Application of apium graveolens extract in preparation of medicine for treating fatty liver disease related to metabolic dysfunction

Abstract

The invention belongs to the field of medicine, and particularly relates to application of apium graveolens extract to preparation of medicine for treating fatty liver diseases related to metabolic dysfunction. The celery and sophora flower extract is an alcohol extract of celery seeds and sophora flower buds in a weight ratio of (1: 1)-(4: 1), preferably (3: 1)-(4: 1), and further preferably (3: 1). The invention provides a new choice for clinical medication for treating fatty liver diseases related to metabolic dysfunction, and has great clinical value and social significance.

Description

Technical Field

[0001] This invention relates to the use of Sophora japonica extract in the preparation of a medicament for treating metabolic dysfunction-associated fatty liver disease (MASLD), and belongs to the pharmaceutical field. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) is a collective term for a group of liver diseases, formerly known as non-alcoholic fatty liver disease (NAFLD) or metabolic-associated fatty liver disease (MAFLD). Metabolic dysfunction-associated steatohepatitis (MASH) is the progressive form of MASLD. Compared to simple hepatic steatosis, MASH is accompanied by hepatocellular damage and liver inflammation, which can lead to progressive liver fibrosis and cirrhosis or promote the development of hepatocellular carcinoma and liver-related death.

[0003] Metabolic liver disease (MASLD) has a complex course and is the most prevalent chronic liver disease worldwide, with a global prevalence of approximately 25.2%, becoming the most common cause of liver disease in children and adults. Currently, there are approximately 250 million people with metabolic liver disease in my country, and the incidence rate is expected to continue to rise with improved living standards, an aging population, and increased obesity. Despite the alarmingly high incidence of MASLD, placing a significant burden on patients and the entire healthcare system, public awareness of the disease is limited. Only 5% of MASLD patients are aware of their condition, and over 12%-14% have metabolic angina (MASH). Approximately 25% of MASH patients can progress to advanced liver fibrosis, cirrhosis, or even hepatocellular carcinoma (HCC). MASLD is a leading cause of end-stage liver disease (liver failure, cirrhosis, and liver cancer). In the coming years, the number of patients undergoing liver transplantation will increase rapidly, making this a growing public health concern.

[0004] The harm of MASLD is not limited to the liver itself; it can affect multiple organs throughout the body and induce and aggravate coronary heart disease, promote the formation of atherosclerosis, and is closely related to various metabolic disorders, including obesity, type 2 diabetes, hypertension, dyslipidemia, and metabolic syndrome. Various metabolic diseases influence each other, forming a vicious cycle.

[0005] Therefore, it is necessary to provide appropriate drug intervention in the early stages of MASLD. Unfortunately, no drugs are currently approved for the treatment of MASLD, either domestically or internationally. Only a few drugs have shown apparent effectiveness against MASLD in clinical trials, but the evidence is insufficient. These include some hypoglycemic agents (pioglitazone, GLP-1RAs, SGLT-2 inhibitors, and metformin) and other drugs (vitamin E, obeticholic acid, statins, pentoxifylline, and Saroglitazar Magnesium). The following sections will describe these drugs currently in clinical trials:

[0006] Pioglitazone: It can promote the transfer of fat from the liver to peripheral tissues, reduce hepatic steatosis, and effectively improve MASH (metastatic angina) complication of type 2 diabetes. 45 mg / day can also improve liver fibrosis. Whether sufficient doses of pioglitazone are necessary to effectively improve liver fibrosis is currently lacking, as studies on the efficacy of different dosages are insufficient. However, adverse reactions during treatment, such as weight gain, edema, bladder cancer, and decreased bone density, limit the use of pioglitazone in this therapeutic area.

[0007] Glucagon-like peptide-1 receptor agonists (GLP-1RAs): Randomized controlled trials and meta-analyses have found that they can reduce weight and improve histological lesions, including liver fibrosis, in patients with MASLD, but gastrointestinal reactions such as loss of appetite may affect the improvement of patients' symptoms.

[0008] Sodium-glucose cotransporter 2 (SGLT2) inhibitors: can reduce liver fat content in patients with MASLD, but currently only small-sample exploratory clinical trials have shown that SGLT2 inhibitors significantly improve the degree of hepatic steatosis, ballooning degeneration and fibrosis in patients with steatohepatitis, and the effect on liver fibrosis still needs further research.

[0009] Metformin can improve insulin resistance and reduce the risk of liver cancer in patients with MAFL in a retrospective, non-cohort observational study, but it cannot improve liver histological lesions in patients with MASLD.

[0010] Vitamin E: Clinical trial results from the United States showed that oral administration of vitamin E (800 IU / day) for two years could normalize serum alanine aminotransferase levels in non-diabetic MASH adults and significantly improve hepatic steatosis and inflammatory damage. However, high-dose vitamin E treatment for chronic hepatitis is not approved in my country, and long-term high-dose use of vitamin E may increase the risk of prostate cancer.

[0011] Obeticholic acid can significantly reduce the degree of liver fibrosis in MASH patients, but it has adverse effects on lipid metabolism, can cause skin itching, and its role in the treatment of MASH has not been confirmed by clinical trials in Japan.

[0012] Statins: They have no beneficial effect on liver histology in patients with MASLD, and there is a lack of strong evidence of pathological improvement in MASH and fibrosis, but their lipid-regulating effect is certain and they can reduce the incidence of cardiovascular disease in patients with MASLD.

[0013] Pentoxothecobalaine: a phosphodiesterase inhibitor with anti-inflammatory effects. Meta-analysis has shown that it can improve hepatic lobular inflammation and NAS scores in patients with MASLD; however, it does not significantly improve hepatic steatosis, ballooning degeneration or fibrosis, or dyslipidemia.

[0014] Saroglitazar Magnesium: In March 2020, Cadila Pharmaceuticals' Saroglitazar Magnesium was approved in India for the treatment of NASH. This drug targets the peroxisome proliferator-activated receptor (PPAR) and is a dual PPARα / γ agonist, primarily activating the PPARα receptor. It is currently undergoing Phase III clinical trials globally. Clinical trials suggest that the drug has limited efficacy in treating hepatocellular damage and fibrosis, and some patients experienced mild weight gain. Its prospects for approval in Europe and the United States for the treatment of NASH are not optimistic.

[0015] In summary, there are currently no specific effective treatments for MASLD in China. Besides the drugs mentioned above, the bicyclol, polyene phosphatidylcholine, silymarin, diammonium glycyrrhizate, and ursodeoxycholic acid, which are widely used in clinical practice in my country, mainly target abnormal liver enzymes and cannot improve the liver pathological changes associated with MASLD. Therefore, there is a significant unmet clinical need for treatment of MASLD, and traditional Chinese medicine is receiving increasing attention for its role in improving MASLD. Summary of the Invention

[0016] In order to address the shortcomings of existing therapeutic drugs and develop drugs for the treatment of MASLD, the inventors have been committed to using new ideas to solve the above-mentioned technical problems.

[0017] Based on the mechanism of action of hepatoprotective and enzyme-lowering drugs, the inventors developed a drug for the treatment of MASLD. Through research, they unexpectedly discovered that extracts of celery seed and sophora japonica buds in different weight ratios produced surprisingly excellent therapeutic effects on MASLD.

[0018] The celery seed and sophora flower bud extract is an alcoholic extract of celery seeds and sophora flower buds in a weight ratio of 1:1–4:1, for example, obtained according to the formulation and method of Chinese Patent ZL201610313303.8. The celery seed and sophora flower bud alcoholic extract of this invention is an extract of celery seeds or sophora flower buds in an alcoholic solvent, with the ratio of celery seeds to sophora flower buds in the extract being 1:1 to 4:1. The alcoholic solvent can be a C1–C4 alcoholic solvent, preferably methanol, ethanol, isopropanol, or n-butanol, or it can be an aqueous alcohol solution, and the concentration of the aqueous alcohol solution is preferably 50%–80% by volume. The celery seed and sophora flower bud extract of this invention can be in the form of tablets, granules, dry suspensions, or capsules, preferably capsules.

[0019] According to literature reports, apigenin, one of the active components of celery seeds, has antioxidant, sedative, analgesic, antitumor, antihypertensive, and lipid-lowering effects. It also has preventive and therapeutic effects on MASLD by interfering with the release of inflammatory mediators, improving insulin sensitivity, regulating the expression of lipid metabolism-related genes, and providing antioxidant effects. Currently, no research has been found on the direct treatment of MASLD with Sophora japonica flower buds. However, quercetin, one of its active components, has a protective effect against metabolic-related fatty liver damage in type 2 diabetic mice, especially showing a good improvement effect on liver fibrosis. This mechanism may be related to the anti-inflammatory activity of quercetin. However, no literature reports that celery seed and Sophora japonica flower bud alcohol extracts have preventive or therapeutic effects on MASLD.

[0020] In vivo experiments were conducted on a rat model of MASLD using celery seed and sophora japonica extract in different mixing ratios. Unexpectedly, we found that celery seed and sophora japonica extract has beneficial effects such as lowering enzymes and lipids, improving liver lipid metabolism and liver steatosis. It can be used to prepare drugs for the treatment of MASLD. Its effects are significantly better than those of using celery seed and sophora japonica extract alone, and it shows a synergistic effect in a ratio of 3:1 to 4:1.

[0021] Therefore, the present invention provides the use of celery and sophora flower bud extract in the preparation of a medicament for treating fatty liver disease associated with metabolic dysfunction, wherein the celery and sophora flower bud extract is an alcoholic extract of celery seeds and sophora flower buds in a weight ratio of 1:1–4:1, preferably an alcoholic extract of celery seeds and sophora flower buds in a weight ratio of 3:1–4:1, and more preferably an alcoholic extract of celery seeds and sophora flower buds in a weight ratio of 3:1.

[0022] In the use of the Sophora japonica extract of the present invention in the preparation of a medicament for treating fatty liver disease related to metabolic dysfunction, the medicament for treating fatty liver disease related to metabolic dysfunction comprises Sophora japonica extract as an active ingredient, which, after being mixed with various pharmaceutically acceptable excipients, can be prepared into a pharmaceutical composition according to the prior art; the pharmaceutical composition is preferably prepared into various oral dosage forms, such as tablets, granules, capsules, dry suspensions, etc., preferably capsules.

[0023] On the other hand, the present invention provides a novel pharmaceutical composition for treating fatty liver disease associated with metabolic dysfunction. The pharmaceutical composition comprises *Sophora japonica* extract as an active ingredient, which, when mixed with various pharmaceutically acceptable excipients, can be prepared according to existing techniques in the art. The *Sophora japonica* extract is an alcoholic extract of celery seeds and *Sophora japonica* flower buds in a weight ratio of 1:1–4:1, preferably an alcoholic extract of celery seeds and *Sophora japonica* flower buds in a weight ratio of 3:1–4:1, and more preferably an alcoholic extract of celery seeds and *Sophora japonica* flower buds in a weight ratio of 3:1. The pharmaceutical composition is preferably formulated into various oral dosage forms, such as tablets, granules, capsules, dry suspensions, etc., preferably capsules.

[0024] The beneficial effect of this invention lies in providing a novel use of *Sophora japonica* extract in the preparation of medicaments for treating fatty liver disease associated with metabolic dysfunction. Experiments have shown that *Sophora japonica* extract has the following excellent effects:

[0025] 1. Significantly reduced the elevation of serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in experimental animals;

[0026] 2. Significantly reduced the elevation of serum total cholesterol (TC) and serum triglycerides (TG) in experimental animals;

[0027] 3. Significantly improves the color and appearance of the liver in experimental animals;

[0028] 4. Significantly reduced liver lipid metabolism indicators (TC, TG) in experimental animals;

[0029] 5. Significantly improves the degree of liver tissue lesions in experimental animals. Attached Figure Description

[0030] Figure 1 Appearance of liver tissue in experimental animals 4 weeks after drug administration (top: normal group; middle: model group; bottom: sample group 1)

[0031] Figure 2 Appearance of liver tissue in experimental animals 4 weeks after drug administration (top: sample group 2; middle: sample group 3; bottom: sample group 4)

[0032] Figure 3 Appearance of liver tissue in experimental animals 8 weeks after drug administration (top: normal group; middle: model group; bottom: sample group 1)

[0033] Figure 4 Appearance of liver tissue in experimental animals 8 weeks after drug administration (top: sample group 2; middle: sample group 3; bottom: sample group 4)

[0034] Figure 5Appearance of liver tissue in experimental animals 12 weeks after drug administration (top: normal group; middle: model group; bottom: sample group 1)

[0035] Figure 6 Appearance of liver tissue in experimental animals 12 weeks after drug administration (top: sample group 2; middle: sample group 3; bottom: sample group 4)

[0036] Figure 7 Liver pathological changes 12 weeks after drug administration (normal group, HE×100)

[0037] Figure 8 Liver pathological changes 12 weeks after drug administration (model group, HE×100)

[0038] Figure 9 Liver pathological changes 12 weeks after drug administration (sample group 1, HE×100)

[0039] Figure 10 Liver pathological changes 12 weeks after drug administration (sample group 2, HE×100)

[0040] Figure 11 Liver pathological changes 12 weeks after drug administration (sample group 3, HE×100)

[0041] Figure 12 Liver pathological changes 12 weeks after drug administration (samples from 4 groups, HE×100)

[0042] Figure 13 Liver pathological changes after 12 weeks of drug administration (celery seed group 1, HE×100)

[0043] Figure 14 Liver pathological changes after 12 weeks of drug administration (celery seed group 2, HE×100)

[0044] Figure 15 Liver pathological changes after 12 weeks of drug administration (celery seed group 3, HE×100)

[0045] Figure 16 Liver pathological changes after 12 weeks of drug administration (celery seed group 4, HE×100)

[0046] Figure 17 Liver pathological changes after 12 weeks of drug administration (Sophora japonica group, HE×100, HE×100) Detailed Implementation

[0047] Example 1:

[0048] We conducted in vivo experiments on a MASLD rat model using celery and sophora flower extracts prepared from celery seeds and sophora flowers in different weight ratios.

[0049] The celery and sophora flower extract used is an alcoholic extract of celery seeds and sophora flower buds in a weight ratio of 1:1–4:1, obtained according to the method of Chinese patent ZL201610313303.8. The operation of the celery and sophora flower extract in this embodiment is as follows:

[0050] Celery seeds and Sophora japonica buds were weighed according to the specified weight ratio, mixed, and then extracted three times with 10 times their weight of 60% (v / v) ethanol aqueous solution at 50-60℃ for 60 minutes each time. The extracts were combined, concentrated under reduced pressure, and dried under vacuum to obtain the celery and Sophora japonica extract.

[0051] Celery seed extract and Sophora japonica flower bud extract were prepared using the same extraction process as the celery and Sophora japonica flower bud extract mentioned above.

[0052] The experimental process and results are as follows:

[0053] 1. Laboratory animals

[0054] Two hundred and thirty-two SPF-grade male SD rats, 4-5 weeks old and weighing 140-160g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. Prior to the experiment, they underwent 5-7 days of quarantine and environmental acclimatization (including the day of receipt) until their weight reached 180-200g.

[0055] 2. Experimental conditions

[0056] Each cage contains 3-5 animals, kept in a clean environment with a temperature of 18-20℃ and a humidity of 50%, following a 12-hour diurnal rhythm.

[0057] 3. Modeling methods

[0058] After passing quarantine, SD rats were randomly divided into a normal group (n=23, including 3 animals for model evaluation) and a model group (n=209, including 9 animals for model evaluation) based on their body weight and triglyceride (TG) levels. A rat model of hepatic steatosis associated with metabolic dysfunction was induced by feeding the rats with a high-fat diet. The high-fat diet formulation is shown in Table 1. Except for the normal group, the model group animals were fed a high-fat diet for 12-16 weeks (after which the high-fat diet was discontinued and replaced with a maintenance diet). Except for the normal group, all other groups received a subcutaneous injection of 40% carbon tetrachloride (CCl4) oil solution (prepared with soybean oil for injection) at 3 mL / kg once in the right hind limb on the second day after model induction.

[0059] Table 1. Composition of high-fat diet

[0060]

[0061] 4. Animal grouping and identification

[0062] During the experiment, samples were collected at different time points to measure blood lipid levels, liver function indicators, visceral lipid metabolism indicators, and liver pathological examination. After confirming the model's establishment, the model group animals were randomly divided according to body weight and triglyceride (TG) levels into 11 groups: model group, 1-4 groups of different proportions of celery and sophora japonica extract (hereinafter referred to as "sample") (weight ratio 1:1, 2:1, 3:1, 4:1), 1-4 groups of celery seed extract (hereinafter referred to as "celery seed"), 1 group of sophora japonica flower extract (hereinafter referred to as "sophora japonica flower"), and normal group, with 20 animals in each group, for a total of 220 animals. The average body weight difference between groups after grouping did not exceed ±20%. Animal cage cards were used to distinguish the groups; the last digit of the cage number was used during the quarantine period, and ear tags were used to identify the animals during the experimental period after grouping.

[0063] Table 2 Animal grouping and drug dosage

[0064]

[0065] 5. Test drugs

[0066] Test formulations: sample groups with different ratios (1:1, 2:1, 3:1, 4:1 groups), celery seed groups 1-4, and sophora japonica flower groups were prepared into suspensions with 0.5% sodium carboxymethyl cellulose (CMC-Na) aqueous solution before use.

[0067] 6. Experimental Grouping

[0068] Animals were randomly divided into a normal group, a model group, sample groups 1-4, celery seed groups 1-4, and a sophora flower group, with 20 animals in each group.

[0069] 7. The route, period, frequency of administration of the test substance and the rationale for its dosage design.

[0070] Administration route and method: oral gavage, the same route as the clinical oral administration of the test substance.

[0071] Administration cycle and frequency: Once the animals undergoing model evaluation have undergone serological and liver histopathological examinations to confirm the presence of steatosis-like lesions (evaluation criteria see 8.1.5, at least grade 1 mild steatosis), the test substance or control substance will be administered once daily, 7 days a week, for 12 consecutive weeks.

[0072] Dosage: The dosage of the test substance was determined based on the results of the preliminary test; both the normal group and the model group were given 0.5% sodium carboxymethyl cellulose.

[0073] Administration volume: The gavage volume for each group was 10 mL / kg. The animal's body weight was measured twice a week, and the administration volume was calculated based on the most recent animal body weight, rounded to one decimal place (using a 5 / 10 mL syringe with an accuracy of 0.2 mL).

[0074] 8. Observation Indicators and Methods

[0075] 8.1 Testing Content

[0076] 8.1.1 Clinical symptom observation

[0077] Observe the animal once a day and record any abnormalities in its appearance, physical signs, and behavior. If any abnormalities are found, the observation frequency can be increased.

[0078] 8.1.2 Weight Measurement

[0079] Weigh the animals twice a week to monitor changes in their weight.

[0080] 8.1.3 Blood lipids and liver function indicators

[0081] Animals were fasted overnight (but allowed water). They were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 mL / 100 g body weight), and 5 mL of blood was collected from the abdominal vein (the animals were euthanized by exsanguination via the femoral artery after blood collection). Serum was separated, and lipid markers (TC, TG) and liver function markers (ALT, AST) were measured.

[0082] 8.1.4 Detection of liver lipid metabolism indicators

[0083] Animals were euthanized by exsanguination of the femoral artery after blood collection. An appropriate amount of liver tissue was taken and homogenized into a 10% liver tissue homogenate under ice bath conditions. The supernatant was collected by centrifugation at 3000 rpm for 10 minutes at 4°C. The lipid metabolism indicators (TC, TG) of the liver tissue were measured using a fully automated blood biochemistry analyzer or a kit.

[0084] 8.1.5 Pathological score of hepatic steatosis

[0085] Liver tissue was taken from the same location, thoroughly fixed in 10% neutral formalin solution, and routinely prepared for pathological examination. Hematoxylin and eosin (HE) staining was performed, and the fatty degeneration was scored. Grade 0 (-): Normal liver lobules, normal hepatocyte structure; Grade 1 (+): Mild fatty degeneration, with hepatocyte fatty degeneration accounting for ≤25% of the liver lobules, predominantly small lipid droplets, with a few medium-sized droplets; Grade 2 (++): Moderate fatty degeneration, with hepatocyte fatty degeneration accounting for 25%-50% of the liver lobules, predominantly small to medium-sized lipid droplets, with a few large lipid droplets; Grade 3 (+++): Severe fatty degeneration, with hepatocyte fatty degeneration accounting for 50%-75% of the liver lobules, predominantly medium to large lipid droplets; Grade 4 (+++): Severe fatty degeneration, with hepatocyte fatty degeneration accounting for more than 75% of the liver lobules, predominantly medium to large lipid droplets.

[0086] 8.2 Indicator Testing Plan

[0087] Blood lipids and liver function indicators: The levels of blood lipids and liver function indicators were measured in each group of animals one day before administration and every four weeks after administration.

[0088] Liver lipid metabolism indicators and pathological examination: Different numbers of animals in each group were collected at 4, 8 and 12 weeks after drug administration for testing (see Table 3).

[0089] Table 3. Frequency of indicator detection and number of animals

[0090]

[0091] 9. Test Results

[0092] 9.1 Clinical symptoms

[0093] From the time the animals were grouped until the end of the administration, except for a few animals in the normal group and sample group 4 who had temporary loose stools, no abnormalities were observed in the appearance, behavior, feeding, or drinking of the animals in any group, and no animals died in any group.

[0094] 9.2 Weight Changes

[0095] Table 4 shows the changes in animal body weight before and during administration at 4-week intervals.

[0096] Table 4. Weight changes in experimental animals during drug administration.

[0097]

[0098] During the administration period, the weight of animals in each group gradually increased, and there was no significant effect on the weight of animals in any of the administration groups.

[0099] 9.3 Liver function and blood lipid levels

[0100] 9.3.1 Liver function indicators

[0101] The serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels of experimental animals are shown in Tables 5-8.

[0102] Table 5. Serum liver function and blood lipid levels of experimental animals before drug administration.

[0103]

[0104] Note: Compared with the normal group, * p < 0.05

[0105] Table 6. Serum liver function and blood lipid levels in experimental animals after 4 weeks of drug administration.

[0106]

[0107] Note: Compared with the normal group, * p < 0.05 ** p < 0.01 *** p < 0.001; compared with the model group, ## p < 0.01 ### p < 0.001.

[0108] Table 7. Serum liver function and blood lipid levels in experimental animals after 8 weeks of drug administration.

[0109]

[0110] Note: Compared with the normal group, * p < 0.05 ** p < 0.01 *** p < 0.001; compared with the model group, # p < 0.05 ## p < 0.01 ### p < 0.001.

[0111] Table 8. Serum liver function and blood lipid levels in experimental animals after 12 weeks of drug administration.

[0112]

[0113] Note: Compared with the normal group, * p < 0.05 ** p < 0.01 *** p < 0.001; compared with the model group, # p < 0.05 ## p < 0.01 ### p < 0.001.

[0114] Results analysis:

[0115] Four weeks after being fed a high-fat diet (before the first administration), serum ALT and AST levels in all other groups were significantly increased compared with the normal group (p<0.05).

[0116] Four weeks after administration, compared with the normal group, the ALT levels in the remaining groups were elevated to varying degrees (p<0.05); compared with the model group, the ALT and AST levels in each administration group were decreased to varying degrees, with the decreases in ALT and AST being greater in sample groups 3 and 4 (p<0.05, p<0.01, or p<0.001); compared with the celery seed group 3 + Sophora japonica bud group, the decrease in AST and ALT in sample group 3 was greater than the sum of the decreases in the two groups, indicating a synergistic effect; compared with the celery seed group 4 + Sophora japonica bud group, the AST and ALT levels in sample group 4 were significantly decreased, and the decrease was greater than the sum of the decreases in the two groups, indicating a synergistic effect.

[0117] After 8 weeks of administration, compared with the normal group, except for the model group, Sophora japonica bud group, and Celery seed group 1 which showed elevated ALT levels, the remaining groups all showed decreased ALT levels. Among them, the ALT levels of sample group 3, sample group 4, and Celery seed group 3 showed statistically significant differences (p<0.05). Compared with the normal group, the AST levels of sample group 3, sample group 4, and Celery seed group 3 were significantly decreased (p<0.001), while no significant changes were observed in the other groups. Compared with the model group, the AST levels of sample group 3, sample group 4, and Celery seed group 3 were also significantly decreased (p<0.01). Compared with the Celery seed group 3 + Sophora japonica bud group, the decrease in AST and ALT in sample group 3 was greater than the sum of the decreases in the Celery seed group 3 + Sophora japonica bud group, indicating a synergistic effect. Compared with the Celery seed group 4 + Sophora japonica bud group, the decrease in AST and ALT in sample group 4 was greater than the sum of the decreases in the Celery seed group 4 + Sophora japonica bud group, indicating a synergistic effect.

[0118] After 12 weeks of drug administration, compared with the normal group, the ALT levels of the remaining groups of animals did not change significantly, but the AST levels of sample groups 3, 4, celery seed group 3, celery seed group 4, and Sophora japonica group all decreased significantly (p<0.05, p<0.01, or p<0.001). Compared with the model group, except for sample groups 1, 2, celery seed group 1, and celery seed group 2, the ALT and AST levels of the other groups decreased to varying degrees (p<0.05, p<0.01). Compared with the celery seed group 3 + Sophora japonica group, the decrease in AST and ALT in sample group 3 was greater than the sum of the decreases in the two groups, indicating a synergistic effect. Compared with the celery seed group 4 + Sophora japonica group, the decrease in AST and ALT in sample group 4 was greater than the sum of the decreases in the two groups, indicating a synergistic effect.

[0119] 9.3.2 Blood lipid indicators

[0120] The blood lipid data of the experimental animals are shown in Tables 5-8 above.

[0121] Results analysis:

[0122] Four weeks after being fed a high-fat diet (before the first administration), compared with the normal group, the serum total cholesterol (TC) and serum triglycerides (TG) in the other groups were increased to varying degrees (p<0.05).

[0123] Four weeks after administration, compared with the normal group, except for sample group 3 (p<0.01), the serum TG and TC levels of the remaining groups of animals were increased to varying degrees. Compared with the model group, TG and TC in sample groups 3 and 4 were significantly decreased (p<0.05, p<0.01, or p<0.001). Compared with the celery seed group 3 + Sophora japonica flower group, the decrease in TG and TC in sample group 3 was greater than the sum of the decreases in the two groups, indicating a synergistic effect. Compared with the celery seed group 4 + Sophora japonica flower group, the decrease in TG and TC in sample group 4 was greater than the sum of the decreases in the two groups, indicating a synergistic effect.

[0124] Eight weeks after administration, compared with the normal group, except for the model group, celery seed group 1, and 2, the serum TC and TG levels of the remaining groups showed a decreasing trend, with the TC and TG levels of sample group 3 showing a significant decrease (p<0.05 or p<0.01). Compared with the model group, the test substance showed a trend of reducing serum TG, with the TC and TG levels of sample group 3 showing a significant decrease (p<0.05). Compared with the celery seed group 3 + Sophora japonica group, the TG decrease in sample group 3 was greater than the sum of the decreases in the two groups, indicating a synergistic effect. Compared with the celery seed group 4 + Sophora japonica group, the TG decrease in sample group 4 was greater than the sum of the decreases in the two groups, indicating a synergistic effect.

[0125] After 12 weeks of drug administration, compared with the normal group, serum TC and TG levels in sample groups 3 and 4 were significantly decreased (p<0.05). Compared with the model group, TC and TG levels in sample groups 3, 4, celery seed group 3, celery seed group 4, and Sophora japonica flower group were all significantly decreased (p<0.05 or p<0.01). Compared with the celery seed group 3 + Sophora japonica flower group, the decrease in TG in sample group 3 was greater than the sum of the decreases in the two groups, indicating a synergistic effect. Compared with the celery seed group 4 + Sophora japonica flower group, the decrease in TG in sample group 4 was greater than the sum of the decreases in the two groups, indicating a synergistic effect.

[0126] The above results indicate that Sophora flavescens extract can reduce serum TC and TG levels in model animals, which helps to correct abnormal lipid metabolism. Sample groups 3 and 4 showed a synergistic effect.

[0127] 9.4 Liver Appearance

[0128] The appearance of the liver of the laboratory animal is shown in the attached figure. Figures 1-6 .

[0129] After 4 weeks of high-fat feeding, the livers of animals in the normal group were bright red and normal in shape, and the liver tissue structure was normal under the microscope. However, the livers of animals in the model group were significantly yellowish, and diffuse mild vacuolar degeneration of hepatocytes was visible under the microscope. The TC and TG contents of liver tissue homogenate were significantly increased and decreased, indicating that the animals developed typical fatty liver after high-fat feeding.

[0130] Four weeks after administration, the livers of animals in the normal group were bright red with sharp edges, while the livers of animals in the model group were yellowish and significantly enlarged. The livers of some animals in the celery extract group showed some improvement in color.

[0131] Eight weeks after administration, the liver tissue of the normal group animals showed normal color and morphology, while the livers of the remaining groups were slightly yellowish, with thick, rounded, and greasy liver lobes. The liver color and morphology of most animals in the *Sophora japonica* extract group showed further improvement.

[0132] After 12 weeks of administration, the livers of animals in the normal group showed normal color and morphology, while the livers of animals in the *Sophora japonica* extract group showed significant improvement compared to 8 weeks, with most livers returning to red color and decreasing in size. The improvement was particularly significant in sample group 3.

[0133] 9.5 Hepatic lipid metabolism

[0134] The lipid metabolism parameters of the liver of experimental animals are shown in Table 9.

[0135] Table 9 Results of liver lipid metabolism parameters in experimental animals after drug administration

[0136]

[0137]

[0138] Note: Compared with the normal group, * p < 0.05 ** p < 0.01 *** p < 0.001; compared with the model group, # p < 0.05 ## p < 0.01 ### p < 0.01; compared with 8 weeks after administration, SS p < 0.01

[0139] Results analysis:

[0140] Four weeks after administration, compared with the model group, the TC and TG levels in sample groups 3, 4, celery seed group 3, and sophora flower group were significantly decreased (p<0.05 or p<0.01). Compared with the celery seed group 3 + sophora flower group, the changes in TC and TG in sample group 3 were greater than the sum of the changes in the indicators of the single-use groups, indicating a synergistic effect. Compared with the celery seed group 4 + sophora flower group, the changes in TC and TG in sample group 4 were greater than the sum of the changes in the indicators of the single-use groups, indicating a synergistic effect.

[0141] Eight weeks after administration, compared with the model group, the TC and TG levels in sample groups 3, 4, celery seed group 3, celery seed group 4, and Sophora japonica bud group were significantly reduced (p<0.01). Compared with the celery seed group 3 + Sophora japonica bud group, the changes in TC and TG in sample group 3 were greater than the sum of the changes in the indicators of the single-use groups, indicating that the two had a synergistic effect; compared with the celery seed group 4 + Sophora japonica bud group, the TC and TG levels in sample group 4 decreased, but the changes were not greater than the sum of the changes in the indicators of the single-use groups.

[0142] After 12 weeks of administration, compared with the model group, the TC and TG levels in sample groups 3, 4, celery seed group 3, and sophora flower group were significantly decreased (p<0.05, p<0.01). Compared with the celery seed group 3 + sophora flower group, the changes in TC and TG in sample group 3 were greater than the sum of the changes in the indicators of the single-use groups, indicating that the two had a synergistic effect; compared with the celery seed group 4 + sophora flower group, the changes in TC and TG in sample group 4 were not greater than the sum of the changes in the indicators of the single-use groups.

[0143] The above results indicate that Sophora japonica extract can inhibit the content of TC and TG in liver tissue, which helps to reduce the severity of hepatic steatosis.

[0144] 9.6 Liver Histopathology

[0145] The results of histopathological examination of the liver tissue of the experimental animals are shown in the figure. Figures 7-17 .

[0146] 9.6.1 Pathological changes in liver tissue

[0147] Twelve weeks after drug administration, the liver tissue structure in the normal group was normal; in the model group, diffuse mild vacuolar degeneration of hepatocytes was observed, with some hepatocytes showing mild vacuolar degeneration. In samples 1 and 2, the structure of the liver lobules and portal areas was normal, with diffuse mild vacuolar degeneration of hepatocytes; in samples 3 and 4, the structure of the liver lobules and portal areas was normal, with scattered mild vacuolar degeneration of hepatocytes. The celery seed and sophora japonica flower groups also showed significant improvement compared to the model group.

[0148] 9.6.2 Pathological scoring of hepatic steatosis

[0149] Table 10 shows the pathological scores of hepatic steatosis 12 weeks after administration.

[0150] Table 10 Pathological Scoring of Hepatic Steatodeformation (HE Staining)

[0151] Group score normal group 0.04±0.07 Model group 1.80±0.54** Sample 1 group 1.45±0.55 2 groups of samples 0.83±0.41 3 groups of samples <![CDATA[0.17±0.05 ## ]]> 4 groups of samples 0.34±0.17 Celery seed 1 set 1.75±0.68 Celery seeds 2 sets 1.66±0.40 Celery seed 3 groups 1.50±0.44 Celery seed 4 groups 1.52±0.70 Sophora japonica group 0.52±0.26

[0152] Compared with the normal group, **p < 0.01; compared with the model group, ## p < 0.01

[0153] Results analysis:

[0154] After 12 weeks of administration, compared with the normal group, the liver steatosis scores of the remaining groups all showed an increasing trend; compared with the model group, the degree of liver steatosis in the test group showed a significant decreasing trend and exhibited a certain dose-response relationship, with sample group 3 showing a significant decrease in liver steatosis score (p<0.01). The changes in sample group 3 were greater than the sum of the changes in the indicators of celery seed group 3 + Sophora japonica flower group, indicating that the two had a synergistic effect.

[0155] The pathological scoring results of hepatic steatosis showed that the hepatic steatosis model was established, and sample groups 3 and 4 had a certain therapeutic effect.

[0156] 10. Conclusion

[0157] Under the conditions of this experiment, a single subcutaneous injection of 40% carbon tetrachloride followed by a high-fat diet for 8 consecutive weeks successfully induced fatty liver-like lesions in SD rats. After 12 consecutive weeks of oral administration of *Sophora japonica* extract, the results showed that *Sophora japonica* extract significantly reduced blood lipid levels TC and TG; significantly reduced serum ALT and AST; significantly improved liver color and appearance; significantly reduced liver lipid metabolism indicators (TC and TG); and significantly improved the degree of liver tissue lesions. Among the different ratios of *Sophora japonica* extract, the preferred weight ratio was 3:1–4:1, with a further preferred weight ratio of 3:1.

[0158] The above results suggest that Sophora flavescens extract can reduce hepatic lipid metabolism, lower liver enzyme levels, and improve hepatic fatty lesions. The development of this product will provide an effective drug treatment for patients with metabolic dysfunction-related fatty liver disease (MASLD).

[0159] Example 2 Formulation Composition

[0160] The Sophora japonica extract used for the purposes described in this application can be prepared into a pharmaceutical composition by mixing it with various pharmaceutically acceptable excipients in accordance with the prior art; preferably, it can be prepared into various oral dosage forms, including tablets, granules, capsules, dry suspensions, etc., for example, it can be prepared with reference to the method and formulation of Chinese Patent ZL201610313303.8; preferably, it can be prepared as a capsule with reference to the method and formulation of Chinese Patent CN202110326111.1.

Claims

1. Use of Sophora japonica extract in the preparation of a treatment for fatty liver disease associated with metabolic dysfunction.

2. The use according to claim 1, characterized in that, The celery and sophora extract is an alcoholic extract of celery seeds and sophora japonica flowers in a weight ratio of 1:1–4:

1.

3. The use according to claim 1, characterized in that, The celery and sophora extract is an alcoholic extract of celery seeds and sophora japonica flowers in a weight ratio of 3:1–4:

1.

4. The use according to claim 1, characterized in that, The celery extract is a celery seed and sophora japonica extract in a weight ratio of 3:

1.

5. The use according to claims 1-4, characterized in that, The drug for treating fatty liver disease associated with metabolic dysfunction comprises Sophora japonica extract as an active ingredient, which is mixed with various pharmaceutically acceptable excipients to obtain a pharmaceutical composition.

6. A pharmaceutical composition for treating fatty liver disease associated with metabolic dysfunction, characterized in that, The pharmaceutical composition contains celery seed extract as an active ingredient, which is an alcoholic extract of celery seeds and sophora flower buds in a weight ratio of 1:1–4:

1.

7. The pharmaceutical composition for treating fatty liver disease related to metabolic dysfunction according to claim 6, characterized in that, The celery and sophora extract is an alcoholic extract of celery seeds and sophora japonica flowers in a weight ratio of 3:1–4:

1.

8. The pharmaceutical composition for treating fatty liver disease related to metabolic dysfunction according to claim 6, characterized in that, The celery and sophora extract is an alcoholic extract of celery seeds and sophora flowers in a weight ratio of 3:

1.

9. The pharmaceutical composition for treating fatty liver disease related to metabolic dysfunction according to claim 6, characterized in that, The pharmaceutical composition is an oral dosage form.

10. The pharmaceutical composition for treating fatty liver disease related to metabolic dysfunction according to claim 6, characterized in that, The pharmaceutical composition is in the form of tablets, granules, capsules, or dry suspensions, preferably capsules.

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