Bovis Folium extractive compound and its extraction method and application in preparation of medicine for preventing or treating non-alcoholic fatty liver disease

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a compound extracted from *Acer buergerianum* (a type of shrub), its extraction method, and its application in the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease. This invention is the first to obtain a natural compound with a novel structure from *Acer buergerianum*, and provides a method for its preparation. Activity studies were conducted, and experimental data show that the compound exhibits low cytotoxicity and no significant cytotoxicity to normal cells. It can inhibit the expression of fatty acid synthases ACC, ACLY, and FAS, thereby inhibiting de novo fatty acid synthesis in cells, suppressing lipid droplet accumulation, and reducing cellular lipid levels. This compound has broad application prospects in the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a compound extracted from *Acer buergerianum*, its extraction method, and its application in the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic syndrome characterized by fat accumulation in the liver due to various causes, without a history of excessive alcohol consumption. It is primarily characterized by hepatocellular steatosis and lipid accumulation. It is estimated that approximately 2.6 billion people worldwide suffer from NAFLD, representing about 32% of the global population, and this number is expected to continue rising. Clinically, mild, moderate, and severe degrees of fatty liver disease are commonly used to describe its severity. The liver possesses a strong regenerative capacity; even in the stage of steatohepatitis, the lesions can be completely reversed through early intervention and aggressive treatment. However, if left untreated, it can progress to cirrhosis, leading to a series of uncomfortable symptoms such as pain in the liver area, nausea, and vomiting. The diseased liver can no longer recover and may eventually develop into hepatocellular carcinoma.

[0003] Currently, the primary first-line treatment for NAFLD is lifestyle modification. This includes adjusting diet, increasing exercise, abstaining from smoking and alcohol, controlling saturated fatty acid intake, and increasing unsaturated fatty acid intake. However, patient adherence to lifestyle modifications is often poor, and long-term effects are not significant, necessitating drug intervention.

[0004] Currently, the clinical treatment options for NAFLD are relatively limited, mainly including statins, pioglitazone, and metformin, which target metabolic syndrome. Statins can effectively lower LDL cholesterol levels in NAFLD and non-alcoholic steatohepatitis (NASH) patients, helping to prevent cardiovascular complications; pioglitazone can improve serum biochemical indicators and liver histological characteristics in NASH patients; while metformin can improve insulin resistance, regulate blood glucose, and aid in weight loss in NAFLD patients with type 2 diabetes. However, given the complex pathological mechanisms and diverse clinical manifestations of NAFLD, the number and variety of existing treatments remain insufficient.

[0005] Therefore, developing more safe and effective new therapeutic drugs is of great significance for meeting clinical needs and optimizing NAFLD treatment strategies. Summary of the Invention

[0006] This invention addresses the shortcomings and deficiencies of existing drugs for treating NAFLD by providing a compound that can inhibit lipid synthesis.

[0007] A second objective of this invention is to provide a method for preparing the compound.

[0008] A third object of the present invention is to provide a pharmaceutical composition comprising the above-described compound or a pharmaceutically acceptable salt thereof.

[0009] A fourth object of the present invention is to provide the use of the said compound or the said pharmaceutical composition in the preparation of a medicament for the treatment or prevention of non-alcoholic fatty liver disease.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention protects a compound having the following structure:

[0012] .

[0013] Furthermore, the compound also includes its pharmaceutically acceptable salt.

[0014] This invention also protects a method for preparing the compound, comprising the following steps:

[0015] S1: The alcohol extract of Acer buergerianum seeds was dissolved in acid and then extracted with ethyl acetate. The aqueous layer was taken and the pH was adjusted to 9-10. It was then extracted with dichloromethane, and the organic layer was taken to obtain the total alkaloids of Acer buergerianum seeds.

[0016] S2: The total alkaloids of the Acer buergerianum seeds obtained in step S1 were separated by non-polar macroporous adsorption resin column chromatography, and were successively eluted with 10 vol%, 30 vol%, 50 vol%, 75 vol%, and 95 vol% ethanol aqueous solution. The eluted fraction of 50 vol% ethanol aqueous solution was collected to obtain the Fr.C fraction.

[0017] S3: The Fr.C fraction obtained in step S2 is separated by glucose gel column chromatography, using a 1:1 volume ratio of dichloromethane-methanol mixed solvent as the eluent to obtain the fraction Fr.C2;

[0018] S4: The Fr.C2 fraction obtained in step S3 is separated by silica gel column chromatography. Gradient elution is performed using a dichloromethane-methanol mixed solvent containing 0.1 vol% diethylamine in volume ratios of 50:1, 20:1, 10:1, 5:1, and 1:0. The fraction eluted by the dichloromethane-methanol mixed solvent containing 0.1 vol% diethylamine in volume ratio of 20:1 is collected to obtain the fraction Fr.C2b.

[0019] S5: The Fr.C2b fraction obtained in step S4 is separated by ODS column chromatography using a gradient elution with methanol-water mixed solvents in volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0. The fraction eluted by the methanol-water mixed solvent in volume ratio of 60:40 is collected to obtain the fraction Fr.C2b3.

[0020] S6: The Fr.C2b3 component obtained in step S5 is purified by high performance liquid chromatography using an acetonitrile-water mixed solvent containing 0.1 vol% ammonia at a volume ratio of 30:70 as the mobile phase to obtain the compound.

[0021] Furthermore, the ethanol extract of Acer buergerianum seeds is prepared by the following steps: after pretreatment of dried Acer buergerianum seeds, they are fully extracted with an ethanol solution of volume concentration ≥85% to remove the solvent, thereby obtaining the ethanol extract of Acer buergerianum seeds.

[0022] Furthermore, the pretreatment includes pulverization.

[0023] Preferably, the concentration of the ethanol solution is 85 vol% to 95 vol.

[0024] More preferably, the concentration of the ethanol solution is 95 vol.

[0025] Furthermore, the fully extracted material is preferably percolation extraction.

[0026] Furthermore, the solvent removal is achieved by vacuum distillation.

[0027] Further, the acid dissolution includes the following steps: dispersing the alcohol extract in water to prepare a suspension, and adjusting the pH to 2-3.

[0028] Furthermore, in the acid dissolution step, the pH adjustment is performed using an acidic reagent.

[0029] Preferably, the acidic reagent is a 1-3 vol% sulfuric acid solution.

[0030] More preferably, the acidic reagent is a 2 vol% sulfuric acid solution.

[0031] Furthermore, in step S1, the pH adjustment is performed using an alkaline reagent.

[0032] Furthermore, the alkaline reagent is preferably sodium carbonate.

[0033] Furthermore, the non-polar macroporous adsorption resin is a D101 type macroporous adsorption resin.

[0034] Furthermore, the glucose gel is Sphadex LH 20 glucose gel.

[0035] Specifically, in step S3, a 1:1 dichloromethane-methanol mixed solvent is used as the eluent, and high performance liquid chromatography is used to remove pigment impurities. The remaining components are the fraction Fr.C2.

[0036] Furthermore, in step S6, the high-performance liquid chromatography purification is performed using a preparative high-performance liquid chromatography column for separation and purification.

[0037] Furthermore, the preparative high-performance liquid chromatography column is preferably an XBridge BEH C18 OBD PrepColumn, 10 mm × 250 mm, 5 μm.

[0038] Furthermore, in step S6, the compound can be distinguished from other impurities by the elution time of the chromatographic peak. In the method of this application, the retention time of the compound is approximately 15 min. Within the determined retention time range, the components corresponding to each chromatographic peak can be collected separately, and then the structures of the obtained components can be confirmed by nuclear magnetic resonance (NMR) technology.

[0039] The present invention also protects a pharmaceutical composition comprising the compound.

[0040] The present invention also protects the use of the compound or the pharmaceutical composition in the preparation of a medicament for the prevention or treatment of non-alcoholic fatty liver disease.

[0041] Furthermore, the cellular administration concentration of the compound is 1–150 μM.

[0042] Preferably, the cellular administration concentration of the compound is 3.125~100 μM.

[0043] More preferably, the cellular administration concentration of the compound is 40-60 μM.

[0044] Furthermore, the prevention or treatment of non-alcoholic fatty liver disease involves inhibiting the accumulation of lipid droplets in cells and reducing lipid levels in cells.

[0045] Furthermore, the prevention or treatment of non-alcoholic fatty liver disease involves inhibiting the expression of fatty acid synthase.

[0046] Furthermore, the lipase includes one or more of ACC, ACLY, and FAS.

[0047] The present invention has the following beneficial effects:

[0048] This invention provides a novel natural compound with a novel structure obtained from *Acer buergerianum* for the first time, along with a method for its preparation. Activity studies were conducted, and experimental data show that the compound exhibits low cytotoxicity and no significant cytotoxicity to normal cells. It can inhibit the de novo synthesis of fatty acids in cells by suppressing the expression of fatty acid synthases ACC, ACLY, and FAS, thereby inhibiting lipid droplet accumulation and reducing lipid levels in cells. This compound has broad application prospects in the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease. Attached Figure Description

[0049] Figure 1 The image shows the 1H NMR spectrum of DBSA-32, an extract of Acer buergerianum.

[0050] Figure 2 The image shows the carbon NMR spectrum of DBSA-32, an extract of Acer buergerianum.

[0051] Figure 3 The graph shows the cytotoxicity results of different concentrations of Acer buergerianum extract DBSA-32 on HepG2 cells. ns represents no significant difference in results.

[0052] Figure 4 The figure shows the effect of the extract DBSA-32 from Acer buergerianum on lipid droplet accumulation in oleic acid-induced HepG2 cells.

[0053] Figure 5 The figure shows the effect of the extract DBSA-32 from Acer palmatum on triglyceride levels in oleic acid-induced HepG2 cells. *** indicates... P <0.001.

[0054] Figure 6 The figure shows the effect of the extract DBSA-32 from Acer palmatum on lipid synthesis-related proteins. Detailed Implementation

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0056] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0057] Experimental materials

[0058] HepG2 cell line was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; DMEM medium was purchased from Gibco (C11995500B); fetal bovine serum was purchased from Sijiqing Biotechnology Co., Ltd. (C13011-8611); penicillin antibody was purchased from Gibco (C15140-122); oleic acid (OA) reagent was purchased from Sigma-Aldrich (C1376); BODIPY reagent was purchased from Invitrogen (C3922); DAPI (4',6-diamidinyl-2-phenylindole) reagent was purchased from Sigma-Aldrich (C8417); triglyceride assay kit was purchased from Beijing Pulilai Gene Technology Co., Ltd. (C1013); and CCK-8 assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. (C1210).

[0059] Example 1: Isolation and extraction of DBSA-32 from Acer palmatum extract

[0060] 50 kg of dried Acer buergerianum seeds were pulverized and then percolated with 95 vol% ethanol solution. After solvent recovery under reduced pressure, 9.1 kg of total extract was obtained. The extract was dispersed in an appropriate amount of pure water to form a suspension. Under continuous stirring, 2 vol% sulfuric acid solution was added dropwise to adjust the pH of the system to 2-3. After thorough stirring to stabilize the pH, acidic lipid-soluble impurities were removed by ethyl acetate extraction. Subsequently, sodium carbonate was added to the aqueous layer to adjust the pH to 9-10, and the solution was enriched by dichloromethane extraction. The organic layer was collected to obtain 250.2 g of total alkaloids. The total alkaloids were further separated by column chromatography using D101 macroporous adsorption resin with a gradient elution of ethanol solutions (10 vol%, 30 vol%, 50 vol%, 75 vol%, 95 vol%). The fractions obtained by elution with 75 vol% ethanol solution and 95 vol% ethanol solution were combined to obtain Fr.D. Finally, four polarity-differentiated fractions (Fr.A~D) were obtained. The fraction obtained by elution with 50 vol% ethanol solution was collected as Fr.C. The Fr.C fraction (60.2 g) was initially separated by Sephadex LH 20 glucose gel column chromatography (dichloromethane-methanol, v / v 1:1). After removing pigment impurities by HPLC analysis, the remaining fraction was Fr.C2. Subsequently, Fr.C2 was further separated by silica gel column chromatography using gradient elution with dichloromethane-methanol solutions containing 1 vol% diethylamine (v / v 50:1, 20:1, 10:1, 5:1, 1:0), yielding five secondary fractions (Fr.C2a~C2e). The fraction eluted with a 20:1 v / v dichloromethane-methanol mixture containing 0.1 vol% diethylamine was collected to obtain the fraction Fr.C2b. Fr.C2b was then subjected to ODS column chromatography with gradient elution using methanol-water solutions (volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0) for further separation. The fractions eluted with methanol-water solutions of 30:70 and 40:60 were combined to form Fr.C2b1, and the fractions eluted with methanol-water solutions of 90:10 and 100:0 were combined to form Fr.C2b6, resulting in a total of 6 fractions (Fr.C2b1~C2b6). The fraction eluted with methanol-water solutions of 60:40 was collected as Fr.C2b3. Finally, Fr.C2b3 was purified by HPLC (acetonitrile:water, volume ratio 30:70, with the addition of 0.1 vol% ammonia) with a retention time of approximately 15 min, successfully yielding compound DBSA-32. Its structure was identified as follows: Figure 1 , Figure 2 .

[0061] Example 2: Application of Acer palmatum extract DBSA-32

[0062] 1. Experimental Methods

[0063] (1) Cell culture

[0064] HepG2 cells were cultured in a 37°C cell culture incubator containing 5% CO2 in high-glucose DMEM (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

[0065] (2) Cytotoxicity test

[0066] Observe cell density under a microscope. Passage the cells when the confluence reaches approximately 80%–90%. Remove the culture medium from the culture dish, wash twice with sterile PBS, add 0.25% trypsin-EDTA solution, and digest at room temperature for 2 minutes. Once the cells can be gently blown off, remove the 0.25% trypsin-EDTA solution and add 1 mL of DMEM culture medium to stop the digestion. After cell counting, transfer the cells to a 1.5 × 10⁶ culture dish. 4 Cells were seeded into 96-well plates and cultured for 24 hours. Serially diluted *Acer buergerianum* extract DBSA-32 (concentrations of 0, 3.125, 6.25, 12.5, 25, 50, and 100 μM) were added to the culture medium, and the cells were cultured for another 24 hours. The culture medium was discarded, and working solution containing 10% CCK-8 reagent was added to the wells. The cells were then incubated in the dark for 2 hours, and the absorbance at 450 nm was measured.

[0067] (3) Cell staining and fluorescence experiments

[0068] Cells were seeded at an appropriate density onto cell slides and, after adhesion, treated with OA and DBSA-32 (a type of Acer buergerianum extract). After treatment, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes. After washing three times with PBS, 2 mg / mL Bodipy dye was diluted 1:5000 with PBS, and 1 mL of dye was added to each well. The cells were incubated at room temperature in the dark for 20 minutes. After staining, the dye was removed, and the cell nuclei were stained with DAPI. Lipid droplet accumulation in the cells was observed using a Leica SP8 confocal microscope.

[0069] (4) Triglyceride content detection

[0070] Cells were seeded at an appropriate density into well plates and, after adhesion, treated with OA and DBSA-32 (a extract of Acer buergerianum). After treatment, the culture medium was discarded, and an appropriate amount of lysis buffer was added for complete lysis. A suitable amount of the lysis buffer was used for BCA protein quantification, and the remainder was used for enzymatic assays, with absorbance measured at 550 nm. The absorbance values ​​were substituted into a standard curve to calculate the triglyceride concentration in the samples, and the triglyceride content was corrected for by protein concentration.

[0071] (5) Western Blot experiment

[0072] Cells were seeded at an appropriate density into well plates and treated with DBSA-32 (a type of pine tree extract) after adhesion. After treatment, the culture medium was discarded, and lysis buffer was added for complete lysis. BCA protein was quantified after lysis, and 20 μg of protein was subjected to SDS-PAGE, followed by membrane transfer. After transfer, the membrane was blocked, incubated with primary antibody, then with secondary antibody, and finally developed.

[0073] 2. Experimental Results

[0074] (1) Cytotoxicity test

[0075] Safety is a primary indicator for evaluating the usability of a compound. To assess the safety of the *Acer buergerianum* extract DBSA-32, this invention designed a concentration gradient assay to detect its cytotoxicity against HepG2 cells. To detect the cytotoxicity of *Acer buergerianum* extract DBSA-32 against HepG2 cells, cell viability was measured using a CCK-8 assay kit. The results are as follows: Figure 3 As shown, treatments with 3.125, 6.25, 12.5, 25, 50, and 100 μM did not show significant cytotoxicity, and 50 μM was selected as the subsequent drug administration concentration.

[0076] (2) Cell staining and fluorescence experiments

[0077] Lipid droplet accumulation in cells is a complex physiological process that plays a crucial role in energy storage and cellular function maintenance. Triglycerides are the main components of lipid droplets in cells. During lipid synthesis, cells synthesize triglycerides and cholesterol from nutrients such as fatty acids and glucose taken from the external environment under the action of a series of enzymes. When cells need energy, triglycerides in lipid droplets are hydrolyzed into fatty acids and glycerol, releasing energy. Therefore, this study used staining methods to evaluate the effect of the extract DBSA-32 from *Acer buergerianum* on cellular lipid accumulation.

[0078] Bodipy staining revealed that the extract DBSA-32 from *Acer buergerianum* significantly inhibited oleic acid-induced lipid droplet accumulation in cells. The results were as follows: Figure 4As shown, intracellular lipid droplet accumulation was low in the negative control CT group, while it significantly increased in the positive control OA treatment group. However, treatment with OA plus *Acer negundo* extract DBSA-32 significantly reduced intracellular lipid droplet accumulation compared to the positive control group, indicating that *Acer negundo* extract DBSA-32 effectively inhibits OA-induced intracellular lipid accumulation in HepG2 cells.

[0079] (3) Triglyceride content detection

[0080] The effect of the extract DBSA-32 from Acer palmatum on cellular lipid accumulation was evaluated using biochemical analysis. The results are as follows: Figure 5 As shown, OA treatment significantly increased intracellular triglyceride levels in HepG2 cells. However, OA treatment combined with DBSA-32 (a type of Maple Leaf Extract) significantly reduced intracellular triglyceride levels compared to the positive control group. The triglyceride content assay further confirmed that DBSA-32 effectively inhibited OA-induced lipid accumulation in HepG2 cells.

[0081] (4) Western Blot experiment

[0082] Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA within cells, which is the first and rate-limiting step in the fatty acid synthesis pathway and plays a crucial role in lipid synthesis metabolism. The main function of ATP-citrate lyase (ACLY) is to cleave citrate into acetyl-CoA and oxaloacetate, providing substrates for fatty acid synthesis. The main function of fatty acid synthase (FAS) is to catalyze the de novo synthesis of fatty acids using acetyl-CoA and malonyl-CoA as raw materials through a series of enzymatic reactions.

[0083] Western blotting results are as follows: Figure 6 As shown, DBSA-32 affects lipid accumulation by inhibiting lipid synthesis. Compared with the control DMSO group, the expression of lipid synthesis-related genes FAS, ACC, and ACLY was significantly downregulated after DBSA-32 treatment.

[0084] The above mechanistic analysis shows that the extract DBSA-32 of Acer palmatum inhibits the expression of fatty acid synthases ACC, ACLY and FAS, thereby inhibiting the de novo synthesis of fatty acids in cells, inhibiting the accumulation of lipid droplets in cells, and reducing the lipid level in cells. It can be used to prepare drugs for the prevention or treatment of non-alcoholic fatty liver disease.

[0085] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A compound, characterized in that, The compound is a compound with the following structure or a pharmaceutically acceptable salt thereof: 。 2. The method for preparing the compound according to claim 1, characterized in that, Includes the following steps: S1: The alcohol extract of Acer buergerianum seeds was dissolved in acid and then extracted with ethyl acetate. The aqueous layer was taken and the pH was adjusted to 9-10. It was then extracted with dichloromethane, and the organic layer was taken to obtain the total alkaloids of Acer buergerianum seeds. S2: The total alkaloids of the Acer buergerianum seeds obtained in step S1 were separated by non-polar macroporous adsorption resin column chromatography, and were successively eluted with 10 vol%, 30 vol%, 50 vol%, 75 vol%, and 95 vol% ethanol aqueous solutions. The eluted fraction of 50 vol% ethanol aqueous solution was collected to obtain the Fr.C fraction. S3: The Fr.C fraction obtained in step S2 is separated by glucose gel column chromatography, using a 1:1 volume ratio of dichloromethane-methanol mixed solvent as the eluent to obtain the fraction Fr.C2; S4: The Fr.C2 fraction obtained in step S3 is separated by silica gel column chromatography. Gradient elution is performed using a dichloromethane-methanol mixed solvent containing 0.1 vol% diethylamine in volume ratios of 50:1, 20:1, 10:1, 5:1, and 1:

0. The fraction eluted by the dichloromethane-methanol mixed solvent containing 0.1 vol% diethylamine in volume ratio of 20:1 is collected to obtain the fraction Fr.C2b. S5: The Fr.C2b fraction obtained in step S4 is separated by ODS column chromatography using a gradient elution with methanol-water mixed solvents in volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:

0. The fraction eluted by the methanol-water mixed solvent in volume ratio of 60:40 is collected to obtain the fraction Fr.C2b3. S6: The Fr.C2b3 component obtained in step S5 is purified by high performance liquid chromatography using an acetonitrile-water mixed solvent containing 0.1 vol% ammonia at a volume ratio of 30:70 as the mobile phase to obtain the compound.

3. The preparation method according to claim 2, characterized in that, The ethanol extract of Acer buergerianum seeds is prepared by the following steps: after pretreatment, dried Acer buergerianum seeds are fully extracted with an ethanol solution of volume concentration ≥85% to remove the solvent, thereby obtaining the ethanol extract of Acer buergerianum seeds.

4. The preparation method according to claim 2, characterized in that, The acid dissolution includes the following steps: dispersing the alcohol extract in water to prepare a suspension, and adjusting the pH to 2-3.

5. The preparation method according to claim 3, characterized in that, The concentration of the ethanol solution is 85 vol% to 95 vol.

6. The preparation method according to claim 3, characterized in that, The extraction described herein is percolation extraction.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of claim 1.

8. Use of the compound of claim 1 or the pharmaceutical composition of claim 7 in the preparation of a medicament for the prevention or treatment of non-alcoholic fatty liver disease.

9. The application as described in claim 8, characterized in that, The cellular administration concentration of the compound is 1–150 μM.

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