Application of oridonin in the preparation of agents to improve muscle function
Oridonin A, by promoting myoblast differentiation and improving insulin sensitivity, addresses the problems of skeletal muscle dysfunction and insulin resistance caused by obesity, thereby improving muscle strength and endurance, reducing lipid accumulation, and enhancing muscle mass and insulin sensitivity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INSTITUTE OF CHINESE MATERIA MEDICA CHINA ACADEMY OF CHINESE MEDICAL SCIENCES
- Filing Date
- 2023-07-04
- Publication Date
- 2026-05-26
Smart Images
Figure CN116570586B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the application of oridonin in the preparation of agents that improve muscle function, and belongs to the field of drug development technology. Background Technology
[0002] Wintergreen herb, also known as icegrass, is a perennial herb or subshrub belonging to the genus *Isodon* in the Lamiaceae family. It is a variety of *Isodon spp.*, named for the thin, butterfly-shaped flakes that form on its plant. It is mainly distributed in the Yellow River basin and areas south of it, with Henan Province being the primary production area. The 2020 edition of the *Chinese Pharmacopoeia* records that it tastes bitter and sweet, and is slightly cold in nature; it enters the lung, stomach, and liver meridians; and has the effects of clearing heat and detoxifying, reducing inflammation and relieving pain, and strengthening the stomach and promoting blood circulation. Clinically, it is mainly used to treat sore throat, tonsillitis, snake and insect bites, and esophageal cancer.
[0003] Oridonin (Ori) is a kauriane-type tetracyclic diterpenoid natural organic compound mainly extracted from *Rabdosia rubescens*. Through continuous research, the various pharmacological effects of oridonin have gradually become known, such as anti-tumor, antibacterial and anti-inflammatory, antioxidant, and immune-enhancing effects. Currently, oridonin has been reported to have anti-cancer activity against various tumors, such as mucosal carcinoma, colon cancer, lung cancer, nasopharyngeal carcinoma, gallbladder cancer, and ovarian cancer. However, in terms of medicinal applications, pharmacological studies of oridonin mainly focus on its anti-tumor and antibacterial / anti-inflammatory effects; whether it can improve skeletal muscle dysfunction or enhance insulin sensitivity has not yet been reported. Summary of the Invention
[0004] The purpose of this invention is to provide the application of oridonin in the preparation of agents that improve muscle function, and to provide a new technical approach for effectively solving the problem of medication for the treatment of insulin resistance and skeletal muscle dysfunction.
[0005] To achieve the above objectives, the technical solution for the application of oridonin A in the preparation of agents that improve muscle function in this invention is as follows:
[0006] The application of oridonin in the preparation of agents that improve muscle function, wherein improving muscle function means improving obesity-induced skeletal muscle dysfunction, promoting myoblast differentiation into myotube cells and / or increasing myoblast insulin sensitivity; the promotion of myoblast differentiation into myotube cells includes increasing the expression of MyOD, myogenin and MyHC.
[0007] The beneficial effects of the above technical solution are as follows: This invention demonstrates, through an obesity-induced skeletal muscle dysfunction model, that oridonin can improve skeletal muscle dysfunction and insulin resistance by increasing muscle strength, muscle endurance, muscle mass, and reducing muscle lipid accumulation. Using an in vitro C2C12 myoblast model, it is demonstrated that oridonin can promote myoblast differentiation and growth, and increase the insulin sensitivity of C2C12 myoblasts.
[0008] As a further improvement, the improvement of obesity-induced skeletal muscle dysfunction is to increase muscle strength, muscle endurance, muscle mass and / or reduce muscle lipid accumulation.
[0009] The beneficial effects of the above technical solution are as follows: This invention has demonstrated through experiments that oridonin can increase the gastrocnemius muscle index and improve muscle function in obese mice.
[0010] As a further improvement, the dosage of oridonin A is 25-50 mg / kg.
[0011] The beneficial effects of the above technical solution are as follows: This invention demonstrates through experiments that using the above-mentioned dose of oridonin to intervene in skeletal muscle dysfunction model mice can improve skeletal muscle dysfunction by increasing muscle strength, muscle endurance, muscle mass and reducing muscle lipid accumulation, and also improve insulin sensitivity and improve insulin resistance.
[0012] As a further improvement, the differentiation includes increasing the expression of MyOD, myogenin, and MyHC.
[0013] The beneficial effects of the above technical solution are as follows: Experiments have demonstrated that, compared with the blank control group, oridonin significantly promotes myotube differentiation, increases the expression of MyOD, myogenin, and MyHC proteins in a dose-dependent manner. MyHC is an important myofibrillar protein in skeletal muscle fibers, containing enzyme activity related to muscle contraction speed, and plays a crucial role in maintaining the integrity of muscle cell structure. Myogenin and MyoD participate in regulating myoblast fusion and differentiation, playing an important role in the regeneration of muscle cells and their growth into mature muscle fibers.
[0014] As a further improvement, the myoblasts are C2C12 cells.
[0015] The beneficial effects of the above technical solution are as follows: through the glucose uptake experiment of C2C12 myotube cells, compared with the blank control group, oridonin can significantly promote the uptake rate of 2-NBDG glucose and increase insulin sensitivity. Attached Figure Description
[0016] Figure 1This is a schematic diagram of the results of the gripping force measurement experiment of each group of mice in Example 1 of the present invention;
[0017] Figure 2 This is a schematic diagram of the exhaustive swimming time of mice in each group in Example 2 of the present invention (in the figure: # indicates a significant difference from the RD group, * and && indicate a significant difference from the HFD model group, # indicates P≤0.05, * indicates P≤0.05, && indicates P≤0.01);
[0018] Figure 3 The effect of oridonin A on lipid accumulation in skeletal muscle of HFD mice in Example 4 of this invention;
[0019] Figure 4 The graph shows the results of the glucose tolerance test and insulin tolerance test in Example 5 of this invention;
[0020] Figure 5 The expression levels of Myogenin, MyHC, and MyoD proteins were detected by Western blot in Example 6 of this invention.
[0021] Figure 6 The figure shows the results of the in vitro C2C12 glucose uptake experiment of oridonin in Example 7 of the present invention (in the figure: # and ** indicate significant differences from group 2, # indicates P≤0.05, and ** indicates P≤0.01). Detailed Implementation
[0022] The present invention will now be further described in conjunction with specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. The equipment and raw materials used are all commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.
[0023] In the following embodiments:
[0024] Experimental reagents
[0025] Oridonin A (Beijing Bettercare: purity over 99%); physiological sodium chloride solution (Sichuan Kelun Pharmaceutical Co., Ltd.: National Drug Approval Number H20083400); sodium carboxymethyl cellulose (Shanghai Maclean Biochemical Technology Co., Ltd.); glucose assay kit (Beijing Box Biotechnology Co., Ltd.); recombinant human insulin (Shanghai Yuanye Biotechnology Co., Ltd.).
[0026] Experimental instruments
[0027] Electronic balance, Mettler Toledo Shanghai Electronic Balance; Benchtop centrifuge, Thermo Scientific ST16R; Electric thermostatic water bath, Shanghai Yiheng Scientific Instruments Co., Ltd.; Tecan microplate reader, manufactured by Tecan GmbH, Switzerland; Microscope, manufactured by OLYMPUS Ltd., Japan.
[0028] laboratory animals
[0029] C57BL / 6 mice, SPF grade, male, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. The animal room temperature was 22±1℃, humidity 40–70%, with a 12-hour diurnal cycle, and free access to food and water.
[0030] Experimental grouping and experimental method of the present invention
[0031] In the following embodiments of the present invention, an obesity-induced skeletal muscle dysfunction model was used to study the relationship between oridonin A and skeletal muscle dysfunction. The specific grouping and administration are as follows:
[0032] 1. Grouping and Dosing
[0033] The mice were divided into three groups: a normal diet control group (RD, n=6) and a high-fat diet model (Research DietsD12492, containing 60% fat) mice, which were randomly divided into three groups: a control group (HFD, n=6), a low-dose oridonin treatment group (OL, n=6), and a high-dose oridonin treatment group (OH, n=6). The administration was continued for 5 weeks. Specifically, the groups were: high-fat diet model group, high-dose oridonin intervention group (50 mg / kg, prepared with 0.5% CMC-Na, 0.1 ml / 10 g orally), low-dose oridonin intervention group (25 mg / kg, prepared with 0.5% CMC-Na, 0.1 ml / 10 g orally), and normal diet control group (RD).
[0034] 2. Route of administration: Oral or gavage.
[0035] 3. Administration method and dosage
[0036] The normal diet control group (RD) and the high-fat model control group (HFD) were orally administered the same volume of physiological saline (0.1 ml / 10 g) via gavage. The high-dose intervention group (50 mg / kg, prepared with 0.5% CMC-Na, 0.1 ml / 10 g) and the low-dose intervention group (25 mg / kg, prepared with 0.5% CMC-Na, 0.1 ml / 10 g) were administered orally. All mice were acclimatized for one week and then continuously intervened for 5 weeks.
[0037] Example 1: Relationship between oridonin A and skeletal muscle strength
[0038] 1. Experimental Procedure
[0039] Thirty minutes after weekly administration, the gripping force of mice in each group was measured using an animal gripping force analyzer. The animal gripping force analyzer was provided by Jiangsu Saions Biotechnology Co., Ltd., product model SANS, SA-417. The gripping force measurement experiment was repeated 3 times and recorded continuously for 5 weeks.
[0040] 2. Experimental Results
[0041] The results of the grip strength test are as follows Figure 1 As shown in the figure. The results indicated statistically significant differences in grip strength measurements between the RD group and the HFD group, as well as between the low-dose group, the high-dose group, and the HFD group. Compared to the RD group, all HFD mice exhibited muscle weakness and a significant decrease in grip strength. The grip strength of the mice in the low-dose and high-dose groups was higher than that of the HFD mice. Therefore, oridonin has the effect of improving muscle function and strength, and preventing, alleviating, or treating muscle function and strength decline caused by obesity.
[0042] Example 2: Relationship between oridonin A and skeletal muscle endurance
[0043] 1. Experimental Procedure
[0044] The muscle endurance of mice was determined using a swimming-to-exhaustion test. The experimental method was as follows: Thirty minutes after the last administration of medication to each group of mice, the swimming-to-exhaustion time was measured in parallel. The start time of the swimming-to-exhaustion time was defined as the time the mouse began swimming after entering the water, and the end time was the time corresponding to ten seconds after the mouse's head submerged underwater. The difference between the start and end times was the swimming-to-exhaustion duration. The water temperature was maintained at 20–22℃ throughout the experiment.
[0045] 2. Experimental Results
[0046] The results of the exhaustive swimming experiment are as follows Figure 2 As shown in the figure. The results showed that there were statistically significant differences in the exhaustive swimming test results between the RD and HFD groups, as well as between the low-dose group, high-dose group, and HFD group. Compared with the RD group, the exhaustive swimming time of mice in the HFD group was significantly shorter, while the exhaustive swimming time of mice in the low-dose group and high-dose group was significantly longer than that of mice in the HFD group. Furthermore, the exhaustive swimming time of mice in the high-dose group was longer than that of mice in the low-dose group.
[0047] Example 3: Relationship between oridonin A and skeletal muscle mass
[0048] 1. Experimental Procedure
[0049] Mice were weighed and euthanized. Muscles were then precisely dissected from various parts of the mouse body. Each muscle was rinsed thoroughly with pre-cooled saline solution, and any remaining water was removed. The gastrocnemius muscle (an important and representative type of skeletal muscle that primarily affects motor function, hence its selection as the research subject) was harvested, weighed precisely, and its weight recorded. The ratio of gastrocnemius muscle mass to body weight was then calculated. The method for calculating this ratio is as follows:
[0050] The ratio of gastrocnemius muscle mass to body weight (gastrocnemius index) = mouse gastrocnemius muscle weight (g) / mouse body weight (g) × 100%.
[0051] 2. Experimental Results
[0052] The gastrocnemius muscle weight and the gastrocnemius muscle mass-to-body weight ratio of each group of mice are shown in Table 1. Table 1 shows that compared to the RD group, the wet muscle weight of the HFD group mice was significantly reduced, but the gastrocnemius muscle mass-to-body weight ratio was significantly decreased, which is due to muscle loss caused by obesity. Compared to the HFD group mice, the gastrocnemius muscle mass-to-body weight ratio was larger in both the low-dose and high-dose groups, and the ratio was greater in the high-dose group than in the low-dose group.
[0053] Table 1. Gastrocnemius muscle weight and the ratio of gastrocnemius muscle mass to body weight in each group of mice.
[0054]
[0055] Note: ## is significantly different from the RD group, and * and & are significantly different from the HFD model group (* and & indicate P≤0.05, ## indicates P≤0.01).
[0056] Example 4: Relationship between oridonin A and skeletal muscle lipid accumulation
[0057] 1. Experimental Procedure
[0058] Muscles from various locations in mice were precisely separated from each group. The muscles were rinsed thoroughly with pre-cooled physiological saline, and residual water was removed before harvesting the gastrocnemius muscle. Frozen tissue blocks embedded at -80℃ were removed and transferred to a cryostat for sectioning. The gastrocnemius muscle was then fixed in 4% paraformaldehyde solution (manufacturer: Beyotime Biotechnology Co., Ltd., product model P0099), followed by Oil Red O staining (manufacturer: Beyotime Biotechnology Co., Ltd., product model C0158M). Finally, the muscle cells and muscle fiber bundles of the gastrocnemius muscle were observed and analyzed using CellSens (Olympus, Tokyo, Japan) microscopy software.
[0059] 2. Experimental Results
[0060] The morphology, diameter, and lipid accumulation of the gastrocnemius muscle fibers in each group of mice are as follows: Figure 3 As shown in the results, the gastrocnemius muscle tissue in the RD group and the HFD group had a dense and orderly arrangement of muscle fibers, while the HFD group showed loose and irregular muscle fibers. HFD also induced lipid accumulation in skeletal muscle. Compared with the HFD group, the low-dose group, the high-dose group, and the HFD group showed more orderly and dense muscle fiber arrangement, and the gastrocnemius muscle fibers were fuller. Oil Red O staining showed that lipid accumulation was significantly reduced in the treatment groups. These results indicate that oridonin improves skeletal muscle dysfunction induced by HFD diet in mice and reduces lipid accumulation and myofibril pathological conditions in skeletal muscle caused by obesity.
[0061] Example 5: Oridonin A improves insulin resistance
[0062] 1. Experimental Procedure
[0063] 1.1 Glucose tolerance test
[0064] After fasting for 16 hours, blood was collected from the tail tip of the mice, and the basal blood glucose concentration was measured using a glucometer. Then, the mice were injected intraperitoneally with a glucose solution of 2 mg / kg body weight. Blood was collected from the tail tip of the mice at 15, 30, 60, 90 and 120 minutes later, and the blood glucose concentration was measured using a glucometer.
[0065] 1.2 Insulin Tolerance Test
[0066] After fasting for 6 hours, blood was collected from the tail tip of the mice, and the basal blood glucose concentration was measured using a glucometer. Then, a human insulin solution of 1.0 U / kg body weight was injected intraperitoneally. Blood was collected from the tail tip of the mice at 15, 30, 60, 90 and 120 minutes later, and the blood glucose concentration was measured using a glucometer.
[0067] 2. Experimental Results
[0068] The results of the glucose tolerance test and insulin tolerance test are as follows: Figure 4 As shown in the figure, oridonin A has excellent hypoglycemic and insulin sensitivity-enhancing effects, exhibiting a certain concentration-dependent effect. Treatment with oridonin A improved insulin resistance, significantly reducing serum insulin levels and fasting blood glucose levels compared to the model group. In glucose tolerance experiments, it accelerated glucose clearance; in insulin tolerance experiments, it enhanced insulin sensitivity.
[0069] Example 6: Oridonin promotes the differentiation of C2C12 myoblasts into myotube cells
[0070] 1. Experimental Procedure
[0071] C2C12 myoblasts in good growth condition were subjected to 10 5 Cells were seeded at a density of 10 cells / well in 96-well plates. When the cells reached 70-80% confluence, the control group received cell induction medium supplemented with 2% horse serum, while the treatment group received cell induction medium supplemented with 2% horse serum, along with different concentrations of oridonin (5-20 μM). Cells were cultured for another 48 hours. After 48 hours of culture, cells were lysed using protein lysis buffer, and the protein lysis buffers from both the control and treatment groups were collected. Western blot analysis was then performed to detect C2C12 cell differentiation regulators (MyoD, Myogenin, MyHC) to assess the differentiation efficiency of C2C12 myoblasts into myotube cells.
[0072] 2. Experimental Results
[0073] The results of the Western blot experiment are shown below. Figure 5 As shown in the figure, compared with the blank control group, oridonin significantly promoted myoblast differentiation, and increased the expression of MyOD, myogenin, and MyHC proteins in a dose-dependent manner. MyHC is an important myofibrillar protein in skeletal muscle fibers, containing enzyme activity related to muscle contraction speed and playing a crucial role in maintaining the integrity of muscle cell structure. Myogenin and MyoD participate in regulating myoblast fusion and differentiation, playing an important role in muscle cell regeneration and growth into mature muscle fibers. Oridonin exhibits good pharmacological activity in promoting myoblast differentiation and growth.
[0074] Example 7: Oridonin increases insulin sensitivity in C2C12 myotube cells
[0075] 1. Experimental Procedure
[0076] C2C12 myoblasts in good growth condition were subjected to 10 5 Cells were seeded at a density of 10 cells / well into 96-well plates. When the cells reached 70-80% confluence, the medium was replaced with cell induction medium containing 2% horse serum. The cells were cultured for another 4-5 days (changing the medium every 2 days). Once the cells differentiated into myotube cells, they were used for experiments. Control group: serum-free medium was added; treatment group: different concentrations of oridonin A (5-20 μM) were added.
[0077] NBDG glucose uptake assay. Cells were washed three times with Krebs-Ringer's phosphate (KRP) buffer (20 mM HEPES, 137 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, and 2 mM pyruvate; pH 7.4), and then cultured and starved for 3 hours with KRP buffer containing 0.2% BSA. Cells were stimulated with 0.1 μM insulin for 30 minutes, washed three times with KRP buffer, and then incubated with 100 μM 2-NBDG (a fluorescent analog of glucose widely used in studies of cellular glucose uptake and metabolism, offering convenient detection and high resolution) to assess glucose uptake capacity. After incubation for 30 minutes and three washes, 2-NBDG uptake was measured at excitation and emission wavelengths of 475 nm and 550 nm, respectively.
[0078] 2. Experimental Results
[0079] See results Figure 6 As shown in the figure, oridonin can improve the sensitivity of cells to insulin and promote glucose uptake. The effect of oridonin on glucose uptake and insulin sensitivity is dose-dependent, and the maximum uptake rate (132.91±6.67%) is reached at 20 μM.
[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the description and drawings of the present invention shall also be included within the scope of protection of the present invention.
Claims
1. The use of oridonin in myoblast cell culture, characterized in that, The application relates to a method for promoting differentiation of myoblasts into myotubes and improving expression of MyOD, myogenin and MyHC proteins by adding 5-20 muM of oridonin, wherein the myoblasts are C2C12 cells.