Obesity inhibition composition, inhibitor and application

Abstract

The invention provides an obesity inhibition composition, an inhibitor and application, and relates to the technical field of obesity inhibitors. The composition provided by the invention comprises urolithin and an MIF inhibitor. The urolithin and the MIF inhibitor are synergistically used, so that the inhibiting and improving effects on obesity can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of obesity inhibitors, and in particular to an obesity inhibitor composition, an inhibitor and applications thereof. Background Art

[0002] Obesity is a chronic metabolic disease caused by the combined effects of multiple factors including genetics, environment and behavior. It is mainly due to the long-term imbalance between energy intake and expenditure, which manifests as excessive accumulation of body fat accompanied by a significant increase in weight. This disease is closely related to a variety of metabolic diseases, including type 2 diabetes, cardiovascular disease, fatty liver, hypertension and certain types of cancer, which seriously affects people's mental health and quality of life.

[0003] The current treatments for obesity mainly include lifestyle intervention, drug therapy and surgical treatment. Lifestyle intervention is the basic treatment method. It can effectively control the weight of low-obese patients by adjusting the dietary structure, increasing exercise and behavioral intervention. However, the effect is extremely limited when facing patients with moderate to severe obesity. Surgical treatment can provide strong intervention for patients with high or severe obesity. Gastric bypass surgery and sleeve gastrectomy can significantly reduce weight, but this method has certain surgical risks.

[0004] Therefore, drug therapy has become a more commonly used method for patients with moderate obesity. Through precise intervention of the neuroendocrine network with drugs, appetite is regulated, fat absorption is inhibited or metabolism is improved, such as semaglutide and telpotide. However, long-term use of drug therapy will also produce certain adverse reactions and limitations, such as unclear efficacy, side effects of drugs and limitations in long-term management. Therefore, there is an urgent need to provide a composition to improve the above problems. Summary of the invention

[0005] The object of the present invention is to provide an obesity suppressing composition, an inhibitor and an application thereof, which can effectively enhance the suppressing and improving effects on obesity by using urolithin in conjunction with a MIF inhibitor.

[0006] In a first aspect, the present invention provides an obesity-suppressing composition comprising urolithin and a MIF inhibitor.

[0007] Optionally, the urolithin comprises urolithin A.

[0008] Optionally, the mixing ratio of the urolithin to the MIF inhibitor is 1:(0.5-2).

[0009] Optionally, the MIF inhibitor comprises 4-IPP.

[0010] In a second aspect, the present invention provides an inhibitor comprising any one of the optional obesity-inhibiting compositions described above.

[0011] Optionally, excipients and / or additives acceptable in the pharmaceutical field are also included.

[0012] Optionally, the dosage form of the inhibitor includes at least one of tablets, capsules, soft capsules, pills, and granules.

[0013] In a third aspect, the present invention further provides use of any of the above optional obesity-suppressing compositions or any of the above optional inhibitors in the preparation of obesity-suppressing drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a four-parameter fitting curve of the drug concentration and the cell inhibition rate of 3T3-L1 cells when 4-IPP and UA are used alone provided by the present invention; wherein A is the four-parameter fitting curve of 4-IPP concentration and cell inhibition rate, and B is the four-parameter fitting curve of UA concentration and cell inhibition rate; Figure 2 This is a graph showing the effect of different concentrations of 4-IPP and UA combined with each other on the viability of 3T3-L1 cells; Figure 3 The cell staining effect diagram and the absorbance change diagram of the cell extract after the blank group and the intervention group of the present invention were treated with 3T3-L1 cells after adipogenic differentiation; wherein A is the cell staining effect diagram, and B is the absorbance change diagram; Figure 4 A graph showing changes in the synergy index when 4-IPP and UA provided by the present invention are used in combination at different concentrations; Figure 5 The present invention is a schematic diagram of the development of PGC-1α, UCP1, and Glut4 proteins expressed by white adipocytes and a graph of the effects of the present invention; wherein A is a schematic diagram of protein development, B is a graph of the effects of the PGC-1α protein, C is a graph of the effects of the UCP1 protein, and D is a graph of the effects of the Glut4 protein; Figure 6 The figures are comparison diagrams of mice pictures and weight change diagrams when the obese model mice are intervened by the present invention; wherein A is the picture of mice in each group after 10 weeks of intervention, and B is the weekly weight change curve of mice in each group during intervention; Figure 7 The present invention is a graph showing changes in blood glucose, triglyceride, cholesterol, alanine aminotransferase, aspartate aminotransferase, creatinine, and urea levels in the serum of mice after the obese model mice were intervened by the present invention; wherein A is a graph showing changes in blood glucose levels, B is a graph showing changes in triglyceride levels, C is a graph showing changes in cholesterol levels, D is a graph showing changes in alanine aminotransferase levels, E is a graph showing changes in aspartate aminotransferase levels, F is a graph showing changes in creatinine levels, and G is a graph showing changes in urea levels; Figure 8The figure is a comparison diagram of the mass, staining morphology and diameter of the mouse fat tissue after the obese model mice are intervened by the present invention; wherein A is a comparison diagram of the bilateral white epididymal fat in the abdominal cavity of the mouse and the weight of the mouse, B is a comparison diagram of the cell staining morphology of the bilateral epididymal white fat tissue in the abdominal cavity of the mouse, C is a comparison diagram of the cell diameter of the white fat tissue of the mouse, D is a comparison diagram of the cell staining morphology of the brown fat tissue of the mouse, and E is a comparison diagram of the cell diameter of the brown fat tissue of the mouse; Fig. 9 The present invention is a schematic diagram of the development of PGC-1α, UCP1, and Glut4 proteins expressed in white adipose tissue cells of mice after the intervention of the obese model mice; wherein A is a schematic diagram of protein development, B is a diagram of the effect change on PGC-1α protein, C is a diagram of the effect change on UCP1 protein, and D is a diagram of the effect change on Glut4 protein. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be the common meanings understood by people with general skills in the field to which the present invention belongs.

[0016] The present invention provides an obesity-suppressing composition, including urolithin and MIF inhibitor. In fact, urolithin is a class of compounds produced by intestinal microbial metabolism, which are converted by intestinal microorganisms after humans eat foods rich in ellagic acid. Macrophage migration inhibitory factor (MIF), as a multifunctional cytokine, can be widely involved in inflammatory response, immune regulation and metabolic balance in the human body.

[0017] In some embodiments, the mixing ratio of urolithin to MIF inhibitor in the obesity-suppressing composition is 1:(0.5-2). In fact, when urolithin and MIF inhibitor work together, they can effectively improve the inhibitory / improving effect on obesity, and at the same time can reduce the side effects produced during the inhibition process, which is beneficial to the long-term management of body weight. Specifically, the urolithin used specifically includes urolithin A, and the MIF inhibitor used specifically includes 4-IPP.

[0018] The present invention also provides an inhibitor comprising any of the above obesity-inhibiting compositions. By preparing the composition into an inhibitor product, it is convenient for obese patients to use it. In addition, the inhibitor also includes excipients and / or additives acceptable in the pharmaceutical field. The dosage form of the inhibitor can be prepared into at least one of tablets, capsules, soft capsules, pills, and granules.

[0019] 1. Drug cytotoxicity analysis: 3T3-L1 cells were seeded into 96-well plates and 1000 cells were seeded in each well. After seeding, the cells were transferred to a carbon dioxide cell culture incubator and cultured normally for 24 hours. Then, 0.1 μmol / L, 1 μmol / L, 10 μmol / L, and 100 μmol / L of culture medium containing urolithin A (UA) (the mother solution was prepared with dimethyl sulfoxide (DMSO) and diluted to the corresponding concentration with culture medium) and 0.1 μmol / L, 1 μmol / L, 10 μmol / L, and 100 μmol / L of culture medium containing 4-IPP (the mother solution was prepared with dimethyl sulfoxide (DMSO) and diluted to the corresponding concentration with culture medium) were added, and an equal amount of blank culture medium was added as a control. After 24 hours of drug intervention, 10 μL of 4-IPP was added to each well. After the CCK-8 reagent was added and shaken evenly, the incubation was continued in the incubator for 4 hours. After the incubation, the absorbance at 450 nm was measured using an ELISA instrument, and the cell inhibition rate was calculated based on the following formula. The fitting curves of the cell inhibition rate, 4-IPP concentration, and urolithin A concentration were drawn using the "four-parameter logistic fitting" as shown in the figure. Figure 1 A in Figure 1 As shown in B.

[0020]

[0021] from Figure 1 It can be seen that the IC50 value of 4-IPP for 3T3-L1 cells is 13.97 μmol / L, R²=0.9997; the IC50 value of UA for 3T3-L1 cells is 48.70 μmol / L, R²=0.9992.

[0022] Using the blank group as a control, 4-IPP and UA were combined according to the concentrations in Table 1, and the above analytical method was used to measure the effect on the viability of 3T3-L1 cells. Figure 2 shown.

[0023] Table 1 Concentration of 4-IPP and UA combined

[0024]

[0025] from Figure 2It can be seen that when the combined use concentrations of 4-IPP and UA were (1μmol / L, 1μmol / L) and (5μmol / L, 5μmol / L), there was no significant effect on the viability of 3T3-L1 cells, indicating that there was no obvious cytotoxicity at these two concentrations. However, when the combined use concentration was (10μmol / L, 10μmol / L), the viability of 3T3-L1 cells was significantly decreased (compared with the concentration (0μmol / L, 0μmol / L) P < 0.05), indicating that there was potential cytotoxicity at this concentration.

[0026] 2. Experiment on decomposition of fat droplets in adipocytes

[0027] Adipogenic induction culture system MDI (Methylisobutylxanthine (MIX) + Dexamethasone (Dex) + Insulin (Ins)) induced 3T3-L1 cells to differentiate into adipocytes. After 7 days of induction culture, a large number of lipid droplets (white adipocytes) could be observed in the cells. After removing MDI, the drug-containing culture medium shown in Table 2 was added for intervention, and an equal amount of normal saline was used as the blank group. After 72 hours of intervention, the culture medium of each well was removed, and the cells were rinsed once with pre-cooled PBS, and then 4% paraformaldehyde was added to fix the cells for 10 minutes. After removing the paraformaldehyde, the cells were rinsed three times with pre-cooled PBS. 60% isopropanol was added to the well plate to cover the cells for 15s-20s, and then the isopropanol was discarded and the water was dried. Then, Oil Red 0 reagent was added to the well plate to cover the cells and stained at room temperature for 30 minutes. After removing Oil Red 0, 60% isopropanol was added for rapid differentiation, and then washed three times with water for 5 minutes each time. Then, hematoxylin stain was added to stain the cell nucleus, washed with water, returned to blue and washed with water again, and then the staining effect was observed under an optical microscope and photographed as shown in Figure 3 As shown in A in the figure; add isopropanol to each well of the well plate to extract the Oil Red 0 dye to obtain an extract, and use a spectrophotometer to measure the absorbance (OD value) of the extract at a wavelength of 500-520 nm, as shown in Figure 3 As shown in B.

[0028] Table 2 Drug concentration in drug-containing culture medium

[0029]

[0030] from Figure 3 It can be seen that both 4-IPP and UA can promote the decomposition of white adipocyte lipid droplets in a dose-dependent manner. At the same concentration, 4-IPP is significantly better than UA in promoting the decomposition of lipid droplets (P < 0.05). The combination of the two drugs has a stronger effect on promoting the decomposition of lipid droplets (compared with 4-IPP or UA alone, P < 0.05).

[0031] based on Figure 3The cell inhibition rate was calculated based on the absorbance in B, and the median concentration ( ) and the slope ( ), for combined drug use, the inverse function of the median effect equation is used to calculate the concentrations required for the two drugs to achieve the same effect when used alone, and finally the synergy index shown below is entered ( ) formula, and the results are shown in Table 3 and Figure 4 shown.

[0032] The intermediate effect equation is:

[0033] Synergy Index Formula:

[0034] in, is the cell inhibition rate when the drug is used in combination, is the actual concentration of the drug when the drug is used in combination. is the median concentration in the log-linear regression equation, is the slope of the log-linear regression equation, is the actual concentration of 4-IPP added, is the concentration of 4-IPP added to achieve the same cell inhibition rate when the drug is used alone, is the actual concentration of UA added, It is the concentration of UA added to achieve the same cell inhibition rate when used alone.

[0035] Table 3 Analysis of synergistic effects when combined with other drugs

[0036]

[0037] From Table 3 and Figure 4 It can be seen that when the drug is used in combination, the synergy index ( ) were 0.486, 0.611 and 0.489, all less than 1, indicating that 4-IPP and UA can produce obvious synergistic effects at these three doses.

[0038] 3. Effects of drugs on proteins expressed in white adipocytes

[0039] 3T3-L1 cells were inoculated into 96-well plates with 1000 cells in each well. After inoculation, the cells were transferred to a carbon dioxide cell culture incubator and cultured normally for 24 hours. A blank solvent was added as a blank group, and the adipogenic differentiation of 3T3-L1 cells was induced by the adipogenic induction culture system MDI (Methylisobutylxanthine (MIX) + Dexamethasone (Dex) + Insulin (Ins)) as the MDI group. The experimental groups included the 4-IPP group (1μmol / L), the UA group (1μmol / L), and the combined drug group (1μmol / L + 1μmol / L). After 72 hours of intervention on white adipocytes, the adipocyte samples were collected and lysed on ice by adding RIPA lysis buffer containing PMSF protease inhibitor, and the supernatant was obtained by centrifugation. The protein was quantified according to the BCA method and adjusted to the same concentration, and then boiled and denatured for later use. A gradient SDS-PAGE gel (about 10%-12%) was prepared, and the samples were loaded for electrophoresis and transferred to a PVDF membrane (wet transfer at 300 mA after methanol activation). 90-120 minutes), Ponceau staining to confirm the transfer efficiency; after blocking with 5% skim milk powder, incubate with primary antibody at 4°C overnight (UCP1 / PGC-1α / Glut4 diluted according to the instructions, GAPDH as internal reference), wash with TBST and incubate with HRP secondary antibody for 1 hour, develop with ECL and analyze the gray value of the bands with ImageJ, and compare the differences between the groups after normalization with the internal reference. Figure 5 As shown in A, and the effects on PGC-1α, UCP1, and Glut4 proteins are shown in Figure 5 B in Figure 5 C in Figure 5 As shown in D.

[0040] from Figure 5 It can be seen that compared with the MDI group, 4-IPP and UA interventions can significantly upregulate the expression of browning markers UCP1, PGC-1α and Glut4 proteins (P < 0.05), while the effect of 4-IPP combined with UA intervention on upregulating the expression of UCP1, PGC-1α and Glut4 proteins is significantly better than that of either single drug treatment (P < 0.05 compared with 4-IPP or UA single treatment).

[0041] 4. Effects of drugs on mice fed a high-fat diet

[0042] Thirty 4-week-old male C57BL / 6 mice were randomly divided into five groups: the normal diet group (ND) was fed with normal growth-maintaining feed; the high-fat diet group (HFD) was fed with high-fat purified feed (Research Diets 60%) to establish an obesity model; the 4-IPP drug group was fed with high-fat purified feed to establish an obesity model and then intraperitoneally injected with 5 mg / kg4-IPP every two days; the UA drug group was fed with high-fat purified feed to establish an obesity model and then gavaged with 160 mg / kg urolithin A every day; the combined drug group was fed with high-fat purified feed to establish an obesity model and then gavaged with 160 mg / kg urolithin A every day and intraperitoneally injected with 5 mg / kg4-IPP every two days. The body weight of mice in each group was weighed once a week and monitored continuously for 10 weeks. The mice were photographed at week 10 as shown in the following figure. Figure 6 As shown in A, the weight changes of mice in each group within 10 weeks are as follows Figure 6 As shown in B.

[0043] All mice were anesthetized by inhalation of isoflurane, and peripheral blood was collected through the heart to separate serum. The levels of blood glucose, triglyceride and cholesterol were tested. The results were as follows: Figure 7 A in Figure 7 B and Figure 7 As shown in C, the liver function indicators alanine aminotransferase and aspartate aminotransferase in the serum of each group of mice were detected as follows Figure 7 D and Figure 7 As shown in E, the serum creatinine and urea levels were measured as Figure 7 F and Figure 7 As shown in G. The mice in each group were dissected and the bilateral epididymal white adipose tissue in the abdominal cavity was completely removed and weighed as shown in Figure 8 As shown in A, the epididymal white fat of mice was fixed with formaldehyde, embedded, and sectioned in paraffin wax, and then stained with hematoxylin-eosin to measure the diameter of the adipocytes. Figure 8 B and Figure 8 As shown in Figure C, HE staining was used to observe the pathological characteristics of brown adipose tissue in the neck of each group of mice. Figure 8 D and Figure 8 As shown in E. The expression levels of UCP1, PGC-1α and Glut4 proteins in epididymal white adipose tissue of each group of mice were detected by Western blotting. Fig. 9 shown.

[0044] from Figure 6It can be seen that the weight of mice in the HFD group increased significantly and rapidly compared with mice on a normal diet (P < 0.05), and they soon showed an obese phenotype. The weight gain of model mice treated with 4-IPP and UA was significantly slower than that of mice in the HFD group (P < 0.05), showing a significant inhibitory effect on obesity. At the same time, it can be seen in the combined drug group that the inhibitory effect on the weight of mice on a high-fat diet was significantly better than any single drug treatment intervention (P < 0.05 vs. 4-IPP or UA). In addition, all mice were in good condition during the experiment, and no obvious abnormalities were found.

[0045] from Figure 7 A to Figure 7 As can be seen from Figure C, compared with mice eating a normal diet, the blood glucose, triglyceride and cholesterol levels of mice in the HFD group were significantly increased (P < 0.05). Compared with mice in the HFD group, the blood glucose, triglyceride and cholesterol levels of obese mice were significantly decreased after intervention with 4-IPP and UA (P < 0.05). In addition, the blood glucose, triglyceride and cholesterol levels of obese mice in combined drug intervention were significantly lower than those in any single drug treatment (compared with 4-IPP or UA single treatment, P < 0.05).

[0046] from Figure 7 D and Figure 7 As can be seen from Figure E, compared with mice on a normal diet, the alanine aminotransferase and aspartate aminotransferase of mice fed a high-fat diet were significantly increased (P < 0.05), indicating liver damage, while the alanine aminotransferase and aspartate aminotransferase were significantly decreased after treatment with 4-IPP or UA (P < 0.05 compared with the normal diet group), indicating that both drug treatments can effectively improve fatty liver in mice induced by a high-fat diet, and the combined treatment of the two drugs is significantly better than either single drug treatment in improving liver function in mice fed a high-fat diet (P < 0.05 compared with single treatment with 4-IPP or UA).

[0047] from Figure 7 F and Figure 7 As can be seen from Figure G, compared with mice fed a normal diet, serum creatinine and urea in obese mice were slightly increased, but there was no significant difference. Neither monotherapy nor combined treatment with 4-IPP and UA had a significant effect on the renal function of mice.

[0048] from Figure 8 As can be seen from Figure A, compared with mice on a normal diet, the white adipose tissue of high-fat fed mice increased significantly (P < 0.05), while the weight of white adipose tissue of obese mice treated with 4-IPP or UA was significantly reduced (P < 0.05 compared with the normal diet group), and the weight of white adipose tissue of obese mice treated with 4-IP combined with UA was significantly less than that of either single drug treatment (P < 0.05 compared with 4-IPP or UA single treatment).

[0049] from Figure 8 B and Figure 8 As can be seen in Figure C, compared with mice on a normal diet, the volume of white adipocytes in mice on a high-fat diet was significantly increased (P < 0.05), accompanied by inflammatory cell infiltration. Treatment with 4-IPP or UA can significantly inhibit the volume of white adipocytes (P < 0.05 compared with the normal diet group) and improve the adipose tissue microenvironment. The combined treatment of the two drugs significantly inhibited the volume of white adipocytes better than either single drug treatment (P < 0.05 compared with 4-IPP or UA single treatment).

[0050] from Figure 8 D and Figure 8 As can be seen from Figure E, compared with mice fed a normal diet, the volume of brown fat cells in mice fed a high-fat diet was significantly increased (P < 0.05), indicating that brown fat cells were "whitened". Treatment with 4-IPP or UA could significantly inhibit the volume of brown fat cells (P < 0.05 compared with the normal diet group), and the combined treatment of the two drugs was significantly better than either single drug treatment in inhibiting the volume of brown fat cells (P < 0.05 compared with single treatment with 4-IPP or UA).

[0051] from Fig. 9 It can be seen that compared with mice fed a normal diet, the expression levels of browning markers UCP1, PGC-1α and Glut4 proteins in white adipose tissue of mice induced by high-fat diet were significantly downregulated (P < 0.05), and the expression levels of UCP1, PGC-1α and Glut4 proteins in white adipose tissue of obese mice treated with 4-IPP or UA were significantly upregulated (P < 0.05 compared with the normal diet group), and the effect of 4-IPP combined with UA treatment on upregulating the expression of UCP1, PGC-1α and Glut4 proteins was significantly better than either single drug treatment (P < 0.05 compared with 4-IPP or UA single treatment).

[0052] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. An obesity-suppressing composition, characterized in that: Including urolithin and MIF inhibitors.

2. The obesity-suppressing composition according to claim 1, characterized in that: The urolithin includes urolithin A.

3. The obesity-suppressing composition according to claim 1, characterized in that: The mixing ratio of the urolithin to the MIF inhibitor is 1:(0.5-2).

4. The obesity-suppressing composition according to claim 3, characterized in that: The mixing ratio of the urolithin to the MIF inhibitor is 1:

1.

5. The obesity-suppressing composition according to claim 1, characterized in that: The MIF inhibitors include 4-IPP.

6. An inhibitor comprising the obesity-suppressing composition according to any one of claims 1 to 5.

7. The inhibitor according to claim 6, characterized in that It also includes excipients and / or additives acceptable in the pharmaceutical field.

8. The inhibitor according to claim 6, characterized in that The dosage form of the inhibitor includes at least one of tablets, capsules, soft capsules, pills and granules.

9. Use of the obesity-suppressing composition according to any one of claims 1 to 5 or the inhibitor according to any one of claims 6 to 8 in the preparation of an obesity-suppressing drug.

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