Application of chest stuffiness relieving composition in preparation of medicine for preventing and / or treating atherosclerosis

Abstract

The invention relates to application of a chest stuffiness relieving composition in preparation of a medicine for preventing and / or treating atherosclerosis. The effective components in the chest stuffiness relieving composition can adjust the pathological processes through various ways, so that the effect of preventing and treating atherosclerosis is achieved. Through repositioning of the chest widening composition, the clinical application range of the chest widening composition is expanded, and a more comprehensive and effective atherosclerosis treatment method is provided.

Description

Technical Field

[0001] The present invention relates to use of a chest-widening composition in preparing a medicine for preventing and / or treating atherosclerosis. Background Art

[0002] Atherosclerosis (AS) is a chronic disease caused by the accumulation of lipids, cholesterol and other substances on the inner wall of the artery to form plaques, causing the arterial wall to thicken and harden. As the plaques gradually form and grow, the arterial lumen becomes narrow and blood flow is obstructed, leading to a series of serious cardiovascular events such as heart attack, stroke and peripheral artery disease. Atherosclerosis is one of the main causes of death and disability worldwide.

[0003] Kuanxiong composition is a clinical drug for treating angina pectoris caused by coronary heart disease. It aims to improve myocardial blood supply, alleviate angina pectoris symptoms, and improve the quality of life of patients through the synergistic effect of multiple active ingredients. Kuanxiong aerosol is a traditional Chinese medicine aerosol developed based on the "aromatic warming and unblocking" theory of "unblocking as supplement, unblocking as the main", which is a known formula preparation in the pharmacopoeia and an effective prescription for rapid analgesia for treating coronary heart disease and angina pectoris. Its main components include asarum oil, sandalwood oil, galangal oil, piper longum oil and borneol. It has the functions of warming and unblocking yang, regulating qi and relieving pain. It is used for chest obstruction caused by yin cold stagnation and qi stagnation, with symptoms of chest tightness and heart pain, cold body and cold limbs, and patients with coronary heart disease and angina pectoris with the above symptoms. The main component of the wide-chest aerosol is volatile oil, which is distributed in a molecular state and can be quickly absorbed into the blood by the human body, dilate coronary blood vessels, significantly improve myocardial ischemia, and quickly relieve angina pectoris symptoms such as chest tightness, chest pain, palpitations, shortness of breath, and fatigue. At the same time, it has no drug resistance, few adverse reactions, and high safety. The composition has proven its effectiveness and safety in improving angina pectoris of coronary heart disease in clinical practice.

[0004] CN202311461513 discloses that the wide-chest composition has the effect of regulating qi and relieving pain, and is currently used to relieve angina pectoris. On this basis, the invention continues to study the new use of the wide-chest composition and expands its use in the preparation of drugs for treating and / or preventing stroke. Among them, "cerebral stroke" (cerebral stroke), also known as "stroke" and "cerebral vascular accident" (cerebralvascular accident, CVA), is an acute cerebrovascular disease, which is a group of diseases caused by brain tissue damage due to sudden rupture of brain blood vessels or blood vessel blockage that prevents blood from flowing into the brain, including ischemic and hemorrhagic stroke.

[0005] CN202011535785 discloses a method for determining multiple components in wide-chest aerosol based on GC-MS / MS, which uses GC-MS / MS to simultaneously determine the contents of 12 components in wide-chest aerosol, including eucalyptol, isoborneol, borneol, methyl eugenol, elemenine, α-pinene, bisabolol, β-pinene, levorotatory camphor, caryophyllene, α-humulene, and trans-nerolidol. It can be quickly absorbed into the blood by the human body, dilate coronary blood vessels, significantly improve myocardial ischemia, and quickly relieve angina pectoris symptoms such as chest tightness, chest pain, palpitations, shortness of breath, and fatigue.

[0006] "Progress in Research on the Chemical Composition and Pharmacological Actions of Piper longum" (Liu Chunlei) disclosed the lipid-lowering effect of unsaponifiable matter and that Piper longum has anti-atherosclerotic effects.

[0007] "An Overview of Research on Piper Longum and Its Active Ingredients in Lowering Lipid and Anti-atherosclerosis" (Ma Chunjie) disclosed that Piper Longum has anti-atherosclerotic effects.

[0008] "Analysis of the mechanism of action of atherosclerosis and intervention of traditional Chinese medicine based on bioinformatics and network pharmacology" (Zhang Wei) disclosed that the thickness of the media of the carotid artery and plaques were reduced after injection of Asarum, and also disclosed the bioinformatics targets of Asarum.

[0009] At present, the methods for preventing and treating atherosclerosis mainly include drug therapy, lifestyle intervention and surgical operation. Drug therapy mainly includes statins, fibrates, niacin and cholesterol absorption inhibitors, etc. These drugs prevent and treat atherosclerosis by reducing blood lipid levels, inhibiting inflammatory responses and improving endothelial function. However, these drugs are usually administered orally, and may have low bioavailability, obvious first-pass effect, and usually have significant side effects. For example, commonly used drugs such as statins may cause side effects such as muscle pain and abnormal liver function, affecting patient compliance. If it is a surgical treatment such as angioplasty and bypass surgery, it is highly invasive and has certain risks and recovery periods. Although surgery can quickly solve the problem of vascular stenosis, the long-term effect is limited and restenosis may occur. Therefore, there is still a demand for drugs with more comprehensive therapeutic effects and lower side effects in the prior art. Summary of the invention

[0010] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and to provide a new indication for the use of a wide-chest composition in the preparation of a drug for preventing and / or treating atherosclerosis. By repositioning the wide-chest composition, its clinical application range is expanded to provide a more comprehensive and effective method for treating atherosclerosis. The discovery of this new indication is not only expected to bring new treatment options to patients with atherosclerosis, but also provides new ideas and directions for the comprehensive prevention and treatment of cardiovascular diseases.

[0011] Specifically, the wide-chest composition can slow down the progression of atherosclerosis through multiple mechanisms such as inhibiting inflammatory response, anti-oxidation, and improving endothelial function. In addition, the good safety and patient compliance of the wide-chest composition make it a promising new treatment option.

[0012] The wide-chest composition of the present invention is intended to overcome the defects of current drugs and methods for preventing and treating atherosclerosis. On the one hand, the sublingual spray administration of the wide-chest composition can improve the absorption efficiency of the drug in the body, thereby improving its bioavailability. On the other hand, existing statins and the like may cause side effects such as muscle pain and abnormal liver function when treating atherosclerosis, affecting the compliance of patients. The present invention reduces the dosage of a single component through the synergistic effect of multiple effective ingredients, thereby reducing drug side effects and improving the safety and compliance of patients. Moreover, the present invention can inhibit vascular inflammatory response and improve vascular endothelial cell function and reduce plaque damage through the comprehensive effect of multiple ingredients, thereby comprehensively treating atherosclerosis from multiple angles and providing a more comprehensive therapeutic effect.

[0013] The functions of the various Chinese medicinal materials used in the Chinese medicine composition of the present invention are as follows: Sandalwood (Scientific name: Santalum album), also known as white sandalwood and white sandalwood, is the heartwood of the trunk of the Santalum tree of the Santalaceae family. It is pungent and warm in nature. It enters the spleen, stomach, heart, and lung meridians. It promotes qi circulation and warms the middle, stimulates appetite and relieves pain. It is used for cold stagnation of qi, chest pain, abdominal pain, stomach pain and loss of appetite; coronary heart disease, angina pectoris.

[0014] Alpinia offinarum Hance, also known as wind ginger and small galangal, is the dried rhizome of the ginger plant. It is dug up in late summer and early autumn, the fibrous roots and remaining scales are removed, washed, cut into sections, and dried in the sun. It is pungent and hot in nature. It enters the spleen and stomach meridians. It is used to warm the stomach and dispel cold, help digestion and relieve pain. It is mainly used to treat cold pain in the abdomen, vomiting due to stomach cold, belching and acid regurgitation.

[0015] Piper longum L., also known as Piper longum and Rat's Tail, is the dried nearly mature or mature fruit clusters of Piper longum, a plant of the Piperaceae family. The fruit clusters are harvested when they turn from green to black, and then the impurities are removed and dried in the sun. It is pungent and hot in nature. It enters the stomach and large intestine meridians. It warms the middle and dispels cold, relieves qi and relieves pain. It is used for cold pain in the abdomen, vomiting, diarrhea, and migraine; it is used externally to treat toothache.

[0016] Asarum sieboldii, also known as Liaoxixin, is harvested in summer when the fruit is ripe or in early autumn, and the whole plant is dried after removing the mud and sand. It is pungent and warm in nature. It enters the heart, lung, and kidney meridians. It is mainly used to dispel wind and cold, open the orifices and relieve pain, warm the lungs and transform fluid. It is used for colds caused by wind and cold, headaches, toothaches, nasal congestion and sinusitis, rheumatic pain, and coughs caused by phlegm and fluid.

[0017] Borneol (scientific name: Borneol) is also known as camphor. It is pungent, bitter and slightly cold. It enters the heart, spleen and lung meridians. It is mainly used to open the mind and refresh the spirit, clear away heat and relieve pain. It is used for coma and convulsions caused by febrile diseases, phlegm syncope caused by stroke, sudden syncope caused by qi stagnation, coma caused by poisoning, red eyes, mouth sores, sore throat and pus in the ear canal.

[0018] The chest-widening composition of the present application is composed of: 70 mL of sandalwood oil, 15 mL of piper longum oil, 32 mL of galangal oil, 23 mL of asarum oil, and 22.5 g of borneol, and the total amount is adjusted to 625 mL with anhydrous ethanol.

[0019] One aspect of the present invention is to provide a use of a wide-chest composition in the preparation of a medicament for preventing and\or treating atherosclerosis.

[0020] In addition to the active ingredient, the drug may contain other pharmaceutically acceptable solvents or excipients that are biocompatible with the above active ingredient or do not affect its stability, such as water, anhydrous ethanol, ethanol, glycerol, propylene glycol, polyethylene glycol, or cell culture medium solution, etc. The drug may also contain a second active ingredient.

[0021] Another aspect of the present invention is to provide a pharmaceutical dosage form of the chest-enlarging composition.

[0022] The drug is in the form of a pharmaceutical preparation.

[0023] The pharmaceutical dosage form is a liquid dosage form, a solid dosage form, a semisolid dosage form or a gas dosage form. Preferably, the pharmaceutical dosage form is an aerosol.

[0024] The pharmaceutical dosage form comprises pharmaceutically acceptable excipients.

[0025] The pharmaceutical composition of the present invention can be prepared into specific dosage forms according to conventional formulation techniques in the art, such as dosage forms for oral administration (e.g., tablets, powders, granules, pills, lozenges, capsules, solutions, emulsions, suspensions, syrups or elixirs), dosage forms for parenteral administration (e.g., sterile injectable solutions, dispersions, suspensions, emulsions, and sterile powders for injection that are re-dissolved in sterile injectable solutions or dispersions before use).

[0026] In order to prepare solid dosage forms for oral administration, solid carriers known in the art can be used, such as lactose, microcrystalline cellulose, sucrose, cyclodextrin, mannitol, gum arabic, sodium carboxymethyl starch, corn starch, potato starch, talc, magnesium stearate, gelatin, etc. In addition, any other carriers commonly used for purposes of flavoring, coloring, preservation, etc. can be used as long as they are compatible with the active ingredients or the ingredients used.

[0027] In order to prepare liquid dosage forms for oral administration, liquid carriers known in the art can be used, such as water, anhydrous ethanol, ethanol, glycerol, propylene glycol, polyethylene glycol and vegetable oils such as peanut oil, sesame oil, soybean oil, olive oil and the like.

[0028] In order to prepare dosage forms for parenteral administration, sterile carriers known in the art can be used, such as water for injection, injection oils such as soybean oil, peanut oil, castor oil, other injection solvents such as ethanol, glycerol, propylene glycol, polyethylene glycol, ethyl oleate, dimethylacetamide, etc. If necessary, various additives such as wetting agents, solubilizers, emulsifiers, suspending agents, buffers, antioxidants, antibacterial agents, isotonicity regulators, etc. can also be used. For sterile powders for injection, fillers and protective agents such as lactose, trehalose, mannitol, glycine and human serum albumin can also be used.

[0029] Specific inhalation preparations can be aerosols, sprays or powders. Aerosols are composed of drugs, optional additives, propellants, pressure-resistant containers and valve systems. In order to prepare aerosols, aerosol excipients known in the art can be used, such as 6 types of aerosols in the second volume of the 2000 edition of the Chinese Pharmacopoeia, and 20 types of aerosols and 6 types of propellants in the 27th edition of the United States Pharmacopoeia. Conventional propellants such as fluorochloroalkanes are generally added with an appropriate amount of ethanol or propylene glycol as a cosolvent.

[0030] The pharmaceutical composition of the present invention can be administered by any suitable route of administration such as oral, pulmonary, topical (including oral and sublingual), transdermal, intracisternal, intraperitoneal and parenteral. Preferably, the pharmaceutical composition of the present invention is administered by sublingual spray.

[0031] To prepare a chest-widening composition, mix the above five ingredients except borneol and the other four ingredients, place in a 40°C water bath, add borneol, heat slightly to dissolve, adjust the total amount to 625 mL with anhydrous ethanol, mix well, and the composition is obtained.

[0032] The present invention relates to a wide-chest composition for preventing and treating atherosclerosis in ApoE- / - mice induced by a high-fat diet. Through multiple pharmacodynamic tests, the wide-chest composition showed significant therapeutic effects in reducing serum CRP and ET-1 levels, reducing the area of ​​arterial plaque damage, and improving lumen stenosis. It significantly improves plaque stability through multiple mechanisms such as anti-inflammatory and improved endothelial function. The method of use is to administer the wide-chest composition sublingually daily at a concentration of 100 μL / L. The prevention group is administered at the same time starting from a high-fat diet. The treatment group is continuously administered for 4 weeks and 8 weeks after the HFD is fed to ApoE- / - mice for 12 weeks to establish a model, and the therapeutic effects under different administration cycles are observed and compared respectively. This scheme provides a new and effective method for the prevention and treatment of atherosclerosis.

[0033] In particular, the wide-chest composition of the present invention is composed of five ingredients: sandalwood oil, piper longum oil, galangal oil, asarum oil and borneol. Although studies have reported that each of its single ingredients has certain pharmacological effects in preventing and treating atherosclerosis, the unique advantage of the wide-chest composition lies in its synergistic effect. In combination, these ingredients can still show significant therapeutic effects at a dosage far lower than that of a single drug. Compared with the use of a single ingredient, the composition not only reduces the dosage of the drug used and reduces the potential risk of adverse reactions, but also improves the overall therapeutic effect, specifically the synergistic effect (see Example 3 or Attachment Figures 7A-7C ). This advantage may be related to the joint action mechanism of multiple components in the combination, including anti-inflammatory, antioxidant, and improvement of endothelial function, and achieving comprehensive therapeutic effects through multi-target regulation. This feature of the wide-chest combination provides new ideas for the development of safer and more effective drugs for the prevention and treatment of atherosclerosis, and further demonstrates the potential and value of traditional Chinese medicine in modern drug research and development.

[0034] Beneficial effects of the present invention: The wide-chest aerosol provided by the present invention was originally used to prevent and treat angina pectoris caused by coronary heart disease, and its main mechanism of action includes improving myocardial blood supply and alleviating angina pectoris symptoms. However, the latest animal experiments have shown that the wide-chest aerosol also shows significant efficacy in the prevention and treatment of atherosclerosis, which is specifically manifested in the following advantages: reducing the formation of atherosclerotic plaques, the wide-chest composition prevents the formation and development of plaques by reducing the deposition of lipids in the arterial wall; the active ingredients in the wide-chest composition can reduce vasoconstriction, protect vascular endothelial cells, and slow down the process of atherosclerosis; the wide-chest composition reduces the infiltration of inflammatory cells in the vascular wall by inhibiting the release and action of inflammatory factors, protects the vascular endothelium, and maintains the normal function of blood vessels. It has the potential advantages of good efficacy, safer, suitable for long-term use, and high clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a design diagram of a pharmacodynamic experiment of the wide-chest composition of the present invention in preventing HFD-induced atherosclerosis in ApoE- / - mice; Figure 2A This is a comparison chart of the C-reactive protein content in the serum of mice in the modeling group and the wide-chest treatment group; Figure 2B This is a comparison chart of endothelin-1 content in the serum of mice in the modeling group and the wide-chest treatment group; Figure 3A This is a comparison of the results of oil red staining of the aorta of mice in the blank group, model group and wide chest treatment group; Figure 3B This is a statistical chart of the percentage of plaque area in the aorta of mice in the blank group, model group and wide-chest treatment group; Figure 4A Comparison of HE, Oil Red and Masson staining of brachiocephalic stem of mice in the blank group, model group and wide chest treatment group; Figure 4B This is a statistical chart of plaque area in brachiocephalic trunk sections of mice in the blank group, model group, and wide-chest treatment group; Figure 4C Comparison of HE, Oil Red and Masson staining of aortic sinus in mice of blank group, model group and wide chest treatment group; Figure 4D The statistical graph of plaque area in aortic sinus sections of mice in the blank group, model group and wide-chest treatment group; Figure 5 This is a design diagram of a drug efficacy verification experiment of the wide-chest composition of the present invention in treating atherosclerotic mice; Fig. 6A This is a comparison of the results of oil red staining of the aorta of atherosclerotic mice after being treated with the wide-chest composition for 1-2 months; Figure 6B This is a statistical graph of the percentage of plaque area in the aorta of atherosclerotic mice after being treated with the wide-chest composition for 1-2 months; Fig. 7A Schematic diagram of the effect of the wide-chest composition of the embodiment on the expression level of HAEC endothelial cell adhesion molecule VCAM1; Figure 7B Schematic diagram of the effect of the wide-chest composition of the embodiment on the expression of HAEC endothelial cell adhesion molecule ICAM1; Figure 7C Schematic diagram of the effect of the wide-chest composition of the example on the expression level of the HAEC endothelial cell adhesion molecule E-Selectin. DETAILED DESCRIPTION

[0036] The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. The present invention provides general and / or specific descriptions of the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible at this point.

[0037] The chest-widening composition in the example is from Zhejiang Suken Pharmaceutical Co., Ltd. (sandalwood oil 70 mL, piper longum oil 15 mL, galangal oil 32 mL, asarum oil 23 mL, borneol 22.5 g, the total amount is adjusted to 625 mL with anhydrous ethanol).

[0038] Example 1 Experimental study on the efficacy of the wide-chest composition in preventing HFD-induced atherosclerosis in ApoE- / - mice 1. Experimental animals: Male ApoE- / - mice with C57BL / 6 background were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. and kept in the SPF barrier environment of the Animal Experiment Center of Zhejiang Suken Pharmaceutical Co., Ltd. The mice were fed with standard rodent food and water in an animal room with a constant temperature and a 12-12h day and night rhythm for one week. All protocols involving animal use were approved by the Ethics Committee of Zhejiang Suken Pharmaceutical Co., Ltd.

[0039] 2. Grouping and modeling and drug administration: 8-week-old male ApoE- / - mice were randomly divided into 3 groups: high-fat feeding group, high-fat feeding + wide-chest combination group, 12 mice in each group, and 10 male mice of the same strain of the same age were used as blank control group. After one week of adaptive feeding, the ordinary feed was replaced with a high-fat diet (HFD) containing 40% fat and 0.15% cholesterol, and the corresponding drug wide-chest combination was sublingually administered at 100 μl / kg / d or an equal amount of normal saline for 12 weeks (see Figure 1 ). Weigh the body weight weekly and adjust the dosage according to the body weight.

[0040] 3. Collection of blood samples and specimens: After the experiment, all animals were fasted for 12 hours but not water, weighed, and their eyeballs were removed after anesthesia to collect blood and centrifuge the supernatant; after the mice in each experimental group were killed, the abdominal and thoracic cavities were dissected open, and the thoracic aorta and abdominal aorta were quickly separated to the bilateral iliac artery branches, fixed with 4% paraformaldehyde and stained with Oil Red O; at the same time, the brachiocephalic artery and heart were fixed with 4% paraformaldehyde, dehydrated with sucrose, and cryo-embedded for staining.

[0041] 4. Testing and indicator observation: 4.1 Effects of the wide-chest combination on hematological parameters of AS mice C-reactive protein (CRP) is extremely sensitive to the body's inflammatory response. When the body is stimulated by inflammation, CRP synthesis increases. Its increased level can induce the production of cell adhesion molecules, promote the expression of tissue factor on the surface of monocytes and the phagocytosis of macrophages, damage vascular endothelial function, and lead to vascular endothelial hyperplasia. Endothelin-1 (ET-1) is a damaging factor that plays an important role in vascular diseases. The increase of ET-1 will disrupt the balance between vasoconstriction and relaxation, leading to vasoconstriction and stenosis. According to the ELISA kit operating procedures, the levels of inflammatory factor CRP and vasoconstrictor factor ET-1 in mouse serum were detected. Figure 2A , 2BAs shown, A is the serum CRP level of mice, and B is the serum ET-1 level of mice; compared with the mice fed with HFD, the serum CRP (p<0.001) and ET-1 (p<0.001) levels of mice in the HFD+KX group were significantly increased, while the administration of the wide-chest composition could significantly reduce the expression levels of serum CRP and ET-1, indicating that the wide-chest composition can significantly reduce the inflammatory level in the serum of AS mice and improve endothelial function.

[0042] 4.2 Effects of the wide-chest composition on the pathological morphology of atherosclerotic plaques in AS mice 4.2.1 Oil red O staining of aorta, brachiocephalic trunk and aortic sinus The tissue sections were placed at room temperature for 30 minutes, transferred to a staining jar and washed three times with PBS, each time for 10 minutes. The sections were placed in a 70% isopropanol solution containing 0.6% Oil red O and stained at room temperature for 20 minutes, washed with water for 10 minutes, drained, stained with hematoxylin dye for 5-10 minutes, and washed with water for 10 minutes. After the sections turned blue, they were taken out and drained, differentiated with a 70% ethanol solution containing 0.5% HCl for 2 seconds, and washed with water for 10 minutes. Drain the water, dip in dilute ammonia water for 8 seconds, and wash with water for 10 minutes. Use filter paper to absorb the surrounding water, seal with a water-soluble sealing agent, and seal with glycerol at the edge of the cover glass and the slide. After drying, observe the image under a microscope.

[0043] Oil Red O is a fat-soluble azo dye, a strong lipid solvent and lipid dye, which can specifically stain neutral triglycerides, lipids, and lipoproteins in tissues and cells. Atherosclerotic plaques are rich in lipids and can be stained red by Oil Red O, so they are often used to evaluate plaque size and lipid content. Figure 3A As shown, the aorta Oil Red O staining results, the red area represents lipid deposition, the lipids in the AS formation area are stained bright red, and the plaques in the HFD group show large areas of red staining, Figure 3B The relative content bar graph of the plaque area in the aorta is given in the figure. The plaque in the HFD feeding group accounts for about 28%, indicating that there is a large amount of lipid deposition in the plaque, while the wide-chest combination treatment group can significantly reduce the content of lipid deposition in the plaque (p < 0.01) ( Figure 3A , 3B ). Figure 4A Brachiocephalic trunk and Figure 4C The results of Oil Red O staining of the aortic sinus showed that the plaques in the HFD group showed large areas of red staining, indicating that there were a large number of lipid deposits in the plaques, while the wide-chest combination group could significantly reduce the lipid content in the plaques.

[0044] 4.2.2 HE and Masson staining of the brachiocephalic trunk and aortic sinus HE staining is to let the tissue sections stand at room temperature for 30 minutes, transfer them to a dyeing jar and wash them three times with PBS, each time for 10 minutes. Add hematoxylin dye to the dyeing jar to cover the sections, dye for 5-10 minutes, and wash with water for 10 minutes. After the sections turn blue, take out the sections, drain the water and put them back into the dyeing jar, differentiate them with a 70% ethanol solution containing 0.5% HCl for 2 seconds, and wash them with water for 10 minutes after differentiation. After draining the water, put them back into the dyeing jar, immerse them in dilute ammonia water for 8 seconds, wash them with water for 10 minutes, put them in eosin solution for staining for 2 minutes, quickly put them into water, and wash them with 70%, 80%, 90% and anhydrous ethanol gradient for 1 minute. Use xylene to make it transparent in the fume hood for 5 minutes, and repeat twice. Take out the sections and place them in the fume hood to dry, add neutral resin to the slide, cover them with a coverslip, and press gently to expel excess air. After drying, observe the image under a microscope.

[0045] Brachiocephalic trunk ( Figure 4A ) and aortic sinus ( Figure 4C ) showed that the HE staining results showed that the relative area bar charts of HE staining lipids were Figure 4B and 4D The cell cytoplasm, erythrocytes, muscle tissue, eosinophilic granules, connective tissue, etc. were stained red or pink to varying degrees. The vascular intima of the brachiocephalic trunk and aortic sinus of the mice in the ND group was smooth and intact, and the various layers of the vascular wall were arranged neatly, with clear structure, few wrinkles and vacuoles, and no AS plaque deposition. In comparison, the intima of the mice in the HFD group was significantly thickened under the induction of a high-fat diet, the lumen was severely narrowed, the plaques increased and deposited, the structure was blurred, and a large number of wrinkles and vacuoles were formed, while the wide-chest combination could significantly reduce the degree of the above-mentioned lesions (p < 0.01).

[0046] For Masson staining, the tissue sections were left at room temperature for 30 min, transferred to a staining jar and washed three times with PBS, each time for 10 min.

[0047] Mordanting was performed in a 60°C incubator for 1 hour, and then washed with water for 10 minutes after dyeing. Put it back into the dyeing tank, dye it with lapis lazuli staining solution for 2-3 minutes, and wash it twice with distilled water, each time for 10-15 seconds. Stain it with Mayer's hematoxylin staining solution for 2-3 minutes, and wash it twice with distilled water, each time for 10-15 seconds. Differentiate it with acidic ethanol differentiation solution for a few seconds until the tissue turns completely red, and wash it with water for 10 minutes. After draining the water, stain it with Ponceau fuchsin staining solution for 10 minutes, wash it twice with water, each time for 10-15 seconds. Treat it with molybdenum phosphate solution for 10 minutes, discard the molybdenum phosphate solution, and add aniline blue dye solution to dye it for 5 minutes. Wash off the aniline blue solution with a weak acid solution and treat it for 2 minutes. Dehydrate it with 95% ethanol and anhydrous ethanol in a gradient manner for 1 minute. Transparent it with xylene in a fume hood twice, each time for 1-2 minutes. Take out the slices and place them in the fume hood to dry, drop neutral resin on the slide, cover it with a coverslip, and press gently to expel excess air. After drying, observe the image under a microscope.

[0048] Brachiocephalic trunk ( Figure 4A ) and aortic sinus ( Figure 4C ) are shown in the figure, collagen fibers are blue, muscle fibers, cellulose, red blood cells, and glial cells are red. The HFD group showed relatively more collagen fiber deposition in the plaque, indicating that the plaque was fibrotic and in a vulnerable state, while the wide-chest combination treatment group could significantly reduce HFD-induced plaque fibrosis and significantly increase plaque stability.

[0049] Example 2 Experimental study on the efficacy of the wide-chest composition in treating atherosclerotic mice 1. Experimental animals: Same as Example 1 2. Group modeling and drug administration: reference Figure 5 8-week-old male ApoE- / - mice were randomly divided into 3 groups: normal feed control group (Baseline), high-fat feeding + saline group (Control), high-fat feeding + wide-chest combination group, 12 mice in each group. After one week of adaptive feeding, the normal feed was replaced with a high-fat diet (containing 40% fat and 0.15% cholesterol) and fed for 12 weeks. After that, the corresponding drug wide-chest combination was administered sublingually by gavage at 100μl / kg / d or by gavage with an equal amount of saline. The mice were weighed weekly and the dosage was adjusted according to the body weight.

[0050] 3. Specimen collection: At 4 and 8 weeks of drug administration, 10 mice were randomly selected from each group and killed. The aorta (from the aortic sinus to the bilateral iliac arteries) was quickly isolated, frozen and sectioned, and then stained with Oil Red O.

[0051] 4. Testing and indicator observation: 4.1 Effects of the wide-chest composition on the pathological morphology of atherosclerotic plaques in AS mice The aorta Oil Red O staining procedure is the same as 4.2.1 of Example 1. After staining, the images were observed and photographed under a light microscope, and the images were calculated using ImageJ software to calculate the lumen area and plaque area, respectively. The total plaque area divided by the lumen area is the relative size of the plaque. The experimental results showed that compared with the model group, the wide-chest composition had a significant effect on improving aortic lipid deposition after one month of treatment (p < 0.05), and after two months of treatment, the wide-chest composition more significantly alleviated the increase in aortic atherosclerotic plaque accumulation in ApoE- / - mice induced by HFD (p < 0.001) ( Fig. 6A , 6B ).

[0052] Example 3 Experimental study on the efficacy of the Kuanxiong composition in inhibiting endothelial inflammation 1. Experimental materials: Test cells: HAEC primary human aortic endothelial cells, purchased from Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center; Main reagents: SteadyPure Universal RNA Extraction Kit II, purchased from Eric Biotechnology; HiScript lllAll-in-one RT SuperMixPerfect for qPCR, purchased from Novozyme Biotechnology; SYBR Green Pro Taq HS Premix qPCR Kit (including ROX), purchased from Eric Biotechnology; 2. Experimental methods: The cultured HAECs were inoculated into two 6-well cell culture dishes (1×106 cells / well) and cultured for 24 hours, with a total of 8 wells plated. The cells were divided into the following groups: ①Blank control group ②TNFα treatment stimulation group ③ Wide-chest combination treatment group ④ Asarum oil treatment group ⑤Sandalwood oil treatment group ⑥ Galangal oil treatment group ⑦ Piper longum oil treatment group ⑧ Borneol treatment group Table 1 Quality standards of finished products of Kuanxiong aerosol

[0053] Table 2 Expression of adhesion molecules in different treatment groups after TNFα stimulation of HAEC endothelial cells

[0054] Place the 6-well plate containing cells in a 37°C, 5% CO 2Cultured overnight in an incubator. On the second day, the culture medium was removed, and cell culture medium containing 5 ng / L TNFα was added to 7 wells, and 0.5 μL DMSO was added to the blank control wells. According to the quality standards of the finished product of the wide-chested combination (see Table 1), the corresponding concentrations of single-component solutions were prepared according to the content ratio (see Table 2), in which the volatile oil was the volume ratio, and the borneol was the mass concentration because it was a solid. 20 μL / L of the wide-chested combination solution or the single-component solution of 5 wide-chested combinations were added to the cell wells of groups ③-⑧ containing TNFα. After continuing to culture for 24 hours, the cell samples were collected, and RNA was extracted according to the instructions of the SteadyPure Universal RNA Extraction Kit II, and the RNA concentration was detected by Nanodrop. Reverse transcription of cDNA was performed with reference to the HiScript lll All-in-one RT SuperMixPerfect for qPCR Kit. Real-time PCR was performed with reference to the instructions of the SYBR Green Pro Taq HS Premixed qPCR Kit, and the gene expression level was analyzed by the 2-△△Ct method with GAPDH as the internal reference.

[0055] 3. Experimental results: In the early stage of atherosclerosis, cytokines in the lesion area induce the expression of adhesion molecules. The expression of adhesion molecules attracts leukocytes in the flow to approach the defective site of endothelial cells and increases the inflammatory response. At the same time, adhesion molecules can also be detected in the blood. VCAM-1 participates in the formation of atherosclerosis by promoting the accumulation of monocytes in the arterial intima. As a prerequisite for leukocyte extravasation at the site of tissue damage, ICAM-1 mediates the adhesion between endothelial cells and leukocytes. This adhesion is one of the early processes of atherosclerosis. In areas susceptible to atherosclerosis, the expression of VCAM-1 and ICAM-1 has been reported to be significantly upregulated. E-selectin mediates the initial rolling and adhesion of leukocytes on the vascular endothelium, which is one of the key steps in the development of atherosclerosis. When endothelial cells are activated by inflammatory factors such as tumor necrosis factor α (TNF-α) or interleukin 1β (IL-1β), the expression of E-selectin increases, promoting the recruitment and migration of leukocytes to the inflammatory site. Therefore, these three detection indicators are often used as one of the gold standards for judging atherosclerosis. The results of this experiment showed that under TNFα pretreatment, the expression levels of VCAM-1, ICAM-1, and E-selectin in the model group increased significantly, while the wide-chest combination treatment group was able to significantly inhibit the expression of the three adhesion factors. At the same time, compared with the five single-component treatment groups of asarum oil, sandalwood oil, galangal oil, piper longum oil, and borneol, the wide-chest combination also showed obvious advantages, while the single components could not effectively inhibit the expression of adhesion factors (see Fig. 7A , 7B, 7C, Table 2). Previous articles have reported that α-santalol 100 mg / kg and sandalwood oil 1g / kg twice a week in mice can effectively resist sugar and oxidation (Biswapriya et al., 2013, Phytomedicine), the effective concentration of galangin for antagonizing endothelial inflammation and the development of atherosclerosis is 50μM / L (equivalent to 13.5mg / L) (Chen Yang et al., 2023, Central South Pharmaceutical Science), and galangin amide can inhibit atherosclerotic plaque formation and vascular smooth muscle cell proliferation at a concentration of 1-5μM (equivalent to 317.34μg-1.59mg / L) (Son et al., 2012, Biochem Biophys Res Commun.), and methyl eugenol, the main component of asarum oil, showed vascular protective effects at a concentration of 10mg / kg (Saadet al., 2004, Life Sci). In addition, 40 mg / kg danshen borneol combined with 20 mg / kg simvastatin showed good effects in the treatment of atherosclerosis (Pu et al., 2016, Analyst). However, we can also see that although a single ingredient has a therapeutic effect on atherosclerosis, the drug concentration is much higher than the concentration of the wide-chest combination here. Therefore, its advantage is that it reduces the dosage of the drug, reduces the potential risk of adverse reactions, and improves the overall treatment effect.

[0056] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-restrictive from any point of view, and the scope of the present invention is defined by the attached technical solutions rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the technical solutions are included in the present invention.

Claims

1. A use of a pharmaceutical composition in the preparation of a drug for preventing and / or treating atherosclerosis, wherein the pharmaceutical composition comprises a wide-chest composition.

2. The use according to claim 1, characterized in that: The chest-widening composition is composed of: 70 mL of sandalwood oil, 15 mL of longan oil, 32 mL of galangal oil, 23 mL of asarum oil, and 22.5 g of borneol, and the total amount is adjusted to 625 mL with anhydrous ethanol.

3. The use according to any one of claims 1 to 2, characterized in that: The pharmaceutical composition also includes pharmaceutically acceptable excipients.

4. The use according to claim 1, characterized in that: The drug is in the form of a pharmaceutical preparation.

5. The use according to claim 4, characterized in that: The preparation is in the form of an aerosol.

6. The use according to claim 1, characterized in that: The drug is used for preventing and treating atherosclerosis in ApoE- / - mice induced by a high-fat diet.

7. A wide-chest composition for preventing and treating atherosclerosis in ApoE- / - mice induced by a high-fat diet, wherein the wide-chest composition reduces serum CRP and ET-1 levels, reduces lipid deposition in arterial plaques, and improves lumen stenosis.

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