Application of eucalyptus fruit extract in preparation of medicine or product for preventing and treating liver lipid metabolism abnormality related diseases

Abstract

The invention belongs to the field of medicines and health-care products, and particularly relates to application of eucalyptus fruit extract in preparation of medicines or products for preventing and treating liver lipid metabolism abnormality related diseases. The abnormal lipid metabolism related diseases are obesity, hyperlipidemia, atherosclerosis (AS), metabolism related fatty liver disease (MAFLD), non-alcoholic steatohepatitis (NASH) and type 2 diabetes mellitus (T2DM). It is found for the first time that the eucalyptus fruit or the extract thereof can reduce anabolism of liver fat and cholesterol, so that liver lipid deposition is reduced, dyslipidemia is improved, and the eucalyptus fruit or the extract thereof can be used for preventing and / or treating dyslipidemia related diseases.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to the application of eucalyptus fruit extract in the preparation of medicines or products for preventing and treating diseases related to abnormal liver lipid metabolism. Background Art

[0002] The liver is an important site for lipid synthesis and transport. The triglycerides, phospholipids, cholesterol and apolipoproteins synthesized by the liver form very low-density lipoproteins, which are transported to organs throughout the body or stored in adipose tissue through the blood. Disorders in fat metabolism in liver cells under physiological and pathological conditions can easily lead to liver fat accumulation and reduced insulin sensitivity, causing liver metabolic diseases such as dyslipidemia, insulin resistance, type 2 diabetes and non-alcoholic fatty liver disease, and also increase the risk of various cancers, such as hepatocellular carcinoma.

[0003] SREBPs are key transcriptional regulators involved in multiple lipid metabolism genes. SREBP1a and SREBP1c can regulate de novo fatty acid synthesis (DNL) in the liver. Since DNL is located at the intersection of glucose and fatty acid processing in the liver, upregulation of the liver DNL pathway has been considered to be one of the important factors in the occurrence and development of various lipid metabolism disorders (hyperlipidemia, MAFLD, insulin resistance and T2DM, etc.). SREBP2 can regulate the mevalonate pathway to affect de novo cholesterol synthesis, and can also regulate the liver's absorption of cholesterol through the low-density lipoprotein receptor (LDLR). Cholesterol is closely related to the metabolism of other lipids, and hypercholesterolemia is recognized as one of the important factors of cardiovascular and cerebrovascular diseases, and is associated with atherosclerosis, heart disease, stroke, etc. (References SREBP Regulation of Lipid Metabolism in Liver Diseases and Treatment Strategies Biomedicine 2023, 11(12), 3280; DOI: 10.3390 / biomedicines11123280).

[0004] The present invention finds that the fruit extract of Eucalyptus, a plant of the genus Eucalyptus of the Myrtaceae family, can reduce lipid accumulation, total cholesterol (TC), and triglyceride (TG) content in the liver of C57 mice induced by a high-fat diet, reduce blood TC, TG, low-density lipoprotein (LDL) content, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicators, and increase blood high-density lipoprotein (HDL) content. In-depth studies have shown that the eucalyptus fruit extract can significantly inhibit the synthesis of free fatty acids (FFA) represented by hexadecanoic acid and octadecanoic acid, TC, TG generation, that is, inhibit the synthesis of DNL and cholesterol in cells. At the same time, inhibiting the gene expression of SREBP1 and SREBP2 is a potential mechanism of action. At present, there is no report on the eucalyptus fruit extract reducing fatty acid and cholesterol synthesis and treating diseases related to abnormal lipid metabolism. Summary of the invention

[0005] Purpose of the Invention

[0006] The invention provides a new use of eucalyptus fruit extract.

[0007] Technical Solution

[0008] The eucalyptus fruit extract is used in the preparation of a medicine or product for preventing and treating diseases related to abnormal liver lipid metabolism, characterized in that the eucalyptus fruit extract is prepared by the following steps: weighing eucalyptus fruit medicinal materials, soaking them in an alcohol-water mixed solvent, a fat-soluble solvent or an alkaline water solvent with a pH value of ≥7, heating to reflux or cold soaking extraction, filtering the extract, concentrating it to a thick extract, drying, and crushing to obtain the extract.

[0009] The alcohol solution is methanol, ethanol or n-butanol aqueous solution; the fat-soluble solvent is petroleum ether, ethyl acetate or acetone; or an alkaline solution with a pH value greater than 7.

[0010] The eucalyptus fruit is derived from the fruit of any eucalyptus of the genus Eucalyptus (Eucalyptus spp.) of the Myrtaceae family.

[0011] The extract is characterized in that the extract contains at least one of the compounds Macrocarpal A (CAS No.: 132951-90-7), Macrocarpal B (CAS No.: 142698-60-0), Macrocarpal D (CAS No.: 142647-71-0), Macrocarpal E (CAS No.: 142628-54-4), Macrocarpal L (CAS No.: 327601-97-8), Macrocarpal M (CAS No.: 327601-96-7), Macrocarpal O (CAS No.: 327622-65-1), and Macrocarpal Q (CAS No.: 1885113-94-9).

[0012]

[0013] Further preferably, the extract solvent is 60%-100% ethanol, methanol or alkaline water with a pH value of 8-12.

[0014] The extract also includes a purification process: the concentrated extract is dissolved in 60%-80% ethanol, insoluble matter is filtered, the ethanol is concentrated until there is no alcohol taste, and petroleum ether or n-hexane is extracted; after the extract is recovered from the solvent, it is dissolved in 30%-50% ethanol, passed through D101 macroporous resin, 60%-80% of the eluate is collected, concentrated, and dried to obtain the product.

[0015] The application is characterized in that the eucalyptus fruit extract is a composition consisting of the eucalyptus fruit extract and pharmaceutically acceptable excipients.

[0016] The application is characterized in that the optional dosage forms prepared include oral dosage forms and non-oral dosage forms; the oral dosage forms include tablets, capsules, powders or granules, and the non-oral dosage forms include suppositories and injections.

[0017] The diseases related to abnormal lipid metabolism are selected from obesity, dyslipidemia, metabolic associated fatty liver disease (MAFLD), atherosclerosis (AS), non-alcoholic steatohepatitis (NASH), type 2 diabetes mellitus (T2DM) and coronary heart disease.

[0018] The dyslipidemia is hyperlipidemia, which is characterized by high cholesterol (TC) content and / or high triglyceride (TG) content and / or high low-density lipoprotein (LDL-C) and / or low high-density lipoprotein (HDL-C) content.

[0019] The dosage of the pharmaceutical composition can be adjusted based on a comprehensive consideration of factors such as the method of administration, severity of illness, age of the patient, and whether or not there is a previous medical history.

[0020] The present invention uses a HepG2 cell model induced by free fatty acids and GC-MS to detect the intracellular fatty acid content. It is found that the eucalyptus fruit extract can reduce lipid accumulation in cells and significantly reduce the fatty acid formation of cells. Further experiments show that the extract can significantly downregulate the gene expression of SREBP-1 and SREBP-2, transcriptional regulators of cell cholesterol and fatty acid synthesis, and significantly upregulate the gene expression of low-density lipoprotein receptor (LDLR). In a high-fat diet-induced lipid metabolism disorder mouse model, the extract significantly reduced the liver index and epididymal fat coefficient of mice; liver tissue pathological staining showed that after drug intervention, liver fat cavitation was significantly improved, lipid droplets were reduced, epididymal fat cell expansion coefficient decreased, and liver TG and TC content were significantly decreased. Serum ALT and AST levels were significantly reduced; the extract administration group achieved a positive regulatory effect on the four blood lipid levels, not only reducing the content of TC, TG, and LDL-C in serum, but also increasing the content of serum HDL-C. In general, the experimental results suggest that the extract of the present invention has good application potential in preventing and / or treating diseases related to abnormal lipid metabolism.

[0021] Beneficial effects: The present invention discovers for the first time that eucalyptus fruit extract can downregulate the expression of SREBP-1 and SREBP-2 genes, thereby inhibiting the synthesis of cellular fatty acids and cholesterol. It can be used to prepare drugs or health products for preventing and treating diseases related to abnormal liver lipid metabolism, providing a new option for combating diseases related to abnormal liver lipid metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 , the HPLC chromatogram of the eucalyptus fruit extract in Example 1, wherein MA is Macrocarpal A, MD is Macrocarpal D, MO is Macrocarpal O, MB is Macrocarpal B, ME is Macrocarpal E, ML is Macrocarpal L, MM is Macrocarpal M, and MQ is Macrocarpal Q;

[0023] Figure 2 , the HPLC chromatogram of the eucalyptus fruit extract in Example 2, wherein MA is Macrocarpal A, MD is Macrocarpal D, MO is Macrocarpal O, MB is Macrocarpal B, ME is Macrocarpal E, ML is Macrocarpal L, MM is Macrocarpal M, and MQ is Macrocarpal Q;

[0024] Figure 3 , the effect of the extract on the lipid droplets of HepG2 cells induced by FFA, where the FFA concentration was 0.5mM, and the extract concentrations were 40μg / mL, 20μg / mL and 10μg / mL, the pictures were taken under an inverted microscope at 200 times, the scale bar is 50μm;

[0025] Figure 4 , the effects of the extract on TC (a) and TG (b) in HepG2 cells induced by FFA; the FFA concentration was 0.5 mM, # indicates compared with the blank group, * indicates compared with the model group, ###p<0.001, **p<0.01, ***p<0.001;

[0026] Figure 5 , Effects of different concentrations of extracts on the fatty acid content of HepG2 cells; A is stearic acid, B is palmitic acid, * indicates that compared with the blank group, **p<0.01, *p<0.05)

[0027] Figure 6 Effects of extracts on the gene expression levels of SREBP-1, SREBP-2 and LDLR in HepG2 cells (# indicates comparison with the blank group, * indicates comparison with the model group, **p<0.01, ***p<0.001)

[0028] Figure 7, extracts reduced HFD-induced liver lipid accumulation, inflammation and fibrosis; paraffin sections of mouse liver were stained with Oil Red O, H&E, Masoon, and Sirius Red, scanned at 40 times with a digital slice scanner, and image analysis was performed using NDP software, with scale bars of 100 μm;

[0029] Figure 8 , the effect of the extract on the expansion of epididymal adipocytes induced by HFD diet in mice; the picture shows the H&E staining results of mouse adipose tissue, which was scanned at 40 times with a digital slide scanner and analyzed using NDP software, and the scale bar is 250mm;

[0030] Fig. 9 , the effects of the extract on the levels of TG (a), TC (b), LDL-C (c), and HDL-C (d) in the serum of mice induced by HFD diet; # indicates compared with the blank group, * indicates compared with the model group, ###p<0.001, *p<0.05, **p<0.01, ***p<0.001;

[0031] Fig.10 , extracts on the serum AST (a), ALT (b) levels of mice induced by HFD diet; # indicates compared with the blank group, * indicates compared with the model group, ###p<0.001, *p<0.05, ***p<0.001; Fig.11 , extracts on HFD diet-induced liver TG (a), TC (b) levels; # indicates compared with the blank group, * indicates compared with the model group, ###p<0.001, *p<0.05);

[0032] Fig.12 , extracts affect liver weight (a), liver coefficient (b), epididymal fat weight (c), and epididymal fat coefficient (d) of mice induced by HFD diet; # indicates compared with the blank group, * indicates compared with the model group, ##p<0.01, ###p<0.001, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0033] The present invention is described in detail below with reference to the accompanying drawings and examples, but the contents described are intended to explain the present invention rather than to limit it.

[0034] Example 1: Extraction and purification method of eucalyptus fruit 1

[0035] The dried eucalyptus fruit was crushed and refluxed with 5 times the volume of 95% ethanol for three times, each time for 3 hours. After filtering and combining the filtrate, it was concentrated to dryness at 50°C under reduced pressure. It was dissolved in 80% ethanol, and the solvent was recovered after filtering the insoluble matter until there was no alcohol taste. It was extracted 3 times with 3 times the volume of petroleum ether, and the petroleum ether was collected and the solvent was recovered. The petroleum ether extract was dissolved in hot ethanol water, and an appropriate amount of D101 macroporous resin was adsorbed overnight. The saturated macroporous resin obtained was loaded with a ratio of 2:1 (unadsorbed macroporous resin weight: saturated resin weight), and eluted with 30%, 60% and 100% ethanol concentration gradients in turn. The 60% ethanol eluate was collected and concentrated under reduced pressure and dried to obtain the eucalyptus fruit extract. The structure of the main components was determined by HPLC-DAD analysis and comparison with the compound standard, and the content of the main compounds was determined by HPLC standard curve method. Chromatographic conditions: Wondasil C18 column (250 mm × 4.6 mm, 5 μm); detection wavelength: 280 nm; column temperature: 25 ° C; flow rate: 1 mL / min. Mobile phase and elution conditions: 0-30 min, 85%-90% MeOH; 30-40 min, 90%-95% MeOH; 40-50 min, 95%-100% MeOH. Liquid phase diagram and quantitative results are shown in Figure 1 The extract contains Macrocarpal A (8.11%, mass percentage, the same below), Macrocarpal B (8.45%), Macrocarpal D (2.08%), Macrocarpal E (1.25%), Macrocarpal L (0.91%), Macrocarpal M (0.31%), Macrocarpal O (0.54%), and Macrocarpal Q (0.23%).

[0036] Subsequent in vitro and in vivo efficacy experiments were conducted using the extract obtained in Example 1.

[0037] Example 2: Extraction and purification method of eucalyptus fruit 2

[0038] The dried eucalyptus fruits were crushed and refluxed and extracted three times with 8 volumes of NaOH solution with a pH of 10, each time for 3 hours. The liquid was replenished during the extraction process to keep the extract alkaline. The extract was adjusted to neutral or weakly acidic with dilute hydrochloric acid, extracted three times with 3 volumes of petroleum ether, the petroleum ether was collected, and the solvent was recovered. The petroleum ether extract was gradient eluted with D101 macroporous resin according to the method in Example 1 to obtain the eucalyptus fruit extract. The structure of the main components was determined by HPLC-DAD analysis and comparison with the compound standard, and the content of the main compounds was determined by the HPLC standard curve method. The HPLC method was the same as above. The liquid phase diagram and quantitative results are shown in Figure 2The extract contains Macrocarpal A (15.60%, mass percentage, the same below), Macrocarpal B (16.22%), Macrocarpal D (4.06%), Macrocarpal E (2.37%), Macrocarpal L (1.76%), Macrocarpal M (0.61%), Macrocarpal O (1.03%), and Macrocarpal Q (0.44%).

[0039] Example 3: Study on the lipid-lowering activity of the extract (using lipid accumulation as an indicator)

[0040] HepG2 cells were cultured at 37°C in an environment of 5% CO2 in DMEM high glucose medium containing 10% heat-inactivated fetal bovine serum, glutamine and antibiotics.

[0041] Take the cells in the logarithmic growth phase, digest them with trypsin to make a cell suspension, then count them and divide them into blank group, model group, drug group (divided into high, medium and low dose groups, i.e. 40μg / mL, 20μg / mL, 10μg / mL), and positive drug group. Cover the bottom of the six-well plate with 1mL complete culture medium and add 1x10 5 Cells. Place in a 5% CO2 incubator to adhere to the wall and grow to about 40%. Take out the six-well plate from the incubator, aspirate the original culture medium, and add the prepared FFA induction solution (1mM sodium oleate solution and 0.5mM sodium palmitate solution) except for the blank control group. Add new complete culture medium to the blank control group without other treatment. After inducing the cells for 24 hours, aspirate all the culture medium, add new complete culture medium to the blank group without treatment, and give the other groups 40μg / mL, 20μg / mL, and 10μg / mL of the extract obtained in Example 1 as three dosage groups, and give the model group an equal volume of DMSO as a control. Put them back into the incubator and continue to culture for 24 hours. After the drug intervention, the culture medium was carefully aspirated and washed twice with PBS to ensure that there was always liquid in the plate and that the lipid droplets were not broken. 2 mL of 4% paraformaldehyde was added to each well for fixation for 30 min. After fixation, the cells were washed twice with distilled water, and then immersed in isopropanol for dehydration twice. Then, Oil Red O stain was added and placed in a 37°C incubator for incubation for 30 min. The stain was then aspirated and rinsed with propylene glycol until the interstitium was clear. The cells were washed with water until there was no excess stain, and hematoxylin was added for counterstaining for 2 min. The stain was discarded and the cells were washed with distilled water until the water was clear. Finally, distilled water was added to the wells and photographs were taken and collected under an inverted fluorescence microscope.

[0042] The experimental results are shown in Figure 3After 24 hours of FFA stimulation, the lipid droplets in the model group increased significantly, and the lipid droplets were large and dense, which was significantly different from the blank control group, indicating that FFA induced lipid accumulation in HepG2 cells. Then DMSO and extracts were given for 24 hours of intervention. The results showed that each extract reduced the lipid accumulation of HepG2 cells, and the number and volume of lipid droplets decreased to varying degrees, and all had significant differences (p<0.001).

[0043] Example 4: Study on the lipid-lowering activity of the extract (taking the intracellular TC and TG contents as indicators)

[0044] Take the cells in the logarithmic growth phase, digest them with trypsin to make a cell suspension, count them, and divide them into blank group, model group, drug group (divided into high, medium and low dose groups), and positive drug group, with three replicate wells in each group. The bottom of the six-well plate was covered with 1 mL of complete culture medium in advance, and 1x10 5 Cells. Place in a 5% CO2 incubator to grow adherently. When the cells grow to about 40%, take out the six-well plate from the incubator, aspirate the original culture medium, and add the pre-prepared FFA induction solution (1mM sodium oleate solution and 0.5mM sodium palmitate solution) to all except the blank control group. Add new complete culture medium to the blank control group without other treatment. After inducing the cells for 24 hours, aspirate all the culture medium, add new complete culture medium to the blank group without treatment, and give the other groups 40μg / mL, 20μg / mL, and 10μg / mL of the extract in Example 1 as three dosage groups, and give the model group an equal volume of DMSO as a control. Put them back into the incubator and continue to culture for 24 hours. After the administration, the six-well plate was taken out, the culture medium of each well was aspirated, and the well was washed twice with PBS. Then, trypsin was added to each well for digestion. The cells were gently scraped with a cell scraper, and then centrifuged at 1000rpm / 5min to enrich the cells at the bottom of the tube. Then, trypsin was carefully aspirated, the cells were redissolved in 1mL PBS, and then continued to centrifuge at 1000rpm / 5min, PBS was aspirated, and then resuspended in 200μL PBS, and quantified according to Nanjing Jiancheng TC and TG kits.

[0045] The experimental results are as follows Figure 4 After 24 hours of free fatty acid (FFA) induction, the TC and TG contents of the model group cells increased significantly, TG increased by 2 times (p < 0.001), and TC increased by 3 times (p < 0.001), proving that the lipid accumulation model was successfully established. After 24 hours of extract treatment, the concentrations of TC and TG in the cells decreased significantly, and as the concentration of the extract increased, the concentrations of TC and TG decreased accordingly. This experiment shows that the extract can dose-dependently reduce the concentrations of TC and TG in HepG2 cells.

[0046] Example 5: Effect of the extract on the fatty acid content in HepG2 cells

[0047] HepG2 cells in the logarithmic growth phase were digested with trypsin to prepare a single cell suspension. After counting with a cell counter, the cell suspension was diluted to 1x 10 5 / mL, add 1mL of cell suspension to each well, add 1mL of complete culture to the 6-well plate in advance to cover the bottom of the plate, add 1mL of cell suspension to each well, put it in the incubator and continue to incubate until the cells adhere to the wall and grow to about 60%. Take out the 6-well plate from the incubator, remove the original culture medium, set the first well as the blank control group, the second well as the DMSO group, add 8μL of DMSO, and add 40μg / mL and 10μg / mL of extracts to the other wells respectively. Continue to incubate in the incubator for 24h, take out the cells, aspirate the culture medium, wash twice with PBS, then put it on ice, scrape the cells, and transfer the cell suspension to a glass tube. Add 1mL of cold water to the culture dish, transfer it to the same tube, then add 1mL of chloroform, vortex for 60s, and store at 4℃ overnight. On the second day, centrifuge at 5000xg and 4℃ for 10 minutes, transfer the lower chloroform phase to a new glass tube, evaporate to dryness under nitrogen at 37℃, treat the sample with 1mL methanol and 50ul sulfuric acid at 100℃ for 2h, cool to room temperature after treatment, and extract the derivatized fatty acids by adding 1mL hydrate and 2mL n-hexane, separate the upper organic phase and dry it under nitrogen flow, then redissolve the dried fatty acids in 100uL n-hexane for GC-MS analysis, quantify by standard curve method. Chromatographic conditions: helium flow rate 1mL / min. Source temperature 230℃, mass spectrometer quadrupole temperature 150℃, interface temperature 280℃, inlet temperature 250℃. GC / MS analysis of fatty acid methyl esters (FAME), 1μL injection. No split. Start at 80℃ for 2 minutes, increase to 280℃ at a rate of 10℃ / min, and maintain for 12 minutes.

[0048] like Figure 5 As shown, the extract significantly reduced the content of palmitic acid and stearic acid in cells and inhibited the synthesis of fatty acids in a dose-dependent manner.

[0049] Example 6: Effects of the extract on lipid metabolism-related genes in HepG2 cells

[0050] HepG2 cells in the logarithmic growth phase were digested with trypsin to form a single cell suspension, and then the cells were diluted to 1x10 5Take a six-well plate and add 1 mL of complete culture medium to each well to cover the bottom of the plate, then add another 1 mL of cell suspension, and continue to culture in a 37°C, 5% CO2 environment for 24 hours. When the cells grow to 60%, remove the original culture medium, add DMSO and different concentrations of drugs to the new culture medium, divided into three concentration gradients (40 mg / mL, 8 mg / mL and 1.6 mg / mL), and continue to culture for 24 hours. After the administration is completed, the original culture medium is aspirated, PBS is added to each well for washing twice, and then 500 μL of lysis solution is added, and the cells are allowed to stand at room temperature for 8 minutes. The cells are gently scraped off with a cell scraper, and all the lysate is transferred to the DNA adsorption column, centrifuged at 12000×g for 30 seconds, and the filtrate is retained. Then 0.5 times the volume of anhydrous ethanol is added to the filtrate, and the solution is blown until the solution is clear without obvious precipitation. Then the whole is transferred to the RNA adsorption column, centrifuged at 12000×g for 30 seconds, and the filtrate is discarded. 700 μL of Buffer RW1 is added to the RNA column, and the column is centrifuged at 12000×g for 30 seconds. The filtrate is discarded, and then 700 μl of Buffer RW2 is added to the RNA column, and the column is centrifuged at 12000×g for 1 minute. After washing twice, the filtrate is discarded, and then the tube is emptied for 1 minute, and the retention tube is discarded. Replace it with a 1.5 mL RNase-free Collection Tubes centrifuge tube, and 50 μL of preheated ddH2O is dripped into the center of the adsorption column, centrifuged for 1 minute, and RNA is eluted. The RNA concentration was then determined using a NanoDrop 2000 / 2000C spectrophotometer.

[0051] First, take out the genomic DNA and proceed according to the following reaction system, and determine the dilution multiple according to the RNA concentration.

[0052] Table 1. Reverse transcription reaction solution ratio

[0053]

[0054] After pipetting evenly, react at 42°C for 2 min.

[0055] Then add 4 mL of 5xHiScriptII qRT SuperMixII to the tube in the first step, pipette evenly, and perform reverse transcription reaction according to the following system.

[0056] Reverse transcription was performed according to the ratio of the reverse transcription reaction system (Table 1), and the cDNA solution was obtained by reaction in the MyCycler Thermal Cycler PCR amplification instrument and stored at -20°C, or directly performed in the subsequent Real Time-PCR reaction. Real Time-PCR was completed using the Light Cycler 480 real-time fluorescence quantitative PCR instrument. The amplification reaction system is shown in Table 2, and the amplification program is shown in Table 3.

[0057] Table 2 PCR amplification reaction system

[0058] Table 3 PCR amplification program

[0059]

[0060] Table 4 Target gene primer sequences

[0061]

[0062]

[0063] The experimental results are shown in Figure 6 The extract at 20 μg / mL significantly upregulated the gene expression of LDLR (up 3 times) and downregulated the gene expression of transcriptional regulators SREBP-1 and SREBP-2.

[0064] Example 7 Effect of the extract on HFD-induced hyperlipidemia mice

[0065] 6-8 week old male C57BL / 6J mice were randomly divided into four groups after adaptive feeding for one week, with 12 mice in each group, namely blank group, model group, three dose groups of extracts (i.e. 20, 40, 60 mg / kg), and positive drug group. The blank group was given normal feed, and the other three groups were given D12492 high-fat feed. After 8 weeks of dietary induction, at the end of the eighth week, 6 mice were randomly selected from each group, blood was collected from the orbits, serum was collected, and serum total cholesterol, triglyceride, and low-density lipoprotein levels were detected. The experimental results showed that the high-fat model was successfully established. After that, the drug was administered orally by gavage every day, and the drug was suspended in 5% sodium carboxymethyl cellulose solvent. The model group was given an equal volume of control solvent. After four weeks of administration, the mice were fasted for 12 hours and their body weights were recorded; the mice were anesthetized, the eyeballs were removed to collect whole blood, and after standing for 2 hours, the supernatant was collected and stored at -80°C at 4000rpm / min for 10 minutes at 4°C; the liver was collected and weighed. The liver tissue fixed with paraformaldehyde, epididymal adipose tissue and liver tissue embedded with frozen embedding solution were sent to the Pharmacology and PDX Efficacy Evaluation Platform of China Pharmaceutical University to make tissue wax blocks and perform HE, Oil Red, Masoon and Sirius Red staining. Then the digital pathology section scanner (NanoZoomer S60) was used to scan and analyze with NDP software. The remaining part was quickly frozen in liquid nitrogen and stored at -80℃.

[0066] like Figure 7As shown in the figure, the results of H&E staining of mouse liver showed that the volume of liver cells in the HFD high-fat model group mice increased several times, the nucleus was displaced, there was no obvious visible cytoplasm, the liver cells were fat vacuolated, ballooning was obvious, the liver plates were disordered, and the hepatic cords and sinusoids were no longer distinguishable; Oil red O staining of the liver showed that the high-fat diet-induced model group mice had obvious liver lipid accumulation, the number of lipid droplets was large and the volume was large; Masson staining of liver tissue showed that the liver of the model group mice showed significant fibrosis. After drug intervention, the liver fat vacuolation was significantly improved, the lipid droplets were reduced, and the hepatocyte fibrosis was improved. All indicators were dose-dependent. Figure 8 As shown, the staining results of epididymal fat showed that the fat cells of the model group mice induced by high-fat diet expanded several times, and the mice had obvious lipid metabolism disorders. After the extract was administered, the expansion coefficient of epididymal fat cells decreased and lipid metabolism was improved.

[0067] Example 8: Effects of the extract on four blood lipid indicators in hyperlipidemia mice

[0068] The activity of TC, TG, LDL-C, HDL-C, ALT and AST in the serum of mice at the end of administration in Example 7 was detected using a kit from Nanjing Jiancheng Bioengineering Institute. Figure 8 As shown, compared with the blank group, the levels of TC, TG, LDL-C, AST, and ALT in the model group mice were significantly increased. After drug treatment, the above indicators decreased significantly and were dose-dependent, indicating that the extract reduced the blood lipid levels of mice and improved liver damage in mice.

[0069] The experimental results are shown in Fig. 9 , Fig.10 After the mice were induced by HFD high-fat diet, the blood lipid levels of the model group mice were significantly increased compared with the blank group mice, and the content of TC and LDL-C increased by about 1.5 times. The TG and HDL-C groups also had significant differences (p<0.001), indicating that the obesity model induced by high-fat diet in mice was successfully established. After four weeks of treatment with different doses of drugs, the three groups of extract administration groups achieved positive regulatory effects on the four blood lipid levels, not only reducing the content of TC, TG, and LDL-C in serum, but also increasing the content of serum HDL-C, and showing dose dependence. In terms of AST and ALT levels, compared with the blank group, the AST and ALT levels of the model group were significantly increased (p<0.001), indicating that the liver of the HFD-induced high-fat model mice was damaged. Compared with the model group, the extract administration group showed an excellent effect of reducing serum transaminase levels, and the three groups of administration groups also showed obvious dose dependence in reducing serum transaminase.

[0070] Example 9: Effect of extract on lipid content in mouse liver

[0071] Determination of TC and TG content in mouse liver at the end of administration in Example 7: weigh about 20 mg of liver tissue, place it in a 2 ml EP tube, add 1 ml of chloroform / methanol (2:1) solution pre-cooled on ice, homogenize in a homogenizer for 2 min until no tissue blocks are visible to the naked eye, transfer the liver homogenate to a glass screw cap tube, add 2 ml of chloroform / methanol solution, and shake at room temperature for 4 h to overnight; add 0.2 times the volume of 0.9% Nacl, vortex, 2000 rpm, 10 min, use a long glass pipette to reach the bottom phase and aspirate the contents into a new glass tube to avoid dirt on the interface, vacuum dry under a nitrogen flow, and after drying, dissolve in n-butanol / TritonX-100 / methanol (0.6 / 0.267 / 0.133), each 10 mg is dissolved in 100 ul of the above solution, and the TC and TG contents in the liver tissue are determined using the Nanjing Jiancheng kit.

[0072] The experimental results are shown in Fig.11 Compared with the blank group, the TC and TG contents in the liver of the high-fat model group mice were significantly increased (p<0.001), indicating that lipid accumulation had occurred in the liver. In terms of liver TC levels, compared with the model group, the liver TC levels in the drug-treated group showed a downward trend, and the medium and high-dose groups significantly reduced the liver TC content compared with the model group (p<0.05), and there was a dose-dependency among the three groups. In terms of TG levels, there was also a downward trend, and the medium and high-dose groups significantly reduced the liver TG content (p<0.05), and there was a dose-dependency among the three groups.

[0073] Example 10: Effects of the extract on general indicators of experimental animals

[0074] During the entire animal experiment in Example 7, the mice were weighed every three days and the body weight was recorded until the end of the experiment. After the animal experiment, the mice were anesthetized and killed by cervical dislocation. The liver was quickly collected, and the epididymal fat was weighed and photographed.

[0075] The results are as follows Fig.12 The liver weight, liver index, epididymal fat weight and epididymal fat coefficient of mice in each group are shown. It can be seen that compared with the blank group, the liver weight, liver index, epididymal fat weight and epididymal fat coefficient of mice in the model group were significantly increased. Compared with the model group, the administration of the extract effectively reduced the liver index and epididymal fat coefficient of mice, and the epididymal white fat was reduced.

Claims

1. The use of eucalyptus fruit extract in the preparation of a medicine or product for preventing and treating diseases related to abnormal liver lipid metabolism, characterized in that: The eucalyptus fruit extract is prepared by the following steps: weighing eucalyptus fruit medicinal materials, adding alcohol-water mixed solvent, fat-soluble solvent or alkaline water solvent with pH≥7 for soaking, heating reflux or cold soaking extraction, filtering the extract, concentrating to a thick extract, drying, and crushing to obtain the extract.

2. The use according to claim 1, characterized in that: The alcohol-water mixed solvent is a mixed solvent of methanol, ethanol or n-butanol; the fat-soluble solvent is petroleum ether, ethyl acetate, acetone or chloroform.

3. The use according to claim 1, characterized in that: The alcohol-water mixed solvent is 60%-100% ethanol or methanol; the alkaline water is alkaline water with a pH value of 8-12.

4. The use according to any one of claims 1 to 3, characterized in that: The extract is dissolved in 60%-80% ethanol, insoluble matter is filtered, ethanol is concentrated until there is no alcohol taste, and petroleum ether or n-hexane is extracted; after the extract is recovered, the solvent is dissolved in 10%-30% ethanol, passed through D101 macroporous resin, 40%-70% eluate is collected, concentrated, and dried to obtain the product.

5. The use according to claim 4, characterized in that The eucalyptus fruit extract contains at least one of Macrocarpal A, Macrocarpal B, Macrocarpal D, Macrocarpal E, Macrocarpal L, Macrocarpal M, Macrocarpal O and Macrocarpal Q.

6. The use according to claim 1, characterized in that The eucalyptus fruit extract is a composition consisting of the eucalyptus fruit extract and pharmaceutically acceptable excipients.

7. The use according to claim 3, characterized in that: The dosage form of the composition includes oral dosage form and non-oral dosage form; the oral dosage form includes tablets, capsules, powders or granules, and the non-oral dosage form includes suppositories and injections.

8. The use according to any one of claims 1 to 7, characterized in that: The disease associated with abnormal liver lipid metabolism is selected from obesity, dyslipidemia, metabolism-related fatty liver disease, atherosclerosis, non-alcoholic steatohepatitis, type 2 diabetes and coronary heart disease.

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