder to fully homogenize. Then add 1 mL Trizol to make the total volume reach 1.3 mL, let it stand on ice for about 5 minutes, and then centrifuge at 4°C and 12,000 rpm for 15 minutes to separate the tissue lysate. Transfer about 1 mL of the supernatant after centrifugation to a new centrifuge tube, add 0.2 mL chloroform at a volume ratio of 5:2, shake vigorously for 30 seconds, let it stand on ice for 2 minutes, and centrifuge again at 4°C and 12,000 rpm for 15 minutes.

[0187] Transfer the supernatant (about 0.5 mL) after centrifugation into a centrifuge tube containing an equal volume of isopropanol, mix gently, and then place it at -20°C for 8 hours to allow the RNA to fully precipitate. After standing, 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, shake it gently by hand to ensure that the precipitate is fully rinsed, and then centrifuge it again at 4°C and 12,000 rpm for 5 minutes.

[0188] After discarding the ethanol, air dry the precipitate for about 5 minutes, being careful to avoid over-drying which may cause difficulty in dissolving. Finally, dissolve the RNA precipitate with an appropriate amount of DEPC water, and use the Agilent 2100 Bioanalyzer to detect the quality and purity of the RNA to ensure that the experimental product is suitable for subsequent analysis.

[0189] ②Reverse transcription reaction

[0190] 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.

[0191] Table 1

[0192] Components volume RT 1μL RI 1μL Primer 1μL dNTP 2μL Buffer 4μL RNA 2μL Enzyme free water Make up to 20 μL

[0193] Table 2 PCR program

[0194] program temperature time Stage 1 25℃ 5min Stage2 42℃ 1h Stage3 70℃ 5min Stage4 16℃ ∞

[0195] ③ Real-time fluorescence quantitative PCR

[0196] The cDNA obtained by reverse transcription was used to Premix Ex Taq TM (Tli RNaseHPlus) 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.

[0197] Table 3

[0198] Components volume Green I 5μL Forward 0.2μL Reverse 0.2μL Rox 0.2μL cDNA 1μL Enzyme free water Make up to 10 μL

[0199] Table 4

[0200]

[0201]

[0202] Table 5 PCR amplification primer sequences

[0203]

[0204] Test results such as Figure 7 As shown, the present application protocol constructed a HFD-induced depression model.

[0205] from Figure 7 As can be seen in Figure B, HFD feeding significantly increased the body weight of mice compared with conventionally fed mice.

[0206] from Figure 7 As can be seen from the CE figure, HFD feeding induced an increase in the immobility time and a decrease in sucrose preference in FST and TST of mice, which was significantly reversed after treatment with PCRCP and PCRCPI. PCRCPII and PCRCPIII could reduce the immobility time in FST to a certain extent, but induced an increase in the immobility time in TST, while PCRCPIII's preference for sugar water decreased, indicating that PCRCP components alleviated HFD-induced depressive-like behavior in mice in a structure-dependent manner.

[0207] from Figure 7 As can be seen in Figure FG, the levels of DA and 5-HT in the plasma of HFD mice were reduced. When treated with PCRCPI, the changes in these neurotransmitters were reversed, especially DA, which reached a level comparable to that of normal mice.

[0208] from Figure 7 As can be seen in the HI graph in Figure , PCRCPI treatment for 18 weeks significantly restored the mRNA expression of DCX (a marker for immature neurons) and Neun (a marker for mature neurons) in neuronal cells in the hippocampus of HFD-fed mice compared to untreated mice.

[0209] from Figure 7 As can be seen in Figure J, PCRCPI significantly increased the mRNA expression level of Iba-1, a classic marker of microglia, which means that PCRCPI can inhibit HFD-induced microglial activation.

[0210] from Figure 7 As can be seen in Figure K, HE staining and Nissl staining were used to observe the changes in hippocampal neurons in each group of mice. The pyramidal cells in the hippocampal DG region of the HFD group mice not only showed obvious neuronal cell loss, neuronal atrophy, and reduced Nissl bodies, unclear stratification, and irregular arrangement. PCRCPI supplementation effectively protected hippocampal neurons from HFD-induced damage. The results showed that HFD can induce depressive-like behavior and cause neural damage in mice, while PCRCPI can improve depressive-like behavior and reverse neural damage.

[0211] The above experiments show that PCRCPI may be the key component of tangerine peel polysaccharide in exerting antidepressant effect. PCRCPI can more effectively alleviate the depressive-like behavior of HFD mice, which is specifically manifested as an increase in the sugar water preference index in the SPT experiment and less immobility time in the TST and FST experiments. PCRCRI can also restore the reduction of DA and 5-HT caused by long-term HFD, increase the RNA expression of DCX and Neun in neuronal cells, and inhibit the activation of microglia. At the same time, HE staining and Nissl staining showed that PCRCPI can effectively protect HFD mice from nerve damage.

[0212] 3. Metabolomics analysis of feces

[0213] In order to study whether the protective effect of PCRCPI on HFD-induced depressive mice is related to changes in metabolomics, this test case used 2. Characteristic components of tangerine peel polysaccharides (PCRCP) differentially improve the depressive-like behavior of HFD-induced mice. The feces of the PCRCPI-treated group of mice intervened for 18 weeks, and non-targeted metabolomics analysis was performed using GC-TOF / MS. The specific analysis steps are as follows:

[0214] 50 mg of feces was homogenized with 600 μL ultrapure water and centrifuged at 13000 rpm for 10 min at 4 °C. The residue was extracted with 600 μL of ice-cold methanol. 80 μL of supernatant was taken and precipitated with 80 μL of methanol containing chloro-d-phenylalanine (100 ng / mL) and ketoprofen (10 ng / mL), followed by centrifugation at 13000 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), which was 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 differential metabolites was performed using the KEGG database.

[0215] The results are as follows Figure 8-12 As shown, from Figure 8 As can be seen in Figures A–C, metabolomics revealed that PCRCPI could significantly change the fecal metabolite content of HFD mice.

[0216] from Fig. 9As can be seen in the figure, KEGG enrichment analysis showed that the differential metabolites in the feces of PCRCPI-treated mice were mainly enriched in the retrograde endocannabinoid signaling pathway, synaptic vesicle cycle, and GABAergic synapse. Changes in these metabolic signaling pathways are often associated with depression in rodents.

[0217] from Fig.10 and Fig.11 As can be seen, PCRCPI significantly upregulated the expression of beneficial metabolites of retrograde endocannabinoid signaling, including 2-arachidonylglycerol (2-AG) and γ-aminobutyric acid (GABA), compared with HFD mice.

[0218] from Fig.12 As can be seen in Figure 2, the correlation between key indicators of depressive-like behavior and differential metabolite changes was investigated by Spearman correlation analysis. The concentration of 2-AG in feces was positively correlated with the sucrose preference index in the SPT and negatively correlated with the immobility time in the TST and FST, indicating that the PCRCPI-enriched metabolite 2-AG may be an active metabolite that helps improve the depressive phenotype.

[0219] Taken together, these results suggest that PCRCPI can significantly modulate gut microbial metabolites in HFD-fed mice with depressive-like behaviors.

[0220] 4. Improvement of intestinal flora and lactic acid bacteria on HFD-induced depressive-like behavior

[0221] To further investigate whether the antidepressant effect of PCRCPI is mediated by the intestinal microbiota, feces from the PCRCPI-treated mice for 18 weeks were transferred to HFD-fed mice with or without microbiota for 15 weeks (animal experimental procedures as shown in Fig.13 (as shown in Figure A).

[0222] The experimental plan is consistent with 2. The characteristic components of tangerine peel polysaccharides (PCRCP) differentially improve HFD-induced depressive-like behavior in mice.

[0223] The experimental results are as follows Fig.13 As shown, from Fig.13 As can be seen in the BE figure, fecal microbiota from PCRCPI-treated mice can ameliorate the development of HFD-induced depression.

[0224] from Fig.13As can be seen in the FJ figure, since PCRCP can significantly enrich Lactobacillus in HFD-induced obese mice, different live Lactobacillus strains, including Lactobacillus reuteri LR-G100 (LR), Lactobacillus johnsonii LJ-G55 (LJ), and Lactobacillus murinus (LM), were colonized into 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.

[0225] from Fig.13 As can be seen in Figure K, histological examinations using HE and Nissl staining showed that supplementation with Lactobacillus strains significantly reduced neuronal damage induced by HFD.

[0226] Taken together, these evidences clearly suggest that the alleviation of depressive-like behaviors in HFD-induced mice by PCRCPI may be associated with the normalization of the gut microbiota composition, especially the restoration of Lactobacillus.

[0227] 5. Lactic acid bacteria differentially enhance the antidepressant effect of PCRCPI

[0228] To further investigate how the interaction between PCRCPI and different Lactobacillus strains affects depressive-like behavior, HFD mice were colonized with 5 × 10 9 CFU / d of LR, LJ and LM live bacteria and 60 mg / kg / day of PCRCPI intervention for 10 weeks (method flow as Fig.14 The study was conducted as shown in Figure A.

[0229] Construction of animal model:

[0230] Male C57BL / 6J mice, SPF grade, 7-8 weeks old, 20±2g; after 1 week of normal feeding in the animal room, the animals were randomly divided into 9 groups, 11 mice in each group; all animals were given ordinary feed (Chow, 13% fat energy supply) or HFD (45% fat energy supply).

[0231] Chow: water / normal feed (13% fat, 3.5 kcal / g);

[0232] HFD: water / high-fat diet (45% fat, 16.8 kcal / g);

[0233] PCRCPI: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / kg / d PCRCPI;

[0234] LR: water / high-fat diet (45% fat, 16.8 kcal / g) / 5×10 9 CFU / d LR;

[0235] LJ: water / high-fat diet (45% fat, 16.8 kcal / g) / 5×10 9 CFU / d LJ;

[0236] LM: water / high-fat diet (45% fat, 16.8 kcal / g) / 5×10 9 CFU / d LM;

[0237] LR-PCRCPI: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / kg / d PCRCPI / 5×10 9 CFU / d LR;

[0238] LJ-PCRCPI: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / kg / d PCRCPI / 5×10 9 CFU / d LJ;

[0239] LM-PCRCPI: water / high-fat diet (45% fat, 16.8 kcal / g) / 60 mg / kg / d PCRCPI / 5×10 9 CFU / d LM.

[0240] The experimental plan is consistent with 2. The characteristic components of tangerine peel polysaccharides (PCRCP) differentially improve HFD-induced depressive-like behavior in mice.

[0241] The results are as follows Fig.14 As shown, from Fig.14 As can be seen in Figures B–E, the combination of Lactobacillus and PCRCPI led to a significant reduction in weight gain and a significant improvement in depressive-like behavior in mice, including a significant reduction in the immobility time in TST and FST and a significant increase in sucrose preference.

[0242] from Fig.14 As can be seen in Figure F, histological analysis by 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.

[0243] from Fig.14 As can be seen in Figures G and H, LM combined with PCRCPI treatment also significantly restored the decrease in DA and 5-HT levels caused by HFD.

[0244] At the same time, from Fig.14As can be seen in the IK figure, 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 the depressive-like behavior induced by HFD feeding and relieved neural damage.

[0245] Therefore, the specific interaction between Lactobacillus and PCRCPI plays an important role in reducing neuronal damage and alleviating depressive-like behaviors in HFD-fed mice.

[0246] 6. LM combined with PCRCPI changes the signal transduction of hippocampal genes

[0247] To further confirm the antidepressant effect and elucidate the potential mechanism by which the combination of LM and PCRCPI improves the depressive-like behavior of HFD-fed mice, we performed unbiased RNA sequencing (RNA-seq) to analyze gene changes in the hippocampus of HFD mice and LM-PCRCPI-treated mice after 10 weeks of intervention.

[0248] Transcriptomic sequencing (RNA_seq) and analysis were performed as follows: Total RNA was extracted from mouse hippocampal tissue using TRIZOL reagent (Invitrogen, Grand Island, NY, USA). The concentration and purity of RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), while the quantity of RNA was assessed using an Agilent Bioanalyzer 2100 system (Agilent Technologies, San Diego, CA, USA). The library was sequenced on an Illumina NovaSeq platform (Beijing Biomarker Technology Co., Ltd., Beijing, China) to generate 150 bp paired-end reads. Differential expression analysis between the two groups was performed using the criteria of P.adj < 0.05 and |FC| ≥ 1.5, and was analyzed and visualized using the BMKCloud platform (www.biocloud.net).

[0249] The results are as follows Figure 15-22 As shown, from Fig.15 As can be seen in Figures A and B, LM-PCR CPI treatment significantly altered gene expression in the hippocampus of HFD-fed mice, with 641 genes upregulated and 544 genes downregulated in LM-PCR CPI-treated mice compared with untreated mice (P < 0.05; |FC| ≥ 1.5).

[0250] from Fig.16 and Fig. 20It can be seen that KOG and KEGG functional enrichment analysis showed that the differentially expressed genes were mainly enriched in functional categories such as signal transduction mechanism, energy production and conversion, and transcription.

[0251] from Fig.17 As can be seen in Figure A, KEGG enrichment analysis showed that these differentially expressed genes were mainly enriched in ribosome, oxidative phosphorylation (OXPHOS), thermogenesis, mitogen-activated protein kinase (MAPK) signaling pathways and retrograde endocannabinoid signaling pathways, among which oxidative phosphorylation, thermogenesis, Parkinson's disease and retrograde endocannabinoid signaling pathways have been identified as potential candidates for promoting targeted prevention and developing personalized treatment strategies.

[0252] from Fig.17 B and Fig.18 As can be seen in Figure B, LM-PCRCPI treatment significantly activated the oxidative phosphorylation signaling pathway (OXPHOS), which is a key process in energy metabolism and ATP production. Impairment of this process can lead to energy metabolism disorders, thereby promoting the development of depression.

[0253] from Fig.18 Figure A and Fig.21 As can be seen in the literature, gene mutations in the retrograde endocannabinoid signaling pathway have also been suggested as potential factors in the pathogenesis of major depressive disorder.

[0254] from Fig.19 As can be seen in Figure B, q-PCR verification showed that LM-PCRCPI treatment significantly increased the expression of mt-Nd3, NDUFs5, ATP5e, etc. in the hippocampal OXPHOS of mice fed with HFD.

[0255] from Fig.19 As can be seen in Figure A, Spearman correlation analysis showed that the mt-Nd3, ATP5e, and COX6c genes were positively correlated with the severity of depression-related indicators.

[0256] from Fig.19 Figure C and Fig. 22 It can be seen that mt-Nd3 has the highest expression level and a higher multiple of difference among the differentially expressed genes in the OXPHOS pathway, and there is a strong molecular binding interaction between 2-AG and mt-Nd3, indicating that enrichment of endogenous 2-AG by LM-PCRCPI may be a potential candidate substance for treating depressive-like behavior in mice by regulating mt-Nd3.

[0257] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of tangerine peel polysaccharide in any of the following (1)-(13): (1) Use in the preparation of products for preventing and / or treating depression; (2) Use in the preparation of products for preventing and / or treating nerve damage; (3) Use in the preparation of products for reducing weight gain; (4) Application in the preparation of products for regulating intestinal flora; (5) Application in the preparation of products for increasing DA content in plasma; (6) Use in the preparation of products for increasing the 5-HT content in plasma; (7) Application in the preparation of products for increasing DCX expression; (8) Application in the preparation of products for improving Neun expression; (9) Use in the preparation of products that reduce Iba-1 expression; (10) Use in the preparation of products for inhibiting HFD-induced microglial activation; (11) Use in the preparation of expression of metabolites that enhance retrograde endocannabinoid signaling; (12) Application in the preparation of products for activating oxidative phosphorylation signaling pathways; (13) Application in the preparation of products for increasing the expression level of genes related to the oxidative phosphorylation pathway.

2. The use according to claim 1, characterized in that: The metabolites of the retrograde endocannabinoid signaling include 2-arachidonoylglycerol and gamma-aminobutyric acid; And / or, the genes related to the oxidative phosphorylation pathway include at least one of COX6c, mt-Nd3, NDUFs5, ATP5e, NDUFb8, SDHB, ATP5e, UQCR11, and UQCRh.

3. The use 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 the 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 comprises the following steps: (1) taking a dried tangerine peel sample, and subjecting it to defatting, water extraction, alcohol precipitation, decolorization, Sevag reagent protein removal, and dialysis to obtain a dried tangerine peel crude polysaccharide; (2) The crude tangerine peel polysaccharide is eluted through a DEAE cellulose column to obtain tangerine peel polysaccharide; the reagent used for the elution includes a NaCl solution with a concentration of 0.1-0.6M.

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

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

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 preventing and / or treating depression; (2) Use in the preparation of products for preventing and / or treating nerve damage; (3) Use in the preparation of products for reducing weight gain; (4) Application in the preparation of products for regulating intestinal flora; (5) Application in the preparation of products for increasing DA content in plasma; (6) Use in the preparation of products for increasing the 5-HT content in plasma; (7) Application in the preparation of products for increasing DCX expression; (8) Application in the preparation of products for improving Neun expression; (9) Use in the preparation of products that reduce Iba-1 expression; (10) Use in the preparation of products for inhibiting HFD-induced microglial activation; (11) Use in the preparation of expression of metabolites that enhance retrograde endocannabinoid signaling; (12) Application in the preparation of products for activating oxidative phosphorylation signaling pathways; (13) Application in the preparation of products for increasing the expression level of genes related to the oxidative phosphorylation pathway.

10. The use according to claim 9, characterized in that: The products include medicines, health products or functional foods; Preferably, the drug further comprises a pharmaceutically acceptable excipient; More preferably, the pharmaceutically acceptable excipients include at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener and a flavoring agent; 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 agents, transdermal microneedles; Preferably, the administration of the drug includes injection or oral administration.

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