Use of silybum marianum pharmaceutical composition in preparation of drugs for treating fatty liver diseases

Abstract

The present application relates to the technical field of medicines, and in particular to a use of a silybum marianum pharmaceutical composition in the preparation of drugs for treating fatty liver diseases. The present application provides a use of a pharmaceutical composition in the preparation of drugs for preventing and / or treating fatty liver diseases. An active ingredient of the pharmaceutical composition comprises a 7-KDCA modulator; the 7-KDCA modulator comprises silymarin, or the 7-KDCA modulator comprises silybin, silychristin, silydianin, and / or isosilybin; the silybin comprises silybin A and / or silybin B; and the isosilybin comprises isosilybin A and / or isosilybin B. Researches show that the pharmaceutical composition can effectively and safely treat fatty liver diseases at a low dosage, exhibits anti-inflammatory activity, and has nerve growth factor-like activity, thereby showing great potential for use in the preparation of drugs for preventing and / or treating fatty liver diseases, inflammations, and senile dementia.

Description

Application of milk thistle medicinal composition in preparing medicine for treating fatty liver disease

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on February 28, 2024, with application number 202410223526.X and invention name “Application of Milk Thistle Pharmaceutical Composition in the Preparation of Drugs for the Treatment of Fatty Liver Disease”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the use of a silymarin pharmaceutical composition in the preparation of a medicament for treating fatty liver disease, and belongs to the field of medical technology. Background Art

[0004] Metabolic dysfunction associated fatty liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is a global liver disease that affects at least 25% of the world's population. MASLD can progress from simple steatosis (lipid accumulation) to metabolic dysfunction associated steatohepatitis (MASH), and further to liver fibrosis, cirrhosis, and liver cancer. The pathogenesis of MASLD is multifactorial, involving genetic, metabolic, and environmental factors. A key feature of MASLD is chronic liver inflammation, which plays a crucial role in the progression from simple steatosis to MASH and beyond. The production of proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-6 (IL-6), can promote the inflammation of MASLD. Another important factor affecting MASLD is the gut-liver axis, which mediates a bidirectional cycle between the gut microbiota and the liver. Alterations in the gut microbiota, increased intestinal permeability, and the resulting excessive influx of microbial metabolites (such as lipopolysaccharide (LPS)) into the liver contribute to the production of TNF and interleukin-1β (IL-1β), leading to liver inflammation and further progression of MASLD.

[0005] Bile acid (BA) metabolism, regulated by the gut microbiota, is also closely associated with MASLD. Microorganisms containing bile salt hydrolases (BSH) can dissociate conjugated bile acids into free bile acids. Some primary bile acids can also be converted into secondary bile acids by the gut microbiota. Therefore, BA composition is influenced by changes in the gut microbiota. As endogenous ligands for the farnesoid X receptor (FXR), bile acids not only affect lipid absorption but also regulate glucose metabolism, lipid metabolism, and energy metabolism by modulating the FXR signaling pathway, playing a role in MASLD. Therefore, many drugs used clinically to treat MASLD are FXR agonists, such as obeticholic acid, vonafexor, PX-104, and TERN-101. However, these drugs currently face challenges. For example, FXR agonist-related drugs are all in the clinical validation stage and have not yet been clinically tested. Furthermore, obeticholic acid, the first FXR agonist to enter Phase III clinical trials, still carries the risk of drug-induced liver injury. Therefore, there is an urgent need to find MASLD treatment drugs with good therapeutic effects, clear mechanisms of action and few side effects.

[0006] Summary of the Invention

[0007] To solve the above problems, the present application provides the use of a pharmaceutical composition in the preparation of a drug, wherein the drug has any of the following functions:

[0008] (a) prevention and / or treatment of fatty liver disease;

[0009] (b) anti-inflammatory;

[0010] (c) prevention and / or treatment of neurodegenerative diseases;

[0011] The active ingredient of the pharmaceutical composition comprises a 7-KDCA modulator; the 7-KDCA modulator includes silymarin, or the 7-KDCA modulator comprises silybin, silybintin, silybinin, and / or isosilybin; the silybin comprises silybin A and / or silybin B; and the isosilybin comprises isosilybin A and / or isosilybin B. Silymarin is a natural active substance extracted from the seeds of milk thistle (Silybum marianum (L.) Gaertn.) and primarily contains flavonolignans (silybin, silybintin, silybinin, and isosilybin) and dihydroquercetin. Silybin (silybin A and silybin B) is the primary active ingredient in silymarin, accounting for 50-70% of the total active ingredient.

[0012] In one embodiment of the present application, the 7-KDCA regulator is silymarin, or the 7-KDCA regulator is composed of silybin and isosilybin A; and the silybin is composed of silybin A and silybin B.

[0013] In one embodiment of the present application, in the drug, the mass ratio of silybin to isosilybin A is 7 to 8:1.

[0014] In one embodiment of the present application, the fatty liver disease includes metabolic dysfunction associated fatty liver disease (MAFLD).

[0015] In one embodiment of the present application, the treating fatty liver disease comprises treating fatty liver disease by regulating bile acid metabolism; the regulating bile acid metabolism comprises reducing the level of 7-KDCA and thereby reducing the binding of 7-KDCA to the farnesoid X receptor (FXR), thereby affecting the stability of the farnesoid X receptor.

[0016] In one embodiment of the present application, the treatment of fatty liver disease includes alleviating inflammation in patients with fatty liver disease, reducing liver weight in patients with fatty liver disease, reducing fat weight in patients with fatty liver disease, reducing the liver weight / body weight ratio in patients with fatty liver disease, improving liver fatty degeneration in patients with fatty liver disease, reducing lipid droplet accumulation in patients with fatty liver disease, improving serum indicators in patients with fatty liver disease, improving liver function indicators in patients with fatty liver disease and / or improving the intestinal flora in patients with fatty liver disease.

[0017] In one embodiment of the present application, the reducing inflammation in patients with fatty liver disease includes reducing inflammation in patients with fatty liver disease by regulating the TLR4 / NF-κB signaling pathway; the improving the intestinal flora in patients with fatty liver disease includes regulating the relative abundance of intestinal flora that produce short-chain fatty acids in the intestine of patients with fatty liver disease, regulating the relative abundance of intestinal flora that produce secondary bile acids (secondary bile acids) in the intestine of patients with fatty liver disease and / or regulating the relative abundance of intestinal flora containing bile salt hydrolase activity in the intestine of patients with fatty liver disease.

[0018] In one embodiment of the present application, the anti-inflammatory effect includes alleviating inflammation in patients with inflammation by regulating the TLR4 / NF-κB signaling pathway.

[0019] In one embodiment of the present application, the treatment of neurodegenerative diseases includes treating neurodegenerative diseases as a nerve growth factor-mimetic active substance.

[0020] In one embodiment of the present application, the neurodegenerative disease includes Alzheimer's disease.

[0021] In one embodiment of the present application, the drug further contains pharmaceutical excipients.

[0022] In one embodiment of the present application, the pharmaceutical excipients include stabilizers, preservatives, disintegrants, sweeteners, glidants and / or diluents.

[0023] In one embodiment of the present application, the dosage form of the drug is capsule, injection, powder, granule, paste and / or tablet.

[0024] The present application also provides a nerve growth factor-mimicking active substance, the ingredients of which include a 7-KDCA regulator; the 7-KDCA regulator includes silymarin, or the 7-KDCA regulator includes silybin, silybintin, silybinin and / or isosilybin; the silybin includes silybin A and / or silybin B; the isosilybin includes isosilybin A and / or isosilybin B.

[0025] In one embodiment of the present application, the 7-KDCA regulator is silymarin, or the 7-KDCA regulator is composed of silybin and isosilybin A; and the silybin is composed of silybin A and silybin B.

[0026] In one embodiment of the present application, in the drug, the mass ratio of silybin to isosilybin A is 7 to 8:1.

[0027] The present application also provides the use of a pharmaceutical composition in the preparation of a nerve growth factor-mimicking active substance, wherein the components of the nerve growth factor-mimicking active substance include a 7-KDCA regulator; the 7-KDCA regulator includes silymarin, or the 7-KDCA regulator includes silybin, silybintin, silybinin and / or isosilybin; the silybin includes silybin A and / or silybin B; the isosilybin includes isosilybin A and / or isosilybin B.

[0028] In one embodiment of the present application, the 7-KDCA regulator is silymarin, or the 7-KDCA regulator is composed of silybin and isosilybin A; and the silybin is composed of silybin A and silybin B.

[0029] In one embodiment of the present application, in the drug, the mass ratio of silybin to isosilybin A is 7 to 8:1.

[0030] The technical solution of this application has the following advantages:

[0031] 1. The present application provides the use of a pharmaceutical composition in the preparation of a medicament for preventing and / or treating fatty liver disease, wherein the active ingredient of the pharmaceutical composition comprises a 7-KDCA regulator; the 7-KDCA regulator includes silymarin, or the 7-KDCA regulator comprises silybin, silybintin, silybinin and / or isosilybin; the silybin comprises silybin A and / or silybin B; the isosilybin comprises isosilybin A and / or isosilybin B.

[0032] Animal experiments have shown that the pharmaceutical composition provided in the present application can reduce inflammation in MASLD model mice, reduce liver weight in MASLD model mice, reduce fat weight in MASLD model mice, reduce the liver weight / body weight ratio in MASLD model mice, improve hepatic fatty degeneration in MASLD model mice, reduce lipid droplet accumulation in MASLD model mice, improve serum indicators in MASLD model mice, improve liver function indicators in MASLD model mice, and improve the intestinal flora in MASLD model mice (including regulating the relative abundance of intestinal flora that produce short-chain fatty acids in the intestine of MASLD model mice). The present invention has the advantages of regulating the relative abundance of intestinal flora that produce secondary bile acids in the intestines of MASLD model mice, regulating the relative abundance of intestinal flora that contain bile salt hydrolase activity in the intestines of MASLD model mice, etc.), and has a good treatment for MAFLD. In addition, animal experiments have shown that the pharmaceutical composition provided by the present application can significantly improve the liver function indicators of MASLD model mice (including reducing the levels of triglycerides, total cholesterol, aminotransferase, aspartate aminotransferase and total bile acid in the serum of MASLD model mice), has a certain liver protection effect, and will not cause liver damage when used for the treatment of MAFLD;

[0033] Mechanistic experiments have demonstrated that the pharmaceutical composition provided herein can alleviate inflammation in MASLD model mice by regulating the TLR4 / NF-κB signaling pathway. Furthermore, the pharmaceutical composition provided herein can reduce the level of 7-KDCA in MASLD model mice, thereby reducing the binding of 7-KDCA to the farnesoid X receptor in MASLD model mice (negative feedback regulation), thereby affecting the stability of the farnesoid X receptor in MASLD model mice, thereby achieving the purpose of treating MASLD.

[0034] Therefore, the pharmaceutical composition provided in the present application can effectively and safely treat fatty liver disease (especially MAFLD), and has great application prospects in the preparation of drugs for preventing and / or treating fatty liver disease (especially MAFLD).

[0035] Furthermore, the 7-KDCA modulator is composed of silybin and isosilybin A; the silybin is composed of silybin A and silybin B; in the drug, the mass ratio of silybin and isosilybin A is 7 to 8:1. Animal experiments have shown that compared with a high dose of silymarin (80 mg / kg / day silymarin), a lower dose of silybin and isosilybin A (44.87 mg / kg / day silybin + 6.09 mg / kg / day isosilybin A) can effectively treat MASLD, and the effect is close to that of a high dose of silymarin (80 mg / kg / day silymarin). Therefore, the pharmaceutical composition provided by the present application can effectively and safely treat fatty liver disease (especially MAFLD) and has great application prospects in the preparation of drugs for preventing and / or treating fatty liver disease (especially MAFLD).

[0036] 2. This application provides the use of a pharmaceutical composition in the preparation of an anti-inflammatory drug, wherein the active ingredient of the pharmaceutical composition comprises a 7-KDCA modulator; the 7-KDCA modulator includes silymarin, or the 7-KDCA modulator comprises silybin, silybintin, silybinin, and / or isosilybin; the silybin comprises silybin A and / or silybin B; and the isosilybin comprises isosilybin A and / or isosilybin B. Cell experiments have demonstrated that the pharmaceutical composition provided herein exhibits excellent anti-inflammatory activity at the cellular level. Animal experiments have demonstrated that the pharmaceutical composition provided herein can alleviate inflammation in MASLD model mice. Mechanistic experiments have demonstrated that the pharmaceutical composition provided herein can alleviate inflammation in MASLD model mice by regulating the TLR4 / NF-κB signaling pathway. Therefore, the pharmaceutical composition provided herein has great application prospects in the preparation of anti-inflammatory drugs.

[0037] 3. The present application provides the use of a pharmaceutical composition in the preparation of a drug for preventing and / or treating a neurodegenerative disease, wherein the active ingredient of the pharmaceutical composition comprises a 7-KDCA modulator; the 7-KDCA modulator includes silymarin, or the 7-KDCA modulator comprises silybin, silybin, silybinin and / or isosilybin; the silybin comprises silybin A and / or silybin B; the isosilybin comprises isosilybin A and / or isosilybin B. Alzheimer's disease is a stubborn disease that seriously threatens the health of the elderly. The drugs currently on the market for treating neurodegenerative diseases such as Alzheimer's disease only play a role in alleviating symptoms, and it is still an incurable disease. It is reported that the onset of neurodegenerative diseases such as Alzheimer's disease is related to nerve cell damage and death, and nerve growth factor (NGF) has an important effect on the growth, survival and function maintenance of neurons. Because NGF is highly hydrophilic and has a large molecular weight, it cannot penetrate the blood-brain barrier, which limits its application in treating neurodegenerative diseases such as Alzheimer's disease. Therefore, looking for small molecule compounds with NGF-like activity that can smoothly pass through the blood-brain barrier and have NGF-like activity has become a research hotspot. Cell experiments have shown that the pharmaceutical composition provided by the application has NGF-like activity, and each component in the pharmaceutical composition is a small molecule compound. Therefore, the pharmaceutical composition provided by the application is a NGF-like active substance and has great application prospects in the preparation of drugs for preventing and / or treating neurodegenerative diseases such as Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1: HPLC analysis results of silymarin.

[0039] Figure 2: Chemical structure of the main components of silymarin.

[0040] Figure 3: Effects of silymarin and its main components on PC12 cells. Figure 3, (A) Images of PC12 cells after sample treatment. (A) In (a), (b) silybin (4.8 μg / mL), (c) silymarin (4.8 μg / mL), (d) silymarin (4.8 μg / mL), (e) silymarin (4.8 μg / mL); (B) Quantification results of Figure A.

[0041] Figure 4: Body weight changes in mice during high-fat diet modeling.

[0042] Figure 5: Effects of silymarin and its main components on body weight, liver weight, and fat weight in HFD-fed mice. In Figure 5, (A) Dosing regimen for mice; (B) Body weight changes of mice before dosing; (C) Body weight changes of mice during dosing; (D) Body weight of mice after dosing; (E) Liver weight of mice after dosing; (F) Epididymal fat weight of mice after dosing; (G) Liver weight to body weight ratio of mice after dosing.

[0043] Figure 6: Effects of silymarin on hepatic steatosis, serum biochemical parameters, and liver function in mice fed a high-flow diet. Figure 6 shows (A) histological staining of mouse organs; (B) quantification of oil red staining of mouse livers; (C) serum triglyceride levels; (D) serum total cholesterol levels; (E) serum ALT levels; (F) serum AST levels; and (G) serum total bile acid levels.

[0044] Figure 7: Effects of silymarin on inflammation in HFD mice. In Figure 7, (A) LPS levels in mouse feces; (B) LPS levels in mouse serum; (C) TNF-α levels in mouse serum; (D) IL-6 levels in mouse serum; (E) LPS levels in mouse liver; (F) TNF-α levels in mouse liver; (G) IL-6 levels in mouse liver; (H) Western blot results of TLR4, p-IKKβ, IKKβ, p-IκBα, IκBα, and βactin in mouse liver; (I) quantification of TLR4 / βactin; (J) quantification of p-IKKβ / IKKβ; (K) quantification of p-IκBα / IκBα.

[0045] Figure 8: Effects of silymarin on the intestinal microbiota of HFD mice. Figure 8 shows (A) α-diversity analysis - Chao1 index; (B) α-diversity analysis - Simpson index; (C) Venn diagram; (D) PCoA analysis; (E) phylum-level cluster analysis; (F) phylum-level relative abundance of intestinal microbiota; (G) Firmicutes / Bacteroidota (F / B); and (H) relative abundance of Desulfobacterota.

[0046] Figure 9: Effect of silymarin on the relative abundance of gut microbiota at the genus level in HFD mice. In Figure 9, (A) Genus-level clustering diagram (heat map); (B) Abundance of gut microbiota positively correlated with NASH; (C) Relative abundance of SCFA-producing gut microbiota; (D) Relative abundance of secondary bile acid-producing gut microbiota; (E) Relative abundance of BSH-containing gut microbiota.

[0047] Figure 10: Effect of silymarin on bile acid metabolism in HFD mice. Figure 10 shows (A) heat map of targeted metabolic analysis of bile acids in mouse feces; (B) bile acid composition and content ratio in mice; and (C) 7-KDCA levels.

[0048] Figure 11: FXR expression levels and the effect of 7-KDCA on FXR stability. Figure 11 shows (A) Western blot analysis of mouse liver FXR; (B) FXR / βactin quantification; (C) CETSA analysis of 7-KDCA and FXR; (D) quantification of Figure A; (E) DARTS analysis of 7-KDCA and FXR; and (F) quantification of Figure C. DETAILED DESCRIPTION

[0049] The following examples are provided to further better understand the present application, but are not limited to the best implementation mode described herein, and do not limit the content and protection scope of the present application. Any product identical or similar to the present application obtained by anyone under the inspiration of the present application or by combining the features of the present application with other prior arts shall fall within the scope of protection of the present application.

[0050] If no specific experimental steps or conditions are specified in the following examples, the experiments were carried out according to the conventional experimental steps or conditions described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional reagents.

[0051] Experimental Example 1: Analysis of the main chemical components of silymarin

[0052] The experimental process is as follows:

[0053] In order to clarify the main chemical components of silymarin, a silymarin sample (purchased from Zhejiang Huisong Pharmaceutical Co., Ltd.) was studied. Silymarin and standard products such as silybin, silybintin, and silybinin were analyzed by HPLC (Cosmosil 5C18-MS-II Packed Column (Ф4.6 / 250mm), flow rate 1mL / min, detection wavelength: 210nm, 50% methanol, 60min), and area normalization calculation was performed. The main components and content proportions of silymarin were determined to be silybin (A, B, 56.09%, t R =27.30min,31.57min), Silybum marianum (24.06%, t R =9.99min), Silymarin (6.85%, t R =12.03min), isosilybin A (7.61%, t R =45.70min), isosilybin B (3.25%, t R =50.63min) and dihydroquercetin (2.14%, t R=6.95min), and its retention time was basically consistent with that of the standard. The HPLC analysis results are shown in Figure 1 and the structure of the main component of silymarin is shown in Figure 2.

[0054] Experimental Example 2: Nerve Growth Factor-like Activity of Silymarin and Its Main Components

[0055] The experimental process is as follows:

[0056] Press 2×10 5 The amount of CFU / well was as follows: PC12 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were added to 24-well plates with 1 mL of DMEM medium (Dublecco's modified Eagle's medium, purchased from Thermo Scientific) containing 10% (v / v) fetal bovine serum, 5% (v / v) horse serum and 1% (w / v, g / 100 mL) premixed antibiotics (purchased from Invitrogen) per well. The 24-well plates were then placed in a cell culture incubator at 37°C and 5% (v / v) CO2 for 24 h. After the culture was completed, fresh DMEM medium was replaced and the 24-well plates were divided into a negative control group (Control), a silybin group (Silybin), a silydianin group (Silydianin), a silychristin group (Silychristin) and a silymarin group (Silymarin), with 3 replicates per group. After grouping, silybin was added to the silybin group, silydianin group, silychristin group and silymarin group, respectively. Silybin, silybin, silymarin, and silymarin were added to a final concentration of 4.8 μg / mL. No substance was added to the negative control group. Silybin was purchased from Chengdu Zhibiao Huapur, silybin and silybin were purchased from Chengdu Aifa Biological, and silymarin was purchased from Zhejiang Huisong Pharmaceutical Co., Ltd. After the addition of the samples, the 24-well plate was placed in a cell culture incubator at 37°C and 5% (v / v) CO2 for 24 hours. After the incubation period, the morphological changes of the cells were observed under a microscope and the cell neurite differentiation rate was recorded (cell neurite differentiation rate = the number of cells with neurites longer than the longest diameter of the cell body / the total number of cells in the selected field of view × 100%). The results showed that compared with the control group, silymarin and its main components significantly increased the proportion of PC12 cells with neurites (Figure 3A and Figure 3B), indicating that silymarin and its main components have nerve growth factor-like activity.

[0057] Experimental Example 3: Effects of Silymarin and Its Main Components on MASLD Model Mice

[0058] The experimental process is as follows:

[0059] 1. Evaluation of the efficacy of silymarin and its main components

[0060] 1.1. Establishment of MASLD mouse model using high-fat diet

[0061] Fifty four-week-old ICR male mice were purchased from the Hangzhou Medical University and housed at the Zhejiang University Experimental Animal Center (room temperature 23°C, 55% humidity, 12-h light / dark cycle). After one week of acclimatization, the mice were divided into a regular chow group (ND group, 10 mice) and a high-fat diet group (HFD group, 40 mice). The ND group was fed a regular chow diet, while the high-fat diet group was fed a high-fat diet for MASLD modeling. Body weight changes were recorded weekly. Regular chow was provided by the Zhejiang University Experimental Animal Center, while the high-fat diet was purchased from Nantong Trophy Feed Technology Co., Ltd. (feed code TP23300, containing 60% fat, 20.6% carbohydrates, and 19.4% protein). After 18 weeks of modeling, significant differences in body weight were observed between the ND and HFD groups. The average body weight gain in the ND group was 25.6 g, while that in the HFD group was 39.3 g (Figure 4).

[0062] 1.2 Effects of silymarin and its main components on liver weight, fat weight, and liver weight / body weight ratio in HFD-induced MASLD mice

[0063] After 18 weeks of modeling, MASLD model mice were randomly divided into four groups: HFD group (given a high-fat diet and water), Silymarin30 group (given a high-fat diet and 30 mg / kg / day silymarin), Silymarin80 group (given a high-fat diet and 80 mg / kg / day silymarin), and Silybin+Iso group (given a high-fat diet and 44.87 mg / kg / day silybin + 6.09 mg / kg / day isosilybin A) (as shown in Figure 5A), with 10 mice in each group, and the drugs were continuously administered by gavage for 8 weeks. The doses of silybin and isosilybin A were calculated by multiplying 80 mg / kg by the proportion of silybin and isosilybin A in silymarin (56.09% and 7.61%). Silybin and isosilybin A were purchased from Chengdu Zhibiao Chemical Purification Co., Ltd., and silymarin was purchased from Zhejiang Huisong Pharmaceutical Co., Ltd. Figure 5 shows changes in body weight during treatment in the ND and HFD groups. After 8 weeks of dosing, mice were weighed and autopsied, and liver and epididymal fat weights were measured. Body weight, liver weight, and epididymal fat weight were significantly higher in the HFD group than in the ND group (Figure 5D to Figure 5F, p<0.001, p<0.01, p<0.05). HFD-induced weight gain was reduced among treatment groups, but no significant differences were found (Figure 5D). However, liver weight was significantly reduced in the silymarin30, silymarin80, and silybin+iso treatment groups compared with the HFD group (Figure 5E, p<0.01, p<0.001, p<0.05), and epididymal fat weight was also significantly reduced (Figure 5F, p<0.01, p<0.05, p<0.05). There was no difference in the liver weight to body weight ratio between the ND and HFD groups, but the liver weight ratios were significantly lower in the three treatment groups compared with the HFD group (Figure 5G, p < 0.05, p < 0.001, and p < 0.05, respectively). These results indicate that silymarin or the combination of silymarin and isosilybin white A significantly reduced the HFD-induced increase in liver and fat weight and reduced the liver to body weight ratio in mice, with the Silymarin 80 group showing the greatest effect.

[0064] 1.3 Effects of silymarin and its main components on hepatic steatosis, lipid droplet accumulation, and serum liver function indices in HFD-induced MASLD mice

[0065] Eight weeks after oral administration, three liver samples were randomly collected from each group for histological staining and microscopic observation. Hematoxylin and eosin (H&E) staining was used to observe hepatocyte morphology. HE staining revealed significant hepatic steatosis and liver damage in the HFD group, which was ameliorated by silymarin 30, silymarin 80, and silybin + Iso (Figure 6A). Oil Red O staining was then used to observe lipid droplet formation. Results showed significant lipid droplet accumulation in the HFD group (Figure 6A and 6B, p < 0.001), while lipid droplet accumulation was reduced in the silymarin 30, silymarin 80, and silybin + Iso groups (Figure 6A and 6B, p < 0.05, p < 0.01, and p < 0.01, respectively). Sirius red and its contrasting dye are both strongly acidic dyes that readily bind to basic groups in collagen molecules, resulting in a red color under light microscopy. Therefore, Sirius red can be used to monitor liver fibrosis. The results showed that a small amount of collagen fibers was observed in the HFD group, but the collagen fibers were reduced in the ND group and the treatment group (Figure 6A), indicating that liver fibrosis was alleviated after silymarin treatment. In addition, after 8 weeks of oral administration, mouse plasma was collected by orbital bleeding. After standing for half an hour, centrifugation (12000 rpm for 30 minutes) was performed to obtain serum. Serum samples (200 μL) of 8 mice were randomly selected from each group to evaluate triglyceride (TG), total cholesterol (TC), aminotransferase (ALT), aspartate aminotransferase (AST), and total bile acid (TBA) levels (detection was completed by Hangzhou Haoke Biological). Compared with the ND group, the HFD group showed significantly increased triglyceride (TG) and total cholesterol (TC) levels (Figure 6C and Figure 6D, p < 0.001, p < 0.001). TG and TC levels were significantly decreased in each treatment group (Figure 6C, p < 0.001, p < 0.001, < 0.01; Figure 6D, p < 0.01, p < 0.001, p < 0.05). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which reflect hepatocellular damage, were significantly increased in the HFD group and significantly decreased in the silymarin80 and silybin+iso groups (Figure 6E, p < 0.01, p < 0.01; Figure 6F, p < 0.001, p < 0.05, p < 0.05). Serum total bile acid (TBA) can reflect the level of hepatocellular damage and hepatic excretion dysfunction. The results showed that the HFD-induced increase in serum TBA levels was reduced in each treatment group (Figure 6G, p < 0.05, p < 0.01, p < 0.01, p < 0.001). These results indicate that silymarin or the combination of silybin and isosilybin white A can not only alleviate HFD-induced hepatic steatosis and reduce serum TG and TC levels, but also improve liver function indicators.

[0066] 1.4 Effects of silymarin and its main components on serum liver function indices in HFD-induced MASLD model mice

[0067] Fifty four-week-old ICR male mice were purchased from the Hangzhou Medical University and housed at the Zhejiang University Experimental Animal Center (room temperature 23°C, 55% humidity, 12-h light / dark cycle). After one week of acclimatization, the mice were divided into a normal chow group (ND group, 5 mice) and a high-fat diet group (HFD group, 45 mice). The normal chow group was fed a normal chow diet, while the high-fat diet group was fed a high-fat diet to establish the MASLD model. Body weight changes of the mice were recorded weekly. The normal chow diet was provided by the Zhejiang University Experimental Animal Center, while the high-fat diet was purchased from Nantong Trophy Feed Technology Co., Ltd. (feed code TP23300, containing 60% fat, 20.6% carbohydrates, and 19.4% protein).

[0068] After 18 weeks of modeling, the MASLD model mice were randomly divided into nine groups: HFD group (given a high-fat diet and water), silymarin group (given a high-fat diet and 80 mg / kg / day silymarin), silybin7 & isosilybinA1 group (given a high-fat diet and 44.87 mg / kg / day silybin + 6.09 mg / kg / day isosilybin A), silybin5 & isosilybinA5 group (given a high-fat diet and 25.48 mg / kg / day silybin). A), silybin group (given a high-fat diet and 44.87 mg / kg / day silybin), isosilybin group (given a high-fat diet and 6.09 mg / kg / day isosilybin), silybin group (given a high-fat diet and 5.48 mg / kg / day silybin), silybin group (given a high-fat diet and 19.25 mg / kg / day silybin), and dihydroquercetin group (given a high-fat diet and 1.71 mg / kg / day dihydroquercetin) (as shown in Table 1), 5 mice in each group, were continuously administered by gavage for 8 weeks. Among them, the dose of the silybin 7 & isosilybin A1 group was obtained by the ratio of silybin to isosilybin A in silymarin, and the dose of the silybin 5 & isosilybin A5 group was obtained by the total weight of the silybin 7 & isosilybin A1 group. Silybin and isosilybin A were purchased from Chengdu Zhibiao Chemical Pure Co., Ltd., silybin, silybinin, and dihydroquercetin were purchased from Chengdu Aifa Biological, and silymarin was purchased from Zhejiang Huisong Pharmaceutical Co., Ltd.

[0069] Eight weeks after oral administration, plasma was collected from the mice by orbital bleeding. After standing for half an hour, the blood was centrifuged (12,000 rpm for 30 minutes) to obtain serum. Serum samples (200 μL) were collected from five mice in each group to assess triglyceride (TG), total cholesterol (TC), aminotransferase (ALT), and aspartate aminotransferase (AST) levels (tested by Hangzhou Haoke Biological). The results further demonstrated that only silymarin or a specific combination of silybin and isosilybin white A in a specific ratio could reduce serum TG and TC levels and improve liver function indicators (Table 1, *p < 0.05, **p < 0.01, ***p < 0.01).

[0070] Table 1 Levels of triglyceride, total cholesterol, aminotransferase and aspartate aminotransferase in serum of mice in different groups

[0071] 2. Research on the mechanism of action of silymarin and its main components

[0072] 2.1 Effects of silymarin and its main components on alleviating inflammation in HFD-fed mice by regulating the TLR4 / NF-κB signaling pathway

[0073] To investigate whether the effects of silymarin and its main components are related to regulating inflammation, ELISA kits were used to detect the levels of pro-inflammatory cytokines in the feces, serum, and liver of mice. For fecal samples, based on Section 1.2, 30 mg of mouse feces collected after 8 weeks of oral administration were taken, and LPS levels were measured using an ELISA kit (purchased from Hefei Laier Bio). For liver and serum samples, based on Section 1.2, 30 mg of mouse liver collected after dissection of mice that had been administered oral administration for 8 weeks were taken, and LPS (lipopolysaccharide), TNF-α (tumor necrosis factor), and IL-6 (interleukin-6) levels in the liver and serum of mice were measured using an ELISA kit (purchased from Suzhou Yikesai Bio). The results of mouse fecal LPS levels showed that silymarin and Silybin + Iso reversed the increase in LPS levels in mouse feces caused by HFD (Figure 7A, p < 0.05, p < 0.05, p < 0.01, p < 0.01). HFD also increased serum LPS, TNF-α, and IL-6 levels (Figure 7B to Figure 7D, p < 0.05, p > 0.05, p < 0.05). Among them, serum LPS levels were significantly reduced in all treatment groups (Figure 7B, p < 0.05, p < 0.05, p < 0.05), but only Silymarin 80 and Silybin + Iso reduced serum TNF-α levels (Figure 7C, p < 0.05, p < 0.01), and only Silymarin 80 reduced serum IL-6 levels (Figure 7D, p < 0.05). The results showed that all treatment groups reduced the HFD-induced increase in liver LPS (Figure 7E, p<0.05, p<0.05, p<0.01, p<0.01), TNF-α (Figure 7F, p<0.05, p<0.05, p<0.05, p<0.01), and IL-6 (Figure 7G, p<0.05, p<0.05, p<0.05, p<0.05) levels. The TLR4 / NF-κB signaling pathway can be activated by LPS and proinflammatory cytokines, so the expression levels of proteins involved in this signaling pathway in mouse liver were evaluated by western blot (WB) analysis. Based on Section 1.2, 8 weeks after oral administration, liver samples from three randomly selected mice in each group were pooled, and 30 mg of the pooled liver sample was collected. The protein concentration of the mouse liver protein solution was measured using a BCA kit (purchased from Kangwei Century), and the test samples were subjected to Western blot analysis. The results showed that the expression levels of TLR4 and phosphorylated IκBα were significantly increased in the HFD group (Figure 7H, Figure 7I, and Figure 7K, p<0.05, p<0.01), and were significantly reduced by Silymarin80 (Figure 7H, Figure 7I, and Figure 7K, p<0.01, p<0.05).The silybin + isopropylamine group significantly increased the phosphorylation level of IKKβ (Figure 7H and Figure 7J, p < 0.05), but reduced the increase in IκBα phosphorylation level caused by HFD (Figure 7H and Figure 7K, p < 0.01, p < 0.05). These results indicate that silymarin reduces the inflammatory signaling pathway by regulating TLR4 / NF-κB and is more effective than the combination of silybin and isosilymarin A.

[0074] 2.2 Silymarin regulates the intestinal microbiota composition of HFD-fed mice

[0075] The gut-liver axis plays a key role in the development of MASLD. To investigate whether silymarin alters the gut microbiota, as described in Section 1.2, 8 weeks after oral administration, the mouse cages were cleaned and disinfected with alcohol, and fecal samples were collected. DNA was extracted from fecal samples (two pellets) of eight randomly selected mice per group. PE250 sequencing of the 16S rDNA V4 region of the samples was performed using a HiSeq 2500 instrument, targeting designated sequencing regions. Alpha-diversity analysis of the mouse gut microbiota is shown in Figure 8, A and Figure 8, B. HFD reduced the total number of microorganisms in mouse feces, while silymarin or silymarin + isopropyl alcohol did not restore the total number of microorganisms (Figure 8, A, p < 0.01). HFD did not alter the richness of microbial species in mouse feces, but species richness was significantly increased in the silymarin 80 group (Figure 8, B, p < 0.05). To examine the differences and similarities between the different groups in more detail, cluster analysis was performed for each group at the phylum level (Figure 8, C). The results showed that the microbial composition of the Silymarin 80 group and the ND group was the most similar, followed by the Silybin + Iso group and the Silymarin 30 group, respectively. The compositional differences between the HFD and ND groups were the greatest (Figure 8, C). These results suggest that the HFD altered the composition of the intestinal microbiota in mice, while 80 mg / kg silymarin partially restored it to the level of the ND group. An increase or decrease in the ratio of Firmicutes to Bacteroidota (F / B) is generally considered to be intestinal dysbiosis, with an increase in F / B often associated with obesity and metabolic disorders, potentially related to increased caloric extraction from the diet, fat deposition and production, and impaired insulin sensitivity. In this experiment, the F / B ratio was significantly increased in the HFD group (Figure 8, D, p < 0.001), while it was decreased in the Silymarin 80 and Silybin + Iso groups (Figure 8, D, p < 0.001, p < 0.05). Furthermore, Desulfovibrio, which is typically negatively correlated with body mass index and triglyceride levels and improves MASLD by producing acetate, decreased in the HFD group, but did not reach a significant level (Figure 8, E). However, the relative abundance of Desulfovibrio increased significantly in each treatment group (Figure 8, E, p < 0.01, p < 0.05, and p < 0.01, respectively). These results suggest that silymarin can regulate the composition of the gut microbiota in HFD-fed mice.

[0076] Silymarin regulates the relative abundance of SCFA- or secondary bile acid-producing microbiota and gut microbiota with BSH activity

[0077] Results of genus-level relative abundance analyses of the intestinal microbiota showed that HFD-induced changes in genus-level relative abundance were partially restored to normal levels by all treatment groups, particularly the silymarin80 group (Figure 9, A and B). Furthermore, 80 mg / kg of silymarin significantly increased the relative abundance of short-chain fatty acid (SCFA)-producing bacteria (i.e., Allobaculum, Bifidobacterium, and Dubosiella), which exert anti-inflammatory, anti-cancer, and intestinal permeability-reducing effects, maintaining homeostasis (Figure 9, C). Bacteria capable of producing secondary bile acids (i.e., Clostridium and Eubacterium, both belonging to the phylum Firmicutes) were also significantly increased in the silymarin80 group (Figure 9, D). However, the relative abundance of bacteria with bile salt hydrolase (BSH) activity showed inconsistent trends among the groups, suggesting that only certain bile acids, but not all bile acids, may play a role in the treatment of MASLD with silymarin. These results suggest that silymarin can regulate the abundance of gut microbiota at the genus level to improve HFD-induced hepatic steatosis.

[0078] 2.4 Silymarin regulates bile acid metabolism and reduces HFD-induced increase in 7-KDCA levels

[0079] To further investigate which bile acids play an important role in improving hepatic steatosis, based on Section 1.2, fecal samples from mice in the ND, HFD, and Silymain80 groups, collected 8 weeks after oral administration, were subjected to bile acid-targeted metabolomics analysis. Four samples were collected from each group, each consisting of a mixture of feces from two mice in the group, with a sample size of 30 mg (fecal samples were sent to Shanghai Zhongke Xinsheng Biotechnology for bile acid-targeted metabolomics analysis). The results showed that HFD altered the levels of different types of bile acids in mouse feces, while Silymarin80 restored the concentrations of some bile acids to ND levels (Figure 10, A). In the ND group, the bile acid with the highest concentration was 12-KLCA, followed by DCA, ApoCA, HDCA, α-MCA, LCA, and 7-KDCA (Figure 10, B). HFD significantly increased the concentrations of 7-KDCA and α-MCA (Figure 10A, p < 0.001, p < 0.05) and decreased the concentration of TCDCA (Figure 10A, p < 0.05). After treatment with 80 mg / kg silymarin, the concentration of 7-KDCA decreased to ND levels (Figure 10A and Figure 10C, p < 0.001), but the concentrations of α-MCA and TCDCA did not return to ND levels. These results suggest that silymarin may exert its effects by regulating 7-KDCA in bile acids.

[0080] 2.5 FXR is a potential target of 7-KDCA

[0081] Farnesoid X receptor (FXR) is an endogenous ligand for bile acids. Therefore, based on Section 1.2, Western blot was used to examine FXR expression in the livers of mice treated with oral gavage for 8 weeks. The results showed that FXR expression levels were significantly elevated in the silymarin30, silymarin80, and silybin+iso groups (Figure 11A and Figure 11B, p<0.001, p<0.001, and p<0.01, respectively). In the bile acid targeted metabolic analysis, 7-KDCA concentrations increased in the HFD group and decreased to normal levels in the silymarin80 group. Therefore, it is speculated that 7-KDCA may negatively regulate FXR through feedback, thereby modulating its downstream signaling pathways and improving MASLD. To verify this hypothesis, based on Section 1.2, proteins were extracted from the liver samples of mice in the ND group (see Section 2.1 for the extraction method) to obtain a mouse liver protein solution; the protein concentration of the mouse liver protein solution was measured using a BCA kit (purchased from Kangwei Century), and then the mouse liver protein solution was diluted with PBS buffer to a protein concentration of 2 μg / μL to obtain a dilution; the dilution was dispensed into Ep tubes (100 μL per tube) and different concentrations (0 μM, 0.001 μM, 0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM) of 7-KDCA (purchased from Sigma-Aldrich) were added to the dilution, and then incubated in a 37°C incubator for 3 h (setting a blank control) for subsequent CETSA and DARTS experiments. For CETSA, the incubated protein solution was heated at 66°C for 3 minutes and then centrifuged (4°C, 12,000 rpm, 20 minutes). The supernatant was collected and 5X SDS-PAGE loading buffer was added to the supernatant at a volume ratio of supernatant:buffer = 4:1. The supernatant was then heated at 100°C for 10 minutes for denaturation to obtain the test sample. The test sample was subjected to Western blot analysis to detect the expression of FXR and β-actin. For DARTS, 1.5% (w / w) pronase (purchased from MCE) was added to the incubated protein solution (TNC buffer was added to the blank control group) and incubated at room temperature (25°C) in the dark for 25 minutes to obtain the incubation solution. 5X SDS-PAGE loading buffer was added to the incubation solution at a volume ratio of incubation solution:buffer = 4:1, and the solution was heat-denatured at 100°C for 10 minutes to obtain the test sample. The test samples were then subjected to Western blot analysis to detect FXR and β-actin expression. The results showed that 7-KDCA dose-dependently increased the thermal stability of FXR when heated at 66°C (Figure 11C and Figure 11D, p < 0.05, p < 0.01, p < 0.01).At the same time, 1.5% (w / w) pronase could degrade FXR and β-actin, while 7-KDCA could dose-dependently inhibit the degradation of FXR by pronase (Figure 11E and Figure 11F, p < 0.01, p < 0.001, p < 0.01). These results indicate that 7-KDCA can bind to FXR and affect its stability, indicating that FXR is a potential target of 7-KDCA.

[0082] In summary, these experimental results indicate that silymarin's therapeutic effects on MASLD primarily occur through targeted regulation of the intestinal microbial metabolites LPS and 7-KDCA, which bind to TLR4 and FXR receptors and modulate downstream signaling pathways. These findings provide new insights into the mechanism of action of silymarin in the treatment of MASLD and clarify the potential role of 7-KDCA in MASLD.

[0083] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of the pharmaceutical composition in preparing a drug, characterized in that: The drug has any of the following functions: (a) prevention and / or treatment of fatty liver disease; (b) anti-inflammatory; (c) prevention and / or treatment of neurodegenerative diseases; The active ingredient of the pharmaceutical composition comprises a 7-KDCA regulator; the 7-KDCA regulator includes silymarin, or the 7-KDCA regulator comprises silybin, silybintin, silybinin and / or isosilybin; the silybin comprises silybin A and / or silybin B; the isosilybin comprises isosilybin A and / or isosilybin B.

2. The use according to claim 1, characterized in that The 7-KDCA regulator is silymarin, or the 7-KDCA regulator consists of silybin and isosilybin A; the silybin consists of silybin A and silybin B.

3. The use according to claim 2, characterized in that In the medicine, the mass ratio of silybin to isosilybin A is 7-8:

1.

4. The use according to any one of claims 1 to 3, characterized in that The fatty liver disease includes fatty liver disease associated with metabolic dysfunction.

5. The use according to any one of claims 1 to 4, characterized in that The treating fatty liver disease includes treating fatty liver disease by regulating bile acid metabolism; the regulating bile acid metabolism includes reducing the level of 7-KDCA and thereby reducing the binding of 7-KDCA to the farnesoid X receptor, thereby affecting the stability of the farnesoid X receptor.

6. The use according to any one of claims 1 to 5, characterized in that The treatment of fatty liver disease includes alleviating inflammation in patients with fatty liver disease, reducing liver weight in patients with fatty liver disease, reducing fat weight in patients with fatty liver disease, reducing the liver weight / body weight ratio in patients with fatty liver disease, improving liver steatosis in patients with fatty liver disease, reducing lipid droplet accumulation in patients with fatty liver disease, improving serum indicators in patients with fatty liver disease, improving liver function indicators in patients with fatty liver disease and / or improving the intestinal flora in patients with fatty liver disease.

7. The use according to claim 6, characterized in that The reducing inflammation in patients with fatty liver disease includes reducing inflammation in patients with fatty liver disease by regulating the TLR4 / NF-κB signaling pathway; the improving the intestinal flora in patients with fatty liver disease includes regulating the relative abundance of intestinal flora that produce short-chain fatty acids in the intestine of patients with fatty liver disease, regulating the relative abundance of intestinal flora that produce secondary bile acids in the intestine of patients with fatty liver disease and / or regulating the relative abundance of intestinal flora that contain bile salt hydrolase activity in the intestine of patients with fatty liver disease.

8. The use according to claim 1, wherein The method for treating neurodegenerative diseases includes treating neurodegenerative diseases as a substance mimicking nerve growth factor.

9. A nerve growth factor-mimicking active substance, characterized in that: The components of the nerve growth factor-mimicking active substance include a 7-KDCA regulator; the 7-KDCA regulator includes silymarin, or the 7-KDCA regulator includes silybin, silybintin, silybinin and / or isosilybin; the silybin includes silybin A and / or silybin B; the isosilybin includes isosilybin A and / or isosilybin B.

10. Use of a pharmaceutical composition in the preparation of a nerve growth factor-mimicking active substance, characterized in that: The components of the nerve growth factor-mimicking active substance include a 7-KDCA regulator; the 7-KDCA regulator includes silymarin, or the 7-KDCA regulator includes silybin, silybintin, silybinin and / or isosilybin; the silybin includes silybin A and / or silybin B; the isosilybin includes isosilybin A and / or isosilybin B.

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