Application of acyl-resorcinol derivatives in the preparation of drugs or health products for the prevention and treatment of non-alcoholic steatohepatitis

Abstract

This invention discloses the application of acyl-resorcinol derivatives in the preparation of drugs or health products for the prevention and treatment of non-alcoholic steatohepatitis (NASH). Studies have shown that in an in vitro model of free fatty acid-induced normal human hepatocytes, these compounds can exert a hepatoprotective effect by reducing lipid accumulation in cells. The representative compound, hyperacmotone A, significantly reduces lipid accumulation in cells in a dose-dependent manner and regulates the expression of genes such as Pparα, Cpt1, and Fapp1. In a methionine-choline deficiency-induced mouse model, administration of hyperacmotone A can inhibit hepatic steatosis, reduce serum alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) levels, while simultaneously reducing hepatic triglyceride levels and increasing hepatic glutathione activity. This suggests a certain preventive and therapeutic effect on the occurrence and development of NASH.

Description

Technical Field

[0001] This invention relates to compounds and their applications, specifically to the extraction, separation, biomimetic semi-synthesis, and hepatoprotective activity studies of acyl-resorcinol derivatives from the medicinal plant *Hymenoplastia lingua*, as well as the application of the representative compound HA in the preparation of drugs or health products for the prevention and treatment of non-alcoholic steatohepatitis. Background Technology

[0002] Non-alcoholic steatohepatitis (NASH) is one of the fastest-progressing forms of non-alcoholic fatty liver disease (NAFLD). Unlike simple fatty liver, NASH involves fat accumulation along with accompanying hepatocellular inflammation, necrosis, and fibrosis. Pathologically, it manifests as hepatic steatosis, ballooning degeneration of hepatocellular cells, and lobular inflammation. NASH development begins with the abnormal accumulation of fat in the liver, which can be caused by various factors, including obesity, type II diabetes, hypertension, hyperlipidemia, metabolic syndrome, and genetic factors. When fat deposition exceeds a certain level, it can lead to inflammatory responses and damage, further resulting in hepatocellular necrosis and fibrosis, thus affecting normal liver function. With improved living conditions, the incidence of NASH has been increasing year by year, becoming the most common liver disease in my country. Current treatments for NASH mainly include lifestyle modifications and drug therapy. Lifestyle modifications include weight loss, a healthy diet, increased physical activity, smoking cessation, and limiting alcohol intake.

[0003] Acyl-phloroglucinol-mixed-terpene derivatives are mainly derived from plants in the genera *Garcinia* and *Hypericum* of the family Guttiferae. They encompass a wide variety of skeletons with unique structures and possess diverse biological activities, including antibacterial, antioxidant, antiviral, antitumor, and antidepressant effects.

[0004] Hypericum beanii N. Robson, a plant belonging to the genus Hypericum in the family Clusiaceae, is primarily used medicinally for its roots, which are known in traditional Chinese medicine as "Huanghuaxiang". It is cold in nature and bitter in taste, possessing properties such as clearing heat and dampness, detoxifying, and dispersing blood stasis. The "Chinese Materia Medica" records that it "treats damp-heat jaundice, gonorrhea, diarrhea, dysentery, impetigo, burns, snake bites, traumatic injuries, and muscle and bone pain."

[0005] To date, there are no approved drug treatments for NASH. Traditional treatment strategies, such as weight loss through diet and exercise, have some effect on NASH, but they are unlikely to reverse liver fibrosis that occurs in the later stages of the disease. Therefore, the development of NASH drugs has become an urgent problem to be solved. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide an acyl-resorcinol derivative with potential therapeutic effects for NASH, which can be used to prepare drugs or health products for the prevention and treatment of non-alcoholic steatohepatitis.

[0007] Technical solution: The acyl-resorcinol derivative or its pharmaceutically acceptable salt described in this invention, wherein the structure of the acyl-resorcinol derivative is shown in the following formula:

[0008]

[0009] R1 is selected from phenyl, C 1-4 alkyl;

[0010] R2 and R3 are each independently selected from hydrogen and C. 1-4 Alkyl, C 3-5 alkenyl, geraniyl, lavenderyl, ω-lavenderyl;

[0011] R4, R5, and R6 are each independently selected from common hydroxyl modifying groups such as hydrogen, methyl, silane ethers, benzoyl, and acetyl.

[0012] The acyl-resorcinol derivative or its pharmaceutically acceptable salt, wherein the silane ethers include TMS, TBDMS, TES, and TIPS.

[0013] The acyl-resorcinol derivative or its pharmaceutically acceptable salt thereof, wherein R2 and R3 are each independently selected from hydrogen, methyl, ethyl, allyl, isopentenyl, geraniyl, lavenderyl, ω-lavenderyl.

[0014] The acyl-resorcinol derivative or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0015]

[0016] The pharmaceutical composition comprises one or more of the acylphloroglucinol derivatives or pharmaceutically acceptable salts thereof, as well as their enantiomers, diastereomers, tautomers, solvates or salts of solvates, and pharmaceutically acceptable excipients.

[0017] The pharmaceutical composition may be in the form of an oral dosage form or a non-oral dosage form; the oral dosage form may be a tablet, capsule, powder or granule, and the non-oral dosage form may be a suppository or an injection.

[0018] The use of the acyl-resorcinol derivative or its pharmaceutically acceptable salt, or the pharmaceutical composition thereof, in the preparation of hepatoprotective drugs or health products.

[0019] The use of the acyl-resorcinol derivative or its pharmaceutically acceptable salt, or the pharmaceutical composition thereof, in the preparation of drugs or health products for the prevention and treatment of non-alcoholic steatohepatitis.

[0020] The method for preparing the acyl-resorcinol derivative or a pharmaceutically acceptable salt thereof includes the following steps:

[0021] (1) Take the root of the seedling flower, slice or crush it, add ethanol to extract it, concentrate and recover the solvent to obtain crude extract, add warm water at 40-70℃ to suspend the crude extract, extract it with petroleum ether, recover the petroleum ether to obtain extract; use hexane / isopropanol as solvent, use dicyclohexylamine to amination salt reaction of petroleum ether layer; then extract it sequentially with petroleum ether, petroleum ether / ethyl acetate with a volume ratio of 4:1 to 6:1, and petroleum ether / ethyl acetate with a volume ratio of 0.5:1 to 1.5:1.

[0022] (2) The petroleum ether:ethyl acetate fraction with a volume ratio of 0.5:1 to 1.5:1 from the previous step was concentrated under reduced pressure to obtain an extract. The extract was subjected to silica gel column chromatography and eluted with petroleum ether / dichloromethane with a volume ratio of 1:1 to 3:1, petroleum ether / dichloromethane with a volume ratio of 1:1 to 1:3, dichloromethane, and ethyl acetate. The ethyl acetate fraction was concentrated under reduced pressure to obtain an extract. The extract was dissolved in methyl tert-butyl ether and acidified. After acidification, the crude product was subjected to medium-pressure ODS reverse column chromatography and eluted with 60 to 80% methanol / water to obtain compounds 1 and 2.

[0023] It may further include one or more of the following steps:

[0024] (3) Compound 1 was dissolved in tetrahydrofuran and PhI(OAc)2 was added under nitrogen protection. After reacting at -5 to 5℃ for 1 to 3 hours, the reaction was quenched by adding saturated sodium bicarbonate aqueous solution. The product was extracted with ethyl acetate, and the organic phase was washed, dried and concentrated under reduced pressure. The crude product was separated and purified by silica gel thin layer preparation to obtain compound 3.

[0025] (4) Compound 2 was dissolved in tetrahydrofuran and protected with nitrogen by adding PhI(OAc)2. After reacting at room temperature for 2-3 hours, the reaction was quenched by adding saturated sodium bicarbonate aqueous solution. The product was extracted with ethyl acetate, and the organic phase was washed, dried and concentrated under reduced pressure. The crude product was separated and purified by silica gel thin-layer chromatography to obtain compound 4.

[0026] This invention involves the extraction, separation, or biomimetic semi-synthesis of multiple acyl-resorcinol derivatives.

[0027] This invention has discovered the hepatoprotective activity of several acyl-resorcinol derivatives.

[0028] This invention provides the application of acyl-resorcinol derivatives, represented by hyperacmotone A (HA, compound 3), in the preparation of drugs or health products for the prevention and treatment of non-alcoholic steatohepatitis.

[0029] In the prevention or treatment of non-alcoholic steatohepatitis (NAH), this invention provides safe and effective oral or non-oral administration of HA or its derivatives. Oral administration can be formulated into any conventional dosage form, such as tablets, capsules, powders, or granules; non-oral administration can be formulated into suppositories, injections, etc.

[0030] In the preparation of drugs or health products for the prevention or treatment of non-alcoholic steatohepatitis, the excipients and preparation methods of this invention can be selected from any pharmaceutically acceptable form.

[0031] The dosage of HA or its derivatives described in this invention can be adjusted according to factors such as dosage form, route of administration, and patient's basic condition.

[0032] This invention first screened acyl-resorcinol derivatives for anti-NASH activity in a free fatty acid-induced L02 cell model, using the degree of lipid accumulation in cells as an evaluation index, and discovered several acyl-resorcinol derivatives that could significantly reduce lipid accumulation in L02 cells. Then, using the highly abundant compound hyperacmotone A (HA, compound 3) as a representative compound, its anti-NASH activity was further evaluated in an MCD mouse model. The results showed that compared with the model group, the treated groups improved the appearance of the liver, and the high-dose HA group (20 mg / kg) significantly reduced the liver index in mice; liver histopathological staining showed that the HA-treated group improved liver cell morphology, significantly reduced fat vacuoles, inflammatory cell infiltration, ballooning degeneration, and lipid accumulation in the liver; serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were significantly reduced; liver triglyceride (TG) content was significantly decreased, and glutathione (GSH) activity was significantly increased. Simultaneously, the expression levels of genes related to liver lipid metabolism were detected. The results showed that HA significantly increased the expression of Pparα and Cpt1 in the liver and decreased the expression of Fap1, suggesting that HA may exert good anti-NASH activity in in vitro and in vivo models by promoting fatty acid oxidation. In summary, the results of this experiment suggest that acyl-resorcinol derivatives, represented by HA, have good application potential in the prevention or treatment of non-alcoholic steatohepatitis.

[0033] The key technology of this invention lies in:

[0034] 1. HA has a significant effect on improving lipid accumulation in mouse liver;

[0035] 2. HA has certain hepatoprotective activity and can be used to prevent or treat NASH-related diseases.

[0036] This invention relates to the application of acyl-resorcinol derivatives. Specifically, using an in vitro model of FFA-induced L02 cells, the hepatoprotective activity of several acyl-resorcinol derivatives was studied. These compounds were found to inhibit lipid accumulation in L02 cells to a certain extent. Then, using compound HA as a representative, preliminary activity verification was performed in vitro using Oil Red O and BODIPY staining and real-time quantitative PCR (RT-qPCR). The results showed that HA inhibited lipid accumulation in L02 cells in a dose-dependent manner, upregulated the expression levels of Pparα and Cpt1 genes, and downregulated the expression level of Fap1 gene. To further investigate its anti-NASH activity, its hepatoprotective ability was evaluated in an MCD diet-induced NASH mouse model. Compared with the model group, the HA treatment group improved the appearance of the liver, and the high-dose HA group significantly reduced the liver index in mice. Histopathological staining of liver tissue showed that the HA treatment group improved hepatocyte morphology and significantly reduced fat vacuoles, inflammatory cell infiltration, ballooning degeneration, and lipid accumulation in the liver. Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were significantly reduced. Hepatic triglyceride (TG) content was significantly decreased, and glutathione (GSH) activity was significantly increased. Simultaneously, the expression levels of hepatic lipid metabolism-related genes were detected, and the results showed that HA significantly increased the expression of Ppara and Cpt1 in the liver and decreased the expression of Fap1. These results suggest that HA may exert good anti-NASH activity in in vitro and in vivo models by promoting fatty acid oxidation. In summary, HA has good application potential in the prevention or treatment of non-alcoholic steatohepatitis.

[0037] This invention utilizes an in vitro model of free fatty acid (FFA)-induced normal human hepatocytes (L02) to study the hepatoprotective activity of acyl-resorcinol derivatives isolated and synthesized from *Phyllostachys edulis*. The study found that these compounds inhibited lipid accumulation in L02 cells to varying degrees, indicating that they possess potential anti-NASH activity. Using compound 3 as a representative compound, this invention further employed a mouse model induced by a choline- and methionine-deficient diet (MCD) to find that HA exhibited good anti-NASH activity both in vivo and in vitro. The anti-NASH activity of acyl-resorcinol derivatives, represented by HA, has not been reported domestically or internationally.

[0038] Beneficial Effects: Compared with the prior art, the present invention has the following advantages: The compound HA described in the present invention can be used as an active ingredient in health products and drugs for liver protection and prevention or treatment of NASH, providing a new option for drugs to combat NASH. Specifically, the application of HA in the preparation of health products and drugs for liver protection; and the application in the preparation of health products and drugs for prevention and treatment of NASH. Attached Figure Description

[0039] Figure 1 The results are the screening results of the hepatoprotective activity of acyl-resorcinol derivatives (based on lipid accumulation).

[0040] Figure 2 It is the effect of HA on lipid accumulation in L02 cells;

[0041] Figure 3 It is the effect of HA on lipid metabolism-related genes in L02 cells;

[0042] Figure 4 The effect of HA on organ toxicity in mice;

[0043] Figure 5 The effects of HA on liver morphology, body weight, liver wet weight, and liver index in mice induced by MCD diet.

[0044] Figure 6 These are the liver pathological staining results of NASH model mice induced by HA and MCD diet.

[0045] Figure 7 The effect of HA on serum ALT and AST levels in mice induced by MCD diet;

[0046] Figure 8 The effect of HA on liver TG and TC levels in mice induced by MCD diet;

[0047] Figure 9The effect of HA on GSH activity in the liver of mice induced by MCD diet;

[0048] Figure 10 This refers to the effect of HA on lipid metabolism-related genes in mouse liver;

[0049] Figure 11 This is the HRMS spectrum of compound 4;

[0050] Figure 12 It is compound 4. 1 H NMR spectrum;

[0051] Figure 13 It is compound 4. 13 C NMR spectrum;

[0052] Figure 14 This is the HMQC spectrum of compound 4;

[0053] Figure 15 This is the HMBC spectrum of compound 4. Detailed Implementation

[0054] The present invention will now be described in detail with reference to the accompanying drawings and examples, but the description is intended to explain rather than limit the invention.

[0055] Example 1: Extraction and separation of compounds 1 and 2

[0056] 20.0 kg of dried *Echeveria elegans* roots were sliced ​​and extracted three times with 70.0 L of 95% ethanol each time for 7 days. After concentration and solvent recovery, a crude extract (1.8 kg) was obtained. The crude extract was suspended in 50°C water and extracted four times with petroleum ether. The petroleum ether was recovered to obtain a paste (753.0 g). The petroleum ether layer was amination to form a salt using dicyclohexylamine in a hexane:isopropanol (9:1) solvent. The extract was then sequentially extracted with petroleum ether, petroleum ether:ethyl acetate (5:1), and petroleum ether:ethyl acetate (1:1).

[0057] The petroleum ether:ethyl acetate (1:1) fraction was concentrated under reduced pressure to obtain an extract (535 g). The extract was subjected to silica gel column chromatography (200-300 mesh), eluted with petroleum ether:dichloromethane (2:1), petroleum ether:dichloromethane (1:2), dichloromethane, and ethyl acetate. The ethyl acetate fraction was concentrated under reduced pressure to obtain 120 g of extract. The 120 g extract was dissolved in 1800 mL of methyl tert-butyl ether and acidified with 1200 mL of citric acid aqueous solution. After acidification, the crude product was subjected to medium-pressure ODS reversed-phase column chromatography, eluted with 73% methanol / water to obtain compounds 1 and 2. Both compounds were recrystallized separately in methanol solution at -20 °C, and eluted with cold methanol to obtain pure compounds 1 (9.0 g) and 2 (3.0 g), respectively.

[0058] Example 2: Biomimetic semi-synthesis of compounds 3 and 4

[0059] Compound 3: Compound 1 (51.6 mg, 0.0996 mmol, 1 equiv) was dissolved in 5 mL of tetrahydrofuran (THF), and PhI(OAc)2 (64.1 mg, 0.1992 mmol, 2 equiv) was added under nitrogen protection. The reaction was carried out at 0 °C for 2 h, and then quenched with saturated sodium bicarbonate aqueous solution. The product was extracted three times with ethyl acetate, and the organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel thin-layer chromatography (petroleum ether:ethyl acetate 7:1) to obtain product compound 3 (12.8 mg, 0.0262 mmol, yield 26.3%).

[0060] Compound 4: Compound 2 (100 mg, 0.2066 mmol, 1 equiv) was dissolved in 10 mL of anhydrous tetrahydrofuran (THF), and PhI(OAc)2 (133 mg, 0.4132 mmol, 2 equiv) was added under nitrogen protection. The reaction was quenched with saturated NaHCO3 aqueous solution after reacting at room temperature for 2.5 h. The product was extracted three times with ethyl acetate, and the organic phase was washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel thin-layer chromatography (petroleum ether:ethyl acetate 9:2) to give compound 4 (9 mg, 0.0198 mmol, yield 9.6%).

[0061] The structures of the above compounds are as follows:

[0062] Compound 1

[0063]

[0064] Compound 2

[0065]

[0066] Compound 3

[0067]

[0068] Compound 4

[0069]

[0070] The structural identification data of the compound are as follows:

[0071] Compound 1 (hypercalin B): Yellow oily substance, ESIMS: m / z 518, molecular formula C 33 H 42 O5.

[0072] 11H NMR (500 MHz, CDCl3): δ H 7.43 (1H, s, H-29), 7.43 (2H, s, H-31 / 33), 7.35 (2H, s, H-30 / 32), 4.94 (1H, t, H-18), 4.89 (1H, brs, H-14a), 4.85 (1H, brs, H-14b), 4.83 (1H, m, H-23), 2.63 (2H, m, H-17), 2.61 (1H, m, H-7a), 2.58 (1H, dd, 7.0 Hz, H-22a), 2.53 (1H, dd, H-22b), 2.41 (1H, td, 7.5 Hz, H-12), 2.15 (1H, dd, 11.9 Hz, H-7b), 1.91 (1H, m, H-10a), 1.82 (1H, m, H-8), 1.78 (3H, s, Me-15), 1.70 (1H, m, H-11a), 1.60 (1H, m, H-10b), 1.60 (3H, s, Me-20), 1.60 (3H, s, Me-25), 1.59 (3H, s, Me-21), 1.56 (3H, s, Me-26), 1.50 (1H, m, H-11b), 1.28 (3H, s, Me-16).

[0073] 13 13C NMR (150 MHz, CDCl3): δ C 196.6 (C-27), 195.4 (C-6), 189.2 (C-2), 170.6 (C-4), 146.3 (C-13), 139.6 (C-28), 134.9 (C-24), 134.1 (C-19), 130.8 (C-31), 127.9 (C-29), 127.9 (C-33), 127.7 (C-30), 127.7 (C-32), 119.8 (C-18), 118.5 (C-23), 111.8 (C-14), 109.7 (C-3), 107.8 (C-1), 81.1 (C-9), 58.0 (C-5), 54.4 (C-12), 50.2 (C-8), 43.5 (C-10), 38.6 (C-17), 37.3 (C-22), 29.3 (C-16), 29.0 (C-11), 26.0 (C-20), 26.0 (C-25), 21.8 (C-7), 18.7 (C-15), 18.2 (C-21), 18.1 (C-26).

[0074] Compound 2 (hypercalin C): white powder, ESIMS: m / z 484, molecular formula C 30 H44 O5.

[0075] 1 H NMR: (600MHz, methanol-d4)δ H 4.84(1H,m,H-14a), 4.82(1H,m,H-23), 4.78(1H,m,H-18), 4.77(1H,m,H-14b), 2.61(1H,m,H-28), 2.60(2 H, m, H-17), 2.52 (1H, m, H-7a), 2.50 (2H, m, H-22), 2.42 (1H, m, H-12), 2.14 (1H, m, H-7b), 1.81 (2H, m, H-10) , 1.77 (1H, m, H-11b), 1.76 (3H, s, H-15), 1.75 (1H, m, H-8), 1.59 (3H, s, H-20), 1.54 (3H, s, H-25), 1.54 (3H, s, H-26), 1.52 (3H, s, H-21), 1.47 (1H, m, H-11a), 1.21 (3H, s, H-16), 1.09 (3H, d, H-29), 1.09 (3H, d, H-30).

[0076] 13 C NMR: (150MHz, methanol-d4)δ c 121 .0(C,C-18),119.7(C,C-23),110.1(C,C-3),107.4(C,C-1),80.6(C,C-9),60.1(C,C-5),55.6(C,C-12),51.7(C,C-8 ), 43.1 (C, C-10), 40.3 (C, C-22), 38.4 (C, C-17), 37.2 (C, C-28), 29.9 (C, C-11), 29.8 (C, C-16), 26.1 (C, C-21), 26.1 ( C, C-25), 22.7 (C, C-7), 19.5 (C, C-30), 19.3 (C, C-29), 18.7 (C, C-15), 18.2 (C, C-20), 18.0 (C, C-26), 11.8 (C, C-14).

[0077] Compound 3 (hyperacmotone A): Deep yellow oily substance, ESIMS: m / z 488, molecular formula C32 H 40 O4。

[0078] 1 H NMR(600MHz,Methanol-d4)δ 2.58(1H,dd,J=13.9,7.3Hz,H-6a),2.32(1H,dd,J=13.9,6.0Hz,H-6b),1.90(1H,ddd,J=10.7,7.3,6.0Hz,H-7),1.64(2H,t,J=7.7Hz,H-9),1.83(1H,m,H-10a),1.37(1H,dd,J=13.1,7.6Hz,H-10b),2.44(1H,m,H-11),4.65(1H,d,J=2.1Hz,H-13a),4.57(1H,t,J=1.7Hz,H-13b),1.58(3H,s,H-14),1.10(3H,s,H-15),2.44(2H,m,H-16),4.97(1H,m,H-17),1.70(3H,s,H-19),1.57(3H,s,H-20),2.43(2H,m,H-21),4.97(1H,m,H-22),1.69(3H,s,H-24),1.56(3H,s,H-25),7.77(2H,d,J=7.2Hz,H-28and H-32),7.55(2H,t,J=7.8Hz,H-29and H-31),7.71(1H,t,J=7.4Hz,H-30).

[0079] 13 C NMR(150MHz,Methanol-d4)δ 155.4(C-1),163.3(C-2),208.1(C-3),56.7(C-4),204.7(C-5),24.8(C-6),51.4(C-7),80.4(C-8),41.7(C-9),28.8(C-10),54.2(C-11),148.1(C-12),112.3(C-13),18.9(C-14),27.4(C-15),33.7(C-16),118.8(C-17),137.6(C-18),26.2(C-19),17.8(C-20),33.7(C-21),118.8(C-22),137.5(C-23),26.3(C-24),17.8(C-25),193.7(C-26),136.9(C-27),130.3(C-28and C-32),130.2(C-29and C-31),136.0(C-30).

[0080] Compound 4: A new compound, a pale yellow oil, HRESIMS: m / z 477.2978 [M+Na] + (calcd forC 29 H 42 NaO4, 477.2975). [α] 25 D +5.4(c 0.07, MeOH); UV(MeOH)λ max (logε)204(3.53), 245(3.21); ECD(MeOH)λmax(Δε)213(-0.31), 226(+0.90), 242(-0.55), 262(+3.93), 296(-0.19)nm; IR(KBr)v max 3442, 2925, 2854, 1733, 1703, 1643, 1599, 1383, 1277cm -1 .

[0081] 1 H NMR(600MHz, CDCl3)δ 2.46 (1H, m, H-6a), 2.41 (1H, m, H-6b), 2.06 (1H, td, J = 11.1, 3.4Hz, H-7), 1.81 (1H, m, H-9a), 1.67 (1H, m, H-9b), 1.90 (1 H, m, H-10a), 1.46 (1H, m, H-10b), 2.57 (1H, m, H-11), 4.85 (1H, m, H-13a), 4.81 (1H, m, H-13b), 1.80 (3H, s, H-14), 1.01 ( 3H, s, H-15), 2.44 (2H, m, H-16), 4.79 (1H, m, H-17), 1.54 (3H, s, H-19), 1.55 (3H, s, H-20), 2.39 (2H, m, H-21), 4.79 (1H, m, H-22), 1.55 (3H, s, H-24), 1.57 (3H, s, H-25), 3.29 (1H, m, H-27), 1.14 (3H, d, J = 6.6, H-29), 1.03 (3H, d, J = 7.3, H-29).

[0082] 13C NMR (150MHz, CDCl3) δ152.0(C-1), 165.3(C-2), 207.5(C-3), 56.1(C-4), 203.2(C-5), 24.4(C-6), 50 .4(C-7), 79.4(C-8), 40.6(C-9), 27.3(C-10), 53.3(C-11), 146.8(C-12), 112.0(C-13), 18.6(C-14), 28.4(C-15), 34.0(C-16), 117.2(C-17), 136.6(C-18), 25.9(C-19), 17.9(C-20), 32.8(C-21), 117.3 (C-22), 136.9(C-23), 26.0(C-24), 17.9(C-25), 206.1(C-26), 40.9(C-27), 16.3(C-2), 17.8(C-29).

[0083] Structural analysis of compound 4: Aside from the difference in the acyl group, compound 4 and compound 3 have similar one-dimensional NMR data. Two-dimensional NMR and crucial HMBC correlations confirm that compound 4 is a structural analog of compound 3, the main difference being that the acyl group in compound 4 is isobutyryl. HRMS of compound 4... 1 H NMR, 13 The C NMR, HMQC, and HMBC spectra are attached. Figure 11-15 .

[0084] Example 3: Study on the hepatoprotective activity of compounds 1-4 (using lipid accumulation as an indicator)

[0085] BODIPY staining: Prepare the BODIPY stock solution by weighing BODIPY powder and dissolving it in PBS to prepare a 1 mg / mL stock solution. Aliquot the solution and store at 4°C protected from light. Digest L02 cells in the logarithmic growth phase with trypsin to prepare a single-cell suspension. After cell counting, filter the suspension at 7 × 10⁶ cells per well. 3 -1×10 4Cells were seeded in 96-well plates. After cell attachment, cells were treated with BSA / FFA (0.5 mM) and incubated with the above compounds at specific doses for 24 h. After the experiment, the culture medium was gently aspirated with a vacuum pump, and PBS was added along the wall to wash away the original culture medium. Cells were fixed with 4% paraformaldehyde for 10 min, washed twice with PBS, diluted with PBS to 0.1 μg / mL, and incubated at room temperature in the dark for 60 min. Cells were washed once with PBS, counterstained with DAPI for 10 min, washed once with PBS, and then infiltrated with PBS. Cells were then imaged using a high-content imaging system and statistically analyzed using Graphpad Prism 8 software. The results showed that all the above compounds had a certain degree of anti-lipid accumulation activity in L02 cells. Figure 1 ).

[0086] Based on the above screening results, compound 3 was selected for further study.

[0087] Example 4: HA-induced Oil Red O and BODIPY staining experiments on FFA-induced L02 cells

[0088] Oil Red O staining: L02 cells in logarithmic growth phase were digested with trypsin to prepare a single-cell suspension. After cell counting, cells were dispensed at a concentration of 1 × 10⁶ cells per well. 5 Cells were seeded in 12-well plates. After cell adhesion, cells were treated with BSA / FFA (0.5 mM) and simultaneously incubated with different doses of HA for 24 h. Oil Red O staining was performed using a kit (G1262) from Beijing Solarbio Science & Technology Co., Ltd., and the cells were observed and photographed under an upright fluorescence microscope. Results showed that HA reduced lipid droplet formation in L02 cells in a dose-dependent manner. Figure 2 -A).

[0089] BODIPY staining: L02 cells in logarithmic growth phase were digested with trypsin to prepare a single-cell suspension. After cell counting, cells were dispensed at 7 × 10⁶ cells per well. 3 -1×10 4 Cells were seeded in 96-well plates. After cell attachment, cells were treated with BSA / FFA (0.5 mM) and incubated with different doses of HA for 24 h. After the experiment, the culture medium was gently aspirated with a vacuum pump, and PBS was added along the cell wall to wash away the original culture medium. Cells were fixed with 4% paraformaldehyde for 10 min, washed twice with PBS, diluted with PBS to 0.1 μg / mL, and incubated at room temperature in the dark for 60 min. Cells were washed once with PBS, counterstained with DAPI for 10 min, washed once with PBS, and then infiltrated with PBS. Cells were then observed and photographed using a high-content imaging system. The results showed that HA reduced lipid accumulation in L02 cells in a dose-dependent manner. Figure 2 -B).

[0090] Example 5: Effects of HA on lipid metabolism-related genes in L02 cells

[0091] Discard the culture medium, wash twice with PBS, add 500 μL of Lysis Buffer and vigorously pipette about 20 times to transfer to a 1.5 ml nuclease-free EP tube. Add an equal volume of anhydrous ethanol and vigorously pipette about 10 times to thoroughly mix the lysis buffer and anhydrous ethanol. Add the mixed liquid to an RNA centrifuge column, centrifuge at 12000 x g for 1 min, discard the waste liquid, add Wash Buffer to the RNA column, centrifuge at 12000 x g for 1 min, discard the waste liquid, and centrifuge the RNA column again. Then transfer the RNA column to a new nuclease-free EP tube, open the cap and let it air dry for 2 min. Add 20-30 μL of RNase-free ddH2O to the center of the RNA column, let it stand at room temperature for 2 min, centrifuge at 12000 x g for 1 min, add the elution buffer back to the column, and centrifuge again to obtain the RNA elution buffer. The RNA concentration was then determined using a NanoDrop2000 / 2000C spectrophotometer. 1 μL of sample was added to the instrument base, and the sample concentration (ng / μL) and OD260 / OD280 values ​​were read. The readings were taken three times, and the average value was calculated. An OD260 / OD280 value in the range of 1.8-2.2 was considered to indicate high RNA purity.

[0092] 1. Preparation ratio of reverse transcription reaction solution

[0093]

[0094] Reverse transcription was performed according to the proportions in the reverse transcription reaction system (Table 1). The resulting cDNA solution was stored at -20°C in a MyCycler Thermal Cycler PCR amplification instrument, or directly used for subsequent Real-Time PCR reactions. Real-Time PCR was performed using a Light Cycler 480 real-time quantitative PCR instrument. The amplification reaction system is shown in Table 2, and the amplification program is shown in Table 3.

[0095] 2. PCR amplification reaction system

[0096]

[0097]

[0098] 3. PCR amplification procedure

[0099]

[0100] 4. Primer sequence of the target gene

[0101]

[0102] like Figure 3 As shown, HA increased the expression levels of Ppara and Cpt1 genes in L02 cells in a dose-dependent manner, and inhibited the expression of Fap1.

[0103] Example 6: Acute toxicity test of HA

[0104] To determine the safety of HA, we conducted an acute toxicity experiment using the single maximum dose method before evaluating its in vivo efficacy. Eight C57BL / 6J mice were randomly divided into two groups of four each. The experimental group was administered HA 200 mg / kg orally by gavage, while the control group was given an equal volume of solvent (corn oil). After one week of observation, the mice were sacrificed, and their hearts, livers, spleens, lungs, and kidneys were collected and fixed in 4% paraformaldehyde. Figure 4 The results showed that a single administration of 200 mg / kg HA had no significant toxicity to mouse organs.

[0105] Example 7: MCD diet-induced mouse NASH model

[0106] Thirty-two 8-week-old male C57BL / 6J mice were randomly divided into four groups of eight mice each after one week of acclimatization: an MCS (methionine-and-choline sufficient diet) group (control group), an MCD group (model group), and low HA (10 mg / kg) and high HA (20 mg / kg) dose groups (experimental groups). The MCS group was fed the MCS diet, while the other three groups were fed the MCD diet. Dietary induction lasted for four weeks, during which the mice were administered the drug daily via oral gavage. The drug was dissolved in physiological saline containing 10% DMSO, while the model group received an equal volume of the control solution. At the end of the experiment, the mice were fasted for 12 hours, and their body weight was recorded. After euthanasia, the eyeballs were enucleated, and whole blood was collected. After standing for 1 hour, the blood was incubated at 4°C and 4000 rpm. -1 Centrifuge for 10 min, collect the supernatant and store at -80℃; collect the liver and weigh it, embed part of it with embedding solution, fix part of it with 4% paraformaldehyde, and freeze the remaining part in liquid nitrogen and store at -80℃.

[0107] Example 8: Detection of general indicators in laboratory animals

[0108] Figure 5 The figures show the body weight, liver weight, liver index, and liver morphology of mice in each group. It is evident that compared to the MCS group, the body weight and wet liver weight of mice in the MCD group were significantly decreased. #### P < 0.0001, ### P < 0.001, liver index significantly increased ( #P < 0.05, indicating that the MCD diet-induced NASH model led to decreased body weight, decreased liver wet weight, and increased liver index in mice. Compared with the MCD group, HA administration failed to improve the decrease in body weight and liver weight (P > 0.05), but the high-dose group effectively reduced the liver index in mice. * (P < 0.05). Furthermore, the morphological appearance of the livers in each group of mice showed that the livers in the MCS group were bright red and glossy, firm and elastic, with sharp edges; the livers in the MCD group were lighter, yellowish, less elastic, and had blunt edges; the livers in the HA group showed improved appearance compared to the MCD group. This indicates that HA has a certain hepatoprotective effect.

[0109] Example 9: Effects of HA on the pathological structure of liver tissue in MCD model mice

[0110] Paraformaldehyde-fixed liver tissue was sent to the Pharmacology and PDX Efficacy Evaluation Platform of China Pharmaceutical University to prepare tissue paraffin blocks for HE, Masson, and Oil Red staining. The tissue was then scanned using a digital pathology slide scanner (NanoZoomer S60) and analyzed using NDP software. The results are as follows: Figure 6 HE staining showed that the hepatocytes of mice in the MCS group had normal morphology and structure, no nucleus displacement, abundant cytoplasm, clearly visible lobular structure, and neatly arranged hepatic cords, with no fatty degeneration or other lesions. In the MCD group, the liver tissue showed obvious fat vacuoles, fragmented hepatocyte morphology, lipid droplet accumulation leading to nucleus displacement, significant ballooning degeneration, and obvious focal inflammatory cell infiltration. The HA-treated group significantly reduced hepatocyte vacuoles, improved cell morphology, and occasionally showed inflammatory cell infiltration, with the high-dose group showing a more significant effect. Oil Red O staining showed no obvious lipid droplet accumulation in the liver tissue of mice in the MCS group; the MCD group had a large number of lipid droplets that further merged and diffused throughout the hepatocytes; the HA group had only a small number of granular lipid droplets, which decreased with increasing dosage. Masson staining showed that the liver structure of mice in the MCS group was normal, with no abnormal fibrous tissue proliferation; the MCD group showed obvious fibrosis characteristics, severe destruction of the lobular structure, and significant collagen deposition; the HA-treated group showed improvement in liver fibrosis, with cell morphology tending towards normal.

[0111] Example 10: Detection of Serum Indicators in Mice

[0112] The activities of ALT and AST in mouse serum were detected using a kit from the Nanjing Jiancheng Biotechnology Institute. The results are as follows: Figure 7 As shown, compared with the MCS group, the serum ALT and AST levels in the MCD group mice were significantly increased. #### (P < 0.0001) After treatment with HA, the levels of ALT and AST decreased significantly, indicating that HA improved liver injury in mice.

[0113] Example 11: Determination of lipid content and glutathione activity in mouse liver

[0114] TG and TC content determination: Weigh approximately 50 mg of liver tissue and add anhydrous ethanol at a ratio of weight (g):volume (mL) = 1:9. Grind twice at 60 Hz for 2 min each time under ice-water bath conditions, then grind at 4℃ and 2500 r·min. -1 Centrifuge for 10 minutes, collect the supernatant, and determine the liver TG and TC content according to the kit instructions. Figure 8 As shown, compared with the MCS group, the TG content in the liver of the MCD group was significantly increased ( ### P < 0.001. Compared with the MCD group, the TG content was significantly lower in both the low- and high-dose HA groups. * P < 0.05 ** P < 0.01); Compared with the MCS group, the liver TC level in the MCD group was increased but not statistically significant; compared with the MCD group, the liver TC level in the low-dose HA group was decreased but not statistically significant; and the liver TC level in the high-dose HA group was significantly decreased. * (P < 0.05). This suggests that HA can improve lipid deposition in the liver.

[0115] GSH activity assay: According to the kit instructions, weigh approximately 20 mg of liver and add reagent one at a ratio of weight (g):volume (mL) = 1:9. Grind twice at 60 Hz under ice-water bath conditions, 2 min each time, then at 4℃ and 3500 rpm. -1 Centrifuge for 10 min, collect the supernatant, add 100 μL of chloroform, vortex to mix, centrifuge again, and use the supernatant to determine GSH activity according to the kit instructions. Figure 9 As shown, the liver GSH level in the MCD group mice was significantly lower than that in the MCS group. ## p < 0.05 indicates a decrease in the body's antioxidant capacity and an increase in oxidative stress, which is consistent with the characteristics of the MCD model. After HA treatment, the liver GSH activity of mice in both the low-dose and high-dose groups was significantly increased (p < 0.05). * P < 0.05 ** P < 0.01, indicating that HA improved the antioxidant capacity of mouse liver.

[0116] Example 12: Effects of HA on lipid metabolism-related genes in mouse liver

[0117] Liver RNA extraction: Weigh 5–10 mg of mouse liver tissue into a 1.5 mL EP tube, add 500 μL of Lysis Buffer, homogenize into a tissue slurry, and let stand at room temperature for 5 min. Vortex for 10 s, centrifuge at 12000 × g for 2 min, and transfer the supernatant to a new 1.5 mL EP tube. Subsequent procedures are the same as in Example 5.

[0118] 5. Primer sequence of the target gene

[0119]

[0120] like Figure 10 As shown, HA increased the expression levels of Ppara and Cpt1 genes in mouse liver in a dose-dependent manner, and inhibited the expression of Fap1. This suggests that HA may exert its anti-NASH activity by promoting hepatic fatty acid oxidation.

Claims

1. An acylphoroglucinol derivative or a pharmaceutically acceptable salt thereof, characterized in that, Selected from the following compounds or their pharmaceutically acceptable salts: 。 2. A pharmaceutical composition, characterized by, It includes one or more of the acyl phloroglucinol derivatives of claim 1 or pharmaceutically acceptable salts thereof, along with pharmaceutically acceptable excipients.

3. The pharmaceutical composition according to claim 2, characterized in that, The dosage forms of the pharmaceutical composition include oral and non-oral dosage forms; the oral dosage forms include tablets, capsules, powders or granules, and the non-oral dosage forms include suppositories and injections.

4. The use of the acyl-resorcinol derivative of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of hepatoprotective medicaments.

5. The use of the acyl-resorcinol derivative of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of a medicament for the prevention and treatment of non-alcoholic steatohepatitis.

6. A method for preparing an acyl-resorcinol derivative of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: (1) Take the root of the seedling flower, slice or crush it, add ethanol to extract it, concentrate and recover the solvent to obtain crude extract, add the crude extract to warm water at 40~70℃ and suspend it, extract it with petroleum ether, recover the petroleum ether to obtain extract; use hexane / isopropanol as solvent, use dicyclohexylamine to amination salt reaction of petroleum ether layer; then extract it with petroleum ether, petroleum ether / ethyl acetate with a volume ratio of 4:1~6:1, and petroleum ether / ethyl acetate with a volume ratio of 0.5:1~1.5:1 in sequence; (2) Take the petroleum ether:ethyl acetate fraction with a volume ratio of 0.5:1 to 1.5:1 from the previous step and concentrate it under reduced pressure to obtain an extract. Perform silica gel column chromatography and elute with petroleum ether / dichloromethane with a volume ratio of 1:1 to 3:1, petroleum ether / dichloromethane with a volume ratio of 1:1 to 1:3, dichloromethane, and ethyl acetate. Concentrate the ethyl acetate fraction under reduced pressure to obtain an extract. Dissolve the extract in methyl tert-butyl ether and acidify it. After acidification, perform medium-pressure ODS reverse column chromatography and elute with 60 to 80% methanol / water to obtain compounds 1 and 2 successively. (3) Compound 1 was dissolved in tetrahydrofuran, PhI(OAc)2 was added, and nitrogen was used for protection. The reaction was carried out at -5~5 ℃ for 1~3 h, and then saturated sodium bicarbonate aqueous solution was added to quench the reaction. The product was extracted with ethyl acetate, and the organic phase was washed, dried and concentrated under reduced pressure. The crude product was separated and purified by silica gel thin layer preparation to obtain compound 3. (4) Compound 2 was dissolved in tetrahydrofuran, PhI(OAc)2 was added, and nitrogen was used for protection. After reacting at room temperature for 2-3 h, saturated sodium bicarbonate aqueous solution was added to quench the reaction. The product was extracted with ethyl acetate, and the organic phase was washed, dried, and concentrated under reduced pressure. The crude product was purified by silica gel thin-layer chromatography to obtain compound 4. The structural formulas of compounds 1 and 2 are as follows: 。

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