A traditional Chinese medicine composition for treating hyperlipidemia, a preparation method and use thereof

Abstract

The present application relates to a kind of traditional Chinese medicine composition for treating hyperlipidemia and its preparation method and purposes, and the traditional Chinese medicine composition is the extract of the mixture of three raw materials of ginseng, hawthorn and sophora flower, in the raw material component, the ratio of ginseng and hawthorn is 2:1-1:2, and the ratio of hawthorn and sophora flower is 2:1-2:2.The present application solves the problems of large toxic side effects of existing lipid-lowering drugs, complex prescription of Chinese patent medicine and strong nature and taste, which is not easy to take long-term, etc.The traditional Chinese medicine composition of the present application has simple components and ideal lipid-lowering effect.

Description

Technical Field

[0001] This invention relates to traditional Chinese medicine compositions, specifically to a traditional Chinese medicine composition for treating hyperlipidemia, its preparation method, and its uses. Background Technology

[0002] Hyperlipidemia, medically termed dyslipidemia, is characterized by elevated serum total cholesterol (TC), decreased serum triglycerides (TG), and decreased levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). Increased levels of non-HDL-C (i.e., serum LDL-C) and residual cholesterol carried by triglyceride-rich lipoproteins are the main type of dyslipidemia worldwide and a major risk factor for atherosclerotic cardiovascular disease (ASCVD). Current clinical treatment for hyperlipidemia primarily focuses on lifestyle modifications and the use of lipid-lowering medications to control blood lipid levels.

[0003] Statins are the clinically recognized first-line drugs for lowering lipids. They work by inhibiting cholesterol synthesis and accelerating the clearance of low-density lipoprotein (LDL). However, according to national adverse drug reaction monitoring data, statins can cause abnormal blood sugar levels within 1-3 months of starting treatment. In addition, patients may experience adverse reactions such as muscle discomfort, neurocognitive impairment, and elevated alanine aminotransferase (ALT). Fibrates are also commonly used to treat hyperlipidemia. They activate lipoprotein hydrolases, increasing the breakdown and metabolism of very low-density lipoprotein (VLDL), thereby lowering triglyceride (TG) levels. Therefore, they are mainly used to treat hypertriglyceridemia or mixed hyperlipidemia with elevated triglyceride levels as the primary symptom. However, their cholesterol-lowering effect is not as strong as statins. Furthermore, during the first 3 months of taking fibrates, adverse reactions such as myalgia, muscle weakness, and liver damage are more likely to occur.

[0004] Currently, there is an increasing number of traditional Chinese medicines (TCMs) for lowering blood lipids. The formulation strategy of these TCMs or modern formulations is to use blood-activating and stasis-removing drugs as the main component, combined with drugs that tonify the liver and kidneys, and reduce turbidity and dampness, depending on the specific indication. For example, Chinese patent CN116211974A discloses a TCM composition for improving human microcirculation. Its characteristic is that the formulation is comprehensive, including cooling blood and activating blood circulation, removing dampness and reducing turbidity, and tonifying qi and nourishing yin. It can improve human microcirculation and also lower blood lipids and blood sugar. According to patent data, when used alone, this composition can significantly reduce TG and LDL-C levels, but its effect on improving TC levels is insufficient. When used in combination with statins and other Western medicines, it can have a synergistic effect, showing better lipid-lowering and blood sugar-lowering efficacy. It can be seen that the lipid-lowering effect of this invention is relatively mild, and the lipid-lowering effect is better when used in combination with Western medicines.

[0005] Chinese patent CN110624077A discloses a traditional Chinese medicine combination liquid for external application, and Chinese patent CN1879792A discloses a traditional Chinese medicine preparation that can treat hypertension and cerebrovascular diseases. The compositions disclosed in the above patents are all formulas with a large number of blood-activating and stasis-removing drugs. However, they contain a lot of blood-activating and stasis-removing drugs such as Polygonum multiflorum, safflower, and Sparganium stoloniferum. Long-term use can cause subcutaneous bleeding symptoms such as rashes. They also contain a lot of traditional Chinese medicines with strong odors, such as Ligusticum chuanxiong, Dalbergia odorifera, and Aucklandia lappa, which can cause gastrointestinal reactions.

[0006] Based on the lipid-lowering compositions in chemical drugs, traditional Chinese medicines and published patents, the main problems with current lipid-lowering drugs include: (1) Chemical drugs have significant toxic side effects. Short-term use may cause muscle pain and muscle weakness, and long-term use may cause liver and kidney damage. Clinical monitoring of liver function is required; (2) Traditional Chinese medicines or compositions contain a lot of traditional Chinese medicines with strong medicinal properties such as blood-activating and qi-regulating drugs. They need to be used according to the symptoms, and long-term use may also cause adverse reactions. Summary of the Invention

[0007] The purpose of this invention is to solve the problems of toxic side effects of lipid-lowering drugs and the fact that traditional Chinese medicine formulas are complicated and have strong properties that make them difficult to take for a long time. This invention provides a traditional Chinese medicine composition for treating hyperlipidemia with simple components and ideal effects, as well as its preparation method and uses.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A traditional Chinese medicine composition for treating hyperlipidemia, comprising an extract of a mixture of ginseng, hawthorn, and sophora japonica. The ginseng-hawthorn ratio, by weight, is 2:1 to 1:2, with examples including 1:1, 1:1.5, 1:2, 1.5:1, and 2:1. The hawthorn-sophora japonica ratio is 2:1 to 2:2, with examples including 2:1, 2:1.3, and 2:2.

[0010] As a preferred embodiment of the present invention, the ratio of ginseng, hawthorn, and sophora japonica flowers by weight is 1-4:2:1-2. Preferably, it is 1:2:1-2, and other possible ratios include 1:2:2, 1:2:1.3, 1:2:1, etc., with 1:2:2 being the most preferred.

[0011] As a preferred technical solution of the present invention, the ginseng is a processed product of ginseng and red ginseng, including ginseng slices and red ginseng slices. According to the Chinese Pharmacopoeia (2020 edition), medicines based on ginseng include ginseng and red ginseng. The content requirements for the processed products, including dried ginseng slices, are as follows: the total amount of ginsenoside Rg1 and ginsenoside Re shall not be less than 0.27%, and ginsenoside Rb1 shall not be less than 0.18%; the content requirements for dried red ginseng slices are as follows: the total amount of ginsenoside Rg1 and ginsenoside Re shall not be less than 0.22%, and ginsenoside Rb1 shall not be less than 0.18%.

[0012] The hawthorn is a processed product of hawthorn, which includes clean hawthorn, stir-fried hawthorn and charred hawthorn. The sophora flower is a processed product of sophora flower, which includes clean sophora flower and stir-fried sophora flower. The content determination requirements for both processed products are that the dried product contains not less than 6.0% rutin.

[0013] As a preferred technical solution of the present invention, the traditional Chinese medicine composition is a solvent (e.g., water or ethanol) extract of ginseng, hawthorn and sophora japonica. Ginseng, hawthorn and sophora japonica are extracted with solvent, concentrated and dried to obtain dry extract, which is then pulverized to obtain the traditional Chinese medicine composition, or suitable excipients are added to make granules, pills, capsules, etc.

[0014] As a preferred technical solution of the present invention, the effective components contained in the traditional Chinese medicine composition include: ginsenosides Re, Rg1, Rb1, and rutin, and the effective component content per unit mass of dry extract is: Rg1+Re≥1.24mg / g; Rb1≥2.13mg / g; rutin≥37.13mg / g.

[0015] A method for preparing a traditional Chinese medicine composition for treating hyperlipidemia is as follows: ginseng, hawthorn and sophora japonica are weighed in proportion, an extraction solvent is added for extraction, the extract is filtered to obtain a medicinal extract, the medicinal extract is concentrated and dried to obtain a dry extract, and then pulverized to obtain the traditional Chinese medicine composition.

[0016] As a preferred technical solution of the present invention, the extraction solvent is water or ethanol, and the amount of the extraction solvent used is 5-10 times the total weight of ginseng, hawthorn and sophora japonica flowers;

[0017] During the extraction process, the extraction solvent is kept at a gentle boil under reflux and extracted 1-5 times, with each extraction lasting 1-3 hours. The solvent is then concentrated under reduced pressure at 50-90℃.

[0018] As a preferred embodiment of the present invention, the extraction solvent is ethanol with a concentration of 30% to 80%. More preferably, it is ethanol with a concentration of 50% to 60%, and most preferably, it is ethanol with a concentration of 50%.

[0019] The use of a traditional Chinese medicine composition for treating hyperlipidemia, wherein the traditional Chinese medicine composition is used in the preparation of a drug for treating hyperlipidemia.

[0020] The minimum dosage of the traditional Chinese medicine composition for mice is 1.0 g / kg; the minimum clinical dosage for a standard adult (60 kg) is 0.08 g / kg.

[0021] Compared to current lipid-lowering drugs, such as statins, which are prone to causing liver damage, and cassia seed lipid-lowering tablets, which have complex formulations, strong properties, and are prone to causing gastrointestinal irritation, and whose long-term use may also burden liver and kidney function, this invention, while ensuring safety and effectiveness, refers to the "List of Items That Are Both Food and Medicine" and its newly added catalog published by the Ministry of Health. It selects three medicinal and edible herbs, namely ginseng, hawthorn, and sophora flower, which participate in different lipid metabolism processes in the body. Among them, the components contained in ginseng can act on PPAR to regulate lipid synthesis and metabolism, the components contained in hawthorn can act on 3-methylglutaryl coenzyme (HMGCR) to regulate cholesterol synthesis, and the components contained in sophora flower can act on LXRα to regulate cholesterol metabolism. The three herbs play a role in different lipid metabolism processes, thus achieving a synergistic lipid-lowering effect.

[0022] This invention selects the L02 cell steatosis model, which has higher reference value. The L02 cell steatosis model has a shorter modeling cycle, a higher induction success rate, and is more stable. It can also better reflect the actual metabolic process in the human body. The L02 cell steatosis model induced by oleic acid-palmitic acid was used to optimize the preparation method and screen the formulation of traditional Chinese medicine composition. Then, the in vivo efficacy and dosage comparison of the traditional Chinese medicine composition were verified by feeding C57BL / 6J mice with a long-term high-fat diet to induce a dietary hyperlipidemia model. The results show that the traditional Chinese medicine composition provided by this invention has excellent lipid-lowering effect and low toxicity and side effects. Attached Figure Description

[0023] Figure 1 Screening for cytotoxicity of different traditional Chinese medicine compositions;

[0024] Figure 2 The effect of different traditional Chinese medicine combinations on the TG level in L02 cells with fatty degeneration;

[0025] Figure 3 Oil Red O staining of L02 cells after administration of different traditional Chinese medicine combinations to induce cell models;

[0026] Figure 4 The cytotoxicity of different ginseng-hawthorn combinations;

[0027] Figure 5 Effects of different ginseng-hawthorn combinations on TG levels in L02 cells;

[0028] Figure 6 Oil Red O staining of L02 cells after different proportions of human ginseng and hawthorn were administered to induce cell modeling;

[0029] Figure 7 The cytotoxicity of different ginseng-hawthorn-sophora flower compositions;

[0030] Figure 8 The effects of different proportions of ginseng, hawthorn and sophora japonica on the TG level in L02 cells used for modeling;

[0031] Figure 9 Oil Red O staining of L02 cells after administration of different ginseng-hawthorn-sophora flower components to induce cell modeling;

[0032] Figure 10 For the determination of lipids in different components by Oil Red O staining;

[0033] Figure 11 The cytotoxicity of the following combinations: ginseng slices-hawthorn-sophora japonica flower composition, red ginseng slices-hawthorn-sophora japonica flower composition, ginseng slices-fried hawthorn-sophora japonica flower composition, ginseng slices-charred hawthorn-sophora japonica flower composition, or ginseng slices-hawthorn-fried sophora japonica flower composition.

[0034] Figure 12 The effects of ginseng slices-hawthorn-sophora japonica flower combination, or red ginseng slices-hawthorn-sophora japonica flower combination, or ginseng slices-fried hawthorn-sophora japonica flower combination, or ginseng slices-charred hawthorn-sophora japonica flower combination, or ginseng slices-hawthorn-fried sophora japonica flower combination on the TG level in L02 cells;

[0035] Figure 13 Oil Red O staining was performed on L02 cells after administration of 200 μg crude drug / mL of the following: ginseng slices-cleaned hawthorn-cleaned sophora japonica flower combination, or red ginseng slices-cleaned hawthorn-cleaned sophora japonica flower combination, or ginseng slices-fried hawthorn-cleaned sophora japonica flower combination, or ginseng slices-cleaned hawthorn-fried sophora japonica flower combination.

[0036] Figure 14 The cytotoxicity of water extract, alcohol extract, and extract extracted first by alcohol and then by water;

[0037] Figure 15 The effects of water extract, alcohol extract, and alcohol-to-water extract on the TG level in L02 cells;

[0038] Figure 16 Oil Red O staining of L02 cells after administration of 200 μg crude drug / mL of the aqueous extract, alcohol extract, and the extract obtained by first alcohol extraction and then aqueous extraction of traditional Chinese medicine composition to cells with fatty degeneration;

[0039] Figure 17 The trend of extraction efficiency of active ingredients at different ethanol concentrations;

[0040] Figure 18 A comprehensive score comparison of the effective components of extracts obtained from different extraction solvents;

[0041] Figure 19 For comparison of blood lipid levels in each group;

[0042] Figure 20 Comparison of liver morphology stained with HE;

[0043] Figure 21 Comparison of liver morphology stained with Oil Red O. Detailed Implementation

[0044] [Medicinal materials, reagents, and instruments]

[0045] The raw materials used in this invention are specifically sourced from:

[0046] Ginseng (Sichuan Xinhehua Traditional Chinese Medicine Pieces Co., Ltd., batch number 2207131, place of origin: Jilin);

[0047] Hawthorn (Sichuan Xinhehua Traditional Chinese Medicine Pieces Co., Ltd., batch number 2211006, place of origin: Hebei);

[0048] Sophora japonica flowers (Sichuan Xinhehua Traditional Chinese Medicine Pieces Co., Ltd., batch number 2206033, place of origin: Hebei);

[0049] Red ginseng (Sichuan Xinhehua Traditional Chinese Medicine Pieces Co., Ltd., batch number 2310103, place of origin: Jilin);

[0050] Fried hawthorn (Sichuan Xinhehua Traditional Chinese Medicine Pieces Co., Ltd., batch number 2309004, place of origin: Hebei);

[0051] Roasted hawthorn (Sichuan Xinhehua Traditional Chinese Medicine Pieces Co., Ltd., batch number 2309049, place of origin: Hebei);

[0052] Stir-fried Sophora japonica flowers (Sichuan Xinhehua Traditional Chinese Medicine Pieces Co., Ltd., batch number 2203103, place of origin: Hebei);

[0053] 95% ethanol (Sinopharm Chemical Reagent Co., Ltd., analytical grade), RPMI-1640 medium, fetal bovine serum, double antibiotics, PBS buffer, L02 cells, triglyceride kit, oleic acid, palmitic acid, bovine serum albumin (BSA), sodium hydroxide, modified Oil Red O staining kit (Beyotime Biotechnology, C0158M).

[0054] Vertical laminar flow clean bench, benchtop large-capacity low-speed centrifuge, 6-well cell culture plate, 96-well culture plate, enzyme-linked immunosorbent assay (ELISA) reader.

[0055] [Preparation of cell drug delivery stock solution, culture medium, and free fatty acid modeling solution]

[0056] Preparation of mother liquor for cell-based drug delivery of traditional Chinese medicine composition: Take an appropriate amount of traditional Chinese medicine composition, dissolve it by sonication with sterile RBS buffer, filter it through a 0.22um filter membrane to obtain a mother liquor with a concentration of about 10mg crude drug / mL, dispense it, and store it at -20℃.

[0057] Preparation of culture medium: Complete culture medium containing 10% fetal bovine serum: Under aseptic conditions, transfer 40 mL of RPMI-1640 medium to a sterilized 50 mL centrifuge tube, add 4.5 mL of fetal bovine serum and 0.45 mL of penicillin and streptomycin, label with the preparation date, and store at 4 °C.

[0058] Preparation of free fatty acid (FFA) molding solution:

[0059] (1) Preparation of 10% bovine serum albumin (BSA): Weigh 5g of BSA and dissolve it in 50mL of RPMI-1640 medium (without fetal bovine serum and antibiotics);

[0060] (2) Preparation of 0.1M sodium hydroxide: Weigh 0.2g of sodium hydroxide into 50mL of sterile deionized water and slowly add it in several batches to dissolve it;

[0061] (3) Preparation of 10mM sodium palmitate (PA) stock solution: Weigh an appropriate amount of PA and dissolve it in 0.1M sodium hydroxide to prepare 100mM PA; take 2mL of 100mM PA and add 18mL of 10% BSA to prepare 20mL of 10mM PA, and store at 4℃.

[0062] (4) Preparation of 20mM sodium oleate (OA) stock solution: Weigh an appropriate amount of OA and dissolve it in 0.1M sodium hydroxide to prepare 100mM OA; take 4mL of 100mM OA and add 16mL of 10% BSA to prepare 20mL of 20mM OA, and store at 4℃.

[0063] (5) Preparation of control group culture medium: Take 1 mL of 0.1 M sodium hydroxide and add 10% BSA to 10 mL, and store at 4℃.

[0064] Preparation of complete medium containing 10% CCK-8: Take 700uL of CCK-8, dissolve it in 7mL of complete medium, and mix well to obtain complete medium containing 10% CCK-8, 110uL per well.

[0065] [Experimental Methods]

[0066] Cell culture: The normal human hepatocyte line L02 was routinely cultured in RPMI 1640 complete medium containing 10% fetal bovine serum at 37°C, with 5% CO2 and saturated humidity. All experiments were performed during the logarithmic growth phase of the cells. When the cells reached 80%–90% confluence, they were passaged by trypsin digestion for subsequent experiments.

[0067] Establishment of a hepatocyte steatosis model: L02 cells were seeded in 96-well plates and cultured. After cell adhesion, when the cells reached 60% confluence, they were induced with 1 mM FFA (OA:PA = 2:1) for 24 hours. Oil Red staining showed a large number of lipid droplets accumulating in the cells and a significant increase in intracellular TG levels without affecting cell viability, indicating successful model establishment.

[0068] Drug cytotoxicity concentration screening: Each screening sample was filtered through a 0.22 μm filter and serially diluted with complete culture medium to prepare complete culture media containing drug at concentrations of 400 μg crude drug / mL, 200 μg crude drug / mL, 100 μg crude drug / mL, 50 μg crude drug / mL, 20 μg crude drug / mL, 10 μg crude drug / mL, 5 μg crude drug / mL, and 2.5 μg crude drug / mL, 100 μL per well. After 24 hours, the drug-containing culture medium was discarded, and complete culture medium containing 10% CCK-8 was added. After incubation at 37°C for 1 hour, the absorbance was calculated using UV detection at 450 nm. Drug concentrations with cell viability >80% were screened.

[0069] Effects of drugs on a hepatocyte steatosis model: L02 cells were seeded in 96-well plates and cultured. After cell adhesion, when the confluence reached 60%, the stock solutions of each screening sample were taken, filtered through a 0.22 μm filter membrane, and serially diluted with complete medium containing 1 mM FFA to prepare a certain concentration, 100 μL per well. The model group was cultured in the control group medium, and the blank group was cultured in normal complete medium. After 24 hours of culture, the cultured cells were collected, washed twice with PBS, and aliquoted. One portion was used for TG measurement, and the other portion was used for Oil Red O staining observation.

[0070] Intracellular TG content determination: The culture plate was removed from the wells and placed on an ice box. It was washed twice with PBS buffer, and cell lysis buffer was added to each well. After a brief incubation, cells were scraped off using a cell scraper, and cell debris and lysis buffer were transferred to tubes. The tubes were incubated on ice with vortexing several times during incubation. The supernatant was collected by centrifugation, and the TG content was determined according to the kit instructions. Total intracellular protein content was determined using the BCA protein concentration assay, and the results were calculated. The final result is expressed as TG content per unit mass of protein (mmol / g). The experiment was repeated three times. The results are expressed as mean ± standard deviation ±. Data were statistically analyzed using SPSS 26.0 software. p < 0.05 was considered statistically significant.

[0071] Oil Red staining to observe intracellular lipid droplet formation: Discard the original culture medium in each well of the 6-well culture plate, wash twice with PBS buffer; fix with 4% paraformaldehyde for 10 min, add 1 mL of modified Oil Red O staining solution, stain at room temperature in the dark for 30 min, and seal; rinse with staining washing solution (or 60% isopropanol) for 30 s until the background is transparent; add 1 mL of sterile PBS to keep the stained cells moist, and observe the Oil Red O staining under a microscope.

[0072] [Preparation of Traditional Chinese Medicine Compositions]

[0073] Hawthorn-Sophora japonica, ginseng-Sophora japonica, ginseng-hawthorn, and ginseng-hawthorn-Sophora japonica were weighed separately, and 8 times the amount of 50% ethanol was added to each. The mixture was heated under reflux for 1.5 hours (slightly boiling) and filtered. The corresponding extraction solvent was added each time, and the extraction was carried out twice in total. The two extracts were combined and concentrated to a certain volume under reduced pressure at 60℃. The extract was dried under reduced pressure to obtain a dry extract, which was then pulverized to obtain the ginseng-hawthorn composition, ginseng-Sophora japonica composition, hawthorn-Sophora japonica, and ginseng-hawthorn-Sophora japonica composition.

[0074] Take appropriate amounts of ginseng-hawthorn combination, ginseng-sophora japonica combination, hawthorn-sophora japonica combination and ginseng-hawthorn-sophora japonica combination, dissolve them by sonication with sterile RBS buffer, filter through a 0.22um filter membrane to obtain a mother liquor with a concentration of about 10mg crude drug / mL, dispense into portions and store at -20℃.

[0075] Experimental results

[0076] 1. Cytotoxicity screening of different traditional Chinese medicine combinations

[0077] Depend on Figure 1 It is known that the hawthorn-sophora japonica, ginseng-sophora japonica, ginseng-hawthorn, and ginseng-hawthorn-sophora japonica compositions had no significant effect on L02 cell activity when the drug concentration was 0–200 μg crude drug / mL. Therefore, the drug concentration for the in vitro efficacy experiment of the traditional Chinese medicine compositions was set at 200 μg crude drug / mL.

[0078] 2. Effects of different traditional Chinese medicine combinations on TG levels in L02 cells with fatty degeneration

[0079] Figure 2 The effect of different traditional Chinese medicine compositions on the TG level in L02 cells with steatosis (compared with the blank group, **p<0.01; compared with the model group, #p<0.05; between groups, ※p<0.05)

[0080] Depend on Figure 2 It was found that, compared with the blank group, the triglyceride level in L02 cells after modeling was significantly increased; compared with the model group, when 200ug / mL of raw drug was administered, the ginseng-hawthorn combination, ginseng-hawthorn combination, ginseng-hawthorn combination and ginseng-hawthorn-hawthorn combination significantly reduced the TG level in L02 cells with fatty degeneration; compared with the ginseng-hawthorn combination, the ginseng-hawthorn-hawthorn combination had a better lipid-lowering effect.

[0081] 3. Oil Red O staining of L02 cells with fatty degeneration after administration of different traditional Chinese medicine combinations at a concentration of 200 μg crude drug / mL.

[0082] Figure 3 Oil Red O staining (×400) was performed on L02 cells after administration of different traditional Chinese medicine combinations at 200 μg crude drug / mL. Group A was the blank control; Group B was the model group; Group C was the hawthorn-sophora japonica combination; Group D was the ginseng-sophora japonica combination; Group E was the ginseng-hawthorn combination; and Group F was the ginseng-hawthorn-sophora japonica combination.

[0083] Depend on Figure 3 It can be seen that, compared with the blank group, the L02 cells after modeling showed dense distribution and aggregation of orange-yellow oil droplets in the cells; after administration of 200ug crude drug / mL hawthorn-sophora flower combination, a large number of lipid droplets were still aggregated and distributed; while after administration of 200ug crude drug / mL ginseng-sophora flower combination, ginseng-hawthorn combination and ginseng-hawthorn-sophora flower combination, the density of lipid droplet distribution in the cells was significantly reduced, and the lipid droplet aggregation phenomenon was significantly reduced.

[0084] The above experiments confirm that ginseng, hawthorn, and sophora japonica can synergistically lower lipid levels. In vitro pharmacodynamic experiments compared the effects of combinations of these three herbs, either in pairs or in all three, on lipid accumulation in L02 cells with fatty degeneration. Based on TG level and Oil Red O staining results, the combination of ginseng, hawthorn, and sophora japonica significantly reduced intracellular TG levels and lipid accumulation in L02 cells with fatty degeneration, demonstrating a good lipid-lowering effect.

[0085] [Determination of the components of the ginseng-hawthorn-sophora japonica composition]

[0086] 1. Preparation of different ginseng-hawthorn compositions and ginseng-hawthorn-sophora japonica compositions

[0087] Weigh out ginseng and hawthorn slices in ratios of 1:1, 1:1.5, 1:2, 1.5:1, and 2:1, respectively. Add 8 times the amount of 50% ethanol, heat under reflux for 1.5 hours (slight boiling), filter, and repeat the extraction twice. Combine the two extracts, concentrate them under reduced pressure at 60°C to a certain volume, dry under reduced pressure to obtain a dry extract, pulverize it, and that is the ginseng-hawthorn composition.

[0088] Weigh out the optimal ratio of ginseng and hawthorn slices, add a certain amount of sophora japonica slices, so that the hawthorn and sophora japonica components are 2:1, 2:1.3, and 1:1 respectively. Add 8 times the amount of 50% ethanol, heat and reflux for 1.5 hours (slight boiling), filter, and extract twice in this way. Combine the two extracts, concentrate them under reduced pressure at 60℃ to a certain volume, dry under reduced pressure to obtain a dry extract, pulverize it, and that is the ginseng-hawthorn-sophora japonica composition.

[0089] 2. Preparation of mother liquor for cell-based drug delivery of traditional Chinese medicine compositions

[0090] Take an appropriate amount of ginseng-hawthorn combination or ginseng-hawthorn-sophora japonica combination, dissolve it by sonication with sterile RBS buffer, filter it through a 0.22um filter membrane to obtain a mother liquor with a concentration of about 10mg crude drug / mL, dispense it, and store it at -20℃.

[0091] Effects of drugs on a hepatocyte steatosis model: L02 cells were seeded in 96-well plates and cultured. After cell adhesion, when the confluence reached 60%, the stock solutions of each screening sample were taken, filtered through a 0.22 μm filter membrane, and serially diluted with complete medium containing 1 mM FFA to prepare a certain concentration, 100 μL per well. The model group was cultured in the control group medium, and the blank group was cultured in normal complete medium. After 24 hours of culture, the cultured cells were collected, washed twice with PBS, and aliquoted. One portion was used for TG measurement, and the other portion was used for Oil Red O staining observation.

[0092] (1) Measurement of intracellular TG content

[0093] Remove the culture plate from the ice bath and place it on an ice pack. Wash twice with PBS buffer. Add cell lysis buffer to each well and incubate briefly. Then scrape off cells using a cell scraper, transfer cell debris and lysis buffer into tubes, and incubate on ice with vortexing 3 times during incubation. Centrifuge and collect the supernatant. Measure the TG content according to the kit instructions. Calculate the total intracellular protein content using the BCA protein concentration assay. The final result is expressed as TG content per unit mass of protein (mmol / g). Repeat the experiment three times. The results are expressed as mean ± standard deviation ±. Statistical analysis was performed using SPSS 26.0 software. p < 0.05 was considered statistically significant.

[0094] (2) Oil Red staining to observe intracellular lipid droplet formation

[0095] Discard the original culture medium in each well of the 6-well culture plate, wash twice with PBS buffer; fix with 4% paraformaldehyde for 10 min, add 1-2 mL of Oil Red O working solution, stain at room temperature in the dark for 30 min, and seal; rinse with 60% isopropanol for 30 s until the background is clear; aspirate the staining solution, add hematoxylin staining solution for counterstaining for 5 min (to stain cell nuclei); wash with water until blue, mount with glycerol gelatin; observe the formation of orange-red lipid droplets in the cells under a light microscope.

[0096] (3) Quantitative determination of intracellular lipids

[0097] Discard the original culture medium from each well of the 6-well plate, wash twice with PBS buffer; fix with 4% paraformaldehyde for 10 min, add 1-2 mL of Oil Red O working solution, stain at room temperature in the dark for 30 min, and seal; rinse with 60% isopropanol for 30 s until the background is clear; dry the plate in a 37℃ incubator, then add 1 mL of isopropanol to each well, shake on a shaker for 10 min, and then add 100 μL to a 96-well plate. Measure the OD value of each well at 510 nm using a microplate reader. Repeat the experiment three times. Results are expressed as mean ± standard deviation ±. Data were statistically analyzed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0098] Experimental results

[0099] Determination of components of ginseng-hawthorn composition

[0100] (1) Screening of cytotoxicity of different ginseng-hawthorn combinations

[0101] Figure 4 For the cytotoxicity of different ginseng-hawthorn combinations, compared with the control group, *p<0.05. Figure 7-11 It was found that ginseng-hawthorn (1:1), ginseng-hawthorn (1:1.5), ginseng-hawthorn (1:2), ginseng-hawthorn (1.5:1), and ginseng-hawthorn (2:1) had no significant effect on L02 cell activity when the drug concentration was 0–200 μg crude drug / mL. However, at a concentration of 400 μg crude drug / mL, ginseng-hawthorn (1:2) had a significant effect on L02 cell activity.

[0102] (2) Effects of different proportions of ginseng-hawthorn composition on TG levels in L02 cells with fatty degeneration

[0103] Figure 5The effects of different ginseng-hawthorn combinations administered at 200 μg crude drug / mL on TG levels in L02 cells with fatty degeneration were investigated. Compared with the control group, *p<0.05; compared with the model group, #p<0.05. Figure 12 It was found that, compared with the blank group, the triglyceride level in L02 cells after modeling was significantly increased. Compared with the model group, when ginseng and hawthorn were administered at a ratio of 200 μg / mL (1:1), (1:1.5), (1:2), (1.5:1), and (2:1), all five ratios of ginseng and hawthorn in the drug solution reduced lipid accumulation in the modeled L02 cells to a certain extent. Among them, ginseng and hawthorn (1:1), (1:2), and (2:1) significantly alleviated lipid accumulation in the modeled L02 cells.

[0104] (3) Different proportions of human ginseng were administered hawthorn extract at a concentration of 200 μg crude drug / mL to induce the formation of L02 cells, which were then stained with Oil Red O.

[0105] Figure 6 Oil Red O staining (×800) was performed on L02 cells after administration of ginseng and hawthorn at ratios of 1:1, 1:1.5, 1:2, 1.5:1, and 2:1 at 200 μg crude drug / mL. Group A was the blank group, Group B was the model group, Group C was ginseng and hawthorn (1:1), Group D was ginseng and hawthorn (1:1.5), Group E was ginseng and hawthorn (1:2), Group F was ginseng and hawthorn (1.5:1), and Group G was ginseng and hawthorn (2:1). It can be seen that, compared with the blank group, the L02 cells after modeling showed a dense distribution and aggregation of orange-yellow oil droplets. Compared with the model group, when ginseng and hawthorn were administered at a ratio of 200ug crude drug / mL (1:1), ginseng and hawthorn (1:1.5), ginseng and hawthorn (1:2), ginseng and hawthorn (1.5:1), and ginseng and hawthorn (2:1), all five ratios of ginseng and hawthorn in the drug solution showed a certain trend of reducing lipid droplet accumulation in the modeled L02 cells. Among them, the reduction in lipid droplet accumulation was most significant with ginseng and hawthorn (1:2).

[0106] The above experiments show that the commonly used ratio of ginseng to hawthorn is 2:1 to 1:2. Therefore, using the L02 cell model of fatty degeneration, the efficacy was compared. Intracellular TG levels and Oil Red O staining were used as in vitro efficacy evaluation indicators for different ginseng-hawthorn combinations. It was found that when 200 μg of crude drug / mL was administered, the ginseng-hawthorn (1:2) combination showed superior efficacy in improving L02 cell fatty degeneration compared to other ginseng-hawthorn combinations. Subsequent experiments will further determine the components of ginseng-hawthorn-sophora japonica at the optimal ratio of ginseng-hawthorn (1:2).

[0107] This invention further identifies the components of ginseng, hawthorn, and sophora japonica, and the specific experimental results are as follows.

[0108] (1) Cytotoxicity screening of different ginseng-hawthorn-sophora flower compositions

[0109] Figure 7 The study investigated the cytotoxicity of different ginseng-hawthorn-sophora flower compositions. It was found that the ginseng-hawthorn-sophora flower (1:2:2) composition, the ginseng-hawthorn-sophora flower (1:2:1.3) composition, and the ginseng-hawthorn-sophora flower (1:2:1) composition had no significant effect on L02 cell activity when the drug concentration ranged from 0 to 400 μg crude drug / mL.

[0110] (2) Effects of different ginseng-hawthorn-sophora flavescens composition on TG levels in modeled L02 cells

[0111] Figure 8 The effects of ginseng, hawthorn, and sophora japonica in ratios of 1:2:2, 1:2:1.3, and 1:2:1 on the TG levels in L02 cells used for modeling were investigated. Compared with the control group, **p<0.01; compared with the model group, #p<0.05. Figure 8 It was found that, compared with the blank group, the triglyceride level in L02 cells after modeling was significantly increased. Compared with the model group, when ginseng, hawthorn and sophora japonica were administered at a ratio of 1:2:2, 1:2:1.3 and 1:2:1 (200 μg / mL of crude drug), the lipid accumulation in the modeled L02 cells was reduced to a certain extent. Among them, ginseng, hawthorn and sophora japonica (1:2:2) and ginseng, hawthorn and sophora japonica (1:2:1) could significantly alleviate the lipid accumulation in the modeled L02 cells.

[0112] (3) L02 cells were modeled by administering ginseng, hawthorn and sophora flower extracts at 200ug crude drug / mL. Oil Red O staining was performed on the cells.

[0113] Figure 9 Oil Red O staining (×400) was performed on L02 cells after administration of 200 μg crude drug / mL of different ginseng-hawthorn-sophora flower compositions. A was the blank group, B was the model group, C was the ginseng-hawthorn-sophora flower (1:2:2) composition, D was the ginseng-hawthorn-sophora flower (1:2:1.3) composition, and E was the ginseng-hawthorn-sophora flower (1:2:1) composition. Figure 10 Oil Red O staining was used to determine lipids in L02 cells after administration of 200 μg crude drug / mL of different ginseng-hawthorn-sophora flower compositions. Compared with the blank group, **p<0.01; compared with the model group, #p<0.05, ##p<0.01; between groups, ※p<0.05.

[0114] Depend on Figure 9 , Figure 10It can be seen that, compared with the blank group, the L02 cells after modeling showed a dense distribution and aggregation of orange-yellow oil droplets in the cells; compared with the model group, when ginseng, hawthorn and sophora japonica were administered at a ratio of 1:2:2, 1:2:1.3 and 1:2:1, the lipid droplet accumulation in the L02 cells was reduced to a certain extent when ginseng-hawthorn-sophora japonica (1:2:2) was administered at a ratio of 200ug crude drug / mL. Among them, the lipid droplet accumulation was reduced most significantly when ginseng-hawthorn-sophora japonica (1:2:2) was administered.

[0115] Based on the above experiments, it can be determined that adding Sophora japonica to the ginseng-hawthorn component in a 1:2 ratio to determine the composition, and based on the changes in TG levels and Oil Red O staining of the model cells after administration, it can be seen that the ginseng-hawthorn-Sophora japonica combination is more effective, especially the ginseng-hawthorn-Sophora japonica (1:2:2) combination has the most significant effect in improving lipid accumulation in L02 cells with fatty degeneration.

[0116] In summary, this invention utilizes the L02 fatty degeneration cell model, using intracellular TG levels and Oil Red O staining as evaluation indicators, to compare the effects of different traditional Chinese medicine combinations—ginseng-hawthorn, hawthorn-sophora japonica, ginseng-sophora japonica, and ginseng-hawthorn-sophora japonica—on improving the L02 fatty degeneration cell model. The results showed that the ginseng-hawthorn-sophora japonica combination exhibited the most significant lipid-lowering effect. Based on this, the optimal ratio of the ginseng-hawthorn combination was determined, and further, the optimal composition of ginseng-hawthorn-sophora japonica was determined to be 1:2:2.

[0117] [Determination of Processed Products for Traditional Chinese Medicine Compositions]

[0118] 1. Preparation of Traditional Chinese Medicine Compositions with Different Processed Products

[0119] Weigh out ginseng slices, hawthorn, and sophora japonica flowers in a ratio of 1:2:2. Add 8 times the amount of 50% ethanol, heat and reflux for 1.5 hours, filter, and repeat the extraction twice. Combine the two extracts, concentrate them under reduced pressure at 60-80℃ to a certain volume, and dry them under reduced pressure at 60-80℃ to obtain a dry extract. Pulverize the extract to obtain the ginseng slice-hawthorn-sophora japonica flower composition.

[0120] Weigh out red ginseng slices, hawthorn berries, and sophora japonica flowers in a ratio of 1:2:2. Add 8 times the amount of 50% ethanol, heat under reflux for 1.5 hours, filter, and repeat the extraction twice. Combine the two extracts, concentrate them under reduced pressure at 60-80℃ to a certain volume, and dry them under reduced pressure at 60-80℃ to obtain a dry extract. Pulverize the extract to obtain the red ginseng slices-hawthorn berries-sophora japonica flower composition.

[0121] Weigh out ginseng slices, stir-fried hawthorn, and cleaned sophora japonica flowers in a ratio of 1:2:2. Add 8 times the amount of 50% ethanol, heat and reflux for 1.5 hours, filter, and repeat the extraction twice. Combine the two extracts, concentrate them under reduced pressure at 60-80℃ to a certain volume, and dry them under reduced pressure at 60-80℃ to obtain a dry extract. Pulverize the extract to obtain the ginseng slices-stir-fried hawthorn-cleansed sophora japonica flower composition.

[0122] Weigh out ginseng slices, charred hawthorn, and cleaned sophora japonica flowers in a ratio of 1:2:2. Add 8 times the amount of 50% ethanol, heat and reflux for 1.5 hours, filter, and repeat the extraction twice. Combine the two extracts, concentrate them under reduced pressure at 60-80℃ to a certain volume, and dry them under reduced pressure at 60-80℃ to obtain a dry extract. Pulverize the extract to obtain the ginseng slices-charred hawthorn-cleansed sophora japonica flower composition.

[0123] Weigh out ginseng slices, hawthorn, and stir-fried sophora japonica flowers in a ratio of 1:2:2. Add 8 times the amount of 50% ethanol, heat and reflux for 1.5 hours, filter, and repeat the extraction twice. Combine the two extracts, concentrate them under reduced pressure at 60-80℃ to a certain volume, and dry them under reduced pressure at 60-80℃ to obtain a dry extract. Pulverize the extract to obtain the ginseng slices-hawthorn-stir-fried sophora japonica flower composition.

[0124] 2. Preparation of mother liquor for cell-based drug delivery of traditional Chinese medicine compositions

[0125] Take an appropriate amount of ginseng slices-hawthorn-sophora japonica flower combination, or red ginseng slices-hawthorn-sophora japonica flower combination, or ginseng slices-fried hawthorn-sophora japonica flower combination, or ginseng slices-charred hawthorn-sophora japonica flower combination, or ginseng slices-hawthorn-fried sophora japonica flower combination, and dissolve them by sonication with sterile RBS buffer. Filter the solution through a 0.22um filter membrane to obtain a mother liquor with a concentration of approximately 10mg crude drug / mL. Dispense the solution and store it at -20℃.

[0126] Effects of drugs on a hepatocyte steatosis model: L02 cells were seeded in 96-well plates and cultured. After cell adhesion, when the confluence reached 60%, the stock solutions of each screening sample were taken, filtered through a 0.22 μm filter membrane, and serially diluted with complete medium containing 1 mM FFA to prepare a certain concentration, 100 μL per well. The model group was cultured in the control group medium, and the blank group was cultured in normal complete medium. After 24 hours of culture, the cultured cells were collected, washed twice with PBS, and aliquoted. One portion was used for TG measurement, and the other portion was used for Oil Red O staining observation.

[0127] (1) Measurement of intracellular TG content

[0128] Remove the culture plate from the ice bath and place it on an ice pack. Wash twice with PBS buffer. Add cell lysis buffer to each well and incubate briefly. Then scrape off cells using a cell scraper, transfer cell debris and lysis buffer into tubes, and incubate on ice with vortexing 3 times during incubation. Centrifuge and collect the supernatant. Measure the TG content according to the kit instructions. Calculate the total intracellular protein content using the BCA protein concentration assay. The final result is expressed as TG content per unit mass of protein (mmol / g). Repeat the experiment three times. The results are expressed as mean ± standard deviation ±. Statistical analysis was performed using SPSS 26.0 software. p < 0.05 was considered statistically significant.

[0129] (2) Oil Red staining to observe intracellular lipid droplet formation

[0130] Discard the original culture medium in each well of the 6-well culture plate, wash twice with PBS buffer; fix with 4% paraformaldehyde for 10 min, add 1-2 mL of Oil Red O working solution, stain at room temperature in the dark for 30 min, and seal; rinse with 60% isopropanol for 30 s until the background is clear; aspirate the staining solution, add hematoxylin staining solution for counterstaining for 5 min (to stain cell nuclei); wash with water until blue, mount with glycerol gelatin; observe the formation of orange-red lipid droplets in the cells under a light microscope.

[0131] Experimental results

[0132] Determination of Processed Products of Traditional Chinese Medicine Compositions

[0133] 1. Cytotoxicity screening of different processed products of traditional Chinese medicine combinations

[0134] Figure 11 Regarding the cytotoxicity of different ginseng-hawthorn combinations, it can be seen that the combination of ginseng slices-cleaned hawthorn-cleaned sophora japonica, or red ginseng slices-cleaned hawthorn-cleaned sophora japonica, or ginseng slices-fried hawthorn-cleaned sophora japonica, or ginseng slices-charred hawthorn-cleaned sophora japonica, or ginseng slices-cleaned hawthorn-fried sophora japonica, at a drug concentration of 0-200 μg crude drug / mL, had no significant effect on L02 cell activity.

[0135] 2. Effects of different processed traditional Chinese medicine compositions on TG levels in model L02 cells.

[0136] Figure 12 The effects of ginseng slices-hawthorn-sophora japonica flower combination, or red ginseng slices-hawthorn-sophora japonica flower combination, or ginseng slices-fried hawthorn-sophora japonica flower combination, or ginseng slices-charred hawthorn-sophora japonica flower combination, or ginseng slices-hawthorn-fried sophora japonica flower combination on the TG level in model L02 cells were compared with the blank group, **p<0.01; compared with the model group, #p<0.05. Figure 12It was found that, compared with the blank group, the triglyceride level in L02 cells after modeling was significantly increased. Compared with the model group, when 200 μg / mL of ginseng slices-hawthorn-sophora japonica flower combination, or red ginseng slices-hawthorn-sophora japonica flower combination, or ginseng slices-fried hawthorn-sophora japonica flower combination, or ginseng slices-charred hawthorn-sophora japonica flower combination, or ginseng slices-hawthorn-fried sophora japonica flower combination, all five ginseng-hawthorn-fried sophora japonica flower combination ratios showed a certain trend of reducing lipid accumulation in modeled L02 cells. Among them, the ginseng slices-hawthorn-sophora japonica flower combination significantly alleviated lipid accumulation in modeled L02 cells. Compared with the ginseng slices-hawthorn-sophora japonica flower combination, there was no difference in the effect of red ginseng slices-hawthorn-sophora japonica flower combination, or ginseng slices-fried hawthorn-sophora japonica flower combination, or ginseng slices-charred hawthorn-sophora japonica flower combination, or ginseng slices-hawthorn-fried sophora japonica flower combination in alleviating lipid accumulation in modeled L02 cells.

[0137] 3. Oil Red O staining of L02 cells with fatty degeneration after administration of 200 μg crude drug / mL of different processed traditional Chinese medicine combinations.

[0138] Figure 13 Oil Red O staining (×400) was performed on L02 cells after administration of different Chinese herbal medicine combinations at 200 μg crude drug / mL. A was the blank group, B was the model group, C was the ginseng slice-hawthorn-sophora japonica flower combination, D was the red ginseng slice-hawthorn-sophora japonica flower combination, E was the ginseng slice-fried hawthorn-sophora japonica flower combination, F was the ginseng slice-charred hawthorn-sophora japonica flower combination, and G was the ginseng slice-hawthorn-fried sophora japonica flower combination.

[0139] Depend on Figure 13 It was found that, compared with the blank group, the L02 cells after modeling showed a dense distribution and aggregation of orange-yellow oil droplets in the cells; after administering 200ug / mL of the following combinations of ginseng slices-hawthorn-sophora japonica, red ginseng slices-hawthorn-sophora japonica, ginseng slices-fried hawthorn-sophora japonica, ginseng slices-charred hawthorn-sophora japonica, or ginseng slices-hawthorn-fried sophora japonica, the density of intracellular lipid droplet distribution showed a certain decreasing trend, among which the ginseng slices-hawthorn-sophora japonica combination showed the most significant relief effect.

[0140] The above experiments confirm that combinations of ginseng, hawthorn, and sophora japonica flowers prepared using different methods all have a certain ameliorative effect on the L02 cell steatosis model. Among them, the lipid-lowering effect of the combination of ginseng slices, hawthorn, and sophora japonica flowers is statistically significant. Therefore, compared with other combinations of prepared products, its lipid-lowering effect is superior.

[0141] [Determination of the preparation process of traditional Chinese medicine composition]

[0142] I. Preparation of Traditional Chinese Medicine Compositions Using Different Extraction Methods

[0143] (1) Aqueous extracts of traditional Chinese medicine compositions

[0144] Weigh out a certain proportion of ginseng, hawthorn and sophora japonica slices, add 8 times the amount of distilled water, heat and reflux for 1.5 hours (slight boiling), filter, and extract twice in this way. Combine the extracts, concentrate them under reduced pressure at 60℃ to a certain volume, dry under reduced pressure to obtain a dry extract, pulverize, and obtain the water extract of the traditional Chinese medicine composition.

[0145] (2) Alcohol extracts of traditional Chinese medicine compositions

[0146] Weigh out a certain proportion of ginseng, hawthorn and sophora japonica slices, add 8 times the amount of 75% ethanol, heat and reflux for 1.5 hours (slight boiling), filter, and extract twice in this way. Combine the extracts and concentrate them to a certain volume under reduced pressure at 60℃. After drying under reduced pressure, obtain a dry extract, pulverize it, and you will get the alcohol extract of the Chinese herbal composition.

[0147] (3) The first alcohol extraction followed by water extraction of traditional Chinese medicine composition

[0148] Weigh out a certain proportion of ginseng, hawthorn and sophora japonica slices, add 8 times the amount of 75% ethanol, heat and reflux for 1.5 hours (slight boiling), and filter; add 8 times the amount of distilled water to the residue, heat and reflux for 1.5 hours (slight boiling), and filter; combine the ethanol extract and the water extract, concentrate under reduced pressure at 60℃ to a certain volume, dry under reduced pressure to obtain a dry extract, pulverize, and obtain the first ethanol extract and then water extract of the traditional Chinese medicine composition.

[0149] Experimental results

[0150] 1. Cytotoxicity screening of aqueous extracts, alcohol extracts, and extracts obtained by first extracting alcohol and then aqueous extracts of traditional Chinese medicine compositions.

[0151] Figure 14 The study examined the cytotoxicity of the aqueous extract, alcohol extract, and the extract obtained by first extracting alcohol and then water from the traditional Chinese medicine composition. It was found that complete culture media containing these extracts, at concentrations of 0–200 μg crude drug / mL, had no significant effect on L02 cell activity. Therefore, subsequent cell administration will be conducted at a concentration of 200 μg crude drug / mL for in vitro efficacy studies.

[0152] 2. Effects of water extract, alcohol extract, and extract obtained by first extracting alcohol and then water extract of traditional Chinese medicine on the TG level in L02 cells with fatty degeneration.

[0153] Figure 15The effects of the aqueous extract, alcohol extract, and the combination of alcohol and aqueous extracts of the traditional Chinese medicine composition on the triglyceride (TG) level in L02 cells with steatosis were investigated. Compared with the control group, **p<0.01; compared with the model group, ##p<0.01. It was found that compared with the control group, the triglyceride level in L02 cells was significantly increased after modeling. Compared with the model group, administration of 200 μg / mL of the aqueous extract, alcohol extract, and the combination of alcohol and aqueous extracts of the traditional Chinese medicine composition tended to reduce lipid accumulation in L02 cells after modeling. Among these, the alcohol extract of the traditional Chinese medicine composition had a significant alleviating effect on lipid accumulation in L02 cells after modeling. There were no significant differences between the groups in the effects of the aqueous extract, alcohol extract, and the combination of alcohol and aqueous extracts of the traditional Chinese medicine composition on the TG level in L02 cells with steatosis.

[0154] 3. Oil Red O staining of L02 cells with 200 μg crude drug / mL of water extract, alcohol extract, and extract obtained by first alcohol extraction and then water extraction of traditional Chinese medicine composition.

[0155] Figure 16 Oil Red O staining (×400) was performed on L02 cells after administration of 200 μg crude drug / mL of the aqueous extract, ethanol extract, and the extract obtained by first ethanol extraction and then aqueous extraction of the traditional Chinese medicine composition. Group A was the blank group, Group B was the model group, Group C was the aqueous extract of the traditional Chinese medicine composition, Group D was the ethanol extract of the traditional Chinese medicine composition, and Group E was the extract obtained by first ethanol extraction and then aqueous extraction of the traditional Chinese medicine composition. It was found that compared with the blank group, the L02 cells after modeling showed a dense distribution of orange-yellow oil droplets and significant lipid droplet aggregation. Compared with the model group, when administered 200 μg crude drug / mL of the aqueous extract, ethanol extract, and the extract obtained by first ethanol extraction and then aqueous extraction of the traditional Chinese medicine composition, the lipid droplet accumulation in the modeling L02 cells showed a certain decreasing trend, manifested as a reduction in oil droplet aggregation and a decrease in oil droplet distribution density.

[0156] As shown above, the extraction solvent of the traditional Chinese medicine composition has a certain impact on its in vitro efficacy. Compared with the model group, the water extract, alcohol extract, and the combination of alcohol extraction followed by water extraction all showed a certain tendency to alleviate intracellular lipid accumulation in L02 cells with steatosis. Among them, the lipid-lowering effect of the alcohol extract was statistically significant. Therefore, the alcohol extraction method is superior to the water extraction method and the combination of alcohol extraction followed by water extraction.

[0157] II. Comparison of extraction efficiency of traditional Chinese medicine compositions with different ethanol concentrations

[0158] Reagents and instruments used: High-performance liquid chromatography instrument (Agilent Technologies (China) Co., Ltd., LC-1200 series, USA); chromatographic column (Agilent Technologies (China) Co., Ltd., ZORBAX Eclipse XDB-C18, Analytical 4.6×250nm 5-Micron, USA); acetonitrile (Sigma-Aldrich (Shanghai) Trading Co., Ltd., chromatographic grade, China); methanol (Sigma-Aldrich (Shanghai) Trading Co., Ltd., chromatographic grade, China); phosphoric acid (Sinopharm Chemical Reagent Co., Ltd., analytical grade, batch number 220220823, China); ginsenoside Rg1 reference standard; ginsenoside Re reference standard; ginsenoside Rb1 reference standard; rutin reference standard; methanol (Sinopharm Chemical Reagent Co., Ltd., analytical grade, China).

[0159] Preparation of the test sample of the traditional Chinese medicine composition: Take a certain proportion of ginseng, hawthorn and sophora japonica slices, add 8 times the amount of ethanol of different concentrations (80% ethanol, 70% ethanol, 60% ethanol, 50% ethanol or 0% ethanol), heat and reflux for 1.5 hours, filter, and extract twice. Combine the filtrates; concentrate under reduced pressure at 60℃ to a certain volume to obtain the extract. Accurately transfer 0.20 mL of the extract, add 30 mL of methanol, extract ultrasonically for 40 minutes, filter, and set aside.

[0160] Preparation of ginsenoside mixed reference standards: Accurately weigh ginsenoside Rg1 reference standard, ginsenoside Re reference standard and ginsenoside Rb1 reference standard, add methanol to prepare a mixed solution containing 0.2 mg of each per 1 mL, shake well, and the solution is obtained.

[0161] Preparation of rutin reference standard: Take an appropriate amount of rutin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.1 mg per 1 mL.

[0162] The detection method for ginsenosides was as follows: octadecylsilane-bonded silica gel was used as the packing material; acetonitrile (A)-water (B) was used as the mobile phase, and gradient elution was performed: 0–12 min, 19% A; 12–60 min, 19% → 36% A; the detection wavelength was 203 nm.

[0163] The detection method for rutin was as follows: octadecylsilane-bonded silica gel was used as the packing material; acetonitrile (A)-0.3% phosphoric acid solution (B) was used as the mobile phase for gradient elution: 0–26 min, 20% → 39.5% A; 26–26.1 min, 39.5% → 20% A; 26.1–30 min, 20% A; the detection wavelength was 360 nm.

[0164] Record the peak areas of ginsenosides Re, Rg1, Rb1 and rutin at their respective retention times, calculate the content of effective components extracted per unit weight of crude drug, and conduct a comprehensive evaluation. The calculation formula is shown in Equation 1-1.

[0165] Formula 1-1: Comprehensive score = Ginsenoside Re content / max (Ginsenoside Re content obtained from different extraction solvents) * 0.2 + Ginsenoside Rg1 content / max (Ginsenoside Rg1 content obtained from different extraction solvents) * 0.2 + Ginsenoside Rb1 content / max (Ginsenoside Rb1 content obtained from different extraction solvents) * 0.2 + Rutin content / max (Rutin content obtained from different extraction solvents) * 0.4

[0166] Experimental results

[0167] Table 1. Content and overall score of ginsenosides Re, Rg1, Rb1 and rutin

[0168]

[0169] Figure 17 The trend of extraction efficiency of active ingredients at different ethanol concentrations; Figure 18 For the comparison of the comprehensive scores of effective components of extracts obtained from different extraction solvents, the inter-group comparisons were conducted, with *p<0.05 and **p<0.01.

[0170] The test results above show that as the ethanol concentration increases, the extraction efficiency of ginsenosides Rg1 and Re increases, while the extraction efficiency of ginsenoside Rb1 decreases. The extraction efficiency of rutin first increases and then decreases, with the optimal extraction efficiency observed at a 50% ethanol concentration. A comprehensive evaluation of the extraction of active ingredients from traditional Chinese medicine compositions using different concentrations of ethanol reveals that, compared to 80%, 70%, 60%, and 0% ethanol, 50%–60% ethanol can better extract the active ingredients from the traditional Chinese medicine compositions. Furthermore, using 50% ethanol to extract the traditional Chinese medicine compositions of this invention maximizes the extraction of ginsenosides Rg1, Re, Rb1, and rutin, as well as other index components listed in the pharmacopoeia.

[0171] Based on the index components and content determination methods of ginseng and sophora japonica in the Chinese Pharmacopoeia, the extraction efficiency of the extraction method for the traditional Chinese medicine composition was comprehensively scored using ginsenosides Rg1, Re, Rb1, and rutin as index components. According to the high-performance liquid chromatography (HPLC) results and the comprehensive score calculation, 50%–60% ethanol showed better extraction efficiency for the above four index components than 70% and 80% ethanol. Among these, using 50% ethanol for extraction of the traditional Chinese medicine composition yielded the best extraction efficiency and economic benefits.

[0172] Therefore, the preferred preparation method of the traditional Chinese medicine composition determined by the present invention is as follows: weigh ginseng, hawthorn and sophora japonica slices, add 8 times the amount of 50% ethanol, heat and reflux for 1.5 hours (slight boiling), filter and extract twice in this way; combine the two extracts, concentrate them under reduced pressure at 60°C to a certain volume, dry them under reduced pressure to obtain a dry extract, pulverize them, and that is the traditional Chinese medicine composition.

[0173] III. Validation Study on the Optimization and Preparation Process of Traditional Chinese Medicine Compositions

[0174] The preferred preparation method of the traditional Chinese medicine composition determined according to this invention is as follows: Weigh out ginseng slices-hawthorn-sophora japonica flower composition, or red ginseng slices-hawthorn-sophora japonica flower composition, or ginseng slices-fried hawthorn-sophora japonica flower composition, or ginseng slices-charred hawthorn-sophora japonica flower composition, or ginseng slices-hawthorn-fried sophora japonica flower composition, with each processed product in a ratio of 1:2:2. Add 8 times the amount of 50% ethanol, heat under reflux for 1.5 hours (slight boiling), filter, and repeat the extraction twice. Combine the two extracts, concentrate under reduced pressure at 60℃ to a certain volume, dry under reduced pressure to obtain a dry extract, pulverize, and that is the traditional Chinese medicine composition. Accurately weigh 0.1g of the traditional Chinese medicine composition, add 5mL of methanol, extract ultrasonically for 40 minutes, centrifuge at 4000r / min for 10 minutes, transfer the supernatant to a 5mL volumetric flask, and dilute to volume with methanol for later use.

[0175] The extraction efficiency of the herbal extracts was compared using the liquid chromatography conditions described in section II, "Comparison of Extraction Efficiency of Herbal Extracts with Different Ethanol Concentrations."

[0176] Record the peak areas of ginsenosides Re, Rg1, Rb1 and rutin at their respective retention times, and calculate the transfer rate of the active ingredients.

[0177] Effective ingredient transfer rate (%) = (mass of Chinese herbal composition × content) / (amount of processed medicinal pieces × content in processed medicinal pieces) = yield of dry extract × content of dry extract / content in processed medicinal pieces;

[0178] Theoretical minimum content of active ingredient = (weight of Sophora japonica flower * minimum content in pharmacopoeia) * component transfer rate / (weight of Sophora japonica flower * yield of dry extract) = minimum content in pharmacopoeia * component transfer rate / yield of dry extract.

[0179] Table 2

[0180]

[0181] Based on the data in Table 2 above, the theoretical minimum content of active ingredients, i.e. the content of active ingredients per unit mass of dry extract, is: Rg1+Re≥1.24mg / g; Rb1≥2.13mg / g; Rutin≥37.13mg / g.

[0182] [In vivo pharmacodynamic experiment of traditional Chinese medicine composition for the treatment of hyperlipidemia]

[0183] Instrument and reagent information

[0184] UV6200H electronic balance (SHIMADZU, d=0.01g, Japan), AUW220D electronic balance (Shimadzu Enterprise Management (China) Co., Ltd., d=0.1mg / 0.01mg, Japan), thermostatic shaker (Taicang Experimental Equipment Factory, Peiying 2-2 thermostatic shaker, China), high-speed refrigerated centrifuge (Thermo Fisher Scientific (China) Co., Ltd., Germany), disposable vacuum blood collection tubes (Hebei Kangweishi Medical Technology Co., Ltd., containing separating gel and coagulant, China), ultrasonic multi-channel grinder (Ningbo Xinzhi Biotechnology Co., Ltd., Scientz-24TD, China), 96-well cell culture plate (Corning Incorporated, ref_3599, China), ELISA reader (BioTek Instruments, model Eon, USA)

[0185] 60% fat-based energy diet (Research Diets, Research) Diets_D12492, batch numbers 22110202, 22120607, USA) Sodium carboxymethyl cellulose 300-800 mPa·s (Sinopharm Chemical Reagent Co., Ltd., chemically pure, China), Total cholesterol test kit (Nanjing Jiancheng Bioengineering Institute, A111-2-1, batch number 20221220, China), Triglyceride test kit (Nanjing Jiancheng Bioengineering Institute, A110-2-1, batch number 20221217, China), High-density lipoprotein cholesterol test kit (Nanjing Jiancheng Bioengineering Institute, A112-2-1, batch number 20221205, China), Low-density lipoprotein cholesterol test kit (Nanjing Jiancheng Bioengineering Institute, A113-2-1, batch number 20221119, China), 4% tissue cell fixative (Sangon Biotech (Shanghai) Co., Ltd., batch number IA11FA0001, China)

[0186] Preparation of the traditional Chinese medicine composition: Take ginseng, hawthorn, and sophora japonica flowers in a ratio of 1:2:2. Mix the three parts of the decoction pieces, add 8 times the weight of 50% ethanol, heat and reflux for 1.5 hours, filter, and extract twice. Combine the filtrates. Rotary evaporate at 60℃ to recover the ethanol, and concentrate under reduced pressure at 60℃ to a thick extract of 1.1g crude drug / mL. This is the traditional Chinese medicine composition. Dispense and store at -20℃.

[0187] Preparation of oral liquid of traditional Chinese medicine composition: Take an appropriate amount of thick extract of traditional Chinese medicine composition, dilute it with 0.5% sodium carboxymethyl cellulose and pulverize it by ultrasonication, and administer it to mice by gavage. Prepare and use immediately.

[0188] Experimental methods

[0189] 1. Experimental animal feeding and model establishment: SPF-grade male C57BL / 6J mice, 5 weeks old, weighing 18 - 20 g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. and were housed in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. They were specially raised by a dedicated person in a feeding environment with a temperature of 22 - 24°C and a relative humidity of 60 - 70% RH, with 12-hour light and dark cycles alternating. Before the formal experiment began, the mice were normally fed for one week to adapt to the feeding environment. The animal use license was SYXK(Shanghai)2020 - 0009, and all experimental animal care and experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai University of Traditional Chinese Medicine.

[0190] 2. Grouping and model establishment: The mice were randomly divided into 7 groups: blank group, model group, high-dose, medium-dose, and low-dose composition groups, with 7 mice in each group, for a total of 35 mice. The blank group was given normal feed, and the model group and each drug administration group were fed a high-fat diet with 60% lipid providing energy.

[0191] 3. Drug administration plan: The mice in each group started drug administration synchronously. The model group and the normal group were given the same volume of 0.5% sodium carboxymethylcellulose. The drug administration for the mice in the traditional Chinese medicine composition group was converted according to the body surface area method and the clinical dosage. The dosage of the dry extract powder of the high-dose composition for mice was 4.0 g / kg, the dosage of the medium-dose composition for mice was 2.0 g / kg, and the dosage of the low-dose composition for mice was 1.0 g / kg. Converted to a standard adult (60 kg), the clinically recommended dosages of the dry extract were: high-dose 0.32 g / kg, medium-dose 0.16 g / kg, and low-dose 0.08 g / kg.

[0192] 4. Pharmacodynamic evaluation of traditional Chinese medicine composition in the treatment of hyperlipidemia

[0193] (1) Blood lipid levels: The main indicators for clinically evaluating hyperlipidemia are serum total cholesterol (TC), serum triglyceride (TG), serum high-density lipoprotein cholesterol (HDL-C), and serum low-density lipoprotein cholesterol (LDL-C). Therefore, the above 4 blood lipid indicators were also used as key evaluation indicators in the pharmacodynamic experiments involved in this invention.

[0194] The food intake of the mice was recorded every three days, and the body weight of the mice was weighed every week. After 4 weeks of model establishment, mice from the blank group and the model group were randomly selected every 2 weeks for blood tests to observe changes in blood lipid levels. When the blood lipid levels of the model mice showed significant changes compared with the blank group (p < 0.05), the model establishment was considered successful. After 10 weeks of synchronous drug administration during actual model establishment, the mice were fasted for 12 hours in advance, then sacrificed. Blood was collected by puncturing the orbital vein, centrifuged at 3000 r / min for 15 minutes, and the upper serum was taken for determination of the above 4 blood lipid indicators. The results of statistical processing were used The values ​​are indicated by p. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicated that the difference was statistically significant.

[0195] (2) Liver tissue HE staining pathological sections: Relevant literature shows that hyperlipidemia may lead to lipid droplet accumulation in the liver. Therefore, liver pathological sections were also used as a reference indicator for the drug efficacy experiment. Liver tissue samples of 1cm × 1cm were taken from mice 10 weeks after modeling and fixed at a fixed location. These samples were fixed with 4% paraformaldehyde fixative, and then subjected to fixation, dehydration, embedding, sectioning, and H&E staining before histopathological analysis and observation.

[0196] (3) Liver tissue Oil Red O staining pathological sections: The liver tissue of mice 10 weeks after modeling was separated into 1cm×1cm sections at fixed locations, fixed with 4% paraformaldehyde fixative, and then subjected to fixation, dehydration, embedding, sectioning, Oil Red O staining and other steps for histopathological analysis and observation.

[0197] Experimental results

[0198] 1. Blood lipid levels

[0199] Table 3. Serum levels of various indicators (LDL-C) in mice of each group (n=7, )

[0200]

[0201] Note: Compared with the control group, **p<0.01; compared with the model group, #p<0.05, ##p<0.01; compared with the model group, ↓ indicates a downward trend, but no significant difference.

[0202] As shown in Table 3, compared with the blank group, the model group showed abnormal blood lipid levels in four indicators after 10 weeks of high-fat diet feeding, namely, TC, TG and LDL-C levels increased significantly, and HDL-C level decreased significantly. Therefore, it is believed that the C57 mouse hyperlipidemia model induced by long-term high-fat diet feeding was successfully established.

[0203] Figure 19 For the comparison of blood lipid levels in each group, compared with the blank group, **p<0.01; compared with the model group, #p<0.05, ##p<0.01. It can be seen that, compared with the model group, all three doses of the composition of the present invention have certain lipid-lowering effects. The efficacy of the low-dose group is mainly reflected in the significant improvement of HDL-C and LDL-C. The medium-dose group can significantly improve TG, HDL-C and LDL-C. The high-dose group has the best efficacy, and all four blood lipid indicators are improved, namely, TC, TG and LDL-C levels are significantly reduced, and HDL-C level is significantly increased.

[0204] 2. Liver sections stained with hematoxylin and eosin (HE)

[0205] Figure 20 The liver morphology was compared using HE staining (×200). A represents the normal group, B the model group, C the high-dose group of the composition, D the medium-dose group of the composition, and E the low-dose group of the composition. The small circles indicated by the red arrows represent lipid droplets. It can be seen that after 10 weeks of modeling with a high-fat diet, compared with the control group, the model group mice showed a large number of lipid droplets throughout their livers, which were large and dense. Compared with the model group, the high-dose, medium-dose, and low-dose groups of the composition showed a significant reduction in lipid droplets in their livers. The liver morphology of the high-dose and medium-dose groups was close to that of the control group, indicating that the traditional Chinese medicine composition can significantly improve hepatic lipid accumulation, with the high dose showing the best lipid-lowering effect.

[0206] 3. Liver sections stained with oil red O

[0207] Figure 21 The comparison of liver morphology stained with Oil Red O (×200) shows that A is the normal group, B is the model group, C is the high-dose group of the composition, D is the medium-dose group of the composition, and E is the low-dose group of the composition. It can be seen that after 10 weeks of modeling by feeding a high-fat diet, compared with the blank group, the livers of mice in the model group stained with Oil Red O showed a large number of lipid droplets, which were dense and aggregated. Compared with the model group, the number of lipid droplets in the livers of the high-dose group, the medium-dose group, and the low-dose group of the composition was significantly reduced, suggesting that the composition has a certain alleviating effect on lipid accumulation in the liver of hyperlipidemic mice.

[0208] Based on the above in vitro cell experiments, the lipid-lowering effect of the ginseng-hawthorn-sophora japonica (1:2:2) alcohol extract was the best. The efficacy of this traditional Chinese medicine composition was verified in animal in vivo experiments.

[0209] A hyperlipidemia model was established in C57BL / 6J mice by feeding them with a high-fat diet. Three doses of a traditional Chinese medicine (TCM) composition (high, medium, and low) were administered simultaneously. After 10 weeks of modeling and administration, the blood lipid levels and liver stained sections of the mice were used as evaluation indicators. It was found that all three doses of the TCM composition could improve abnormal blood lipid levels and liver lipid accumulation to a certain extent. Among them, the high dose of the TCM composition had the best efficacy, significantly reducing TC, TG, and LDL-C levels, significantly increasing HDL-C levels, and significantly reducing lipid accumulation in the liver.

[0210] In summary, based on the above series of in vitro experimental results, the ginseng-hawthorn-sophora japonica (1:2:2) composition with lipid-lowering potential was used to induce a hyperlipidemia model in C57BL / 6J mice by feeding them with a high-fat diet to verify its lipid-lowering efficacy. After 10 weeks of simultaneous drug administration during modeling, the lipid-lowering effect of the ginseng-hawthorn-sophora japonica (1:2:2) composition was verified using TC, TG, HDL-C, LDL-C levels and liver pathological staining sections as evaluation indicators. The efficacy of the composition at different dosages was compared. The results showed that low-dose (pharmacopoeia-specified), medium-dose (1.2 times the pharmacopoeia-specified), and high-dose (2.4 times the pharmacopoeia-specified) of the herbal composition all improved abnormal blood lipid levels and liver lipid accumulation to some extent. The lipid-lowering efficacy of the low- and medium-dose herbal composition was mainly reflected in improving TG, HDL-C, and LDL-C levels, and it also had a certain effect in alleviating liver lipid accumulation. The high-dose herbal composition showed the best efficacy, significantly reducing TC, TG, and LDL-C levels, significantly increasing HDL-C levels, and significantly reducing lipid accumulation in the liver. Based on the lipid-lowering effects of the low- and medium-dose herbal composition in in vivo experiments, long-term use at low doses is recommended.

[0211] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for treating hyperlipidemia, characterized in that, This traditional Chinese medicine composition is made from Chinese medicinal materials, namely ginseng, hawthorn, and sophora japonica flowers; The proportions of the Chinese medicinal materials, namely ginseng, hawthorn, and sophora japonica, are 1:2:2 by weight.

2. The traditional Chinese medicine composition for treating hyperlipidemia according to claim 1, characterized in that, The ginseng in question refers to ginseng slices and red ginseng slices; The hawthorn mentioned includes clean hawthorn, stir-fried hawthorn, and charred hawthorn; The locust flowers mentioned are clean locust flowers and stir-fried locust flowers.

3. The traditional Chinese medicine composition for treating hyperlipidemia according to claim 1, characterized in that, The traditional Chinese medicine composition is a solvent extract of ginseng, hawthorn and sophora japonica. Ginseng, hawthorn and sophora japonica are extracted with solvent, concentrated and dried to obtain a dry extract, which is then pulverized to obtain the traditional Chinese medicine composition.

4. The traditional Chinese medicine composition for treating hyperlipidemia according to claim 3, characterized in that, Add appropriate excipients to make granules, pills or capsules.

5. The traditional Chinese medicine composition for treating hyperlipidemia according to claim 3, characterized in that, The effective components contained in the traditional Chinese medicine composition include: ginsenosides Re, Rg1, Rb1, and rutin. The effective component content per unit mass of dry extract is: Rg1+Re≥1.24mg / g; Rb1≥2.13mg / g; rutin≥37.13mg / g.

6. The method for preparing a traditional Chinese medicine composition for treating hyperlipidemia as described in claim 1, characterized in that, The following method was used to prepare the herbal composition: ginseng, hawthorn and sophora japonica were weighed in proportion, and an extraction solvent was added for extraction. The extract was filtered to obtain a medicinal extract. The medicinal extract was concentrated and dried to obtain a dry extract. The dry extract was then pulverized to obtain the herbal composition.

7. The method for preparing a traditional Chinese medicine composition for treating hyperlipidemia according to claim 6, characterized in that, The extraction solvent is water and / or ethanol, and the amount of the extraction solvent used is 5-10 times the total weight of ginseng, hawthorn and sophora japonica flowers; During the extraction process, the extraction solvent is kept at a gentle boil under reflux and extracted 1-5 times, with each extraction lasting 1-3 hours. The solvent is then concentrated under reduced pressure at 50-90℃.

8. The method for preparing a traditional Chinese medicine composition for treating hyperlipidemia according to claim 7, characterized in that, The extraction solvent is ethanol with a concentration of 30% to 80%.

9. The use of the traditional Chinese medicine composition for treating hyperlipidemia as described in claim 1 in the preparation of a drug for treating hyperlipidemia.

Images