Application of kushenol O in preparation of anti-hepatic fibrosis drugs
The drug prepared by using matrine O inhibits the liver fibrosis process, solving the problem of the lack of effective anti-liver fibrosis drugs in the existing technology, and achieving effective inhibition of liver fibrosis and tissue repair.
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. Existing technologies cannot effectively inhibit the progression of liver fibrosis, which affects liver metabolic function and may lead to cirrhosis, liver failure, and liver cancer.
Matrine O is used as the active ingredient to prepare an anti-liver fibrosis drug. It inhibits liver tissue fibrosis by suppressing the proliferation and activation of hepatic stellate cells and reducing the levels of fibronectin, α-smooth muscle actin, type I collagen, alanine aminotransferase, aspartate aminotransferase and hydroxyproline.
Matrine O significantly inhibited the liver fibrosis process, reduced the expression of fibrosis markers in liver tissue and the increase of serum biochemical indicators, and has good drug development potential and clinical application prospects.
Smart Images
Figure CN122056904A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of matrine O 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 the progression of chronic and acute liver disease 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 O in the preparation of anti-hepatic fibrosis drugs.
[0006] Application of matrine O in the preparation of anti-hepatic fibrosis drugs.
[0007] Furthermore, the matrine O has the molecular formula C0. 27 H 30 O 13 Its molecular weight is 562.
[0008] Furthermore, the specific structural formula of matrine O 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 proliferation or activation of hepatic stellate cells. Furthermore, the 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 aforementioned anti-liver fibrosis drug is a drug that inhibits the increase of serum alanine aminotransferase (ALT) levels.
[0017] Furthermore, the aforementioned anti-liver fibrosis drug is a drug that inhibits the increase of serum aspartate aminotransferase (AST) levels.
[0018] Furthermore, the aforementioned anti-hepatic fibrosis drug is a drug that inhibits the increase of hydroxyproline (HYP) levels in liver tissue.
[0019] 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.
[0020] The beneficial effects of this invention are as follows: This invention creatively proposes the use of compound KO in the preparation of drugs for the prevention or treatment of liver fibrosis. For example, it can be used in the preparation of drugs for the prevention or treatment of hepatocellular fibrosis and mouse liver tissue fibrosis. It can also be used to prepare drugs that inhibit the proliferation or activation of hepatic stellate cells, inhibit liver fibrosis in vivo, inhibit the increase of fibronectin (FN) levels, inhibit the increase of α-smooth muscle actin (α-SMA) levels, inhibit the increase of type I collagen (COLI) levels, inhibit the increase of alanine aminotransferase (ALT) levels in mouse serum, inhibit the increase of aspartate aminotransferase (AST) levels in mouse serum, and inhibit the increase of hydroxyproline (HYP) levels in mouse liver tissue. It has good drug development potential and broad development, transformation, and clinical application value and prospects. Attached Figure Description
[0021] Figure 1 Example 1 uses LX-2 cells as a model to detect the cytotoxicity of the compound kushenol O (KO) isolated and purified from the ethyl acetate fraction of Sophora flavescens. The effect of different concentrations of KO on the cytotoxicity of LX-2 cells is shown in the figure. Figure 2 Figure 2 shows the effect of KO on the expression levels of fibrosis marker proteins FN and collagen I in a TGF-β1-induced LX-2 cell fibrosis model. a) is the SDS-PAGE electrophoresis result, and b) is the quantitative analysis statistical graph. Figure 3 The figure shows the effect of KO on the transcriptional levels of ACTA2 (a), COL1A1 (b), and FN (c) in the TGF-β1-induced LX-2 cell fibrosis model in Example 3. Figure 4 For Example 4, hematoxylin and eosin staining (H&E) and Masson trichrome staining were performed on the liver tissue structure of a mouse model of CCl4-induced liver fibrosis.
[0022] Figure 5 The figure shows the results of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and liver fibrosis marker hydroxyproline (HYP) levels in the mouse liver fibrosis model induced by CCl4 in Example 4.
[0023] Figure 6 The figure shows the effect of KO on the expression levels of fibrosis markers FN and α-SMA in mouse liver tissue in a CCl4-induced mouse liver fibrosis model in Example 5; a is the SDS-PAGE electrophoresis result, and b is the quantitative analysis statistical graph. Detailed Implementation
[0024] 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.
[0025] The structural formula of compound KO provided by this invention is as follows: .
[0026] 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.
[0027] Example 1 The effects of different concentrations of compound KO on the viability of hepatic stellate cells (LX-2 cells) were tested using a CCK-8 assay kit. (1) Experimental materials and methods To verify whether KO would be toxic to normally growing LX-2 cells, 4 × 10⁶ cells were seeded per well in a 96-well plate. 3 LX-2 cells were collected and, after reaching 90% cell growth, were treated according to the experimental groups: Blank zeroing group: Only DMEM complete culture medium was added, without cells.
[0028] Normal control group: LX-2 cells cultured normally and DMEM complete medium were added.
[0029] Model group: TGF-β1 (10 ng / mL) stimulation for 24 h.
[0030] Drug administration groups: TGF-β1 (10 ng / mL) stimulation for 24 h and KO (0 μmol / L, 2.5 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, 80 μmol / L, 100 μmol / L) culture for 24 h.
[0031] Positive control group: TGF-β1 (10 ng / ml) stimulation for 24 h and silybin (20 μmol / L) culture for 24 h. Then, 10 μL of CCK-8 reagent was added to each well for reagent incubation. After gentle shaking and mixing, the cells were incubated in the dark for 1–4 h. Absorbance was then measured. The absorbance of the 96-well plates after incubation was measured at 450 nm using a microplate reader, and the experimental data were recorded. Finally, cell viability was calculated. The net absorbance of the experimental and control groups was calculated by subtracting the absorbance of the blank wells from the baseline. Quantitative results were obtained using the formulas: "Cell viability (%) = (Net absorbance of experimental group - Net absorbance of blank group) / (Net absorbance of control group - Net absorbance of blank group) × 100%" and "Cell inhibition rate (%) = 100% - Cell viability". The above results, such as Figure 1 As shown.
[0032] (2) Experimental results The effect of KO on LX-2 cell cytotoxicity was detected by the CCK-8 assay. The results were obtained by... Figure 1It can be seen that the cell viability did not change significantly with the increase of KO concentration, that is, the dose of 80 μM and below had little effect on cell viability. Therefore, subsequent experiments determined 80 μM as the maximum dose (n=3, *P<0.05, **P<0.01, ns, no statistical significance).
[0033] Example 2 Effects of KO on the expression levels of fibrosis markers FN and collagen I in a TGF-β1-induced LX-2 cell fibrosis model.
[0034] (1) Experimental methods LX-2 cells were passaged one day in advance. After cell adhesion, 10 ng / mL TGF-β1 (prepared in 10% complete medium) was added to construct an LX-2 cell fibrosis model, and the drug was administered simultaneously. The cells were then cultured in an incubator (37℃, 5% CO2) according to the following regimens for at least 24 h: ① Blank group (10% complete medium) ② Model group (10 ng / mL TGF-β1) ③ Positive drug group (20 μM silybin) ④ Drug-treated groups (KO (10 μM, 20 μM, 40 μM)) for 24 h. Cells were removed, and an appropriate amount of RIPA lysis buffer was added. Lysis was performed on ice for 30 min, with shaking every 5 min. The cells were centrifuged at 12000 r / min for 15 min at 4℃, and the supernatant was collected. Protein concentration was determined using the BCA method. An equal volume of protein sample was taken, and 5×SDS loading buffer was added. The sample was boiled for 5 min to denature the protein. SDS-PAGE electrophoresis was performed to transfer the protein to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk for 2 h, washed three times with TBST, and then incubated overnight at 4℃ with primary antibody. The next day, the membrane was washed three times with TBST for 10 min each time, and then incubated at room temperature for 2 h with secondary antibody. After washing three times with TBST, ECL chemiluminescence was used for color development. The gray values of the bands were analyzed using ImageJ software. Using GAPDH as an internal control, the relative expression level of the target protein was calculated, and a quantitative analysis statistical graph was generated.
[0035] The above results, such as Figure 2 As shown.
[0036] (2) Experimental results Depend on Figure 2 It can be seen that, compared with the model group, the KO treatment group can reduce the expression levels of fibrosis marker proteins FN and collagen I in a dose-dependent manner, indicating that KO has the activity of improving LX-2 cell fibrosis.
[0037] Example 3 Effects of KO at the gene level on the transcriptional levels of ACTA2 (a), COL1A1 (b), and FN (c) in a TGF-β1-induced LX-2 cell fibrosis model.
[0038] 1. Incubate LX-2 cells in DMEM medium containing 10% FBS at a density of 4 × 10⁶ cells per well. 5 LX-2 cells were seeded in 6-well plates. After cell attachment, LX-2 cells were stimulated with 10 ng / mL TGF-β1 and co-cultured with KO (10 μmol / L, 20 μmol / L, 40 μmol / L) and silybin for 24 h. Total RNA was then extracted for RT-qPCR. Total RNA was isolated from cells using the Total RNA Isolation Kit. cDNA was synthesized using FastKing gDNA elimination RT ultramixing buffer, and RT-qPCR was performed using TB Green premixed buffer Ex Taq II. Relative mRNA levels were normalized to GAPDH levels. All primer sequences are listed in Table 1. Table 1 Primer sequence listing
[0039] Example 4 This study investigated the effects of KO on hematoxylin-eosin staining and Masson's trichrome staining of liver tissue in a CCl4-induced mouse model of liver fibrosis, as well as on serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and hydroxyproline (HYP) levels in liver tissue. 1.1 Experimental Materials: Six-week-old SPF-grade male C57BL mice were purchased from the Experimental Animal Center of Guizhou Medical University. They were provided with 12 hours of light and 12 hours of darkness, kept at a constant temperature, and fed normal food and water. After acclimatizing to a sterile environment for 4 days, experiments were conducted.
[0040] Reagents and Instruments: Reagents: Matrine O (purity ≥98%), monomeric compound of Sophora flavescens; Modeling agents: CCl4 analytical alcohol and olive oil; Positive reagent: Silybin. Instruments: Electronic balance (for weighing and drug preparation); 1.2 Experimental Methods 1.2.1 Animal grouping and administration: Mice were randomly divided into 5 groups (n=8): 1) Control group: Daily intraperitoneal injection of pure olive oil + intraperitoneal injection of solvent control; 2) Model group: Daily intraperitoneal injection of 20% CCl4 olive oil solution; 3) Positive control group: Daily intraperitoneal injection of 20% CCl4 olive oil solution + intraperitoneal injection of Silybin 20 mg / kg; 4) Low-dose KO group: Daily intraperitoneal injection of 20% CCl4 olive oil solution + intraperitoneal injection of matrine O (5 mg / kg); 5) High-dose KO group: Daily intraperitoneal injection of 20% CCl4 olive oil solution + intraperitoneal injection of matrine O (20 mg / kg).
[0041] 1.2.2 Establishment and treatment of liver fibrosis model: Except for the blank control group, mice in the other groups were injected intraperitoneally with 20% CCl4 olive oil solution (5 mL / kg) twice a week for 6 consecutive weeks to induce fibrosis; from the 4th week, each treatment group was injected intraperitoneally with the corresponding treatment drug daily for 3 weeks; and the weight changes of mice in each group were recorded every Monday.
[0042] 1.2.3 Sample Collection and Detection: H&E and Masson Staining: After treatment (i.e., at the end of week 6), mice were euthanized, blood was collected from the eyeballs, and liver tissue was harvested after cardiac perfusion. A portion of the tissue was fixed in 4% paraformaldehyde for 24 hours. The fixed mouse liver tissue was sent to Sewell Technology Co., Ltd. for Masson and H&E staining. Masson and H&E staining helps to assess liver inflammation and collagen fiber deposition. Blood samples were allowed to stand at room temperature for 2 hours, then centrifuged at 8000 g for 15 minutes at 4°C to collect serum. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum and the hydroxyproline (HYP) level in liver tissue were detected using a Solarbio reagent kit.
[0043] 1.3 Experimental Results Depend on Figure 4Images of liver tissue sections from different groups of mice were compared using H&E staining and Masson staining. H&E staining showed that the liver tissue structure of the normal group mice was normal, while in the CCl4 model group mice, the hepatocytes around the central vein lacked normal cell morphology, were enlarged, and some cells showed ballooning degeneration, with inflammatory cell infiltration. Compared with the CCl4 model group, the lesions in the positive control group, low-dose KO group, and high-dose KO group were all reduced to some extent, with the positive control group and high-dose KO group showing relatively significant reductions, and the low-dose KO group showing slight reductions, consistent with the results of blood biochemistry experiments. Masson trichrome staining confirmed that CCl4 can induce collagen production, while KO can inhibit collagen production. Compared with the blank group mice, the CCl4 model group mice had increased liver fibrosis, while the liver fibrosis of mice treated with intraperitoneal injection of KO was significantly reduced.
[0044] Depend on Figure 5 It is known that damaged hepatocytes release serum biochemical markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The concentration of these enzymes in serum reflects the degree of liver damage. After administration of KO, these biochemical markers showed a decreasing trend, especially in the high-dose KO group, with effects comparable to positive control drugs. Hydroxyproline (HYP) in liver tissue is one of the important indicators reflecting liver fibrosis. After administration, HYP levels decreased significantly compared to the model group, further indicating that KO has an anti-liver fibrosis effect.
[0045] Example 5 The effect of KO on the expression levels of FN and α-SMA, markers of liver fibrosis in a CCl4-induced mouse model.
[0046] Mouse liver tissue was ground and subjected to Western blotting. An appropriate amount of RIPA lysis buffer was added, and the mixture was lysed on ice for 30 min, with shaking every 5 min. After centrifugation at 12000 r / min for 15 min at 4℃, the supernatant was collected, and protein concentration was determined using the BCA method. An equal volume of protein sample was taken, and 5×SDS loading buffer was added. The protein was boiled for 5 min to denature it. SDS-PAGE electrophoresis was performed to transfer the protein to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk for 2 h, washed three times with TBST, and then incubated overnight at 4℃ with primary antibody. The next day, the membrane was washed three times with TBST for 10 min each time, and then incubated for 2 h at room temperature with secondary antibody. After washing three times with TBST, ECL chemiluminescence was used for color development. The gray values of the bands were analyzed using ImageJ software. Using GAPDH as an internal control, the relative expression level of the target protein was calculated, and then quantitative analysis was performed. Figure 6 .
[0047] Statistical analysis of the experimental data from Examples 1-5 showed that KO has a good anti-fibrotic effect.
[0048] In summary, this invention creatively proposes the application of matrine O (KO), a flavonoid compound obtained by isolating and purifying the ethyl acetate fraction of matrine, in the preparation of drugs for treating liver fibrosis. It can also be used to prepare drugs that inhibit the upregulation of FN, α-SMA, and COL-1 protein levels, as well as drugs that inhibit the upregulation of FN1, ACTA2, and COL1A transcriptional levels. Furthermore, it can be used to reduce the release of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice, and the fibrosis marker hydroxyproline (HYP) in mouse liver tissue. This invention possesses excellent drug development potential and broad value and prospects for development, transformation, and clinical application.
[0049] In summary, this invention provides for the first time the application of matrine O 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 matrine O in the preparation of anti-hepatic fibrosis drugs.
2. The application of matrine O as described in claim 1 in the preparation of anti-hepatic fibrosis drugs, characterized in that, The matrine O has the molecular formula C0. 27 H 30 O 13 Its molecular weight is 562.
3. The application of matrine O as described in claim 1 in the preparation of anti-hepatic fibrosis drugs, characterized in that, The specific structural formula of matrine O is as follows: 。 4. The application of matrine O 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 O 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 O 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 O 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 or activation of hepatic stellate cells.
8. The application of matrine O 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 or activation of hepatic stellate cells.
9. The application of matrine O 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 O 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.