Hypecoum erectum composition for treating non-alcoholic fatty liver disease and preparation method of hypecoum erectum composition

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a hypecoum erectum composition for treating a non-alcoholic fatty liver disease and a preparation method of the hypecoum erectum composition. The hypecoum erectum composition is prepared from the following components: hypecoum erectum ketone and juglantin, wherein the mass ratio of the hypecoum erectum ketone to the juglantin is 1: (0.3-0.5). Experimental results show that the hypecoum erectum composition can improve liver function and liver lesion degree by reducing blood fat level, liver lipid accumulation and slowing down liver inflammation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, and particularly relates to an anise composition for treating non-alcoholic fatty liver disease and its preparation method. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease characterized by hepatic steatosis and closely associated with metabolic syndrome. Its disease spectrum is broad, progressing from simple fatty liver to nonalcoholic steatohepatitis (NASH), and further developing into cirrhosis and even hepatocellular carcinoma. Currently, NAFLD treatment is based on lifestyle interventions, with only a few targeted drugs included in clinical guidelines, resulting in limited overall treatment options. Therefore, developing safe and effective drugs is of great significance.

[0003] Anise is an annual herb belonging to the genus Anise in the Papaveraceae family. It is cool in nature, bitter and pungent in taste, and enters the lung, large intestine, and liver meridians. It has the effects of clearing heat and detoxifying, relieving cough and pain. The *Henan Traditional Chinese Medicine Handbook* records that it can "clear heat, reduce inflammation, and relieve pain," and is commonly used in traditional medicine to treat colds with fever, cough, sore throat, liver heat with red eyes, hepatitis, cholecystitis, dysentery, joint pain, etc. Patent CN111012847A discloses a Mongolian medicine compound for treating non-alcoholic fatty liver disease (NAFLD). This formula consists of various medicinal materials such as anise, violet, safflower, Mongolian astragalus, cassia seed, gardenia, gentian, sophora flavescens, licorice, and artificial bezoar. Through the synergistic effect of its components, it shows effects in lowering blood lipids, improving liver function damage and insulin resistance, and has a certain antioxidant capacity, which can reduce oxidative stress and lipid peroxidation, thereby playing a role in treating NAFLD.

[0004] However, current research on anise in the treatment of NAFLD is still in its early stages and requires further in-depth exploration. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the first objective of the present invention is to provide an anise composition for treating non-alcoholic fatty liver disease, which can improve liver function and the degree of liver lesions by reducing blood lipid levels, liver lipid accumulation and alleviating liver inflammation.

[0006] The second objective of this invention is to provide a simple method for preparing an anise composition for treating non-alcoholic fatty liver disease.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A senna composition for treating non-alcoholic fatty liver disease, comprising the following components: senna ketone and juglone, wherein the mass ratio of senna ketone to juglone is 1:(0.3-0.5). The structural formula of the anethole ketone is: .

[0008] Furthermore, the preparation method of the anethole includes the following steps: (1) Add fennel powder to an 80-85% ethanol solution for ultrasonic extraction, filter to obtain ultrasonic extract and filter cake; reflux extract the filter cake with a 90-95% ethanol solution 3-4 times to obtain reflux extract; combine the ultrasonic extract and the reflux extract, adjust the pH with hydrochloric acid solution and concentrate to a thick paste, dry and sieve to obtain fennel extract; (2) Take the anise extract, adjust the pH with ammonia, and then extract with petroleum ether 3-4 times to obtain a petroleum ether layer; extract the petroleum ether layer with a mixed solvent 5-6 times, combine the extract phases and remove the solvent to obtain a mixed solvent layer; (3) Mix the mixed solvent layer with silica gel and then perform a first gradient elution to obtain components Fr.1-Fr.4. Take component Fr.2 for later use. (4) Mix the component Fr.2 with silica gel and then perform a second gradient elution to obtain components Fr.2.1-Fr.2.8. Take component Fr.2.2 for later use; (5) After concentrating the component Fr.2.2, perform a third gradient elution, take the fraction Fr.2.2.4 at gradient 80:20, freeze dry to obtain the fenestrin.

[0009] Furthermore, in step (1), during ultrasonic extraction, the ratio of anise powder to ethanol solution is 10 kg: (4-6) L; the amount of ethanol aqueous solution used in each reflux extraction is 1-2 L; and the concentration of hydrochloric acid solution is 2-3 mol / L.

[0010] Furthermore, in step (1), the ultrasonic extraction time is 40-60 min; the pH is adjusted to 3-4; the concentration temperature is 75-85℃; the relative density of the thick paste is 1.1-1.5; and the sieve mesh size is 60 mesh.

[0011] Furthermore, in step (2), the pH is adjusted to 9-10; the mixed solvent is composed of dichloromethane, n-butanol and methanol in a volume ratio of 8.5:1:0.5.

[0012] Furthermore, in step (2), the amount of petroleum ether added each time during the petroleum ether extraction process is 1-2L; and in the mixed solvent extraction process, the amount of mixed solvent added each time is 2-3L.

[0013] Further, in step (3), the mass ratio of the mixed solvent layer to the silica gel is 1:(1.5-2); the first gradient elution is carried out sequentially with a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 90:10, 70:30, 50:50, and 0:100, and the elution time for each gradient is 8-10 h, and the flow rate is 15-20 mL / min.

[0014] Further, in step (4), the mass ratio of component Fr.2 to silica gel is 1:(1.5-2); the second gradient elution is performed sequentially with a dichloromethane-n-butanol mixed solvent in volume ratios of 100:0, 95:5, 90:10, 80:20, 70:30, 60:40, 50:50, and 0:100, with each gradient elution lasting 10-12 hours and a flow rate of 8-12 mL / min.

[0015] Furthermore, the third gradient elution in step (5) is performed sequentially using a water-acetonitrile mixed solvent with volume ratios of 95:5, 90:10, 85:15, 80:20, 70:30, 60:40, and 50:50. Each gradient elution lasts 8-10 minutes, with a flow rate of 3-4 mL / min. The chromatographic column is a Fisher Wharton Xbridge C10 ... 18 Chromatographic column.

[0016] The preparation method of the above-mentioned anise composition for treating non-alcoholic fatty liver disease includes the following steps: Mix the chalcone and juglone evenly according to the mass ratio.

[0017] Compared with the prior art, the main advantages of the present invention are as follows: This invention provides a fennel composition comprising anisole and juglone. Experimental results show that the synergistic effect of anisole and juglone can improve liver function and the degree of liver lesions by reducing blood lipid levels, liver lipid accumulation, and alleviating liver inflammation. Attached Figure Description

[0018] Figure 1 The image shows the thin-layer chromatography results of components Fr.2.1-Fr.2.8 obtained in step (4) of Example 1; Figure 2 The 1H NMR spectrum of anethole obtained in Example 1; Figure 3 This is the carbon spectrum of anethole obtained in Example 1; Figure 4 This is the mass spectrum of the anethole obtained in Example 1; Figure 5 This is a graph showing the cytotoxicity results of the acetophenone obtained in Example 1 of this invention. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0020] Example 1 A type of anethole, the preparation method includes the following steps: (1) Take fennel seeds, dry them in an oven at 60°C for 14 hours, pulverize them and pass them through a 60-mesh sieve to obtain fennel powder; according to the ratio of fennel powder to ethanol solution of 2 kg: 1 L, add fennel powder to an 82% ethanol solution and extract it with ultrasonic power of 140 W for 50 min. After filtration, obtain ultrasonic extract and filter cake; reflux extract the filter cake with a 93% ethanol solution (3 × 1 L) to obtain reflux extract; combine the ultrasonic extract and the reflux extract, adjust the pH to 3 with a 2.5 mol / L hydrochloric acid solution, concentrate it at 80°C to a thick paste with a relative density of 1.4, dry it at 60°C, pulverize it into fine powder and pass it through a 60-mesh sieve to obtain fennel extract; (2) Take the anise extract, adjust the pH to 9.5 with ammonia, and then extract with petroleum ether (3×1L). Combine the extract phases and remove the solvent to obtain the petroleum ether layer; use a mixed solvent (V) to extract the petroleum ether layer. 二氯甲烷 V 正丁醇 V 甲醇 Extraction was performed using a ratio of 8.5:1.0:0.5 (5×2L). The extracted phases were combined, and the solvent was removed to obtain the mixed solvent layer. (3) Add 1.5 times the amount of silica gel to the mixed solvent layer and mix. Then, perform gradient elution with a petroleum ether-ethyl acetate mixed solvent system with a volume ratio of 90:10, 70:30, 50:50 and 0:100. The elution time for each gradient is 9 h and the flow rate is 17 mL / min. After removing the solvent, obtain components Fr.1-Fr.4. Take component Fr.2 for later use. (4) Mix the component Fr.2 with 1.5 times the amount of silica gel, and then perform gradient elution with a dichloromethane-n-butanol mixed solvent with volume ratios of 100:0, 95:5, 90:10, 80:20, 70:30, 60:40, 50:50 and 0:100. The elution time for each gradient is 11 h and the flow rate is 10 mL / min. After removing the solvent, components Fr.2.1-Fr.2.8 are obtained. Take component Fr.2.2. (5) The component Fr.2.2 was concentrated and purified by high performance liquid chromatography. Gradient elution was performed using water-acetonitrile mixed solvents with volume ratios of 95:5, 90:10, 85:15, 80:20, 70:30, 60:40, and 50:50. The elution time for each gradient was 9 min, the flow rate was 3 mL / min, and the chromatographic column was Fisher Wharton Xbridge C10. 18 The chromatographic column was used, and the fraction Fr.2.2.4 with a gradient of 80:20 was lyophilized to obtain the anethole ketone.

[0021] Example 2 A type of anethole, the preparation method includes the following steps: (1) Take fennel seeds, dry them in an oven at 60°C for 14 hours, pulverize them and pass them through a 60-mesh sieve to obtain fennel powder; according to the ratio of fennel powder to ethanol solution of 5 kg: 2 L, add fennel powder to an 80% ethanol solution and extract it by ultrasonic extraction at 140 W ultrasonic power for 40 min. After filtration, obtain ultrasonic extract and filter cake; reflux extract the filter cake with a 90% ethanol solution (4 × 2 L) to obtain reflux extract; combine the ultrasonic extract and the reflux extract, adjust the pH to 4 with a 3 mol / L hydrochloric acid solution, concentrate it at 75°C to a thick paste with a relative density of 1.1, dry it at 60°C, pulverize it into fine powder and pass it through a 60-mesh sieve to obtain fennel extract; (2) Take the anise extract, adjust the pH to 9 with ammonia, and then extract with petroleum ether (4×2L). Combine the extract phases and remove the solvent to obtain the petroleum ether layer; use a mixed solvent (V 二氯甲烷 V 正丁醇 V 甲醇 Extraction was performed using a ratio of 8.5:1.0:0.5 (6×3L). The extracted phases were combined, and the solvent was removed to obtain the mixed solvent layer. (3) Add twice the amount of silica gel to the mixed solvent layer and mix. Then, perform gradient elution with petroleum ether-ethyl acetate mixed solvent with volume ratios of 90:10, 70:30, 50:50 and 0:100. The elution time for each gradient is 8 h and the flow rate is 15 mL / min. After removing the solvent, obtain components Fr.1-Fr.4. Take component Fr.2 for later use. (4) Mix the component Fr.2 with twice the amount of silica gel, and then perform gradient elution with a dichloromethane-n-butanol mixed solvent with volume ratios of 100:0, 95:5, 90:10, 80:20, 70:30, 60:40, 50:50, and 0:100. The elution time for each gradient is 10 h, and the flow rate is 8 mL / min. After removing the solvent, components Fr.2.1-Fr.2.8 are obtained. Take component Fr.2.2. (5) The component Fr.2.2 was concentrated and purified by high performance liquid chromatography. Gradient elution was performed using water-acetonitrile mixed solvents with volume ratios of 95:5, 90:10, 85:15, 80:20, 70:30, 60:40, and 50:50. The elution time for each gradient was 8 min, the flow rate was 3 mL / min, and the chromatographic column was Fisher Wharton Xbridge C10. 18 The chromatographic column was used, and the fraction Fr.2.2.4 with a gradient of 80:20 was lyophilized to obtain the anethole ketone.

[0022] Example 3 A type of anethole, the preparation method includes the following steps: (1) Take fennel seeds, dry them in an oven at 60°C for 14 hours, pulverize them and pass them through a 60-mesh sieve to obtain fennel powder; according to the ratio of fennel powder to ethanol solution of 5 kg: 4 L, add fennel powder to an 85% ethanol solution and extract it by ultrasonic extraction at 140 W ultrasonic power for 60 min. After filtration, obtain ultrasonic extract and filter cake; reflux extract the filter cake with a 95% ethanol solution (3 × 2 L) to obtain reflux extract; combine the ultrasonic extract and the reflux extract, adjust the pH to 4 with a 3 mol / L hydrochloric acid solution, concentrate it at 85°C to a thick paste with a relative density of 1.5, dry it at 60°C, pulverize it into fine powder and pass it through a 60-mesh sieve to obtain fennel extract; (2) Take the anise extract, adjust the pH to 10 with ammonia, and then extract with petroleum ether (3×2L). Combine the extract phases and remove the solvent to obtain the petroleum ether layer; use a mixed solvent (V) to extract the petroleum ether layer. 二氯甲烷 V 正丁醇 V 甲醇 Extraction was performed using a ratio of 8.5:1.0:0.5 (6×3L). The extracted phases were combined, and the solvent was removed to obtain the mixed solvent layer. (3) Add 1.5 times the amount of silica gel to the mixed solvent layer and mix. Then, perform gradient elution with petroleum ether-ethyl acetate mixed solvent with volume ratios of 90:10, 70:30, 50:50 and 0:100. The elution time for each gradient is 10 h and the flow rate is 20 mL / min. After removing the solvent, obtain components Fr.1-Fr.4. Take component Fr.2 for later use. (4) Mix the component Fr.2 with 1.5 times the amount of silica gel, and then perform gradient elution with dichloromethane-n-butanol mixed solvents with volume ratios of 100:0, 95:5, 90:10, 80:20, 70:30, 60:40, 50:50 and 0:100. The elution time for each gradient is 12 h and the flow rate is 12 mL / min. After removing the solvent, components Fr.2.1-Fr.2.8 are obtained. Take component Fr.2.2. (5) The component Fr.2.2 was concentrated and purified by high performance liquid chromatography. Gradient elution was performed using water-acetonitrile mixed solvents with volume ratios of 95:5, 90:10, 85:15, 80:20, 70:30, 60:40, and 50:50. The elution time for each gradient was 10 min, the flow rate was 4 mL / min, and the chromatographic column was Fisher Wharton Xbridge C10. 18 The chromatographic column was used, and the fraction Fr.2.2.4 with a gradient of 80:20 was lyophilized to obtain the anethole ketone.

[0023] Example 4 An anise composition for treating non-alcoholic fatty liver disease, comprising aniseone and juglone in a mass ratio of 1:0.4.

[0024] The preparation method of the above-mentioned anise composition for treating non-alcoholic fatty liver disease includes the following steps: Mix anisole and juglone evenly according to the stated mass ratio.

[0025] Example 5 A fennel composition for treating non-alcoholic fatty liver disease, comprising fennelone and juglone in a mass ratio of 1:0.3.

[0026] The preparation method of the above-mentioned anise composition for treating non-alcoholic fatty liver disease includes the following steps: Mix anisole and juglone evenly according to the stated mass ratio.

[0027] Example 6 A fennel composition for treating non-alcoholic fatty liver disease, comprising fennelone and juglone in a mass ratio of 1:0.5.

[0028] The preparation method of the above-mentioned anise composition for treating non-alcoholic fatty liver disease includes the following steps: Mix anisole and juglone evenly according to the stated mass ratio.

[0029] Comparative Example 1 The difference between Comparative Example 1 and Example 4 is that walnutin is omitted.

[0030] Experimental Example 1 Sixty 8-week-old male C57BL / 6J mice were randomly divided into six groups: a control group, a model group, and groups Fr.1-Fr.4. The control group was fed a standard diet, the model group was fed a CDAHFD high-fat diet, and the other groups were fed a CDAHFD high-fat diet. Simultaneously, groups Fr.1-Fr.4 were administered the drug via gavage at a dose of 10 g / kg for 10 consecutive weeks. After the experiment, blood was collected from the orbital sinus of each group, and serum was obtained. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were detected using appropriate kits. The results are shown in Table 1.

[0031] Table 1 ; As shown in Table 1, compared with the control group, the serum AST and ALT levels of mice in the model group were significantly increased after 10 weeks of continuous high-fat diet. Compared with groups Fr.1, Fr.3, and Fr.4, group Fr.2 significantly reduced the AST and ALT levels in mice. Therefore, Fr.2 was selected for subsequent purification and separation.

[0032] Experimental Example 2 Thin-layer chromatography (TLC) was performed on the components Fr.2.1-Fr.2.8 obtained in step (4) of Example 1, with V as the developing solvent. 二氯甲烷 V 甲醇 =10:1, examined under a 254nm UV lamp, results are shown below. Figure 1 .

[0033] Figure 1 The TLC plates from left to right represent the TLC detection results of components Fr.2.1-Fr.2.8 obtained in step (4) of Example 1. Observation Figure 1 It was observed that Fr.2.2 presented a single, distinct UV absorption spot; the spot Fr.2.1 was located at the chromatographic front and could not be effectively separated under the given development conditions; spots were detected in Fr.2.3 through Fr.2.8, but they exhibited high polarity, with the Fr.2.8 spot concentrated at the origin and showing significant tailing. Therefore, based on the TLC results, fraction Fr.2.2 was selected for further separation and purification.

[0034] Experimental Example 3 The structure of the anethole obtained in Example 1 of this invention was identified. 1 HNMR spectra as follows Figure 2 As shown, 13 CNMR spectra as follows Figure 3 As shown, the HRMS map is as follows Figure 4 As shown, the specific data is as follows: 1 HNMR (C 19 H 13 NO4, 400MHz, DMSO): δ8.58(s,1H),8.36(s,1H),7.63(s,1H),7.42(s,1H),6.89-6.85(m,2H),6.06(s,2H),3.88(s,3H),3.23(d,2H); 13 CNMR(C 19 H 13 NO4,100MHz,DMSO)δ:183.9,161.2,152.8,147.7,140.5,138.7,138.4,136.0,130 .9,130.4,129.8,122.4,121.8,119.5,106.9,101.3,100.3,37.9,30.4;HRMS(ESI + ):[M+H] + The calculation yields 320.08, and the value is found to be 320.08.

[0035] HR-ESI-MS yielded a quasi-molecular ion peak at m / z 320.08 [M+H]+ (calculated value 320.08), which, combined with its... 13 C-NMR and 1 H-NMR determined its molecular formula to be C. 19 H 13 NO4, with a calculated unsaturation degree of 14, has the following structure: .

[0036] This compound 1 HNMR (C 19 H 13 The NO4, 400MHz, DMSO spectra showed hydrogen signals in four aromatic regions: two groups of tetrasubstituted aromatic hydrogens with δH 8.58 (C). 7 H-,s,1H),δH8.36(C 14 H-,s,1H),δH7.63(C 19 H-,s,1H) and δH7.42(C 4 H-, s, 1H); shows a group of olefin hydrogen signals δH 6.89-6.85 (C 10-11 H-,m,2H); shows one methylenedioxy hydrogen signal δH 6.06 (C 2 H-,s,2H); shows one N-methyl hydrogen signal δH 3.88 (C 16 H-,s,2H); shows one methylene hydrogen signal δH3.23 (C 12 H-,d,1H,). Analyze its 13C-NMR (C 19 H 13 The NO4, 100MHz, DMSO spectrum shows that the compound contains 19 carbon atoms. In addition to the 9 carbon atoms present in the structural units mentioned above, the compound also contains one aromatic carbonyl group δC183.9 (-C). 9 O), 1 lactam carbonyl carbon δC161.2(-C 17 O), two aromatic carbons linked to alkoxy groups δC152.8(-C 3 -O), δC147.7(-C 1 -O), an aromatic carbon with one nitrogen atom bonded to it, δC140.5(-C 15 N), an aromatic carbon with 5 bonded C atoms δC138.7(-C 13 ), δC130.4(-C 18 ), δC129.8(-C 8 ), δC121.8(-C 6 ), δC119.5(-C 5 ). HRMS analysis revealed 320.08 (M+H), 319.10 (M), 304.06 (M-N-methyl), 291.1 (M-aromatic ketone), and 289.05 (M-methylenedioxane CH2O).

[0037] Experiment Example 4 Add 100 μL of a 1×10⁻⁶ solution to a 96-well plate. 4 L929 fibroblasts in logarithmic growth phase were cultured in DMEM complete medium at a density of 1 / mL. The 96-well plates were then incubated at 37°C with 5% CO2 for 12 hours. After discarding the medium, 100 μL of DMEM complete medium containing 10 μmol / L of the acetylcholine obtained in Example 1 was added, and the cells were cultured for another day. Cell morphology was then observed under a microscope. The control group used serum-free DMEM medium, and the positive control group used DMEM complete medium supplemented with 5% phenol. Results are shown below. Figure 5 .

[0038] Figure 5 This figure shows the cytotoxicity results of the anisole obtained in Example 1 of this invention. In the figure, A represents the cytotoxicity results of the positive control group, B represents the cytotoxicity results of the control group, and C represents the cytotoxicity results of the anisole obtained in Example 1. (Observation) Figure 5 It can be seen that the anisole obtained in Example 1 of the present invention has no obvious cytotoxicity.

[0039] Experimental Example 5 5.1 Grouping Take 1 mL of HepG2 cells in the logarithmic growth phase (1×10⁻⁶). 4Cells were seeded in 6-well plates with DMEM medium containing 10% FBS and cultured at 37°C with 5% CO2 for 24 hours. After cell adhesion, the medium was discarded, and the cells were divided into the following groups for subsequent experiments: control group, model group, lovastatin positive control group, and anethole group. The control and model groups received 1 mL of DMEM medium containing 10% FBS, the lovastatin positive control group received 1 mL of 10 μmol / L lovastatin, and the anethole group received 1 mL of 10 μmol / L anethole. Except for the control group, 1 mL of 0.75 μmol / L free fatty acids (FFA: oleic acid: palmitic acid = 2:1) was added to each well. The cells were cultured at 37°C with 5% CO2 for another 24 hours. Each experiment was repeated three times with six replicates.

[0040] 5.2 Oil Red O Staining Cells from each group were fixed with 4% paraformaldehyde for 30 min, then differentiated with 60% isopropanol for 10 min. Freshly prepared Oil Red O staining solution (saturated Oil Red O staining solution: water = 3:2) was added for staining for 30 min. Excess dye was washed away, and the cells were rinsed several times with distilled water before being observed and photographed under a microscope. ImageJ was used for quantitative analysis of the images; the results are shown in Table 2.

[0041] 5.3 Biochemical index detection Cells from each group were lysed using high-efficiency RIPA cell lysis buffer, and the supernatant was obtained after centrifugation. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of each group of cells were detected using a kit. The results are shown in Table 2.

[0042] Table 2 ; Table 2 shows that, compared with the control group, the accumulation of red lipids in the model group cells was significantly increased; compared with the model group, the accumulation of lipids in the cells of the squalene group was significantly decreased. These results indicate that squalene can improve FFA-induced lipid metabolism disorders and lipid accumulation in HepG2 cells.

[0043] Table 2 shows that, compared with the control group, the levels of AST and ALT in the model group cells were significantly increased, indicating that FFA induced stem cell damage; compared with the model group, the serum levels of AST and ALT in the squalene group cells were decreased. These results indicate that squalene can alleviate FFA-induced HepG2 cell damage.

[0044] Experimental Example 6 Sixty 8-week-old male C57BL / 6J mice were randomly divided into six groups: normal control group, model group, Example 1 group, Example 2 group, Example 3 group, and comparative example 1 group. The normal control group was fed a standard diet, the model group was fed a CDAHFD high-fat diet, and the other groups were fed a CDAHFD high-fat diet while simultaneously receiving anethole at a dose of 80 mg / kg / day for 10 consecutive weeks.

[0045] (1) After the experiment, blood was collected from the orbits of mice in each group. After separation, serum was obtained. The levels of ALT, AST, total cholesterol (TC), total triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in the serum were detected using the corresponding kits. The results are shown in Table 3.

[0046] (2) After blood collection, mice in each group were sacrificed, and livers were dissected. 50 mg of liver from each group of mice was weighed and the contents of TG and TC in the liver were detected according to the instructions of the triglyceride (TG) and total cholesterol (TC) assay kit. The results are shown in Table 4.

[0047] Table 3 ; Table 4 ; As shown in Tables 3-4, compared with the normal control group, after 10 weeks of continuous intake of a high-fat diet, the serum levels of AST, ALT, TC, TG, LDL-c, and HDL-c in the model group mice were significantly increased, and the liver levels of TC and TG were also significantly increased. Compared with Comparative Example 1, the anise compositions obtained in Examples 1-3 of this invention can significantly reduce hyperlipidemia, liver lipid accumulation, and alleviate liver inflammation in mice. The above results indicate that the synergistic effect of aniseone and juglone can improve liver function and the degree of liver lesions by reducing blood lipid levels, liver lipid accumulation, and alleviating liver inflammation.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A fennel composition for treating non-alcoholic fatty liver disease, characterized in that, It includes the following components: anisole and juglone, wherein the mass ratio of anisole to juglone is 1:(0.3-0.5). The structural formula of the anethole ketone is: .

2. The anise composition for treating non-alcoholic fatty liver disease according to claim 1, characterized in that, The method for preparing the anethole includes the following steps: (1) Add fennel powder to an 80-85% ethanol solution for ultrasonic extraction, filter to obtain ultrasonic extract and filter cake; reflux extract the filter cake with a 90-95% ethanol solution 3-4 times to obtain reflux extract; combine the ultrasonic extract and the reflux extract, adjust the pH with hydrochloric acid solution and concentrate to a thick paste, dry and sieve to obtain fennel extract; (2) Take the anise extract, adjust the pH with ammonia, and then extract with petroleum ether 3-4 times to obtain a petroleum ether layer; extract the petroleum ether layer with a mixed solvent 5-6 times, combine the extract phases and remove the solvent to obtain a mixed solvent layer; (3) Mix the mixed solvent layer with silica gel and then perform a first gradient elution to obtain components Fr.1-Fr.

4. Take component Fr.2 for later use. (4) Mix the component Fr.2 with silica gel and then perform a second gradient elution to obtain components Fr.2.1-Fr.2.

8. Take component Fr.2.2 for later use; (5) After concentrating the component Fr.2.2, perform a third gradient elution, take the fraction Fr.2.2.4 at gradient 80:20, freeze dry to obtain the fenestrin.

3. The anise composition for treating non-alcoholic fatty liver disease according to claim 2, characterized in that, In step (1), during ultrasonic extraction, the ratio of anise powder to ethanol solution is 10 kg: (4-6) L; the amount of ethanol solution used in each reflux extraction is 1-2 L; and the concentration of hydrochloric acid solution is 2-3 mol / L.

4. The anise composition for treating non-alcoholic fatty liver disease according to claim 2, characterized in that, The ultrasonic extraction time in step (1) is 40-60 min; the pH is adjusted to 3-4; the concentration temperature is 75-85℃; the relative density of the thick paste is 1.1-1.5; and the sieve mesh size is 60 mesh.

5. The anise composition for treating non-alcoholic fatty liver disease according to claim 2, characterized in that, The pH is adjusted to 9-10 in step (2); the mixed solvent is composed of dichloromethane, n-butanol and methanol in a volume ratio of 8.5:1:0.

5.

6. The anise composition for treating non-alcoholic fatty liver disease according to claim 2, characterized in that, In step (2), the amount of petroleum ether added each time during the petroleum ether extraction process is 1-2L; in the mixed solvent extraction process, the amount of mixed solvent added each time is 2-3L.

7. The anise composition for treating non-alcoholic fatty liver disease according to claim 2, characterized in that, In step (3), the mass ratio of the mixed solvent layer to the silica gel is 1:(1.5-2); the first gradient elution is carried out sequentially with a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 90:10, 70:30, 50:50, and 0:100, with each gradient elution lasting 8-10 hours and a flow rate of 15-20 mL / min.

8. The anise composition for treating non-alcoholic fatty liver disease according to claim 2, characterized in that, In step (4), the mass ratio of component Fr.2 to silica gel is 1:(1.5-2); the second gradient elution is carried out sequentially with a dichloromethane-n-butanol mixed solvent with volume ratios of 100:0, 95:5, 90:10, 80:20, 70:30, 60:40, 50:50, and 0:100, with each gradient elution lasting 10-12 hours and a flow rate of 8-12 mL / min.

9. The anise composition for treating non-alcoholic fatty liver disease according to claim 2, characterized in that, The third gradient elution in step (5) involves sequentially eluting with a water-acetonitrile mixed solvent at volume ratios of 95:5, 90:10, 85:15, 80:20, 70:30, 60:40, and 50:

50. Each gradient elution lasts 8-10 minutes at a flow rate of 3-4 mL / min. The chromatographic column used for this third gradient elution is a Fisher Wharton Xbridge C10 ... 18 Chromatographic column.

10. A method for preparing the anise composition for treating non-alcoholic fatty liver disease according to any one of claims 1-9, characterized in that, Includes the following steps: Mix the chalcone and juglone evenly according to the mass ratio.

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