Traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention belongs to the technical field of pharmaceutical compositions, and provides a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response as well as a preparation method and application of the traditional Chinese medicine composition. The traditional Chinese medicine composition provided by the invention adopts radix bupleuri, radix paeoniae alba, fructus aurantii stir-fried with bran, angelica sinensis, peach kernels, safflower carthamus, ligusticum wallichii, radix rehmanniae, fructus gardeniae praeparatus, rhizoma atractylodis macrocephalae stir-fried with bran, poria cocos, leech, earthworm and honey-fried licorice root as main raw materials, can inhibit serum inflammation, reduce the blood fat level, improve the chronic inflammation state of an organism, stabilize atherosclerotic plaque and delay the pathological progress of the atherosclerotic plaque, and has a good application prospect. The traditional Chinese medicine composition can reduce acute cardiovascular and cerebrovascular accident risks, can relieve symptoms and signs of chest distress, chest pain, unsmooth emotion, uncomfortable hypochondrium, pale and dark tongue with ecchymosis and astringent pulse of patients with atherosclerosis and coronary heart disease, and improves life quality. In addition, the traditional Chinese medicine composition provided by the invention also has the characteristics of simplicity in preparation, low cost, safety, no toxic or side effect, convenience in carrying, convenience in taking and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical compositions, and more particularly, to a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response, and its preparation method and application. Background Art

[0002] Modern medical treatment measures for atherosclerosis (AS) and coronary heart disease mainly include controlling risk factors (such as smoking cessation, diet control, blood lipid regulation, blood pressure regulation, blood sugar regulation, etc.) and using prevention and treatment drugs. Currently, commonly used prevention and treatment drugs include statins, lipid-lowering drugs, antiplatelet drugs (such as aspirin, clopidogrel, and ticagrelor), etc. Among them, statins have side effects such as liver and kidney function damage, muscle soreness, and rhabdomyolysis, which limit the scope of applicable patients and easily result in poor patient compliance. Fibrate lipid-lowering drugs can increase the risk of cholelithiasis, elevated liver transaminases, cause myalgia and myositis, gastrointestinal discomfort, etc. And in the case of applying high-intensity statins and lipid-lowering drugs, the AS disease course of 50% of patients still shows adverse progression. Antiplatelet drugs inhibit platelet aggregation, but can lead to a decline in the body's hemostatic function, and in severe cases, can cause internal organ bleeding, such as gastrointestinal bleeding, cerebral hemorrhage, etc., and even endanger life. Common adverse reactions also include peptic ulcer, gastrointestinal discomfort, liver and kidney function damage, and allergic reactions. In addition, in the current strategy of using colchicine for anti-inflammatory treatment in the treatment of AS, a number of clinical studies have shown that the side effects of colchicine cannot be ignored. Colchicine is prone to cause poisoning, and clinically, it is also necessary to avoid combined use with a variety of foods and drugs. Patients with impaired liver and kidney function also need to use it with caution, and the incidence of its 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 for treatment. Although chemical prevention and treatment drugs have certain effects clinically, it is difficult to effectively prevent the progression of AS, and the treatment cost is high, accompanied by significant side effects, resulting in poor patient compliance, which in turn affects the drug efficacy and the treatment effect is not good. In traditional Chinese medicine, although there are already traditional Chinese medicine preparations for anti-atherosclerosis applied clinically, the main mechanism of action is still mainly to regulate blood lipids. In terms of formula selection and drug use, the current traditional Chinese medicine formula ideas and treatment methods still mainly focus on simple blood-activating methods and phlegm-resolving methods, and the clinical efficacy is not significant enough. Most traditional Chinese medicine preparations for preventing and treating AS ignore 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, has significant efficacy and few side effects. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art described above. To this end, the present invention provides a traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response, its preparation method and application. The traditional Chinese medicine composition provided by the present invention takes the method of regulating the liver as the core prescription treatment method, is used to inhibit chronic inflammation, enhance the stability of AS plaques, has a significant effect on treating AS, and the main raw materials used are all traditional Chinese medicine herbs, with little side effect 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 response comprises the following raw materials:

[0008] Bupleurum chinense, Paeonia lactiflora, Fructus Aurantii Immaturus stir-fried with bran, Angelica sinensis, Prunus persica, Carthamus tinctorius, Ligusticum wallichii, Rehmannia glutinosa, Gardenia jasminoides Ellis stir-fried until charred, Atractylodes macrocephala Koidz stir-fried with bran, Poria cocos, Hirudo, Pheretima, Glycyrrhiza uralensis Fisch stir-fried.

[0009] The traditional Chinese medicine composition provided by the present invention takes regulating the liver qi, dispelling stasis and phlegm, and dredging the heart vessels as the main treatment methods, and is used for inhibiting inflammation, regulating blood lipids, and enhancing the stability of AS plaques. Among them, Bupleuri Radix and Fructus Aurantii Immaturus stir-fried with bran soothe the liver qi and relieve depression to assist the "function of the liver", Angelicae Sinensis Radix and Paeoniae Radix Alba nourish the liver and soften the liver to tonify the "body of the liver", Persicae Semen, Carthami Flos, and Chuanxiong Rhizoma activate blood circulation to remove stasis, Rehmanniae Radix and Gardeniae Fructus stir-fried with coke nourish yin, clear heat, and cool blood, Atractylodis Macrocephalae Rhizoma stir-fried with bran and Poria cocos strengthen the spleen and resolve phlegm-dampness, and Glycyrrhizae Radix Preparata harmonizes various herbs. In the course of emotional stress promoting the progression of AS vulnerable plaques, although the disease location is in the "heart", it is inseparable from the "liver". The pathogenesis is that "liver qi stagnation leads to heart qi deficiency", and "liver failure to disperse" is the key hub in the pathogenesis: "liver governing dispersion" is the most important in emotional regulation. Long-term chronic stress is likely to cause "liver failure to disperse", which can induce abnormal emotions. "Sorrow, worry, fear will damage the heart", so it interferes with the function of "heart governing mind", and further affects the function of "heart governing blood vessels", making the heart unable to normally regulate blood circulation and blood vessels. Insufficient blood circulation fails to nourish the "heart spirit", and the dysfunction of the two forms a vicious cycle, exacerbating the damage of blood vessels and poor blood circulation, thus promoting the progression of AS. In view of the above, based on the principle of "when the liver qi is unobstructed, the heart qi is harmonious; when the liver qi is stagnant, the heart qi is deficient. For this kind of heart disease, seek the root cause in the liver and clear its source", the present invention closely adheres to the key pathogenesis of "liver failure to disperse", and combines the transmission law of this disease of "liver qi stagnation - transformation into heat due to stagnation - coagulation of phlegm and stasis due to qi stagnation" to obtain the following compound prescription: In the formula, Bupleuri Radix and Paeoniae Radix Alba are used as the monarch herbs, combined with the ministerial herbs Fructus Aurantii Immaturus stir-fried with bran and Angelicae Sinensis Radix to soothe the liver qi and relieve depression to assist the "function of the liver", nourish the liver and soften the liver to tonify the "body of the liver", strengthen the root ministerial herbs Poria cocos and Atractylodis Macrocephalae Rhizoma stir-fried with bran to strengthen the spleen and resolve phlegm-dampness, assist the herbs Persicae Semen, Carthami Flos, and Chuanxiong Rhizoma to activate blood circulation to remove stasis and relieve pain, the guiding herbs Rehmanniae Radix and Gardeniae Fructus stir-fried with coke enter the blood aspect to nourish yin, clear heat, and cool blood, the guiding herbs Hirudo and Pheretima enter the liver meridian and guide the herbs into the collaterals to remove stasis and dredge collaterals, and Glycyrrhizae Radix Preparata harmonizes various herbs. The whole prescription takes regulating the key hub of the pathogenesis of "liver failure to disperse" as the core treatment method. First, soothe the liver and soften the liver to regulate qi movement to make the "liver qi unobstructed", and then combine the methods of promoting blood circulation and resolving phlegm, and cooling blood to achieve "heart qi harmony" so that blood circulation is unobstructed and blood vessels are unobstructed, playing a role in inhibiting inflammation and stabilizing AS plaques.

[0010] Preferably, calculated by weight, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 5-25 parts of Bupleuri Radix, 5-25 parts of Paeoniae Radix Alba, 5-20 parts of Fructus Aurantii Immaturus stir-fried with bran, 5-20 parts of Angelicae Sinensis Radix, 5-25 parts of Persicae Semen, 5-25 parts of Carthami Flos, 5-20 parts of Chuanxiong Rhizoma, 5-20 parts of Rehmanniae Radix, 5-20 parts of Gardeniae Fructus stir-fried with coke, 5-20 parts of Atractylodis Macrocephalae Rhizoma stir-fried with bran, 7.5-30 parts of Poria cocos, 1.5-8 parts of Hirudo, 5-20 parts of Pheretima, and 3-12 parts of Glycyrrhizae Radix Preparata.

[0011] Further preferably, by weight, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 10-25 parts of Bupleuri Radix, 10-25 parts of Paeoniae Radix Alba, 10-20 parts of Fructus Aurantii Immaturus Praeparatus, 10-20 parts of Angelicae Sinensis Radix, 10-25 parts of Persicae Semen, 10-25 parts of Carthami Flos, 10-20 parts of Chuanxiong Rhizoma, 10-20 parts of Rehmanniae Radix, 10-20 parts of Gardeniae Fructus Praeparatus, 10-20 parts of Atractylodis Macrocephalae Rhizoma Praeparatus, 15-30 parts of Poria, 3-8 parts of Hirudo, 10-20 parts of Lumbricus, 6-12 parts of Glycyrrhizae Radix Preparata.

[0012] More preferably, by weight, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 10-20 parts of Bupleuri Radix, 10-20 parts of Paeoniae Radix Alba, 10-15 parts of Fructus Aurantii Immaturus Praeparatus, 10-15 parts of Angelicae Sinensis Radix, 10-20 parts of Persicae Semen, 10-20 parts of Carthami Flos, 10-15 parts of Chuanxiong Rhizoma, 10-15 parts of Rehmanniae Radix, 10-15 parts of Gardeniae Fructus Praeparatus, 10-15 parts of Atractylodis Macrocephalae Rhizoma Praeparatus, 15-20 parts of Poria, 3-6 parts of Hirudo, 10-15 parts of Lumbricus, 6-8 parts of Glycyrrhizae Radix Preparata.

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

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

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

[0016] Mix the raw materials with a solvent, extract, and obtain the traditional Chinese medicine composition.

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

[0018] Preferably, the dosage of the solvent is 2-3 times the total mass of the raw material components.

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

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

[0021] An 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 application of the traditional Chinese medicine composition in the preparation of a drug for inhibiting inflammation in atherosclerotic plaques and improving the vulnerability of atherosclerotic plaques.

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

[0024] A drug for treating atherosclerosis, coronary heart disease, hypertension, hyperlipidemia, diabetes, obesity or fatty liver, comprising the 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 comprises excipients, and the excipients are one or more of dextrin, silicon dioxide and flavoring agents.

[0027] Preferably, the drug is a drug for inhibiting inflammation in atherosclerotic plaques and improving 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 the present invention uses bupleurum root, white peony root, stir-fried immature bitter orange, angelica sinensis, peach kernel, safflower, chuanxiong rhizome, raw rehmannia root, stir-fried gardenia jasminoides Ellis, stir-fried atractylodes macrocephala koidz, poria cocos, leech, earthworm and roasted licorice root as main raw materials, which can inhibit serum inflammation and reduce blood lipid levels, improve the chronic inflammation state of the body, stabilize atherosclerotic plaques and delay their pathological progression, reduce the risk of acute cardiovascular and cerebrovascular accidents, and can also relieve the symptoms and signs of chest tightness, chest pain, emotional discomfort, hypochondriac discomfort, pale and dull tongue with ecchymosis and stringy and unsmooth pulse in patients with atherosclerosis and coronary heart disease, and improve the quality of life. In addition, the traditional Chinese medicine composition provided by the present invention also has the characteristics of simple production, low cost, safe and non-toxic side effects, convenient to carry and convenient to take. Description of the Drawings

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

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

[0032] Figure 3 Serum TC, TG, LDL and HDL levels of mice in Ctrl, HFD, MTG, and Comparative Examples 1-3 groups in animal experiments (n = 8); 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 of mice in Ctrl, HFD, MTG, and Examples 2-4 groups in animal experiments (n = 8); 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 Lp(a), IL1β, IL18, IL6 and TNF-a inflammatory index levels of mice in Ctrl, HFD, LTG, MTG, HTG, and ATO groups in animal experiments (n = 8); compared with the HFD group, P < 0.001, ##P < 0.01, #P < 0.05, ns P > 0.05;

[0035] Figure 6 Serum Lp(a), IL1β, IL18, IL6 and TNF-a inflammatory index levels of mice in Ctrl, HFD, MTG, and Comparative Examples 1-3 groups in animal experiments (n = 8); 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, ns P > 0.05;

[0036] Figure 7Graph showing the levels of inflammatory indicators Lp(a), IL1β, IL18, IL6, and TNF-a in the sera of mice in each group of Ctrl, Model, MTG, and Examples 2-4 in animal experiments (n = 8); 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 Graph showing the oil red O staining areas of aortic sinus pathological sections of mice in each group of Ctrl, HFD, LTG, MTG, HTG, and ATO in animal experiments (n = 8); compared with the HFD group, P < 0.001, ##P < 0.01, #P < 0.05, ns P > 0.05;

[0038] Figure 9 Graph showing the oil red O staining areas of aortic sinus pathological sections of mice in each group of Ctrl, HFD, MTG, and Comparative Examples 1-3 in animal experiments (n = 8); 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 Graph showing the oil red O staining areas of aortic sinus pathological sections of mice in each group of Ctrl, HFD, MTG, and Examples 2-4 in animal experiments (n = 8); 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 Graph showing the HE staining (hematoxylin-eosin staining method) areas of aortic sinus pathological sections of mice in each group of Ctrl, HFD, LTG, MTG, HTG, and ATO in animal experiments. Compared with the HFD group, P < 0.001, ##P < 0.01, #P < 0.05, ns P > 0.05;

[0041] Figure 12It is a graph showing the HE staining area of the aortic sinus pathological sections of mice in each group of Ctrl, HFD, MTG, and Comparative Examples 1-3 in animal experiments (n = 8); 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 It is a graph showing the HE staining area of the aortic sinus pathological sections of mice in each group of Ctrl, HFD, MTG, and Examples 2-4 in animal experiments (n = 8); 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 It is a graph showing the results of multiple immunofluorescence staining (CD68 / NLRP3 / ASC) of the aortic sinus of mice in each group of Ctrl, HFD, LTG, MTG, HTG, and ATO in animal experiments;

[0044] Figure 15 It is a graph showing the Oil Red O staining (n = 3) of RAW macrophages in each group of Ctrl, Model, LTG, MTG, HTG, and siCD36 in cell experiments;

[0045] Figure 16 It is a graph showing the flow cytometry detection of RAW macrophages in each group of Ctrl, Model, LTG, MTG, HTG, and siCD36 in cell experiments (n = 3). Compared with the Model group, P < 0.001, ##P < 0.01, #P < 0.05, ns P > 0.05;

[0046] Figure 17 It is a graph showing the levels of inflammatory indexes of IL1β, IL18, IL6, and TNF-a of RAW macrophages in each group of Ctrl, Model, LTG, MTG, HTG, and siCD36 in cell experiments (n = 3); compared with the Model group, P < 0.001, ##P < 0.01, #P < 0.05, ns P > 0.05;

[0047] Figure 18 It is a graph showing the expression level of NLRP3 inflammasome detected by immunofluorescence of RAW macrophages in each group of Ctrl, Model, LTG, MTG, HTG, and siCD36 in cell experiments. Detailed implementation manners

[0048] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the following embodiments are listed for illustration. It should be noted that the following embodiments do not limit the scope of protection required by the present invention.

[0049] In the following embodiments, the raw materials, reagents or devices used can be obtained from conventional commercial channels or can be obtained by existing known methods without special instructions.

[0050] Example 1

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

[0052] The preparation method of the above 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 Fructus aurantii are stir-fried with bran, and Gardenia jasminoides Ellis is stir-fried until charred. The rest of the traditional Chinese medicines are used as conventional decoction pieces;

[0054] (2) Stir-fry Atractylodes macrocephala with bran: The dried Atractylodes macrocephala slices have a 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 frying pan, place the frying pan on the fire, heat it with medium heat until the temperature of the pan reaches 120°C, and the preheating time is about 3 minutes; pour the wheat bran into the preheated frying pan, continuously stir the wheat bran with a spatula to make it evenly heated, and stir-fry until the wheat bran smokes and emits a burnt fragrance (about 1-2 minutes); pour the Atractylodes macrocephala slices into the pan and mix them with the wheat bran, quickly and evenly stir-fry with a spatula to ensure that each slice of Atractylodes macrocephala is coated with wheat bran, keep the frying temperature at 120°C, and the frying time is about 8 minutes; observe the color, stir-fry until the surface of Atractylodes macrocephala is golden yellow or dark yellow and emits a burnt fragrance, and pay attention to avoiding over-frying; quickly pour the fried Atractylodes macrocephala into a sieve to sieve out the wheat bran, spread the Atractylodes macrocephala on a tray or container to cool, and after the Atractylodes macrocephala is completely cooled, put it into a sealed container and store it in a cool and dry place. After stir-frying Atractylodes macrocephala with bran, with the assistance of wheat bran, its effect of strengthening the spleen and stopping diarrhea is enhanced, and it is more suitable for those with weak spleen and stomach;

[0055] (3)Stir-fry Fructus Aurantii Immaturus with bran: Take dried Fructus Aurantii Immaturus and wheat bran (without mildew and insect damage). The ratio of Fructus Aurantii Immaturus to wheat bran is 1000 g: 100 g (10:1). Wash the Fructus Aurantii Immaturus, dry it in the air or by low-temperature drying, and cut it into uniform slices or pieces. Sieve the wheat bran to remove coarse grains and impurities. Preheat the frying pan to 150 °C for about 5 minutes. Pour the wheat bran into the pan and stir-fry until it smokes and turns dark yellow. Quickly add the Fructus Aurantii Immaturus and mix well with the wheat bran. Keep stir-frying quickly over medium heat (150 °C) for 10 minutes. Observe the color of the Fructus Aurantii Immaturus. Stir-fry until the surface of the Fructus Aurantii Immaturus turns slightly yellow, the wheat bran turns dark yellow, and a fragrance is emitted. Separate the stir-fried Fructus Aurantii Immaturus from the wheat bran with a sieve and take out the Fructus Aurantii Immaturus. Spread out the stir-fried Fructus Aurantii Immaturus and let it cool naturally in a ventilated place to avoid moisture. Put the cooled Fructus Aurantii Immaturus stir-fried with bran into a dry and sealed container and store it in a cool and dry place to prevent moisture and mildew. After being processed by stir-frying with bran, the cold nature and gastrointestinal irritation of Fructus Aurantii Immaturus are reduced. At the same time, it can not only enhance its effect of strengthening the spleen and promoting digestion and accumulation, but also retain its effect of promoting qi circulation and relieving distension, and is more suitable for long-term use by those with weak spleen and stomach;

[0056] (4)Carbonize Gardenia Fruit: Take dried Gardenia Fruit, remove impurities. Preheat the frying pan to 180 °C for 5 minutes, put in the Gardenia Fruit, and stir-fry evenly over high heat (180 °C) for 12 minutes. Observe the change in the surface color of the Gardenia Fruit until it turns charred black but not carbonized, and the inside is yellowish-brown, and a burnt fragrance is emitted. The frying is completed, then quickly take out the carbonized Gardenia Fruit. Spread out the fried carbonized Gardenia Fruit and let it cool naturally in a ventilated place for 20 minutes. Put it into a dry container, seal it, and store it in a cool and dry place to prevent moisture and mildew. After being carbonized, Gardenia Fruit can better reduce its cold nature, its medicinal property becomes milder, reduce gastrointestinal irritation and adverse reactions, and retain its effect of guiding the medicine into the blood aspect and cooling blood to stop bleeding, which is convenient for long-term taking;

[0057] (5)Mix all the components, then soak the components in drinking water (about 400 mL) for 30 minutes. After heating to boiling, maintain the water temperature at nearly 90 °C over low heat and decoct for 30 minutes, filtering out about 200 mL of the medicinal liquid. Add an appropriate amount of warm water (about 300 mL) to the medicinal residues for the second decoction. After heating to boiling, maintain the water temperature at nearly 90 °C over low heat and decoct for 20 minutes, filtering out about 150 mL of the medicinal liquid. Mix the two decoctions.

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

[0059] Example 2

[0060] A traditional Chinese medicine composition, which is different from Example 1 in that the dosages of Pheretima and Hirudo are increased to 15 g and 6 g respectively.

[0061] Example 3

[0062] A traditional Chinese medicine composition, which is different from Example 1 in that the dosages of Bupleuri Radix and Paeoniae Radix Alba are increased to 20 g and 20 g respectively.

[0063] Example 4

[0064] A traditional Chinese medicine composition, which is different from that in Example 1 in that the dosages of peach kernels and safflower are increased to 20 g and 20 g respectively.

[0065] The raw material components and their dosages of each example are shown in the following table:

[0066] Table 1 Raw material components and contents of Examples 1-4 (unit: g)

[0067] Raw material components Example 1 Example 2 Example 3 Example 4 Bupleurum chinense 10 10 20 10 White Peony Root 10 10 20 10 Stir-fried Fructus Aurantii 10 10 10 10 Angelica sinensis 10 10 10 10 Peach Kernel 10 10 10 20 Safflower 10 10 10 20 Rhizoma Chuanxiong 10 10 10 10 Radix Rehmanniae 10 10 10 10 Fried Gardenia jasminoides Ellis 10 10 10 10 Stir-fried Atractylodes macrocephala Koidz 10 10 10 10 Poria cocos 15 15 15 15 Whitmania pigra Whitman 3 6 3 3 Pheretima aspergillum 10 15 10 10 Fried Licorice Root 6 6 6 6

[0068] Comparative Example 1

[0069] A traditional Chinese medicine composition, which is different from that in Example 1 in that bupleurum root and white peony root are respectively replaced with equal masses of Cyperus rotundus L. and radix paeoniae rubra.

[0070] Comparative Example 2

[0071] A traditional Chinese medicine composition, which is different from that in Example 1 in that earthworm and leech are respectively replaced with equal masses of ground beetle and gadfly.

[0072] Comparative Example 3

[0073] A traditional Chinese medicine composition, which is different from that in Example 1 in that peach kernels and safflower are respectively replaced with equal masses of salvia miltiorrhiza and cortex moutan.

[0074] The raw material components and their dosages of Example 1 and Comparative Examples 1-3 are shown in the following table:

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

[0076]

[0077]

[0078] Verification of application effect

[0079] (I) Animal experiment

[0080] I. Animal grouping and experiment

[0081] Animals: 40 apolipoprotein E gene knockout (ApoE - / - ) mice and 8 wild-type C57BL / 6J mice with the same genetic background were selected. All were male, 5-6 weeks old, and weighed 19-22 g. They were all purchased from Changzhou Cavens Experimental Animal Co., Ltd. The animal feeding conditions were an SPF-class animal house at a temperature of 23-25 °C, a 12 h / 12 h light-dark circadian rhythm, unrestricted food and water, and an adaptive feeding period of 1 week.

[0082] The traditional Chinese medicine decoction obtained from each example and comparative example (in example 1, the traditional Chinese medicine composition is divided into three groups: low dose, medium dose, and high dose. The medium dose refers to the traditional Chinese medicine concentration obtained by converting the conventional daily dose of traditional Chinese medicine for a 70 kg adult according to the body surface area method to the traditional Chinese medicine concentration administered to 20 g mice per day. 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. That is, the traditional Chinese medicine concentrations of the traditional Chinese medicine decoctions in each group are 8.71 g / kg, 17.42 g / kg, and 34.84 g / kg respectively) were used to conduct the following experiments: C57BL / 6J mice were fed with basal diet for 16 weeks as ① blank control group (Ctrl, basal diet + intragastric administration of normal saline). After 40 ApoE - / - mice were fed with a simple high-fat diet for 8 weeks, they were randomly divided into the following groups according to body weight stratification randomization method: ② high-fat model group (HFD, that is, the animal model group, high-fat diet + intragastric administration of normal saline), ③ low-dose traditional Chinese medicine preparation group (LTG, high-fat diet + intragastric administration of 8.71 g / kg traditional Chinese medicine decoction), ④ medium-dose traditional Chinese medicine preparation group (MTG, high-fat diet + intragastric administration of 17.42 g / kg traditional Chinese medicine decoction of example 1), ⑤ high-dose traditional Chinese medicine preparation group (HTG, high-fat diet + intragastric administration of 34.84 g / kg traditional Chinese medicine decoction), ⑥ positive control group (ATO, high-fat diet + intragastric administration of 2.6 mg / kg atorvastatin calcium aqueous solution), ⑦ traditional Chinese medicine formula of example 2 (high-fat diet + intragastric administration of 18.46 g / kg traditional Chinese medicine decoction), ⑧ traditional Chinese medicine formula of example 3 (high-fat diet + intragastric administration of 21.06 g / kg traditional Chinese medicine decoction), ⑨ traditional Chinese medicine formula of example 4 (high-fat diet + intragastric administration of 21.06 g / kg traditional Chinese medicine decoction), ⑩ traditional Chinese medicine formula of comparative example 1 (high-fat diet + intragastric administration of 17.42 g / kg traditional Chinese medicine decoction), traditional Chinese medicine formula of comparative example 2 (high-fat diet + intragastric administration of 17.42 g / kg traditional Chinese medicine decoction), traditional Chinese medicine formula of comparative example 3 (high-fat diet + intragastric administration of 17.42 g / kg traditional Chinese medicine decoction).

[0083] The basal diet (1010086) and the high-fat diet (D09100301, containing 40 kcal% fat, 800 kcal% fructose, 384 kcal% sucrose, and 18 kcal% cholesterol) were both purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. Fructus Aurantii Immaturus stir-fried with bran (21041391), Semen Persicae (21062001), Radix Paeoniae Alba (21072361), Poria (21102081), Radix Bupleuri (21071491), Rhizoma Chuanxiong (21072931), and Pheretima (21074511) were purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd. Radix Angelicae Sinensis (1099221), Atractylodes Macrocephala Koidz stir-fried with bran (1092061), Radix Rehmanniae (1109011), and Hirudo (9110821) were purchased from Guangdong Yifang Pharmaceutical Co., Ltd. Flos Carthami (2109067) was purchased from Sichuan New Green Pharmaceutical Technology Development Co., Ltd. Fructus Gardeniae carbonized (2109003W) and Radix Glycyrrhizae prepared with honey (2109032C) were purchased from China Resources Sanjiu Medical & Pharmaceutical Co., Ltd. Atorvastatin calcium tablets (EP8439) were purchased from Pfizer Inc., USA. Normal saline was provided by Shenzhen Traditional Chinese Medicine Hospital.

[0084] Starting from the 9th week, the corresponding intervention measures were carried out for 8 weeks according to each group. The total time for modeling and grouped intervention was 16 weeks, and body weight was monitored weekly.

[0085] At the end of the experiment, the mice were fasted overnight. The next day, whole blood was collected by orbital blood sampling and placed in a centrifuge tube. Serum was obtained by centrifuging at 3000 rpm for 10 min using a centrifuge, and the supernatant was placed in a clean centrifuge tube and stored in a -80°C refrigerator. The contents of serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density cholesterol (HDL) were detected using an automatic biochemical analyzer (Roche, Germany, cobas 8000, HITACHI). The levels of inflammatory indicators interleukin-1β (IL-1β, ab197742), interleukin-18 (IL-18, ab216165), interleukin-6 (IL-6, ab203360), and tumor necrosis factor-α (TNF-α, ab208348) were measured using ELISA kits, all purchased from Abcam Reagent Company, UK.

[0086] The mice were anesthetized and then killed. After the blood samples were extracted, the mice were cut open along the midline of the abdomen to the sternal angle with tissue scissors to expose the heart, and physiological saline was gently injected for lavage to remove blood accumulation in the heart cavity and blood vessels. The aorta was dissected from the origin of the aorta to the bifurcation of the common iliac artery of the abdominal aorta, and the left brachiocephalic artery and innominate artery were separated upward at the aortic arch, and the heart and blood vessels were removed; the aorta was carefully removed from the separated aorta with ophthalmic scissors to remove the fat and connective tissue of the aortic adventitia; the heart was then placed in 10% neutral buffered formalin tissue fixative (formaldehyde mass fraction of 10%) for fixation; the aorta used for Sudan IV macroscopic staining was placed in 10% neutral buffered formalin tissue fixative for fixation, and the remaining aorta was quickly placed in liquid nitrogen and transferred to a -80°C refrigerator as soon as possible for storage.

[0087] Preparation of paraffin sections of the aortic root: fixed heart and aorta specimens were obtained, and the heart was cut from the coronal plane below the auricle. After that, the heart was dehydrated with gradient ethanol and made transparent with xylene. The section of the ascending aorta root of the heart was embedded downward, and the cross section of the aorta attachment part was obtained. Six sections were cut continuously, each with a thickness of 4 μm, for subsequent Oil Red O and HE staining. The immunofluorescence multiple staining method 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 immunofluorescence labeled after baking, dewaxing, antigen repair, blocking endogenous peroxidase, serum blocking, primary antibody and secondary antibody addition steps. The corresponding indicator antibodies CD68 (GB115723), NLRP3 (GB114320), and ASC (GB115270) were used for fluorescent labeling and staining for a total of three times. The above antibodies were purchased from Wuhan Saiweier Biotechnology Co., Ltd. Finally, the residual liquid on the glass slide that had been multi-stained and labeled was absorbed with filter paper, and 10-20 μL of glycerol / PBS mounting medium was dropped on a clean glass slide for sealing. The sections were placed under a pathological section scanner (Pannoramic MIDI: 3Dhistech) to collect images.

[0088] 2. Experimental Results

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

[0090] Depend on Figure 1 The results showed that at week 16, the body weights of mice in the Ctrl, MTG and HTG groups were significantly lower than those in the HFD group (P < 0.001), and there was no significant difference in the body weights of mice in the LTG and ATO groups (P > 0.001). From week 9 to week 16, the body weights of mice in the LTG and ATO groups showed a stable trend, while the body weights of mice in the MTG and HTG groups showed a decreasing trend. This shows that the Chinese medicine composition of Example 1 of the present invention can control or even reduce the body weight of high-fat model mice.

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

[0092] As shown by Figure 2 , compared with the HFD group, the LTG, MTG, HTG and ATO groups could all significantly reduce the levels of TC, TG and LDL (P < 0.01 or P < 0.001), and there was no significant difference in the HDL level among the LTG, MTG, HTG and ATO groups (P > 0.05). It indicates that the traditional Chinese medicine composition of Example 1 of the present invention can reduce the levels of TC, TG and LDL in high-fat model mice.

[0093] As shown by Figure 3 , compared with the HFD group, MTG could significantly reduce the levels of TC, TG and LDL (P < 0.001), and Comparative Example 3 could significantly reduce the TC level (P < 0.01). There was no significant difference in the HDL level among the Ctrl, MTG, Comparative Example 1, Comparative Example 2 and Comparative Example 3 groups (P > 0.05). Compared with the MTG group, the levels of TC, TG and LDL in the Comparative Example 1, Comparative Example 2 and Comparative Example 3 groups were higher (P < 0.05; P < 0.01; P < 0.001). The results prove that the traditional Chinese medicine composition of Example 1 of the present invention can reduce the levels of TC, TG and LDL in high-fat model mice, and the lipid-lowering effect of Example 1 of the present invention is significantly better than that of Comparative Example 1, Comparative Example 2 and Comparative Example 3.

[0094] As shown by Figure 4 , compared with the HFD group, MTG and the groups of Examples 2-4 could significantly reduce the levels of TC, TG and LDL (P < 0.001). There was no significant difference in the HDL level among the Ctrl, MTG and the groups of Examples 2-4 (P > 0.05). Compared with the MTG group, there was no significant difference in the levels of TC, TG, LDL and HDL in the groups of Examples 2-4 (P > 0.05). The results prove that the MTG group of Example 1 of the present invention can reduce the levels of TC, TG and LDL in high-fat model mice, and there is no significant difference between MTG and the groups of Examples 2-4 in improving blood lipid.

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

[0096] As shown by Figure 5 , compared with the HFD group, MTG, HTG and ATO groups could all significantly reduce the inflammatory index levels of IL1β, IL18 and IL6 (P < 0.001), and the LTG, MTG, HTG and ATO groups could all significantly reduce the inflammatory index levels of Lp(a) and TNF-a (P < 0.05 or P < 0.001). It indicates that Example 1 of the present invention can inhibit the inflammatory index levels of Lp(a), IL1β, IL18, IL6 and TNF-a in high-fat model mice.

[0097] As shown byFigure 6 As shown, compared with the HFD group, the MTG group could significantly reduce the levels of inflammatory indexes such as Lp(a), IL1β, IL18, TNF-α, and IL6 (P < 0.001). The comparative example 1 group could reduce the levels of inflammatory indexes such as Lp(a), IL1β, TNF-α, and IL6 (P < 0.05; P < 0.01; P < 0.001). The comparative example 2 group could reduce the levels of inflammatory indexes such as Lp(a), IL1β, and TNF-α (P < 0.05; P < 0.01; P < 0.001). The comparative example 3 group could reduce the levels of inflammatory indexes such as Lp(a), IL1β, IL18, TNF-α, 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 the comparative example 1, comparative example 2, and comparative example 3 groups were still relatively high (P < 0.05; P < 0.01; P < 0.001). The results proved that the traditional Chinese medicine composition of Example 1 of the present invention could significantly reduce the levels of inflammatory indexes such as Lp(a), IL1β, IL18, TNF-α, and IL6 in high-fat model mice, and the effect of reducing inflammatory indexes was significantly better than that of comparative example 1, comparative example 2, and comparative example 3.

[0098] As Figure 7 shown, compared with the HFD group (model model group), the MTG group and the groups of Examples 2-4 could significantly reduce the levels of inflammatory indexes such as Lp(a), IL1β, IL18, TNF-α, and IL6 (P < 0.001). Compared with the MTG group, there was no significant difference in the levels of inflammatory indexes such as Lp(a), IL1β, IL18, TNF-α, and IL6 in the groups of Examples 2-4 (P > 0.05). The results proved that the MTG group of the traditional Chinese medicine composition of Example 1 of the present invention could reduce the levels of inflammatory indexes such as Lp(a), IL1β, IL18, TNF-α, and IL6 in high-fat model mice, and there was no significant difference between MTG and Examples 2-4 in improving the levels of inflammatory indexes.

[0099] 4. Oil red O staining area of aortic sinus

[0100] As Figure 8 shown, compared with the HFD group, the LTG group, MTG group, HTG group, and ATO group could all significantly reduce the percentage of the oil red staining area of AS plaque in the total vascular area (P < 0.01 or P < 0.001). It was shown that the traditional Chinese medicine composition of Example 1 of the present invention could reduce AS plaques in high-fat model mice.

[0101] As Figure 9As shown, compared with the HFD group, the MTG group, Comparative Example 1, Comparative Example 2, and Comparative Example 3 groups were all able to significantly reduce the percentage of the oil red staining area of the AS plaque in the total vascular area (P < 0.001). However, compared with the MTG group, the percentage of the oil red staining area of the AS plaque in the total vascular area of the Comparative Example 1, Comparative Example 2, and Comparative Example 3 groups was still relatively high (P < 0.01; P < 0.001). The results prove that the traditional Chinese medicine composition of Example 1 of the present invention can reduce the AS plaque in high-fat model mice, and the effect of inhibiting the AS plaque is significantly better than that of Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0102] As Figure 10 shown, compared with the HFD group, MTG and Examples 2-4 were all able to significantly reduce the percentage of the oil red staining area of the AS plaque in the total vascular area (P < 0.001). Compared with the MTG group, there was no significant difference in the percentage of the oil red staining area of the AS plaque in the total vascular area of the Examples 2-4 groups (P > 0.05). The results prove that the MTG group of the traditional Chinese medicine preparation of Example 1 of the present invention can reduce the AS plaque in high-fat model mice, and there is no significant difference between MTG and Examples 2-4 in terms of improving the percentage of the oil red staining area of the AS plaque in the total vascular area.

[0103] 5. Inflammatory necrosis area of aortic sinus

[0104] As Figure 11 shown, compared with the HFD group, MTG, HTG, and ATO groups were all able to significantly reduce the percentage of the inflammatory necrosis area of the AS plaque in the total plaque area (P < 0.01 or P < 0.001). It shows that the traditional Chinese medicine composition of Example 1 of the present invention can inhibit the inflammatory necrosis of the AS plaque in high-fat model mice.

[0105] As Figure 12 shown, compared with the HFD group, MTG was able to significantly reduce the percentage of the inflammatory necrosis area of the AS plaque in the total vascular area (P < 0.001). However, compared with the MTG group, the percentage of the inflammatory necrosis area of the AS plaque in the total vascular area of the Comparative Example 1, Comparative Example 2, and Comparative Example 3 groups was still relatively high (P < 0.05; P < 0.01). The results prove that Example 1 of the present invention can significantly inhibit the inflammatory necrosis of the AS plaque in high-fat model mice, and the effect of inhibiting the inflammatory necrosis of the AS plaque is significantly better than that of Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0106] As Figure 13As shown, compared with the HFD group, the MTG, Example 2-4 groups could significantly reduce the percentage of the area of AS plaque inflammation and necrosis in the total vascular area (P < 0.001). Compared with the MTG group, there was no significant difference in the percentage of the area of AS plaque inflammation and necrosis in the total vascular area in the Example 2-4 groups (P > 0.05). The results proved that the traditional Chinese medicine preparation of the present invention could inhibit AS plaque inflammation and necrosis in high-fat model mice, and there was no significant difference among the MTG, Example 2-4 groups in terms of improving the percentage of the area of AS plaque inflammation and necrosis in the total vascular area.

[0107] 6. Detection of the expression of CD68 / NLRP3 / ASC inflammasome in the aortic sinus by multiplex immunofluorescence staining

[0108] As Figure 14 shown, compared with the HFD group, the LTG, MTG, HTG and ATO groups could all significantly inhibit the expression of CD68 / NLRP3 / ASC inflammasome in AS plaques. It was shown that the traditional Chinese medicine composition of Example 1 of the present invention could inhibit the expression of CD68 / NLRP3 / ASC inflammasome in AS plaques of high-fat model mice.

[0109] (2) Cell experiments

[0110] I. Part of cell experiments

[0111] 1. Preparation of medicated serum

[0112] Forty male SD rats, all 5-6 weeks old, weighing 220-250 g, of SPF grade, were all purchased from Zhuhai BestBio Technology Co., Ltd. The animals were housed in an SPF animal room at a temperature of 23-25 °C, with a 12h / 12h light-dark circadian rhythm, unrestricted food and water. After 1 week of adaptive feeding, they were randomly divided into 4 groups according to the body weight stratification random method: ① blank control group (intragastric administration of normal saline), ② low-dose traditional Chinese medicine preparation group (intragastric administration of 6.66 g / kg traditional Chinese medicine decoction), ③ medium-dose traditional Chinese medicine preparation group (intragastric administration of 12.32 g / kg traditional Chinese medicine decoction), ④ high-dose traditional Chinese medicine preparation group (intragastric administration of 26.64 g / kg traditional Chinese medicine decoction). Among them, the low, medium and high doses of the decoction preparation of Example 1 of the present invention were set at 6.66 g / kg, 12.32 g / kg and 26.64 g / kg respectively. After continuous administration for 2 weeks, all groups were given normal diet with normal feed during the administration period, and there were no differences in the remaining feeding conditions among groups.

[0113] The Chinese herbal pieces stir-fried with bran Fructus Aurantii (21041391), Semen Persicae (21062001), Radix Paeoniae Alba (21072361), Poria (21102081), Radix Bupleuri (21071491), Rhizoma Chuanxiong (21072931), Pheretima (21074511) were purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd. Angelica sinensis (1099221), Atractylodes macrocephala Koidz stir-fried with bran (1092061), Radix Rehmanniae (1109011), Hirudo (9110821) were purchased from Guangdong Yifang Pharmaceutical Co., Ltd. Flos Carthami (2109067) was purchased from Sichuan New Green Pharmaceutical Technology Development Co., Ltd. Fructus Gardeniae carbonized (2109003W), Radix Glycyrrhizae preparata (2109032C) were purchased from China Resources Sanjiu Medical & Pharmaceutical Co., Ltd.

[0114] The rats were fasted for 12 h before the last intragastric administration, with water not restricted. Blood samples were collected at 1 h, 2 h, and 4 h after the last administration. Blood was collected from the orbital venous plexus at 1 h and 2 h, and from the abdominal aorta at 4 h. The blood samples were centrifuged at 4 °C and 3000 r / min for 10 min to obtain serum. The serum of rats in the same group was collected and mixed (divided into normal control serum, low-, medium-, and high-dose Chinese medicine preparation-containing serum), centrifuged at 4 °C and 3000 r / min for 10 min to fully remove red blood cells, and the supernatant was collected; inactivated in a water bath at 56 °C for 30 min, filtered and sterilized through a 0.22 μm filter membrane, 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 the logarithmic growth phase were cultured in DMEM and 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin, and cultured loosely capped in a constant temperature incubator under the conditions of 5% CO2, 37 °C, and saturated humidity. The medium was changed every 1 - 2 days, and the cells were digested and passaged when they reached 80% confluence. The experimental groups were as follows:

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

[0118] ② Model group (Model): Added 50 μg / mL ox-LDL (oxidized modified low-density lipoprotein) + normal control serum, intervention time 48 h;

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

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

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

[0122] ⑥ CD36-siRNA group (siCD36): Add 50 μg / mL ox-LDL + CD36-siRNA + DMEM, with an intervention time of 48 h. 3. Observe the lipid accumulation of RAW macrophages by Oil Red O staining

[0123] Slowly aspirate the cell culture medium, wash once with PBS, add 4% paraformaldehyde fixative and fix for 10 min, and rinse twice with PBS; Prepare the Oil Red O staining working solution (C0157, Beyotime Biotechnology Co., Ltd.); Add an appropriate amount of staining washing solution to cover the cells for 20 s; Aspirate the staining washing solution, 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 washing solution, let stand for 30 s, then remove the staining washing solution, and wash with PBS for 20 s; The cell nucleus can be counterstained with hematoxylin staining solution (C0107); Add an appropriate amount of PBS to evenly cover the cells, and observe and take pictures under the microscope.

[0124] 4. Detection of cell apoptosis rate

[0125] Dilute 10× Binding Buffer (binding buffer) to 1× Binding Buffer with deionized water; After digestion and collection with trypsin without EDTA, centrifuge at 2000 rpm for 5 - 10 min at room temperature to collect cells; Cell washing: Resuspend the cells once with pre-cooled 1× PBS (4°C), centrifuge at 2000 rpm for 5 - 10 min to wash the cells; Add 300 μL of 1× BindingBuffer to suspend the cells; Annexin V-FITC labeling: Add 5 μL of Annexin V-FITC, mix well, protect from light, and incubate at room temperature for 15 min; PI labeling: Add 5 μL of PI staining 5 min before loading. Before loading, add 200 μL of 1× Binding Buffer. The Annexin V-FITC / PI apoptosis kit (KGA108-2) is purchased from Nanjing KeyGen Biotech Co., Ltd., and the flow cytometer (BD, model C6).

[0126] 5. Detection of the levels of inflammatory indexes IL1β, IL18, IL6, TNF-a by ELISA

[0127] After the intervention of each group of cells, the supernatant was collected, and an ELISA kit was used to measure the levels of inflammatory indexes such as interleukin-1β (IL-1β, ab216165), interleukin-18 (IL-18, ab216165), interleukin-6 (IL-6, ab203360), and tumor necrosis factor-α (TNF-α, ab208348), all of which were purchased from Abcam reagent company in the UK.

[0128] 6. Immunofluorescence detection of NLRP3 expression

[0129] The expression of NLRP3 inflammasome in RAW macrophages was observed by immunofluorescence staining. Take the glass slide with RAW macrophages to be tested, and perform immunofluorescence labeling through steps such as baking the slide, dewaxing, antigen repair, blocking endogenous peroxidase, serum blocking, adding primary antibody and secondary antibody. Among them, NLRP3 (27458-1-AP, purchased from proteintech company) was used for fluorescence labeling and staining with the corresponding index antibody, and the TSAPLus fluorescence triple-labeling and four-color staining kit (G1236-100T) was purchased from Wuhan Sevier Biotechnology Co., Ltd. Finally, the residual liquid on the slide that had been multiply stained and labeled was blotted dry with filter paper, and 10-20 μL of glycerol / PBS mounting medium was dropped on a clean glass slide for mounting. The section was placed under a pathological section scanner (Pannoramic MIDI: 3Dhistech) to collect images.

[0130] II. Results of cell experiments

[0131] 1. Oil red O staining of cells

[0132] As Figure 15 shown, compared with the Model group, the LTG, MTG, HTG, and siCD36 groups could all significantly reduce lipid accumulation in RAW macrophages. It shows that the traditional Chinese medicine composition of Example 1 of the present invention can inhibit lipid accumulation in RAW macrophages induced by ox-LDL.

[0133] 2. Detection by flow cytometry

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

[0135] 3. Levels of inflammatory indicators of IL1β, IL18, IL6, and TNF-a in cells

[0136] As shown by Figure 17 compared with the Model group, the LTG, MTG, HTG, and siCD36 groups could all significantly reduce the level of the IL1β inflammatory indicator in cells (P < 0.01 or P < 0.001), and the MTG, HTG, and siCD36 groups could all significantly reduce the levels of the IL18, IL6, and TNF-a inflammatory indicators in cells (P < 0.01 or P < 0.001). The traditional Chinese medicine composition in Example 1 of the present invention can inhibit the levels of IL1β, IL18, IL6, and TNF-a inflammatory indicators in ox-LDL-induced RAW macrophages.

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

[0138] As shown by Figure 18 compared with the Model group, the LTG, MTG, HTG, and siCD36 groups could all significantly reduce the fluorescence intensity of the NLRP3 inflammasome. It indicates that the traditional Chinese medicine composition in 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 experiments and cell experiments, the results prove that the traditional Chinese medicine composition provided by the present invention can at least achieve:

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

[0141] 2. The traditional Chinese medicine composition provided by the present invention can inhibit the inflammatory indicators (IL1β, IL18, IL6, TNF-a) of macrophages induced by oxidized low-density lipoprotein;

[0142] 3. The traditional Chinese medicine composition provided by the present invention can inhibit the expression of NLRP3 protein in macrophages induced by oxidized low-density lipoprotein; 4. The traditional Chinese medicine composition provided by the present 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 the present invention can inhibit the levels of serum TC, TG, and LDL-C;

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

[0145] 7. The traditional Chinese medicine composition provided by the present invention can inhibit the activation of ASC and NLRP3 inflammasome in atherosclerotic 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] Based on the core idea of regulating the liver, the whole formula takes regulating the key hub of pathogenesis, "liver qi stagnation", as the core treatment method, and combines Bupleurum chinense, Paeonia lactiflora, Fructus Aurantii Immaturus stir-fried with bran, Angelica sinensis, Prunus persica, Carthamus tinctorius, Ligusticum wallichii, Rehmannia glutinosa, Gardenia jasminoides Ellis stir-fried, Atractylodes macrocephala Koidz stir-fried with bran, Poria cocos, Hirudo, Pheretima, and Glycyrrhiza uralensis Fisch stir-fried. First, it soothes the liver and softens the liver to regulate qi movement to make "liver qi unobstructed", and then combines promoting blood circulation and resolving phlegm, and cooling blood method to achieve "heart qi harmony" so that blood circulation is unimpeded and the blood vessels are smooth, playing a role in inhibiting inflammation and stabilizing AS plaques. It can inhibit serum inflammation and reduce blood lipid levels, inhibit the activation of NLRP3 inflammasome, improve the chronic inflammatory state of the body, stabilize atherosclerotic plaques and delay their pathological progression, reduce the risk of acute cardio-cerebrovascular accidents, and also relieve the symptoms and signs of chest tightness, chest pain, emotional discomfort, hypochondriac discomfort, pale and dull tongue with ecchymosis, and stringy and unsmooth pulse in patients with atherosclerosis and coronary heart disease, and improve the quality of life. In addition, the traditional Chinese medicine composition provided by the present invention also has the characteristics of simple preparation, low cost, safe and non-toxic side effects, convenient to carry, and convenient to take.

Claims

1. A traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response, characterized in that, It includes the following raw materials: Bupleurum chinense, Paeonia lactiflora, Fructus Aurantii Praeparatus, Angelica sinensis, Prunus persica, Carthamus tinctorius, Ligusticum wallichii, Rehmannia glutinosa, Gardenia jasminoides Ellis, Atractylodes macrocephala Koidz. Praeparata, Poria cocos, Hirudo, Pheretima aspergillum, and Radix Glycyrrhizae Preparata.

2. The traditional Chinese medicine composition according to claim 1, wherein By weight, the traditional Chinese medicine composition includes the following raw materials in parts by weight: 5-25 parts of Bupleurum chinense, 5-25 parts of Paeonia lactiflora, 5-20 parts of Fructus Aurantii Praeparatus, 5-20 parts of Angelica sinensis, 5-25 parts of Prunus persica, 5-25 parts of Carthamus tinctorius, 5-20 parts of Ligusticum wallichii, 5-20 parts of Rehmannia glutinosa, 5-20 parts of Gardenia jasminoides Ellis, 5-20 parts of Atractylodes macrocephala Koidz. Praeparata, 7.5-30 parts of Poria cocos, 1.5-8 parts of Hirudo, 5-20 parts of Pheretima aspergillum, and 3-12 parts of Radix Glycyrrhizae Preparata.

3. The traditional Chinese medicine composition according to claim 2, wherein By weight, the traditional Chinese medicine composition includes the following raw materials in parts by weight: 10-25 parts of Bupleurum chinense, 10-25 parts of Paeonia lactiflora, 10-20 parts of Fructus Aurantii Praeparatus, 10-20 parts of Angelica sinensis, 10-25 parts of Prunus persica, 10-25 parts of Carthamus tinctorius, 10-20 parts of Ligusticum wallichii, 10-20 parts of Rehmannia glutinosa, 10-20 parts of Gardenia jasminoides Ellis, 10-20 parts of Atractylodes macrocephala Koidz. Praeparata, 15-30 parts of Poria cocos, 3-8 parts of Hirudo, 10-20 parts of Pheretima aspergillum, and 6-12 parts of Radix Glycyrrhizae Preparata.

4. The traditional Chinese medicine composition according to claim 3, wherein, By weight, the traditional Chinese medicine composition includes the following raw materials in parts by weight: 10-20 parts of Bupleurum chinense, 10-20 parts of Paeonia lactiflora, 10-15 parts of Fructus Aurantii Praeparatus, 10-15 parts of Angelica sinensis, 10-20 parts of Prunus persica, 10-20 parts of Carthamus tinctorius, 10-15 parts of Ligusticum wallichii, 10-15 parts of Rehmannia glutinosa, 10-15 parts of Gardenia jasminoides Ellis, 10-15 parts of Atractylodes macrocephala Koidz. Praeparata, 15-20 parts of Poria cocos, 3-6 parts of Hirudo, 10-15 parts of Pheretima aspergillum, and 6-8 parts of Radix Glycyrrhizae Preparata.

5. The preparation method of the traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response according to any one of claims 1-4, characterized in that, It includes the following steps: Mix the above raw materials with a solvent, extract, and obtain the traditional Chinese medicine composition.

6. The preparation method of the traditional Chinese medicine composition according to claim 5, wherein The solvent is water and / or ethanol.

7. The preparation method of the traditional Chinese medicine composition according to claim 5, wherein, The dosage of the solvent is 2-3 times the total mass of the raw materials.

8. The preparation method of the traditional Chinese medicine composition according to claim 5, characterized in that, The extraction temperature is 70-100 °C, and / or the extraction time is 10-60 minutes, and / or the extraction times is 1-2 times.

9. Use of the traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response according to any one of claims 1-4 in the preparation of drugs for treating atherosclerosis, coronary heart disease, hypertension, hyperlipidemia, diabetes, obesity, or fatty liver.

10. A drug for treating atherosclerosis, coronary heart disease, hypertension, hyperlipidemia, diabetes, obesity, or fatty liver, comprising the traditional Chinese medicine composition for treating atherosclerosis and improving inflammatory response according to any one of claims 1-4.

11. The drug according to claim 10, characterized in that, The dosage form of the drug is at least one of pills, capsules, tablets, ointments, aerosols, suspensions, granules, powders, solutions, creams, lozenges, or films.

12. The drug according to claim 10, wherein The drug further includes excipients, and the excipients are one or more of dextrin, silicon dioxide, and flavoring agents.

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