Application of formyl phloroglucinol mixed-source terpenoid derivative or pharmaceutically acceptable salt thereof in preparation of medicine for preventing and treating liver lipid metabolism abnormality related diseases

Abstract

The invention discloses application of formyl phloroglucinol mixed-source terpene derivatives or pharmaceutically acceptable salts thereof in preparation of drugs for preventing and treating liver lipid metabolism abnormality related diseases, and relates to terpenes. The liver lipid metabolism disorder related diseases are obesity, hyperlipidemia, atherosclerosis (AS), metabolism related fatty liver disease (MAFLD), non-alcoholic steatohepatitis (NASH) and type 2 diabetes mellitus (T2DM). The formyl phloroglucinol mixed-source terpenoid derivative is found to be capable of reducing anabolism of liver fatty acid and cholesterol for the first time, so that liver lipid deposition is reduced, dyslipidemia is improved, and the formyl phloroglucinol mixed-source terpenoid derivative can be used for preventing and treating liver lipid metabolism disorder related diseases.

Description

Technical Field

[0001] The present invention relates to terpenes, and particularly to the use of formylphloroglucinol mixed-source terpenoid derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs for preventing and treating diseases related to abnormal liver lipid metabolism. Background Art

[0002] In recent years, with the rapid development of economy and the change of people's eating habits, the incidence of lipid metabolic diseases such as obesity, cardiovascular disease, metabolic associated fatty liver disease (MAFLD), non-alcoholic steatohepatitis (NASH), type 2 diabetes mellitus (T2DM) has increased significantly. These diseases not only seriously threaten human health, but also impose a huge burden on the social health insurance system.

[0003] Cholesterol is a type of lipid that plays an important role in human life activities. Most of it is produced by the liver, and a small part is ingested from food. Excessive synthesis, uptake disorders or increased outflow of cholesterol in hepatocytes will not only cause abnormal accumulation of cholesterol in the liver, but also lead to the occurrence of hypercholesterolemia, which in turn induces cardiovascular diseases such as atherosclerosis, seriously threatening human health. SREBP2 is a key regulator of cholesterol synthesis and metabolism. It can regulate the mevalonate pathway to affect cholesterol synthesis, and can also regulate the liver low-density lipoprotein receptor (LDLR) to affect cholesterol absorption. Triglycerides are another important lipid, which is a fat molecule formed by long-chain fatty acids and glycerol. In recent years, it has been found that fatty acids in the liver are energy substrates, structural components and signaling molecules necessary for human survival. Cells have therefore evolved a mechanism to produce fatty acids from alternative carbon sources, a process called de novo lipogenesis (DNL). The liver is the main site of DNL. The newly generated lipids in the liver DNL are stored or secreted by hepatocytes and play a key role in maintaining liver energy homeostasis. SREBP1a and SREBP1c are key regulators of liver DNL. 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 development of various lipid metabolism disorders (hyperlipidemia, MAFLD, insulin resistance and T2DM, etc.). Inhibition of DNL-related enzymes, such as ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), is a very promising treatment strategy for related diseases. Several new inhibitors of DNL-related enzymes have entered the clinical development stage and may become new treatments for diseases related to liver lipid metabolism disorders. (SREBP regulation of lipid metabolism in liver disease and therapeutic strategies, Biomedicine, 2023, 11(12), 3280, DOI: 10.3390 / biomedicines11123280; Lipogenesis inhibitors: therapeutic opportunities and challenges, Nature Reviews Drug Discovery, 2022, 21, 283–305 DOI: 10.1038 / s41573-021-00367-2)

[0004] Formylphloroglucinol mixed terpenoids (FPMs) are a class of plant secondary metabolites that are only widely found in plants of the genera Eucalyptus and Guava of the Myrtaceae family, and have many reports of biological activities in antibacterial, antiviral and antitumor aspects. Currently, there are no reports on the FPMs reducing DNL and treating diseases related to abnormal liver lipid metabolism. Summary of the invention

[0005] Purpose of the invention: The present invention provides a new use of a class of formylphloroglucinol mixed-source terpene derivatives.

[0006] Technical solution:

[0007] A type of formylphloroglucinol mixed terpene derivative or a pharmaceutically acceptable salt thereof for use in the preparation of a drug for preventing and treating diseases related to abnormal liver lipid metabolism, characterized in that the structure of the formylphloroglucinol mixed terpene derivative is shown in the following formula:

[0008]

[0009] R is independently selected from the following structures:

[0010]

[0011] R1 is independently selected from hydrogen, alkyl groups such as methyl, ethyl, allyl, and isopentenyl; and acyl groups such as formyl, acetyl, and propionyl.

[0012] The formylphloroglucinol mixed terpene derivative or a pharmaceutically acceptable salt thereof is selected from any one of the following compounds:

[0013] Macrocarpal A (CAS No.: 132951-90-7), Eucarobustol E (CAS No.: 2650180-04-2), Macrocarpal D (CAS No.: 142647-71-0), Macrocarpal O (CAS No.: 327622-65-1), Macrocarpal B (CAS No.: 142698-60-0), Macrocarpal E (CAS No.: 142628-54-4), Macrocarpal L (CAS No.: 327601-97-8), Macrocarpal M (CAS No.: 327601-96-7), Macrocarpal Q (CAS No.: 1885113-94-9) or a pharmaceutically acceptable salt thereof.

[0014]

[0015] The pharmaceutical composition comprises any one or more of the formylphloroglucinol mixed-source terpene derivatives or pharmaceutically acceptable salts thereof, as well as their enantiomers, diastereomers, tautomers, solvates or salts of solvates and pharmaceutically acceptable excipients.

[0016] The pharmaceutical composition includes 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 dosage of the pharmaceutical composition can be adjusted based on a comprehensive consideration of factors such as the method of administration, severity of the disease, age of the patient, and whether or not there is a previous medical history.

[0018] Beneficial effects:

[0019] The present invention first discovered that formylphloroglucinol mixed terpenes with a specific mother core structure can downregulate the expression of SREBP-1 and SREBP-2 genes, thereby inhibiting the synthesis of cellular fatty acids and cholesterol, and can be used to prepare medicines or 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, as follows:

[0020] The present invention uses a HepG2 cell model induced by free fatty acids and GC-MS to detect the intracellular fatty acid content. The experiment found that the p-formyl phloroglucinol mixed terpene derivatives with a specific skeleton structure can reduce the degree of lipid accumulation in cells and significantly reduce the fatty acid and cholesterol production of HepG2. Further experiments show that this type of component can significantly downregulate the gene expression of SREBP-1 and SREBP-2, transcriptional regulators of cholesterol and fatty acid synthesis, and significantly upregulate the gene expression of low-density lipoprotein receptor (LDLR). Subsequently, the compound Macrocarpal A was used as a representative to evaluate the effects of this type of compound on the liver and blood lipid levels of mice with lipid metabolism disorders induced by a high-fat diet. The results showed that MacrocarpalA (15 mg / kg) significantly reduced the liver index and epididymal fat coefficient of mice; liver tissue pathology staining showed that after MacrocarpalA administration, liver fat cavitation was significantly improved, lipid droplets were reduced, liver inflammation was reduced, and fibrosis was reduced; the expansion coefficient of epididymal adipocytes decreased, and the content of TG and TC in the liver was significantly reduced. Serum ALT and AST levels were significantly reduced; the Macrocarpal A administration group achieved positive regulatory effects on the four blood lipid levels, not only reducing the levels of TC, TG, and LDL-C in serum, but also increasing the level of serum HDL-C. In general, the experimental results suggest that the formylphloroglucinol mixed-source terpene derivatives of the present invention have good application potential in preventing and treating diseases related to abnormal liver lipid metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Effects of mixed terpene derivatives of formylphloroglucinol on lipid droplets of HepG2 cells induced by FFA In Figure A, MA is Macrocarpal A, EE is Eucarobustol E, MD is Macrocarpal D, MO is Macrocarpal O, MB is Macrocarpal B, ME is Macrocarpal E, ML is Macrocarpal L, MM is Macrocarpal M, MQ is Macrocarpal Q, FFA concentration is 0.5mM, compound concentration is 40μM, pictures were taken under an inverted microscope at 200 times, scale bar is 50μm; Figure B is the quantitative processing of lipid droplets in Figure A using imageJ, and GraphPad Prism 8.0 was used to analyze the data; # indicates compared with the blank group, * indicates compared with the model group ###p<0.001, ***p<0.001;

[0022] Figure 2 is the result of in vitro lipid-lowering activity screening of formylphloroglucinol mixed terpenoid derivatives (with intracellular TC and TG contents as indicators); a is the effect of FPMs compounds on TC in HepG2 cells induced by FFA, b is the effect of FPMs compounds on TG in HepG2 cells induced by FFA (the compound concentration is 40 μM, the FFA concentration is 0.5 mM, # indicates compared with the blank group, * indicates compared with the model group, ###p<0.001, **p<0.01, ***p<0.001);

[0023] Figure 3 The effect of different formyl phloroglucinol mixed terpenoid derivatives on the fatty acid content of HepG2 cells; a is stearic acid, b is palmitic acid, * indicates **p<0.01, *p<0.05, ***p<0.001 compared with the blank group;

[0024] Figure 4 is the screening result of MacrocarpalA's lipid-lowering activity in vitro (with lipid accumulation as an indicator); a is the effect of different concentrations of MacrocarpalA (MA) on FFA-induced HepG2 cell lipid droplets (the picture was taken under an inverted microscope at 200 times, the scale bar is 50μm, and the FFA concentration is 0.5mM); b is the effect of different concentrations of MacrocarpalA (MA) on FFA-induced HepG2 cell lipid droplets (imageJ was used to quantify pictures 2-4a, and GraphPad Prism 8.0 was used to analyze the data. # indicates compared with the blank group, * indicates compared with the model group ###p<0.001, ***p<0.001);

[0025] Figure 5The effect of different concentrations of MacrocarpalA (MA) on TC (a) and TG (b) in HepG2 cells induced by FFA; (FFA concentration is 0.5 mM, # indicates compared with the blank group, * indicates compared with the model group, ###p<0.001, ***p<0.001);

[0026] Figure 6 is the effect of MacrocarpalA (MA) on the gene levels of LDLR (a), SREBP-1 (b), and SREBP-2 (c) in HepG2 cells (* indicates compared with the model group, *p<0.05, **p<0.01, ***p<0.001);

[0027] Figure 7 is the effect of Macrocarpal A (MA) on the levels of TC (a), TG (b), LDL-C (c), and HDL-C (d) in mouse serum (# indicates compared with the blank group, * indicates compared with the model group, ###p<0.001, *p<0.05, **p<0.01, ***p<0.001);

[0028] Figure 8 is the effect of Macrocarpal A (MA) on serum AST (a) and ALT (b) levels (# indicates compared with the blank group, * indicates compared with the model group, ###p<0.001, *p<0.05, ***p<0.001);

[0029] Fig. 9 is the effect of Macrocarpal A (MA) on the levels of TC (a) and TG (b) in the liver (# indicates compared with the blank group, * indicates compared with the model group, ###p<0.001, *p<0.05, ***p<0.001);

[0030] Fig.10 is the effect of Macrocarpal A (MA) on mouse liver weight (a), liver coefficient (b), epididymal fat weight (c), and epididymal fat coefficient (d) (# indicates compared with the blank group, * indicates compared with the model group, ##p<0.01, ###p<0.001, **p<0.01, ***p<0.001);

[0031] Fig.11 Macrocarpal A (MA) alleviates the lipid accumulation of mice induced by HFD diet (the first row of pictures are H&E staining of mouse liver paraffin sections, the second row are Oil Red O staining of mouse liver frozen sections, and the third row are H&E staining of mouse epididymal fat paraffin sections, scanned at 40× on a digital slide scanner, and image analysis was performed using NDP software, and the scale bars are all 250 μm);

[0032] Fig.12 MacrocarpalA (MA) reduces HFD diet-induced liver fibrosis and macrophage infiltration in mice (the first row of pictures are Masoon staining of mouse liver paraffin sections, the second row are Sirius Red staining of mouse liver, scanned at 40ⅹ with a digital slide scanner, and image analysis was performed using NDP software, with scale bars of 100μm; the third row are F4 / 80 staining of mouse liver, image analysis was performed at 40ⅹ with SlideViewer software, with scale bars of 100μm). 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: Study on the lipid-lowering activity of formylphloroglucinol mixed terpenoid derivatives (using lipid accumulation as an indicator)

[0035] 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, suck out the original culture medium, set the first well as the blank control group, add the complete culture medium containing 1% BSA, add the newly prepared 0.5mM FFA solution to the remaining wells, 2mL of the corresponding solution per well, continue to incubate in the incubator for 24h, and the modeling is completed. After the modeling is completed, take out the six-well plate, suck out the original culture medium, add DMSO or different drugs to each well of the culture plate, and set them as the blank control group (CON, complete culture medium), model group (MOD, add corresponding concentration of DMSO), compound group (40μM), and the drug is diluted in the complete culture medium in advance for standby use. Three replicate wells were set up for each group and the cells were placed in the incubator for another 24 h. After the drug intervention, the culture medium was carefully aspirated and PBS was added for washing to ensure that there was always liquid in the plate and that the lipid droplets were not broken. The cells were washed twice with PBS and fixed with 4% paraformaldehyde solution for 30 min, 2 mL per well. During cell fixation, the oil red stain was filtered and preheated in a 37°C water bath before use. After fixation, the cells were washed twice with ddH2O and dehydrated with 100% propylene glycol. The cells were placed at room temperature for 10 min, the propylene glycol was discarded, the oil red stain was added, and the cells were placed in a 37°C incubator for incubation for 30 min. The oil red stain was aspirated and replaced with counterstain. The cells were counterstained for 2 min, and the counterstain was washed with ddH2O until the water was clear. The water was retained for the last time to ensure that there was always liquid in the wells. The cells were photographed and collected under an inverted fluorescence microscope.

[0036] Experimental results:

[0037] like Figure 1 As shown in the figure, after 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 formyl phloroglucinol mixed terpene derivatives were given for 24 hours of intervention. The results showed that each formyl phloroglucinol mixed terpene derivative 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).

[0038] Example 2: Study on the lipid-lowering activity of formylphloroglucinol mixed terpenoid derivatives (with intracellular TC and TG content as indicators)

[0039] HepG2 cells in the logarithmic growth phase were taken, digested with trypsin to make a single cell suspension, counted with a cell counter, and diluted with complete culture medium to 1x 105 / mL. 1 mL of cell suspension was added to each well. 1 mL of complete culture medium was added to the 6-well plate to cover the bottom of the plate in advance, and 1 mL of cell suspension was added to each well. The plate was placed in an incubator and continued to be incubated until the cells adhered to the wall and grew to about 60%. The 6-well plate was taken out of the incubator, the original culture medium was aspirated, the first well was set as a blank control group, and complete culture medium containing 1% BSA was added. Freshly prepared 0.5 mM FFA solution was added to the remaining wells, 2 mL of the corresponding solution per well, and the plate was incubated in the incubator for 24 hours to complete the modeling. After the modeling is completed, the six-well plate is taken out, the original culture medium is sucked off, and DMSO or different drugs are added to each well of the culture plate to set up a blank control group (CON, complete culture medium), a model control group (MOD, adding corresponding concentrations of DMSO), and a compound group (40 μM). Three replicate wells are set for each group and continued to be placed in the incubator for incubation for 24 hours. The culture plate after drug administration is taken out, the culture medium is carefully sucked off, and PBS is added for washing. The above operation is repeated until the residual culture medium is completely washed away, and trypsin is added. 100 μL of trypsin is added to each well to digest the cells. The cells are gently scraped off with a cell scraper and transferred to a new 1.5 mL nuclease-free centrifuge tube. Centrifuge at room temperature at 1000 rpm for 5 minutes, slowly suck off the trypsin and add 200 μL PBS buffer, centrifuge at 1000 rpm for 5 minutes, gently suck off the culture medium, and add 100 μL PBS buffer was added, and small steel balls were added for grinding. The grinder was ground 5 times at 60Hz, 60s each time, and 10s were paused between each two times. Finally, the small steel balls were removed, and the cell homogenate was centrifuged at 4℃ and 12000rpm for 10min. The supernatant was taken and the total protein content in the supernatant was determined using the Biyuntian BCA protein content determination kit. Subsequent experiments were carried out according to the total cholesterol and triglyceride kits of Nanjing Jiancheng Company.

[0040] Experimental results:

[0041] like Figure 2 As shown in the figure, after 24 hours of free fatty acid (FFA) induction, the intracellular TC and TG contents of the model group increased significantly, nearly two times compared with the blank group (P<0.001), thus confirming the successful establishment of the FFA-induced lipid accumulation model. After 24 hours of intervention with five mixed terpenoid derivatives of formylphloroglucinol (all at a concentration of 40 μM), the intracellular TC and TG contents showed a downward trend, and all showed significant differences (p<0.01).

[0042] Example 3: Effects of compounds on fatty acid content produced in HepG2 cells

[0043] Take HepG2 cells in the logarithmic growth phase, digest them with trypsin and make a single cell suspension. After counting with a cell counter, dilute the cell suspension to 1x 105 / mL with complete culture medium, add 1mL of cell suspension to each well, add 1mL of complete culture medium 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 grow to about 60% by adhering to the wall, 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 8μL of the 2.5uM drug prepared in advance to the other wells. Continue to incubate in the incubator for 24h, take out the cells, absorb 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 it at 4℃ overnight. The next day, centrifuge at 5000xg and 4°C for 10 minutes, transfer the lower chloroform phase to a new glass tube, evaporate to dryness under nitrogen at 37°C, treat the sample with 1mL methanol and 50ul sulfuric acid at 100°C for 2h, cool to room temperature after treatment, and extract the derivatized palmitic acid by adding 1ml hydrate and 2mL n-hexane, separate the upper organic phase and dry it under nitrogen flow, then redissolve the dried palmitic acid in 100uL n-hexane for GC-MS analysis.

[0044] Chromatographic conditions: Helium flow rate maintained at 1 mL / min. Source temperature maintained at 230°C, mass spectrometer quadrupole temperature maintained at 150°C, interface temperature at 280°C, inlet temperature at 250°C. GC / MS analysis of fatty acid methyl esters (FAME) with 1 μL injection. No split. The column was started at 80°C for 2 minutes, then increased to 280°C at a rate of 10°C / min and held for 12 minutes.

[0045] Experimental results:

[0046] like Figure 3 As shown, the tested formylphloroglucinol mixed terpene derivatives significantly reduced the content of palmitic acid and stearic acid in cells at 20 μM and inhibited the synthesis of fatty acids.

[0047] Based on the above screening results, the compound MacrocarpalA was selected for further study.

[0048] Example 4: Study on the lipid-lowering activity of Macrocarpal A (MA) (using lipid accumulation as an indicator)

[0049] The experimental steps were the same as in Example 1, and the MA concentrations were set to 10 μM, 20 μM, and 40 μM.

[0050] Experimental results:

[0051] like Figure 4 As shown in the figure, after 24 hours of FFA stimulation, the lipid droplets in the model group increased significantly, indicating that FFA induced lipid accumulation in HepG2 cells. After 24 hours of intervention with DMSO and different concentrations of MA, Oil Red O staining showed a significant decrease in red lipid droplets. Compared with the model group, the number of intracellular lipid droplets in the four drug-treated groups was significantly reduced, and the higher the MA concentration, the more significant the reduction in intracellular lipid droplets, showing a clear dose-dependency.

[0052] Example 5: Study on the lipid-lowering activity of Macrocarpal A (with intracellular TC and TG content as indicators)

[0053] The experimental steps were the same as those in Example 2, and the MA concentrations were set to 10 μM, 20 μM, and 40 μM.

[0054] Experimental Results

[0055] like Figure 5 As shown in the figure, 24 hours after FFA induction, the total cholesterol in the model group increased by 1.5 times (P<0.001) and the triglyceride increased by 2.5 times (P<0.001) compared with the blank group, which verified the successful establishment of the FFA-induced lipid accumulation model. After administration of DMSO and different concentrations of MA, the total cholesterol and triglyceride in the low, medium and high dose MA groups decreased compared with the model group. With the increase of MA concentration, the decrease of total cholesterol and triglyceride also increased. The results showed that MA can reduce the TC and TG content in HepG2 cells in a dose-dependent manner.

[0056] Example 6: Effect of MacrocarpalA on lipid metabolism-related genes in HepG2 cells

[0057] 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 solution 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, aspirate the original culture medium, add DMSO or different concentrations of MA to each well of the culture plate, set them as blank control, MA low, medium and high dose groups (0.01, 0.1, 1μM) in turn, add the corresponding drug-containing culture medium, continue to incubate in the cell incubator for 8h, discard the culture medium, wash twice with PBS, add 500ul lysis solution to each well, let it stand for 8min, scrape all the cells with a cell scraper and transfer them to the DNA adsorption column, 12000xg, 30s, retain the filtrate, add 0.5 times the volume of anhydrous ethanol to the filtrate, mix thoroughly, transfer the mixed liquid to the RNA centrifugal column, 12000xg, 30s, discard the filtrate, add 700ul Wash to the RNA column Buffer A, 12000xg, 30s, discard the filtrate, add 700ul WashBufferB to the RNA column, 12000xg, 30s, discard the filtrate, add 500ul Wash BufferB to the RNA column, 12000xg, 1min, discard the filtrate, centrifuge the RNA column again, 12000xg, 1min, discard the retention tube, replace with a new 1.5ml enzyme-free centrifuge tube, add 60ul preheated DEPC water to the center of the RNA column, let it stand for 2min, 12000xg, 1min, get the RNA eluate. Then use NanoDrop2000 / 2000C spectrophotometer to determine the RNA concentration, take 1μL sample and drop it into the instrument base, read the sample concentration (ng / μL) and OD260 / OD280 value, read three times, calculate the average, OD260 / OD280 value in the range of 1.8-2.2 is considered to be high in RNA purity.

[0058] Table 1. Reverse transcription reaction solution ratio

[0059]

[0060] 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.

[0061] Table 2 PCR amplification reaction system

[0062]

[0063] Table 3 PCR amplification program

[0064]

[0065] Table 4 Target gene primer sequences

[0066]

[0067]

[0068] Experimental results:

[0069] like Figure 6 As shown, MA at a concentration of 1 μM significantly upregulated the gene expression of LDLR (upregulated 2.4 times), while downregulated the gene expression of transcriptional regulators SREBP-1 (downregulated 0.45 times) and SREBP-2 (downregulated 0.55 times).

[0070] Example 7: Effect of Macrocarpal A on HFD-induced hyperlipidemia mice

[0071] 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, MA low-dose group, and MA high-dose 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 eye sockets, serum was collected, and serum total cholesterol, triglyceride, and low-density lipoprotein levels were tested. The experimental results showed that the high-fat model was successfully established. After that, the drug was administered orally by gavage every day. 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 weight was recorded. The mice were anesthetized, and the eyeballs were removed to collect whole blood. After standing for 2 hours, the blood was centrifuged at 4000 rpm / min at 4°C for 10 minutes. The supernatant was collected and stored at -80°C. The liver weight was collected and weighed, and a part was taken for frozen section for subsequent Oil Red O staining, a part was fixed with 4% paraformaldehyde for subsequent HE section staining, and the remaining part was quickly frozen in liquid nitrogen and stored at -80°C.

[0072] The kit of Nanjing Jiancheng Bioengineering Institute was used to detect the activity of TC, TG, LDL-C, HDL-C, ALT and AST in the serum of mice at the end of drug administration. Compared with the blank group, the levels of TC, TG, LDL-C, AST and ALT in the model group mice were significantly increased. After MA treatment, the above indicators were significantly decreased in a dose-dependent manner, indicating that MA reduced the blood lipid level of mice and improved the liver damage of mice.

[0073] Experimental results:

[0074] like Figure 7 , Figure 8 As shown in the figure, 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 3 times. The TG and HDL-C groups also had significant differences (P<0.001), indicating that lipid metabolism disorders had occurred in the mice. After the mice were treated with different doses of MA for four weeks, the two MA-administered groups achieved positive regulation 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 obvious dose dependence. At a dose of 15 mg / kg, MA down-regulated TC levels by 30%, TG levels by 42%, LDL-C levels by 40%, and up-regulated HDL-C content by 35%. In terms of AST and ALT levels, compared with the blank group, the AST and ALT levels in 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 MA-administered group showed an excellent effect of reducing serum transaminase levels, and the two MA-administered groups also showed obvious dose-dependency in reducing serum transaminase levels. The low-dose MA group reduced AST levels by 18% and ALT levels by 37.5%, while the high-dose MA group reduced AST levels by 32% and ALT levels by 55%.

[0075] Example 8: Effect of Macrocarpal A on Lipid Content in Mouse Liver

[0076] 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.

[0077] Experimental results:

[0078] like Fig. 9 As shown in the figure, the TC and TG levels in the liver of the high-fat model group mice were significantly higher than those in the blank group (P<0.001), indicating that lipid accumulation has occurred in the liver. Figure 3-4 a), compared with the model group, the liver TC levels of the drug-treated group showed a downward trend, and the high-dose MA group significantly down-regulated the liver TC content compared with the model group (P<0.05), and there was a dose-dependent relationship between the two MA groups. In terms of TG level, the MA group showed a downward trend, but there was no significant difference in the low-dose MA group. The high-dose MA group significantly down-regulated the liver TG content (P<0.001), and there was a dose-dependent relationship between the two MA-treated groups.

[0079] Example 9: Effects of Macrocarpal A on General Indicators of Experimental Animals

[0080] 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.

[0081] Experimental Results

[0082] like Fig.10 As shown in the figure, the liver weight, liver index, epididymal fat weight and epididymal fat coefficient of mice in each group showed 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 MA effectively reduced the liver index and epididymal fat coefficient of mice. In addition, the liver weight of mice in the model group increased, the elasticity decreased, the granularity was obvious, and the edges were blunt; the appearance of mice in the MA group was significantly improved compared with the model group. This shows that MA can reduce liver lipid synthesis.

[0083] Example 10: Effect of Macrocarpal A on the pathological structure of liver tissue in hyperlipidemia model mice

[0084] After the administration in Example 7, different tissues were taken, and the liver tissue fixed with paraformaldehyde, the epididymal adipose tissue, and the liver tissue embedded in the frozen embedding solution were submitted to the Pharmacology and PDX Efficacy Evaluation Platform of China Pharmaceutical University to make tissue wax blocks for HE, Oil Red, Masoon, Sirius Red, and F4 / 80 staining. Then, the digital pathology slide scanner (NanoZoomer S60) was used to scan and analyze using NDP software.

[0085] Experimental results:

[0086] like Fig.11 As 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 increased several times, the nucleus was displaced, there was no obvious visible cytoplasm, the liver cell fat vacuolation, ballooning was obvious, the liver plates were arranged in disorder, and the liver cords and sinusoids could no longer be distinguished; Oil red O staining of the liver showed that the mice in the model group induced by the high-fat diet had obvious liver lipid accumulation, with a large number of lipid droplets and a large volume; H&E staining of epididymal fat showed that the fat cells expanded several times, and the mice had obvious lipid metabolism disorders. After MA administration, the liver fat vacuolation was significantly improved, the lipid droplets were reduced, and the expansion coefficient of epididymal fat cells decreased. All indicators were dose-dependent.

[0087] The results are as follows Fig.12 As shown in the figure, the results of mouse liver Masoon showed that the interstitial spaces between liver cells of mice in the HFD high-fat model group were filled with blue collagen fibers and collagen, which was consistent with the results of Sirius red staining, indicating that HFD mice had obvious liver fibrosis. In addition, inflammatory response is a prerequisite for fibrosis after liver damage. Macrophages are key regulators of the fibrosis cascade and are essential for liver damage, the initiation, maintenance and regression of fibrosis. As shown in the results of mouse liver F4 / 80 staining, the nuclei of liver cells in the HFD high-fat model group mice were surrounded by a large number of macrophages stained red. The liver cells of the surface HFD mice suffered severe erosion by inflammatory macrophages, and their fibrosis was aggravated. After MA administration intervention, liver fibrosis subsided, inflammatory damage was reduced, and all indicators were dose-dependent.

Claims

1. Use of a type of formylphloroglucinol mixed terpene derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs for preventing and treating diseases related to abnormal liver lipid metabolism, characterized in that: The structure of the formyl phloroglucinol mixed terpene derivative is shown in the following formula: R is independently selected from the following structures: R1 is each independently selected from hydrogen, methyl, ethyl, allyl, prenyl, formyl, acetyl or propionyl.

2. The use according to claim 1, characterized in that: The formylphloroglucinol mixed terpene derivative is selected from the following compounds:

3. Use of a pharmaceutical composition in the preparation of a drug for preventing and treating diseases related to abnormal liver lipid metabolism, characterized in that: Any one or more of the formylphloroglucinol mixed terpene derivatives or pharmaceutically acceptable salts thereof as described in claim 1 or 2, and their enantiomers, diastereomers, tautomers, solvates or salts of solvates and pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that: The dosage forms of the pharmaceutical composition 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.

5. The use according to any one of claims 1 to 4, characterized in that: The disease related to abnormal liver lipid metabolism is selected from obesity, dyslipidemia, metabolism-related fatty liver disease, atherosclerosis, non-alcoholic steatohepatitis, type 2 diabetes or coronary heart disease.

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