Broccoli leaf extract and application thereof in preparation of products for preventing and treating metabolism-related fatty liver diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a broccoli leaf extract and application thereof in preparation of products for preventing and treating metabolism-related fatty liver diseases. According to the invention, the active ingredients in the broccoli leaves are extracted by adopting a solvent, and the obtained broccoli leaf extract shows remarkable blood fat reduction, oxidation resistance and anti-inflammatory activity, which proves that BLCE can relieve MAFLD by inhibiting liver lipid accumulation, oxidative stress and inflammation; therefore, a new application that the broccoli leaf extract can be used as a dietary supplement for relieving MAFLD is developed.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a broccoli leaf extract and an application thereof, in particular to an application in preparing a product for preventing and treating metabolism-related fatty liver disease. Background Art

[0002] As a nutritious vegetable, broccoli is loved by people and widely grown around the world. However, during the harvesting and processing of broccoli, broccoli leaves are usually regarded as by-products. Traditionally, a large number of broccoli leaves are discarded in the field or simply treated as low-value feed, resulting in a waste of resources. Some leaves used as feed have a low utilization rate due to their high fiber content and poor palatability. At present, the processing and utilization of broccoli leaves not only increases the processing costs of growers and processing companies, but also creates potential pressure on the environment, such as stacking and rotting to produce greenhouse gases and occupy land resources.

[0003] At present, the utilization technology and related research on broccoli leaves are extremely limited, and mainly focus on two aspects: first, using chemical means to extract certain active ingredients therein to obtain active ingredients with certain functions, such as patent CN111011589A and patent CN101993480A, etc., all of which are to extract protein components in broccoli leaves to obtain feed components that are easier for animals to absorb, and the utilization rate is limited; second, the efficacy of broccoli leaf extracts is studied, such as patent CN107173805A discloses that the alcohol extract of broccoli leaves has a certain lung cancer inhibitory effect.

[0004] Metabolic-associated fatty liver disease (MAFLD) is currently the most common liver and metabolic disease in the world. Unhealthy lifestyle habits such as sitting for long periods of time and lack of exercise, and unhealthy eating habits such as excessive dietary calories and unreasonable dietary structure are closely related to the increasing incidence of MAFLD.

[0005] The treatment of MAFLD currently mainly includes lifestyle changes and personalized drug treatment. Among them, personalized Western medicine treatments include insulin sensitizers, vitamin E, weight loss and lipid-lowering drugs, liver protection and enzyme-lowering drugs, etc. However, while Western medicine is taking effect, it can also transport lipids to the liver, aggravating liver lipid metabolism, and may further aggravate liver fatty degeneration; while traditional Chinese medicine and its combination have fewer side effects and good effects, becoming a hot research direction for the treatment of MAFLD.

[0006] At present, there are no relevant studies and reports on the application of broccoli leaf extract in the prevention and treatment of metabolism-related non-fatty liver disease. Summary of the invention

[0007] To solve the above technical problems, the present invention provides a broccoli leaf extract and its application, especially in the preparation of products for preventing and treating metabolic associated fatty liver disease.

[0008] Metabolic associated fatty liver disease is a chronic liver disease characterized by liver fat accumulation and metabolic abnormalities, and its pathology is closely related to lipid metabolism disorders, chronic inflammation and oxidative stress.

[0009] The present invention identified the active ingredients in broccoli leaf extract (BLCE) by HPLC-MS / MS and chemical methods, confirmed its significant antioxidant activity through three in vitro tests, and in oleic acid (OA)-induced HepG2 cells, BLCE attenuated the excessive production of reactive oxygen species (ROS), restored antioxidant markers (SOD, MDA and GSH), and reduced intracellular TC and TG; in LPS-induced RAW264.7 cells, BLCE significantly inhibited the production of inflammatory factors (TNF-α, IL-6, IL-1β), showing an anti-inflammatory effect; in a high-fat diet (HFD)-induced MAFLD mouse model, BLCE reversed MAFLD by reducing serum and liver lipid metabolism, restoring liver function and morphology, and reducing liver injury, inflammation and oxidative stress. Therefore, BLCE can be used as a dietary supplement to alleviate MAFLD by inhibiting liver lipid accumulation, oxidative stress and inflammation.

[0010] The present invention provides a new application of broccoli leaf extract, that is, the application of broccoli leaf extract in the preparation of products for preventing and treating metabolic associated fatty liver disease.

[0011] Preferably, the product is any one of food and medicine. Preferably, the broccoli leaf extract is obtained by extracting the leaves of broccoli with a solvent, and the solvent includes at least one of water and alcohol solvents.

[0012] More preferably, the solvent is water.

[0013] Preferably, the form of the extract includes any one of an extract obtained by directly extracting with a solvent, a concentrated solution obtained by concentrating the extract, and a freeze-dried preparation obtained by adding freeze-drying excipients to the extract.

[0014] More preferably, the preparation method of the broccoli leaf extract is as follows:

[0015] (1) Select, wash broccoli leaves, vacuum freeze-dry at a temperature of -80 ± 5 °C, pulverize, and store for later use;

[0016] (2) Take the powder in (1), add water at a mass-to-volume ratio of 1 g: 20 - 50 mL, mix evenly, stir and extract at 60 - 80 °C for 1.5 - 3 h, centrifuge to obtain the supernatant, and concentrate to obtain the broccoli leaf extract described above.

[0017] Furthermore, in the application provided by the present invention, the broccoli leaf extract plays a role in preventing and treating metabolic associated fatty liver disease through at least one of the following ways: reducing oleic acid-induced lipid accumulation and oxidative stress in HepG2 cells, decreasing the levels of inflammatory factors, and weakening the inflammatory response of LPS-induced RAW264.7 cells and improving liver fibrosis.

[0018] The beneficial effects of the present invention are as follows:

[0019] (1) It provides a new use of broccoli leaves, that is, applying broccoli leaves to the preparation of products for treating metabolic associated fatty liver disease, improving the utilization rate of broccoli leaves, and endowing broccoli leaves with extremely high medicinal value;

[0020] (2) Using water as the extraction solvent to extract broccoli leaves, the obtained BLCE exhibits significant lipid-lowering, antioxidant and anti-inflammatory activities, proving that BLCE can alleviate MAFLD by inhibiting hepatic lipid accumulation, oxidative stress and inflammation, and can be used as an effective dietary supplement.

[0021] (3) Experimental results show that: BLCE can significantly reduce the levels of TC and TG in HepG2 cells, reduce the levels of TC, TG and LDL-C in the serum and liver of MAFLD mice, and increase the level of HDL-C, showing good lipid-lowering efficacy; in addition, the oil red O staining results show that BLCE further confirms its lipid-lowering effect by reducing the number of lipid droplets;

[0022] In addition, in vitro experiments measured by DPPH, ABTS and FRAP prove the significant antioxidant activity of BLCE, and BLCE significantly reduces the levels of ROS and MDA in HepG2 cells, and increases the levels of SOD and GSH. In MAFLD mice, BLCE significantly reduces the level of MDA, while increasing the levels of SOD, GSH and CAT in the liver;

[0023] Moreover, the treatment of BLCE significantly reduces the levels of inflammatory factors TNF-α, IL-6 and IL-1β in LPS-induced RAW264.7 cells, and the supplementation of BLCE also significantly reduces the levels of TNF-α, IL-6 and IL-1β in the serum and liver of MAFLD mice;

[0024] In addition, mouse experiments show that BLCE can restore the liver function of MAFLD mice and reduce liver damage. Brief Description of the Drawings

[0025] Figure 1 This shows the effect of BLCE on lipid accumulation and oxidative stress in OA-induced HepG2 cells in Example 3 of the present invention;

[0026] Figure 2 This shows the effect of BLCE on the inflammatory response of lipopolysaccharide-induced RAW264.7 cells in Example 4 of the present invention;

[0027] Figure 3 This shows the effect of BLCE on the physiological indexes of MAFLD mice in Example 5 of the present invention;

[0028] Figure 4 This shows the effect of BLCE on the serum and liver lipid levels of MAFLD mice in Example 6 of the present invention;

[0029] Figure 5 This shows the effect of BLCE on the liver function and histopathology of MAFLD mice in Example 7 of the present invention;

[0030] Figure 6 This shows the effect of BLCE on liver inflammatory factors and liver fibrosis in MAFLD mice in Example 8 of the present invention. Detailed Description of the Invention

[0031] In order to enable those skilled in the art to better understand the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0032] Example 1

[0033] The broccoli leaves used in this example are produced in Jiyang District, Jinan City, Shandong Province.

[0034] A broccoli leaf extract (BLCE) was prepared as follows:

[0035] Take the broccoli waste, pick out the broccoli leaves therein, screen them, wash them, and then place the broccoli leaves in a vacuum at -80°C and freeze-dry them to a constant weight, crush them, and store them at -20°C for later use.

[0036] Take the frozen broccoli leaf powder, add water at a ratio of 1 g:30 mL, mix evenly, stir at 70°C for 2 h, then centrifuge at a centrifugal force of 4000 g for 10 min, collect the supernatant, concentrate it under reduced pressure at 40°C to obtain BLCE, freeze-dry it, and store it at a temperature of -20°C.

[0037] Identification of the active ingredients in BLCE of Example 2 The BLCE extract prepared in Example 1 was dissolved in water to prepare a 1.0 mg / mL solution.

[0038] The active substances in the extract were identified and quantified by HPLC-MS / MS, and the test results are shown in Table 1 below.

[0039] In addition, in the present invention, the antioxidant capacity of the prepared BLCE was comprehensively evaluated by three methods: DPPH radical scavenging activity, ABTS radical scavenging activity, and FRAP. The results are also shown in Table 1.

[0040] Table 1 Content of active ingredients and antioxidant capacity of BLCE

[0041]

[0042] Table 1 shows that the BLCE prepared by the method of the present invention is rich in components such as polysaccharides and proteins, and exhibits good antioxidant performance.

[0043] In addition, in the present invention, HPLC-MS / MS was used to analyze the chemical components of BLCE in the negative ionization mode, and the detailed information of the compounds identified from BLCE is shown in Table 2 below.

[0044] Table 2 Chemical composition of BLCE in the negative mode of UHPLC-MS / MS

[0045]

[0046]

[0047] Table 2 shows that a total of 35 compounds were identified from BLCE, including 6 flavonoid compounds, 6 cinnamic acids and their derivatives, 5 coumarins and their derivatives, 3 fatty acid acyl groups, 3 isoflavones, 3 organic hydroxy compounds, 2 pentenol lipids, and 7 other compounds (organic nitrogen compounds, sulfoxides, pyrans, indenes and their derivatives, imidazopyrimidines, cinnamaldehyde, benzene and its substituted derivatives).

[0048] Among the above-mentioned compounds, the content of 5-hydroxymethyl-2-furaldehyde is the highest (12.732%), followed by choline (11.825%). In addition, the unique compounds of the cruciferous family, such as sulforaphane (0.084%), choline sinapate (0.467%), and sinapic acid (0.049%), were also detected in BLCE by the HPLC-MS / MS method in the present invention. These results also indicate that the BLCE prepared by the present invention is rich in various bioactive compounds such as polysaccharides, polyphenols, and flavonoids.

[0049] Example 3

[0050] Effect of BLCE on lipid accumulation and oxidative stress in HepG2 cells induced by oleic acid (OA).

[0051] In vitro cell culture: The culture medium for HepG2 and RAW264.7 cells was DMEM medium supplemented with 1% antibiotic mixture (100 U / mL penicillin, 100 mg / mL streptomycin) and 10% (v / v) heat-inactivated fetal bovine serum, and cultured in an incubator at 37 °C with 5% CO2. The culture medium was changed every 3 days until the cell confluence reached 90%, and then subcultured at a ratio of 1:3.

[0052] Cell viability assay: HepG2 cells were seeded in 96-well plates at a density of 1×10 4 cells per well, and RAW264.7 cells were seeded in 96-well plates at a density of 5×10 4 cells per well and cultured for 24 h.

[0053] The cytotoxicity of OA (0 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 4 mM), LPS (0 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL) and BLCE (0 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL) was measured using the CCK-8 method to determine the optimal concentration for modeling and drug administration. The specific operation was as follows: LPS and OA were first diluted with DMSO and then further diluted with the culture medium to the required concentration. The final concentration of DMSO in each group was 0.1%. The blank group received only the culture medium, while the negative control group received 0.1% DMSO.

[0054] Determination of TC and TG in HepG2 cells: HepG2 cells were seeded in 6-well plates at a density of 1×10 5 cells per well and cultured for 24 h. 1 mM OA was used to induce excessive lipid synthesis and accumulation to construct a fatty liver HepG2 cell model, and the levels of total cholesterol (TC) and triglyceride (TG) in the cells were detected. The treatment groups were co-treated with different concentrations of BLCE and 1 mM OA for 24 h. At the end of the experiment, HepG2 cells were collected, and the contents of TC and TG were measured using an appropriate kit. The experiment was repeated six times.

[0055] Analysis of oxidative stress level in HepG2 cells: HepG2 cells were treated as above. At the end of the experiment, the cells were collected, and the contents of ROS, SOD, MDA and GSH in HepG2 cells were measured according to the kit instructions.

[0056] Lipid accumulation and oxidative stress are considered key factors in metabolic - associated fatty liver disease (MAFLD). In this invention, a cell model of metabolic - associated fatty liver disease was constructed, and the lipid - lowering and antioxidant activities of BLCE were evaluated through OA - induced HepG2 cells. OA, as a monounsaturated fatty acid, exists in the form of glyceride in all animal and vegetable oils. Glyceride is the most ingested fatty acid in the daily diet.

[0057] Figure 1 The effects of BLCE on lipid accumulation and oxidative stress in OA - induced HepG2 cells were shown. Figure 1 In it, Figure A shows the effect of OA on the viability of HepG2 cells, Figure B shows the effect of BLCE on the viability of HepG2 cells, Figure C shows the effect of OA and BLCE on the viability of HepG2 cells, Figure D shows the ROS level, Figure E shows the SOD activity, Figure F shows the MDA content, Figure G shows the GSH activity, Figure H shows the TC content in HepG2 cells, and Figure I shows the TG content in HepG2 cells.

[0058] First, the CCK - 8 method was used to determine the OA concentration. As shown in Figure A of Figure 1 , when the OA concentration was less than 1 mM, the survival rate of HepG2 cells remained above 98%, indicating no cytotoxicity. When the concentration was greater than 1 mM, the survival rate decreased significantly. Therefore, 1 mM OA was selected to treat HepG2 cells to establish a MAFLD cell model.

[0059] Subsequently, the CCK - 8 method was used to determine the cytotoxicity and optimal concentration of BLCE. As shown in Figures B - C of Figure 1 , no obvious cytotoxicity was observed when the BLCE concentration was lower than 1 mg / mL. Concentrations of 0.25, 0.5, and 1 mg / mL were selected for further experiments.

[0060] According to the measured OA and BLCE concentrations, HepG2 cells were cultured for 24 h, and markers of oxidative stress and lipid accumulation were measured. The levels of oxidative factors (ROS, SOD, MDA, GSH) and TC, TG in HepG2 cells treated with OA increased significantly, indicating that OA induced oxidative stress and lipid accumulation.

[0061] As shown in Figure 1As shown in D - G, BLCE (0.25, 0.5, and 1 mg / mL) reversed OA - induced oxidative stress in a dose - dependent manner, resulting in a significant decrease in ROS and MDA levels and a significant increase in SOD and GSH levels (P < 0.05). Similarly, BLCE (0.25, 0.5, and 1 mg / mL) also improved OA - induced lipid accumulation, with significant decreases in TC and TG levels in BLCE - treated HepG2 cells (P < 0.05), as shown in Figures H - I. Therefore, BLCE can alleviate oxidative stress and lipid accumulation in OA - induced HepG2 cells.

[0062] Example 4

[0063] Effect of BLCE on the inflammatory response of lipopolysaccharide - induced RAW264.7 cells.

[0064] Determination of inflammatory factors in RAW 264.7 cells: A cell inflammation model was induced by stimulating RAW 264.7 cells with 500 ng / mL of LPS for 24 h.

[0065] RAW 264.7 cells were seeded in 96 - well plates at a density of 5×10 4 and cultured for 24 h. The treatment groups were treated with different concentrations of BLCE for 1 h and then co - incubated with 500 ng / mL of LPS for 24 h. The NO content was measured using a Griess kit.

[0066] In addition, RAW264.7 cells were seeded in 24 - well plates at a density of 2×10 5 and cultured as described above. At the end of the experiment, the cell culture supernatant was collected, and the concentrations of TNF - α, IL - 6, and IL - 1β were measured according to the instructions of the ELISA kit. The experimental results are shown in Figure 2 Figure.

[0067] Appendix Figure 2 In it, Figure A shows the effect of LPS on the viability of RAW264.7 cells, Figure B shows the effect of BLCE on the viability of RAW264.7 cells, Figure C shows the effect of LPS and BLCE on the viability of HepG2 cells, Figure D shows the NO production, Figure E shows the TNF - α production, Figure F shows the IL - 6 production, and Figure G shows the IL - 1β production.

[0068] Effect of BLCE on the inflammatory response of lipopolysaccharide - induced RAW264.7 cells Macrophages protect organisms from harmful external substances through phagocytosis and play a key role in immune responses, allergies, and inflammation. In this invention, LPS - induced RAW264.7 cells were used to evaluate the anti - inflammatory activity of BLCE.

[0069] Figure 2 The results showed that after treating RAW264.7 cells with LPS at a dose of 0 - 500 ng / mL for 24 h, there was no cytotoxicity or reduction in cell viability. Similarly, BLCE had no cytotoxicity at a dose of 0 - 1 mg / mL and could be used for subsequent experiments.

[0070] Then, in the present invention, RAW264.7 cells were treated with 0.25, 0.5, and 1 mg / mL BLCE for 1 h, then 500 ng / mL LPS was added to stimulate the cells, and the anti - inflammatory activity of BLCE was evaluated by measuring the levels of NO, TNF - α, IL - 6, and IL - 1β. The results showed that after treatment with LPS, the production of NO, TNF - α, IL - 6, and IL - 1β increased significantly (P < 0.05); however, after administration of BLCE, the production of NO, TNF - α, IL - 6, and IL - 1β decreased significantly in a dose - dependent manner.

[0071] The above results indicate that BLCE can attenuate the inflammatory response of LPS - induced RAW264.7 cells by reducing the levels of inflammatory factors.

[0072] Example 5 In vitro experiments

[0073] Animal experiment design: 50 male C57BL / 6J mice (SPF, 8 - week - old, body weight 20 ± 2 g) were purchased from the Shandong Laboratory Animal Center. All animal procedures during the experiment were carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" of Shandong University of Technology and were approved by the Animal Ethics Committee of Shandong University of Technology.

[0074] Initially, the mice were acclimatized to a 12 - h light / dark cycle at 28 ± 2 °C for 1 week, and then they were randomly divided into 5 groups of 8 mice each. Except for the blank control group (CON group), the other groups were fed a normal diet, and the other groups were fed a high - fat diet (HFD).

[0075] The HFD consisted of 24.2% protein (19.8 kcal%), 27.4% fat (48.5 kcal%), and 40.1% carbohydrates (31.7 kcal%), with a total energy content of 4.7 kcal / g;

[0076] The control group was fed a standard diet (14.2% protein, 73.1% carbohydrates, and 4% fat; SYSE Bio - Tech, Changzhou, China).

[0077] After adapting to a normal diet for one week, the CON group continued to receive a normal diet and was gavaged with 0.9% normal saline daily. The other groups were fed with HFD: the HFD group was gavaged with 0.9% normal saline daily, the BL-H group was gavaged with 200 mg / kg / d BLCE, the BL-M group was gavaged with 100 mg / kg / d BLCE, and the BL-L group was gavaged with 50 mg / kg / d BLCE.

[0078] After the experiment started, the body weights of the mice were recorded once a week. Before the end of the experiment, blood glucose, food intake, and water intake were recorded respectively. At the end of the experiment, all the mice were fasted for 16 h and allowed free access to water, and then they were euthanized with CO2. Blood was collected with an anticoagulant tube and centrifuged at 4000×g for 10 min at 4°C to obtain serum. The liver and epididymal white adipose tissue (eWAT) were collected, weighed, and the percentages of liver weight and eWAT were calculated. Then the liver was dissected and processed. A part of the liver was sectioned and fixed in 10% formalin solution, and the other part was stored in liquid nitrogen.

[0079] Biochemical index analysis: Commercial kits were used to measure the levels of TC, TG, LDLC, HDLC, ALT, and AST in serum and liver. According to the kit instructions, the liver oxidative stress was evaluated by measuring SOD, MDA, CAT, and GSH in the liver. Commercial ELISA kits were used to detect the levels of TNF-α, IL-6, and IL-1β in serum and liver to evaluate the inflammatory response.

[0080] Histopathological study: After the liver tissue specimens were fixed in formalin solution for 24 h, they were paraffin-embedded, sectioned, and stained for histopathological analysis. The liver sections were stained with hematoxylin-eosin (H&E) for histological evaluation, Sirius red staining for liver fibrosis analysis, and Oil Red O staining to evaluate the degree of liver lipid accumulation.

[0081] The effects of BLCE on the physiological indexes of MAFLD mice are shown in the Figure 3 appendix.

[0082] Figure 3 In it, Figure A is the grouping of the animal experiment, Figure B is the change in the body weight of the mice, Figure C is the body weight of the mice before euthanasia, Figure D is the change in blood glucose, Figure E is the liver weight, Figure F is the weight of the epididymal white adipose tissue (eWAT), Figure G is the food intake of the mice, and Figure H is the water intake of the mice.

[0083] The appendix Figure 3 shows that during the 12-week experiment, the body weights of the mice in all groups increased steadily, and the mice fed with HFD had the most significant increase in body weight. In addition, the supplementation of BLCE led to a dose-dependent decrease in the body weight gain rate of the mice.

[0084] Compared with the initial body weight, the mice in the CON group had the lightest body weight, while the mice in the HFD group had the heaviest body weight. The mice in the BL-H group had the largest decrease in body weight, with no significant difference from the CON group (P<0.05). Followed by the mice in the BL-M group and the BL-L group. However, there were no significant differences in fasting blood glucose level, 2-hour postprandial blood glucose level, and water intake between the two groups (P<0.05). The trends of liver weight and eWAT percentage were similar. The percentage in the CON group was the lowest, followed by the BL-H, BL-M, and BL-L groups, and the percentage in the HFD group was the highest.

[0085] Finally, the food intakes of all HFD-fed groups were similar (P<0.05), and the mice in the CON group had the most food intake.

[0086] These results indicate that BLCE reduced the body weight, liver weight percentage, and eWAT percentage in MAFLD mice, but did not affect fasting blood glucose, 2-hour postprandial blood glucose, food intake, or water intake.

[0087] Example 6

[0088] Effect of BLCE on serum and liver lipid levels in MAFLD mice.

[0089] The effects of BLCE on serum and liver lipid levels in MAFLD mice are shown in the appendix Figure 4 as shown, in the appendix Figure 4 In it, Figure A is the serum TC content, Figure B is the serum TG content, Figure C is the serum LDLC content, Figure D is the serum HDLD content, Figure E is the liver TC content, Figure F is the liver TG content, Figure G is the liver LDLC content, and Figure H is the liver HDLC content.

[0090] MAFLD is closely associated with dyslipidemia and hepatic lipid abnormalities. The results of serum and hepatic lipid levels in each group of mice showed that compared with the CON group, the levels of TC, TG, and LDLC in HFD-fed mice were significantly increased (P<0.05). The levels of serum TC, TG, and LDLC in the BL-H, BL-M, and BL-L groups showed an obvious downward trend compared with the HFD group, and there was an obvious dose-dependence (P<0.05). Among these groups, the levels of TC, TG, and LDLC in the BL-H group decreased most significantly, followed by the BL-M group and the BL-L group (P<0.05). In contrast to the levels of TC, TG, and LDLC, the serum HDLC level was opposite. Compared with the CON group and the three BLCE treatment groups, the HDLC level in the HFD group was significantly decreased, and with the increase in the BLCE concentration, the HDLC level was significantly increased (P<0.05). A similar pattern was also observed in the liver. In HFD-fed mice, the levels of TC, TG, and LDLC were significantly increased (P<0.05). After BLCE intervention, this phenomenon was reversed and decreased with the increase in the BLCE concentration. Finally, compared with the CON group, the HDLC level in the liver of MALFD mice was significantly decreased. In contrast, the hepatic HDLC levels in the BL-H, BL-M, and BL-L groups were significantly increased, with the BL-H group being the highest, the BL-M group being the second, and the BL-L group being the lowest (P<0.05).

[0091] These results indicate that BLCE significantly improved the blood lipid profile of HFD-fed mice and decreased the hepatic blood lipid levels (P<0.05).

[0092] Example 7

[0093] Effect of BLCE on liver function and histopathology of MAFLD mice.

[0094] The effect of BLCE on liver function and histopathology of MAFLD mice is shown in the appendix Figure 5 as follows. In the appendix Figure 5 , A: serum ALT, B: serum AST, C: hepatocyte vacuolization-like change area, D: Oil Red positive area, E: HE staining and quantitative graph, F: Oil Red staining and quantitative graph.

[0095] HFD can induce MAFLD in mice, leading to abnormal lipid metabolism and impaired liver function. Currently, improving liver function is the cornerstone of MAFLD treatment.

[0096] In this invention, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were measured to evaluate the effect of BLCE on the liver function of MAFLD mice. Compared with the CON group, the levels of ALT and AST in the serum of mice fed with HFD were significantly increased. Compared with the HFD group, the levels of ALT and AST in the BL-H, BL-M, and BL-L groups were significantly decreased (P<0.05). Histopathological analysis of liver tissues stained with H&E showed that compared with the CON group, the hepatocytes of mice fed with HFD exhibited cell swelling and extensive vacuolization. Administration of BLCE could reduce the vacuoles and cell swelling in MAFLD mice.

[0097] The percentage results of the vacuolated area of hepatocytes in liver tissue cells showed that compared with the CON group, vacuolated liver tissue cells were more common in mice fed with HFD. Among the mice fed with HFD, the HFD group had the highest percentage of vacuoles, followed by BL-L, BL-M, and BL-H. In addition, oil red staining showed that compared with the CON group, the number of lipid droplets in the liver of mice fed with HFD increased, and administration of BL-H reversed this phenomenon. The specific number of lipid droplets is shown in the figure, where the HFD group had the most, followed by BL-L, BL-M, and BL-H, and the CON group had the least.

[0098] These above results indicate that BLCE can improve the liver function and histopathological changes in MAFLD mice.

[0099] Example 8

[0100] Effect of BLCE on liver inflammatory factors and liver fibrosis in MAFLD mice.

[0101] Appendix Figure 6 Shows the effect of BLCE on liver inflammatory factors and liver fibrosis in MAFLD mice, where A: LPS in serum, B: TNF-α in serum, C: IL-6 in serum, D: IL-1β in serum, E: TNF-α in liver, F: IL-6 in liver, G: IL-1β in liver, H: Sirius red staining.

[0102] The progression of MAFLD and other chronic liver diseases to cirrhosis is jointly driven by liver inflammation and fibrosis. In this process, metabolic dysfunction leads to hepatocyte damage and death, resulting in the recruitment and activation of immune cells and fibrotic cells. Therefore, in this invention, the anti-inflammatory activity of broccoli leaf extract in vivo was evaluated by measuring the levels of LPS and inflammatory factors (TNF-α, IL-6, and IL-1β), and the anti-liver fibrosis activity of BLCE in vivo was confirmed by analyzing Sirius red-stained liver tissue sections.

[0103] The results showed that the LPS level in the serum of MAFLD mice was significantly higher than that in the CON group (P<0.05). However, these elevated LPS levels were improved by BLCE and decreased in a dose-dependent manner (P<0.05). In addition, the levels of TNF-α, IL-6, and IL-1β in the serum and liver followed a similar pattern, being significantly higher in the HFD-fed mice than in the CON group. Compared with the HFD group, the levels of TNF-α, IL-6, and IL-1β in the serum and liver of the BL-H group decreased the most (P<0.05).

[0104] Sirius red staining showed that the hepatic tissue morphology of the mice in the CON group was normal, with uniform distribution of hepatocytes and no collagen deposition. In contrast, the hepatocytes in the HFD group were scattered, with severe collagen deposition and obvious fibrosis. The high-dose BL-H, medium-dose BL-M, and low-dose BL-L groups all alleviated hepatic fibrosis and collagen deposition in MAFLD mice to varying degrees.

[0105] It can be seen that BLCE can improve hepatic fibrosis in mice and effectively alleviate MAFLD.

Claims

1. Use of broccoli leaf extract in the preparation of a product for preventing and treating metabolic associated fatty liver disease.

2. The application according to claim 1, characterized in that The broccoli leaf extract is obtained by extracting the leaves of broccoli with a solvent, and the solvent includes at least one of water and alcohol solvents.

3. The application according to claim 2, characterized in that, The solvent is water.

4. The application according to claim 1, characterized in that The form of the extract includes any one of an extract obtained by directly extracting with a solvent, a concentrated solution obtained by concentrating the extract, and a freeze-dried preparation obtained by adding a freeze-drying adjuvant to the extract and then freeze-drying.

5. The application according to claim 1, characterized in that, The preparation method of the broccoli leaf extract is as follows: (1) Select, wash broccoli leaves, vacuum freeze-dry at a temperature of -80 ± 5 °C, pulverize, and store for later use; (2) Take the powder in (1), add water at a mass-to-volume ratio of 1 g: 20 - 50 mL, mix evenly, stir and extract at 60 - 80 °C for 1.5 - 3 h, centrifuge to obtain the supernatant, and concentrate to obtain the broccoli leaf extract.

6. The application according to claim 1, characterized in that, The broccoli leaf extract plays a role in preventing and treating metabolic associated fatty liver disease by at least one of the following ways: reducing oleic acid-induced lipid accumulation and oxidative stress in HepG2 cells, decreasing the levels of inflammatory factors, weakening the inflammatory response of LPS-induced RAW264.7 cells, and improving liver fibrosis.

7. The application according to claim 1, wherein The product is any one of food and medicine.

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