RU2806632C1 - Composition of traditional chinese medicine, method of its preparation and its use - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions relates to compositions of traditional Chinese medicine for the prevention or treatment of elevated blood lipid levels, elevated triglyceride levels, elevated cholesterol levels, hyperlipidemia and atherosclerosis. A traditional Chinese medicine composition for the prevention or treatment of elevated blood lipid levels, elevated triglyceride levels, elevated cholesterol levels, hyperlipidemia and atherosclerosis, wherein the traditional Chinese medicine composition is prepared from the following raw materials, in the following parts by weight: 20–30 parts of Scutellaria root, 10–20 parts of unripe orange fruit, 5–15 parts of rhubarb root, 5–10 parts of Baikal skullcap root, 5–15 parts of white peony root, 5–10 parts of dried ginger rhizome, 7–15 parts of leeches, 5–15 parts of licorice root, 5–15 parts of dried codonopsis root, 5–15 parts of Atractylodes macrocephala rhizome and 10–20 parts of pinellia tuber. A traditional Chinese medicine composition for the prevention or treatment of elevated blood lipids, elevated triglycerides, elevated cholesterol, hyperlipidemia and atherosclerosis, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 20–30 parts of Bupleurum root, 10–20 parts of unripe orange fruit, 5–15 parts of rhubarb root, 5–10 parts of Baikal skullcap root, 5–15 parts of white peony root, 5–10 parts dried ginger rhizome, 7–15 parts leeches, 5–15 parts of licorice root, 5–15 parts of dried codonopsis root, 5–15 parts of Atractylodes macrocephala rhizome, 10–20 parts of pinelli tuber and 10–35 parts of oyster shell. A traditional Chinese medicine composition for the prevention or treatment of elevated blood lipid levels, elevated triglyceride levels, elevated cholesterol levels, hyperlipidemia and atherosclerosis, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 20–30 parts of Bupleurum root, 10–20 parts of unripe orange fruit, 5–15 parts of rhubarb root, 5–10 parts of Baikal skullcap root, 5–15 parts of white peony root, 5–10 parts of dried ginger rhizome, 7–15 parts of leeches, 5–15 parts of licorice root, 5–15 parts of dried codonopsis root, 5–15 parts of Atractylodes macrocephala rhizome, 10–20 parts of pinellia tuber, 10–35 parts of oyster shell and 5–12 parts of red fermented rice.

EFFECT: above-described compositions are effective for the prevention or treatment of elevated blood lipids, elevated triglycerides, elevated cholesterol, hyperlipidemia and atherosclerosis.

8 cl, 8 dwg, 15 tbl, 6 ex

Description

TECHNICAL FIELD

The present invention relates to the field of traditional Chinese medicine and, in particular, to a traditional Chinese medicine composition, a method for its preparation and the use of a traditional Chinese medicine composition in the preparation of a medicament for preventing or treating high blood lipid levels, high triglyceride levels, high cholesterol levels, hyperlipidemia and atherosclerosis.

BACKGROUND OF THE ART

Blood lipids are a general term for various lipid substances in the blood, the most important of which are cholesterol and triglycerides (also called neutral fat). Other important lipids include lipoproteins, which include low-density lipoproteins and high-density lipoproteins. The average blood cholesterol level in healthy people is 2.8–5.17 mmol/L, and the average blood triglyceride level is 0.56–1.7 mmol/L. Either elevated cholesterol levels, elevated triglyceride levels, or a combination of these two pathological conditions can be called hyperlipidemia.

The "Chronic Disease and Nutrition Report of the Chinese People" published by the National Health and Family Planning Commission in early 2015 indicated that the incidence of dyslipidemia in 2012 was 40.40% among Chinese adults, a significant increase compared with 2002. g., when the incidence of hypercholesterolemia was 4.9%, and the incidence of hypertriglyceridemia was 13.1%. These findings indicate a widespread condition of dyslipidemia among residents, which poses a major challenge to the prevention and treatment of dyslipidemia in China.

Atherosclerosis (AS) is the leading cause of coronary heart disease, ischemic stroke and peripheral vascular disease. Hyperlipidemia is the pathological basis of atherosclerosis and is characterized by the accumulation of lipids and complex carbohydrates, hemorrhage, thrombosis and subsequent hyperplasia of fibrous tissue and calcareous deposits starting from the inner lining of the arteries, and gradual breakdown and calcification in the tunica media of the affected arteries, ultimately leading to thickening and hardening of the arterial wall and narrowing of the lumen of blood vessels. Such pathological changes often affect the large and medium-sized muscular arteries, and once the changes progress to the point of occluding the lumen of the arteries, the tissues or organs supplied by such arteries are subject to ischemia or necrosis. Lipids that accumulate on the inner lining of the arteries are yellow in color and have an atheromatous appearance, so their accumulation is called atherosclerosis.

Atherosclerotic cardiovascular disease is known as a “major killer” in developed countries, and its incidence is rapidly increasing in developing countries. Atherosclerosis is responsible for the majority of deaths from myocardial infarction and stroke. By 2035, more than 130 million adults (45.1%) in the United States are expected to suffer from some form of cardiovascular disease, with a total financial cost of up to $1.1 trillion. The incidence and mortality of cardiovascular diseases in China are still on the rise. In China, cardiovascular diseases affect about 290 million people and rank first in mortality, accounting for more than 30% of deaths from diseases. Of course, hyperlipidemia does not necessarily mean cardiovascular disease, but active treatment of hyperlipidemia is one of the important measures to prevent cardiovascular disease.

Proper diet and proper exercise are effective and necessary measures to control and prevent elevated blood lipid levels and thus reduce the harm of atherosclerotic cardiovascular diseases. However, when diet and exercise are ineffective, pharmacotherapy becomes the inevitable choice.

Currently, various chemical lipid-lowering agents are used in clinical practice, which mainly include statins and fibrates. Although the specific mechanisms of action of these drugs vary, they generally work by directly targeting fat synthetic and metabolic pathways. Due to the high rate of recurrence of elevated blood lipids after treatment cessation, synthetic chemical lipid-lowering agents require long-term use, resulting in a loss of economic resources and significantly increasing the likelihood of toxicity and adverse effects. For example, long-term use is often associated with adverse effects of varying severity, including muscle pain, liver and kidney dysfunction, etc. In addition, the use of chemical lipid-lowering agents cannot fundamentally prevent and control the development and progression of vascular plaque growth.

Lipid-lowering agents based on natural preparations of traditional Chinese medicine include Dantian Jiangzhi Wan (Hongxing, Guangdong), Zhibituo (DIAO, Chengdu), Xuezhikang (Weixin, Beijing), etc. As can be seen from the instructions for these drugs, they have certain adverse effects to varying degrees. For example, the adverse effects of Xuezhikang include gastrointestinal discomfort such as abdominal pain, bloating, heartburn, etc., and may sometimes include a reversible increase in serum aminotransferase and creatine phosphokinase levels. In addition, these drugs are generally recommended in the literature for combination use with chemical lipid-lowering agents or for use in the elderly, etc.

Therefore, there is still a strong clinical need for traditional Chinese medicine drugs to effectively prevent and treat elevated blood lipids, elevated triglycerides, elevated cholesterol, and hyperlipidemia. Meanwhile, there are currently no methods for preventing and controlling vascular plaques and treating atherosclerosis based on traditional Chinese medicine. Therefore, there is great social and market demand for them.

SUMMARY OF THE INVENTION

The present invention is directed to a traditional Chinese medicine composition, a method of preparation and use for producing a medicament for preventing or treating high blood lipid levels, high triglyceride levels, high cholesterol levels, hyperlipidemia, atherosclerosis and the like. Animal model studies and clinical studies have shown that the traditional Chinese medicine composition described herein has excellent effects in lowering blood lipids, lowering cholesterol, and in particular lowering blood triglyceride levels. Meanwhile, animal model studies and clinical studies have also shown that the traditional Chinese medicine composition described herein has good effects in slowing down the formation of atherosclerotic plaques and removing them.

The inventor of the present invention proposed a treatment method after long-term study of the theory of traditional Chinese medicine and based on clinical practice, which is significantly different from the theories of modern medicine regarding the prevention and treatment of high blood lipids, high triglycerides, high cholesterol, hyperlipidemia and atherosclerosis. It is believed that the underlying cause of hyperlipidemia and subsequent atherosclerosis can be summarized using the logic and language of traditional Chinese medicine as follows: phlegm-fluid retention in the blood. This is similar to the concept of increased viscosity in the context of modern medicine. Increased viscosity is not only the cause of hyperlipidemia and atherosclerosis, but also a disorder manifested by the retention of phlegm-fluid in the blood. Since phlegm-fluid retention in the blood in traditional Chinese medicine refers to the concept of hyperviscosity in modern medicine, types of modulating therapy are considered to be the main and effective means of preventing and treating hyperlipidemia and atherosclerosis.

"Phlegm-fluid retention" is defined as moisture leaving the meridians in The Yellow Emperor 's Classic of Internal Medicine , i.e. body fluid in circulatory metabolism. Thicker body fluids are called phlegm, and thinner ones are called fluid, which are collectively called phlegm-fluid retention. “Phlegm-fluid retention” in traditional Chinese medicine mainly refers to thick or thin fluid in the digestive tract, lungs and respiratory tract, which is difficult to metabolize and can be continuously formed in the digestive tract or respiratory tract even if excreted from the body. Such thick fluids can also flow from the digestive tract and respiratory tract and remain under the skin, muscles, bone joints and even internal organs and the brain, leading to various formations, cysts and polyps. Even tumors are considered to be associated with the retention of phlegm-fluid in the body. Traditional Chinese Medicine believes that many diseases are associated with such thick fluids that are difficult to eliminate or metabolize from the body. However, traditional Chinese medicine does not discuss that the retention of phlegm-fluid in the blood causes its increased viscosity.

In modern medicine, the concept of “increased viscosity” includes dozens of biochemical indicators. For example, it may include some measures of hyperlipidemia (serum total cholesterol, low-density cholesterol, high-density cholesterol, translipoproteins, triglycerides), some measures of hyperglycemia (fasting glucose, glycated hemoglobin, fasting insulin, C-peptide, glycated serum protein, fructosamine ), platelet indicators (platelet count, platelet adhesion, platelet sedimentation, etc.), some indicators of erythrocytes (erythrocyte count, erythrocyte sedimentation, erythrocyte adhesion, hematocrit, erythrocyte deformability, erythrocyte size), indicators of increased viscosity (apparent viscosity, relative viscosity, reduced viscosity, specific viscosity, etc.) and some other relevant indicators (fibrinogen, immunoglobulin, etc.). These indicators have a relatively similar effect on increased viscosity, but still not all. Increased viscosity can have various manifestations. Modern medicine has not yet identified a suitable and generally accepted pathogenetic cause, and treatments for hyperviscosity are still indicator-oriented therapy, such as oral drugs for the treatment of hyperlipidemia and hyperglycemia. However, since the problem of increased viscosity is not fundamentally addressed, the lipid-lowering and hypoglycemic effects are short in duration and not curable, requiring long-term administration of drugs.

The retention of phlegm-fluid in the blood causes its increased viscosity, which significantly increases the risk of hyperlipidemia and hyperglycemia, which further causes atherosclerosis, blocks blood vessels, and then causes heart, brain and kidney diseases. Based on this theory, in medical clinical practice, the authors have developed a traditional Chinese medicine composition to effectively and lastingly lower blood lipid levels, particularly lower triglyceride levels, and effectively prevent and treat hyperlipidemia and atherosclerosis.

The present invention proposes a composition of traditional Chinese medicine containing the following raw materials in parts by weight: 10–35 parts of scutellaria root, 5–25 parts of unripe orange fruit, 3–20 parts of rhubarb root, 3–20 parts of Scutellaria baicalensis root, 3–20 parts white peony root, 3–15 parts of dried ginger, 5–20 parts of leech, 3–20 parts of licorice root, 3–20 parts of codonopsis, 3–20 parts of atractylodes bighead rhizome and 3–25 parts of pinellia tuber.

Preferably, the composition for traditional Chinese medicine contains the following raw materials in parts by weight: 20-30 parts of scutellaria root, 10-20 parts of unripe orange fruit, 5-15 parts of rhubarb root, 5-10 parts of Scutellaria baicalensis root, 5-15 parts of peony root white, 5–10 parts of dried ginger, 7–15 parts of leech, 5–15 parts of licorice root, 5–15 parts of codonopsis, 5–15 parts of Atractylodes bighead rhizome and 10–20 parts of pinellia tuber.

More preferably, the traditional Chinese medicine composition described herein further contains the following raw materials in parts by weight: 3-45 parts, preferably 10-35 parts, oyster shell. More preferably, on this basis, the traditional Chinese medicine composition described herein further contains the following raw materials in parts by weight: 3-15 parts, preferably 5-12 parts, red fermented rice.

Preferably, the traditional Chinese medicine composition described herein is prepared from raw materials in parts by weight.

More preferably, the traditional Chinese medicine composition described herein is prepared from the following raw materials in parts by weight: 25 parts of capillary root, 15 parts of unripe orange fruit, 10 parts of rhubarb root, 6 parts of skullcap root, 10 parts of white peony root, 6 parts dried ginger, 10 parts of leech, 10 parts of licorice root, 10 parts of codonopsis, 10 parts of rhizome of Atractylodes bighead and 15 parts of pinellia tuber.

More preferably, the traditional Chinese medicine composition described herein is prepared from the following raw materials in parts by weight: 25 parts of capillary root, 15 parts of unripe orange fruit, 10 parts of rhubarb root, 6 parts of skullcap root, 10 parts of white peony root, 6 parts dried ginger, 10 parts leech, 10 parts licorice root, 10 parts codonopsis, 10 parts Atractylodes bighead rhizome, 15 parts pinellia tuber and 30 parts oyster shell.

More preferably, the traditional Chinese medicine composition described herein is prepared from the following raw materials in parts by weight: 25 parts of capillary root, 15 parts of unripe orange fruit, 10 parts of rhubarb root, 6 parts of skullcap root, 10 parts of white peony root, 6 parts dried ginger, 10 parts leech, 10 parts licorice root, 10 parts codonopsis, 10 parts Atractylodes bighead rhizome, 15 parts pinellia tuber, 30 parts oyster shell and 10 parts red fermented rice.

In the composition described above, a portion is a portion by weight, and the weight is based on the crude drug. During the manufacturing process, weights can be proportionally increased or decreased. For example, in large-scale production, the weight can be expressed in kilograms or tons, it can be increased or decreased, but the ratio of medicinal raw material weights between the components remains unchanged.

The introduction of related raw materials according to the present invention is carried out as follows.

Bupleurum root is the dried root of Bupleurum Chinense DC. or Bupleurum scorzonerifolium Willd. It harmonizes the external and internal aspects, calms the liver and increases yang.

Unripe orange (or unripe bitter orange, unripe sweet orange, fructus aurantii immaturus) is the dried young fruit of Citrus aurantium L. and its cultivated varieties or Citrus × sinensis (family Rutaceae). It has an effect on eliminating stagnation of phlegm and relieving the feeling of constriction in the chest and abdomen.

Rhubarb root is the dried root and rhizome of Rheum palmatum L., Rheum tanguticum Maxim. ex Balf. or Rheum officinale Baill. (family Polygonaceae). It has the effects of reducing fever, relaxing the intestines, lowering blood temperature, counteracting toxicity, eliminating blood stasis and stimulating menstruation.

Scutellaria baicalensis root is the dried root of Scutellaria baicalensis Georgi (family Labiatae). It has the effects of relieving wet-heat syndrome, reducing fever, counteracting toxins, stopping bleeding, and preventing abortion.

White peony root is the dried root of Paeonia lactiflora Pall. (family Ranunculaceae). It soothes the liver, relieves pain, nourishes the blood and regulates menstruation, stops excessive sweating, etc.

Dried ginger is the dried rhizome of Zingiber officinale (Willd.) Rosc. (family Zingiberaceae). Ginger is harvested in winter, removing fibrous roots and dirt, and dried in the sun or at low temperatures. The pieces cut before drying are called "dried ginger pieces". Ginger warms the body from the inside, dispels cold, promotes recovery after collapse, eliminates excess moisture, dissolves phlegm, etc.

The leech is the dried organism of Whitmania pigra Whitman, Hirudo mipponica Whitman, or Whitmania acranulata Whitman (family Hirudinidae). Leeches are caught in summer and autumn, killed with boiling water and dried in the sun or at low temperature. They have blood thinning effects, eliminate blood stasis and stimulate menstruation.

Licorice root is the dried root and rhizome of Glycyrrhiza uralensis Fisch. (herbaceous perennial from the Leguminosae family). It is used to enhance spleen function, replenish qi , remove heat, counteract toxicity, remove phlegm and relieve cough, relieve spasmodic pain and regulate the action of drugs in the gastrointestinal tract.

Codonopsis is the dried root of Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) LT Shen or Codonopsis tangshen Oliv. (family Campanulaceae). It has the effect of toning qi and activating the function of the spleen and lungs.

Atractylodes macrocephala rhizome is the dried rhizome of Atractylodes macrocephala Koidz. (family Compositae). It has the effects of enhancing spleen function, replenishing qi , eliminating excess moisture through diuresis, and stopping excessive sweating.

Pinellia tuber is the dried tuber of Pinellia ternata (Thunb.) Breit. (family Araceae). It has the effects of relieving excess moisture by dissolving phlegm, directing qi downward to relieve nausea and vomiting, and relieving constriction in the chest and epigastric region.

The oyster shell is the shell of Ostrea gigas Thunberg, Ostrea talienwhanensis Crosse or Ostrea rivularis Gould (family Osteridae). It has a calming effect, nourishes yin and suppresses excess yang , softens hard masses and eliminates nodules.

Red fermented rice is produced using the mycelium of Monascus purpureus Went. (family Aspergillaceae), parasitizing Japanese rice grains. It has a strengthening effect on the function of the spleen, promotes digestion, activates blood circulation and eliminates blood stasis.

The recipe of the present invention is developed by combining the experience of clinical practice based on the complete research described in the Treatise on Cold Damage Diseases and the corresponding medicinal substances. In the recipe of the present invention, capillary root is used as the main medicine, and capillary root cleanses liver qi , so that liver function can be restored to normal levels, which is the key to fat metabolism. Volupus root is also a very good remedy for immunity, reducing inflammation, reducing fever and cooling. It can regulate the immune mechanism of the whole body, play a key role in restoring some key hormones to re-degrade and balance blood lipids, and also have a good effect on the functional restoration of thyroxine. Rhubarb root is a medicinal product that plays a key role in cleansing the gastrointestinal tract and eliminating inflammation and harmful flora in the gastrointestinal tract. The unripe orange fruit is also a supportive medicinal product that can be used with capillary root to achieve proper functioning of the upper and lower internal organs, as well as with rhubarb root to improve the functions of the gastrointestinal tract and eliminate inflammation and harmful flora in the intestinal tract. Scutellaria baicalensis root is also a supportive medicine that is used to reduce stomach acid secretion and gastrin levels, as well as eliminate inflammation in the stomach and harmful flora in the intestinal tract. Licorice root, codonopsis, white peony root, rhizome of Atractylodes bighead and pinelli tuber are auxiliary medicines that are used to remove excess mucus in the intestines and stomach, reduce the entry of viscous substances into the blood and reduce blood viscosity. Dried ginger instead of ginger is used in the formulation to reduce the gastrointestinal reaction to rhubarb, so that the formulation is suitable for medium to long-term use, and the requirements of the dosage period for the treatment of hyperlipidemia and arterial plaque are met. The leech contained in the formulation is used to activate the blood, eliminate stasis, remove the accumulation and viscosity of various platelets and red blood cells in the blood vessels, which helps eliminate plaque in the blood vessels. You can add oyster shell to the recipe, which is used to digest accumulated and insufficiently digested substances in the human intestines and stomach. At the same time, oyster shell can help calm an overworked liver and better digest liver fat. You can add red fermented rice to the recipe, which is used to enhance the effect of cholesterol breakdown, but it is not a key component.

The active ingredient of the traditional Chinese medicine composition described herein can be obtained by extraction or other methods known in the art. The active ingredient can be obtained by separate extraction of natural raw materials of a compound drug or by joint extraction of natural raw materials of a compound drug.

Preferably, it is obtained by an aqueous extraction method. More preferably, the method of preparing the traditional Chinese medicine composition described herein includes: weighing the raw materials, adding 4 to 12 times the amount of raw materials with water, and boiling at 75 to 95 °C for 30 to 120 minutes to obtain first decoction; adding water in an amount 3–10 times higher than the amount of raw materials, and boiling at a temperature of 75–95 ° C for 30–120 minutes to obtain a second decoction; and combining the two decoctions, filtering and combining the filtrates. More preferably, the method of preparing the traditional Chinese medicine composition described herein includes: weighing the raw materials, adding 8 times the amount of raw materials with water, and boiling at 80°C for 60 minutes to obtain the first decoction; adding water in an amount 6 times greater than the amount of raw materials, and boiling at a temperature of 80 ° C for 60 minutes to obtain a second decoction; and combining the two decoctions, filtering and combining the filtrates. The resulting extract can be further concentrated into an ointment extract, which can be a dry ointment extract or a liquid ointment extract. Preferably it is concentrated under reduced pressure (-0.05 MPa, 60 °C) to a relative density of 1.05–1.15 (60 °C).

The traditional Chinese medicine composition described herein can be formulated by adding pharmaceutically acceptable carriers as required. The weight percentage of the extracted or processed active ingredient of the traditional Chinese medicine composition described herein in the preparation is 0.1% to 99.9%, with the remainder being pharmaceutically acceptable carriers.

The traditional Chinese medicine composition described herein may be any pharmaceutically acceptable dosage form, including: decoction, tablet, sugar-coated tablet, film-coated tablet, enteric-coated tablet, capsule, hard capsule, soft capsule, liquid oral, lozenge, granule, electuary, pill, fine powder, ointment, pellets, suspension, powder, solution, injection, suppository, ointment, patch, cream, spray, drops and bandage. Preferably, the composition is in oral dosage form and even more preferably in the form of a decoction, capsule, tablet, oral liquid, granule, pill, fine powder, pellet, ointment and the like. Most preferably, the composition is in the form of a decoction, granules, capsules and tablets.

The present invention further relates to the use of a traditional Chinese medicine composition for the preparation of a medicament for preventing or treating elevated blood lipid levels.

The present invention further relates to the use of a traditional Chinese medicine composition for the preparation of a medicament for preventing or treating elevated triglyceride levels.

The present invention further relates to the use of a traditional Chinese medicine composition for the preparation of a medicament for preventing or treating high cholesterol.

The present invention further relates to the use of a traditional Chinese medicine composition for the preparation of a medicament for preventing or treating hyperlipidemia.

The present invention further relates to the use of a traditional Chinese medicine composition for the preparation of a medicament for preventing or controlling vascular plaque.

The present invention further relates to the use of a traditional Chinese medicine composition for the preparation of a medicament for preventing or treating atherosclerosis.

The present invention further relates to the use of a traditional Chinese medicine composition for the preparation of a medicament for preventing or treating chronic metabolic diseases.

The present invention further provides a method for preventing or treating elevated blood lipid levels, comprising: administering to a person in need thereof a prophylactically or therapeutically effective amount of a traditional Chinese medicine composition described herein as described above.

The present invention further provides a method for preventing or treating elevated triglyceride levels, comprising: administering to a person in need thereof a prophylactically or therapeutically effective amount of a traditional Chinese medicine composition described herein as described above.

The present invention further provides a method for preventing or treating high cholesterol, comprising: administering to a person in need thereof a prophylactically or therapeutically effective amount of a traditional Chinese medicine composition described herein as described above.

The present invention further provides a method for preventing or treating hyperlipidemia, comprising: administering to a person in need thereof a prophylactically or therapeutically effective amount of a traditional Chinese medicine composition described herein as described above.

The present invention further provides a method for preventing or controlling vascular plaque, comprising: administering to a person in need thereof a prophylactically or therapeutically effective amount of a traditional Chinese medicine composition described herein as described above.

The present invention further provides a method for preventing or treating atherosclerosis, comprising: administering to a person in need thereof a prophylactically or therapeutically effective amount of a traditional Chinese medicine composition described herein as described above.

The present invention further provides a method for preventing or treating chronic metabolic diseases, comprising: administering to a person in need thereof a prophylactically or therapeutically effective amount of a traditional Chinese medicine composition described herein as described above.

The traditional Chinese medicine composition described herein can be administered one to three times a day, and when prepared as a unit dose, 1 to 10 unit doses are administered each time. After decoction of the traditional Chinese medicine composition described herein in water, the decoction can be concentrated and dried to prepare a powder to obtain a dry ointment extract. An appropriate amount of excipients was added to prepare the granular formulation (each dose contains 1250 mg of powder to obtain a dry ointment extract, equivalent to 20 g of raw material). The granular formulation can be taken one to three times a day, 1-2 doses each time. Alternatively, the dry ointment extract powder can also be capsuled or prepared in tablet form (each dose contains 312.5 mg of dry ointment extract powder, equivalent to 5 g of raw material). Capsules or tablets can be taken one to three times a day, 2-8 capsules or tablets each time. Specific use and dosage may be adjusted according to the patient's specific condition.

The traditional Chinese medicine formulation of the present invention is an innovative design based on the theory and practice of "phlegm-fluid retention in the blood" and the " Treatise on Cold-Induced Diseases ". The inventor of the present invention has further optimized the ratio of components based on complete research and consideration of the properties and effectiveness of the components in a given formulation. In particular, the formulation of the present invention creatively contains dried ginger and leech in proportions optimized for clinical practice. The traditional Chinese medicine composition described herein has a very significant effect on hyperviscosity and optimizes overall blood viscosity in two ways. The first way is to reduce the viscous substance - retention of phlegm-liquid in the intestines and stomach and reduce the cause of increased viscosity by improving digestion in the intestines and stomach, as well as excretion. The second way is to directly metabolize highly viscous organic components, including blood lipids, by stimulating metabolism. The traditional Chinese medicine composition described herein can also reduce the aggregation degree, sedimentation degree and cutting degree of red blood cells, and also reduce the aggregation degree, sedimentation degree and cutting degree of platelets. Clinical studies have demonstrated that the traditional Chinese medicine formulation of the present invention has excellent effectiveness in lowering blood lipids, lowering cholesterol, and in particular lowering blood triglycerides, with a response rate of more than 90%. The drug has a significant effect on a strong increase in blood triglyceride levels (triglycerides >5.6 mmol/l), which can be reduced by 90% or more in a short time (14 days, one course of treatment). The traditional Chinese medicine formulation of the present invention has excellent efficacy, which has also been confirmed by animal model studies conducted by third parties. In addition, studies have demonstrated that the traditional Chinese medicine composition described herein also has good efficacy in specifically inhibiting the formation of atherosclerotic plaques and removing them.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Embodiments of the present invention are described in detail below with reference to the accompanying drawings.

FIG. 1: Comparison of full-length aorta stained with Oil Red O from ApoE ^−/− mice in the negative control group versus the atherosclerotic plaque simulation control group. A: Representative pattern of Oil Red O staining in the negative control group; B: Representative pattern of Oil Red O staining in the simulation control group after pathology simulation.

FIG. 2: comparison of the effect of the traditional Chinese medicine composition described herein on the formation of atherosclerotic plaques in ApoE ^-/- mice (full-length aorta stained with Oil Red O). A: Representative pattern of Oil Red O staining in the negative control group; B: representative pattern of Oil Red O staining in the simulation control group; C: Representative Oil Red O staining pattern in the group receiving the 833 mg/kg traditional Chinese medicine formulation; D: Representative pattern of Oil Red O staining in the group receiving the 1677 mg/kg traditional Chinese medicine formulation.

FIG. 3: comparison of the effect of the traditional Chinese medicine composition described herein on the formation of atherosclerotic plaques in ApoE ^-/- mice (aortic arch section stained with HE) in the negative control group, simulation control group, positive control group, traditional composition treated group Chinese medicine at 833 mg/kg, and the group receiving the traditional Chinese medicine composition at 1677 mg/kg.

FIG. 4: comparison of the effect of the traditional Chinese medicine composition described herein on the formation of atherosclerotic plaques in ApoE ^-/- mice (aortic arch section, Sirius stained) in the negative control group, simulation control group, positive control group, traditional composition treated group Chinese medicine at 833 mg/kg, and the group receiving the traditional Chinese medicine composition at 1677 mg/kg.

FIG. 5: Comparison of the effect of the traditional Chinese medicine composition described herein on the formation of atherosclerotic plaques in ApoE ^-/- mice (carotid artery section stained with Oil Red O) in the negative control group, simulation control group, positive control group, treatment group the traditional Chinese medicine composition at 833 mg/kg, and the group receiving the traditional Chinese medicine composition at 1677 mg/kg.

FIG. 6: The result of treatment with the traditional Chinese medicine composition described herein in 14 patients with elevated total cholesterol and elevated triglyceride levels. A: analysis of total cholesterol before and after treatment; B: Triglyceride analysis before and after treatment.

FIG. 7: The result of treatment with the traditional Chinese medicine composition described herein in 11 patients with hypercholesterolemia and the analysis of total cholesterol before and after treatment.

FIG. 8: The result of treatment with the traditional Chinese medicine composition described herein in 88 patients with hypertriglyceridemia and analysis of triglycerides before and after treatment.

DETAILED DESCRIPTION

The present invention will be further described in detail below with reference to specific embodiments, and examples are given only to illustrate the present invention and not to limit its scope.

All test methods in the following examples are conventional methods unless otherwise noted; All raw materials, reagents, substances, etc. used in the following examples are commercially available products unless otherwise noted.

In the description of this application, unless otherwise indicated, the amount of solvent during the preparation of each study drug is a multiple of the volume based on the weight of the drug substances.

Example 1 Preparation of the traditional Chinese medicine composition described herein

Example 1-1:

The following raw materials were weighed: 25 g of volodushka root, 15 g of unripe orange fruits, 10 g of rhubarb root, 6 g of Baikal skullcap root, 10 g of white peony root, 6 g of dried ginger, 10 g of leech, 10 g of licorice root, 10 g of codonopsis, 10 g of Atractylodes bighead rhizome, 15 g of pinellia tuber and 30 g of oyster shell. The raw materials were boiled twice for extraction in a closed container. For the first time, water was added in an amount 8 times higher than the amount of raw material, and the mixture was boiled at 80 °C for 60 minutes to obtain a decoction. Then, water was added a second time in an amount 6 times higher than the original raw material, and the mixture was boiled at 80 °C for 60 minutes to obtain a second decoction. The two decoctions were combined and filtered, and the filtrates were pooled. The filtrate was concentrated under reduced pressure (-0.05 MPa, 60 °C) to a relative density of 1.05–1.15 (60 °C) and lyophilized under reduced pressure to obtain powder A for dry ointment extract with a yield of 25% relative to the raw material .

To the resulting dry ointment extract powder A, β-cyclodextrin was added in a ratio of 4:1 to obtain granules of dry ointment extract powder A. The granules were dried, cooled and packaged into a granular formulation (each sachet contained 1250 mg of powder to produce a dry ointment extract of traditional Chinese medicine, equivalent to 20 g of raw material).

Example 1-2:

The following raw materials were weighed: 25 g of volodushka root, 15 g of unripe orange fruits, 10 g of rhubarb root, 6 g of Baikal skullcap root, 10 g of white peony root, 6 g of dried ginger, 10 g of leech, 10 g of licorice root, 10 g of codonopsis, 10 g of Atractylodes macrocephala rhizome and 15 g of Pinellia tuber. The raw materials were boiled twice for extraction in a closed container. For the first time, water was added in an amount 8 times higher than the amount of raw material, and the mixture was boiled at 80 °C for 60 minutes to obtain a decoction. Then, water was added a second time in an amount 6 times higher than the original raw material, and the mixture was boiled at 80 °C for 60 minutes to obtain a second decoction. The two decoctions were combined and filtered, and the filtrates were pooled. The filtrate was concentrated under reduced pressure (-0.05 MPa, 60 °C) to a relative density of 1.05–1.15 (60 °C) and lyophilized under reduced pressure to obtain powder B for dry ointment extract with a yield of 25% relative to the raw material .

To the resulting dry ointment extract powder B, β-cyclodextrin was added in a ratio of 4:1 to obtain dry ointment extract powder B granules. The granules were dried, cooled and packaged into a granular formulation (each sachet contained 1250 mg of powder to produce a dry ointment extract of traditional Chinese medicine, equivalent to 20 g of raw material).

Example 1-3:

The following raw materials were weighed: 25 g of volodushka root, 15 g of unripe orange fruits, 10 g of rhubarb root, 6 g of Baikal skullcap root, 10 g of white peony root, 6 g of dried ginger, 10 g of leech, 10 g of licorice root, 10 g of codonopsis, 10 g of Atractylodes bighead rhizome, 15 g of pinellia tuber, 10 g of red fermented rice and 30 g of oyster shell. The raw materials were boiled twice for extraction in a closed container. For the first time, water was added in an amount 8 times higher than the amount of raw material, and the mixture was boiled at 80 °C for 60 minutes to obtain a decoction. Then, water was added a second time in an amount 6 times higher than the original raw material, and the mixture was boiled at 80 °C for 60 minutes to obtain a second decoction. The two decoctions were combined and filtered, and the filtrates were pooled. The filtrate was concentrated under reduced pressure (-0.05 MPa, 60 °C) to a relative density of 1.05–1.15 (60 °C) and lyophilized under reduced pressure to obtain powder C for dry ointment extract with a yield of 25% relative to the raw material .

To the resulting dry ointment extract powder C was added β-cyclodextrin in a ratio of 4:1 to obtain dry ointment extract powder C granules. The granules were dried, cooled and packaged into a granular formulation (each sachet contained 1250 mg of powder to produce a dry ointment extract of traditional Chinese medicine, equivalent to 20 g of raw material).

Example 2 Study of the effect of the traditional Chinese medicine composition described herein in a rat model of hybrid hyperlipidemia

2.1 Objectives : To investigate the effect of the traditional Chinese medicine composition described herein in a rat model of hybrid hyperlipemia.

2.2 Test compound: Powder A for dry ointment extract obtained in Example 1-1. Appearance: brown powder; storage conditions: 2–8 °C, in a dry place.

2.3 Reference compound : Atorvastatin calcium tablets (Lipitor). Appearance: white oval film-coated tablets; dose: 10 mg/tablet; packaging: aluminum/aluminum contour-cell packaging; manufacturer: Pfizer Inc.; storage conditions: sealed packaging.

2.4 Carrier, emulsifier and other media:

2.4.1 Sodium carboxymethylcellulose

Storage conditions: room temperature;

Method for preparing 0.5% sodium carboxymethylcellulose solution: 5.0 g of CMC-Na was accurately weighed and slowly added to a beaker containing about 800 ml of purified water, and the mixture was stirred with a magnetic stirrer at room temperature until CMC-Na dissolved. The solution was left overnight at 2–8 °C and diluted to 1000 ml. The resulting solution was thoroughly mixed and stored at 2–8 °C for subsequent use.

2.4.2 Name: purified water

Manufacturer: Laboratory of Comparative Medicine, Guangdong Province Laboratory Animal Medical Center.

2.5 Basic instruments and reagents

Electronic analytical balance BS-3000A, sensitivity: 0.1 g, Shanghai Yousheng Weighing Apparatus Co., Ltd.;

electronic analytical balance BS224S, sensitivity: 0.1 mg, Sartorius Scientific Instruments (Beijing) Co., Ltd.;

desktop high-speed centrifuge 5418, EPPENDORF, Germany;

automatic biochemical analyzer 7020, Hitachi High-tech Corporation;

Isoflurane: Lot No. 217171101, Expiry Date: November 1, 2020, Shenzhen RWD Life Science Co., Ltd.;

Urethane: Lot No. 20160920, Expiry Date: September 20, 2021, Sinopharm Chemical Reagent Co., Ltd.;

Sodium Chloride Injection: Lot No. H18052802-2, Guangdong Kelun Pharmaceutical Co., Ltd.;

20% urethane solution for anesthesia: 20.0 g of urethane was added to 100 ml of sodium chloride injection solution, the mixture was thoroughly mixed and sterilized through a filter membrane with pores of 0.2 microns;

TC (Total Cholesterol) Test Kit: Lot No. 20180722 and 20190222, Expiry Date: January 10, 2020 and August 20, 2020, Shanghai Kehua Bio-Engineering Co., Ltd.;

TG (Triglyceride) Test Kit: Lot No. 20180822 and 20190222, Expiry Date: February 23, 2020 and August 25, 2020, Shanghai Kehua Bio-Engineering Co., Ltd.;

HDL-C (High Density Lipoprotein Cholesterol) Test Kit: Lot No. 20181012, Expiry Date: October 11, 2019, Shanghai Kehua Bio-Engineering Co., Ltd.;

LDL-C (Low Density Lipoprotein Cholesterol) Test Kit: Lot No. 20190212, Expiry Date: February 26, 2020, Shanghai Kehua Bio-Engineering Co., Ltd.

2.6 Experimental system

Species: SD rat; status: SPF status; males 190.2–223.9 g;

Source and Certificates: Purchased from Guangdong Provincial Laboratory Animal Medical Center, Animal Production License No. SCXK (Guangdong) 2018-0002, Animal Certificate No. 44007200066274;

Identification: Animals were numbered with saturated picric acid by dyeing the fur, and the fur on different areas of the animal's body surface was stained with dye spots to indicate different numbers. Animals were identified by skin coloring and labels on their cages.

Animal Welfare: The tests and procedures associated with the animal experimentation conducted in this study complied with relevant laws and regulations for the use and treatment of experimental animals, as well as relevant institutional ethics committee rules for animal research, to ensure the welfare of experimental animals.

Euthanasia: excluded animals were killed by carbon dioxide inhalation after the simulation. After the study, the animals were anesthetized by intraperitoneal injection of a 20% urethane solution at a rate of 6 ml/kg body weight and then killed by exsanguination. Before burial, the corpses were temporarily stored in a freezer.

Quarantine: acquired rats were quarantined for 3 days and observed once a day, and no unhealthy animals were found.

Housing and handling: Animals were housed in the SPF animal facility of Guangdong Provincial Laboratory Animal Medical Center. Laboratory Animal Use License No. SYXK (Guangdong) 2018-0002; Certificate of experiment on animals No. 00219646. Conditions for keeping animals: group housing, 5 animals/cage; temperature and humidity: 20–26 °C, 40–70%; light/dark cycle: 12 h/12 h; The condition of the living space was always maintained stable to ensure the reliability of the experimental results. During the experiment, animals were fed appropriate pelleted diets in accordance with the experimental requirements and were provided with free access to food and water.

2.7 Doses and division into groups

Doses: In a preliminary test, 4 SD rats were intragastrically administered the test compound at doses of 2000 mg/kg body weight and 5000 mg/kg body weight. No animals died within 72 hours. The dose of the test compound for an adult is 5 g/day. Based on 60 kg adult body weight, 5 times and 10 times the recommended human dose were used as study doses, and the study doses were 417 mg/kg body weight and 833 mg/kg body weight, respectively. The clinical dose of the reference drug, atorvastatin calcium tablets, was 10 mg/day. Based on 60 kg of adult body weight. The study dose of the reference drug was 20 times the recommended human dose, i.e. the study dose of the reference drug, atorvastatin calcium tablets, was 3.3 mg/kg body weight.

Separation into groups: after quarantine, the animals were randomized into a negative control group of 10 animals, and the remaining animals into the modeling groups. After the simulation groups were fed the simulation diet for 2 weeks, the animals were anesthetized with isoflurane inhalation without fasting. Blood was collected from the orbital venous sinus and centrifuged at 3000 rpm for 10 min to separate the serum. Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were determined. Animals with lower TG levels were excluded. 40 animals were recruited and randomized into a simulation control group, a positive control group, and 417 mg/kg and 833 mg/kg test compound groups according to TG levels, 10 animals each.

Table 1. Doses and division into groups Group n Dose
(mg/kg body weight) Equivalent recommended human dose Route of administration and volume Solution concentration (mg/ml) Negative control group 10 - - Intragastric, 1 time per day
10 ml/kg body weight - Simulation control group 10 - - Intragastric, 1 time per day
10 ml/kg body weight - Positive control group 10 3.3 20 times Intragastric, 1 time per day
10 ml/kg body weight 0.33 Group receiving test compound 417 mg/kg 10 417 5 times Intragastric, 1 time per day
10 ml/kg body weight 41.7 Group receiving test compound 833 mg/kg 10 833 10 times Intragastric, 1 time per day
10 ml/kg body weight 83.3

2.8 Methodology

2.8.1 Modeling feed with high fat and cholesterol content: maintenance feed containing 20.0% sucrose, 5% lard, 1.0% cholesterol, 0.1% sodium cholate, appropriate amounts of casein, calcium bicarbonate, crushed lime, etc. In addition to crude fat, in the model feed, all parameters of moisture content, crude protein, crude fat, crude fiber, crude ash, calcium, phosphorus and calcium:phosphorus ratio corresponded to the national standard for maintenance feed. Food was provided by Guangdong Provincial Laboratory Animal Medical Center.

2.8.2 Preparation of a solution of the test compound: the required amount of the test compound was added to a volumetric flask, ground and dissolved in a small amount of purified water. The mixture was brought to a given volume with purified water and stirred until completely dissolved to obtain a concentration of 166.7 mg/ml. The solution was serially diluted to concentrations of 83.3 mg/ml and 41.7 mg/ml.

2.8.3 Preparation of reference drug solution: 1 tablet of atorvastatin calcium (dose: 10 mg/tablet) was added to a volumetric flask, ground and dissolved in 0.5% CMC-Na solution. The mixture was brought to 60 ml with 0.5% CMC-Na solution, giving a concentration of about 0.33 mg/ml. To ensure thorough mixing, the solution was shaken before use.

2.8.4 Simulation: The negative control group received the maintenance food, and the remaining groups received the simulation food until the end of the study. Except for the negative control group, the other groups were fed a high-fat Western diet from days 30 to 36 during the treatment period.

2.8.5 Introduction: after completion of the simulation, the positive control group and the groups receiving the test compound at 417 mg/kg and 833 mg/kg, with the test samples, were intragastrically administered an appropriate dose of therapeutic fluid of 10 ml/kg body weight daily, and the negative control group and the simulation control group were administered purified water in the same volume for 67–68 days (the first 5 animals in each group were administered for 67 days, and the last 5 animals in each group were administered for 68 days).

2.8.6 Measurements

2.8.6.1 General condition: The clinical condition of the animals was observed once a day until the end of the study.

2.8.6.2 Body weight: The body weight of animals was measured once at the beginning and end of the study and once a week during the study.

2.8.6.3 Blood lipids: After 2, 4, 7 and 9 weeks of treatment (day 14, day 28, day 52 and day 65), animals were anesthetized by isoflurane inhalation without fasting. Blood was collected from the orbital venous sinus and centrifuged at 3000 rpm for 10 min to separate the serum. TC, TG, LDL-C and HDL-C levels were determined.

2.8.6.4 The first 5 animals in each group were fasted overnight on day 66, and the last 5 animals were fasted on day 67. Animals were treated, weighed and anesthetized the next day with an intraperitoneal injection of 20% urethane solution at a rate of 6 ml/kg body weight. Blood was collected from the abdominal aorta, and serum was separated and cryopreserved. Animals were killed by bleeding. Testicles and perirenal adipose tissue were weighed. Part of the liver was fixed in neutral formaldehyde, the rest of the liver was cryopreserved in liquid nitrogen.

2.9 Statistics: data presented ( $$\overline{x}\pm s$$ ). Statistical analysis was performed using SPSS 21.0; Pairwise comparisons were performed using one-way analysis of variance with a control level of α = 0.05.

2.10 Results

2.10.1 General observation: During the study, the animals did not exhibit any abnormal reactions in body shape, hair, skin, feces, muscle tension, gait, mood, breathing, etc.

2.10.2 Body weight (see Table 2): There was no statistically significant difference (P > 0.05) in the body weight of animals in each group during the study.

2.10.3 Blood lipids (see tables 3–7)

2.10.3.1 Blood lipids at baseline after simulation (see Table 3): Compared with the negative control group, the increase in serum TC, TG and LDL-C values in the simulation control group at baseline after simulation was statistically significant (P < 0 .05 or .01); compared with the simulation control group, the four blood lipid measurements in each group at baseline after simulation were not statistically significant (P > 0.05).

2.10.3.2 Blood lipids at week 2 of treatment (see Table 4): Compared with the negative control group, the increase in serum TC, TG and LDL-C values in the simulation control group at week 2 of treatment was statistically significant (P < 0.05 or 0.01); Compared with the simulation control group, the decrease in serum TC, TG, HDL-C, and LDL-C values in the positive control group at week 2 of treatment was not statistically significant (P > 0.05); Compared with the simulation control group, serum measurements in the 417 mg/kg and 833 mg/kg study compound groups at week 2 of treatment were not statistically significant (P > 0.05). Compared with the simulation control group, the mean TG value was reduced from 3.22 mmol/L in the simulation control group to 2.89 mmol/L in the 417 mg/kg test compound group and to 2.51 mmol/L in the simulation control group. group receiving the test compound at 833 mg/kg, but this decrease was not statistically significant (P value was between 0.05 and 0.1).

2.10.3.3 Blood lipids at week 4 of treatment (see Table 5): Compared with the negative control group, the increase in serum TC, TG and LDL-C values in the simulation control group at week 4 of treatment was statistically significant (P < 0.05 or 0.01); compared with the simulation control group, the decrease in serum TC and LDL-C values in the positive control group at week 4 of treatment was close to statistically significant (P = 0.10 for TC and P = 0.05 for LDL-C); Compared with the simulation control group, the decrease in TG and LDL-C values in the 833 mg/kg test compound group at week 4 of treatment was statistically significant (P < 0.05). In the 417 mg/kg test compound group, TG and LDL-C values showed a decreasing trend at week 4 of treatment, which was not statistically significant (P > 0.05). TG levels decreased from 3.03 mmol/L in the simulation control group to 2.32 mmol/L (P = 0.05). At the same time, the test compound also showed a tendency to reduce TC levels. TC levels decreased from 3.44 mmol/L in the simulation control group to 2.72 mmol/L (group receiving 417 mg/kg test compound, P = 0.08) and 2.75 mmol/L (group receiving test compound at 833 mg/kg, P = 0.09).

2.10.3.4 Blood lipids at week 7 of treatment (see Table 6): Compared with the negative control group, the increase in serum TC and LDL-C values in the simulation control group at week 7 of treatment was statistically significant (P < 0.01) , and the TG value showed an increasing trend, which was not statistically significant (P > 0.05); The serum TC value of the positive control group at week 7 of treatment was 3.03 mmol/L, and compared with the simulation control group (3.55 mmol/L), the decrease was not statistically significant (P = 0.10); Compared with the simulation control group, the decrease in serum TG levels in the 417 mg/kg and 833 mg/kg study compound groups at week 7 of treatment had statistically significant or nearly significant statistical differences (P values for study compound groups at 417 mg/kg and 833 mg/kg were P = 0.05 and P = 0.04, respectively). Additionally, there was no statistically significant difference (P < 0.05) in the reduction in LDL-C levels in the 833 mg/kg test compound group.

2.10.3.5 Blood lipids at week 9 of treatment (see Table 7): Compared with the negative control group, the increase in serum TC, TG and LDL-C values in the simulation control group at week 9 of treatment was statistically significant (P < 0.01 ); Compared with the simulation control group, the decrease in serum TC, TG and LDL-C values in the positive control group at week 9 of treatment was not statistically significant (P > 0.05); Compared with the simulation control group, four serum lipid measurements in the 417 mg/kg test compound group at week 9 of treatment were not statistically significant (P > 0.05). However, the decrease in TC, TG, and LDL-C in the 833 mg/kg test compound group was statistically significant (P < 0.05) at week 9 of treatment.

2.10.4 Weight and organ/tissue ratio (see Table 8): Compared with the negative control group, the increase in weight and liver ratio of rats in the simulation control group was statistically significant (P < 0.01); Compared with the simulation control group, the liver weight and coefficient in the positive control group did not have a statistically significant difference (P < 0.05 or 0.01); Compared with the simulation control group, the masses and organ/tissue ratios in the groups receiving the test compound at 417 mg/kg and 833 mg/kg did not have a statistically significant difference (P > 0.05). The organs were preserved.

Table 2.
Group Before modeling Day 1 of treatment Day 7 of treatment Day 14 of treatment Day 21 of treatment Day 28 of treatment Negative control group 210.8 ± 7.1 345.8 ± 18.4 386.7 ± 22.6 423.1 ± 26.2 456.7 ± 32.4 471.3 ± 31.0 Simulation control group 208.7 ± 9.4 330.1 ± 12.5 364.1 ± 13.1 404.8 ± 17.8 435.1 ± 19.7 468.9 ± 23.6 Positive control group 212.3 ± 8.3 331.5 ± 21.2 363.9 ± 21.2 401.4 ± 17.7 424.8 ± 24.5 454.2 ± 30.7 Group receiving test compound 417 mg/kg 208.2 ± 6.6 335.0 ± 24.7 366.9 ± 26.2 405.9 ± 31.7 429.1 ± 31.7 463.3 ± 37.1 Group receiving test compound 833 mg/kg 210.1 ± 4.9 342.7 ± 16.3 375.0 ± 18.8 410.5 ± 18.9 443.0 ± 22.2 475.7 ± 26.6

Note: Statistical analysis is based on repeated measurements.

Table 2 (continued).
Group Day 35 of treatment Day 42 of treatment Day 51 of treatment Day 56 of treatment Day 63 of treatment Negative control group 487.3 ± 29.8 512.2 ± 29.1 548.2 ± 34.4 556.4 ± 35.8 578.7 ± 36.8 Simulation control group 500.2 ± 26.6 531.4 ± 29.5 563.4 ± 30.4 578.3 ± 32.3 586.5 ± 31.8 Positive control group 485.8 ± 33.6 523.4 ± 42.1 557.4 ± 48.0 566.7 ± 52.5 577.3 ± 55.9 Group receiving test compound 417 mg/kg 489.8 ± 40.8 518.0 ± 56.0 554.1 ± 53.6 559.4 ± 52.3 578.7 ± 56.5 Group receiving test compound 833 mg/kg 509.0 ± 28.9 546.3 ± 34.5 583.9 ± 34.7 592.5 ± 37.8 610.5 ± 40.7

Note: Statistical analysis is based on repeated measurements.

Table 3.
Group TC TG HDL-C LDL-C Negative control group 1.96 ± 0.19 2.43 ± 0.54 0.71 ± 0.08 0.35 ± 0.03 Simulation control group 4.19 ± 0.47 ^▲▲ 3.08±0.74 ^▲ 0.69 ± 0.07 1.17 ± 0.31 ^▲▲ Positive control group 4.11 ± 0.51 3.09 ± 0.75 0.77 ± 0.12 1.04 ± 0.33 Group receiving test compound 417 mg/kg 4.01 ± 0.58 3.17 ± 0.84 0.68 ± 0.22 1.03 ± 0.27 Group receiving test compound 833 mg/kg 4.25 ± 0.64 3.11 ± 0.85 0.78 ± 0.11 1.07 ± 0.17

Note: comparison between groups, independent sample T-test; “ ^▲ ” indicates P < 0.05, “ ^▲▲ ” indicates P < 0.01, “ ^# ” indicates P between 0.05 and 0.10.

Table 4.
Group TC TG HDL-C LDL-C Negative control group 1.79 ± 0.22 2.51 ± 0.32 0.61 ± 0.07 0.38 ± 0.05 Simulation control group 3.79 ± 0.68 ^▲▲ 3.22 ± 0.93 ^▲ 0.60 ± 0.09 1.51 ± 0.46 ^▲▲ Positive control group 3.84 ± 0.57 3.02 ± 0.91 0.63 ± 0.07 1.33 ± 0.47 Group receiving test compound 417 mg/kg 3.80 ± 0.61 2.89 ± 0.76 0.60 ± 0.08 1.37 ± 0.28 Group receiving test compound 833 mg/kg 4.07 ± 0.44 2.51±0.68 ^# 0.68±0.09 ^# 1.37 ± 0.22

Note: comparison between groups, independent sample T-test; “ ^▲ ” indicates P < 0.05, “ ^▲▲ ” indicates P < 0.01, “ ^# ” indicates P between 0.05 and 0.10.

Table 5.
Group TC TG HDL-C LDL-C Negative control group 1.42 ± 0.18 2.27 ± 0.66 0.53 ± 0.05 0.19 ± 0.03 Simulation control group 3.44 ± 0.99 ^▲▲ 3.03±0.83 ^▲ 0.53 ± 0.13 1.18 ± 0.46 ^▲▲ Positive control group 2.75±0.77 ^# 3.02 ± 1.23 0.50 ± 0.10 0.79±0.36 ^# Group receiving test compound 417 mg/kg 2.72±0.71 ^# 2.32±0.70 ^# 0.44±0.07# 0.89±0.28 ^# Group receiving test compound 833 mg/kg 2.75±0.72 ^# 2.33±0.63 ^▲ 0.45 ± 0.13 0.85±0.15 ^▲

Note: comparison between groups, independent sample T-test; “ ^▲ ” indicates P < 0.05, “ ^▲▲ ” indicates P < 0.01, “#” indicates P between 0.05 and 0.10.

Table 6.
Group TC TG HDL-C LDL-C Negative control group 2.08 ± 0.33 2.66 ± 0.51 0.71 ± 0.07 0.26 ± 0.02 Simulation control group 3.55 ± 0.66 ^▲▲ 3.39 ± 1.12 0.69 ± 0.11 0.79 ± 0.27 ^▲▲ Positive control group 3.03 ± 0.68 3.30 ± 1.40 0.65 ± 0.07 0.65 ± 0.25 Group receiving test compound 417 mg/kg 3.23 ± 0.72 2.39±1.01 ^# 0.66 ± 0.07 0.64 ± 0.20 Group receiving test compound 833 mg/kg 3.17 ± 0.51 2.49 ± 0.66 ^▲ 0.67 ± 0.11 0.56 ± 0.08 ^▲

Note: comparison between groups, independent sample T-test; “ ^▲ ” indicates P < 0.05, “ ^▲▲ ” indicates P < 0.01, “ ^# ” indicates P between 0.05 and 0.10.

Table 7.
Group TC TG HDL-C LDL-C Negative control group 1.93 ± 0.27 2.69 ± 0.53 0.63 ± 0.04 0.35 ± 0.09 Simulation control group 3.66 ± 0.55 ^▲▲ 3.62±0.83 ^▲ 0.65 ± 0.11 1.19 ± 0.40 ^▲▲ Positive control group 3.58 ± 0.88 3.78 ± 1.76 0.69 ± 0.10 1.23 ± 0.48 Group receiving test compound 417 mg/kg 3.29 ± 0.87 3.34 ± 1.47 0.65 ± 0.08 1.11 ± 0.47 Group receiving test compound 833 mg/kg 3.19 ± 0.41 ^▲ 2.59 ± 0.67 ^▲▲ 0.68 ± 0.12 0.82±0.14 ^▲

Note: comparison between groups, independent sample T-test; “ ^▲ ” indicates P < 0.05, “ ^▲▲ ” indicates P < 0.01.

Table 8.
Group Autopsy day
Body weight (g) Liver weight (g) Perirenal fat
Weight (g) Mass of fat in the testicle and around the epididymis (g) Liver coefficient Perirenal fat
Coefficient Fat ratio in the testicle and around the epididymis Negative control group 563.7 ± 36.8 14.21 ± 1.55 12.59 ± 2.79 10.75 ± 2.80 2.52 ± 0.18 2.22 ± 0.43 1.90 ± 0.45 Simulation control group 577.5 ± 28.3 22.16 ± 3.43 ^▲▲ 15.13 ± 5.03 12.62 ± 3.20 3.84 ± 0.57 ^▲▲ 2.61 ± 0.85 2.18 ± 0.53 Positive control group 570.9 ± 56.2 20.15 ± 3.80 14.81 ± 4.58 12.07 ± 5.00 3.51 ± 0.42 2.65 ± 1.01 2.06 ± 0.68 Group receiving test compound 417 mg/kg 568.5 ± 57.7 20.66 ± 4.16 13.88 ± 5.11 10.41 ± 3.86 3.62 ± 0.50 2.40 ± 0.69 1.81 ± 0.53 Group receiving test compound 833 mg/kg 604.0 ± 40.5 22.07 ± 2.68 17.48 ± 2.33 13.42 ± 1.92 3.65 ± 0.38 2.90 ± 0.37 2.22 ± 0.31

Note: comparison between groups, independent sample T-test; “ ^▲ ” indicates P < 0.05, “ ^▲▲ ” indicates P < 0.01.

2.11 Summary:

1) There was no statistically significant difference (P > 0.05) in the body weight of animals in each group.

2) Compared with the negative control group, serum TC, TG and LDL-C values in the simulation control group at baseline, after simulation and at weeks 2, 4 and 9 of treatment were increased (P < 0.05 or 0.01 ). A model of hybrid hyperlipemia in rats was created.

3) Compared with the simulation control group at each time point, the positive control group showed some effects on reducing serum total cholesterol in rats, which, however, were not significant, indicating that the reference drug atorvastatin did not have a significant effect effect on lowering blood lipid levels in a rat model of hybrid hyperlipidemia and is not an ideal therapeutic agent.

4) The traditional Chinese medicine composition described herein began to show the effect of reducing TG levels in week 2 of treatment, especially in the group receiving 833 mg/kg, and the effects of reducing TG levels in weeks 4, 7 and 9 of treatment were statistically significant ( P < 0.05). Meanwhile, the 833 mg/kg group also showed a preliminary effect of reducing TC levels, and in particular, at week 9 of treatment, the reduction in TC levels was statistically significant (P < 0.05). The TG-lowering effect in the 417 mg/kg group at weeks 4, 7, and 9 of treatment was the critical point for statistical difference in the statistical tests (P value between 0.05 and 0.10).

5) Compared with the negative control group, the liver weight and coefficient in the simulation control group were increased (P < 0.01); Compared to the simulation control group, the weight and organ/tissue ratio in the groups receiving the test compound did not change statistically significantly (P > 0.05). The organs were stored for further delivery to the sponsor.

6) Conclusion: Under experimental conditions, animal studies have demonstrated that the traditional Chinese medicine composition described herein has statistically significant effects on lowering blood lipids and lowering cholesterol levels, and in particular has a statistically significant effect on lowering blood triglyceride levels . Meanwhile, the effect on lowering blood lipids has a certain dose dependence.

Example 3 Study of the effect of the traditional Chinese medicine composition described herein on the formation of atherosclerotic plaques in ApoE mice ^-/-

3.1 Objectives: To investigate the effect of the traditional Chinese medicine composition described herein on the formation of atherosclerotic plaques in the ApoE ^-/- mouse model of atherosclerotic plaques.

3.2 Test compound: Powder A for dry ointment extract obtained in Example 1-1. Appearance: brown powder; storage conditions: 2–8 °C, in a dry place.

3.3 Reference drug: rosuvastatin calcium tablets (Crestor); appearance: pink film-coated tablet; dose/purity: 10 mg/tablet; packaging: aluminum-plastic contour-cell packaging; manufacturer: AstraZeneca Pharmaceuticals (China) Co., Ltd.; Storage conditions: sealed and in a dry place.

3.4 Carrier, emulsifier and other media:

3.4.1 Sodium carboxymethylcellulose

Storage conditions: room temperature;

Method for preparing 0.5% sodium carboxymethylcellulose solution: 5.0 g of CMC-Na was accurately weighed and slowly added to a beaker containing about 800 ml of purified water, and the mixture was stirred with a magnetic stirrer at room temperature until CMC-Na dissolved. The solution was left overnight at 2–8 °C and diluted to 1000 ml. The resulting solution was thoroughly mixed and stored at 2–8 °C for subsequent use.

3.4.2 Name: purified water

Manufacturer: Laboratory of Comparative Medicine, Guangdong Province Laboratory Animal Medical Center.

3.5 Basic instruments and reagents

Electronic analytical balance BS-3000A, sensitivity: 0.1 g, Shanghai Yousheng Weighing Apparatus Co., Ltd.;

electronic analytical balance BS224S, sensitivity: 0.1 mg, Sartorius Scientific Instruments (Beijing) Co., Ltd.;

desktop high-speed centrifuge 5418, EPPENDORF, Germany;

automatic biochemical analyzer 7020, Hitachi High-tech Corporation;

Isoflurane: Lot No. 217171101, Expiry Date: November 1, 2020, Shenzhen RWD Life Science Co., Ltd.;

Urethane: Lot No. 20160920, Expiry Date: September 20, 2021, Sinopharm Chemical Reagent Co., Ltd.;

Sodium Chloride Injection: Lot No. H18052802-2, Guangdong Kelun Pharmaceutical Co., Ltd.;

20% urethane solution for anesthesia: 20.0 g of urethane was added to 100 ml of sodium chloride injection solution, the mixture was thoroughly mixed and sterilized through a filter membrane with pores of 0.2 microns;

TC, TG, HDL-C, LDL-C kits: Shanghai Kehua Bio-Engineering Co., Ltd.;

Quality Control Serum: Randox Laboratories Limited

3.6 Experimental system

Types and status: SPF status, ApoE mouse ^-/- ; 6 ApoE mice, female, 28–35 days old; 48 ApoE mice, half male and half female, 49–56 days old.

Identification: Mice were identified by ear tags. Before perforation, the animal's ear and the perforation device were disinfected. The animal's ear was then perforated and an ear tag with the animal's number was attached.

Animal Welfare: The tests and procedures associated with the animal experimentation conducted in this study complied with relevant laws and regulations for the use and treatment of experimental animals, as well as relevant institutional ethics committee rules for animal research, to ensure the welfare of experimental animals.

Euthanasia: After completion of the study, animals were anesthetized with isoflurane inhalation. Blood was collected from the eyeball, mice were sacrificed by cervical dislocation, and samples were collected. Before burial, the corpses were temporarily stored in a freezer.

Quarantine: acquired mice were quarantined for 7 days. During this period, animals were examined once a day. If unhealthy animals were found, they were immediately removed and healthy ones were added.

Housing and handling: Animals were housed in the SPF animal facility of Guangdong Provincial Laboratory Animal Medical Center. Laboratory Animal Use License No. SYXK (Guangdong) 2018-0002. Conditions for keeping animals: 1 individual/cage; temperature and humidity: 20–26 °C, 40–70%; light/dark cycle: 12 h/12 h; The condition of the living space was always maintained stable to ensure the reliability of the experimental results. During the study, the animals were fed appropriate pelleted food according to the experimental requirements, and the food was provided by the Guangdong Provincial Laboratory Animal Medical Center. Animals were given free access to food and water.

3.7 Doses and division into groups

Doses: In a preliminary test, 4 SD rats (half female, half male) were intragastrically administered test compound at doses of 2000 mg/kg body weight and 5000 mg/kg body weight. No animals died within 72 hours. According to information provided by the sponsor, the adult dose of the study compound is 5 g/day. Based on 60 kg adult body weight, the study doses were 10 times and 20 times the recommended human dose and these doses were 833 mg/kg body weight and 1667 mg/kg body weight for the test compound groups 1 and 2, respectively. The maximum clinical daily dose of the reference drug rosuvastatin calcium tablets is 20 mg. Based on 60 kg of adult body weight. The study dose of the reference drug was 20 times the recommended dose for the human body, that is, the study dose of the reference drug, rosuvastatin calcium tablets, was 6.6 mg/kg body weight.

Separation into groups: after quarantine, mice were kept for 1 week in the usual way. At the first group assignment (simulation period), 6 ApoE ^−/− mice were randomized into the negative control group and received maintenance food. The remaining 48 ApoE ^−/− mice were randomized into model groups and fed a high 21% fat diet for 1 month. Blood was collected to determine four blood lipid parameters. In the second group division (treatment period), ApoE ^−/− mice were randomized into a simulation control group, a positive control group, an 833 mg/kg test compound group, or a 1667 mg/kg test compound group, according to TC levels , 12 mice per group, half male and half female.

Table 9. Doses and division into groups Group n Dose
(mg/kg body weight) Floor Equivalent recommended human dose Route of administration and volume Solution concentration (mg/ml) Negative control group 6 - ♂ - Intragastrically, 1 time per day, 20 ml/kg body weight. - Simulation control group 12 - ♂/♀ - Intragastrically, 1 time per day, 20 ml/kg body weight. - Positive control group 12 3.3 ♂/♀ 20 times Intragastrically, 1 time per day, 20 ml/kg body weight. 0.33 Group receiving test compound 833 mg/kg 12 833 ♂/♀ 10 times Intragastrically, 1 time per day, 20 ml/kg body weight. 41.65 Group receiving study compound 1667 mg/kg 12 1667 ♂/♀ 20 times Intragastrically, 1 time per day, 20 ml/kg body weight. 83.35

3.8 Methodology

3.8.1 Modeling feed with high fat and cholesterol content: maintenance feed containing 20.0% sucrose, 5% lard, 1.0% cholesterol, 0.1% sodium cholate, appropriate amounts of casein, calcium bicarbonate, crushed lime, etc. In addition to crude fat, in the model feed, all parameters of moisture content, crude protein, crude fat, crude fiber, crude ash, calcium, phosphorus and calcium:phosphorus ratio corresponded to the national standard for maintenance feed. Food was provided by Guangdong Provincial Laboratory Animal Medical Center.

3.8.2 Preparation of a solution of the test compound: the required amount of the test compound was crushed and dissolved in a small amount of purified water. The mixture was transferred to a volumetric flask, diluted to the specified volume with purified water and stirred until completely dissolved to obtain a solution for group 2, which received the test compound at 83.35 mg/ml. The solution was diluted 2-fold to about 41.65 mg/ml for group 1 receiving the test compound.

3.8.3 Preparation of a solution of the reference drug: 1 tablet of rosuvastatin calcium (dose: 10 mg/tablet) was ground and dissolved in a 0.5% CMC-Na solution. The mixture was transferred to a volumetric flask and diluted to 30 ml with 0.5% CMC-Na solution, giving a concentration of about 0.33 mg/ml. To ensure thorough mixing, the solution was shaken before use.

3.8.4 Simulation: The negative control group received a maintenance diet and the remaining groups received a high 21% fat diet until the end of the study.

3.8.5 Administration: mice in the positive control group, the 833 mg/kg test compound-treated group, and the 1667 mg/kg test compound-treated group were intragastrically administered the corresponding drug solution at a dose of 20 ml/kg body weight daily, and mice in the negative control group and the simulation control group were administered purified water in the same volume once a day for 60 days.

3.8.6 Measurements:

1) General condition: the general clinical condition of the mice was observed and recorded once a day.

2) Body weight: Body weight was measured once at the beginning and end of the study and once a week during the study. Body weight gain was calculated on Day 30 of treatment (Day 29 of treatment - Day 1 of treatment) and Day 60 of treatment (Day 60 of treatment - Day 1 of treatment).

3) TC, TG, LDL-C and HDL-C levels: After the end of the modeling period, animals were anesthetized with isoflurane inhalation without fasting. Blood was collected from the orbital venous plexus and centrifuged at 3000 rpm at low temperature for 10 min. Serum was separated for determination of total cholesterol TC, triglycerides TG, high-density lipoprotein HDL-C and low-density lipoprotein LDL-C levels, and the remaining serum samples were stored in a freezer at −80°C.

4) Organ weight: The weight of the liver, left kidney, left perinephric fat, right kidney and right perirenal fat was measured, and the organ coefficient was calculated using the following formula. The liver and right kidney were weighed and stored at -80°C.

Organ coefficient = Organ weight (g) Animal body weight on Day 60 of the treatment period (g)

5) Pathological examination: After the end of the study, the mice were fixed in the supine position, the chest cavities of the mice were quickly opened, perfused with saline throughout the body from the left ventricle, and then perfused with 4% paraformaldehyde for fixation. The entire artery from the heart to the inferior branch of the iliac artery was removed, preserved in 4% paraformaldehyde fixative, processed and stained with Oil Red O. The aortic arch, along with the heart and common carotid artery (right), was quickly isolated and fixed with 4% paraformaldehyde. The common carotid artery was stained with Oil Red O, and the aortic arch was stained with HE and Sirius.

3.9 Statistics: data presented ( $$\overline{x}\pm s$$ ). Statistical analysis was performed using SPSS 21.0; Pairwise comparisons were performed using one-way analysis of variance with a control level of α = 0.05.

3.10 Results

3.10.1 General clinical observation: during the study, animal No. 2 in the negative control group died on day 30 of treatment. Anatomical examination demonstrated an enlarged left kidney, with the upper half of the left kidney being milky white and the lower half dark red. In the left kidney, longitudinal dissection revealed a large cavity containing milky fluid and clear fluid. The right kidney was shriveled and atrophied, with aggregated blood clots found. In the bladder, a milky-white fat-like fluid was detected, which accumulated at the outlet of the bladder, accounting for about half of the intraluminal volume of the bladder. There were no visible abnormalities in the gross anatomical structure of the remaining abdominal organs, no visible abnormalities in the gross anatomical structure of the thoracic cavity, and no plaques were observed in the thoracic aorta. Discussion: In vivo cholesterol accumulation and hyperlipidemia are normal manifestations in ApoE knockout mice, and the cause of death in mice is believed to be due to individual differences, impaired lipid metabolism, and excess cholesterol accumulation. No abnormalities in body shape, fur, skin, feces, muscle tension, gait, mood, breathing, etc. were observed in the animals.

3.10.2 Data during the simulation period

3.10.2.1 Body weight during the simulation period (Table 10): There was no statistical difference ( p > 0.05) in the body weight of animals in each group during the simulation period.

3.10.2.2 Blood lipid measurements during the simulation period (Table 11): Compared with the negative control group (♂), the serum levels of TC, TG, HDL-C and LDL-C in the simulation group (♂) increased ( p < 0. 01), and serum TC levels in the simulation group (♂) increased ( p < 0.05).

3.10.3 Data during the treatment period

3.10.3.1 Body weight during the treatment period (Table 12): there was no statistical difference ( p > 0.05) in the body weight of animals between groups during the treatment period; compared with the negative control group (♂), body weight gain in the simulation control group (♂) increased on day 30 and day 60 of treatment ( p <0.05); Compared with the simulation control group (♂), body weight gain in the positive control group (♂), the 833 mg/kg test compound group (♂), and the 1667 mg/kg test compound group (♂) decreased on day 30 and day 60 of treatment ( p < 0.05).

3.10.3.2 Masses and coefficients for organs on day 60 of treatment (Tables 13 and 14): compared with the negative control group (♂), the masses and coefficients for the liver and perirenal fat on the left in the simulation control group (♂) increased ( p < 0. 05 or 0.01), the coefficients for the left kidney and the right kidney decreased ( p < 0.05 or 0.01), and the weights of the left kidney and the right kidney and the coefficient for the left kidney in the simulation control group (♀) decreased ( p < 0.05 or 0.01); compared with the simulation control group (♂), the weight of the left kidney in the positive control group (♂) and the group receiving the test compound 833 mg/kg (♂) decreased ( p <0.05); Compared to the simulation control group (♀), the weight of the right kidney in the group receiving the test compound at 833 mg/kg (♀) decreased ( p < 0.05).

3.10.4 Pathological findings: Full-length Oil Red O staining of the aorta showed that red plaque-like substances were found in the bifurcation of the aorta of ApoE ^-/- mice fed a high-fat diet, and the number of atherosclerotic plaques in simulation group was significantly increased compared to that in the negative control group (FIG. 1). Meanwhile, compared with the simulation control group, Oil Red O staining of the aorta with the test compound in the 833 mg/kg and 1677 mg/kg groups showed that the formation of atherosclerotic plaques was clearly reduced after 60 days of treatment (FIG. 2 ). HE-stained sections of the aortic arch demonstrated that certain histological changes were observed in the simulation control group compared to the negative control group and were manifested mainly by the accumulation of foam cells under the inner lining in the aortic arch and the formation of plaques on the inner lining (FIG. 3). The positive control group showed no significant difference compared to the simulation control group. However, in the 833 mg/kg and 1667 mg/kg test compound groups, only foam cell aggregation was observed under the inner lining in the aortic arch, and no plaques were formed on the inner lining (FIG. 3). Sections of the aortic arch stained with Sirius dye demonstrated that the simulation control group had significant hyperplasia of collagen fibers in the plaque portion of the tunica media and tunica adventitia compared with the negative control group. In the test compound groups, especially in the 1667 mg/kg group, only milder collagen fiber hyperplasia was detected in the tunica adventitia, and no collagen fiber hyperplasia was observed in the tunica interna (FIG. 4). As shown in FIG. 5, Oil Red O staining of the carotid arteries showed that red lipid droplets formed within the lumen, and the 833 mg/kg and 1667 mg/kg test compound groups were significantly superior to the simulation control group and the statin treatment group. In the group receiving the test compound at 1667 mg/kg, only a small amount of red lipid droplets were observed in the lumen of the carotid arteries (FIG. 5).

Group Floor n D1 D8 D15 D22 D29 Negative control group ♂ 6 20.6 ± 1.5 22.0 ± 0.5 24.3 ± 0.6 25.3 ± 0.5 26.8 ± 0.6 Modeling group ♂ 24 25.7 ± 1.5 26.2 ± 1.7 28.0 ± 2.1 28.7 ± 3.0 30.7 ± 2.8 ♀ 24 19.1 ± 1.0 19.3 ± 1.3 20.9 ± 1.3 21.4 ± 1.3 22.2 ± 1.7

Group Floor n TC TG HDL-C LDL-C Negative control group ♂ 6 20.03 ± 3.40 2.39 ± 0.93 3.16 ± 0.67 6.41 ± 2.07 Modeling group ♂ 24 40.00 ± 4.29 ^## 3.91±1.57 ^## 4.49±0.62 ^## 23.99±3.90 ^## ♀ 24 23.81±1.96 ^# 1.59 ± 0.36 2.76 ± 0.28 11.51 ± 6.10 Note: Compared to the negative control group (♂), ^# indicates p < 0.05 and ^## indicates p < 0.01.

Group Floor n D1 D8 D15 D22 D29 Weight gain on day 30 of treatment Negative control group ♂ 5 26.2 ± 0.6 27.5 ± 0.6 27.9 ± 0.9 28.3 ± 1.1 28.4 ± 2.3 2.1 ± 2.2 Simulation control group ♂ 6 31.2 ± 3.5 32.2 ± 3.3 33.1 ± 3.8 33.3 ± 3.9 34.3 ± 3.9 3.2±1.0 ^# ♀ 6 22.3 ± 1.2 23.0 ± 1.4 23.4 ± 1.4 24.0 ± 1.9 24.4 ± 1.8 2.0 ± 1.6 Positive control group ♂ 6 30.1 ± 2.8 30.1 ± 2.9 29.9 ± 2.9 30.4 ± 2.8 30.9 ± 3.7 0.7 ± 1.8 ^■ ♀ 6 21.7 ± 2.4 22.5 ± 2.5 22.7 ± 2.2 23.2 ± 2.9 23.2 ± 3.1 1.5 ± 1.0 Group receiving test compound 833 mg/kg ♂ 6 30.2 ± 2.8 31.0 ± 2.9 30.7 ± 3.0 30.9 ± 3.6 31.8 ± 3.6 1.6 ± 0.9 ^■ ♀ 6 21.1 ± 1.1 22.2 ± 1.1 22.1 ± 1.0 22.5 ± 0.8 22.9 ± 1.0 1.9 ± 0.7 Group receiving study compound 1667 mg/kg ♂ 6 30.6 ± 2.9 30.8 ± 2.7 31.0 ± 3.3 31.5 ± 3.4 32.2 ± 3.5 1.6 ± 1.0 ^■ ♀ 6 21.2 ± 1.0 21.9 ± 0.8 22.6 ± 0.7 22.4 ± 0.8 22.7 ± 1.0 1.5 ± 1.5

Group Floor n D36 D43 D50 D57 D60 Weight gain on day 60 of treatment Negative control group ♂ 5 28.5 ± 1.0 29.3 ± 1.2 30.0 ± 1.4 30.1 ± 1.2 30.4 ± 1.0 4.2 ± 0.8 Simulation control group ♂ 6 34.7 ± 4.0 35.6 ± 4.2 36.3 ± 4.4 36.3 ± 4.4 37.4 ± 4.1 6.2±1.5 ^# ♀ 6 24.4 ± 2.0 25.1 ± 2.4 25.3 ± 2.6 25.3 ± 2.6 25.5 ± 2.4 3.2 ± 2.1 Positive control group ♂ 6 30.5 ± 3.9 31.6 ± 4.3 31.8 ± 4.0 31.8 ± 4.0 32.6 ± 4.4 2.5 ± 2.5 ^■ ♀ 6 23.3 ± 3.3 23.9 ± 4.0 24.2 ± 3.7 24.2 ± 3.7 24.4 ± 2.8 2.7 ± 0.7 Group receiving test compound 833 mg/kg ♂ 6 32.1 ± 3.8 33.0 ± 4.0 33.4 ± 4.0 33.9 ± 3.8 34.2 ± 4.1 4.1 ± 1.7 ^■ ♀ 6 22.8 ± 0.8 25.3 ± 4.8 23.5 ± 1.1 24.1 ± 0.8 24.2 ± 0.7 3.1 ± 0.9 Group receiving study compound 1667 mg/kg ♂ 6 32.0 ± 3.6 32.9 ± 3.7 33.5 ± 3.9 34.2 ± 4.0 34.0 ± 4.1 3.4 ± 1.3 ^■ ♀ 6 22.7 ± 1.1 23.5 ± 1.1 23.8 ± 1.5 24.0 ± 1.4 24.1 ± 1.9 2.9 ± 2.8

Note: Compared to the negative control group (♂), ^# indicates p <0.05; compared to the simulation control group (♂) ^■ indicates p < 0.05.

Group Floor n Organ weight (g) Liver Left kidney (10 ^-1 ) Right kidney (10 ^-1 ) Perirenal fat on the left (10 ^-1 ) Perirenal fat on the right (10 ^-1 ) Negative control group ♂ 5 1.31 ± 0.07 1.76 ± 0.09 1.85 ± 0.19 2.86 ± 3.40 1.18 ± 0.45 Simulation control group ♂ 6 1.83±0.28 ^## 1.90 ± 0.13 1.86 ± 0.18 3.85 ± 1.10 3.54±0.93 ^## ♀ 6 1.17 ± 0.15 1.31±0.23 ^## 1.42±0.09 ^## 1.13 ± 0.61 1.09 ± 0.57 Positive control group ♂ 6 1.56 ± 0.28 1.67 ± 0.19 ^■ 1.79 ± 0.18 2.54 ± 1.27 2.67 ± 1.37 ♀ 6 1.05 ± 0.17 1.26 ± 0.18 1.32 ± 0.18 1.10 ± 0.89 0.89 ± 0.66 Group receiving test compound 833 mg/kg ♂ 6 1.56 ± 0.41 1.77 ± 0.07 ^■ 1.87 ± 0.16 2.83 ± 1.51 2.72 ± 1.40 ♀ 6 1.03 ± 0.03 1.27 ± 0.06 1.27 ± 0.07 ^* 0.81 ± 0.22 0.69 ± 0.23 Group receiving study compound 1667 mg/kg ♂ 6 1.32 ± 0.52 1.76 ± 0.21 1.84 ± 0.17 2.99 ± 1.28 2.96 ± 1.41 ♀ 6 1.06 ± 0.04 1.30 ± 0.06 1.35 ± 0.05 0.95 ± 0.63 0.78 ± 0.53

Note: Compared to the negative control group (♂), ^# indicates p < 0.05, ^## indicates p <0.01; compared to the simulation control group (♂) ■ indicates p <0.05; compared to the simulation control group (♀) * indicates p < 0.05.

Group Floor n Organ coefficient Liver Left kidney (10 ^-1 ) Right kidney (10 ^-1 ) Perirenal fat on the left (10 ^-1 ) Perirenal fat on the right (10 ^-1 ) Negative control group ♂ 5 4.30 ± 0.20 5.80 ± 0.28 6.09 ± 0.67 9.42 ± 11.22 3.87 ± 1.35 Simulation control group ♂ 6 4.88±0.39 ^# 5.13±0.49 ^# 4.99±0.28 ^## 10.17 ± 1.86 9.37±1.40 ^## ♀ 6 4.56 ± 0.23 5.12±0.61 ^# 5.58 ± 0.40 4.30 ± 1.89 4.21 ± 2.05 Positive control group ♂ 6 4.77 ± 0.35 5.16 ± 0.59 5.51 ± 0.52 7.43 ± 3.22 7.81 ± 3.49 ♀ 6 4.28 ± 0.29 5.14 ± 0.19 5.42 ± 0.34 4.30 ± 2.80 3.45 ± 2.03 Group receiving test compound 833 mg/kg ♂ 6 4.51 ± 0.78 5.20 ± 0.46 5.51 ± 0.53 7.99 ± 3.33 7.69 ± 3.05 ♀ 6 4.27 ± 0.24 5.26 ± 0.28 5.25 ± 0.31 3.36 ± 0.90 2.88 ± 0.97 Group receiving study compound 1667 mg/kg ♂ 6 3.87 ± 1.32 5.24 ± 0.84 5.46 ± 0.58 8.57 ± 2.52 8.45 ± 2.89 ♀ 6 4.43 ± 0.32 5.40 ± 0.30 5.61 ± 0.44 3.83 ± 2.10 3.17 ± 1.84

Note: Compared to the negative control group (♂), ^# indicates p < 0.05, ^## indicates p <0.01; compared to the simulation control group (♂) ■ indicates p <0.05; compared to the simulation control group (♀) * indicates p < 0.05.

3.11 Summary

The traditional Chinese medicine composition described herein has a significant effect on reducing the formation of atherosclerotic plaques and reducing weight gain in animal models.

Example 4: Clinical Trials for the Treatment of Hyperlipidemia

The subjects were from Zhejiang Huzhou Ruibo Traditional Chinese Medicine Clinic (No. 322, Meizhou Road, Huzhou City, Zhejiang Province) and Beijing Tongyitang Clinic (1st Floor, Building 4, Zhongguancun Biological Sciences Park, Changping District, Beijing).

Treatment data were collected from two clinics as described above using powder A, B or C for the dry ointment extract of the present invention in these subjects, with 14 subjects having elevated total cholesterol levels (>6.2 mmol/L ) and triglycerides (>1.7 mmol/L), 11 subjects had elevated total cholesterol levels and 88 subjects had elevated triglyceride levels (Table 15).

Patients received one to three 14-day courses of treatment. Patients were administered the drug twice a day, each time 1 or 2 packets of granular formulation depending on the severity of the disease, each packet of granular formulation containing 1250 mg of powder A, B or C for dry ointment extract.

With a 10% reduction in total cholesterol or triglycerides as the criterion for effectiveness, in 14 patients with elevated total cholesterol and triglycerides, nearly 79% of patients had effective reduction in total cholesterol, 93% of patients had effective reduction in triglycerides, and 10 patients effective reductions in both indicators were observed (Table 15 and FIG. 6).

The traditional Chinese medicine composition described herein has an effective rate of reducing elevated total cholesterol levels of 45% (shown in Table 15 and FIG. 7) and an effective rate of reducing hypertriglyceridemia close to 90% (shown in Table 15 and FIG. 7). FIGURE 8). The traditional Chinese medicine composition described herein has excellent efficacy in patients with severe hyperlipidemia, especially in subjects with high blood triglyceride levels (triglycerides >5.6 mmol/L).

Table 15. Summary of data regarding the level of total cholesterol and triglycerides in the blood before and after treatment in 113 patients with hyperlipidemia Number of subjects Drugs
(quantity) Total cholesterol (TC > 6.2 mmol/l) Triglycerides (TG > 1.7 mmol/l) Before treatment (mean ± std, mmol/l) After treatment (mean ± std, mmol/l) Coeff. reduction by 10% or more Before treatment (mean ± std, mmol/l) After treatment (mean ± std, mmol/l) Coeff. reduction by 10% or more Patients with hypercholesterolemia (HC) eleven A(3),C(8) 6.78 ± 0.68 6.08±0.68 ^▲ 45.0% Patients with hypertriglyceridemia (TG) 88 A(71),B(17) 2.95 ± 2.19 1.69 ± 1.07 ^▲▲ 88.7% Patients with hyperlipemia (increased TC and TG levels) 14 A(7),B(4),C(3) 7.18 ± 1.14 5.8 ± 1.59 ^▲▲ 78.6% 8.67 ± 10.73 1.54 ± 0.75 ^▲▲ 92.8%

Note: comparison between groups, independent sample T-test; “ ^▲ ” indicates P < 0.05, “ ^▲▲ ” indicates P < 0.01.

Example 5. Typical cases of treatment of hyperlipidemia

Example 5-1:

Shao, a male patient, visited a doctor at Zhejiang Huzhou Ruibo Traditional Chinese Medicine Clinic (No. 322 Meizhou Road, Huzhou City, Zhejiang Province) in 2016. Diagnostic testing demonstrated that the blood was milky white, blood triglycerides were 33.4 mmol/L, and total blood cholesterol was 6.71 mmol/L. The granular formulation was administered twice daily, 2 packets each time, each packet containing 1250 mg of powder A for dry ointment extract. After one course of treatment (14 days), triglycerides dropped to 0.62 mmol/L and total cholesterol dropped to 4.41 mmol/L, both of which returned to normal levels.

Example 5-2:

Chen, a female patient, visited a doctor at Zhejiang Huzhou Ruibo Traditional Chinese Medicine Clinic (No. 322 Meizhou Road, Huzhou City, Zhejiang Province) in 2016. Chen was taking a long-term lipid-lowering statin drug at the time of her visit, and diagnostic testing demonstrated that her blood triglyceride level was 8.31 mmol/L and her total cholesterol level was 6.59 mmol/L, both of which were higher than normal values. In particular, blood triglyceride levels were much higher than normal levels. The granular formulation was administered twice daily, 2 packets each time, each packet containing 1250 mg of powder A for dry ointment extract. After one course of treatment (14 days), triglycerides dropped to 1.01 mmol/L and total cholesterol dropped to 4.77 mmol/L, both of which returned to normal levels.

Example 5-3:

Huang, a male patient, visited a doctor at Zhejiang Huzhou Ruibo Traditional Chinese Medicine Clinic (No. 322 Meizhou Road, Huzhou City, Zhejiang Province) in 2017. When Dai visited the doctor, diagnostic testing showed his blood triglyceride levels were 8.05 mmol/L and total cholesterol levels were 5.51 mmol/L, both of which were higher than normal. Triglyceride levels were especially elevated. The granular formulation was administered twice daily, 1 packet each time, each packet containing 1250 mg of Powder B for dry ointment extract. After two courses of treatment, triglyceride levels decreased to 1.45 mmol/L and returned to normal levels.

Example 5-4:

Yu, a male patient, visited a doctor at Zhejiang Huzhou Ruibo Traditional Chinese Medicine Clinic (No. 322 Meizhou Road, Huzhou City, Zhejiang Province) in 2018. When Chen visited the doctor, diagnostic testing showed his blood triglyceride level was 7.13 mmol/L and total cholesterol level was 4.51 mmol/L, indicating hypertriglyceridemia. The granular formulation was administered twice daily, 1 packet each time, each packet containing 1250 mg of Powder B for dry ointment extract. After two courses of treatment, triglyceride levels decreased to 1.75 mmol/L and returned to normal levels. Total cholesterol levels did not change.

Example 5-5:

Shen, a male patient, visited a doctor at Zhejiang Huzhou Ruibo Traditional Chinese Medicine Clinic (No. 322 Meizhou Road, Huzhou City, Zhejiang Province) in 2017. When Xu visited the doctor, diagnostic testing showed that his blood triglyceride level was 0.95 mmol/L and his total cholesterol level was 7.22 mmol/L, indicating hypercholesterolemia. The granular formulation was administered twice daily, 1 packet each time, each packet containing 1250 mg of C powder for dry ointment extract. After three courses of treatment, the level of total cholesterol decreased to 4.91 mmol/l and returned to normal levels. Triglyceride levels did not change significantly.

Example 6. Typical cases of treatment of atherosclerosis

Example 6-1:

Yang, a male patient, visited a doctor at Beijing Tongyitang Hospital (1st Floor, Building 4, Zhongguancun Life Science Park, Changping District, Beijing) in 2019. At the visit, diagnostic examination demonstrated that atherosclerotic plaques had formed in the common carotid artery and right subclavian artery on both sides, and multiple plaques had formed in the right common carotid artery, the largest of which was 16.5 mm in length and 3.9 mm in thickness; At the same time, arteriosclerotic plaques formed in the right subclavian artery. In the left common carotid artery, numerous plaques with a maximum length of 3.5 mm and a thickness of 1.6 mm had formed; the diameter of the right vertebral artery was 2.8 mm, the mean blood flow velocity was 0.21 m/s, and the blood flow volume was 77 ml/min.

The granular formulation was administered twice daily, 2 packets each time, each packet containing 1250 mg of powder A for dry ointment extract. After eight courses of treatment and four months of treatment, sclerotic plaques in the right common carotid artery decreased in size and disappeared, the largest of which was 10.0 mm long and 1.8 mm thick. Atherosclerotic plaques in the upper part of the right subclavian artery disappeared, the diameter of the vertebral artery increased (3.2 mm), blood flow velocity (0.21 m/s) and blood flow volume (up to 135 ml/min after treatment for impaired blood flow volume) significantly improved. .

Example 6-2:

Wang, a 40-year-old male patient, visited a doctor at Beijing Tongyitang Hospital (1st Floor, Building 4, Zhongguancun Life Science Park, Changping District, Beijing) in 2018. When visiting a doctor, a diagnostic examination demonstrated that arteriosclerotic plaques measuring about 1.6 mm had formed in the common carotid artery on both sides.

The granular formulation was administered twice daily, 2 packets each time, each packet containing 1250 mg of powder A for dry ointment extract. After six courses of treatment (3 months of treatment), no obvious plaques had formed in the left and right common carotid arteries, and the original sclerotic plaques had disappeared.

Claims (8)

1. A traditional Chinese medicine composition for the prevention or treatment of high blood lipid levels, high triglyceride levels, high cholesterol levels, hyperlipidemia and atherosclerosis, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 20-30 parts of capillary root, 10-20 parts of unripe orange fruit, 5-15 parts of rhubarb root, 5-10 parts of skullcap Baikal root, 5-15 parts of white peony root, 5-10 parts of dried ginger rhizome, 7-15 parts of leech, 5-15 parts of root licorice, 5-15 parts of dried codonopsis root, 5-15 parts of Atractylodes macrocephala rhizome and 10-20 parts of pinellia tuber. 2. The composition of traditional Chinese medicine according to claim 1, wherein the specified composition of traditional Chinese medicine is prepared from the following raw materials in parts by weight: 25 parts of volodushka root, 15 parts of unripe orange fruits, 10 parts of rhubarb root, 6 parts of Baikal skullcap root, 10 parts of root white peony, 6 parts of dried ginger rhizome, 10 parts of leech, 10 parts of licorice root, 10 parts of dried codonopsis root, 10 parts of Atractylodes macrocephala rhizome and 15 parts of pinellia tuber. 3. A traditional Chinese medicine composition for the prevention or treatment of high blood lipid levels, high triglyceride levels, high cholesterol levels, hyperlipidemia and atherosclerosis, where the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 20-30 parts of volodushi root, 10 -20 parts of unripe orange fruit, 5-15 parts of rhubarb root, 5-10 parts of skullcap Baikal root, 5-15 parts of white peony root, 5-10 parts of dried ginger rhizome, 7-15 parts of leech, 5-15 parts of licorice root , 5-15 parts of dried codonopsis root, 5-15 parts of Atractylodes macrocephala rhizome, 10-20 parts of pinellia tuber and 10-35 parts of oyster shell. 4. The composition of traditional Chinese medicine according to claim 3, wherein the specified composition of traditional Chinese medicine is prepared from the following raw materials in parts by weight: 25 parts of volodushka root, 15 parts of unripe orange fruits, 10 parts of rhubarb root, 6 parts of Baikal skullcap root, 10 parts of root white peony, 6 parts dried ginger rhizome, 10 parts leech, 10 parts licorice root, 10 parts dried codonopsis root, 10 parts Atractylodes macrocephala rhizome, 15 parts pinelli tuber and 30 parts oyster shell. 5. A traditional Chinese medicine composition for the prevention or treatment of elevated blood lipid levels, elevated triglyceride levels, elevated cholesterol levels, hyperlipidemia and atherosclerosis, where the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 20-30 parts of volodushi root, 10 -20 parts of unripe orange fruit, 5-15 parts of rhubarb root, 5-10 parts of skullcap Baikal root, 5-15 parts of white peony root, 5-10 parts of dried ginger rhizome, 7-15 parts of leech, 5-15 parts of licorice root , 5-15 parts dried codonopsis root, 5-15 parts Atractylodes macrocephala rhizome, 10-20 parts pinellia tuber, 10-35 parts oyster shell and 5-12 parts red fermented rice. 6. The composition of traditional Chinese medicine according to claim 5, wherein the specified composition of traditional Chinese medicine is prepared from the following raw materials in parts by weight: 25 parts of volodushka root, 15 parts of unripe orange fruits, 10 parts of rhubarb root, 6 parts of Baikal skullcap root, 10 parts of root white peony, 6 parts dried ginger rhizome, 10 parts leech, 10 parts licorice root, 10 parts dried codonopsis root, 10 parts Atractylodes macrocephala rhizome, 15 parts pinellia tuber, 30 parts oyster shell and 10 parts red fermented rice. 7. The composition of traditional Chinese medicine according to any one of paragraphs. 1-6, wherein said traditional Chinese medicine composition is an oral composition. 8. The traditional Chinese medicine composition according to claim 7, wherein said traditional Chinese medicine composition is a decoction, granule, tablet or capsule.