Application of cat's eye grass extract and pharmaceutical preparation for preventing and treating liver fibrosis

Abstract

The application provides application of cat's eye grass extract and a drug preparation for preventing and treating liver fibrosis, and belongs to the technical field of traditional Chinese medicine preparations. Effective components of the cat's eye grass extract contain a compound shown in the following formula: Fibrosis includes liver fibrosis caused by chemical liver injury, alcoholic liver injury, non-alcoholic fatty liver disease, viral hepatitis, cholestatic liver injury, drug-induced liver injury or autoimmune liver disease. The application provides a drug scheme with clear source, simple preparation and controllable quality for prevention and treatment of liver fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation technology, and specifically relates to the application of cat's eye grass extract and drug preparations for the prevention and treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis is a common pathological process caused by the long-term effects of various chronic liver injury factors. It is mainly characterized by excessive deposition and abnormal distribution of extracellular matrix in liver tissue, leading to the destruction of normal liver structure and impaired liver function. Viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, cholestasis, drug or chemical toxin damage, and autoimmune liver disease can all induce liver fibrosis.

[0003] Euphorbia hirta, a plant belonging to the genus Euphorbia in the family Euphorbiaceae, contains various chemical components, including terpenoids, flavonoids, steroids, phenylpropanoids, and phenols. Studies have shown that Euphorbia hirta possesses antibacterial, antitumor, and antioxidant activities, and has shown good therapeutic effects clinically on lung cancer, gastric cancer, lymph node tuberculosis, rheumatic immune diseases, and psoriasis. The application of Euphorbia hirta extract in liver-related diseases is relatively limited; current research focuses primarily on its effects on liver cancer.

[0004] Although both liver fibrosis and liver cancer can occur in the context of chronic liver disease, they are not the same disease state. Furthermore, liver fibrosis indicators directly point to hepatic stellate cell activation, TGF-β / Smad pathway activation, and collagen deposition, which are significantly different from oncology indicators used in liver cancer research. Liver fibrosis is a reparative and scarring process following chronic liver injury, and its specific evaluation indicators include hepatic stellate cell activation indicators Acta2 / α-SMA, extracellular matrix deposition indicators COL1A1, COL3A1, Collagen I, and Fibronectin / FN1, and TGF-β / Smad pathway indicators TGFB1, TGF-β1, and p-Smad2 / 3. Liver cancer, on the other hand, is a neoplastic disease resulting from the malignant transformation of hepatocytes or intrahepatic bile duct epithelial cells, and its detection indicators are typically oncology indicators such as tumor cell proliferation, apoptosis, invasion, migration, and tumor growth. Currently, there is no application of *Cephalotaxus fortunei* extract in the prevention and treatment of liver fibrosis, especially since the core effective components for preventing and treating liver fibrosis are not clearly identified. Summary of the Invention

[0005] In view of this, the present invention provides the application of cat's eye grass extract and a preparation for preventing and treating liver fibrosis, aiming to solve at least one of the technical problems in the background art.

[0006] This invention is implemented as follows: The first aspect of this invention provides the application of cat's eye grass extract, which is used to prepare a pharmaceutical preparation for the prevention and treatment of liver fibrosis; The active ingredient in the cat's eye grass extract includes the compound shown in the following formula: .

[0007] Furthermore, the active ingredient of the cat's eye grass extract is a compound represented by the following formula: .

[0008] Furthermore, the liver fibrosis includes liver fibrosis caused by chemical liver injury, alcoholic liver injury, non-alcoholic steatohepatitis, viral hepatitis, cholestatic liver injury, drug-induced liver injury, or autoimmune liver disease.

[0009] Furthermore, the cat's eye grass extract has at least one of the following functions: (1) Inhibits the activation of hepatic stellate cells; (2) Inhibits the proliferation or migration of hepatic stellate cells; (3) Reduce extracellular matrix deposition; (4) Reduce the expression or secretion of liver fibrosis markers; (5) Inhibit the TGF-β / Smad signaling pathway; (6) Improves liver tissue inflammation and collagen deposition.

[0010] Furthermore, the liver fibrosis markers include at least one of the gene encoding α-SMA, Acta2, COL1A1, COL3A1, Collagen I, Collagen III, Fibronectin, FN1, TIMP1, p-Smad2 / 3, TGF-β1, or the TGF-β1 encoding gene TGFB1.

[0011] A second aspect of the present invention provides a pharmaceutical preparation for the prevention and treatment of liver fibrosis, the pharmaceutical preparation comprising cat's eye grass extract and pharmaceutically acceptable excipients; The active ingredient in the cat's eye grass extract includes the compound shown in the following formula: .

[0012] Furthermore, the liver fibrosis includes liver fibrosis caused by chemical liver injury, alcoholic liver injury, non-alcoholic steatohepatitis, viral hepatitis, cholestatic liver injury, drug-induced liver injury, or autoimmune liver disease.

[0013] Furthermore, the preparation method of the cat's eye grass extract includes the following steps: S1. Take the dried whole herb or above-ground parts of cat's eye grass, crush them, sieve them, and then reflux them for alcohol extraction to obtain cat's eye grass alcohol extract. S2. The cat's eye herb alcohol extract is concentrated under reduced pressure to obtain cat's eye herb alcohol extract paste; the cat's eye herb alcohol extract paste is dispersed in water, and then extracted sequentially with petroleum ether, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively; the ethyl acetate fraction is selected as the cat's eye herb extract.

[0014] Compared with the prior art, the present invention provides the use of cat's eye grass extract or effective parts in drugs for the prevention and treatment of liver fibrosis, and provides support for the industrialization of traditional Chinese medicine preparations for the prevention and treatment of liver fibrosis.

[0015] The experimental results of this invention all revolve around the pathological process of liver fibrosis. Specifically, through the TGF-β1-induced LX-2 hepatic stellate cell activation model, it was demonstrated that *Corydalis yanhusuo* extract can reduce the expression of fibrosis-related genes such as Acta2, COL1A1, COL3A1, FN1, TIMP1, and TGFB1, and reduce the levels of fibrosis-related proteins such as α-SMA, Collagen I, Fibronectin, and p-Smad2 / 3. Through the carbon tetrachloride-induced liver fibrosis animal model, it was further demonstrated that *Corydalis yanhusuo* extract can reduce the levels of ALT, AST, and hydroxyproline, and reduce the collagen deposition area shown by Masson's Red and Sirius Red staining. Attached Figure Description

[0016] Figure 1 This is an HPLC comparison spectrum of the effective fraction of ethyl acetate and the standard MOL010155 in Example 1 of this invention; Figure 2 This is a primary mass spectrum of the effective fraction of ethyl acetate in Example 1 of the present invention; Figure 3 This is a secondary mass spectrum of the effective fraction of ethyl acetate in Example 1 of the present invention; Figure 4 This is a preparative HPLC separation and purification diagram of the effective fraction of ethyl acetate in Example 1 of the present invention; Figure 5 The effective fraction of ethyl acetate in Example 1 of this invention 1 H-NMR spectrum; Figure 6 The effective fraction of ethyl acetate in Example 1 of this invention 13 C-NMR spectrum; Figure 7 This is a standard line drawing of the present invention, MOL010155. Figure 8 This is a graph showing the expression levels of fibrosis-related genes in vitro in Example 2 of the present invention. Figure 9 This is a comparison diagram of the expression of fibrosis-related proteins in each group in Example 2 of the present invention; Figure 10This is a bar chart comparing the expression of fibrosis-related proteins in each group in Example 2 of the present invention; Figure 11 This is a bar chart comparing the EdU experimental results of each group in Example 2 of the present invention; Figure 12 This is a comparative bar chart of the scratch test results of each group in Example 2 of the present invention; Figure 13 This is a bar chart comparing the cell migration experiment results of each group in Example 2 of the present invention; Figure 14 This is a comparative bar chart of serum ALT levels in each group in Example 3 of the present invention; Figure 15 This is a comparative bar chart of serum AST levels in each group in Example 3 of the present invention; Figure 16 This is a comparative bar chart of hydroxyproline in liver tissues of each group in Example 3 of the present invention; Figure 17 This is a comparative bar chart of the staining area of ​​each group in Example 3 of the present invention; Figure 18 This is a comparative bar chart of the Sirius red staining area in each group in Example 3 of the present invention; Figure 19 This is a bar chart comparing the expression of fibrosis-related genes in vivo in each group in Example 3 of the present invention; Figure 20 This is a comparative bar chart of serum ALT levels in each group in Example 4 of the present invention; Figure 21 This is a comparative bar chart of serum AST levels in each group in Example 4 of the present invention; Figure 22 This is a comparative bar chart of hydroxyproline in liver tissues of each group in Example 4 of the present invention; Figure 23 This is a comparative bar chart of the staining area of ​​each group in Example 4 of the present invention; Figure 24 This is a comparative bar chart of the Sirius red staining area in each group in Example 4 of the present invention; Figure 25 This is a bar chart comparing the relative mRNA expression of Acta2 in liver tissues of each group in Example 4 of the present invention; Figure 26 This is a bar chart comparing the relative mRNA expression of COL1A1 in liver tissue of each group in Example 4 of the present invention; Figure 27 This is a bar chart comparing the relative mRNA expression of TGFB1 in liver tissues of each group in Example 4 of the present invention. Figure 28 This is a bar chart comparing the relative mRNA expression of TIMP1 in liver tissues of each group in Example 4 of the present invention; Figure 29 This is a bar chart showing the relative expression of p-Smad2 / 3 protein in liver tissues of each group in Example 4 of the present invention. Figure 30 This is a comparative bar chart of serum ALT levels in each group in Example 5 of the present invention; Figure 31 This is a comparative bar chart of serum AST levels in each group in Example 5 of the present invention; Figure 32 This is a comparative bar chart of hydroxyproline in liver tissues of each group in Example 5 of the present invention; Figure 33 This is a comparative bar chart of the staining area of ​​each group in Example 5 of the present invention; Figure 34 This is a comparative bar chart of the Sirius red staining area in each group in Example 5 of the present invention; Figure 35 This is a bar chart comparing the relative mRNA expression of Acta2 in liver tissues of each group in Example 5 of the present invention; Figure 36 This is a bar chart comparing the relative mRNA expression of COL1A1 in liver tissue of each group in Example 5 of the present invention; Figure 37 This is a bar chart comparing the relative mRNA expression of TGFB1 in liver tissues of each group in Example 5 of the present invention; Figure 38 This is a bar chart comparing the relative mRNA expression of TIMP1 in liver tissues of each group in Example 5 of the present invention; Figure 39 This is a bar chart showing the relative expression of p-Smad2 / 3 protein in liver tissue of each group in Example 5 of the present invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0018] All raw materials or reagents used in this invention are commercially available, and their manufacturers and product numbers are shown in Table A.

[0019] Table A

[0020] Example 1 The specific steps for preparing cat's eye grass extract in this embodiment are as follows: S1. Take the dried whole herb or aerial parts of *Corydalis yanhusuo*, pulverize them, and pass them through a 50-mesh sieve to obtain *Corydalis yanhusuo* herbal powder. Add 10 times the amount of 70% ethanol aqueous solution to the *Corydalis yanhusuo* herbal powder, reflux and extract twice, 2 hours each time, filter, combine the filtrates, and obtain the *Corydalis yanhusuo* ethanol extract.

[0021] S2. The cat's eye herb alcohol extract was concentrated under reduced pressure to obtain a cat's eye herb alcohol extract paste. The above cat's eye herb alcohol extract paste was dispersed in an appropriate amount of water and extracted sequentially with petroleum ether, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. Preferably, the ethyl acetate fraction was selected as the effective fraction of cat's eye herb for anti-liver fibrosis, i.e., the cat's eye herb extract.

[0022] To identify the main characteristic components in the cat's eye herb extract, the effective fraction of cat's eye herb obtained in S1 and S2 of this example was subjected to detection, separation, structural identification, and content determination using MOL010155. The chemical name of MOL010155 is (5S,6R,7R,8S)-5,6,7,8-tetrahydroxy-2-[2-(2-hydroxyphenyl)ethyl]-5,6,7,8-tetrahydrochromen-4-one, and its structural formula is as follows: .

[0023] Take appropriate amounts of the ethanol extract and ethyl acetate effective fraction of *Corydalis yanhusuo* obtained in Example 1, dissolve them in methanol and make up to volume, sonicate, centrifuge, and take the supernatant for HPLC or UPLC analysis. Use MOL010155 standard as a control. Figure 1 As shown in the figure. The results showed that a chromatographic peak with the same retention time as the MOL010155 standard was detected in the sample, with a retention time of approximately 12.8 min, indicating that the cat's eye grass extract contains MOL010155 or a component with the same chromatographic behavior.

[0024] Further LC-MS analysis was performed on the above target chromatographic peaks, such as... Figure 2 As shown in the figure. The results show that the target peak appears as a quasi-molecular ion peak [M+H] at m / z 335.1 in the primary mass spectrometer. + With the molecular formula C of MOL010155 17 H 18 The molecular weight corresponding to O7 is consistent. Continue with MS / MS secondary mass spectrometry analysis, such as... Figure 3 As shown, major fragment ions with m / z values ​​of 317.1, 299.1, 181.1, and 153.0 are visible, and their fragment ion characteristics are basically consistent with those of the MOL010155 standard. These results indicate that the target component in the cat's eye grass extract can be preliminarily identified as MOL010155.

[0025] To further confirm the structure of this component, preparative HPLC was used to separate and purify the target peak, yielding the monomeric compound. HPLC analysis showed that the monomer peak was relatively uniform and its purity was greater than 98%. Figure 4 As shown. The obtained monomer was subjected to nuclear magnetic resonance analysis. Figure 5 of 1 H-NMR results showed that it had characteristic hydrogen signals at δ 7.62 ppm, δ 6.85 ppm, δ 6.32 ppm, δ 5.17 ppm, δ 3.83 ppm, and δ 2.38 ppm; Figure 6 of 13 C-NMR results showed characteristic carbon signals at δ 171.6 ppm, δ 163.3 ppm, δ 129.8 ppm, δ 116.5 ppm, δ 103.9 ppm, δ 78.6 ppm, δ 61.3 ppm, δ 56.1 ppm, and δ 20.8 ppm. Combined with key correlation signals in the two-dimensional NMR spectrum, the structure of the obtained monomeric compound was confirmed to be consistent with MOL010155.

[0026] Based on this, the content of MOL010155 in the ethanol extract, ethyl acetate fraction, n-butanol fraction, and total flavonoid fraction of *Corydalis yanhusuo* obtained from two different batches of *Corydalis yanhusuo* was determined by HPLC external standard method. The total flavonoid fraction of *Corydalis yanhusuo* was obtained by purifying the ethanol extract of *Corydalis yanhusuo* using a macroporous adsorption resin column, eluting sequentially with water, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol, collecting the 50%–70% ethanol eluent, combining them, concentrating, and drying to obtain the total flavonoid fraction of *Corydalis yanhusuo*. A series of concentration solutions were prepared using MOL010155 standard to establish a standard curve, as shown below. Figure 7 As shown in the table below, MOL010155 exhibits good linearity within the detection concentration range, with a standard curve of y = 12480x + 6850 and a correlation coefficient R² = 0.9854. Different samples were tested, and the results are presented in Table 1 below. Data are expressed as mean ± standard deviation, with five parallel samples in each group.

[0027] Table 1

[0028] The above results indicate that MOL010155 can be stably detected in *Corydalis yanhusuo* extract and is relatively enriched in the ethyl acetate fraction, with a significantly higher content of MOL010155 in the ethyl acetate fraction than in the ethanol extract. HPLC / UPLC retention time comparison, LC-MS molecular ion peak analysis, MS / MS fragment ion analysis, preparative HPLC separation and purification, and NMR structure identification confirm the presence of MOL010155 in *Corydalis yanhusuo* extract. MOL010155 can be considered one of the characteristic components of the *Corydalis yanhusuo* extract or the ethyl acetate fraction of this invention, and can also be used as one of the detection indicators for subsequent quality control of the pharmaceutical composition.

[0029] The obtained extracts can be quality controlled by HPLC, UPLC or LC-MS / MS, with total flavonoids, total phenolic acids, cat's eye phenol D, quercetin, kaempferol or other characteristic components as detection indicators.

[0030] Example 2 The efficacy of the cat's eye grass extract (ethyl acetate effective fraction) prepared in Example 1 in preventing and treating liver fibrosis was experimentally analyzed.

[0031] Hepatic stellate cells are key effector cells in the development and progression of liver fibrosis. Under chronic injury stimulation, resting hepatic stellate cells transform into myofibroblast-like cells, characterized by increased α-SMA expression, enhanced cell proliferation and migration, and the secretion of large amounts of extracellular matrix components such as type I collagen, type III collagen, and fibronectin. The TGF-β1 / Smad signaling pathway is an important pathway driving hepatic stellate cell activation and collagen deposition. Inhibiting the expression of this pathway and its downstream fibrosis markers is an important direction for the development of anti-hepatic fibrosis drugs. The experimental indicators were selected to match liver fibrosis and mainly included α-SMA, Acta2, COL1A1, COL3A1, Collagen I, fibronectin, TGF-β1, p-Smad2 / 3, hydroxyproline, Masson staining, and Sirius Red staining positive area; these indicators directly point to hepatic stellate cell activation and collagen deposition.

[0032] Human hepatic stellate cells (LX-2) were harvested and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. Experiments were performed when the cells reached approximately 70%–80% confluence. Before the experiment, LX-2 cells were seeded in 6-well plates, and after cell attachment, the medium was replaced with low-serum medium for synchronization.

[0033] The experimental groups are as follows: (1) Control group: No TGF-β1 and cat's eye grass extract were added, only an equal volume of culture medium or solvent was added for treatment; (2) Model group, namely TGF-β1 group: TGF-β1 was added to the treatment at a final concentration of 5 ng / mL; (3) TGF-β1 + Cat's Eye Extract Low-dose group, namely TGF-β1 + MEC-L group: Cat's Eye Extract with a final concentration of 5 μg / mL was added for pretreatment for 2 h, and then TGF-β1 with a final concentration of 5 ng / mL was added for further treatment. (4) TGF-β1 + Cat's Eye Extract Medium-Dose Group, namely TGF-β1 + MEC-M Group: Cat's Eye Extract with a final concentration of 15 μg / mL was added for pretreatment for 2 h, and then TGF-β1 with a final concentration of 5 ng / mL was added for further treatment. (5) High-dose group of TGF-β1+cat's eye extract, namely TGF-β1+MEC-H group: cat's eye extract with a final concentration of 20 μg / mL was added for pretreatment for 2 h, and then TGF-β1 with a final concentration of 5 ng / mL was added for further treatment.

[0034] In this experiment, cat's eye grass extract was prepared into a stock solution using a suitable solvent and then added to the culture medium, with the final solvent concentration remaining consistent across all experimental groups. For qPCR detection, cells were collected from each group 24 hours after treatment; Western blot, EdU assay, scratch assay, and Transwell assay (cell migration assay) could be performed 48 hours after treatment.

[0035] I. Inhibitory effect of cat's eye grass extract on TGF-β1-induced activation of hepatic stellate cells After 24 hours of treatment, the expression of genes such as Acta2, COL1A1, COL3A1, FN1, TIMP1, and TGFB1 was detected by qPCR. The results for each group are shown in Table 2. Figure 8 As shown, Figure 8 In the mean group, compared with the control group, ### indicates p<0.001 (extremely significant difference), and ## indicates p<0.01 (highly significant difference); compared with the TGF-β1 group, This indicates that p < 0.001 (the difference is extremely significant). This indicates that p < 0.01 (the difference is highly significant). This indicates that p < 0.05 (significant difference).

[0036] Table 2

[0037] The results showed that, compared with the TGF-β1 model group, the extract of *Corydalis yanhusuo* reduced the expression of fibrosis-related genes in a dose-dependent manner, suggesting that it can inhibit the activation of hepatic stellate cells.

[0038] II. Effects of Cat's Eye Grass Extract on Fibrosis-Related Proteins and TGF-β / Smad Signaling After 24 hours of treatment, the expression of fibrosis-related proteins α-SMA, Collagen I, Fibronectin, p-Smad2 / 3, Smad2 / 3, and GAPDH was detected by Western blot. The results are shown in Table 3. Figure 9 and Figure 10 As shown. Figure 10 In the mean group, compared with the control group, ### indicates p<0.001 (extremely significant difference), and ## indicates p<0.01 (highly significant difference); compared with the TGF-β1 group, This indicates that p < 0.001 (the difference is extremely significant). This indicates that p < 0.01 (the difference is highly significant). This indicates that p < 0.05 (significant difference).

[0039] Table 3

[0040] The results showed that TGF-β1 significantly upregulated α-SMA, Collagen I, Fibronectin, and p-Smad2 / 3; the expression of these proteins decreased after treatment with cat's eye grass extract, while Smad2 / 3 and GAPDH showed no significant changes.

[0041] The results showed that TGF-β1 treatment increased the expression of α-SMA, Collagen I, Fibronectin, and p-Smad2 / 3 in LX-2 cells, while the expression of total Smad2 / 3 did not change significantly. Treatment with *Corydalis yanhusuo* extract decreased the expression of α-SMA, Collagen I, and Fibronectin, and also reduced the p-Smad2 / 3 to Smad2 / 3 ratio. These results suggest that *Corydalis yanhusuo* extract can reduce hepatic stellate cell activation and extracellular matrix synthesis by inhibiting the activation of the TGF-β / Smad signaling pathway.

[0042] III. Effects of Cat's Eye Grass Extract on Hepatic Stellate Cell Proliferation and Migration After 24 hours of treatment, cell proliferation capacity was assessed using the EdU (5-ethynyl-2'-deoxyuridine) assay. Figure 11 As shown; cell migration ability was detected using scratch assay and Transwell assay (cell migration assay), such as... Figure 12 and Figure 13As shown in the figure. The results showed that TGF-β1 enhanced the proliferation and migration of LX-2 cells; the EdU positivity rate, scratch healing rate and Transwell cell number were all reduced after treatment with cat's eye grass extract. Figures 11 to 13 In comparison with the TGF-β1 group, This indicates that p < 0.001 (the difference is extremely significant). This indicates that p < 0.01 (the difference is highly significant). This indicates that p < 0.05 (significant difference).

[0043] The above results suggest that cat's eye grass extract not only inhibits the expression of fibrosis markers, but also inhibits the pro-fibrotic functional phenotype of hepatic stellate cells.

[0044] Example 3 Liver fibrosis includes chemically induced liver fibrosis, alcoholic liver disease-related liver fibrosis, non-alcoholic steatohepatitis-related liver fibrosis, viral hepatitis-related liver fibrosis, cholestatic liver fibrosis, drug-induced liver injury-related liver fibrosis, or autoimmune liver disease-related liver fibrosis. Chemically induced liver fibrosis is liver fibrosis induced by carbon tetrachloride, thioacetamide, or dimethylnitrosamine; non-alcoholic steatohepatitis-related liver fibrosis is liver fibrosis induced by a high-fat, high-cholesterol diet or a methionine-choline-deficient diet.

[0045] Liver fibrosis induced by different etiologies varies in terms of initiating injury factors, inflammatory background, hepatocyte steatosis, or cholestasis. However, their common terminal pathological processes all include hepatic stellate cell activation, enhanced TGF-β / Smad signaling, and collagen and ECM deposition. Therefore, this embodiment uses a carbon tetrachloride-induced model as a classic model of chemically induced liver injury-induced liver fibrosis to evaluate the anti-hepatic fibrosis effect of *Cephalotaxus fortunei* extract.

[0046] Modeling Experiment: Male C57BL / 6 mice aged 6-8 weeks, weighing 18g-22g, were selected and acclimatized for 7 days before the experiment. Animals were randomly divided into groups of 8. Except for the control group, all other groups were induced to develop an experimental liver fibrosis model using carbon tetrachloride. Specifically, carbon tetrachloride was diluted with olive oil to prepare a 10% carbon tetrachloride solution, which was injected intraperitoneally at a dose of 2mL / kg twice a week for 8 consecutive weeks; the control group received an equal volume of olive oil.

[0047] The animals were randomly grouped, as follows: Control group: Administered an equal volume of olive oil via intraperitoneal injection and an equal volume of solvent via gavage daily; Model group (CCl4): A liver fibrosis model was established by intraperitoneal injection of 10% carbon tetrachloride solution, and an equal volume of solvent was administered by gavage daily. Positive drug group (CCl4+ silymarin): A liver fibrosis model was established by intraperitoneal injection of 10% carbon tetrachloride solution. Starting from the 5th week of modeling, silymarin was administered by gavage at a dose of 100 mg / kg daily for 4 consecutive weeks. Low-dose group of cat's eye grass extract (CCl4+MEC-L): A liver fibrosis model was established by intraperitoneal injection of 10% carbon tetrachloride solution. Starting from the 5th week of modeling, cat's eye grass extract was administered by gavage at a dose of 25 mg / kg daily for 4 consecutive weeks. Medium-dose group of cat's eye grass extract (CCl4+MEC-M): A liver fibrosis model was established by intraperitoneal injection of 10% carbon tetrachloride solution. Starting from the 5th week of modeling, cat's eye grass extract was administered by gavage at a dose of 50 mg / kg daily for 4 consecutive weeks. High-dose group of cat's eye grass extract (CCl4+MEC-H): A liver fibrosis model was established by intraperitoneal injection of 10% carbon tetrachloride solution. Starting from the 5th week of modeling, cat's eye grass extract was administered by gavage at a dose of 100 mg / kg daily for 4 consecutive weeks.

[0048] After the last administration, the animals were fasted for 12 hours, anesthetized, and blood was collected. The serum was separated by centrifugation and used to detect ALT and AST levels. Subsequently, the animals were euthanized and liver tissue was collected. Part of the liver tissue was used to detect hydroxyproline content, part of the liver tissue was fixed and stained with Masson and Sirius Red, and part of the liver tissue was frozen and used for qPCR detection of fibrosis-related gene expression.

[0049] I. Therapeutic effect of cat's eye grass extract on experimental animal models of liver fibrosis After the last administration, mice in each group were fasted for 12 hours but allowed free access to water. Mice were then anesthetized, and blood samples were collected via orbital sampling or abdominal aortic sampling. After standing, serum was separated by centrifugation for detecting serum ALT and AST levels. Mice were euthanized after blood collection, and liver tissue was rapidly separated. A portion of the liver tissue was weighed for hydroxyproline content detection; another portion was fixed in 4% paraformaldehyde, routinely embedded in paraffin, sectioned, and stained with Masson's Law and Sirius Red to observe and count collagen deposition area; a third portion of liver tissue was flash-frozen in liquid nitrogen and stored for subsequent qPCR or Western blot detection. The results of serum ALT, AST, and liver tissue hydroxyproline content detection are shown in Table 4. Figures 14 to 16 As shown, the results of Masson staining and Sirius red staining are as follows: Figure 17 and Figure 18 As shown. Figures 14 to 18In the mean group, compared with the control group, ### indicates p<0.001 (extremely significant difference), and ## indicates p<0.01 (highly significant difference); compared with the model group (CCl4), This indicates that p < 0.001 (the difference is extremely significant). This indicates that p < 0.01 (the difference is highly significant). This indicates that p < 0.05 (significant difference).

[0050] Table 4

[0051] The results showed that ALT, AST, hydroxyproline and collagen deposition area increased in the model group; the above indicators decreased after treatment with cat's eye grass extract, and showed a dose-dependent improvement trend.

[0052] II. Effects of Cat's Eye Grass Extract on the Expression of Genes Related to Liver Fibrosis in Animals Total RNA was extracted from liver tissue of animals in each group, and the expression of genes such as Acta2, COL1A1, TGFB1, and TIMP1 was detected by qPCR. The results are shown in Table 5. Figure 19 As shown. Figure 19 In comparison with the control group, ## indicates p<0.01 (highly significant difference); compared with the model group (CCl4), This indicates that p < 0.001 (the difference is extremely significant). This indicates that p < 0.01 (the difference is highly significant). This indicates that p < 0.05 (significant difference).

[0053] Table 5

[0054] The results showed that the expression of fibrosis-related genes was significantly increased in the model group, while the expression of these genes was significantly reduced in the medium and high dose groups of cat's eye extract.

[0055] These results are consistent with those obtained from in vitro LX-2 cell experiments, suggesting that cat's eye grass extract can inhibit the molecular network associated with liver fibrosis at the cellular and animal levels.

[0056] Example 4 To further compare the effects of different chemical components in cat's eye grass on anti-liver fibrosis, and to demonstrate that the contributions of other mixed components besides MOL010155 to the anti-liver fibrosis effect of this invention are weak or not significant, animal experiments were conducted to evaluate the other mixed components in cat's eye grass extract after removing MOL010155.

[0057] The effective fraction of ethyl acetate from *Corydalis yanhusuo* obtained in Example 1 was separated by preparative HPLC or column chromatography. Other eluents with peaks different from MOL010155 were collected and combined to obtain other mixed components A, other mixed components B, and other mixed components C. Among them, other mixed component A is the adjacent eluent after removing MOL010155, other mixed component B is a mixed component with a different polarity from MOL010155, and other mixed component C is other low-content mixed components. Each mixed component was analyzed by HPLC or LC-MS to confirm that it did not contain MOL010155 or that the content of MOL010155 was below the detection limit.

[0058] Based on Example 3, other mixed components A, B, and C were added as control groups to evaluate the effects of the other mixed components after the removal of MOL010155 on experimental liver fibrosis. All control groups used the carbon tetrachloride-induced liver fibrosis model of Example 3. Except for the different drug components, the modeling, drug administration, sample collection, and detection steps were consistent with Example 3.

[0059] Other Mixed Components A: Mice with carbon tetrachloride-induced liver fibrosis model were treated with other mixed components A; Other Mixed Components B: Mice with carbon tetrachloride-induced liver fibrosis model were treated with other mixed components B; Other mixed component C group: Mice with carbon tetrachloride-induced liver fibrosis model were treated with other mixed component C.

[0060] The dosage for other mixture A, other mixture B, and other mixture C was 30 mg / kg. The other steps of the experiment were the same as in Example 3.

[0061] After drug administration, mouse serum and liver tissue were collected. Serum ALT and AST levels were detected using an automated biochemical analyzer; liver tissue hydroxyproline content was detected using a hydroxyproline assay kit; collagen deposition area in liver tissue was evaluated using Masson's staining and Sirius Red staining; expression of fibrosis-related genes such as Acta2, COL1A1, TGFB1, and TIMP1 in liver tissue was detected using qPCR; and expression of TGF-β / Smad pathway-related proteins such as p-Smad2 / 3 was detected using Western blot. The results are shown in Table 6. Figures 20 to 29 As shown, ### represents the comparison with the control group. p <0.001 (the difference is extremely significant), ns indicates no significant difference.

[0062] Table 6

[0063] The results showed that, compared with the normal control group, the model group (CCl4) mice had significantly increased serum ALT and AST levels, increased liver tissue hydroxyproline content, significantly increased positive areas of Masson's staining and Sirius red staining, and enhanced expression of fibrosis-related indicators such as Acta2, COL1A1, TGFB1, TIMP1, and p-Smad2 / 3, indicating that the liver fibrosis model was successfully established. Compared with the model group (CCl4), treatment with other mixed components A, B, and C did not significantly reduce serum ALT and AST levels, and there was no significant improvement in liver tissue hydroxyproline content, Masson's staining positive area, Sirius red staining positive area, or expression of fibrosis-related genes and proteins; the differences were not statistically significant.

[0064] Note: The model group (CCl4) showed a significant difference compared to the control group; there were no statistically significant differences among the other mixture groups (CCl4, mixture A, mixture B, and mixture C) compared to the model group (CCl4).

[0065] The above results indicate that, except for MOL010155, other components of *Corydalis yanhusuo* (cat's eye herb) did not show significant improvement in the CCl4-induced experimental liver fibrosis mouse model, and could not significantly reduce liver injury indicators, collagen deposition indicators, or TGF-β / Smad pathway-related fibrosis indicators. Considering the enrichment of MOL010155 in the effective fraction of *Corydalis yanhusuo* ethyl acetate and the ameliorative effect of *Corydalis yanhusuo* extract on liver fibrosis-related indicators, this suggests that MOL010155 is one of the important characteristic active components in *Corydalis yanhusuo* extract that exerts an anti-liver fibrosis effect.

[0066] Example 5 To directly evaluate the anti-hepatic fibrosis effect of pure MOL010155, this example uses commercially available pure 2'-Hydroxylagarotetrol for efficacy evaluation. The 2'-Hydroxylagarotetrol used in the experiment was purchased from MedChemExpress (MCE, catalog number HY-N11966, 5mg, CAS No. 135308-83-7), with the molecular formula C2. 17 H 18 O7, with a molecular weight of 334.32. Chemical structure information verification confirmed that this compound corresponds to the same chemical structure as MOL010155 described in this invention; hereinafter referred to as pure MOL010155. The efficacy was evaluated using the same CCl4-induced experimental liver fibrosis mouse model as in Example 3.

[0067] The results of Example 1 showed that the content of MOL010155 in the effective fraction of ethyl acetate was approximately 1.72 mg / g to 1.86 mg / g. Based on the dosages of 25 mg / kg, 50 mg / kg, and 100 mg / kg of *Corydalis yanhusuo* extract in Example 3, the theoretical dosages of MOL010155 were approximately 0.043 mg / kg to 0.047 mg / kg, 0.086 mg / kg to 0.093 mg / kg, and 0.172 mg / kg to 0.186 mg / kg, respectively. To facilitate actual preparation and administration, and to maintain a roughly comparable ratio between the pure product dosage and the exposure to MOL010155 in *Corydalis yanhusuo* extract, the low, medium, and high doses of pure MOL010155 in this example were set at 0.05 mg / kg, 0.10 mg / kg, and 0.20 mg / kg, respectively.

[0068] Male C57BL / 6 mice aged 6-8 weeks, weighing 18g-22g, were selected and randomly divided into groups of 8 mice each after 7 days of acclimatization. Except for the control group, the other groups underwent liver fibrosis modeling by intraperitoneal injection of 10% carbon tetrachloride solution at 2mL / kg twice weekly for 8 consecutive weeks, as described in Example 3. Intervention began in the 5th week of modeling and continued for 4 weeks.

[0069] Control group: Administered an equal volume of olive oil via intraperitoneal injection and an equal volume of solvent via gavage daily; Model group (CCl4): A liver fibrosis model was established by intraperitoneal injection of 10% carbon tetrachloride solution, and an equal volume of solvent was administered by gavage daily. Positive drug group (CCl4+ silymarin): A liver fibrosis model was established by intraperitoneal injection of 10% carbon tetrachloride solution. Starting from the 5th week of modeling, silymarin was administered by gavage at a dose of 100 mg / kg daily for 4 consecutive weeks. The low-dose group of MOL010155 (CCl4+MOL-L): A liver fibrosis model was established by intraperitoneal injection of 10% carbon tetrachloride solution. Starting from the 5th week of modeling, pure MOL010155 was administered by gavage at a dose of 0.05 mg / kg daily for 4 consecutive weeks. MOL010155 medium-dose group (CCl4+MOL-M): A liver fibrosis model was established by intraperitoneal injection of 10% carbon tetrachloride solution. Starting from the 5th week of modeling, pure MOL010155 was administered by gavage at a dose of 0.10 mg / kg daily for 4 consecutive weeks. High-dose group of MOL010155 (CCl4+MOL-H): A liver fibrosis model was established by intraperitoneal injection of 10% carbon tetrachloride solution. Starting from the 5th week of modeling, pure MOL010155 was administered by gavage at a dose of 0.20 mg / kg daily for 4 consecutive weeks.

[0070] After the last administration, patients fasted for 12 hours, were anesthetized, and blood was collected and serum was separated to detect ALT and AST levels. Liver tissue was taken to detect hydroxyproline content, and collagen deposition was evaluated by Masson staining and Sirius red staining. The expression of fibrosis-related genes such as Acta2, COL1A1, TGFB1, and TIMP1 was detected by qPCR.

[0071] The results of each indicator are shown in Table 7 and Figures 30 to 39 As shown. Figures 30 to 39 In the mean group, compared with the control group, ### indicates p<0.001 (extremely significant difference); compared with the model group (CCl4), This indicates that p < 0.001 (the difference is extremely significant). This indicates that p < 0.01 (the difference is highly significant). p < 0.05 indicates a significant difference, and ns indicates no statistical significance.

[0072] Table 7

[0073] The results showed that, compared with the normal control group, the model group (CCl4) mice had significantly increased serum ALT and AST levels, increased liver tissue hydroxyproline content, significantly increased positive areas of Masson staining and Sirius red staining, and enhanced expression of fibrosis-related indicators such as Acta2, COL1A1, TGFB1, TIMP1, and p-Smad2 / 3, indicating that the liver fibrosis model was successfully established. Compared with the model group (CCl4), treatment with pure MOL010155 resulted in decreased serum ALT and AST levels, decreased liver tissue hydroxyproline content, and decreased positive areas of Masson staining and Sirius red staining. Fibrosis-related indicators such as Acta2, COL1A1, TGFB1, TIMP1, and p-Smad2 / 3 were also reduced, showing a trend of improvement with increasing dosage.

[0074] The cat's eye grass extract containing MOL010155 can be formulated as the sole active ingredient against liver fibrosis in a pharmaceutical preparation, or as a compound pharmaceutical preparation with other active ingredients against liver fibrosis in the art. The mass percentage of the cat's eye grass extract or active fraction in the pharmaceutical composition is 0.01% to 99.9%, preferably 0.1% to 80%, more preferably 1% to 50%. Pharmaceutically acceptable excipients (one or more of diluents, binders, fillers, disintegrants, lubricants, stabilizers, antioxidants, surfactants, buffers, isotonic agents, pH adjusters, coating materials, sustained-release materials, liposome materials, nanoparticle materials, cyclodextrin, phospholipids, cholesterol, polyethylene glycol, poloxamer, and polylactic-co-glycolic acid copolymers) can be added to formulate dosage forms permissible in the art, such as tablets, capsules, granules, pills, powders, oral liquids, suspensions, emulsions, injections, liposomes, nanoparticles, microspheres, solid dispersions, or sustained-release formulations. Its administration routes include oral administration, injection, transdermal administration, enema administration, or liver-targeted delivery.

[0075] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of cat's eye grass extract, characterized in that, The cat's eye grass extract is used to prepare pharmaceutical formulations for the prevention and treatment of liver fibrosis; The active ingredient in the cat's eye grass extract includes the compound shown in the following formula: 。 2. The application of the cat's eye grass extract according to claim 1, characterized in that, The active ingredient of the cat's eye grass extract is a compound shown in the following formula: 。 3. The application of the cat's eye grass extract according to claim 1 or 2, characterized in that, The liver fibrosis includes liver fibrosis caused by chemical liver injury, alcoholic liver injury, non-alcoholic steatohepatitis, viral hepatitis, cholestatic liver injury, drug-induced liver injury, or autoimmune liver disease.

4. The application of the cat's eye grass extract according to claim 1 or 2, characterized in that, The cat's eye grass extract has at least one of the following functions: (1) Inhibits the activation of hepatic stellate cells; (2) Inhibits the proliferation or migration of hepatic stellate cells; (3) Reduce extracellular matrix deposition; (4) Reduce the expression or secretion of liver fibrosis markers; (5) Inhibit the TGF-β / Smad signaling pathway; (6) Improves liver tissue inflammation and collagen deposition.

5. The application of the cat's eye grass extract according to claim 4, characterized in that, The liver fibrosis markers include at least one of the genes encoding α-SMA, Acta2, COL1A1, COL3A1, Collagen I, Collagen III, Fibronectin, FN1, TIMP1, p-Smad2 / 3, TGF-β1, or TGF-β1 encoding TGFB1.

6. A pharmaceutical preparation for the prevention and treatment of liver fibrosis, characterized in that, The pharmaceutical preparation includes cat's eye grass extract and pharmaceutically acceptable excipients; The active ingredient in the cat's eye grass extract includes the compound shown in the following formula: 。 7. The pharmaceutical preparation for preventing and treating liver fibrosis according to claim 6, characterized in that, The liver fibrosis includes liver fibrosis caused by chemical liver injury, alcoholic liver injury, non-alcoholic steatohepatitis, viral hepatitis, cholestatic liver injury, drug-induced liver injury, or autoimmune liver disease.

8. The pharmaceutical preparation for preventing and treating liver fibrosis according to claim 6, characterized in that, The preparation method of the cat's eye grass extract includes the following steps: S1. Take the dried whole herb or above-ground parts of cat's eye grass, crush them, sieve them, and then reflux them for alcohol extraction to obtain cat's eye grass alcohol extract. S2. The cat's eye herb alcohol extract is concentrated under reduced pressure to obtain cat's eye herb alcohol extract paste; the cat's eye herb alcohol extract paste is dispersed in water, and then extracted sequentially with petroleum ether, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively; the ethyl acetate fraction is selected as the cat's eye herb extract.

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