Application of isorhamnetin in preparation of medicine for preventing and treating fatty liver diseases related to metabolic dysfunction

Abstract

The invention relates to the technical field of biological medicines, in particular to application of isorhamnetin in preparation of a medicine for preventing and treating fatty liver diseases related to metabolic dysfunction. The invention provides application of isorhamnetin or pharmaceutically acceptable salts, esters or derivatives thereof in preparation of drugs for preventing and treating fatty liver diseases related to metabolic dysfunction. The invention also provides application of isorhamnetin or pharmaceutically acceptable salts, esters or derivatives thereof in preparation of drugs for preventing and treating diseases caused by intestinal flora disorder. The isorhamnetin can recover the diversity and richness of intestinal flora of mice with fatty liver diseases related to metabolic dysfunction and recover the balance of the intestinal flora, and the high-dose (50 mg / kg.d) donor flora transplantation of the isorhamnetin can partially reproduce metabolism improvement. The fatty liver diseases related to obesity or metabolic dysfunction are relieved through an'intestinal flora-bile acid-lipid metabolism 'axis.

Description

Application of isorhamnetin in the preparation of drugs for the prevention and treatment of fatty liver disease associated with metabolic dysfunction. Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of isorhamnetin in the preparation of a drug for the prevention and treatment of fatty liver disease associated with metabolic dysfunction. Background Technology

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is a disease characterized by excessive accumulation of triglycerides in the liver in the presence of at least one cardiometabolic risk factor. MASLD is closely related to obesity, insulin resistance, and dyslipidemia, and can manifest as hepatic lipid deposition, inflammation, and abnormal liver function. It can further progress to metabolic dysfunction-associated steatohepatitis (MASH), liver fibrosis, and even cirrhosis, seriously endangering public health and increasing the risk of cardiovascular events and the medical burden. Globally, the prevalence of MASLD is as high as 25% and continues to rise. In China, with the prevalence of obesity and metabolic syndrome, MASLD has become the leading chronic liver disease and the primary cause of abnormal liver biochemical indicators in health checkups, placing a heavy burden on the healthcare economy.

[0003] Current intervention strategies for MASLD are based on lifestyle management, namely reducing dietary energy intake and increasing exercise intensity, but long-term adherence is limited. Furthermore, many existing clinical drugs or candidate drugs require long-term use, some with risks of gastrointestinal discomfort, metabolic disorders, liver and kidney burden, or other adverse reactions, and their efficacy varies significantly among different populations, making it difficult to meet the needs of "long-term, safe, and sustainable" chronic disease management, and hindering continuous intervention for mild to moderate MASLD individuals and those in the health management stage. Existing technologies either lean towards drug intervention and are limited by safety concerns, or lean towards nutritional supplementation and have unsatisfactory therapeutic effects. Therefore, a solution is needed that can stably improve key MASLD phenotypes while ensuring gentleness and safety. In addition, existing MASLD intervention products are mostly concentrated in the pharmaceutical system, making it difficult to cover the daily health management needs of a wider population; therefore, a technological solution is needed that can be used to prepare drugs and can also be extended to food additives, functional foods, or dietary supplements to meet the application needs of different populations and different stages. Summary of the Invention

[0004] In view of this, the present invention provides the use of isorhamnetin in the preparation of a drug for the prevention and treatment of MASLD.

[0005] To achieve the above objectives, the present invention provides the following solution: one of the technical solutions of the present invention is the application of isorhamnetin or its pharmaceutically acceptable salts, esters or derivatives in the preparation of a drug for the prevention and treatment of MASLD.

[0006] Isorhamnetin improves MASLD-related phenotypes by regulating lipid metabolism and improving hepatic steatosis; isorhamnetin improves MASLD by regulating gut microbiota and bile acid metabolism and enhancing farnesoid X receptor (FXR)-related signaling; isorhamnetin alters the composition of the bile acid pool and promotes enterohepatic bile acid circulation flux.

[0007] The second technical solution of the present invention is a drug for preventing and treating MASLD, the active ingredient of which includes isorhamnetin or its pharmaceutically acceptable salt, ester or derivative.

[0008] The drug used to prevent and treat MASLD is an oral preparation, and the dosage of isorhamnetin is 12.5 mg / kg·d to 50 mg / kg·d.

[0009] The third technical solution of the present invention is the use of isorhamnetin or its pharmaceutically acceptable salts, esters or derivatives in the preparation of drugs for the prevention and treatment of diseases caused by intestinal flora imbalance.

[0010] Isorhamnetin can restore the diversity and richness of the gut microbiota in MASLD mice, restore the balance of the gut microbiota, and transplantation of donor microbiota with high doses of isorhamnetin (50 mg / kg·d) can partially reproduce metabolic improvement.

[0011] The fourth technical solution of the present invention is a drug for preventing and treating diseases caused by intestinal flora imbalance, wherein the active ingredient includes isorhamnetin or its pharmaceutically acceptable salt, ester or derivative.

[0012] In a preferred embodiment of the present invention, the medicament for preventing and treating diseases caused by intestinal flora imbalance also includes pharmaceutically acceptable excipients.

[0013] The fifth technical solution of the present invention is the application of isorhamnetin in the preparation of drugs, health foods or food supplements for controlling body fat, lowering blood lipids, protecting the liver or regulating bile acids.

[0014] Compared with existing technologies, this invention has the following beneficial effects: To solve the technical problems in the treatment of MASLD, this invention provides a product and its application method with isorhamnetin as the core ingredient, which can be used to prevent and / or improve MASLD. This is a safe, mild, and suitable solution for long-term use. It can be further expanded to applications such as food additives, functional foods, or dietary supplements, thereby improving compliance and cure rates in the MASLD population, reducing the risk of disease progression, and improving overall management benefits. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 shows the reduction of obesity signs in MASLD mice by isorhamnetin; where A is the experimental mouse grouping and feeding process; B is the mouse weight line graph; C is the final weight of mice in each group; D is the weight gain of mice within 18 weeks; E is the average daily food intake; there are significant differences between different letters (p<0.05) (n=3), the same below.

[0017] Figure 2 shows the results of isorhamnetin-induced reduction of hepatic steatosis in MASLD mice; where A represents liver morphology observation: HE staining of liver tissue (200×, scale bar: 100 μm; 400×, scale bar: 50 μm; yellow arrow: steatosis; blue arrow: inflammatory cell infiltration; green arrow: ballooning degeneration; red arrow: congestion) and Oil Red O staining (400×, scale bar: 50 μm); B represents the liver weight of mice in each group at week 18; C represents the ratio of lipid droplet staining area; and D represents the NAS score.

[0018] Figure 3 shows the results of isorhamnetin reducing the weight of adipose tissue in MASLD mice; where A is the morphology of epididymal white adipose tissue (eWAT), HE staining of eWAT tissue (200×, scale bar: 100 μm) and distribution of adipocyte diameter; B is the average adipocyte area; C is the weight of eWAT; and D is the weight of inguinal white adipose tissue (iWAT).

[0019] Figure 4 shows the results of isorhamnetin reducing blood lipid levels in MASLD mice; where A represents serum triacylglycerol (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c); and B represents liver TG, TC, HDL-c, and LDL-c levels.

[0020] Figure 5 shows the results of isorhamnetin reducing liver damage and serum free fatty acid (FFA) levels in MASLD mice; where A represents serum aspartate transaminase (AST) and alanine transaminase (ALT) levels; and B represents serum FFA levels.

[0021] Figure 6 shows the results of isorhamnetin improving lipid metabolism in MASLD mice; where A / B are the protein imaging patterns and relative protein expression levels of sterol regulatory element-binding protein 1 (SREBP1c), fatty acid synthase (FASn), stearoyl-CoA desaturase 1 (SCD1), diacylglycerol acyltransferase 2 (DGAT2), cluster of differentiation 36 (CD36), carnitine palmitoyltransferase 2 (CPT2), and hormone-sensitive lipase (HSL) in mouse liver; C is the relative mRNA expression levels of Srebp1c, Fasn, Scd1, Dgat2, Cd36, Cpt2, and Hsl in mouse liver.

[0022] Figure 7 shows the effect of isorhamnetin at a dose of 50 mg / kg·d on the gut microbiota of MASLD mice; where A is the Venn diagram of the distribution of Operational Taxonomic Units (OTUs); B is the α diversity index; C is the principal component analysis (PCA) based on β diversity; and D is the phylum-level composition analysis of the gut microbiota.

[0023] Figure 8 shows the results of isorhamnetin improving the bile acid content in the cecal contents of MASLD mice; where A is a violin plot of differential bile acids in the cecal contents of mice; B is a heatmap of differential bile acid clustering; C is the ratio of total bile acid (CD) to total chenodeoxycholic acid (CDCA); and D is the ratio of primary bile acid to secondary bile acid.

[0024] Figure 9 shows the results of isorhamnetin regulating the expression of the FXR pathway in the liver of MASLD mice; where A / B are the protein imaging and relative expression levels of liver FXR, small heterodimer partner (SHP), Na+ / taurocholate cotransporting polypeptide (NTCP), cytochrome P450 family 7 subfamily A member 1 (CYP7A1), and cytochrome P450 family 27 subfamily A member 1 (CYP27A1); C is the relative mRNA expression levels of liver Nr1h4, Nr0b2, Slc10a1, Abcb11, Cyp7a1, and Cyp27a1; D is the total bile acid content in the liver, ileum, feces, and serum.

[0025] Figure 10 shows the results of isorhamnetin reducing intestinal lipid absorption in MASLD mice; where A represents the lipase content in intestinal contents; B represents the TG and TC content in feces; and C represents the relative expression levels of Niemann-Pick C1-Like 1 (NPC1L1), Microsomal Triglyceride Transfer Protein (MTTP), and Solute Carrier Family 27 Member 4 (SLC27A4) mRNA in the jejunum and ileum. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] This invention proposes an intervention strategy for MASLD based on the natural flavonoid compound isorhamnetin. Isorhamnetin is widely available, obtained from various edible plants and traditional Chinese medicinal resources, such as sea buckthorn and ginkgo, offering a good safety profile and broad dietary sources. This study demonstrates that appropriate doses of isorhamnetin can improve lipid metabolism disorders, hepatic steatosis, and related inflammatory damage without significant drug side effects. Furthermore, it can synergistically regulate lipid metabolism at multiple targets through pathways such as adjusting gut microbiota and bile acid metabolism, making it suitable for long-term management and widespread application.

[0032] This invention demonstrates through experiments that plant-derived isorhamnetin has a high safety profile and can lower blood lipids and alleviate hepatic steatosis. It exerts its lipid-lowering effect through the gut microbiota-bile acid metabolism axis, thus complementing and improving upon the shortcomings of existing therapeutic drugs and food supplements. This addresses or improves problems such as numerous side effects, insufficient long-term tolerability, and poor adherence in existing drug treatments.

[0033] This invention provides applications of isorhamnetin in reducing fat deposition in mice, improving bile acid and lipid metabolism in mice, activating the FXR pathway, and restoring gut microbiota homeostasis, including one or more of these effects: 1) Dose-dependent improvement of obesity and liver lipid deposition: This invention is the first to clearly demonstrate that isorhamnetin has a significant therapeutic effect on MASLD. Based on a high-fat diet induction, isorhamnetin intervention can gradually reduce indicators such as body weight, liver weight, and body fat, and this is independent of changes in food intake.

[0034] 2) Significantly improves blood lipid and liver function damage indicators: reduces serum TG, TC, LDL-c and ALT / AST, increases beneficial indicators and reduces liver tissue TG / TC levels.

[0035] 3) Reprogramming lipid metabolism pathways: downregulating lipid production (SREBP1c, etc.) and upregulating fatty acid oxidation (PPARα, CPT1A, HSL, etc.).

[0036] 4) Restoring gut microbiota homeostasis: Isorhamnetin can restore the diversity and richness of gut microbiota in MASLD mice, restore gut microbiota balance, and high-dose isorhamnetin donor microbiota transplantation can partially reproduce metabolic improvement.

[0037] 5) Remodel the bile acid pool: Increase the ratio of primary bile acids to conjugated bile acids.

[0038] 6) Activate the liver FXR axis: Isorhamnetin can activate the expression of the liver FXR pathway, promote bile acid circulation flux, and regulate bile acid metabolism.

[0039] 7) Reduces intestinal lipid absorption: Isorhamnetin can reduce intestinal absorption of fatty acids by inhibiting lipid transmembrane uptake and loading.

[0040] 8) Mild effects, suitable for long-term intervention and has potential for food application: Isorhamnetin is derived from edible plants, has a good safety profile and a relatively low potential toxic side effects burden, and can be expanded to food additives / functional foods / nutritional supplements application scenarios.

[0041] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0042] The isorhamnetin used in the embodiments of the present invention was purchased from Tiancui Biotechnology Co., Ltd. (Wuxi, China) (batch number: AZCA0807) with a purity of 98.84%.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1: Isorhamnetin can reduce obesity signs and hepatic steatosis in mice. 1.1 Isorhamnetin can reduce obesity signs in MASLD mice. Technical scheme: 36 male C57BL / 6 mice, 5 weeks old, weighing 20±2.5 g, were housed in a mouse house with a temperature of 25±1℃, humidity of 45%±5%, and natural light and dark cycles, and were provided with sufficient feed and water. After one week of acclimatization, mice were randomly divided into six groups: a normal diet (ND) group fed a normal diet, a high-fat diet (HFD) group, a positive control (PC) group, a low-dose isorhamnetin treatment group (ISOL) (12.5 mg / kg / d), a medium-dose isorhamnetin treatment group (ISOM) (25 mg / kg / d), and a high-dose isorhamnetin treatment group (ISOH) (50 mg / kg / d), all fed a high-fat diet for 12 weeks. After confirming that the high-fat diet mice weighed 15%–25% more than the normal diet mice and that their serum TG and TC levels were significantly elevated, thus confirming successful model establishment, the PC group was administered atorvastatin orally at 10 mg / kg / day daily; the ISOL, ISOM, and ISO-H groups were administered the corresponding doses of isorhamnetin orally daily; and the ND and HFD groups were given double-distilled water for 6 weeks. The feeding and grouping of the experimental animals are shown in Figure 1A (in Figure 1, there are significant differences between different letters (p<0.05) (n=3), the same below). During the feeding period, the mice's weight and food intake were monitored weekly.

[0045] As shown in Figure 1B, the mice in the HFD group gained significantly more body weight than the ND group over 12 weeks. However, under continuous intervention with atorvastatin and different doses of isorhamnetin, the mice's body weight gradually decreased. At the end of the experiment (18 weeks), the final body weight and weight gain of the mice in the HFD group were significantly higher than those in the ND group. Atorvastatin and different doses of isorhamnetin significantly reduced these parameters, with no difference compared to the ND group (Figure 1C / D). There was no significant change in food intake among the groups, indicating that the decrease in body weight and liver weight was not due to reduced energy intake (Figure 1E).

[0046] 1.2 Technique for reducing hepatic steatosis in MASLD mice using isorhamnetin: After the experiment, mice were sacrificed, the thoracic cavity was dissected, and the liver was removed, weighed, and a portion was stored at -80℃, while another portion was fixed in 4% paraformaldehyde solution for 36 h. After gradient dehydration with ethanol, xylene clearing, paraffin embedding, and HE staining, the pathological degree of the tissue was assessed (NAS score). The steatosis grade was divided into 0-3 (Grade 0: <5%; Grade 1: 5%-33%; Grade 2: 34%-66%; Grade 3: >66% of hepatocytes showed steatosis), and the inflammation grade was divided into 0-3 (Grade 0: 0 inflammatory lesions; Grade 1: <2 inflammatory lesions; Grade 2: 2-4 inflammatory lesions; Grade 3: >4 inflammatory lesions). After freezing and sectioning the liver tissue, it was stained with Oil Red O working solution and hematoxylin, and the distribution of lipid droplets was observed under a microscope.

[0047] As shown in Figure 2A / B, histopathological examination revealed that atorvastatin and isorhamnetin intervention effectively reduced liver weight and reversed HFD-induced liver jaundice and hypertrophy. The ISODL group showed a gradual recovery trend from the central vein to the lobular margins, suggesting that isorhamnetin has a progressive ameliorative effect on HFD-induced liver injury. HE staining results showed that the livers of mice in the HFD group exhibited significant macrovesicular steatosis, hepatocyte ballooning degeneration, and inflammatory cell infiltration. The degree of lesions improved in a dose-dependent manner in each group after isorhamnetin intervention, with the high-dose group showing near-normal liver pathological characteristics. NAS scores indicated a tendency for mice in the HFD group to develop NASH. After drug treatment, hepatocyte steatosis and ballooning degeneration significantly improved, and inflammatory foci decreased (Figure 2D). Oil Red O staining results (Figure 2A / C) showed that hepatocytes in the HFD group contained a large number of diffusely distributed red lipid droplets. The lipid droplet area decreased in the PC group and the isorhamnetin treatment group, with the smallest lipid droplet area in the ISODL / M group, significantly lower than that in the PC group.

[0048] 1.3 Technique for reducing fat weight in MASLD mice by isorhamnetin: After the experiment, the mice were sacrificed, the abdominal cavity was opened, and eWAT and iWAT were removed, weighed, and fixed in 4% paraformaldehyde solution for 36 h. After ethanol gradient dehydration, xylene clearing, paraffin embedding, sectioning and HE staining were performed.

[0049] As shown in Figure 3 (AD), intervention with atorvastatin and isorhamnetin significantly reduced the weight of eWAT and iWAT in mice and decreased the mean area and diameter of eWAT adipocytes. The mean area of ​​eWAT adipocytes in the ISOL group was significantly higher than that in the ISOM / H group, suggesting that isorhamnetin can dose-dependently inhibit peripheral fat accumulation in MASLD mice.

[0050] Example 2: Isorhamnetin regulates lipid metabolism and reduces blood lipid levels in MASLD mice. 2.1 Isorhamnetin reduces blood lipid levels in MASLD mice. Technical procedure: After the feeding experiment as described in Example 1, the mice were sacrificed, and the liver was removed from the thoracic cavity. Blood was collected from the eyeballs, and the blood was placed in a 4°C refrigerator overnight. Then, it was centrifuged at 3000 r / min for 5 min at 4°C to separate the serum. The levels of TG, TC, HDL-c, and LDL-c in the serum and liver were measured using a kit.

[0051] As shown in Figure 4A / B: Compared with HFD, the serum and liver TG, TC, and LDL-c levels in the PC and isorhamnetin intervention groups were significantly reduced, while HDL-c levels were significantly increased. The serum and liver TG and TC levels in the ISH group were significantly lower than those in the ISH / M group, and there was no difference compared to the ND and PC groups. This indicates that isorhamnetin reduces blood lipid levels in MASLD mice in a dose-dependent manner.

[0052] 2.2 Technical procedure for reducing liver damage and FFA content in MASLD mice using isorhamnetin: After the feeding experiment as described in Example 1, mice were sacrificed, and blood was collected from the eyeballs. The blood was placed in a 4°C refrigerator overnight, then centrifuged at 3000 r / min for 5 min at 4°C to separate the serum. The levels of AST, ALT, and FFA in the serum were determined using a kit.

[0053] As shown in Figure 5A, isorhamnetin intervention significantly reduced serum ALT and AST levels, effectively reducing mild liver damage caused by HFD. Furthermore, isorhamnetin intervention significantly reduced serum FFA levels and decreased the body's fatty acid content (as shown in Figure 5B).

[0054] 2.3 Isorhamnoside can improve lipid metabolism in MASLD mice. The technical protocol was as follows: qPCR detection: Total RNA was extracted from the liver, and after detecting the RNA concentration, it was reverse transcribed into cDNA. The relative gene expression level was detected using real-time quantitative qPCR. The reaction conditions were: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, followed by denaturation at 60°C for 1 min, for 40 cycles; finally, a melting reaction was performed. β-actin protein was used as a control to quantify mRNA expression levels. The calculation method used was 2... -ΔΔCt .

[0055] Western blot assay: Liver tissue was lysed and quantified using protein lysis buffer containing protease inhibitors and phosphatase inhibitors. SDS-PAGE was used for separation. The molecular weight regions of target proteins indicated by pre-stained markers were excised and transferred to a PVDF membrane. The membrane was blocked with skim milk powder at room temperature for 2 h. At the end of blocking, primary antibody was added and the membrane was incubated overnight at 4°C. The primary antibody was washed off, followed by incubation with secondary antibody for 1 h, then washed off again. After exposure, the developed protein bands were quantified, and the relative expression levels of the measured proteins were compared with the gray values ​​of the internal reference gene β-actin.

[0056] Western blot and qPCR results, as shown in Figure 6 (AC), indicate that isorhamnetin significantly inhibited the upregulation of adipogenesis-related genes and proteins (SREBP1c, FASn, ACC1, SCD1, and DGAT2), with the ISH group showing significantly stronger inhibition than the ISOM and ISOL groups, and some indicators approaching the ND level. Furthermore, isorhamnetin intervention significantly upregulated the expression levels of genes and proteins regulating fatty acid breakdown (PPARα, CPT1A, and HSL), while significantly downregulated CD36, which regulates fatty acid uptake. These results suggest that all three doses of isorhamnetin can balance hepatic lipid metabolism and lipid synthesis, but the degree of improvement is dose-dependent, with the highest dose of isorhamnetin showing the most significant effect.

[0057] Example 3: A technique for improving the gut microbiota composition of MASLD mice using isorhamnetin: Twenty-four 5-week-old male C57BL / 6 mice (20±2.5 g) were randomly divided into ND, HFD, ISONH, and fecal microbiota transplantation (FMT) groups. Mice were fed according to the method in Example 1. After 12 weeks of HFD administration, FMT mice were given daily oral antibiotics (ampicillin 150 mg / kg, vancomycin 75 mg / kg, metronidazole 150 mg / kg, neomycin trisulfate 150 mg / kg) for one week. Fresh feces from ISONH mice were then collected daily and dissolved thoroughly in sterile PBS at a 1:10 mass / volume ratio. The solution was vortexed and centrifuged (800×g, 4°C, 3 min), and the supernatant was provided to FMT (recipient) mice for 6 weeks.

[0058] Mice were euthanized after feeding, and cecal contents were collected. Genomic DNA was extracted according to the kit instructions. The V3–V4 hypervariable region of the bacterial 16S rRNA gene (approximately 480 bp in length) was sequenced and amplified. After paired-end sequencing, the raw data underwent splitting and quality control, sequence assembly, and dechimeric extraction. DADA2 was used for quality filtering and noise reduction to obtain characteristic sequences. Subsequently, OTU clustering analysis, α-diversity analysis, and β-diversity analysis were performed on the QIIME2 platform.

[0059] As shown in Figure 7A, Venn diagram analysis revealed significant differences in gut microbiota composition among the groups, with only 54 OTUs shared by all four groups. Combined with α-diversity analysis (Figure 7B), this further confirmed that HFD reduces gut microbiota richness, diversity, and evenness, while ISOH intervention restores these to normal levels. In contrast, the number and diversity of gut microbiota colonized after FMT were limited. PCA analysis (Figure 7C) showed that the ND, ISOH, and FMT groups were all significantly separated from the HFD group in terms of community structure. The ISOH group showed a significant convergence towards the ND group, while the FMT group was close to the ISOH group in terms of community structure. This indicates that ISOH can effectively correct HFD-induced gut microbiota dysbiosis, while FMT can only partially transfer donor microbiota characteristics, with limited restoration. The relative abundance at the phylum level (D in Figure 7) showed that the relative abundance of Bacteroidota was reduced and Firmicutes was increased in the HFD group, resulting in an increase in the F / B ratio; this imbalance was corrected after ISH intervention; FMT replicated this change, with the F / B ratio approaching that of the ND group.

[0060] Example 4: Isorhamnetin can improve bile acid metabolism in MASLD mice. 4.1 Isorhamnetin can improve bile acid content in the cecal contents of MASLD mice. Technical scheme: After feeding the animals according to Example 3 and obtaining the cecal contents, a mixed internal standard working solution was added. After vortex mixing and centrifugation, the supernatant was collected and concentrated. Ultra-high performance liquid chromatography coupled with tandem mass spectrometry was used for qualitative and quantitative analysis of bile acids in the sample.

[0061] The downregulation of multiple bile acids induced by HFD included tauro-β-muricholic acid (Tβ-MCA), taurocholic acid (TCA), tauro-α-muricholic acid (Tα-MCA), taurohyocholic acid (THCA), chenodeoxycholic acid 3-sulfate disodium salt (CDCA-3S), taurochenodeoxycholic acid (TCDCA), lithocholic acid 3-sulfate (LCA-3S), glycochenodeoxycholic acid (GCDCA), and tauroursodeoxycholic acid. The increases in TUDCA, taurohyodeoxycholic acid (THDCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), and isochenodeoxycholic acid (isoCDCA) were particularly significant. Furthermore, the total CA / total CDCA ratio was significantly increased in the HFD group (Figure 8, C), indicating a possible shift in the bile acid synthesis pathway. Notably, in the FMT group, apart from a significant increase in TCA-3S, most secondary bile acids, such as deoxycholic acid (DCA), 3β-deoxycholic acid (3β-DCA), ω-muricholic acid (ω-MCA), and lithocholic acid (LCA), and their proportions were significantly lower than in the ISOH group (Figure 8, D). This may be a result of gut microbiota regulation, and this change can reduce the content of highly hydrophobic bile acids and their stimulation of the intestine, thus helping to maintain intestinal homeostasis.

[0062] 4.2 Technique for regulating the expression of the FXR pathway in MASLD mice by isorhamnetin: After feeding animals according to Example 3 and obtaining liver, ileum, feces, and serum, the total bile acid content was detected. The relative expression levels of liver proteins and mRNA were extracted and detected according to the method in Example 2.

[0063] As shown in Figure 9 (AC): Compared with the ND group, the HFD group showed significantly decreased protein and gene expression levels of FXR (Nr1h4), SHP (Nr0b2), Abcb11, and CYP27A1 (Cyp27a1, alternative pathway bile acid synthase) in the liver, while significantly increased expression levels of NTCP (Slc10a1) and CYP7A1 (Cyp7a1, classical pathway bile acid synthase). All these changes were reversed after ISOH treatment. The results indicate that ISOH can activate the FXR signaling pathway in the liver of MASLD mice, accelerating the efflux of bile acids from the liver, reducing hepatic bile acid reabsorption, and alleviating tissue bile acid load. Furthermore, bile acid synthesis shifts from the classical pathway to the alternative pathway, thereby altering the composition of the bile pool. Compared with ISOH, FMT showed differences only in the expression of SHP and NTCP proteins and the genes Nr0b2 and Abcb11 in the liver, suggesting that the gut microbiota plays an important role in ISOH-mediated FXR-bile acid regulation. As shown in Figure 9D, HFD significantly disrupted bile acid distribution, with a significant increase in total bile acids in mouse serum and liver, and a significant decrease in total bile acids in feces. ISOH intervention promoted bile acid efflux and reduced the bile acid burden on the liver. The overall trend of change in the FMT group was consistent with that of ISOH, indicating that gut microbiota plays an important role in ISOH-mediated bile acid redistribution.

[0064] Example 5: A technique for reducing intestinal lipid absorption in MASLD mice using isorhamnetin: Animals were fed according to Example 3, and jejunum, ileum, feces, and intestinal contents were obtained. The levels of lipase in the intestinal contents and TG and TC in the feces were then detected. The relative expression levels of mRNA in the jejunum and ileum were extracted and detected according to the method in Example 2.

[0065] As shown in Figure 10A, the lipase content in the jejunum and ileum of the HFD group was significantly decreased, suggesting impaired pancreatic exocrine secretion or intestinal enzyme environment. ISOH intervention significantly increased the intestinal lipase content, and FMT also showed an increasing trend, but the magnitude was weaker. However, the fecal TG and TC content in both the ISOH and FMT groups increased (Figure 10B), suggesting that the net absorption of TG and TC by the small intestine was actually reduced. To determine the rate-limiting link of lipid absorption, genes related to lipid transport / loading in the small intestine were detected (Figure 10C): ISOH significantly downregulated Cd36, Slc27a4, Npc1l1, and Mttp in the jejunum and ileum, and their expression levels were close to those in the ND group; the inhibitory effect in the FMT group was weaker overall compared to the ISOH group. The results indicate that ISOH can reduce intestinal absorption flux and alleviate the body's lipid load by inhibiting lipid transmembrane uptake and loading.

[0066] This invention provides the use of isorhamnetin or a pharmaceutically acceptable derivative thereof in the preparation of products for the prevention and / or treatment of obesity or MASLD, and provides corresponding dosing / usage regimens. The preferred usage regimen is: oral administration of isorhamnetin to MASLD subjects at a dose of 12.5 mg / kg·d to 50 mg / kg·d for 6 consecutive weeks.

[0067] (1) Active ingredient: isorhamnetin, with a purity of ≥98%.

[0068] (2) Applicable subjects: Applicable to subjects with metabolic dysfunction and accompanying liver lipid deposition; preferably subjects with HFD-induced MASLD or subjects with abnormal MASLD-related indicators.

[0069] (3) Mechanism chain verification: FXR signal related indicators were detected, the composition of bile acid pool and changes in gut microbiota structure were evaluated, and the disease was confirmed to be relieved through the "gut microbiota-bile acid-lipid metabolism" axis.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of isorhamnetin or its pharmaceutically acceptable salts, esters or derivatives in the preparation of medicaments for the prevention and treatment of fatty liver disease associated with metabolic dysfunction.

2. A drug for the prevention and treatment of fatty liver disease related to metabolic dysfunction, characterized in that, The active ingredient includes isorhamnetin or its pharmaceutically acceptable salts, esters or derivatives.

3. The use of isorhamnetin or its pharmaceutically acceptable salts, esters or derivatives in the preparation of drugs for the prevention and treatment of diseases caused by intestinal flora imbalance.

4. A drug for preventing and treating diseases caused by intestinal flora imbalance, characterized in that, The active ingredient includes isorhamnetin or its pharmaceutically acceptable salts, esters or derivatives.

5. The drug for preventing and treating diseases caused by intestinal flora imbalance according to claim 4, characterized in that, It also includes pharmaceutically acceptable excipients.

6. The application of isorhamnetin in the preparation of drugs, health foods or food supplements for lowering blood lipids.

7. Application of isorhamnetin in the preparation of drugs, health foods or food supplements for liver protection.

8. The application of isorhamnetin in the preparation of drugs, health foods or food supplements for regulating bile acids.

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