An obesity inhibitory composition, inhibitor and application

Abstract

The present invention provides an obesity inhibitory composition, an inhibitor and an application, relating to the technical field of obesity inhibitors. The composition provided by the present invention comprises urolithin and an MIF inhibitor. By using urolithin and an MIF inhibitor in combination, the present invention can effectively improve the inhibitory and ameliorating effects on obesity.

Description

Technical Field

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

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

[0003] Currently, the main treatment methods for obesity are lifestyle intervention, drug treatment and surgical treatment. Among them, lifestyle intervention is the basic treatment method. By adjusting the diet structure, increasing exercise and behavioral intervention, the weight of low obesity patients can be effectively controlled. However, the effect is extremely limited when facing medium and high obesity patients. Surgical treatment can strongly intervene in high and severe obesity patients. Using gastric bypass surgery and sleeve gastrectomy can significantly reduce body weight, but this method has certain surgical risks.

[0004] Therefore, drug treatment has become a more commonly used method for moderate obesity patients. By precisely intervening in the neuroendocrine network with drugs, regulating appetite, inhibiting fat absorption or improving metabolism, such as semaglutide, tirzepatide, etc. However, long-term use of drug treatment will also produce certain adverse reactions and limitations, such as the uncertainty of efficacy, the side effects of drugs and the 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 purpose of the present invention is to provide an obesity inhibitory composition, an inhibitor and an application thereof, which can effectively improve the inhibitory and improvement effects on obesity by synergistically using urolithin and MIF inhibitor.

[0006] In a first aspect, an obesity inhibitory composition provided by the present invention includes urolithin and an MIF inhibitor.

[0007] Optionally, the urolithin includes urolithin A.

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

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

[0010] In a second aspect, an inhibitor provided by the present invention includes any one of the above optional obesity inhibitory compositions.

[0011] Optionally, it further includes excipients and / or additives acceptable in the pharmaceutical field.

[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 also provides the use of any one of the above-mentioned optional obesity inhibitory compositions or any one of the above-mentioned optional inhibitors in the preparation of obesity inhibitory drugs. Description of the Drawings

[0014] Figure 1 It is a four-parameter fitting curve of the drug concentration of 4-IPP and UA when used alone and the cell inhibition rate on 3T3-L1 cells provided by the present invention; wherein, A is the four-parameter fitting curve of the 4-IPP concentration and the cell inhibition rate, and B is the four-parameter fitting curve of the UA concentration and the cell inhibition rate;

[0015] Figure 2 It is a graph showing the change in the viability of 3T3-L1 cells when 4-IPP and UA are combined at different concentrations provided by the present invention;

[0016] Figure 3 It is a graph of the cell staining effect diagram and the absorbance change diagram of the cell extract after treating the differentiated 3T3-L1 cells with the blank group and the intervention group of the present invention; wherein, A is the cell staining effect diagram, and B is the absorbance change diagram;

[0017] Figure 4 It is a graph of the change in the synergy index when 4-IPP and UA are combined at different concentrations provided by the present invention;

[0018] Figure 5 It is a schematic diagram and a graph of the change in the imaging of PGC-1α, UCP1, and Glut4 proteins expressed in white adipocytes provided by the present invention; wherein, A is the schematic diagram of protein imaging, B is the graph of the change in the effect on PGC-1α protein, C is the graph of the change in the effect on UCP1 protein, and D is the graph of the change in the effect on Glut4 protein;

[0019] Figure 6 It is a comparison diagram of mouse pictures and a graph of body weight change when intervening in obese model mice provided by the present invention; wherein, A is the photo of each group of mice after 10 weeks of intervention, and B is the weekly body weight change curve of each group of mice during intervention;

[0020] Figure 7Graph showing the changes in the levels of blood glucose, triglyceride, cholesterol, alanine aminotransferase, aspartate aminotransferase, creatinine, and urea in the serum of mice after intervention with the present invention on obese model mice; wherein, A is the graph of blood glucose level change, B is the graph of triglyceride level change, C is the graph of cholesterol level change, D is the graph of alanine aminotransferase level change, E is the graph of aspartate aminotransferase level change, F is the graph of creatinine level change, and G is the graph of urea level change;

[0021] Figure 8 Graph showing the comparison of the mass, staining morphology, and diameter of adipose tissue of mice after intervention with the present invention on obese model mice; wherein, A is the comparison graph of bilateral white epididymal fat in the abdominal cavity of mice and the body weight of mice, B is the comparison graph of the cell staining morphology of bilateral epididymal white adipose tissue in the abdominal cavity of mice, C is the comparison graph of the cell diameter of white adipose tissue of mice, D is the comparison graph of the cell staining morphology of brown adipose tissue of mice, and E is the comparison graph of the cell diameter of brown adipose tissue of mice;

[0022] Figure 9 Graph showing the developed image schematic diagram and influence change graph of PGC-1α, UCP1, and Glut4 proteins expressed by white adipose tissue cells of mice after intervention with the present invention on obese model mice; wherein, A is the developed image schematic diagram of the protein, B is the influence change graph of PGC-1α protein, C is the influence change graph of UCP1 protein, and D is the influence change graph of Glut4 protein. Detailed implementation manners

[0023] To make the objectives, 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 some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings understood by those of ordinary skill in the art belonging to the field of the present invention.

[0024] The present invention provides an obesity inhibitory composition, including urolithin and an MIF inhibitor. In fact, urolithin is a class of compounds produced by intestinal microbiota metabolism, which are compounds obtained by the transformation of human consumption of foods rich in ellagic acid through the action of intestinal microbiota. Macrophage migration inhibitory factor (MIF), as a multifunctional cytokine, can widely participate in inflammatory reactions, immune regulation, and metabolic balance in the human body.

[0025] In some embodiments, the mixing ratio of urolithin to MIF inhibitor in the obesity inhibitory composition is 1:(0.5 - 2). In fact, when urolithin and MIF inhibitor cooperate, they can effectively improve the efficacy of inhibiting / improving obesity, and at the same time can reduce the side effects generated 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.

[0026] The present invention also provides an inhibitor comprising any of the above obesity inhibitory compositions. Making the composition into an inhibitor product is beneficial for obese patients to use. In addition, the inhibitor also includes excipients and / or additives acceptable in the pharmaceutical field, and the dosage form of the inhibitor can be made into at least one of tablets, capsules, soft capsules, pills, and granules.

[0027] 1. Drug cytotoxicity analysis:

[0028] Inoculate 3T3-L1 cells into a 96-well plate, with 1000 cells inoculated in each well. After inoculation, transfer them to a carbon dioxide cell incubator and culture them normally for 24 h. Then, add culture media containing 0.1 μmol / L, 1 μmol / L, 10 μmol / L, and 100 μmol / L of urolithin A (UA) (prepare the stock solution with dimethyl sulfoxide (DMSO) and dilute it to the corresponding concentration with the culture medium), and culture media containing 0.1 μmol / L, 1 μmol / L, 10 μmol / L, and 100 μmol / L of 4-IPP (prepare the stock solution with dimethyl sulfoxide (DMSO) and dilute it to the corresponding concentration with the culture medium), and add an equal amount of blank culture medium as a control. After adding drug intervention for 24 h, add 10 μL of CCK-8 reagent to each well and shake evenly, and then continue to incubate in the incubator for 4 h. After the incubation ends, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 450 nm for each well, calculate the cell inhibition rate based on the following formula, and use "four-parameter logistic fitting" to draw the fitting curves of the cell inhibition rate versus the 4-IPP concentration and the urolithin A concentration, as shown in Figure 1 A in Figure 1 and B in

[0029]

[0030] As can be seen from Figure 1 the IC50 value of 4-IPP for 3T3-L1 cells = 13.97 μmol / L, R² = 0.9997; the IC50 value of UA for 3T3-L1 cells = 48.70 μmol / L, R² = 0.9992.

[0031] The blank group was used as a control. After combining 4-IPP and UA according to the concentrations in Table 1, the above analysis method was used to measure the effect on the viability of 3T3-L1 cells as Figure 2 shown.

[0032] Table 1 Combined concentrations of 4-IPP and UA

[0033]

[0034] It can be seen from Figure 2 that when the combined drug concentrations of 4-IPP and UA were (1 μmol / L, 1 μmol / L) and (5 μmol / L, 5 μmol / L), there was no significant effect on the viability of 3T3-L1 cells, indicating no obvious cytotoxicity at these two concentrations. However, when the combined drug concentration was (10 μmol / L, 10 μmol / L), the viability of 3T3-L1 cells decreased significantly (P < 0.05 compared with the concentration of (0 μmol / L, 0 μmol / L)), indicating potential cytotoxicity at this concentration.

[0035] 2. Experiment on the decomposition of lipid droplets in adipocytes

[0036] The adipogenic induction culture system MDI (Methylisobutylxanthine (MIX) + Dexamethasone (Dex) + Insulin (Ins)) was used to induce the adipogenic differentiation of 3T3-L1 cells. 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 medium shown in Table 2 was added for intervention, and an equal amount of normal saline was used as the blank group; after 72 h of intervention, the medium in each well was removed, and the cells were rinsed once with pre-cooled PBS and then fixed with 4% paraformaldehyde for 10 min. After removing paraformaldehyde, the cells were rinsed three times with pre-cooled PBS. 60% isopropanol was added to the well plate to cover the cells for 15 s - 20 s, and then the isopropanol was aspirated and the cells were dried. Oil Red O reagent was added to the well plate to cover the cells and stained at room temperature for 30 min. After removing Oil Red O, 60% isopropanol was added for rapid differentiation and then washed three times with water, 5 min each time. Then hematoxylin stain was added to stain the cell nuclei, washed with water, blued and washed with water again, and then the staining effect was observed under an optical microscope and photographed as shown in A in Figure 3 ; The extraction solution was obtained by adding isopropanol to each well of the well plate to extract the Oil Red O dye, and the absorbance (OD value) of the extraction solution at a wavelength of 500 - 520 nm was measured using a spectrophotometer, as shown in B in Figure 3 shown.

[0037] Table 2 Drug concentrations in the drug-containing medium

[0038]

[0039] It can be seen from Figure 3 that both 4-IPP and UA can promote the decomposition of lipid droplets in white adipocytes in a dose-dependent manner. Among them, at the same concentration, the effect of 4-IPP on promoting lipid droplet decomposition is significantly better than that of UA (P < 0.05). After the two drugs are combined, a stronger effect on promoting lipid droplet decomposition is obtained (P < 0.05 compared with the single treatment of 4-IPP or UA).

[0040] Based on Figure 3 the absorbance in B, the cell inhibition rate was calculated, and after fitting the data of the drugs acting alone with the median-effect equation shown below, the median concentration ( ), and the slope ( ) were obtained by logarithmic linear regression. For the combined drugs, the concentrations required for the two drugs to achieve the same effect when used alone were calculated through the inverse function of the median-effect equation. Finally, the formula of the combination index ( ) shown below was substituted, and the results are shown in Table 3 and Figure 4 as follows.

[0041] Median-effect equation:

[0042] Combination index formula:

[0043] Among them, is the cell inhibition rate during combined drug use, is the actual concentration of the drug during combined drug use, is the median concentration in the logarithmic linear regression equation, is the slope in the logarithmic linear regression equation, is the actual concentration of 4-IPP added, is the added concentration of 4-IPP when the same cell inhibition rate is achieved during single drug use, is the actual concentration of UA added, is the added concentration of UA when the same cell inhibition rate is achieved during single drug use.

[0044] Table 3 Analysis of synergistic effect during combined drug use

[0045]

[0046] It can be seen from Table 3 and Figure 4 that during combined drug use, the combination indices ( ) are 0.486, 0.611, and 0.489 respectively, all of which are less than 1, indicating that 4-IPP and UA can produce an obvious synergistic effect at these three doses.

[0047] 3. Effects of drugs on the expression of proteins in white adipocytes

[0048] 3T3-L1 cells were seeded into 96-well plates at a density of 1000 cells per well. After seeding, the plates were transferred to a carbon dioxide incubator and cultured normally for 24 h. Then, a blank solvent was added as the blank group. An adipogenic induction culture system MDI (Methylisobutylxanthine (MIX) + Dexamethasone (Dex) + Insulin (Ins)) was used to induce adipogenic differentiation of 3T3-L1 cells as the MDI group. After 72 h of intervention with white adipocytes in the experimental groups, namely the 4-IPP group (1 μmol / L), UA group (1 μmol / L), and combined drug group (1 μmol / L + 1 μmol / L), adipocyte samples were collected and lysed on ice with RIPA lysis buffer containing PMSF protease inhibitor. The supernatant was obtained by centrifugation. Protein was quantified according to the BCA method and adjusted to equal concentrations, and then boiled for denaturation for standby. A gradient SDS-PAGE gel (about 10%-12%) was prepared. After loading and electrophoresis, the gel was transferred to a PVDF membrane (wet transfer at 300 mA for 90 - 120 minutes after methanol activation). Ponceau S staining was used to confirm the transfer efficiency. After blocking with 5% skim milk powder, the membrane was incubated with the primary antibody overnight at 4 °C (UCP1 / PGC-1α / Glut4 were diluted according to the instructions, and GAPDH was used as an internal reference). After washing the membrane with TBST, it was incubated with the HRP secondary antibody for 1 h. ECL imaging was performed, and the band gray values were analyzed by ImageJ. After normalization with the internal reference, the differences between groups were compared. As shown in Figure 5 A in Figure 5 and the effects on PGC-1α, UCP1, and Glut4 proteins were respectively as shown in Figure 5 B in Figure 5 and

[0049] C in Figure 5 and

[0050] 4. Effects of drugs on high-fat diet mice

[0051] Thirty 4-week-old male C57BL / 6 mice were randomly divided into five groups: The normal diet group (ND) was fed with a normal growth maintenance diet; the high-fat diet group (HFD) was fed with a high-fat purified diet (Research Diets 60%) to establish an obesity model; the 4-IPP drug group was fed with a high-fat purified diet to establish an obesity model and then intraperitoneally injected with 5 mg / kg 4-IPP every two days; the UA drug group was fed with a high-fat purified diet to establish an obesity model and then gavaged with 160 mg / kg urolithin A every day; the combined drug group was fed with a high-fat purified diet to establish an obesity model and then gavaged with 160 mg / kg urolithin A every day and intraperitoneally injected with 5 mg / kg 4-IPP every two days. The body weights of the mice in each group were measured once a week for 10 consecutive weeks, and the mice were photographed at the 10th week as shown in Figure 6 A in Figure 6 . The changes in the body weights of the mice in each group within 10 weeks are shown in

[0052] All the mice were anesthetized by inhaling isoflurane, and then peripheral blood was collected from the heart to isolate serum. The blood glucose, triglyceride, and cholesterol levels were detected, and the results are shown in Figure 7 A, Figure 7 B in Figure 7 and Figure 7 C in Figure 7 respectively. The alanine aminotransferase and aspartate aminotransferase, which are liver function indicators in the serum of the mice in each group, were detected as shown in Figure 7 D and Figure 7 E in Figure 7 respectively. The serum creatinine and urea contents were detected as shown in Figure 7 F and Figure 7 G in Figure 8 respectively. Each group of mice was dissected, and the bilateral epididymal white adipose tissues in the abdominal cavity were completely dissected and weighed as shown in Figure 8 A. After the epididymal white adipose tissue of the mice was fixed with formaldehyde, embedded, sectioned with paraffin, hematoxylin-eosin staining was used for analysis, and the diameters of adipocytes were measured as shown in Figure 8 B and Figure 8 C in Figure 8 respectively. HE staining was used to observe the pathological characteristics of the brown adipose tissue in the necks of the mice in each group, as shown in Figure 8 D and Figure 8 E in Figure 9 respectively. Western blotting was used to detect the protein expression levels of UCP1, PGC-1α, and Glut4 in the epididymal white adipose tissue of the mice in each group as shown in Figure 9 .

[0053] From Figure 6It can be seen that compared with the mice on normal diet, the body weight of the mice in the HFD group increased significantly and rapidly (P < 0.05), and they quickly showed an obese phenotype. In the model mice intervened with 4-IPP and UA, compared with the mice in the HFD group, the increase in body weight was significantly slowed down (P < 0.05), showing a significant obesity inhibitory effect. At the same time, it can be seen in the combination drug group that the inhibitory effect on the body weight of the mice on high-fat diet was significantly better than that of any single drug treatment intervention (P < 0.05 vs. 4-IPP or UA). In addition, during the experiment, all the mice were in good condition and no obvious abnormalities were found.

[0054] From Figure 7 A to Figure 7 C in it, it can be seen that compared with the mice on normal diet, the blood glucose, triglyceride and cholesterol levels of the mice in the HFD group were all significantly increased (P < 0.05). Compared with the mice in the HFD group, after intervention with 4-IPP and UA, the blood glucose, triglyceride and cholesterol levels of the obese mice were all significantly decreased (P < 0.05). In addition, the blood glucose, triglyceride and cholesterol levels of the obese mice intervened with the combination drug were significantly lower than those of any single drug treatment (P < 0.05 compared with the single treatment of 4-IPP or UA).

[0055] From Figure 7 D and Figure 7 E in it, it can be seen that compared with the mice on normal diet, the alanine aminotransferase and aspartate aminotransferase of the mice fed with high fat were significantly increased (P < 0.05), indicating liver injury. After treatment with 4-IPP or UA, the alanine aminotransferase and aspartate aminotransferase were both significantly decreased (P < 0.05 compared with the normal diet group), indicating that both drugs can effectively improve the fatty liver of mice caused by high-fat diet. The effect of the combination of the two drugs in improving the liver function of the mice on high-fat diet was significantly better than that of any single drug treatment (P < 0.05 compared with the single treatment of 4-IPP or UA).

[0056] From Figure 7 F and Figure 7 G in it, it can be seen that compared with the mice on normal diet, the serum creatinine and urea of the obese mice were slightly increased, but there was no significant difference. The single drug treatment or combined treatment of 4-IPP and UA had no obvious effect on the renal function of the mice.

[0057] From Figure 8 A in it, it can be seen that compared with the mice on normal diet, the white adipose tissue of the mice fed with high fat was significantly increased (P < 0.05). The weight of the white adipose tissue of the obese mice treated with 4-IPP or UA was significantly decreased (P < 0.05 compared with the normal diet group). The weight of the white adipose tissue of the obese mice treated with the combination of 4-IP and UA was significantly smaller than that of any single drug treatment (P < 0.05 compared with the single treatment of 4-IPP or UA).

[0058] From Figure 8 B in and Figure 8 C in, it can be seen that compared with the normal diet mice, the volume of white adipocytes in the high-fat diet mice was significantly increased (P < 0.05), and there was infiltration of inflammatory cells. Both 4-IPP or UA treatment could significantly inhibit the volume of white adipocytes (P < 0.05 compared with the normal diet group), and improve the adipose tissue microenvironment. The effect of the combined treatment of the two drugs in inhibiting the volume of white adipocytes was significantly better than that of either single drug treatment (P < 0.05 compared with the treatment with 4-IPP or UA alone).

[0059] From Figure 8 D in and Figure 8 E in, it can be seen that compared with the normal diet mice, the volume of brown adipocytes in the high-fat diet mice was significantly increased (P < 0.05), suggesting "whitening" of brown adipocytes. Both 4-IPP or UA treatment could significantly inhibit the volume of brown adipocytes (P < 0.05 compared with the normal diet group), and the effect of the combined treatment of the two drugs in inhibiting the volume of brown adipocytes was significantly better than that of either single drug treatment (P < 0.05 compared with the treatment with 4-IPP or UA alone).

[0060] From Figure 9 it can be seen that compared with the normal diet mice, the protein expression levels of the browning markers UCP1, PGC-1α, and Glut4 in the white adipose tissue of the high-fat diet-induced obese mice were significantly down-regulated (P < 0.05). After treatment with 4-IPP or UA, the protein expression levels of UCP1, PGC-1α, and Glut4 in the white adipose tissue of the obese mice were significantly up-regulated (P < 0.05 compared with the normal diet group), and the effect of the combined treatment of 4-IPP and UA in up-regulating the protein expression of UCP1, PGC-1α, and Glut4 was significantly better than that of either single drug treatment (P < 0.05 compared with the treatment with 4-IPP or UA alone).

[0061] Although the embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes all fall within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein can have other embodiments, and can be implemented or realized in various ways.

Claims

1. An obesity-inhibiting composition, characterized in that, It includes urolithin A and 4-IPP with a mixing ratio of 1:

1.

2. An inhibitor comprising the obesity inhibitory composition according to claim 1.

3. The inhibitor according to claim 2, wherein It also includes excipients and / or additives acceptable in the pharmaceutical field.

4. The inhibitor according to claim 2, wherein The dosage form of the inhibitor includes at least one of tablets, capsules, pills, and granules.

5. Use of the obesity inhibitory composition according to claim 1 or the inhibitor according to any one of claims 2 to 4 in the preparation of an obesity inhibitory drug.

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