Application of 5, 7-dimethoxy coumarin in preparation of medicine for treating metabolic syndrome

Abstract

The invention relates to application of 5, 7-dimethoxy coumarin in preparation of a medicine for treating metabolic syndrome, and belongs to the technical field of biological medicine. The invention discloses a new application of 5, 7-dimethoxy coumarin in treatment of metabolic syndrome for the first time, and the 5, 7-dimethoxy coumarin can regulate intestinal flora composition and serum metabolic spectrum, mainly relates to key pathways such as steroid hormone biosynthesis, taurine and hypotaurine metabolism and the like, so that the alleviation or treatment of metabolic disorder is realized in multiple aspects, and the treatment effect is good. A brand new medicine development strategy is provided for comprehensive prevention and treatment of the metabolic syndrome.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of 5,7-dimethoxycoumarin in the preparation of drugs for treating metabolic syndrome. Background Technology

[0002] With changes in lifestyle and dietary structure, metabolic disorders induced by high-fat diets have become a global health problem, with clinical manifestations including obesity, insulin resistance, dyslipidemia, non-alcoholic fatty liver disease, and chronic low-grade inflammation. Currently, the main drugs used clinically to treat these metabolic diseases include insulin sensitizers, lipid-lowering drugs, and weight-loss drugs; however, these drugs generally have significant side effects, limited efficacy, or are prone to drug resistance.

[0003] Coumarins are a class of aromatic oxygen-containing heterocyclic compounds with benzo-α-pyranone as the parent structure. Based on the type of substituents and cyclization mechanism, they can be divided into subclasses such as simple coumarins, furanocoumarins, and pyranocoumarins. 5,7-Dimethoxycoumarin is a simple coumarin, and studies have reported its anti-tumor, antidepressant, and anti-colitis effects; however, no systematic research has yet revealed its role in high-fat diet-induced metabolic disorders. Summary of the Invention

[0004] The purpose of this invention is to address the current situation where drugs for treating high-fat diet-induced metabolic syndrome generally have side effects and limited efficacy, and to provide a novel drug development strategy. To this end, this invention provides the application of 5,7-dimethoxycoumarin in the preparation of drugs for treating metabolic syndrome.

[0005] This invention provides the use of 5,7-dimethoxycoumarin in the preparation of medicaments for treating metabolic syndrome.

[0006] Preferably, the metabolic syndrome includes metabolic disorders induced by a high-fat diet.

[0007] Preferably, the metabolic disorder includes one or more of the following: glucose metabolism disorder, lipid metabolism disorder, and metabolic hormone disorder.

[0008] Preferably, the glucose metabolism disorder includes abnormal fasting blood glucose and / or impaired oral glucose tolerance; The lipid metabolism disorder includes abnormal serum total cholesterol levels and / or abnormal low-density lipoprotein cholesterol levels; The metabolic hormone disorders include one or more of the following: abnormal insulin levels, abnormal adiponectin levels, and abnormal leptin levels.

[0009] The present invention also provides the use of 5,7-dimethoxycoumarin in the preparation of medicaments for treating obesity.

[0010] Preferably, the obesity includes obesity induced by a high-fat diet.

[0011] Preferably, the obesity includes visceral fat accumulation and / or fat cell hypertrophy.

[0012] The present invention also provides the use of 5,7-dimethoxycoumarin in the preparation of medicaments for treating non-alcoholic fatty liver disease.

[0013] Preferably, the non-alcoholic fatty liver disease includes non-alcoholic fatty liver disease induced by a high-fat diet.

[0014] Preferably, the application includes regulating gut microbiota composition and / or improving serum metabolic profiles.

[0015] Beneficial effects: This invention provides the application of 5,7-dimethoxycoumarin in the preparation of drugs for treating metabolic syndrome, including metabolic disorders induced by a high-fat diet. This invention reveals for the first time a novel application of 5,7-dimethoxycoumarin in the treatment of metabolic syndrome. This substance can alleviate or treat metabolic disorders in multiple ways by regulating the composition of the gut microbiota and serum metabolic profiles, mainly involving key pathways such as steroid hormone biosynthesis and taurine and alpha-taurine metabolism. Verification through examples shows that the 5,7-dimethoxycoumarin described in this invention exhibits multi-target and comprehensive beneficial effects in the treatment of metabolic syndrome, significantly improving glucose metabolism disorders, effectively reducing fasting blood glucose and improving oral glucose tolerance. Simultaneously, it significantly reduces serum total cholesterol and low-density lipoprotein cholesterol levels. Furthermore, it significantly increases the level of the beneficial hormone adiponectin, reduces leptin levels, and improves insulin sensitivity, profoundly regulating the balance of metabolic hormones. The solution described in this invention provides a novel drug development strategy for the comprehensive prevention and treatment of metabolic syndrome. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The figure shows the effect of 5,7-dimethoxycoumarin on the body weight of high-fat diet-induced obese mice; among them... Figure 1 In the graph, 'a' represents the mouse's weight change curve. Figure 1 In the graph, b represents the total weight gain of mice. Figure 1 In the figure, 'c' represents the average food intake of mice; the significance of the markers in the figure is as follows: express p <0.05, express p <0.01, expressp <0.001; # (compared with the HFD model group) indicates p <0.05, ## indicates p <0.01; Figure 2 The figure shows the effect of 5,7-dimethoxycoumarin on glucose metabolism in high-fat diet-induced obese mice; among them... Figure 2 In the figure, 'a' represents the result of fasting blood glucose testing in mice. Figure 2 In the figure, b represents the oral glucose tolerance curve. Figure 2 In the figure, 'c' represents the calculated area under the oral glucose tolerance curve; the significance markers in the figure have the following meanings: express p <0.05, express p <0.01, express p <0.001; # (compared with the HFD model group) indicates p <0.05; Figure 3 The figure shows the effect of 5,7-dimethoxycoumarin on lipid metabolism in high-fat diet-induced obese mice; among them... Figure 3 In the figure, 'a' represents the result of total cholesterol level detection in mice. Figure 3 In the image, b represents the results of low-density lipoprotein cholesterol level detection in mice. Figure 3 In the image, 'c' represents the result of high-density lipoprotein cholesterol level detection in mice. Figure 3 The 'd' in the graph represents the results of mouse triglyceride level detection; the significance of the markers in the graph is as follows: express p <0.001; Figure 4 The figure shows the effects of 5,7-dimethoxycoumarin on liver function and pathological damage in high-fat diet-induced obese mice; among them... Figure 4 In the figure, 'a' represents the results of mouse liver quality testing. Figure 4 In the figure, b represents the results of the detection of triglyceride content in mouse liver. Figure 4 In the figure, 'c' represents a schematic diagram of mouse liver morphology and corresponding HE-stained and Oil Red O-stained sections. HE staining scale bar: 100 μm; Oil Red O staining scale bar: 50 μm. The meanings of the salient markers in the figure are as follows: express p <0.001; Figure 5 The figure shows the effects of 5,7-dimethoxycoumarin on adipose tissue accumulation and pathological damage in high-fat diet-induced obese mice; among them... Figure 5 In the figure, 'a' represents the results of mouse pWAT quality testing. Figure 5 In the figure, b represents the results of mouse eWAT quality testing. Figure 5 In the figure, 'c' represents the result of mouse eWAT cell area detection. Figure 5 In the figure, 'd' represents the morphology of mouse eWAT and an HE-stained section. The HE staining scale bar is 100 μm. The meanings of the salient markers in the figure are as follows: express p <0.01, express p <0.001; Figure 6 The figure shows the effect of 5,7-dimethoxycoumarin on serum metabolic hormone levels in high-fat diet-induced obese mice; among them... Figure 6 In the graph, 'a' represents the result of mouse insulin level detection. Figure 6 In the image, b represents the result of mouse adiponectin level detection. Figure 6 In the graph, 'c' represents the results of mouse leptin level detection; the significance of the markers in the graph is as follows: express p <0.05, express p <0.01, express p <0.001; Figure 7 The figure shows the effect of 5,7-dimethoxycoumarin on the gut microbiota of high-fat diet-induced obese mice; among them... Figure 7 In the figure, 'a' represents the PCoA analysis results based on ASV levels (HFD vs. HFD-5, 7DMC, based on Bray-Curtis distance). Figure 7 In the figure, b represents the LEfSe analysis results at the genus level (HFD vs. HFD-5,7DMC, LDA>2.0). Figure 7 c in the figure represents the results of the top 20 differentially abundant bacterial genera by mean abundance (HFD vs. HFD-5, 7DMC). Figure 8 The figure shows the effect of 5,7-dimethoxycoumarin on the serum metabolic profile of mice induced by a high-fat diet; among them... Figure 8 In the figure, 'a' represents the OPLS-DA score plot based on serum metabolites (HFD vs. HFD-5,7DMC). Figure 8 b in the figure represents the differential metabolite volcano plot (HFD vs. HFD-5,7DMC). Figure 8 In the diagram, c represents the metabolic pathway enrichment and topology analysis (HFD vs. HFD-5, 7DMC). Detailed Implementation

[0018] This invention provides the use of 5,7-dimethoxycoumarin in the preparation of medicaments for treating metabolic syndrome.

[0019] As one embodiment, the chemical formula of the 5,7-dimethoxycoumarin of the present invention is C 11 H 10 O4, CAS number 487-06-9, purity ≥98%. As one embodiment, the structural formula of the 5,7-dimethoxycoumarin of this invention is shown in Formula I: , Formula I.

[0020] In one embodiment, the 5,7-dimethoxycoumarin of the present invention is a white crystalline powder, soluble in methanol, ethanol, or dimethyl sulfoxide. In another embodiment, the dosage form of the drug of the present invention is an oral preparation. In one embodiment, the dosage of the 5,7-dimethoxycoumarin of the present invention is 1.5-3 mg per kg body weight per day. In yet another embodiment, the dosage of the 5,7-dimethoxycoumarin of the present invention is 1.62 mg per kg body weight per day.

[0021] In one embodiment, the metabolic syndrome described in this invention includes metabolic disorders induced by a high-fat diet.

[0022] As one embodiment, the metabolic disorder of the present invention includes one or more of the following: glucose metabolism disorder, lipid metabolism disorder, and metabolic hormone disorder.

[0023] As one implementation method, the glucose metabolism disorder of the present invention includes abnormal fasting blood glucose and / or abnormal oral glucose tolerance; The lipid metabolism disorder includes abnormal serum total cholesterol levels and / or abnormal low-density lipoprotein cholesterol levels; The metabolic hormone disorder includes one or more of the following: abnormal insulin levels, abnormal adiponectin levels, and abnormal leptin levels. As one embodiment, the 5,7-dimethoxycoumarin of this invention can significantly reverse metabolic hormone disorders in experimental mice, including significantly increasing serum insulin and serum adiponectin levels and significantly decreasing serum leptin levels in obese mice.

[0024] The present invention also provides the use of 5,7-dimethoxycoumarin in the preparation of medicaments for treating obesity.

[0025] In one embodiment, the obesity described in this invention includes obesity induced by a high-fat diet. In another embodiment, the 5,7-dimethoxycoumarin described in this invention can significantly inhibit weight gain.

[0026] In one embodiment, the obesity described in this invention includes visceral fat accumulation and / or adipocyte hypertrophy. In another embodiment, the 5,7-dimethoxycoumarin described in this invention can significantly alleviate visceral fat accumulation and improve adipocyte hypertrophy in experimental mice.

[0027] The present invention also provides the use of 5,7-dimethoxycoumarin in the preparation of medicaments for treating non-alcoholic fatty liver disease.

[0028] In one embodiment, the non-alcoholic fatty liver disease described in this invention includes non-alcoholic fatty liver disease induced by a high-fat diet. In another embodiment, the 5,7-dimethoxycoumarin described in this invention can significantly alleviate liver pathological damage in experimental mice, including significantly alleviating hepatic hypertrophy and hepatic fat accumulation in obese mice.

[0029] In one embodiment, the application of this invention includes regulating gut microbiota composition and / or improving serum metabolic profiles. In one embodiment, regulating gut microbiota composition includes upregulating the relative abundance of one or more of the following bacterial groups: Oscillibacter , Anaerotruncus norank_f_norank_o_ Clostridia _UCG-014、 Bilophila , Family_XIII _AD3011_group、 Alloprevotella norank_f_UCG-010 Odoribacter , Harryflintia , LachnospiraceaeFCS020_group, Eubacteriumxylanophilum_ group,NK4A214_group,unclassified_f_Atopobiaceae , Clostridium _sensu_stricto_1、 Eubacterium _brachy_group and Parasutterella As one implementation method, the regulation of gut microbiota composition according to the present invention includes downregulating the relative abundance of one or more of the following bacterial groups: Lachnospiraceae _NK4A136_group、 Lactobacillus , Erysipelatoclostridium , Negativibacillus , Dielma , Muribaculum , Anaerovorax , Frisingicoccus and Streptococcus As one embodiment, the improvement of serum metabolic profile described in this invention includes regulating steroid hormone biosynthesis pathways and / or taurine and taurine metabolic pathways.

[0030] To further illustrate the present invention, the application of 5,7-dimethoxycoumarin provided by the present invention in the preparation of drugs for treating metabolic syndrome is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 Effects of 5,7-Dimethoxycoumarin on body weight in high-fat diet-induced obese mice This embodiment aims to evaluate the effect of 5,7-dimethoxycoumarin on the body weight of high-fat diet-induced obese mice using high-fat diet-induced obese mice as experimental subjects.

[0032] 1. Experimental Methods Male C57BL / 6J mice (6-8 weeks old) were acclimatized for one week in a specific pathogen-free (SPF) laboratory at a temperature maintained at 23 ± 3°C, with a 12 h / 12 ​​h light-dark cycle, and provided with ample food and water for free access. After acclimatization, the mice were randomly divided into three groups of eight mice each, ensuring no significant difference in body weight among the groups at the start of the experiment. Mice were given free access to a basal maintenance diet (12.4% fat energy content) or a high-fat diet (60% fat energy content), and were simultaneously administered pure water or a 5,7-dimethoxycoumarin aqueous solution by gavage six times a week for eight weeks. The specific groupings are as follows: ① Normal control group (Chow): Free access to basal maintenance feed and gavage with pure water; ② High-fat diet model group (HFD): Free access to high-fat feed and gavage with pure water; ③ High-fat diet-5,7-dimethoxycoumarin group (HFD-5,7DMC): Mice were allowed free access to a high-fat diet and were administered 5,7-dimethoxycoumarin aqueous solution by gavage at a dose of 20 mg / kg·d (human equivalent dose 1.62 mg / kg·d). The body weight and food intake of mice in each group were recorded weekly.

[0033] 2. Experimental Results The effects of 5,7-dimethoxycoumarin on the body weight of high-fat diet-induced obese mice were measured as follows: Figure 1 As shown. According to Figure 1 It can be seen that there was no significant difference in the initial body weight of the mice in each group, indicating that the grouping was randomized and balanced. As the experiment progressed, the body weight and weight gain of the mice in the HFD group were consistently significantly higher than those in the Chow group. At the end of the experiment, the body weight of the mice in the HFD group (31.98 ± 1.02 g) was 1.24 times that of the Chow group (25.70 ± 0.51 g). p <0.001, and the weight gain (12.88 ± 0.70 g) was 1.73 times that of the Chow group (7.43 ± 0.40 g). p <0.001), indicating the successful establishment of the obesity model. Compared with the HFD group, the rate of weight gain in HFD-5,7DMC mice was significantly slower. From week 6 onwards, the body weight of HFD-5,7DMC mice remained significantly lower than that of the HFD group. At the end of the experiment, the body weight of the HFD-5,7DMC group (26.70 ± 0.28 g) was 83.49% of that of the HFD group (…).p <0.01), and the weight gain was 32.39% lower than that in the HFD group (8.71 ± 0.31 g). p <0.001). Furthermore, there was no significant difference in food intake among the HFD groups, indicating that the regulation of mouse body weight by 5,7-dimethoxycoumarin is not achieved by affecting energy intake.

[0034] The results above indicate that 5,7-dimethoxycoumarin has a significant inhibitory effect on weight gain in obese mice.

[0035] Example 2 Effects of 5,7-Dimethoxycoumarin on glucose metabolism in high-fat diet-induced obese mice This embodiment aims to evaluate the effect of 5,7-dimethoxycoumarin on glucose metabolism in high-fat diet-induced obese mice using high-fat diet-induced obese mice as experimental subjects.

[0036] 1. Experimental Methods The establishment and experimental treatment of a high-fat diet-induced obese mouse model are described in Example 1. At week eight, mice were fasted for 12 hours but allowed free access to water. Fasting blood glucose levels were measured, and an oral glucose tolerance test was performed. The specific procedures were as follows: Mouse weight was measured, and blood was collected via tail vein sampling and dripped onto blood glucose test strips. The blood glucose level was recorded as the initial blood glucose value (0 min). Mice were administered glucose solution by gavage at a dose of 3.0 g / kg body weight (BW). Timing was started immediately after gavage, and blood glucose levels were measured at 30 min, 60 min, 90 min, and 120 min after gavage. An oral glucose tolerance curve was plotted with time on the x-axis and blood glucose level on the y-axis, and the area under the curve (AUC) was calculated.

[0037] 2. Experimental Results The effects of 5,7-dimethoxycoumarin on glucose metabolism in high-fat diet-induced obese mice were measured as follows: Figure 2 As shown. According to Figure 2 It can be seen that the fasting blood glucose level of the HFD group mice was significantly higher than that of the Chow group ( p <0.05, indicating that HFD impairs the glucose regulation capacity of mice. At the end of the experiment, the fasting blood glucose level of mice in the HFD-5,7DMC group (7.39 ± 0.25 mmol / L) was significantly lower than that in the HFD group (8.48 ± 0.38 mmol / L), a decrease of 12.83%. p <0.05. The results of the OGTT showed that the blood glucose levels in the HFD group were significantly higher than those in the Chow group at all time points after the start of the trial. p <0.05), and the AUC value of the HFD group was significantly higher than that of the Chow group (p <0.001), further confirming that HFD significantly impairs glucose tolerance in mice. Compared with the HFD group, the blood glucose levels of mice in the HFD-5 and 7DMC groups were significantly lower at 60 min and 120 min after gavage administration of glucose solution ( ). p <0.05, and its AUC value was also significantly lower than that of the HFD group ( p <0.05).

[0038] The above results indicate that 5,7-dimethoxycoumarin has a significant effect on improving glucose metabolism disorders in obese mice, including significantly reducing fasting blood glucose and significantly improving oral glucose tolerance in obese mice.

[0039] Example 3 Effects of 5,7-Dimethoxycoumarin on lipid metabolism in high-fat diet-induced obese mice This embodiment aims to evaluate the effect of 5,7-dimethoxycoumarin on lipid metabolism in high-fat diet-induced obese mice using high-fat diet-induced obese mice as experimental subjects.

[0040] 1. Experimental Methods The establishment and experimental treatment of a high-fat diet-induced obese mouse model are described in Example 1. At the end of the experiment, the mice were fasted but allowed free water for 12 hours. The whiskers of both sides of the mice were cut off with dry scissors, and blood samples were collected by enucleation. After standing at 4°C for 6 hours, the samples were centrifuged at 3,500 rpm for 10 minutes at 4°C. The collected serum was stored at −80°C for subsequent analysis. The levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG) in the mouse serum were measured using commercial reagent kits (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.) according to the product instructions.

[0041] 2. Experimental Results The effects of 5,7-dimethoxycoumarin on lipid metabolism in high-fat diet-induced obese mice were measured as follows: Figure 3 As shown. According to Figure 3 It was found that the serum TC (5.57 ± 0.10 mmol / L) and LDL-C (4.34 ± 0.17 mmol / L) levels in the HFD group were significantly higher than those in the Chow group (TC: 2.91 ± 0.10 mmol / L, LDL-C: 1.58 ± 0.19 mmol / L). p <0.001), indicating significant lipid metabolism abnormalities in obese mice. However, there were no significant differences in serum TG and HDL-C levels among the groups. Compared with the HFD group, intervention with 5,7-dimethoxycoumarin significantly reduced serum TC (4.64 ± 0.13 mmol / L).p <0.001) and LDL-C (3.10 ± 0.23 mmol / L, p The level is <0.001).

[0042] The results above indicate that 5,7-dimethoxycoumarin has a significant effect on improving lipid metabolism disorders in obese mice, including significantly reducing serum TC and LDL-C levels in obese mice.

[0043] Example 4 Effects of 5,7-Dimethoxycoumarin on liver function and pathological damage in high-fat diet-induced obese mice This embodiment aims to evaluate the effects of 5,7-dimethoxycoumarin on liver function and pathological damage in high-fat diet-induced obese mice.

[0044] 1. Experimental Methods The establishment and experimental treatment of the high-fat diet-induced obese mouse model are described in Example 1. After the experiment, the mice were euthanized by cervical dislocation, dissected, and liver tissue was collected, weighed, and cut. A portion of the tissue was immersed in 4% paraformaldehyde fixative for subsequent histological evaluation, and the remaining tissue was aliquoted into cryovials, flash-frozen in liquid nitrogen, and stored at −80℃ for subsequent experimental analysis.

[0045] The specific procedure for hematoxylin and eosin (HE) staining of liver tissue is as follows: Mouse livers were fixed in 4% paraformaldehyde for one week, followed by dehydration, clearing, paraffin embedding, and sectioning. The paraffin sections were then dewaxed using the following steps: sections were sequentially immersed in xylene I (8 min) → xylene II (8 min) → xylene III (8 min) → anhydrous ethanol I (5 min) → anhydrous ethanol II (5 min) → 85% ethanol (5 min) → 75% ethanol (5 min) → tap water (2 min). HE staining was then performed using the following steps: hematoxylin staining for 5 min, differentiation with hydrochloric acid solution for 2 s, blueing with ammonia solution for 15-30 s, and washing with water. The sections were then dehydrated in 95% ethanol and then stained with eosin for 5-8 s. Finally, the slides were dehydrated and mounted using the following steps: The slides were sequentially immersed in anhydrous ethanol I (30 s) → anhydrous ethanol II (2.5 min) → anhydrous ethanol III (2.5 min) → xylene I (2.5 min) → xylene II (2.5 min). The cleared tissue slides were then removed, allowed to air dry slightly, and mounted with neutral resin. Microscopic examination and image acquisition and analysis were then performed.

[0046] The specific procedure for Oil Red O staining of liver tissue is as follows: Frozen sections are thawed and fixed in fixative for 5 minutes. Then, the frozen sections, now at room temperature, are gently immersed in freshly prepared Oil Red O working solution, covered, and protected from light for 8-10 minutes. Next, background differentiation is performed. The sections are removed, held for 3 seconds, and then sequentially immersed in 60% isopropanol I (8-10 seconds) → 60% isopropanol II (8-10 seconds) → pure water I (10 seconds) → pure water II (10 seconds). The sections are then stained with hematoxylin for 3-5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, and then blued with a blueing solution, followed by rinsing with running water. Finally, the sections are mounted with glycerol-gelatin mounting medium, examined under a microscope, and images are acquired for analysis.

[0047] The specific procedure for determining the TG content in liver tissue is as follows: Weigh approximately 100 mg of thawed liver tissue and add ice-cold PBS buffer (1×) at a ratio of tissue mass (g): homogenization medium (mL) = 1:9. Homogenize thoroughly using a high-speed homogenizer, centrifuge at 2,500 rpm for 10 min, and collect the supernatant for analysis. The TG content in the supernatant is determined using a commercial kit (Nanjing Jiancheng Bioengineering Institute Co., Ltd.) according to the manufacturer's instructions. The final result is expressed as μmol TG / g liver tissue.

[0048] 2. Experimental Results The effects of 5,7-dimethoxycoumarin on liver function and pathological damage in high-fat diet-induced obese mice were measured as follows: Figure 4 As shown. According to Figure 4 It can be seen that although the liver quality of the HFD group was higher than that of the Chow group, the difference was not statistically significant. p >0.05); while the liver quality of the HFD-5,7DMC group was significantly lower ( p <0.001. Liver lipid assay results showed that the liver TG level in the HFD group (70.14 ± 5.97 μmol / g liver) was significantly higher than that in the Chow group (36.51 ± 2.17 μmol / g liver). p <0.001), while the HFD-5,7DMC group (44.74 ± 3.03 μmol / g liver, p The liver TG level (<0.001) was significantly lower in the group than in the HFD group.

[0049] Morphological observation of the livers showed that the livers in the Chow group were dark red, firm, and normal in shape. In contrast, the livers in the HFD group were lighter in color and larger in size, suggesting possible fatty infiltration. The livers in the HFD-5,7DMC group were similar in color and size to those in the Chow group. HE staining of the livers showed that the hepatocytes in the Chow group were neatly arranged with homogeneous cytoplasm and no obvious lipid droplets, vacuoles, or inflammatory cell infiltration. In contrast, the hepatocytes in the HFD group were disordered, with diffuse lipid droplets and vacuoles in the cytoplasm, and localized inflammatory cell infiltration, indicating severe hepatocellular steatosis. In contrast, the vacuolar area in the HFD-5,7DMC group was significantly reduced, with occasional small lipid droplets. Oil Red O staining showed that only a few scattered lipid droplets were observed in the hepatocytes of the Chow group, while the HFD group showed diffuse lipid droplet deposition, presenting a "red patch" phenomenon, indicating severe lipid accumulation. In contrast, the lipid droplet area in the HFD-5,7DMC group was significantly reduced.

[0050] The results above indicate that 5,7-dimethoxycoumarin has a significant effect in alleviating pathological liver damage in obese mice, including significantly alleviating liver hypertrophy and hepatic fat accumulation in obese mice.

[0051] Example 5 Effects of 5,7-Dimethoxycoumarin on adipose tissue accumulation and pathological damage in high-fat diet-induced obese mice This embodiment aims to evaluate the effects of 5,7-dimethoxycoumarin on adipose tissue accumulation and pathological damage in high-fat diet-induced obese mice.

[0052] 1. Experimental Methods The establishment and experimental treatment of the high-fat diet-induced obesity mouse model are described in Example 1. After the experiment, mice were euthanized by cervical dislocation, and dissected to collect epididymal white adipose tissue (eWAT) and perirenal white adipose tissue (pWAT). The tissues were weighed, cut, and a portion was immersed in 4% paraformaldehyde fixative for subsequent histological evaluation. The area of ​​adipocytes in the eWAT sections was measured using ImageJ (USA).

[0053] 2. Experimental Results The effects of 5,7-dimethoxycoumarin on adipose tissue accumulation and pathological damage in high-fat diet-induced obese mice were measured as follows: Figure 5 As shown. According to Figure 5It was found that the eWAT mass (1447.00 ± 153.00 mg) and pWAT mass (464.00 ± 54.00 mg) in the HFD group were significantly higher than those in the Chow group (eWAT: 376.50 ± 26.99 mg, pWAT: 100.00 ± 9.28 mg). p <0.001 indicates significant accumulation of visceral fat in obese mice. In contrast, treatment with 5,7-dimethoxycoumarin significantly reduced the quality of white fat ( p <0.01).

[0054] Morphological observation of eWAT showed that the adipose tissue in each group was light pink. The eWAT in the Chow group was slender and smaller, while the eWAT in the HFD group was significantly larger. The adipose tissue in the HFD-5,7DMC group was smaller than that in the HFD group. Histopathological evaluation showed that the adipocytes in the Chow group were regular in shape, uniformly polygonal, with densely arranged intracellular lipid droplets and clearly visible nuclei. In contrast, the adipocytes in the HFD group showed significant hypertrophic changes, with a significant decrease in cell number at the same magnification. The adipocytes in the HFD-5,7DMC group were smaller than those in the HFD group and their morphology was closer to normal. Quantitative analysis of adipocyte area showed that the adipocyte area in both the Chow group and the HFD-5,7DMC group was significantly smaller than that in the HFD group. p <0.001).

[0055] The results above indicate that 5,7-dimethoxycoumarin significantly alleviated visceral fat accumulation in obese mice and improved adipocyte hypertrophy.

[0056] Example 6 Effects of 5,7-Dimethoxycoumarin on serum metabolic hormone levels in high-fat diet-induced obese mice Insulin, adiponectin, and leptin are important hormones that regulate glucose and lipid metabolism in the body. This study aims to evaluate the effects of 5,7-dimethoxycoumarin on serum metabolic hormone levels in high-fat diet-induced obese mice.

[0057] 1. Experimental Methods The establishment and experimental treatment of a high-fat diet-induced obese mouse model are described in Example 1, and the serum collection method is described in Example 3. Commercial ELISA kits (Wuhan Sanying Biotechnology Co., Ltd.) were used, and the levels of insulin, leptin, and adiponectin in mouse serum were measured according to the product instructions.

[0058] 2. Experimental Results The effects of 5,7-dimethoxycoumarin on serum metabolic hormone levels in high-fat diet-induced obese mice were measured as follows: Figure 6 As shown. According to Figure 6 It can be seen that, compared with the Chow group (adiponectin: 36.77 ± 3.15 μg / mL; leptin: 1.67 ± 0.01 ng / mL), the serum adiponectin level in the HFD group (19.97 ± 1.53 μg / mL) was significantly lower. p The level of leptin (5.09 ± 0.84 ng / mL) was significantly reduced (<0.01). p The level of insulin was significantly increased (<0.001), while there was no significant difference in insulin levels between the two groups.

[0059] Compared with the HFD group (2.52 ± 0.28 ng / mL), the HFD-5,7DMC group (5.67 ± 0.58 ng / mL) had significantly lower concentrations. p <0.001) significantly increased serum insulin levels. Intervention with 5,7-dimethoxycoumarin significantly reversed the HFD-induced decrease in adiponectin levels, causing them to significantly recover to 38.38 ± 4.61 μg / mL ( p <0.01). Simultaneously, 5,7-dimethoxycoumarin significantly reversed the HFD-induced increase in leptin levels, causing them to significantly decrease to 3.13 ± 0.33 ng / mL ( p <0.05).

[0060] The results above indicate that 5,7-dimethoxycoumarin significantly reversed the metabolic hormone disorders in obese mice, including significantly increasing serum insulin and adiponectin levels and significantly decreasing serum leptin levels in obese mice.

[0061] Example 7 Effects of 5,7-Dimethoxycoumarin on gut microbiota in high-fat diet-induced obese mice This embodiment aims to evaluate the effect of 5,7-dimethoxycoumarin on the gut microbiota of high-fat diet-induced obese mice using high-fat diet-induced obese mice as experimental subjects.

[0062] 1. Experimental Methods The establishment and experimental treatment of the high-fat diet-induced obese mouse model are described in Example 1. During the eighth week of the experiment, each mouse was placed individually in a new sterile cage, and 3-5 fresh fecal pellets were collected in sterile centrifuge tubes and immediately flash-frozen in liquid nitrogen at −80°C for subsequent 16S rRNA gene sequencing analysis.

[0063] Total genomic DNA of the microbial community was extracted from mouse feces using the EZNA® soil DNA kit. The quality of the extracted genomic DNA was assessed by 1% agarose gel electrophoresis, and the concentration and purity of the DNA were determined using a NanoDrop2000. Using the extracted DNA as a template, PCR amplification of the V3-V4 variable region of the 16S rRNA gene was performed using upstream primer 338F (SEQ ID NO.1: 5'-ACTCCTACGGGAGGCAGCAG-3') with a barcode sequence and downstream primer 806R (SEQ ID NO.2: 5'-GGACTACHVGGGTWTCTAAT-3'). The PCR reaction system was as follows: 4 μL of 5× TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 μM), 0.8 μL of downstream primer (5 μM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and the volume was brought to 20 μL. The amplification program was as follows: 95℃ pre-denaturation for 3 min, 27 cycles (95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s), followed by a stable extension at 72℃ for 10 min, and finally storage at 4℃. The PCR amplification products were purified using the AxyPrep DNA Gel Extraction Kit, and the recovered products were quantified using the Quantus™ Fluorometer. The purified PCR products were used to construct libraries and sequenced using the Miseq PE300 platform (Illumina, USA).

[0064] The raw paired-end sequencing sequences were quality-controlled using FASTP software to obtain high-quality clean data for subsequent analysis. Sequence assembly was performed using FLASH software. The optimized sequences after quality-controlled assembly were denoised using the DADA2 plugin in Qiime2 to obtain amplicon sequence variants (ASVs). All sample sequences were flattened according to the minimum number of sequences. Based on the Sliva 16S rRNA gene database (v138), the Naive Bayes classifier in Qiime2 was used for species taxonomy analysis of ASVs, with a classification confidence of 0.7. PCoA analysis based on the Bray-Curtis distance algorithm was used to examine the similarity of microbial community structure among samples, and the PERMANOVA nonparametric test was used to analyze whether the differences in microbial community structure between sample groups were significant.

[0065] 2. Experimental Results The effects of 5,7-dimethoxycoumarin on the gut microbiota of high-fat diet-induced obese mice were measured as follows: Figure 7 As shown. According to Figure 7 The PCoA analysis based on Bray-Curtis distance showed significant differences in the overall gut microbiota structure between the HFD group and the HFD-5,7DMC group. The samples from the two groups were significantly separated on the PC1 axis (explaining 24.12% of the variance) (PERMANOVA: p = 0.001). We then used LEfSe analysis to screen for genera with significant differences between the two groups (LDA>2.0). Compared with the HFD group, the HFD-5 and 7DMC groups induced significant changes in the relative abundance of 25 genera (16 upregulated and 9 downregulated).

[0066] The results above indicate that 5,7-dimethoxycoumarin significantly altered the gut microbiota composition of obese mice.

[0067] Example 8 Effects of 5,7-Dimethoxycoumarin on serum metabolic profiles in high-fat diet-induced obese mice This embodiment aims to evaluate the effect of 5,7-dimethoxycoumarin on the serum metabolic profile of high-fat diet-induced obese mice using high-fat diet-induced obese mice as experimental subjects.

[0068] 1. Experimental Methods The establishment and experimental treatment of a high-fat diet-induced obese mouse model are described in Example 1, and the serum collection method is described in Example 3. 50 μL of thawed mouse serum sample was transferred to a new centrifuge tube. 300 μL of 20% acetonitrile-methanol internal standard extraction buffer was added, vortexed for 3 min, and then centrifuged at 12,000 rpm for 10 min at 4°C. After centrifugation, 200 μL of the supernatant was transferred to a new centrifuge tube and incubated at −20°C for 30 min. After incubation, the tube was centrifuged at 12,000 rpm for 3 min at 4°C. After centrifugation, 180 μL of the supernatant was transferred to the corresponding sample vial liner for analysis.

[0069] Ultra-high performance liquid chromatography (ExionLC AD) and tandem mass spectrometry (QTRAP®) were used for qualitative and quantitative detection of metabolites in mouse serum. Chromatographic acquisition conditions were as follows: a Waters ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm, Waters, USA) was used as the solid phase. A linear gradient elution was employed with ultrapure water containing 0.1% formic acid (phase A) and acetonitrile containing 0.1% formic acid (phase B). The elution gradient was: 0 min: 95% A; 11.0 min: 10% A; 12.0 min: 10% A; 12.1 min: 95% A; 14 min: 95% A. The flow rate was 0.4 mL / min, the column temperature was 40 °C, and the injection volume was 2 μL. The mass spectrometry acquisition conditions were as follows: electrospray ionization source temperature 500℃, mass spectrometry voltage 5500 V and −4500 V, ion source gas I 55 psi, gas II 60 psi, curtain gas 25 psi, and collision-induced ionization parameters set to high. In a triple quadrupole, each ion pair was scanned and detected according to the optimized declustering voltage and collision energy.

[0070] Qualitative analysis was performed based on the target standard database built by Wuhan Metawell Biotechnology Co., Ltd., according to the retention time, mother-daughter ion pair information, and secondary spectroscopic data of the detected substances. Metabolite quantification was performed using the multiple reaction monitoring mode of triple quadrupole mass spectrometry. Mass spectrometry data were processed using Analyst 1.6.3 software. Orthogonal partial least squares-discriminant analysis (OPLS-DA) was used to reveal the main trends and structures in the metabolomics data. MetaboAnalyst 6.0 (https: / / www.metaboanalyst.ca / ) was used to perform pathway enrichment and pathway topology analysis on differentially expressed metabolites based on the KEGG database (Kyoto Encyclopedia of Genes and Genomes).

[0071] 2. Experimental Results The effects of 5,7-dimethoxycoumarin on the serum metabolic profile of high-fat diet-induced obese mice were measured as follows: Figure 8 As shown. According to Figure 8 As can be seen from the OPLS-DA score plot, the HFD group samples and the HFD-5,7DMC group samples (explanation 21.7%, R) 2 Y = 0.996, Q 2= 0.909) were significantly separated on the Tscore[1] axis of the predicted principal components, indicating that the model has high explanatory power and predictive ability. Based on variable importance projection (VIP ≥ 1.0), difference fold value (|Log2FC| ≥ 1.0) and statistical significance ( p <0.05) Differential metabolites were screened between groups. Results showed that, compared to the HFD group, the HFD-5,7DMC group induced significant changes in 63 metabolites (18 upregulated and 45 downregulated). Topology maps generated using MetaboAnalyst further revealed the enriched metabolic pathways under HFD and 5,7-dimethoxycoumarin intervention. Compared to the HFD group, the differentially metabolites induced by 5,7-dimethoxycoumarin intervention were mainly involved in steroid hormone biosynthesis and taurine and hypotaurine metabolism.

[0072] The results above indicate that 5,7-dimethoxycoumarin significantly altered the serum metabolic profile of obese mice.

[0073] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of 5,7-dimethoxycoumarin for the manufacture of a medicament for the treatment of metabolic syndrome.

2. Use according to claim 1, characterized in that, The metabolic syndrome includes high-fat diet-induced metabolic disorder.

3. Use according to claim 1, characterized in that, The metabolic disorder includes one or more of glucose metabolism disorder, lipid metabolism disorder, and metabolic hormone disorder.

4. Use according to claim 3, characterized in that, The glucose metabolism disorder includes one or more of fasting glucose abnormality and / or oral glucose tolerance abnormality. The lipid metabolism disorder includes one or more of serum total cholesterol abnormality and / or low-density lipoprotein cholesterol abnormality. The metabolic hormone disorder includes one or more of insulin level abnormality, adiponectin level abnormality, and leptin level abnormality.

5. Use of 5,7-dimethoxycoumarin for the manufacture of a medicament for the treatment of obesity.

6. Use according to claim 5, characterized in that, The obesity includes high-fat diet-induced obesity.

7. Use according to claim 5, characterized in that, The obesity includes one or more of visceral fat accumulation and / or adipocyte hypertrophy.

8. Use of 5,7-dimethoxycoumarin for the manufacture of a medicament for the treatment of non-alcoholic fatty liver disease.

9. Use according to claim 8, characterized in that, The non-alcoholic fatty liver disease includes high-fat diet-induced non-alcoholic fatty liver disease.

10. The use according to any one of claims 1 to 8, characterized in that, The use includes one or more of modulating gut microbiota composition and / or improving serum metabolic profile.

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