Use of ginseng soup in the preparation of a drug for preventing and / or treating liver fibrosis

Abstract

The application belongs to the technical field of medicines, and relates to application of ginseng decoction in preparation of medicines for preventing and / or treating liver fibrosis. The ginseng decoction is composed of dried ginger, ginseng, white atractylodes rhizome and fried licorice, and the mass ratio of the dried ginger, the ginseng, the white atractylodes rhizome and the fried licorice is 1:1:1:1. The application provides a brand-new medicine selection for treating liver fibrosis. The application first discovers that the ginseng decoction has significant anti-liver fibrosis efficacy, provides a brand-new candidate medicine with natural source, small toxic and side effects and great development potential for treating liver fibrosis, and solves the urgent needs of clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of ginseng decoction in the preparation of drugs for the prevention and / or treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis (LF) is a repair response to excessive deposition of extracellular matrix (ECM) in the liver under the influence of various chronic liver injury factors. It is characterized by a prolonged course and potential reversibility, and belongs to the early pathological stage of cirrhosis. The core mechanism of liver fibrosis lies in the activation and transdifferentiation of hepatic stellate cells (HSCs), leading to an imbalance in the dynamic balance between ECM synthesis and degradation. Under continuous stimulation by liver injury factors, damaged hepatocytes and activated Kupffer cells release various pro-inflammatory and pro-fibrotic cytokines, including transforming growth factor-β1 (TGF-β1), platelet-derived growth factor (PDGF), and tumor necrosis factor-α (TNF-α), activating resting HSCs and transforming them into myofibroblast-like cells, resulting in excessive synthesis of ECM, primarily composed of type I and type III collagen.

[0003] Currently, clinical treatment strategies for liver fibrosis mainly revolve around etiological control: antiviral therapy for viral hepatitis (such as nucleoside analogs and direct-acting antiviral drugs), alcohol cessation guidance for alcoholic liver disease, lifestyle interventions (weight loss, exercise) and regulation of metabolic disorders for NAFLD, and immunosuppressive therapy for autoimmune liver disease. While these measures can delay or even partially reverse fibrosis to some extent, they have significant limitations: First, etiological treatment has limited effectiveness in reversing existing fibrotic tissue, especially after progressing to cirrhosis, where the reversal rate decreases significantly; second, some patients cannot achieve ideal etiological control, such as due to viral resistance, drug intolerance, or poor treatment adherence; finally, no specific drugs targeting fibrosis itself have been successfully developed, and many clinical trials of inhibitors targeting TGF-β1, LOXL2, and CCR2 / 5 have failed in recent years. Therefore, there is an urgent clinical need for adjuvant therapies that can intervene in the fibrosis process, promote ECM degradation, or repair liver damage.

[0004] Ginseng Decoction originates from *Synopsis of Prescriptions of the Golden Chamber* by Zhang Zhongjing of the Han Dynasty. It consists of four herbs: ginseng, dried ginger, atractylodes macrocephala, and prepared licorice root. It is primarily used to treat spleen and stomach weakness and insufficient yang in the middle burner. The formula uses dried ginger to warm the middle burner and assist yang, ginseng to tonify qi and strengthen the spleen, atractylodes macrocephala to strengthen the spleen and dry dampness, and licorice root to tonify qi and assist yang. The entire formula works synergistically to warm and tonify the middle burner, invigorate spleen yang, and strengthen the spleen and stomach, thus restoring the yang qi of the middle burner, enabling the spleen and stomach to function properly, and restoring the physiological functions of the middle burner in receiving qi, extracting essence, and transforming blood. Therefore, current technology only records that Ginseng Decoction can be used to treat spleen and stomach weakness...

[0005] There is no record of ginseng decoction being used to treat liver fibrosis due to deficiency of the middle yang.

[0006] In recent years, researchers have discovered significant differences in the heterogeneity of hepatic fibrosis cells (HSCs), the characteristics of immune microenvironment remodeling, and intercellular communication networks among liver fibrosis patients with different etiologies. Therefore, a deeper understanding of the role of the gut microenvironment in liver fibrosis is crucial for developing intervention strategies based on gut microenvironment regulation. Against this backdrop, traditional Chinese medicine (TCM), with its unique advantages of multi-target, multi-pathway, and holistic regulation, offers new possibilities for the treatment of liver fibrosis. Summary of the Invention

[0007] This invention discovers a new function of ginseng decoction, specifically its application in the preparation of drugs for the prevention and / or treatment of liver fibrosis, filling the gap in drugs specifically for the treatment of liver fibrosis. This invention finds that ginseng decoction can nourish the liver and replenish liver blood, thereby fundamentally treating liver fibrosis.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] The application of ginseng decoction in the preparation of drugs for the prevention and / or treatment of liver fibrosis, wherein the ginseng decoction is composed of dried ginger, ginseng, atractylodes macrocephala, and prepared licorice root, and the mass ratio of dried ginger, ginseng, atractylodes macrocephala, and prepared licorice root is 1:1:1:1.

[0010] The *Synopsis of Prescriptions of the Golden Chamber* states that the entire formula of Ginseng Decoction works to warm and tonify the middle jiao, invigorate spleen yang, and strengthen the spleen and stomach. This invention utilizes Ginseng Decoction for the prevention and / or treatment of liver fibrosis; the mechanism is analyzed as follows:

[0011] Liver fibrosis falls under the categories of "hypochondriac pain, accumulation, abdominal masses, and jaundice" in Traditional Chinese Medicine. Its development is related to external pathogenic factors such as damp-heat, improper diet, emotional imbalance, overwork, and prolonged illness leading to physical weakness. All of these factors can cause liver dysfunction and stagnation of Qi. The liver governs the free flow of Qi; insufficient free flow leads to stagnation, which in turn weakens the body's ability to propel blood circulation, resulting in blood stasis. Prolonged blood stasis can obstruct the liver's collaterals, further depleting the body's vital energy and causing accumulation. This disease is a complex condition characterized by deficiency in the root and excess in the branch, a combination of deficiency and excess.

[0012] The liver is yin in substance but yang in function. The liver's function of regulating qi relies on liver blood as its material basis; insufficient liver blood leads to impaired liver function. The spleen and stomach in the middle jiao receive and absorb the essence of food and water, which is then processed, transformed, and vaporized to generate blood. This is the source of blood production in the human body. The blood stored in the liver relies on the blood transformed by the middle jiao for nourishment. Ginseng decoction can strengthen the middle jiao, ensuring a source for qi and blood production, nourishing the liver, and replenishing liver blood. This fundamentally addresses the pathogenesis of spleen and stomach weakness and insufficient yang qi, leading to insufficient liver blood production and impaired liver function.

[0013] In a further embodiment, the ginseng decoction is administered orally.

[0014] The ginseng soup is prepared by the following steps:

[0015] S1. Add the Chinese medicinal herbs dried ginger, ginseng, atractylodes macrocephala, and prepared licorice to pure water for reflux extraction, and then pour out the filtrate.

[0016] S2. Concentrate the filtrate under reduced pressure and cool it to obtain ginseng soup.

[0017] In a further step, the mass of pure water added is 8-10 times that of the medicinal herbs. Dried ginger, ginseng, atractylodes macrocephala, and prepared licorice root are subjected to reflux extraction at least twice, with each reflux extraction lasting 1 hour. The concentration of the concentrated solution after vacuum concentration is 0.3 g of raw medicinal material per mL of solution.

[0018] Furthermore, based on molecular docking and simulation studies, this invention investigated the interaction between the active ingredients of ginseng decoction and ALDH2. It was found that six components—Neoliquiritin, Licochalcone B, Formononetin, Isoliquiritigenin, Shogaol, and Umbelliferone—are potential active ingredients acting on ALDH2. Neoliquiritin exhibits the strongest binding affinity to ALDH2, and the binding is the most stable. Therefore, this invention provides the application of neoliquiritin in the preparation of drugs for the prevention and / or treatment of liver fibrosis.

[0019] The beneficial effects of this invention are as follows:

[0020] 1. This invention provides a novel application of ginseng decoction in the treatment of liver fibrosis. This invention is the first to discover that ginseng decoction has significant anti-liver fibrosis effects, providing a novel candidate drug with a natural source, few toxic side effects, and great development potential for the current treatment of liver fibrosis, thus addressing an urgent clinical need.

[0021] 2. The drug action pathway of this invention is novel and precise. Ginseng decoction works by regulating the intestinal microenvironment, which is in line with the latest research on the close relationship between liver fibrosis and the intestine, and provides a new approach for the treatment of liver fibrosis.

[0022] 3. This invention has demonstrated through in vivo experiments that ginseng decoction can significantly improve the pathological scores of animal models of liver fibrosis, reduce collagen deposition, and restore liver function, with clear effects.

[0023] 4. The research results of this invention have a high conversion rate and low development risk. Through cross-validation of multiple cutting-edge technologies such as multi-omics analysis, molecular docking, and molecular dynamics simulation, and finally through in vivo animal experiments, the causal relationship of "target-action link-efficacy" was revealed, forming a solid and reliable chain of evidence, which significantly reduces the uncertainty and failure risk of subsequent new drug development.

[0024] 5. This invention provides a model for the modernization of traditional Chinese medicine, elucidates the mechanism of action of compound traditional Chinese medicine, and provides a highly valuable template for the international promotion and secondary development of traditional Chinese medicine. Attached Figure Description

[0025] Figure 1 This is a representative diagram illustrating the anti-liver fibrosis effect of the ginseng decoction in mice according to the present invention;

[0026] Figure 2 The following are UPLC-Q-TOF / MS total ion chromatograms of the ginseng decoction extract of this invention: A: Chromatogram of the ginseng decoction extract in negative ion mode; B: Chromatogram of the ginseng decoction extract in positive ion mode.

[0027] Figure 3 The following are examples of the effects of ginseng decoction on the gut microbiota of a liver fibrosis model in this invention: A: ACE index of mouse gut microbiota; B: Chao index of mouse gut microbiota; C: Obseerved index of mouse gut microbiota; D: Simpson index of mouse gut microbiota; E: PCA analysis of mouse gut microbiota; F: Heatmap of differential gut microbiota in mice; G: KEGG analysis of mouse gut microbiota.

[0028] Figure 4 This invention evaluates the effects of ginseng decoction on the intestinal microenvironment of an in vivo liver fibrosis model.

[0029] Figure 5 To evaluate the effects of ginseng decoction on the metabolism of an in vivo liver fibrosis model, the following analyses were conducted: A: PCA analysis of mouse serum metabolomics (positive ion mode); B: PCA analysis of mouse serum metabolomics (negative ion mode); C: Metabolite heatmap of mouse serum metabolomics; D: KEGG analysis of differentially metabolized metabolites in mouse serum; E: Heatmap of related metabolites in tryptophan metabolism; F: Changes in the abundance of indoleacetic acid in mouse intestinal metabolites.

[0030] Figure 6To evaluate the effects of ginseng decoction on indoleacetic acid metabolism and the downstream AHR-NFκB pathway in this embodiment of the invention; A: Immunofluorescence detection of PV-1 and CD31 expression in mouse colon; B: Immunofluorescence detection of AHR and ARNT expression in mouse liver; C: Western blot detection of AHR and NFκB expression in nucleus and cytoplasm; D: Statistical analysis of nuclear AHR protein expression; E: Statistical analysis of cytoplasmic AHR protein expression; F: Statistical analysis of NFκB protein expression.

[0031] Figure 7 This invention presents the network pharmacology of the active ingredients in ginseng decoction and the molecular docking results with ALDH2, an upstream metabolic enzyme of indoleacetic acid; A: Active ingredients of ginseng decoction and corresponding potential targets; B: Visualization of the molecular docking results between key active ingredients of ginseng decoction and ALDH2; C: Statistical analysis of the binding energy between active ingredients of ginseng decoction and ALDH2.

[0032] Figure 8 This is a 100 ns molecular dynamics simulation diagram of the binding of the key active ingredient of the ginseng decoction of the present invention with ALDH2; A: RMSD; B: RMSF; C: Radius; D: H-bonds. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] The Chinese medicinal herbs used in this example—dried ginger, ginseng, atractylodes macrocephala, and prepared licorice root—were purchased from the Traditional Chinese Medicine Pharmacy of the First Affiliated Hospital of Zhengzhou University. Male C57BL / 6 mice were purchased from Henan Skebest Biotechnology Co., Ltd.

[0035] Example 1

[0036] The preparation method of ginseng soup includes:

[0037] The Chinese medicinal herbs dried ginger, ginseng, atractylodes macrocephala, and prepared licorice root in a mass ratio of 1:1:1:1 were added to 10 times the volume of pure water and refluxed for 1 hour. The filtrate was poured out, and then 8 times the volume of pure water was added and refluxed for another hour. The two filtrates were combined and concentrated under reduced pressure to 0.3 g of raw medicinal material / mL of solution for subsequent animal experiments and UPLC-Q-TOF / MS detection and identification of the solution components.

[0038] Animal experiments:

[0039] P1. A mouse model of liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4). Specifically, 8-week-old male C57BL / 6 mice were intraperitoneally injected twice a week with 25% CCl4 (CCl4:olite = 1:3) for 12 consecutive weeks.

[0040] P2. Male C57BL / 6 mice were divided into four groups: normal group (Ctr, no model establishment or drug treatment), model group (Mod, model establishment but no drug treatment), high-dose ginseng decoction group (RSTH, model establishment and high-dose ginseng decoction 2.34 g / kg / day treatment), and low-dose ginseng decoction group (RSTL, model establishment and low-dose ginseng decoction 1.17 g / kg / day treatment).

[0041] P3. Starting from week 7 of model establishment, both RSTH and RSTL mice were given ginseng decoction for intervention: once a day by gavage for 6 weeks. Then, Masson staining and Sirius Red staining were used to detect liver pseudolobules, and H&E staining was used to assess liver inflammation.

[0042] Figure 1 The results show the HE staining, Masson staining, and SiriusRed staining of the livers of mice with liver fibrosis treated with ginseng decoction. These results indicate that, compared to the model group, the high- and low-dose ginseng decoction groups showed significantly thinner and fewer fibrous septa, a significantly reduced collagen deposition area, a significantly decreased α-SMA, and reduced liver inflammation.

[0043] Figure 2 The figure shows the total ion chromatogram of the ginseng decoction extract in UPLC-Q-TOF / MS positive and negative ion modes, which characterizes the potential active ingredients in the ginseng decoction extract.

[0044] The above results all demonstrate that the ginseng decoction prepared in this invention can effectively improve liver fibrosis and exert anti-inflammatory effects.

[0045] Example 2

[0046] Evaluating the effects of ginseng decoction on the intestinal environment of a liver fibrosis model using 16S RNA sequencing technology:

[0047] P1. Fresh feces were collected from the four groups of mice in Example 1, and the composition and abundance of the bacterial community were detected using 16sRNA sequencing technology.

[0048] P2. Liver fibrosis mouse model was established using the method described in Example 1;

[0049] P3. Male C57BL / 6 mice were divided into four groups: normal group (Ctr, no model establishment or drug treatment), model group (Mod, model establishment but no drug treatment), ginseng decoction group (RST, model establishment and high-dose ginseng decoction 2.34 g / kg / day treatment), and ginseng decoction + antibiotic group (RSTA, model establishment and high-dose ginseng decoction 2.34 g / kg / day treatment, quadruple antibiotics (vancomycin, 100 mg / kg; neomycin sulfate, 200 mg / kg; metronidazole, 200 mg / kg; and ampicillin, 200 mg / kg), antibiotics were administered by gavage daily for 5 consecutive days before ginseng decoction administration).

[0050] P4. Starting from week 7 of modeling, the RSTA group was given antibiotics by gavage for 5 consecutive days. Then, both the RST and RSTA groups were given ginseng decoction by gavage for 12 weeks. Subsequently, the pseudolobules and inflammation of the liver were assessed by α-SMA detection and H&E staining.

[0051] The results are as follows Figure 3 As shown, the composition and abundance of gut microbiota changed in mice with liver fibrosis after intervention with ginseng decoction. Further KEGG pathway enrichment analysis of the microbiota revealed that the tryptophan metabolism pathway was significantly altered. These results suggest that RST intervention changes the intestinal microenvironment of mice with liver fibrosis, and regulating intestinal tryptophan metabolism may be a pathway by which RST improves liver fibrosis in mice.

[0052] like Figure 4 As shown, in order to further clarify the mechanism of action of ginseng decoction, the intestinal flora of mice was cleared and then treated with ginseng decoction. The results showed that after the flora was cleared, ginseng decoction did not improve the liver fibrosis index of mice.

[0053] The above results all demonstrate that the anti-liver fibrosis effect of ginseng decoction depends on the gut microbiota.

[0054] Example 3

[0055] Evaluate the effects of ginseng decoction on metabolism and downstream AHR-NFκB of indoleacetic acid in a liver fibrosis model using non-targeted metabolomics:

[0056] P1. Collect serum and feces from the four groups of mice constructed in step P3 of Example 2 for metabolomics detection;

[0057] P2. Immunofluorescence was used to detect the intestinal vascular barrier and liver AHR and ARNT in four groups of mice, and Western blot was used to detect the expression levels of liver AHR and NFκB in four groups of mice.

[0058] The results are as follows Figure 5 As shown, serum metabolomics results indicated differences in the composition and content of metabolites in the serum of the normal group, model group, and ginseng decoction group. KEGG pathway enrichment revealed tryptophan metabolism and other pathways as significant enrichment pathways. Among them, the key differential metabolite indoleacetic acid (IAA) is a product of a key metabolic step in tryptophan metabolism and is closely related to the gut microbiota, which is consistent with the previous microbiota analysis. In addition, the indoleacetic acid content in the feces of the ginseng decoction group was significantly higher than that of the model group and the ginseng decoction + antibiotic group.

[0059] like Figure 6 As shown, immunofluorescence assays revealed increased expression of CD31 and PV-1 in the intestinal tissue of model mice, indicating disruption of the intestinal vascular barrier, which allows harmful substances to enter the bloodstream and accelerate disease progression. After intervention with ginseng decoction, the expression of CD31 and PV-1 in the mouse intestinal tissue decreased, suggesting that ginseng decoction can improve the vascular barrier in mice and inhibit the entry of harmful components into the bloodstream. Indoleacetic acid can activate downstream AHR and translocate into the nucleus, thereby inhibiting NFκB and exerting anti-inflammatory and anti-hepatic fibrosis effects. Immunofluorescence and Western blot assays revealed no significant change in AHR in the nuclei of liver tissue cells in the model group mice, but a significant increase in AHR in the cytoplasm. After intervention with ginseng decoction, the AHR in the nuclei significantly increased, while the AHR in the cytoplasm significantly decreased. Mice with depleted gut microbiota showed no significant change after RST administration. Furthermore, NFκB significantly decreased after ginseng decoction intervention, suggesting that ginseng decoction exerts its anti-hepatic fibrosis effect by mediating AHR nuclear translocation and inhibiting NFκB.

[0060] The above results all indicate that ginseng decoction can enter the bloodstream by mediating the production of indoleacetic acid in tryptophan metabolism in intestinal flora, promote the translocation of AHR to the nucleus of hepatocytes, inhibit NFκB, reduce liver tissue inflammation, and thus inhibit liver fibrosis.

[0061] Example 4

[0062] The interaction between the active ingredients of ginseng decoction and ALDH2 was studied based on molecular docking and simulation.

[0063] ALDH2 is a protein associated with the production of indoleacetic acid. Network pharmacology analysis was performed on the components identified from the ginseng decoction, and ALDH2 was identified as a potential target for 6 components. Then, the components in the ginseng decoction were molecularly docked with the protein ALDH2 using AutoDock v1.5.7 software. The results were visualized using PyMol v3.1.3 software, and the component with the strongest binding energy was subjected to molecular dynamics simulation with ALDH2.

[0064] The results are as follows Figure 7 , Figure 8As shown, Neoliquiritin, Licochalcone B, Formononetin, Isoliquiritigenin, Shogaol, and Umbelliferone are six potential active ingredients that act on ALDH2.

[0065] Molecular docking results showed that Neoliquiritin had the strongest binding affinity to ALDH2, and molecular dynamics simulations further confirmed that the binding between Neoliquiritin and ALDH2 was more stable.

[0066] All of the above results indicate that ginseng decoction can promote the translocation of hepatocyte AHR to the nucleus by mediating the production of indoleacetic acid in tryptophan metabolism in intestinal flora, thereby inhibiting liver fibrosis, while the action of neoliquiritin on ALDH2 may be its mechanism of action.

[0067] This invention aims to protect the novel use of ginseng decoction in the preparation of drugs for the prevention and / or treatment of liver fibrosis, and further protect the application of ginseng decoction in treating liver fibrosis by regulating the intestinal microenvironment, and also protect the application of ginseng decoction in treating liver fibrosis by acting on the specific molecular target ALDH2 to promote the production of indoleacetic acid in tryptophan metabolism.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Ginseng Decoction in the preparation of drugs for the prevention and / or treatment of liver fibrosis.

2. The application according to claim 1, characterized in that, The ginseng soup is composed of dried ginger, ginseng, atractylodes macrocephala, and prepared licorice root, with a mass ratio of 1:1:1:

1.

3. The application according to claim 1, characterized in that, The ginseng decoction is administered orally.

4. The application according to claim 1, characterized in that, The ginseng soup is prepared by the following steps: S1. Add the Chinese medicinal herbs dried ginger, ginseng, atractylodes macrocephala, and prepared licorice to pure water for reflux extraction, and then pour out the filtrate. S2. Concentrate the filtrate under reduced pressure and cool it to obtain ginseng soup.

5. The application according to claim 4, characterized in that, In step S1, the mass of the added pure water is 8-10 times that of the Chinese medicinal materials.

6. The application according to claim 4, characterized in that, In step S1, dried ginger, ginseng, atractylodes macrocephala, and prepared licorice are subjected to reflux extraction more than twice, with each reflux extraction lasting 1 hour.

7. The application according to claim 4, characterized in that, In step S2, the concentration of the concentrated medicinal solution after vacuum concentration is 0.3g of raw medicinal material / mL of medicinal solution.

8. Application of neoglycyrrhizin in the preparation of drugs for the prevention and / or treatment of liver fibrosis.

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