Novel spirobenzylisoquinoline alkaloid hypecoum erectum ketobase C extracted from hypecoum erectum as well as preparation method and application thereof

Abstract

The invention relates to a novel spirobenzylisoquinoline alkaloid hypecoum erectum ketone base C extracted from hypecoum erectum as well as a preparation method and application of the novel spirobenzylisoquinoline alkaloid hypecoum erectum ketone base C, which can be used for effectively extracting and preparing the novel spirobenzylisoquinoline alkaloid hypecoum erectum ketone base C from the hypecoum erectum and realizing the application in preparing a medicine for preventing and treating a non-alcoholic fatty liver disease. Carrying out heating reflux extraction on the dried hypecoum erectum by using ethanol, carrying out acid extraction and alkali precipitation, and sequentially extracting by using chloroform and n-butyl alcohol to obtain an extract of a corresponding part; and mixing the target extract with silica gel, eluting, concentrating under reduced pressure, detecting, combining the same fractions, eluting on a column, mixing the silica gel with the sample, eluting, collecting the fractions with retention time, concentrating and drying to obtain the anise keto alkali C. The invention also discloses a preparation method of the anise keto alkali C. The invention also discloses a preparation method of the anise keto alkali C. The compound can significantly improve free fatty acid-induced HepG2 cell lipid metabolism disorder and lipid accumulation, meanwhile alleviate FFA-induced HepG2 cell damage, can be prepared into various drug dosage forms, is convenient and effective in clinical use, has wide development and application prospects, and has significant economic and social benefits.

Description

Technical Field

[0001] The invention relates to the field of medicine, in particular to a novel 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 liver cells caused by excessive energy intake, obesity, lipid metabolism disorders, etc. It is also the main inducing factor for liver fibrosis, cirrhosis and liver cancer. With the influence of multiple factors such as changes in people's diet and lifestyle, the incidence of NAFLD has increased sharply around the world. However, there is no drug officially approved by the FDA for the treatment of NAFLD worldwide. Therefore, it is of great significance to find new drugs for the treatment of NAFLD.

[0003] Hypecoum erectum L. is the whole herb of Hypecoum erectum L., a plant of the genus Hypecoum in the family Papaveraceae. It is also known as throat grass, wheat yellow grass, yellow flower grass, etc. It is widely distributed in Northeast my country, North China and Northwest China. It is mainly used to treat sore throat, hepatitis, bacterial dysentery and red and swollen eyes. It is a commonly used Chinese herbal medicine for the treatment of sore throat and icteric hepatitis. Chemical composition studies have shown that Hypecoum erectum contains a large number of alkaloid compounds, among which isoquinoline alkaloids are the most common. At present, literature reports have confirmed that total alkaloids of fennel have significant protective effects on acute liver injury induced by carbon tetrachloride in rats and acute liver injury induced by thioacetamide in mice [Ke Jun, Zhang Guiqing, Weng Shiai, et al., Study on the effect of throat grass on liver, Chinese Pharmacology and Clinical Medicine, 1985, (1): 185-186; Guo Jie, Zhang Xide, Huang Wei, Screening of effective parts of fennel, Journal of Shaanxi College of Traditional Chinese Medicine, 2006, 29 (2): 58-60], suggesting that total alkaloids of fennel have potential application value in the treatment of acute liver injury. However, there are no literature reports on its use in the treatment of NAFLD, and there are no public reports 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 novel spirobenzylisoquinoline alkaloid anisone C extracted from fennel and a preparation method and application thereof, which can effectively solve the problem of extracting and preparing the novel spirobenzylisoquinoline alkaloid anisone C from fennel and realize its application in the preparation of drugs for the prevention and treatment of non-alcoholic fatty liver disease (NAFLD).

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

[0006]

[0007] The preparation method is:

[0008] The dried fennel is extracted by heating and refluxing with ethanol 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 is mixed with an equal amount of alkaline silica gel, and the sample is eluted with a gradient ratio of dichloromethane-methanol, each fraction is concentrated under reduced pressure, and a thin layer spot plate is used for identification, and the same fractions are combined to obtain multiple first group components, and a target first component is selected from the multiple first components;

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

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

[0012] The target third group 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 a novel spirobenzylisoquinoline alkaloid anisone C extracted from anise prepared by the above method in preparing a drug for preventing and treating non-alcoholic fatty liver disease (NAFLD).

[0014] The novel spirobenzylisoquinoline alkaloid anisone C extracted from aniseed by the method of the present invention can significantly improve lipid metabolism disorder and lipid accumulation of HepG2 cells induced by free fatty acids (FFA), and can also reduce HepG2 cell damage induced by FFA, thereby providing a new chemical structure type for the development of novel NAFLD therapeutic drugs, and can be conveniently prepared into a variety of pharmaceutical dosage forms, which are convenient and effective for clinical use, have broad development and application prospects, and have 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 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 nuclear magnetic resonance spectrum of anisone alkaloid C of the present invention;

[0019] Figure 5 This is a result diagram of the improvement of lipid accumulation of HepG2 cells induced by free fatty acid (FFA) by anisone alkaloid C of the present invention, wherein Oil Red O staining; 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, ***P<0.001;

[0020] Figure 6 This is a graph showing the results of anisone C of the present invention improving free fatty acid (FFA)-induced HepG2 cell damage, wherein, compared with the control group, ## is P < 0.01, # is P < 0.05; compared with the model group, **P < 0.01, *P < 0.05. DETAILED DESCRIPTION

[0021] The specific implementation modes of the present invention are described in detail below in combination with embodiments and specific situations.

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

[0023] Embodiment 1:

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

[0025]

[0026] The preparation method is:

[0027] (1) 10 kg of dried fennel was crushed and extracted with 95% ethanol by volume for 3 times by condensation reflux. The extraction temperature was 90-95° C. and the amount used each time was 3-5 times the weight volume of fennel. The extraction time was 2 hours. 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 an acid aqueous solution and insoluble matter; adding petroleum ether to the acid aqueous solution for extraction 5-8 times, 2.8-4 L each time, 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 turn for 5-8 times, 2.8-4 L each time, 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, respectively. 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 each fraction was analyzed by silica gel thin layer chromatography and analyzed by GF 254 Thin layer plate, using dichloromethane-methanol with a volume ratio of 10:1 and 5:1 as developing solvent, respectively, under ultraviolet lamp 254nm detection analysis, using improved potassium bismuth iodide solution as a color developer, according to the thin layer chromatography detection results, respectively merge fractions 1-10, fractions 11-27, fractions 28-48, fractions 49-72, fractions 73-93, fractions 94-108, fractions 109-123, and fractions 124-130 to obtain components Fr.A, Fr.B...Fr.H;

[0030] (4) Component Fr.B was passed through an ODS column and eluted with methanol:water in a gradient 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 and the flow rate was 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 turn. 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 plate was used, and petroleum ether:ethyl acetate with a volume ratio of 2:1 was used as the developing solvent. The analysis was detected at 254 nm of ultraviolet lamp. According to the thin layer detection results, fractions 1-4, fractions 5-9 and fractions 10-13 were respectively 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 methanol:water mixed solvent system with a volume ratio of 65:35 and a Fisher Wharton Xbridge C column. 18 The 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 (about 9.7 mg).

[0033] Embodiment 2:

[0034] The present invention provides a method for preparing a novel spirobenzylisoquinoline alkaloid anisone C extracted from anise, wherein 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] Embodiment 3:

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

[0037] Embodiment 4:

[0038] The present invention provides a method for preparing a novel 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] Embodiment 5:

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

[0041] Embodiment 6:

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

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

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

[0045] The medicine is a tablet, capsule, injection, powder injection, granule, powder, pill, fat emulsion, microcapsule, dripping pill, ointment, sustained-release agent or controlled-release dosage form 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 given embodiment is only used to illustrate the specific implementation mode of the present invention, rather than to limit the protection scope of the present invention. The technical core protected by the present invention is the compound, which is extracted from fennel to achieve the application in the preparation of treatment of non-alcoholic fatty liver disease (NAFLD).

[0047] The anisone alkaloid C of the present invention is prepared from anise, and can improve lipid metabolism disorder and lipid accumulation of HepG2 cells induced by free fatty acids (FFA), and can also reduce HepG2 cell damage induced by FFA. The experiment has achieved very good beneficial technical effects, and the relevant data are as follows (taking Example 1 as an example):

[0048] 1. Instruments and test drugs

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

[0050] Column chromatography fillers: 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 reagents used were produced by Tianjin Siyou Fine Chemicals Co., Ltd., and the analytical reagents used were produced by Beijing Chemical Plant and Tianjin Third Chemical Reagent Factory. Fennel was collected in Henan and identified as the whole herb of Fennel (Hypecoum erectum L.).

[0051] 2. Structural Identification

[0052] The anisone alkaloid C of the present invention is a light yellow solid, and the spectrum is shown in 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 shows 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 3 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 2 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 show that the compound has 21 carbon atoms. In addition to the carbon atoms in the above structural units, the compound also has a carbonyl carbon and a quaternary carbon: (δ C :108.2).

[0054] In the HMBC spectrum, the hydrogen on the nitrogen methyl group is correlated with C-6 and C-8, H-5 is correlated with C-1a, C-4, C-4a and C-6, and C-4 is remotely correlated with C-8, indicating that the compound contains an isoquinoline group. The two methoxy groups are located at C-2 and C-3, respectively. H-16 is correlated 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 both shifted to the low field, which is speculated that C-8 is connected to C-16 through an oxygen atom. Based on the molecular weight and unsaturation of the compound, it is speculated that the 9th position contains a carbonyl group, and forms an oxygen-containing six-membered heterocyclic ring with C-8, C-16, C-15 and C-10, which is directly connected to the benzene ring through C-15 and C-10. Considering that the carbon at position 8 is a quaternary carbon, the isoquinoline group is connected to the six-membered oxygen-containing heterocyclic ring in the form of a spirocycle through the carbon at position 8. Compound 1 was searched by Scifinder, and the structure was not reported in the literature. It was named anisone C, and the molecular structure formula 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 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 (Soleb); high-efficiency RIPA cell lysis buffer (Soleb); oleic acid (OA) and palmitic acid (PA) were purchased from Xi'an Kunchuang Biotechnology Development Co., Ltd.; saturated oil red O staining solution (Soleb), 4% paraformaldehyde (Seville), isopropanol (Hengxing Reagent); CCK-8 cell viability detection reagent (Soleb); 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 a DMEM high-glucose medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%, the cells were digested with 0.25% trypsin and collected for passage, and cells in the logarithmic growth phase were used for the experiment.

[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 collected and cultured at 5×10 4 / ml was inoculated on a 96-well plate, with 100μL of cell suspension per well, and cultured for 4h in a 37℃, 5% CO2 incubator with DMEM culture medium containing 10% fetal bovine serum. After the cells adhered to the wall, the original culture medium was discarded, and serum-free culture medium was added for 24h. After synchronization, the original culture medium was discarded, and 100μL (10μmol / L) of the prepared drug was added. At the same time, a control group containing no drug, culture medium and cells was set up for intervention for 24h. After 24h, the original culture medium was discarded, and 100μL of culture medium containing 10% CCK-8 was added. A blank group (no cells, only culture medium containing 10% CCK-8) was set up. After incubation for 1h in a 37℃, 5% CO2 incubator, the absorbance (A) value at 450nm was detected with an enzyme marker. Six duplicate wells were set up for each group of the experiment, and repeated 3 times. The relative activity of cells in 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 each well was evenly inoculated in a 6-well plate and cultured at 37°C, 5% CO2 for 24 hours. After the cells adhered to the wall, the original culture medium was discarded, and the control group, model group, positive drug lovastatin (10 μmol / L) group and anisone alkaloid C (10 μmol / L) group were set up. 1 ml of basal culture medium was added to the blank group and the model group, 1 ml of lovastatin with a concentration of 10 μmol / L was added to the positive drug group, and 1 ml of anisone alkaloid C with a concentration of 10 μmol / L was added to the anisone alkaloid C group. At the same time, 1 ml of free fatty acids with a concentration of 0.75 μmol / L (FFA is OA:PA=2:1) was added to each well except the control group, and cultured at 37°C, 5% CO2 for 24 hours, and then oil red O staining was performed to observe the accumulation of intracellular lipid droplets. Three replicate wells were set up for each group of the experiment, and repeated 3 times.

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

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

[0075] 2.5 Biochemical reagents for determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) content

[0076] The cell modeling and drug administration were the same as in 2.3. Then the cells of the control group, model group, positive drug and anisone alkaloid group C were collected and lysed with cell lysis buffer, 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] GraphPad Prism 8 software was used for data analysis, and all data were in accordance with normal distribution. One-way analysis of variance (between multiple groups) and Dunnett's t test (between two groups) were used to analyze the data. P < 0.05 was considered statistically significant.

[0079] 3. Experimental results

[0080] 3.1 Effects of compounds on HepG2 cell activity

[0081] The CCK-8 kit was used to detect the effect of the compound on the activity of HepG2 cells. 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 model group cells 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 indicates that anisidine C can improve FFA-induced lipid metabolism disorders and lipid accumulation in HepG2 cells.

[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 higher than those in the control group (P < 0.05, P < 0.01), indicating that FFA induced liver cell damage. The levels of AST and ALT in the anisone C group were significantly lower than those in the model group (P < 0.05, P < 0.01). This indicates that anisone C can significantly reduce FFA-induced HepG2 cell damage.

[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 repeated here.

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

[0088] 1. The present invention explores new medical uses for anise, expands the application field of anise, 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 alkaloid 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 is convenient to use.

[0090] 3. Through active experimental studies, it was found that the product discovered, anisone C, has a brand-new compound structure. At a concentration of 10 μmol / L, it can significantly improve FFA-induced HepG2 cell lipid metabolism disorders and lipid accumulation, and reduce cell damage. It can prevent and treat non-alcoholic fatty liver disease by improving cell 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, expands the medicinal value of aniseed, and opens up a new way for the preparation of drugs for the treatment and prevention of non-alcoholic fatty liver disease (NAFLD). It has broad development and application prospects and significant economic and social benefits.

Claims

1. A novel spirobenzylisoquinoline alkaloid anisone C extracted from anise, the chemical molecular structure of which is:

2. The novel spirobenzylisoquinoline alkaloid anisone C extracted from anise according to claim 1, wherein the preparation method thereof is: (1) 10 kg of dried fennel was crushed, and 95% ethanol was used for condensation reflux extraction three times at a temperature of 90-95° C., and the amount used each time was 3-5 times the weight volume of fennel, and each time was 2 hours, where the weight volume refers to the solid in kg and the liquid in L, and the three extracts were combined; the extracts were concentrated under reduced pressure to obtain an ethanol extract; (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 an acid aqueous solution and insoluble matter; adding petroleum ether to the acid aqueous solution for extraction 5-8 times, 2.8-4 L each time, 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 turn 5-8 times, 2.8-4 L each time, for 3-5 hours, recovering 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, respectively. 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 each fraction was analyzed by silica gel thin layer chromatography and analyzed by GF 254 Thin layer plate, using dichloromethane-methanol in a volume ratio of 10:1 and 5:1 as developing solvent, respectively, under ultraviolet lamp 254nm detection analysis, using improved potassium bismuth iodide solution as a color developer, according to the thin layer chromatography detection results, respectively merge the flow fractions 1-10, flow fractions 11-27, flow fractions 28-48, flow fractions 49-72, flow fractions 73-93, flow fractions 94-108, flow fractions 109-123, flow fractions 124-130 to obtain components Fr.A, Fr.B...Fr.H; (4) Component Fr.B was passed through an ODS column and eluted with methanol:water in a gradient 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 and the flow rate was 20-35 mL / h. Each elution gradient was a fraction, and 10 subcomponents 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 turn. 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 plate was used, and petroleum ether:ethyl acetate with a volume ratio of 2:1 was used as the developing solvent. The analysis was detected at 254 nm of ultraviolet lamp. According to the thin layer detection results, fractions 1-4, fractions 5-9 and fractions 10-13 were combined to obtain three components Fr.B7-1, Fr.B7-2 and Fr.B7-3. (6) Component Fr.B7-1 was purified by preparative high performance liquid chromatography using a methanol:water mixed solvent system with a volume ratio of 65:35 and a Fisher Wharton Xbridge C column. 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 novel spirobenzylisoquinoline alkaloid anisone C according to claim 1 in the preparation of drugs for preventing and treating non-alcoholic fatty liver disease.

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

5. The use of the novel spirobenzylisoquinoline alkaloid anisone C in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease according to claim 3, characterized in that: The medicine is a tablet, capsule, injection, powder injection, granule, powder, pill, fat emulsion, microcapsule, dripping pill, ointment, sustained-release agent or controlled-release dosage form prepared from anisone alkaloid C and a pharmaceutically acceptable carrier.

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