Preparation method of traditional Chinese medicine composition and application of traditional Chinese medicine composition in preparation of medicine for preventing and treating hypertrophic cardiomyopathy

Abstract

The invention discloses a preparation method of a traditional Chinese medicine composition and application of the traditional Chinese medicine composition in preparation of a medicine for preventing and treating hypertrophic cardiomyopathy, and belongs to the technical field of traditional Chinese medicines. The technical problem to be solved is to provide the traditional Chinese medicine for treating dilated cardiomyopathy, atherosclerotic plaque, myocardial fibrosis, ventricular remodeling, senile cardiac metabolic disorder, hypertrophic cardiomyopathy, hypertrophic cardiomyopathy and hypertrophic cardiomyopathy aiming at the defects of the prior art in preparation of medicines for preventing and treating hypertrophic cardiomyopathy. The invention also discloses application of the traditional Chinese medicine composition in preparation of medicines for treating hypertrophic cardiomyopathy, wherein the traditional Chinese medicine composition has the effects of improving cardiac degeneration of mice with myocardial hypertrophy induced by blood constriction. According to the key point of the technical scheme, the invention provides the application of the traditional Chinese medicine composition in preparing the medicine for preventing and treating hypertrophic cardiomyopathy. The traditional Chinese medicine composition comprises the following raw materials: American ginseng, ginseng, astragalus membranaceus, spina date seeds, salvia miltiorrhiza, pseudo-ginseng, fingered citron and mangnolia officinalis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a method for preparing a traditional Chinese medicine composition and its application in the preparation of drugs for the prevention and treatment of hypertrophic cardiomyopathy. Background Technology

[0002] Hypertrophic cardiomyopathy (HCM) is a myocardial disease of unknown cause, characterized by asymmetrical thickening of the ventricular walls, often involving the interventricular septum, resulting in a smaller ventricular cavity, obstructed left ventricular filling, and decreased left ventricular diastolic compliance. Based on the presence or absence of left ventricular outflow tract obstruction, it is classified into obstructive and non-obstructive hypertrophic cardiomyopathy, possibly related to genetics. The annual cardiovascular mortality rate for HCM patients is 1%-2%. Clinically, a left ventricular outflow tract gradient (LVOTG) ≥30 mm Hg at rest or after provocation is defined as hypertrophic obstructive cardiomyopathy (HOCM). Statistics show that approximately 75% of HCM patients are obstructive. Compared to non-obstructive hypertrophic cardiomyopathy patients, HOCM patients have a worse long-term survival rate, and relieving outflow tract obstruction helps reduce mortality. Hypertrophic cardiomyopathy carries a risk of sudden death and is one of the causes of exercise-induced sudden death.

[0003] Hypertrophic cardiomyopathy (HCM) refers to cardiac dysfunction that occurs in patients with hypertension and ischemic coronary artery disease. Early manifestations include left ventricular diastolic dysfunction, but the ejection fraction may be normal. As the disease progresses, pathological features such as myocardial calcium overload, myocardial hypertrophy, and myocardial fibrosis appear. The pathogenesis of HCM is complex, involving mitochondrial energy metabolism disorders, oxidative stress, glucose and lipid toxicity, and calcium homeostasis imbalance. Unfortunately, there are currently no effective methods in clinical practice to prevent or halt the progression of HCM.

[0004] Due to the complex pathogenesis and clinical phenotype of hemolytic ileus (HCM), effective treatments are currently lacking. In recent years, the clinically accepted optimal targeted intervention strategy for HCM is coronary artery dilation, increasing coronary blood flow, and improving myocardial circulation. While new drugs for treating cognitive impairment have emerged and new targets have been discovered, drugs with good efficacy and minimal side effects are scarce. Traditional Chinese medicine (TCM) is a natural medicine whose effects are mostly multi-pathway and multi-systemic. Moreover, TCM has fewer side effects than chemical drugs. Currently, the medicinal activity and efficacy of TCM are a research hotspot both domestically and internationally. Ginseng and American ginseng are known for "nourishing the five internal organs, calming the mind, and invigorating the spirit," and are rich in ginsenosides, various sugars, and vitamins. They have a bidirectional regulatory effect on the heart in various animals, exhibiting both excitatory and inhibitory effects. Magnolia officinalis, isolated from Magnolia officinalis, enhances myocardial contractility, increases cardiac output, and improves cardiac pumping function. Tanshinone IIA sodium sulfonate and tanshinone in Salvia miltiorrhiza can dilate coronary arteries, increase coronary blood flow, improve myocardial circulation, and regulate heart rate. Panax notoginseng tablets can increase glomerular filtration rate and promote renal blood flow, thus having a diuretic effect. Panax notoginseng alcohol extract has a cardiotonic effect, enhancing myocardial contractility, slowing heart rate, and increasing cardiac output in weakened hearts. Astragalus polysaccharides in Astragalus membranaceus can inhibit oxidative stress and enhance and regulate cardiac function. Jujube seed saponins in Ziziphus jujuba var. spinosa have antioxidant, sedative, and hypnotic effects, possessing powerful anti-inflammatory activity that reduces the production of inflammatory factors in the heart and protects cardiac function. Bergamot lactone isolated from Citrus medica reduces lipid peroxides and reactive oxygen species in myocardial cells, inhibits oxidative stress, promotes angiogenesis, improves cardiac microcirculation, and enhances cardiac pumping function.

[0005] In recent years, American ginseng, ginseng, astragalus, jujube seed, salvia miltiorrhiza, notoginseng, citron, and magnolia bark have been widely used in cardiovascular diseases. However, these herbs also have potential applications in cerebrovascular diseases. Related studies report that the bioavailability of American ginseng, ginseng, astragalus, jujube seed, salvia miltiorrhiza, notoginseng, citron, and magnolia bark when taken orally alone is not high.

[0006] Relevant patent documents retrieved: This document, published in China (CN103285322A) on September 11, 2013, discloses a traditional Chinese medicine preparation for treating hypertrophic cardiomyopathy and its preparation method. The herbal composition includes turmeric, myrrh, leech, sandalwood, ophiopogon japonicus, schisandra chinensis, thistle, polygala tenuifolia, pearl, glehnia littoralis, borneol, bezoar, pinellia ternata, astragalus membranaceus, and alisma plantago-aquatica. The invention aims to prevent and treat hypertrophic cardiomyopathy by increasing angiogenesis; improving myocardial contractile function and reducing diastolic dysfunction; reducing myocardial tissue pathological damage and inflammatory infiltration; increasing endogenous antioxidant capacity; and improving cardiac microcirculation.

[0007] This document, published in China (CN103263574A) on August 28, 2013, discloses a traditional Chinese medicine composition for the prevention or treatment of hypertrophic cardiomyopathy and its uses. The composition comprises *Spatholobus suberectus*, *Ligusticum chuanxiong*, *Taenia sibiricum*, *Typha orientalis*, *Lilium brownii*, *Schisandra chinensis*, *Cirsium japonicum*, *Ziziphus jujuba* seed, dragon bone, *Adenophora stricta*, borneol, cinnamon twig, *Pinellia ternata*, *Astragalus membranaceus*, and *Alisma plantago-aquatica*. The composition alleviates systemic metabolic disorders in mice with hypertrophic cardiomyopathy, improves myocardial microcirculation, thereby inhibiting myocardial hyperplasia and hypertrophy, as well as the proliferation of endocardial fibrosis, and has a protective effect against myocardial structural changes in a hypertrophic cardiomyopathy model mouse.

[0008] Relevant non-patent literature retrieved: The journal *Tianjin Journal of Traditional Chinese Medicine* published an article titled "Modern Research Progress on TCM Syndromes and Mechanisms of Action of Traditional Chinese Medicine in Myocardial Hypertrophy," Volume 40, dated June 30, 2023. This article discloses that myocardial hypertrophy is an abnormal enlargement of cardiomyocytes caused by pressure or volume overload, which in turn affects cardiac gene transcription, protein synthesis, metabolism, oxidative stress, autophagy, and other pathways, ultimately leading to chronic heart failure. Traditional Chinese medicine (TCM) has unique advantages in the prevention, treatment, and rehabilitation of cardiovascular diseases. TCM syndrome differentiation can more comprehensively, profoundly, and accurately reflect the essence of diseases. This article reviews four TCM syndromes of myocardial hypertrophy: Qi deficiency and blood stasis syndrome, phlegm and blood stasis syndrome, Qi and Yin deficiency syndrome, and Yang deficiency and water retention syndrome, along with their pathogenesis, and the research progress on the application of traditional Chinese medicine in the treatment of myocardial hypertrophy based on syndrome differentiation, aiming to provide new ideas for the clinical prevention and treatment of myocardial hypertrophy.

[0009] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: (1) The composition of the drug lacks specificity and does not focus on the core pathological needs of HCM. The relevant evidence is: Publication No. CN103285322A. The composition of the drug in this literature contains 16 kinds of Chinese medicine such as turmeric, myrrh, and leech. The composition is complex and does not clearly target the core pathological links of HCM such as myocardial hypertrophy, inflammation, and mitochondrial dysfunction.

[0010] (2) Excessive number of medicinal ingredients increases the risk of component interactions. Relevant evidence is provided in publication number CN103285322A, which states that unknown component interactions may occur when multiple Chinese medicinal ingredients coexist. For example, the cholic acid in bezoar and the alkaloids in pinellia may undergo a complexation reaction, reducing the activity of both. The volatile components of borneol may accelerate the oxidation of resinous components in myrrh, producing toxic metabolites.

[0011] (3) The cost of raw material procurement and storage is high. The relevant evidence is: Publication No. CN103285322A. The supply of raw materials is unstable. Pearls require special storage conditions (moisture-proof and light-proof), and the storage cost is high. In industrial production, the more types of raw materials there are, the greater the pressure on procurement channel management and inventory turnover, which can easily lead to a surge in production costs.

[0012] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: The search for mechanisms that can act on multiple pathological aspects of hepatocellular carcinoma (HCM), such as improving mitochondrial energy metabolism, inhibiting oxidative stress, regulating calcium homeostasis, reducing glucose and lipid toxicity, and myocardial fibrosis, aims to achieve multi-target regulation of HCM and holistic treatment. This involves regulating the body's internal environment, dilating coronary arteries, increasing coronary blood flow, and enhancing myocardial cell function and tolerance, thereby delaying HCM progression and reducing the risk of sudden death. This requires synergistic cooperation among the components of traditional Chinese medicine such as American ginseng and ginseng to compensate for the low bioavailability of individual herbs and achieve the aforementioned effects. Summary of the Invention

[0013] The purpose of this invention is to provide: A method for preparing a traditional Chinese medicine composition and its application in the preparation of drugs for the prevention and treatment of hypertrophic cardiomyopathy, and related technologies, to solve technical problems such as improving left ventricular systolic and diastolic function, regulating abnormal cardiac function, anti-inflammation, and regulating angiogenesis disorders in mice with hypertrophic cardiomyopathy, or combinations thereof.

[0014] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0015] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0016] Definitions of standard chemical terms can be found in the references Chinese Pharmacopoeia 2025 edition and Complete Explanation of Pharmacological Terminology.

[0017] Unless otherwise stated, conventional methods within the scope of the art, such as thin-layer chromatography (TLC), infrared spectroscopy (IR), high-performance liquid chromatography (HPLC), and in vitro antibacterial experiments, shall be used.

[0018] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0019] The term "American ginseng" used in this article refers to the dried root of the American ginseng plant (Panax quinquefolius), a member of the Araliaceae family. It is commonly used as a tonic ingredient in pharmaceutical preparations. In these preparations, it is often sliced, pulverized, or extracted before use. Its core functions are to replenish qi and nourish yin, clear heat and promote the production of body fluids. It is commonly found in oral liquids, capsules, granules, and other preparations, and is used to improve symptoms such as fatigue, dry mouth, and sore throat caused by qi and yin deficiency.

[0020] The term "ginseng" as used in this article refers to the dried root and rhizome of Panax ginseng, a plant belonging to the genus Panax in the family Araliaceae. In pharmaceutical preparations, it can be directly pulverized and used (e.g., in tablets or powders), or used as an extraction raw material to obtain effective components such as ginsenosides. Its main effects are to greatly replenish vital energy, restore pulse and consolidate the body, and tonify the spleen and lungs. It is suitable for the development of preparations for symptoms such as qi deficiency and impending collapse, cold limbs and weak pulse, and spleen deficiency with poor appetite.

[0021] The term "Astragalus" used in this article refers to the dried root of Astragalus membranaceus, a legume, which is one of the core ingredients for replenishing Qi in traditional Chinese medicine preparations. In pharmaceutical production, it can be used raw (sliced ​​or crushed) or after being processed with honey. Raw Astragalus is more effective in consolidating the exterior and stopping sweating, and promoting diuresis and reducing swelling, while processed Astragalus is more effective in replenishing Qi and raising Yang. It is often used in granules, oral liquids, injections, etc., to improve symptoms such as Qi deficiency and fatigue, poor appetite and loose stools, edema and oliguria.

[0022] The term "sour jujube seed" used in this article refers to the dried, mature seeds of the sour jujube plant (Ziziphus jujuba), a member of the Rhamnaceae family. It is a core ingredient in traditional Chinese medicine preparations used for calming the mind and body. In preparations, it is typically processed through pulverization and extraction (such as alcohol or water extraction). Its core functions include nourishing the heart and liver, calming the mind and soothing the nerves, reducing sweating, and promoting body fluid production. It is commonly found in calming tablets, capsules, and compound preparations, used to improve symptoms such as insomnia due to deficiency and restlessness, palpitations and excessive dreaming, and excessive sweating due to physical weakness.

[0023] The term "Sanqi" as used in this article refers to the dried root and rhizome of Panax notoginseng, a plant belonging to the genus Panax notoginseng in the family Araliaceae. In pharmaceutical preparations, it can be directly pulverized and used as medicine (such as in powders and capsules) or as an extraction raw material. Its core functions are to disperse blood stasis, stop bleeding, reduce swelling, and relieve pain. It is commonly used in hemostatic granules, tablets, and external patches to improve symptoms such as hemoptysis, hematemesis, traumatic bleeding, and swelling and pain from falls.

[0024] The term "Buddha's Hand" as used in this article refers to the dried fruit of the citrus plant (Citrus medica var. sarcodactylis), also known as Buddha's Hand Citrus or Hand Citrus. It has a pungent, bitter, and sour taste, is warm in nature, and enters the liver, spleen, stomach, and lung meridians. It has the effects of soothing the liver and regulating qi, harmonizing the stomach and relieving pain, and drying dampness and resolving phlegm. It is mainly used to treat symptoms such as liver and stomach qi stagnation, chest and rib pain, epigastric fullness, poor appetite and vomiting, and cough with excessive phlegm.

[0025] The term "Buddha's Hand" used in this article refers to the dried bark of the trunk, root, and branches of *Magnolia officinalis* or *Magnolia davidii*, both belonging to the Magnoliaceae family. It is also known as Thick Bark, Red Bark, and Fierce Bark. It has a bitter and pungent taste, is warm in nature, and enters the spleen, stomach, lung, and large intestine meridians. It has the functions of drying dampness and resolving phlegm, lowering qi and relieving fullness, and relieving asthma. It can be used for symptoms such as dampness stagnation in the middle jiao, epigastric fullness and vomiting / diarrhea, food stagnation and qi stagnation, abdominal distension and constipation, and phlegm-dampness cough and wheezing.

[0026] The term "processed Buddha's Hand" used in this article refers to the dried fruit of the Buddha's Hand plant (Citrus medica var. sarcodactylis), a member of the Rutaceae family, after processing (such as steaming, stir-frying, or honey-processing). It is a raw material for regulating qi in traditional Chinese medicine preparations. Processing reduces its irritation and enhances its qi-regulating and stomach-harmonizing effects. In preparations, it is often used after being pulverized or extracted. Its effects include soothing the liver and regulating qi, harmonizing the stomach and relieving pain, and drying dampness and resolving phlegm. It is commonly found in qi-regulating compound preparations, tablets, and pills, and is used to improve symptoms such as liver and stomach qi stagnation, chest and rib distension and pain, and epigastric fullness.

[0027] The term "Jiang Houpu" used in this article refers to the dried bark, root bark, and branch bark of the Magnolia officinalis plant (Magnolia officinalis), processed with ginger, alum, and other excipients (such as ginger-processed or ginger-boiled). It is a core ingredient in traditional Chinese medicine preparations for regulating qi and drying dampness. Processing can mitigate its harshness and enhance its warming and drying effects. In preparations, it is often used after being pulverized or extracted. Its effects include drying dampness and resolving phlegm, and relieving qi stagnation and fullness. It is commonly used in qi-regulating and dampness-drying pills, tablets, granules, etc., to improve symptoms such as dampness stagnation in the middle jiao, epigastric fullness and vomiting, diarrhea, food stagnation, and qi stagnation.

[0028] The term "solvent" as used in this article refers to a class of chemical substances used to extract, separate, and purify the active ingredients in medicinal materials, or to dissolve and disperse materials to prepare finished formulations. It is a key medium connecting raw materials and formulations, and its selection directly affects the yield of active ingredients, the stability of the formulation, and its safety.

[0029] The term "extraction" as used in this article refers to the core unit operation in the production of traditional Chinese medicine preparations. It refers to the process of separating the active ingredients (or effective fractions) from medicinal materials or processed medicinal slices using suitable solvents (such as water, ethanol, methanol, etc.) and processes (such as decoction, reflux, percolation, ultrasound, microwave, etc.). Its purpose is to enrich the active ingredients, remove ineffective impurities, and provide qualified intermediate products (such as extracts, extracts, and dry powders) for subsequent purification and shaping. The optimization of the extraction process directly affects the quality and efficacy of the preparation.

[0030] The term "mixing" as used in this article refers to a key unit operation in the production of traditional Chinese medicine preparations. It refers to the process of uniformly dispersing two or more materials (such as medicinal powders, extract powders, excipients, etc.) using appropriate equipment and processes. Its core requirement is to ensure the homogeneity of the mixed materials to avoid fluctuations in the preparation content due to uneven component distribution, which could affect efficacy or cause safety risks. It is commonly used in the preparation of solid dosage forms such as powders, tablets, and capsules.

[0031] The term "macroporous resin enrichment and purification" used in this article refers to a purification technique that utilizes the porous structure and selective adsorption capacity of macroporous adsorption resins to separate impurities and enrich effective components (such as saponins, flavonoids, alkaloids, etc.) from medicinal extracts. It is one of the core methods for refining the effective parts of traditional Chinese medicine, which can significantly improve the purity of target components and reduce the dosage of preparations.

[0032] In a first aspect, the present invention provides: the application of a traditional Chinese medicine composition in the preparation of a drug for the prevention and treatment of hypertrophic cardiomyopathy, wherein the raw materials of the traditional Chinese medicine composition include American ginseng, ginseng, astragalus, jujube seed, salvia miltiorrhiza, notoginseng, citron and magnolia bark.

[0033] This includes technical features: traditional Chinese medicine composition and application.

[0034] Preferably, the Buddha's Hand is a type of hand-made Buddha's Hand. Preferably, the magnolia bark is ginger-processed magnolia bark; More preferably, the raw materials of the traditional Chinese medicine composition, by weight, include 2-12 parts American ginseng, 4-18 parts ginseng, 5-40 parts astragalus, 5-30 parts jujube seed, 5-30 parts salvia miltiorrhiza, 4-18 parts notoginseng, 3-20 parts citron and 3-20 parts magnolia bark. For example, the raw materials of the traditional Chinese medicine composition, by weight, include: 2-6 portions, 6-12 portions, 8-10 portions, 3-8 portions, 4-9 portions, 5-11 portions, 7-10 portions, 3-11 portions, or 4-12 portions of American ginseng. 4-17 portions, 5-18 portions, 6-18 portions, 9-15 portions, 7-10 portions, 8-15 portions, 7-17 portions, or 5-10 portions of ginseng. 5-30 portions, 5-35 portions, 5-25 portions, 5-10 portions, 10-15 portions, 25-40 portions, 30-40 portions, 6-40 portions, or 10-40 portions of Astragalus membranaceus. 5-25 portions, 5-20 portions, 5-15 portions, 5-10 portions, 6-30 portions, 7-25 portions, 8-25 portions, or 10-30 portions of jujube seed. 5-25 portions, 5-20 portions, 5-15 portions, 5-10 portions, 6-30 portions, 7-25 portions, 8-25 portions, or 10-30 portions of Salvia miltiorrhiza; 4-17 portions, 5-18 portions, 6-18 portions, 9-15 portions, 7-10 portions, 8-15 portions, 7-17 portions, or 5-10 portions of Panax notoginseng; 3-19 servings, 4-20 servings, 3-15 servings, 3-10 servings, 10-20 servings, 15-20 servings, 5-10 servings, or 5-20 servings of Buddha's Hand; And 3-19 parts, 4-20 parts, 3-15 parts, 3-10 parts, 10-20 parts, 15-20 parts, 5-10 parts or 5-20 parts of Magnolia officinalis.

[0035] As a further example, the raw materials of the traditional Chinese medicine composition, by weight, include: 2, 3, 4, 5, 6, 7, 8, 9, or 12 portions of American ginseng. 4, 5, 6, 7, 8, 9, 10, 15, or 18 portions of ginseng. 5, 6, 7, 8, 9, 10, 15, 20, or 40 portions of Astragalus membranaceus. 5, 6, 7, 8, 9, 10, 15, 20, or 30 portions of jujube seeds. 5, 6, 7, 8, 9, 10, 15, 20 or 30 portions of Salvia miltiorrhiza; 4, 5, 6, 7, 8, 9, 10, 15, or 18 portions of Panax notoginseng. 3, 4, 5, 6, 7, 8, 9, or 20 servings of Buddha's Hand citron. And 3, 4, 5, 6, 7, 8, 9 or 20 parts of Magnolia officinalis.

[0036] More preferably, the raw materials of the traditional Chinese medicine composition, by weight, include 2-10 parts American ginseng, 4-10 parts ginseng, 5-15 parts astragalus, 5-10 parts jujube seed, 5-10 parts salvia miltiorrhiza, 4-10 parts notoginseng, 3-10 parts citron and 3-10 parts magnolia bark. More preferably, the raw materials of the traditional Chinese medicine composition, by weight, include 3 parts American ginseng, 4.5 parts ginseng, 10 parts astragalus, 7.5 parts jujube seed, 7.5 parts salvia miltiorrhiza, 4.5 parts notoginseng, 5 parts citron and 5 parts magnolia bark.

[0037] Preferably, the method for preparing the traditional Chinese medicine composition includes the steps of: dissolving each medicinal material separately in a solvent, extracting and then mixing them to obtain the traditional Chinese medicine composition.

[0038] This includes technical features such as solvent, extraction, and mixing.

[0039] The solvent is selected from at least one of water, ethanol, ethyl acetate, methanol, and isopropanol; The solvent is preferably at least one of water and ethanol; The solvent is further preferably ethanol; The solvent is more preferably 50wt%-70wt% ethanol; The solvent is preferably 60 wt% ethanol.

[0040] The ratio of the medicinal material to the solvent is 1:20-80. The preferred material-to-solvent ratio of the medicinal material to the solvent is 1:20-60; The preferred ratio of the medicinal material to the solvent is 1:20-50; The ratio of the medicinal material to the solvent is more preferably 1:30; Preferably, the medicinal material is further pulverized before being dissolved in the solvent; More preferably, the pulverizing method is selected from at least one of the following: high-speed electric grinder, traditional Chinese medicine crusher, high-speed universal pulverizer, hammer mill, vibratory mill, and air jet mill; A further preferred method is to use a traditional Chinese medicine crusher for pulverizing.

[0041] More preferably, the medicinal material is pulverized and then passed through a 50-mesh sieve; The extraction method is selected from at least one of the following: hot reflux extraction, ultrasonic-assisted extraction, percolation extraction, cold soaking extraction, supercritical fluid extraction, microwave-assisted extraction, and accelerated solvent extraction. The extraction method is preferably at least one of hot reflux extraction, ultrasonic-assisted extraction, and percolation extraction. The extraction method is further preferably hot reflux extraction; Preferably, the reaction conditions for the hot reflux extraction are: heating temperature of 60-95℃ and reaction time of 1-3h; More preferably, the reaction conditions for the hot reflux extraction are: heating temperature of 60-85℃ and reaction time of 1-2h; More preferably, the reaction conditions for the hot reflux extraction are: heating temperature of 80°C and reaction time of 1.5 h.

[0042] Preferably, the extraction operation further includes a purification step; More preferably, the purification method is selected from at least one of precipitation, adsorption, and membrane separation. The purification method is further preferably adsorption; The purification method is more preferably at least one of macroporous resin enrichment and purification and activated carbon adsorption. The preferred purification method is macroporous resin enrichment and purification. The mixing method is selected from at least one of container rotary mixing, stirring mixing, pipeline mixing, and dispersion mixing; The preferred mixing method is at least one of container rotary mixing, stirring mixing, and dispersion mixing; The mixing method is further preferably a stirring-type mixing; The mixing ratio of the extracts of the medicinal materials is as follows: American ginseng extract, ginseng extract, astragalus extract, jujube seed extract, salvia miltiorrhiza extract, notoginseng extract, citron extract and magnolia bark extract are mixed in a volume ratio of 1:1-5:1-5:1-5:1-5:1-5:1-5:1-5:1-5:1-5 by volume. Preferably, the mixing ratio is as follows: American ginseng extract, ginseng extract, astragalus extract, jujube seed extract, salvia miltiorrhiza extract, notoginseng extract, citron extract and magnolia bark extract are mixed in a volume ratio of 1:1-3:3-5:1-3:1-3:1-3:1-3:1-3:1-3 by volume. More preferably, the mixing ratio is as follows: American ginseng extract, ginseng extract, astragalus extract, jujube seed extract, salvia miltiorrhiza extract, notoginseng extract, citron extract and magnolia bark extract are mixed in a volume ratio of 1:1.5:3.3:2.5:2.5:1.5:1.67:1.67.

[0043] Preferably, the mixing process further includes a drying step; More preferably, the drying method includes, but is not limited to, at least one of freeze drying, spray drying, oven drying, air drying, blow drying, natural drying, and vacuum drying; More preferably, the drying method is oven drying.

[0044] Preferably, the preparation method of the traditional Chinese medicine composition includes the following steps: (1) Weigh the Chinese medicinal materials according to the weight ratio, crush them, and dissolve them in 60wt% ethanol solution at a material-to-liquid ratio of 1:30; heat and reflux at 80℃ for 1.5h to obtain the extract of each medicinal material; (2) The extract obtained in step (1) is mixed with American ginseng extract, ginseng extract, astragalus extract, jujube seed extract, salvia miltiorrhiza extract, notoginseng extract, citron extract and magnolia bark extract in a volume ratio of 1:1.5:3.3:2.5:2.5:1.5:1.67:1.67, and dried to obtain the Chinese herbal composition.

[0045] Preferably, the application is that the traditional Chinese medicine composition has the effect of treating dilated cardiomyopathy, atherosclerotic plaques, myocardial fibrosis, ventricular remodeling, and senile cardiac metabolic disorders, and improving cardiac degeneration in mice with myocardial hypertrophy induced by blood flow constriction; More preferably, the application is that the traditional Chinese medicine composition has the effects of dispelling pathogens and strengthening the body's resistance, invigorating the heart and promoting blood circulation, regulating qi and replenishing qi, and nourishing the heart and calming the mind. It can promote the absorption of Panax notoginseng saponins, ginseng saponins, bergapten lactone, magnolol, jujube seed saponins, tanshinone, astragalus polysaccharides, etc., and improve their bioavailability in the body. Therefore, the combination of American ginseng, ginseng, astragalus, jujube seed, tanshinone, Panax notoginseng, bergapten, and magnolia bark will exert their medicinal effects to a greater extent.

[0046] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred embodiment: the raw materials of the traditional Chinese medicine composition, by weight, include 2-12 parts American ginseng, 4-18 parts ginseng, 5-40 parts astragalus, 5-30 parts jujube seed, 5-30 parts salvia miltiorrhiza, 4-18 parts notoginseng, 3-20 parts citron and 3-20 parts magnolia bark; preferably, the raw materials of the traditional Chinese medicine composition, by weight, include 2-10 parts American ginseng, 4-10 parts ginseng, 5-15 parts astragalus, 5-10 parts jujube seed, 5-10 parts salvia miltiorrhiza, 4-10 parts notoginseng, 3-10 parts citron and 3-10 parts magnolia bark; more preferably, the raw materials of the traditional Chinese medicine composition, by weight, include 3 parts American ginseng, 4.5 parts ginseng, 10 parts astragalus, 7.5 parts jujube seed, 7.5 parts salvia miltiorrhiza, 4.5 parts notoginseng, 5 parts citron and 5 parts magnolia bark. This technical solution, having already addressed the technical problem of "prevention and treatment of hypertrophic cardiomyopathy," further addresses the technical problem of "further prevention and treatment of hypertrophic cardiomyopathy."

[0047] The second preferred option is as follows: the mixing ratio of the extracts from the various medicinal herbs is: American ginseng extract, ginseng extract, astragalus extract, jujube seed extract, salvia miltiorrhiza extract, notoginseng extract, citron extract, and magnolia bark extract are mixed in a volume ratio of 1:1-5:1-5:1-5:1-5:1-5:1-5:1-5:1-5; preferably, the mixing ratio is: American ginseng extract, ginseng extract, astragalus extract, jujube seed extract, salvia miltiorrhiza extract, and notoginseng extract... The extracts of ginseng, citronella, and magnolia bark are mixed in a volume ratio of 1:1-3:3-5:1-3:1-3:1-3:1-3:1-3:1-3; more preferably, the mixing ratio is: American ginseng extract, ginseng extract, astragalus extract, jujube seed extract, salvia miltiorrhiza extract, notoginseng extract, citronella extract, and magnolia bark extract are mixed in a volume ratio of 1:1.5:3.3:2.5:2.5:1.5:1.67:1.67. This technical solution, while addressing the technical problem of "prevention and treatment of hypertrophic cardiomyopathy," further addresses the technical problem of "further prevention and treatment of hypertrophic cardiomyopathy."

[0048] The third preferred option: the purification method is selected from at least one of precipitation, adsorption, and membrane separation; the preferred purification method is adsorption; the further preferred purification method is at least one of macroporous resin enrichment purification and activated carbon adsorption; the most preferred purification method is macroporous resin enrichment purification. This technical solution, while addressing the technical problem of "prevention and treatment of hypertrophic cardiomyopathy," further addresses the technical problem of "further prevention and treatment of hypertrophic cardiomyopathy."

[0049] The fourth preferred option is that the extraction method is selected from at least one of the following: hot reflux extraction, ultrasound-assisted extraction, percolation extraction, cold maceration extraction, supercritical fluid extraction, microwave-assisted extraction, and accelerated solvent extraction; preferably, at least one of hot reflux extraction, ultrasound-assisted extraction, and percolation extraction; and more preferably, hot reflux extraction. This technical solution, while addressing the technical problem of "prevention and treatment of hypertrophic cardiomyopathy," further addresses the technical problem of "further prevention and treatment of hypertrophic cardiomyopathy."

[0050] The embodiments of this invention at least support the protection scope of the technical features "traditional Chinese medicine composition", "solvent", "extraction", "mixing" and "application".

[0051] The technical feature "traditional Chinese medicine composition" is summarized from the corresponding technical features in the foregoing explanation and / or examples, such as "the raw materials of the traditional Chinese medicine composition include American ginseng, ginseng, astragalus, jujube seed, salvia miltiorrhiza, notoginseng, citronella, and magnolia bark," and "the raw materials of the traditional Chinese medicine composition, by weight, include 3 parts American ginseng, 4.5 parts ginseng, 10 parts astragalus, 7.5 parts jujube seed, 7.5 parts salvia miltiorrhiza, 4.5 parts notoginseng, 5 parts citronella, and 5 parts magnolia bark." Therefore, those skilled in the art can reasonably presume that the technical feature "traditional Chinese medicine composition," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace "traditional Chinese medicine composition" within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0052] The technical feature "solvent" is derived from the aforementioned explanation and / or examples of the corresponding technical feature "at least one of water, ethanol, ethyl acetate, methanol, and isopropanol," and is summarized by the common feature "a class of chemical substances used to extract, separate, and purify the effective components in medicinal materials, or to dissolve and disperse materials to prepare finished formulations; it is a key medium connecting raw materials and formulations, and its selection directly affects the yield of effective components, the stability of the formulation, and its safety." Therefore, those skilled in the art can reasonably infer that the technical feature "solvent," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace "solvent" within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of this invention. For example, replacing "solvent" with "ethanol," "purified water," etc., while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0053] The technical feature "extraction" is derived from the corresponding technical features in the foregoing explanation and / or examples, such as "at least one of hot reflux extraction, ultrasonic-assisted extraction, percolation extraction, cold maceration extraction, supercritical fluid extraction, microwave-assisted extraction, and accelerated solvent extraction," which are summarized by the common feature "a core unit operation in the production of traditional Chinese medicine preparations, referring to the process of separating the effective components (or effective parts) from medicinal materials or processed medicinal slices using suitable solvents and processes." Therefore, those skilled in the art can reasonably presume that the technical feature "extraction," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace "extraction" within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of this invention. For example, replacing "extraction" with "reflux extraction" or "extraction" while keeping other technical features unchanged still falls within the protection scope of this invention.

[0054] The technical feature "mixing" is derived from the corresponding technical features "at least one of container rotary mixing, stirring mixing, pipeline mixing, and dispersion mixing" explained above and / or in the embodiments, and is summarized by the common feature "the process of uniformly dispersing two or more materials (such as medicinal powder, extract powder, excipients, etc.) using suitable equipment and processes." Therefore, those skilled in the art can reasonably presume that the technical feature "mixing," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace "mixing" based on existing technology and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing "mixing" with "stirring mixing" while keeping other technical features unchanged still falls within the protection scope of this invention.

[0055] The technical feature "application" is summarized from the foregoing explanation and / or the corresponding technical feature in the embodiments, such as "the traditional Chinese medicine composition has the efficacy of treating dilated cardiomyopathy, atherosclerotic plaques, myocardial fibrosis, ventricular remodeling, and senile cardiac metabolic disorders, and improving cardiac degeneration in mice with induced myocardial hypertrophy due to blood flow constriction." Therefore, those skilled in the art can reasonably presume that the technical feature "application," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0056] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: (1) This invention provides a new traditional Chinese medicine product that is effective, safe and inexpensive for the prevention and / or treatment of hypertrophic cardiomyopathy.

[0057] (2) This compound Chinese medicine can improve the left ventricular systolic and diastolic function, regulate abnormal cardiac function, reduce inflammation, and regulate angiogenesis disorder in hypertrophic cardiomyopathy mice. Attached Figure Description

[0058] Figure 1 A chart showing the cardiac function of mice and statistical graphs of left ventricular ejection fraction, fractional shortening, diastolic diameter, and systolic diameter (compared with the HCM group, #: p<0.05; compared with the sham group, *: P<0.05). Figure 2 Image showing the volume and appearance of a mouse heart; Figure 3 Statistical plots of the ratios of heart weight to body weight and heart weight to tibia length in mice (compared with the HCM group, #: p<0.05; compared with the sham group, *: p<0.05). Figure 4 Statistical graph of BNP and β-MHC expression in mouse myocardium (compared with HCM group, #: p<0.05; compared with sham group, *: P<0.05); Figure 5 This is a statistical graph showing the expression of HE in mice. Figure 6 A statistical graph showing WGA expression in mice; Figure 7 Statistical graph of apoptosis-related protein expression in mice (compared with HCM group, #: p<0.05; compared with sham group, *: P<0.05). Figure 8 Statistical graph of expression of mitochondrial function-related proteins in mice (compared with HCM group, #: p<0.05; compared with sham group, *: P<0.05). Detailed Implementation

[0059] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0060] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0061] Specific raw material information is shown in Table 1: Table 1. Raw Material Information

[0062] Example 1 (1) Weigh out the Chinese medicinal materials according to the following weight ratios: 3 parts American ginseng, 4.5 parts ginseng slices, 10 parts astragalus, 7.5 parts jujube seed, 7.5 parts salvia miltiorrhiza, 4.5 parts notoginseng slices, 5 parts prepared Buddha's hand, and 5 parts ginger magnolia bark. Crush the medicinal materials and dissolve them in 60wt% ethanol solution at a material-to-liquid ratio of 1:30. Heat and reflux at 80℃ for 1.5h to obtain the extracts of each medicinal material. (2) The extract obtained in step (1) is mixed with American ginseng extract, ginseng slice extract, astragalus extract, jujube seed extract, salvia miltiorrhiza extract, notoginseng slice extract, prepared citron extract and magnolia bark extract in a volume ratio of 1:1.5:3.3:2.5:2.5:1.5:1.67:1.67, and dried to obtain the Chinese herbal composition.

[0063] Example 1: The traditional Chinese medicine composition improved cardiac function in mice with hypertrophic cardiomyopathy. 1. Establish a mouse model of hypertrophic cardiomyopathy. Eight-week-old male SPF-grade C57BL / 6N mice were housed in a standard animal facility (temperature 22±1℃, humidity 55±5%) for one week to acclimatize. The mice were then randomly divided into a control group and a model group. A hypertrophic cardiomyopathy model was induced in the mice using aortic arch coarctation surgery.

[0064] 2. Group administration of drugs to animals Mice that successfully developed the model were divided into a model group and a drug-treated group. The experiment consisted of a total of 6 groups, with 15 mice in each group. The specific grouping is as follows: Sham group (control group): normal mice, without any drugs; HCM group (model group): Mice with hypertrophic cardiomyopathy were not given drugs; Low-dose group: For mice with hypertrophic cardiomyopathy, the traditional Chinese medicine composition prepared in Example 1 was administered by gavage at a dose of 60 mg / kg / day for 4 consecutive weeks. Medium dose group: For mice with hypertrophic cardiomyopathy, the traditional Chinese medicine composition prepared in Example 1 was administered by gavage at a dose of 120 mg / kg / day for 4 consecutive weeks; High-dose group: For mice with hypertrophic cardiomyopathy, the traditional Chinese medicine composition prepared in Example 1 was administered by gavage at a dose of 180 mg / kg / day for 4 consecutive weeks. Captopril group: Mice with hypertrophic cardiomyopathy were administered the positive control drug Captopril by gavage at a dose of 25 mg / kg / day for 4 consecutive weeks. The sham group and HCM group were given corresponding doses of dispersible excipients by gavage, and the diet of each group remained unchanged for 4 consecutive weeks.

[0065] 3. Doppler ultrasound detection of cardiac function in mice Four weeks after the successful establishment of the hypertrophic cardiomyopathy model, the cardiac function of mice was assessed using a small animal ultrasound device, Vevo 2100. First, the mice were weighed according to the need for ultrasound examination. An anesthetic agent (0.1 mL / g body weight) of azedarach (2.7 g tribromoethanol + 1.43 mL tert-amyl alcohol + 100 mL physiological saline, stored protected from light) was injected intraperitoneally into the mice. After the mice were comatose, hair removal cream was applied to the chest area to remove hair. After hair removal, the mice were secured to the operating table with medical tape. The isoflurane gas anesthesia canister was turned on, and the mice were put on gas anesthesia masks. Conductive adhesive was applied thoroughly to the hair-removed chest area. The Doppler ultrasound probe was vertically aimed at the mouse's chest area, and dynamic and static images were captured after accurate positioning. The probe was then rotated 90 degrees clockwise to the short axis to obtain dynamic and static echocardiograms of the mice. The instrument's internal analysis system was used to calculate various cardiac function indicators. Each indicator for each mouse was manually located three times consecutively, and the average value was taken. The static images captured during the experiment were manually processed using a Doppler ultrasound instrument. The instrument automatically calculated various indicators of the mouse's cardiac function based on the selected location. Each indicator for each mouse was manually located three times, and the average value was taken.

[0066] 4. Statistical processing Statistical analysis of all experimental data was performed using GraphPad Prism 8.0 software. Quantitative data were presented as mean ± standard deviation, and all experiments were repeated at least three times. A t-test was used to compare the means of two independent samples, and a one-way ANOVA was used to compare the means of multiple samples. A p-value < 0.05 was considered statistically significant.

[0067] 5. Heart function test results Clinically, Doppler ultrasound is mainly used to assess cardiac systolic function and the degree of disease in diabetic patients by measuring left ventricular ejection fraction (LVEF%), fractional shortening (FS%), left ventricular internal dimension in systole (LVIDs), and left ventricular internal dimension in diastole (LVIDd). Echocardiographic examination showed that hypertrophic cardiomyopathy mice exhibited cardiac dysfunction, and low-dose, medium-dose, and high-dose groups all significantly improved their cardiac function. Figure 1 ).

[0068] Example 2: The traditional Chinese medicine composition improved myocardial hypertrophy in mice with hypertrophic cardiomyopathy. 1. Comparison of mouse heart size Four weeks after the successful establishment of the hypertrophic cardiomyopathy model, mouse hearts were harvested, and the appearance changes of each mouse heart were observed and photographed. At the same time, the weight of the mouse heart and the length of the tibia were measured.

[0069] 2. PCR detection of changes in hypertrophy gene expression (1) Extraction of total RNA from tissues Take approximately 50 μg of mouse heart tissue from each group, frozen at -80℃, and place it in an enzyme-free EP tube. Add 1 mL of Trizol and let it stand at room temperature for 5-10 min to allow complete lysis. Transfer the lysate to a 1.5 mL enzyme-free EP tube. Add 200 µL of chloroform to each 1 mL of Trizol and homogenize until no tissue fragments are visible to the naked eye. Let it stand at room temperature for 5 min and then centrifuge at 12000 rpm for 15 min (4℃). Transfer 450 µL of the supernatant to a new EP tube containing 600 µL of frozen isopropanol, mix well, let it stand for 5 min, and then centrifuge at 12000 rpm for 10 min (4℃). Discard the supernatant, add 1 mL of frozen 75% ethanol, and centrifuge at 7500 rpm for 10 min (4℃). Discard the ethanol, air dry the EP tube, and after the ethanol has evaporated, add 10-20 µL of DEPC water. Label the tube and store at -80℃.

[0070] (2) Determination of RNA purity and concentration by ultraviolet spectrophotometry Follow the instructions for using the Nanophotometer ultra-micro spectrophotometer. Before measurement, zero the instrument using DEPC-treated water for dissolving RNA. Determine RNA purity using the A260 / A280 ratio, and obtain the RNA concentration from the reading at 260 nm.

[0071] (3) Reverse transcription reaction Table 2. Reagents used in the reverse transcription reaction

[0072] After the sample is added, centrifuge and mix it, then place it in a reverse transcriptometer, select the Toyobo program, and the reverse transcription process takes about 20 minutes. After reverse transcription, the RNA is converted into cDNA and stored at -80℃.

[0073] (4) System preparation and loading: The total system added to each well is 20 μL. Total system = SYBR Green Master + R / F primers + ddH2O + cDNA; SYBR Green Master = 10μL; 1 μL each of R / F primers; 7 μL of ddH2O; 1 μL of cDNA.

[0074] Finally, cDNA was amplified by qRT-PCR. Forty amplification cycles were performed using a pre-defined chain amplification program (95℃, 10 min; 95℃, 15 s; 60℃, 30 s; 72℃, 30 s). During this process, the cDNA in the PCR system repeatedly replicated the same target gene DNA fragment. Each cycle, the DNA fragment was amplified exponentially and labeled with a fluorescent signal, which was captured by the ABI 7500 fast system. The fewer cycles required for the captured fluorescence signal to reach the pre-defined fluorescence threshold (i.e., cycle value CT), the higher the expression level of the target gene corresponding to the primers in the cDNA sample. Therefore, by substituting the obtained CT value into the 2-ΔΔCT algorithm, the target gene in the tissue sample can be semi-quantitatively analyzed after standardization with an internal reference gene, thereby determining whether the expression level of the target gene has changed and the extent of such changes.

[0075] 3. WGA staining was used to observe the morphology of myocardial tissue in mice with hypertrophic cardiomyopathy.

[0076] (1) Cut 5µm sections from the paraffin block, retrieve the sections, and mount them; (2) Place the slices in the oven and bake at 56℃ for 30 minutes; (3) Routine dewaxing to water: Place the sections in xylene I for 10 minutes, xylene II for 10 minutes, and xylene III for 10 minutes respectively, followed by immersion in 100%, 100%, 90%, 80%, and 70% alcohol for 5 minutes each to remove xylene, rinse with ultrapure water for 5 minutes to remove alcohol; (4) Antigen retrieval: Place a 1000 mL beaker in a water bath, add 490 mL of double-distilled water and heat to 95°C. Then, place 10 mL of EDTA antigen retrieval solution and the slide into the beaker in sequence, boil for 20 min, and then cool to room temperature. It can be washed 3 times in PBS, 5 min each time. (5) Wipe away excess liquid around the tissue with filter paper, circle the tissue with an immunohistochemical pen, add 20 μL of 0.5% Triton-X 100 penetration solution to each tissue, penetrate for 30 min, and then rinse 3 times with PBS. (6) Remove PBS, place in a light-proof box, add 20 μL of WGA staining solution (1:200) to each tissue section, and incubate at room temperature for 2 h; (7) Rinse 3 times with PBS; mount with anti-fluorescence quenching mounting medium (containing DAPI, water-soluble); (8) Observation of mouse heart tissue morphology under an Olympus optical microscope.

[0077] 4. Statistical processing Statistical analysis of all experimental data was performed using GraphPad Prism 8.0 software. Quantitative data were presented as mean ± standard deviation, and all experiments were repeated at least three times. A t-test was used to compare the means of two independent samples, and a one-way ANOVA was used to compare the means of multiple samples. A p-value < 0.05 was considered statistically significant.

[0078] 5. Test Results Previous studies have shown that hypertrophic cardiomyopathy patients and mice exhibit significant cardiac structural changes, mainly characterized by increased heart volume, ventricular wall thickening, ventricular remodeling, and cardiomyocyte hypertrophy. Heart weight / tibia length and heart weight / body weight are used to reflect myocardial hypertrophy. Results showed that compared to the control group, the model group mice had significantly increased heart volume, and the low-dose, medium-dose, and high-dose groups all showed significant recovery (…). Figure 2 Furthermore, compared with the control group, the ratios of heart weight / tibia length and heart weight / body weight were significantly increased in the model group, while these ratios were significantly restored in the low-dose, medium-dose, and high-dose groups. Figure 3 Furthermore, compared to the control group, the ratio of hypertrophy genes in the model group was significantly increased, while the low-dose, medium-dose, and high-dose groups all showed significant recovery. Figure 4 Furthermore, compared to the control group, the ratio of hypertrophic morphological surface area was significantly increased in the model group, while the low-dose, medium-dose, and high-dose groups all showed significant recovery. Figure 5 ).

[0079] The inhibitory effect of the traditional Chinese medicine composition described in Example 3 on myocardial inflammation in mice with hypertrophic cardiomyopathy was detected. 1. Observation of myocardial tissue morphology in hypertrophic cardiomyopathy mice using H&E staining.

[0080] (1) Cut 5µm sections from the paraffin block, retrieve the sections, and mount them; (2) Place the slices in the oven and bake at 56℃ for 30 minutes; (3) Routine dewaxing to water: Place the sections in xylene I for 10 minutes, xylene II for 10 minutes, and xylene III for 10 minutes respectively, followed by immersion in 100%, 100%, 90%, 80%, and 70% alcohol for 5 minutes each to remove xylene, rinse with ultrapure water for 5 minutes to remove alcohol; (4) Stain with hematoxylin and argyroum extract for 10-15 minutes, then rinse the slide with pure water to remove excess stain. (5) Differentiate with 0.5% hydrochloric acid ethanol for a few seconds, then rinse with running water for 3-5 minutes; (6) Stain with eosin solution for 4 minutes, then rinse with pure water for 3 minutes; (7) Dehydration: Soak in 100% alcohol for 5 minutes each; (8) Clear the specimen with xylene I for 10 minutes and xylene II for 10 minutes to make it clear; (9) Sealing: Add an appropriate amount of neutral resin and cover with a coverslip; (10) Morphological observation of mouse heart tissue under an Olympus optical microscope ( Figure 6 ).

[0081] 2. Statistical processing Statistical analysis of all experimental data was performed using GraphPad Prism 8.0 software. Quantitative data were presented as mean ± standard deviation, and all experiments were repeated at least three times. A t-test was used to compare the means of two independent samples, and a one-way ANOVA was used to compare the means of multiple samples. A p-value < 0.05 was considered statistically significant.

[0082] 3. H&E Experiment Results H&E staining results showed that the myocardial tissue of mice in the Sham group had a compact morphology and structure, while the myocardial tissue of mice in the HCM group was damaged, with disrupted structure, significant cell nucleus aggregation, and inflammation accumulation. The low-dose, medium-dose, and high-dose groups significantly inhibited the levels of these inflammatory factors and reduced myocardial tissue damage in mice. These results suggest that the protective effect of the traditional Chinese medicine composition against cardiac dysfunction in hypertrophic cardiomyopathy mice may be partly due to its inhibition of inflammation levels. Figure 6 ).

[0083] The inhibitory effect of the traditional Chinese medicine composition described in Example 4 on mitochondrial apoptosis of cardiomyocytes in hypertrophic cardiomyopathy mice was detected. 1. Western blot detection of cardiomyocyte-related protein expression Total protein samples were extracted from each group of cells. Gels were prepared according to the specified ratio and poured into the assembled gel casting plate. After the gels coagulated, the casting plate was placed in the electrophoresis tank, and the electrophoresis buffer was also poured in. The comb was removed, and the denatured protein samples were loaded sequentially at 90 µg, with 1–5 µl of protein marker added to one well on either the left or right side. During electrophoresis, the stacking gel was charged at 70 V, and the voltage was changed to 110 V when the samples reached the separating gel. The appropriate electrophoresis time was determined based on the separation state of the marker bands and the molecular weight of the target protein. NC membranes and filter papers of the same size as the gels to be transferred were extracted and immersed in the electrophoresis buffer for approximately 10 min to equilibrate. After electrophoresis, the sponges were placed in the electrophoresis tank in the following order: sponge – 3 layers of filter paper – NC membrane – separating gel – 3 layers of filter paper – sponge. Air bubbles were to be eliminated between each layer, and the upper and lower filter papers should not touch. With the gel negative and the membrane positive, transfer the membrane at 300 mA for 50-110 min. After transfer, block the membrane: remove the NC membrane with tweezers, pour in the prepared 5% skim milk powder, and shake at room temperature for 2 h; discard the milk powder, add PBST, and wash the membrane 3 times, 5 min each time. Primary antibody incubation: dilute β-actin antibody (1:1000), Bax antibody (1:500), Bcl-2 antibody (1:400), and Caspase-3 (1:500) with TBS, and incubate overnight (or more than 16 h) at 4℃; recover the primary antibody, and wash the membrane 3 times with PBST, 5 min each time. Secondary antibody incubation: select the corresponding secondary antibody according to the primary antibody, and prepare the secondary antibody reaction solution (1:8000) with PBS; immerse the NC membrane in the secondary antibody reaction solution and incubate at room temperature for 50 min; discard the secondary antibody, wash the membrane 3 times with PBST, 5 min each time; develop the membrane.

[0084] 2. Statistical processing Statistical analysis of all experimental data was performed using GraphPad Prism 8.0 software. Quantitative data were presented as mean ± standard deviation, and all experiments were repeated at least three times. A t-test was used to compare the means of two independent samples, and a one-way ANOVA was used to compare the means of multiple samples. A p-value < 0.05 was considered statistically significant.

[0085] 3. Western blot experimental results Compared with the control group, the ratio of apoptosis protein expression levels in the model group was significantly increased, while it was significantly restored in the low-dose, medium-dose, and high-dose groups. The ratio of anti-apoptotic protein expression levels in the model group was significantly decreased, while it was significantly restored in the low-dose, medium-dose, and high-dose groups. Figure 7 Compared with the control group, the ratio of mitochondrial functional protein expression levels in the model group was significantly increased, while the low-dose, medium-dose, and high-dose groups all showed significant recovery. Figure 8 ).

[0086] Verification of technical effectiveness and / or analysis of technical problem solving This invention establishes a mouse model of hypertrophic cardiomyopathy induced by aortic arch constriction and studies the effects of the traditional Chinese medicine composition on cardiac dysfunction, left ventricular diastolic and systolic dysfunction, increased inflammatory factors, and angiogenesis disorders caused by hypertrophic cardiomyopathy in mice.

[0087] Pharmacological studies have shown that the Chinese herbal composition can significantly improve left ventricular systolic and diastolic function in hypertrophic cardiomyopathy mice, regulate abnormal cardiac function, reduce inflammation, and regulate angiogenesis disorders.

[0088] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of drugs for the prevention and treatment of hypertrophic cardiomyopathy, characterized in that, The raw materials of the traditional Chinese medicine composition include American ginseng, ginseng, astragalus, jujube seed, salvia miltiorrhiza, notoginseng, citronella and magnolia bark.

2. The application according to claim 1, characterized in that, The raw materials of the Chinese herbal composition, by weight, include 2-12 parts American ginseng, 4-18 parts ginseng, 5-40 parts astragalus, 5-30 parts jujube seed, 5-30 parts salvia miltiorrhiza, 4-18 parts notoginseng, 3-20 parts citron and 3-20 parts magnolia bark. Preferably, the raw materials of the traditional Chinese medicine composition, by weight, include 2-10 parts of American ginseng, 4-10 parts of ginseng, 5-15 parts of astragalus, 5-10 parts of jujube seed, 5-10 parts of salvia miltiorrhiza, 4-10 parts of notoginseng, 3-10 parts of citron and 3-10 parts of magnolia bark. More preferably, the raw materials of the traditional Chinese medicine composition, by weight, include 3 parts American ginseng, 4.5 parts ginseng, 10 parts astragalus, 7.5 parts jujube seed, 7.5 parts salvia miltiorrhiza, 4.5 parts notoginseng, 5 parts citron and 5 parts magnolia bark.

3. The application according to any one of claims 1-2, characterized in that, The preparation method of the traditional Chinese medicine composition includes the following steps: dissolving each medicinal material in a solvent, extracting and then mixing them to obtain the traditional Chinese medicine composition.

4. The application according to claim 3, characterized in that, The solvent is selected from at least one of water, ethanol, ethyl acetate, methanol and isopropanol; preferably at least one of water and ethanol; more preferably ethanol; even more preferably 50wt%-70wt% ethanol; and most preferably 60wt% ethanol.

5. The application according to any one of claims 3-4, characterized in that, The ratio of the medicinal material to the solvent is 1:20-80; preferably 1:20-60; more preferably 1:20-50; and even more preferably 1:

30. Preferably, the medicinal material is further pulverized before being dissolved in the solvent; More preferably, the pulverization method is selected from at least one of a high-speed electric grinder, a traditional Chinese medicine crusher, a high-speed universal pulverizer, a hammer mill, a vibratory mill, and an air jet mill; even more preferably, it is pulverized using a traditional Chinese medicine crusher; and even more preferably, the pulverized medicinal materials are passed through a 50-mesh sieve.

6. The application according to claim 3, characterized in that, The extraction method is selected from at least one of hot reflux extraction, ultrasound-assisted extraction, percolation extraction, cold soaking extraction, supercritical fluid extraction, microwave-assisted extraction, and accelerated solvent extraction; preferably at least one of hot reflux extraction, ultrasound-assisted extraction, and percolation extraction; more preferably hot reflux extraction. Preferably, the reaction conditions for the hot reflux extraction are: heating temperature of 60-95℃ and reaction time of 1-3h; more preferably, heating temperature of 60-85℃ and reaction time of 1-2h; and even more preferably, heating temperature of 80℃ and reaction time of 1.5h.

7. The application according to claim 3 or claim 6, characterized in that, The extraction process further includes a purification step; the purification method is selected from at least one of precipitation, adsorption and membrane separation; preferably adsorption; more preferably at least one of macroporous resin enrichment and purification and activated carbon adsorption; and even more preferably macroporous resin enrichment and purification.

8. The application according to claim 3, characterized in that, The mixing method is selected from at least one of container rotary mixing, stirring mixing, pipeline mixing and dispersion mixing; preferably at least one of container rotary mixing, stirring mixing and dispersion mixing; more preferably stirring mixing.

9. The application according to claim 3, characterized in that, The mixing ratio of the extracts of the medicinal materials is as follows: American ginseng extract, ginseng extract, astragalus extract, jujube seed extract, salvia miltiorrhiza extract, notoginseng extract, citron extract and magnolia bark extract are mixed in a volume ratio of 1:1-5:1-5:1-5:1-5:1-5:1-5:1-5:1-5:1-5 by volume. Preferably, the mixing ratio is as follows: American ginseng extract, ginseng extract, astragalus extract, jujube seed extract, salvia miltiorrhiza extract, notoginseng extract, citron extract and magnolia bark extract are mixed in a volume ratio of 1:1-3:3-5:1-3:1-3:1-3:1-3:1-3:1-3 by volume. More preferably, the mixing ratio is as follows: American ginseng extract, ginseng extract, astragalus extract, jujube seed extract, salvia miltiorrhiza extract, notoginseng extract, citron extract and magnolia bark extract are mixed in a volume ratio of 1:1.5:3.3:2.5:2.5:1.5:1.67:1.

67.

10. The application according to claim 3, characterized in that, The mixing process further includes a drying step; preferably, the drying is at least one of freeze drying, spray drying, oven drying, air drying, blow drying, natural drying, and vacuum drying; more preferably, the drying method is oven drying.

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