Traditional Chinese medicine composition for improving dyslipidemia and preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition, belongs to the technical field of traditional Chinese medicines, and aims to solve the problems of dyslipidemia and liver lipid deposition caused by chronic high fat diet. The composition comprises two formulas, wherein the formula A consists of astragalus membranaceus, lucid ganoderma and schisandra chinensis according to the weight ratio of 1: 1: 1; the formula B is composed of schisandra chinensis and liquorice according to the weight ratio of 3: 1, and can significantly activate an NRF2 / ARE pathway, up-regulate expression of antioxidant genes such as NQO1 and HO-1, inhibit H2O2-induced ROS generation and cell apoptosis, and improve related symptoms from the molecular level; meanwhile, the provided preparation method can improve the solubility of active ingredients and the intestinal absorption efficiency, avoids ingredient degradation caused by high-temperature extraction, ensures long-acting stable release of the medicine in vivo, and is mainly used for improving dyslipidemia and liver lipid deposition caused by chronic high-fat diet.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine compositions, and more specifically, to a traditional Chinese medicine composition for improving dyslipidemia, and a preparation method and application thereof. Background Art

[0002] With the changes in modern lifestyle, the incidence of dyslipidemia and related metabolic diseases (such as hyperlipidemia, fatty liver, etc.) caused by chronic high-fat diet has increased year by year.

[0003] While Western medications can currently regulate blood lipids in the short term, long-term use can easily lead to side effects such as liver and kidney damage and gastrointestinal reactions, resulting in low patient compliance. Traditional Chinese medicine, with its multi-target and low toxicity profile, shows potential for improving blood lipid metabolism. However, existing Chinese herbal formulas suffer from unclear compatibility ratios and low bioavailability of active ingredients, limiting the stability of their clinical efficacy. Existing studies have reported the antioxidant and lipid metabolism-regulating effects of various single Chinese herbs (such as Astragalus, Ganoderma lucidum, and Schisandra chinensis), but the efficacy of individual ingredients is limited, and there is a lack of systematic combination strategies for dyslipidemia caused by a chronic high-fat diet. Summary of the Invention

[0004] An object of the present invention is to provide a Chinese medicine composition for improving dyslipidemia, which is composed of one of the following two formulas A and B: A: Astragalus, Ganoderma lucidum, and Schisandra chinensis, the weight ratio of the three being 1:1:1; B: Schisandra chinensis and Licorice root are composed in a weight ratio of 3:1.

[0005] Preferably, the method comprises the following steps: S1. Grind the raw materials into 80-100 mesh respectively, mix them, add 6 times the mass of distilled water, and reflux extract at 85-90℃ for 2 times, each time for 2 hours, and combine the extracts; S2, concentrating the extract under reduced pressure at 45-50° C. to obtain a concentrated solution; S3. Freeze the concentrated solution at -80°C overnight, and then freeze-dry to obtain freeze-dried powder of the traditional Chinese medicine composition.

[0006] Preferably, the concentrated liquid after the reduced pressure concentration in step S2 needs to be subjected to nano-processing, specifically as follows: After adjusting the pH value of the concentrate to 5.5-6.0, the concentrate was treated 5-8 times with a pressure of 200-250 MPa using high-pressure microfluidization technology to form a nanosuspension. In the nanosuspension, astragalus polysaccharide, Ganoderma lucidum triterpenes and schisandrin in formula A or schisandrin and glycyrrhizic acid in formula B form stable micelles through self-assembly.

[0007] Preferably, the method for forming stable micelles of astragalus polysaccharide, ganoderma triterpenes and schisandrin in formula A or schisandrin and glycyrrhizic acid in formula B comprises the following steps: The concentrated solution after reduced pressure concentration was adjusted to a pH of 5.5-6.0, and stirred at a constant temperature of 45-50° C. for 30 min to obtain a mixed solution; The mixed solution was cooled to 25°C at a rate of 0.5-2°C / min, and a sodium chloride solution accounting for 0.1% of the volume of the concentrated solution was added; Then, the samples were sonicated at 40 kHz and 100 W for 5 min; High-pressure microfluidization technology was then used for shearing treatment to control the micelle particle size to 20-50 nm to obtain a nanosuspension. During this process, the temperature was maintained at 25±2°C.

[0008] Preferably, the concentration of the sodium chloride solution is 0.05-0.15 wt%.

[0009] Preferably, the method further comprises spray drying the nanosuspension at an inlet temperature of 160-170°C and an outlet temperature of 70-75°C.

[0010] Preferably, the preparation is in the form of tablets, capsules, granules, injections, aerosols, suppositories, patches, ointments, oral liquid preparations or powders.

[0011] Preferably, the tablet is an enteric-coated tablet, a sustained-release tablet or an effervescent tablet, the capsule is a hard capsule or a soft capsule, and the oral liquid preparation is a syrup, a mixture or a tincture.

[0012] Provided is an application of the traditional Chinese medicine composition in preparing a medicament for treating fatty liver and hyperlipidemia.

[0013] Provided is a use of the traditional Chinese medicine composition in preparing a medicament for improving abnormal blood lipid elevation caused by a chronic high-fat diet.

[0014] The present invention has at least the following beneficial effects: First, the present invention significantly activates the NRF2 / ARE pathway, upregulates the expression of antioxidant genes such as NQO1 and HO-1, inhibits H2O2-induced ROS generation and cell apoptosis, and improves dyslipidemia and hepatic lipid deposition caused by a chronic high-fat diet at the molecular level through the scientific combination of Formula A (Astragalus, Ganoderma lucidum, and Schisandra chinensis in a weight ratio of 1:1:1) or Formula B (Schisandra chinensis and Licorice in a weight ratio of 3:1).

[0015] Second, high-pressure microfluidization nanotechnology and self-assembly micelle technology are used to self-assemble astragalus polysaccharides, Ganoderma lucidum triterpenes, schisandrin or schisandrin and glycyrrhizic acid into stable micelles. Compared with the traditional water extraction process, this technology improves the solubility and intestinal absorption efficiency of the active ingredients, while avoiding the degradation of ingredients caused by high-temperature extraction, ensuring long-term and stable release of drugs in the body.

[0016] Third, the present invention further enhances the stability and bioavailability of drugs by precisely controlling process parameters (such as temperature, pH value, pressure, etc.).

[0017] Fourth, this invention rationally combines ingredients based on the Traditional Chinese Medicine theory of monarch, minister, assistant, and envoy. Astragalus nourishes qi and raises yang, strengthens the exterior and stops perspiration, while Ganoderma lucidum nourishes the heart qi and calms the mind, and Schisandra chinensis astringes and strengthens qi and promotes fluid production. Through multi-target synergistic action, it not only enhances the lung-tonifying and qi-invigorating effects, but also regulates lipid metabolism, protects liver cells, and significantly improves oxidative stress damage caused by a chronic high-fat diet.

[0018] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The cytotoxicity screening results of different Chinese medicines of the present invention are shown, where A to H are the test results of ginseng, ganoderma lucidum, wolfberry, astragalus, schisandra chinensis, codonopsis pilosula, licorice, and jujube on cell proliferation activity or toxicity, respectively; Figure 2 The figures are the experimental results of the antioxidant mechanism and dosage determination of the traditional Chinese medicine composition of the present invention, wherein A is the detection result of Ganoderma lucidum, Schisandra chinensis, Codonopsis pilosula, Astragalus membranaceus, and Licorice on NRF2-ARE luciferase activity at 0 min; B is the effect of different concentrations of TCM5 on cell activity detected by CCK8 method; C-1 is the qRT-PCR detection of the promoting effect of TCM5 on NQO1mRNA expression; C-2 is the qRT-PCR detection of the promoting effect of TCM5 on HO-1mRNA expression; C-3 is the qRT-PCR detection of the promoting effect of TCM5 on GCLMmRNA expression; C-4 is the qRT-PCR detection of the promoting effect of TCM5 on MRP1mRNA expression; D is the result of the hydrogen peroxide detection kit; E is the detection result of Ganoderma lucidum, Schisandra chinensis, Codonopsis pilosula, Astragalus membranaceus, and Licorice on NRF2-ARE luciferase activity at 10 min; Figure 3The results of the verification of the mechanism of action of the Chinese medicine composition of the present invention at the cellular level are shown, wherein A-1 is a bar graph of WB detection of NRF2 protein expression in L-02 cells; A-2 is a bar graph of the relative expression of NRF2 protein; A-3 is the immunofluorescence staining result of NRF2 in L-02 cells; B is the spectrophotometric detection of ROS generation level in L-02 cells in the NC group; C is the flow cytometry detection of cell apoptosis rate; Figure 4 The figure shows the regulation of NRF2 expression by the Chinese herbal medicine combination. A is the NRF2 expression detection result of different groups at 0 min; B is the NRF2 expression detection result of different groups at 10 min. Figure 5 The macroscopic index results of the TCM5 animal experiment, among which A-1 is the weight change curve of mice in each group at different culture time; A-2 is the quantitative comparison of weight gain; B-1 is the bar graph of liver index of mice in each group; B-2 is the bar graph of kidney index of mice in each group; C-1 to C-10 are the inter-group comparisons of blood routine indicators (WBC, LY, RBC, HGB, PLT); D-1 to D-15 are the inter-group comparisons of serum biochemical indicators (ALT, AST, CHOL, TRIG, UA, CREA, BUN, β2-MG); Figure 6 Figure 5 shows the liver pathological results (HE staining) of the TCM5 animal experiment. A shows the HE staining sections of the livers of mice in the normal control group, high-fat diet group, Chinese medicine alone group, simultaneous drug administration group, post-drug administration group, and pre-drug administration group, respectively. B shows the results of Western Blot detection of target proteins in the liver tissues of mice in each group and the grayscale quantitative analysis of Western Blot bands. C shows the results of qRT-PCR detection of the relative expression levels of MPO1 gene mRNA in the liver tissues of mice in each group. D shows the results of qRT-PCR detection of the relative expression levels of CK1 gene mRNA in the liver tissues of mice in each group. E shows the results of qRT-PCR detection of mRNA content in the liver tissues of mice in each group. F shows the results of qRT-PCR detection of the relative expression levels of GSH-RSSG related gene mRNA in the liver tissues of mice in each group. Figure 7 Figure 2 shows the macroscopic index results of the TCM2 animal experiment. A is the weight change curve of mice in each group at different culture times; B-1 to B-5 are the inter-group comparisons of blood routine indicators (WBC, LY, RBC, HGB, PLT); C-1 to C-10 are the inter-group comparisons of serum biochemical indicators (ALT, AST, CHOL, TRIG, TBIL, UA, CREA, UREA, β2-MG, DBIL); Figure 8These are the liver pathological results (HE staining) of the TCM2 group animal experiment. A to D are HE-stained sections of the livers of mice in the normal control group, the Chinese medicine group alone, the high-fat diet group, and the post-drug administration group, respectively. DETAILED DESCRIPTION

[0020] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0021] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0022] <Chinese medicine screening experiment> The preparation method of the Chinese medicine component comprises the following steps: 100 g of different Chinese medicinal ingredients were ground into 100 mesh, and then added with distilled water (6 times the weight), and refluxed at 90 ° C for 2 times, each time for 2 hours. The extracts were combined and filtered to obtain the filtrate; Among them, the Chinese medicinal ingredients are: ginseng, ganoderma lucidum, wolfberry, astragalus, schisandra chinensis, codonopsis pilosula, and liquorice; The filtrate was concentrated under reduced pressure at 50°C to obtain a concentrate; The concentrate was adjusted to pH 5.5, stirred at 50°C for 30 min, cooled to 25°C at a rate of 0.5°C / min, and 0.15 wt% sodium chloride solution (0.1% by volume of the concentrate) was added. After ultrasonic treatment (40 kHz, 100 W, 5 min), a nanosuspension was prepared using high-pressure microfluidization technology (pressure 250 MPa, 8 cycles). The nanosuspension was spray-dried (inlet temperature 170°C, outlet temperature 75°C) to obtain the corresponding traditional Chinese medicine components.

[0023] Different concentrations of TCM components were prepared using cell culture medium. The CCK8 method (Cell Counting Kit-8) was used to evaluate the effects of different TCM components with Qi-tonifying effects on the proliferation activity or cytotoxicity of HEK293T cells. This experiment was mainly used to screen the effective concentration range of Qi-tonifying drugs and verify their regulatory effects on cell activity. The results are shown in Figure 2. Figure 1 As shown, from Figure 1 It can be seen that the dosage concentrations of Chinese herbal medicine components in cell experiments are as follows: ginseng: 117.19 µg / mL, Ganoderma lucidum: 117.19 µg / mL, Lycium barbarum: 1875 µg / mL, Astragalus: 117.19µg / mL, Schisandra chinensis: 1875 µg / mL, Codonopsis pilosula: 1875 µg / mL, Licorice: 7500 µg / mL, and Jujube: 1875µg / mL.

[0024] The NRF2-ARE luciferase activity was detected by immunofluorescence and WB methods respectively at the concentrations of the above-mentioned Chinese medicinal components. The expression of NRF2 was detected at the initial administration (0 min) and 10 min after administration. Among them, the blank group without any addition was used as the blank group, and the single administration of hydrogen peroxide was used as the reference. The results are as follows: Figure 2 As shown in the results, it was found that Ganoderma lucidum, Schisandra chinensis, Codonopsis pilosula, Astragalus membranaceus, and Licorice root could significantly increase the expression of NRF2 at the initial administration (0 min) and 10 min after administration.

[0025] Based on the NRF2-ARE luciferase activity detection experiment, the present invention screened out Ganoderma lucidum, Schisandra chinensis, Codonopsis pilosula, Astragalus membranaceus, and Licorice as Qi-invigorating drugs that can maintain the body's homeostatic state, and confirmed through experiments that the above-mentioned drugs can significantly activate the NRF2-ARE signaling pathway, thereby exerting the effect of maintaining the body's homeostatic state.

[0026] Based on the theory of Chinese medicine compatibility, the above-mentioned drugs were scientifically combined to obtain a Chinese medicine composition: Formula A (TCM5) consists of Astragalus membranaceus, Ganoderma lucidum, and Schisandra chinensis, with a weight ratio of 1:1:1; Formula B (TCM2) consists of Schisandra chinensis and Licorice, with a weight ratio of 3:1.

[0027] In Formula A (TCM5) of the TCM composition, Astragalus membranaceus is sweet and slightly warm in nature, entering the spleen and lung meridians. It serves as the monarch herb, tonifying qi and raising yang, strengthening the exterior and stopping perspiration. It stimulates qi throughout the body, playing a leading role in the core efficacy of tonifying the lungs and replenishing qi, laying the foundation for the formula's qi-tonifying effect. Furthermore, Astragalus membranaceus serves as an adjunct herb, guiding the herb to its meridians.

[0028] Lingzhi is sweet and neutral in nature, entering the Heart, Lung, Liver, and Kidney meridians, serving as a ministerial herb. Lingzhi nourishes the heart and warms the lungs. Working synergistically with Astragalus, it not only enhances the lung-tonifying and Qi-invigorating effects, but also calms the mind and nerves, harmonizing Qi and blood. This complements the principal herb's lack of calming properties, allowing it to simultaneously tonify Qi and achieve peace of mind.

[0029] Schisandra chinensis, with its sour and sweet flavors and warm nature, enters the lung, heart, and kidney meridians, acting as an adjuvant. Its astringent, firming, and Qi-boosting properties can prevent the excessive Qi-boosting and dispersing effects of Astragalus membranaceus, which can deplete Qi and damage Yin. Simultaneously, Schisandra chinensis enhances the calming and tranquilizing effects of Ganoderma lucidum. Together, these three ingredients enhance the lung-tonifying and Qi-boosting effects, providing a more gentle and lasting balance and maintaining the body's homeostasis.

[0030] In Formula B (TCM2) of the traditional Chinese medicine composition, Schisandra chinensis is sour and sweet, warm in nature, and enters the lung, heart, and kidney meridians. It is the main herb and has the remarkable power of astringing, strengthening, and replenishing qi and promoting body fluid. It can astringe the lungs and nourish the kidneys, astringe essence, and stop diarrhea. It plays a leading role in the core efficacy of strengthening, astringing, and replenishing qi, laying the foundation for the prescription's astringent properties. Licorice is sweet in taste and neutral in nature. It enters the heart, lung, spleen and stomach meridians. It is both an adjuvant and a guiding drug. Licorice can replenish qi, tonify the middle, clear away heat and detoxify, and harmonize various drugs. It works together with Schisandra chinensis to enhance the effect of Schisandra chinensis in replenishing qi and promoting body fluid, and to alleviate the astringent nature of Schisandra chinensis to avoid excessive astringency. At the same time, it guides the drugs to the meridians and harmonizes the various drugs in the prescription, making the function of consolidating and replenishing qi more gentle and appropriate, and together they achieve the effect of both astringing and tonifying, and harmonizing the body.

[0031] <Example 1> The preparation method of the Chinese medicine composition comprises the following steps: Take 100 g each of Astragalus, Ganoderma lucidum, and Schisandra chinensis (1:1:1), grind them into 100 mesh, mix them, add 1800 mL of distilled water (6 times the weight), and reflux extract at 90°C twice, each time for 2 hours. Combine the extracts and filter to obtain the filtrate; The filtrate was concentrated under reduced pressure at 50°C to obtain a concentrate; The concentrate was adjusted to pH 5.5, stirred at 50°C for 30 min, cooled to 25°C at a rate of 0.5°C / min, and 0.15 wt% sodium chloride solution (0.1% by volume of the concentrate) was added. After ultrasonic treatment (40 kHz, 100 W, 5 min), a nanosuspension was prepared using high-pressure microfluidization technology (pressure 250 MPa, 8 cycles). The nanosuspension was spray-dried (inlet temperature 170°C, outlet temperature 75°C) to obtain a traditional Chinese medicine composition (TCM5).

[0032] <Example 2> The preparation method of the Chinese medicine composition comprises the following steps: Take 100 g each of Astragalus, Ganoderma lucidum, and Schisandra chinensis (1:1:1), grind them into 90 mesh size, mix them, add 1800 mL of distilled water (6 times the weight), and reflux extract at 87.5°C twice, each time for 2 hours. Combine the extracts and filter to obtain the filtrate. The filtrate was concentrated under reduced pressure at 47.5°C to obtain a concentrate; The concentrate was adjusted to pH 5.8, stirred at 47.5°C for 30 min, cooled to 25°C at a rate of 1°C / min, and 0.10 wt% sodium chloride solution (0.1% by volume of the concentrate) was added. After ultrasonic treatment (40 kHz, 100 W, 5 min), a nanosuspension was prepared using high-pressure microfluidization technology (pressure 225 MPa, 6 cycles). The nanosuspension was spray-dried (inlet temperature 165° C., outlet temperature 72° C.) to obtain a traditional Chinese medicine composition.

[0033] <Example 3> The preparation method of the Chinese medicine composition comprises the following steps: Take 100 g each of Astragalus, Ganoderma lucidum, and Schisandra chinensis (1:1:1), grind them into 80 mesh, mix them, add 1800 mL of distilled water (6 times the weight), and reflux extract at 85°C twice, each time for 2 hours. Combine the extracts and filter to obtain the filtrate; The filtrate was concentrated under reduced pressure at 45°C to obtain a concentrate; The concentrate was adjusted to pH 6.0, stirred at 45°C for 30 min, cooled to 25°C at a rate of 2°C / min, and 0.05 wt% sodium chloride solution (0.1% by volume of the concentrate) was added. After ultrasonic treatment (40 kHz, 100 W, 5 min), a nanosuspension was prepared using high-pressure microfluidization technology (pressure 200 MPa, 5 cycles). The nanosuspension was spray-dried (inlet temperature 160° C., outlet temperature 70° C.) to obtain a traditional Chinese medicine composition.

[0034] Comparative Example 1 The preparation method of the Chinese medicine composition comprises the following steps: Take 100 g each of Astragalus, Ganoderma lucidum, and Schisandra chinensis (1:1:1), grind them into 100 mesh, mix them, add 1800 mL of distilled water (6 times the weight), and reflux extract at 90°C twice, each time for 2 hours. Combine the extracts and filter to obtain the filtrate; The filtrate was concentrated under reduced pressure at 50°C to obtain a concentrate; The concentrated solution was frozen at -80°C overnight and then freeze-dried to obtain freeze-dried powder of the traditional Chinese medicine composition.

[0035] The particle size, in vitro dissolution rate, and absorption efficiency of Examples 1 to 3 and Comparative Example 1 were investigated. The in vitro dissolution rate was determined using the paddle method of the Chinese Pharmacopoeia (rotation speed 50 rpm, medium pH 6.8 phosphate buffer), and the cumulative dissolution rate was measured at 37° C. for 4 hours. The absorption efficiency was determined using a Caco-2 cell monolayer model to simulate intestinal absorption. The apparent permeability coefficient of the drug was calculated and expressed as relative bioavailability. The results are shown in Table 1 below: Table 1 In vitro dissolution and absorption efficiency of Examples 1 to 3 and Comparative Example 1 Group Example 1 Example 2 Example 3 control group Particle size (nm) 25.3 32.8 43.6 358 In vitro dissolution (%) 92.3±1.5 85.6±1.8 78.4±2.1 48.5±3.0 Absorption efficiency (%) 88.5±2.0 80.2±2.3 70.8±2.5 38.2±3.5 According to the data in Table 1, the in vitro dissolution rates of Examples 1 to 3 decreased with the decrease of the nanonization pressure and the number of cycles. The high-pressure microfluidization in Example 1 formed the smallest particle size, the largest specific surface area, and the fastest dissolution rate. In the control group without nanonization treatment, the drug existed in the form of ordinary freeze-dried powder, and the dissolution rate was only 48.5%, which was significantly lower than that of Examples 1 to 3. The absorption efficiency of Examples 1 to 3 was positively correlated with the dissolution rate. The nanomicelles in Example 1 had the best permeability in the Caco-2 cell model due to their small particle size and uniform dispersion. The control group without nanonization treatment had the lowest absorption efficiency, only 43% of that in Example 1, due to its coarse particles and poor solubility. The above data show that the nanonization process (high-pressure microfluidization technology) of the present invention can significantly improve the in vitro dissolution rate and absorption efficiency of the traditional Chinese medicine composition.

[0036] <Example 4> Preparation method of traditional Chinese medicine composition, comprising the following steps: Take each raw material of Formulation B (composed of Schisandra chinensis and Glycyrrhiza uralensis, and the weight ratio of the two is 3:1), respectively pulverize to 100 meshes, mix and add 6 times the mass of distilled water, reflux and extract at 90 °C for 2 times, each time for 2 hours, combine the extracts, filter, and obtain a filtrate; Concentrate the filtrate under reduced pressure at 50 °C and under vacuum to obtain a concentrated solution; Adjust the pH of the concentrated solution to 5.5, stir at 50 °C for 30 min, cool to 25 °C at a rate of 0.5 °C / min, add 0.15 wt% sodium chloride solution (accounting for 0.1% of the volume of the concentrated solution), after ultrasonic treatment (40 kHz, 100 W, 5 min), use high-pressure microfluidization technology (pressure 250 MPa, cycle 8 times) to obtain a nano-suspension; Perform spray drying on the nano-suspension (inlet temperature 170 °C, outlet temperature 75 °C) to obtain a traditional Chinese medicine composition (TCM2).

[0037] <CCK8 experiment> Prepare solutions with different concentrations of the traditional Chinese medicine composition (TCM5) prepared in Example 1, and determine the dosage of the traditional Chinese medicine composition by the CCK8 method (Cell Counting Kit-8). The results are as Figure 2 shown in B, and it is found according to Figure 2 in B that the dosage of the traditional Chinese medicine composition is 3600 μg / mL. Compared with the non-administered group in the figure, * P <0.05, ** P <0.01, *** P <0.001.

[0038] <Antioxidant experiment> The expression levels of NQO1, HO-1, GCLM, and MRP1 were detected by qRT-PCR. Among them, the group without any intervention was used as the blank group (NC group), 1.2 mM H2O2 was used as the oxidative stress model group (H2O2 group), and 3600 μg / mL of the traditional Chinese medicine composition was used as the traditional Chinese medicine formula intervention group (TCM5 group). The results are as Figure 2 shown in C-1 to C-4. It was found that TCM5 significantly promoted the expression of NQO1, HO-1, GCLM, and MRP1 mRNAs.

[0039] To understand whether TCM5 directly reacts with H2O2 to exert antioxidant effects, the hydrogen peroxide detection kit from Beyotime was used to detect the H2O2 concentrations in the NC group, H2O2 group, TCM5 group, TCM2 group, and H2O2 + TCM5 group (containing both 1.2 mM H2O2 and 3600 μg / mL of the traditional Chinese medicine composition). The results are as Figure 2 shown in D. It was found that TCM2 and TCM5 do not directly react with H2O2 to exert antioxidant effects and do not affect the H2O2 concentration.

[0040] <Experiment on the expression of NRF2> The NC group, H2O2 group, TCM2 group, and TCM5 group were respectively applied to L-02 cells, and the expression of NRF2 in L-02 cells was detected. The results are as Figure 3 shown in A-1 to A-3. It was found that TCM2 and TCM5 could significantly increase the expression of NRF2 in L-02 cells.

[0041] <ROS experiment> The synthesis and distribution of ROS were detected by spectrophotometry. The results are as Figure 3 shown in B. It was found that TCM5 could significantly reduce the H2O2-induced ROS generation in L-02 cells.

[0042] <Anti-cell apoptosis experiment> Flow cytometry was used to detect the cell apoptosis status. The results are as Figure 3 shown in C. It was found that TCM5 could significantly reduce the H2O2-induced cell apoptosis According to the above experimental results, TCM5 (Astragalus membranaceus - Ganoderma lucidum - Schisandra chinensis, 1:1:1) and TCM2 (Schisandra chinensis - Glycyrrhiza uralensis, 3:1) can significantly promote the expression of NRF2 and its downstream genes NQO1, HO-I, GCLM, and MRP1, and significantly inhibit the H2O2-induced ROS generation and cell apoptosis.

[0043] <Comparative example 2> The preparation method of the traditional Chinese medicine composition includes the following steps: Take 300 g of different Chinese herbal ingredients, grind them into 100 mesh, mix them, add 1800 mL of distilled water (6 times the weight), and reflux extract them at 90°C twice, each time for 2 hours. Combine the extracts and filter to obtain the filtrate; Among them, the Chinese herbal ingredients are: A: Astragalus; B: Ganoderma lucidum; C: Schisandra chinensis; D: Licorice; The filtrate was concentrated under reduced pressure at 50°C to obtain a concentrate; The concentrate was adjusted to pH 5.5, stirred at 50°C for 30 min, cooled to 25°C at a rate of 0.5°C / min, and 0.15 wt% sodium chloride solution (0.1% by volume of the concentrate) was added. After ultrasonic treatment (40 kHz, 100 W, 5 min), a nanosuspension was prepared using high-pressure microfluidization technology (pressure 250 MPa, 8 cycles). The nanosuspension was spray-dried (inlet temperature 170°C, outlet temperature 75°C) to obtain different traditional Chinese medicine compositions.

[0044] The corresponding solutions were prepared from groups A to D and the prepared Chinese medicine compositions in Example 1, Example 4 and Comparative Example 2. Immunofluorescence and WB methods were used to detect NRF2-ARE luciferase activity, and the expression of NRF2 was detected at the initial administration (0 min) and 10 min after administration. The blank group was set without any addition. The results are as follows: Figure 4 As shown in the results, it was found that TCM2 and TCM5 could significantly increase the expression of NRF2 at the initial administration (0 min) and 10 min after administration.

[0045] <Animal Experiments> 1.1 Experimental animals Sixty SPF-grade male C57BL / 6J mice, aged 6-8 weeks and weighing 22-25 g, were selected and provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd. They were fed with standard mouse pellet feed or high-fat feed according to the groups, which were provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and Sibeifu (Beijing) Biotechnology Co., Ltd., respectively.

[0046] 1.2 Grouping, Dosage Method, and Experimental Period A normal control group (NC), a high-fat diet group (HFD), a traditional Chinese medicine group (TCM5), a pre-administration group (TCM5-HFD), a simultaneous administration group (HFD+TCM5), and a post-administration group (HFD-TCM5) were set up, wherein TCM5 here and below refers to the traditional Chinese medicine composition prepared in Example 1.

[0047] After three days of adaptive feeding, starting at 8:00 a.m. on the fourth day, mice on the HFD, HFD+TCM5, and HFD-TCM5 were fed a high-fat diet, while the rest were fed a normal pelleted diet. TCM5 and TCM5-HFD mice were gavaged with 3.90 mg / kg TCM5, while the rest were gavaged with drinking water. This treatment lasted for 30 days. On the evening of the 33rd day, mice on the NC (partial), HFD, TCM5, and HFD+TCM5 groups were fasted with no water, and samples were collected on the morning of the 34th day. From the 34th day onwards, mice on the TCM5-HFD group were fed a high-fat diet, while the rest were fed a normal pelleted diet. HFD-TCM5 mice were gavaged with 3.90 mg / kg TCM5, while the rest were gavaged with drinking water. This treatment lasted for 30 days. On the evening of the 63rd day, mice on the NC (remaining), TCM5-HFD, and HFD-TCM5 groups were fasted with no water, and samples were collected on the morning of the 64th day.

[0048] 1.3 Collection of materials Whole blood and serum: 20 μL of blood was collected from the eyeball and placed in an anticoagulant tube on ice. The remaining blood was placed in an EP tube and allowed to stand for 4 h before centrifugation at 3000 rpm for 20 min. The supernatant (i.e., serum) was collected by pipette and placed in an EP tube and stored in a -80°C refrigerator.

[0049] Liver and kidney: The mouse peritoneal cavity was opened, and part of the liver and kidney were taken and placed in 4% paraformaldehyde solution and paraffin sectioned; the remaining liver was placed in a cryovial and stored in a -80°C freezer.

[0050] 1.4 Observation indicators and methods Flow cytometry was used to detect blood cell classification and count in whole blood. A blood biochemistry analyzer was used to measure liver and kidney function indicators in plasma, including ALT, AST, TBIL, DBIL, IBIL, CHOL, TRIG, UA, CREA, BUN, and β2-MG. HE staining was used to observe liver pathological changes. qRT-PCR and Western blotting were used to detect the expression levels of NRF2, NQO1, HO-1, GCLM, and MRP1 in liver tissue. The Beyotime lipid oxidation (MDA) detection kit and GSH / GSSG detection kit were used to detect the levels of MDA and GSH / GSSG in liver tissue. RNAseq was used to detect liver mRNA transcription profiles. LC-MS was used to detect the non-targeted metabolome of the liver.

[0051] 1.5 Statistics Differentially expressed genes were screened using P < 0.05 and |log2FoldChange (FC)| > 1 as criteria, and the differentially expressed genes were enriched in the Kyoto Encyclopedia of Genes and Genomes (https: / / www.genome.jp / kegg / ) database to obtain relevant pathway information; differential metabolites were screened using P < 0.05 and |log2FC| > 0 as criteria, and the differential metabolites were enriched in the KEGG database to obtain relevant pathway information; the rest of the procedures were the same as before.

[0052] 1.6 Results 1.6.1 Weight During the treatment period, the mice in each group were measured regularly. Figure 5 As shown in A-1 and A-2, it was found that concurrent administration of TCM5 significantly reduced the weight gain induced by a high-fat diet, and TCM5 significantly reduced the weight gain of mice induced by a chronic high-fat diet.

[0053] 1.6.2 Organ Index The organ index results are as follows Figure 5 As shown in Figures B-1 and B-2, it was found that the liver index of the TCM5 pre-administered group was significantly increased, and TCM5 significantly alleviated the liver enlargement caused by chronic high-fat diet.

[0054] 1.6.3 Routine blood test During the experiment, the mice were tested for blood routine. The results were as follows: Figure 5 As shown in Figures C-1 to C-10, it was found that concurrent administration of TCM5 could significantly reduce the increase in white blood cell (WBC) count in mice caused by HFD, and pre-administration of TCM5 could significantly reduce the number of white blood cells (WBC), lymphocytes (LY) and platelets (PLT) in mice; post-administration of TCM5 could significantly reduce the number of lymphocytes (LY) in mice, indicating that TCM5 significantly improved the whole blood cell disorder caused by chronic high-fat diet.

[0055] 1.6.4 Liver and kidney function Liver and kidney function test results Figure 5 As shown in Figures D-1 to D-15, concurrent administration of TCM5 significantly reduced HFD-induced increases in triglycerides (TRIG) in mice, while pre-administration of TCM5 significantly reduced blood urea nitrogen (BUN) levels in mice. TCM5 significantly improved the dyslipidemia induced by a chronic high-fat diet.

[0056] 1.6.5 Liver pathology Liver histopathological findings Figure 6As shown in center A, pre- and post-administration of TCM5 alleviated HFD-induced fat vacuolation, whereas concurrent administration of TCM5 had no significant effect. TCM5 significantly improved hepatic lipid accumulation induced by a chronic high-fat diet.

[0057] Animal experiments have shown that the Chinese herbal combination formulas TCM2 and TCM5 have similar effects, such as Figure 7 As shown in Figure A, TCM5 administration at the same time significantly inhibited the weight gain induced by a high-fat diet, while TCM2 significantly reduced the weight gain of mice induced by a chronic high-fat diet; Figure 7 Data from B-1 to B-5 showed that the liver index of the TCM2 pre-administration group increased significantly, indicating that the formula can significantly reduce liver enlargement caused by chronic high-fat diet; Figure 7 Results from C-1 to C-10 showed that concurrent administration of TCM2 significantly reduced the increase in white blood cell (WBC) count in mice induced by a high-fat diet (HFD); pre-administration significantly reduced the number of WBC, lymphocytes (LY) and platelets (PLT) in mice; and post-administration significantly reduced the number of LY, confirming that TCM2 has a significant improvement effect on the whole blood cell disorder caused by a chronic high-fat diet. Figure 8 The results showed that pre-administration and post-administration of TCM2 could alleviate HFD-induced fat vacuolation, while the effect of concurrent administration did not reach a significant level. Overall, it was confirmed that TCM2 has a significant improvement effect on hepatic lipid accumulation caused by chronic high-fat diet.

[0058] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A Chinese medicine composition for improving dyslipidemia, characterized in that: It is composed of one of the following two formulas: A or B: A: Astragalus, Ganoderma lucidum, and Schisandra chinensis, the weight ratio of the three being 1:1:1; B: Schisandra chinensis and Licorice root are composed in a weight ratio of 3:

1.

2. The method for preparing the Chinese medicine composition according to claim 1, wherein The following steps are involved: S1. Grind the raw materials into 80-100 mesh respectively, mix them, add 6 times the mass of distilled water, and reflux extract at 85-90℃ for 2 times, each time for 2 hours, and combine the extracts; S2, concentrating the extract under reduced pressure at 45-50° C. to obtain a concentrated solution; S3. Freeze the concentrated solution at -80°C overnight, and then freeze-dry to obtain freeze-dried powder of the traditional Chinese medicine composition.

3. The method for preparing the Chinese medicine composition as claimed in claim 2, characterized in that: The concentrated liquid after the reduced pressure concentration in step S2 needs to be subjected to nano-processing, specifically as follows: After adjusting the pH value of the concentrate to 5.5-6.0, the concentrate was circulated 5-8 times at a pressure of 200-250 MPa using high-pressure microfluidization technology to form a nanosuspension. In the nanosuspension, astragalus polysaccharide, Ganoderma lucidum triterpenes and schisandrin in formula A or schisandrin and glycyrrhizic acid in formula B form stable micelles through self-assembly.

4. The method for preparing the Chinese medicine composition according to claim 3, wherein: The method for forming stable micelles of astragalus polysaccharide, ganoderma triterpenes and schisandrin in formula A or schisandrin and glycyrrhizic acid in formula B comprises the following steps: The concentrated solution after reduced pressure concentration was adjusted to a pH of 5.5-6.0, and stirred at a constant temperature of 45-50° C. for 30 min to obtain a mixed solution; The mixed solution was cooled to 25°C at a rate of 0.5-2°C / min, and a sodium chloride solution accounting for 0.1% of the volume of the concentrated solution was added; Then, the samples were sonicated at 40 kHz and 100 W for 5 min; High-pressure microfluidization technology was then used for shearing treatment to control the micelle particle size to 20-50 nm to obtain a nanosuspension. During this process, the temperature was maintained at 25±2°C.

5. The method for preparing the Chinese medicine composition according to claim 4, wherein: The concentration of the sodium chloride solution is 0.05-0.15 wt%.

6. The method for preparing the Chinese medicine composition according to claim 4, wherein: The method also includes spray drying the nanosuspension with an inlet temperature of 160-170°C and an outlet temperature of 70-75°C.

7. The Chinese medicine composition according to claim 1, wherein The preparation forms are tablets, capsules, granules, injections, aerosols, suppositories, patches, ointments, oral liquid preparations or powders.

8. The Chinese medicine composition according to claim 7, characterized in that The tablets are enteric-coated tablets, sustained-release tablets or effervescent tablets, the capsules are hard capsules or soft capsules, and the oral liquid preparations are syrups, mixtures or tinctures.

9. Use of the traditional Chinese medicine composition according to claim 1 in preparing medicines for treating fatty liver and hyperlipidemia.

10. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of a medicament for improving dyslipidemia caused by a chronic high-fat diet.

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