Use of compound Resinacein S or its derivatives in the preparation of a medicament for preventing or treating obesity or related metabolic complications

Abstract

The present invention relates to the technical field of medicinal chemistry, and particularly relates to the use of the compound Resinacein S or its derivatives in the preparation of a medicament for preventing or treating obesity or related metabolic complications. The present invention provides the use of the compound Resinacein S or its derivatives in the preparation of a medicament for preventing or treating obesity or related metabolic complications; the present invention has found through research that the compound Resinacein S has the effect of preventing and treating obesity, and the mechanism of action is to reduce fat accumulation by increasing whole-body energy metabolism, so as to reduce body weight and prevent and treat obesity. Compared with traditional weight-loss drugs that reduce body weight by suppressing appetite, the compound Resinacein S does not prevent and treat obesity by interfering with appetite, thus avoiding side effects such as gastrointestinal discomfort and cardiovascular risks caused by appetite interference.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and particularly to the use of compound Resinacein S or its derivatives in the preparation of a medicament for preventing or treating obesity or related metabolic complications. Background Art

[0002] The occurrence of obesity is the result of the imbalance of the body's energy metabolism. That is, when the body's energy intake exceeds energy consumption, the remaining energy is almost entirely converted into triglycerides and stored in white adipose tissue (WAT) throughout the body. People consume excessive amounts of food while reducing physical activity and exercise, leading to an imbalance in the body's energy metabolism, and obesity has become an inevitable problem for humans.

[0003] Studies have shown that adipose tissue plays a central role in the occurrence and development of obesity and is crucial for metabolic health. Currently, it is known that there are three main types of adipose tissue in the human body: white fat, brown fat, and beige fat. White fat mainly stores energy in the form of triglycerides, while brown fat and beige fat consume energy by generating heat. Beige fat exists within white fat and has the characteristics of brown fat, and can be regarded as a transitional form between white fat and brown fat. The phenomenon of the presence of beige fat in white adipose tissue is called the browning of white fat. Although beige fat and brown fat are located in different parts and have different developmental origins, they both have similar thermogenic functions and energy consumption potential, so they are collectively referred to as thermogenic adipose tissue. Under physiological conditions, thermogenic adipose tissue is usually activated through two pathways: cold exposure and dietary intake (especially high-calorie foods). Under cold exposure, thermogenic adipose tissue is activated to maintain a constant body temperature; during dietary intake, the activation of thermogenic adipose tissue helps to relieve a transient energy peak, thereby maintaining metabolic stability.

[0004] In recent years, the differentiation, development, functional regulation, and clinical application of thermogenic adipose tissue have become research hotspots in the field of metabolism. Population surveys have found an association between active thermogenic adipose tissue and a lower risk of obesity and metabolic-related diseases such as type 2 diabetes. Some drugs that improve metabolism, such as berberine and salicylsalicylic acid, partially achieve their effects by activating thermogenic adipose tissue. In animal models, activating thermogenic brown fat can control body weight and improve metabolism, whether by altering related genes (knockout or overexpression) or through drug stimulation (such as β-adrenergic receptor agonists). Therefore, beige adipose tissue, as the thermogenic adipose tissue in the human body, has the potential to consume excess energy and can be activated under specific conditions. Activating thermogenic adipose tissue, whether by enhancing the function of brown fat or promoting the browning of white fat, can achieve the purpose of controlling body weight and improving metabolism. To date, no compounds or drugs specifically targeting thermogenic adipose tissue to improve metabolism have been discovered, and high-throughput screening for this target has not been implemented.

[0005] The reason thermogenic adipose tissue can convert the chemical energy stored in fatty acids into heat energy and release it is that it has a special molecule, namely uncoupling protein 1 (UCP1). UCP1 is located on the inner mitochondrial membrane and, when activated, can cut off the coupling between the electron transport chain and ATP synthesis, thereby directly converting chemical energy into heat energy. UCP1 is mainly present in thermogenic adipose tissue. In this special tissue localization, UCP1 is not only a functional molecule of thermogenic adipose tissue but also its phenotypic marker. In addition to UCP1, molecules such as PGC1 and PRDM16 are also important factors involved in the browning process.

[0006] Ganoderma triterpenoids are a class of active compounds extracted from Ganoderma lucidum, belonging to polyterpenoid compounds. Ganoderma lucidum is a traditional medicinal mushroom widely used in Asia. Ganoderma triterpenoids are considered to be one of the most active components in Ganoderma lucidum and have various benefits for human health. Their physiological activities are very extensive, including antioxidant, anti-inflammatory, anti-tumor, hypoglycemic, and cardiovascular protection. Research has shown that these compounds can act through multiple mechanisms, such as inhibiting key enzymes in the inflammatory response, increasing the activity of antioxidant enzymes, inhibiting the proliferation of tumor cells, and inducing their apoptosis. However, there has been no relevant report on the use of the Ganoderma triterpenoid compound Resinacein S and its analogs in the preparation of agents for preventing and treating obesity and improving glucose metabolism disorders. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide the use of compound Resinacein S or its derivatives in the preparation of drugs for preventing or treating obesity or related metabolic complications.

[0008] To this end, the present invention provides the following technical solutions:

[0009] Use of compound Resinacein S or its derivative in the preparation of a drug for preventing or treating obesity or related metabolic complications.

[0010] Optionally, the obesity includes weight gain and / or increase in fat mass.

[0011] Use of compound Resinacein S or its derivative in the preparation of a drug for preventing or treating glucose metabolism disorders.

[0012] Optionally, the glucose metabolism disorders include diabetes and / or improvement of insulin resistance.

[0013] Optionally, the derivative of the compound includes its cis-trans isomers, its diastereoisomers, its geometric isomers, its racemates, its solvates, its pharmaceutically acceptable salts or its prodrugs.

[0014] A pharmaceutical preparation for preventing and treating obesity or glucose metabolism disorders, comprising the compound Resinacein S or its derivative, and a pharmaceutically acceptable excipient.

[0015] Optionally, the form of the pharmaceutical preparation includes liquid preparation, semi-solid preparation or solid preparation.

[0016] Optionally, the pharmaceutical preparation is an oral preparation or a topical preparation.

[0017] Optionally, the dosage form of the pharmaceutical preparation is powder, powder for injection, injection, tablet, capsule, granule, ointment, paint, lotion, tincture, liniment, film-forming agent, ointment, gel, paste, patch or aerosol.

[0018] Optionally, the excipient includes any one or a combination of at least two of carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant.

[0019] The technical solution of the present invention has the following advantages:

[0020] 1. Use of the compound Resinacein S or its derivatives provided by the present invention in the preparation of a drug for preventing or treating obesity or related metabolic complications; the present invention has found through research that the compound Resinacein S has the effect of preventing and treating obesity, and its mechanism of action is to reduce fat accumulation by increasing whole-body energy metabolism, so as to reduce body weight and prevent and treat obesity. Compared with traditional weight-loss drugs that reduce body weight by suppressing appetite, the compound Resinacein S does not prevent and treat obesity by interfering with appetite, thus avoiding side effects such as gastrointestinal discomfort and cardiovascular risks brought about by appetite intervention.

[0021] 2. Use of the compound Resinacein S or its derivatives provided by the present invention in the preparation of a drug for preventing or treating glucose metabolism disorders; the present invention further finds through research that the compound Resinacein S not only has the effect of preventing and treating obesity, but also has the effect of preventing and treating glucose metabolism disorders. Compared with existing anti-obesity drugs that generally have a significant effect only on preventing and treating obesity, but have limited effects on improving metabolic diseases such as insulin resistance and diabetes, and have relatively single functions, the compound Resinacein S of the present invention has a dual therapeutic effect of preventing and treating obesity and treating glucose metabolism disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 It is the experimental result that the compound Resinacein S (abbreviated as Res S) in Example 1 of the present invention prevents obesity caused by a high-fat diet by increasing whole-body energy metabolism; in the figure, Figure A is the weight change curve graph of each group of mice within eight weeks; Figure B is the weight graph of each group of mice within the eighth week; Figure C is the fat mass graph of each group of mice in the 7th week; Figure D is the lean body mass graph of each group of mice in the 7th week; Figure E is the oxygen consumption (VO2) result of each group of mice in the 7th week; Figure F is the oxygen consumption (VO2) result of each group of mice during the day in the 7th week; Figure G is the oxygen consumption (VO2) result of each group of mice at night in the 7th week; Figure H is the total oxygen consumption (VO2) result of each group of mice in the 7th week; Figure I is the energy consumption graph of each group of mice after adjusting the weight by ANCOVA; Figure J is the average energy consumption graph of each group of mice; Figure K is the physical activity graph of each group of mice; Figure L is the food intake graph of each group of mice;

[0024] Figure 2It is the result graph of Res S treatment in Example 2 of the present invention for improving the whole-body glucose homeostasis in obese mice; in the figure, Figure A shows the fasting blood glucose of each group of mice; Figure B shows the fasting insulin of each group of mice; Figure C shows the glucose tolerance of each group of mice; Figure D is a bar graph of the area under the curve of the glucose tolerance test plotted in Figure C; Figure E shows the insulin tolerance of each group of mice; Figure F is a bar graph of the area under the curve of the insulin tolerance test shown in Figure E;

[0025] Figure 3 It is the result that Res S treatment in Example 3 of the present invention promoted the thermogenic activity of BAT in HF diet-induced obese mice; in the figure, Figure A shows the HE staining of BAT adipose tissue of each group of mice; Figure B shows the quantitative result of lipid content in the BAT adipose tissue in Figure A; Figure C shows the immunohistochemical staining of UCP1 in each group of mice; Figure D shows the quantitative result of the abundance of UCP1 protein in Figure C; Figure E shows the mRNA expression levels of genes related to thermogenesis (Ucp1, Pgclα, Prdm16, Dio2, Cideα, AdipoQ) in each group of mice; Figure F shows the mRNA expression levels of genes related to lipolysis (Atg1 and Hsl) in each group of mice; Figure G shows the mRNA expression levels of genes related to fatty acid oxidation (Ppara, Cptlα, Mcad and Lcad) in each group of mice; Figure H shows the mRNA expression levels of genes related to mitochondrial biogenesis factors (Nrf1, Nrf2, Tfam1) related to BAT in each group of mice; Figure I shows the mitochondrial DNA copy number in each group of mice;

[0026] Figure 4 It is the result that Res S treatment in Example 4 of the present invention induced the formation of brown adipocytes in IngWAT of mice fed with HF diet; in the figure, Figure A shows the HE staining of IngWAT adipose tissue of each group of mice; Figure B shows the quantitative result of lipid content in the IngWAT adipose tissue in Figure A; Figure C shows the immunohistochemical staining of UCP1 in IngWAT of each group of mice; Figure D shows the quantitative result of the abundance of UCP1 protein in Figure C; Figure E shows the mRNA expression levels of genes related to thermogenesis (Ucp1, Pgclα, Fgf21), fatty acid oxidation (Ppara, Cptlα, Elovl3), and lipolysis (Atg1 and Hsl) in each group of mice; Figure F shows the mRNA expression levels of genes related to mitochondrial biogenesis (Nrf1, Nrf2, Tfam1) in each group of mice; Figure G shows the mRNA expression levels of genes related to beige cell-specific markers (Tmem26, Cd137, Tbxl) in each group of mice; Figure H shows the OCR results of ex vivo IngWAT of each group of mice. Detailed implementation manners

[0027] The following embodiments are provided to better understand the present invention further. It is not limited to the best embodiment, and does not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other prior arts falls within the protection scope of the present invention.

[0028] For those embodiments in which specific experimental steps or conditions are not indicated, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase.

[0029] The CAS number of the compound Resinacein S is 2259327-71-2, and its molecular formula is C 30 H 44 O8, and its structural formula is as follows:

[0030]

[0031] The compound Resinacein S is a commercially available product.

[0032] Example 1 The compound Resinacein S (abbreviated as Res S) prevents high-fat diet-induced obesity by increasing whole-body energy metabolism

[0033] Research reports that the occurrence of obesity and metabolic diseases is the result of the combined action of living environment and genetic factors. High-fat diet-induced obese mice are commonly used animal models in the laboratory to study the occurrence and development mechanisms of obesity and metabolic diseases. Therefore, in this example, high-fat diet-induced obese mice were used for the experiment.

[0034] 1. Experimental methods and grouping

[0035] Five-week-old male C57BL / 6 mice (purchased from Beijing Vital River Laboratories Co., Ltd.) were taken. After one week of environmental adaptation, they were fed a high-fat diet (Research Diets, USA) or a normal diet (Research Diets, USA) for 4 weeks. Subsequently, the mice were randomly divided into four groups, with 6 mice in each group:

[0036] Normal diet group (NC): Regularly fed a normal diet, and at the same time, intraperitoneally injected with corn oil once every two days, and the injection volume was the same as that of a single injection of Res S solution in the NCR group or HFR group;

[0037] Normal diet plus Res S group (NCR): Regularly fed a normal diet, and at the same time, intraperitoneally injected with Res S (4 mg / kg body weight of the mouse, the solvent was corn oil) solution once every two days;

[0038] High-fat diet group (HF): Mice were fed a high-fat diet routinely and intraperitoneally injected with corn oil once every two days. The injection volume was the same as that of a single injection of Res S solution in the NCR group or HFR group.

[0039] High-fat diet plus Res S group (HFR): Mice were fed a high-fat diet routinely and intraperitoneally injected with Res S (4 mg / kg body weight of mice, with corn oil as the solvent) solution once every two days.

[0040] Each group was administered drugs according to the above treatment methods for eight weeks. The body weight of the mice was recorded weekly.

[0041] In the seventh week, first, a MesoQMR 23-060H-I system (Newmai Company, China) was used to analyze the body composition of each mouse to measure its fat and lean body mass. Then, the mice were placed in the main system of the TSE laboratory to continuously monitor their metabolic activities, and the oxygen consumption (VO2), carbon dioxide production (VCO2), and food intake of the mice were recorded. The Opto-M3 beam technology (Columbus Company, USA) was used to monitor the dynamic activities of each mouse within a 12-hour light-dark cycle, and the activity frequency and intensity were recorded. According to the collected gas exchange data, the respiratory exchange ratio (RER, VCO2 / VO2) and total energy expenditure (EE) were calculated, and the metabolic rate and activity pattern of the mice were further analyzed in combination with the dynamic activity data.

[0042] 2. Data processing: One-way analysis of variance (ANOVA) was used to compare the differences in indicators such as body weight, fat percentage, RER, and EE among the groups of mice. If there were significant differences, multiple comparison tests were performed. The paired t-test was used to compare the changes in the same group of mice at different time points, and correlation analysis was used to evaluate the relationship between variables. Finally, the analysis results were visualized through charts to intuitively present the differences and trends of the data in each group, so as to comprehensively evaluate the effect of Res S on mouse metabolism.

[0043] 3. Experimental results

[0044] The results are as Figure 1 shown. Res S treatment significantly inhibited the weight gain of mice induced by HF diet ( Figure 1 Figures A and B in Figure 1 ). Further body composition analysis showed that the weight loss of HF diet-fed mice was usually attributed to the reduction of fat mass by Res S ( Figure 1 Figure C in Figure 1 ). However, the treatment with Res S had no obvious effect on weight gain and lean body mass in NC diet-fed mice. Under HF diet conditions, Res S treatment significantly increased the energy expenditure ( Figure 1 Figures I and J in Figure 1 and oxygen intake ( Figure 1 Figures E - G in Figure 1 ) of HF diet-fed mice, but did not affect physical activity and food intake ( Figure 1in the K and L figures). At the same time, mice treated with Res S showed a lower respiratory exchange ratio (RER), indicating a higher dependence on fat-mediated oxidation( Figure 1 in the H figure).

[0045] 4. Experimental conclusions

[0046] In summary, the Res S treatment group significantly increased energy consumption and oxygen intake under high-fat diet conditions, while reducing the respiratory exchange ratio (RER), indicating that it increases energy metabolism by promoting fat oxidation( Figure 1 in I-J, Figure 1 in E-G, Figure 1 in the H figure). The above results indicate that Res S reduces fat accumulation through natural metabolic pathways, prevents HF diet-induced weight gain, and avoids direct intervention in appetite. Compared with traditional weight loss drugs that often achieve weight loss by suppressing appetite and are often accompanied by significant side effects such as gastrointestinal discomfort and cardiovascular risks, the Res S of the present invention reduces potential side effects.

[0047] Example 2 Res S treatment improves whole-body glucose homeostasis in obese mice

[0048] Numerous studies have shown that obesity is often accompanied by severe insulin resistance. Therefore, an obese mouse model was constructed and grouped according to the "1. Experimental methods and grouping" in Example 1. Mice administered drugs for 6-7 weeks were subjected to the following tests:

[0049] 1.1 Intraperitoneal glucose tolerance test (IPGTT):

[0050] Mice administered drugs for 6 weeks were fasted overnight, weighed, and then intraperitoneally injected with 1.5 g / kg body weight of glucose. After injection, blood glucose levels were measured using a blood glucose meter at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min respectively.

[0051] Data processing: Record the blood glucose concentration at each time point, and calculate the area under the curve (AUC) to evaluate glucose clearance efficiency. Then use one-way analysis of variance (ANOVA) to compare the AUC differences between groups. Finally, display the blood glucose change trend and AUC value through charts to evaluate the effect of different treatments on glucose metabolism. And calculate the area under the curve (AUC) to evaluate glucose clearance efficiency. The AUC is integrated through the blood glucose levels at time points. The smaller the AUC value, the faster the glucose metabolism. Compare the fasting blood glucose levels to evaluate the effect of Res S on blood glucose control, and use one-way analysis of variance (ANOVA) or t-test to compare the differences in AUC and fasting blood glucose levels between groups. The results are visualized through line charts and bar charts to intuitively show the improvement effect of Res S on glucose metabolism.

[0052] The results are as Figure 2 shown in Figures A, C, and D below:

[0053] The fasting blood glucose of obese mice in the HF group was significantly higher than that of mice treated with Res S, indicating that Res S can effectively control hyperglycemia in obese mice. Figure 2 Figure D below shows a bar graph of the area under the curve of the glucose tolerance test plotted according to Figure 2 Figure C below. It shows that the area under the curve of the Res S group was significantly smaller than that of the control group (HF group), indicating that Res S can significantly improve glucose clearance.

[0054] 1.2 Insulin Tolerance Test (ITT):

[0055] After fasting the mice for 4 hours 7 weeks after drug administration, weighing them, and then intraperitoneally injecting insulin at a dose of 0.7 IU / kg body weight, blood glucose levels were measured using a blood glucose meter at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after injection.

[0056] Data processing: Record the blood glucose concentration at each time point and calculate the area under the curve (AUC) to evaluate insulin sensitivity. One-way analysis of variance (ANOVA) was performed on the fasting insulin levels and AUC of each group of mice to compare the differences between groups. Finally, the trend of blood glucose changes was shown through a line graph, and the comparison results of AUC values and fasting insulin levels were shown using a bar graph to evaluate the effect of Res S on insulin tolerance.

[0057] The results are as Figure 2 shown in Figures B, E, and F below:

[0058] The fasting insulin of obese mice in the HF group was significantly higher than that of mice treated with Res S, indicating that Res S can effectively control insulin in obese mice. Figure 2 Figure F below shows a bar graph of the area under the curve of the insulin tolerance test plotted according to Figure 2 Figure E below. It shows that the area under the curve of the Res S group was significantly smaller than that of the control group (HF group), indicating that Res S can significantly reduce insulin levels.

[0059] In summary, Res S significantly improved the glucose tolerance and insulin sensitivity of obese mice. Specifically, the fasting blood glucose and fasting insulin levels of mice in the Res S treatment group were significantly reduced. The results of both the Intraperitoneal Glucose Tolerance Test (IPGTT) and Insulin Tolerance Test (ITT) showed that Res S significantly increased glucose clearance and insulin sensitivity. Figure 2In Figures A-F). This indicates that Res S not only can prevent and treat obesity, but also can effectively improve glucose metabolism disorders (insulin resistance) caused by obesity, having a dual therapeutic effect. Compared with many existing anti-obesity drugs that can reduce body weight but have limited effects on improving metabolic diseases such as insulin resistance and diabetes, Res S of the present invention has a dual therapeutic effect of preventing and treating obesity and treating glucose metabolism disorders.

[0060] Example 3 Res S treatment promoted the thermogenic activity of BAT in HF diet-induced obese mice

[0061] Construct an obese mouse model and group according to the "1. Experimental methods and grouping" in Example 1. After 8 weeks of drug administration, the mice were sacrificed by cervical dislocation, and the brown adipose tissue (BAT, classical brown adipose tissue) in the interscapular region of the mice was dissected, weighed, fixed, and entrusted to Wuhan Sevier Company for histological analysis of adipose tissue, including fixation, paraffin embedding, sectioning, HE staining, and immunohistochemical staining of UCP1. Three representative adipose tissues were taken out for analysis.

[0062] Data processing: Sections of brown adipose tissue (BAT) were stained by HE, and the size and lipid content of adipocytes were measured using image analysis software to quantitatively analyze the differences between the Res S treatment group and the control group of mice. At the same time, the expression level of UCP1 protein was detected by immunohistochemical staining, and the staining intensity was quantitatively analyzed using integrated optical density (IOD). Then, one-way analysis of variance (ANOVA) was used to compare the differences in adipocyte size, lipid content, and UCP1 protein expression among groups. Finally, the results were presented by histograms to evaluate the effect of Res S on BAT metabolic activity and UCP1 protein expression.

[0063] The results are as Figure 3 shown in Figures A-D of

[0064] Under HF diet conditions, the lipid content in BAT of mice treated with Res S was significantly reduced, and the size of adipocytes was significantly reduced ( Figure 3 in Figures A and B), indicating that the metabolic activity of fat was enhanced. In addition, through immunohistochemical detection, it was found that the abundance of UCP1 protein in BAT of mice treated with Res S was significantly up-regulated ( Figure 3In Figures C and D), it was further verified that the activity of BAT was enhanced. The enhanced activity of brown adipocytes indicated an increase in their metabolic activity, which was specifically manifested as a decrease in lipid content in adipocytes, a reduction in adipocyte size, and an upregulation of UCP1 protein expression. This means that brown fat accelerated energy consumption through thermogenesis (non-shivering thermogenesis), converting more fat into heat through oxidative metabolism, thereby promoting the reduction of body fat. Therefore, it can be concluded from the experiment that Res S plays a role in weight loss by activating brown adipocytes and increasing energy consumption.

[0065] RNA extraction:

[0066] Take appropriate BAT samples from each group, add 1 ml of Trizol for sufficient lysis, and after thorough mixing, add 200 μl of chloroform. After vigorous shaking, let it stand at room temperature for 15 min, and then centrifuge at 4 °C with a centrifugation condition of 12,000 g for 10 min. At this time, due to chromatography, the lysate is divided into three layers. Carefully aspirate the upper aqueous phase solution and transfer it to a new centrifuge tube. Add 500 μl of isopropanol, gently mix, and let it stand at room temperature for 10 minutes. Then centrifuge at 4 °C, 12,000 g for 10 min, discard the supernatant. A milky white translucent precipitate can be seen at the bottom of the tube. Wash the precipitate 3 times with 75% ethanol diluted with DEPC water, centrifuge at 4 °C, 7,500 g for 5 min, discard the supernatant, air-dry at room temperature, and dissolve the precipitate in 60 μl of DEPC water. Use a NanoDrop2000 ultra-micro spectrophotometer to detect the concentration of the RNA solution.

[0067] Reverse transcription of cDNA:

[0068] Reverse transcribe it according to the manufacturer's instructions using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas K1622): Melt and mix the total RNA solution and each reagent in the reverse transcription kit, and then centrifuge. Add the components in Table 1:

[0069] Table 1 shows the reagent components and dosages used for reverse transcription of cDNA

[0070]

[0071]

[0072] After gently mixing, centrifuge using a microcentrifuge for 30 s.

[0073] React at 42 °C for 15 min using a conventional PCR instrument, heat at 85 °C for 5 s. After the reaction, obtain cDNA, take out the reaction product cDNA and place it on ice.

[0074] RT-PCR reaction:

[0075] (1) RT-PCR was performed using SYBR Green I Master Mix reagent (Roch) on a real-time PCR system (Roche). Primers: According to the gene sequences provided by NCBI, primers were designed on the online primer design website "NCBI Primer-BLAST". The primers are shown in Table 3, and the designed primers were synthesized by Bomed Co., Ltd.

[0076] (2) Add the components shown in Table 2 to a 96-well PCR plate:

[0077] Table 2 shows the reagent components and dosages for RT-PCR reaction

[0078] Component Volume cDNA 1.5 μL Forward primer (10 μmol / L) 1 μL Reverse primer (10 μmol / L) 1 μL 2×TransTaq HiFi PCR SuperMixⅡ 10 μL ddH2O 6.5 μL Total volume 20 μL

[0079] Table 3. Primers

[0080]

[0081]

[0082] After gently mixing, centrifuge for 30 s using a microcentrifuge.

[0083] Place the 96-well PCR plate in a PCR instrument and perform the amplification reaction under the following conditions: Each cycle includes: 94 °C for 5 min (pre-denaturation) → 94 °C for 30 s (denaturation) → 56 °C for 30 s (annealing) → 72 °C for 30 s (extension); This amplification reaction consists of 40 cycles; Finally, 72 °C for 7 min.

[0084] Calculate the relative expression levels of mRNAs of genes related to thermogenesis, fat-related genes, and mitochondrial biogenesis factors in brown adipose tissue.

[0085] The results are as Figure 3 shown in E-I:

[0086] In the Res S treatment group of mice, the mRNA expressions of genes related to thermogenesis (Ucp1, Pgclα, Prdm16, Dio2, Cideα, AdipoQ) ( Figure 3 in Figure E) were significantly increased. At the same time, the mRNA expressions of genes related to lipolysis (Atg1 and Hsl) ( Figure 3 in Figure F) and fatty acid oxidation (Ppara, Cptlα, Mcad, and Lcad) ( Figure 3 in Figure G) were significantly increased. In addition, the expressions of BAT-related mitochondrial biogenesis factors (Nrf1, Nrf2, Tfam1) in the Res S treatment group were also significantly increased ( Figure 3 in Figure H), Figure 3Figure I shows a bar graph of the effect of Res S on the number of mitochondria in obese mice. The number of mitochondrial DNA copies in the Res S group of mice was significantly higher than that in the mitochondria of high-fat-induced obese mice, which further indicated that the energy metabolism level of the mice in the Res S group was higher than that of the control group (mice in the HF group).

[0087] In summary, traditional weight-loss drugs have limited activation effects on BAT, and the activity of BAT is closely related to energy consumption and weight management. Res S of the present invention significantly promoted the thermogenic activity of BAT in obese mice. The above experimental results showed that the lipid content of BAT in the Res S treatment group of mice decreased, the size of adipocytes shrank, and the protein abundance of UCP1 was significantly up-regulated ( Figure 3 in Figures A-D). In addition, the mRNA expression of genes related to thermogenesis and fat metabolism in BAT of the Res S treatment group increased significantly ( Figure 3 in Figures E-I). These results indicated that Res S increased energy consumption in the body by activating BAT and further inhibited fat accumulation.

[0088] Example 4 Res S treatment induced the formation of brown adipocytes in IngWAT of mice fed a HF diet

[0089] An obese mouse model and grouping were constructed according to the "1. Experimental methods and grouping" in Example 1. After 8 weeks of drug administration, the mice were sacrificed by cervical dislocation. The inguinal fat (IngWAT, subcutaneous fat) of the mice was dissected, weighed, fixed, and entrusted to Wuhan Sevier Company for histological analysis of adipose tissue, including fixation, paraffin embedding, sectioning, HE staining, and immunohistochemical staining of UCP1. Three representative pieces of adipose tissue were taken for analysis.

[0090] Data processing: Sections of inguinal fat (IngWAT) tissue were stained by HE, and the size and lipid content of adipocytes were measured using image analysis software to quantitatively analyze the differences between the Res S treatment group and the control group of mice. At the same time, the expression level of UCP1 protein was detected by immunohistochemical staining, and the staining intensity was quantitatively analyzed using the integrated optical density (IOD). Then, one-way analysis of variance (ANOVA) was used to compare the differences in adipocyte size, lipid content, and UCP1 protein expression among groups.

[0091] The results are as shown in Figure 4 Figures A and B:

[0092] Under HF diet conditions, mice treated with Res S showed browning of IngWAT. Compared with the control group, lipid accumulation in IngWAT of the Res S treatment group decreased and the size of adipocytes shrank.

[0093] Extract the total RNA of IngWAT, reverse transcribe it into cDNA, and detect the mRNA levels related to thermogenesis, fat-related genes, mitochondrial synthesis, and beige cell markers using Real time qPCR. The above operations were carried out according to Example 3.

[0094] The above Res S treatment significantly promoted the browning of inguinal white adipose tissue (IngWAT) in high-fat diet (HF)-induced mice. Compared with the control group (HF group), lipid accumulation in the IngWAT of mice in the Res S treatment group decreased, and the size of adipocytes significantly shrank, indicating that Res S accelerated the decomposition and oxidation of fat by promoting fat metabolism, thereby enhancing the metabolic activity of adipocytes. This browning phenomenon shows the potential role of Res S in transforming white fat into thermogenic beige adipocytes, contributing to increased energy consumption and having the potential for weight loss and improved metabolic health.

[0095] The results are as Figure 4 shown in C–F in

[0096] Figure 4 C–F in are the real-time fluorescence quantitative bar graphs showing the mRNA expression of thermogenesis, fat-related genes, and mitochondrial biogenesis factors in IngWAT of high-fat-induced obese mice treated with Res S. The abundance of UCP1 protein in the IngWAT of mice in the Res S treatment group increased ( Figure 4 in C and D), and the mRNA expression of genes related to thermogenesis (Ucp1, Pgclα, Fgf21), fatty acid oxidation (Ppara, Cptlα, Elovl3), lipolysis (Atg1 and Hsl) ( Figure 4 in E) and mitochondrial biogenesis (Nrf1, Nrf2, Tfam1) ( Figure 4 in F) increased significantly. It can be concluded from this that Res S plays a role in improving obesity and promoting fat loss by enhancing fatty acid oxidation and mitochondrial biogenesis through promoting thermogenesis and energy consumption in adipose tissue. The upregulation of these genes further supports the positive effect of Res S on fat browning, metabolic regulation, and energy balance. In addition, the beige cell-specific markers (Tmem26, Cd137, Tbxl) in the Res S treatment group ( Figure 4The mRNA expression of G) was significantly upregulated. It was thus concluded that Res S promoted the transformation of inguinal white adipose tissue (IngWAT) into beige adipocytes in high-fat diet-induced mice. Beige adipocytes have a thermogenic function similar to brown fat and can increase energy consumption. Tmem26, Cd137, and Tbxl are specific markers of beige adipocytes, and their significant upregulation indicates that ResS enhances the thermogenic and metabolic capabilities of adipose tissue by promoting the browning process of white fat (i.e., the transformation of white fat into beige fat). This further supports the potential role of Res S in improving energy metabolism, obesity, and metabolic health.

[0097] Wash the IngWAT, cut it into small pieces, and place them in a Seahorse XF microplate. Add serum-free medium and equilibrate the tissue pieces for 30 - 60 minutes. Calibrate the sensor probe and load it onto the microplate. Prepare Oligomycin (purchased from Sigma-Aldrich), FCCP (purchased from Sigma-Aldrich), Rotenone (purchased from Sigma-Aldrich), and Antimycin A (purchased from Sigma-Aldrich) reagents and load them into the injection ports. Load the microplate into the analyzer, set the experimental protocol, and start it to monitor the oxygen consumption rate (OCR) in real time.

[0098] The results were as Figure 4 shown in

[0099] During HF feeding, the OCR of the isolated IngWAT of Res S-treated mice increased significantly.

[0100] These results indicate that Res S treatment induced the browning of IngWAT under HF diet conditions, which may complement the activation of BAT and jointly limit weight gain.

[0101] In summary, the existing browning of WAT (i.e., the transformation of white adipocytes into brown adipocytes with thermogenic function) is a potential weight loss pathway discovered in recent years, but there is little research and application of existing drugs in this regard. In the present invention, it was verified that Res S treatment induced the browning of inguinal white adipose tissue (IngWAT) in high-fat diet mice. The above experimental results showed that the abundance of UCP1 protein in the IngWAT of Res S-treated mice increased, lipid accumulation decreased, and adipocyte size decreased ( Figure 4 in A - D). In addition, the mRNA expression of genes related to thermogenesis, fatty acid oxidation, lipolysis, and mitochondrial biogenesis in the IngWAT of the Res S-treated group increased significantly ( Figure 4 in C - F). These results indicate that Res S further enhanced energy consumption in the body and prevented weight gain by inducing WAT browning.

[0102] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to exhaustively list all implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of the compound Resinacein S as the sole active ingredient in the preparation of a drug for preventing or treating diabetes.

2. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

3. The use according to claim 2, characterized in that: The form of the drug includes liquid preparation, semisolid preparation or solid preparation.

4. The use according to claim 2 or 3, characterized in that The medicine is an oral preparation or an external preparation.

Images