Intestinal flora derivative indolepropionic acid capable of relieving obesity and application thereof

Abstract

The invention relates to an intestinal flora derivative indolepropionic acid capable of relieving obesity and application thereof, and discloses new application of indolepropionic acid in obesity treatment. Indolepropionic acid is prepared through a microbial fermentation method, the purity is higher than that of indolepropionic acid prepared through a chemical synthesis method, multi-target metabolism regulation is achieved by activating a PPAR-gamma signal channel, and the effects of fat metabolism remodeling, liver injury repairing, glycometabolism regulation, inflammation inhibition and the like are achieved; compared with the prior art, the probiotic strain dependence is broken through, a single and definite active component, namely indolepropionic acid, is provided, and the complexity and effect fluctuation of flora intervention are eliminated; the multi-target metabolism regulation is realized, the increase of white fat browning and the reduction of liver fat decomposition are synchronously promoted, and the problem that the fat type cannot be converted by the existing metabolite is solved; the industrial feasibility is optimized, the high-purity indolepropionic acid is prepared through microbial fermentation, the cost is reduced compared with that of a chemical synthesis method, and the purity and production cost bottlenecks are broken through.

Description

Technical Field

[0001] This invention lies at the intersection of microbial metabolite development and biomedicine, specifically the novel application of indolepropionic acid, a compound derived from intestinal flora, in regulating fat metabolism. In particular, the invention reveals for the first time the core mechanism by which this compound redistributes adipose tissue through activation of the PPAR-γ signaling pathway and develops a multi-faceted industrial application system encompassing pharmaceutical formulations, functional foods, and detection kits. Background Art

[0002] According to the "Dietary Guidelines for Adult Obesity (2024 Edition)," the obesity rate among Chinese residents aged 18 and over has reached 16.4%. Metabolic syndrome (such as non-alcoholic fatty liver disease and insulin resistance) caused by overweight and obesity has become a major public health issue. Traditional medications such as orlistat have limited efficacy in improving hepatic steatosis (reducing liver disease by only 1.2 pathological grades) and are associated with side effects such as diarrhea. There is an urgent need to develop safer, multi-targeted obesity treatments.

[0003] The prior art has the following defects:

[0004] (1) Limitations of probiotic therapy:

[0005] Current probiotic-based obesity interventions are significantly strain-dependent, with weight loss rates varying by 40%-60% between different strains. Furthermore, the effectiveness of interventions is significantly influenced by the baseline state of the host's intestinal flora (e.g., differences in flora diversity can lead to fluctuations in effect exceeding 35%). Furthermore, probiotic preparations rely on live bacterial colonization, leading to poor storage stability and complex mechanisms of action.

[0006] (2) Deficiencies in metabolite research:

[0007] Although known metabolites such as short-chain fatty acids (such as butyrate) can reduce some blood lipid indicators (such as serum triglycerides 15%-20%), they cannot achieve the transformation of adipose tissue types (such as browning of white fat) and lack a significant improvement effect on liver fat deposition.

[0008] (3) Deficiencies of chemical synthesis pathways:

[0009] Indolepropionic acid prepared by chemical synthesis has insufficient purity (usually <93%), high production cost (about three times that of microbial fermentation), and its specific role in the regulation of fat metabolism has not been explored. Summary of the Invention

[0010] (1) Technical problems solved

[0011] To solve the above problems, the present invention provides an intestinal flora derivative indolepropionic acid that can alleviate obesity and its application, which breaks through the dependence of probiotic strains, realizes multi-target metabolic regulation, and optimizes industrial feasibility.

[0012] (2) Technical solution

[0013] To achieve the above object, the present invention provides the following technical solutions:

[0014] An application of indolepropionic acid, a gut flora derivative that can alleviate obesity, wherein the application is for preparing an obesity intervention product to achieve adipose tissue metabolism regulation by activating the PPAR-γ signaling pathway.

[0015] Preferably, the obesity intervention product is selected from pharmaceutical preparations, functional foods, microbial preparations or animal feed additives, and its dosage forms include tablets, capsules, sustained-release microspheres or liquid suspensions.

[0016] Preferably, the pharmaceutical preparation comprises a therapeutically effective amount of indolepropionic acid, a derivative of intestinal flora, and pharmaceutically acceptable excipients, wherein the excipients include microcrystalline cellulose, sodium carboxymethyl starch or hydroxypropyl methylcellulose, and the daily dosage is 50-200 mg / kg.

[0017] Preferably, the adipose tissue metabolism regulation includes:

[0018] (a) Reduce subcutaneous fat content by 20%-40% and visceral fat content by 25%-45% in obese individuals;

[0019] (b) promoting an increase in brown adipose tissue content to 115%-135% of normal levels;

[0020] (c) Adjust serum triglyceride, total cholesterol, and low-density lipoprotein concentrations to 65%-85% of baseline values.

[0021] Preferably, the metabolic regulation is achieved by inhibiting the activities of AST and ALT in liver tissue, reducing the enzyme activities by 30%-50%.

[0022] A composition comprising indolepropionic acid, a gut flora derivative capable of alleviating obesity, further comprising a synergistic component selected from inulin-type prebiotics, β-glucan dietary fiber, or eicosapentaenoic acid, and wherein the mass ratio of the indolepropionic acid to the synergistic component is 1:0.5-1:2.

[0023] Preferably, the fasting blood glucose level can be reduced by 15%-25% and the HOMA-IR index can be improved by 30%-50%.

[0024] A kit for detecting the efficacy of indolepropionic acid, a gut flora derivative that can alleviate obesity, comprises an antibody pair that specifically detects the degree of activation of the PPAR-γ pathway, wherein the antibody pair comprises a monoclonal antibody against the N-terminal domain of the PPAR-γ protein and a corresponding HRP-labeled secondary antibody.

[0025] Preferably, in an animal model of metabolic syndrome induced by a high-fat diet, the degree of liver steatosis is reduced by 2-3 pathological grades.

[0026] (3) Beneficial effects

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] It breaks through the dependence on probiotic strains and provides a single clear active ingredient, indolepropionic acid, eliminating the complexity and effect fluctuations of microbial intervention;

[0029] It achieves multi-target metabolic regulation, simultaneously promoting the browning of white fat (UCP1↑) and liver fat decomposition (SREBP-1c↓), solving the problem that existing metabolites cannot convert fat types;

[0030] The feasibility of industrialization has been optimized. High-purity indolepropionic acid is prepared by microbial fermentation at a lower cost than chemical synthesis, breaking through the bottleneck of purity and production cost.

[0031] In addition, after activating the PPAR-γ signaling pathway, the fat metabolism remodeling effect of indolepropionic acid is better than that of butyric acid (only lipid lowering, no fat conversion); the liver pathology improvement effect of indolepropionic acid is significantly better than that of orlistat (improvement rate of hepatic steatosis↑); the synergistic regulation effect of indolepropionic acid on glucose metabolism breaks through the limitation of metformin alone in glucose control; the mechanism of indolepropionic acid's inflammation inhibition effect is clear (PPAR-γ inhibits NF-κB phosphorylation); indolepropionic acid also has industrial advantages, which solves the problems of low purity (<93%) and high cost of chemical synthesis method. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0033] Figure 1 The figure shows the schematic diagram of the experimental animal grouping and intervention treatment process of the present invention;

[0034] Figure 2 Shown is a graph showing changes in weight and fat content of mice according to the present invention;

[0035] Figure 3 Shown is a comparative diagram of liver histopathology HE staining of the present invention;

[0036] Figure 4 The heat map of gene expression of the PPAR-γ signaling pathway of the present invention is shown;

[0037] Figure 5 Shown is the Western blot protein expression band diagram of the present invention;

[0038] Figure 6 The schematic diagram of the IPA mechanism of action and metabolic regulation network of the present invention is shown;

[0039] Figure 7 The results show the differences among the NC group, Obesity group and IPA group in the present invention, which directly reflects the improvement effect of IPA on obesity, fatty liver, sugar metabolism and inflammation. DETAILED DESCRIPTION

[0040] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] Example 1:

[0042] See attached Figure 6 The embodiment of the present invention discloses an application of indolepropionic acid, a gut flora derivative that can alleviate obesity, for preparing obesity intervention products to achieve adipose tissue metabolism regulation by activating the PPAR-γ signaling pathway.

[0043] Through the design of the above technical solution, namely, activating the PPAR-γ signaling pathway to regulate fat metabolism, the technical problems of the existing obesity treatment methods, such as unclear mechanism of action and poor targeting (such as probiotics relying on the host microbiota status), are solved; the core mechanism of the above technical solution is: 1. Molecular mechanism: IPA, as an endogenous ligand of PPAR-γ, induces UCP1 expression after binding to the receptor (Western blot data: 3.2±0.4 times upregulation), promoting the browning of white fat (see the instructions). Figure 4 ); 2. Effect verification: The reduction in fat content (subcutaneous fat↓) embodied in claim 4 is directly related to the level of PPAR-γ activation.

[0044] See attached Figure 6 , the following design is also carried out in this embodiment. Specifically, the obesity intervention product is selected from pharmaceutical preparations, functional foods, microbial preparations or animal feed additives, and its dosage form includes tablets, capsules, sustained-release microspheres or liquid suspensions.

[0045] The design of the above technical solution improves the accuracy of drug delivery. For example, the tablets are granulated by wet method to ensure uniform dispersion of IPA. The parameter is based on the following: the range of "50-200 mg / kg / day" in claim 3 is based on a dose escalation experiment in mice (50 mg / kg is ineffective and 200 mg / kg reaches a plateau).

[0046] Furthermore, for functional foods, the addition of inulin (claim 6) can synergistically enhance intestinal absorption (fluorescence tracer experiments show an increase in absorption rate).

[0047] See attached Figure 1 and attached Figure 2 , the following design is also carried out in this embodiment. Specifically, the pharmaceutical preparation contains a therapeutically effective amount of intestinal flora derivative indolepropionic acid and pharmaceutically acceptable excipients, the excipients include microcrystalline cellulose, sodium carboxymethyl starch or hydroxypropyl methylcellulose, and the daily dosage is 50-200 mg / kg.

[0048] The above technical solution provides a dosage range and excipients, and the dosage setting principle is: lower limit 50 mg / kg: no significant change in fat content is observed below this dose; upper limit 200 mg / kg: above this dose, a diminishing marginal effect occurs (the slope of the dose-effect curve decreases by 40%).

[0049] Furthermore, regarding excipient selection: Microcrystalline cellulose (MCC PH102): Optimal fluidity at 35% (angle of repose 28.7° vs. ordinary MCC 35.2°); Sodium carboxymethyl starch (CMS-Na): Disintegration time ≤ 5 minutes (Chinese Pharmacopoeia 2020 edition standard)

[0050] See attached Figure 2 In this embodiment, the following designs are also made. Specifically, the regulation of adipose tissue metabolism includes: (a) reducing the subcutaneous fat content of obese individuals by 20%-40% and the visceral fat content by 25%-45%; (b) promoting the increase of brown adipose tissue content to 115%-135% of the normal level; and (c) regulating serum triglyceride, total cholesterol, and low-density lipoprotein concentrations to 65%-85% of the baseline value.

[0051] The above technical solution achieves the quantification of biological effects, and its fat redistribution mechanism is as follows: subcutaneous fat reduction: by activating AMPK phosphorylation (Western blot data: p-AMPK / AMPK ratio ↑2.1 times), lipid synthesis is inhibited; visceral fat reduction: PPAR-γ-mediated lipolytic enzyme (ATGL) activity is increased (enzyme activity detection: ↑).

[0052] See attached Figure 3 and attached Figure 5, the following design is also carried out in this embodiment. Specifically, metabolic regulation is achieved by inhibiting the activities of AST and ALT in liver tissue, reducing the enzyme activity by 30%-50%.

[0053] Through the design of the above technical scheme, a protective effect is achieved on the liver; the mechanism of AST / ALT activity inhibition is: IPA reduces hepatocyte inflammatory damage by inhibiting the NF-κB pathway (ELISA detection: TNF-α↓, IL-6↓); the GSH level in liver tissue returns to 95±6μmol / g in the normal group (vs 52±5μmol / g in the obese group).

[0054] See attached Figure 5 and attached Figure 6 This embodiment also discloses a composition comprising indolepropionic acid, a derivative of intestinal flora that can alleviate obesity, and also includes a synergistic component, which is selected from inulin-type prebiotics, β-glucan-type dietary fiber or eicosapentaenoic acid, and the mass ratio of indolepropionic acid to the synergistic component is 1:0.5-1:2.

[0055] In the above technical solution, the inulin-based prebiotic promotes intestinal colonization of IPA (16S sequencing shows an increase in Bifidobacterium abundance); a synergistic ratio of 1:1 results in the highest hepatic triglyceride clearance (76.3±5.2% vs. 58.4±4.1% in the IPA alone group). β-glucan increases IPA's intestinal retention time by increasing viscosity (MRI imaging shows an increase in retention time).

[0056] See attached Figure 5 , this embodiment also carries out the following design, specifically, can reduce fasting blood sugar level by 15%-25%, and improve HOMA-IR index by 30%-50%.

[0057] Through the design of the above technical solution, the regulation of glucose metabolism was achieved; the mechanism of fasting blood glucose reduction was: IPA activated the PI3K / Akt pathway (Western blot data: p-Akt / Akt ratio↑), promoted skeletal muscle GLUT4 transport, and inhibited hepatic gluconeogenesis (PEPCK mRNA expression↓).

[0058] See attached Figure 4 and attached Figure 5 This embodiment also discloses a kit for detecting the efficacy of indolepropionic acid, a gut flora derivative that can alleviate obesity, comprising an antibody pair that specifically detects the degree of activation of the PPAR-γ pathway, the antibody pair comprising a monoclonal antibody against the N-terminal domain of the PPAR-γ protein and a corresponding HRP-labeled secondary antibody.

[0059] In the above technical solution, the antibody pair is designed as follows: 1. Target selection: N-terminal domain of PPAR-γ protein (antigenic epitope: amino acid residues 50-70); 2. Verification data: The antibody pair has a binding affinity with PPAR-γ of KD = 1.2 nM (SPR detection data).

[0060] See attached Figure 3 In this embodiment, the following design is also carried out. Specifically, in an animal model of metabolic syndrome induced by a high-fat diet, the degree of liver steatosis is reduced by 2-3 pathological grades.

[0061] In the above technical solution, the metabolic syndrome model is: pathological grading: NAS score dropped from 5.3±0.6 to 2.8±0.4 (p<0.01), which meets the "reduction of 2-3 levels" in claim 9; cross-species verification: the liver fat content of the crab-eating macaque model dropped from 28.4±3.1% to 12.7±2.5% (p<0.05).

[0062] Example 2 (implementation steps are as follows):

[0063] 1. Experimental model construction (see Appendix Figure 1 、 2 )

[0064] Preparation of high-fat feed (supporting claims 4-5):

[0065] Fat energy ratio 60% (lard:soybean oil = 3:1, peroxide value <5meq / kg), supplemented with 2% cholesterol and 0.5% bile salt;

[0066] Carbohydrate composition: maltodextrin (20%) + sucrose (10%), mixing uniformity RSD < 3% (double-helix conical mixer, rotation speed 30 rpm, mixing time 45 min).

[0067] Animal grouping intervention (supporting claims 3-5):

[0068] C57 / Bl 6J mice (7 weeks old) were divided into three groups (NC group, Obesity group, and IPA group), with 2 cages in each group (n=8);

[0069] IPA intervention dose: 100 mg / kg / day (oral volume 0.2 mL, suspension formula: 0.5% sodium carboxymethylcellulose), intervention period 8 weeks.

[0070] 2. Standardized processing of tissue samples (supporting claims 4-5, 9)

[0071] Fat tissue collection (if attached) Figure 2 ):

[0072] Inguinal subcutaneous fat: longitudinally incise the skin along the linea alba, and bluntly separate the edges of the fat pad. The weighing error should be less than 0.1 g.

[0073] Epididymal visceral fat: The fat capsule surrounding the epididymis was completely removed to avoid residual blood vessels (rinse with pre-cooled PBS 3 times immediately after sampling).

[0074] Liver sample processing (supporting claims 5, 8-9):

[0075] Tissue blocks of 10 × 10 × 3 mm were obtained from the right lobe of the liver and fixed with 4% paraformaldehyde for 24 h (4°C);

[0076] The remaining liver tissue was divided into 50 mg / tubes, quickly frozen in liquid nitrogen, and stored at -80°C (storage period ≤ 6 months, avoid repeated freezing and thawing).

[0077] 3. Detection system optimization (supporting claims 5-9)

[0078] Serum index detection (combined with Figure 5 ):

[0079] GSH determination (claims 5 and 7):

[0080] DTNB colorimetric assay (Kit #CS0260, Sigma), detection limit 0.5 μM, intra-assay coefficient of variation <5%;

[0081] Data verification: GSH 5.2±0.8μM in the obesity group → 8.1±0.9μM in the IPA group (p<0.01).

[0082] HDL-C detection (claim 5):

[0083] Magnesium phosphotungstate precipitation method (kit Abcam #ab65390), recovery rate 98.2±3.5%;

[0084] Data verification: HDL-C increased from 0.8±0.1mmol / L to 1.4±0.2mmol / L (p<0.05).

[0085] Quantification of inflammatory response (supporting claims 5 and 9):

[0086] Serum CRP detection (ELISA kit Abcam #ab157712):

[0087] Obesity group CRP 12.3±1.5 ng / mL→IPA group 5.1±0.7 ng / mL (p<0.01);

[0088] Liver NAS score (see attached Figure 7 ):

[0089] Steatosis score: 2.8±0.3→0.7±0.2 (p<0.01);

[0090] Inflammatory infiltration score: 2.1±0.4→0.5±0.1 (p<0.01).

[0091] 4. Synergistic Implementation of Composition (Supporting Claims 6-7)

[0092] Preparation of formula A (IPA: inulin = 1:1):

[0093] Inulin pretreatment: 80 mesh sieving followed by vacuum drying at 60°C for 4 hours (moisture content ≤ 3%);

[0094] V-type mixer parameters: rotation speed 25 rpm, mixing time 30 min, uniformity RSD < 2.5%.

[0095] Preparation of Formulation B (IPA:β-glucan=1:0.5):

[0096] β-glucan activation: dissolved in deionized water at 50°C (10% w / v) and spray dried (inlet temperature 160°C);

[0097] Accelerated stability: 6-month content retention rate>93% (HPLC detection conditions are the same as in Example 1).

[0098] 5. Development of detection kits (supporting claims 8-9)

[0099] PPAR-γ activation detection (combined with attached Figure 4 、 5 ):

[0100] Antibody screening: 1A2 monoclonal antibody (antigenic peptide Met1-Glu50) was obtained using phage display technology;

[0101] Detection process: liver lysate → coating antibody (2 μg / mL) → HRP-labeled secondary antibody (1:5000) → TMB color development;

[0102] Data verification: activation increased from 32.7% to 89.5% (p<0.001, as shown in the attached Figure 5 histogram).

[0103] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0104] In addition, it should be noted that the use of terms such as "first" and "second" to limit components is only for the convenience of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore cannot be understood as limiting the scope of protection of this application.

[0105] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An application of indolepropionic acid, a derivative of intestinal flora, for alleviating obesity, characterized in that: The application is to prepare obesity intervention products to achieve adipose tissue metabolism regulation by activating the PPAR-γ signaling pathway.

2. The use according to claim 1, characterized in that The obesity intervention product is selected from pharmaceutical preparations, functional foods, microbial preparations or animal feed additives, and its dosage forms include tablets, capsules, sustained-release microspheres or liquid suspensions.

3. The use according to claim 2, characterized in that The pharmaceutical preparation comprises a therapeutically effective amount of indolepropionic acid, a derivative of intestinal flora, and pharmaceutically acceptable excipients, wherein the excipients include microcrystalline cellulose, sodium carboxymethyl starch or hydroxypropyl methylcellulose, and the daily dosage is 50-200 mg / kg.

4. The use according to claim 1, characterized in that The adipose tissue metabolism regulation includes: (a) Reduce subcutaneous fat content by 20%-40% and visceral fat content by 25%-45% in obese individuals; (b) promoting an increase in brown adipose tissue content to 115%-135% of normal levels; (c) Adjust serum triglyceride, total cholesterol, and low-density lipoprotein concentrations to 65%-85% of baseline values.

5. The use according to claim 4, characterized in that The metabolic regulation is achieved by inhibiting the activities of AST and ALT in liver tissue, reducing the enzyme activities by 30%-50%.

6. A composition comprising indolepropionic acid, a derivative of intestinal flora, which can alleviate obesity, characterized in that: It also includes a synergistic component, which is selected from inulin-type prebiotics, beta-glucan dietary fiber or eicosapentaenoic acid, and the mass ratio of the indolepropionic acid to the synergistic component is 1:0.5-1:

2.

7. The composition according to claim 6, characterized in that It can reduce fasting blood sugar levels by 15%-25% and improve the HOMA-IR index by 30%-50%.

8. A kit for detecting the efficacy of indolepropionic acid, a derivative of intestinal flora that can alleviate obesity, characterized in that: The invention comprises an antibody pair for specifically detecting the degree of activation of the PPAR-γ pathway, wherein the antibody pair comprises a monoclonal antibody against the N-terminal domain of the PPAR-γ protein and a corresponding HRP-labeled secondary antibody.

9. The use or product according to any one of claims 1 to 8, characterized in that: In an animal model of metabolic syndrome induced by a high-fat diet, the degree of liver steatosis was reduced by 2-3 pathological grades.

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