High-efficiency extraction and purification method of luteolin from portulaca oleracea and application thereof in improving metabolic-related fatty liver

Abstract

The application discloses a high-efficiency extraction and purification method of luteolin in Portulaca oleracea L. and application of the luteolin in improving metabolic-associated steatohepatitis. The extraction and purification method takes Portulaca oleracea L. extract (PO) as raw material, and sequentially goes through four steps of macroporous resin separation and enrichment, normal-phase silica gel chromatographic separation and purification, medium-pressure preparation chromatography purification and dextran gel chromatography purification, combines with high-performance liquid chromatography (HPLC) detection and purity verification, and finally obtains luteolin monomer with purity of greater than or equal to 97%, which is identified as luteolin by NMR. Experimental verification shows that the method has good linear relationship (R 2 =0.9993), high precision (RSD=0.502%), is simple, efficient and stable, and can realize large-scale preparation of luteolin. Meanwhile, the application finds that the luteolin prepared by the method can significantly improve MASH liver pathological conditions such as steatosis and lobular inflammation, reduce lipid level, and inhibit expression of inflammatory factors, thereby providing a new natural active substance and related application scheme for treatment of MASH.

Description

Technical Field

[0001] This invention belongs to the fields of natural product chemistry and biomedicine, specifically relating to a method for efficiently extracting and purifying luteolin from purslane, and the application of the extracted luteolin in the preparation of drugs or functional foods for improving metabolic dysfunction-associated steatohepatitis (MAS). Background Technology

[0002] Metabolic dysfunction-associated steatohepatitis (MASH) is a critical stage in the progression of metabolic dysfunction-associated steatotic liver disease (MASLD). If left untreated, severe cases can progress to liver fibrosis, cirrhosis, and even hepatocellular carcinoma. MASH carries approximately a 10-fold increased risk of advanced liver fibrosis compared to MASLD, but its progression time is shortened by about half. With the global prevalence of metabolic risk factors such as obesity, type 2 diabetes, and dyslipidemia, the incidence of MASLD is rising annually, currently exceeding 30% globally, affecting over 1.2 billion people, of whom approximately one-quarter have developed MASH. In my country, the overall prevalence of MASLD has reached 29.6%, affecting nearly 200 million people, and the incidence continues to increase, now surpassing viral hepatitis as the leading chronic liver disease in my country. Currently, only one drug targeting MASH—the THRβ agonist resmetirom (Rezdiffra; Madrigal Pharmaceuticals)—has recently been approved by the US FDA. However, it is still in clinical trials and has not yet been widely adopted. Clinical treatment remains primarily based on lifestyle interventions, and effective treatment methods are still lacking. Furthermore, the critical intervention window for MASH often lasts for years or even decades, and long-term drug treatment may increase the metabolic burden on the liver and even lead to drug-induced hepatitis. Therefore, given the limitations of drugs and the large number of people suffering from MASH and its progressive harm, finding safe and effective natural products to improve MASH has become a research hotspot.

[0003] Purslane (Portulaca oleracea L.), also known as longevity vegetable or Chinese purslane, belongs to the genus Portulaca in the family Portulacaceae. It is a widely distributed plant used for both medicinal and culinary purposes. It has a sour taste and is considered to be cold in nature. Traditional Chinese medicine believes it enters the liver meridian and has a protective effect on the liver. Modern research shows that it contains various bioactive components such as flavonoids, polysaccharides, and alkaloids, possessing diverse medicinal and nutritional values, and is widely used in traditional Chinese medicine and food processing. Luteolin (LO) is a natural flavonoid compound with the structural formula shown in Formula I. It is widely found in various plants and possesses various pharmacological activities such as antioxidant, anti-inflammatory, and anti-tumor effects. However, the content of luteolin in purslane is usually low, and it coexists with other flavonoid compounds and impurities, making it difficult to directly isolate and obtain a high-purity product. Existing methods for extracting and purifying luteolin often suffer from problems such as complex operation, low purification efficiency, low product purity, and high cost, making it difficult to meet the needs of large-scale production and pharmaceutical applications. Meanwhile, there are currently no clear patent reports on the specific role of luteolin derived from purslane in improving MASH, which limits its application in the pharmaceutical field.

[0004]

[0005] Formula I

[0006] Therefore, developing a simple, efficient, stable, and scalable method for preparing high-purity purslane luteolin, and clarifying its application in improving MASH, has significant theoretical and practical value. Summary of the Invention

[0007] This invention aims to address the problems of low efficiency and low purity in the extraction and purification of luteolin from purslane in existing technologies, and to provide a highly efficient and stable extraction and purification method. Furthermore, this invention further verifies the significant effect of the obtained luteolin in improving metabolic-associated steatohepatitis (MASH), providing a scientific basis for its application in the pharmaceutical field.

[0008] The research process of this invention is as follows: First, using purslane extract as raw material, the extract undergoes four steps sequentially: macroporous resin separation and enrichment, normal-phase silica gel chromatography separation and purification, medium-pressure preparative chromatography purification, and dextran gel chromatography purification. Combined with high-performance liquid chromatography (HPLC) detection and purity verification, a luteolin monomer with a purity ≥97% is finally obtained, and NMR identification confirms it as luteolin. Subsequently, a MASH animal model is constructed, and the effects on the normal control group (ND), model group (MCD), and luteolin intervention group (MCD+LO) are compared and analyzed. Luteolin intervention significantly improves the presence of numerous fatty vacuolar structures and inflammatory cell aggregation in the liver of the model group. The levels of triglycerides (TG), cholesterol (TC), and inflammatory factors TNF-α and IL-1β in the model group are also significantly lower than those in the model group, indicating that luteolin derived from purslane has a protective effect on the liver and can effectively improve MASH.

[0009] Based on the above research, the technical solution to be protected by this invention is as follows:

[0010] In a first aspect, the present invention provides a method for the efficient extraction and purification of luteolin from purslane. This method is stable and efficient, and can obtain pure luteolin with a purity of ≥97%, meeting the needs of large-scale production.

[0011] Preferably, the extraction and purification method of luteolin from purslane described above is as follows:

[0012] S1. Preparation of purslane extract: Dried purslane was soaked in 50% ethanol overnight, and the soaking solution was collected (stored at 4℃); the remaining powder obtained by filtration was then ultrasonically extracted with another new 50% ethanol solution for 2 hours, and the extraction was repeated 2-3 times; the extracts were combined and filtered, and concentrated under reduced pressure to remove ethanol to obtain purslane extract.

[0013] S2. Macroporous resin adsorption and desorption: The purslane extract obtained in S1 was dissolved in water, filtered, and loaded onto an activated macroporous resin column. After complete adsorption, the column was eluted sequentially with water, 60% ethanol, and 95% ethanol. The 60% ethanol eluent was collected and concentrated to obtain crude product 1.

[0014] S3. Purification by normal phase silica gel chromatography: The crude product 1 obtained in S2 was separated by normal phase silica gel column chromatography, with gradient elution using an ethyl acetate-methanol system, and thin layer chromatography (TLC) for follow-up detection. The components containing luteolin were collected and concentrated to obtain crude product 2.

[0015] S4. Medium-pressure preparative chromatographic purification: The crude pure product 2 obtained in S3 was dissolved in a solvent, filtered, and then loaded onto a C18 medium-pressure preparative chromatographic column. Gradient elution was performed using methanol-0.1% formic acid aqueous solution as the mobile phase. The target peak fraction at 350 nm was collected and concentrated to obtain crude pure product 3 with a purity ≥90%.

[0016] S5. Dextran gel chromatography purification: The crude pure product 3 obtained in S4 was dissolved in a solvent and loaded onto a dextran gel chromatography column. It was eluted with 5% methanol aqueous solution, and the target peak fraction at 350 nm was collected. The single fractions were combined, concentrated and dried to obtain high-purity luteolin with a purity ≥97%.

[0017] Furthermore, in step S1, when performing ultrasonic extraction, 50% ethanol is used for ultrasonic extraction at 50~60℃; when performing vacuum concentration, the extract is subjected to vacuum recovery of the solvent at 50~60℃ to prevent solvent boiling.

[0018] Further, in step S2, the macroporous resin used is type D-101, and its activation method includes soaking in ethanol, eluting until no alcohol odor is detected, and then treating with acid and alkali in sequence. The preferred activation method is as follows: the D-101 macroporous resin is soaked in 95% ethanol for 20-30 hours and then wet-packed into a column. It is then eluted with 95% ethanol until no alcohol odor is detected and the eluent does not become cloudy when water is added. It is then rinsed with pure water until no alcohol odor is detected. Then, it is soaked in 5% HCl for 4 hours and washed with pure water until neutral. It is then soaked in 2% NaOH for 4 hours and washed with pure water until neutral. After activation, it is stored in pure water for later use.

[0019] Preferably, the purslane extract is dissolved in water, filtered through a 0.45 μm filter membrane, loaded at a flow rate of 20 mL / min, and adsorbed at a low temperature of 4 °C.

[0020] Further, in step S3, the gradient elution procedure is as follows: elution is performed sequentially with a gradient of ethyl acetate:methanol at ratios of 40:1, 25:1, 20:1, and 15:1, followed by TLC monitoring. The components containing luteolin are combined and concentrated under reduced pressure to obtain crude product 2.

[0021] Further, in step S4, crude pure product 2 is dissolved in 60% methanol and filtered through a 0.45μm filter membrane;

[0022] The C18 medium-pressure preparative chromatographic column has a column temperature of 30℃, a detection wavelength of 350nm, and a flow rate of 20ml / min;

[0023] A gradient elution was performed using 0.1% formic acid aqueous solution and methanol as phase A and phase B, respectively: 0-5 min, 20% B; 5-15 min, 20%-30% B; 15-25 min, 30%-40% B; 25-35 min, 40%-50% B; 35-45 min, 50%-60% B; 45-50 min, 60%-20% B; 50-55 min, 20% B. The target peak fraction at 350 nm was collected, and after detection and verification, the components containing the target analyte were combined and concentrated under reduced pressure to obtain crude pure product 3 with a purity ≥90%.

[0024] Furthermore, in step S5, the dextran gel is G-10, which needs to be fully swollen and washed with 5% methanol aqueous solution before being packed into the column to separate the crude pure product 3.

[0025] The crude pure product 3 was dissolved in a 5% methanol aqueous solution, filtered through a 0.45μm filter membrane, and then loaded onto the sample. It was continuously eluted with a 5% methanol aqueous solution, and the target peak fraction at 350nm was collected. The fractions with high absorbance and single TLC fluorescence were combined, concentrated under reduced pressure at 40-60℃, and freeze-dried to obtain a pure product of the target substance with a purity ≥97%.

[0026] In a second aspect, the present invention provides the use of luteolin derived from purslane in the preparation of drugs or functional foods for the prevention or relief of MASH.

[0027] Preferably, the improvement in metabolic-associated steatohepatitis (MASH) is manifested in one or more of the following aspects:

[0028] a) Improve MASH pathological conditions in MASH model animals, such as hepatic steatosis, lobular inflammation, and hepatocyte ballooning degeneration;

[0029] b) Reduce triglyceride (TG) or cholesterol (TC) levels in MASH model animals;

[0030] c) Reduce the levels of pro-inflammatory cytokines TNF-α or IL-1β in the liver.

[0031] In a third aspect, the present invention provides a pharmaceutical composition for preventing or alleviating metabolic-associated steatohepatitis, characterized in that luteolin, obtained by extraction and purification using any of the methods described above, is used as the active component.

[0032] The role and effect of invention

[0033] This invention establishes a complete and efficient process route for the extraction and purification of luteolin from purslane. This route comprehensively utilizes macroporous adsorption resin, normal-phase silica gel, medium-pressure preparative chromatography, and dextran gel chromatography techniques, successfully separating high-purity (≥97%) luteolin monomers from purslane extract with extremely low concentrations (approximately 0.0122%). This method is stable and reliable, providing a material basis for the large-scale production and efficacy studies of luteolin.

[0034] This invention is the first to verify the efficacy of luteolin derived from purslane in the prevention and treatment of MASH (muscular atrophy). Experimental results show that luteolin prepared using the extraction method of this invention can improve MASH liver pathological conditions such as hepatic steatosis, lobular inflammation, and hepatocyte ballooning degeneration, while also reducing triglyceride, cholesterol, and inflammatory factor levels such as TNF-α and IL-1β, thus improving lipid metabolism and inflammatory response in MASH.

[0035] The luteolin derived from purslane in this invention has a significant effect on improving MASH and can be used to prepare medicines and functional foods for the prevention and treatment of MASH, showing broad prospects for development and utilization. Attached Figure Description

[0036] Figure 1 The HPLC chromatogram for the determination of the target substance (luteolin) in purslane extract;

[0037] Figure 2 This is the HPLC chromatogram of the luteolin target obtained by the method of the present invention after separation and purification;

[0038] Figure 3 The NMR spectrum for structural identification of luteolin, the target compound obtained by the method of this invention, is shown below, where A is... 1 1H NMR (hydrogen spectroscopy), B is... 13 C1 NMR (carbon spectrum);

[0039] Figure 4 Representative images of mouse liver appearance and histopathological examination are shown, where ND is the normal control group, MCD is the MASH model group, and MCD+LO is the luteolin intervention group.

[0040] Figure 5 The graph shows the changes in triglyceride (TG, Figure A) and cholesterol (TC, Figure B) levels in the liver of mice. ND represents the normal control group, MCD represents the MASH model group, and MCD+LO represents the luteolin intervention group.

[0041] Figure 6 The graph shows the changes in the levels of tumor necrosis factor α (TNF-α, Figure A) and interleukin 1β (IL-1β, Figure B) in the liver of mice. ND represents the normal control group, MCD represents the MASH model group, and MCD+LO represents the luteolin intervention group. Detailed Implementation

[0042] The following embodiments and experimental examples further illustrate the present invention and should not be construed as limiting the invention. The embodiments do not include a detailed description of conventional methods, which are well known to those skilled in the art and described in numerous publications.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention, and the preferred embodiments and materials described in the specific implementation are for illustrative purposes only.

[0044] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0045] Example 1: Extraction, separation and purification of luteolin from purslane

[0046] 1. Preparation of Portulaca oleracea extract

[0047] Dried purslane was soaked in 50% ethanol overnight, and the soaking solution was collected (stored at 4℃). The remaining powder obtained after filtration was then extracted with a fresh 50% ethanol solution at 50-60℃ using ultrasound for 2 hours, and the extraction was repeated 2-3 times. The extracts were combined and filtered, and concentrated under reduced pressure at 50-60℃ to remove the ethanol, yielding the purslane extract. The HPLC chromatogram for the determination of luteolin content in the purslane extract is shown below. Figure 1 .

[0048] 2. Separation and purification methods

[0049] 2.1 Separation and Enrichment of Macroporous Resins

[0050] D-101 macroporous resin was soaked in 95% ethanol for 24 hours and then wet-packed into a column. Elution was performed with 95% ethanol at a flow rate of 2 BV / h until no alcohol odor was detected and the eluent did not become cloudy upon addition of water. The eluent was then rinsed with pure water until no alcohol odor remained. The resin was then soaked in 5% HCl for 4 hours, followed by washing with pure water until neutral; and then soaked in 2% NaOH for 4 hours, followed by washing with pure water until neutral. After activation, the resin was stored in pure water for later use.

[0051] Purslane extract was dissolved in water and filtered through a 0.45 μm filter membrane. The sample was loaded at a flow rate of 20 mL / min and adsorbed at 4 °C. After complete adsorption, the sample was eluted sequentially with water, 60% ethanol, and 95% ethanol. HPLC analysis confirmed that luteolin was mainly concentrated in the 60% ethanol eluent. The eluent from this fraction was collected, concentrated under reduced pressure, and crude product 1 was obtained.

[0052] 2.2 Normal-phase silica gel chromatography separation and purification

[0053] The silica gel for column chromatography was activated at 100-120℃ for 1 hour, cooled, and then packed into a column using a wet packing method. Crude product 1 was dissolved in 80% ethanol, and twice the amount of silica gel was added and mixed thoroughly. After evaporating the solvent, the solution was evenly spread onto the silica gel column. Elution was performed sequentially using a gradient of ethyl acetate:methanol at ratios of 40:1, 25:1, 20:1, and 15:1, with TLC monitoring. The fractions containing luteolin were combined and concentrated under reduced pressure to obtain crude product 2.

[0054] 2.3 Medium-pressure preparative chromatographic purification

[0055] Crude product 2 was dissolved in 60% methanol and filtered through a 0.45 μm filter membrane. A C18 glass column was prepared under medium pressure at 30 °C, a detection wavelength of 350 nm, and a flow rate of 20 mL / min. Gradient elution was performed using phase A (0.1% formic acid in water) and phase B (methanol): 0–5 min, 20% B; 5–15 min, 20%–30% B; 15–25 min, 30%–40% B; 25–35 min, 40%–50% B; 35–45 min, 50%–60% B; 45–50 min, 60%–20% B; 50–55 min, 20% B. The target peak fraction at 350 nm was collected, and after TLC verification, the fractions containing the target analyte were combined and concentrated under reduced pressure to obtain crude product 3 (purity ≥90%).

[0056] 2.4 Dextran purification by gel chromatography

[0057] Take dextran gel G-10, add 5% methanol aqueous solution, and allow it to swell at room temperature for 24 hours, stirring several times during this period. Discard the fine particles in the upper layer, and wash 2-3 times with 5% methanol aqueous solution. After wet packing, dissolve the crude pure product 3 in 5% methanol aqueous solution, filter through a 0.45 μm filter membrane, and load the sample. Elute continuously with 5% methanol aqueous solution, collecting the target peak fraction at 350 nm. Combine the fractions with high absorbance and single TLC fluorescence, concentrate under reduced pressure at 40-60℃, and freeze-dry to obtain the pure target product.

[0058] 3. Results and Analysis

[0059] The luteolin content in purslane extract was extremely low, at 0.0122%. A yellow crystalline powder was obtained using this method, and its structure was identified as luteolin (C...). 15 H 10 O6) monomer, with a purity of 97.3%. The HPLC chromatogram of the luteolin target obtained after separation and purification by the method of this invention is shown below. Figure 2 The NMR chromatogram for the structural identification of luteolin, obtained by the method of this invention, after separation and purification, is shown below. Figure 3 .

[0060] Example 2: Study on the prevention and treatment of metabolic-related fatty liver disease using luteolin derived from purslane.

[0061] 1. Materials and Equipment

[0062] 1.1 Experimental Materials

[0063] The intervention drug used in the experiment was luteolin derived from purslane, as described in Example 1.

[0064] 1.2 Experimental Reagents

[0065] The main reagents used in the experiment are shown in Table 1:

[0066] Table 1 Main Reagents

[0067]

[0068] 1.3 Experimental Instruments and Equipment

[0069] The main instruments and equipment involved in the experiment included: a vortex mixer (TYXH-I), a benchtop high-speed refrigerated centrifuge (TGL-16MS), an upright optical microscope (Nikon Eclipse E100), and an ELISA reader (Thermo Fisher).

[0070] 2. Experimental Methods

[0071] 2.1 Establishment of a metabolic-related steatohepatitis model and animal grouping and drug administration

[0072] Male C57BL / 6N mice, 6-8 weeks old and weighing 20-25g, were selected. Mice were housed in an animal room at (22 ± 2)℃ and (50 ± 5)% humidity, with free access to food and water, and a 12h light / 12h dark cycle. Experiments began after one week of acclimatization. After the acclimatization period, mice were randomly divided into a normal control group (ND), a model group (MCD), and a luteolin intervention group (MCD+LO), with 8 mice in each group. Except for the control group, which received normal food, all other groups received MCD food. Food and water were changed every 3 days, and mouse weight and food intake were recorded weekly. The luteolin intervention group received the corresponding drug via gavage in addition to the MCD diet, once a day, 0.2ml, at a dose of 100mg / kg. The control group received the same dose of physiological saline via gavage. All groups were housed for a total of 8 weeks. After the last administration, mice were euthanized after a 16-hour fast with free access to water, and blood and liver samples were collected.

[0073] 2.2 Histopathological examination of mouse liver tissue

[0074] Take 1cm of the largest lobe of the liver 3 Small pieces were fixed in 4% paraformaldehyde solution for 48 hours, then embedded in paraffin, and sectioned to a thickness of 5 μm. The tissue structure was observed and photographed using an optical microscope after hematoxylin-eosin (H&E), oil red O, and F4 / 80 staining.

[0075] 2.3 Detection of biochemical indicators in mice

[0076] Take 20-30 mg of liver tissue, add the lysis buffer from the kit, quantify BCA protein, and then measure the absorbance according to the kit instructions to calculate the concentrations of TC and TG.

[0077] 2.4 Detection of inflammatory factors in mouse liver tissue

[0078] 20-30 mg of liver tissue was added to 1 ml of Trizol to extract RNA. The RNA was reverse transcribed into cDNA, and then subjected to real-time quantitative PCR to detect the levels of inflammatory factors. The primer sequences used in the detection process are shown in Table 2.

[0079] Table 2 PCR Primer Sequence List

[0080]

[0081] 2.5 Data Analysis

[0082] R 4.3.2 and IBM SPSS Statistics 27 software were used, with a significance level of α=0.05. For normally distributed continuous data, (x ± s) were described using t-tests; for non-normally distributed data, the median (interquartile range) was used, and a nonparametric rank-sum test was used. For categorical data, n (%) was used for statistical description, and a chi-square test was used for analysis.

[0083] 3. Results and Analysis

[0084] Liver pathological hematologic staining can directly reflect the effect of luteolin derived from purslane on MASH, such as... Figure 4 As shown. Compared with the normal control group, the model group had a large number of fatty vacuolar structures, lobular inflammation, and hepatocyte ballooning degeneration in the liver, which were significantly improved after luteolin intervention. The TG and TC levels in the luteolin intervention group were significantly lower than those in the model group ( Figure 5 A, 5B). The levels of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the mouse liver were also significantly lower than those in the model group. Figure 6 A, 6B) indicate that luteolin has a protective effect on the liver and can effectively improve MASH.

[0085] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A method for extracting and purifying luteolin from purslane, characterized in that, Includes the following steps: S1. Preparation of purslane extract: Purslane extract was obtained by extracting from dried purslane with 50% ethanol and then concentrating under reduced pressure. S2. Macroporous resin adsorption and desorption: The purslane extract prepared in step S1 was dissolved in water and filtered. It was then loaded onto an activated macroporous resin column at low temperature and adsorbed. Gradient elution was performed sequentially with water, 60% ethanol, and 95% ethanol. The 60% ethanol eluent was collected and concentrated to obtain crude pure product 1. S3. Purification by normal phase silica gel chromatography: The crude pure product 1 obtained in S2 was dissolved in 80% ethanol, separated by normal phase silica gel column chromatography, and eluted with an ethyl acetate-methanol system. The component containing luteolin was collected and concentrated to obtain crude pure product 2. S4. Medium-pressure preparative chromatographic purification: The crude pure product 2 obtained in S3 was dissolved in 60% ethanol and filtered through a filter membrane. Then, it was loaded onto a C18 medium-pressure preparative chromatographic column and subjected to gradient elution with methanol and acidic aqueous solution as the mobile phase. The target peak fraction at 350 nm was collected and concentrated to obtain crude pure product 3. S5. Dextran gel chromatography purification: The crude pure product 3 obtained in S4 was dissolved in methanol aqueous solution and filtered through a filter membrane. The sample was loaded onto a dextran gel chromatography column, eluted with methanol aqueous solution, and the target peak fraction at 350 nm was collected. The fractions were combined, concentrated, and dried to obtain high-purity luteolin.

2. The method for extracting and purifying luteolin from purslane according to claim 1, characterized in that, In step S1, the preparation method of purslane extract is as follows: After soaking dried purslane in 50% ethanol overnight, the soaking solution is collected by filtration and stored at 4°C; the remaining powder obtained by filtration is then subjected to ultrasonic extraction again with another fresh 50% ethanol solution for 2-3 hours, the soaking solution and extract are combined and filtered, and concentrated under reduced pressure to remove ethanol to obtain purslane extract.

3. The method for extracting and purifying luteolin from purslane according to claim 2, characterized in that, Ultrasonic extraction with 50% ethanol at 50-60℃ was performed, and the solvent was recovered from the extract under reduced pressure at 50-60℃.

4. The method for extracting and purifying luteolin from purslane according to claim 1, characterized in that, In step S2, the macroporous resin used is D-101 macroporous resin, and the activation method is as follows: D-101 macroporous resin is soaked in 95% ethanol for 20-30 hours and then wet-packed into a column. It is then eluted with 95% ethanol until there is no alcohol odor and the eluent is not cloudy when water is added. It is then rinsed with pure water until there is no alcohol odor. Then it is soaked in 5% HCl for 4 hours and washed with pure water until neutral. Soak in 2% NaOH for 4 hours, wash with pure water until neutral, activate, and store in pure water for later use. The purslane extract was dissolved in water, filtered through a 0.45 μm filter membrane, loaded at a flow rate of 20 mL / min, and adsorbed at a low temperature of 4 °C.

5. The method for extracting and purifying luteolin from purslane according to claim 1, characterized in that, In step S3, the gradient elution procedure is as follows: elution is performed sequentially with ethyl acetate:methanol at ratios of 40:1, 25:1, 20:1, and 15:1, with TLC monitoring. The fractions containing luteolin are combined and concentrated under reduced pressure to obtain crude product 2.

6. The method for extracting and purifying luteolin from purslane according to claim 1, characterized in that, In step S4, crude pure product 2 is dissolved in 60% methanol and filtered through a 0.45μm filter membrane; The C18 medium-pressure preparative chromatographic column was used at a column temperature of 30℃, a detection wavelength of 350nm, and a flow rate of 20ml / min. Gradient elution was performed using 0.1% formic acid aqueous solution and methanol as phase A and phase B, respectively: 0-5min, 20% B; 5-15min, 20%-30% B; 15-25min, 30%-40% B; 25-35min, 40%-50% B; 35-45min, 50%-60% B; 45-50min, 60%-20% B; 50-55min, 20% B. The target peak fraction at 350nm was collected, and after verification, the components containing the target analyte were combined and concentrated under reduced pressure to obtain crude product 3 with a purity ≥90%.

7. The method for extracting and purifying luteolin from purslane according to claim 1, characterized in that, In step S5, the dextran gel is G-10, which is swollen and washed with 5% methanol aqueous solution to separate crude pure product 3. The crude pure product 3 was dissolved in a 5% methanol aqueous solution, filtered through a 0.45μm filter membrane, and then loaded onto the sample. It was continuously eluted with a 5% methanol aqueous solution, and the target peak fraction at 350nm was collected. The fractions with high absorbance and single TLC fluorescence were combined, concentrated under reduced pressure at 40-60℃, and freeze-dried to obtain a pure product of the target substance with a purity ≥97%.

8. The use of luteolin extracted and purified by the method according to any one of claims 1-7 in the preparation of drugs or functional foods for improving metabolic-related fatty liver disease.

9. The application according to claim 8, characterized in that, The luteolin improves metabolic-associated fatty liver disease in one or more of the following ways: a) Improve MASH pathological conditions in MASH model animals, such as hepatic steatosis, lobular inflammation, and hepatocyte ballooning degeneration; b) Reduce triglyceride (TG) or cholesterol (TC) levels in MASH model animals; c) Reduce the levels of pro-inflammatory cytokines TNF-α or IL-1β in the liver.

10. A pharmaceutical composition for preventing or alleviating metabolic-associated steatohepatitis, characterized in that, Luteolin extracted and purified by the method described in any one of claims 1-7 is used as the active component.

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