Traditional Chinese medicine compound for treating fatty liver

Abstract

The invention relates to the field of Chinese herbal compounds, and discloses a Chinese herbal compound for treating fatty liver, which is prepared from the following raw materials in parts by mass: 28-32 parts of gynostemma pentaphylla, 13-17 parts of oriental wormwood, 11-14 parts of cassia seed, 6-12 parts of turmeric and 7-11 parts of red yeast rice. The traditional Chinese medicine compound, namely the trickle food fat-reducing formula, has the effects of activating the spleen, calming the heart, dissolving turbidity and removing blood stasis, is remarkable in curative effect, not only remarkably reduces the body weight index and the blood fat level of an NASH patient, but also can improve the liver injury and fibrosis degree.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine compound prescriptions, and in particular to a traditional Chinese medicine compound prescription for treating fatty liver. Background Art

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD), formerly known as non-alcoholic fatty liver disease (NAFLD), has become the most common chronic liver disease. Its disease spectrum includes metabolic-associated fatty liver, metabolic-associated steatohepatitis (MASH), metabolic-associated steatohepatitis, and metabolic-associated steatohepatitis. Studies have shown that about 25% of patients with metabolic-associated fatty liver will progress to MASH (formerly known as NASH), which is an inflammatory disease state of MAFLD and is associated with persistent liver cell damage. When MASH develops to the stage of liver fibrosis, the condition often becomes difficult to reverse, and the risk of cirrhosis and liver cancer increases significantly. Given the large prevalence of MASH, serious health hazards, and strong clinical needs, studying its intervention drugs and therapeutic targets is a key issue that needs to be urgently addressed in the field of life sciences. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a Chinese medicine compound for treating fatty liver, aiming to solve the problem that existing prescriptions are difficult to cope with the huge number of MASH patients and difficult to intervene and treat fatty liver.

[0004] The present invention solves the above technical problems by the following technical means:

[0005] A traditional Chinese medicine compound for treating fatty liver comprises the following raw materials in terms of mass ratio: 28-32 gynostemma pentaphyllum, 13-17 capillaris, 11-14 cassia seed, 6-12 turmeric, and 7-11 red yeast rice.

[0006] Furthermore, the raw material composition of the traditional Chinese medicine compound in terms of mass ratio is: 30 gynostemma pentaphyllum, 15 capillaris, 12 cassia seed, 10 turmeric, and 12 red yeast rice.

[0007] Furthermore, a preparation suitable for oral administration is used.

[0008] Beneficial effects:

[0009] 1. Under the guidance of the basic theories of traditional Chinese medicine, combined with the theory of "liver is the governance of qi", through long-term clinical practice, it is concluded that the treatment of fatty liver should focus on the key pathogenesis of phlegm and spleen qi not functioning properly, phlegm turbidity disturbing the heart, and qi stagnation and obstruction of the collaterals. The traditional Chinese medicine compound for the treatment of fatty liver of the present invention - Juanyu Xiaozhi Fang was formulated, which has the effects of regulating the spleen and calming the heart, resolving turbidity and removing blood stasis, with significant therapeutic effects, and animal experiments have fully verified its effectiveness.

[0010] 2. The Juanyu Xiaozhi prescription of the present invention has significant therapeutic effects. It not only significantly reduces the body mass index and blood lipid levels of NASH patients, but also improves liver damage and fibrosis.

[0011] 3. The present invention discovered through metabolomics that UDP-N-acetylglucosamine (UDP-GlcNAc), a key metabolite of the hexosamine biosynthetic pathway (HBP), was significantly downregulated after the intervention of the JYF formula. Combined with preliminary experiments, it was found that the JYF formula intervention could significantly downregulate the O-linked β-N-acetylglucosamine (O-linked β-N-acetylglucosamine, O-GlcNAc) glycosylation modification level and the expression of the key enzyme OGT mediated by the HBP pathway; further glycosylation modification omics found that the O-GlcNAc modification of TGF-β-activated kinase 1 / MAP3K7-binding protein 2 (TAB2) of the MAPK pathway was significantly downregulated. It can be seen from this that the specific molecular mechanism by which the Juanyu Xiaozhi recipe of the present invention inhibits the O-GlcNAc modification of TAB2 protein mediated by the HBP pathway, leading to the inactivation of the MAPK signaling pathway, and further inhibits the progression of NASH will not only help the clinical application and promotion of the Juanyu Xiaozhi recipe and promote the transformation of research results, but also will provide a new pharmacological target with Chinese medicine characteristics for the treatment of NASH and provide new ideas for the design of innovative drugs.

[0012] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The treatment with JYXF significantly inhibited liver damage in NASH mice;

[0014] Figure 2 The JXF treatment inhibited lipid deposition and fibrosis in NASH mice;

[0015] Figure 3 This is the positive and negative ion diagram of the traditional Chinese medicine solution of Junyu Xiaozhi Recipe;

[0016] Figure 4 To detect differential metabolites after the intervention of JYXF prescription by non-targeted metabolomics;

[0017] Figure 5.1KEGG analysis of the effective active ingredients of the Junyu Xiaozhi recipe;

[0018] Figure 5.2 It is a compound-target-signaling pathway network diagram;

[0019] Figure 5.3 Molecular docking model diagram of active compound quercetin (left, binding energy -8.5) or curcumin (right, binding energy -8.45) with OGT;

[0020] Figure 6 The intervention of JYXF significantly down-regulated the O-GlcNAc modification in liver tissue of NASH mice;

[0021] Figure 7 The intervention of serum containing the Juanyu Xiaozhi Recipe significantly inhibited the lipid deposition in fatty cells;

[0022] Figure 8 Treatment with serum containing the JYXF formula significantly inhibited lipid deposition in fatty cells and down-regulated O-GlcNAc modification;

[0023] Fig. 9 This is the O-GlcNAc modification omics detection after the intervention of the Junyu Xiaozhi Recipe;

[0024] Fig.10 The enrichment of differentially modified peptides and proteins after the intervention of Junyu Xiaozhi Recipe;

[0025] Fig.11 The O-GlcNAc modification and MAPK pathway of TAB2 were significantly downregulated after intervention with the Junyu Xiaozhi recipe. DETAILED DESCRIPTION

[0026] The following describes the implementation of the present invention through specific specific embodiments, and those skilled in the art can understand the advantages and effects of the present invention from the contents disclosed in this specification. It should be noted that the illustrations provided in the following embodiments are only for exemplary descriptions, and only schematic diagrams are shown, not physical diagrams, and cannot be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts in the figures may be omitted, enlarged or reduced, and do not represent the size of the actual product; for those skilled in the art, it is understandable that some well-known structures and their descriptions may be omitted in the figures.

[0027] The same or similar numbers in the figures of the embodiments of the present invention correspond to the same or similar parts. In the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "back", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the figure. It is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the figures are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to the specific circumstances.

[0028] This embodiment discloses a traditional Chinese medicine compound for treating fatty liver, the raw materials of which are as follows: 30 g of Gynostemma pentaphyllum, 15 g of Artemisia capillaris, 12 g of Cassia seed, 10 g of Curcuma longa, and 12 g of Monascus.

[0029] Experimental analysis

[0030] (1) The Junyu Xiaozhi recipe can significantly reduce lipid deposition and fibrosis in NASH mice and improve liver function

[0031] Preparation of decoction: The dosage of Junyu Xiaozhi Recipe is converted into an equivalent dosage ratio according to the body surface area of ​​humans and rats, and then soaked in 20 times of water for 30 minutes, boiled, filtered to obtain the filtrate, and then boiled in 15 times of water for 30 minutes, repeated the above steps, and filtered and combined the filtrates three times.

[0032] Low-dose slimming formula: The drug content in the liquid medicine is 5g / kg;

[0033] Medium-dose juanyuxiaozhi prescription: the drug content in the liquid medicine is 10g / kg;

[0034] High-dose slimming formula: The drug content in the liquid medicine is 20g / kg;

[0035] In the in vivo study of this example, low, medium and high doses of JYXZF were administered intragastricly to NASH mice fed with high fat and high sugar, the negative control group was administered intragastricly with normal saline, and the positive control group was administered intragastricly with atorvastatin. Figure 1 As shown in Table 1, after 12 weeks of treatment, it was found that compared with the negative control group, the weight of the medium and high doses of Xiaozhi Fang gavage group was significantly reduced, the liver specific gravity decreased, and the levels of ALT, AST, FFA, TG, TC, etc. in the serum were significantly reduced, indicating that the degree of liver damage was reduced and lipid metabolism disorders were improved (see Table 1). In addition, HE staining, Oil Red O staining, F4 / 80 staining and Sirius red staining experiments confirmed that the intervention of Juanyu Xiaozhi Fang significantly inhibited the lipid accumulation, inflammatory response and fibrosis in the liver of NASH mice. Figure 2 Shown, scale bar = 100 μm.

[0036] Figure 1 In the figure, A is a schematic diagram of the NASH diet-fed mouse model; B is the mouse body weight curve; C is the mouse food intake; DF is the mouse body weight, liver weight and liver weight to body weight ratio; GH is the ALT and AST levels in serum; IJ is the determination of blood glucose values ​​in ITT and GTT experiments (NCD normal control group, HFD high-fat group, JYXZF high-fat + Juanyu Xiaozhi prescription intervention group, ATO high-fat + atorvastatin intervention group, n=6, *P<0.05, **P<0.01, ***P<0.001).

[0037] Table 1 Treatment with the JYXF formula inhibits lipid deposition in NASH mice

[0038]

[0039] Serum TG / TC / FFA / Blood Glucose levels (NCD normal control group, HFD high-fat group, JYXZF high-fat + Juanyu Xiaozhi prescription intervention group, ATO high-fat + atorvastatin intervention group, n=6, *P<0.05, **P<0.01, ***P<0.001, ###P<0.001).

[0040] (2) Identification of ingredients in the Junyu Xiaozhi recipe.

[0041] Blood was collected from rats 7 days after intragastric administration of the fat-removing prescription to obtain the drug-containing serum of the fat-removing prescription. The main components of the fat-removing prescription were detected by chromatography. A total of 2831 components of the traditional Chinese medicine liquid of the fat-removing prescription and 591 components of the drug-containing serum of the fat-removing prescription were found. The positive and negative ion diagrams are shown in Figure 2. Figure 3 shown.

[0042] (3) Metabolomics combined with network pharmacology to screen and predict HBP pathway as the key target of the Junyu Xiaozhi formula

[0043] The serum of NASH patients who took and did not take the Junyu Xiaozhi recipe were tested by non-targeted metabolomics, such as Figure 4 The results showed that compared with the untreated group, the HBP pathway metabolites F6P, GlcNAc-1-P and UDP-GlcNAc (the final product of HBP, the substrate for O-GlcNAc modification) in the treated group were significantly reduced. Figure 4In the figure, NASH means the group that did not take the fat-removing prescription, XZF means the group that took the fat-removing prescription, Fructose 6-phosphate (F6P), Acetyglucosamine 1-phosphate (GlcNAc-1-P), UDP-N-acetylglucosamine (UDP-GlcNAc). At the same time, this example predicts the targets of the fat-removing prescription by network pharmacology. KEGG analysis shows that the sugar metabolism pathway is significantly enriched. The compound-target-signal pathway network diagram also shows that the main components of the fat-removing prescription may target the key enzyme OGT ( Figures 5.1 to 5.3 ), molecular docking also showed that the active ingredients quercetin and curcumin in the compound had high binding energy with OGT. The above results suggest that O-GlcNAc modification mediated by the HBP pathway and its key enzyme OGT may be the key targets of the Junyu Xiaozhi recipe.

[0044] (4) JYXF significantly down-regulated O-GlcNAc modification and key enzyme expression in NASH mice

[0045] In order to clarify the changes in O-GlcNAc modification in NASH mice after treatment with the Juanyu Xiaozhi recipe, the liver tissue of mice after oral administration in the previous step was tested in this example. The results of immunohistochemical staining and Western blot experiments showed that compared with the control group, the O-GlcNAc modification level in the liver tissue of NASH mice decreased in a dose-dependent manner as the concentration of Xiaozhi recipe was increased ( Figure 6 AB). In addition, targeted metabolomics detection found that the level of UDP-GlcNAc, a key metabolite modified by O-GlcNAc, also decreased significantly ( Figure 6 C). Further detection of changes in key enzyme levels revealed that after intervention with Xiaozhi Fang, the protein and mRNA levels of OGT, a key enzyme for O-GlcNAc modification, were downregulated. It is speculated that the Juanyu Xiaozhi Fang may regulate the protein O-GlcNAc modification mediated by the HBP pathway by downregulating the expression of the metabolite UDP-GlcNAc and the key enzyme OGT ( Figure 6 DE).

[0046] Figure 6 A is the immunohistochemical staining of O-GlcNAc modification, scale bar = 100 μm; B is the O-GlcNAc modification level of liver tissue in NASH mice; C is the metabolite UDP-GlcNAc level; D is the protein level of key enzymes OGA, OGT and GFPT1; E is the mRNA level of key enzymes OGA, OGT and GFPT1 (NCD normal control group, HFD high-fat group, JYXZF high-fat + Juanyu Xiaozhi prescription intervention group, n = 6, *P < 0.05, **P < 0.01, ***P < 0.001).

[0047] (5) In vitro treatment with serum containing the JYX formula significantly inhibited lipid deposition in steatotic cells and downregulated O-GlcNAc modification

[0048] In order to verify the effect of the JYXF formula in vitro, the present example used the JYXF formula-containing serum to treat FFA-induced human normal hepatocyte MIHA, and the HBP pathway key enzyme GPFT1 inhibitor DON was used as a positive control. First, different concentrations of serum containing the drug were used to treat, and the optimal concentration of 10% ( Figure 7 A). Then, Oil Red O staining revealed that treatment with drug-containing serum significantly reduced fat accumulation in MIHA cells induced by FFA, and the TG and TC contents in the supernatant also decreased significantly ( Figure 7 BC), indicating that the serum containing JYXF can significantly inhibit the lipid deposition in adipose cells.

[0049] Figure 7 In the figure, A shows FFA-induced MIHA cells treated with different concentrations of serum containing JYXZF formula, and cell viability was detected by CCK8 method; BC shows the cells treated with the optimal concentration of serum containing JYXZF formula, and the fat accumulation of cells was detected by Oil Red O staining (B), and the levels of TG and TC in the cell supernatant were detected by ELISA (C); among them, BSA normal control group, FFAs steatosis induction group, JYXZF steatosis induction + JYXZF formula serum intervention group, DON steatosis induction + DON intervention group, n=3, *P<0.05, **P<0.01, ***P<0.001.

[0050] Then, the results of Western blot experiments showed that compared with the control group, the O-GlcNAc modification levels of cells in the JYXZF group and the DON group were significantly decreased ( Figure 8 A), target metabolic detection also found that the level of UDP-GlcNAc, a key metabolite modified by O-GlcNAc, also decreased significantly ( Figure 8 B). Furthermore, this example detected the changes in key enzyme levels and found that the results were consistent with the in vivo animal experiment results: after the intervention of the fat-removing recipe, the expression level of the key enzyme OGT for O-GlcNAc modification was downregulated, further confirming our hypothesis that the fat-removing recipe regulates the protein O-GlcNAc modification mediated by the HBP pathway by downregulating the expression of the metabolite UDP-GlcNAc and the key enzyme OGT ( Figure 8 CD).

[0051] Figure 8A is the total cellular O-GlcNAc modification level; B is the metabolite UDP-GlcNAc level; C is the protein level of key enzymes OGA, OGT, GFPT1 and p-GFPT1; D is the mRNA level of key enzymes OGA, OGT and GFPT1 (BSA normal control group, FFAs steatosis induction group, JYXZF steatosis induction + Juanyu Xiaozhi prescription drug-containing serum intervention group, DON steatosis induction + DON intervention group, n=3, *P<0.05, **P<0.01, ***P<0.001).

[0052] (6) Glycosylation modification omics depicts the differential modification profile of NASH mice after intervention with the JYX formula

[0053] In order to further describe the differential modification profile after the intervention of the Juanyu Xiaozhi Recipe, this example performed a label-free O-GlcNAc modification analysis on the liver tissue of NASH mice treated with the Xiaozhi Recipe ( Fig. 9 A). In the six samples, we detected a total of 488 unique peptides from 416 proteins, including 1236 O-GlcNAc-modified protein sites, of which 569 quantifiable O-GlcNAc peptides were found on 299 modified proteins ( Fig. 9 B). Next, the Venn diagram showed that there were 342 modified peptides overlapped between the two groups, and the overlap was good ( Fig. 9 C). Fig. 9 In the figure, A is the proteomic enrichment and identification flow chart of O-GlcNAc modified peptides; B is the statistical bar chart of the identification and quantification results of all modified peptides; C is the Venn diagram of O-GlcNAc peptides identified between the groups (A: saline gavage group, B: Junyu Xiaozhi recipe gavage group)

[0054] In order to analyze the O-GlcNAc peptides with different expression levels among different groups, we quantified the experimental results. The results showed that compared with the control group, there were 51 peptides with downregulated modification levels in the Xiaozhi prescription intervention group ( Fig.10 AB). We further performed KEGG analysis on the proteins to which the downregulated differentially modified peptides belonged. The results showed that the MAPK pathway, NF-kB pathway, and HIF-1 pathway were significantly enriched. These pathways are all related to lipid regulation, suggesting that the Juanyu Xiaozhi recipe does inhibit the progression of NASH by affecting protein O-GlcNAc modification ( Fig.10 C). Fig.10A is a bar graph of the quantitative differences in O-GlcNAc peptides between groups; B is a volcano plot of O-GlcNAc differentially modified peptides between groups, with significantly downregulated modified peptides marked in blue, significantly upregulated modified peptides marked in red, and no difference in modified peptides in gray, FC≤0.67 or FC≥1.5, P<0.05; C is the KEGG analysis of the proteins to which the differentially expressed peptides belong, P<0.05 (A: saline gavage group, B: Juanyu Xiaozhi recipe gavage group).

[0055] (7) O-GlcNAc modification of TAB2 and downstream MAPK pathway were significantly downregulated after intervention with JYF

[0056] This example selected TAB2, a differentially modified protein in the MAPK pathway, for further study. We first used Co-IP experiments to confirm that TAB2 interacted with the key enzyme OGT modified by O-GlcNAc, and then sWGA affinity experiments confirmed that TAB2 was O-GlcNAc-modified, and that its modification level was significantly reduced after the intervention of the Junyu Xiaozhi recipe ( Fig.11 AB). TAB2 is a binding protein of TAK1, a kinase in the MAPK pathway. Therefore, we detected the MAPK pathway activity in the liver tissue of NASH mice after treatment with JYXF. The results showed that compared with the saline gavage group, p-TAK1, p-p38 and p-65 (NF-kB) in the high-fat group were significantly upregulated, while the above indicators decreased significantly after JYXF intervention ( Fig.11 C), and inflammatory factors such as TNF-α and IL-6 were also significantly downregulated after treatment ( Fig.11 D), indicating that the activities of MAPK and NF-kB mediated by TAK1 were significantly decreased after the intervention of JYXF.

[0057] Fig.11 In the figure, A is the Co-IP experiment of TAB2 and OGT; B is the sWGA affinity experiment of TAB2, and anti-TAB2 detects the O-GlcNAc modification level of TAB2 protein; C is the protein level of MAPK and NF-kB pathway-related indicators; D is ELISA to detect the levels of TNF-α and IL-6 in the liver of NASH mice (NCD normal control group, HFD high-fat group, JYXZF high-fat + Juanyu Xiaozhi prescription intervention group, n=6, *P<0.05, **P<0.01, ***P<0.001).

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. A Chinese medicinal compound for treating fatty liver, characterized in that: The raw materials of the traditional Chinese medicine compound are as follows according to the mass ratio: 28-32 of gynostemma pentaphyllum, 13-17 of capillaris, 11-14 of cassia seed, 6-12 of turmeric, and 7-11 of red yeast rice.

2. A Chinese medicinal compound for treating fatty liver according to claim 1, characterized in that: The raw material composition of the traditional Chinese medicine compound according to mass ratio is: 30 gynostemma pentaphyllum, 15 capillaris, 12 cassia seed, 10 turmeric, and 12 red yeast rice.

3. A Chinese medicinal compound for treating fatty liver according to claim 1, characterized in that: A formulation suitable for oral administration is employed.

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