Spirobenzylisoquinoline alkaloid anisone C extracted from anise, and its preparation method and application

Abstract

A novel spirobenzylisoquinoline alkaloid anisone C extracted from anise, as well as its preparation method and application, can effectively solve the problem of extracting and preparing the novel spirobenzylisoquinoline alkaloid anisone C from anise, and realize its application in the preparation of drugs for the prevention and treatment of non-alcoholic fatty liver disease. Dried anise is extracted with ethanol by heating and refluxing, followed by acid extraction and alkali precipitation, and sequentially extracted with chloroform and n-butanol to obtain an extract of the corresponding parts. The target extract is mixed with silica gel, eluted, concentrated under reduced pressure, and identified. Fractions of the same size are combined, loaded onto a column, eluted, mixed with silica gel, eluted, and fractions with the same retention time are collected, concentrated, and dried to obtain anisone C. This compound can significantly improve lipid metabolism disorders and lipid accumulation in HepG2 cells induced by free fatty acids, while also reducing FFA-induced HepG2 cell damage. It can be prepared into a variety of pharmaceutical dosage forms for convenient and effective clinical use, with broad development and application prospects and significant economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to a spirobenzylisoquinoline alkaloid ketobase C extracted from aniseed, and a preparation method and application thereof. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is a metabolic disease characterized by excessive lipid accumulation in hepatocytes due to factors such as excess energy intake, obesity, and dyslipidemia. It is also a major risk factor for liver fibrosis, cirrhosis, and liver cancer. The incidence of NAFLD has increased dramatically worldwide due to multiple factors, including changes in diet and lifestyle. However, there are no FDA-approved drugs for the treatment of NAFLD worldwide. Therefore, the search for novel therapeutic agents for NAFLD is of great importance.

[0003] Hypecoum erectum L., also known as "throat grass," "yellow wheat grass," and "yellow flower grass," is the whole herb of Hypecoum erectum L., a plant of the Papaveraceae family. Widely distributed in Northeast, North, and Northwest my country, it is used to treat sore throats, hepatitis, bacterial dysentery, and red and swollen eyes. It is a popular folk herbal remedy for sore throats and icteric hepatitis. Chemical composition studies have shown that Hypecoum erectum L. contains a large number of alkaloids, with isoquinoline alkaloids being the most prevalent. Current literature reports have confirmed that total alkaloids from fennel have a significant protective effect on acute liver injury in rats induced by carbon tetrachloride and acute liver injury in mice induced by thioacetamide [Ke Jun, Zhang Guiqing, Weng Shiai, et al., Study on the effect of pharyngeal herb on the liver, Pharmacology and Clinic of Traditional Chinese Medicine, 1985, (1): 185-186; Guo Jie, Zhang Xide, Huang Wei, Screening of effective fractions from fennel, Journal of Shaanxi University of Traditional Chinese Medicine, 2006, 29(2): 58-60]. This suggests that total alkaloids from fennel have potential application value in the treatment of acute liver injury. However, there has been no literature report on their use in the treatment of NAFLD, and there has been no public report on the use of a new type of spirobenzylisoquinoline alkaloid isolated from fennel in the prevention and treatment of NAFLD. Summary of the Invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a spirobenzylisoquinoline alkaloid anisone C extracted from fennel and its preparation method and application, which can effectively solve the problem of extracting and preparing spirobenzylisoquinoline alkaloid anisone C from fennel and realize its application in the preparation of drugs for preventing and treating non-alcoholic fatty liver disease (NAFLD).

[0005] The technical solution provided by the present invention is a spirobenzylisoquinoline alkaloid anisone C extracted from anise, the chemical molecular structure of which is:

[0006]

[0007] The preparation method is:

[0008] The dried aniseed is extracted with ethanol under heating and reflux to obtain a total extract; the total extract is subjected to acid extraction and alkali precipitation, and then extracted with petroleum ether, chloroform and n-butanol in sequence to obtain a first extract of the chloroform part and a second extract of the n-butanol part;

[0009] The second extract was mixed with an equal amount of alkaline silica gel, and eluted with a gradient of dichloromethane-methanol. Each fraction was concentrated under reduced pressure, and the fractions were analyzed by thin layer spot plate. The same fractions were combined to obtain multiple first fractions, and the target first fraction was selected from the multiple first fractions.

[0010] The target first component is loaded onto an ODS column and eluted with a gradient ratio of methanol:water to obtain multiple second components, and the target second component is selected from the multiple second components;

[0011] The target second component is mixed with alkaline silica gel, and eluted with a gradient ratio of petroleum ether to ethyl acetate to obtain multiple third components, and the target third component is selected from the multiple third components;

[0012] The target third group of components was eluted with methanol-water at a flow rate of 3 mL / min and a volume of 1 L. The retention time t R The fraction at 13.8 min was concentrated and dried to obtain anisone base C.

[0013] The invention discloses an application of spirobenzylisoquinoline alkaloid anisone C extracted from anise prepared by the above method in the preparation of drugs for preventing and treating non-alcoholic fatty liver disease (NAFLD).

[0014] The spirobenzylisoquinoline alkaloid anisone C extracted from aniseed by the method of the present invention can significantly improve lipid metabolism disorders and lipid accumulation in HepG2 cells induced by free fatty acids (FFA), and can also reduce FFA-induced HepG2 cell damage. This provides a new chemical structure type for the development of new NAFLD therapeutic drugs, and can be easily prepared into a variety of pharmaceutical dosage forms for convenient and effective clinical use. It has broad development and application prospects and significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The chemical molecular formula structure diagram of anisone alkaloid C of the present invention;

[0016] Figure 2 This is a high-resolution mass spectrum of anisone alkaloid C of the present invention;

[0017] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of anisone alkaloid C of the present invention;

[0018] Figure 4 is the carbon NMR spectrum of anisone alkaloid C of the present invention;

[0019] Figure 5 This is a graph showing the effect of anisone C of the present invention on improving lipid accumulation in HepG2 cells induced by free fatty acids (FFA), wherein the image is stained with Oil Red O; scale bar = 100 μm; n=3, (A) and (B) Oil red O staining and quantitative analysis; compared with the control group, ### is P < 0.001; compared with the model group, *** is P < 0.001;

[0020] Figure 6 The figure shows the results of anisone C improving the free fatty acid (FFA)-induced HepG2 cell damage, where compared with the control group, ## indicates P < 0.01, # indicates P < 0.05; compared with the model group, ** indicates P < 0.01, * indicates P < 0.05. DETAILED DESCRIPTION

[0021] The specific implementation methods of the present invention are described in detail below with reference to examples and specific situations.

[0022] The present invention can be specifically implemented by the following examples:

[0023] Example 1:

[0024] The present invention discloses a spirobenzylisoquinoline alkaloid anisone C extracted from anise, and its chemical molecular structure is as follows:

[0025]

[0026] The preparation method is:

[0027] (1) 10 kg of dried fennel was crushed and extracted three times with 95% ethanol by volume at a condensation reflux temperature of 90-95° C., with each extraction amount being 3-5 times the weight volume of the fennel, for 2 hours each time. The weight volume refers to the solid in kg and the liquid in L. The extracts were filtered and combined. The extracts were concentrated under reduced pressure to obtain an ethanol extract (about 1.5 kg).

[0028] (2) adding 2% hydrochloric acid aqueous solution with a weight volume of ten times the amount of the ethanol extract to dissolve, filtering to obtain the acid aqueous solution and insoluble matter; adding petroleum ether to the acid aqueous solution and extracting 5-8 times, each time 2.8-4 L, for 3-5 hours, recovering the solvent to obtain a petroleum ether layer and an acid aqueous layer; adding ammonia water to the acid aqueous layer to adjust the pH value to 9-10 to obtain an alkaline aqueous solution; extracting the alkaline aqueous solution with chloroform and n-butanol in sequence 5-8 times, each time 2.8-4 L, for 3-5 hours, recovering the solvent to obtain a chloroform fraction (about 70 g) and an n-butanol fraction (about 65 g);

[0029] (3) The chloroform fraction was mixed with an equal amount of alkaline silica gel and gradient eluted with a dichloromethane-methanol mixed solvent system with a volume ratio of 100:0, 100:2, 100:5, 100:10, 100:20, 100:50, 100:100, and 0:100. Each gradient used 13-16 L of eluent, the flow rate was 10-15 mL / min, and each 850-1000 ml volume was a fraction. 130 fractions were collected and analyzed by silica gel thin layer chromatography. 254 Thin layer plates were developed using dichloromethane-methanol in a volume ratio of 10:1 and 5:1, respectively. Detection and analysis were performed at 254 nm using a UV lamp and a modified potassium bismuth iodide solution as a developer. Based on the thin layer chromatography results, fractions 1-10, 11-27, 28-48, 49-72, 73-93, 94-108, 109-123, and 124-130 were combined to obtain components Fr.A, Fr.B…Fr.H.

[0030] (4) Component Fr.B was passed through an ODS column and eluted with a gradient of methanol:water at a volume ratio of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0. Each gradient used 6-8 L of eluent at a flow rate of 20-35 mL / h. Each elution gradient was a fraction, and 10 subcomponents Fr.B1…Fr.B7…Fr.B10 were collected.

[0031] (5) The subcomponent Fr.B7 was mixed with an equal amount of 100-200 mesh alkaline silica gel and eluted with petroleum ether, petroleum ether-ethyl acetate mixture and pure ethyl acetate in sequence. The volume ratio of petroleum ether-ethyl acetate mixture was 8:1, 5:1, 2:1 and 1:1. The amount of each eluent was 100-200 mL, the flow rate was 10-15 ml / min, and each 50 mL was a fraction. 13 fractions were collected and analyzed by silica gel thin layer chromatography using GF254 thin layer plates with petroleum ether:ethyl acetate in a volume ratio of 2:1 as the developing solvent. The analysis was performed under an ultraviolet lamp at 254 nm. According to the thin layer detection results, fractions 1-4, 5-9 and 10-13 were combined to obtain three components Fr.B7-1, Fr.B7-2 and Fr.B7-3.

[0032] (6) Component Fr.B7-1 was purified by preparative high performance liquid chromatography using a mixed solvent system of methanol and water with a volume ratio of 65:35. The column was a Fisher Wharton Xbridge C 18 The column was used at a flow rate of 3 mL / min, and the chromatographic peak with a retention time of 13.8 min was collected. The solvent was recovered to obtain anisone base C (about 9.7 mg).

[0033] Example 2:

[0034] The present invention provides a method for preparing spirobenzylisoquinoline alkaloid anisone C extracted from anise, comprising the following steps: in the step (1), the condensation reflux extraction temperature is 92° C., and the amount used is 3.5, 4, or 4.5 times the weight volume of anise;

[0035] Example 3:

[0036] The present invention provides a method for preparing spirobenzylisoquinoline alkaloid anisone base C extracted from anise, comprising the following steps: in the step (2), adding ammonia water to the acidic aqueous layer to adjust the pH value to 9.5;

[0037] Example 4:

[0038] The present invention provides a method for preparing spirobenzylisoquinoline alkaloid anisone C extracted from anise, wherein in the step (3), each gradient of the gradient elution uses 14.5 L of eluent, the flow rate is 12.5 mL / min, and each 900 ml volume is a fraction;

[0039] Example 5:

[0040] The present invention provides a method for preparing spirobenzylisoquinoline alkaloid anisone C extracted from anise, wherein in the step (4), each gradient of the gradient elution uses 7 L of eluent at a flow rate of 28 mL / h;

[0041] Example 6:

[0042] The present invention provides a method for preparing spirobenzylisoquinoline alkaloid anisone C extracted from anise, wherein in the step (5), the amount of each eluent used is 150 mL, and the flow rate is 12 ml / min;

[0043] The invention relates to an application of the isoquinoline alkaloids extracted from fennel prepared by the above method in the preparation of drugs for preventing and treating non-alcoholic fatty liver disease (NAFLD).

[0044] The medicine is a medicine containing anisone alkaloid C, or a pharmaceutically acceptable salt or halide for preventing and treating non-alcoholic fatty liver disease (NAFLD).

[0045] The medicine is a medicine in the form of tablets, capsules, injections, powder injections, granules, powders, pills, fat emulsions, microcapsules, dripping pills, ointments, sustained-release preparations or controlled-release preparations prepared from anisone alkaloid C and a pharmaceutically acceptable carrier.

[0046] According to the preparation method given in the above embodiment, any amount of compound can be prepared as needed. The examples given are only used to illustrate the specific embodiments of the present invention, and are not used to limit the scope of protection of the present invention. The technical core protected by the present invention is the compound, which is extracted from fennel to achieve its application in the preparation and treatment of non-alcoholic fatty liver disease (NAFLD).

[0047] The present invention, anisone alkaloid C, is prepared from anise, and can improve lipid metabolism disorders and lipid accumulation in HepG2 cells induced by free fatty acids (FFA), while also alleviating FFA-induced damage to HepG2 cells. Experimental results have yielded excellent technical results, as shown in the following (using Example 1 as an example):

[0048] 1. Instruments and reagents

[0049] Bruker AVANCEⅢ500 nuclear magnetic resonance spectrometer (TMS internal standard) (Bruker), Nicolet is10 Microscope Spectrometer (Thermo Scientific, USA) infrared spectrometer, Bruker maxis HDmass spectrometer high-resolution mass spectrometer, Shimadzu UV-2401PC apparatus ultraviolet spectrometer, Waters Alliance series 2695 high-performance liquid chromatography system equipped with 2998 diode array detector, Empower3 chromatography data workstation, LC50 high-pressure preparative liquid chromatograph, UV200 ultraviolet detector (Sepures (Beijing) Technology Co., Ltd.), Fisher Wharton Xbridge C 18 The chromatographic column was 250 × 20 mm, 5 μm. Other instruments included an N-1100 rotary evaporator (Shanghai Airang Instrument Co., Ltd.), an A-1000S water flow aspirator (Shanghai Airang Instrument Co., Ltd.), an N-1111 chilled water circulation device (Shanghai Airang Instrument Co., Ltd.), an FDU-2110 freeze dryer (Shanghai Airang Instrument Co., Ltd.), a DFZ-60508 vacuum drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.), and an AB204-N 1 / 10,000 precision analytical balance (METTLER TOLEDO).

[0050] Column chromatography packing materials were Diaion HP-20, MCI Gel CHP-20 (Mitsubishi Chemical Corporation, Japan), Toyopearl HW-40 (TOSOH Corporation, Japan), Sephadex LH-20 (Parmacia Biotech Corporation), silica gel H (100-200 mesh, 200-300 mesh) for column chromatography, thin layer chromatography silica gel (GF 254 ) was produced by Qingdao Ocean Chemical Plant. The chromatographic-grade reagents used were produced by Tianjin Siyou Fine Chemicals Co., Ltd., and the analytical-grade reagents used were produced by Beijing Chemical Plant and Tianjin Third Chemical Reagent Plant. The fennel was collected in Henan and identified as the whole herb of Hypecoum erectum L.

[0051] 2. Structural Identification

[0052] The anisone base C of the present invention is a light yellow solid, and the spectrum is shown in FIG. Figure 2-5 , the specific data are as follows:

[0053] HR-ESI-MS gave a quasi-molecular ion peak m / z 384.1431 [M+H] + (The calculated value is 384.1441), combined with its 13 C-NMR and1 H-NMR confirmed that its molecular formula is C 21 H 21 NO6, the calculated unsaturation is 12. 1 H-NMR (500MHz, CDCl3) spectrum showed four aromatic hydrogen proton signals including a group of 1,2,3,4-tetrasubstituted aromatic hydrogen protons: [δ H :6 . 70 (1H, d, J = 8.1 Hz, H-13) and 6.53 (1H, d, J = 8.1 Hz, H-14)], a group of 1,2,4,5-tetrasubstituted aromatic hydrogen protons: [δ H :6.88 (2H, s, H-1) and 6.69 (2H, s, H-4). In addition, there are three methylene hydrogen signals: [δ H :3.25 (1H, m, H-5a) / 3.08 (1H, ddd, J = 15.9, 9.4, 3.2 Hz, H-5b); 3.39 (1H, d, J = 13.9, 9.2 Hz, H-6a) / 3.20 (1H, m, H-6b); 5.17 (1H, d, J = 12.4 Hz, H-16a) / 5.13 (1H, d, J = 12.4 Hz, H-16b)], 1 methylenedioxy hydrogen signal: [δ H :6.00(1H,d,J=1.2Hz,11a-OCH2O-) / 5.96(1H,d,J=1.2Hz,11b-OCH2O-)], 1 N methyl hydrogen signal: [δ H :2.31(3H,s,7-NH3)], and two methoxy hydrogen signals: [δ H :3.79 (3H, s, 2-OCH3); 3.93 (3H, s, 3-OCH3)]. 13 C-NMR (125MHz, CDCl3), DEPT and HSQC spectra showed that the compound had 21 carbon atoms. In addition to the carbon atoms in the above structural units, the compound also had one carbonyl carbon and one quaternary carbon: (δ C :108.2).

[0054] In the HMBC spectrum, the hydrogen of the nitrogen methyl group correlates with C-6 and C-8, H-5 correlates with C-1a, C-4, C-4a, and C-6, and C-4 has a long-range correlation with C-8, indicating that the compound contains an isoquinoline group. Two methoxy groups are located at C-2 and C-3, respectively. H-16 correlates with C-8, C-10, C-11, C-12, C-13, C-14, and C-15, and the chemical shifts of C-16 and C-8 are shifted downfield, suggesting that C-8 and C-16 are connected through an oxygen atom. Furthermore, based on the molecular weight and degree of unsaturation of the compound, it is inferred that the 9-position contains a carbonyl group, which forms an oxygen-containing six-membered heterocyclic ring with C-8, C-16, C-15, and C-10, directly connected to the benzene ring through C-15 and C-10. Considering that the 8-position carbon is a quaternary carbon, the isoquinoline group is connected to the six-membered oxygen-containing heterocycle in the form of a spirocycle through the 8-position carbon. A Scifinder search revealed that compound 1 had no literature reports and was named anisone C. The molecular structure is:

[0055]

[0056] Table 1. 1 H NMR (500 MHz) and 13 C NMR(125MHz)data of compounds 1

[0057]

[0058]

[0059] 3. Activity Experiment

[0060] Effects of anisone C on a free fatty acid-induced nonalcoholic fatty liver cell model

[0061] 1. Experimental reagents and instruments

[0062] Human hepatocellular carcinoma HepG2 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences;

[0063] Fetal bovine serum (Sijiqing); DMEM high-glucose medium (Solybold); high-efficiency RIPA cell lysis buffer (Solybold); oleic acid (OA) and palmitic acid (PA) were purchased from Xi'an Kunchuang Biotechnology Development Co., Ltd.; saturated Oil Red O staining solution (Solybold), 4% paraformaldehyde (Seville), and isopropanol (Hengxing Reagent); CCK-8 cell viability assay reagent (Solybold); lovastatin (LOV) (Aladdin); aspartate aminotransferase (AST) and alanine aminotransferase (ALT) kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0064] Airtech clean bench (Suzhou Antai Air Technology Co., Ltd.); FORMA SERIES Ⅱ WATER JACKET CO2 cell culture incubator (Thermo Fisher Scientific, USA); inverted fluorescence microscope (Nikon, Japan); Multiskan GO full-wavelength microplate reader (Thermo Fisher Scientific, USA); Centrifuge 5424R high-speed refrigerated centrifuge (Eppendorf, Germany).

[0065] 2. Experimental Methods

[0066] 2.1 Cell culture

[0067] HepG2 cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Depending on the cell growth status, when the cell confluence reached 80%, the cells were harvested using 0.25% trypsin and passaged. Cells in the logarithmic growth phase were used for experiments.

[0068] 2.2 CCK-8 assay to detect the effects of compounds on HepG2 cell activity

[0069] HepG2 cells with good growth status and 90% confluence were taken and 5×10 4 Cells were seeded into 96-well plates at 400 μL / ml, with 100 μL of cell suspension per well. The cells were cultured in DMEM supplemented with 10% fetal bovine serum for 4 hours in a 37°C, 5% CO2 incubator. After cell attachment, the original culture medium was discarded and serum-free culture medium was added for 24 hours. After synchronization, the original culture medium was discarded and 100 μL of the prepared drug (10 μmol / L) was added. A control group containing no drug but culture medium and cells was also established for 24 hours. After 24 hours, the original culture medium was discarded and 100 μL of culture medium supplemented with 10% CCK-8 was added. A blank group (containing only culture medium with 10% CCK-8 but no cells) was established. After incubation for 1 hour in a 37°C, 5% CO2 incubator, the absorbance (A) at 450 nm was measured using a microplate reader. The experiment was repeated three times with six replicate wells per group. The relative cell viability of each group was calculated based on the A value.

[0070] Relative cell activity = (A drug - A blank) / (A control - A blank)

[0071] 2.3 Cell modeling and drug administration

[0072] HepG2 cells with good growth status and 80% to 90% confluence were cultured at a cell density of 5×10 51 ml of culture medium was evenly seeded into each well of a 6-well plate and incubated at 37°C, 5% CO₂ for 24 hours. After cell attachment, the original culture medium was discarded. A control group, a model group, a positive drug lovastatin (10 μmol / L) group, and anisoneline C (10 μmol / L) group were set up. 1 ml of basal medium was added to the blank and model groups, 1 ml of 10 μmol / L lovastatin was added to the positive drug group, and 1 ml of 10 μmol / L anisoneline C was added to the anisoneline C group. Simultaneously, 1 ml of 0.75 μmol / L free fatty acids (FFA: OA:PA = 2:1) was added to each well except the control group. The cells were incubated at 37°C, 5% CO₂ for 24 hours. Oil Red O staining was then performed to observe intracellular lipid droplet accumulation. Three replicate wells were set up for each group, and the experiment was repeated three times.

[0073] 2.4 Oil Red O staining to observe cell lipid accumulation

[0074] Cells were fixed with 4% paraformaldehyde for 30 minutes, differentiated with 60% isopropanol for 10 minutes, and stained with freshly prepared Oil Red O staining solution (saturated Oil Red O staining solution: deionized water = 3:2) for 30 minutes. Excess dye was washed off, and the cells were rinsed several times with distilled water before observation and photography under a microscope. Images were quantitatively analyzed using ImageJ.

[0075] 2.5 Determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels using biochemical reagents

[0076] The cell modeling and drug administration were the same as in 2.3. The cells of the control group, model group, positive drug group, and anisone alkaloid C group were collected and lysed with cell lysis buffer. The cells were centrifuged at low speed and the supernatant was collected to detect the protein content and the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT).

[0077] 2.6 Statistical analysis

[0078] Data were analyzed using GraphPad Prism 8 software, and all data conformed to a normal distribution. Data differences were analyzed using one-way analysis of variance (between multiple groups) and Dunnett's t-test (between two groups). P < 0.05 was considered statistically significant.

[0079] 3. Experimental Results

[0080] 3.1 Effects of compounds on HepG2 cell activity

[0081] The effects of the compounds on HepG2 cell activity were detected using a CCK-8 kit. The results showed that compound anisone C had no cytotoxicity.

[0082] 3.2 Oil Red O staining results

[0083] Oil red O staining results ( Figure 5 ) showed that compared with the blank group, the accumulation of red lipid droplets in the cells of the model group was significantly increased (P<0.001); compared with the model group, the accumulation of intracellular lipids in the positive drug and anisidine C groups was significantly reduced (P<0.001), which indicated that anisidine C could improve the lipid metabolism disorder and lipid accumulation in HepG2 cells induced by FFA.

[0084] 3.3 Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) results

[0085] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) test results ( Figure 6 ) showed that the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the model group were significantly increased compared with the control group (P < 0.05, P < 0.01), indicating that FFA induced hepatocyte damage. The levels of AST and ALT in the anisidine C group were significantly decreased compared with the model group (P < 0.05, P < 0.01). This suggests that anisidine C can significantly alleviate FFA-induced damage in HepG2 cells.

[0086] While conducting experiments on Example 1, the same experiments were also conducted on other examples, and the same or similar results were obtained, which will not be detailed here.

[0087] As can be seen from the above, the beneficial effects of the present invention are:

[0088] 1. The present invention explores new medical uses for aniseed, expands the application field of aniseed, and provides the use of anisone C in the preparation of drugs for intervening in non-alcoholic fatty liver disease.

[0089] 2. The raw material sources of anisone base C of the present invention are abundant, the preparation process is simple, and it can be made into tablets, capsules, powders, granules, injections, etc., which are easy to use.

[0090] 3. Through activity experimental studies, it was found that the product discovered, anisone alkaloid C, has a brand-new compound structure. At a concentration of 10 μmol / L, it can significantly improve FFA-induced lipid metabolism disorders and lipid accumulation in HepG2 cells, and reduce cell damage. It can prevent and treat non-alcoholic fatty liver disease by improving cellular lipid metabolism disorders and lipid accumulation and reducing liver cell damage.

[0091] Experiments have fully demonstrated that anisone C has potential activity in preventing and treating non-alcoholic fatty liver disease. Therefore, the present invention provides a new compound for the development of new drugs for preventing and treating non-alcoholic fatty liver disease, explores the medicinal value of anise, and opens up a new path for the preparation of drugs for treating and preventing non-alcoholic fatty liver disease (NAFLD). It has broad development and application prospects and significant economic and social benefits.

Claims

1. A spirobenzylisoquinoline alkaloid anisone C extracted from anise, having the chemical molecular structure: 。 2. The spirobenzylisoquinoline alkaloid anisone C extracted from aniseed according to claim 1, wherein the preparation method is: (1) 10 kg of dried fennel was crushed and extracted with 95% ethanol by volume for three times under condensation reflux at a temperature of 90-95°C. The extraction amount was 3-5 times the weight volume of the fennel each time, and the extraction time was 2 hours each time. The weight volume refers to the solid in kg and the liquid in L. The three extracts were filtered and combined. The extracts were concentrated under reduced pressure to obtain an ethanol extract. (2) Add 2% hydrochloric acid aqueous solution with a weight volume of ten times that of the ethanol extract to dissolve, filter to obtain the acid aqueous solution and insoluble matter; add petroleum ether to the acid aqueous solution and extract 5-8 times, 2.8-4 L each time, for 3-5 hours, recover the solvent to obtain a petroleum ether layer and an acid aqueous layer; add ammonia water to the acid aqueous layer to adjust the pH value to 9-10 to obtain an alkaline aqueous solution; extract the alkaline aqueous solution with chloroform and n-butanol in sequence 5-8 times, 2.8-4 L each time, for 3-5 hours, recover the solvent to obtain a chloroform fraction and an n-butanol fraction; (3) The chloroform fraction was mixed with an equal amount of alkaline silica gel and gradient eluted with a dichloromethane-methanol mixed solvent system with a volume ratio of 100:0, 100:2, 100:5, 100:10, 100:20, 100:50, 100:100, and 0:

100. Each gradient used 13-16 L of eluent at a flow rate of 10-15 mL / min. Each 850-1000 ml volume was a fraction. 130 fractions were collected and analyzed by silica gel thin layer chromatography. 254 Thin layer plates were developed using dichloromethane-methanol in a volume ratio of 10:1 and 5:1, respectively. Detection was performed at 254 nm using an ultraviolet lamp and a modified potassium bismuth iodide solution as a color developer. Based on the thin layer chromatography results, fractions 1-10, 11-27, 28-48, 49-72, 73-93, 94-108, 109-123, and 124-130 were combined to obtain components Fr.A, Fr.B…Fr.H. (4) Component Fr.B was passed through an ODS column and eluted with a gradient of methanol:water at a volume ratio of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:

0. Each gradient used 6-8 L of eluent at a flow rate of 20-35 mL / h. Each elution gradient was a fraction, and 10 subfractions Fr.B1…Fr.B7…Fr.B10 were collected. (5) The subcomponent Fr.B7 was mixed with an equal amount of 100-200 mesh alkaline silica gel and eluted with petroleum ether, petroleum ether-ethyl acetate mixture and pure ethyl acetate in sequence. The volume ratio of petroleum ether-ethyl acetate mixture was 8:1, 5:1, 2:1 and 1:

1. The amount of each eluent was 100-200 mL, the flow rate was 10-15 ml / min, and each 50 mL was a fraction. 13 fractions were collected and analyzed by silica gel thin layer chromatography. GF254 thin layer plates were used, and petroleum ether:ethyl acetate with a volume ratio of 2:1 was used as the developing solvent. The analysis was performed at 254 nm of an ultraviolet lamp. According to the thin layer detection results, fractions 1-4, 5-9 and 10-13 were combined to obtain three components Fr.B7-1, Fr.B7-2 and Fr.B7-3. (6) The component Fr.B7-1 was purified by preparative high performance liquid chromatography using a mixed solvent system of methanol and water with a volume ratio of 65:35 and a column of Fisher Wharton Xbridge C 18 The chromatographic column was used with a flow rate of 3 mL / min, and the chromatographic peak with a retention time of 13.8 min was collected. The solvent was recovered to obtain anisone base C.

3. Use of the spirobenzylisoquinoline alkaloid anisone C according to claim 1 in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease.

4. The use of the spirobenzylisoquinoline alkaloid anisone C according to claim 3 in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease, characterized in that: The medicine is a medicine for preventing and treating non-alcoholic fatty liver disease in the form of a pharmaceutically acceptable salt.

5. The use of the spirobenzylisoquinoline alkaloid anisone C according to claim 3 in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease, characterized in that: The medicine is a medicine in the form of tablets, capsules, injections, powder injections, granules, powders, pills, fat emulsions, microcapsules, dripping pills, ointments, sustained-release preparations or controlled-release preparations prepared from anisone alkaloid C and a pharmaceutically acceptable carrier.

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