Application of rhododendron dauricum extract in treatment of metabolic dysfunction related fatty liver disease
By using the Monkey Extract, the problem of poor efficacy in the treatment of MASLD was solved in the prior art, and the effect of reducing lipid accumulation and improving liver function was achieved, providing new possibilities for the treatment of MASLD.
Patent Information
- Application Number
- CN202510459776.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-14
- Publication Date
- 2025-06-20
AI Technical Summary
The prior art has poor efficacy in the treatment of metabolic dysfunction-associated fatty liver disease (MASLD) and has limited treatment options.
The marquis extract was used as a drug for treating MASLD, and the marquis extract was obtained through 80% ethanol extraction and verified in HepG2 cells and MASLD mouse models, showing that it can reduce lipid accumulation, increase the GSH content of liver tissue, and reduce the serum TC, TG, AST, ALT and MDA levels.
The extract of the leucorrhea significantly reduces the lipid accumulation of HepG2 cells and MASLD mice, improves liver function, and reduces inflammation indicators, providing a new method to treat MASLD.
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Figure CN120168538A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of rhododendron dauricum extract in the treatment of metabolic dysfunction-associated steatotic liver disease. Background Art
[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic progressive liver disease caused by nutritional excess, insulin resistance, and their related metabolic dysfunctions in genetically susceptible individuals. MASLD includes a series of progressive fatty liver diseases, ranging from isolated hepatic steatosis to metabolic dysfunction-associated steatohepatitis (MASH), with varying degrees of liver fibrosis, which may progress to cirrhosis. In fact, MASLD is not only associated with liver complications, but also closely related to the occurrence of various extrahepatic manifestations such as cardiovascular diseases, chronic kidney diseases, obesity, type 2 diabetes, and certain types of extrahepatic cancers.
[0003] MASLD is caused by the complex interaction of genetic, environmental, and metabolic factors. The key driver of MASLD is metabolic disorder, especially insulin resistance. When insulin signaling is impaired, adipose tissue releases excessive free fatty acids, which accumulate in the liver and lead to hepatic steatosis and lipid metabolism imbalance. Over time, it may even evolve into MASH, which is characterized by increased oxidative stress and lipotoxicity. Obesity further exacerbates this process by promoting the release of inflammatory cytokines and adipokines, which can cause systemic inflammation and liver injury. In addition, dyslipidemia characterized by high triglycerides and low high-density lipoprotein cholesterol plays a key role in hepatic fat accumulation, promoting steatosis and inflammation. Genetic susceptibility also significantly promotes the development of steatosis, MASH, and liver fibrosis. Individuals with a family history of metabolic diseases such as obesity, type 2 diabetes, or liver disease are at higher risk of developing from simple steatosis to more severe stages of liver disease (including MASH and fibrosis).
[0004] Although existing therapies such as thyroid hormone receptor-β (THR-β) agonists, PPAR agonists, FXR agonists, and GLP-1 receptor agonists show good prospects, these drugs have shown varying degrees of efficacy in the treatment of MASLD, but also have risks, and their effectiveness is limited by the complexity of the metabolic, inflammatory, and fibrotic pathways driving the progression of MASLD. Current treatment options are limited and the efficacy is uneven, and there is still a great demand for drugs for the treatment of MASLD clinically.
[0005] Compared with the single target of traditional chemical drugs, traditional Chinese medicine has the characteristics of multi-level, multi-target and overall regulation, and has significant advantages in the treatment of metabolic diseases such as MASLD that affect the whole body. In the clinical trials of MASLD drugs, the active ingredients of traditional Chinese medicine, resveratrol and curcumin, have completed phase III clinical studies, while berberine has been approved to enter phase IV clinical studies, indicating that the active ingredients of traditional Chinese medicine have good prospects for the treatment of MASLD.
[0006] Folium Rhododendri Daurici is the dried leaf of Rhododendron dauricum L. of the genus Rhododendron in the family Ericaceae. It is a traditional Chinese medicine in China. It is cold in nature, bitter and pungent in taste, and has the effects of relieving cough, reducing phlegm, and relieving asthma. Clinically, it is mainly used for the treatment of respiratory diseases such as acute and chronic bronchitis and bronchial asthma. The chemical constituents of Folium Rhododendri Daurici mainly include flavonoids, coumarins, volatile oils and organic acids. Modern pharmacological studies have shown that Folium Rhododendri Daurici has anti-inflammatory, antioxidant, analgesic and anti-cancer effects. However, the mechanism of action of Folium Rhododendri Daurici is not clear, and its therapeutic effect needs to be discussed. Summary of the Invention
[0007] The present invention discovers the application of the extract of Folium Rhododendri Daurici in the treatment of metabolic dysfunction-related fatty liver disease, and is used for preparing a drug for treating metabolic dysfunction-related fatty liver disease.
[0008] Furthermore, the extract of Folium Rhododendri Daurici of the present invention is an 80% ethanol extract.
[0009] Furthermore, the extract of Folium Rhododendri Daurici of the present invention has the effect of reducing lipid accumulation in HepG2 cells.
[0010] Furthermore, the extract of Folium Rhododendri Daurici of the present invention has the effect of reducing lipid accumulation in the liver of MASLD mice.
[0011] Furthermore, the extract of Folium Rhododendri Daurici of the present invention has the effect of increasing the content of GSH in the liver tissue of MASLD mice.
[0012] Furthermore, the extract of Folium Rhododendri Daurici of the present invention has the effect of reducing TC and TG in the serum of MASLD mice.
[0013] Furthermore, the extract of Folium Rhododendri Daurici of the present invention has the effect of reducing AST and ALT in the serum of MASLD mice.
[0014] Furthermore, the extract of Folium Rhododendri Daurici of the present invention has the effect of reducing the level of MDA in the serum of MASLD mice. Brief Description of the Drawings
[0015] Figure 1 It is the Oil Red O staining map of lipid droplets in HepG2 cells after the treatment of Example 3;
[0016] Figure 2 It is a measurement chart of the contents of TC and TG in HepG2 cells after the treatment in Example 4;
[0017] Figure 3 It is an Oil Red O staining chart of lipid droplets in the liver of mice after the treatment in Example 6;
[0018] Figure 4 It is a measurement chart of the contents of TC and TG in the liver of mice after the treatment in Example 7;
[0019] Figure 5 It is a measurement chart of the content of GSH in the liver of mice after the treatment in Example 7.
[0020] Figure 6 It is a measurement chart of the contents of TC and TG in the serum of mice after the treatment in Example 8;
[0021] Figure 7 It is a measurement chart of the contents of AST and ALT in the serum of mice after the treatment in Example 8;
[0022] Figure 8 It is a measurement chart of the content of MDA in the serum of mice after the treatment in Example 8;
[0023] # It indicates that compared with the normal group, # p < 0.05, ## p < 0.01; * It indicates that compared with the model group, * p < 0.05, ** p < 0.01. Abbreviations: TC: total cholesterol; TG: triglyceride; AST: aspartate aminotransferase; ALT: alanine aminotransferase; MDA: malondialdehyde; GSH: reduced glutathione. Detailed implementation manners
[0024] The present invention is further described by the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change that is easily achieved by those of ordinary skill in the art will fall within the scope of the claims of the present invention.
[0025] Materials: Rhododendron dauricum was purchased from Jilin, Oil Red O powder was purchased from Sigma-Aldrich, product number: 215-295-3; SPF male C57BL / 6 mice, weighing 20±2 g, were provided by Liaoning Changsheng Biotechnology Co., Ltd., and the experimental animal production license number was SCXK2010-0001; the high-fat diet was purchased from Dietz Biotechnology, product number: HF60; the measurement kits for TG, TC, LDL-C, HDL-C, ALT, and AST were purchased from Nanjing Jiancheng Bioengineering Institute; the MDA content detection kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: BC0025; the GSH content detection kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: BC1175.
[0026] Example 1: Preparation of Rhododendron dauricum extract (TFRE)
[0027] ① Extraction of active ingredients: The dried leaves of Rhododendron dauricum were ground and passed through a 65-mesh sieve, soaked in 80% ethanol at a solid-liquid ratio of 1:25 (g / mL) for 12 h, and sonicated at 80 W for 1 h at room temperature to extract the active ingredients of Rhododendron dauricum. The supernatant was collected, filtered through a 0.22-μm filter, concentrated by rotary evaporation and then dried, and the concentrated solution was evaporated to remove excess water and then dried, ground into powder, and the obtained Rhododendron dauricum extract was stored in a dry and cool place.
[0028] ② Preparation of formulations for use: The Rhododendron dauricum extract powder was dissolved in DMSO to obtain a 50-mg / mL TFRE solution and dissolved in normal saline to obtain a 100-mg / mL TFRE solution, which were used for cell and animal medications, respectively.
[0029] Example 2: HepG2 cell culture
[0030] ① Preparation of culture medium: DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 4.5 g / L glucose;
[0031] ② Cell seeding: HepG2 cells were seeded in 6-well plates at a density of 1×10 6 cells / well and placed in an incubator at 37°C containing 5% carbon dioxide for subsequent experiments;
[0032] ③ Starvation of cells: After culturing the cells for 12 h, the culture medium was replaced with DMEM medium containing only 4.5 g / L glucose to starve the cells for 3 h;
[0033] ④ Experimental grouping: Dilute TFRE dissolved in DMSO with PBS to a TFRE solution with a concentration of 1 mg / mL. Incubate the cells with the 25 μg / mL TFRE solution for 1 h, and then add oleic acid (OA) (660 μΜ) and palmitic acid (PA) (330 μM), and incubate for a total of 24 h. Divide into a blank group (cultured with DMEM), a model group (OA (660 μΜ) + PA (330 μM)), a treatment group (25 μg / mL TFRE + OA (660 μΜ) + PA (330 μM)), and a drug control group (25 μg / mL TFRE).
[0034] Example 3: Oil Red O staining of HepG2 cells
[0035] Discard the culture medium and wash the cells 2 times with PBS; Fix the cells with 4% paraformaldehyde for 30 min, then discard the fixative and wash the cells 2 times with PBS; Infiltrate with 0.1% Triton-X for 10 min to perforate the cells; Wash the cells 2 times with PBS, infiltrate the cells with 60% isopropanol for 1 - 2 s, and then stain with a 0.5% Oil Red O staining solution prepared with 60% isopropanol for 30 min; Discard the staining solution and wash the cells multiple times with PBS until the Oil Red O residue is washed away; Stain the cell nuclei with hematoxylin for 2 min, then discard the staining solution and wash the cells 3 times with PBS; Add ammonia water for 1 min, discard it, and add PBS for use; After sealing with glycerol, observe the lipid droplet situation under an optical microscope at a magnification of 400 times.
[0036] The experimental results are shown in the appendix Figure 1 . The Oil Red O staining results showed that OA (660 μΜ) + PA (330 μM) could significantly increase the formation of lipid droplets in HepG2 cells; while the intervention of TFRE could significantly reduce the lipid droplet accumulation induced by OA (660 μΜ) + PA (330 μM).
[0037] Example 4: Determination of TC and TG contents in HepG2 cells
[0038] Collect cells: Use a cell scraper to collect the cells into a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and obtain cell pellets;
[0039] Cell lysis: Add 0.2 mL of lysis buffer to each tube, vortex thoroughly, and place on ice for 45 min;
[0040] According to the kit instructions, use an enzyme-linked immunosorbent assay (ELISA) colorimetric method to measure the absorbance, and calculate the TC and TG contents in HepG2 cells of each group.
[0041] The measured TC and TG contents are shown in the appendix Figure 2。The experimental results showed that, compared with the normal group, OA (660 μM) + PA (330 μM) could significantly increase the levels of TC and TG in HepG2 cells (p < 0.01); while the addition of TFRE decreased the levels of TC and TG in HepG2 cells (p < 0.01).
[0042] Example 5: Establishment of a MASLD mouse model
[0043] The mice were housed in an SPF environment at room temperature with a relative humidity of 40% - 80%. Twenty-four SPF-grade male C57BL / 6 mice weighing 20 ± 2 g were randomly divided into groups of 6 mice each: blank group, model group (HFD + 100 mg / mL sugar water), treatment group (HFD + 100 mg / mL sugar water, 100 mg / kg TFRE), and drug control group (100 mg / kg TFRE). Among them, the normal group and the drug control group were fed a maintenance diet, while the model group and the treatment group were fed a high-fat and high-cholesterol diet and high-fructose drinking water starting from the first day of the experiment to establish a MASLD mouse model. From the 9th week of the experiment, the mice in the treatment group and the drug control group were intragastrically administered TFRE, and the mice in the blank group and the model group were intragastrically administered an equal volume of normal saline for 4 consecutive weeks. After 12 weeks of the experiment, blood was collected from the mice in each group, and the liver tissues were collected after the mice were sacrificed.
[0044] Example 6: Oil Red O staining of mouse hepatocytes
[0045] The mouse liver was taken, fixed with 4% paraformaldehyde for 4 h, then transferred to a 200 g / L sucrose-PB solution for dehydration. After 24 h, it was changed to a 300 g / L sucrose-PB solution. After 24 h, the tissue was placed in an embedding agent. The liver tissue sections were left to air-dry naturally at room temperature for 20 min. After the water vapor on the glass slides disappeared, Oil Red O staining as in Example 3 was performed.
[0046] The experimental results are shown in the appendix Figure 3 。High-fat feeding could significantly increase lipid accumulation in the mouse liver. Compared with the model group, TFRE in the treatment group could significantly alleviate the level of lipid accumulation in the liver tissue.
[0047] Example 7: Determination of TC, TG, and GSH contents in mouse liver tissue
[0048] The mouse liver was removed, 900 μL of normal saline was added per 100 mg, and mechanical homogenization was performed. The supernatant was taken for determination of the contents of TC and TG; 900 μL of GSH reagent I was added per 100 mg, and ice-bath homogenization was performed. After centrifugation at 12,000 g for 10 min at 4 °C, the supernatant was taken for determination. The contents of TC, TG, and GSH were detected according to the kit instructions, and the absorbance values of each well were measured using an enzyme-linked immunosorbent assay reader.
[0049] The experimental results are shown in the appendix Figure 4 、 5The results showed that, compared with the normal group, the contents of TC and TG in the liver tissues of mice fed with high-fat diet were significantly increased, while the content of GSH was significantly decreased (p < 0.01). TFRE treatment could significantly down-regulate the contents of TC and TG in the liver tissues of mice and increase the content of GSH (p < 0.01).
[0050] Example 8: Determination of the Contents of TC, TG, AST, ALT and MDA in Mouse Serum
[0051] Blood was collected from the orbital cavities of mice. The collected blood was placed at room temperature. After the serum was separated out, it was centrifuged at 3000 rpm for 5 min, and the upper-layer serum was taken. The contents of various indicators in the mouse serum were detected using the instruction manual for the detection of the contents of TC, TG, AST, ALT and MDA, and the absorbance values of each well were measured using an enzyme-linked immunosorbent assay reader (ELISA reader).
[0052] The experimental results are shown in Appendix Figure 6 、 7 、8. The experimental results showed that, compared with the normal group, the contents of ALT and MDA in the serum of mice fed with high-fat diet were significantly increased. TFER treatment could significantly reduce the levels of TC, TG, AST, ALT and MDA in the serum of mice (p < 0.01).
[0053] One-way analysis of variance was used to perform multiple comparisons and statistical significance between different groups. Statistical analysis was performed using GraphPad Prism 9.
Claims
1. Application of Rhodiola rosea extract in the preparation of drugs for the treatment of fatty liver disease associated with metabolic dysfunction.
2. The use according to claim 1, characterized in that: The dried leaves of R. mandshurica were ground and passed through a 65-mesh sieve, and then immersed in 80% ethanol at a solid-liquid ratio of 1:25 (g / mL) for 12 hours, and ultrasonically treated at 80W for 1 hour at room temperature to extract the effective ingredients of R. mandshurica. The supernatant was collected, filtered with a 0.22μm filter, concentrated by rotary evaporation and then evaporated to dryness. The concentrate was evaporated to remove excess water and then dried. After grinding, R. mandshurica extract powder was obtained for the preparation of drugs for the treatment of fatty liver disease associated with metabolic dysfunction.
3. The use according to claim 1, characterized in that: The Rhodiola rosea extract has the function of reducing the lipid level in HepG2 cells.
4. The use according to claim 1, characterized in that: The Rhizoma Achyranthis Bidentatae extract has the function of reducing lipid accumulation in the liver of MASLD mice.
5. The use according to claim 1, characterized in that: The Rhizoma Adenophorae extract can increase the level of GSH in the liver of MASLD mice.
6. The use according to claim 1, characterized in that: The Rhodiola rosea extract has the function of reducing the TC and TG contents in the serum of MASLD mice.
7. The use according to claim 1, characterized in that: The Rhizoma Adenophorae extract can reduce the levels of ALT and AST in the serum of MASLD mice.
8. The use according to claim 1, characterized in that: The Rhizoma Adenophorae extract has the function of reducing the MDA content in the serum of MASLD mice.