Use of 6-methoxybenzazolinone for regulating lipid metabolism and improving non-alcoholic fatty liver disease and metabolic syndrome

Abstract

The invention provides an application of 6-methoxybenzolinone in regulating lipid metabolism and improving non-alcoholic fatty liver disease and metabolic syndrome. The 6-methoxybenzolinone disclosed by the invention achieves the effects of regulating lipid metabolism and improving non-alcoholic fatty liver diseases and metabolic syndromes by virtue of a plurality of efficacy experiments.

Description

Technical Field

[0001] The present invention relates to the use of 6-methoxybenzoxazolinone (6-MBOA) for regulating lipid metabolism, improving non-alcoholic fatty liver disease (NAFLD) and metabolic syndrome. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a disease characterized by fatty deposits in the liver due to causes other than alcohol. NAFLD is the most common liver disease in developed countries. Non-alcoholic fatty liver disease (NASH) is the most serious form of NAFLD, which may lead to liver inflammation, fibrosis, cirrhosis, chronic liver failure and hepatocellular carcinoma (HCC). Currently, there is no approved therapy for the treatment of NASH or NAFLD. Therefore, the need for new treatments for NAFLD and NASH has not yet been met.

[0003] Metabolic syndrome refers to the accumulation of cardiovascular disease risk factors at the physiological metabolic level, including fat accumulation, excessive fat synthesis, hypertension, hyperlipidemia, hyperglycemia and obesity. Body fat generation is positively correlated with obesity. When body fat is excessively generated or the fat metabolism rate slows down, it is easy to cause fat accumulation in the liver to form fatty liver. Excess fat will also accumulate in adipose tissue, causing obesity. When fatty liver occurs, it is easy to cause liver damage, which in turn increases the liver index in the blood.

[0004] At present, the clinical drug treatment of non-alcoholic fatty liver disease and metabolic syndrome has limited effects and serious side effects, and many patients cannot continue treatment. More importantly, the drug only relieves symptoms but fails to fundamentally solve the problem. Therefore, how to develop a new drug that can effectively improve non-alcoholic fatty liver disease, metabolic syndrome and regulate lipid metabolism is an important issue that the present invention aims to solve. Adenosine 5'-monophosphate-activated protein kinase (AMPK) is an energy sensor that plays a key role in regulating cellular energy metabolism.

[0005] In order to solve the above problems, technicians in this field are in urgent need of developing novel compositions for improving non-alcoholic fatty liver disease, metabolic syndrome and regulating lipid metabolism to benefit the vast population in need. Summary of the invention

[0006] In view of this, an object of the present invention is to provide a use of 6-methoxybenzoxazolinone (6-MBOA) for preparing a composition for regulating lipid metabolism.

[0007] In one embodiment of the invention, the 6-MBOA downregulates de novo lipogenesis in a subject in need of regulated lipid metabolism.

[0008] In one embodiment of the invention, the 6-MBOA upregulates lipid metabolism and fatty acid β-oxidation in a subject in need of regulated lipid metabolism.

[0009] In one embodiment of the present invention, the 6-MBOA does not produce cytotoxicity to immortalized primary human hepatocytes at a concentration of 25-400 μM.

[0010] In one embodiment of the invention, the 6-MBOA reduces lipid accumulation in a subject in need of regulated lipid metabolism.

[0011] In one embodiment of the present invention, the 6-MBOA increases the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) in an individual whose lipid metabolism needs to be regulated.

[0012] In one embodiment of the present invention, the lipid metabolism is regulated by regulating the phosphorylation of acetyl-CoA carboxylase (ACC), while down-regulating the expression of fatty acid synthase FAS and up-regulating the expression of adipose triglyceride lipase (ATGL) and carnitine palmitoyl-transferase 1 (CPT1).

[0013] Another object of the present invention is to provide a use of 6-methoxybenzoxazolinone (6-MBOA) for preparing a composition for improving non-alcoholic fatty liver disease (NAFLD) and metabolic syndrome.

[0014] In one embodiment of the present invention, the 6-MBOA prevents weight gain in a subject in need of improvement of non-alcoholic fatty liver disease and metabolic syndrome.

[0015] In one embodiment of the present invention, the 6-MBOA improves adipose tissue hypertrophy and dyslipidemia in a subject in need of improvement of non-alcoholic fatty liver disease and metabolic syndrome.

[0016] In one embodiment of the present invention, the 6-MBOA improves glucose homeostasis, glucose tolerance and insulin resistance (HOMA-IR) in a subject in need of improvement of non-alcoholic fatty liver disease and metabolic syndrome.

[0017] In one embodiment of the present invention, the 6-MBOA improves hepatic fat deposition and inflammation induced by a high fat diet (HFD).

[0018] In one embodiment of the present invention, the 6-MBOA alleviates high-fat diet-induced renal injury.

[0019] In one embodiment of the present invention, the composition is a pharmaceutical composition, a food composition or a topical composition.

[0020] In one embodiment of the present invention, the pharmaceutical composition is in a dosage form for oral administration.

[0021] In one embodiment of the present invention, the pharmaceutical composition is in a dosage form for parenteral administration.

[0022] In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, adjuvant and / or food additive.

[0023] In one embodiment of the present invention, the composition has a powder, a granule, a solution, a gel, or a paste form.

[0024] In summary, the efficacy of 6-MBOA of the present invention is that it has been proven through various efficacy experiments that it can effectively regulate lipid metabolism, improve non-alcoholic fatty liver disease, improve blood sugar homeostasis and metabolic syndrome.

[0025] The following will further illustrate the implementation mode of the present invention. The following examples are used to illustrate the present invention, not to limit the scope of the present invention. Anyone skilled in the art may make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the definition of the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of 6-methoxybenzoxazolinone (6-MBOA, represented by CX in the figure) on the viability of HuS-E / 2 cells is shown. HuS-E / 2 cells were treated with 25-1600 μM CX for 24 hours, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the viability at the indicated concentrations. Data (n=3) are shown as the mean ± standard error of the mean (SEM). One-way ANOVA was performed using Dunnett's multiple comparison test to determine the statistical significance of the parameter data between the control group and other groups, which are indicated by asterisks (*, p<0.05; **, p<0.01; ***, p<0.001).

[0027] Figure 2A and 2B Shown is the effect of CX on lipid accumulation in HuS-E / 2 cells induced by oleic acid (OA). Figure 2A Micrographs of HuS-E / 2 cells treated with or without OA and CX. OA was treated at 0.1 mM, and CX was treated at the indicated concentrations. Scale bar is 100 μM. Micrographs were magnified 200 times. Figure 2B Lipid content of HuS-E / 2 cells treated with or without OA and CX is shown. OA was treated at 0.1 mM and CX was treated at the indicated concentrations. Data (n=3) are shown as mean ± SEM. One-way ANOVA and Tukey's multiple comparison test were performed to determine the statistical differences between each group and the other groups, where groups with different letters are statistically significant (p<0.05) and groups with the same letters are not statistically significant (p≥0.05).

[0028] Figure 3A and 3BThe effects of CX on the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in OA-induced HuS-E / 2 cells were shown. Phosphorylation of AMPK (AMPK converted to pAMPK) and ACC (ACC1 converted to pACC1) were determined by Western blot analysis, while the levels of fatty acid synthase (FAS) were inhibited and adipose triglyceride lipase (ATGL) and carnitine palmitoyl-transferase 1 (CPT1) were increased. Data (n=3) are shown as mean ± SEM. One-way analysis of variance and Tukey's multiple comparison test were performed to determine the statistical differences between each group and the other groups, where groups with different letters were statistically significant (p < 0.05), and groups with the same letters were not statistically significant (p ≥ 0.05). Control represents the control group.

[0029] Figures 4A to 4C The effects of CX on body weight, weight gain, food intake and food efficiency ratio in high fat diet (HFD)-induced obese mice are shown. Figure 4A showed weight gain during the study period. Figure 4B Displays food intake. Figure 4C Food efficiency ratio (FER) is shown as weight gain (g) / food intake (g). Data are shown as mean ± SEM (n = 8 per group). Based on one-way analysis of variance with Tukey post hoc test, the bars with different letters above represent statistically significant results (P < 0.05), while the bars with the same letters correspond to results that do not show statistically significant differences. ND represents the normal diet group, EGCG represents epigallocatechin gallate, and Coixol represents CX.

[0030] Figures 5A to 5G Show the effects of CX on fat deposition and lipid levels in HFD-induced obese mice. Figure 5A Shown is hematoxylin and eosin (H&E) staining of adipocytes in epididymal white adipose tissue (eWAT). Figure 5B Displays the weight of the eWAT. Figure 5C Displays adipocyte diameter. Figure 5D Displays the level of serum triglyceride (TG). Figure 5E Displays the level of serum total cholesterol (TC). Fig. 5F Displays the level of serum high-density lipoprotein-cholesterol (HDL-C). Figure 5G Shows the level of serum low-density lipoprotein-cholesterol (LDL-C). The scale bar is 100 μM. Data are shown as mean ± SEM. Based on the one-way analysis of variance with Tukey's post hoc test, the bars with different letters above represent statistically significant results (P < 0.05), while the bars with the same letter correspond to results that do not show statistically significant differences. If there are two letters above the bar, each letter should be compared with the letters of the other bars to determine whether the results show statistically significant differences.

[0031] Figures 6A to 6D The effects of CX on fasting blood glucose, intraperitoneal glucose tolerance, and insulin resistance in HFD-induced obese mice were shown. Fig. 6A The results showed that after 6 weeks of HFD-induced obese mice, the fasting blood glucose level gradually increased (### indicates that the difference between the HFD group and the control group is extremely significant p<0.001). However, after drug (CX) intervention, the fasting blood glucose level was able to maintain normal from 0 to 12 weeks (*** indicates that the difference between the CX group and the HFD group is extremely significant p<0.001). Figure 6B The area under the curve (AUC) of the intraperitoneal glucose tolerance test (IPGTT) is shown. Glucose concentration was measured by tail vein sampling after intraperitoneal injection of glucose at a concentration of 1.0 g / kg body weight. Data are shown as mean ± SEM. Unpaired one-tailed Student's t-test was used for analysis. Fig. 6A Differences in fasting blood glucose (ND vs. HFD, ###p<0.001, HFD vs. drug, ***p<0.001). Bars with different letters above indicate statistically significant results (P<0.05), while bars with the same letter correspond to results that did not show statistically significant differences, based on one-way ANOVA with Tukey post hoc test. If there are two letters above a bar, each letter should be compared with the letters of the other bars to determine whether the results show statistically significant differences. ( Figure 6C ) is the fasting insulin concentration. Fig.6D ) Homeostasis model assessment index of insulin resistance (HOMA-IR). HOMA-IR was calculated as [fasting insulin concentration (mU / L) × fasting glucose concentration (mmol / L)] / 22.5. Data (n=8) are presented as mean ± standard error. ND: normal diet; HFD: high-fat diet; EGCG: epigallocatechin gallate. Different letters (e.g., a, b) indicate statistically significant differences (p<0.05).

[0032] Figures 7A to 7H showed the effects of CX on hepatic fat deposition and inflammation in HFD-induced obese mice. Fig. 7A Liver weights are shown. Figure 7B Shows liver triglyceride levels. Figure 7C Shows liver cholesterol levels. Fig.7D H&E staining of liver cross sections is shown (original magnification x200). Serum levels of liver injury markers. Fig. 7E Displays glutamic oxaloacetic transaminase (GOT). Figure 7F Displays alanine transaminase (glutamic pyruvic transaminase, GPT). The serum level of liver biochemical markers is based on serum albumin (albumin, ALB). Obesity will increase the ALB value, while liver and kidney diseases will reduce the ALB value. Figure 7G Displays albumin (ALB). Figure 7H Liver weight ratios are shown. Scale bar is 100 μM. Bars with different letters above represent statistically significant results (P<0.05) based on one-way ANOVA with Tukey post hoc test, while bars with the same letter correspond to results that did not show statistically significant differences. If there are two letters above a bar, each letter should be compared with the letters of the other bars to determine whether the results show statistically significant differences.

[0033] Fig. 8A and 8B The effects of CX on renal function in HFD-induced obese mice are shown. Serum creatinine (CRE) (8A) and uric acid (UA) (8B) levels were used as indicators of renal function. Data are shown as mean ± SEM. Bars with different letters above represent statistically significant results (P < 0.05) based on one-way analysis of variance with Tukey's post hoc test, while bars with the same letter correspond to results that did not show statistically significant differences. If there are two letters above a bar, each letter should be compared with the letters of the other bars to determine whether the results show statistically significant differences. DETAILED DESCRIPTION

[0034] definition

[0035] The numerical values ​​used in this article are approximate values, and all experimental data are expressed in the range of ±20%, preferably in the range of ±10%, and most preferably in the range of ±5%.

[0036] Unless otherwise specified herein, the terms “a”, “an”, “the” and similar terms used in this specification (especially in the claims) should be understood to include both the singular and the plural.

[0037] According to the present invention, 6-methoxybenzoxazolinone (6-MBOA) (CX) (chemical formula C 8 H 7 NO 3 ) is a lactam compound in the Gramineae family, which has been confirmed by the present invention to have anti-lipid synthesis, lipid decomposition promotion, anti-diabetes and AMPK, ACC protein kinase regulation activities.

[0038] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for parenteral or oral administration using techniques well known to those skilled in the art, including, but not limited to: injections [e.g., sterile aqueous solutions or dispersions], sterile powders, tablets, troches, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries and the like.

[0039] The pharmaceutical composition according to the present invention can be administered via a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, intralesional injection, sublingual administration and transdermal administration.

[0040] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier widely used in drug manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of: solvent, emulsifier, suspending agent, decomposer, binder, excipient, stabilizer, chelating agent, diluent, gelling agent, preservative, lubricant, absorption delaying agent, liposome and the like. The selection and amount of these agents fall within the professional qualities and routine technical scope of those skilled in the art.

[0041] According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of: water, normal saline, phosphate buffered saline (PBS), sugar solution, aqueous solution containing alcohol, and combinations thereof.

[0042] According to the present invention, the process of statistical analysis of Examples 1 to 3 below is as follows. GraphPad Prism 7.03 (GraphPad, USA) was used to analyze and plot data. One-way ANOVA was performed using Dunnett's multiple comparison test to determine the statistical significance of parameter data between the control group and other groups, indicated by asterisks (*, p<0.05; **, p<0.01; ***, p<0.001). One-way ANOVA and Tukey's multiple comparison test were performed to determine the statistical differences between each group and other groups, where groups with different letters were statistically significant (p<0.05), and groups with the same letters were not statistically significant (p≥0.05).

[0043] According to the present invention, the process of statistical analysis of Examples 4 to 8 below is as follows. Data obtained from all experiments are shown as mean ± standard error of the mean (SEM). Unpaired single-tailed Student's t-test was used to evaluate differences in body weight and fasting blood glucose. Data sets involving more than two groups were evaluated by analysis of variance (ANOVA) using Tukey's post hoc test. A P value of 0.05 was considered statistically significant. In the figure, data with different superscript letters were significantly different based on post-ANOVA statistics.

[0044] The present invention is further illustrated by the following examples, which are provided for illustration only and are not intended to limit the scope of the present invention, which is defined by the claims.

[0045] Example 1. 6-Methoxybenzoxazolinone (6-MBOA) (CX) has little cytotoxicity in HuS-E / 2 cells

[0046] To determine the cytotoxicity of 6-methoxybenzoxazolinone (6-MBOA) CX (Cat. 543551) (Sigma-Aldrich, USA), HuS-E / 2 cells were treated with the indicated concentrations of CX for 24 hours. CX stock solution was 200 mM dissolved in dimethyl sulfoxide (DMSO; Cat. 15578544, JTBaker, USA) and stored at -20°C.

[0047] The culture process of immortalized primary human hepatocyte HuS-E / 2 cell line is as follows. HuS-E / 2 cell line was kindly provided by Dr. Shimotohno (Kyoto University, Japan) and maintained in a medium containing 20mM HEPES, 10% fetal bovine serum, 15μg / mL L-proline, 0.25μg / mL insulin, 50nM dexamethasone, 44mM NaHCO 3, 10 mM nicotinamide, 5 ng / mL epidermal growth factor (EGF), 0.1 mM Asc-2P, 100 IU / mL penicillin, 100 μg / mL streptomycin, 10 μg / mL gentamicin, and 1 μg / mL plasmocin (dissolved in high glucose Dulbecco's modified Eagle medium (DMEM)). The cells were incubated at 37°C in an atmosphere supplemented with 5% CO 2 cultured in an incubator.

[0048] The stock solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Cat. M5655) (Sigma-Aldrich, USA) was 5 mg / mL dissolved in 1× phosphate buffered saline (PBS) and stored at -20°C.

[0049] The procedure for cell viability is as follows. HuS-E / 2 cells were cultured at 4.5×10 4 Cells / well were seeded in a 96-well plate and treated with 0-1600 μM CX for 24 hours. MTT was added to each well to a final concentration of 0.5 μg / mL and incubated at 37°C for 1 hour before removal. MTT-formazan crystals formed by the metabolism of living cells were dissolved in 200 μL DMSO and stained using a TECAN, Switzerland, manufacturer's product. The absorbance was measured at 550 nm using a Multimode microplate analyzer. The 50% cytotoxic concentration (CC) was calculated using the normalized response (variable slope) model provided by GraphPad Prism 7.03 (USA). 50 ).

[0050] Figure 1Effect of CX on HuS-E / 2 cell viability is shown. HuS-E / 2 cells were treated with 25-1600 μM CX for 24 hours, and MTT assay was performed to determine viability at the indicated concentrations. Data (n=3) are shown as mean ± standard error of the mean (SEM). One-way ANOVA was performed using Dunnett's multiple comparison test to determine the statistical significance of parameter data between the control group and other groups, indicated by asterisks (*, p<0.05; **, p<0.01; ***, p<0.001).

[0051] like Figure 1 As shown, CX CC 50 The value was 1431.5±169.6 μM. In particular, 25-400 μM of CX had no significant effect on the viability of HuS-E / 2 cells. In conclusion, treatment with up to 400 μM of CX did not cause cytotoxicity to HuS-E / 2 cells.

[0052] Example 2. CX alleviates oleic acid (OA)-induced lipid accumulation in HuS-E / 2 cells

[0053] To evaluate the effect of CX on lipid accumulation, HuS-E / 2 cells were treated with or without 0.1 mM OA (Cat. O1383) in the presence or absence of CX for 18 h. OA stock solution was 3.15 M in DMSO and stored at -20°C.

[0054] The procedure for oil red O staining is as follows. Oil red O stain (Cat.O0625) was purchased from Sigma-Aldrich (USA). The stock solution of oil red O stain was dissolved in 3 mg / mL isopropanol and stored at room temperature. For microscopic observation, cells were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde, fixed at room temperature for 30 minutes, stained with oil red O staining working solution (60% oil red O stock solution and 40% distilled water) at room temperature for 1 hour, and then rinsed with water. Micrographs were taken under a microscope. For quantitative analysis of cell lipids, cells were washed three times with ice-cold PBS, fixed with 10% formalin for 1 hour, washed, stained with oil red O solution at room temperature for 1 hour, and then washed with water to remove excess dye. The cell-bound oil red O dye was dissolved in isopropanol at room temperature and shaken for 5 minutes. The absorbance at 510 nm was measured in a spectrophotometer. Relative lipid content was calculated by setting the readings receiving vehicle to 0% and the readings receiving free fatty acids only to 100%.

[0055] Figure 2A and 2B Shown are the effects of CX on OA-induced lipid accumulation in HuS-E / 2 cells. Figure 2A Micrographs of HuS-E / 2 cells treated with or without OA and CX. OA was treated at 0.1 mM, and CX was treated at the indicated concentrations. Scale bar is 100 μM. Micrographs were magnified 200 times. Figure 2B Lipid content of HuS-E / 2 cells treated with or without OA and CX is shown. OA was treated at 0.1 mM and CX was treated at the indicated concentrations. Data (n=3) are shown as mean ± SEM. One-way ANOVA and Tukey's multiple comparison test were performed to determine the statistical differences between each group and the other groups, where groups with different letters are statistically significant (p<0.05) and groups with the same letters are not statistically significant (p≥0.05).

[0056] like Figure 2A As shown, HuS-E / 2 cells treated with 0.1 mM OA alone showed lipid accumulation compared with the control group. Meanwhile, CX reduced lipid accumulation in OA-induced HuS-E / 2 cells in a dose-dependent manner. On the other hand, the lipid content of HuS-E / 2 cells was determined at the same time. Figure 2B As shown, CX reduced lipid content in OA-induced HuS-E / 2 cells, which corresponds to the observations in the micrographs. In conclusion, CX improved lipid accumulation in OA-induced HuS-E / 2 cells.

[0057] Example 3. CX increases AMPK phosphorylation to downregulate OA-induced de novo lipogenesis and upregulate lipolysis and fatty acid β-oxidation in HuS-E / 2 cells

[0058] In order to determine how CX improves lipid accumulation, this example studies signal pathways related to lipid metabolism. Among them, adenosine 5'-monophosphate-activated protein kinase (AMPK) is an energy sensor that plays a key role in regulating cellular energy metabolism.

[0059] The procedure for Western blot analysis is as follows: For Western blot analysis, equal amounts of protein from each sample were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a poly(vinylidene fluoride) (PVDF) membrane. Primary antibodies against AMPK (Cat. GTX103487), adipose triglyceridelipase (ATGL) (Cat. GTX109941), carnitine palmitoyl-transferase 1 (CPT1) (Cat. GTX114337) and β-actin (Cat. GTX109639) were from GeneTex, and primary antibodies against pAMPK (Cat. AP0432), phospho acetyl-CoAcarboxylase (pACC) (Cat. AP0298), ACC (Cat. A15606) and fatty acid synthase (FAS) (Cat. A21182) were from ABclonal. Horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Jackson ImmunoResearch Laboratories, Inc., USA. ECL kit (Advansta Inc., USA) was used for protein visualization. Protein expression levels were quantified by densitometry analysis.

[0060] Figure 3A The effect of CX on the expression of AMPK, ACC, and lipid metabolism-related enzymes in OA-induced HuS-E / 2 cells was shown. The levels of pAMPK, AMPK, pACC1, ACC1, FAS, ATGL, and CPT1 protein expression were determined by Western blot analysis. Data (n=3) are shown as mean ± SEM. One-way ANOVA and Tukey's multiple comparison test were performed to determine the statistical differences between each group and other groups, where groups with different letters were statistically significant (p<0.05), groups with the same letters were not statistically significant (p≥0.05), and control represents the control group.

[0061] like Figure 3BAs shown in the results, treatment with 0.1 mM OA alone significantly reduced AMPK phosphorylation compared with the control group. At the same time, treatment with 100-400 μM CX significantly increased AMPK phosphorylation in OA-induced HuS-E / 2 cells. Regarding proteins associated with lipid accumulation, acetyl-CoA carboxylase (i.e., ACC1 and ACC2) is the rate-limiting enzyme in lipid production, and ACC and fatty acid synthase (FAS) participate in the biosynthesis of long-chain saturated fatty acids. Phosphorylation of the ACC enzyme (conversion of ACC to pACC) reduces ACC activity and, in turn, reduces fatty acid synthesis. In response to upregulated AMPK phosphorylation, 50-400 μM CX reduces ACC1 activity by increasing ACC1 phosphorylation, while 100-400 μM CX downregulates FAS expression, and both inhibit fatty acid biosynthesis. In contrast to ACC1 and FAS, adipose triglyceride lipase (ATGL) is involved in the catalysis of lipid droplet degradation, while carnitine palmitoyl-transferase 1 (CPT1) is involved in fatty acid β-oxidation. Consistently, 50-400 μM CX significantly increased ATGL expression, while 50-400 μM CX also increased CPT1 levels. In summary, CX improves lipid accumulation by downregulating lipogenic enzymes and upregulating enzymes that degrade lipolysis and fatty acid β-oxidation in HuS-E / 2 cells. Specifically, CX regulates cellular energy homeostasis through AMPK phosphorylation. Accordingly, CX increases ACC1 phosphorylation and decreases FAS expression to reduce fatty acid synthesis, while promoting ATGL and CPT1 expression to promote fatty acid degradation.

[0062] Example 4. CX intervention prevents weight gain and increases food efficiency ratio in high fat diet (HFD)-induced obese mice

[0063] To determine the effects of CX (Sigma, Inc., USA) on obesity and non-alcoholic fatty liver disease (NAFLD) in vivo, this example established an experimental method using HFD-induced obese C57BL / 6J mice. CX was suspended in 0.5% methylcellulose solution for oral administration.

[0064] The animals used in the examples are described as follows. Male C57BL / 6J mice (5 weeks old) were purchased from the Taiwan Experimental Animal Center (Taipei, Taiwan Province, China) and housed in a temperature-controlled room with a 12-h light-dark cycle. They were kept in cages with four mice per cage and had free access to food and drinking water. The mice, which were fed a standard diet and acclimatized for 1 week, were then randomly divided into five groups. The normal diet (ND) group (n=8) continued to use the same diet, while the other four groups (n=8 in each group) switched to HFD (494 kcal / 100 g, 45% energy as fat; TestDiet Inc., USA). Of the four HFD groups, three groups began to receive 10 mg / kg / day of CX (CX10 group), 30 mg / kg / day of CX (CX30 group), and 30 mg / kg / day of epigallocatechin gallate (EGCG) (Sigma, Inc., USA) (EGCG30 group) by oral gavage. EGCG was suspended in 0.5% methylcellulose solution for oral administration. Food consumption and body weight gain were measured daily and weekly, respectively. After 12 weeks, all mice were sacrificed. Serum samples, liver tissue, epididymal adipose tissue, and feces were collected for further analysis. Figures 4A to 4C The effects of CX on body weight, weight gain, food intake, and food efficiency ratio in HFD-induced obese mice are shown. Figure 4A showed weight gain during the study period. Figure 4B Displays food intake. Figure 4C Food efficiency ratio (FER; weight gain (g) / food intake (g)) is shown. Data are shown as mean ± SEM (n = 8 per group). Based on Tukey's post hoc test one-way analysis of variance, the bars with different letters above represent statistically significant results (P < 0.05), while the bars with the same letters correspond to the results that do not show statistically significant differences.

[0065] Compared with ND control mice, 12 weeks of HFD feeding resulted in a significant increase in body weight and food efficiency ratio (FER) ( Figure 4A and 4C ). EGCG was used as the drug control group. Figure 4A As shown in Figure 2, CX and EGCG significantly prevented weight gain. In addition, the FER of the CX10 and CX30 groups was greatly reduced compared with the HFD group, and food intake was almost equal ( Figure 4B and 4C ), which means less weight gain when eating the same weight of food after CX treatment.

[0066] Example 5. Adipose tissue hypertrophy and dyslipidemia in high-fat diet-induced obese mice were improved after CX intervention

[0067] A well-known feature of metabolic syndrome is increased lipid accumulation in the trunk region, resulting in excessive visceral fat deposition. In order to confirm that the weight gain is indeed an increase in fat mass, epididymal white adipose tissue (eWAT) was isolated after autopsy of mice in this example.

[0068] The procedure for immunohistochemical tissue characterization is as follows. During mouse dissection, epididymal adipose tissue and liver were isolated, weighed, and then fixed in PBS containing 10% triformaldehyde. After overnight fixation, the tissues were embedded in paraffin for hematoxylin and eosin (H&E) staining. All specimens were observed under a microscope (Carl Zeiss Inc., Germany) at 200x magnification. H&E staining of paraffin sections showed that the adipocyte size in the HFD group was larger than that in the ND group.

[0069] The procedure for biochemical characterization is as follows. Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), glutamic oxaloacetic transaminase (GOT), alanine transaminase (GPT), albumin (ALB), uric acid (UA), and creatinine (CRE) levels were measured using an enzyme assay kit with a FUJIDRI-CHEM analyzer (Fujifilm, Tokyo, Japan). LDL-C levels were calculated as [(TC)-(HDL-C)-(TG / 5)].

[0070] Figures 5A to 5G Show the effects of CX on fat deposition and lipid levels in HFD-induced obese mice. Figure 5A H&E staining of adipocytes in eWAT is shown. Figure 5B Displays the weight of the eWAT. Figure 5C Displays adipocyte diameter. Figure 5D Displays the level of serum triglyceride (TG). Figure 5EDisplays the level of serum total cholesterol (TC). Fig. 5F Displays the level of serum high-density lipoprotein-cholesterol (HDL-C). Figure 5G Shows the level of serum low-density lipoprotein-cholesterol (LDL-C). The scale bar is 100 μM. Data are shown as mean ± SEM. Based on the one-way analysis of variance with Tukey's post hoc test, the bars with different letters above represent statistically significant results (P < 0.05), while the bars with the same letter correspond to results that do not show statistically significant differences. If there are two letters above the bar, each letter should be compared with the letters of the other bars to determine whether the results show statistically significant differences.

[0071] The size of adipocytes after CX or EGCG administration was similar to that of the ND group ( Figure 5A ).like Figure 5B As shown, HFD-induced eWAT mass increase was improved by CX or EGCG intervention. In addition, this example found that compared with the adipocytes in the HFD group, the cell diameter and cell volume of the CX and EGCG groups were smaller ( Figure 5C The serum triglyceride (TG) and total cholesterol (TC) levels in the HFD group were significantly higher than those in the ND group ( Figure 5D and 5E ). After CX or EGCG treatment, serum TG and TC levels were significantly lower than those in the HFD group. After CX or EGCG intervention, HDL cholesterol (HDL-C) levels were not significantly different from those in the HFD group ( Fig. 5F ). The LDL-C level in the HFD group was also significantly higher than that in the ND group, but was improved by supplementation with CX or EGCG ( Figure 5G ). This suggests that CX can reduce fat deposition and suppress hypertriglyceridemia and high cholesterol levels in the HFD mouse model.

[0072] Example 6. CX improves glucose homeostasis and insulin resistance in HFD-induced obese mice

[0073] Studies have shown that NAFLD is closely related to insulin resistance, as 70% to 80% of obese and diabetic patients suffer from NAFLD. Obesity is the main risk factor for insulin resistance, which causes liver cells to convert glycogen into glucose and release glucose into the blood. And reduce the effect of fat and muscle on glucose absorption. In addition, insulin resistance also inhibits the β-oxidation of free fatty acids, further promoting liver fat accumulation. In this regard, in addition to measuring the fasting blood glucose of mice every two weeks, this embodiment also analyzes the amount of insulin in their serum after the mice are sacrificed. First, this embodiment measures fasting blood glucose, which is directly related to impaired insulin sensitivity.

[0074] The procedures for blood glucose and intraperitoneal glucose tolerance tests (IPGTTs) are as follows. After fasting for 16 hours overnight, whole blood glucose was measured using a glucose analyzer. Serum insulin concentration was determined using an enzymatic assay (Cisbio, USA). Twelve weeks after the start of the study, all mice underwent intraperitoneal glucose tolerance tests (IPGTTs). Mice fasting for 16 hours were intraperitoneally injected with 1.0 g / kg body weight of glucose, and tail vein blood glucose values ​​were measured at 0, 30, 60, 90, 120, and 150 minutes.

[0075] Fig. 6A and 6B Show the effect of CX on glucose metabolism in HFD-induced obese mice. Fig. 6A Shows fasting blood glucose levels from week 0 to week 12 after drug intervention. Figure 6B The area under the IPGTT curve (AUC) is shown. Data are shown as mean ± SEM. Unpaired one-tailed Student's t-test was used for analysis. Fig. 6A Fasting blood glucose differences in ND vs. HFD, ###p<0.001, HFD vs. medication, ***p<0.001). Bars with different letters above indicate statistically significant results (P<0.05) based on Tukey's post hoc test one-way ANOVA, while bars with the same letter correspond to results that did not show statistically significant differences. If there are two letters above a bar, each letter should be compared with the letters of the other bars to determine whether the results show statistically significant differences.

[0076] First, blood samples were drawn every two weeks during the feeding process to analyze the fasting blood glucose level. The fasting blood glucose level of the HFD group was significantly higher than that of the ND group starting from the second week, and increased rapidly after the eighth week, and was significantly higher than that of the control ND group (p<0.001). The fasting blood glucose levels of the CX10 group, CX30 group, and EGCG30 group were stable and significantly lower than those of the HFD group after the second week ( Fig. 6A), extremely significant differences were observed in the eighth to twelfth week (p<0.001). In addition, a glucose tolerance test was performed at the twelfth week to measure blood glucose changes at 0, 30, 60, 90, 120 and 150 minutes and calculate the area under the curve (AUC). In terms of the AUC of IPGTT, the HFD group was significantly higher than the ND group, indicating that the intraperitoneal injected glucose could not be effectively removed. The AUC of the CX10 group and the CX30 group were significantly lower than that of the HFD group, indicating that the glucose tolerance of the drug-treated group was better than that of the HFD group ( Figure 6B ). In terms of insulin expression, the HFD group was significantly higher than the ND group, while the CX10 group and CX30 group had significantly lower insulin expression than the HFD group ( Figure 6C ). Then, the insulin resistance index HOMA-IR was calculated and analyzed through blood glucose and insulin levels. It was also observed that the insulin resistance index of the HFD group was significantly higher than that of the ND group, while the indexes of the CX10 group and CX30 group were significantly lower than those of the HFD group ( Fig.6D ). To summarize the above experimental results, HFD not only causes hyperglycemia in mice and reduces the ability to metabolize glucose, but also causes insulin resistance. All of the above phenomena were significantly improved in the group treated with CX.

[0077] Example 7. CX improves the severity of NAFLD in HFD-induced obese mice

[0078] One of the prominent features of metabolic syndrome is NAFLD, which is characterized by the accumulation of triglycerides in hepatocytes. To examine the effect of CX on hepatic lipid deposition, the present example weighed the liver and measured the hepatic triglyceride and cholesterol levels of mice.

[0079] The procedures for triglyceride and cholesterol analysis of liver tissue are as follows: For triglyceride and cholesterol determination, mouse liver tissue was extracted and analyzed using triglyceride and cholesterol quantification kits (Abcam, UK), respectively, according to the manufacturer's instructions.

[0080] Figures 7A to 7H showed the effects of CX on hepatic fat deposition and inflammation in HFD-induced obese mice. Fig. 7A Liver weights are shown. Figure 7B Shows liver triglyceride levels. Figure 7C Shows liver cholesterol levels. Fig.7D H&E staining of liver cross sections is shown (original magnification ×200). Fig. 7E Displays glutamic oxaloacetictransaminase (GOT), Figure 7FAlanine aminotransferase (glutamic pyruvic transaminase, GPT), serum GOT and GPT levels are shown as indicators of liver damage. Figure 7G Display serum albumin (ALB) as a liver biochemical indicator. Figure 7H Liver to body weight ratio is shown. Scale bar is 100 μM. Bars with different letters above represent statistically significant results (P<0.05) based on one-way ANOVA with Tukey post hoc test, while bars with the same letter correspond to results that did not show statistically significant differences. If there are two letters above a bar, each letter should be compared with the letters of the other bars to determine whether the results show statistically significant differences.

[0081] The results showed that after drug intervention, the liver weights of the CX and EGCG groups were significantly lower than those of the HFD group ( Fig. 7A ). In addition, the levels of hepatic triglyceride (TG) and cholesterol (TC) were higher after HFD feeding but lower after CX and EGCG treatment ( Figure 7B and 7C ). H&E and Oil Red O staining showed considerable lipid deposition in the liver tissue of the HFD group, while CX or EGCG treatment significantly improved lipid deposition ( Fig.7D ), while maintaining the normal morphology of liver cells. Next, this example studies liver damage markers, including alanine aminotransferase (glutamic pyruvic transaminase, GPT) and aspartate aminotransferase (glutamic oxaloacetic transaminase, GOT). Serum GOT and GPT levels were higher in HFD mice, but these high levels were significantly prevented by CX in both supplements, indicating that liver damage was improved ( Fig. 7E and 7F ). In addition, the level of albumin, a clinical biochemical marker produced by the liver, was significantly increased in the HFD group, and there was no difference in albumin levels between the ND group, CX group, and EGCG group ( Figure 7G ), indicating that CX can maintain normal liver biochemical metabolism. The liver-to-body weight ratio did not differ among all groups ( Figure 7H ). These results suggest that CX can improve the severity of HFD-induced NAFLD, maintain normal liver function, and prevent liver hypertrophy.

[0082] Example 8. CX improves HFD-induced renal injury

[0083] Studies have shown that a high-fat diet can promote renal impairment and lead to kidney damage. Therefore, this example studies common biochemical indicators of renal function, including creatinine (CRE) and uric acid (UA).

[0084] Fig. 8A and 8B The effects of CX on renal function in HFD-induced obese mice were shown. Serum creatinine (CRE) (8A) and uric acid (UA) (8B) were used as indicators of renal function. Creatinine (CRE) is a decomposition product of creatine in human muscle and is filtered out by the glomerulus. When the glomerular filtration rate decreases, the CRE in the blood will increase. Uric acid (UA) is a metabolite of purine. High concentrations of uric acid (UA) represent low renal metabolic function. Therefore, the levels of CRE and UA in serum are reverse indicators of renal function. Data are shown as mean ± SEM. Based on one-way analysis of variance with Tukey's post hoc test, bars with different letters above represent statistically significant results (P < 0.05), while bars with the same letter correspond to results that do not show statistically significant differences. If there are two letters above a bar, each letter should be compared with the letters of the other bars to determine whether the results show statistically significant differences.

[0085] It has been reported that the natural compound EGCG can protect against renal injury or other kidney damage. Therefore, the renal injury protective effect of CX was compared with EGCG as a reference. Compared with the ND group, the CRE and UA levels in the HFD group were significantly increased, indicating that a high-fat diet can cause renal damage. Interestingly, the CRE and UA levels decreased after CX treatment, and CX significantly improved ( Fig. 8A and 8B There was no significant difference in CRE level between the CX30 group and the ND group and the EGCG group, and there was no significant difference in UA between the CX10-30 group and the ND group and the EGCG group, suggesting that CX can alleviate HFD-induced renal injury.

[0086] In summary, the 6-MBOA of the present invention has been proven through various efficacy experiments to be able to effectively regulate lipid metabolism, improve non-alcoholic fatty liver disease, prevent excessive insulin secretion, and improve insulin resistance and metabolic syndrome.

[0087] The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or changes made thereto without departing from the spirit and scope of the present invention shall be included in the scope defined by the claims.

Claims

1. Use of 6-methoxybenzoxazolinone (6-MBOA) for preparing a composition for regulating lipid metabolism.

2. The use according to claim 1, characterized in that The 6-MBOA downregulates de novo lipogenesis in individuals in whom lipid metabolism needs to be regulated.

3. The use according to claim 1, characterized in that The 6-MBOA upregulates lipid breakdown and fatty acid β-oxidation in individuals in need of regulated lipid metabolism.

4. The use according to claim 1, characterized in that The 6-MBOA does not produce cytotoxicity to immortalized primary human hepatocytes at 25 to 400 μM.

5. The use according to claim 1, characterized in that: The 6-MBOA reduces lipid accumulation in an individual in need of regulated lipid metabolism.

6. The use according to claim 1, characterized in that: The 6-MBOA increases the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), an enzyme required for regulating lipid metabolism.

7. The use according to claim 1, characterized in that The lipid metabolism is regulated by regulating the phosphorylation of acetyl-CoA carboxylase (ACC), while down-regulating the expression of fatty acid synthase FAS and up-regulating the expression of adipose triglyceride lipase (ATGL) and carnitine palmitoyl-transferase 1 (CPT1).

8. Use of 6-methoxybenzoxazolinone (6-MBOA) for preparing a composition for improving non-alcoholic fatty liver disease (NAFLD) and metabolic syndrome.

9. The use according to claim 8, characterized in that The 6-MBOA prevents weight gain in an individual in need of improvement of non-alcoholic fatty liver disease and metabolic syndrome.

10. The use according to claim 8, characterized in that The 6-MBOA improves adipose tissue hypertrophy and dyslipidemia in an individual in need of improvement of non-alcoholic fatty liver disease and metabolic syndrome.

11. The use according to claim 8, characterized in that The 6-MBOA improves glucose homeostasis, glucose tolerance and insulin resistance (HOMA-IR) in an individual in need of improvement of non-alcoholic fatty liver disease and metabolic syndrome.

12. The use according to claim 8, characterized in that The 6-MBOA improves high fat diet (HFD)-induced hepatic fat deposition and inflammation.

13. The use according to claim 8, characterized in that The 6-MBOA alleviates high-fat diet-induced renal injury.

14. The use according to claim 1 or 8, characterized in that: The composition is a pharmaceutical composition, a food composition or an external use composition.

15. The use according to claim 14, characterized in that The pharmaceutical composition is in a dosage form for oral administration.

16. The use according to claim 14, characterized in that The pharmaceutical composition is in a dosage form for parenteral administration.

17. The use according to claim 14, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, adjuvant and / or food additive.

18. The use according to claim 1 or 8, characterized in that: The composition has a powdery, granular, solution, gel, or paste form.

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