Silybin derivatives and their use in the preparation of a medicament for the treatment of non-alcoholic fatty liver disease

Abstract

This invention discloses silybin derivatives or their stereoisomers, and pharmaceutically acceptable salts, with structures as shown in Formula I: R1, R2, R3, R4, and R6 are independently selected from hydrogen, and R5 is selected from methyl; excluding: R1 selected from hydrogen, R2 selected from hydrogen, R3 selected from hydrogen, R4 selected from hydrogen, R6 selected from hydrogen, and R5 selected from methyl. This invention discloses the use of silybin derivatives or their stereoisomers, and pharmaceutically acceptable salts, in the preparation of medicaments for the prevention and / or treatment of non-alcoholic fatty liver disease. This invention discloses the use of silybin derivatives or their stereoisomers, and pharmaceutically acceptable salts, in the preparation of medicaments for the prevention and / or treatment of non-alcoholic steatohepatitis. Silybin derivatives, at significantly lower doses than silybin alone or in combination with royal jelly acid, have shown significant effects on liver damage, fatty liver, inflammation, and liver fibrosis in non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention relates to silymarin derivatives and their use in the preparation of medicaments for treating non-alcoholic fatty liver disease. Background Technology

[0002] There are many types of liver-damaging diseases, affecting a large population. Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH, now also known as MASH) are the most common. NAFLD is a general term for metabolic-related liver diseases characterized primarily by hepatocyte fat accumulation. Broadly speaking, it includes a full spectrum of lesions, from simple fatty liver (without inflammation) to steatohepatitis (with inflammation), liver fibrosis, cirrhosis, and even hepatocellular carcinoma. Its core pathology is hepatocyte fat accumulation, and it can also include hepatocyte steatosis, hepatocyte damage, and inflammation that occur during the non-alcoholic steatohepatitis stage. Early clinical presentation is often asymptomatic, although some patients may experience fatigue and right upper quadrant pain. Laboratory tests may reveal mild to moderate elevations in transaminases (ALT / AST) (AST / ALT often <1), dyslipidemia (high triglycerides, low HDL-C), insulin resistance, or metabolic syndrome such as type 2 diabetes. The disease typically progresses slowly. NASH is the progressive inflammatory stage of NAFLD, characterized by hepatocellular steatosis, hepatocellular damage (ballooning degeneration), and inflammation, possibly accompanied by liver fibrosis. Typically, some (approximately 15%-25%) of NAFLD patients progress to the more severe NASH. NASH usually progresses faster than NAFLD, with patients often exhibiting persistently elevated liver enzymes (ALT / AST levels reaching 2-5 times the normal value) during physical examinations, and some may develop hepatosplenomegaly; both can progress to cirrhosis (ascites, palmar erythema, hypersplenism) or liver cancer in advanced stages. Unfortunately, there are currently no highly effective and safe drugs available for the treatment of NAFLD and NASH.

[0003] Silybin (CAS No. 36804-17-8) is a flavonoid compound extracted and isolated from the fruit of the chrysanthemum plant *Silybum marianum*. Silybin is usually not a single compound, but rather a mixture of two diastereomers in equal amounts: silybin A (2R, 3R, 10R, 11R, Silybin A / Silibinin A, CAS No. 22888-70-6) and silybin B (2R, 3R, 10S, 11S, Silybin B / Silibinin B, CAS No. 142797-34-0). Silybin A and B have highly homologous chemical structures; their only difference is the diastereomeric relationship with opposite configurations of the chiral carbon atom at position 11. Both have similar pharmacological and pharmacodynamic effects. In terms of liver protection, silymarin reduces hepatocyte damage by stabilizing hepatocyte membranes and inhibiting lipid peroxidation, while promoting hepatocyte DNA synthesis and regeneration. It can also inhibit the expression of liver fibrosis-related factors and reduce transaminase levels, thus improving alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver injury, and chronic hepatitis. Regarding its anti-inflammatory effects, it primarily inhibits the activation of the NF-κB signaling pathway, reduces the release of pro-inflammatory factors such as TNF-α and IL-1β, and alleviates inflammatory responses in the liver and systemic tissues, especially targeting inflammation-mediated organ damage. In terms of antioxidant and cell protection, it can significantly increase the content of antioxidants such as glutathione, scavenge free radicals, block oxidative stress-induced cell damage, maintain the body's redox balance, and protect cells from oxidative damage. Furthermore, silymarin can inhibit the proliferation and migration of tumor cells such as liver cancer and breast cancer, induce cancer cell apoptosis, regulate lipid metabolism, reduce low-density lipoprotein, protect cardiovascular endothelial cells, and has potential neuroprotective value.

[0004]

[0005]

[0006] Although studies have shown that silymarin has hepatoprotective effects at higher doses, it has not yet been used as a primary drug in the clinical treatment of NAFLD and NASH, suggesting that there are persistent weaknesses in the use of silymarin for the treatment of NAFLD and NASH that need to be overcome. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention tackles the problems of silybin's poor water solubility, low oral bioavailability, high dosage, and unsatisfactory efficacy. A carboxyl and hydroxyl esterification synthesis process was employed to successfully synthesize silybin derivatives. Through investigation of their lipid-lowering, anti-inflammatory, anti-fibrotic, and hepatoprotective effects in NAFLD and NASH models, it was unexpectedly discovered that silybin derivatives, with significantly reduced dosages, achieved unexpectedly and significantly better effects than silybin alone or in combination with royal jelly acid (also known as 10-hydroxy-2-decenoic acid), demonstrating promising applications in hepatoprotection, anti-fibrosis, lipid-lowering, and anti-inflammatory effects.

[0008] The technical solution of the present invention is as follows:

[0009] Silymarin derivatives or their stereoisomers as shown in Formula I, and pharmaceutically acceptable salts thereof:

[0010] ;

[0011] Among them, R1, R2, R3, R4, and R6 are independently selected from hydrogen, 10-hydroxy-2-decenoyl, and other groups, respectively. R5 is selected from methyl;

[0012] However, it does not include: R1 selected from hydrogen, R2 selected from hydrogen, R3 selected from hydrogen, R4 selected from hydrogen, R6 selected from hydrogen, and R5 selected from methyl.

[0013] As a preferred embodiment of the present invention, the following are silymarin derivatives or their stereoisomers, or pharmaceutically acceptable salts:

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020] Another object of the present invention is to provide the use of the silymarin derivatives thereof or their stereoisomers, pharmaceutically acceptable salts, in the preparation of medicaments for the prevention and / or treatment of non-alcoholic fatty liver disease.

[0021] Preferably, the use is the use of the silymarin derivative or its stereoisomer, or a pharmaceutically acceptable salt, in the preparation of a medicament for the prevention and / or treatment of hyperlipidemia, inflammation, and liver damage caused by non-alcoholic fatty liver disease.

[0022] Another object of the present invention is to provide the use of the silymarin derivatives thereof or their stereoisomers, pharmaceutically acceptable salts, in the preparation of medicaments for the prevention and / or treatment of non-alcoholic steatohepatitis.

[0023] Preferably, the use is the use of silymarin derivatives or their stereoisomers, or pharmaceutically acceptable salts, in the preparation of medicaments for the prevention and / or treatment of liver fibrosis and liver injury caused by non-alcoholic steatohepatitis.

[0024] Another object of the present invention is to provide the use of the silymarin derivatives thereof or their stereoisomers, pharmaceutically acceptable salts, in the preparation of medicaments for the prevention and / or treatment of liver fibrosis.

[0025] Another object of the present invention is to provide a pharmaceutical composition wherein the silymarin derivative or its stereoisomer, or a pharmaceutically acceptable salt thereof, is used as an active ingredient or a major active ingredient, and is formulated into a pharmaceutical dosage form with pharmaceutically acceptable excipients.

[0026] Preferably, the dosage form is a tablet, capsule, granule, suspension, liposome, or droplet.

[0027] When the dosage form of the pharmaceutical composition is tablets, it is made from the following raw and excipient materials in parts by weight: 2.5 to 300 parts of the silymarin derivative or its stereoisomer, pharmaceutically acceptable salt, 60 parts of microcrystalline cellulose, 30 parts of lactose, 0.5 parts of sodium carboxymethyl cellulose, and 0.5 parts of magnesium stearate.

[0028] When the dosage form of the pharmaceutical composition is a capsule, it is made into granules from the following raw and excipient materials in parts by weight: 2.5 to 90 parts of the silymarin derivative or its stereoisomer, a pharmaceutically acceptable salt, 60 parts of microcrystalline cellulose, 60 parts of lactose, and 0.5 parts of sodium carboxymethyl cellulose, and then the granules are filled into capsules.

[0029] When the dosage form of the pharmaceutical composition is granules, the granules are made from the following raw and excipient materials in parts by weight: 2.5 to 300 parts of the silymarin derivative or its stereoisomer, pharmaceutically acceptable salt, 90 parts of microcrystalline cellulose, 120 parts of corn starch, 90 parts of sucrose, and 0.5 parts of sodium carboxymethyl cellulose.

[0030] When the pharmaceutical composition is in liposome form, it is prepared from the following raw and excipient materials in parts by weight: 3-9 parts of the silybin derivative or its stereoisomer, a pharmaceutically acceptable salt, 12 parts of soybean lecithin, 7 parts of cholesterol, and 0.6 parts of vitamin E; the silybin derivative or its stereoisomer, a pharmaceutically acceptable salt, soybean lecithin, cholesterol, and vitamin E are added to anhydrous ethanol, and sonicated to dissolve, forming a homogeneous solution; the organic solvent is removed by rotary evaporation to form a phospholipid film; PBS buffer solution is added, hydrated, and sonicated in an ice bath to obtain a liposome solution, which is then dispensed.

[0031] When the dosage form of the pharmaceutical composition is drop pills, it is made from the following raw and excipient materials: the silybin derivative or its stereoisomer, a pharmaceutically acceptable salt, polyethylene glycol 4000 (PEG4000), Span 80 or Span 85, and polyethylene glycol 400 (PEG400); the weight ratio of the silybin derivative or its stereoisomer, a pharmaceutically acceptable salt to polyethylene glycol 4000 is (15-45):(150-450); the weight ratio of the silybin derivative or its stereoisomer, a pharmaceutically acceptable salt to Span 80 or Span 85 is (15-45):(2-6); and the amount ratio of the silybin derivative or its stereoisomer, a pharmaceutically acceptable salt to polyethylene glycol 400 is (15-45):(10-30) g / m³. L; Silybin derivatives or their stereoisomers, and pharmaceutically acceptable salts are passed through a 60-mesh sieve, and PEG4000 is passed through an 80-mesh sieve; PEG4000 is heated to melt, Span 80 or Span 85 and polyethylene glycol 400 are added, stirred evenly, silybin derivatives or their stereoisomers, and pharmaceutically acceptable salts are added, stirred, and allowed to stand until the foam dissipates, then poured into the storage tank of the pelleting machine (keeping it at 70-80℃), adjusting the dripping rate (50-60 drops / minute), and dripping it into dimethyl silicone oil at a temperature of 10-15℃, which is then cooled into pellets; the surface condensate is absorbed by filter paper, dried, and qualified pellets are screened (roundness ≥0.95, hardness ≥20KN), and the size and weight of the pellets are adjusted, with each 5 pellets containing approximately 10-30 mg of silybin derivatives or their stereoisomers, and pharmaceutically acceptable salts.

[0032] When the pharmaceutical composition is in the form of a suspension, it is prepared from the following raw and excipient materials in parts by weight: 5-30 parts of the silybin derivative or its stereoisomer, pharmaceutically acceptable salt, 10-60 parts of cyclodextrin, 0.6 parts of sodium carboxymethyl cellulose, and 1000 parts of purified water; the cyclodextrin is passed through an 80-mesh sieve and dissolved in hot water at 50-60°C to obtain a cyclodextrin aqueous solution; the silybin derivative or its stereoisomer, pharmaceutically acceptable salt is added to the cyclodextrin aqueous solution and ultrasonically mixed and dispersed to obtain solution A; sodium carboxymethyl cellulose is dispersed in solution A and ultrasonically dissolved and dispersed evenly; it is dispensed in 10 mL vials, each containing approximately 50-300 mg of the silybin derivative or its stereoisomer, pharmaceutically acceptable salt.

[0033] Another object of the present invention is to provide the use of the silymarin derivative or its stereoisomer, or a pharmaceutically acceptable salt thereof, in the preparation of health foods that have an adjunctive protective effect against chemically induced liver injury.

[0034] The beneficial effects of this invention are:

[0035] Compared to silybin or royal jelly acid, the silybin derivative of this invention, at significantly lower dosages than silybin or royal jelly acid alone or in combination, has achieved unexpectedly and significantly better effects on liver injury, fatty liver, inflammation, and liver fibrosis in non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver fibrosis. Therefore, the novel silybin derivative of this invention can be used to prepare drugs for treating liver injury, fatty liver, and liver fibrosis. Attached Figure Description

[0036] Figure 1 The 1H NMR spectrum of compound AH-SW01 is shown.

[0037] Figure 2 Mass spectra of compounds AH-SW02, AH-SW03, AH-SW04, and AH-SW07.

[0038] Figure 3 The 1H NMR spectrum of compound AH-SW06 is shown.

[0039] Figure 4 This is the carbon NMR spectrum of compound AH-SW06.

[0040] Figure 5 The 1H NMR spectrum of compound AH-SW07 is shown.

[0041] Figure 6 The results of pathological examination of liver sections in NAFLD model mice are shown for compounds AH-SW01 and AH-SW06.

[0042] Figure 7 This study investigated the lipid-lowering and hepatoprotective effects of compound AH-SW01 on a mouse model of non-alcoholic fatty liver disease (NAFLD). The values ​​were: A: AST; B: liver weight; C: liver TG; D: liver TC; E: ALT; F: liver-to-body weight ratio; G: serum TG; H: serum TC; Control: normal control group; Model: model group; AH-SW01-L: low-dose AH-SW01 group (6 mg / kg); AH-SW01-H: high-dose AH-SW01 group (12 mg / kg); FJB-3H: triple-dose silymarin group (26.7 mg / kg, three times the dose of the AH-SW01-H high-dose group); WJS-3H: triple-dose royal jelly acid group (10.4 mg / kg, three times the dose of the AH-SW01-H high-dose group). All results were compared with the model group using paired groups of students. T-test analysis: *: p<0.05; **: p<0.01; ***: p<0.001; ns: p>0.05, no statistically significant difference.

[0043] Figure 8 This study investigated the lipid-lowering and hepatoprotective effects of compound AH-SW06 on a mouse model of non-alcoholic fatty liver disease (NAFLD). Wherein, A: AST; B: liver weight; C: liver TG; D: liver TC; E: ALT; F: liver-to-body weight ratio; G: serum TG; H: serum TC; Control: normal control group; Model: model group; AH-SW06-L: low-dose AH-SW06 group (6 mg / kg); AH-SW06-H: high-dose AH-SW06 group (12 mg / kg); FJB-H: high-dose silymarin group (8.9 mg / kg, corresponding to AH-SW06). -H high-dose group equimolar amount); FJB-3H: silymarin triple high-dose group (26.7 mg / kg, corresponding to three times the dose of AH-SW06-H high-dose group, and six times the dose of AH-SW06-L low-dose group); WJS-H: royal jelly acid high-dose group (3.5 mg / kg, corresponding to equimolar amount of AH-SW06-H high-dose group); WJS-3H: royal jelly acid triple high-dose group (10.4 mg / kg, corresponding to three times the dose of AH-SW06-H high-dose group, and six times the dose of AH-SW06-L low-dose group); silymarin triple high-dose combined with royal jelly acid triple high-dose combined group (F+W 3H); all were analyzed using the independent students' t-test compared with the model group. *: p<0.05; **: p<0.01; ***: p<0.001; ns: p>0.05, no statistically significant difference.

[0044] Figure 9The results of H&E staining of liver pathological sections from mice with non-alcoholic steatohepatitis (NASH) caused by compounds AH-SW01 and AH-SW06 are shown. Among them, AH-SW01-H: high-dose AH-SW01 treatment group (12 mg / kg); AH-SW06-H: high-dose AH-SW06 treatment group (12 mg / kg); FJB-3H: three times high-dose silymarin treatment group (26.7 mg / kg); WJS-3H: three times high-dose royal jelly acid treatment group (10.4 mg / kg).

[0045] Figure 10 Oil Red O staining results of liver pathological sections from mice with non-alcoholic steatohepatitis (NASH) model, using compounds AH-SW01 and AH-SW06; where AH-SW01-H: high-dose AH-SW01 treatment group (12 mg / kg); AH-SW06-H: high-dose AH-SW06 treatment group (12 mg / kg); FJB-3H: three times the high dose of silymarin (26.7 mg / kg); WJS-3H: three times the high dose of royal jelly acid (10.4 mg / kg).

[0046] Figure 11 This study investigated the hepatoprotective effects of compounds AH-SW01 and AH-SW06 on non-alcoholic steatohepatitis (NASH) in mice, focusing on their anti-inflammatory and anti-fibrotic effects. The study included the following groups: A: TNF-α; B: IL-6; C: Cxcl1; D: Ccl2; E: Col1α1; F: Tgf-β; G: Timp1; H: Acta2; Control: normal control group; Model: model group; AH-SW01-H: high-dose AH-SW01 group (12 mg / kg); AH-SW06-H: high-dose AH-SW06 group (12 mg / kg); FJB-3H: triple-dose silymarin group (26.7 mg / kg); WJS-3H: triple-dose royal jelly acid group (10.4 mg / kg). All groups were compared with the model group. T-test analysis: *: p<0.05; **: p<0.01; ***: p<0.001; ns: p>0.05, no statistically significant difference.

[0047] Figure 12 The efficacy of compounds AH-SW02, AH-SW03, AH-SW04, and AH-SW07 on palmitic acid-induced intracellular lipid accumulation in AML12 cells was investigated.

[0048] Figure 13 The efficacy of compounds AH-SW02, AH-SW03, AH-SW04, and AH-SW07 on palmitic acid-induced intracellular lipid accumulation in HepG2 cells was investigated. Detailed Implementation

[0049] The technical solution of the present invention will be further described below with reference to the embodiments. The embodiments can enable those skilled in the art to have a more comprehensive understanding of the present invention, but do not limit the present invention to the scope of the embodiments described.

[0050] Unless otherwise specified, all chemicals purchased from commercial suppliers were used as is. Silymarin was obtained from Shanghai Bid Pharmaceutical Technology Co., Ltd. (CAS No.: 36804-17-8, purity >98.0%). All chemical reagents used were commercially available chemically pure or analytically pure, and all solvents were reagent grade. Purification and drying were performed according to standard methods when necessary. The reaction progress was monitored by thin-layer chromatography on GF254 silica gel plates (Shanghai Haohong Biomedical Technology Co., Ltd.) using petroleum ether (boiling range 60-90℃) and ethyl acetate, dichloromethane, and methanol as developing solvents. Ultraviolet absorption was detected by irradiation using a ZF-20D darkroom ultraviolet analyzer (Shanghai Baoshan Gucun Electro-Optical Instrument Factory). Solvent concentration was performed using an EYELA Instruments N-1300 rotary evaporator (at 40°C). Column chromatography used 200-300 mesh silica gel (Qingdao Ocean Chemical Plant Branch). Eluents included petroleum ether (boiling range 60-90°C), ethyl acetate, dichloromethane, and methanol. Melting point determination was performed using a Mel-TEMP II melting point apparatus without calibration. 1 H-NMR, 13 C10-NMR spectra were determined using a Bruker AV300 (300 MHz) NMR spectrometer. Tetramethylsilane (TMS) was used as an internal standard. Chemical shifts (δ) were expressed using the residual solvent resonance method, with units of ppm (CDCl3: 1 The H-NMR value was 7.26 ppm. 13 C-NMR was 77.16 ppm, DMSO: 1 The H-NMR value was 2.5 ppm. 13 C-NMR was 39.5 ppm. Multiplicity is abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplyt. Mass spectrometry was performed using an Agilent 1946A-MSD mass spectrometer (ESI-MS) and a Water Q-Tof mass spectrometer (HRMS). Compound purity was determined by HPLC using an Agilent C18 (4.6 mm × 250 mm, 5 μm) reversed-phase column. The mobile phase was a mixture of methanol and water, and the flow rate was 0.8 mL / min. UV absorbance was monitored at 254 nm. Yield refers to the percentage of the compound obtained through chromatographic and spectroscopic purification relative to the theoretical yield.

[0051] Example 1

[0052] Preparation of compound AH-SW01

[0053]

[0054] Step 1, Synthesis of Intermediate 1: 10-hydroxy-2-decenoic acid (3.00 g, 16.11 mmol) and tert-butyldimethylsilylchlorosilane (2.67 g, 17.72 mmol) were added sequentially to dichloromethane, followed by imidazole (2.75 g, 40.26 mmol). The mixture was stirred at room temperature for 24 hours under nitrogen protection, and the reaction was monitored by TLC until complete (eluent: PE:EA = 1:1 V / V). The reaction was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, allowed to stand, concentrated under reduced pressure, and separated by normal-phase silica gel column chromatography (eluent: PE:EA = 20:1 V / V) to obtain 3.87 g of Intermediate 1 (English name: (E)-10-((tert-butyldimethylsilyl)oxy)dec-2-enoic acid). acid, Chinese name: (E)-10-((tert-butyldimethylsilyl)oxy)dec-2-enoic acid, white solid, yield 80%). 1 H NMR (300 MHz, DMSO-d6) δ 12.10(s, 1H), 6.80 (dt, J = 15.6, 6.9 Hz, 1H), 5.74 (dt, J = 15.5, 1.5 Hz, 1H), 3.56 (t, J = 6.2 Hz, 2H), 2.16 (q, J = 6.8 Hz, 2H), 1.49–1.34 (m, 4H), 1.34–1.20 (m, 6H), 0.86 (s, 9H), 0.02 (s, 6H); ESI-m / z: 299.51 [M+H] + .

[0055] Step 2, Synthesis of Intermediate 2: Intermediate 1 (3.50 g, 11.65 mmol) was dissolved in N,N-dimethylformamide (DMF), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 8.86 g, 23.30 mmol) and N,N-diisopropylethylamine (DIPEA, 4.52 g, 34.95 mmol) were added. The mixture was activated for 30 minutes with stirring in an ice bath, and silymarin (6.18 g, 12.81 mmol) was added. The reaction was carried out at room temperature for 12 hours, and TLC monitoring showed that the reaction was complete at this point (evolving solvent: DCM:MeOH = 20:1). (V / V); quenched with water, extracted three times with ethyl acetate, the organic phase was collected, washed three times with saturated sodium chloride solution, the organic phase was collected again, dried over anhydrous sodium sulfate, allowed to stand, concentrated under reduced pressure, and the residue was separated by normal-phase silica gel column chromatography (eluent: DCM:MeOH = 20:1V / V) to give 3.30 Intermediate 2 (English name: (2R,3R)-3,5-dihydroxy-2-((2R,3R)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-oxochroman-7-yl(E)-10-((tert-butyldimethylsilyl)oxy)dec-2-enoate, Chinese name: (2R,3R)-3,5-dihydroxy-2-((2R,3R)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[b][1,4]dioxacyclohexene-6-yl)-4-carbonylchroman-7-yl(E)-10-((tert-butyldimethylsilyl)oxy)dec-2-enoate, white solid, yield 37%). 1HNMR (300 MHz, DMSO-d6) δ 11.69 (s, 1H), 9.15 (s, 1H), 7.20–6.94 (m, 5H),6.92–6.75 (m, 2H), 6.38 (dt, J = 9.7, 1.9 Hz, 2H), 6.07 (d, J = 15.6 Hz, 1H),5.96 (d, J = 6.2 Hz, 1H), 5.24 (d, J = 11.7 Hz, 1H), 4.99–4.87 (m, 2H), 4.85–4.72 (m, 1H), 4.18 (s, 1H), 3.78 (s, 3H), 3.57 (t, J = 6.2 Hz, 3H), 2.27 (d,J = 7.0 Hz, 2H), 1.54–1.38 (m, 4H), 1.35-1.27 (m, 6H), 0.86 (s, 9H), 0.02 (s,6H); ESI-m / z: 763.94 [M+H] + 。

[0056] Step 3, Synthesis of Compound AH-SW01: Intermediate 2 was dissolved in a mixed solvent of 30 mL water, tetrahydrofuran, and acetic acid (water:tetrahydrofuran:acetic acid = 1:1:2 V / V / V), and stirred at room temperature for 4 hours. After the reaction was completed, a small amount of water was added to quench the reaction, followed by washing with saturated sodium chloride solution, extraction three times with ethyl acetate, collection of the organic phase, drying with anhydrous sodium sulfate, standing, filtration under reduced pressure, and normal-phase silica gel column chromatography (eluent: ethyl acetate:petroleum ether = 1:2 V / V) to obtain 2.33 g of crude product E rich in compound AH-SW01. Further purification was performed using preparative liquid chromatography, including: dissolving the sample in methanol (25 mg / mL), injecting 1.0 mL into a Waters RCM Nova PakHR C18 Cartridge (300 mm × 25 mm, 6 μm) column, using methanol-water (30:70 V / V) as the mobile phase, a flow rate of 20 mL / min, and a detection wavelength of 272 nm. nm yielded compound AH-SW01 (English name: (2R,3R)-3,5-dihydroxy-2-((2R,3R)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-oxochroman-7-yl(E)-10-hydroxydec-2-enoate, Chinese name: (2R,3R)-3,5-dihydroxy-2-((2R,3R)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[b][1,4]dioxacyclohexene-6-yl)-4-carbonylchroman-7-yl(E)-10-hydroxydec-2-enoate, solid, yield 83%).

[0057] mp 237.2-238.5 ℃. 11H NMR (300 MHz, DMSO-d6) δ 11.69 (s, 1H), 9.16(s, 1H), 7.18–6.93 (m, 5H), 6.84 (q, J = 8.2 Hz, 2H), 6.38 (d, J = 9.9 Hz,2H), 6.02 (d, J = 15.6 Hz, 1H), 5.96 (d, J = 6.2 Hz, 1H), 5.24 (d, J = 11.7Hz, 1H), 5.00–4.87 (m, 2H), 4.83-4.73 (m, 1H), 4.34 (s, 1H), 4.18 (s, 1H),3.78 (s, 3H), 3.61–3.49 (m, 1H), 3.2-3.11 (m, 1H),2.27 (q, J = 7.0, 6.6 Hz,2H), 1.42 (s, 4H), 1.28 (s, 6H). 13 13C NMR (75 MHz, DMSO-d6) δ 199.98, 163.75,162.41, 162.29, 158.45, 153.74, 148.12, 147.50, 144.27, 143.77, 130.15,127.96, 121.90, 120.99, 120.14, 117.16, 116.87, 115.80, 112.15, 105.25,103.37, 102.14, 83.25, 78.61, 76.33, 72.24, 61.17, 60.65, 56.17, 32.95,32.18, 29.12, 27.71, 27.68, 25.85. HRMS (ESI): calcd. For C 35 H 38 O 12 [M+H] + m / z651.2436, found 651.2467. HPLC (80 % methanol in water with 1‰ HCOOH ): t R =2.806 min, 96.330%.

[0058] Example 2

[0059]

[0060]

[0061]

[0062] Following the synthesis method of compound AH-SW01 in Example 1, crude product E (containing compounds AH-SW02, AH-SW03, AH-SW04, etc.) rich in compound AH-SW01 was obtained. The product was collected and fractionated by preparative chromatography, followed by recrystallization purification to obtain compounds AH-SW02, AH-SW03, and AH-SW04. The method is as follows:

[0063] The compound-rich AH-SW01 solid product was dissolved in a methanol-chloroform solvent (70:30 V / V, approximately 25 mg / mL). First, normal-phase silica gel column chromatography (200-300 mesh) was performed with gradient elution using chloroform:methanol (90:10, 80:20, 70:30 V / V) as eluents. The chloroform:methanol 80:20 V / V eluent fraction was then purified using a Waters C18 low-pressure column (Waters RCM Nova PakHR C18 Cartridge, packing particle size 40–63 μm, 200 g) with 30% methanol. Following polyamide thin-layer chromatography (UV 365 nm), further purification was achieved using preparative liquid chromatography, including: dissolving the sample in methanol (25 mg / mL) and injecting 1.0 mL into a Waters RCM Nova PakHR C18 Cartridge (300 mm × 25 mm, 6...). A μm column was used, with methanol-water (30:70 V / V) as the mobile phase, a flow rate of 20 mL / min, and a detection wavelength of 272 nm. Elution fractions containing compounds AH-SW02 (11.5–11.9 min), AH-SW03 (13.4–13.9 min), and AH-SW04 (13.0–13.4 min) were collected by repeated injections, dried under reduced pressure, and the resulting compounds AH-SW02 (off-white powder), AH-SW03 (off-white powder), and AH-SW04 (off-white powder) were scanned by time-of-flight liquid chromatography-mass spectrometry (LC / TOF-MS) in negative ion mode. The obtained mass spectra are shown below. Figure 2 A, 2B, 2C.

[0064] The quasi-molecular ion [MH] of compound AH-SW02 - The value is m / z 649.1859, and the characteristic fragment ions are m / z 631.1810, m / z 481.0928, m / z 463.1191, m / z 463.0979, m / z 445.0943, m / z 317.0591, m / z 309.0431, m / z 283.0349, and m / z 179.0020.

[0065] The quasi-molecular ion [MH] of compound AH-SW03 - The value is m / z 649.2295, and the characteristic fragment ions are m / z 481.1137, m / z 463.1044, m / z 453.1204, m / z 301.0362, and m / z 125.0250.

[0066] The quasi-molecular ion [MH] of compound AH-SW04 - The value is m / z 649.2279, and the characteristic fragment ions are m / z 481.1168, m / z 463.0976, m / z 301.0384, m / z 357.0462, m / z 178.9998, and m / z 125.0278.

[0067] Example 3

[0068] Preparation of compound AH-SW06

[0069]

[0070] Under nitrogen protection, silymarin (482.4 mg, 1.0 mmol) was dissolved in 10 mL of a dry mixture of acetonitrile and dichloromethane (acetonitrile:dichloromethane = 1:1 V / V). Under ice bath conditions, royal jelly acyl chloride (204.7 mg, 1.0 mmol) and boron trifluoride diethyl ether (340.6 mg, 1.2 mmol, 50% V / V) were slowly added dropwise, and the mixture was stirred for about 2 h. Then, boron trifluoride diethyl ether (48% by mass, solvent:dichloromethane:acetonitrile = 1:1 V / V, 354.8 mg, 1.2 mmol) was added, and the mixture was stirred at room temperature for about 12 h. After the reaction was completed, 2 mL of saturated sodium bicarbonate aqueous solution was added and stirred for about 10 min to quench the reaction. The mixture was extracted with dichloromethane and water, and the organic phase was collected. The organic phase was dried with anhydrous sodium sulfate and filtered under reduced pressure to obtain the filter cake of crude silymarin derivative (rich in compounds AH-SW06 and AH-SW07). The mixture was purified by normal phase silica gel column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:2 V / V) to obtain compound AH-SW06 (white solid, 522.8 mg, yield 80.3%).

[0071] 1H NMR (300 MHz, Chloroform-d) δ 11.23 (s, 1H), 7.18-6.86 (m, 7H), 6.03 (d, J = 2.1 Hz, 1H), 5.95 (d, J = 2.3 Hz, 1H), 5.86 (d, J = 16.0 Hz, 2H), 4.98 (d, J = 11.9 Hz, 1H), 4.88 (dd, J = 8.1, 3.4 Hz, 1H), 4.44 (ddd, J= 51.8, 12.1, 2.6 Hz, 2H), 4.29-4.21 (m, 1H), 4.06 (ddd, J = 12.2, 4.5, 2.2Hz, 1H), 3.89 (s, 3H), 3.66 (s, 1H), 3.55 (t, J = 6.7 Hz, 2H), 2.24 (q, J =7.2 Hz, 2H), 1.80 (q, J = 7.0 Hz, 2H), 1.50-1.33 (m, 8H); 13 C NMR (75 MHz, Chloroform-d) δ 195.61, 166.64, 166.02, 163.71, 163.04, 151.33, 147.01,146.51, 143.96, 143.75, 129.33, 127.42, 120.79, 120.25, 117.50, 116.54,114.81, 109.38, 100.65, 97.14, 96.03, 82.92, 75.91, 72.30, 62.90, 56.00,45.14, 32.54, 32.29, 29.00, 28.63, 27.77, 26.73.

[0072] Example 4

[0073]

[0074] Following the synthetic method of compound AH-SW06 in Example 3, filter cake of crude silymarin derivative was obtained. The cake was collected by preparative chromatographic fractionation and then purified by recrystallization to obtain compound AH-SW07. The method is as follows:

[0075] The filter cake of crude silymarin derivative was dissolved in methanol-chloroform solvent (70:30 V / V, approximately 25 mg / mL). Normal-phase silica gel column chromatography (200-300 mesh) was performed with gradient elution using chloroform:methanol (90:10, 80:20, 70:30 V / V) as eluent. The chloroform:methanol 80:20 V / V eluent fraction was then introduced into a Waters C18 low-pressure column (Waters RCMNova PakHR C18 Cartridge, packing particle size 40–63 μm, 200 g), eluted with 30% methanol. The resulting fraction was then subjected to polyamide thin-layer chromatography and confirmed under UV at 365 nm. Further purification was performed using preparative chromatography: the sample was dissolved in methanol (25 mg / mL), and 1.0 mL was injected into a Waters RCM Nova PakHR C18 Cartridge (300 mm × 25 mm, 6 μm) column. The mobile phase was methanol-water (30:70 V / V), the flow rate was 20 mL / min, and the detection wavelength was 272 nm. The fraction collected between 16.6 and 17.3 min was repeatedly injected and dried under reduced pressure to obtain a white powder of compound AH-SW07. The mass spectrum was obtained by time-of-flight liquid chromatography-mass spectrometry (LC / TOF-MS) in negative ion mode. (See attached image). Figure 2 D.

[0076] The quasi-molecular ion [MH] of compound AH-SW07 - The m / z value is 649.2290, and the characteristic fragment ions are m / z 621.2346, m / z 481.1138, m / z 463.1045, m / z 453.1198, m / z 319.1198, m / z 301.0362, m / z 293.1403, m / z 273.0415, m / z 257.0458, m / z 173.9993, and m / z 125.0248.

[0077] Example 5

[0078] Efficacy study of AH-SW01 and AH-SW06 in lipid-lowering and liver-protective effects in a high-fat diet-induced non-alcoholic fatty liver disease (NAFLD) model animal.

[0079] The efficacy of lipid-lowering and liver-protective drugs was studied by inducing a non-alcoholic fatty liver disease (NAFLD) model using a high-fat methionine and choline-deficient diet (MCD).

[0080] Animal model establishment: C57BL / 6J mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., male, 6-7 weeks old, weighing 20-22 g) were purchased and acclimatized under standard conditions (temperature 25 ± 2 ℃, relative humidity 50 ± 10%, simulating a 12-hour day-night cycle). During the acclimatization period, mice had free access to food and water. After one week of acclimatization, the normal control group was fed a methionine-choline-sufficient diet (MCS), while the model group and the treatment group were fed an MCD diet. Samples were taken approximately one week later to confirm successful model establishment. Mice had free access to water throughout the experiment.

[0081] Grouping and administration method:

[0082] In the efficacy study of AH-SW01, NAFLD model mice were randomly divided into 5 groups: model group, low-dose AH-SW01 group (AH-SW01-L, 6 mg / kg), high-dose AH-SW01 group (AH-SW01-H, 12 mg / kg), triple-dose silymarin group (FJB-3H, 26.7 mg / kg, three times the high-dose AH-SW01 group and six times the low-dose AH-SW01-L group), and triple-dose 10-hydroxy-2-decenoic acid group (WJS-3H, 10.4 mg / kg, three times the high-dose AH-SW01 group and six times the low-dose AH-SW01-L group). Samples from each group were dispersed in 0.5% CMC-Na solution and administered by gavage. The normal control group and model group were given an equal volume of 0.5% CMC-Na solution.

[0083] In the efficacy study of AH-SW06, NAFLD model mice were randomly divided into 8 groups: model group, low-dose AH-SW06 treatment group (AH-SW06-L, 6 mg / kg), high-dose AH-SW06 treatment group (AH-SW06-H, 12 mg / kg), high-dose silymarin group (FJB-H, 8.9 mg / kg, corresponding to the high-dose AH-SW06 group), triple-dose silymarin group (FJB-3H, 26.7 mg / kg, three times the high-dose AH-SW06 group and six times the low-dose AH-SW06-L group), high-dose 10-hydroxy-2-decenoic acid group (WJS-3H, 3.5 mg / kg, corresponding to the high-dose AH-SW06 group), and triple-dose royal jelly acid group (WJS-3H, 10.4 mg / kg). The drug was administered at three times the dose of the AH-SW06-H high-dose group and six times the dose of the AH-SW06-L low-dose group (mg / kg). A combination of three times the high dose of silymarin and three times the high dose of royal jelly acid was used (silymarin dose 26.7 mg / kg, 10-hydroxy-2-decenoic acid dose 10.4 mg / kg), i.e., the F+W 3H group. All drugs were dispersed in 0.5% CMC-Na solution and administered by gavage. The normal control group and the model group received an equal volume of 0.5% CMC-Na solution.

[0084] Animal appearance / behavior and histopathological examination:

[0085] The results showed that the normal control group mice were more active and had shiny fur; the model group mice experienced continuous weight loss, wet and disheveled fur, and lethargy; the AH-SW01-H and AH-SW06-H treatment groups improved activity levels and fur color, with the AH-SW01-H and AH-SW06-H treatment groups showing significant improvement. Examination of the liver revealed that the normal control group mice had clearly defined lobed structures, a reddish and shiny color, a smooth surface, and a soft and elastic texture; the NAFLD model mice had yellowish-brown livers with a rough, granular surface and increased volume. Figure 6 ).

[0086] The results of H&E staining of liver pathological sections are shown in the figure. Figure 6 The results showed that in the normal control group, mouse liver cells were morphologically intact and structurally compact, with blue-stained nuclei located in the center of the cell. In the model group, mouse liver cells were disordered, with loose cytoplasm and obvious ballooning degeneration. Administration of AH-SW01-H and AH-SW06-H significantly improved the ballooning degeneration induced by the model. Silymarin also showed some effect, but the improvement was not as significant as with AH-SW01 and AH-SW06. This indicates that compounds AH-SW01 and AH-SW06 were more effective than high-dose silymarin or royal jelly acid alone.

[0087] Oil Red O is a lipid-soluble dye that stains neutral fats such as triglycerides in tissues, making lipid droplets appear red and cell nuclei appear blue. The results of Oil Red O staining are shown in the figure. Figure 6 The results showed that lipids accumulated in the hepatocytes of model mice, with larger and more numerous red lipid droplets. The AH-SW01 and AH-SW06 treatment groups showed a significant decrease in both lipid droplet area and number. This indicates that the compounds AH-SW01 and AH-SW06 were more effective than high-dose silymarin or royal jelly acid alone.

[0088] In summary, compounds AH-SW01 and AH-SW06 significantly improved the appearance, behavior, liver histopathology, hepatocyte lipid accumulation, and steatosis in mice with non-alcoholic fatty liver disease (NAFLD). This demonstrates that compounds AH-SW01 and AH-SW06 were more effective than high-dose silymarin or royal jelly acid alone.

[0089] Liver function and total triglycerides (TG) and total cholesterol (TC) tests: Compared with the normal control group, serum ALT and AST levels were significantly increased in NAFLD model mice, and liver and serum triglyceride (TG) and total cholesterol (TC) levels were also increased; compared with the model group, administration of low-dose or high-dose AH-SW01 and AH-SW06 significantly reduced ALT, AST, liver TG, and TC levels. Figure 7 , Figure 8 ), administering three times the dose of silymarin also significantly reduced ALT and AST levels ( Figure 7 A, 7E, Figure 8 (A, 8E), among which AH-SW01 and AH-SW06 showed significant advantages, outperforming other groups, including the silymarin triple-dose group (FJB-3H), the royal jelly acid triple-dose group (WJS-3H), and the silymarin triple-dose combined with royal jelly acid triple-dose group (F+W 3H). This suggests that AH-SW01 and AH-SW06 have good lipid-lowering and hepatoprotective effects. This demonstrates that the effects of compounds AH-SW01 and AH-SW06 are more significant than those of high-dose silymarin or royal jelly acid alone.

[0090] Example 6

[0091] Efficacy study of AH-SW01 and AH-SW06 in the antifibrotic effect of a methionine and choline-deficient diet-induced non-alcoholic steatohepatitis (NASH) model.

[0092] The efficacy of liver protection and anti-fibrosis was studied using a non-alcoholic steatohepatitis (NASH) model induced by a combination of high-fat methionine and choline-deficient fertilizer (MCD) and carbon tetrachloride.

[0093] Animal model establishment: C57BL / 6J mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., male, 6-7 weeks old, weighing 20-22 g) were purchased and acclimatized under standard feeding conditions (temperature 25 ± 2 ℃, relative humidity 50 ± 10%, simulating a 12-hour day-night cycle). During the acclimatization period, mice had free access to food and water. After one week of acclimatization, the normal control group was fed a methionine-choline-sufficient diet (MCS), while the model group and four treatment groups were fed a high-fat methionine and choline-deficient diet (MCD) for four weeks. On days 4 and 7 of the fourth week, mice were intraperitoneally injected with a CCl4 solution (CCl4:mineral oil = 1:12 V / V, injected at 10 mL / kg). Samples were taken 24 hours after the last intraperitoneal injection for analysis.

[0094] Grouping and administration: NASH model mice were randomly divided into 5 groups: model group, AH-SW01 high-dose administration group (AH-SW01-H, 12 mg / kg), AH-SW06 high-dose administration group (AH-SW06-H, 12 mg / kg), silymarin triple high-dose group (FJB-3H, 26.7 mg / kg), and royal jelly acid triple high-dose group (WJS-3H, 10.4 mg / kg). The drugs in each group were dispersed in 0.5% CMC-Na solution and administered by gavage. The normal control group and model group were given an equal volume of 0.5% CMC-Na solution.

[0095] The results of H&E staining of liver pathological sections are shown in the figure. Figure 9 The results showed that in the normal control group, mouse liver cells were morphologically intact and structurally compact, with blue-stained nuclei located in the center of the cell. In the model group, mouse liver cells were disordered, with loose cytoplasm and obvious ballooning degeneration. Administration of high doses of AH-SW01 and AH-SW06 significantly improved these conditions, alleviating the ballooning degeneration induced by the model. This indicates that compounds AH-SW01 and AH-SW06 were more effective than high doses of silymarin or royal jelly acid alone.

[0096] Oil Red O is a lipid-soluble dye that stains neutral fats such as triglycerides in tissues, making lipid droplets appear red and cell nuclei appear blue. The results of Oil Red O staining are shown in the figure. Figure 10 The results showed that lipids accumulated in the hepatocytes of model mice, and the number of red lipid droplets increased. The lipid droplet area and number were significantly reduced in the AH-SW01 and AH-SW06 treatment groups. This indicates that the effects of compounds AH-SW01 and AH-SW06 were more significant than those achieved by using high-dose silymarin or royal jelly acid alone.

[0097] In conclusion, AH-SW01 and AH-SW06 significantly improved the appearance, behavior, histopathology, hepatocyte lipid accumulation, and steatosis in mice with non-alcoholic steatohepatitis (NASH). This demonstrates that compounds AH-SW01 and AH-SW06 were more effective than high-dose silymarin or royal jelly acid alone.

[0098] Tests for indicators related to inflammation and fibrosis:

[0099] Compared with the normal control group, NASH model mice showed significantly increased levels of inflammatory factors such as TNF-α, IL-6, Cxcl1, and Ccl2. Figure 11 A, 11B, 11C, 11D), and fibrosis-related factors Col1α1, Tgf-β, Timp1, and Acta2 were all significantly increased. Figure 11 (E, 11F, 11G, 11H). High doses of AH-SW01 and AH-SW06 significantly reduced the aforementioned inflammatory factors and fibrosis-related indicators, with AH-SW06 showing a more significant effect; while silymarin (FJB-3H) and royal jelly acid (WJS-3H) showed weaker regulatory effects at three times the dose. This suggests that AH-SW01 and AH-SW06 have good anti-inflammatory and anti-fibrotic effects, and the effects of compounds AH-SW01 and AH-SW06 are more significant than those of high-dose silymarin or royal jelly acid alone.

[0100] Conclusion: The silymarin derivatives of this invention achieved significantly higher efficacy at much lower doses than silymarin or royal jelly acid alone, exceeding the efficacy of silymarin or royal jelly acid at three times the dose, achieving unexpected results. This demonstrates that compounds AH-SW01 and AH-SW06 show more significant improvement than high doses of silymarin or royal jelly acid alone.

[0101] Example 7

[0102] Pharmacodynamic study of silymarin derivatives against palmitic acid-induced lipid accumulation in AML12 and HepG2 cells

[0103] A palmitic acid-induced hepatic cell lipid accumulation model was used, and silymarin derivatives AH-SW02, AH-SW03, AH-SW04, and AH-SW07 were administered to investigate their efficacy against intracellular lipid accumulation.

[0104] Preparation of drug delivery solutions: Accurately weigh approximately 5 mg of each of AH-SW02, AH-SW03, AH-SW04, and AH-SW07, and dissolve them in 45 μL of DMSO to prepare drug stock solutions with a concentration of approximately 200 mM. Dilute the drug stock solutions with DMEM-F12 medium to a drug stock solution with a concentration of 100 μM, and further dilute to a final concentration of 10 μM in the medium for cell drug delivery.

[0105] Cell culture and lipid accumulation cell model: AML12 and HepG2 cells were used. After spotting, cell density reached 70%-80% before modeling / drug administration. Blank culture medium was added to the control wells. The model group (Model group) was treated with palmitic acid solution (PA, used to induce lipid accumulation in AML12 and HepG2 cells) diluted with DMEM medium. The PA stimulation concentration was 150 μM for AML12 cells and 200 μM for HepG2 cells. The drug treatment groups were treated with 10 μM and 50 μM silybin derivatives and 10 μM and 50 μM silybin simultaneously with palmitic acid solution. Cells were collected 24 h after drug administration (density approximately 1 × 10⁻⁶ cells / well). 6 Centrifuge at 1000 g for 3-5 minutes at 4℃, discard the supernatant; wash cells twice with PBS for 5 minutes each time; resuspend cells in 1 mL of BODIPY working solution (2 µg / mL), incubate at room temperature in the dark for 15-30 minutes; after incubation, centrifuge at 400 g for 3-4 minutes at 4℃, discard the dye-containing supernatant, wash cells twice with PBS. Resuspend cells in 1 mL of DAPI working solution (0.5 µg / mL), incubate at room temperature in the dark for 10-15 minutes; after incubation, centrifuge at 400 g for 3-4 minutes at 4℃, discard the dye-containing supernatant, wash cells twice with PBS; wash cells twice with PBS, resuspend cells in an appropriate amount of serum-free culture medium or PBS, mount with anti-fluorescence quenching mounting medium, and observe the results under a fluorescence microscope (BODIPY green channel, DAPI UV / blue channel).

[0106] The results of the fluorescent staining are shown in Figure 12 , Figure 13 The results showed that, compared with the control group, AML12 cells ( Figure 12 ) and HepG2 cells ( Figure 13The model group treated with PA showed significantly enhanced green fluorescence signal (BODIPY), indicating a significant increase in the number of intracellular lipid droplets, suggesting that PA successfully induced cellular lipid accumulation. Compared with the model group, after administration of AH-SW03, AH-SW04, AH-SW07, and AH-SW02, the intracellular green fluorescence intensity (BODIPY) of both groups was reduced, and the number of lipid droplets decreased, significantly lower than that of the model group. Among them, AH-SW03 and AH-SW04 showed more significant reductions, suggesting that they could alleviate PA-induced lipid accumulation. DAPI staining results showed that the nuclei of cells in each group were basically intact, with no obvious morphological abnormalities such as nuclear pyknosis or fragmentation, indicating that within the experimental concentration range, the treatments in each group did not cause significant damage to the cell nucleus morphology. In conclusion, AH-SW03, AH-SW04, AH-SW07, and AH-SW02 can all significantly inhibit PA-induced intracellular lipid accumulation, showing good efficacy in treating non-alcoholic fatty liver disease.

[0107] Example 8

[0108] Tablet preparation

[0109] prescription:

[0110] AH-SW01 or AH-SW06 in 2.5, 5.0, 10, 20, 30, 60, 90, 150 or 300 g quantities.

[0111] 60 g of microcrystalline cellulose

[0112] 30 g of lactose

[0113] 60 mL of 0.5% sodium carboxymethyl cellulose

[0114] Magnesium stearate 0.5 g.

[0115] Preparation process: Weigh out compounds AH-SW01 or AH-SW06, microcrystalline cellulose, and lactose according to the prescribed amounts, pass them through an 80-mesh sieve, and mix them evenly. Separately, take 0.5g of sodium carboxymethyl cellulose, disperse it in 100 mL of water, and sonicate to dissolve and disperse evenly. Prepare a soft mass using conventional methods, granulate it through a 20-mesh sieve, dry it in flowing air at 60℃ for 2 hours, add magnesium stearate (passed through a 120-mesh sieve), mix well, and compress into tablets. Prepare 1000 tablets, each containing approximately 2.5, 5.0, 10, 20, 30, 60, 90, 150, or 300 mg of AH-SW01 or AH-SW06.

[0116] Example 9

[0117] Capsule preparation

[0118] prescription:

[0119] AH-SW01 or AH-SW06, 2.5, 5.0, 10, 20, 30, 60 or 90 g.

[0120] 60 g of microcrystalline cellulose

[0121] 60 g of lactose

[0122] 60 mL of 0.5% sodium carboxymethyl cellulose

[0123] 1000 capsules.

[0124] Preparation process: Weigh out compounds AH-SW01 or AH-SW06, microcrystalline cellulose, and lactose according to the above prescription, pass them through an 80-mesh sieve, and mix them evenly; separately take 0.5g of sodium carboxymethyl cellulose, disperse it in 100mL of aqueous solution, and sonicate to dissolve and disperse evenly. Prepare a soft mass using conventional methods, granulate it through a 20-mesh sieve, dry it in flowing air at 60℃ for 2 hours, then granulate it and fill it into No. 3, No. 2, No. 1, or No. 0 ordinary capsules. Each capsule contains approximately 2.5, 5.0, 10, 20, 30, 60, or 90 mg of AH-SW01 or AH-SW06, respectively.

[0125] Example 10

[0126] Granule preparation

[0127] prescription:

[0128] AH-SW01 or AH-SW06 in 5.0, 10, 20, 30, 60, 90, or 300 g quantities.

[0129] 90 g of microcrystalline cellulose

[0130] 120 g of corn starch

[0131] 90 g of sucrose

[0132] 70 mL of 0.5% sodium carboxymethyl cellulose.

[0133] Preparation process: Weigh AH-SW01 or AH-SW06, corn starch, microcrystalline cellulose, and sucrose according to the above prescription, pass them through an 80-mesh sieve, and mix them evenly; separately, take 0.5g of sodium carboxymethyl cellulose, disperse it in 100mL of aqueous solution, and sonicate to dissolve and disperse evenly. Prepare the soft mass using conventional methods, granulate it through a 20-mesh sieve, dry it in flowing air at 60℃ for 2 hours, and then package it after granulation. Each bag contains approximately 5.0, 10, 20, 30, 60, 90, or 300 mg of AH-SW01 or AH-SW06.

[0134] Example 11

[0135] Preparation of liposomes

[0136] prescription:

[0137] AH-SW01 or AH-SW06 3, 6 or 9g,

[0138] 12 g of soybean lecithin

[0139] Cholesterol 7.0g

[0140] Vitamin E 0.6 g,

[0141] 60 mL of ethanol

[0142] 240 mL of PBS buffer solution (pH 7.4).

[0143] Weigh out AH-SW01 or AH-SW06, soybean lecithin, cholesterol, and vitamin E according to the above prescription, add them to anhydrous ethanol, and sonicate for 3 minutes to dissolve and form a homogeneous solution; evaporate at 40°C to remove the organic solvent, forming a phospholipid film on the inner wall of a round-bottom flask. Add PBS buffer solution (pH 7.4), and hydrate at 35°C for 30 minutes to hydrate the phospholipid film and form a coarse suspension; sonicate in an ice bath for 10 minutes (200W, 40Hz) to refine the particle size and obtain a liposome solution. Dispense into 1mL vials, each containing approximately 10, 20, or 30 mg of AH-SW01 or AH-SW06.

[0144] Example 12

[0145] Preparation of droplets

[0146] prescription:

[0147] AH-SW01 or AH-SW06, 15, 30, or 45 g.

[0148] Polyethylene glycol 4000 (PEG4000) 150, 300 or 450 g,

[0149] Span 80 or Span 85 2, 4 or 6 g,

[0150] Polyethylene glycol 400 (PEG400) 10, 20 or 30 mL.

[0151] Weigh the drugs and excipients according to the above prescription. Pass AH-SW01 or AH-SW06 through a 60-mesh sieve, and excipient PEG4000 through an 80-mesh sieve. Heat PEG4000 to melt (60-70℃), add Span 80 or Span 85 and polyethylene glycol 400, stir evenly, then add fine powder of AH-SW01 or AH-SW06, stir for 15 minutes, let the mixture stand for 0.5-1 hour until the foam dissipates, pour the mixture into the storage tank of the pelleting machine (keep it at 70-80℃), adjust the dripping rate (50-60 drops / minute), and drip it into dimethyl silicone oil at 10-15℃, allowing it to condense into pellets. Use filter paper to absorb the surface condensate, dry, and screen the qualified pellets (roundness ≥0.95, hardness ≥20KN). Adjust the size and weight of the droplets, each 5 drops containing approximately 10, 20 or 30 mg of AH-SW01 or AH-SW06.

[0152] Example 13

[0153] Suspension preparation

[0154] prescription:

[0155] AH-SW01 or AH-SW06, 5, 10, 15, 20, 25 or 30 g.

[0156] Cyclodextrin 10, 20, 30, 40, 50 or 60 g,

[0157] Sodium carboxymethyl cellulose 0.6 g,

[0158] 1000 mL of purified water.

[0159] Preparation process: Weigh the drugs and excipients according to the above formula. Pass the cyclodextrin through an 80-mesh sieve and dissolve it in 1000 mL of hot water at 50-60℃ to obtain a cyclodextrin aqueous solution. Add AH-SW01 / or AH-SW06 to the above cyclodextrin aqueous solution and ultrasonically mix and disperse to obtain solution A. Separately, take 0.6 g of sodium carboxymethyl cellulose and disperse it in the above-mentioned solution A, which has been allowed to stand and cool, and ultrasonically dissolve and disperse it evenly. Dispense into 10 mL vials, each containing approximately 50, 100, 150, 200, 250, or 300 mg of AH-SW01 or AH-SW06.

Claims

1. Silybin derivatives or their stereoisomers as shown in Formula I, and pharmaceutically acceptable salts thereof: ； in, R1, R2, R3, R4, and R6 are each independently selected from hydrogen, R5 is selected from methyl; However, it does not include: R1 selected from hydrogen, R2 selected from hydrogen, R3 selected from hydrogen, R4 selected from hydrogen, R6 selected from hydrogen, and R5 selected from methyl.

2. Silybin derivatives or their stereoisomers, or pharmaceutically acceptable salts, with the structures shown below: ， ， ， ， ， 。 3. Use of the silymarin derivative or its stereoisomer, or a pharmaceutically acceptable salt, as described in any one of claims 1-2 in the preparation of a medicament for the prevention and / or treatment of non-alcoholic fatty liver disease.

4. The use according to claim 3, characterized in that: The use of the silymarin derivative or its stereoisomer, or a pharmaceutically acceptable salt, in the preparation of medicaments for the prevention and / or treatment of hyperlipidemia, inflammation, and liver damage caused by non-alcoholic fatty liver disease.

5. Use of the silymarin derivative or its stereoisomer, or a pharmaceutically acceptable salt, as described in any one of claims 1-2 in the preparation of a medicament for the prevention and / or treatment of non-alcoholic steatohepatitis.

6. The use according to claim 5, characterized in that: The use described is the use of silymarin derivatives or their stereoisomers, or pharmaceutically acceptable salts, in the preparation of medicaments for the prevention and / or treatment of liver fibrosis and liver injury caused by non-alcoholic steatohepatitis.

7. Use of the silymarin derivative or its stereoisomer, or a pharmaceutically acceptable salt, as described in any one of claims 1-2 in the preparation of a medicament for the prevention and / or treatment of liver fibrosis.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises, as an active ingredient or main active ingredient, a silymarin derivative or its stereoisomer as described in any one of claims 1-2, or a pharmaceutically acceptable salt, and is formulated into a pharmaceutical dosage form with pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that: The dosage form is tablet, capsule, granule, suspension, liposome, or droplet.

10. The use of the silymarin derivative or its stereoisomer, or a pharmaceutically acceptable salt, as described in any one of claims 1-2 in the preparation of a health food product that has an adjunctive protective effect against chemically induced liver injury.

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