A traditional Chinese medicine composition for treating atherosclerosis and a preparation method thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine preparation, and specifically discloses a traditional Chinese medicine composition for treating atherosclerosis and a preparation method thereof. The traditional Chinese medicine composition is composed of the following raw medicinal materials: Herba Epimedii, Panax Notoginseng, Salvia Miltiorrhiza, Panax Quinquefolium, Crataegus Pinnatifida, Hirudo, Chuanxiong Rhizome, Poria Cocos, Radix Pseudostellariae, Radix Astragali, Ramulus Cinnamomi, Resina Olibanii, Typha Angustifolia Pollen, Sparganium Stoloniferum, Curcuma, Radix Aucklandiae and Alisma Orientalis, which are matched according to specific weight ratios. The preparation method comprises the following steps: grouping based on the differences in the physicochemical properties of the raw medicinal materials, and respectively adopting the supercritical fluid extraction, low-temperature water extraction-alcohol precipitation coupling ultrasonic-assisted extraction, microbial conversion fermentation and vacuum freeze-drying process steps to prepare the traditional Chinese medicine composition with high bioavailability and rich in active ingredients. The traditional Chinese medicine composition has the comprehensive effects of reducing blood lipids, resisting inflammation, resisting oxidation, improving blood rheology, stabilizing and subsiding atherosclerotic plaques, and is safe and less likely to relapse, thereby providing a new traditional Chinese medicine scheme for treating atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, and in particular to a traditional Chinese medicine composition for treating atherosclerosis and its preparation method. Background Technology

[0002] Atherosclerosis (AS) is the most common pathological basis of cardiovascular disease. Its characteristic pathological changes include subintimal lipid deposition, inflammatory cell infiltration, smooth muscle cell proliferation, and fibrous cap formation, ultimately leading to luminal narrowing or even occlusion. This disease is a major cause of fatal cardiovascular and cerebrovascular events such as coronary heart disease, myocardial infarction, and stroke, seriously threatening human health.

[0003] Currently, clinical treatment of atherosclerosis mainly relies on statins, which are effective in lowering LDL cholesterol. However, long-term use can lead to adverse reactions such as liver damage, muscle pain, and elevated blood sugar, and their effects on plaque stabilization and reversal are limited in some patients. In recent years, traditional Chinese medicine has demonstrated unique advantages in the prevention and treatment of atherosclerosis through its multi-target, multi-pathway, and holistic regulatory approaches, and has gradually become a research hotspot.

[0004] However, existing traditional Chinese medicine compound preparations generally have the following shortcomings: First, the formulations are complex, with numerous ingredients, unclear effective components, and difficult quality control; second, the extraction processes are crude, mostly using simple decoction or alcohol extraction, resulting in low retention rates of heat-sensitive and fat-soluble components and poor bioavailability; third, there is a lack of systematic pharmacodynamic verification and rigorous clinical trial data support; fourth, the synergistic mechanism between different medicinal materials is not studied in depth, making it difficult to elucidate its pharmacological basis at the molecular level.

[0005] To address the aforementioned issues, it is necessary to develop a traditional Chinese medicine composition with a scientifically sound formulation, advanced extraction process, definite efficacy, and high safety to better meet the clinical needs of patients with atherosclerosis. Summary of the Invention

[0006] In view of this, the present invention provides a traditional Chinese medicine composition for treating atherosclerosis and a method for preparing the same, in order to solve or alleviate one of the technical problems existing in the prior art, and at least provide a beneficial alternative.

[0007] The technical solution of this invention is implemented as follows: A traditional Chinese medicine composition for treating atherosclerosis, comprising the following raw materials in the indicated weight ratios: Epimedium 4-20 parts, Panax notoginseng 4-20 parts, Salvia miltiorrhiza 4-20 parts, American ginseng 5-15 parts, hawthorn 5-15 parts, leech 2-8 parts, Ligusticum chuanxiong 4-20 parts, Poria cocos 4-20 parts, Pseudostellaria heterophylla 5-15 parts, Astragalus membranaceus 10-20 parts, Cinnamomum cassia 4-20 parts, Trogopterus xanthipes 5-15 parts, Typha orientalis 5-15 parts, Sparganium stoloniferum 5-15 parts, Curcuma zedoaria 5-15 parts, Aucklandia lappa 5-15 parts, Alisma plantago-aquatica 5-15 parts; The raw materials are divided into three groups according to their physicochemical properties: the first group is the fat-soluble active ingredient group, including leech, five-spice powder, turmeric, sparganium, chuanxiong, costus root, and cinnamon twig; the second group is the water-soluble active ingredient group, including astragalus, codonopsis, alisma, American ginseng, poria, salvia miltiorrhiza, and cattail pollen; the third group is the heat-sensitive active ingredient group, including epimedium, notoginseng, and hawthorn.

[0008] As a preferred option, the raw materials are composed of the following ingredients in the indicated weight ratios: Epimedium 10-15 parts, Panax notoginseng 10-15 parts, Salvia miltiorrhiza 10-15 parts, American ginseng 8-12 parts, Crataegus pinnatifida 8-12 parts, Hirudo medicinalis 4-6 parts, Ligusticum chuanxiong 10-15 parts, Poria cocos 10-15 parts, Pseudostellaria heterophylla 8-12 parts, Astragalus membranaceus 12-18 parts, Cinnamomum cassia 10-15 parts, Trogopterus xanthipes 8-12 parts, Typha orientalis 8-12 parts, Sparganium stoloniferum 8-12 parts, Curcuma zedoaria 8-12 parts, Aucklandia lappa 8-12 parts, Alisma plantago-aquatica 8-12 parts.

[0009] As a preferred option, the raw materials are composed of the following ingredients in the indicated weight ratios: Epimedium 12 parts, Panax notoginseng 12 parts, Salvia miltiorrhiza 12 parts, American ginseng 10 parts, Crataegus pinnatifida 10 parts, Hirudo medicinalis 5 parts, Ligusticum chuanxiong 12 parts, Poria cocos 12 parts, Pseudostellaria heterophylla 10 parts, Astragalus membranaceus 15 parts, Cinnamomum cassia 12 parts, Trogopterus xanthipes 10 parts, Typha orientalis 10 parts, Sparganium stoloniferum 10 parts, Curcuma zedoaria 10 parts, Aucklandia lappa 10 parts, Alisma plantago-aquatica 10 parts.

[0010] Preferably, the following steps are included: (1) Pretreatment: Weigh each raw material according to the weight ratio, clean, dry, pulverize, and sieve to obtain coarse powder of each raw material for later use; (2) First group extraction: The crude powder of the first group of raw materials was mixed and extracted using supercritical fluid extraction technology to obtain a fat-soluble extract and extraction residue; (3) Second extraction: The crude powder of the second group of raw materials was combined with the above extraction residue, and an aqueous solvent was added. Dynamic countercurrent extraction was carried out under ultrasonic assistance. The extract was purified and concentrated by membrane separation to obtain a water-soluble extract. (4) Third group extraction: The crude powder of the third group of raw materials was added to an aqueous solvent, and enzymatic hydrolysis was first performed using a compound enzyme to destroy the plant cell wall structure. Then, alcohol-water gradient extraction was performed to obtain a heat-sensitive extract. (5) Combining and biotransformation: The above three extracts are combined and inoculated with microbial fermentation strains to carry out biotransformation reaction to obtain fermentation conversion broth; (6) Post-processing: The fermentation conversion liquid is freeze-dried, and the resulting solid is pulverized and sieved to obtain the Chinese medicine composition.

[0011] Preferably, the process conditions for supercritical fluid extraction in step (2) are as follows: using supercritical CO2 as the extraction solvent, the flow rate is 20-30 L / h, the extraction temperature is 40-50℃, the extraction pressure is 25-35 MPa, and the extraction time is 2-4 h; the supercritical fluid may also contain an entrainer with a volume fraction of 5%-15%, and the entrainer is selected from one or more of ethanol, methanol or ethyl acetate.

[0012] Preferably, the amount of aqueous solvent added in step (3) is 10-20 times the total mass of the crude raw material powder and extraction residue of the second group; the conditions for ultrasonic-assisted dynamic countercurrent extraction are: ultrasonic frequency 20-40kHz, ultrasonic power 300-600W, extraction temperature 50-70℃, system pH value 6.0-7.0, and extraction time 2-3h; the membrane separation uses an ultrafiltration membrane with a molecular weight cutoff of 5-15kDa for concentration and purification.

[0013] Preferably, the composite enzyme in step (4) is composed of cellulase and pectinase at an enzyme activity ratio of 1:6-12, and the amount of composite enzyme added is 2%-3% of the mass of the crude powder of the third group of raw materials; the enzymatic hydrolysis conditions are: temperature 35-45℃, pH value 4.5-5.5, time 1-3h; in the alcohol-water gradient extraction after enzymatic hydrolysis, the volume fraction of ethanol is 55%-65%, the extraction temperature is 20-30℃, the extraction process is accompanied by stirring, the speed is 80-120rpm, and the extraction time is 2-3h.

[0014] Preferably, the microbial fermentation strain in step (5) is *Saccharomyces cerevisiae*, which has been genetically engineered to overexpress β-glucosidase; the fermentation conditions are as follows: the concentration of viable bacteria in the fermentation system is controlled at 10... 6 -10 8 CFU / mL, fermentation temperature 25-35℃, aeration and stirring speed 180-220rpm, fermentation time 36-48h; pH value naturally maintained at 4.0-6.0 during fermentation.

[0015] Preferably, the freeze-drying conditions in step (6) are: cold trap temperature -40℃ to -55℃, vacuum degree 4-6kPa, drying temperature 20-40℃, and drying time 4-8h; the dried product is pulverized and passed through a 100-150 mesh sieve to obtain a fine powdered traditional Chinese medicine composition.

[0016] Preferably, the drug further includes pharmaceutically acceptable excipients, and the dosage form of the drug is one of decoction, powder, granules, pills, tablets, capsules or injection.

[0017] The embodiments of the present invention have the following advantages due to the adoption of the above technical solutions: First, this invention selects seventeen Chinese medicinal herbs, with Panax notoginseng, Salvia miltiorrhiza, leeches, and Ligusticum chuanxiong as the core herbs for promoting blood circulation and removing blood stasis; Epimedium, Astragalus membranaceus, and American ginseng as auxiliary herbs for tonifying qi and strengthening the body; Poria cocos and Alisma plantago-aquatica as adjuvant herbs; and Aucklandia lappa and Cinnamomum cassia as guiding herbs, forming a complete prescription system that combines attack and tonification, regulates qi and blood, and treats phlegm and blood stasis. The various herbs work synergistically to comprehensively intervene in atherosclerosis through multiple pathways, including lowering blood lipids, anti-inflammation, anti-oxidation, improving blood rheology, and protecting vascular endothelium.

[0018] Secondly, based on the differences in the physicochemical properties of the active ingredients in each raw material, this invention scientifically divides the seventeen medicinal materials into three groups, each employing the most suitable extraction process: (a) For medicinal materials rich in fat-soluble components, supercritical CO2 fluid extraction is used, avoiding the residue problems of traditional organic solvent extraction, resulting in high extraction efficiency and good selectivity; (b) For medicinal materials rich in water-soluble polysaccharides and saponins, ultrasound-assisted dynamic countercurrent extraction combined with membrane separation technology is used. Low-temperature operation effectively protects heat-sensitive components, and the ultrasonic cavitation effect significantly improves cell wall permeability, allowing for more complete release of active ingredients; (c) For medicinal materials containing heat-sensitive flavonoids and saponins, enzymatic hydrolysis and cell wall breaking are first performed using a compound enzyme (cellulase + pectinase), followed by low-temperature alcohol extraction, effectively overcoming the defects of traditional high-temperature decoction leading to degradation of active ingredients. The combined use of these three extraction processes maximizes the retention and enrichment of the effective components in each medicinal material.

[0019] Third, this invention combines the three extracts and inoculates them with genetically engineered Saccharomyces cerevisiae that overexpresses β-glucosidase for fermentation and transformation. This process has dual benefits: on the one hand, yeast fermentation can convert macromolecular glycosides (such as icariin) in the original medicinal materials into small molecule aglycones (such as icariin), significantly reducing polarity, improving intestinal absorption and bioavailability, and enhancing pharmacological activity several times over; on the other hand, the fermentation process can effectively degrade allergenic proteins and other toxic components that may be present in animal-derived drugs such as leeches, improving medication safety. In addition, yeast metabolism can also produce a variety of beneficial trace active substances, further enhancing the overall efficacy.

[0020] Fourth, the traditional Chinese medicine composition prepared in this invention can significantly reduce the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in atherosclerotic model mice, increase the level of high-density lipoprotein cholesterol (HDL-C), and significantly reduce the area of ​​aortic intima plaques. Clinical observations have also confirmed that after taking the medicine, the plaque area of ​​patients was significantly reduced, the symptoms were significantly improved, and no obvious adverse reactions or recurrence were observed during follow-up. Detailed Implementation

[0021] Example 1: Preparation of Traditional Chinese Medicine Composition (Low-Dose Group)

[0022] 1. Raw material preparation

[0023] Weigh the following raw materials according to the following weights: Epimedium 4g, Panax notoginseng 4g, Salvia miltiorrhiza 4g, American ginseng 5g, hawthorn 5g, leech 2g, Ligusticum chuanxiong 4g, Poria cocos 4g, Codonopsis pilosula 5g, Astragalus membranaceus 10g, Cinnamomum cassia 4g, Trogopterus xanthipes 5g, Typha orientalis 5g, Sparganium stoloniferum 5g, Curcuma zedoaria 5g, Aucklandia lappa 5g, Alisma plantago-aquatica 5g.

[0024] Each raw material was cleaned separately to remove impurities and non-medicinal parts. It was then dried in a 40℃ hot air circulating oven to constant weight (moisture content ≤8%). After being removed and cooled to room temperature, it was pulverized separately using a high-speed universal pulverizer and passed through a 60-mesh sieve. The coarse powder of each herb was collected, sealed, and stored for later use.

[0025] 2. Supercritical CO2 extraction of the first group of medicinal materials

[0026] Take the coarse powders of leeches, five-spice powder, turmeric, sparganium, chuanxiong, costus root, and cinnamon twig, weigh them according to the prescription, and mix them thoroughly to obtain mixture A (total mass approximately 30g). Place mixture A into a supercritical CO2 extraction vessel and set the extraction conditions as follows: CO2 flow rate 20L / h, extraction temperature 40℃, extraction pressure 25MPa, extraction time 2h; the entrainer is anhydrous ethanol, and the entrainer flow rate is 8% (v / v) of the CO2 flow rate. During the extraction process, the CO2 fluid penetrates into the interior of the herbal powder particles under high pressure, dissolving the fat-soluble active ingredients (including ligustrazine, ligustilide, costus root lactone, cinnamon twig volatile oil, leech fatty acid components, etc.). After flowing out of the extraction vessel with the fluid, the mixture is separated under reduced pressure, and the collected extract is the fat-soluble extract (extract A). The remaining solid residue in the extraction vessel is retained for later use.

[0027] 3. Ultrasonic-assisted dynamic countercurrent extraction of the second group of medicinal materials

[0028] Take the coarse powders of Astragalus membranaceus, Codonopsis pilosula, Alisma plantago-aquatica, Panax quinquefolius, Poria cocos, Salvia miltiorrhiza, and Typha orientalis. Weigh them according to the prescription amount and mix them thoroughly to obtain mixture B1 (total mass approximately 38g). Combine mixture B1 with the extraction residue retained in step 2 above and mix evenly to obtain total mixture B (total mass approximately 68g). Add 10 times the mass (680mL) of deionized water to total mixture B, stir evenly, and transfer to a dynamic countercurrent extraction unit. Set the extraction conditions as follows: ultrasonic frequency 25kHz, ultrasonic power 350W, extraction temperature 50℃, system pH 6.0 (adjusted with 0.1mol / L phosphate buffer), extraction time 2h, and countercurrent stage 5. During the extraction process, under the dual drive of ultrasonic cavitation effect and concentration gradient, the water-soluble active ingredients (including Astragalus membranaceus polysaccharide, Panax quinquefolius saponins, Salvia miltiorrhiza phenolic acid, Poria cocos polysaccharide, Codonopsis pilosula polysaccharide, Typha orientalis flavonoids, Alisma plantago-aquatica terpenoid lactones, etc.) of the medicinal particles are fully dissolved. After extraction, the extract is separated and concentrated through a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The permeate is collected and concentrated to an appropriate volume, which is the water-soluble extract (extract B).

[0029] 4. Enzymatic hydrolysis coupled with alcohol extraction of the third group of medicinal materials

[0030] Take coarse powders of Epimedium, Panax notoginseng, and hawthorn, weigh them according to the prescription, and mix them thoroughly to obtain mixture C (total mass approximately 13g). Add 10 times the mass (130mL) of ultrapure water to mixture C, stir well, adjust the temperature to 35℃, adjust the pH to 4.5 with citrate-sodium citrate buffer, add 2% of the mass of mixture C of a compound enzyme (cellulase to pectinase activity ratio of 1:6), and enzymatically hydrolyze for 1 hour at 120 rpm in a constant temperature shaker. During the enzymatic hydrolysis, cellulase and pectinase work synergistically to hydrolyze the cellulose-hemicellulose network and pectin matrix in the cell walls of the medicinal materials, destroying the cell wall structure and allowing the full release of heat-sensitive active ingredients such as epimedium glycoside, Panax notoginsenoside R1, ginsenoside Rg1, and hawthorn flavonoids.

[0031] After enzymatic hydrolysis, anhydrous ethanol was added to the system to bring the final ethanol volume fraction to 55%. The temperature was lowered to 20°C, and extraction was continued for 2 hours at 80 rpm on a thermostatic magnetic stirrer. After extraction, the mixture was centrifuged at 4000 rpm for 15 minutes, and the supernatant was collected, which is the heat-sensitive extract (extraction solution C).

[0032] 5. Combine the extract with microbial fermentation and transformation

[0033] Extracts A (lipid-soluble extract), B (water-soluble extract), and C (heat-sensitive extract) were combined and mixed thoroughly to obtain a total mixture. Saccharomyces cerevisiae ATCC 18824 (this strain was genetically engineered to overexpress the β-glucosidase gene from almonds under the regulation of the GAL1 promoter) was inoculated into the total mixture, and the viable cell concentration in the fermentation system was controlled at 10⁻⁶. 6 The concentration of CFU / mL was placed in a constant temperature shaking incubator, and the fermentation conditions were set as follows: temperature 25℃, rotation speed 180 rpm, fermentation time 36 h, and natural pH (initial pH approximately 5.5).

[0034] During fermentation, yeast cells proliferate rapidly and secrete β-glucosidase extracellularly. This enzyme specifically hydrolyzes the glycosyl moiety of flavonoid glycosides such as icariin, converting them into smaller aglycones such as icariin, significantly improving the product's lipid solubility and transmembrane absorption capacity. Simultaneously, yeast metabolism consumes some endotoxins and allergenic proteins, further purifying the product. After fermentation, the fermentation conversion broth is obtained.

[0035] 6. Freeze-drying and finished product preparation

[0036] The fermentation broth was transferred to a freeze dryer, and the freeze-drying conditions were set as follows: pre-freezing temperature -40℃, cold trap temperature -50℃, vacuum degree 4kPa, drying temperature 20℃, and drying time 4h. After freeze-drying, a loose and porous solid product was obtained. The solid product was pulverized using a high-speed pulverizer, passed through a 100-mesh sieve, and the fine powder was collected. This fine powder is the finished traditional Chinese medicine composition prepared in this embodiment. It was then sealed in packaging and stored in a cool, dark place.

[0037] 7. Product Characterization

[0038] The traditional Chinese medicine composition prepared in this example was qualitatively and quantitatively analyzed by high performance liquid chromatography (HPLC). Chromatographic conditions: Waters XBridge C18 column (4.6 × 250 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution gradient elution; flow rate: 1.0 mL / min; detection wavelength: 270 nm; column temperature: 30 °C. Results showed that the content of icariin I was 12.5 mg / g, notoginsenoside R1 was 8.3 mg / g, tanshinone B was 15.6 mg / g, astragaloside A was 6.8 mg / g, total flavonoids were 42.3 mg / g, and total saponins were 58.7 mg / g.

[0039] Example 2: Preparation of Traditional Chinese Medicine Composition (Medium Dosage / Preferred Group)

[0040] 1. Raw material preparation

[0041] Weigh the following raw materials according to the following weights: Epimedium 12g, Panax notoginseng 12g, Salvia miltiorrhiza 12g, American ginseng 10g, Crataegus pinnatifida 10g, Hirudo medicinalis 5g, Ligusticum chuanxiong 12g, Poria cocos 12g, Codonopsis pilosula 10g, Astragalus membranaceus 15g, Cinnamomum cassia 12g, Trogopterus xanthipes 10g, Typha orientalis 10g, Sparganium stoloniferum 10g, Curcuma zedoaria 10g, Aucklandia lappa 10g, Alisma plantago-aquatica 10g.

[0042] Each raw material was individually cleaned to remove impurities, moldy particles, and non-medicinal parts. The cleaned materials were then placed in a vacuum low-temperature drying oven and dried at 45℃ and a vacuum of 0.08 MPa until the moisture content was ≤6%. After drying, they were removed and cooled to room temperature. Each material was then pulverized using an air jet mill, passed through a 70-mesh sieve, and the ultrafine powder was collected and stored in a sealed, light-protected container for later use. Air jet milling yields powders with more uniform particle size and a larger specific surface area, which is beneficial for the dissolution of active ingredients during subsequent extraction processes.

[0043] 2. Supercritical CO2 extraction of the first group of medicinal materials

[0044] Take the ultrafine powders of leeches, trogopterus dung, turmeric, sparganium, chuanxiong, costus root, and cinnamon twig, weigh them according to the prescription, and mix them thoroughly to obtain mixture A (total mass approximately 74g). Evenly load mixture A into a supercritical CO2 extraction vessel and set the extraction conditions as follows: CO2 flow rate 25L / h, extraction temperature 45℃, extraction pressure 30MPa, extraction time 3h; the entrainer is 10% (v / v) food-grade ethanol, which is continuously injected into the CO2 flow path using a metering pump.

[0045] Under these conditions, supercritical CO2 fluid possesses liquid-like dissolving power and gas-like diffusion rate, enabling it to efficiently penetrate the micropores of medicinal powders, selectively dissolving and carrying out lipid-soluble active ingredients. Specifically, target components such as tetramethylpyrazine and ferulic acid in Ligusticum chuanxiong, turmeric alcohol and β-elemene in Curcuma zedoaria and Sparganium stoloniferum, costus lactone and dehydrocostus lactone in Aucklandia lappa, cinnamaldehyde and cinnamic acid in Cinnamomum cassia, fatty acids and steroidal components in Hirudo medicinalis, and trogopterin in Trogopterus xanthipes were all effectively extracted. The extract was then subjected to vacuum separation in a two-stage separation vessel (first stage separation pressure 8 MPa, temperature 50℃; second stage separation pressure 4 MPa, temperature 40℃). The two phases were collected and combined to obtain the lipid-soluble extract (extract A). The remaining solid residue in the extraction vessel was retained for later use.

[0046] 3. Ultrasonic-assisted dynamic countercurrent extraction of the second group of medicinal materials

[0047] Take ultrafine powders of Astragalus membranaceus, Codonopsis pilosula, Alisma plantago-aquatica, Panax quinquefolius, Poria cocos, Salvia miltiorrhiza, and Typha orientalis, weigh them according to the prescription amount, and mix them thoroughly to obtain mixture B1 (total mass approximately 79g). Combine mixture B1 with the extraction residue retained in step 2 above, mix thoroughly, and obtain total mixture B (total mass approximately 153g).

[0048] Add 15 times the mass (approximately 2295 mL) of deionized water to the total mixture B, stir well, and then transfer to an ultrasound-assisted dynamic countercurrent extraction system. This system consists of six extraction tanks connected in series. The herbal powder slurry flows countercurrently through each tank along with the extraction solvent. The extraction conditions are set as follows: ultrasonic frequency 30 kHz, ultrasonic power 500 W (independent ultrasonic probe for each tank), extraction temperature 60℃, system pH 6.5 (adjusted with phosphate buffer), residence time in each tank 25 min, and total extraction time 2.5 h.

[0049] Under the synergistic effect of ultrasonic cavitation and dynamic countercurrent concentration gradient, the water-soluble active ingredients in the second group of medicinal materials were fully dissolved: Astragalus polysaccharides and Codonopsis polysaccharides in Astragalus and Codonopsis, ginsenosides Rb1 and Rg1 in American ginseng, poria polysaccharides and poria acid in Poria, tanshinone B and rosmarinic acid in Salvia miltiorrhiza, quercetin and isorhamnetin in Typha pollen, and alismaol A and alismaol B in Alisma plantago-aquatica were all efficiently transferred to the aqueous extract.

[0050] After extraction, the extract was pumped into an ultrafiltration system for separation and purification. The ultrafiltration membrane had a molecular weight cutoff of 10 kDa, an operating pressure of 0.3 MPa, and a transmembrane flux controlled at 15-20 L / (m²·h). The ultrafiltration process effectively removed impurities such as large molecular weight proteins, starch, and suspended particles, while retaining active polysaccharides and oligosaccharides with molecular weights in the range of 1-10 kDa. The permeate was collected and concentrated under reduced pressure to an appropriate volume, yielding the water-soluble extract (extract B).

[0051] 4. Enzymatic hydrolysis coupled with alcohol extraction of the third group of medicinal materials

[0052] Take the ultrafine powders of Epimedium, Panax notoginseng, and hawthorn, weigh them according to the prescription amount, and mix them thoroughly to obtain mixture C (total mass approximately 34g). Add 15 times the mass (approximately 510mL) of ultrapure water to mixture C, place it in a constant temperature water bath, adjust the temperature to 40℃, and adjust the pH value to 5.0 with acetate-sodium acetate buffer solution.

[0053] Add 2.5% (by mass of mixture C) of a compound enzyme preparation (cellulase to pectinase activity ratio of 1:9), and carry out enzymatic hydrolysis at 150 rpm in a constant temperature air bath shaker for 2 hours. During enzymatic hydrolysis, cellulase specifically hydrolyzes β-1,4-glycosidic bonds in the cellulose microfibrils of the cell wall, while pectinase specifically hydrolyzes pectin polysaccharides in the middle lamella and cell wall. The synergistic effect of both causes the cell wall structure to relax and disintegrate completely, thereby releasing the intracellular active components encapsulated by the cell wall.

[0054] After enzymatic hydrolysis, the temperature was raised to 50℃ and maintained for 10 min to denature and inactivate the enzyme protein (enzyme inactivation step), then cooled to room temperature. Anhydrous ethanol was added to the system to bring the final ethanol volume fraction to 60%, and the temperature was controlled at 25℃. Extraction was carried out on a mechanical stirrer at 100 rpm for 2.5 h. After ethanol extraction, the mixture was centrifuged at 5000 rpm for 20 min, and the supernatant was collected, which is the heat-sensitive extract (extract C). HPLC analysis showed that the extraction rates of icariin, notoginsenoside R1, and hyperoside from hawthorn reached 92.3%, 88.7%, and 85.4%, respectively.

[0055] 5. Combine the extract with microbial fermentation and transformation

[0056] Extracts A, B, and C were combined and thoroughly mixed to obtain a total mixture (approximately 3.0 L). Genetically engineered *Saccharomyces cerevisiae* ATCC 18824 was inoculated into the total mixture. This strain was modified with plasmid pYES2-βG to carry the amygdalin β-glucosidase gene, which can overexpress the enzyme under galactose induction. The initial viable cell concentration in the fermentation system was controlled at 10⁻⁶. 7 The CFU / mL sample was placed in a 10L fermenter for fermentation. The conditions were set as follows: temperature 30℃, aeration rate 1.5vvm, stirring speed 200rpm, fermentation time 42h, and pH value was naturally regulated by cell metabolism (maintained in the range of 4.5-5.5).

[0057] During fermentation, yeast cells proliferate rapidly (reaching a final concentration of 10). 9 (CFU / mL level), β-glucosidase is continuously expressed and secreted extracellularly. This enzyme catalyzes the following key conversion reactions: (a) hydrolysis of icariin to remove one molecule of rhamnose, generating icariside I, which has significantly enhanced lipid solubility and cell permeability by about 5 times; (b) partial conversion of notoginsenoside R1 to ginsenosides Rg1 and Rb1, enhancing the anti-inflammatory and vascular endothelial protective effects of saponins; (c) conversion of hyperoside in hawthorn to quercetin aglycone, which significantly enhances its free radical scavenging ability.

[0058] Simultaneously, the yeast fermentation process also produced beneficial synergistic effects: (a) β-glucan components from the yeast cell wall were released into the fermentation broth, exhibiting immunomodulatory and lipid-lowering effects; (b) the small amounts of ethanol and organic acids produced during fermentation could act as natural preservatives; and (c) small molecule allergens that might be present in the leech residue were degraded into non-toxic short peptides and amino acids by yeast proteases. After fermentation, a dark brown, homogeneous, and stable fermentation conversion broth was obtained.

[0059] 6. Freeze-drying and finished product preparation

[0060] The fermentation broth was dispensed into freeze-drying trays, with a liquid level of approximately 1.5 cm, and placed in a freeze dryer for drying. The freeze-drying program was set as follows: in the pre-freezing stage, the temperature was lowered from room temperature to -40°C at a rate of 1°C / min and held for 2 hours; in the sublimation drying stage, the vacuum degree was 5 kPa, and the shelf temperature was linearly increased from -40°C to 0°C, taking 6 hours; in the desorption drying stage, the vacuum degree was maintained at 5 kPa, and the shelf temperature was increased from 0°C to 30°C, taking 4 hours; the total drying time was approximately 14 hours (including pre-freezing), and the actual vacuum drying time was approximately 10 hours.

[0061] After freeze-drying, a honeycomb-like, loosely textured solid block is obtained. This block is then pulverized using a high-speed grinder and passed through a 120-mesh sieve to collect a uniform fine powder, which is the finished traditional Chinese medicine composition prepared in this embodiment. It is then sealed in packaging and stored in a desiccator away from light.

[0062] 7. Product Characterization and Quality Evaluation

[0063] The product of this example was quantitatively analyzed by HPLC using multiple indicators, under the same chromatographic conditions as in Example 1. The results showed that the content of icariin I was 28.6 mg / g (conversion rate of 87.5%), the content of notoginsenoside R1 was 15.2 mg / g, the content of ginsenoside Rg1 was 9.8 mg / g, the content of tanshinone B was 32.4 mg / g, the content of astragaloside A was 14.5 mg / g, the content of total flavonoids was 78.9 mg / g, and the content of total saponins was 105.3 mg / g.

[0064] The antioxidant activity of the product was determined using the DPPH free radical scavenging method, with vitamin C as a positive control. The results showed that the IC50 of this product was [missing information]. 50 The concentration was 12.3 μg / mL, indicating significant free radical scavenging ability. The antithrombotic activity was determined using an in vitro platelet aggregation inhibition assay. The results showed that the product inhibited ADP-induced platelet aggregation by 68.5% (concentration 100 μg / mL), indicating good antithrombotic effect.

[0065] Example 3: Preparation of Traditional Chinese Medicine Composition (High-Dose Group)

[0066] 1. Raw material preparation

[0067] Weigh the following raw materials according to the following weights: Epimedium 20g, Panax notoginseng 20g, Salvia miltiorrhiza 20g, American ginseng 15g, Crataegus pinnatifida 15g, Hirudo medicinalis 8g, Ligusticum chuanxiong 20g, Poria cocos 20g, Codonopsis pilosula 15g, Astragalus membranaceus 20g, Cinnamomum cassia 20g, Trogopterus xanthipes 15g, Typha orientalis 15g, Sparganium stoloniferum 15g, Curcuma zedoaria 15g, Aucklandia lappa 15g, Alisma plantago-aquatica 15g.

[0068] Each raw material was individually cleaned to remove impurities, insect-damaged grains, and non-medicinal parts. The cleaned materials were then placed in a microwave vacuum dryer and dried at 50°C and a vacuum of 0.09 MPa until the moisture content was ≤5%. After drying, the materials were removed and cooled. Each powder was then pulverized to 80 mesh using a ball mill. The powders were collected, sealed, and stored in a light-proof container for later use. Microwave vacuum drying allows for rapid dehydration at lower temperatures, effectively protecting heat-sensitive active ingredients from damage.

[0069] 2. Supercritical CO2 extraction of the first group of medicinal materials

[0070] Take the fine powders of the above-mentioned leeches, five-spice powder, turmeric, sparganium, chuanxiong, costus root, and cinnamon twig, weigh them according to the prescription amount, and mix them thoroughly to obtain mixture A (total mass approximately 133g). Divide mixture A into two batches and load them into a supercritical CO2 extraction vessel (approximately 66.5g per batch). Set the extraction conditions as follows: CO2 flow rate 30L / h, extraction temperature 50℃, extraction pressure 35MPa, extraction time 4h; the entrainer is 12% (v / v) food-grade ethanol, injected through a high-pressure metering pump.

[0071] During the extraction process, supercritical CO2 has a stronger solubility under high pressure and high temperature conditions, which can more fully extract the fat-soluble active ingredients in the first group of medicinal materials. Specifically, phthalide compounds such as ligustilides, butylphthalide, and ferulic acid coniferyl ester from Ligusticum chuanxiong; sesquiterpenoids such as curcumol, curdione, and β-elemene from Curcuma zedoaria; saponins and volatile oils from Sparganium stoloniferum; sesquiterpenoid lactones such as costunolide and saussurealactone from Aucklandia lappa; volatile oils such as cinnamaldehyde, cinnamic acid, and cinnamic acetate from Cinnamomum cassia; fatty acids, steroids, and polypeptides from Hirudo medicinalis; and active ingredients such as wulingzhic acid and urease from Trogopterus xanthipes were all efficiently extracted.

[0072] The extract was purified through a three-stage separation process: In the first stage, the high-boiling-point lipid-soluble components were collected at a pressure of 10 MPa and a temperature of 55°C; in the second stage, the medium-boiling-point components were collected at a pressure of 6 MPa and a temperature of 45°C; and in the third stage, the low-boiling-point volatile components were collected at a pressure of 3 MPa and a temperature of 35°C. The products from these three stages were combined to obtain the lipid-soluble extract (extract A), and the remaining solid residue in the extraction vessel was retained for later use. The total lipid-soluble component extraction rate of this process reached 91.2%.

[0073] 3. Ultrasonic-assisted dynamic countercurrent extraction of the second group of medicinal materials

[0074] Take the fine powders of Astragalus membranaceus, Codonopsis pilosula, Alisma plantago-aquatica, Panax quinquefolius, Poria cocos, Salvia miltiorrhiza, and Typha orientalis, weigh them according to the prescription amount, and mix them thoroughly to obtain mixture B1 (total mass approximately 120g). Combine mixture B1 with all the extraction residues retained in step 2 above, mix thoroughly, and obtain total mixture B (total mass approximately 253g).

[0075] Add 20 times the mass (approximately 5060 mL) of deionized water to the total mixture B, stir thoroughly, and then transfer to a large-scale ultrasonic-assisted dynamic countercurrent extraction unit. This unit consists of 8 extraction tanks connected in series, each with a volume of 2 L, equipped with an independent ultrasonic generator and temperature control system. The extraction conditions were set as follows: ultrasonic frequency 40 kHz, ultrasonic power 600 W, extraction temperature 70 °C, system pH 7.0 (adjusted with Tris-HCl buffer), residence time per tank 22.5 min, total extraction time 3 h, and 8 countercurrent stages.

[0076] Ultrasonic cavitation creates a localized high-temperature, high-pressure microenvironment on and inside the surface of medicinal material particles, causing physical rupture of the cell walls and accelerating the dissolution rate and amount of active ingredients. In dynamic countercurrent mode, the fresh solvent and the medicinal material to be extracted maintain the maximum concentration gradient difference, significantly improving mass transfer driving force and extraction efficiency.

[0077] After extraction, the extract was purified using a two-stage membrane separation process: first, it was passed through a microfiltration membrane with a molecular weight cutoff of 50 kDa to remove large suspended solids and bacterial fragments; then, it was concentrated and separated through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to remove small molecule inorganic salts and water, while retaining active polysaccharides and oligopeptides in the range of 1-10 kDa. The ultrafiltration permeate was collected and concentrated under reduced pressure to approximately 1.5 L, which is the water-soluble extract (extract B). The total polysaccharide content in this extract was determined to be 38.6 mg / mL using the phenol-sulfuric acid method.

[0078] 4. Enzymatic hydrolysis coupled with alcohol extraction of the third group of medicinal materials

[0079] Take the fine powders of Epimedium, Panax notoginseng, and hawthorn, weigh them according to the prescription, and mix them thoroughly to obtain mixture C (total mass approximately 55g). Add 20 times the mass (approximately 1100mL) of ultrapure water to mixture C, place it in a large constant temperature water bath, adjust the temperature to 45℃, and precisely adjust the pH value to 5.5 using citrate-sodium citrate buffer solution.

[0080] Add 3% (by mass of mixture C) of a compound enzyme preparation (cellulase to pectinase activity ratio of 1:12) and carry out enzymatic hydrolysis in a large air bath shaker at 180 rpm for 3 hours. During the enzymatic hydrolysis, the compound enzyme system with high enzyme content and high enzyme activity ratio deeply hydrolyzes the cell walls of the medicinal materials: cellulase degrades cell wall microfibrils into cellobiose and glucose, and pectinase degrades pectin polysaccharides in the intercellular layer into galacturonic acid and oligogalacturonic acid. The two work together to completely disintegrate the cell wall structure, and the heat-sensitive active ingredients in the cells, such as icariin, icariin II, ascorbic acid A / B / C, notoginsenosides R1 / R2 / R3, ginsenosides Rg1 / Rb1 / Rd, hyperoside, quercetin-3-O-rhamnoside, and ursolic acid, are fully released into the aqueous phase.

[0081] After enzymatic hydrolysis, the system was heated to 55℃ and held for 15 minutes to inactivate the enzyme, then rapidly cooled to room temperature. Anhydrous ethanol was added to the system to achieve a final ethanol volume fraction of 65%, and the temperature was maintained at 30℃. Extraction was carried out for 3 hours at 120 rpm on a large mechanical stirrer. After ethanol extraction, the mixture was centrifuged at 6000 rpm for 20 minutes, and the supernatant was collected as the heat-sensitive extract (extract C). HPLC analysis showed that the extraction rates of icariin, citric acid C, and notoginsenoside R1 reached 95.1%, 89.3%, and 91.8%, respectively.

[0082] 5. Combine the extract with microbial fermentation and transformation

[0083] Extracts A, B, and C were combined and thoroughly mixed to obtain a total mixture (approximately 5.5 L). The total mixture was inoculated with genetically engineered *Saccharomyces cerevisiae* ATCC 18824. This strain was constructed by cloning the amygdalin β-glucosidase gene from *Saccharomyces cerevisiae* BY4741 into the expression vector pPICZαA, transforming it into *Pichia pastoris* X-33 to obtain a high-expression strain, and then undergoing adaptive evolution to obtain an engineered strain that stably expresses the gene in a *Saccharomyces cerevisiae* background. The initial viable cell concentration in the fermentation system was controlled at 10⁻⁶. 8 CFU / mL was placed in a 20L fermenter for large-scale fermentation. The conditions were set as follows: temperature 35℃, aeration rate 2.0 vvm, stirring speed 220 rpm, fermentation time 48h, and pH value maintained naturally (4.0-5.5).

[0084] During fermentation, the engineered yeast rapidly proliferated in a rich nutrient substrate, with β-glucosidase expression reaching 3200 U per liter of fermentation broth. This enzyme catalyzed the following key biotransformations: (a) a stepwise hydrolysis reaction of icariin → icariin I → icariin, with the final product exhibiting significantly better cell permeability and receptor binding activity than the prototype compound; (b) a cascade transformation of astragalin C → icariin → icariin I; (c) partial deglycosylation of Panax notoginseng saponins, generating low-glycosylated active saponin derivatives; and (d) transesterification of tanshinone phenolic acids, generating more lipid-soluble phenolic acid derivatives.

[0085] After 48 hours of fermentation, samples were taken and the conversion rate was measured to reach a plateau (icariin I conversion rate >90%). Fermentation was then stopped, yielding a dark brown, homogeneous, and stable fermentation broth. Thin-layer chromatography (TLC) and HPLC analysis of the fermentation broth showed no detectable bands of protohirudin protein, indicating that the leech-derived allergen had been effectively degraded.

[0086] 6. Freeze-drying and finished product preparation

[0087] The fermentation broth was dispensed into large freeze-drying trays, with a liquid level of approximately 2 cm, and then placed in an industrial-grade freeze dryer for drying. The freeze-drying program was set as follows: in the pre-freezing stage, the temperature was lowered from room temperature to -45°C at a rate of 0.5°C / min and held for 3 hours; in the sublimation drying stage, the vacuum degree was 6 kPa, and the shelf temperature was linearly increased from -45°C to 5°C, taking 8 hours; in the desorption drying stage, the vacuum degree was maintained at 6 kPa, and the shelf temperature was increased from 5°C to 40°C, taking 6 hours; the total drying time was approximately 20 hours (including pre-freezing), and the actual vacuum drying time was approximately 14 hours.

[0088] After freeze-drying, a dark brown, loose, porous solid block was obtained. This block was then pulverized using an air jet mill and passed through a 150-mesh sieve to collect the ultrafine powder, which is the finished traditional Chinese medicine composition prepared in this embodiment. The particle size D of this powder is... 50 With a surface area of ​​approximately 75 μm, it has a large specific surface area, which is beneficial for rapid disintegration and dissolution in subsequent formulations. After sealing the packaging, store in a desiccator protected from light.

[0089] 7. Product Characterization and Quality Evaluation

[0090] HPLC was used to quantitatively determine the components of the product in this embodiment. The results showed that the content of icariin was 8.6 mg / g, the content of icariin I was 35.2 mg / g, the content of notoginsenoside R1 was 22.8 mg / g, the content of ginsenoside Rg1 was 15.6 mg / g, the content of tanshinone B was 48.3 mg / g, the content of astragaloside A was 22.4 mg / g, the content of total flavonoids was 105.6 mg / g, and the content of total saponins was 148.7 mg / g.

[0091] In vitro experiments were used to evaluate the product's pharmacodynamics in multiple dimensions: (a) Antioxidant activity (DPPH method): IC50 50 =8.7 μg / mL; (b) Nitric oxide (NO) scavenging activity: IC50 = 8.7 μg / mL; 50 =15.3 μg / mL; (c) Protective effect against ox-LDL-induced vascular endothelial cell injury: at a concentration of 50 μg / mL, the endothelial cell survival rate recovered from 52.3% in the model group to 89.6%; (d) Platelet aggregation inhibition rate (ADP-induced): 78.2% at a concentration of 100 μg / mL. The above results indicate that the traditional Chinese medicine composition prepared in this embodiment has comprehensive and significant pharmacological activities.

[0092] Comparative Example 1: Traditional water extraction and alcohol precipitation method (comparison process 1)

[0093] This comparative example uses the same raw materials and proportions as Example 2 (i.e., the medium-dose group formulation), but uses the conventional extraction process of water decoction combined with alcohol precipitation to compare the advantages of the segmented differentiated extraction process of the present invention.

[0094] The specific steps are as follows: Mix all seventeen medicinal herbs in coarse powder, add 15 times their weight of water, and extract twice by boiling under normal pressure, 1.5 hours each time. Combine the decoctions. Concentrate the decoction to a relative density of 1.15 (measured at 60℃), add 95% ethanol to achieve an alcohol content of 60%, and let stand for 24 hours to allow complete precipitation. Collect the supernatant, recover the ethanol, and concentrate to obtain the water-extracted alcohol-precipitated extract. Directly freeze-dry the extract (under the same conditions as in Example 2), pulverize it through a 120-mesh sieve, and obtain control sample 1.

[0095] HPLC analysis revealed that the control sample contained 5.2 mg / g of icariin (significantly lower than the 28.6 mg / g of icariin I in Example 2), 12.8 mg / g of tanshinone B (lower than the 32.4 mg / g in Example 2), and 25.3 mg / g of total flavonoids (lower than the 78.9 mg / g in Example 2). This indicates that the traditional water extraction and alcohol precipitation process is far less effective than the segmented differentiated extraction process of this invention in retaining heat-sensitive and fat-soluble active ingredients.

[0096] Comparative Example 2: Single Supercritical Extraction Method (Control Process 2)

[0097] This comparative example also uses the same raw materials and proportions as Example 2, but all medicinal materials are extracted using supercritical CO2 extraction without grouping or differentiating extraction.

[0098] The specific steps are as follows: All seventeen medicinal herbs were mixed in coarse powder, and 20% ethanol was added as an entrainer. Supercritical extraction was performed for 3 hours at a CO2 flow rate of 25 L / h, a temperature of 45℃, and a pressure of 30 MPa. The extract was then collected. While supercritical CO2 has a good extraction effect on non-polar lipid-soluble components, its ability to extract highly polar polysaccharides and saponins is limited, resulting in a large amount of water-soluble active ingredients remaining in the extraction residue and failing to be effectively extracted. The extract was directly freeze-dried (under the same conditions as in Example 2), pulverized, and passed through a 120-mesh sieve to obtain control sample 2.

[0099] HPLC analysis revealed that the control sample contained high levels of lipid-soluble components (such as tetramethylpyrazine and cinnamaldehyde), but only 3.2 mg / g of tanshinone B was detected, and astragaloside A was not detected (<0.5 mg / g). The total polysaccharide content was extremely low (<5 mg / g). This indicates that a single supercritical fluid extraction process cannot simultaneously extract water-soluble active ingredients, thus validating the necessity of the grouped extraction strategy of this invention.

[0100] Comparative Example 3: Microbial fermentation step omitted (Control Process 3)

[0101] This comparative example uses the same raw materials, proportions, and segmented extraction process as Example 2, but the microbial fermentation and transformation step is omitted in step 5, and the three extracts are directly combined and freeze-dried.

[0102] The specific steps are as follows: Prepare extracts A, B and C according to steps 1-4 of Example 2, combine them and freeze-dry them directly without yeast fermentation (under the same conditions as Example 2), and pulverize them through a 120-mesh sieve to obtain control sample 3.

[0103] HPLC analysis showed that the icariin content in the control sample was 18.5 mg / g (not converted to icariin I), and icariin was not detected. In vitro cell experiments showed that this sample had a protection rate of 56.8% against oxidative damage to endothelial cells (far lower than the 89.6% in Example 2), and a DPPH free radical scavenging IC50 value. 50 The concentration was 28.5 μg / mL (lower than the 12.3 μg / mL in Example 2). This fully demonstrates the crucial role of the microbial fermentation transformation step in enhancing drug efficacy.

[0104] Comparative Example 4: Commercially available similar traditional Chinese medicine preparations (positive control)

[0105] This comparative study used a commercially available traditional Chinese medicine preparation for the treatment of atherosclerosis (Tongxinluo Capsules, produced by Shijiazhuang Yiling Pharmaceutical Co., Ltd.) as a positive control. Its formula contains a variety of traditional Chinese medicine ingredients such as ginseng, leech, scorpion, ground beetle, centipede, cicada molting, and red peony root.

[0106] In subsequent pharmacodynamic experiments, this product was used as a positive control drug and compared in parallel with the product of Example 2 of the present invention to objectively evaluate the technical advantages of the present invention.

[0107] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A traditional Chinese medicine composition for treating atherosclerosis, characterized in that, Composed of active pharmaceutical ingredients in the following weight ratios: Epimedium 4-20 parts, Panax notoginseng 4-20 parts, Salvia miltiorrhiza 4-20 parts, American ginseng 5-15 parts, hawthorn 5-15 parts, leech 2-8 parts, Ligusticum chuanxiong 4-20 parts, Poria cocos 4-20 parts, Pseudostellaria heterophylla 5-15 parts, Astragalus membranaceus 10-20 parts, Cinnamomum cassia 4-20 parts, Trogopterus xanthipes 5-15 parts, Typha orientalis 5-15 parts, Sparganium stoloniferum 5-15 parts, Curcuma zedoaria 5-15 parts, Aucklandia lappa 5-15 parts, Alisma plantago-aquatica 5-15 parts; The raw materials are divided into three groups according to their physicochemical properties: the first group is the fat-soluble active ingredient group, including leech, five-spice powder, turmeric, sparganium, chuanxiong, costus root, and cinnamon twig; the second group is the water-soluble active ingredient group, including astragalus, codonopsis, alisma, American ginseng, poria, salvia miltiorrhiza, and cattail pollen; the third group is the heat-sensitive active ingredient group, including epimedium, notoginseng, and hawthorn.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The raw materials are composed of the following ingredients in the indicated weight ratios: Epimedium 10-15 parts, Panax notoginseng 10-15 parts, Salvia miltiorrhiza 10-15 parts, American ginseng 8-12 parts, Crataegus pinnatifida 8-12 parts, Hirudo medicinalis 4-6 parts, Ligusticum chuanxiong 10-15 parts, Poria cocos 10-15 parts, Pseudostellaria heterophylla 8-12 parts, Astragalus membranaceus 12-18 parts, Cinnamomum cassia 10-15 parts, Trogopterus xanthipes 8-12 parts, Typha orientalis 8-12 parts, Sparganium stoloniferum 8-12 parts, Curcuma zedoaria 8-12 parts, Aucklandia lappa 8-12 parts, Alisma plantago-aquatica 8-12 parts.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The raw materials are composed of the following ingredients in the indicated weight ratios: Epimedium 12 parts, Panax notoginseng 12 parts, Salvia miltiorrhiza 12 parts, American ginseng 10 parts, Crataegus pinnatifida 10 parts, Hirudo medicinalis 5 parts, Ligusticum chuanxiong 12 parts, Poria cocos 12 parts, Pseudostellaria heterophylla 10 parts, Astragalus membranaceus 15 parts, Cinnamomum cassia 12 parts, Trogopterus xanthipes 10 parts, Typha orientalis 10 parts, Sparganium stoloniferum 10 parts, Curcuma zedoaria 10 parts, Aucklandia lappa 10 parts, Alisma plantago-aquatica 10 parts.

4. A method for preparing the traditional Chinese medicine composition according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Pretreatment: Weigh each raw material according to the weight ratio, clean, dry, pulverize, and sieve to obtain coarse powder of each raw material for later use; (2) First group extraction: The crude powder of the first group of raw materials was mixed and extracted using supercritical fluid extraction technology to obtain a fat-soluble extract and extraction residue; (3) Second extraction: The crude powder of the second group of raw materials was combined with the above extraction residue, and an aqueous solvent was added. Dynamic countercurrent extraction was carried out under ultrasonic assistance. The extract was purified and concentrated by membrane separation to obtain a water-soluble extract. (4) Third group extraction: The crude powder of the third group of raw materials was added to an aqueous solvent, and enzymatic hydrolysis was first performed using a compound enzyme to destroy the plant cell wall structure. Then, alcohol-water gradient extraction was performed to obtain a heat-sensitive extract. (5) Combining and biotransformation: The above three extracts are combined and inoculated with microbial fermentation strains to carry out biotransformation reaction to obtain fermentation conversion broth; (6) Post-processing: The fermentation conversion liquid is freeze-dried, and the resulting solid is pulverized and sieved to obtain the Chinese medicine composition.

5. The preparation method according to claim 4, characterized in that, The process conditions for supercritical fluid extraction in step (2) are as follows: supercritical CO2 is used as the extraction solvent, the flow rate is 20-30 L / h, the extraction temperature is 40-50℃, the extraction pressure is 25-35 MPa, and the extraction time is 2-4 h; the supercritical fluid may also contain an entrainer with a volume fraction of 5%-15%, and the entrainer is selected from one or more of ethanol, methanol or ethyl acetate.

6. The preparation method according to claim 4, characterized in that, The amount of aqueous solvent added in step (3) is 10-20 times the total mass of the crude raw material powder and extraction residue of the second group; the conditions for ultrasonic-assisted dynamic countercurrent extraction are: ultrasonic frequency 20-40kHz, ultrasonic power 300-600W, extraction temperature 50-70℃, system pH value 6.0-7.0, and extraction time 2-3h; the membrane separation uses an ultrafiltration membrane with a molecular weight cutoff of 5-15kDa for concentration and purification.

7. The preparation method according to claim 4, characterized in that, The complex enzyme mentioned in step (4) is composed of cellulase and pectinase at an enzyme activity ratio of 1:6-12. The amount of complex enzyme added is 2%-3% of the mass of the crude powder of the third group of raw materials. The enzymatic hydrolysis conditions are: temperature 35-45℃, pH value 4.5-5.5, time 1-3h. In the alcohol-water gradient extraction after enzymatic hydrolysis, the volume fraction of ethanol is 55%-65%, the extraction temperature is 20-30℃, the extraction process is accompanied by stirring, the speed is 80-120rpm, and the extraction time is 2-3h.

8. The preparation method according to claim 4, characterized in that, The microbial fermentation strain mentioned in step (5) is *Saccharomyces cerevisiae*, which has been genetically engineered to overexpress β-glucosidase; the fermentation conditions are as follows: the concentration of viable cells in the fermentation system is controlled at 10... 6 -10 8 CFU / mL, fermentation temperature 25-35℃, aeration and stirring speed 180-220rpm, fermentation time 36-48h; pH value naturally maintained at 4.0-6.0 during fermentation.

9. The preparation method according to claim 4, characterized in that, The freeze-drying conditions described in step (6) are: cold trap temperature -40℃ to -55℃, vacuum degree 4-6kPa, drying temperature 20-40℃, and drying time 4-8h; the dried product is pulverized and passed through a 100-150 mesh sieve to obtain a fine powdered traditional Chinese medicine composition.

10. The use of the traditional Chinese medicine composition according to any one of claims 1-3 in the preparation of a drug for treating atherosclerosis, characterized in that, The drug also includes pharmaceutically acceptable excipients, and the dosage form of the drug is one of the following: decoction, powder, granules, pills, tablets, capsules or injection.