Daphniphyllum calycinum extraction compound, extraction method thereof and application of daphniphyllum calycinum extraction compound in preparation of medicine for preventing or treating non-alcoholic fatty liver disease

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a daphniphyllum calycinum extract compound, an extraction method thereof and application of the daphniphyllum calycinum extract compound in preparation of a medicine for preventing or treating non-alcoholic fatty liver diseases. A natural compound with a novel structure is obtained from daphniphyllum calycinum for the first time, a preparation method of the compound is provided, activity research is carried out on the compound, and experimental data shows that the compound is low in cytotoxicity and has no obvious cytotoxicity to normal cells, and the compound can be used for preparing the compound by inhibiting expression of fatty acid synthetases ACC, ACLY and FAS. The compound inhibits the de novo synthesis of fatty acid in cells, thereby inhibiting the accumulation of lipid droplets in the cells and reducing the lipid level in the cells, and has wide application prospects in the field of preparation of drugs for preventing or treating non-alcoholic fatty liver diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More particularly, it relates to a compound extracted from Fatsia japonica and an extraction method thereof and application of the compound in preparation of a medicine for preventing or treating non-alcoholic fatty liver disease. BACKGROUND

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic syndrome characterized by accumulation of fat in the liver, with hepatocyte steatosis and lipid accumulation as the main features, caused by various reasons without excessive alcohol consumption. It is estimated that the global prevalence of NAFLD is about 2.6 billion, accounting for about 32% of the global population, and this number will continue to rise. Clinically, mild, moderate and severe are often used to describe the severity of fatty liver. The liver has strong regenerative capacity, and even at the stage of fatty hepatitis, the lesion can be completely reversed through early intervention and active treatment. However, if not taken seriously, it can also evolve into cirrhosis, with symptoms such as liver pain, nausea, vomiting and other discomforts. The diseased liver cannot recover as before, and eventually develops into hepatocellular carcinoma.

[0003] Currently, the first-line treatment for NAFLD is mainly to improve the lifestyle. Adjusting the dietary structure, strengthening exercise, avoiding smoking and alcohol, controlling the intake of saturated fatty acids and increasing the intake of unsaturated fatty acids. However, improving the lifestyle often has low patient compliance, and the long-term effect is not obvious, so drug intervention is still needed.

[0004] Currently, the clinical treatment drug selection for NAFLD is relatively limited, mainly including statins, pioglitazone and metformin and other drugs for metabolic syndrome. Statins can effectively reduce the low-density lipoprotein cholesterol level of NAFLD and non-alcoholic steatohepatitis (NASH) patients, which helps to prevent cardiovascular complications; pioglitazone can improve the serum biochemical indicators and liver histological characteristics of NASH patients; and metformin has the effects of improving insulin resistance, regulating blood glucose and assisting weight loss for NAFLD patients combined with type 2 diabetes. However, in the face of the complex pathological mechanism and diversified clinical manifestations of NAFLD, the existing treatment drugs are still insufficient in quantity and variety.

[0005] Therefore, it is of great significance to develop more safe and effective new treatment drugs to meet the clinical needs and optimize the treatment strategy for NAFLD. SUMMARY

[0006] The present application provides a compound that can inhibit lipid synthesis to overcome the defects and deficiencies of the existing drugs for treating NAFLD.

[0007] The second object of the present application is to provide a preparation method of the compound.

[0008] The third object of the present application is to provide a pharmaceutical composition comprising the compound or the pharmaceutically acceptable salt of the compound.

[0009] The fourth object of the present application is to provide an application of the compound or the pharmaceutical composition in the preparation of a drug for treating or preventing non-alcoholic fatty liver.

[0010] The above objects of the present application are achieved by the following technical solutions. The present application protects a compound, which has the following structure: .

[0011] Further, the compound also includes a pharmaceutically acceptable salt thereof.

[0012] The present application also protects a preparation method of the compound, which comprises the following steps: S1: The alcohol extract of the seed of Millettia pachycarpa Benth. is extracted with ethyl acetate after acid dissolution, the water layer is adjusted to pH 9-10, and then extracted with dichloromethane, and the organic layer is obtained to obtain total alkaloids of the seed of Millettia pachycarpa Benth.; S2: The total alkaloids of the seed of Millettia pachycarpa Benth. obtained in step S1 are separated by non-polar macroporous adsorption resin column chromatography, and gradient elution is performed with 10vol%, 30vol%, 50vol%, 75vol% and 95vol% ethanol aqueous solution in sequence, and the elution part of 50vol% ethanol aqueous solution is collected to obtain Fr.C component; S3: The Fr.C component obtained in step S2 is separated by Sephadex column chromatography, and a mixed solvent of dichloromethane-methanol with a volume ratio of 1:1 is used as an eluent to obtain a fraction Fr.C2; S4: The Fr.C2 component obtained in step S3 is separated by silica gel column chromatography, and a mixed solvent of dichloromethane-methanol containing 0.1vol% diethylamine with a volume ratio of 50:1, 20:1, 10:1, 5:1 and 1:0 is used as an eluent for gradient elution, and the part eluted by the mixed solvent of dichloromethane-methanol containing 0.1vol% diethylamine with a volume ratio of 20:1 is collected to obtain a fraction Fr.C2b; S5: The Fr.C2b component obtained in step S4 is separated by ODS column chromatography, and a mixed solvent of methanol-water with a volume ratio of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 and 100:0 is used as an eluent for gradient elution, and the part eluted by the mixed solvent of methanol-water with a volume ratio of 60:40 is collected to obtain a fraction Fr.C2b3; S6: The Fr.C2b3 component obtained in step S5 is purified by high performance liquid chromatography to obtain the compound, using a mixed solvent of acetonitrile containing 0.1 vol% ammonia water and water in a volume ratio of 30:70 as a mobile phase.

[0013] Further, the alcohol extract of the seed of the Aesculus chinensis is prepared by the following steps: the seed of the Aesculus chinensis is pretreated, and then is sufficiently extracted by using an ethanol solution with a volume concentration of ≥85%, and the solvent is removed to obtain the alcohol extract of the seed of the Aesculus chinensis.

[0014] Further, the pretreatment comprises pulverization.

[0015] Preferably, the concentration of the ethanol solution is 85vol%-95vol%.

[0016] More preferably, the concentration of the ethanol solution is 95vol%.

[0017] Further, the sufficient extraction is preferably percolation extraction.

[0018] Further, the removal of the solvent is performed by removing the solvent by distillation under reduced pressure.

[0019] Further, the acid dissolution comprises the following steps: the alcohol extract is dispersed in water to form a suspension, and the pH is adjusted to 2-3.

[0020] Further, in the acid dissolution step, the adjustment of the pH is performed by using an acidic reagent.

[0021] Preferably, the acidic reagent is a 1-3vol% sulfuric acid solution.

[0022] More preferably, the acidic reagent is a 2vol% sulfuric acid solution.

[0023] Further, in step S1, the adjustment of the pH is performed by using an alkaline reagent.

[0024] Further, the alkaline reagent is preferably sodium carbonate.

[0025] Further, the non-polar macroporous adsorption resin is a D101 type macroporous adsorption resin.

[0026] Further, the glucose gel is a Sphadex LH 20 glucose gel.

[0027] Specifically, in step S3, a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1 is used as an eluent, and the remaining component is the fraction Fr.C2 after removing the pigment impurities by high performance liquid chromatography analysis.

[0028] Further, in step S6, the high performance liquid chromatography purification is performed by using a preparative high performance liquid chromatography column for separation and purification.

[0029] Further, the preparative high performance liquid chromatography column is preferably XBridge BEH C18 OBD Prep Column, 10mm*250mm, 5um.

[0030] Further, in step S6, the compound can be distinguished by the peak time of the chromatographic peak and other impurities. In the method of the present application, the peak retention time of the compound is about 15 min, and within the determined retention time range, the components corresponding to each chromatographic peak can be collected for subsequent structure confirmation by nuclear magnetic resonance technology.

[0031] The present application also protects a pharmaceutical composition comprising the compound.

[0032] The present application also protects the use of the compound or the pharmaceutical composition in the preparation of a drug for preventing or treating non-alcoholic fatty liver.

[0033] Further, the cell administration concentration of the compound is 1-150um.

[0034] Preferably, the cell administration concentration of the compound is 3.125-100um.

[0035] More preferably, the cell administration concentration of the compound is 40-60um.

[0036] Further, the prevention or treatment of non-alcoholic fatty liver is to inhibit the accumulation of lipid droplets in cells and reduce the lipid level in cells.

[0037] Further, the prevention or treatment of non-alcoholic fatty liver is to inhibit the expression of fatty acid synthase.

[0038] Further, the fatty acid synthase includes one or more of ACC, ACLY, and FAS.

[0039] The present application has the following beneficial effects: The present application first obtains a natural compound with a novel structure from the roots of Disporum megalonyx, and provides a preparation method of the compound, and carries out activity research on it. Experimental data shows that the compound has low cytotoxicity and no obvious cytotoxicity to normal cells, can inhibit the de novo synthesis of fatty acids in cells by inhibiting the expression of fatty acid synthases ACC, ACLY and FAS, thereby inhibiting the accumulation of lipid droplets in cells and reducing the lipid level in cells, and has a broad application prospect in the field of preparing drugs for preventing or treating non-alcoholic fatty liver. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1The nuclear magnetic resonance hydrogen spectrum of the extract DBSA-32 from the leaves of Symplocos paniculata.

[0041] Figure 2 The nuclear magnetic resonance carbon spectrum of the extract DBSA-32 from the leaves of Symplocos paniculata.

[0042] Figure 3 The results of the cytotoxicity of the extract DBSA-32 from the leaves of Symplocos paniculata on HepG2 cells at different concentrations, ns represents no significant difference.

[0043] Figure 4 The results of the effect of the extract DBSA-32 from the leaves of Symplocos paniculata on the accumulation of lipid droplets in the HepG2 cells induced by oleic acid.

[0044] Figure 5 The results of the effect of the extract DBSA-32 from the leaves of Symplocos paniculata on the triglyceride level in the HepG2 cells induced by oleic acid, *** represents P <0.001.

[0045] Figure 6 The results of the effect of the extract DBSA-32 from the leaves of Symplocos paniculata on the proteins related to lipid synthesis. DETAILED DESCRIPTION

[0046] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0048] Experimental materials The HepG2 cell line was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, the DMEM culture medium was purchased from Gibco Company (item number: C11995500B), the fetal bovine serum was purchased from Sijiqing Company (item number: 13011-8611), the double antibody was purchased from Gibco Company (item number: 15140-122), the oleic acid (Oelic acid, OA) reagent was purchased from Sigma Company (item number: L1376), the BODIPY reagent was purchased from Invitrogen Company (item number: D3922), the DAPI (4', 6-diamidino-2-phenylindole) reagent was purchased from Sigma Company (item number: D8417), the triglyceride assay kit was purchased from Beijing Puli Lei Gene Technology Co., Ltd. (item number: E1013), and the CCK-8 kit was purchased from Beijing Solabio Technology Co., Ltd. (item number: CA1210).

[0049] Example 1 Separation and extraction of the extract DBSA-32 from the leaves of Symplocos paniculata After 50 kg of dried seed of Aesculus chinensis Bge. was crushed, it was extracted by percolation with 95 vol% ethanol solution. After the solvent was recovered under reduced pressure, 9.1 kg of total extract was obtained. The extract was dispersed in appropriate amount of pure water to form a suspension, and 2 vol% sulfuric acid solution was added dropwise under continuous stirring to adjust the pH of the system to 2-3. After the pH of the solution was stabilized by sufficient stirring, the acidic fat-soluble impurities were removed by extraction with ethyl acetate. Then, sodium carbonate was added to the water layer to adjust the pH to 9-10, and the alkaloids were enriched by extraction with dichloromethane. The organic layer was obtained, and 250.2 g of total alkaloids was obtained. Further, the total alkaloids were separated by column chromatography using D101 macroporous adsorption resin, and eluted with gradient ethanol solution (10 vol%, 30 vol%, 50 vol%, 75 vol%, 95 vol%). The components eluted with 75 vol% ethanol solution and 95 vol% ethanol solution were combined to obtain Fr. D. Finally, four components with different polarities (Fr. A-D) were obtained, and the component eluted with 50 vol% ethanol solution was collected as Fr. C. Fr. C (60.2 g) was preliminarily separated by Sephadex LH 20 glucose gel column chromatography (dichloromethane-methanol, volume ratio 1:1). After HPLC analysis, the remaining component was Fr. C2 after removing the impurities containing pigments. Then, Fr. C2 was further separated by silica gel column chromatography, and eluted with gradient dichloromethane-methanol solution containing 1 vol% diethylamine (volume ratio 50:1, 20:1, 10:1, 5:1, 1:0). Five sub-components (Fr. C2a-C2e) were obtained by further separation. The fraction eluted with 20:1 volume ratio of dichloromethane-methanol mixed solvent containing 0.1 vol% diethylamine was collected to obtain Fr. C2b. Then, Fr. C2b was further separated by ODS column chromatography, and eluted with gradient methanol-water solution (volume ratio 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0). The components eluted with 30:70 volume ratio of methanol-water solution and the components eluted with 40:60 volume ratio of methanol-water solution were combined as Fr. C2b1. The components eluted with 90:10 volume ratio of methanol-water solution and the components eluted with 100:0 volume ratio of methanol-water solution were combined as Fr. C2b6. Six fractions (Fr. C2b1-C2b6) were obtained by further separation, and the components eluted with 60:40 volume ratio of methanol-water solution were collected as Fr. C2b3. Finally, Fr. C2b3 was purified by HPLC (XBridge BEH C18 OBD Prep Column, 10 mm x 250 mm, 5 μm) (acetonitrile: water, volume ratio 30:70, with 0.1 vol% ammonia water), and the retention time was about 15 min. Compound DBSA-32 was successfully obtained, and its structure was identified as Figure 1 ,Figure 2 .

[0050] Example 2 Application of DBSA-32 of Sycopsis sinensis Extract 1. Experimental method (1) Cell culture HepG2 cells were used, and high-sugar DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% fetal bovine serum (FBS) and 1% double-antibiotic (penicillin / streptomycin) was used for culture in a 37°C cell culture box containing 5% CO2.

[0051] (2) Cytotoxicity experiment The cell density was observed under a microscope, and when the confluence rate reached about 80%~90%, the cells were passaged. The culture medium in the culture dish was removed, and the cells were washed twice with sterile PBS, then 0.25% trypsin-EDTA solution was added, and the cells were digested at room temperature for 2 min. When the cells could be blown down gently, the 0.25% trypsin-EDTA solution was removed, and 1 mL of DMEM medium was added to stop the digestion. After counting the cells, 1.5×10 4 cells were inoculated into a 96-well plate and cultured in the incubator for 24 hours. DBSA-32 of Sycopsis sinensis extract (concentrations were 0, 3.125, 6.25, 12.5, 25, 50 and 100 μM, respectively) was added to the culture medium in continuous dilution, and the cells were cultured for another 24 hours. The culture medium was discarded, and the working solution containing 10% CCK-8 reagent was added to the wells, and the cells were cultured in the dark for 2 hours. Then the absorbance at 450 nm was detected.

[0052] (3) Cell staining and fluorescence experiment The cells were inoculated into cell slides at an appropriate density, and after adhering, OA and DBSA-32 of Sycopsis sinensis extract were given. After the treatment was completed, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes. After washing with PBS for three times, 2 mg / mL Bodipy dye was diluted with PBS at a ratio of 1:5000, and 1 mL of the dye was added to each well for incubation at room temperature in the dark for 20 minutes. After the staining was completed, the dye was removed, and the cell nucleus was stained with DAPI. The accumulation of lipid droplets in the cells was observed using a Leica SP8 confocal microscope.

[0053] (4) Triglyceride content detection The cells were inoculated into the wells at an appropriate density, and after adhering, OA and DBSA-32 of Sycopsis sinensis extract were given. After the treatment was completed, the culture medium was discarded, and an appropriate amount of lysis solution was added for complete lysis. An appropriate amount of lysis solution was taken for BCA protein quantification, and the rest was subjected to enzymatic determination, and the absorbance at 550 nm was measured. The absorbance value was substituted into the standard curve to calculate the triglyceride concentration of the sample, and the triglyceride content was corrected with the protein concentration.

[0054] (5) Western Blot experiment Cells were seeded into the well plate at an appropriate density, and after adhering, they were treated with DBSA-32. After treatment, the culture medium was discarded, and an appropriate amount of lysis solution was added for complete lysis. After lysis, BCA protein quantification was performed, and 20 ug of protein sample was subjected to SDS-PAGE, followed by membrane transfer. After membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and development were performed.

[0055] 2. Experimental results (1) Cytotoxicity experiment Safety is the primary indicator for evaluating whether a compound can be used. To evaluate the safety of DBSA-32, different concentration gradients were designed to detect its cytotoxicity to HepG2 cells. To detect the cytotoxicity of DBSA-32 to HepG2 cells, the CCK-8 kit was used to determine cell viability, and the results are shown in Figure 3 There was no significant cytotoxicity after treatment with 3.125, 6.25, 12.5, 25, 50, and 100 μM, and 50 μM was selected as the subsequent drug concentration.

[0056] (2) Cell staining and fluorescence experiment Lipid droplet accumulation in cells is a complex physiological process that plays an important role in energy storage, cell function maintenance, and other aspects. Triglycerides are the main component of lipid droplets in cells. When cells synthesize lipids, they will take up fatty acids, glucose, and other nutrients from the outside world, and under the action of a series of enzymes, they will synthesize triglycerides and cholesterol; when cells need energy, the triglycerides in the lipid droplets will be hydrolyzed into fatty acids and glycerol and release energy. Therefore, the effect of DBSA-32 on cell lipid accumulation was evaluated using staining methods.

[0057] Bodipy staining showed that DBSA-32 could significantly inhibit the accumulation of lipid droplets in cells induced by oleic acid, and the results are shown in Figure 4 The accumulation of lipid droplets in cells was less in the negative control CT group, and the accumulation of lipid droplets in cells was significantly increased in the positive control OA treatment group. After OA+DBSA-32 treatment, the accumulation of lipid droplets in cells was significantly reduced compared with the positive control group, indicating that DBSA-32 could effectively inhibit the accumulation of lipids in HepG2 cells caused by OA.

[0058] (3) Triglyceride content detection Biochemical analysis was used to evaluate the effect of DBSA-32 on cell lipid accumulation, and the results are shown in Figure 5As shown, the triglyceride level in HepG2 cells was significantly increased after OA treatment, and the triglyceride level in HepG2 cells was significantly decreased after OA+DBSA-32 treatment compared with the positive control group. The triglyceride content determination results further confirmed that DBSA-32 can effectively inhibit the lipid accumulation in HepG2 cells caused by OA.

[0059] (4) Western Blot experiment Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA in cells, which is the first step 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 from acetyl-CoA and malonyl-CoA through a series of enzymatic reactions.

[0060] The results of the Western Blot experiment are shown in Figure 6 , which proves that 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 down-regulated after DBSA-32 treatment.

[0061] The above mechanism analysis shows that DBSA-32 can be used for the preparation of a drug for preventing or treating non-alcoholic fatty liver disease by inhibiting the expression of fatty acid synthase ACC, ACLY and FAS, inhibiting the de novo synthesis of fatty acids in cells, thereby inhibiting the accumulation of lipid droplets in cells and reducing the lipid level in cells.

[0062] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A compound, characterized in that, The compound has the following structure: 。 2. The compound according to claim 1, characterized in that, The compounds also include their pharmaceutically acceptable salts.

3. 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 solution. 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 a 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.

4. The preparation method according to claim 3, 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.

5. The preparation method according to claim 3, 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.

6. The preparation method according to claim 4, characterized in that, The concentration of the ethanol solution is 85 vol% to 95 vol.

7. The preparation method according to claim 4, characterized in that, The extraction described herein is percolation extraction.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of claim 1 or 2.

9. Use of the compound of claim 1 or 2 or the pharmaceutical composition of claim 8 in the preparation of a medicament for the prevention or treatment of non-alcoholic fatty liver disease.

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

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