A traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response, and a preparation method and application thereof

Abstract

The present application belongs to the technical field of pharmaceutical compositions, and provides a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response, and a preparation method and application thereof.The traditional Chinese medicine composition provided by the present application uses Radix Bupleuri, Radix Paeoniae Alba, Fructus Aurantii Immaturus Stir-baked with Wheat Bran, Radix Angelicae Sinensis, Semen Persicae, Flos Carthami, Rhizoma Chuanxiong, Radix Rehmanniae, Fructus Gardeniae Charred, Radix Paeoniae Alba Stir-baked with Wheat Bran, Poria, Hirudo, Lumbricus, and Radix Glycyrrhizae as main raw materials, can inhibit serum inflammation and reduce blood lipid level, improve chronic inflammatory state of the body, stabilize atherosclerotic plaque and delay pathological progression, reduce the risk of acute cardiovascular and cerebrovascular accidents, can also relieve symptoms and signs such as chest distress, chest pain, emotional discomfort, discomfort of the lateral ribs, pale tongue with blood stasis spots, and wiry and astringent pulse of atherosclerosis and coronary heart disease patients, and improve the quality of life.In addition, the traditional Chinese medicine composition provided by the present application also has the characteristics of simple preparation, low cost, safety, no toxic side effects, easy to carry, convenient to take and the like.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical composition technology, and more specifically, to a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response, its preparation method, and its application. Background Technology

[0002] Modern medicine's treatment of atherosclerosis (AS) and coronary heart disease mainly includes controlling risk factors (such as smoking cessation, dietary control, regulating blood lipids, blood pressure, and blood sugar) and using preventive and therapeutic drugs. Currently, commonly used preventive and therapeutic drugs include statins, lipid-lowering drugs, and antiplatelet drugs (such as aspirin, clopidogrel, and ticagrelor). Among these, statins have side effects such as liver and kidney damage, muscle pain, and rhabdomyolysis, limiting their use and leading to poor patient adherence. Fibrates, lipid-lowering drugs, increase the risk of gallstones, elevated liver transaminases, and cause myalgia, myositis, and gastrointestinal discomfort. Furthermore, even with high-intensity use of statins and lipid-lowering drugs, 50% of patients still experience adverse progression of AS. Antiplatelet drugs inhibit platelet aggregation, but can lead to decreased hemostasis, potentially causing internal bleeding such as gastrointestinal bleeding and cerebral hemorrhage, even endangering life. Common adverse reactions include peptic ulcers, gastrointestinal discomfort, liver and kidney damage, and allergic reactions. Furthermore, the current strategy of using colchicine for anti-inflammatory treatment in AS has been studied by multiple clinical studies, and its side effects are not negligible. Colchicine is prone to poisoning, and its combined use with various foods and drugs should be avoided. Patients with impaired liver and kidney function should also use it with caution, as the incidence of gastrointestinal adverse reactions is as high as 10%.

[0003] As can be seen from the above, AS, as a chronic disease, requires long-term medication. Although chemotherapeutic drugs have some clinical efficacy, they are difficult to effectively stop the progression of AS, and the treatment is costly, accompanied by significant side effects, leading to poor patient compliance, which in turn affects drug efficacy and results in poor treatment outcomes. In terms of traditional Chinese medicine, although there are anti-atherosclerotic Chinese herbal preparations used clinically, their main mechanism of action is still primarily through regulating blood lipids. Regarding the selection of prescriptions and medications, current TCM prescription approaches and treatment methods still mainly focus on simple blood-activating and phlegm-resolving methods, with insufficient clinical efficacy. Most Chinese herbal preparations for the prevention and treatment of AS neglect the role of mental and emotional stress in AS.

[0004] Therefore, there is an urgent need to develop a traditional Chinese medicine preparation that can treat atherosclerosis and improve inflammatory response with significant efficacy and few side effects. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory responses, its preparation method, and its application. The traditional Chinese medicine composition provided by this invention uses liver-regulating therapy as its core treatment principle, and is used to inhibit chronic inflammation, enhance the stability of atherosclerotic plaques, and has a significant therapeutic effect on atherosclerosis. Moreover, the main raw materials used are all traditional Chinese medicinal herbs, resulting in minimal side effects on the human body.

[0006] The first aspect of the present invention provides a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response.

[0007] Specifically, a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory responses includes the following ingredients:

[0008] Bupleurum, white peony root, stir-fried bitter orange peel, angelica, peach kernel, safflower, chuanxiong rhizome, rehmannia root, charred gardenia fruit, stir-fried atractylodes rhizome, poria cocos, leech, earthworm, and prepared licorice root.

[0009] The traditional Chinese medicine composition provided by this invention mainly uses the principles of regulating liver qi, removing blood stasis and phlegm, and unblocking the heart vessels to inhibit inflammation, regulate blood lipids, and enhance the stability of AS plaques. Among them, Bupleurum and stir-fried Citrus aurantium soothe the liver, regulate qi, relieve depression, and assist the liver's function; Angelica sinensis and Paeonia lactiflora nourish blood, soften the liver, and replenish the liver's body; Prunus persica, Carthamus tinctorius, and Ligusticum chuanxiong invigorate blood and remove blood stasis; Rehmannia glutinosa and stir-fried Gardenia jasminoides nourish yin, clear heat, and cool blood; stir-fried Atractylodes macrocephala and Poria cocos strengthen the spleen and remove phlegm and dampness; and roasted Glycyrrhiza uralensis harmonizes the various herbs. In the course of AS vulnerable plaques promoted by emotional stress, although the location of the disease is in the "heart", it is inseparable from the "liver". The pathogenesis is "stagnation of liver qi leads to deficiency of heart qi", and "liver dysfunction" is the key pivot in the pathogenesis: "liver governs the free flow of qi" is the most important in emotional regulation. Long-term chronic stress can easily cause "liver dysfunction", which can induce emotional abnormalities. "Worry, fear and anxiety injure the heart", thus interfering with the function of "heart governs the mind", which in turn affects the function of "heart governs blood vessels". The heart is unable to regulate blood circulation and blood vessels normally. Insufficient blood circulation cannot nourish the "mind". The dysfunction of the two forms a vicious cycle, which aggravates the damage to blood vessels and poor blood circulation, thereby promoting the course of AS. In response to the above, this invention, based on the principle that "when liver qi flows smoothly, heart qi is harmonious; when liver qi stagnates, heart qi is deficient; therefore, heart disease should first be addressed by clearing the liver and addressing its source," focuses on the key pathogenesis of "liver qi stagnation." Combining this with the disease's progression of "liver qi stagnation—stagnation leading to heat—qi stagnation leading to phlegm and blood stasis," the following compound formula is derived: Bupleurum and white peony root are used as the principal herbs, combined with the assistant herbs stir-fried bitter orange peel and angelica root to soothe the liver, regulate qi, relieve stagnation, assist "liver function," nourish blood, soften the liver, and replenish "liver constitution." The assistant herbs Poria and stir-fried atractylodes macrocephala strengthen the spleen and eliminate phlegm. The formula addresses dampness by using adjuvant herbs such as peach kernel, safflower, and chuanxiong to invigorate blood, remove blood stasis, and relieve pain. It also uses raw rehmannia root and charred gardenia fruit to nourish yin, clear heat, and cool the blood. Leeches and earthworms are guided to the liver meridian and collaterals to expel blood stasis and invigorate the collaterals. Prepared licorice root harmonizes the other herbs. The core treatment principle of this formula is to regulate the key pathogenesis of "liver dysfunction." First, it soothes and softens the liver to regulate qi, thus ensuring "liver qi flows smoothly." Then, it combines blood-activating, phlegm-resolving, and blood-cooling methods to achieve "heart qi harmony," ensuring unimpeded blood flow and unobstructed blood vessels. This helps to inhibit inflammation and stabilize AS plaques.

[0010] Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: Bupleurum chinense 5-25 parts, Paeonia lactiflora 5-25 parts, Citrus aurantium (fried with wheat bran) 5-20 parts, Angelica sinensis 5-20 parts, Prunus persica 5-25 parts, Carthamus tinctorius 5-25 parts, Ligusticum chuanxiong 5-20 parts, Rehmannia glutinosa 5-20 parts, Gardenia jasminoides (fried with wheat bran) 5-20 parts, Atractylodes macrocephala (fried with wheat bran) 5-20 parts, Poria cocos 7.5-30 parts, Hirudo medicinalis 1.5-8 parts, Pheretima aspergillum 5-20 parts, and Glycyrrhiza uralensis (processed with wheat bran) 3-12 parts.

[0011] More preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: Bupleurum chinense 10-25 parts, Paeonia lactiflora 10-25 parts, Citrus aurantium (fried with wheat bran) 10-20 parts, Angelica sinensis 10-20 parts, Prunus persica 10-25 parts, Carthamus tinctorius 10-25 parts, Ligusticum chuanxiong 10-20 parts, Rehmannia glutinosa 10-20 parts, Gardenia jasminoides (fried with wheat bran) 10-20 parts, Atractylodes macrocephala (fried with wheat bran) 15-30 parts, Hirudo medicinalis 3-8 parts, Pheretima aspergillum 10-20 parts, and Glycyrrhiza uralensis (processed with wheat bran) 6-12 parts.

[0012] More preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 10-20 parts Bupleurum chinense, 10-20 parts Paeonia lactiflora, 10-15 parts stir-fried Citrus aurantium, 10-15 parts Angelica sinensis, 10-20 parts Prunus persica, 10-20 parts Carthamus tinctorius, 10-15 parts Ligusticum chuanxiong, 10-15 parts Rehmannia glutinosa, 10-15 parts Gardenia jasminoides, 10-15 parts stir-fried Atractylodes macrocephala, 15-20 parts Poria cocos, 3-6 parts Hirudo medicinalis, 10-15 parts Pheretima aspergillum, and 6-8 parts prepared Glycyrrhiza uralensis.

[0013] Preferably, the traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response is a traditional Chinese medicine composition that treats vulnerable atherosclerotic plaques by lowering blood lipid levels and inhibiting inflammation within atherosclerotic plaques. The traditional Chinese medicine composition provided by the present invention can lower blood lipid levels and inhibit inflammation within atherosclerotic plaques, thereby treating vulnerable atherosclerotic plaques.

[0014] A second aspect of the present invention provides a method for preparing a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response.

[0015] A method for preparing a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response includes the following steps:

[0016] The raw materials are mixed with a solvent and extracted to obtain the traditional Chinese medicine composition.

[0017] Preferably, the solvent is water or ethanol.

[0018] Preferably, the amount of solvent used is 2-3 times the total mass of each raw material component.

[0019] Preferably, the extraction temperature is 70-100℃, and / or the extraction time is 10-60 minutes, and / or the extraction is performed 1-2 times.

[0020] A third aspect of the present invention provides the application of a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory responses.

[0021] The application of a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response in the preparation of drugs for treating atherosclerosis, coronary heart disease, hypertension, hyperlipidemia, diabetes, obesity or fatty liver.

[0022] Preferably, the traditional Chinese medicine composition is used in the preparation of drugs that inhibit inflammation within atherosclerotic plaques and improve the vulnerability of atherosclerotic plaques.

[0023] A fourth aspect of the present invention provides a medicament for treating atherosclerosis, coronary heart disease, hypertension, hyperlipidemia, diabetes, obesity, or fatty liver.

[0024] A medicine for treating atherosclerosis, coronary heart disease, hypertension, hyperlipidemia, diabetes, obesity, or fatty liver, comprising the aforementioned traditional Chinese medicine composition.

[0025] Preferably, the dosage form of the drug is at least one of pills, capsules, tablets, ointments, aerosols, suspensions, granules, powders, solutions, creams, lozenges, or films.

[0026] Preferably, the drug further includes excipients, which are one or more of dextrin, silicon dioxide, and flavoring agents.

[0027] Preferably, the drug is a drug that inhibits inflammation within atherosclerotic plaques and improves the vulnerability of atherosclerotic plaques.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] The traditional Chinese medicine composition provided by this invention uses Bupleurum chinense, Paeonia lactiflora, Citrus aurantium (fried with wheat bran), Angelica sinensis, Prunus persica, Carthamus tinctorius, Ligusticum chuanxiong, Rehmannia glutinosa, Gardenia jasminoides (fried with wheat bran), Atractylodes macrocephala (fried with wheat bran), Poria cocos, Hirudo medicinalis, Pheretima aspergillum, and Glycyrrhiza uralensis (processed) as main raw materials. It can inhibit serum inflammation and lower blood lipid levels, improve the body's chronic inflammatory state, stabilize atherosclerotic plaques and delay their pathological progression, reduce the risk of acute cardiovascular and cerebrovascular accidents, and alleviate symptoms and signs such as chest tightness, chest pain, emotional distress, hypochondriac discomfort, pale and dark tongue with ecchymosis, and wiry and hesitant pulse in patients with atherosclerosis and coronary heart disease, thus improving their quality of life. Furthermore, the traditional Chinese medicine composition provided by this invention is simple to prepare, low in cost, safe and non-toxic, easy to carry, and convenient to take. Attached Figure Description

[0030] Figure 1 Figure 1 shows the body weight (n=8) of mice in the Ctrl, HFD, LTG, MTG, HTG, and ATO groups at 16 weeks in animal experiments; compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, nsP>0.05;

[0031] Figure 2Figure 1 shows the serum TC, TG, LDL and HDL levels (n=8) in mice in the Ctrl, HFD, LTG, MTG, HTG and ATO groups in animal experiments; compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, ns P>0.05;

[0032] Figure 3 Figure 1 shows the serum TC, TG, LDL, and HDL levels (n=8) in mice in the Ctrl, HFD, MTG, and comparative groups 1-3. Compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, ns P>0.05; compared with the MTG group, ***P<0.001, **P<0.01, *P<0.05, ns P>0.05.

[0033] Figure 4 Serum TC, TG, LDL, and HDL levels in mice in the Ctrl, HFD, MTG, and Example 2-4 groups (n=8) were measured in animal experiments. Compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, ns P>0.05; compared with the MTG group, ***P<0.001, **P<0.01, *P<0.05, ns P>0.05.

[0034] Figure 5 Serum levels of inflammatory markers Lp(a), IL1β, IL18, IL6, and TNF-α in mice of the Ctrl, HFD, LTG, MTG, HTG, and ATO groups (n=8) were measured in animal experiments. Compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, and nsP>0.05.

[0035] Figure 6 Figure 1 shows the serum levels of inflammatory markers Lp(a), IL1β, IL18, IL6, and TNF-α in mice of the Ctrl, HFD, MTG, and comparative groups 1-3 (n=8) in animal experiments. Compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, nsP>0.05; compared with the MTG group, ***P<0.001, **P<0.01, *P<0.05, nsP>0.05.

[0036] Figure 7Figure 1 shows the serum levels of inflammatory markers Lp(a), IL1β, IL18, IL6, and TNF-α in mice of the Ctrl, Model, MTG, and Example 2-4 groups (n=8) during animal experiments. Compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, ns P>0.05; compared with the MTG group, ***P<0.001, **P<0.01, *P<0.05, ns P>0.05.

[0037] Figure 8 The image shows the Oil Red O staining area (n=8) of aortic sinus pathological sections from mice in the Ctrl, HFD, LTG, MTG, HTG, and ATO groups in animal experiments; compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, nsP>0.05;

[0038] Figure 9 The image shows the Oil Red O staining area (n=8) of aortic sinus pathological sections from mice in the Ctrl, HFD, MTG, and Comparative Examples 1-3 groups during animal experiments. Compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, ns P>0.05; compared with the MTG group, ***P<0.001, **P<0.01, *P<0.05, ns P>0.05.

[0039] Figure 10 The image shows the Oil Red O staining area (n=8) of aortic sinus pathological sections from the Ctrl, HFD, MTG, and Example 2-4 groups in animal experiments. Compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, ns P>0.05; compared with the MTG group, ***P<0.001, **P<0.01, *P<0.05, ns P>0.05.

[0040] Figure 11 Figure 1 shows the area (n=8) of HE staining (hematoxylin-eosin staining method) of aortic sinus pathological sections in mice of the Ctrl, HFD, LTG, MTG, HTG, and ATO groups in animal experiments. Compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, ns P>0.05;

[0041] Figure 12Figure 1 shows the HE staining area (n=8) of aortic sinus pathological sections from mice in the Ctrl, HFD, MTG, and Comparative Examples 1-3 groups during animal experiments. Compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, ns P>0.05; compared with the MTG group, ***P<0.001, **P<0.01, *P<0.05, ns P>0.05.

[0042] Figure 13 Figure 1 shows the HE staining area (n=8) of aortic sinus pathological sections from mice in the Ctrl, HFD, MTG, and Example 2-4 groups during animal experiments. Compared with the HFD group, ###P<0.001, ##P<0.01, #P<0.05, ns P>0.05; compared with the MTG group, ***P<0.001, **P<0.01, *P<0.05, ns P>0.05.

[0043] Figure 14 The images show the results of multiple immunofluorescence staining of the aortic sinus in mice of the Ctrl, HFD, LTG, MTG, HTG, and ATO groups in animal experiments (CD68 / NLRP3 / ASC).

[0044] Figure 15 The image shows the Oil Red O staining results (n=3) of RAW macrophages in the Ctrl, Model, LTG, MTG, HTG, and siCD36 groups during cell experiments.

[0045] Figure 16 Figure 1 shows the flow cytometry results of RAW macrophages in the Ctrl, Model, LTG, MTG, HTG, and siCD36 groups (n=3) during cell experiments. Compared with the Model group, ###P<0.001, ##P<0.01, #P<0.05, nsP>0.05;

[0046] Figure 17 Figure 1 shows the levels of IL1β, IL18, IL6, and TNF-α inflammatory markers in RAW macrophages from the Ctrl, Model, LTG, MTG, HTG, and siCD36 groups (n=3) in cell experiments; compared with the Model group, ###P<0.001, ##P<0.01, #P<0.05, nsP>0.05;

[0047] Figure 18 This figure shows the expression level of NLRP3 inflammasome in RAW macrophages in the Ctrl, Model, LTG, MTG, HTG, and siCD36 groups, as detected by immunofluorescence in cell experiments. Detailed Implementation

[0048] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0049] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0050] Example 1

[0051] A traditional Chinese medicine composition comprising the raw material components and dosages shown in Table 1.

[0052] The preparation method of the above-mentioned traditional Chinese medicine composition includes the following steps:

[0053] (1) Weigh the raw materials, remove impurities, wash and dry them. Among them, Atractylodes macrocephala and Citrus aurantium are stir-fried with wheat bran, Gardenia jasminoides is charred, and the rest of the Chinese herbs are used as conventional decoction pieces;

[0054] (2) Stir-frying Atractylodes macrocephala with wheat bran: Use dried Atractylodes macrocephala slices of uniform thickness. The weight ratio of wheat bran to Atractylodes macrocephala is 1000g:100g to 1000g:200g (10:1 to 10:2). First, preheat the wok. Place the wok on the fire and heat it over medium heat until the wok temperature reaches 120℃, which takes about 3 minutes. Pour the wheat bran into the preheated wok and stir it constantly with a spatula to ensure even heating. Stir-fry until the wheat bran smokes and emits a caramel aroma (about 1-2 minutes). Pour the sliced ​​Atractylodes macrocephala into a pot, mix it with wheat bran, and stir-fry quickly and evenly with a spatula, ensuring that each slice is coated with wheat bran. Maintain the stir-frying temperature at 120℃ for about 8 minutes. Observe the color; stir-fry until the surface of the Atractylodes macrocephala turns golden yellow or dark yellow and emits a caramelized aroma, taking care to avoid burning. Quickly pour the stir-fried Atractylodes macrocephala into a sieve to remove the wheat bran. Spread the Atractylodes macrocephala on a tray or container to cool. Once the Atractylodes macrocephala is completely cooled, store it in an airtight container in a cool, dry place. Stir-frying Atractylodes macrocephala with wheat bran enhances its spleen-strengthening and diarrhea-stopping effects, making it more suitable for those with weak spleen and stomach.

[0055] (3) Stir-frying Citrus aurantium with wheat bran: Use dried Citrus aurantium and wheat bran (free from mold and insects), with a ratio of 1000g Citrus aurantium to 100g wheat bran (10:1). Wash and air-dry the Citrus aurantium, or dry it at low temperature, and cut it into uniform slices or pieces. Sift the wheat bran to remove coarse particles and impurities. Preheat the stir-frying pan to 150℃ for about 5 minutes. Pour the wheat bran into the pan and stir-fry until it smokes and turns a dark yellow color. Quickly add the Citrus aurantium. Mix the bitter orange peel evenly with wheat bran and stir-fry quickly over medium heat (150℃) for 10 minutes. Observe the color of the bitter orange peel; stir-fry until the surface of the bitter orange peel is slightly yellow, the wheat bran is dark yellow, and a fragrant aroma is emitted. Use a sieve to separate the stir-fried bitter orange peel from the wheat bran and remove the bitter orange peel. Spread the stir-fried bitter orange peel out and place it in a ventilated place to cool naturally, avoiding moisture. Place the cooled stir-fried bitter orange peel in a dry, sealed container and store it in a cool, dry place to prevent moisture and mold. After being processed by stir-frying with wheat bran, the cold medicinal properties and gastrointestinal irritation of the bitter orange peel are reduced. At the same time, it can enhance its spleen-strengthening and digestion-aiding effects while retaining its qi-regulating and stomach-soothing effects, making it more suitable for long-term use by those with weak spleen and stomach.

[0056] (4) Gardenia scorching: Remove impurities from dried gardenia fruits, preheat the wok to 180℃ for 5 minutes, add the gardenia, and stir-fry evenly over high heat (180℃) for 12 minutes. Observe the color change of the gardenia surface until it turns charred black but not carbonized, and the inside is yellowish-brown, emitting a charred aroma. The scorching is complete. Then quickly remove the charred gardenia, spread it out, and let it cool naturally in a ventilated place for 20 minutes. Place it in a dry container, seal it, and store it in a cool, dry place to prevent moisture and mold. After scorching, the cold and cooling properties of gardenia can be reduced, making it more mild, reducing gastrointestinal irritation and adverse reactions, while retaining its effects of guiding the medicine into the blood, cooling the blood and stopping bleeding, making it convenient for long-term use.

[0057] (5) Mix all the components together, then soak the components in drinking water (about 400 mL) for 30 minutes, heat to boiling, and simmer over low heat at a temperature of nearly 90°C for 30 minutes. Filter out about 200 mL of the decoction. Add an appropriate amount of warm water (about 300 mL) to the dregs and simmer for a second time. Heat to boiling, and simmer over low heat at a temperature of nearly 90°C for 20 minutes. Filter out about 150 mL of the decoction and mix the two decoctions together.

[0058] The preparation methods for the other embodiments and comparative examples are the same as those in Example 1.

[0059] Example 2

[0060] A traditional Chinese medicine composition differs from Example 1 in that the amounts of earthworm and leech are increased to 15g and 6g, respectively.

[0061] Example 3

[0062] A traditional Chinese medicine composition differs from Example 1 in that the dosages of Bupleurum and White Peony are increased to 20g and 20g, respectively.

[0063] Example 4

[0064] A traditional Chinese medicine composition differs from Example 1 in that the amounts of peach kernel and safflower are increased to 20g and 20g, respectively.

[0065] The raw material components and their amounts for each embodiment are shown in the table below:

[0066] Table 1. Raw material components and content (unit: g) for Examples 1-4

[0067] raw material components Example 1 Example 2 Example 3 Example 4 Bupleurum 10 10 20 10 White peony root 10 10 20 10 Fried Bran-Fried Fructus Aurantii Immaturus 10 10 10 10 Angelica sinensis 10 10 10 10 peach kernel 10 10 10 20 safflower 10 10 10 20 Chuanxiong 10 10 10 10 birthplace 10 10 10 10 Gardenia 10 10 10 10 Stir-fried Atractylodes macrocephala 10 10 10 10 Poria 15 15 15 15 leech 3 6 3 3 earth dragon 10 15 10 10 Prepared licorice 6 6 6 6

[0068] Comparative Example 1

[0069] A traditional Chinese medicine composition differs from Example 1 in that Bupleurum and White Peony are replaced with equal masses of Cyperus and Red Peony, respectively.

[0070] Comparative Example 2

[0071] A traditional Chinese medicine composition differs from Example 1 in that earthworms and leeches are replaced with equal masses of ground beetles and horseflies, respectively.

[0072] Comparative Example 3

[0073] A traditional Chinese medicine composition differs from Example 1 in that peach kernel and safflower are replaced with equal masses of salvia miltiorrhiza and peony bark.

[0074] The raw material components and their amounts in Examples 1 and Comparative Examples 1-3 are shown in the table below:

[0075] Table 2. Components and dosages of Examples 1 and Comparative Examples 1-3

[0076]

[0077]

[0078] Application effect verification

[0079] (I) Animal Experiments

[0080] I. Animal grouping and experimentation

[0081] Animals: Apolipoprotein E gene knockout (ApoE) was selected. - / - Forty male C57BL / 6J mice, aged 5-6 weeks and weighing 19-22g, and with the same genetic background, were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. The animals were housed in an SPF-grade animal room at a temperature of 23-25℃, with a 12h / 12h light / dark diurnal rhythm, and were kept unrestricted by food or water. Adaptation was carried out for one week.

[0082] Using the herbal decoctions prepared in each embodiment and comparative example (where the herbal composition in Example 1 was divided into three groups: low-dose, medium-dose, and high-dose; the medium-dose group refers to the herbal concentration of a 70kg adult who takes herbal medicine daily, converted by the body surface area method to obtain the herbal concentration of a 20g mouse given herbal medicine daily; the low-dose group is 1 / 2 times that of the medium-dose group, and the high-dose group is 2 times that of the medium-dose group, i.e., the herbal concentrations of the herbal decoctions in each group are 8.71g / kg, 17.42g / kg, and 34.84g / kg, respectively, to explore the effect of dosage on efficacy), the following experiments were conducted: C57BL / 6J mice were fed a basal diet for 16 weeks as ① a blank control group (Ctrl, basal diet + physiological saline by gavage), and 40 ApoE mice were fed a basal diet by gavage. - / - Mice were fed a high-fat diet alone for 8 weeks and then randomly divided into the following groups according to body weight: ② High-fat model group (HFD, i.e., animal model group, high-fat diet + physiological saline by gavage), ③ Low-dose traditional Chinese medicine preparation group (LTG, high-fat diet + 8.71 g / kg traditional Chinese medicine decoction by gavage), ④ Medium-dose traditional Chinese medicine preparation group (MTG, high-fat diet + 17.42 g / kg traditional Chinese medicine decoction from Example 1 by gavage), ⑤ High-dose traditional Chinese medicine preparation group (HTG, high-fat diet + 34.84 g / kg traditional Chinese medicine decoction). The following were administered via gavage: ⑥ Positive control group (ATO, high-fat diet + 2.6 mg / kg atorvastatin calcium solution via gavage); ⑦ Example 2: Traditional Chinese medicine formula (high-fat diet + 18.46 g / kg traditional Chinese medicine decoction via gavage); ⑧ Example 3: Traditional Chinese medicine formula (high-fat diet + 21.06 g / kg traditional Chinese medicine decoction via gavage); ⑨ Example 4: Traditional Chinese medicine formula (high-fat diet + 21.06 g / kg traditional Chinese medicine decoction via gavage); ⑩ Comparative Example 1: Traditional Chinese medicine formula (high-fat diet + 17.42 g / kg traditional Chinese medicine decoction via gavage). Comparative Example 2: Traditional Chinese Medicine Formula (High-fat feed + 17.42 g / kg of traditional Chinese medicine decoction administered by gavage) Comparative Example 3: Traditional Chinese Medicine Formula (high-fat diet + 17.42 g / kg of traditional Chinese medicine decoction administered by gavage).

[0083] The basal feed (1010086) and the high-fat feed (D09100301, containing 40kcal% fat, 800kcal% fructose, 384kcal% sucrose, and 18kcal% cholesterol) were both purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. Fried Citrus aurantium (21041391), Prunus persica (21062001), Paeonia lactiflora (21072361), Poria cocos (21102081), Bupleurum chinense (21071491), Ligusticum chuanxiong (21072931), and Pheretima aspergillum (21074511) were purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd.; Angelica sinensis (1099221), fried Atractylodes macrocephala (1092061), Rehmannia glutinosa (1109011), and Hirudo medicinalis (9110821) were purchased from Guangdong Yifang Pharmaceutical Co., Ltd.; Carthamus tinctorius (2109067) was purchased from Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd.; Gardenia jasminoides (2109003W) and Glycyrrhiza uralensis (2109032C) were purchased from China Resources Sanjiu Pharmaceutical Co., Ltd.; Atorvastatin calcium tablets (EP8439) were purchased from Pfizer Inc.; and saline solution was provided by Shenzhen Traditional Chinese Medicine Hospital.

[0084] Starting from week 9, interventions were implemented for 8 weeks according to the corresponding intervention measures for each group. The total time for modeling and group intervention was 16 weeks, with weight monitoring conducted weekly.

[0085] At the end of the experiment, mice were fasted overnight. The next day, whole blood was collected using the ocular blood sampling method and placed in centrifuge tubes. The blood was centrifuged at 3000 rpm for 10 minutes to collect serum. The supernatant was collected and placed in clean centrifuge tubes and stored at -80°C. Serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels were measured using a fully automated biochemical analyzer (Roche, cobas 8000, HITACHI, Germany). The levels of inflammatory markers, including interleukin-1β (IL-1β, ab197742), interleukin-18 (IL-18, ab216165), interleukin-6 (IL-6, ab203360), and tumor necrosis factor-α (ab208348), were measured using ELISA kits purchased from Abcam, UK.

[0086] Mice were euthanized after anesthesia, and blood samples were extracted. The mice were dissected along the midline of the abdomen to the sternal angle using tissue scissors to expose the heart. The heart chambers and blood vessels were gently irrigated with saline to remove blood accumulation. Dissection began at the origin of the aorta, separating the aorta from the bifurcation of the abdominal aorta to the common iliac artery. The left brachiocephalic trunk and brachiocephalic artery were separated upwards at the aortic arch. The heart and blood vessels were removed. The separated aorta was carefully cleaned of fat and connective tissue from the adventitia using ophthalmic scissors. The heart was then fixed in 10% neutral buffered formalin (10% formaldehyde). The aorta used for Sudan IV gross staining was fixed in 10% neutral buffered formalin. The remaining aorta was quickly immersed in liquid nitrogen and then transferred to a -80°C freezer for storage.

[0087] Preparation of paraffin sections of the aortic root: Take a fixed heart and aortic specimen, cut the heart from the coronary plane below the auricle, then dehydrate it with ethanol of varying concentrations, clear it with xylene, embed the cut surface of the ascending aortic root of the heart downward, take a transverse section of the aortic attachment, and cut 6 sections in succession, each section 4μm thick, for subsequent Oil Red O and HE staining. Immunofluorescence multiplex staining was used to observe the recruitment status of NLRP3 inflammasomes (CD68, NLRP3, ASC) in macrophages of aortic sinus tissue. Aortic sinus tissue sections were prepared and subjected to immunofluorescence staining through the following steps: baking, dewaxing, antigen retrieval, blocking of endogenous peroxidase, serum blocking, and addition of primary and secondary antibodies. The corresponding indicator antibodies CD68 (GB115723), NLRP3 (GB114320), and ASC (GB115270) were used for fluorescent staining three times. All antibodies were purchased from Wuhan Saiwei Biotechnology Co., Ltd. Finally, the residual liquid on the multiplexed slides was blotted dry with filter paper, and 10-20 μL of glycerol / PBS mounting medium was added to clean slides for mounting. The sections were then placed under a pathological slide scanner (Pannoramic MIDI: 3Dhistech) to acquire images.

[0088] II. Experimental Results

[0089] 1. Body weight of mice in each group during the feeding period

[0090] Depend on Figure 1 As shown, at week 16, compared with the HFD group, the body weight of mice in the Ctrl, MTG, and HTG groups was significantly lower (P < 0.001), while there was no significant difference in body weight between the LTG and ATO groups (P > 0.001). From week 9 to 16, the body weight of mice in the LTG and ATO groups showed a stable trend, while the body weight of mice in the MTG and HTG groups showed a decreasing trend. This indicates that the traditional Chinese medicine composition of Example 1 of the present invention can control or even reduce the body weight of hyperlipidemic model mice.

[0091] 2. Serum TC, TG, LDL and HDL levels

[0092] Depend on Figure 2 The results showed that, compared with the HFD group, the LTG, MTG, HTG, and ATO groups significantly reduced TC, TG, and LDL levels (P < 0.01 or P < 0.001), while there was no significant difference in HDL levels among the LTG, MTG, HTG, and ATO groups (P > 0.05). This indicates that the traditional Chinese medicine composition of Example 1 of the present invention can reduce TC, TG, and LDL levels in hyperlipidemic model mice.

[0093] Depend on Figure 3 The results showed that, compared with the HFD group, MTG significantly reduced TC, TG, and LDL levels (P < 0.001), and Comparative Example 3 significantly reduced TC levels (P < 0.01). There were no significant differences in HDL levels among the Ctrl, MTG, Comparative Example 1, Comparative Example 2, and Comparative Example 3 groups (P > 0.05). Compared with the MTG group, the TC, TG, and LDL levels in Comparative Example 1, Comparative Example 2, and Comparative Example 3 groups were higher (P < 0.05; P < 0.01; P < 0.001). These results demonstrate that the traditional Chinese medicine composition of Example 1 of this invention can reduce TC, TG, and LDL levels in hyperlipidemic model mice, and the lipid-lowering effect of Example 1 of this invention is significantly better than that of Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0094] Depend on Figure 4 The results showed that, compared with the HFD group, the MTG and Examples 2-4 groups significantly reduced TC, TG, and LDL levels (P < 0.001), while there was no significant difference in HDL levels among the Ctrl, MTG, and Examples 2-4 groups (P > 0.05). Compared with the MTG group, the TC, TG, LDL, and HDL levels in Examples 2-4 groups were slightly higher, but there was no significant difference (P > 0.05). These results demonstrate that the MTG group of Example 1 of this invention can reduce TC, TG, and LDL levels in hyperlipidemic model mice, while there was no significant difference in the improvement of blood lipids between the MTG and Examples 2-4 groups.

[0095] 3. Serum levels of inflammatory markers Lp(a), IL1β, IL18, IL6, and TNF-α

[0096] Depend on Figure 5 The results showed that, compared with the HFD group, the MTG, HTG, and ATO groups significantly reduced the levels of inflammatory markers IL1β, IL18, and IL6 (P < 0.001), and the LTG, MTG, HTG, and ATO groups significantly reduced the levels of inflammatory markers Lp(a) and TNF-a (P < 0.05 or P < 0.001). This indicates that Example 1 of the present invention can inhibit the levels of inflammatory markers Lp(a), IL1β, IL18, IL6, and TNF-a in high-fat model mice.

[0097] Depend on Figure 6 The results showed that, compared with the HFD group, the MTG group significantly reduced the levels of inflammatory markers Lp(a), IL1β, IL18, TNF-a, and IL6 (P < 0.001), the comparative group 1 reduced the levels of inflammatory markers Lp(a), IL1β, TNF-a, and IL6 (P < 0.05; P < 0.01; P < 0.001), the comparative group 2 reduced the levels of inflammatory markers Lp(a), IL1β, and TNF-a (P < 0.05; P < 0.01; P < 0.001), and the comparative group 3 reduced the levels of inflammatory markers Lp(a), IL1β, IL18, TNF-a, and IL6 (P < 0.05; P < 0.01; P < 0.001). However, compared with the MTG group, the levels of IL1β, IL18, TNF-α, and IL6 in Comparative Examples 1, 2, and 3 were still higher (P < 0.05; P < 0.01; P < 0.001). The results demonstrate that the traditional Chinese medicine composition of Example 1 of this invention can significantly reduce the levels of inflammatory markers Lp(a), IL1β, IL18, TNF-α, and IL6 in hyperlipidemic model mice, and the effect of reducing inflammatory markers is significantly better than that of Comparative Examples 1, 2, and 3.

[0098] Depend on Figure 7 The results showed that, compared with the HFD group (model group), the MTG and Examples 2-4 groups significantly reduced the levels of inflammatory markers Lp(a), IL1β, IL18, TNF-a, and IL6 (P < 0.001). Compared with the MTG group, there were no significant differences in the levels of inflammatory markers Lp(a), IL1β, IL18, TNF-a, and IL6 in the Examples 2-4 groups (P > 0.05). These results demonstrate that the herbal composition MTG group in Example 1 of this invention can reduce the levels of inflammatory markers Lp(a), IL1β, IL18, TNF-a, and IL6 in high-fat model mice, while there were no significant differences in the improvement of inflammatory marker levels between the MTG and Examples 2-4 groups.

[0099] 4. Area of ​​Oil Red O staining in the aortic sinus

[0100] Depend on Figure 8 The results showed that, compared with the HFD group, the LTG, MTG, HTG, and ATO groups all significantly reduced the percentage of AS plaque area stained with Oil Red dye to the total vascular area (P < 0.01 or P < 0.001). This indicates that the traditional Chinese medicine composition of Example 1 of the present invention can reduce AS plaques in hyperlipidemic model mice.

[0101] Depend on Figure 9The results showed that, compared with the HFD group, the MTG, Comparative Example 1, Comparative Example 2, and Comparative Example 3 groups all significantly reduced the percentage of AS plaque area stained with Oil Red Red ink to the total vascular area (P < 0.001). However, compared with the MTG group, the percentage of AS plaque area stained with Oil Red Red ink to the total vascular area in Comparative Example 1, Comparative Example 2, and Comparative Example 3 groups was still higher (P < 0.01; P < 0.001). These results demonstrate that the traditional Chinese medicine composition of Example 1 of this invention can reduce AS plaques in hyperlipidemic model mice, and its inhibitory effect on AS plaques is significantly better than that of Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0102] Depend on Figure 10 The results showed that, compared with the HFD group, MTG and Examples 2-4 significantly reduced the percentage of AS plaque area stained with Oil Red Red ink to the total vascular area (P < 0.001). Compared with the MTG group, there was no significant difference in the percentage of AS plaque area stained with Oil Red Red ink to the total vascular area between Examples 2-4 (P > 0.05). These results demonstrate that the herbal preparation MTG in Example 1 of this invention can reduce AS plaques in high-fat model mice, and there was no significant difference between MTG and Examples 2-4 in improving the percentage of AS plaque area stained with Oil Red Red ink to the total vascular area.

[0103] 5. Area of ​​inflammatory necrosis in the aortic sinus

[0104] Depend on Figure 11 The results showed that, compared with the HFD group, the MTG, HTG, and ATO groups all significantly reduced the percentage of inflammatory necrosis area in AS plaques to the total plaque area (P < 0.01 or P < 0.001). This indicates that the traditional Chinese medicine composition of Example 1 of the present invention can inhibit inflammatory necrosis in AS plaques in hyperlipidemic model mice.

[0105] Depend on Figure 12 The results showed that, compared with the HFD group, MTG significantly reduced the percentage of inflammatory necrosis area in AS plaques relative to the total vascular area (P < 0.001). However, compared with the MTG group, the percentage of inflammatory necrosis area in AS plaques relative to the total vascular area in Comparative Examples 1, 2, and 3 was still higher (P < 0.05; P < 0.01). These results demonstrate that Example 1 of the present invention can significantly inhibit AS plaque inflammation and necrosis in high-fat model mice, and its inhibitory effect on AS plaque inflammation and necrosis is significantly better than that of Comparative Examples 1, 2, and 3.

[0106] Depend on Figure 13The results showed that, compared with the HFD group, the MTG and Examples 2-4 groups significantly reduced the percentage of AS plaque inflammatory necrosis area to total vascular area (P < 0.001). Compared with the MTG group, there was no significant difference in the percentage of AS plaque inflammatory necrosis area to total vascular area between the Examples 2-4 groups (P > 0.05). These results demonstrate that the traditional Chinese medicine preparation of this invention can inhibit AS plaque inflammatory necrosis in high-fat model mice, and there was no significant difference in the improvement of the percentage of AS plaque inflammatory necrosis area to total vascular area between the MTG and Examples 2-4 groups.

[0107] 6. Multiplex immunofluorescence staining to detect CD68 / NLRP3 / ASC inflammasome expression in the aortic sinus.

[0108] Depend on Figure 14 The results showed that, compared with the HFD group, the LTG, MTG, HTG, and ATO groups all significantly inhibited the expression of CD68 / NLRP3 / ASC inflammasomes in AS plaques. This indicates that the traditional Chinese medicine composition of Example 1 of the present invention can inhibit the expression of CD68 / NLRP3 / ASC inflammasomes in AS plaques of high-fat model mice.

[0109] (II) Cell Experiments

[0110] I. Cellular Experiment Section

[0111] 1. Preparation of drug-containing serum

[0112] Forty male SD rats, aged 5-6 weeks and weighing 220-250g, SPF grade, were selected and purchased from Zhuhai Baishitong Biotechnology Co., Ltd. The animals were housed in an SPF-grade animal room at a temperature of 23-25℃, with a 12h / 12h diurnal rhythm, and without food or water restrictions. After one week of acclimatization, they were randomly divided into four groups according to body weight: ① blank control group (saline by gavage), ② low-dose traditional Chinese medicine preparation group (6.66g / kg of traditional Chinese medicine decoction by gavage), ③ medium-dose traditional Chinese medicine preparation group (12.32g / kg of traditional Chinese medicine decoction by gavage), and ④ high-dose traditional Chinese medicine preparation group (26.64g / kg of traditional Chinese medicine decoction by gavage). The low, medium, and high doses of the decoction preparation in Example 1 of this invention were set at 6.66g / kg, 12.32g / kg, and 26.64g / kg, respectively. Administration was continued for two weeks. During the administration period, all groups were fed a normal diet, and other feeding conditions were similar among the groups.

[0113] The following Chinese medicinal herbs were purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd.: stir-fried Citrus aurantium (21041391), peach kernel (21062001), white peony root (21072361), Poria cocos (21102081), Bupleurum chinense (21071491), Ligusticum chuanxiong (21072931), and earthworm (21074511). Angelica sinensis (1099221), stir-fried Atractylodes macrocephala (1092061), Rehmannia glutinosa (1109011), and leech (9110821). Carthamus tinctorius (2109067) was purchased from Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd. Gardenia jasminoides (2109003W) and prepared licorice root (2109032C) were purchased from China Resources Sanjiu Pharmaceutical Co., Ltd.

[0114] The rats were fasted for 12 hours before the last gavage administration, but water was allowed. Blood samples were collected at 1 hour, 2 hours, and 4 hours after the last administration. Blood was collected from the orbital venous plexus at 1 hour and 2 hours, and from the abdominal aorta at 4 hours. The blood samples were centrifuged at 3000 rpm for 10 minutes at 4°C to obtain serum. The serum from the same group of rats was collected and mixed (divided into normal control serum, low, medium, and high dose serum containing the Chinese medicine preparation). The mixture was centrifuged at 3000 rpm for 10 minutes at 4°C to remove red blood cells. The supernatant was collected, inactivated by water bath at 56°C for 30 minutes, filtered through a 0.22 μm filter membrane for sterilization, and then aliquoted into 2 mL centrifuge tubes and stored at -20°C for later use.

[0115] 2. Cell Culture and Grouping

[0116] RAW 264.7 macrophages in logarithmic growth phase were cultured in DMEM 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin. The cells were loosely capped and cultured in a constant temperature incubator at 37°C and saturated humidity with 5% CO2. The medium was changed every 1-2 days. Cells were passaged after digestion when they reached 80% confluence. Experimental groups were as follows:

[0117] ① Blank control group (Ctrl): DMEM, intervention time 48h;

[0118] ② Model group: 50 μg / mL ox-LDL (oxidatively modified low-density lipoprotein) + normal control serum were added, and the intervention time was 48 h;

[0119] ③Low-dose traditional Chinese medicine preparation group (LTG): 50 μg / mL ox-LDL + serum containing low-dose traditional Chinese medicine preparation was added, and the intervention time was 48 h;

[0120] ④ Medium-dose traditional Chinese medicine preparation group (MTG): 50 μg / mL ox-LDL + medium-dose traditional Chinese medicine preparation containing serum was added, and the intervention time was 48 h;

[0121] ⑤ High-dose traditional Chinese medicine preparation group (HTG): 50 μg / mL ox-LDL + high-dose traditional Chinese medicine preparation containing serum was added, and the intervention time was 48 h;

[0122] ⑥ CD36-siRNA group (siCD36): 50 μg / mL ox-LDL + CD36-siRNA + DMEM were added, and the intervention time was 48 h. 3. Oil Red O staining was used to observe lipid accumulation in RAW macrophages.

[0123] Slowly aspirate the cell culture medium, wash once with PBS, fix with 4% paraformaldehyde fixative for 10 min, and rinse twice with PBS; prepare Oil Red O staining working solution (C0157, Beyotime Biotechnology Co., Ltd.); add an appropriate amount of staining wash buffer to cover the cells for 20 seconds; aspirate the staining wash buffer, add an appropriate amount of Oil Red O staining working solution, and stain for 10-20 min; remove the Oil Red O staining working solution, add an appropriate amount of staining wash buffer, let stand for 30 seconds, then remove the staining wash buffer and wash with PBS for 20 seconds; hematoxylin staining solution (C0107) can be used for counterstaining the cell nuclei; add an appropriate amount of PBS to evenly cover the cells, and observe and photograph under a microscope.

[0124] 4. Apoptosis rate detection

[0125] Dilute 10×Binding Buffer with deionized water to make 1×Binding Buffer; collect cells by trypsin digestion with EDTA-free enzyme, centrifuge at 2000 rpm for 5-10 min at room temperature; wash cells: resuspend cells once with pre-chilled 1×PBS (4℃), centrifuge at 2000 rpm for 5-10 min, and wash cells; add 300 μL of 1×Binding Buffer to resuspend cells; Annexin V-FITC labeling: add 5 μL of Annexin V-FITC, mix well, and incubate at room temperature for 15 min in the dark; PI labeling: add 5 μL of PI staining 5 min before flow cytometry. Before flow cytometry, add 200 μL of 1×Binding Buffer. The Annexin V-FITC / PI apoptosis kit (KGA108-2) was purchased from Nanjing Kaiji Biotechnology, and the flow cytometer was (BD Biosciences, model C6).

[0126] 5. ELISA method for detecting inflammatory markers IL1β, IL18, IL6, and TNF-α.

[0127] After cell intervention, supernatants were collected from each group, and the levels of interleukin-1β (IL-1β, ab216165), interleukin-18 (IL-18, ab216165), interleukin-6 (IL-6, ab203360), and tumor necrosis factor-α (ab208348) inflammatory markers were measured using ELISA kits. All kits were purchased from Abcam Reagents Ltd., UK.

[0128] 6. Immunofluorescence detection of NLRP3 expression

[0129] The expression of NLRP3 inflammasomes in RAW macrophages was observed by immunofluorescence staining. Slides containing RAW macrophages were prepared and subjected to immunofluorescence staining following the steps of baking, dewaxing, antigen retrieval, blocking of endogenous peroxidase, serum blocking, and addition of primary and secondary antibodies. The corresponding indicator antibody NLRP3 (27458-1-AP, purchased from ProteinTech) was used for fluorescent staining. The TSAPLUS fluorescent three-color four-color staining kit (G1236-100T) was purchased from Wuhan Saiwei Biotechnology Co., Ltd. Finally, residual liquid on the multi-stained slides was blotted dry with filter paper, and 10-20 μL of glycerol / PBS mounting medium was added to a clean slide for mounting. The slides were then placed under a pathological section scanner (PannoramicMIDI: 3Dhistech) to acquire images.

[0130] II. Cell Experiment Results

[0131] 1. Cell Oil Red O staining

[0132] Depend on Figure 15 The results showed that, compared with the Model group, the LTG, MTG, HTG, and siCD36 groups all significantly reduced lipid accumulation in RAW macrophages. This indicates that the traditional Chinese medicine composition of Example 1 of the present invention can inhibit ox-LDL-induced lipid accumulation in RAW macrophages.

[0133] 2. Flow cytometry findings

[0134] Depend on Figure 16 As shown, compared with the Model group, the LTG, MTG, HTG, and siCD36 groups all significantly reduced the apoptosis rate and increased the cell survival rate (P < 0.01 or P < 0.001). This indicates that the traditional Chinese medicine composition of Example 1 of the present invention can reduce the apoptosis rate of ox-LDL-induced RAW macrophages and increase the cell survival rate.

[0135] 3. Cellular inflammatory markers levels of IL1β, IL18, IL6, and TNF-α

[0136] Depend on Figure 17 The results showed that, compared with the Model group, the LTG, MTG, HTG, and siCD36 groups significantly reduced the levels of cellular IL1β inflammatory markers (P < 0.01 or P < 0.001), and the MTG, HTG, and siCD36 groups significantly reduced the levels of cellular IL18, IL6, and TNF-α inflammatory markers (P < 0.01 or P < 0.001). The traditional Chinese medicine composition of Example 1 of this invention can inhibit the levels of IL1β, IL18, IL6, and TNF-α inflammatory markers induced by ox-LDL in RAW macrophages.

[0137] 4. Immunofluorescence detection of NLRP3 inflammasome expression level

[0138] Depend on Figure 18 The results showed that, compared with the Model group, the LTG, MTG, HTG, and siCD36 groups all significantly reduced the fluorescence intensity of the NLRP3 inflammasome. This indicates that the traditional Chinese medicine composition of Example 1 of the present invention can reduce the expression of the NLRP3 inflammasome in ox-LDL-induced RAW macrophages.

[0139] Based on the above animal and cell experiments, the results demonstrate that the traditional Chinese medicine composition provided by this invention can at least achieve the following:

[0140] 1. The traditional Chinese medicine composition provided by this invention can reduce lipid accumulation in macrophages induced by oxidized low-density lipoprotein;

[0141] 2. The traditional Chinese medicine composition provided by this invention can inhibit macrophage inflammatory markers (IL1β, IL18, IL6, TNF-α) induced by oxidized low-density lipoprotein;

[0142] 3. The traditional Chinese medicine composition provided by this invention can inhibit the expression of NLRP3 protein in macrophages induced by oxidized low-density lipoprotein; 4. The traditional Chinese medicine composition provided by this invention can reduce the apoptosis rate of macrophages induced by oxidized low-density lipoprotein and inhibit pyroptosis.

[0143] 5. The traditional Chinese medicine composition provided by this invention can inhibit serum TC, TG, and LDL-C levels;

[0144] 6. The traditional Chinese medicine composition provided by this invention can inhibit the levels of serum inflammatory markers Lp(a), IL1β, IL18, IL6, and TNF-a;

[0145] 7. The traditional Chinese medicine composition provided by this invention can inhibit the activation of ASC and NLRP3 inflammasomes in AS plaques;

[0146] 8. The traditional Chinese medicine composition provided by the present invention can inhibit inflammatory necrosis and lipid accumulation in AS plaques and enhance plaque stability.

[0147] This invention is based on the core concept of regulating the liver. The whole formula takes regulating the key link of "liver dysfunction" as the core treatment method. It combines Bupleurum, white peony root, stir-fried bitter orange peel, angelica, peach kernel, safflower, chuanxiong, rehmannia root, charred gardenia, stir-fried atractylodes macrocephala, poria, leech, earthworm, and prepared licorice root. First, it soothes the liver and regulates the flow of qi to make "liver qi flow smoothly". Then, it combines blood-activating and phlegm-resolving and blood-cooling methods to achieve "heart qi harmony" so that blood circulation is unimpeded and the pulse is unobstructed. It can inhibit inflammation and stabilize atherosclerotic plaques. It can inhibit serum inflammation and lower blood lipid levels, inhibit NLRP3 inflammasome activation, improve the body's chronic inflammatory state, stabilize atherosclerotic plaques and delay their pathological progression, reduce the risk of acute cardiovascular and cerebrovascular accidents, and relieve the symptoms and signs of chest tightness, chest pain, emotional distress, hypochondriac discomfort, pale and dark tongue with ecchymosis, and wiry and hesitant pulse in patients with atherosclerosis and coronary heart disease, thus improving their quality of life. In addition, the traditional Chinese medicine composition provided by this invention has the characteristics of being simple to prepare, low in cost, safe and free of toxic side effects, easy to carry and convenient to take.

Claims

1. A traditional Chinese medicine composition for treating atherosclerosis, characterized in that, The traditional Chinese medicine composition is composed of the following raw materials by weight parts: Radix Bupleuri 10-25 parts, Radix Paeoniae Alba 10-25 parts, Fructus Aurantii Immaturus 10-20 parts, Radix Angelicae Sinensis 10-20 parts, Semen Persicae 10-25 parts, Flos Carthami 10-25 parts, Rhizoma Chuanxiong 10-20 parts, Radix Rehmanniae 10-20 parts, Fructus Gardeniae 10-20 parts, Radix Paeoniae Alba 10-20 parts, Poria 15-30 parts, Hirudo 3-8 parts, Earthworm 10-20 parts, and Radix Glycyrrhizae 6-12 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is composed of the following raw materials by weight parts: Radix Bupleuri 10-20 parts, Radix Paeoniae Alba 10-20 parts, Fructus Aurantii Immaturus 10-15 parts, Radix Angelicae Sinensis 10-15 parts, Semen Persicae 10-20 parts, Flos Carthami 10-20 parts, Rhizoma Chuanxiong 10-15 parts, Radix Rehmanniae 10-15 parts, Fructus Gardeniae 10-15 parts, Radix Paeoniae Alba 10-15 parts, Poria 15-20 parts, Hirudo 3-6 parts, Earthworm 10-15 parts, and Radix Glycyrrhizae 6-8 parts.

3. The method for preparing the traditional Chinese medicinal composition for treating atherosclerosis according to claim 1 or 2, characterized in that, The method comprises the following steps: The raw materials are mixed with the solvent to extract the traditional Chinese medicine composition.

4. The preparation method of the traditional Chinese medicine composition according to claim 3, characterized in that, The solvent is water and / or ethanol.

5. The preparation method of the traditional Chinese medicine composition according to claim 3, characterized in that, The solvent is used in an amount of 2-3 times the total mass of the raw materials.

6. The preparation method of the traditional Chinese medicine composition according to claim 3, characterized in that, The extraction is performed at a temperature of 70-100°C, for a time of 10-60 minutes, and / or for 1-2 times.

7. Use of the traditional Chinese medicine composition for treating atherosclerosis according to claim 1 or 2 in the preparation of a medicine for treating atherosclerosis.

8. A medicine for treating atherosclerosis, comprising the traditional Chinese medicine composition for treating atherosclerosis according to claim 1 or 2.

9. The medicament according to claim 8, characterized in that, The medicine is in the form of at least one of a pill, a capsule, a tablet, a paste, an aerosol, a suspension, a granule, a powder, a solution, a lozenge, or a film.

10. The medicament according to claim 8, characterized in that, The medicine further comprises an excipient, which is one or more of dextrin, silicon dioxide, and a flavoring agent.

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