A preparation of erdun-wurige optimized based on traditional mongolian medicine formula

Abstract

The application discloses a Eerduan-Wurige optimized preparation based on a traditional Mongolian medicine prescription and belongs to the technical field of medicines. The Eerduan-Wurige optimized preparation based on the traditional Mongolian medicine prescription comprises the following raw materials in a mass ratio: 1000 parts of pearl powder, 200 parts of gardenia, 100 parts of safflower, 40 parts of saffron, 140 parts of cubeb, 80 parts of liquidambar altinge, 59 parts of artificial ox gall, 1 part of artificial musk and 80 parts of silver pearl. The Eerduan-Wurige optimized preparation based on the traditional Mongolian medicine prescription can effectively reduce the efflux of cholesterol, and the effect is equivalent to that of simvastatin; and the Eerduan-Wurige optimized preparation based on the traditional Mongolian medicine prescription can reduce the LDLR ‑ / ‑ The effect on the area of atherosclerotic plaque of a mouse.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an optimized formulation of Erdun-Urile based on traditional Mongolian medicine. Background Technology

[0002] Lipid metabolism disorders are closely related to the occurrence and development of cardiovascular diseases. Hyperlipidemia is one of the core risk factors for atherosclerosis, and elevated serum total cholesterol is an important warning indicator of atherosclerosis. Hyperlipidemia is defined as one or more lipid components in plasma exceeding the normal reference range due to abnormalities in lipid synthesis, metabolism, or transport function. Because lipids are poorly soluble in water, they need to bind with proteins under physiological conditions and be transported in the body as lipoproteins. Therefore, hyperlipidemia is also often called hyperlipoproteinemia, mainly including three types: hypercholesterolemia, hypertriglyceridemia, and a combination of both. Studies have found that hyperlipidemia accounts for more than 60% of patients with coronary heart disease, and about half of the coronary heart disease patients also have hyperlipidemia; the prevalence of hypertriglyceridemia is as high as about 80% among patients with myocardial infarction. At the same time, hyperlipidemia is also an important trigger for various cerebrovascular diseases.

[0003] Atherosclerosis is the most common and serious type of atherosclerotic vascular disease, primarily originating in the arterial intima. Its pathological process involves abnormal deposition of lipids and complex carbohydrates, accompanied by local hemorrhage, thrombosis, fibrosis, and calcium salt deposition. Simultaneously, the arterial media gradually undergoes degenerative changes and calcification. The lesions mainly affect elastic arteries and large to medium-sized muscular arteries. As the disease progresses, it can cause luminal narrowing or even occlusion, leading to ischemia and even necrosis of the corresponding blood-supplying tissues and organs. Because the lipids deposited in the arterial intima have a yellow, porridge-like appearance, it is named atherosclerosis. Atherosclerosis is a crucial pathological basis for cardiovascular and cerebrovascular diseases. The coronary heart disease and stroke caused by atherosclerosis are characterized by high incidence, high disability rate, high mortality rate, and high recurrence rate, seriously threatening human physical and mental health and considered a major public health hazard. Therefore, strengthening the prevention and intervention of atherosclerosis is a core element in reducing the incidence and mortality risks of cardiovascular and cerebrovascular diseases. The core pathogenesis of acute cardiovascular and cerebrovascular events mainly stems from the rupture of vulnerable atherosclerotic plaques, which subsequently induces platelet activation and thrombus formation. Therefore, stabilizing atherosclerotic plaques is crucial for preventing and controlling acute cardiovascular and cerebrovascular events.

[0004] Currently, there are various Western and traditional Chinese medicines available for treating hyperlipidemia. Western medicines include statins and fibrates, while traditional Chinese medicines include lipoprotein lipase and lipoprotein lipase. However, these medications have drawbacks such as poor therapeutic efficacy and significant side effects. Therefore, there is an urgent need to find effective drugs for treating hyperlipidemia while avoiding the occurrence of side effects. Summary of the Invention

[0005] The purpose of this invention is to provide an optimized Erdun-Urile formula based on traditional Mongolian medicine to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is an optimized formula of Erdun-Urile based on traditional Mongolian medicine, comprising the following raw materials in parts by weight: 1000 parts pearl powder, 200 parts gardenia, 100 parts safflower, 40 parts saffron, 140 parts long pepper, 80 parts styrax resin, 59 parts artificial bezoar, 1 part artificial musk, and 80 parts silver beads.

[0007] The second technical solution of the present invention: an optimized Erdun-Urile formula based on traditional Mongolian medicine, comprising the above-mentioned optimized Erdun-Urile formula based on traditional Mongolian medicine.

[0008] Preferably, the formulation comprises pills.

[0009] The third technical solution of the present invention: an optimized Erdun-Urile formula pill based on the traditional Mongolian medicine formula, comprising the above-mentioned optimized Erdun-Urile formula based on the traditional Mongolian medicine formula.

[0010] The fourth technical solution of the present invention: a method for preparing the above-mentioned Erdun-Urile optimized pill, comprising the following steps: The raw materials, excluding the silver beads, are crushed and mixed to form pills. Then, silver beads are added for coating and polishing to obtain the Erdun-Urile optimized pill formulation.

[0011] Preferably, the step of pulverizing and mixing the raw materials other than silver beads and then pelletizing them includes: Saffron, artificial bezoar and artificial musk were pulverized and sieved to obtain saffron powder, artificial bezoar powder and artificial musk powder respectively. Pearl powder, gardenia, safflower, long pepper, and styrax resin are initially crushed and then mixed together, followed by secondary crushing and sieving to obtain a fine mixed powder. The saffron powder, artificial bezoar powder, artificial musk powder, and mixed fine powder are mixed evenly, then ground and made into pills.

[0012] Preferably, the sieve used for sieving has a mesh size of 120.

[0013] Preferably, the diameter of the pellets obtained by pelletizing is 5-7 mm.

[0014] The fifth technical solution of the present invention: the application of the above-mentioned Erdun-Urile optimized formula, Erdun-Urile optimized formula preparation or Erdun-Urile optimized formula pills in the preparation of a drug for treating atherosclerosis.

[0015] The sixth technical solution of the present invention: the application of the above-mentioned Erdun-Urile optimized formula, Erdun-Urile optimized formula preparation or Erdun-Urile optimized formula pills in the preparation of lipid-lowering drugs.

[0016] The present invention discloses the following technical effects: The Erdun-Urile optimized formula based on traditional Mongolian medicine of this invention can effectively reduce cholesterol excretion, with effects comparable to simvastatin. Furthermore, the Erdun-Urile optimized formula based on traditional Mongolian medicine of this invention can reduce LDLR. - / - The effect of atherosclerotic plaque area on mice.

[0017] The Erdun-Urile optimized formula based on traditional Mongolian medicine of the present invention has a better therapeutic effect on hyperlipidemia than the traditional Erdun-Urile formula. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The results of Oil Red O staining of the full-length aorta of LDLR- / - mice after 8 weeks of administration in Example 1 are shown. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0026] In a first aspect, the present invention provides an optimized formula of Erdun-Urile based on a traditional Mongolian medicine formula, comprising the following raw materials in parts by weight: 1000 parts pearl powder, 200 parts gardenia, 100 parts safflower, 40 parts saffron, 140 parts long pepper, 80 parts styrax resin, 59 parts artificial bezoar, 1 part artificial musk, and 80 parts silver beads.

[0027] In a second aspect, the present invention provides an optimized Erdun-Urile formula based on a traditional Mongolian medicine formula, comprising the aforementioned optimized Erdun-Urile formula based on a traditional Mongolian medicine formula.

[0028] Preferably, the formulation includes pills.

[0029] In a third aspect, the present invention provides an optimized Erdun-Urile formula pill based on a traditional Mongolian medicine formula, comprising the aforementioned optimized Erdun-Urile formula based on a traditional Mongolian medicine formula.

[0030] In a fourth aspect, the present invention provides a method for preparing the above-mentioned Erdun-Urile optimized pill, comprising the following steps: (1) Pretreatment and pulverization of medicinal materials Separate pulverization: Take saffron, artificial bezoar and artificial musk and pulverize them separately. After pulverization, pass them through a 120-mesh sieve to make extremely fine powder. The resulting saffron powder, artificial bezoar powder and artificial musk powder should be sealed and stored away from light to prevent the volatilization of active ingredients or moisture.

[0031] Mixed pulverization: First, the five medicinal materials, pearl powder, gardenia, safflower, long pepper, and styrax resin, are initially crushed to remove impurities, and then pulverized together. After pulverization, they are also passed through a 120-mesh sieve to obtain a mixed fine powder, which is then sealed for later use.

[0032] (2) Mixing of medicinal powders Saffron powder, artificial bezoar powder, artificial musk powder, and mixed fine powder are placed together in a mixing device and thoroughly stirred to ensure that each powder is evenly distributed, with no obvious color difference or particle separation. After mixing, the mixture is sealed and stored for later use.

[0033] (3) Grinding and pelletizing The uniformly mixed powder is accurately weighed, and the amount of powder required for starting the mill is calculated based on the total amount of powder. Grinding process: Take an appropriate amount of purified water and wet the millstone in small amounts several times. Then add a small amount of medicine powder one by one, shaking it while adding the medicine powder. Repeat the process until a suitable sized clump of medicine is ground (grinding is up to standard). Mechanical pelleting: After the powder has been ground to the required standard, the pellets are placed into a pelleting machine. The remaining powder and purified water are added in small, frequent batches, maintaining uniform operation throughout the process. Periodically, the pellets are sieved with a pellet sieve to remove unevenly sized pellets. The small pellets are then shaped into uniform sizes and placed back into the pelleting machine to continue pelleting, ensuring that the pellet size is consistent.

[0034] (4) Coating and polishing After pelleting is completed, the pellets are transferred to a coating and polishing equipment, where silver beads are added for coating and polishing until the pellet surface is smooth, the color is uniform, and the silver beads are evenly coated on the pellet surface without significant shedding.

[0035] (5) Screening and drying Screening and grading: The coated and polished pellets are screened using a pellet sieve with a 5mm aperture. Pellets that meet the specifications are selected, while broken pellets, irregularly shaped pellets, and pellets that do not match the size are removed.

[0036] Low-temperature drying: The selected qualified pellets are spread evenly on a drying tray and placed in a 30℃ constant temperature drying equipment for drying until the moisture content of the pellets meets the standard and the texture is firm.

[0037] (6) Finished product inspection and harvesting After drying, inspect the specifications of the pills to ensure that every 10 pills weigh 2g, and that the pills are round, uniform in color, without sticking together or mold, meeting the quality standards for water pills, thus obtaining the Erdun-Urile optimized formula pills.

[0038] Preferably, the diameter of the pellets obtained from pelleting is 5-7 mm.

[0039] In a fifth aspect, the present invention provides the use of the above-mentioned Erdun-Urile optimized formula, Erdun-Urile optimized formula preparation or Erdun-Urile optimized formula pills in the preparation of a drug for treating atherosclerosis.

[0040] In a sixth aspect, the present invention provides the use of the above-mentioned Erdun-Urile optimized formula, Erdun-Urile optimized formula preparation or Erdun-Urile optimized formula pills in the preparation of cholesterol-lowering drugs.

[0041] The Erdun-Urile, also known as Zhenbao Pill, used in this invention was purchased from Inner Mongolia Mongolian Medicine Co., Ltd.

[0042] Example 1 A method for preparing Erdun-Urile optimized pills based on traditional Mongolian medicine formulas: The Erdun-Urile optimized formula, based on traditional Mongolian medicine, consists of the following ingredients: 1000g pearl powder, 200g gardenia, 100g safflower, 40g saffron, 140g long pepper, 80g styrax resin, 59g artificial bezoar, 1g artificial musk, and 80g silver beads.

[0043] (1) Pretreatment and pulverization of medicinal materials Separate pulverization: Take saffron, artificial bezoar and artificial musk and pulverize them separately. After pulverization, pass them through a 120-mesh sieve to make extremely fine powder. The resulting saffron powder, artificial bezoar powder and artificial musk powder should be sealed and stored away from light to prevent the volatilization of active ingredients or moisture.

[0044] Mixed Grinding: First, the five medicinal materials, pearl powder, gardenia, safflower, long pepper, and styrax resin, are initially crushed to remove impurities. Then, they are ground together and passed through a 120-mesh sieve to obtain a mixed fine powder, which is then sealed for later use.

[0045] (2) Mixing of medicinal powders Saffron powder, artificial bezoar powder, artificial musk powder, and mixed fine powder are placed together in a mixing device and thoroughly stirred to ensure that each powder is evenly distributed, with no obvious color difference or particle separation. After mixing, the mixture is sealed and stored for later use.

[0046] (3) Grinding and pelletizing Weigh the well-mixed powder precisely (1000g), and calculate the amount of powder needed for starting the grinding process based on the total amount of powder.

[0047] Grinding process: Take 150mL of purified water and wet the grinding mill in small amounts several times. Then add small amounts of powder while shaking to mix. Repeat this process until a 2-3cm clump of powder is formed (grinding is up to standard).

[0048] Mechanical pelleting: After the powder has been ground to the required standard, place the pellets into a pelleting machine. Continue to add the remaining powder and purified water (650mL) in small, frequent increments, maintaining uniform operation throughout the process. Periodically sift the pellets with a pellet sieve to remove those of uneven size. After the small pellets are sized uniformly, they are returned to the pelleting machine to continue pelleting, ensuring that the pellet size is consistent (approximately 5-7mm in diameter).

[0049] (4) Coating and polishing After pelleting is completed, the pellets are transferred to a coating and polishing equipment, where silver beads (5~15μm in diameter) are added for coating and polishing until the pellet surface is smooth and uniform in color, and the silver beads are evenly coated on the pellet surface without significant shedding.

[0050] (5) Screening and drying Screening and grading: The coated and polished pellets are initially screened using a pellet sieve with a 5mm aperture to remove small pellets with a diameter of less than 5mm; then, an oversized pellet is removed using a pellet sieve with a 7mm aperture, and pellets with a diameter of 5-7mm and uniform shape are selected as qualified pellets.

[0051] Low-temperature drying: The selected pellets are spread evenly on a drying tray and placed in a 30℃ constant temperature drying equipment for drying until the moisture content of the pellets reaches the standard (7~8%) and the texture is firm.

[0052] (6) Finished product inspection and harvesting After drying, inspect the specifications of the pills to ensure that every 10 pills weigh 2g, and that the pills are round, uniform in color, without sticking together or mold, meeting the quality standards for water pills, thus obtaining the Erdun-Urile optimized formula pills.

[0053] Application Example 1 Atherosclerotic plaque experiment: (a) Materials (1) Laboratory animals Twelve male C57BL / 6 mice (normal control group), aged 12 weeks; LDLR - / - Seventy-five male mice, 12 weeks old, were provided by Changzhou Cavens Laboratory Animal Co., Ltd.

[0054] (2) Test sample Erdun-Urile optimized pill preparation prepared in Example 1.

[0055] (3) Reagents Distilled water, Hangzhou Wahaha Group Co., Ltd.; Sodium pentobarbital, Union Chem. Inc.; Physiological saline, Shandong Hualu Pharmaceutical Co., Ltd.; 10% neutral formalin fixative, Inlani International Trading (Shanghai) Co., Ltd.; Isopropanol, Sinopharm Chemical Reagent Co., Ltd.; Oil Red O, Sigma-Aldrich.

[0056] (4) Instruments Balance, Tianmei Instruments Laboratory Equipment (Shanghai) Co., Ltd.; Benchtop High-Speed ​​Low-Temperature Centrifuge, Merrick Instruments (Shanghai) Co., Ltd.; Shaker, Dalong Xingchuang Laboratory Instruments (Beijing) Co., Ltd.; Low-Temperature Refrigerator, Changhong Meiling Co., Ltd.; Stereo Microscope, Olympus (China) Co., Ltd.; Imaging System, Canon Corporation.

[0057] (II) Methods (1) High-fat diet induces LDLR - / - Atherosclerotic plaque formation in mice Give LDLR - / - Mice were fed a high-fat, high-cholesterol diet. C57BL / 6 mice were given a normal diet. After 8 weeks of modeling, the animals were fasted overnight. 200 μL of blood was collected from the inner canthus of the eye, allowed to stand at room temperature for 30 min, centrifuged at 4000×g, and the serum was separated, aliquoted, and stored at -80℃ for later use. Three mice on the high-fat diet were randomly selected for LDLR testing. - / - In mice, the aortic root was isolated, embedded in OCT, and frozen at -20°C for later use; the entire length of the mouse aorta was isolated, fixed in 10% neutral formalin solution, and stored at 4°C for later use.

[0058] (2) LDLR - / - Mouse full-length aorta Oil Red O staining Preparation of Oil Red O stock solution (0.5% isopropanol solution): Weigh 0.5g of Oil Red O powder, dilute to 100mL with isopropanol solution, dissolve by shaking on a shaker at room temperature in the dark for 6h, and store at room temperature in the dark.

[0059] Preparation of Oil Red O working solution: On the day of staining, take Oil Red O stock solution (0.5% isopropanol solution) and distilled water, dilute them at a ratio of stock solution:water = 3:2 (v:v), and filter them through a 0.22μm filter membrane to obtain Oil Red O working solution.

[0060] Cleaning and trimming of the entire aorta: The entire aorta fixed with 10% neutral formalin was cleaned with tap water. Under a stereomicroscope, the perivascular adipose tissue was removed, and the remaining branch vessels were trimmed, except for the branch vessels at the aortic arch location, which were preserved for about 3 mm.

[0061] Staining of the entire aorta: After rinsing with tap water, place the entire aorta into a 2 mL EP tube, add distilled water, and wash on a shaker for 5 min, repeating 3 times. Discard the distilled water, replace with 60% isopropanol, and wash with shaking for 5 min, repeating 3 times. Aspirate the residual liquid on the vessel, add 2 mL of Oil Red O working solution, and stain with shaking on a shaker for 8 h. Remove the entire aorta and aspirate the residual staining solution, place it into a new 5 mL EP tube, add 60% isopropanol for washing and differentiation, and shake for 10 min, repeating 3 times. Rinse the differentiated entire aorta with tap water for later use.

[0062] Dissection and photography of the entire aorta: The aorta was transferred to a petri dish approximately 5.5 cm in diameter lined with a black silicone pad. Distilled water was added, and the main vessel trunk was fixed with acupuncture needles. Residual fatty tissue stained with Oil Red O was removed from the vessel wall under a stereomicroscope. The three branches of the aortic arch (brachiocephalic trunk, left common carotid artery, and left subclavian artery) were cut along the lateral side of the distal end of the aortic arch to a level 2 mm below the left clavicle, ensuring the aortic arch and bifurcation vessels were flattened. The acupuncture needles were used for orderly fixation and spreading. The inner surface of the aortic arch and plaques were fully exposed. From the opening of the lesser bend of the aorta, the entire vessel was cut along the longitudinal axis of the aorta using microsurgical scissors, pointing directly towards the bifurcation of the iliac artery. The cut vessel was fixed with acupuncture needles. A full-length photograph of the inner surface of the aorta was obtained using a DSLR camera equipped with a macro lens.

[0063] Total aortic plaque area statistics: ImageJ software was used to count the total inner surface area of ​​the aorta. At the same time, the area of ​​atherosclerotic plaques stained with Oil Red O was circled, and the value of (plaque area / inner surface area of ​​blood vessel) × 100% was calculated.

[0064] (3) Animal grouping, administration and sampling Eight weeks after modeling, the high-fat diet was replaced with a regular diet, and LDLR was introduced. - / -Mice were randomly divided into a model group, a simvastatin group, an Erdun-Urile (i.e., Zhenbao Pill, purchased from Inner Mongolia Mongolian Medicine Co., Ltd.) group, and high, medium, and low dose groups of the Erdun-Urile optimized formula (i.e., the Erdun-Urile optimized formula pill prepared in Example 1, ground and administered by gavage), with 12 mice in each group. C57BL / 6 mice and the model control group were given the control solvent (distilled water for drug preparation), while the other groups were administered the corresponding drugs by gavage. Four weeks after drug administration, the animals were fasted overnight, and 200 μL of blood was collected from the inner canthus of the eye. The serum was separated, aliquoted, and frozen for later use. Eight weeks after drug administration, the animals were fasted overnight, and 0.8 mL of blood was collected from the inner canthus of the eye after anesthesia with sodium pentobarbital. The serum was separated, aliquoted, and frozen for later use. The mouse heart and full-length aorta were rapidly separated. The heart was severed 2 mm from the aortic root towards the apex, and the aorta was cut 1 mm towards the aorta. The entire aortic root was placed in an OCT embedding mold, ensuring the ascending aortic opening was upright. After flash freezing in liquid nitrogen, the aorta was stored at -20°C for later use. The full-length aorta of 6 mice from each group was fixed in 10% neutral formalin for Oil Red O staining. The full-length aorta of the remaining 6 mice was flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0065] (III) Statistical Analysis Statistical analysis was performed using IBM SPSS Statistics 26 software. For continuous variables, the Shapiro-Wilk test for normality was first used. If the p-value was greater than 0.05, it indicated that the variable conformed to a normal distribution, and the result was expressed as mean ± standard deviation. If the data did not conform to a normal distribution (including some groups not conforming to a normal distribution), the median (interquartile range) was used.

[0066] Levene's test is used to test for homogeneity of variance. A p-value > 0.05 indicates homogeneity of variance among the data. If all groups are normally distributed and have homogeneous variances, the Bonferroni test is used for comparisons among multiple groups. If the groups are normally distributed but have unequal variances, the Tammhenko test is used. If the groups do not conform to a normal distribution, the Kruskal-Wallis H test is used for comparisons among multiple groups. Paired samples that do not conform to a normal distribution are analyzed using the Wilcoxon test (signed-rank test). A p-value < 0.05 is considered statistically significant.

[0067] (iv) Results (1) LDLR after 8 weeks of administration - / - Results of Oil Red O staining of the full-length aorta of mice are shown in the figure. Figure 1 .

[0068] from Figure 1As can be seen, large areas of Oil Red O positive staining were visible in the aortic arch intima of each group of animals, and scattered positive staining plaques of varying degrees were visible from the descending aorta to the abdominal aorta. The plaque formation was different in different drug administration groups.

[0069] Table 1. Erdun-Urile optimization formula for LDLR - / - Effect of the proportion of atherosclerotic plaque area in mice Compared with the model group, ** , P<0.01 ; *** , P<0.001 .

[0070] Statistical results showed that the area of ​​Oil Red O positive staining in the entire aorta of the model group accounted for 6.7% of the total vascular intima area. Numerous atherosclerotic plaques were observed at the bifurcation of the aortic arch, brachiocephalic trunk, left common carotid artery, and left subclavian artery in the model group animals; significant plaque distribution was also observed at the lesser curvature of the aortic arch, the thoracic aorta, and the abdominal aorta, suggesting that atherosclerotic lesions persisted even after 8 weeks of high-fat diet modeling.

[0071] Compared with the model group, the simvastatin group (24 mg / kg) showed a significant reduction in the Oil Red O positive area of ​​the aortic intima, with plaques mainly limited to the bifurcation and lesser curvature of the aortic arch, accounting for 2.8% of the total plaque area.

[0072] Compared with the model group, the Erdun-Urile group (400 mg / kg) showed a significant reduction in the number of plaques in the thoracic and abdominal aortas, with a plaque area ratio of 4.8%.

[0073] The number of plaques in the thoracic and abdominal aortas was significantly reduced in the Erdun-Uryl optimized formula groups at 400 mg / kg and 800 mg / kg; the plaque area in the aortic arch of the 800 mg / kg group showed a decreasing trend. The atherosclerotic area was 3.9% and 3.1% at the two dosages, respectively.

[0074] The above results indicate that the Erdun-Urile optimization formula can reduce LDLR. - / - The effect of atherosclerotic plaque area on mice.

[0075] Application Example 2 Experiment on cholesterol efflux: (a) Materials (1) Laboratory animals Twelve male C57BL / 6 mice (normal control group), aged 12 weeks; LDLR - / - Seventy-two male mice, 12 weeks old, were provided by Changzhou Cavens Laboratory Animal Co., Ltd.

[0076] (2) Test sample Erdun-Urile optimized pill preparation prepared in Example 1.

[0077] (3) Reagents Total bile acid assay kit, Sinopharm Group Chemical Reagent Co., Ltd.; NaOH, Sinopharm Group Chemical Reagent Co., Ltd.; HCl, Sinopharm Group Chemical Reagent Co., Ltd.; Ethanol, Sinopharm Group Chemical Reagent Co., Ltd.; Triton X-100, Sinopharm Group Chemical Reagent Co., Ltd.; Methanol, Merck AG, Germany; n-Hexane, Merck AG, Germany; Ethyl acetate, Merck AG, Germany; d7-cholesterol, Tanmo Quality Inspection Technology Co., Ltd.; d4-codasteinol, Tanmo Quality Inspection Technology Co., Ltd.; Methyl tert-butyl ether (MTBE), Merck AG, Germany; Hexafluoroisopropanol (HFIP), Merck AG, Germany; N,N-dimethylglycine (DMG), Shanghai Aladdin Biochemical Technology Co., Ltd.; 2-Methyl-6-nitrobenzoic anhydride (MNBA), Shanghai Aladdin Biochemical Technology Co., Ltd.; 4-Dimethylaminopyridine (DMAP), Shanghai Aladdin Biochemical Technology Co., Ltd.; Triethylamine (TEA), Shanghai Aladdin Biochemical Technology Co., Ltd.; Dimethylformamide (DMF), Merck AG, Germany.

[0078] (4) Instruments Balance, Tianmei Instruments Laboratory Equipment (Shanghai) Co., Ltd.; Benchtop High-Speed ​​Low-Temperature Centrifuge, Merrick Instruments (Shanghai) Co., Ltd.; Microplate Reader, Bertek Instruments, Inc.; Phenomenex Kinetex Biphenyl, 2.1×100mm, 1.7μm Biphenyl Column, Finnemax, Inc.; Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS), Agilent Technologies Inc.

[0079] (II) Methods (1) High-fat modeling, animal grouping and administration Give LDLR - / - Mice were fed a high-fat, high-cholesterol diet, followed by a normal diet for C57BL / 6 mice. After 8 weeks of modeling, the high-fat diet was replaced with a normal diet, and LDLR was introduced. - / - Mice were randomly divided into a model group, a simvastatin administration group, an Erdun-Urile administration group, and high, medium, and low administration groups of the Erdun-Urile optimized formula (i.e., the Erdun-Urile optimized formula pills prepared in Example 1), with 12 mice in each group and 4 mice per cage. C57BL / 6 mice and the model control group were given the control solvent (distilled water for drug preparation), while the other administration groups were given the corresponding drugs by gavage.

[0080] (2) Collection of mouse feces Animals were transferred to cages without bedding at 2, 4, 6, and 8 weeks after administration. Feces were collected from each cage at these four time points after an 8-hour fast, and stored at -20°C for later use. Before use, the feces were freeze-dried at -50°C for 48 hours, the dry weight was recorded, and the feces were ground into a fine powder (passed through a 60-mesh sieve). Two 50 mg portions were weighed for subsequent testing.

[0081] Calculate the average dry weight of mouse feces over 8 hours, W (g) = total dry weight of feces per cage / 4; (3) Extraction and detection of total cholesterol (cholesterol, fecal sterol, bile acids) excreted in feces. ① Extraction and detection of fecal neutral sterols (cholesterol and fecal sterol) Extraction: Add 50 mg of dried fecal powder to a 2 mL screw-cap centrifuge tube. Add a deuterated internal standard mixture containing 10 μg d7-cholesterol and 10 μg d4-fecal sterol, and vortex for 10 s. Add 1.0 mL of MTBE / HFIP (1:1, v / v) solvent, and sonicate for 30 min (ice-water bath). Add 0.3 mL of ultrapure water and vortex vigorously for 1 min. Centrifuge at 12000 × g, 4 °C for 10 min, and transfer the upper organic phase to a new centrifuge tube. Repeat the extraction once more with 0.5 mL of MTBE, and combine the organic phases. Add a small amount of anhydrous sodium sulfate to the organic phase for dehydration, centrifuge, collect the supernatant, and dry under nitrogen.

[0082] Derivatization: Add 100 μL of DMG derivatization mixture (DMG 10 mg / mL, MNBA 10 mg / mL, DMAP 2 mg / mL, TEA 5 μL / mL, dissolved in anhydrous DMF) to the dried residue, vortex thoroughly, and then react in a sealed container at 60 °C for 30 min. After the reaction is complete, cool to room temperature, add 900 μL of acetonitrile / water (1:1, v / v) to dilute, vortex thoroughly, filter through a 0.22 μm filter membrane, and transfer to a sample vial for LC-MS / MS analysis.

[0083] LC-MS / MS detection: Biphenyl column; column temperature, 40℃; mobile phase A - water (containing 2 mM ammonium formate), mobile phase B - methanol (containing 2 mM ammonium formate); gradient: 0~1 min 60% B; 1~8 min 60→98% B; 8~12 min 98% B; 12~12.1 min 98→60% B; 12.1~15 min 60% B; flow rate: 0.3 mL / min; injection volume: 5 μL; mass spectrometry mode: parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM), electrospray ionization (ESI) + ).

[0084] ② Extraction and detection of fecal bile acids Extraction: Add 2.2 mL of 0.08 M NaOH ethanol solution to 50 mg of dried fecal powder and heat at 95 °C for 2 h. After cooling, extract three times with 5 mL of hexane, then acidify with 2.5 mL of 0.16 M HCl, and extract five times with 5 mL of ethyl acetate. Dry the extract. Dissolve the dried extract in a 20% methanol solution of 1.25% Triton X-100.

[0085] Detection: Enzymatic detection was performed according to the instructions of the total bile acid assay kit.

[0086] (4) Data processing and calculation Standard curve: Prepare 6 concentrations using the corresponding standards, add an equal amount of internal standard, and then degenerate / extract according to the same procedure before injection.

[0087] Plot a calibration curve with the target peak area / internal standard peak area as the ordinate and concentration as the abscissa.

[0088] Sample quantification: The concentration (μg / mL extract) is calculated by substituting the peak area ratio of the target analyte to the internal standard into the standard curve.

[0089] Excretion volume per mouse over 8 hours (μg) = [(C×V) / 0.05g]×W.

[0090] C represents the concentration of the extract (μg / mL), V represents the volume of reconstitution (mL), and W represents the average dry weight of mouse feces over 8 hours (g).

[0091] Total fecal cholesterol excretion in 8 hours = fecal neutral sterols + fecal bile acids.

[0092] (III) Statistical Analysis Statistical analysis was performed using IBM SPSS Statistics 26 software. For continuous variables, the Shapiro-Wilk test for normality was first used. If the p-value was greater than 0.05, it indicated that the variable conformed to a normal distribution, and the result was expressed as mean ± standard deviation. If the data did not conform to a normal distribution (including some groups not conforming to a normal distribution), the median (interquartile range) was used.

[0093] Levene's test is used to test homogeneity of variance. A p-value > 0.05 indicates homogeneity of variance among the data. If all groups are normally distributed and their variances are homogeneous, the Bonferroni test is used for comparisons among multiple groups. If the groups are normally distributed but their variances are unequal, the Tammhenko test is used. If the groups do not conform to a normal distribution, the Kruskal-Wallis H test is used for comparisons among multiple groups. Paired samples that do not conform to a normal distribution are analyzed using the Wilcoxon test (signed-rank test). A p-value < 0.05 is considered statistically significant.

[0094] (iv) Results Table 2 Total cholesterol excretion in mouse feces over 8 hours (μg) Compared with the normal control group, ### , P<0.001 Compared with the model group, * , P<0.05 , ** , P<0.01 , *** , P< 0.001 .

[0095] Compared with the normal control group, the fecal total cholesterol excretion of mice in the model group was significantly increased at all time points (p<0.001), reaching a peak at 2 weeks (approximately 19045 μg), and then decreasing in a time-dependent manner, reaching approximately 3983 μg at 8 weeks. Simvastatin (24 mg / kg) administration group showed increased LDLR from week 4. - / - Cholesterol excretion in mice was significantly lower than in the model group (p<0.01, P<0.001); at week 8, it decreased to 1492 μg, approaching the level of the normal control group. The Erdun-Urile (400 mg / kg) administration group only showed a significant reduction at week 8 (approximately 3235 μg, p<0.05). The optimized Erdun-Urile formula at 200 mg / kg showed an effect on LDLR at various time points. - / - No significant effect was observed on cholesterol excretion in mice; however, 400 mg / kg significantly reduced cholesterol excretion in mice only at 8 weeks (3045 μg, p < 0.01); the 800 mg / kg group treated with the Erdun-Uryl optimized formula significantly reduced LDLR from 4 weeks onwards. - / - Cholesterol excretion in mice decreased to 3829 μg and 1999 μg at 6 and 8 weeks, respectively (p<0.05) (p<0.01 and p<0.001), with the effect at 8 weeks comparable to that in the simvastatin group. These results suggest that high-fat diet-induced LDLR... - / - Mice showed a significant increase in cholesterol efflux, and the high dose of the Erdun-Uryl optimized formula effectively reduced this efflux, with an effect comparable to simvastatin.

[0096] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An optimized formula of Erdun-Urile based on traditional Mongolian medicine, characterized in that, The ingredients include the following parts by weight: 1000 parts pearl powder, 200 parts gardenia, 100 parts safflower, 40 parts saffron, 140 parts long pepper, 80 parts styrax resin, 59 parts artificial bezoar, 1 part artificial musk, and 80 parts silver beads.

2. An optimized formula of Erdun-Urile based on traditional Mongolian medicine, characterized in that, Includes the Erdun-Urile optimized formula based on traditional Mongolian medicine as described in claim 1.

3. The Erdun-Urile optimized formula preparation according to claim 2, characterized in that, The formulation includes pills.

4. An optimized formula of Erdun-Urile pills based on traditional Mongolian medicine, characterized in that, Includes the Erdun-Urile optimized formula based on traditional Mongolian medicine as described in claim 1.

5. A method for preparing the Erdun-Urile optimized pill formula according to claim 4, characterized in that, Includes the following steps: The raw materials, excluding the silver beads, are crushed and mixed to form pills. Then, silver beads are added for coating and polishing to obtain the Erdun-Urile optimized pill formulation.

6. The preparation method according to claim 5, characterized in that, The process of pulverizing and mixing raw materials other than silver beads and then making pellets includes: Saffron, artificial bezoar and artificial musk were pulverized and sieved to obtain saffron powder, artificial bezoar powder and artificial musk powder respectively. Pearl powder, gardenia, safflower, long pepper, and styrax resin are initially crushed and then mixed together, followed by secondary crushing and sieving to obtain a fine mixed powder. The saffron powder, artificial bezoar powder, artificial musk powder, and mixed fine powder are mixed evenly, then ground and made into pills.

7. The preparation method according to claim 6, characterized in that, The sieve used for sieving has a mesh size of 120.

8. The preparation method according to claim 5, characterized in that, The diameter of the pellets obtained by pelleting is 5-7 mm.

9. The use of the Erdun-Urile optimized formula according to claim 1, the Erdun-Urile optimized formula preparation according to any one of claims 2-3, or the Erdun-Urile optimized formula pill according to claim 4 in the preparation of a medicament for treating atherosclerosis.

10. The use of the Erdun-Urile optimized formula according to claim 1, the Erdun-Urile optimized formula preparation according to any one of claims 2-3, or the Erdun-Urile optimized formula pill according to claim 4 in the preparation of lipid-lowering drugs.

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