Traditional Chinese medicine composition for treating atherosclerosis as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines and disease treatment, and particularly relates to a traditional Chinese medicine composition for treating atherosclerosis and a preparation method and application thereof. The composition is prepared by taking rhizoma pinellinae praeparata, rhizoma coptidis, pericarpium trichosanthis, radix angelicae sinensis, rhizoma chuanxiong, ground beeltle, radix salviae miltiorrhizae and radix ginseng as raw medicinal materials and adding sucralose and maltodextrin. Wherein berberine and palmatine in coptis chinensis are core active components, and play a role in regulating blood fat, inhibiting inflammation and blocking foam cell formation. Researches show that the particles can significantly reduce the serum total cholesterol, triglyceride and low-density lipoprotein cholesterol levels of model animals, increase the high-density lipoprotein cholesterol level, reduce the aortic plaque area and inhibit RAW264.7 cell foaming, the curative effect is equivalent to that of simvastatin, and a new choice is provided for prevention and treatment of atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine composition technology, specifically relating to a traditional Chinese medicine composition for treating atherosclerosis, its preparation method, and its application. Background Technology

[0002] Atherosclerosis (AS) is the most common degenerative disease of the cardiovascular system. Its pathogenesis is complex, involving the synergistic effects of multiple factors, including endothelial dysfunction, lipid metabolism disorders, chronic inflammatory responses, and oxidative stress. Early lesions are characterized by lipid deposition in the vascular intima and inflammatory cell infiltration. As the disease progresses, abnormal proliferation of vascular smooth muscle cells, foam cell formation, and fibrosis occur, ultimately leading to thickening of the vessel wall and narrowing of the lumen, inducing serious cardiovascular and cerebrovascular events such as myocardial infarction and cerebral infarction. Among these factors, lipid metabolism disorders and abnormal release of inflammatory factors are the core driving factors of disease progression. Therefore, regulating lipid metabolism, inhibiting inflammatory responses, and blocking foam cell formation have become key targets for AS treatment.

[0003] While statins, commonly used in clinical practice, can lower blood lipid levels and slow plaque progression, long-term use can easily lead to adverse reactions such as liver and kidney damage and muscle toxicity, limiting their clinical application. Traditional Chinese medicine (TCM), based on the principles of "holistic view and syndrome differentiation and treatment," demonstrates unique advantages in the treatment of ankylosing spondylitis (AS) through safety and multi-target regulation. Huoxue Huatan Granules, derived from the TCM theory of "treating phlegm and blood stasis simultaneously," are composed of herbs such as Pinellia ternata, Coptis chinensis, Trichosanthes kirilowii peel, Angelica sinensis, Ligusticum chuanxiong, Eupolyphaga sinensis, Salvia miltiorrhiza, and Panax ginseng. They possess the effects of clearing heat and resolving phlegm, breaking up blood stasis and eliminating masses, and invigorating qi and unblocking collaterals. Clinical studies have confirmed their efficacy in treating phlegm-blood stasis syndromes. However, systematic reports on the product application, mechanism of action, and quality control standards of these granules in AS treatment have not yet been found, necessitating further in-depth research and development. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes a traditional Chinese medicine composition for treating atherosclerosis, its preparation method, and its application. This application is based on apolipoprotein E gene knockout mice induced by a high-fat diet (ApoE). - / - The study was conducted using mouse atherosclerosis models and oxidized low-density lipoprotein (ox-LDL)-induced RAW264.7 cell foaming models. Simvastatin was used as a positive control drug for efficacy comparison and verification. Based on the TCM theory of "treating phlegm and blood stasis together", the drug achieved multi-target regulation and showed excellent performance in terms of safety and efficacy stability, which can meet the application needs of clinical treatment of atherosclerosis.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A traditional Chinese medicine composition for treating atherosclerosis, said composition is prepared from the following raw materials in parts by weight: Raw medicinal materials: 8-10 parts of Pinellia ternata, 5-7 parts of Coptis chinensis, 13-17 parts of Trichosanthes kirilowii peel, 9-11 parts of Angelica sinensis, 13-17 parts of Ligusticum chuanxiong, 9-11 parts of Eupolyphaga sinensis, 27-33 parts of Salvia miltiorrhiza, and 9-11 parts of Panax ginseng; Excipients: sucralose and maltodextrin, wherein the amount of sucralose is 0.25%-0.28% of the total weight of the raw materials, and the amount of maltodextrin is 1.0-1.3 times the total weight of the raw materials; The raw medicinal materials are decocted in water, concentrated and dried, and then mixed with excipients to form granules to obtain the blood-activating and phlegm-resolving granules.

[0006] Preferably, the traditional Chinese medicine composition is used in the preparation of a drug for treating atherosclerosis, wherein the traditional Chinese medicine composition is the main active ingredient of the drug.

[0007] Preferably, the dosage form of the drug is an oral dosage form, selected from one or more of granules, tablets, capsules, pills and oral liquids.

[0008] Preferably, the carrier used in preparing the drug is selected from one or more of water, ethanol, buffer solution, cellulose, starch, sucrose and lactose; the carrier is used for the dissolution, dispersion or shaping of the composition.

[0009] Preferably, when preparing the tablets and capsules, one or more of stearic acid and micronized silica gel may be added as suitable excipients.

[0010] Preferably, when the drug is in granule form, each 1 g contains 1-5 mg of berberine derived from Coptis chinensis, and the berberine is determined by high performance liquid chromatography.

[0011] Preferably, when the drug is a tablet, the weight percentage of the blood-activating and phlegm-reducing granules is 50%-80%, and it is prepared with the help of starch and stearic acid.

[0012] Due to the adoption of the above technical solution, the beneficial effects of this invention are: this invention expands the application field of blood-activating and phlegm-resolving granules, provides a new use for them in the preparation of drugs for treating atherosclerosis, and thus constructs a new traditional Chinese medicine treatment plan for atherosclerosis. Based on clinical needs and existing basic research theories, this invention constructs a high-fat diet-induced Apoe - / -This study established an in vivo mouse model of atherosclerosis and an in vitro model of oxLDL-induced foaming in RAW264.7 cells. The model animals and cells were treated with low-dose (15.9 g crude drug / kg) and high-dose (47.7 g crude drug / kg) administration of Huoxue Huatan granules, with simvastatin as a positive control. Oil Red O staining of the aorta and cells was used to analyze the effects of the granules on inhibiting atherosclerotic plaque formation and cell foaming. Simultaneously, the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in mouse serum were measured to comprehensively clarify the preventive and therapeutic effects of Huoxue Huatan granules on atherosclerosis. Attached Figure Description

[0013] Figure 1 This is a flowchart of the preparation process of the blood-activating and phlegm-resolving granules of the present invention; Figure 2 For each group of Apoe - / - Oil Red O staining results of mouse aorta: Control (normal diet), HF (high-fat diet), HHG (treated with blood-activating and phlegm-reducing granules), SV (simvastatin); Figure 3 For each group of Apoe - / - Statistical results of serum TC, TG, LDL-C, and HDL-C levels in mice; Figure 4 Oil Red O staining results and statistical analysis of RAW264.7 cells in each group: Control (control group), Model (50 μg / mL ox-LDL), HHG-L (2.5% HHG-containing serum), HHG-M (5% HHG-containing serum), HHG-H (10% HHG-containing serum); Figure 5 This is a high-performance liquid chromatogram of Coptis chinensis (berberine, palmatine) in the blood-activating and phlegm-resolving granules of the present invention. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0015] Example 1: This Example 1 describes a traditional Chinese medicine composition for treating atherosclerosis, prepared from the following raw materials in parts by weight: Raw medicinal materials: 9 parts of Pinellia ternata, 6 parts of Coptis chinensis, 15 parts of Trichosanthes kirilowii peel, 10 parts of Angelica sinensis, 15 parts of Ligusticum chuanxiong, 10 parts of Eupolyphaga sinensis, 30 parts of Salvia miltiorrhiza, and 10 parts of Panax ginseng; Excipients: sucralose and maltodextrin, wherein the amount of sucralose is 0.25% of the total weight of the raw materials and the amount of maltodextrin is 1.0 times the total weight of the raw materials; This embodiment describes a method for preparing a traditional Chinese medicine composition for treating atherosclerosis, with the following specific preparation steps: S1. Weigh the raw medicinal materials, namely Pinellia ternata, Coptis chinensis, Trichosanthes kirilowii peel, Angelica sinensis, Ligusticum chuanxiong, Eupolyphaga sinensis, Salvia miltiorrhiza, and Panax ginseng. Grind all the medicinal materials into powder and pass them through a 20-mesh sieve for later use. S2. Add 6 times the amount of water to the pulverized medicinal materials, decoct for 30 minutes, and filter with gauze to obtain decoction I; S3. Collect the dregs from S2, add 4 times the amount of water, continue to decoct for 20 minutes, and filter with gauze to obtain decoction II; S4. Combine decoction I and decoction II, concentrate the combined decoction to a relative density of 1.20 (60℃), and spray dry (inlet air temperature 170℃, outlet air temperature 80℃) to obtain a dry extract with a moisture content ≤5%. S5. Add sucralose and maltodextrin to the dry extract, mix well, and then granulate. S6. Dry the prepared wet granules at 55℃ for 45 min until the granule moisture content is 3%-5%, and then pass them through a 16-mesh sieve to granulate them, thus obtaining the blood-activating and phlegm-resolving granules (HHG).

[0016] Example 2: Blood-activating and phlegm-resolving granules inhibit Apoe induced by high-fat diet in vivo. - / - Atherosclerotic lesions in mice: S1. Animal grouping and model establishment: Eight-week-old male Apoe were selected - / - Fifty mice were randomly divided into five groups of 10 each. Control group: fed with regular feed; Model group: fed a high-fat diet; Low-dose group of Huoxue Huatan granules: fed with high-fat diet and administered Huoxue Huatan granules (15.9 g crude drug / kg) by gavage. High-dose group of Huoxue Huatan granules: fed with high-fat diet and administered Huoxue Huatan granules (47.7 g crude drug / kg) by gavage. Simvastatin positive control group: fed a high-fat diet and administered simvastatin (5 mg / kg) by gavage. All mice were fed for 16 weeks to establish an atherosclerosis model; S2, drug intervention After 16 weeks of modeling, each drug group (groups 3, 4, and 5 in S1) began to receive the corresponding drugs once a day by gavage, while the control group (group 1 in S1) and the model group (group 2 in S1) were given an equal volume of physiological saline by gavage. The intervention continued for 8 weeks. S3. Sample Collection and Processing After the last administration, mice were fasted for 12 hours but allowed free access to water. Blood was then collected via abdominal aortic puncture after anesthesia. The blood samples were allowed to stand at 4°C for 2 hours, then centrifuged at 3000 rpm for 15 minutes. The supernatant was used as the serum sample and stored at -80°C for later testing. At the same time, mouse aortic tissue was collected.

[0017] S4. Aortic Oil Red O staining and plaque area analysis The collected mouse aortic tissue was stained with Oil Red O, and the area of ​​atherosclerotic plaques was observed and statistically analyzed. S5, Serum lipid level detection Strictly follow the instructions of the test kit to detect the expression levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in serum samples; Example 3: The blood-activating and phlegm-resolving granules inhibited oxLDL-induced foaming in RAW264.7 cells in vitro. S1. Preparation of drug-containing serum Twenty male SD rats were randomly divided into two groups of 10 each. Blank serum group: Double distilled water was administered by gavage twice daily for 7 consecutive days; HHG drug-containing serum group: 47.7 g crude drug / kg of Huoxue Huatan granules solution was administered by gavage twice daily for 7 consecutive days.

[0018] One hour after the last administration, rats were anesthetized and blood was collected by puncture of the abdominal aorta. The serum was separated, inactivated, and stored at -80°C for later use.

[0019] S2, RAW264.7 cell culture and grouping RAW264.7 cells were divided into 3 groups for culture: Control group: Routine culture, without the addition of ox-LDL and drug-containing serum; ox-LDL group (model group): 50 μg / mL ox-LDL was added to induce cell foaming; HHG-containing serum group: 50 μg / mL ox-LDL was added, and HHG-containing serum at concentrations of 2.5%, 5%, and 10% was added at the same time; All cells were cultured for 48 h. S3, Oil Red O staining and observation Cells from each group were stained with Oil Red O after 48 h of culture to observe and analyze intracellular lipid droplet deposition. Results are shown in [Table / Reference]. Figure 4 ; Example 4: Preparation and evaluation of granules: S1. Determine the composition of granule raw materials. 100 g of dry extract of blood-activating and phlegm-resolving granules, 125 g of maltodextrin, and sucralose (0.25% of the total weight of the raw materials). S2, Preparation of Granules The preparation method of the granules in this embodiment is the same as that in Example 1. The dried granules are packaged into 11 g / bags to obtain granules for the prevention and treatment of atherosclerosis. S3. In vivo experimental evaluation Apoe is established according to the method in Example 2. - / - A mouse model of atherosclerosis was established by administering different doses of the above-mentioned granules via gavage to the model mice. Serum lipid levels (TC, TG, LDL-C, HDL-C) and aortic plaque area were measured to evaluate the in vivo anti-atherosclerotic effect of the granules. S4. In vitro experimental evaluation A foaming model of RAW264.7 cells induced by oxLDL was established. Drug-containing serum was added to the model cells for intervention. Intracellular lipid droplet deposition was detected to evaluate the effect of the granules on inhibiting cell foaming in vitro. S5. Results and Conclusions In vitro and in vivo experimental results show that the granules prepared in this invention with blood-activating and phlegm-resolving granules as the active ingredient have significant anti-atherosclerotic effects, confirming that blood-activating and phlegm-resolving granules have the ability to treat atherosclerosis, and can provide a material basis for the research and development of related preventive and therapeutic drugs.

[0020] Performance testing 1. In vitro pharmacodynamic testing: Inhibition of cell foaming experiment (refer to Example 3) Test Groups: Twenty male SD rats were randomly divided into two groups of 10 each. Control group: RAW264.7 cells were cultured routinely without ox-LDL or drug-containing serum; Model group: 50 μg / mL ox-LDL was added to induce cell foaming; Experimental group: 50 μg / mL ox-LDL + 2.5%, 5%, and 10% HHG-containing serum were added (concentration gradients were set up to verify dose-effect relationship); Test steps: S1. RAW264.7 cells were seeded in 6-well plates at a density of 5 × 10⁶ cells / well. 5 Cells / well, incubated at 37°C in a 5% CO2 incubator for 24 h; S2. Add the corresponding reagents according to the groups and continue culturing for 48 h; S3. After the culture is completed, discard the culture medium, wash twice with PBS buffer, and fix with 4% paraformaldehyde for 30 min. S4. Follow the instructions for the Oil Red O staining kit. After staining, observe the intracellular lipid droplet deposition under a microscope and count the percentage of positive lipid droplet area. The lower the value, the better the inhibition of foaming.

[0021] Results analysis: Compared with the control group, the intracellular lipid droplet deposition in the ox-LDL group was significantly increased (#p<0.05), indicating that the cell foaming model was successfully constructed. Compared with the model group, HHG-containing serum treatment significantly reduced intracellular lipid droplet deposition in a concentration-dependent manner, suggesting that Huoxue Huatan granules can inhibit oxLDL-induced RAW264.7 cell foaming, thereby inhibiting the progression of atherosclerosis.

[0022] 2. In vivo pharmacodynamic testing: Animal experiments on anti-atherosclerosis (corresponding to Example 2) Test Groups: Eight-week-old male Apoe were selected - / - Fifty mice were randomly divided into five groups of 10 each. Control group: fed with normal feed and administered an equal volume of physiological saline by gavage; Model group: An atherosclerosis model was established by feeding a high-fat diet for 16 weeks and then administering an equal volume of physiological saline by gavage; HHG low-dose group: After modeling on a high-fat diet, HHG solution of 15.9 g crude drug / kg was administered by gavage; HHG high-dose group: After modeling on a high-fat diet, HHG solution of 47.7 g crude drug / kg was administered by gavage; Positive control group: After modeling on a high-fat diet, simvastatin solution of 5 mg / kg was administered by gavage; Test steps: S1. Modeling phase: The control group was fed a normal diet, while the other groups were fed a high-fat diet for 16 consecutive weeks. S2, Drug administration phase: After successful modeling, each drug group was administered the drug once daily by gavage, while the control group and model group were administered an equal volume of physiological saline by gavage. This intervention continued for 8 weeks. S3. Sample collection: After the last administration, mice were fasted but allowed to drink water for 12 hours. Blood was collected from the abdominal aorta after anesthesia, incubated at 4°C for 2 hours, centrifuged at 3000 rpm for 15 minutes, and the serum was separated (stored at -80°C). The aortic tissue of the mice was dissected and separated. Results analysis: (1) Compared with the model group, the aortic plaque area was significantly reduced after treatment with Huoxue Huatan granules (p<0.05), indicating that Huoxue Huatan granules can significantly inhibit atherosclerotic lesions in mice. The results are shown in the figure. Figure 2 ; (2) Compared with the control group, the serum TC, TG, and LDL-C levels in the model group were significantly increased, while the HDL-C level was significantly decreased (#p<0.05). Compared with the model group, both low-dose and high-dose treatment with Huoxue Huatan Granules significantly reduced the serum TC, TG, and LDL-C levels in mice and significantly increased the HDL-C level (*p<0.05), with the high-dose group showing a more significant effect, comparable to the simvastatin group. The results are shown in the table below. Figure 3 .

[0023] 3. Determination of active ingredient content (HPLC method): Determination of berberine hydrochloride and palmatine content in Coptis chinensis. Preparation of reference solutions: Accurately weigh 10.0 mg of berberine hydrochloride reference standard and 5.0 mg of palmatine hydrochloride reference standard, place them in a 100 mL volumetric flask, dissolve them in methanol and dilute to the mark, shake well to obtain a 100 μg / mL berberine hydrochloride reference standard stock solution and a 50 μg / mL palmatine hydrochloride reference standard stock solution; accurately measure 1 mL of each of the above stock solutions, place them in a 10 mL volumetric flask, dilute to the mark with methanol, shake well to obtain a mixed reference standard working solution (10 μg / mL berberine hydrochloride and 5 μg / mL palmatine hydrochloride), filter through a 0.45 μm organic phase membrane for later use; Preparation of test solution: Accurately weigh 1.0 g of the blood-activating and phlegm-resolving granules sample and place it in a 50 mL centrifuge tube. Add 25 mL of methanol and sonicate (500 W power, 40 kHz frequency) for 30 min. After cooling to room temperature, centrifuge at 5000 rpm for 10 min. Take the supernatant and place it in a 50 mL volumetric flask. Dilute to the mark with methanol and shake well. Accurately measure 2 mL of the supernatant and place it in a 10 mL volumetric flask. Dilute to the mark with methanol and filter through a 0.45 μm organic phase filter membrane to obtain the test solution. Chromatographic conditions: An Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm) was used as the separation column. The mobile phase was acetonitrile-0.05 mol / L potassium dihydrogen phosphate solution (containing 0.02 mol / L SDS) (volume ratio 40:60, pH adjusted to 4.0 with phosphoric acid). The flow rate was set at 1.0 mL / min, the detection wavelength was 345 nm, the column temperature was controlled at 30℃, and the injection volume was 10 μL. Standard curve: Accurately measure 0.2 mL, 0.5 mL, 1.0 mL, 2.0 mL, and 5.0 mL of the mixed reference standard stock solution and place them in five 10 mL volumetric flasks respectively. Dilute to the mark with methanol and shake well to obtain a series of mixed reference standard solutions of different concentrations. Under the chromatographic conditions set above, accurately pipette 10 μL of each concentration of the mixed reference standard solution and inject it into the high-performance liquid chromatograph. Record the chromatogram and peak area. Perform linear regression analysis with the concentration of each reference standard as the abscissa and the corresponding peak area as the ordinate to plot the standard curve. Sample content test: Take 3 batches of test solution, inject and analyze them under the above chromatographic conditions, record the peak area, and substitute it into the standard curve to calculate the content of berberine hydrochloride and palmatine hydrochloride per gram of granules.

[0024] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A traditional Chinese medicine composition for treating atherosclerosis, characterized in that, The traditional Chinese medicine composition is blood-activating and phlegm-resolving granules prepared from the following raw materials by weight: Raw medicinal materials: 8-10 parts of Baijiangxi, 5-7 parts of Huanglian, 13-17 parts of Gualoupi, 9-11 parts of Danggui, 13-17 parts of Chuanqiong, 9-11 parts of Tuobiechong, 27-33 parts of Danshen, and 9-11 parts of Renshen; Auxiliary materials: sucralose and maltodextrin, wherein the amount of sucralose is 0.25%-0.28% of the total weight of raw medicinal materials, and the amount of maltodextrin is 1.0-1.3 times the total weight of raw medicinal materials; The raw medicinal materials are decocted with water, concentrated and dried, and then mixed with auxiliary materials to granulate to obtain the blood-activating and phlegm-resolving granules.

2. The Chinese medicinal composition for treating atherosclerosis according to claim 1, characterized in that, The traditional Chinese medicine composition is used in the preparation of a medicine for treating atherosclerosis, and is the main active ingredient of the medicine.

3. The use of a traditional Chinese medicine composition for treating atherosclerosis according to claim 2, characterized in that, The dosage form of the medicine is an oral dosage form, selected from one or more of granules, tablets, capsules, pills, and oral liquids.

4. The use of a traditional Chinese medicine composition for treating atherosclerosis according to claim 2, characterized in that, The carrier used in the preparation of the medicine is selected from one or more of water, ethanol, buffer, cellulose, starch, sucrose, and lactose; and the carrier is used for the dissolution, dispersion, or molding of the composition.

5. The use of a traditional Chinese medicine composition for treating atherosclerosis according to claim 2, characterized in that, In the preparation of the tablet and capsule medicines, one or more of stearic acid and micronized silica gel can be additionally added as suitable auxiliary materials.

6. The use of a traditional Chinese medicine composition for treating atherosclerosis according to claim 3, characterized in that, When the medicine is in the form of granules, 1-5 mg of berberine from Huanglian is contained in 1 g, and the berberine is determined by high-performance liquid chromatography.

7. The use of a traditional Chinese medicine composition for treating atherosclerosis according to claim 3, characterized in that, When the medicine is in the form of tablets, the blood-activating and phlegm-resolving granules account for 50%-80% by weight, and are prepared with the addition of starch and stearic acid.

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