Application of combination of ginsenoside-containing component medicinal materials and bile acid-containing medicinal materials in preparation of anti-hepatic fibrosis medicines
By combining ginseng saponin with bile acids, the problem of low bioavailability of ginseng saponin in the prior art has been solved, and the inhibitory effect on liver stellate cell activation and liver fibrosis has been significantly enhanced, providing a better treatment strategy.
Patent Information
- Application Number
- CN202510391563.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art lacks direct anti-fibrotic drugs in the treatment of liver fibrosis, and the bioavailability of ginseng saponins is low, making it difficult to effectively inhibit liver stellate cell activation and liver fibrosis.
Ginseng saponin is combined with bile acids to improve the pharmacological effects of ginseng saponin and inhibit the activation of hepatic stellate cells and liver fibrosis.
Through combination, the inhibitory effect on hepatic stellate cell activation and liver fibrosis is significantly enhanced, providing a better therapeutic strategy.
Smart Images
Figure BDA0005337572110000051 
Figure BDA0005337572110000052 
Figure BDA0005337572110000061
Abstract
Description
Technical Field
[0001] The present invention relates to a new use of medicines, and in particular to the use of a medicinal material containing ginsenoside components in combination with a medicinal material containing bile acid in the preparation of an anti-liver fibrosis medicine. Background Art
[0002] Liver fibrosis is a pathological process caused by chronic liver damage, characterized by excessive extracellular matrix deposition, which may eventually lead to cirrhosis and liver failure. Globally, the incidence of liver fibrosis varies by region and etiology. The main causative factors include chronic hepatitis B and C, alcoholic liver disease, and non-alcoholic fatty liver disease (NAFLD). In recent years, the prevalence of NAFLD has increased significantly and has become one of the main causes of liver fibrosis in developed countries.
[0003] The core mechanism of liver fibrosis is the activation of hepatic stellate cells (HSC). Under chronic liver damage, HSC transform from a quiescent state to a myofibroblast-like phenotype, leading to excessive production of extracellular matrix such as collagen. Currently, there are limited therapeutic drug options for liver fibrosis. Existing therapies mainly focus on removing pathogenic factors, such as antiviral treatment, alcohol abstinence, and improving lifestyle. However, there are no direct anti-fibrotic drugs for clinical use.
[0004] Ginsenosides, especially Rg3 and Rd, have been studied for the treatment of liver fibrosis. Studies have shown that these compounds have anti-inflammatory, antioxidant and HSC activation inhibitory effects. However, their efficacy is still limited in clinical applications, which may be related to low bioavailability and pharmacokinetic properties. Bile acids are important components of bile and play an important role in fat metabolism, playing a complex role in liver fibrosis. Under normal physiological conditions, bile acids contribute to fat digestion and absorption. However, during liver fibrosis, dysregulation of bile acid metabolism may aggravate liver damage and fibrosis. Summary of the invention
[0005] Purpose of the invention: To solve the above technical problems, the present invention provides the use of ginsenosides in combination with bile acids in the preparation of anti-liver fibrosis drugs, and experiments have confirmed that the combination of ginsenosides and bile acids has a significantly enhanced anti-fibrosis effect.
[0006] Technical solution: Use of ginsenoside-containing drugs or medicinal materials in combination with bile acid-containing drugs or medicinal materials in the preparation of anti-liver fibrosis drugs.
[0007] In the application, the ginsenosides contained in the ginsenoside-containing medicine or medicinal material include ginsenoside Rd and ginsenoside Rg3.
[0008] In the application, the ginsenoside-containing drugs or medicinal materials include Panax notoginseng, ginseng, and American ginseng.
[0009] The application, the ginsenoside-containing medicine or medicinal material can inhibit the activation of hepatic stellate cells and liver fibrosis.
[0010] In the application, the bile acid-containing drugs or medicinal materials include drugs or medicinal materials containing swine bile acid.
[0011] In the application, the bile acid can promote the inhibitory effect of ginsenoside on hepatic stellate cell activation and liver fibrosis.
[0012] The application is the application of the ginsenoside-containing drug in combination with bile acid in the preparation of anti-liver fibrosis drugs.
[0013] The pharmaceutical composition is prepared by using ginsenoside-containing drugs or medicinal materials and bile acid as active ingredients, with or without pharmaceutically acceptable excipients.
[0014] The pharmaceutical composition, the pharmaceutically acceptable excipients include diluents, adhesives, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, and adsorption materials.
[0015] The pharmaceutical composition is an oral preparation.
[0016] The pharmaceutical composition and the oral preparation include granules, powders, tablets, capsules, pills, oral liquids, decoctions, and dripping pills.
[0017] More preferably, ginsenosides and bile acids are used in combination for the preparation of anti-liver fibrosis drugs.
[0018] The ginsenosides include saponins of various structural types, and include ginsenosides of natural origin, semi-synthetic and fully chemically synthesized. More preferably, the ginsenosides are selected from Panax notoginseng.
[0019] The ginsenosides can inhibit the activation of hepatic stellate cells and the expression of target genes such as liver fibrosis.
[0020] The bile acid includes bile acids of various structural types, and includes naturally derived, semisynthetic, and chemically synthesized bile acids.
[0021] The medicine is a medicine prepared by taking ginsenoside and bile acid as active ingredients.
[0022] Alternatively, the drug is a drug prepared by using ginsenoside and bile acid as active ingredients and adding pharmaceutically acceptable excipients.
[0023] The pharmaceutically acceptable excipients described in the present invention refer to various conventional excipients required for the preparation of different dosage forms, such as diluents, adhesives, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorbents, etc., which are prepared into any commonly used oral preparations by conventional preparation methods, such as granules, powders, tablets, capsules, pills, oral liquids, decoctions, and drop pills.
[0024] Beneficial effects: Compared with the prior art, the present invention discloses the application of ginsenosides combined with bile acids in the preparation of anti-liver fibrosis drugs. Ginsenosides have a certain inhibitory effect on HSC activation under the action of high concentration for a long time; however, ginsenosides are easily hydrolyzed by intestinal flora, have low bioavailability, and are absorbed into the liver at a low content and concentration, and have weaker effects on HSC activation and liver fibrosis; the research results of the present invention show that bile acid can significantly promote the inhibitory effect of ginsenosides on HSC activation and liver fibrosis. Therefore, the present invention combines ginsenosides with bile acids to prepare anti-liver fibrosis drugs, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a pharmacokinetic analysis of the rapid metabolism of ginsenosides by intestinal flora, where: *P<0.05, * * P<0.01, ** * P < 0.001;
[0026] Figure 2 The mRNA level analysis of the effect of ginsenoside Rg3 on Col1a1, Col1a2, Col3a1, Acta2, and Timp2 in hepatic stellate cells, among which: *P<0.05, ¨P<0.01, ¨*P<0.001;
[0027] Figure 3 The mRNA level analysis of the effect of ginsenoside Rd on Colla1, Col1a2, Col3a1, Acta2, and Timp2 in hepatic stellate cells, among which: *P<0.05, ¨P<0.01, ¨*P<0.001;
[0028] Figure 4 The mRNA level analysis of the effect of Panax notoginseng on Col1a1, Col1a2, Col3a1, Acta2, and Timp2 in hepatic stellate cells, among which: *P<0.05, ¨P<0.01, ¨*P<0.001;
[0029] Figure 5 This is the mRNA level analysis of the effects of ginsenoside Rg3 and HCA alone / combined on Col1a1, Col1a2, Col3a1, Acta2, and Timp2 in hepatic stellate cells, among which: *P<0.05, ¨P<0.01, ¨*P<0.001;
[0030] Figure 6 This is an analysis of the effects of ginsenoside Rd and HCA alone or in combination on the mRNA levels of Col1a1, Col1a2, Col3a1, Acta2, and Timp2 in hepatic stellate cells, among which: *P<0.05, ¨P<0.01, ¨*P<0.001. DETAILED DESCRIPTION
[0031] The present invention provides a composition containing ginsenosides, wherein the composition comprises ginsenosides and bile acid. The present invention has no special limitation on the mass ratio of ginsenosides to bile acid, and any mass ratio is acceptable.
[0032] The applicant of the present invention found in previous studies that after long-term administration of high-concentration ginsenosides in an activated state of hepatic stellate cells, the expression levels of genes such as Col1a1, Col1a2, Col3a1, Acta2, and Timp2 were significantly downregulated; however, ginsenosides have low bioavailability and can be rapidly metabolized by intestinal flora after oral administration, and the drug content absorbed into the liver is low, making it difficult to exert an anti-stellate cell activation effect; in the research of the present invention, a combination of ginsenosides and bile acids is provided, and it is found that when bile acids are administered, the pharmacological effects of ginsenosides are more significant, providing a better strategy for the treatment of liver fibrosis.
[0033] The present invention is further described below in conjunction with specific examples. The biological materials (experimental animals) and reagents used in the examples of the present invention can be obtained from commercial channels unless otherwise specified.
[0034] Example 1 Study on the metabolism of ginsenosides by intestinal flora
[0035] 1 Experimental Materials
[0036] The ginsenoside Rg3 used in the present invention was purchased from Taoshu Company, and the ginsenoside Rd was purchased from MacLean Company.
[0037] 2 Experimental methods
[0038] 2.1 Preparation of intestinal flora
[0039] When cutting the mouse ileocecal valve in the clean bench, cut more of the end of the ileum and the proximal end of the colon to prevent the cecal contents from being exposed to the air. Gently wipe the mouse ileocecal valve with PBS in the clean bench. Cut the end of the ileocecal valve with sterilized scissors, and use sterilized tweezers to take part of the ileocecal valve contents into a weighed 1.5mL EP tube. Close the centrifuge tube lid tightly, and weigh and record it on a balance. Add 5ml of M9 culture medium (add 20% glycerol) for every 1g of the weight of the cecal contents. Mix evenly, but not too violently.
[0040] 2.2 Intestinal flora incubation
[0041] After sterilizing the M9 medium, add 0.5% cysteine, add 100ul of the above medium to the sterilized 1.5mL EP tube in the primary clean bench, and add 100ul of bacterial solution (final concentration is 1:10 dilution) to the tube after filtering the bacterial solution with a 70um mesh, and then add 10ug / ml (DMSO dissolved, DMSO final concentration is one thousandth) ginsenoside, open the lid and put it in the incubation box with anaerobic bag, and then place the anaerobic box in a 37℃ shaker at 120rpm to react the bacterial colony. Collect samples after different incubation times (0min, 10 min, 20min, 30min, 60min, 120min, 360min).
[0042] 2.3 Sample processing and testing
[0043] When collecting the sample, add 5 volumes of methanol (chromatographic grade) to terminate the reaction. Then centrifuge at 18000rpm for 5min, take 1ml of the supernatant, centrifuge at 18000rpm for 5min again to take 800ul of the supernatant, blow dry with nitrogen, and store in a -80℃ refrigerator. Add 0.1ml of 50% methanol to re-dissolve, vortex and oscillate for 10min until completely dissolved, centrifuge at 18000rpm for 5min, transfer 80ul of the supernatant to the injection bottle, and wait for injection.
[0044] 3 Experimental results
[0045] Rg3 metabolism experiment ( Figure 1 A) shows that the initial concentration of Rg3 is about 11000 ng / ml, which decreases in a time-dependent manner as the incubation time with the intestinal flora increases, decreasing to more than half at 60 min and being almost completely metabolized at 360 min, indicating that Rg3 is rapidly metabolized in the simulated intestinal environment.
[0046] Rd metabolism experiment ( Figure 1 B) shows that the initial concentration of Rd was about 8000 ng / ml, which decreased time-dependently with the extension of incubation time with intestinal flora, decreased to more than half at 30 min, and was almost completely metabolized at 360 min.
[0047] Example 2 Study on the activation effect of ginsenosides on hepatic stellate cells
[0048] 1 Experimental Materials
[0049] The ginsenoside Rg3 used in the present invention was purchased from Taoshu Company, and the ginsenoside Rd was purchased from MacLean Company.
[0050] 2 Experimental methods
[0051] 2.1 Extraction and isolation of primary HSC cell lines
[0052] 1) Select rats aged 8 weeks or above, disinfect the abdomen of the rats, expose the abdominal cavity, separate the portal vein, insert the cannula and perfuse, and perfuse the perfusion fluids ①, ② and ③ according to the perfusion fluid sequence. Stop the perfusion when the liver becomes soft, translucent and earthy yellow.
[0053] 2) Collect all the livers into a sterile dish filled with Hanks, remove the liver capsule, fully disperse the liver and transfer it to a bottle, add the same volume of 2x enzyme digestion solution A, add 1% (v / v) enzyme digestion solution B, shake in a 37°C water bath for 15 minutes to obtain a cell suspension.
[0054] 3) Filter the cell suspension with a 70μm filter into a 50ml centrifuge tube, add cold GBSS B solution to 50ml, incubate at 4℃, 580g, 10min, keep 10ml and add 130μl DNase solution, add cold GBSS B solution to 50ml and resuspend, incubate at 4℃, 580g, 10min, discard the supernatant and keep 10ml and add 130μl DNase solution to resuspend and mix. Add cold GBSS B to the tube to make it 15ml, and mix the above liquids.
[0055] 4) Add 30 ml of 18% Nycodenz solution to the above cell suspension, mix the liquid in the tube, and slowly overlay 5 ml of cold GBSS B on the mixed solution at 4°C, 1380 g, for 17 min (adjust the rising speed to level 1 and the falling speed to level 0).
[0056] 5) Take out the white cell layer at the junction of the liquid surface, centrifuge at 4°C, 50g, 4 min to remove the hepatocytes, transfer the supernatant to a new 50 ml centrifuge tube, and finally centrifuge at 4°C, 580g, 10 min to obtain the precipitate, which is the hepatic stellate cells.
[0057] 6) Resuspend with appropriate amount of DMEM containing 20% FBS (use trypan blue staining to count and detect cell viability), inoculate at a density of 2 x 105 cells / well in a 12-well plate, change the medium the next day, and then change the medium every 2 days. After the HSC cells are cultured to an appropriate density, administer the drug.
[0058] GBSS A:
[0059]
[0060] GBSS B:
[0061]
[0062] D-Hank's:
[0063]
[0064] Hank's:
[0065]
[0066] Perfusion buffer 1:
[0067]
[0068] Perfusion buffer 2:
[0069]
[0070]
[0071] Perfusion buffer 3:
[0072]
[0073] Enzyme digestion solution A:
[0074]
[0075] Enzyme digestion solution B:
[0076]
[0077]
[0078] Nycodenz (18%):
[0079]
[0080] 2.2 Cell culture and drug treatment
[0081] After culturing for 12 hours until the cells adhered to the wall, the cells in the blank control group were collected and frozen at -80°C, and the cells in the other groups were cultured for 48 hours to allow them to be naturally activated to form a pHHSCs self-activation model. Subsequently, the corresponding dosing solution was added according to the grouping, and the cells were collected after 2 hours to detect HSCs activation-related indicators.
[0082] 2.3 Real-time quantitative PCR
[0083] 2.3.1 Total RNA extraction from cell samples
[0084] 1) Wash the cells with PBS and add 1 ml of Trizol reagent. Use a pipette to pipette repeatedly to mix well and then transfer to an EP tube.
[0085] 2) Add 200 μl of chloroform, shake vigorously for 15 seconds, place at room temperature for 5 minutes, and centrifuge at 13,000 g for 15 minutes. The sample is divided into three layers: the bottom layer is a yellow organic phase, the upper layer is a colorless aqueous phase, and an intermediate layer.
[0086] 3) Carefully transfer 400 μl of the upper aqueous phase to a new tube, add 400 μl of isopropanol, mix thoroughly by inversion, leave at room temperature for 10 min, centrifuge at 13,000 g for 10 min, and discard the supernatant;
[0087] 4) Wash the RNA precipitate with 800 μl of pre-cooled 75% ethanol, then centrifuge at 13,000 g for 5 min, discard the supernatant to obtain total RNA, and re-dissolve it with 5 μl of DEPC water, and dilute to 0.5 μg / μl after quantification.
[0088] 2.3.2 Reverse transcription
[0089] According to the system ratio required by the instructions, the RNA solution and the kit components were prepared into a system with a total volume of 20 μl and the program temperature was set for reverse transcription. The specific ratio requirements are shown in Table 1:
[0090] Table 1 Reverse transcription ratio
[0091]
[0092] The conditions for reverse transcription were as follows:
[0093] Stage 1: reverse transcription at 37°C for 15 min;
[0094] Stage 2: Denaturation at 85℃ for 5s.
[0095] 2.3.3PCR
[0096] The PCR system is shown in Table 2:
[0097] Table 2 PCR system
[0098]
[0099] The PCR conditions were as follows:
[0100] Stage 1: Pre-denaturation at 95°C for 1 min;
[0101] Stage 2: PCR reaction at 95℃ for 15sec; 60℃ for 30sec for 40cycles; 72℃ for 30sec;
[0102] Stage 3: Melting curve analysis: 65-95°C, 0.5°C / 5s.
[0103] The primer sequences of the genes to be tested are shown in Table 3:
[0104] Table 3 Primer sequences
[0105]
[0106] 3 Experimental results
[0107] 3.1 Effects of ginsenoside Rg3 on hepatic stellate cells
[0108] According to the results of Example 1, ginsenoside Rg3 can be rapidly metabolized by intestinal flora, with metabolism exceeding 50% at 60 min. Therefore, in this section, the effect of short-term (2 h) incubation of Rg3 with hepatic stellate cells on their activation was investigated. The results showed that ginsenoside Rg3 had no significant effect on the mRNA expression levels of Col1a1, Col1a2, Col3a1, Acta2 and Timp2 ( Figure 2 ). This suggests that short-term low-concentration exposure to ginsenoside Rg3 has no significant effect on HSC activation.
[0109] 3.2 Effects of ginsenoside Rd on hepatic stellate cells
[0110] According to the results of Example 1, similar to the results of Rg3, ginsenoside Rd can also be rapidly metabolized by intestinal flora, with metabolism exceeding 50% at 30 minutes. In this part of the study, the effect of short-term (2h) incubation of Rd with hepatic stellate cells on their activation was investigated. The results showed that ginsenoside Rg3 had no significant effect on the mRNA expression levels of Col1a1, Col1a2, Co13a1, Acta2 and Timp2 ( Figure 3 ). This suggests that short-term low-concentration exposure to ginsenoside Rd has no significant effect on HSC activation.
[0111] 3.3 Effects of Panax notoginseng on hepatic stellate cells
[0112] Ginsenosides Rd and Rg3 are widely found in medicinal materials such as Panax notoginseng, ginseng, and American ginseng. In this part of the study, Panax notoginseng was used as a representative to investigate its inhibitory effect on HSC. According to the results of PCR ( Figure 4 ), in the activated HSC model, Panax notoginseng had no significant effect on the mRNA expression levels of Col1a1, Col1a2, Col3a1, Acta2 and Timp2, and had no obvious effect on the activation of HSCs.
[0113] Example 3 Study on the effect of bile acid HCA combined with ginsenoside on hepatic stellate cells
[0114] 1 Experimental Materials
[0115] The bile acid HCA used in the present invention was purchased from Taoshu Company, the ginsenoside Rg3 was purchased from Taoshu Company, and the ginsenoside Rd was purchased from Maclean Company.
[0116] 2 Experimental methods
[0117] 2.1 Extraction and isolation of primary HSC cell lines
[0118] The experimental method is the same as that in Example 1.
[0119] 2.2 Cell culture and drug treatment
[0120] After culturing for 12 hours until the cells adhered to the wall, the cells in the blank control group were collected and frozen at -80°C, and the cells in the other groups were cultured for 48 hours to allow them to be naturally activated to form a pHSCs self-activation model. Subsequently, the corresponding dosing solution was added according to the grouping, and the cells were collected after 12 hours to detect HSCs activation-related indicators.
[0121] 2.3 Real-time quantitative PCR
[0122] The experimental method is the same as that in Example 1.
[0123] 3 Experimental results
[0124] 3.1 Effects of bile acid HCA combined with ginsenoside Rg3 on hepatic stellate cells
[0125] The above results show that short-term administration of ginsenoside Rg3 has no significant effect on HSCs activation. In this part of the study, it is intended to investigate the composition that can promote ginsenosides to inhibit HSCs activation. After screening, it was found that in the activated hepatic stellate cell model, bile acid HCA combined with ginsenoside Rg3 had a significant effect on HSCs activation ( Figure 5 ).from Figure 5 It can be seen that HCA has no obvious effect on HSCs activation, ginsenoside Rg3 has no obvious effect on HSCs activation, and the combination of ginsenoside Rg3 and bile acid HCA has a synergistic inhibitory effect on HSCs activation.
[0126] 3.2 Effects of bile acid HCA combined with ginsenoside Rd on hepatic stellate cells
[0127] The above results show that short-term administration of ginsenoside Rd has no significant effect on HSCs activation. In this part of the study, it is intended to investigate the composition that can promote ginsenosides to inhibit HSCs activation. After screening, it was found that in the activated hepatic stellate cell model, bile acid HCA combined with ginsenoside Rd had a significant effect on HSCs activation ( Figure 6 ).from Figure 6 It can be seen that HCA has no obvious effect on the activation of HSCs, ginsenoside Rd has no obvious effect on the activation of HSCs, and the combination of ginsenoside Rd and bile acid HCA has a synergistic inhibitory effect on the activation of HSCs.
Claims
1. The use of ginsenoside-containing drugs or medicinal materials in combination with bile acid-containing drugs or medicinal materials in the preparation of anti-liver fibrosis drugs.
2. The use according to claim 1, characterized in that: The ginsenosides contained in the ginsenoside-containing drugs or medicinal materials include ginsenoside Rd and ginsenoside Rg3.
3. The use according to claim 1, characterized in that: The ginsenoside-containing drugs or medicinal materials include Panax notoginseng, ginseng, and American ginseng.
4. The use according to claim 1, characterized in that: The ginsenoside-containing medicine or medicinal material can inhibit the activation of hepatic stellate cells and liver fibrosis.
5. The use according to claim 1, characterized in that: The bile acid-containing drugs or medicinal materials include drugs or medicinal materials containing swine bile acid.
6. The use according to claim 1, characterized in that: The bile acid can promote the inhibitory effect of ginsenoside on hepatic stellate cell activation and liver fibrosis.
7. A pharmaceutical composition, characterized in that It is made of ginsenoside-containing drugs or medicinal materials and bile acid as active ingredients, with or without pharmaceutically acceptable excipients.
8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutically acceptable excipients include diluents, binders, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, and adsorption materials.
9. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition is an oral preparation.
10. The pharmaceutical composition according to claim 9, characterized in that The oral preparations include granules, powders, tablets, capsules, pills, oral liquids, decoctions, and dripping pills.