Application of ganoderic acid A as intestinal farnesic acid X receptor inhibitor in preparation of product for preventing and / or treating hyperlipidemia
By using ganoderic acid A as a naturally derived FXR inhibitor, specific problems that have not been effectively addressed in existing technologies are solved. By using ganoderic acid A as a naturally derived FXR inhibitor, active sites for the prevention and/or treatment of hyperlipidemia are prepared. This solves the problems of significant side effects and high cost of existing drugs for the treatment of hyperlipidemia, and achieves a significant reduction in hyperlipidemia.
Patent Information
- Application Number
- CN202410885536.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-03
- Publication Date
- 2026-01-06
AI Technical Summary
Existing treatments for hyperlipidemia have significant side effects and are expensive. Furthermore, there is a lack of effective enterofenadine X receptor inhibitors in the current technology for the preparation of drugs to prevent and/or treat hyperlipidemia. It is necessary to find new therapeutic targets and develop relevant therapeutic drugs suitable for long-term use.
Using ganoderic acid A as a naturally derived enterofenadine X receptor inhibitor, through binding to FXR inhibitors, ganoderic acid A, as a naturally derived FXR inhibitor, is applied to the active site for the prevention and/or treatment of hyperlipidemia, and is used to prepare products for the prevention and/or treatment of hyperlipidemia.
Ganoderma lucidum acid A significantly reduces lipid disorders induced by a high-fat diet by inhibiting FXR activity, improves hyperlipidemia, and provides a new, natural intestinal FXR inhibitor for the preparation of functional products or drugs. It significantly reduces the protein expression of FXR target genes, reduces lipid absorption, and alleviates hyperlipidemia.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of ganoderic acid A as an enterofarnic acid X receptor inhibitor in the preparation of products for the prevention and / or treatment of hyperlipidemia. Background Technology
[0002] Hyperlipidemia is characterized by elevated levels of total cholesterol and triglycerides in the blood. While it often presents with few clinical symptoms and is easily overlooked, it is a significant risk factor for cardiovascular disease and can induce various metabolic disorders. However, current medications for hyperlipidemia have significant side effects and are expensive. As a chronic condition, hyperlipidemia requires long-term treatment. Therefore, there is an urgent need to identify new therapeutic targets and develop suitable long-term medications.
[0003] Intestinal farnesoid X receptors (FXRs) play a crucial and complex role in regulating energy and metabolic homeostasis, particularly in intestinal FXR signaling, which modulates the enterohepatic bile acid cycle, lipid metabolism, and glucose metabolism. Intestinal FXRs influence bile acid composition, which in turn significantly affects lipid absorption; therefore, intestinal FXRs are closely related to lipid digestion and absorption. Under a high-fat diet, when the amount of bile acids (agonists of endogenous FXRs) in the intestine decreases, lipid absorption decreases, suggesting that reduced intestinal FXR activity may inhibit lipid absorption. Conversely, activation of intestinal FXRs leads to disordered intestinal bile acid reabsorption, which can stimulate excessive lipid absorption and induce hyperlipidemia. Ileal bile acid-binding protein (IBABP), fibroblast growth factor 15 (FGF15; human FGF19), and small heterodimer chaperone (SHP) are target genes of intestinal FXRs, and increased expression of these three factors can indicate FXR activation. Increased expression of IBABP, in particular, promotes lipid absorption and increases the risk of hyperlipidemia. Furthermore, FXR is a bile acid receptor, and bile acids can feedback regulate FXR activity. Tauroursodeoxycholic acid (TUDCA) and glycoursodeoxycholic acid (GUDCA), two conjugated bile acids, can bind to intestinal FXR and inhibit its activity, thereby improving metabolic diseases related to lipid metabolism disorders. In conclusion, inhibiting intestinal FXR activity is an effective approach to the prevention and treatment of hyperlipidemia.
[0004] Ganoderic acid A is a natural product extracted from Ganoderma lucidum (reishi mushroom) and possesses various pharmacological effects, such as anti-inflammatory, antioxidant, and anti-tumor properties. Ganoderic acid A is a tetracyclic triterpenoid compound composed of 30 carbon atoms with a lanane skeleton, structurally similar to the bound bile acid GUDCA, which can inhibit FXR activity. Furthermore, ganoderic acid A reaches its highest concentration in the intestine after oral administration, making it a promising candidate as a novel intestinal FXR inhibitor for targeted treatment of hyperlipidemia.
[0005] The chemical structural formulas of ganoderic acid A(I) and bile acid GUDCA(II) are as follows:
[0006] (I) (II) Summary of the Invention To address the problems existing in the prior art, the present invention provides an application of a naturally derived FXR inhibitor in the prevention and treatment of hyperlipidemia.
[0007] One object of the present invention is to provide the use of ganoderic acid A as a naturally sourced FXR inhibitor, or the use of ganoderic acid A in the preparation of FXR inhibitors.
[0008] Another object of the present invention is to provide the use of ganoderic acid A in the preparation of products for the prevention and / or treatment of hyperlipidemia.
[0009] In the aforementioned application, ganoderic acid A is a product targeting FXR to prevent or treat hyperlipidemia.
[0010] Another object of the present invention is to provide the use of compositions containing ganoderic acid A in the preparation of products for the prevention and / or treatment of hyperlipidemia.
[0011] In the described applications, the composition further comprises pharmaceutically acceptable excipients and / or drugs compatible with ganoderic acid A.
[0012] In this invention, the above-mentioned products may be pharmaceuticals, health products and / or functional foods.
[0013] The drug contains an effective dose of ganoderic acid A. The effective dose is a unit dose (e.g., the content in one tablet) or a unit dose for the treated patient (e.g., a dose per unit body weight). In this invention, the drug is used to treat humans or mammals (e.g., mice).
[0014] The dosage form of the drug described in this invention is not limited, as long as it enables the active ingredient to be effectively delivered into the body, including: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, lozenges, granules, powders, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches, etc.
[0015] This invention regulates lipid absorption by binding ganoderic acid A to FXR and inhibiting its activity and the expression of its target genes, thereby alleviating hyperlipidemia.
[0016] This invention uses Autodock software to analyze and predict the binding capacity of ganoderic acid A, the FXR agonist GW4064, and the enterogenic FXR antagonist glycoursodeoxycholic acid (GUDCA) to FXR. The results show that ganoderic acid A has a high binding energy score to FXR protein, and its binding position is similar to that of GW4064 and GUDCA, with similar binding pockets, and can occupy the active site of FXR.
[0017] This invention utilizes a Caco2 cell model to conduct a thermal drift experiment, detecting the binding effect of ganoderic acid A with FXR protein extracted from Caco2 cells, as well as its direct binding with FXR in living cells. The results showed that ganoderic acid A can significantly inhibit the denaturation and precipitation of FXR protein, suggesting that ganoderic acid A can bind to FXR.
[0018] Specifically, the ganoderic acid A can penetrate the cell membrane and bind to FXR in living Caco2 cells.
[0019] This invention analyzes the binding kinetics of FXR with different concentrations of ganoderic acid A through surface plasmon resonance experiments, showing that the ganoderic acid A described in this invention has a good ability to bind FXR.
[0020] This invention uses surface plasmon resonance experiments to detect the effect of adding ganoderic acid A on the binding of FXR endogenous agonists chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA). The results show that ganoderic acid A can significantly increase the equilibrium dissociation constant (KD value) of CDCA / GCDCA with FXR, suggesting that ganoderic acid A of this invention can competitively inhibit the binding of endogenous FXR agonists with FXR.
[0021] Specifically, the ganoderic acid A has the function of binding FXR and can competitively inhibit the binding of endogenous FXR agonists to FXR.
[0022] This invention utilizes a high-fat diet-induced C57BL6J mouse model and employs immunohistochemistry to detect the protein expression levels of FXR target genes in the ileum tissue of mice in the blank group, high-fat group, and 20 mg / kg ganoderic acid A treatment group. The results show that, compared with the high-fat group, the protein expression levels of FXR target genes in the ileum of mice treated with ganoderic acid A were significantly reduced, indicating that ganoderic acid A inhibits FXR activity in the ileum.
[0023] Specifically, the ganoderic acid A can reduce the protein expression levels of FXR target genes ileal bile acid binding protein (IBABP), fibroblast growth factor 15 (FGF15; human FGF19) and small heterodimer chaperone (SHP).
[0024] This invention measured the blood biochemical indicators of mice in the blank group, high-fat group, and 20 mg / kg ganoderic acid A treatment group. The results showed that ganoderic acid A can significantly reduce the concentration of triglycerides and cholesterol in the serum of mice on a high-fat diet, suggesting that ganoderic acid A can improve lipid disorders induced by a high-fat diet and thus alleviate hyperlipidemia.
[0025] The beneficial effects of this invention are as follows: (1) Ganoderma lucidum acid A has a significant inhibitory effect on FXR activity and can be used as a new, naturally sourced intestinal FXR inhibitor to prepare functional products or drugs for the prevention and treatment of hyperlipidemia.
[0026] (2) Ganoderma lucidum acid A has a significant effect on improving hyperlipidemia induced by a high-fat diet.
[0027] (3) Ganoderma lucidum acid A can reduce lipid absorption by inhibiting intestinal FXR activity and thereby reducing the protein expression of FXR target genes, thus improving dyslipidemia caused by high-fat diet. It can be used to prevent and treat hyperlipidemia in a variety of ways. Attached Figure Description
[0028] Figure 1 This is a schematic diagram of the docking analysis of ganoderic acid A and FXR.
[0029] Figure 2 This is a schematic diagram of the predicted binding site between ganoderic acid A and FXR.
[0030] Figure 3 To analyze the binding interaction between ganoderic acid A and FXR extracted from Caco2 cells using thermal drift analysis.
[0031] Figure 4 To analyze the binding of ganoderic acid A to FXR in live Caco2 cells using thermal drift analysis.
[0032] Figure 5 To analyze the binding of ganoderic acid A and FXR on the surface plasmon resonance.
[0033] Figure 6 This study aimed to competitively inhibit the binding of CDCA / GCDCA to FXR by ganoderic acid A.
[0034] Figure 7 The effect of ganoderic acid A on the expression of FXR protein in mouse ileum tissue.
[0035] Figure 8 The effect of ganoderic acid A on the expression of IBABP protein in mouse ileum tissue.
[0036] Figure 9 The effect of ganoderic acid A on the expression of SHP protein in mouse ileum tissue.
[0037] Figure 10 The effect of ganoderic acid A on the expression of FGF15 protein in mouse ileum tissue.
[0038] Figure 11 The effect of ganoderic acid A on the concentrations of triglycerides and cholesterol in the blood of mice. Detailed Implementation
[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0041] Example 1: The effect of ganoderic acid A binding with FXR Molecular docking analysis: Autodock software was used to analyze and predict the binding energy and binding conformation of GW4064 (FXR agonist), glycoursodeoxycholic acid (GUDCA, enterogenic FXR antagonist), and ganoderic acid A with FXR.
[0042] Experimental results: see Figure 1-2 The binding energies of GW4064 and GUDCA to FXR proteins were -13.1 and -9.15, respectively. The binding energy of ganoderic acid A to FXR was -8.6. The binding site of ganoderic acid A is similar to that of GW4064 and GUDCA, exhibiting a similar binding pocket. The possible binding site of ganoderic acid A to FXR was also predicted, showing that ganoderic acid A forms a hydrogen bond with the TYR (tyrosine) 369 amino acid residue of FXR, with a hydrogen bond length of 2.4 Å. These results suggest that ganoderic acid A can bind to FXR and, like GUDCA, can occupy the active site of FXR. The possible binding amino acid site for ganoderic acid A to FXR is TYR369.
[0043] Thermal drift assay: First, total protein was extracted from Caco2 cells. The cell protein was incubated with ganoderic acid A at room temperature for 30 min, and then heated at 37 ℃, 38.5 ℃, 41.3 ℃, 45.7 ℃, 51 ℃, 55.1 ℃, 58.1 ℃, and 60 ℃ respectively to obtain the binding products. Western blot analysis was performed, with DMSO as a control. The binding effect of ganoderic acid A on FXR protein extracted from Caco2 cells was examined. Furthermore, the ability of ganoderic acid A to penetrate the cell membrane and bind to FXR in live Caco2 cells was investigated. Caco2 cells were suspended and incubated with ganoderic acid A (100 μM) for 2 h. Total protein was then extracted from Caco2 cells, and the binding products were heated at 37℃, 38.5℃, 41.3℃, 45.7℃, 51℃, 55.1℃, 58.1℃, and 60℃. The results were then detected by Western blot, with DMSO used as a control.
[0044] Experimental results: see Figure 3-4 It can be seen that ganoderic acid A significantly inhibited the denaturation and precipitation of FXR protein extracted from Caco2 cells at 55.1 ℃, suggesting that ganoderic acid A can bind to FXR. Simultaneously, ganoderic acid A inhibited the denaturation and precipitation of FXR protein in living Caco2 cells at 45.7-58.1 ℃, suggesting that ganoderic acid A can penetrate the cell membrane and bind to FXR in living Caco2 cells. Figure 3-4 ).
[0045] Example 2: Ganoderic acid A can competitively inhibit the binding of endogenous FXR agonists to FXR. Surface plasmon resonance (SPR) experiment: Human recombinant FXR protein was immobilized on a CM5 sensor chip. To analyze binding kinetics, different concentrations of ganoderic acid A diluted in HBS-EP buffer were injected into the FXR sensor chip.
[0046] Experimental results: see Figure 5 The experimental KD value was 6.28 e -5 This indicates that ganoderic acid A can bind to FXR, and the binding ability is good.
[0047] In another SPR experiment, we first measured the KD values of the endogenous FXR agonists chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA) with FXR. Then, we allowed ganoderic acid A to fully bind with FXR before adding either CDCA or GCDCA, and examined the effect of adding ganoderic acid A on the KD values of CDCA / GCDCA with FXR.
[0048] Experimental results: see Figure 6Ganoderic acid A can significantly increase the KD values of CDCA / GCDCA and FXR, with the KD value of CDCA increasing from 1.28 e -5 Increased to 2.74 e -4 The KD value of GCDCA is 8.87 e -6 Increased to 1.59 e -4 The above demonstrates that ganoderic acid A can competitively inhibit the binding of endogenous FXR agonists to FXR.
[0049] Example 3: Ganoderma lucidum acid A inhibits the activity of FXR in the midgut of mice with high-fat diet-induced hyperlipidemia. Experimental grouping and administration: Eighteen 8-week-old C57BL / 6J mice were acclimatized for one week and then randomly divided into three groups of six each: a control group, a high-fat group, and a high-fat + ganoderic acid A group. The control group was fed a normal diet, while the high-fat and high-fat + ganoderic acid A groups were fed a high-fat diet. The normal and model groups were administered 0.5% sodium carboxymethyl cellulose saline solution by gavage, and the ganoderic acid A group was administered ganoderic acid A (20 mg / kg, dissolved in 0.5% sodium carboxymethyl cellulose saline solution) by gavage once daily for 16 weeks. The normal and high-fat diets were purchased from Beijing Keao Xieli Feed Co., Ltd.
[0050] Immunohistochemical detection of protein expression: The following steps were performed: (1) Dewaxing paraffin sections to water; (2) Antigen retrieval; (3) Blocking endogenous peroxidase; (4) Serum blocking; (5) Primary antibody incubation; (6) Secondary antibody incubation; (7) DAB staining; (8) Counterstaining cell nuclei; (9) Dehydration and mounting; (10) Microscopic examination and image acquisition and analysis.
[0051] Experimental results: see Figure 7-10 Ganoderic acid A did not significantly alter the expression level of FXR protein in mouse ileum tissue. However, ganoderic acid A reduced the expression of downstream genes of FXR in the ileum. Ibabp , Shp and Fgf15 The expression of ganoderic acid A at the protein level indicates that ganoderic acid A inhibits FXR activity in the ileum.
[0052] Example 4: The effect of ganoderic acid A in alleviating hyperlipidemia induced by a high-fat diet Detection of triglyceride, cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol content: After the growth index experiment in Example 3 was completed, mice were fasted for 12 h, blood was collected, centrifuged at 3000 r / min for 10 min, serum was separated for testing, and the content of triglycerides, cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in serum was measured.
[0053] Experimental results: see Figure 11Ganoderma lucidum acid A significantly reduced the elevated levels of triglycerides, cholesterol, and LDL cholesterol caused by a high-fat diet, while increasing HDL cholesterol levels. This indicates that Ganoderma lucidum acid A significantly improved hyperlipidemia induced by a high-fat diet.
[0054] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. Use of ganoderic acid A in the preparation of a product for preventing and / or treating hyperlipidemia.
2. Use of a composition comprising ganoderic acid A in the preparation of a product for preventing and / or treating hyperlipidemia.
3. Use according to claim 2, characterized in that: The composition further comprises pharmaceutically acceptable excipients and / or drugs compatible with ganoderic acid A.
4. Use according to any one of claims 1 to 3, characterized in that: The product for preventing and / or treating hyperlipidemia targets intestinal farnesoid X receptor (FXR).
5. Use according to any one of claims 1 to 3, characterized in that: The ganoderic acid A can bind to FXR and occupy the active site of FXR; And / or, the ganoderic acid A can pass through the cell membrane and directly bind to intracellular FXR; And / or, the ganoderic acid A can competitively inhibit the binding of FXR endogenous agonist to FXR, thereby inhibiting its activity; And / or, the ganoderic acid A can significantly inhibit the expression of FXR target genes ileal bile acid binding protein (IBABP), fibroblast growth factor 15 (FGF15) and small heterodimeric partner (SHP).
6. Use according to any one of claims 1 to 3, characterized in that: The ganoderic acid A can reduce the increase of triglyceride, cholesterol and low-density lipoprotein cholesterol levels in serum caused by hyperlipidemia, and increase the content of high-density lipoprotein cholesterol.
7. The use according to any one of claims 1 to 6, characterized in that: The hyperlipidemia is hyperlipidemia induced by high-fat diet.
8. Use according to any one of claims 1 to 7, characterized in that: The product can be a drug and / or health product and / or functional food.
9. Use of ganoderic acid A as an FXR inhibitor, or use of ganoderic acid A in the preparation of an FXR inhibitor.
10. Use of ganoderic acid A in the prevention and / or treatment of hyperlipidemia; or, use of a composition comprising ganoderic acid A in the prevention and / or treatment of hyperlipidemia.