Anti-hepatic fibrosis natural active substance and application thereof
By using rhubarb glycoside to inhibit the activation of hepatic stellate cells, the problem of limited efficacy of existing drugs has been solved, achieving significant anti-hepatic fibrosis effects and improving liver function, which has potential clinical application value.
Patent Information
- Application Number
- CN202511771955.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-28
- Publication Date
- 2026-02-17
AI Technical Summary
Existing anti-liver fibrosis drugs have limited efficacy and significant side effects, and the active anti-liver fibrosis components in privet fruit are unclear.
Using rhubarb glycoside as a natural active substance for anti-liver fibrosis, it inhibits the activation of hepatic stellate cells, reduces the expression levels of α-SMA, COL1α1 and TGF-β1, while promoting the expression of GFAP and reducing collagen fiber deposition.
Rhubarb glycosides significantly improve liver function and reduce the degree of liver fibrosis. They are more effective than colchicine, have higher safety, and have good prospects for clinical application.
Smart Images
Figure CN121534068A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a natural active substance that combats liver fibrosis and its application in the treatment of liver fibrosis. Background Technology
[0002] Hepatic fibrosis is a common pathological change in the liver and an important pathological stage in the development of various chronic liver diseases. After liver damage caused by factors such as alcohol consumption, viral infection, and parasitic infection, hepatic stellate cells (HSCs) in the liver tissue are activated and transformed into myofibroblasts, secreting a large amount of extracellular matrix (ECM). This leads to excessive deposition of ECM in the liver interstitium, eventually forming scar tissue, i.e., fibrosis.
[0003] The core event in liver fibrosis is the activation of HSCs. Under normal circumstances, HSCs are in a resting state, expressing resting markers such as glial fibrillary acidic protein (GFAP). When the liver is damaged, HSCs are activated and transform into myofibroblasts, expressing activation markers such as α-smooth muscle actin (α-SMA) and type I collagen (COL1α1), while also secreting profibrotic factors such as transforming growth factor-β1 (TGF-β1).
[0004] Currently, the main drugs used in clinical treatment of liver fibrosis include colchicine, silymarin, and glycyrrhizic acid, but these drugs all have limited efficacy and significant side effects. Therefore, the development of highly effective and low-toxicity anti-liver fibrosis drugs is of great clinical significance.
[0005] Privet fruit possesses various biological activities, including immunomodulatory and antioxidant effects. Previous studies have shown that privet fruit has certain therapeutic effects on liver fibrosis induced by CCl4 and Echinococcus multilocularis infection, but the specific active components have not yet been fully identified. Summary of the Invention
[0006] The purpose of this invention is to provide a natural active substance for anti-liver fibrosis, clarify its chemical composition and mechanism of action, and provide its application in the treatment of liver fibrosis, thus offering a new drug option for the clinical treatment of liver fibrosis. Specific experiments have shown that rhubarb glycosides can significantly inhibit the activation of hepatic stellate cells, significantly reduce CCl4-induced liver fibrosis, and effectively improve liver function.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] A natural active substance for combating liver fibrosis, wherein the active substance is rosin, and its molecular formula is C. 27 H 30 O 14The CAS number is 17306-46-6.
[0009] Preferably, the rhubarb glycoside can inhibit the activation of hepatic stellate cells, specifically by reducing the expression levels of α-smooth muscle actin (α-SMA), type I collagen (COL1α1), and transforming growth factor-β1 (TGF-β1), while promoting the expression of glial fibrillary acidic protein (GFAP).
[0010] By inhibiting hepatic stellate cell activation, reducing the expression levels of α-SMA, COL1α1, and TGF-β1, and promoting GFAP expression, the deposition of collagen fibers in liver tissue is reduced, thereby improving liver function.
[0011] Preferably, the use of rhubarb glycoside in the preparation of a drug for treating liver fibrosis.
[0012] Preferably, the drug is administered via intraperitoneal injection at a dose of 5 mg / kg and at a frequency of once every 10 days.
[0013] Preferably, the liver fibrosis is carbon tetrachloride (CCl4)-induced liver fibrosis.
[0014] This invention utilizes ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to determine the components of Ligustrum lucidum. Based on this, using a database of traditional Chinese medicine systems pharmacology and an integrated platform for traditional Chinese medicine pharmacology, and employing oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 as screening criteria, the components (with CAS numbers) in Ligustrum lucidum were searched. Known components including astragaloside and corosolic acid were identified from Ligustrum lucidum.
[0015] The English name of rhoifolin is Rhoifolin, CAS number is 17306-46-6, and molecular formula is C. 27 H 30 O 14 The molecular structure is:
[0016]
[0017] The inhibitory effect of rhubarb glycoside on the activation of hepatic stellate cells was then analyzed in vitro. Rhubarb glycoside significantly inhibited the expression of α-SMA, COL1α1, and TGF-β1 in activated hepatic stellate cells, but significantly promoted the expression of GFAP, suggesting that rhubarb glycoside can inhibit the activation of hepatic stellate cells.
[0018] The application of rhubarb glycoside in the treatment of CCl4-induced liver fibrosis was further validated through experiments. First, a liver fibrosis model was established in mice by intraperitoneal injection of a CCl4-ol olive oil solution (CCl4:ol olive oil = 2:8 (vol:vol)). Based on this, a control group, a rhubarb glycoside group, and a colchicine group were set up. The rhubarb glycoside group was administered intraperitoneally at 5 mg / kg once every 10 days, while the colchicine group was administered by gavage at 0.1 mg / kg / day. Treatment was observed for a total of 30 days. The expression levels of α-SMA and COL1α1, serum AST and ALT levels, and the degree of liver fibrosis were analyzed using RT-qPCR and Masson staining. The experimental results confirmed that rhubarb glycoside significantly reduced the degree of CCl4-induced liver fibrosis in mice and significantly improved liver function. Furthermore, considering various indicators such as body weight, AST and ALT levels, the efficacy of rhubarb glycoside was superior to that of colchicine.
[0019] The beneficial effects of this invention are as follows:
[0020] 1. The core active ingredient in privet fruit that exerts its anti-liver fibrosis effect has been identified as rosin, filling the research gap of unclear active ingredients in privet fruit for anti-liver fibrosis.
[0021] 2. Rhus chinensis glycosides exert their effects by directly inhibiting the activation of hepatic stellate cells, a core component of liver fibrosis. The mechanism of action is clear, and the anti-liver fibrosis effect is significant. It can effectively reduce collagen fiber deposition and improve liver function.
[0022] 3. In vivo experiments have confirmed that the anti-hepatic fibrosis efficacy of rhubarb glycoside is superior to that of colchicine, a commonly used clinical drug, and it has less impact on the body weight of experimental animals, suggesting that it is safer and has good prospects for clinical application.
[0023] 4. Rhus glycosides are natural active substances with relatively wide sources, providing new candidate compounds for the research and development of anti-liver fibrosis drugs, and have important medicinal value and market potential. Attached Figure Description
[0024] Figure 1 Effects of rhubarb glycosides on JS-1 cell proliferation;
[0025] Figure 2 Effects of rhubarb glycosides on the expression of fibrosis-related genes in TGF-β1-activated JS-1 cells;
[0026] Figure 3 The effect of rhubarb glycoside treatment on collagen deposition in mouse liver tissue;
[0027] Figure 4 Effects of rhubarb glycoside treatment on serum AST and ALT levels in mice;
[0028] Figure 5 Effects of rhubarb glycoside treatment on the expression of liver fibrosis-related proteins. Detailed Implementation
[0029] The following embodiments are further illustrations of the present invention, but not the only limitations thereof.
[0030] Example 1: Identification of components in privet fruit
[0031] Experimental techniques: The privet fruit purchased from a Chinese medicine store was ground into powder, extracted with alcohol or alcohol solution, filtered through a microporous membrane, and then analyzed by UPLC-MS / MS.
[0032] Mass spectrometry analysis employed electrospray ionization (ESI) as the ionization method, with the operating temperature controlled at 500℃. In positive ion detection mode, the ion spray voltage was set to 5500V, while in negative ion mode, a voltage of -4500V was used. To obtain optimal ionization efficiency, the gas path system was configured as follows: ion source gas I (GSI) pressure 50 psi, ion source gas II (GSII) pressure 60 psi, and curtain gas (CUR) pressure 25 psi, with the collision-induced ionization intensity adjusted to a high-sensitivity state. During detection, triple quadrupole mass spectrometry was used in multiple reaction monitoring (MRM) mode, with nitrogen at medium pressure purged into the collision chamber as the collision gas. The declustering voltage and collision energy parameters were optimized for different metabolites to ensure optimal ionization efficiency for each target compound. Based on the differences in retention time of metabolites on the chromatographic column, the entire analysis process was divided into multiple time periods. Each time period selectively monitored its corresponding characteristic ion pairs, enabling accurate quantitative analysis of multiple components in complex samples simultaneously.
[0033] Using the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and the Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine (ITCM), the components of Ligustrum lucidum (with CAS numbers) were searched based on oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18.
[0034] Experimental results: Several components were identified from privet fruit, including quercetin (CAS: 17306-46-6), quercetin (CAS: 117-39-5), and neoprivetin (CAS: 96382-91-1).
[0035] Example 2: Inhibitory effect of rhubarb glycoside on hepatic stellate cell activation
[0036] Experimental techniques and methods:
[0037] 1. Drug treatment: Mouse hepatic stellate cells (JS-1) that had been resuscitated and normally passaged were digested with trypsin and counted to adjust the cell concentration to 1×10⁻⁶. 5 Add 2 mL of the sample per well to a 6-well cell culture plate, with three replicates per well. Incubate the plates at 37°C in a 5% CO2 incubator.
[0038] When the cells reached the logarithmic growth phase, complete culture medium containing 10 ng / mL TGF-β1 and different concentrations of arvense glycoside (0, 0.9375, 1.875, 3.75, 7.5, 15 mg / mL) was added to each well. Blank control wells without TGF-β1 were also set up. The cells were placed in a 37℃, 5% CO2 cell culture incubator and cultured for 24 h or 36 h.
[0039] 2. Gene Expression Detection: After the cells were cultured, the culture medium was discarded, and the cells were washed three times with PBS. Total RNA was extracted from each group of cells according to the FastPure Cell / Tissue Total RNA Isolation Kit V2 instructions. RNA was quantified and reverse transcribed according to the Hiscript III All-in-one RT SuperMix Perfect for qPCR kit instructions. The reverse transcription program was: 50℃ for 15 min, 85℃ for 5 s. The reverse transcription product was diluted 5-fold with RNase-free H2O. The qPCR reaction system was prepared according to Table 1. The qPCR reaction conditions were: 95℃ for 2 min; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles. GAPDH was used as an internal control, and 2... -ΔΔCt Calculate the relative expression levels of each target gene.
[0040] Table 1 qPCR reaction system
[0041]
[0042] 3. Statistical Analysis: Data were analyzed using GraphPad Prism 8.0 statistical software. One-way ANOVA was used to analyze the differences between three or more groups. Based on this, the Bonferroni test was used for post-hoc testing; a p-value < 0.05 was considered statistically significant.
[0043] Experimental results: Rhus glycosides at concentrations of 0-10 μM had no significant effect on cell viability. Figure 1 In TGF-β1-activated JS-1 cells, 5 μM of rosin significantly upregulated the mRNA transcription level of GFAP, but at concentrations of 5-10 μM, rosin significantly downregulated the mRNA transcription levels of α-SMA and COL1α1 (P < 0.001). Figure 2 The results suggest that rosin can inhibit TGF-β1-induced JS-1 activation.
[0044] Example 3: The therapeutic effect of rhubarb glycosides on liver fibrosis in mice
[0045] Experimental techniques and methods:
[0046] 1. Establishment of a mouse liver fibrosis model: A mouse liver fibrosis model was established by intraperitoneal injection of CCl4-ol olive oil solution (CCl4:ol olive oil = 2:8 (vol:vol)). After one week of acclimatization feeding, mice were randomly divided into a blank control group and a model group. The model group received intraperitoneal injections of CCl4-ol olive oil solution, 2 mL / kg / time, twice a week for 4 consecutive weeks.
[0047] 2. Drug treatment regimen in mice: Diseased mice were randomly divided into three groups: control group, colchicine group, and rhubarb glycoside group. The colchicine group was administered colchicine by gavage at 0.1 mg / kg / day, the rhubarb glycoside group was administered intraperitoneally at 5 mg / kg / time once every 10 days, and the control group was administered the same amount of DMSO intraperitoneally once every 10 days.
[0048] 3. Index Analysis: Liver tissue sections were weighed every 5 days. Serum ALT and AST levels were measured according to the instructions of the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) detection kits. Masson and HE staining kits were used for staining, followed by clearing in fresh xylene, mounting, and observation and photography under a microscope. For immunofluorescence staining, liver tissue sections were dewaxed and then subjected to antigen retrieval. They were washed three times with 1×TBST for 10 min each time. COL1α1 and α-SMA primary antibodies were added, and the sections were incubated overnight at 4°C. After washing as before, fluorescently labeled secondary antibody was added, and the sections were incubated at room temperature for 1 h in a dark box. DAPI staining solution was then added, and the sections were incubated at room temperature in the dark for 30 min before mounting, observation under a fluorescence microscope, and analysis of relative fluorescence intensity using ImageJ software.
[0049] Experimental results: During the entire treatment period, the body weight of mice in the control group remained basically unchanged. The body weight of mice in the colchicine group began to increase after 20 days of treatment, while the body weight of mice in the rhubarb glycoside group increased significantly before 10 days of treatment. Moreover, the increase in body weight of mice in the rhubarb glycoside group was more significant compared with that in the colchicine group (P < 0.05).
[0050] HE staining results showed that the liver tissue of the control group mice had a large number of inflammatory cell infiltrations and disordered liver tissue structure, while the liver tissue of the colchicine group and the rhubarb glycoside group mice had relatively fewer inflammatory cells and the hepatocytes were more neatly arranged. Masson staining results also showed that the proportion of collagen fibers in the liver tissue of the colchicine group and the rhubarb glycoside group mice was significantly lower than that in the control group (P < 0.01). Figure 3 ).
[0051] At day 10 of treatment, the serum AST level in the rosin group was significantly lower than that in the control group (P < 0.05). Furthermore, at day 15 of treatment, the serum ALT level in the rosin group was also significantly lower than that in the control group (P < 0.05). Figure 4 ).
[0052] Compared with the control group, the relative fluorescence intensity of COL1α1 and α-SMA in the liver of mice in both the colchicine group and the rhubarb glycoside group was significantly decreased (P < 0.0001). Figure 5 ).
[0053] The above examples illustrate that rhubarb glycosides can significantly alleviate CCl4-induced liver fibrosis and significantly improve liver function, thus possessing potential medicinal value.
[0054] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A natural active substance against liver fibrosis, characterized in that, The active substance is anacrotine, whose molecular formula is C 27 H 30 O 14 , and the CAS number is 17306-46-6.
2. The natural active substance for anti-hepatic fibrosis according to claim 1, characterized in that, The wild lacquer tree glycoside can inhibit the activation of liver stellate cells, specifically by reducing the expression levels of alpha-smooth muscle actin, collagen type I and transforming growth factor beta 1, and promoting the expression of glial fibrillary acidic protein.
3. The natural active substance for anti-hepatic fibrosis according to claim 1, characterized in that, The wild lacquer tree glycoside is applied to the preparation of a medicine for treating liver fibrosis.
4. The application of the natural active substance for anti-liver fibrosis according to claim 3, characterized in that, The administration mode of the medicine includes intraperitoneal injection, the administration dose is 5mg / kg, and the administration frequency is once every 10 days.
5. The use of a natural active substance against liver fibrosis according to claim 3 or 4, characterized in that, The liver fibrosis is carbon tetrachloride-induced liver fibrosis.