Use of alpha-hydroxyisocaproic acid in weight loss and lipid reduction

Abstract

This invention discloses the application of α-hydroxyisocaproic acid in weight loss and lipid reduction, belonging to the fields of biopharmaceutical manufacturing, biomedical applications, and functional food development. This invention confirms the application of α-hydroxyisocaproic acid in weight loss; this compound can inhibit weight gain in high-fat diet-induced obese mouse models, reduce body fat, and decrease the area of ​​adipocytes in high-fat diet-induced obese mouse models. Compared to GLP-1 receptor weight-loss drugs that reduce weight by suppressing appetite, the compound α-hydroxyisocaproic acid intervenes in and prevents obesity by accelerating fat metabolism, thereby achieving weight reduction. This invention provides a safe and effective new strategy for weight loss and lipid reduction.

Description

Technical Field

[0001] This invention belongs to the fields of biopharmaceutical manufacturing and the development of biomedical applications and functional foods, specifically involving the application of α-hydroxyisocaproic acid in weight loss and lipid reduction. Background Technology

[0002] In recent years, with improved living standards and changes in dietary structure, the global incidence of overweight and obesity has continued to rise, and is showing a trend towards affecting younger people. Obesity not only significantly increases the risk of chronic diseases such as type 2 diabetes, fatty liver disease, and cardiovascular disease, but is also a major factor leading to preventable diseases and disability, becoming a serious public health and socioeconomic burden. Therefore, developing safe, effective, and long-term suitable nutritional intervention programs is of great significance for controlling weight and improving related metabolic disorders.

[0003] Currently, some drugs are approved for obesity management, such as orlistat and smegglutide, but they generally carry certain side effects, limiting their feasibility for long-term use. For example, orlistat can cause gastrointestinal discomfort and abnormal liver function, while GLP-1 receptor agonist peptide drugs are often accompanied by adverse reactions such as nausea, diarrhea, and dizziness, and pose a risk of hypoglycemia in some individuals. Therefore, there is an increasing demand for functional ingredients with higher safety profiles suitable for pharmaceutical or functional food applications. Summary of the Invention

[0004] To address the risk of side effects associated with existing weight loss drugs, this invention provides α-hydroxyisocaproic acid (HIC) as an active ingredient for regulating lipid metabolism disorders in organisms.

[0005] α-Hydroxyisocaproic acid (HIC) is a small molecule compound naturally found in many fermented foods, such as certain cheeses, soy sauces, and wines, thus its use as a dietary source has a certain safety basis. Some studies have shown that HIC plays a beneficial role in exercise recovery and muscle protection (Mero et al, J Int SocSports Nutr. 2010;7:1.; Zhang Y et al, Cell Metabolism. 2024;36(8):1823-1838.e6.), and some literature indicates that it can be used to delay the aging process (CN110870859B). Furthermore, some literature suggests that HIC can significantly upregulate intestinal lipid absorption and enhance the uptake of fatty acids in white adipose tissue (WAT), thereby inducing fat accumulation (Nature Metabolism, 2022;4:1713-1731). This report observed an association between HIC and enhanced lipid absorption in a specific dietary recovery / microbiome metabolism context; however, the present invention unexpectedly found that under the dosage, administration method and obesity model conditions described in this application, HIC exhibited the opposite fat reduction and weight loss effect.

[0006] α-Hydroxyisohexanoic acid, also known as 2-hydroxyisohexanoic acid (HIC), 2-hydroxy-4-methylpentanoic acid, squalane, or DL-leucine, has the molecular formula C6H2O. 12 O3, its molecular structural formula is .

[0007] This invention provides the use of α-hydroxyisocaproic acid or a pharmaceutically acceptable derivative thereof in the preparation of medicaments for treating or preventing obesity, overweight and / or reducing body fat.

[0008] The present invention further provides the use of α-hydroxyisohexanoic acid or a food-acceptable derivative thereof in the preparation of foods for weight management, reducing body fat and / or reducing visceral fat.

[0009] The present invention further provides α-hydroxyisocaproic acid or a food-acceptable derivative thereof to reduce body weight and improve obesity-related lipid metabolism disorders; said lipid metabolism includes improving liver indicators (including liver weight, ALT) and blood lipid indicators (TG and LDL-c).

[0010] In embodiments of the present invention, the food- or pharmaceutically acceptable derivative is selected from: its food- or pharmaceutically acceptable salts, optical isomers, racemates, solvates, or ester precursor compounds.

[0011] In embodiments of the present invention, the food- or pharmaceutically acceptable salt is an inorganic salt or an organic salt; the inorganic salt is selected from sodium salt, calcium salt, potassium salt, and magnesium salt; the organic salt is selected from meglumine salt, tromethamine salt, diethylamine salt, lysine salt, choline salt, arginine salt, tert-butylamine salt, and N,N-dibenzylethylenediamine salt.

[0012] In embodiments of the present invention, the drug contains α-hydroxyisocaproic acid as the active component and also includes pharmaceutical or food-grade excipients.

[0013] In embodiments of the present invention, the pharmaceutical or food-grade excipient comprises a carrier system, which includes, but is not limited to, liposomes, emulsions, solid lipid nanoparticles, microcapsules, hydrogels, micelles, and chylomicron mimics.

[0014] In embodiments of the present invention, the pharmaceutical or food-grade excipients include, but are not limited to, conventional excipients such as solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, and fragrances.

[0015] In embodiments of the present invention, the dosage form of the drug includes, but is not limited to, tablets, capsules, elixirs, syrups, lozenges, inhalers, sprays, injections, films, patches, powders, granules, blocks, emulsions, suppositories, etc.

[0016] In this invention, the organism refers to a human individual, a mammal, or its tissue cells. The intended use as a medicine or food is applicable to human individuals and / or non-human mammals.

[0017] In this invention, HIC reduces body fat content and / or weight in obese or overweight individuals. The drug containing α-hydroxyisocaproic acid or a pharmaceutically acceptable derivative thereof is administered via oral, intravenous, subcutaneous, transdermal absorption, or nasal spray methods, with oral administration being preferred; the food containing α-hydroxyisocaproic acid or a food-acceptable derivative thereof is taken orally.

[0018] In this invention, the food includes nutritional compositions suitable for obese / overweight individuals, foods for special medical purposes (medical foods), and health foods; the food also includes animal foods, feeds, or feed additives suitable for obese / overweight non-human mammals. When consumed by humans, foods containing α-hydroxyisocaproic acid or its food-acceptable derivatives are consumed in solid, liquid, or semi-solid forms, including solid beverages, packaged powders, liquid beverages, and powder products. When consumed by non-human mammals, foods containing α-hydroxyisocaproic acid or its food-acceptable derivatives are in the form of feed, or in the form of feed additives combined with feed, or dissolved in drinking water for animal ingestion.

[0019] In this invention, HIC reduces body fat content and / or weight in obese or overweight organisms, characterized by increasing fat consumption, thereby reducing fat content and weight. At the molecular level, HIC upregulates the expression of uncoupling protein 1 (UCP1) in adipose tissue, enhancing thermogenesis, thereby increasing energy expenditure, promoting lipolysis, and reducing fat content and / or weight, thus achieving the goal of weight loss.

[0020] The present invention further provides a method for regulating energy metabolism using α-hydroxyisohexanoic acid (HIC) or a derivative thereof. The method comprises administering an effective amount of the compound to an organism requiring treatment, wherein the dosage is 0.1-300 mg / kg body weight daily, preferably 5-200 mg / kg body weight. The route of administration of the compound is selected from oral, intravenous, subcutaneous, or intramuscular injection, with oral administration being the preferred method.

[0021] The method is particularly applicable to human subjects or mammalian individuals, including but not limited to obese or overweight patients induced by a high-fat diet. Obesity refers to excessive total body fat and / or increased localized fat content and abnormal distribution, and is a chronic metabolic disease caused by the combined effects of genetic and environmental factors. According to the World Health Organization, a body mass index (BMI) greater than or equal to 30 is considered obese, and a BMI greater than or equal to 25 is considered overweight. In practical applications, the administration period of the compound is usually no less than 4 consecutive weeks, preferably 8-16 weeks, and most preferably 12 weeks. The dosing frequency is generally 1-2 times daily, and the specific dosing regimen can be appropriately adjusted according to the patient's blood drug concentration monitoring results, changes in body fat percentage, and liver function test indicators.

[0022] The core innovation of this invention lies in the discovery and confirmation that HIC can effectively improve body fat and reduce weight.

[0023] Compared with the prior art, the present invention has the following significant advantages: Compared to GLP-1 receptor agonist peptide weight-loss drugs such as smegglutinin, which have significant side effects, appetite suppression, low oral bioavailability, and easy inactivation, the small molecule compound HIC of this invention has clear weight loss and body fat reduction effects after oral administration, significant weight reduction, and significant improvement in glucose and lipid metabolism-related indicators. In addition, since HIC is a small molecule compound that is naturally found in food, compared with traditional weight-loss drugs or chemically synthesized drugs, this technical solution has greater potential for further development as an active ingredient in pharmaceuticals or functional foods. Attached Figure Description

[0024] Figure 1 The results of HIC's effects on inhibiting obesity and weight loss are shown in the figure.

[0025] Figure 2 The figure shows the results of intervention on mice with different concentrations of HIC and high-fat diet-induced obesity (DIO).

[0026] Figure 3 The figure shows the results of HIC intervention on ob / ob mice.

[0027] Figure 4 The figure shows the results of HIC upregulation of mitochondrial brown adipose uncoupling protein 1 (UCP1) in subcutaneous fat (iWAT) and brown adipose fat (BAT) in DIO mice and its promotion of heat production.

[0028] Figure 5 The figure shows the results of HIC in reducing lipid droplets in primary adipocytes of mice and upregulating heat-related proteins.

[0029] Figure 6 Figure showing the results of acute oral toxicity and 90-day oral toxicity evaluation of HIC administration in mice. Detailed Implementation

[0030] 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.

[0031] 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 the upper and lower limits of the range and each intermediate value between them are 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, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] 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 to which this invention is described. 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. Unless otherwise stated, “%” means percentage based on weight.

[0033] The term "heat production" as used in this article can be used interchangeably with the terms "thermogeneity," "heat generation," "thermogeneous effect," and "thermogenic effect." Similarly, "weight loss," "weight reduction," "lipid reduction," "fat reduction," and "weight loss" can be used interchangeably. In this article, thermogenic effect refers to the process by which mammals, including humans, regulate heat production.

[0034] Example 1: Effect of oral administration of HIC on weight loss and lipid reduction in high-fat diet-induced obese mice 1. Experimental Materials and Methods 1.1 Mice Animal experiments were conducted in accordance with the ethical guidelines and protocols approved by Dalian University of Technology. All experiments used sex- and age-matched mice, which were randomly assigned to groups. Mice were housed in a specific, sterile animal facility with controlled temperature and humidity (25 °C, 12:12 hour light:dark cycle) and free access to food and water. The conditions for mice used in subsequent examples were the same.

[0035] Dietary-induced obesity (DIO) mouse modeling process: 4-6 week old male C57BL / 6J mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. After one week of acclimatization, the mice were fed a high-fat diet (XTHF60, Synergistic Bio) for 4 weeks. Subsequently, an intervention was implemented, maintaining a high-fat diet throughout the intervention period.

[0036] 1.2 Enzyme-linked immunosorbent assay (ELISA) Mouse insulin, leptin, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and growth differentiation factor 15 (GDF15) were detected using a commercial ELISA kit (Jianglai Biotechnology) according to the instructions for use.

[0037] 1.3 Histology Fat or liver tissue was fixed overnight in 4% paraformaldehyde at 4 °C and embedded in paraffin before sectioning. Sections were stained with hematoxylin and eosin (H&E) and photographed under a bright-field microscope.

[0038] 1.4 Intervention with 2-hydroxyisohexanoic acid Diet-induced obesity (DIO) mice were treated by gavage with a HIC dose of 100 mg / kg for 8 weeks.

[0039] 1.5 Statistical Analysis R software (version 4.5.0) was used for plotting and statistical analysis. All statistical tests are fully described in the legends of the graphs and conform to the criteria of a normal distribution with similar variances. No statistical methods were used to predetermine the sample size. t-tests were used for comparisons between two groups. Repeated measures ANOVA was performed for assessment data of relevant samples. One-way ANOVA and multiple comparisons were used for assessments of more than two groups. Two-way ANOVA with multiple comparisons was used for assessments of two independent variables. The log-rank test was used for survival analysis. Linear regression analysis was used for correlation analysis. Otherwise, data are expressed as mean ± standard error (Mean ± SEM) unless otherwise stated. p < 0.05 was considered statistically significant, denoted as *p < 0.05, **p < 0.01, ***p < 0.001, NS, not significant. The statistical methods used in subsequent examples are the same.

[0040] 2. Experimental Results Figure 1 A graph showing how HIC inhibits obesity and improves glucose homeostasis. Figure 1 A) Experimental Design Flowchart: After a 1-week acclimatization period, mice were randomly divided into two groups (n = 8 / group): a control group (HFD group) and a 100 mg / kg HIC intervention group (HIC group). Mice were fed a high-fat diet for 4 weeks before the intervention, during which they were continuously fed a high-fat diet (XTHF60, Synergy Biotech) for 8 weeks. The experimental group received 100 mg / kg HIC orally via gavage daily, while the control group received an equal volume of the solution. Food intake was monitored weekly during the intervention period. Figure 1 B) and weight changes ( Figure 1 C). Conducted 8 weeks after the intervention ( Figure 1 D) Glucose tolerance test (oral administration of 1 g / kg glucose) and ( Figure 1 E) Insulin tolerance test (intraperitoneal injection of 0.5 U / kg insulin). Figure 1 F) Body composition (lean body mass and fat mass) analyzed by low-field nuclear magnetic resonance. Tissue samples collected post-euthanasia: macroscopic morphology of epididymal fat, subcutaneous fat, and scapular brown fat. Figure 1 G), weight ( Figure 1 H) and microscopic morphology examined by H&E section microscopy ( Figure 1 I) (Scale bar: 100 μm). Figure 1 J) Fasting blood glucose and serum test indicators: ( Figure 1 K) Insulin Resistance Index (HOMA-IR), Figure 1 L) triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), and ( Figure 1 M) Leptin levels. Figure 1N) Liver weight and ( Figure 1 O) Microscopic morphology of liver tissue examined by H&E section microscopy (scale bar: 50 μm). Figure 1 P) Alanine aminotransferase (ALT), aspartate aminotransferase (AST), Figure 1 Q) Liver triglycerides and ( Figure 1 R) Hepatic lipid peroxidation level. Figure 1 Changes in serum levels of appetite-suppressing hormones GLP-1, GIP, and GDF15 within 0–4 hours after oral administration of 100 mg / kg HIC (n=5 / group). Data are expressed as mean ± standard error (Mean ± SEM) for biologically independent samples. Statistical significance was indicated by *p<0.05, **p<0.01, ***p<0.001 (two-tailed t-test).

[0041] 3. Conclusion The experimental results showed that although the mice in the 100 mg / kg HIC treatment group exhibited a decreasing trend in food intake (lower than the control group, p = 0.15), the difference was not statistically significant; however, their body weight was significantly reduced (p < 0.05). Figure 1 AC). Metabolic function analysis showed that HIC treatment also significantly improved glucose tolerance and insulin sensitivity in mice. Figure 1 DE). Low-field nuclear magnetic resonance imaging (NMR) analysis revealed that the fat mass of mice in the HIC group was significantly reduced by 33.5% compared to the control group (p = 0.007), while the lean meat mass did not change significantly (p = 0.19). Figure 1 F). Further histological analysis showed that HIC intervention significantly reduced the weight of various adipose tissues: epididymal fat (-31.2%, p = 0.01), subcutaneous fat (-29.3%, p = 0.03), and scapular brown fat (-30.2%, p = 0.004). Figure 1 GH). Histological examination confirmed that the volume of lipid droplets in the aforementioned adipose tissue was significantly reduced. Figure 1 I). Regarding glucose metabolism, HIC treatment significantly reduced fasting blood glucose levels and the HOMA-IR index. Regarding lipid metabolism, lipid indicators (TG and LDL-c, p < 0.05) significantly decreased / improved, but no statistically significant differences were observed in total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-c). Figure 1 JM). Furthermore, regarding liver function, HIC intervention significantly reduced liver weight (p<0.05). Figure 1 N), reducing the amount of lipid droplets in the liver ( Figure 1 O). It reduced liver function indicators such as ALT (p<0.05), liver triglyceride levels, and liver lipid peroxidation levels (p<0.05). Figure 1 PR). Serum levels of appetite-suppressing hormones GLP-1, GIP, and GDF15 did not increase within 0-4 hours after oral administration of 100 mg / kg HIC, indicating that HIC does not improve obesity in mice by upregulating these three hormones. Figure 1 (SU). In summary, HIC can effectively alleviate the obesity phenotype induced by a high-fat diet, and its mechanism of action is achieved through the following pathways: selectively reducing fat deposition; and promoting glucose and lipid metabolism. These findings provide experimental evidence for the development of HIC as a potential anti-obesity drug.

[0042] Example 2: Effects of different concentrations of HIC on DIO and ob / ob mice 1. Experimental Materials and Methods 1.1 Different doses of 2-hydroxyisocaproic acid were used to treat DIO mice DIO mice were constructed according to the modeling method described in Example 1. Diet-induced obese (DIO) C57BL / 6J mice were treated by gavage with HIC doses of 50, 100, and 200 mg / kg for 4 weeks. Mice fed low-fat (XTCON50J, Synergistic Bio) and high-fat (XTHF60, Synergistic Bio) diets served as controls. Body weight and food intake were recorded weekly.

[0043] 1.2 HIC intervention on the metabolic phenotype of ob / ob mice ob / ob male mice (leptin-deficient obesity model): Four-week-old ob / ob mice were purchased from Cyagen Biosciences. HIC intervention was performed one week after acclimatization.

[0044] 2. Experimental Results Figure 2 The figure shows the effects of different concentrations of HIC on DIO mice. After a one-week acclimatization period, mice were randomly divided into two groups (n = 8 / group): a low-fat diet control group (WT group), a high-fat diet control group, a 50 mg / kg HIC intervention group (HIC_50 group), a 100 mg / kg HIC intervention group (HIC_100 group), and a 200 mg / kg HIC intervention group (HIC_200 group). Intervention was initiated after four weeks of high-fat diet feeding, during which the mice were continuously fed a high-fat diet (XTHF60, Synergistic Bio) for four weeks. Figure 2 A) Body weight changes in DIO mice after four weeks of HIC intervention with 50, 100, and 200 mg / kg. Figure 2 B) Changes in food intake in DIO mice after four weeks of HIC intervention at doses of 50, 100, and 200 mg / kg.

[0045] Figure 3 The effect of HIC on the metabolic phenotype of ob / ob mice. Figure 3A) Experimental design diagram: Mice were divided into four groups (n=6 / group): wild-type control group (WT group), wild-type + HIC 100 mg / kg (WT_HIC group), ob / ob model control group (ob / ob group), and ob / ob + HIC 100 mg / kg (ob / ob_HIC group). Weekly monitoring was conducted during the intervention period. Figure 3 B) Food intake and ( Figure 3 C) Weight change. This was assessed after week 8 of the intervention. Figure 3 D) Glucose tolerance test (oral administration of 1 g / kg glucose) and ( Figure 3 E) Insulin tolerance test (intraperitoneal injection of 0.5 U / kg insulin). Figure 3 F) Tissue samples collected after euthanasia: weight of liver, epididymal fat, subcutaneous fat, and brown fat of the scapula. Serum biochemical indicators: ( Figure 3 G) HOMA-IR index, ( Figure 3 H) Glycated hemoglobin (GHbA1c), ( Figure 3 I) Triglycerides (TG), ( Figure 3 J) Total cholesterol (TC), ( Figure 3 K) High-density lipoprotein cholesterol (HDL-c), ( Figure 3 L) Low-density lipoprotein cholesterol (LDL-c), ( Figure 3 M) alanine aminotransferase (ALT) and ( Figure 3 N) Aspartate aminotransferase (AST). Data are expressed as mean ± standard error (Mean ± SEM) of independent biological samples. Statistical analysis was performed using two-way ANOVA or t-test, with significance indicated by *p<0.05, **p<0.01, ***p<0.001.

[0046] 3. Experimental Conclusions HIC administered via gavage for four weeks significantly reduced the body weight of obese mice induced by a high-fat diet (p<0.05), in a dose-dependent manner. In week four, the average body weight loss was 1.7 g in the 50 mg / kg HIC intervention group, 2.95 g in the 100 mg / kg HIC intervention group, and 3.31 g in the 200 mg / kg HIC intervention group. Figure 2 A). On the other hand, during the intervention period, 200 mg / kg HIC significantly reduced food intake in mice (p<0.05). Figure 2 B).

[0047] To elucidate the mechanism of action of HIC in alleviating obesity, this invention first investigates whether it regulates body weight through a leptin-dependent pathway. In ob / ob mice (a leptin-deficient obesity model), after 8 weeks of intervention with 100 mg / kg HIC: HIC treatment had no significant effect on food intake in either wild-type (WT_HIC) or ob / ob mice (OB_HIC) (p>0.05). Figure 3 B). Despite no change in food intake, HIC significantly reduced the body weight of WT and ob / ob mice (p<0.05). Figure 3 C). Regarding glucose metabolism, ob / ob mice, after HIC intervention, showed improved oral glucose tolerance ( Figure 3 D) and insulin sensitivity ( Figure 3 E) were significantly increased. Furthermore, it was found that HOMA-IR and GHbA1c values ​​in mice decreased significantly after intervention (p<0.05, Figure 3 GH was used to confirm its ability to reverse metabolic disorders caused by leptin deficiency. Regarding lipid metabolism, serum TC levels in mice were significantly reduced after intervention (p<0.05). Figure 3 J) and HDL were significantly elevated (p<0.05, Figure 3 K). Regarding liver function indicators, HIC significantly reduced ALT in mice (p<0.05, K). Figure 3 In summary, HIC can still improve body weight and glucose and lipid metabolism phenotypes in leptin-deficient obesity models, suggesting that its effects are independent of leptin signaling.

[0048] Example 3: Molecular Mechanism of HIC in Reducing Weight and Fat 1. Experimental Methods 1.1 Transcriptomics RNA was extracted from tissues or cells using standard extraction methods, followed by rigorous quality control of the RNA samples. Quality control was primarily achieved using an Agilent 2100 bioanalyzer to precisely detect RNA integrity. Library construction and sequencing were then performed.

[0049] 1.2 RNA extraction and gene expression analysis Total RNA was extracted from frozen tissues using RNAiso EASY extraction reagent (Takara, Dalian) and quantified using a Nanodrop micro-UV-Vis spectrophotometer (Thermo). cDNA was extracted using PrimeScript. TM The RTReagent kit (Takara, Dalian) was used to prepare total RNA (1 μg) via reverse transcription polymerase chain reaction. cDNA was analyzed using TB Green's assay. TM Premix EX Taq TMThe II kit (TAKARA) was used on a QuantStudio 3 real-time quantitative PCR system (Thermo), with mouse Actb and Rplp0 as internal controls, and the results were obtained via ΔΔC. t The fold change in expression was calculated using this method. The primer sequences are as follows: Actb: forward-CATCCGTAAAGACCTCTATGCCAAC (SEQ ID NO.1), reverse-ATGGAGCCACCGATCCACA (SEQ ID NO.2); Prdm16: forward-CAGAGGTGTCATCCCAGGAG (SEQ ID NO.3), reverse-ACGGATGTACTTGAGCCAGC (SEQ ID NO.4); Ppargc1α: forward-TGTAGCGACCAATCGGAAAT (SEQ ID NO.5), reverse-TGAGGACCGCTAGCAAGTTT (SEQ ID NO.6); Cidea: forward-CAGTGATTTAAGAGACGCGG (SEQ ID NO.7), reverse-TCTGCAATCCCATGAATGTC (SEQ ID NO.8); Ucp1: forward-TCAGCTGTTCAAAGCACACA (SEQ ID NO.9), reverse-GTACCAAGCTGTGCGATGTC (SEQ ID NO.10); Nrf1: forward-TAGTCCTGTCTGGGGAAACC (SEQ ID NO.11), reverse-CTGGTACATGCTCACAGGGA (SEQ ID NO.12); Tfam: forward-CACCCAGATGCAAAACTTTCAG (SEQ ID NO.13), reverse-CTGCTCTTTATACTTGCTCACAG (SEQ ID NO.14); Cytb: forward-ACCTCCTATCAGCCATCCCA (SEQ ID NO.15), reverse-AGCGAAAGAATCGGGTCAAGG (SEQ ID NO.16); Cox7a: forward-GCTCTGGTCCGGTCTTTTAGC (SEQ ID NO.17), reverse-GTACTGGGAGGTCATTGTCGG (SEQ ID NO.18); Cox8b: forward-GAACCATGAAGCCAACGACT (SEQ ID NO.19), reverse-GCGAAGTTCACAGTGGTTCC (SEQ ID NO.20); Cpt1b: forward-GCTGCTTGCACATTTGTGTT (SEQ ID NO.21), reverse-TGAGTGACTGGTGGGAAGAA (SEQ ID NO.22); Nampt: forward-GAATGTCTCCTTCGGTTCTGG (SEQ ID NO.23), reverse-TCAGCAACTGGGTCCTTAAAC (SEQ ID NO.24); Rplp0: forward-TGACATCGTCTTTAAACCCCG (SEQ ID NO. 25), reverse-TGTCTGCTCCCACAATGAAG (SEQ ID NO. 26).

[0050] 1.3 Enzyme-linked immunosorbent assay The UCP1 protein content in mice was detected using a commercial ELISA kit according to the instructions for use.

[0051] 1.4 Protein Blotting Tissue lysates or whole-cell lysates were extracted using RIPA lysis buffer (Beyotime, China) supplemented with complete protein inhibitors (Beyotime, China) and phosphatase inhibitors (Beyotime, China), and the supernatant was used for subsequent analysis. Proteins were diluted in loading dye, heated at 95°C for 10 min, and run on a 10–12% polyacrylamide gel. Proteins were transferred to polyvinylidene fluoride membranes and Western blotted using the aforementioned commercial antibodies.

[0052] 1.5 Immunohistochemistry Immunohistochemistry for UCP1 was performed using the rabbit-specific HRP / DAB (ABC) IHC detection kit (ab64261, Abcam) according to the manufacturer's instructions. Lipid droplets in mouse primary adipocytes were stained with BODIPY 493 / 503 (Solepro) and observed and photographed using a laser scanning confocal microscope (LEICA).

[0053] 1.6 Preparation of primary beige adipocytes Subcutaneous adipose tissue from the groin was minced and digested in PBS containing collagenase II (1 mg / ml) at 37 °C for 45 min. The tissue suspension was filtered through a 100 μm cell filter and centrifuged at 600 × g for 5 min to granulate the stromal vascular fraction (SVF). After granulation, it was further filtered through a 40 μm cell filter and placed on collagen-coated plates. After overnight culture, the supernatant containing non-adherent cells was removed. Preadipocytes were grown and fused in DMEM containing 10% FBS + insulin (5 μg / ml). Dexamethasone (1 μM) was used in 3... Isobutyl 1 Methylxanthine (IBMX, 0.5 mM), insulin (5 μg / ml), indomethacin (125 nM), and rosiglitazone (1 μM) were used to induce fusion cell differentiation for 2 days, followed by 5 days of treatment with insulin (5 μg / ml) and triiodothyronine (T3, 1 nM) alone. On day 7, 2-hydroxyisohexanoic acid (100 μg / ml) was added for 12 days of treatment. 24 hours for protein expression level detection.

[0054] 1.7 Laser confocal microscopy for observing lipid droplets in adipocytes Before starting the experiment, prepare BODIPY 493 / 503 staining solution according to the instructions, preparing it fresh each time. Primary adipocytes were then stained at 5 × 10⁻⁶ staining sites. 4 1 mL of the sample was added to each well of a 20 mm laser confocal culture dish (5 × 10⁻⁶ cells / mL). 4Cells were induced for 2 days in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin solution) containing 0.5 mM IBMX, 1 μM dexamethasone (DEX), 125 nM indomethacin, 850 nM insulin, 1 nM triiodothyronine, and 1 μM rosiglitazone for 4 days. The medium was then replaced with DMEM complete medium for another 2 days. The original medium was discarded, and the cells were washed twice with PBS and incubated with 100 µg / mL HIC for 48 hours. After the incubation, the medium was removed, and the cells were washed three times with PBS. 500 µL of 2 µM BODIPY493 / 503 staining solution was added to each well, and the cells were incubated at 37°C in the dark for 15 minutes. After incubation, the cells were washed twice with PBS to remove the staining solution. Add 200 µL of anti-fluorescence quenching sealing solution (containing DAPI) to each well, and perform laser confocal imaging with a maximum excitation wavelength of 493 nm and a maximum emission wavelength of 503 nm.

[0055] 2. Experimental Results Figure 4 The graph shows the results of HIC upregulation of UCP1 promoting thermogenesis. The subcutaneous fat in the HIC-treated group and the control group (… Figure 4 A) and brown fat in the scapula ( Figure 4 C) GO analysis of tissues; subcutaneous fat ( Figure 4 B) and scapular brown fat ( Figure 4 D) Thermograph of thermograms showing the expression of heat-related genes; qPCR detection of subcutaneous fat ( Figure 4 E) and scapular brown fat ( Figure 4 F) mRNA levels of heat-related genes in subcutaneous fat; immunohistochemical detection of subcutaneous fat (F) Figure 4 G) and scapular brown fat ( Figure 4 I) UCP1 protein expression (scale bar: 100 μm); ELISA quantitative analysis of subcutaneous fat ( Figure 4 H) and scapular brown fat ( Figure 4 UCP1 protein content in J); Western Blot analysis of subcutaneous fat (J) Figure 4 K) and scapular brown fat ( Figure 4 L) Expression of Pgc1α and UCP1 proteins, and relative quantification of protein bands using ImageJ. Data are expressed as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 (n = 6-8 / group).

[0056] Figure 5 A diagram illustrating how HIC reduces lipid droplets in primary mouse adipocytes and upregulates thermogenesis-related proteins. Figure 5A) Representative images showing the reduction of lipid droplets in primary mouse adipocytes by different doses of HIC. Figure 5 B) Different doses of HIC upregulated the expression of Pgc1α and UCP1 proteins in primary adipocytes of mice.

[0057] 3. Conclusion HIC intervention for 8 weeks significantly activated the thermogenesis program in adipose tissue of high-fat diet-induced obese mice. Transcriptome analysis showed that the expression of thermogenesis-related genes was significantly upregulated in subcutaneous fat and brown fat of the scapula. Figure 4 AD). qPCR validation revealed that the expression levels of key thermogenic genes such as Pgc1α, Ucp1, Cytb, Cox8b, and Cpt1b were upregulated 2-6 times in subcutaneous adipose tissue (p<0.05), while the expression levels of the same genes were upregulated 1-2 times in brown adipose tissue of the scapula (p<0.05). Figure 4 EF). At the protein level, immunohistochemistry showed increased UCP1 expression, ELISA detected elevated UCP1 protein levels, and Western blot analysis further confirmed this result. Figure 4 These results indicate that HIC activates the adipose-derived thermogenesis program in a tissue-specific manner, with a more pronounced activation effect on subcutaneous fat. Furthermore, HIC can reduce lipid droplets in mouse primary adipocytes (GL). Figure 5 A), and upregulated the expression levels of the heat-related proteins Pgc1α and Ucp1 ( Figure 5 B). These results demonstrate that HIC effectively activates brown adipose tissue and promotes thermogenesis in beige adipocytes by significantly upregulating Ucp1 gene expression and protein levels, thereby reducing subcutaneous fat deposition and lowering body weight.

[0058] Example 4: HIC Toxicological Evaluation 1. Experimental Methods 1.1 Acute oral toxicity evaluation of HIC The acute toxicity test method was based on GB 15193.3-2014 National Food Safety Standard - Acute Oral Toxicity Test. The Horn method was used, with doses set at 1 g / kg, 2.15 g / kg, 4.64 g / kg, and 10 g / kg, using five male and five female C57BL / 6J mice in each group.

[0059] 1.2 Oral toxicity evaluation at 90 days of HIC The chronic toxicity test method was based on GB 15193.13-2015, the national food safety standard for 90-day oral toxicity testing. Doses were set at 100 mg / kg, 200 mg / kg, and 300 mg / kg. Eight male C57BL / 6J mice were used in each group.

[0060] 2. Experimental Results Figure 6 A graph evaluating the acute oral toxicity and 90-day oral toxicity of HIC in mice. Figure 6 A) Acute toxicity experiment design: 4-6 week old C57BL / 6J mice (n = 5 / dose group / sex) were orally administered HIC (1, 2.15, 4.64, 10 g / kg) and observed for 7 days. Figure 6 B) The dose-mortality curve shows the sex-specific median lethal dose (LD50) and 95% confidence interval. Figure 6 C) 90-day subchronic toxicity experiment design: Mice were administered 0 (control), 100, 200, and 300 mg / kg HIC (n = 8 / group), respectively. Weekly weight gain curves were monitored during the intervention period. Figure 6 D) and food intake ( Figure 6 E) / Water intake ( Figure 6 F). ( Figure 6 G) Weight of terminal anatomical organs (brain / heart / liver / spleen / lung / kidney / testis / epididymis fat / subcutaneous fat / brown fat / quadriceps / gastrocnemius). Figure 6 H) Fasting blood glucose and serum biochemical indicators: ( Figure 6 IL) lipid metabolism (triglycerides TG, total cholesterol TC, high-density lipoprotein HDL-c, low-density lipoprotein LDL-c), Figure 6 M, N, O) Liver function (alanine aminotransferase ALT, aspartate aminotransferase AST, alkaline phosphatase ALP), Figure 6 PQ) Renal function (blood urea nitrogen BUN, creatinine Cr), Figure 6 R, S) protein metabolism (total protein, albumin) and ( Figure 6 T) Calcium content.

[0061] 3. Experimental Conclusions Acute toxicity tests showed that the oral median lethal dose (LD50) of HIC in C57BL / 6J mice differed between sexes: the LD50 was 1.26 g / kg (95% CI: 0.775–2.05) in male mice and 1.71 g / kg (95% CI: 1.26–2.33) in female mice. Figure 6 B).

[0062] Subchronic toxicity studies showed that continuous administration of 100-300 mg / kg HIC for 90 days dose-dependently inhibited body weight gain in mice (p<0.05). Figure 6 D). Among them, the highest dose group (300 mg / kg) reduced the food intake of mice in the early and middle stages (within 2 months), while having no significant effect on the overall water intake. Figure 6EF). At the end of the experiment, a gross examination was performed on the mice, and the brain, heart, liver, spleen, lungs, kidneys, testes, epididymal fat, subcutaneous fat, brown fat, quadriceps femoris muscle, and gastrocnemius muscle were weighed. The results showed that HIC reduced the weight of the mouse liver in a dose-dependent manner, while having no significant effect on other tissues or organs. Serum biochemical index analysis ( Figure 6 The IT (internal medicine) results indicate that this dose can affect the animal's basal metabolism and physiological functions, specifically manifested as elevated total cholesterol (TC) and alkaline phosphatase (ALP) (p<0.05), and significantly decreased levels of triglycerides (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, and calcium (Ca) (p<0.05). Notably, this dose range did not cause organic lesions in vital organs (heart, brain, lungs, reproductive organs, etc.), and the changes in blood biochemical indicators remained within physiologically adjustable ranges. In summary, HIC showed good overall tolerability within the intervention dose range described in this application, suggesting no direct evidence of significant damage to vital organs, providing a safety reference for its further application.

[0063] Comparative Example 1 The effects of different small molecule compounds, such as leucine (Leu), α-ketoisocaproic acid (KIC), isoleucine (Ile), 3-methyl-2-oxovaleric acid (KMVA), valine (Val), 3-methyl-2-oxobutyric acid (KIVA), β-hydroxy-β-methylbutyric acid (HMB), and α-hydroxyisocaproic acid (HIC), on reducing body weight in high-fat diet-induced obese mice were compared.

[0064] Referring to Example 1, after a 1-week acclimatization period, mice were randomly divided into 9 groups (n = 6 / group): control group (HFD group), 100 mg / kg leucine intervention group (Leu group), 100 mg / kg α-ketoisocaproic acid intervention group (KIC group), 100 mg / kg isoleucine intervention group (Ile group), 100 mg / kg 3-methyl-2-oxovaleric acid intervention group (KMVA group), 100 mg / kg valine intervention group (Val group), 100 mg / kg 3-methyl-2-oxobutyric acid intervention group (KIVA group), 100 mg / kg β-hydroxy-β-methylbutyric acid HMB intervention group (HMB group), and 100 mg / kg α-hydroxyisocaproic acid intervention group (HIC group). Other parameters remained unchanged. After 8 weeks of intervention, changes in body weight, epididymal fat, subcutaneous fat, and brown fat were measured. The results are shown in Table 1.

[0065] Table 1

[0066] Note: Tukey's HSD multiple comparison analysis showed that different letters indicated significant differences between groups (p<0.05).

[0067] The comparison revealed that 100 mg / kg HIC significantly reduced the body weight, subcutaneous fat, epididymal fat, and brown fat in DIO mice (p<0.05), while there was no significant difference in weight between Leu and HMB (p>0.05). Notably, although 100 mg / kg KIC significantly reduced the body weight of DIO mice, 33.33% of the mice died during the intervention period, indicating that KIC at this dose had toxic side effects on mice, and the weight reduction was not a beneficial effect. Referring to the results of Example 4, 100 mg / kg HIC was non-toxic to mice.

[0068] While some existing studies have shown that certain small molecule compounds have some influence on body fat regulation, such as β-carotene... hydroxyl β Methylbutyric acid (HMB) is thought to improve lipid metabolism (US Pat. No. 5,360,613). However, combined with the comparative experiment in Example 1, it can be seen that these substances do not significantly reduce weight and lipids in a diet-induced obesity (DIO) mouse model.

[0069] Conversely, studies have shown that small molecule compounds such as 2-hydroxy-3-methylbutyric acid (HMB) and 2-hydroxyisocaproic acid (HIC) can significantly upregulate intestinal lipid absorption and enhance fatty acid uptake in white adipose tissue (WAT), thereby inducing fat accumulation (Nature Metabolism, 2022;4:1713-1731). It is evident that the mechanisms of action of these small molecule compounds in the regulation of lipid metabolism are currently highly controversial, complex, and unclear.

[0070] Therefore, the core innovation of this invention lies in the discovery and mechanistic verification that 2-hydroxyisocaproic acid (HIC) can effectively improve body fat and reduce weight, and has good oral efficacy, providing a new strategy with potential applications for obesity or overweight intervention. 2-hydroxyisocaproic acid (HIC), as a key active ingredient, can be applied in pharmaceuticals, health foods, special medical foods, dietary supplements / nutritional supplements, and mammalian foods.

[0071] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. The use of the small molecule compound α-hydroxyisocaproic acid or a food- or pharmaceutically acceptable derivative thereof in the preparation of a medicament for treating or preventing obesity, overweight, and / or reducing body fat, characterized in that, The medicine for treating or preventing obesity, overweight, and / or reducing body fat contains α-hydroxyisocaproic acid or a food- or pharmaceutically acceptable derivative thereof; the structure of the α-hydroxyisocaproic acid is shown below: 。 2. The use of the small molecule compound α-hydroxyisohexanoic acid or a food-acceptable derivative thereof in the preparation of foods for weight management, reducing body fat and / or reducing visceral fat, characterized in that, The food contains α-hydroxyisohexanoic acid or a food-acceptable derivative thereof.

3. The use according to claims 1-2, characterized in that, The stated uses and effects involve reducing body fat content, increasing body fat consumption, and reducing body weight through α-hydroxyisohexanoic acid or its food- or pharmaceutically acceptable derivatives.

4. The use according to any one of claims 1–3, wherein, The uses also include improving obesity-related lipid metabolism disorders.

5. The use according to any one of claims 1–4, wherein, The intended use applies to humans and / or non-human mammals.

6. The use according to any one of claims 1-4, characterized in that, The derivatives are selected from: food or pharmaceutically acceptable salts, optical isomers, racemates, solvates, and carboxyl-terminated ester derivatives.

7. The use according to claim 6, characterized in that, The food- or pharmaceutically acceptable salts are inorganic or organic salts; the inorganic salts include sodium salts, calcium salts, potassium salts, and magnesium salts; the organic salts include meglumine salts, tromethamine salts, diethylamine salts, lysine salts, choline salts, arginine salts, tert-butylamine salts, and N,N-dibenzylethylenediamine salts.

8. The use according to any one of claims 1-4, characterized in that, In addition to α-hydroxyisocaproic acid as the active ingredient, it also contains pharmaceutical or food-grade excipients.

9. The use according to claim 8, characterized in that, The pharmaceutical or food-grade excipients comprise a carrier system, which includes liposomes, emulsions, nanoparticles, microcapsules, hydrogels, micelles, and chylomicron mimics; the pharmaceutical or food-grade excipients comprise any one or more of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, and flavorings.

10. The use according to claims 1-2, characterized in that, The dosage form of the drug is selected from: tablets, capsules, elixirs, syrups, lozenges, inhalers, sprays, injections, films, patches, powders, granules, blocks, emulsions, and suppositories; the food exists in solid, liquid, or semi-solid form; the food includes nutritional compositions suitable for obese / overweight individuals, special medical foods, health foods, and animal foods, feeds, or feed additives suitable for obese / overweight non-human mammals.

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