Application of N-trans-feruloyltyramine in treatment of non-alcoholic fatty liver disease, verification method and medicine

Abstract

The invention discloses an application of N-trans-feruloyltyramine in treatment of a non-alcoholic fatty liver disease. The invention further discloses a verification method for proving that the N-trans-feruloyltyramine is used for treating the non-alcoholic fatty liver disease. The invention further discloses a medicine for treating the non-alcoholic fatty liver disease. The invention discloses N-trans-feruloyl tyramine which can be applied to prevention or treatment of NAFLD (non-alcoholic fatty liver disease). In-vitro cell experiments find that N-trans-feruloyltyramine can reduce lipid deposition and oxidative stress damage of HepG2 cells induced by OA / PA, reduce the level of TC and TG in the cells by promoting efflux of TC and TG in the cells, reduce expression of MDA in the cells and promote expression of SOD, CAT and GSH in the cells; in-vivo animal experiments show that the N-trans-feruloyltyramine can reduce the levels of TC, TG and LDL-C in serum, reduce the levels of TC, TG, LPO, ALT and AST in liver, and relieve infiltration of inflammatory cells in liver tissue.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of N-trans-feruloyltyramine in treating non-alcoholic fatty liver disease, a verification method and a medicine. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD), also known as metabolic dysfunction-associated fatty liver disease (MASLD), is a leading cause of chronic liver disease worldwide. Its progression ranges from non-alcoholic simple fatty liver disease (NAFL) to non-alcoholic steatohepatitis (NASH), fatty liver fibrosis, cirrhosis, and hepatocellular carcinoma. Currently, the global prevalence of NAFLD is approximately 32%, and the prevalence of NAFLD in adults in my country is approximately 29%. It is characterized by excessive accumulation of triglycerides (TG) in hepatocytes. An imbalance between TG synthesis and clearance leads to hepatic steatosis, which, once progressing to NASH, causes irreversible damage to the liver. In terms of clinical treatment, Resmetirom, approved by the US Food and Drug Administration in 2024, is the first drug currently available for NASH. However, it is only effective in a select group of patients, and there are no approved drugs for the clinical treatment of NAFLD in China. This situation highlights the urgent need to explore new therapeutic targets and drugs for NAFLD. NAFLD has yet to establish a unified name in Traditional Chinese Medicine (TCM). In clinical practice, it is primarily categorized based on its clinical manifestations and symptom profile, including "fat and gas," "flank pain," "fullness," "liver obstruction," "accumulation," "accumulation syndrome," "phlegm turbidity," and "liver distension." Traditional Chinese medicine (TCM) has a long history of treating NAFLD with significant efficacy, demonstrating its synergistic regulatory effects across multiple components, targets, and pathways. Developing safer and more effective treatment strategies from TCM is a current research hotspot.

[0003] N-trans-feruloyltyramide is an alkaloid found in traditional Chinese medicines such as tangerine peel, hemp seed, buza leaf, taro, and ground peach blossom. It has pharmacological effects such as anti-inflammatory, lipid-lowering, anti-melanin, antiviral, and antibacterial properties, but its role in the treatment of NAFLD remains unclear. Cell membrane immobilized chromatography (CMIC) is a screening technique based on bioaffinity. It works by incubating drug extracts with specific cells, allowing potential active molecules to bind to targets on the cell membrane. Unbound free components are then washed away, and bound compounds are then dissociated and analyzed using appropriate methods to screen for bioactive substances. This technique combines the advantages of cell biology and chromatographic analysis and can be used to efficiently identify active ingredients in drugs. Based on this, the present invention establishes the HepG2-CMIC method to verify that N-trans feruloyltyramide is a membrane-bound component, and then verifies the effect of N-trans feruloyltyramide in treating NAFLD through in vitro and in vivo experiments, providing a research basis for the development of drugs for treating NAFLD with N-trans feruloyltyramide, and also providing new ideas for the screening of active ingredients. Summary of the Invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, provide a method for verifying the use of N-trans feruloyl tyramide in preventing and treating NAFLD by combining cell membrane solid phase chromatography technology with in vivo and in vitro experiments, and further increase the medicinal value of N-trans feruloyl tyramide.

[0005] In response to the above technical problems, the present invention provides a use of N-trans-feruloyltyramine in treating non-alcoholic fatty liver disease.

[0006] The above-mentioned N-trans feruloyl tyramine is used to treat non-alcoholic fatty liver disease, wherein the N-trans feruloyl tyramine reduces the levels of TC and TG in hepatocytes by promoting the efflux of TC and TG in hepatocytes.

[0007] The above-mentioned use of N-trans-feruloyltyramine in the treatment of non-alcoholic fatty liver disease, wherein the N-trans-feruloyltyramine alleviates non-alcoholic fatty liver disease by improving oxidative stress damage, and its mechanism is:

[0008] Reduce intracellular malondialdehyde levels;

[0009] Increase superoxide dismutase and catalase activities;

[0010] Increases intracellular glutathione levels.

[0011] The above-mentioned use of N-trans-feruloyltyramine in the treatment of non-alcoholic fatty liver disease, wherein the N-trans-feruloyltyramine alleviates non-alcoholic fatty liver disease by improving abnormal lipid metabolism, and its mechanism is:

[0012] Reduce the levels of TC, TG, and low-density lipoprotein cholesterol in serum, or reduce the levels of TC, TG, lipid peroxides, alanine aminotransferase, and aspartate aminotransferase in the liver.

[0013] A method for demonstrating that N-trans-feruloyltyramine can treat non-alcoholic fatty liver disease, the method comprising the following steps:

[0014] Step 1, establishment of HepG2 cell membrane chromatography and identification of binding components;

[0015] Step 2: In vitro cell experiments were performed to verify the anti-NAFLD activity of N-trans-feruloyltyramide;

[0016] Step 3: In vivo animal experiments were conducted to verify the anti-NAFLD activity of N-trans-feruloyltyramide.

[0017] The above-mentioned verification method for proving that N-trans feruloyltyramide can treat non-alcoholic fatty liver disease, wherein the step 1 verifies that N-trans feruloyltyramide is a membrane-bound component by establishing a HepG2-CMIC method.

[0018] The above-mentioned verification method for demonstrating the effect of N-trans-feruloyltyramide on the treatment of non-alcoholic fatty liver disease, wherein the specific method of step 2 includes: establishing an OA / PA-induced lipid deposition model, staining and observing intracellular lipid droplets, and measuring the levels of TC and TG inside and outside the cells, as well as measuring the intracellular oxidative stress indicators glutathione, superoxide dismutase, malondialdehyde and catalase.

[0019] The above-mentioned verification method for demonstrating the effect of N-transferuloyltyramide on the treatment of non-alcoholic fatty liver disease, wherein the specific method of step 3 includes: replicating the Triton WR-1339 acute NAFLD model, observing liver damage, measuring the levels of TC, TG and low-density lipoprotein cholesterol in serum, and measuring the expression of TG, TC, lipid peroxides, aspartate aminotransferase and alanine aminotransferase in the liver.

[0020] A medicine for treating non-alcoholic fatty liver disease, comprising N-trans-feruloyltyramide.

[0021] The above-mentioned drug for treating non-alcoholic fatty liver disease, wherein the concentration of the N-trans-feruloyltyramine in the drug is 1 to 50 μM.

[0022] The beneficial effects of the present invention include:

[0023] (1) The present invention establishes a HepG2-CMIC (HepG2-cell solid phase chromatography) method to verify that N-trans-feruloyltyramide is a membrane-bound component;

[0024] (2) In vitro cell experiments revealed that N-trans-feruloyltyramine could reduce lipid deposition and oxidative stress damage in HepG2 cells induced by OA / PA (2:1), reduce the intracellular levels of TC and TG by promoting the efflux of intracellular TC and TG, reduce the expression of intracellular MDA, and promote the expression of intracellular SOD, CAT, and GSH;

[0025] (3) In vivo animal experiments showed that N-trans-feruloyltyramide can reduce serum TC, TG, and LDL-C levels, reduce liver TC, TG, LPO, ALT, and AST levels, and alleviate the infiltration of inflammatory cells in liver tissue;

[0026] (4) N-trans-feruloyltyramide can be used to prevent or treat NAFLD, providing a research basis for the development of NAFLD therapeutic drugs based on N-trans-feruloyltyramide. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below with reference to the accompanying drawings and examples.

[0028] Figure 1 In the present invention, N-trans-feruloyltyramide is determined to be a component that specifically binds to HepG2 cells by using cell solid phase chromatography;

[0029] Figure 2 The in vitro cell experiments in the present invention verify the therapeutic effect of N-trans feruloyltyramide on NAFLD (A: Oil Red O staining; B: cell viability; C: biochemical index determination);

[0030] Figure 3 This is the in vivo animal experiment in the present invention to verify the therapeutic effect of N-trans feruloyltyramide on NAFLD (A: HE staining; B: biochemical index determination). DETAILED DESCRIPTION

[0031] The present invention is further described below through specific examples to make the technical solution of the present invention easier to understand and grasp, rather than to limit the present invention.

[0032] In this embodiment, unless otherwise specified, all reagents can be purchased on the market.

[0033] In this embodiment, unless otherwise specified, the methods used are all conventional methods.

[0034] The detection drug used in the present invention is N-trans-Feruloyltyramine, the molecular formula of which is C 18 H 19NO4, CAS No. 66648-43-9, has the following structure:

[0035]

[0036] The present invention uses cell membrane solid phase chromatography to verify that N-trans feruloyl tyramide specifically binds to HepG2 cell membranes, and N-trans feruloyl tyramide is a membrane-bound component; in vivo and in vitro experiments are used to verify the anti-NAFLD activity of N-trans feruloyl tyramide, proving that N-trans feruloyl tyramide can prevent and treat NAFLD. The method comprises the following steps:

[0037] Step 1, establishment of HepG2 cell membrane chromatography method and identification of bound components: N-trans-feruloyltyramide was verified as a membrane-bound component by establishing a HepG2-CMIC method;

[0038] Step 2: In vitro cell experiments were conducted to verify the anti-NAFLD activity of N-trans-feruloyltyramide: an OA / PA (2:1)-induced lipid deposition model was established, intracellular lipid droplets were stained and observed, and intracellular and extracellular TC and TG levels were measured. In addition, intracellular oxidative stress indicators such as glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) were measured.

[0039] Step 3: In vivo animal experiments were performed to verify the anti-NAFLD activity of N-trans-feruloyltyramide: the Triton WR-1339 acute NAFLD model was replicated, liver damage was observed, serum TC, TG, and low-density lipoprotein cholesterol (LDL-C) levels were measured, and the expression of TG, TC, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the liver was measured.

[0040] Specifically, the specific method of step 1 includes:

[0041] Step 1-1, screening the safe concentration of N-trans-feruloyltyramide by CCK8 method;

[0042] Steps 1-2, N-trans-feruloyltyramide binds to the HepG2 cell membrane;

[0043] Steps 1-3, elution of unbound components / non-specifically bound components;

[0044] Steps 1-4, N-trans-feruloyltyramide dissociates from HepG2 cell membranes;

[0045] Step 1-5: The active ingredient dissociation solution obtained in step 1-4 is subjected to UPLC-Q-TOF-MS / MS analysis and compared with the N-trans-feruloyltyramide reference substance to confirm that N-trans-feruloyltyramide can specifically bind to the HepG2 cell membrane.

[0046] Specifically, the specific method of step 2 includes:

[0047] Step 2-1: Cells were seeded into 12-well plates at a certain cell density overnight. After modeling and drug treatment, the cells were stained with Oil Red O for 12 hours to identify intracellular lipid droplets, and the cells were observed and photographed under a microscope.

[0048] In step 2-2, cells were seeded into 6-well plates at a certain cell density overnight. After modeling and drug treatment, 12 hours later, the intracellular and extracellular TC and TG levels, as well as the levels of intracellular oxidative stress indicators glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) were measured according to the instructions of the TC, TG, SOD, MDA, and CAT kits.

[0049] Specifically, the specific method of step 3 includes:

[0050] Step 3-1: After 7 days of adaptive feeding, C57BL / 6 mice were treated with the drug twice daily for 5 consecutive days. On the 4th day, except for the blank group, TritonWR-1339 (0.1 mL / 20 g) was injected intramuscularly to induce an acute NAFLD model. One hour after the last administration on the 5th day, the mice were anesthetized, and orbital blood and liver samples were collected.

[0051] Step 3-2: According to the instructions of the TC, TG, LDL-C, LPO, AST, ALT and other kits, the levels of TC, TG and LDL-C in serum, and the levels of TC, TG, lipid peroxide (LPO), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the liver were determined.

[0052] Example 1: Establishment of HepG2 cell membrane chromatography and identification of binding components

[0053] (1) Determination of the dosage concentration of N-trans-feruloyltyramine

[0054] HepG2 cells were cultured at 1×10 5 Cells were seeded at a density of 100 μL / well in a 96-well plate. After the cells grew to 50-60% (24 h), they were treated with a series of drug concentrations (400, 200, 100, 50 μM) for 24 hours (100 μL / well), in parallel 3 times. Then, 10 μL of CCK-8 solution was added to each well, and cell viability was measured after incubation in an incubator for 2 h. The absorbance was measured at 450 nm using a microplate reader. To evaluate the drug's in vitro safety and screen the drug's maximum safe concentration, which will serve as the drug concentration for subsequent experiments.

[0055] N-trans-feruloyltyramide showed an increased inhibitory effect on HepG2 cell growth with increasing concentrations. At concentrations above 50 μM, HepG2 cell viability was less than 80%. Therefore, to ensure the normal progress of subsequent experiments, 50 μM was selected as the administration concentration of N-trans-feruloyltyramide.

[0056] (2) Drug configuration

[0057] Weigh an appropriate amount of N-trans-feruloyltyramide into a sterile brown EP tube, add DMSO to make a 100 mM stock solution, and store at -20°C. The complete cell culture medium used in this example consists of MEM-α basal medium, 10% fetal bovine serum, and 1% double-antibody. Dilute the stock solution with complete culture medium to the desired concentration.

[0058] Solution A: Accurately weigh 2.12 g of citric acid and dilute to 100 mL with ultrapure water in a volumetric flask. Set aside.

[0059] Solution B: Accurately weigh 2.96 g of trisodium citrate and dilute to a 100 mL volumetric flask with ultrapure water. Set aside.

[0060] Dissociation solution preparation: Measure 52.4 mL of solution A and 27.6 mL of solution B, mix well, adjust the pH to 4.0, filter through a 0.22 μm microporous filter membrane, and set aside.

[0061] (3) Cellular administration

[0062] Cells were seeded in 10 cm cell culture dishes, and blank and drug-treated groups were set up. When the cell confluency reached 90%, the culture medium was aspirated and the cells were washed three times with PBS. The drug-treated group was treated with 5 mL of complete culture medium containing 50 mM N-trans-feruloyltyramide, while the blank group was treated with an equal amount of drug-free culture medium. The cells were then incubated in an incubator (37°C, 5% CO2) for 1 hour.

[0063] (4) Elute unbound components / non-specific binding components

[0064] After incubation for 1 hour, the culture medium was discarded and 5 mL of basal culture medium was added for 5 washes. Each wash required the entire culture dish to be soaked and the culture dish to be gently shaken for thorough washing. The cells were digested with trypsin and transferred to a 15 mL centrifuge tube. The cells were centrifuged at 800 rpm for 5 minutes. The supernatant was discarded and the cells were washed 3 times with PBS. The last wash solution was collected.

[0065] (5) Dissociation of N-trans-feruloyltyramine from HepG2 cell membranes and enrichment of N-trans-feruloyltyramine

[0066] After washing away any nonspecifically bound components, dissociate the cells in 3 mL of dissociation buffer (pH 4.0) in an incubator for 2 hours to inactivate the effector targets. Collect the dissociation buffer, enrich and purify it using SPE, blow dry with nitrogen, and reconstitute it in 500 μL of 50% acetonitrile. Vortex for 3 minutes, centrifuge at 12,000 rpm for 15 minutes, and remove the supernatant for later use.

[0067] SPE enrichment method: Install a C18 SPE solid-phase extraction column on a solid-phase extraction device, rinse the filler with 5 mL of methanol and 4 mL of ultrapure water in sequence for activation and equilibrium treatment, add the sample to the filler at a flow rate of 1 mL / min, let it stand for 1 min, rinse with 5 mL of ultrapure water to wash away interferences such as inorganic salts, and elute with 5 mL of eluent acetonitrile: water (1:1) solution, and collect the eluate.

[0068] (6)UPLC-Q-TOF-MS / MS analysis

[0069] The final washing solution sample, blank group dissociation solution sample and drug-treated group dissociation solution sample were analyzed by UPLC-Q-TOF-MS / MS respectively.

[0070] Chromatographic conditions: AB SCIEX Exion LC ultra-high performance liquid chromatograph was used, the chromatographic column was a Waters CORTECS UPLC C18 column (2.1 mm × 150 mm, 1.6 μm), 0.1% formic acid aqueous solution was used as mobile phase A, methanol was used as mobile phase B, gradient elution was performed, and the flow rate was 0.3 mL min -1 , column temperature was 40℃, injection volume was 1μL; gradient elution program was: 0-4min, 14%-30% B; 4-16min, 30%-58% B; 16-25min, 58-78% B.

[0071] Mass spectrometry conditions: An AB SCIEX X500R QTOF mass spectrometer was used with an electrospray ionization (ESI) source in negative ion mode with a scan range of m / z 100 to 1500, a nebulizer gas pressure of 282.1 kPa, an auxiliary gas pressure of 282.1 kPa, a curtain gas pressure of 206.8 kPa, a nebulizer temperature of 500°C, and an electrospray ionization (ESI) source. In negative ion mode, the capillary voltage was -5.5 kV, the fragmentor voltage was -80 V, and the collision energy was 35±15 eV.

[0072] Table 1 Mass spectrometry information of specific binding components

[0073]

[0074] Based on the comparison and identification of lysis behavior, reference material information and HepG2 cell-specific binding components, it was determined that N-trans-feruloyltyramide can specifically bind to the HepG2 cell membrane and is a HepG2 cell membrane-specific binding component.

[0075] Example 2: In vitro cell experiment to verify the anti-NAFLD activity of N-trans-feruloyltyramide

[0076] (1) Determination of TC, TG, SOD, MDA and CAT contents

[0077] HepG2 cells were counted and 1×10 6 Cells were seeded at a density of 100 μL / mL in 6-well plates at 2 mL / well and allowed to grow overnight. After cell attachment, all cells, except the blank group, were treated with induction interventions (Control, 0.5 mM OA / PA, 0.5 mM OA / PA + N-trans-feruloyltyramide), with triplicate wells per group. After 24 hours, the remaining culture medium from each group was collected and set aside. The cells were trypsinized, 500 μL of PBS was added, and ultrasonic disruption was performed to obtain a cell extract, which was then placed on ice for analysis. Intracellular and extracellular TC and TG, as well as intracellular GSH, SOD, MDA, and CAT were measured according to the kit instructions.

[0078] N-trans-feruloyltyramide promotes the efflux of TC and TG in HepG2 cells to reduce their levels, improves OA / PA-induced lipid accumulation in HepG2 cells, and can increase the levels of intracellular oxidative stress indicators GSH, SOD and CAT, and reduce MDA levels.

[0079] (2) Oil Red O staining

[0080] HepG2 cells were counted at 1×10 5 Cells were seeded at a density of 0.5 mL / well in a 12-well plate at 100 μg / mL. After the cells adhered, all groups except the blank group were treated with induction intervention (Control, 0.5 mM OA / PA, 0.5 mM OA / PA + N-trans-feruloyltyramide), with 3 replicates per group. After 24 hours, the culture medium was discarded and the cells were stained and photographed according to the Oil Red O staining instructions.

[0081] After Oil Red O staining, it was observed that compared with the blank group, the red lipid droplets in the OA / PA group were significantly increased. N-trans-feruloyltyramide intervention treatment significantly inhibited the increase of lipid droplets in HepG2 cells and alleviated OA / PA-induced lipid accumulation in HepG2 cells.

[0082] Example 3: In vivo animal experiment to verify the anti-NAFLD activity of N-trans-feruloyltyramide

[0083] (1) Preparation of drugs and dosage setting

[0084] Preparation of Triton WR-1339 solution: Weigh the corresponding amount of Triton WR-1339, add the corresponding amount of normal saline, and directly stir with a glass rod to dissolve it into a solution with a concentration of 0.12 g / mL.

[0085] Fenofibrate group: The taking method of fenofibrate capsules is oral. The daily dosage for adults in the instruction manual is 200 mg. According to the "body surface area ratio" in "Methodology of Traditional Chinese Medicine Pharmacological Research", the clinically equivalent dose of fenofibrate capsules is 26.0 mg / kg. During the experiment, it is prepared according to the dose with normal saline and made into a mass concentration of 2.6 mg / mL.

[0086] N-trans-feruloyltyramine group: Referring to the administration method of fenofibrate and its clinically equivalent dose for mice, 26.0 mg / kg is selected as the clinical administration dose for mice in the N-trans-feruloyltyramine group. Weigh an appropriate amount of N-trans-feruloyltyramine compound and prepare it with normal saline to make a solution with a mass concentration of 2.6 mg / mL.

[0087] (2) Animal grouping, model establishment and administration

[0088] Twenty-four SPF-grade mice (animal source: Guangdong Provincial Center for Medical Laboratory Animals; facility use license number: SCXK (Guangdong) 20220-0059; experimental animal quality certificate number: 44007200143334) that have been adaptively fed for 7 days are randomly divided into 4 groups: blank, model, fenofibrate, and N-trans-feruloyltyramine groups. The mice in the administration groups are continuously administered for 5 days, twice a day. The model and blank groups are given the corresponding volume of normal saline. At 5:00 pm on the 3rd day of administration, except for the blank group, the mice in the other groups are intramuscularly injected with Triton WR-1339 (0.1 mL / 20 g) to induce an acute NAFLD model. The blank group is intramuscularly injected with the same volume of normal saline in the same way. After model establishment, the mice in each group are allowed to eat normally. At 9:00 pm on the 4th day, the mice in each group start to fast for 12 hours without water. At 9:00 on the 5th day, that is, 1 hour after the last administration, the mice in each group are sacrificed by eyeball blood collection, centrifuged at 3000 rpm for 10 minutes, the serum is taken and stored in a -80°C refrigerator; the mouse liver is taken and stored in a -80°C refrigerator, and part of the liver is stored in 4% paraformaldehyde.

[0089] (3) Determination of biochemical indexes in liver and serum

[0090] According to the requirements of the kit instruction manual, determine the levels of TC, TG, and LDL-C in the serum of mice, and the levels of TC, TG, AST, ALT, and LPO in the liver.

[0091] N-trans-feruloyltyramide can reduce the levels of TC, TG and LDL-C in serum, reduce the levels of TC, TG, LPO, ALT and AST in the liver, and improve TritonWR-1339-induced lipid accumulation and liver function damage.

[0092] (4) Pathological section observation

[0093] The liver was fixed and preserved in 4% paraformaldehyde, and then fixed, dehydrated, transparent, immersed in wax, embedded and sliced, dewaxed, and stained with HE. The pathological morphological changes of the liver were observed under a microscope.

[0094] Histopathological examination of liver tissue from each group of mice revealed that in the acute NAFLD model induced by Triton WR-1339, hepatocytes were disorganized, infiltrated by numerous inflammatory cells, and contained small lipid droplets and vacuoles. However, treatment with N-trans-feruloyltyramide normalized the hepatocytes, resulting in a more regular arrangement and reduced inflammatory infiltration.

[0095] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the scope of the present invention, utilize the above-disclosed technical means and technical content to make many possible variations and modifications to the present invention, or modify it into equivalent embodiments with equivalent variations. Therefore, any equivalent variations made in accordance with the shape, structure, and principle of the present invention that do not depart from the content of the present invention's technical solution should be included in the scope of protection of the present invention.

Claims

1. Application of N-trans-feruloyltyramide in the treatment of non-alcoholic fatty liver disease.

2. The use of N-trans-feruloyltyramine in treating non-alcoholic fatty liver disease according to claim 1, characterized in that: The N-trans feruloyltyramide reduces the levels of TC and TG in the liver cells by promoting the efflux of TC and TG in the liver cells.

3. The use of N-trans-feruloyltyramine in treating non-alcoholic fatty liver disease according to claim 1, characterized in that: The N-trans feruloyltyramine alleviates non-alcoholic fatty liver disease by improving oxidative stress damage, and its mechanism is: Reduce intracellular malondialdehyde levels; Increase superoxide dismutase and catalase activities; Increases intracellular glutathione levels.

4. The use of N-trans-feruloyltyramine in treating non-alcoholic fatty liver disease according to claim 1, characterized in that: The N-trans feruloyltyramine alleviates non-alcoholic fatty liver disease by improving abnormal lipid metabolism, and its mechanism is: Reduce the levels of TC, TG, and low-density lipoprotein cholesterol in serum, or reduce the levels of TC, TG, lipid peroxides, alanine aminotransferase, and aspartate aminotransferase in the liver.

5. A method for demonstrating that N-trans-feruloyltyramine can treat non-alcoholic fatty liver disease, characterized in that: The verification method comprises the following steps: Step 1, establishment of HepG2 cell membrane chromatography and identification of binding components; Step 2: In vitro cell experiments were performed to verify the anti-NAFLD activity of N-trans-feruloyltyramide; Step 3: In vivo animal experiments were conducted to verify the anti-NAFLD activity of N-trans-feruloyltyramide.

6. The method for verifying that N-trans-feruloyltyramine can treat non-alcoholic fatty liver disease according to claim 5, characterized in that: In step 1, the HepG2-CMIC method is established to verify that N-trans feruloyltyramide is a membrane-bound component.

7. The method for verifying that N-trans-feruloyltyramine can treat non-alcoholic fatty liver disease according to claim 5, characterized in that: The specific method of step 2 includes: establishing an OA / PA-induced lipid deposition model, staining and observing intracellular lipid droplets, measuring the levels of TC and TG inside and outside the cells, and measuring the intracellular oxidative stress indicators glutathione, superoxide dismutase, malondialdehyde and catalase.

8. The method for verifying that N-trans-feruloyltyramine can treat non-alcoholic fatty liver disease according to claim 5, characterized in that: The specific method of step 3 includes: replicating the TritonWR-1339 acute NAFLD model, observing liver damage, measuring the levels of TC, TG and low-density lipoprotein cholesterol in serum, and measuring the expression of TG, TC, lipid peroxides, aspartate aminotransferase and alanine aminotransferase in the liver.

9. A drug for treating non-alcoholic fatty liver disease, characterized in that: The drug includes N-trans-feruloyltyramide.

10. The drug for treating non-alcoholic fatty liver disease according to claim 9, characterized in that: The concentration of the N-trans feruloyltyramine in the drug is 1 to 50 μM.

Images