Application of isohesperidin in preparation of medicine for preventing and treating obesity or related metabolic syndrome thereof

Abstract

The invention belongs to the field of biological medicines, and relates to application of isohesperidin or pharmaceutically acceptable salts thereof in preparation of medicines for preventing and / or treating obesity or related metabolic syndromes thereof. The invention finds that the natural compound isohesperidin can obviously induce adipocyte browning and improve the expression level and the cell oxygen consumption rate of uncoupling protein 1, so that the weight gain is effectively inhibited and the glucose homeostasis and the insulin sensitivity are improved in an obesity mouse model, and meanwhile, the isohesperidin can be used for inhibiting the adipocyte browning. No obvious cytotoxicity and animal toxicity are observed in an effective dose of isohesperidin, so that the isohesperidin has better safety and is expected to be used for preventing and / or treating obesity or related metabolic syndromes thereof.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of isoobtusilactone in the preparation of drugs for preventing and treating obesity or its related metabolic syndrome. BACKGROUND

[0002] Obesity and its related metabolic syndrome (such as type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease, etc.) have become a major global public health problem. The fundamental cause is the imbalance between energy intake and consumption. White adipose tissue (WAT) is responsible for energy storage, while brown adipose tissue (BAT) consumes energy through heat production.

[0003] Fat browning refers to the process of white fat transforming into beige fat similar to brown fat under certain stimuli (such as cold, exercise, or drugs). Beige fat also highly expresses the key heat-producing protein uncoupling protein 1 (Ucp1) and can efficiently consume energy to produce heat. Therefore, finding compounds that can safely and effectively promote fat browning to reduce fat accumulation by increasing energy consumption has become a new strategy for treating obesity and its related metabolic diseases.

[0004] Currently, some compounds have been reported to have the potential to induce fat browning, such as β-adrenergic receptor agonists (such as CL316,243). However, such drugs often have cardiovascular side effects (such as tachycardia, hypertension, etc.), limiting their clinical application. Therefore, developing new, safe, and efficient fat browning inducers has important clinical significance and market demand. SUMMARY

[0005] The inventors found that the natural compound isoobtusilactone (ISM) can significantly induce fat cell browning, increase the relative expression level of Ucp1 and cell oxygen consumption rate, thereby effectively inhibiting weight gain, improving glucose homeostasis and insulin sensitivity in an obesity mouse model. At an effective dose, isoobtusilactone was not observed to have significant cytotoxicity and animal toxicity, and was safe.

[0006] The technical solution provided by the present application is as follows: The present application provides the application of isoobtusilactone or its pharmaceutically acceptable salt in the preparation of a drug for preventing and / or treating obesity or its related metabolic syndrome.

[0007] In some embodiments, the obesity at least includes one of the following symptoms: overweight, excessive body mass index, excessive body fat rate, and abnormal fat distribution.

[0008] In some embodiments, the obesity is caused by excessive energy intake or reduced metabolic rate leading to reduced energy consumption.

[0009] In some embodiments, the drug also has the following therapeutic effects: inhibiting weight gain, slowing down fat accumulation, improving glucose metabolism, and increasing insulin sensitivity.

[0010] In some embodiments, reducing fat accumulation includes reducing fat accumulation in at least one of the following areas: liver, groin, and epididymis.

[0011] In some embodiments, the associated metabolic syndrome includes at least one of type 2 diabetes, insulin resistance, non-alcoholic fatty liver disease, and hyperlipidemia.

[0012] In some embodiments, the drug comprises: A therapeutically effective amount of isohesperidin or a pharmaceutically acceptable salt thereof; and Pharmaceutically acceptable carrier.

[0013] In some embodiments, the dosage form of the drug is an oral preparation or an injection; the oral preparation is one or more of tablets, capsules, granules, and oral liquids; the injection is an injection solution and / or lyophilized powder for injection.

[0014] In some embodiments, the dosage form of the drug is a sustained-release tablet or a sustained-release capsule.

[0015] In some embodiments, the drug also comprises CL316243.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: This invention utilizes isohesperidin, a natural compound, to achieve safe, effective, and side-effect-free obesity prevention and fat reduction. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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.

[0018] Figure 1 : A program for isohesperidin to activate the expression of thermogenic genes in mouse white adipocytes; wherein: A: The structure of ISM; B: CCK8 assay of cell viability in primary inguinal adipocytes treated with different concentrations of ISM (n=3). C: Relative expression level of Ucp1 mRNA in mature primary inguinal adipocytes treated with different concentrations of ISM (n=3). D: Representative immunoblot images of Ucp1 in mature primary inguinal adipocytes treated with different concentrations of ISM; E: Statistical graph of quantitative analysis of relative protein expression levels of Ucp1 in mature primary inguinal adipocytes treated with different concentrations of ISM (n=3). F: Representative immunoblot images of Pgc1α in mature primary inguinal adipocytes in the control group and the ISM (50 μM) treatment group; G: Statistical graph of quantitative analysis of the relative expression level of Pgc1α in mature primary inguinal adipocytes in the control group and the ISM (50 μM) treatment group (n=3). H: Relative mRNA expression levels of thermogenic genes, mitochondrial genes and β-oxidation-related genes in mature primary inguinal adipocytes in the control group and the ISM (50 μM) treatment group (n=3).

[0019] Figure 2 Isohexolone promotes mitochondrial biosynthesis and enhances its function in mouse white adipocytes; among which: A: MitoTracker staining representation of mature primary inguinal adipocytes in the control group and the ISM (50 μM) treatment group, scale bar = 50 μm; B: Quantitative analysis of MitoTracker staining (n=3); C: Oxygen consumption rate (OCR) of mature primary inguinal adipocytes in the control group and the ISM (50 μM) treatment group; D: Quantitative analysis of OCR (n=3).

[0020] Figure 3 Isohexolone promotes lipolysis in mouse white adipocytes; among which: A: Oil Red O staining representation of mature primary inguinal adipocytes in the control group and the ISM (50 μM) treatment group, scale bar = 50 μm; B: Quantitative analysis of Oil Red O staining (n=3); C: Relative content of non-esterified fatty acids (NEFA) in the culture medium of mature primary inguinal adipocytes in different groups (n=3). D: Representative immunoblot images of lipolysis proteins from mature primary inguinal adipocytes in different groups; E: Quantitative analysis of immunoblotting of lipolysis proteins (n=3).

[0021] Figure 4Isohexolone can improve obesity induced by a high-fat diet (HFD) in mice; among which: A: Body weight of mice in different groups after being fed a high-fat diet (n=6); B: Gross morphological representation of the mice at the experimental endpoint; C: A representative diagram of the gross morphology of mouse adipose tissue (iWAT, eWAT, and BAT); D: Weight of mouse tissues and organs (iWAT, eWAT, BAT, and liver) (n=6).

[0022] Figure 5 Isohexolone can improve lipid metabolism and liver fat deposition in HFD-induced obese mice; among which: A: Serum lipid (triglycerides, total cholesterol, low-density cholesterol lipoprotein, high-density cholesterol lipoprotein) levels after 13 weeks of high-fat diet feeding (n=6); B: A representative diagram of the gross morphology of a mouse liver; C: Representative image of H&E staining of liver sections, scale bar = 50 μm; D: Quantitative analysis of serum ALT levels in mice from different groups (n=6); E: Quantitative analysis of serum AST levels in mice from different groups (n=6).

[0023] Figure 6 Isohesperidin can improve glucose tolerance and insulin sensitivity in HFD-induced obese mice; among which: A: Glucose tolerance test 11 weeks after HFD (n=6); B: Quantitative analysis of glucose tolerance test (n=6); C: Insulin tolerance test 12 weeks after HFD (n=6); D: Quantitative analysis of insulin tolerance test (ITT) (n=6); E: Quantitative analysis of serum creatinine (CREA) levels in mice from different groups (n=6).

[0024] Figure 7 Isohesperidin enhanced the thermic capacity of HFD-induced obese mice; among which: A: Food intake of mice (n=6); B: Average physical activity of mice (n=6); C: Whole-body condition of mice under basal conditions V O2 (n=6); D: In Figure C V Quantitative analysis of O2 (n=6); E: Whole-body function of mice under basal conditions VCO2 (n=6); F: In Figure E V Quantitative analysis of CO2 (n=6); G: Respiratory exchange rate (RER, n=6) in mice. H: Quantitative analysis of RER in Figure I (n=6); I: Rectal temperature of mice under different treatments was measured at 4°C for a specified time (n=6).

[0025] Figure 8 Isohexolone promoted the browning of subcutaneous white adipose tissue in HFD-induced obese mice; among which: A: Relative expression level of Ucp1 mRNA in BAT (n=6); B: Representative immunoblot diagram of Pgc1α and Ucp1 in BAT; C: Quantitative analysis of the relative expression levels of the proteins shown in Figure B (n=6); D: Relative expression level of Ucp1 mRNA in eWAT (n=6); E: Representative immunoblot image of Pgc1α and Ucp1 in eWAT; F: Quantitative analysis of the relative expression levels of the proteins shown in Figure E (n=6); G: Relative mRNA expression levels of thermogenesis and lipogenesis genes in iWAT (n=6). H: Representative immunoblot images of Pgc1α, Ucp1, and Fabp4 in iWAT; I: Quantitative analysis of the relative expression levels of the proteins shown in Figure H (n=6). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] Isomeranzin, also known as 7-methoxy-8-(3-methyl-2-oxobutyl)coumarin and 7-methoxy-8-(3-methyl-2-oxobutyl)-2H-pyran-2-one, has a molecular weight of 260.29 and a molecular formula of C2. 15 H 16 O4, CAS No.: 1088-17-1, structural formula as follows Figure 1As shown in Figure A, isohesperidin, a common coumarin-type natural compound, is widely found in various medicinal plants, primarily in the peel and juice of citrus fruits. It has also been extracted from medicinal plants such as Leonurus japonicus and Murraya paniculata. Current literature reports that isohesperidin mainly exerts anti-inflammatory effects, and some reports suggest it may be a potential D2 dopamine receptor antagonist. However, there are currently no reports on isohesperidin promoting fat browning or its use in treating obesity.

[0028] This invention investigated ISM intervention in an obese mouse model fed a high-fat diet. The results showed that ISM could slow weight gain induced by a high-fat diet (Example 4), reduce hepatic fat deposition (Example 5), and significantly improve glucose tolerance and insulin sensitivity in the obese mouse model (Example 6), indicating that ISM can be used to prevent obesity, fatty liver, and diabetes. Furthermore, this invention found that ISM does not exhibit nephrotoxicity when exerting its therapeutic effect (Example 6), confirming its biocompatibility and suggesting its potential as a replacement for previously reported drugs that induce fat browning.

[0029] This invention provides the use of isohesperidin or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of obesity or related metabolic syndromes.

[0030] In some embodiments, the obesity includes at least one of the following symptoms: Overweight: Weight exceeding the normal range for the same sex, age, and height. Usually, a body mass index (BMI) ≥ 30 is used as the diagnostic criterion for obesity. Exceeding the standard body mass index (BMI): BMI ≥ 30 is a medically recognized diagnostic criterion for obesity; Excessive body fat percentage: Men with a body fat percentage ≥25% and women with a body fat percentage ≥32% (according to the American College of Sports Medicine standards); Abnormal fat distribution: Significant accumulation of abdominal fat, manifested as excessive waist circumference (≥90cm for men, ≥85cm for women). Metabolic abnormalities: such as hyperglycemia, hyperinsulinemia, dyslipidemia, and other metabolic disorders.

[0031] In some embodiments, the obesity is caused by excessive energy intake or reduced energy expenditure due to a decreased metabolic rate. Specifically, this is due to either excessive energy intake from a long-term high-fat, high-sugar diet or reduced energy expenditure due to a decreased metabolic rate. Obesity is primarily characterized by abnormal accumulation of adipose tissue, particularly an abnormal increase in visceral and subcutaneous fat. The drug of this invention, when treating this type of obesity, increases energy expenditure and reduces fat accumulation by promoting the browning of adipocytes, transforming white adipose tissue into beige adipose tissue.

[0032] In some embodiments, the drug simultaneously has the following therapeutic effects: Inhibits weight gain: By promoting the browning of fat cells and increasing energy expenditure, the rate of weight gain is significantly lower than that in the control group. Figure 4 AB); Improve blood glucose metabolism: enhance glucose tolerance ( Figure 6 AB), to lower fasting blood glucose levels; Improve insulin sensitivity: Enhance tissue response to insulin and improve insulin resistance. Figure 6 CD); Slow down fat accumulation, especially reducing fat accumulation in the following areas: Liver: Reduces the formation of fatty liver ( Figure 5 C), improves symptoms of non-alcoholic fatty liver disease ( Figure 4 D、 Figure 5 B); Groin: Reduces subcutaneous fat accumulation (Example 4), improves appearance and metabolic function; Epididymis: Reduces visceral fat accumulation (Example 5), lowering metabolic risk.

[0033] The related metabolic syndromes described in this invention include, but are not limited to, at least one of the following diseases: Type 2 diabetes: Effectively control type 2 diabetes by improving insulin sensitivity and glucose metabolism; Insulin resistance: Reversing insulin resistance by increasing the sensitivity of fat cells and muscle tissue to insulin; Non-alcoholic fatty liver disease: Improves fatty liver symptoms by reducing fat accumulation in the liver; Hyperlipidemia: Improves dyslipidemia by lowering blood triglyceride and cholesterol levels; Metabolic syndrome: Meeting at least 3 of the following criteria: abdominal obesity, hypertriglyceridemia, low HDL cholesterol, hypertension, and hyperglycemia.

[0034] The drug of the present invention comprises: (1) a therapeutically effective amount of isohesperidin or a pharmaceutically acceptable salt thereof: the effective dose range is 1 to 50 mg / kg body weight, and the specific dose is adjusted according to the patient's weight and condition. The experiment in Example 6 showed that no obvious cytotoxicity or animal toxicity was observed in isohesperidin within the range of 1 to 15 mg / kg body weight; (2) a pharmaceutically acceptable carrier: including but not limited to common pharmaceutical excipients such as starch, lactose, microcrystalline cellulose, and hydroxypropyl methylcellulose.

[0035] In some embodiments, the dosage form of the drug is an oral preparation, which is one or more of tablets, capsules, granules, and oral liquids; preferably: sustained-release tablets, which use special coating or matrix technology to allow isohesperidin to be slowly released in the body and maintain an effective blood drug concentration for a longer period of time; or sustained-release capsules, which use microencapsulation technology to achieve a sustained-release effect of the drug.

[0036] In some embodiments, the dosage form of the drug is an injection, specifically an injectable solution or lyophilized powder for injection, suitable for patients requiring rapid onset of action or those with poor oral absorption.

[0037] In some embodiments, the drug also comprises CL316243. Figure 3 CE indicates that ISM can enhance the lipolysis stimulated by CL316243 and improve the fat breakdown effect, and the two have a synergistic fat reduction effect.

[0038] The technical solution of the present invention will be described in detail below through specific embodiments. Unless otherwise stated, the raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods.

[0039] Unless otherwise specified, the concentrations in the following examples refer to the final concentrations in the culture medium, incubation solution, or treatment solution.

[0040] The CL316243 used in this embodiment is a highly potent selective β3-adrenergic receptor agonist with an EC50 of 3 nM. It is an effective lipolysis stimulant for adipocytes, increasing thermogenesis and metabolic rate in brown adipose tissue, and has the potential to treat obesity, diabetes, and urge incontinence.

[0041] Example 1: Program for activating the expression of thermogenic genes in mouse white adipocytes using isohesperidin Cell culture and differentiation induction: Mouse vascular stromal component cells (SVF cells) were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum). Two days after cell confluence, they were induced to differentiate into mature adipocytes using the standard "cocktail" method (3-isobutyl-1-methylxanthine, dexamethasone, and insulin).

[0042] Drug treatment: After differentiation, the cells were divided into experimental group (2 μM, 10 μM, and 50 μM isohesperidin DMSO solution added respectively) and control group (equal volume of DMSO added), and incubated at 37°C for 48 hours.

[0043] CCK8 assay for cytotoxicity: Mouse SVF cells were seeded at a density of 5000 cells per well in 96-well plates. The cells were then incubated with 0.1% (v / v) DMSO and DMSO solutions of different concentrations of isohesperidin (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM). After 48 hours, 10 μL of CCK8 reagent was added and incubated for 2 hours. The absorbance at 450 nm was measured using a microplate reader.

[0044] qRT-PCR assay: Total RNA was extracted from cells and reverse transcribed into cDNA. Quantitative PCR was performed using the SYBR Green assay to detect the mRNA expression levels of key browning marker genes (Ucp1, Cidea, Pgc1α, etc.). 18S rRNA was used as an internal reference gene, and a 2... -ΔΔCt The relative expression level is calculated using this method.

[0045] Western Blot analysis: Total protein was extracted from cells and its concentration was determined using the dioctanine acid assay (BCA method). Equal amounts of protein were subjected to SDS-PAGE electrophoresis. After transfer to a membrane, the membrane was incubated with primary antibodies against Ucp1 and Pgc1α, and corresponding horseradish peroxidase (HRP)-labeled secondary antibodies to enhance chemiluminescence (ECL) imaging.

[0046] Results: To assess the biosafety of isohesperidin, a CCK8 cytotoxicity assay was performed. The experimental design was as follows: SVF cells were isolated from mouse inguinal white adipose tissue (iWAT) and co-incubated with different concentrations of ISM. Cell viability was assessed after 48 hours. The results showed that even at a high concentration of 50 μM, ISM did not have toxicity to mouse SVF cells. Figure 1 B). To investigate the optimal concentration of ISM for its best effect, the experiment was designed as follows: First, mouse SVF cells were induced to differentiate into mature mouse primary inguinal adipocytes. Then, these cells were incubated with ISM at different concentration gradients. The optimal concentration was determined by detecting the efficacy of ISM in promoting the expression of thermogenic genes in mature white adipocytes at each concentration. The results showed that ISM dose-dependently increased the mRNA abundance of Ucp1 in mouse adipocytes (…). Figure 1 C) and protein abundance ( Figure 1 DE). Next, after treating mouse adipocytes with ISM at a maximum concentration of 50 μM, the protein expression levels of thermogenic genes were observed ( Figure 1 FG) and mRNA expression levels ( Figure 1 Both H) increased. These results indicate that ISM activates the expression program of thermogenic genes in mouse white adipocytes.

[0047] Example 2: Isohexolone promotes mitochondrial biosynthesis and enhances function in mouse white adipocytes. Cell preparation and treatment: Same as in Example 1, mature adipocytes were treated with 50 μM isohesperidin or DMSO for 48 hours.

[0048] MitoTracker staining: The culture medium was removed from differentiated mature adipocytes, and then a staining solution containing 200 nM MitoTracker Red CMXRos, preheated to 37°C, was added. Cells and probes were incubated for 30 minutes under standard culture conditions. Images were acquired using a fluorescence microscope after staining. Quantitative analysis was performed using Image-J software.

[0049] Oxygen consumption rate (OCR) measurement: Mitochondrial respiratory function of cells was detected using a Seahorse XF cellular energy metabolism analyzer. Oligomycin (ATP synthase inhibitor), FCCP (uncoupling agent), and Rotenone / Antimycin A (complex I / III inhibitor) were injected sequentially, and basal respiration, ATP production, proton leakage, maximum respiratory capacity, respiratory reserve, and non-mitochondrial respiration were measured, respectively.

[0050] Results: The abundance of mitochondria in adipocytes was significantly increased in the ISM-treated group. Figure 2 AB), and its basal respiration, proton leakage, maximum respiratory capacity, and respiratory reserve were all significantly higher than those of the control group (AB). Figure 2 CD). This directly demonstrates that isohesperidin treatment promotes mitochondrial biosynthesis and enhances function in mouse white adipocytes.

[0051] Example 3: Isohexolone promotes lipolysis in mouse white adipocytes Cell preparation and treatment: Same as in Example 1, mature mouse primary adipocytes were treated with 50 μM isohesperidin or DMSO for 24 hours, followed by simultaneous addition of PBS or 1 μM CL316243, and treatment continued for another 24 hours before lipid breakdown was assessed. Grouping is shown in Table 1. Table 1

[0052] Oil Red O staining: Adipocytes were fixed with 4% paraformaldehyde for 10 minutes, washed twice with phosphate-buffered saline (PBS), and then stained with Oil Red O solution for 30 minutes. Subsequently, the cells were differentiated with 60% isopropanol for 5 seconds, followed by washing three times with pure water. Oil Red O staining images were recorded under a microscope and analyzed using Image-J software.

[0053] NEFA assay: The NEFA level in the culture medium is determined using a specific kit, following the manufacturer's instructions.

[0054] Western Blot detection: Same as in Example 1, except that the detection antibody is replaced with a primary antibody against lipolysis protein and its phosphorylation site.

[0055] Results: Lipid droplets in adipocytes of mice in the ISM group were significantly "fragmented" ( Figure 3 A), and the fat content was significantly reduced ( Figure 3 B). Furthermore, ISM significantly increased the NEFA content in the culture medium of adipocytes (B). Figure 3 C), and promotes the phosphorylation of the lipolytic protein hormone-sensitive lipase (HSL). Figure 3 DE), which directly demonstrates that ISM treatment promotes lipolysis in mouse white adipocytes. Simultaneously, ISM also enhanced the lipolysis stimulated by CL316243 (DE). Figure 3 CE).

[0056] Example 4: Isohexolone can improve obesity induced by a high-fat diet (HFD) in mice. Establishment and treatment of obese mouse model: Twenty-four 8-week-old male C57BL / 6J mice were randomly and equally divided into 4 groups. The mice were fed for 13 weeks and simultaneously injected intraperitoneally. The grouping and treatment details are shown in Table 2. Table 2

[0057] All mice were administered the medication once daily. Food intake and body weight were measured weekly.

[0058] Mice underwent glucose tolerance tests (GTT) and insulin tolerance tests (ITT) after 11 and 12 weeks of continuous feeding, respectively. One week later, metabolic parameters were measured using the Comprehensive Laboratory Animal Monitoring System (CLAMS). At 13 weeks, mice were euthanized under anesthesia, and serum and tissue samples were collected for further analysis.

[0059] Results: Compared with the control group, the mice in the ISM treatment group gained body weight more slowly. Figure 4 After sampling and weighing, it was found that the inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) of the ISM-treated mice were smaller in volume and lighter in weight, but there was no significant difference in brown adipose tissue (BAT) compared with the control group. Figure 4 CD). This indicates that isohesperidin can improve obesity induced by a high-fat diet in mice.

[0060] Example 5: Isohexolone can improve lipid metabolism and hepatic steatosis in HFD-induced obese mice. Establishment and treatment of the obese mouse model: Same as in Example 4.

[0061] Blood biochemistry assays: Using specific kits, the levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CREA) in mouse serum were measured according to the manufacturer’s instructions.

[0062] HE staining: Liver tissue was fixed overnight at room temperature with 4% paraformaldehyde, dehydrated with a gradient of ethanol, infiltrated with xylene, and embedded in paraffin. Serial sections of 5 μm thickness were prepared from the paraffin-embedded tissue and stained with hematoxylin and eosin (H&E). The tissue was observed and photographed under a light microscope.

[0063] Results: Compared with the HFD-Control group, the plasma concentrations of TG, TC, LDL-C, and HDL-C in mice in the HFD-ISM group all showed a significant decreasing trend. Figure 5 A). Meanwhile, it was also observed that the liver weight of mice in the HFD-ISM group was reduced compared to the HFD-Control group ( Figure 4 D), the volume decreased, and the degree of fatty degeneration was alleviated ( Figure 5 B). Furthermore, HE staining of liver tissue showed that, compared to the HFD-Control group, the number of lipid droplets in the livers of mice in the HFD-ISM group was significantly reduced (B). Figure 5 C). These findings collectively demonstrate that ISM treatment effectively improves lipid metabolism in HFD mice and reduces hepatic fat deposition.

[0064] Given that hepatic fat deposition may adversely affect normal liver metabolic function, leading to impaired liver function and elevated plasma transaminase levels, this invention further detected transaminase levels in mouse plasma ( Figure 5 D、 Figure 5 (E) Compared with the SCD group, the transaminase levels in the HFD group were significantly increased. However, compared with the HFD-Control group, the plasma ALT and AST concentrations in the HFD-ISM group were significantly decreased, suggesting that ISM treatment effectively improved the abnormal liver function in HFD mice. Furthermore, it is noteworthy that there was no significant difference in transaminase levels between the SCD-Control group and the SCD-ISM group, further indicating that ISM had no significant adverse effects on liver function in mice, thus verifying the biosafety of ISM in vivo.

[0065] Example 6: Isohexolone can improve glucose tolerance and insulin sensitivity in HFD-induced obese mice. Establishment and treatment of the obese mouse model: Same as in Example 4.

[0066] Glucose tolerance test (GTT): Mice were starved for 16-18 h and then injected intraperitoneally with glucose (2 g / kg body weight). Blood glucose levels were measured at 0, 15, 30, 60, 90 and 120 minutes.

[0067] Insulin Tolerance Test (ITT): The insulin tolerance test is performed one week after the GTT. Insulin (0.75 U / kg body weight) is injected intraperitoneally after a 4-hour fast, and blood glucose levels are measured at 0, 15, 30, 60, 90 and 120 minutes after insulin injection.

[0068] Results: GTT results showed that, compared with the HFD-Control group, mice in the HFD-ISM group exhibited lower fasting blood glucose levels, significantly reduced blood glucose elevation, and a faster recovery to baseline blood glucose levels. These findings collectively indicate that ISM treatment significantly improves glucose tolerance in HFD mice. Figure 6 AB).

[0069] Furthermore, the ITT results revealed that, compared with the HFD-Control group, mice in the HFD-ISM group also exhibited lower fasting blood glucose levels, with a faster rate and greater magnitude of blood glucose decline. This strongly suggests that ISM treatment enhances insulin sensitivity in HFD mice. Figure 6 CD).

[0070] Given the importance of biosafety, this invention also monitored CREA levels in mouse plasma to assess the potential nephrotoxicity of ISM. The results showed that, regardless of the feeding method, ISM treatment had no significant effect on mouse plasma CREA levels. Figure 6 E). This finding suggests that a 15 mg / kg dose of ISM may not be nephrotoxic when exerting a therapeutic effect in mice, thus further confirming its biocompatibility.

[0071] Example 7: Isohexole enhances thermogenesis in HFD-induced obese mice. Establishment and treatment of the obese mouse model: Same as in Example 4.

[0072] Metabolic cages: Mice were subjected to 24-hour metabolic analysis at 26°C using the Integrated Laboratory Animal Monitoring System (CLAMS) under a 12-hour light / dark cycle per day.

[0073] Cold exposure test: Basal rectal temperature was measured at room temperature. Mice were then individually housed in a 4°C cold room for 4 hours. Rectal temperature was measured hourly.

[0074] Results: The results showed that the food intake of mice in each group ( Figure 7 A) and activity level ( Figure 7 B) There was no significant difference. However, compared with the HFD-Control group, the HFD-ISM group mice showed a higher oxygen consumption ( Figure 7 CD) and carbon dioxide production ( Figure 7 EF), but RER did not increase ( Figure 7 These results support the idea that ISM prevents obesity by increasing energy expenditure through enhanced thermogenesis. Similarly, when exposed to low temperatures (4 °C), ISM-treated mice maintained body temperature better than control mice, directly indicating that ISM enhanced thermogenesis in mice. Figure 7 I).

[0075] Example 8: Isohexolone promoted the browning of subcutaneous white adipose tissue in HFD-induced obese mice. Mouse model establishment and treatment: Same as in Example 4.

[0076] qRT-PCR and Western Blot assays: Same as in Example 1. The cell samples were replaced with tissue samples.

[0077] Results: Compared with the HFD-Control group, the expression level of thermogenic genes at the mRNA level in the BAT of mice in the HFD-ISM group was significantly lower. Figure 8 A) No significant changes were observed. Furthermore, Western blot results also indicated that, compared to the HFD-Control group, no significant difference was observed in the expression of thermogenic genes at the protein level in the BAT of mice in the HFD-ISM group. Figure 8 BC). Based on this, it is speculated that ISM may not promote thermogenesis through activation of mouse BAT. Subsequent studies focused on the expression of thermogenic genes in the WAT of HFD mice. First, the expression of thermogenic genes in visceral WAT, i.e., eWAT, was examined. Although the eWAT fat was reduced in the ISM-treated group mice (BC). Figure 4 A), but no significant changes were observed in the expression of thermogenic genes in eWAT ( Figure 8 This is presumably attributed to the fact that ISM treatment enhances energy expenditure in HFD mice, leading to a reduction in the volume of energy-storing WATs. Next, the expression of thermogenic genes in the iWATs of HFD mice was examined. Unlike eWATs, ISM treatment significantly upregulated the expression of thermogenic genes on mRNA in iWATs (DF). Figure 8 G) and protein levels ( Figure 8The expression of HI was also found. Furthermore, this invention also found that ISM treatment had no significant effect on the expression of adipogenesis-related genes in iWAT. Figure 8 (GI). In summary, ISM can activate thermogenic genes in iWAT of HFD mice, but has no significant effect on the expression of thermogenic genes in eWAT. These results indicate that ISM promotes thermoproduction by inducing browning of subcutaneous white adipose tissue in HFD mice.

[0078] In summary, isohesperidin can improve obesity and obesity-related metabolic disorders in mice by promoting subcutaneous fat browning, increasing thermogenesis, and improving obesity in mice.

[0079] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. The use of isohesperidin or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of obesity or its associated metabolic syndrome.

2. The application according to claim 1, characterized in that: The obesity condition includes at least one of the following symptoms: being overweight, having an excessive body mass index, having an excessive body fat percentage, or having abnormal fat distribution.

3. The application according to claim 1, characterized in that: Obesity is defined as obesity caused by excessive energy intake or a decrease in metabolic rate leading to reduced energy expenditure.

4. The application according to claim 1, characterized in that: The drug also has the following therapeutic effects: inhibiting weight gain, slowing down fat accumulation, improving blood glucose metabolism, and increasing insulin sensitivity.

5. The application according to claim 1, characterized in that: The reduction of fat accumulation includes reducing fat accumulation in at least one of the following areas: liver, groin, and epididymis.

6. The application according to claim 1, characterized in that: The relevant metabolic syndrome includes at least one of type 2 diabetes, insulin resistance, non-alcoholic fatty liver disease, and hyperlipidemia.

7. The application according to claim 1, characterized in that: The drug contains: A therapeutically effective amount of isohesperidin or a pharmaceutically acceptable salt thereof; and Pharmaceutically acceptable carrier.

8. The application according to claim 1, characterized in that: The dosage form of the drug is an oral preparation or an injection; the oral preparation is one or more of tablets, capsules, granules, and oral liquids; the injection is an injection solution and / or lyophilized powder for injection.

9. The application according to claim 8, characterized in that: The drug is in the form of sustained-release tablets or sustained-release capsules.

10. The application according to claim 1, characterized in that: The drug also contains CL316243.

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