Application of kushenol T in preparation of anti-hepatic fibrosis drugs
By using matrine T to prepare an anti-liver fibrosis drug, the problem of the lack of effective drugs to inhibit liver fibrosis in the existing technology has been solved, and effective inhibition of liver fibrosis has been achieved. It has good drug development potential and clinical application prospects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUIZHOU MEDICAL UNIV
- Filing Date
- 2026-03-26
- Publication Date
- 2026-05-19
AI Technical Summary
Currently, there are no effective anti-liver fibrosis drugs in clinical practice, and existing technologies cannot effectively inhibit the abnormal activation of myofibroblasts and the excessive synthesis of extracellular matrix during liver fibrosis.
Using matrine T (compound KT) as the active ingredient, an anti-hepatic fibrosis drug was prepared by inhibiting the proliferation and activation of hepatic stellate cells and reducing the levels of fibronectin, α-smooth muscle actin and type I collagen.
It effectively inhibits the process of liver fibrosis, showing good drug development potential and broad prospects for development and clinical application, and possesses the potential to inhibit liver fibrosis.
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Figure CN122056868A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of kushenol T in the preparation of anti-hepatic fibrosis drugs. Background Technology
[0002] Sophora flavescens is a legume plant (Sophora flavescens). Sophora flavescens The dried root of *Sophora flavescens* (Ait.), first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), has a long history of clinical application and significant therapeutic effects. *Sophora flavescens* is bitter and cold in nature, and enters the heart, liver, stomach, large intestine, and bladder meridians. It has the effects of clearing heat and drying dampness, killing parasites, and promoting diuresis. It is used for dysentery, hematochezia, jaundice, urinary retention, leukorrhea, vulvar swelling and itching, eczema, damp sores, pruritus, scabies, and leprosy; externally, it is used to treat trichomonal vaginitis. According to modern pharmacological studies, *Sophora flavescens* extract has antioxidant and anti-inflammatory activities.
[0003] Hepatic fibrosis (HF) is a key factor leading to the deterioration of liver disease and patient mortality. Its core pathological mechanism lies in the abnormal activation of myofibroblasts (MFs) during liver damage repair, resulting in excessive synthesis and deposition of extracellular matrix (ECM), which in turn triggers abnormal proliferation of connective tissue and the formation of fibrous scars. This process severely impairs liver metabolic function and is an inevitable pathway for chronic and acute liver diseases to progress to cirrhosis, portal hypertension, liver failure, and even liver cancer, thereby affecting normal liver metabolic function.
[0004] However, there is still a lack of effective anti-fibrotic drugs in clinical practice. Therefore, finding and developing more effective new anti-liver fibrosis drugs has become an important issue in the current medical field. Summary of the Invention
[0005] To address the aforementioned problems, this invention provides the application of matrine T in the preparation of anti-hepatic fibrosis drugs.
[0006] Application of kushenol T in the preparation of anti-hepatic fibrosis drugs.
[0007] Furthermore, the matrine T has the molecular formula C0.05. 25 H 30 O6 has a molecular weight of 426.
[0008] Furthermore, the specific structural formula of matrine T is as follows: .
[0009] Furthermore, the aforementioned anti-hepatic fibrosis drug is a drug for the prevention or treatment of hepatocellular fibrosis.
[0010] Furthermore, the hepatocyte fibrosis mentioned is hepatic stellate cell fibrosis.
[0011] Furthermore, the aforementioned anti-liver fibrosis drug is a drug for the prevention or treatment of liver tissue fibrosis.
[0012] Furthermore, the aforementioned anti-hepatic fibrosis drug is a drug that inhibits the proliferation or activation of hepatic stellate cells.
[0013] Furthermore, the aforementioned anti-hepatic fibrosis drug is a drug that inhibits the increase of fibronectin (FN) levels.
[0014] Furthermore, the anti-liver fibrosis drug is a drug that inhibits the increase of α-smooth muscle actin (α-SMA) levels.
[0015] Furthermore, the aforementioned anti-hepatic fibrosis drug is a drug that inhibits the increase of type I collagen (COLI) levels.
[0016] Furthermore, the anti-hepatic fibrosis drug comprises pharmaceutically acceptable conventional carriers and / or excipients. These pharmaceutically acceptable conventional carriers and / or excipients are at least one of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants.
[0017] The beneficial effects of this invention are as follows: This invention creatively proposes that matrine T (compound KT) can be used to prepare drugs for the prevention or treatment of liver fibrosis, such as its application in the preparation of drugs for the prevention or treatment of hepatocellular fibrosis. It can also be used to prepare drugs that inhibit the proliferation or activation of hepatic stellate cells, inhibit the increase of fibronectin levels, inhibit the increase of α-smooth muscle actin levels, or inhibit the increase of type I collagen levels. It has good drug development potential and broad development, transformation and clinical application value and prospects. Attached Figure Description
[0018] Figure 1 Screening of safe concentrations for the anti-liver fibrosis activity of matrine T (compound KT) in Example 1 and determination of the IC50 of matrine T (compound KT). 50 Value results graph; Figure 2 This is a diagram showing the results of the LX-2 cell scratch assay in Example 2; Figure 3 The figure shows the experimental results of matrine T (compound KT) in Example 3, which inhibited the levels of liver fibrosis markers Fibronectin, α-SMA, and collagen I at the gene and protein levels. Figure 4 The figure shows the experimental results of apoptosis induced in LX-2 cells by matrine T (compound KT) in Example 4. Detailed Implementation
[0019] The specific embodiments of the present invention will be described in further detail below. However, the present invention is not limited to these embodiments. Any improvements or substitutions based on the basic spirit of these embodiments shall still fall within the scope of protection claimed by the claims of the present invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.
[0020] The structural formula of matrine T (compound KT) provided by this invention is as follows: .
[0021] This compound can be extracted from the root of Sophora flavescens, and the extraction method can refer to relevant existing technologies. In addition, the above compound can also be extracted or isolated from other substances containing this compound.
[0022] Example 1 Screening experiment on safe concentration of matrine T (compound KT) for anti-liver fibrosis activity (1) Experimental materials and methods A liver fibrosis model was established using TGF-β1-induced hepatic stellate cells (LX-2). Silybin was used as a positive control. Hepatic stellate cells, after stimulation and activation by TGF-β1, proliferate abnormally and secrete extracellular matrix components including collagen, fibronectin, and α-smooth muscle actin (α-SMA), further promoting liver fibrosis. Inhibiting excessive proliferation of LX-2 cells is a crucial strategy for mitigating liver fibrosis at its source. The CCK-8 assay was used to determine the cell number by detecting the absorbance (A) of the cells, thus determining the anti-fibrotic activity of the compounds. The following groups were established: a blank group (containing only culture medium, no cells), a control group (containing normally growing cells), a model group (treated only with TGF-β1), a positive control group (treated with TGF-β1 + slybin), and a test group (treated with TGF-β1 + different concentrations of matrine T (compound KT)).
[0023] LX-2 cells were cultured in 10% fetal bovine serum medium and incubated at 37 ℃ in a 5% carbon dioxide incubator. Once the cells reached 90% confluence, they were incubated at 2 × 10⁻⁶ cells / year. 3 Inoculate the cells at a density of 10 cells / mL into 96-well plates and incubate for 24 h. Remove the complete culture medium and add blank culture medium to the modeling drug, positive control drug, and matrine T (compound KT).
[0024] Experimental results are as follows Figure 1 As shown (2) Experimental results Screening was conducted using a TGF-β1-stimulated LX-2 cell model, and the results showed that the compound (kushenol T, KT) had no significant toxicity to LX-2 cells, while exhibiting significant inhibitory activity against LX-2 cell proliferation, with an IC50 value of [missing information]. 50 The concentration was 4.80 uM, so we further explored its anti-liver fibrosis activity and mechanism of action.
[0025] Example 2 LX-2 cell scratch assay (1) Experimental materials and methods Select cells in the logarithmic growth phase, at a ratio of 5 × 10⁻⁶ 4 Cells were seeded at a density of 1 cell / mL in 6-well plates, with 2 ml of DMEM complete medium added to each well. After reaching the target cell density, a scratching operation was performed on the surface of the cell monolayer, and the original medium was removed. Serum-free DMEM medium was added, and according to the experimental groups, positive control agents and different concentrations of KT (1 μM, 2 μM, 4 μM) were added to each well. Cells were observed and photographed using an inverted microscope at 0 h, 24 h, and 48 h after scratching, with the same field of view selected for each photograph. Finally, the width of the scratch was measured using ImageJ software, and the cell migration rate was calculated.
[0026] (2) Experimental results like Figure 2 As shown, compared with the blank group, the KT group can significantly inhibit the migration of activated LX-2 cells, and the inhibitory effect at 4 uM is almost equivalent to that of the positive drug at 60 uM, indicating that KT has strong anti-liver fibrosis activity.
[0027] Example 3 KT inhibits the levels of liver fibrosis markers Fibronectin, α-SMA, and collagen I at both the gene and protein levels. (1) Experimental materials and methods.
[0028] Cells in logarithmic growth phase were treated with the drug according to the experimental groups. After culturing for 24 h, the culture medium was discarded, and 1 mL of TRIzol reagent was added to each well to lyse the cells. Total RNA was extracted according to the TRIzol reagent instructions, and cDNA was synthesized according to the reverse transcription kit instructions. Real-time quantitative PCR amplification was performed using the cDNA as a template. Reaction conditions: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ annealing for 30 s; for a total of 40 cycles. GAPDH was used as an internal control, and the relative expression level of the target gene was calculated using the 2^-ΔΔCt method. All primer sequences are listed in Table 1.
[0029] Table 1 Primer sequence listing
[0030] 2. Cells in the logarithmic growth phase were selected and treated with drugs according to experimental groups. After 24 hours of culture, protein samples were collected and subjected to SDS-PAGE electrophoresis. After electrophoresis, the proteins were transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk. Then, rabbit anti-human α-SMA, rabbit anti-human Collagen I, rabbit anti-human Fibronectin monoclonal antibodies, and rabbit anti-human GAPDH monoclonal antibodies were added and incubated overnight at 4°C. After incubation, goat anti-rabbit IgG-HRP secondary antibody was added and incubated at room temperature for 2 hours. ECL chemiluminescence was used for color development. Finally, the gray values of the bands were analyzed using ImageJ software, and the relative expression level of the target protein was calculated using GAPDH as an internal control.
[0031] The above results are as follows: Figure 3 As shown in (A) and (B).
[0032] (2) Experimental results RT-qPCR results showed that, compared with the model group, the mRNA expression levels of α-SMA, Fibronectin, and Collagen I were significantly reduced in the KT-treated group (P<0.05), and this reduction exhibited a clear dose-dependent effect. That is, with increasing KT dosage, the mRNA expression levels of these genes gradually decreased.
[0033] Western blot results showed that the protein expression levels of α-SMA, Fibronectin, and Collagen I in the KT-treated group were significantly lower than those in the model group (P<0.05), and this also showed a dose-dependent effect. These results indicate that KT can inhibit the abnormal proliferation and migration of activated hepatic stellate cells and reduce the RT-qPCR and protein expression of liver fibrosis markers, demonstrating its potential for anti-liver fibrosis.
[0034] Example 4 Matrine T (compound KT) induces apoptosis in LX-2 cells. (1) Experimental materials and methods.
[0035] This study used flow cytometry to investigate this phenomenon. Cells in the logarithmic growth phase were obtained and analyzed at a concentration of 5 × 10⁶ cells / mL. 4Cells were seeded at a density of [number] cells / mL into 6-well plates. After 24 h of culture, the corresponding drug was added to each well according to the experimental grouping plan, and co-incubation continued for another 24 h. After culture, cells were collected. The cells were centrifuged, and the cell pellet was resuspended in 500 μL of Binding Buffer. Then, 5 μL of Annexin V-FITC and 10 μL of PI were added, mixed thoroughly, and incubated in the dark for 15 min. Finally, flow cytometry was used to detect cell apoptosis and analyze the proportion of early and late apoptotic cells in the total cell count.
[0036] The results are as follows Figure 4 As shown.
[0037] (2) Experimental results The results showed that, compared with the early and late apoptosis rates of 13.59% in the model group, the proportion of apoptotic cells in the low-to-high-dose KT group increased from 22.65% to 34.53%, a significant increase that was dose-dependent and even superior to the 28.18% in the positive control group. This clearly demonstrates that KT can induce apoptosis in LX-2 cells, thereby reducing activated cells, providing important evidence for elucidating the mechanism of action of KT in combating liver fibrosis.
[0038] In summary, this invention creatively proposes that matrine T (compound KT) can be used to prepare drugs for the prevention or treatment of liver fibrosis, such as its application in the preparation of drugs for the prevention or treatment of hepatocellular fibrosis. It can also be used to prepare drugs that inhibit the proliferation or activation of hepatic stellate cells, inhibit the increase of fibronectin levels, inhibit the increase of α-smooth muscle actin levels, or inhibit the increase of type I collagen levels. It has good drug development potential and broad development, transformation, and clinical application value and prospects.
[0039] In summary, this invention provides for the first time the application of matrine T in the preparation of anti-hepatic fibrosis drugs, which has good drug development potential and broad value and prospects for development, transformation and clinical application.
Claims
1. Application of kushenol T in the preparation of anti-hepatic fibrosis drugs.
2. The application of matrine T as described in claim 1 in the preparation of anti-hepatic fibrosis drugs, characterized in that, The matrine T has the molecular formula C 25 H 30 O6 has a molecular weight of 426.
3. The application of matrine T as described in claim 1 in the preparation of anti-hepatic fibrosis drugs, characterized in that, The specific structural formula of matrine T is as follows: 。 4. The application of matrine T as described in claim 1 in the preparation of anti-hepatic fibrosis drugs, characterized in that, The aforementioned anti-hepatic fibrosis drug is a drug used to prevent or treat hepatocyte fibrosis.
5. The application of matrine T as described in claim 4 in the preparation of anti-hepatic fibrosis drugs, characterized in that, The hepatocyte fibrosis mentioned is hepatic stellate cell fibrosis.
6. The application of matrine T as described in claim 1 in the preparation of anti-hepatic fibrosis drugs, characterized in that, The aforementioned anti-liver fibrosis drug is a drug used to prevent or treat liver tissue fibrosis.
7. The application of matrine T as described in claim 1 in the preparation of anti-hepatic fibrosis drugs, characterized in that, The aforementioned anti-hepatic fibrosis drug is a drug that inhibits the proliferation of hepatic stellate cells.
8. The application of matrine T as described in claim 1 in the preparation of anti-hepatic fibrosis drugs, characterized in that, The aforementioned anti-hepatic fibrosis drug is a drug that inhibits the activation of hepatic stellate cells.
9. The application of matrine T as described in claim 1 in the preparation of anti-hepatic fibrosis drugs, characterized in that, The anti-liver fibrosis drug includes pharmaceutically acceptable conventional carriers and / or excipients.
10. The application of matrine T as described in claim 9 in the preparation of anti-hepatic fibrosis drugs, characterized in that, The pharmaceutically acceptable conventional carrier and / or excipient is at least one of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbents, and lubricants.