Use of sporidesmin for the preparation of a slimming medicament

Abstract

The present application relates to the use of parvuline in the preparation of a weight loss drug. It has been found that parvuline B can reduce the weight of mammals. Further research has found that it can reduce the weight of mammals because it mainly reduces the blood lipid level and the lipid accumulation in adipocytes by hydrolyzing the triglyceride in adipocytes into free fatty acid and glycerol through enhancing the activity of key rate-limiting enzyme responsible for lipid metabolism in adipocytes, thereby reducing the mass of adipose tissue to achieve weight loss.

Description

Technical Field

[0001] This invention relates to the field of heterocyclic compounds, and particularly to the use of oligosporins in the preparation of weight-loss drugs. Background Technology

[0002] Obesity is a chronic disease characterized by abnormal or excessive accumulation of body fat. It is closely related to cardiovascular disease, type 2 diabetes, non-alcoholic fatty liver disease, psychosomatic disorders, and even certain types of cancer, seriously threatening human health and imposing a huge burden on medical and health resources and the socio-economic system. The global prevalence of obesity has become a widely concerned public health issue. Finding safer, more effective, and convenient anti-obesity treatments can overcome the limitations of existing treatments. Developing new anti-obesity drugs is an inevitable requirement for addressing obesity and related metabolic diseases, filling existing treatment gaps, and ultimately reducing the global disease burden. Summary of the Invention

[0003] This invention provides the use of oligosporin B in the preparation of weight-loss drugs.

[0004] In one specific embodiment, oligosporin B is used to induce weight loss in humans or other animals. Other animals may be pets such as cats or dogs, or mice or rats.

[0005] In one specific embodiment, the dosage of oligosporin B is from 15 mg oligosporin B / kg body weight to 30 mg oligosporin B / kg body weight.

[0006] In one specific implementation, the drug is administered orally.

[0007] Beneficial effects of the present invention: The present invention discovers that oligosporin B can reduce weight in mammals. Further research found that its ability to reduce weight in mammals is mainly due to its ability to reduce blood lipid levels and lipid accumulation in adipocytes by enhancing the activity of key rate-limiting enzymes responsible for lipid metabolism in adipocytes, thereby reducing the mass of adipose tissue and achieving weight loss. Attached Figure Description

[0008] Figure 1 The changes in body weight of mice in each group from day 0 to 30 are shown in Example 1. * indicates that other groups were significantly different from the high-fat diet group. p There is a significant difference at the <0.05 level.

[0009] Figure 2 The table shows the daily food intake of mice in each group during days 0 to 30 of Example 1. ns indicates no significant difference compared to the high-fat diet group; * indicates a significant difference compared to the high-fat diet group. pThere is a significant difference at the <0.05 level.

[0010] Figure 3 The daily water intake of mice in each group in Example 1 is shown from day 0 to 30. ns indicates no significant difference compared to the high-fat diet group.

[0011] Figure 4 The figures show the mass of epididymal white adipose tissue and inguinal white adipose tissue in each group of mice in Example 2. A shows the mass of epididymal white adipose tissue in each group of mice in Example 2, and B shows the mass of inguinal white adipose tissue in each group of mice in Example 2. * indicates the mass compared to the high-fat diet group. p Significant differences exist at levels <0.05; ns indicates no significant difference compared to the high-fat diet group.

[0012] Figure 5 Microscopic images of hematoxylin-eosin stained epididymal and inguinal white adipose tissue from each group of mice in Example 2 are shown.

[0013] Figure 6 The table shows the levels of triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the serum of mice in each group of Example 3. Specifically, A shows the triglyceride level in the serum of mice in each group of Example 3, B shows the total cholesterol level, C shows the HDL-C level, and D shows the LDL-C level. * indicates the level compared to the high-fat diet group. p Significant differences exist at levels <0.05; ns indicates no significant difference compared to the high-fat diet group.

[0014] Figure 7 Microscopic images of Oil Red O staining of cells from each group in Example 4 are shown, along with the OD values ​​of Oil Red O dye extracted from each group of cells. 510 Values, A shows microscopic images of Oil Red O staining of cells from each group in Example 4, and B shows the OD values ​​of Oil Red O dye extracted from cells from each group in Example 4. 510 Value. * indicates the distance between the two groups indicated by the line segments in the diagram. p A difference <0.05 indicates a significant difference; ns indicates that there is no significant difference between the two groups indicated by the line segment in the graph.

[0015] Figure 8 The figures show the content of free fatty acids and glycerol in the supernatants of cell cultures in each group of Example 4. A shows the content of free fatty acids in the supernatants of cell cultures in each group of Example 4, and B shows the content of glycerol in the supernatants of cell cultures in each group of Example 4. * indicates that the two groups are different. pA difference <0.05 indicates a significant difference; ns indicates that there is no significant difference between the two groups indicated by the line segment in the graph.

[0016] Figure 9 The results show the levels of triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and hormone-sensitive lipase with serine phosphorylation at position 565 (p-HSL) in the cells of each group in Example 5. Ser565 Immunoblotting results of proteins ) and β-tubulin.

[0017] Figure 10 The levels of ATGL, HSL, and p-HSL in cells from each group in Example 5 are shown. Ser565 Relative expression levels of proteins and p-HSL Ser565 The fold increase in HSL compared to the model group is shown in Figure A, where A represents the relative expression level of ATGL protein in each group of cells in Example 5, B represents the relative expression level of HSL protein in each group of cells in Example 5, and C represents the relative expression level of p-HSL protein in each group of cells in Example 5. Ser565 The relative expression levels of the protein, D shows the p-HSL levels in each group of cells in Example 5. Ser565 / HSL improvement factor compared to the model group. * indicates the difference between the two groups indicated by the line segments in the graph. p A difference <0.05 indicates a significant difference; ns indicates that there is no significant difference between the two groups indicated by the line segment in the graph. Detailed Implementation

[0018] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0019] Unless otherwise specified, the reagents and other materials used in the embodiments of this invention can be purchased commercially.

[0020] The chemical structural formula of oligosporin B used in this invention is shown in Formula I).

[0021] Formula I).

[0022] For details on the synthesis of fluorescein B, please refer to the published article "Unexpected biosynthesis of fluorescein-like arthrocolins against resistant strains in an engineered Escherichiacoli". Org. Lett. 2019, 21, 6499−6503.

[0023] C57BL / 6J mice are sensitive to high-fat diets and are prone to obesity after being fed a high-fat diet. The model is stable and has a clear genetic background.

[0024] C57BL / 6J male mice (6 weeks old) were purchased from the Experimental Animal Center of Yunnan University.

[0025] The entire mouse experiment was supervised by the Experimental Animal Welfare and Ethics Committee of Yunnan University. The experimental animals were strictly housed in a standard animal facility with the following conditions: temperature 22±3 degrees Celsius, humidity 40% to 65%, and a 12-hour light-dark cycle. After one week of acclimatization to standard feeding, 32 mice were randomly assigned to a high-fat D12492 diet (fat providing 60% of the total energy, purchased from Research Diets) and an aqueous solution containing 15% (w / v) fructose (purchased from Beijing Solarbio Science & Technology Co., Ltd.); the other 8 mice were fed a normal diet and water. All mice were weighed after 8 weeks of feeding. The mice fed the standard diet had an average weight of less than 30 grams, while the mice fed the high-fat, high-sugar diet had an average weight of 40 grams, indicating a successful establishment of an obesity model, resulting in 36 obese mice.

[0026] 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (ATCC).

[0027] Example 1: Effect of oligosporin B on body weight in obese mice

[0028] Oligosporin B solvent: It consists of three components: dimethyl sulfoxide, polyethylene glycol 300 and corn oil, with the volume percentages of the three components being 5%, 30% and 65% respectively.

[0029] Oligosporin B solution: Oligosporin B was dissolved in the above-mentioned oligosporin B solvent to prepare a 3 mg / mL oligosporin B solution.

[0030] Orlistat solution: Dissolve orlistat in physiological saline to prepare an orlistat solution of 10 micrograms / mL.

[0031] Eight normally fed mice were designated as the normal diet group, and 32 obese mice were randomly divided into four groups of eight each. The specific administration methods for each group are described below.

[0032] (1) Normal diet group: The feed was ordinary feed and the drinking water was ordinary water without sugar for 30 days.

[0033] (2) High-fat diet group: The mice were fed D12492 high-fat diet and drinking water containing 15% (w / v) fructose. The mice were administered oligosporin B solution by gavage daily for 30 days. The gavage dose of oligosporin B solution was 200 μL per mouse.

[0034] (3) Orlistat positive control group: The mice were fed D12492 high-fat diet and drinking water containing 15 (w / v)% fructose. Orlistat solution was administered to the mice by gavage daily for 30 days. The dosage per mouse was 50 mg orlistat / kg body weight.

[0035] (4) Low-dose oligosporin B treatment group: The mice were fed with D12492 high-fat diet and drinking water containing 15 (w / v)% fructose. The mice were administered oligosporin B solution by gavage at a dose of 3 mg / mL for 30 days. The dosage per mouse was 15 mg oligosporin B / kg body weight.

[0036] (5) High-dose oligosporin B treatment group: The mice were fed with D12492 high-fat diet and drinking water containing 15 (w / v)% fructose. The mice were administered oligosporin B solution by gavage at a dose of 3 mg / mL for 30 days. The dosage per mouse was 30 mg oligosporin B / kg body weight.

[0037] For the above 5 groups, mouse body weight, food intake, and water consumption were recorded every 5 days for 30 days. The average body weight of mice in each group at each time point is shown in the figure. Figure 1 The food intake of each group of mice is shown in the figure. Figure 2 The water intake of each group of mice is shown in the figure. Figure 3 .

[0038] Figure 1The results showed that the body weight of mice in the normal diet group remained essentially unchanged over 30 days, while the body weight of mice in the high-fat diet group gradually increased, indicating that a high-fat diet can induce weight gain (obesity) in mice. When mice on a high-fat diet were treated with orlistat for 10 days, their body weight was significantly lower than that of the high-fat diet group, and remained essentially unchanged for the following 20 days. This indicates that orlistat can reduce the body weight of obese mice. Similar to the orlistat positive control group, when mice on a high-fat diet were treated with low-dose (15 mg / kg) or high-dose (30 mg / kg) oligosporin B for 10 days, both groups of mice also showed a significant reduction in body weight compared to the high-fat diet group, and maintained essentially unchanged body weight for the following 20 days. This indicates that both low-dose (15 mg / kg) and high-dose (30 mg / kg) oligosporin B can reduce the body weight of obese mice, with effects comparable to the existing weight-loss drug orlistat. It is noteworthy that the dosage of oligosporin B is lower than that of orlistat.

[0039] at the same time, Figure 2 Food intake and Figure 3 The results of water intake analysis showed that mice in the orlistat positive control group, the low-dose (15 mg / kg) or high-dose (30 mg / kg) oligosporin B treatment group did not show significant differences in daily food intake and daily water intake compared with mice in the high-fat diet group. This indicates that oral administration of oligosporin B did not significantly affect the food intake and water intake of mice, and that oligosporin B did not exert its weight loss effect by inhibiting diet.

[0040] Example 2: Effects of oligosporin B on white adipose tissue in obese mice

[0041] After the experiment in Example 1 lasted for 30 days, blood was collected from the mice, and the mice were euthanized.

[0042] The epididymal white adipose tissue and the inguinal white adipose tissue were separated, weighed, and the results are as follows: Figure 4 As shown. The two types of adipose tissue were then fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The tissues were observed and photographed under a microscope. The results are shown below. Figure 5 As shown.

[0043] Depend on Figure 4 It was found that, compared with the normal diet group, the weight of epididymal and inguinal white adipose tissue in mice in the high-fat diet group was significantly increased. After treatment with a high dose (30 mg / kg) of oligosporin B, the weight was significantly reduced to a level comparable to that of the orlistat positive control group, indicating that oligosporin B can effectively reduce the accumulation level of white adipose tissue in mice.

[0044] Depend on Figure 5It was found that, compared with the normal diet group, the diameter of adipocytes in the epididymal and inguinal white adipose tissues of mice in the high-fat diet group was significantly larger, while high-dose (30 mg / kg) administration of oligosporin B significantly reduced the size of adipocytes. Since the size of adipocytes is mainly determined by the content of lipids such as triglycerides stored within them, smaller adipocytes mean a decrease in the amount of lipids stored in a single adipocyte, which is associated with increased lipolysis.

[0045] Example 3: Effects of oligosporin B on blood lipid levels in obese mice

[0046] The mouse blood collected in Example 2 was centrifuged at 4°C and 2,000×g for 15 minutes, and then the serum was collected. The levels of triglycerides, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol in the mouse serum were measured using a triglyceride, total hepatic cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol assay kit (purchased from Nanjing Jiancheng Biotechnology Institute). Figure 6 As shown, compared with the normal diet group, the serum levels of triglycerides, total cholesterol, and low-density lipoprotein cholesterol in mice in the high-fat diet group were significantly increased, indicating that a high-fat diet can increase blood lipid levels in mice. Compared with the high-fat diet group, the serum levels of total cholesterol and low-density lipoprotein cholesterol in mice in the orlistat positive control group were significantly decreased. The serum levels of triglycerides, total cholesterol, and low-density lipoprotein cholesterol in mice treated with low-dose (15 mg / kg) or high-dose (30 mg / kg) oligosporin B were significantly decreased. Orlistat positive control, low-dose oligosporin B treatment, and high-dose oligosporin B treatment did not affect the serum level of high-density lipoprotein cholesterol in mice. These results indicate that oligosporin B can regulate blood lipid levels and improve lipid metabolism disorders.

[0047] Example 4: Effects of oligosporin B on lipid metabolism in mature adipocytes

[0048] Oligosporin B solution: Oligosporin B was dissolved in dimethyl sulfoxide to prepare oligosporin B solutions with concentrations of 10 and 20 mmol / L.

[0049] Induction of differentiation of 3T3-L1 preadipocytes: 3T3-L1 preadipocytes were cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum at 37°C, 5% CO2, and 95% ± 5% humidity. After the cells reached 100% confluence, they were starved for two days. Then, the medium was removed, and differentiation reagent I (10% fetal bovine serum, 10 μg / mL insulin (INS), 1 μmol / L dexamethasone (DEX), 0.5 mmol / L 3-isobutyl-1-methylxanthine (IBMX), and 90% DMEM) was added. The cells were then treated at 37°C, 5% CO2, and 95% ± 5% humidity for 3 days. Afterward, differentiation reagent I was removed, and differentiation reagent II (10% fetal bovine serum, 10 μg / mL insulin, and 90% DMEM) was added, followed by treatment for another 4 days. At this point, the 3T3-L1 preadipocytes have been induced to differentiate into mature adipocytes.

[0050] 3T3-L1 preadipocytes were set as the undifferentiated group, and mature adipocytes were set as the four experimental groups.

[0051] (1) Undifferentiated group: 3T3-L1 preadipocytes were cultured in DMEM supplemented with 10% fetal bovine serum. After the cells reached 100% confluence, they were starved for two days. Then the culture medium was removed, and DMEM supplemented with 10% fetal bovine serum was added and cultured for another 7 days. Then the culture medium was replaced with fresh DMEM supplemented with 10% fetal bovine serum and cultured for 48 hours. The cells were separated from the culture medium to obtain undifferentiated group test cells and undifferentiated group test supernatant.

[0052] (2) Model group: Mature adipocytes were cultured in DMEM containing 10% fetal bovine serum for 48 hours. The cells were then separated from the culture medium to obtain the model group test cells and the model group test supernatant.

[0053] (3) Low-dose oligosporin B treatment group: Oligosporin B solution was added to DMEM containing 10% fetal bovine serum to obtain DMEM containing 10 μmol / L oligosporin B and 10% fetal bovine serum. Mature adipocytes were cultured in DMEM containing 10 μmol / L oligosporin B and 10% fetal bovine serum for 48 hours. The cells were separated from the culture medium to obtain the test cells in the low-dose oligosporin B treatment group and the test supernatant in the low-dose oligosporin B treatment group.

[0054] (4) High-dose oligosporin B treatment group: Oligosporin B solution was added to DMEM containing 10% fetal bovine serum to obtain DMEM containing 20 μmol / L oligosporin B and 10% fetal bovine serum. Mature adipocytes were cultured in DMEM containing 20 μmol / L oligosporin B and 10% fetal bovine serum for 48 hours. The cells were separated from the culture medium to obtain the test cells in the high-dose oligosporin B treatment group and the test supernatant in the high-dose oligosporin B treatment group.

[0055] (5) Solvent control group: Dimethyl sulfoxide (DMSO) was added to DMEM containing 10% fetal bovine serum (the volume of which was the same as that of the DMSO in the low-dose oligosporin B (10 μmol / L) and high-dose oligosporin B (20 μmol / L) treatment groups) to make the final concentration of DMSO 0.1% (v / v). Mature adipocytes were cultured in DMEM containing DMSO and 10% fetal bovine serum for 48 hours. The cells were then separated from the culture medium to obtain the test cells of the solvent control group and the test supernatant of the solvent control group.

[0056] Oil Red O staining was used to detect intracellular lipid accumulation. Cells in each group were gently washed twice with phosphate-buffered saline (PBS), then fixed at room temperature for 30 minutes with 10% formaldehyde solution (formaldehyde:PBS volume ratio 1:9). The formaldehyde solution was discarded, and the cells were permeated with 60% isopropanol solution (isopropanol:double-distilled water volume ratio 6:4) for 30 seconds. The isopropanol solution was discarded, and Oil Red O staining solution was added to stain the cells at room temperature for 10 minutes. The cells were then washed four times with double-distilled water. Cell morphology and Oil Red O staining were observed using an inverted fluorescence microscope, and photographs were taken. Results are shown below. Figure 7 (A). After observation, 500 μL of 100% isopropanol solution was added to the cells to extract Oil Red O dye. The OD value of the extracted Oil Red O dye was measured at a wavelength of 510 nm using a multi-functional microplate reader. The results are shown in [Figure 1]. Figure 7 (B). The main component of lipids that can be stained with Oil Red O in cells is triglycerides. Therefore, the OD value of Oil Red O dye can reflect the lipid content and accumulation in cells.

[0057] Figure 7 Oil Red O staining results showed that, compared with the undifferentiated group, cells in the model group and solvent control group exhibited a large number of red lipid droplets after Oil Red O staining (Figure A). 510nm The value was significantly increased (Figure B), indicating that 3T3-L1 cells had been successfully induced to differentiate into mature adipocytes. The number of lipid droplets and OD in cells of the model group and the solvent control group were significantly increased. 510nmThe values ​​showed no significant difference, indicating that the solvent did not affect intracellular lipid accumulation. When differentiated and mature adipocytes were treated with low-dose (10 μmol / L) or high-dose (20 μmol / L) oligosporin B for 48 hours, the number of lipid droplets stained red by Oil Red O was significantly reduced, and the OD value... 510nm The value decreased significantly, indicating that oligosporin B has a degrading effect on lipids accumulated in adipocytes.

[0058] Intracellular triglyceride hydrolysis in adipocytes produces free fatty acids and glycerol, which are released extracellularly. Therefore, the content of free fatty acids and glycerol in the supernatant can be used to evaluate the intracellular triglyceride hydrolysis status. Methods for determining free fatty acids and glycerol: The supernatants of each group were tested using a free fatty acid and glycerol detection kit (purchased from Nanjing Jiancheng Bioengineering Institute). The content of free fatty acids and glycerol in the supernatant was determined according to the kit instructions. Results are shown below. Figure 8 . Figure 8 The results showed that, compared with the solvent control group, the content of free fatty acids and glycerol in the supernatant was significantly increased after treating cells with oligosporin B, indicating that oligosporin B can promote the decomposition of triglycerides in adipocytes.

[0059] Example 5: Effects of oligosporin B on the activity of lipid metabolism enzymes in adipocytes

[0060] Cell lysis buffer RIPA (Radio-Immunoprecipitation Assay Buffer): 1.5 mol / L sodium chloride, 0.25 mol / L tris(hydroxymethyl)aminomethane, 0.5% (w / v) sodium deoxycholate, 0.1% (w / v) sodium dodecyl sulfate, 1% Triton X-100 (v / v), 0.1% (v / v) protease inhibitor, deionized water. The protease inhibitor was purchased from Beyotime Biotechnology Co., Ltd., catalog number: P1050.

[0061] Adipose triglyceride lipase (ATGL) is responsible for catalyzing the first step of the hydrolysis of triglycerides (TG), breaking them down into diacylglycerols (DAG) and releasing one molecule of free fatty acid. It is a key rate-limiting enzyme in intracellular lipolysis. The antibody (primary antibody) used to detect this enzyme is a rabbit-derived anti-adipose triglyceride lipase antibody, purchased from ProteinTech, catalog number 55190-1-AP.

[0062] Hormone-Sensitive Lipase (HSL): In adipocytes, this enzyme is primarily responsible for catalyzing the further hydrolysis of diacylglycerol (DAG) into monoacylglycerol (MAG) and free fatty acids, acting as the "rate-limiting enzyme of the second step" in the lipolysis cascade. The activity of this enzyme is regulated by phosphorylation modifications at specific amino acid sites on its protein. Hormone-sensitive lipase phosphorylated at serine position 565 is abbreviated as p-HSL. Ser565 The primary antibody used to detect hormone-sensitive lipase was a rabbit-derived anti-hormone-sensitive lipase antibody, purchased from Cell Signaling Technology, catalog number 4107T; the primary antibody used to detect hormone-sensitive lipase with phosphorylation at serine position 565 was a rabbit-derived anti-hormone-sensitive lipase with phosphorylation at serine position 565, purchased from Cell Signaling Technology, catalog number 4137T.

[0063] β-Tubulin: A constitutively expressed housekeeping protein, its expression level remains relatively constant in most eukaryotic cells. β-Tubulin can be used as an internal reference protein to correct for loading errors and operational deviations during experiments, ensuring the accuracy and reliability of the target protein quantification results. The antibody (primary antibody) used to detect β-tubulin is a rabbit-derived anti-β-tubulin antibody, purchased from Proteintech, catalog number 10094-1-AP.

[0064] The secondary antibody used to bind to rabbit-derived anti-triglyceride lipase antibody, rabbit-derived anti-hormone-sensitive lipase antibody, rabbit-derived anti-hormone-sensitive lipase antibody modified by phosphorylation of serine at position 565, and rabbit-derived anti-β-tubulin antibody was a goat anti-rabbit immunoglobulin G (IgG) antibody conjugated with horseradish peroxidase, purchased from Thermo Fisher Scientific, catalog number 31460.

[0065] The horseradish peroxidase substrate reaction solution used for protein development was purchased from Proteintech, catalog number PK10002.

[0066] 3T3-L1 preadipocytes were set as the undifferentiated group; mature adipocytes were obtained by the procedure in Example 4 and subsequently set as 4 experimental groups.

[0067] (1) Undifferentiated group: 3T3-L1 adipocytes were cultured in DMEM supplemented with 10% fetal bovine serum. After the cells reached 100% confluence, they were starved for two days. Then the culture medium was removed, and DMEM supplemented with 10% fetal bovine serum was added and cultured for another 7 days. Then the culture medium was replaced with fresh DMEM supplemented with 10% fetal bovine serum and cultured for 48 hours. Cells were collected, cell lysis buffer RIPA was added to the ice bath, and the cells were placed on ice for 30 minutes. Then the cells were centrifuged at 4 degrees Celsius and 10,000×g for 10 minutes to obtain the supernatant of the undifferentiated group.

[0068] (2) Model group: Mature adipocytes were cultured in DMEM containing 10% fetal bovine serum for 48 hours, and the supernatant of the model group was obtained by the same operation as the undifferentiated group.

[0069] (3) Solvent control group: Dimethyl sulfoxide was added to DMEM containing 10% fetal bovine serum to make the final concentration of dimethyl sulfoxide 0.1% (v / v). Mature adipocytes were cultured in DMEM containing dimethyl sulfoxide and 10% fetal bovine serum for 48 hours. The supernatant of the solvent control group was obtained by the same operation as the undifferentiated group.

[0070] (4) Low-dose oligosporin B treatment group: Oligosporin B solution was added to DMEM containing 10% fetal bovine serum to obtain DMEM containing 10 μmol / L oligosporin B and 10% fetal bovine serum. Mature adipocytes were cultured in DMEM containing 10 μmol / L oligosporin B and 10% fetal bovine serum for 48 hours. The supernatant of the low-dose oligosporin B treatment group was obtained by the same operation as the undifferentiated group.

[0071] (5) High-dose oligosporin B treatment group: Oligosporin B solution was added to DMEM containing 10% fetal bovine serum to obtain DMEM containing 20 μmol / L oligosporin B and 10% fetal bovine serum. Mature adipocytes were cultured in DMEM containing 20 μmol / L oligosporin B and 10% fetal bovine serum for 48 hours. The supernatant of the high-dose oligosporin B treatment group was obtained by the same operation as the undifferentiated group.

[0072] The total protein content in the supernatant of each group was determined using a protein quantification kit (Thermo Science) to determine the sample loading amount for Western blotting detection. Western blotting was performed using polyvinylidene fluoride (PVDF) membranes (Millipore) as samples from each group. Specifically, triglyceride lipase (ATGL) and the internal control β-tubulin were simultaneously hybridized with primary antibodies on a single membrane; hormone-sensitive lipase (HSL) and hormone-sensitive lipase phosphorylated at serine position 565 (p-HSL) were also tested.Ser565 The PVDF membrane containing the secondary antibody and the internal control β-tubulin were simultaneously hybridized with the primary antibody on a separate membrane. The PVDF membrane incubated with the secondary antibody was placed in a multi-functional imaging system, and substrate reaction solution was added to the PVDF membrane. The resulting blue fluorescence signal was then acquired and imaged using a multi-functional microplate reader to display the target proteins (ATGL, HSL, p-HSL). Ser565 The location and fluorescence intensity of β-tubulin on the PVDF membrane are shown in the figure. Figure 9 ImageJ software was used to analyze the grayscale of protein images, with the grayscale value of the internal reference β-tubulin protein used as calibration. ATGL, HSL, and p-HSL in each sample were calculated. Ser565 The ratio of the gray value of the protein band to the gray value of the β-tubulin band in the same sample, with the corresponding ratios being ATGL, HSL, and p-HSL. Ser565 The calibration values ​​of the protein band grayscale were calculated. Then, using the model group as a reference, the ratio of the calibration value of ATGL protein grayscale in each group to the calibration value of ATGL protein grayscale in the model group was calculated. This ratio represents the relative expression level of ATGL protein in each group relative to ATGL protein in the model group. The ratio of the calibration value of HSL protein grayscale in each group to the calibration value of HSL protein grayscale in the model group was also calculated. This ratio represents the relative expression level of HSL protein in each group relative to HSL protein in the model group. The p-HSL protein in each group was also calculated. Ser565 The calibration value of protein grayscale and p-HSL in the model group Ser565 The ratio of the calibrated values ​​of protein grayscale; this ratio is the p-HSL of each group of cells. Ser565 Protein relative to p-HSL in the model group Ser565 Relative expression levels of proteins; calculation of p-HSL in cells of each group. Ser565 The ratio of the calibrated gray value of the protein band to the calibrated gray value of the HSL protein band is used to obtain the p-HSL in each group of cells. Ser565 The ratio of p-HSL was calculated, and then, using the model group as a reference, the p-HSL ratio in each group was calculated. Ser565 The ratio of / HSL to p-HSL in the model group Ser565 Divide the ratio of p-HSL to p-HSL in each group of cells to obtain the p-HSL ratio. Ser565 The ratio of / HSL increased by a factor compared to the ratio in the model group. See the results below. Figure 10 .

[0073] Figure 9 and Figure 10 The results showed that, compared with the solvent control group, treatment of adipocytes with oligosporin B (especially high-dose oligosporin B) for 48 hours increased the protein expression level of triglyceride lipase (ATGL) and significantly enhanced hormone-sensitive lipase (p-HSL) modified by serine phosphorylation at position 565.Ser565 Treatment of adipocytes with low-dose (10 μmol / L) or high-dose (20 μmol / L) oligosporin B for 48 hours significantly increased the proportion of activated hormone-sensitive lipase in total hormone-sensitive lipase (p-HSL). Ser565 The above results indicate that oligosporin B can enhance the activity of key rate-limiting enzymes responsible for lipid metabolism in adipocytes, thus promoting lipolysis.

Claims

1. The use of oligosporin B in the preparation of weight-loss drugs, wherein the chemical structural formula of oligosporin B is as follows: 。 2. The use according to claim 1, characterized in that, Oligosporin B is used for weight loss in humans or other animals.

3. The use according to claim 1, characterized in that, The dosage of oligosporin B is from 15 mg oligosporin B / kg body weight to 30 mg oligosporin B / kg body weight.

4. The use according to any one of claims 1 to 3, characterized in that, Administered orally.

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