Traditional Chinese medicine composition for preventing and treating fatty liver diseases related to metabolic dysfunction as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention discloses a traditional Chinese medicine composition for preventing and treating fatty liver diseases related to metabolic dysfunction as well as a preparation method and application of the traditional Chinese medicine composition, and belongs to the technical field of biological medicines and functional foods. The composition contains medicinal and edible ingredients of radix puerariae, rhizoma polygonati or walnut kernels. In-vitro AML-12 cell experiments prove that the composition can be used for remarkably improving lipid metabolism and oxidative stress states of MASLD model cells; zebra fish animal experiments prove that the Liuwei Dihuang pill can obviously improve the liver function, lipid metabolism and liver form of MASLD model juvenile fish, and the treatment effect of the Liuwei Dihuang pill is superior to that of the traditional Liuwei Dihuang pill. The traditional Chinese medicine composition can be used for preparing medicines for treating the MASLD or food additives or functional foods for preventing the MASLD.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and functional food technology, specifically relating to a traditional Chinese medicine composition, its preparation method, and its application in the preparation of drugs or functional foods for the prevention and treatment of fatty liver disease related to metabolic dysfunction. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) has become one of the most prevalent chronic liver diseases worldwide. Its pathological process involves multiple processes, including excessive lipid deposition in the liver, insulin resistance, oxidative stress, and chronic inflammation, seriously threatening human health. Current clinical prevention and treatment methods have significant shortcomings: long-term use of chemical drugs is prone to side effects such as hepatotoxicity and nephrotoxicity, and metabolic disorders; although traditional Chinese medicine formulas have the potential to regulate metabolism holistically, most compound formulas have weak efficacy and are difficult to meet clinical needs.

[0003] Liuwei Dihuang Wan, a classic formula for nourishing kidney yin, has been proven by modern research to have metabolic regulation and antioxidant effects by its main components, Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita, and Poria cocos. However, there are two key problems in the clinical application of this formula: First, long-term use may lead to toxic side effects such as damage to the heart and kidneys, acidosis, and hyperkalemia (Xi Jiangqin, Modern Medicine and Health, 2024, 40(12):2155-2157.), and its toxic components are mainly related to Paeonia suffruticosa and Alisma plantago-aquatica; Second, its regulatory effect on liver lipid metabolism disorders is relatively weak (Feng Jiahua, Shanghai Journal of Traditional Chinese Medicine, 2019, 53(11):71-75.), and it cannot be used for the prevention and treatment of MASLD.

[0004] Existing research on modified versions of Liuwei Dihuang Wan (Six-Ingredient Rehmannia Pill) mainly focuses on "adding ingredients" for optimization. This involves adding additional Chinese medicinal herbs such as Danshen (Salvia miltiorrhiza), Huangqi (Astragalus membranaceus), and Danggui (Angelica sinensis) to increase efficacy or expand indications. These modified versions typically contain more than six types of herbs, which not only fails to reduce the toxicity of the original formula but also increases the risk of toxic side effects due to the introduction of new herbs. More importantly, existing Liuwei Dihuang Wan and its modified versions contain non-food-medicine homologous ingredients (such as Moutan bark and Alisma plantago-aquatica), thus they can only be used as medicines and cannot be used as food additives for the daily prevention of MASLD (Massage and Disease Syndrome).

[0005] Currently, there is still a lack of traditional Chinese medicine compound formulas in clinical practice that can reduce the number of medicinal materials and reduce toxic side effects, while also improving the efficacy of MASLD prevention and treatment, and possessing both pharmaceutical and food additive properties. Summary of the Invention

[0006] This invention addresses the problems existing in the prior art by providing a simplified version of Liuwei Dihuang Pills, a traditional Chinese medicine composition with high safety and significant efficacy. By optimizing the combination of core efficacy components, it achieves both preventive and therapeutic effects against MASLD, while simultaneously improving the safety of the composition and meeting the application requirements of pharmaceuticals and functional foods.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a traditional Chinese medicine composition for the prevention and treatment of fatty liver disease related to metabolic dysfunction, made from the following medicinal and edible plant raw materials: Rehmannia glutinosa (processed), Cornus officinalis, Dioscorea opposita, Poria cocos, and one selected from Pueraria lobata, Polygonatum sibiricum, and walnut kernel. The traditional Chinese medicine composition is a derivative of the simplified Liuwei Dihuang Wan (Six-Ingredient Rehmannia Pill) composed of these five medicinal materials. All components comply with the relevant provisions of the Pharmacopoeia of the People's Republic of China (2020 Edition) and the Catalogue of Medicinal and Edible Products.

[0008] Furthermore, the weight ratio of Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita, and Poria cocos is 8:4:4:3 (totaling 19 parts), and the weight ratio of their total weight to that of Pueraria lobata, Polygonatum sibiricum, or walnut kernels is 19:3-12; preferably, the weight ratio of their total weight to that of Pueraria lobata, Polygonatum sibiricum, or walnut kernels is 19:6-12; and most preferably, the weight ratio of their total weight to that of Pueraria lobata is 19:6.

[0009] Furthermore, the preparation method of the traditional Chinese medicine composition includes the following steps: S1 Raw material pretreatment: Take the above 5 medicinal materials, remove impurities, wash them and dry them at 60℃ for 4 hours, pulverize them and pass them through an 80-100 mesh sieve to obtain raw material coarse powder; S2 Extraction: Add 8-10 times the amount of purified water to the raw material powder and soak for 30 minutes. After boiling over high heat, simmer over low heat for 60 minutes. Filter and collect the filtrate. Add 5-7 times the amount of purified water to the residue and simmer over low heat for 30 minutes. Combine the two decoctions. S3 Concentration: The combined decoctions are concentrated to an extract with a relative density of 1.1-1.2.

[0010] Furthermore, the above-described traditional Chinese medicine composition can be used to prepare a drug for treating metabolic dysfunction-associated fatty liver disease (MASLD), which can reduce lipid accumulation in the liver and alleviate hepatic steatosis.

[0011] Furthermore, the above-mentioned traditional Chinese medicine composition can be used to prepare functional foods or food additives for the prevention of metabolic dysfunction-associated fatty liver disease (MASLD), and can be added to beverages, dairy products, pastries, and health products.

[0012] Furthermore, the above-mentioned traditional Chinese medicine composition can be used to prepare drugs or functional foods that improve lipid metabolism and liver function, thereby reducing the levels of total cholesterol (TC), triglycerides (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and low-density lipoprotein cholesterol (LDL-C), while increasing the level of high-density lipoprotein cholesterol (HDL-C).

[0013] The beneficial effects of this invention are as follows:

[0014] Significantly enhanced efficacy: The traditional Chinese medicine composition designed in this invention can significantly improve lipid accumulation and oxidative stress in MASLD model AML-12 cells in in vitro cell experiments, with a significantly better effect than Liuwei Dihuang Pills; in zebrafish in vivo experiments, its effect on improving hepatic steatosis and lipid metabolism disorders is also significantly better than Liuwei Dihuang Pills.

[0015] Enhanced safety: All components in the composition of this invention are medicinal and edible plants, reducing toxic side effects. In vitro cell experiments have demonstrated that this invention is safer than the traditional Liuwei Dihuang Wan (Six-Ingredient Rehmannia Pill).

[0016] Wide range of applications: It has the properties of both medicine and food additive, which can provide treatment options for MASLD patients and meet the daily prevention needs of healthy people, thus overcoming the limitation of traditional Liuwei Dihuang Pills being used only as a medicine. Attached Figure Description

[0017] Figure 1 The effects of this invention and Liuwei Dihuang Pill on the survival rate of juvenile zebrafish;

[0018] Figure 2 This invention improves lipid over-accumulation in AML-12 cells, a model of metabolic dysfunction-associated fatty liver disease (MASLD).

[0019] Figure 3 This invention relates to the ameliorative effect of the present invention on oxidative stress in AML-12 cells, a model of metabolic dysfunction-associated fatty liver disease (MASLD).

[0020] Figure 4 This invention improves lipid over-accumulation in zebrafish juveniles, a model of metabolic dysfunction-associated fatty liver disease (MASLD).

[0021] Figure 5 This invention describes the ameliorative effects of the invention on total cholesterol (TC), triglycerides (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in zebrafish juveniles with a metabolic dysfunction-associated fatty liver disease (MASLD).

[0022] Figure 6 This invention relates to the HE staining results of the livers of zebrafish juveniles, a model of metabolic dysfunction-associated fatty liver disease (MASLD). Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments and related drawings, but the scope of protection of the present invention is not limited thereto.

[0024] Unless otherwise specified in the examples, the experimental methods used are all conventional methods, and the reagents and instruments used are all commercially available products. Unless otherwise specified, all extraction reagents used in this invention are analytical grade reagents.

[0025] Examples and Materials and Methods Used in Experiments

[0026] 1. Raw materials for the example

[0027] The raw material information used in the examples is shown in Table 1:

[0028] Table 1

[0029] ,

[0030] 2. Experimental subjects and pretreatment

[0031] The experimental subjects and related materials are shown in Table 2:

[0032] Table 2

[0033] ,

[0034] 2.1 AML-12 cell culture

[0035] 2.1.1 Cell resuscitation

[0036] Remove the cryovials of AML-12 cells from the liquid nitrogen tank and quickly thaw them in a 37 ℃ water bath. After thawing, transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 r / min for 5 min, discarding the supernatant. Add 5 mL of DMEM / F12 complete culture medium (containing 10% FBS and 1% penicillin-streptomycin) to the centrifuge tube, gently pipette the cells until a homogeneous suspension is formed, transfer the suspension to a T25 cell culture flask, and add complete culture medium to a final volume of 10 mL. Place the culture flask in a 37 ℃, 5% CO2 incubator for static culture.

[0037] 2.1.2 Cell passage

[0038] Observe cell growth status daily. When the AML-12 cell density reaches 80%-90%, perform passage. Discard the old culture medium in the culture flask, add 3 mL of sterile PBS buffer to rinse the cell surface twice to completely remove residual culture medium. Add 1 mL of 0.25% trypsin and incubate in an incubator for 2-3 min. When cells are observed to shrink and detach under an inverted microscope, immediately add 2 mL of complete culture medium to stop digestion. Gently pipette the adherent cells with a sterile, enzyme-free Pasteur pipette to completely detach the cells from the flask wall and disperse them into a single-cell suspension. Transfer the cell suspension to a new T25 culture flask at a ratio of 1:3 or 1:4, add complete culture medium to 10 mL, pipette evenly, and incubate in a 37 ℃, 5% CO2 incubator for further static culture.

[0039] 2.1.3 Cell cryopreservation

[0040] Digest the cells into complete culture medium according to the cell passage procedure in 2.1.2. Transfer the culture medium to a centrifuge tube and centrifuge at 1000 r / min for 5 min. Discard the supernatant after centrifugation. Add 1 mL of cell cryopreservation solution to the centrifuge tube, gently pipette to resuspend the cells, and quickly transfer them to a sterile cryopreservation tube. Place the cryopreservation tube in a gradient cryopreservation box and store it overnight at -80 ℃. The next day, transfer it to a liquid nitrogen tank for long-term storage.

[0041] 2.2 Zebrafish embryo pretreatment

[0042] Zebrafish embryos were cultured in embryo culture medium and relied on the yolk sac for nutrition before hatching, requiring no additional feeding. The rearing environment was controlled at a temperature of 28±0.5 ℃ and a light-dark cycle of 14 h:10 h to ensure normal growth and development of the zebrafish.

[0043] To minimize the impact of environmental stress on the experimental results, zebrafish embryos underwent a two-day acclimatization period before the formal experiment, during which the embryo culture medium was changed daily and the water quality was kept clean and stable. All procedures in this experiment complied with the relevant guidelines and requirements of the Animal Ethics Committee.

[0044] 3. Experimental Chemicals and Reagents

[0045] Detailed information on the laboratory chemicals and reagents is shown in Table 3:

[0046] Table 3

[0047] ,

[0048] 4 Main Equipment

[0049] The main equipment used is shown in Table 4.

[0050] Table 4

[0051] ,

[0052] 5. Statistical Analysis

[0053] Statistical analysis was performed using GraphPad Prism 9.5.1 and IBM SPSS Statistics 27.0. Data are expressed as mean ± standard error of mean. Statistical differences between groups were assessed using one-way ANOVA, followed by Tukey's post-hoc test (LSR) at 95% confidence intervals. A p-value < 0.05 was considered statistically significant.

[0054] Example 1: Preparation of the traditional Chinese medicine composition containing kudzu root of the present invention (19:6)

[0055] 1.1 Raw material formula: The weight ratio of Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita and Poria cocos is 8:4:4:3, and the weight ratio of the total weight of the above raw materials to that of Pueraria lobata is 19:6.

[0056] 1.2 Preparation steps: (1) Pretreatment: Remove impurities from the above-mentioned medicinal materials, wash them, dry them at 60℃ for 4 hours, pulverize them into coarse powder and pass them through an 80-mesh sieve; (2) Extraction: Weigh 50 g of coarse powder and place it in a stainless steel decoction pot. Add 500 ml of purified water (meeting the requirements of the Chinese Pharmacopoeia for pharmaceutical water) and soak for 30 min. Bring to a boil over high heat (900 W) and then simmer over low heat (500 W) for 60 min. Filter and collect the filtrate. Add 350 mL of purified water to the residue and simmer over low heat for 30 min. Combine the two decoctions. (3) Concentration: The decoction is concentrated at 60 °C using a rotary evaporator to obtain an extract with a relative density of 1.10.

[0057] Example 2: Preparation of the walnut kernel-containing traditional Chinese medicine composition of the present invention (19:6)

[0058] 2.1 Raw material formula: The weight ratio of Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita and Poria cocos (the four original ingredients of Liuwei Dihuang Pill) is 8:4:4:3, and the weight ratio of the total weight of the above raw materials to the weight of walnut kernels is 19:6.

[0059] 2.2 Preparation steps: Refer to the pretreatment, extraction and concentration steps in Example 1.

[0060] Example 3: Preparation of the Polygonatum-containing Chinese herbal composition of the present invention (19:6)

[0061] 3.1 Raw material formula: The weight ratio of Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita and Poria cocos is 8:4:4:3, and the total weight of the above raw materials is 19:6 compared with the weight of Polygonatum sibiricum.

[0062] 3.2 Preparation steps: Refer to the pretreatment, extraction and concentration steps in Example 1.

[0063] Example 4: Preparation of the traditional Chinese medicine composition containing kudzu root of the present invention (19:3)

[0064] 4.1 Raw material formula: The weight ratio of Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita and Poria cocos is 8:4:4:3, and the total weight of the above raw materials is 19:3 compared with the weight of Pueraria lobata.

[0065] 4.2 Preparation steps: Refer to the pretreatment, extraction and concentration steps of Example 1, except that during extraction, 8 times the amount of purified water is added for the first decoction, and 5 times the amount of purified water is added for the subsequent filter residue for decoction.

[0066] Example 5: Preparation of the kudzu-containing composition of the present invention (19:9)

[0067] 5.1 Raw material formula: The weight ratio of Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita and Poria cocos is 8:4:4:3, and the weight ratio of the total weight of the above raw materials to that of Pueraria lobata is 19:9.

[0068] 5.2 Preparation steps: Refer to the pretreatment, extraction and concentration steps of Example 1, except that during pretreatment, the powder is crushed into coarse powder and passed through a 100-mesh sieve.

[0069] Example 6: Preparation of the kudzu-containing composition of the present invention (19:12)

[0070] 6.1 Raw material formula: The weight ratio of Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita and Poria cocos is 8:4:4:3, and the weight ratio of the total weight of the above raw materials to that of Pueraria lobata is 19:12.

[0071] 6.2 Preparation steps: Refer to the pretreatment, extraction and concentration steps of Example 1, except that during concentration, the decoction is concentrated to an extract with a relative density of 1.20.

[0072] Comparative Example: Preparation of Liuwei Dihuang Pills

[0073] 1.1 Raw material formula: The weight ratio of Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita, Poria cocos, Paeonia suffruticosa, and Alisma plantago-aquatica is 8:4:4:3:3:3.

[0074] 1.2 Preparation steps: Refer to the pretreatment, extraction and concentration steps of Example 1.

[0075] Experimental Example 1: In vitro safety evaluation (cell viability assay)

[0076] 1.1 Experimental Methods:

[0077] AML-12 cells in the logarithmic growth phase with a density of 80%-90% were collected, and after adjusting the cell concentration, 1 × 10⁶ cells were used per well. 5Cells were seeded at a density of 100 μL into each well of a 96-well cell culture plate. The plates were incubated at 37°C with 5% CO2 for 24 h until the cells were fully adhered to the plate, at which point they were divided into groups for further processing.

[0078] The grouping is as follows: Experimental group (As): Discard the supernatant of each well and add 100 μL of complete culture medium containing 62.5 μg / mL, 125 μg / mL, 250 μg / mL and 500 μg / mL of traditional Chinese medicine composition extract, respectively. Set up 3 replicates for each concentration. Control group (Ac): Discard the supernatant and add 100 μL of complete culture medium without the Chinese medicine composition to each well; Blank group (Ab): Add 100 μL of complete culture medium containing no cells and Chinese medicine composition to each well.

[0079] After grouping, the 96-well plate was returned to the incubator for 24 h of further culture. Then, 100 μL of culture medium containing 10% CCK-8 reagent was added to each well, the plate was gently shaken to mix evenly, and incubated at 37℃ in a 5% CO8 incubator for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader, and the absorbance of the experimental group (As), the control group (Ac), and the blank group (Ab) were recorded. Cell viability was calculated according to the following formula (1):

[0080] (1)

[0081] In the formula: As - absorbance of experimental wells (including cells, culture medium, CCK8 solution and traditional Chinese medicine composition solution); Ac-control well absorbance (including cells, culture medium, and CCK8 solution); Ab - Absorbance of blank wells (including culture medium and CCK8 solution).

[0082] 1.2 Experimental Results:

[0083] Table 5 shows the effects of different herbal compositions and drug concentrations on cell viability. As can be seen from the results in Table 5, the cell viability of each herbal composition group of the present invention was significantly higher than that of the Liuwei Dihuang Pill group. Especially at a high concentration of 500 μg / mL, the cell viability of Example 1 remained above 93%, confirming that its in vitro safety was significantly better than that of the Liuwei Dihuang Pill group.

[0084] Table 5

[0085] ,

[0086] Experiment Example 2: In vivo safety evaluation (survival rate test of zebrafish juveniles)

[0087] 2.1 Experimental method: Hatched zebrafish fry were randomly divided into groups of 20 fry each after 5 days of adaptation training. The survival of the fry was observed and recorded every 12 hours.

[0088] The grouping is as follows: Normal control group (NC group): Normal juvenile fish culture medium, fed standard feed twice a day, 2 mg each time; Liuwei Dihuang Pill Group (DG Group): The comparative extract concentrations were 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL and 400 μg / mL, respectively. The different concentrations of extract were added to the culture medium of juvenile fish and fed with standard feed twice a day, 2 mg each time. The herbal composition group (EG group) of the present invention: The extract concentrations of the herbal composition of Example 1 were 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL and 400 μg / mL, respectively. The extracts of different concentrations were added to the culture medium of juvenile fish and fed with standard feed twice a day, 2 mg each time.

[0089] 2.2 Experimental Results and Analysis:

[0090] Experimental results are as follows Figure 1 As shown. By Figure 1 It was found that the survival rate of zebrafish larvae in the EG group was significantly higher than that in the DG group at all concentrations, and the survival rate advantage became more obvious with increasing drug concentration, proving that the in vivo safety of the herbal composition extract of this invention is superior to that of traditional Liuwei Dihuang pills. In the NC group, larvae died on day 9 of the experiment, while in the EG group, when the drug concentration was in the range of 12.5-100 μg / mL, all larvae survived to day 10 of the experiment. This result indicates that the herbal composition extract of this invention not only did not produce additional toxicity, but also reduced the normal mortality rate of larvae to a certain extent, indirectly demonstrating its protective effect and potential preventive value on the organism, which is consistent with the safety result of higher cell viability in the EG group in the in vitro cell activity experiment.

[0091] Experimental Example 3: In vitro pharmacodynamic evaluation (lipid accumulation and oxidative stress state experiment)

[0092] This experiment used AML-12 cells as the research subject, constructing a MASLD cell model by induction with 200 μmol / L palmitic acid (PA). A "drug and inducer co-treatment" mode was adopted, adding extracts of various traditional Chinese medicine compositions along with 200 μmol / L palmitic acid (PA) to the cell wells of the corresponding experimental groups, and culturing for 24 h. After culture, cell staining was observed using Oil Red O staining to determine the effect of the tested drugs on improving excessive lipid accumulation in cells. The regulatory effect of the tested drugs on cellular oxidative stress was evaluated using reactive oxygen species (ROS) level detection.

[0093] 3.1 Oil Red O Staining Experimental Method

[0094] AML-12 cells in the logarithmic growth phase with a density of 80%-90% were collected, and after adjusting the cell concentration, they were injected at a rate of 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 100 μL into 24-well cell culture plates and incubated at 37°C with 5% CO2 for 24 h. After the cells were fully adhered, the supernatant was discarded, and the cells were divided into groups. Each well was then replenished with DMEM / F12 medium to a total volume of 100 μL. Each group was configured with 3 replicates to ensure experimental reproducibility.

[0095] The grouping is as follows: Normal control group (NC group): containing only DMEM / F12 culture medium; Model group (MG group): DMEM / F12 medium containing a final concentration of 200 μmol / L PA; Liuwei Dihuang Pill Group (DG Group): DMEM / F12 culture medium containing a final concentration of 200 μmol / L PA and Liuwei Dihuang Pill extracts prepared in different concentration proportions; The herbal composition group (EG group) of this invention consists of DMEM / F12 culture medium containing a final concentration of 200 μmol / L PA and extracts of herbal compositions prepared in different concentration examples.

[0096] The specific steps for performing the Oil Red O staining experiment are as follows: (1) Discard the culture medium in each well, and gently rinse the cell surface twice with PBS buffer to remove residual culture medium; (2) Add 500 μL of 4% paraformaldehyde solution to each well and fix the cells for 30 min; (3) Discard the fixative, rinse the cells twice with deionized water to remove any residual fixative; (4) Add 500 μL of 60% isopropanol to each well and rinse the cells for 30 s; (5) Discard 60% isopropanol, add 500 μL of freshly prepared Oil Red O staining solution (saturated Oil Red O staining agent and deionized water are mixed at a volume ratio of 3:2) to each well, place the 24-well plate on a shaker and stain for 15 min; (6) After staining, discard the staining solution, rinse the cells with 60% isopropanol for 30 seconds to remove background staining, and then rinse slowly with deionized water 2-5 times until there is no excess staining solution remaining in the washing solution. (7) Add 500 μL of Mayer hematoxylin staining solution to each well for 1 min to counterstain, discard the staining solution, and rinse slowly with deionized water 2-5 times.

[0097] Qualitative analysis: Add 500 μL of deionized water to each well, observe cell staining under a microscope, take pictures and save them, and judge the degree of lipid accumulation by the area and intensity of the red staining area;

[0098] Quantitative analysis: The Mayer hematoxylin staining step can be omitted. 250 μL of isopropanol can be added directly to each well, and the staining is destained in a shaker for 15 min. After the staining is completely dissolved, the supernatant is taken, and the absorbance value of each well is measured at a wavelength of 490 nm using an ELISA reader to quantitatively reflect the lipid accumulation level. Taking the lipid accumulation degree of the MG group as 100%, the relative efficacy of the experimental group is calculated using the following formula (2):

[0099] (2)

[0100] 3.2 Qualitative analysis of the effect of the extract of the traditional Chinese medicine composition of the present invention on lipid accumulation

[0101] This experiment qualitatively investigated the effect of the herbal composition extract (EG group) of this invention on lipid over-accumulation in MASLD model cells, and compared it with the Liuwei Dihuang Pill group. The herbal composition extract obtained in Example 1 was used as the EG group for Oil Red O staining experiments. The experiment included a normal control group (NC group), a model group (MG group), a Liuwei Dihuang Pill group (DG group), and a herbal composition group (EG group). The Liuwei Dihuang Pill group was set at a concentration of 500 μg / mL, while the herbal composition group was set at three concentration gradients: 125 μg / mL, 250 μg / mL, and 500 μg / mL.

[0102] Experimental results are as follows Figure 2 As shown, from Figure 2 The staining results clearly show that the staining depth of the MG group cells was significantly deeper than that of the NC group after induction, confirming the successful construction of the MASLD lipid over-accumulation model in AML-12 cells.

[0103] The staining results of the drug administration groups of the traditional Chinese medicine composition extract of Example 1 at different concentrations showed that as the drug concentration increased, the staining intensity of the red staining area in the cells continuously decreased, and the degree of excessive lipid accumulation in the cells was significantly improved. This proves that the traditional Chinese medicine composition can effectively alleviate the excessive lipid accumulation in MASLD model cells, and the improvement effect is enhanced with increasing concentration, and is significantly better than that of Liuwei Dihuang Pill group.

[0104] 3.3 Quantitative analysis of the effects of extracts from traditional Chinese medicine compositions with different compositions on lipid accumulation

[0105] In this experiment, the effects of different compositions of the traditional Chinese medicine composition of this invention and Liuwei Dihuang Pill on the improvement of excessive lipid accumulation in cells were quantitatively compared in an MASLD model constructed from AML-12 cells. The experiment included a comparative Liuwei Dihuang Pill administration group and extracts of the traditional Chinese medicine composition obtained in Examples 1, 2, and 3 as EG administration groups for Oil Red O staining experiments. Three concentration gradients of 125 μg / mL, 250 μg / mL, and 500 μg / mL were set for each administration group, and the degree of lipid accumulation (%) was calculated according to formula (2) in 3.1. The experimental results are shown in Table 6.

[0106] Table 6

[0107] ,

[0108] As shown in Table 6, the lipid accumulation levels of the herbal extracts with different compositions of the present invention at all concentration levels were significantly lower than those of the Liuwei Dihuang Pills group in the comparative example. The results indicate that the herbal extracts of the present invention are more effective than the Liuwei Dihuang Pills group in treating excessive cellular lipid accumulation, and exhibit better efficacy in treating MASLD. In particular, the effect of Example 1 group was especially outstanding, proving that the herbal composition containing kudzu root of the present invention has a better effect in treating excessive cellular lipid accumulation.

[0109] 3.4 Quantitative analysis of the effects of extracts from traditional Chinese medicine compositions with different contents on lipid accumulation

[0110] In this experiment, the effects of different concentrations of the herbal composition of this invention and Liuwei Dihuang Pill on the improvement of excessive lipid accumulation in cells were quantitatively compared in an MASLD model constructed from AML-12 cells. The experiment included a comparative group treated with Liuwei Dihuang Pill, and the extracts of the herbal compositions obtained in Examples 1, 4, 5, and 6 were used as EG groups for Oil Red O staining. Three concentration gradients of 125 μg / mL, 250 μg / mL, and 500 μg / mL were set for each group, and the degree of lipid accumulation (%) was calculated according to formula (2) in 3.1. The experimental results are shown in Table 7.

[0111] Table 7

[0112] ,

[0113] As shown in Table 7, the kudzu root in the herbal composition of this invention, at a weight ratio of 3, already exhibited superior effects compared to Liuwei Dihuang Pills at 125 μg / mL. Furthermore, at weight ratios of 6-12, it showed superior effects compared to Liuwei Dihuang Pills at all experimental concentrations. These results demonstrate that the extracts from the herbal composition of this invention at different weight ratios have good therapeutic effects on MASLD.

[0114] 3.5 Measurement of ROS levels in cells

[0115] AML-12 cells were seeded in 15 mm glass-bottomed culture dishes and cultured in a 37°C, 5% CO2 incubator. After the cells were fully attached to the culture dish, they were divided into groups.

[0116] The grouping is as follows: Normal control group (NC group): containing only DMEM / F12 culture medium; Model group (MG group): DMEM / F12 medium containing a final concentration of 200 μmol / L PA; Positive drug group (PC group): DMEM / F12 medium containing a final concentration of 200 μmol / L PA and metformin containing 5 mmol / L; Liuwei Dihuang Pill Group (DG Group): DMEM / F12 medium containing a final concentration of 200 μmol / L PA and Liuwei Dihuang Pill extract prepared in the comparative ratio at a concentration of 250.00 μg / mL; The herbal composition group of this invention (EG group): DMEM / F12 culture medium containing a final concentration of 200 μmol / L PA and the herbal composition extract prepared in Example 1 at a concentration of 250.00 μg / mL.

[0117] After co-treatment with the drug and inducer according to the above grouping scheme, the culture medium was removed, and the cells were carefully washed twice with PBS buffer. 4% paraformaldehyde was added to the culture dishes, and the cells were fixed at room temperature for 20 min. The cells were then washed twice with PBS to completely remove any residual fixative. 0.5 mmol / L DCFH-DA staining solution was added to each culture dish, and the cells were stained in the dark at room temperature for 60 min. The staining solution was discarded, and the cells were carefully washed twice with PBS to remove any remaining staining solution. Finally, the glass-bottomed culture dishes were placed under an inverted fluorescence microscope to observe and photograph the cell fluorescence expression.

[0118] 3.6 Analysis of the effect of the extract of the traditional Chinese medicine composition of the present invention on the regulation of oxidative stress state

[0119] This experiment measured intracellular ROS levels and investigated the regulatory effects of various test drugs on cellular oxidative stress. The experimental results are as follows: Figure 3 As shown in the figure, we can see that the fluorescence intensity of the MG group cells is significantly higher than that of the NC group. This indicates that after PA induction, a large amount of ROS is generated in AML-12 cells, and the oxidative stress model is successfully constructed, which is consistent with the pathological characteristics of MASLD cells.

[0120] As can be seen from the figure, compared with the MG group, the fluorescence intensity of cells in the PC group, DG group, and EG group was significantly reduced, indicating that these three types of drugs can effectively inhibit the generation of intracellular ROS and exert certain antioxidant effects. Among them, although the DG group can reduce the cell ROS level, the fluorescence intensity is still higher than that of the EG group, while the EG group has the weakest cell fluorescence signal, and its ROS level is close to that of the NC group, showing a particularly outstanding antioxidant effect.

[0121] The above results demonstrate that the extract of the traditional Chinese medicine composition in Example 1 can effectively remove excessive ROS in MASLD model cells, significantly alleviate PA-induced cellular oxidative stress, thereby reducing the inflammatory response caused by oxidative stress and improving the pathological damage of MASLD. Compared with the Liuwei Dihuang Pill group, the antioxidant effect of the traditional Chinese medicine composition of this invention is more significant, which also reflects its good potential therapeutic value in alleviating MASLD-related oxidative damage.

[0122] Experiment Example 4: In vivo pharmacodynamic evaluation (zebrafish model experiment)

[0123] This experiment used wild-type AB strain zebrafish embryos (5 dpf) as the research subject to construct a MASLD model by feeding them a high-fat diet. The experimental zebrafish were housed in embryo culture medium, fed twice daily, with the culture medium water kept clear, for 4 days. Then, the staining of liver cells was observed using zebrafish oil red O staining to assess the effect of the tested drug on improving hepatic lipid deposition. Next, serum TC, TG, ALT, AST, LDL-C, and HDL-C levels were measured using kits to further evaluate liver function and lipid metabolism. Finally, the pathological morphology of the zebrafish liver was observed using HE staining.

[0124] The zebrafish were randomly divided into groups of 20 each, and the experiment was repeated three times.

[0125] The grouping is as follows: Normal control group (NC group): Zebrafish were housed in normal embryo culture medium and fed standard diet twice a day, 2 mg each time; Model group (MG group): Zebrafish were raised in normal embryo culture medium and fed a high-fat diet twice a day, 10mg each time. The medium was changed after 30-60 minutes of feeding to ensure that the zebrafish culture medium water was clear. Positive drug group (PC group): Zebrafish were housed in embryo culture medium containing 10 μmol / L bezafibrate and fed a high-fat diet twice a day, 10 mg each time. The medium was changed after 30-60 minutes of feeding to ensure that the zebrafish culture medium was clear. Liuwei Dihuang Pill Group (DG Group): Zebrafish were raised in embryo culture medium containing 25 μg / mL of Liuwei Dihuang Pill extract. They were fed a high-fat diet twice a day, 10 mg each time. The medium was changed after 30-60 minutes of feeding to ensure that the zebrafish culture medium was clear. Low-dose group of Chinese herbal composition extract (EG-L group): Zebrafish were raised in embryo culture medium containing 25 μg / mL of Chinese herbal composition extract from Example 1. They were fed a high-fat diet twice a day, 10 mg each time. The medium was changed after 30-60 minutes of feeding to ensure that the zebrafish culture medium was clear. High-dose group of traditional Chinese medicine composition extract (EG-H group): Zebrafish were raised in embryo culture medium containing 100 μg / mL of traditional Chinese medicine composition extract from Example 1. They were fed a high-fat diet twice a day, 10 mg each time. The medium was changed after 30-60 minutes of feeding to ensure that the zebrafish culture medium water was clear.

[0126] 4.1 Zebrafish Oil Red O Staining Experiment Method

[0127] After grouping and feeding according to the above grouping scheme for 4 days, zebrafish oil red O staining experiments were performed. This experiment was modified based on the cell oil red O staining procedure and tailored to the characteristics of zebrafish. The specific operation steps are as follows: (1) Take zebrafish from each group and place them in a 24-well plate. Add an appropriate amount of zebrafish anesthetic to each well. After the juvenile fish are completely anesthetized, proceed with the subsequent operations. (2) Discard the anesthetic and gently wash the zebrafish twice with distilled water to remove impurities from its body surface; (3) Add 4% paraformaldehyde solution to each well and fix the 24-well plate at 4°C overnight; (4) Discard the fixative and wash the zebrafish twice with PBS buffer to remove residual fixative; (5) Add 60% isopropanol and soak the zebrafish for 30 min; (6) Discard 60% isopropanol, add Oil Red O staining solution, and stain at room temperature for 4 h; (7) Discard the staining solution, wash the zebrafish with 60% isopropanol for 3 min, and then wash once with PBS buffer. (8) Fix the stained zebrafish in a 24-well plate, place it under an inverted microscope to observe the liver lipid staining, and take pictures to record the results.

[0128] 4.2 Detection of liver function and lipid metabolism indicators in zebrafish

[0129] Zebrafish from each group with a 9dpf surviving period were anesthetized and euthanized at low temperature. The whole fish were mechanically crushed and centrifuged, and the supernatant was collected, mixed evenly, and used for index detection.

[0130] (1) TG content determination: Take a portion of the supernatant and use the kit to determine the TG content using the GPO-PAP method. Take another portion of the supernatant and use the BCA method to determine the protein concentration to correct the TG content. The final TG content is calculated using the following formula (3):

[0131] (3)

[0132] Where: OD sample - absorbance value of experimental group; OD blank - absorbance value of pure water; OD calibration - absorbance value after adding standard.

[0133] (2) TC content determination: The determination method is the same as that for TG content.

[0134] (3) Determination of ALT and AST content: Take the supernatant and determine the ALT and AST content in the supernatant according to the instructions of the ALT / GPT kit and the AST / GOT kit.

[0135] (4) Determination of LDL-C and HDL-C content: Take the supernatant and determine the LDL-C and HDL-C content in the supernatant according to the methods in the kit instructions for low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C).

[0136] 4.3 HE staining experiment in zebrafish

[0137] Zebrafish from the NC, MG, DG, and EG-L groups with a 9dpf liver were anesthetized and euthanized at low temperature. After fixation of the whole fish, they were routinely embedded in paraffin, sectioned (5μm thick), stained with hematoxylin and eosin (HE), and their liver pathological changes were observed under a light microscope.

[0138] 4.4 Analysis of the effect of the extract of the traditional Chinese medicine composition of the present invention on excessive lipid accumulation in the MASLD zebrafish model

[0139] This experiment investigated the effect of the extract of the traditional Chinese medicine composition of this invention on lipid over-accumulation in a MASLD zebrafish model using zebrafish oil red O staining, and compared it with Liuwei Dihuang Wan (a traditional Chinese medicine formula). The experimental results are shown below. Figure 4 .

[0140] fromFigure 4 The results of zebrafish oil red O staining clearly showed that after 4 days of high-fat diet feeding, the zebrafish in the MG group showed significant abdominal obesity, and the red staining range in the liver area was greatly expanded and the staining depth was significantly deepened, which was significantly different from the NC group. This confirmed that the high-fat diet feeding successfully constructed the MASLD zebrafish lipid over-accumulation model.

[0141] After bezafibrate intervention, the abdominal obesity symptoms of zebrafish in the PC group were alleviated, and the red staining intensity in the liver area was significantly reduced, indicating that the positive control drug can effectively reduce lipid accumulation in the model zebrafish, verifying the effectiveness of the experimental system. Compared with the MG group, the red staining of zebrafish in the EG-L and EG-H groups was significantly weakened, and the degree of abdominal obesity was also improved, indicating that the herbal extract of Example 1 can effectively improve the problem of lipid accumulation in zebrafish induced by high-fat diet. At the same time, the red staining intensity of the EG-H group was significantly lower than that of the EG-L group, and the improvement effect on liver lipid deposition was more significant and better than that of the DG group.

[0142] In summary, the herbal composition extract of Example 1 can effectively reduce lipid accumulation in the MASLD zebrafish model, and the improvement effect is better with high-dose intervention, further confirming that the composition also has a good regulatory effect on MASLD lipid metabolism disorder in vivo.

[0143] 4.5 Analysis of the effects of the extract of the traditional Chinese medicine composition of the present invention on liver function and lipid metabolism in the MASLD zebrafish model

[0144] Zebrafish from the model group, control group, and experimental group were used to detect liver function and lipid metabolism-related indicators, and the data were visualized and analyzed to further investigate the regulatory effect of the herbal composition extract from Example 1 on lipid metabolism disorder and protective effect against liver injury in the MASLD zebrafish model. The experimental results are shown in […]. Figure 5 .

[0145] Depend on Figure 5The data show that, compared with the NC group, the TG level in the MG group of zebrafish exhibited a sharp upward trend, which is the most direct evidence of hepatocyte steatosis, indicating that triglyceride synthesis and accumulation in the zebrafish liver have reached pathological levels. Simultaneously, the significant increases in TC and LDL-C, and the decrease in HDL-C, indicate systemic lipid metabolism disorder. LDL-C, as "bad cholesterol," is responsible for transporting cholesterol from outside the liver to tissues; its increase exacerbates the cholesterol load on the liver. Conversely, the decrease in HDL-C weakens its ability to reverse the transport of peripheral cholesterol back to the liver for metabolism, further worsening the lipid homeostasis imbalance. At the level of hepatocyte damage, the significant increases in AST and ALT activities indicate obvious inflammation and necrosis of hepatocytes. In the zebrafish model, this damage is mainly caused by "lipotoxicity" induced by lipid accumulation in the liver. Since ALT is mainly found in the hepatocyte cytoplasm, its increase usually specifically indicates hepatocyte membrane damage; while AST is present in both the cytoplasm and mitochondria, and its significant increase often suggests more severe hepatocyte damage, possibly involving mitochondrial dysfunction. The simultaneous increase in both strongly demonstrates that the model zebrafish has progressed from simple hepatic steatosis to the stage of metabolic-associated steatohepatitis (MASH) with liver damage, indicating that the MASLD model was successfully constructed and has typical pathological features.

[0146] Based on the intervention effect of the herbal composition extract in Example 1, it showed significant regulatory and protective effects on lipid metabolism and liver function in the MASLD zebrafish model.

[0147] Regarding lipid metabolism regulation, after intervention with the herbal extract from Example 1, the total cholesterol (TC), triglycerides (TG), and atherogenic low-density lipoprotein cholesterol (LDL-C) in model zebrafish all showed a significant decreasing trend. This not only directly confirms its powerful lipid-lowering effect but also corroborates the results of reducing lipid accumulation in cell experiments, forming a complete chain of evidence from cells to living organisms. Most importantly, its positive effect on high-density lipoprotein cholesterol (HDL-C) suggests that this extract may promote cholesterol reverse transport by increasing "good cholesterol" levels, thereby reshaping a healthy lipid metabolism profile through multiple targets.

[0148] Regarding liver damage protection, the significant reduction in alanine aminotransferase (AST) and aspartate aminotransferase (ALT) levels provides direct evidence of the restoration of hepatocyte membrane integrity and the containment of liver damage. This strongly suggests that the hepatoprotective effect of this traditional Chinese medicine composition extract is not only established in in vitro cells but also effective in complex in vivo environments.

[0149] In summary, the zebrafish model experiments systematically demonstrated that the extract of the traditional Chinese medicine composition of this invention exhibits significant and broad therapeutic potential in a high-fat diet-induced MASLD zebrafish model. Furthermore, these experiments proved that the traditional Chinese medicine composition of this invention can effectively combat the disease progression of MASLD through a synergistic mechanism of "lipid regulation and liver protection," providing solid preclinical experimental evidence for its subsequent development and application.

[0150] 4.6 Analysis of the effects of the extract of the traditional Chinese medicine composition of the present invention on liver degeneration in the MASLD zebrafish model

[0151] Nine dpf MASLD zebrafish from each model group, control group, and experimental group were used to prepare whole fish tissue sections. The liver tissue morphology was observed by HE staining of zebrafish. The effect of the extract of the Chinese herbal composition in Example 1 (EG-L group) on the liver characteristics of the MASLD zebrafish model was further investigated. The experimental results are shown in Figure 6.

[0152] The histological results showed that, compared with the liver tissue of the NC group, the hepatocytes of the zebrafish in the MG group were loosely arranged, with increased nuclei, sparse chromatin distribution, and significantly enlarged nucleoli, indicating abnormal hepatocyte proliferation. The cytoplasm showed eosinophilic changes, and the intercytoplasmic spaces were enlarged, reflecting enhanced cellular metabolic activity or abnormal accumulation of intracellular substances. Simultaneously, some hepatocytes exhibited irregular morphology and heterogeneous size, further confirming that excessive lipid accumulation in the liver led to metabolic disorders. These pathological changes are consistent with the histological characteristics of MASLD liver disease, indicating that the high-lipid-induced zebrafish liver degeneration model was successfully established.

[0153] After intervention with Liuwei Dihuang Pill extract, the above-mentioned pathological abnormalities in the DG group were not significantly improved, indicating that Liuwei Dihuang Pill has a limited effect on improving liver degeneration in MASLD zebrafish.

[0154] In the EG group, intervention with the extract of the traditional Chinese medicine composition from Example 1 significantly improved the aforementioned pathological characteristics. The liver tissue structure was normal, with clear lobular morphology, tightly packed hepatocytes, and no obvious cellular atypia or hyperplasia. The hepatocyte nuclei were regular, with uniform chromatin, and no pathological changes such as nuclear atypia or nuclear membrane enlargement were observed. The cytoplasm showed moderate eosinophilia, without excessive basophilia or eosinophilia. The liver sinusoidal structure was intact, with no obvious vasodilation or hemorrhage. The liver tissue morphology was close to the normal level of the NC group, and the overall liver tissue and cellular state were basically restored to normal.

[0155] In summary, the herbal composition extract of this invention can effectively improve the pathological degeneration of MASLD-induced liver disease in zebrafish and repair the normal tissue morphology and cell structure of the liver. In contrast, Liuwei Dihuang Wan (a traditional Chinese medicine formula) showed no significant effect in improving liver degeneration. This further confirms that the herbal composition of this invention is significantly superior to Liuwei Dihuang Wan in improving MASLD liver tissue damage and reversing liver degeneration.

[0156] Application Example 1: Preparation of Food Additives

[0157] Take 100 g of the powdered product obtained by drying the extract obtained in Example 1, add 400 g of food-grade maltodextrin, mix evenly to obtain 500 g of food additive powder, and add 0.5%-5% of the total weight of the food.

[0158] Application Example 2: Preparation of Functional Foods

[0159] Take 25 g of the food additive powder obtained in Application Example 1, add it to 500 g of pastry flour, mix thoroughly and evenly, and process according to conventional pastry making process to obtain functional pastry.

Claims

1. A traditional Chinese medicine composition for the prevention and treatment of fatty liver disease related to metabolic dysfunction, characterized in that, It is made from the following medicinal and edible plant materials: Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita, Poria cocos, and one of the following: Pueraria lobata, Polygonatum sibiricum, and walnut kernel.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The weight ratio of Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita, and Poria cocos is 8:4:4:3, and the weight ratio of their total weight to that of Pueraria lobata, Polygonatum sibiricum, or walnut kernels is 19:3-12.

3. The traditional Chinese medicine composition according to claim 2, characterized in that, The total weight ratio of Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita, and Poria cocos to the weight ratio of Pueraria lobata, Polygonatum sibiricum, or walnut kernels is 19:6-12.

4. The traditional Chinese medicine composition according to claim 3, characterized in that, The total weight ratio of Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita, and Poria cocos to Pueraria lobata is 19:

6.

5. A method for preparing the traditional Chinese medicine composition according to claim 2, characterized in that, Includes the following steps: S1 Raw material pretreatment: Take the above 5 medicinal materials, remove impurities, wash them and dry them at 60℃ for 4 hours, pulverize them and pass them through an 80-100 mesh sieve to obtain raw material coarse powder; S2 Extraction: Add 8-10 times the amount of purified water to the raw material powder and soak for 30 minutes. After boiling over high heat, simmer over low heat for 60 minutes. Filter and collect the filtrate. Add 5-7 times the amount of purified water to the residue and simmer over low heat for 30 minutes. Combine the two decoctions. S3 Concentration: The combined decoctions are concentrated to an extract with a relative density of 1.1-1.

2.

6. The use of any one of the traditional Chinese medicine compositions described in claims 1-4 in the preparation of a medicament for treating metabolic dysfunction-related fatty liver disease.

7. The use of any one of the traditional Chinese medicine compositions described in claims 1-4 in the preparation of functional foods or food additives for the prevention of metabolic dysfunction-related fatty liver disease.

8. The use of any one of the traditional Chinese medicine compositions described in claims 1-4 in the preparation of a drug or functional food for improving lipid metabolism and liver function.

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