Medicine for treating atherosclerosis and preparation method thereof

Abstract

The invention relates to a medicine for treating atherosclerosis and a preparation method thereof, and belongs to the technical field of medicines, and the medicine comprises the following components: a tremella aurantialba mycelium extracellular extract, a cicada slough extract, a siraitia grosvenorii seed extract, a ribes spinosa extract, panax notoginseng saponins, tanshinone IIA and puerarin. The extracellular extract of the tremella aurantialba mycelium is obtained by purifying a fermentation product obtained by inducing the mycelium of the tremella aurantialba by using an inducer through a DEAE-52 cellulose gel column and a propylene sephadex S-400HR chromatographic column. The cicada slough extract is obtained by purifying a product obtained by carrying out step-by-step enzymolysis on cicada slough by virtue of papain, chitinase and ginger protease by virtue of an HPD300 macroporous adsorption resin column. The siraitia grosvenorii seed extract is an oil-phase product and an ethyl acetate-phase product obtained by subcritical water extraction and ethyl acetate extraction of siraitia grosvenorii. The Ribes spinosa extract is an ethanol water solution extract of Ribes spinosa and is obtained by purifying the ethanol water solution extract of Ribes spinosa through a polyamide chromatographic column. The effects of lowering lipid and stabilizing atheromatous plaques are synergistically realized.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a drug for treating atherosclerosis and its preparation method. Background Technology

[0002] Cerebral arteriosclerosis is the core pathological basis for cerebrovascular diseases such as ischemic stroke and vascular dementia. Essentially, it involves lipid deposition, inflammatory responses, and fibrous tissue proliferation in the cerebral artery walls, forming atherosclerotic plaques that gradually lead to vascular stenosis, hemodynamic abnormalities, and ultimately, cerebral ischemia and hypoxia. Against the backdrop of a rapidly aging global population and the high incidence of metabolic diseases such as hypertension, hyperlipidemia, and diabetes, the incidence of cerebral arteriosclerosis continues to rise, seriously threatening patients' lives and health, and imposing a heavy medical burden on families and society. Therefore, the development of safe and effective therapeutic drugs is of significant clinical importance.

[0003] From a pathological perspective, cerebral arteriosclerosis is a chronic progressive process involving multiple stages and factors. The core components include: First, vascular endothelial cells are damaged by factors such as hypertension, hyperglycemia, and oxidative stress, leading to low-density lipoprotein cholesterol in the blood penetrating the endothelial barrier, depositing under the arterial intima, and being oxidized to oxidized low-density lipoprotein. Second, oxidized low-density lipoprotein activates endothelial cells to release inflammatory factors, recruiting monocytes to migrate to the intima and differentiate into macrophages. Macrophages engulf oxidized low-density lipoprotein to form foam cells, further exacerbating lipid deposition and inflammatory responses. Finally, foam cells undergo apoptosis, the lipid core expands, and vascular smooth muscle cells proliferate and synthesize collagen fibers to form a fibrous cap, which wraps around the lipid core to form atherosclerotic plaques. As the plaques gradually enlarge, they can lead to vascular stenosis. If the fibrous cap ruptures, it can trigger thrombosis, instantly blocking the blood vessel and causing stroke.

[0004] In the development of drug therapy, interventions for cerebral arteriosclerosis have gradually evolved from "symptomatic relief" to "targeting the pathological mechanisms." Early treatment focused on improving symptoms, such as using calcium channel blockers to dilate cerebral blood vessels and relieve dizziness and headaches caused by cerebral ischemia. However, these drugs could not stop plaque progression and could only improve blood flow in the short term. With a deeper understanding of the pathological mechanisms, lipid-lowering drugs have become the core intervention. Among them, statins (such as atorvastatin and rosuvastatin) significantly reduce low-density lipoprotein cholesterol by inhibiting cholesterol synthesis in the liver, while also having anti-inflammatory and plaque-stabilizing effects, making them the current first-line drugs in clinical practice. In addition, antiplatelet drugs (such as aspirin and clopidogrel) reduce the risk of thrombus formation after plaque rupture by inhibiting platelet aggregation and are often used in combination with statins to prevent stroke.

[0005] However, existing treatments still have significant limitations: on the one hand, some patients experience a "dose ceiling" effect with statins, meaning that even at the maximum tolerated dose, LDL cholesterol cannot be lowered to the target level, and long-term use may cause side effects such as abnormal liver function and muscle damage; on the other hand, these drugs have limited effectiveness in controlling inflammation in existing atherosclerotic plaques, only slowing progression or stabilizing the plaques, and even these effects are not ideal. Therefore, there is a need to develop more effective, anti-inflammatory, and safer drugs to improve treatment outcomes. Summary of the Invention

[0006] To address the shortcomings of existing drugs for treating atherosclerosis, such as poor efficacy and insufficient safety, this invention provides a drug for treating atherosclerosis and its preparation method. Through the synergistic effect of multiple components—extracellular extracts of *Auricularia auricula-judae* mycelium, cicada molting extract, *Siraitia grosvenorii* seed extract, *Scirpus arvensis* fruit extract, total saponins of Panax notoginseng, tanshinone IIA, and puerarin—it achieves comprehensive therapeutic effects including lipid-lowering, anti-inflammatory, antioxidant, vascular endothelial protection, and plaque stabilization. Its core advantage lies in overcoming the limitations of existing drugs, such as "single target, limited plaque control, and risk of side effects." The specific technical solution is as follows:

[0007] A drug for treating atherosclerosis, comprising the following components in a mass ratio of (2-3): (1-2): (0.8-1.5): (0.8-1.5): (1.5-2): (0.3-0.5): (1-2): extracellular extract of Auricularia auricula-judae mycelium, cicada molting extract, Siraitia grosvenorii seed extract, Curcuma longa extract, total saponins of Panax notoginseng, tanshinone IIA and puerarin.

[0008] The preparation of the extracellular extract of *Auricularia auricula-judae* mycelium includes inducing fermentation of *Auricularia auricula-judae* mycelium with an inducer containing trehalose and salicylic acid to obtain an induced bacterial broth; adding an ethanol aqueous solution, allowing it to stand, centrifuging, collecting the precipitate, diluting it with deionized water, loading it onto a DEAE-52 cellulose gel column, rinsing with 0.08M–0.1M NaCl aqueous solution to remove impurities, eluting with 0.3M–0.35M NaCl aqueous solution, and collecting eluent A; loading it onto an propylene dextran gel S-400HR chromatography column, eluting with 0.15M–0.2M NaCl aqueous solution, collecting eluent B, concentrating under reduced pressure, dialyzing, and freeze-drying to obtain the extracellular extract of *Auricularia auricula-judae* mycelium.

[0009] The preparation of the cicada molting extract includes enzymatic hydrolysis of cicada molting by papain and chitinase, followed by enzymatic hydrolysis by ginger protease. The product is then adsorbed onto an HPD300 macroporous adsorption resin column, washed with deionized water to remove impurities, eluted with 40 vol%–45 vol% ethanol aqueous solution, the eluent is collected, concentrated under reduced pressure, and freeze-dried to obtain the cicada molting extract.

[0010] The preparation of the monk fruit seed extract includes subcritical water extraction of monk fruit, collection of the extract, standing and separation of layers, separation of the oil phase and aqueous phase, extraction of the aqueous phase with ethyl acetate to obtain ethyl acetate extract, combination with the oil phase, concentration under reduced pressure, and vacuum drying to obtain monk fruit seed extract.

[0011] The preparation of the burdock fruit extract includes ultrasonic extraction of burdock fruit in an ethanol aqueous solution at pH 2.0–3.0, followed by purification of the extract by a polyamide chromatography column to obtain the burdock fruit extract.

[0012] The preparation method of the above-mentioned extracellular extract of *Auricularia auricula-judae* mycelium includes the following steps: Seed culture of *Auricularia auricula-judae* is inoculated into a fermentation medium and cultured to obtain a proliferating culture; an inducing agent aqueous solution containing 0.5wt%–0.8wt% trehalose and 0.1wt%–0.15wt% salicylic acid is added to the proliferating culture to induce fermentation, obtaining an induced culture; the culture is centrifuged, the supernatant is collected, filtered through a microfiltration membrane, the filtrate is collected, an ethanol aqueous solution is added, the culture is allowed to stand, centrifuged, the precipitate is collected, the precipitate is diluted with deionized water, and loaded onto a DEAE-52 cellulose gel column. The column is first eluted with 0.08M–0.1M NaCl aqueous solution for 1 BV–2 BV to remove impurities, then eluted with 0.3M–0.35M NaCl aqueous solution for 4 BV–5 BV, and the eluent A is collected; the eluent A is loaded onto an propylene dextran gel S-400HR column and eluted with 0.15M–0.2M NaCl aqueous solution. Elute with NaCl aqueous solution for 4-5 BV, collect eluent B, concentrate under reduced pressure, pack into a dialysis bag, dialyze, and freeze-dry to obtain extracellular extract of Auricularia auricula mycelium.

[0013] In the above method for preparing the extracellular extract of *Auricularia auricula-judae* mycelium, the mycelial concentration of the seed liquid is above 0.8 g / L; the inoculation amount of the seed liquid is 8%–10% of the fermentation medium volume; the fermentation medium contains 3 wt%–4 wt% corn flour, 1 wt%–2 wt% sucrose, and 0.5 wt%–0.8 wt% yeast extract; the proliferation culture is carried out at 25℃–28℃, with a sterile air flow rate of 1.0 vvm–1.5 vvm and stirring at 150 rpm–200 rpm for 4–5 days; the flow rate of the inducer aqueous solution is 1%–1.5% of the volume of the proliferation culture liquid added every 24 hours; the induced fermentation is… Fermentation was induced for 3 to 4 days at 18℃~20℃, with a sterile air flow rate of 0.4vvm~0.6vvm and stirring at 80rpm~100rpm; the microfiltration membrane was a 0.22μm pore size microfiltration membrane; the ethanol concentration of the ethanol aqueous solution was 90vol%~95vol%; the amount of ethanol aqueous solution added was 4 to 4.5 times the volume of the filtrate; the standing was carried out at 4℃~6℃ for 18h~24h; the centrifugation was carried out at 4000rpm~5000rpm for 10min~15min; the molecular weight cutoff of the dialysis bag was 1000Da; the dialysis was performed with flowing deionized water for 24h~30h.

[0014] The preparation method of the above-mentioned cicada molting extract includes the following steps: Pulverize dried cicada molting to obtain powder, add 8 to 12 times the weight of the powder in deionized water, add papain and chitinase, adjust the pH to 5.5 to 6.0, and enzymatically hydrolyze at 50℃ to 55℃ for 1.5 to 2 hours. Then adjust the pH to 6.4 to 6.8, add ginger protease, and enzymatically hydrolyze at 50℃ to 55℃ for 1 to 1.5 hours to inactivate the enzyme. Cool, centrifuge, collect the supernatant, concentrate under reduced pressure, and load the sample onto an HPD300 macroporous adsorption resin column. First, rinse with deionized water for 4 BV to 5 BV to remove impurities, then elute with 40 vol% to 45 vol% ethanol aqueous solution for 4 BV to 5 BV. Collect the eluent, concentrate under reduced pressure, and freeze-dry to obtain the cicada molting extract.

[0015] In the above method for preparing cicada molting extract, the particle size of the powder is sieved through a 40-60 mesh sieve; the amount of papain added is 0.8%-1.5% of the powder mass; the amount of chitinase added is 0.8%-1.5% of the powder mass; the amount of ginger protease added is 0.8%-1.5% of the powder mass; the enzyme inactivation is performed at 85℃-90℃ for 10-15 minutes; and the centrifugation is performed at 7000rpm-7500rpm for 10-15 minutes.

[0016] The preparation method of the above-mentioned monk fruit seed extract includes the following steps: drying and pulverizing monk fruit seeds, performing subcritical water extraction, collecting the extract, allowing it to stand and separate into layers, and taking the upper oil phase and the lower aqueous phase separately; extracting the aqueous phase with ethyl acetate to obtain ethyl acetate extract, combining it with the oil phase, concentrating under reduced pressure, and drying under vacuum to obtain monk fruit seed extract.

[0017] In the above method for preparing monk fruit seed extract, the subcritical water extraction parameters are: temperature 140℃~160℃, pressure 4MPa~4.5MPa, static extraction time 20min~30min, followed by dynamic extraction, with the extract collected at a flow rate of 4mL / min~6mL / min; the temperature for static separation is 4℃~6℃; and the aqueous phase is extracted 3 to 4 times with 1 to 1.2 times the volume of ethyl acetate.

[0018] The preparation method of the above-mentioned burdock fruit extract includes the following steps: homogenizing the burdock fruit, mixing it with 10 to 12 times the volume of 60 vol% to 70 vol% ethanol aqueous solution, adjusting the pH to 2.0 to 3.0, ultrasonically extracting, centrifuging, collecting the supernatant, concentrating under reduced pressure, loading the sample onto a polyamide chromatography column, first rinsing with deionized water for 4 BV to 6 BV to remove impurities, then eluting with eluent for 4 BV to 6 BV, collecting the eluent, concentrating under reduced pressure, and freeze-drying to obtain the burdock fruit extract.

[0019] In the above-mentioned method for preparing the extract of *Rhizophora stylosa*, the ultrasonic extraction is performed under nitrogen protection at 20℃~25℃ and 500W~600W for 15min~20min; the centrifugation is performed at 4000rpm~5000rpm for 10min~15min; and the eluent is a mixture of 4wt%~5wt% ammonia solution and 80vol%~90vol% ethanol solution at a volume ratio of (1~2):(8~9).

[0020] The preparation method of the above-mentioned drug for treating atherosclerosis includes the following steps: compounding the extracellular extract of Auricularia auricula mycelium, cicada molting extract, Siraitia grosvenorii seed extract, Curcuma longa extract, total saponins of Panax notoginseng, tanshinone IIA and puerarin in a mass ratio to obtain a drug composition.

[0021] This invention provides a drug for treating atherosclerosis and its preparation method, the beneficial effects of which include: I. This invention is guided by the traditional Chinese medicine theory of "strengthening the body's resistance and eliminating pathogenic factors, resolving phlegm and removing blood stasis," and integrates modern medical understanding of the core mechanisms of atherosclerosis, namely "lipid metabolism-inflammation and immunity-vascular endothelial function." Through the synergistic effect of multiple components, it achieves comprehensive therapeutic effects of "lowering lipids, anti-inflammation, anti-oxidation, protecting vascular endothelium, and stabilizing atherosclerotic plaques." Its core advantage lies in overcoming the limitations of existing drugs, such as "single target, limited plaque control effect, and risk of side effects." It intervenes simultaneously at multiple key links in the pathological mechanism, both alleviating symptoms and delaying or even blocking disease progression. Furthermore, based on the combination of natural extracts and mature medicinal ingredients, it has higher safety.

[0022] II. Extracellular extract of Auricularia auricula-judae mycelium: The inducer (trehalose + salicylic acid) activates the stress metabolic pathway of Auricularia auricula-judae mycelium, promoting the synthesis of antioxidant and anti-inflammatory active ingredients (including polysaccharide derivatives); the combination purification of DEAE-52 cellulose gel and S-400HR chromatography column accurately adsorbs active polysaccharides and removes small molecule impurities, ensuring the high activity and high purity of the extract.

[0023] III. Cicada molting extract: The synergistic enzymatic hydrolysis of papain, chitinase and ginger protease efficiently degrades the chitin-protein complex structure of cicada molting, releasing and activating anti-inflammatory components such as chitin derivatives and active peptides; the polarity and specific surface area of ​​HPD300 macroporous adsorption resin match the active ingredients, further removing impurities, enhancing anti-inflammatory and synergistic efficacy, and improving safety.

[0024] IV. Monk fruit seed extract: Subcritical water extraction combined with ethyl acetate extraction, the ethyl acetate extract is combined with the oil phase, retaining the fat-soluble components, and can effectively intervene in the lipid deposition process.

[0025] V. Ricinus officinalis extract: Acidic ethanol ultrasonic extraction can stabilize polyphenolic antioxidant and anti-inflammatory components and prevent their oxidative degradation; polyamide chromatography column specifically adsorbs polyphenolic components, removes ineffective impurities such as sugars and organic acids, and enhances the anti-inflammatory and foam cell inhibition effects.

[0026] VI. Panax notoginseng total saponins, tanshinone IIA, and puerarin: As mature medicinal components, when combined with other extracts, they can further improve the drug's action network through "complementary targets" (such as Panax notoginseng total saponins improving microcirculation, tanshinone IIA inhibiting inflammatory factors, and puerarin protecting vascular endothelium).

[0027] VII. The various components work together in specific proportions to cover the entire pathological chain of "lipid deposition-inflammatory response-plaque formation," forming a "multi-target synergistic intervention" mode of action, avoiding the limitations of single-target intervention. Antioxidant components (including auricularia auricula-judae extracellular polysaccharides and burdock fruit polyphenols) can reduce oxidative stress damage to vascular endothelium, creating a favorable vascular microenvironment for anti-inflammatory components (including cicada molting active peptides) to exert their effects; anti-inflammatory components can inhibit macrophage activation, reduce foam cell formation, and reduce the interference of inflammatory factors on lipid metabolism, assisting lipid-lowering components (including limon fruit seed fat-soluble components) in enhancing their effects; vascular protective components (including puerarin) can enhance vascular endothelial barrier function, reduce LDL-C penetration into the intima, and reduce lipid deposition from the source. These mechanisms promote and complement each other, achieving a synergistic effect of "1+1>2," forming a stable synergistic efficacy network based on molecular interactions between components (such as the synergistic activation or inhibition of the same pathway by active ingredients). Detailed Implementation

[0028] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0029] Example 1 A drug for treating atherosclerosis, comprising extracellular extract of Auricularia auricula-judae mycelium, cicada molting extract, Siraitia grosvenorii seed extract, Curcuma longa extract, total saponins of Panax notoginseng, tanshinone IIA and puerarin in a mass ratio of 2.5:1.5:1.2:1:1.8:0.4:1.5.

[0030] The preparation method of extracellular extract of Auricularia auricula-judae mycelium includes the following steps: Activation of the mycelium and preparation of the seed culture: Auricularia auricula-judae mycelium is inoculated onto PDA slant medium and cultured at 26℃ for 7 days. Mycelial blocks are then picked and inoculated into a seed culture medium (2.5wt% glucose, 1.5wt% soybean meal, 0.15wt% KH₂PO₄, 0.06wt% MgSO₄, with the remainder being water). The culture is then incubated on a shaker at 26℃ and 180rpm for 6 days to obtain the seed culture, with a mycelial concentration of ≥0.8g / L (dry weight). The seed culture is then inoculated into a fermentation medium (3.5wt% corn flour, 1.5wt% sucrose, 0.6wt% yeast extract, with the remainder being water, pH at rest) at 9% volume. The temperature is controlled at 26℃, the stirring speed at 180rpm, and the sterile air flow rate at 1.2vvm. The culture is then incubated for 5 days to obtain the proliferating bacterial culture. An inducing agent aqueous solution containing 0.6 wt% trehalose and 0.12 wt% salicylic acid was added to the proliferating bacterial culture (the addition rate was 1.2% of the proliferating bacterial culture volume every 24 hours), and the culture temperature was lowered to 19℃. At the same time, the stirring speed was reduced to 90 rpm, the sterile air flow rate was adjusted to 0.5 vvm, and fermentation was continued for 4 days to obtain the induced bacterial culture. The induced bacterial culture was centrifuged at 4500 rpm for 12 min, and the supernatant was collected and filtered through a 0.22 μm microfiltration membrane. The filtrate was collected, and 4.2 times the volume of 92 vol% ethanol aqueous solution was added. The mixture was allowed to stand at 5 °C for 20 h, centrifuged at 4500 rpm for 12 min, and the precipitate was collected. The precipitate was diluted with deionized water to 2.5 times its mass and loaded onto a DEAE-52 cellulose gel column. The column was first eluted with 0.09 M NaCl aqueous solution for 1.5 BV to remove impurities, and then eluted with 0.32 M NaCl aqueous solution for 4.5 BV. The eluent A was collected. The eluent A was loaded onto an propylene dextran gel S-400HR chromatography column and eluted with 0.18 M NaCl aqueous solution for 4.5 BV. The eluent B was collected, concentrated under reduced pressure at 48 °C, and placed into a dialysis bag with a molecular weight cutoff of 1000 Da. The mixture was dialyzed with flowing deionized water for 26 h and then freeze-dried to obtain the extracellular extract of Auricularia auricula mycelium.

[0031] The preparation method of cicada molting extract includes the following steps: dried cicada molting is pulverized to pass through a 40-mesh sieve to obtain powder. Deionized water at 10 times the powder mass is added, along with 1% papain and 1.2% chitinase. The pH is adjusted to 5.8 with 0.12M citrate-disodium hydrogen phosphate buffer, and enzymatic hydrolysis is performed at 52℃ for 2 hours. Then, the pH is adjusted to 6.5 with 0.9M NaOH aqueous solution, and ginger protease at 1% of the powder mass is added. Enzymatic hydrolysis is performed at 52℃ for 1 hour, followed by enzyme inactivation at 88℃ for 12 minutes. After cooling, the mixture is centrifuged at 7200 rpm for 12 minutes, and the supernatant is collected. The supernatant is concentrated under reduced pressure at 48℃ to 25% of its volume and loaded onto an HPD300 macroporous adsorption resin column. The column is first washed with deionized water for 4.5 BV to remove impurities, then eluted with 42 vol% ethanol aqueous solution for 4.5 BV. The eluent is collected, concentrated under reduced pressure at 48℃ to remove ethanol, and then freeze-dried to obtain the cicada molting extract.

[0032] The preparation method of monk fruit seed extract includes the following steps: drying monk fruit seeds at 48℃, pulverizing them through a 120-mesh sieve, and performing subcritical water extraction. The extraction parameters are set as follows: temperature 150℃, pressure 4.2MPa, static extraction time 25min, followed by dynamic extraction. The extract is collected at a flow rate of 5mL / min and allowed to stand at 5℃ to separate the layers. The upper oil phase and the lower aqueous phase are taken separately. The aqueous phase is extracted three times with 1.1 times the volume of ethyl acetate to obtain the ethyl acetate extract, which is combined with the oil phase. The extract is concentrated under reduced pressure at 48℃ to remove the ethyl acetate and then vacuum dried at 48℃ to constant weight to obtain the monk fruit seed extract.

[0033] The preparation method of the extract of *Scirpus spp.* includes the following steps: homogenize the fruit of *Scirpus spp.*, mix with 11 times the volume of 65 vol% ethanol aqueous solution, adjust the pH to 2.5 with 0.12 M HCl aqueous solution, and extract by ultrasonication at 22℃ and 550W intermittently (working for 2 seconds and pausing for 3 seconds) for 18 min under nitrogen protection. Centrifuge at 4500 rpm for 13 min, collect the supernatant, concentrate under reduced pressure at 42℃ to remove ethanol, load the sample onto a polyamide chromatography column, first wash with deionized water to remove 5 BV of impurities, then elute with eluent (4.5 wt% ammonia aqueous solution and 85 vol% ethanol aqueous solution mixed at a volume ratio of 1.5:8.5) to remove 5 BV, collect the eluent, concentrate under reduced pressure at 43℃, and freeze-dry to obtain the extract of *Scirpus spp.*.

[0034] Example 2 A drug for treating atherosclerosis, comprising extracellular extract of Auricularia auricula-judae mycelium, cicada molting extract, Siraitia grosvenorii seed extract, Curcuma longa extract, total saponins of Panax notoginseng, tanshinone IIA and puerarin in a mass ratio of 2:1:1.5:0.8:1.5:0.3:1.

[0035] The preparation method of extracellular extract of Auricularia auricula-judae mycelium includes the following steps: Activation of the mycelium and preparation of the seed culture: Auricularia auricula-judae mycelium is inoculated onto PDA slant medium and cultured at 25℃ for 8 days. Mycelial blocks are then picked and inoculated into a seed culture medium (2wt% glucose, 2wt% soybean meal, 0.1wt% KH₂PO₄, 0.08wt% MgSO₄, with the remainder being water). The culture is then incubated on a shaker at 25℃ and 200 rpm for 5 days to obtain the seed culture, with a mycelial concentration of ≥0.8 g / L (dry weight). The seed culture is then inoculated into a fermentation medium (3wt% corn flour, 2wt% sucrose, 0.5wt% yeast extract, with the remainder being water, pH at rest) at a volume of 10%. The temperature is controlled at 28℃, the stirring speed at 150 rpm, and the sterile air flow rate at 1.5 vvm. The culture is then incubated for 4 days to obtain the proliferating bacterial culture. An inducing agent aqueous solution containing 0.8 wt% trehalose and 0.1 wt% salicylic acid was added to the proliferating bacterial culture (the addition rate was 1.5% of the proliferating bacterial culture volume every 24 hours), and the culture temperature was lowered to 18℃. At the same time, the stirring speed was reduced to 100 rpm, the sterile air flow rate was adjusted to 0.4 vvm, and fermentation was continued for 4 days to obtain the induced bacterial culture. The induced bacterial culture was centrifuged at 4000 rpm for 15 min, and the supernatant was collected and filtered through a 0.22 μm microfiltration membrane. The filtrate was collected, and 4 times the volume of 95 vol% ethanol aqueous solution was added. The mixture was allowed to stand at 4℃ for 24 h, centrifuged at 4000 rpm for 15 min, and the precipitate was collected. The precipitate was diluted with deionized water to 2 times its mass and loaded onto a DEAE-52 cellulose gel column. The column was first eluted with 0.1 M NaCl aqueous solution for 1 BV to remove impurities, and then eluted with 0.35 M NaCl aqueous solution for 4 BV. The eluent A was collected. The eluent A was loaded onto an propylene dextran gel S-400HR chromatography column and eluted with 0.2 M NaCl aqueous solution for 4 BV. The eluent B was collected and concentrated under reduced pressure at 50℃. The eluent was placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with flowing deionized water for 24 h. The eluent B was then freeze-dried to obtain the extracellular extract of Auricularia auricula mycelium.

[0036] The preparation method of cicada molting extract includes the following steps: dried cicada molting is pulverized to pass through a 60-mesh sieve to obtain powder. Eight times the weight of the powder in deionized water is added, along with 1.5% papain and 0.8% chitinase. The pH is adjusted to 5.5 with 0.15M citrate-disodium hydrogen phosphate buffer, and the mixture is enzymatically hydrolyzed at 55℃ for 1.5 h. Then, the pH is adjusted to 6.4 with 1M NaOH aqueous solution, and 1.5% ginger protease is added. The mixture is enzymatically hydrolyzed at 50℃ for 1.5 h, and then the enzyme is inactivated at 85℃ for 15 min. After cooling, the mixture is centrifuged at 7000 rpm for 15 min, and the supernatant is collected. The supernatant is concentrated under reduced pressure at 45℃ to 30% of its volume and loaded onto an HPD300 macroporous adsorption resin column. The column is first washed with deionized water for 4 BV to remove impurities, then eluted with 45 vol% ethanol aqueous solution for 4 BV. The eluent is collected, concentrated under reduced pressure at 50℃ to remove ethanol, and then freeze-dried to obtain the cicada molting extract.

[0037] The preparation method of monk fruit seed extract includes the following steps: drying monk fruit seeds at 45℃, pulverizing them through a 150-mesh sieve, and performing subcritical water extraction. The extraction parameters are set as follows: temperature 140℃, pressure 4.5MPa, static extraction time 20min, followed by dynamic extraction. The extract is collected at a flow rate of 6mL / min and allowed to stand at 4℃ to separate the layers. The upper oil phase and the lower aqueous phase are taken separately. The aqueous phase is extracted three times with 1.2 times the volume of ethyl acetate to obtain the ethyl acetate extract, which is combined with the oil phase. The ethyl acetate is removed by vacuum concentration at 50℃, and the extract is dried under vacuum at 45℃ to constant weight to obtain monk fruit seed extract.

[0038] The preparation method of the extract of *Scirpus spp.* includes the following steps: homogenize the fruit of *Scirpus spp.*, mix with 12 times the volume of 60 vol% ethanol aqueous solution, adjust the pH to 2.0 with 0.15 M HCl aqueous solution, and extract by ultrasonication at 25°C and 500W intermittently (working for 2 seconds and pausing for 3 seconds) for 20 min under nitrogen protection. Centrifuge at 4000 rpm for 15 min, collect the supernatant, concentrate under reduced pressure at 40°C to remove ethanol, load the sample onto a polyamide chromatography column, first wash with deionized water to remove 6 BV of impurities, then elute with eluent (4 wt% ammonia aqueous solution and 90 vol% ethanol aqueous solution mixed at a volume ratio of 1:9) to remove 4 BV, collect the eluent, concentrate under reduced pressure at 45°C, and freeze-dry to obtain the extract of *Scirpus spp.*.

[0039] Example 3 A drug for treating atherosclerosis, comprising extracellular extract of Auricularia auricula-judae mycelium, cicada molting extract, Siraitia grosvenorii seed extract, Curcuma longa extract, total saponins of Panax notoginseng, tanshinone IIA and puerarin in a mass ratio of 3:2:0.8:1.5:2:0.5:2.

[0040] The preparation method of extracellular extract of Auricularia auricula-judae mycelium includes the following steps: Activation of the mycelium and preparation of the seed culture: Auricularia auricula-judae mycelium is inoculated onto PDA slant medium and cultured at 28℃ for 7 days. Mycelial blocks are then picked and inoculated into a seed culture medium (3wt% glucose, 1wt% soybean meal, 0.2wt% KH₂PO₄, 0.05wt% MgSO₄, with the remainder being water). The culture is then incubated on a shaker at 28℃ and 150 rpm for 7 days to obtain the seed culture, with a mycelial concentration of ≥0.8 g / L (dry weight). The seed culture is then inoculated into a fermentation medium (4wt% corn flour, 1wt% sucrose, 0.8wt% yeast extract, with the remainder being water, pH at rest) at an inoculation rate of 8%. The temperature is controlled at 25℃, the stirring speed at 200 rpm, and the sterile air flow rate at 1.0 vvm. The culture is then incubated for 5 days to obtain the proliferating bacterial culture. An inducing agent aqueous solution containing 0.5 wt% trehalose and 0.15 wt% salicylic acid was added to the proliferating bacterial culture (the addition rate was 1% of the proliferating bacterial culture volume every 24 hours), and the culture temperature was lowered to 20℃. At the same time, the stirring speed was reduced to 80 rpm, the sterile air flow rate was adjusted to 0.6 vvm, and fermentation was continued for 3 days to obtain the induced bacterial culture. The induced bacterial culture was centrifuged at 5000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm microfiltration membrane. The filtrate was collected, and 4.5 times the volume of 90 vol% ethanol aqueous solution was added. The mixture was allowed to stand at 6 °C for 18 h, centrifuged at 5000 rpm for 10 min, and the precipitate was collected. The precipitate was diluted with deionized water to 3 times its mass and loaded onto a DEAE-52 cellulose gel column. The column was first eluted with 0.08 M NaCl aqueous solution for 2 BV to remove impurities, and then eluted with 0.3 M NaCl aqueous solution for 5 BV. The eluent A was collected. The eluent A was loaded onto an propylene dextran gel S-400HR chromatography column and eluted with 0.15 M NaCl aqueous solution for 5 BV. The eluent B was collected and concentrated under reduced pressure at 45 °C. The eluent was placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with flowing deionized water for 30 h. The eluent B was then freeze-dried to obtain the extracellular extract of Auricularia auricula mycelium.

[0041] The preparation method of cicada molting extract includes the following steps: dried cicada molting is pulverized to pass through a 40-mesh sieve to obtain powder. Deionized water at 12 times the powder mass is added, along with 0.8% papain and 1.5% chitinase. The pH is adjusted to 6.0 with 0.1M citrate-disodium hydrogen phosphate buffer, and enzymatic hydrolysis is performed at 50℃ for 2 hours. Then, the pH is adjusted to 6.8 with 0.8M NaOH aqueous solution, and ginger protease at 0.8% of the powder mass is added. Enzymatic hydrolysis is performed at 55℃ for 1 hour, followed by enzyme inactivation at 90℃ for 10 minutes. After cooling, the mixture is centrifuged at 7500 rpm for 10 minutes. The supernatant is collected and concentrated under reduced pressure at 50℃ to 20% of its volume. The supernatant is loaded onto an HPD300 macroporous adsorption resin column. The column is first washed with deionized water (5 BV) to remove impurities, then eluted with 40 vol% ethanol aqueous solution (5 BV). The eluent is collected, concentrated under reduced pressure at 45℃ to remove ethanol, and then freeze-dried to obtain the cicada molting extract.

[0042] The preparation method of monk fruit seed extract includes the following steps: drying monk fruit seeds at 50℃, pulverizing them through a 100-mesh sieve, and performing subcritical water extraction. The extraction parameters are set as follows: temperature 160℃, pressure 4MPa, static extraction time 30min, followed by dynamic extraction. The extract is collected at a flow rate of 4mL / min and allowed to stand at 6℃ to separate the layers. The upper oil phase and the lower aqueous phase are taken separately. The aqueous phase is extracted four times with 1 volume of ethyl acetate to obtain an ethyl acetate extract, which is combined with the oil phase. The ethyl acetate is removed by vacuum concentration at 45℃, and the extract is dried under vacuum at 50℃ to constant weight to obtain monk fruit seed extract.

[0043] The preparation method of the extract of *Scirpus spp.* includes the following steps: homogenize the fruit of *Scirpus spp.*, mix it with 10 times the volume of 70 vol% ethanol aqueous solution, adjust the pH to 3.0 with 0.1 M HCl aqueous solution, and extract by ultrasonication at 20℃ and 600W intermittently (working for 2 seconds and pausing for 3 seconds) for 15 min under nitrogen protection. Centrifuge at 5000 rpm for 10 min, collect the supernatant, concentrate under reduced pressure at 45℃ to remove ethanol, load the sample onto a polyamide chromatography column, first wash with deionized water for 4 BV to remove impurities, then elute with eluent (5 wt% ammonia aqueous solution and 80 vol% ethanol aqueous solution mixed at a volume ratio of 2:8) for 6 BV, collect the eluent, concentrate under reduced pressure at 40℃, and freeze dry to obtain the extract of *Scirpus spp.*

[0044] The preparation method of the drug in the above embodiments includes the following steps: compounding the extracellular extract of Auricularia auricula-judae mycelium, cicada molting extract, Siraitia grosvenorii seed extract, Curcuma longa extract, total saponins of Panax notoginseng, tanshinone IIA and puerarin in a mass ratio to obtain a drug composition; mixing the drug composition with pharmaceutically available excipients to prepare a drug for preventing, stabilizing, delaying or treating atherosclerosis.

[0045] Comparative Example 1 The difference from Example 1 is that the mass ratio of the extracellular extract of Auricularia auricula-judae mycelium, cicada molting extract, monk fruit seed extract, burdock fruit extract, total saponins of Panax notoginseng, tanshinone IIA and puerarin is 0.5:3.5:1.2:1:1.8:0.4:1.5.

[0046] Comparative Example 2 The difference from Example 1 is that the mass ratio of the extracellular extract of Auricularia auricula-judae mycelium, cicada molting extract, monk fruit seed extract, burdock fruit extract, total saponins of Panax notoginseng, tanshinone IIA and puerarin is 2.5:1.5:0.2:2:1.8:0.4:1.5.

[0047] Comparative Example 3 The difference from Example 1 is that the mass ratio of the extracellular extract of Auricularia auricula-judae mycelium, cicada molting extract, monk fruit seed extract, burdock fruit extract, total saponins of Panax notoginseng, tanshinone IIA and puerarin is 2.5:0.5:2.2:1:1.8:0.4:1.5.

[0048] Comparative Example 4 The difference from Example 1 is that no inducing agent aqueous solution is added in the preparation method of the extracellular extract of Auricularia auricula mycelium.

[0049] Comparative Example 5 The difference from Example 1 is that salicylic acid is not added in the preparation method of the extracellular extract of Auricularia auricula mycelium.

[0050] Comparative Example 6 The difference from Example 1 is that in the preparation method of the extracellular extract of Auricularia auricula-judae mycelium, DEAE-52 cellulose gel is replaced with DEAE-23 cellulose gel.

[0051] Comparative Example 7 The difference from Example 1 is that in the preparation method of the extracellular extract of Auricularia auricula mycelium, the propylene dextran gel S-400HR is replaced with propylene dextran gel S-100HR.

[0052] Comparative Example 8 The difference from Example 1 is that ginger protease is not added in the preparation method of the cicada molting extract. Comparative Example 9 The difference from Example 1 is that in the preparation method of cicada molting extract, papain is replaced by bromelain.

[0053] Comparative Example 10 The difference from Example 1 is that in the preparation method of cicada molting extract, HPD300 macroporous adsorption resin is replaced with HPD600 macroporous adsorption resin.

[0054] Comparative Example 11 The difference from Example 1 is that in the preparation method of Luo Han Guo seed extract, the oil phase is discarded, and the aqueous phase is not extracted with ethyl acetate. Instead, the aqueous phase is directly concentrated under reduced pressure and dried to obtain the product.

[0055] Comparative Example 12 The difference from Example 1 is that in the preparation method of the burdock fruit extract, polyamide is replaced with macroporous adsorption resin D101.

[0056] The raw materials used in the above embodiments and comparative examples are as follows: Panax notoginseng total saponins are from Xi'an Lvteng Biotechnology Co., Ltd., health product grade, 80% purity. Tanshinone IIA is from Nanjing Guangrun Biological Products Co., Ltd., pharmaceutical grade, 98% purity. Puerarin is from Xi'an Tianfeng Biotechnology Co., Ltd., pharmaceutical grade, 98% purity. Soybean meal powder is from Lixin County Juxin Agricultural Co., Ltd. Yeast extract is from Shandong Xinfuwo Bioengineering Co., Ltd. Trehalose is from Shandong Pingju Biotechnology Co., Ltd. Salicylic acid is from Henan Qiande Pharmaceutical Co., Ltd. DEAE-52 cellulose gel is from Wuhan Kanos Technology Co., Ltd., DEAE cellulose DE-52. Acrylic dextran gel S-400HR is from Shanghai Zeye Biotechnology Co., Ltd. Cicada molts are from cicadas. Papain is from Shaanxi Shengyuantai Biotechnology Development Co., Ltd., enzyme activity 100,000 U / g. Chitinase is from Guangdong Yuanfeng Chemical Reagent Co., Ltd., enzyme activity 100,000 U / g. Ginger protease is from Shanghai Yimiao Chemical Technology Co., Ltd., enzyme activity 400 U / mg. HPD300 macroporous adsorption resin is from Zhengzhou Aino Chemical Technology Co., Ltd. Ethyl acetate is from Shanghai Yaokan Chemical Co., Ltd. Polyamide is from Shanghai Mairui Biochemical Technology Co., Ltd., 100-200 mesh grade, model M87011. DEAE-23 cellulose gel is from Shanghai Chunshi Biotechnology Co., Ltd., DEAE cellulose DE-23. Acrylic dextran gel S-100HR is from Shanghai Zeye Biotechnology Co., Ltd. Bromelain is from Shanxi Lanyuan Biotechnology Co., Ltd., enzyme activity 100,000 U / g. HPD600 macroporous adsorption resin is from Zhengzhou Aino Chemical Technology Co., Ltd. Macroporous adsorption resin D101 is from Shanghai Huzheng Biotechnology Co., Ltd.

[0057] The following tests were performed on the pharmaceutical composition (pure pharmaceutical, without excipients).

[0058] I. Hemolytic toxicity test: Sample dilution preparation: Take 20 mg of the drug and prepare a 1000 μg / mL stock solution with physiological saline. Filter the solution through a 0.22 μm filter membrane for sterilization, and then dilute it with physiological saline to prepare test solutions of 400 μg / mL, 200 μg / mL, and 100 μg / mL.

[0059] The detection method includes the following steps: Fresh anticoagulated blood (sodium citrate anticoagulation) from healthy individuals is collected, centrifuged at 1500 rpm for 10 min, the supernatant is discarded, and red blood cells are washed three times with physiological saline to prepare a 2% red blood cell suspension. 10 mL centrifuge tubes are used, and 2 mL of different concentrations of detection solution are added to each tube to create experimental groups; a positive control group (distilled water) and a negative control group (physiological saline) are also included, with three replicates per group. 2 mL of 2% red blood cell suspension is added to each tube, gently mixed, incubated in a 37°C water bath for 60 min, and centrifuged at 1500 rpm for 10 min. The supernatant is collected, and the OD value is measured at 545 nm using a spectrophotometer.

[0060] Evaluation index: Hemolysis rate = (OD value of experimental group - OD value of negative control group) / (OD value of positive control group - OD value of negative control group) × 100%.

[0061] Table 1 Hemolysis rate

[0062] The above results are averages.

[0063] II. Detection of Low-Density Lipoprotein Cholesterol (LDL-C) Oxidation Inhibition Rate: Sample dilution preparation: Take 5 mg of PBS buffer (pH 7.4) to prepare a 500 μg / mL stock solution, and then dilute it to prepare 400 μg / mL, 200 μg / mL, and 100 μg / mL test solutions.

[0064] The detection method includes the following steps: 100 μL of human serum LDL-C (concentration 1 mg / mL) is added to 100 μL of detection solutions of different concentrations to prepare experimental groups; simultaneously, blank groups (LDL-C + PBS) and model groups (LDL-C + 5 μmol / L CuSO4) are set up, with 3 replicates for each group, and incubated at 37℃ for 24 h. The LDL-C content of each group is determined using the cholesterol oxidase-peroxidase endpoint method.

[0065] Evaluation index: LDL-C oxidation inhibition rate = (LDL-C content in experimental group - LDL-C content in model group) / (LDL-C content in blank group - LDL-C content in model group) × 100%.

[0066] Table 2 LDL-C oxidation inhibition rate

[0067] The above results are averages.

[0068] III. Detection of foam cell formation inhibition (Oil Red O staining method): Sample dilution preparation: Take the sterilized drug stock solution (1000 μg / mL, dissolved in DMEM medium containing 10% fetal bovine serum) after filtration through a 0.22 μm filter membrane and dilute it to prepare test solutions of 400 μg / mL, 200 μg / mL, and 100 μg / mL.

[0069] The detection method includes the following steps: THP-1 cells (human monocytes) are obtained and induced to differentiate into macrophages with 100 nmol / L PMA for 48 hours; then, at a rate of 2 × 10⁻⁶ cells / year... 4 Seeds were inoculated into 24-well plates and cultured for 24 h. The old culture medium was discarded, and culture medium containing 80 μg / mL oxidized low-density lipoprotein (ox-LDL) and different concentrations of detection solution (500 μL total per well) was added to prepare the experimental group. Simultaneously, a blank group (no ox-LDL, no drug) and a model group (ox-LDL, no drug) were set up, with 3 replicates per group, and cultured for 48 h. The supernatant was discarded, and the sample was washed twice with PBS (pH 7.4). The sample was fixed with 4% paraformaldehyde solution (pH 7.4) for 30 min, the fixative was discarded, and the sample was washed twice with PBS (pH 7.4). The sample was stained with 0.5% Oil Red O staining solution (prepared with isopropanone) for 30 min, the staining solution was discarded, and the sample was differentiated with 70% ethanol for 10 s. The sample was washed three times with distilled water. 100 μL of isopropanol was added to each well, and the sample was shaken for 10 min. The OD value was measured at 510 nm using a microplate reader.

[0070] Evaluation index: Foam cell formation inhibition rate = (OD value of model group - OD value of experimental group) / OD value of model group × 100%, where the OD value of each group is the OD value after deducting the blank group; at the same time, observe the red lipid droplets in the cells, and the lipid droplet area is significantly smaller than that of the model group to be effective.

[0071] Table 3. Foam cell formation inhibition rate

[0072] The above results are averages.

[0073] IV. Detection of inflammatory factors (TNF-α, IL-6): Sample dilution preparation: The drug was dissolved in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin, and diluted to 400 μg / mL, 200 μg / mL, and 100 μg / mL test solutions, and then filtered through a 0.22 μm filter membrane for sterilization.

[0074] Detection method and steps: Take mouse RAW264.7 macrophages, and use 1×10 5Seeds were seeded per well in a 24-well plate, with 500 μL of RPMI-1640 medium containing 10% fetal bovine serum added to each well. The plates were incubated at 37°C and 5% CO2 for 24 h. The old medium was discarded, and 500 μL of medium containing 1 μg / mL LPS and different concentrations of detection solutions were added to each well to create experimental groups. A blank group (no LPS, no drug) and a model group (containing LPS, no drug) were also prepared, with three replicates per group. The plates were incubated for 24 h. The supernatant from each well was collected, centrifuged at 12000 rpm for 5 min, and the concentrations of inflammatory factors (TNF-α, IL-6) in the supernatant were measured according to the ELISA kit instructions.

[0075] Evaluation index: Inflammatory factor inhibition rate = (factor concentration in model group - factor concentration in experimental group) / factor concentration in model group × 100%, where the factor concentration in each group is the factor concentration after deducting the blank group.

[0076] Table 4 Inhibition rate of inflammatory factors

[0077] The above results are averages.

[0078] V. Detection of vascular smooth muscle cell (VSMC) proliferation inhibition: Sample dilution preparation: The drug was dissolved in DMEM medium (containing 5% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin) and diluted to 400 μg / mL, 200 μg / mL, and 100 μg / mL test solutions, and then filtered through a 0.22 μm filter membrane for sterilization.

[0079] Detection method and steps: Take VSMCs from the thoracic aorta of SD rats, and use 5×10 3 Cells were seeded per well in a 96-well plate, and 100 μL of DMEM medium containing 5% fetal bovine serum was added. The plates were incubated for 24 h until cell adhesion occurred. The old medium was discarded, and 100 μL of medium containing 10 ng / mL PDGF-BB and different concentrations of detection solution were added per well to create experimental groups. A blank group (no PDGF-BB, no drug) and a model group (containing PDGF-BB, no drug) were also set up, with three replicates per group. Cells were incubated for 48 h. 10 μL of 10 μmol / L BrdU solution was added to each well, and the plates were incubated for another 4 h. The OD value was measured at 450 nm using a microplate reader according to the BrdU detection kit instructions.

[0080] Evaluation index: VSMC proliferation inhibition rate = (OD value of model group - OD value of experimental group) / OD value of model group × 100%, where the OD value of each group is the OD value after deducting the blank group.

[0081] Table 5. VSMC proliferation inhibition rate

[0082] The above results are averages.

[0083] The test results above show that the drugs in Examples 1 to 3 performed well in tests for hemolysis, lipid-lowering and antioxidant effects, anti-inflammatory effects, inhibition of foam cell formation, and vascular smooth muscle cell (VSMC) proliferation, demonstrating both safety and potent therapeutic effects. They exhibit good formulation synergy, covering the core pathological links of atherosclerosis: lipid metabolism, inflammation and immunity, and vascular endothelial function; they can lower lipids, provide antioxidant effects, inhibit foam cell formation, and also have anti-inflammatory effects, protect the vascular endothelium, and stabilize plaques.

[0084] Comparative Examples 1 to 3 (Imbalance of Raw Material Ratio): The intervention of the drug on atherosclerosis relies on the synergistic effect of seven raw materials. Each raw material has a different focus and synergistic effect. When the mass ratio deviates, it will lead to the absence of intervention in one or more pathological links, disrupting the overall efficacy balance. At the same time, the imbalance of raw material ratio will damage the compatibility between components, increase the risk of hemolysis, and the excess of imbalanced components will trigger cellular stress response.

[0085] Comparative Example 4 (Preparation of *Auricularia auricula-judae* extract without inducers): Inducers (trehalose + salicylic acid) play a "signal regulation" role in the fermentation of *Auricularia auricula-judae* mycelium: Trehalose can activate the stress protection mechanism of the mycelium and promote the synthesis of antioxidant active substances (including polysaccharide derivatives); salicylic acid, as a signaling molecule, induces the mycelium to secrete specific extracellular products with anti-inflammatory and lipid metabolism-regulating properties. Without inducers, *Auricularia auricula-judae* mycelium only undergoes basal metabolism and cannot efficiently synthesize highly active products, resulting in a weakening of its core functions such as antioxidation (inhibition of LDL-C oxidation) and anti-inflammation (reduction of TNF-α and IL-6 secretion). At the same time, the basal metabolites contain trace amounts of small molecule impurities (such as ineffective metabolic waste), which increase the risk of hemolysis (disruption of erythrocyte membrane stability).

[0086] Comparative Example 5 (Preparation of *Auricularia auricula-judae* extract without salicylic acid): Salicylic acid is a key signaling molecule in the synthesis of *Auricularia auricula-judae* mycelium. It also regulates the extracellular secretion pathway of mycelium, improving the secretion efficiency of active ingredients. Without salicylic acid, trehalose alone cannot fully activate the metabolic pathway, leading to a reduction in the types and amounts of active ingredients in the *Auricularia auricula-judae* extract. This directly affects the inhibitory efficacy against foam cell formation and VSMC proliferation. Furthermore, the product purity is slightly reduced, and trace impurities may slightly increase the risk of hemolysis.

[0087] Comparative Example 6 (DEAE-23 replaced DEAE-52 for purification of Auricularia auricula-judae extract): The "ion exchange capacity" and "pore structure" of DEAE-cellulose gel determine the purification effect: DEAE-52 has a higher ion exchange capacity and a pore size that is more matched to the active polysaccharides in the Auricularia auricula-judae extracellular extract, which can accurately adsorb active ingredients and remove impurities (such as small molecule metabolic waste and ineffective proteins); while DEAE-23 has a low ion exchange capacity and a small pore size, which cannot completely adsorb active ingredients and is prone to leaving impurities.

[0088] Comparative Example 7 (Purification of Auricularia auricula-judae extract using S-100HR instead of S-400HR): S-400HR can accurately separate active macromolecular polysaccharides from Auricularia auricula-judae extract and remove small molecule ineffective components; while S-100HR has a low separation efficiency for macromolecular polysaccharides due to its limited retention range, easily leading to mixing of active polysaccharides with small molecule impurities. Impurities can interfere with the efficacy process (e.g., when inhibiting LDL-C oxidation, impurities compete for binding sites, reducing the efficiency of the active ingredient), resulting in a decrease in LDL-C oxidation inhibition rate and foam cell formation inhibition rate.

[0089] Comparative Example 8 (Preparation of Cicada Slough Extract without Ginger Protease): The effective components of cicada slough (including chitin derivatives and bioactive peptides) are encapsulated in a chitin-protein complex structure, requiring the synergistic degradation of multiple enzymes: papain and chitinase mainly degrade chitin and some proteins, while ginger protease can further degrade proteins, promoting the cleavage and activation of bioactive peptides to produce bioactive peptides. Without ginger protease, the release and generation rates of active components decrease, and more impurities remain. This directly leads to a decline in anti-inflammatory efficacy (insufficient bioactive peptides make it difficult to inhibit the secretion of inflammatory factors) and VSMC proliferation inhibition efficacy (low chitin derivative content makes it unable to effectively regulate smooth muscle cell proliferation).

[0090] Comparative Example 9 (Preparation of Cicada Slough Extract with Bromelain Replacing Papain): Papain exhibits high specificity for hydrolyzing the "chitin-protein cross-linking bonds" in cicada slough, effectively breaking the glycosidic bonds between chitin and protein and efficiently releasing the encapsulated active ingredients; while bromelain's active sites are mismatched, tending to hydrolyze simple proteins, resulting in low degradation efficiency of cross-linking bonds; furthermore, the peptide structures generated by enzymatic hydrolysis with ginger protease are different, leading to different active substance effects; the efficacy is reduced, and residual impurity proteins bind to the red blood cell membrane, slightly increasing the risk of hemolysis.

[0091] Comparative Example 10 (Cicada molting extract purification using HPD600 instead of HPD300): The specific surface area and polarity of the HPD300 macroporous adsorption resin are better matched to the active ingredients in cicada molting, enabling efficient adsorption of active peptides, chitin derivatives, and other active ingredients from cicada molting. After replacement with HPD600, the purity of active ingredients in the cicada molting extract decreased, and the impurity content increased. Insufficient active ingredients led to a decrease in the inhibition rate of foam cell formation and the inhibition rate of inflammatory factors.

[0092] Comparative Example 11 (Luo Han Guo Seed Extract Lacking Lipid-Soluble Active Ingredients): Without lipid-soluble components, Luo Han Guo seed extract cannot effectively intervene in the lipid deposition process. It is neither able to efficiently inhibit the oxidation of LDL-C to ox-LDL nor effectively prevent foam cell formation. Furthermore, the water-soluble components contain trace amounts of saponin impurities, which have some hemolytic activity and may slightly increase the risk of hemolysis.

[0093] Comparative Example 12 (Purification of *Scirpus spp.* extract using D101 instead of polyamide): Polyamide exhibits high adsorption specificity for polyphenolic active ingredients in *Scirpus spp.*, effectively removing impurities such as sugars and organic acids. In contrast, D101 macroporous adsorption resin is a non-polar resin, its adsorption of polyphenols relies on hydrophobic interactions, resulting in low specificity and the potential for residual sugar impurities. After replacement, the content of polyphenolic active ingredients in the *Scirpus spp.* extract decreased, while impurities increased. Insufficient polyphenols led to a decline in anti-inflammatory efficacy (reduced TNF-α inhibition rate) and foam cell formation inhibition rate.

Claims

1. A drug for treating atherosclerosis, characterized in that, The drug is composed of extracellular extracts of Auricularia auricula-judae mycelium, cicada molting extract, Siraitia grosvenorii seed extract, Curcuma longa extract, total saponins of Panax notoginseng, tanshinone IIA and puerarin in a mass ratio of (2-3):(1-2):(0.8-1.5):(0.8-1.5):(1.5-2):(0.3-0.5):(1-2); The preparation of the extracellular extract of Auricularia auricula mycelium includes: inducing fermentation of Auricularia auricula mycelium with an inducer containing trehalose and salicylic acid to obtain an induced bacterial broth; adding an ethanol aqueous solution, allowing it to stand, centrifuging, collecting the precipitate, diluting it with deionized water, loading it onto a DEAE-52 cellulose gel column, rinsing with 0.08M-0.1M NaCl aqueous solution to remove impurities, eluting with 0.3M-0.35M NaCl aqueous solution, collecting eluent A; loading it onto an propylene dextran gel S-400HR chromatography column, eluting with 0.15M-0.2M NaCl aqueous solution, collecting eluent B, concentrating under reduced pressure, dialyzing, and freeze-drying to obtain the extracellular extract of Auricularia auricula mycelium; The preparation of the cicada molting extract includes the following steps: cicada molting is enzymatically hydrolyzed by papain and chitinase, followed by enzymatic hydrolysis by ginger protease. The product is then adsorbed onto an HPD300 macroporous adsorption resin column, washed with deionized water to remove impurities, eluted with 40 vol% to 45 vol% ethanol aqueous solution, the eluent is collected, concentrated under reduced pressure, and freeze-dried to obtain the cicada molting extract. The preparation of the monk fruit seed extract includes subcritical water extraction of monk fruit, collection of the extract, standing and separation of layers, taking the oil phase and the aqueous phase, extracting the aqueous phase with ethyl acetate to obtain the ethyl acetate extract, combining it with the oil phase, concentrating under reduced pressure, and drying under vacuum to obtain the monk fruit seed extract. The preparation of the burdock fruit extract includes ultrasonic extraction of burdock fruit in an ethanol aqueous solution at pH 2.0–3.0, followed by purification of the extract by a polyamide chromatography column to obtain the burdock fruit extract.

2. The drug for treating atherosclerosis according to claim 1, characterized in that, The preparation method of the extracellular extract of *Auricularia auricula-judae* mycelium includes the following steps: Seed culture of *Auricularia auricula-judae* is inoculated into a fermentation medium and cultured to obtain a proliferating culture; an inducing agent aqueous solution containing 0.5wt%–0.8wt% trehalose and 0.1wt%–0.15wt% salicylic acid is added to the proliferating culture to induce fermentation, obtaining an induced culture; the culture is centrifuged, the supernatant is collected, filtered through a microfiltration membrane, the filtrate is collected, an ethanol aqueous solution is added, the culture is allowed to stand, centrifuged, the precipitate is collected, the precipitate is diluted with deionized water, and the precipitate is loaded onto a DEAE-52 cellulose gel column. First, 1 BV–2 BV of 0.08M–0.1M NaCl aqueous solution is used to remove impurities, then 4 BV–5 BV of 0.3M–0.35M NaCl aqueous solution is used to elute, and eluent A is collected; eluent A is loaded onto an propylene dextran gel S-400HR column and eluted with 0.15M–0.2M NaCl aqueous solution. Elute with NaCl aqueous solution for 4-5 BV, collect eluent B, concentrate under reduced pressure, pack into a dialysis bag, dialyze, and freeze-dry to obtain extracellular extract of Auricularia auricula mycelium.

3. The drug for treating atherosclerosis according to claim 2, characterized in that, The mycelial concentration of the seed culture is above 0.8 g / L; the inoculation amount of the seed culture is 8%–10% of the fermentation medium volume; the fermentation medium contains 3 wt%–4 wt% corn flour, 1 wt%–2 wt% sucrose, and 0.5 wt%–0.8 wt% yeast extract; the proliferation culture is carried out at 25℃–28℃ with sterile air flow of 1.0 vvm–1.5 vvm and stirring at 150 rpm–200 rpm for 4–5 days; the flow rate of the inducer aqueous solution is 1%–1.5% of the volume of the proliferation culture liquid added every 24 hours; the induced fermentation is carried out at 18℃–20℃ with sterile air flow of 1.0 vvm–1.5 vvm and stirring at 150 rpm–200 rpm for 4–5 days; Fermentation was induced for 3 to 4 days under sterile air volume of 0.4 vvm to 0.6 vvm and stirring at 80 rpm to 100 rpm; the microfiltration membrane was a 0.22 μm pore size microfiltration membrane; the ethanol concentration of the ethanol-water solution was 90 vol% to 95 vol%; the amount of ethanol-water solution added was 4 to 4.5 times the volume of the filtrate; the standing was carried out at 4℃ to 6℃ for 18 to 24 hours; the centrifugation was carried out at 4000 rpm to 5000 rpm for 10 to 15 minutes; the molecular weight cutoff of the dialysis bag was 1000 Da; the dialysis was performed with flowing deionized water for 24 to 30 hours.

4. The drug for treating atherosclerosis according to claim 1, characterized in that, The preparation method of the cicada molting extract includes the following steps: Pulverize dried cicada molting to obtain powder, add 8 to 12 times the weight of the powder in deionized water, add papain and chitinase, adjust the pH to 5.5 to 6.0, and enzymatically hydrolyze at 50℃ to 55℃ for 1.5 to 2 hours. Then adjust the pH to 6.4 to 6.8, add ginger protease, and enzymatically hydrolyze at 50℃ to 55℃ for 1 to 1.5 hours to inactivate the enzyme. Cool, centrifuge, collect the supernatant, concentrate under reduced pressure, and load the sample onto an HPD300 macroporous adsorption resin column. First, rinse with deionized water for 4 BV to 5 BV to remove impurities, then elute with 40 vol% to 45 vol% ethanol aqueous solution for 4 BV to 5 BV. Collect the eluent, concentrate under reduced pressure, and freeze-dry to obtain the cicada molting extract.

5. The drug for treating atherosclerosis according to claim 4, characterized in that, The particle size of the powder is such that it passes through a 40-60 mesh sieve; the amount of papain added is 0.8%-1.5% of the powder mass; the amount of chitinase added is 0.8%-1.5% of the powder mass; the amount of ginger protease added is 0.8%-1.5% of the powder mass; the enzyme inactivation is performed at 85℃-90℃ for 10-15 minutes; the centrifugation is performed at 7000rpm-7500rpm for 10-15 minutes.

6. The drug for treating atherosclerosis according to claim 1, characterized in that, The preparation method of the monk fruit seed extract includes the following steps: drying and pulverizing monk fruit seeds, performing subcritical water extraction, collecting the extract, allowing it to stand and separate into layers, and taking the upper oil phase and the lower aqueous phase separately; extracting the aqueous phase with ethyl acetate to obtain ethyl acetate extract, combining it with the oil phase, concentrating under reduced pressure, and drying under vacuum to obtain monk fruit seed extract.

7. The medicament for treating atherosclerosis according to claim 6, characterized in that, The parameters for the subcritical water extraction are: temperature 140℃~160℃, pressure 4MPa~4.5MPa, static extraction time 20min~30min, followed by dynamic extraction, with the extract collected at a flow rate of 4mL / min~6mL / min; the temperature for static separation is 4℃~6℃; and the aqueous phase is extracted 3 to 4 times with 1 to 1.2 times the volume of ethyl acetate.

8. The drug for treating atherosclerosis according to claim 1, characterized in that, The preparation method of the burdock fruit extract includes the following steps: homogenizing the burdock fruit, mixing it with 10 to 12 times the volume of 60 vol% to 70 vol% ethanol aqueous solution, adjusting the pH to 2.0 to 3.0, ultrasonically extracting, centrifuging, collecting the supernatant, concentrating under reduced pressure, loading the sample onto a polyamide chromatography column, first rinsing with deionized water for 4 BV to 6 BV to remove impurities, then eluting with eluent for 4 BV to 6 BV, collecting the eluent, concentrating under reduced pressure, and freeze-drying to obtain the burdock fruit extract.

9. The medicament for treating atherosclerosis according to claim 8, characterized in that, The ultrasonic extraction is performed under nitrogen protection at 20℃~25℃ and 500W~600W for 15min~20min; the centrifugation is performed at 4000rpm~5000rpm for 10min~15min; the eluent is a mixture of 4wt%~5wt% ammonia solution and 80vol%~90vol% ethanol solution at a volume ratio of (1~2):(8~9).

10. A method for preparing a medicament for treating atherosclerosis according to any one of claims 1 to 9, characterized in that, The process includes the following steps: compounding the extracellular extract of Auricularia auricula-judae mycelium, cicada molting extract, Siraitia grosvenorii seed extract, Curcuma longa extract, total saponins of Panax notoginseng, tanshinone IIA and puerarin in a certain mass ratio to obtain a pharmaceutical composition.

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