Application of baicalein-7-methyl ether in hepatic fibrosis
By using baicalin-7-methyl ether to reduce the expression of α-SMA and Collagen-I, inhibit the activation of hepatic stellate cells, solve the problem of treatment of liver fibrosis, and achieve the recovery of liver morphology and effective prevention and treatment of liver fibrosis.
Patent Information
- Application Number
- CN202510669732.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-23
- Publication Date
- 2025-08-08
AI Technical Summary
There is no effective drug or therapy in the prior art that can effectively treat liver fibrosis, resulting in liver fibrosis progression into cirrhosis and even liver failure and liver cancer. The patient has a poor prognosis and it is necessary to seek anti-hepatic fibrosis drugs.
Baicalin-7-methyl ether is used to prevent and treat liver fibrosis by reducing the expression of α-SMA and Collagen-I, and inhibit the activation of hepatic stellate cells and restore liver morphology.
Baicalin-7-methyl ether can effectively reduce liver fibrosis, restore liver morphology, and reduce the expression of α-SMA and Collagen-I. As a drug to prevent and treat liver fibrosis, it has significant therapeutic effects.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical technology, and in particular to application of baicalein-7-methyl ether in treating liver fibrosis. Background Art
[0002] Liver fibrosis is a disease process characterized by scarring and tissue remodeling of the liver, typically caused by long-term liver damage. This damage can result from a variety of factors, including viral hepatitis, alcohol abuse, fatty liver disease, drug toxicity, or autoimmune diseases. Liver fibrosis is essentially the body's process of repairing abnormal liver tissue damage. During this process, hepatic stellate cells (HSCs) in the liver are activated, releasing various inflammatory factors and extracellular matrix (ECM) components, promoting fibrosis formation. This disrupts the liver's normal architecture and gradually declines liver function. Mild liver fibrosis progresses to severe cirrhosis, even leading to liver failure and liver cancer, resulting in poor prognosis and high mortality. Effective treatment of the fibrotic stage can halt disease progression and even reverse fibrosis, but pathological changes after cirrhosis are difficult to reverse. However, the clinical treatment of liver fibrosis remains a significant challenge, with no single drug or therapy specifically effective for the treatment of liver fibrosis. Therefore, the search for effective anti-fibrotic drugs and active ingredients is crucial. Summary of the Invention
[0003] To address the above problems, the present invention provides the use of baicalein-7-methyl ether in liver fibrosis. Baicalein-7-methyl ether can restore liver morphology by reducing the expression of α-SMA and Collagen-I, effectively alleviating liver fibrosis, and can be used as a drug for preventing and / or treating liver fibrosis.
[0004] The present invention provides a use of baicalein-7-methyl ether in the preparation of a medicament for preventing and / or treating liver fibrosis. The structural formula of the baicalein-7-methyl ether is as follows:
[0005]
[0006] The above-mentioned baicalein-7-methyl ether has a molecular weight of 284.26 and a molecular formula of C 16 H 12O5 is a flavonoid compound. The present invention uses zebrafish larvae and activated rat hepatic stellate cells (HSC-T6) as experimental models for pharmacological efficacy studies. In vitro experiments show that baicalein-7-methyl ether can reduce the activation of HSC-T6 cells and reduce the expression of α-SMA and Collagen-I; at the same time, baicalein-7-methyl ether can effectively reduce liver fibrosis in zebrafish, reduce the expression of Collagen-I, and restore liver morphology. Therefore, baicalein-7-methyl can be used as a drug for the prevention and / or treatment of liver fibrosis.
[0007] In one embodiment, the baicalein-7-methyl ether exerts its efficacy in its original form after entering the blood.
[0008] Studies have shown that after baicalein-7-methyl ether enters the blood and reaches the target, it exerts its effects as the prototype component (such as Figure 1 It is not metabolically converted in the body.
[0009] In one embodiment, the efficacy includes: restoring liver morphology by reducing the expression of α-SMA and Collagen-I and inhibiting the activation of HSC-T6 cells.
[0010] In one embodiment, the drug further comprises pharmaceutically acceptable excipients and / or pharmaceutical ingredients compatible with baicalein-7-methyl ether.
[0011] In one embodiment, the excipients include at least one of a solvent, a disintegrant, a flavoring agent, a colorant, a lubricant, an antioxidant, a preservative, a binder, a filler or a thickener.
[0012] In one embodiment, the working concentration of baicalein-7-methyl ether is ≥1.5 μM.
[0013] The working concentration is the concentration at which the above-mentioned ingredients can achieve the expected effect when used. It is understandable that those skilled in the art can prepare a mother solution or stock solution with a higher concentration when preparing the above-mentioned medicine containing baicalein-7-methyl ether, and then dilute it when it is used. The mother solution or stock solution and its concentration are all within the scope of protection of the present invention.
[0014] In one embodiment, the dosage form of the drug includes capsules, granules, tablets, oral liquids, pills, injections, ointments, liposome nanoparticles, sustained-release agents, controlled-release agents or dispersible tablets.
[0015] In one embodiment, the administration route of the drug includes oral administration or intravenous injection.
[0016] In one embodiment, the liver fibrosis is caused by chronic liver disease, which includes at least one of viral hepatitis, steatohepatitis, alcoholic hepatitis, autoimmune liver disease, and Wilson's disease.
[0017] The present invention also provides a medicine for preventing and / or treating liver fibrosis, comprising a medicine active ingredient and excipients, wherein the medicine active ingredient is baicalein-7-methyl ether.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The application of baicalein-7-methyl ether in liver fibrosis of the present invention can restore liver morphology by reducing the expression of α-SMA and Collagen-I, effectively reducing liver fibrosis, and can be used as a drug for preventing and / or treating liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is a schematic diagram of the process by which baicalein-7-methyl ether takes effect after entering the blood and reaching the target;
[0021] Figure 2 Graph showing the CCK8 detection results of the HSC-T6 cell line control group (0 μM) and the drug administration groups (0, 4, 8, 10, 20, 40 μM) in the examples;
[0022] Figure 3 Graph showing the WB results of α-SMA in the HSC-T6 cell line control group (NC) and drug-treated group (NE) in the examples;
[0023] Figure 4 Graph showing the immunofluorescence detection results of α-SMA and Collagen-I in the HSC-T6 cell line control group (Control) and the drug-treated group (NE) in the examples;
[0024] Figure 5 The figures are H&E pathological staining results of the zebrafish control group (NC), model group (0.06% TAA), low-concentration drug administration group (0.78 μmol / L), medium-concentration drug administration group (1.56 μmol / L), and high-concentration drug administration group (3.125 μmol / L) in the examples;
[0025] Figure 6 Figure 2 is a Collagen-I immunofluorescence staining image of the zebrafish control group (NC), model group (0.06% TAA), low-concentration drug administration group (0.78 μmol / L), medium-concentration drug administration group (1.56 μmol / L), and high-concentration drug administration group (3.125 μmol / L) in the examples;
[0026] Figure 7Graph showing the statistical results of Collagen-I immunofluorescence staining of the zebrafish control group (NC), model group (0.06% TAA), low-concentration drug administration group (0.78 μmol / L), medium-concentration drug administration group (1.56 μmol / L), and high-concentration drug administration group (3.125 μmol / L) in the examples. DETAILED DESCRIPTION
[0027] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.
[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0029] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; and the experimental methods, unless otherwise specified, are all conventional experimental methods in the art.
[0030] Example
[0031] 1. Experimental materials and methods.
[0032] Baicalein-7-methyl ether was purchased from TargetMol with a purity of 99.2%, CAS No.: 29550-13-8.
[0033] Rat hepatic stellate cells: HSC-T6 cell line.
[0034] Wild-type zebrafish (AB strain), a transgenic line with liver-specific eGFP expression [Tg(lfabp10α-eGFP)], and AB strain wild-type fish.
[0035] 2. Effect of baicalein-7-methyl ether on the growth and proliferation of T6 cells.
[0036] T6 cells in the logarithmic growth phase were evenly plated into 96-well culture plates (3000-5000 cells / well). After incubation in a cell culture incubator overnight, the culture medium in each well was discarded. Subsequently, 100 μL of baicalein-7-methyl ether solution of different concentrations was added to each well. The sample concentrations were set to 0, 4, 8, 10, 20, and 40 μM. Five replicates were added and the cells were incubated in a cell culture incubator for 48 hours. After incubation, 10 μL of CCK8 solution was added to each well. The cells were then placed in a cell culture incubator and incubated in the dark for 2-4 hours. The absorbance of each well was then measured using a full-wavelength microplate reader with a wavelength set to 450 nm. The viability of the cells in each well was calculated according to the following formula: Cell Viability (%) = As / Ab * 100%, where As and Ab represent the absorbance of the sample treatment and the blank control, respectively.
[0037] Results: As Figure 2 As shown, after drug treatment for 48 hours, baicalein-7-methyl ether had an inhibitory effect on the growth of T6 cells within a certain concentration range, proving that it has a certain effect of inhibiting the activation of hepatic stellate cells.
[0038] 3. Effect of baicalein-7-methyl ether on HSC-T6 cell activation.
[0039] 1. Western blot: Protein samples are extracted and then separated by polyacrylamide gel electrophoresis (PAGE), separating proteins according to their molecular weight. The separated proteins are transferred to a polyvinylidene fluoride membrane (PVDF membrane) using electroblotting. Nonspecific binding sites are blocked (using milk or BSA, etc.), and then a primary antibody against α-SMA is added for incubation. After the primary antibody binds to the protein, it is incubated with a secondary antibody. Finally, the immunoreaction product is detected by chemiluminescence to determine the presence and expression of α-SMA before and after drug treatment. The NE concentration is 40 μM.
[0040] 2. Immunofluorescence: During subculture, cells were seeded onto culture dishes pre-placed with cell slides. After treatment with baicalein-7-methyl ether (NE, 40 μM), the cells were fixed and permeabilized. The cells were then blocked with blocking buffer and incubated with the primary antibody at room temperature for 1 hour or overnight at 4°C. The secondary antibody was incubated at room temperature in the dark for 1 hour, then the slides were mounted and observed under a fluorescence microscope.
[0041] Results: As Figure 3 、 Figure 4 As shown in the figure, we used WB to detect the changes in the protein levels of α-SMA and Collagen-I by IF. Compared with the blank group, the expression of α-SMA and Collagen-I in HSC-T6 cells in the group supplemented with baicalein-7-methyl ether solution was significantly decreased (P<0.01).
[0042] 4. Zebrafish modeling and drug administration.
[0043] One day in advance, transgenic zebrafish [Tg(lfabp10α-eGFP)] zebrafish and AB strain wild fish were selected in a 1:1 ratio of male to female and placed in a mating box. The next day, the partitions were removed for mating. After spawning, the eggs were collected, and the transgenic zebrafish eggs and AB fish eggs that specifically expressed green fluorescence in the liver were collected using an 180 μm pore filter. The eggs were washed 2-3 times with culture water. The embryos were placed in a 90 mm dish filled with culture water containing methylene blue at a density of 50-60 eggs / dish, and foreign matter and unfertilized eggs were removed. At 2 dpf, zebrafish juveniles with fluorescent livers and AB strain juveniles were randomly selected and randomly divided into a control group, a model group, a low-concentration drug group, a medium-concentration drug group, and a high-concentration drug group, and placed in a six-well plate, with 30 eggs per well in each group. Except for the control group, each group was added with 8 mL of fish culture system water containing 0.06% thioacetamide (TAA) and the exposure was continued for 6 days. On the third day of exposure, the low-concentration drug administration group, the medium-concentration drug administration group and the high-concentration drug administration group were added with 0.06% TAA modeling system water containing 0.78 μM, 1.56 μM and 3.125 μM baicalein-7-methyl ether, respectively. The control group and the model group did not receive any treatment. The death of zebrafish was recorded every day. The modeling was terminated 3 days after drug administration, and the above-mentioned zebrafish larvae were collected.
[0044] 5. Effect of baicalein-7-methyl ether on the morphology of zebrafish liver tissue.
[0045] The zebrafish larvae were fixed with 4% paraformaldehyde (PFA) and refrigerated at 4°C overnight. The next day, they were dehydrated with 80% ethanol for 2 hours, 90% ethanol for 2 hours, and 95% ethanol overnight. The next day, they were dehydrated with 100% ethanol I for 0.5 hours, 100% ethanol II for 0.5 hours, and 100% ethanol III for 0.5 hours. They were then treated with xylene I, II, and III for 0.5 hours each, and immersed in paraffin I for 0.5 hours, paraffin II for 0.5 hours, and paraffin III for 1 hour. The zebrafish embryos were removed from the paraffin, placed in an embedding cassette, and embedded in paraffin. The embryos were then sectioned on a microtome with a thickness of 4 μm. The slices were gently placed in a 60°C water bath. Once fully developed, they were transferred to glass slides and dried in a 60°C incubator for approximately 2 hours.
[0046] The paraffin sections prepared above were dewaxed with xylene I, II, and III for 3 minutes each, anhydrous ethanol I and II for 2 minutes each, 95% ethanol, 90% ethanol, and 80% ethanol for 1 minute each, stained with hematoxylin for 10 minutes, rinsed with water, differentiated with 1% hydrochloric acid alcohol for 15 seconds, soaked in water for 10 minutes, stained with eosin for 3 minutes, 80% ethanol and 90 ethanol for 10 seconds each, 95% ethanol for 1-2 minutes, anhydrous ethanol I, II, and III for 3 minutes each, xylene I, II, and III for 3 minutes each, and finally sealed with neutral gum and photographed under a microscope.
[0047] Results: As Figure 5 As shown in the figure, the liver tissue structure of the model group was disordered, with liver cells showing swelling, degeneration, a large number of vacuoles, and some nodular formation. The liver tissue structure of zebrafish in the high-concentration drug group was normal, with liver cells arranged neatly.
[0048] 6. Effect of baicalein-7-methyl ether on the expression of Collagen-I in zebrafish with liver fibrosis.
[0049] Liver-specific fluorescent transgenic zebrafish (Tg(lfabp10α-eGFP)) were collected and fixed with 4% paraformaldehyde (PFA) overnight at 4°C. The next day, the fish were dehydrated with 30% sucrose for three days, until all the fish sank to the bottom of the tube. The dehydrated zebrafish were removed with forceps, embedded in OCT, and cooled to -80°C. The slices were then sliced on a cryostat at a thickness of 14 μm and stored at -20°C.
[0050] The frozen sections were briefly washed with distilled water, permeabilized with 0.1% Triton X-100 in PBS at room temperature for 1 hour, washed three times with PBS for 5 minutes each time, blocked with 5% blocking solution (PBS + 5% goat serum + 0.1% Triton X-100) at room temperature for two hours, and then incubated with Collagen-I (1:200 dilution) at 4°C overnight. The next day, the sections were washed three times with PBS for 5 minutes each time, incubated with fluorescent rabbit secondary antibody at room temperature in the dark for 2 hours, washed three times with PBS for 5 minutes each time, stained with DAPI in the dark for 10 minutes, washed three times with PBS for 5 minutes each time, dried, mounted with anti-fluorescence quencher, and photographed.
[0051] Results: As Figure 6 、 Figure 7 As shown, thioacetamide (TAA) induced a large amount of expression of Collagen-I. Compared with the model group, the medium-concentration and high-concentration administration groups significantly reduced the amount of Collagen-I in the liver.
[0052] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0053] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.
Claims
1. Use of baicalein-7-methyl ether in the preparation of a medicament for preventing and / or treating liver fibrosis, wherein the structural formula of the baicalein-7-methyl ether is as follows:
2. The use according to claim 1, characterized in that The baicalein-7-methyl ether exerts its efficacy in its original form after entering the blood.
3. The use according to claim 2, characterized in that The efficacy includes: restoring liver morphology by reducing the expression of α-SMA and Collagen-I and inhibiting the activation of HSC-T6 cells.
4. The use according to claim 1, characterized in that The medicine further comprises pharmaceutically acceptable excipients and / or medicinal ingredients compatible with baicalein-7-methyl ether.
5. The use according to claim 4, characterized in that The excipients include at least one of a solvent, a disintegrant, a flavoring agent, a colorant, a lubricant, an antioxidant, a preservative, a binder, a filler or a thickener.
6. The use according to claim 1, characterized in that The working concentration of baicalein-7-methyl ether is ≥1.5 μM.
7. The use according to claim 1, characterized in that The dosage forms of the drug include capsules, granules, tablets, oral liquids, pills, injections, ointments, liposome nanoparticles, sustained-release agents, controlled-release agents or dispersible tablets.
8. The use according to claim 1, characterized in that The administration routes of the drug include oral administration or intravenous injection.
9. The use according to any one of claims 1 to 8, characterized in that The liver fibrosis is caused by chronic liver disease, which includes at least one of viral hepatitis, steatohepatitis, alcoholic hepatitis, autoimmune liver disease, and Wilson's disease.
10. A drug for preventing and / or treating liver fibrosis, characterized in that: The invention comprises active pharmaceutical ingredients and excipients, wherein the active pharmaceutical ingredient is baicalein-7-methyl ether.