Application of compound Resinacein S or derivative thereof in preparation of medicine for preventing or treating obesity or related metabolic complications

Abstract

The invention relates to the technical field of medicinal chemistry, in particular to application of a compound Resinacein S or a derivative thereof in preparation of a medicine for preventing or treating obesity or related metabolic complications. The invention also provides an application of the compound Resinacein S or the derivative thereof in preparation of medicines for preventing or treating obesity or related metabolic complications. Researches find that the compound Resinacein S has the effect of preventing and treating obesity, the action mechanism is that fat accumulation is reduced by increasing energy metabolism of the whole body, the effects of reducing body weight and preventing and treating obesity are achieved, and compared with a traditional weight-reducing medicine for reducing body weight by inhibiting appetite, the compound Resinacein S does not prevent and treat obesity by interfering with appetite, and the effect is obvious. And side effects such as gastrointestinal discomfort and cardiovascular risk caused by appetite intervention are avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to use of a compound, Resinacein S, or a derivative thereof in preparing a drug for preventing or treating obesity or related metabolic complications. Background Art

[0002] Obesity is the result of imbalanced energy metabolism, that is, when energy intake exceeds energy expenditure, the remaining energy is almost entirely converted into triglycerides and stored in white adipose tissue (WAT) throughout the body. Excessive food intake and reduced physical activity and exercise lead to an imbalance in energy metabolism, and obesity has become an unavoidable problem for humans.

[0003] Studies have shown that adipose tissue plays a central role in the occurrence and development of obesity and is essential for metabolic health. It is currently 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 inside white fat and has the characteristics of brown fat. It can be regarded as a transitional form between white fat and brown fat. The phenomenon of beige fat in white adipose tissue is called browning of white fat. Although beige fat and brown fat are located in different parts and have different developmental origins, they both have similar heat-generating functions and energy consumption potentials, 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; when dietary intake occurs, the activation of thermogenic adipose tissue helps to alleviate transient energy peaks, thereby maintaining metabolic stability.

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

[0005] The reason why thermogenic adipose tissue can convert chemical energy stored in fatty acids into heat energy is because it has a special molecule, namely uncoupling protein 1 (UCP1). UCP1 is located on the inner membrane of mitochondria. When activated, it 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. Under this special tissue localization, UCP1 is not only a functional molecule of thermogenic adipose tissue, but also a phenotypic marker. In addition to UCP1, molecules such as PGC1 and PRDM16 are also important factors involved in the browning process.

[0006] Ganoderma triterpenes are a class of active compounds extracted from Ganoderma lucidum. They belong to the polyterpenoid class and are a traditional medicinal fungus widely used in Asia. Ganoderma triterpenes are considered to be one of the most active components of Ganoderma lucidum and have multiple benefits for human health. Their physiological activities are very extensive, including antioxidant, anti-inflammatory, anti-tumor, hypoglycemic, and cardiovascular and cerebrovascular protection. Studies have shown that these compounds can exert their effects through multiple mechanisms, such as inhibiting key enzymes in inflammatory responses, increasing the activity of antioxidant enzymes, inhibiting the proliferation of tumor cells, and inducing their apoptosis. However, there have been no reports on the use of Ganoderma triterpenoid compound Resinacein S and its analogs in the preparation of preparations 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 a use of the compound Resinacein S or its derivatives in the preparation of a drug for preventing or treating obesity or related metabolic complications.

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

[0009] Use of the compound Resinacein S or its derivatives in preparing medicines for preventing or treating obesity or related metabolic complications.

[0010] Optionally, the obesity includes increased body weight and / or increased fat mass.

[0011] Use of the compound Resinacein S or its derivatives in preparing a medicine for preventing or treating glucose metabolism disorders.

[0012] Optionally, the carbohydrate metabolism disorder includes diabetes and / or improving insulin resistance.

[0013] Optionally, the derivatives of the compound include cis-trans isomers, diastereomers, geometric isomers, racemates, solvates, pharmaceutically acceptable salts or prodrugs thereof.

[0014] A pharmaceutical preparation for preventing and treating obesity or glucose metabolism disorder comprises the compound Resinacein S or its derivatives, and pharmaceutically acceptable excipients.

[0015] Optionally, the pharmaceutical preparation is in the form of a liquid preparation, a semisolid preparation or a solid preparation.

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

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

[0018] Optionally, the auxiliary materials include any one or a combination of at least two of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, and colorants.

[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 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, thereby reducing body weight and preventing and treating 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 intervening on appetite, thus avoiding the side effects caused by appetite intervention such as gastrointestinal discomfort, cardiovascular risks, etc.

[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; further research by the present invention found 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, which generally only have significant effects 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 implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The present invention 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; Figure A is a weight change curve of each group of mice in eight weeks; Figure B is a weight graph of each group of mice in the eighth week; Figure C is a fat mass graph of each group of mice in the 7th week; Figure D is a 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 daytime 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 their 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 2Graph showing the results of improving systemic glucose homeostasis of obese mice by Res S treatment in Example 2 of the present invention; Graph A shows fasting blood glucose of each group of mice; Graph B shows fasting insulin of each group of mice; Graph C shows glucose tolerance of each group of mice; Graph D shows a bar graph showing the area under the curve of the glucose tolerance test drawn from the glucose tolerance test shown in Graph C; Graph E shows the insulin tolerance of each group of mice; Graph F shows a bar graph showing the area under the curve of the insulin tolerance test shown in Graph E;

[0025] Figure 3 is Res in Example 3 of the present invention S treatment promoted the thermogenic activity of BAT in HF diet-induced obese mice; Figure A shows the HE staining of BAT adipose tissue of each group of mice; Figure B shows the quantitative results of lipid content in BAT adipose tissue in Figure A; Figure C shows the immunohistochemical staining of UCP1 in each group of mice; Figure D shows the quantitative results 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 number of mitochondrial DNA copies in each group of mice;

[0026] Figure 4 The results of the formation of brown adipocytes in the IngWAT of mice fed a HF diet induced by Res S treatment in Example 4 of the present invention; Figure A is HE staining of the IngWAT adipose tissue of each group of mice; Figure B is the quantitative result of the lipid content in the IngWAT adipose tissue of Figure A; Figure C is the immunohistochemical staining of UCP1 in the IngWAT of each group of mice; Figure D is the quantitative result of the abundance of UCP1 protein in Figure C; Figure E is the quantitative result of thermogenesis (Ucp1, Pgclα, Fgf21), fatty acid oxidation in each group of mice Figure 2 shows the mRNA expression levels of genes related to mitochondrial biogenesis (Ppara, Cptlα, Elovl3) and lipolysis (Atg1 and Hsl) in each group of mice; Figure 3 shows the mRNA expression levels of genes related to mitochondrial biogenesis (Nrf1, Nrf2, Tfam1) in each group of mice; Figure 4 shows the mRNA expression levels of genes related to beige cell-specific markers (Tmem26, Cd137, Tbxl) in each group of mice; Figure 5 shows the results of OCR of isolated IngWAT in each group of mice. DETAILED DESCRIPTION

[0027] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to 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 other prior arts shall fall within the protection scope of the present invention.

[0028] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

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

[0030]

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

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

[0033] Research reports show that the occurrence of obesity and metabolic diseases is the result of the combined effects of living environment and genetic factors. Obese mice induced by a high-fat diet are commonly used animal models in laboratories to study the occurrence and development mechanisms of obesity and metabolic diseases. Therefore, this embodiment uses obese mice induced by a high-fat diet to conduct experiments.

[0034] 1. Experimental methods and grouping

[0035] Five-week-old C57BL / 6 male mice (purchased from Beijing Weitong Lihua Company) were taken and acclimatized for one week. They were then 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 feed group (NC): Rats were fed with a normal feed and injected intraperitoneally with corn oil once every two days, with the same injection volume as the volume of Res S solution injected once in the NCR group or HFR group;

[0037] Normal diet plus Res S group (NCR): The mice were routinely fed with normal diet and injected intraperitoneally with Res S (4 mg / kg mouse body weight, solvent: corn oil) solution once every two days;

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

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

[0040] Each group was treated with the above treatment for eight weeks, and the body weight of mice was recorded every week.

[0041] In the seventh week, the body composition of each mouse was first analyzed using the MesoQMR 23-060H-I system (Newmai, China) to measure its fat and lean body mass. Then, the mice were placed in the TSE laboratory main system to continuously monitor their metabolic activity and record the mice's oxygen consumption (VO2), carbon dioxide production (VCO2), and food intake. The dynamic activity of each mouse was monitored during a 12-hour light and dark cycle using Opto-M3 beam technology (Columbus, USA), and the activity frequency and intensity were recorded. Based on 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 body weight, fat percentage, RER, EE and other indicators among the groups of mice. If significant, multiple comparison tests were performed. Paired t-tests were 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, thereby comprehensively evaluating the effects of Res S on mouse metabolism.

[0043] 3. Experimental results

[0044] The results are as follows Figure 1 As shown, Res S treatment significantly inhibited the weight gain of mice induced by HF diet ( Figure 1 Further body composition analysis showed that the weight loss in HF diet mice was generally attributed to the reduction of fat mass by Res S ( Figure 1 However, Res S treatment did not significantly change the weight gain and lean body mass in mice fed the NC diet. Res S treatment significantly increased the energy expenditure of mice fed the HF diet ( Figure 1 I and J) and oxygen intake ( Figure 1 E to G in the middle), but did not affect physical activity and food intake ( Figure 1Meanwhile, Res S-treated mice showed a lower respiratory exchange ratio (RER), indicating a higher reliance on fat-mediated oxidation ( Figure 1 (Fig. H).

[0045] 4. Experimental Conclusion

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

[0047] Example 2 Res S treatment improves systemic glucose homeostasis in obese mice

[0048] A large number of studies have shown that obesity is often accompanied by severe insulin resistance. Therefore, the obese mouse model was constructed and grouped according to "1. Experimental methods and grouping" in Example 1. The following experiments were performed on mice that were administered for 6-7 weeks:

[0049] 1.1 IPGTT:

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

[0051] Data processing: The blood glucose concentration at each time point was recorded, and the area under the curve (AUC) was calculated to evaluate the glucose clearance efficiency. Then, one-way analysis of variance (ANOVA) was used to compare the AUC differences among the groups, and finally the blood glucose change trend and AUC value were displayed in a graph to evaluate the effect of different treatments on glucose metabolism. And the area under the curve (AUC) was calculated to evaluate the glucose clearance efficiency. AUC is integrated by the blood glucose level at the time point, and the smaller the AUC value, the faster the glucose metabolism. Fasting blood glucose levels were compared to evaluate the effect of Res S on blood glucose control, and one-way analysis of variance (ANOVA) or t-test was used to compare the differences in AUC and fasting blood glucose levels among the groups. The results were visualized by line graphs and bar graphs to intuitively show the improvement of Res S on glucose metabolism.

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

[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 shows the Figure 2 The bar graph of the area under the curve of the glucose tolerance test drawn in Figure C shows that the area under the curve of the Res S group is significantly smaller than that of the control group (HF group), indicating that Res S can significantly improve the clearance of blood glucose.

[0054] 1.2. Insulin tolerance test (ITT):

[0055] The mice after 7 weeks of drug administration were fasted for 4 hours, weighed and injected intraperitoneally with 0.7 IU / kg body weight of insulin. After injection, blood glucose levels were measured with a blood glucose meter at 0 min, 15 min, 30 min, 60 min, 90 min and 120 min.

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

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

[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 the insulin of obese mice; Figure 2 Figure F shows the Figure 2 The bar graph of the area under the curve of the insulin tolerance test drawn in Figure E shows that the area under the curve of the Res S group is 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 glucose tolerance and insulin sensitivity in obese mice. Specifically, the fasting blood glucose and fasting insulin levels of mice in the Res S treatment group were significantly reduced, and the results of the glucose tolerance test (IPGTT) and insulin tolerance test (ITT) showed that Res S significantly increased glucose clearance and insulin sensitivity ( Figure 2AF). This shows that Res S can not only prevent and treat obesity, but also effectively improve glucose metabolism disorders (insulin resistance) caused by obesity, and has a dual therapeutic effect. Compared with many existing anti-obesity drugs that can reduce weight but have limited effects on improving metabolic diseases such as insulin resistance and diabetes, the 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 promotes the thermogenic activity of BAT in HF diet-induced obese mice

[0061] The obese mouse model and grouping were constructed according to "1. Experimental methods and grouping" in Example 1. The mice were killed by cervical dislocation after 8 weeks of administration, and the brown fat (BAT, classic brown fat) in the interscapular region of the mice was removed by dissection, weighed and fixed, and Wuhan Sewell Company was commissioned to perform histological analysis of the adipose tissue, including fixation, paraffin embedding, sectioning, HE staining and immunohistochemical staining of UCP1, and three representative adipose tissues were taken out for analysis.

[0062] Data processing: Brown adipose (BAT) tissue was sliced ​​by HE staining, and the size and lipid content of adipocytes were measured using image analysis software, and the differences between the Res S-treated group and the control group were quantitatively analyzed. 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 the groups. Finally, the results were presented in a bar graph to evaluate the effects of Res S on BAT metabolic activity and UCP1 protein expression.

[0063] The results are as follows Figure 3 As shown in Figures A to D:

[0064] Under HF diet conditions, the lipid content of BAT in Res S-treated mice was significantly reduced, and the size of adipocytes was significantly reduced ( Figure 3 Figures A and B in the figure), indicating that the metabolic activity of fat was enhanced. In addition, immunohistochemistry revealed that the abundance of UCP1 protein in BAT of mice treated with Res S was significantly upregulated ( Figure 3Figures C and D in the middle further verified that the activity of BAT was enhanced. The enhanced activity of brown fat cells indicated that their metabolic activity was improved, which was specifically manifested by a decrease in lipid content in fat cells, a decrease in fat cell size, and an upregulation of UCP1 protein expression. This means that brown fat accelerates energy consumption through thermogenesis (non-shivering thermogenesis), converting more fat into heat energy through oxidative metabolism, thereby promoting the reduction of body fat. Therefore, the experiment can conclude that Res S plays a role in weight loss by activating brown fat cells and increasing energy consumption.

[0065] RNA extraction:

[0066] Take appropriate amount of BAT samples from each group, add 1ml Trizol to fully lyse, mix thoroughly, add 200ul of chloroform, shake vigorously, let stand at room temperature for 15min, centrifuge at 4℃, the centrifugation condition is 12000g, 10min. At this time, due to the chromatography, the lysate is divided into three layers. Carefully aspirate the upper aqueous phase solution and transfer it to a new centrifuge tube, add 500ul of isopropanol, mix gently and let stand at room temperature for 10 minutes, centrifuge at 4℃, 12000g, 10min, discard the supernatant, and a milky white translucent precipitate can be seen at the bottom of the tube. Wash the precipitate three times with 75% ethanol diluted with DEPC water, centrifuge at 4℃, 7500g, 5min, discard the supernatant, dry at room temperature, dissolve the precipitate with 60μl DEPC water, and use NanoDrop2000 ultramicro spectrophotometer to detect the concentration of RNA solution.

[0067] Reverse transcription of cDNA:

[0068] Reverse transcribe it using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas K1622) according to the manufacturer: melt the total RNA solution and each reagent in the reverse transcription kit, mix well and 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 gentle mixing, centrifuge in a microcentrifuge for 30 seconds.

[0073] A common PCR instrument was used to react at 42°C for 15 min and heated at 85°C for 5 s. After the reaction was completed, cDNA was obtained and the reaction product cDNA was taken out and placed 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: Primers were designed using the online primer design website “NCBIPrimer-BLAST” based on the gene sequence provided by NCBI. The primers are listed in Table 3. The designed primers were synthesized by Bomed Co., Ltd.

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

[0077] Table 2 shows the reagent components and dosages used in RT-PCR reactions

[0078] Components volume cDNA 1.5μL Upstream primer (10 μmol / L) 1μL Downstream primer (10 μmol / L) 1μL 2×TransTaq HiFi PCR SuperMixⅡ 10μL ddH2O 6.5μL Total volume 20μL

[0079] Table 3. Primers

[0080]

[0081]

[0082] After gentle mixing, centrifuge in a microcentrifuge for 30 seconds.

[0083] Place a 96-well plate dedicated to PCR in a PCR instrument and perform 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); the amplification reaction is performed for a total of 40 cycles; finally, 72°C for 7 min.

[0084] The relative expression levels of mRNA of thermogenesis, fat-related genes, and mitochondrial biogenesis factor-related genes in brown fat were calculated.

[0085] The results are as follows Figure 3 EI shows:

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

[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 BAT lipid content of mice treated with Res S was reduced, the size of adipocytes was reduced, and the abundance of UCP1 protein was significantly upregulated ( Figure 3 In addition, the mRNA expression of genes related to thermogenesis and fat metabolism in BAT of the Res S-treated group was significantly increased ( Figure 3 These results suggest that Res S increases energy expenditure in vivo and further inhibits fat accumulation by activating BAT.

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

[0089] The obese mouse model and grouping were constructed according to "1. Experimental methods and grouping" in Example 1. The mice were killed by cervical dislocation after 8 weeks of administration, and the inguinal fat (IngWAT, subcutaneous fat) of the mice was dissected out, weighed and fixed, and Wuhan Sewell Company was commissioned to perform histological analysis of the adipose tissue, including fixation, paraffin embedding, sectioning, HE staining and immunohistochemical staining of UCP1, and three representative adipose tissues were taken out for analysis.

[0090] Data processing: Inguinal adipose tissue (IngWAT) was sliced ​​by HE staining, and the size and lipid content of adipocytes were measured using image analysis software, and the differences between the Res S-treated group and the control group were quantitatively analyzed. 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 the groups.

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

[0092] Under HF diet conditions, Res S-treated mice showed browning of IngWAT. Compared with the control group, lipid accumulation in IngWAT of the Res S-treated group was reduced, and adipocyte size was reduced.

[0093] Total RNA of IngWAT was extracted and reverse transcribed into cDNA, and the mRNA levels related to thermogenesis, fat-related genes, mitochondrial synthesis, and beige cell markers were detected by real time qPCR. The above operation was performed according to Example 3.

[0094] The above Res S treatment significantly promoted the browning of inguinal white adipose tissue (IngWAT) of mice induced by a high-fat diet (HF). Compared with the control group (HF group), the lipid accumulation in IngWAT of mice in the Res S treatment group was reduced, and the size of adipocytes was significantly reduced, 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 converting white fat into beige adipocytes with thermogenic function, which helps to increase energy consumption and has the potential to lose weight and improve metabolic health.

[0095] The results are as follows Figure 4 As shown in C~F:

[0096] Figure 4 C to F are real-time fluorescence quantitative bar graphs showing the thermogenic effect of Res S on the mRNA expression of fat-related genes and mitochondrial biogenesis factors in IngWAT of high-fat-induced obese mice, among which the abundance of UCP1 protein in IngWAT of mice treated with Res S increased ( Figure 4 C and D), and are involved in thermogenesis (Ucp1, Pgclα, Fgf21), fatty acid oxidation (Ppara, Cptlα, Elovl3), lipolysis (Atg1 and Hsl) ( Figure 4 E) and mitochondrial biogenesis (Nrf1, Nrf2, Tfam1) ( Figure 4 The mRNA expression of genes related to (F) in the middle was significantly increased. It was concluded that this indicates that Res S enhances fatty acid oxidation and mitochondrial generation by promoting thermogenesis and energy consumption of adipose tissue, thereby improving obesity and promoting fat loss by increasing metabolic rate. The upregulation of these genes further supports the positive effects of Res S on fat browning, metabolic regulation and energy balance. In addition, beige cell-specific markers (Tmem26, Cd137, Tbxl) ( Figure 4The mRNA expression of Res S (in G) was significantly upregulated. It was concluded that Res S promoted the transformation of inguinal white adipose tissue (IngWAT) into beige adipocytes induced by a high-fat diet in mice. Beige adipocytes have a thermogenic function similar to that of brown fat and can increase energy expenditure. Tmem26, Cd137 and Tbxl are specific markers of beige adipocytes. Their significant upregulation indicates that Res S enhances the thermogenic and metabolic capacity 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, improving obesity and metabolic health.

[0097] IngWAT was washed, cut into small pieces and placed in a Seahorse XF microplate. Serum-free medium was added and the tissue pieces were equilibrated for 30-60 minutes. The sensor probe was calibrated and loaded onto the microplate, and Oligomycin (purchased from Sigma-Aldrich), FCCP (purchased from Sigma-Aldrich), Rotenone (purchased from Sigma-Aldrich) and Antimycin A (purchased from Sigma-Aldrich) reagents were prepared and loaded into the injection port. The microplate was loaded into the analyzer, and the experimental protocol was set up and started to monitor the oxygen consumption rate (OCR) in real time.

[0098] The results are as follows Figure 4 As shown in H:

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

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

[0101] In summary, the existing WAT browning (i.e., the transformation of white fat cells into brown fat cells with thermogenic function) is a potential way to lose weight discovered in recent years, but there are few studies and applications 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) of mice on a high-fat diet. The above experimental results showed that the abundance of UCP1 protein in IngWAT of mice treated with Res S increased, lipid accumulation decreased, and the size of fat cells decreased ( Figure 4 In addition, the mRNA expression of genes related to thermogenesis, fatty acid oxidation, lipolysis, and mitochondrial biogenesis in IngWAT of the Res S-treated group was significantly increased ( Figure 4 These results suggest that Res S further enhances energy expenditure and prevents body weight gain by inducing WAT browning.

[0102] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

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

2. The use according to claim 1, characterized in that Obesity includes increased body weight and / or increased fat mass.

3. Use of the compound Resinacein S or its derivatives in the preparation of drugs for preventing or treating glucose metabolism disorders.

4. The use according to claim 3, characterized in that The glucose metabolism disorder includes diabetes and / or improving insulin resistance.

5. The use according to any one of claims 1 to 4, characterized in that: The derivatives of the compound include cis-trans isomers, diastereomers, geometric isomers, racemates, solvates, pharmaceutically acceptable salts or prodrugs thereof.

6. A pharmaceutical preparation for preventing and treating obesity or glucose metabolism disorder, characterized in that: The invention comprises the compound Resinacein S or its derivatives as described in any one of claims 1 to 5, and pharmaceutically acceptable excipients.

7. The pharmaceutical preparation according to claim 6, characterized in that The pharmaceutical preparation may be in the form of a liquid preparation, a semisolid preparation or a solid preparation.

8. The pharmaceutical preparation according to claim 6 or 7, characterized in that The pharmaceutical preparation is an oral preparation or an external preparation.

9. The pharmaceutical preparation according to any one of claims 6 to 8, characterized in that The dosage form of the pharmaceutical preparation is powder, powder injection, injection, tablet, capsule, granule, paste, paint, lotion, tincture, liniment, coating, ointment, gel, paste, patch or aerosol.

10. The use according to any one of claims 6 to 9, characterized in that: The auxiliary materials include any one of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, and colorants, or a combination of at least two of them.

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